Characterization of Fusobacterium necrophorum subsp. necrophorum outer membrane proteins.

Liver abscesses are of major economic importance to the cattle industry. These are mainly associated with the presence of Fusobacterium necrophorum, a non-spore forming and Gram-negative anaerobe. There are two main subspecies, F. necrophorum subspecies necrophorum and subsp. funduliforme, and they differ molecularly, morphologically, biochemically and in virulence. Previous studies have shown that the outer membrane proteins (OMP) of F. necrophorum subsp. necrophorum are important for its successful binding to immobilized bovine adrenal gland capillary endothelial (EJG) cells. In this study, a 42.4 kDa OMP of F. necrophorum subsp. necrophorum with the highest binding capacity to EJG cells was characterized. The gene was cloned into pFLAG-CTS vector and the proteins were subsequently expressed on the surface of E. coli BL21 DE3 cells. When E. coli carrying the recombinant plasmid (SM 2013) was induced using IPTG, there was significant enhancement in the binding to immobilized EJG cells compared to both uninduced SM 2013 and the E. coli carrying control vector only. When fixed EJG cells were incubated with purified native OMP, SM 2013 showed lowered levels of binding, compared to the uninduced SM 2013 and the E. coli carrying control vector only. Pre-incubation of induced SM 2013 with polyclonal antibodies made against the OMP reduced the binding to immobilized EJG cells to uninduced SM 2013 levels. This gain of function by recombinant E. coli confirms the ability of this protein to act as an adhesion to help binding of F. necrophorum subsp. necrophorum to host cells.

Conditional reprogramming of pediatric airway epithelial cells: A new human model to investigate early-life respiratory disorders.

BACKGROUND: Airway epithelial cells (AEC) are quite difficult to access in newborns and infants. It is critically important to develop robust life-extended models to conduct translational studies in this age group. We propose the use of a recently described cell culture technology (conditionally reprogrammed cells-CRC) to generate continuous primary cell cultures from nasal and bronchial AEC of young children. METHODS: We collected nasal and/or bronchial AEC from a total of 23 subjects of different ages including newborns/infants/toddlers (0-2 years; N = 9), school-age children (4-11 years; N = 6), and a group of adolescent/adult donors (N = 8). For CRC generation, we used conditioned medium from mitotically inactivated 3T3 fibroblasts and Rho-associated kinase (ROCK) inhibitor (Y-27632). Antiviral immune responses were studied using 25 key antiviral genes and protein production of type III epithelial interferon (IFN λ1) after double-stranded (ds) RNA exposure. RESULTS: CRC derived from primary AEC of neonates/infants and young children exhibited: (i) augmented proliferative capacity and life extension, (ii) preserved airway epithelial phenotype after multiple passages, (iii) robust immune responses characterized by the expression of innate antiviral genes and parallel nasal/bronchial production of IFN λ1 after exposure to dsRNA, and (iv) induction of airway epithelial inflammatory and remodeling responses to dsRNA (eg, CXCL8 and MMP9). CONCLUSION: Conditional reprogramming of AEC from young children is a feasible and powerful translational approach to investigate early-life airway epithelial immune responses in humans.

Multicenter evaluation of the BD max enteric bacterial panel PCR assay for rapid detection of Salmonella spp., Shigella spp., Campylobacter spp. (C. jejuni and C. coli), and Shiga toxin 1 and 2 genes.

Diarrhea due to enteric bacterial pathogens causes significant morbidity and mortality in the United States and worldwide. However, bacterial pathogens may be infrequently identified. Currently, culture and enzyme immunoassays (EIAs) are the primary methods used by clinical laboratories to detect enteric bacterial pathogens. We conducted a multicenter evaluation of the BD Max enteric bacterial panel (EBP) PCR assay in comparison to culture for the detection of Salmonella spp., Shigella spp., Campylobacter jejuni, and Campylobacter coli and an EIA for Shiga toxins 1 and 2. A total of 4,242 preserved or unpreserved stool specimens, including 3,457 specimens collected prospectively and 785 frozen, retrospective samples, were evaluated. Compared to culture or EIA, the positive percent agreement (PPA) and negative percent agreement (NPA) values for the BD Max EBP assay for all specimens combined were as follows: 97.1% and 99.2% for Salmonella spp., 99.1% and 99.7% for Shigella spp., 97.2% and 98.4% for C. jejuni and C. coli, and 97.4% and 99.3% for Shiga toxins, respectively. Discrepant results for prospective samples were resolved with alternate PCR assays and bidirectional sequencing of amplicons. Following discrepant analysis, PPA and NPA values were as follows: 97.3% and 99.8% for Salmonella spp., 99.2% and 100% for Shigella spp., 97.5% and 99.0% for C. jejuni and C. coli, and 100% and 99.7% for Shiga toxins, respectively. No differences in detection were observed for samples preserved in Cary-Blair medium and unpreserved samples. In this large, multicenter study, the BD Max EBP assay showed superior sensitivity compared to conventional methods and excellent specificity for the detection of enteric bacterial pathogens in stool specimens.

Outcome of Treatment in Locally Advanced Upper Alveolar Carcinoma Extending to Infra-temporal Fossa.

Squamous cell carcinoma of oral cavity has a high prevalence in our region. Upper alveolar cancers are uncommon but present with locally advanced disease extending to infra temporal fossa. The outcome of treatment in these tumors is poor. Surgery followed by adjuvant therapy is the mainstay of treatment. We are presenting a retrospective analysis of outcome of treatment in 20 patients with locally advanced (T4b) upper alveolar carcinoma treated by infra-structure maxillectomy with compartment resection of infra-temporal fossa and neck dissection followed by adjuvant radiotherapy or radiotherapy with chemotherapy. Study period was from 2013 to 2018. Minimum follow up was 12 months and mean follow up 30 months. 12 patients are alive and disease free, 6 patients had local recurrence and 2 patients had regional recurrence with one having pulmonary metastasis. We observed that positive or close margins (< 5 mm after formalin fixation) predisposed to early recurrence. Erosion of pterygoid plates was a poor prognostic factor. Cervical lymph node metastasis occurred late in disease, but carried poor prognosis. Compartment resection of infra temporal fossa gave better outcome compared to other studies which reported outcome of treatment in upper alveolar cancers.

Outcome of Treatment in Verrucous Carcinoma of Oral Cavity: A Tertiary Rural Hospital Experience.

Verrucous carcinoma of oral cavity is a highly well differentiated variant of squamous cell carcinoma with a low potential for invasion and metastasis. It is prevalent in the tobacco quid chewing population in our region. In this observational study, we reviewed the medical case records of 58 patients treated for oral verrucous carcinoma staged T2 to T4a. All patients underwent wide excision of tumour which included marginal mandibulectomy in 22 and hemimandibulectomy in 23 patients along with neck dissection saving the accessory nerve and internal jugular vein. 5 patients were found to have bone involvement along the alveolar sockets. 11 patients had other associated premalignant lesions in oral cavity. Only 2 patients had lymph node metastasis without extra nodal spread in submandibular region. With a mean follow up of 6 years and minimum follow up of 1 year, 3 patients had local recurrence. All these 3 patients had bone involvement and 2 of them had lymph node metastasis on histopathological examination. 3 patients who had associated premalignant lesions developed second primary in oral cavity after 3 years. In our experience, verrucous carcinoma has good prognosis when treated by surgery. Bone involvement along alveolar sockets and associated oral premalignant lesions adversely affect the outcome. There was no difference in the outcome between selective and modified radical neck dissection. Therefore selective neck dissection (supraomohyoid) would be adequate in treating these patients. Adjuvant radiotherapy can be reserved for T4a lesions or for positive margins.

Impact of isolation precautions on quality of life: a meta-analysis.

Impact of isolation precautions on psychological wellbeing of patients has yet to be fully quantified. To assess the impact of isolation precautions on patients' health-related quality of life and depression or anxiety scales and estimate per day cost of anxiety and depression. Literature pertaining to impact of isolation precautions was searched on EMBASE and PubMed databases and Google Scholar. A two-step independent screening of the articles was performed. Articles that compared isolated and non-isolated patients using different quality of life and psychological burden scales were included. A meta-analysis was conducted using the Hospital Anxiety and Depression Scales (HADS-A and HADS-D). Psychological burden measures from selected literature were presented in a graph as effect sizes. Per day cost of anxiety and depression was estimated using pooled mean difference from meta-analysis. Out of 106 articles, 94 were excluded due to inclusion criteria, leaving 12 for full text review. After review of full text of the articles, seven articles were shortlisted for empirical analysis and four out of these seven for meta-analysis. The pooled mean difference estimates for HADS-A was -1.4 (P=0.15) and that for HADS-D was -1.85 (P=0.09). In the empirical analysis of psychological burden scales, the effect in all studies except one was negative. Results from meta-analysis and empirical analysis of psychological burden implied that isolated patients are worse off in general. The implied estimated per day cost of anxiety and depression in terms of quality-adjusted life years (QALYs) is approximately US$10.

Lethal Midline Granuloma: An Enigma in Diagnosis-A Rare Case Report.

Lethal midline granuloma is a rare disease, characterised by progressive unrelenting necrosis and mutilation of nose, midline facial tissues and other respiratory passages. This particular type of disease is heterogeneous in its pathogenesis, non-specificity of symptoms obscures timely and correct diagnosis and is responsible for delay in of treatment which can be detrimental as this disease calls for immediate intervention. We present a case report of 60 year old female who gave short one-month history of clinical symptoms.

A new twist on an old problem: a case of pediatric meningitis caused by multidrug-resistant Streptococcus pneumoniae serotype 19A.

Recent studies show that multidrug-resistant Streptococcus pneumoniae serotype 19A continues to emerge as a cause of invasive pneumococcal disease after the introduction of Prevnar. We report a case of multidrug-resistant S. pneumoniae serotype 19A meningitis successfully treated with vancomycin and levofloxacin. This case reinforces the need for the empiric use of vancomycin in meningitis and the need for alternative treatments.

Pelvic Ewing's sarcoma: a review from Scottish Bone Tumour Registry.

PURPOSE: To review various treatment modalities on outcome of patients with pelvic Ewing's sarcoma. METHODS: Between January 1948 and December 2004, 18 male and 15 female patients aged 3 to 48 (mean, 20) years with pelvic Ewing's sarcoma were retrospectively reviewed from the Scottish Bone Tumour Registry. RESULTS: The commonest site involved was the ilium. The main presenting symptom was pain, followed by swelling and restricted hip movements. Treatments included surgery, radiotherapy, chemotherapy, or any of them in combination. Patients who had no metastasis at presentation (p<0.01) and underwent chemotherapy with ifosfamide (p<0.01) had significantly longer survival. Age (p=0.09), gender (p=0.95), delay in presentation (p=0.31), tumour site (p=0.9), surgery (p=0.73), and radiotherapy (p=0.23) were not predictive of survival in the univariate analysis. The 5-year event-free survival rate in patients with no metastasis at presentation was 35%. CONCLUSION: Intense multi-agent neo-adjuvant chemotherapy including ifosfamide followed by wide excision and postoperative radiation are recommended. Local therapy should not take precedence over or interfere with systemic chemotherapy.

Endotypes of difficult-to-control asthma in inner-city African American children.

African Americans have higher rates of asthma prevalence, morbidity, and mortality in comparison with other racial groups. We sought to characterize endotypes of childhood asthma severity in African American patients in an inner-city pediatric asthma population. Baseline blood neutrophils, blood eosinophils, and 38 serum cytokine levels were measured in a sample of 235 asthmatic children (6-17 years) enrolled in the NIAID (National Institute of Allergy and Infectious Diseases)-sponsored Asthma Phenotypes in the Inner City (APIC) study (ICAC (Inner City Asthma Consortium)-19). Cytokines were quantified using a MILLIPLEX panel and analyzed on a Luminex analyzer. Patients were classified as Easy-to-Control or Difficult-to-Control based on the required dose of controller medications over one year of prospective management. A multivariate variable selection procedure was used to select cytokines associated with Difficult-to-Control versus Easy-to-Control asthma, adjusting for age, sex, blood eosinophils, and blood neutrophils. In inner-city African American children, 12 cytokines were significant predictors of Difficult-to-Control asthma (n = 235). CXCL-1, IL-5, IL-8, and IL-17A were positively associated with Difficult-to-Control asthma, while IL-4 and IL-13 were positively associated with Easy-to-Control asthma. Using likelihood ratio testing, it was observed that in addition to blood eosinophils and neutrophils, serum cytokines improved the fit of the model. In an inner-city pediatric population, serum cytokines significantly contributed to the definition of Difficult-to-Control asthma endotypes in African American children. Mixed responses characterized by TH2 (IL-5) and TH17-associated cytokines were associated with Difficult-to-Control asthma. Collectively, these data may contribute to risk stratification of Difficult-to-Control asthma in the African American population.

Comparison of Mannheimia haemolytica isolates from an outbreak of bovine respiratory disease.

The objective of this study was to determine the clonal relatedness of Mannheimia haemolytica isolates responsible for an outbreak of bovine respiratory disease in a commercial feedlot. The isolates were obtained from the lungs of 21 calves with fatal pneumonia that were part of a group of 206 total calves. All isolates were serotyped and analyzed by pulsed-field gel electrophoresis (PFGE) and for antibiotic sensitivity patterns. ELISA and immunoblotting assays were performed to compare serum antibody levels to M. haemolytica antigens in calves with fatal pneumonia to those calves that survived the outbreak. Isolates were categorized into 14 different PFGE groups based on 90% similarity. Two Group D isolates (1 and 6), and 3 Group H isolates (14, 15, and 16) were characterized as 100% similar. Antimicrobial susceptibility profiles defined 8 groups based on differences in patterns of resistance between isolates. The two 100% similar isolates from PFGE Group D were both in susceptibility Group 1. All but isolate 14 from PFGE Group H (3, 15, 16, and 19) were in susceptibility Group 4a. Serum antibody levels to M. haemolytica antigens in the dead calves were not different than the antibody levels in the 185 calves that survived the outbreak. Immunoblots of selected isolates from each of the PFGE groups demonstrated only minimal differences in antigenic profiles between strains when reacted with serum from calves that either died from or survived the outbreak. Based on the characteristics of these isolates, multiple strains of M. haemolytica were responsible for fatal pneumonia during this outbreak.

Production of the beta-subunit of human chorionic gonadotropin in Escherichia coli and its export mediated by the heat-labile enterotoxin chain-B signal sequence.

The PCR-amplified beta-subunit of the human chorionic gonadotropin structural gene (betahCG) was cloned under the control of the tac promoter and the heat-labile enterotoxin chain B (LTB) signal sequence (LTBss). BetahCG was successfully produced, processed and exported to the periplasmic space in Escherichia coli. Expression of betahCG was confirmed by immunoblot analysis using an anti-betahCG polyclonal antibody. The processing of the protein was very efficient, as only the processed band could be detected at all time points during the course of induction. Expression was evident soon after the addition of the lactose analogue, IPTG. These results demonstrate that E. coli cells can synthesize, process and export betahCG using the LTBss.

High-level production and one-step purification of biologically active human growth hormone in Escherichia coli.

A plasmid has been constructed to direct the synthesis of recombinant human growth hormone (re-hGH) in Escherichia coli as a fusion protein containing a His6 tag at the N-terminus under the control of the T5 promoter. The re-hGH was synthesized in large amounts and accumulated in the form of inclusion bodies upon induction with IPTG. Inclusion bodies were solubilized in 6 M guanidine.HCl and the re-hGH was purified by single-step affinity chromatography on Ni(2+)-nitrilotriacetic acid (NTA) agarose. At the shake flask level, the purified re-hGH was obtained with a yield of 30 mg/l of culture. The re-hGH was biologically active in a node rat lymphoma (Nb2) cell bioassay.

Translational fusion of heat labile enterotoxin chain B and beta-subunit of human chorionic gonadotropin: periplasmic expression in Escherichia coli and its immunogenicity.

A fusion gene was constructed consisting of heat labile enterotoxin chain B (LTB) of E. coli genetically linked at its C-terminus to the beta-subunit of human chorionic gonadotropin in translational fusion, under the control of tac promoter and LTB signal sequence. Expression of the fusion gene (about 5 microgram/ml) in E. coli was confirmed by immunoblot analysis using both anti-LTB and anti-betahCG polyclonal antibodies. The fusion protein was efficiently processed and exported to the periplasmic space. LTB in the fusion protein retained its ability to bind to GM1 ganglioside receptor. Mice immunized with the fusion protein produce antibodies that recognize recombinant betahCG and the native hCG suggesting its potential use as a contraceptive vaccine.

Recent developments in amoebiasis:the Gal/GalNAc lectins of Entamoeba histolytica and Entamoeba dispar.

Amoebiasis is responsible for 50000-100000 deaths annually. Invasive amoebic disease begins with the attachment of Entamoeba histolytica trophozoites to colonic mucin, a process mediated by the amoebic Gal/GalNAc lectin. The non-pathogenic counterpart, E. dispar, is morphologically identical but genetically distinct. Investigations comparing the Gal/GalNac lectin from these two organisms are under way.

Effects of carbidopa and of intravenous saline infusion into normal and hypertensive subjects on urinary free and conjugated dopamine.

OBJECTIVE: To investigate the possible role played by endogenous dopamine as a modulator of renal sodium (Na+) reabsorption after a combined Na+ and volume load. DESIGN: A randomized placebo-controlled study. METHODS: Ten healthy volunteers and four hypertensive patients were subjected to intravenous infusions of 21 0.9% saline (308 mmol Na+) administered from 1000 to 1300 h after oral administration of placebo or of carbidopa, a dopamine decarboxylase inhibitor. RESULTS: Studies on control subjects after placebo showed that natriuresis occurred during the 6 h after commencement of the saline infusion, with falls in plasma albumin concentration, plasma renin activity and plasma aldosterone concentration; in comparison with results of mock infusion (6 mmol Na+) there was no change in the urinary excretion of dopamine and noradrenaline (In their free or conjugated forms). There was, however, a marked surge in excretion of urinary conjugated dopamine and in the dopamine: noradrenaline ratio from 1300 to 1600 h, after either type of infusion. Administration of carbidopa before the saline infusion resulted in a marked decrease in excretion of urinary free dopamine, but had no effect on the surge in excretion of urinary conjugated dopamine. Saline infusion decreased proximal fractional Na+ reabsorption. Administration of carbidopa delayed but did not prevent this decrease. The effects of saline infusion and of carbidopa on the urinary excretion of dopamine and noradrenaline from hypertensive patients were similar to those observed with the healthy volunteers. CONCLUSIONS: These findings indicate that volume expansion by intravenous saline infusion has no appreciable effect on the urinary free dopamine excretion from normal or hypertensive humans; with any apparent increase, it is important to exclude the possibility of conversion of conjugates to free dopamine in vitro. Furthermore, that carbidopa administration did not inhibit the afternoon surge of conjugated dopamine suggests that administration of carbidopa is deficient as a tool to investigate the functional role of the renal dopamine system.

The "lung shift" in treated schistosomiasis. Bronchoalveolar lavage evidence of eosinophilic pneumonia.

There is conflicting and incomplete information in the literature on the pulmonary reaction which can occur following treatment of schistosomiasis. We examined the pulmonary function, bronchoalveolar lavage profile, and lung histopathology of a patient with pneumonia and peripheral eosinophilia following oxaminquine chemotherapy for intestinal Schistosoma mansoni infection. Spirometry revealed restrictive rather than obstructive impairment, and lavage showed eosinophil prominence which was also seen in the interstitial and alveolar-filling process in transbronchial biopsies.

Parasight F test compared with the polymerase chain reaction and microscopy for the diagnosis of Plasmodium falciparum malaria in travelers.

Imported malaria is an increasing problem worldwide. A rapid and accurate test for Plasmodium falciparum infection would facilitate the diagnosis of malaria in the returned traveler. The ParaSight F antigen capture assay (dipstick test) is a new diagnostic test for P. falciparum based on detection of circulating histidine-rich protein-2 antigen. We performed a blinded evaluation of this assay compared with microscopy and the polymerase chain reaction (PCR) for the detection of P. falciparum infection in 151 febrile travelers. Compared with the PCR, the dipstick test had a sensitivity of 88% and a specificity of 97%. The ability of the dipstick test to detect P. falciparum was similar with that of microscopy (88% versus 83%) since the species of Plasmodium in 14 of 133 malaria-infected patients could not be determined by microscopy due to low parasite numbers. The dipstick test was 40% sensitive for infections with < 50 parasites/microliter, 89% with 50-100 parasites/microliter, and > or = 93% with > 100 parasites/microliter. Circulating antigen was detectable in 68% of the patients seven days after initiation of treatment and in 27% at day 28. The dipstick test represents a simple and accurate test for the diagnosis of P. falciparum infection in the returned traveler.

Quality assurance for biogenic amines with authentic patient specimens.

The measurement of biogenic amines and metabolites is essential for diagnosis and follow-up of neural crest tumors. A Quality Assurance programme involving the distribution of urine specimens obtained from patients with neural crest and related tumors was conducted by the Australian Association of Clinical Biochemists' Working Party on Biogenic Amines in 1988. Fifty laboratories participated in the programme and measured a number of analytes with a variety of methods. These included high performance liquid chromatography with electrochemical detection (HPLC-EC), spectrophotometry, fluorescence, fluorescence polarization immunoassay (Abbott TDX) and gas chromatography--mass spectroscopy (GC-MS). The results of this survey indicated that fluorimetric methods for catecholamines are unreliable as they are subject to interference particularly by Labetalol. Twice as many laboratories utilized catecholamines rather than metanephrines for detection of pheochromocytoma. 5-HIAA appears to be the analyte measured with the least accuracy, particularly with spectrophotometric methods of analysis. Several laboratories would have failed to diagnose some neural crest tumors and need to improve their performance.

Alpha 1-antitrypsin serum levels in widespread bronchiectasis.

This study found significantly higher serum levels of alpha 1-antitrypsin (alpha 1-AT) in 35 patients with widespread bronchiectasis compared with healthy controls. The results are in contradiction with a previous study and four case reports which found an association between alpha 1-AT deficiency and bronchiectasis. However, the validity of the association has been questioned in the literature on the grounds that most of the patients in those reports had other possible aetiologies for their bronchiectasis. We believe the raised level of alpha 1-AT in our patients represents a non-specific acute phase response to bronchial infection. Further studies are needed to clarify whether bronchiectasis, like emphysema, can be a manifestation of alpha 1-AT deficiency.