7954

Saturday 7 February I976

THERAPY WITH PARENT’S LYMPHOCYTETRANSFER FACTOR IN CHILDREN WITH

INFECTION AND MALNUTRITION

DAVID G. JOSE GEOFFREY W. FORD

Royal Children’s Hospital Research Foundation,Melbourne, 3052, Australia

JOHN S. WELCHQueensland Institute of Medical Research,

Brisbane, 4006

Summary Transfer factor (T.F.) prepared from5x108 lymphoid cells from 500 ml of a

parent’s blood was given to 40 Australian aboriginalchildren aged 2-46 months who had been in hospitalwith acute infection. Many had protein-calorie malnu-trition. These and a control group of 35 similar childrenwere assessed blind for at least 12 months. InT.F.-treated children there were significantly fewer epi-sodes of diarrhœal disease for periods in excess of 26weeks. Recurrent moderate diarrhœal disease was par-ticularly reduced, and the onset of severe gastroenteritismay have been delayed. There was no protection againstchest, middle-ear, or skin infection.

Introduction

GLOBALLY, protein-calorie malnutrition (P.C.M.) withinfection is believed to be the commonest cause ofchronic disability and death in childhood.l Infections ofthe bowel and respiratory tract are major causes of mor-tality and long-term morbidity in underdeveloped areasand may precipitate undernourished children into severep.C.M.1-3 Passively acquired maternal antibodies andbreast feeding provide considerable protection againstdiarrhoeal disease in the first months of life,4 but wean-ing is often followed by diarrhrea and months of growthfailure, undernutrition, and immunodeficiency markedby recurrent and persistent infections which may befatal.’-’2 In older children and adults recurrent infec-tion becomes progressively less frequent-presumablybecause immunity is acquired through repeated expo-sure to local organisms. On aboriginal settlements incentral and northern Australia, moderate P.C.M. is seenin 10-60% of children under 5 years old.3 9 13 Rapidevacuation of sick children to hospital has reduced mor-tality, but medical services have been less effective inpreventing recurrent bowel and respiratory infections.We have assessed the effect of parents’ transfer factor(T.F.)—i.e., passive transfer of acquired cellular im-

munity-upon the frequency of bowel and respiratoryinfections in aboriginal children.

Lawrencel4 15 and others’6 reported transfer of anti-gen-specific delayed skin reactivity from immune to non-immune individuals; and recipients ofT.F. (a crude dia-lysate of disrupted lymphoid cells) have been made moreresistant to specific infections such as candidiasis,leprosy, tuberculosis, and viral diseases.’s 17-19 An effectin malnourished children was reported by Brown andKatz, 20 who by means of a crude lysate of lymphocytesfrom a tuberculin-positive donor secured Mantoux con-version in 11 cases out of 12. Walker et al .21 gave T.F.,each dose derived from 109 leucocytes from a healthyGuatemalan adult, to malnourished/infected children atan early stage of rehabilitation but there was no effecton dermal reactivity or nutritional recovery. It seemedto us that the end of a period in hospital after a courseof antibiotics, in relatively clean surroundings and withnutritional recovery, when loads of bowel organismswere likely to be low, was the best time to try immunetransfer. This paper reports such an investigation, withclinical follow-up and results offsecal examinations.

Patients and Methods

75 children aged 2-46 months were selected for study afteradmission to Alice Springs Hospital with acute infection.

Many had P.C.M. Criteria for selection were a past history ofrespiratory or bowel infection; absence of other disease in childor parent; a high probability of the family being accessible forfollow-up for at least 12 months; agreement of a parent todonate blood; and parents’ informed consent to administrationof lymphocyte transfer factor to their child. Parents under-stood that they were "giving part of the strength in their bloodto their child to help fight infection", and that the risk of side-effects was probably lower than with a blood-transfusion.Children selected were randomised to receive either their

parent’s lymphocyte transfer factor (T.F.) or saline from codedvials administered on the day of discharge from hospital. Atthis stage children were clinically free of infection, and nutri-tional rehabilitation was well advanced. A second dose of T.F.or saline was administered about three months later.

Children were followed for at least 12 months by a nursingsister or medical officer at 12 settlements in semi-desert

country within 500 km of Alice Springs. Each episode of infec-tion and each regular 2-weekly examination was recorded forevery child on standard forms. About every three months allchildren were examined by one medical officer; body-weightswere checked on standardised scales; and samples of faeces andblood were examined. Any child readmitted to a base hospitalwas re-examined and the hospital course was carefullyrecorded. Neither field nor hospital staff knew which childrenhad received transfer factor. Chest infection was diagnosedwhen a child presented with fever, malaise, cough, and chestsigns. Bowel infection was diagnosed when a child presentedwith more than two fluid, offensive bowel actions a daytogether with malaise, anorexia, and abdominal pain or vomit-ing or dehydration. Occasional loose motions accompanying

264

obvious throat or respiratory infection were not classed asbowel infection.

Preparation of Transfer Factor500 ml venous blood was taken into sterile plastic blood-

bags containing 10 u/ml of preservative-free heparin.Mononuclear leucocytes, approximately 5 x 108 per donor,were purified by ’Ficoll’/’Hypaque’ discontinuous density-gra-dient centrifugation, washed three times in saline, packed intoa sterile plastic tube, and transported to Melbourne in liquidnitrogen. The packed cells were freeze-thawed twelve timesand were dialysed overnight at 4°C with 100 ml sterile Waterfor Injection B.P. The dialysate was lyophilised, reconstitutedin 2 ml of water for injection, filtered through a 0.22 p’m Milli-pore filter into two sterile 1 ml glass vials, lyophilised, andsealed. Control vials contained lyophilised Saline for InjectionB.P. After return to Alice Springs the T.F. or saline was givenas a 1 ml subcutaneous injection. Batches of T.F. were testedusing skin-test reactivity in laboratory volunteers, but neitherdonors nor child recipients were subjected to multiple skin-tests. Each dose of T.F. was derived from between 2 x 108 and5 x 108 blood lymphoid cells.

Groups Selected

Analysis after randomisation showed that the T.F. groupcontained 40 children with 24 males and mean age 17.8 8months and the control group had 35 children with 17 malesand mean age 16.3 months. Age distribution was:

Age (mo) 2-6 7-12 13-18 19-24 25-30 31-46 TotalT.F.-treated 4 7 10 6 7 6 40Control 6 7 10 5 4 3 35

The reason for hospital admission had been gastroenteritis in52 patients (26 selected for T.F., 26 for control) and pneumoniain 19 patients (11 T.F., 8 control), with 4 patients having mal-nutrition and infections at other sites. At randomisation meanstandard weight for age (S.W.F.A.) relative to Harvard growthscale22 was 86-67% in T.F. children and 86.71% in controls. 3children in each group had body-weights less than 75%S.W.F.A. at discharge. Numbers of children returning to eachof the twelve central Australian communities ranged from2 to 15 per settlement, but the distribution of T.F. and controlgroups was approximately equal for all communities and nogeographic bias could be detected. All children selected intp thestudy at randomisation are included in the follow-up analysis.

Laboratory results (means±S.D.) on children at randomisa-tion were: hemoglobin (g/dl) T.F. 11.4±11 ·3, control11.4i2.01; leucocyte-count (xl03/p.l blood) T.F. 14.5±5.3,control 13-5±3-6; erythrocyte-sedimentation rate (mm/lst h)T.F. 25±19, control 29±23; serum-albumin (g/dl) T.F.

3-8±0.6, control 3’9±0-5.

Follow-up

Complete follow-up data for 4 consecutive 3-month(13-week) periods were obtained for 36 out of 40 T.F. children(90%) and 32 out of 35 (91%) control children. The proportionof time in which follow-up was inadequate was the same (4%)in the two groups.

Results

InfectionsApart from transient mild irritation at the injection

site in 3 children, T.F. seemed to have no side-effects.554 episodes of infection were recorded in 75 childrenduring the 12 months after randomisation. 40T.F.-treated children had 238 infections and 35 controlshad 316 infections. Bowel or chest infection accountedfor 66% of recorded episodes (table i).

Table n shows the numbers of children with one ormore episodes of bowel or chest infection during each of

TABLE I-REPORTED EPISODES OF BOWEL AND CHEST INFECTIONS

TABLE II-NUMBERS OF CHILDREN WITH EPISODES OF BOWEL OR CHEST

INFECTION DURING 4 CONSECUTIVE 13-WEEK PERIODS. I ..

*Not statistically significant

four consecutive 13-week periods after randomisation.In the first 13 weeks numbers of T.F .-treated childrenwith bowel infection were significantly reduced (8 9;p<0-003) compared with control children. These dif-ferences between groups were still present up to 40weeks from the first dose of T.F. (about 30 weeks fromthe second dose of T.F.). There were no differencesbetween groups in numbers of children with chest, mid-dle-ear, or skin infections. 6 children in the T.F.-treatedgroup had localised pharyngitis or tonsillitis (not seen incontrols). No other clinically obvious differences in in-fections were noted.Numbers of episodes of infection per child in each

13-week period ranged from zero to seven. In each13-week period during the first 6 months the numbers ofT.F. children having two or more separate episodes ofbowel infection were significantly reduced (X2=5-8i !i;p<0-02) compared with controls. No differences werefound in numbers of children with recurrent chest infec-tions (table in).

Growth

Weight percentiles were maintained or improved in 31out of 40 (78%) and 35 out of 38 (92%) of T.F.-treatedchildren during the first and second 26-week follow-upperiods, respectively. Numbers of control children main-taining or improving weight during equivalent periodswere 20 out of 35 (57%) and 25 out of 33 (76%). OverTABLE !H&mdash;NUMBERS OF CHILDREN IN EACH GROUP WITH TWO OR MORE

EPISODES OF INFECTION PER 13-WEEK PERIOD

.p<O.02

265

the full 12-month follow-up 3 of 38 T.F .-treated childrenand 8 of 31 control children lost more than 5% ofS.W.F.A. These differences were not statistically signifi-cant.

Hospital Readmissions:

During the 12 months follow-up 21 (52%) T.F.-treatedand 16 (46%) control children were readmitted to hospi-tal with infection. 3 children, all T.F.-treated, were re-admitted within one week of randomisation with recur-rence of pneumonia. Exclusion of these recurrences

would make readmission-rates between the two groupssimilar (45% T.F., 46% control) over the remaining 12months. During the first 16 weeks no T.F. children wereadmitted with gastroenteritis compared with 6 controlchildren. 8 T.F. children were admitted with gastroenter-itis 17-27 weeks (median 19 weeks) after T.F. dose 1 and2-20 weeks (median 8 weeks) after T.F. dose 2; 5 con-trol children were admitted with gastroenteritis duringthis time. These children were separated both chronolo-gically and geographically prior to admission. From the27th to the 52nd weeks of follow-up 8 T.F. and 10 controlchildren were readmitted with gastroenteritis.

15 children were readmitted more than once during12 months: 19 readmissions were in 8 T.F. children, allwith pneumonia, and 19 readmissions were in 12 controlchildren, mostly with gastroenteritis. 1 child in the con-trol group died with marasmic kwashiorkor and infec-tion during the third readmission after 42 weeks’ fol-low-up. This was the only death among the childrenselected. During the first 26 weeks of follow-up 17T.F.-treated children were readmitted for a mean of 3.6weeks each while 13 control children were readmittedfor a mean of 7.6 weeks each.

Faecal Bacteria

Cultures for fxcal bacteria during 37 readmissions tohospital for gastroenteritis yielded a Shigella in five

specimens and an Escherichia coli in three. No bacterialpathogens were found in the remaining specimens.Intestinal Parasites

Ninety-two fresh fsecal specimens were examined from63 children (32 T.F., 31 control) on two occasions aboutthree and eight months through the follow-up period.Prevalence among all children of the organisms was:Giardia lamblia 39%, Trichomonas hominis 33%,

TABLE IV-DISTRIBUTION OF INFESTATION AND APPROXIMATE MEAN

LOADS OF ALL PARASITES IN INFECTED CHILDREN IN TREATED ORCONTROL GROUPS

’iKtOt

Entamaeba histolytica 13%, Entamaeba coli 4%,Endolimax nana 1%, Strongyloides- stercoralis 10%,Hymenolepis nana 8%, and Enteromonas hominis 9%.No differences in the prevalence of any one species werefound between groups. The approximate load of eachspecies in faeces was graded 0 to +++++. The approxi-mate total mean load of all parasites seemed to be lessin the T.F. children (table iv).

Discussion

Lymphocyte transfer factor prepared from parentsand administered prophylactically to their childrenseemed to reduce the number of episodes of diarrhoealdisease for periods in excess of 26 weeks. The effect wasgreater in preventing mild or recurrent episodes ofdiarrhoea than for severe gastroenteritis, although hospi-tal admissions for gastroenteritis may have been delayedfor several weeks in T.F.-treated children. No protectionagainst chest, ear, or skin infection was apparent,although recurrence of pneumonia, development of loca-lised pharyngo-tonsillar inflammation, and a shortermean period of hospital admission for infection inT.F.-treated children was suggestive of enhanced im-munity. Other favourable trends in T.F. treated childrenwere the maintenance of normal growth rates and pos-sibly lower total loads of faecal parasites.The mechanism of the apparent enhanced resistance

to diarrhoeal disease in the T.F.-treated children is notknown. Passive transfer of cellular immunity specific fora particular enteric organism, or for a group oforganisms, is a possible explanation. Cellular immunitymay protect against viruses, intracellular bacteria,fungi, and some protozoa.23 The seemingly muchshorter duration of effect of the second dose of T.F. mayhave been related to inactivation upon storage or torecolonisation of recipients with high loads of entericorganisms.

Recurrent diarrhceal disease is a consistent feature ofmalnourished children in underdeveloped areas. Preven-tion has not been achieved by long-term antimicrobial 24or antiparasitic therapy, 25 or by intermittent hospitaladmission26 without major changes in the socio-econo-mic environment. Agents known to cause diarrhoea! dis-ease in young children, other than enteropathogenicbacteria, are adenoviruses, echo viruses,27 28 and therecently described duovirus (orbivirus).29 3o Childrenwith P.C.M. have increased upper-intestinal loads of bac-teria, of Candida species, and of parasites (particularlyGiardia lamblia), with associated unfavourable effectsupon intestinal epithelial structure and function.26 31-34Transfer factor has been effective, in our hands, againstrecurrence of infection when prepared from a highly im-mune donor and administered prophylactically. Furtherstudies to assess the combined effect of antibiotic andantiparasitic drugs in combination with T.F. therapymay prove rewarding.We are grateful to the Director and officers of the Departments of

Health and Aboriginal Affairs, Northern Territory and South Austra-lia, and to the medical and nursing staff of Alice Springs Hospital.Particular contributions were made by the Aerial Medical Serviceand Settlement sisters. The interest and cooperation of the abori-ginal elders, parents, and children made this study possible.

This work was supported by grants from the National Health andMedical Research Council of Australia, the Wellcome Trust, and theWorld Health Organisation.

References overleaf

266

DR JOSE AND OTHERS: REFERENCES

1. Scrimshaw, N. S., Taylor, C. E., Gordon, J. E. Wld Hlth Org. Monogr Ser.no. 57, 1968.

2. Mata, L. J., Urrutia, J. J., Albertazzi, C., Pellecer, O., Arellano, E. Am. J.clin. Nutr. 1972, 25, 1267.

3. Jose, D. G., Welch, J. S. Med. J. Aust. 1970, i, 349.4. Mata, L. J., Wyatt, R. Am. J. clin. Nutr. 1971, 24, 976.5. Trowell, H. C., Davies, J. N. P., Dean, R. F. A. Kwashiorkor. London, 1954.6. McKenzie, D., Hansen, J. D. L., Becker, W. Archs Dis. Childh. 1959, 34,

250.7. Phillips, I., Wharton, B. Br. med. J. 1968, i, 407.8. Vitale, J. J. Am. J. clin. Nutr. 1971, 24, 248.9. Jose, D. G., Shelton, M., Tauro, G. P., Belbin, R., Hosking, C. S. Med. J.

Aust. 1975,ii, 699.10. Harland, P. S., Brown, R. E. E. Afr. med. J. 1965, 42, 233.11. Smythe, P. M., Schonland, M., Brereton, S. G. G., Coovadia, H. M., Grace,

H. J., Loening, W. E. K., Mafoyane, A., Parent, M. A., Vos, G. H. Lancet1971, ii, 939.

12. Chandra, R. K. J. Pediat. 1972, 81, 1194.13. Kirke, D. K., Vorbach, E. A., Newman, D. M. Med. J. Aust. 1970, i, 187.14. Lawrence, H. S. J. clin. Invest. 1955, 34, 219.15. Lawrence, H. S. Adv. Immun. 1969,11, 195.16. Rapaport, F. T., Lawrence, H. S., Miller, J. W., Pappagianis, D., Smith, C.

E. J. Immun. 1960, 84, 358.17. Kirkpatrick, C. H. J. Allergy clin. Immun. 1974, 55, 411.18. Kirkpatrick, C. H., Gallin, J. I. Oncology, 1974, 29, 46.19. Spitter, L. E., Levin, A. S., Fudenberg, H. H. in Immunodeficiency in Man

and Animals (Birth Def. orig. Art. Ser. Vol. xi) (edited by D. Bergsma,R. A. Good, and J. Finstad); p. 449 National Foundation&mdash;March ofDimes, Sinauer, Mass., 1975.

20. Brown, R. E., Katz, M. J. Pediat. 1967, 70, 126.21. Walker, A. M., Garcia, R., Pate, P., Mata, L. J., David, J: R. Cellular

Immun. 1975, 15, 372.22. Jelliffe, D. B. Wld Hlth Org. Monogr Ser, no. 53, 1966.23. Good R. A., Finstad, J., Gatti, R. A. in Infectious Agents and Host Reactions

(edited by S. Mudd); p. 76. Philadelphia, 1970.24. Heyworth, B. Med. Offr, 1970, 123, 349.25. Welch, J. S., Stuart, J. E. Med. J. Aust. 1975, i, 14.26. Gracey, M. Aust. N. Z. J. Med. 1973, iii, 576.27. Cramblett, H. G., Azimi, P., Haynes, R. E. Ann. N.Y. Acad. Sci. 1971, 176,

80.28. Blacklow, N. R., Dolin, R., Fedson, D. S., Dupont, H., Northrup, R. S.,

Hornick, R. B., Chanock, R. M. Ann. intern. Med. 1972, 76, 993.29. Bishop, R. F., Davidson, G. P., Holmes, I. H., Ruck, B. J. Lancet, 1973, ii,

1281.30. Sexton, M., Davidson, G. P., Bishop, R. F., Townley, R. R. W., Holmes, I.

H., Ruck, B. J. ibid. 1974, ii, 355.31. Barbieri, D., De Brito, T., Hoshino, S., Nascimento, O. B., Martins Campos,

J. V., Quarente, G., Marcondes, E. Archs Dis. Childh. 1970, 45, 466.32. Gracey, M., Burke, V., Thomas, J. A., Stone, D. E. Am. J. clin. Nutr. 1975,

28, 841.33. Gracey, M., Stone, D. E., Suharjono, Sunoto. ibid. 1974, 27, 345.34. Heyworth, B., Brown, J. Archs dis. Childh. 1975, 50, 27.

APATITE DEPOSITION DISEASE

A New Arthropathy

P. A. DIEPPEP. CROCKER

E. C. HUSKISSOND. A. WILLOUGHBY

Departments of Rheumatology and Experimental Pathology,St. Bartholomew’s Hospital, London EC1A 7BE

Summary A method for identifying particles of

crystalline calcium hydroxyapatite in

synovial fluids and biopsy material has been developedwith, high-resolution scanning electron microscopy andan energy-dispersive micro-analytical system. Particlesof hydroxyapatite were identified in the joints of six pa-tients diagnosed as having osteoarthritis, three of whomhad acute inflammatory episodes with effusions into thejoints. Apatite was not identified in joints affected byrheumatoid arthritis and other types of arthropathy.Animal studies showed that hydroxyapatite crystals cancause an acute inflammatory reaction, and this has beenconfirmed by experimental studies in man. It is sug-gested that a third type of crystal-deposition diseaseshould be recognised&mdash;namely, calcium-hydroxyapatitecrystal-deposition disease.

Introduction

THE use of polarising microscopy has led to easy iden-tification of urate and pyrophosphate crystals in syno-vial fluids. Needle-shaped crystals of sodium biuratecan be found in practically every case of gout,2 and thepositive identification of crystals of calcium pyrophos-phate dihydrate led to the recognition of the "pseudo-gout syndrome". 3 Both types of crystal can cause acuteinflammation when injected into normal tissue,’-’ andthe phrase "crystal-deposition disease" has been coinedfor these conditions. 8

In addition to urate and pyrophosphate deposits, twoother crystal types have been identified in human carti-lage and in extra-articular calcific material; they are cal-cium hydroxyapatite and calcium orthophosphatedihydrate.9 10 Periarticular hydroxyapatite may be as-

sociated with acute inflammation," raising the possibi-lity that it may also be important in joint diseases. How-ever, indentification of apatite is difficult because the

particles are usually too small to be seen by light micros-copy, and are not birefringent. In this study a methodfor the indentification of apatite particles in synovialfluid has been developed; hydroxyapatite has beenshown to be capable of causing inflammation, and aclinical syndrome associated with deposition of apatitein joints has been identified.

Methods

Synovial fluids were aspirated from joints and immediatelyexamined by light and polarising microscopy for the presenceof crystals and to calculate the total white-cell count. Remaining fluid was centrifuged at 3000 r.p.m. for 15 minutes, :washed once in deionised water, and resuspended in deionised !water. A drop of this suspension was applied to carbon-coated ’copper electron microscope grids. Calcific deposits were pre-pared similarly and applied to grids for examination.The grids were examined in a Jeol 100C electron microscope

fitted with an attachment for high-resolution scanningtransmission electron microscopy, and secondary electron

scanning mode. The system also has an energy-dispersivemicro-analytical system providing analysis of areas of less than20 nm in diameter. Results obtained on the energy-dispersivesystem, operating at 80 kV with a count-time of 200 seconds,were analysed, and the ratios of the different elements in 6cparticles being examined were calculated. Particles with mor-phology similar to that of pure crystalline calcium hydmxya-patite were sought, and examined, and those found to containonly calcium and phosphorus counted so that the ratio of thesetwo elements within the specimen was obtained. These ratioswere compared with those obtained from pure standard sam-ples of calcium hydroxyapatite (Ca,OH[P04h-H;zO), calciumorthophosphate dihydrate (CaHP(B-2HO), and calcium

pyrophosphate dihydrate (CaPO-2HO), examined in thesame way. The average of three ratios from each individuiparticle was recorded in each case.

Pure crystalline calcium hydroxyapatite was supplied byProf. H. Fleisch and the size of the particles of aggregatecrystals varied from 0. 5 to 10 {im diameter A suspension of10 mg in z2 ml sterile saline was injected intradermally in theforearm of 12 normal volunteers, and the resulting erythema.and induration measured over the next 72 hours. 10 mg Gapatite, suspended in 1 ml of saline, was injected intrapleurallyin male Wistar rats, and the animals were killed 6, 12, 24, and48 hours thereafter. The resulting inflammatory exudate washarvested in Medium 199, and the volume and total andferential white-cell counts estimated.

Results

Apatite particles were not seen on light microscopy of