Pathogenesis of Providencia alcalifaciens-induced diarrhea.

Providencia alcalifaciens is a member of the family Enterobacteriaceae. There are reports that P. alcalifaciens can cause diarrhea, but the mechanism(s) by which it causes diarrhea is known. We studied P. alcalifaciens isolated from a child and two adults with diarrhea for enteropathogenicity. The three isolates did not exhibit any characteristic adherence to cultured HEp-2 cell monolayers, and they did not produce enterotoxins, cytotoxins, or keratoconjunctivitis in the Sereny test. Two isolates invaded cultured HEp-2 cell monolayers, producing localized bacterial clusters and actin condensation. The pattern of actin condensation was different from that produced by enteropathogenic Escherichia coli but similar to that produced by Shigella flexneri. Invasion and actin condensation were poor for the third isolate. Histology of adult rabbit small intestinal loops inoculated with all three isolates revealed bacterial attachment to, penetration of, and microulcer formation on the surface epithelium and hyperemia, edema, and polymorphonuclear cell infiltration of lamina propria. All the isolates produced diarrhea in rabbits with removable intestinal ties, and some of these rabbits developed hindlimb paralysis. Intestinal histology of the rabbits with removable intestinal ties which developed diarrhea showed changes similar to that in adult rabbits on which ileal loop assays had been performed. Transmission electron microscopy of intestinal tissues also confirmed tissue penetration by the isolates. Nerve tissue histology of two rabbits that developed hindlimb paralysis showed focal mononuclear cell infiltration around peripheral nerve sheaths. It is concluded that some strains of P. alcalifaciens are enteropathogenic and that they cause diarrhea by invading the intestinal mucosal epithelium. However, the relevance to human disease of the hindlimb paralysis observed in this animal model is not clear.     

Antimicrobial susceptibilities and plasmid contents of Neisseria gonorrhoeae isolates from commercial sex workers in Dhaka, Bangladesh: emergence of high-level resistance to ciprofloxacin

Commercial sex workers (CSWs) serve as the most important reservoir of sexually transmitted diseases (STD), including gonorrhea. Periodic monitoring of the antimicrobial susceptibility profile of Neisseria gonorrhoeae in a high-risk population provides essential clues regarding the rapidly changing pattern of antimicrobial susceptibilities. A study concerning the prevalence of gonococcal infection among CSWs was conducted in Bangladesh. The isolates were examined with regards to their antimicrobial susceptibility to, and the MICs of, penicillin, tetracycline, ciprofloxacin, cefuroxime, ceftriaxone, and spectinomycin by disk diffusion and agar dilution methods. The total plasmid profile of the isolates was also analyzed. Of the 224 CSWs, 94 (42%) were culture positive for N. gonorrhoeae. There was a good correlation between the results of the disk diffusion and agar dilution methods. Some 66% of the isolates were resistant to penicillin, and 34% were moderately susceptible to penicillin. Among the resistant isolates, 23.4% were penicillinase-producing N. gonorrhoeae (PPNG). 60.6% of the isolates were resistant and 38.3% were moderately susceptible to tetracycline, 17.5% were tetracycline-resistant N. gonorrhoeae, 11.7% were resistant and 26.6% had reduced susceptibility to ciprofloxacin, 2.1% were resistant and 11.7% had reduced susceptibility to cefuroxime, and 1% were resistant to ceftriaxone. All PPNG isolates contained a 3.2-MDa African type of plasmid, and a 24.2-MDa conjugative plasmid was present in 34.1% of the isolates. Since quinolones such as ciprofloxacin are recommended as the first line of therapy for gonorrhea, the emergence of significant resistance to ciprofloxacin will limit the usefulness of this drug for treatment of gonorrhea in Bangladesh.     

Integrin alpha2beta1 affects mechano-transduction in slowly and rapidly adapting cutaneous mechanoreceptors in rat hairy skin

The role of a transmembrane protein, integrin alpha2beta1, to modulate the neural responses of cutaneous mechanoreceptors to mechanical indentation was examined using an isolated skin-nerve preparation in a rat model. Skin and its intact innervation were harvested from the medial thigh of the hindlimb and placed in a dish containing synthetic interstitial fluid. Using a standard teased nerve preparation, the neural responses of single slowly or rapidly adapting mechanoreceptors (SA or RA, respectively) were identified and the afferents categorized according to standard protocols (i.e. response to constant stimuli). The most sensitive spot of a mechanoreceptor's receptive field was identified and then stimulated using controlled compressive stress (constant or dynamic loads between threshold and saturation load for SAs and RAs, respectively). Loads were applied before, during, and after passive diffusion into the skin of a function-blocking anti-integrin alpha2 monoclonal antibody (FBmAb) or one of two types of control antibodies (immunoglobulin G or a FBmAb conjugated with a secondary antibody). The sensitivities of both SA and RA mechanoreceptors were profoundly reduced in the presence of the FBmAb, while not changing the waveforms of their action potentials or their adaptation properties. Both control antibodies had no significant effect on mechanoreceptors' sensitivities. Following removal of the FBmAb, the effects in some neurons were partially reversible. Taken together, the data from this study support the hypothesis that integrin alpha2beta1 plays a significant role in modulating mechanoreceptive response to compressive indentation.     

Ocular measurements in fetal alcohol spectrum disorders

Fetal alcohol spectrum disorders (FASD) describe a range of physical, behavioral, and neurologic deficits in individuals exposed to alcohol prenatally. Reduced palpebral fissure length is one of the cardinal facial features of FASD. However, other ocular measurements have not been studied extensively in FASD. Using the Fetal Alcohol Syndrome Epidemiologic Research (FASER) database, we investigated how inner canthal distance (ICD), interpupillary distance (IPD), and outer canthal distance (OCD) centiles differed between FASD and non‐FASD individuals. We compared ocular measurement centiles in children with FASD to non‐FASD individuals and observed reductions in all three centiles for ICD, IPD, and OCD. However, when our non‐FASD children who had various forms of growth deficiency (microcephaly, short‐stature, or underweight) were compared to controls, we did not observe a similar reduction in ocular measurements. This suggests that reductions in ocular measurements are a direct effect of alcohol on ocular development independent of its effect on growth parameters, which is consistent with animal models showing a negative effect of alcohol on developing neural crest cells. Interpupillary distance centile appeared to be the most significantly reduced ocular measure we evaluated, suggesting it may be a useful measure to be considered in the diagnosis of FASD.     

Differentiating molecular etiologies of Angelman syndrome through facial phenotyping using deep learning

Angelman syndrome (AS) is caused by several genetic mechanisms that impair the expression of maternally‐inherited UBE3A through deletions, paternal uniparental disomy (UPD), UBE3A pathogenic variants, or imprinting defects. Current methods of differentiating the etiology require molecular testing, which is sometimes difficult to obtain. Recently, computer‐based facial analysis systems have been used to assist in identifying genetic conditions based on facial phenotypes. We sought to understand if the facial‐recognition system DeepGestalt could find differences in phenotype between molecular subtypes of AS. Images and molecular data on 261 individuals with AS ranging from 10 months through 32 years were analyzed by DeepGestalt in a cross‐validation model with receiver operating characteristic (ROC) curves generated. The area under the curve (AUC) of the ROC for each molecular subtype was compared and ranked from least to greatest differentiable phenotype. We determined that DeepGestalt demonstrated a high degree of discrimination between the deletion subtype and UPD or imprinting defects, and a lower degree of discrimination with the UBE3A pathogenic variants subtype. Our findings suggest that DeepGestalt can recognize subclinical differences in phenotype based on etiology and may provide decision support for testing.     

Development and evaluation of NucliSens basic kit NASBA for diagnosis of parainfluenza virus infection with 'end-point' and 'real-time' detection

New methods for the detection of human parainfluenza viruses (HPIVs) were developed. These were based on nucleic acid sequence-based amplification (NASBA) and utilised the NucliSens Basic Kit. Primers and probes were selected from the haemagglutinin neuraminidase (HN) gene of HPIV1, HPIV2 and HPIV3, and from the phosphoprotein (P) of HPIV4a and -4b. Synthetic RNA, titrated control virus stocks and respiratory specimens (n=44) were utilised to evaluate performance of the assays. Detection of NASBA products was by probe hybridisation and electrochemiluminescence (ECL) ('end-point' detection) or using molecular beacons ('real-time' detection). The assays using ECL detection proved to be both sensitive and specific. Typically, less than or equal to 100 RNA copies or one TCID(50) input was detectable with no cross-reaction between the specific HPIV assays and other respiratory viruses. Results for clinical samples were concordant with those obtained by 'conventional' procedures by classical viral diagnostic methods. 'Real-time' detection utilised probes specific for either HPIV1 or HPIV3 with similar performance characteristics to the assays with 'end-point' detection. The feasibility of multiplexing targets together was confirmed using a combined HPIV1 and HPIV3 assay with good results for ECL and molecular beacon detection on control material and clinical samples.     

Cigarette smoke prevents apoptosis through inhibition of caspase activation and induces necrosis

Emphysema is characterized by enlargement of the distal airspaces in the lungs due to destruction of alveolar walls. Alveolar endothelial and epithelial cell apoptosis induced by cigarette smoke is thought to be a possible mechanism for this cell loss. In contrast, our studies show that cigarette smoke condensate (CSC) induces necrosis in alveolar epithelial cells and human umbilical vein endothelial cells. Furthermore, study of the cell death pathway in a model system using Jurkat cells revealed that in addition to inducing necrosis, CSC inhibited apoptosis induced by staurosporine or Fas ligation, with both effects prevented by the antioxidants glutathione and dithiothreitol. Time course experiments revealed that CSC inhibited an early step in the caspase cascade, whereby caspase-3 was not activated. Moreover, cell-free reconstitution of the apoptosome in cytoplasmic extracts from CSC-treated cells, by addition of cytochrome-c and dATP, did not result in activation of caspases-3 or -9. Thus, smoke treatment may alter the levels of pro- and antiapoptogenic factors downstream of the mitochondria to inhibit active apoptosome formation. Therefore, unlike previous studies, cell death in response to cigarette smoke by necrosis and not apoptosis may be responsible for the loss of alveolar walls and inflammation observed in emphysema.