Idiopathic subglottic stenosis in Saskatchewan Hutterite population.

BACKGROUND: By studying the odds of developing idiopathic subglottic stenosis in the isolated and genetically unique Hutterite population, this study sought to strengthen the hypothesis that an underlying genetic predisposition may exist for its development. METHODS: A retrospective chart review examined the medical records of all adult patients treated for idiopathic subglottic stenosis in Saskatchewan between 2008 and 2018. Cases were segregated into Hutterite and non-Hutterite. RESULTS: Four out of 36 cases of idiopathic subglottic stenosis occurred among Hutterites. The odds of a Hutterite developing idiopathic subglottic stenosis are 21.89 times higher than for non-Hutterites. Positive family history was only observed in the Hutterite population. CONCLUSION: The study strengthens the hypothesis that genetics may play a role in the aetiology of idiopathic subglottic stenosis by demonstrating that the genetically and socially unique Hutterites are more likely to develop this rare disease. This study is the first to demonstrate that a specific subpopulation is at a higher risk for developing idiopathic subglottic stenosis.

Histoid Leprosy Presenting with Keloid Like Lesions.

A 42 year old male presented with multiple, discrete, hyperpigmented, firm, non elastic, non tender papules and plaques on the posterior trunk of 5 months duration, resembling keloid. The patient had also a few skin colored papules on the anterior trunk and face. The sensations over the skin lesions were intact. The patient had glove and stocking type of anesthesia and bilaterally thickened, non tender peripheral nerve trunks. The slit skin smear for acid fast bacilli from the ear lobes, skin lesions and normal skin were highly positive for Mycobacterium leprae. A skin biopsy showed a well defined collection of spindle shaped histiocytes in the dermis packed with acid fast bacilli. We are presenting here a case of histoid leprosy presenting with keloid like lesions, probably the rarest presentation of histoid leprosy.

Resimulation of noise: a precision estimator for least square error curve-fitting tested for axial strain time constant imaging.

Recent elastography techniques focus on imaging information on properties of materials which can be modeled as viscoelastic or poroelastic. These techniques often require the fitting of temporal strain data, acquired from either a creep or stress-relaxation experiment to a mathematical model using least square error (LSE) parameter estimation. It is known that the strain versus time relationships for tissues undergoing creep compression have a non-linear relationship. In non-linear cases, devising a measure of estimate reliability can be challenging. In this article, we have developed and tested a method to provide non linear LSE parameter estimate reliability: which we called Resimulation of Noise (RoN). RoN provides a measure of reliability by estimating the spread of parameter estimates from a single experiment realization. We have tested RoN specifically for the case of axial strain time constant parameter estimation in poroelastic media. Our tests show that the RoN estimated precision has a linear relationship to the actual precision of the LSE estimator. We have also compared results from the RoN derived measure of reliability against a commonly used reliability measure: the correlation coefficient (CorrCoeff). Our results show that CorrCoeff is a poor measure of estimate reliability for non-linear LSE parameter estimation. While the RoN is specifically tested only for axial strain time constant imaging, a general algorithm is provided for use in all LSE parameter estimation.

A robust spike and wave algorithm for detecting seizures in a genetic absence seizure model.

Animal Models are used extensively in basic epilepsy research. In many studies, there is a need to accurately score and quantify all epileptic spike and wave discharges (SWDs) as captured by electroencephalographic (EEG) recordings. Manual scoring of long term EEG recordings is a time-consuming and tedious task that requires inordinate amount of time of laboratory personnel and an experienced electroencephalographer. In this paper, we adapt a SWD detection algorithm, originally proposed by the authors for absence (petit mal) seizure detection in humans, to detect SWDs appearing in EEG recordings of Fischer 334 rats. The algorithm is robust with respect to the threshold parameters. Results are compared to manual scoring and the effect of different threshold parameters is discussed.

The inability of a bacteriophage to infect Staphylococcus aureus does not prevent it from specifically delivering a photosensitizer to the bacterium enabling its lethal photosensitization.

OBJECTIVES: It has been demonstrated that the efficiency of lethal photosensitization can be improved by covalently binding photosensitizing agents to bacteriophage. In this study we have investigated whether a bacteriophage requires the capacity to infect the bacterium to enhance lethal photosensitization when linked to a photosensitizer. METHODS: Tin (IV) chlorin e6 (SnCe6) was conjugated to bacteriophage Phi11, a transducing phage that can infect Staphylococcus aureus NCTC 8325-4, but not epidemic methicillin-resistant S. aureus (EMRSA)-16. The conjugate and appropriate controls were incubated with these bacteria and either exposed to laser light at 632.8 nm or kept in the dark. RESULTS: The SnCe6/Phi11 conjugate achieved a statistically significant reduction in the number of viable bacteria of both 8325-4 and EMRSA-16 strains by 2.31 log(10) and 2.63 log(10), respectively. The conjugate could not however instigate lethal photosensitization of Escherichia coli. None of the other combinations of controls, such as an equivalent concentration of SnCe6 only, an equivalent titre of bacteriophage only or experiments conducted without laser light, yielded significant reductions in the number of viable bacteria recovered. CONCLUSIONS: The inability of a bacteriophage to infect S. aureus does not prevent it from specifically delivering a photosensitizer to a bacterium enabling its lethal photosensitization.

Quantum dots as enhancers of the efficacy of bacterial lethal photosensitization.

Because of the increasing resistance of bacteria to antibiotics there is considerable interest in light-activated antimicrobial agents (LAAAs) as alternatives to antibiotics for treating localized infections. The purpose of this study was to determine whether CdSe/ZnS quantum dots (QD) could enhance the antibacterial activity of the LAAA, toluidine blue O (TBO). Suspensions of Staphylococcus aureus and Streptococcus pyogenes were exposed to white light (3600 lux) and TBO (absorbance maximum = 630 nm) in the presence and absence of 25 nm diameter QD (emission maximum = 627 nm). When the TBO:QD ratio was 2667:1, killing of Staph. aureus was enhanced by 1.72log(10) units. In the case of Strep. pyogenes, an enhanced kill of 1.55log(10) units was achieved using TBO and QD in the ratio 267:1. Singlet oxygen and fluorescence measurements showed that QD suppress the formation of singlet oxygen from TBO and that QD fluorescence is significantly quenched in the presence of TBO (70-90%). Enhanced killing appears to be attributable to a non-Förster resonance energy transfer mechanism, whereby the QD converts part of the incident light to the absorption maximum for TBO; hence more light energy is harvested, resulting in increased concentrations of bactericidal radicals. QD may, therefore, be useful in improving the efficacy of antimicrobial photodynamic therapy.

ABSENCE SEIZURES AS RESETTING MECHANISMS OF BRAIN DYNAMICS.

To understand the increase in age-related incidence and frequency of absence seizures in the rat brain, we investigated the effect of these seizures on brain dynamics. This paper puts forward the hypothesis that age-related differences in the expression of absence seizures are associated with the ability of the seizures to reset brain dynamics.

The interaction between staphylococcal superantigen-like proteins and human dendritic cells.

Staphylococcus aureus produce a family of exotoxins (staphylococcal superantigen like proteins, SSLs) with structural, but not functional, homology to superantigens. These proteins have previously been shown to interact selectively with antigen presenting cells, including dendritic cells. The functional consequences of this interaction are now explored. SSL7 and 9 had no effect on viability or morphology of dendritic cells. The proteins did not induce dendritic cell maturation, as measured by cell surface phenotype. Exposure to SSL did not alter the ability of dendritic cells to take up FITC-dextran. Finally, exposure to SSLs did not impair the ability of the dendritic cells to stimulate allogeneic or antigen specific T cell responses. However, dendritic cells loaded with SSL7 or 9 were able to stimulate a T cell proliferative response in 3/8 healthy individuals tested. Sera from nine out of 10 individuals tested contained antibodies against both SSL7 and SSL9, and the response to each SSL was specific and not cross-reactive. The results demonstrate that SSLs are immunogenic in humans at both the B and T cell level, but it remains unclear whether this response is to the benefit of the bacterium or the host.

Protective immune response against foot-and-mouth disease virus challenge in guinea pigs vaccinated with recombinant P1 polyprotein expressed in Pichia pastoris.

Vaccination of the susceptible livestock with potent, safe and cost effective vaccine is the primary requirement to control foot-and-mouth disease (FMD) in an endemic country. In this study, an alternative approach was used in which structural protein genes of all the four serotypes of FMDV (O, Asia 1, A22 and C) were expressed separately in methylotrophic yeast Pichia pastoris. The recombinant polyproteins (P1) were characterized by SDS-PAGE and in Western Blot analysis. Partially purified protein was used for immunization in guinea pigs with different adjuvant formulations and immune response studied. Ninety micrograms of the recombinant protein per monovalent dose was used for immunization. A single injection of a monovalent or polyvalent vaccine was given to guinea pigs with various adjuvant combinations viz., Monovalent recombinant protein either adjuvanted with Montanide-ISA50V or Indigenous oil, Monovalent recombinant protein mixed with 1/10th dose of inactivated oil-adjuvanted virus vaccine and Polyvalent recombinant protein with Montanide ISA50V. FMDV specific humoral immune response was observed at about 28th day post vaccination. The immune response as assessed by indirect ELISA and Serum neutralization test titres was found to be 320-640 and 16-32, respectively. When challenged with virulent homologous type 'O' virus, the guinea pigs showed protective C index of 2.01,1.81, 2.56 and 2.48, respectively, with above said adjuvant combinations. The study has shown that yeast-expressed FMDV P1 polyprotein in a single dose could elicit a protective immune response in guinea pigs, and this could be a possible future vaccine candidate in homologous host.

Clinico-epidemiological study of HIV patients in Trivandrum.

A retrospective descriptive study of 121 HIV patients in the Department was carried out. The male/female ratio was 2.3:1. The maximum number of patients were seen in age group 21-40 (77.68%). Skilled workers constituted the maximum (13.2%). Sexual route was the commonest mode of transmission (78.5%). Cutaneous manifestations were present in 57% of patients, oral condidiasis being the commonest (16.5%). Pulmonary tuberculosis was the commonest systemic manifestation (13.2%). 37 patients (30.57%) had other STD's, syphilis being the commonest (12.39%). 22 patients had AIDS defining conditions.

Expression of the S. aureus hysA gene in S. carnosus from a modified E. coli-staphylococcal shuttle vector.

We have modified an E. coli-staphylococcal shuttle vector for use in the general cloning and expression of genes from pathogenic staphylococci in Staphylococcus carnosus. As S. carnosus is non-pathogenic, this expression system will facilitate the study of the roles of individual gene products in the disease process. To evaluate the use of this expression system, a DNA fragment containing the Staphylococcus aureus hyaluronate lyase (hysA) gene was cloned into the modified vector, pNW21, and introduced into S. carnosus. Hyaluronate lyase was both produced and secreted by S. carnosus. In addition, the secreted HysA protein was enzymatically active, as determined using a zymographic assay.

Staphylococcus aureus fibronectin binding proteins A and B possess a second fibronectin binding region that may have biological relevance to bone tissues.

Staphylococcus aureus is a major human pathogen that has a propensity for targeting to bone tissues and thereby causing bone disease. A plausible hypothesis is that S. aureus targets to bone using the MSCRAMM family of surface proteins possessed by this organism. Two such proteins that have recently been shown to be important in bone infections are the S. aureus fibronectin binding proteins (FnBP) A and B. To identify fibronectin-binding domains from S. aureus that have biological relevance to bone, a phage display library of S. aureus genomic DNA was constructed and panned sequentially against immobilized fibronectin and cultured osteoblasts. Using this system, phage displaying a second fibronectin-binding region within the N-terminal part of FnBPA and FnBPB, which is distinct from the primary fibronectin-binding domain located within the D repeat region of these proteins, was isolated. Phage displaying this second region bound to both immobilized fibronectin and to osteoblasts and/or the extracellular matrix synthesized by these cells, thereby suggesting a biological relevance for these regions in S. aureus binding to bone tissues. Analysis of these binding regions for their ability to bind to other extracellular matrix proteins revealed a preference for fibronectin, with slight binding to fibrinogen and no binding to collagen or laminin.

Early antibody responses of cattle for foot-and-mouth disease quadrivalent double oil emulsion vaccine.

Foot-and-mouth disease (FMD) is an economically important disease of cloven-hoofed animals. The multiplicity of FMDV serotypes in animals poses a central problem in the policy of vaccination and is of much concern to health authorities. Hence it is the practice of vaccination with polyvalent vaccine for prophylactic measure. In the present report, we analysed the early antibody responses elicited by FMDV quadrivalent (FMDV O, A, C and Asia 1 serotypes) double emulsion (Montanide ISA 206) vaccines in cattle. We observed variations between various viral serotypes in eliciting early antibody response although neutralizing antibody response against all the four serotypes were detected as early as fourth day following vaccination. The duration of immunity also appeared to maintain for long period. The neutralizing antibody titres were maintained well above 2log(10) even after 6 months of vaccination irrespective of serotypes. Thus, allows the possibilities of two vaccinations per year for the maintenance of herd immunity.

Recombinant Actinobacillus actinomycetemcomitans cytolethal distending toxin proteins are required to interact to inhibit human cell cycle progression and to stimulate human leukocyte cytokine synthesis.

It has recently been discovered that Actinobacillus actinomycetemcomitans, an oral bacterium causing periodontitis, produces cytolethal distending toxin (CDT), a cell cycle-modulating toxin that has three protein subunits: CdtA, CdtB, and CdtC. In this study, we have cloned and expressed each toxin gene from A. actinomycetemcomitans in Escherichia coli and purified the recombinant Cdt proteins to homogeneity. Individual Cdt proteins failed to induce cell cycle arrest of the human epithelial cell line HEp-2. The only combinations of toxin proteins causing cell cycle arrest were the presence of all three Cdt proteins and the combination of CdtB and CdtC. A similar experimental protocol was used to determine if recombinant Cdt proteins were able to induce human peripheral blood mononuclear cells (PBMCs) to produce cytokines. The individual Cdt proteins were able to induce the synthesis by PBMCs of interleukin-1beta (IL-1beta), IL-6, and IL-8 but not of tumor necrosis factor alpha, IL-12, or granulocyte-macrophage colony-stimulating factor, with CdtC being the most potent and CdtB being the least potent cytokine inducer. There was evidence of synergism between these Cdt proteins in the stimulation of cytokine production, most markedly with gamma interferon, which required the minimum interaction of CdtB and -C to stimulate production.

Staphylococcus aureus accessory regulators: expression within biofilms and effect on adhesion.

Many of the genes encoding the virulence factors for Staphylococcus aureus are controlled by the accessory gene regulator (agr) and staphylococcal accessory regulator (sar). This regulation may be affected by the environment in which the organisms are grown. In the majority of ecosystems, bacteria grow attached to surfaces and form biofilms. We used S. aureus strains containing mutations inactivating agr and sar to determine whether the presence of these genes influences the attachment of the bacterium to a surface. We also used strains harbouring reporter constructs of the agr and sar operons to determine their expression in biofilms. The attachment study results showed that the sarA mutant strain adhered better to glass than did the agrA mutant or the wild type. There was an increased adherence to fibronectin-coated glass for all three strains compared to glass. Thus, these adhesion studies demonstrate that agr and sar have pleiotrophic effects on the surface expression of molecules responsible for binding to different substrata. In the biofilms higher numbers of bacteria and the greatest expression were observed at the base, but there were no observable differences between the reporter constructs. Expression of the agr and sar reporter fusions was significantly higher in the deepest layers of the biofilms where the greatest numbers of bacteria were also observed, perhaps as one might expect for genes that are regulated in a cell density dependent fashion.

Identification of photolabile outer membrane proteins of Porphyromonas gingivalis.

As the prevalence of antibiotic-resistant strains of bacteria increases, novel ways of treating infections need to be developed. This is particularly pertinent with respect to the periodontal diseases--the most common chronic bacterial infections of man. The use of a photosensitizer in combination with red light has been demonstrated to be effective in killing several human pathogens, including the oral bacterium, Porphyromonas gingivalis, a major pathogen in periodontitis. Killing was associated with alterations in the molecular masses of several outer membrane and plasma membrane proteins and these may be therapeutic targets for photodynamic therapy and other antimicrobial approaches. To identify these photolabile proteins, we have used a panel of monoclonal antibodies raised to whole P. gingivalis. A number of the antibodies recognized various photolabile proteins. Using a combination of Western blotting and protein sequencing the predominant photolabile proteins in P. gingivalis have been identified as the major secreted/cell surface proteases--Lys and Arg gingipain.

Analysis of the effect of changing environmental conditions on the expression patterns of exported surface-associated proteins of the oral pathogen Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans has been specifically implicated in the aetiology of one or more of the periodontal diseases, conditions in which inflammation of the gums is associated with destruction of the alveolar bone supporting the teeth. In these diseases there is loss of attachment of the gums (gingivae) to the teeth forming a periodontal pocket. The microenvironment of this pocket is extremely complex and it is likely that there will be substantial variation in the environmental conditions operating in this habitat. The aim of the current investigation was to study the effect of disease-relevant environmental factors on the production and release of secreted surface- associated proteins of A. actinomycetemcomitans. These secreted proteins contain many of the virulence determinants of this organism. A range of environmental conditions were investigated: growth in a CO(2)-enriched aerobic atmosphere vs anaerobic growth, presence of serum or blood, biofilm vs planktonic mode of growth and iron depletion. Differential expression of a number of the secreted surface-associated proteins was observed under different growth conditions and these included the glycolytic enzyme triose phosphate isomerase. An ability to adapt to prevailing environmental conditions may facilitate the survival of the organism in the changing microIenvironment of the periodontal pocket.

Staphylococcus aureus fibronectin binding proteins are essential for internalization by osteoblasts but do not account for differences in intracellular levels of bacteria.

Staphylococcus aureus is a major pathogen of bone that has been shown to be internalized by osteoblasts via a receptor-mediated pathway. Here we report that there are strain-dependent differences in the uptake of S. aureus by osteoblasts. An S. aureus septic arthritis isolate, LS-1, was internalized some 10-fold more than the laboratory strain 8325-4. Disruption of the genes for the fibronectin binding proteins in these two strains of S. aureus blocked their ability to be internalized by osteoblasts, thereby demonstrating the essentiality of these genes in this process. However, there were no differences in the capacity of these two strains to bind to fibronectin or osteoblasts. Analysis of the kinetics of internalization of the two strains by osteoblasts revealed that strain 8325-4 was internalized only over a short period of time (2 h) and to low numbers, while LS-1 was taken up by osteoblasts in large numbers for over 3 h. These differences in the kinetics of uptake explain the fact that the two strains of S. aureus are internalized by osteoblasts to different extents and suggest that in addition to the fibronectin binding proteins there are other, as yet undetermined virulence factors that play a role in the internalization process.

Identification of the exported proteins of the oral opportunistic pathogen Actinobacillus actinomycetemcomitans by using alkaline phosphatase fusions.

A phoA fusion library of Actinobacillus actinomycetemcomitans genomic DNA has been screened to identify genes encoding exported and secreted proteins. A total of 8,000 colonies were screened, and 80 positive colonies were detected. From these, 48 genes were identified with (i) more than half having homology to known or hypothetical Haemophilus influenzae genes, (ii) 14 having no ascribed function, and (iii) 4 having very limited or no homology to known genes. The proteins encoded by these genes may, by virtue of their presence on the cell surface, be novel virulence determinants.