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Biomedical subjects

D R Howard

Publications and source records attributed to D R Howard.

At least 19 recordsLinked to original sources
PubMed1992

Deoxyribonucleic acid analysis by flow cytometry of uterine leiomyosarcomas and smooth muscle tumors of uncertain malignant potential.

OBJECTIVES: The histologic distinction of uterine benign leiomyomas from leiomyosarcomas is difficult. Ploidy analysis and measurement of the proliferative rate were examined to determine if they could distinguish malignant from benign tumors and if they were independent prognostic factors. STUDY DESIGN: Paraffin-embedded blocks were recut and prepared for flow cytometry with the technique of Hedley. Mitotic counts and tumor grading were performed on an adjacent hematoxylin and eosin-stained section. Statistical analysis was carried out with chi 2 life-table, and Cox model analysis. RESULTS: There were 33 patients with deoxyribonucleic acid histograms that were acceptable for analysis. Fourteen tumors were diploid and 19 were aneuploid. There were no significant differences in the clinical characteristics between the patients with diploid tumors and those with aneuploid tumors. Aneuploid tumors were more likely to have cellular atypia (p = 0.085). There was a strong correlation between the percentage of cells in the S phase and the mitotic count (p = 0.0001). Increasing mitotic count, increasing S phase, presence of extrauterine disease, and postmenopausal status were all adverse prognostic factors. However, when multivariant analysis with a Cox model was used, only S phase and presence of extrauterine disease were adverse factors. Diploid tumors have a better overall survival (p = 0.0658) but a similar disease-free survival. In those patients who ultimately have relapses, diploid tumors have a significantly longer interval from relapse to death (p = 0.0045). CONCLUSIONS: Neither ploidy analysis nor measurement of the proliferative rate will distinguish a benign from a malignant course in an individual patient; however, ploidy is predictive of survival from time of disease progression and proliferative rate is the strongest predictor of overall survival. The time-proven reliability of mitotic count in the diagnosis of smooth muscle tumors reflects its ability to predict proliferative rate.

DNA, Neoplasm↗
PubMed1992

Kinetochore-staining of spermatid micronuclei: studies of mice treated with X-radiation or acrylamide.

The rodent spermatid micronucleus (MN) assay was used in conjunction with immunofluorescent techniques to distinguish kinetochores in MN following exposure of mice to X-radiation or acrylamide. After either treatment, modest increases in kinetochore-positive MN were observed. Spermatids which had been exposed during meiotic prophase to X-rays (400 cGy) had approximately 10-fold increases in MN compared to controls; up to 15% of the MN observed were kinetochore-positive. Following acrylamide treatment of meiotic prophase cells, there was a doubling of spermatid MN over baseline levels, approximately one-third of which were kinetochore-positive.

Acrylamide↗
PubMed1991

Evidence that methanol inhalation does not induce chromosome damage in mice.

Mice were exposed by inhalation to 800 or 4000 ppm methanol for 5 days, and cytogenetic effects were analyzed in blood erythrocytes, lung cells, and testicular germ cells. The results were uniformly negative; no increased frequencies of micronuclei in blood cells, of sister-chromatid exchanges, chromosome aberrations, or micronuclei in lung cells, or of synaptonemal complex damage in spermatocytes were found. From the standpoint of risk assessment, these experimental studies do not reveal any evidence of a cytogenetic hazard associated with inhalation of methanol.

Administration, Inhalation↗
PubMed1990

Transfection of a human alpha-(1,3)fucosyltransferase gene into Chinese hamster ovary cells. Complications arise from activation of endogenous alpha-(1,3)fucosyltransferases.

In order to isolate a human gene encoding an alpha-(1,3)fucosyltransferase (alpha-(1,3)Fuc-T), genomic DNA from HL-60 cells was transfected by several methods into Chinese hamster ovary (CHO) cells. Colonies expressing alpha-(1,3)Fuc-T activity were identified by their ability to bind a monoclonal antibody (anti-SSEA-1) that recognizes the carbohydrate product of alpha-(1,3)Fuc-T action. CHO cells do not express alpha-(1,3)Fuc-T activity but contain at least two, silent alpha-(1,3)Fuc-T genes previously identified by their activation in the rare, dominant mutants LEC11 and LEC12. These CHO enzymes were shown to be distinguishable from the alpha-(1,3)Fuc-T activity of HL-60 cells by the latter's comparative inability to transfer fucose to paragloboside and fetuin. Based on these criteria, only 11 isolates from more than 70 putative transfectants examined were found to stably express an alpha-(1,3)Fuc-T activity typical of HL-60 cells. Genomic DNA from two of these isolates was used to generate five independent secondary transfectants with HL-60-like alpha-(1,3)Fuc-T activity. Southern analysis revealed a common DNA fragment that hybridized to an Alu probe in each secondary, providing evidence that a human alpha-(1,3)Fuc-T gene had been transfected. However, in all transfection experiments, isolates that expressed alpha-(1,3)Fuc-T activities similar to CHO-encoded enzymes were also obtained. Several lines of evidence indicated that these cells arose from activation of endogenous CHO alpha-(1,3)Fuc-T genes as a consequence of DNA transfection. These false positives complicated the identification of transfectants expressing a human alpha-(1,3)Fuc-T gene and represent an important consideration in experiments to transfect other glycosyltransferase genes.

Animals↗
PubMed1990

Genotoxicity of inhibitors of DNA topoisomerases I (camptothecin) and II (m-AMSA) in vivo and in vitro.

The present study was designed to determine and compare the clastogenicity of m-AMSA and camptothecin (CAMP) in vivo in mouse bone marrow and peripheral blood lymphocytes (PBLs), and in vitro in mouse lymphoma L5178Y cells. m-AMSA interferes with topoisomerase II to induce double-strand DNA breaks. CAMP interferes with topoisomerase I to induce single-strand DNA breaks. Thus, we expected the two drugs to induce different types of chromosomal aberrations (CAs). However, both drugs produced quantitatively and qualitatively similar numbers and types of aberrations under similar experimental conditions. In mouse bone marrow exposed over and 18-h period, both drugs (3 mg/kg) induced approximately 30 damaged cells, with an average of 0.4 chromatid breaks per cell (in 100 cells analyzed/mouse). In addition, both drugs induced only chromatid-type aberrations in mouse bone marrow in vivo when exposure occurred during G2. Cell cycle specificity was indicated by the absence of CAs when exposure to the drugs occurred in vivo in mouse PBLs during G0. In L5178Y cells, m-AMSA was considerably more potent for the induction of mutations and somewhat more potent for the induction of CAs than CAMP was. In contrast to the in vivo bone marrow results, the drugs induced high levels of both chromatid- and chromosome-type aberrations in vitro. The ultimate types of chromosomal damage induced by m-AMSA and CAMP result from a complex interaction of (i) cell cycle specific variations in topoisomerase enzyme levels, (ii) the abilities of these drugs to interfere with the orderly DNA breakage/reunion associated with topoisomerase activity, and (iii) the processing of the damage resulting from these interactions.

Amsacrine↗
PubMed1990

Intramuscular vaccination of skunks and raccoons against rabies.

Live-captured striped skunks (Mephitis mephitis) and raccoons (Procyon lotor) were immunized with inactivated rabies vaccine by intramuscular injection and released at the point of capture during a rabies control program in Metropolitan Toronto (Ontario, Canada). Serum samples collected prior to and following vaccination revealed that 100% of the skunks and 98% of the raccoons seroconverted. Rabies antibody was still detectable 314 to 757 days postvaccination. Five of six skunks vaccinated in the laboratory survived challenge with rabies virus 90 days postvaccination. To our knowledge, this is the first documentation of the successful seroconversion of skunks and raccoons vaccinated against rabies in the field.

Animals↗
PubMed1990

Angiomyolipoma of kidney with lymph node involvement. DNA flow cytometric analysis.

We performed DNA flow-cytometric analysis in three cases of angiomyolipoma of the kidney with lymph node involvement. The primary tumors of the kidneys and tumors in the lymph nodes of all patients contained diploid DNA. All three patients have been monitored for longer than 1 year and have no evidence of recurrence or metastasis. The lack of aneuploidy in both the kidney and the lymph nodes in our patients, although not excluding malignant neoplasia, supports the benign and multifocal nature of this lesion. Larger studies with long-term follow-up are reported to confirm this conclusion.

DNA, Neoplasm↗
PubMed1987

The GDP-fucose:N-acetylglucosaminide 3-alpha-L-fucosyltransferases of LEC11 and LEC12 Chinese hamster ovary mutants exhibit novel specificities for glycolipid substrates.

Previous studies have shown that the GDP-fucose:N-acetylglucosaminide 3-alpha-L-fucosyltransferase (alpha (1,3) fucosyltransferase (Fuc-T)) activities expressed by the Chinese hamster ovary cell mutants LEC11 (Fuc-TI) and LEC12 (Fuc-TII) are different enzymes and indicated that Fuc-TI might act on sialylated lactosamine sequences (Campbell, C., and Stanley, P. (1984) J. Biol. Chem. 259, 11208-11214). In this paper we show that CSLEX-1, a monoclonal antibody specific for NeuNac alpha (2,3)Gal beta (1,4)(Fuc alpha (1,3))GlcNAc beta 1 sequences, bound to LEC11 cells but not to LEC12 cells. Direct evidence that Fuc-TI could act on sialylated substrates was sought with a series of glycolipid acceptors. Optimal assay conditions in crude cell extracts were determined with nLc4, a glycolipid which accepted fucose with both Fuc-TI and Fuc-TII to generate the Lex antigenic determinant. The two enzymes differed in their detergent sensitivities, pH optima, Mn2+ requirements, and apparent Km values for nLc4. When sialylated glycolipids were examined as substrates, Fuc-TI added fucose to IV3NeuNAcnLc4 but not to IV6NeuNAcnLc4, whereas Fuc-TII was unable to utilize either glycolipid as a substrate. Further studies showed that Fuc-TI and Fuc-TII possess novel specificities for glycolipids containing two lactosamine sequences as potential fucose acceptors. Fuc-TI exhibited good activities with VI3NeuNAcnLc6 and VI6NeuNAcnLc6 whereas Fuc-TII had very low activity with both substrates. Glycosidase digestions of the labeled products showed that Fuc-TI added fucose primarily to the internal N-acetylglucosamine of both glycolipids. The same preference for the internal N-acetylglucosamine was shown by Fuc-TI when nLc6 was the acceptor. In contrast, Fuc-TII preferred to transfer fucose to the external acceptor site of nLc6, consistent with the low activities of Fuc-TII with sialylated nLc6 derivatives. Thus the two enzymes preferentially add fucose to different N-acetylglucosamines in the same substrate, nLc6. This indicates that the biosynthetic pathway for fucosylation of polylactosamine sequences in glycolipids and glycoproteins will vary depending upon the particular alpha (1,3)fucosyltransferase present.

Animals↗
PubMed1987

Comparative prevalence of rabies antibodies among household and unclaimed/stray dogs as determined by the immune adherence haemagglutination assay.

The immune adherence haemagglutination assay (IAHA), widely used for human viral disease diagnosis, has been adapted for detection of rabies virus antibodies in dog sera. Rabies virus antibody titres obtained by the IAHA correlated well with those obtained by the currently accepted test for rabies antibody determination, the rapid-fluorescent-focus-inhibition test (RFFIT). Although it is not known if the antibodies detected in IAHA test represent neutralizing antibodies against rabies, IAHA has several advantages over the RFFIT: the IAHA is rapid, requiring about seven hours for results to be available; it is relatively inexpensive and easy to perform; uses reagents commonly available in any routine virology laboratory; and uses inactivated rabies virus, thus eliminating hazards associated with the use of live virus in RFFIT. Using this test we found that rabies antibody titres were significantly higher, and at the same time more prevalent, among household dogs than among the unclaimed/stray dogs. The results re-emphasize the increased hazard associated with unclaimed/stray dogs and the need for vaccination of all dogs.

Animals↗
PubMed1986

Lymphocyte function-associated antigen (LFA-1) is involved in B cell activation.

Human LFA-1 is a widely expressed leukocyte antigen present on cells of myeloid and lymphoid lineage. Monoclonal antibodies to LFA-1 have been shown to inhibit in vitro T cell immune functions. However, a role for LFA-1 in B cell activation has not been documented. To investigate this possibility, we examined the distribution of LFA-1 on normal, neoplastic, and EBV-transformed B cells as well as the ability of a monoclonal anti-LFA-1 antibody (NB-107) to inhibit B cell mitogenesis. NB-107 immunoprecipitates a noncovalently linked heterodimer of approximately 170,000 and 95,000 daltons. Sequential immunoprecipitation and cross-blocking studies showed that NB-107 identified a distinct epitope on the LFA-1 molecule. NB-107-defined LFA-1 was present on peripheral blood mononuclear cells (PBMC) from all normal individuals (N = 27) and on EBV-transformed cell lines (N = 9), but was absent from four of seven neoplastic B lymphoma lines. NB-107 was observed to profoundly inhibit the response of PBMC to the B cell mitogens anti-IgM (mean 71% inhibition) and lipopolysaccharide (mean 80% inhibition). In order to investigate the mechanism of inhibition, B cells were sequentially purified from PBMC by using a combination of E rosette depletion of T cells, monocyte removal by glass adherence, and finally cell sorting. These extensively enriched populations of B cells, although still responding to anti-mu, showed no evidence of inhibition by NB-107. Growth of EBV-transformed cell lines, cultured in the presence of NB-107, also was not inhibited by this antibody. When tested in assays for T cell function, NB-107 was shown to inhibit the mixed lymphocyte response, but had no effect on phytohemagglutinin stimulation of PBMC nor on the clonal growth and differentiation of granulopoietic, erythropoietic, and pluripotent progenitor cells. We conclude that anti-LFA-1 monoclonal antibody inhibits B cell mitogens via indirect effects on monocytes and/or T cells, rather than by a direct antiproliferative effect on B cells.

Animals↗
PubMed1986

High-frequency transfection of CHO cells using polybrene.

High-frequency transfection of CHO cells has been achieved for several plasmids, a cosmid library, and genomic DNA using Polybrene and dimethyl sulfoxide. All plasmid transfectants examined were stable and exhibited plasmid sequences in genomic DNA. The method is simple, reproducible, and succeeded with several independent CHO clones in the presence or the absence of carrier DNA, even at very low concentrations of plasmid DNA.

Animals↗
PubMed1986

Monoclonal antibodies to HLA-DP, DQ, and DR determinants: functional effects on the activation and proliferation of normal and EBV-transformed B cells.

To investigate the function of HLA class II molecules in B-cell activation, we generated three new anti-HLA class II monoclonal antibodies with differing specificities for DP, DQ, and DR determinants. These were tested for their ability to inhibit various B- and T-lymphocyte responses. Each of these antibodies (NB-29, DH-84, and DH-224) immunoprecipitates a heterodimer of approximately 35,000 and 28,000 mol wt from 125I-surface-labeled B-lymphoma cells, as shown by SDS-PAGE. NB-29 (IgG1) detects a polymorphic DQ determinant, while DH-224 (IgG1) is reactive with monomorphic DR determinants, and DH-84 (IgG2a) has specificity for DP, DQ, and DR. Both DH-224 and DH-84, but not NB-29, were found to inhibit significantly the stimulation of peripheral blood mononuclear cells (PBMC) by anti-mu (70%-90% inhibition) and by lipopolysaccharide (80%-90% inhibition), as measured by incorporation of tritiated thymidine. When added to highly purified populations of peripheral blood B cells, none of these anti-class II monoclonal antibodies inhibited anti-mu-induced stimulation. This suggests that the inhibitory effect that DH-224 and DH-84 have on the stimulation of unfractionated PBMC may be due to their ability to interfere with the action of accessory cells. Epstein-Barr-virus (EBV)-transformed B-cell lines, in contrast, showed substantial inhibition of growth when cultured in the presence of any of the three antibodies. With respect to T cells, DH-84 and DH-224 strongly inhibited the mixed lymphocyte response; NB-29 did not. None of these antibodies inhibited stimulation of PBMC by phytohemagglutinin (PHA). These findings suggest that DQ and DR HLA class II molecules have differing roles in B-cell activation and document a direct antiproliferative effect of anti-HLA class II monoclonal antibodies on the growth of EBV-transformed cell lines.

Animals↗
PubMed1985

Tn, a carcinoma-associated antigen, reacts with anti-Tn of normal human sera.

Tn antigen is the immediate precursor of the carcinoma (CA)-associated T antigen; both are masked in non-CA tissues. Tn antigen was detected by absorption of human anti-Tn antibody in 46 of 50 primary breast CAs and in all 6 metastases originating from Tn-positive primary CAs. Thirteen of 25 (52%) anaplastic CAs, but only 2 of 15 (13%) well differentiated CAs had more Tn than T; 1 anaplastic CA had neither antigen. Eighteen of 20 benign breast lesions had no Tn; the 2 positive lesions were premalignant. All 19 breast CAs, studied immunohistochemically, reacted strongly with human polyclonal anti-Tn; benign or normal glandular tissues had minimal or no reactivity. Among live cancer cell lines, the most malignant sublines had more Tn than T on their cell surfaces. Preliminary studies with rodent monoclonal anti-Tn and anti-T antibodies gave immunohistochemical reactivity patterns similar to those of the polyclonal antibodies, but the former were less sensitive in absorption tests. Tn is a CA marker that promises to be useful in tumor detection.

Animals↗
PubMed1985

Two monoclonal antibodies defining unique antigenic determinants on human B-lymphoma cells.

The non-Hodgkin's lymphomas are a clinically, morphologically, and immunologically heterogeneous group of diseases. Why lymphoma cells are unresponsive to normal regulatory growth controls and how they differ from normal lymphocytes are not well understood. In order to address these questions we have raised monoclonal antibodies to neoplastic B-cells. Two of these, LM-26 and LM-155, show a high degree of specificity for B-cell lymphomas. When tested by fluorescence activated cell sorter analysis, LM-26 reacted with 80% (18 of 23) of B-cell lymphomas freshly explanted from patients and LM-155 reacted with 20% (5 of 23). The antigenic determinant detected by LM-26 was also found to be present on four of seven neoplastic large cell B-lymphoma lines. LM-155 detected a determinant present on all seven of these lines. For neither monoclonal antibody was there any association between antibody reactivity and the morphological subtype of lymphoma examined or the type of cell surface immunoglobulin expressed. LM-155 reacted with one case of B-cell acute lymphoblastic leukemia. Neither antibody reacted with normal B-cell blasts, normal peripheral blood mononuclear or marrow cells, T-cell leukemias or lymphomas, or chronic lymphocytic leukemia cells. Lymphocytes from reactive lymphoid hyperplasias involving lymph nodes, spleen, peripheral blood, and lung were also negative for LM-26 and LM-155 binding or showed only a small percentage of cells positive (4-8%). Both monoclonals were unreactive with non-B-lymphoid neoplastic cell lines, nine of ten Epstein-Barr virus transformed B-cell lines, and cells freshly explanted from patients with cancers of diverse cellular origins. Fluorescence activated cell sorter analysis of the expression of the antigens defined by LM-26 and LM-155 on lymphoma cells and normal B-cell blasts suggests that they are not normal differentiation antigens associated with lymphocyte activation or proliferation. The highly restricted expression of detectable levels of antigens reactive with monoclonal antibodies LM-26 and LM-155 on non-Hodgkin's lymphoma cells suggests a possible relation to their neoplastic properties. From a practical viewpoint these monoclonals may prove useful in the diagnosis, classification, detection of residual disease, and treatment of the non-Hodgkin's lymphomas.

Antibodies, Monoclonal↗
PubMed1984

Differentiation of chronic lymphocytic leukemia from reactive lymphocytosis by the mouse red cell rosette assay.

The differential diagnosis of chronic lymphocytic leukemia (CLL) from other neoplastic and nonneoplastic lymphocytoses may be difficult, even when optimal clinical and laboratory data are available. The mouse red cell (M) rosette assay has been valuable in distinguishing normal and lymphomatous lymphocytes from those of CLL. Not yet tested is the ability of this test to separate reactive (benign) lymphocytes from morphologically similar but neoplastic cells of CLL. In the present study, lymphocytes of 58 subjects with CLL, reactive lymphocytosis, and healthy controls were evaluated for M rosette formation, in addition to the usual B and T cell markers. Lymphocytes from patients with CLL showed a marked increase in the ability to form M rosettes in comparison to those from normal controls and patients with reactive lymphocytosis. The formation of M rosettes was a more reliable marker for CLL than surface membrane immunoglobulin.

Adult↗