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Biomedical subjects

M Khanna

Publications and source records attributed to M Khanna.

At least 19 recordsLinked to original sources
PubMed2006

Anti-filarial activity of novel formulations of albendazole against experimental brugian filariasis.

The study was aimed at developing better orally active albendazole (ALB) formulations. Six formulations (ALB-1 to ALB-6) were prepared and tested against Brugia malayi in Mastomys coucha and jird (Meriones unguiculatus) at 200 mg/kg, orally, for 5 consecutive days. The anti-filarial efficacy was assessed against microfilariae (mf), adult worms and female reproductive potential. Three of the 6 ALB formulations showed greatly improved female worm sterilizing potential (ALB-1: 90%; ALB-3: 63%; ALB-4: 77% of untreated control) in B. malayi - M. coucha model. Sterilization efficacy of ALB-1 was also better than that shown by pure-ALB (P<0.001) or its marketed tablet formulation, Zentel (P<0.01), while that of ALB-4 was better than pure-ALB (P<0.05). The activity of ALB-3, pure-ALB and Zentel was, however, comparable. ALB-1 also showed late microfilaricidal activity with a maximum of 78% fall in microfilarial count. In contrast, neither the pure ALB nor Zentel showed any microfilaricidal activity. In the jird - B. malayi model, ALB-1 and ALB-4 showed marginal sterilizing efficacy whereas pure ALB or Zentel were ineffective. In conclusion the anti-filarial efficacy of ALB-1 was found to be superior to pure-ALB or Zentel.

Albendazole↗
PubMed2005

The pneumoplex assays, a multiplex PCR-enzyme hybridization assay that allows simultaneous detection of five organisms, Mycoplasma pneumoniae, Chlamydia (Chlamydophila) pneumoniae, Legionella pneumophila, Legionella micdadei, and Bordetella pertussis, and its real-time counterpart.

Respiratory disease caused by atypical bacteria remains an important cause of morbidity and mortality for adults and children, despite the widespread use of effective antimicrobials agents. Culture remains the "gold standard" for the detection of these agents. However, culture is labor-intensive, takes several days to weeks for growth, and can be very insensitive for the detection of some of these organisms. Newer singleplex PCR diagnostic tests are sensitive and specific, but multiple assays would be needed to detect all of the common pathogens. Therefore, we developed the Pneumoplex assays, a multiplex PCR-enzyme hybridization assay (the standard assay) and a multiplex real-time assay to detect the most common atypical pathogens in a single test. Primer and probe sequences were designed from conserved regions of specific genes for each of these organisms. The limits of detection were as follows: for Bordetella pertussis, 2 CFU/ml; for Legionella pneumophila (serotypes 1 to 15) and Legionella micdadei, 9 and 80 CFU/ml, respectively; for Mycoplasma pneumoniae, 5 CFU/ml; and for Chlamydia (Chlamydophila) pneumoniae, 0.01 50% tissue culture infective doses. Recombinant DNA controls for each of these organisms were constructed, and the number of copies for each DNA control was calculated. The Pneumoplex could detect each DNA control down to 10 copies/ml. The analytical specificity demonstrated no cross-reactivity between 23 common respiratory pathogens. One hundred twenty-five clinical bronchoalveolar lavage fluid samples tested by the standard assay demonstrated that the Pneumoplex yielded a sensitivity and a specificity of 100 and 98.5%, respectively. This test has the potential to assist clinicians in establishing a specific etiologic diagnosis before initiating therapy, to decrease hospital costs, and to prevent inappropriate antimicrobial therapy.

Adult↗
PubMed2003

Etanercept therapy in patients with autoimmunity and hepatitis C.

Accumulated data suggest that etanercept may be a therapeutic option in patients with hepatitis C and coexisting autoimmune disorders such as rheumatoid arthritis, psoriasis, psoriatic arthritis and ankylosing spondylitis. Additionally, etanercept may actually be of benefit, when used in combination with standard treatments, for hepatitis C.

Animals↗
PubMed2001

Occupational contact dermatitis to textile dyes in airline personnel.

BACKGROUND: Reports of textile dye allergic contact dermatitis are becoming frequent in the literature. Occupational exposure to textile dyes has been reported, but less frequently. OBJECTIVE: To report 2 cases of allergic contact dermatitis to Disperse Blue dyes 106 and 124 occurring in airline personnel. METHODS: The patients were patch tested to the European or North American standard series, a textile dye series, and 1 patient was tested with pieces of textile from an airplane seat. RESULTS: Patch testing elicited in both patients a 2+ reaction to Disperse Blue dyes 106 and 124, and a 1+ reaction to paraphenylenediamine in one patient. CONCLUSION: We describe 2 cases of occupational textile dye allergy occurring in airline personnel. Both cases showed the utility of Disperse Blue dyes 106 and 124 to serve as the screening allergens for textile dermatitis. Mandatory uniforms might be an occupational hazard in certain professions.

Aircraft↗
PubMed2001

Clustered charge-to-alanine mutagenesis of the vaccinia virus A20 gene: temperature-sensitive mutants have a DNA-minus phenotype and are defective in the production of processive DNA polymerase activity.

Although the vaccinia virus DNA polymerase is inherently distributive, a highly processive form of the enzyme exists within the cytoplasm of infected cells (W. F. McDonald, N. Klemperer, and P. Traktman, Virology 234:168-175, 1997). In the accompanying report we outline the purification of the 49-kDa A20 protein as a stoichiometric component of the processive polymerase complex (N. Klemperer, W. McDonald, K. Boyle, B. Unger, and P. Traktman, J. Virol. 75:12298-12307, 2001). To complement this biochemical analysis, we undertook a genetic approach to the analysis of the structure and function of the A20 protein. Here we report the application of clustered charge-to-alanine mutagenesis of the A20 gene. Eight mutant viruses containing altered A20 alleles were isolated using this approach; two of these, tsA20-6 and tsA20-ER5, have tight temperature-sensitive phenotypes. At the nonpermissive temperature, neither virus forms macroscopic plaques and the yield of infectious virus is <1% of that obtained at the permissive temperature. Both viruses show a profound defect in the accumulation of viral DNA at the nonpermissive temperature, although both the A20 protein and DNA polymerase accumulate to wild-type levels. Cytoplasmic extracts prepared from cells infected with the tsA20 viruses show a defect in processive polymerase activity; they are unable to direct the formation of RFII product using a singly primed M13 template. In sum, these data indicate that the A20 protein plays an essential role in the viral life cycle and that viruses with A20 lesions exhibit a DNA(-) phenotype that is correlated with a loss in processive polymerase activity as assayed in vitro. The vaccinia virus A20 protein can, therefore, be considered a new member of the family of proteins (E9, B1, D4, and D5) with essential roles in vaccinia virus DNA replication.

Amino Acid Sequence↗
PubMed2001

Evaluation of influenza virus detection by direct enzyme immunoassay (EIA) and conventional methods in asthmatic patients.

Throat gargle specimens of fifty-seven acute asthmatic patients (age range 18-40 years) were collected for the study. Thirty-four patients were found influenza virus positive in acute asthma cases. Influenza virus was isolated by conventional culture method on MDCK cell-line and by enzyme immunoassay test (EIA). The EIA negative specimens were retested after virus amplification on MDCK cell-line. Virus shedding and virus surface receptors assay was carried out to determine influenza virus titre. Airway functions were measured by spirometry. A good relationship was observed between the degree of airflow limitation and presence of influenza virus infection (p < 0.001; r = 0.85). A comparable difference in % FEV1 was observed in relation to the symptoms. The patients with greater viral antigen load had lower % FEV1. Two specimens, which were EIA negative, turned out to be positive after amplification on MDCK cell-line. The sensitivity was 98% and specificity was 100%. It was concluded that EIA method is a useful diagnostic tool as it detects influenza viral antigen quickly as compared to conventional methods.

Adolescent↗
PubMed2000

3'-deoxyribonucleosides and their derivatives as anti-amoebic agents.

In general nucleoside analogues were found to possess in vitro amoebicidal property against trophozoites of Entamoeba histolytica. The acid-labile nature of these compounds completely destroyed their ability to cure caecal amoebiasis of rats. However the therapeutic efficacy of one of these compounds yielded most promising results, with 10% cures when it was administered as enteric coated formulation.

Amebicides↗
PubMed2000

The importance of homologous recombination in the generation of large deletions in hybrid plasmids in Amycolatopsis mediterranei.

The cloning vector pRL60 was developed previously as a tool for genetic manipulations in Amycolatopsis mediterranei, which produces the commercially and medicinally important antibiotic rifamycin. Here, a method based on intraplasmid recombinations is described for the construction of smaller plasmids in A. mediterranei, which also helped in delimiting the origin of replication (pA-rep) of the parent plasmid. The strategy involved the cloning of a selectable marker, erythromycin resistance gene (ermE), onto plasmids pULAM2 and pULVK2A (derivatives of pRL1), followed by selection of the hybrid or concatemeric plasmids pRL50 and pRL80 (with large homologous repeats) in Escherichia coli GM2163. These hybrid plasmids were then transferred to A. mediterranei DSM 40773 by electroporation, with selection in the presence of different antibiotics. During the process of transformation and selection in A. mediterranei, pRL50 and pRL80 underwent intraplasmid recombinations, yielding derivatives that retained a common region essential for maintenance and replication, as well as the selected resistance genes. This approach produced several smaller plasmids designated pRL51, pRL52, pRL53, pRL60, pRL81, and pRL82. These plasmids, isolated from A. mediterranei DSM 40773, could be transferred to different Amycolatopsis strains at transformation efficiencies ranging from 0.7 x 10(2) to 4 x 10(4) transformants/microg DNA. The electroporation parameters under which maximum transformation efficiencies were obtained varied from strain to strain. Since the isolation of plasmid DNA from Amycolatopsis strains were extremely difficult, a convenient and rapid method of direct transfer of plasmid DNA, i.e., electroduction, was also developed in which the above-described shuttle plasmids were transferred directly from A. mediterranei to E. coli. In addition, the sequence of the minimal (pA-rep, approximately 1.0 kb) of plasmid pRL51 was determined. The nucleotide base sequence of the pA-rep region did not have any clear similarity to the DNA or amino acid sequences in various databases, suggesting that it is unique.

Actinomycetales↗
PubMed2000

Allergic contact dermatitis to tea tree oil with erythema multiforme-like id reaction.

The commercial production of tea tree oil, extracted from Melaleuca alternifolia Cheel, has considerably increased over the past 15 years in response to a strong demand for natural remedies and aromatic substances. The number of case reports that describe allergic contact dermatitis (ACD) to this essential oil is also on the rise. We report an additional case of ACD to tea tree oil that presented with an extensive erythema multiforme-like reaction. A skin biopsy was performed from a targetlike lesion distant from the site of the initial dermatitis. The patient was treated with systemic and topical corticosteroids. Five months later, he was patch tested to the North American standard series, to his own tea tree oil, to a fresh batch of tea tree oil, and to some related allergens. The skin biopsy showed a spongiotic dermatitis without histological features of erythema multiforme. Patch testing elicited a 3+ reaction to old, oxidized tea tree oil, a 2+ reaction to fresh tea tree oil, a 2+ reaction to colophony, a 1+ reaction to abitol, and a 1+ reaction to balsam of Peru. We believe this is the first report of erythema multiforme-like reaction secondary to ACD from tea tree oil. Other interesting features are the stronger reaction to oxidized than to fresh tea tree oil, and concomitant reactivity to colophony, abitol, and balsam of Peru.

Allergens↗
PubMed1999

Multiplex PCR/LDR for detection of K-ras mutations in primary colon tumors.

Point mutations in codons 12, 13, and 61 of the K-ras gene occur early in the development of colorectal cancer and are preserved throughout the course of tumor progression. These mutations can serve as biomarkers for shed or circulating tumor cells and may be useful for diagnosis of early, curable tumors and for staging of advanced cancers. We have developed a multiplex polymerase chain reaction/ligase detection reaction (PCR/LDR) method which identifies all 19 possible single-base mutations in K-ras codons 12, 13, and 61, with a sensitivity of 1 in 500 wild-type sequences. In a blinded study, 144 paraffin-embedded archival colon carcinomas were microdissected and K-ras mutations determined by both dideoxy-sequencing and multiplex PCR/LDR. Results were concordant for 134 samples. The ten discordant samples were re-evaluated using higher sensitivity uniplex PCR/LDR, and the original multiplex PCR/LDR result was confirmed in nine of these ten cases. Multiplex PCR/LDR was able to identify mutations in solid tumors or paraffin-embedded tissues containing a majority of wild-type stromal cells, with or without microdissection. The technique is well suited for large scale studies and for analysis of clinical samples containing a minority population of mutated cells.

Colorectal Neoplasms↗
PubMed1999

Tyrosine phosphorylation of A17 during vaccinia virus infection: involvement of the H1 phosphatase and the F10 kinase.

Vaccinia virus encodes two protein kinases (B1 and F10) and a dual-specificity phosphatase (VH1), suggesting that phosphorylation and dephosphorylation of substrates on serine/threonine and tyrosine residues are important in regulating diverse aspects of the viral life cycle. Using a recombinant in which expression of the H1 phosphatase can be regulated experimentally (vindH1), we have previously demonstrated that repression of H1 leads to the maturation of noninfectious virions that contain several hyperphosphorylated substrates (K. Liu et al., J. Virol. 69:7823-7834). In this report, we demonstrate that among these is a 25-kDa protein that is phosphorylated on tyrosine residues in H1-deficient virions and can be dephosphorylated by recombinant H1. We demonstrate that the 25-kDa phosphoprotein represents the product of the A17 gene and that A17 is phosphorylated on serine, threonine, and tyrosine residues during infection. Detection of phosphotyrosine within A17 is abrogated when Tyr(203) (but not Tyr(3), Tyr(6), or Tyr(7)) is mutated to phenylalanine, suggesting strongly that this amino acid is the site of tyrosine phosphorylation. Phosphorylation of A17 fails to occur during nonpermissive infections performed with temperature-sensitive mutants defective in the F10 kinase. Our data suggest that this enzyme, which was initially characterized as a serine/threonine kinase, might in fact have dual specificity. This hypothesis is strengthened by the observation that Escherichia coli induced to express F10 contain multiple proteins which are recognized by antiphosphotyrosine antiserum. This study presents the first evidence for phosphotyrosine signaling during vaccinia virus infection and implicates the F10 kinase and the H1 phosphatase as the dual-specificity enzymes that direct this cycle of reversible phosphorylation.

Animals↗
PubMed1998

Selection of suitable marker genes for the development of cloning vectors and electroporation in different strains of Amycolatopsis mediterranei.

To select suitable genetic markers for optimizing electroporation efficiency in Amycolatopsis mediterranei, thiostrepton (tsr), erythromycin (ermE) and apramycin (am) resistance genes were used. Although tsr could not be suitably expressed in A. mediterranei, the cloning of ermE in pRL1 or its derivative (containing am) resulted in the development of cloning vectors pRLM20, pRLM30 and pRL90. In contrast to tsr and km (kanamycin resistance gene), ermE and am were suitably expressed in A. mediterranei strains and no spontaneous mutants were observed among transformants. Under optimum conditions, maximum electroporation efficiency of 1.2 x 10(4) transformants/micrograms DNA was achieved for A. mediterranei DSM 40,773. These plasmids could also be effectively transferred in other strains of A. mediterranei including F1/24 and T-195. With the cloning of ermE and am and their expression in different strains of Amycolatopsis, we have overcome the problem of the choice of suitable selectable markers for A. mediterranei and related species.

Actinomycetaceae↗
PubMed1998

Development of an improved cloning vector and transformation system in Amycolatopsis mediterranei (Nocardia mediterranei).

A versatile plasmid cloning vector pRL60 carrying kanamycin/neomycin resistance (km/neo), erythromycin resistance (ermE) and alpha-amylase (alpha-amy) marker genes that replicates in various Amycolatopsis mediterranei strains and Escherichia coli has been constructed. This cloning vector has been derived from a hybrid plasmid pRL50, which was developed by cloning ermE from pIJ4026 into a pRL1 derivative pULAM2. While cloning ermE into the BamHI site of pULAM2, only a hybrid plasmid pRL50 with an additional copy of pULAM2 was selected. Thus pRL50 (18.7 kb) contained two copies each of the km/neo, alpha-amy, and one copy of ermE. When pRL50 was transformed into A. mediterranei DSM 40773 through electroporation and selected under erythromycin resistance, the plasmid underwent a spontaneous deletion of 8.5 kb fragment resulting in the formation of plasmid pRL60. pRL60 (10.2 kb) is a shuttle vector between A. mediterranei and E. coli with three marker genes: km/neo, ermE and alpha-amy. ermE is expressed in A. mediterranei thus allowing good selection of transformants. The alpha-amy gene of pRL60 is also expressed in A. mediterranei DSM 40773 and its activity can be easily detected on starch containing medium after iodine staining. Most critical parameters evaluated for electrotransformation using pRL60 in A. mediterranei were growth phase, electrical field strength, pulse length, pretreatment of mycelia with lysozyme and use of salt free water. At optimized parameters, a transformation efficiency of 4.0 x 10(4) transformants/microg DNA was reproducibly achieved for A. mediterranei DSM 40773. pRL60 could also be transformed into A. mediterranei DSM 43304, DSM 46095, MTCC-17 and in mutants F1/24 and T-195, (derived from an industrial strain of A. mediterranei N813). The alpha-amy of pRL60 conferred an amylolytic phenotype to all these strains. With the development of pRL60 and a reproducible transformation protocol, the application of recombinant DNA techniques to these industrial microorganisms has now become feasible.

Actinobacteria↗
PubMed1996

Engineering antibiotic producers to overcome the limitations of classical strain improvement programs.

Improvement of the antibiotic yield of industrial strains is invariably the main target of industry-oriented research. The approaches used in the past were rational selection, extensive mutagenesis, and biochemical screening. These approaches have their limitations, which are likely to be overcome by the judicious application of recombinant DNA techniques. Efficient cloning vectors and transformation systems have now become available even for antibiotic producers that were previously difficult to manipulate genetically. The genes responsible for antibiotic biosynthesis can now be easily isolated and manipulated. In the first half of this review article, the limitations of classical strain improvement programs and the development of recombinant DNA techniques for cloning and analyzing genes responsible for antibiotic biosynthesis are discussed. The second half of this article addresses some of the major achievements, including the development of genetically engineered microbes, especially with reference to beta-lactams, anthracyclines, and rifamycins.

Anthracyclines↗
PubMed1996

Release of superoxide anion from activated mouse peritoneal macrophages during Mycobacterium tuberculosis infection.

Mouse peritoneal macrophage monolayers infected with M. tuberculosis were cultured in RPMI up to 7 days. Release of superoxide was assayed on different days in presence or absence of Phorbol myristate acetate (PMA), a known stimulator of NADPH oxidase which is involved superoxide production. Basal level of superoxide release was significantly higher in M. tuberculosis infected peritoneal mouse macrophages (P < 0.01) as compared to normal mouse macrophages. When normal and tuberculoid macrophage cultures were stimulated with PMA, increased superoxide anion release was observed in both the cultures but the increase of superoxide was significantly higher in normal macrophages as compared to tuberculoid stimulated macrophages. Superoxide release was maximum in 4 day old cultured macrophages and gradually it declined in older cultures by day 7, both in vitro and in vivo. A defective macrophage function in killing of M. tuberculosis bacilli was observed after 4 days of in vitro and in vivo cultures.

Animals↗