Australian experience with total pancreatectomy with islet autotransplantation to treat chronic pancreatitis.

BACKGROUND: This study aimed to describe the clinical outcomes of total pancreatectomy with islet autotransplantation (TP-IAT) in Australia. METHODS: Individuals selected for TP-IAT surgery according to the Minnesota Criteria (Appendix) without evidence of diabetes were evaluated including time to transplantation from pancreatectomy, islet numbers infused and post-transplantation HbA1c, C-peptide, total daily insulin and analgesic requirement. RESULTS: Sixteen individuals underwent TP-IAT from Australia and New Zealand between 2010 and 2020. Two recipients are deceased. The median islet equivalents/kg infused was 4244 (interquartile range (IQR) 2290-7300). The median C-peptide 1 month post-TP-IAT was 384 (IQR 210-579) pmol/L and at median 29.5 (IQR 14.5-46.5) months from transplant was 395 (IQR 139-862) pmol/L. Insulin independence was achieved in eight of 15 (53.3%) surviving recipients. A higher islet equivalents transplanted was most strongly associated with the likelihood of insulin independence (P < 0.05). Of the 15 surviving recipients, 14 demonstrated substantial reduction in analgesic requirement. CONCLUSION: The TP-IAT programme in Australia has been a successful new therapy for the management of individuals with chronic pancreatitis including hereditary forms refractory to medical treatment to improve pain management with 50% insulin independence rates.

IGF-2 coated porous collagen microwells for the culture of pancreatic islets.

Islet transplantation, the only curative therapy for type I diabetes, requires isolation of the graft in highly specialized facilities for its later dispatch to remote transplantation centres. During transport and culture, many valuable cells are lost due to several factors such as mechanical stress, islet aggregation and dissociation. Here, we evaluate a porous microwell array sheet made of natural collagen type I extracellular matrix (ECM) protein as a novel islet culture substrate. This culture platform can be coated with IGF-2, a growth factor favorable for islet survival, and allows segregation of the islets within the porous microwell sheet, preventing aggregation. This design shows promising results for improving human pancreatic islets viability and function during culture and could form a novel paradigm for the transport of islets between isolation and transplantation centres.

Multicenter Australian trial of islet transplantation: improving accessibility and outcomes.

Whilst initial rates of insulin independence following islet transplantation are encouraging, long-term function using the Edmonton Protocol remains a concern. The aim of this single-arm, multicenter study was to evaluate an immunosuppressive protocol of initial antithymocyte globulin (ATG), tacrolimus and mycophenolate mofetil (MMF) followed by switching to sirolimus and MMF. Islets were cultured for 24 h prior to transplantation. The primary end-point was an HbA1c of <7% and cessation of severe hypoglycemia. Seventeen recipients were followed for ≥ 12 months. Nine islet preparations were transported interstate for transplantation. Similar outcomes were achieved at all three centers. Fourteen of the 17 (82%) recipients achieved the primary end-point. Nine (53%) recipients achieved insulin independence for a median of 26 months (range 7-39 months) and 6 (35%) remain insulin independent. All recipients were C-peptide positive for at least 3 months. All subjects with unstimulated C-peptide >0.2 nmol/L had cessation of severe hypoglycemia. Nine of the 17 recipients tolerated switching from tacrolimus to sirolimus with similar graft outcomes. There was a small but significant reduction in renal function in the first 12 months. The combination of islet culture, ATG, tacrolimus and MMF is a viable alternative for islet transplantation.

Survival of macroencapsulated allogeneic parathyroid tissue one year after transplantation in nonimmunosuppressed humans.

The use of immunoisolation devices may allow transplantation without need for immunosuppression and could widen the indications for cell transplantation. In this study, we evaluated the survival of encapsulated parathyroid tissue in nonimmunosuppressed humans. Autologous parathyroid implants: Seven patients undergoing parathyroidectomy had devices containing small pieces of their own parathyroid tissue implanted SC. These devices were explanted after 2-4 weeks for histological evaluation. Allogeneic parathyroid implants: Four patients with chronic hypoparathyroidism were transplanted with one to three large (40 microl) and one small (4.5 microl) device filled with meshed parathyroid tissue and implanted SC. The small devices were explanted at 4 weeks, while the large ones were explanted 8.5 to 14 months after implantation. In both studies, control implants were placed in nude mice. Autologous study results: At explantation, the grafts consisted of 22 +/- 6% endocrine tissue and 63 +/- 7% fibrosis, while 15 +/- 5% of the grafts were necrotic. Allogeneic study results: In devices explanted from the patients at 4 weeks, fibrosis dominated and only 1%, 5%, and 23% of the grafts consisted of endocrine tissue. A similar histological appearance was found in grafts from nude mice. In devices explanted at 8.5-14 months, histologically intact endocrine tissue was found in all patients. However, nearly all the tissue consisted of fibrosis. There was no detectable increase in the parathormone (PTH) level in all patients. Macroencapsulated human allogeneic parathyroid tissue can survive up to 1 year after transplantation into nonimmunosuppressed patients. However, marked fibroblast overgrowth occurred, especially in the allogeneic implant study, using meshed parathyroid tissue. This was probably not related to the allo-response, because similar findings were observed in the nude mouse implants. In future studies, better tissue preparation and improvements in the physiological milieu inside the device may help to reduce fibroblast overgrowth and increase survival of the parathyroid cells.

In vivo delivery of recombinant human growth hormone from genetically engineered human fibroblasts implanted within Baxter immunoisolation devices.

Continuous delivery of therapeutic peptide to the systemic circulation would be the optimal treatment for a variety of diseases. The Baxter TheraCyte system is a membrane encapsulation system developed for implantation of tissues, cells such as endocrine cells or cell lines genetically engineered for therapeutic peptide delivery in vivo. To demonstrate the utility of this system, cell lines were developed which expressed human growth hormone (hGH) at levels exceeding 1 microgram per million cells per day. These were loaded into devices which were then implanted into juvenile nude rats. Significant levels of hGH of up to 2.5 ng/ml were detected in plasma throughout the six month duration of the study. In contrast, animals implanted with free cells showed peak plasma levels of 0.5 to 1.2 ng four days after implantation with no detectable hGH beyond 10 days. Histological examination of explanted devices showed they were vascularized and contained cells that were viable and morphologically healthy. After removal of the implants, no hGH could be detected which confirmed that the source of hGH was from cells contained within the device. The long term expression of human growth hormone as a model peptide has implications for the peptide therapies for a variety of human diseases using membrane encapsulated cells.

Correction of diabetic nod mice with insulinomas implanted within Baxter immunoisolation devices.

Insulin replacement by injection is clearly not a cure for Insulin Dependent Diabetes Mellitus (IDDM). Replacement of the destroyed islets by pancreas or islet allograft transplantation can achieve the good metabolic control required to prevent diabetic complications, but tissue supply is limited. The problem of islet supply to treat the 1 million IDDM patients in the USA could be overcome by using immortalized islet beta-cells as a donor source. However, before either allogeneic or xenogeneic immortalized beta-cells are used, some major problems have to be overcome: control of immortalized cell growth, allograft or xenograft rejection and recurrence of autoimmunity. To tackle these problems we have used a cell impermeable immunoisolation device containing mouse insulinoma cells. Transplantation of devices with insulinomas from NOD mice carrying the Rat-insulin promoter regulated SV40 T-Antigen transgene (RIP-TAg), normalized the blood glucose levels of diabetic NOD mice. Insulinomas from allogeneic CBA/NOD-RIP-TAg mice were also capable of normalizing diabetic NOD mice. Not only were non-fasting blood glucoses normalized but when given an intraperitoneal injection of glucose, the corrected mice had a near normal clearance of glucose from the blood. When the devices were removed from normalized mice they became diabetic again, demonstrating that the immunoisolation device was capable of protecting against both alloimmune and autoimmune destruction. The results with allogeneic mouse beta-cells suggest the possibility that immortalized human beta-cells could be an effective source of tissue to correct diabetes in IDDM patients without the use of immunosuppression.

CD4+ T cell mediated destruction of xenografts within cell-impermeable membranes in the absence of CD8+ T cells and B cells.

Xenogeneic cells encapsulated in cell-impermeable diffusion chambers die within 3 weeks when implanted into immunocompetent animals but not when implanted into immunodeficient animals. To determine which cells are necessary for this observation, we depleted normal mice in vivo of either CD4+ or CD8+ T cells using monoclonal antibodies. We also reconstituted the immune system of athymic CBA mice (T-lymphocyte deficient) and C.B17 SCID mice (T- and B-lymphocyte deficient) with different cell subsets from normal CBA and BALB/C mice, respectively. Depleted or reconstituted mice were implanted with a diffusion chamber containing COS (monkey kidney) cells. Membrane enclosed xenografts survived in CD4+ T cell depleted mice but not in CD8+ T cell depleted or nondepleted control mice. Encapsulated xenografts survived when implanted into either athymic or SCID mice but were destroyed in reconstituted athymic and SCID mice. Furthermore, encapsulated xenogeneic cells were destroyed in athymic or SCID mice reconstituted with CD4+ cell preparations depleted of CD8+ cells and/or B cells. In contrast, encapsulated xenogeneic cells were not destroyed in athymic or SCID mice reconstituted with CD8+ cell preparations depleted of CD4+ cells. These studies highlight the critical role of CD4+ T cells, in the absence of CD8+ cells and B cells, in the processes leading to the ultimate destruction of encapsulated xenografts. Because of the use of cell-impermeable membranes in these studies, the most likely involvement of CD4+ T cells is in the indirect antigen recognition by these cells and subsequent stimulation of inflammatory cells.

Reactivity to human islets and fetal pig proislets by peripheral blood mononuclear cells from subjects with preclinical and clinical insulin-dependent diabetes.

A simple, direct assay for T-lymphocyte reactivity to islet antigen(s) in human insulin-dependent diabetes mellitus (IDDM) should facilitate preclinical diagnosis and the evaluation of intervention therapy to avert autoimmune-mediated beta-cell destruction. In subjects with preclinical or clinical IDDM, we measured the reactivity of peripheral blood mononuclear cells (PBMCs) incubated over 6 days with either adult human islets or fetal pig proislets, or other fetal pig tissues, and with human insulin. With islets, the stimulation index (SI) of [3H]thymidine uptake by PBMCs exceeded the mean + 2SD of control subjects in 6 of 6 preclinical subjects (SI 8.7 +/- 3.7), 7 of 11 clinical subjects (SI 5.2 +/- 3.4), and 1 of 12 control subjects (SI 2.7 +/- 1.7); with insulin, the responses were less in frequency and magnitude, being 4 of 6 (2.7 +/- 1.6), 3 of 11 (2.2 +/- 1.1), and 0 of 12 (1.20 +/- 0.55), respectively. The mean responses to islets of PBMCs from preclinical and clinical subjects differed significantly from control subjects (P less than 0.02 by 2-tailed Kruskal-Wallis test). Secretion of granulocyte macrophage colony-stimulating factor by PBMCs over 6 days was assayed in the preclinical group and generally paralleled the uptake of [3H]thymidine. PBMC reactivity to islets appeared to be at least as sensitive a marker of preclinical IDDM as autoantibodies to a 64,000-Mr protein, presumably the enzyme glutamic acid decarboxylase, in fetal pig proislets. In conclusion, islet-reactive T lymphocytes in subjects with preclinical and clinical IDDM can be identified in bulk culture of PBMCs.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic susceptibility of chicken macrophages to in vitro infection with infectious laryngotracheitis virus.

Chicken macrophages are susceptible to infection with infectious laryngotracheitis virus (ILTV), the level of infection being dependent, in part, on the genotype of the host cell. Following infection in vitro a greater proportion of macrophages from the ILTV-resistant J1(B113/113) and N1(B114/114) inbred lines of chickens were found to be positive for ILTV antigens, than macrophages from the ILTV-susceptible M1(B15/15) chickens. The proportion of ILTV-positive macrophages was found to be genetically regulated, in part by the chicken major histocompatibility complex (MHC), although alloantisera to class I and class II MHC antigens did not reduce the number of macrophages infected. Similarly, the phagocytic activity of the cultured macrophages from chickens of different MHC genotype did not correlate with their susceptibility to infection with ILTV.

Genetic resistance to infectious laryngotracheitis in inbred lines of White Leghorn chickens.

The susceptibility of inbred lines of chickens to graded levels of infection with a virulent isolate (wild-strain) of infectious laryngotracheitis virus (ILTV) was investigated. An association was found between the level of resistance to ILTV and the line of inbred chickens, with chickens from the J1 inbred line being more resistant to clinical ILTV than M1 and N1 inbred chickens. The N1 chickens, however, became susceptible with higher doses of ILTV, while J1 chickens remained relatively resistant to clinical disease. Studies using other inbred lines of chicken which had the same major histocompatibility complex (MHC) antigens showed similar levels of resistance or susceptibility, suggesting a possible association of the chicken MHC with resistance or susceptibility to ILTV.

The major histocompatibility complex and genetic control of antibody response to sheep red blood cells in chickens.

The genetic association of the B-F/B-L region of the chicken major histocompatibility complex (MHC) with the antibody response to sheep red blood cells (SRBC) was investigated using three inbred lines of White Leghorn chickens. Two out of the three inbred lines of chickens. J1 and N1, were found to be a low responder to SRBC. Planned matings between the three inbred lines of chickens showed that the genetic regulation of the magnitude of the antibody response to SRBC was associated with the MHC. The low antibody response to SRBC was inherited as a recessive trait linked to the B15 MHC phenotype of the M1 inbred line.

The major histocompatibility complex and genetic control of antibody response to synthetic antigens in chickens.

The genetic association of the B-F/B-L region of the chicken major histocompatibility complex (MHC) with the antibody responses to the hapten 2,4-dinitrophenol (DNP) conjugated to human gamma globulin and to the synthetic antigens, GAT and (T,G)-A-L were investigated using five inbred lines of White Leghorn chickens. Two of the five inbred lines of chickens J1 and M1, were found to be high responders to DNP and (T,G)-A-L, whilst L1, M4 and N1 chickens were found to be relatively low responders to both antigens. Chickens from all five inbred lines were low responders to GAT. Matings between the inbred lines. J1, M1 and N(1), showed that the magnitude of the antibody response to (T,G)-A-L in backcross chickens was regulated by a dominant gene or set of genes linked to the MHC. High antibody responsiveness to (T,G)-A-L was inherited as a dominant trait linked to the B113 and the B15 MHC alleles of the J1 and M1 inbred lines respectively. The similarity of the anti-(T,G)-A-L responses in chickens from the three B113 homozygous inbred lines L1, M4 and N1, also suggests that control of this response was linked to the MHC. The magnitude of the antibody response to DNP in backcross chickens from matings between the inbred lines N1 and J1 or M1 was also found to be regulated by a gene or set of genes linked to the MHC. Again, high antibody responsiveness to DNP was inherited as a dominant trait linked to the B113 and the B15 MHC alleles of the J1 and M1 inbred lines respectively.

The chicken major histocompatibility complex and resistance to Rous sarcoma virus tumours.

Chickens from an Australorp line which had two major histocompatibility complex (MHC) alleles, B3A and B(9A) , segregating within the line, were inoculated with Rous sarcoma virus and the resulting tumours were monitored over time. In the majority of chickens possessing the B8A allele, the developing tumours regressed, while in all chickens without B8A allele (i.e. B 9A/9A homozygotes) the tumours progressed and eventually led to their death.