Comparative sperm protein profiling in bulls differing in fertility and identification of phosphatidylethanolamine-binding protein 4, a potential fertility marker.

This study aimed to identify sperm proteomic signatures regulating sperm functions and fertility by: (i) comparing the sperm electrophoretic protein profiles and identifying the differentially abundant proteins among breeding bulls differing in fertility status and (ii) elucidating the possible role of one of the identified novel proteins, PEBP4 on sperm function and fertility. The grouping of bulls as fertile (n = 6) and low fertile (n = 6) was performed based on bull fertility index and infertile (n = 6) based on semen rejection rate (>33%). The sperm motility, fructolysis index, acrosomal reaction, intracellular calcium levels, and seminal plasma fructose and calcium levels were studied among fertility groups. The differentially expressed sperm proteins observed in single- and two-dimensional gel electrophoresis (2DE) were identified using Nano-LC-MS/MS. In the fertile bulls, the expression levels of calmodulin (CALM1), spermadhesinZ13 (SPADH2), and phosphatidylethanolamine-binding protein 4 (PEBP4) were significantly (p < 0.05) higher than in other fertility groups. In bovine, expression of PEBP4 a novel seminal protein was not observed in spermatozoa of infertile bulls. When the bulls were grouped based on the presence (n = 8) or absence (n = 10) of PEBP4 protein in spermatozoa, a positive significant (p < 0.05) association of this protein with the percentage of motile, type-A spermatozoa, and sperm fructose uptake was observed. Further, PEBP4 was localized in elongated spermatids, Leydig cells, excurrent duct system, and principal piece of spermatozoa. These findings suggest a crucial role for the PEBP4 protein in spermiogenesis, epididymal sperm maturation, and sperm motility. This first study in bovine indicates the positive association of PEBP4 in regulating sperm maturation, functions, and fertility and could be a potential marker for predicting semen quality and fertility.

Direct incorporation of the NKT-cell activator α-galactosylceramide into a recombinant Listeria monocytogenes improves breast cancer vaccine efficacy.

BACKGROUND: Immune suppression in the tumour microenvironment remains a major limitation to successful immunotherapy of cancer. In the current study, we analysed whether the natural killer T cell-activating glycolipid α-galactosylceramide could overcome immune suppression and improve vaccination against metastatic breast cancer. METHODS: Mice with metastatic breast cancer (4T1 model) were therapeutically treated with a Listeria monocytogenes-based vaccine expressing tumour-associated antigen Mage-b followed by α-galactosylceramide as separate agents, or as a complex of α-galactosylceramide stably incorporated into Listeria-Mage-b. Effects on metastases, tumour weight, toxicity and immune responses were determined. RESULTS: Sequential treatments of mice with established 4T1 breast carcinomas using Listeria-Mage-b followed by α-galactosylceramide as a separate agent was highly effective at reducing metastases, but was accompanied by severe liver toxicity. In contrast, combined therapy using Listeria-Mage-b modified by incorporation of α-galactosylceramide resulted in nearly complete elimination of metastases without toxicity. This was associated with a significant increase in the percentage of natural killer T cells in the spleen, and an increase in natural killer cell activity and in T cell responses to Mage-b. CONCLUSIONS: Our results suggest that direct incorporation of α-galactosylceramide into a live bacterial vaccine vector is a promising non-toxic new approach for the treatment of metastatic breast cancer.

Role of the BACTEC radiometric method in the evaluation of patients with clinically probable tuberculous meningitis.

BACKGROUND: Confirmation of tuberculous meningitis (TBM) and initiation of treatment are often delayed due to limitations in isolating Mycobacterium tuberculosis from cerebrospinal fluid (CSF). OBJECTIVES: To evaluate the role of the BACTEC radiometric method in a clinical setting for the early diagnosis of TBM. MATERIALS AND METHODS: Patients meeting criteria for clinically probable TBM over a 3 year period were included. Clinical features, results of CSF investigations (protein, glucose, cell count, Ziehl-Neelsen staining, culture in Löwenstein-Jensen (LJ) medium and BACTEC) and brain CT imaging were reviewed. Drug sensitivity was tested using BACTEC. Patients were started on standard treatment and functional outcome, and response at discharge and follow-up were assessed. Patients were divided according to whether or not M. tuberculosis was isolated by BACTEC and the clinical, radiological, and laboratory features compared. RESULTS: Sixty patients were evaluated. The mean age was 30 years +/- 11.7 years. Headache and fever were the most common symptoms and the mean duration was 26 days. CT findings were hydrocephalus (n=21), basal exudates (n=16), and tuberculoma (n=14). In 40 patients, M. tuberculosis was isolated by BACTEC and average 15 days was required for detection, whereas it was 30 days in LJ medium. Results of drug-sensitivity testing (n=32) were obtained average 7 days after isolation. Patients from whom M. tuberculosis had been isolated by BACTEC more often had tuberculomas in CT imaging (P=0.018). CONCLUSION: Use of the BACTEC method allows early confirmation in patients with clinically probable TBM. It can guide clinicians in the rational use of anti-tuberculosis treatment by confirming diagnosis and identifying drug- sensitivity.

Comparative evaluation of BACTEC 460TB system and Lowenstein-Jensen medium for the isolation of M. tuberculosis from cerebrospinal fluid samples of tuberculous meningitis patients.

PURPOSE: To evaluate the role of the radiometric BACTEC 460TB system and the conventional Lowenstein-Jensen (LJ) medium for isolation of M. tuberculosis from cerebrospinal fluid (CSF) samples of tuberculous meningitis (TBM) patients. METHODS: CSF specimens (n=2325) from suspected TBM patients were processed for isolation of mycobacteria by inoculating BACTEC 12B medium and the LJ medium. The isolation of mycobacteria in both media was confirmed by microscopy and biochemical identification. Drug sensitivity testing for the anti-TB drugs was carried out by BACTEC radiometric method. RESULTS: Among the total 2325 CSF specimens processed by both methods, M. tuberculosis was isolated from 256 specimens. The isolation rates were 93% and 39% for the BACTEC system and LJ medium respectively. Both the media supported growth in 32% of the culture-positive specimens. BACTEC system alone yielded growth in 61% and LJ alone in 7%, of the culture-positive specimens. Among 205 isolates tested for drug susceptibility 81% were sensitive to all the drugs tested and 19% were resistant. CONCLUSIONS: The BACTEC 460TB system provides a highly sensitive and rapid tool for the isolation and drug susceptibility testing of M. tuberculosis, from CSF of TBM patients. Use of a solid medium in conjunction with the BACTEC 12B medium is essential for optimal recovery for M. tuberculosis from CSF specimens.

Role of IS6110 uniplex PCR in the diagnosis of tuberculous meningitis: experience at a tertiary neurocentre.

SETTING: Tuberculous meningitis (TBM) is the commonest form of neurotuberculosis in the Indian subcontinent. Rapid laboratory confirmation of TBM is important for the institution of early treatment and to avoid associated morbidity and mortality. The polymerase chain reaction (PCR) is the most widely applied alternative rapid diagnostic technique for TBM. OBJECTIVE: To evaluate the efficacy of an in-house developed IS6110 uniplex PCR (uPCR) in the diagnosis of TBM. DESIGN: A prospective, blinded study was conducted in a large sample base of 677 cerebrospinal fluid samples from 677 patients with clinically suspected TBM. RESULTS: All culture-positive samples (n = 136) were positive (100%) by the PCR assay. The assay was found to be positive in 70% (n = 541) of the samples with a clinical diagnosis of TBM. The assay had an observed sensitivity of 76.37% (negative predictive value 59.90%) and a specificity of 89.18% (positive predictive value 94.69%). A diagnostic accuracy of 80% (kappa 0.57) was seen in patients with a clinical diagnosis of TBM. Statistical significance was observed, as patients with a clinical diagnosis of TBM were found to be 9.38 times more likely to be PCR-positive (OR 9.38, chi2 = 149.94, P < 0.001). CONCLUSION: The performance of the in-house IS6110 uPCR assay merits its use as a sensitive and specific tool for the rapid diagnosis of TBM.