Cholesterol oxidase is required for virulence of Mycobacterium tuberculosis

Recent reports have indicated that cholesterol plays a crucial role during the uptake of mycobacteria by macrophages. However, the significance of cholesterol modification enzymes encoded by Mycobacterium tuberculosis for bacterial pathogenicity remains unknown. Here, the authors explored whether the well-known cholesterol modification enzyme, cholesterol oxidase (ChoD), is important for virulence of the tubercle bacillus. Homologous recombination was used to replace the choD gene from the M. tuberculosis genome with a nonfunctional copy. The resultant mutant (delta choD) was attenuated in peritoneal macrophages. No attenuation in macrophages was observed when the same strain was complemented with an intact choD gene controlled by a heat shock promoter (delta choDP(hsp)choD). The mice infection experiments confirm the significance of ChoD in the pathogenesis of M. tuberculosis.     

Cardiovascular events in early RA are a result of inflammatory burden and traditional risk factors: a five year prospective study

Introduction  

Co-morbidity and mortality due to cardiovascular disease (CVD) are increased in patients with rheumatoid arthritis (RA). Most published studies in this field are retrospective or cross sectional. We investigated the presence of traditional and disease related risk factors for CVD at the onset of RA and during the first five years following diagnosis. We also evaluated their potential for predicting a new cardiovascular event (CVE) during the five-year follow-up period and the modulatory effect of pharmacological treatment.     

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses^1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)³. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity⁴, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method⁵. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses^6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide⁶, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)⁷. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera^7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only. Download video file.^{(80M, mov)}     

Infections caused by RSV among children and adults during two epidemic seasons

Respiratory Syncytial Virus (RSV) is one of the most common causes of lower respiratory tract infections in young children, immunocompromised patients (children and adults), patients with chronic respiratory diseases and elderly people. Reinfections occur throughout the life, but the severity of disease decreased with subsequent infection. The aim of this study was to analyze the frequency of RSV infections in two selected subpopulations: young children (below 5 y.) and adults with chronic respiratory diseases (25-87 y.). Nasopharyngeal swabs (334) collected from October 2008 to March 2010 were examined. The presence of RSV genome was determined by RT-PCR and the presence of RSV antigen by quick immunochromatographic test. Positive results of RT-PCR were found in 45.2% of all swabs: 48.6% samples in 2008; 41.5% in 2009; 50.8% in 2010. The highest frequency of RSV-positive samples was in fall-winter months, but differences in RSV epidemic seasons were found. In the first season (2008-2009) an increased number of RSV infections was observed from November 2008, but in the second season--from January 2010. Generally, the frequency of RSV-positive RT-PCR among children was 53%, among adults 25%. The highest difference was observed in the first three-month period of 2010. RT-PCR positive samples were found in 68.5% of children and 5.9% of adults. However, the RSV antigen was found in 44.4% of samples collected from adults in this period. Our results indicate that the contribution of RSV infections during epidemic season of respiratory tract infections in Poland was really high among children and adults.     

Fluorescence imaging to quantify the fluorescent microspheres in cardiac tissue

To quantify the fluorescent microsphere (FM) content in cardiac tissue, which is an indicative of blood flow, fluorescence imaging of both sides of the pig heart slice was employed. Despite the light scattering inside the tissue and contributions from multiple tissue layers to the total emission, it is shown that the fluorescence intensity at any pixel is proportional to the FM content and the fluorescence image may be transformed to the image of the FM concentration. A convenient standard for the emission‐FM concentration transformation is proposed. The approach has several advantages in comparison with the traditional “digestion & extraction” method such as: non‐destructiveness, high spatial resolution, high throughput, repeatability and simplicity of operation. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Cyclosporine a induces growth arrest or programmed cell death of human glioma cells

Human malignant gliomas are highly resistant to current therapeutic approaches. We previously demonstrated that cyclosporine A (CsA) induces an apoptotic cell death in rat C6 glioma cells. In the present study, we found the induction of growth arrest or cell death of human malignant glioma cells exposed to CsA. In studied glioma cells, an accumulation of p21Cip1/Waf1 protein, a cell cycle inhibitor, was observed following CsA treatment, even in the absence of functional p53 tumour suppressor. CsA induced a senescence-associated growth arrest, in U87-MG glioma cells with functional p53, while in U373 and T98G glioma cells with mutated p53, CsA treatment triggered cell death associated with alterations of cell morphology, cytoplasm vacuolation, and condensation of chromatin. In T98G cells this effect was completely abolished by simultaneous treatment with an inhibitor of protein synthesis, cycloheximide (CHX). Moreover, CsA-induced cell death was accompanied by activation of executory caspases followed by PARP cleavage. CsA treatment did not elevate fasL expression and had no effect on mitochondrial membrane potential. We conclude that CsA triggers either growth arrest or non-apoptotic, programmed cell death in human malignant glioma cells. Moreover, CsA employs mechanisms different to those in the action of radio- and chemotherapeutics, and operating even in cells resistant to conventional treatments. Thus, CsA or related drugs may be an effective novel strategy to treat drug-resistant gliomas or complement apoptosis-based therapies.     

Strains of genus Salmonella isolated from extraintestinal infections

Every year in Poland from tens to more than hundred bacteriologically verified extraintestinal infections caused by Salmonella species have been registered. These unusually located infections have substantially heavy course and in many cases hospitalisation and antibiotic therapy have to be involved. Cases of extraintestinal infections with these Gram-negative rods, which were described in the literature, concerned: pneumonias, lung abscesses and thoracic empyemas, and infections of: blood, bones and joints, wounds, fistulas and urinary tract. The aim of this study was to set extraintestinal Salmonella infections and to analyze a susceptibility of isolated strains to antimicrobial agents. Between 01.07.2002 and 31.12.2004 (30 months) 13 strains of Salmonella genus have been isolated, including 11 S. enteritidis and 2 S. Hadar. In general, with one exception, isolated strains were susceptible to tested antibiotics/chemotherapeutics. ESBL - positive strains were not detected. The tendency of Salmonella strains to cause extraintestinal infections has been noticed. The problem is still escalating, especially in group of patients chronically treated, with immunodeficiency and immunosuppression, after complicated medical procedures, also in the group of small children and aged persons. Therefore, it is necessary to evaluate a susceptibility to antibiotics/chemotherapeutics of every strain from confirmed case of Salmonella extraintestinal infection and it is important to apply a guided therapy.     

Cannabinoids down-regulate PI3K/Akt and Erk signalling pathways and activate proapoptotic function of Bad protein

Cannabinoids were shown to induce apoptosis of glioma cells in vitro and tumor regression in vivo, but mechanisms of their antiproliferative action remain elusive. In the present studies, C6 cells were exposed to a synthetic cannabinoid, WIN 55,212-2, which produced down-regulation of the Akt and Erk signalling pathways prior to appearance of any sign of apoptosis. We hypothesized that cannabinoid-induced cell death may be mediated by a Bcl-2 family member--Bad, whose function is hampered by these kinases due to control of its phosphorylation state. Using Western blot analysis, we found that levels of phosphorylated Bad, but not total Bad protein, decreased under exposure to WIN 55,212-2. WIN 55,212-2 treatment further resulted in mitochondrial depolarization and activation of caspase cascade. Thus, we suggest that the increase of proapoptotic Bad activity is an important link between the inhibition of survival pathways and an onset of execution phase of cannabinoid-induced glioma cell death.     

The concentrations of soluble HLA-G protein are elevated during mid-gestation and decreased in pre-eclampsia

The aim of our study was to investigate the dynamics of the alterations of soluble human leukocyte antigen-G (sHLA-G) concentrations in sera of healthy non-pregnant women, as well as healthy pregnant women and patients with pre-eclampsia. Thirty five patients with pre-eclampsia, 52 healthy pregnant women, and 24 healthy non-pregnant women were included in the study. Sera concentrations of sHLA-G protein were determined using the immunoenzymatic ELISA method. Statistical analysis was performed using ANOVA and Mann-Whitney U tests. The concentrations of sHLA-G protein in sera of pregnant women in the first, as well as the second and third, trimesters of normal pregnancy were significantly higher in comparison with healthy nonpregnant women. The sera concentrations of sHLA-G in pregnant women in the second trimester of pregnancy were significantly higher compared to the first and third trimesters. The concentrations of sHLA-G in sera of patients with pre-eclampsia were significantly lower than in pregnant women in the third trimester of physiological pregnancy. The results of our study suggest that normal physiological pregnancy is associated with elevated sera concentrations of sHLA-G molecule. The increased concentrations of sHLA-G molecule in mid-gestation could suggest a role for the protein in the second phase of a physiological invasion of extravillous cytotrophoblast to spiral arteries. Furthermore, the results could suggest a role for the decreased sera concentrations of sHLA-G in the pathogenesis of pre-eclampsia.