Preclinical assessment of the immunosuppressive properties of an anti-CD4 monoclonal antibody (MAB) in an allogeneic foetal rat pancreatic transplantation model

Muller, Christo John Frederick (2004-12)

Dissertation (PhD)--Stellenbosch University, 2004.

Thesis

ENGLISH ABSTRACT: Introduction Despite advances in insulin therapy, the side effects associated with diabetes mellitus still remain. Pancreas transplantation has benefited diabetics with end-stage renal failure by reversing the diabetic state and preventing or reversing the progression of diabetes associated diseases. Currently the side effects associated with lifelong immunosuppression preclude pancreas transplantation as a viable treatment option for both type I and II diabetics. In the laboratory, transplanted rat foetal pancreata have been shown to be able to reverse the clinical signs of streptozotocin-induced diabetes in an isogeneic model. Reversal of diabetes by allogeneic foetal rat pancreas transplantation, although possible has proved to be more difficult due to fierce rejection of the grafts and the diabetogenic effects of conventional immunosuppressants. Aims One of the goals, focus and intentions of this laboratory study in rodents, is to contribute new information to the scientific literature. The potential to “reverse” the diabetic state by allogeneic foetal pancreatic transplantation, was the main stimulus for this study. Methods Foetal pancreata of 16-18 days gestation were transplanted into a surgically prepared renal subcapsular space. Immunosuppressive protocols used to prevent rejection of the allogeneic foetal rat pancreata included donor specific transfusion (DST), cyclosporine [a calcineurin inhibitor (CsA)], mycophenolate mofetil [a purine syntase inhibitor (MMF)], and a mouse anti-rat CD4 monoclonal antibody (W3/25). Immunosuppressants were used as monotherapies and in combination. Results Isogeneic foetal rat pancreas transplantation resulted in the growth and development of mature insulin producing islets of Langerhans at the site of engraftment. Allogeneic foetal pancreatic transplantation without immunosuppression resulted in complete rejection of the grafts at 14 days post-transplantation. Histological assessment of allografts at 14 and 30 days post-transplantation showed that CsA was able to prevent acute rejection in our rat models although graft scores and survival were improved if CsA was combined with MMF. Intraperitoneal anti-CD4 monoclonal injections were well tolerated, and if given daily effectively prolonged graft survival up to 30 days. Combining DST with anti-CD4 and CsA induction therapy provided long-term graft survival without daily immunosuppression. This combination, together with allogeneic foetal rat pancreas transplantation, was effective in reversing the clinical signs of experimentally induced diabetes. To my knowledge these are the first published results in which reversal of streptozotocin induced diabetes was achieved by fully MHC mismatched foetal rat pancreatic transplantation. Conclusion Foetal rat pancreatic transplantation is a potential source of endocrine replacement, which, with effective immunosuppression allows for the development of functional islets able to reverse the clinical signs of experimentally induced diabetes in an allogeneic rat model. An unique immunosuppressive protocol, with potential clinical relevance in the human, combines anti-CD4 mAb, CsA and DST induction therapy, which alleviates the burden of daily immunosuppression and associated side effects.

AFRIKAANSE OPSOMMING: Inleiding Ten spyte van die vordering met modeme insulienterapie bly die newe-effekte, waarmee diabetes mellitus geassosieer is, steeds ‘n probleem vir diabete. Diabetiese pasiente met eindstadium nierversaking trek geweldig voordeel uit nier-pankreasoorplantings wat die diabetes omkeer en die progressie van diabetesverwantesiektes voorkom of selfs omkeer. Die newe-effekte van lewenslange immuunonderdrukking skakel pankreasoorplanting uit as ‘n lewensvatbare behandelingsopsie vir tipe I of II diabete. In ‘n streptozotosien-gei'nduseerde diabetiese rotmodel kan isogenei'ese fetale pankreasoorplanting die kliniese tekens van diabetes omkeer. Die omkering van streptozotosien-gei'nduseerde diabetes deur allogeneiese fetale pankreasoorplanting behoort moontlik te wees indien verwerping en die diabetogeniese newe-effekte van konvensionele immuunonderdrukkers oorkom word. Doelstellings Een van die mikpunte, fokusse en oogmerke van hierdie laboratorium studie in knaagdiere, is om ‘n betekenisvolle bydrae tot nuwe kennis in die wetenskaplike literatuur, te maak. Die potensiaal om die diabetiese toestand deur allogeneiese fetale pankeasoorplanting om te keer, was die hoof stimulus vir die studie. Metodes Fetale rotpankreata van 16-18 dae gestasie was in ‘n chirurgies voorbereide spasie onder die nierkapsel oorgeplant. Immuunonderdrukkende protokolle, vir die voorkomming van verwerping van die allogeneiese fetale pankreasoorplantings, het donorspesifiekeoortappings (DST), siklosporien [‘n kalsineurien inhibitor (CsA)], mikofenolaat mofetiel [‘n purien sintase inhibitor (MMF)] en ‘n anti-rot CD4 monoklonale antiliggaam (W3/25) ingesluit. Die immuunonderdrukkers is as mono- of as kombinasieterapie gebruik. Resultate IsogeneTese fetale rotpankreasoorplanting het tot die ontwikkeling van volwasse insulienproduseerende eilande van Langerhans gelei, wat die kliniese tekens van streptozotosien-gei'nduseerde diabetes kon omkeer. Allogenei'ese fetale rotpankreasoorplanting sonder immuunonderdrukking het tot algehele verwerping van die oorplanting binnel4 dae na oorplanting gelei. Histologiese beoordeling van die oorplantings 14 en 30 dae na oorplanting het getoon dat CsA akute verwerping van fetale pankreasoorplantings in die rotmodelle voorkom. Indien CsA met MMF gekombineer word, word die oorplantings-telling en oorlewing verbeter. Intraperitoneale anti-CD4 monoklonale inspuitings was goed verdra, en indien daagliks toegedien, het dit die oorlewing van die pankreasoorplantings effektief tot 30 dae verleng. Die kombinasie van DST, anti-CD4 en CsA induksieterapie het tot langtermyn oorlewing van die pankreasoorplantings gelei sonder verdere daaglikse immuunonderdrukking. Die induksieterapie in kombinasie met allogenei'ese fetale pankreasoorplanting was effektief in die omkering van die kliniese tekens van streptozotosien-gei'nduseerde diabetes in die rot. Hierdie is, sover ek weet, die eerste keer dat omkering van streptozotosien-gei'nduseerde diabetes suksesvol met ‘n volledige MHC onverenigbare allogenei'ese fetale pankreasoorplanting behaal is. Gevolgtrekkings Fetale rotpankreasoorplanting is ‘n potensiele bron vir endokrien vervangingsterapie, wat met effektiewe immuunonderdrukking tot die ontwikkeling van funksionele eilande van Langerhans lei, wat die vermoe het om die kliniese tekens van experimenteel-ge'induseerde diabetes in ‘n allogeneiese rotmodel om te keer. ‘n Unieke immuunonderdrukkingsprotokol, met kliniese relevansie, kombineer DST met anti-CD4 mAb en CsA induksieterapie wat die las van daaglikse immuunonderdrukking en die geassosieerde newe-effekte van konvensionele immuunonderdrukking verlig.

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