Seroprevalence of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato and <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> in Danish horses

Background

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are able to infect horses. However, the extend to which Danish horses are infected and seroconvert due to these two bacteria is unknown. The aim of the present study was to evaluate the seroprevalence of B. burgdorferi sensu lato and A. phagocytophilum in Danish horses.

Methods

A total of 390 blood samples collected from all major regions of Denmark and with a geographical distribution corresponding to the density of the Danish horse population were analyzed. All samples were examined for the presence of antibodies against B. burgdorferi sensu lato and A. phagocytophilum by the use of the SNAP^®4DX ^® ELISA test.

Results

Overall, 29.0% of the horses were seropositive for B. burgdorferi sensu lato whereas 22.3% were seropositive for A. phagocytophilum.

Conclusions

Antibodies against B burgdorferi sensu lato and A. phagocytophilum are commonly found among Danish horses thus showing that Danish horses are frequently infected by these organisms.

     

Enhanced anti-tumor activity of interferon-alpha in SOCS1-deficient mice is mediated by CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cells

Interferon-alpha (IFN-α) is an immunomodulatory cytokine that is used clinically for the treatment of melanoma in the adjuvant setting. The cellular actions of IFN-α are regulated by the suppressors of cytokine signaling (SOCS) family of proteins. We hypothesized that the anti-tumor activity of exogenous IFN-α would be enhanced in SOCS1-deficient mice. SOCS1-deficient (SOCS1^−/−) or control (SOCS1^+/+) mice on an IFN-γ^−/− C57BL/6 background bearing intraperitoneal (i.p.) JB/MS murine melanoma cells were treated for 30 days with i.p. injections of IFN-A/D or PBS (vehicle). Log-rank Kaplan-Meier survival curves were used to evaluate survival. Tumor-bearing control SOCS1^+/+ mice receiving IFN-A/D had significantly enhanced survival versus PBS–treated mice (P = 0.0048). The anti-tumor effects of IFN-A/D therapy were significantly enhanced in tumor-bearing SOCS1^−/− mice; 75% of these mice survived tumor challenge, whereas PBS-treated SOCS1^−/− mice all died at 13-16 days (P = 0.00038). Antibody (Ab) depletion of CD8⁺ T cells abrogated the anti-tumor effects of IFN-A/D in SOCS1^−/− mice as compared with mice receiving a control antibody (P = 0.0021). CD4⁺ T-cell depletion from SOCS1^−/− mice also inhibited the effects of IFN-A/D (P = 0.0003). IFN-A/D did not alter expression of CD80 or CD86 on splenocytes of SOCS1^+/+ or SOCS1^−/− mice, or the proportion of T regulatory cells or myeloid-derived suppressor cells in SOCS1^+/+ or SOCS1^−/− mice. An analysis of T-cell function did reveal increased proliferation of SOCS1-deficient splenocytes at baseline and in response to mitogenic stimuli. These data suggest that modulation of SOCS1 function in T-cell subsets could enhance the anti-tumor effects of IFN-α in the setting of melanoma.     

Estimates of DNA and protein sequence divergence: an examination of some assumptions

Some of the assumptions underlying estimates of DNA and protein sequence divergence are examined. A solution for the variance of these estimates that allows for different mutation rates and different population sizes in each species and for an arbitrary structure in the initial population is obtained. It is shown that these conditions do not strongly affect estimates of divergence. In general, they cause the variance of divergence to be smaller than a binomial variance. Thus, the binomial variance that is usually assumed for these estimates is safely conservative. It is shown that variability in the mutation rate among sites can have an effect as large as or larger than variability in the mutation rate among bases. Variability in the mutation rate among bases and among sites causes the number of substitutions between two sequences to be underestimated. Protein and DNA sequences from several species are collected to estimate the variability in mutation rates among sites. When many homologous sequences are known, standard methods to estimate this variability can be used. The estimates of this variability show that this factor is important when considering the spectrum of spontaneous mutations and is strongly reflected in the divergence of sequences. Smaller variability is found for the third position of codons than for the first and second codon positions. This may be because of less selective constraints on this position or because the third position has been saturated with mutations for the sequences examined.      

Novel vaccine strategies to T-independent antigens

T cell independent antigens do not require T cell help to induce an immune response, and are characterized by a lack of immunologic memory. These antigens can be divided into two classes, TI-1 or TI-2. TI-1 antigens, such as bacterial lipopolysaccharide, are potent B-cell mitogens, capable of non-specific, polyclonal activation of B cells. In contrast, TI-2 antigens can only activate mature B cells and consist of highly repetitive structures, such as capsular polysaccharides (CPS) from bacteria. Many vaccines currently in use consist of purified capsular polysaccharides from pathogenic bacteria such as Streptococcus pneumoniae and Neisseria meningitidis. These vaccines are efficacious in immune-competent adults, however, due to their TI-2 nature, are not effective in children <2 years of age. Converting polysaccharides into T cell dependent (TD) antigens, allows children, <2, to produce an effective immune response. This review focuses on various strategies used to convert the immune response to polysaccharide antigens from TI-2 to a TD response. Conjugate vaccines, anti-idiotypic antibodies, phage display library technology and DNA vaccines are discussed.     

Distinct myeloid suppressor cell subsets correlate with plasma IL-6 and IL-10 and reduced interferon-alpha signaling in CD4<Superscript>+</Superscript> T cells from patients with GI malignancy

Interferon-alpha (IFN-α) promotes anti-tumor immunity through its actions on immune cells. We hypothesized that elevated percentages of myeloid-derived suppressor cells (MDSC) and increased pro-inflammatory cytokines in peripheral blood would be associated with impaired response to IFN-α in patients with gastrointestinal (GI) malignancies. This study evaluated relationships between plasma IL-6, IL-10, circulating MDSC subsets, and IFN-α-induced signal transduction in 40 patients with GI malignancies. Plasma IL-6 and IL-10 were significantly higher in patients versus normal donors. CD33⁺HLADR⁻CD11b⁺CD15⁺ and CD33⁺HLADR^−/lowCD14⁺ MDSC subsets were also elevated in patients versus normal donors (P < 0.0001). Plasma IL-6 was correlated with CD33⁺HLADR⁻CD15⁺ MDSC (P = 0.008) and IL-10 with CD33⁺HLADR⁻CD15⁻ MDSC (P = 0.002). The percentage of CD15⁺ and CD15⁻ but not CD14⁺ MDSC subsets were inversely correlated with IFN-α-induced STAT1 phosphorylation in CD4⁺ T cells, while co-culture with in vitro generated MDSC led to reduced IFN-α responsiveness in both PBMC and the CD4⁺ subset of T cells from normal donors. Exploratory multivariable Cox proportional hazards models revealed that an increased percentage of the CD33⁺HLADR⁻CD15⁻ MDSC subset was associated with reduced overall survival (P = 0.049), while an increased percentage of the CD33⁺HLADR^−/lowCD14⁺ subset was associated with greater overall survival (P = 0.033). These data provide evidence for a unique relationship between specific cytokines, MDSC subsets, and IFN-α responsiveness in patients with GI malignancies.     

Novel Small Molecule XPO1/CRM1 Inhibitors Induce Nuclear Accumulation of TP53, Phosphorylated MAPK and Apoptosis in Human Melanoma Cells

XPO1/CRM1 is a key nuclear exporter protein that mediates translocation of numerous cellular regulatory proteins. We investigated whether XPO1 is a potential therapeutic target in melanoma using novel selective inhibitors of nuclear export (SINE). In vitro effects of SINE on cell growth and apoptosis were measured by MTS assay and flow cytometry [Annexin V/propidium iodide (PI)], respectively in human metastatic melanoma cell lines. Immunoblot analysis was used to measure nuclear localization of key cellular proteins. The in vivo activity of oral SINE was evaluated in NOD/SCID mice bearing A375 or CHL-1 human melanoma xenografts. SINE compounds induced cytostatic and pro-apoptotic effects in both BRAF wild type and mutant (V600E) cell lines at nanomolar concentrations. The cytostatic and pro-apoptotic effects of XPO1 inhibition were associated with nuclear accumulation of TP53, and CDKN1A induction in the A375 cell line with wild type TP53, while pMAPK accumulated in the nucleus regardless of TP53 status. The orally bioavailable KPT-276 and KPT-330 compounds significantly inhibited growth of A375 (p<0.0001) and CHL-1 (p = 0.0087) human melanoma cell lines in vivo at well tolerated doses. Inhibition of XPO1 using SINE represents a potential therapeutic approach for melanoma across cells with diverse molecular phenotypes by promoting growth inhibition and apoptosis.