Showing papers in "Molecular Medicine in 1994"

Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair.

Abstract: The host response to tissue injury requires a complex interplay of diverse cellular, humoral, and connective tissue elements. Fibroblasts participate in this process by proliferating within injured sites and contributing to scar formation and the long-term remodeling of damaged tissue. Fibroblasts present in areas of tissue injury generally have been regarded to arise by recruitment from surrounding connective tissue; however this may not be the only source of these cells. Long-term culture of adherent, human, and murine leukocyte subpopulations was combined with a variety of immunofluorescence and functional analyses to identify a blood-borne cell type with fibroblast-like properties. We describe for the first time a population of circulating cells with fibroblast properties that specifically enter sites of tissue injury. This novel cell type, termed a “fibrocyte,” was characterized by its distinctive phenotype (collagen+/vimentin+/CD34+), by its rapid entry from blood into subcutaneously implanted wound chambers, and by its presence in connective tissue scars. Blood-borne fibrocytes contribute to scar formation and may play an important role both in normal wound repair and in pathological fibrotic responses.

Lipopolysaccharide Signals Activation of Tumor Necrosis Factor Biosynthesis Through the Ras/Raf-1/MEK/MAPK Pathway

Abstract: Lipopolysaccharide (LPS) is known to activate macrophages, causing the release of toxic cytokines that may provoke inflammation and shock. One of the most important and best studied of these cytokines is tumor necrosis factor (TNF). Details of the signaling pathway leading to TNF biosynthesis remain unclear. The pathway is branched in the sense that TNF gene transcription and TNF mRNA translation are both strongly stimulated by LPS. Recent evidence has indicated that MAP kinase homologs become phosphorylated in LPS-stimulated cells, suggesting their possible involvement in signal transduction. We sought to test this hypothesis. Measurements of LPS-induced MEK and ERK2 activity were undertaken in LPS-sensitive and LPS-insensitive cells. Transfection studies, in which dominant inhibitors of ras and raf-1 were used to block signaling to the level of MAP kinase, were carried out in order to judge whether the TNF gene transcription and TNF mRNA translation are modulated through this pathway. In RAW 264.7 mouse macrophages, both ERK2 and MEK1 activity are induced by LPS treatment. In the same cell line, dominant negative inhibitors of ras and raf-1 block LPS-induced activation of the TNF promoter, as well as derepression of the translational blockade normally imposed by the TNF 3′-untranslated region. A constitutively active form of raf-1 (raf-BXB) was found to augment, but not replace, the LPS signal. In LPS-insensitive cells (RAW 264.7 × NIH 3T3 fusion hybrid cells and primary macrophages derived from C3H/HeJ mice), ERK2 activity was found to be refractory to induction by LPS. The ras/raf-1/MEK/MAPK pathway is chiefly responsible for transduction of the LPS signal to the level of the TNF gene and mRNA. raf and raf-1 lie upstream from (or actually represent) the physical branchpoints of the transcriptional and translation activation signals generated by LPS. The lesions that prevent LPS signaling in macrophages from C3H/HeJ mice, or in RAW 264.7 × NIH 3T3 fusion hybrid cells, occupy a proximal position in the signaling pathway.

High prion and PrPSc levels but delayed onset of disease in scrapie-inoculated mice heterozygous for a disrupted PrP gene.

Abstract: It has been proposed that the prion, the infectious agent of transmissible spongiform encephalopathies, is PrPSc, a post-translationally modified form of the normal host protein PrPC. We showed previously that mice devoid of PrPC (Prn-p0/0) are completely resistant to scrapie. We now report on the unexpected response of heterozygous (Prn-p0/+) mice to scrapie infection. Prn-p0/+, Prn-p0/0 and Prn-p+/+ mice were obtained from crosses of Prn-p0/+ mice. Mice were inoculated intracerebrally with mouse-adapted scrapie agent and the clinical progression of the disease recorded. Mice were sacrificed at intervals, PrPSc was determined as protease-resistant PrP and the prion titer by the incubation time assay. Prn-p0/+ mice, which have about half the normal level of PrPC in their brains, show enhanced resistance to scrapie, as manifested by a significant delay in onset and progression of clinical disease. However, while in wild type animals an increase in prion titer and PrPSc levels is followed within weeks by scrapie symptoms and death, heterozygous Prn-p0/+ mice remain free of symptoms for many months despite similar levels of scrapie infectivity and PrPSc. Our findings extend previous reports showing an inverse relationship between PrP expression level and incubation time for scrapie. However, contrary to expectation, overall accumulation of PrPSc and prions to a high level do not necessarily lead to clinical disease. These findings raise the question whether high titers of prion infectivity could also persist for long periods under natural circumstances in the absence of clinical symptoms.

Role of macrophage oxidative burst in the action of anthrax lethal toxin.

Abstract: Major symptoms and death from systemic Bacillus anthracis infections are mediated by the action of the pathogen’s lethal toxin on host macrophages. High levels of the toxin are cytolytic to macrophages, whereas low levels stimulate these cells to produce cytokines (interleukin-1β and tumor necrosis factor-α), which induce systemic shock and death. Experiments were performed to assess the possibility that the oxidative burst may be involved in one or both of lethal toxin’s effects on macrophages. Toximediated cell lysis, superoxide anion and cytokine production were measured. Effects of antioxidants and macrophage mutations were examined. RAW264.7 murine macrophages treated with high levels of toxin released large amounts of superoxide anion, beginning at about 1 hr, which correlates with the onset of cytolysis. Cytolysis could be blocked with various exogenous antioxidants or with N-acetyl-L-cysteine and methionine, which promote production of the endogenous antioxidant, glutathione. Mutant murine macrophage lines deficient in production of reactive oxygen intermediates (ROIs) were relatively insensitive to the lytic effects of the toxin, whereas a line with increased oxidative burst potential showed elevated sensitivity. Also, cultured blood monocyte-derived macrophages from a patient with Chronic Granulomatous Disease, a disorder in which the phagocyte’s oxidative burst is disabled, were totally resistant to toxin, in contrast to control monocytes. These results imply that the cytolytic effect of the toxin is mediated by ROIs. Additionally, cytokine production and consequent pathologies showed partial dependence on macrophage ROIs. Antioxidants moderately inhibited toxin-induced cytokine production in vitro, and BALB/c mice pretreated with N-acetyl-L-cysteine or mepacrine showed partial protection against lethal toxin. Thus ROIs are involved in both the cytolytic action of anthrax lethal toxin and the overall pathologic process in vivo.

Glucocerebrosidase mutations in Gaucher disease.

Abstract: Thirty-six mutations that cause Gaucher disease, the most common glycolipid storage disorder, are known. Although both alleles of most patients with the disease contain one of these mutations, in a few patients one or both disease-producing alleles have remained unidentified. Identification of mutations in these patients is useful for genetic counseling. The DNA from 23 Gaucher disease patients in whom at least one glucocerebrosidase allele did not contain any of the 36 previously described mutations has been examined by single strand conformation polymorphism (SSCP) analysis, followed by sequencing of regions in which abnormalities were detected. Eight previously undescribed mutations were detected. In exon 3, a deletion of a cytosine at cDNA nt 203 was found. In exon 6, three missense mutations were identified: a C→A transversion at cDNA nt 644 (Ala176→Asp), a C→A transversion at cDNA nt 661 that resulted in a (Pro182→Thr), and a G→A transition at cDNA nt 721(Gly202→Arg). Two missense mutations were found in exon 7: a G→A transition at cDNA nt 887 (Arg257→Gln) and a C→T at cDNA nt 970 (Arg285→Cys). Two missense mutations were found in exon 9: a T→G at cDNA nt 1249 (Trp378→Gly) and a G→A at cDNA nt 1255 (Asp380→Asn). In addition to these disease-producing mutations, a silent C→G transversion at cDNA nt 1431, occurring in a gene that already contained the 1226G mutation, was found in one family. The mutations described here and previously known can be classified as mild, severe, or lethal, on the basis of their effect on enzyme production and on clinical phenotype, and as polymorphic or sporadic, on the basis of the haplotype in which they are found. Rare mutations such as the new ones described here are sporadic in nature.

X chromosome inactivation patterns correlate with fetal-placental anatomy in monozygotic twin pairs: implications for immune relatedness and concordance for autoimmunity

Abstract: Monozygotic (MZ) twinning is a poorly understood phenomenon that may result in subtle biologic differences between twins, despite their identical inheritance. These differences may in part account for discordant expression of disease in MZ twin pairs. Due to their stochastic nature, differences in X chromosome inactivation patterns are one source of such variation in female MZ twins. We investigated X chromosome inactivation patterns in the blood of 41 MZ twin pairs based on methylation of the androgen receptor gene using a Hpa II-PCR assay. Twenty-six female MZ twin pairs with autoimmune disease (rheumatoid arthritis or multiple sclerosis) were studied. In addition, we studied 15 newborn female MZ twin pairs who were characterized at birth with respect to the anatomy of chorionic membranes (dichorionic versus monochorionic). We found a strong correlation between dichorionic fetal anatomy and differences in X chromosome inactivation patterns between members of an MZ twin pair. In contrast, all monochorionic twin pairs had closely correlated patterns of X chromosome inactivation. X chromosome inactivation patterns did not distinguish between MZ twin pairs who were concordant or discordant for autoimmune disease. The highly similar patterns of X chromosome inactivation among monochorionic twin pairs may result from their shared placental blood supply during intrauterine life. Alternatively, these patterns may indicate that X chromosome inactivation occurs before the twinning event in this anatomic subgroup of MZ twins. The data further suggest that these factors do not make a major contribution to the high discordance rates for autoimmune disease in MZ twin pairs.

Pentoxifylline and other protein kinase C inhibitors down-regulate HIV-LTR NF-kappa B induced gene expression.

Abstract: This investigation deals with the molecular mechanism of anti-human immunodeficiency virus type 1 (HIV-1) action of pentoxifylline (PTX) [1-(5′-oxohexyl)-3,7-dimethylxanthine] a drug widely used for the treatment of conditions involving defective regional microcirculation. The inhibition by PTX of protein kinase C (PKC) or cAMP-dependent protein kinase (PKA)-mediated activation by phorbol ester (PMA) and tumor necrosis factor alpha (TNF-α) of HIV-1-LTR-regulated reporter gene expression was studied in human CD4+ T lymphocytes (Jurkat) and human embryo kidney cells (293-27-2). A protein kinase C is involved in activation of NF-κB in whole cells, identified by using inhibitors specific for PKC- or PKA-catalyzed NF-κB activation in whole cell and cell-free systems. PTX inhibited PKC- or PKA-catalyzed activation of NF-κB in cytoplasmic extracts from unstimulated Jurkat or 293-27-2 cells, but not interaction of preactivated NF-κB with its motifs. Calphostin C, a specific inhibitor of PKC, inhibited NF-κB activation and HIV-1 LTR-driven reporter gene expression in both PMA- and TNF-α-treated cells. In contrast, although H88 specifically inhibited PKA activity in the cell-free extract, it did not affect NF-κB action in PMA- or TNF-α-treated cells. The mechanism of inhibitory action of PTX on virus replication and NF-κB-induced trans-activation of HIV-1 gene expression has been elucidated as due to blocking PKC-dependent PMA- or TNF-α-induced activation of NF-κB in Jurkat and 293-27-2 cells. Other protein kinase inhibitors may be useful in down regulating transcription of HIV-1 provirus and thereby virus replication in HIV-infected patients.

The evolutionarily conserved ribosomal protein L23 and the cationic urease beta-subunit of Yersinia enterocolitica O:3 belong to the immunodominant antigens in Yersinia-triggered reactive arthritis: implications for autoimmunity.

Abstract: Reactive arthritis (ReA) is a T cell mediated inflammatory process. The immune response is primarily directed against a triggering organism, although autoimmunity has been invoked in long-lasting, antibiotic-resistant disease. Although a variety of different species are known to trigger Reactive arthritis, the clinical manifestations are strikingly similar as well as closely associated to the HLA-B27 (70%). Various antigenic fractions and single antigens of Yersinia enterocolitica were prepared, and their immunological activity was assessed by proliferation of synovial fluid mononuclear cells from 10 Reactive arthritis patients. The gene encoding one hitherto unknown antigen has been sequenced. Nonapeptides deduced from sequences of the target antigens were tested in an assembly assay. Two immunodominant proteins of Yersinia enterocolitica were found, one being the urease β-subunit and the other the 50 S ribosomal protein L23. The latter has been sequenced and belongs to the evolutionarily conserved ribosomal proteins with homology to procaryotes and eucaryotes. One nonapeptide derived from the urease β-subunit was identified as a possible epitope for HLA-B27-restricted cytotoxic T cells by its high affinity. This epitope is also highly conserved. Sharing of conserved immunodominant proteins between different disease triggering microorganisms could provide an explanation of the shared clinical picture in Reactive arthritis. Moreover, autoimmunity in Reactive arthritis might be mediated by antigen mimicry between evolutionarily conserved epitopes of ribosomal proteins and their host analogs.

Gene Therapy: Hopes, Hypes, and Hurdles

Abstract: The problem is vast, first, because there is hardly any form of major restriction on research. After all, as far as science is concerned, new knowledge is always a good thing and there is hardly anyone who doubts that its applications are, all in all, more beneficial than harmful. Because of the unlimited possibilities of application, any restriction must be agreed internationally: no country will voluntarily create competitive disadvantages by dispensing with basic research. Further, applications can be harmful if their basis comes from another country. The decisions on each application must naturally be made by each country individually. Then, the question arises on each occasion: who can guarantee that the decisions are good decisions? Or, put another way: can we reach a situation where knowing is better than not knowing?

Molecular Medicine: The Future of Biomedical Science and Clinical Practice

Abstract: The last several decades have seen great advances in the biomedical sciences with the burgeoning and integration of fields such as molecular and structural biology, biochemistry and immunology. They have provided both a new perspective and powerful new tools which are permeating medical research. We have now reached the point where \"molecular medicine\" can be defined as a discipline concerned with understanding the pathogenesis of disease at the molecular level, and, based on that knowledge, the designing of specific molecular tools for diagnosis, treatment and prevention. Furthermore, this new field, which integrates the tools and concepts of science at the molecular level, has provided a common language and understanding among clinical groups as diverse as virologists and oncologists, dermatologists and neurologists. The time is ripe, therefore, for the introduction of a journal wholly devoted to molecular medicine. Since this is a new field which has captured the imagination of many of the best biomedical and clinical scientists, from those at the beginnings of their careers to established investigators at their peak, this journal aims to be the forum for the best work at all levels. Our remarkable board of contributing editors is fully supportive of our plan to scale the heights of modern biomedical science, not only by contributing their own papers, but by facilitating the publication of superb science by others who want to join in this grand endeavor. Working together, the editors and publishers will do everything possible to ensure the most rapid publication of the best work with the least amount of pain. In order to maintain the rigor of peer review, while keeping the process down to two weeks, the editors will only accept papers of the highest quality that require minimal changes. Including editing and printing time, most papers will appear in approximately three months from the time of submission. This will be facilitated by the requirement that manuscripts must be accompanied by floppy discs using any standard word processing program. Communication between all parties will be expedited by fax and e mail, with rapid development of a fully electronic system. As can be seen by the broad range of interests of the contributing editors and of the papers in this premier issue of Molecular Medicine, the journal aims to cover all of clinical medicine at the molecular level. While the publication of original articles is our most important goal, we will be developing the journal as a forum for the field, including editorials, reviews, databases and news items. As we move forward, we hope that you will seize the opportunity to suggest to us innovative ways of improving the publication process and other aspects of the quality of the journal. We ask you to join us in the belief that Molecular Medicine wil be the vanguard of a new millenium, in which science will truly complement the art of medicine.

The Molecular Basis of Increased Glomerulosclerosis after Blockade of the Renin Angiotensin System in Growth Hormone Transgenic Mice

Abstract: Angiotensin converting enzyme inhibitor (ACEi) therapy delays the onset of renal failure in diabetic nephropathy and inhibits or delays the onset of proteinuria in several animal models. We examined this question using a transgenic model of chronic glomerulosclerosis caused by an excess production of growth hormone (GH) in which there is progressive glomerular scarring leading to uremia. In addition, since GH mice do not have systemic hypertension or an elevated glomerular filtration rate, we could address the question of whether ACEi or angiotensin II receptor antagonists (AII RA) had an effect on the development of glomerulosclerosis under these conditions. Since excess matrix accumulates in glomerulosclerosis because of alterations in the balance between its synthesis and degradation, we examined the effect of ACEi and AII RA on these parameters. Systemic blood pressure was unaffected by ACEi treatment, but the glomerular filtration rate decreased 85%. ACEi-treated mice had increased mesangial deposition of type I collagen and decreased 105 kD complex collagenase activity. In addition, ACEi-treated GH mice had increased glomerular α1 type I collagen, α1 type IV collagen, and α-smooth muscle cell actin mRNAs. No changes were noted in β actin, or 72 kD metalloproteinase mRNAs. The result of these changes was a net increase in sclerosis. Surprisingly, GH mice treated with ACEi or AngII RA developed marked renal arteriolar lesions.

Clonal hematopoiesis and acquired thalassemia in common variable immunodeficiency.

Abstract: Common variable immunodeficiency (CVID) is defined by hypogammaglobulinemia and increased susceptibility to infections. The gene defect responsible for CVID remains unknown. During the course of their CVID disease, a female and three male patients developed microcytic anemia. The investigation of this anemia forms the basis for this report. Reticulocyte globin chain synthesis studies revealed the abnormal α/β ratios that are pathognomonic of thalassemia. Through transcriptional analysis of the glucose-6-phosphate-dehydrogenase (G6PD) locus of the active X-chromosome in blood cells, we determined that the female patient has clonal reticulocytes, platelets, granulocytes, and B and T lymphocytes. The simultaneous presence of globin synthesis abnormalities and panhypogammaglobulinemia suggests that a common insult at the stem cell level could contribute to the development of CVID and acquired thalassemia.