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K R Mittal

Publications and source records attributed to K R Mittal.

At least 37 records · Page 2Linked to original sources
PubMed1996

Identification and characterization of genus-specific epitopes of Serpulina species using monoclonal antibodies.

Four murine monoclonal antibodies (mAbs) designated as C9E8, A10, G12, and G8 which recognized both Serpulina hyodysenteriae and S. innocens were produced and characterized. The mAbs reacted with whole cell antigens in ELISA, indirect immunofluorescence and immunoblot assays. The mAbs did not show any cross reactivity in rapid dot ELISA or immunoblot assay with Leptospira icterohemorrhagiae, Campylobacter jejuni and Escherichia coli. Treatment of whole cell suspension with proteinase K and sodium periodate indicated that the reacting epitopes of the mAbs were protein in nature. The genus-specific antigens were identified as heat-stable proteins with molecular weight in the range of 26 to 45 kDa. Immunofluorescence and immunogold labelling studies showed that the antibody-binding epitopes were exposed on the outer-surface of the spirochaetal cell wall. The mAbs inhibited growth of reference strains of both S. hyodysenteriae and S. innocens in vitro but failed to cause agglutination. The detection of spirochaetal forms directly in fecal smears or paraffin-embeded tissue sections from experimentally infected pigs indicated that such mAbs were potentially useful for the diagnosis of swine spirochaetosis. This is the first report of mAbs identifying and characterizing common antigens of porcine Serpulina.

Animals↗
PubMed1996

Biochemical and antigenic relationships between porcine and equine isolates of Actinobacillus suis.

A total of 50 Actinobacillus suis isolates were studied for their biochemical and antigenic characteristics. Of them, 40 isolates originated from different tissues of diseased pigs, and the other ten isolates were from horses with respiratory problems. There was no major biochemical difference among equine and porcine A. suis isolates. Results of tube agglutination tests showed that porcines isolates were antigenically homogeneous while equine isolates were heterogeneous.

Actinobacillus↗
PubMed1996

Characterization of an outer membrane protein of Pasteurella multocida belonging to the OmpA family.

The outer membrane vesicle and N-lauroylsarcosine-insoluble protein preparations of Pasteurella multocida 656 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A major outer membrane protein (OMP) was found to be heat-modifiable, having a molecular mass of 28 kDa when the OMP preparation was solubilized at 60 degrees C and a molecular mass of 37 kDa when it was solubilized at 100 degrees C. A monoclonal antibody, designated mAb MT4.1, was generated against heat-modifiable OMP of P. multocida. This mAb reacted with the heat-modifiable OMP irrespective of the temperature at which it was solubilized, as demonstrated by immunoblot results. The heat-modifiable OMP of P. multocida showed a significant N-terminal amino acid sequence homology with OmpA family. Immunoelectron microscopic study revealed that the mAb Mt4.1 epitope was not surface exposed on the intact bacterium. The mAb MT4.1 reacted with all the reference strains of 5 capsular and 16 somatic serotypes, as well as with 75 field strains of P. multocida in immunoblot assay. This mAb MT4.1 also reacted with strains of various other Pasteurella species such as P. stomatis, P. aerogenes P. gallinarum, P. betti, P. sp, B, P. SP-g and P. canis, but not with strains of 12 other Gram-negative bacteria. These results indicated that this protein carried a genus-specific epitope and mAb MT4.1 may be useful for identification of Pasteurella species. This is the first report in which a major heat-modifiable OMP has been identified and characterized using a mAb, and has been shown belonging to the OmpA family.

Amino Acid Sequence↗
PubMed1996

Comparison of p53 and MIB1 expression in benign and borderline areas of ovarian serous tumors.

Ovarian serous tumors of borderline malignancy frequently show morphologically benign and borderline areas within the same tumor. This study was undertaken to determine if these two morphologically disparate areas differ in their proliferative activity and p53 expression. Formalin-fixed, paraffin-embedded archival tissue from 17 ovarian serous borderline tumors with morphologically benign and borderline areas were immunostained with monoclonal antibodies against p53 and MIB1. The percentage of positive cells was determined by counting 100 consecutive cells for each stain in the most intensely stained areas in morphologically benign and borderline portions of these tumors. There was a significantly increased proliferation (MIB1 expression) in borderline areas compared with benign areas (37.05 +/- 15.3 versus 12.88 +/- 6.7, p = 0.0001). More than 30% of cells were positive for MIB1 in 13/17 borderline areas compared with none of the 17 benign areas (p < 0.0001). The expression of p53 was also higher in borderline areas compared with benign areas (7.12 +/- 8.8 versus 2.94 +/- 4.46, p = 0.0078). More than 10% of cells were p53 positive in 5/17 borderline areas compared with 1/17 benign areas (p = 0.08). However, there was no significant correlation between p53 expression and MIB1 expression in either the benign or borderline areas (p = 0.4 and 0.2, respectively). In summary, morphologically borderline areas show significantly higher p53 expression and proliferation compared with morphologically benign areas in ovarian serous borderline tumors. Alterations of p53 may play a pathogenetic role in some ovarian serous borderline tumors. The lack of correlation between p53 expression and MIB1 expression, however, suggests involvement of other factors, in addition to p53, in determining the proliferative rate of ovarian serous borderline tumors.

Antigens, Nuclear↗
PubMed1996

The 32 kDa major outer-membrane protein of Pasteurella multocida capsular serotype D.

The major outer-membrane protein (MOMP) of Pasteurella multocida serotype D strain P210, with an apparent molecular mass of 32 kDa, was purified and characterized. The purification method involved selective extraction of MOMP with N-lauroylsarcosine and SDS, followed by immunoaffinity chromatography using a murine monoclonal antibody (mAb). The N-terminal sequence and amino acid composition of the MOMP showed considerable similarity to other Gram-negative bacterial porins, notably to the 37 kDa MOMP (porin H) of P. multocida. Immunoelectron microscopy and colony blotting assays were used to demonstrate the surface localization of the 32 kDa MOMP on bacterial cells. The colony blotting assay provided a simple, sensitive and rapid screening method for visualizing accessibility of the antibody on the cells. In a Western blot assay, murine polyclonal hyperimmune serum against the purified 32 kDa MOMP recognized both serotype B and D strains bearing either a 32 kDa or a 37 kDa MOMP, whereas the mAb recognized only serotype D strains bearing a 32 kDa but not a 37 kDa MOMP. The present data indicate that the 32 kDa MOMPs of P. multocida are antigenically heterogeneous and possess both specific and cross-reacting epitopes. Detection of type-specific epitopes on the 32 kDa MOMP using an mAb may have potential implications regarding the feasibility of developing a serotyping system for P. multocida.

Amino Acid Sequence↗
PubMed1996

Development of an experimental model allowing discrimination between virulent and avirulent isolates of Serpulina (Treponema) hyodysenteriae.

Variation in virulence among different strains of Serpulina hyodysenteriae was studied by oral inoculation of specific pathogen free piglets and CD-1 mice. Piglets infected with serotype 2 reference strain B204 and an untypable field strain LHV-90-9-I had severe diarrhea tainted intermittently with mucus and fresh blood. The piglets inoculated with B169, B8044, B6933, and ACK300-8 reference strains representing serotypes 3, 5, 6, and 7 respectively developed moderate diarrhea. However, reference strains B234 and A-1 of serotypes 1 and 4, respectively, failed to cause any diarrhea. None of the S. hyodysenteriae strains caused diarrhea in mice. The results indicate a great variation in virulence among strains of different serotypes of S. hyodysenteriae. Mice were less susceptible to infection with S. hyodysenteriae.

Animals↗
PubMed1995

Cervical squamous dysplasias and carcinomas with immunodetectable p53 frequently contain HPV.

Studies using cervical carcinoma cell lines usually show mutated p53 in cases without detectable HPV, and wild-type p53 in cases with detectable HPV. These findings suggest that loss of p53 function, either by mutation or by binding to HPV E6, is required for cervical carcinogenesis. Because mutated p53 is usually detectable immunohistochemically, one would predict an inverse relationship between the presence of HPV and detectable p53. In this study we examined 88 formalin-fixed paraffin-embedded clinical specimens of cervix for the presence of HPV and p53 expression. All cases were studied for the presence of p53 using immunohistochemical methods. The antibody used was mouse monoclonal PAb1801 (Biogenex). The presence of HPV was detected by PCR. Twenty-six specimens showed foci of p53 expression (0/7 normal, 1/8 (13%) condylomas, 1/6 (17%) CIN I, 3/7 (43%) CIN II, 6/20 (30%) CIN III, 13/22 (59%) SCC, 2/5 (40%) adenosquamous carcinomas, and 0/13 adenocarcinomas). p53 expression was more frequent in SCC than with CIN (P = 0.026). HPV was present in 15 of 24 cases with detectable p53 and 22 of 48 cases without detectable p53. No correlation was seen between HPV status and detection of p53. With the exception of one case, p53 expression was seen in less than 10% of cells. p53 expression was not detected in any of the 13 adenocarcinomas examined (P = 0.0016 vs SCC). Our results show that alterations of p53 may play a role in the pathogenesis of cervical squamous carcinoma. However, p53 expression was neither sufficient nor required for cervical carcinogenesis, irrespective of HPV status.

Adenocarcinoma↗
PubMed1995

Immunohistochemical profile of ovarian inclusion cysts in patients with and without ovarian carcinoma.

The expression of cytokeratin, epithelial membrane antigen, Leu-M1, B72.3, carcinoembryonic antigen, human placental lactogen, proliferating cell nuclear antigen, p53, and ovarian carcinoma-associated antigen OC-125 was evaluated in inclusion cysts in contralateral ovaries of patients with unilateral ovarian carcinoma. The findings were compared with the findings in inclusion cysts in ovaries of patients without ovarian carcinoma. Although there was more frequent expression of tumour markers B72.3 and CEA in patients with ovarian carcinoma, these differences did not reach statistical significance.

Biomarkers, Tumor↗
PubMed1995

Evaluation of different serological tests for detection of antibodies against Serpulina hyodysenteriae in pig sera.

Swine dysentery is a mucohemorrhagic diarrheal disease caused by S. hyodysenteriae. The detection of asymptomatic carriers in herds is possible by serological tests. However, cross-reactions between S. hyodysenteriae and S. innocens pose a major problem in serological diagnosis. Several serological tests were evaluated for detection of antibodies to S. hyodysenteriae such as: indirect hemagglutination, passive hemolysis, conglutination and microagglutination tests. Among the tests used, only the microagglutination test was able to detect antibodies to S. hyodysenteriae. 70 to 95% of the pigs were invariably seropositive in a single dilution of 1:10 in actively infected herds whereas the number of seropositives did not exceed 10% in presumably non-infected herds. The test was found to be simple, and reliable to be used with confidence for detection of herd infection using boiled cell suspension as an antigen.

Animals↗
PubMed1995

Identification and characterization of outer membrane proteins of Pasteurella multocida serotype D by using monoclonal antibodies.

Monoclonal antibodies (MAbs) against Pasteurella multocida serotype D were obtained by fusion of spleen cells from BALB/c mice immunized with outer membrane proteins (OMPs) with SP2/0-Ag 14 murine myeloma cells. Desirable MAbs were selected by enzyme-linked immunosorbent assay (ELISA) with OMP as the antigen. MAbs MT1 and MT2 identified two different proteins (H [heavy] and W [weak]), each with a molecular mass of 32 kDa, in Western blots (immunoblots). Treatment of the OMPs with proteolytic enzymes and sodium periodate indicated that the binding sites of MAbs MT1 and MT2 are of protein and glycoprotein natures, respectively. The epitopes reactive with MAbs were surface exposed, as visualized by immunoelectron microscopy. Among field isolates of P. multocida serotype D, two distinct OMP patterns were recognized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and these patterns were designated types I and II. In both the ELISA and the Western blot, MAb MT1 recognized only type I isolates, whereas MAb MT2 recognized both type I and II isolates. Neither MAb MT1 nor MAb MT2 reacted with either reference strains of capsular serotypes A, B, E, and F or field isolates of capsular serotype A of P. multocida. This is the first report of MAbs identifying the serotype D-specific OMP of P. multocida.

Animals↗
PubMed1995

Production and characterization of monoclonal antibodies against Serpulina hyodysenteriae and S. innocens and their use in serotyping.

Murine monoclonal antibodies (MAbs) directed against serotypes 1, 2, 8, and 9 of Serpulina hyodysenteriae and strain B256 of Serpulina innocens were produced and characterized. A serological classification of 96 field strains of S. hyodysenteriae and 28 field strains of S. innocens isolated from pigs showing clinical signs of swine dysentery was performed by rapid dot enzyme-linked immunosorbent assay (ELISA) with the MAbs. The results indicated that the majority of the field strains of S. hyodysenteriae (69%) belonged to serotypes 8, 1, and 9, whereas only 31% of the S. innocens strains were recognized by MAb 9H7, indicating the presence of antigenic heterogeneity among S. innocens isolates. Rapid dot ELISA with type-specific MAbs was found to be specific, sensitive, and easy to perform and thus to be suitable for routine serotyping of S. hyodysenteriae and S. innocens isolates. This is the first report of MAbs being used for serotyping clinical isolates of S. hyodysenteriae and S. innocens.

Antibodies, Monoclonal↗
PubMed1994

Increased expression of transforming growth factor beta isoforms and basic fibroblast growth factor in complex hyperplasia and adenocarcinoma of the endometrium: evidence for paracrine and autocrine action.

Endometrial carcinoma is associated with antecedent simple and complex hyperplasia, and the endometrium is a target tissue for the action of cytokines and growth factors. Transforming growth factor (TGF)-beta s are potent cellular growth and differentiation regulatory factors. Therefore, we investigated the potential role for TGF-beta s in the normal proliferative endometrium and its possible involvement in the transition to complex hyperplasia and progression to endometrial carcinoma. The angiogenic and mitogenic growth factor, basic fibroblast growth factor, was used for comparison. Differential TGF-beta isoform-specific immunoreactivity was observed in the normal endometrium, which is composed of glandular and stromal cells. There was an increase in TGF-beta 3 but not TGF-beta 1 or TGF-beta 2 in the glandular epithelium from the proliferative to the secretory phase of the menstrual cycle. Immunostaining for TGF-beta 2 was more intense in the stroma than the glands. In contrast, TGF-beta 1 and TGF-beta 3 were near equal intensity in these two endometrial compartments, TGF-beta 3 being the most intense. The glandular epithelium demonstrated a statistically significant stepwise increase in the expression of all three TGF-beta s progressing from the normal proliferative endometrium to simple hyperplasia and on to complex hyperplasia. However, the stromal cells maintained approximately the same level of immunoreactivity for TGF-beta in all these samples. In comparing proliferative endometrium with complex hyperplasia, there was a 5.1-, 3.4-, and 2.6-fold increase in immunostaining in the glands for TGF-beta 1, TGF-beta 2, and TGF-beta 3, respectively (P < or = 0.001). There was no further increase in immunoreactivity with progression from preneoplastic complex hyperplasia to carcinoma. Immunoreactive basic fibroblast growth factor was slight in normal endometrium and simple hyperplasia. There was a 4.6- and 4.2-fold increase in immunostaining observed in complex hyperplasia compared with proliferative endometrium in the glandular (P < or = 0.0054) and stromal (P < or = 0.0053) cells, respectively, with no further increase in carcinoma. By in situ hybridization, an increase in mRNA for all TGF-beta isoforms paralleled TGF-beta immunoreactivity. However, in contrast to the increased immunostaining in the glands in complex hyperplasia, there was remarkably more mRNA in the stromal cell compartment. The discordant expression of mRNA and protein was only observed in the pathological endometrium since both were more highly expressed in the stromal cells in normal proliferative endometrium.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenocarcinoma↗
PubMed1994

Evaluation of microagglutination test for differentiation between Serpulina (Treponema) hyodysenteriae and S. innocens and serotyping of S. hyodysenteriae.

Swine dysentery is a mucohemorrhagic diarrheal disease caused by the anaerobic spirochete Serpulina hyodysenteriae. At present, the serotyping is done by immunodiffusion testing with lipopolysaccharide (LPS) extract as antigen and rabbit hyperimmune sera produced against different serotypes of S. hyodysenteriae. Since the preparation of LPS is time-consuming and requires a large quantity of bacteria, it is desirable to use a serotyping method which does not require the extraction of LPS. In the present investigation, microagglutination was evaluated by using both formalinized whole- and boiled-cell suspensions as antigens and rabbit hyperimmune sera produced against formalinized whole-cell suspensions of reference strains of S. hyodysenteriae and S. innocens B256. Use of boiled cell suspension as antigen permitted the differentiation between isolates of S. hyodysenteriae and S. innocens as well as serotyping of S. hyodysenteriae strains accurately. A total of 18 isolates were identified as S. hyodysenteriae, and 3 isolates were identified as S. innocens. The microagglutination test was found specific, sensitive, and easy to perform; thus, it was judged suitable for routine identification and serotyping of S. hyodysenteriae isolates.

Agglutination Tests↗
PubMed1993

Endometrial adenocarcinoma involving adenomyosis without true myometrial invasion is characterized by frequent preceding estrogen therapy, low histologic grades, and excellent prognosis.

We reviewed cases of endometrial adenocarcinoma to characterize the carcinomas that involved foci of adenomyosis in contrast to the carcinoma that invaded into the myometrium. We encountered 18 cases in which tumor involved foci of adenomyosis without myometrial invasion. There were no cancer-related deaths in these 18 cases over a minimum follow-up period of 5 years. Myometrial invasive tumor was present in 43 cases. Eight of these 43 patients died within 5 years. History of postmenopausal use of estrogen of longer than 6 months duration was obtained in 9 of the 18 patients (50%) with carcinoma confined to adenomyosis compared with 8 of the 43 (19%) with myometrial invasive carcinoma. Only 1 of the 18 cases with adenocarcinoma involving adenomyosis had architectural or nuclear grade III carcinoma compared to 12 of the 43 patients with myometrial invasive carcinoma. Adenocarcinomas involving adenomyosis were characterized by frequent preceding estrogen use, low histologic grades, and excellent prognosis.

Adenocarcinoma↗
PubMed1993

Serological characterisation of Actinobacillus pleuropneumoniae strains of serotypes 1, 9 and 11.

Various serological tests such as agglutination, coagglutination, indirect haemagglutination, immunodiffusion and counterimmunoelectrophoresis were used to characterise serologically Actinobacillus pleuropneumoniae isolates of serotypes 1, 9 and 11 using rabbit hyperimmune sera against serotypes 1 to 12. The rapid slide agglutination test using whole-cell suspension and the indirect haemagglutination test using whole-cell saline extract as antigens gave type specific reactions for serotypes 1 and 9. Antigens comprising saline extracts of boiled or autoclaved cells demonstrated common epitopes among the isolates of all three serotypes in the indirect haemagglutination test. Quantification of the type and group-specific antigens by the coagglutination test. Quantification of the type and group-specific antigens by the coagglutination test distinguished serotypes 1 and 9 strains from those of serotype 11. Results of absorption studies in immunodiffusion tests indicated that the cross-reactivity encountered among strains of serotypes 1, 9 and 11 might be due to common epitopes associated with cell-wall antigens. However, they certainly also have type-specific epitopes, possibly associated with superficially located, capsular antigens.

Actinobacillus Infections↗
PubMed1993

Proliferating cell nuclear antigen (cyclin) expression in normal and abnormal cervical squamous epithelia.

Expression of Proliferating cell nuclear antigen (PCNA) was evaluated in formalin-fixed, paraffin-embedded normal and abnormal cervical squamous epithelia using immunoperoxidase stains and PC10 monoclonal antibody to PCNA. PCNA was exclusively expressed in the parabasal and basal layers of normal ectocervix and a similar pattern was seen in nine of the 11 cases with squamous metaplasia. Examples of cervical dysplasia showed expression in higher layers of cervical epithelium, corresponding to the degree of dysplasia. Increased staining was seen in condylomas and markedly reduced staining with atrophy. The percentage of basal cells that stained increased progressively from atrophic to normal, to condylomatous, to dysplastic epithelia. Proliferative activity can be satisfactorily assessed in formalin-fixed cervical epithelia using PC10 PCNA antibody. This assessment can be of potential diagnostic use in difficult cases.

Atrophy↗