Estimation of the von Willebrand factor-cleaving protease in plasma using monoclonal antibodies to vWF

A protease present in plasma cleaves von Willebrand factor (vWF) at the peptide bond 842Tyr-843Met of the mature subunit. To quantify this vWF-cleaving protease activity in plasma we have developed a simple method based on the estimation by IRMA of the degradation of a constant amount of wild type recombinant vWF used as substrate, by serial dilutions of test plasma used as protease provider. vWFAg was estimated by two-site IRMA using as first coating antibody a monoclonal antibody (MoAb) whose epitope is localized on the C-terminal side of the cleavage site, and as second labeled antibody a pool of MoAbs specific for the N-terminal side. Because the proteolytic process leads to the progressive separation of the C- and N-terminal portions of the vWF subunit such an IRMA also shows a progressive apparent loss of vWFAg. In contrast, the levels of vWFAg estimated after proteolysis by regular IRMA remained essentially constant. Results obtained with this new method were compared with the analysis by SDS-agarose gel electrophoresis of the multimeric pattern of proteolyzed WT-rvWF and no significant difference was noted testing a series of 28 plasmas. As compared with normal pooled plasma, 14 normal individuals and 13 patients with various types of vWD had normal levels of protease activity (44-178%) by both methods. The validity of the method was confirmed by showing a lack of detectable protease activity in a patient with chronic relapsing thrombotic thrombocytopenic purpura. In conclusion our method appears as a useful tool for the quantification of the vWF-cleaving protease activity in plasma. Its sensitivity and specificity are similar to those of SDS-gel electrophoresis. However, this new IRMA has the major advantages of being much simpler and faster, and open to most research laboratories in the field.     

Response to 5% carbon dioxide in children and adolescents: relationship to panic disorder in parents and anxiety disorders in subjects

BACKGROUND: Carbon dioxide (CO(2)) sensitivity is postulated to be a familial risk marker of panic disorder (PD). Exaggerated responses to CO(2) inhalation have been reported in adults with PD and their unaffected adult relatives, as well as in clinic-referred children with anxiety disorders. OBJECTIVE: To test in a family-based design whether CO(2) hypersensitivity is a familial risk marker for PD and associated with current anxiety disorders in children and adolescents. SETTING AND PARTICIPANTS: One hundred forty-two offspring (aged 9-19 years) of parents with PD, major depressive disorder, or no disorder. Forty-five (32%) had a current anxiety disorder, excluding specific phobia. DESIGN AND MAIN OUTCOME MEASURES: Parents and offspring received diagnostic assessments. Offspring underwent 5% CO(2) inhalation at home. Panic symptoms and panic attacks were rated with the Acute Panic Inventory at baseline, while anticipating CO(2) delivery ("threat"), and during CO(2) inhalation. Respiratory rate and volume were measured with spirometry. RESULTS: No group differences were found in Acute Panic Inventory ratings at baseline or in respiratory measures during threat. Risk for PD was not associated with CO(2) sensitivity (panic symptoms and respiratory physiologic response). During CO(2) inhalation, offspring with anxiety disorders, relative to offspring without anxiety disorders, experienced significantly more panic symptoms and panic attacks, as well as elevated respiratory rates. During threat, panic symptoms were significantly and independently associated with both parental PD and offspring anxiety disorders. CONCLUSIONS: No support was obtained for CO(2) hypersensitivity as a familial risk marker for PD in children and adolescents. Links between childhood anxiety disorders and CO(2) sensitivity were replicated. Familial risk for PD in children and adolescents may be associated with vulnerability to anticipatory anxiety.     

Antibacterial diterpenes from Calceolaria pinifolia

Two new isopimaranes, 19-methylmalonyloxy-ent-isopimara-8(9),15-diene (5) and 19-malonyloxy-ent-isopimara-8(9),15-diene (6), were isolated using bioassay-guided fractionation of the CH(2)Cl(2)-MeOH (1:1) extract of the aerial part of Calceolaria pinifolia along with eight other diterpenes (1-4, 7-10) and two triterpenes (11, 12). All compounds were assayed against Staphylococcus aureus (SA), methicillin-resistant S. aureus (MRSA), Bacillus subtilis (BS), and Escherichia coli (EC). 4-Epi-dehydroabietinol (2) and ent-isopimara-9(11),15-diene-19-ol (8) were found to be active against MRSA with MIC values of 8 and 2 microgram/mL, respectively. Mechanistic studies of 8 in BS suggested rapid and nonspecific inhibition of uptake and incorporation of radiolabeled precursors into DNA, RNA, and protein consistent with membrane-damaging effects in bacteria. Compound 8 did not afford protection against an acute infection with SA in mice.     

Development of a nucleic acid sequence-based amplification assay that uses gag-based molecular beacons to distinguish between human immunodeficiency virus type 1 subtype C and C' infections in Ethiopia

A gag-based molecular beacon assay utilizing real-time nucleic acid sequence-based amplification technology has been developed to differentiate between the two genetic subclusters of human immunodeficiency virus type 1 (HIV-1) subtype C (C and C') circulating in Ethiopia. Of 41 samples, 36 could be classified as C or C' by sequencing of the gag gene. All 36 isolates were correctly identified by the gag beacon test. Three isolates with genomes that were recombinant in gag were unambiguously typed as belonging to the C' subcluster. Further analysis revealed that these contained the most sequence homology with a reference subcluster C' sequence in the target region of the beacon and hence were correct for the analyzed region. For one sample, sequencing and gag molecular beacon results did not match, while another isolate could not be detected at all by the beacon assay. Overall, high levels of sensitivity and specificity were achieved for both beacons (90.5% sensitivity and 100% specificity for the C beacon and 100% sensitivity and 95.2% specificity for the C' beacon). The availability of a diagnostic test which can quickly and reliably discriminate between C and C' HIV-1 infections in Ethiopia is an important first step toward studying their respective biological characteristics. As the assay is specific to the Ethiopian HIV-1 subtype C epidemic, it will contribute to characterizing the circulating viruses in this population, thereby generating the information necessary for the development of a potential efficacious HIV-1 vaccine appropriate for the Ethiopian context.     

Bacterial Uropathogens,Antimicrobial Susceptibility Profile and Associated Factors among Pediatric Patients in Bahir Dar,Northwest Ethiopia

BackgroundUrinary tract infection (UTI) in the pediatric group may lead to end-stage renal dysfunction later in life. Tracking the type of the isolates and their antimicrobial resistance pattern would impact the management of UTI in these group. The aim of this study was to describe the distribution of bacterial uropathogenes, their antimicrobial susceptibility profile and factors associated with significant bacteriuria (SBU) among pediatric patients at selected facilities in Bahir Dar, Northwest Ethiopia.MethodsA cross-sectional study was conducted from 1^st February to 30^th June 2020. About 5–10ml of urine samples were collected from pediatrics presumptive for UTI and a urine sample was considered positive for SBU if a single organism was grown at a concentration of ≥10⁴cfu/ml. Antimicrobial sensitivity testing was performed using the Kirby-Bauer disc diffusion technique. Logistic regression was used to identify factors associated with SBU and statistical significance was set at p-value < 0.05.ResultsOf the total 299 study participants, the majority 173 (57.9%) were females. The mean age of the participants was 6.6 years. The proportion of significant bacteriuria was at 49(16.4%). Most, 37 (75.5%) of the isolates were Gram-negative. The most predominant isolate was E. coli, 21(42.9%) followed by P. aeruginosa, 6(12.2%) and coagulase negative staphylococci, 6(12.2%). The level of multi-drug resistance among Gram-positive and Gram-negatives was at 50% and 78.4%, respectively. Participants'' sex, circumcision status, having a flank pain and being malnourished were statistically associated with significant bacteriuria.ConclusionActions to minimize antimicrobial resistance should be strengthened to reduce the impact of UTI among the pediatric group.     

Predictors of adherence to antiretroviral therapy among HIV-infected persons: a prospective study in Southwest Ethiopia

Background  

The devastating impact of AIDS in the world especially in sub-Saharan Africa has led to an unprecedented global effort to ensure access to antiretroviral (ARV) drugs. Given that medication-taking behavior can immensely affect an individual's response; ART adherence is now widely recognized as an 'Achilles heel' for the successful outcome. The present study was undertaken to investigate the rate and predictors of adherence to antiretroviral therapy among HIV-infected persons in southwest Ethiopia.     

Distinctive cerebellar immunoreactivity for the prion protein in familial (E200K) Creutzfeldt-Jakob disease

We have compared the immunomorphological spectrum of the deposition of the disease-associated prion protein (PrP(Sc)) in the cerebral and cerebellar cortex of 32 Creutzfeldt-Jakob disease (CJD) patients with the PrP gene (PRNP) E200K mutation to 45 sporadic CJD and 14 other genetic prion disease cases. PrP deposits correlate with the genotype at the methionine/valine (MV) polymorphic codon 129. While the diffuse/synaptic and patchy/perivacuolar PrP deposits and PrP plaques have a similar distribution and correlation with the genotype at codon 129 as in sporadic CJD, an additional peculiar PrP immunostaining pattern occurs in the cerebellum in 81% E200K mutation brains including 93% of M129M, 71% of M129V, but not in the single V129V case. It is localized to the molecular layer and consists of coarse granular PrP deposits arranged in a stripe-like manner predominantly perpendicular to the surface, closely resembling the parasagittal arborization of climbing fibers. Our results suggest that (1) the type of PrP deposits in the cerebellum may suggest genetic disease and the need for genetic testing; and (2) the peculiar stripes of PrP deposits might reflect selective vulnerability of cerebellar structures.