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OBJECTIVE: To determine the effects of varying O(2) on pH homeostasis, based on the hypothesis that the function of articular chondrocytes is best understood at realistic O(2) tensions. METHODS: Cartilage from equine metacarpophalangeal/tarsophalangeal joints was digested with collagenase to isolate chondrocytes, and then loaded with the pH-sensitive fluorophore 2',7'-bis-2-(carboxyethyl)-5(6)-carboxylfluorescein. The radioisotope(22)Na(+) was used to determine the kinetics of Na(+)/H(+) exchange (NHE) and the activity of the Na(+)/K(+) pump, and ATP levels were assessed with luciferin assays. Levels of reactive oxygen species (ROS) were determined using 2',7'-dichlorofluorescein diacetate. RESULTS: The pH homeostasis was unaffected when comparing tissue maintained at 20% O(2) (the level in water-saturated air at 37 degrees C) with that at 5% O(2) (which approximates the normal level in healthy cartilage); however, an O(2) tension of <5% caused a fall in intracellular pH (pH(i)) and slowed pH(i) recovery following acidification, an effect mediated via inhibition of NHE activity (likely through acid extrusion by NHE isoform 1). The Na(+)/K(+) pump activity and intracellular ATP concentration were unaffected by hypoxia, but the levels of ROS were reduced. Hypoxic inhibition of NHE activity and the reduction in ROS levels were reversed by treatment with H(2)O(2), Co(2+), or antimycin A. Treatment with calyculin A also prevented hypoxic inhibition of NHE activity. CONCLUSION: The ability of articular chondrocytes to carry out pH homeostasis is compromised when O(2) tensions fall below those normally experienced, via inhibition of NHE. The putative signal is a reduction in levels of ROS derived from mitochondria, acting via altered protein phosphorylation. This effect is relevant to both physiologic and pathologic states of lowered O(2), such as in chronic inflammation. [ABSTRACT FROM AUTHOR]

Background: Evidence is sparse on the effects of Medicare medication therapy management (MTM) on racial/ethnic disparities in medication adherence among patients with Alzheimer's disease and related dementias., Objectives: This study examined the Medicare MTM program's effects on racial/ethnic disparities in the adherence to antidementia medications among patients with Alzheimer's disease and related dementias., Methods: This is a retrospective analysis of 100% of 2010-2017 Medicare Parts A, B, and D data linked to Area Health Resources Files. The study outcome was nonadherence to antidementia medications, and intervention was defined as new MTM enrollment in 2017. Propensity score matching was conducted to create intervention and comparison groups with comparable characteristics. A difference-in-differences model was employed with logistic regression, including interaction terms of dummy variables for the intervention group and racial/ethnic minorities., Results: Unadjusted comparisons revealed that Black, Hispanic, and Asian/Pacific Islander patients were more likely to be nonadherent than non-Hispanic White (White) patients in 2016. Differences in odds of nonadherence between Black and White patients among the intervention group were lower in 2017 than in 2016 by 27% (odds ratios [OR]: 0.73, 95% confidence interval [CI]: 0.65-0.82). A similar lowering was seen between Hispanic and White patients by 26% (OR: 0.74, 95% CI: 0.63-0.87). MTM enrollment was associated with reduced disparities in nonadherence for Black-White patients of 33% (OR: 0.67, 95% CI: 0.57-0.78) and Hispanic-White patients of 19% (OR: 0.81, 95% CI: 0.67-0.99)., Discussion: The Medicare MTM program was associated with lower disparities in adherence to antidementia medications between Black and White patients, and between Hispanic and White patients in the population with Alzheimer's disease and related dementias., Conclusions: Expanding the MTM program may particularly benefit racial/ethnic minorities in Alzheimer's disease and related dementia care., Competing Interests: The authors list the following conflicts of interest: Xiaobei Dong, Chi Chun Steve Tsang, Jim Y. Wan, Christopher K. Finch, Jack W. Tsao, Jamie A. Browning, Joseph Garuccio, Rose Zeng: None; Marie A. Chisholm-Burns: Received funding from Carlos and Marguerite Mason Trust; Junling Wang: Received funding from AbbVie, Curo, Bristol Myers Squibb, Pfizer, and Pharmaceutical Research and Manufacturers of America (PhRMA), and serves on the Health Outcomes Research Advisor Committee of the PhRMA Foundation., (© 2024 The Authors. Published by Elsevier Inc.)

Objective: Medicare Part D Star Ratings are instrumental in shaping healthcare quality improvement efforts. However, the calculation metrics for medication performance measures for this program have been associated with racial/ethnic disparities. In this study, we aimed to explore whether an alternative program, named Star Plus by us that included all medication performance measures developed by Pharmacy Quality Alliance and applicable to our study population, would reduce such disparities among Medicare beneficiaries with diabetes, hypertension, and/or hyperlipidemia., Method: We conducted an analysis of a 10% random sample of Medicare A/B/D claims linked to the Area Health Resources File. Multivariate logistic regressions with minority dummy variables were used to examine racial/ethnic disparities in measure calculations of Star Ratings and Star Plus, respectively., Results: Adjusted results indicated that relative to non-Hispanic Whites (Whites), racial/ethnic minorities had significantly lower odds of being included in the Star Ratings measure calculations: the odds ratios (ORs) for Blacks, Hispanics, Asians, and Others were 0.68 (95% confidence interval [CI] = 0.66-0.71), 0.73 (CI = 0.69-0.78), 0.88 (CI = 0.82-0.93), and 0.92 (CI = 0.88-0.97), respectively. In contrast, every beneficiary in the sample was included in Star Plus. Further, racial/ethnic minorities had significantly higher increase in the odds of being included in measure calculation in Star Plus than Star Ratings. The ORs for Blacks, Hispanics, Asians, and Others were 1.47 (CI = 1.41-1.52), 1.37 (CI = 1.29-1.45), 1.14 (CI = 1.07-1.22), and 1.09 (CI = 1.03-1.14), respectively., Conclusions: Our study demonstrated that racial/ethnic disparities may be eliminated by including additional medication performance measures to Star Ratings.

Objectives: Equity and effectiveness of the medication therapy management (MTM) program in Medicare has been a policy focus since its inception. The objective of this study was to evaluate the cost-effectiveness of the Medicare MTM program in improving medication utilization quality across racial and ethnic groups., Methods: This study analyzed 2017 Medicare data linked to the Area Health Recourses File. A propensity score was used to match MTM enrollees and nonenrollees, and an incremental cost-effectiveness ratio between the 2 groups was calculated. Effectiveness was measured as the proportion of appropriate medication utilization based on medication utilization measures developed by Pharmacy Quality Alliance. Net monetary benefits were compared across racial and ethnic groups at various societal willingness-to-pay (WTP) thresholds. The 95% confidence intervals were obtained by nonparametric bootstrapping., Results: MTM dominated non-MTM among the total sample (N = 699 992), as MTM enrollees had lower healthcare costs ($31 135.89 vs $32 696.69) and higher proportions of appropriate medication utilization (87.47% vs 85.31%) than nonenrollees. MTM enrollees had both lower medication costs ($10 681.21 vs $11 003.08) and medical costs ($20 454.68 vs $21 693.61) compared with nonenrollees. The cost-effectiveness of MTM was higher among Black patients than White patients across the WTP thresholds. For instance, at a WTP of $3006 per percentage point increase in effectiveness, the net monetary benefit for Black patients was greater than White patients by $2334.57 (95% confidence interval $1606.53-$3028.85)., Conclusions: MTM is cost-effective in improving medication utilization quality among Medicare beneficiaries and can potentially reduce disparities between Black and White patients. Expansion of the current MTM program could maximize these benefits., (Copyright © 2023. Published by Elsevier Inc.)

Objectives: Racial/ethnic disparities have been found in prior literature examining enrolment in Medicare medication therapy management programs. However, those studies were based on various eligibility scenarios because enrolment data were unavailable. This study tested for potential disparities in enrolment using actual MTM enrolment data., Methods: Medicare Parts A&B claims, Medication Therapy Management Data Files, and the Area Health Resources File from 2013 to 2014 and 2016 to 2017 were analysed in this retrospective analysis. An adjusted logistic regression compared odds of enrolment between racial/ethnic minorities and non-Hispanic Whites (Whites) in the total sample and subpopulations with diabetes, hypertension, or hyperlipidaemia. Trends in disparities were analysed by including interaction terms in regressions between dummy variables for race/ethnic minority groups and period 2016-2017., Key Findings: Disparities in MTM enrolment were detected between Blacks and Whites with diabetes in 2013-2014 (Odds Ratio = 0.78, 95% Confidence Interval = 0.75-0.81). This disparity improved from 2013-2014 to 2016-2017 for Blacks (Odds Ratio=1.08, 95% Confidence Interval = 1.04-1.11) but persisted in 2016-2017 (Odds Ratio = 0.84, 95% Confidence Interval = 0.81-0.87). A disparity was identified between Blacks and Whites with hypertension in 2013-2014 (Odds Ratio = 0.92, 95% Confidence Interval = 0.89-0.95) but not in 2016-2017. Enrolment for all groups, however, declined between periods. For example, in the total sample, the odds of enrolment declined from 2013-2014 to 2016-2017 by 22% (Odds Ratio=0.78, 95% Confidence Interval=0.75-0.81)., Conclusions: Racial disparities in MTM enrolment were found between Blacks and Whites among Medicare beneficiaries with diabetes in both periods and among individuals with hypertension in 2013-2014. As overall enrolment fell between periods, concerns about program enrolment remain., Competing Interests: Joseph Garuccio: None. Chi Chun Steve Tsang: None. Jim Y. Wan: None. Ya Chen Tina Shih: Received consulting fees, travel, and accommodations for serving on a grants review panel for Pfizer Inc and an advisory board for AstraZeneca in 2019. Marie A. Chisholm-Burns: Received funding from Carlos and Marguerite Mason Trust. Samuel Dagogo-Jack: Led clinical trials for AstraZeneca, Boehringer Ingelheim, and Novo Nordisk, Inc., received consulting fees from AstraZeneca, Boehringer Ingelheim, Janssen, Merck & Co. Inc., and Sanofi, and has equity interests in Jana Care, Inc. and Aerami Therapeutics. William C. Cushman: Received grant funding from Eli Lilly. Xiaobei Dong: None. Jamie Browning: None. Rose Zeng: None. Junling Wang: Received funding from AbbVie, Curo, Bristol Myers Squibb, Pfizer, and Pharmaceutical Research and Manufacturers of America (PhRMA), and serves on the Heath Outcomes Research Advisor Committee of the PhRMA Foundation., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The Medicare Part D medication therapy management (MTM) program has positive effects on medication and health service utilization. However, little is known about its utilization, much less so about the use among racial and ethnic minorities., Objective: To examine MTM service utilization among older Medicare beneficiaries and to identify any racial and ethnic disparity patterns., Methods: A retrospective cross-sectional analysis of 2017 Medicare administrative data, linked to the Area Health Resources Files. Fourteen outcomes related to MTM service nature, initiation, quantity, and delivery were examined using logistic, negative binomial, and Cox proportional hazards regression models., Results: Racial and ethnic disparities were found with varying patterns across outcomes. For example, compared with White patients, the odds of opting out of MTM were 8% higher for Black patients (odds ratio [OR] = 1.08, 95% confidence interval [CI] = 1.03-1.14), 57% higher for Hispanic patients (OR = 1.57, 95% CI = 1.42-1.72), and 57% higher for Asian patients (OR = 1.57, 95% CI = 1.33-1.85). The odds of continuing MTM from the previous years were 12% lower for Black patients (OR = 0.88, 95% CI = 0.86-0.90) and 3% lower for other patients (OR = 0.97, 95% CI = 0.95-0.99). In addition, the probability of being offered a comprehensive medication review (CMR) after MTM enrollment was 9% lower for Hispanic patients (hazard ratio [HR] = 0.91, 95% CI = 0.85-0.97), 9% lower for Asian patients (HR = 0.91, 95% CI = 0.87-0.94), and 3% lower for other patients (HR = 0.97, 95% CI = 0.95-0.99). Hispanic and Asian patients were more likely to have someone other than themselves receive a CMR., Conclusions: Racial and ethnic disparities in MTM service utilization were identified. Although the disparities in specific utilization outcomes vary across racial/ethnic groups, it is evident that these disparities exist and may result in vulnerable communities not fully benefiting from the MTM services. Causes of the disparities should be explored to inform future reform of the Medicare Part D MTM program., Competing Interests: Xiaobei Dong: None. Chi Chun Steve Tsang: None. Jamie A. Browning: None. Joseph Garuccio: None. Jim Y. Wan: None. Ya Chen Tina Shih: None. Marie A. Chisholm-Burns: Received funding from Carlos and Marguerite Mason Trust. Samuel Dagogo-Jack: Led clinical trials for AstraZeneca, Boehringer Ingelheim, and Novo Nordisk, Inc., received consulting fees from AstraZeneca, Boehringer Ingelheim, Janssen, Merck & Co. Inc., and Sanofi, and has equity interests in Jana Care, Inc. and Aerami Therapeutics. William C. Cushman: Received grant funding from Eli Lilly. Junling Wang: Received funding from AbbVie, Curo, Bristol Myers Squibb, Pfizer, and Pharmaceutical Research and Manufacturers of America (PhRMA), and serves on Heath Outcomes Research Advisor Committee of the PhRMA Foundation., (© 2023 The Authors.)

Objective: Previous analysis of policy scenarios reported potential disparities in eligibility in the Medicare Medication Therapy Management (MTM) program. With recently released MTM data, this study aimed to determine if racial/ethnic disparities exist in MTM enrollment among Medicare beneficiaries with Alzheimer's disease and related dementias (ADRD)., Methods: Medicare claims/records (from 2013-2014 and 2016-2017) linked to the Area Health Resources File were examined. Included individuals were patients with ADRD and diabetes, hypertension or hyperlipidemia. The proportions of MTM enrollment were compared between non-Hispanic White (White) patients and racial/ethnic minority groups in descriptive analysis. Racial/ethnic disparities were then examined using a logistic regression adjusting for patient and community characteristics. Disparities across study periods were compared by estimating a logistic regression model with interaction terms between dummy variables for each racial/ethnic minority group and 2016-2017., Results: In unadjusted analyses, minorities had higher enrollment proportions than Whites. In 2016-2017, for example, enrollment percentages for Whites, Blacks, Hispanics, Asian/Pacific Islanders (Asians) and Others were respectively 14.44%, 16.71%, 19.83%, 16.66%, and 17.78%. In adjusted analyses, Blacks had lower enrollment odds than Whites within all cohorts. In the entire study sample in 2016-2017, for example, Blacks with ADRD had 9% lower odds of MTM enrollment (odds ratio 0.91, 95% confidence interval [CI] = 0.86-0.97) than Whites. These disparities decreased over time among the ADRD sample and all sub-groups. The interaction term between Blacks and 2016-2017, for instance, indicated that disparities were lowered by 11% (odds ratio 1.11, 95% CI = 1.05-1.16) across study periods among those with ADRD., Conclusions: Blacks with ADRD, and diabetes, hypertension or hyperlipidemia have lower likelihood of MTM enrollment than Whites. Racial disparities were reduced over time but not eliminated.

Background: Alzheimer's Disease (AD) is the mostcommon cause of dementia, a neurological disorder characterized by memory loss and judgment impairment. Hyperlipidemia, a commonly co-occurring condition, should be treated to prevent associated complications. Medication adherence may be difficult for individuals with AD due to the complexity of AD management. Comprehensive Medication Reviews (CMRs), a required component of Medicare Part D Medication Therapy Management (MTM), have been shown to improve medication adherence. However, many MTM programs do not target AD. Additionally, racial/ethnic disparities in MTM eligibility have been revealed. Thus, this study examined the effects of CMR receipt on reducing racial/ethnic disparities in the likelihood of nonadherence to hyperlipidemia medications (statins) among the AD population., Methods: This retrospective study used 2015-2017 Medicare data linked to the Area Health Resources Files. The likelihood of nonadherence to statin medications across racial/ethnic groups was compared between propensity-score-matched CMR recipients and non-recipients in a ratio of 1 to 3. A difference-in-differences method was utilized to determine racial/ethnic disparity patterns using a logistic regression by including interaction terms between dummy variables for CMR receipt and each racial/ethnic minority group (non-Hispanic Whites, or Whites, as reference)., Results: The study included 623,400 Medicare beneficiaries. Blacks and Hispanics had higher statin nonadherence than Whites: Compared to Whites, Blacks' nonadherence rate was 4.53% higher among CMR recipients and 7.35% higher among non-recipients; Hispanics' nonadherence rate was 2.69% higher among CMR recipients and 7.38% higher among non-recipients. Differences in racial/ethnic disparities between CMR recipients and non-recipients were significant for each minority group (p < 0.05) except Others. The difference between Whites and Hispanics in the odds of statin nonadherence was 11% lower among CMR recipients compared to non-recipients (OR = 0.89; 95% Confidence Interval = 0.85-0.94 for the interaction term between dummy variables for CMR and Hispanics). Interaction terms between dummy variables for CMR and other racial/ethnic minorities were not significant., Conclusions: Receiving a CMR was associated with a disparity reduction in nonadherence to statin medications between Hispanics and Whites among patients with AD. Strategies need to be explored to increase the number of MTM programs that target AD and promote CMR completion., (© 2022. The Author(s).)

Objective: Older patients with Alzheimer's disease (AD) are challenged with adhering to complex medication regimens. We examined effects of Comprehensive Medication Review (CMR), a required Medicare Part D Medication Therapy Management (MTM) program component, on medication adherence among AD patients., Methods: This retrospective study analyzed 100% of 2016-2017 Medicare claims covering the entire United States, linked to Area Health Resources Files. Medicare beneficiaries aged ≥65 years were included. Propensity score matching identified comparable intervention and comparison groups with the intervention defined as receiving a CMR in 2017. A difference-in-differences analysis included in multivariate logistic regressions an interaction term between CMR receipt and year 2017. The outcome measured was nonadherence to diabetes, hypertension and hyperlipidemia medications, with nonadherence defined as proportion of days covered <80% for study medications., Results: Unadjusted comparisons indicated the proportion of nonadherence for intervention group members decreased from 2016 to 2017 but increased for the comparison group. In adjusted analyses, reduction in medication nonadherence among the intervention group remained higher: odds ratios for the interaction term were 0.62 (95% confidence interval [CI] = 0.54-0.71), 0.54 (95% CI = 0.50-0.58) and 0.50 (95% CI = 0.47-0.53) respectively for diabetes, hypertension and hyperlipidemia medications. This suggests that the likelihood of nonadherence in the intervention group was respectively reduced by 38%, 46% and 50% more than the comparison group., Conclusions: CMR was found to reduce nonadherence to diabetes, hypertension and hyperlipidemia medications among older Medicare beneficiaries with AD. This provides evidence that the MTM program is effective for a population with unique medication compliance challenges.

Harmful algal bloom (HAB) toxins have severe negative impacts on marine mammals, particularly for Florida bottlenose dolphins (Tursiops truncatus) which frequently experience mass mortality events. Dolphins on the Florida Atlantic coast inhabit a region endemic to two HAB species, Karenia brevis and Pyrodinium bahamense, which produce the neurotoxins brevetoxin (PbTx) and saxitoxin (STX), respectively. Although toxic HABs and associated dolphin mortality events have been reported from this region, there is a lack of available data necessary for comparing toxin exposure levels between bloom ('exposed') conditions and non-bloom ('baseline') conditions. Here we present a 10-year dataset of PbTx and STX concentrations detected in dolphins stranding in this region, and compare the toxin loads from HAB-exposed dolphins to those detected in dolphins recovered in the absence of a HAB. We analyzed liver tissue samples from dead-stranded dolphins (n = 119) recovered and necropsied between 2002-2011, using an enzyme-linked immunosorbent assay (ELISA) modified for use with mammalian tissues. For dolphins recovered during baseline conditions, toxin-positive samples ranged in concentration from 0.27 to 1.2 ng/g for PbTx and from 0.41 to 1.9 ng/g for STX. For K. brevis-exposed dolphins, concentrations of up to 12.1 ng PbTx/g were detected, and for P. bahamense-exposed dolphins, concentrations of up to 9.9 ng STX/g were detected. Baseline PbTx values were similar to those reported in other regions where K. brevis blooms are more frequent and severe, but HAB-exposed PbTx values were considerably lower relative to these other regions. Since no baseline STX dolphin data exist for any region, our data serve as a first step towards establishing reference STX values for potential dolphin mortality events associated with STX-producing blooms in the future. This study demonstrates that although HABs in eastern Florida are only infrequently associated with dolphin mortalities, the presence of toxins in these animals may pose significant health risks in this region., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In hypertrophic cardiomyopathy (HC), atrial fibrillation (AF) is an important determinant of clinical deterioration due to heart failure or embolic stroke. This study characterizes left atrial (LA) structural and functional parameters to establish markers predictive of AF risk, using cardiovascular magnetic resonance (CMR) imaging. We studied 427 consecutive patients with HC in sinus rhythm with CMR (age 44±18 years), including 41 who developed clinically overt AF after study entry (2.6±2.1 years), 49 patients with AF before CMR, 337 patients with HC but without AF, and 244 normal controls. LA chamber was assessed for absolute and indexed end-diastolic volume (LAEDV), end-systolic volume, and percent ejection fraction (LAEF). In the 41 prospectively studied patients with HC who developed AF during follow-up, LAEDV was significantly greater than in patients without AF (146±48 vs 107±37 ml) or in normal controls (81±24 ml, p<0.001). Percent LAEF was lower in patients developing AF (36±10%) than without AF (46±12%) or controls (55±9%, p<0.001). Multivariate analysis identified LAEF (<38%), LAEDV (≥118 ml), and age (≥40 years) as independently associated with AF occurrence. In conclusion, CMR measures of LA remodeling and dysfunction reliably identified patients with HC at risk for future development of AF. Decrease in LAEF represents a strong novel marker of susceptibility to AF in this disease., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Abstract  Red blood cells (RBCs) from patients with sickle cell disease (SCD) lyse in deoxygenated isosmotic non-electrolyte solutions. Haemolysis has features which suggest that it is linked to activation of the pathway termed Psickle. This pathway is usually described as a non-specific cationic conductance activated by deoxygenation, HbS polymerisation and RBC sickling. The current work addresses the hypothesis that this haemolysis will provide a novel diagnostic and prognostic test for SCD, dependent on the altered properties of the RBC membrane resulting from HbS polymerisation. A simple test represented by this haemolysis assay would be useful especially in less affluent deprived areas of the world where SCD is most prevalent. RBCs from HbSS and most HbSC individuals showed progressive lysis in deoxygenated isosmotic sucrose solution at pH 7.4 to a level greater than that observed with RBCs from HbAS or HbAA individuals. Cytochalasin B prevented haemolysis. Haemolysis was temperature- and pH-dependent. It required near physiological temperatures to occur in deoxygenated sucrose solutions at pH 7.4. At pH 6, haemolysis occurred even in oxygenated samples. Haemolysis was reduced in patients on long-term (>5 months) hydroxyurea treatment. Several manoeuvres which stabilise soluble HbS (aromatic aldehydes o-vanillin or 5-hydroxymethyl, and urea) reduced haemolysis, an effect not due to increased oxygen affinity. Conditions designed to elicit HbS polymerisation in cells from sickle trait patients (deoxygenated hyperosmotic sucrose solutions at pH 6) supported their haemolysis. These findings are consistent with haemolysis requiring HbS polymerisation and support the hypothesis that this may be used as a test for SCD.

Objectives: We hypothesized that older patients in a regional ST-elevation myocardial infarction (STEMI) transfer program would attain comparable treatment to younger patients., Background: Older patients have been either excluded or underrepresented in STEMI clinical trials. Observational studies suggest that these patients are less likely to receive adjunctive pharmacologies and reperfusion therapy-thrombolysis or percutaneous coronary intervention (PCI)-and therapy is frequently delayed., Methods: We identified a consecutive series of 2,262 STEMI patients (March 2003-December 2008) who either presented or were transferred to Abbott Northwestern Hospital for PCI (<65 years [n = 1285], 65-74 years [n = 436], 75-84 years [n = 381], and ≥85 years [n = 160]). Main outcome measures included time-to-reperfusion therapy, adjunctive medications received, and all-cause mortality., Results: Overall time-to-reperfusion therapy was similar across age strata-94 minutes (<65 years), 101 minutes (65-74 years), 106 minutes (75-84 years), and 103 minutes (≥85 years). No difference in adjunctive antiplatelet or anticoagulant medications was seen at hospital admission, and only slight differences in standard post-myocardial infarction medication use were seen by age at hospital discharge. Age was an independent predictor of in-hospital and yearly mortality up to 5 years (1-year mortality 3.4% [<65 years], 9.2% [65-74 years], 15.2% [75-84 years], and 28.9% [≥85 years]; P < .0001)., Conclusions: Older patients receive similar care to younger patients when treated in a regional STEMI transfer program. Although all-cause mortality in the elderly is increased, the absolute rates are lower than previously established. Our data suggest primary PCI (including transfer) can be applied to all appropriate STEMI patients, regardless of age., (Copyright © 2011 Mosby, Inc. All rights reserved.)

Individuals heterozygous for HbS and HbC (HbSC) represent about 1/3(rd) of sickle cell disease (SCD) patients. Whilst HbSC disease is generally milder, there is considerable overlap in symptoms with HbSS disease. HbSC patients, as well as HbSS ones, present with the chronic anaemia and panoply of acute vaso-occlusive complications that characterize SCD. However, there are important clinical and haematological differences. Certain complications occur with greater frequency in HbSC patients (like proliferative retinopathy and osteonecrosis) whilst intravascular haemolysis is reduced. Patients with HbSC disease can be considered as a discrete subset of SCD cases. Although much work has been carried out on understanding the pathogenesis of SCD in HbSS homozygotes, including the contribution of altered red blood cell permeability, relatively little pertains directly to HbSC individuals. Results reported in the literature suggest that HbSC cells, and particularly certain subpopulations, present with similar permeability to HbSS cells but there are also important differences - these have not been well characterized. We hypothesise that their unique cell transport properties accounts for the different pattern of disease in HbSC patients and represents a potential chemotherapeutic target not shared in red blood cells from HbSS patients. The distinct pattern of clinical haematology in HbSC disease is emphasised here. We analyse some of the electrophysiological properties of single red blood cells from HbSC patients, comparing them with those from HbSS patients and normal HbAA individuals. We also use the isosmotic haemolysis technique to investigate the behaviour of total red blood cell populations. Whilst both HbSS and HbSC cells show increased monovalent and divalent (Ca(2+)) cation conductance further elevated upon deoxygenation, the distribution of current magnitudes differs, and outward rectification is greatest for HbSC cells. In addition, although Gd(3+) largely abolishes the cation conductance of both HbSS and HbSC cells, only in HbSS ones are currents inhibited by the aminoglycosides like streptomycin. This distinction is retained in isosmotic lysis experiments where both HbSS and HbSC cells undergo haemolysis in sucrose solutions but streptomycin significantly inhibits lysis only in HbSS cells. These findings emphasise similarities but also differences in the permeability properties of HbSS and HbSC cells, which may be important in pathogenesis., (Copyright 2010 Elsevier Inc. All rights reserved.)

A number of situations that result in abnormal permeability pathways in human red blood cells (RBCs) have been investigated. In sickle cell disease (SCD), RBCs contain HbS, rather than the normal HbA. When deoxygenated, an abnormal conductance pathway, termed P(sickle), is activated, which contributes to cell dehydration, largely through allowing Ca(2+) entry and subsequent activation of the Gardos channel. Whole-cell patch-clamp recordings from sickle RBCs show a deoxygenated-induced conductance, absent from normal RBCs, which shares some of the properties of P(sickle): equivalent Na(+) and K(+) permeability, significant Ca(2+) conductance, partial inhibition by DIDS and also Zn(2+). Gd(3+) markedly attenuates conductance in both normal and sickle RBCs. In addition, deoxygenated sickle cells, but not oxygenated ones or normal RBCs regardless of the oxygen tension, undergo haemolysis in isosmotic non-electrolyte solutions. Non-electrolyte entry was confirmed radioisotopically whilst haemolysis was inhibited by DIDS. These findings suggest that under certain circumstances P(sickle) may also be permeable to non-electrolytes. Finally, RBCs from certain patients with hereditary stomatocytosis have a mutated band 3, which appears able to act as a conductance pathway for univalent cations. These results extend our understanding of the abnormal permeability pathways of RBCs.

Aims: In mineralizing tissues such as growth plate cartilage extracellular organelles derived from the chondrocyte membrane are present. These matrix vesicles (MV), possess membrane transporters that accumulate Ca(2+) and inorganic phosphate (P(i)), and initiate the formation of hydroxyapatite crystals. MV are also present in articular cartilage, and hydroxyapatite crystals are believed to promote cartilage degradation in osteoarthritic joints. This study characterizes P(i) transport in MV derived from articular cartilage., Methods: Matrix vesicles were harvested from collagenase digests of bovine articular cartilage by serial centrifugation. P(i) uptake by MV was measured using radioactive phosphate ((33)[P]HPO(4)(2-)). The Na(+) dependence, pH sensitivity and effects of P(i) analogues that inhibit P(i) transport were determined., Results: P(i) uptake was temperature-sensitive and comprised Na(+)-dependent and Na(+)-independent components. The Na(+)-dependent component saturated at high extracellular P(i) concentrations, with a K(m) of 0.16 mM. In Na(+)-free solutions, uptake did not fully saturate implying that carrier-mediated uptake is supplemented by a diffusive pathway. Uptake was inhibited by phosphonoacetate and arsenate, although a fraction of Na(+)-independent P(i) uptake persisted. Total P(i) uptake was maximal at pH 6.5, and reduced at more acidic or alkaline values, representing inhibition of both components., Conclusion: These properties are highly similar to those of P(i) uptake by chondrocytes, suggesting that MV inherit P(i) transporters of the chondrocyte membrane from which they are derived. Na(+)-independent P(i) uptake has not previously been described in MV from growth plate cartilage and is relatively uncharacterized, but warrants further attention in articular cartilage, given its likely role in initiating inappropriate mineral formation.

Objective: Chondrocyte behavior is very sensitive to culture environment such as physical and biochemical conditions. As extracellular pH (pHo) and the existence of bicarbonate could affect the chondrocyte fate, hence, the purpose of this study is to investigate the buffer system effect on chondrocyte fate during relatively long-term culture., Methods: In order to examine whether effects seen were due to bicarbonate or to pHo, we had to devise a system which could differentiate between the two effects. Culture media buffered by N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (HEPES) only and the combination of HEPES and bicarbonate were used. Bovine articular chondrocytes were cultured in alginate beads for up to 12 days. pHo was kept constant by culture of 3 beads in 2 ml culture medium. Cell density, intracellular pH (pHi) and glycosaminoglycan (GAG) were measured at day 5 and day 12. Cell morphology, distribution and viability in alginate beads were monitored over 12 days of culture., Results: Compared to culture in the absence of bicarbonate, a higher proliferation rate of chondrocytes was observed in the presence of bicarbonate. pHi was more alkaline, about 0.2 pH unit, in the presence of bicarbonate than that in the absence of bicarbonate. About 50% more GAG was deposited in alginate beads when chondrocytes were cultured in the combination of HEPES and bicarbonate, compared to chondrocytes cultured in the absence of NaHCO3 at the end of 12 days of culture., Conclusion: The presence of bicarbonate results in more alkaline in the pHi of bovine chondrocytes after long-term culture. The combination of bicarbonate and HEPES in culture medium improves cell growth, matrix production in three-dimensional alginate beads.

Red blood cells from patients with sickle cell disease (SCD) exhibit increased electrogenic cation permeability, particularly following deoxygenation and hemoglobin (Hb) polymerisation. This cation permeability, termed P(sickle), contributes to cellular dehydration and sickling, and its inhibition remains a major goal for SCD treatment. Nevertheless, its characteristics remain poorly defined, its molecular identity is unknown, and effective inhibitors have not been established. Here, patch-clamp methodology was used to record whole-cell currents in single red blood cells from healthy individuals and patients with SCD. Oxygenated normal red blood cells had a low membrane conductance, unaffected by deoxygenation. Oxygenated HbS cells had significantly increased conductance and, on deoxygenation, showed a further rise in membrane conductance. The deoxygenation-induced pathway was variable in magnitude. It had equal permeability to Na(+) and K(+), but was less permeable to NMDG(+) and Cl(-). Conductance to Ca(2+) was also of a similar magnitude to that of monovalent cations. It was inhibited by DIDS (100 microM), Zn(2+) (100 microM), and by Gd(3+) (IC(50) of approximately 2 microM). It therefore shares some properties with P(sickle). These findings represent the first electrical recordings of single HbS cells and will facilitate progress in understanding altered red blood cell cation transport characteristics of SCD.

In mineralising tissues such as growth plate cartilage extracellular organelles derived from the chondrocyte membrane are present. These matrix vesicles (MV) possess membrane transporters that accumulate Ca(2+) and inorganic phosphate (P(i)), and initiate the formation of hydroxyapatite crystals. MV are also present in articular cartilage, and hydroxyapatite crystals are believed to promote cartilage degradation in osteoarthritic joints. In the present study, P(i) transport pathways in isolated bovine articular chondrocytes have been characterised. P(i) uptake was temperature-sensitive and could be resolved into Na(+)-dependent and Na(+)-independent components. The Na(+)-dependent component saturated at high concentrations of extracellular P(i), with a K(m) for P(i) of 0.17 mM. In solutions lacking Na(+), uptake did not fully saturate, implying that under these conditions carrier-mediated uptake is supplemented by a diffusive pathway. Both Na(+)-dependent and Na(+)-independent components were sensitive to the P(i) transport inhibitors phosphonoacetate and arsenate, although a fraction of Na(+)-independent P(i) uptake was resistant to these anions. Total P(i) uptake was optimal at pH 7.4, and reduced as pH was made more acidic or more alkaline, an effect that represented reduced Na(+)-dependent influx. RT-PCR analysis confirmed that two members of the NaPi III family, Pit-1 and Pit-2, are expressed, but that NaPi II transporters are not.

Red cells from patients with sickle cell disease contain HbS rather than the normal HbA (here termed HbS cells). On deoxygenation, HbS cells exhibit a distinctive solute permeability pathway, P(sickle), activated stochastically, and partially inhibited by DIDS and dipyridamole. It is often referred to as a cation channel although its permeability characteristics remain vague and its molecular identity is unknown. We show that, in contrast to normal red cells, a proportion of HbS cells underwent haemolysis when deoxygenated in isosmotic non-electrolyte solutions. Haemolysis was stochastic: cells unlysed after an initial deoxygenation pulse showed lysis when harvested, reoxygenated and subsequently exposed to a second period of deoxygenation. O(2) dependence of haemolysis was similar to that of P(sickle) activation. Haemolysis was accompanied by high rates of sucrose influx, and both haemolysis and sucrose influx were inhibited by DIDS and dipyridamole. Sucrose influx was only detected as ionic strength was reduced below 80 mM. These findings are consistent with the postulate that deoxygenation of HbS cells, under certain conditions, activates a novel non-electrolyte pathway. Their significance lies in understanding the nature of the deoxygenation-induced permeability in HbS cells, together with its relationship with novel pathways induced by a variety of manipulations in normal red cells., (Copyright 2007 S. Karger AG, Basel.)

In the current work, we review three situations where red cell volume changes are important. Red cell apoptosis (eryptosis) accounts for the removal of ageing and damaged erythrocytes from the circulation by macrophages. Amongst other cellular responses, eryptosis is associated with net cytosolic KCl loss and concomitant cell shrinkage. KCl efflux is mediated by activation of Ca(2+)-activated K(+) (Gardos) channels, permitting downhill movement of K(+) and electrically obliged Cl(-) through, as yet, incompletely described pathways. Red cells from patients suffering from sickle cell disease demonstrate progressive dehydration. Osmolyte loss is accounted for by the activation of two separate pathways. KCl cotransport, normally quiescent in red cells from HbA individuals, is activated under deoxygenated conditions and mediates net KCl efflux. Furthermore, intracellular Ca(2+) is elevated, probably as a result of Ca(2+) influx through a deoxygenation induced non-selective cation pathway termed P(sickle). This results in Gardos channel activation coupled indirectly with Cl(-) loss. Finally, a number of red cell stomatocytoses have been described where alterations to erythrocyte volume are the result of increased membrane cation permeability, in particular to Na(+) and K(+). The emerging significance of non-selective cation pathways is common to each of these conditions, and, although differences exist between their properties, particularly with regard to activation and ion selectivity, it is conceivable that they represent activation of closely related pathways. The recent finding that many hereditary stomatocytoses are caused by mutations to band 3 (AE-1) raises the possibility that modifications to this transporter could account for altered cation fluxes under different conditions.

The proinflammatory cytokine interleukin-1 (IL-1) promotes the degradation of articular cartilage by inhibiting matrix synthesis and stimulating degradative enzyme activity. Generation of nitric oxide (NO) in response to IL-1 is implicated in these actions. The catabolic actions of IL-1 can be inhibited by manoeuvres which are predicted to dissipate H+ gradients across the chondrocyte plasma membrane. In the present study, the effects of IL-1 on H+ extrusion from bovine articular chondrocytes were investigated. pH was measured using the H+-sensitive fluorescent dye BCECF. Cells were acidified by ammonium rebound and the contribution of the Na+-H+ exchanger (NHE) and of the vacuolar H+-ATPase to acid extrusion was characterised by ion substitution and inhibitor studies. Overnight (18 h) exposure to IL-1 stimulated acid extrusion in a dose-dependent fashion. This effect represented stimulation of both NHE and the ATPase. Characterisation of the timecourse of this response indicated that, while stimulation of acid extrusion was rapid, effects on the ATPase were only apparent after greater than 8h incubation with the cytokine. In keeping with this observation, the protein synthesis inhibitor cycloheximide abolished the stimulatory effect of IL-1 on ATPase-mediated extrusion. The upregulation of ATPase activity by IL-1 was inhibited by the NOS inhibitor L-NAME and by the NO scavenger PTIO. In cells which had not been exposed to IL-1, treatment with the NO donor SNAP also stimulated acid extrusion by the ATPase. In contrast, NHE activity was not altered by any of these compounds. Taken together, these results imply that IL-1 can stimulate acid extrusion in chondrocytes and that this reflects rapid upregulation of NHE with slower induction of H+-ATPase activity which requires elevated levels of NO. While ATPase induction involves protein synthesis, this process may not constitute synthesis of ATPase proteins per se, but rather of some associated regulatory process., (Copyright (c) 2005 S. Karger AG, Basel.)

We have studied the effects of anti-GLUT1 antibodies on the uptake of glucose into erythrocytes. Glucose transport into human erythrocyte ghosts was measured directly using 3H-2-deoxy-glucose, or indirectly by monitoring associated volume changes using light scattering. The uptake of glucose was significantly inhibited in ghosts resealed in solutions containing specific antibodies against GLUT1. Such an effect was not observed when an antibody against the oestrogen receptor, lacking specificity towards GLUT1, was employed instead. The antibodies were also without effect on the efflux of preloaded glucose from erythrocyte ghosts. The demonstration that anti-GLUT antibodies can inhibit glucose uptake is support for the hypothesis that they exaggerate the cytoplasmic barrier to glucose uptake created by endofacial segments of GLUT1.

The maintenance of chondrocyte pH is an important parameter controlling cartilage matrix turnover rates. Previous studies have shown that, to varying degrees, chondrocytes rely on Na(+)/H(+) exchange to regulate pH. HCO(3)(-)-dependent buffering and HCO(3)(-)-dependent acid-extrusion systems seem to play relatively minor roles. This situation may reflect minimal carbonic anhydrase activity in cartilage cells. In the present study, the pH regulation of the human chondrocyte cell line, C-20/A4 has been characterised. Intracellular pH (pH(i)) was measured using the H(+)-sensitive fluoroprobe BCECF. In solutions lacking HCO(3)(-)/CO(2), pH(i) was approximately 7.5, and the recovery from intracellular acidification was predominantly mediated by a Na(+)-dependent, amiloride- and HOE 694-sensitive process. A small additional component which was sensitive to chloro-7-nitrobenz-2-oxa-1,3-diazole, an inhibitor of the V-type H(+)-ATPase, was also apparent. In solutions containing HCO(3)(-)/CO(2), pH(i) was approximately 7.2. Comparison of buffering capacity in the two conditions showed that this variable was not significantly augmented in HCO(3)(-)/CO(2)-containing media. The recovery from intracellular acidification was more rapid in the presence of HCO(3)(-)/CO(2), although under these conditions it was again largely dependent on Na(+) ions and inhibited by amiloride and HOE 694. A small component was inhibited by SITS, although this effect did not reach the level of statistical significance. These findings indicate that HCO(3)(-)-dependent processes play only a minimal role in pH regulation in C-20/A4 chondrocytes. pH regulation instead relies heavily on the Na(+)/H(+) exchanger together with a H(+)-ATPase. The absence of extrinsic (HCO(3)(-)/CO(2)) buffering is likely to reflect the low levels of carbonic anhydrase in these cells. In addition to providing fundamental information about a widely-used cell line, these findings support the contention that the unusual nature of pH regulation in chondrocytes reflects the paucity of carbonic anhydrase activity in these cells.

The intracellular milieu of chondroctyes is regulated by an array of proteins in the cell membrane which operate as transport pathways, allowing ions and nutrients such as glucose and amino acids and metabolites such as lactate to cross the plasma membrane. Here we investigated the influence of hydrostatic pressure on intracellular calcium concentrations ([Ca(2+)](i)) in isolated bovine articular chondrocytes. We found that short applications of high hydrostatic pressures led to a significant increase in [Ca(2+)](i). The pressure-induced rise was abolished for long (240 sec) but not short (30 sec) pressure applications by removal of extracellular Ca(2+). The rise in pressure was also blocked by the inhibitors neomycin and thapsigargin confirming that pressure, by generating IP(3), led to an increase in [Ca(2+)](i) by mobilising the pool of Ca(2+) ions contained within intracellular stores. We also found that intracellular [Na(+)] was affected by a rise in osmotic pressure and further affected by application of hydrostatic pressure. The effect of hydrostatic pressure on sulphate incorporation depended strongly on extracellular osmolality. Since significant gradients in extracellular osmolality exist across intact cartilage, the results imply that responses of chondrocytes to the same pressure will vary depending on location in the joint. The results also indicate that hydrostatic pressures can affect several different transporter systems thus influencing the intracellular milieu and chondrocyte metabolism.

In this study, bovine articular and human chondrocytes from the C-20/A4 cell line were tested for the functional activity and molecular presence of the enzyme carbonic anhydrase. This enzyme is classically considered to be important in the maintenance of high cellular buffering capacity by catalysing the slow attainment of equilibrium between CO(2) and HCO(3)(-). The first functional assay measured the rate of pH equilibration after administration of a fixed dose of CO(2) solution to cell lysates. Compared to positive controls (human erythrocytes, murine M1 cells and purified carbonic anhydrase), chondrocyte lysates attained equilibrium at a significantly slower rate, similar to the rate obtained with a negative control (Xenopus oocytes). A second functional assay studied CO(2) hydration kinetics in intact C-20/A4 cells, using a pH-sensitive fluorescent dye, as the CO(2) content of the extracellular solution was changed. It was shown that C-20/A4 cells accelerate hydration only to a small degree. Hydration kinetics were reduced to the spontaneous rate in the presence of acetazolamide. Western immunoblotting with isoform-nonspecific antibodies to carbonic anhydrase demonstrated weak staining in both bovine and human chondrocytes., (Copyright 2002 Elsevier Science Inc.)

Intracellular pH (pH(i)) is a well-established determinant of cartilage matrix metabolism. Changes to chondrocyte pH(i), and therefore matrix turnover rates, arise following joint loading. It is not yet clear whether pH changes exert their effects on matrix metabolism directly, or by changing the concentration of another, as yet unidentified, intracellular factor. In this study the effect of intracellular alkalinisation on intracellular [Ca(2+)] has been examined using the human chondrocyte C-20/A4 cell line. pH(i) was manipulated by the addition of weak bases to suspensions of chondrocytes and fluorimetric techniques were employed to measure pH(i) and [Ca(2+)](i). The effect of pH(i) changes on intracellular inositol 1,4,5-trisphosphate (IP(3)) levels was also determined. The pH-sensitive properties of the Ca(2+)-sensitive fluoroprobe employed in this study, Fura-2, were investigated such that artefactual effects of pH changes upon the dye could be discounted. It was demonstrated that, for dye loaded into cells, alkalinisation resulted in a small increase in the affinity of the dye for Ca(2+) ions. Intracellular alkalinisation elicited by treatment with either of the weak bases trimethylamine or ammonium chloride initiated a rise in [Ca(2+)](i). This effect was too large to be explicable by the effects of pH changes on Fura-2 and was not dependent on the presence of extracellular Ca(2+) ions. Prior depletion of intracellular Ca(2+) stores by treatment with thapsigargin inhibited alkalinisation-induced increases in [Ca(2+)](i) and intracellular alkalinisation was also associated with increased levels of intracellular IP(3). These results confirm that alkaline pH(i) changes associated with dynamic loading of cartilage also result in knock-on alterations to [Ca(2+)](i). Given the sensitivity of cartilage matrix metabolism to [Ca(2+)](i) it is likely that this signalling cascade forms an important part of the mechanotransduction pathway that determines the response of chondrocytes to applied load.

The effects of raised hydraulic pressure on D-glucose exit from human red cells at 25 degrees C were determined using light scattering measurements in a sealed pressurized spectrofluorimeter cuvette. The reduction in the rates of glucose exit with raised pressure provides an index of the activation volume, deltaV++ (delta ln k/deltaP)(T) = -deltaV++/RT. Raised pressure decreased the rate constant of glucose exit from 0.077 +/- 0.003 s(-1) to 0.050 +/- 0.002 s(-1) (n = 5, P < 0.003). The Ki for glucose binding to the external site was 2.7 +/- 0.4 mm (0.1 MPa) and was reduced to 1.45 +/- 0.15 mm (40 MPa), (P < 0.01, Student's t test). Maltose had a biphasic effect on deltaV++. At [maltose] <250 microM, deltaV++ of glucose exit increased above that with [maltose = 0 mM], at >1 mm maltose, deltaV++ was reduced below that with [maltose = 0 mM]. Pentobarbital (2 mM) decreased the deltaV++ of net glucose exit into glucose-free solution from 30 +/- 5 ml mol(-1) (control) to 2 +/- 0.5 ml mol(-1) (P < 0.01). Raised pressure had a negligible effect on L-sorbose exit. These findings suggest that stable hydrated and liganded forms of GLUT with lower affinity towards glucose permit higher glucose mobilities across the transporter and are modelled equally well with one-alternating or a two-fixed-site kinetic models.

1. This study was aimed at identifying the signalling pathways involved in the activation of volume-regulatory mechanisms of human cervical cancer cells. 2. Osmotic swelling of human cervical cancer cells induced a substantial increase in intracellular Ca2+ ([Ca2+]i) by the activation of Ca2+ entry across the cell membrane, as well as Ca2+ release from intracellular stores. This Ca2+ signalling was critical for the normal regulatory volume decrease (RVD) response. 3. The activation of swelling-activated ion and taurine transport was significantly inhibited by tyrosine kinase inhibitors (genistein and tyrphostin AG 1478) and potentiated by the tyrosine phosphatase inhibitor Na3VO4. However, the Src family of tyrosine kinases was not involved in regulation of the swelling-activated Cl- channel. 4. Cell swelling triggered mitogen-activated protein (MAP) kinase cascades leading to the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2) and p38 kinase. The volume-responsive ERK1/ERK2 signalling pathway linked with the activation of K+ and Cl- channels, and taurine transport. However, the volume-regulatory mechanism was independent of the activation of p38 MAP kinase. 5. The phosphorylated ERK1/ERK2 expression following a hypotonic shock was up-regulated by protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and down-regulated by PKC inhibitor staurosporine. The response of ERK activation to hypotonicity also required Ca2+ entry and depended on tyrosine kinase and mitogen-activated/ERK-activating kinase (MEK) activity. 6. Considering the results overall, osmotic swelling promotes the activation of tyrosine kinase and ERK1/ERK2 and raises intracellular Ca2+, all of which play a crucial role in the volume-regulatory mechanism of human cervical cancer cells.

The change of intracellular pH of erythrocytes under different experimental conditions was investigated using the pH-sensitive fluorescent dye BCECF and correlated with (ouabain + bumetanide + EGTA)-insensitive K(+) efflux and Cl(-) loss. When human erythrocytes were suspended in a physiological NaCl solution (pH(o) = 7.4), the measured pH(i) was 7.19 + or - 0.04 and remained constant for 30 min. When erythrocytes were transferred into a low ionic strength (LIS) solution, an immediate alkalinization increased the pH(i) to 7.70 + or - 0.15, which was followed by a slower cell acidification. The alkalinization of cells in LIS media was ascribed to a band 3 mediated effect since a rapid loss of approximately 80% of intracellular Cl(-) content was observed, which was sensitive to known anion transport inhibitors. In the case of cellular acidification, a comparison of the calculated H(+) influx with the measured unidirectional K(+) efflux at different extracellular ionic strengths showed a correlation with a nearly 1:1 stoichiometry. Both fluxes were enhanced by decreasing the ionic strength of the solution resulting in a H(+) influx and a K(+) efflux in LIS solution of 108.2 + or - 20.4 mmol (l(cells) hr)(-1) and 98.7 + or - 19.3 mmol (l(cells) hr)(-1), respectively. For bovine and porcine erythrocytes, in LIS media, H(+) influx and K(+) efflux were of comparable magnitude, but only about 10% of the fluxes observed in human erythrocytes under LIS conditions. Quinacrine, a known inhibitor of the mitochondrial K(+)(Na(+))/H(+) exchanger, inhibited the K(+) efflux in LIS solution by about 80%. Our results provide evidence for the existence of a K(+)(Na(+))/H(+) exchanger in the human erythrocyte membrane.

The effects of hyperosmotic shock on intracellular pH (pHi) have been characterized in bovine articular chondrocytes. Osmotic shock is one of a variety of physicochemical stimuli experienced by chondrocytes upon cartilage loading. Cells were isolated from their extracellular matrix, and loaded with the pH-sensitive fluorophore 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Hyperosmotic shocks were imposed by addition of KCl or sucrose to the extracellular medium. For cells at steady-state pHi, resuspension in hyperosmotic solutions elicited an alkalinization, which was significantly inhibited by removal of extracellular Na+ ions, or treatment with amiloride (1 mM) or HOE-694 (10 microM), both inhibitors of Na+ x H+ exchange. For cells acidified by ammonium rebound, recovery of pHi towards resting levels was significantly stimulated by exposure to hyperosmotic solutions, and the effect was again attenuated by inhibition of Na+ x H+ exchange. Determination of the rate of acid extrusion at different levels of acidification indicated that the affinity of acid extrusion systems for H+ ions was increased by hypertonic shock. The response to hyperosmotic media could be abolished by treatment of chondrocytes with the non-specific kinase inhibitor staurosporine (10 nM), while the phosphatase inhibitor okadaic acid (1 mM) was able to augment recovery rates to values similar to those measured under hyperosmotic conditions. The osmotic sensitivity of recovery was unaffected by exposure to the protein kinase C inhibitor calphostin C, but was abolished in cells treated with ML-7, a specific inhibitor of myosin light chain kinase. These results confirm that - as for other components of mechanical load - increased osmolarity can modulate the activity of Na+ x H+ exchange, in this case by altered patterns of phosphorylation of transporter-associated myosin. The changes of pHi which will result dictate in part the rate of cartilage macromolecule synthesis.

Rationale: Investigators have postulated that neuroleptic medications may affect the motor system through the creation of free radicals. Also, structural brain changes related to oxidative damage may disrupt normal striatal function., Objective: The goals of this study were to examine whether an antioxidant diet reduced the abnormal movements caused by long-term neuroleptic exposure and to examine structural effects within specific striatal regions in rats., Methods: Rats were given a basal diet or a diet high in antioxidants for 4 months, and treated with 10 mg/kg fluphenazine decanoate or sesame seed oil IM every 2 weeks. At baseline and after treatment, head movements were quantified by accelerometry, and immunocytochemically stained cholinergic neurons in the ventrolateral, mediodorsal, and ventromedial regions of the striatum were quantified., Results: Rats treated with fluphenazine had significantly lower neuron densities than those that did not receive antioxidants. Rats exposed to a diet consisting of antioxidants had significantly higher neuron densities than those that did not receive antioxidants in each of the three regions tested. Rats treated with fluphenazine had a greater increase in the number of accelerometric peaks recorded per minute compared with untreated animals. The increase in the number of accelerometric peaks recorded per minute was lower for animals exposed to antioxidant diets compared with unexposed animals. Lastly, there was a significant correlation between the accelerometric peak change score and cholinergic neuron density in all three regions., Conclusions: Our results suggest that long-term neuroleptic treatment is associated with an increase in head movements and a reduction in ChAT-stained striatal cholinergic neurons and that these abnormalities are reduced by antioxidants.

The effects of load on articular cartilage are complex. Dynamic loading of cartilage is associated with slight cell and tissue deformation as well as cyclical fluctuations in the hydrostatic pressure of cartilage and in fluid movement. Static loading results in expression of fluid from the tissue, concentrating extracellular matrix macromolecules and consequently increasing the concentrations of cations, reducing extracellular pH and increasing extracellular osmolarity. Each of these alterations is implicated in regulating the synthetic response of chondrocytes to load. However, the mechanisms by which these changes affect matrix turnover are poorly understood. In this review we consider how load may affect chondrocyte behaviour through its influence on membrane transport processes and thus on the intracellular environment.

Background: Pulmonary embolism (PE) produces ventilation/perfusion mismatch that may be manifested in various variables of the volume-based capnogram (VBC). We hypothesized that a neural network (NN) system could detect changes in VBC variables that reflect the presence of a PE., Methods: A commercial VBC system was used to record multiple respiratory variables from consecutive expiratory breaths. Data from 12 subjects (n = 6 PE+ and n = 6 PE-) were used as input to a fully connected back-propagating NN for model development. The derived model was tested in a prospective, observational study at an urban teaching hospital. Volumetric capnograms were then collected on 53 test subjects: 30 subjects with PE confirmed by pulmonary angiography or diagnostic scintillation lung scan, and 23 subjects without PE based on pulmonary angiography. The derived NN model was applied to VBC data from the test population., Results: Seventeen VBC variables were used by the derived NN model to generate a numeric probability of PE. When the derived NN model was applied to VBC data from the 53 test subjects, PE was detected with a sensitivity of 100% (95% CI = 89% to 100%) and a specificity of 48% (95% CI = 27% to 69%). The likelihood ratio positive [LR(+)] for the VBC-NN test was 1.82 and the LR (-) was 0.1., Conclusion: This study demonstrates the feasibility of developing a rapid, noninvasive breath test for diagnosing PE using volumetric capnography and NN analysis.

The effects of increased hydrostatic pressure on Na+ x H+ exchange activity in bovine articular chondrocytes have been characterized. Chondrocytes were isolated from the cartilage matrix and the cells were loaded with the pH-sensitive fluorophore BCECF. Cells were acidified by ammonium rebound and the rate of recovery of pHi back to control levels was determined using cuvette fluorimetry. The application of hydrostatic pressure (1-300 atm) to cells within the fluorimeter was found to stimulate the rate of recovery from acidification, recorded as proton fluxes, in MOPS buffered media. This increase was dependent on the presence of extracellular Na+ ions and was inhibited by the Na+ x H+ exchange inhibitor EIPA. The pressure-stimulated increase in H+ flux is therefore mediated completely by Na+ x H+ exchange. In addition, the stimulation could be abolished by the kinase inhibitor staurosporine, was not additive with the stimulation of Na+ x H+ exchange elicited by the addition of serum and was unaffected by low concentrations of the myosin light chain kinase inhibitor ML-7. We therefore conclude that hydrostatic pressure activates Na+ x H+ exchange in this cell type by a pathway which involves direct phosphorylation of the transporter protein itself. This is the first demonstration of the activation of Na+ x H+ exchange by hydrostatic pressure and the relevance of this finding to the biology of cartilage tissue is discussed.

Free radicals are highly reactive chemical species with an unpaired electron, and their formation is catalyzed by transition metals like iron, copper, and manganese. There have been numerous studies linking free radical damage with neuropsychiatric illnesses, including several psychiatric and motor disorders, raising the possibility that antioxidant strategies might serve a neuroprotective role for some conditions. The illnesses studied include tardive dyskinesia, schizophrenia, Parkinson's disease, and Alzheimer's disease. Although oxidative mechanisms may play a role in these conditions, further studies are necessary to define their involvement, and to determine the extent to which antioxidants may partially alleviate or prevent some of these conditions.

An outbreak of measles occurred in Tucson, AZ, in 1985; 112 of the 225 cases were among students at two large high schools. A review of the immunization records of all students at both schools was undertaken in order to assess the risk of a person contracting measles in relation to that person's immunization status. Two factors, the lack of an immunization record and immunization prior to 12 months of age, showed a positive association with contracting measles. The association was statistically significant at one high school but not the other. At the first high school, students who were immunized at 12 to 14 months of age had a greater risk of infection than those immunized at 15 months or older. However, age at immunization of 12 to 14 months was not associated with a significantly higher risk when persons with multiple doses of vaccine were excluded from the analysis. Students of both schools showed a lower attack rate for those who had received multiple doses of vaccine, but the difference was not statistically significant.

Investigation of 189 cases of shigellosis reported to the Pima County, Arizona Health Department in 1986 revealed that 23 per cent of cases could be attributed to travel to Mexico, and 10 per cent to day care attendees and their household contacts. No source of infection or high-risk activity could be demonstrated for 43 per cent of the cases. Households in which S. flexneri occurred were more likely to be characterized by crowded living situations and to have no known source of infection.