P glycoprotein and multidrug resistance]

Drug resistance has been shown to be associated with the expression of P-glycoprotein (P-gp), the product of mdr-1 gene. One of the suggested mechanisms for the phenomenon called Multidrug Resistance (MDR) is related to the overexpression and amplification of the mdr-1 gene. The product of this gene is called P-gp and it has been considered as a potential marker for drug resistance. Transfection of the mdr-1 gene into drug-sensitive cells confers the role of mdr-1 gene in developing MDR phenotype. Structural analysis of homologous cDNA, responsible for production of transport proteins, from: MDR cell lines, bacteria and yeast, revealed high similarity. Molecular structure analysis indicate that P-gp has nucleotide binding sites. It has been established that P-gh has an internal ATP-ase activity, thus it can act as energy dependent transport protein. The expression of P-gp has been found in neoplastic and normal tissues (as adrenal glands, kidney, liver, pancreas, jejunum and large intestine), as well as in several cell lines which have been induced to become resistant to cytostatics. The aim of present study was to review the role of P-gp expression in laboratory and clinic.     

Regional distribution of antibodies to herpes simplex virus type 1 (HSV-1) and HSV-2 in men and women in Ontario,Canada

This study estimated the regional and age- and gender-specific seroprevalences of herpes simplex virus type 1 (HSV-1) and HSV-2 in Ontario, Canada. Stored serum specimens from subjects aged 15 to 44 years, including men (n = 979), women not under prenatal care (n = 638), and women under prenatal care (n = 701) submitted for routine viral serology were randomly selected according to regional population size from public health laboratories. HSV-1 and HSV-2 testing was done with the MRL enzyme immunoassay (EIA) (Focus Technologies), and HSV-2 was also tested by the Gull/Meridian EIA. Specimens discordant for HSV-2 antibodies between the two EIAs were resolved by a recombinant immunoblot assay (Focus Technologies). The overall age- and gender-standardized seroprevalences of HSV-1 and HSV-2 were 51.1% (95% confidence interval [CI], 50.1 to 52.1) and 9.1% (95% CI, 8.6 to 9.7), respectively. The seroprevalence of HSV-1 antibodies increased from 26.9 to 54.7% in men between 15 to 16 and 40 to 44 years of age, from 32.0 to 88.7% in women not under prenatal care, and from 55.2 to 69.2% in women under prenatal care. The seroprevalence of HSV-2 increased from 3.8 to 21.3% in men between 15 to 16 and 40 to 44 years of age, from 0 to 18.9% in women not under prenatal care, and from 3.4 to 23.1% in women under prenatal care. HSV-2 results were discordant for 3.3% (76 of 2,318) of specimens. Both types of HSV antibodies appeared to be acquired earlier among women under prenatal care than among men and women not under prenatal care. Antibodies were more prevalent among people in northern Ontario (72.9% of subjects [range, 68.4 to 77.4%] for HSV-1 and 13.7% of subjects [95% CI, 10.2 to 17.2%] for HSV-2) than elsewhere.     

Does spaced education improve clinical knowledge among Family Medicine residents? A cluster randomized controlled trial

Advances in Health Sciences Education - Spaced education is a learning strategy to improve knowledge acquisition and retention. To date, no robust evidence exists to support the utility of spaced...     

The conventional versus automated measurement of blood pressure in the office (CAMBO) trial: masked hypertension sub-study

OBJECTIVES:: To determine the prevalence of masked hypertension [normal office blood pressure (BP) with high awake ambulatory BP] using automated office BP (AOBP) or conventional manual office BP (MOBP) measurement in routine clinical practice. METHODS:: The prevalence of masked hypertension was evaluated on three consecutive visits during a median 6 months period in patients with systolic hypertension randomized to management with AOBP (n?=?140) or continued conventional MOBP (n?=?112) in routine primary care practice. AOBP was recorded using the automated BpTRU device with the patient resting alone in a quiet examining room. All patients underwent 24-h ambulatory BP monitoring. RESULTS:: The prevalence of masked hypertension on any one of three visits calculated using only SBP varied between 12 and 17% in the AOBP group compared with 19 and 22 % in the MOBP group. Masked hypertension was present on both of the first two visits in 7 and 12% and on all three visits in 6 and 7% of AOBP and MOBP patients, respectively. The prevalence for masked hypertension based upon both SBP and DBP was similar being 11-15% for AOBP and 19-20% for MOBP patients on single visits, but decreased to 6 and 10% when readings from the first two visits were used and to 4 and 6% when all three visits were used for the AOBP and MOBP groups, respectively. CONCLUSION:: The prevalence of masked hypertension is lower with AOBP compared with MOBP. The number of patients with masked hypertension decreases if the criteria for having this condition need to be met on multiple visits.     

A framework for discussion on how to improve prevention, management, and control of hypertension in Canada

Increased blood pressure is a leading risk for premature death and disability. The causes of increased blood pressure are intuitive and well known. However, the fundamental basis and means for improving blood pressure control are highly integrated into our complex societal structure both inside and outside our health system and hence require a comprehensive discussion of the pathway forward. A group of Canadian experts was appointed by Hypertension Canada with funding from Public Health Agency of Canada and the Heart and Stroke Foundation of Canada, Canadian Institute for Health Research (HSFC-CIHR) Chair in Hypertension Prevention and Control to draft a discussion Framework for prevention and control of hypertension. The report includes an environmental scan of past and current activities, proposals for key indicators, and targets to be achieved by 2020, and what changes are likely to be required in Canada to achieve the proposed targets. The key targets are to reduce the prevalence of hypertension to 13% of adults and improve control to 78% of those with hypertension. Broad changes in government policy, research, and health services delivery are required for these changes to occur. The Hypertension Framework process is designed to have 3 phases. The first includes the experts' report which is summarized in this report. The second phase is to gather input and priorities for action from individuals and organizations for revision of the Framework. It is hoped the Framework will stimulate discussion and input for its full intended lifespan 2011-2020. The third phase is to work with individuals and organizations on the priorities set in phase 2.     

Design of immunogens to elicit broadly neutralizing antibodies against HIV targeting the CD4 binding site

A vaccine which is effective against the HIV virus is considered to be the best solution to the ongoing global HIV/AIDS epidemic. In the past thirty years, numerous attempts to develop an effective vaccine have been made with little or no success, due, in large part, to the high mutability of the virus. More recent studies showed that a vaccine able to elicit broadly neutralizing antibodies (bnAbs), that is, antibodies that can neutralize a high fraction of global virus variants, has promise to protect against HIV. Such a vaccine has been proposed to involve at least three separate stages: First, activate the appropriate precursor B cells; second, shepherd affinity maturation along pathways toward bnAbs; and, third, polish the Ab response to bind with high affinity to diverse HIV envelopes (Env). This final stage may require immunization with a mixture of Envs. In this paper, we set up a framework based on theory and modeling to design optimal panels of antigens to use in such a mixture. The designed antigens are characterized experimentally and are shown to be stable and to be recognized by known HIV antibodies.

Vaccines are the most important medical countermeasure for protecting entire populations against viruses, of which smallpox and measles vaccines are successful examples. In fact, a safe and effective HIV vaccine is considered to be the best way to end the global AIDS epidemic (1). However, how to produce a universal vaccine for highly antigenically variable viruses like HIV is a daunting and yet unsolved problem. The high variability of this virus allows it to elude the immune system, making the produced antibodies ineffective; that is, they are generally specific for a given strain of the virus but not for other strains resulting from mutation. In some cases, HIV-infected patients can elicit antibodies that can recognize and neutralize a broad range of different viral strains (2, 3). These broadly neutralizing antibodies (bnAbs) usually take a long time to appear naturally in infected patients and then only in a subset of such individuals.The reason that bnAbs can arise is that even highly variable pathogens have regions with a well-defined, relatively conserved structure, which is required for their function. In HIV, entry depends on the trimeric spike exposed on the external lipid membrane of the virion, a heterotrimer formed by the gp120 and gp41 glycoproteins produced by posttranslational cleavage of a gp160 precursor. This protein binds to the CD4 coreceptor on CD4 T lymphocytes during HIV infection, and it has some relatively conserved regions that can be used as a target for bnAbs. Indeed, many bnAbs target the CD4 binding site (CD4bs) (4–8). If naive B cells that can bind to one of these relatively conserved regions can be expanded upon exposure to different variants of the virus, antibodies could evolve to better recognize the conserved portions, while avoiding the variable ones. The resulting antibodies can acquire breadth in this way, thereby becoming bnAbs. A successful vaccine would contain immunogens that can guide the immune system to produce bnAbs, rather than strain-specific antibodies.In the past, numerous approaches for the development of an effective HIV vaccine have been tried. They include the use of cleverly chosen natural HIV proteins, the design of a consensus (9) or “center-of-tree” (10) antigens, and the creation of a mosaic protein from different HIV strains (11). All these methods used a single optimized antigen in the vaccine and were shown to be ineffective at eliciting bnAbs (12, 13). One possible reason for this is that, when exposed to a single antigen, the immune system will produce antibodies specific for that particular antigen, and neutralization escape variants can easily develop. A possible solution is to use more than one antigen in a vaccination protocol. This raises a number of questions: How many antigens are necessary? How different should they be from each other? And in what temporal order should they be administered? Answering such questions is far from trivial, in particular due to the limited mechanistic understanding of affinity maturation (AM) in vivo. Another problem is that bnAbs have an unusually high number of somatic mutations, not only in the complementarity-determining regions (CDRs) but also in the immunoglobulin framework regions (7, 14). Recent computational data on the flexibility of the antibody and the need for framework mutations in the simulated AM showed how important it is for a vaccination protocol to have a specific antigen that can prime a good antibody precursor B cell receptor (BCR) (15). Moreover, it has been shown that putative precursors of known classes of bnAbs are generally not able to neutralize HIV or recognize envelope (Env), often due to clashes of the antibody with the glycosylation shield that protects the HIV Env protein (8, 16–18). For example, VRC01-class bnAbs are known to introduce a deletion or a mutation to a flexible glycine in the CDRL1 loop to avoid the glycan at N276 (19, 20).The above discussion led to the proposal of a vaccination strategy consisting of three steps. First, a special purpose antigen is used to activate the correct naïve or precursor B cell (17, 21). Since this precursor will generally not bind to native HIV, as a second step, one or more antigens are used as intermediates to induce somatic mutations and to allow recognition of the native virus. In the third step, one or more antigens are used in a mixture or in sequence to increase the breadth of the antibody population (19, 22, 23). Implementations of the first and second steps have already been shown to be promising in experiments (16, 21, 24–27). However, much less is known about the third step. Some insights into this question can be obtained by in silico simulations of AM. Using coarse-grained models, it has been shown that, while administering a single mixture containing multiple antigens may induce too much frustration to lead to bnAbs formation, a sequential approach, in which antigens are administered one after another, seems to be more effective (23). It was also observed that the number of antigens required in a mixture is correlated with their sequence dissimilarity, and optimal breadth is obtained at an optimal number of antigens and dissimilarity (28). Given the coarse-grained nature of these studies, the actual antigen sequences to use in experiments cannot be obtained from them.In this work, we focus on the third step of the proposed vaccination protocol. In particular, we derive a set of empirical rules and protocols to select an optimal panel of antigens to maximize the breadth of the produced antibodies upon AM. To be able to do so, it is essential to understand, at an atomistic level of detail, the role of each antigen amino acid in the antibody/antigen interaction. This aspect will be presented in the next section based on an analysis of the available crystallographic structures of bnAbs bound to the gp160 Env glycoprotein. However, the structures do not provide information concerning HIV stability and function. For example, generating antigen sequences by introducing purely random mutations will likely lead to sequences that are lethal for the virus and/or are not representative of HIV in vivo. To overcome this problem, it is useful to consider the structural data together with a model of the gp160 fitness landscape (29), which is a measure of the ability of HIV to tolerate mutations in its gp160 sequence to escape immune pressure. Structural and fitness information together provide a classification of the antibody/antigen interface and indicate the residues to mutate and the amino acids that are more probable at those positions.While this analysis helps to reduce the number of antigen sequences to consider by highlighting the “hot spots” of antibody/antigen binding, it leaves open the question of how to select a combination of antigen sequences for use in a vaccine. Given rules of optimal sequence dissimilarity and optimal fitness according to the HIV landscape, a Pareto frontier approach will be described. It is able to select, from all possible panels of antigen sequences, the few that are predicted to best elicit antibodies with a broad activity spectrum. Experimental evidence of the viability of the designed antigens and of their immunogenic properties is presented in the final section.     

Calcium currents in GH3 cultured pituitary cells under whole-cell voltage-clamp: inhibition by voltage-dependent potassium currents.

To isolate inward Ca2+ currents in GH3 rat pituitary cells, an inward Na+ current as well as two outward K+ currents, a transient voltage-dependent current (IKV) and a slowly rising Ca2+-activated current (IKCa), must be suppressed. Blockage of these outward currents, usually achieved by replacement of intracellular K+ with Cs+, reveals sustained inward currents. Selective blockage of either K+ current can be accomplished in the presence of intracellular K+ by use of quaternary ammonium ions. When IKCa and Na+ currents are blocked, the net current elicited by stepping the membrane potential (Vm) from -60 to 0 mV is inward first, becomes outward and peaks in 10-30 msec, and finally becomes inward again. Under this condition, in which both IKV and Ca2+ currents should be present throughout the duration of the voltage step, the Ca2+ current was not detected at the time of peak outward current. That is, plots of peak outward current vs. Vm are monotonic and are not modified by nisoldipine or low external Ca2+ as would be expected if Ca2+ currents were present. However, similar plots at times other than at peak current are not monotonic and are altered by nisoldipine or low Ca2+ (i.e., inward currents decrease and plots become monotonic). When K+ channels are first inactivated by holding Vm at -30 mV, a sustained Ca2+ current is always observed upon stepping Vm to 0 mV. Furthermore, substitution of Ba2+ for Ca2+ causes blockage of IKV and inhibition of this current results in inward Ba2+ currents with square wave kinetics. These data indicate that the Ca2+ current is completely inhibited at peak outward IKV and that Ca2+ conductance is progressively disinhibited as the transient K+ current declines due to channel inactivation. This suggests that in GH3 cells Ca2+ channels are regulated by IKV.     

Comparison of Bayesian and classical methods in the analysis of cluster randomized controlled trials with a binary outcome: The Community Hypertension Assessment Trial (CHAT)

Background  

Cluster randomized trials (CRTs) are increasingly used to assess the effectiveness of interventions to improve health outcomes or prevent diseases. However, the efficiency and consistency of using different analytical methods in the analysis of binary outcome have received little attention. We described and compared various statistical approaches in the analysis of CRTs using the Community Hypertension Assessment Trial (CHAT) as an example. The CHAT study was a cluster randomized controlled trial aimed at investigating the effectiveness of pharmacy-based blood pressure clinics led by peer health educators, with feedback to family physicians (CHAT intervention) against Usual Practice model (Control), on the monitoring and management of BP among older adults.