Cell-based interventions to halt autoimmunity in type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) results from death of insulin-secreting ß cells mediated by self-immune cells, and the consequent inability of the body to maintain insulin levels for appropriate glucose homeostasis. Probably initiated by environmental factors, this disease takes place in genetically predisposed individuals. Given the autoimmune nature of T1DM, therapeutics targeting immune cells involved in disease progress have been explored over the last decade. Several high-cost trials have been attempted to prevent and/or reverse T1DM. Although a definitive solution to cure T1DM is not yet available, a large amount of information about its nature and development has contributed greatly to both the improvement of patient's health care and design of new treatments. In this study, we discuss the role of different types of immune cells involved in T1DM pathogenesis and their therapeutic potential as targets and/or modified tools to treat patients. Recently, encouraging results and new approaches to sustain remnant ß cell mass and to increase ß cell proliferation by different cell-based means have emerged. Results coming from ongoing clinical trials employing cell therapy designed to arrest T1DM will probably proliferate in the next few years. Strategies under consideration include infusion of several types of stem cells, dendritic cells and regulatory T cells, either manipulated genetically ex vivo or non-manipulated. Their use in combination approaches is another therapeutic alternative. Cell-based interventions, without undesirable side effects, directed to block the uncontrollable autoimmune response may become a clinical reality in the next few years for the treatment of patients with T1DM.

Cotransplantation of marginal mass allogeneic islets with 3D culture-derived adult human skin cells improves glycemia in diabetic mice.

Islet transplantation represents a therapeutic option for type 1 diabetes (T1D). Long-term viability of transplanted islets requires improvement. Mesenchymal stromal cells (MSCs) have been proposed as adjuvants for islet transplantation facilitating grafting and functionality. Stem cell aggregation provides physiological interactions between cells and enhances the in situ concentration of modulators of inflammation and immunity. We established a hanging-drop culture of adult human skin fibroblast-like cells as spheroids, and skin spheroid-derived cells (SphCs) were characterized. We assessed the potential of SphCs in improving islet functionality by cotransplantation with a marginal mass of allogeneic islets in an experimental diabetic mouse model and characterized the secretome of SphCs by mass spectrometry-based proteomics. SphCs were characterized as multipotent progenitors and their coculture with anti-CD3 stimulated mouse splenocytes decreased CD4+ T cell proliferation with skewed cytokine secretion through an increase in the Th2/Th1 ratio profile. SphCs-conditioned media attenuated apoptosis of islets induced by cytokine challenge in vitro and importantly, intratesticular SphCs administration did not show tumorigenicity in immune-deficient mice. Moreover, SphCs improved glycemic control when cotransplanted with a marginal mass of allogeneic islets in a diabetic mouse model without pharmacological immunosuppression. SphCs' protein secretome differed from its paired skin fibroblast-like counterpart in containing 70% of up- and downregulated proteins and biological processes that overall positively influenced islets such as cytoprotection, cellular stress, metabolism, and survival. In summary, SphCs improved the performance of transplanted allogeneic islets in an experimental T1D model, without pharmacological immunosuppression. Future research is warranted to identify SphCs-secreted factors responsible for islets' endurance.

Recombinant human prolactin promotes human beta cell survival via inhibition of extrinsic and intrinsic apoptosis pathways.

AIMS/HYPOTHESIS: Transplantation of pancreatic islets constitutes a promising alternative treatment for type 1 diabetes. However, it is limited by the shortage of organ donors. Previous results from our laboratory have demonstrated beneficial effects of recombinant human prolactin (rhPRL) treatment on beta cell cultures. We therefore investigated the role of rhPRL action in human beta cell survival, focusing on the molecular mechanisms involved in this process. METHODS: Human pancreatic islets were isolated using an automated method. Islet cultures were pre-treated in the absence or presence of rhPRL and then subjected to serum starvation or cytokine treatment. Beta cells were labelled with Newport green and apoptosis was evaluated using flow cytometry analysis. Levels of BCL2 gene family members were studied by quantitative RT-PCR and western blot. Caspase-8, -9 and -3 activity, as well as nitric oxide production, were evaluated by fluorimetric assays. RESULTS: The proportion of apoptotic beta cells was significantly lowered in the presence of rhPRL under both cell death-induced conditions. We also demonstrated that cytoprotection may involve an increase of BCL2/BAX ratio, as well as inhibition of caspase-8, -9 and -3. CONCLUSIONS/INTERPRETATION: Our study provides relevant evidence for a protective effect of lactogens on human beta cell apoptosis. The results also suggest that the improvement of cell survival may involve, at least in part, inhibition of cell death pathways controlled by the BCL2 gene family members. These findings are highly relevant for improvement of the islet isolation procedure and for clinical islet transplantation.

Pleth variability index combined with passive leg raising-induced pulse pressure variation to detect hypovolemia in spontaneously breathing patients.

The combination of Pleth Variability Index (PVI) and passive leg raising (PLR)-induced pulse pressure variation may help to diagnose hypovolemia in spontaneously breathing patients. In 44 subjects, PVI and Pulse Pressure (PP) variation after PLR were measured before and after induced hypovolemia (blood gift or hemodialysis session). PVI values were significantly greater after hemodialysis session or blood gift (22% vs 18%, P = 0.03); in contrast PP variation did not change significantly (7% vs 4%, P = 0.49). The accuracy of these parameters or of their combination to identify the "after hypovolemia induction" period was weak. In spontaneous ventilation, PVI value is greater after induced hypovolemia, whereas PP variation does not change significantly. The combination of PVI and PLR does not improve the accuracy of the detection of induced hypovolemia.

Successful treatment with voriconazole of prolonged Paecilomyces lilacinus fungemia in a chronic hemodialyzed patient.

Paecilomyces lilacinus is a saprophytic mould which rarely causes infection in humans. We report a case of Paecilomyces lilacinus catheter-related fungemia in a chronic hemodialyzed patient. Blood cultures remained positive for 8 weeks. The infection was cured after eventual acceptance by the patient of oral voriconazole treatment for 6 weeks and removal of the tunneled catheter. The literature on Paecilomyces fungemia in humans is reviewed.

Tenofovir-related acute kidney injury and proximal tubule dysfunction precipitated by diclofenac: a case of drug-drug interaction.

We describe an HIV1-positive patient under long-term tenofovir treatment who developed a severe, biopsy-proven, acute tubular necrosis with proximal tubule (PT) dysfunction, precipitated by the very recent start of diclofenac, a nonsteroidal antiinflammatory drug (NSAID). Recent studies show that NSAIDs not only alter glomerular filtration but also multidrug resistance protein (MRP) 4-mediated PT secretion of several substrates. Since the patient tolerated tenofovir well for several years prior to diclofenac use, our observation suggests that diclofenac interfered with tenofovir clearance, thereby favoring its nephrotoxicity. NSAIDs should be avoided in patients under tenofovir.

Where do we aspire to publish? A position paper on scientific communication in biochemistry and molecular biology.

The scientific publication landscape is changing quickly, with an enormous increase in options and models. Articles can be published in a complex variety of journals that differ in their presentation format (online-only or in-print), editorial organizations that maintain them (commercial and/or society-based), editorial handling (academic or professional editors), editorial board composition (academic or professional), payment options to cover editorial costs (open access or pay-to-read), indexation, visibility, branding, and other aspects. Additionally, online submissions of non-revised versions of manuscripts prior to seeking publication in a peer-reviewed journal (a practice known as pre-printing) are a growing trend in biological sciences. In this changing landscape, researchers in biochemistry and molecular biology must re-think their priorities in terms of scientific output dissemination. The evaluation processes and institutional funding for scientific publications should also be revised accordingly. This article presents the results of discussions within the Department of Biochemistry, University of São Paulo, on this subject.

An easily overlooked iatrogenic cause of renal failure.

We report moderate renal failure in a 50-year-old man with a history of recent colonoscopy after oral sodium phosphate purgative use. We initially missed the correct diagnosis, but renal biopsy revealed signs of acute phosphate nephropathy. The patient had residual renal impairment at 8-month follow-up. Greater awareness of this complication is needed amongst health care professionals. The preventive strategies are discussed.

Cotransplantation of marginal mass allogeneic islets with 3D culture-derived adult human skin cells improves glycemia in diabetic mice

Islet transplantation represents a therapeutic option for type 1 diabetes (T1D). Long-term viability of transplanted islets requires improvement. Mesenchymal stromal cells (MSCs) have been proposed as adjuvants for islet transplantation facilitating grafting and functionality. Stem cell aggregation provides physiological interactions between cells and enhances the in situ concentration of modulators of inflammation and immunity. We established a hanging-drop culture of adult human skin fibroblast-like cells as spheroids, and skin spheroid-derived cells (SphCs) were characterized. We assessed the potential of SphCs in improving islet functionality by cotransplantation with a marginal mass of allogeneic islets in an experimental diabetic mouse model and characterized the secretome of SphCs by mass spectrometry-based proteomics. SphCs were characterized as multipotent progenitors and their coculture with anti-CD3 stimulated mouse splenocytes decreased CD4+ T cell proliferation with skewed cytokine secretion through an increase in the Th2/Th1 ratio profile. SphCs-conditioned media attenuated apoptosis of islets induced by cytokine challenge in vitro and importantly, intratesticular SphCs administration did not show tumorigenicity in immune-deficient mice. Moreover, SphCs improved glycemic control when cotransplanted with a marginal mass of allogeneic islets in a diabetic mouse model without pharmacological immunosuppression. SphCs' protein secretome differed from its paired skin fibroblast-like counterpart in containing 70% of up- and downregulated proteins and biological processes that overall positively influenced islets such as cytoprotection, cellular stress, metabolism, and survival. In summary, SphCs improved the performance of transplanted allogeneic islets in an experimental T1D model, without pharmacological immunosuppression. Future research is warranted to identify SphCs-secreted factors responsible for islets' endurance.

Prolactin-induced changes in protein expression in human pancreatic islets.

Ex vivo islet cell culture prior to transplantation appears as an attractive alternative for treatment of type 1 diabetes. Previous results from our laboratory have demonstrated beneficial effects of human prolactin (rhPRL) treatment on human islet primary cultures. In order to probe into the molecular events involved in the intracellular action of rhPRL in these cells, we set out to identify proteins with altered expression levels upon rhPRL cell treatment, using two-dimensional (2D) gel electrophoresis and mass spectrometry (MS). An average of 300 different protein spots were detected, 14 of which were modified upon rhPRL treatment (p<0.01), of which 12 were successfully identified using MS and grouped according to their biological functions. In conclusion, our study provides, for the first time, information about proteins that could be critically involved in PRL's action on human pancreatic islets, and facilitate identification of new and specific targets involved in islet cell function and proliferation.

Interactions between progestins and heregulin (HRG) signaling pathways: HRG acts as mediator of progestins proliferative effects in mouse mammary adenocarcinomas.

The present study addressed links between progestin and heregulin (HRG) signaling pathways in mammary tumors. An experimental model of hormonal carcinogenesis, in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female Balb/c mice, was used. MPA induced an in vivo up-regulation of HRG mRNA expression in progestin-dependent (HD) tumor lines. Mammary tumor progression to a progestin-independent (HI) phenotype was accompanied by a high constitutive expression of HRG. The HRG message arose from the tumor epithelial cells. Primary cultures of malignant epithelial cells from a HD tumor line were used to investigate HRG involvement on cell proliferation. HRG induced a potent proliferative effect on these cells and potentiated MPA mitogenic effects. Blocking endogenous HRG synthesis by antisense oligodeoxynucleotides (ASODNs) to HRG mRNA inhibited MPA-induced cell growth, indicating that HRG acts as a mediator of MPA-induced growth. High levels of ErbB-2 and ErbB-3 expression and low ErbB-4 levels were found in HD cells. Treatment of these cells with either MPA or HRG resulted in tyrosine phosphorylation of both ErbB-2 and ErbB-3. Furthermore, both HRG and MPA proliferative effects were abolished when cells were treated with ASODNs to ErbB-2 mRNA, providing evidence for a critical role of ErbB-2 in HRG-induced growth. Finally, blocking type I insulin-like growth factor receptor (IGF-IR) expression with ASODN resulted in the complete inhibition of HRG proliferative effect, demonstrating that a functional IGF-IR is required for HRG mitogenic activity. These results provide the first evidence of interactions between progestins and HRB/ErbB signal transduction pathways in mammary cancer and the first demonstration that IGF-IR is required for HRG proliferative effects.

Activation of ErbB-2 via a hierarchical interaction between ErbB-2 and type I insulin-like growth factor receptor in mammary tumor cells.

The present study focused on interactions between signaling pathways activated by progestins and by type I and II receptor tyrosine kinases (RTKs) in mammary tumors. An experimental model in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in Balb/c mice was used. MPA-stimulated proliferation, both in vivo and in vitro, of progestin-dependent tumors induced up-regulation of ErbB-2 protein levels and tyrosine phosphorylation of this receptor. Combinations of antisense oligodeoxynucleotides (ASODNs) directed to ErbB-2 mRNA with ASODNs directed to the insulin-like growth factor-I receptor (IGF-IR) were used to study the effect of the simultaneous block of these receptors on the MPA-induced proliferation of epithelial cells from the progestin-dependent C4HD line. Neither synergistic nor additive effects on the inhibition of MPA-induced proliferation of C4HD cells were observed as a result of the combination of these ASODNs. Suppression of IGF-IR expression by ASODNs resulted in complete abrogation of MPA-induced phosphorylation of ErbB-2 in C4HD cells, whereas blockage of ErbB-2 did not affect IGF-IR phosphorylation. These results show the existence of a hierarchical interaction between IGF-IR and ErbB-2, by means of which IGF-IR directs ErbB-2 phosphorylation. We demonstrated, for the first time, that this hierarchical interaction involves physical association of both receptors, resulting in the formation of a heteromeric complex. Furthermore, confocal laser microscopy experiments demonstrated that MPA was able to induce co-localization of ErbB-2 and IGF-IR. This hetero-oligomer was also found in MCF-7 human breast cancer cells in which association of IGF-IR and ErbB-2 was induced by heregulin and IGF-I. Oncogene (2001) 20, 34 - 47.

Massive amoxycillin crystalluria causing anuric acute renal failure.

The authors report the case of a 45-year-old woman admitted for pneumonia who presented anuric acute renal failure after 12 days of intravenous amoxycillin-clavulanate treatment. Acute renal failure resolved rapidly and completely after antibiotic withdrawal. Analysis of the first post-anuric urine specimen showed many crystals. Infrared spectrophotometry revealed that the crystals were composed of trihydrated amoxycillin. The possibility of intrarenal obstruction due to massive drug crystalluria should not be overlooked in the face of abrupt anuria.

Where do we aspire to publish? A position paper on scientific communication in biochemistry and molecular biology

The scientific publication landscape is changing quickly, with an enormous increase in options and models. Articles can be published in a complex variety of journals that differ in their presentation format (online-only or in-print), editorial organizations that maintain them (commercial and/or society-based), editorial handling (academic or professional editors), editorial board composition (academic or professional), payment options to cover editorial costs (open access or pay-to-read), indexation, visibility, branding, and other aspects. Additionally, online submissions of non-revised versions of manuscripts prior to seeking publication in a peer-reviewed journal (a practice known as pre-printing) are a growing trend in biological sciences. In this changing landscape, researchers in biochemistry and molecular biology must re-think their priorities in terms of scientific output dissemination. The evaluation processes and institutional funding for scientific publications should also be revised accordingly. This article presents the results of discussions within the Department of Biochemistry, University of São Paulo, on this subject.

Clostridium perfringens hip arthritis in a haemodialysis patient.

Haemodialysis patients have acquired immunity disturbances, co-morbidities and a vascular access, factors predisposing them to infection and bacteraemia. Clostridium perfringens is an anaerobic bacterium potentially causing severe infections, including rarely septic arthritis. We report the first case of Clostridium perfringens septic arthritis in a haemodialysis patient and suggest a haematogenous spread. After rapid joint lavage combined with appropriate anti-microbial therapy, the patient recovered.

Epithelial-mesenchymal transition: implications in cancer progression and metastasis.

During the past few years, Epithelial-Mesenchymal Transition (EMT) has emerged as one of the most hot spots in clinical research. Its existence in human tumors can form the basis for explaining characteristics of cancer progression and metastasis, as well as certain cases of drug resistance and relapses after treatment. These cellular responses are tightly regulated by intracellular signaling pathways evoked by humoral factors that include growth factors, chemokines and cytokines. Indeed, several gene regulatory programs known to promote EMT during development have recently been discovered to play key roles in cancer progression. A deeper understanding of the cellular and molecular basis of these different programs should aid in both the development of better diagnosis methods, as well as of specific treatments for invasive cancer. In this review we set out to summarize recent novel insights into the molecular players underlying EMT and its relation with cancer progression and metastasis.