Nuria Somoza

Interferon Expression in the Pancreases of Patients With Type I Diabetes

We have used a reverse transcriptase–polymerase chain reaction (RT-PCR) protocol to examine the expression of cytokines in the pancreases and islets of patients with type I diabetes. We detect a significant increase in the level of expression of interferon (IFN)-α in the pancreases of the diabetic patients as compared with the control pancreases. In contrast, IFN-β was detected at comparable levels in both groups, while IFN-γ was detected in three of four control pancreases and one of four pancreases from the diabetic individuals. The IFN-α cDNAs generated by the RT-PCR were cloned and sequenced to determine which α-subtypes were being expressed. We found that the repertoire of subtypes was quite limited in any one individual (diabetic or not), although each individual was different with respect to the pattern of subtypes expressed. We also examined these pancreases for the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-2, IL-4, and IL-6. We found no detectable expression of TNF-α or IL-2 in any pancreases, and the expression of the other cytokines was variable, with no pattern emerging from the comparison of the diabetic and nondiabetic individuals. We conclude that, of the cytokines examined, only IFN-α was significantly increased in the diabetic patients, a result that is consistent with the possibility that this cytokine is directly involved in the development of type I diabetes.

Evidence of expression of endotoxin receptors CD14, toll‐like receptors TLR4 and TLR2 and associated molecule MD‐2 and of sensitivity to endotoxin (LPS) in islet beta cells

CD14, a GPI‐linked membrane protein, is a component of the lipopolysaccharide (LPS) receptor complex, one of the pattern‐recognizing receptors (PRR) expressed by myeloid lineage cells. Here we report that CD14, the functionally linked toll‐like receptor molecules, TLR2 and TLR4, and the associated molecule MD‐2 are expressed in endocrine cells of the human pancreatic islets. CD14 expression in human pancreatic islets was determined by immunofluorescence staining of tissue sections and primary cultures, and confirmed by flow cytometry of dispersed normal islets and SV40‐transformed islet cells (HP62). The latter cells synthesized and secreted CD14 in response to lipopolysaccharide (LPS) in a time‐ and dose‐dependent manner. Reverse transcription polymerase chain reaction (RT‐PCR)‐Southern was positive for CD14, TLR2, TLR4 and MD‐2 in human pancreas, purified islets and HP62 cells. In vitro experiments using rat islets (also positive for CD14 by RT‐PCR) and HP62 cells showed that LPS regulates glucose‐dependent insulin secretion and induces inflammatory cytokines [interleukin (IL)‐1α, IL‐6 and tumour necrosis factor (TNF)‐α]. The functional expression of CD14 and associated molecules in islet β cells adds a new pathway that islet cells may follow to adjust their function to endotoxaemia situations and become vulnerable to the inflammatory events that occur during diabetogenic insulitis.

Endotoxin contamination may be responsible for the unexplained failure of human pancreatic islet transplantation.

Clinical transplantation of human islets has a disappointingly low rate of success. We report here the identification of a possible causative factor: endotoxin present in the collagenase preparations used to disperse the pancreatic tissue before islet purification and transplantation. Supporting evidence includes (1) detection of unexpectedly high levels of endotoxin in most collagenase solutions currently used to digest human pancreases; (2) demonstration that supernatants generated during islet separation are able to induce the inflammatory cytokines interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha (TNF-alpha) in macrophages; and (3) induction of IL-1, IL-6, and TNF-alpha in the islets during the separation procedure. Cytokine expression was assessed by reverse transcription-polymerase chain reaction and, for TNF-alpha, confirmed by enzyme-linked immunoabsorbent assay. It is proposed that endotoxin and locally induced cytokines carried over with the graft activate the endothelium and promote lymphomonocytic infiltration of grafted islets and surrounding liver tissue favoring primary nonfunction and early rejection. These results also have implications for the numerous experimental procedures that use collagenase, and they point to possible ways to improve islet preparation and transplantation protocols.

Adhesion Molecules in Human Islet β-cells: De Novo Induction of ICAM-1 but Not LFA-3

Understanding how T lymphocytes recognize β-cell autoantigens is essential forthe elucidation of the pathogenesis of insulin-dependent diabetes mellitus. The increased and ectopic expression of HLA class I and II molecules detected in human β-cells may facilitate this interaction. T-lymphocyte recognition of surface antigens also involves adhesion accessory molecules: intercellular adhesion molecule 1 (ICAM-1) and lymphocyte function—associated antigen 3 (LFA-3). These molecules not only allow cell contact but can also provide costimulatory signals for T-lymphocyte activation. Levels of ICAM-1 and LFA-3 expression in normal human islet cells and regulation of their expression by cytokines interferon-γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6 have been studied by two-color immunofluorescence staining of pancreatic cryostat sections and fluorescence-activated cell sorter analysis. Neither ICAM-1 nor LFA-3 could be demonstrated in sections or in fresh cell preparations, but after 18 h of culture, β-, α-, and δ-cells expressed spontaneously moderate levels of ICAM-1 (but not LFA-3). IFN-γ and TNF-α alone or in combination strongly enhanced this spontaneous expressionof ICAM-1 in a time- and/or dose-dependent and additive manner but had no effect on LFA-3. An SV40-transformed islet cell line showed high basal levels of both ICAM-1 and LFA-3, but the response to cytokines followed the same pattern as primary cultures.Theabsence of constitutive expression of adhesion molecules in β-cells supports the concept that they are immunologically silent cells and may also explain the failure of inducing autoimmune diabetes in transgenic mice by the sole expression of class II in theβ-cells via genetic manipulation. The induction of ICAM-1 by the same cytokines that can induce HLA class I and class II expression suggests that, in the diabetic pancreas, remaining β-cells probably express ICAM-1 concomitantly with HLA products.

Expression of Transporter Associated With Antigen Processing–1 in the Endocrine Cells of Human Pancreatic Islets: Effect of Cytokines and Evidence of Hyperexpression in IDDM

A possible role of transporter associated with antigen processing (TAP)-l in the pathogenesis of IDDM has been investigated by examining the level of TAP-1 expression in the islets of IDDM pancreas and by studying in vitro the effect of interferon (IFN)-γ, IFN-α, and tumor necrosis factor-α in TAP-1 expression by cultured islet cells. A remarkable hyperexpression of TAP-1 has been found in the endocrine cells (β and non-β) of IDDM islets, which constitutes first evidence of hyperexpression of this molecule in the target organ of an autoimmune disease. TAP-1 hyperexpression correlated clearly with HLA class I hyperexpression but only very partially with HLA class II ectopic expression. IFN-γ and IFN-α, both cytokines putatively implicated in IDDM pathogenesis, were capable of inducing TAP-1 protein (as assessed by immunofluorescence flow cytometry) and message (by Northern blot analysis and reverse transcription polymerase chain reaction). These findings suggest that under the influence of cytokines (most probably IFN-α) β-cells may express in their surface a high density of HLA class I–peptide complexes that may facilitate their recognition and lysis by low-affinity CD8+ T-cells.

Expression of glutamic acid decarboxylase (GAD) in the α, β and δ cells of normal and diabetic pancreas: implications for the pathogenesis of type I diabetes

One of the paradoxes of insulin‐dependent diabetes mellitus is that the destruction of the pancreatic islets’ endocrine cells is restricted to the insulin‐producing β cells, whereas the main autoantibodies, islet cell antibodies (ICA), are directed against all endocrine islet cells. GAD has recently been proposed as the main target of the humoral and cellular autoimmune attack to the islets, and since in rat pancreas this enzyme was expressed only in the β cells, this provided an explanation for the cell specificity of the destructive process. The finding of GAD‐positive cells in the islets of two diabetic patients, one of whom had completely lost the β cells, led us to study in detail thedistribution of GAD in normal human islet ceils using a panel of GAD aniiscra and the double indirect immunofluorescence technique on cryostat sections, monolayer cultures and cytosmears. The results showed that GAD is present not only in the cytoplasm of β cells but also in 69% of the α and 27% of the δ cells. GAD was not present, however, on the surface of the islet cells. These results suggest that the cellular distribution of GADcan not by itself explain the selectivity of β cell destruction in insulin‐dependent diabetes mellitus.

Endotoxin activity of collagenase and human islet transplantation

Vol 350 • August 30, 1997 641 which ranged from 10 to 26 times per day before treatment, decreased to 6–12 times per day (33%–58%), and this effect was detected as soon as the first day after treatment. Three patients were incontinent and became dry most days. Improvement was sustained up to 3 months, the longest follow-up available. A rise in maximum cystometric capacity (MCC) occurred in four of these patients, with maximum increment ranging from 76 mL to 596 mL (51% and 900% of pretreatment MCC, respectively). In a sixth patient, who had 18 mL of MCC and collected urine on pads, no clinical improvement occurred despite a continuous increase in MCC which reached 76 mL in one occasion (330% rise). The only patient (incontinent after transverse myelitis) who did not respond clinically or urodynamically to RTX had experienced a similar failure after capsaicin. We gave RTX (50 nmol/L) to two patients who have never received capsaicin. Both emptied their bladders by intermittent catheterisation but still leaked due to nonvoluntary bladder contractions. Again, discomfort evoked by RTX was minimum. In one patient, who was taking 5 mg oxybutynin twice daily without success, the addition of RTX brought continence on most days and increased MCC from 200 mL to 256 mL.

Advantages of using a cell separator and metrizamide gradients for human islet purification.

Human islet transplantation has a high rate of failure, often due to primary nonfunction, which suggests that islets are damaged during the processing of the pancreas. The preparation of human islets for transplantation is still a complex process that requires large teams of surgical and laboratory personnel. To overcome this problem, we have adopted the use of the IBM 2991 COBE cell separator and a metrizamide/Ficoll density medium that is easy to prepare. Twenty-seven pancreatic glands have been processed using the COBE cell separator, 23 of which were purified in metrizamide/Ficoll gradients and 4 in bovine serum albumin gradients. The results show an improvement of recovery and viability in these preparations when compared retrospectively with manual gradients. More importantly, the time required for purification was shortened to one fourth the usual time and total processing time is about half as long. Moreover, a team of two laboratory staff was regularly able to prepare islets for transplantation, reducing the separation time from 7 hr to 3.5 hr. We conclude that the automatic cell separator and metrizamide-based separation medium are useful modifications of current islet purification methods.

Reevaluation of Autoantibodies to Islet Cell Membrane in IDDM: Failure to Detect Islet Cell Surface Antibodies Using Human Islet Cells as Substrate

Since their demonstration in 1975, ICSAs have been proposed as serological markers and pathogenic elements in IDDM. ICSAs are detected in the sera of most newly diagnosed IDDM patients by indirect IFL that uses viable preparations of rat islet or insulinoma cells as substrate, but they also can be detected by using human insulinoma or fetal islet cells. We have tried to demonstrate ICSAs in the sera of 31 newly diagnosed diabetic patients, including 6 positive samples on human fetal islet cells, which used their natural target for the first time: normal human islet cells. In spite of using different types of preparations of these cells (i.e., freshly dispersed cell suspensions, monolayer cultures, or dispersed islets after culture), ICSAs could not be detected by IFL under the UV microscope, nor by flow cytometry. In contrast, 9 of 29 of the sera gave a positive staining on the RIN rat insulinoma cells. In an attempt to establish whether the putative ICSA autoantigen is present in the surface of human islet cells in the diabetic pancreas, the insulitis microenvironment was emulated by exposing the islets to three types of stress: 1) cytokines (IFN-γ and TNF-α); 2) heat shock; and 3) hyperglycemia. However, diabetic sera failed again to recognize membrane antigens on the islet cells after either of these treatments. Neither were islet cells from a newly diagnosed diabetic patient stained by its autologous serum (ICA titer >80 JDF U). These results suggest that ICSA autoantigen is not expressed in the membrane of human islet cells and therefore raises doubts about their proposed pathogenic role.

Biological safety in the storage and transport of biological specimens from patients with respiratory diseases used in research settings

Abstract Major advances in genomics and proteomics have prompted the creation of biological specimen collections and biobanks for use in biomedical research. These specimen collections and the wealth of data they generate will allow longitudinal studies to be conducted and subproducts such as DNA or RNA to be obtained. They may even be used in future studies. To ensure specimen integrity, from the outset it is necessary to define procedures for sampling, transport and storage, the subproducts to be obtained, and the end purpose, as well as to address biosafety issues and arrange for suitable equipment monitoring. Strict control of these conditions will confer added value on the specimens, as quality and traceability would be assured. This article aims to provide a general overview of the recommendations concerning biological safety, transport, and storage of biological specimens for biomedical research into respiratory diseases in accordance with current legislation.