Activity In Culture Supernatants Research Articles

Oxygen dependency of epidermal growth factor receptor binding and DNA synthesis of rat hepatocytes

Background/Aims: Changes in oxygen availability modulate replicative responses in several cell types, but the effects on hepatocyte replication remain unclear. We have studied the effects of transient nonlethal hypoxia on epidermal growth factor receptor binding and epidermal growth factor-induced DNA synthesis of rat hepatocytes. Methods: Lactase dehydrogenase activity in culture supernatant, intracellular adenosine triphosphate content, 25I-epidermal growth factor specific binding, epidermal growth factor receptor protein expression, and 3H-thymidine incorporation were compared between hepatocytes cultured in hypoxia and normoxia. Results: Hypoxia up to 3 caused no significant increase in lactate dehydrogenase activity in the culture supernatant, while intracellular adenosine triphosphate content decreased time-dependently and was restored to normoxic levels by reoxygenation (nonlethal hypoxia). Concomitantly, 125I-epidermal growth factor specific binding to hepatocytes decreased time-dependent (to 54.1% of normoxia) and was restored to control levels by reoxygenation, although 125I-insulin specific binding was not affected. The decrease in 125I-epidermal growth factor specific binding was explained by the decrease in the number of available epidermal growth factor receptors (21.37±3.08 to 12.16±1.42 fmol/10 5 cells), while the dissociation constant of the receptor was not affected. The change in the number of available receptors was not considered to be due to receptor degradation-resynthesis, since immunodetection of the epidermal growth factor receptor revealed that the receptor protein expression did not change during hypoxia and reoxygenation, and since neither actinomycin D nor cycloheximide affected the recovery of 125I-epidermal growth factor binding by reoxygenation. Inhibition of epidermal growth factor-induced DNA synthesis after hypoxia (to 75.4% of normoxia by 3 h hypoxia) paralleled the decrease in 125I-epidermal growth factor binding. Conclusions: Transient hypoxia, which caused no increase in lactase dehydrogenase leakage but affected intracellular adenosine triphosphate levels, did, however, modulate the number of available epidermal growth factor receptors without affecting the receptor protein expression, and inhibit the epidermal growth factor-induced DNA synthesis of hepatocytes. This suggests that even transient nonlethal hypoxia affects the epidermal growth factor-induced DNA synthesis of rat hepatocytes through reversible changes in the epidermal growth factor receptor molecule, which depends on oxygen availability.

A Novel Interferon-Inducible Gene Expressed during Myeloid Differentiation

ABSTRACT: The acute promyelocytic leukemia cell line, NB4, can be induced to differentiate to mature granulocytes by retinoic acid treatment. A novel retinoic acid-inducible cDNA clone, designated RI58, was isolated from a cDNA library constructed from retinoic acid-treated NB4 cells by differential hybridization. RI58 cDNA encodes a protein of 58kDa which has a similarity in its amino acids sequence to interferon (IFN)-inducible proteins. In addition, RI58 was induced by recombinant human IFN-α (rhIFN-α) in NB4 cells. RI58 was detectable within 4 hours post-stimulation with rhIFN-α, while it took as long as 1day after retinoic acid stimulation. Culture supernatant from retinoic acid-treated NB4 cells also induced RI58 expression similarly as rhIFN-α. This activity in culture supernatant was inhibited by anti-leukocyte IFN antiserum which showed specific reactivity to rhIFN-α. These results indicate that RI58 is induced by retinoic acid stimulation through autocrinally secreted IFN-α from NB4 cells. In the retinoic acid-treated NB4 cells, the expression of RI58 was increased along the process of differentiation. On the other hand, it was expressed constitutively in untreated non-hematopoietic cell lines and mature hematopoietic cell lines.

The effect of iron limitation growth conditions on the cell and extracellular components of the fish pathogen Pasteurella piscicida

The effect of iron limitation, using the iron‐chelating agent 2,2 dipyridyl, on the electrophoretic profiles of outer membrane proteins (OMPs) and extracellular products (ECPs) from 21 Pasteurella piscicida strains isolated from Europe and Japan was investigated. In addition, the effect of iron‐limited and iron‐surplus growth conditions on caseinase activity in culture supernatants of the pathogen was examined. The majority of P. piscicida strains, from Greece, Italy and France, cultured under iron‐limited conditions, produced four novel OMPs (63 and three at and above 200 kDa). In contrast, iron‐regulated outer membrane proteins were not induced in Japanese strains. Electrophoretic analysis of the ECPs from the pathogen grown under iron surplus and iron limitation revealed a large range of products and additional high molecular mass (MM) bands were evident under iron‐limited conditions. When culture supernatants were analysed for their activity, most of the bacteria tested showed elevated activities under iron limited conditions. Finally, neither hydroxamate nor phenolate type siderophores could be detected with any of the chemical assays used.

Cases of streptococcal toxic shock syndrome in Germany since 1989, toxin (mitogen) expression, content of toxin genes and relation to M-serotypes.

A worldwide increase of severe invasive Streptococcus pyogenes infections beginning from the skin or soft tissue infections (cellulitis, fasciitis, myositis) has been reported during the last decade. This so-called Streptococcal Toxic Shock Syndrome (STSS) is considered as a resurgence of the historically known fulminant or toxic scarlet fever. It is characterized by fever, multiorgan failure, skin desquamation and occurs mainly in young/middle-aged, otherwise healthy adults within hours of the onset of symptoms. The onset of STSS seems to be related to the efficacy of bacterial products and host factors (particularly the immune state) of the patients. Bacterial products which are believed to be causally involved in these infections are the M-protein, different enzymes and the various erythrogenic toxins (ET or SPE), the streptococcal mitogenicity factor MF, the streptococcal superantigen (SSA), low molecular weight proteins (LMP) and the cell bound streptococcal membrane protein (CAP) (1). Our interest in this investigation has been focused on the examination of STSS strains isolated in Germany since 1989, when the first case was reported. More than 70 S. pyogenes strains isolated from cases of STSS in Germany and a group of control strains isolated from healthy people or from patients suffering from harmless streptococcal diseases were typed and investigated with regard to content of speA, speB, speC, and MF genes, toxin expression (ETA and ETC) and for mitogenic activity in culture supernatants. The results of this investigation are presented and discussed.KeywordsBacterial ProductSoft Tissue InfectionMitogenic ActivityToxin GeneGene DetectionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Tumor necrosis factor alpha inhibits entry of human immunodeficiency virus type 1 into primary human macrophages: a selective role for the 75-kilodalton receptor.

The proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) is readily detected after human immunodeficiency virus type 1 (HIV-1) infection of primary macrophages in vitro and is present in plasma and tissues of patients with AIDS. Previous studies have shown that human recombinant TNFalpha (hrTNFalpha) enhances HIV replication in both chronically infected promonocytic and T-lymphoid cell lines in vitro. We report here that in contrast to untreated tissue culture-differentiated macrophages (TCDM), in which the proviral long terminal repeat (LTR) could be detected as soon as 8 h postinfection by a PCR assay, TCDM pretreatment for 3 days by hrTNFalpha markedly delayed its appearance until 72 h after infection with the HIV-1 Ada monocytotropic strain. Moreover the inhibition of formation of the proviral LTR in HIV-1-infected TCDM was directly proportional to the concentration of hrTNFalpha used. To determine if the inhibition of LTR formation results from blockade of viral entry, we performed a reverse transcription PCR assay to detect intracellular genomic viral RNA as early as 2 h after infection. Pretreatment of primary TCDM by hrTNFalpha for 3 days and even for only 2 h inhibits 75% of the viral entry into the cells. The inhibition of viral entry by hrTNFalpha was totally abolished by the use of anti-human TNFalpha monoclonal antibody. By using TNFalpha mutants specific for each human TNFalpha receptor, we showed that the inhibition of HIV-1 entry into TCDM was mediated not through the 55-kDa TNF receptor but through the 75-kDa TNF receptor. Although prolonged (1 to 5 days) TNFalpha treatment can downregulate CD4 expression in primary human TCDM, surface CD4 levels were not reduced by 2 h of treatment and was therefore not a limiting step for HIV-1 entry. In contrast to the inhibition of viral entry into primary TCDM, pretreatment with hrTNFalpha did not modify HIV-1 entry into phytohemagglutinin A-activated peripheral blood lymphocytes. TNFalpha-pretreatment inhibited HIV-1 replication in primary TCDM but not in phytohemagglutinin A-activated peripheral blood lymphocytes as assessed by decreased reverse transcriptase activity in culture supernatants. These results demonstrate that TNFalpha is able to enhance host cellular resistance to HIV-1 infection and that selective inhibition of HIV-1 entry into primary TCDM by TNFalpha involves the 75-kDa TNF receptor but not the 55-kDa TNF receptor.

Measurement of virulence of aeromonads using a suckling mouse model of infection

Virulent and avirulent strains of Aeromonas spp. were identified and virulence quantified using an animal model. Virulence was measured by determining a 50% lethal dose (LD 50) 43 h after oral administration of live bacteria. The LD 50 of virulent Aeromonas isolates ranged from log 10 7.53 (mean) organisms to log 10 8.88 (mean). Some isolates were avirulent in this model. Detection of cytotoxic activity in culture supernatants correlated with virulence (Fisher exact test, P = 0.0029). There was no correlation between LD 50 and the source of the isolate, β-haemolysis or lipopolysaccharide (LPS) banding profile on SDS-PAGE. In this animal model, virulence was multifactorial in that: (i) bacterial multiplication in the gut was associated with fatal infection; (ii) the increase in bacterial numbers in the gut of mice administered a lethal dose of bacteria was accompanied by accumulation of fluid; and (iii) there was evidence of extraintestinal spread of infection. Protection of suckling mice by rabbit antiserum to Aeromonas cell envelopes was observed.

Measurement of virulence of aeromonads using a suckling mouse model of infection.

Virulent and avirulent strains of Aeromonas spp. were identified and virulence quantified using an animal model. Virulence was measured by determining a 50% lethal dose (LD50) 43 h after oral administration of live bacteria. The LD50 of virulent Aeromonas isolates ranged from log10 7.53 (mean) organisms to log10 8.88 (mean). Some isolates were avirulent in this model. Detection of cytotoxic activity in culture supernatants correlated with virulence (Fisher exact test, P = 0.0029). There was no correlation between LD50 and the source of the isolate, beta-haemolysis or lipopolysaccharide (LPS) banding profile on SDS-PAGE. In this animal model, virulence was multifactorial in that: (i) bacterial multiplication in the gut was associated with fatal infection; (ii) the increase in bacterial numbers in the gut of mice administered a lethal dose of bacteria was accompanied by accumulation of fluid; and (iii) there was evidence of extraintestinal spread of infection. Protection of suckling mice by rabbit antiserum to Aeromonas cell envelopes was observed.

Fluorometric determination of acid proteinase activity in vulvovaginal candidosis.

Vulvovaginal candidosis is one of the most frequent disorders in obstetrics and gynaecology. Candida albicans is commonly considered to be the true vaginopathic agent. The secreted acid proteinase might be especially relevant in the pathogenesis of vulvovaginal candidosis. A fluorometric determination of acid proteinase activity of clinical C. albicans isolates was carried out during the present work using fluorescamine. L-Leucyl-L-alanine was included as an internal standard and the results were expressed as nmoles of leucylalanine equivalents h-1 per 2 x 10(4) cells. The 13 isolates were taken from non-diabetic, non-pregnant women aged 22-35 years with vulvovaginal candidosis. Candida albicans ATCC 44858 was used as a control. The proteinase activity in culture supernatants was detectable starting from the mid- to late- exponential phase of growth, peaked between 30 and 46 h, and then declined. The control had an activity of 2.72 nmol h-1 per 2 x 10(4) cells, whereas eight of the samples had a lower activity (1.05 nmol h-1 per 2 x 10(4) cells on average) and five of the samples had a higher activity (6.53 nmol h-1 per 2 x 10(4) cells on average). The fluorometric determination of acid proteinase activity was found to be more reproducible and sensitive than the previously used spectrophotometric determinations.

Ecto-Sialyltransferase of Human B Lymphocytes Reconstitutes Differentiation Markers in the Presence of Exogenous CMP-N-Acetyl-Neuraminic Acid

The existence of an ecto-sialyltransferase (ecto-ST) on B lymphocytes with increasing activity at late maturation stages is shown using a novel flow cytometric enzyme assay. This ecto-ST is effective in reconstituting different surface glycoconjugates on desialylated B cells in the presence of exogenous CMP-NeuAc. We found that this ecto-ST is distinct in its activity from soluble ST released into the culture supernatant. Surface sialylation was independent of the amount of ST secreted into the culture supernatant and followed different kinetics than sialylation of exogenous substrate by soluble ST. Four human B-cell lines representing different maturation stages were analyzed for secreted and ecto-ST activity. The myeloma cell line U266 and the lymphoblastoid cell line JOK-1 showed higher activity of both ST forms than the acute lymphoblastic leukemia B-cell line Nalm-6. ST activity in culture supernatants of U266, JOK-1, and Nalm-6 cells consisted predominantly of the alpha 2,6 ST type with specificity for N-linked oligosaccharides. As an exception, the myeloma cell line IM-9, deficient of alpha 2,6 ST activity, secreted only small amounts of ST and showed low activity of ecto-ST. Sialylation of surface-expressed glycoconjugates by ecto-ST was measured by incubating B-cell lines in the presence of fluorescent CMP-sialic acid. Surface structures labeled with fluorescent sialic acid under this condition were visualized by confocal laser scanning microscopy and fluorescent label was quantitatively assessed by flow cytometric analysis on live cells. Incubation of cells in acidified culture medium, to release possibly receptor-bound ST, did not alter the intensity of cell surface sialylation. Inhibition of internalization and membrane traffic by various approaches (reduced incubation temperature and chloroquine or brefeldin A treatment) did not block surface sialylation. Together, these observations point to cell surface sialylation in B lymphocytes mediated by a cell surface-expressed ecto-ST distinct from the secreted ST form. On desialylated JOK-1 cells, ecto-ST in the presence of exogenous CMP-NeuAc was able to resialylate the B-cell surface sialoglycans CDw75 and HB-6 and major surface glycoproteins of B cells, such as HLA class I and II antigens, transferrin receptor, and surface IgM. In contrast, cell surface glycans of coincubated desialylated erythrocytes were not sialylated by the B-cell ecto-ST. Ecto-alpha 2,6 ST of B cells may be involved in the sialylation of distinct differentiation glycan antigens.

Plasmid-liposome transfer of the alpha 1 antitrypsin gene to cystic fibrosis bronchial epithelial cells prevents elastase-induced cell detachment and cytokine release.

Human neutrophil elastase (NE) stimulates release of neutrophil chemotactic activity by a bronchial epithelial cell line and from nasal epithelial cells. In this article, we show that NE stimulates the production of neutrophil chemotactic activity by 2CFSMEo-cells, a transformed cystic fibrosis bronchial epithelial cell line. The production of chemotactic activity is dose- and time-dependent and can be blocked by preincubation of NE with alpha 1 antitrypsin (alpha1AT). Incubation of the NE-stimulated culture supernatant with neutralizing concentrations of rabbit anti-human interleukin 8 antibody completely neutralizes the chemotactic activity. Transfection of 2CFSMEo- cells with the eukaryotic expression vector pCMV4alpha1AT, complexed to cationic liposomes in a 1:3 wt/wt ratio, results in at least a 10-fold increase in measured human alpha1AT protein in culture supernatant. Detection of human alpha1AT mRNA by reverse transcriptase polymerase chain reaction in total RNA from transfected, but not untransfected cells, confirms successful gene transfer. Compared with untransfected cells, transfer of the human alpha1AT gene decreases chemotactic activity in culture supernatant and prevents cell detachment after NE exposure. Our data indicate that alpha1AT gene transfer is capable of blocking at least some of the biological effects of free elastase on cultured epithelial cells.

IL-12 produced by antigen-presenting cells induces IL-2-independent proliferation of T helper cell clones.

We investigated the role of IL-12 in proliferation of various Th cell clones (class II-alloreactive (4-86 and 4-55) and keyhole limpet hemocyanin + self I-Ek-reactive (9-16)) following stimulation with Ag on APCs. These clones proliferated in response to stimulation with rIL-2, rIL-12, or Ag/APC. The proliferation induced by Ag/APC stimulation was not affected by anti-IL-2 Ab but was markedly inhibited by anti-IL-12 Abs. Consistent with this finding was the absence of detectable IL-2 activity in culture supernatants 12 to 48 h after Ag/APC stimulation, and the detection of significant levels of IL-12 in an Ab-capture bioassay. IL-12 was produced within 12 h after Ag/APC stimulation, reaching a peak after 18 to 24 h. The production of IL-12 in cultures of Th clones and APC contrasted with the production of IL-2 but not IL-12 upon allostimulation of primary T cells and the inhibition of their proliferation exclusively by anti-IL-2 Abs. Analysis of the expression of IL-12-binding sites on Th cells revealed low levels of IL-12 receptors in resting Th clones but high IL-12R levels 2 to 3 days after Ag/APC stimulation, declining gradually thereafter. The changes in IL-12R expression levels correlated closely with the IL-12 responsiveness of Th populations at various times after Ag/APC stimulation; Th populations obtained 3 and 10 days after Ag/APC stimulation exhibited very high and weak or marginal responsiveness to rIL-12, respectively, whereas the responses to rIL-2 were comparable in both Th populations. These results indicate that the Ag/APC-stimulated proliferation of terminally differentiated Th clones, in contrast to naive T cells, depends on the production of IL-12 by APC and on the simultaneous up-regulation of IL-12R on Th cells rather than on an IL-2 autocrine mechanism.

ThePasteurella haemolytica O-Sialoglycoprotein Endopeptidase Is Inhibited by Zinc Ions and Does Not Cleave Fetuin

Culture supernatants ofPasteurella haemolyticaA1 contain anO-sialoglycoprotein endopeptidase that cleaves human glycophorin A. This enzyme is inhibited by micromolar concentrations of Zn2+. It can be separated from a neuraminidase activity in culture supernatants by ion-exchange chromatography. The neuraminidase activity can cause the de-sialation of the bovine soluble sialoglycoprotein, fetuin. However fetuin is not cleaved proteolytically either by culture supernatants fromP. haemolyticaA1 or by chromatographically purifiedO-sialoglycoprotein endopeptidase or neuraminidase.

The human immunodeficiency virus type 1 5' packaging signal structure affects translation but does not function as an internal ribosome entry site structure

The role of the RNA secondary structure in the 5' packaging signal region of human immunodeficiency virus type 1 (HIV-1) in initiating translation of gag mRNA has been investigated both in vitro and in the presence of cellular cofactors in vivo. Heat denaturation of the structure and mutagenic deletion both lead to an increase in levels of translated products, indicating that the structure is a significant inhibitor of translation. The proximity of the gag AUG to the packaging signal structure suggested that it might function as an internal ribosome entry site. However, in both a cell-free system and eukaryotic cells, translation will initiate at a novel upstream initiation codon introduced within the 5' noncoding region. This codon is utilized exclusively, resulting in gag protein products with an extra 11 amino acids at the amino terminus, which, when expressed in T lymphocytes, are confined intracellularly, probably because of the lack of an N-terminal glycine myristoylation signal. Deletion of the secondary structure abolishes gag production even in the presence of tat and rev in trans. Using dicistronic constructs containing the HIV-1 5' leader cloned between two heterologous open reading frames, we were unable to detect any significant expression of the second open reading frame that would have been supportive of an internal ribosome entry site mechanism. Using mutant proviruses either lacking the entire packaging signal structure region or containing the introduced upstream initiation codon in long-term replication studies, we were unable to detect reverse transcriptase activity in culture supernatants. The 5' packaging signal structure of HIV-1 does not serve as an internal ribosome entry site. The translation of gag is consistent with ribosomal scanning. However, the packaging signal structure causes significant translational inhibition.

Inhibition of the induction of collagenase by interleukin-1 ? in cultured rabbit synovial fibroblasts after treatment with the poly(ADP-ribose)-polymerase inhibitor 3-aminobenzamide

3-Aminobenzamide is an inhibitor of poly-(ADP-ribosyl)ation. In concentrations from 3 to 10 mM it reduced the collagenase activity in culture supernatants of interleukin-1 beta-stimulated rabbit synovial fibroblasts. 3-Aminobenzoate, not an inhibiter of poly(ADP-ribosyl)ation, had no effect on collagenase activity at a concentration of 10 mM. We concluded that poly(ADP-ribosyl)ation plays a role in the induction of the expression of collagenase and that 3-aminobenzamide can inhibit this process.

Regulation and kinetic modeling of galactose oxidase secretion

Production of extracellular galactose oxidase by Fusarium sp. NRRL 2903 appears to be best in medium containingl-sorbose, which acts as a paramorphogen for several filamentous fungal species. Analysis of galactose oxidase activity in culture supernatants and the occurrence of gaoA mRNA, during growth in media containing eitherl-sorbose or glycerol as the sole source of carbon, indicates that the two carbon sources result in high and low levels of extracellular galactose oxidase activity, respectively, which is in parallel with the level of transcription. The influence of altering the carbon source for growth on the regulation of galactose oxidase synthesis and secretion appears to be mainly at the level of gene expression rather than pH or cell density, although these factors are also known to have some regulatory effect on the level of secretion. Kinetics of galactose oxidase secretion, evaluated on the basis of altering the pH of the growth medium, agrees with the Luedeking-Piret equation and suggests that the growth associated and the non-growth associated product formation rates are changed in expense of each other.

Anti-viral and immunomodulatory effects of interferon-α on cultured lymphocytes from patients with human T lymphotropic virus type I-associated myelopathy (HAM/TSP)

In contrast to therapeutic benefits of interferon-α (IFN-α) in patients with human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), little is known about the mechanisms underlying its clinical efficacy. To investigate the anti-viral and/or immunomodulatory properties of IFN-α in HTLV-I infection, the effects of IFN-α on HTLV-I-induced in vitro phenomena were evaluated. In vitro activation of HTLV-I in fractionated CD4 + T lymphocyte-rich cells (CD4 + cells) could be demonstrated by increased thymidine incorporation into the cells, detection of proviral HTLV-I and viral RNA, and by assays of reverse transcriptase activities in culture supernatants. T cell immune responses were evaluated by thymidine incorporation into CD8 + T lymphocyte-rich cells (CD8 + cells) responding to cultured and irradiated autologous CD4 + cells possessing HTLV-1 antigens. It could be shown that IFN-α suppressed both the in vitro activation of HTLV-I and the CD8 + cell response. Moreover, 1 day supplementation of IFN-α as a pretreatment was sufficient for the induction of these properties. These findings, together with the clinical efficacy of IFN-α administration in patients with HAM/TSP, support the view that viral activation and T cell responses are critical components in the pathogenic processes involved in HAM/TSP.

Chronic Alcohol Intoxication Enhances the Expression of CD18 Adhesion Molecules on Rat Neutrophils and Release of a Chemotactic Factor by Kupffer Cells

Chronic alcohol intoxication has been associated with increased migration of inflammatory leukocytes to the liver that may contribute to the development of alcoholic hepatitis in susceptible individuals. Thus, this work was performed to examine the mechanism by which neutrophils [polymorphonuclear neutrophils (PMNS)] are sequestered in the liver during prolonged consumption of alcohol. Male Sprague-Dawley rats were fed with Sustacal supplemented by 36% alcohol, or isocaloric diet for 16 weeks. Circulating blood PMNs were collected and examined for CD18 (beta 2-integrin) adhesion molecule expression. Monoclonal antibody 1F12, an anti-CD18 antibody and potent neutropenic agent, was used to detect CD18 on PMNs. More than 97% of neutrophils obtained from pair and ethanol-fed rats were positive for the antibody. Fluorescence intensity of fluorescein isothiocyanate-1F12 binding to PMNs from ethanol-fed rat was significantly enhanced 2-fold compared with the pair-fed controls. The release of chemoattractant and free radical-generating activity in culture supernatants of Kupffer cells was also examined. Twenty-four hr culture supernatants of Kupffer cells from chronic alcoholic rats enhanced the migration and superoxide anion generation by normal PMNs, compared with those of the pair-fed rats. Antirat interleukin-8 antiserum inhibited chemotactic activity and superoxide generating capacity of culture supernatants. These results suggest that upregulation of adhesion molecules on PMNs and chemotactic factor release from Kupffer cells may contribute, at least in part, to enhanced migration of inflammatory leukocytes to the liver during chronic alcohol intoxication.

Streptokinase activity among group A streptococci in relation to streptokinase genotype, plasminogen binding, and disease manifestations

Certain genotypic variants of streptokinase (ska) of beta-hemolytic streptococci group A have been associated with acute post-streptococcal glomerulonephritis (APSGN). In our earlier studies on strains isolated from Ethiopian children with various streptococcal disease manifestation, we reported an even distribution of streptokinase genotypes with no association to disease patterns. Considering the possibility that strains could differ in their ability to secrete the protein, levels of streptokinase activity in culture supernatants of these strains were determined by a plasminogen activation assay using a synthetic tripeptide, H-D-valyl-leucyl-lysin-p-nitroaniline, as a substrate. Of the 53 streptococcal group A strains, ten (19%), which belonged to genotype ska4 and ska8, did not activate human plasminogen. These strains did not activate bovine, sheep, horse, rabbit or porcine plasminogens either. They represented at least five M protein and non-typeable serotypes, and were characterized by high human plasminogen binding activity. Six of the 53 strains (11%) harbouring genotype ska3 and ska7 showed low levels of human plasminogen activation. Strains of ska1 and ska2, 37/53, activated human plasminogen at a higher level (p < 0.005). Levels of plasminogen activation were not significantly different among the ska1 and ska2 strains associated with various streptococcal disease manifestations. Antibody levels against streptokinase were higher (p < 0.05) in convalescent sera from acute rheumatic fever and APSGN patients in comparison with sera from other patient categories and healthy controls. Streptokinase genotype and in vitro streptokinase production do not correlate directly to streptococcal disease manifestation, indicating a probable significance of additional streptococcal and/or host factors in the initiation of APSGN.

Streptokinase activity among group a streptococci in relation to streptokinase genotype, plasminogen binding, and disease manifestations

Certain genotypic variants of streptokinase (ska) of beta-hemolytic streptococci group A have been associated with acute post-streptococcal glomerulonephritis (APSGN). In our earlier studies on strains isolated from Ethiopian children with various streptococcal disease manifestation, we reported an even distribution of streptokinase genotypes with no association to disease patterns. Considering the possibility that strains could differ in their ability to secrete the protein, levels of streptokinase activity in culture supernatants of these strains were determined by a plasminogen activation assay using a synthetic tripeptide, H-D-valyl-leucyl-lysin-p-nitroaniline, as a substrate. Of the 53 streptococcal group A strains, ten (19%), which belonged to genotype ska4 and ska8, did not activate human plasminogen. These strains did not activate bovine, sheep, horse, rabbit or porcine plasminogens either. They represented at least five M protein and non-typeable serotypes, and were characterized by high human plasminogen binding activity. Six of the 53 strains (11%) harbouring genotype ska3 and ska7 showed low levels of human plasminogen activation. Strains of ska1 and ska2, 37/53, activated human plasminogen at a higher level (p < 0.005). Levels of plasminogen activation were not significantly different among the ska1 and ska2 strains associated with various streptococcal disease manifestations. Antibody levels against streptokinase were higher (p < 0.05) in convalescent sera from acute rheumatic fever and APSGN patients in comparison with sera from other patient categories and healthy controls. Streptokinase genotype and in vitro streptokinase production do not correlate directly to streptococcal disease manifestation, indicating a probable significance of additional streptococcal and/or host factors in the initiation of APSGN.

Predictive values of cord blood IgE and cord blood lymphocyte responses to food antigens in allergic disorders during infancy

Proliferative response of cord blood lymphocytes stimulated twice by food antigens and cord blood IgE concentration were measured in 131 full-term newborn infants for the prediction of allergic disorders. Through the follow-up study for 2 1 2 years, the value of stimulation index in proliferative response of cord blood lymphocytes to ovalbumin or bovine serum albumin was greater than 1.5 in 17 (sensitivity 53.1%) of 32 infants in whom allergic disorders developed and less than 1.5 in 81 (specificity 81.8%) of 99 infants who had no allergic disorders (cutoff limit of stimulation index 1.5). The sensitivity was increased (71.9%) by the combination of the cord blood IgE concentration (cutoff limit 1.0 IU/ml) and proliferative response of cord blood lymphocytes to food antigens (cutoff limit of stimulation index 1.5). The combination of the cord blood IgE concentration and proliferative response of cord blood lymphocytes to food antigens is useful for the prediction of allergic disorders. Interleukin-2 production of cord blood lymphocytes stimulated with food antigens was also measured in 24 newborn infants. Interleukin-2 activity in culture supernatants of ovalbumin- or bovine serum albumin-stimulated cord blood lymphocytes correlated well with proliferative response of cord blood lymphocytes to ovalbumin or bovine serum albumin.