Porin of Shigella dysenteriae activates mouse peritoneal macrophage through Toll-like receptors 2 and 6 to induce polarized type I response

Porin of Shigella dysenteriae type 1 coexpressed Toll-like receptor (TLR) 2 and TLR6 on peritoneal cavity (PerC) macrophages (MPhi) of C57BL/6 mice implicating that both the TLRs are essential as a combinatorial repertoire to recognize the protein. Besides TLRs, mRNA for MyD88 and TRAF6, and nuclear translocation of NF-kappaB were enhanced that indicate their involvement in tandem in the activity of porin. The protein selectively up-regulated CD80 on the activated MPhi together with MHC class II molecule and CD40, and had no effect on CD86 expression. The porin-induced profile of MIP-1alpha, MIP-1beta and RANTES showed strong bias for chemokines correlated with M1 polarization. Intracellular expression and release of TNF-alpha and IL-12 in presence of porin was found to be TLR2 and NF-kappaB dependent. Induction of TNF-alpha and IL-12 along with the chemokine profile suggests type I polarization of the MPhi that would influence Th1-type response.     

A class of Covariate-Adjusted Response-Adaptive Allocation Designs for Multitreatment Binary Response Trials

A class of covariate-adjusted response-adaptive randomization procedures is developed for binary treatment outcomes in a phase III clinical trial set up involving multiple treatments. The target allocation is developed by combining the ethical aspects with statistical precision under the existence of treatment covariate interaction. Relevant measures of the performance for the proposed allocation designs are studied and compared.     

Molecular mechanisms of resistance to antibiotics in Bartonella bacilliformis

OBJECTIVES: Bartonella bacilliformis is the aetiological agent of Carrion's disease. Although ciprofloxacin, rifampicin and erythromycin have been successfully used in the treatment of the disease, failures and relapses have been reported. The objective of our study was to select in vitro mutants resistant to antibiotics in order to determine the frequency of mutations and to characterize the mechanism of resistance at the molecular level. METHODS: Antibiotic-resistant mutants were selected by serial passages of bacteria on blood agar plates containing antibiotics. Candidate genes involved in resistance were amplified and sequenced and compared in order to look at mutations associated with antibiotic resistance. RESULTS: Ciprofloxacin-, rifampicin- and erythromycin-resistant mutants were obtained after five, three and four passages, respectively. Conversely, no mutant was obtained with either gentamicin or doxycycline even after 16 passages. The ciprofloxacin mutant contained an amino acid change at position 87 (Asp --> Asn) in its quinolone resistance-determining region of the DNA gyrase protein, whereas the rifampicin-resistant strain had an amino acid change at position 531 (Ser --> Phe) in the rifampicin resistance-determining region of the rpoB gene. Similarly, the erythromycin-resistant mutant showed an A2058G mutation in the 23S rRNA gene. CONCLUSIONS: According with the current knowledge on the treatment of human bartonellosis, we believe that doxycycline in association with gentamicin may be the preferred regimen for the treatment of the acute and eruptive stages of Carrion's disease, but clinical trials are warranted to support our findings.     

Detection of HSV-1 variants highly resistant to the helicase-primase inhibitor BAY 57-1293 at high frequency in 2 of 10 recent clinical isolates of HSV-1

OBJECTIVES: BAY 57-1293 is a helicase-primase inhibitor (HPI) from a new class of antivirals that are highly efficacious in herpes simplex virus (HSV)-1 animal infection models. Resistant mutants with point mutations in the helicase (UL5) were reported to be present in laboratory isolates at a low frequency of approximately 10(-6). In contrast, we have shown elsewhere that some laboratory isolates contain resistant variants at higher frequency (10(-4)). Therefore, we screened 10 recent clinical isolates of HSV-1 for BAY 57-1293-resistant virions. METHODS: Clinical isolates were screened by a plaque reduction assay in Vero cells to determine the frequency of occurrence of BAY 57-1293-resistant variants. The helicase gene for the resistant variants was sequenced. RESULTS: One isolate contained highly resistant variants at 10(-4) and another at 10(-5). Both variants contained a previously reported BAY 57-1293 resistance mutation (K356N) in UL5 and were >5000-fold resistant. CONCLUSIONS: Occurrence of HPI-resistant viruses at high frequency in a clinical isolate is intriguing. Two alternative hypotheses are proposed to explain this phenomenon. It is also surprising that two unrelated clinical isolates contain an identical HPI resistance mutation. These results have important implications for HPI drug-resistance monitoring during subsequent clinical trials.     

Microsatellite markers within --SEA breakpoints for prenatal diagnosis of HbBarts hydrops fetalis

BACKGROUND: We sought to develop a rapid prenatal diagnostic test for simultaneous detection of HbBarts hydrops fetalis and exclusion of maternal contamination. METHODS: We developed a multiplex quantitative fluorescent PCR (QF-PCR) test that detects the presence/ absence of 2 microsatellite markers (16PTEL05/16PTEL06) located within breakpoints of the Southeast Asia ((-SEA)) deletion. HbBarts hydrops fetalis ((-SEA/-SEA)) is diagnosed by absence of both markers, and maternal contamination of fetal DNA is excluded by absence of noninherited maternal alleles. Fetal and parental DNA samples from 50 families were analyzed in a blinded clinical validation study, and QF-PCR results were compared with their respective molecular genotypes. RESULTS: The multiplex QF-PCR results included correct diagnoses of HbBarts hydrops fetalis in 11 of the fetuses tested, correct verification as unaffected in 20 fetuses, and correct identification as either carriers (alphaalpha/(-SEA)) or unaffected homozygotes in 18. Misidentification as unaffected occurred for 1 carrier. Sensitivity for diagnosis of HbBarts hydrops fetalis was 100% [lower 95% confidence interval, 76.2%], and specificity was 100% (lower 95% confidence interval, 92.6%). None of the samples tested showed any traces of noninherited maternal alleles; thus false-positives because of maternal contamination were eliminated. CONCLUSIONS: In this QF-PCR method, detection of maternally and paternally inherited fetal alleles allowed diagnosis of the double-deletion syndrome, and the ability to differentiate between these alleles allowed simultaneous exclusion of maternal contamination of the fetal genetic material. This novel strategy using cell-free fetal DNA in maternal plasma could form the basis for noninvasive testing for HbBarts hydrops fetalis.     

MYD88 L265P mutation in intraocular lymphoma: A potential diagnostic marker

Purpose:Vitreoretinal lymphoma (VRL) is the most common intraocular lymphoma (IOL). This can be either primary or secondary to the central nervous system lymphoma. The diagnosis of primary intraocular lymphoma (PIOL) currently relies on clinical diagnosis and cytological analysis of the vitreous or subretinal biopsy. Although most cases are diagnosed without much issue, the limited amount of vitreous fluid, subjectivity in cytological reporting, and special expertise in ocular pathology make the diagnosis challenging. MYD88 L265P mutation has been implicated to have diagnostic utility in PIOL. In this study, we screened consecutive vitreous biopsies for the presence of MYD88 L265P mutation to understand its diagnostic utility compared to conventional cytological analysis.Methods:Cytological analysis and MYD88 L265P mutation by PCR-based sequencing and restriction fragment length polymorphism (RFLP) were carried out on consecutive vitreous and subretinal biopsies collected from 21 patients. The diagnostic utility of the cytology and MYD88 L265P mutation analysis were compared.Results:Out of the 21 patients, 15 had clinical suspicion of having PIOL. Out of these suspected cases of PIOL, nine were confirmed on follow-up, while six were diagnosed as other intraocular pathologies. Diagnostic utility of MYD88 L265P mutation analysis revealed a sensitivity of 88.9%, specificity of 91.6%, positive and negative predictive value of 88.9% and 91.7%, respectively. Diagnostic accuracy of 90.5% was achieved with the mutation analysis that shows the superiority of MYD88 in both ruling in and ruling out PIOL. The diagnostic utility of MYD88 L265P mutation was superior to conventional cytological analysis.Conclusion:The analysis of MYD88 L265P mutation is reliable and efficient in the diagnosis of PIOL.