Single acute stress-induced progesterone and ovariectomy alter cardiomyocyte contractile function in female rats

Aim

To assess how ovarian-derived sex hormones (in particular progesterone) modify the effects of single acute stress on the mechanical and biochemical properties of left ventricular cardiomyocytes in the rat.

Methods

Non-ovariectomized (control, n = 8) and ovariectomized (OVX, n = 8) female rats were kept under normal conditions or were exposed to stress (control-S, n = 8 and OVX-S, n = 8). Serum progesterone levels were measured using a chemiluminescent immunoassay. Left ventricular myocardial samples were used for isometric force measurements and protein analysis. Ca²⁺-dependent active force (F_active), Ca²⁺-independent passive force (F_passive), and Ca²⁺-sensitivity of force production were determined in single, mechanically isolated, permeabilized cardiomyocytes. Stress- and ovariectomy-induced alterations in myofilament proteins (myosin-binding protein C [MyBP-C], troponin I [TnI], and titin) were analyzed by sodium dodecyl sulfate gel electrophoresis using protein and phosphoprotein stainings.

Results

Serum progesterone levels were significantly increased in stressed rats (control-S, 35.6 ± 4.8 ng/mL and OVX-S, 21.9 ± 4.0 ng/mL) compared to control (10 ± 2.9 ng/mL) and OVX (2.8 ± 0.5 ng/mL) groups. F_active was higher in the OVX groups (OVX, 25.9 ± 3.4 kN/m² and OVX-S, 26.3 ± 3.0 kN/m²) than in control groups (control, 16.4 ± 1.2 kN/m² and control-S, 14.4 ± 0.9 kN/m²). Regarding the potential molecular mechanisms, F_active correlated with MyBP-C phosphorylation, while myofilament Ca²⁺-sensitivity inversely correlated with serum progesterone levels when the mean values were plotted for all animal groups. F_passive was unaffected by any treatment.Conclusion Stress increases ovary-independent synthesis and release of progesterone, which may regulate Ca²⁺-sensitivity of force production in left ventricular cardiomyocytes. Stress and female hormones differently alter Ca²⁺-dependent cardiomyocyte contractile force production, which may have pathophysiological importance during stress conditions affecting postmenopausal women.The relation between stress, gender, and cardiovascular diseases is well established (1-4). Some of the known risk factors for cardiovascular disease such as smoking, unhealthy diet, and behavioral and psychosocial stress have deleterious effects on the cardiovascular system via activation of the sympathetic nervous system and the hypothalamic-pituitary-adrenal (HPA) axis (5-8). Acute restraint stress is a preferred and widely used method to induce physical stress in animal models (9). Moreover, restraint and immobilization are important as models for psychological stress, which was shown to adversely affect ovarian function (10) and to play a pivotal role in the pathomechmanism of Takotsubo (stress) cardiomyopathy in postmenopausal women (11).Gender is a very important factor in the development of cardiovascular diseases. Premenopausal women have better lipid profile, endothelial function (12), and a lower risk to develop coronary artery disease and myocardial infarction (MI) than men. These advantages of female gender, however, are abolished after menopause, which is associated with increased prevalence of left ventricular (LV) hypertrophy, decreased LV ejection fraction, and LV contractility (13). One of the explanations for the distinct myocardial responses is the cardioprotective effect of female sex hormones (eg, estrogens) (14,15).Progesterone performs several actions on the heart: it exerts an antiarrhythmic effect by accelerating cardiac repolarization (16) and has a preventive role in ischemia-reperfusion injury via reducing inflammatory response (17). It has been shown to inhibit cardiomyocyte apoptosis (18), induce vasodilation, and reduce blood pressure via increasing nitric oxide (NO) levels in normotensive and hypertensive patients (19). Importantly, progesterone is produced by the both ovaries and the adrenal gland: Moreover, the adrenal progesterone content is similar or even larger than that in the ovaries (20). Adrenal progesterone production and secretion increase along with corticosterone regardless of gender and estradiol under stress conditions (21). Progesterone, being an indirect precursor of cortisol (22), increases in response to adrenocorticotrophic hormone (ACTH) stimulation (23).In the heart, there are multiple estrogen hormone receptor types (24). The expression of aromatase in the heart suggests that estrogen may be synthesized also within the cardiomyocyte to exert autocrine/paracrine actions (25). Myocyte contractility seems to be modulated by systemic estrogen levels and altered in cardiomyocytes derived from ovariectomized (OVX) rats (26). In particular, myofilament Ca²⁺-sensitivity is increased in isolated myofibrillar preparations from OVX rats, and restored to the basal levels with estrogen supplementation (27,28).Activation of the sympathetic nervous system plays a central role in the regulation of cardiomyocyte contractile function and myofilament Ca²⁺-sensitivity through beta-adrenergic receptor stimulation, activating the protein kinase A (PKA). PKA-mediated phosphorylation of Ca²⁺-handling and myofilament proteins (myosin binding protein-C [MyBP-C], troponin I [TnI], titin) were shown to alter cardiomyocyte contractile function (29,30). It has been suggested that female cardiomyocytes operate at lower levels of intracellular Ca²⁺ than those of males, particularly under inotropic conditions (31). This difference in Ca²⁺ homeostasis may be related to the fact that estrogen suppresses the L-type Ca²⁺ current (32,33) and may reduce the amount of Ca²⁺ released from the sarcoplasmic reticulum (SR) (34), which was shown to be larger in myocytes from OVX rats (35). Not only cardiomyocyte contraction, but relaxation may also be affected by estrogen via altered Ca²⁺ re-uptake into the SR and modified Ca²⁺ efflux via increased sarcolemmal Na⁺/Ca²⁺ exchange (36). Interestingly, despite similar SR Ca²⁺ content in males and females (37), studies using OVX models report conflicting results concerning changes in the expression and activity of the SR Ca²⁺-ATPase and its regulator protein phospholamban (38-41). Much less is known about the possible effect of progesterone on cardiomyocyte contractile function. We hypothesized that progesterone affected force production of single isolated cardiomyocytes. Therefore, in the present study we aimed to investigate how sex hormones (particularly progesterone) and single acute restraint stress altered cardiomyocyte contractile function and to identify the consequent posttranslational myofilament protein modifications in OVX rats.     

Distribution of KIR genes in the Croatian population

The KIR locus with genes involved in immune processes is among the most polymorphic and structurally diverse human loci. KIR genes encode activating and inhibitory receptors that differ in specificity for HLA class I ligands and signaling potential. These receptors are expressed principally by natural killer (NK) cells and subpopulations of T cells. This study represents the first report of the distribution of KIR genes, KIR genotypes and KIR/HLA pairs in 121 unrelated healthy Croatian individuals. Twenty-three different genotypes were observed in the Croatian population and all 16 KIR genes known to date were found. The most frequent KIR genotype was the AA genotype. All individuals had at least one inhibitory KIR/HLA pair with the majority of individuals with three inhibitory KIR/HLA pairs. The most frequent KIR/HLA pair was the KIR2DL3/C1 group. Our results demonstrated the similarity of the Croatian population’s KIR repertoire with other Caucasian populations reported so far.     

Slaying the dragon myth: an ethnographic study of receptionists in UK general practice

Background

General practice receptionists fulfil an essential role in UK primary care, shaping patient access to health professionals. They are often portrayed as powerful ‘gatekeepers’. Existing literature and management initiatives advocate more training to improve their performance and, consequently, the patient experience.

Aim

To explore the complexity of the role of general practice receptionists by considering the wider practice context in which they work.

Design and setting

Ethnographic observation in seven urban general practices in the north-west of England.

Method

Seven researchers conducted 200 hours of ethnographic observation, predominantly in the reception areas of each practice. Forty-five receptionists were involved in the study and were asked about their work as they carried out their activities. Observational notes were taken. Analysis involved ascribing codes to incidents considered relevant to the role and organising these into related clusters.

Results

Receptionists were faced with the difficult task of prioritising patients, despite having little time, information, and training. They felt responsible for protecting those patients who were most vulnerable, however this was sometimes made difficult by protocols set by the GPs and by patients trying to ‘play’ the system.

Conclusion

Framing the receptionist–patient encounter as one between the ‘powerful’ and the ‘vulnerable’ gets in the way of fully understanding the complex tasks receptionists perform and the contradictions that are inherent in their role. Calls for more training, without reflective attention to practice dynamics, risk failing to address systemic problems, portraying them instead as individual failings.     

Construction and Immunological Evaluation of Multivalent Hepatitis B Virus (HBV) Core Virus-Like Particles Carrying HBV and HCV Epitopes

A multivalent vaccine candidate against hepatitis B virus (HBV) and hepatitis C virus (HCV) infections was constructed on the basis of HBV core (HBc) virus-like particles (VLPs) as carriers. Chimeric VLPs that carried a virus-neutralizing HBV pre-S1 epitope corresponding to amino acids (aa) 20 to 47 in the major immunodominant region (MIR) and a highly conserved N-terminal HCV core epitope corresponding to aa 1 to 60 at the C terminus of the truncated HBcΔ protein (N-terminal aa 1 to 144 of full-length HBc) were produced in Escherichia coli cells and examined for their antigenicity and immunogenicity. The presence of two different foreign epitopes within the HBc molecule did not interfere with its VLP-forming ability, with the HBV pre-S1 epitope exposed on the surface and the HCV core epitope buried within the VLPs. After immunization of BALB/c mice, specific T-cell activation by both foreign epitopes and a high-titer antibody response against the pre-S1 epitope were found, whereas an antibody response against the HBc carrier was notably suppressed. Both inserted epitopes also induced a specific cytotoxic-T-lymphocyte (CTL) response, as shown by the gamma interferon (IFN-γ) production profile.Genetically engineered virus-like particle (VLP)-based vaccines are one of the most promising tools in modern vaccinology. VLPs from almost all classes of viruses are being evaluated now or have just been adopted to use as carriers for presentation of foreign immunological epitopes (for a review, see references 29 and 31). VLP technologies possess obvious advantages for the generation of safe and efficacious vaccines. First, the repetitive antigenic structure of VLPs makes them highly immunogenic. Second, VLPs lack viral genomes or genes and are noninfectious, although they mimic infectious viruses in their structural and immunological features. Third, VLPs are generated by highly efficient heterologous expression of the cloned viral structural genes with subsequent quantitative in vivo or in vitro self-assembly of their products. Fourth, VLPs can be obtained by simple and efficient purification procedures. VLPs can be used for a broad range of applications, but the generation of vaccines against hepatitis B virus (HBV) and hepatitis C virus (HCV) infections is of special interest.The HBV core (HBc) protein was first reported as a promising VLP carrier in 1986 and was published in 1987 (6, 10, 24). In many ways, HBc occupies a unique position among the VLP carriers because of its high-level synthesis and efficient self-assembly in virtually all known homologous and heterologous expression systems, including bacteria (for a review, see references 29 to 31). The major HBc B-cell epitopes (c and e1) are localized within the major immunodominant region (MIR), whereas the next important epitope, e2, is localized around amino acid position 130, close to the C-terminal histone-like region (for a review, see reference 30).The high-resolution spatial structure of HBc icosahedrons (11, 43) shows that the MIR is located on the tip of the spike, around the most protruding region between amino acids (aa) 78 and 82. For this reason, the MIR is generally accepted as the target site of choice for insertion of foreign epitopes (30). The other widely accepted site for insertions is C-terminal position 144, a short stretch after the e2 epitope. For C-terminal insertions, so-called HBcΔ vectors lacking a 39-aa-long positively charged C-terminal histone-like fragment are preferred for their high insertion capacities (up to 741 amino acid residues) (30).Here, we present the construction and preliminary immunological characterization of a first multivalent HBV and HCV vaccine candidate. As an HBV epitope, we chose the pre-S1 sequence aa 20 to 47, which alone is able to elicit HBV-neutralizing and protective antibodies (23), for insertion into the HBc MIR. Concurrently, we inserted at the C terminus of the HBcΔ vector the N-terminal 60-aa fragment of the HCV core, which is highly conserved among various HCV genotypes with amino acid homology exceeding 95% (12, 14) and therefore is an attractive target for the generation of an HCV vaccine (19, 41). Such a combination of foreign epitopes did not prevent correct self-assembly of chimeric HBc-based particles and provided them with specific HBV and HCV antigenicity and immunogenicity in mice.     

XbaI polymorphism of the estrogen receptor alpha gene influences the effect of raloxifene on the endothelial function

Objectives

Cardiovascular disease is the leading cause of death in postmenopausal women and estrogen deficiency may be an important factor in its development. The selective estrogen receptor modulator, raloxifene, exerts a part of its actions through the estrogen receptor alpha (ESR1) activation. We explored if polymorphisms of the ESR1 modify the effects of 6 months raloxifene treatment on endothelial function.

Methods

A total of 53 postmenopausal women, mean age 59.7 ± 6.2, finished the prospective clinical trial. The PvuII, XbaI, and P325P polymorphisms of the ESR1 gene were analyzed. In all subjects endothelium-dependent flow mediated dilatation (FMD) and cell adhesion molecules (CAM) ICAM-1, VCAM-1 and E-selectin were measured before and after 6 months of raloxifene treatment.

Results

There was no difference in FMD between the ESR1 genotypes, at baseline. After raloxifene treatment, the FMD was significantly greater in subjects with XX genotype of XbaI polymorphism compared to xx (p = 0.03) and borderline greater when compared to Xx genotype (p = 0.053). The FMD increased significantly with raloxifene treatment in women with Xx genotype of XbaI and Pp genotype of PvuII polymorphisms (p = 0.027 and p = 0.034, respectively). The P325P polymorphism did not influence the FMD after raloxifene. None of the ESR1 gene polymorphisms had any impact on the levels of CAM before or after the treatment. When analysing the whole group, a significant decrease in E-selectin (p < 0.001) and a small increase in ICAM-1 levels (p = 0.029) was observed with raloxifene treatment, but no influence on VCAM-1 levels or FMD overall was seen.

Conclusion

Our data suggest that XbaI and possibly PvuII polymorphisms of the ESR1 gene influence the impact of raloxifene treatment on endothelial function. This effect could be of pharmacogenomic and clinical importance.     

Identification and characterization of a conserved,stage-specific gene product of Plasmodium falciparum recognized by parasite growth inhibitory antibodies

We have identified a novel conserved protein of Plasmodium falciparum, designated D13, that is stage-specifically expressed in asexual blood stages of the parasite. The predicted open reading frame (ORF) D13 contains 863 amino acids with a calculated molecular mass of 99.7 kDa and displays a repeat region composed of pentapeptide motives. Northern blot analysis with lysates of synchronized blood stage parasites showed that D13 is highly expressed at the mRNA level during schizogony. The first N'-terminal 138 amino acids of D13 were expressed in Escherichia coli and the purified protein was used to generate anti-D13 monoclonal antibodies (MAbs). Using total lysates of blood stage parasites and Western blot analysis, these MAbs stained one single band of approximately 100 kDa, corresponding to the predicted molecular mass of ORF D13. Western blot analysis demonstrated further that D13 is expressed during schizogony, declines rapidly in early ring stages and is undetectable in trophozoites. D13 protein is localized in individual merozoites in a distinct area, as demonstrated by indirect immunofluorescence analysis. After subcellular fractionation, D13 was confined to the pelleted fraction of the parasite lysate and its extraction by alkaline carbonate buffer treatment indicated that D13 is not a membrane-integral protein. Inclusion of certain anti-D13 MAbs into in vitro cultures of blood stage parasites resulted in considerable reduction in parasite growth. The N'-terminal domain encompassing 158 amino acids is 94 and 95%, respectively, identical at the amino acid level between Plasmodium knowlesi, Plasmodium yoelii, and P. falciparum. In summary, we describe a novel stage-specifically expressed, highly conserved gene product of P. falciparum that is recognized by parasite growth inhibitory antibodies.     

An outbreak of HBV and HCV infection in a paediatric oncology ward: epidemiological investigations and prevention of further spread

Hospital-acquired hepatitis B (HBV) and C virus (HCV) infections continue to occur despite increased awareness of this problem among the medical community. One hundred six patients were infected in a haematology oncology ward for children, over the time period 1996 to 2000. Serum samples from 45 such patients and 3 from infected medical personnel were used for nucleic acid amplification. HBV core, as well as HCV core and hypervariable region 1 (HVR1) nucleotide sequences, were analysed by phylogenetic tree analysis, in order to characterise the epidemiological pattern of viral transmission on the ward. Samples from 32 patients were positive for HBV-DNA or HCV-RNA by PCR. Ten patients were positive for both markers. Seventeen out of twenty-three HCV core gene sequences were found to be evolutionarily related and clustered separately from other local sequences in the phylogenetic tree, indicating nosocomial transmission. This was confirmed by analysis of HVR1 gene sequences. One nurse and one physician from the ward were HCV RNA positive, but their HCV sequences were not related evolutionarily to those of the patient cluster. Fifteen out of nineteen HBV core gene sequences were also clustered together and were positioned separately in the relevant tree. Epidemiological investigation excluded a common source infection and indicated that spread of infection was most likely due to inappropriate infection control measures on the ward. No obvious risk factors for transmission were identified during the retrospective survey in patients with related sequences, except use of multidose vials for saline and poor staff compliance with routine hand hygiene procedures. The preventive measures that were introduced reduced the incidence of infection significantly. No new cases of HBV infection and only three anti-HCV seroconversions occurred over a period of 19 months. The introduction and maintenance of strict prevention measures over a 2 year period, combined with HBV vaccination, reduced significantly the incidence of new HCV and HBV infections.     

Immunohistochemical demonstration of mitochondria in routinely processed tissue using a monoclonal antibody

We report results obtained using the monoclonal antibody M-II 68, which recognizes inner mitochondrial membrane in routinely processed (formalin-fixed and paraffin-embedded) tissue by light microscopic immunohistochemistry. In ten normal brains, the range of immunoreactivity in various cell types and locations was defined. The most intense staining was observed in Purkinje cells, in neurons of cranial nerve nuclei, pons and substantia nigra, as well as in choroid plexus epithelial cells. By comparison with this control group, one case of primary mitochondrial encephalomyopathy exhibited increased staining of endothelial and vascular smooth muscle cells, choroid plexus epithelial cells, and neurons of various locations. Scattered ragged-red fibres were heavily labelled in one case of mitochondrial myopathy, while ten muscles without mitochondriopathy were left unstained. Our method is able to detect accumulations of mitochondria and increases in mitochondrial cristae density. It could prove useful for differential diagnosis of routine biopsy material and for clarification of cell types involved in mitochondrial cytopathies.     

Proton MR spectroscopy and diffusion-weighted imaging of intracranial germ cell tumors: Implications for differentiation from other lesions

Introduction

Intracranial germ cell tumors (GCT) are extremely rare brain neoplasms, with imaging characteristics being often non-specific. Considering such imaging limitation in establishing their exact histology, the aim of this paper is to integrate conventional, physiologic and metabolic magnetic resonance imaging (MRI) with histology of these tumors, with the goal of proposing some new insights that could aid in their diagnosis.

Materials and methods

We have retrospectively analyzed preoperative imaging findings in 9 patients with histology proven intracranial germ cell tumors. In all patients conventional magnetic resonance imaging in conjunction with diffusion-weighted imaging (DWI) and proton magnetic resonance spectroscopy (MRS) was performed.

Results

Based on imaging characteristics on conventional sequences germinoma cannot be differentiated from nongerminoma. DWI offered the potential for differentiation of germinomas from non-germinomas since solid part of germinomas demonstrated restricted diffusion with ADC values under 1.0 × 10⁻³ mm²/s, whereas nongerminoma showed elevated diffusion with ADC values above 1.3 × 10⁻³ mm²/s. Short echo MRS demonstrated characteristic spectra in germinomas: increased Cho, decreased NAA and prominent broad lipid resonances.

Conclusion

DWI and MRS imaging findings provide additional information that can facilitate evaluation of intracranial germ cell tumors.