The human non-gastric H,K-ATPase has a different cation specificity than the rat enzyme

The primary sequence of non-gastric H,K-ATPase differs much more between species than that of Na,K-ATPase or gastric H,K-ATPase. To investigate whether this causes species-dependent differences in enzymatic properties, we co-expressed the catalytic subunit of human non-gastric H,K-ATPase in Sf9 cells with the beta(1) subunit of rat Na,K-ATPase and compared its properties with those of the rat enzyme (Swarts et al., J. Biol. Chem. 280, 33115-33122, 2005). Maximal ATPase activity was obtained with NH(4)(+) as activating cation. The enzyme was also stimulated by Na(+), but in contrast to the rat enzyme, hardly by K(+). SCH 28080 inhibited the NH(4)(+)-stimulated activity of the human enzyme much more potently than that of the rat enzyme. The steady-state phosphorylation level of the human enzyme decreased with increasing pH, [K(+)], and [Na(+)] and nearly doubled in the presence of oligomycin. Oligomycin increased the sensitivity of the phosphorylated intermediate to ADP, demonstrating that it inhibited the conversion of E(1)P to E(2)P. All three cations stimulated the dephosphorylation rate dose-dependently. Our studies support a role of the human enzyme in H(+)/Na(+) and/or H(+)/NH(4)(+) transport but not in Na(+)/K(+) transport.     

Determinants and Consequences of Failure of Linkage to Antiretroviral Therapy at Primary Care Level in Blantyre, Malawi: A Prospective Cohort Study

Background

Poor rates of linkage from HIV diagnosis to ART initiation are a major barrier to universal coverage of ART in sub-Saharan Africa, with reasons for failure poorly understood. In the first study of this kind at primary care level, we investigated the pathway to care in the Malawian National Programme, one of the strongest in Africa.

Methods and Findings

A prospective cohort study was undertaken at two primary care clinics in Blantyre, Malawi. Newly diagnosed HIV-positive adults (>15 years) were followed for 6-months to assess completion of eligibility assessments, initiation of ART and death. Two hundred and eighty participants were followed for 82.6 patient-years. ART eligibility assessments were problematic: only 134 (47.9%) received same day WHO staging and 121 (53.2%) completed assessments by 6-months. Completion of CD4 measurement (stage 1/2 only) was 81/153 (52.9%). By 6-months, 87/280 (31.1%) had initiated ART with higher uptake in participants who were ART eligible (68/91, 74.7%), and among participants who received same-day staging (52/134 [38.8%] vs. 35/146 [24.0%] p = 0.007). Non-completion of ART eligibility assessments (adjusted hazard ratio: 0.11, 95% CI: 0.06–0.21) was associated with failure to initiate ART. Retention in pre-ART care for non-ART initiators was low (55/193 [28.5%]). Of the 15 (5.4%) deaths, 11 (73.3%) occurred after ART initiation.

Conclusions

Although uptake of ART was high and prompt for patients with known eligibility, there was frequent failure to complete eligibility assessment and poor retention in pre-ART care. HIV care programmes should urgently evaluate the way patients are linked to ART. In particular, there is a critical need for simplified, same-day ART eligibility assessments, reduced requirements for hospital visits, and active defaulter follow-up.     

Prediction of muscle energy states at low metabolic rates requires feedback control of mitochondrial respiratory chain activity by inorganic phosphate

The regulation of the 100-fold dynamic range of mitochondrial ATP synthesis flux in skeletal muscle was investigated. Hypotheses of key control mechanisms were included in a biophysical model of oxidative phosphorylation and tested against metabolite dynamics recorded by ³¹P nuclear magnetic resonance spectroscopy (³¹P MRS). Simulations of the initial model featuring only ADP and Pi feedback control of flux failed in reproducing the experimentally sampled relation between myoplasmic free energy of ATP hydrolysis (ΔG_p = ΔG_p ^o′+RT ln ([ADP][Pi]/[ATP]) and the rate of mitochondrial ATP synthesis at low fluxes (<0.2 mM/s). Model analyses including Monte Carlo simulation approaches and metabolic control analysis (MCA) showed that this problem could not be amended by model re-parameterization, but instead required reformulation of ADP and Pi feedback control or introduction of additional control mechanisms (feed forward activation), specifically at respiratory Complex III. Both hypotheses were implemented and tested against time course data of phosphocreatine (PCr), Pi and ATP dynamics during post-exercise recovery and validation data obtained by ³¹P MRS of sedentary subjects and track athletes. The results rejected the hypothesis of regulation by feed forward activation. Instead, it was concluded that feedback control of respiratory chain complexes by inorganic phosphate is essential to explain the regulation of mitochondrial ATP synthesis flux in skeletal muscle throughout its full dynamic range.     

Stavudine Toxicity in Adult Longer-Term ART Patients in Blantyre, Malawi

Background

Stavudine is an effective and inexpensive antiretroviral drug, but no longer recommended by WHO for first-line antiretroviral regimens in resource-limited settings due to toxicity concerns. Because of the high cost of alternative drugs, it has not been feasible to replace stavudine in most adults in the Malawi ART programme. We aimed to provide policy makers with a detailed picture of stavudine toxicities in Malawians on longer-term ART, in order to facilitate prioritization of stavudine replacement among other measures to improve the quality of ART programmes.

Methods

Prospective cohort of Malawian adults who had just completed one year of stavudine containing ART in an urban clinic, studying peripheral neuropathy, lipodystrophy, diabetes mellitus, high lactate syndromes, pancreatitis and dyslipidemia during 12 months follow up. Stavudine dosage was 30 mg irrespective of weight. Cox regression was used to determine associations with incident toxicities.

Results

253 patients were enrolled, median age 36 years, 62.5% females. Prevalence rates (95%-confidence interval) of toxicities after one year on stavudine were: peripheral neuropathy 21.3% (16.5–26.9), lipodystrophy 14.7% (2.4–8.1), high lactate syndromes 0.0% (0–1.4), diabetes mellitus 0.8% (0–2.8), pancreatitis 0.0% (0–1.5). Incidence rates per 100 person-years (95%-confidence interval) during the second year on stavudine were: peripheral neuropathy 19.8 (14.3–26.6), lipodystrophy 11.4 (7.5–16.3), high lactate syndromes 2.1 (0.7–4.9), diabetes mellitus 0.4 (0.0–1.4), pancreatitis 0.0 (0.0–0.2). Prevalence of hypercholesterolemia and hypertriglyceridemia increased from 12.1% to 21.1% and from 29.5% to 37.6% respectively between 12 and 24 months. 5.5% stopped stavudine, 1.3% died and 4.0% defaulted during follow up. Higher age was an independent risk factor for incident peripheral neuropathy and lipodystrophy.

Conclusion

Stavudine associated toxicities continued to accumulate during the second year of ART, especially peripheral neuropathy and lipodystrophy and more so at increasing age. Our findings support investments for replacing stavudine in first-line regimens in sub-Saharan Africa.     

Hypertension in sub-Saharan Africa: cross-sectional surveys in four rural and urban communities

Background

Cardiovascular disease (CVD) is the leading cause of adult mortality in low-income countries but data on the prevalence of cardiovascular risk factors such as hypertension are scarce, especially in sub-Saharan Africa (SSA). This study aims to assess the prevalence of hypertension and determinants of blood pressure in four SSA populations in rural Nigeria and Kenya, and urban Namibia and Tanzania.

Methods and Findings

We performed four cross-sectional household surveys in Kwara State, Nigeria; Nandi district, Kenya; Dar es Salaam, Tanzania and Greater Windhoek, Namibia, between 2009–2011. Representative population-based samples were drawn in Nigeria and Namibia. The Kenya and Tanzania study populations consisted of specific target groups. Within a final sample size of 5,500 households, 9,857 non-pregnant adults were eligible for analysis on hypertension. Of those, 7,568 respondents ≥18 years were included. The primary outcome measure was the prevalence of hypertension in each of the populations under study.The age-standardized prevalence of hypertension was 19.3% (95%CI:17.3–21.3) in rural Nigeria, 21.4% (19.8–23.0) in rural Kenya, 23.7% (21.3–26.2) in urban Tanzania, and 38.0% (35.9–40.1) in urban Namibia. In individuals with hypertension, the proportion of grade 2 (≥160/100 mmHg) or grade 3 hypertension (≥180/110 mmHg) ranged from 29.2% (Namibia) to 43.3% (Nigeria). Control of hypertension ranged from 2.6% in Kenya to 17.8% in Namibia. Obesity prevalence (BMI ≥30) ranged from 6.1% (Nigeria) to 17.4% (Tanzania) and together with age and gender, BMI independently predicted blood pressure level in all study populations. Diabetes prevalence ranged from 2.1% (Namibia) to 3.7% (Tanzania).

Conclusion

Hypertension was the most frequently observed risk factor for CVD in both urban and rural communities in SSA and will contribute to the growing burden of CVD in SSA. Low levels of control of hypertension are alarming. Strengthening of health care systems in SSA to contain the emerging epidemic of CVD is urgently needed.     

Defining adult asthma endotypes by clinical features and patterns of volatile organic compounds in exhaled air

Background

Several classifications of adult asthma patients using cluster analyses based on clinical and demographic information has resulted in clinical phenotypic clusters that do not address molecular mechanisms. Volatile organic compounds (VOC) in exhaled air are released during inflammation in response to oxidative stress as a result of activated leukocytes. VOC profiles in exhaled air could distinguish between asthma patients and healthy subjects. In this study, we aimed to classify new asthma endotypes by combining inflammatory mechanisms investigated by VOC profiles in exhaled air and clinical information of asthma patients.

Methods

Breath samples were analyzed for VOC profiles by gas chromatography–mass spectrometry from asthma patients (n = 195) and healthy controls (n = 40). A total of 945 determined compounds were subjected to discriminant analysis to find those that could discriminate healthy from asthmatic subjects. 2-step cluster analysis based on clinical information and VOCs in exhaled air were used to form asthma endotypes.

Results

We identified 16 VOCs, which could distinguish between healthy and asthma subjects with a sensitivity of 100% and a specificity of 91.1%. Cluster analysis based on VOCs in exhaled air and the clinical parameters FEV1, FEV1 change after 3 weeks of hospitalization, allergic sensitization, Junipers symptoms score and asthma medications resulted in the formation of 7 different asthma endotype clusters. We identified asthma clusters with different VOC profiles but similar clinical characteristics and endotypes with similar VOC profiles, but distinct clinical characteristics.

Conclusion

This study demonstrates that both, clinical presentation of asthma and inflammatory mechanisms in the airways should be considered for classification of asthma subtypes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0136-8) contains supplementary material, which is available to authorized users.     

Measurement of serum levels of natalizumab, an immunoglobulin G4 therapeutic monoclonal antibody

Human immunoglobulin G4 (IgG4) is a poor trigger of effector functions and, therefore, is the preferred subclass for therapeutic monoclonal antibodies that merely aim to block their in vivo targets. An example is natalizumab, a recombinant IgG4 antibody directed against α4-integrin and used for treatment of multiple sclerosis. Efficient treatment requires that the pharmacokinetics of therapeutic monoclonal antibodies can be accurately monitored. For natalizumab, this requires special precautions due to recently reported structural peculiarities of human IgG4. Here we describe the development of an assay to determine serum levels of natalizumab. Compared with other IgG subclasses, human IgG4 possesses unique structural properties that influence its interactions in both in vivo and in vitro settings. Thus, IgG4 undergoes Fab arm exchange in vivo, resulting in effectively monovalent antibodies. Furthermore, IgG4 is able to bind to other human and nonhuman IgG via Fc interactions. We demonstrate how these features can interfere with measurement of specific IgG4 and describe how we addressed these issues, resulting in an assay that is not sensitive to Fab arm exchange by natalizumab or to IgG4 Fc interactions.     

Temporary Treatment during Primary HIV Infection Does Not Affect Virologic Response to Subsequent Long-Term Treatment

Temporary cART during primary HIV-infection (PHI) did not select for drug resistance mutations after treatment interruption and did not affect the subsequent virological response to long-term cART. Our data demonstrate that fear of drug resistance development is not a valid argument to refrain from temporary early treatment during PHI.