Swine Flu and LPS Bacteria – a deadly combination? Scientific reasons why swine flu needs to be accepted seriously

Liubov Shynkarenko Sichel, PhD, Pure Research Products, LLC, Boulder, Colorado

In response to the rise in the number of confirmed cases of swine influenza A (H1N1) reported in the United States, Mexico and around the world, the Department of Health and Human Services declared a public health emergency in the United States1. About real threat to develop human influenza epidemic caused by novel subtypes of human- swine-avian reassortment viruses community have been told by European scientists already in 2002 2-9

In the past the Centers for Disease Control and Prevention (CDC) received reports of approximately one human swine influenza virus infection every one to two years in the U.S., but from December 2005 through February , 2009, 12 cases of human infection with swine influenza have been reported.

In that time scientists found 50 cases of apparent zootic swine influenza virus infection on period in April 2006 to March 2007, 37 of which involved civilians and 13 of which involved military personnel, with a case-fatality rate of 14% (7 of 50 persons)10

Researchers have been warned about probable genetic and antigenic evolution of Swine Influenza Viruses on the grounds of the pigs nature. The pig is susceptible to both avian and human influenza viruses and can serve as intermediate host in influenza virus ecology. Zoonotic agents may emerge in pigs following the modification of an established swine strain, adaptation of a strain of avian origin to the mammalian host, or reassortment between human and avian influenza viruses11,12.

On June 11, 2009, SNN reported :The World Health Organization raised the swine flu alert Thursday June 9, 2009, to its highest level , saying the H1N1 virus has spread to enough countries to be considered a global pandemic13. Increasing the alert to Phase 6 does not mean that the disease is deadlier or more dangerous that before, just that it has spread to more countries, the WHO said.

As of June 9, 2009, the virus has spread to 74 countries, there were 28,774 confirmed cases and 144 deaths. The United States had 13,217 cases and 27 deaths.

” We are moving into the early days of the first flu pandemic of the 21st century, WHO Director General Margaret Chan said . Phase 6 is meant as a signal to countries to recalibrate their strategies to minimize the harm from swine flu. The virus is not stoppable, she said.”I would advise them to maintain vigilance, enhance surveillance and be prepared for the arrival of the novel H1N1 in their country”13

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The swine Influenza A (H1N1) viruses characterized in this outbreak have not been previously detected in pigs or humans. The viruses so far characterized have been sensitive to the prescription antiviral drugs oseltamivir (Tamiflu®) and zanamivir (Relenza®), but resistant to both amantadine and rimantadine14

Types of Influenza Viruses

According to the National Center for Immunization and Respiratory Diseases (NCIRD), the Coordinating Center for Infectious Diseases (CCID) and the CDC, there are three types of influenza viruses: Influenza A, Influenza B and Influenza C. Influenza A and B viruses cause seasonal epidemics of disease almost every winter in America. Influenza type C infections cause mild respiratory illnesses and can cause local epidemics15.

Influenza A viruses are divided into subtypes based on two proteins on the surface of the virus: the hemagglutinin (H) and the neuraminidase (N). There are 16 different hemagglutinin subtypes and 9 different neuraminidase subtypes. Influenza A viruses can be further broken down into different strains.

The current subtypes of influenza A viruses are A (H1N1) and A (H3N2) and since 1977, these two virus subtypes have circulated widely among humans. Influenza B viruses are not divided into subtypes, but they are further broken down into different strains.

A new A(H1N2) strain was been reported in February of 2002 and was isolated from humans in England, Israel and Egypt. The H1N2 strain appears to have resulted from the reassortment of the genes of the currently circulating influenza A (H1N1) and A (H3N2) subtypes.

The hemagglutinin protein of the influenza A (H1N2) virus is similar to that of the currently circulating A(H1N1) viruses, and the neuraminidase protein is similar to that of the current A(H3N2) viruses. Each of one of the viruses Influenza A (H1N1), A (H3N2), and one Influenza B strains are included in each year’s influenza vaccine.

Because the current (2008/2009) influenza vaccine contains strains with both H1 and N2 proteins similar to those in new strain, the current vaccine should provide good protection against the new A(H1N2) virus. No unusual levels of disease have been associated with this virus and, at this time, it is uncertain if the A (H1N2) virus will persist and circulate widely.

Getting a flu vaccine can protect against influenza A and B viruses, but does not offer protection against influenza C viruses.This year’s influenza vaccine contains three new influenza virus strains:

A/Brisbane/59/2007(H1N1)-like virus;

A/Brisbane/10/2007 (H3N2)-like virus;

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B/Florida/4/2006-like virus.

The 2008-09 influenza vaccine can offer protection against the three above mentioned viruses, or it can reduce the adverse symptoms of a related but different influenza virus strain.

Swine Influenza

The classical swine flu virus (an influenza type A (H1N1) virus was first isolated from a pig in 1930. Swine flu viruses cause high level of illness, but low death rates in pigs. Like all influenza viruses, swine flu viruses change constantly. Pigs can also be infected by avian influenza and human influenza viruses. When influenza viruses from different species infect pigs, the viruses can reassort (i.e. swap genes) and new viruses that are a mix of swine, human and/or avian influenza viruses can emerge. Over the years different variations of swine flu viruses have emerged and at this time there are four main influenza type A virus subtypes that have been isolated in pigs; H1N1, H1N2, H3N2 and H3N1. Most of the recently isolated influenza viruses from pigs have been H1N1 viruses, but these viruses are antigenically very different from human H1N1 viruses and as such, human vaccines for seasonal flu would not provide protection against H1N1 swine flu virus16.

The current swine flu strain is H1N1 which is the same virus type that is thought to have caused the Spanish flu in 1918, killing approximately 50-100 million people worldwide. The Avian Influenza virus (i.e. “bird flu”) has a serotype of H5N1.(CDC-Updated Interim Guidance for Laboratory Testing of Persons with Suspected Infection with Highly Pathogenic Avian Influenza A(H5N1) Virus in the United States;Feb.20, 2009,http://www.cdc.gov/flu/avian/professional/guidance-labtesting.htm) .

The CDC has confirmed that U.S. A (H1N1) cases were found to be made up of genetic elements from four different flu viruses; North American swine influenza, North American avian influenza, human influenza, and swine influenza virus typically found in Asia and Europe . Pigs have been shown to act as a potential "mixing vessel" in which reassortment can occur between flu viruses of several species.

This new strain appears to be a result of the reassortment of two swine influenza viruses, which themselves are descended from previous reassortments in pigs. Influenza viruses readily undergo reassortment because their genome is split between eight pieces of RNA. The A (H1N1) virus was resistant to adamantane and ridomantadine but susceptible to oseltamivir (Tamiflu®) and zanamivir (Relenza®)17.

Gene sequences of every viral gene were made available through the Global Initiative on Sharing Avian Influenza Data (GISAID). Preliminary genetic characterization found that the hemagglutinin gene was similar to that of swine flu viruses present in American pigs since 1999, but the neuraminidase (NA) and matrix protein (M) genes resembled versions present in

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European swine flu isolates. The six genes from American swine flu are themselves mixtures of swine flu, bird flu, and human flu viruses18.

While viruses with this genetic makeup had not previously been found to be circulating in humans or pigs, there is no formal national surveillance system to determine what viruses are circulating in pigs in the U.S19.

The CDC does not fully understand why the U.S. cases generated mild symptomatology while the Mexican cases resulted in multiple deaths. Research on previous pandemic strains has suggested that mortality can vary widely between different countries, with mortality being concentrated in the developing world. Differences in the viruses or co-infection are also being considered as possible causes. Of the fourteen initial samples from Mexico tested by the CDC, seven were found to match the American strain. The virus likely passes through several cycles of infection with no known linkages between patients in Texas and California, and the CDC concluded that containment of the virus is "not very likely"

Whereas most influenza strains affect the elderly and young children worst, this strain has primarily caused deaths in people between the ages- healthy young adults20.

In the past, sporadic human infections with swine flu have occurred. Most commonly, these cases affected persons with direct exposure to pigs. For today known very little evidence for human-to-human transmission21,22 .

The most well known swine flu outbreak occurred in 1976 among soldiers in Fort Dix, New Jersey. More than 200 cases were reported with serious illness in several people and one death. All of the patients had previously been healthy. The source of the virus, the exact time of its introduction into Fort Dix, and factors limiting its spread and duration are unknown. The Fort Dix outbreak may have caused by introduction of an animal virus into a stressed human population in close contact in crowded facilities during the winter. The swine virus collected from a Fort Dix soldier was named A/New Jersey/76 (Hsw1N1).

An outbreak of apparent swine flu infection in pigs in Wisconsin in 1988 resulted in multiple human infections. In the past, CDC received reports of approximately one human swine influenza infection every one to two years in the U.S., but from December 2005 through February 2009 12 cases of human infection with swine influenza have been reported.

The symptoms of swine flu in people are expected to be similar to the symptoms of regular human seasonal influenza and include fever, lethargy, lack of appetite and coughing. Some people with swine flu also have reported runny nose, sore throat, nausea, vomiting and diarrhea.

Human-to-human transmission of swine flu can also occur in the same way as seasonal flu occurs in people, which is mainly person-to-person transmission through coughing or

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sneezing of people infected with the A(H1N1) influenza virus. People may become infected by touching something with flu viruses on it and then touching their mouth or nose.

To diagnose swine influenza A infection, a respiratory specimen would generally need to be collected within the first 4 to 5 days of illness (when an infected person is most likely to be shedding the virus). However, some people may shed the virus for 10 days and longer. Identification of swine flu A virus requires special genetic testing with PCR amplification.

Antiviral Drugs for Swine Flu

There are four different antiviral drugs that are licensed for use in the U.S. for the treatment of influenza: amantadine, rimantadine, oseltamivir and zanamivir. Antiviral drugs are prescription medicines with activity against influenza viruses, including swine influenza viruses. These medications must be prescribed by licensed medical practitioners and/or specialists. When considering the use of influenza antiviral medications, clinicians must consider the patient’s age, weight, and renal function, presence of other medical conditions, indication for use and the potential for interaction with other medications and test the level of the virus susceptibility to the drug.

The most recent swine flu viruses isolated from human are resistant to amantadine and rimantadine so these drugs will not work against swine influenza viruses. At this time, the swine influenza A (H1N1) virus is sensitive (susceptible) to the neuraminidase inhibitors. As such, the CDC recommends the use of oseltamivir or zanamivir for the treatment and /or prevention of infection with swine influenza virus23.

Empiric antiviral treatment is recommended for any ill person suspected to have swine influenza A (H1N1) virus infection. Antiviral treatment with either zanamivir alone or with a combination of oseltamivir should be initiated as soon as possible after the onset of symptoms. Recommended duration of treatment is five days. Antiviral doses and schedules recommended for treatment of swine influenza A (H1N1) virus infection are the same as recommended for seasonal influenza.

Side Effects and Drug Resistance

Generally speaking, antiviral drugs can reduce the intensity of the virus and hasten recovery. They may also prevent serious influenza complications. Antiviral medications work best if started as soon after getting sick as possible as they may not work if treatment commences more than 48 hours after initial infection.

On their website, the CDC has documented a list side effects and adverse reactions for the main antiviral medications24. Oseltamivir (Tamiflu®): nausea and vomiting were reported more frequently among adults receiving oseltamivir for treatment than among persons receiving placebo. Among children treated with oseltamivir, 14% had vomiting, compared with 8.5% of

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placebo recipients. Similar types and rates of adverse events were reported in studies of oseltamivir chemoprophylaxis. Nausea and vomiting might be less severe if oseltamivir is taken with food. Transient neuropsychiatric events (self-injury or delirium) have been reported post marketing among persons taking seltamivir. FDA advises that persons receiving oseltamivir be monitored closely for abnormal behavior.

Zanamivir (Relenza ®): Limited data are available about the safety or efficacy of zanamivir for persons with underlying respiratory disease or for persons with complications of acute influenza. Zanamivir is licensed only for use in persons without underlying respiratory or cardiac disease. During post marketing surveillance, cases of respiratory function deterioration after inhalation of zanamivir have been reported. Because of the risk for serious adverse events and because efficacy has not been demonstrated among this cohort, zanamivir is not recommended for treatment for patients with underlying airway disease. Allergic reactions, including oropharyngeal or facial edema, also have been reported during post marketing surveillance.

Special Considerations for Children: Aspirin or aspirin-containing products (e.g. bismuth subsalicylate – Pepto-Bismol) should not be administered to any confirmed or suspected ill case of swine influenza A (H1N1) virus infection in persons aged 18 years or younger due to the risk of Reye syndrome. For relief of fever, other anti-pyretic medications are recommended such as acetaminophen or non steroidal anti-inflammatory drugs25.

Drug Resistance

One of the most serious problems that can be faced with swine flu is the emergence of oseltamivir resistance in influenza A(H1N1) viruses as was the case with human influenza A(H1N1) viruses during last 3-5 years.

Oseltamivir-resistant influenza virus strains are both communicable and pathogenic, and the intranasal live attenuated influenza vaccine has limitations. When compared, clinical symptoms and outcomes are similar between oseltamivir-resistant cases and oseltamivir –susceptible cases26.

In February of 2006, the CDC reported that 92.3% of the circulating influenza A (H3N2) at that time was resistant to the adamantanes (adamantadine and remantadine), 1 of 2 pharmacological classes available for the treatment of influenza. Although resistance to adamantanes increased to 14.5% in the prior year, the dramatic increase in 2005-2006 came as a shock to both the medical and scientific communities and the public27.

At the time, it was confidently held that influenza was unlikely to develop similar resistance to neuraminidase inhibitors (oseltamivir and zanamivir), the second class of influenza-directed agents. In clinical trials of oseltamivir, resistance was relatively rare (0.32% in adults and 4.1% in children) and the few oseltamivir-resistant isolates recovered during treatment

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seemed clinically unimportant. Nevertheless, studies by Dharan et al and Gooskens et al demonstrated that change was in the air28,29.

On December 19, 2008, the CDC reported that nearly all cases of influenza A (H1N1), the predominant circulating strain for the season thus far, were resistant to oseltamivir30.

Of all influenza isolates typed by the CDC this season, approximately 55% are oseltamivir-resistant H1N1 strains. The oseltamivir- resistant H1N1, which is now circulating on all major continents, is similar to the A/Brisbane/59/2007 strain that circulated during the 2007-2008 season and is included in the 2008-2009 influenza vaccines31.

Studies by Dharan with colleagues demonstrated that oseltamivir resistance compromises virulence. Four of the 142 patients (2.8%) with oseltamivir-resistant H1N1 isolates submitted to the CDC during the 2007-2008 influenza season died of influenza. In a comparison between 99 oseltamivir-resistant cases and 182 matched oseltamivir-susceptible cases, there were no discernible differences in the predisposing factors, clinical symptoms or complications related to influenza infection32. At least 3 patients who were immunocompromised acquired influenza from the index case, each developed pneumonia and 2 died33.

The widespread belief that oseltamivir would retain activity against epidemic influenza strains has crumbled now, but this should come as no surprise. Global and regional surveillance networks have tracked the incidence of neuraminidase-inhibitor resistance among circulating influenza for several years. The available epidemiology indicated that oseltamivir resistance was increasing on a trajectory that precisely paralleled the surge of adamantane resistance 3 years earlier34.

During the 2007-2008 season, only 10.9% of H1N1 viruses tested in the United States were oseltamivir resistant (Centers for Disease Control and Prevention (CDC). Influenza activity –United States and worldwide, 2007-2008 season. MMWR Morb Mortal Wkly Rep. 2008; 57 (25) :692-697.) Even higher rates were reported elsewhere, including Canada (26%), Europe (25%), and Hong Kong (12%). The highest rate of oseltamivir resistance was in Norway -67.3%, where oseltamivir can only be acquired with a prescription and is rarely used. During the summer of 2008, H1N1 strains related to A/Brisbane/59/2007 predominated in the Southern hemisphere and 100% of the H1N1 isolates in South Africa were resistant to oseltamivir35

The World Health Organization Report from 18 March 2009 “Influenza A (H1N1) virus resistance to oseltamivir -2008/2009 influenza season, North hemisphere – demonstrated that a total 30 countries from all WHO regions have oseltamivir resistance for 1291 of 1362 A (H1N1) viruses analyses – 94.78%. The prevalence of oseltamivir resistance was very high in following countries: Canada -52 of 52 tested -100%, USA -237 of 241 tested -98,3%; Japan – 420 of 422 tested – 99.5%; Korea – 268 of 269 tested – 99.5%; Germany – 66 of 67 tested – 98.5%; UK -61 of 62 tested – 98.4%; Honk Kong – 72 of 80 tested – 90%. Resistance prevalence was relatively low in China – 6 of 44 tested -13.6 %36. There is not enough time to develop a vaccine,

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administer it, and wait for the person’s body to manufacture antibodies for the swine flu. Generally, flu vaccines are manufactured based upon the strain predicted to be a problem in the coming year. Lifelong immunity to Influenza A is virtually impossible because the virus, being so primitive, often replicates inefficiently or incorrectly (mutation) causing a novel new virus to develop.

A new strain of flu is something to which we do not have natural immunity, nor do we have a vaccine for, which means that many more people are vulnerable to infection and the virus can more readily spread.When we know more about the H1N1 virus we will be able to give better predictions about who is at greater risk, and who is at risk of developing serious complications.

Cellular Immunity and Influenza

Respiratory syncytial virus (RSV) is a negative- strand, non-segmented RNA virus and a member of the family Paramyxoviridae, genus Pneumovirus.

Respiratory tract disease caused by RSV imposes a significant burden on health care, and all age groups are infected. The most significant disease, however, occurs in young infants, aged adults, and patients with immunological abnormalities who are immunocompromised.

It is estimated that lower respiratory tract (LRT) disease caused by RSV is responsible for 90% of bronchiolitis in infancy and 50% of all cases of pneumonia during the first 2 years of life.

Cellular immunity has been described as the defense mechanism for viral dysfunctions. An imbalance in the Th1/Th2 cytokine immune response has been related to pathogenesis of the respiratory syncytial virus (RSV) bronchiolitis and to the severity of the infection. Th1 cells produce IFN-gamma, an essential cytokine in the viral cell-mediated immune response.

Glucocorticosteroids have a role in inhibiting the IFN-gamma response, acting directly on T cell or indirectly through IL12. In this way an increase in plasma cortisol would induce a decrease in the Th1 products with the imbalance between Th1/Th2 cytokines and a shift to Th2 response. IL-12 is a potent inducer of NK and cytolytic T cell activity, IFN–γ production, and T cell proliferation, and is necessary for differentiation of naïve T cells to the Th1 subset37.

Respiratory epithelial cells are the primary target cell for replication of influenza viruses. Human respiratory epithelial cells respond to viral infections by mounting a cytokine response that contributes both to the innate and adaptive host defenses. Human influenza A viruses have been previously reported to induce an IFN-gamma –inducible protein-10 (IP-10), interferon –β, IL-6, IL-8 and RANTES (regulated on activation, normal T cell expressed and secreted) in vitro from the transformed bronchial epithelial cell line38. Acute RSV infection in infancy may produce some asthma-like symptoms and may be followed by a recurrent wheeze later in

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childhood. Studies have showed that the cells from RSV-infected infants produced more IL-4 and less IFN-γ than those from healthy controls. IL-4 production was more frequent in CD8 than in CD4 cells, and the bias toward IL-4 production was greatest in infants with mild infections, whereas IFN-γ production increased with disease severity39.

Patients with influenza A subtype H5NI had unusually high serum levels of IP-10 (IFN-gamma –inducible protein-10), which suggests that cytokine dysregulation may play a role in pathogenesis of influenza A subtype H5NI disease.

The differential up-regulation of IL-6 expression in human respiratory epithelial cells and the cytokines induced in macrophages by H5N1 (IL-1, TNF-alpha) viruses may contribute synergistically to the pathogenesis of human H5N1 disease.

Immunizations for an immunodominant epitope in the influenza virus nucleoprotein (NP) were conducted using DNAs expressing the HA of influenza virus A/PR/8/34 (H1N1) fused at the C terminus to H-2b –restricted class II and class I NP epitopes. Recently, it has been demonstrated that DNA-based immunizations could be used to elicit responses that varied from type 1 responses with predominantly IFN-gamma- producing CD4 and CD8 T cells and complement-dependent antibody, through mixed type 1 and type 2 responses, to type 2 responses in which most of the CD4 and CD8 cells produced IL-4 and the associated antibody was complement independent40.

Swine influenza virus (SIV), porcine respiratory coronavirus (PRCV) and porcine reproductive and respiratory syndrome virus (PRRSV) are enzootic viruses causing pulmonary infections in pigs. SIV is a primary respiratory pathogen; PRCV and PRRSV, on the contrary, tend to cause subclinical infections if uncomplicated but they appear to be important contributors to multifactorial respiratory diseases. The exact mechanisms whereby these viruses cause symptoms and pathology, however, remain unresolved. Classical studies of pathogenesis have revealed different lung cell tropisms and replication kinetics for each of these viruses and they suggest the involvement of different lung inflammatory responses or mediators. Nasal and bronchial biopsy specimens reveal degeneration and desquamation of epithelial cells and, initially, a marked infiltration of neutrophils in the airways.

These neutrophils play an important role in the pathophysiology of influenza, as they cause narrowing of terminal bronchi/bronchioli and correlate with the onset of fever in experimental animal models.

There is now growing evidence that the so-called ‘early’ cytokines produced at the site of infection mediate many of the clinical and pathological manifestations of respiratory viral infections. Early cytokines are produced by (non-immune) cells at the site of infection and they are responsible for local inflammatory reactions, as well as some systemic effects.

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The pro-inflammatory cytokines interferon- α (IFN- α), tumour necrosis factor- α (TNF- α) and interleukin-1 (IL-1) α and β have been shown to play key roles in several respiratory disease conditions and stand most proximal in the early cytokine cascade. IFN-α, TNF-α, IL-1 and interleukin-6 (IL-6) are typical examples of cytokines with multifunctional activities of which all have been associated with fever, excessive sleepiness and anorexia41.

TNF-α and IL-1 are well known for their profound stimulating effects on neutrophil and macrophage functions. Both cytokines strongly upregulate leukocyte adhesion molecules on the vascular endothelium, thereby mediating the first essential step for sequestration of neutrophils and /or macrophages into the respiratory tract42,43.

Here again, most cytokines occurred at much lower levels in plasma or serum than in respiratory tract secretions. IFN-α and IL-6, in particular, seem to be responsible for much of the early symptom formation44.

Although studies in mice, pigs and humans dealt with different experimental conditions and clinic-pathological outcomes, all of them assert a role of multiple cytokines in influenza symptoms and pathology. It is even reasonable to assume that additive and synergistic interactions between these cytokines will occur. On the other hand, production of most cytokines during influenza is transient and particularly short-lasting (not sure point trying to convey)45. Cytokines involved in the down-regulation of the inflammatory response, notably IFN-γ and IL-10, have also been demonstrated early in murine and human influenza46,47 .

It seems that in uncomplicated influenza at least, upregulation of early cytokines is tightly controlled and escalation of the cytokine response is an exception rather than the rule.

It is necessary to remember, that many of these early response cytokines have overlapping and synergistic activities, and can even induce their own production or that of other cytokines. TNF-α and IL-1, for example, stimulate the release of IL-6 and some chemokines. Both, the production and action of cytokines are thus critically dependent on the context in which they occur48.

The following points regarding the role of cytokines during influenza deserve attention:A multitude of cytokines are involved, and it remains to be seen whether some are more important than others.Direct viral cytopathic effects to the respiratory tract will at least in part account for the pathological changes; virus, probably, uses different mechanisms for the induction of different cytokines, i.e. TNF-α production could only be induced by infectious virus49. The same cytokines that exacerbate inflammation and disease also have a central role in resolution of the infection – IFN-α, TNF-α, IL-1 and IL-6 are known to participate in non-specific and specific antiviral immune responses.

The cytokine ‘microenvironment’ seems to have a profound effect on the magnitude of the virus-induced cytokine response. Exposure to granulocyte macrophage-colony stimulating

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factor (GM-CSF) markedly enhanced production of IFN-α, TNF-α, IL-1 and IL-6 in response to influenza virus; synergism between influenza virus and lipopolysaccharide from gram-negative bacteria was detected for TNF-α production50.

In single SIV infections, typical signs of swine "flu'' were tightly correlated with an excessive and coordinate production of the 3 cytokines examined. Porcine respiratory coronavirus (PRCV or PRRSV) infections, in contrast, were subclinical and did not induce production of all 3 cytokines. Combined infections with these 2 subclinical respiratory viruses failed to potentiate disease or cytokine production. After combined inoculation with PRCV followed by bacterial lipopolysaccharide, both clinical respiratory disease and TNF-α/IL-1 production were markedly more severe than those associated with the respective single inoculations51.

In this experiments pigs were inoculated intratracheally with a porcine respiratory coronavirus (PRCV), with LPS from E.coli O111:B4 (20µg /kg ) or with a combination of the two and than killed at set times after inoculation. The effects of separate virus or LPS inoculations were subclinical and failed to induce high and sustained cytokine levels.

When pigs were inoculated with virus and then with LPS 24 hours later, severe respiratory disease and significantly enhanced TNF-α titers (5- 40 times higher) were seen during the first 12 hours after LPS inoculations. In striking contrast to the effects of the single viral inoculations, the combined PRCV-24h-LPS inoculation induced a marked tachypnoea and dyspnoea- with labored, jerky abdominal breathing – alone with anorexia and dullness. All pigs were without symptoms prior to the LPS inoculation and developed clinical symptoms within 2 hours after inoculation of LPS. Lung pathological changes and broncho-alveolar lavage cell profiles resembled a combination of the effects induced by the single swine flu virus and LPS inoculation. On histopathology, excessive neutrophil infiltration of the alveoli and terminal airways was most prominent. Many alveoli were filled with fibrin stands and proteinaceous debris, and accumulations of erythrocytes. The cytokine response was significantly enhanced in magnitude and duration when compared with that induced by LPS only.

In the case when pigs were inoculated with the virus and subsequently with LPS at various time intervals ranging from 0 to 24 hours, production of TNF-α was dependent on the time interval between inoculations and tightly correlated with disease. These data show that exposure to high endotoxin concentrations in swine buildings can precipitate respiratory disease in PRCV-infected pigs, and that TNF-α is probably an important mediator of these effects51.

Thus, the mechanisms whereby viruses can trigger lung cells for an enhanced cytokine secretion remain to be defined. Interestingly, the influenza virus infection alone was shown to induce a massive TNF-α mRNA accumulation in leucocytes, but efficient translation into bioactive protein occurred only upon further stimulation by LPS52,53.

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TNF-α and IL-1 are the part of a complex network with potent feedback regulations involved in their synthesis and biological effects. The combination of both cytokines may lead to a secondary cytokine response that is 250-fold greater than that seen with each cytokine alone54. It has been shown that TNF-α and IL-1 separately and synergistically depress human myocardial function. Therapeutic strategies to reduce production or signaling of either TNF-α or IL-1 beta may limit myocardial dysfunction in sepsis55.

In addition, there appears to be a significant individual receptiveness among man to inhale endotoxin LPS. Not everyone exposed to high concentrations of LPS can develop airway symptomatology and exacerbations of asthma. About 40% of individuals were sensitive to LPS and they were more often female. Hyporesponsive subjects on the other hand were more often male. Peripheral blood monocytes from hyporesponsive subjects, compared with sensitive subjects, released less Il-6 and IL-8. These findings demonstrate that a LPS phenotype can be reproducibly elicited in humans, which creates an opportunity to identify genes involved in this response to inhaled LPS56. The response to inhaled LPS is dose-related57.

Furthermore, massive amounts of endotoxins are released locally in the lungs during infections with gram-negative bacteria, which often complicate pulmonary virus infections in animals and man. This data supports the idea that the complications of combined influenza virus and bacterial infections may be partially due to an excessive TNF-α production and subsequent “cytokine-storm” effect.

Other Possible Mechanisms of Defense

On the basis of the LPS nature that it is one of the PAMPS – “pathogen associated molecular patterns” that are the microbial principles that trigger an innate immune response, possible to create ideology of the defense from swine flu infections and complications that can be provoked by accompanied infections, first of all by gram-negative pathogenic bacteria.

Innate immunity is an evolutionary ancient system that provides multicellular organisms with immediately available defense mechanisms against a wide variety of pathogens without requiring prior exposure. Recognition of microbial determinants by elements of the innate immune system sets in motion early humoral and cellular mechanisms for defense against pathogens. The rapid inflammatory reaction that follows infection is principally mediated by monocytes neutrophils, and endothelial cells and can be reproduced in vitro in the absence of components of the adaptive immune response.

Bacterial products directly elicit the upregulation of adhesion molecules on vascular endothelial cells, contributing to the recruitment of leukocytes to the focus of infection. The activation of leukocytes that occurs upon ligation of adhesion-promoting receptors stimulates the production of reactive oxygen intermediates that contribute to the clearance of bacteria from tissues. Bacterial products also induce the synthesis and release of pro-inflammatory cytokines, such as TNF and IL-1 that amplify the response to infection. Innate immune recognition induces

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secretion of effector cytokines, such as IL-12, that control CD-4-T cell differentiation, upregulation of co-stimulatory molecules and antigen-presenting cells that are necessary for T-cell activation, and signals that are necessary for B-cell proliferation. Thus, the innate response to microbial challenge controls and instructs the adaptive immune response through special mechanisms with system of the receptors – PRR – pathogen recognition receptors, that include toll-like receptors (TLRs) and nucleotide-binding oligomerization domains ( NODs).

The first observations for a direct role of TLRs in mediating the innate immune response to lipopolysaccharide (LPS, endotoxin) were reported by Kirschning et al and Yang58,59.

TLR2 has since been shown to confer responsiveness to a wide variety of gram-positive bacterial cell wall components as well as to lipoproteins that are found in gram-positive and gram-negative bacteria and Mycoplasma spp60-62.

However , LPS preparations from E.coli and Salmonella lose their ability to induce TLR2- dependent responses after removal of contaminant proteins by multiple phenol extractions, suggesting that the lipopeptides rather than the LPS are responsible for cellular activation via TLR263. In the last 10 years studies have confirmed that LPS from other organisms, with as yet unknown structural properties could trigger an innate immune response through TLR2.

The importance of TLR4 for responses to LPS and gram-negative bacteria was also confirmed in studies by Hoshino and others64. This research showed that hyporesponsiveness to LPS in a small group of humans is also linked to a missense mutation in TLR465. Given the importance of TLR4 for responses to LPS in vivo, it was shown additionally by the discovery that coexpression with MD2, a soluble protein tethered to cell surface through its physical association with human TLR4, allowed activation of NF-kB by TLR4 in response to LPS to levels well above those observed in the absence of bacterial agonists. Human TLR4 thus associates with MD2 to form a complex that is responsive to LPS66.

The intriguing results for TLR-1, TLR6 and TLR2 contribute to the growing notion that different TLRs could combine to recognize foreign agonists. Such combinations could alter not only the specificity of the determinants recognized but also the nature and intensity of the signals generated. Nine very well known TLRs forming either homo-or heterodimers give rise to more than 500, possible combinations. Evan more, TLRs could potentially associate into complexes larger than dimmers, thus opening up the possibility for a very large combinational repertoire. There is also evidence that TLRs can be activated by nonbacterial products, such as yeasts67, respiratory syncytial virus68, and heat shock protein6069.

A recent study demonstrated that three proteins, RP105, NOD1 and NOD2, are structurally related to TLRs and have been suggested to play a role in responses to LPS as TLR2 and -4. In addition, NOD1 and NOD2 are both cytosolic proteins that could act as intracellular

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functional equivalents of TLRs. NOD1 and -2 were recently shown to endow 293 cells with the ability to activate NF-kB in response to LPS independently of TLR470.

Transfection with NOD2 also enables the cells to respond to peptidoglycan (PGN) while transfection with NOD1 did not, suggesting that the proteins may confer a specificity to the response. NOD family members may thus function as proteins that activate innate immune response to a wide array of products from pathogens and was shown that mutations in human Nod 2 have been linked with susceptibility to Crohn’s disease, an inflammatory bowel disease thought to be due to an abnormal inflammatory response to enteric microbiota71.

Recent data that were published by research group lead by Tomohiro Watanabe clearly showed that one of the bacterial derivates – muramyl dipeptides (MDP) from cell wall peptidoglycan has ability to activate NOD2 and negatively regulates TLR2 responses and that absence of such regulations leads to heightened Th1 responses. Researchers showed that administration of MDP protects mice from the development of experimental colitis by down regulating multiple TLR responses, not just TLR272.

Researchers suggested to use muramyl dipeptide as a therapeutic agent for inflammation associated with the innate immune response caused by the activation of TLR-2, which ultimately provokes inflammation. Activation of NOD2 by MDP negatively regulates the activity of TLR2 and thus reduces inflammation. It was shown also that MDP reduces pro-inflammatory cytokine production from multiple TLRs and in case of MDP pretreatment MDP has a remarkable inhibitory effect on not only TLR2 and IL-12, but also other types of TLR responses and other cytokine responses73.

This conclusion was based on in vivo studies that showed that administration of MDP to mice led to the amelioration that was a direct result of a down regulatory effect on multiple TLR pathways responsible for the production of key inflammatory cytokines such as IL-12 and IL-6. It was also based on in vivo studies that demonstrated first that NOD2 (MDP) pre-stimulation of human monocyte-derived dentritic cells (DCs) is followed by a greatly diminished capacity of TLR2, TLR3, TLR4, TLR5 and TLR9 ligands to induced production of IL-12, IL-6 and second that NOD2 pre-stimulation of human DCs abolishes the subsequent ability of MDP to synergize with ligands of TLR3 and TLR9 in the induction of IL-12, IL-6 and TNF.

That these “tolerogenic” effects of MDP pre-stimulation were in fact mediated by NOD2 provides strong evidence that NOD2 pre-stimulation of antigen presenting cells (APCs) negatively regulates inflammatory responses induced by a broad range of TLR ligands74 .

It is necessary to emphasize that these natural regulatory mechanisms are dedicated to balancing the immune response and protecting the body against immune over reactivity (i.e. the “cytokine storm”). As such, these regulatory mechanisms can be one of the solutions for neutralizing the immune response for swine flu virus infection and can be combined in some

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subjects with LPS exposure from gram-negative accompany infections, some cardiovascular problems and asthma conditions.

In this case special drugs and dietary supplements that are capable of breaking down structural components of bacterial peptidoglycan could be one of the main tools to protect against the next waves of swine virus influenza, especially for persons with different types of compromising immune systems.

Probiotics could be “ideal” suppliers of MDP for regulating and balancing Immune System

Additional powerful tools for swine virus influenza defense could be to use of some Probiotics with high level expression of immunomodulating activity. This suggestion is based on the knowledge of the interaction between intestinal microflora and the host organism immune system. Native inhabitants are those organisms that colonise or fill specific niches in the body and protect the host from invading harmful bacteria and viruses and regulate the local immune system.

The intestinal microflora also has an effect on the expression of glycoconjugates present on epithelial cells. The sugar moiety of these glycoconjugates can serve as reseptors for several microorganisms, and can influence the binding possibilities of bacterial pathogens. The binding possibility can be or enhanced, increasing the susceptibility for disease or be decrease, giving the pathogen no chance to effect the host75.

The mucosal flora can also produce toxins that can destroy certain pathogens and regulate the quantity and quality of microorganisms present.The gut-associated lymphoid tissue (GALT) is part of the specialised mucosa-associated immune system, and represents the largest mass of lymphoid tissue in the entire human body. The immune response occurs in different physiologic compartments of this tissue: aggregated in follicles and Peyer’s patches, and distributed within the mucosa, the intestinal epithelium, and secretory sites76.

Intestinal epithelial cells termed ‘M’ (microfold) cells, due to the numerous microfolds on their luminal surface, carry out this particular function. M cells are able to absorb and transport antigens and, possibly, process and present them to subepithelial lymphoid cells. An in addition, microbial entry into epithelial cells results in the production and release of proinflammatory cytokines. Therefore in response to a bacterial invasion an acute mucosal inflammatory response is initiated77.

As now known regulatory pathways in the immune system include the dichotomy of Th1 and Th2 activity. T cells that developed cellular responses are characterized by interleukin production such as IFN-γ or IL-2 and are designated T-helper 1(Th1) cells. Other T helper cells, so called T helper 2 cells, are characterized rather by IL-4 production , and provide help to the development of antibody production. These two T helper cell types regulate each others activity

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as a form of feed back, so that usually a proper balance exist between the two. However, it should be realized that during Th1 or Th2 dominated responses both Th subsets seem to be involved, at least partially or indirectly. Under physiological conditions, it is likely that cytokines only transiently shift the balance along the Th1-Th2 axis, without permanently fixing the Th phenotype. More extrim Th1 or Th2 phenotype is only seen in certain pathological conditions78. For instance, in case of swine influenza virus with combination with LPS from Gr- infections we will see predominantly Th1 type of immune response which can lead to the “cytokine storm” effect.

Intestinal epithelial cells produce various pattern-recognition receptors (PRRs) that recognize microbial motifs, referred to as pathogen-associated molecular patterns (PAMPs). Because PAMPs are evolutionary highly conserved and invariable in microorganisms of the same class, mammals can recognize almost all microorganisms with a small number of PRRs.

Probiotics are naturally joined with human organisms and are the supplier of immune active molecular patterns that completely deprive of pathogenicity - MAMPs – microbial –associated molecular patterns. MDP is the smallest immune active structure of cell wall peptidoglycan and plays leading role in host immune response as MAMPs.

It is clear that probiotics might have immunomodulatory effects, but it is still unknown yet how these effects are achieved and their mechanisms of immune activity. There have been several reports recently describing the effects of probiotics on balancing immune parameters. Most of the known probiotics have activate the production of inflammation cytokines - IFNs, TNF, IL-6 and activate Th1 pathway.

In the case of swine flu virus with combination of LPS from Gr- bacteria need to be used probiotics with high level induction activity for regulatory suppressive cytokines including IL-4, IL-10 and especially transforming growth factor β1 (TGF β1) for reduction of high level of inflammation as a results of upgrading production of TNF-α .

Petra Winkler and colleagues presented analytical data on the induction of immune modulating molecules and effector substances by lactic acid bacteria79.It was shown that some probiotics strains demonstrated ability to reduce production of TNF –α or increase production of IL-10 and have strain-specific anti-inflammatory effects80-85.

In the study that was represented by86 researchers used two potentially anti-inflammatory strains – Bifidobacterium and Propionibacterium strain and a well studied L.rhamnosus GG strain as a reference probiotics. Their data indicates that in vivo probiotics differ in their ability to induce anti-inflammatory and cytokine responses and may have a weak, genera-specific anti-inflammatory effect reflected as a decrease in serum highly sensitive particle-enhanced immunoturbidimetric C-reactive protein (hsCRP) level in healthy adults. In addition, it was showed that during the intervention, S.pyogenes- induced TNF-α responses and

16

influenza A virus-induced IL-2 responses in in vitro cultured PBMC were reduced, indicating a clear anti-inflammatory potential of some probiotic bacteria.

This is the first and only one yet study to show that probiotics may reduce serum hsCRP levels in healthy adults in a randomized , double-blind, placebo-controlled setting. Researchers suggested to use CRP –test as a sensitive marker of inflammation and provides an easy way to measure the anti- inflammation potential of probiotics and other biological or pharmacological substances87,88.

It is of interest that a combination of four probiotic bacteria (L.rhamnosus GG, L.rhamnosus Lc705, B.breve99, P.freudenreichii ssp.shermanii JS) did not have an effect on sensitive CRP in the same clinical setting with allergic children. This data correlated with our results that were demonstrated reduction of immunomodulation properties of the probiotic composition which contains live cells of five probiotics strains with high individual level of cytokines induction89. From represented here data it has become clear that different strains of probiotics induce very different immunological effects. In addition, effects seen in a certain human population with one strain of bacteria can often not be reproduced. It is very difficult to compare the effects due to the differences in measurement technique, the different patient materials (healthy vs various diseases) and the different probiotic strains that have been used. It seems that age, the immunological status of the individual and the probiotic strain used in the study has a great impact on imunomodulatory effects.

For creation the natural safety effective remedy for treatment the swine influenza virus infection and prevention ‘cytokine storm’ serious immune reaction novel probiotics may have a strain-specific ability to lower serum CRP levels, also able to reduce pro-inflammatory TNF-α production and may have a beneficial effects on the host Th1/Th2 balance thus having anti-inflammatory effects in apparently healthy adults and in patients suffering from different inflammatory conditions.

Thereby, Probiotics could be one of the best candidates for development the algorithm for swine influenza virus treatment and prevention serious complications to be based on their safety nature, ability to restore normal flora that is essential for controlled inflammation reactions, ability to balance immune reactions, and their MAMPs nature – to be “ideal” suppliers of structural cell fragments that regulate immune responses through PRRs – pattern recognition receptors – like TLRs and DODs.

Realization this great opportunity also demands for further quest of potentially immunoregulatory functional pobiotics strains as a candidates for special investigations.

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CDC recommendations for prevention swine flu virus infection

Try to avoid close contact with sick people.

Influenza is thought to spread mainly person-to-person through coughing or sneezing of infected people.

If you get sick, CDC recommends that you stay home from work or school and limit contact with others to keep from infecting them.

There are everyday actions people can take to stay healthy.

Cover your nose and mouth with a tissue when you cough or sneeze. Throw the tissue in the trash after you use it.

Wash your hands often with soap and water, especially after you cough or sneeze. Alcohol-based hands cleaners are also effective.

Avoid touching your eyes, nose or mouth. Germs spread that way.

Boost your Immune System FLU STRAINS COMPARED

H1N1 (seasonal flu/swine flu)

Spreads easily through coughing and sneezing

Less severe symptoms, but can be deadly

H5N1 (avian flu)

Can mutate rapidly

Causes severe illness and can trigger pneumonia

Spreads easily between birds but human transmission rare

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