Association and expression of the antigen-processing gene PSMB8, coding for low-molecular-mass protease 7, with vitiligo in North India: case-control study.

BACKGROUND: Vitiligo is a multifactorial, autoimmune, depigmenting disorder of the skin where aberrant presentation of autoantigens may have a role. OBJECTIVES: To study the association of two antigen-processing genes, PSMB8 and PSMB9, with vitiligo. METHODS: In total 1320 cases of vitiligo (1050 generalized and 270 localized) and 752 healthy controls were studied for the PSMB9 exon 3 G/A single-nucleotide polymorphism (SNP), PSMB8 exon 2 C/A SNP and PSMB8 intron 6 G/T SNP at site 37 360 using polymerase chain reaction (PCR)-restriction fragment length polymorphism. Real-time PCR was used for transcriptional expression of PSMB8 and cytokines. Expression of ubiquitinated proteins and phosphorylated-p38 (P-p38) was studied by Western blotting. RESULTS: Significant increases in PSMB8 exon 2 allele A (P < 2.07 × 10-6 , odds ratio 1·93) and genotypes AA (P < 1.03 × 10-6 , odds ratio 2·51) and AC (P < 1.29 × 10-6 , odds ratio 1·63) were observed in patients with vitiligo. Interferon-γ stimulation induced lower expression of PSMB8 in peripheral blood mononuclear cells of cases compared with controls, suggesting impaired antigen processing, which was confirmed by accumulation of ubiquitinated proteins in both lesional and nonlesional skin of patients with vitiligo. Expression of proinflammatory cytokines - interleukin (IL)-6, IL-1ß and IL-8 - was higher in the lesional skin. P-p38 expression was variable but correlated with the amount of ubiquitinated proteins in the lesional and nonlesional skin, suggesting that the inflammatory cytokine responses in lesional skin could be a result of both P-p38-dependent and -independent pathways. CONCLUSIONS: The PSMB8 exon 2 SNP is significantly associated with vitiligo. Accumulation of ubiquitinated proteins in skin of cases of vitiligo suggests their aberrant processing, which may promote the development of the disease.

High-Sensitivity Measurement of Density by Magnetic Levitation.

This paper presents methods that use Magnetic Levitation (MagLev) to measure very small differences in density of solid diamagnetic objects suspended in a paramagnetic medium. Previous work in this field has shown that, while it is a convenient method, standard MagLev (i.e., where the direction of magnetization and gravitational force are parallel) cannot resolve differences in density <10(-4) g/cm(3) for macroscopic objects (>mm) because (i) objects close in density prevent each other from reaching an equilibrium height due to hard contact and excluded volume, and (ii) using weaker magnets or reducing the magnetic susceptibility of the medium destabilizes the magnetic trap. The present work investigates the use of weak magnetic gradients parallel to the faces of the magnets as a means of increasing the sensitivity of MagLev without destabilization. Configuring the MagLev device in a rotated state (i.e., where the direction of magnetization and gravitational force are perpendicular) relative to the standard configuration enables simple measurements along the axes with the highest sensitivity to changes in density. Manipulating the distance of separation between the magnets or the lengths of the magnets (along the axis of measurement) enables the sensitivity to be tuned. These modifications enable an improvement in the resolution up to 100-fold over the standard configuration, and measurements with resolution down to 10(-6) g/cm(3). Three examples of characterizing the small differences in density among samples of materials having ostensibly indistinguishable densities-Nylon spheres, PMMA spheres, and drug spheres-demonstrate the applicability of rotated Maglev to measuring the density of small (0.1-1 mm) objects with high sensitivity. This capability will be useful in materials science, separations, and quality control of manufactured objects.

Pathology of experimental infection by Pasteurella multocida serotype A: 1 in buffalo calves.

Pasteurella multocida serotype A:3 has been mostly implicated in pneumonic pasteurellosis in ruminants. In contrast, our previous studies have reported that both serotypes A:1 and A:3 were responsible for respiratory diseases in cattle and buffaloes. However, the pathology and pathogenesis of P. multocida serotype A:1 (Pm A:1) infection have not been studied in ruminants. In the present study, 12- to 15-week-old buffalo calves (Bubalus bubalis) infected by Pm A:1 had fibrinous and suppurative bronchopneumonia with focal areas of coagulation necrosis typical of pneumonic pasteurellosis. For the first time, this study reports the lung pathology and pathogenecity of Pm A:1 infection in calves.

Complete larval development of the Brachyuran crab (Epixanthus Frontalis H. Milne Edwards, 1834) (Crustacea, Decapoda, Eriphioidea, Oziidae) under laboratory conditions.

The complete larval development of the oziid crab Epixanthus frontalis (H. Milne Edwards, 1834) hatched from ovigerous specimens collected from Saso Island, southern Red Sea, was obtained under laboratory conditions. Four zoeae, one additional zoea and a megalopa were obtained and these are described and illustrated in detail for the first time. Larvae of this species can be differentiated from those of other oziid species based on a combination of characters such as the number of aesthetascs and setae of the antennule, and the coxal and basial setal numbers and patterns of the maxilla and maxillule.

Trend in decline in leprosy disabilities of a LEPRA project in Malkangiri district, Odisha, India.

This a retrospective analysis of the changes in 646 disabilities occurred amongst 3979 cases registered during 19 years from 1992 to 2010 in Malkangiri district. This amounted to 16.2% of cases with disability segregated to 310 (48%) Grade 1 and 336 (52%) Grade 2. In this project, managed by LEPRA India, POD care was in practice from the year 1992 and records were updated regularly. An analysis of the annual records showed that the next year-end balance increased up to the year 2001 followed by gradual decline. Within this period the total cases with disabilities declined by about 369 (57%) due to death by aging 204 (55%), migration from the area 77 (21%) and reversing to normal 88 (24%) in cases. Deletion due to recovery to normal especially with sensory impairment is fairly good with or without steroid. Disability percentage in new cases declined steadily especially Grade 2 from 30% to 1%, initial high rate attributed mostly to backlog cases. In later years the rate is erratic high amongst low number of new cases. Absolute number indicates the situation better. Such study helps to roughly extrapolate the existing disability load in a particular area and assists in planning for care and prevention.

Dermal metastases in oral cancer after curative treatment: a single institution cohort study.

Dermal metastasis (DM) is, by definition, the involvement of the skin by cancer cells that originate from cancer elsewhere in the body. The skin is considered a rare site of distant failure in head and neck cancer and DM is the bearer of a poor outcome. Literature about it is limited so this study was undertaken to analyse the factors associated with its incidence and outcomes. A prospectively maintained database on operated cases of oral cancer at a tertiary cancer centre was analysed, and patients who developed dermal metastases during follow up were evaluated. Factors that contributed to early DM and predicted survival after its development were studied. A total of 68 patients (2.8%) had DM as the first presentation of recurrence after a median disease-free period of five months. Early DM was significantly associated with skin involvement by the primary tumour at the time of presentation (p=0.06), extracapsular extension of nodes (p=0.004), and with those who required adjuvant chemotherapy in view of aggressive histology (p=0.021). Median (range) survival after the detection of DM was 97 (5-328) days (3.25 months). Surgical excision of isolated cases was associated with significantly increased survival after detection (p=0.05). Whenever it is feasible without too much morbidity, solitary DM should be excised.

Descriptions of the first zoeas of ten xanthid crabs (Crustacea: Decapoda: Xanthoidea) from the Gulf of Aqaba, Red Sea.

Ovigerous females of 10 species of xanthid crabs (Xanthidae MacLeay, 1838), from five subfamilies, namely, Pseudoliomera speciosa (Dana, 1852) (Actaeinae), Chlorodiella cytherea (Dana, 1852), Chl. laevissima (Dana, 1852), Chl. nigra (Forskål, 1775), Cyclodius granulosus (De Man, 1888) (Chlorodiellinae), Danielea noelensis (Ward, 1942) (Euxanthinae), Liomera rugata (H. Milne Edwards, 1834), Lio. tristis (Dana, 1852) (Liomerinae), Lachnopodus subacutus (Stimpson, 1858) and Leptodius sanguineus (H. Milne Edwards, 1834) (Xanthinae), were collected from the Gulf of Aqaba, and the zoea I obtained from them have been described herein. Six species, viz. Chl. cytherea, Chl. laevissima, Cyc. granulosus, Lio. rugata, Lio. tristis and Lep. sanguineus, are described for the first time, and Lac. subacutus and D. noelensis are re-described. Spinulations of dorsal and rostral spines of cephalothorax, length of rostral spine of cephalothorax to protopod of antenna, setations of antennule, ratio of antennal exopod to protopod and setations of exopod of antenna are important characters that distinguish xanthid larvae from their congeners and other closely related species at subfamilial levels.

Morphology of the complete larval stages of Portunus segnis (Forskål, 1775) (Crustacea: Brachyura: Portunidae) from the Gulf of Aqaba, Saudi Arabia.

Portunus pelagicus (Linnaeus, 1758) sensu lato has been recognized as a species complex comprising four species. Of these four species, the larval stages of all except Portunus segnis (Forskål, 1775), have been described. The larvae of P. segnis, hatched from an ovigerous female, caught in the Gulf of Aqaba, were cultured in the laboratory up to the megalopa stage. All the larval stages are described herein for the first time. The number of aesthetascs of the antennules of all the zoeal stages of P. segnis differs from those of the larvae of the other species of the P. pelagicus species complex. In the telson forks of zoea I-IV of P. segnis, there is a pair of ventral spines and two pairs of dorsal spines, whereas in the other P. pelagicus species complex larvae, there is a pair each of ventral and dorsal spines. Another unique feature, in the megalopa of P. segnis, are two endopod hooks in pleonites I-V. Different zoeal and megalopal stages of P. segnis can be distinguished clearly from the other P. pelagicus species complex larvae based on the number of setae and patterns of different appendages.

Molecular characterization of avian strains of Pasteurella multocida serogroup-A:1 based on amplification of repetitive regions by PCR.

Repetitive extragenic palindromic (REP)-PCR (polymerase chain reaction), enterobacterial repetitive intergenic consensus (ERIC)-PCR, and single primer PCR assays were employed to characterize 66 strains of Pasteurella multocida serogroup A:1 isolated from avian species belonging to different regions of India. REP-PCR resulted in amplification of REP sequences from the genome which were in the range of approximately 200 to approximately 3000 bp and accounted for a total of 54 distinguishing profiles (D=0.99). ERIC-PCR analysis also generated amplified products in the range of approximately 200 to approximately 3200 bp categorizing strains into a total of 50 different profiles (D=0.98). Amplification of repetitive regions using a microsatellite primer (GTG)(5), resulted in clear distinctive bands ranging from approximately 200 to approximately 2400 bp. Strains were assigned to 43 profiles (D=0.96). No correlation could be drawn between genotypic profiles and avian hosts with their geographical area of origin. Avian strains of P. multocida serogroup A:1 were found to be highly heterogeneous with diverse profiles. REP-PCR was found to be highly discriminatory and simple method for differentiation of phenotypically similar strains. The present study also indicated that PCR based amplification of repetitive regions of P. multocida is a rapid technique with good discrimination and could be employed directly for routine typing of field isolates from fowl cholera outbreaks.

Comparative sequence analysis of 16S rRNA gene of Pasteurella multocida serogroup B isolates from different animal species.

The phylogenetic relationships of five isolates of Pasteurella multocida serotype B:2 belonging to buffalo, cattle, pig, sheep and goat were investigated by comparative sequence analysis of 16S rRNA gene. The 1468bp fragment of 16S rRNA gene sequence comparison showed that the isolates of cattle (PM75), pig (PM49) and sheep (PM82) shared 99.9% homology with the buffalo isolate (vaccine strain P52) whereas, the goat isolate (PM86) shared 99.8% homology with the vaccine strain. The 16S rRNA gene sequences of these isolates were also found monophyletic with type B reference strain NCTC 10323 of P. multocida subsp. multocida. The present study indicated the close relationships of haemorrhagic septicaemia causing P. multocida serotype B:2 isolates of buffalo and cattle with other uncommon hosts (pig, sheep and goat).

Evaluation of a recombinant LipL41 antigen of Leptospira interrogans serovar canicola in ELISA for serodiagnosis of bovine leptospirosis.

The efficacy of a recombinant leptospiral lipoprotein LipL41 as an antigen for conducting enzyme-linked immunosorbent assay (ELISA) for diagnosis of bovine leptospirosis was evaluated. Using known positive and known negative cattle sera the recombinant antigen was found to be highly reactive in the concentration of 100 ng/well. Using a total of 321 field cattle sera the sensitivity of ELISA as compared to microscopic agglutination test (MAT) was calculated to be 100% whereas the specificity was 85.3%. The seropositivity of leptospirosis among bovine population was found to be 21.18% having the predominance of serovars Sejroe and Pomona. It was concluded that rLipL41 protein could be a putative diagnostic candidate for serodiagnosis of bovine leptospirosis.

Characterization of avian strains of Pasteurella multocida by restriction endonuclease and amplified fragment length polymorphism.

Avian strains of Pasteurella multocida were typed by employing restriction endonuclease analysis (REA) and single enzyme-amplified fragment length polymorphism (AFLP) to evaluate their applicability for epidemiological studies of fowl cholera outbreaks. A total of 72 strains isolated from different avian species (chicken, duck, turkey, quail and goose) belonging to various geographical regions of India were characterized. REA using two different enzymes HhaI and HpaII produced 9 and 18 clusters respectively, whereas Single enzyme-AFLP recognized 32 patterns out of 72 strains typed. The study indicated that REA using HpaII is a simple and resource efficient method, however, further typing with more stringent and rapid method like Single enzyme-AFLP, could drastically enhance investigation in epidemiological studies of fowl cholera outbreaks.

Identification of avian strains of Pasteurella multocida in India by conventional and PCR assays.

The prevalence of capsular and somatic serotypes were studied among 123 Pasteurella multocida strains isolated from chickens (n=94), ducks (22), quails (4), turkeys (2) and geese (1) from different geographical regions of India. All strains exhibited similar cultural and morphological characteristics. Ninety-two of the isolates belonged to serotype A:1, the most prevalent serotype, with serotypes A:3, A:1,3, D:3 and F:3 having two isolates each. Only one isolate was positive for serotypes A:4 and D:1. Twenty isolates were untyped. A multiplex capsular PCR assay generated amplicons of sizes approximately 460, approximately 1044, approximately 657 and approximately 854 bp in 106 isolates identified as capsular serotype-A, 15 in serotype D and two in serotype F. Capsular types B and E were not detected in any of the avian isolates studied. The present findings suggest that a multiplex capsular PCR assay may be suitable for the rapid initial identification serotypes P. multocida during epidemiological studies of fowl cholera.

Detection of Pasteurella multocida in experimentally infected embryonated chicken eggs by PCR assay.

Applicability of polymerase chain reaction (PCR) assay to detect Pasteurella multocida in experimentally infected embryonated chicken egg was assessed in the present study. PCR assay rapidly and specifically detected the genome of P. multocida in amniotic fluid, allantoic fluid and homogenates of infected embryo and its membranes. The sensitivity of detection was as low as 20 bacterial cells/ml of allantoic or amniotic fluids. Detection of P. multocida in dead embryos by PCR was possible up to 6 and 30 days or more following storage of dead embryos at 37 degrees C, and at 4 degrees C as well as at -20 degrees C, respectively. The study revealed that PCR assays could be employed directly for detection and confirmation of P. multocida infection in experimentally infected chicken embryos.

REP-PCR analysis of Pasteurella multocida isolates from wild and domestic animals in India.

Repetitive extragenic palindromic sequence-based PCR (REP-PCR) was used to characterize 67 field isolates of Pasteurella multocida originating from different animal species and geographical regions of India. REP-PCR was found to be rapid and reproducible (three repeats were done). These isolates yielded different 23 profiles which were clustered into eight groups. The discrimination index was moderate (D value 0.83). Somatic and antigenic typing of the isolates did not reveal any correlation with REP-PCR profiles. There was no host-specific, type-specific, region-specific or pathenogenicity-specific pattern. The REP profiles of isolates obtained from wild animals were similar to those obtained from domestic animals. Two common bands were present in all the isolates irrespective of somatic or antigenic types. The results were not comparable with earlier findings, which had shown high discrimination index and correlation with disease presentation.

Premedication in an autistic, combative child: Challenges and nuances.

Children with autistic spectrum disorders are often encountered in anesthesia practice mainly for outdoor procedural sedation or anesthesia in endoscopy and magnetic resonance imaging suites. We describe a case of a 7-year-old autistic boy who required management of dental caries. He had a phobia to intravenous cannulation, displayed increasing anxiety and became combative on the day of surgery. With parental involvement and distraction, we succeeded in giving oral midazolam by concealing it, with the intent of avoiding intramuscular injection or unnecessary restraint. Lack of knowledge about the medical condition of such a patient can lead to inadequate preoperative preparation and use of restraint on the patient, which might cause anxiety or panic attacks in the operative room. To effectively manage children with special needs one needs to have clear guidelines on the management of uncooperative children, involve parents perioperatively, plan ahead with an emphasis on perioperative analgesia and sometimes incorporate the ethical use of restraint.

Cost and efficiency of HIV voluntary counselling and testing centres in Andhra Pradesh, India.

BACKGROUND: [corrected] As part of the effort to control HIV/AIDS, the number of HlV voluntarycounselling and testingcentres (VCTCs) is increasing rapidly in the public health system of the Indian state of Andhra Pradesh, which is estimated to have one of the highest rates of HIV infection in India. However, systematic data on the cost and efficiency of providing VCT services in India are not available to help guide efficient use of resources for these services. METHODS: We used standardized methods to obtain detailed cost and output data for the 2002-03 fiscal year from written records and interviews in 17 VCTCs in the public health system in Andhra Pradesh. We calculated the economic cost per client receiving VCT services, and analysed the variation and determinants of total and unit costs across VCTCs. We used multivariate regression techniques to estimate incremental unit costs. We assessed hurdles towards serving an optimal number of clients by VCTCs. RESULTS: In the 2002-03 fiscal year, 32 413 clients received the complete sequence of services at the 17 VCTCs, including post-HIV test counselling. The number of clients served by each VCTC ranged from 334 to 7802 (median 979). The overall HIV-positive rate in post-test counselled clients was 20.5% (range 5.4%-52.6%). The cost per client for the complete VCT sequence varied 6-fold between VCTCs (range Rs 141.5-829.6 [US 2.92-17.14 dollars], median Rs 363.5 [US 7.51 dollars]). The cost per client was significantly lower at VCTCs with more clients (p < 0.001, R2 = 0.83; power function) due to substantial fixed costs. Personnel made up the largest component of cost (53.7%). The cost per client had a significant direct relation with percent personnel cost for VCTCs (p < 0.001, R2 = 0.58; exponential function). A multiple regression model revealed that the incremental cost of providing complete VCT services to each HIV-positive and -negative client was Rs 123.5 (US 2.54 dollars) and Rs 59.2 (US 1.22 dollars), respectively. Fourteen VCTCs (82.4%) reported that they could serve more clients with the available personnel and infrastructure, and that inadequate demand for their services was the main hurdle towards achieving this. CONCLUSION: These data suggest that the efforts of the National AIDS Control Organisation of India and the Andhra Pradesh State AIDS Control Society in increasing VCTCs could yield even higher benefit if the demand for these services was enhanced, as this would increase the number of clients served and reduce the cost per client. Ongoing systematic cost-efficiency analysis is necessary to help guide efficient use of HIV-control resources in India.

Ribotyping of Indian isolates of Pasteurella multocida based on 16S and 23S rRNA genes.

The applicability of ribotyping based on 16S and 23S rRNA was evaluated for molecular epidemiological studies. Forty-eight isolates of Pasteurella multocida isolated from different hosts and geographical locations and one reference isolate were ribotyped. Only four ribotypes were found. All the isolates including reference isolate from wild carnivores had the same ribotype, though they had different serotypes. The isolate from a tiger had one band in addition to the bands present in the major ribotype. The isolates from lions represented two ribotypes; of these ribotypes, one (r2) had an additional band of 3.6 kbp, which was absent in all other ribotypes. The second ribotype (r4) from a lion had one band missing (6 kbp) that was present in the other ribotypes. These isolates were further typed using ERIC-PCR and REP-PCR. With ERIC-PCR and REP-PCR, higher D values of 0.83 and 0.89 were obtained. The current study revealed that ribotyping is not a very efficient typing tool for use in molecular epidemiology for differentiation of isolates.

Bone marrow transplant in adrenoleukodystrophy.

An allogeneic bone marrow transplant (BMT) from a normal HLA identical sibling donor was performed in a 13-year-old boy with rapidly progressive adrenoleukodystrophy (ALD). Engraftment and complete hematologic recovery occurred within 4 weeks, but neurologic deterioration continued. The patient died of an adenovirus infection 141 days after BMT. ALD is characterized by abnormally high plasma levels of very long chain fatty acids (VLCFA) as a result of impaired capacity to degrade them. Ten days after BMT, the white blood cell VLCFA levels and enzyme activity became normal; after 3 months, there was progressive reduction of plasma VLCFA to levels only slightly above normal.

Methotrexate analogues. 20. Replacement of glutamate by longer-chain amino diacids: effects on dihydrofolate reductase inhibition, cytotoxicity, and in vivo antitumor activity.

Chain-extended analogues of methotrexate were synthesized by condensation of 4-amino-4-deoxy-N10-methylpteroic acid with esters of L-alpha-aminoadipic, L-alpha-aminopimelic, and L-alpha-aminosuberic acids, followed by ester hydrolysis with acid or base. Coupling was accomplished in up to 85% yield by the use of the peptide bond forming reagent diethyl phosphorocyanidate at room temperature. The products were found to bind bacterial (Lactobacillus casei) and mammalian (L1210 mouse leukemia) dihydrofolate reductase with an affinity comparable to methotrexate and were also equitoxic to L1210 cells in culture. Cytotoxicity increased up to 3-fold as the number of CH2 groups in the amino acid side chain was extended from two to five. The alpha-aminoadipate and alpha-aminopimelate analogues were poor substrates for carboxypeptidase G1, confirming that this enzyme has a strict requirement for a C-terminal L-glutamic acid residue. The in vivo antitumor activity of the chain-extended analogues against L1210 leukemia in mice was comparable to that of the parent drug on the qd X 9 schedule, but higher doses were required to achieve the same increase in survival. The results were consistent with findings, reported separately, that these compounds are poor substrates for folate polyglutamate synthetase and therefore would not be expected to form gamma-polyglutamates once they enter a cell. This distinctive property has potential therapeutic implications for the treatment of certain MTX-resistant tumors whose resistance may be associated with a lower than normal capacity to form gamma-polyglutamates in comparison with proliferative tissues such as intestinal mucosa or marrow.