Phase III, placebo-controlled, randomized, double-blind trial of tableted, therapeutic TB vaccine (V7) containing heat-killed M. vaccae administered daily for one month.

OBJECTIVE: Immunotherapy of tuberculosis (TB) to shorten treatment duration represents an unmet medical need. Orally delivered, tableted TB vaccine (V7) containing heat-killed Mycobacterium vaccae (NCTC 11659) has been demonstrated in prior clinical studies to be safe and fast-acting immune adjunct. METHODS: The outcome of Phase III trial of V7 containing 10 µg of hydrolyzed M. vaccae was evaluated in 152 patients randomized at 2:1 ratio: V7 (N = 100), placebo (N = 52). Both arms received conventional 1st or 2nd line TB drugs co-administered with daily pill of V7 or placebo. RESULTS: After one month mycobacterial clearance was observed in 68% (P < 0.0001) and 23.1% (P = 0.04) of patients on V7 and placebo. Stratified conversion rates in V7 recipients with drug-sensitive and multidrug-resistant TB were 86.7% and 55.6% vs 27.2% and 15% in placebo. Patients on V7 gained on average 2.4 kg (P < 0.0001) vs 0.3 kg (P = 0.18) in placebo. Improvements in hemoglobin levels, erythrocyte sedimentation rate and leukocyte counts were significantly better than in controls. Liver function tests revealed that V7 can prevent chemotherapy-induced hepatic damage. CONCLUSION: Oral M. vaccae is safe, can overcome TB-associated weight loss and inflammation, reduce hepatotoxicity of TB drugs, improve sputum conversion three-fold OR 3.15; 95%CI (2.3,4.6), and cut treatment length by at least six-fold. Longer follow-up studies might be needed to further substantiate our findings (Clinicaltrials.gov: NCT01977768).

Double-blind, placebo-controlled, 1:1 randomized Phase III clinical trial of Immunoxel honey lozenges as an adjunct immunotherapy in 269 patients with pulmonary tuberculosis.

AIM: Safer and shorter antituberculosis treatment (ATT) regimens represent the unmet medical need. PATIENTS & METHODS: The patients were randomly assigned into two arms: the first (n = 137) received once-daily sublingual honey lozenge formulated with botanical immunomodulator Immunoxel and the second (n = 132) received placebo lozenges along with conventional ATT. Immunoxel and placebo arms were demographically similar: 102 versus 106 had drug-susceptible TB; 28 versus 20 multidrug-resistant TB (MDR-TB); 7 versus 7 extensively drug-resistant TB (XDR-TB); and 22 versus 20 TB-HIV. The primary end point was sputum smear conversion. RESULTS: After 1 month 87 out 132 (65.9%) of Immunoxel recipients became sputum smear negative, whereas 32 out of 127 (25.2%) in placebo group had converted (p < 0.0001). Sputum clearance produced by Immunoxel was equally effective across all forms of TB. In the immunotherapy arm the average weight gain was 2 kg, but placebo recipients gained only 0.6 kg. Immunoxel reduced TB-associated inflammation as evidenced by defervescence and normalization of elevated leukocyte counts and erythrocyte sedimentation rate. No adverse effects were seen at any time. The liver function tests indicate that ATT-caused hepatotoxicity was counteracted by Immunoxel. These results are in agreement with prior 20 trials of Immunoxel conducted over the past 17 years. CONCLUSION: Immunoxel is affordable, safe, effective, fast-acting, commercially available immunotherapeutic intervention to supplement conventional TB chemotherapy. Clinicaltrials.gov ID: NCT01061593.

Association of interleukins genes polymorphisms with multi-drug resistant tuberculosis in Ukrainian population.

INTRODUCTION: Multi-drug resistant tuberculosis (MDR TB) is a significant health problem in some parts of the world. Three major cytokines involved in TB immunopathogenesis include IL-2, IL-4 and IL-10. The susceptibility to MDR TB may be genetically determined. The aim of the study was to assess the association of IL-2, IL-4, IL-10 gene polymorphisms with multi-drug resistant tuberculosis (MDR TB) in Ukrainian population. MATERIAL AND METHODS: We observed 140 patients suffering from infiltrative pulmonary tuberculosis (PT) and 30 apparently healthy subjects. The patients were assigned to two groups whether they suffer or do not suffer from pulmonary MDR TB. Interleukin gene (IL) polymorphisms, particularly T330G polymorphism in the IL-2 gene, C589T polymorphism in the IL-4 gene and G1082A polymorphism in the IL-10 gene were studied through polymerase chain reaction. Circulating levels of IL-2, IL-4 and IL-10 in venous blood were estimated using ELISA. RESULTS: Prior to treatment, patients with PT showed significant increase of IL-2 levels and decrease of IL-4 and IL-10 levels compared to apparently healthy subjects. Circulating IL-4 and IL-10 levels were significantly decreased whilst serum IL-2 level was significantly increased in patients with MDR TB compared to non-MDR TB. Low IL-4 and IL-10 secretion and considerable IL-2 alterations were shown to be significantly associated with mutations of homozygous and heterozygous genotypes affecting C589T polymorphism in the IL-4 gene, G1082A polymorphism in the IL-10 gene and T330G polymorphism in the IL-2 gene in patients with PT. CONCLUSIONS: Heterozygous genotype and mutations homozygous genotypes gene in polymorphisms determining specified cytokines' production is a PT risk factor and may lead to disease progression into chronic phase. Heterozygous genotype of aforementioned cytokine genetic polymorphisms was significantly the most frequent in patients with MDR TB.

ATP-gated P2X3 receptors constitute a positive autocrine signal for insulin release in the human pancreatic beta cell.

Extracellular ATP has been proposed as a paracrine signal in rodent islets, but it is unclear what role ATP plays in human islets. We now show the presence of an ATP signaling pathway that enhances the human beta cell's sensitivity and responsiveness to glucose fluctuations. By using in situ hybridization, RT-PCR, immunohistochemistry, and Western blotting as well as recordings of cytoplasmic-free Ca(2+) concentration, [Ca(2+)](i), and hormone release in vitro, we show that human beta cells express ionotropic ATP receptors of the P2X(3) type and that activation of these receptors by ATP coreleased with insulin amplifies glucose-induced insulin secretion. Released ATP activates P2X(3) receptors in the beta-cell plasma membrane, resulting in increased [Ca(2+)](i) and enhanced insulin secretion. Therefore, in human islets, released ATP forms a positive autocrine feedback loop that sensitizes the beta cell's secretory machinery. This may explain how the human pancreatic beta cell can respond so effectively to relatively modest changes in glucose concentration under physiological conditions in vivo.

Possible role of an ischemic preconditioning-like response mechanism in K(ATP) channel opener-mediated protection against streptozotocin-induced suppression of rat pancreatic islet function.

Potassium channel openers (KCOs) decrease insulin secretion from beta-cells. Some KCOs also protect against damage to beta-cell function and type 1 diabetes in animal models. Previously we have found that the KCO NNC 55-0118 counteracted islet cell dysfunction, and this was associated with a lowering of the mitochondrial membrane potential (Deltapsi). Presently we aimed to explore whether inhibition of insulin secretion per se or rather inhibition of mitochondrial function correlates to counteraction of beta-cell suppression. For this we used two novel KCOs (NNC 55-0321 and NNC 55-0462), which at certain concentrations have different actions regarding insulin secretion and the Deltapsi, with NNC 55-0321 being a potent inhibitor of Deltapsi and NNC 55-0462 being a potent inhibitor of insulin secretion. At 10 microM NNC 55-0321, but not with NNC 55-0462, the islet ATP content and ATP/ADP ratio was acutely decreased. This was accompanied by a complete protection against streptozotocin-induced suppression of islet insulin secretion using the former KCO. In cardiac research KCOs have been used to induce an ischemic preconditioning (IPC) response. In line with an IPC-like mechanism we found that NNC 55-0321 induced an initial free oxygen radical formation, PKC-epsilon isoform activation and a subsequent phosphorylation of the survival promoting factor Akt. Thus, KCOs may elicit mitochondrial events that resemble classical IPC seen in cardiomyocytes, and this could explain the enhanced islet cell function observed. KCOs with this property may be particularly interesting compounds to study as a rescue therapy during acute episodes of beta-cell suppression/destruction.

Transforming growth factor-beta-activated protein kinase 1-binding protein (TAB)-1alpha, but not TAB1beta, mediates cytokine-induced p38 mitogen-activated protein kinase phosphorylation and cell death in insulin-producing cells.

Previous studies have indicated that the p38 MAPK participates in signaling events that lead to the death of the insulin-producing beta-cell. The aim of the present study was to elucidate the role of the TGF-beta-activated protein kinase 1-binding protein 1 (TAB1) in the cytokine-induced activation of p38. Levels of TAB1 mRNA and protein were analyzed by real-time PCR and immunoblotting, and TAB1 expression in mouse and human islet cells was down-regulated using lipofection of diced-small interfering RNA. TAB1 overexpression in beta-TC6 cells was achieved by transient transfections followed by fluorescence activated cell sorting. Phosphorylation of p38, c-Jun N-terminal kinase, and ERK was assessed by immunoblotting, and viability was determined using vital staining with bisbenzimide and propidium iodide. We observed that TAB1 is expressed in insulin-producing cells. Cytokine (IL-1beta + interferon-gamma)-stimulated p38 phosphorylation was significantly increased by TAB1alpha overexpression, but not TAB1beta overexpression, in beta-TC6 cells. The TAB1alpha-augmented p38 phosphorylation was paralleled by an increased cell death rate. Treatment of islet cells with diced-small interfering RNA specific for TAB1, but not for TGF-beta-activated kinase 1, resulted in lowered cytokine-induced p38 phosphorylation and protection against cell death. The cytokine-induced phosphorylation of c-Jun N-terminal kinase and ERK was not affected by changes in TAB1 levels. Finally, TAB1 phosphorylation was decreased by the p38 inhibitor SB203580. We conclude that TAB1alpha, but not TAB1beta, plays an important role in the activation of p38 in insulin-producing cells and therefore also in cytokine-induced beta-cell death.

Imatinib mesylate (Gleevec) protects against streptozotocin-induced diabetes and islet cell death in vitro.

The tyrosine kinase inhibitor imatinib mesylate (Gleevec) has been demonstrated to protect various cell types from death by inhibition of Abelson tyrosine kinase (c-Abl). The aim of the present study was to establish whether imatinib protects the insulin producing beta-cell from the different apoptosis promoting agents in vitro and whether imatinib counteracts streptozotocin-induced diabetes in NMRI mice. We observe that imatinib attenuated the actions of several different death promoting substances. In addition, mice injected with streptozotocin did not develop diabetes when given imatinib. The beneficial effects of imatinib may be related to inhibition of the pro-apoptotic MAP kinase JNK. We conclude that imatinib protects against beta-cell death and that this may contribute to the previously reported anti-diabetic actions of imatinib.

Role of MKK3 and p38 MAPK in cytokine-induced death of insulin-producing cells.

The aim of the present investigation was to elucidate further the importance of p38 MAPK (mitogen-activated protein kinase) in nitric oxide- and cytokine-induced beta-cell death. For this purpose, isolated human islets were treated with d-siRNA (diced small interfering RNA) and then exposed to the nitric oxide donor DETA/NONOate [2,2'-(hydroxynitrosohydrazono)bis-ethanamine]. We observed that cells treated with p38alpha-specific d-siRNA, but not with d-siRNA targeting GL3 (a firefly luciferase siRNA plasmid) or PKCdelta (protein kinase Cdelta), were protected against nitric oxide-induced death. This was paralleled by an increased level of Bcl-XL (B-cell leukaemia/lymphoma-X long). For an in-depth study of the mechanisms of p38 activation, MKK3 (MAPK kinase 3), MKK6 and their dominant-negative mutants were overexpressed in insulin-producing RIN-5AH cells. In transient transfections, MKK3 overexpression resulted in increased p38 phosphorylation, whereas in stable MKK3-overexpressing RIN-5AH clones, the protein levels of p38 and JNK (c-Jun N-terminal kinase) were decreased, resulting in unaffected phospho-p38 levels. In addition, a long-term MKK3 overexpression did not affect cell death rates in response to the cytokines interleukin-1beta and interferon-gamma, whereas a short-term MKK3 expression resulted in increased cytokine-induced RIN-5AH cell death. The MKK3-potentiating effect on cytokine-induced cell death was abolished by a nitric oxide synthase inhibitor, and MKK3-stimulated p38 phosphorylation was enhanced by inhibitors of phosphatases. Finally, as the dominant-negative mutant of MKK3 did not affect cytokine-induced p38 phosphorylation, and as wild-type MKK3 did not influence p38 autophosphorylation, it may be that p38 is activated by MKK3/6-independent pathways in response to cytokines and nitric oxide. In addition, it is likely that a long-term increase in p38 activity is counteracted by both a decreased expression of the p38, JNK and p42 genes as well as an increased dephosphorylation of p38.

Role of TAB1 in nitric oxide-induced p38 activation in insulin-producing cells.

The aim of present study was to elucidate the role of TAB1 in nitric oxide-induced activation of p38 MAPK. For this purpose we over-expressed TAB1 in insulin-producing beta-TC6 cells. We observed in cells transiently over-expressing TAB1 that p38 activation was enhanced in response to DETA/NONOate. A lowering of TAB1 levels, using the siRNA technique, resulted in the opposite effect. The DETA/NONOate-induced cell death rate was increased in cells transiently overexpressing TAB1. In stable beta-TC6 cell clones with very high TAB1 levels p38 phosphorylation was enhanced also at basal conditions. DETA/NONOate increased also the phosphorylation of JNK and ERK in beta-TC6 cells, but these events were not affected by TAB1. Interestingly, the inhibitory effect of SB203580 on p38 phosphorylation was paralleled by a stimulatory effect on JNK phosphorylation and an inhibitory effect on ERK phosphorylation. In summary, we propose that TAB1 promotes nitric oxide-induced p38 autophosphorylation. In addition, nitric oxide-induced p38 activation seems to promote JNK inhibition and ERK activation, but this effect appears to not require TAB1. A better understanding of how the TAB1/p38 pathway promotes beta-cell death in response to nitric oxide might help in the development of novel pharmacological approaches in the treatment of diabetes.

Effect of cytokines on ICAM-1 and ZO-1 expression on human airway epithelial cells.

The presence of adhesion molecules on airway epithelial cells may be important in recruiting leukocytes to the epithelium. The study aimed at investigating the effects of interleukin (IL)-4, IL-8, IL-13 and interferon-gamma (IFN-gamma) on cell viability and intracellular adhesion molecule (ICAM)-1 and zonula occludens protein (ZO)-1 expression on cultured human basal and columnar airway epithelial cells. Cycloheximide (CHX) induced cell death in both cell lines. The cytokines IL-4, IL-8, IL-13 and IFN-gamma had only minor effects on cell proliferation in the columnar 16HBE14o-cells, and inhibited the effects of CHX on cell death. IFN-gamma increased ICAM-1 expression in both cell lines. Western blot analysis showed that CHX inhibited both ICAM-1 and ZO-1 expression in the basal cell line. A combination of IL-4 and IFN-gamma appeared to break the tight junctions. IL-4 and IL-13 potentiated CHX-induced apoptosis in basal cells but not in columnar cells, possibly due to low levels of the IL-4 receptor. It is concluded that cytokines produced by airway epithelium may have a role in regulating sequestering of leukocytes to the airways during airway inflammation.

Distinct organization of the candidate tumor suppressor gene RFP2 in human and mouse: multiple mRNA isoforms in both species- and human-specific antisense transcript RFP2OS.

In the present study, we describe the human and mouse RFP2 gene structure, multiple RFP2 mRNA isoforms in the two species that have different 5' UTRs and a human-specific antisense transcript RFP2OS. Since the human RFP2 5' UTR is not conserved in mouse, these findings might indicate a different regulation of RFP2 in the two species. The predicted human and mouse RFP2 proteins are shown to contain a tripartite RING finger-B-box-coiled-coil domain (RBCC), also known as a TRIM domain, and therefore belong to a subgroup of RING finger proteins that are often involved in developmental and tumorigenic processes. Because homozygous deletions of chromosomal region 13q14.3 are found in a number of malignancies, including chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), we suggest that RFP2 might be involved in tumor development. This study provides necessary information for evaluation of the role of RFP2 in malignant transformation and other biological processes.

Overexpression of the Shb SH2 domain-protein in insulin-producing cells leads to altered signaling through the IRS-1 and IRS-2 proteins.

BACKGROUND: Overexpression of the Src homology 2 domain protein Shb in beta-cells of transgenic mice has been shown to promote an increased beta-cell mass. To investigate the mechanisms by which Shb controls the beta-cell mass, we have presently studied the effects of Shb overexpression on the IRS-1-induced signaling pathway in mouse islet beta-cells and in insulin-producing RINm5F cells and correlated these effects to growth and death patterns. MATERIALS AND METHODS: Shb overexpression was achieved in RINm5F cells by selection of stable clones or by FACS purification of transiently transfected cells. For Shb overexpression in primary mouse islet cells, a Shb-transgene mouse was used. Cell proliferation and death rates were determined using flow cytometry. Serum-, insulin-, and IGF-1-stimulated signaling events were studied by immunoblot, immunoprecipitation, and in vitro kinase procedures. RESULTS: Transient Shb overexpression in RINm5F cells resulted in increased proliferation. Both Shb-overexpressing RINm5F cells and islet cells from transgenic mice (islet Shb) exhibited increased basal tyrosine phosphorylation of IRS-1. Shb overexpression resulted also in the assembly and activation of a multiunit complex consisting of at least Shb, IRS-1, IRS-2, FAK, and PI3K. Consequently, the phosphorylation of Akt was enhanced under basal conditions in Shb overexpressing cells. Finally, Shb overexpression did not affect insulin-induced phosphorylation of the PI3K-antagonist PTEN. CONCLUSION: It is concluded that the Shb-induced alterations in the IRS-1/PI3K/Akt pathway may be relevant to the understanding of growth and death patterns of insulin-producing cells.