Allele-specific and peptide-dependent recognition of swine leukocyte antigen class I by human cytotoxic T-cell clones.

BACKGROUND: The T-cell mediated immune responses play a major role in xenograft rejection. However, the mechanisms behind human T-cell recognition of porcine xenoantigens remain to be elucidated. METHODS: Human CD8+ T-cell lines were generated against porcine aortic endothelial cells (PAECs) from y/y and z/z haplotypes of Yucatan inbred swine. T-cell clones were obtained by limiting dilution. The human T-cell receptor (TCR)-swine leukocyte antigen (SLA) class I interaction was characterized. RESULTS: The human CD8+ T-cell mediated direct recognition of PAECs was SLA haplotype-specific. The haplotype specificity was restricted by the SLA class I allelic polymorphism. To characterize the role of SLA-bound peptides in the human TCR-SLA class I interaction, we stripped peptides from SLA molecules by a brief acid treatment. Using z/z-specific CD8+ T cells as effectors, we demonstrated that the acid-treatment, which stripped SLA molecules of bound peptides, decreased the lysis of PAECs by 72%. Addition of peptides eluted from affinity purified z/z SLA class I molecules, but not from the irrelevant y/y SLA class I, restored the lysis of acid-treated z/z PAECs. In addition, the lysis of a human HLA class I negative cell line, 721.221, transfected with a relevant SLA class I allele derived from the z/z haplotype, was significantly increased with the addition of relevant z/z peptides. These experiments indicated that both SLA class I and bound peptides were required for recognition by human CD8+ T cells. Cloning studies identified two groups of xenoreactive T-cell clones. Group I clones recognized distinct porcine peptides in the context of SLA class I molecules, whereas group II clones recognized human endogenous cross-reactive peptides presented by SLA class I. CONCLUSIONS: Our results demonstrated that, despite the differences in MHC molecules between species, human T-cell recognition of porcine MHC is similar to direct allo-recognition, that is, human TCR recognizes xenogeneic SLA-peptide complexes.     

Engraftment of Cells from Porcine Islets of Langerhans and Normalization of Glucose Tolerance Following Transplantation of Pig Pancreatic Primordia in Nonimmune-Suppressed Diabetic Rats

Transplantation therapy for human diabetes is limited by the toxicity of immunosuppressive drugs. However, even if toxicity can be minimalized, there will still be a shortage of human donor organs. Xenotransplantation of porcine islets may be a strategy to overcome these supply problems. Xenotransplantation in mesentery of pig pancreatic primordia obtained very early during organogenesis [embryonic day 28 (E28)] can obviate the need for immune suppression in rats or rhesus macaques. Here, in rats transplanted previously with E28 pig pancreatic primordia in the mesentery, we show normalization of glucose tolerance in nonimmune-suppressed streptozotocin-diabetic LEW rats and insulin and porcine proinsulin mRNA-expressing cell engraftment in the kidney following implantation of porcine islets beneath the renal capsule. Donor cell engraftment was confirmed using fluorescent in situ hybridization for the porcine X chromosome and electron microscopy. In contrast, cells from islets did not engraft in the kidney without prior transplantation of E28 pig pancreatic primordia in the mesentery. This is the first report of prolonged engraftment and sustained normalization of glucose tolerance following transplantation of porcine islets in nonimmune-suppressed, immune-competent rodents. The data are consistent with tolerance induction to a cell component of porcine islets induced by previous transplantation of E28 pig pancreatic primordia.In that pigs are plentiful and because porcine insulin works well in humans, the pig has been suggested to be a pancreas organ donor for humans with diabetes. The severity of humoral rejection due to pre-existing natural antibodies effectively precludes their use as whole pancreas donors in nonhuman primates or humans.^1,2,3,4 However, isolated islets of Langerhans (islets) can be transplanted into nonhuman primates^2,3 or humans with diabetes¹ without initiating humoral rejection. Unfortunately, recent experience with pig to primate islet² or neonatal islet³ transplantation shows that sustained insulin independence can be achieved, but only through the use of immune suppressive agents that are not approved for human use or would result in an unacceptable level of morbidity in humans.^2,3We have shown that glucose tolerance can be normalized in streptozotocin (STZ)-diabetic (type 1) LEW rats or ZDF (type 2) diabetic rats within 4 weeks following transplantation in mesentery of pig pancreatic primordia obtained very early during embryogenesis [on embryonic day 28 (E28)—just after the organ differentiates and before the time dorsal and ventral anlagen fuse] without host immune suppression.^5,6,7,8 No rat insulin can be detected in STZ-treated rats. Porcine insulin circulates posttransplantation of E28 pig pancreatic primordia (embryonic pancreas) and levels increase after a glucose load.^6,7 Cells expressing insulin and porcine proinsulin mRNA with β cell morphology engraft in host mesentery, mesenteric lymph nodes, liver, and pancreas posttransplantation.^5,6,7,8 Cells originating from E28 pig pancreatic primordia engraft similarly in nonimmune-suppressed STZ-diabetic rhesus macaques.⁹Long-term engraftment of cells originating from E28 pig pancreatic primordia in non-immune suppressed immune competent hosts reflects tolerance to a cellular component present in the primordia or that differentiates in situ following implantation.^5,6,7,8,9 To ascertain whether tolerance might extend to the same or similar cell component present in porcine islets from adult swine (adult islets), we implanted adult porcine islets beneath the renal capsule of rats that previously had been transplanted with E28 pig pancreatic primordia in mesentery but remained glucose intolerant. Intact porcine islets do not engraft following renal subcapsular implantation. However, a population of cells originating from donor islets with β cell morphology that express insulin and porcine proinsulin mRNA engraft in kidneys of rats transplanted previously with E28 pig pancreatic primordia. Glucose tolerance is normalized in these animals. Our observations are consistent with induction of tolerance to a cell component of adult porcine islets by previous transplantation of E28 pig pancreatic primordia in rats.     

Hypothyroidism in the developing rat brain is associated with marked oxidative stress and aberrant intraneuronal accumulation of neurofilaments.

The effects of hypothyroidism on parameters of oxidative stress and on intraneuronal distribution of neurofilaments have been investigated in the developing rat brain. Progressive hypothyroidism during the first 4 weeks of postnatal development led to an increase in superoxide dismutase and catalase activity, decline in the level of glutathione and mitochondrial cytochrome c oxidase activity and increase in the level of .OH radical along with enhanced protein carbonylation and lipid peroxidation. Immunocytochemical staining of cryostat sections of normal and hypothyroid cerebella from 25 day postnatal rats with anti neurofilament (NF) light chain (L) antibody showed aberrant accumulation of neurofilaments in the perikaryon of the hypothyroid Purkinje neurons in contrast to relatively uniform distribution in the controls. The morphological and biochemical alterations in the neurons of the developing hypothyroid brain are comparable to those seen in several neurodegenerative diseases.     

Characterization of a monoclonal antibody, KR-P8, that detects a new prostate-specific marker

This report characterizes a prostate-specific monoclonal antibody, KP-P8, which was prepared against the human prostate cell line PC3. The antigen detected by KR-P8 was identified on the surfaces of 90% of cells of the PC3 line, as well as on 67% of cells of the Du-145 prostate line, but it was absent from the surfaces of normal peripheral blood leukocytes and cells of a number of lymphoblastoid lines. As judged by immunoperoxidase staining techniques, KR-P8 reacted with the glandular epithelium of all specimens of normal, benign hypertrophic, and malignant prostate glands tested. However, no reactivity was noted with numerous other human tissues including normal bladder, lung, liver, kidney, testis, colon, parotid gland, thyroid gland, and spleen. These results indicate that the antigen detected by KR-P8 is prostate organ-specific. Competitive blocking studies showed that the antibody did not recognize the previously described prostate-specific antigen or the alpha-Pro-3 antigen described by other investigators. The KR-P8 antibody also did not bind to purified prostatic acid phosphatase. The presence of the KR-P8 antigen was demonstrated in cell-free preparations of dilute seminal plasma by radioimmunoassay, indicating that this antigen is secreted by the glandular cells of the prostate gland. The clinical significance of this marker was demonstrated by its ability to identify prostate metastases of the lymph node.     

Development of immune response to tissue‐restricted self‐antigens in simultaneous kidney‐pancreas transplant recipients with acute rejection

Simultaneous kidney‐pancreas transplantation (SKP Tx) is a treatment for end‐stage kidney disease secondary to diabetes mellitus. We investigated the role of immune responses to donor human leukocyte antigens (HLA) and tissue‐restricted kidney and pancreas self‐antigens (KSAgs and PSAgs, respectively) in SKP Tx recipients (SKP TxRs). Sera collected from 39 SKP TxRs were used to determine de novo Abs specific for KSAgs (collagen‐IV, Col‐IV; fibronectin, FN) and PSAgs (insulin, islet cells, glutamic acid decarboxylase, and pancreas‐associated protein‐1) by ELISA. KSAg‐specific IFN‐γ, IL‐17, and IL‐10 cytokines were enumerated by ELISpot. Abs to donor HLA classes I and II were determined by Luminex assay. Abs to KSAgs and PSAgs were detectable in recipients with rejection compared with stable recipients (P<.05). Kidney‐only rejection recipients had increased Abs against KSAgs compared with stable (P<.05), with no increase in Abs against PSAgs. Pancreas‐only rejection recipients showed increased Abs against PSAgs compared to stable (P<.05), with no Abs against KSAgs. SKP TxRs with rejection showed increased frequencies of KSAg‐specific IFN‐γ and IL‐17 with reduction in IL‐10‐secreting cells. SKP TxRs with rejection developed Abs to KSAgs and PSAgs demonstrated increased frequencies of kidney or pancreas SAg‐specific IFN‐γ and IL‐17‐secreting cells with reduced IL‐10, suggesting loss of peripheral tolerance to SAgs.     

Protective role of bortezomib in steatotic liver ischemia/reperfusion injury through abrogation of MMP activation and YKL-40 expression

Steatotic liver grafts tolerate ischemia–reperfusion (I/R) injury poorly, contributing to poor survival following transplantation. However the molecular mechanisms leading to I/R injury still remain to be defined. We have previously reported that the protective effect of bortezomib towards inhibiting cold induced I/R injury in obese rat liver transplant model is through NF-κB down modulation. In this report using an orthotopic liver transplant (OLT) model in Zucker rats (from obese, leptin deficient donor, to lean recipient) we defined the mechanisms of steatotic liver injury, and characterized the role of bortezomib in inhibiting MMP activation and YKL-40, both of which are involved in extracellular matrix deposition and fibrosis, the key pathological features of liver allograft failure. Obese donor rats were treated with bortezomib (i.v., 0.1 mg/kg immediately prior to liver procurement) to assess the role of MMP and YKL-40 in steatotic liver I/R injury. I/R injury in steatotic livers resulted in significant increases in expression of YKL-40 (9 fold), and activation of MMP-2 (15 fold)/MMP-9 (12 fold). Bortezomib treatment reduced the expression of YKL-40 and MMP to basal levels. Bortezomib also inhibited the pro-fibrotic (VEGF, HGF, bFGF, TGF-β) and pro-inflammatory (IL-1β, TNF-α and IFN-γ) cytokines significantly in comparison to untreated animals with I/R injury. These results demonstrate that I/R injury in steatotic livers following transplantation are associated with MMP activation and YKL-40 upregulation resulting in pro-fibrotic and pro-inflammatory cytokine release. Administration of the proteosomal inhibitor, bortezomib, effectively attenuated the I/R injury by inhibiting MMP and YKL-40 expression and therefore support the clinical utility of this drug in donor management for preventing I/R injury and its sequelae.     

Newer trends in tubercular uveitis: a case series with systemic correlation

AIM: To determine the spectrum of uveitis, causes of visual loss in systemic tuberculosis, role of investigations and outcome after anti-tuberculosis treatment (ATT). METHODS: A retrospective study was conducted on 250 patients with systemic tuberculosis at a referral center in Chennai, South India from April 2016 to May 2019. Systemic workup comprised of Mantoux, chest X-ray, polymerase chain reaction (PCR) and QuantiFERON (QFT) TB Gold. Aqueous humor analysis by nested PCR or real time PCR (RT-PCR) and ancillary ophthalmic investigations such as fundus fluorescein angiography, optical coherence tomography were performed. RESULTS: Multifocal choroiditis and vasculitis were the most common manifestations and had a higher risk of recurrence. Pulmonary tuberculosis was more frequently associated with uveitis. Among those with extra-pulmonary tuberculosis miliary, bone and abdominal tuberculosis had uveitis. Complications such as cystoid macular edema, choroidal neovascular membranes and macular scarring caused visual loss. Aqueous humor analysis detected mycobacterium tuberculosis antigen. Collectively, systemic investigations such as chest X-ray, Mantoux test and those performed on blood samples such as PCR and QFT were positive in 39% of patients. In inconclusive patients, nested PCR and/or RT-PCR were done on aqueous humor samples and was diagnostic in 96%. A combination of tests was diagnostic in 92%. ATT in isolation in 71% and combined with corticosteroids in 29% was used for treatment of which signs of resolution and improvement in vision started as early as 6weeks in those who were started immediately on CS and ATT and longer than 3mo in those on ATT alone. Vision improved in 69%. Complete resolution occurred in 75% and worsening in 12%. CONCLUSION: A combination of investigations guided by clinical suspicion helps in precise diagnosis. In diagnostic dilemmas analysis of ocular samples is reliable and confirmatory. Prompt treatment with ATT and corticosteroids improved vision in 23% of our patients within 2mo. Vitritis with choroiditis causes cystoid macular oedema and requires longer duration of ATT. Screening all patients and a multidisciplinary approach in tuberculosis (active, healed or during treatment) is recommended.