Search

Introduction: Fluid overload on Extracorporeal Membrane Oxygenation (ECMO) is associated with worse outcomes. Previous in vitro studies have attempted to quantify oxygenator-related insensible losses, as failure to account for this fluid loss may lead to inaccurate fluid balance assessment and potentially harmful clinical management, such as unnecessary exposure to diuretics, slow continuous ultrafiltration (SCUF), or continuous kidney replacement therapy (CKRT). We performed a novel in vivo study to measure insensible fluid losses in pediatric ECMO patients., Methods: Pediatric ECMO patients were approached over eleven months in the pediatric and cardiac intensive care units. The water content of the oxygenator inflow sweep gas and exhaust gas were calculated by measuring the ambient temperature and relative humidity at frequent intervals and various sweep flow., Results and Discussion: Nine subjects were enrolled, generating 431 data points. The cohort had a median age of 11 years IQR [0.83, 13], weight of 23.2 kg IQR [6.48, 44.28], and body surface area of 0.815 m 2 IQR [0.315, 1.3725]. Overall, the cohort had a median sweep of 2.5 L/min [0.9, 4], ECMO flow of 3.975 L/m 2 /min [0.75, 4.51], and a set ECMO temperature of 37 degrees Celsius [36.6, 37.2]. The calculated net water loss per L/min of sweep was 75.93 ml/day, regardless of oxygenator size or patient weight. There was a significant difference in median documented vs. calculated fluid balance incorporating the insensible fluid loss, irrespective of oxygenator size (pediatric oxygenator: 7.001 ml/kg/day [-12.37, 28.59] vs. -6.11 ml/kg/day [-17.44, 13.01], respectively, p  = 0.005 and adult oxygenator: 14.36 ml/kg/day [1.54, 25.77] and 9.204 ml/kg/day [-1.28, 22.05], respectively, p  = <0.001). We present this pilot study of measured oxygenator-associated insensible fluid losses on ECMO. Our results are consistent with prior in vitro methods and provide the basis for future studies evaluating the impact of incorporating these fluid losses into patients' daily fluid balance on patient management and outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Suttles, Poe, Neumayr and Said.)

Neurologic injury continues to be a debilitating worldwide disease with high morbidity and mortality. The systemic sequelae of a neural insult often lead to prolonged hospital stays and challenging nutritional demands that contribute to poorer prognoses. Clinical management of a given condition should prioritize preserving the homeostatic parameters disrupted by inflammatory response cascades following the primary insult. This focused review examines the reciprocal relationship between electrolyte disturbance and neurologic injury. A prolonged electrolyte imbalance can significantly impact morbidity and mortality in neurologic injuries. A detailed overview of the major electrolytes and their physiologic, iatrogenic, and therapeutic implications are included. The pathophysiology of how dysnatremias, dyskalemias, dyscalcemias, and dysmagnesemias occur and the symptoms they can induce are described. The manifestations in relation to traumatic brain injury, status epilepticus, and acute ischemic stroke are addressed. Each type of injury and the strength of its association with a disruption in either sodium, potassium, calcium, or magnesium is examined. The value of supplementation and replacement is highlighted with an emphasis on the importance of early recognition in this patient population. This review also looks at the current challenges associated with correcting imbalances in the setting of different injuries, including the relevant indications and precautions for some of the available therapeutic interventions. Based on the findings of this review, there may be a need for more distinct clinical guidelines on managing different electrolyte imbalances depending on the specified neurologic injury. Additional research and statistical data on individual associations between insult and imbalance are needed to support this potential future call for context-based protocols., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Oncolytic viral therapy is quickly emerging as a promising subset of immunotherapy, which theoretically can target tumor cells while sparing surrounding healthy cells by harnessing the replication machinery of viruses with tropism for tumor cells, resulting in direct oncolysis, and by transforming immunologically "cold" tumor into areas that elicit the host's immune response. This review provides an overview of oncolytic viral therapy until the present day, starting with the original concept in 1912. The general mechanism of oncolytic viruses (OVs) depends on selectively integrating them into tumor cells based on genetic engineering of viral genomic material, inducing oncolysis and eliciting the host's innate immune response. Moreover, a major component of oncolytic viral therapy has been herpes simplex virus, with talimogene laherparepvec being the only FDA-approved oncolytic viral therapy for the treatment of melanomas. This review explores the characteristics, advantages, disadvantages, and therapeutic uses of several DNA and RNA viral families. A snapshot of the oncolytic viral treatments used in the most recent and advanced clinical trials is also provided. Lastly, the challenges of implementing oncolytic viral therapy are explored, both at a molecular and clinical level, with a highlight of promising future directions. In particular, the lack of an optimal delivery method based on tumor type for oncolytic viral therapy poses a significant obstacle, even in clinical studies. Intrathecal continuous delivery of OVs is a promising prospect, potentially by adapting the novel continuous irrigation and drainage IRRAflow catheter. Further exploration and testing of the IRRAflow catheter should be undertaken.

Fibromuscular dysplasia (FMD) is a nonatherosclerotic, noninflammatory vasculopathy with no identifiable underlying cause. Clinical manifestations of the disease typically occur at the site of occurrence. Ocular manifestations of fibromuscular dysplasia are rare but can occur in the form of central or branched retinal artery occlusions, which can cause painless monocular vision loss. We present the case of a 71-year-old female patient with FMD presenting with worsening visual acuity due to suspected right branch retinal artery occlusion. Pathology and imaging findings were consistent with classic FMD, and given our initial concerns for this patient, the rare ocular manifestations of this disease are highlighted., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2022, Mehkri et al.)

Lhermitte-Duclos disease (LDD) is a rare cerebellar lesion characterized by a hamartomatous lesion of the cerebellum. Mainly diagnosed by MRI, the clinical presentation is usually made of neurological symptoms. Modern neuroimaging techniques such as MRI have led to accurate diagnosis of this disease in both its pre- and post-operative periods. We present the case of a 68-year-old male with a past medical history of cardiac stenting and coronary artery disease who originally presented to the emergency department as a transfer for evaluation of possible obstructing hydrocephalus and left posterior inferior cerebellar artery (PICA) infarct. Based on the clinical presentation and imaging, the favored diagnosis of his left cerebellar abnormality was LDD rather than an unusual acute/subacute infarct or a metastatic lesion. The rapid progression of symptoms with rapidly progressive cytotoxic edema on serial CTs helped exclude LDD, which is nearly always more of a chronic process. The classic neuroimaging findings and clinical presentation of LDD are also discussed., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2022, Mehkri et al.)

What motivates faculty teaching gateway courses to consider adopting an evidence-based classroom intervention? In this nationally representative study of biology faculty members in the United States (N = 422), we used expectancy-value-cost theory to understand three convergent motivational processes the faculty members' underlying intentions to adopt an exemplar evidence-based classroom intervention: the utility value intervention (UVI). Although the faculty members perceived the intervention as valuable, self-reported intentions to implement it were degraded by concerns about costs and lower expectancies for successful implementation. Structural equation modeling revealed that the faculty members reporting lower intentions to adopt it tended to be White and to identify as male and had many years of teaching or were from a more research-focused university. These personal, departmental, and institutional factors mapped onto value, expectancies, and cost perceptions uniquely, showing that each process was a necessary but insufficient way to inspire intentions to adopt the UVI. Our findings suggest multifaceted, context-responsive appeals to support faculty member motivation to scale up adoption of evidence-based classroom interventions., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Institute of Biological Sciences.)

Objectives: Academia is grappling with how to address persistent underrepresentation and reduce inequities. With so many diversity-enhancing initiatives underway, some within the academic community might experience "diversity fatigue," a construct we use to understand majority groups' feelings of weariness toward diversity efforts., Method: For our testing purposes, we focused on ethnic and minority underrepresentation, and collected data in four studies from 473 White American students and faculty. Using exploratory and confirmatory factor analysis and latent profile analysis, we develop and confirm the single factor structure of the final 6-item Diversity Fatigue scale. We measured associations with other established measures and examined the strength of the association between diversity fatigue and faculty's support for a diversity-enhancing intervention., Results: Results demonstrated scale reliability, convergent validity with system-justifying beliefs, and offer suggestive evidence of discriminant validity with inclusion concerns and implicit race-based associations. Although mean levels of diversity fatigue were low overall, diversity fatigue scores were related to concerns about the effort involved with diversity work and were significantly associated with faculty's motivation to adopt a diversity-enhancing classroom activity., Conclusions: Diversity fatigue in academia is a dampening in people's response to or enthusiasm for efforts that improve the experience of underrepresented people. This state experience is connected to system-justifying beliefs and is related to concerns about the effort required to do diversity interventions. Understanding and measuring this construct has implications for the psychology of intergroup relations, as well as practical implications for campus communities committed to diversity programs. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Study Design: Retrospective analysis of administrative billing data., Objective: To evaluate the extent to which a metric of serious complications determined from administrative data can reliably profile hospital performance in spine fusion surgery., Summary of Background Data: While payers are increasingly focused on implementing pay-for-performance measures, quality metrics must reliably reflect true differences in performance among the hospitals profiled., Methods: We used State Inpatient Databases from nine states to characterize serious complications after elective cervical and thoracolumbar fusion. Hierarchical logistic regression was used to risk-adjust differences in case mix, along with variability from low case volumes. The reliability of this risk-stratified complication rate (RSCR) was assessed as the variation between hospitals that was not due to chance alone, calculated separately by fusion type and year. Finally, we estimated the proportion of hospitals that had sufficient case volumes to obtain reliable (>0.7) complication estimates., Results: From 2010 to 2017 we identified 154,078 cervical and 213,133 thoracolumbar fusion surgeries. 4.2% of cervical fusion patients had a serious complication, and the median RSCR increased from 4.2% in 2010 to 5.5% in 2017. The reliability of the RSCR for cervical fusion was poor and varied substantially by year (range 0.04-0.28). Overall, 7.7% of thoracolumbar fusion patients experienced a serious complication, and the RSCR varied from 6.8% to 8.0% during the study period. Although still modest, the RSCR reliability was higher for thoracolumbar fusion (range 0.16-0.43). Depending on the study year, 0% to 4.5% of hospitals had sufficient cervical fusion case volume to report reliable (>0.7) estimates, whereas 15% to 36% of hospitals reached this threshold for thoracolumbar fusion., Conclusion: A metric of serious complications was unreliable for benchmarking cervical fusion outcomes and only modestly reliable for thoracolumbar fusion. When assessed using administrative datasets, these measures appear inappropriate for high-stakes applications, such as public reporting or pay-for-performance.Level of Evidence: 3., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Alternating Hemiplegia of Childhood (AHC) is a devastating autosomal dominant disorder caused by ATP1A3 mutations, resulting in severe hemiplegia and dystonia spells, ataxia, debilitating disabilities, and premature death. Here, we determine the effects of delivering an extra copy of the normal gene in a mouse model carrying the most common mutation causing AHC in humans, the D801N mutation. We used an adeno-associated virus serotype 9 (AAV9) vector expressing the human ATP1A3 gene under the control of a human Synapsin promoter. We first demonstrated that intracerebroventricular (ICV) injection of this vector in wild-type mice on postnatal day 10 (P10) results in increases in ouabain-sensitive ATPase activity and in expression of reporter genes in targeted brain regions. We then tested this vector in mutant mice. Simultaneous intracisterna magna and bilateral ICV injections of this vector at P10 resulted, at P40, in reduction of inducible hemiplegia spells, improvement in balance beam test performance, and prolonged survival of treated mutant mice up to P70. Our study demonstrates, as a proof of concept, that gene therapy can induce favorable effects in a disease caused by a mutation of the gene of a protein that is, at the same time, an ATPase enzyme, a pump, and a signal transduction factor.

Structural racism is a multilevel system of ideologies, institutions, and processes that have created and reified racial/ethnic inequities. As a system, it works in concert across institutions to propagate racial injustice. Thus, efforts to address structural racism and its implications for health inequity require transdisciplinary collaboration. In this article, we begin by describing the process through which we have leveraged our discipline-specific training -- spanning education, epidemiology, social work, sociology, and urban planning -- to co-construct a transdisciplinary analysis of the determinants of racial health inequity. Specifically, we introduce the underlying theories that guide our framework development and demonstrate the application of our integrated framework through a case example. We conclude with potential research and policy implications., Competing Interests: Competing Interests: None declared., (Copyright © 2020, Ethnicity & Disease, Inc.)

Executive Summary: Quality improvement, regulatory, and payer organizations use various definitions of hospital mortality as clinical outcome measures. In this prospective study, the authors evaluated a multicomponent intervention aimed at reducing inpatient mortality in a multistate healthcare delivery system. The project was initiated because of a statistically nonsignificant upward trend in mortality suggested by a six-quarter rise in the observed/expected mortality ratio generated by the Vizient Clinical Data Base and Resource Manager. The design of the mortality reduction plan was influenced by the known limitations of using hospital-wide mortality as a quality improvement measure. The primary objective was to reduce mortality through focused care redesign. The project leadership team attempted to implement standardized system-wide improvements while allowing individual hospitals to simultaneously pursue site-specific practice redesign opportunities. Between Q3, 2015, and Q4, 2017, system-wide mortality reduced from 1.78 to 1.53 (per 100 admissions; p = .01). The actual plan implemented in Mayo Clinic's hospitals is included as Appendix A to this article, published online as Supplemental Digital Content. The authors included it to allow comparison with similar efforts at other healthcare systems, as well as to stimulate criticism and discussion by readers.

Background: Historically, older people who inject drugs (PWID) have had the highest hepatitis C virus (HCV) burden; however, young PWID now account for recent increases. We assessed factors associated with past or present HCV infection (HCV antibody [anti-HCV] positive) among young (≤35 years) and older (>35 years) PWID., Methods: We calculated adjusted prevalence ratios (aPRs) and 95% confidence intervals (CIs) to examine sociodemographic and past 12-month injection behaviors associated with HCV infection., Results: Of 4094 PWID, 55.2% were anti-HCV positive. Among young PWID, anti-HCV prevalence was 42.1% and associated with ≤high school diploma/General Education Development diploma (GED) (aPR, 1.17 [95% CI, 1.03-1.33]), receptive syringe sharing (aPR, 1.37 [95% CI, 1.21-1.56]), sharing injection equipment (aPR, 1.16 [95% CI, 1.01-1.35]), arrest history (aPR, 1.14 [95% CI, 1.02-1.29]), and injecting speedball (aPR, 1.37 [95% CI, 1.16-1.61]). Among older PWID, anti-HCV prevalence was 62.2% and associated with ≤high school diploma/GED (aPR, 1.08 [95% CI, 1.02-1.15]), sharing injection equipment (aPR, 1.08 [95% CI, 1.02-1.15]), high injection frequency (aPR, 1.16 [95% CI, 1.01-1.34]), and injecting speedball (aPR, 1.09 [95% CI, 1.01-1.16])., Conclusions: Anti-HCV prevalence is high among PWID and varies with age. Scaling up direct-acting antiviral treatment, syringe service programs, and medication-assisted therapy is critical to mitigating transmission risk and infection burden., (Published by Oxford University Press for the Infectious Diseases Society of America 2019.)

The rate of drug and opioid overdose deaths in the United States has more than tripled over the past 15 years. The ability to conduct public health surveillance on nonfatal overdoses is limited. The current study used National HIV Behavioral Surveillance (NHBS) data to estimate recent and lifetime history of nonfatal overdose events in persons who inject drugs in 7 cities. Recent and lifetime experience of overdose events ranged from 3% to 20% and from 29% to 63%, respectively. Adapting systems such as NHBS may be useful in responding to and monitoring emergent public health problems such as the overdose epidemic.

Men who have sex with men (MSM), particularly racial/ethnic minority MSM, are disproportionately affected by HIV in the United States and Texas. Bareback sex or condomless anal intercourse (CAI) can be a high HIV risk behavior. Despite this, a majority of MSM continues to engage in barebacking. Research suggests racial/ethnic differences in barebacking exist; however, these conclusions remain unclear due to insufficient sample sizes to compare racial/ethnic groups. Our cross-sectional correlational design explores barebacking correlates (substance use during sex, safe sex fatigue, and optimistic HIV treatment beliefs) within and between racial/ethnic groups among 366 MSM. Regression models are significant for Latino and African-American MSM alone and for all MSM combined, though not significant for European-American and Other Race/Ethnicity MSM alone. Our findings suggest motivations and behaviors underlying barebacking among MSM vary by racial/ethnic membership with clinical implications for informing culturally sensitive HIV interventions and prevention programs for target racial/ethnic groups.

This analysis presents a method for estimating the population of men who have sex with men (MSM) at the county and metropolitan area level in Texas. Surveillance data consistently demonstrate that MSM experience a high burden of HIV and other sexually transmitted infections (STIs). Numerous studies have shown that MSM are also vulnerable to many other health concerns such as suicide, substance abuse, domestic violence and assault, homelessness, and mental illness. However, compilation of rates of HIV, STIs, and other health issues is dependent on estimation of population denominators. In the absence of systematic, consistent, and direct assessment of sexual orientation and gender identity in national surveys, it is difficult to estimate the size of at-risk populations. Previous estimates at the national and state level have been calculated using varied methodologies. However, to date, statewide estimates at the county level have only been produced for the state of Florida. County-level and metropolitan area estimates of MSM population were produced using three modified models developed by Lieb et al. These models used data on population and same-sex households from the US Census, along with estimates of sexual behavior from the National Survey on Family Growth. These models produce an estimate of 599,683 MSM in Texas (6.4 % of the adult male population). Metropolitan areas with the highest percentage of MSM population include Dallas and Austin (10.3 and 9.8 %, respectively). County-level estimates of MSM population range from 1.0 to 12.9 %. These local estimates are critical to targeting vulnerable populations and effective allocation of resources for prevention and treatment programs.

We investigated the extent of lymphocytic choriomeningitis virus (LCMV) infection in employees and rodents at 3 commercial breeding facilities. Of 97 employees tested, 31 (32%) had IgM and/or IgG to LCMV, and aseptic meningitis was diagnosed in 4 employees. Of 1,820 rodents tested in 1 facility, 382 (21%) mice (Mus musculus) had detectable IgG, and 13 (0.7%) were positive by reverse transcription PCR; LCMV was isolated from 8. Rats (Rattus norvegicus) were not found to be infected. S-segment RNA sequence was similar to strains previously isolated in North America. Contact by wild mice with colony mice was the likely source for LCMV, and shipments of infected mice among facilities spread the infection. The breeding colonies were depopulated to prevent further human infections. Future outbreaks can be prevented with monitoring and management, and employees should be made aware of LCMV risks and prevention.

Background: Female pattern hair loss affects many women; its pathogenetic basis has been held to be similar to men with common baldness., Objective: The objective of this study was to determine the role of immunity and inflammation in androgenetic alopecia in women and modulate therapy according to inflammatory and immunoreactant profiles., Materials and Methods: 52 women with androgenetic alopecia (AA) underwent scalp biopsies for routine light microscopic assessment and direct immunofluroescent studies. In 18 patients, serologic assessment for antibodies to androgen receptor, estrogen receptor and cytokeratin 15 was conducted., Results: A lymphocytic folliculitis targeting the bulge epithelium was observed in many cases. Thirty-three of 52 female patients had significant deposits of IgM within the epidermal basement membrane zone typically accompanied by components of complement activation. The severity of changes light microscopically were more apparent in the positive immunoreactant group. Biopsies from men with androgenetic alopecia showed a similar pattern of inflammation and immunoreactant deposition. Serologic assessment for antibodies to androgen receptor, estrogen receptor or cytokeratin 15 were negative. Combined modality therapy with minocycline and topical steroids along with red light produced consistent good results in the positive immunoreactant group compared to the negative immunoreactant group., Conclusion: A lymphocytic microfolliculitis targeting the bulge epithelium along with deposits of epithelial basement membrane zone immunoreactants are frequent findings in androgenetic alopecia and could point toward an immunologically driven trigger. Cases showing a positive immunoreactant profile respond well to combined modality therapy compared to those with a negative result.

The α(2)δ auxiliary subunits (α(2)δ-1 and α(2)δ-2) of voltage-sensitive calcium channels are thought to be the site of action of pregabalin (Lyrica), a drug that has been shown to be anxiolytic in clinical trials for generalized anxiety disorder. Pregabalin and the chemically related drug gabapentin have similar binding and pharmacology profiles, demonstrating high-affinity, in vitro binding to both α(2)δ-1 and α(2)δ-2 subunits. Two independent point mutant mouse strains were generated in which either the α(2)δ-1 subunit (arginine-to-alanine mutation at amino acid 217; R217A) or the α(2)δ-2 subunit (arginine-to-alanine mutation at amino acid 279; R279A) were rendered insensitive to gabapentin or pregabalin binding. These strains were used to characterize the activity of pregabalin in the Vogel conflict test, a measure of anxiolytic-like activity. Pregabalin showed robust anticonflict activity in wild-type littermates from each strain at a dose of 10 mg/kg but was inactive in the α(2)δ-1 (R217A) mutants up to a dose of 320 mg/kg. In contrast, pregabalin was active in the α(2)δ-2 (R279A) point mutants at 10 and 32 mg/kg. The positive control phenobarbital was active in mice carrying either mutation. These data suggest that the anxiolytic-like effects of pregabalin are mediated by binding of the drug to the α(2)δ-1 subunit.

Less than optimal outcomes for hypoplastic left heart syndrome may be related to unstable in-parallel circulation. Mechanical lung assist by (1) superior vena cava to right atrium pump with oxygenation (oxygenator assist), or (2) superior vena cava to pulmonary artery pump (pump assist) may permit successful neonatal in-series palliation. Nineteen 15-day-old piglets underwent single ventricle and bidirectional cavopulmonary shunt (BCPC) creation without mechanical lung assist (n = 8), with pump assist (n = 5), or with oxygenator assist (n = 6). Baseline hemodynamic measurements were not different between groups. Median survival for the cavopulmonary shunt alone, pump assist, and oxygenator assist groups was 28, 180, and 180 minutes, respectively (p = 0.0006). No differences in arterial oxygen concentration or bicarbonate levels were detected. Arterial carbon dioxide (p < or = 0.007) was higher in the cavopulmonary shunt alone versus lung assist groups. Cavopulmonary shunt alone animals had decreased mean arterial pressure (p < 0.02) and cerebral perfusion pressure (p = 0.029) and elevated left atrial pressure compared with lung assist groups (p < 0.05). This data demonstrates creation of a novel translational neonatal BCPC model in which mechanical lung assist augments survival. Early BCPC death was related to poor ventricular function and an inability to ventilate, issues that were improved with both types of lung assist.

Background: Chitosan glutamate and polyacrylic acid (e.g., carbomer 974P) are known to modulate the tight junctions in the intestinal wall and increase permeability and blood exposure of drugs absorbed orally by the paracellular route., Aim: To assess the impact of chitosan glutamate and carbomer 974P on the absorption of paracellularly absorbed model drug, acyclovir, in vitro and in rat in vivo., Methods: The influence of chitosan glutamate and carbomer 974P (alone and in combination with EDTA-Na2) on the in vitro Caco-2 permeability and oral pharmacokinetic profile in the rat of acyclovir was investigated., Results: In the presence of chitosan glutamate, the apparent permeability of acyclovir across Caco2 monolayer increased 4.1 times relative to control. This increase was accompanied by a significant ( approximately 60%) decrease in transepithelial electrical resistance values indicating opening of the tight junctions in the cell monolayer. In rat, chitosan glutamate doubled oral bioavailability of acyclovir and tripled the amount of acyclovir excreted unchanged into urine. In contrast, the effect of carbomer 974P was not statistically significant at 5% level., Conclusions: In conclusion, chitosan glutamate (1-3%) and chitosan glutamate (1%)/EDTA-Na2 (0.01%) are effective excipients to increase permeability of acyclovir across Caco-2 cell monolayers and the oral absorption in the rat in vivo.

Preliminary studies in our laboratory demonstrated low oral bioavailability of Drug X in male Sprague Dawley rats. However, the factors responsible for the observed poor bioavailability were not well understood. The objective of this study was to investigate the contribution of cytochrome P450(s) metabolism to the observed poor oral bioavailability of Drug X in male Sprague-Dawley rats in the presence of 1-aminobenzotriazole, a non-specific irreversible inhibitor of cytochrome P450s. Male Sprague-Dawley rats were pre-treated with or without oral 1-aminobenzotriazole (50 mg/kg) two hours prior to receiving a single intravenous or oral dose of Drug X (3 mg/kg). Blood samples were collected from animals at different time points over six hours following Drug X dosing. Plasma concentrations of Drug X were determined using LC/MS/MS. Pharmacokinetic data obtained from an intravenous dose study in rats suggested that Drug X exhibited a high clearance (55 mL/min/kg) and moderate volume of distribution (1.3 L/kg) with short half-life in rats (0.7 hr). Oral dosing of Drug X to rats resulted in low oral bioavailability (19%). 1-aminobenzotriazole pre-treatment of male Sprague Dawley rats followed by an intravenous dose of Drug X resulted in a decrease in plasma clearance by 71% and an increase in half-life by 100%, without affecting the volume of distribution. Furthermore, the oral bioavailability of Drug X increased markedly with 1-aminobenzotriazole pre-treatment. However, the fraction absorbed of Drug X did not significantly change with 1-aminobenzotriazole pre-treatment. The results of this study indicated that CYP-mediated metabolism played a major role in limiting the oral bioavailability of Drug X in rats. The data suggests that 1-aminobenzotriazole can be used as an effective tool in assessing the factors contributing to the poor oral bioavailability of drugs.

Primary creation of a bidirectional cavopulmonary shunt (BCPS), which provides a stable in-series circulation, might improve survival in patients with hypoplastic left heart syndrome. However, pulmonary vascular immaturity and high pulmonary vascular resistance must be overcome to achieve a successful primary neonatal BCPS. This study tested the hypothesis that a novel mechanical lung assist (MLA) system would permit a primary BCPS in juvenile pigs. A BCPS (1(1/2) ventricle repair physiology) was created in nine 4-5-week-old pigs (mean weight 17.9 kg), followed by MLA with a miniature centrifugal pump and a hand-made double-lumen cannula for 2 hours. Systemic and cerebral hemodynamic and metabolic data were evaluated. A mean BCPS flow of 331 +/- 56 ml/min was obtained with a mean pump speed of 3899 +/- 306 rpm at 30 minutes after MLA initiation. Adequate oxygenation, ventilation, and hemodynamics were maintained. Transcranial Doppler ultrasound demonstrated consistent pre- and postoperative peak systolic and diastolic velocities of the middle cerebral artery, indicating sustained cerebral perfusion pressure. This study demonstrated that the MLA is able to sustain a high-resistance BCPS and provides a strategy or rationale for developing a primary neonatal cavopulmonary connection in appropriately selected neonates with congenital heart disease.

The objective of the study was to establish primary cultured porcine brain microvessel endothelial cells (PBMECs) as an in vitro model to predict the blood-brain barrier (BBB) permeability in vivo. The intercellular tight junction formation of PBMECs was examined by electron microscopy and measured by transendothelial electrical resistance (TEER). The mRNA expression of several BBB transporters in PBMECs was determined by reverse transcriptionpolymerase chain reaction analysis. The in vitro permeability of 16 structurally diverse compounds, representing a range of passive diffusion and transporter-mediated mechanisms of brain penetration, was determined in PBMECs. Except for the perfusion flow rate marker diazepam, the BBB permeability of these compounds was determined either in our laboratory or as reported in literature using in situ brain perfusion technique in rats. Results in the present study showed that PBMECs had a high endothelium homogeneity, an mRNA expression of several BBB transporters, and high TEER values. Culturing with rat astrocyte-conditioned medium increased the TEER of PBMECs, but had no effect on the permeability of sucrose, a paracellular diffusion marker. The PBMEC permeability of lipophilic compounds measured under stirred conditions was greatly increased compared with that measured under unstirred conditions. The PBMEC permeability of the 15 test compounds, determined under the optimized study conditions, correlated with the in situ BBB permeability with an r2 of 0.60. Removal of the three system L substrates increased the r2 to 0.89. In conclusion, the present PBMEC model may be used to predict or rank the in vivo BBB permeability of new chemical entities in a drug discovery setting.

Hck is a member of the Src protein-tyrosine kinase family and is expressed strongly in macrophages, an important HIV target cell. Previous studies have shown that Nef, an HIV-1 accessory protein essential for AIDS progression, binds and activates Hck through its SH3 domain. Structural analysis suggests that Nef forms oligomers in vivo, which may bring multiple Hck molecules into close proximity and promote autophosphorylation. Using bimolecular GFP fluorescence complementation, we show for the first time that Nef oligomerizes in living cells and that the oligomers localize to the plasma membrane. To test the role of Nef oligomerization in Hck activation, we fused Nef to the hormone-binding domain of the estrogen receptor (Nef-ER), allowing us to control its dimerization with 4-hydroxytamoxifen (4-HT). In Rat-2 fibroblasts co-expressing Nef-ER and Hck, 4-HT treatment induced Nef-ER dimer and tetramer formation, leading to Hck kinase activation and cellular transformation. The number of transformed foci observed with Nef-ER plus Hck in the presence of 4-HT was markedly greater than that observed with wild-type Nef plus Hck, suggesting that enforced oligomerization enhances activation of Hck by Nef in vivo. Enhanced transformation correlated with increased Hck/Nef complex formation at the plasma membrane. In complementary experiments, we observed that a Nef mutant defective for Hck SH3 domain binding (Nef-PA) suppressed Hck kinase activation and transformation by the wild-type Hck/Nef complex. This effect correlated with the formation of a ternary complex between wild-type Nef, Nef-PA, and Hck, suggesting that Nef-PA suppresses Hck activation by blocking wild-type Nef oligomerization. Finally, Nef-ER induced Hck activation in a 4-HT-dependent manner in the macrophage precursor cell line TF-1, suggesting that oligomerization is essential for signaling through Hck in a cell background relevant to HIV infection. Together, these data demonstrate that Nef oligomerization is critical to the activation of Hck in vivo, and suggest that inhibitors of oligomerization may suppress Nef signaling through Hck in HIV-infected macrophages, slowing disease progression.

B lymphocytes are critically regulated by signals transduced through the CD19-CD21 cell surface receptor complex, where complement C3d binding to CD21 supplies an already characterized ligand. To determine the extent that CD19 function is controlled by complement activation, CD19-deficient mice (that are hyporesponsive to transmembrane signals) and mice overexpressing CD19 (that are hyperresponsive) were crossed with CD21- and C3-deficient mice. Cell surface CD19 and CD21 expression were significantly affected by the loss of CD21 and C3 expression, respectively. Mature B cells from CD21-deficient littermates had approximately 36% higher cell surface CD19 expression, whereas CD21/35 expression was increased by approximately 45% on B cells from C3-deficient mice. Negative regulation of CD19 and CD21 expression by CD21 and C3, respectively, may be functionally significant because small increases in cell surface CD19 overexpression can predispose to autoimmunity. Otherwise, B cell development and function in CD19-deficient and -overexpressing mice were not significantly affected by a simultaneous loss of CD21 expression. Although CD21-deficient mice were found to express a hypomorphic cell surface CD21 protein at low levels that associated with mouse CD19, C3 deficiency did not significantly affect B cell development and function in CD19-deficient or -overexpressing mice. These results, and the severe phenotype exhibited by CD19-deficient mice compared with CD21- or C3-deficient mice, collectively demonstrate that CD19 can regulate B cell signaling thresholds independent of CD21 engagement and complement activation.

CD19 and the Src family protein tyrosine kinases (PTKs) are important regulators of intrinsic signaling thresholds in B cells. Regulation is achieved by cross-talk between Src family PTKs and CD19; Lyn is essential for CD19 phosphorylation, while CD19 establishes an Src family PTK activation loop that amplifies kinase activity. However, CD19-deficient (CD19(-/-)) B cells are hyporesponsive to transmembrane signals, while Lyn-deficient (Lyn(-/-)) B cells exhibit a hyper-responsive phenotype resulting in autoimmunity. To identify the outcome of interactions between CD19 and Src family PTKs in vivo, B cell function was examined in mice deficient for CD19 and Lyn (CD19/Lyn(-/-)). Remarkably, CD19 deficiency suppressed the hyper-responsive phenotype of Lyn(-/-) B cells and autoimmunity characterized by serum autoantibodies and immune complex-mediated glomerulonephritis in Lyn(-/-) mice. Consistent with Lyn and CD19 each regulating conventional B cell development, B1 cell development was markedly reduced by Lyn deficiency, with further reductions in the absence of CD19 expression. Tyrosine phosphorylation of Fyn and other cellular proteins induced following B cell Ag receptor ligation was dramatically reduced in CD19/Lyn(-/-) B cells relative to Lyn(-/-) B cells, while Syk phosphorylation was normal. In addition, the enhanced intracellular Ca(2+) responses following B cell Ag receptor ligation that typify Lyn deficiency were delayed by the loss of CD19 expression. BCR-induced proliferation and humoral immune responses were also markedly inhibited by CD19/Lyn deficiency. These findings demonstrate that while the CD19/Lyn amplification loop is a major regulator of signal transduction thresholds in B lymphocytes, CD19 regulation of other Src family PTKs also influences B cell function and the development of autoimmunity.

B lymphocyte development and function depend upon the activity of intrinsic and B cell antigen receptor (BCR)-induced signals. These signals are interpreted, amplified, fine-tuned, or suppressed through the precise actions of specialized cell surface coreceptors, or "response regulators," that inform B cells of their extracellular environment. Important cell surface response regulators include the CD19/CD21 complex, CD22, and CD72. CD19 establishes a novel Src-family protein tyrosine kinase (PTK) amplification loop that regulates basal signaling thresholds and intensifies Src-family PTK activation following BCR ligation. In turn, CD22 limits the intensity of CD19-dependent, BCR-generated signals through the recruitment of potent phosphotyrosine and phosphoinositide phosphatases. Herein we discuss our current understanding of how CD19/CD21 and CD22 govern the emergence and intensity of BCR-mediated signals, and how alterations in these tightly controlled regulatory activities contribute to autoimmunity in mice and humans.

CD19 regulates constitutive and antigen receptor-induced signaling thresholds in B lymphocytes through its unique cytoplasmic domain. Herein, we demonstrate a novel molecular mechanism where interactions between CD19 and Lyn amplify basal and antigen receptor-induced Src family kinase activation. Lyn expression was required for CD19 tyrosine phosphorylation in primary B cells. Experiments with purified proteins demonstrated that CD19-Y513 was Lyn's initial phosphorylation and binding site. This led to processive phosphorylation of CD19-Y482, which recruited a second Lyn molecule, allowing for transphosphorylation and amplification of Lyn activation. In vivo, CD19 deficiency impaired, and CD19 overexpression enhanced, Lyn kinase activity. Thus, CD19 functions as a specialized adapter protein for the amplification of Src family kinases that is crucial for intrinsic and antigen receptor-induced signal transduction.