CMV infection of trabecular meshwork cells

Purpose: Human cytomegalovirus (HCMV) infections can cause endotheliitis which is associated with an elevation of the intraocular pressure (IOP). However, the mechanism of the IOP elevation has not been determined. The purpose of this study was to determine whether HCMV strains which are capable of infecting corneal endothelial cells can also replicate, induce anti-viral responses, and can reorganize the actin cytoskeleton in trabecular meshwork cells. Study design: Experimental study design Methods: Cultured primary human trabecular meshwork cells (HTMCs) were infected with the Towne or TB40/E strains of HCMV. TB40/E is trophic for vascular endothelial and corneal endothelial cells. Real-time PCR, western blot, and fluorescent immunostaining have been used to determine whether HCMV-infected HTMCs will support the expression of viral mRNA and protein, allow viral replication, and elicit anti-viral host responses. We also determined whether lytic replication was present after an HCMV infection. Results: HCMV infection led to the expression of viral mRNA and proteins of IE1, glycoprotein B(gB), and pp65. TB40/E infection induced interferon-β, a sign of host anti-viral immune response and MCP-1. Together with the induction of the regulators of actin cytoskeleton, myosin phosphatase Rho interacting protein (MPRIP) and monocyte chemotactic protein-1 (MCP-1), TB40/E induced a high level of expression of viral proteins, including IE1, gB, and pp65 as well as actin stress fiber formation, and achieved pathogenically high viral titers. Conclusions: Human trabecular meshwork cells support the replication of endotheliotropic TB40/E strain of HCMV which indicates that this strain may have high virulence for trabecular meshwork

Real-time PCR for VZV keratitis

Pupose: To determine the efficacy of real-time PCR for the diagnosis and prognosis of varicella-zoster virus (VZV) keratitis. Study design: Retrospective case series. Methods: Patients: 545 consecutive patients with keratitis were examined to quantify copy numbers of VZV DNA by real-time PCR. Association of copy numbers of DNA of VZV to clinical signs and disease course was assessed using logistic regression analysis and Cox proportional hazard model. Results: Of the 545 eyes, 38 eyes (6.9%) were diagnosed as VZV keratitis. The copy numbers of the DNA of VZV (median: 104.19 copy) was significantly associated with diagnosis of VZV keratitis with the highest odds ratio (OR) of 3390 (for median copy) compared to clinical signs. Diagnostic accuracy of the VZV DNA copy indicated good diagnostic value of area under the curve (0.92) by receiver operating characteristic analysis, and detection of unrelated VZV DNA from the cornea was very rare (0.2%). When the VZV DNA copy and clinical signs were assessed for association with the disease course after herpes zoster ophthalmicus, the disease duration was significantly prolonged in VZV keratitis cases with higher numbers of VZV DNA copies, iritis, and history of recurrences. The amount of VZV DNA led to a continuous risk to prolong disease duration until the ocular inflammation subsides (hazard ratio (HR) 0.17, 95%CI: 0.07 - 0.42, for median copies). Conclusions: Higher VZV DNA copy numbers are associated with the refractoriness of VZV keratitis, and its evaluation may be a clinically useful way to diagnose and manage VZV keratitis

Functional characterization of the regulators of calcineurin in Candida glabrata

The serine-threonine-specific protein phosphatase calcineurin is a key mediator of various stress responses in fungi. Herein, we characterized functions of the endogenous regulators of calcineurin (RCNs), Rcn1 and Rcn2, in the pathogenic fungus Candida glabrata. Rcn1 exerted both inhibitory and stimulatory effects on calcineurin signaling, but Rcn2 displayed only inhibitory activity. Phenotypic analyses of C. glabrata strains lacking either RCNs, calcineurin, or both revealed that calcineurin requires Rcn1, but not Rcn2, for antifungal tolerance in C. glabrata

Two-surgeon technique using saline-linked electric cautery and ultrasonic surgical aspirator in living donor hepatectomy: its safety and efficacy

Background: Saline-linked electric cautery (SLC) is introduced as an effective device to reduce blood loss in liver surgery. The aim of this study is to evaluate the safety and efficacy of two-surgeon technique using SLC and Cavitron Ultrasonic Surgical Aspirator (CUSA) in living donor hepatectomy. Methods: Forty-three living donor right hepatectomies were enrolled in this study. The first 28 cases underwent liver transection with CUSA alone (CUSA group), while additional SLC was applied in the current 15 cases (two-surgeon technique, TS group). Results: Blood loss was significantly reduced by two-surgeon technique (1115.2±652.9g in CUSA group vs 732.3±363.6g in TS group, p<0.05). In the TS group, there was no bile leakage from the cut surface. The early graft function and postoperative recipient survival were not significantly different between the groups. Conclusions: According to our single center experience, blood loss and donor complications were significantly reduced by two-surgeon technique using CUSA and SLC, with maintaining the graft viability

The glycosylphosphatidylinositol-linked aspartyl protease Yps1 is transcriptionally regulated by the calcineurin-Crz1 and Slt2 MAPK pathways in Candida glabrata.

In the pathogenic fungus Candida glabrata, the YPS1 gene, which encodes a glycosylphosphatidylinositol-linked aspartyl protease, is required for cell wall integrity and virulence. Although the expression of YPS1 has been studied in Saccharomyces cerevisiae, the transcriptional regulation of this gene in C. glabrata is not well understood. Here, we report that C. glabrata Yps1 is required for cell growth at elevated temperatures, and that the heat-induced expression of YPS1 is regulated predominantly by the calcineurin-Crz1 pathway and partially by the Slt2 MAPK pathway. Although a total of 11 YPS genes are present in the C. glabrata genome, the loss of transcriptional induction in a calcineurin mutant was observed only for YPS1. The results of a YPS1 promoter-lacZ reporter assay using a series of constructs with mutated promoter elements indicated that the transcription factor Crz1 binds to multiple sites in the promoter region of YPS1. To date, as none of the putative Crz1 targets in C. glabrata have been characterized using a Δcrz1 mutant, monitoring the expression of YPS1 represents an effective method for measuring the activity of the calcineurin-Crz1 signaling pathway in this fungus

Detection of significant antiviral drug effects on COVID-19 with reasonable sample sizes in randomized controlled trials : a modeling study

Background Development of an effective antiviral drug for Coronavirus Disease 2019 (COVID-19) is a global health priority. Although several candidate drugs have been identified through in vitro and in vivo models, consistent and compelling evidence from clinical studies is limited. The lack of evidence from clinical trials may stem in part from the imperfect design of the trials. We investigated how clinical trials for antivirals need to be designed, especially focusing on the sample size in randomized controlled trials. Methods and findings A modeling study was conducted to help understand the reasons behind inconsistent clinical trial findings and to design better clinical trials. We first analyzed longitudinal viral load data for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) without antiviral treatment by use of a within-host virus dynamics model. The fitted viral load was categorized into 3 different groups by a clustering approach. Comparison of the estimated parameters showed that the 3 distinct groups were characterized by different virus decay rates (p-value < 0.001). The mean decay rates were 1.17 d−1 (95% CI: 1.06 to 1.27 d−1), 0.777 d−1 (0.716 to 0.838 d−1), and 0.450 d−1 (0.378 to 0.522 d−1) for the 3 groups, respectively. Such heterogeneity in virus dynamics could be a confounding variable if it is associated with treatment allocation in compassionate use programs (i.e., observational studies). Subsequently, we mimicked randomized controlled trials of antivirals by simulation. An antiviral effect causing a 95% to 99% reduction in viral replication was added to the model. To be realistic, we assumed that randomization and treatment are initiated with some time lag after symptom onset. Using the duration of virus shedding as an outcome, the sample size to detect a statistically significant mean difference between the treatment and placebo groups (1:1 allocation) was 13,603 and 11,670 (when the antiviral effect was 95% and 99%, respectively) per group if all patients are enrolled regardless of timing of randomization. The sample size was reduced to 584 and 458 (when the antiviral effect was 95% and 99%, respectively) if only patients who are treated within 1 day of symptom onset are enrolled. We confirmed the sample size was similarly reduced when using cumulative viral load in log scale as an outcome. We used a conventional virus dynamics model, which may not fully reflect the detailed mechanisms of viral dynamics of SARS-CoV-2. The model needs to be calibrated in terms of both parameter settings and model structure, which would yield more reliable sample size calculation. Conclusions In this study, we found that estimated association in observational studies can be biased due to large heterogeneity in viral dynamics among infected individuals, and statistically significant effect in randomized controlled trials may be difficult to be detected due to small sample size. The sample size can be dramatically reduced by recruiting patients immediately after developing symptoms. We believe this is the first study investigated the study design of clinical trials for antiviral treatment using the viral dynamics model

Clinical features of pulmonary cryptococcosis in non-HIV patients in Japan

OBJECTIVE: To clarify the clinical features of pulmonary cryptococcosis in Japanese non-HIV population.METHODS: Retrospective investigation of 151 pulmonary cryptococcosis cases between 1977 and 2012 was executed. The underlying disease (UDs), aggravating factors, radiological characteristics, and treatment were examined.RESULTS: Sixty-seven patients (44.4%) had no UDs. The common UDs were diabetes (32.1%) followed by hematologic disease (22.6%), and collagen disease (22.6%). Peripherally distributed pulmonary nodules/masses were most commonly seen. Lesions in the right middle lobe (p = 0.01) and air bronchogram (P = 0.05) were significantly more frequent, respectively, in patients with UDs than patients without them. Azoles were mainly selected for the patients without meningoencephalitis. Mean treatment duration for patients with and without UDs was 6.64 and 2.87 months, respectively. Patients whose pulmonary nodules improved after treatment continued to experience gradual reduction of cryptococcosis antigen titers, even if antigen titers were positive at the time of treatment cessation. The average time for antigen titers to become negative after treatment cessation was 13.1 and 10.7 months for patients with and without UDs, respectively. When groups were compared according to the presence of meningoencephalitis complications, deaths, and survivals, factors contributing to cryptococcosis prognosis included higher age, hypoproteinemia, hypoalbuminemia, steroid use, high C-reactive protein levels, and meningoencephalitis complications.CONCLUSIONS: It is crucial to consider the presence of UDs and meningoencephalitis for the choice of antifungals and treatment duration for cryptococcosis in non-HIV patients. Three- and six months-administration of azoles for pulmonary cryptococcosis with or without UDs, respectively is reasonable