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Experience with HLA and sex randomized human fetal tissue transplant in adult diseases: a preliminary report.

Abstract

The art of transplant surgery has come a long way in establishing itself as an important discipline with the support of immunology, molecular biology, etc., for the restoration of the function of a failing organ. Today, there is a major discrepancy in the demand and supply of adult organ grafts. The aim of the present study is to examine whether fetal tissue/organ, with its intrinsic advantage of hypo-antigenicity as well as its rich organ specific stem cell content, can survive in a HLA and sex randomized host in a heterotopic site without any immunosuppressive support.

KEY WORDS: Fetal tissue growth; HLA randomized adult's axilla.

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In the year 1977, in the course of certain experimentations on the acquisition of immunocompetence of the human fetus, we were surprised to note that intraamniotic instillation of an antigen, that is, tetanus toxoid, could cause abortion. (1). That the effect is not specific to tetanus toxoid was confirmed when abortion was induced as a result of the instillation of other antigens like double antigen, maternal buffy coated leucocyte of 10ml. blood, etc. (2). While antigen stimulation of the developing fetal humoral immune system can provoke abortion, stimulation of the developing fetal cellular immune system by intraamniotic instillation of BCG can cause dissolution and autoabsorption of the human fetus. (3, 4, 5,6). In the case of stimulation by instillation of intraamniotic 2cc. tetanus toxoid, we observed that the induction-abortion interval varied widely, i.e., 92.8 percent of the mothers in our study aborted within one month, which fell to 86.2 percent within 21 days, 72.4 percent within two weeks and 52.8 percent within one week. All consenting mothers were admitted for hysterotomy and ligation and the experimental protocol passed through the ethical committee of the institute. One major problem which remains to be solved with this intraamniotic tetanus toxoid experimentation is the wide fluctuation in the induction-abortion interval.

Why the allogenic fetus is not rejected by the mother remains a mystery. The possible reasons (7) could be summarized as: (a) a mild state of immunosuppression in pregnancy, (b) poor allo-antigenic expression of the trophoblast, (c) the trophoblast contains some strong locally acting cytokines and growth factors which suppress the immune response mechanism, (d) the placenta is resistant to attack by the maternal antibody or cell mediated damage due to the presence of a non-specific blocking antibody.

On the basis of our experiences over the past two decades on the reactions of the fetal immune system to an intraamniotic antigenic assault, we can say that the pre-immune (within! 6 weeks) and hypo-immune (after 16 weeks) developing fetal immune systems provide certain intrinsic advantages of the fetal tissue as a graft for transplants. We have earlier reported on the safety of (174 units) umbilical cord whole blood transplantation in 62 patients (8), which we have been following up for the last two years. What is worthy of note is the fact that after following the standard blood transfusion protocol, there was no immunological or non-immunological reaction in any patient. In a subsequent set of experiments on orthotopic fetal thymus transplant in the axilla of cancer patients, we noted with surprise that there was growth, differentiation and even interaction with the hosts' immune status. (9).

Materials And Methods:

Whether the privileges of the hypoantigenicity of the human fetal tissue can be used for universal cell or tissue transplant in case of need for the restoration of the functions of a diseased organ, is the rationale behind the present transplantation protocol. In the current set of experimentations on a simple orthotopic transplantation site at the axilla under local 1 percent xylocaine infiltration anaesthesia, a little fetal tissue/organ is placed in a vascular subcutaneous 2x1cm. dissected space. The experiments were preceded by written informed consent by the patient/guardian and after the completion of the necessary formalities of the hospital ethical committee. A second consent was taken from the donor mothers admitted for hysterotomy and ligation who contributed the fetal tissues. Hysterotomy was conducted in the O.T. under general anaesthesia following standard pre-operative, operative and post-operative text book protocol and the fetus with the intact sac was taken out judiciously by the first group of surgeons. Care must be taken not to rupture the amniotic cavity. In the same O.T., simultaneously in an adjacent table, a second group of surgeons prepared the recipients' axilla, after infiltrating subcutaneously one percent xylocaine, and after repeatedly washing with rectified spirit and betadine. All other standard protocol of antiseptic/ aseptic dressing, draping were followed meticulously. Then, the second group of surgeons dissected a 2cm. x 1cm. space in the subcutaneous tissue of the prepared axilla and controlled the bleeding adequately. The second group of surgeons then waited for the first group of surgeons to hand over the fetus with the intact amniotic sac and placenta. Within a minute, the second group of surgeons then dissected the fetus and took out the tissue/organ from the thymus/liver/lung/pancreas/cartilage/heart, as per decisions of transplantation. After the placement of the fetal tissue graft in the adult axilla, the skin was closed with interrupted (000) atraumatic vicryl suture and a small dressing was applied to cover the incision site. After an one month observation period, the transplanted fetal tissue was taken out by an elliptical incision, 2cm x 1cm, under local anaesthesia following the standard antiseptic/aseptic protocol of surgery in the O.T. A periodic assessment of Hb, Tc, Dc, ESR was done at the pre- and post-transplant phases, as reported by us earlier. (10).

Results And Analysis:

The following table depicts our experience with pre-immune and hypo-immune fetal tissue transplant at the axillary site of HLA and sex randomized adults with neo-plastic and non-neo-plastic disease backgrounds.

These transplants were conducted on our patients in Bijoygarh State Hospital. As per the ethical committee's strict protocol, in no case was the primary care and treatment of the patient jeopardized as a result of the experiment protocol. We strictly followed duty based ethics and the traditional concept of doctor-patient relationship relying on the principle of (a) do no harm, (b) try to do good, (c) respect for autonomy, (d) justice, also keeping in mind the concept of utilitarianism in a broader view.

In all these 14 cases, though the axillary transplant site was healthy, we removed the transplanted tissue after one month with an elliptical incision under local anaesthesia and subsequently stained the removed tissue with haematoxylin and eosin (HE), to examine the impact of transplantation of the fetal tissue (sex randomized) in the adult host and vice versa.

We know that the cornea and cartilage are readily accepted as grafts. We also know that the deciduas of the uterus provides a certain degree of environmental privilege for the growth of the allogenic fetus, but the axilla has never been cited in medical literature as a privileged site. The serial studies of the histological specimens did not reveal features of acute rejection and there was no sign of ischaemic damage of the graft like thrombosis, mononuclear invasion, endothelial disruption or endarteritites, etc. On the contrary, there was clinical evidence of increased vascularity of the graft and its surroundings. The question is, why this is so. Fetal growth is dependent on an unique symbiotic environment where the mother provides all the necessary factors and environment for growth, proliferation and differentiation. The fetal micro-environment is distinctly different from the adult neuro-endocrine and metabolic micro-environment.(11). Hence, it is possible that the fetal transplanted graft tissue adjusts its own micro-environment to an altered metabolic environment, i.e., in the adult, using the advantage of its hypo-immune or pre-immune status in order to survive, grow, proliferate and differentiate further. Another interesting point to note is the perfect healing of the transplant site surgical wound. Why and how a transplanted fetal tissue in sex and HLA randomized adults escapes immunological recognition and becomes a human homologous chimera still remains a mystery.

In addition, a study of the histological material did not suggest any foreign body giant cell or other similar response. Sequential haemoglobin, Tc, Dc, ESR studies on the recipients of transplants did not show any major variation when comparing the pre- and post-transplant results. There was no gross variation in the pre- and post-transplant leucocyte or lymphocyte counts.

Discussion:

The basis for transplant immunology was laid by Sir Peter Medwar in a series of experiments from 1944 onwards using mainly skin transplant techniques. He formulated the basic tenets of transplant immunology like donor specificity of graft rejection, cell mediated first set response and antibody mediated second set response.(12). The rapid development of clinical organ transplantation over the past 40 years has often outstripped our knowledge of the mechanisms involved. The immune systems' need to differentiate between self and non-self (eg., bacteria) with distorted or perverted self (tumour or virally infected self), allows the organism to survive in this hostile environment. The molecular mechanism of self and non-self recognition resides in specialized cell-surface molecules known as major histocompatibility antigen (MHC). The first MHC was recognized in 1958 with a serological identification attempted in the line of ABO blood group system. Hence, it was promptly renamed as "Human Leucocytic Antigen" (HLA).(13). This is now known as HLA-A2 and can be traced in the short arm of chromosme-6. The MHC has two sub-sets, i.e., the Class-I region and the Class-II region.(14). The Class-I region is divisible into HLA-A,B,C, allelic series (as well as E,F,G,and H) (15), of which A and B are thought to be important for transplantation purposes. Class-II is divided into DR, DP, and DQ loci (16), though only DR is used for matching purposes. Between Class-I and Class-II, there is a region known as Class-III which contains more than 70 genes to control the complement system (C2, C4, tumour necrotic factor alpha, tumour necrotic factor beta, etc.).(17).

While the molecular mechanisms discussed above in brief can explain the mechanism of transplant rejection of an adult organ, the phenomenon is far from clear so far as the hypo-immune or pre-immune fetal tissue transplant survival mechanism in adults is concerned, at least over a one month period of observation in the present set of experiments in HLA randomized hosts without any concommitent immunosuppressive drug support to delay rejection. From the HE stained histological photomicrographs of the thymic tissue, liver tissue, cardiac tissue, pancreas, lung tissue of fetuses (within 20 weeks) transplanted into adult axillary folds' incision sites, there did not appear to be any feature of rejection within the one month observation period.

What, then, is the importance of this study? What are the futuristic implications of the research? According to one report from the United States, there is an increasing disparity between the demand and supply of organs needed for patients awaiting organ transplantation. While the number of patients awaiting transplants increases at approximately 15 percent per year, the number of cadaveric organ donors increases at only one to three percent per year. Actually, the number of cadaveric organ donors in 1999 increased less than one percent over that in 1998. (18). Whether fetal tissue/organ/cellular transplant, with its intrinsic advantages of hypo-antigenicity and hypo-immune status, can fill up the existing gap in the demand and supply of organs required for transplants, is a matter under intense scientific study. The preliminary report of our study indicates a positive direction in this field, in our long quest to understand the dimensions of the acquisition of immunocompetence by the human fetus and its manipulation in health and disease.

Another interesting development in the past 50 years is the attempted use of bio-ionic devices and solid organ transplants as a replacement for a diseased or failing tissue or organ, based on the principle of cell transplant and regeneration in vivo with bio-artificial hypo-antigenic synthetic support. Cell transplants have been used successfully in Parkinsonism, to restore articular cartilage, etc. With the support and wisdom of the bio-engineering department, ther have been attempts to control uncontrolled diabetes by implanting a bio-artificial pancreas consisting of islet cells micro-encapsulated in alginate. There is a fascinating review by Stocum D.L., on the topic, "Regeneration Biology and Engineering: Strategies for Tissue Restoration".(19). In fine, if the entire medical community unites on this very complex issue of fetal tissue-adult tissue transplant interaction in health and disease, there may be a major breakthrough in this hitherto vastly unknown and fascinating field.

Photograph No. 1,2,3:

These are HE stained microphotographs showing 10 weeks and 14 weeks fetal liver tissue under Low power (Microphotograph 5, 10 weeks fetus) and scan power (Microphotograph , 14 weeks) and Low power (Microphotograph 7, 14 weeks). Here, too, the photographs of the fetal transplanted liver tissue in the adult host do not suggest any features of rejection. In this connection, it is worth mentioning that haematopoisis of the fetal liver starts from 7 to 10 weeks., and bile formation starts from the tenth week. Bile itself can provoke an inflammatory response, which is not visible in these pictures.

[ILLUSTRATIONS OMITTED]

Photographs No. 4,5

These are HE stained microphotographs showing 20 weeks fatal lung tissue transplanted under the adult axilla and viewed under Low Power (Microphotograph 4) and Scan power (Microphotograph 5). At 20 weeks, the fetal lung development passes through the canalicular period (16 to 24 weeks). In these pictures, once again, the features of rejection of the fetal tissue transplant are not observed.

[ILLUSTRATIONS OMITTED]

Photographs No. 6,7,8,9

These are HE stained microphotographs showing the fate of a 20 weeks total fetal pancreas transplant in a HLA randomized host suffering from diabetic gangrene. Microphotograph No. 6 is a low power depiction of the pancreas . Microphotographs Nos. 7 to 9 show proximal (No. 7), middle (No. 8) and distal (No. 9), parts of the transplanted pancreas. The classical features of immunological rejection are not present also in these pictures.

[ILLUSTRATIONS OMITTED]

References:

(1.) Bhattacharya N, Chaudhury N, Banerjee S, Mukherjee K L, "Intraamniotic Tetanus Toxoid as a Safe Abortifaecient", Indian Journal of Medical Research, 70, Sept. 1979 : 435-39.

(2.) Bhattacharya N, Letter to the Editor, Clinical and Experimental Obstetrics and Gynecology, vol. XXIII, no. 4,1996 :272-75.

(3.) Bhattacharya N, "Intraamniotic Instillation of Tetanus Toxoid : A Safe, Cheap, Effective Abortifaecient in the Light of Our Experiences with Different Intraamniotic Instillation of Antigens for Alteration of Pregnancy Immunotolerence-A Study from 1978-1996", in R L Tambiraja and N K Ho, eds., Relevance and Excellence in Perinatal Care, Proceedings of the Ninth Congress of the Federation of the Asia and Oceania Perinatal Societies, Singapore, 10-14 November, 1996, Monduzzi Editore, Bologna, Italy :193-200.

(4.) Bhattacharya N, "Dissolution of the Fetus : A New Experience with Intraamniotic BCG Instillation", Ibid. :201-206.

(5.) Bhattacharya N, "Study of the Aborted Fetuses After Intraamniotic Instillation of Tetanus Toxoid", Ibid. :187-192.

(6.) Bhattacharya N, "A Study on the Intraamniotic Instillation of Tetanus Toxoid on A Growing Human Fetus", Dissertation submitted to the Calcutta University (India), for the degree of Doctor of Science( D.Sc.), Faculty of Medicine (2001).

(7.) Halloran P F, "Tissue Transplantation", in J B Walter, I C Talbolt, eds., Walter and Israel General Pathology, 7th edition, Churchill Livingstone, New York, 1996 :281.

(8.) Bhattacharya N, Mukherjee K, Chettri M K, Banerjee T, Mani U, Bhattacharya S, "A Study Report of 174 Units of Placental Umbilical Cord Whole Blood Transfusion in 62 Patients as A Rich Source of Fetal Hemoglobin Supply in Different Indications of Blood Transfusion", Clinical and Experimental Obstetrics and Gynecology, vol. XXVIII, no. 1, 2001 : 47-52.

(9.) Bhattacharya N, Mukherjee K L, Chettri M K, Banerjee T, Bhattacharya S, Ghosh A B, Bhattacharya M, "An Unique Experience with Human Pre-immune (12weeks) and Hypo-immune (16 weeks) Fetal Thymus Transplant in A Vascular Subcutaneous Axillary Fold in Patients with Advanced Cancer-A Report of Two Cases", European Journal of Gynecological Oncology, June-July, 2001 (in press).

(10.) Bhattacharya N et al, Ibid.

(11.) Miller R K, "Fetal Drug Therapy :Principles and Issues", in M R Pitkin and J R Scott, eds., Clinics in Obstetrics and Gynecology, June 1991,34 (2) : 241-50.

(12.) Medwar P B, "The Behaviour and Fate of Skin Autografts and Skin Homografts in Rabbits", Journal of Anatomy, 1944, 78 : 176-99.

(13.) Dausset J, "Iso-leuco-anticorps (iso-leuko-antibodies)", Acta Haematologica, 1958, 20 : 156-66.

(14.) Trowsdale J, Ragoussis J, Campbell R D, "Map of Human MHC", Immunology Today, 1991, 12 : 433-36.

(15.) Koller B H, Geraghty D E, Demars R, Duvick L, Rich S S, Orr H T, "Chromosomal Organization of the Human Major Histocompatibility Class I Gene Family", Journal of Experimental Medicine, 1989, 169 : 469-80.

(16.) Dunham J, Sargent C A, Dawkins R L, Campbell R D, "An Analysis of Variations in the Long Range Genomic Organization of the Human Major Histocompatibility Class II Region by Pulse Field Gel Electrophoresis", Genomics, 1989,5 : 787-96.

(17.) Trowsdale et al, no. 14.

(18.) Howard R J, "Transplantation", Comments in E M Copeland III , K I Bland , E A Ditch , T J Eberlin , R J Howard , E A Luce , J M Seeger , W W Souba , D J Sugarbaker , eds., Yearbook Surgery 2000 (Mosby Inc., 2000) : 93.

(19.) Stocun D L, "Regenerative Biology and Engineering : Stategies for Tissue Restoration", Comment in Wound Repair and Regeneration, 1998, Jul-Aug, 6(4) : 273-5.

Niranjan Bhattacharya

Bijoygarh State Hospital, Kolkata-70032, India.
Patient/Age Suffering Type of Serial study of
 from fetal grafted Hb/Tc/Dc/ESR
 tissue at on 1st/3rd/5th/
 adult axilla 7th/14th/21st/
 28th days post-
 transplant

1. Mrs. L.D. (45) Cancer 14 weeks Very little change
 Gall Bladder, fetal liver from pre-transplant
 Stage IV tissue level
2. Mrs. M.S.(46) Infiltrating 14 weeks No major change
 duct fetal total (more than 20%)
 carcinoma, thymus from pretrans-
 plant level
3. Mrs. S.M.(65) Adeno- Total No major shift
 carcinoma thymus of
 ovary, Stage 20 weeks
 IV fetus
4. Mr. S.D.(40) Adeno- Lung tissue -do-
 carcinoma from a 20
 lung, Stage weeks fetus
 IV
5. Mrs. PM.(39) Infiltrating Liver tissue -do-
 duct from a 10
 carcinoma, weeks fetal
 Stage III liver
6. Mr. K.K.C.(82) Diabetic Total -do-
 gangrene with Pancreas
 poor GC and spleen
 from 20
 weeks fetus
7. Mr. T.H.(79) COPD with Cardiac -do-
 ischaemic tissue from
 heart disease a 20 weeks
 fetus
8. Mr. G.N.(54) Disc prolapse Thymus of a -do-
 20 weeks

9. Mr. M.S.(38) Amoebic 14 weeks -do-
 liver abscess fetal liver
 tissue
10. Mr. T.D.(49) Ischaemic 10 weeks -do-
 heart disease fetal heart
 with old tissue
 infarction
11. Mr. A.S. (45) Diabetic Fetal -do-
 gangrene thymus, 20
 weeks
12. Mr. R.T.(49) Diabetic Fetal -do-
 gangrene thymus, 20
 weeks
13. Mrs. S.T.(49) Ischaemic Fetal heart -do-
 heart disease tissue, 16
 weeks
14. Mrs. K.B.(55) Rheumatoid 14 weeks, -do-
 arthritis fetal thymus
 with
 deformity

Patient/Age Other supportive Comments
 treatments on the local
 axillary
 transplant
 site

1. Mrs. L.D. (45) Cephalexin and Healthy
 NS-AID *
2. Mrs. M.S.(46) Cephalexin and Healthy
 NS-AID *
3. Mrs. S.M.(65) -do- Healthy
4. Mr. S.D.(40) -do- Healthy
5. Mrs. PM.(39) -do- Healthy
6. Mr. K.K.C.(82) Act-rapid human Healthy
 insulin,
 pentoxyphylline
 drip, vitamins,
 cephalexine and
 other supports
7. Mr. T.H.(79) Salmeterol Healthy
 inhalation with
 nitrates and
 calcium blocker
8. Mr. G.N.(54) Traction, Healthy
 vitamins,
 NS-AID *
9. Mr. M.S.(38) Anti-amoebic Healthy
 drug (ornida-
 zole) and tetra-
 cycline etc.
10. Mr. T.D.(49) Nitrate, beta- Healthy
 blocker, aspirin
11. Mr. A.S. (45) Act-rapid human Healthy
 insulin, anti-
 biotics, pentoxy-
 phylline drip
12. Mr. R.T.(49) -do- Healthy
13. Mrs. S.T.(49) Nitrates and Healthy
 betablocker,
 aspirin
14. Mrs. K.B.(55) NS-AID * and Healthy
 chloroquine
 with physiothe-
 rapy

* = Non-steroidal anti-inflammatory drug.
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Article Details
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Title Annotation:Human Leukocyte Antigen
Author:Bhattacharya, Niranjan
Publication:Trends in Biomaterials and Artificial Organs
Article Type:Clinical report
Geographic Code:1USA
Date:Jan 1, 2004
Words:3316
Previous Article:Experience in combating lymphopaenia in advanced cancer with human fetal thymus transplant.
Next Article:A preliminary experience with placental umbilical cord whole blood transfusion as an emergency alternative of adult whole blood transfusion.
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