129 REVIEW ARTICLE GLAUCOMA: CURRENT AND

EUROPEAN JOURNAL OF INFLAMMATION

1721-727X (2006)Copyright © by BIOLIFE, s.a.s.

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REVIEW ARTICLEGLAUCOMA: CURRENT AND DEVELOPING CONCEPTS FOR INFLAMMATION,

PATHOGENESIS AND TREATMENT

N.A. KUMARASAMY, F.S. LAM, A.L. WANG and T.C. THEOHARIDES1

Department of Biology, Tufts University, Medford, Massachusetts, 1Departments of Pharmacology and Experimental Therapeutics, Internal Medicine and Biochemistry, Tufts University School of

Medicine, Boston, Massachusetts, USA

The first three authors contributed equally to the manuscript

Received January 18, 2006 – Accepted October 4, 2006

Glaucoma is a prevalent neurodegenerative disorder of the eye. However, the mechanism leading to the disease is still unclear. Increased intraocular pressure (IOP) and subsequent retinal ganglion cell (RGC) death leading to the loss of visual field characterizes the pathology of primary open angle glaucoma, which is the most common form. Possible factors leading to glaucoma include glutamate-induced neurotoxicity, nitric oxide (NO) based damage, disruption of neurotrophic factor transport and immune-induced neurodestruction. Current treatment options primarily aim at decreasing IOP by utilizing pharmacological agents, laser therapy and surgery. Developing treatments target neuroprotection with vaccines, the inhibition of NO synthesis and apoptosis. Gaining a better understanding of the pathogenesis can aid in the development of new treatment options and, perhaps, even a cure for glaucoma.

Mailing address: Normandan A. Kumarasamy,Columbia University,Mailman School of Public Healt,Healt Policy and Management50 Haven Avenue, New York, NY 10021, USATel: +1 617 216 9887 - Fax: +1 732 422 3604e-mail: [email protected]

Key words: glaucoma, glutamate neurotoxicity, autoimmunity, neuroprotection, erythropoietin

Glaucoma is characterized by the slow, progressive degeneration of retinal ganglion cells (RGCs) and optic nerve axons. The disease affects over 66 million people worldwide, causing bilateral blindness in 6.8 million (1). The most common form of the disease is primary open angle glaucoma (POAG), with a prevalence ranging from 0.58% in Asian Americans and as high as 6.7% in African Americans (2). African-Americans have a greater prevalence of the disease, contributed by optic nerve damage occurring at an earlier age, incurring more severe damage than other ethnic groups and experiencing less surgical repair successes. There are several recognized risk factors for glaucoma, such as an increased intraocular

pressure (IOP), aging, family history, high myopia, systemic hypertension, cardiovascular disease, migraine headaches, peripheral vasospasm and prior nerve damage (1, 3-4). The visual damage incurred from glaucoma is considered irreversible. However, it can be treated if diagnosed at an early enough stage. Since blindness is ultimately induced by the loss of visual field caused by neuronal cell death, understanding the pathology that leads to increased IOP, and subsequently to neuronal cell death, is crucial to the development of effective treatments.

Current understanding of pathologyPOAG is typically diagnosed by the presence

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130 131Eur. J. Inflamm.

of the “classical triad”: characteristic pattern of visual field defects, morphological loss of optic disk substance and increased IOP (2). Central visual acuity is relatively resistant to glaucomatous damage in the initial stages of the disease, with the peripheral vision mainly affected. Selective perimetry and visual field testing can effectively identify changes to the peripheral field. Examination of the optic disk is perhaps the most valuable method in early diagnosis, characterized by a concomitant decrease in the width of the neuroretinal rim and enlargement of the optic disc cup (1). Increased IOP of greater than 21 mm Hg (mean adult of 15-16 mm Hg) is not required for diagnosis, though it has traditionally been suspected to cause glaucoma (5). Patients with POAG typically exhibit increased resistance to the outflow of aqueous humor through the trabecular meshwork, which can result in an increase in IOP and subsequent cell death from compression of the optic nerve axons (1).

Genetically, no single Mendelian mode of inheritance can describe POAG (1). Rather, it seems to be determined by the interactions of multiple genes which may either increase or decrease the risk of glaucoma. In contrast, other forms of glaucoma, including congenital glaucoma and Reiger syndrome, can be mapped to specific genes leading to deformation of the anterior chamber of the eye (6). Recently, genomic mutations that are associated with POAG have been identified; one example is myocilin (MYOC), which may be responsible for 2-4% of POAG in the world (6). The mutation exhibits an autosomal dominant pattern of inheritance. MYOC itself is expressed in the human trabecular meshwork and several other ocular tissues, though its cellular function is still unknown (6)

DEVELOPING CONCEPTS OF PATHOLOGY NeurotoxicityGlutamate ExcitotoxicityActivation of glutamate receptors may contribute

to cellular apoptosis and neuronal cell death. In glaucoma, there is a chronic elevation of glutamate concentrations in the inner eye (7). Glutamate receptors are either metabotrophic (mGluR) or ionotropic (8).

The effects of mGluRs have been less characterized in glaucoma, but studies suggest a

relationship between increased IOP and mGluR (8-9). There is a significant increase in the expression of several mGluR subunits, in glaucomatous mice when compared to the control mice (8). However, it is unknown if these observed changes in mGluR contribute directly to cellular apoptosis or occur as a result of apoptosis (8).

Neurotoxicity resulting from ionotropic receptors has been better characterized. In glaucoma, it is thought that activation of both NMDA and non-NMDA ionotropic receptors increases intracellular calcium, stimulating a variety of intracellular enzymes, which ultimately leads to RGC death (10). However, a recent study suggests RGCs are in fact invulnerable to glutamate excitotoxicity; the RGC layer has equal numbers of RGCs and displaced amacrine cells, suggesting that past experiments may have misinterpreted cell death in the RGC layer without proper distinction between RGCs and amacrine cells (7). Past studies may also have only observed cell death in prolonged exposures (>24 hours), where cell death of RGCs is observable. However, this may be due to trophic deprivation from loss of amacrine signals, rather than delayed excitotoxicity (7).

NO NeurotoxicityNO is known as a second messenger, important

in the relaxation of smooth muscle. Excess NO is associated with stroke, Parkinson’s disease, Alzheimer’s disease and multiple sclerosis (11). Nitric oxide synthase-2 (iNOS-2) is an inducible form of NOS that is not normally present in tissue, but it makes an excessive quantity of NO when present (12). In glaucoma, intense labeling of iNOS-2 in reactive astrocytes of severely damaged regions was observed. Stimulation of the human lamina cribrosa astrocyte culture with cytokines results in intense labeling of iNOS-2, with subsequent excess NO production inducing neurotoxicity (13).

Disrupted transport of neurotrophic factorsNeurotrophins, or neurotrophic factors (NTFs), are

polypeptides that regulate the growth, differentiation and death of neurons. They originate from the superior calliculus of the central nervous system (CNS), and their transport may be disrupted by high IOP (14). NTFs can also be made locally in the retina, lamina

N.A. KUMARASAMY ET AL.


cribosa or optic nerve body, and act in an autocrine or paracrine fashion (15). The lack of NTFs has been suggested to be a factor that causes RGC apoptosis. These factors bind and phosphorylate Trk receptors, resulting in an anti-apoptotic effect. The expression of Trk receptor families (TrkA, B and C) also partially regulates p75 receptors, which induce apoptosis when activated (14).

Under high IOP, transient expression of TrkA receptors and truncated TrkC receptors in the glial cells have been observed (14). Transient expression of TrkA receptors may not provide sufficient neurotrophic support, and ineffective truncated TrkC receptors could competitively inhibit the TrkC receptor, which further decreases the available neurotrophins to the RGC since NTFs act in an autocrine or paracrine fashion locally. Furthermore, since p75 receptors are partially controlled by Trk receptors, truncated TrkC receptors may contribute to p75 activation. Under increased IOP, p75 was upregulated (14).

Neurinflammation Long term presence of lymphocytes and antigens

from sustained neurodegeneration can lead to increased T-cells. Cytokines are subsequently released, resulting in secondary antigen-mediated neurotoxicity similar to that in autoimmune diseases. A 67% increase in cytokine receptors has been detected in normal pressure glaucoma (NPG) patients, and a 77% increase in POAG patients, suggesting T-cell over-activation in glaucomatous patients (16).

In support of autoimmune neuropathy in NPG, there has been an epidemiological association of immune-related disease with 30% of NPG patients, versus 8% of control patients (16). Autoantigens in NPG are similar to those found in autoimmune thyroid disease, rheumatoid arthritis and type 1 diabetes (17). This may provide the link to the increased association of diabetes and glaucoma. An example of molecular mimicry that has been suggested as a pathogenic factor in glaucoma is the antibodies for heat shock proteins (hsps) (17). Hsps assist in protein folding and assembly, and are initially helpful by responding to stressors increased IOP. However, they become harmful when they persist and can induce neuronal apoptosis (18).

Glaucoma-stressed neurons can also lead to cell death via immune response surveillance. Cell death occurs by the activation of a protease called caspase, which depletes the cell of energy sources, NAD and ATP, eventually cleaving and unraveling DNA. Ischemia and the excessive production of hsp antibodies have been found to activate caspase (19). Thus, sustained optic neuropathy can result from normal immune surveillance repairing infected neuronal cells (Fig. 1).

NeuroprotectionT-cells can protect neurons from axonal injury by

destroying damaged cells and removing pathogenic agents, referred to as protective immunity (18). When splenocytes from rats with neurons resistant to damage caused by IOP were transferred to rats with susceptible neurons, the susceptible rats became resistant to damage caused by high IOP (20). This suggests that the susceptibility of neuronal damage to high IOP is immune-dependent.

Some degree of T-cell activity may be necessary for the survival of neurons at an increased IOP state. A recent study investigated whether methylprednisolone can eliminate T-cell induced inflammation due to CNS insult, resulting in the destruction of neurons (21). Researchers injected methylprednisolone to glaucomatous rat eyes and found that not only did it fail to protect RGC from inflammation-induced death, but it also caused RGC death, suggesting the presence of T-cells might be necessary for neuroprotection (21) .

CURRENT TREATMENT OPTIONSThe goal of treating glaucoma lies primarily on

preventing or delaying the loss of visual field (13). Since neuronal cell death is irreversible, no cure is available once the visual field is lost. However, since IOP is the primary risk factor causing the loss of RGCs, the strategies of treatment mostly involve lowering IOP (13). Other important factors such as cost, convenience and safety should also be considered (22). Current treatments for glaucoma include medication, laser use and surgery.

MedicationsMedications involve inhibiting the inflow of

aqueous humor, enhancing the outflow of aqueous


humor, protecting the optic nerves (23) and manipulating the osmotic pressure between plasma and the eyes (5).

α2 adrenoreceptor agonists and β1 receptor antagonists lower IOP by inhibiting the inflow of aqueous humor to the eye. Timolol, which is the most prescribed drug, and betaxolol, which has the fewest systemic side effects, are both β1 receptor blockers (23). A third type of drug that inhibits the inflow of humor is carbonic anhydrase inhibitors, such as acetazolamide and dorzolamide. Such drugs are often formulated together as in Cosopt (dorzolamide hydrochloride and timolol maleate) (22).

Another method of reducing IOP is by enhancing the outflow of humor from the eyes through the use of muscarinic acetylcholine receptor agonists (23-24). This mechanism is indirect, but involves a muscarinic acetylcholine receptor (M3)-mediated contraction of the ciliary muscle (23). The contraction causes the widening of the spaces in the trabecular meshwork. The newest class of drugs using this strategy is the prostaglandin F2α derivatives which enhance the uveoscleral outflow (25). Bimatoprost falls under this category and is considered the most effective anti-glaucoma drug (23).

Laser and SurgeryA secondary choice of treatment of glaucoma

is the use of laser therapy. The primary strategy involves “burning” holes in various areas within the eyes including the ciliary and the pigmented trabecular meshwork cells (24). The benefits include being noninvasive, needing less patient compliance and lowering the possibility of infection or bleeding. The IOP of most patients can decrease about 20-30%, but the treatment effect wears off 5-10% every year. In combination with timolol, the two year IOP lowering success rate is 70%, compared with the laser alone (44%) and timolol alone (30%) (24).

A common form of surgery is trabeculectomy, which creates a guarded channel allowing aqueous humor to flow from the anterior chamber inside the eye to sub-Tenon’s and subconjunctival space (24-25). The benefits of surgery include stabilizing IOP and bypassing the requirements for strict patient compliance and continuous drug costs (24). Surgery is considered as the last resort because failure of surgery can result in immediate blindness due to

complications such as choroidal effusion, hypotonic maculopathy, suprachoroidal hemorrhage and optic nerve snuffing (24).

FUTURE TREATMENT OPTIONSBased on new knowledge gained from research

on the pathology of neuronal apoptosis, there are several forms of new treatments. Many of these treatments focus on neuroprotection through anti-apoptosis of RGCs.

Neuroprotective VaccinesSince resistance to high IOP is immune-

dependent, T-cell induced neuroprotection may vaccinate the RGC from apoptosis. For example, copolymer-1 may be used as a vaccine because it is an antigen that cross-reacts with a wide range of T-cells, and can provoke a protective immune response to protect RGC from cell death caused by toxins or increased IOP (21).

A crucial consideration for designing neuroprotective vaccines is the site of protection. Some have suggested that the target should be in the RGC and not in the optic nerves because during the early glaucomatous stage cell death induced by increased IOP occurs in the RGC, not in the optic nerve (21). R16, a peptide derived from the RGC, is one example of a neuroprotective vaccine. Although R16 can cause slight RGC death for those without glaucoma, the benefit from this treatment far exceeded the damage from untreated glaucoma conditions (21).

NMDA Receptor AntagonistsMemantine is a NMDA receptor antagonist that

blocks excessive NMDA receptor activity. This drug has been approved for clinical use in Europe for the treatment of Alzheimer’s disease and vascular dementia, though its effectiveness in preventing glaucomatous RCG degeneration is still unknown (26).

iNOS-2 InhibitorsSince the upregulation of iNOS-2 is harmful to

neurons, its inhibition might have a neuroprotective effect. An inhibitor such as aminoguanidine can prevent the death of about 75% of RGCs during 6 months of induced elevated IOP and prevent further loss of RGC (12).



Although an unregulated level of NO can cause neuronal degeneration via apoptosis, a small amount of NO could inhibit apoptosis. Survival of serum deprived peochromocytoma PC-12 cells was observed when treated with S-nitroso-N-acetylpenicillamine (Nipradilol), which is an NO donor (27). Nipradilol is unique because it can decrease both IOP and protect neurons by releasing a small amount of NO.

Nutritional SupplementsAn interesting alternative to aminoguanidine is

Ginkgo biloba extract (EGb 761). Ginkgo biloba is used as a nutritional supplement for the reduction of platelet aggregation, vasodilation and reduction of blood viscosity (28). EGb 761 contains 24% flavonoid glycosides and 6% terpenoids, which may inhibit toxicity and NO free radical accumulation by inhibiting iNOS (29). In experiments when IOP is elevated in rats by cautery of episcleral vessels, feeding the rats 30 mg of EGb 761 per day for 5 months decreased RGC loss from 29.9% to 4.6% (n=5) (29). Further investigation monitoring the modulation of NO in response to EGb 761 treatment could provide insight into its neuroprotective mechanism.

In human models, EGb 761 was able to significantly improve the visual field indices in patients with NPG and pre-existing visual field

damage (30). However, a discontinuation of EGb 761 resulted in a reversion of visual field indices, suggesting that EGb 761 may require continuous administration (28). While EGb 761 may be appealing because it is a nutritional supplement and not a prescription drug, care should be taken because the effects of EGb 761 are not fully understood and there have been some reports of toxicity.

Calcium Channel BlockersMost apoptotic mechanisms involve rising

intracellular calcium levels. Flunarizine, a calcium channel blocker, has been shown to significantly enhance RGC survival in rat and rabbit models by reducing IOP ((31-32). However, the exact mechanism of this and other calcium channel blockers needs to be elucidated.

STAT-3 ActivationAnother target is the signal transducers and

activators of transcription protein-3 (STAT-3). They play an important role in cell growth and differentiation and are of interest because the mRNA of this protein is upregulated in rats with glaucoma (33).

The activation of STAT-3 pathway might inhibit apoptosis by suppressing caspase-3. One molecule that has been investigated is ciliary neurotrophic factor (CNTF), which is an interleukin-6 cytokine.

Fig. 1. Potential roles of the immune system in facilitating glaucomatous damage (19).

Glaucoma 18

Fig. 1. Potential roles of the immune system in facilitating glaucomatous damage

(19).

Immunesystem

Autoimmune mechanism

Immunesurveillance

Antibodies injure ganglion cells

Ganglion cell injury via molecular mimicry

Arbiter of ganglion cell death following stress

direct

indirect

Evidence

Monoclonal gammopathy, small heart shock protein

antibodies

Evidence

Rhodopsin antibodies,heat shock protein 60

antibodies, GAG antibodies

Stressors

IOP/mechanical stress, neurotrophin withdrawal,

ischemia, glutamate toxicity, NO synthase, reactive oxygen species

Direct Indirect

Immunesystem


The injection of CNTF into rat eyes with increased IOP reduces apoptosis, phosphorylates STAT-3, and reduces the activity of caspase-3 (34). Interleukin-10 also has neuroprotective activity through the STAT-3 pathway (35).

Caspase InhibitorsInhibitors of apoptosis protein (IAP) can also

reduce apoptosis by inhibiting caspase. Baculoviral IAP repeat-containing protein-4 (BIRC-4) is a direct inhibitor of caspase 3, 7 and 9. BIRC-4, transducted into the eye, can inhibit apoptosis of optic nerve axons (36).

A pharmacological approach for apoptosis

inhibition is the use of minocycline, which can inhibit caspase-3-induced apoptosis (37). It increases the survival rate of RGC exposed to the harmful effects of glutamate. Furthermore, it can function synergistically with MK-801, an antagonist to NMDA receptors, to increase RGC survival rate.

Heat Shock ProteinsGeranylgeranylacetone (GGA) is an acyclic

polyisoprenoid currently used in Japan as an anti-ulcer drug. The neuroprotective effects of the drug are facilitated through the use of hsps. In particular, HSP72 seems to act as an anti-apoptotic chaperone protein that interferes with multiple stages in the

Glaucoma 19

Table I. Overview of glaucoma pathophysiology, treatment and method of action.

Pathophysiology Treatment Method of Action Brimonidine �2-adrenoreceptor agonists

Timolol, Betatoxol �1-receptor antagonists Increased IOP (reduce aqueous

inflow) Acetozolamide, Dorzolamide Carbonic anhydrase inhibitors

Pilocarpine Muscarinic acetylcholine receptor agonists

Latanoprost, Travoprost, Bimatoprost Prostanoid receptor agonists

Laser therapy

CurrentTreatments

Increased IOP (increase aqueous

outflow) Surgery (e.g.

trabeculectomy or artificial drainage implants)

Better drainage through the trabecular meshwork

Copolymer-1 Immune protection through cross-reactivity with T-cells

Memantine NMDA receptor antagonist aminoguanidine, Gingko biloba extract (EGb 761) NOS inhibitor

Flunarizine Calcium channel blocker ciliary neurotrophic factor

(CNTF)STAT-3 activation � caspase-3

inhibition BIRC-4 Direct inhibition of caspases

Minocycline Caspase-3 inhibitor Geranylgeranylacetone Hsps mediation

Develop

RGC apoptosis (neuroprotection)

Erythropoietin PI-3kinase/Akt pathway

REFERENCES

1. Weinreb R.N. and P.T. Khaw. 2004. Primary open-angle glaucoma. Lancet

363:1711.

Table I. Overview of glaucoma pathophysiology, treatment and method of action.


Cur

rent

Tre

atm

ents

Dev

elop

ing

Trea

tmen

ts

Prostaglandin F2α analogues


apoptotic pathway. Mechanistically, the drug is believed to activate Heat Shock Factor 1 (HSF-1), a transcription factor for hsps, which oligomerizes in the cytosol and translocates into the nucleus when exposed to stressors (38).

ErythropoietinErythropoietin (EPO) is a hematopoietic factor

that stimulates differentiation and proliferation of erythroid progenitor cells. It is also neuroprotective and may prevent further RGC death by inhibiting apoptosis (39). EPO receptors are found on cell bodies and dendrites of RGCs. Intravitreal injection of EPO in rats with axotomized RGCs enhances RGC survival by 92% compared to those without EPO injection. However, RGC survival rate is dependent on the dosage. Dosage less than 4 μl of 0.1M EPO decreases its efficiency, but does not have the toxic effects of higher doses (39).

The suggested mechanism of neuroprotection is by the PI-3kinase/Akt pathway, which inhibits apoptosis by maintaining mitochondrial membrane potential, preventing cytochrome c release, and consequently inhibiting caspase (40). A membrane potential indicator shows that the addition of EPO maintains the function of RGCs by decreasing depolarization caused by anoxia or excessive NO. In addition, EPO reduces caspase activity, indicated by a decrease in the absorbance of colorimetric caspase substrates (40).

While axotomy, neurotrophic deprivation and increased NO mimic the conditions of glaucoma, it is important to note that IOP was not increased in these experiments, and thus the application to glaucoma treatment is not perfect. The injection of EOP does not regenerate RGCs, but it can prevent neuronal injury. EPO decreases DNA fragmentation caused by anoxia or NO, indicated by terminal deoxynucleotidyl transferase nick end labeling. Care should also be taken to ensure that erythrocytes from EOP injection do not aggregate in the retina and cause occlusion (40).

CONCLUSION

Glaucoma is a common neurodegenerative disorder characterized by loss of RGCs, ultimately leading to reduction of the visual field and potential

blindness. New and emerging research has provided a number of insights about the pathology, identifying new underlying factors including glutamate excitotoxicity, NO neurotoxicity, disruption of neurotrophic factor transport and aberrant autoimmune responses. As a result, novel targets for treatment are now in development with the primary objective of neuroprotection through anti-apoptotic mechanisms. Examples include neuroprotective vaccines, NMDA receptor antagonists, calcium channel blockers, NOS-2 inhibitors, erythropoietin and a number of drugs and supplements that inhibit the caspase pathway. It is, however, important to note that these factors are not independent of each other. Treatment interactions and their effects on other tissues should also be researched, especially since results from various experiments seem contradictory. For example, excess NO causes neuronal death, but less might inhibit apoptosis. Aberrant immune responses may lead to neuronal damage, but their absence might also promote neuronal death. While all current research is geared towards treatment options, the possibility of RGC regeneration should be explored to search for a glaucoma cure.

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HEREDITARY HEMORRHAGIC TELANGIECTASIA (HHT) IN DENTISTRY:A LITERATURE REVIEW

A. BALLINI1,2, S. CANTORE2, G. DE FRENZA2, C. FOTI3, C. CASULLI3, L. SALINI4

M. RAPANI5, F. MASTRANGELO, S. TETÈ5 and F.R. GRASSI2

1Department of Internal Medicine and Public Health, Division of Medical Genetics, University of Bari; 2Department of Odontostomatology and Surgery, University of Bari; 3 Department of Internal

Medicine, Immunology and Infectious Diseases, Unit of Dermatology, University of Bari, Bari; 4D.B.S. - A.S.L. of Chieti; 5Department of Oral Sciences, University G. d’Annunzio, Chieti, Italy

Received August 2, 2006 – Accepted October 10, 2006

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal-dominantly-inherited vascular dysplasia characterized by age-dependant incomplete penetrance and variable expressivity, with clini-cal manifestations consisting in epistaxis, mucocutaneous telangiectases, gastrointestinal bleeding and arteriovenous malformations (AVMs), which affects approximately 1⁄2 million people world-wide. It af-fects males and females of all racial and ethnic groups. Up to 1/3 of HHT patients have multiple organ involvement, which can be disabling and/or life threatening. HHT can be treated successfully if correctly diagnosed. Morbidity of HHT is often due to complications of AVMs, such as stroke or haemorrhage, also known to occur in children. Many authors have reported successful new therapeutical options for AVMs, which have resulted in a significant decrease of life-threatening complications and HHT mor-bidity. Since early diagnosis permits an appropriate care of affected subjects, a very sensitive mutation screening technique is required to identify the mutation carriers among all at-risk individuals belong-ing to HHT-families. There may be one or more genes that cause HHT but, if so, they are quite rare. Currently, scientists are trying to better understand exactly how the abnormal gene can interfere with normal blood vessel formation and promote the phenotype of HHT, so that better treatments for the symptoms of HHT can be developed.

Mailing address: Dr Filiberto Mastrangelo,Department of Oral Science,University of Chieti,Via dei Vestini, 31,66100 Chieti, ItalyTel: +39 0871 3554122 - Fax: +39 0871 3554095e-mail: [email protected]

Key words: hereditary hemorragic telangiectasia, Rendu-Osler-Weber disease, hht, endoglin, alk1, vascular malformation, gene expression, tgf-beta1 polymorphisms

Hereditary hemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber syndrome, is an autosomal-dominant disorder in which abnormal communications between arteries and veins, the so-called teleangiectases, occur in skin, mucosal surfaces and solid organs. Telangiectases often “break” and bleed after slight trauma. Spontaneous and recurrent epistaxis is the most common symptom. It begins by the age of 10 years and tends

to be increasingly frequent as the time goes by.It may compromise/damage the patient’s

quality of life and cause anemia, requiring oral iron supplementation or blood transfusions. Moreover, affected patients can suffer from chronic blood loss, hypoxemia, hepatic dysfunction and high-output cardiac failure. HHT is an uncommon disease with a prevalence of 1:8000 (1), even if it is probably underdiagnosed in affected individuals because of

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the various spectrum of its clinical manifestations.HHT is characterized by age-dependant

phenotypic penetrance, nearly complete by the age of 40. The expression of HHT is extremely variable with a great disparity of clinical manifestations between affected individuals and even among members of the same family. Genetic and epigenetic factors have been postulated to account for this diversity.

HHT: clinical signs The clinical manifestations are caused by

direct arteriovenous connections without an intervening capillary bed. This can result in a range of malformations from smaller mucocutaneous telangiectases to large visceral arteriovenous malformations (AVM).

Clinical manifestations of HHT include recurrent epistaxis, multiple mucocutaneous telangiectases at characteristic sites (lips, oral cavity, facial skin, hands including fingers, auricles, thorax and nose) (2), visceral lesions, such as gastrointestinal hemorrhages, pulmonary arteriovenous malformations (PAVMs), cerebral arteriovenous malformations (CAVMs) and hepatic arteriovenous malformations (HAVMs) (3). Recurrent epistaxis is usually the first symptom and is present in more than 90% of patients with HHT (3). The cutaneous and mucosal telangiectases occur more often in typical sites (fingertips, lips, oral cavity, conjunctivae, nasal mucosa) and begin generally by the age of 30 and 50 years. They are small raised lesions, bright red or purple-red in colour, which are very fragile and thus bleed easily, but this bleeding is rarely clinically important (Fig. 1).

The dimension of telangiectases varies in time due to their slow but progressive enlargement and generally they also tend to increase in number (Fig. 2). Telangiectases can be detected in more than 80% of HHT patients (3), but sometimes they are not visible or are barely visible with the naked eye. For this reason, capillary microscopy is a valuable tool in the diagnosis of HHT(4). The alterations that can be observed are giant loops, often with tortuous configuration and prominent enlargement of the draining limb between normal capillaries.

Telangiectases are the hallmark of the disease, but may be difficult to distinguish from other diseases, like angiokeratoma corporis diffusum

(Fabry syndrome), systemic sclerosis and ataxia-telangiectasia. Other problems to be considered are pregnancy, spider angioma in chronic alcoholism, calcinosis, Raynaud phenomenon, esophageal motility disorder, sclerodactily and teleangectasia (CREST syndrome). Treatment is rarely needed for skin lesions, but cosmetically disturbing or frequently bleeding lesions can be treated with laser therapy.

Arteriovenous malformations affect lung, brain, liver and, more rarely, spinal cord. (2). Patients develop life-threatening complications such as severe gastrointestinal bleeding and arteriovenous malformations (AVMs) (direct connection between a dilated venule and arteriole, bypassing the capillary network) of the liver, lung or brain. The shunting of blood through pulmonary or cerebral AVMs can lead to hypoxemia, stroke, brain abscess, heart failure and fatal hemorrhage (3). Gastrointestinal (GI) bleeding from intestinal telangiectatic lesions usually does not start before the fifth decade and may cause severe anemia (5). GI bleeding is possible, generally in the form of chronic stillicidium, due to telangiectases in the esophageal gastric and intestinal mucosa (6). The most common site of AVM is the lung. Pulmonary arteriovenous malformations are estimated to develop in more then 20% of the patients (3). The presence of pulmonary arteriovenous fistulae (PAVMs) (3) is a relatively frequent event in HHT1. PAVMs result in a right to left shunt and thus hypoxemia, and may cause serious complications such as bleeding (hemothorax), bypass of emboli or bacteria causing serious systemic complications (7) such as stroke and cerebral abscess (3). Because of these serious complications, treatment of PAVMs is indicated, even when asymptomatic. Cerebral arteriovenous malformations (CAVMs) are less common, but are probably under-recognized, and are present in 10–15% of the patients (8). Although they are often silent, they can cause headache, seizure, ischemia, bleeding, epilepsy and hemorrhagic stroke (break of aneurysms), sometimes with disabling consequences.(5, 9-12).

Hepatic arteriovenous fistulae (HAVMs) are very often asymptomatic and as a result, their treatment is not generally necessary and is applied only when they cause complications (13). Organ transplantation is the treatment of choice for hepatic arteriovenous

A. BALLINI ET AL.


malformations in the presence of meaningful symptomatology (14).

Clinical diagnostic criteria for HHTCurrently the diagnosis relies on strict association

between molecular screening and clinical investigation, considering signs and symptoms, that allow the prompt identification and timely treatment of severe vascular alterations observed in gene mutation carriers in a pre-symptomatic stage.

According to the Curacao criteria (2), the diagnosis of HHT is considered definite in an individual if three or more of the following four criteria are present and suspected if two of the following four criteria are present: 1) epistaxis - spontaneous and recurrent; 2) telangiectases - multiple, at characteristic sites, including lips, oral cavity, fingers and nose; 3) internal telangiectases or AVM- lung, brain, GI, liver or spinal; 4) family history - parent, sibling or child with HHT according to these criteria (2).

Mutidisciplinary approach is the gold standard in HHT investigation. The most commonly used instrumental tests to investigate HHT patients are: an echocolor-Doppler of the portal system/area used to detect the survey of hepatic fistulae; arterial blood gas analysis and echocardiography with c.m. used to inquire into pulmonary shunts; thorax- abdomen multislice-Computed Tomography (CT) used as a check test; brain MRI performed in order to locate cerebral arteriovenous fistulae whereas esophagogastroduodenoscopy (EGDS) is performed in order to diagnose telangiectases in the gastric and duodenal mucosa (15); capillary microscopy can be used to detect telangectases of the nail fold (4).

Dentistry in HHT patientsSince the most common immunological route

for the entry of bacteria into the blood stream is from the gingival mucosa, it is very important to inform the dentist before any dental work, including dental cleaning. If pulmonary AVMs are present, the bacteria, once in the bloodstream, can pass through them and may lodge in the brain and cause a brain abscess. A brain abscess is an extremely serious medical emergency and could be life threatening. If an HHT patient has not been specifically screened for lung AVMs, to prevent a brain abscess antibiotics must be taken prior to any dental procedure (1 hour

before dental work). It is essential that patients with HHT receive antibiotics before dental cleaning or dental work of any kind.

It is important that the HHT patient also discusses with the dentist and dental hygienist any homecare procedures or devices that have been recommended, such as oral irrigation, or any procedures that could introduce bacteria into the bloodstream (American Heart Association Guidelines).

Furthermore, there are medications that should not be routinely prescribed for HHT patients. Dental workers should be aware of these. Anti-inflammatory agents should generally not be taken by individuals with HHT because these medications can increase bleeding. Often these medications are prescribed following dental procedures. Telangiectases of the gingival mucosa, hard and soft palate, lips and tongue, are often mistakenly called red spots upon oral examination. They can vary in size (Fig. 3, 4). Procedures involving minor tissue manipulation such as having teeth cleaned, brushing teeth and using dental floss at home can cause bleeding. The dental worker should pay attention to the bleeding and rule out gingivitis, periodontal diseases or bacterial infection. Telangiectases related to HHT in the mouth and lips can be acquired in adulthood. The dental health worker is therefore in a position to identify telangiectases (7).

Pathogenesis of HHTIn normal skin, arterioles in the papillary

dermis are connected to venules through multiple capillaries. These vessels arise from larger arterioles and venules at the junction of the dermis and fat. In the earliest stage of cutaneous telangiectasia, a single venule becomes dilated but it is still connected to an arteriola through one or more capillaries.

Two genes are responsible for HHT, ENG (Endoglin) (Online Mendelian Inheritance in Man), (OMIM*131195) mutated in HHT 1 (16) (OMIM*187300) map along the long arm of the chromosome 9 (9 q33-q34.1) and ACVRL1 (activin receptor-like kinase 1, also known as ALK-1) (OMIM*601284), mutated in HHT 2 (17). (OMIM*600376), located on the long arm of the chromosome 12 (12 q11-q14) HHT 1 is associated with a higher incidence of pulmonary AVMs than HHT 2, which generally has a later onset (3).


Both genes are expressed predominantly in endothelial cells. To date, more than 100 different mutations have been identified in the ENG gene (18-20), and the number of different mutations reported in the ALK-1 gene is also more than 100 (20-21). Almost all the mutations reported are unique for a particular family. So far, numerous mutations of ENG and ALK-l gene have been described and, with few exceptions, mutations tend to be unique and family specific (18). The severity of HHT

is not correlated with the type of mutation or its position (22). Mutated ENG is rarely expressed in HHT 1 patients and, if expressed, it is only as an intracellular species (23). The current model for HHT 1 is haploinsufficiency, because of reduced levels of functional endoglin at the surface of endothelial cells (19, 23-26). Haploinsufficiency in ALK-I also seems to be associated with HHT 2 (27).

Murine models have been obtained by engineering mice lacking one or both copies of endoglin (End) or ALK1 gene. A crucial role for endoglin and activin-receptor like kinase 1 in angiogenesis was demonstrated in mice deficient in both copies of Endoglin or ALK1 gene respectively, which showed multiple vascular and cardiac defects leading to death in early embryos (28-31). The murine model of HHT reproduces the human disease and can help to elucidate the mechanisms that lead to the initiation and progression of vascular abnormalities. For example, cutaneous telangiectases are found in 90% of heterozygous (End+/-) mice with HHT. A telangiectasia arises

Fig. 1a

Fig. 1b

Fig. 1.a) Typical facial telangiectase;b) Telangiectases on the left cheek and the left auricle.

Fig. 2. Magnification of a large vascular lesion, consisting of singular dilated vascular loop.

Fig. 3. Multiple patch of telangiectases on palate mucose.

Fig. 4. Telangiectases on the upper lip and tongue.

A. BALLINI ET AL.


from the dilation of a postcapillary venule and direct fusion with an arteriole, bypassing the capillary network (32). This implies that regulation of the normal angiogenic process of vessel branching is altered in HHT. The 50% reduction in endoglin or Alk-1 present in endothelial cells of all vessels of individuals with mutation of one of these genes (9, 11-12 ), and in mice engineered to express a single allele of these genes, therefore predisposes vessels to dilation. However, additional genetic and environmental factors seem necessary to trigger the development of vascular abnormalities as suggested by their heterogeneity in humans and mice with a single functional copy of endoglin or ALK-l gene.

Both endoglin and ALK-l are components of the transforming growth factor (TGF-β) superfamily of receptors that are predominantly expressed on vascular endothelium. TGF-β1 superfamily members bind to heteromeric complexes of type I and II transmembrane serine/threonine kinase receptors (33). Upon ligand binding, type Il receptors recruit and transphosphorylate type I receptors, which subsequently activate downstream signal mediators, Smads. Only one TGF-β type II receptor (TR-II) is present in mammals, whereas two type I receptors appear to serve as TGF-β type I receptors. Activin receptor-like kinase 5 (ALK-5) is a TGF- β type I receptor ubiquitously expressed in TGF- β responsive cells. ALK-5 mediates TGF- β signaling through phosphorylatìng Smad2/Smad3. In addition, another type I receptor, ALK-l, is expressed in endothelial cells and binds to TGF- β and possibly other ligands (34). ALK-l signals are transduced through Smadl/SmadS, which are major downstream mediators of BMPs. Thus, in endothelial cells, ALK-5 and ALK-l mediate TGF- β signaling through distinct transcriptional regulation of target genes. TGF-β l plays a very important role in the development of the vascular system, affecting functions of endothelial cells (35). The cellular responses mediated by TGF- β l are complex and can be stimulatory or inhibitory, depending on cell type and conditions. TGF- β l generally inhibits proliferation and migration of endothelial cells, but potently stimulates extracellular matrix (ECM) production. Importantly, TGF-β l is essential for the recruitment and regulation of pericytes and smooth muscle cells, which lead to vascular maturation and stabilization (36). TGF-β l

acts by autocrine and paracrine mechanisms that are likely relevant in the pathogenesis of HHT.

TGF-β l stimulates the activity of its own promoter, and this autoregulation might explain the prolongation of secretion and autocrine action of TGF-β l after an initial stimulus (37). It has been demonstrated (38) that endothelial cells from End+/- mice secrete less TGF-β l than normal cells, and it has been proposed that the level of endoglin could control this autoregulatory pathway. Less endoglin would then lead to reduced autocrine effects of TGF-β l. A decrease in both local and circulating TGF-β l levels will lead to unstable cellular interactions in the vessel wall, dilated vessels and vascular abnormalities. Such alterations could impair other angiogenic regulatory mechanisms and lead to deterioration of the vascular network associated with the progression of HHT. Reduced TGF-β l levels hence play a role in the vascular remodelling of cerebral and pulmonary human AVMs which leads to extremely dilated and tortuous vessels with variable, and sometimes excessive, layers of smooth-muscle cells without elastic fibers, disorganized adventitia and active angiogenesis (12). A dysregulation in the mechanisms responsible for maintaining interactions between intimal, medial, and adventitial layers of vessels can therefore likely cause the progression and expansion of vascular lesions.

Endoglin and ALK-1 genes are both essential for normal angiogenesis and vasculogenesis but the molecular mechanisms regulating the expression of them are not well understood. Recently (39) it was observed that ENG gene is actively transcribed in vascular smooth muscle cells whose proliferation is dramatically altered in HHT patients, the ENG protein also being present, although at low levels.

To date, there are no data on ENG and ALK-1 gene expression patterns in normal subjects nor in HHT patients. Similarly, little is known about intracellular proteins able to interact with ALK-1 and ENG gene products. A recent paper (40) reports an ALK-1 dependent upregulation of ENG (40) Id1 and Id2 (41) genes in the endothelial cells. Finally, even if the presence of modifier genes has been hypothesized, so far no candidates have been identified, therefore the genetic factors correlating with the wide variation, both intra- and inter-familial, in clinical symptoms remain unknown. However,


within families there is considerable inter- and intra-familial variability with respect to age-related penetrance and pattern of clinical expression of the disease. Recently, mutations in the MADH4 gene in patients that show clinical features of both HHT and juvenile polyposis was reported (42).

MADH4 codes for the protein SMAD4 and is expressed in a variety of cell types. The protein has a role in the TGF-β pathway, like ENG and ALK-1, as well as in the bone-morphogenic-protein pathway. HHT, as part of this syndrome, can thus be explained by MADH4 mutations.

Alternatively, more extensive locus heterogeneity may play a role. A few families have been described that show no significant linkage to either ENG or ALK-1 (43-45). After re-evaluating all the members in the family studied by Piantanida and Buscarini, the evidence for exclusion of chromosome 12 was no longer considered significant and analysis revealed an ALK-1 mutation (p.R67W) (46). However, the presence of a third locus cannot be ruled out. In this respect, the finding of a new locus (HHT-3) maps to chromosome 5 in screened HHT families with no mutations in ENG or ALK-1, but with clinical manifestations, renders it a new candidate gene (47).

CONCLUSIONS

HHT is an autosomal-dominantly-inherited vascular dysplasia characterized by age-dependant incomplete penetrance and variable expressivity, with clinical manifestations consisting in epistaxis, gastrointestinal bleeding and AVMs. Morbidity of HHT is often due to complications of AVMs, such as stroke or haemorrhage, also known to occur in children. Many authors have reported successful treatments of AVMs, which resulted in a significant decrease of life-threatening complications and HHT morbidity. Since early diagnosis permits an appropriate care of affected subjects, a very sensitive mutation screening technique is required to identify the mutation carriers among all at-risk individuals belonging to HHT-families. For this purpose, many different techniques have been utilized for mutation screening of endoglin and ALK1 genes, responsible for HHT-1 and HHT-2, respectively.

The identification of the disease-causing mutations in a large panel of HHT families provides

us with a good basis for studying in detail the phenotype–genotype relationship for both ENG and ALK-1 within and between families. In this way it is possible to gain more insight into the variability of clinical expression and the factors contributing to HHT. The nature of the practice of odonstomatology necessitates a broad understanding of the systemic diseases reflected in the immune system during normal dental practice. Hereditary hemorrhagic telangiectasia is one such disease.

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INFLAMMATORY THERMOPHILIC FUNGI ARE USED IN BIOTECHNOLOGY APPLICATIONS

Y.B. SHAIK

Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, Boston, USA

Received May 28, 2006 - Accepted September 28, 2006

Mailing address: Yazdani B. Shaik Ph.D.Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, 650 Albany Street, Room 630, Boston, MA 02118. USA. Tel: +1 617 414 5257 - Fax: +1 617 414 5280email: [email protected]

Key words: thermophiles, enzymes, invertases, mesophiles, Thermomyces lanuginosus

Thermophilic microorganisms which can colonize at extreme ecological niches are known as extre-mophiles. Because of their capacity to withstand high temperatures, enzymes from these organisms are relatively heat stable. The versatile enzyme properties of these organisms make them excellent candidates in biotechnology. In general, fungi have been widely used for the production of proteins and enzymes, since they can grow rapidly in a low cost media and they secrete proteins into the extra-cel-lular medium. Recently, these organisms have also been used in large scale fermentation as host for the expression of heterologous proteins in industrial applications. However, little is known about the regula-tion and genetic manipulations of these fungi. We have previously shown the regulation of gene expres-sion in a thermophilic fungus, Thermyces lanuginosus, using an inducible invertase system. The aim of this review is to elucidate the recent advances of thermophilic fungi, and their implications in industrial applications are discussed.

Organisms which can grow at extreme environments are known as extremophiles. The study of these organisms has lead to an understanding of the absolute limits of life in terms of environmental parameters such as temperature, pressure, pH, salinity, ionic strength, etc. Among extremophiles, thermophilic organisms are of particular interest, because of their thermostable enzymes, which have potential applications in biotechnology. Thermophily has been extensively studied in bacteria, and these bacteria have been subsequently screened for thermostable enzymes, e.g., Taq polymerase from Thermus aquaticus and vent polymerases from other hyper thermophilic organism which are extensively used in polymerase chain reaction (PCR). It is interesting to note that several enzymes from the genus Pyrococcus furiosus were found to be optimally active at

temperatures between 95°C and 115°C with a half-life of about 20 h, hence they occupied a prominent place in modern biotechnology (1) (Table I).

Several such extremozymes have been isolated from Archaea and consequently, microorganisms belonging to this kingdom have been exploited for their genetic, physiological and biochemical prospective applications. Not much information is available on the thermophilic eukaryotic organisms. Among eukaryotes, thermophilicity is well defined only in fungi (2). Fungi that grow at maximum temperature of 50°C or above, with a minimum growth temperature of 20°C, have been arbitrarily defined as thermophilic fungi. According to this definition, about 23 species of thermophilic fungi are known today (2) (Table II).

Generally, these thermophilic fungi have been isolated from wastes of domestic animals, compost,

Vol. 4, no. 3, 147-155 (2006)

REVIEW ARTICLE

148 149Eur. J. Inflamm.Y. B. SHAIK

municipal wastes, stored agricultural products, nesting materials of birds, soil, etc., suggesting that they have a ubiquitous distribution (3). The maximum temperature at which thermophilic fungi can grow is close to 60°C, and this temperature is considered as being the upper limit for eukaryotic organisms (4). In spite of their unique thermophilic nature, these fungi have not yet been as defined as certain prokaryotic organisms found growing at 99-100°C in hot springs (3).

Various species of thermophilic fungi have been commonly isolated using selective media and appropriate temperatures from the same soil sample. Furthermore, these fungi are very easy to cultivate in chemically-defined media (5-7). Moreover, these fungi also exhibit rapid growth rates (7-8). A number of thermo tolerant and thermophilic fungi have also been isolated from shipments of food-aid grains. Thermo tolerant fungi include Aspergillus candidus, A. fumigatus, A. flavus and Paecilomyces varioti. Thermophilic fungi include Thermomyces lanuginosus, Rhizomucor pusillers, Thermoascus aurantiacus, T. crustaceous and Chetomium thermophile (9-10).

These fungi served as a model system for comparative biochemical investigations on the adaptations of eukaryotic microorganisms at high temperatures. Studies on the thermophilic strains of fungi revealed that they can produce thermostable proteins at a higher rate than mesophilic organisms (11-12). Furthermore, the use of thermophilic microorganisms has also reduced the cost of production of such proteins on a large scale, since they do not require any cooling system for the maintenance of the desired temperature, as in mesophilic counter parts. Hence, these thermophilic fungi have been exploited as industrial microorganisms.

Enzymes that convert different substrates have been identified and characterized from a variety of thermophilic fungi. Most of the extra cellular enzymes are found to be thermostable. For example, xylanase from Paecilomyces varioti has been purified and crystallized (13). Similarly, an extra cellular amylase activity was found in other thermophilic fungi Rhizomucor pusillers, Humicola lanuginosa (14). Enzymes from these thermophilic fungi that are used in industrial applications include glucanases

(15), cellulases (16), Cellobiose dehydrogenases (17), Galactomannases (18), invertases (19-23), lactases (24), lipases (25), pectinolytic and hemicellulolytic enzymes (21, 26-27). But the presently accepted nomenclature for this fungus is Thermomyces lanuginosus (28- 29). T. lanuginosus belongs to the deuteromycetes group of fungi. T. lanuginosus is one of the best characterized thermophilic fungi because of the uniform conidial production in the submerged cultures (30). About 90% of synchronous germination was achieved within a period of 3 h, in submerged cultivation when it was incubated at 50°C, whereas the same percentage of germination takes about 5 h on agar plate, indicating its ability for germination in submerged cultures (31). Hence, T. lanuginosus can be handled as easily as bacteria. This property encouraged researchers to use this fungus as a model system.

Many enzymes have been characterized from T. lanuginosus because of their widespread occurrence and potential economic importance. These include enzymes such as β-galactosidases (32), extra cellular α-glucosidases (33), invertases (19-21, 23), pectinases (20-21, 23), trehalases (7) and xylanases (34-37). Apart from the production of thermostable enzymes, thermophilic fungi are also exploited for the cultivation of the edible Agaricus bisporus as co-cultivator. Experimental data suggest that the carbon dioxide produced by thermophilic fungi during decomposition enhances the hyphal extension rate of the mushroom mycelium (38-39). Furthermore, they are also utilized in the bio-leaching of metals, e.g., thermophilic fungus Talaromyces emersonii CBS814.7 is used to recover uranium from ores (40).

Invertase has been reported from a variety of mesophilic fungi. Interestingly, an inducible invertase and sucrose permease was reported in the thermophilic fungi T. lanuginosus for the first time by Palanivelu (30). Invertase biosynthesis in T. lanuginosus is induced by sucrose and repressed by glucose and fructose. In contrast, invertase production is constitutive in all the mesophilic fungi so far reported, but it is subjected to glucose repression (41-43). Apart from the physiological differences in growth temperature, we have demonstrated that the invertase from T. lanuginosus also exhibited few unique features such as activation by proteins and phospholipids (19-21, 23, 44) and unusually


strong interactions with hydrophobic matrixes. We also examined gene regulation in the thermophilic fungus T. lanuginosus, at the molecular level, using an inducible invertase as a model system (22).

Not much information is available on gene expression, especially foreign genes in thermophilic fungi at the molecular level, in spite of their potential industrial application. This is probably due to the following reasons. 1. Regulation of the gene expression is not clearly understood. 2. Very few inducible promoters are available to construct expression vectors for cloning in fungi. 3. Transformation system is not well standardized. 4. Very few selectable markers are available for cloning in the fungi.

However, recently, a gene encoding actin has been cloned in the thermophilic fungus T. lanuginosus and expressed (29). Another gene that encodes for laccase, an extra cellular enzyme, also cloned from the thermophilic fungus Myceliophthora thermophila into Aspergillus oryzae and characterized. The deduced amino acid sequence was found to have homology to laccases from diverse fungal genera (45). Similarly, phytase gene has been cloned from another thermophilic fungus called Talaromyces thermophilus. Phytase (EC. 3.1.3.8) belongs to the histidine group of acid phosphatases, and sequence analysis suggests that 21 amino acids within the active site are conserved among all fungal phytases (46-47).

Similarly, phyA gene encoding an extra cellular phytase has also been characterized from the thermophilic fungus T. lanuginosus. The phytase

gene was cloned into an expression vector under the transcriptional control of trypsin promoter from Fusarium oxysporum and used for transformation of F. venenatum (45). The cloned phyA gene encodes a primary transcript of 425 amino acids, which includes a putative signal peptide (34) and a propeptide (48). The deduced amino acid sequence of PhyA shows less sequence identity (47%) with A. niger phytase. Similarly, cloning of genes encoding for lipase (49) and xylanase (50) were also reported from T. lanuginosus. Recently, attempts have been made to develop a transformation system in the thermophilic fungus Talaromyces sp. using phleomycin resistance gene as a dominant selectable marker (51) and in Humicola grisea var. thermoidea using hygromycin B resistance gene as a selectable marker (52). Furthermore, a detailed study has been carried out on the amino acid sequence of ribosomal subunits of T. lanuginosus and found that it is comparable to those sequences from other eukaryotic organisms indicating the common origin of ribosomal genes in these organisms. Increasing interest in these fungi probably reveals additional information on the adaptation and molecular evolution of these organisms (53-54).

Thermophilic nature has been well pronounced in fungi as compared to other eukaryotes. Attempts have therefore been made to evaluate the factors that are involved in thermophilicity, but it has yet to be established completely. Recently the effect of growth temperature on the fatty acid composition of the phospholipids of the thermophilic fungus

11

Table I. Temperature optima of enzymes from a hyper thermophile.

Source Activity Topt

�-glucosidase 110-115 �-glucosidase 105 �-amylase 100 Protease >100 Amylopullulanase 110 Aldehyde oxidoreductase >95 Glutamate dehydrogenase 95 Polysulphide dehydrogenase >95 Hydrogenase >95

Pyrococcus furiosus

Sucrose �-glucohydrolase 105

Table I. Temperature optima of enzymes from a hyper thermophile.


Talaromyces thermophilus was investigated and it was found that the fungus was unable to increase the degree of unsaturation of its fatty acids when it was shifted from high to low temperature. These data suggested that thermophily is due to metabolic limitations that restrict its ability to regulate

membrane fluidity (55).Studies of the structural basis of protein thermo

stability have produced a confusing picture. Small sets of proteins have been analyzed from a variety of thermophilic species, suggesting different structural features as being responsible for protein thermo

12

Table II. List of thermophilic fungi and their growth temperatures.

Growth temperature Fungi

Minimum Optimum Maximum

Phycomycetes

Mucor miehei 25 42.5-47.5 57

Mucor pusillus 20 40-45 55

Ascomycetes

Chaetomium thermophile La Touche 27 50-52.5 55

Talaromyces thermophilus Stolk Syn. Penicillium duponti

27 47.5 59

Talaromyces emersonii Stolk Syn Penicillium emersonii

30 47.5 60

Thermoascus aurantiacus 22 52.5 55 Thermoascus crustaceus 20 37 55 Thielavia thermophila 20 45 56 Thielavia australiensis 20 40.5 50 Thielavia albomyces Syn. Myriococcum albomyces

26 45 57

Thielavia terrestris Syn. Allescheria terrestris Apinis

<28 42.5-47.5 >48

DeuteromycetesAcremonium alabamensis 25 37 >50 Humicola grisea var. Thermoidea

24 47.5 56

Humicola insolens 23 45-47.5 55 Thermomyces lanuginosus <30 47.5-52.5 60 Humicola stellata 22 37.5 50 Malbranchea pulchella var. Sulfurea 27 47.5 56 Paecilomyces sp. 30 45-50 55-60 Sporotrichium thermophile 24 42.5-45 55-60 Stilbella thermophila 25 35-50 55 Thermomyces ibadanensis 31-35 42-47 60-61 Torula thermophila 23 42.5-47.5 58 Mycelia sterilia Papulaspora thermophila

29-30 45 52

Table II. List of thermophilic fungi and their growth temperatures.

Y. B. SHAIK


xylanase. Furthermore, use of low cost inducers have been used in the production these enzymes (58-59). Recently the use of filamentous fungi became very attractive for the high level protein production in a cost effective way. A heat-stable subtilisin-type serine protease aqualysin I is cloned from an extreme thermophile Thermus aquaticus in the methylotrophic yeast, Pichia pastoris (60). An attempt has also been made to increase the thermostability of the enzymes Thermus thermophilus by genetic mutations that alter the amino acid composition of the protein (61-62). These studies open up potential avenues for designer protein with high thermostability for industrial applications which is cost effective due the advantage of eliminating extensive downstream processing.

Attempts are now being made to transform the phenomenon of thermophilicity from thermophilic organisms into mesophiles by various genetic approaches. Transformation of DNA from thermophilic archaebacteria into mesophilic Bacillus sp. made the latter exhibit an increased stability at higher temperatures. The mechanism whereby the thermophilic phenotype in the recipient is affected is unknown. Studies using phenotypically stable transformants indicated that the thermostability of some cellular components significantly increase both in the vegetative cells and spores. Similarly, enzyme thermostability appears to be associated with an increased use of hydrophobic amino acids (19-21, 48, 63). However, the biochemical mechanism for the physiological alteration of the transformation remains unknown. Thus, molecular mechanism involved in the thermophily is still an intriguing topic for investigation.

In conclusion, thermophilic organisms offer several advantages in comparison with their mesophilic counterparts. Furthermore, thermophilic fungi offer additional features such as high levels of protein expression and secretion of the proteins into the extracellular medium. However, little is known about the gene regulation and expression, which is hindering the use of these organisms in industrial applications. We have recently demonstrated the regulation of gene expression using a novel inducible invertase system in a thermophilic Fungus T. lanuginosus. We have cloned the invertase gene along with complete promoter elements and

stability. Recently there has been great interest in the understanding of molecular mechanisms behind thermo stability of the proteins from these microorganisms. It has been demonstrated that there is some chemical and structural variation in the elongation factor from mesophilic and thermophilic organisms. It has also been shown that proteins from mesophilic or thermophilic organisms have the same macromolecular packing, while the water-exposed surface area is larger for the thermophile. A larger coordination with water is probably due to a peculiar corrugation of the exposed surface of this species. From an enthalpy point of view, the thermophile shows a larger number of intra-molecular hydrogen bonds, stronger electrostatic interactions and a flatter free-energy landscape. Overall, the data suggest that the specific hydration state enhances macromolecular fluctuations but, at the same time, increases thermal stability (56). A comprehensive analysis of the relationship between structure and function of the protein was recently analyzed using a hyperthermophilic bacterium Thermotoga maritima, and those of close homologs from mesophilic bacteria (57). These studies suggest that oligomerization order, hydrogen bonds and secondary structure play minor roles in adaptation to hyperthermophily in bacteria. On the other hand, the data exhibited very significant increases in the density of salt-bridges and in compactness for proteins from T. maritima. These studies provide a clear picture of protein thermostability in one species and a framework for future studies of thermal adaptation.

Recent advances in genetic engineering made the protein production from heterologous source easy by gene manipulations. Gram negative bacterium Escherichia coli is routinely used as an expression host for the production of heterologous proteins. Recently, few proteins have been expressed in E. coli using modern genetic engineering technology. Overexpression of a xylanase, from the thermophile Rhodothermus marinus was achieved by cloning under the control of the strong T7/lac promoter and cultivated in a chemically defined medium. The stability and the yield enhanced by the addition of some selected nutrient feed additives such as TCA cycle intermediates (succinic acid or malic acid) resulted in an increase in production of approximately 40% of the heterologous thermostable


sequenced it. The long term goal of this project is to construct sucrose inducible vectors for cloning in fungi for the expression of foreign proteins from any eukaryotic or prokaryotic origin in large quantities. Finally, thermophilic fungi, particularly T. lanuginosus, would be good candidates for the expression and production of heterologous protein using fermenters in industrial applications.

ACKNOWLEDGEMENTS

I would like thank Prof. P. Palanivelu and Dr. D. Kempuraj for the discussions, and also Dr. R. Jayakumar for his help while preparing this article.

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HSP70 MODULATES THE ENHANCED PRODUCTION OF REACTIVE INTERMEDIATE METABOLITES AND A PROINFLAMMATORY CYTOKINE

TNF-α EXPRESSION IN A T CELL LYMPHOMA

S. KUMAR, P. DEEPAK and A. ACHARYA

Immunology Laboratory, Department of Zoology, Faculty of Science, Banaras Hindu University, Varanasi, India

Received March 7, 2006 – Accepted September 28, 2006

Heat shock proteins are intracellular soluble proteins expressed consecutively in all cells. They are immunogenic proteins able to activate antigen-presenting cells by binding through the CD91 receptor and activate both CD4+ and CD8+ T-cells. Macrophage plays a pivotal role in innate immune response and secretes a number of regulatory molecules upon activation. In the present study, we investigate the activation of normal and tumor-associated macrophage to produce the effector molecules which have a role in immunomodulation, especially in the killing of the transformed or tumor cells. In vitro and in vivo treatment of NMO and TAMs (from T-Cell Lymphoma) with optimum dose 10 μg of hsp70 produce effector molecules such as nitric oxide (NO), hydrogen peroxide (H2O2) and tumor necrosis factor-α (TNF α). The results of our experiments reveal that the production of effector molecules is dose-dependent, and the result of immunoblots also confirms the increased expression of iNOS. These findings suggest that autologous hsp70 are highly immunogenic and potent activating agents for the enhanced production of effector molecules in NMO and TAMs in a T-cell lymphoma.

Mailing Address:Dr Arbind Acharya,Department of Zoology,Faculty of Science,Banaras Hindu University,Varanasi-221005, U.P., IndiaTel: +91 542 2307149 - Fax: +91 542 2368965e-mail: [email protected]

Heat Shock Proteins (HSPs) are expressed in nearly all prokaryotic and eukaryotic cells. They are phyllogenetically highly conserved molecules performing important functions in folding and unfolding and in the translocation of proteins (1). Because of these functions, HSPs have been termed molecular chaperones. HSPs are expressed inducibly as well as consecutively under different conditions such as stress, nutrient deficiency, infection and even in tumors. Inducible forms of HSPs protect the cells from the damage incurred due to stress, thereby exerting a cytoprotective role

by inhibition of apoptosis. The HSP family consists of low molecular weights of proteins, HSP40, HSP60, HSP70 HSP90 and HSP110. Among HSP families, hsp70 is highly conserved (2), highly immunogenic and capable of inducing antibody production and T-cell activation. Hsp70 also plays an important role in the antigen presentation and cross-presentation of tumor antigens (3-5). It is well documented that hsp70 induces the production of Th1 cytokines such as TNF-α, IL-1, IL-6, IL-12 (6-9) and the release of nitric oxide, chemokines by monocytes, and dendritic cells that induce tumor

Key words: Hsp70, TAMs, RNI, ROI, TNF-α, Τ−cell lymphoma, iNOS

Vol. 4, no. 3, 157-169 (2006)


immunity by activating both innate and adaptive immune responses (10-15). Through the cytokine productions and their functions, HSPs may serve as a danger signal to the innate immune system at the site of tissue injury. Macrophages are most abundant at a tumor site and play a vital role in innate immune response. Upon activation, macrophages secrete several effector molecules, cytokines and chemokines for the upregulation of the immune network and the killing of transformed cells. However, the macrophages infiltrated into tumor sites have been shown to perform a pleiotrophic function and differ largely from normal counterparts in terms of receptor expression, cytokine responsiveness and other functions. Tumor-associated macrophages (TAMs) are termed as type II polarized macrophages or M2 phenotype. These TAMs have been shown to produce an extremely low level of NO (16) and a reactive oxygen intermediate (ROI) (17) as compared with normal macrophages.

The present investigation reports the role of Hsp70 in the modulation of TAMs function in a spontaneous transplantable T-cell lymphoma designated as Dalton’s Lymphoma. Amazingly, Hsp70 induces the expression of inducible nitric oxide synthase (iNOS) and the subsequent production of Nitric Oxide in TAMs, whereas, normally it is arginase-dependent (18) which accounts for the constitutive low production of NO. It also reports the enhanced production of other non-specific effector molecules such as reactive oxygen intermediate (ROI) and TNF-α by TAMs in vitro and in vivo.

MATERIALS AND METHODS

Mice and tumor systemPathogen-free adult mice of BALB/c (H-2d) strain,

comprising male and female, were used at 8-12 weeks of age. The mice were maintained mainly by human care in an approved animal room facility of the Department of Zoology. The mice were transplanted with Dalton’s Lymphoma in ascitic form at 6-8 weeks of age, which became full-grown DL-bearing host about 18 days after transplantation. DL-cell line was also maintained in vitro in cell culture system by serial passage and in a cryopreserved state for reference. The mice were challenged with 1.5x106 cells/mouse by intraperitoneal injection.

Reagents The macrophages and DL-cells were cultured in

RPMI 1640 tissue culture medium (Hi Media, India), supplemented with 10% fetal calf serum (Hyclone, Utah). All the reagents were determined endotoxin-free by Limulus Amoebocyte lysate assay (sensitivity limit is 0.1 ng/ml) and maintained sterile. The recombinant TNF-α and IFN-γ were obtained from NIBSC, UK, and the monoclonal antibodies for TNF-α (anti-mouse) were obtained from IMGENEX, San Diego. A secondary antibody conjugated with alkaline phosphatase was obtained from Bangalore Genie (India). The antibody for Hsp70 was obtained from Sigma Chemicals.

Isolation and purification of hsp70The hsp70 were isolated and purified from the

culture of DL-cells grown in a short period of thermal shock condition. The DL-cell cultures were lysed in dehydrogenated glassware and clear supernatants were loaded onto a DEAE sephacel column (Amersham Pharmacia Biotech.). Elution of hsp70 was achieved by applying a 25-50 mM NaCl gradient. Fractions containing hsp70 were loaded again onto an ATP-agarose column (Sigma Co. St. Louis), and elution was achieved by applying a 0.1 mM ATP gradient. Final fractions were precipitated with 80% ammonium sulphate; the precipitates were redissolved in low salt-buffer containing magnesium acetate, dialyzed and stored. The purified hsp70 was tested for Limulus Amoebocyte assay (sensitivity limit is 0.1 ng/ml).

Isolation and activation of peritoneal macrophagesMacrophages from healthy mice of 8-12 weeks of age

were prepared by a standard method. Peritoneal exudate cells (PEC) were harvested by peritoneal lavage using chilled serum-free culture medium RPMI 1640. The PEC were then transferred into a vented plastic tissue culture flask (Tarson, India) for culture at 37oC in CO2 incubator (Sheldon, Oregon). The non-adherent cells were removed by washing three times with lukewarm serum-free culture medium with gentle flushing. More than 95% of the adherent cell population was that of macrophages, as determined by morphology and non-specific esterase staining. The macrophages were then detached from the tissue culture flask using a cell-scraper (Corning, USA) resuspended in serum-free culture medium and seeded in 96-well flat-bottom culture plates (Tarson, India) at a cell density of 1.5x106 per well in the presence or absence of hsp70.

Isolation and activation of tumor associated macrophages

Tumor Associated Macrophages (TAMs) were isolated, and the monolayer was prepared from DL-bearing mice after 7days of DL-cell transplantation. Tumor-bearing mice were killed by cervical dislocation and PEC was harvested as

S. KUMAR ET AL.


described above. Adherent purified TAMs were seeded in a 96-well flat bottom culture plate at the density of 1.5x106 cells/well in the presence or absence of hsp70 for 24 hrs before assay.

In vivo stimulation of macrophagesHealthy and DL-bearing BALB/c mice of 6-8 weeks

of age were administered intraperitoneally with PBS alone and PBS containing hsp70 at 1.0, 5.0, 10.0, 50.0 and 100.0 µg, and after two days a booster dose was given. After 24 hrs, the PEC were isolated from the mice of both groups by peritoneal lavage after cervical dislocation as described above, and the macrophages were harvested in a plastic tissue culture flask for later incubation in CO2 incubator for 24 hrs, again in complete medium. Finally, adherence purified PEC were seeded in a 96-well flat-bottom culture plate for assay.

Nitrite assay for estimation of nitric oxide productionNitrite concentration in culture supernatants was

determined by a standard micro-plate assay. In brief, 50 µl samples were harvested from the conditioned medium and treated with an equal volume of Griess reagent (1% sulfanilamide, 0.1% Naphthalene-ethylene-diamine dihidrochloride and 2.5% H3PO4) at room temperature for 10 min. The absorbance at 540 nm was monitored with a micro plate reader (UV/VIS spectrometer, Lambda2, Norwalk, Canada). Nitrite concentration was determined by using sodium nitrite as a standard. The chemicals used for the preparation of Griess reagent were obtained from Sigma Chemical Co., St. Louis.

Assay for reactive oxygen intermediate (ROI) production

The ROI production was estimated by the standard method (19). Briefly, 50 µl samples were harvested from the conditioned medium and treated with an equal volume of red phenol solution containing 140 mM NaCl, 10 mM K2HPO4, 5.5 mM dextrose and 5.5 nM horseradish peroxidase. After treatment with red phenol solution, adherent cells at the cell density of 1.5x106 were incubated for 1 hr in CO2 incubator. Thereafter, 10 µl of 1M NaOH was added and the absorbances were measured at 620 nm on ELISA plate reader.

TNF-α measurementTNF-α in culture supernatant of PEC was determined

by Sandwich ELISA (20). Briefly, a 96-well microtitre plate was coated overnight with 50 μl of rabbit anti-mouse TNF-α mAb (5 µg/ml) at 4oC in coating buffer (0.1 mM NaHCO3, pH8.2). The coating buffer was then discarded and the plate was washed thrice with washing buffer (PBS/ 0.05% Tween20, pH7.4). The 50 µl of culture supernatants were added in triplicate and, for standard graded solution of recombinant TNF were also added to the wells and

incubated for 1 hr at 37oC. The plates were washed three times and the unbound sites were blocked with 50 µl blocking buffer (PBS/2% BSA) for 1 hr at 37oC. Thereafter, the plates were again washed three times with washing buffer, followed by incubation with secondary antibodies conjugated with alkaline phosphatase at a dilution of 1:5000. Plates were then incubated at 37oC for 60 min followed by the addition of 50 µl of p-nitro-phenyl phosphate (NPP) (1 mg/ml in enzyme substrate buffer). Finally, the absorbance was read at 405 nM after 10-20 min on ELISA plate reader.

Western blotting For Western blotting of hsp70, the ingredients isolated

from tumor cell lysates were separated by 12% SDS-Polyacrylamide gel electrophoresis and the proteins were then transferred to nitrocellulose filter paper. The transferred bands were immunoblotted with an anti-hsp70 (Sigma) at 1:2000 dilution, followed by incubation with alkaline phosphatase conjugated polyclonal antibody at the dilution of 1:5000. Finally, the blots were incubated with nitro blue tetrazolium reagent for 10 min at 37oC.

Similarly, for iNOS gene expression, macrophages (105) were lysed in ice-cold buffer containing 25 mM monosodium phosphate (pH 7.4), 75 mM NaCl, 5 mM EDTA, 1%Triton X-100, 100 µg/ml phenylmethyl sulfonyl fluoride (Merck), leupeptin, 10 µg/ml pepstatin, 20 µg/ml aprotinin and 10 µg/ml tyrosine (Sigma, St. Louis) inhibitor and centrifuged at 50,000g for 20 min at 4oC. The cytosolic proteins (6 µg/lane) were separated by 12% SDS-polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose filter paper and then immunoblotted with a rabbit anti-iNOS monoclonal antibody followed by incubation with alkaline phosphatase conjugated antibody (Bangalore Genie, India) at a dilution of 1:5000. Colour was developed with blots that were incubated with NPP for 10-20 min. The colour was developed by using nitro blue tetrazolium reagent as described above.

Statistical analysisEach value represents the mean SEM of three

independent experiments in each group except for in vivo stimulation experiments where two independent experiments were conducted. Data are analyzed by using two-tailed student’s t-test on statistical software package Sigma Plot, version 5.0. Differences were considered statistically significant at P < 0.05.

RESULTS

Purification of Hsp70Homologous preparations of hsp70 were purified

from the culture supernatant of Dalton’s Lymphoma


(DL) cells isolated from DL-bearing mice and cultured under thermal stress conditions as described in Materials and Methods. The protein content of the preparations was measured by Lowry’s Method and 6 µg per lane of hsp70 preparations were separated by SDS-PAGE. The hsp70 purity is estimated to be greater than 95% in this preparation, as determined by bands obtained in silver staining of SDS-PAGE of purified samples. The bands obtained in silver staining were also checked and confirmed by immunoblotts (Fig. 1) using hsp70 specific monoclonal antibodies.

Induction of NO2 production by hsp70 in murine macrophages

To determine whether autologous hsp70 preparation has any effect on inducible NO formation in murine macrophages, experiments were conducted (three times in triplicate) examining the effect of hsp70 on NO2

- production, including their dose-dependency and time course. As shown in Fig. 2, normal macrophages (NMO) and tumor-associated macrophages (TAMs) were treated with graded concentration of hsp70 (0.1 µg to 10 µg) for 24 hrs. Treated cells showed the release of a significant amount of NO2

- production after incubation in the concentration range of 1 µg to 10 µg (4.712 ± 0.45, 6.549 ± 0.45 and 10.198 ± 0.89 for NMO at the concentration of 1 µg, 5 µg and 10 µg respectively), whereas, in treatment with 0.1 µg of hsp70, NO2

- production was not found to be induced. In comparison to NMO, TAM showed enhanced production of NO2

- at the same concentration (6.913 ± 0.50, 9.23 ± 0.60 and 13.479 ± 1.05). The effect of hsp70 totally disappeared when the cultures were treated with heat-treated hsp70 preparations (data not shown). LPS-treated macrophages were taken as a control group and a significant amount of NO2

- release (slightly lower than 10 µg of hsp70) was found. The treatment with Hsp70+Polymixin-B did not affect the release of NO2

-, excluding the probability of any endotoxin contaminant in Hsp70 preparations. The experiment for dose-dependency shows that the lowest concentration for NO2

- production for both NMO and TAMs is 1 µg, therefore increasing concentration leads to a steady rise in NO2- production.

Subsequently, the time course study for NO2-

production was carried out on both NMO and TAMs. The NO2

- produced in both groups of culture supernatants was demonstrated only after incubation for 12 hrs (Fig. 3). After the incubation of culture of both groups for 12 hrs on, a steady rise in NO2

- production was shown until the end of the 48 hr observation period. TAMs showed an ever-increasing production of NO2

- in the entire situation.

Induction of H2O2 production by hsp70 in murine macrophages

It was observed that hsp70 treated macrophages and TAMs show an enhanced production of H2O2 in the culture supernatants. Here, we observed dose-dependency also in H2O2 production. When macrophages were treated with 0.1 µg of hsp70, no H2O2 was found to be accumulated in the supernatant, whereas, on treatment with lowest effective concentration (1 µg/ml, as determined by previous experiment), a demonstrable quantity of H2O2 (0.288 ± 0.02 nM in NMO and 0.397 ± 0.02 nM in TAMs) was accumulated in the culture supernatants (Fig. 4). An increase in the concentration of hsp70 led to a steady rise in H2O2 production and at 10 µg (taken as standard optimal dose) a very high quantity (0.419 ± 0.03 in NMO and 0.523 ± 0.03 in TAMs) of H2O2 was observed. LPS treatment induces the H2O2 production at almost similar magnitude. In all cases, TAMs were found to be more responsive for H2O2 production in comparison to NMO at the same concentration of hsp70, except that TAMs were found to be low responders of LPS.

When time courses for the production of H2O2 were followed, we observed, as others, that H2O2 was demonstrable in the culture supernatant of both groups after incubation of at least 2 hrs. Also, on later study, after the incubation of 6, 12, 24 and 48 hrs, a substantial and steady rise in H2O2 production was observed (Fig. 5). On LPS treatment, only after 12 hrs of incubation, H2O2 production was observed, which slowly dropped after 24 hrs of co-incubation.

Induction of TNF-α production by hsp70 in murine macrophages

When macrophage culture was treated with hsp70 for 24 hrs TNF-α in culture supernatant was demonstrated at the lowest concentration of 1 µg/ml of hsp70 (1.39 ± 0.125 ng/ml). No TNF-α was found

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at the concentration of 0.1 µg/ml of hsp70, whereas on increasing the concentration of hsp70 from 1.0 µg, substantial and steady rises in TNF-α production were observed (2.098 ± 0.125 and 4.797 ± 0.145 at 5 µg and 10 µg respectively) for NMO. Release of TNF-α by TAMs was not found in similar magnitude as compared to NMO but showed higher production of TNF-α (Fig. 6). For TAMs also, the lowest amount of hsp70 for TNF-α production was 1 µg (2.281 ± 0.125), and increasing concentration lead to the increased production of TNF-α (3.793 ± 0.145 and 7.498 ± 0.172 at 5 µg and 10 µg, respectively). Heat treatment completely destroyed hsp70 activity in both groups (data not shown). In this manner, TNF-α secretion in response to hsp70 was found to be dose-dependent, with1 µg/ml being the lowest effective concentration. On treatment with LPS, secretion of TNF-α was observed but lower than the optimal dose of hsp70.

For time course study of TNF-α, we analyzed the culture supernatants of the groups at the time intervals of 2, 6, 12, 24 and 48 hrs. The TNF-α in supernatants was demonstrated as early as 2 hrs of incubation with

hsp70 (taking an optimum dose 10 µg/ml) (Fig. 7). The TNF-α production was found to be highest after incubation for 24 hrs (5.491 ± 0.555 for NMO and 7.01 ± 0.723 for TAMs) but thereafter a decline in TNF-α production was observed. However, TNF-α was still well demonstrated after 48 hrs in both groups. The LPS treated followed a similar path, with lower expression of TNF-α than the optimal dose of hsp70 by both groups of macrophages.

Post-immunization enhancement of TAMs responsiveness

TAMs harvested from mice immunized with different concentrations of hsp70 (5.0 μg, 10.0 μg, 50.0 μg, and 100.0 μg) showed comparatively more reactivity with LPS and IFN-γ than the TAMs obtained before immunization at 24 hrs of incubation at 37oC. Before immunization, the culture supernatants of TAMs incubated with LPS were found to release 0.211 ± 0.035 µΜ and with IFN-γ, 0.234 ± 0.04 µΜ NO2, while after immunization the nitrite level was found to be very high (1.077 ± 0.175 µΜ), (Fig. 8A, B, C). Similar effects were

Fig. 1. Identification of Hsp70 by SDS-PAGE and immunoblotting. Purified fraction of Hsp70 obtained from 24 hr cultured DL-cells. ADS-PAGE was run along with molecular marker isolated for the confirmation of Hsp70. The bands obtained were confirmed by immunoblotting with specific monoclonal antibody of Hsp70 as described in Materials and Methods.

Fig. 1 a Fig. 1 b


observed in the production of H2O2 and TNF-α. The maximum level of all theses mediators (NO2, H2O2, TNF-α) were found in culture supernatants of macrophages isolated from pre-immunized tumor-bearing host with 50.0 μg hsp70 (data not shown), but at a similar concentration, the production of NO2, H2O2 and TNF-α were found to be low in in vitro study of TAMs obtained before immunization.

Effect of Hsp70 on iNOS gene expressionFig.10 shows a Western blot analysis of

inducible nitric oxide synthase in both NMO and TAMs. Immunoblotting shows a band with an estimated molecular weight of 130 kD (the known molecular mass of inducible nitric oxide synthase) in macrophages (NMO and TAMs). An

identical molecular mass was found on blotting against purified iNOS protein. Inducible nitric oxide synthase protein was increased in contrast to medium and further iNOS protein was found more expressed in TAMs in respect to NMO.

DISCUSSION

Our findings show that hsp70 directly induces the production of reactive metabolic intermediates in normal and altered macrophages (TAMs). Therefore, the abrogated effect shown by heat denatured hsp70 preparation (data not shown) excluded the possibility of any contaminants. Hsp70 induces enhanced production of NO, H2O2 and TNF-α in TAMs in vivo. The effect of IFN-γ treatment on

Fig. 2. Dose-Dependent nitrite production. NMO and TAMs at the cell density of 1.5x106 cells were incubated with medium alone or medium containing LPS or indicated concentration of Hsp70 or Hsp70 + PmB for 24 hours and nitrite level was measured in culture supernatants as described in Materials and Methods. The data represent the mean concentrations of nitrite ± SEM of three independent experiments in triplicate. * P<0.05 vs values for NMO incubated in medium alone # P<0.05 vs values for NMO incubated with LPS $ P<0.05 vs values for TAMs incubated in medium alone.

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the in vivo stimulated TAMs indicates that a possible synergism in the function of the two molecules might exist. However, in this regard, the similar synergistic effect has been well-reported in in vitro experiment (21). Macrophages have long been known to produce non-specific effector mediators NO, H2O2 and TNF-α upon stimulation. It has been reported that activation of macrophages by HSPs also modulates the production of effector molecules. Macrophages are considered to be the first line of defense in conferring innate immunity against infection and malignancies. However, tumor growth simultaneously modulates the function of macrophages (22). These tumor-associated macrophages are activated or polarized M2 macrophage population, which eventually have a tumor protective function. Usually, TAMs produce cytokines of immunosuppressive function or tumor growth promoting function, such as IL-10 and TGF-β and other suppressive mediators such as

prostaglandin, with an extremely low level of NO. In this investigation, we observed higher levels of production of nitric oxide, hydrogen peroxide and tumor necrosis factor (TNF)-α in T-cell lymphoma model (cytotoxicity data not shown). NO is produced by activated macrophages through the enzymatic action of iNOS on terminal guanidinonitrogen of L-arginine that yield L-citrulline as co-product, while NO is constitutively produced by TAMs at a lower level. Therefore, the release of these NO2, H2O2 and TNF-α are considered as major tumoricidal mechanisms of activated macrophages in vitro (23-25) and in vivo (26-27).

The pathophysiological relevance of the TAMs in response to either microbial or autologous hsp70 has not yet been determined in any tumor cell types including T-cell lymphoma. TAMs have different characteristics with normal macrophages due to association with tumor. Since TAMs

Fig. 3. Time kinetics of nitrite production. 1.5x106 cells of NMO and TAMs were incubated with medium containing 10 ng of LPS or 10 µg of hsp70 for different time periods as indicated, and nitrite level in culture supernatants was measured. The data represent the nitrite level ± SEM of three independent experiments in triplicate. * P<0.05 vs values for NMO incubated for 2 hrs # P<0.05 vs values for NMO incubated with LPS for 48 hrs.


Fig. 4. Dose-dependent H2O2 production. 1.5x106 cells (NMO and TAMs) were incubated with medium alone or medium containing LPS or indicated concentration of hsp70 or Hsp70 + PmB, and H2O2 level for 24 hrs was measured in culture supernatants. The data represent mean H2O2 concentration ± SEM of three independent experiments done in triplicate. * P<0.05 vs values for NMO incubated in medium alone# P<0.05 vs values for NMO incubated with LPS$ P<0.05 vs values for TAMs incubated in medium alone.

Fig. 5. Time Kinetics of H2O2 production. 1.5x106 cells of NMO and TAMs were incubated with medium containing 10 ng of LPS or 10 µg of hsp70 for different time periods, as indicated. The data represent mean H2O2 production ± SEM of three independent experiments in triplicate. * P<0.05 vs values for NMO incubated for 2 hrs # P<0.05 vs values for NMO incubated with LPS for 48 hrs.

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Fig. 6. Dose-dependent TNF-α produc-tion. 1.5x106 cells of NMO and TAMs were incubated for 24 hrs with medium alone or medium containing LPS, indicated concentration of Hsp70 or Hsp70 + PmB, and TNF-α level was measured in culture supernatants. The data represent the mean TNF-α concentration in culture superna-tants ± SEM of three independent experi-ments in triplicate.* P<0.05 vs values for NMO incubated in medium alone# P<0.05 vs values for NMO incubated with LPS$ P<0.05 vs values for TAMs incubated in medium alone.

Fig. 7. Time kinetics of TNF-α production. NMO and TAMs (1.5x106

cells) were incubated with medium containing 10 ng of LPS or 10 µg of hsp70 for indicated time period, and TNF-α level in culture supernatants was measured. The data represent the TNF-α level ± SEM of three independent experiments in triplicate. * P<0.05 vs values for NMO incubated for 2 hrs# P<0.05 vs values for NMO incubated with LPS for 48 hrs.

show enhanced activity upon activation hsp70, as our observations confirm, this might be pre-activated or raged macrophages, which could be of relevance in immunotherapeutic protocols against malignancies. Normally, most of the hsp70 that are constitutively synthesized and localized in the cytosol are expressed and induced in inflammatory conditions (28-29, 30) such as in malignancies and other diseases. Necrosis of these inflamed cells

provides highly local concentration of hsp70 in extra cellular microenvironment for stimulation of an immune response. Consequently, in peritoneal fluids from females with endometriosis, the increased expression of hsp60 and cytokines has been reported (31). Unstimulated macrophages do not show any changes at the synthesis level, nor in the production of reactive intermediates. Therefore, it can be concluded that hsp70 must exert a stimulatory effect


Fig. 8. Post immunization increase in TAMs responsiveness. 1.5x106 cells TAMs harvested from DL-bearing mice before and after immunization were treated with medium alone or medium containing 10 ng of LPS, and NO2 (A), H2O2 (B) and TNF-α (C) were measured in culture supernatants. The data represents the mean concentration in supernatants ± SEM of two independent experiments in triplicate.* P<0.05 vs values for TAMs harvested from non-immunized DL-bearing mice and incubated with LPS.

Fig. 8 a

Fig. 8 b

Fig. 8 c

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Fig. 9. Analysis of Expression of iNOS by SDS-PAGE and Western blotting. Tumor-associated macrophages (TAMs) from DL-bearing BALB/c mice injected with medium alone or medium containing different concentration of hsp70 were recovered and after 24 hrs of incubation their culture supernatants were analyzed by SDS-PAGE. The bands obtained were immunoblotted with iNOS specific monoclonal antibody and color was developed as described in Materials and Methods.

at the transcriptional and expression level of the proteins; the immunoblot confirms this assumption. We observed a sharp increase in the expression of iNOS gene products that is consistent with the previous observations (16). We also observed a sharp increase in iNOS gene product in TAMs treated with hsp70 in comparison to normal macrophages (NMO) at the same condition. The intracellular signaling events involved in RNI, ROI and TNF-α production are not well understood. However, the expression of iNOS in murine macrophage has been shown to be dependent on NFκB activation (8-9) that is linked to TLR-signaling pathway conferred by LPS.

The enhanced production of reactive nitrogen intermediate (RNI), reactive oxygen intermediate (ROI) and TNF-α by ΤΑΜs indicates the possibility that TAMs are more responsive to hsp70, or there might be a possibility that TAMs show enhanced cell surface expression of receptor that are thought to be specific for hsp70. In this regard, the enhanced production of NO, H2O2 and TNF-α by hsp70 activated TAMs is likely to have consequences for the control of tumor. Our results also indicate that enhanced production of RNI, ROI and TNF-α by TAMs causes higher levels of these non-specific mediators that may contribute to a decreased susceptibility to tumorigenesis. Therefore, these immunomodulatory properties of hsp70 can be amalgamated in preparation of immunotherapeutic protocol based on hsp70 to fight against malignancies (32). Further investigation is needed

on hsp70 mediated signal transduction pathways of the complex immune network.

ACKNOWLEDGEMENTS

We thank Dr S.M. Singh for his valuable suggestions in preparing the manuscript, and for the financial support of U.G.C. New Delhi.

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25. Cui S., J. Reichner, R. Mates and J. Albina. 1994. Activated murine macrophages induces apoptosis in tumor cells through nitric-oxide dependent or independent mechanism. Cancer Res. 54:2462.

26. Yim C., N. Bastian, J.C. Smith, J.J. Hibbs and W. Samlowski. 1993. Macrophage nitric oxide synthesis delays expression of ultraviolet light induced murine skin cancer. Cancer Res. 15:5507.

27. Farias-Eisner R., M. Sherman, E. Aeberhard and G. Chaudhuri. 1994. Nitric oxide is an important mediator of tumoricidal activity in vivo. Proc. Natl. Acad. Sci. USA 91:9407.

28. Soltys B.J. and R.S. Gupta. 1997. Cell surface localization of the 60kDa heat shock chaperonin protein (hsp60) in mammalian cells. Cell Biol. Int. 21:315.

29. Soltys B.J. and R.S. Gupta. 1996. Immunoelectron microscopic localization of the 60kDa heat shock protein (hsp60) in mammalian cells. Exp. Cell Res.

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222:16.30. Laplante A.F., V. Moulin, F.A. Auger, J. Landry, H.

Lie, G. Morrow, R.M. Tanguay and L. Germain. 1998. Expression of heat shock proteins in mouse skin during wound healing. J. Histochem. Cytochem. 46:1291.

31. Kligman I., J.A. Grifo and S.S. Witkin. 1996.

Expression of the 60kD heat shock protein in peripheral fluids from women with endometriosis: implications for endometriosis associated infertility. Hum. Reprod. 11:2736.

32. Deepak P., S. Kumar and A. Acharya. 2006. Heat shock protein (HSP); future trends in immunotherapy. Eur. J. Inflamm. 4:1.









ETANERCEPT IN RHEUMATOID ARTHRITIS: LONG TERM ANTI-INFLAMMATORY EFFICACY IN CLINICAL PRACTICE

E. CACACE, C. ANEDDA, V. RUGGIERO, D. FORNASIER, A. DENOTTI and G. PERPIGNANO

Rheumatology - Department of Internal Medicine “Mario Aresu” University of Cagliari,

Policlinico Monserrato, Cagliari, Italy

Received March 13, 2006 - Accepted October 9, 2006

Rheumatoid Arthritis (RA) is a chronic inflammatory disease of erosive nature that tends to progress to juxta-articular destruction and ankylosis. The pathological events that lead to joint destruction are due, in part, to an enhanced expression of Tumour Necrosis Factor (TNF). It has been shown that TNF-α blocking agents significantly reduce joint inflammation and slow down the progression of radiographic joint damage. Etanercept is a biological drug obtained through recombinant DNA techniques that acts by inhibiting the cellular response mediated by TNF. The aim of this study is to evaluate the efficacy and tolerance of Etanercept in patients affected by active Rheumatoid Arthritis non- responsive to standard traditional therapies. All patients presented an improvement in the illness. Our study concords with the most recent data from literature that Etanercept acts rapidly and offers a quick and long-lasting reduction of illness activity, as well as an improvement in functional capability and quality of life in patients affected by active RA.

Key words: rheumatoid arthritis, Etanercept

Mailing address:Dr. E. Cacace, Policlinico Universitario,Reumatologia I, SS554 Bivio Sestu, Monserrato Cagliari, ItalyTel: +39 070 51096610 - Fax: +39 070 512020E-mail: [email protected]

Rheumatoid Arthritis (RA) is a chronic inflammatory disease of unknown aetiology and of an erosive nature that tends to progress to juxta-articular destruction and ankylosis. The pathological events that lead to joint destruction are due, in part, to an enhanced expression of Tumour Necrosis Factor (TNF) (1-2). It has been shown in numerous clinical studies that TNF-α blocking agents significantly reduce joint inflammation and slow down the progression of radiographic joint damage.

An essential pathogenetic role is played by TNF-α in both synovitis and osteo-cartilaginous erosive lesions by modulating cellular growth and differentiation, by stimulating lipolysis and activating cellular apoptosis and by inducing the

synthesis of other citochemical molecules such as IL-2 and IL-18. T-helper 1 responses are amplified, NO synthesis is induced, as is the expression of adhesion molecules, thus favouring cellular migration to the inflamed sites, fibroblastic proliferation of the fibroblast-like synoviocytes and neoangiogenesis (3-7). A TNF-α inhibiting drug is an efficient therapy in the suppression of joint inflammation and prevention of joint damage in RA (8).

Etanercept is a biological drug obtained through recombinant DNA techniques: it is a dimer of a chimerical protein obtained through the fusion of a soluble receptor for TNF (p75R) and the Fc fragment from human immunoglobin IgG1 (9-11). Etanercept is well-suited to soluble and membrane

Vol. 4, no. 3, 171-180 (2006)


TNF-α; it acts by inhibiting the linkage of TNF to the surface receptors rendering the TNF biologically inactive and inhibiting cellular response that it mediates (12).

The aim of the study is to evaluate the efficacy and tolerance of Etanercept (25 mg subcutaneously every 3 days) in patients affected by Rheumatoid Arthritis non-responsive to standard traditional therapies.


23 patients affected by active RA (mean age 45.74 ± 14.16 yrs, 21 – 75yrs, 4 males - 19 females) were enrolled on the study. All patients were resistant to traditional therapy. 15 patients were found positive for IgM Rheumatoid Factor (Table I).

The following enrolment criteria were satisfied by all patients prior to the start of the study:- confirmed diagnosis of Rheumatoid Arthritis

according to ACR criteria (13)- resistance to combined therapy with at least two

DMARDs, one of which was methotrexate- exclusion of specific pathologies (both on-going and

previous)- exclusion of infections and/or tumours.

All patients were evaluated on enrolment, at the 14th, 22nd and 54th weeks as follows:- laboratory tests: inflammation indexes (Erythrocyte

Sedimentation rate, PCR), Rheumatoid Factor (RA-test and Waaler-Rose), anti-nuclear factors (ANA, ENA, Anti-DNA), antibodies to cyclic citrullinated peptide (anti-CCP)

- general objective and articular tests including:a) evaluation of number of painful and swollen joints

(0-44)b) Ritchie joint index (0-78)c) VAS paind) VAS illness activity doctor/patiente) General health condition (GH)f) Health Assessment Questionnaireg) Disease Activity Score.

The study was approved by the Ethical Committee of the centre, and all enrolled patients provided a written informed consent. On enrolment, previous therapies were suspended and Etanercept (25 mg) was administered to patients subcutaneously twice weekly for the duration of the study. Statistical analysis was performed using commercially available software (STATISTICA 6.0 Stat. Soft Inc. USA) and a p value < 0.05 was considered significant.

RESULTS

All patients underwent a basal check and at the 14th week, 19 at the 22nd week and 12 at the 54th week. No difficulty in performing subcutaneous injections was reported by the patients who confirmed they had followed instructions given and had rotated the sites recommended for the injections. Analysis of the variables in the study showed that all patients presented an improvement in the illness showing a reduction of all the parameters studied. Mean inflammation index values progressively dropped, but the difference was statistically significant only in VES values between the basal and 22nd weeks (p<0.05) (Table II).

The reduction in the number of painful and swollen joints and in the Ritchie joint index were already statistically significant at the 14th week and remained so until the 54th week (Table III). VAS pain (39.78 ±19.57) at the 14th week showed a statistically significant reduction (p<0.001) compared to basal values (71.74±17.75); this reduction was maintained at the 22nd week (27.74±17.52) and at the 54th (30.00±31.11 ) (Table III).

Among other parameters taken into consideration, VAS illness activity evaluated by the physician progressively dropped from the basal (70.00±9.80) to the 14th week (32.39±16.23) and more evidently to the 22nd (22.10±15.02) and to the 54th (20.00±18.22) weeks. The same trend was seen in the VAS activity evaluated by the patient: basal (71.52±13.43), 14th week (36.30±17.91), 22nd week (24.74±19.54) and 54th week (21.67±21.36); a statistically significant reduction (p<0.001) was found for both VAS (Table III).

An improvement in GH values was observed in all tests after the basal (38.04±15.13): 14th week (64.56±16.98), 22nd week (74.27±19.85) and 54th week (78.75±18.96). The statistical significance (p<0.001) was achieved at the 14th week (Table III).

Mean illness activity values evaluated with DAS (Disease Activity Score) showed a statistically significant drop (p<0.001) in all tests following enrolment: basal (5.16±0.88), 14th week (4.21±0.70), 22nd week (3.70±0.92), 54th week (3.59±0.90) (Table III). Clinical improvement evaluated according to ACR criteria showed that at 14 weeks 47.8% (11/23 patients) obtained an ACR 20 response, 30.4% (7/23) an ACR50 response and 13% (3/23) an ACR70

E. CACACE ET AL.


response (Fig. 1). An ACR20 response was reached by 15.8% (3/19), ACR50 by 47.4% (9/19) and ACR70 by 36.8% (7/19) at the 22nd week (Fig. 1). At the end of the study (54th week), responses were: ACR20 8.3% (1/12), ACR50 16.7% (2/12) and ACR70 66.7% (8/12) (Fig. 1). Non-responders evaluated at the same intervals (14th, 22nd, 54th weeks) were 8.85% (2/23) 0% (0/19) and 8.3% (1/12) respectively (Fig. 1). One patient had to interrupt therapy at the 22nd week due to a lung infection (lobular pneumonia) which was clinically resolved with specific antibiotic treatment. One patient had to interrupt therapy at the 22nd week due to increased anti-dsDNA values.

DISCUSSION

A reduction in illness activity was observed at the 14th week in all patients treated with Etanercept, showing a significant statistical drop in the number of painful and swollen joints, in the Ritchie joint index, in VAS pain and VAS illness activity evaluated by both patient and physician, in General Health Condition and DAS. Important reductions in inflammation indexes (Erythrocyte Sedimentation rate – PCR) were obtained throughout the study. However, a statistically important drop was only seen in Erythrocyte

Sedimentation rate at the 22nd week. In line with other international clinical trials (10-

12, 14-15), important improvements in functional capacity, measured using the HAQ, and reductions in pain symptoms were also observed in our patients. These improvements were confirmed by the evaluation of illness activity by patient and physician (VAS).

Good illness management was obtained by all patients, with important statistical improvements of the variables examined maintained throughout the study. This is confirmed by the higher ACR responses of 20, 50 and 70 obtained by our patients in mono-therapy at the 22nd week compared to data reported in literature (14-16). It is currently believed that the number of patients affected by RA under therapy with biologic drugs varies between 5 and 30% depending on the reference centre and country. High production costs and the Italian Society of Rheumatology guidelines, mean that biologic drugs are currently prescribed only to those patients affected by aggressive RA (with negative prognosis factors) and resistant to combined therapies that include MTX, considered the “anchor-drug” in RA. The use of anti-TNF in early stages of the illness, once the use of 2 months therapy with high doses of DMARDs has been shown to be inefficient, is

Table I. Patients� demographic characteristics.

PATIENTS EXAMINED n° AGE(mean+SD)

range

DURATION (YRS) OF THE DISEASE

(mean+SD) range

TOTAL 23 45.74 � 14.16 (21-75)

8.70 � 4.72 (3-19)

FEMALES 19 44.84 � 14.57 (29-73)

8.26 � 4.61 (3-19)

MALES 4 50 � 22.23 (21-75)

10.75 � 5.06 (9-17)

Table II. Inflammation index values (mean + SD).

Parameters Basal 14th week 22nd week 54th week

n° of patients 23 23 19 12

ESR (mm 1st hr) 28.30 16.92 18.52 16.30 17.00 13.46 p<0.05

20.67 15.10

PCR (mg/dl) 4.58 14.38 0.98 1.25 0.52 0.45 0.42 0.43

Table I. Patients’ demographic characteristics.

Table I. Patients� demographic characteristics.

PATIENTS EXAMINED n° AGE(mean+SD)

range

DURATION (YRS) OF THE DISEASE

(mean+SD) range

TOTAL 23 45.74 � 14.16 (21-75)

8.70 � 4.72 (3-19)

FEMALES 19 44.84 � 14.57 (29-73)

8.26 � 4.61 (3-19)

MALES 4 50 � 22.23 (21-75)

10.75 � 5.06 (9-17)




ESR (mm 1st hr) 28.30 16.92 18.52 16.30 17.00 13.46 p<0.05

20.67 15.10

PCR (mg/dl) 4.58 14.38 0.98 1.25 0.52 0.45 0.42 0.43



Table III. Disease activity index (mean + SD) in patients examined. Differences were statistically significant (p<0,001).



VAS pain 71.74 17.75 39.78 19.57 24.74 17.52 30.00 31.11

VAS glob (ph) 70.00 9.80 32.39 16.23 22.10 15.02 20.00 18.22

VAS glob (pat) 71.52 13.43 36.30 17.91 24.74 19.54 21.67 21.36

painful joints (n°) 18.09 8.91 9.96 7.94 4.16 3.11 2.33 3.47

swollen joints (n°) 10.00 9.18 2.39 3.59 0.74 1.24 0.75 1.42

Ritchie�s index 16.35 5.87 7.96 4.33 4.26 2.70 2.33 1.97

Global Health 38.04 15.13 64.56 16.98 74.27 19.85 78.75 18.96

H.A.Q 1.78 0.47 1.08 0.63 0.80 0.51 0.63 0.43

D.A.S 5.16 0.88 4.21 0.70 3.70 0.92 3.59 0.90

Table III. Disease activity index (mean + SD) in patients examined. Differences were statistically significant (p<0,001).

0

10

20

30

40

50

60

70

14th week 22nd week 54th week

Fig. 1. Frequency of ACR responders.

ACR non respondersACR 20 respondersACR 50 respondersACR 70 responders

REFERENCES

1. Arend W.P. and C. Gabay. 2000. Network citochinico. In: Artrite Reumatoide (Ed.

Italiana). G. S. Firestein, G. S. Panayi, F. A.Wollheim, ed. Masson, p. 171.

2. Choy E.H.S. and G.S. Panayi. 2001. Cytokine pathways and joint inflammation in

rheumatoid arthritis. N. Engl. J. Med. 344:907.

3. Feldmann M., F.M. Brennan and R.N. Maini. 1996. Role of cytokines in rheumatoid

arthritis. Ann. Rev. Immunol. 14:397.

4. Aggrarwal B.B. 2000. Tumor necrosis factors receptor associated signalling molecules

and their role in activaction of apoptosis, JNK and NF-kB. Ann. Rheum. Dis. 59:6.

5. Tetta C., G. Camussi, V. Modena, C. Di Vittorio and C. Baglioni. 1990. Tumour

necrosis factor in serum and synovial fluid of patients with active and severe rheumatoid

arthritis. Ann. Rheum. Dis. 49:665.

6. Husby G. and R.C. Williams Jr. 1998. Synovial localization of tumor necrosis factor in

patients with rheumatoid arthritis. J. Autoimmun. 1:363.

Fig. 1. Frequency of ACR responders.

E. CACACE ET AL.


foreseen by the new therapeutic algorithms (17). It has been noticed that TNF is involved in both

the early and late stages of RA. Early treatment with anti-TNF agents in the initial stages of the illness can delay or prevent the inflammatory process that provokes and maintains the pathological process, consequently reducing future risk of bone erosion, joint destruction and deformities (16). Side effects were noticed in only one of the patients examined who presented a lobular pneumonia after 22 weeks of therapy. This was rapidly resolved by suspending the drug and with appropriate antibiotic treatment. This therapy is therefore on the whole safe and well tolerated, as many studies carried out after the drug was commercially available have demonstrated.

The severe side effects most frequently reported in literature are granulomatous infections, especially tuberculosis (18-20). This is normally a reactivation of latent TBC that can occur in the first few months of therapy; a distinct drop in the frequency of this complication has been noticed recently because patients destined to use Etanercept are specifically screened to exclude previous exposure to Koch mycobacterium. For this purpose, our clinical experience foresees carrying out the Mantoux test with 10 U of PPD under the skin in the volar part of the forearm with readings after 48 and 72 hours, as well as chest radiography.

The role of biologic drugs in favouring a relapse or the ex-novo origin of autoimmune illnesses such as Lupus or Multiple Sclerosis is more controversial. Statistics show that the percentage of patients on anti-TNF therapy that develops anti-dsDNA antibodies varies on average between 4 and 10%, though a drug-induced LES is extremely rare (21). Both the anti-nucleus antibodies and anti-dsDNA were evaluated in our patients on enrolment and at the end of the study: only 5 women among the patients enrolled showed a significant basal ANA (>1:80), with anti-dsDNA within the normal range. These values did not change in the following weeks with the exception of one patient whose anti-dsDNA value increased (11UI/ml) compared to reference values (4 UI/ml).

No patient developed lupus like syndrome.The most frequent side effects reported in

literature are local, with allergic reactions where the injection was performed. These are normally

erythematous lesions, with or without itching, pain or bruising, that normally appear within the first month of therapy. Therapy of any sort, either systemic or local (anti-histamines and/or cortisone for topic use) is not normally required.

In conclusion, this study concords with the most recent data from literature that Etanercept acts rapidly and offers a quick and long-lasting reduction of illness activity as well as an improvement in functional capability and quality of life in patients affected by active RA. The good tolerance levels suggest that laboratory monitoring would not appear to be particularly necessary.

Etanercept is fundamentally a well-tolerated drug. However, careful patient screening in the initial phase and regular checks during treatment are required for good illness management due to the risk of severe infection (from germs, tubercular mycobacteria and the hepatitis B + C virus) and tumours.

REFERENCES

1. Arend W.P. and C. Gabay. 2000. Network citochinico. In: Artrite Reumatoide (Ed. Italiana). G.S. Firestein, G.S. Panayi, F.A.Wollheim, ed. Masson, p. 171.

2. Choy E.H.S. and G.S. Panayi. 2001. Cytokine pathways and joint inflammation in rheumatoid arthritis. N. Engl. J. Med. 344:907.

3. Feldmann M., F.M. Brennan and R.N. Maini. 1996. Role of cytokines in rheumatoid arthritis. Ann. Rev. Immunol. 14:397.

4. Aggrarwal B.B. 2000. Tumor necrosis factors receptor associated signalling molecules and their role in activaction of apoptosis, JNK and NF-kB. Ann. Rheum. Dis. 59:6.

5. Tetta C., G. Camussi, V. Modena, C. Di Vittorio and C. Baglioni. 1990. Tumour necrosis factor in serum and synovial fluid of patients with active and severe rheumatoid arthritis. Ann. Rheum. Dis. 49:665.

6. Husby G. and R.C. Williams Jr. 1998. Synovial localization of tumor necrosis factor in patients with rheumatoid arthritis. J. Autoimmun. 1:363.

7. Goldring S.R. and F.M. Gravallese. 2000. Pathogenesis of bone erosions in rheumatoid





arthritis. Curr. Opin. Rheumatol. 12:195.8. Chikanza I.C., S. Jawed, D. Naughton and D.R.

Blake. 1998. Why do we need new treatment for Rheumatoid arthritis? J. Pharm. Pharmacol. 50:357.

9. Murray K.M. and S.L. Dahl. 1997. Recombinant human tumor necrosis factor receptor (p75) Fc fusion protein (TNFR:Fc) in rheumatoid arthritis. Ann. Pharmacother. 31:1335.

10. Moreland L.W., G. Maargolies, L.W. Heck Jr, A. Saway, C. Blosch, R.Hanna and W.J. Koopman. 1996. Recombinant soluble TNFR (p80) fusion protein: toxicity and dose finding trial in refractory rheumatoid arthritis. J. Rheumatol. 23:1849.

11. Moreland L.W., S.W. Baumgartner, M.H. Schjiff, E.A. Tindall, R.M. Fleishmann, A.L.Weaver, R.E. Ettlinger, S. Cohen, W.J. Koopman, K. Mohler, M.B. Widmer and C.M. Blosch. 1997. Treatment of rheumatoid arthritis with a recombinant human TNFR (p75)- Fc fusion protein. N. Engl. J. Med. 33:141.

12. Moreland L.W., M.H. Schiff, S.W. Baumgartner, et al. 1999. Etanercept therapy in rheumatoid arthritis. Ann. Inter. Med. 130:478.

13. Arnett F.C., S.M. Edworthy, D.A. Bloch et al. 1988. The American Rheumatism Association 1987 revised criteria for classification of rheumatoid arthritis. Arthritis Rheum. 31:315.

14. Moreland L.W., S.B. Cohen, S.W. Baumgartner, E.A. Tindall, K. Bulpitt, R. Martin et al. 2001. Long term safety and efficacy of Etanercept in patients with rheumatoid arthritis. J. Rheumatol. 28:1238.

15. Lyseng-Williamson K.A. and G.L. Plosker. 2004.

Etanercept. A pharmacoeconomic review of its use in Rheumatoid Arthritis. Pharmacoeconomics 22:1071.

16. Finckh A., J.F. Simard, J. Duryea, M.H. Liang, J. Huang, S. Daneel, A. Forster, C. Gabay and P.A. Guerne. 2006. The effectiveness of anti-tumor necrosis factor therapy in preventing progressive radiographic joint damage in rheumatoid arthritis: A population-based study. Arthritis Rheum. 54:54.

17. De Vries-Bouwstra J.K., B.A. Dijkmans and F.C. Breedveld. 2005. Biologics in early rheumatoid arthritis. Rheum. Dis. Clin. North. Am. 31:745.

18. Listing J., A. Strangfeld, S. Kary, R. Rau, U. Von Hinueber, M. Stoyanova-Scholz, E. Gromnica-Ihle, C. Antoni, P. Herzer, J. Kekow, M. Schneider and A. Zink. 2005. Infections in patients with rheumatoid arthritis treated with biologic agents. Arthritis Rheum. 52:3403.

19. Mufti A.H., B.W. Toye, R.R. Mckendry and J.B. Angel. 2005. Mycobacterium abscessus infection after use of tumor necrosis factor alpha inhibitor therapy: case report and review of infectious complications associated with tumor necrosis factor alpha inhibitor use. Diagn. Microbiol. Infect. Dis. 53:233.

20. Giles J.T. and J.M. Bathon. 2004. Serious infections associated with anticytokine therapies in the rheumatic diseases. J. Intensive. Care. Med. 19:320.

21. Mohan A.K., E.T. Edwards, T.R. Cotè, J.N. Siegel and M.M. Brawn. 2002. Drug-induced systemic lupus erythematosus and TNFα blockers. Lancet 360:646.

E. CACACE ET AL.





SPOTTED FEVER GROUP RICKETTSIAE IN IXODES RICINUS AND HAEMAPHYSALIS PUNCTATA TICKS IN ITALY

A. CIERVO, J. SIMEONI1, C. KHOURY, F. MANCINI and L. CICERONI

Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Rome; 1Servizio Igiene e Sanità Pubblica, Azienda Sanitaria di Bolzano, Bolzano, Italy

Received June 26, 2006 - Accepted October 4, 2006

In this study Ixodes ricinus and Haemaphysalis punctata ticks are examined. For the first time we detected Rickettsia conorii in I. ricinus and H. punctata, and Rickettsia sibirica in I. ricinus. Our results raise the question of whether other spotted fever group rickettsiae, in addition to R. conorii subsp. conorii and R. conorii subsp. israelensis, are involved in bacterial diseases in Italy and whether I. ricinus and H. punctata can act as new vectors for these rickettsiae.

Mailing address: Dr. Lorenzo Ciceroni, Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, ItalyTel: +39 06 49902741 - Fax: +39 06-49387112 e-mail: [email protected]

Key words: spotted fever group rickettsiae, Ixodes ricinus, Haemaphysalis puntata, Italy

Over fifteen pathogenic species of spotted fever group (SFG) rickettsiae have been recognized worldwide, nine since 1991. In Italy, until 2002, Rickettsia conorii was thought to be the only SFG rickettsia present, yet five other tick-transmitted rickettsiae that are pathogenic for humans have since been described: Rickettsia helvetica in Ixodes ricinus ticks (1); the Israeli spotted fever agent (R. conorii subsp. israelensis) in Rhipicephalus sanguineus ticks (2); Rickettsia slovaca in Dermacentor marginatus and Haemaphysalis punctata ticks (3); and Rickettsia africae and Rickettsia aeschlimannii in Hyalomma marginatum ticks (3). Moreover, in 2004, three people who developed a mild, non-eruptive disease, showed serological evidence of R. helvetica infection (4). More recently, Giammanco et al. (5) used molecular-sequence-based identification techniques to retrospectively study clinical isolates obtained from 24 Mediterranean spotted fever (MSF) cases occurring in Western Sicily from 1987 to 2001. Sequence analysis of the ompA gene identified 5 clinical isolates as R. conorii subsp. israelensis and

demonstrated the occurrence of Israeli spotted fever in Sicily. The remaining 19 of the 24 isolates were R. conorii subsp. conorii.


From June to July 2001, we conducted a tick-spirochete and tick-TBE survey in 41 sites in the region of Alto Adige (northeast Italy). A total of 330 adult ticks and 140 nymphae of the Ixodidae family were collected by flagging over vegetation; 308 ticks were identified as I. ricinus and 162 as H. punctata, according to standard taxonomic keys (6-7). Ticks were pooled according to species and the area and date of collection (40 pools of I. ricinus and 9 pools of H. punctata). They were rinsed in sterile distilled water and homogenised with sterile glass pestles in 2 ml of Hank’s medium containing 7.5% BSA.

As a side experiment, a sample of each extract was tested by PCR for SFG rickettsiae after DNA extraction with a QIAmp tissue kit (QIAGEN, Hilden, Germany), following the manufacturer’s instructions. PCR amplification was performed as previously described, using a rickettsial citrate synthase gene (gltA) primer pair (Rp.CS.877p and Rp.CS.1258n) and an SFG rickettsial

Vol. 4, no. 3, 177-180 (2006)

178 179Eur. J. Inflamm.A. CIERVO ET AL.

190-kDa surface antigen gene (rompA) primer pair (Rr.190.70p and Rr.190.602n) (8-9). Each positive PCR product was purified using a QIA Quick Gel extraction kit (QIAGEN), following the manufacturer’s instructions, and cloned in the pGEM-T easy vector (Promega, Madison, Wis.). Sequencing was performed with a commercial T7 sequencing kit (Amersham Biosciences, Uppsala, Sweden) with M13 forward and reverse primers, and the results were analysed on a Pharmacia Biotech ALFExpress automated DNA sequencer. The sequences obtained were compared to others using the BLAST search tool (http://www.nbc.n/m.nib.gov/BLAST/).

To confirm the identity of the tick species in the positive rickettsia samples, the complete 18S rRNA was obtained by PCR using primers A and G as previously described (10). The PCR products were sequenced and the nucleotide sequences were matched up in the GenBank with BLAST search tool (http://www.nbc.n/m.nib.gov/BLAST/).

RESULTS

Rickettsial DNA was detected in 5 (10.2%) of the 49 tick samples studied (Table I). The identity

of the tick species in the positive rickettsia samples was confirmed with the 18S rRNA sequence analysis (10). Sequences displayed 100% identity to I. ricinus accession no. Z74479 and H. punctata accession no. Z74478.

A rickettsia exhibiting 100% gltA (GenBank accession no. U59734) and rompA (GenBank accession no. U43807) identity to Rickettsia sibirica was found in 2 samples of I. ricinus (Table I, Fig. 1). R. sibirica is the etiological agent of Siberian tick typhus which is mainly transmitted by Dermacentor sylvarum and Haemaphysalis concinna ticks.

A rickettsia exhibiting 100% gltA (GenBank accession no. U59730) and rompA (GenBank accession no. U43806) identity to R. conorii (the agent of MSF) was identified in 1 sample of I. ricinus and in 2 samples of H. punctata (Table I, Fig. 1).

DISCUSSION

The Siberian Tick TIPHUS was first recognized in 1932 and has been extensively documented in Siberia

Table 1. Identification of Rickettsia spp. in tick samples from June to July 2001, in the region of Alto Adige (Northeast Italy).

Fig. 1.

M

500-400-

300-

bp1 2 3 4 5 1 2 3 4 5

I. ricinus H. pu

nctata

I. ricinus H. pu

nctata

gltA rompA

Fig. 1. PCR experiments with primers Rp.CS.877p/Rp.CS.1258n and Rr.190.70p/Rr.190.602n for gltA and rompA genes respectively. Lanes 1, 2 and 3 amplicons of I. ricinus samples. Lanes 4 and 5 amplicons of H. punctata samples.

14. Psaroulaki A., A. Germanakis, A. Gikas, E. Scoulica and Y. Tselentis. 2005.

Simultaneous detection of �Rickettsia mongolotimonae� in a patient and in a tick in Greece.

J. Clin. Microbiol. 43:3558.

15. de Sousa R., C. Barata, L. Vitorino, M. Santos-Silva, C. Carrapato, J. Torgal, D.

Walker and F. Bacillar. 2006. Rickettsia sibirica isolation from a patient and detection in

ticks, Portugal. Emerg. Infect. Dis. 12:1103.

16. Sanogo Y.O., P. Parola, S. Shpynov, J.L. Camicas, P. Brouqui, G. Caruso and D.

Raoult. 2003. Genetic diversity of bacterial agents detected in ticks removed from

asymptomatic patients in northeastern Italy. Ann. N.Y. Acad. Sci. 990:182.

Table 1. Identification of Rickettsia spp. in tick samples from June to July 2001, in the region of

Alto Adige (northeast Italy).

Tick species identified (n° examined /n° pools)

Pool infection rate (%) N° Rickettsia spp. identified

(% identity with gltA/% identity with rompA)

Ixodes ricinus (308/40) 3/40 (7.5) 2 Rickettsia sibirica (100/100)1 Rickettsia conorii (100/100)

Haemaphysalis punctata (162/9) 2/9 (22.2) 2 Rickettsia conorii (100/100)

Legend to figure

Fig. 1. PCR experiments with primers Rp.CS.877p/Rp.CS.1258n and Rr.190.70p/Rr.190.602n for gltA and rompA genes respectively. Lanes 1, 2 and 3 amplicons of I. ricinus samples. Lane 4 and 5 amplicons of H. punctata samples.


and western China. To the best of our knowledge, this is the first report of R. sibirica (or a closely related rickettsia) in ticks in Italy and of its presence in I. ricinus ticks. The first evidence of a rickettsia closely related to R. sibirica in a European country was found in 1996 in southern France (11-12). This rickettsia (Rickettsia sibirica mongolotimonae) belongs to the R. sibirica species yet exhibits specific serotypic and ecological characteristics. While North Asian tick typhus is confined to Siberia and western China, R. sibirica mongolotimonae infection has been found in Hyalomma ticks in Inner Mongolia, Niger and Greece, in a Rhipicephalus pusillus tick in Portugal, and in humans in France, Greece, Portugal and in northern and southern Africa. To date, only 12 human cases of Rickettsia sibirica mongolotimonae infection have been described (13-15). However, there was 98% similarity in the nucleotide sequence of rompA gene between the rickettsia found in the 2 I. ricinus samples and the Rickettsia sibirica mongolotimonae strain HA-91 (GenBank accession no. U43796). Consequently, the rickettsia we found is likely to be R. sibirica.

Although the finding of R. conorii in Alto Adige is not a novelty - in that cases of MSF, though infrequent, have been reported - this species has never been found in I. ricinus or H. punctata ticks. However, whether I. ricinus and H. punctata ticks can act as vectors of MSF remains to be determined. MSF is transmitted by R. sanguineus and is prevalent in countries around the Mediterranean and Black Seas; in Sicily, it has also been detected in Rhipicephalus turanicus ticks (3). In Italy, as in other European countries, I. ricinus is the vector of Borrelia burgdorferi sensu lato, tick-borne encephalitis virus, Anaplasma phagocytophilum and possibly R. helvetica (1). It is widespread and represents more than 90% of all the ticks removed from humans in northern Italy (16). H. punctata has been found to be infected with R. slovaca in Sicily (3).

The results of this study raise the question of whether other SFG rickettsiae, in addition to R. conorii subsp. conorii and R. conorii subsp. israelensis, are involved in bacterial diseases in Italy and whether I. ricinus and H. punctata can act as vectors for R. conorii, and I. ricinus as a vector for R. sibirica. Since rickettsiae are fastidious and culturing them is very complex, serology is the most widely used microbiological means of diagnosing SFG

rickettsioses, with indirect immunofluorescence assay (IFA) being the most common method. However, given that IFA cannot identify rickettsiae at the species level (because of cross-reactivity among SFG rickettsiae), microbiological confirmation could be performed using a multiple–antigen IFA and/or cross-adsorption and western blotting. PCR on eschar biopsy samples has a high probability of detecting rickettsial DNA. In any case, clinicians need to be aware of emerging tick-borne diseases in Italy, in particular, infections due to R. sibirica.

REFERENCES

1. Beninati T., N. Lo, H. Noda, F. Esposito, A. Rizzoli, G. Favia and C. Genchi. 2002. First detection of spotted fever group rickettsiae in Ixodes ricinus from Italy. Emerg. Infect. Dis. 8:983.

2. Giammanco G., S. Mansueto, P. Ammatuna and G. Vitale. 2003. Israeli spotted fever Rickettsia in Sicilian Rhipicephalus sanguineus ticks. Emerg. Infect. Dis. 9:892.

3. Beninati T., C. Genchi, A. Torina, S. Caracappa, C. Bandi and N. Lo. 2005. Rickettsiae in Ixodid ticks, Sicily. Emerg. Infect. Dis. 11:509.

4. Fournier P.E., C. Allombert, Y. Supputamongkol, G. Caruso, P. Brouqui and D. Raoult. 2004. Aneruptive fever associated with antibodies to Rickettsia helvetica in Europe and Thailand. J. Clin. Microbiol. 42:816.

5. Giammanco G.M., G. Vitale, S. Mansueto, G. Capra, M.P. Caleca and P. Ammatuna. 2005. Presence of Rickettsia conorii subsp. israelensis, the causative agent of Israeli spotted fever, in Sicily, ascertained in a retrospective study. J. Clin. Microbiol. 43:6027.

6. Starkoff O. 1958. Ixodoidea d’Italia. ed Il Pensiero Scientifico. Rome, Italy. p. 385

7. Khoury C. and C. Lezzerini. 1980. Guida per il riconoscimento dei più comuni Ixodidi (Acarina Ixodoidea) Italiani. In Rapporti ISTISAN N° 21. ed. Istituto Superiore di Sanità ed.. Rome, Italy p. 1.

8. Regnery R.L., C.L. Spruill and B.D. Plikaytis. 1991. Genotypic identification of rickettsiae and estimation of intraspecies divergence for portions





of two rickettsial genes. J. Bacteriol. 173:1576.9. Roux V., E. Rydkina, M. Eremeeva and D. Raoult.

1997. Citrate synthase gene comparison, a new tool for phylogenetic analysis, and its application for the rickettsiae. Int. J. Syst. Bacteriol. 47:252.

10. Mangold A.J., M.D. Bargues and S. Mas-Coma. 1998. 18S rRNA gene sequences and phylogenetic relationships of European hard-tick species (Acari: Ixodidae). Parasitol. Res. 84:31.

11. Raoult D., P. Brouqui and V. Roux. 1996. A new spotted-fever-group rickettsiosis. Lancet 348:412.

12. Fournier P.E., H. Tissot-Dupont, H. Gallais and D. Raoult. 2000. Rickettsia mongolotimonae: a rare pathogen in France. Emerg. Infect. Dis. 6:290.

13. Fournier P.E., F. Gouriet, P. Brouqui, F. Lucht and D. Raoult. 2005. Lymphangitis-associated rickettsiosis, a new rickettsiosis caused by Rickettsia

sibirica mongolotimonae: seven new cases and review of the literature. Clin. Infect. Dis. 40:1435.

14. Psaroulaki A., A. Germanakis, A. Gikas, E. Scoulica and Y. Tselentis. 2005. Simultaneous detection of “Rickettsia mongolotimonae” in a patient and in a tick in Greece. J. Clin. Microbiol. 43:3558.

15. de Sousa R., C. Barata, L. Vitorino, M. Santos-Silva, C. Carrapato, J. Torgal, D. Walker and F. Bacillar. 2006. Rickettsia sibirica isolation from a patient and detection in ticks, Portugal. Emerg. Infect. Dis. 12:1103.

16. Sanogo Y.O., P. Parola, S. Shpynov, J.L. Camicas, P. Brouqui, G. Caruso and D. Raoult. 2003. Genetic diversity of bacterial agents detected in ticks removed from asymptomatic patients in northeastern Italy. Ann. N.Y. Acad. Sci. 990:182.

A. CIERVO ET AL.





SINGLE-BLIND EVALUATION OF POST-TONSILLECTOMY PAIN TREATMENT WITH AN EUTECTIC MIXTURE OF LOCAL ANESTHETICS

G. NERI, C. BAFFA, F. VITULLO, R. DI MASCIO1 and E. BALLONE1

Surgical Experimental and Clinical Sciences Department, ENT Division, “G. d’Annunzio” University of Chieti; 1Medicine and Ageing Science Department, Laboratory of Biostatistics,

University of Chieti, Chieti, Italy

Received March 17, 2006 – Accepted October 5, 2006

The efficacy of an eutectic mixture of local anesthetics (lidocaine and prilocaine) in ointment for topical use (EMLA) in the treatment of post-tonsillectomy pain in both pediatric and adult patients was evaluated. Ninety patients affected by recurring tonsillitis and refractory to antibiotic therapy were submitted to tonsillectomy by dissection. Of the ninety patients 45 random subjects received topical treatment and the remaining 45 did not receive any treatment in the emptied tonsillar compartment. Pain intensity was evaluated at 3, 6 and 9 hours after surgical treatment using a visual subjective evalu-ation scale (VAS for adults and FES for children). The percentage of adults who referred pain of minor intensity was greater in the treated patients than in the controls: 51.4% vs 14.3% (p<0.001) at the third hour, 71.4% vs 2.9% (p<0.001) at the sixth hour and 88.6% vs 14.3% (p<0.001) at the ninth hour. In the pediatric groups, 80.0% of the treated subjects referred moderate pain at the third hour, compared to 40% of the control group. At the ninth hour all children (100%) referred moderate pain compared to 20% of the control group. No treated patient required analgesic therapy during the post-operative observation period. The application of EMLA in the compartment after tonsillectomy determined an extremely significant reduction of postoperative pain in terms of intensity and duration.

Mailing address: Giampiero Neri,c/o Ospedale SS Annunziata, Via dei Vestini, 66013 Chieti, ItalyTel/Fax: +39 0871 552033e-mail: [email protected]

Key words: tonsillectomy, pain, lidocaine, prilocaine

Even though tonsillar pathology can be treated with medical therapy, tonsillectomy, in the child or adult, is without doubt the most frequent surgical procedure in otorhinolaryngology. In Italy, its incidence is approximately three times higher than average and is inferior only to appendectomy (1). The frequency of tonsillectomy is probably linked to the fact that tonsillar pathology strongly influences the quality of life of those affected and their families, in terms of economic and biological costs, by limiting work, scholastic, social, and recreational activities. During the pre-operative evaluation, these details

prevail over the modifications of the local-regional immune capacity and the unpleasant postoperative aspects (for example, pain and inflammatory state due to tissue trauma).

Pain after tonsillectomy is thought to be due to a combination of nerve irritation, inflammation and pharyngeal muscle spasm. Primary hyperalgesia (2), due to the liberation of vasoactive, chemotactic and proinflammation substances (bradykinin, serotonin, prostaglandin, etc.), represents the immediate response to the surgical injury and is an alteration of the sensitivity that, reducing pain

Vol. 4, no. 3, 181-189 (2006)


threshold, causes an abnormal increase of the pain sensibility (3) induced by a stimulus(4-5). The persistence of pain in time is linked to a “secondary hyperalgesia” whose origin is complex and not yet perfectly clarified Therefore, the treatment of acute pain, especially postoperative pain, besides having an ethical aspect, assumes fundamental importance because it enormously improves patient outcome, significantly reducing mortality and morbidity along with noted systemic effects (neuroendocrine response, incretion of catabolic hormones, inhibition of anabolic substances, modification of A.N.S. and modifications of the cardiovascular and respiratory system) (6-13).

Many studies concerning the treatment of post-tonsillectomy pain have been conducted through local application of medicines (anesthetic and non) injected or sprayed or otherwise applied in the compartment, immediately before or after the abscission of the tonsils. Nevertheless, a publication in the Cochrane register concerning the treatment of pain with local anesthetics after tonsillectomy, concluded that there was no proof that the use of local anesthetic in patients subjected to tonsillectomy improved postoperative pain control (14).

According to the literature, the use of an eutectic mixture of local anesthetics (EMLA) in ointment for topical use in the treatment of post-tonsillectomy pain has never been considered. This is a basic oleaginous emulsion with a 5% concentration where two local anesthetics, lidocaine and prilocaine, are present in equal proportions. Their mechanism of action is to block Na+ channels at the axonal membrane level, preventing the transit of nociceptive impulses at the cortical level. This result is obtained by reducing the action potential of the nerve fibers and by increasing the refractory period, avoiding the wind-up phenomenon responsible for post-surgical hyperalgesia.

We evaluate the efficacy of EMLA in the treatment of post-tonsillectomy pain in both pediatric and adults patients.


Ninety patients were enlisted, equally divided by sex, and affected by recurring tonsillitis and refractory to antibiotic therapy. Of these, 70 were

adults (average age 27.3 years, range 18-47) and 20 children (average age 8 years, range 4-13). After a series of preoperative “routine” tests, all patients were informed of the aim of the study, and personal consent was given by the person directly interested or his/her parents (in case of minors). The patients underwent tonsillectomy by dissection in the Rose position, in general anesthesia with assisted ventilation and tracheal intubation. Thirty minutes preceding the surgical treatment, the adult patients were medicated with atropine 0.5 mg and promethazine 0.5 mg im and the children received midazolam 0.5 mg/kg sublingual.

In the emptied tonsillar compartment, the patients randomly received, at the end of the operation and before waking up, 2 ml of an association of lidocaine-prilocaine in ointment (EMLA) applied in the region with the use of a 5 ml syringe equipped with plastic needle cannula (35 adults and 10 children) or no treatment (35 patients). The same methodology was used in pediatric patients, of whom 10 received EMLA treatment and 10 received no treatment (control group). Pain intensity was evaluated for all the patients in the study at 3, 6 and 9 hours after surgical treatment using a visual subjective evaluation scale already amply validated in literature.

For the adults we used a visual analogical scale (VAS) from 0 to 10 points, while in the children pain intensity was evaluated using a facial expressions scale (FES) with up to 6 points. All the patients were requested to assess their pain level on the respective evaluation scale (15-18). The VAS was then categorized into three groups of pain intensity: light <4 points; moderate between 4 and 7 points; and heavy >7 points; the FES was categorized into two groups of pain: moderate ≤4 points and heavy >4 points.

Statistical analysisThe adult sample size was calculated on the

basis of a 30% difference in percentage of adult patients with pain intensity >7 at 6 hours assuming a prevalence of 40% among controls. To reach a power of 80% with a type I error of 5%, a minimum of 32 subjects must be enrolled for each adult group.

The randomization of patients into the two groups was determined by sequentially alternating

G. NERI ET AL.


their assignment following the scheduled order (determined by an administrative office) of the surgical procedures. The study was single-blind since only the patients did not know to which group they were assigned.

Initially, a summary analysis of the ordinal variables of pain intensity (VAS and FES) was performed by mean score and Standard Deviation (SD). Frequencies of VAS and FES <4 for each treated patient categorized values that were compared to their respective control group at 3, 6 and 9 hours using Chi-squared test or Fisher exact test, when appropriate.

The ordinal values obtained for VAS and FES did not have a normal distribution, therefore, we applied a non-parametric test. The Mann-Whitney test was used to compare the treated group with the control group and the Wilcoxon test to compare the different time points for each group.

Statistical analysis was performed using SPSS Advanced StatisticalTM 10.0 software (SPSS Inc., Chicago, Ill).

RESULTS

No statistically significant differences were seen between the treated and control groups of both adult and pediatric groups in terms of age and gender (Table I). Patients not treated required analgesic therapy during the post-operative observation period; while 9 adult patients (25.7%) and 4 pediatric patients (40%) in both control groups required analgesic therapy which was administered during the first three post-operative hours after awakening (Table II).

No adult patient in the treated group indicated that they had pain >7 on the VAS scale during the 9 hours of observation (Fig. 1A) compared to 34.3%, 40.0% and 11.4% of the control group at the third, sixth and ninth hours, respectively (Fig. 1B). The percentage of patients that referred pain of a light intensity (VAS<4) was greater in the treated patients than in the controls: 51.4% vs 14.3% (p<0.001) at the third hour, 71.4% vs 2.9% (p<0.001) at the sixth hour and 88.6% vs 14.3% (p<0.001) at the ninth hour (Fig. 1A and 1B).

In the pediatric groups, 80.0% of the children referred pain with an intensity ≤ 4 (FES) at the third hour, compared to 40% of the control group, at the

sixth and ninth hour all children (100%) referred pain with an intensity ≤ 4; in the control group the children referred pain with an intensity > 4 in 60% of subjects at the third hour in 60% at the sixth hour (p<0.05) and in 80% at the ninth hour (p<0.01) (Fig. 2A and 2B).

In the adults, the mean values (± SD) of pain intensity of the VAS at the third, sixth and ninth hours were 4.07 (1.66), 3.27 (1.43) and 2.51 (1.06) in treated patients and 6.19 (1.32), 6.35 (1.25) and 5.35 (1.10) in the control group, respectively. A comparison of the means indicated a statistically significant difference between the treated group and the control group at each time point (Mann-Whitney test, p<0.01) (Fig. 3). A significant reduction in pain intensity was already registered at the sixth hour for the treated group and at the ninth hour for both groups (Wilcoxon test, p<0.001).

In the children, the mean values (±SD) of pain intensity of the VAS at the third, sixth and ninth hours were 3.1 (0.87), 2.2 (0.63) and 1.7 (0.67) in the treated patients and 4.7 (1.16), 5.0 (0.82) and 5.0 (0.67) in the control group. A comparison of the means indicated a statistically significant difference between treated patients and the control group at each time point (Mann-Whitney test, p<0.05 at the third hour and p<0.01 at both the sixth and ninth hours) (Fig. 4) with a statistically significant reduction in pain intensity (Wilcoxon test, p<0.001) over the observation time in the treated group. We did not observe any collateral effects in the treated patients.

DISCUSSION

Pain has been defined by the International Association for the study of pain as “an unpleasant sensory and emotional experience, associated to potential or real tissue damage or described in terms of such damage” (19-20). “Pathological” pain and its correlated problems constitute a serious problem in postoperative management; because an operation determines two types of pain:1. intraoperative pain: extremely violent due to

surgical maneuvers; 2. postoperative pain: following surgery, due to

tissue lesions and produced by the stimulation of peripheral receptors.


The results obtained in the present study demonstrate that the application of EMLA cream in the tonsillar compartment at the end of the operation significantly reduces post-tonsillectomy pain. In both treated groups (adults and children), from the

first registration, pain was progressively reduced, such that at the ninth hour all the patients treated presented pain referable to the lowest VAS or FES range (Fig. 1A, 2A); whereas, in the control group, postoperative pain was significantly greater than that

G. NERI ET AL.

B

0

20

40

60

80

100

3 6 9Postoperative Hour

Adult patients (%)

A

0

20

40

60

80

100

3 6 9

Postoperative Hour

Adu

lt pa

tient

s (%

) ***

***

**

Fig. 1. Percentage of adult patients in 3 classes of VAS at 3, 6 and 9 postoperativehours.** p<0.05; *** p<0.001; Chi squared test, A compared with B. Non-treated patients(A) referred pain > 7 and pain is reduced progressively in all patients while in thecontrol group (B) at the sixth hour the moderate and strong pain increased while thelight pain is reduced. At the ninth hour a reduction of heavy pain corresponds to anincrease in moderate and light pain.

Fig. 1. Percentage of adult patients in 3 classes of VAS at 3, 6 and 9 postoperative hours. ** p<0.05; *** p<0.001; Chi squared test, A compared with B. Non-treated patients (A) referred pain > 7 and pain is reduced progressively in all patients while in the control group (B) at the sixth hour the moderate and strong pain increased while the light pain is reduced. At the ninth hour a reduction of heavy pain corresponds to an increase in moderate and light pain.


B

0

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40

60

80

100

3 6 9

A

0

20

40

60

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3 6 9Child patients

Postoperative Hour

* **

ns.

Fig. 2. Percentage distribution of children in two classes of FES at 3, 6 and 9 postoperative hours. A: treated with EMLA; B: control group. N.S. Not significant; *P<0.05; **P<0.01; Fisher exact test, A compared with B. In thetreated patients at the third hour 80.0% referred moderate pain and at the sixth and ninthhour all children (100%) referred moderate pain; in the control group only 40% of thechildren referred pain with an intensity <4 that remained stationary at the third hour andincreased in ninth hour.

FES < 4

FES > 4

%

FES < 4

FES > 4

%

Child patientsPostoperative Hour

Fig. 2. Percentage distribution of children in two classes of FES at 3, 6 and 9 postoperative hours. A: treated with EMLA; B: control group.n.s. not significant; *p<0.05; **p<0.01; Fisher exact test, A compared with B. In the treated patients at the third hour 80.0% referred moderate pain and at the sixth and ninth hour all children (100%) referred moderate pain; in the control group only 40% of the children referred pain with an intensity <4 that remained stationary at the third hour and increased in ninth hour.


0

1

2

3

4

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3 6 9

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FES

Sco

re

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***

**

0

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4

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8

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3 6 9

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VA

S S

core

Emla

No Emla

******

Fig. 3. Mean ± SD of VAS for adult patients.** p<0.01; Mann-Whitney test, treated group versus control group. A comparison of the means indicated a statistically significant differencebetween the treated group and the control group at each time point(Mann-Whitney test, p<0.01).

Fig. 4. Mean±SD of FES for children. * p<0.05; ** p<0.01; Mann-Whitney test, treated group versus controlgroup. A comparison of the means indicated a statistically significantdifference between treated patients and the control group at each timepoint (Mann-Whitney test, p<0.05 at the third hour and p<0.01 at boththe sixth and ninth hours) with a statistically significant reduction in painintensity (Wilcoxon test, p<0.001) over the observation time in thetreated group.

Fig. 3. Mean ± SD of VAS for adult patients. ** p<0.01; Mann-Whitney test, treated group versus control group.A comparison of the means indicated a statistically significant difference between the treated group and the control group at each time point (Mann-Whitney test, p<0.01).

0

1

2

3

4

56

3 6 9

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FES

Sco

re

EmlaNo Emla

***

**

0

2

4

6

8

10

3 6 9

Postoperative Hour

VA

S S

core

Emla

No Emla

******

Fig. 3. Mean ± SD of VAS for adult patients.** p<0.01; Mann-Whitney test, treated group versus control group. A comparison of the means indicated a statistically significant differencebetween the treated group and the control group at each time point(Mann-Whitney test, p<0.01).

Fig. 4. Mean±SD of FES for children. * p<0.05; ** p<0.01; Mann-Whitney test, treated group versus controlgroup. A comparison of the means indicated a statistically significantdifference between treated patients and the control group at each timepoint (Mann-Whitney test, p<0.05 at the third hour and p<0.01 at boththe sixth and ninth hours) with a statistically significant reduction in painintensity (Wilcoxon test, p<0.001) over the observation time in thetreated group.

Fig. 4. Mean±SD of FES for children. * p<0.05; ** p<0.01; Mann-Whitney test, treated group versus control group. A comparison of the means indicated a statistically significant difference between treated patients and the control group at each time point (Mann-Whitney test, p<0.05 at the third hour and p<0.01 at both the sixth and ninth hours) with a statistically significant reduction in pain intensity (Wilcoxon test, p<0.001) over the observation time in the treated group.

G. NERI ET AL.


significantly improve the control of postoperative pain (14). In the other studies, in fact, generally only one type of anesthetic (bupivacaine or lidocaine), and not an association, was used.

Bupivacaine is characterized by a long-lasting effects (120-300 minutes for a dosage of 2.5 mg/Kg) but a slow action time. Lidocaine has a rapid action time but a brief duration (30-60 minutes for a dosage rate of 7 mg/kg if used with adrenaline). These two drugs have never been used together in any previous study. In our study, we used an association of lidocaine and prilocaine that presents a longer action time than lidocaine alone and a duration slightly inferior to that of bupivacaine alone (60-240 minutes for a dose of 2 gr).

In some studies the anesthetic was sprayed into the tonsillar region at the end of operation, and this probably determined a dispersion and consequently the reduction of local anesthetic concentration (14). In other studies the anesthetic was injected into the compartment before the removal of tonsils (17, 21) or at the end of the operation (14, 22). In the first case, the rich vascularization of the tonsillar region

indicated by the treated group at the first registration, and with the progression of the postoperative period, pain increased (Fig. 1B, 2B). Moreover, of the control group, 25.7% of adults and 40.0% of children asked for an analgesic therapy whereas this therapy was not requested by any of the treated patients. This data confirms, even if indirectly, the effectiveness of the postoperative treatment effectuated and explains how at the ninth hour the intensity of pain was significantly reduced with respect to the preceding observations. Moreover, excluding those patients that had assumed the analgesic, the modification of pain in time did not vary. The diversity between the results in our experience and those in literature could be due to the pharmacological characteristics of the preparations used or to the diverse modality of application.

In the literature, many studies have been conducted on the treatment of post-tonsillectomy pain through the local use of anesthetic, not injected, in the tonsillar region immediately before or after the removal of tonsils, or sprayed or otherwise applied in the compartment only after the surgical procedure. In all cases it has been observed that this did not

Table I . The distribution of patients by age and sex in all groups considered.

Adults Children

Emla (n=35) No Emla(n=35) Emla (n=10) No Emla

(n=10)Age (mean�SD) 26.91�7.36 27.09�7.40 7.50�3.10 8.70�2.45

Male 17 (48.6) 18 (51.4) 4 (50.0) 4 (50.0) Sex n (%)

Female 18 (51.4) 17 (48.6) 6 (50.0) 6 (50.0)

Table II . The distribution of patients that requested analgesic therapy. Nine adults, (5 males and 4 females) and 4 children (2 males and 2 females requested analgesics in the postoperative period. It is evident that treatment with Emla prevents the necessity of administering additional analgesics.

Adults Children


(n=10)Male 0 5 (14.3) 0 2 (20)

Sex n (%) Female 0 4 (11.4) 0 2 (20)



Adults Children


(n=10)Age (mean�SD) 26.91�7.36 27.09�7.40 7.50�3.10 8.70�2.45

Male 17 (48.6) 18 (51.4) 4 (50.0) 4 (50.0) Sex n (%)

Female 18 (51.4) 17 (48.6) 6 (50.0) 6 (50.0)

Table II . The distribution of patients that requested analgesic therapy. Nine adults, (5 males and 4 females) and 4 children (2 males and 2 females requested analgesics in the postoperative period. It is evident that treatment with Emla prevents the necessity of administering additional analgesics.

Adults Children


(n=10)Male 0 5 (14.3) 0 2 (20)

Sex n (%) Female 0 4 (11.4) 0 2 (20)

Table II . The distribution of patients that requested analgesic therapy.Nine adults, (5 males and 4 females) and 4 children (2 males and 2 females requested analgesics in the postoperative period. It is evident that treatment with Emla prevents the necessity of administering additional analgesics.


could have favored the rapid displacement of the drug through the vessel network with a consequent reduction of local concentration. In the second case, the slowness of bupivacaine action, the only one experimented in literature, would not permit the inhibition of the wind-up phenomenon with subsequent postoperative hyperalgesia.

In our study we used EMLA cream applied to the tonsillar compartment, in direct contact with the muscular fascia, after tonsillectomy. This permitted the anesthetics to penetrate without causing excessive modifications of their concentration. The pharmacological characteristics and the application method used in our study definitely permitted a rapid action on the genesis of postoperative pain. The association of lidocaine and prilocaine, taking into consideration action time and the diverse duration of these drugs, permitted us to achieve a winning “combination” against postoperative pain. Moreover, the reduction of pain in the first hours favors the precocious recovery of muscular activity. This phenomenon is translated in a rapid removal by the lymphatic pathway of degradation products and of phlogistic neurotransmitters that are at the basis of postoperative pain. This explains the velocity of pain reduction verified at the 6th and 9th hour (Fig. 3, 4).

In conclusion, on the basis of our experience, the application of EMLA in the compartment after tonsillectomy, determined an extremely significant reduction of postoperative pain, either as absolute intensity or duration of pain. In fact, in all patients a significant difference in the intensity of pain was observed between the treated patients and the control group. These differences increased in the subsequent registration, such that in the treated group a progressive reduction of pain was observed whereas in the group not treated pain tended to increase in the post-operative period. This resulted in greater patient comfort and a reduction of economic and biological postoperative costs. In fact, better tolerability of postoperative pain reduces the request of analgesic drugs, which on one hand increases health care costs, while on the other can be the cause of allergic and metabolic collateral effects.

REFERENCES

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129 REVIEW ARTICLE GLAUCOMA: CURRENT AND