Teresa Rodriguez-Calvo

Increased immune cell infiltration of the exocrine pancreas: a possible contribution to the pathogenesis of type 1 diabetes

Type 1 diabetes (T1D) results from a complex interplay between genetic susceptibility and environmental factors that have been implicated in the pathogenesis of disease both as triggers and potentiators of β-cell destruction. CD8 T cells are the main cell type found in human islets, and they have been shown in vitro to be capable of killing β-cells overexpressing MHC class I. In this study, we report that CD8 T cells infiltrate the exocrine pancreas of diabetic subjects in high numbers and not only endocrine areas. T1D subjects present significantly higher CD8 T cell density in the exocrine tissue without the presence of prominent insulitis. Even T1D donors without remaining insulin-containing islets and long disease duration show elevated levels of CD8 T cells in the exocrine compartment. In addition, higher numbers of CD4+ and CD11c+ cells were found in the exocrine tissue. Preliminary data in type 2 diabetic (T2D) subjects indicate that overall, there might be a spontaneous inflammatory infiltration of the exocrine tissue, common to both T1D and T2D subjects. Our study provides the first information on the precise tissue distribution of CD8 T cells in pancreata from T1D, T2D, autoantibody-positive, and healthy control subjects.

Establishment of a bluetongue virus infection model in mice that are deficient in the alpha/beta interferon receptor.

Bluetongue (BT) is a noncontagious, insect-transmitted disease of ruminants caused by the bluetongue virus (BTV). A laboratory animal model would greatly facilitate the studies of pathogenesis, immune response and vaccination against BTV. Herein, we show that adult mice deficient in type I IFN receptor (IFNAR(−/−)) are highly susceptible to BTV-4 and BTV-8 infection when the virus is administered intravenously. Disease was characterized by ocular discharges and apathy, starting at 48 hours post-infection and quickly leading to animal death within 60 hours of inoculation. Infectious virus was recovered from the spleen, lung, thymus, and lymph nodes indicating a systemic infection. In addition, a lymphoid depletion in spleen, and severe pneumonia were observed in the infected mice. Furthermore, IFNAR(−/−) adult mice immunized with a BTV-4 inactivated vaccine showed the induction of neutralizing antibodies against BTV-4 and complete protection against challenge with a lethal dose of this virus. The data indicate that this mouse model may facilitate the study of BTV pathogenesis, and the development of new effective vaccines for BTV.

Heterologous prime boost vaccination with DNA and recombinant modified vaccinia virus Ankara protects IFNAR(-/-) mice against lethal bluetongue infection.

Recent recombinant DNA technology has provided novel approaches to develop marker and safe vaccines against bluetongue virus (BTV). To develop new vaccination strategies against BTV infection we have engineered naked DNAs and recombinant modified vaccinia virus Ankara (rMVA) expressing VP2, VP5 and VP7 proteins from BTV-4. IFNAR(-/-) mice inoculated with DNA/rMVA-VP2, -VP5, -VP7 in an heterologous prime boost vaccination strategy generated significant levels of neutralizing antibodies against BTV-4 and they were completely protected against BTV-4 challenge. Interestingly, VP2 and VP7 proteins expressed in the DNA/rMVA vaccines induced a specific BTV T-cell response that might contribute to the protection of IFNAR(-/-) mice against challenge with BTV-4. In addition, antibodies against VP2, VP5, and VP7, but not NS3 were detected in the sera of DNA/rMVA-VP2, -VP5, -VP7 immunized mice confirming the DIVA (differentiating infected from vaccinated animals) properties of this vaccine. Overall, our results show that the heterologous prime boost vaccination with DNA/rMVA expressing VP2, VP5, and VP7 proteins protects against BTV-4 infection.

Immunosuppression during Acute Infection with Foot-and-Mouth Disease Virus in Swine Is Mediated by IL-10

Foot-and-mouth disease virus (FMDV) is one of the most contagious animal viruses, causing a devastating disease in cloven-hoofed animals with enormous economic consequences. Identification of the different parameters involved in the immune response elicited against FMDV remains unclear, and it is fundamental the understanding of such parameters before effective control measures can be put in place. In the present study, we show that interleukin-10 (IL-10) production by dendritic cells (DCs) is drastically increased during acute infection with FMDV in swine. In vitro blockade of IL-10 with a neutralizing antibody against porcine IL-10 restores T cell activation by DCs. Additionally, we describe that FMDV infects DC precursors and interferes with DC maturation and antigen presentation capacity. Thus, we propose a new mechanism of virus immunity in which a non-persistent virus, FMDV, induces immunosuppression by an increment in the production of IL-10, which in turn, reduces T cell function. This reduction of T cell activity may result in a more potent induction of neutralizing antibody responses, clearing the viral infection.

T cell responses to bluetongue virus are directed against multiple and identical CD4+ and CD8+ T cell epitopes from the VP7 core protein in mouse and sheep.

Bluetongue virus (BTV), an economically important orbivirus of the Reoviridae family, is a non-enveloped, dsRNA virus that causes a haemorrhagic disease mainly in sheep, but little is known of the cellular immunity elicited against BTV. We observed that vaccination of interferon type I-deficient mice (IFNAR((-/-))), or inoculation of the wild type C57BL/6 strain with BTV-8, induced a strong T cell response. Therefore, we proceeded to identify some of the T cell epitopes targeted by the immune system. We selected, using H-2(b)-binding predictive algorithms, 3 major histocompatibility complex (MHC)-class II-binding peptides and 7 MHC-class I binding peptides from the BTV-8 core protein VP7, as potential T cell epitopes. Peptide binding assays confirmed that all 7 MHC-class I predicted peptides bound MHC-class I molecules. Three MHC-class I and 2 MHC-class II binding peptide consistently elicited peptide-specific IFN-γ production (as measured by ELISPOT assays) in splenocytes from C57BL/6 BTV-8-inoculated mice and IFNAR((-/-))-vaccinated mice. The functionality of these T cells was confirmed by proliferation and cytotoxicity assays. Flow cytometry analysis demonstrated that CD8(+) T cells responded to MHC-class I binding peptides and CD4(+) T cells to MHC-class II binding peptides. Importantly, these 5 epitopes were also able to induced IFN-γ production in sheep inoculated with BTV-8. Taken together, these data demonstrate the activation of BTV-specific T cells during infection and vaccination. The characterisation of these novel T cell epitopes may also provide an opportunity to develop DIVA-compliant vaccination approach to BTV encompassing a broad-spectrum of serotypes.

Increase in Pancreatic Proinsulin and Preservation of Beta Cell Mass in Autoantibody Positive Donors prior to Type 1 Diabetes Onset

Type 1 diabetes is characterized by the loss of insulin production caused by β-cell dysfunction and/or destruction. The hypothesis that β-cell loss occurs early during the prediabetic phase has recently been challenged. Here we show, for the first time in situ, that in pancreas sections from autoantibody-positive (Ab+) donors, insulin area and β-cell mass are maintained before disease onset and that production of proinsulin increases. This suggests that β-cell destruction occurs more precipitously than previously assumed. Indeed, the pancreatic proinsulin-to-insulin area ratio was also increased in these donors with prediabetes. Using high-resolution confocal microscopy, we found a high accumulation of vesicles containing proinsulin in β-cells from Ab+ donors, suggesting a defect in proinsulin conversion or an accumulation of immature vesicles caused by an increase in insulin demand and/or a dysfunction in vesicular trafficking. In addition, islets from Ab+ donors were larger and contained a higher number of β-cells per islet. Our data indicate that β-cell mass (and function) is maintained until shortly before diagnosis and declines rapidly at the time of clinical onset of disease. This suggests that secondary prevention before onset, when β-cell mass is still intact, could be a successful therapeutic strategy.

Heterogeneity and Lobularity of Pancreatic Pathology in Type 1 Diabetes during the Prediabetic Phase.

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells are destroyed in the islets of Langerhans. One of its main pathological manifestations is the hyper-expression of Major Histocompatibility Complex I (MHC-I) by beta cells, which was first described over 3 decades ago yet its cause remains unknown. It might not only be a sign of beta cell dysfunction but could also render the cells susceptible to autoimmune destruction; for example, by islet-infiltrating CD8 T cells. In this report, we studied pancreas tissue from a 22-year-old non-diabetic male cadaveric organ donor who had been at high risk of developing T1D, in which autoantibodies against GAD and IA-2 were detected. Pancreas sections were analyzed for signs of inflammation. Multiple insulin-containing islets were identified, which hyper-expressed MHC-I. However, islet density and MHC-I expression exhibited a highly lobular and heterogeneous pattern even within the same section. In addition, many islets with high expression of MHC-I presented higher levels of CD8 T cell infiltration than normal islets. These results demonstrate the heterogeneity of human pathology that occurs early during the pre-diabetic, autoantibody positive phase, and should contribute to the understanding of human T1D.

Abnormalities of the Exocrine Pancreas in Type 1 Diabetes

Type 1 diabetes (T1D) is considered a pancreatic beta cell-specific disease that results in absolute insulin deficiency. Nevertheless, clinical studies from 1940 onwards showed that patients with T1D had an abnormal exocrine pancreas due to the presence of subclinical exocrine insufficiency and acinar atrophy. Exocrine abnormalities are an important, and mostly neglected, characteristic associated with T1D. It is however still unclear whether the exocrine dysfunction in T1D is a primary damage caused by the same pathogenic event that led to beta cell destruction or secondary to beta cell loss. In this review, we collect evidence supporting the hypothesis that T1D is a combined endocrine-exocrine disease in which the loss of functional beta cell mass is most clinically apparent.

Enterovirus Infection and Type 1 Diabetes: Closing in on a Link?

The famous French writer Victor Hugo (1802–1885) once said: “Perseverance, secret of all triumphs!” (1). Due to the divergence seen in monozygotic twin cohorts, we know that both genetic and environmental factors must contribute to the development of human type 1 diabetes (T1D) (2,3). For many decades, we have searched for environmental triggers, but it has remained difficult to define the key players. In addition to viral infections, nutritional and hygiene conditions as well as sunlight exposure and geographical locations have all been investigated but often with inconclusive results (4). Now, we might be getting closer to identifying a key culprit, which could be enteroviral infections. In this issue of Diabetes, Krogvold et al. (5) document the presence of a low-grade enteroviral infection in pancreata from recently diagnosed patients. The importance of this study resides on the uniqueness of the six specimens examined, which were collected via partial pancreatectomy from volunteers with very recently diagnosed diabetes (3–9 weeks) as part of the Diabetes Virus Detection study (DiViD) (6). Interestingly, hyperexpression of major histocompatibility complex (MHC-I) molecules and the presence of the enteroviral capsid protein 1 (VP1) were detected in the islets from all the patients. Additional confirmation was obtained by sequencing techniques in four of the six cases. These findings indicate that infections with enteroviruses could, at least in some cases, be a precipitating key factor for the onset of T1D. The natural history tells us that T1D is an inflammatory disease affecting specifically the pancreatic islets of Langerhans, where autoantibodies to various islet antigens in many cases precede clinical diagnosis and autoreactive CD8 T cells can be documented in inflamed islets (7). Viruses have been traditionally implicated in the etiopathogenesis of the disease with no definitive proof so far. Enteroviruses are one of the main candidates because …

Recent advances in understanding Type 1 Diabetes

Type 1 diabetes is a multifactorial disease in which genetic and environmental factors play a key role. The triggering event is still obscure, and so are many of the immune events that follow. In this brief review, we discuss the possible role of potential environmental factors and which triggers are believed to have a role in the disease. In addition, as the disease evolves, beta cells are lost and this occurs in a very heterogeneous fashion. Our knowledge of how beta cell mass declines and our view of the disease’s pathogenesis are also debated. We highlight the major hallmarks of disease, among which are MHC-I (major histocompatibility complex class I) expression and insulitis. The dependence versus independence of antigen for the immune infiltrate is also discussed, as both the influence from bystander T cells and the formation of neo-epitopes through post-translational modifications are thought to influence the course of the disease. As human studies are proliferating, our understanding of the disease’s pathogenesis will increase exponentially. This article aims to shed light on some of the burning questions in type 1 diabetes research.