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S C Watkins

Publications and source records attributed to S C Watkins.

At least 181 records · Page 10Linked to original sources
PubMed1994

Fibroblasts genetically engineered to secrete interleukin 12 can suppress tumor growth and induce antitumor immunity to a murine melanoma in vivo.

Interleukin 12 (IL-12), a disulfide-linked heterodimeric cytokine produced primarily by macrophages, is composed of light (p35) and heavy (p40) chains. It binds to a receptor on T-cells and natural killer cells, promoting the induction of primarily a TH1 response in vitro and in vivo. To determine whether paracrine IL-12 secretion can alter tumor cell growth or promote antitumor immunity, we have developed a delivery system using genetically engineered fibroblasts in murine tumor models. NIH3T3 cells were stably transfected to express 100-240 units/10(6) cells/48 h of IL-12 using expression plasmids carrying both the murine p35 and p40 genes of murine IL-12. The effects of paracrine secretion of IL-12 on tumor establishment and vaccination models were examined using the poorly immunogenic murine melanoma cell line (BL-6) in C57BL/6 mice. To determine the effects of IL-12 on tumor formation, nonirradiated BL-6 cells were inoculated s.c. into C57BL/6 mice admixed with NIH3T3 cells transfected with both subunits of mIL-12 (3T3-IL-12) or with cells transfected with only the neomycin phosphotransferase gene (3T3-Neo). Compared to mice given injections of BL-6 alone, the day of emergence of detectable tumors was significantly delayed in mice given injections of BL-6 admixed with 3T3-IL-12, but not in mice with BL-6 admixed with 3T3-Neo. Effectiveness in this system was related to the amount of IL-12 expressed by the 3T3-IL-12. To determine the ability of locally secreted IL-12 at the tumor site to induce antitumor immunity, 10(6) irradiated tumor cells mixed with 3T3-IL-12 or 3T3-Neo were injected as a vaccine, and the response to a tumor challenge was subsequently examined. With a tumor challenge of less than 1 x 10(5) nonirradiated BL-6 cells, significant delay of establishment of tumor was noted with a relatively small amount of IL-12 secretion (1.2 units/5 x 10(5) cells/48 h). Larger amounts of secreted IL-12 provided no additional therapeutic benefit. Histological examination of tumor inoculum with 3T3-IL-12 secreting a high level of IL-12 showed peritumoral accumulation of macrophages, a characteristic capsule around the tumor composed of palisades of fibroblasts, and decreased numbers of CD4+ cells in the tumor. These results suggest that local delivery of IL-12 inhibits tumor growth in a dose dependent manner but leads to the development of an antitumor immune response when IL-12 is expressed at the tumor site at the relatively small amount indicated above.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗
PubMed1994

Quantitative and morphologic analysis of bacterial translocation in neonates.

OBJECTIVE: To elucidate the mechanisms of bacterial translocation in animals fed a conventional formula by correlating transmucosal bacterial passage in vitro with the structural characteristics of the neonatal intestinal mucosa. DESIGN: Newborn rabbits were randomized to receive a conventional formula or breast milk. Bacterial translocation to the mesenteric lymph nodes, liver, and spleen was quantitated after 7 days, and transmucosal passage of bacteria was measured in vitro using the Ussing chamber. The mucosal membranes were examined by light, transmission electron, and confocal laser scanning microscopy. RESULTS: Bacterial passage was rarely seen in the breast milk-fed animals in contrast to the formula-fed animals. Unlike the normal-appearing membranes from breast milk-fed animals, the epithelial cells of formula-fed animals were vacuolated but healthy, with normal polarization and microvillus border by confocal laser scanning microscopy. Villi of formula-fed animals were less densely packed than those of the breast milk-fed animals. Bacterial adhesion, internalization, and transmucosal passage were seen only in membranes from formula-fed animals. Transmission electron microscopy demonstrated bacteria incorporating into the epithelial surface through an active phagocytic process, with rearrangement of the actin cytoskeleton. Once internalized, these bacteria were seen within the cytoplasmic vacuoles and subsequently in the submucosa. No bacteria passed between epithelial cells. CONCLUSION: Morphological changes in the intestinal mucosa of formula-fed newborn rabbits may increase permeability to bacteria.

Animals↗
PubMed1994

Nongranular proteolytic enzymes of rat IL-2-activated natural killer cells. II. Purification and identification of rat A-NKP 1 and A-NKP 2 as constituents of the multicatalytic proteinase (proteasome) complex.

We have recently described nongranular, cytosolic, high-molecular-weight trypsin-like (A-NKP 1) and chymotrypsin-like (A-NKP 2) proteases of interleukin-2-activated rat natural killer (A-NK) cells. A functional correlation between the inactivation of A-NKP 2 and the inhibition of rat A-NK cell-mediated cytotoxicity was found. Herein we describe the 6,000-fold purification of A-NKP 2 to apparent homogeneity following: isopycnic sucrose gradient fractionation of postnuclear supernatants, molecular sieve chromatography, and heparin-Sepharose chromatography. We also report the novel finding that A-NKP 2 as well as A-NKP 1, derived from either rat A-NK cells or the rat NK leukemic cell line CRNK-16, are constituents of the multicatalytic proteinase (MCP/proteasome) complexes of these cells. Characteristic biochemical, biophysical, and electron microscopic/ultrastructural similarity to the rat liver proteasome was observed. However, Western blot analysis using polyclonal antibodies to the rat liver proteasome clearly indicated differences in the rat hepatic proteasome and the CRNK-16-derived proteasomal subunits. The identification, characterization, and purification of A-NKP 1 and A-NKP 2, described herein, now allow for further investigation of the potential role of these proteasome components in NK cell function. Moreover, the proteasome of NK and A-NK cells can now be compared and contrasted to the granzymes of lytic granules with respect to their role in cell-mediated cytotoxicity.

Animals↗
PubMed1994

Breast milk protects the neonate from bacterial translocation.

Clinical and experimental evidence suggests that breast milk enhances the neonatal gut barrier. Using bacterial translocation (BT) as a measure of gut barrier function, a series of experiments was designed to explore the relationship between the neonatal gut barrier and breast milk as well as the factors associated with the feeding of breast milk. Full-term newborn rabbits were assigned to one of four groups: formula-fed (group I), fed with colostrum plus formula (group II), breast-fed with breast milk (group III), and fed with colostrum plus stored breast milk (group IV). At 7 days of age, body weights were obtained, the rabbits were killed, and the mesenteric lymph nodes (MLN), liver, and spleen were quantitatively cultured for translocating bacteria. The cecum was cultured for aerobic and anaerobic enteric organisms. Distal ileal tissues were examined by light and transmission electron microscopy and compared among groups. The viability of cells in the stored, frozen breast milk was assessed by Trypan blue staining. Group I rabbits had significantly lower mean body weights compared with the other groups. The animals breast-fed breast milk had no BT to the MLN or liver and had a 9% incidence of BT to the spleen. There was no difference between BT in groups III and IV. The stored breast milk contained no viable cells. The incidence of BT to all three areas was significantly lower than in groups I and II. The animals fed with formula alone had the highest incidence of BT to the MLN (88%), liver (60%), and spleen (32%). BT in this group was significantly higher compared with groups III and IV.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Characterization and kinetics of MHC class I-restricted presentation of a soluble antigen delivered by liposomes.

We describe in this report the characterization and kinetics of MHC class I-restricted presentation of a soluble antigen delivered by liposomes. pH-sensitive liposomes delivered ovalbumin (OVA) to the class I pathway more efficiently than pH-insensitive liposomes; the latter showed competence in the class I-restricted sensitization only when a large antigen loading dose was used. Such sensitization of EL4 cells resulted from substantial delivery of the ingested liposomal OVA to the cytosolic compartment as revealed with immunogold electron microscopy. Most of the ingested liposomal OVA were rapidly catabolized and released into the extracellular medium. The residual processed antigen took about 2 h to egress to the cell surface for recognition by CTL. The liposome-mediated antigen presentation exhibited a transient kinetics which was manipulable with antigen dose. Brefeldin A, an ER-to-Golgi transport inhibitor, strongly inhibited such presentation. The antigens displayed on the cell surface could also be removed by a brief acid wash of the cells and subsequently reappeared on the surface if an intracellular pool of antigen existed at the time of acid wash.

Animals↗
PubMed1994

The size and symmetry of B capsids of herpes simplex virus type 1 are determined by the gene products of the UL26 open reading frame.

Herpes simplex virus type 1 (HSV-1) B capsids are composed of seven proteins, designated VP5, VP19C, 21, 22a, VP23, VP24, and VP26 in order of decreasing molecular weight. Three proteins (21, 22a, and VP24) are encoded by a single open reading frame (ORF), UL26, and include a protease whose structure and function have been studied extensively by other investigators. The protease encoded by this ORF generates VP24 (amino acids 1 to 247), a structural component of the capsid and mature virions, and 21 (residues 248 to 635). The protease also cleaves C-terminal residues 611 to 635 of 21 and 22a, during capsid maturation. Protease activity has been localized to the N-terminal 247 residues. Protein 22a and probably the less abundant protein 21 occupy the internal volume of capsids but are not present in virions; therefore, they may form a scaffold that is used for B capsid assembly. The objective of the present study was to isolate and characterize a mutant virus with a null mutation in UL26. Vero cells were transformed with plasmid DNA that encoded ORF UL25 through UL28 and screened for their ability to support the growth of a mutant virus with a null mutation in UL27 (K082). Four of five transformants that supported the growth of the UL27 mutant also supported the growth of a UL27-UL28 double mutant. One of these transformants (F3) was used to isolate a mutant with a null mutation in UL26. The UL26 null mutation was constructed by replacement of DNA sequences specifying codons 41 through 593 with a lacZ reporter cassette. Permissive cells were cotransfected with plasmid and wild-type virus DNA, and progeny viruses were screened for their ability to grow on F3 but not Vero cells. A virus with these growth characteristics, designated KUL26 delta Z, that did not express 21, 22a, or VP24 during infection of Vero cells was isolated. Radiolabeled nuclear lysates from infected nonpermissive cells were layered onto sucrose gradients and subjected to velocity sedimentation. A peak of radioactivity for KUL26 delta Z that sedimented more rapidly than B capsids from wild-type-infected cells was observed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the gradient fractions showed that the peak fractions contained VP5, VP19C, VP23, and VP26. Analysis of sectioned cells and of the peak fractions of the gradients by electron microscopy revealed sheet and spiral structures that appear to be capsid shells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Cross-species graft-versus-host-disease is accompanied by a donor-derived cellular immune response.

Acute graft-versus-host-disease is classically described as reactivity of donor lymphocytes to recipient alloantigens. To date there is debate as to whether GVHD can be induced across a species barrier. We recently reported the induction of stable xenogeneic chimerism (rat-->mouse) and donor-specific transplantation tolerance using the transplantation of untreated rat bone marrow cells into B10 mouse recipients. Survival of chimeras was excellent, and there was no evidence of GVHD. We now describe the induction of xenogeneic GVHD by transplanting large numbers of donor rat spleen cells with the bone marrow inoculum. All chimeras that received bone marrow and untreated spleen cells developed an external appearance compatible with GVHD and had a median survival time of 14 days. Mice that received equivalent numbers of untreated rat bone marrow alone appeared healthy, had no evidence for GVHD, and survived > 90 days. The usual epithelial target tissues for allogeneic GVHD in those mice that received xenogeneic bone marrow and spleen cells showed the presence of tissue injury and histologic features compatible with GVHD. Donor rat MHC class I and class II positive cells were prominent cell types present in the tongues of mice that developed features of GVHD, and this was accompanied by a significant inflammatory tissue response with loss of the dermal-epidermal architecture. In contrast, fully xenogeneic chimeras without GVHD had no evidence for tissue injury or pathologic cellular infiltrates when examined by immunohistochemical analysis. These data suggest that although fully xenogeneic chimeras resist GVHD, GVHD can be induced across a species barrier if sufficient numbers of donor rat spleen cells are added to the bone marrow inoculum. Further comparisons of these models may provide an approach to study the mechanisms responsible for xenoreactivity in vivo.

Animals↗
PubMed1993

Nuclear localization of bacterial Streptoalloteichus hindustanus bleomycin resistance protein in mammalian cells.

Prokaryotes produce a variety of toxins that affect genomic function of both eukaryotes and prokaryotes. The 375-base pair bacterial gene Streptoalloteichus hindustanus (Sh) ble encodes a small protein, Streptoalloteichus hindustanus bleomycin resistance protein (BRP), that inhibits in vitro DNA cleavage by the prokaryotic glycopeptide bleomycin, which is a clinically used anticancer drug. NIH/3T3 cells infected with a retroviral vector containing Sh ble (SH-9 cells) were highly resistant to the cytotoxicity of bleomycin-like drugs but not to the cytotoxicity of other, structurally unrelated, DNA-cleaving agents. Expression of BRP did not markedly alter total cellular content or distribution of bleomycin-like compounds. Fluorescently labeled bleomycin was primarily localized in cytoplasmic vesicles in NIH/3T3 and SH-9 cells, whereas BRP, which has no established nuclear localization sequence, was segregated to the nucleus and more specifically to euchromatin. This karyophilic BRP may intercept bleomycin in the nucleus.

3T3 Cells↗
PubMed1992

Negative inotropic effects of cytokines on the heart mediated by nitric oxide.

The direct effects of pro-inflammatory cytokines on the contractility of mammalian heart were studied. Tumor necrosis factor alpha, interleukin-6, and interleukin-2 inhibited contractility of isolated hamster papillary muscles in a concentration-dependent, reversible manner. The nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) blocked these negative inotropic effects. L-Arginine reversed the inhibition by L-NMMA. Removal of the endocardial endothelium did not alter these responses. These findings demonstrate that the direct negative inotropic effect of cytokines is mediated through a myocardial nitric oxide synthase. The regulation of pro-inflammatory cytokines and myocardial nitric oxide synthase may provide new therapeutic strategies for the treatment of cardiac disease.

Animals↗
PubMed1992

The subcellular distribution of chromosome 6-encoded dystrophin-related protein in the brain.

Chromosome 6-encoded dystrophin-related-protein (DRP) shows significant structural similarities to dystrophin at the carboxyl terminus, though the two proteins are encoded on different chromosomes. Both DRP and dystrophin are expressed in muscle and brain and show some similarity in their subcellular localization. For example, in skeletal muscle both are expressed at neuromuscular and myotendinous junctions. However, while dystrophin is absent or severely reduced in Duchenne/Becker muscular dystrophy, DRP continues to be expressed. Within the brain, dystrophin is enriched at the postsynaptic regions of specific subsets of neurons, while the distribution of DRP is yet to be described. In this study we demonstrate a distinct though highly specific pattern of distribution of DRP in the brain. DRP is enriched in the choroid plexus, pia mater, intracerebral vasculature, and ependymal lining. Within the parenchyma proper, DRP is located at the inner plasma face of astrocytic foot processes at the abluminal aspect of the blood-brain barrier. The distribution of DRP is conserved across a large evolutionary distance, from mammals to elasmobranchs, suggesting that DRP may play a role in the maintenance of regional specializations in the brain.

Animals↗
PubMed1992

Association of murine CD31 with transmigrating lymphocytes following antigenic stimulation.

Human CD31 is a recently characterized molecule present on leukocytes, platelets, and endothelium. Its function is not known. Because it is a member of the immunoglobulin superfamily and structurally homologous to carcinoembryonic antigen, a putative intercellular adhesion molecule, it is believed that CD31 may function also as an adhesion molecule. In this report, we characterize the cellular reactivity of a monoclonal antibody to a murine protein that is homologous to CD31. To delineate the cellular reactivity of the murine CD31 homologue recognized by our monoclonal antibody, we used immunoperoxidase and immunoelectron microscopic techniques. The most striking finding was that the putative murine homolog of CD31 is expressed in particularly high amounts on endothelium-adherent lymphocytes transmigrating across sinusoidal or venular vascular boundaries. Such a distribution was apparent in draining murine lymph nodes during the peak of an immune response after immunization with a protein antigen in adjuvant, a situation in which there are many transmigrating lymphocytes. Immunoelectron microscopic analysis also shows that CD31 is predominantly distributed on portions of transmigrating lymphocytes that are in contact with or adjacent to areas of contact with endothelial cells. These findings suggest a previously undescribed role for CD31 in lymphocyte recruitment and transmigration.

Amino Acid Sequence↗
PubMed1991

Platelet activation by a synthetic hydrophobic polymer, polymethylmethacrylate.

Platelets adhere to artificial surfaces in the initial stage of thrombus formation, but the subsequent steps in signal transduction that lead to platelet activation by artificial surfaces are not understood. When 0.325-micron diameter beads composed of a hydrophobic polymer, polymethylmethacrylate (PMMA), were added to gel-filtered aequorin-loaded platelets suspended in media containing Ca2+, the platelets aggregated; addition of fibrinogen was not required. Platelet aggregation was preceded by an increase in cytoplasmic Ca2+ and was accompanied by phosphorylation of the 47-Kd substrate of protein kinase C (PKC), 5-hydroxytryptamine (5-HT) release, and accumulation of phosphatidic acid. All these effects were partially inhibited by apyrase and aspirin. Monoclonal antibodies (MoAbs) 7E3 and M148 and the synthetic peptides RGDS and fibrinogen gamma chain fragment 400-411, all of which bind to the platelet fibrinogen receptor glycoprotein IIb-IIIa (GPIIb-IIIa) and inhibit fibrinogen binding, prevented PMMA-induced aggregation but did not inhibit the Ca2+ increase. Chymotrypsin-treated platelets aggregated after addition of fibrinogen, but not PMMA. We conclude that platelets interact initially with PMMA at membrane sites other than those required for fibrinogen binding, leading to activation of membrane phospholipases and PKC, an increase in cytoplasmic Ca2+, release of 5-HT, ADP, and fibrinogen from storage granules, and to platelet aggregation.

Adenosine Diphosphate↗
PubMed1991

Intracellular pathway of a mucin-type membrane glycoprotein in mouse mammary tumor cells.

Epiglycanin, a mucin-type glycoprotein, was found by immunoelectron microscopy to be located in cytoplasmic compartments, as well as at the surface of the TA3-Ha mammary carcinoma ascites cell. The glycoprotein was identified by means of gold-labeled secondary antibody bound to a primary anti-epiglycanin monoclonal antibody or by lectins specific for carbohydrate structures in epiglycanin. The primary antibody recognized a glycopeptide component containing a beta-D-(1----3)-D-GalNAc chain attached to a serine or threonine residue. Two routes to the cell surface from epiglycanins's first-recognized location in the trans-Golgi reticulum were suggested. Its presence in vesicles, which fuse with the cell surface, would explain the presence of epiglycanin as an integral membrane protein. Some of these observed vesicles, however, may be endocytotic in character. Epiglycanin was also found in large multivesiculate sacs which were observed on occasion to be open to the extracellular milieu. This finding, as well as the observed fusion of small vesicles from the trans-Golgi network with the sacs, strongly suggested exocytotic migration for the large sacs. Endocytotic migration may also be possible, although incubation of viable cells with gold-labeled antiepiglycanin antibody resulted in minimal uptake within the intracellular sacs, and incubation with [125I]-epiglycanin under metabolic conditions resulted in no detectable uptake of radiolabel by the cells.

Animals↗
PubMed1991

Immunolocalization and developmental expression of dystrophin related protein in skeletal muscle.

Dystrophin Related Protein is the recently identified protein product of a large autosomal transcript, showing significant similarity to dystrophin at the carboxyl terminus. Dystrophin related protein and dystrophin share a similar abundance and molecular weight, however, they differ both in their tissue distribution and expression in Duchenne/Becker muscular dystrophy. Here we define the immunolocalization of dystrophin related protein to neuromuscular and myotendinous junctions, along with peripheral nerves and vasculature of skeletal muscle. Groups of regenerating muscle fibres as well as embryonic and neonatal muscle express far greater amounts of dystrophin related protein compared with adult mdx mice. These findings may explain the paradoxical labelling seen using dystrophin antibodies in Duchenne patients and dystrophin deficient mdx mice. Finally, no abnormalities of dystrophin related protein expression were detected in three patients with Duchenne-like autosomal recessive muscular dystrophy.

Animals↗
PubMed1991

The subcellular distribution of dystrophin in mouse skeletal, cardiac, and smooth muscle.

We use a highly specific and sensitive antibody to further characterize the distribution of dystrophin in skeletal, cardiac, and smooth muscle. No evidence for localization other than at the cell surface is apparent in skeletal muscle and no 427-kD dystrophin labeling was detected in sciatic nerve. An elevated concentration of dystrophin appears at the myotendinous junction and the neuromuscular junction, labeling in the latter being more intense specifically in the troughs of the synaptic folds. In cardiac muscle the distribution of dystrophin is limited to the surface plasma membrane but is notably absent from the membrane that overlays adherens junctions of the intercalated disks. In smooth muscle, the plasma membrane labeling is considerably less abundant than in cardiac or skeletal muscle and is found in areas of membrane underlain by membranous vesicles. As in cardiac muscle, smooth muscle dystrophin seems to be excluded from membrane above densities that mark adherens junctions. Dystrophin appears as a doublet on Western blots of skeletal and cardiac muscle, and as a single band of lower abundance in smooth muscle that corresponds most closely in molecular weight to the upper band of the striated muscle doublet. The lower band of the doublet in striated muscle appears to lack a portion of the carboxyl terminus and may represent a dystrophin isoform. Isoform differences and the presence of dystrophin on different specialized membrane surfaces imply multiple functional roles for the dystrophin protein.

Animals↗