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Biomedical subjects

S Ratner

Publications and source records attributed to S Ratner.

At least 19 recordsLinked to original sources
PubMed1997

Microtubule retraction into the uropod and its role in T cell polarization and motility.

Spherical circulating T cells must polarize to extravasate. We have found that the polarization process includes a drastic reconfiguration of the tubulin cytoskeleton. In spherical T cells, the nucleus is surrounded by microtubules radiating from the microtubule organizing center (MTOC). During polarization the uropod (a slender posterior appendage) forms at the site of the MTOC. As the uropod buds out, the MTOC is carried in its distal tip. The attached microtubules retract into the uropod lumen, collapsing like the spokes of an umbrella into a compact sheaf. Experiments with microtubule inhibitors show that the retracted microtubules do not support the uropod or produce motive force. Instead, the data suggest that retraction of the relatively rigid microtubules into the streamlined uropod increases T cell deformability, thereby facilitating migration through constricted spaces. Microtubule retraction, therefore, may prove to be a strategy for accelerating extravasation without disassembly of the microtubule-based transport system.

Cell Movement↗
PubMed1996

Modulation of the development of humoral immunity by topically applied acetone, ethanol, and 12-O-tetradecanoylphorbol-13-acetate.

The effects of topically applied 12-O-tetradecanoylphorbol-13-acetate (TPA), acetone, and ethanol on systemic immune function were analyzed in SSIN mice. A total of one, four, or eight dorsal applications of solvent (50-300 microl/treatment, 2x/week) affected neither the overall cellularity of the spleen nor the relative proportions of splenic cells expressing CD4, CD8, or Ig surface markers. In contrast, overall cellularities of the spleen increased and relative T cell content of the spleen decreased in mice treated multiple times with TPA (2 microg/application in 0.2 ml acetone). The development of splenic B cells secreting IgM against sheep red blood cells (SRBC, a T-cell-dependent antigen) was retarded, and the overall duration of IgM synthesis was decreased, in mice immunized 1 hr after the last of four applications (>200 microl) of either solvent. Comparable retardations occurred in mice immunized as late as 7 days after termination of solvent treatment. However, solvent effects on the development of antibody-forming cells were not observed after eight topical applications or when TNP-LPS (a T-cell-independent antigen) was used as the immunogen. The effects of TPA on the development of IgM-secreting B cells were indistinguishable from those of the solvent used for its application. Although serum hemagglutination titers to SRBC correlated with the relative numbers of splenic B cells producing IgM, in vitro proliferative responses to B and T cell mitogens were not predictive of the effects of solvents or TPA on the development of antibody-secreting cells. Collectively, these studies demonstrate that topically applied acetone and ethanol can systemically modulate humoral immunity and emphasize the need for inclusion of nontreated controls when assessing the potential immunomodulatory activities of agents dissolved in acetone or ethanol.

Acetone↗
PubMed1996

Phorbol ester effects on splenic lymphocyte composition and cytotoxic T cell activities of SSIN mice: a strain deficient in CD8+ T cells.

SSIN mice are considerably more sensitive to the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) in two-stage skin carcinogenesis protocols than are most other strains and stocks of mice. Experiments were performed to determine whether there was an immunological basis for this sensitivity. SSIN mice were haplotyped and found to be H-2q. T cells represented approximately 31% of the splenic cellularity of non-treated SSIN mice, but approximately 44% in BALB/c, C57BL/6, B6C3F1 and SENCAR mice. Splenic CD4+/CD8+ T cell ratios were approximately 4.2, 2.9, 2.4, 1.8 and 1.7 in SSIN, SENCAR, BALB/c, B6C3F1 and C57BL/6 mice, respectively. The unusually high ratio in SSIN spleens was the consequence of reductions in CD8+ T cells. The ratio of CD4+/CD8+ T cells in SSIN thymocytes was similiar to that measured in the spleen. The splenic cytotoxic T lymphocyte (CTL) activities of the various murine strains inversely correlated with their splenic CD4+/CD8+ ratios and their sensitivities in two-stage skin carcinogenesis protocols. Repeated in vivo topical treatment of SSIN mice with TPA caused significant decreases in splenic T cell contents, but affected neither the splenic CD4+/CD8+ T cell ratio nor the development of a CTL response upon allogeneic tumor challenge. SSIN mice also had very low splenic natural killer cell activities. Furthermore, relative to the other strains of mice, SSIN mice were poor responders upon alloantigen challenge in mixed lymphocyte response assays. These findings demonstrate that SSIN mice differ markedly from other murine stocks and strains in their splenic lymphocyte composition and in their abilities to mount some MHC-restricted and non-restricted immunosurveillance processes.

Animals↗
PubMed1995

Breast cancer in males: DNA content and sex chromosome constitution.

The infrequent occurrence of breast cancer in males may reflect different etiologic factors and decreased hormonal dependence in comparison with the disease in females. We have attempted to define genetic differences in tumors of males that might reflect alterations in pathogenesis, by examining 22 tumors and 11 examples of gynecomastia for ploidy in relation to X- and Y- chromosome copy number by fluorescence in situ hybridization. Ploidy values were obtained for 15 infiltrating ductal and four papillary tumors (three invasive and one intraductal), two ductal carcinomas in situ, one papilloma, and 11 cases of gynecomastia by flow cytometry of disaggregated cells from the paraffin blocks. The malignant papillary lesions and 14 of the ductal tumors were examined by fluorescence in situ hybridization. DNA probes for pericentromeric regions of the X and Y chromosomes were reacted with formalin-fixed paraffin-embedded sections using a combination of avidin-biotin, digoxigenin-antidigoxigenin, and direct-labeling techniques. Seven multimodal tumors, including one carcinoma in situ, were clearly aneuploid by flow cytometry; the remaining 14 malignant tumors, the papilloma, and 11 examples of gynecomastia were within the diploid range. In two cases, aneuploid-tetraploid clones in tumors that were not recognized by flow cytometry were detected as subpopulations because of extra copies of the X and Y chromosomes. Two cases with aneuploid subpopulations by fluorescence in situ hybridization showed evidence of excess X-chromosome copy number, suggestive of preferential increase of this chromosome within the tumor, and the fluorescence in situ hybridization results also supported exclusion of constitutional Klinefelter's syndrome, although mosaicism could not be excluded.

Adult↗
PubMed1994

Chromosomal aberrations in two adrenocortical tumors, one with a rearrangement at 11p15.

Adrenocortical tumors are detected with increasing frequency, but symptomatic cases with excessive hormone production are rare. We investigated cytogenetically one benign aldosterone-producing tumor (Conn Syndrome)(case 1) and one malignant cortisol-producing tumor (Cushing Syndrome)(case 2). Radioimmunoassay of cell culture supernatant of case 2 detected cortisol secretion during 2 months in culture. Flow cytometry of spill-out cells from case 2 showed a bimodal pattern (DNA Index 1.0, 1.4). Case 1 revealed a marker chromosome in 4/25 cells analyzed; the marker was a long acrocentric partially derived from chromosome 2,der(2q). In case 2, a cytogenetic harvest was achieved after prolonged culture time (6 weeks) and a marker chromosome, add(11)(p15), was detected in 16/22 cells. A breakpoint of 11p13, as well as loss of heterozygosity of alleles on 11p15, has been reported in the literature for other malignant adrenocortical cancers.

Adrenal Cortex Neoplasms↗
PubMed1993

Chromosomal aberrations in two sporadic gastrinomas.

Results of cell culture and cytogenetic analysis (standard and fluorescent in situ hybridization, FISH) of two sporadic gastrinomas are reported. Maintenance of hormonal activity was assessed by detection of gastrin levels during the first 3 months in culture. Case 1 showed clonal aberrations consisting of two marker chromosomes: marker 1 is a large metacentric chromosome and marker 2 is a small acrocentric chromosome. Case 2 showed a constitutional polymorphism with chromosome 15p+ and a clone in the tumor cell culture with trisomy for chromosome 3. To our knowledge, this is the first cytogenetic report of sporadic gastrinomas (Zollinger-Ellison syndrome).

Aged↗
PubMed1992

Lymphocyte development of adherence and motility in extracellular matrix during IL-2 stimulation.

To extravasate into normal and neoplastic tissue, lymphocytes must migrate through the subendothelial basement membrane and underlying interstitium, structures rich in extracellular matrix (ECM). We have performed a time-course study of the development of motility in ECM by murine lymphocytes during in vitro exposure to high titers of IL-2 (1000 Cetus units/ml). This protocol generates immunotherapeutic lymphocyte populations expressing lymphokine-activated killer activity. Spontaneous motility was measured in three-dimensional gels of type I (interstitial) collagen or Matrigel, a model basement membrane. A newly developed assay permitted not only the measurement of distance traveled by the leading cell front, but also the separation of lymphocytes on the basis of three types of behavior. The motile fraction consisted of lymphocytes that penetrated beneath the ECM gel surface during an 18-h migration period. There were also two nonmotile fractions: the nonadherent fraction, which failed to bind to the gel surface; and the adherent fraction, which bound but did not penetrate during the assay period. During a 3- to 5-day exposure to high titer IL-2, both adherence and motility increased significantly. In type I collagen, cells of the NK lineage developed greater surface adherence and less motility than cells of the T lineage. The surface-adherent fraction expressed higher lymphokine-activated killer and NK activity than did the nonadherent or motile fractions. Under prolonged IL-2 stimulation (7 to 12 days), there was a decline in the percentage of cells exhibiting motility in both types of ECM, and an increase in the percentage of surface-adherent cells. The findings indicate that the behavior of an IL-2-stimulated lymphocyte population in ECM is profoundly influenced by the duration of IL-2 exposure. Furthermore, lack of lymphocyte motility may reflect two different behaviors, nonadherence and adherence without motility. The nonadherent and surface-adherent populations may differ in phenotypic distribution and function. The motility system described in this report will be useful in separating and studying the mechanisms that produce lymphocyte adherence and motility, and in understanding the in vivo implications of these behaviors.

Animals↗
PubMed1992

Motility of IL-2-stimulated lymphocytes in neutral and acidified extracellular matrix.

The migration of lymphocytes through extracellular matrix (ECM) is an essential feature of the infiltration process. In the course of their extravasation into poorly perfused neoplastic lesions, lymphocytes often encounter regions of acidified ECM. This study was designed to determine whether lymphocyte adherence and motility in ECM are influenced by ambient pH. Murine splenic lymphocytes, activated by culture with high-titer IL-2, were allowed to migrate into three-dimensional gels of Type I collagen, a major component of interstitial stroma, or into Matrigel, a basement membrane model. After 18 hr at pH 7.1, the leading cell front traveled a mean distance of approx 475 microM into Type I collagen gel. Approx 50% of the cells remained nonadherent, 25% adhered to the gel surface, and 25% were motile (penetrated beneath the surface). At pH 6.7, the leading-front distance increased significantly, by a factor of 1.4X, but there was little change in the proportion of cells exhibiting nonadherence, surface adherence, or motility. The relative motilities of CD3+ and AsGM1+ subsets were also unaltered. It therefore appears that acidification of collagen matrix increases the locomotory activity of motile lymphocytes, but causes little recruitment of nonmotile lymphocytes into the motile pool. Similar results were obtained in experiments with Matrigel. The increased motility observed at pH 6.7 did not reflect breakdown or relaxation of matrix lattices, as measured by the passive diffusion of latex beads of defined diameter. Preincubation of lymphocytes at pH 6.7 did not alter their subsequent motility in pH 7.1 gels. The findings establish ambient pH as a microenvironmental variable which can influence lymphocyte migration through ECM. The weak acidity characteristic of certain tumor microenvironments may be a factor which encourages lymphocyte infiltration through tissue matrix. Treatments which alter intratumor pH could potentially be used to manipulate the infiltration process for immunotherapeutic benefit.

Animals↗
PubMed1992

Lymphocyte migration through extracellular matrix.

The movement of lymphocytes through extracellular matrix (ECM) is an essential component of normal traffic and infiltration into inflammatory sites. This review surveys current knowledge of the mechanisms of lymphocyte migration through ECM, most of which was derived from work with in vitro models of basement membranes, interstitial stroma, or their constituent components. Normal lymphocyte motility is an extremely plastic property. Naive lymphocytes tend to be unresponsive to ECM components and many chemoattractants, but when exposed to antigens, artificial mitogens and certain lymphokines, they rapidly acquire locomotory capacity, which is expressed as increased polarity, adhesiveness, invasiveness and chemotactic response. Acquisition of locomotory capacity is associated with the G0/G1 transition, and activation of protein kinase C appears to be a key event. Preliminary evidence indicates that mitogenesis and differentiation to the memory phenotype trigger a long-lasting, possibly permanent elevation of locomotory response to ECM. Receptors for fibronectin, laminin and collagens I and IV have been implicated as mediators of lymphocyte motility, but these receptors have not been characterized in detail. Heparanases facilitate T cell movement through the basement membrane, but the role of proteases has not yet been defined. Major gaps remain in our understanding of the connection between in vitro models and specific stages of the infiltration process in vivo and of motility regulation at the molecular level.

Animals↗
PubMed1992

Dental fellowships in developmental disabilities help broaden care of disabled.

Continuation of the national trend toward deinstitutionalization and community placement for persons with developmental disabilities, physical handicaps and other medical problems will mean increased demand for dentists trained to care for this segment of the population. The New York State Office of Mental Retardation and Developmental Disabilities now offers dental fellowships in developmental disabilities to help fill the learning gap.

Academic Medical Centers↗
PubMed1991

Genetic markers in thyroid tumors.

Tissue from nine patients with malignant tumors and two with benign tumors was cultured briefly before cytogenetic analysis. The tumors included one goiter and one Hürthle cell adenoma, one lymphoma, one medullary carcinoma, two Hürthle cell cancers, and five papillary cancers, varying widely in clinical staging and histologic differentiation. When assessed, DNA content was aneuploid in two of six malignant tumors. Various culture conditions (oxygen levels, dissociation methods, and media) were evaluated; the end points were growth, cell differentiation, and time to first harvest. Clonal aberrations were detected in one of four successfully harvested papillary cancers: they consisted of trisomy 7 and a rearrangement of chromosome 10. The rea (10) seen in 22 of 27 cells involved bands q11-21. Two other papillary tumors and a medullary cancer (a family member with multiple endocrine neoplasia type IIA) showed tetraploidy and nonclonal numerically aberrant cells. A lymphoma and two benign lesions showed no cytogenetic abnormality. The tumor with rea (10) is of special interest because abnormalities of 10q have been reported repeatedly in thyroid tumors, including two cases of papillary thyroid tumors with a structural aberration similar to that of the presented case. This rearrangement could affect the ret-proto-oncogene, localized to 10q11.2 which is activated in some papillary thyroid carcinomas.

Adult↗
PubMed1990

Lymphocytes stimulated with recombinant human interleukin-2: relationship between motility into protein matrix and in vivo localization in normal and neoplastic tissues of mice.

Murine splenic lymphocytes nonspecifically stimulated with recombinant human interleukin-2 in vitro were fractionated according to their ability to migrate through type I collagen gel during a 24-hour period. After labeling with 111In and adoptive transfer into hosts of subcutaneous mammary tumors, motile fractions exhibited approximately twofold greater tumor localization and approximately twofold lower lung localization than nonmotile fractions. The results suggest that lymphocyte properties associated with motility through extracellular matrix also influence patterns of lymphocyte localization in vitro. It should be possible to identify these properties by comparative analysis of motile and nonmotile fractions obtained from various matrices.

Animals↗
PubMed1989

In vivo localization of lymphocytes labelled with low concentrations of Hoechst 33342.

Hoechst 33342 (HO 33342) is a fluorescent dye which binds specifically to DNA and can be used to label lymphocytes for in vivo migration studies. Lymphocytes were treated with varying concentrations of HO 33342 and assayed in vitro for effects on viability, mitogen-stimulated proliferation, and motility. In vivo traffic studies were performed to determine a dye concentration with minimal toxicity for lymphocytes, but sufficient fluorescence for detection of cells in frozen sections. The concentration reported to yield quantitative staining of nuclear DNA (10.7 microM, or 6 micrograms/ml) reduced motility and proliferative response, and resulted in an altered lymphocyte migration pattern compared to untreated lymphocytes. A concentration of 0.25 microM, however, produced no toxicity in the in vitro assays, and an in vivo migration pattern similar to that of untreated cells; lymphocytes stained with 0.25 microM HO 33342 for 30 min were readily observable in histological sections. This study indicates that the concentration of HO 33342 optimal for DNA staining may exert deleterious effects on in vivo lymphocyte traffic studies, and that far lower dye concentrations are more suitable for such studies.

Animals↗
PubMed1988

Motility and tumoricidal activity of interleukin-2-stimulated lymphocytes.

The motility of murine splenic lymphocytes stimulated nonspecifically by recombinant interleukin 2 (RIL-2) was studied in a three-dimensional collagen-gel system. Nonadherent BALB/c splenic lymphocytes were cultured in medium containing Cetus RIL-2 (700 to 1000 units/ml) or excipient control. They were then allowed to locomote randomly for 16 to 18 h into slabs of type I rat tail collagen gel. The gels were digested with collagenase, and total lymphocyte populations and motile subpopulations were collected and compared with respect to their lymphokine-activated killer activity (measured as 4-h cytotoxicity against the natural killer-resistant mammary adenocarcinoma line 410.4), their natural killer activity (measured as 4-h cytotoxicity versus lymphoma YAC-1), and their subset distribution (defined by immunofluorescence). Some of the slabs were not digested but fixed for measurement of leading-front distance. RIL-2-stimulated lymphocyte populations displayed greater motility than unstimulated populations; the mean leading front distance was 2.4 times greater, and the percentage of cells exhibiting motility was approximately doubled. The most motile RIL-2-stimulated cells, however, were not the most tumoricidal. Motile subpopulations displayed approximately 25 to 60% lower lymphokine-activated killer activity than did the total populations from which they were derived. Natural killer activity followed a similar pattern. Motile subpopulations contained a lower proportion of asialo-GM1+ and T-null cells than did total populations and a higher proportion of L3T4+ cells. Chemokinetic stimulation with alpha-interferon increased overall motility, but the lymphokine-activated killer activity of the motile subpopulation was still lower than that of the total population. Lymphocyte motility is important in the infiltration of tumors and other inflammatory lesions. The results indicate that the most tumoricidal lymphocytes in RIL-2-stimulated populations may not be the best tumor infiltrators, and that the tumoricidal activity of circulating lymphocytes may be a misleading indicator of the effectiveness of immunotherapy.

Animals↗
PubMed1988

Motility of murine lymphocytes during transit through cell cycle. Analysis by a new in vitro assay.

The relationship between the basal (spontaneous) motility of murine lymphocytes and their position in the cell cycle was examined in a new collagen gel motility assay system. Concanavalin A-stimulated or control lymphocytes were allowed to locomote into slabs of type I collagen gel. The assay configuration permitted extraction of both total populations and locomotory subpopulations as viable, single-cell suspensions suitable for phenotypic and cell analysis. Concanavalin A stimulation resulted in a significant increase in the mean distance traveled by the leading cell front in 4 hr, from 23 microns (controls) to 67 microns. The estimated percentage of motile cells increased from 0.9 to 2.8%. Similar increases were observed after 18 hr of locomotion. The SIg+, Thy-1+, L3T4+, and Ly-2+ subsets exhibited equivalent increases in motility. Total populations and locomotory subpopulations were allowed to incorporate 5-bromo-2'-deoxyuridine, and their cell cycle profiles were compared by dual parameter anti-5-bromo-2'-deoxyuridine, propidium iodide fluorescence analysis. Total population and locomotory subpopulations did not differ significantly with respect to the ratio G0/G1:S, indicating that lymphocytes in these two phases exhibited approximately equal motility. Cells in late S and G2 + M were significantly less motile; locomotory subpopulations contained 60 to 75% fewer G2 + M cells than the total populations from which they were derived. Taken together, the results indicate that the concanavalin A-induced increase in motility commences before S phase and that motility diminishes shortly before or during G2 + M.

Animals↗
PubMed1987

A new assay to measure monoclonal antibody-dependent complement or macrophage-mediated tumor growth inhibition.

A new in vitro assay has been developed to measure the inhibition of tumor growth by antibody and complement or by antibody and macrophages. Tumor cells (1 X 10(5) cells) or a mixture of tumor cells (1.5 X 10(4) cells) and macrophages (1.5 X 10(5) cells) are immobilized in a 1 microliter agarose droplet. Antibody is added to the medium bathing the agarose droplet. Complement is also added to wells containing droplets with tumor cells alone. The area covered by tumor cell monolayer is measured non-destructively from day 0 to day 7 with a split image tracing device. Cell growth is expressed by the increase in the square root of the measured area. This assay does not require isotopes and can be used to test tumor cells freshly dissociated from solid tissues. It permits cell-cell interactions which may change the sensitivity of tumor cells to various treatments. The extended period of observation allows the testing of multiple treatments. Surviving cells can also be recovered for further study. This assay may be useful for testing the efficacy of monoclonal antibody treatment on solid tumors.

Animals↗
PubMed1987

Reaction of argininosuccinase with bromomesaconic acid: role of an essential lysine in the active site.

We have undertaken studies on bovine liver argininosuccinase (L-argininosuccinate arginine-lyase, EC 4.3.2.1) with the active site-directed reagent bromo[U-14C]mesaconic acid, an analogue of fumaric acid. Reactivity, measured by enzyme inactivation, followed pseudo-first-order kinetics, and the rate increased with reagent concentration. Argininosuccinate completely protected the enzyme against inactivation, but neither arginine nor fumarate was protective. A plot of the degree of inactivation as a function of alkyl groups incorporated was extrapolated to 4 mol per mol of enzyme, or 1 mol per active site. After large-scale alkylation of the enzyme (and digestion with trypsin), two 14C-labeled tryptic peptides were isolated. These were chemically sequenced by the Edman method. The amino acid sequences proved to be identical with regions of the deduced amino acid sequences or argininosuccinases from human and yeast sources [O'Brien, W. E., McInnes, R., Kalumuck, K. & Adcock, M. (1986) Proc. Natl. Acad. Sci. USA 83, 7211-7215; Beacham, I. R., Schweitzer, B. W., Warrick, H. M. & Carbon, J. (1984) Gene 29, 271-279]. The 14C-labeled tryptic peptide in the active site region had the sequence Gly-Leu-Glu-Xaa-Ala-Gly-Leu-Leu-Thr-Lys; Xaa represents an unknown phenylthiohydantoin derivative detected in cycle 4. The corresponding amino acid was identified as lysine-51 on the basis of sequence similarity with human and yeast amino acid sequences in this region. The reaction of the enzyme with the alkylating agent and the specific protection against inactivation by argininosuccinate suggest that this lysine residue has an essential role in the binding of argininosuccinate to the enzyme and, consequently, is essential for catalysis.

Amino Acid Sequence↗