Fecal microbiomes of laboratory beagles receiving antiparasitic formulations in an experimental setting.

Here, we describe the fecal microbiome of laboratory beagles in a non-invasive experiment designed to contrast in vivo versus in vitro bioequivalence in response to antiparasitic drug administration. The experiment provided a unique opportunity to evaluate metagenomic profiles of canine feces before and after anti-parasitic drug exposure.

Successful reconstruction of full-thickness skin defects in a swine model using simultaneous split-thickness skin grafting and composite collagen microstructured dermal scaffolds.

Reconstitution of normal skin anatomy after full-thickness skin loss may be accomplished using a combination of a dermal regeneration template (DRT) and a split thickness skin graft (STSG). However, because of the relatively low rate of cell infiltration and vascularisation of currently available DRTs, reconstruction is almost always performed in a two-step procedure over the course of several weeks, resulting in multiple dressing changes, prolonged immobilisation and increased chance of infection. To mitigate the potential complications of this prolonged process, the collagen-based dermal template DermiSphere™ was developed and tested in a single-step procedure wherein DermiSphere and STSG were implanted simultaneously. When evaluated in a porcine, full thickness, excisional wound model, DermiSphere successfully supported simultaneous split thickness skin graft take and induced functional neodermal tissue deposition. When compared to a market leading product Integra Bilayer Wound Matrix, which was used in a multistep procedure (STSG placed 14 days after product implantation according to the product IFU), DermiSphere induced a similar moderate and transient inflammatory response that produced similar neodermal tissue maturity, thickness and vascularity, despite being implanted in a single surgical procedure leading to wound closure 2 weeks earlier. These data suggest that DermiSphere may be implanted in a single-step procedure with an STSG, which would significantly shorten the time course required for the reconstruction of both dermal and epidermal components of skin after full thickness loss.

Greening automation: Wash and Re-use of disposable 384-well liquid handling tips to enable sustainable high-throughput vaccine development.

Laboratory automation uses large amounts of plastic consumables, generating substantial single-use plastic waste. Automated ELISAs are an indispensable analytical tool in vaccine formulation and process development. Current workflows, however, rely on disposable liquid handling tips. In progress toward sustainability, we developed workflows for washing 384-well format liquid handling tips, using nontoxic reagents, for re-use during ELISA testing. We estimate that this workflow reduces plastic and cardboard waste in our facility by 989 kg/year and 202 kg/year, respectively, without introducing new chemicals into our waste steam.

Identifying access barriers faced by rural and dispersed communities to better address their needs: implications and lessons learned for rural proofing for health in the Americas and beyond.

INTRODUCTION: Universal access to health services and universal health coverage are needed to achieve good health for all, yet rural communities face a variety of access barriers. As part of an effort to 'rural proof' health systems, it is therefore imperative to identify and act on the factors limiting access to health services by rural and indigenous communities. This article provides a comprehensive overview of the wide range of access barriers faced by rural and remote communities in two countries where barrier assessments were conducted. It also discusses the potential for barrier assessments to contribute evidence for rural proofing of national health policies, strategies, plans and programs. METHODS: The study applied a concurrent triangulation design to collect and analyze data obtained from narrative-style literature reviews, in-depth interviews with local health authorities, and secondary analyses of existing household data on Guyana and Peru. These two countries were selected because they have some of the largest rural and indigenous populations in Latin America and the Caribbean, and have national policies in place for providing free, essential health services for these communities. Both quantitative and qualitative data were collected separately, and results were interpreted together. The main objective was to corroborate and cross-validate findings looking for convergence between the separate data analyses. RESULTS: Seven dominant themes were identified across the two countries: use of traditional medicine and practice; decision making, gender, and family power dynamics; ethnicity and trust; knowledge and health literacy; geographic accessibility, health personnel and intercultural skills; and financial accessibility. The findings suggest that the interaction between these barriers may be as important as the singular role played by each factor, thereby highlighting the complex and multifactorial nature of accessing services in rural settings. Issues with limited availability of human resources for health were compounded by inadequate supplies and infrastructure. Financial barriers were often linked to the indirect costs of transport and geographic location, and further exacerbated by reduced socioeconomic status of rural communities, a majority of which are indigenous and have a strong preference for traditional medicines. Importantly, rural and indigenous communities experience considerable non-financial barriers related to issues of acceptability, which requires adaptation of health personnel and health service delivery models to the context-specific needs and realities of each rural community. CONCLUSION: This study presented an approach for data collection and analysis that is both feasible and effective for evaluating access barriers in rural and remote communities. While this study explored access barriers through general health services in two rural settings, the issues identified reflect the structural deficiencies of many health systems. These challenges and singularities require adaptive organizational models for the provision of health services that respond to the specific characteristics of rural and indigenous communities. This study indicates the potential relevance of conducting assessments of barriers to health services as part of a wider approach to rural proofing and supports the notion that a mixed-methods approach, linking secondary analysis of existing relevant national survey data with focused key-informant interview data, may be an effective and efficient way to transform data into the knowledge policymakers need to rural proof health policies.

Accelerated vascularization of a novel collagen hydrogel dermal template.

Full thickness skin loss is a debilitating problem, most commonly reconstructed using split thickness skin grafts (STSG), which do not reconstitute normal skin thickness and often result in suboptimal functional and esthetic outcomes that diminish a patient's quality of life. To address the minimal dermis present in most STSG, engineered dermal templates were developed that can induce tissue ingrowth and the formation of neodermal tissue. However, clinically available dermal templates have many shortcomings including a relatively slow rate and degree of neovascularization (∼2-4 weeks), resulting in multiple dressing changes, prolonged immobilization, and susceptibility to infection. Presented herein is a novel composite hydrogel scaffold that optimizes a unique scaffold microarchitecture with native hydrogel properties and mechanical cues ideal for promoting neovascularization, tissue regeneration, and wound healing. In vitro analysis demonstrated the unique combination of improved mechanical attributes with native hydrogel properties that promotes cell invasion and remodeling within the scaffold. In a novel 2-stage rat model of full thickness skin loss that closely mimics clinical practice, the composite hydrogel induced rapid cell infiltration and neovascularization, creating a healthy neodermis after only 1 week onto which a skin graft could be placed. The scaffold also elicited a gradual and favorable immune response, resulting in more efficient integration into the host. We have developed a dermal scaffold that utilizes simple but unique collagen hydrogel architectural cues that rapidly induces the formation of stable, functional neodermal tissue, which holds tremendous promise for the treatment of full thickness skin loss.

Elucidation of the Central Serotonin Metabolism Pathway in Rhesus Macaques (Macaca mulatta) with Self-injurious Behavior.

Macaques with self-injurious behavior (SIB) have been used as a model of human SIB and have previously been shown to respond to treatments targeting enhancement of central serotonin signaling, whether by supplementation with tryptophan, or by inhibiting synaptic reuptake. Decreased serotonin signaling in the brain has also been implicated in many human psychopathologies including major depression disorder. A disturbance in tryptophan metabolism that moves away from the production of serotonin and toward the production of kynurenine has been proposed as a major etiological factor of depression. We hypothesized that in macaques with SIB, central tryptophan metabolism would be shifted toward kynurenine production, leading to lower central serotonin (5-hydroxytryptamine). We analyzed tryptophan metabolites in the cerebral spinal fluid (CSF) of macaques with and without SIB to determine whether and where tryptophan metabolism is altered in affected animals as compared with behaviorally normal controls. We found that macaques with SIB had lower CSF concentrations of serotonin than did behaviorally normal macaques, and that these deficits were inversely correlated with the severity of abnormal behavior. However, our results suggest that this decrease is not due to shifting of the tryptophan metabolic pathway toward kynurenine, as concentrations of kynurenine were also low. Concentrations of IL6 were elevated, suggesting central inflammation. Determining the mechanism by which serotonin function is altered in self-injurious macaques could shed light on novel therapies for SIB and other disorders of serotonin signaling.

Spinocerebellar ataxia type 12: clues to pathogenesis.

PURPOSE OF REVIEW: Spinocerebellar ataxia type 12 (SCA12) is a rare autosomal dominant neurodegenerative disease characterized by tremor, gait abnormalities, and neuropsychiatric syndromes. The location of the causative CAG/CTG expansion mutation in PPP2R2B, a gene encoding regulatory units of the protein phosphatase 2A, may provide unique insights into the pathogenesis of neurodegeneration. RECENT FINDINGS: The first neuropathological examination of a brain from an SCA12 patient revealed both cerebellar and cerebral cortical atrophy, with a noted loss of Purkinje cells and no evidence of polyglutamine aggregates. Molecular investigations have demonstrated considerable complexity of PPP2R2B, which appears to encode at least eight isoforms each with a different N-terminal region. The repeat potentially influences PPP2R2B expression, and is itself included in several splice variants, falling within an open reading frame of at least one of these variants. SUMMARY: The current data suggest at least two nonmutually exclusive hypotheses of SCA12 neurodegeneration. First, the repeat may influence PPP2R2B expression, by altering promoter activity, splicing, or transcript stability. This hypothesis would predict that the mutation changes the regulation of protein phosphatase 2A, with implications for the phosphoproteome. Alternatively, the repeat itself may be expressed and have toxic properties, though perhaps not through polyglutamine tracts. Either hypothesis may provide novel insight into the pathogenesis of neurodegeneration.

Evidence that exposure to progesterone alone is a sufficient stimulus to cause a precipitous rise in the immunomodulatory protein the progesterone induced blocking factor (PIBF).

PURPOSE: To determine if exposure to progesterone alone is sufficient to increase the production of the immunomodulatory protein known as the progesterone induced blocking factor (PIBF). Also to determine what method of progesterone delivery or form of P best stimulates PIBF secretion. METHODS: Serum samples from patients with infertility and paid volunteers were evaluated for both PIBF and progesterone at various times during the follicular phase and the luteal phase in both natural cycles and cycles involving embryo transfer after endogenous and exogenous progesterone exposure and after various synthetic progestins. PIBF was measured by a non-commercial research ELISA assay. Comparisons were made of serum PIBF before and after exposure to progesterone, 17-hydroxyprogesterone, and oral contraceptives. PIBF was also measured before and after transfer of embryos. RESULTS: Progesterone alone without exposure to the fetal allogeneic stimulus was able to produce a marked increase in serum PIBF. Neither a synthetic progestin (19-nortestosterone derivative) nor 17-hydroxyprogesterone caused an increase in PIBF. Some PIBF is generally detected even in the follicular phase. CONCLUSIONS: A previous concept considered that an allogeneic stimulus, e.g., from the fetal semi-allograft, was necessary to induce de novo progesterone receptors in gamma delta T cells, which, in turn, when exposed to a high concentration of progesterone, would secrete high levels of PIBF. These data show that exposure to an allogeneic stimulus is not needed to cause a marked rise in PIBF, merely progesterone alone is sufficient.

Neuropathology and Cellular Pathogenesis of Spinocerebellar Ataxia Type 12.

OBJECTIVE: SCA12 is a progressive autosomal-dominant disorder, caused by a CAG/CTG repeat expansion in PPP2R2B on chromosome 5q32, and characterized by tremor, gait ataxia, hyperreflexia, dysmetria, abnormal eye movements, anxiety, depression, and sometimes cognitive impairment. Neuroimaging has demonstrated cerebellar and cortical atrophy. We now present the neuropathology of the first autopsied SCA12 brain and utilize cell models to characterize potential mechanisms of SCA12 neurodegeneration. METHODS: A fixed SCA12 brain was examined using gross, microscopic, and immunohistochemical methods. The effect of the repeat expansion on PPP2R2B Bß1 expression was examined in multiple cell types by transient transfection of constructs containing the PPP2R2B Bß1 promoter region attached to a luciferase reporter. The neurotoxic effect of PPP2R2B overexpression was examined in transfected rat primary neurons. RESULTS: Neuropathological investigation revealed enlarged ventricles, marked cerebral cortical atrophy and Purkinje cell loss, less-prominent cerebellar and pontine atrophy, and neuronal intranuclear ubiquitin-positive inclusions, consistent with Marinesco bodies, which did not stain for long polyglutamine tracts, alpha-synuclein, tau, or transactive response DNA-binding protein 43. Reporter assays demonstrated that the region of PPP2R2B containing the repeat functions as a promoter, and that promoter activity increases with longer repeat length and is dependent on cell type, repeat sequence, and sequence flanking the repeat. Overexpression of PPP2R2B in primary cortical neurons disrupted normal morphology. CONCLUSIONS: SCA12 involves extensive, but selective, neurodegeneration distinct from Alzheimer's disease, synucleinopathies, tauopathies, and glutamine expansion diseases. SCA12 neuropathology may arise from the neurotoxic effect of repeat-expansion-induced overexpression of PPP2R2B.

Nuclear retention of full-length HTT RNA is mediated by splicing factors MBNL1 and U2AF65.

Huntington's disease (HD) is caused by a CAG repeat expansion in the huntingtin (HTT) gene. Recent evidence suggests that HD is a consequence of multimodal, non-mutually exclusive mechanisms of pathogenesis that involve both HTT protein- and HTT RNA-triggered mechanisms. Here we provide further evidence for the role of expanded HTT (expHTT) RNA in HD by demonstrating that a fragment of expHTT is cytotoxic in the absence of any translation and that the extent of cytotoxicity is similar to the cytotoxicity of an expHTT protein fragment encoded by a transcript of similar length and with a similar repeat size. In addition, full-length (FL) expHTT is retained in the nucleus. Overexpression of the splicing factor muscleblind-like 1 (MBNL1) increases nuclear retention of expHTT and decreases the expression of expHTT protein in the cytosol. The splicing and nuclear export factor U2AF65 has the opposite effect, decreasing expHTT nuclear retention and increasing expression of expHTT protein. This suggests that MBNL1 and U2AF65 play a role in nuclear export of expHTT RNA.

Evidence that Mifepristone, a progesterone receptor antagonist, can cross the blood brain barrier and provide palliative benefits for glioblastoma multiforme grade IV.

BACKGROUND: Mifepristone, a progesterone receptor antagonist, has been found to provide palliative benefits for various types of spontaneous murine cancer in randomized controlled trials and in anecdotal reports from a variety of advanced metastatic human cancer not known to be associated with progesterone receptors. The theory of its mechanism is that it prevents the secretion of a progesterone-induced immunomodulatory protein in the tumor microenvironment, or in the tumor cell itself, called the progesterone-induced blocking factor, which inhibits natural killer cells from attacking the cancer cell. Many anticancer chemotherapeutic agents fail to cross the blood-brain barrier and thus prove ineffective for brain cancer. The objective of the present study was to determine if mifepristone could provide palliative benefits to a patient with end-stage stage IV glioblastoma multiforme. CASE REPORT: A 43-year-old male with end-stage stage IV glioblastoma multiforme was exclusively treated with mifepristone 200 mg orally daily. RESULTS: The patient showed definite palliative effects for several weeks and his life was significantly extended beyond pre-treatment predictors. CONCLUSION: It appears that mifepristone does cross the blood-brain barrier and could be considered for palliative therapy of other patients with chemotherapy-resistant brain cancer.

Mifepristone causing complete remission of rapidly advancing leukemia with measurement of progesterone-induced blocking factor.

BACKGROUND: Mifepristone has been demonstrated to cause palliation from murine and human cancer, even in cancers not known to be positive for expression of progesterone receptors. The aim of the present study was to determine if rapidly advancing chronic lymphocytic leukemia responds to mifepristone therapy, and if so, is this effect related to increased expression of the progesterone-induced blocking factor? CASE REPORT: An 81-year-old woman with chronic lymphocytic leukemia whose condition progressed to the acute rapidly progressing stage agreed to be exclusively treated orally with 200 mg mifepristone daily. RESULTS: The patient showed a dramatic improvement after a short exposure time to mifepristone. Complete remission has persisted so far for 12 months on exclusive mifepristone therapy. Her PIBF levels were normal before mifepristone therapy and did not change after treatment. CONCLUSION: Mifepristone can provide marked improvement of human leukemia even in the absence of increased serum PIBF levels.

The role of progesterone and the progesterone receptor in human reproduction and cancer.

Insufficient progesterone, effect possibly more on immune factors rather than adequate endometrial development, can be an easy remedial cause of infertility by simply supplementing the luteal phase with either vaginal or intramuscular or oral (dydrogesterone) progesterone. Progesterone will also help to reduce miscarriage rates when follicle maturing drugs are used for those with regular menses but follicular maturation defects, or women with recurrent miscarriages. One mechanism of action seems to be related to production of an immunomodulatory protein, the progesterone-induced blocking factor either in the cytoplasm or in the circulation. PIBF inhibits cytotoxicity of natural killer cells. Cancer cells may 'borrow' the same mechanism to escape NK cell immunosurveillance.

Live delivery and implantation rates of donor oocyte recipients in their late forties are similar to younger recipients.

OBJECTIVE: To determine if there is a certain age when the uterus is somewhat less receptive to successful pregnancy despite the transfer of embryos from donated oocytes. STUDY DESIGN: We conducted a retrospective evaluation of donor oocyte recipient cycles according to specific ages. The recipients used an oral/vaginal graduated estradiol regimen followed by intramuscular and vaginal progesterone. Only recipients sharing oocytes with either the donor or another recipient were included. RESULTS: Evaluating the pregnancy rate by each year of age from 40-49 following transfer of embryos derived from donor oocytes showed no trend for lower pregnancy rates up to age 49. In fact the highest live delivery pregnancy rates (though not significant) were found at ages 47 (64.3%) and 49 (63.6%). The live delivered pregnancy rates for recipients < or = 39 was 52.5% vs. 55.6% for women > or = age 46. The live delivered pregnancy rate was 34.6% for women > or = age 50. The pregnancy and implantation rates were similar whether the source was infertile women sharing half their oocytes or compensated donors. CONCLUSION: The uterus does not seem to have a diminished capacity for implantation up to the age of 49, but it may be slightly less receptive after age 50.

Sexual socialization in lesbian-parent families: an exploratory analysis.

Few studies have examined the sexual socialization of children within lesbian-parent families, despite evidence that these children may experience benefits in this regard. Qualitative interviews were conducted with 10 partnered, lesbian mothers to explore what and how participants taught their children about sexuality-related issues. Themes related to what participants taught their children about these topics included diverse notions of sexual orientation and reproduction. Themes related to how participants taught these concepts included tag-teaming with their partners (i.e., participants shared with their partners the task of teaching their children about sexuality-related issues), although differences in how the partners carried out this task were identified. This study demonstrated a diversity of experiences of lesbian-parent families, often seemingly in relation to family contextual factors (e.g., how children were conceived), and has potential implications for family researchers, practitioners, and, perhaps, all parents.

Pharmacokinetics of oral gabapentin in greyhound dogs.

The purpose of this study was to assess the pharmacokinetics of gabapentin in healthy greyhound dogs after single oral doses targeted at 10 and 20mg/kg PO. Six healthy greyhounds were enrolled (3 males, 3 females). Blood was obtained at predetermined times for the measurement of gabapentin plasma concentrations by liquid chromatography/mass spectrometry. Pharmacokinetic parameters were determined with computer software. The actual mean (and range) doses administered were 10.2 (9.1-12.0) mg/kg and 20.5 (18.2-24) mg/kg for the 10mg/kg and 20mg/kg targeted dose groups. The mean C(MAX) for the 10 and 20mg/kg groups were 8.54 and 13.22 microg/mL at 1.3 and 1.5h, and the terminal half-lives were 3.3 and 3.4h, respectively. The relative bioavailability of the 10mg/kg group was 1.13 compared to the 20mg/kg group. Gabapentin was rapidly absorbed and eliminated in dogs, indicating that frequent dosing is needed to maintain minimum targeted plasma concentrations.

Cysteinyl-leukotriene receptor antagonism blunts the acute hypotensive response to endotoxin in cats.

This study evaluated the effects of a cysteinyl-leukotriene-1 (cys-LT(1)) receptor antagonist, zarfirlukast, during feline endotoxemia. Six adult, sexually intact male cats received either placebo or zarfirlukast (10mg, PO) and endotoxin (2 µg/kg/h q 6h) in a cross-over design. Rectal temperature, heart rate, systolic arterial blood pressure, plasma tumor necrosis factor (TNF) activity, interleukin (IL)-6 concentration and urine cys-LT to creatinine ratio were evaluated. The rectal temperature, plasma TNF activity and IL-6 concentrations were significantly higher and systolic arterial blood pressure and heart rate significantly lower after endotoxin infusion. Cats treated with zafirlukast had a significantly higher blood pressure at 4h (P=0.002) compared to placebo. Urine cys-LT to creatinine ratio was significantly greater in the cats treated with zafirlukast compared to placebo (P=0.02). Zafirlukast administration ameliorated the acute hypotensive response to endotoxin in cats, but failed to significantly alter rectal temperature, heart rate or production of TNF and IL-6.

Efficacy of the progesterone receptor antagonist mifepristone for palliative therapy of patients with a variety of advanced cancer types.

BACKGROUND: Mifepristone has been demonstrated to improve longevity and quality of life in mice with spontaneous murine cancer without progesterone receptors and in human colon cancer. The present study evaluated the palliative effect of mifepristone in a variety of different types of human cancer. PATIENTS AND METHODS: Mifepristone was given at 200 mg daily orally with permission from the Food and Drug Administration to people with widely metastatic human cancer no longer responsive to other chemotherapy regimens. RESULTS: Improvement in pain and energy and/or length of life was found in thymic epithelial cell carcinoma, transitional cell carcinoma of the renal pelvis, leiomyosarcoma, pancreatic carcinoma, malignant fibrous histiocytoma and another case of adenocarcinoma of the colon. CONCLUSION: Our data demonstrate a palliative role for the use of mifepristone in cancer therapy. Progesterone receptor antagonists should be given a therapeutic trial in larger controlled studies of various malignancies in humans.