The use of human organs for transplantation was the initial trend, but due to the shortage of human organs xenotransplantation (xeno- foreign) came into picture. From an immunological perspective nonhuman animals would be better donors for transplantation of organs however, most of these species are either endangered or are too small to provide organs for the transplantation. There is also a risk of pathogens as most of these nonhuman animals are caught in the wild or have been housed for a long time. Genetically modifying these animals is also a herculean task. In order to avoid these issues pig organs were considered as they are physiologically and physically similar to human organs. Selective breeding program and genetic modification of pigs are much easier to do in comparison to the other animals.

BARRIERS DURING XENOTRANSPLANTATION (XT) & THEIR SOLUTIONS
The problem of organ shortage can be solved if effective XTs can be done, however there are certain barriers which make the XTs very difficult. The barriers are:
1. Immunological Problems
2. Microbial Problems
3. Ethical Problems
4. Pathophysiological Problems

Immunological barriers should be over come to make the transplantation process effective. The immunological barriers in xenotransplantations are more than that compared to the allo transplantations (allo- same).

I. HAR (Hyper Acute Rejection)
HAR develops within few minutes to few hours of the transplant. HAR is mediated by Antibody- mediated xenograft rejection and by performed natural antibodies (PFNAs) which bind to the endothelial cells thus activating complement and causes coagulation. Humans develop natural antibodies (Nabs) against Galα1- 3Galα1- 4GlcNAc (α1, 3Gal) and these are present on viruses, bacteria and viruses. This is a major xenoepitope structure is found as the terminal sugar on glycolipids and glycoproteins that are present on porcine vascular endothelium. The functional enzyme α1, 3- galactosyltransferase (α1, 3GalT also called GGTA1) synthesizes the Gal epitope using uridine diphoshate galactose (UDP-Gal) .The second enzyme is isoglobotriaosylceramide synthase (iGb3) can synthesise α1, 3 Gal on N-acyetyl lactosamine. Natural α 1, 3 Gal experiment owing to activity of iGb3 synthase in humans is unclear.

This 1, 3 Gal is functional in all except some humans and old monkeys. It is a possibility that humans have been previously exposed to this specific natural antibody. When α1,3 Gal specific Natural Antibodies bind to endothelium of vascularised xenografts the complement system is activated which leads to activation of coagulation cascade and rapid graft rejection process which is called HAR.

The complement-regulatory proteins (CRPs) which are present on the donor endothelium regulate the activity of complement. The CPRs present on the pig endothelium do not function well to limit the action of the human complement system this severely damages the endothelium and also causes thrombosis and graft loss. This does not occur in case of allografts, where the presence of antibody prior to the transplant does not result in HAR this may be due to the local control of complement activation.

Xeno antibodies (Xabs) bind to the α1, 3Gal via fragment antigen binding (FAB) and via their fragment crystallisable (Fc) region they bind to the C1q this activates the classical pathway. The alternative pathway can be activated in the absence of Xabs, by damaged tissues, polysaccharides. Thus, finally both these pathways form the membrane attack complex (MAC) causing destruction of the graft. In HAR there is massive interstitial haemorrage, odema and thrombosis of small vessel.

There are many ways to combat the problems associated with HAR.
i. Depletion of α1, 3 Gal natural Abs or complement inhibition can effectively prevent HAR.
ii. HAR can also be prevented by absorbing α1,3 Gal specific antigen ex vivo and in vivo or by inactivation of complement.
iii. Intravenous infusion of soluble glycoconjugates is also performed. In this technique synthetic Gal molecules are conjugated to either a polyethylene-glycol or poly l-lysine backbone or to bovine serum albumin carrier.
iv. α1,3 Gal gene knockout pigs that do not express the gal epitope was produced in order to sort the problem of HAR.
v. Complement in HAR plays a role where complement deletion with cobra venom or use of rodent recipients.

Even if HAR is prevented there is AXHR which is still caused which is the second stage of rejection.

II. AXHR (Acute Humoral Rejection)

AXHR also called as delayed xenograft rejection occurs in 24 hours and destroys the graft within days. This occurs mainly due to low levels of α1, 3 Gal natural Abs or due to lack of α1, 3 Gal T deficient pigs. A recent study using the transplantation of a vascularized cardiac allograft from mice deficient in the complement regulatory protein, decay-accelerating factor (DAF; also known as CD55) into α1, 3GalT-deficient recipient mice showed that low levels of natural antibody can cause AHXR when complement regulation is. These results prove that complement independent mechanisms contribute to pathogenesis of AXHR. Activation of cytokine genes, expression of adhesion molecules, and changes from an anticoagulant to a procoagulant phenotype on the endothelial cell surface may all play a role.

AXHR is the first humoral immune response which is elicited. The humoral response is characterized by the occurrence of Thrombotic microangiopathy and disseminated intra vascular coagulation (DIC). This early humoral mechanism is known as instant bloodmediated inflammatory reaction (IBMIR).

a) Coagulation & Thrombosis
Coagulation & thrombosis is associated with xenograft rejection where it was seen that there is formation of platelets, aggregation and endothelial dysfunction resulting from activation of platelets and coagulation pathway. Thrombosis along with structural integrity and also loss of function results from prothrombic stage. The platelet aggregation can be seen from the histological exams. Pigs and humans have many molecular incompatibilities which might give rise to coagulation which further develops into thrombosis and then finally results in AXHR.
Fibrinogen- Fg12 plays a role in thrombosis. Conversion of pro thrombin to thrombin occurs due to this.

b) Natural Killer cells (NK)
NK cells are said to play a dominant role in solid organ xenografts in comparison to allografts. NK cells promote xenotransplantation via three separate pathways. NK inhibitory receptors fail to recognize xenogenic MHC class I products on xenografts resulting in NK cell activation and direct perforin mediated damage or cytokine production that increase in xenoreactive T cell activity. NK cells can activate xenogenic endothelium, including expression of procoagulant factors and adhesion molecules that promote immune cell invasion. NK cells participate in xeno graft rejections through antibody dependent cell mediated toxicity (ADCC) induced by xenoreactive antibody bound to epitopes like α Gal on xenograft. Interactions of Fcα R III (CD16) on NK cells with anti Gal antibody also increase NK cell infiltration of xenograft. Contact between NK cells and endothelial cells results in activation of endothelium to express E selectin adhesion molecule and IL-8. Cytotoxic lysis of porcine endothelial cells by human NK cells is also dependent on interaction with activating NK receptors such as NKG2D which binds to ULBP-1 and NKp44.

c) Macrophages
Macrophages cause almost immediate rejection of xenogenic bone marrow. Human macrophages phagocytose porcine cells in an antibody complement independent manner. Certain xenogenic receptors can activate macrophages eg: Galectin 3 on human monocytes is a receptor for α 1,3 gal on porcine cells. Galectin 3 binds to α 1,3 gal to activate human monocytes. Macrophages are also activated by T cell derived cytokines and play a role in rejection of cellular xenografts. Signal regulatory protein α (SIRP α; also called CD172a pr SHPS1) important macrophage inhibitory receptor binds to CD47 prevents autologous phagocytosis.

d) Neutrophils
Neutrophils are said to activate xenogenic endothelium in invitro in the absence of XAbs or complement. Adhesion of human neutrophils to porcine endothelium under flow condition was shown to occur independently of αGal and ICAM-1. The role of neutrophils for graft rejection is a secondary phenomenon and this happens by specific direct recognition mechanisms.

Ways to overcome AXHR:
Costimulatory blockade with anti-CD154 monoclonal antibody (Mab) with a pharmacological agent inhibits the induced antibody response in many cases, although it does not suppress the baseline production of anti-Gal Nabs. This is however associated with thrombosis making clinical applicability problematic.

Induction of immunological tolerance in the donor would provide a reliable basis for long-term xenograft survival. Induction of tolerance is by mixed hematopoietic chimerism by stem cell transplantation which has been shown in experimental models of allotransplantation to provide robust T- and B-cell transplantation tolerance and these observations in experimental models have provided the rationale for clinical induction of allograft tolerance using hematopoietic stem cell grafts.
Elimation of antigen targeted by xenoreactive antibodies and this could be done by knocking out α1, 3-galactosyltransferase, which catalyses the synthesis of Galα1-3Gal, which has been shown to be the target of some of the antibodies that cause acute vascular rejection.

Accommodation is another way to overcome humoral responses. The graft endothelium appears to become resistant to the actions of antibody; this phenomenon may develop in pig-to-primate transplants. There are no definitive data to suggest that accommodation can develop in a pig-to-primate transplant.

A possible way to prevent NK cell mediated rejection of xenografts is generation of pigs deficient in porcine ULBP-1 but expressess transgene for HLA-E/b2 microglobulin. Transgene expression of HLA-E/b2 microglobulin by porcine endothelium cells suppresses human NK cell mediated IFN-γ secretion. CD40L blocking antibodies in non human primates effects NK cells.

Genetic manipulation of porcine cells for human CD47 expression could provide a novel approach for preventing macrophage derived mediated xenograft rejection.

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