bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Ca2+ ATPase Inhibitors on MCP-1 Release from Bone Marrow-Derived Mast Cells and the Involvement of p38 MAP Kinase Activation
Authors: Teshima R. , Onose J. , Okunuki H. , Sawada J.
Published: April 2003

The effect of two Ca2+ ATPase inhibitors, cyclopiazonic acid (CPA) and 2,5-di-(tert-butyl)-1,4-hydroquinone (DTBHQ), on the release of MCP-1 from bone marrow-derived mast cells (BMMCs) were investigated. CPA and DTBHQ increased the intracellular free Ca2+ concentration ([Ca2+]i) and induced MCP-1 release in a dose-dependent manner. These Ca2+ ATPase inhibitors induced MCP-1 release in the absence of phorbol ester, in contrast to their induction of TNF-α. MCP-1 release reached a maximum at 6–9 h. It was inhibited by treatment with actinomycin D, the immunosuppressant cyclosporin A, and the cytosolic Ca2+ chelator BAPTA-AM. Furthermore, RT-PCR showed a time-dependent increase of MCP-1 mRNA. Thus MCP-1 release seems to depend on Ca2+-dependent transcriptional activation. MCP-1 release was dose-dependently inhibited by the p38 MAP kinase inhibitor SB202190, but not by the p44/42 MAP kinase inhibitor PD98059. Therefore, transcriptional activation of MCP-1 production and its release seem to be dependent on the nuclear factor of activated T cells and p38 MAP kinase activation. This is the first report to show the regulation of MCP-1 production in BMMCs.
Sequential Development of Airway Hyperresponsiveness and Acute Airway Obstruction in a Mouse Model of Allergic Inflammation
Authors: Neuhaus-Steinmetz Ulrich , Glaab Thomas , Daser Angelika , Braun Armin , Lommatzsch Marek , Herz Udo , Kips Johan , Alarie Yves , Renz Harald
Published: April 2003

Background: Mouse models have been established mirroring key features of human bronchial asthma including airway hyperresponsiveness (AHR). Acute airway obstruction in response to an allergen challenge, however, remains to be demonstrated in these models. Objective: A mouse model of allergic lung inflammation was employed to analyze the development of specific (allergen-induced) and nonspecific (methacholine-induced) airway obstruction. Methods: Mice were sensitized to ovalbumin (OVA) and challenged with OVA aerosol twice each week during four weeks. Changes in lung functions were determined by noninvasive head-out body plethysmography. The development of acute airway obstruction after OVA challenge and AHR after methacholine aerosol application were assessed by a decrease in the mid-expiratory flow rate (EF50). Results: Two airway challenges were sufficient to induce AHR (5.7 vs. 15 mg/ml methacholine). Further OVA challenges reduced the baseline EF50 from 1.85 to 1.20 ml/s (4th week) and induced acute airway obstruction. The OVA-induced obstruction was maximal in the 4th week (EF50 = 0.91 ml/s). Conclusion: The development of acute airway obstruction in allergen-sensitized mice was demonstrated by means of head-out body plethysmography. In our model, AHR was observed before the development of airway obstruction.
Latex Allergy in Children
Authors: Niggemann Bodo , Breiteneder Heimo
Published: April 2003

Natural rubber is a component of the latex of the tropical Hevea brasiliensis tree which is widely used in the manufacturing of medical devices and a large variety of articles for everyday use. Over a dozen allergens have been identified in the latex of H. brasiliensis. The allergens Hev b 1, Hev b 3, Hev b 6, and Hev b 7 are proteins that are involved in the biosynthesis of rubber or the coagulation of latex. Allergens that are part of the plant’s defense system are represented by Hev b 2 and class I endochitinases. The allergens Hev b 4, Hev b 5, and Hev b 8–10 were classified as either structural or housekeeping proteins. Immediate-type hypersensitivity reactions to proteins present in Hevea latex were first described in 1927. Since then, natural rubber latex (NRL) allergy has become an important medical problem for an increasing number of individuals. Sensitization mainly occurs by wound or mucosal contact with NRL devices during surgery or by inhalation of airborne allergens released from powdered latex gloves. The number of surgical interventions and an atopic disposition are the most important risk factors for developing latex allergy, especially in children with spina bifida. Exposure to NRL products should be carefully avoided for individuals who belong to high-risk groups. Initial studies on establishing a latex-free environment for surgery in all spina bifida patients have reported on a decrease in sensitization and allergy to NRL.
Effect of Suplatast Tosilate (IPD-1151T) on a Mouse Model of Asthma: Inhibition of Eosinophilic Inflammation and Bronchial Hyperresponsiveness
Authors: Zhao Guang-Dong , Yokoyama Akihito , Kohno Nobuoki , Sakai Kimiko , Hamada Hironobu , Hiwada Kunio
Published: April 2003

Background: Suplatast tosilate (IPD) is a newly developed ‘anti-allergic’ drug. It seems to be a unique compound because of its ability to suppress IgE but not IgG or IgM production in vivo and cytokine production from type 2 helper T cells (Th2) in vitro. However, information on its in vivo effect on an animal model of asthma is limited. Method: BALB/c mice sensitized to ovalbumin (3 times, 2-week interval) were challenged with ovalbumin by inhalation (50 mg/ml for 20 min, once a day for 6 days). In this study, we explored the influence of IPD on eosinophil infiltration into the airways, bronchial hyperresponsiveness (BHR) to methacholine, specific IgE antibody production, and cytokines in bronchoalveolar lavage fluid (BALF) using this murine model. Results: Treatment with IPD significantly reduced the number of total cells and eosinophils in BALF (around –40%) and almost completely inhibited the development of antigen-induced BHR. Histological findings confirmed the reduction of submucosal cell infiltration in the lung, and disclosed the marked inhibition of bronchial epithelial cell damage. Ovalbumin-specific IgE was slightly but significantly reduced. The levels of IL-4, IL-5 and IL-13 in BALF were significantly decreased in mice treated with the compound compared to those in untreated mice. Conclusion: These results suggest that IPD is capable of inhibiting the production of Th2 cytokines, which inhibit eosinophil infiltration into the murine airway, IgE synthesis, and development of BHR, in a murine model of asthma.
β-Estradiol Suppresses T Cell-Mediated Delayed-Type Hypersensitivity through Suppression of Antigen-Presenting Cell Function and Th1 Induction
Authors: Salem Mohamed Labib , Matsuzaki Goro , Kishihara Kenji , Madkour Gamal A. , Nomoto Kikuo
Published: April 2003

Background: Although an immunomodulatory role for estrogens has long been demonstrated by experimental and clinical observations, the mechanism by which estrogens exert their effect on T cells has not been clearly defined. Methods: In this study we analyzed the effects of β-estradiol (E2), at its contraceptive dose, on the delayed-type hypersensitivity (DTH) to purified protein derivatives (PPD) and associated immune response in female mice. Results: E2 treatment decreased PPD-specific DTH response, which coincided with a decrease in the leukocytes numbers in the draining lymph nodes (DLN) and spleen compared with control mice. E2 treatment also suppressed the in vitro PPD-specific proliferative response of DLN and spleen cells from PPD-primed mice. The analysis of production and gene expression of cytokines by DLN cells demonstrated that E2 treatment suppressed IL-2 and IFN-γ production in response to PPD in vitro. In contrast, IL-4 and IL-10 gene expression by DLN cells of E2-treated mice, taken 24 h after in vivo restimulation of mice with PPD, was enhanced. Furthermore, we found that spleen APC from E2-treated mice failed to induce optimum proliferation of the PPD-primed T cells in response to PPD in vitro. The impaired APC function by E2 was not due to induction of suppressor cell activity because addition of the normal spleen APC to APC from E2-treated mice restored the proliferative response of the PPD-primed T cells in response to PPD. Conclusion: Our results suggest that the E2-mediated inhibition of DTH reaction is due to a combination of the down regulation of APC function and deviation of the immune response from Th1-type to Th2-type.
Enhancing Effect of Dietary Oil Emulsions on Immune Responses to Protein Antigens Fed to Mice
Authors: Kaneko Tetsuo , Terasawa Yuka , Senoo Yukiko , Nagata Masashi , Kuwata Tamotsu
Published: April 2003

Repeated intragastric administration of β-lactoglobulin (β-Lg) with emulsified soybean oil elicited an antigen-specific, systemic humoral immune response in different strains of mice. The antibody response was enhanced as the dose of oil was increased and the particle size of emulsions was decreased. Feeding of aqueous β-Lg could induce the antibody response only when emulsified oil was fed almost simultaneously. However, the emulsion-driven humoral immune response was not observed when mice were treated with anti-CD40 ligand antibody or in athymic mice. It is likely that the intestinal coexistence of emulsified oil with dietary antigens modulates the immune system to crucially support B cell response. A practical application of the present results to the prevention of cow’s milk protein sensitization in infants is proposed.
Isolation of a Lactose-Binding Protein with Monocyte/Macrophage Chemotactic Activity
Authors: Yamanaka Tohru , Saita Naoki , Kawano Osamu , Matsumoto Mitsuhiro , Kohrogi Hirotsugu , Suga Moritaka , Ando Masayuki , Hirashima Mitsuomi
Published: April 2003

Background: We established a T cell line, STO-5, which constitutively produced monocyte/macrophage chemotactic activity via human T cell lymphoma-leukemia-virus-induced transformation of normal human T cells. Methods: We isolated and purified a lactose-binding protein, MCF-pl5-L (MW of about 50 kD, pl of about 5) from a conditioned medium of STO-5. By using highly purified MCF-pl5-L, its biological and physicochemical properties were elucidated in comparison with C5a and MCP-1. Results: MCF-pl5-L exhibited an evident dose-dependent monocyte chemotactic activity (MCA). MCF-pl5-L had no or little affinity for heparin unlike chemokines such as MCP-1. We further found that MCF-pl5-L exhibited potent chemotactic activity not only for monocytes but also for alveolar macrophages. In contrast, C5a and MCP-1 failed to show evident chemotactic activity for alveolar macrophages though they did show MCA. MCF-pl5-L failed to exhibit evident eosinophil and neutrophil chemotactic activities, indicating its chemotactic activity is selective for monocytes/macrophages. Regarding the biological functions of MCF-pl5-L other than MCA and chemotactic activity for alveolar macrophages, we found that MCF-pl5-L but not C5a and MCP-1 could prolong the life span of alveolar macrophages, probably by inhibiting apoptosis of macrophages, and stimulate the production of TNF-α from macrophages. Conclusions: These results suggest that MCF-pl5-L plays a role as an immune modulator for monocytes/macrophages in the site.
Humoral and Cellular Autoimmunity in Autoimmune Bullous Skin Disorders
Authors: Hertl Michael
Published: April 2003

Autoimmune bullous skin diseases, such as pemphigus vulgaris (PV) and bullous pemphigoid (BP), are severe, frequently life-threatening skin disorders. Immunologically, they are characterized by the presence of serum autoantibodies (auto-Ab) targeting distinct adhesion molecules of the epidermis or dermoepidermal basement membrane zone. Antibody (Ab) binding interferes with the adhesive function of these molecules, leading to detachment and subsequently blister formation. PV is the classical example of an Ab-mediated autoimmune disease affecting epidermal adhesion. Auto-Ab against the desmosomal adhesion molecule, desmoglein 3 (Dsg3), are critical in the pathogenesis of this disease, since the transfer of serum IgG Ab reactive with Dsg3 into newborn mice induces a bullous skin disease resembling PV. Autoreactive T cell responses to Dsg3 may be critical in the pathogenesis of PV because: (1) Ab production generally requires T cell help; (2) the involvement of CD4+ T lymphocytes in PV has been suggested by the strong association with distinct HLA class II alleles, and (3) T cell recognition of epitopes of Dsg3 may be crucial for the initiation and perpetuation of the production of Dsg3-specific auto-Ab by B cells. In PV and BP, autoreactive CD4+ T cells recognize distinct epitopes of the extracellular portions of Dsg3 and BP180 [BP antigen 2 (BPAG2) or type XVII collagen], respectively, and produce preferentially T helper type 2 (TH2) cytokines. Auto-Ab of the TH2-dependent IgG4 subtype are preferentially seen in the active stages of both PV and BP, while auto-Ab of the TH1-dependent IgG1 subclass are predominant during the chronic course of these disorders. These observations suggest that autoreactive TH2 cells may provide targets to specifically modulate the T cell-dependent production of pathogenic auto-Ab in these disorders.
Ultrastructural Autoradiographic Analysis of RNA in Isolated Human Lung Mast Cells during Secretion and Recovery from Secretion
Authors: Dvorak Ann M. , Morgan Ellen S. , Lichtenstein Lawrence M. , Schleimer Robert
Published: April 2003

Background: Previous work has implicated isolated, control human lung mast cell granules in RNA metabolism using multiple methods of high-magnification imaging based on different mechanistic principles. These methods have demonstrated ribosomes, RNA, U1snRNP and uridine in, around and attached to secretory granules. Methods: Here, we have extended these studies using ultrastructural autoradiography of radiolabeled uridine incorporation in degranulating and recovering mast cells. Results: We found that control cells incorporated uridine into granules, with values that decreased dramatically in conjunction with stimulated histamine secretion and granule extrusion, and that granule stores of tritiated uridine increased together with the reconstitution of secretory granules in recovering mast cells. Conclusion: These findings support a possible new role for secretory granules in RNA metabolism in mast cell biology.
Purification and Partial Characterization ofa 67-kD Cross-React ive Allergen from Imperata cylindrica Pollen Extract
Authors: Verma Jyotsna , Singh B.P. , Gangal Sharad V. , Arora Naveen , Sridhara Susheela
Published: April 2003

Background: Grass pollens are known to induce type I allergic reactions in a large number of genetically predisposed individuals. Earlier studies have recognized Imperata cylindrica (Ic) pollen as an important source of aeroallergen which contained 7 IgE binding proteins in the MW range of 85–16 kD. Objectives: To isolate, purify and characterize a cross-reactive allergenic protein from Ic pollen extract for diagnosis and therapy of grass pollen allergy. Methodology: Ic pollen extract was fractionated using DEAE Sephadex A-50, Sephadex G-200 and Mono Q column. Allergenic activity of the fractions was checked by ELISA, skin tests, ELISA inhibition and immunoblot using sera of Ic-sensitive patients. A 67-kD protein was purified to homogeneity from Ic-VIII. The allergenic determinants of this protein were identified by SDS-PAGE and immunoblot after CNBr treatment. Results: Among Ic fractions, Ic-VIII was highly potent by ELISA, skin tests and showed cross-reactivity with 4 other tropical grasses by immunoblot and ELISA inhibition. The subfraction Ic-VIIIe1 of Ic-VIII showed a band at 67 kD on SDS-PAGE. On CNBr treatment, it gave 7 peptides, 3 of which were found to be allergenic. Conclusion: A 67-kD protein (Ic-VIIIe1) was isolated, purified to homogeneity and partially characterized. It showed cross-reactivity with tropical grasses tested and contained at least three allergenic determinants.
Reduced Frequencies of Peripheral Interferon-γ-Producing CD4+ and CD4– Cells during Acute Kawasaki Disease
Authors: Hahn Youn-Soo , Kim Yeom , Jo Seong-Ok , Han Heon-Seok
Published: April 2003

Background: This study was performed to analyze the frequencies of peripheral interferon (IFN)-γ-producing cells at the single-cell level and to determine concentrations of circulating IFN-γ in the acute and subacute phases of Kawasaki disease (KD). Methods: Ten patients with KD were studied and seven healthy children were selected as control subjects. Using immunofluorescent detection of intracellular IFN-γ in CD4-positive and CD4-negative cells, the frequencies of IFN-γ-producing cells in peripheral blood mononuclear cells were studied. Circulating IFN-γ levels were measured by enzyme-linked immunosorbent assay. Results: The frequencies of peripheral blood CD4+ and CD4– IFN-γ-producing cells in acute-phase KD patients were significantly lower than in subacute-phase KD patients and control children (p < 0.05). CD4– cells, thought to be mainly composed of CD8+ cells, appeared to be more responsible for the reduced frequencies of total IFN-γ-producing cells than CD4+ cells. There were, however, no differences in the frequencies of IFN-γ-producing cells between KD patients in the subacute phase and control children. In contrast, serum IFN-γ levels were higher in KD patients in the acute phase than in the subacute phase (p < 0.05). Conclusions: The above results show increased levels of circulating IFN-γ and decreased emergence of peripheral IFN-γ-producing cells in acute KD patients, suggesting transient infiltration of activated IFN-γ-producing cells into the inflammatory sites during acute KD. These findings also support the hypothesis that IFN-γ plays an important role in the pathogenesis of KD-related vasculitis.
Significance of Autoantibodies in Neonatal Lupus Erythematosus
Authors: Dörner Thomas , Feist Eugen , Pruss Axel , Chaoui Rabija , Göldner Burkhard , Hiepe Falk
Published: April 2003

Autoantibodies produced by the mother and transported into the fetal circulation are of significant importance in the diagnosis of neonatal lupus syndromes. These humoral autoimmune findings provide an unique opportunity to assess the pathogenic role of autoantibodies against the Ro(SS-A)/La(SS-B) complex, most notably for congenital heart block. Current knowledge about the involved autoantibody-autoantigen systems, including recent therapeutic concepts of these autoimmune syndromes, is summarized.
Effects of Mizolastine in vitro on Human Immunocompetent and Airway Cells: Evidence for Safety and Additional Property
Authors: Oddera S. , Cagnoni F. , Dellacasa P. , Canonica G.W.
Published: April 2003

Background: Mizolastine is a potent, peripherally acting, selective H1-receptor antagonist with potential anti-inflammatory properties. The aim of the study was to evaluate the in vitro effects of mizolastine on the expression of adhesion molecules by primary human airway epithelial and stromal cultures; moreover, the activity of mizolastine on parameters which reflect the immune response efficacy was investigated. Methods: Airway epithelial and stromal cells were collected from hypereosinophilic subjects by enzymatic digestion of polyps or turbinates. Cells were stimulated with interferon (IFN)-γ (500 IU/ml) in the presence of various mizolastine concentrations (6 × 10–8–6 × 10–6 M) for 24 h and the expression of CD106, CD54, CD58 and HLA class I was evaluated. Peripheral blood mononuclear cells from healthy volunteers were incubated with 1% phytohemagglutinin or anti-CD3 monoclonal antibody (20 ng/ml) in the presence of mizolastine, then T lymphocyte proliferation, HLA-DR expression and T cell subpopulations were evaluated. Results: Both in epithelial and stromal cultures, IFN-γ significantly upregulated all of the tested surface molecules (p < 0.05). The highest dose of mizolastine (6 × 10–6 M), corresponding to 10-fold the peak plasma level after a single oral administration of 10 mg, was able to act on fibroblasts, significantly downregulating the expression of CD54 (p < 0.05). Regarding T lymphocyte proliferation, the addition of mizolastine did not induce any significant change; furthermore, mizolastine was ineffective at all of the tested concentrations on both HLA-DR expression and CD4+/CD8+ ratio. Conclusions: This study demonstrated that mizolastine is able to selectively downregulate CD54 expression on stimulated stromal but not epithelial cells without impairing the immune system effectors. The possible clinical significance of these results are an antiallergic property and CD54 modulation on fibroblasts with a good safety profile as far as the lymphocyte response is concerned.
Anti-IgE Therapy in Asthma: Rationale and Therapeutic Potential
Authors: Barnes Peter J.
Published: April 2003

Airway inflammation is found in virtually all individuals with asthma symptoms. The factors contributing to asthma-related airway inflammation are multiple and involve a number of different inflammatory cells and mediators. Allergic responses at the level of the respiratory system are mostly mediated by IgE-dependent mechanisms. After sensitization of a susceptible individual and the synthesis and binding of allergen-specific IgE to target cells, atopic individuals respond immunologically to common, naturally occurring allergens by releasing mast cell-derived mediators. Subsequent allergen exposure in susceptible individuals produces a characteristic cascade of events orchestrated by immune effector cells, most prominently, mast cells, T lymphocytes and eosinophils. A new strategy, neutralizing IgE antibodies, inhibits expression of allergic symptoms by preventing the initial trigger of the allergic reaction, IgE binding to IgE-receptor bearing cells. rhuMAb-E25 is a recombinant humanized monoclonal anti-IgE antibody currently under investigation that has been shown to reduce allergic responses in atopic individuals and to improve symptoms and reduce rescue medication and corticosteroid use in patients with allergic asthma. Thus, the clinical effectiveness of rhuMAb-E25 supports the central role of IgE in allergic reactions and the viability of anti-IgE therapy as a potentially effective treatment option for asthma.
Immunologic Stimulation of Mast Cells Leads to the Reversible Exposure of Phosphatidylserine in the Absence of Apoptosis
Authors: Martin Sophie , Pombo Isabel , Poncet Pascal , David B. , Arock M. , Blank Ulrich
Published: April 2003

Background: Loss of phospholipid asymmetry represents one of the hallmarks of apoptosis and results in the surface exposure of phosphatidylserine (PS) which can be indirectly monitored by the calcium-dependent binding of annexin V. Methods and Results: Here, we provide evidence that the IgE-dependent stimulation of a rat mast cell line, as well as murine and human nontransformed mast cells, leads to the exposure of PS at the plasma membrane. The appearance of PS was quantitatively related to allergic mediator release. Pharmacological agents that prevent stimulus-secretion coupling blocked PS cell surface exposure and calcium ionophore-induced PS appearance, suggesting that it is a direct consequence of exocytosis rather than early signaling events initiated by the aggregation of the high-affinity IgE receptor (FcεRI). The surface exposure of PS in mast cells was reversible even in the continuous presence of stimulus and was not associated with the appearance of apoptotic nuclei, demonstrating that it was independent of physiological cell death. Conclusions: In addition to providing a means of monitoring exocytosis at the single cell level, our results indicate that PS externalization in mast cells is not necessarily related to apoptosis but could be an important feature of the degranulation process.
Metastatic Dissemination of Human Ovarian Epithelial Carcinoma Is Promoted by α2β1-Integrin-Mediated Interaction with Type I Collagen
Authors: Fishman David A. , Kearns Alicia , Chilukuri Krishna , Bafetti Lisa M. , O’Toole Edel A. , Georgacopoulos Joanne , Ravosa Matthew J. , Stack M. Sharon
Published: April 2003

Metastatic dissemination of epithelial ovarian carcinoma is thought to be mediated via tumor cell exfoliation into the peritoneal cavity, followed by adhesion to and invasion through the mesothelium which overlies the contents of the peritoneal cavity. In this study, we have utilized short-term primary cultures to analyze the effect of specific extracellular matrix proteins on properties of human ovarian epithelial carcinoma cells which contribute to the invasive phenotype. Analysis of cell:matrix adhesive profiles indicated that ovarian carcinoma cells adhere preferentially to type I collagen. Immunoprecipitation analyses demonstrated the presence of the collagen-binding α2β1 integrin in biotin-labeled ovarian carcinoma cell membranes, and cellular adhesion was inhibited by blocking antibodies directed against the α2 and β1 integrin subunits. The α2β1-binding peptide Asp-Gly-Glu-Ala (DGEA) was also moderately effective at blocking adhesion to collagen relative to the control peptide Ala-Gly-Glu-Ala (AGEA). Analysis of cell motility on protein-coated colloidal gold coverslips demonstrated that ovarian carcinoma cells migrate preferentially on type I collagen coated surfaces. Type I collagen promoted migration in a concentration-dependent, saturable manner, with maximal migration observed at a collagen-coating concentration of 50 μg/ml. Migration on collagen was inhibited by antibodies directed against the α2 and β1 integrin subunits and by DGEA peptide, providing evidence for the role of the α2β1 integrin in ovarian carcinoma cell motility. Culturing ovarian carcinoma cells on type I collagen gels led to a significant increase in conversion of the matrix metalloproteinase 2 zymogen to the 66-kD form, suggesting that adhesion to collagen also influences matrix-degrading proteinases. These data suggest that α2β1-integrin-mediated interaction of ovarian carcinoma cells with type I collagen, a protein prevalent both in the mesothelial extracellular matrix and in the peritoneal cavity of ovarian carcinoma patients, may function on multiple levels to promote metastatic dissemination of ovarian carcinoma cells.
Expression of Adhesion Factors and Degrading Proteins in Primary and Secondary Glioblastomas and Their Precursor Tumors
Authors: Tews Dominique S. , Nissen Anja
Published: April 2003

In tumor tissue specimens of 27 primary and 17 secondary glioblastomas and the precursor lesions, the immunohistochemical expression patterns of the membrane protein CD44s, the basal lamina proteins laminin, collagen IV, and fibronectin, the lectin galectin-3 recognizing tenascin and N-CAM as well as of the matrix-degrading enzymes matrix metalloproteinase MMP-2 and MMP-9, and cathepsin D were studied. Besides expression of basal lamina proteins in vessels, all glioblastomas and the precursor lesions showed strong immunoreactivity of CD44s, tenascin, galectin-3, and N-CAM which were restricted to solid tumor masses. Present in solid tumor areas, MMP-2, MMP-9 and cathepsin D were also strongly expressed by single tumors cells invading adjacent brain tissue at the infiltrative margin. Neither the expression pattern in primary and secondary glioblastomas nor in the precursor tumors revealed significant differences. There was also no intraindividual constant expression pattern during glioma progression or correlation with malignancy. Restricted expression of CD44s, galectin-3, tenascin and N-CAM in solid tumor masses seems to contribute to homotypic tumor cell adhesion while single tumor cells abolish this expression profile and acquire invasive activities by expression of cathepsin D, MMP-2 and MMP-9.
Quantification of Viral Load: Clinical Relevance for Human Immunodeficiency Virus, Hepatitis B Virus and Hepatitis C Virus Infection
Authors: Berger Annemarie , Braner Jens , Doerr Hans Wilhelm , Weber Bernard
Published: April 2003

Quantitative determination of viral load using nucleic acid amplification techniques represents the most accurate prognostic marker for human immunodeficiency virus type 1 (HIV-1) infection, independently of CD4+ cell count. Overall, the different methods for HIV-1 RNA determination (RT-PCR, nucleic acid sequence-based amplification, branched DNA) show a good reproducibility (0.5 log), however for low copy numbers and in HIV-1-infected children the variability may exceed 0.7 log. In non-HIV-1 subtype B infections the copy number is underestimated. While serology permits an accurate follow-up of hepatitis B virus (HBV) infection, HBV DNA quantification is used for monitoring of antiviral therapy, determination of infectiosity and in combination with serological markers for the resolution of unusual profiles, i.e. isolated anti-HBc reactivity. The prognostic relevance of hepatitis C virus (HCV) RNA determination is of limited value for the long-term prognosis of chronic hepatitis C, however the viral load may predict the outcome of antiviral therapy. Genetic diversity represents a challenge for HCV RNA quantification.
Parvovirus B19 Infection in Pregnancy
Authors: Eis-Hübinger Anna Maria , Dieck Dorothea , Schild Ralf , Hansmann Manfred , Schneweis Karl Eduard
Published: April 2003

Transplacental transmission of human parvovirus B19 (B19 virus) to the fetus is an important cause of intrauterine death, abortion, stillbirth, and nonimmune hydrops fetalis. Adverse outcome of pregnancy can occur after symptomatic and asymptomatic maternal infection. Only rare cases of congenital malformations and fetal disease in live-born infants have been associated with intrauterine B19 virus infection. Laboratory results obtained from paired maternal and fetal cord blood samples indicate that a reliable diagnosis of fetal B19 virus infection should be based on detection of parvovirus B19 DNA.
Placental and Fetal Pathology in Intrauterine Viral Infections
Authors: Horn L.-C. , Röse Ingeborg
Published: April 2003

Intrautrine viral infections show a wide range of fetal involvement with variable fetal outcome. The histopathologic changes of the placenta during viral infection show a wide spectrum. The most active processes are proliferative with a variety of cells involved. Vascular changes, including inflammatory and obliterative lesions, are frequently found leading ultimately to fibrosis of the villous stroma. Abnormalities of villous maturation occur, especially in early onset infections, sometimes with edematous features. In cases of fetal injury, intrauterine death and nonimmunologic fetal hydrops may occur, viral infection must be suspected. In several cases placental examination using conventional and molecular techniques may explain the clinical findings.
Examination of Potential Inhibitors of Hepatitis A Virus Uncoating
Authors: Bishop Naomi E.
Published: April 2003

Hepatitis A virus (HAV) replication in BS-C-1 cells was studied in the presence of ten potential uncoating inhibitors. Strong inhibition of HAV replication was only observed in the presence of the phenothiazine compound chlorpromazine and the lysosomotropic agent chloroquine, but not by other lysosomotropic agents. Chlorpromazine and chloroquine were found to prevent virus uncoating. Chlorpromazine is known to inhibit endocytosis of non- clathrin-coated vesicles. Chloroquine is a weak base amine, and thought to inhibit virus replication by preventing endosomal acidification. These results therefore suggest that entry of HAV in BS-C-1 cells does not depend on the low pH encountered in the clathrin-coated endocytic entry pathway. A possible role of calcium ions in mediating viral uncoating is discussed, as calcium ions were found to destabilize HAV particles in vitro.
Hepatitis Delta Virus
Authors: Taylor John M.
Published: April 2003

The purpose of this article is to give a concise update on our understanding of hepatitis delta virus (HDV). For more extensive background information the reader is directed to published reviews [1-5].
Sequence Analysis of Porcine Adenovirus Type 3 E1 Region, pIX and pIVa2 Genes, and Two Novel Open Reading Frames
Authors: Aggarwal Neeraj , Mittal Suresh K.
Published: April 2003

The porcine adenovirus type 3 (PAd3) genome between map units 0 and 13.7 was sequenced and compared with similar regions of other adenoviruses. This region consists of the left inverted terminal repeat sequences involved in DNA packaging, the entire early region 1 (E1) and the protein IX (pIX) transcription unit. The lower strand contains the C-terminal end of IVa2 of the E2A transcriptional unit and two novel open reading frames (ORFs). The E1 transcription unit consists of ORFs for proteins homologous to the E1A, E1B-17k and E1B-55k of both human adenovirus type 5 (HAd5) and bovine adenovirus type 3 (BAd3). The predicted PAd3 pIX demonstrated homology with the N-terminal portion of the pIXs of HAd5 and BAd3. On the lower strand, immediately after the putative IVa2 ORF, there are two unique ORFs of 208 and 203 amino acid residues that showed homology with Epstein-Barr virus nuclear antigens and other cellular transcription factors.
Pregnancy-Induced Decrease in Evoked Excitatory Junction Potentials in Guinea Pig Uterine Artery
Authors: Tare Marianne , Parkington Helena C. , Tonta Mary Anne , Coleman H.A.
Published: April 2003

The effects of stimulating the intramural nerves on the membrane potential and tension in the uterine artery of virgin guinea pigs were compared with the responses during pregnancy. In all tissues the amplitude of the excitatory junction potential (EJP) increased as the stimulus voltage was increased. The rate of increase in EJP amplitude in tissues from virgin animals greatly exceeded that recorded in late pregnant tissues. EJPs were abolished by tetrodotoxin but were resistant to blockade by α-adrenoceptor antagonists. Stimulation of the nerves also evoked a slow depolarization and contraction which were abolished by both tetrodotoxin and α-adrenoceptor antagonists. The amplitudes of the depolarizations and contractions were not correlated. The role of EJPs and α-adrenoceptor activation in the control of vascular function is discussed. Fluorescence histochemistry revealed a decrease in the density of the catecholamine innervation that was correlated with a decrease in catecholamine content as pregnancy progressed. In addition, there appeared to be a difference in the arrangement of the fluorescent varicosities, with a shift from varicosities that were close to the outer layer of smooth muscle in virgin tissues to those that were more distantly dispersed in the adventitia during late pregnancy. The changes would be expected to reduce the effectiveness of vasoconstrictor drive to the uterine artery as pregnancy progresses.
Coexpression of mRNAs for P2X1, P2X2 and P2X4 Receptors in Rat Vascular Smooth Muscle: An in situ Hybridization and RT-PCR Study
Authors: Nori Stefania , Fumagalli Lorenzo , Bo Xuenong , Bogdanov Yuri , Burnstock Geoffrey
Published: April 2003

The expression of mRNAs for three P2X receptor subtypes (PX21, P2X2, P2X4) in the rat vascular system was studied by in situ hybridization and RT-PCR. In heart sections mRNAs transcripts for all three receptors were colocalized in smooth muscle cells of coronary vessels, while no specific positivity was apparent in myocardium. Coexpression of P2X receptor mRNA transcripts were also observed in other peripheral vessels, including aorta, pulmonary artery, internal and external iliac arteries, renal artery and femoral artery. By contrast, no mRNA transcripts of the above receptors were found in the superior mesenteric artery. RT-PCR performed on microdissected tissues (coronary arteries, aorta and myocardium from various heart areas) confirmed the presence of P2X1, P2X2 and P2X4 receptor mRNAs. Furthermore, in the same tissues two splice variants of the P2X2 receptor were identified. These results reveal an important molecular heterogeneity of P2X receptors, thus substantiating the possibility of a heteropolymeric assembly of ATP-gated ion channels in the cardiovascular system.
The Impact of Contralateral Cooling on Skin Capillary Blood Cell Velocity in Patients with Diabetes mellitus
Authors: Haak Eva , Haak Thomas , Grözinger Yael , Krebs Günter , Usadel Klaus Henning , Kusterer Klaus
Published: April 2003

In healthy volunteers, cooling of the contralateral hand leads to a rapid decrease in the ipsilateral capillary perfusion via a nerval reflex arc. The aim of this study was to investigate whether this reflex arc after contralateral cooling might be altered in patients with diabetes mellitus with and without peripheral neuropathy. Therefore, 12 patients with diabetic neuropathy (4 IDDM, diabetes duration 17.2 ± 2.9 (SD) years, age 60.8 ± 4.0 years, HbA1c 6.5 ± 0.3%) and 12 patients with diabetes mellitus but without neuropathy (6 IDDM, diabetes duration 15.1 ± 2.7 years, age 55.9 ± 4.5 years, HbA1c 5.4 ± 0.1%) were investigated by nailfold capillaroscopy. Twelve healthy volunteers (age 56.8 ± 3.1 years, HbA1c 4.8 ± 0.2%) served as controls. Contralateral skin capillary blood cell velocity was determined at rest and during the following 20 min after cooling of the hand (3 min at 15°C). Blood pressure, heart rate and local skin temperature were examined regularly during the investigation. Resting capillary blood cell velocity did not differ between patients and controls. While contralateral cooling resulted in a decrease in capillary blood cell velocity (CBV) in controls (0.29 ± 0.05 vs. 0.42 ± 0.05 mm/s, p < 0.03), CBV remained unchanged or was delayed in patients. These results demonstrate that in diabetic patients nerval reflex arcs are impaired. A long-term follow-up in a larger number of patients is required to evaluate whether these findings might serve as a very early diagnostic tool for the diagnosis of developing diabetic neuropathy.
Insulin-Induced Arteriolar Dilation after Tyrosine Kinase and Nitric Oxide Synthase Inhibition in Hamster Cheek Pouch Microcirculation
Authors: Bertuglia S. , Colantuoni A.
Published: April 2003

We investigated the effects of tyrosine kinase (TK) and nitric oxide synthase (NOS) inhibition on insulin-induced dilation of arterioles. We determined the arteriolar diameter, red blood cell velocity (VRBC) and blood flow changes in hamster cheek pouch microcirculation as affected by insulin in presence of TK and NOS inhibitors, genistein, piceatannol and NG-monomethyl-L-arginine (L-NMMA). Microvessels were visualized by a fluorescent microscopy technique. Arteriolar diameter and VRBC were measured after topical application of insulin and genistein or piceatannol or L-NMMA. Insulin (10 µU/ml) induced diameter and VRBC increase in A3 and A2 arterioles by 30 ± 5 and 123 ± 4%, 16 ± 4 and 102 ± 3%, as percent of baseline values, respectively. After genistein or piceatannol prior to insulin A3 and A2 arterioles dilated by 10 ± 4, 5 ± 2% and 9 ± 4, 2 ± 1%, respectively. After L-NMMA prior to insulin A2 and A3, arteriole diameters increased by 12 ± 3 and 7 ± 2%, respectively. VRBC increased significantly in all the cases. TK and NOS inhibitors applied together abolished insulin-induced dilation with a reduction in VRBC and blood flow. In conclusion, full insulin-induced dilation of hamster cheek pouch arterioles requires TK signaling pathways. Furthermore, activation of insulin receptors, as well as other TK receptors, appears to be required for vasomotor tone regulation.
Voltage-Gated Sodium Channels in Human Aortic Smooth Muscle Cells
Authors: Cox R.H. , Zhou Z. , Tulenko T.N.
Published: April 2003

Whole-cell, voltage clamp methods were used to study inward currents in human aortic smooth muscle cells in culture. Cells were plated on glass coverslips, cultured in supplemented M-199 media with 5% serum and studied as primary cells and at passages 2–5. Inward currents were measured with a pipette solution containing Cs+ and TEA+ to block K+ currents and with 2.5 mM [Ca2+]₀ in the perfusate. Inward currents activated at about –50 mV, peaked at about –15 mV and reversed at about +30 mV. Values of peak inward current averaged 14.7 ± 3.3 pA/pF and cell capacitance averaged 124 ± 10 pF (n = 35). These currents activated rapidly with a time-to-peak current of 2.4 ± 0.3 ms at a test potential of –10 mV from a holding potential of –80 mV. The current also inactivated rapidly with a time course that could be described by two components with time constants of 1.8 ± 0.2 and 17.8 ± 3.5 ms at –10 mV. The currents decreased when extracellular Na+ was reduced and were completely inhibited by 50 nM tetrodotoxin (TTX), suggesting that they represented voltage-gated Na+ currents (INa). Activation curves were characterized with a V0.5 = –16.6 ± 2.4 mV and a slope factor k = –5.2 ± 0.2 mV while inactivation curves were characterized with a V0.5 = –60.9 ± 1.7 mV and a slope factor k = 8.8 ± 0.4 mV. Lowering external [Ca2+] to zero increased the maximum INa, shifted its voltage dependence in the hyperpolarizing direction and increased the rate of INa inactivation. Increasing external [Ca2+] or [Mg2+]decreased INa and slowed its rate of inactivation. These studies demonstrate the presence of voltage-gated Na+ channels with high TTX sensitivity that are modulated by extracellular divalent cations in human aortic smooth muscle cells maintained in cell culture. Window currents were found in the voltage range of –50 to –20 mV, suggesting that these channels could contribute to the resting membrane potential.
Evidence for the Involvement of Protein Kinase C in Depression of Endothelium-Dependent Vascular Responses in Spontaneously Hypertensive Rats
Authors: Soloviev A.I. , Parshikov A.V. , Stefanov A.V.
Published: April 2003

The goal of the present study was to evaluate the role of protein kinase C (PKC) in the depression of endothelium-dependent vacular response in spontaneously hypertensive Okamoto rats (SHR). Aortae from SHR demonstrated a decreased relaxant response to acetylcholine (Ach) as compared to aortae from normotensive Wistar-Kyoto (WKY) rats, while papaverine lowered the force of aorta to a similar degree in both strains of rats. PKC inhibitors, H-7 (5 × 10–6 M) and chelerythrine chloride (10–6 M), produced a greater decrease in the force developed by the aortae from SHR vs. WKY rats both in intact and chemically permeabilized tissues. In SHR aortae PKC inhibitors enhanced relaxation to Ach to a greater extent as compared to WKY aortae. Furthermore, in the presence of PKC inhibitors, the constrictor responses of SHR aortae to Ach were transformed into relaxant responses, and the concentration-response curve to Ach was shifted to the left. The sensitivity of aortae from SHR to authentic nitric oxide (NO) was lowere compared to WKY rats. EC50s for authentic NO in SHR and WKY rat aortae were different: –2.9 ± 0.15 × 10–6 M and 4.58 ± 0.1 × 10–7 M (n = 15, p < 0.001), respectively. Bioassay experiments using SHR aortae showed that the addition of chelerythrine (10–6 M) to the detector superfusate caused relaxation during treatment of the donor segment with Ach, indicating that the sensitivity of the aortae to NO had been restored. When SHR detector ring was substituted for denuded aortae from WKY rats and PKC inhibitors were not added to the detector superfusate, the relaxation of the detector aortae was also close to the normal Ach-induced relaxation. WKY aortae demonstrated a positive relationship between Ach-stimulated NO release and relaxant response amplitudes (correlation coefficient r = 0.905, p < 0.001, n = 10). In contrast, there was a significant negative correlation in SHR aortae (r = –0.712, p < 0.05, n = 10). Detection of NO release by chemiluminescence showed no significant difference in NO release in SHR and WKY aortae. Taken together, these data suggest that the blunted endothelium-dependent relaxations seen in SHR aortae are mainly due to a decreased sensitivity of vascular smooth muscle to EDRF/NO resulting from an increased PKC activity.
Sterically Inhomogenous Viscoelastic Behavior of Human Saccular Cerebral Aneurysms
Authors: Tóth Mária , Nádasy György L. , Nyáry István , Kerényi Tibor , Orosz Miklós , Molnárka Gyözö , Monos Emil
Published: April 2003

To clarify the mechanism leading to the development and rupture of intracranial aneurysms, tensile strength and viscoelastic parameters of 22 human saccular aneurysms were investigated. Meridional and circumferential strips from the thin and the thick part of the aneurysm sack and 18 control strips from the basilar artery of 8 patients with pathologies not affecting the cerebral arterial system were studied. The length of the strips was increased in 200-µm steps, while distending force was recorded. Tensile strength and viscoelastic parameters were computed. In both directions, tensile strength of thick strips was significantly lower than that of controls. In the meridional direction, tensile strength of thin strips was significantly larger than that of thick ones (14.5 ± 4.1 × 106 vs. 7.5 ± 2.0 × 106 dyn/cm2, p < 0.05). In the circumferential direction, thin strips tore at lower strain values than thick ones (29 ± 4 vs. 55 ± 16%, p < 0.05). Viscoelastic parameters changed in parallel. In circumferential direction, values of thick and thin strips were significantly lower than those of controls. In the meridional direction, values of thin strips were significantly higher than those of the thick ones. These observations show that characteristic mechanical deterioration and steric inhomogeneities accompany the loss of smooth muscle cells and the derangement of connective tissue elements in the wall of intracranial aneurysms, which may explain certain steps in their initiation, enlargement and rupture.
The Emergence of Physiological Genomics
Authors: Cowley Jr. A.W.
Published: April 2003

‘Physiological genomics’ represents a research paradigm shift emerging to define the functions of tens of thousands of newly discovered genes which are expected to emerge from the sequencing of the human genome and other model organisms. Genomic tools, which will allow a higher efficiency of identification of gene function, are being developed at remarkable speed. This article discusses some of the genomic and bioinformatic tools currently available or under development to provide the infrastructure for mapping and identification of gene function in simple organisms (bacteria, zebrafish, fly, worm) and complex mammalian organisms (mouse and rat). The problems facing the scientific community in the implementation of this functional approach are discussed as it is now evident that new technological and organizational infrastructures are emerging to link genes to overall function of whole organisms.
Thrombomodulin Activity of Fat-Derived Microvascular Endothelial Cells Seeded on Expanded Polytetrafluorethylene
Authors: Hedeman Joosten P.Ph.A. , Verhagen H.J.M. , Heijnen-Snyder G.J. , Sixma J.J. , de Groot Ph.G. , Eikelboom B.C. , Elisen M.G.L.M.
Published: April 2003

Lining the luminal surface of prosthetic vascular grafts with endothelial cells (cell seeding) will lower its thrombogenicity. Commonly used macrovascular human adult endothelial cells (HAEC) require in vitro cultivation before large enough numbers are obtained to cover grafts confluently. Fat-derived microvascular endothelial cells (MVEC) prove to be a good alternative as they can be harvested in much larger numbers while showing similar antithrombotic and fibrinolytic characteristics. An important anticoagulant function of macrovascular endothelial cells is due to the activity of thrombomodulin (TM) on their surface. In this study, the presence and functional activity of TM on fat-derived microvascular cells used in cell seeding was investigated. The expression and localization of TM on MVEC was studied using immunohistochemistry. Functional activity of TM on MVEC was measured by the generation of activated protein C (APC) and was compared to human umbilical vein endothelial cells (HUVEC). TM activity was studied in MVEC seeded on expanded polytetrafluorethylene (ePTFE) vascular prostheses and compared to blank prostheses. We found that TM was expressed on the surface of MVEC, both in vivo and vitro. TM-dependent generation of APC differed significantly between MVEC and HUVEC (3.98 ± 1.2 vs. 3.0 ± 0.7 nM, respectively). After seeding MVEC on vascular prostheses, TM activity did not change. APC generation was significantly higher on MVEC-seeded vascular grafts compared to blank grafts (4.0 ± 0.7 vs. 1.7 ± 0.5 nM, respectively). We conclude that TM is present and highly active on cultured MVEC. When seeded on ePTFE, MVEC retain the possibility to inhibit thrombin coagulant activity and to activate protein C. Therefore, since MVEC are readily available, the anticoagulant properties demonstrated here indicate that this cell type is suitable for cell seeding of vascular prostheses.