Molecular Characterization and Seasonal Variation in Expression of HSP70.1 Gene in Gangatiri Cattle and Its Comparison with Buffalo.

Under tropical climate heat stress is a major challenge for livestock production. HSP70.1 is a ubiquitously expressed protein maintaining cellular machinery through proper folding of denatured proteins and prevents cellular apoptosis and protect cell from heat stress. Therefore, present investigation was undertaken to explore genetic variability in HSP70.1 gene in Gangatiri cattle, its comparison with buffalo sequences and differential expression in different season. The allelic variant was identified by sequencing amplified PCR product of HSP70.1 gene by primer walking. Season-wise total RNA samples was prepared for differential expression study. Brilliant SYBR Green QPCR technique was used to study the expression kinetics of this gene. DNA sequencing by primer walking identified four allelic variants in Gangatiri cattle. Sequence alignment study revealed four, six and one substitutions in the 5' untranslated region (5'UTR), coding and 3' untranslated region ((3'UTR) of HSP70.1 gene, respectively. Comparative analysis of HSP70.1 gene revealed that Cattle has shorter 5'UTR and 3' UTR than the buffalo. In Gangatiri cattle, summer season has significantly higher (P ≤ 0.05) expression of HSP70.1 than the spring and winter. The relative expression of HSP70.1 was increased by more than six folds in summer and nearly 1.5 folds higher in winter in comparison to the spring season. Therefore, HSP70.1 may be considered to have a critical role in the development of thermal tolerance in Gangatiri cattle.

Morphometric characteristics and microsatellite markers based diversity and differentiation recognizes the first prospective cattle breed from the Jharkhand state of India.

India is bestowed with immense cattle biodiversity with 50 registered breeds. However, the majority (59.3%) is yet not characterized. Identification and characterization are the gateways to the management of prized indigenous resources. Present research described a unique cattle population of Jharkhand state, managed under a traditional low-input, low-output system. It was characterized by morphological traits, performance parameters, and management practices. Animals have the characteristic pre-scapular location of the hump. Genetic variation within this population and its differentiation with the six closely distributed cattle breeds were evaluated using FAO recommended microsatellite markers. Jharkhandi cattle have substantial genetic variation based on gene diversity (>0.6) and the average number of alleles per locus (>8). The population did not suffer from a genetic bottleneck in the recent past. Pairwise Nei's genetic distance, phylogenetic relationship, population differentiation, and the correct assignment of all the animals to self group substantiated its separate genetic identity. Since gene flow (Nm = 2.8-7.32) was identified and admixture was indicated by the Bayesian analysis there is a pressing need for scientific management of this population. Results endow authorities with critical information for registering a new Indian cattle breed (Medini) that contributes to the food security, livelihood, and economic sustainability of rural tribal households.

Activated integrins identify functional antigen-specific CD8+ T cells within minutes after antigen stimulation.

Immediate ß2-integrin activation upon T cell receptor stimulation is critical for effective interaction between T cells and their targets and may therefore be used for the rapid identification and isolation of functional T cells. We present a simple and sensitive flow cytometry-based assay to assess antigen-specific T cells using fluorescent intercellular adhesion molecule (ICAM)-1 multimers that specifically bind to activated ß2-integrins. The method is compatible with surface and intracellular staining; it is applicable for monitoring of a broad range of virus-, tumor-, and vaccine-specific CD8+ T cells, and for isolating viable antigen-reacting cells. ICAM-1 binding correlates with peptide-MHC multimer binding but, notably, it identifies the fraction of antigen-specific CD8+ T cells with immediate and high functional capability (i.e., expressing high levels of cytotoxic markers and cytokines). Compared with the currently available methods, staining of activated ß2-integrins presents the unique advantage of requiring activation times of only several minutes, therefore delivering functional information nearly reflecting the in vivo situation. Hence, the ICAM-1 assay is most suitable for rapid and precise monitoring of functional antigen-specific T cell responses, including for patient samples in a variety of clinical settings, as well as for the isolation of functional T cells for adoptive cell-transfer immunotherapies.

Metastatic leiomyosarcoma of the thyroid: A rare entity.

Cancer metastasis to the thyroid gland from non-thyroid sites is a rare presentation in clinical practice. The most frequent primary cancers that metastasise to the thyroid are renal cell carcinoma, followed by colorectal, lung and breast. We report a case of a 64-year-old Malay lady who presented with anterior neck swelling 4 years after an initial diagnosis of uterine leiomyosarcoma. She had undergone a hysterectomy procedure four years ago. Fine needle aspiration cytology of the thyroid mass suggested undifferentiated thyroid carcinoma. After multi-disciplinary discussion, the patient underwent thyroidectomy and the final histopathological diagnosis was metastatic leiomyosarcoma of the thyroid. The diagnosis was aided by an immunohistochemistry panel of positive myogenic markers, negative epithelial markers as well as the previous medical history of uterine leiomyosarcoma. Metastatic leiomyosarcoma of the thyroid may mimic primary undifferentiated (anaplastic) thyroid carcinoma (UTC) with a sarcomatoid pattern, medullary thyroid carcinoma (MTC) with spindle cells morphology and spindle cell tumour with thymus-like differentiation (SETTLE). Hence, a multidisciplinary approach must be practised by pathologists, surgeons and radiologists to consider metastatic lesions of the thyroid gland, especially when a previous history of cancer exists or is suspected.

Chimera-like states induced by additional dynamic nonlocal wirings.

We investigate the existence of chimera-like states in a small-world network of chaotically oscillating identical Rössler systems with an addition of randomly switching nonlocal links. By varying the small-world coupling strength, we observe no chimera-like state either in the absence of nonlocal wirings or with static nonlocal wirings. When we give an additional nonlocal wiring to randomly selected nodes and if we allow the random selection of nodes to change with time, we observe the onset of chimera-like states. Upon increasing the number of randomly selected nodes gradually, we find that the incoherent window keeps on shrinking, whereas the chimera-like window widens up. Moreover, the system attains a completely synchronized state comparatively sooner for a lower coupling strength. Also, we show that one can induce chimera-like states by a suitable choice of switching times, coupling strengths, and a number of nonlocal links. We extend the above-mentioned randomized injection of nonlocal wirings for the cases of globally coupled Rössler oscillators and a small-world network of coupled FitzHugh-Nagumo oscillators and obtain similar results.

Chimera states in coupled logistic maps with additional weak nonlocal topology.

We demonstrate the occurrence of coexisting domains of partially coherent and incoherent patterns or simply known as chimera states in a network of globally coupled logistic maps upon addition of weak nonlocal topology. We find that the chimera states survive even after we disconnect nonlocal connections of some of the nodes in the network. Also, we show that the chimera states exist when we introduce symmetric gaps in the nonlocal coupling between predetermined nodes. We ascertain our results, for the existence of chimera states, by carrying out the recurrence quantification analysis and by computing the strength of incoherence. We extend our analysis for the case of small-world networks of coupled logistic maps and found the emergence of chimeralike states under the influence of weak nonlocal topology.

Personalized peptide vaccine-induced immune response associated with long-term survival of a metastatic cholangiocarcinoma patient.

BACKGROUND & AIMS: We report a novel experimental immunotherapeutic approach in a patient with metastatic intrahepatic cholangiocarcinoma. In the 5year course of the disease, the initial tumor mass, two local recurrences and a lung metastasis were surgically removed. Lacking alternative treatment options, aiming at the induction of anti-tumor T cells responses, we initiated a personalized multi-peptide vaccination, based on in-depth analysis of tumor antigens (immunopeptidome) and sequencing. METHODS: Tumors were characterized by immunohistochemistry, next-generation sequencing and mass spectrometry of HLA ligands. RESULTS: Although several tumor-specific neo-epitopes were predicted in silico, none could be validated by mass spectrometry. Instead, a personalized multi-peptide vaccine containing non-mutated tumor-associated epitopes was designed and applied. Immunomonitoring showed vaccine-induced T cell responses to three out of seven peptides administered. The pulmonary metastasis resected after start of vaccination showed strong immune cell infiltration and perforin positivity, in contrast to the previous lesions. The patient remains clinically healthy, without any radiologically detectable tumors since March 2013 and the vaccination is continued. CONCLUSIONS: This remarkable clinical course encourages formal clinical studies on adjuvant personalized peptide vaccination in cholangiocarcinoma. LAY SUMMARY: Metastatic cholangiocarcinomas, cancers that originate from the liver bile ducts, have very limited treatment options and a fatal prognosis. We describe a novel therapeutic approach in such a patient using a personalized multi-peptide vaccine. This vaccine, developed based on the characterization of the patient's tumor, evoked detectable anti-tumor immune responses, associating with long-term tumor-free survival.

Delayed Presentation of Jejuno-Jejunal Fistula With Stricture After Physical Child Abuse.

Small intestinal injury is seldom described in the context of child abuse. Signs and symptoms are subtle, often leading to delays in diagnosis. We describe a 3-year-old boy initially admitted with severe blunt abdominal trauma from physical child abuse. He was successfully managed nonoperatively. The child was then hospitalized several times for nonspecific abdominal symptoms until diagnostic laparoscopy discovered a jejunal stricture with a proximal jejuno-jejunal fistula. Symptoms fully resolved after resection. Delayed presentation of small intestinal injury should remain on the differential diagnosis in the evaluation of persistent abdominal symptoms in a child with a prior history of physical abuse, even if imaging studies do not reveal specific abnormalities.

Omega-3 fatty acid concentrate from Dunaliella salina possesses anti-inflammatory properties including blockade of NF-κB nuclear translocation.

The health benefits of omega-3 polyunsaturated fatty acids (ω-3 PUFA), mainly eicosapentaenoic acid (EPA 20:5) and docosahexaenoic acid (DHA, 22:6), have been long known. Although various studies have demonstrated the health benefits of ω-3 PUFA, the mechanisms of action of ω-3 PUFAs are still not completely understood. While the major commercial source is marine fish oil, in this study we suggest the marine micro algae, Dunaliella salina as an alternate source of omega-3 fatty acids. Treatment with this algal omega-3 fatty acid concentrate (Ds-ω-3 FA) resulted in significant down-regulation of LPS-induced production of TNF-α and IL-6 by peripheral blood mononuclear cells (PBMCs). The concentrate was also found to be a potent blocker of cyclooxygenase (COX-2) and matrix metalloproteinase (MMP-2 and MMP-9) expression. The present study reveals the anti-inflammatory properties of Ds-ω-3 FA concentrate including the inhibition of NF-κB translocation.

Primary closure or rhomboid excision and Limberg flap for the management of primary sacrococcygeal pilonidal disease? A meta-analysis of randomized controlled trials.

AIM: Sacrococcygeal pilonidal disease is a common condition afflicting the young male working and student population, resulting in considerable pain, embarrassment and loss of work days. Controversy surrounds the most appropriate surgical approach to achieve low recurrence rates whilst minimizing morbidity and permitting an early return to work. This study aims to review the published literature comparing excision followed by either primary suture or rhomboid flap repair. METHODS: PubMed, EMBASE, MEDLINE and The Cochrane Library were systematically reviewed, by two independent investigators, for relevant randomized controlled trials. Keywords and MeSH terms included 'pilonidal disease', 'primary suture/repair', 'rhomboid flap' and 'limberg/modified Limberg flap'. 'Related study' function and manuscript bibliographies were searched for further relevant studies. Study quality was assessed using the Jadad score. Meta-analysis was performed on pooled data, utilizing a random effects model when heterogeneity was high and a fixed effects model when heterogeneity was low. The primary end-point assessed was disease recurrence. Secondary end-points included wound dehiscence, pain scores, hospital stay and return to work. RESULTS: Six studies were eventually included for pooled analysis following exclusion of randomized controlled trials with poor methodology. Two studies compared 'off-midline' (Karydakis) primary suture with the Limberg flap repair. Six hundred and forty-one patients were included (331 flap repairs). Rhomboid flap excision demonstrated a trend towards less disease recurrence (P = 0.07), lower wound infection (P = 0.001) and dehiscence (P = 0.01). However, no significant difference was found for pain scores, hospital stay or return to work. CONCLUSION: The current published literature supports the use of the rhomboid flap excision and the Limberg flap-repair procedures over primary midline suture techniques for the elective management of primary pilonidal disease. Further high-quality studies are necessary to compare flap with off-midline repairs.

Cannabinoid Hyperemesis Syndrome and Hypophosphatemia in Adolescents.

We report 3 adolescents with cannabis hyperemesis syndrome and recurrent hypophosphatemia complicating their clinical course with potential for significant consequences. They serve as reminders for providers to consider the diagnosis of cannabis hyperemesis syndrome and to monitor serum electrolytes closely in the setting of adolescent hyperemesis.

A specific miRNA signature in the peripheral blood of glioblastoma patients.

The prognosis of patients afflicted by glioblastoma remains poor. Biomarkers for the disease would be desirable in order to allow for an early detection of tumor progression or to indicate rapidly growing tumor subtypes requiring more intensive therapy. In this study, we investigated whether a blood-derived specific miRNA fingerprint can be defined in patients with glioblastoma. To this end, miRNA profiles from the blood of 20 patients with glioblastoma and 20 age- and sex-matched healthy controls were compared. Of 1158 tested miRNAs, 52 were significantly deregulated, as assessed by unadjusted Student's t-test at an alpha level of 0.05. Of these, two candidates, miR-128 (up-regulated) and miR-342-3p (down-regulated), remained significant after correcting for multiple testing by Benjamini-Hochberg adjustment with a p-value of 0.025. The altered expression of these two biomarkers was confirmed in a second cohort of glioblastoma patients and healthy controls by real-time PCR and validated for patients who had received neither radio- nor chemotherapy and for patients who had their glioblastomas resected more than 6 months ago. Moreover, using machine learning, a comprehensive miRNA signature was obtained that allowed for the discrimination between blood samples of glioblastoma patients and healthy controls with an accuracy of 81% [95% confidence interval (CI) 78-84%], specificity of 79% (95% CI 75-83%) and sensitivity of 83% (95% CI 71-85%). In summary, our proof-of-concept study demonstrates that blood-derived glioblastoma-associated characteristic miRNA fingerprints may be suitable biomarkers and warrant further exploration.

Ectonucleotidases CD39 and CD73 on OvCA cells are potent adenosine-generating enzymes responsible for adenosine receptor 2A-dependent suppression of T cell function and NK cell cytotoxicity.

The ectonucleotidases CD39 and CD73 degrade immune stimulatory ATP to adenosine that inhibits T and NK cell responses via the A(2A) adenosine receptor (ADORA2A). This mechanism is used by regulatory T cells (T(reg)) that are associated with increased mortality in OvCA. Immunohistochemical staining of human OvCA tissue specimens revealed further aberrant expression of CD39 in 29/36 OvCA samples, whereas only 1/9 benign ovaries showed weak stromal CD39 expression. CD73 could be detected on 31/34 OvCA samples. While 8/9 benign ovaries also showed CD73 immunoreactivity, expression levels were lower than in tumour specimens. Infiltration by CD4(+) and CD8(+) T cells was enhanced in tumour specimens and significantly correlated with CD39 and CD73 levels on stromal, but not on tumour cells. In vitro, human OvCA cell lines SK-OV-3 and OaW42 as well as 11/15 ascites-derived primary OvCA cell cultures expressed both functional CD39 and CD73 leading to more efficient depletion of extracellular ATP and enhanced generation of adenosine as compared to activated T(reg). Functional assays using siRNAs against CD39 and CD73 or pharmacological inhibitors of CD39, CD73 and ADORA2A revealed that tumour-derived adenosine inhibits the proliferation of allogeneic human CD4(+) T cells in co-culture with OvCA cells as well as cytotoxic T cell priming and NK cell cytotoxicity against SK-OV3 or OAW42 cells. Thus, both the ectonucleotidases CD39 and CD73 and ADORA2A appear as possible targets for novel treatments in OvCA, which may not only affect the function of T(reg) but also relieve intrinsic immunosuppressive properties of tumour and stromal cells.

Whole blood-derived miRNA profiles as potential new tools for ovarian cancer screening.

BACKGROUND: Screening is an unsolved problem for ovarian cancer (OvCA). As late detection is equivalent to poor prognosis, we analysed whether OvCA patients show diagnostically meaningful microRNA (miRNA) patterns in blood cells. METHODS: Blood-borne whole miRNome profiles from 24 patients with OvCA and 15 age- and sex-matched healthy controls were biostatistically evaluated. RESULTS: Student's t-test revealed 147 significantly deregulated miRNAs before and 4 after Benjamini-Hochberg adjustment. Although these included miRNAs already linked to OvCA (e.g., miR-16, miR-155), others had never before been connected to specific diseases. A bioinformatically calculated miRNA profile allowed for discrimination between blood samples of OvCA patients and healthy controls with an accuracy of >76%. When only cancers of the serous subtype were considered and compared with an extended control group (n=39), accuracy, specificity and sensitivity all increased to >85%. CONCLUSION: Our proof-of-principle study strengthens the hypothesis that neoplastic diseases generate characteristic miRNA fingerprints in blood cells. Still, the obtained OvCA-associated miRNA pattern is not yet sensitive and specific enough to permit the monitoring of disease progression or even preventive screening. Microarray-based miRNA profiling from peripheral blood could thus be combined with other markers to improve the notoriously difficult but important screening for OvCA.

A new synthetic toll-like receptor 1/2 ligand is an efficient adjuvant for peptide vaccination in a human volunteer.

BACKGROUND: We previously showed that the bacterial lipopeptide Pam3Cys-Ser-Ser, meanwhile established as a toll-like receptor (TLR) 1/2 ligand, acts as a strong adjuvant for the induction of virus specific CD8+ T cells in mice, when covalently coupled to a synthetic peptide. CASE PRESENTATION: We now designed a new water-soluble synthetic Pam3Cys-derivative, named XS15 and characterized it in vitro by a TLR2 NF-κB luciferase reporter assay. Further, the capacity of XS15 to activate immune cells and stimulate peptide-specific CD8+ T and NK cells by 6-sulfo LacNAc+ monocytes was assessed by flow cytometry as well as cytokine induction using immunoassays. The induction of a functional immune response after vaccination of a volunteer with viral peptides was assessed by ELISpot assay and flow cytometry in peripheral blood cells and infiltrating cells at the vaccination site, as well as by immunohistochemistry and imaging. XS15 induced strong ex vivo CD8+ and TH1 CD4+ responses in a human volunteer upon a single injection of XS15 mixed to uncoupled peptides in a water-in-oil emulsion (Montanide™ ISA51 VG). A granuloma formed locally at the injection site containing highly activated functional CD4+ and CD8+ effector memory T cells. The total number of vaccine peptide-specific functional T cells was experimentally assessed and estimated to be 3.0 × 105 in the granuloma and 20.5 × 106 in peripheral blood. CONCLUSION: Thus, in one volunteer we show a granuloma forming by peptides combined with an efficient adjuvant in a water-in-oil-emulsion, inducing antigen specific T cells detectable in circulation and at the vaccination site, after one single vaccination only. The ex vivo T cell responses in peripheral blood were detectable for more than one year and could be strongly boosted by a second vaccination. Hence, XS15 is a promising adjuvant candidate for peptide vaccination, in particular for tumor peptide vaccines in a personalized setting.

Differential effects of cannabinoid receptor agonists on regional brain activity using pharmacological MRI.

BACKGROUND AND PURPOSE: Activation of cannabinoid CB1 and/or CB2 receptors mediates analgesic effects across a broad spectrum of preclinical pain models. Selective activation of CB2 receptors may produce analgesia without the undesirable psychotropic side effects associated with modulation of CB1 receptors. To address selectivity in vivo, we describe non-invasive, non-ionizing, functional data that distinguish CB1 from CB2 receptor neural activity using pharmacological MRI (phMRI) in awake rats. EXPERIMENTAL APPROACH: Using a high field (7 T) MRI scanner, we examined and quantified the effects of non-selective CB1/CB2 (A-834735) and selective CB2 (AM1241) agonists on neural activity in awake rats. Pharmacological specificity was determined using selective CB1 (rimonabant) or CB2 (AM630) antagonists. Behavioural studies, plasma and brain exposures were used as benchmarks for activity in vivo. KEY RESULTS: The non-selective CB1/CB2 agonist produced a dose-related, region-specific activation of brain structures that agrees well with published autoradiographic CB1 receptor density binding maps. Pretreatment with a CB1 antagonist but not with a CB2 antagonist, abolished these activation patterns, suggesting an effect mediated by CB1 receptors alone. In contrast, no significant changes in brain activity were found with relevant doses of the CB2 selective agonist. CONCLUSION AND IMPLICATIONS: These results provide the first clear evidence for quantifying in vivo functional selectivity between CB1 and CB2 receptors using phMRI. Further, as the presence of CB2 receptors in the brain remains controversial, our data suggest that if CB2 receptors are expressed, they are not functional under normal physiological conditions.