bluuebunny/crossref_metadata_2025 huggingface dataset:
High–Molecular–Weight Allergens of the House Dust Mite: An Apolipophorin–Like cDNA Has Sequence Identity with the Major M–177 Allergen and the IgE–Binding Peptide Fragments Mag1 and Mag3
Authors: Epton M.J. , Dilworth R.J. , Smith W. , Hart B.J. , Thomas W.R.
Published: April 2003

Background: A 349–residue recombinant polypeptide of Dermatophagoides farinae, Mag 3, has been shown to represent part of a larger 177–kD (M–177) allergen with very high IgE–binding activity. Methods: Cloning and sequencing of cDNA from the house dust mites Dermatophagoides pteronyssinus and Euroglyphus maynei was used to characterise the polypeptide containing the Mag 3 sequence. Results: cDNA clones containing the complete sequence of the E. maynei homologue of the M–177 allergen were isolated and analysed. The translation contained not only an amino acid sequence with 90% identity to the 349–residue Mag–3 fragment but also a further sequence with 90% identity to another IgE–binding recombinant D. farinae polypeptide designated Mag 1. The complete sequence encoded a mature polypeptide of 1,650 residues and a molecular mass of 189.5 kD. cDNA clones from D. pteronyssinus also encoded sequences equivalent to the Mag 1 and 3 polypeptides. The M–177 sequence showed strong similarity to the lipid transport apolipophorins found in insect lipophorins. Conclusions: cDNA sequence data show that the D. pteronyssinus and E. maynei homologues of the M–177 high–molecular–weight D. farinae allergen contain sequences equivalent to both the Mag 1 and Mag 3 recombinant IgE–binding fragments. The N–terminal sequence of the full–length 1,650 amino–acid allergen showed strong similarity to the insect apolipophorins which are poorly soluble in aqueous extracts and exist in the lipid transport particles in haemolymph. It is proposed that presentation in lipid particles could be a factor which enhances the immunogenicity of this group of allergens.
Antigen–Specific, IgE–Selective Unresponsiveness Induced by Antigen–Liposome Conjugates
Authors: Nakano Yoshio , Mori Masahito , Nishinohara Shouichi , Takita Yusaku , Naito Seishiro , Horino Atsuko , Kato Hiroshi , Taneichi Maiko , Ami Yasushi , Suzaki Yuriko , Komuro Katsutoshi , Uchida Tetsuya
Published: April 2003

Background: We have previously reported that ovalbumin (OVA) coupled with liposome via glutaraldehyde (GA) induced OVA–specific– and IgE–selective unresponsiveness in mice. Methods: In this study, OVA–liposome conjugates were made using four different coupling protocols: via GA, N–(6–maleimidocaproyloxy) succinimide (EMCS), disuccinimidyl suberate (DSS) and N–succimidyl–3(2–pyridyldithio)propionate (SPDP) and the induction of antigen–specific IgG and IgE antibody production was investigated for each. In addition, antigen–specific cytokine production by spleen cells of mice immunized either with OVA–liposome or with OVA adsorbed with aluminum hydroxide was investigated. Results: OVA–liposome conjugates coupled via GA or DSS did not induce anti–OVA IgE antibody production but induced substantial anti–OVA IgG antibody production. On the other hand, the induction of anti–OVA IgE unresponsiveness by OVA–liposome conjugates coupled via EMCS or SPDP was incomplete. The amount of interleukin 4 (IL–4) produced by spleen cells stimulated in vitro with OVA correlated well with anti–OVA IgE antibody production in donor mice. However, the production of no other cytokine, i.e., IL–2, IL–5, IL–10 or interferon–γ, was correlated with in vivo IgE antibody production. Conclusion: OVA–liposome coupled via GA or DSS induced complete suppression of anti–OVA IgE production. The results in this study further suggest that the regulation of IgE antibody production does not neccessarily correlate with so–called Th1 cytokine production.
The Effect of Antigen Stimulation on α4, β1 and β7 Chain Integrin Expression and Function in CD4+ Cells
Authors: Tarkowski Maciej , Pacheco Karin A. , Rosenwasser Lanny J.
Published: April 2003

Background: The α4 integrin, as α4β1 (VLA-4) or α4β7, is critical for T cell migration and proliferation, although its functional modulation remains poorly understood. We hypothesized that increased receptor density, based on new receptor chain synthesis, was one such mechanism. We examined the surface receptor density of the α4 and β1 chains on CD4+CD45RO+ cells, and the mRNA expression of these and the β7 chain in response to allergen and nonallergen antigen stimulation. Methods: Flow-cytometric analyses for CD49d, CD29, and CD45RO were performed on T cell lines specific for timothy, tetanus, and Candida from atopic and nonatopic donors. RNA was extracted from cells sorted to select CD4+/CD49d-positive cells before and after stimulation. Equivalent amounts of cDNA for β-actin, α4, β1 and β7 were used in PCR, and the products were quantified using phosphoimaging. Results: CD49d expression is heterogeneous on T cell lines and is upregulated by antigen stimulation on CD4+ T cells. The surface expression on CD4+CD45RO+ timothy allergen or tetanus toxoid T cell lines is at least double that found on CD45RO– cells. Antigen stimulation upregulated CD49d expression on the CD4+CD45RO+ subpopulation of both cell lines although it was not as significant as in the case of all CD4+ T cells. CD29 surface expression behaves similarly. Candida had no effect on CD49d or CD29. Messenger RNA expression for the α4 chain (CD49d) is significantly upregulated 48 h following the addition of timothy or tetanus. β7 chain expression also rises significantly on both cell lines. β1 chain expression increases, but not significantly. Conclusions: The surface expression of the CD49d is heterogeneous and much higher on CD4+CD45RO+ cells than on CD4+RO– T cells. The CD49d integrin chain on CD4+ T cells is upregulated following antigen exposure. However, the CD4+CD45RO+ subpopulation is only partially responsible for this increase suggesting other T cells to have this receptor expression upregulated. CD29 expression behaves similarly. Messenger RNA expression increases coordinately for α4, β7, and not significantly for β1 in these cells. These observations provide a potential mechanism for the selective accumulation of T cells at sites of inflammation, and suggest an important point of intervention for allergic and inflammatory disease.
Modulation of Allergen-Specific Immune Responses to the Major Shrimp Allergen, Tropomyosin, by Specific Targeting to Scavenger Receptors on Macrophages
Authors: Rajagopal Deepa , Ganesh Kolumam A. , Subba Rao Pillarisetti V.
Published: April 2003

Background: Tropomyosin from shrimp is the major cross-reacting crustacean food allergen. Earlier studies have led to the purification and immunochemical characterization of the major IgE binding epitopes of the allergen. Maleylated proteins are known to be specifically targeted to scavenger receptors on macrophage. Since antigens processed and presented by macrophages are known to elicit Th1 type of responses and allergic responses are characterized by polarization towards Th2 phenotype, the possibility of modulation of allergen-specific immune responses by targeting of tropomyosin to macrophage via scavenger receptor was explored. Methods: The IgG and IgE binding potential of the native maleylated form of tropomyosin was carried out by ELISA and immunoblot. The ability of the native and maleylated form of allergen to induce in vitro proliferation of splenocytes from BALB/C mice immunized with both forms of allergen was tested. The in vitro production of IL-4 and IFN-γ by splenocytes from mice immunized with the two forms of allergen was determined from culture supernatants. The in vivo production of serum IgG1 and IgG2a antibodies following immunization with native and modified allergens was monitored by ELISA. Results: The maleylated form of tropomyosin was found to have reduced antigenicity and allergenicity as compared to its native counterpart. The modified allergen was, however, found to elicit a cellular response similar to native tropomyosin in vitro. Analysis of the cytokine profiles showed a modulation from an IL-4-dominant, proallergic, Th2 phenotype to an IFN-γ-dominant, antiallergic, Th1 phenotype that could also be correlated to a modulation in the in vivo antibody isotype. Conclusion: The results suggest the possible potential for modulating allergic responses in vivo by selective targeting to macrophages.
Soluble CD30 and Cyclosporine in Severe Atopic Dermatitis
Authors: Caproni M. , Salvatore E. , Cardinali C. , Brazzini B. , Fabbri P.
Published: April 2003

Background: Allergen-reactive Th2-like cells expressing membrane CD30 are present in the circulation of atopic dermatitis (AD) patients during seasonal allergen exposure. Moreover, CD30+ T cells are present in the lesional skin of AD patients and high levels of soluble CD30 (sCD30) are found in the serum of the same atopic patients. To investigate the immunosuppressive capacity of cyclosporin A (CsA) in AD patients, the sCD30 serum level was determined before and after CsA treatment (5 mg/kg/day) in 10 patients with severe, refractory AD. The sCD30 serum levels before and after CsA therapy together with other serum parameters were correlated with disease activity. Methods: sCD30 serum levels were detected using a commercial sandwich ELISA; serum eosinophil cationic protein (ECP) levels were determined using a radioimmunoassay (RIA). Results: In all AD patients sCD30 serum levels were increased ranging from 36 to 300 U/ml, with a mean value equal to 135.7 U/ml. After 6 weeks of CsA treatment, not only was there a significant difference between serum sCD30 levels before (mean 135.7) and after (mean 96.2) treatment but even the serum ECP levels before (mean 57.78) and after (mean 18.69) therapy showed an important reduction. Moreover, no significant difference was found between the mean of serum IgE levels before and after treatment, although the values showed a correlation (p = 0.0003). No significant correlations could be demonstrated between sCD30 levels and serum IgE or between sCD30 and ECP serum levels nor between sCD30 levels and blood eosinophil count after CsA treatment. Moreover, a positive correlation (p = 0.001) was instead documented between sCD30 and the severity of the disease. Conclusions: In this study, CsA therapy results in clinical improvement together with a statistically significant reduction in sCD30 and ECP serum levels in AD patients.
The Role of Sphingosine Kinase in the Signaling Initiated at the High-Affinity Receptor for IgE (FcεRI) in Mast Cells
Authors: Baumruker Thomas , Prieschl Eva E.
Published: April 2003

Over the last few years, sphingolipids have emerged as an additional class of lipids participating in signaling events in various cell types. The best-investigated examples so far are ceramide and sphingosine-1-phosphate. Ceramide-activated protein kinase and sphingosine kinase are two enzymes which respond to and generate these mediators. In particular, sphingosine kinase, its substrate sphingosine and the product sphingosine-1-phosphate have recently been implicated in the signaling cascades initiated at the FcεRI of mast cells. High intracellular levels of sphingosine seem to serve as an ‘intracellular’ inhibitor which is ‘deactivated’ by the action of sphingosine kinase, due to the conversion to sphingosine-1-phosphate. One mode of action of the inhibitory process in this cell type is prevention of the activation of the mitogen-activated protein (MAP) kinase pathway. Sphingosine-1-phosphate itself, the product of this enzymatic reaction, is believed to lead to Ca2+ mobilization and to stimulate the MAP kinase pathway. The existence and function of this second messenger explains the ‘IP3 gap’ described in mast cells after FcεRI activation. Therefore, a picture emerges whereby the balance of these two lipid molecules seems to be decisive for the activation of mast cells by IgE plus antigen, with sphingosine kinase acting as a permissive switch for stimulation.
Activation-Induced Apoptosis in T Cells from Young and Old Fischer 344 Rats
Authors: Pahlavani Mohammad A. , Vargas Danial A.
Published: April 2003

Background: Activation-induced apoptosis is believed to limit cell proliferation and eliminate the high number of activated cells during an immune response. Methods: Activation-induced apoptosis was investigated in splenic T cells isolated from young (6 months) and old (24 months) male Fischer 344 rats. The cells were incubated with anti-CD3, concanavalin A or staphylococcal enterotoxin B (primary stimulus) for 72 h, followed by restimulation with anti-CD3 or concanavalin A (secondary stimulus). Apoptosis was assessed by DNA fragmentation assay and DNA gel electrophoresis. The expression of the apoptotic marker CD95 was analyzed by flow cytometry and the relative levels of CD95 ligand, Bcl-2 and Bax protein were measured by immunoblotting. Results: It was shown that DNA fragmentation was very low in the unstimulated T cells from both young and old rats. However, the level of DNA fragmentation was 45–55% greater in the activated T cells from old rats than in the activated T cells from young rats. The increase in DNA fragmentation was paralleled by an increase in the proportion of cells expressing the CD95 molecule. The proportion of CD95+ cells was approximately 40% higher in T cells from old rats than in T cells from young rats. In addition, it was found that the expression of CD95 ligand and Bax increased and Bcl-2 decreased in the activated T cells from old rats compared to the activated T cells from young rats. Conclusion: Our data suggest that the increase in sensitivity of T cells to apoptosis with age may contribute to age-associated immune dysfunction and disorders.
Eosinophil Activation Status and Corticosteroid Responsiveness in Severe Asthma
Authors: Bossé M. , Audette M. , Laflamme G. , Ferland C. , Boulet L.P. , Laviolette M.
Published: April 2003

Background: Since eosinophils are implicated in asthma pathogenesis, we investigated whether these cells were activated in severe asthma. Methods: Twenty-six asthmatics with different clinical responses to oral corticosteroid (CS), i.e. sensitive [change in forced expiratory volume in 1 s (ΔFEV1) ≥ 25% after oral methylprednisolone, 40 mg daily, for 14 days, n = 7], resistant (ΔFEV1 ≤ 15%, n = 9) and dependent (≥ 20 mg oral prednisone daily for acceptable asthma control, n = 10), were studied. Results: Calcium ionophore-induced leukotriene (LT) C4 release of purified blood eosinophils was similar in the three groups. Cell incubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced ionophore-induced LTC4 release, and this effect was higher in CS-sensitive (5-fold) than in CS-resistant subjects (1.7-fold) (p = 0.02). CS treatment decreased blood eosinophil counts in these two groups of subjects (p ≤ 0.02) and decreased GM-CSF-enhanced LTC4 release in CS-sensitive asthmatics only (p = 0.04). In contrast, despite a high mean daily dose of oral CS (35 ± 8 mg), blood counts of eosinophils from CS-dependent subjects were higher (p = 0.03) and GM-CSF enhancement of LTC4 release was greater (2.8-fold) than in CS-sensitive (2.1-fold) and CS-resistant (1.7-fold) subjects (p = 0.04). Interestingly, serum from CS-resistant subjects reduced GM-CSF enhancement of LTC4 release by eosinophils of CS-sensitive asthmatics (p = 0.001). Conclusions: Eosinophils from CS-dependent asthmatics have an impaired response to CS, whereas serum from CS-resistant subjects contains an inhibitor of eosinophil response to GM-CSF.
Transepithelial Migration of Neutrophils
Authors: Huber Denise , Balda Maria S. , Matter Karl
Published: April 2003

Neutrophils are a key cell type of nonadaptive immune system and are the first phagocytic cell type that reaches mucosal inflammatory sites. On the last stage of their journey from the blood stream to a mucosal surface, neutrophils cross a generally sealed epithelium by migrating along the paracellular pathway to the luminal side of the epithelial layer. This last step involves a specific receptor-mediated adhesion event of the neutrophil to the epithelium, followed by a rapid and highly coordinated reversible opening of the epithelial intercellular junctions that allows the transmigration of the neutrophils. Although we do not yet understand the molecular mechanisms that mediate this transmigration process, the last years witnessed the discovery of the first neutrophil and epithelial cell surface proteins critically involved in transepithelial migration of neutrophils.
Accumulation of Genetic Alterations in Brain Metastases of Sporadic Breast Carcinomas Is Associated with Reduced Survival after Metastasis
Authors: Hampl Monika , Hampl Jürgen Anton , Schwarz Peter , Frank Stephan , Hahn Matthias , Schackert Gabriele , Saeger Hans-Detlev , Schackert Hans Konrad
Published: April 2003

Tumor progression is characterized by stepwise accumulation of genetic alterations. To identify alterations associated with breast cancer metastasis, an analysis of comparative loss of heterozygosity (LOH) was performed on 38 primary sporadic breast carcinomas and 16 distant metastases. Two loci at 5q21 and 18q21 were chosen because of their reported increased deletion frequency in metastatic tumors. LOH at 17q21, 13q12–13, 17p13.1 and 11q22–23 was analyzed to determine whether there is a specific involvement of these breast cancer-associated gene loci in the metastatic process. Our data show that distant metastases are characterized by markedly increased LOH frequency at all loci examined. In both gene locus groups, significantly more distant metastases are affected by combined LOH. Furthermore, a significantly reduced postmetastatic survival time has been observed in patients with brain metastases affected by synchronous allelic loss at the four breast cancer-associated gene loci. Our results suggest that cumulative LOH of breast cancer-related gene loci is associated with a more aggressive phenotype of metastatic breast tumors.
Tissue-Specific Posttranscriptional Downregulation of Expression of the S100A4(mts1) Gene in Transgenic Animals
Authors: Ambartsumian N. , Klingelhöfer J. , Grigorian M. , Karlstrøm O. , Sidenius N. , Georgiev G. , Lukanidin E.
Published: April 2003

The S100A4(mts1) is a gene associated with generation of metastatic disease. In order to analyze the consequences of alteration of the pattern of expression of the S100A4(mts1) gene we obtained strains of transgenic mice bearing the S100A4(mts1) gene under the control of a ubiquitous and constitutive 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) gene promoter. In transgenic animals the expression of the transgene RNA was detected in all organs, but only some of the organs showed elevated levels of the protein. Expression of the S100A4(Mts1) protein was downregulated in the organs that normally do not express the gene in the wild-type animal. The transgene RNA is detected in the polysomes indicating that it could be translated into the S100A4(Mts1) protein. The specificity of the S100A4(Mts1) protein expression is determined by a complex mechanism including regulation of translation and/or posttranslational degradation.
Cyclosporin A Upmodulates the α-Subunit of the Interleukin-2 Receptor and the Metastatic Ability of Murine B16F10 Melanoma Cells
Authors: Boyano María Dolores , de Galdeano Alicia García , García-Vázquez María Dolores , Alvarez Antonia , Luz Cañavate María
Published: April 2003

In the present study, the effect of in vitro cyclosporin A (CsA) treatment on IL-2R expression and the metastatic behavior of B16F10 melanoma cells has been reported. CsA treatment was found to increase the percentage of B16F10 cells expressing the α-subunit of IL-2R on the cell surface and also at the mRNA level. Moreover, CsA treated B16F10 cells also express the β-subunit of IL-2. In vivo experiments showed that CsA increases the affinity of B16F10 metastazing cells for the liver and decreases that for the lung. CsA modulated the expression of MHC class I and class II antigens, but no significant differences in the resistance of CsA-treated B16F10 cells to NK lysis were observed. Finally, proliferation of B16F10 cells in the presence of several doses of CsA did not vary and CsA increased the amount of IL-1β mRNA expression. These results suggest that CsA, through the modulation of cytokines and MHC antigen expression on B16F10 cells, could have an effect upon the metastatic progression of the B16F10 melanoma.
In vitro Invasiveness of Human Breast Cancer Cells Is Promoted by Low Density Lipoprotein Receptor-Related Protein
Authors: Li Yonghe , Wood Nick , Grimsley Philip , Yellowlees David , Donnelly Peter K.
Published: April 2003

Low density lipoprotein receptor-related protein/α2-macroglobulin receptor (LRP) is a surface membrane endocytic receptor, one of whose many functions is the regulation of plasminogen activator-mediated cell migration. LRP is known to have a role in migration and invasion, but its direct involvement has been demonstrated only in non-tumour cells. We investigated six breast cancer cell lines and a normal mammary epithelial cell clone for surface and total cellular LRP expression, and confirmed that its presence corresponds to the ability to invade and migrate in vitro. We showed that LRP in the tumour cell lines is expressed at a wide range of levels: from ∼300 to ∼6,300 sites per cell. Four of the breast cancer cell lines expressed LRP at over 1,000 sites/cell and were markedly invasive in our assay, the remainder of the cell lines and the normal clone having far fewer LRP sites and lacking invasive ability. We further showed that the migratory and invasive abilities of a highly invasive breast cancer cell line are both inhibited by receptor-associated protein, a unique LRP ligand which normally has a solely intracellular distribution but which, when added to culture medium, can inhibit all other ligand interactions with this receptor.
Clustering of Murine Lung Metastases Reflects Fractal Nonuniformity in Regional Lung Blood Flow
Authors: Kendal Wayne S.
Published: April 2003

In the experimental metastasis assay certain animals, from groups of similarly treated animals, develop more lung metastases than expected from random chance alone. This clustering of metastases is characterized by a power function relationship, σ2 = aμb, between the variance, σ2, and mean, μ, of the numbers of lung metastases per animal (a and b are constants). To determine whether this clustering could be an artifact of experimental metastasis, whether it could be influenced by different experimental conditions, and to attempt to clarify its cause, 22 published data sets from experimental metastasis utilizing 2,145 mice, as well as 8 data sets from spontaneous metastasis utilizing 1,020 mice were analyzed. In these experiments cell cloning, cell-cell fusion, treatment with a protein kinase C inhibitor, treatment with cell adhesion compounds, and transfection with either the ras oncogene, the sialidase gene, or the urokinase sense and antisense genes were used to influence metastasis. They employed 14 different cell lines and 6 different strains of inbred mice. Clustering of metastasis was evident in animals from the spontaneous metastasis assays as well as from the experimental metastasis assays. It was apparent whether mice were injected with tumor cells derived from clones or from cell lines. Clustering was demonstrated within each data set, regardless of the experimental conditions employed. A single variance to mean power function (with a = 2.2 and b = 1.51) characterized the clustering in the 30 data sets. The regional distribution of blood flow through lungs and other organs is nonuniform, exhibiting a fractal symmetry on change of scale. This symmetry implies that the variance of a region’s blood flow is related to its mean by the same power function as was observed with metastasis. Indeed, measurements of blood flow from isolated canine lungs yield b = 1.56, similar to the corresponding figure from murine lung metastasis. These findings lend support to the hypothesis that the observed clustering of metastases is a consequence of fractal variations in lung blood flow.
Improvement of the Specificity of Enzyme Immunoassays for the Detection of Rotavirus and Adenovirus in Fecal Specimens
Authors: Rabenau Holger , Knoll Bianca , Allwinn Regina , Doerr Hans Wilhelm , Weber Bernard
Published: April 2003

A variable rate of false-positive results may be observed with commercial enzyme immunoassays (EIAs) for the detection of rotavirus and adenovirus antigen in stool specimens, depending on the quality of the reagents and the presence of potentially interfering substances in stool samples. The present study was performed in an attempt to improve the specificity of current commercial rotavirus and adenovirus EIAs without significant loss of sensitivity by optimizing the cut-off value. A collective of 174 stool samples obtained from children suffering from acute gastroenteritis was tested. Electron microscopy (EM) and PAGE were used as reference methods for rotavirus detection. For the evaluation of the adenovirus kits, virus isolation in cell culture and the polymerase chain reaction served as reference standards. The highest sensitivity for rotavirus and adenovirus detection was achieved by the Ridascreen® Rotavirus and Ridascreen® Adenovirus. However, the Ridascreen® Rotavirus and Ridascreen® Adenovirus produced the highest number of false-positive results (n = 9) for each rotavirus and adenovirus detection. Cross-reactivities to coronaviruses and reoviruses were observed with the rotavirus antigen EIAs. For Rotazyme II, Ridascreen Rotavirus and Ridascreen Adenovirus, the specificity could be markedly increased without loss of sensitivity by doubling the cut-off value. For the alternative immunoassays, which were overall more specific, it was not possible to significantly decrease the rate of false-positive results without impairment of sensitivity by raising the cut-off value. In conclusion, at least for some rotavirus and adenovirus antigen EIAs, the cut-off value set by the manufacturer may not permit an optimal differentiation between true-positive and -negative samples. By raising the cut-off value from 50 to 100%, the specificity of two rotavirus antigen and one adenovirus antigen EIA can be improved markedly without significant loss of sensitivity.
Herpes simplex Virus Type 1 DNase: Functional Analysis of the Enzyme Expressed by Recombinant Baculovirus
Authors: Kehm Elke , Göksu Mehmet-Ali , Bayer Stefanie , Knopf Charles W.
Published: April 2003

Herpes simplex virus type 1 DNase (HSV-1 DNase) was expressed in insect cells by recombinant baculovirus (NPVUL12) and purified by a combination of anionic exchanger chromatography and gel filtration. Two polypeptides of 85 and 75 kD, whose ratio varied during purification, were induced 24 h after infection. The 75-kD protein was isolated and shown to possess catalytic activity. Gel filtration analysis indicated that the active form of the enzyme at an ionic strength of I = 0.3 is a dimeric protein with an apparent molecular weight of 130,000. The recombinant enzyme exhibited the overall characteristics of the native enzyme such as 5′-3′ exonuclease and endonuclease activities with a preferred degradation of DNA. In the absence of extraneously added Mg2+, the enzyme was capable of removing mononucleotides from 5′-end-labeled DNA, but not from RNA and 3′-end-labeled DNA. The peculiar mechanism of double-strand DNA degradation suggests a specific role of HSV-1 DNase in DNA recombination processes during viral replication.
Cytomegalovirus Infection in Pregnancy: A Still Complicated Diagnostic Problem
Authors: Lazzarotto T. , Spezzacatena P. , Pradelli P. , Abate D.A. , Gabrielli L. , Varani S. , Landini M.P.
Published: April 2003

The diagnostic problems linked to human cytomegalovirus (HCMV) in pregnancy are many and not all have been fully defined. In long-term seropositive women there is a tacit agreement that no laboratory testing for HCMV should be carried out. In seronegative women a test for HCMV-specific IgG should be performed at least twice during the first 4 months of pregnancy, and if the seronegativity persists, further follow-up might be stopped. On the other hand, if a seropositivity appears the diagnosis of a primary HCMV infection is established and prenatal diagnosis should be offered to the mother. Finally, in the case of a pregnant woman with unknown serological status, the diagnosis of HCMV infection is a complex problem and several different questions need to be addressed. In our opinion they should be screened with a reliable IgM test (confirmed by blot if necessary) followed, in the case of positivity, by an avidity assay. Pregnant women undergoing a primary HCMV infection should be encouraged to seek prenatal diagnosis to be performed by PCR and virus isolation from amniotic fluid at the 21st to 23rd week of gestation.
Herpes simplex Virus Infection in Pregnancy: Diagnosis and Significance
Authors: Desselberger Ulrich
Published: April 2003

Herpes simplex viruses (HSV) cause genital infections, and HSV infection during pregnancy can result in infection of the neonate. Here HSV structure and replication,viral pathogenesis, human immune responses to HSV, the epidemiology, details of HSV diagnosis with emphasis on typing, clinical features and antiviral treatment are reviewed. The importance of appropriate diagnostic procedures in relation to correct risk assessment of neonatal infection is emphasized.
Human Immunodeficiency Virus and Hepatitis B Virus Infection in Pregnancy: Diagnostic Potential of Viral Genome Detection
Authors: Berger Annemarie , Doerr Hans W. , Weber Bernard
Published: April 2003

Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) account for millions of cases of vertical infections worldwide. Laboratory diagnosis is essential for the detection of potentially infectious carriers. HBeAg represents the best serological marker for HBV replication. Since 10% of HBeAg-negative carriers transmit the virus to their children, determination of viral DNA is more reliable for the assessment of the risk to vertical infection. Risk assessment of vertical HIV transmission and monitoring AZT therapy during pregnancy are achieved by determination of HIV-1 viral load and CD4+ cell count. HIV-1 RNA or cDNA detection permits a nearly 100% sensitive diagnosis of congenital HIV infection already 2 weeks after birth. While qualitative HBV DNA determination should be limited only to anti-HBe carriers in order to assess infectiosity, HIV-1 RNA measurement represents in combination with the CD4+ cell count the best prognostic marker for vertical HIV infection and for the follow-up of infected children.
Functional Characterization of Bovine Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase and Fusion Proteins Expressed by Adenovirus Recombinants
Authors: Mittal Suresh K. , Tikoo Suresh K. , van den Hurk J.V. , Breker-Klassen Michelle M. , Yoo Dongwan , Babiuk Lorne A.
Published: April 2003

We constructed replication-competent human adenovirus type 5 (HAd5) recombinants (HAd5-HN and HAd5-F) containing the bovine parainfluenza virus type 3 (BPIV3) hemagglutinin-neuraminidase (HN) or fusion (F) gene under the control of the simian virus 40 (SV40) regulatory sequences. These genes were inserted in the early region 3 (E3) of the HAd5 genome in the E3 parallel orientation. Expression of HN or F in HAd5-HN- or HAd5-F-infected cell extracts, respectively, was observed by immunoprecipitation using a BPIV3-specific polyclonal antiserum. Our results suggest that HN and F expressed by HAd5 recombinants were functionally similar to the native HN and F expressed in BPIV3-infected cells.
A Precipitation Reaction Found in Patients with Hepatitis C as a Marker for the Purification of Virus-Like Particles
Authors: Kim Chung Yong , Yoon Jung-Hwan , Kim Joo Sung , Kim Yong Tae , Jung Hyun Chae , Lee Hyo-Suk , Yoon Yong Bum , Song In Sung
Published: April 2003

Even after the molecular cloning of the hepatitis C virus (HCV), an HCV-specific precipitation reaction has not yet been identified. We attempted to develop a precipitation system exclusively for anti-HCV-positive sera as a first step in finding an HCV-specific antigen and HCV-associated particles. In some patients being in a final stage of different liver diseases, we found sera (179/132,761; designated ‘a-CK’) which specifically precipitated with anti-HCV- and HCV-RNA-positive sera (designated ‘CK’). When CK-positive sera were searched for in patients with various liver diseases using standard a-CK-positive plasma, CK was detected in 420 (57.9%) of 726 anti-HCV-positive sera and in none of the 1,630 anti-HCV-negative ones. The nature of CK and a-CK has not been fully clarified yet; CK demonstrated inter-βγ mobility, whereas a-CK showed β-globulin mobility; CK was not detected in cryoprecipitate, but HCV RNA was present in precipitates of CK-positive plasma incubated with one that was a-CK positive. Transmission electron microscopy revealed two size ranges of particles in the precipitate of CK- and a-CK-positive plasmas, 23–38 nm and 48–65 nm. We have found a novel precipitation system which is exclusive to anti-HCV-positive sera and which specifically precipitates an HCV-RNA-containing serum fraction and particles. This system can be useful for the purification and characterization of the circulating particles. Furthermore, it may be a new approach to the nature of HCV-RNA-carrying material.
Immunoglobulin G and M Hepatitis C Virus Core Antibody (JCC.2) Response in Chimpanzees Infected Experimentally with Hepatitis C Virus
Authors: Shioda Atsuo , Moriyama Mitsuhiko , Hayashi Nakanobu , Sugitani Masahiko , Uchida Toshikazu , Shikata Toshio , Nishizono Akira , Abe Kenji , Arakawa Yasuyuki
Published: April 2003

Objective: To evaluate changes in serum immunoglobulin M (IgM) and G (IgG) hepatitis C virus (HCV) core antibodies after HCV infection in acute hepatitis C. Methods: Serum HCV RNA and IgM and IgG HCV core antibodies were investigated using sera sequentially sampled from three chimpanzees experimentally infected with HCV. Serum IgG HCV core antibody titer was measured using a JCC.2 enzyme-linked immunosorbent assay (ELISA) kit (Chemo-Sera-Therapeutic Research Center, Kumamoto, Japan). IgM core antibody titer was measured using horseradish peroxidase-labeled monoclonal anti-human IgM as the secondary antibody for the JCC.2 ELISA kit. Serum HCV RNA was detected using the 5′ noncoding region as the primer according to the reverse transcriptase (RT) nested polymerase chain reaction (PCR) and competitive RT-PCR method. Results: IgM JCC.2 antibody was detected when alanine aminotransferase (ALT) peaked, showing the closest correlation with the changes in ALT. A period during which IgM JCC.2 antibody was positive but HCV RNA as determined by RT-nested PCR was negative was observed after the elevation of ALT level. Conclusion: These results indicate the usefulness of detection of serum IgM JCC.2 antibody in making a definitive diagnosis of acute hepatitis C and the follow-up observation of hepatitis C.
Transcriptional Pattern of Varicella-Zoster Virus Glycoproteins E and I
Authors: Rahaus Markus , Wolff Manfred H.
Published: April 2003

Glycoproteins I (gI) and E (gE) of the Varicella-zoster virus are encoded by the neighbouring open reading frames 67 and 68. From earlier work it is known that both genes are transcribed into several transcript species which differ in size. From gI, three transcripts of 1.65, 2.7 and 3.6 kb and from gE, two transcripts of 2.15 and 3.6 kb in size are known. We present further northern analysis and show that these various transcript species appear in different amounts at different times after infection. 12 h after infection, transcripts of 1.65 kb (gI) and 2.15 kb (gE) were clearly detectable, whereas the other transcripts appeared later on. RT-PCR experiments using a set of seven different primers provided clear evidence that gI and gE are transcribed both, mono- and bicistronically with predominance on the respective monocistronic transcript.
mRNAs Coding for the Calcium–Sensing Receptor along the Rat Nephron: Effect of a Low–Phosphate Diet
Authors: Caride Ariel J. , Chini Eduardo N. , Homma Sumiko , Dousa Thomas P. , Penniston John T.
Published: April 2003

We investigated the localization of mRNA encoding the calcium–sensing receptor (CaSR) along the rat nephron. For this purpose, we combined microdissection of nephron segments and RT–PCR techniques. The results indicate that mRNA encoding rat CaSR is present in rat glomeruli and distal segments (medullary thick ascending limb, cortical thick ascending limb, distal convoluted tubule and cortical collecting duct), whereas it was not detected in proximal convoluted tubules or proximal straight tubules. We also studied whether the CaSR transcription in kidney cortex was modified in response to low dietary phosphate. No significant changes were detected. Given the fact that a low–phosphate diet increased Ca2+ excretion by more than 50–fold, the results suggest that if the CaSR regulates Ca2+ reabsorption, it does so through receptor occupancy by Ca2+ rather than by changes in receptor expression.
How Does Podocyte Damage Result in Tubular Damage?
Authors: Kriz W. , Elger M. , Hosser H. , Hähnel B. , Provoost A. , Kränzlin B. , Gretz N.
Published: April 2003

Severe podocyte damage including detachment from the GBM leads to adhesion of the glomerular tuft to Bowman’s capsule, thus to a local loss of the separation of the tuft from the interstitium. Perfused capillaries contained in the tuft adhesion deliver their filtrate no longer into Bowman’s space but into the interstitium. In response, interstitial fibroblasts create a cellular cover around the focus of misdirected filtration, interpreted teleologically, aiming at preventing the entry of this fluid into the interstitium. This results in the formation of a crescent–shaped, fluid–filled paraglomerular space overarching the segmental glomerular lesion. Extension of this space over the entire glomerulus leads to global sclerosis; extension of this space via the urinary pole onto the outer aspect of the corresponding tubule leads to the degeneration of the tubule. Since, as we postulate, such misdirected filtration and filtrate spreading is the crucial mechanism of damage progression in 'classic’ focal segmental glomerulosclerosis (FSGS), the most characteristic structural injury of FSGS is the merger of the tuft with the interstitium, represented by a tuft adhesion, later a synechia. Therefore, histopathologically, 'classic’ FSGS is best defined by an adhesion/synechia of the tuft to Bowman’s capsule.
Protection against Hydrogen Peroxide Induced Injury in Renal Proximal Tubule Cell Lines by Inhibition of Poly(ADP–Ribose) Synthase
Authors: Jung Jin Sup , Park Mi Young , Lee Ryang Hwa , Jun Jin Sook , Kim Yong Keun
Published: April 2003

Radicals including superoxide anions, hydrogen peroxide or hydroxyl radicals and NO or peroxynitrite cause the breakage of DNA strands and activation of poly–(ADP–ribose) synthase (PARS). Recent studies showed that inhibition of PARS activity reduces the tissue injury after exposure to oxidative stress. However, the role of PARS in renal injury by oxidants has not been examined. In this study effect of a PARS inhibitor, 3–aminobenamide (3–AB), on injury of opossum kidney or LLC–PK1 cells by hydrogen peroxide or tert–butyl hydroperoxide (t–BHP) was examined. The exposure of opossum kidney cells to hydrogen peroxide activated PARS and decreased cellular adenosine triphosphate levels in a concentration–dependent manner. Inhibition of PARS with 3–AB prevented the cell death induced by hydrogen peroxide and also prevented adenosine triphosphate depletion. 3–AB did not have hydroxyl radical scavenging effect. In contrast, t–BHP did not affect the PARS activity. The decrease in cellular adenosine triphosphate levels by t–BHP was less than that by hydrogen peroxide. 3–AB failed to prevent the cell death induced by t–BHP. PARS activation after exposure of hydrogen peroxide was inhibited by addition of t–BHP. However, t–BHP showed an additive effect on cell death with hydrogen peroxide. These results indicate that activation of PARS plays an important role in hydrogen peroxide induced injury in opossum kidney cells and that hydrogen peroxide and t–BHP induce cell injury by different mechanisms.
Heme Metabolism and Lipid Peroxidation in Rat Kidney Hexachlorobenzene–Induced Porphyria: A Compartmentalized Study of Biochemical Pathogenic Mechanisms
Authors: Fernández-Tomé María del Carmen , Billi de Catabbi Silvia C. , Aldonatti Carmen , San Martin de Viale Leonor C. , Sterin-Speziale Norma B.
Published: April 2003

In the present study, the effects of hexachlorobenzene (HCB) on lipid peroxidation and heme metabolism in the different constitutive suborgans of the kidney were determined. For this purpose, conjugated diene and malondialdehyde levels, as lipid peroxidation parameters, and porphyrin accumulation, uroporphyrinogen decarboxylase activity, and its inhibitor formation, as measures of heme metabolism, were determined in renal cortex, medulla, and papilla. Adult Wistar rats were treated with HCB during 1, 2, 3, or 4 weeks. A significant increase in cortical conjugated dienes was observed from the 1st week of treatment. The malondialdehyde levels rose by 47, 34, and 28% after 2, 3, and 4 weeks of intoxication, respectively. The porphyrin content showed a tenfold increase after 4 weeks of treatment, and the uroporphyrinogen decarboxylase activity was reduced by 26 and 58% with respect to control values after 3 and 4 weeks of treatment, respectively. The results demonstrate a direct correlation between the oxidative environment and the effect elicited by the drug on heme metabolism in the renal cortex. In contrast, in papilla and medulla, where the antioxidant systems were higher, HCB showed no porphyrinogenic effect.
Two Types of K+ Channels are Present in the Apical Membrane of the Thick Ascending Limb of the Mouse Kidney
Authors: Lu Ming , Wang Wenhui
Published: April 2003

We used the patch–clamp technique to study apical K+ channels in the split–opened thick ascending limb (TAL) of mouse kidneys. We observed a low–conductance K+ channel in 8 patches and an intermediate–conductance K+ channel in 11 patches from a total of 135 cell–attached patches with GΩ seals in the apical membrane of the mouse TAL. Open probability (Po) of the low–conductance K+ channel was 0.85 and was not voltage–dependent between –60 mV and 0 mV. Po of the intermediate–conductance was 0.6 at a spontaneous cell membrane potential and decreased to 0.4 by 40 mV hyperpolarization. Both the low–conductance and intermediate–conductance K+ channels were an inwardly rectifying K+ channel with inward slope conductances of 26 pS and 74 pS between –20 and 20 mV, respectively. The 74–pS K+ channel was inhibited by 1 mM Ba2+ and 10 mM tetraethylammonium when they were applied to the bath facing the cytosolic surface of inside–out patches. Furthermore, addition of 1 mM Mg–ATP reversibly reduced the activity of the 74–pS K+ channel by 90% within 1 min in inside–out patches. A decrease in bath pH from 7.4 to 6.5 completely blocked the 74–pS K+ channel in inside–out patches. We conclude that two types of K+ channels are present in the apical membrane of the mouse TAL and that the biophysical properties of the apical 74–pS K+ channel are identical to those in the rat TAL.
Native and Oxidized Low–Density Lipoproteins Enhance Superoxide Production from Diabetic Rat Glomeruli
Authors: Chen Hung-Chun , Tan Mian-Shin , Guh Jinn-Yuh , Tsai Juei-Hsiung , Lai Yung-Hsiung
Published: April 2003

Oxygen free radicals have been implicated in mediating diabetic complications, and patients with diabetic nephropathy frequently show increased levels of circulating and oxidized low–density lipoproteins (LDL). In the present study, we measured the superoxide production of glomeruli isolated from poorly controlled diabetic (streptozotocin) rats sacrificed 1 week and 1, and 3 months after the induction of diabetes. The animals were stimulated with native and oxidized LDL isolated from normal humans with normolipidemia. The superoxide ion was measured by using a spectrophotometer. The results demonstrated that the poorly controlled diabetic rat glomeruli showed a significantly higher production of superoxide than normal glomeruli under basal conditions, and this production increased further with the progression of diabetes. Stimulation with either LDL or oxidized LDL enhanced superoxide production by diabetic glomeruli, with oxidized LDL being more potent than LDL. Our results suggest that oxidized LDL may play important roles in the pathogenesis of diabetic nephropathy through enhanced generation of oxygen free radicals.
Cigarette Smoking Causes Impairment of Spermatozoal Quality: Andrological and Biochemical Evaluation
Authors: Omu Alexander E. , Dashti Hussein , Mohammed Asiya T. , Mattappallil Alice Babu
Published: April 2003

The effect of cigarette smoking on spermatozoal parameters was studied in 40 known cigarette smokers and 40 non-smokers by evaluating sperm parameters, semen and serum levels of cadmium, zinc, copper, selenium and magnesium, circulating antisperm antibodies, and FSH, LH, prolactin and testosterone. Non-smokers and light smokers had better sperm parameters than heavy smokers as indicated by the sperm count (p < 0.03), active motility (p < 0.03), asthenozoospermia (p < 0.05), and positive hypo-osmotic swelling (p < 0.05) tests. The number of smokers with higher titres of circulating antisperm antibodies was larger than that of non-smokers (≥1:128; p < 0.01). Serum testosterone level was higher in non-smokers (18.4 ± 2.1 pmol/l) than in smokers (14.3 ± 2.5 pmol/l; p < 0.05). Cadmium levels were higher in the semen and serum of smokers (p < 0.05) when compared to non-smokers (p = 0.05). Cigarette smoking impairs fertility through the putative effect of cadmium in causing testicular endothelial injury and production of antisperm antibodies.
Chronic Obstructive Pulmonary Disease
Authors: Goldstein Roger S.
Published: April 2003

Chronic obstructive pulmonary disease is a widely prevalent condition usually associated with cigarette consumption. It has a worldwide impact on the allocation of health care resources. The diagnosis is made by history and physical examination supported by measures of airflow and lung volume. The levels of impairment, disability and handicap dictate the requirements for medical care. Smoking cessation, immunization, maximal pharmacologic therapy including the use of supplemental oxygen and prompt attention to exacerbations, represent the basic principles of management. Pulmonary rehabilitation has been shown in a number of randomized clinical trials to improve functional exercise capacity and health-related quality of life. Many interesting approaches, such as lung volume reduction, lung transplantation, nutritional support and mechanical ventilation for acute exacerbations, are in various stages of clinical use and evaluation.