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Biomedical subjects

J Roman

Publications and source records attributed to J Roman.

At least 37 records · Page 2Linked to original sources
PubMed2000

Novel type of BCR-ABL transcript in a chronic myelogenous leukaemia patient relapsed after bone marrow transplantation.

We identified a novel BCR-ABL transcript in a chronic myelogenous leukaemia (CML) patient who relapsed after bone marrow transplantation (BMT), containing a fusion between part of BCR exon 3, 44 nucleotides derived from ABL intron 1b and ABL exon 2. The breakpoints were located within BCR exon 3 on chromosome 22 and within the ABL intron 1b on chromosome 9, and the transcript derives from a splicing of ABL exon 2 to a putative splicing acceptor site 44 nucleotides downstream to the breakpoint on chromosome 9. The patient's clinical course strengthens the idea that short forms of BCR-ABL transcripts are associated with a more aggressive disease.

Adult↗
PubMed2000

Dietary separation of sympatric carnivores identified by molecular analysis of scats.

We studied the diets of four sympatric carnivores in the flooding savannas of western Venezuela by analysing predator DNA and prey remains in faeces. DNA was isolated and a portion of the cytochrome b gene of the mitochondrial genome amplified and sequenced from 20 of 34 scats. Species were diagnosed by comparing the resulting sequences to reference sequences generated from the blood of puma (Puma concolor), jaguar (Panthera onca), ocelot (Leopardus pardalus) and crab-eating fox (Cerdocyon thous). Scat size has previously been used to identify predators, but DNA data show that puma and jaguar scats overlap in size, as do those of puma, ocelot and fox. Prey-content analysis suggests minimal prey partitioning between pumas and jaguars. In field testing this technique for large carnivores, two potential limitations emerged: locating intact faecal samples and recovering DNA sequences from samples obtained in the wet season. Nonetheless, this study illustrates the tremendous potential of DNA faecal studies. The presence of domestic dog (Canis familiaris) in one puma scat and of wild pig (Sus scrofa), set as bait, in one jaguar sample exemplifies the forensic possibilities of this noninvasive analysis. In addition to defining the dietary habits of similar size sympatric mammals, DNA identifications from faeces allow wildlife managers to detect the presence of endangered taxa and manage prey for their conservation.

Animal Nutritional Physiological Phenomena↗
PubMed2000

Cytokine message and protein expression during lung granuloma formation and resolution induced by the mycobacterial cord factor trehalose-6,6'-dimycolate.

Trehalose-6,6'-dimycolate (TDM), or cord factor, is a mycobacterial cell wall component that induces granuloma formation and proinflammatory cytokine production in vivo and in vitro. The purpose of this work was to better understand the mechanisms by which TDM promotes lung granuloma formation. This was accomplished by characterizing cytokine mRNA expression during TDM-induced alveolitis culminating in cohesive granuloma development. A single intravenous injection of TDM given to C57BL/6 mice produced lung granulomas that peaked in number 5 days after challenge and were nearly resolved by 14 days. mRNA in whole lung preparations was quantitated by bioluminescent RT-PCR. Tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 were significantly elevated during granuloma development and decreased during granuloma resolution. There were no detectable changes in mRNA for interferon-y (IFN-y), IL-2, IL-4, IL-5, IL-10, and IL-12(p40). The level of TNF-alpha protein extracted from lung minces highly correlated with morphologic indices of granulomatous inflammation, indicating that it may be an important modulator of the inflammatory intensity induced by TDM. TDM may interact specifically with macrophages in vivo, as evidenced by induction of TNF-alpha, IL-1beta, and IL-6, but not IFN-gamma, protein in bone marrow-derived macrophages from C57BL/6 mice. TDM may therefore play an important role early in macrophage activation during the host granulomatous response to mycobacteria.

Adjuvants, Immunologic↗
PubMed2000

Phorbol ester-induced U-937 differentiation: effects on integrin alpha(5) gene transcription.

Lung injury is accompanied by increased deposition of fibronectin (FN) matrices. Activated monocytic cells recruited to sites of lung injury express integrin receptors for FN that mediate their interaction with this matrix. One such integrin, alpha(5)beta(1), mediates many of the biological effects of FN, and its expression may be important for immune cell function at sites of lung injury. Herein, we examine the expression of alpha(5)beta(1) in response to the tumor promoter phorbol 12-myristate 13-acetate (PMA) in the human promonocytic cell line U-937. We demonstrate that PMA enhanced the adherence of U-937 cells to FN by increasing the expression of both the alpha(5)- and beta(1)-subunit mRNAs and the surface expression of the protein. In U-937 cells transfected with an alpha(5) promoter-reporter gene, we found that PMA induced the transcription of the alpha(5) gene by acting on very specific promoter sequences other than activator protein-1 in a protein kinase C-dependent manner. Lipopolysaccharide had a similar effect. Modulation of alpha(5)beta(1) expression may be important for regulation of monocytic cell function in lung inflammation after injury.

Antigens, CD↗
PubMed1999

Sequence specificity of in vivo reverse splicing of the Tetrahymena group I intron.

Reverse splicing of group I introns is proposed to be a mechanism by which intron sequences are transferred to new genes. Integration of the Tetrahymena intron into the Escherichia coli 23S rRNA via reverse splicing depends on base pairing between the guide sequence of the intron and the target site. To investigate the substrate specificity of reverse splicing, the wild-type and 18 mutant introns with different guide sequences were expressed in E. coli. Amplification of intron-rRNA junctions by RT-PCR revealed partial reverse splicing at 69 sites and complete integration at one novel site in the 23S rRNA. Reverse splicing was not observed at some potential target sites, whereas other regions of the 23S rRNA were more reactive than expected. The results indicate that the frequency of reverse splicing is modulated by the structure of the rRNA. The intron is spliced 10-fold less efficiently in E. coli from a novel integration site (U2074) in domain V of the 23S rRNA than from a site homologous to the natural splice junction of the Tetrahymena 26S rRNA, suggesting that the forward reaction is less favored at this site.

Animals↗
PubMed1999

Prevention of graft-versus-host disease in high risk patients by depletion of CD4+ and reduction of CD8+ lymphocytes in the marrow graft.

From March 1994 to September 1997, 30 patients with hematological malignancies (12 ANLL, 10 CML, four ALL and four multiple myeloma) received HLA-identical allogeneic bone marrow transplants with the marrow graft selectively depleted of CD4+ lymphocytes and the CD8+ cell content adjusted to 1x10(6)/kg. Total depletion of CD4+ and partial depletion of CD8+ lymphocytes was carried out by an immunomagnetical method. All patients were considered as having high risk for developing GVHD by at least one of the following criteria: patient age >35 years; donor age >35 years; donor multiparity or marrow from an unrelated donor. Twenty-four cases received marrow from an identical sibling and six from an unrelated donor. In order to assess the role of methotrexate (MTX) in addition to cyclosporin A (CsA) after transplant, patients were randomly assigned to received either CsA alone (n = 15) or CsA plus a short course of MTX (n = 15). No case of primary graft failure was observed, but two patients developed late graft failure. Six patients presented grade II acute GVHD and no case of severe III-IV GVHD was seen. The actuarial probability of developing grade II-IV acute GVHD was 25.9+/-9.6% for the entire population. Patients receiving post-transplant CsA + MTX had significantly less probability of acute GVHD than those receiving CsA exclusively (6.7+/-6.4% vs. 50.5+/-17.8%, P = 0.03) and the schedule of post-transplant immunosuppression was the only factor associated with the incidence of acute GVHD in a multivariate analysis. The actuarial incidence of chronic GVHD for the entire population was 31.8+/-12.5, and there was no significant difference between both groups with additional prophylaxis. Four patients with CML and three with ANLL relapsed: the actuarial probability of remaining in complete remission for all patients was 53.6+/-17.3%. For patients with acute leukemia, the probability of remaining in complete remission did not differ significantly between those transplanted in first complete remission and those receiving a transplant in more advanced phases of the disease (87.5+/-11.6% vs. 72.9+/-16.5%; P = 0.44). The incidence of mixed chimerism assessed by PCR was 34%. Nineteen patients are alive between 2 and 43 months post-transplant, the probability of overall survival being 57.8+/-10.4%. Our data indicate that this method of selective T cell depletion is very effective in preventing acute GVHD in high risk patients, particularly when used in combination with post-transplant CsA + MTX.

Adolescent↗
PubMed1999

Differential modes of regulation of interleukin-1beta expression by extracellular matrices.

Extracellular matrices (ECMs) can stimulate human monocytic cells to express interleukin-1beta (IL-1beta), a proinflammatory cytokine implicated in the regulation of tissue inflammation. In this study, we explored the intracellular mechanisms responsible for ECM induction of IL-1beta using human promonocytic U937 cells transfected with the full-length human IL-1beta gene promoter connected to a reporter gene. Using this system, we demonstrated that the ECM molecules fibronectin (FN), type I collagen (Coll), fibrin (Fb) and laminin (Lm) induced transcription of the IL-1beta gene (which was associated with a modest increase in IL-1beta protein secretion) in suspended cells, when used in their soluble monomeric form. This effect was mimicked or blocked by anti-integrin monoclonal antibodies (mAbs) and was dependent on the activation of protein kinase C (PKC). Three of the ECMs tested (FN, Coll and Fb) induced the activation of mitogen-activated protein kinases (MAPKs), whereas Lm had no effect. FN, Coll and Fb (but not Lm) also induced DNA binding of the transcription factor activator protein-1 (AP-1), but not that of nuclear factor-kappaB. Co-transfection of U937 cells with a competing AP-1 oligomer blocked the IL-1beta response induced by FN, but not that induced by the other ECMs. By inhibiting AP-1 translocation, glucocorticoids also blocked the FN-induced response, but not that of the other ECMs. These studies suggest that the signalling pathways which mediate ECM induction of IL-1beta expression in human monocytic cells converge at the level of PKC activation. However, they diverge in other aspects, as demonstrated by the differential activation of MAPKs and the need for diverse transcription factors.

Administration, Topical↗
PubMed1999

Ethanol ingestion increases activation of matrix metalloproteinases in rat lungs during acute endotoxemia.

Previously we reported that alcohol abuse increases the incidence of the acute respiratory distress syndrome (ARDS) in septic patients, and that chronic ethanol ingestion in rats depletes alveolar epithelial glutathione and increases endotoxin-mediated lung edema. In this study we examined a potential mechanism by which ethanol-induced glutathione depletion could predispose to acute lung injury. We hypothesized that glutathione depletion activates matrix metalloproteinases (MMPs), thereby increasing degradation of the alveolar extracellular matrix (ECM) during sepsis. Ethanol-fed rats (20% vol/vol in water for 6 wk) were given endotoxin (2 mg/kg, intraperitoneally) followed 2 h later by lung isolation and ex vivo perfusion with n-formyl-methionyl-leucyl-phenylalanine (fMLP) (10(-)(7) M). Ethanol ingestion increased (p < 0.05) MMP-9 and MMP-2 activity, as determined by zymography, in the lung tissue and lavage fluid compared with control-fed rats, and increased (p < 0.05) levels of the 7S fragment of type IV collagen in the lung lavage fluid. Ethanol ingestion increased activation, but not production, of the MMP-9 and MMP-2 zymogens. Finally, although concomitant ingestion of N-acetylcysteine had no effect (p > 0.05) on MMP production, it increased (p > 0.05) lung glutathione levels, blocked (p < 0.05) MMP-9 and MMP-2 activation, and decreased (p < 0.05) levels of the 7S fragment of type IV collagen. We conclude that chronic ethanol ingestion, via glutathione depletion, activates MMPs during sepsis, thereby increasing degradation of the alveolar epithelial ECM. Lois M, Brown LAS, Moss IM, Roman J, Guidot DM. Ethanol ingestion increases activation of matrix metalloproteinases in rat lungs during acute endotoxemia.

Acetylcysteine↗
PubMed1999

Transcriptional regulation of the interleukin-1beta promoter via fibrinogen engagement of the CD18 integrin receptor.

Fibrinogen, with or without its conversion to fibrin, in the extravascular spaces of injured and inflamed lung tissues is thought to promote inflammatory responses that can eventually lead to pulmonary fibrosis. One of these responses likely involves the elaboration of the proinflammatory cytokine interleukin (IL)- 1beta. We reported that both fibrinogen and fibrin stimulated production of IL-1beta message and protein by binding to CD18 integrin receptors on normal human monocytes (J. Immunol., 1995;154:1879-1887). The purpose of the current work was to extend our previous observations by characterizing the transcriptional regulation of fibrinogen-induced IL-1beta expression. Our model was the human monocytic cell line U937 transfected with the human IL-1beta promoter connected to reporter genes. We found that fibrinogen induced the IL-1beta promoter and that induction could be blocked by anti-CD18 antibody. Transfection with deletion constructs of the promoter and DNA electrophoresis mobility gel shift assays suggested that sequences containing activator protein (AP)-1, cyclic adenosine monophosphate response element (CRE), and nuclear factor (NF)-kappaB cis-acting motifs regulate IL-1beta gene expression by fibrinogen. In combination with competitive cotransfection studies using consensus oligonucleotides mimicking these motifs, we conclude that transactivation of an NF-kappaB-like sequence is necessary for induction of the IL-1beta gene, that activation of CRE may repress induction of the gene, and that AP-1 potentially modulates induction and repression of the gene induced by fibrinogen. This study begins to define the molecular mechanisms by which fibrin(ogen) promotes and regulates expression of the IL-1beta gene and further substantiates a role for fibrin(ogen) in tissue injury and inflammation.

Base Sequence↗
PubMed1999

Declining bone mass in men with chronic pulmonary disease: contribution of glucocorticoid treatment, body mass index, and gonadal function.

BACKGROUND: Men with chronic lung disease (CLD) are at risk for osteoporosis, but the relative contributions of their chronic pulmonary disease, glucocorticoid therapy, and other factors toward loss of bone has not been established. Understanding the relative importance of these factors would assist in selecting patients for bone densitometry screening and in policy decisions regarding Medicare reimbursement. OBJECTIVE: To identify patients with CLD who are most likely to benefit from bone densitometry screening based on clinical and biochemical measures. DESIGN: Cross-sectional medical survey. PATIENTS: Patients with CLD who were treated with either oral, inhaled, or no glucocorticoid therapy. A control group without lung disease was recruited from the same clinic population. MEASUREMENTS: Dual-energy X-ray absorptiometry was obtained for each group, and the association between bone mass and clinical variables, glucocorticoid use, gonadal hormones, and biochemical markers of bone metabolism was determined. RESULTS: Osteoporosis (a T score < -2.5 at the hip or spine) was five times as likely in patients with CLD as in control subjects. Although the prevalence of osteoporosis was higher (ninefold) after chronic glucocorticoid therapy, patients with CLD who had never been treated with glucocorticoids had a substantial (fourfold) risk of osteoporosis. Chronic inhaled glucocorticoid therapy offered no protection from bone loss compared to treatment with oral glucocorticoids. Of the clinical and biochemical measures that were obtained, bone mass was weakly correlated with body mass index (BMI), serum estradiol-17beta, and N-telopeptide, but not with testosterone, alkaline phosphatase, bone-specific alkaline phosphatase, or osteocalcin. CONCLUSION: Patients with CLD should be considered for bone densitometry screening regardless of glucocorticoid use. Those patients with a low BMI and/or decreased serum estradiol-17beta comprise a subgroup with increased risk for osteoporosis.

Adult↗
PubMed1998

Integration of the Tetrahymena group I intron into bacterial rRNA by reverse splicing in vivo.

Horizontal gene transfer is thought to contribute to the wide distribution of group I introns among organisms. Integration of an intron into foreign RNA or DNA by reverse self-splicing, followed by reverse transcription and recombination, could lead to its transposition. Reverse self-splicing of group I introns has been demonstrated in vitro, but not in vivo. Here we report RNA-dependent integration of the Tetrahymena intron into the 23S rRNA in Escherichia coli. Analysis of products by Northern blot and reverse transcription-PCR amplification revealed precise intron insertion into a site homologous to the natural splice junction. Products are sensitive to treatment with RNase but not DNase and depend on the splicing activity of the intron. Partial reaction with 11 novel sites in the 23S RNA that are complementary to the guide sequence of the intron illustrates lower specificity than intron homing. Reverse splicing of the Tetrahymena intron in bacteria demonstrates the possibility of RNA-catalyzed transposition of group I introns in foreign hosts.

Animals↗
PubMed1998

Identification of genes differentially expressed in Mycobacterium tuberculosis by differential display PCR.

RNA arbitrarily-primed differential display PCR (RAP-PCR) was used to identify and isolate genes differentially expressed between attenuated (H37Ra) and virulent (H37Rv, Erdman) laboratory strains of Mycobacterium tuberculosis (Mtb). Using this method, cDNA fragments showing homology to three known mycobacterial genes and six putative novel genes in mycobacterial cosmid vectors were identified. Among the putative novel Mtb genes identified, we found: (1) gene MTV041.29, containing multiple tandem repetitive sequences and encoding a putative Gly-, Ala, Asn-rich protein (PPE family); (2) gene MTV004.03, containing the AT10S repetitive gene sequence; (3) gene MTV028.09, encoding a hypothetical protein of unknown function; (4) genes MTCY78.20,21, possibly encoding two hypothetical proteins of unknown function; (5) gene MTCY01A6.09, encoding a putative novel ferrodoxin dependent glutamate synthase; and (6) gene MTCY31.20, encoding a putative cyclohexanone monooxygenase. Using gene specific primers in a second differential display PCR and by RT-PCR amplification, novel genes 1, 2, 3 and 4 were shown to be differentially up-regulated in the attenuated Mtb strain H37Ra compared to H37Rv and Erdman strain. Overall, we demonstrated that RAP-PCR, as a first step, is a quick and sensitive method for the identification and isolation of novel genes expressed in Mtb. Because of limitations inherent to the lack of specificity of arbitrary primers in the RAP-PCR method, a second differential display PCR and RT-PCR amplification with gene-specific primers was necessary in order to confirm differential expression of the identified genes.

Cloning, Molecular↗
PubMed1998

Pregnancy outcomes by mode of delivery among term breech births: Swedish experience 1987-1993.

OBJECTIVE: To study the influence of mode of delivery on infant mortality, neonatal morbidity, and maternal morbidity in pregnancies with nonmalformed term singleton infants presented in breech. METHODS: We studied all nonmalformed live-born singleton infants, delivered at term (at 37 weeks or later) in breech position in Sweden between 1987 and 1993 (n = 15,818). The pregnancy outcomes analyzed were neonatal and infant mortality, low Apgar score (less than 7) at 5 minutes, birth injury, and neonatal convulsions. Severe perineal or vaginal lacerations, wound rupture, infections, and thrombosis were used as measures of maternal morbidity. Logistic regression analysis was used to determine risks of infant mortality and morbidity, after adjusting for a number of potential confounders. RESULTS: Compared with infants delivered by elective cesarean, infants delivered vaginally were at significantly higher risk for infant mortality (odds ratio [OR] 2.5). Infants delivered vaginally were at increased risk for birth injury (OR 12.2), and infants delivered by emergency cesarean were at increased risk for neonatal convulsions (OR 4.1). Infants delivered vaginally or by emergency cesarean were at increased risk for a low Apgar score at 5 minutes. Maternal morbidity was highest among women who delivered by emergency cesarean (2.8%), whereas lower rates were obtained among women who delivered vaginally and those who delivered by elective cesarean (1.8 and 1.7%, respectively). CONCLUSION: Vaginal delivery of term infants presented in breech is associated with higher risks of neonatal mortality and morbidity compared with delivery by elective cesarean. We conclude that term singleton infants presented in breech would benefit from an elective cesarean delivery.

Adult↗
PubMed1998

Coexisting Sjögren's syndrome and sarcoidosis in the lung.

CONTEXT: Sjögren's syndrome (SS) and sarcoidosis are diseases of unknown origin that are considered to result from abnormal regulation of the immune system. Pulmonary involvement by SS and sarcoidosis may have similar clinical and radiographic manifestations, making it difficult for the clinician to distinguish between these diseases. OBJECTIVES: This study was undertaken to analyze the characteristics of SS and sarcoidosis in the lung to identify distinguishing features that may assist clinicians in the differentiation of these conditions. DESIGN: We present two cases with severe pulmonary impairment in which the distinction between SS and sarcoidosis required lung tissue biopsy. The literature regarding the pulmonary manifestations of these diseases is reviewed. RESULTS: The clinical, pathological, radiographic, and physiological characteristics of lung disease in the setting of SS and sarcoidosis can be very similar, preventing a diagnosis solely on clinical grounds. This is exemplified in the two cases reported. In one patient who carried the diagnosis of sarcoidosis, examination of lung tissue revealed lymphocytic interstitial pneumonitis consistent with SS. In the other patient, who had previously been diagnosed with SS on clinical grounds, examination of lung tissue showed lymphocytic interstitial pneumonitis with scattered noncaseating granulomas, suggesting the possibility of coexisting SS and sarcoidosis. A literature review indicated that lung involvement by SS may be difficult to distinguish from that of sarcoidosis. Furthermore, several cases have been reported in which both diseases coexisted. CONCLUSIONS: Because SS and sarcoidosis may coexist and present with similar pulmonary manifestations, aggressive evaluation including tissue biopsy may be required. However, even tissue biopsy may not distinguish between these entities unless noncaseating granulomas are seen (in the case of sarcoidosis) or isolated lymphocytic interstitial pneumonitis is detected (in the case of SS). When both features (ie; noncaseating granuloma and lymphocytic interstitial pneumonitis) are encountered in the same organ, we believe these diseases are coexisting. Distinguishing both conditions may have prognostic implications, because sarcoidosis may present as an autolimiting process and frequently resolves spontaneously without significant residual functional impairment. In contrast, pulmonary involvement with SS often leads to permanent defects and may progress to incapacitating disease.

Aged↗
PubMed1998

Differential effects of protein kinase C inhibitors on fibronectin-induced interleukin-beta gene transcription, protein synthesis and secretion in human monocytic cells.

Human monocytic cells express interleukin-1beta (IL-1beta) when stimulated with the extracellular matrix glycoprotein, fibronectin (FN). Protein kinase C (PKC) activation is considered important for this process; however, the metabolic steps at which PKC acts upon to mediate the FN-induced IL-1beta response remain unclear. We performed an analysis of the mechanisms by which two PKC inhibitors, Calphostin C and Staurosporine, prevent the FN-induced IL-1beta response. Both inhibitors blocked the secretion of IL-1beta protein into the media of peripheral blood mononuclear cells exposed to FN. Immunoprecipitation analysis revealed that under these circumstances, Calphostin C inhibited the production of IL-1beta protein, whereas Staurosporine allowed protein production, but inhibited its secretion. To determine the mechanisms responsible for these differences, we turned to human U937 promonocytic cells. U937 cells transfected with the human full-length IL-1beta promoter connected to a luciferase reporter gene were submitted to transcription assays, Northern blotting, and DNA electrophoresis mobility gel shift assays. These studies revealed that Calphostin C inhibited the nuclear translocation of the transcription factor activator protein-1 (AP-1) which is considered necessary for FN induction of IL-1beta gene transcription, and prevented the transcription of the IL-1beta gene. In contrast, Staurosporine alone induced AP-1 translocation and stimulation of the gene. Overall, our data indicate that Calphostin C prevents the transcription of the IL-1beta gene thereby inhibiting protein synthesis. Based on the high specificity of this compound for PKC, we conclude that PKC is necessary for FN-induced IL-1beta protein production. In contrast, Staurosporine prevented secretion of IL-1beta by unknown mechanisms.

Blotting, Northern↗
PubMed1998

Heparin inhibits lung branching morphogenesis: potential role of smooth muscle cells in cleft formation.

Lung branching morphogenesis is the process by which the embryonic lung undergoes repetitive branching to form the bronchial tree. This process occurs during the pseudoglandular stage of lung development and requires epithelial-mesenchymal interactions. Coinciding with lung branching morphogenesis is the appearance of parabronchial smooth muscle cells (PSMCs) and the accumulation of extracellular matrices (ECMs) around the developing airways. The authors previously reported in preliminary form that heparin prevents the branching of murine lung explants (Roman et al., Am Rev Respir Dis. 1991; 143:A401); this article corroborates those early observations and expands them by demonstrating that heparin results in disruption of PSMC distribution and abnormal organization of ECMs around the developing airways. These changes were associated with inhibition of lung branching morphogenesis in the absence of effects on cell proliferation. The data provide further support for the role of ECMs in lung branching morphogenesis, and points to PSMCs as potential players in this process.

Animals↗