**3.1 Genes can impact the effectiveness of islet graft**

The effectiveness of the islet graft depends both on beta cell function as well as the interaction between the graft and the host, and most importantly, these are governed by the expression of specific islet genes [27]. Components of specific cytokine pathways are upregulated in *bad* islet preparations (those which failed to reverse diabetes after transplantation). And these include tumor necrosis factor (TNF) machinery such as the TRAIL receptor TNFRSF10B that engage in β-cell death induced by T cells [28, 29]. The FAS and its ligand, FASL, can induce beta cell apoptosis [30, 31], and these are hiked in *bad* islets, suggesting that islet death-related pathways are already activated in these preparations even before transplantation. Adding on to apoptosis is the activation of NFκB and AP-1 transcription factors, which up-regulate expression of inflammatory cytokines [32]. A local proinflammatory environment is promoted by CCL2 (MCP1), which associates with islet death and diabetes [33, 34]. Also seen in b*ad* islets are higher expressions of the pattern recognition receptor TLR3, which relates with islet dysfunction and increased cytokine expression [35]. An elevated expression of tissue factor (F3) is pro-inflammatory and inhibits islet graft function [33, 36]. On the contrary, the TGFB2 and its receptor TGFBR1, and the IL13 receptor, OSMR, are other elevated chemokines which can initiate protective signals for islet cells [37–39]. Similarly, SERPINA3, also known as alpha-1-antichymotrypsin is upregulated and may promote wound healing [40, 41]. The SEPT9 gene is upregulated in bad islets and has recently been shown to be upregulated in islets of type 2 diabetics [42]. So it appears that the pathways leading to islet dysfunction are already triggered before transplantation, but that there is also the initiation of some counteractive measures.

A list of genes that were preferentially upregulated in *good* islet preparations (those which failed to reverse diabetes after transplantation) were relatable with the development and regeneration of pancreas. Hence a prior initiation of repair/ regeneration pathways in damaged islets would prove effective after transplantation. Several such genes including ONECUT1 (HNF6) [43, 44], MNX1 (HB9) [45, 46], NKX2-2 [47, 48], INSM1 [49, 50], NKX6-1 [47, 51], FOXA2 [52–54], and PTCH1 [55, 56] interact in regulatory networks aiding embryonic pancreas development and regeneration following an injury. On the other hand, the NOTCH2 gene, is predominantly expressed in the *bad* islet preparations, possibly because of its significant role in expansion of the progenitor cell population through suppression of neurogenin3-dependent endocrine cell differentiation [57]. Gene encoding the rectifying potassium channel KCNMA1 which is upregulated in good islets has been shown to be important for repolarization of the membrane following insulin secretion. Loss of KCNMA1 suppresses insulin secretion and increases susceptibility to oxidative stress and apoptosis [58].

In the recipient inducing protective genes like heme oxygenase-1 (HO-1), A20/ tumor necrosis factor alpha inducible protein3 (tnfaip3), biliverdin reductase (BVR), Bcl2, and others could synergistically improve islet graft survival and function. A similar effect is seen on administration of one or more of the products of HO-1 to the donor [59].

In heme degradation, HO-1 is the rate-limiting enzyme that produces equal molar amounts of carbon monoxide (CO), biliverdin, and iron. Biliverdin's rapid conversion by biliverdin reductase to bilirubin sequesters iron into ferritin. Being an ubiquitous stress protein, HO-1 gets induced in several cell types by various stimuli. Evidence

piles up supporting HO-1 induction to offer cellular protection against transplant rejection. Induction of HO-1 pharmacologically or via gene transfer protects islets from stress-induced apoptosis in both the *in vitro* and the *in vivo* settings. HO-1 induction in *β* cell lines, or human islets protects against apoptosis induced by TNF-*α* and cyclohexamide (CHX), interleukin-1*β* (IL-1*β*), and Fas. In recepients, HO-1 induction with cobalt protoporphyrin (CoPP) pharmacologically improves islet function in a rodent model with marginal mass islet transplantation, wherein fewer islets are required to achieve normoglycemia when transplanted into a sygeneic recipient, whose been rendered diabetic by streptozotocin (STZ) treatment [60].

A20, also known as the TNF-*α*-induced protein 3 (TNFAIP3), is a zinc-ring finger protein. As a negative regulator of nuclear factor kappa B (NF-*κ*B) activation, A20 is recognized as a central and ubiquitous regulator of inflammation and as a potent antiapoptotic gene in certain cell types, including *β* cells. Islets can be protected against apoptosis induced by IL-1*β*/INF-*γ* and Fas through adenovirus-mediated gene transfer causing overexpression of A20. A higher percentage of cure was seen after transplantation in recipients with suboptimal number of islets overexpressing A20 compared to control islets. Islets expressing A20 preserved functional *β* cell mass and are resistant to cell death. In *β* cells, expression of A20 renders a dual-protective effect through antiapoptosis and antiinflammation. The antiapototic effect of A20 attributes to its cytoprotective properties and is dependent on the abrogation of cytokine-induced NO (nitric oxide) production due to transcriptional blockade of iNOS induction [61–63].

Bilirubin administration reduced apoptosis and improved insulin secretion in an *in vitro* model in INS-1 cells when challenged with nonspecific inflammation induced by cytokines. Protective genes like HO-1 and bcl-2 were strongly expressed seen in in freshly isolated islets from bilirubin-treated donors. Also noticed was an evident suppression in the proinflammatory and proapoptotic genes including caspase-3, caspase-8, and MCP-1. Such a protective effect rendered by bilirubin reduces *β*-cell destruction post- transplantation, minimizes macrophage infiltration, and suppresses expression of MCP-1, BID, caspase-3, -8, and -9, TNF-*α*, iNOS, Fas, TRAIL-R, and CXCL10 in the graft after allogeneic transplantation [64, 65].

Exposing human islets to the nonpeptidyl low molecular weight radical scavenger IAC [bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidiny) decanedioate dihydrochloride] on isolated human islet cells protected them from isolation and culture-induced oxidative stress. Transduction of NOD islets with the antioxidative gene thioredoxin (TRX, reactive oxygen species scavenger and antiapoptotic) using a lentiviral vector before transplantation prolonged islet graft survival. Anthocyanins present in Chinese Bayberry have the potential to upregulate HO-1, and thus protect *β* cells against hydrogen-peroxide-induced necrosis and apoptosis. Islets' viability and function improved with adenoviral transfection as X-linked inhibitor of apoptosis provided protection from inflammatory cytokines [66].
