Combining Antigen-Based Therapy with GABA Treatment Synergistically Prolongs Survival of Transplanted ?-Cells in Diabetic NOD Mice

Antigen-based therapies (ABTs) very effectively prevent the development of type 1 diabetes (T1D) when given to young nonobese diabetic (NOD) mice, however, they have little or no ability to reverse hyperglycemia in newly diabetic NOD mice. More importantly, ABTs have not yet demonstrated an ability to effectively preserve residual ß-cells in individuals newly diagnosed with type 1 diabetes (T1D). Accordingly, there is great interest in identifying new treatments that can be combined with ABTs to safely protect ß-cells in diabetic animals. The activation of γ-aminobutyric acid (GABA) receptors (GABA-Rs) on immune cells has been shown to prevent T1D, experimental autoimmune encephalomyelitis (EAE) and rheumatoid arthritis in mouse models. Based on GABA''s ability to inhibit different autoimmune diseases and its safety profile, we tested whether the combination of ABT with GABA treatment could prolong the survival of transplanted ß-cells in newly diabetic NOD mice. Newly diabetic NOD mice were untreated, or given GAD/alum (20 or 100 µg) and placed on plain drinking water, or water containing GABA (2 or 6 mg/ml). Twenty-eight days later, they received syngenic pancreas grafts and were monitored for the recurrence of hyperglycemia. Hyperglycemia reoccurred in the recipients given plain water, GAD monotherapy, GABA monotherapy, GAD (20 µg)+GABA (2 mg/ml), GAD (20 µg)+GABA (6 mg/ml) and GAD (100 µg)+GABA (6 mg/ml) about 1, 2-3, 3, 2-3, 3-8 and 10-11 weeks post-transplantation, respectively. Thus, combined GABA and ABT treatment had a synergistic effect in a dose-dependent fashion. These findings suggest that co-treatment with GABA (or other GABA-R agonists) may provide a new strategy to safely enhance the efficacy of other therapeutics designed to prevent or reverse T1D, as well as other T cell-mediated autoimmune diseases.     

Expression and Aggregation of Recombinant Human Consensus Interferon-α Mutant by Pichia pastoris

A recombinant human consensus interferon-α mutant (cIFN) was expressed in Pichia pastoris. The maximum dry cell weight, cIFN concentration and antiviral activity were 160 g l⁻¹, 1.24 g l⁻¹ and 4.1 × 10⁷ IU ml⁻¹, respec tively. The cIFN secreted into the medium was in the form of aggregates dominantly by non-covalent interaction and partially by disulphide bond. When the fermentation supernatant was disaggregated with 6 M guanidine hydrochloride, the antiviral activity of cIFN achieved 2.2 × 10⁸ IU ml⁻¹.     

Interferon-γ Induces Expression of MHC Class II on Intestinal Epithelial Cells and Protects Mice from Colitis

Immune responses against intestinal microbiota contribute to the pathogenesis of inflammatory bowel diseases (IBD) and involve CD4⁺ T cells, which are activated by major histocompatibility complex class II (MHCII) molecules on antigen-presenting cells (APCs). However, it is largely unexplored how inflammation-induced MHCII expression by intestinal epithelial cells (IEC) affects CD4⁺ T cell-mediated immunity or tolerance induction in vivo. Here, we investigated how epithelial MHCII expression is induced and how a deficiency in inducible epithelial MHCII expression alters susceptibility to colitis and the outcome of colon-specific immune responses. Colitis was induced in mice that lacked inducible expression of MHCII molecules on all nonhematopoietic cells, or specifically on IECs, by continuous infection with Helicobacter hepaticus and administration of interleukin (IL)-10 receptor-blocking antibodies (anti-IL10R mAb). To assess the role of interferon (IFN)-γ in inducing epithelial MHCII expression, the T cell adoptive transfer model of colitis was used. Abrogation of MHCII expression by nonhematopoietic cells or IECs induces colitis associated with increased colonic frequencies of innate immune cells and expression of proinflammatory cytokines. CD4⁺ T-helper type (Th)1 cells - but not group 3 innate lymphoid cells (ILCs) or Th17 cells - are elevated, resulting in an unfavourably altered ratio between CD4⁺ T cells and forkhead box P3 (FoxP3)⁺ regulatory T (Treg) cells. IFN-γ produced mainly by CD4⁺ T cells is required to upregulate MHCII expression by IECs. These results suggest that, in addition to its proinflammatory roles, IFN-γ exerts a critical anti-inflammatory function in the intestine which protects against colitis by inducing MHCII expression on IECs. This may explain the failure of anti-IFN-γ treatment to induce remission in IBD patients, despite the association of elevated IFN-γ and IBD.     

Activation of Myocardial Phosphoinositide-3-Kinase p110α Ameliorates Cardiac Dysfunction and Improves Survival in Polymicrobial Sepsis

Phosphoinositide-3-kinase (PI3K)/Akt dependent signaling has been shown to improve outcome in sepsis/septic shock. There is also ample evidence that PI3K/Akt dependent signaling plays a crucial role in maintaining normal cardiac function. We hypothesized that PI3K/Akt signaling may ameliorate septic shock by attenuating sepsis-induced cardiac dysfunction. Cardiac function and survival were evaluated in transgenic mice with cardiac myocyte specific expression of constitutively active PI3K isoform, p110α (caPI3K Tg). caPI3K Tg and wild type (WT) mice were subjected to cecal ligation/puncture (CLP) induced sepsis. Wild type CLP mice showed dramatic cardiac dysfunction at 6 hrs. Septic cardiomyopathy was significantly attenuated in caPI3K CLP mice. The time to 100% mortality was 46 hrs in WT CLP mice. In contrast, 80% of the caPI3K mice survived at 46 hrs after CLP (p<0.01) and 50% survived >30 days (p<0.01). Cardiac caPI3K expression prevented expression of an inflammatory phenotype in CLP sepsis. Organ neutrophil infiltration and lung apoptosis were also effectively inhibited by cardiac PI3k p110α expression. Cardiac high mobility group box–1 (HMGB-1) translocation was also inhibited by caPI3K p110α expression. We conclude that cardiac specific activation of PI3k/Akt dependent signaling can significantly modify the morbidity and mortality associated with sepsis. Our data also indicate that myocardial function/dysfunction plays a prominent role in the pathogenesis of sepsis and that maintenance of cardiac function during sepsis is essential. Finally, these data suggest that modulation of the PI3K/p110α signaling pathway may be beneficial in the prevention and/or management of septic cardiomyopathy and septic shock.     

Expression of GDNF and GDNFR-α mRNAs in Muscles of Patients with Motor Neuron Diseases

The mRNA expression levels of GDNF, GDNFR- and RET were examined in the muscles of amyotrophic lateral screlosis (ALS) and X-linked spinal and bulbar muscular atrophy (SBMA). GDNF mRNA levels were significantly elevated to variable extent in the diseased muscles compared to control muscles, although they were not specific to the type of the diseases. The diseased muscles also have a different expression pattern of GDNF mRNA isoforms from controls. GDNF mRNA expression, however, tended to reduce in advanced muscle pathology. On the other hand, GDNFR- mRNA levels were not changed significantly on expression levels in the diseased muscles. In situ hybridization study revealed that GDNF and GDNFR- mRNAs were localized in subsarcolemmal space of muscle cells. RET mRNA was not detected in control nor diseased muscles. These results suggest that the elevated muscle GDNF acts as a trophic signal for motor neurons of motor neuron diseases, implying a possible therapeutic implication of GDNF to this type of diseases.(Drs. Yamamoto, N. Mitsuma, Inukai, Ito, Li, Sobue)     

l-3-n-Butylphthalide Activates Akt/mTOR Signaling,Inhibits Neuronal Apoptosis and Autophagy and Improves Cognitive Impairment in Mice with Repeated Cerebral Ischemia–Reperfusion Injury

l-3-n-Butylphthalide (l-NBP) exerts neuroprotective effects in animal models of cerebral ischemia, but its potential benefits in repeated cerebral ischemia–reperfusion (RCIR) injury remain unknown. We investigated the effect of l-NBP on cognitive impairment induced by RCIR in mice. Male C57Bl/6 mice received sham surgery or bilateral common carotid artery occlusion (3 times, 20 min each) and were orally administered preoperative l-NBP (30 mg/kg/day, 7 days), postoperative l-NBP (30 or 60 mg/kg/day, 28 days) or postoperative vehicle (28 days). Learning and memory were assessed by the Morris water maze task and step-down passive avoidance test. Nissl staining was used to identify pathologic changes in the hippocampal CA1 region. The expressions of proteins associated with signaling, apoptosis and autophagy were assessed by quantitative PCR and western blot. RCIR induced deficits in learning and memory that were alleviated by preoperative or postoperative l-NBP administration. Pathologic lesions in the hippocampal CA1 region induced by RCIR were less severe in mice treated with l-NBP. Preoperative or postoperative l-NBP administration in mice receiving RCIR promoted hippocampal expression of phospho-Akt and phospho-mTOR (suggesting activation of Akt/mTOR signaling), increased the Bcl-2/Bax ratio (indicating suppression of apoptosis) and reduced the LC3-II/LC3-I ratio (implying inhibition of autophagy). Preoperative or postoperative l-NBP administration also depressed hippocampal levels of beclin-1 mRNA (indicating suppression of autophagy). These findings suggest that the effect of l-NBP to alleviate learning and memory deficits in mice following RCIR may involve activation of Akt/mTOR signaling and regulation of the expressions of proteins related to apoptosis and autophagy.

     

The Tolerogenic Peptide,hCDR1, Down-Regulates the Expression of Interferon-α in Murine and Human Systemic Lupus Erythematosus

Background

The tolerogenic peptide, hCDR1, ameliorated manifestations of systemic lupus erythematosus (SLE) via the immunomodulation of pro-inflammatory and immunosuppressive cytokines and the induction of regulatory T cells. Because type I interferon (IFN-α) has been implicated to play a role in SLE pathogenesis, we investigated the effects of hCDR1 on IFN-α in a murine model of SLE and in human lupus.

Methodology/Principal Findings

(NZBxNZW)F1 mice with established SLE were treated with hCDR1 (10 weekly injections). Splenocytes were obtained for gene expression studies by real-time RT-PCR. hCDR1 down-regulated significantly IFN-α gene expression (73% inhibition compared to vehicle treated mice, p = 0.002) in association with diminished clinical manifestations. Further, hCDR1 reduced, in vitro, IFN-α gene expression in peripheral blood mononuclear cells (PBMC) of 10 lupus patients (74% inhibition compared to medium, p = 0.002) but had no significant effects on the expression levels of IFN-α in PBMC of primary anti-phospholipid syndrome patients or of healthy controls. Lupus patients were treated for 24 weeks with hCDR1 (5) or placebo (4) by weekly subcutaneous injections. Blood samples collected, before and after treatment, were frozen until mRNA isolation. A significant reduction in IFN-α was determined in hCDR1 treated patients (64.4% inhibition compared to pretreatment expression levels, p = 0.015). No inhibition was observed in the placebo treated patients. In agreement, treatment with hCDR1 resulted in a significant decrease of disease activity. IFN-α appears to play a role in the mechanism of action of hCDR1 since recombinant IFN-α diminished the immunomodulating effects of hCDR1 on IL-1β, TGFβ and FoxP3 gene expression.

Conclusions/Significance

We reported previously that hCDR1 affected various cell types and immune pathways in correlation to disease amelioration. The present studies demonstrate that hCDR1 is also capable of down-regulating significantly (and specifically to lupus) IFN-α gene expression. Thus, hCDR1 has a potential role as a novel, disease specific treatment for lupus.     

Characterization and Identification of Subpopulations of Mononuclear Preosteoclasts Induced by TNF-α in Combination with TGF-β in Rats

Osteoclasts are unique multinucleated cells formed by fusion of preosteoclasts derived from cells of the monocyte/macrophage lineage, which are induced by RANKL. However, characteristics and subpopulations of osteoclast precursor cells are poorly understood. We show here that a combination of TNF-α, TGF-β, and M-CSF efficiently generates mononuclear preosteoclasts but not multinucleated osteoclasts (MNCs) in rat bone marrow cultures depleted of stromal cells. Using a rat osteoclast-specific mAb, Kat1, we found that TNF-α and TGF-β specifically increased Kat1⁺c-fms⁺ and Kat1⁺c-fms⁻ cells but not Kat1⁻c-fms⁺ cells. Kat1⁻c-fms⁺ cells appeared in early stages of culture, but Kat1⁺c-fms⁺ and Kat1⁺c-fms⁻ cells increased later. Preosteoclasts induced by TNF-α, TGF-β, and M-CSF rapidly differentiated into osteoclasts in the presence of RANKL and hydroxyurea, an inhibitor of DNA synthesis, suggesting that preosteoclasts are terminally differentiated cells. We further analyzed the expression levels of genes encoding surface proteins in bone marrow macrophages (BMM), preosteoclasts, and MNCs. Preosteoclasts expressed itgam (CD11b) and chemokine receptors CCR1 and CCR2; however, in preosteoclasts the expression of chemokine receptors CCR1 and CCR2 was not up-regulated compared to their expression in BMM. However, addition of RANKL to preosteoclasts markedly increased the expression of CCR1. In contrast, expression of macrophage antigen emr-1 (F4/80) and chemokine receptor CCR5 was down-regulated in preosteoclasts. The combination of TNF-α, TGF-β, and M-CSF induced Kat1⁺CD11b⁺ cells, but these cells were also induced by TNF-α alone. In addition, MIP-1α and MCP-1, which are ligands for CCR1 and CCR2, were chemotactic for preosteoclasts, and promoted multinucleation of preosteoclasts. Finally, we found that Kat1⁺c-fms⁺ cells were present in bone tissues of rats with adjuvant arthritis. These data demonstrate that TNF-α in combination with TGF-β efficiently generates preosteoclasts in vitro. We delineated characteristics that are useful for identifying and isolating rat preosteoclasts, and found that CCR1 expression was regulated in the fusion step in osteoclastogenesis.     

Rate of Primary Refractory Disease in B and T-Cell Non-Hodgkin’s Lymphoma: Correlation with Long-Term Survival

Background

Primary refractory disease is a main challenge in the management of non-Hodgkin’s Lymphoma (NHL). This survey was performed to define the rate of refractory disease to first-line therapy in B and T-cell NHL subtypes and the long-term survival of primary refractory compared to primary responsive patients.

Methods

Medical records were reviewed of 3,106 patients who had undergone primary treatment for NHL between 1982 and 2012, at the Hematology Centers of Torino and Bergamo, Italy. Primary treatment included CHOP or CHOP-like regimens (63.2%), intensive therapy with autograft (16.9%), or other therapies (19.9%). Among B-cell NHL, 1,356 (47.8%) received first-line chemotherapy with rituximab. Refractory disease was defined as stable/progressive disease, or transient response with disease progression within six months.

Results

Overall, 690 (22.2%) patients showed primary refractory disease, with a higher incidence amongst T-cell compared to B-cell NHL (41.9% vs. 20.5%, respectively, p<0.001). Several other clinico-pathological factors at presentation were variably associated with refractory disease, including histological aggressive disease, unfavorable clinical presentation, Bone Marrow involvement, low lymphocyte/monocyte ration and male gender. Amongst B-cell NHL, the addition of rituximab was associated with a marked reduction of refractory disease (13.6% vs. 26.7% for non-supplemented chemotherapy, p<0.001). Overall, primary responsive patients had a median survival of 19.8 years, compared to 1.3 yr. for refractory patients. A prolonged survival was consistently observed in all primary responsive patients regardless of the histology. The long life expectancy of primary responsive patients was documented in both series managed before and after 2.000. Response to first line therapy resulted by far the most predictive factor for long-term outcome (HR for primary refractory disease: 16.52, p<0.001).

Conclusion

Chemosensitivity to primary treatment is crucial for the long-term survival in NHL. This supports the necessity of studies aimed to early identify refractory disease and to develop different treatment strategies for responsive and refractory patients.     

Cytoplasmic Expression of Pontin in Renal Cell Carcinoma Correlates with Tumor Invasion,Metastasis and Patients’ Survival

Renal cell carcinoma (RCC) is the most lethal of all genitourinary malignancies. Distant metastasis represents the major cause of death in patients with RCC. Recent studies have implicated the AAA+ ATPase pontin in many cellular activities that are highly relevant to carcinogenesis. In this study, we demonstrate for the first time that pontin was up-regulated in RCC, and plays a previously unknown pro-invasive role in the metastatic progression of RCC through epithelial-to-mesenchymal transition (EMT) pathway. 28 pairs of freshly frozen clear cell RCC samples and the matched normal renal tissues analyzed by quantitative RT-PCR and western blotting demonstrated that pontin was up-regulated in clear cell RCC tissues than in normal renal tissues. In addition, immunohistochemistry was used to evaluate subcellular pontin expression in 95 RCC patients, and found that overexpression of pontin in cytoplasm positively correlated with the metastatic features, predicting unfavorable outcomes of RCC patients. Furthermore, in vitro experiments show pontin was predominantly expressed in cytoplasm of RCC cell lines, and a significant suppression of cell migration and invasion in pontin siRNA treated RCC cell lines was observed. Mechanistic studies show that pontin depletion up-regulated the E-cadherin protein and down-regulated vimentin protein, and decreased nuclear β-catenin expression, suggesting the involvement of EMT in pontin induced metastatic progression. Together, our data suggest pontin as a potential prognostic biomarker in RCC, and provide new promising therapeutic targets for clinical intervention of kidney cancers.     

CXCR4 Expression and Treatment with SDF-1α or Plerixafor Modulate Proliferation and Chemosensitivity of Colon Cancer Cells

BACKGROUND: Signaling through stromal cell-derived factor-1α (SDF-1α), strongly secreted by bone marrow stromal cells and the CXC chemokine receptor 4 (CXCR4) exposed on tumor cells has pivotal roles in proliferation, metastasis, and tumor cell “dormancy.” Dormancy is associated with cytostatic drug resistance and is probably a property of tumor stem cells and minimal residual disease. Thus, hampering the SDF-1α/CXCR4 cross talk by a CXCR4 antagonist like Plerixafor (AMD3100) should overcome tumor cell dormancy bymobilization of tumor cells from “sanctuary” niches. Our aim was to elucidate the direct effects exerted by SDF-1α and Plerixafor on proliferation, chemosensitivity, and apoptosis of CXCR4-expressing tumor cells. METHODS: The ability of SDF-1α and Plerixafor to regulate intracellular signaling, proliferation, and invasion was investigated using two colon cancer cell lines (HT-29 and SW480) with either high endogenous or lentiviral expression of CXCR4 compared to their respective low CXCR4-expressing counterparts as a model system. Efficacy of Plerixafor on sensitivity of these cell lines against 5-fluorouracil, irinotecan, or oxaliplatin was determined in a cell viability assay as well as stroma-dependent cytotoxicity and apoptosis assays. RESULTS: SDF-1α increased proliferation, invasion, and ERK signaling of endogenously and lentivirally CXCR4-expressing cells. Exposure to Plerixafor reduced proliferation, invasion, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. Combination of chemotherapy with Plerixafor showed an additive effect on chemosensitivity and apoptosis in CXCR4-overexpressing cells. An SDF-1-secreting feeder layer provideda“protective niche” for CXCR4-overexpressing cells resulting in decreased chemosensitivity. CONCLUSION: CXCR4-antagonistic therapy mobilizes and additionally sensitizes tumor cells toward cytoreductive chemotherapy.     

Copper Deficiency Leads to Anemia,Duodenal Hypoxia,Upregulation of HIF-2α and Altered Expression of Iron Absorption Genes in Mice

Iron and copper are essential trace metals, actively absorbed from the proximal gut in a regulated fashion. Depletion of either metal can lead to anemia. In the gut, copper deficiency can affect iron absorption through modulating the activity of hephaestin - a multi-copper oxidase required for optimal iron export from enterocytes. How systemic copper status regulates iron absorption is unknown. Mice were subjected to a nutritional copper deficiency-induced anemia regime from birth and injected with copper sulphate intraperitoneally to correct the anemia. Copper deficiency resulted in anemia, increased duodenal hypoxia and Hypoxia inducible factor 2α (HIF-2α) levels, a regulator of iron absorption. HIF-2α upregulation in copper deficiency appeared to be independent of duodenal iron or copper levels and correlated with the expression of iron transporters (Ferroportin - Fpn, Divalent Metal transporter – Dmt1) and ferric reductase – Dcytb. Alleviation of copper-dependent anemia with intraperitoneal copper injection resulted in down regulation of HIF-2α-regulated iron absorption genes in the gut. Our work identifies HIF-2α as an important regulator of iron transport machinery in copper deficiency.     

Changes in the Anatomic and Microscopic Structure and the Expression of HIF-1α and VEGF of the Yak Heart with Aging and Hypoxia

The study aimed to identify the changes of anatomic and microscopic structure and the expression and localization of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) in the myocardium and coronary artery of the yak heart adapted to chronic hypoxia with aging. Thirty-two yaks (1 day, 6 months, 1 year, 2 years, and 5 year old) were included, and immunoelectronmicroscopy, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) were used. Right ventricular hypertrophy was not present in yaks with aging. There was no intima thickening phenomenon in the coronary artery. The ultrastructure of myofibrils, mitochondria, and collagen fibers and the diameter and quantity of collagen changed significantly with aging. The enzymatic activity of complexes I, II, and V increased with age. Immunogold labeling showed the localization of HIF-1α protein in the cytoplasm and nuclei of endothelial cells and cytoplasm of cardiac muscle cells, and VEGF protein in the nuclei and perinuclei areas of smooth muscle cells of coronary artery, and in the cytoplasm and nuclei of endothelial cells. ELISA results showed that HIF-1α secretion significantly increased in the myocardium and coronary artery from an age of 1 day to 2 years of yaks and decreased in old yaks. However, VEGF protein always increased with aging. The findings of this study suggest that 6 months is a key age of yak before which there are some adaptive changes to deal with low-oxygen environment, and there is a maturation of the yak heart from the age of 6 months to 2 years.     

Daily Rhythms of TNFα Expression and Food Intake Regulate Synchrony of Plasmodium Stages with the Host Circadian Cycle

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Suppression of NF-κB and NF-κB-Regulated Gene Expression by Apigenin through IκBα and IKK Pathway in TRAMP Mice

Aberrant Nuclear Factor-κappaB (NF-κB) activation due to rapid IκBα turnover and high basal IκBα kinase (IKK) activity has been frequently observed in prostate cancer. Apigenin, a naturally occurring plant flavone, exhibits anti-proliferative, anti-inflammatory and anti-carcinogenic activities by inhibiting NF-κB pathway, through a mechanism not fully understood. We found that apigenin feeding in microgram doses (bioavailable in humans) inhibited prostate tumorigenesis in TRAMP mice by interfering with NF-κB signaling. Apigenin feeding to TRAMP mice (20 and 50 μg/mouse/day, 6 days/week for 20 weeks) exhibited significant decrease in tumor volumes of the prostate and completely abolished metastasis, which correlated with inhibition of NF-κB activation and binding to the DNA. Apigenin intake blocked phosphorylation and degradation of IκBα by inhibiting IKK activation, which in turn led to suppression of NF-κB activation. The expression of NF-κB-regulated gene products involved in proliferation (cyclin D1, and COX-2), anti-apoptosis (Bcl-2 and Bcl-xL), and angiogenesis (vascular endothelial growth factor) were also downregulated after apigenin feeding. These events correlated with the induction of apoptosis in tumor cells, as evident by increased cleaved caspase-3 labeling index in the dorsolateral prostate. Our results provide convincing evidence that apigenin inhibits IKK activation and restores the expression of IκBα, preventing it’s phosphorylation in a fashion similar to that elicited by IKK and proteasomal inhibitors through suppression of NF-κB signaling pathway.