Elevated serum receptor activator of NFκB ligand (RANKL), osteoprotegerin (OPG), matrix metalloproteinase (MMP)3, and ProMMP1 in patients with juvenile idiopathic arthritis

We studied the serum levels of receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), pro-matrix metalloproteinase (MMP) 1, MMP3, and tissue inhibitor of metalloproteinase (TIMP) 1 in patients with juvenile idiopathic arthritis (JIA) and correlated these with different disease variables. Sera of 70 patients with JIA (ILAR 2001 criteria) and 33 age- and sex-matched controls were assayed by enzyme-linked immunosorbent assay. Nonparametric tests were used for analysis of data. The subtype distribution of the JIA patients was: enthesitis-related arthritis (ERA) 24, polyarticular 22, systemic onset 13, oligoarticular 8, and others 3. The median level of RANKL, OPG, pro-MMP1, MMP3, and TIMP-1 were elevated in JIA patients as compared to controls (p < 0.001). There was no difference in levels among different types of JIA. RANKL/OPG ratio was elevated in all subtypes of JIA. MMP3/TIMP-1 ratio correlated with measures of disease activity including swollen and tender joint count, erythrocyte sedimentation rate, and disease activity score (rS 0.28, p < 0.05). Ours is the first study to show elevated RANKL in serum of patients with JIA. Further, our data suggest that patients with ERA have similar levels to other forms of JIA. Association of the MMP3/TIMP-1 ratio with disease activity suggests that it may be a useful biomarker for follow-up.     

Prevalence-correlates-awareness-treatment and control of hypertension in kumarakom, kerala: baseline results of a community-based intervention program

BACKGROUND, Hypertension is one of the major causes of cardiovascular morbidity and mortality. However, awareness, treatment, and control of hypertension remain major challenges worldwide. In this article, we present the baseline prevalence of hypertension from an ongoing intervention program for its control in a community-based sample in Kerala, Southern India. METHODS, We measured blood pressure, body weight, and height of 4955 individuals above the age of 30 yers (men;2159:mean-age: 50 years) and collected information on alcohol use, tobacco use, and other demographic variables using a pre-tested structured questionnaire. RESULTS, The overall prevalence of hypertension (JNC-VII) was 36.7% ( 95% CI:35.5-38.0; men: 36.0% and women 37.2% ) in multipile logistic regression analysis, a body mass index of >/=25 kg/m(2) was associated with a 1.65-fold (95% CI:1.37-1.98) prevalence of hypertension compared to a body mass index <25kg/m(2). Individuals with diabetes mellitus had 2.10 higher odds of hypertension prevalence (95% CI: 1.62-2.73) compared to people wihtout diabetes mellitus. Participants with increased waist circumference (90 cm in men, 85 cm in women) were 1.84 times more likely to be hypertensive compared to those with normal waist circumference (95% CI: 1.55-2.19). Among hypertensives, 24% were aware of the condition, 20% were on treatment, and 6.4% achieved effective blood pressure control. CONCLUSION, A higher body mass index, increased waist circumference, and self-reported diabetes mellitus were the important correlates of hypertension in our community-based sample. Our data emphasize the importance of educational interventions and appropriate lifestyle modifications that target increased body mass index and waist circumference to reduce the community burden of hypertension.     

Role of polymorphic N-acetyl transferase2 and cytochrome P4502E1 gene in antituberculosis treatment-induced hepatitis

Background and Aim: Antituberculosis drugs, isoniazid and rifampicin, in combination, are known to develop drug‐induced hepatotoxicity (DIH). A higher risk of DIH during antituberculosis treatment (ATT) has been reported in the Indian subcontinent compared to its Western counterparts. The role of genetic factors in a higher incidence of ATT hepatotoxicity in the Indian population is still unclear. The present study was aimed at investigating the role of the N‐acetyltransferase2 (NAT2) and cytochrome P4502E1 (CYP2E1) gene polymorphisms in ATT hepatotoxicity. Methods: The study population included 218 pulmonary tuberculosis patients who were started on ATT and followed up for the occurrence of ATT‐induced hepatitis. The genetic polymorphisms of the NAT2 and CYP2E1 genes were studied by polymerase chain reaction–restriction fragment length polymorphism. Results: The occurrence of DIH was 18.8% (41/218). There was a higher prevalence of NAT2 slow‐acetylator genotypes in DIH (70.73%) compared to non‐DIH (44.63%; P < 0.05). The frequency of the NAT2*5/*7 and NAT2*6/*7 genotypes was significantly higher in DIH than non‐DIH (19.51% vs 6.78%, and 19.51% vs 5.08%). No association of the CYP2E1 RsaI polymorphism could be demonstrated with DIH. However, the DraI C/D genotype of the CYP2E1 gene was mostly prevalent in DIH (85.37%), compared to non‐DIH (64.41%) (P < 0.05). Slow‐acetylator status and the CYP2E1 C/D or C/C genotype together showed a higher frequency in DIH (65.85%) compared to non‐DIH (28.81%) (P < 0.0001). Conclusion: The study demonstrates for the first time a possible association between the DraI polymorphism of the CYP2E1 gene and the risk of ATT hepatotoxicity. The genotyping of the NAT2 and CYP2E1 genes could possibly identify the groups at highest risk of developing ATT‐induced hepatitis prior to medication.     

Influenza hemagglutinin stem-fragment immunogen elicits broadly neutralizing antibodies and confers heterologous protection

Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vaccination. Current influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies (bnAbs), thereby limiting their efficacy. Although several bnAbs bind to the conserved stem domain of HA, focusing the immune response to this conserved stem in the presence of the immunodominant, variable head domain of HA is challenging. We report the design of a thermotolerant, disulfide-free, and trimeric HA stem-fragment immunogen which mimics the native, prefusion conformation of HA and binds conformation specific bnAbs with high affinity. The immunogen elicited bnAbs that neutralized highly divergent group 1 (H1 and H5 subtypes) and 2 (H3 subtype) influenza virus strains in vitro. Stem immunogens designed from unmatched, highly drifted influenza strains conferred robust protection against a lethal heterologous A/Puerto Rico/8/34 virus challenge in vivo. Soluble, bacterial expression of such designed immunogens allows for rapid scale-up during pandemic outbreaks.Seasonal influenza outbreaks across the globe cause an estimated 250,000–500,000 deaths annually (1). Current influenza vaccines need to be updated every few years because of antigenic drift (2). Despite intensive monitoring, strain mismatch between vaccine formulation and influenza viruses circulating within the population has occurred in the past (2). Public health is further compromised when an unpredictable mixing event among influenza virus genomes leads to antigenic shift facilitating a potential pandemic outbreak. These concerns have expedited efforts toward developing a universal influenza vaccine.Neutralizing antibodies (nAbs) against hemagglutinin (HA) are the primary correlate for protection in humans and hence HA is an attractive target for vaccine development (3). The precursor polypeptide, HA0, is assembled into a trimer along the secretory pathway and transported to the cell surface. Cleavage of HA0 generates the disulfide-linked HA1 and HA2 subunits. Mature HA has a globular head domain which mediates receptor binding and is primarily composed of the HA1 subunit, whereas the stem domain predominantly comprises the HA2 subunit. The HA stem is trapped in a metastable state and undergoes an extensive low-pH-induced conformational rearrangement in the host-cell endosomes to adopt the virus–host membrane fusion-competent state (4, 5).The antigenic sites on the globular head of HA are subjected to heightened immune pressure resulting in escape variants, thereby limiting the breadth of head-directed nAbs (6). However, extensive efforts have resulted in the isolation of monoclonal antibodies (mAbs) that bind within the globular head and inhibit receptor attachment, which neutralize drifted variants of an HA subtype or heterosubtypic HA (7–16). The HA stem is targeted by several broadly neutralizing antibodies (bnAbs) with neutralizing activity against diverse influenza A virus subtypes (17). The epitopes of these bnAbs in the HA stem are more conserved across different influenza HA subtypes compared with the antigenic sites in the HA globular head (18).During a primary infection, the immunodominant globular head domain suppresses the response toward the conserved stem. Several efforts have been made to circumvent this problem. Repeated immunizations with full-length, chimeric HAs (cHAs) in a protracted vaccination regimen have been shown to boost stem-directed responses in mice (19). Alternatively, full-length HA presented on nanoparticles (np) has been shown to elicit stem-directed nAbs (20). Attempts have also been made to steer the immune response toward the conserved HA stem by hyperglycosylating the head domain (21). Although the aforementioned strategies need to be further evaluated and provide novel alternatives, detrimental interference from the highly variable immunodominant head domain in eliciting a broad functional response cannot be completely evaded. A “headless” stem domain immunogen offers an attractive solution. However, early attempts at expressing the HA2 subunit independently in a native, prefusion conformation were unsuccessful. In the absence of the head domain, the HA2 subunit expressed in Escherichia coli spontaneously adopted the low-pH conformation (22) in which the functional epitopes of stem-directed bnAbs are disrupted. More recently, the entire HA stem region has been expressed in a prefusion, native-like conformation in both prokaryotic and eukaryotic systems adopting multiple strategies (23–26).Design of independently folding HA stem fragments which adopt the prefusion HA conformation presents another approach to elicit bnAbs against influenza (27, 28). The A helix of the HA2 subunit contributes substantial contact surface to the epitope of stem-directed bnAbs such as CR6261, F10, and others. Although multivalent display of A helix on the flock house virus as a virus-like particle platform elicited cross-reactive antibodies, it conferred only minimal protection (20%) against virus challenge in mice (29).We report the design and characterization of engineered headless HA stem immunogens based on the influenza A/Puerto Rico/8/34 (H1N1) subtype. H1HA10-Foldon, a trimeric derivative of our parent construct (H1HA10), bound conformation-sensitive, stem-directed bnAbs such as CR6261 (30), F10 (31), and FI6v3 (32) with a high-affinity [equilibrium dissociation constant (K_D) of 10–50 nM]. The designed immunogens elicited broadly cross-reactive antiviral antibodies which neutralized highly drifted influenza virus strains belonging to both group 1 (H1 and H5 subtypes) and 2 (H3 subtype) in vitro. Significantly, stem immunogens designed from unmatched, highly drifted influenza strains conferred protection against a lethal (2LD₉₀) heterologous A/Puerto Rico/8/34 virus challenge in mice. Our immunogens confer robust subtype-specific and modest heterosubtypic protection in vivo. In contrast to previous stem domain immunogens (23–25), the designed immunogens were purified from the soluble fraction in E. coli. The HA stem-fragment immunogens do not aggregate even at high concentrations and are cysteine-free, which eliminates the complications arising from incorrect disulfide-linked, misfolded conformations. The aforementioned properties of the HA stem-fragment immunogens make it amenable for scalability at short notice which is vital during pandemic outbreaks.     

Salivary levels of isoniazid and rifampicin in tuberculous patients

Concentrations of isoniazid and rifampicin were determined in time-matched samples of saliva and serum from 30 tuberculous patients (18 with pulmonary tuberculosis and 12 with intestinal tuberculosis), comprising 18 slow and 12 rapid acetylators of isoniazid, following administration of isoniazid 300 mg and rifampicin 12 mg/kg. The diffusion of isoniazid into saliva was quite rapid and the salivary concentrations were similar to those in serum, suggesting that saliva could be used in place of serum for all pharmacokinetic studies with isoniazid. The salivary concentrations of rifampicin were much lower than those in serum, the mean peak concentrations being 0.9 and 8.5 microgram/ml, respectively. Further, there was evidence of a significant delay in the diffusion of rifampicin from serum to saliva.     

Inhalation of a dry powder tobramycin PulmoSphere formulation in healthy volunteers

STUDY OBJECTIVES: To evaluate the efficiency and reproducibility of pulmonary delivery of an investigational tobramycin PulmoSphere formulation (PStob) [Inhale Therapeutic Systems; San Carlos, CA] by a passive dry powder inhaler, and to compare serum concentrations and whole-lung deposition with a commercial nebulized tobramycin product (TOBI; Chiron Corporation; Seattle, WA). DESIGN: A five-period, open-label, nonrandomized crossover study. PARTICIPANTS: Fourteen healthy volunteers were studied, and 12 completed the study. INTERVENTIONS: PStob powder was manufactured using lipid-based PulmoSphere technology, producing highly dispersible porous particles. PStob was radiolabeled with (99m)Tc, and in vitro experiments confirmed it as a valid drug marker. To identify whole-lung distribution via scintigraphy, subjects inhaled contents of a single capsule (72 L/min) containing 25 mg of (99m)Tc-labeled PStob (13.5 mg of tobramycin free base) in periods 1 to 3. In period 4, subjects received (99m)Tc nebulized tobramycin, approximately 2.5 mL of 300 mg/5 mL. Deposition and blood samples were obtained. In period 5, six 25-mg doses of unlabeled PStob (81 mg of tobramycin base) were inhaled and blood samples were collected. Measurements and results: Mean whole-lung deposition of PStob was 34 +/- 6% and nebulized tobramycin was 5 +/- 2%. Peak tobramycin concentration in serum (Cmax) values were 0.6 microg/mL with PStob and 0.28 microg/mL after nebulized tobramycin. Serum area under the curve was 4.4 microg x h/mL vs 2.1 micro g x h/mL for nebulized tobramycin. Median time to Cmax for PStob was comparable to nebulized tobramycin. CONCLUSIONS: The aerosol doses of PStob (25 mg and 150 mg) were well dispersed and tolerated. Serum drug concentrations matched scintigraphy data and were roughly twice that of the comparator. Intrasubject dose variability for three equivalent periods did not exceed 18% relative SD. PStob Cmax (0.6 microg/mL) was well below the toxic threshold (2 micro g/mL).     

Fetal programming: excess prenatal testosterone reduces postnatal luteinizing hormone, but not follicle-stimulating hormone responsiveness, to estradiol negative feedback in the female

Exposure of female sheep fetuses to excess testosterone (T) during early to midgestation produces postnatal hypergonadotropism manifest as a selective increase in LH. This hypergonadotropism may result from reduced sensitivity to estradiol (E2) negative feedback and/or increased pituitary sensitivity to GnRH. We tested the hypothesis that excess T before birth reduces responsiveness of LH and FSH to E2 negative feedback after birth. Pregnant ewes were treated with T propionate (100 mg/kg in cotton seed oil) or vehicle twice weekly from d 30-90 gestation. Responsiveness to E2 negative feedback was assessed at 12 and 24 wk of age in the ovary-intact female offspring. Our experimental strategy was first to arrest follicular growth and reduce endogenous E2 by administering the GnRH antagonist (GnRH-A), Nal-Glu (50 microg/kg sc every 12 h for 72 h), and then provide a fixed amount of exogenous E2 via an implant. Blood samples were obtained every 20 min at 12 wk and every 10 min at 24 wk before treatment, during and after GnRH-A treatment both before and after E2 implant. GnRH-A ablated LH pulsatility, reduced FSH by approximately 25%, and E2 production diminished to near detection limit of assay at both ages in both groups. Prenatal T treatment produced a precocious and selective reduction in responsiveness of LH but not FSH to E2 negative feedback, which was manifest mainly at the level of LH/GnRH pulse frequency. Collectively, these findings support the hypothesis that prenatal exposure to excess T decreases postnatal responsiveness to E2 inhibitory feedback of LH/GnRH secretion to contribute to the development of hypergonadotropism.     

Aspirin Use in Women: Current Perspectives and Future Directions

Purpose of Review

This review examines the recent literature on the use of low-dose aspirin (LDA) for primary and secondary prevention of cardiovascular disease in women, use of LDA for pre-eclampsia prevention in pregnancy, and the underutilization of aspirin therapy in women as compared to men.

Recent Findings

While men and women should not differ with respect to aspirin use for secondary prevention, its role in primary prevention remains unclear for both sexes, with particular uncertainty in women. Reflective of this are conflicting recommendations in current guidelines for primary prevention and thus investigations of primary prevention aspirin use are ongoing and will play an important role in elucidating its efficacy. While there is significant heterogeneity in studies to date of LDA for pre-eclampsia prevention, based on recent meta-analyses suggesting promising results, guidelines now recommend initiation in high risk women after the 12th week of gestation. Finally, studies consistently reveal that aspirin therapy is underutilized in women as compared to men, suggesting a need to better educate physicians and the general public about its use in women.

Summary

Further research is needed to better elucidate the role of aspirin in women for primary prevention of cardiovascular disease and for pre-eclampsia in high risk pregnant women. In addition, further investigation into the factors that lead to the current underutilization of aspirin in women are required in order to ensure that patients of both sexes are optimally treated, with the goal of improving cardiovascular outcomes in all patients.