Bisphenol A (BPA) aggravates multiple low-dose streptozotocin-induced Type 1 diabetes in C57BL/6 mice.

Type 1 diabetes (T1D) is a T-cell-mediated autoimmune disorder characterized by destruction of insulin-producing pancreatic ß-cells. Whereas epidemiological data implicate environmental factors in the increasing incidence of T1D, their identity remains unknown. Though exposure to bisphenol A (BPA) has been associated with several disorders, no epidemiologic evidence has linked BPA exposure and T1D. The goal of this study was to elucidate diabetogenic potentials of BPA and underlying mechanisms in the context of T-cell immunity, in a multiple low-dose streptozotocin (MLDSTZ)-induced autoimmune mouse T1D model. C57BL/6 mice were orally exposed to 1 or 10 mg BPA/L starting at 4 wk of age; diabetes was induced at 9 wk of age with STZ. T-cell composition, function, and insulitis levels were studied at Days 11 and 50 during diabetes development (i.e. post-first STZ injection). Results showed both BPA doses increased diabetes incidence and affected T-cell immunity. However, mechanisms of diabetogenic action appeared divergent based on dose. Low-dose BPA fits a profile of an agent that exhibits pro-diabetogenic effects via T-cell immunomodulation in the early stages of disease development, i.e. decreases in splenic T-cell subpopulations [especially CD4+ T-cells] along with a trend in elevation of splenic T-cell formation of pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-6). In contrast, high-dose BPA did not affect T-cell populations and led to decreased levels of IFN-γ and TNF-α. Both treatments did not affect insulitis levels at the disease early stage, but aggravated it later on. By the study end, besides decreasing T-cell proliferative capacity, low-dose BPA did not affect other T-cell-related parameters, including cytokine secretion, comparable to the effects of high-dose BPA. In conclusion, this study confirmed BPA as a potential diabetogenic compound with immunomodulatory mechanisms of action - in the context of T-cell immunity - that seemed to be dose dependent in the early immunopathogenesis of a MLDSTZ-induced model of T1D.

Exposure to polychlorinated biphenyl-153 decreases incidence of autoimmune Type 1 diabetes in non-obese diabetic mice.

Type 1 diabetes (T1D) incidence has been steadily rising across the globe. Exposure to persistent organic pollutants (POP) has been implied as one potential cause of increased T1D occurrence. Since data regarding the role of POP polychlorinated biphenyl-153 (PCB-153) in autoimmune T1D development in experimental animal models are lacking, this study sought to evaluate the effect of PCB-153 exposure on T1D development in a non-obese diabetic (NOD) mouse model. As T1D is an autoimmune, T-cell-dependent disease, PCB-153 effects on T-cells were studied as well. Pre-diabetic 8-9-week-old NOD mice were exposed to intraperitoneal injections of PCB-153 in a 10-day short- (subacute exposure; 0.5 or 50 mg/kg) or 16-week long-term (subchronic exposure; 0.125 or 12.5 mg/kg) fashion. A significant decrease in incidence of T1D was observed in both low- and high-dose subchronically exposed mice. Analysis of various immune parameters, including T-cell types and subtypes, T-cell proliferative responses - as well as their cytokine secretions, revealed that both short- and long-term exposure to PCB-153 caused significant immunosuppression. PCB-153-induced immunosuppression was reflected in reductions in levels of T helper (TH)-type cells and their functions after subacute treatment with low- and high-dose PCB-153. In agreement, decreased levels of TH cells, reduced proliferation and IL-2 secretion seemed to be a mechanism of PCB-153 action in the development of T1D in subchronically exposed mice. In conclusion, this study for the first time revealed the effects of PCB-153 on development of T1D, bridging the existing experimental knowledge gap regarding the association of non-dioxin-like PCBs and T1D.

Exposure to DDT metabolite p,p'-DDE increases autoimmune type 1 diabetes incidence in NOD mouse model.

The incidence of autoimmune Type 1 diabetes (T1D) has been steadily rising in developed countries. Although the exact cause of T1D remains elusive, it is known that both genetics and environmental factors play a role in its immunopathogenesis. Whereas a positive association between p,p'-DDE, a DDT metabolite, and Type 2 diabetes (T2D) has been well established, its role in T1D development in an experimental animal model has never been elucidated. This study seeks to investigate the effects of DDE exposure on the development of T1D in a NOD mouse model. As T1D is a T-cell-mediated disease, the underlying mechanism of DDE action on T-cells was studied in vitro and, in the context of acute and chronic DDE exposure, in vivo by investigating lymphocytes' viability, proliferation, their subsets and cytokine profiles. Chronic high-dose DDE treatment, initiated in pre-diabetic 8-week-old NOD females administered twice weekly intraperitoneally with 50 mg/kg DDE, significantly increased diabetes incidence and augmented disease severity in treated animals. Whereas T-cell proliferation and cell viability in the spleens of treated mice were not affected, diabetogenic action of chronic DDE exposure was associated with a decrease in regulatory T-cells and a suppression of secretion of protective cytokines, such as IL-4 and IL-10. Interestingly, an acute high-dose in vivo treatment of 8-week-old NOD males with 100 mg DDE/kg, administered intraperitoneally every other day over a period of 10 days, increased T-cell proliferation and potentiated pro-inflammatory and TH1-type cytokine secretion, without affecting the splenocytes viability and the T-cell sub-populations. These results confirm that high-dose DDE treatments affect the immune system, in particularly T-cell function. In conclusion, this study shows for the first time that high-dose chronic DDE exposure exhibits a diabetogenic potential, with an underlying immunomodulatory mechanism of action, in the development of T1D in an experimental mouse NOD model.

Complex mixtures, complex responses: Assessing pharmaceutical mixtures using field and laboratory approaches.

Pharmaceuticals are present in low concentrations (<100 ng/L) in most municipal wastewater effluents but may be elevated locally because of factors such as input from pharmaceutical formulation facilities. Using existing concentration data, the authors assessed pharmaceuticals in laboratory exposures of fathead minnows (Pimephales promelas) and added environmental complexity through effluent exposures. In the laboratory, larval and mature minnows were exposed to a simple opioid mixture (hydrocodone, methadone, and oxycodone), an opioid agonist (tramadol), a muscle relaxant (methocarbamol), a simple antidepressant mixture (fluoxetine, paroxetine, venlafaxine), a sleep aid (temazepam), or a complex mixture of all compounds. Larval minnow response to effluent exposure was not consistent. The 2010 exposures resulted in shorter exposed minnow larvae, whereas the larvae exposed in 2012 exhibited altered escape behavior. Mature minnows exhibited altered hepatosomatic indices, with the strongest effects in females and in mixture exposures. In addition, laboratory-exposed, mature male minnows exposed to all pharmaceuticals (except the selective serotonin reuptake inhibitor mixture) defended nest sites less rigorously than fish in the control group. Tramadol or antidepressant mixture exposure resulted in increased splenic T lymphocytes. Only male minnows exposed to whole effluent responded with increased plasma vitellogenin concentrations. Female minnows exposed to pharmaceuticals (except the opioid mixture) had larger livers, likely as a compensatory result of greater prominence of vacuoles in liver hepatocytes. The observed alteration of apical endpoints central to sustaining fish populations confirms that effluents containing waste streams from pharmaceutical formulation facilities can adversely impact fish populations but that the effects may not be temporally consistent. The present study highlights the importance of including diverse biological endpoints spanning levels of biological organization and life stages when assessing contaminant interactions.

In vivo pharmacokinetic features, toxicity profile, and chemosensitizing activity of alpha-cyano-beta-hydroxy-beta- methyl-N-(2,5-dibromophenyl)propenamide (LFM-A13), a novel antileukemic agent targeting Bruton's tyrosine kinase.

The purpose of the present study was to examine the in vivo pharmacokinetics and activity of alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide (LFM-A13), a novel antileukemic agent targeting Bruton's tyrosine kinase (BTK). We have applied an analytical high-performance liquid chromatography method for the quantitative detection of LFM-A13 in plasma samples. Our findings indicate that LFM-A13 is quickly absorbed, with the time required to reach the maximum plasma drug concentration (t(max)) being 10-18 min after i.p. administration with nearly complete bioavailability. LFM-A13 had an elimination half-life of 17-32 min after i.p. administration at dose levels of 10-50 mg/kg. LFM-A13 exhibited a dose-dependent and significant increase in the values of normalized area under the curve and maximum concentration (C(max)) as well as a dose-dependent and significant decrease in clearance values, suggesting a saturable clearance mechanism. LFM-A13 was not toxic to mice when administered systemically at dose levels ranging from 10 to 80 mg/kg. Highly effective BTK-inhibitory and apoptosis-promoting plasma concentrations of LFM-A13 could be achieved in mice without toxicity. LFM-A13 exhibited a favorable pharmacokinetic behavior that was not adversely affected by the standard chemotherapy drugs vincristine, methylprednisolone, or L-asparaginase (when used as combination treatment, VPL) and significantly improved the chemotherapy response and survival outcome of mice challenged with BCL-1 leukemia cells. Whereas only 14% of mice treated with the standard triple-drug combination VPL became long-term survivors, 41% of mice treated with this combination plus LFM-A13 survived long-term. LFM-A13 prolonged the median survival time of VPL-treated mice from 37 to 58 days. Our results confirm and extend previous studies regarding the role of BTK chemotherapy resistance of B-lineage leukemic cells (S. Mahajan et al., J. Biol. Chem., 274: 9587-9599, 1999). BTK inhibitors such as LFM-A13 may be useful as a new class of chemosensitizing and apoptosis-promoting antileukemic agents for treatment of patients with chemotherapy-resistant B-lineage leukemias or lymphomas.

The family of toxin-related ecto-ADP-ribosyltransferases in humans and the mouse.

ADP-ribosyltransferases including toxins secreted by Vibrio cholera, Pseudomonas aerurginosa, and other pathogenic bacteria inactivate the function of human target proteins by attaching ADP-ribose onto a critical amino acid residue. Cross-species polymerase chain reaction (PCR) and database mining identified the orthologs of these ADP-ribosylating toxins in humans and the mouse. The human genome contains four functional toxin-related ADP-ribosyltransferase genes (ARTs) and two related intron-containing pseudogenes; the mouse has six functional orthologs. The human and mouse ART genes map to chromosomal regions with conserved linkage synteny. The individual ART genes reveal highly restricted expression patterns, which are largely conserved in humans and the mouse. We confirmed the predicted extracellular location of the ART proteins by expressing recombinant ARTs in insect cells. Two human and four mouse ARTs contain the active site motif (R-S-EXE) typical of arginine-specific ADP-ribosyltransferases and exhibit the predicted enzyme activities. Two other human ARTs and their murine orthologues deviate in the active site motif and lack detectable enzyme activity. Conceivably, these ARTs may have acquired a new specificity or function. The position-sensitive iterative database search program PSI-BLAST connected the mammalian ARTs with most known bacterial ADP-ribosylating toxins. In contrast, no related open reading frames occur in the four completed genomes of lower eucaryotes (yeast, worm, fly, and mustard weed). Interestingly, these organisms also lack genes for ADP-ribosylhydrolases, the enzymes that reverse protein ADP-ribosylation. This suggests that the two enzyme families that catalyze reversible mono-ADP-ribosylation either were lost from the genomes of these nonchordata eucaryotes or were subject to horizontal gene transfer between kingdoms.

Treatment of post-bone marrow transplant acute graft-versus-host disease with a rationally designed JAK3 inhibitor.

Here we show that the Janus kinase 3 (JAK3) inhibitor 4-(3'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (JANEX-3) exhibits potent anti-GVHD activity and consequently improves the post-BMT survival outcome of C57BL/6 (H-2b) recipient mice transplanted with allogeneic bone marrow/splenocyte (BM/S) grafts from MHC disparate BALB/c mice (H-2d). One hundred percent of the vehicle-treated allograft recipients developed severe GVHD and died with a median survival of 41 days. Treatment of recipient mice with JANEX-3 (30 mg/kg/day, 3 x/day) after the onset of rapidly progressive severe GVHD in the 3rd week after BMT significantly improved the survival of BMT recipients with GVHD and prolonged the median survival time to 78 days (P < 0.0001, log-rank test). The probability of survival at two and three months post-BMT was 6 +/- 6% and 0 +/- 0% for vehicle-treated control mice and 100 +/- 0% and 38 +/- 17% for mice treated with JANEX-3. These results prompted the hypothesis that JAK3 plays a pivotal role in the pathophysiology of GVHD. To test this hypothesis, we examined if mice transplanted with allogeneic BM/S grafts from Jak3 knockout mice Jak3-/- develop GVHD. The allografts from (Jak3-/-) C57BL/6 (H-2b) mice rescued MHC-disparate recipient BALB/c mice (H-2d) of the lethal toxicity of TBI without causing fatal GVHD. Taken together, these observations establish JAK3 as a key mediator of severe GVHD after allogeneic BMT in the context of a major-HLA disparity.

Targeting Janus kinase 3 in the treatment of leukemia and inflammatory diseases.

Janus tyrosine kinases (JAKs) are cytoplasmic protein tyrosine kinases that play a crucial role in the initial steps of cytokine signaling. JAK3, a member of JAK kinase family of four (JAK1, JAK2, JAK3 and TYK2), is abundantly expressed in lymphoid cells. JAK3 has been found to initiate signaling of interleukin (IL)-2, IL-4, IL-7, IL-9, IL-13 and IL-15. Indispensable role of JAK3 in lymphocyte development and function has been revealed recently. Because of the involvement of JAK3 in T cell activation and proliferation, and the documented genetic evidence for the role of JAK3 in autoimmune or transplant -induced inflammatory disorders, the selective targeting of JAK3 in T cells may potentially be clinically beneficial in T cell-derived pathologic disorders. In this review we discuss inhibitors of JAK3 as a new class of immunomodulatory agents with immunosuppressive, anti-inflammatory, anti-allergic, and anti-leukemic properties. Preclinical data from multiple experimental model systems of autoimmune diabetes, allergy, solid organ transplantation, pancreatic islet transplantation and bone marrow transplantation are discussed in the context of the clinical need for new immunomodulatory agents with such properties.

Targeting Janus tyrosine kinase 3 (JAK3) with an inhibitor induces secretion of TGF-ß by CD4+ T cells.

Regulatory T cells (Tregs) are critical for the peripheral maintenance of the autoreactive T cells in autoimmune disorders such as type 1 diabetes (T1D). Pharmacological inhibition of Janus tyrosine kinase 3 (JAK3) has been proposed as a basis for new treatment modalities against autoimmunity and allogeneic responses. Targeting JAK3 with an inhibitor has previously been shown to exhibit protective action against the development of T1D in non-obese diabetic (NOD) mice. As the mechanism of such preventative action has been unknown, we hypothesized that JAK3 inhibition induces generation of Tregs. Here, we show that the JAK3 inhibitor 4-(4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P131) suppresses proliferation of short-term cultured NOD CD4(+) T cells through induction of apoptosis, while promoting survival of a particular population of long-term cultured cells. It was found that the surviving cells were not of the CD4(+)CD25(+)FoxP3(+) phenotype. They secreted decreased amounts of IL-10, IL-4 and interferon (IFN)-γ compared to the cells not exposed to the optimal concentrations of JAK3 inhibitor. However, an elevated transforming growth factor (TGF)-ß secretion was detected in their supernatants. In vivo treatment of prediabetic NOD mice with WHI-P131 did not affect the frequency and number of splenic and pancreatic lymph node CD4(+)FoxP3(+) Tregs, while generating an elevated numbers of CD4(+)FoxP3(-) TGF-ß-secreting T cells. In conclusion, our data suggest an induction of TGF-ß-secreting CD4(+) T cells as the underlying mechanism for antidiabetogenic effects obtained by the treatment with a JAK3 inhibitor. To our knowledge, this is the first report of the JAK3 inhibitor activity in the context of the murine Tregs.

Effect of targeted disruption of signal transducer and activator of transcription (Stat)4 and Stat6 genes on the autoimmune diabetes development induced by multiple low doses of streptozotocin.

The MLDS (multiple low doses of streptozotocin) model of diabetes was induced in Stat4(-/-), Stat6(-/-), and double-deficient Stat4(-/-)/6(-/-) mice to examine the role of STAT4/STAT6 deficiency in development of autoimmune diabetes. Cytokine production of T-cells from Stat4(-/-) mice confirmed a predominantly Th2-type immune response. Stat4(-/-) mice exhibited delayed onset and reduced severity of disease compared to wild-type (WT) mice. In contrast, STAT6 deficiency, with a predominant Th1 response, did not influence the kinetics or severity of MLDS-induced autoimmune diabetes. Interestingly, Stat4(-/-)/6(-/-) mice, with a prominent Th1-type response, experienced an accelerated and aggravated course of diabetes after MLDS, implicating a STAT4-independent Th1 response in the immunopathogenesis of MLDS-induced autoimmune diabetes. The sensitivity of islet cells from Stat4(-/-) or Stat4(-/-)/6(-/-) mice to cytokines and STZ was not different from that of islet cells of WT mice. Hence, the observed effects of STAT4 and STAT4/6 deficiency on MLDS-induced autoimmune diabetes are likely due to their effects on T-cell responses.

Dual targeting of Bruton's tyrosine kinase and Janus kinase 3 with rationally designed inhibitors prevents graft-versus-host disease (GVHD) in a murine allogeneic bone marrow transplantation model.

The purpose of the present study was to evaluate the effectiveness of targeting Bruton's tyrosine kinase (BTK) with a specific BTK inhibitor, alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)-propenamide (LFM-A13), for prevention of acute fatal graft-versus-host disease (GVHD) in a murine model of allogeneic bone marrow transplantation (BMT). Vehicle-treated control C57BL/6 mice receiving bone marrow/splenocyte grafts from allogeneic BALB/c donors developed severe multi-organ acute GVHD and died after a median survival time (MST) of 40 d. LFM-A13 treatment (25 mg/kg/d) significantly prolonged the MST of the BMT recipients to 47 d. The probability of survival at 2 months after BMT was 2 +/- 2% for vehicle-treated control mice and 22 +/- 6% for mice treated with LFM-A13 (P = 0.0008). Notably, the combination regimen of LFM-A13 plus the standard anti-GVHD drug methotrexate (MTX) (10 mg/m(2)/d) was more effective than LFM-A13 alone, while the combination regimen of LFM-A13 plus the novel anti-GVHD drug JANEX-1 (60 mg/kg/d), targeting Janus kinase 3, was more effective than LFM-A13, JANEX-1 or MTX alone. More than 70% of recipients receiving this most effective GVHD prophylaxis (LFM-A13 + JANEX-1) remained alive throughout the 80-d observation period with an MST of >80 d. Taken together, these results indicate that targeting BTK with the chemical inhibitor LFM-A13 may attenuate the severity of GVHD, especially when it is combined with other anti-GVHD drugs, such as MTX and JANEX-1.

Prevention of islet allograft rejection in diabetic mice by targeting Janus Kinase 3 with 4-(4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (JANEX-1).

Janus kinase (JAK) 3-deficient mice were not able to reject allogeneic islet allografts. The JAK3 inhibitor 4-(4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (CAS 202475-60-3, JANEX-1, WHI-P131) prevented the rejection of islet allografts in mice with a normal JAK3 expression status. The combination of JANEX-1 and cyclosporin A (CAS 59865-13-3) was more effective than either agent alone.

Janus kinase 3 inhibitor WHI-P131/JANEX-1 prevents graft-versus-host disease but spares the graft-versus-leukemia function of the bone marrow allografts in a murine bone marrow transplantation model.

The purpose of the present study was to evaluate the effects of graft-versus-host disease (GVHD) prophylaxis with the Janus kinase 3 (JAK3) inhibitor WHI-P131/JANEX-1 on the graft-versus-leukemic (GVL) function of marrow allografts in mice undergoing bone marrow transplantation (BMT) after being challenged with an otherwise invariably fatal dose of BCL-1 leukemia cells. GVHD prophylaxis using WHI-P131 markedly improved the survival outcome after BMT. The probability of survival at 30 days after BMT was 11% +/- 6% for vehicle-treated recipients (median survival time, 25 days) versus 63% +/- 12% for recipients treated with WHI-P131 (median survival time, 36 days; P <.0001). Because WHI-P131 is devoid of antileukemic activity against BCL-1 leukemia cells, this marked improvement in survival outcome was due to reduced incidence of GVHD-associated fatalities combined with sustained GVL function of the allografts in the WHI-P131 group. Notably, adoptive transfer experiments demonstrated that the spleens of WHI-P131-treated allograft recipients contained less than 0.001% BCL-1 cells. Notably, GVHD prophylaxis with WHI-P131 plus methotrexate resulted in 100% survival of mice receiving allotransplants challenged with an otherwise invariably fatal dose of BCL-1 leukemia. Taken together, our results provide strong experimental evidence that GVHD prophylaxis using WHI-P131 does not impair the GVL function of the allografts and consequently contributes to an improved post-BMT survival outcome of the recipient mice.

Targeting JAK3 with JANEX-1 for prevention of autoimmune type 1 diabetes in NOD mice.

Here we show that Janus kinase (JAK) 3 is an important molecular target for treatment of autoimmune insulin-dependent (type 1) diabetes mellitus. The rationally designed JAK3 inhibitor JANEX-1 exhibited potent immunomodulatory activity and delayed the onset of diabetes in the NOD mouse model of autoimmune type 1 diabetes. Whereas 60% of vehicle-treated control NOD mice became diabetic by 25 weeks, the incidence of diabetes at 25 weeks was only 9% for NOD females treated with daily injections of JANEX-1 (100 mg/kg/day) from Week 10 through Week 25 (P = 0.007). Furthermore, JANEX-1 prevented the development of insulitis and diabetes in NOD-scid/scid females after adoptive transfer of splenocytes from diabetic NOD females. Chemical inhibitors such as JANEX-1 may provide the basis for effective treatment modalities against human type 1 diabetes. To our knowledge, this is the first report of the immunosuppressive activity of a JAK3 inhibitor in the context of an autoimmune disease.