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Background: Studies of targeted therapy resistance in lung cancer have primarily focused on single-gene alterations. Based on prior work implicating apolipoprotein b mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) mutagenesis in histological transformation of epidermal growth factor receptor (EGFR)-mutant lung cancers, we hypothesized that mutational signature analysis may help elucidate acquired resistance to targeted therapies., Patients and Methods: APOBEC mutational signatures derived from an Food and Drug Administration-cleared multigene panel [Memorial Sloan Kettering Cancer Center Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT)] using the Signature Multivariate Analysis (SigMA) algorithm were validated against the gold standard of mutational signatures derived from whole-exome sequencing. Mutational signatures were decomposed in 3276 unique lung adenocarcinomas (LUADs), including 93 paired osimertinib-naïve and -resistant EGFR-mutant tumors. Associations between APOBEC and mechanisms of resistance to osimertinib were investigated. Whole-genome sequencing was carried out on available EGFR-mutant lung cancer samples (10 paired, 17 unpaired) to investigate large-scale genomic alterations potentially contributing to osimertinib resistance., Results: APOBEC mutational signatures were more frequent in receptor tyrosine kinase (RTK)-driven lung cancers (EGFR, ALK, RET, and ROS1; 25%) compared to LUADs at large (20%, P < 0.001); across all subtypes, APOBEC mutational signatures were enriched in subclonal mutations (P < 0.001). In EGFR-mutant lung cancers, osimertinib-resistant samples more frequently displayed an APOBEC-dominant mutational signature compared to osimertinib-naïve samples (28% versus 14%, P = 0.03). Specifically, mutations detected in osimertinib-resistant tumors but not in pre-treatment samples significantly more frequently displayed an APOBEC-dominant mutational signature (44% versus 23%, P < 0.001). EGFR-mutant samples with APOBEC-dominant signatures had enrichment of large-scale genomic rearrangements (P = 0.01) and kataegis (P = 0.03) in areas of APOBEC mutagenesis., Conclusions: APOBEC mutational signatures are frequent in RTK-driven LUADs and increase under the selective pressure of osimertinib in EGFR-mutant lung cancer. APOBEC mutational signature enrichment in subclonal mutations, private mutations acquired after osimertinib treatment, and areas of large-scale genomic rearrangements highlights a potentially fundamental role for APOBEC mutagenesis in the development of resistance to targeted therapies, which may be potentially exploited to overcome such resistance., (Copyright © 2022 European Society for Medical Oncology. Published by Elsevier Ltd. All rights reserved.)

Background: Although evidence suggests relationships between some crude oil components and glycemic dysregulation, no studies have examined oil spill-related chemical exposures in relation to type 2 diabetes mellitus (DM) risk. This study examined the relationship between total hydrocarbon (THC) exposure among workers involved in the 2010 Deepwater Horizon (DWH) oil spill and risk of DM up to 6 years afterward., Methods: Participants comprised 2660 oil-spill cleanup or response workers in the prospective GuLF Study who completed a clinical exam and had no self-reported DM diagnosis prior to the spill. Maximum THC exposure was estimated with a job-exposure matrix based on interview data and personal measurements taken during cleanup operations. We defined incident DM by self-reported physician diagnosis of DM, antidiabetic medication use, or a measured hemoglobin A1c value ≥ 6.5%. We used log binomial regression to estimate risk ratios (RRs) for DM across ordinal categories of THC exposure. The fully adjusted model controlled for age, sex, race/ethnicity, education, employment status, and health insurance status. We also stratified on clinical body mass index categories., Results: We observed an exposure-response relationship between maximum daily ordinal THC exposure level and incident DM, especially among overweight participants. RRs among overweight participants were 0.99 (95% CI: 0.37, 2.69), 1.46 (95% CI: 0.54, 3.92), and 2.11 (95% CI: 0.78, 5.74) for exposure categories 0.30-0.99 ppm, 1.00-2.99 ppm, and ≥3.00 ppm, respectively (p trend  = 0.03)., Conclusion: We observed suggestively increasing DM risk with increasing THC exposure level among overweight participants, but not among normal weight or obese participants., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background & Aims: Glecaprevir/pibrentasvir is approved for treating adults infected with HCV genotypes 1-6. In clinical trials, glecaprevir/pibrentasvir was associated with high rates of sustained virologic response at post-treatment week 12 (SVR12) and was well tolerated. A systematic review and meta-analysis of the real-world effectiveness and safety of glecaprevir/pibrentasvir were undertaken., Methods: Real-world studies reporting SVR12 in adults with HCV infection (n ≥20) treated with glecaprevir/pibrentasvir were identified in journal publications from January 1, 2017, to February 25, 2019, and congress presentations through April 14, 2019. Random-effects meta-analysis was used to determine SVR12 rates using data from ≥2 cohorts; intention-to-treat (ITT) analyses included patients treated with glecaprevir/pibrentasvir who had SVR12 data available, discontinued early, or were lost to follow-up; modified ITT (mITT) analyses excluded those with non-virologic failure. Naïve pooling was used to calculate adverse event (AE) rates., Results: Overall, 12,531 adults were treated with glecaprevir/pibrentasvir (18 cohorts). Of patients with post-treatment week 12 data, SVR12 rates were 96.7% (95% CI 95.4-98.1) in the ITT population (n = 8,583, 15 cohorts) and 98.1% (95% CI 97.1-99.2) in the mITT population (n = 7,001, 14 cohorts). SVR12 rates were ≥95% across subgroups (HCV genotype, cirrhosis status, treatment history, treatment duration, on-label treatment, and subgroups of interest). AEs were reported in 17.7% (1,271/7,199) of patients (8 cohorts). Serious AEs were reported in 1.0% (55/5,522) of patients (6 cohorts). The most frequent AEs were pruritus, fatigue, and headache. AE-related treatment discontinuations were reported in 0.6% (33/5,595) of patients (6 cohorts)., Conclusions: Consistent with clinical trials, real-world evidence indicates that glecaprevir/pibrentasvir is a well-tolerated and highly effective pangenotypic treatment for a broad range of HCV-infected patients., Lay Summary: It is important to assess treatments for hepatitis C virus (HCV) in the real world, as patient populations tend to be more diverse and potentially less adherent to treatment compared to those in clinical trials. Results from 18 studies performed in real-world clinics were pooled and analyzed to investigate the effectiveness and safety of a direct-acting antiviral combination (glecaprevir/pibrentasvir) in routine clinical practice. This analysis showed that glecaprevir/pibrentasvir is highly effective and well tolerated across all HCV genotypes and patient groups studied. It also showed that results seen in the real world are similar to the results seen in clinical trials, even in patients historically considered more challenging to treat., Competing Interests: Conflict of interest Pietro Lampertico: Speaker/advisor for Bristol-Myers Squibb, Roche, Gilead Sciences, GSK, AbbVie, MSD, Arrowhead, Alnylam, Janssen, MYR Pharma, Eiger BioPharmaceuticals. Jose A Carrión: Speaker for AbbVie, Gilead, and MSD; advisor for AbbVie. Michael P Curry: Consultant for Trio Health; advisor for Gilead Sciences, AbbVie, and Bristol-Myers Squibb; research support from Gilead. Juan Turnes: Speaker/consultant for AbbVie, Gilead, and MSD; research support from Gilead. Markus Cornberg: Speaker/advisor for AbbVie, Gilead, and Merck (MSD) related to this project; personal fees from AbbVie, Bristol-Myers Squibb, Gilead, Janssen-Cilag, Roche, MSD, Biogen, Falk Foundation, Boehringer Ingelheim, Siemens, Spring Bank outside submitted work. Francesco Negro: Advisor for AbbVie, Merck, and Gilead; research support from Gilead Sciences. Ashley Brown: Investigator, advisor and speaker for AbbVie, Gilead, and Merck; received financial support for attending academic meetings. Marcello Persico: Consultant for Gilead, MSD, and AbbVie. Nicole Wick: Trio Health employee; research support from AbbVie, Gilead, and Merck. Heiner Wedemeyer: Grants from Abbott, AbbVie, Bristol-Myers Squibb, Eiger, Falk and Falk Foundation, Gilead, Novartis, Merck/MSD, Roche, Roche Diagnostics; investigator in clinical trials for Transgene; advisor, committee chair, and investigator for MyrmbH. Ariel Porcalla, Andreas Pangerl, Eric Crown, Lois Larsen, and Yao Yu: Employees (or former employees) of AbbVie and may hold stock or share options. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Patient survival after pancreas after kidney transplant (PAK) has been reported to be inferior to patient survival after simultaneous pancreas-kidney transplant (SPK). The authors examine national data to further explore allograft (kidney and pancreas) and patient survival after PAK. Kaplan-Meier and Cox proportional hazard models were used to analyze Organ Procurement and Transplantation Network data from 1995 to 2010. The analysis compared PAK and SPK candidates and recipients. Kaplan-Meier analysis results showed that PAK after either a living or a deceased donor kidney transplant is associated with increased kidney graft survival compared with recipients with type 1 diabetes who received only a kidney. The best kidney allograft survival was for patients who received a living donor kidney followed by PAK. Receiving a living donor kidney was associated with increased pancreas allograft survival compared with receiving a deceased donor kidney. PAK transplant recipients who receive both organs have a survival advantage compared with uremic candidates who receive neither (SPK waitlist). Compared with uremic diabetic waitlist patients, SPK and PAK recipients showed similar overall patient survival. Successful PAK offers a survival advantage compared with receiving neither a kidney nor a pancreas transplant. These data also suggest that receiving a pancreas (after kidney) transplant may have a protective effect on the kidney allograft., (© 2018 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Since 1976, melanoma-prone families have been followed at the National Cancer Institute to identify etiologic factors for melanoma. We compared risks of melanoma and other cancers in 1,226 members of 56 families followed for up to 4 decades with population rates in the Surveillance, Epidemiology, and End Results program. All families were tested for mutations in CDKN2A and CDK4; 29 were mutation-positive and 27 mutation-negative. We compared rates of invasive melanomas, both first and second, by family mutation status, with Surveillance, Epidemiology, and End Results program. Comparing three calendar periods of the study, risk of first primary melanoma decreased slightly. Risks of melanoma after first examination, however, were approximately one-third the risks prior to the first examination in both mutation-positive and mutation-negative families. Among patients with melanoma, risk of a second melanoma was increased 10-fold in all families; risk was somewhat higher in mutation-positive families. Risks of other second cancers were increased only for pancreatic cancer after melanoma in mutation-positive families. Over 4 decades, prospective risk of melanoma has decreased substantially in both mutation-positive and mutation-negative families, when melanoma has greatly increased in the general population., Trial Registration: NCI 02-C-0211, ClinicalTrials.gov ID NCT00040352., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The number of pancreas transplants performed in the United States increased by 7.0% in 2016 over the previous year, the first such increase in more than a decade, largely attributable to an increase in simultaneous kidney pancreas transplants. Transplant rates increased in 2016, and mortality on the waiting list decreased. The declining enthusiasm for pancreas after kidney (PAK) transplants persisted. The uniform definition of graft failure was approved by the OPTN Board of Directors in 2015 and will be implemented in early 2018. Meanwhile, SRTR continues to refrain from reporting pancreas graft failure data. The OPTN/UNOS Pancreas Transplantation Committee is seeking to broaden allocation of pancreata across compatible ABO blood types in a proposal out for public comment July 31 to October 2, 2017. A new initiative to provide guidance on the benefits of PAK transplants is also out for public comment., (.)

The availability of direct-acting antiviral agents for the treatment of hepatitis C virus (HCV) infection has resulted in a profound shift in the approach to the management of this infection. These changes have affected the practice of solid organ transplantation by altering the framework by which patients with end-stage organ disease are managed and receive organ transplants. The high level of safety and efficacy of these medications in patients with chronic HCV infection provides the opportunity to explore their use in the setting of transplanting organs from HCV-viremic patients into non-HCV-viremic recipients. Because these organs are frequently discarded and typically come from younger donors, this approach has the potential to save lives on the solid organ transplant waitlist. Therefore, an urgent need exists for prospective research protocols that study the risk versus benefit of using organs for hepatitis C-infected donors. In response to this rapidly changing practice and the need for scientific study and consensus, the American Society of Transplantation convened a meeting of experts to review current data and develop the framework for the study of using HCV viremic organs in solid organ transplantation., (© 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The number of pancreas transplants performed in the United States stabilized over the last 3 years after nearly a decade of steady decline. Numbers of new additions to the list also stabilized during the same period. Notably, the persistent decline in pancreas after kidney transplants also seems to have abated, at least for now. The first full year of data after implementation of the new pancreas allocation system revealed no change in the distribution of organs between simultaneous pancreas-kidney (SPK) transplant and pancreas transplant alone. The percentage of kidneys used in SPK transplants was also unchanged. While a uniform definition of pancreas graft failure was approved in June 2015, it is awaiting implementation. Meanwhile, SRTR will refrain from publishing pancreas graft failure data in the program-specific reports. Therefore, it is difficult to track trends in outcomes after pancreas transplant over the past 2 years. New initiatives by the OPTN/UNOS Pancreas Transplantation Committee include facilitated pancreas allocation and broadened allocation of pancreata across compatible ABO blood types to increase organ utilization., (.)

PMCA2 overexpression in some breast cancers suggests that this calcium pump isoform may play a role in breast pathophysiology. To investigate PMCA2 as a potential drug target for breast cancer therapy, we assessed the functional consequence of PMCA2 silencing on cell death pathways and calcium signals in the basal-like MDA-MB-231 breast cancer cell line. Silencing PMCA2 expression alone has no effect on MDA-MB-231 cell viability, however, PMCA2 silencing promotes calcium-induced cell death initiated with the calcium ionophore ionomycin. Assessment of cytoplasmic calcium responses generated with various agents including ionomycin demonstrates that in MDA-MB-231 cells, PMCA2 does not play a major role in shaping global calcium signals. We also examined the ability of PMCA2 silencing to modulate caspase-dependent cell death triggered by a Bcl-2 inhibitor that is in clinical development for the treatment of various cancers, ABT-263 (Navitoclax). Despite the lack of effect on global calcium responses, PMCA2 silencing augmented Bcl-2 inhibitor (ABT-263)-mediated MDA-MB-231 breast cancer cell death. These studies provide evidence that PMCA2 inhibitors could sensitize PMCA2-positive breast cancers to cell death initiators that work through mechanisms involving the Bcl-2 survival pathway., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Traumatic brain injury (TBI) results in an estimated 1.7 million emergency department visits each year in the United States. These injuries frequently occur outside, leaving injured individuals exposed to environmental temperature extremes before they are transported to a hospital., Objective: Evaluate the existing literature for evidence that exposure to high temperatures immediately after TBI could result in elevated body temperatures (EBTs), and whether or not EBTs affect patient outcomes., Discussion: It has been clear since the early 1980s that after brain injury, exposure to environmental temperatures can cause hypothermia, and that this represents a significant contributor to increased morbidity and mortality. Less is known about elevated body temperature. Early evidence from the Iraq and Afghanistan wars indicated that exposure to elevated environmental temperatures in the prehospital setting may result in significant EBTs, however, it is unclear what impact these EBTs might have on outcomes in TBI patients. In the hospital, EBT, or neurogenic fever, is thought to be due to the acute-phase reaction that follows critical injury, and these high body temperatures are associated with poor outcomes after TBI., Conclusion: Hospital data suggest that EBTs are associated with poor outcomes, and some preliminary reports suggest that early EBTs are common after TBI in the prehospital setting. However, it remains unclear whether patients with TBI have an increased risk of EBTs after exposure to high environmental temperatures, or if this very early "hyperthermia" might cause secondary injury after TBI., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Achievement of a sustained virologic response (SVR) with antiviral therapy significantly improves graft survival in hepatitis C virus (HCV) monoinfected liver transplant (LT) patients. Risks and benefits of HCV therapy in HCV-human immunodeficiency virus (HIV) coinfected LT recipients are not well established. Among 89 HCV-HIV LT recipients in the HIVTR cohort, 39 (23% Black, 79% genotype 1, 83% fibrosis stage ≤ 1) were treated with peginterferon-a2a or a2b plus ribavirin for a median 363 days (14-1373). On intent-to-treat basis, 22% (95% CI: 10-39) and 14% (95% CI: 5-30) achieved an end-of-treatment response (EOTR) and SVR, respectively. By per-protocol analysis (completed 48 weeks of therapy ± dose reductions), 42% and 26% had EOTR and SVR, respectively. Severe adverse events occurred in 85%, with 26% hospitalized with infections and 13% developing acute rejection. Early discontinuations and dose reductions occurred in 38% and 82%, respectively, despite use of growth factors in 85%. Eighteen of 39 treated patients (46%) subsequently died/had graft loss, with 10 (26%) attributed to recurrent HCV. In conclusion, SVR rates are low and tolerability is poor in HCV-HIV coinfected transplant recipients treated with peginterferon and ribavirin. These results highlight the critical need for better tolerated and more efficacious HCV therapies for HCV-HIV coinfected transplant recipients., (© Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Rapid allograft infection complicates liver transplantation (LT) in patients with hepatitis C virus (HCV). Pegylated interferon-α and ribavirin therapy after LT has significant toxicity and limited efficacy. The effect of a human monoclonal antibody targeting the HCV E2 glycoprotein (MBL-HCV1) on viral clearance was examined in a randomized, double-blind, placebo-controlled pilot study in patients infected with HCV genotype 1a undergoing LT. Subjects received 11 infusions of 50 mg/kg MBL-HCV1 (n=6) or placebo (n=5) intravenously with three infusions on day of transplant, a single infusion on days 1 through 7 and one infusion on day 14 after LT. MBL-HCV1 was well-tolerated and reduced viral load for a period ranging from 7 to 28 days. Median change in viral load (log10 IU/mL) from baseline was significantly greater (p=0.02) for the antibody-treated group (range -3.07 to -3.34) compared to placebo group (range -0.331 to -1.01) on days 3 through 6 posttransplant. MBL-HCV1 treatment significantly delayed median time to viral rebound compared to placebo treatment (18.7 days vs. 2.4 days, p<0.001). As with other HCV monotherapies, antibody-treated subjects had resistance-associated variants at the time of viral rebound. A combination study of MBL-HCV1 with a direct-acting antiviral is underway., (© Copyright 2013 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Utilization of livers from expanded criteria donors (ECD) is one strategy to overcome the severe organ shortage. The decision to utilize an ECD liver is complex and fraught with uncertainty for both providers and patients. We assessed patients' willingness to accept ECD liver transplantation (LTx) and acceptable 1-year mortality risk. One hundred eight patients listed for LTx were asked to rate their willingness to accept ECD LTx and the associated 1-year mortality risk they were willing to accept. Also, patients completed the SF-36v2 and sociodemographic and health information was gathered from their medical records. Patients reported significantly higher willingness to accept standard criteria donor (SCD) versus ECD LTx (t = 13.8, p < 0.001), with more than one-third of patients reporting low willingness to accept ECD LTx. Relative to our center's 10% SCD LTx 1-year mortality rate, most patients (71%) were willing to accept moderately or substantially higher 1-year mortality risk for ECD LTx. In multivariable analyses, higher lab MELD score and white race were significant independent predictors of both ECD willingness and ECD increased mortality risk acceptability. Findings highlight the importance of assessing patients' willingness to pursue ECD LTx and the relative mortality risks they are willing to accept., (©2011 The Authors Journal compilation©2011 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Liver transplantation (LT) is the treatment of choice for end-stage liver disease, but is controversial in patients with human immunodeficiency virus (HIV) infection. Using a prospective cohort of HIV-hepatitis B virus (HBV) coinfected patients transplanted between 2001-2007; outcomes including survival and HBV clinical recurrence were determined. Twenty-two coinfected patients underwent LT; 45% had detectable HBV DNA pre-LT and 72% were receiving anti-HBV drugs with efficacy against lamivudine-resistant HBV. Post-LT, all patients received hepatitis B immune globulin (HBIG) plus nucleos(t)ide analogues and remained HBsAg negative without clinical evidence of HBV recurrence, with a median follow-up 3.5 years. Low-level HBV viremia (median 108 IU/mL, range 9-789) was intermittently detected in 7/13 but not associated with HBsAg detection or ALT elevation. Compared with 20 HBV monoinfected patients on similar HBV prophylaxis and median follow-up of 4.0 years, patient and graft survival were similar: 100% versus 85% in HBV mono- versus coinfected patients (p = 0.08, log rank test). LT is effective for HIV-HBV coinfected patients with complications of cirrhosis, including those who are HBV DNA positive at the time of LT. Combination HBIG and antivirals is effective as prophylaxis with no clinical evidence of HBV recurrence but low-level HBV DNA is detectable in approximately 50% of recipients.

Background: There is ample evidence that both intimate-partner violence (IPV) and childhood abuse adversely affect the physical and mental health of adult women over the long term., Objective: The authors assessed the associations between abuse, symptoms, and mental health utilization., Method: The authors performed a cross-sectional survey of 380 adult female, internal-medicine patients., Results: Although both IPV and childhood abuse were associated with depressive and physical symptoms, IPV was independently associated with physical symptoms, and childhood abuse was independently associated with depression. Women with a history of childhood abuse had higher odds, whereas women with IPV had lower odds, of receiving care from mental health providers., Conclusion: IPV and childhood abuse may have different effects on women's symptoms and mental health utilization.

Background/aims: Chronic hepatitis B virus (HBV) disease is caused by both necroinflammation and active viral replication. The role of ALT levels as a predictor of liver injury has recently been questioned. The aim of the study was to determine whether normal ALT is associated with liver injury in a cohort of HBV patients undergoing liver biopsy., Methods: This is a retrospective review of chronic HBV patients divided into 3 groups; (1) persistently normal ALT (PNALT); (2) ALT 1-1.5X ULN and (3) ALT>1.5X ULN. Multiple clinical, biochemical, virological variables were evaluated., Results: One hundred and ninety-two patients met the inclusion criteria, 59 with PNALT, 26 with ALT 1-1.5X ULN, and 107 with ALT>1.5X ULN. Increasing age, higher ALT, higher grade of inflammation on biopsy, and HBeAg positivity predicted fibrosis. 18% of patients with PNALT had stage 2+ fibrosis and 34% had grade 2 or 3 inflammation. Overall 37% of patients with PNALT had significant fibrosis or inflammation. Subgroup analysis showed the majority with fibrosis belonged to the high normal ALT group and that only a minority who were young and immune tolerant had significant findings on biopsy., Conclusions: There is significant fibrosis and inflammation in 37% of patients with PNALT and a liver biopsy should be considered in patients older than 40 with high normal ALT.

PNF following liver transplantation (LT) is an infrequent but life-threatening complication. Liver allocation under MELD is based upon recipient severity of illness, a known risk factor for the occurrence of PNF. The incidence of PNF since the application of MELD has not previously been reported. The SRTR database was studied since inception of MELD until September 2004 for all adult recipients of deceased donor LT. PNF was defined as graft loss or death within 14 days of LT secondary to PNF or without defined cause. A total of 10545 transplants met inclusion criteria and PNF occurred in 613 (5.81%) of recipients. Univariate analysis demonstrated donor age, serum creatinine >1.5 mg/mL, hypertension and CVA as risk factors for PNF. Recipient factors included life support, mechanical ventilation, use of inotropes, hemodialysis, initial status 1 and use of a shared transplant. In the multivariate model only donor age and recipient serum creatinine, bilirubin, on life support and status 1 at transplant were significant risk factors for PNF. In this analysis of PNF in the MELD era the incidence of PNF does not appear to have increased from prior reports. Risk factors for PNF are related to donor age and severity of recipient illness.

Published values for sperm membrane water permeability (L(p)) obtained using a time-to-lysis methodology have produced anomalous results when used to model optimal cooling rates for cryopreservation of spermatozoa. As the lysis method is dependent on potentially questionable assumptions, we describe an alternative method for measuring sperm L(p). Spermatozoa were exposed to hypo- and hyperosmotic conditions using a stopped-flow apparatus and the time course of resulting volume changes was measured using concentration-dependent self-quenching of the entrapped fluorophore, carboxyfluorescein (CF). L(p) was measured for boar, rabbit, and ram spermatozoa using a range of osmotic stresses (+/-50-100 mOsm). Values for exosmotic and endosmotic flow showed no evidence of rectification. Mean L(p) values were 0.84 microm/min/atm (boar), 0.28 microm/min/atm (rabbit), and 2.79 microm/min/atm (ram). These values are lower than the lysis method estimates, with the ram value reduced by approximately two-thirds using the current methodology. The value for boar spermatozoa showed good agreement with published values obtained using an electronic cell-sizing technique. Substitution of the revised values for L(p) into the model for optimal cooling rates brings the calculated optimal rate closer to the lower empirically observed value but does not fully account for the previously reported discrepancies., (Copyright 2000 Academic Press.)

Murine and human studies have demonstrated that the normal liver contains significant numbers of resident lymphocytes that have functions distinct from those found in blood and other organs. To characterize these cells requires the isolation of viable lymphocytes that can be analysed by flow cytometry and in functional assays. The techniques classically used to isolate single cell suspensions of hepatic lymphocytes for phenotypic and functional studies involve mechanical and/or enzymatic dissociation of liver tissue. The aim of this study was to determine the effect of these procedures on surface molecule expression and lymphocyte function and to optimise an isolation technique that minimises these effects. Mechanical homogenisation of liver tissue alone resulted in low viable lymphocyte yields but these were improved by the combined use of mechanical and enzymatic techniques. A mean yield of 2.3 x 10(6) lymphocytes with a mean viability was 88.8% was obtained from 200 mg wedge biopsy samples of normal adult human liver using a combination of gentle mechanical dissociation followed by digestion with collagenase type IV and DNase I. These cells were suitable for phenotypic characterisation by flow cytometry. They also retained their ability to grow in vitro, to respond to cytokines and activation stimuli, to mediate cytotoxic killing of target cells, and to produce inflammatory and regulatory cytokines.

Background/aims: Hepatitis C virus (HCV) infection is associated with the development of chronic liver disease and extra-hepatic manifestations, which include autoantibody production, immune-mediated diseases such as cryoglobulinaemia and B-cell lymphoproliferation. Recent identification of intra-hepatic clonal B cells capable of rheumatoid factor production, selective infection of B cells over T cells and of an HCV receptor on B lymphocytes strongly supports a central role for these cells in the immune response to HCV infection. In particular, CD5+ B cells which are capable of producing natural antibodies with autoreactive specificities are likely to be important in the development of HCV-associated autoimmunity and lymphoproliferation., Methods: We have investigated the presence of CD5+ B cells in a unique cohort of HCV-infected women who were infected with a single inoculum of HCV genotype 1b following immunisation with contaminated anti-D immunoglobulin in 1977., Results: CD5+ B cells are significantly increased in chronic HCV infection (37.66+/-1.92%) as compared with those with resolved infection (25.33+/-1.90%). High levels of CD5+ B cells were associated with the production of rheumatoid factor. The number of peripheral blood CD5+ B cells correlated negatively with histological activity index., Conclusions: The expansion of this B cell population in patients with active HCV infection may give rise to immune-mediated sequelae associated with HCV infection. This expanded population of CD5+ B cells may protect against the development of progressive liver disease.

Objective: To describe the association between abuse during pregnancy and substance use and psychosocial stress., Design: Prospective study of pregnant women., Setting: Urban prenatal clinics., Participants: 1,937 predominately low-income, ethnically diverse women., Main Outcome Measures(s): Three questions from the Abuse Assessment Screen were used to measure abuse. For the total sample, 25.7% reported physical abuse in the past year, 10.5% physical abuse since pregnancy, and 4.5% sexual abuse in the past year. Adolescents were significantly more likely to report any abuse (37.6%) than were adults (22.6%) (chi-square = 44.94; df = 1; p < 0.001). White abused women were significantly more likely to report use of tobacco (chi-square = 17.34; df = 1; p < 0.001) and alcohol (chi-square = 5.65; df = 1, p < 0.01). Abused Asian women were more likely to smoke (chi-square = 12.13; df = 1, p < 0.001), as were women ethnically described as "other" (chi-square = 8.39; df = 1, p < 0.001). There was a higher, but not statistically significant, rate of substance use between abused and nonabused African-American, Native-American, and Hispanic women. Abused women of all races reported higher stress, less support from partners, less support from others, and lower self-esteem., Conclusions: Abuse during pregnancy is associated with an increased incidence of substance use and psychosocial stress. These relationships must be incorporated into the clinical care of abused pregnant women.

Dithiothreitol (DTT) activation of the adhesive function of several different integrins suggests the existence of a common DTT-sensitive integrin regulatory element. Ui11/E3, a natural killer (NK) cell-resistant murine target cell line genetically engineered to constitutively express human intercellular adhesion molecule-1 (ICAM-1; CD54) was used in a flow cytometric experimental model to evaluate DTT effects on the NK cell integrin adhesion molecule, leukocyte function antigen-1 (LFA-1; alpha L beta 2, CD11a/CD18). DTT and several structurally related dithiol compounds elicited a dramatic elevation in conjugate formation that was dependent on target cell ICAM-1 expression, was blocked by LFA-1 alpha L or beta 2 chain-specific antibodies, and occurred in the absence of Ui11/E3 target cell exposure to DTT or quantitative changes in NK cell membrane LFA-1 expression. This avidity modulation of LFA-1 by DTT required actin polymerization, was abrogated by the protein kinase C inhibitor calphostin C, involved activities of calyculin A- and okadaic acid-sensitive serine/threonine protein phosphatases PP-1 and/or PP-2A but not geldanamycin-sensitive tyrosine kinases, and differed with respect to kinetics and enzyme inhibitor sensitivity from LFA-1 activation promoted by cross-linking of NK cell CD16 or phorbol ester treatment. A key structural feature of DTT was the presence of two thiol groups, both reduced but not physically adjacent as in the nonstimulatory dithiol, 2,3-dimercaptopropanol. LFA-1 activation was not because of DTT chelation of Ca2+ or Zn2+. Immunoblotting studies identified multiple NK cell plasma membrane-associated proteins to be reduced by DTT under LFA-1-activating conditions, but similar effects were also promoted by reducing agent treatments that failed to alter adhesive function. Direct chemical modification of LFA-1 seemed an unlikely basis of activation because (1) DTT activated LFA-1 in HSB2 T cells without detectable disulfide reduction in LFA-1 alpha L or beta 2 chains immunoprecipitated from these cells and (2) DTT treatment of NK cells did not hinder binding of KIM127 and KIM185, monoclonal antibodies that recognize epitopes in the potentially DTT-susceptible cysteine-rich domain of the beta 2 chain. Thus, these results extended the range of DTT-activatible integrins to include NK cell LFA-1 and characterized for the first time signaling-associated enzymatic activities involved in DTT activation of NK cell LFA-1. Moreover, they suggested that structural features of DTT, particularly SH group spatial positioning, are important in LFA-activation for reasons other than cation chelation or disulfide reduction.(ABSTRACT TRUNCATED AT 400 WORDS)

Critical tonicity (tonicity at which 50% of cells swell beyond their maximum volume-to-surface area ratio and lyse) of rabbit spermatozoa was measured as 45.6 mOsm at 22 degrees C. To determine the temperature effect on critical tonicity, cells were equilibrated to 15, 25, and 35 degrees C and critical tonicity was measured as 52.9, 42.2, and 32.4 mOsm, respectively. The time taken for rabbit sperm to achieve lysis at these temperatures was measured by exposing cells for increasing times to distilled water. From these results values for the permeability coefficient of rabbit spermatozoa to water (Lp) and its activation energy (Ea) were calculated. At 25 degrees C, Lp was 0.63 micron/min/atm and was clearly temperature dependent; Ea was 17.8 kcal/mol. Rabbit spermatozoa appear to have a low Lp and high Ea, the opposite of the situation seen with spermatozoa from all other species examined to date which have high Lp and low Ea. Nevertheless, the values obtained permit modeling of cooling rates for cell survival during cryopreservation in keeping with cooling rates commonly employed.

The effects of hyposmotic and hyperosmotic stresses on ram and human spermatozoa were examined. Human spermatozoa exhibited a precipitate decline in survival at osmolalities below 90 mOsm caused by cells swelling beyond their maximum volume-to-surface area ratio and lysing. Ram spermatozoa exhibited a progressive decline in cell survival at relatively small hyposmotic stresses before exceeding their maximum volume-to-surface area ratio; this prelytic cell loss could be prevented by decreasing the osmolality in a series of 25-mOsm steps. Repeated hyposmotic stress experiments indicated that cells sensitive to prelytic damage constitute a discrete subpopulation within the ram ejaculate. Spermatozoa of both species were apparently resistant to hyperosmotic stresses; human spermatozoa maintained membrane integrity when subject to stresses up to 2.5 Osm and ram spermatozoa up to 1 Osm. However, ram spermatozoa suffered an almost complete and irreversible loss of motility above 600 mOsm. Spermatozoa of both species exposed to hyperosmotic stress and returned to isosmotic conditions exhibited significant cell damage, although ram spermatozoa were the more vulnerable. These observations are related to cryopreservation of spermatozoa.

The earliest muscle in zebrafish arises from iterated sets of two to six cells in each somite, the muscle pioneers (MP). MP develop synchronously in young trunk myotomes adjacent to the notochord, precisely where the horizontal myoseptum will form. They elongate without cell fusion and differentiate hours earlier than surrounding cells, thus providing a simple and accessible system for in vivo study of myogenesis and muscle patterning. Before the MP form definitive myofibrils they assemble long bundles of actin-containing filaments, similar to "stress-fiber-like structures" reported by others. In fub-1 mutants, in which myofibrils are disorganized in all skeletal muscle cells, the MP appear and elongate normally, but ordered actin filament bundles are not seen. This defect could underlie the later myofibrillar ones, consistent with the proposal that actin filament bundles are essential for proper formation of the muscle contractile apparatus.

To assess the potential use of plasma apolipoprotein B (ApoB) as a risk factor for coronary artery disease, this apolipoprotein was quantified by electroimmunoassay in 161 male patients with angiographically documented coronary atherosclerosis and 72 male patients with normal coronary arteries. In addition to ApoB, the analyzed lipoprotein profile included plasma total cholesterol, plasma triglyceride and VLDL-, LDL- and HDL-cholesterol levels. Age, plasma cholesterol, triglyceride, and VLDL- and LDL-cholesterol were significantly greater (P less than 0.05) for patients with coronary artery disease than for patients with normal arteries. In contrast, there was no difference in the mean levels of HDL-cholesterol between these 2 groups of patients. However, patients with HDL-cholesterol les than 40 mg/dl had a higher rate of coronary artery disease than those with HDL-cholesterol greater than 40 mg/dl (P less than 0.05). The results of multivariate analysis showed that age and plasma cholesterol were the 2 variables most significantly (P less than 0.01) related to the presence of coronary artery disease. However, in a subgroup of patients with plasma cholesterol less than 265 mg/dl, the most reliable variable was ApoB (P less than 0.01). For patients under 50 years of age ApoB and LDL-cholesterol were the most significant variables (P less than 0.05), whereas for patients at 50 years of age or older VLDL-cholesterol was the most significant variable (P less than 0.01). Results of this study indicate that measurement of ApoB may offer important predictive value for coronary artery disease, especially at lower levels of plasma cholesterol. Whether this and other conclusions also apply to general population, remains to be established in future studies.

The excluded fraction from Sephadex G-25 separation of human seminal plasma has been found to suppress lymphocyte transformation and DNA synthesis [7]. We have shown that most of its in vitro activity is due to contamination by spermine which binds tenaciously to, and co-elutes with the protein.