Increased expression of IL-18 in the serum and islets of type 1 diabetics.

Type 1 diabetes (T1D) is a chronic disease characterized by autoimmune-mediated destruction of pancreatic insulin-producing beta cells. Interleukin (IL)-18 is a pro-inflammatory cytokine implicated in the pathogenesis of a number of inflammatory diseases. Here, we analyzed IL-18 levels in the plasma of juveniles with T1D. Compared to control subjects, IL-18 levels were significantly elevated in patients with T1D. On the other hand, levels of IL-18 binding protein (IL-18BP) and IL-37, two negative regulators of IL-18 function, remained unchanged when comparing T1D to control samples. Notably, however, although IL-18BP levels were not elevated, IL-18 and IL-18BP were found to be positively correlated in type 1 diabetics. Even so, free, unbound IL-18 remained significantly increased in diabetic patients. Additionally, correlation studies also revealed that IL-18 and IL-18BP are positively correlated with HbA1c levels in T1D patients, suggesting a potential link between IL-18 and metabolic control in these patients. Finally, we observed a significant increase in IL-18 protein expression within human pancreatic islet specimens collected from type 1 diabetics. These results further expand our knowledge of the role of IL-18 in T1D, may give insight into common pathogenic mechanisms associated with metabolic control in both T1D and T2D, and suggest that targeting this cytokine may improve therapeutic outcomes for T1D patients.

CD28⁻ CD8⁺ T cells are significantly reduced and correlate with disease duration in juveniles with type 1 diabetes.

Type 1 diabetes (T1D) is a chronic disease caused by autoimmune destruction of insulin-producing pancreatic ß-cells. T1D is typically diagnosed in children, but information regarding immune cell subsets in juveniles with T1D is scarce. Therefore, we studied various lymphocytic populations found in the peripheral blood of juveniles with T1D compared to age-matched controls (ages 2-17). One population of interest is the CD28(-) CD8(+) T cell subset, which are late-differentiated cells also described as suppressors. These cells are altered in a number of disease states and have been shown to be reduced in adults with T1D. We found that the proportion of CD28(-) cells within the CD8(+) T cell population is significantly reduced in juvenile type 1 diabetics. Furthermore, this reduction is not correlated with age in T1D juveniles, although a significant negative correlation between proportion CD28(-) CD8(+) T cells and age was observed in the healthy controls. Finally, correlation analysis revealed a significant and negative correlation between the proportion of CD28(-) CD8(+) T cells and T1D disease duration. These findings show that the CD28(-) CD8(+) T cell population is perturbed following onset of disease and may prove to be a valuable marker for monitoring the progression of T1D.

Oncolytic vesicular stomatitis virus and bortezomib are antagonistic against myeloma cells in vitro but have additive anti-myeloma activity in vivo.

Multiple myeloma cells are highly sensitive to the oncolytic effects of vesicular stomatitis virus (VSV), which specifically targets and kills cancer cells. Myeloma cells are also exquisitely sensitive to the cytotoxic effects of the clinically approved proteasome inhibitor bortezomib. Therefore, we sought to determine whether the combination of VSV and bortezomib would enhance tumor cell killing. However, as shown here, combining these two agents in vitro results in antagonism. We show that bortezomib inhibits VSV replication and spread. We found that bortezomib inhibits VSV-induced NF-κB activation and, using the NF-κB-specific inhibitor BMS-345541, that VSV requires NF-κB activity to spread efficiently in myeloma cells. In contrast to other cancer cell lines, viral titer is not recovered by BMS-345541 when myeloma cells are pretreated with interferon ß. Thus, inhibiting NF-κB activity, either with bortezomib or BMS-345541, results in reduced VSV titers in myeloma cells in vitro. However, when VSV and bortezomib are combined in vivo in two syngeneic, immunocompetent myeloma models, the combination reduces tumor burden to a greater degree than VSV does as a single agent. Intratumoral VSV viral load is unchanged when mice are treated concomitantly with bortezomib compared to VSV treatment alone. To our knowledge, this report is the first to analyze the combination of VSV and bortezomib in vivo. Although antagonism between VSV and bortezomib is seen in vitro, analyzing these cells in the context of their host environment shows that bortezomib enhances VSV response, suggesting that this combination will also enhance response in myeloma patients.

Bortezomib salvage followed by a Phase I/II study of bortezomib plus high-dose melphalan and tandem autologous transplantation for patients with primary resistant myeloma.

We conducted a Phase 1/2 study of bortezomib administered in combination with high-dose melphalan followed by tandem autologous transplants in patients with primary resistant multiple myeloma. Thirty patients received two cycles of salvage bortezomib followed by stem cell mobilization with granulocyte colony-stimulating factor and harvest. Melphalan 100 mg/m(2) per day on two consecutive days was administered, immediately followed by one dose of bortezomib (dose escalation) and stem cell infusion. The median beta 2-microglobulin was 4·35 mg/l (range: 1·8-11·4); albumin was 37 g/l (range: 3·1-4·9); high-risk karyotypes were noted in 45% of patients. The maximum planned dose of bortezomib at 1·3 mg/m(2) was well tolerated and a formal maximum tolerated dose was not determined. The peak of best overall response (≥partial response) and complete response rates after tandem transplants were 84% and 36%, respectively. With a median follow-up of 48 months, the median progression-free survival was 15 [95% confidence interval (CI): 11-21] months and the median overall survival was 35 (95% CI: 22-43) months. Correlative studies demonstrated decreased expression of BRCA2 (P = 0·0072) and FANCF (P = 0·0458) mRNA following bortezomib treatment. Bortezomib combined with high-dose melphalan is a well-tolerated conditioning with some activity in patients with resistant myeloma.

Targeting the Fanconi anemia/BRCA pathway circumvents drug resistance in multiple myeloma.

The Fanconi anemia/BRCA (FA/BRCA) DNA damage repair pathway plays a pivotal role in the cellular response to replicative stress induced by DNA alkylating agents and greatly influences drug response in cancer treatment. We recently reported that FA/BRCA genes are overexpressed and causative for drug resistance in human melphalan-resistant multiple myeloma cell lines. However, the transcriptional regulation of the FA/BRCA pathway is not understood. In this report, we describe for the first time a novel function of the NF-kappaB subunits, RelB/p50, as transcriptional activators of the FA/BRCA pathway. Specifically, our findings point to constitutive phosphorylation of IkappaB kinase alpha and subsequent alterations in FANCD2 expression and function as underlying events leading to melphalan resistance in repeatedly exposed multiple myeloma cells. Inhibiting NF-kappaB by small interfering RNA, blocking the IkappaB kinase complex with BMS-345541, or using the proteasome inhibitor bortezomib drastically reduced FA/BRCA gene expression and FANCD2 protein expression in myeloma cells, resulting in diminished DNA damage repair and enhanced melphalan sensitivity. Importantly, we also found that bortezomib decreases FA/BRCA gene expression in multiple myeloma patients. These results show for the first time that NF-kappaB transcriptionally regulates the FA/BRCA pathway and provide evidence for targeting Fanconi anemia-mediated DNA repair to enhance chemotherapeutic response and circumvent drug resistance in myeloma patients.

Beta1 integrin adhesion enhances IL-6-mediated STAT3 signaling in myeloma cells: implications for microenvironment influence on tumor survival and proliferation.

The bone marrow microenvironmental components interleukin (IL)-6 and fibronectin (FN) individually influence the proliferation and survival of multiple myeloma (MM) cells; however, in vivo, these effectors most likely work together. We examined signaling events, cell cycle progression, and levels of drug response in MM cells either adhered to FN via beta1 integrins, stimulated with IL-6, or treated with the two combined. Although G(1)-S cell cycle arrest associated with FN adhesion was overcome when IL-6 was added, the cell adhesion-mediated drug resistance (CAM-DR) was maintained in the presence of IL-6. Concomitant exposure of MM cells to IL-6 and FN adhesion revealed a dramatic increase in signal transducers and activators of transcription 3 (STAT3) phosphorylation, nuclear translocation, and DNA binding, compared with either IL-6 or FN adhesion alone in four MM cell lines. Importantly, this increase in STAT3 activation correlated with a novel association between STAT3 and gp130 in cells adhered to FN before stimulation with IL-6, relative to nonadherent cells. Taken together, these results suggest a mechanism by which collaborative signaling by beta1 integrin and gp130 confers an increased survival advantage to MM cells.