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Biomedical subjects

G G Miller

Publications and source records attributed to G G Miller.

At least 73 records · Page 4Linked to original sources
PubMed1995

Necrotizing fasciitis in childhood.

Necrotizing fasciitis is a rare entity in the pediatric population. Five cases of this soft tissue infection were treated at the authors' institution between January and December 1993. Three of the children were profoundly neutropenic secondary to chemotherapy. All five were treated with aggressive surgical debridement, frequent dressing changes, broad-spectrum antibiotics, and nutritional support. In addition, the patients with neutropenia received a combination of granulocyte-colony stimulating factor and granulocyte transfusions. One child died of overwhelming sepsis and bone marrow graft failure. The others eventually made a complete recovery. Necrotizing fasciitis may be becoming a more common problem in children. Aggressive chemotherapeutic regimens and more frequent use of bone marrow transplantation could be a factor in this. Early diagnosis and aggressive surgical therapy is critical. However, mortality may be significant, especially in patients with neutropenia. Leukocyte response to the infection may be a prognostic marker. Pseudomonas and enteric gram-negative organisms are seen frequently in immunocompromised children with necrotizing fasciitis. Antimicrobial selection should supply adequate coverage of these organisms.

Adolescent↗
PubMed1995

Uptake kinetics and intracellular localization of hypocrellin photosensitizers for photodynamic therapy: a confocal microscopy study.

Hypocrellins are naturally occurring compounds with photosensitizing properties in biological systems. We have prepared synthetic derivatives of hypocrellin B, which have promise as photosensitizers in the clinical application of photodynamic therapy. The intracellular localization and uptake kinetics of hypocrellin B and several selected hypocrellin congeners were determined semiquantitatively by fluorescence confocal microscopy in monolayer cultures of EMT6/Ed murine tumor cells. Each compound had unique uptake kinetics. Although no compound tested to date has demonstrated nuclear labeling, most could be detected in lysosomes, Golgi, endoplasmic reticulum and, to a minor extent, in cellular membranes. No two compounds gave identical labeling distributions. The differences are assumed to originate in physicochemical properties characteristic of each compound, which may ultimately impact upon the primary modality of phototoxicity.

Animals↗
PubMed1995

Interleukin-8 response of gastric epithelial cell lines to Helicobacter pylori stimulation in vitro.

Gastric infection with Helicobacter pylori activates a mucosal inflammatory response by mononuclear cells and neutrophils that includes expression of cytokines interleukin-1 beta (IL-1 beta), IL-6, tumor necrosis factor alpha, and IL-8. In this study, we analyzed the IL-8 response of human gastric cancer cell lines (Kato III, AGS, and MKN28) to H. pylori infection in vitro. IL-8 mRNA expression was detected by reverse transcription-PCR amplification of RNA extracted from epithelial cells after incubation with different H. pylori wild-type and mutant strains, and IL-8 secretion was measured by an enzyme-linked immunosorbent assay. Exposure to viable H. pylori induced IL-8 mRNA and protein synthesis in all three gastric cell lines but not in nongastric epithelial cell lines. Heat-killed H. pylori and a crude cytotoxin preparation did not induce significant IL-8 secretion. IL-8 mRNA peaked between 2 and 4 h postinfection, and IL-8 protein production was maximal 24 h postinfection. Exposure of gastric carcinoma cells to other gastrointestinal bacteria, such as Pseudomonas aeruginosa, Campylobacter jejuni, and Escherichia coli, but not Campylobacter fetus, induced IL-8 synthesis. Wild-type strains that expressed the vacuolating cytotoxin (Tox+) and a cytotoxin-associated gene (cagA) product (CagA+) induced significantly more IL-8 than did CagA- Tox- strains. However, there was no decrease in IL-8 induction by isogenic mutants of CagA-, Tox-, or Cag- Tox- strains or by a mutant lacking the urease subunits. These results indicate that exposure to H. pylori and other gram-negative organisms that do not colonize the gastric mucosa induces IL-8 production by gastric carcinoma cells in vitro. Although the CagA+ Tox+ phenotype of H. pylori is associated with enhanced IL-8 production by gastric cell lines, other bacterial constituents are clearly essential.

Antigens, Bacterial↗
PubMed1995

Detection of Helicobacter pylori gene expression in human gastric mucosa.

Mucosal and systemic immunologic recognition of cagA by Helicobacter pylori-infected individuals is associated with peptic ulcer disease; however, in the laboratory, expression of cagA is subject to artificial conditions which may not accurately reflect the conditions in host tissues. Gastric antral and body biopsy specimens and serum for anti-H. pylori immunoglobulin G serology were obtained from 42 patients. Biopsy specimens were studied by histology, culture, and reverse transcription PCR (RT-PCR). Oligonucleotide primers specific for H. pylori (16S rRNA, ureA, and cagA) were used to detect bacterial mRNA in gastric biopsy specimens. PCR was performed on DNA from corresponding H. pylori isolates to detect genomic 16S rRNA, ureA, and cagA. Of the 42 patients from whom clinical specimens were obtained, 25 were infected with H. pylori on the basis of both serology and histology or culture (i.e., tissue positive); 13 were negative by serology, histology, and culture; and 4 were positive by serology only. RT-PCR with 16S rRNA primers detected 24 of 25 tissue-positive and 0 of 17 tissue-negative patients (P < 0.001). RT-PCR with ureA primers detected 16 of 25 tissue-positive and 0 of 17 tissue-negative patients (P < 0.001). CagA mRNA was detected by RT-PCR in 14 of 25 gastric biopsy specimens in the tissue-positive group and in 0 of 17 gastric biopsy specimens in the tissue-negative group. PCR of genomic DNA for the presence of the cagA gene in the corresponding bacterial isolates correlated absolutely with cagA gene expression in gastric tissue. These results indicate that RT-PCR is a sensitive and specific method for the detection of the presence of H. pylori and the expression of H. pylori genes in human gastric tissue. Detection of H. pylori gene expression in vivo by this approach may contribute to improving the diagnosis and understanding the pathogenesis of H. pylori infections.

Antigens, Bacterial↗
PubMed1995

Heightened inflammatory response and cytokine expression in vivo to cagA+ Helicobacter pylori strains.

BACKGROUND: Helicobacter pylori strains that possess the cytotoxin-associated gene (cagA) are highly associated with peptic ulcer disease, but the role of cagA in pathogenesis is unknown. EXPERIMENTAL DESIGN: To test the hypothesis that cagA+ stains elicit a greater proinflammatory cytokine response in the gastric mucosa than cagA- strains, gastric biopsies were obtained from 52 patients and studied by histology, culture, enzyme-linked immunosorbent assay, and reverse transcription polymerase chain reaction. RESULTS: Of 52 patients, 32 (62%) were infected with H. pylori based upon both serology and histology or culture, 16 (31%) were negative by serology, histology, and culture, and four (7%) were positive by serology only. Of 15 H. pylori-infected patients with peptic ulceration, 14 (92%) were infected with cagA+ strains compared with 8 (50%) of 16 patients with gastritis alone, and those infected with cagA+ strains had significantly higher grades of inflammation in the gastric mucosa. Antral inflammation score was significantly associated with IL-8 production. Antral biopsies from infected patients, compared with uninfected patients, significantly more often demonstrated IL-1 beta, IL-2, and IL-8 expression, and those infected with cagA+ compared with cagA- strains significantly more often expressed IL-1 alpha and IL-1 beta and showed elevated antral IL-8 protein levels. Similarly, patients with ulcer disease significantly more often expressed antral IL-1 alpha and IL-8 than those without ulceration. CONCLUSION: These results indicate that infection with cagA+ H. pylori strains is associated with higher grades of gastric inflammation, correlating with enhanced mucosal levels of IL-8, and increased risk of peptic ulceration.

Antigens, Bacterial↗
PubMed1994

Detection of T cells responsive to a vascular growth factor in rheumatoid arthritis.

The primary lesion in rheumatoid arthritis (RA) is a destructive synovitis characterized by proliferation of endothelial cells, fibroblasts, and vascular smooth muscle cells, and with perivascular lymphocyte aggregates. A nonhematopoietic growth factor, acidic fibroblast growth factor (aFGF), may induce many of the biological features found in rheumatoid synovium, including T cell activation. To determine if aFGF-responsive T cells are increased in RA, we developed an assay to measure the frequency of peripheral blood T cells that are costimulated by aFGF. The data indicate that the frequency of aFGF-responsive T cells is increased in RA and may change with disease activity and treatment.

Arthritis, Rheumatoid↗
PubMed1994

Humoral and cellular immune recognition of Helicobacter pylori proteins are not concordant.

Helicobacter pylori is a major cause of chronic antral gastritis and peptic ulcer disease. Further definition is needed of the factors that determine whether infected individuals remain asymptomatic, or ultimately develop ulceration of the mucosa or transformation to malignancy. To explore the possibility that host response to H. pylori may play a role in the outcome of this infection, we have examined humoral and cellular recognition of several H. pylori proteins by seropositive and seronegative persons. A complex mixture of water-extractable cell proteins, which did not include lipopolysaccharide (LPS), was recognized by serum antibodies only in seropositive or infected individuals. IgG from seropositive subjects also bound to urease and to a heat shock protein (hsp)60 that is homologous to the 65-kD mycobacterial heat shock protein, while sera from uninfected individuals were negative. Although antibody responses to these antigens were restricted to seropositive subjects, T cell recognition of the same proteins was found in both seropositive and seronegative subjects. The water extract of H. pylori stimulated peripheral blood mononuclear cells (PBMC) from all subjects, while purified proteins activated lymphocytes of only some seropositive and seronegative subjects. PBMC that were activated by the H. pylori hsp60 did not respond to the autologous human p60 heat shock protein. These results demonstrate that, in contrast to antibody responses, T cell recognition of H. pylori proteins may occur in non-infected persons. In addition, the data suggest that in these subjects, peripheral lymphocytes that are activated by bacterial heat shock proteins do not mediate tissue damage by recognition of human heat shock homologues.

Adult↗
PubMed1994

Induction of acidic fibroblast growth factor and full-length platelet-derived growth factor expression in human cardiac allografts. Analysis by PCR, in situ hybridization, and immunohistochemistry.

BACKGROUND: Further understanding of cardiac allograft vasculopathy (CAV) is needed to improve long-term survival after cardiac transplantation. The diffuse hyperplasia of coronary intima characteristic of CAV suggests that growth factors may play a role in the development of CAV. Fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) are potent mitogens for smooth muscle cells (SMCs), and PDGF is an important cofactor in the pathogenesis of native coronary atherosclerosis. METHODS AND RESULTS: Reverse transcriptase/polymerase chain reaction (RT/PCR), in situ hybridization, and immunohistochemistry were used to determine whether transplantation results in increased cardiac expression of acidic (a) FGF, basic (b) FGF, and PDGF-A and -B chains. Sixty-eight myocardial biopsies from 36 heart transplant recipients and 7 normal hearts were analyzed by PCR. aFGF mRNA was present in 54 of 61 allograft biopsies and was not found in any normal heart. In situ hybridization and immunohistochemistry demonstrated diffuse, intense expression of a FGF mRNA and protein in allograft biopsies, predominantly in myocytes and vascular walls. Only scattered aFGF expression was observed in normal hearts. mRNA for the full-length isoform of PDGF-A chain was found in 43 of 61 allograft biopsies and was not detected in any normal heart. In situ hybridization and immunohistochemistry confirmed that full-length PDGF-A chain mRNA and PDGF protein were present in myocytes and vascular walls. CONCLUSIONS: Expression of aFGF and PDGF-A chain is significantly increased in cardiac allografts. Cardiac myocytes and vascular walls are the predominant sources of aFGF and PDGF. Diffuse expression of these growth factors in cardiac allografts may be important in the pathogenesis of CAV.

Adult↗
PubMed1994

Modification of alternative messenger RNA splicing of fibroblast growth factor receptors in human cardiac allografts during rejection.

Accelerated coronary atherosclerosis in cardiac transplants (cardiac allograft vasculopathy, CAV) is characterized by coronary intimal hyperplasia. Acidic fibroblast growth factor (aFGF) is a potent mitogen for vascular smooth muscle cells and endothelial cells, and its expression is increased in cardiac allografts, suggesting it may play a role in the pathogenesis of CAV. The activity of aFGF is dependent on binding to transmembrane receptors. To investigate whether receptors for aFGF are also induced after transplantation, polymerase chain reaction, in situ hybridization, and immunohistochemistry were used to analyze expression of four receptors for aFGF (FGFR1-FGFR4). Expression of mRNA encoding extracellular immunoglobulin-like domains of FGFR1 was increased 35-fold in cardiac allografts compared with normal hearts and was predominantly present in cardiac myocytes and vascular structures. Alternatively spliced mRNA that encodes transmembrane forms of FGFR1, which contain the signal-transducing tyrosine kinase domains, was induced in allografts during rejection, in infiltrating cells, vascular structures, and myocytes. In vitro experiments showed that differential expression of FGF receptor isoforms was induced by aFGF, and also by IL-6 and TGF-beta, which are expressed in cardiac allografts during rejection. The results show that expression of both aFGF and its receptors is altered in cardiac allografts and suggest that these events are important in the pathogenesis of CAV.

Adolescent↗
PubMed1993

The expression of acidic fibroblast growth factor (heparin-binding growth factor-1) and cytokine genes in human cardiac allografts and T cells.

The purpose of this study was to investigate the role of cytokines and growth factors in cardiac allograft rejection and vasculopathy (CAV). The polymerase chain reaction (PCR) was used to detect the expression of IL-1, IL-2, IL-4, IL-5, IL-6, TNF-alpha, IFN-gamma, TGF-beta, TCR-beta chain and aFGF genes in 21 myocardial biopsies obtained from 9 heart transplant patients. There was no statistically significant correlation between cytokine gene expression and rejection, although a trend toward increased IL-6 and TGF-beta expression was noted with rejection (6 of 10 biopsies with vs. 1 of 7 without rejection, and 4 of 9 biopsies with vs. none of 7 without rejection, respectively). IL-2 gene expression was detected in only 2 of 21 biopsies, both positive for rejection. IL-1, IL-4, IL-5, CD8, IFN-gamma, and TNF-alpha were not detected in any of the biopsies. TCR-beta chain mRNA was found in all biopsies, indicating the invariable presence of T cells regardless of histologic diagnosis of rejection. The aFGF gene was expressed in the majority (18 of 21) of biopsies, and its presence was not correlated with rejection. In addition to mRNA for the complete coding sequence of aFGF, two alternatively spliced mRNAs for aFGF were present in myocardial biopsies. Because aFGF and TCR beta genes were expressed in most biopsies, we determined whether aFGF mRNA was expressed in T cells; aFGF transcripts were found in 2 of 5 T-cell clones examined. Thus, aFGF mRNA in cardiac allografts may have been induced within the myocardium or elaborated by infiltrating T cells. The presence of mRNA for aFGF, a potent endothelial and smooth muscle cell mitogen, in allograft myocardium suggests that aFGF may play a role in the pathogenesis of CAV.

Alternative Splicing↗
PubMed1993

Expression of cytokine genes in human cardiac allografts: correlation of IL-6 and transforming growth factor-beta (TGF-beta) with histological rejection.

Cytokines may play critical roles in allograft rejection. Currently, a clear pattern of cytokine production that correlates with rejection has not emerged. Our preliminary studies suggested a trend toward increased IL-6 and TGF-beta gene expression in cardiac allografts during rejection. We have extended these studies using reverse transcriptase/polymerase chain reaction (RT/PCR) to detect the expression of IL-6, TGF-beta, and T cell receptor beta chain constant region (TCR-beta) genes in 21 additional consecutive myocardial biopsies obtained from six heart transplant patients and from five pre-transplant donor hearts. Cytokine gene expression was compared with histological diagnosis of rejection. There was strong correlation between IL-6 as well as TGF-beta gene expression, and histological rejection (6/8 biopsies with versus 0/7 without rejection (P = 0.006) and 7/9 biopsies with versus 0/7 without rejection (P = 0.003) respectively). Neither IL-6 nor TGF-beta transcripts were detected in any pre-transplant donor heart. TCR-beta chain mRNA was found in all allograft biopsies regardless of the presence of rejection, but was absent in pre-transplant donor hearts. Our results indicate that expression of IL-6 and TGF-beta is highly correlated with allograft rejection and thus may play an important role in regulation of cardiac allograft rejection. T cell infiltration of allografted myocardium is invariably detected by PCR regardless of histological rejection. The long-term functional significance of these cells in transplanted hearts needs further investigation.

Base Sequence↗
PubMed1993

A novel superantigen isolated from pathogenic strains of Streptococcus pyogenes with aminoterminal homology to staphylococcal enterotoxins B and C.

Streptococcus pyogenes (group A Streptococcus) has re-emerged in recent years as a cause of severe human disease. Because extracellular products are involved in streptococcal pathogenesis, we explored the possibility that a disease isolate expresses an uncharacterized superantigen. We screened culture supernatants for superantigen activity with a major histocompatibility complex class II-dependent T cell proliferation assay. Initial fractionation with red dye A chromatography indicated production of a class II-dependent T cell mitogen by a toxic shock-like syndrome (TSLS) strain. The amino terminus of the purified streptococcal superantigen was more homologous to the amino termini of staphylococcal enterotoxins B, C1, and C3 (SEB, SEC1, and SEC3), than to those of pyrogenic exotoxins A, B, C or other streptococcal toxins. The molecule, designated SSA, had the same pattern of class II isotype usage as SEB in T cell proliferation assays. However, it differed in its pattern of human T cell activation, as measured by quantitative polymerase chain reaction with V beta-specific primers. SSA activated human T cells that express V beta 1, 3, 15 with a minor increase of V beta 5.2-bearing cells, whereas SEB activated V beta 3, 12, 15, and 17-bearing T cells. Immunoblot analysis of 75 disease isolates from several localities detected SSA production only in group A streptococci, and found that SSA is apparently confined to only three clonal lineages as defined by multilocus enzyme electrophoresis typing. Isolates of one of these lineages, (electrophoretic type 2) are strongly associated with TSLS. The data identify SSA as a novel streptococcal superantigen that appears to be more related structurally to staphylococcal enterotoxins than to streptococcal exotoxins. Because abundant SSA production is apparently confined to only three streptococcal clonal lineages, the data also suggest that the SSA gene has only recently been acquired by S. pyogenes.

Amino Acid Sequence↗
PubMed1992

Identification of human T cells that require zinc for growth.

Zinc is an essential trace element required for normal function of the immune system. Deficiency of zinc results in marked thymic atrophy in experimental animals, and in man immunodeficiency is a recognized complication of zinc deprivation. Although numerous proteins require zinc as a cofactor, its precise functions in the immune system remain unknown. The mechanism by which metals stimulate lymphocytes, whether all T cells are responsive, and the relationship to zinc requirements have not been determined. We unexpectedly isolated a number of human T-cell lines that have a highly specific requirement for zinc. The ability to respond to zinc resides in only a subset of T cells since antigen-specific clones are not stimulated by zinc. Although proliferation requires the presence of antigen-presenting cells and is restricted by class II MHC antigens, antigen-presenting cells could not be pulsed with zinc to induce T-cell activation. Our results suggest that zinc-dependent T cells are a subset of CD4+ cells present in all normal individuals and that zinc stimulates their growth by novel mechanisms.

Antigen-Presenting Cells↗
PubMed1992

[Detection of Mycoplasma and its antibodies in the blood of HIV-positive patients and in healthy persons].

The comparative study of HIV-positive and clinically healthy persons has indicated that the antigens of M. pneumoniae, M. fermentans and U. urealyticum are encountered nearly twice more frequently in the blood of HIV-infected patients than in that of healthy individuals. Mycoplasma antibodies are detected in HIV-positive persons 12 times more frequently than in healthy ones. Among the HIV-infected persons there are those who have simultaneously antigens of some Mycoplasma species.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Isolation and characterization of a novel streptococcal superantigen.

The involvement of S. pyogenes (group A Streptococcus) in severe invasive disease, toxic-shock-like syndrome, and episodes of rheumatic fever led us to explore the possibility that these strains produce a novel superantigen. By using a superantigen-specific assay, we purified a 28-kDa protein from culture supernatants that stimulated T cells in an MHC class II-dependent, V beta-specific manner and designated it SSA, streptococcal superantigen. The amino terminus of SSA showed striking resemblance to SEB, SEC1, and SEC3. The structural homology exhibited by SSA to SEB was reflected functionally in that both of these superantigens bound the same class II isotypes. In contrast, SSA differed from SEB and other known bacterial superantigens with respect to its pattern of V beta-specific T-cell activation. SSA stimulated human T cells that expressed V beta 1, 3, 15, and perhaps V beta 5.2. Using SSA-specific antibodies in an immunoblot assay, we screened 26 strains of Lancefield group A Streptococcus and 16 strains of group B, C, and G Streptococcus. We found that SSA was expressed with high frequency in group A strains, but was absent from all other groups tested. These data establish SSA as a novel superantigen secreted by S. pyogenes. Further study of the structure and expression of SSA may reveal a role for this molecule in current episodes of severe streptococcal diseases.

Antibodies, Bacterial↗
PubMed1992

Collagen metabolism in the murine colon following X irradiation.

Female CBA mice, aged 16 weeks, were irradiated to the total pelvic region with either single doses (5-20 Gy) or two equal fractions (10- to 30-Gy total dose, 24-h interval) of 240 kV X rays. Total protein and collagen synthesis rates, collagen breakdown, and net collagen content of the colon were measured at various times postirradiation using a radioisotope incorporation method and HPLC analysis. Immunohistochemical staining and computerized image analysis were used to assess the relative amounts of collagen types I and III at various times postirradiation, in various regions of the colon. Total protein and collagen synthesis rates were elevated above control levels at 4 and 8 weeks postirradiation, as was collagen degradation. Values had returned to control levels by 16 weeks postirradiation, and there were no further changes up to 71 weeks postirradiation. The net amount of collagen in the colon did not change relative to controls at any time during the investigation. There was, however, increased immunohistochemical staining for collagen type I 52 weeks postirradiation in all regions of the colon and decreased staining of type III in the circular muscle layer and villi. Altered ratios of these two collagen isotypes are consistent with changes in mechanical properties of the tissue.

Animals↗
PubMed1991

Similarities in long-term cultures of blood and bone marrow from patients with acute myelogenous leukemia.

Dexter-type long-term cultures (LTC) were initiated with peripheral blood (PB) and/or bone marrow cells from 11 patients with acute myelogenous leukemia (AML), and 2 with myelodysplastic syndrome in blastic transformation. Marrow and PB cells from normal subjects served as controls. Assessment of nucleated cells and clonogenic progenitors in the adherent and nonadherent fractions of LTC revealed active hemopoiesis for greater than 5 wks in 4 of 8 cultures of AML blood, and 4 of 7 of AML marrow. The morphology and kinetics of nucleated cells and progenitors with putative normal (granulocyte-macrophage colony-forming units or CFU-gm), and abnormal (blast) phenotype in LTC from AML blood were similar to those from AML marrow, and adherent cells positive for collagen I and III and vimentin were found in both types of LTC. Growth of CFU-gm colonies ceased by wk 5-8 in AML cultures, significantly earlier than in LTC of normal marrow cells (survival of greater than 10 wks), which may indicate derivation of the CFU-gm from a transformed clone or deficiency of stromal function in the leukemic state. In most AML blood and AML marrow LTCs, growth of abnormal (blast) colonies continued until wk 4-6. This study demonstrates certain similarities of morphology and function between LTC of AML blood and AML marrow cells. LTC may provide a useful model for further analysis of circulating primitive hemopoietic progenitor cells in leukemic states.

Blood Cells↗