A novel enzyme-linked immunosorbent assay method for the detection of human neutrophil antigen-2a antibodies

BACKGROUND: Antibodies to human neutrophil antigen (HNA)-2a are responsible for a number of immune-mediated neutropenia disorders. Although several methods exist for the identification of anti-HNA-2a, all these methods have several limitations. In this study, a solid-phase enzyme-linked immunosorbent assay (ELISA) using recombinant HNA-2a antigen (rHNA-2a) allowing rapid detection of HNA-2a antibodies was developed.
STUDY DESIGN AND METHODS: Soluble rHNA-2 was generated by transfection of insect cells with CD177 vector. Purified rHNA-2a was immobilized on microtiter wells coated with anti-CD177 and was applied to analyze 10 sera containing HNA-2a antibodies. For the evaluation of the ELISA method, results were compared with the standard assay, MAIGA (monoclonal antibody antigen capture assay) for detection of neutrophil antibodies.
RESULTS: The specificity of HNA-2a antibodies in all sera was confirmed by immunoblotting. Sera were then tested simultaneously in ELISA and MAIGA assays. Nine of 10 sera showed positive reactions in ELISA, whereas only 9 of 10 sera reacted in the standard MAIGA assay. All HNA-2a antibodies were detectable in MAIGA when diluted sera were applied. No reaction was observed with different sera containing neutrophil-reactive antibodies (6 anti-HNA-1a, 4 anti-HNA-1b, and 20 anti-HLA Class I and II) in ELISA. All HNA-2a antibodies were detectable in MAIGA when diluted sera were applied. Notably, sera containing anti-proteinase 3 (PR3) from patients with Wegener's granulomatosis reacted in MAIGA. In contrast, this antibody showed no reaction in ELISA with purified rHNA-2a.
CONCLUSIONS: These results demonstrated that ELISA with rHNA-2a provides a good method for detecting HNA-2a antibodies in human serum. This assay enables to exclude the presence of autoantibody against PR3 in patient's sera, which cannot be differentiated from anti HNA-2a with current serologic methods.     

Relationship Between Serum Macrophage Migration Inhibitory Factor Level and Insulin Resistance,High-Sensitivity C-Reactive Protein and Visceral Fat Mass in Prediabetes

Background

Growing evidence suggest that macrophage migration inhibitory factor (MIF) plays a vital role in glucose metabolism. We aimed to ascertain whether MIF levels are altered in subjects with prediabetes and also to determine the relationship between MIF and metabolic parameters as well as visceral fat mass.

Urinary tract infections following radical cystectomy and urinary diversion: a review of 1133 patients

Objective

To investigate the incidence and microbiology of urinary tract infection (UTI) within 90 days following radical cystectomy (RC) and urinary diversion.

Methods

We reviewed 1133 patients who underwent RC for bladder cancer at our institution between 2003 and 2013; 815 patients (72%) underwent orthotopic diversion, 274 (24%) ileal conduit, and 44 (4%) continent cutaneous diversion. 90-day postoperative UTI incidence, culture results, antibiotic sensitivity/resistance and treatment were recorded through retrospective review. Fisher’s exact test, Kruskal–Wallis test, and multivariable analysis were performed.

Results

A total of 151 urinary tract infections were recorded in 123 patients (11%) during the first 90 days postoperatively. 21/123 (17%) had multiple infections and 25 (20%) had urosepsis in this time span. Gram-negative rods were the most common etiology (54% of positive cultures). 52% of UTI episodes led to readmission. There was no significant difference in UTI rate, etiologic microbiology (Gram-negative rods, Gram-positive cocci, fungi), or antibiotic sensitivity and resistance patterns between diversion groups. Resistance to quinolones was evident in 87.5% of Gram-positive and 35% of Gram-negative bacteria. In multivariable analysis, Charlson Comorbidity Index > 2 was associated with higher 90-day UTI rate (OR = 1.8, 95% CI 1.1–2.9, p = 0.05) and Candida UTI (OR 5.6, 95% CI 1.6–26.5, p = 0.04).

Conclusions

UTI is a common complication and cause of readmission following radical cystectomy and urinary diversion. These infections are commonly caused by Gram-negative rods. High comorbidity index is an independent risk factor for postoperative UTI, but diversion type is not.

     

Sarcoptic mange of gazelle (<Emphasis Type="Italic">Gazella subguttarosa</Emphasis>) and its medical importance in Iran

Sarcoptes scabiei infestation was diagnosed in four freshly dead and three net-captured gazelle while ranging freely. The captured animals presented with an alopecic pruritic skin disease with signs of crusted skin lesions, numerous small nodules which first appeared on the lips or nostrils and then it also extended towards the eyelids, around the ears, and, in some cases, over entire face, neck, trunk, and legs. Skin over the affected area gradually became bald, thick and hard, being dry and doughy to the touch, and serous fluid or sometimes blood oozes from the lesions which had a severe malodor. Skin scrapings confirmed the presence of the mite S. scabiei. Histopathology of lesions demonstrated marked acanthosis, hyperkeratosis, and parakeratosis. Microscopical examination also revealed all stages of S. scabiei, which were located mainly in the stratum corneum and also in the stratum granulosum. During the capture and sampling of the animals, four persons ranging in age from 25 to 62 years were exposed to scabies. Two relatives of one of them have been also affected by familiar contact. Clinical signs appeared within 9 days of exposure. They developed several pruretic erythematous papules with intense itching.     

Use of D11S2179 and D11S1343 as markers for prenatal diagnosis of ataxia telangiectasia in Iranian patients

Ataxia telangiectasia (AT) is an autosomal recessive disorder with an estimated prevalence of 1/40,000 to 1/100,000 in reported populations. There is a 25% possibility for having an affected child when parents are carriers for the ATM gene mutation. There is no cure available for this disease and prenatal testing is strongly recommended for prevention of this disease. Although the preferred method is the direct mutation analysis of the ATM gene, the large size of the ATM gene with 63 exons and the large number of possible mutations in patients considerably limit efficiency of mutation analysis as a diagnostic choice. Indirect method is a better tool when parents are not carriers of founder mutation and pass different mutations to their children. Indirect molecular diagnosis using ATM-related molecular markers facilitates prenatal diagnosis of AT children. In this study, four molecular markers: D11S2179, D11S1787, D11S535, D11S1343 are genotyped in 19 unrelated families from different regions of Iran. Those markers are amplified using extracted sequence primers from the Gene Bank with their described PCR conditions. Amplified products were separated using denaturing PAGE gels, and data were analyzed to detect their pattern of inheritance in each family. In all families, segregation of alleles was according to Mendelian inheritance, and affected chromosomes were distinguishable from unaffected ones. All carriers and affected patients were diagnosed accurately. Thus, this method is effectively useful in prenatal diagnosis of AT.     

Low molecular weight heparin improves peritoneal ultrafiltration and blocks complement and coagulation.

OBJECTIVES: Clinical studies have demonstrated that the intraperitoneal (IP) complement and coagulation systems are activated in peritoneal dialysis (PD) patients. In animal models, low molecular weight heparin (LMWH) was seen to inhibit peritoneal angiogenesis, and related compounds have increased ultrafiltration volumes after repeated administration to PD patients. The present study evaluated the effects of LMWH on ultrafiltration, coagulation, and complement activation during a single PD dwell. DESIGN: Rats were exposed to a single dose of 20 mL 2.5% glucose-based, filter-sterilized PD fluid, with or without supplementation with LMWH. The PD fluid was administered either as an IP injection or as an infusion through an indwelling catheter. The dwell fluid was analyzed 2 hours later concerning activation of the complement and coagulation cascades, chemotactic activity, neutrophil recruitment, ultrafiltration volume, and glucose and urea concentrations. RESULTS: Exposure to PD fluid induced activation of IP complement [formation of C3a (desArg) and increase of C5a-dependent chemotactic activity] and coagulation (formation of thrombin-antithrombin complex) and recruitment of neutrophils. In the case of IP injection, neutrophil recruitment and complement activation were inhibited by LMWH. In both models, LMWH inhibited thrombin formation, reduced complement-dependent chemotactic activity, and increased the IP fluid volume, indicating an improved ultrafiltration. CONCLUSIONS: The acute inflammatory reaction to PD fluid involves the complement and coagulation cascades. Addition of LMWH to the PD fluid improves ultrafiltration, inhibits formation of thrombin, and potentially blocks C5a activity. The present results motivate further investigations of the IP cascade systems in PD.     

Development of a physical shoulder simulator for the training of basic arthroscopic skills

Background

Orthopaedic training programs are incorporating arthroscopic simulations into their residency curricula. There is a need for a physical shoulder simulator that accommodates lateral decubitus and beach chair positions, has realistic anatomy, allows for an objective measure of performance and provides feedback to trainees.

Methods

A physical shoulder simulator was developed for training basic arthroscopic skills. Sensors were embedded in the simulator to provide a means to assess performance. Subjects of varying skill level were invited to use the simulator and their performance was objectively assessed.

Results

Novice subjects improved their performance after practice with the simulator. A survey completed by experts recognized the simulator as a valuable tool for training basic arthroscopic skills.

Conclusions

The physical shoulder simulator helps train novices in basic arthroscopic skills and provides objective measures of performance. By using the physical shoulder simulator, residents could improve their basic arthroscopic skills, resulting in improved patient safety.     

Coping strategy in infertile couples undergoing assisted reproduction treatment

Objective

The purpose of the study was to determine whether infertility is associated with coping processes and is there a difference between infertile women and men in the use of coping strategies?