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Progressive multifocal leukoencephalopathy (PML) is a disease of the central nervous system caused by neuropathogenic prototypes of ubiquitous community-acquired JC virus (JCV). The disease became of particular concern following its association with certain therapies that modulate immune system function without heavy immunosuppression. Due to lack of prophylactic/treatment options and poor outcomes, which often include severe disability or death, PML is a considerable concern for development of new drugs that interfere with immune system functions. In this review of clinical and research findings, we discuss the evidence that deficiencies in CD4+ T helper cells, cytotoxic CD8+ T cells, and interferon gamma are of crucial importance for development of PML under a variety of circumstances, including those associated with use of various drugs, regardless of differences in their mechanisms of action. These deficiencies apparently enable transformation of the harmless JCV archetype into neuropathogenic prototypes, but the site(s), and the mechanisms, of this transformation are yet to be elucidated. Here we discuss the evidence for brain as one of the sites of this transformation, and propose a model of PML pathogenesis that emphasizes the central role of T cell deficiencies in the two life cycles of the JCV, one non-pathogenic and one neuropathogenic. Finally, we conclude that the development of clinical grade T cell functional tests and more consistent use of already available laboratory tests for T cell subset analysis would greatly aid the effort to more accurately predict and assess the magnitude of PML risk for concerned therapeutic interventions.

Progressive multifocal leukoencephalopathy (PML) is a rare but debilitating and frequently fatal viral disease of the central nervous system, primarily affecting individuals with chronically and severely suppressed immune systems. The disease was relatively obscure until the outbreak of HIV/AIDS, when it presented as one of the more frequent opportunistic infections in this immune deficiency syndrome. It attracted additional attention from the medical and scientific community following the discovery of significant PML risk associated with natalizumab, a monoclonal antibody used for treatment of relapsing–remitting multiple sclerosis. This was followed by association of PML with other immunosuppressive or immunomodulating drugs. PML is currently untreatable disease with poor outcomes, so it is a significant concern when developing new immunotherapies. Current prophylaxis and treatment of PML are focused on immune reconstitution, restoration of immune responses to JC virus infection, and eventual suppression of immune reconstitution inflammatory syndrome. This approach was successful in reducing the incidence of PML and improved survival of PML patients with HIV infection. However, the outcome for the majority of PML patients, regardless of their medical history, is still relatively poor. There is a high unmet need for both prophylaxis and treatment of PML. The aim of this review is to discuss potential drug candidates for prophylaxis and treatment of PML with a critical review of previously conducted and completed PML treatment studies as well as to provide perspectives for future therapies.

Background: Pre-natal porcine endocrine islet cell grafts were recently shown to contain immature beta cells with a marked potential for growth and differentiation following transplantation, and hence for a progressive and long-term correction of diabetes in immune-incompetent mice. The present study investigates whether these grafts are also capable of correcting hyperglycemia in immune-competent mice receiving a short treatment with anti-CD4–CD8 antibodies. Methods: Pure endocrine islet cell grafts with 0.5 to 1.0 million beta cells were prepared from pre-natal pigs and transplanted under the kidney capsule of alloxan-diabetic CBA/Ca mice. Survival, growth and function of implanted beta cells were followed by measuring plasma porcine C-peptide and glucose, and graft insulin content at start and at post-transplant (PT) week 35. The effect was studied of a 5-day treatment with non-depleting anti-CD4 YTS177 and depleting anti-CD8 YTS169 antibody, either without or with transient insulin injections. Results: Without antibody treatment, all graft recipients remained porcine C-peptide negative and died. Antibody treatment decreased CD4-expression and percentage CD8 cells for 10 and 18 weeks respectively. It resulted in a 30 week-survival of nine out of 14 graft recipients; all nine had progressively become C-peptide positive but only one proceeded to normoglycemia. When antibody treatment was combined with transient insulin injections, 11 out of 14 graft recipients survived long-term, eight became C-peptide positive and six were normoglycemic at PT week 30. In both groups, surviving recipients exhibited a graft insulin content that was 6- to 9-fold higher than at implantation. Conclusions: Pre-natal porcine beta cells grow and differentiate when transplanted in diabetic immune-competent mice that have been transiently immune suppressed with anti-CD4 and anti-CD8 monoclonal antibodies. They develop metabolic control when recipients are also transiently treated with insulin injections.

Introduction. Anal atresia is a congenital anomaly, very life threatening and urgent. Surgical treatment of this anomaly consists of colostomy first, and then of anal reconstruction. Case report. We presented a 31-year old female with the surgery treatment of anal atresia in the early childhood. In the reproductive period, due to tubal infertily, the patient was included in the program of in vitro fertilization (IVF), in the Clinic for Gynecology and Obstetrics 'Narodni front', Belgrade. Within this program a long protocol of ovarian stimulation was performed. Ultrasonographic and color Doppler monitoring of the patient was applied by the use of an ultrasonographic apparatus type Siemens Acuson X 150, while any hormonal examinations were performed by an Architect Abbott unit. During the IVF program, the growth of follicules was controlled by the use of ultrasonography, microcirculation of the ovaries and the uterus was marked by a power-pulsating color Doppler, and hormonal examination was performed starting from the day of stimulation up to the day of injecting Pregnyl?. The patient was administered Suprefact? (buserelin) sc from the 21st day of the menstrual cycle, as well as from the 3rd day of the cycle, for totally 11 days. The patient was given 29 ampoules of Gonal F? (recombinant human FSH) 75 IJ im and 15 ampoules of Menopur? (menotrophin) im. Due to a modified pelvic anatomy, the left ovary aspiration was disabled, while the right ovary aspired seven oocytes successfully. Three embryos were inserted in the uterus. The delivery was performed by cesarean section. Conclusion. In the reported patient with a modified pelvic anatomy due to four corrective surgeries of anal atresia, and tubal infertility in the reproductive period, the method of choice for the realization of pregnancy was the IVF procedure. The realized pregnancy and the delivery could be considered highly successful in regard to possible risks.

Whereas nitric oxide (NO) production is associated with the toxic effect of cytokines on rodent pancreatic beta-cells, cytokine-induced apoptosis in human islets may occur independently of NO. The cysteine protease interleukin (IL)-1 converting enzyme (ICE) is a key proapoptotic caspase. Our aim was therefore to analyze the effect of cytokines on ICE expression in human, rat, and mouse islets and rat insulinoma cells. ICE messenger RNA (mRNA) expression was highly up-regulated after 6-, 24-, and 72-h exposure of human islets to interferon (IFN)gamma, tumor necrosis factor (TNF)alpha + IFNgamma or IL-1beta + TNFalpha + IFNgamma, paralleled by increased iNOS (the inducible form of NO synthase) expression and NO production after exposure to the combined cytokines but not to IFNgamma or TNFalpha + IFNgamma. Cytokine-induced NO-independent ICE transcription was confirmed using iNOS inhibitors. Exposure of rat and mouse islets, or rat insulinoma cells, for 24 h to IFNgamma alone or in combination with the two other cytokines also resulted in a highly significant ICE mRNA expression. ICE transcription was not inducible in islets from IFN regulatory factor-1 knock-out mice, suggesting a key-role of this transcription-factor in cytokine-mediated ICE expression in pancreatic islets. In conclusion, cytokines and IFNgamma in particular increase ICE mRNA expression in pancreatic islet cells and beta-cell lines, independently of NO synthesis, suggesting that ICE up-regulation may be involved in cytokine-induced NO-independent apoptosis of human islets.

Infiltration of immunocytes into pancreatic islets precedes loss of beta cells in type 1 diabetes. It is conceivable that local release of cytokines affects the function of beta cells before their apoptosis. This study examines whether the elevated proinsulin levels that have been described in prediabetes can result from exposure of beta cells to cytokines. Human beta-cell preparations were cultured for 48 or 72 hours with or without IL-1beta, TNF-alpha, or IFN-gamma, alone or in combination. None of these conditions were cytotoxic, nor did they reduce insulin biosynthetic activity. Single cytokines did not alter medium or cellular content in insulin or proinsulin. Cytokine combinations, in particular IL-1beta plus IFN-gamma, disproportionately elevated medium proinsulin levels. This effect expresses an altered functional state of the beta cells characterized by preserved proinsulin synthesis, a slower hormone conversion, and an increased ratio of cellular proinsulin over insulin content. The delay in proinsulin conversion can be attributed to lower expression of PC1 and PC2 convertases. It is concluded that disproportionately elevated proinsulin levels in pre-type 1 diabetic patients might result from exposure of their beta cells to cytokines released from infiltrating immunocytes. This hormonal alteration expresses an altered functional state of the beta cells that can occur independently of beta-cell death.

Interleukin (IL)-1beta is known to cause beta-cell death in isolated rat islets. This effect has been attributed to induction of nitric oxide (NO) synthase in beta-cells and subsequent generation of toxic NO levels; it was not observed, however, in dispersed rat beta-cells. The present study demonstrates that IL-1beta induces NO-dependent necrosis in rat beta-cells cultured for 3 days at high cell density or in cell aggregates but not as single cells. Its cytotoxic condition is not explained by higher NO production rates but might result from higher intercellular NO concentrations in statically cultured cell preparations with cell-to-cell contacts; nitrite levels in collected culture medium are not a reliable index for these intercellular concentrations. Absence of IL-1-induced necrosis in rat alpha-cells or in human beta-cells is attributed to the cytokine's failure to generate NO in these preparations, not to their reduced sensitivity to NO: the NO donor GEA 3162 (15 min, 50-100 micromol/l) exerts a comparable necrotic effect in rat and human alpha- or beta-cells. In preparations in which IL-1beta does not cause beta-cell necrosis, its combination with gamma-interferon (IFN-gamma) results in NO-independent apoptosis, starting after 3 days and increasing with the duration of exposure. Because IFN-gamma alone was apoptotic for rat alpha-cells, it is proposed that IL-1beta can make beta-cells susceptible to this effect, conceivably through altering their phenotype. It is concluded that IL-1beta can cause NO-dependent necrosis or NO-independent apoptosis of islet cells, depending on the species and on the environmental conditions. The experiments in isolated human beta-cell preparations suggest that these cells may preferentially undergo apoptosis when exposed to IL-1beta plus IFN-gamma unless neighboring non-beta-cells produce toxic NO levels.

In type 1 diabetes, autoimmune destruction of pancreatic beta-cells has been attributed to cytokines released from infiltrating immunocytes. Exposure of isolated islets to cytokines leads to nitric oxide (NO) production, which can damage beta-cells. Because ductal cells are closely associated with human beta-cells, we examined whether they can contribute to this process. Isolated human ductal cells were cultured for 48 h with various cytokines. The combination of interleukin-1beta (IL-1beta) plus interferon-gamma (IFN-gamma) increased nitric oxide production 12-fold while stimulating mRNA expression of inducible nitric oxide synthase (iNOS). In this condition, 10-20% of cells positive for the cytokeratin-19 duct marker also stained positive for iNOS protein, whereas no positive cells were found in control preparations. Comparison of the magnitude of iNOS mRNA expression and nitric oxide production in these cells with that in isolated human islets suggests that >50% of total islet nitric oxide production might originate from associated ductal cells. It is concluded that ductal cells are a potential source of nitric oxide production in human islets infiltrated by cytokine-releasing immunocytes.

The normal pancreatic beta-cell population exhibits intercellular differences in its responsiveness to glucose. This cellular heterogeneity allows glucose to regulate, in a dose-dependent manner, total rates of insulin synthesis and release. It may also predispose to intercellular differences in susceptibility to dysregulating agents. The present study examines whether this is the case for interleukin 1beta (IL-1beta), which is known to suppress glucose-induced insulin synthesis and release. The effects of the cytokine were compared on beta-cell subpopulations with, respectively, high and low sensitivity to glucose. These subpopulations were separated on the basis of differences in the cellular metabolic responsiveness to an intermediate glucose concentration (7.5 mmol/liter) and then cultured for 20 h at 5 or 20 mmol/liter with or without IL-1beta. The suppressive action of IL-1beta (0.1 ng/ml) occurred predominantly in glucose-activated beta cells, reducing their high rates of insulin synthesis and release by more than 80%. Glucose-unresponsive cells became subject to a similar inhibition after their activation during culture at 20 mmol/liter glucose. On the other hand, IL-1beta induced or enhanced the expression of several noninsulin proteins in both subpopulations. The IL-1beta-stimulated expression of inducible nitric oxide synthase (iNOS) and heat shock protein 70 was more marked in the glucose-responsive subpopulation; that of heme oxygenase and Mn superoxide dismutase was comparable in the two subpopulations. Exposure to IL-1beta resulted in 10-fold higher medium nitrite levels in both subpopulations; this effect was prevented by the iNOS blocker, N(G)-methyl-L-arginine, which also prevented the IL-1beta-induced suppression in the glucose-responsive subpopulation. This study demonstrates that the cellular heterogeneity in glucose responsiveness predisposes to intercellular differences in the IL-1-induced suppression of insulin synthesis and release. While the cytokine induces the expression of noninsulin proteins such as iNOS in both glucose responsive and unresponsive cells, the subsequent nitric oxide production appears to predominantly affect glucose-stimulated functions in the glucose-activated cells.