Persistence of Ebola virus in semen among Ebola virus disease survivors in Sierra Leone: A cohort study of frequency, duration, and risk factors.

BACKGROUND: Sexual transmission chains of Ebola virus (EBOV) have been verified and linked to EBOV RNA persistence in semen, post-recovery. The rate of semen persistence over time, including the average duration of persistence among Ebola virus disease (EVD) survivors, is not well known. This cohort study aimed to analyze population estimates of EBOV RNA persistence rates in semen over time, and associated risk factors in a population of survivors from Sierra Leone. METHODS AND FINDINGS: In this cohort study from May 2015 to April 2017 in Sierra Leone, recruitment was conducted in 2 phases; the first enrolled 100 male participants from the Western Area District in the capital of Freetown, and the second enrolled 120 men from the Western Area District and from Lungi, Port Loko District. Mean age of participants was 31 years. The men provided semen for testing, analyzed by quantitative reverse transcription PCR (qRT-PCR) for the presence of EBOV RNA. Follow-up occurred every 2 weeks until the endpoint, defined as 2 consecutive negative qRT-PCR results of semen specimen testing for EBOV RNA. Participants were matched with the Sierra Leone EVD case database to retrieve cycle threshold (Ct) values from the qRT-PCR analysis done in blood during acute disease. A purposive sampling strategy was used, and the included sample composition was compared to the national EVD survivor database to understand deviations from the general male survivor population. At 180 days (6 months) after Ebola treatment unit (ETU) discharge, the EBOV RNA semen positive rate was 75.4% (95% CI 66.9%-82.0%). The median persistence duration was 204 days, with 50% of men having cleared their semen of EBOV RNA after this time. At 270 days, persistence was 26.8% (95% CI 20.0%-34.2%), and at 360 days, 6.0% (95% CI 3.1%-10.2%). Longer persistence was significantly associated with severe acute disease, with probability of persistence in this population at 1 year at 10.1% (95% CI 4.6%-19.8%) compared to the probability approaching 0% for those with mild acute disease. Age showed a dose-response pattern, where the youngest men (≤25 years) were 3.17 (95% CI 1.60, 6.29) times more likely to be EBOV RNA negative in semen, and men aged 26-35 years were 1.85 (95% CI 1.04, 3.28) times more likely to be negative, than men aged >35 years. Among participants with both severe acute EVD and a higher age (>35 years), persistence remained above 20% (95% CI 6.0%-50.6%) at 1 year. Uptake of safe sex recommendations 3 months after ETU discharge was low among a third of survivors. The sample was largely representative of male survivors in Sierra Leone. A limitation of this study is the lack of knowledge about infectiousness. CONCLUSIONS: In this study we observed that EBOV RNA persistence in semen was a frequent phenomenon, with high population rates over time. This finding will inform forthcoming updated recommendations on risk reduction strategies relating to sexual transmission of EBOV. Our findings support implementation of a semen testing program as part of epidemic preparedness and response. Further, the results will enable planning of the magnitude of testing and targeted counseling needs over time.

The M694V mutation in Armenian-Americans: a 10-year retrospective study of MEFV mutation testing for familial Mediterranean fever at UCLA.

Familial Mediterranean fever (FMF), inherited in an autosomal recessive manner, is a systemic auto-inflammatory disorder characterized by recurrent attacks of fever with peritonitis, pleuritis, synovitis and erysipeloid rash. The marenostrin-encoding fever (MEFV) gene, located on chromosome 16p13.3, is the only gene in which mutations are currently known to cause FMF. To correlate specific genotypes with adverse phenotypes of affected populations residing in the Western United States, a retrospective case series review was conducted of all MEFV gene mutation testing completed at UCLA Clinical Molecular Diagnostic Laboratory between February 2002 and February 2012, followed by clinical chart review of all subjects who either have a single or double mutation. All 12 common mutations in the MEFV gene were analyzed and the M694V variant was found to be associated with an adverse FMF clinical outcome in the Armenian-American population, manifested by earlier onset of disease, increased severity of disease, and renal amyloidosis.

Dose escalation with proton radiation therapy for high-grade meningiomas.

Purpose of this study was to determine the toxicity and treatment outcome after dose escalation with proton radiation therapy for patients with World Health Organization (WHO) grade II and grade III meningiomas. Between 1997 and 1999, 6 patients with newly diagnosed or recurrent grade II or III meningioma were treated on a Phase I/II dose escalation trial with combined proton-photon radiotherapy at the Harvard Cyclotron Laboratory/Massachusetts General Hospital. The median age was 46. The sites were sphenoid wing in 2 patients, parasagittal/falcine in 2, parasellar in 1, and olfactory groove in 1. The median gross total volume (GTV) at the time of radiation was 13.3 cc (range: 4.0-129.5). The total dose to the GTV for the grade II and III meningiomas was 68.4 and 72.0 Gy (RBE) in 1.8 Gy (RBE), respectively. The median percentage of proton was 80%. All patients tolerated radiation treatment without any treatment break. None of the patients required steroids or hospitalization during radiation. There was no acute grade 3 to 5 toxicity. With a median follow-up period of 145 months for all surviving patients, one patient developed local recurrence. For the 5 patients with tumor controlled at the primary sites, 3 patients developed new meningioma(s) distantly from the primary sites at a median time of 25 months (range, 9-79). The median survival for grade II and grade III tumors was 145 months and 28 months, respectively. One patient developed late grade 1 dry eye. Two patients developed late grade 2 hypothyroidism and two developed grade 2 hypogonadism. There was no late grade 3-5 toxicity. Dose escalation with proton radiation therapy resulted in low toxicity and high local control rate in patients with high-grade meningiomas. Development of distant meningioma(s) intracranially was the main pattern of failure. Larger prospective studies are necessary to confirm our results.

HIV is hyperendemic among men who have sex with men in San Francisco: 10-year trends in HIV incidence, HIV prevalence, sexually transmitted infections and sexual risk behaviour.

OBJECTIVES: To evaluate trends in the HIV epidemic among men who have sex with men (MSM) in San Francisco and the implications for HIV prevention. METHODS: An ecological approach assessed temporal trends in sexual risk behaviour, sexually transmitted infections (STI), HIV incidence and prevalence from multiple data sources between 1998 and 2007. RESULTS: By 2007, there were over 13 000 HIV-infected MSM living in San Francisco. No consistent upward or downward temporal trends were found in HIV incidence, newly reported HIV cases, AIDS deaths, proportion of AIDS cases using antiretroviral therapy, rectal gonorrhoea or primary and secondary syphilis cases among MSM during the study period. Trends in indicators of sexual risk behaviour among MSM were mixed. Overall, unprotected anal intercourse (UAI) increased in community-based surveys. Among HIV-positive MSM, no significant trends were noted for UAI. Among HIV-negative MSM, UAI with unknown serostatus partners decreased but increased with potentially discordant serostatus partners. Among MSM seeking HIV testing, increases were noted in insertive UAI at anonymous testing sites and at the STI clinic, in receptive UAI at anonymous test sites and in receptive UAI with a known HIV-positive partner at the STI clinic. CONCLUSIONS: Temporal trends in multiple biological and behavioural indicators over the past decade describe a hyperendemic state of HIV infection among MSM in San Francisco, whereby prevalence has stabilised at a very high level. In the absence of new, effective prevention strategies this state will persist.

Canadian survey of clinical status at dialysis initiation 1998-1999: a multicenter prospective survey.

AIMS: The current growth in end-stage kidney disease populations has led to increased efforts to understand the impact of status at dialysis initiation on long-term outcomes. Our main objective was to improve the understanding of current Canadian nephrology practice between October 1998 and December 1999. METHODS: Fifteen nephrology centers in 7 provinces participated in a prospective data collection survey. The main outcome of interest was the clinical status at dialysis initiation determined by: residual kidney function, preparedness for chronic dialysis as measured by presence or absence of permanent peritoneal or hemodialysis access, hemoglobin and serum albumin. Uremic symptoms at dialysis initiation were also recorded, however, in some cases these symptom data were obtained retrospectively. RESULTS: Data on 251 patients during 1-month periods were collected. Patients commenced dialysis at mean calculated creatinine clearance levels of approximately 10 ml/min, with an average of 3 symptoms. 35% of patients starting dialysis had been known to nephrologists for less than 3 months. These patients are more likely to commence without permanent access and with lower hemoglobin and albumin levels. Even of those known to nephrologists, only 66% had permanent access in place. CONCLUSIONS: Patients commencing dialysis in Canada appear to be doing so in relative concordance with published guidelines with respect to timing of initiation. Despite an increased awareness of kidney disease, a substantial number of patients continues to commence dialysis without previous care by a nephrologist. Of those who are seen by nephrologists, clinical and laboratory parameters are suboptimal according to current guidelines. This survey serves as an important baseline for future comparisons after the implementation of educational strategies for referring physicians and nephrologists.

The relevance of tissue angiotensin-converting enzyme: manifestations in mechanistic and endpoint data.

Angiotensin-converting enzyme (ACE) is primarily localized (>90%) in various tissues and organs, most notably on the endothelium but also within parenchyma and inflammatory cells. Tissue ACE is now recognized as a key factor in cardiovascular and renal diseases. Endothelial dysfunction, in response to a number of risk factors or injury such as hypertension, diabetes mellitus, hypercholesteremia, and cigarette smoking, disrupts the balance of vasodilation and vasoconstriction, vascular smooth muscle cell growth, the inflammatory and oxidative state of the vessel wall, and is associated with activation of tissue ACE. Pathologic activation of local ACE can have deleterious effects on the heart, vasculature, and the kidneys. The imbalance resulting from increased local formation of angiotensin II and increased bradykinin degradation favors cardiovascular disease. Indeed, ACE inhibitors effectively reduce high blood pressure and exert cardio- and renoprotective actions. Recent evidence suggests that a principal target of ACE inhibitor action is at the tissue sites. Pharmacokinetic properties of various ACE inhibitors indicate that there are differences in their binding characteristics for tissue ACE. Clinical studies comparing the effects of antihypertensives (especially ACE inhibitors) on endothelial function suggest differences. More comparative experimental and clinical studies should address the significance of these drug differences and their impact on clinical events.

Genetic manipulation of the renin-angiotensin system.

The renin-angiotensin system is widely known for its importance in control of blood pressure, electrolyte homeostasis and volume regulation. Recently, renin-angiotensin system function was studied using homologous recombination in embryonic stem cells to manipulate the mouse genome. Angiotensinogen, angiotensin-converting enzyme and angiotensin II receptors were each eliminated in separate lines of mice. These null animals share similar phenotypes, such as a lowering of blood pressure, abnormal renal development, malfunction of the kidney and, unexpectedly, a decrease in hematocrit. In addition, angiotensin-converting enzyme null male mice sire far smaller litters than male wild-type mice. This suggests an unexplored role for angiotensin-converting enzyme in conception. Future studies with these and other genetically engineered mice lines will reveal novel physiological effects of angiotensin II.

Tyrosine kinase activation by the angiotensin II receptor in the absence of calcium signaling.

The angiotensin II type 1 (AT(1)) receptor signals via heterotrimeric G-proteins and intracellular tyrosine kinases. Here, we investigate a modified AT(1) receptor, termed M5, where the last five tyrosines (residues 292, 302, 312, 319, and 339) within the intracellular carboxyl tail have been mutated to phenylalanine. This receptor did not elevate cytosolic free calcium or inositol phosphate production in response to angiotensin II, suggesting an uncoupling of the receptor from G-protein activation. Despite this, the M5 receptor still activated tyrosine kinases, induced STAT1 tyrosine phosphorylation, and stimulated cell proliferation. We also studied another AT(1) mutant receptor, D74E, stably expressed in Chinese hamster ovarian cells and a fibroblast cell line from mice with a genetic inactivation of Galpha(q/11). Both cell lines have a deficit in calcium signaling and in G-protein activation, and yet in both cell lines, angiotensin II induced the time-dependent tyrosine phosphorylation of STAT1. These studies are the first to show the ability of a seven-transmembrane receptor to activate intracellular tyrosine kinase pathways in the absence of a G-protein-coupled rise in intracellular calcium.

The angiotensin II-dependent nuclear translocation of Stat1 is mediated by the Jak2 protein motif 231YRFRR.

In response to angiotensin II, Jak2 autophosphorylates and binds the angiotensin II AT(1) receptor. By studying a variety of Jak2 deletion proteins, we now show that the Jak2 protein motif (231)YRFRR is required for the co-association of this kinase with the AT(1) receptor. We also used a full-length Jak2 protein containing a (231)FAAAA amino acid substitution. Although this protein still autophosphorylated in response to angiotensin II, it did not co-associate with the AT(1) receptor. This uncoupling indicates that AT(1)/Jak2 co-association is not necessary for angiotensin II-induced Jak2 autophosphorylation and that Jak2 autophosphorylation per se is insufficient for AT(1) receptor co-association. In response to angiotensin II, the Jak2-(231)FAAAA mutant will tyrosine phosphorylate Stat1. However, in the absence of AT(1)/Jak2 co-association, Stat1 did not translocate into the cell nucleus and failed to mediate gene transcription. This notable result indicates that Stat1 tyrosine phosphorylation alone is insufficient for Stat1 nuclear translocation. In summary, we now show that, although Jak2-mediated tyrosine phosphorylation of Stat1 is independent of receptor co-association, Jak2-mediated recruitment of Stat1 to the AT(1) receptor is critical for Stat1 nuclear translocation and subsequent gene transcription.

The role of Ca2+ mobilization and heterotrimeric G protein activation in mediating tyrosine phosphorylation signaling patterns in vascular smooth muscle cells.

This work investigated the role of Ca2+ mobilization and heterotrimeric G protein activation in mediating angiotensin II-dependent tyrosine phosphorylation signaling patterns. We demonstrate that the predominant, angiotensin II-dependent, tyrosine phosphorylation signaling patterns seen in vascular smooth muscle cells are blocked by the intracellular Ca2+ chelator BAPTA-AM, but not by the Ca2+ channel blocker verapamil. Activation of heterotrimeric G proteins with NaF resulted in a divergent signaling effect; NaF treatment was sufficient to increase tyrosine phosphorylation levels of some proteins independent of angiotensin II treatment. In the same cells, NaF alone had no effect on other cellular proteins, but greatly potentiated the ability of angiotensin II to increase the tyrosine phosphorylation levels of these proteins. Two proteins identified in these studies were paxillin and Jak2. We found that NaF treatment alone, independent of angiotensin II stimulation, was sufficient to increase the tyrosine phosphorylation levels of paxillin. Furthermore, the ability of either NaF and/or angiotensin II to increase tyrosine phosphorylation levels of paxillin is critically dependent on intracellular Ca2+. In contrast, angiotensin II-mediated Jak2 tyrosine phosphorylation was independent of intracellular Ca2+ mobilization and extracellular Ca2+ entry. Thus, our data suggest that angiotensin II-dependent tyrosine phosphorylation signaling cascades are mediated through a diverse set of signaling pathways that are partially dependent on Ca2+ mobilization and heterotrimeric G protein activation.

Lack of angiotensin II-facilitated erythropoiesis causes anemia in angiotensin-converting enzyme-deficient mice.

While nephrologists often observe reduced hematocrit associated with inhibitors of angiotensin-converting enzyme (ACE), the basis for this effect is not well understood. We now report that two strains of ACE knockout mice have a normocytic anemia associated with elevated plasma erythropoietin levels. (51)Cr labeling of red cells showed that the knockout mice have a normal total blood volume but a reduced red cell mass. ACE knockout mice, which lack tissue ACE, are anemic despite having normal renal function. These mice have increased plasma levels of the peptide acetyl-SDKP, a possible stem cell suppressor. However, they also show low plasma levels of angiotensin II. Infusion of angiotensin II for 2 weeks increased hematocrit to near normal levels. These data suggest that angiotensin II facilitates erythropoiesis, a conclusion with implications for the management of chronically ill patients on inhibitors of the renin-angiotensin system.

Antibiotic-heparin lock: in vitro antibiotic stability combined with heparin in a central venous catheter.

Long-term hemodialysis frequently requires vascular access through central venous catheters (CVCs). Infection related to these catheters is a significant complication. The use of an antibiotic-heparin lock could decrease the risks associated with infected permanent catheters. As an initial step in developing an antibiotic-heparin lock, we investigated the in vitro stability of antibiotic-heparin combinations in CVCs. Initially, cefazolin, vancomycin, ceftazidime, ciprofloxacin 10 mg/ml each, and gentamicin 5 mg/ml were incubated separately in glass test tubes in the dark at 37 degrees C for 72 hours. Samples were analyzed spectrophotometrically for stability at 24-hour intervals. The procedure was repeated with the addition of heparin (final concentration 5000 U/ml in glass test tubes), and the combination was also examined in CVCs. High-performance liquid chromatography analysis was conducted on the antibiotic-heparin combinations at 72 hours to confirm the spectrophotometric results. Ciprofloxacin produced an immediate precipitate with the addition of heparin and was not analyzed further. Absorbance values decreased for all antibiotics, with the greatest decreases at 72 hours for cefazolin (27.4%), vancomycin (29.7%), ceftazidime (40.2%), and gentamicin (8%) when combined with heparin. These decreases were postulated to be secondary to adsorption of the antibiotics to the luminal surface of the catheters because submitting the catheters to ultrasound with 1% sodium bicarbonate and analyzing the resulting solution for absorbance revealed that some of the drug was recovered. Although free antibiotic in CVC solution was reduced, the concentration should be sufficient (approximately 5 mg/ml) to decrease the frequency of infections associated with CVCs. We conclude that the concentrations of vancomycin, ceftazidime, cefazolin, or gentamicin used in our study should be sufficient for an antibiotic-heparin lock.

Jak2 acts as both a STAT1 kinase and as a molecular bridge linking STAT1 to the angiotensin II AT1 receptor.

Angiotensin II activates the Jak-STAT pathway via the AT(1) receptor. We studied two mutant AT(1) receptors, termed M5 and M6, that contain Y to F substitutions for the tyrosine residues naturally found in the third intracellular loop and the carboxyl terminus. After binding ligand, both the M5 and M6 AT(1) receptors trigger STAT1 tyrosine phosphorylation equivalent to that observed with the wild type receptor, indicating that angiotensin II-mediated phosphorylation of STAT1 is independent of these receptor tyrosine residues. In response to angiotensin II, Jak2 autophosphorylates on tyrosine, and Jak2 and STAT1 physically associate, a process that depends on the SH2 domain of STAT1 in vitro. Evaluation of the wild type, M5, and M6 AT(1) receptors showed that angiotensin II-dependent AT(1) receptor-Jak2-STAT1 complex formation is dependent on catalytically active Jak2, not on the receptor tyrosine residues in the third intracellular loop and carboxyl tail. Immunodepletion of Jak2 virtually eliminated the ligand-dependent binding of STAT1 to the AT(1) receptor. These data indicate that the association of STAT1 with the AT(1) receptor is not strictly bimolecular; it requires Jak2 as both a STAT1 kinase and as a molecular bridge linking STAT1 to the AT(1) receptor.

The experience of acupuncture for treatment of substance dependence.

PURPOSE: To explore the meaning of substance abusers' experience while receiving acupuncture as a part of the treatment for substance dependence. DESIGN: Phenomenology. Eight participants with diagnosis of substance dependence were selected for the study. The participants were in an inpatient setting with detoxification and rehabilitation programs provided to substance abusers. METHODS: In 1997, eight substance abusers who had accepted acupuncture as a part of their treatment for substance dependence were interviewed about their experiences with acupuncture treatment. Data collection included interviews with each of the subjects, the researcher's field notes, and demographic data obtained from the participants' medical records. The verbatim transcripts of each of the interviews were analyzed using Giorgi's modification of phenomenological method. FINDINGS: Analysis of the data after one treatment with acupuncture indicated seven major themes: anticipation of pain, apprehension concerning a new experience, mood elevation, inability to describe the experience, physical sensation, relaxation, and improved sleep. CONCLUSIONS: The substance abusers' experiences with acupuncture revealed an acceptance of a new treatment method, and demonstrated potential for healing through integration and balance. Further research is indicated to assess the effects of repeated treatments.

Insights derived from ACE knockout mice.

The evaluation of ACE knockout mice has illustrated the tremendous physiologic importance of the RAAS. We have discussed how interruption of this system influences blood pressure, renal function, renal development, serum and urine electrolyte composition, haematocrit and male reproductive capacity. This body of data underlines the modelling of the RAAS as a type of biological machine that is positioned to respond to environmental insult and to maintain a homeostasis of blood pressure, blood volume and electrolyte composition. These data also emphasise Harry Goldblatt's seminal observation that the kidney and the RAAS are intimately linked in the regulation of normal blood pressure.

A catalytically active Jak2 is required for the angiotensin II-dependent activation of Fyn.

Recent work with interleukins has shown a convergence of tyrosine phosphorylation signal transduction cascades at the level of the Janus and Src families of tyrosine kinases. Here we demonstrate that activation of the seven-transmembrane AT(1) receptor by angiotensin II induces a physical association between Jak2 and Fyn, in vivo. This association requires the catalytic activity of Jak2 but not Fyn. Deletion studies indicate that the region of Jak2 that binds Fyn is located between amino acids 1 and 240. Studies of the Fyn SH2 and SH3 domains demonstrate that the SH2 domain plays the primary role in Jak2/Fyn association. Not surprisingly, this domain shows a marked preference for tyrosine-phosphorylated Jak2. Surface plasmon resonance estimated the dissociation equilibrium constant (K(d)) of this association to be 2.36 nM. Last, in vivo studies in vascular smooth muscle cells show that, in response to angiotensin II, Jak2 activation is required for Fyn activation and induction of the c-fos gene. The significance of these data is that Jak2, in addition to serving as a critical angiotensin II activated signal transduction kinase, also functions as a docking protein and participates in the activation of Fyn by providing phosphotyrosine residues that bind the SH2 domain of Fyn.

Oral squamous cell carcinoma in a coyote (Canis latrans).

A 19-yr-old spayed female coyote (Canis latrans) was evaluated for an elliptical swelling of the skin beneath its right eye and an elevated mass that involved the soft and hard palate and gingivae around the upper right carnassial tooth and molars. Histopathologic analysis revealed a squamous cell carcinoma, and a postmortem examination revealed no evidence of vascular invasion or dissemination to the regional lymph nodes or viscera. This report describes the biology and progresion of an oral squamous cell carcinoma in an aged captive coyote.

Targeting genes for self-excision in the germ line.

A procedure is described that directs the self-induced deletion of DNA sequences as they pass through the male germ line of mice. The testes-specific promoter from the angiotensin-converting enzyme gene was used to drive expression of the Cre-recombinase gene. Cre was linked to the selectable marker Neor, and the two genes flanked with loxP elements. This cassette was targeted to the Hoxa3 gene in mouse ES cells that were in turn used to generate chimeric mice. In these chimeras, somatic cells derived from the ES cells retained the cassette, but self-excision occurred in all ES-cell-derived sperm.