John W. Fabre

THE DETAILED DISTRIBUTION OF MHC CLASS II ANTIGENS IN NORMAL HUMAN ORGANS

In a previous article we described the detailed tissue distribution of MHC class I antigens. In this study, we have used a monoclonal antibody, NFK1, to study the tissue distribution of MHC class II antigens. This antibody, which detects a monomorphic determinant common to the DR, SB, and DC molecules, was used to stain frozen sections of normal tissues from throughout the human body by a sensitive peroxidase-antiperoxidase immunohistological technique. Although previous studies, both in animal models and in human beings, have shown that class II antigens are expressed on a limited number of nonlymphoid tissues, our study has extended the spectrum of tissues on which this class of antigens is detectable. Epithelial cells in a number of organs were positively stained--these include the tongue, tonsils, epiglottis, trachea, small intestine, urethra, epididymis, and proximal renal tubules. Lymphatics throughout the body appeared to express class II antigens. Capillaries in brain, testis, and placenta appeared not to express class II antigens, but in the rest of the body they showed strong and uniform staining. These and other observations, and their implications, are discussed in relation to previously published studies.

The detailed distribution of HLA-A, B, C antigens in normal human organs.

We have used a monoclonal antibody, PA2.6, directed against the heavy chain of HLA-ABC antigens to study the detailed tissue distribution of MHC class I antigens. Normal tissues from throughout the human body were obtained fresh from organ donors or operative specimens and were snap-frozen in liquid nitrogen within 1-2 hr of removal. Frozen sections were then studied using a sensitive peroxidase-antiperoxidase immunohistological technique. The results of our study show that class I antigens could be detected on most, but not all, the nucleated cells in the body. They were only weakly detectable in several tissues including endocrine cells in the thyroid, parathyroid, pituitary and islets of Langerhans in the pancreas and on gastric mucosa, the myocardium, skeletal muscle, and hepatocytes in some of the specimens. Spermatozoa were positively stained in the testis, but as they moved up into the epididymis class I antigens were no longer detectable. We found that class I antigens were not detectable on corneal endothelium, some Brunners glands in the duodenum, villous trophoblast, central nervous system neurones, the exocrine portion of the pancreas, and acinar cells in the parotid. We conclude, therefore, that class I antigens are not ubiquitous, as previously thought.

Evidence for the occurrence of O-glycosidically linked oligosaccharides of poly-N-acetyllactosamine type on the human leucocyte common antigen

High molecular weight glycoproteins of human B and T lymphocytes known as leucocyte common antigen or T200 have been shown to carry O- and N-glycosidically linked, sialylated, carbohydrate chains. The O-linked chains are polydisperse and those of B rather than T cell type are highly susceptible to degradation by endo-beta-galactosidase. These differences among lymphocytes that are functionally distinct raise the possibility that the oligosaccharides may contribute to the functions of these differentiation molecules as well as to their electrophoretic diversity.

The membrane antigens of human colorectal cancer cells: Demonstration with monoclonal antibodies of heterogeneity within and between tumours and of anomalous expression of HLA-DR

The membrane antigens of fifteen colorectal tumours were studied using a number of monoclonal antibodies and the immunoperoxidase technique on frozen sections. With this approach we could easily demonstrate differences in the membrane structure of the cancer cells within the tumour mass of given patients and also readily demonstrate differences between tumours that were indistinguishable by histological and other standard criteria. An unexpected finding was the patchy expression of HLA-DR antigens on cancer cells, in spite of the absence of HLA-DR on normal colorectal epithelium. These findings have interesting theoretical and clinical implications.

Biochemical Characterisation and Localization in Brain of a Human Brain-Leucocyte Membrane Glycoprotein Recognised by a Monoclonal Antibody

Abstract: The F10‐44‐2 monoclonal antibody was originally shown to interact with a determinant found predominantly in human brain and leucocytes. In this study we demonstrate by quantitative absorption analysis with homogenates of the head of the caudate nucleus, putamen, thalamus, cerebral grey matter, cerebral white matter, corpus callosum and cerebellar folia that the determinant is restricted to the white matter of the CNS. Immunofluorescence studies on frozen sections of the above brain subregions confirm the absorption analyses, showing staining only of white matter. In addition, and unexpectedly, we found very bright staining around blood vessels, particularly in the cerebellum. Biochemical studies established that the molecule in white matter bearing the F10‐44‐2 determinants is a sialylated membrane glycoprotein with an apparent molecular weight of 90,000, which is similar to but slightly smaller than the T lymphocyte form of the antigen. Developmental studies comparing 16‐week foetal and adult cerebrum showed a fivefold increase in F10‐44‐2 antigen content. Thus, in the human CNS, the F10‐44‐2 antigen is a medium‐sized glycoprotein which is restricted to white matter and shows a marked increase in concentration during development. No such molecule has been described previously.

Distribution of Thy-1 in human brain: Immunofluorescence and absorption analyses with a monoclonal antibody

A monoclonal antibody to human Thy-1 has been used to study the anatomical localization of Thy-1 in human brain and to quantitate the relative amounts of Thy-1 in different brain subregions. Quantitative absorption analyses using homogenates of carefully dissected brain subregions, together with an [125I]anti-immunoglobulin binding assay using brain homogenate as target, established that Thy-1 was present in large amounts throughout human brain, but the grey matter of cerebrum (cortical grey matter, caudate nucleus, putamen and thalamus) had 5-10 times as much Thy-1 as white matter. Grey matter of cerebellum (cerebellar cortex and dentate nucleus) also had higher amounts of Thy-1 than white matter, but the total amount of Thy-1 in cerebellum was less than in the cerebrum. Immunofluorescence studies gave interesting results and demonstrated in particular: (a) the outlining of some neuronal cell bodies and their processes (particularly the Purkinje cells of the cerebellar cortex) by spots of fluorescence; (b) staining of what appeared to be cell bodies of satellite cells in areas of grey matter; (c) granular staining in grey but not white matter; (d) staining of what appeared to be fibre tracts in the basal ganglia and thalamus, the tracts appearing duller than the surrounding grey matter of the nuclei; (e) staining of only some fibres in sciatic nerve; and (f) absence of staining of the adrenal gland.

A study of three protocols of blood transfusion before renal transplantaion in the dog.

SUMMARY Three protocols of blood transfusion were evaluated in a canine model for (1) the strength and breadth of leukocytotoxin induction, (2) the induction of cell-mediated immunity against the blood donors, (3) haemagglutinin production, and (4) any effect on kidney graft survival. At the end of the transfusion schedule, each dog received a kidney graft and was given azathioprine and prednisolone postoperatively. All dogs were unrelated and blood donors were not used as kidney donors. All three transfusion protocols, comprising i.v. injections of blood twice weekly or every 2 weeks from one or three donors, induced unacceptably strong and broad leukocytotoxins. All transplants performed across a positive crossmatch failed to function. However, where a negative crossmatch was available, the trend of results was that the transfused dogs had better graft survival than nontransfused animals similarly treated with azathioprine and prednisolone. Only one dog produced haemagglutinins. Several animals had positive cell-mediated immunity against the blood donors, but the response was not strong and was frequently not sustained. The problems posed by blood transfusions in clinical renal transplantation are complex, and have been a subject of debate from the time of the earliest transplants. A number of early studies comparing graft survival in patients, who had received few transfusions with those that had received many before transplantation, showed that graft outcome either was not different in the two groups (19) or that it might have been better in the patients given the most transfusions (7). That blood transfusions before transplantation could induce a state of specific unresponsiveness to a subsequent organ allograft was clearly demonstrated in the rat by a number of groups ( 10, 17, 18). The more exacting clinical comparison of a group of nontransfused patients with a transfused group was not made until 1974 when Opelz and Terasaki (21) published data showing that graft survival was substantially worse in nontransfused patients. Their work has been supported by further clinical reports ( 14, 15, 22, 27) and by experimental work in the rhesus monkey (26) and the dog (1). Although the clinical results from our unit show that nontransfused and transfused patients have essentially similar and excellent graft survival (P. J. Morris et al., in preparation), the retrospective studies from most, but not all, units suggest that blood transfusions before transplantation do lead to better graft survival in those patients that receive a transplant. The problem that arises in patients given transfusions before transplantation is that any beneficial effect in terms of graft protection for those that come to transplantation might be offset by the relegation to life-long dialysis of those that become highly sensitized (3, 24). The clinical problem, then, is the choice of a protocol for transfusion that is most likely to result in graft protection and least likely to result in the production of lymphocytotoxins to HLA-A, B, and C antigens. The balance between an overall harmful or beneficial effect of transfusions almost certainly depends on a number of variables, such as the number of different blood donors, the frequency of transfusion, the volume of blood transfused, the use of frozen blood, the age of the blood, the interval between the last transfusion and transplantation, the degree of histocompatibility between blood donor and recipient, the genotype of the recipient, and the degree of nonspecific immunosuppression induced by chronic renal failure. The experiments reported in this paper attempt to evaluate some of these factors in the dog.

Use of cyclophosphamide and enhancing serum to suppress renal allograft rejection in the rat.

Cyclophosphamide was tested for its interaction with passive enhancement in suppressing the rejection of kidney allografts in the (DA x Lewis)F1 to Lewis rat strain. Dose response studies with cyclophosphamide showed that 10 mg/kg/day for 14 days was necessary for complete suppression of rejection and indefinite graft survival. Doses of 5 and 3.5 mg/kg/day had only a marginal effect on graft function and survival, although the lymphocytotoxin response to the graft was completely or very substantially suppressed by these smaller doses. The use of passive enhancement with cyclophosphamide at the 5- and 3.5-mg/kg/day doses resulted in a favourable interaction with improved graft function and survival. Interestingly, passive enhancement in combination with 5 mg/kg/day of cyclophosphamide resulted in indefinite graft survival only if cyclophosphamide was given for 28 days. If cyclophosphamide was given for 14 days, rejection was suppressed only during the period of cyclophosphamide treatment.