The posttraumatic response of CD4+ regulatory T cells is modulated by direct cell-cell contact via CD40L- and P-selectin-dependent pathways.

CD4+ FoxP3+ regulatory T cells (CD4+ Tregs) are important for the posttraumatic anti-inflammatory host response. As described previously, platelets are able to modulate CD4+ Treg activity in a reciprocally activating interaction following injury. The underlying mechanisms of the posttraumatic interaction between platelets and CD4+ Tregs remain unclear. We investigated the potential influence of CD40L and P-selectin, molecules known to be involved in direct cell contact of these cell types. In a murine burn injury model, the potential interaction pathways were addressed using CD40L- and P-selectin-deficient mice. Draining lymph nodes were harvested following trauma (1 h) and following a sham procedure. Early rapid activation of CD4+ Tregs was assessed by phospho-flow cytometry (signaling molecules (p)PKC-δ and (p)ZAP-70). Platelet function was analyzed performing rotational thromboelastometry (ROTEM). We hypothesized that disruption of the direct cell-cell contact via CD40L and P-selectin would affect posttraumatic activation of CD4+ Tregs and influence the hemostatic function of platelets. Indeed, while injury induced early activation of CD4+ Tregs in wild-type mice (ZAP-70: p = 0.13, pZAP-70: p < 0.05, PKC-δ: p < 0.05, pPKC-δ: p < 0.05), disruption of CD40L-dependent interaction (ZAP-70: p = 0.57, pZAP-70: p = 0.68, PKC-δ: p = 0.68, pPKC-δ: p = 0.9) or P-selectin-dependent interaction (ZAP-70: p = 0.78, pZAP-70: p = 0.58, PKC-δ: p = 0.81, pPKC-δ: p = 0.73) resulted in reduced posttraumatic activation. Furthermore, hemostatic function was impaired towards hypocoagulability in either deficiency. Our results suggest that the posttraumatic activation of CD4+ Tregs and hemostatic function of platelets are affected by direct cell-cell-signaling via CD40L and P-selectin.

Horizontal Gene Transfer and Tandem Duplication Shape the Unique CAZyme Complement of the Mycoparasitic Oomycetes Pythium oligandrum and Pythium periplocum.

Crop protection strategies that are effective but that reduce our reliance on chemical pesticides are urgently needed to meet the UN sustainable development goals for global food security. Mycoparasitic oomycetes such as Pythium oligandrum and Pythium periplocum, have potential for the biological control of plant diseases that threaten crops and have attracted much attention due to their abilities to antagonize plant pathogens and modulate plant immunity. Studies of the molecular and genetic determinants of mycoparasitism in these species have been less well developed than those of their fungal counterparts. Carbohydrate-active enzymes (CAZymes) from P. oligandrum and P. periplocum are predicted to be important components of mycoparasitism, being involved in the degradation of the cell wall of their oomycete and fungal prey species. To explore the evolution of CAZymes of these species we performed an in silico identification and comparison of the full CAZyme complement (CAZyome) of the two mycoparasitic Pythium species (P. oligandrum and P. periplocum), with seven other Pythium species, and four Phytophthora species. Twenty CAZy gene families involved in the degradation of cellulose, hemicellulose, glucan, and chitin were expanded in, or unique to, mycoparasitic Pythium species and several of these genes were expressed during mycoparasitic interactions with either oomycete or fungal prey, as revealed by RNA sequencing and quantitative qRT-PCR. Genes from three of the cellulose and chitin degrading CAZy families (namely AA9, GH5_14, and GH19) were expanded via tandem duplication and predominantly located in gene sparse regions of the genome, suggesting these enzymes are putative pathogenicity factors able to undergo rapid evolution. In addition, five of the CAZy gene families were likely to have been obtained from other microbes by horizontal gene transfer events. The mycoparasitic species are able to utilize complex carbohydrates present in fungal cell walls, namely chitin and N-acetylglucosamine for growth, in contrast to their phytopathogenic counterparts. Nonetheless, a preference for the utilization of simple sugars for growth appears to be a common trait within the oomycete lineage.

Extradigital glomus tumor of the anterior knee.

The benign glomus tumor is an uncommon cause of crippling pain most commonly associated with the fingers. This sheep in wolf's clothing is identified by careful examination, confirmed by MRI, and often resolved with a simple procedure. Here we present a patient with chronic knee pain of 21 years duration.

Evidence for safe tourniquet use in 500 consecutive upper extremity procedures.

BACKGROUND: Although pneumatic tourniquets are widely used in upper extremity surgery, further evidence is needed to support their safe use. Excessive pressure and prolonged ischemic time can cause soft-tissue injury. The purpose of this study was to determine the safety of tourniquet use in a yearlong, consecutive series of patients. METHODS: A retrospective review of all patients who underwent upper extremity surgery by two board-certified hand surgeons over a 1-year period was performed. Demographic variables, comorbidities, and complications were noted along with tourniquet parameters, including application site, ischemic pressure, and time. RESULTS: A total 505 patients were included in the study because a tourniquet was used during their operation. Patients ranged in age from 3 months to 90 years old (mean 40.1 years). More than half of the population was overweight (mean body mass index (BMI) 27.1), and 77.1 % of adults had at least one cardiac risk factor. No immediate or delayed tourniquet-related injuries were identified. The average operative time was 35.9 min, with an average tourniquet time of 33.1 min. Tourniquet inflation pressure of 250 or 225 mmHg was utilized in 78 and 21 % of adult patients, respectively; no patients had a pressure setting exceeding 275 mmHg. CONCLUSION: In this series of more than 500 operations, there were no immediate or delayed tourniquet-related events using parameters determined perioperatively by the attending surgeon. Tourniquet pressures of 250 mmHg or less in adult patients with less than 2 h of ischemic time appear to be safe, even in the elderly and patients with multiple medical comorbidities.

Candida glabrata tryptophan-based pigment production via the Ehrlich pathway.

Pigments contribute to the pathogenicity of many fungi, mainly by protecting fungal cells from host defence activities. Here, we have dissected the biosynthetic pathway of a tryptophan-derived pigment of the human pathogen Candida glabrata, identified key genes involved in pigment production and have begun to elucidate the possible biological function of the pigment. Using transcriptional analyses and a transposon insertion library, we have identified genes associated with pigment production. Targeted deletion mutants revealed that the pigment is a by-product of the Ehrlich pathway of tryptophan degradation: a mutant lacking a tryptophan-upregulated aromatic aminotransferase (Aro8) displayed significantly reduced pigmentation and a recombinantly expressed version of this protein was sufficient for pigment production in vitro. Pigment production is tightly regulated as the synthesis is affected by the presence of alternative nitrogen sources, carbon sources, cyclic AMP and oxygen. Growth of C. glabrata on pigment inducing medium leads to an increased resistance to hydrogen peroxide, an effect which was not observed with a mutant defective in pigmentation. Furthermore, pigmented yeast cells had a higher survival rate when exposed to human neutrophils and caused increased damage in a monolayer model of human epithelia, indicating a possible role of pigmentation during interactions with host cells.

Mutually antagonistic effects of androgen and activin in the regulation of prostate cancer cell growth.

Activin, a member of the TGFbeta superfamily, is expressed in the prostate and inhibits growth. We demonstrate that the effects of activin and androgen on regulation of prostate cancer cell growth are mutually antagonistic. In the absence of androgen, activin induced apoptosis in the androgen-dependent human prostate cancer cell line LNCaP, an effect suppressed by androgen administration. Although activin by itself did not alter the cell cycle distribution, it potently suppressed androgen- induced progression of cells into S-phase of the cell cycle and thus inhibited androgen-stimulated growth of LNCaP cells. Expression changes in cell cycle regulatory proteins such as Rb, E2F-1, and p27 demonstrated a strong correlation with the mutually antagonistic growth regulatory effects of activin and androgen. The inhibitory effect of activin on growth was independent of serine, serine, valine, serine motif phosphorylation of Smad3. Despite their antagonistic effect on growth, activin and androgen costimulated the expression of prostate-specific antigen through a Smad3-mediated mechanism. These observations indicate the existence of a complex cross talk between activin and androgen signaling in regulation of gene expression and growth of the prostate.

Persistent Mullerian duct syndrome caused by both a 27-bp deletion and a novel splice mutation in the MIS type II receptor gene.

BACKGROUND: Persistent Mullerian duct syndrome (PMDS) is a rare form of male pseudohermaphroditism that is characterized by the persistence of Mullerian derivatives in otherwise normally virilized males. Mutations of the Mullerian inhibiting substance (MIS) gene or the MIS type II receptor (MISRII) gene have been identified in PMDS patients with autosomal recessive transmission. We analyzed a compound heterozygote PMDS patient who had a 27-bp deletion in exon 10 in one allele and a novel mutation in intron 5 in the other allele of the MISRII gene. METHODS: Whole blood and tissue samples were obtained from a one-month-old 46,XY male with persistent PMDS and the MISRII gene was sequenced and compared to his mother's genomic DNA and that of 22 normal individuals. Serum MIS and the reproductive hormones were measured by standard immunoassays. RESULTS: The patient's hormone levels were normal but the gene for MISRII contained several mutations, a 27-bp deletion in exon 10 on one allele (one of the most common mutations in PMDS) and a novel mutation in intron 5 in the other allele that altered splicing, resulting in retention of the intron and a frameshift, introducing a stop codon. Other mutations in introns 6 and 9 and in exon 11 might not be functionally significant. CONCLUSIONS: This case reveals a novel mutation in the MISRII gene involving intronic sequences, which when coexisting with the already identified 27-bp deletion in exon 10, leads to PMDS.

Highly purified müllerian inhibiting substance inhibits human ovarian cancer in vivo.

PURPOSE: Müllerian inhibiting substance (MIS) causes Müllerianduct regression in mammalian, avian, and reptilian embryos; MIS also inhibits growth in vitro of Müllerian-derived cell lines and primary cells from human ovarian carcinomas. We hypothesize that highly purified MIS delivered parenterally inhibits ovarian cancer in vivo. EXPERIMENTAL DESIGN: To test the efficacy of highly purified MIS against ovarian cancer cell lines in vivo, we treated immunosuppressed mice with MIS after implanting OVCAR 8 or IGROV 1 human ovarian cancer cells beneath the renal capsules and measured tumor volume over time. Animals were treated with daily injections of 10 micro g of purified exogenous recombinant human MIS or by endogenous MIS secreted from cells growing on biodegradable mesh. RESULTS: The average graft size ratio (change in size compared with starting size) of the OVCAR 8 tumor implants was larger in the control animals than in animals treated for 2 weeks (P < 0.019) or 3 weeks (P < 0.001) with parenteral MIS, or after treating with MIS produced from transfected cells, which impregnated the biodegradable mesh (P = 0.02). The average graft size ratio of the IGROV 1 tumors was also larger in the control animals than in those treated with injected MIS (P = 0.0174). CONCLUSIONS: Highly purified recombinant human MIS, delivered parenterally, or MIS produced endogenously causes inhibition of human ovarian cancer cell lines in vivo, providing convincing preclinical evidence to support the use of MIS as a parenteral agent for the treatment of ovarian cancer.

Early follicular serum müllerian-inhibiting substance levels are associated with ovarian response during assisted reproductive technology cycles.

OBJECTIVE: To test the hypothesis that the concentration of early follicular phase serum müllerian-inhibiting substance (MIS) is associated with ovarian response in women undergoing ovulation induction in preparation for assisted reproductive technology (ART). DESIGN: Retrospective analysis of frozen day 3 serum samples. SETTING: Academic ART program. PATIENT(S): One sample of frozen day 3 serum from women with < or = 6 retrieved oocytes (n = 28) compared with women with > or = 11 oocytes retrieved (n = 79) in preparation for IVF. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Comparison of day 3 serum MIS levels between two groups of women. Other comparisons included maximum serum E(2) concentrations, number of retrieved oocytes, and percentage of mature oocytes between groups. RESULT(S): Mean serum MIS concentrations were 1.0 +/- 0.4 ng/mL compared with 2.5 +/- 0.3 ng/mL, or more than a 2.5-fold greater serum concentration of MIS in the group with > or = 11 oocytes retrieved compared with in the group with < or = 6 retrieved oocytes. CONCLUSION(S): These data demonstrate an association between early follicular phase serum MIS and the number of retrieved oocytes. Higher day 3 serum MIS concentrations were associated with greater number of retrieved oocytes.