Norman H. Altman

A low interleukin-2 receptor signaling threshold supports the development and homeostasis of T regulatory cells.

Interleukin-2 receptor (IL-2R) signaling is essential for T regulatory (Treg) cell development and homeostasis. Here, we show that expression of IL-2Rbeta chains that lack tyrosine residues important for the association of the adaptor Shc and the transcription factor STAT5 in IL-2Rbeta-deficient mice resulted in production of a normal proportion of natural Treg cells that suppressed severe autoimmunity related with deficiency in IL-2 or IL-2R. These mutant IL-2Rbeta chains supported suboptimal and transient STAT5 activation that upregulate the transcription factor Foxp3 to normal amounts in natural, but not induced, Treg cells. Nevertheless, gene expression profiling revealed many targets in peripheral natural Treg cells that were IL-2 dependent and a substantial overlap between the Treg cell IL-2-dependent gene program and the Treg cell transcriptional signature. Collectively, these findings demonstrate that a critical, and perhaps minor, subset of IL-2-dependent targets is indexed to a low IL-2R signaling threshold and that a substantial proportion of the Treg cell gene program is regulated by IL-2.

ANTITUMOR ACTIVITY OF HYALURONIC ACID SYNTHESIS INHIBITOR 4-METHYLUMBELLIFERONE IN PROSTATE CANCER CELLS

4-Methylumbelliferone (4-MU) is a hyaluronic acid (HA) synthesis inhibitor with anticancer properties; the mechanism of its anticancer effects is unknown. We evaluated the effects of 4-MU on prostate cancer cells. 4-MU inhibited proliferation, motility, and invasion of DU145, PC3-ML, LNCaP, C4-2B, and/or LAPC-4 cells. At IC(50) for HA synthesis (0.4 mmol/L), 4-MU induced >3-fold apoptosis in prostate cancer cells, which could be prevented by the addition of HA. 4-MU induced caspase-8, caspase-9, and caspase-3 activation, PARP cleavage, upregulation of Fas-L, Fas, FADD and DR4, and downregulation of bcl-2, phosphorylated bad, bcl-XL, phosphorylated Akt, phosphorylated IKB, phosphorylated ErbB2, and phosphorylated epidermal growth factor receptor. At IC(50), 4-MU also caused >90% inhibition of NF-kappaB reporter activity, which was prevented partially by the addition of HA. With the exception of caveolin-1, HA reversed the 4-MU-induced downregulation of HA receptors (CD44 and RHAMM), matrix-degrading enzymes (MMP-2 and MMP-9), interleukin-8, and chemokine receptors (CXCR1, CXCR4, and CXCR7) at the protein and mRNA levels. Expression of myristoylated-Akt rescued 4-MU-induced apoptosis and inhibition of cell growth and interleukin-8, RHAMM, HAS2, CD44, and MMP-9 expression. Oral administration of 4-MU significantly decreased PC3-ML tumor growth (>3-fold) when treatment was started either on the day of tumor cell injection or after the tumors became palpable, without organ toxicity, changes in serum chemistry, or body weight. Tumors from 4-MU-treated animals showed reduced microvessel density ( approximately 3-fold) and HA expression but increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells and expression of apoptosis-related molecules. Therefore, the anticancer effects of 4-MU, an orally bioavailable and relatively nontoxic agent, are primarily mediated by inhibition of HA signaling.

Potentiation of metastasis by cell surface sialomucin complex (rat MUC4), a multifunctional anti-adhesive glycoprotein

Sialomucin complex (SMC), a rat homologue of the human mucin MUC4, is a large membrane‐bound mucin complex, originally isolated from highly metastatic ascites 13762 mammary adenocarcinoma cells. When overexpressed, SMC exerts potent anti‐adhesive effects, which sterically disrupt molecular interactions for cell‐cell and cell‐ECM adhesions. SMC similarly suppresses anti‐tumor immunity by inhibition of interactions between cytotoxic lymphocytes and target tumor cells. Previously, recombinant cDNAs for SMC were transfected and inducibly expressed in A375 human melanoma cells using a tetracycline‐responsive expression system. In the current studies, we investigated the role of MUC4/SMC in tumor metastasis by regulating SMC expression of tumor transplants in vivo. Intravenous injection of SMC‐overexpressing cells resulted in substantially greater lung metastasis than injection of SMC‐repressed cells. Injection of SMC‐overexpressing cells followed by in vivo downregulation of SMC did not lower the frequency of lung metastasis. Growth of the micrometastatic lesions was the same for all 3 cases in short‐term (3‐week) assays. Further, subcutaneous injection of A375 cells followed by in vivo induction of SMC overexpression within the solid tumor resulted in spontaneous distant metastasis. These studies suggest that SMC potentiates metastasis by contributing to the establishment of metastatic foci. These studies directly demonstrate for the first time that tumor metastasis can be modulated by the regulation of MUC4/SMC expression. Int. J. Cancer 87:480–486, 2000.

Targeted and controlled anticancer drug delivery and release with magnetoelectric nanoparticles.

It is a challenge to eradicate tumor cells while sparing normal cells. We used magnetoelectric nanoparticles (MENs) to control drug delivery and release. The physics is due to electric-field interactions (i) between MENs and a drug and (ii) between drug-loaded MENs and cells. MENs distinguish cancer cells from normal cells through the membrane’s electric properties; cancer cells have a significantly smaller threshold field to induce electroporation. In vitro and in vivo studies (nude mice with SKOV-3 xenografts) showed that (i) drug (paclitaxel (PTX)) could be attached to MENs (30-nm CoFe2O4@BaTiO3 nanostructures) through surface functionalization to avoid its premature release, (ii) drug-loaded MENs could be delivered into cancer cells via application of a d.c. field (~100 Oe), and (iii) the drug could be released off MENs on demand via application of an a.c. field (~50 Oe, 100 Hz). The cell lysate content was measured with scanning probe microscopy and spectrophotometry. MENs and control ferromagnetic and polymer nanoparticles conjugated with HER2-neu antibodies, all loaded with PTX were weekly administrated intravenously. Only the mice treated with PTX-loaded MENs (15/200 μg) in a field for three months were completely cured, as confirmed through infrared imaging and post-euthanasia histology studies via energy-dispersive spectroscopy and immunohistochemistry.

Host innate recognition of an intestinal bacterial pathogen induces TRIF-dependent protective immunity

TRIF signaling triggers the amplification of macrophage bactericidal activity sufficient to eliminate invading intestinal pathogens through the sequential induction of IFN-β and IFN-γ from macrophages and NK cells, respectively.

Diagnosis of Enteritis and Enterotoxemia due to Clostridium Difficile in Captive Ostriches (Struthio Camelus)

petted normal ranges. Seven of the expired birds were necropsied immediately after death. Two live ostriches with clinical signs were euthanized, and tissues were obtained for histopathology, bacterial culture, and viral isolation. At necropsy, all of the ostriches examined had similar gross lesions that differed only in severity. The colon and cecum were markedly dilated and diffusely hemorrhagic and contained no formed feces. The lungs were variably congested, and the liver was frequently pale yellow in color. Two of the birds had hemorrhagic foci in the proventriculus. No other significant gross lesions were observed in any of the birds. Microscopic examination of the tissues revealed an acute, severe, necrotizing typhlitis and colitis in all birds, with massive numbers of large gram-positive bacilli present within glands and in the intestinal lumen (Figs. 1, 2). There was moderate, diffuse hepatocellular vacuolar change in 6 birds and multifocal lymphoid necrosis in the bursa of Fabricius of 2 of the ostriches. No viral inclusions were noted. Moderate to severe congestion was present in the lungs of 7 of 9 birds. Two of the birds had a mild necrotizing proventriculitis with mild hemorrhage. Sections of the intestine were submitted for aerobic and anaerobic culture. Enrichment for Salmonella in selenite broth for 12 hours yielded negative results, and no clinically significant aerobic pathogenic bacteria were found. Two different species of Clostridium were anaerobically cultured from the intestine. They were tentatively identified as C. hastiforme and C. clostridiiforme by API 20A biochemical assay. b After isolation and subculture of the original cultures in cycloserine-cefoxitin-fructose agar and additional biochemical analysis by An-IDENT biochemical assay, b the C. hastiforme culture was shown to be C. difficile. The API-20A biochemical assay was then repeated on the pure culture, and it too indicated the presence of C. difficile. The intestine was submitted for clostridial enterotoxin assay c by enzyme-linked immunosorbent assay (ELISA). Extracts of multiple sections

CD4(+) CD25(+) Foxp3(+) T regulatory cells with limited TCR diversity in control of autoimmunity.

The importance of high TCR diversity of T regulatory (Treg) cells for self-tolerance is poorly understood. To address this issue, TCR diversity was measured for Treg cells after transfer into IL-2Rβ−/− mice, which develop lethal autoimmunity because of failed production of Treg cells. In this study, we show that high TCR diversity of pretransferred Treg cells led to selection of therapeutic Treg cells with lower TCR diversity that prevented autoimmunity. Pretransferred Treg cells with lower diversity led to selection of Treg cells through substantial peripheral reshaping with even more restricted TCR diversity that also suppressed autoimmune symptoms. Thus, in a setting of severe breakdown of immune tolerance because of failed production of Treg cells, control of autoimmunity is achieved by only a fraction of the Treg TCR repertoire, but the risk for disease increased. These data support a model in which high Treg TCR diversity is a mechanism to ensure establishing and maintaining self-tolerance.

Donor T cells lacking Fas ligand and perforin retain the capacity to induce severe GvHD in minor histocompatibility antigen mismatched bone-marrow transplantation recipients.

Graft-versus-host disease (GvHD) is a frequent impediment to therapeutically successful allogeneic bone-marrow transplantation (BMT). This investigation further examines the roles of two potential donor cytotoxic effector mechanisms previously implicated in tissue pathogenesis. Cytotoxically double deficient (B6-cdd) T cells (lacking functional fas ligand and perforin) and wild-type (B6-wt) donor T-cell transplantation in a minor antigen-mismatched BMT model (C57BL/6 → C3H.SW) resulted in similar mortality and weight loss. Histopathologic findings revealed mononuclear infiltrates and cellular atrophy in GvHD target tissues (liver, stomach) in recipients of B6-wt and B6-cdd donor T cells. Both recipients also exhibited GvH-associated lymphohematopoietic compartment (LHC) alterations as evidenced by inverted CD4:CD8 ratios and B-cell hypoplasia. Notably, transplants using recombinant inbred mHAg disparate recipients demonstrated that B6-cdd T cells induced lethal GvHD in CXBE but not CXBG recipients: the same pattern induced by B6-wt T cells. This observation is consistent with previous findings that cytotoxic T lymphocyte (CTL) responses against CXBG and CXBE antigens did not correlate with GvH responses in these strains. In contrast with the typical pattern of donor T-cell expansion and contraction, T cells lacking perforin and FasL function exhibited extensive expansion postBMT. In summary, these findings support the notion that donor anti-host cytotoxicity by way of the two major pathways is not a prerequisite for induction of GvHD. In addition, the results suggest that this model will be useful to investigate the regulation of allogeneic donor T-cell expansion after major histocompatibility complex-matched allogeneic BMT.

Dietary Supplement 4-Methylumbelliferone: An Effective Chemopreventive and Therapeutic Agent for Prostate Cancer

BACKGROUND Prevention and treatment of advanced prostate cancer (PCa) by a nontoxic agent can improve outcome, while maintaining quality of life. 4-methylumbelliferone (4-MU) is a dietary supplement that inhibits hyaluronic acid (HA) synthesis. We evaluated the chemopreventive and therapeutic efficacy and mechanism of action of 4-MU. METHODS TRAMP mice (7-28 per group) were gavaged with 4-MU (450mg/kg/day) in a stage-specific treatment design (8-28, 12-28, 22-28 weeks). Efficacy of 4-MU (200-450mg/kg/day) was also evaluated in the PC3-ML/Luc(+) intracardiac injection and DU145 subcutaneous models. PCa cells and tissues were analyzed for HA and Phosphoinositide 3-kinase (PI-3K)/Akt signaling and apoptosis effectors. HA add-back and myristoylated Akt (mAkt) overexpression studies evaluated the mechanism of action of 4-MU. Data were analyzed with one-way analysis of variance and unpaired t test or Tukeys multiple comparison test. All statistical tests were two-sided. RESULTS While vehicle-treated transgenic adenocarcinoma of the prostate (TRAMP) mice developed prostate tumors and metastases at 28 weeks, both were abrogated in treatment groups, without serum/organ toxicity or weight loss; no tumors developed at one year, even after stopping the treatment at 28 weeks. 4-MU did not alter the transgene or neuroendocrine marker expression but downregulated HA levels. However, 4-MU decreased microvessel density and proliferative index (P < .0001,). 4-MU completely prevented/inhibited skeletal metastasis in the PC3-ML/Luc(+) model and DU145-tumor growth (85-90% inhibition, P = .002). 4-MU also statistically significantly downregulated HA receptors, PI-3K/CD44 complex and activity, Akt signaling, and β-catenin levels/activation, but upregulated GSK-3 function, E-cadherin, and apoptosis effectors (P < .001); HA addition or mAkt overexpression rescued these effects. CONCLUSION 4-MU is an effective nontoxic, oral chemopreventive, and therapeutic agent that targets PCa development, growth, and metastasis by abrogating HA signaling.

Immunoblastic malignant lymphoma in dolphins: Histologic, ultrastructural, and immunohistochemical features

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