bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential susceptibility to Mycoplasma pulmonis intranasal infection in X-linked immunodeficient (xid), severe combined immunodeficient (scid ), and immunocompetent mice
Authors: SANDSTEDT K , BERGLÖF A , FEINSTEIN R , BÖLSKE G , EVENGÅRD B , SMITH C I E
Published: November 2003

Mice with the scid mutation are highly susceptible to Mycoplasma pulmonis infection and develop a disseminated disease. In order to study the contribution of humoral immunity to the immune response, M. pulmonis was inoculated intranasally to X-linked immunodeficient (xid) mice. Severe combined immunodeficient (scid) and immunocompetent CBA mice were used as controls. The mice were killed and necropsied at day 30 or 37 post-infection. Samples from the nose, lungs and joints were taken for bacteriological and histological examination. Rhinitis was observed in all mouse strains. Chronic purulent bronchopneumonia was diagnosed in some of the CBA mice. Xid mice did not show severe lung lesions, despite the presence of numerous mycoplasma organisms in the lungs, in contrast to immunocompetent mice, which developed lung pathology. Scid mice showed less signs of pneumonia, but unlike in xid and CBA mice, there was spread of mycoplasmas from the respiratory tract and severe pathological changes in the joints. Our results indicate that B and/or T lymphocytes protect against dissemination of M. pulmonis from the airways. Innate immune reactions and/or bacterial virulence factors seem to contribute to the development of joint lesions, whereas IgG3 and IgM antibodies might be involved in lung pathology.
Premature removal of uninfected erythrocytes during malarial infection of normal and immunodeficient mice
Authors: SALMON M G , DE SOUZA J B , BUTCHER G A , PLAYFAIR J H L
Published: November 2003

Anaemia during blood-stage plasmodial infections is known to be due to at least three mechanisms: direct destruction of erythrocytes by the intra-erythrocytic parasite, inhibition of erythropoiesis, and premature removal of uninfected erythrocytes. The removal of the uninfected erythrocytes is considered by many to be dependent on an antibody-mediated mechanism. Our investigations involving normal, severe combined immunodeficient (SCID) and nude BALB/c mice and the murine malaria parasite Plasmodium yoelii, indicate that this mechanism is unlikely. The process of removal of uninfected erythrocytes was reduced in SCID mice but could not be enhanced by the passive transfer of serum from infected immunocompetent mice. Macrophage activation, as judged by the removal of xenogeneic erythrocytes, did not differ in the three strains of mice. Changes in the erythrocytes themselves may be responsible for their shortened lifespan.
Jak3 activation in human lymphocyte precursor cells
Authors: SHARFE N , DADI H K , O'SHEA J J , ROIFMAN C M
Published: November 2003

Although expression of the Jak3 tyrosine kinase in T lymphocytes has been thought to be restricted to mature, activated cells, mutations of Jak3 can lead to the development of a human severe combined immunodeficiency (SCID) characterized by an absence of peripheral T lymphocytes. We therefore examined in detail the expression of Jak3 throughout human T cell differentiation and show that Jak3 is in fact present throughout the entire developmental process, with high levels expressed in thymocytes. Jak3 is highly expressed in double negative (CD4−CD8−) cells, one of the earliest stages of thymocyte differentiation, and can be activated via the IL-7 receptor. IL-7 is known to stimulate thymocyte proliferation and initiate re-arrangement of the T cell receptor (TCR) β gene, suggesting that the failure of mutated Jak3 proteins to transduce this signal may be responsible for failures in T cell development. While Jak3 SCID patients possess mature peripheral B cells, we demonstrate that the Jak3 tyrosine kinase is also expressed in human pre-B cells and can be activated by the pre-B cell growth factor IL-7.
IL-5 expressed by CD4+ lymphocytes from Echinococcus multilocularis-infected patients
Authors: JENNE L , KILWINSKI J , SCHEFFOLD W , KERN P
Published: November 2003

IL-5 is a major factor inducing differentiation of B lymphocytes into immunoglobulin-producing cells as well as a main regulator of eosinophils. Recently, we have shown that peripheral blood mononuclear cells (PBMC) from patients with alveolar echinococcosis (AE) express IL-5 mRNA after stimulation with crude Echinococcus multilocularis (E.m.) antigen. To characterize the observed response in lymphocyte subpopulations, we cultured patients' PBMC in the presence of E.m. crude antigen for 18 h. PBMC were separated from seven patients by fluorescence-activated cell sorting (EPICSorter) into CD4+ and CD8+ subpopulations and from an additional seven patients by magnetic cell sorting (MACS) into CD4+, CD8+ and the CD4+/CD8+ depleted fractions. mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) for the cytokines IFN-γ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, as well as for β-actin as control. IL-4 and IFN-γ expression was positive in all of the patients in the stimulated CD4+ subgroup. IL-5 mRNA expression was detected in eight out of 14 CD4+ samples (58%) and not observed in the other subpopulations, or the unstimulated and healthy controls. Co-expression of other Th2 cytokines in the eight patients expressing IL-5 mRNA was found in five patients for IL-3 and in seven for IL-10. Expression of IL-5 and both Th2 cytokines (IL-3 and IL-10) was only observed in patients judged as critically ill. Out of the six patients who were regarded as cured after radical operation or as stabilized with or without chemotherapy, only two expressed IL-5. Out of those eight patients considered as critically ill, six expressed IL-5 mRNA and five of these co-expressed IL-3 and IL-10. Thus, we conclude that specific antigenic challenge of PBMC from patients with active or previous AE induces an IL-5 response of CD4+ lymphocytes. The expression of Th2-type interleukin mRNA is significantly more frequent in patients clinically judged as progressive. Furthermore, IgE was elevated only in patients regarded as critically ill (six out of eight). In none of the patients were eosinophils elevated. These data support a Th2-type immune response in patients with chronic E. multilocularis infection.
Binding and internalization of human IgG by living cultured endothelial cells
Authors: RONDA N , GATTI R , ORLANDINI G , BORGHETTI A
Published: November 2003

Interactions between circulating IgG and endothelial cells (EC) in humans have been described only in conditions associated with pathologic immunoglobulins and/or activated or damaged EC. In this study we provide evidence that normal human IgG includes one/some antibody species that bind to and are internalized by living EC in culture. This novel function of EC and natural autoantibodies is of potential importance for the understanding of physiologic interactions between vessels and the immune system and for the clarification of pathogenesis of vasculitis and mechanisms of action of pooled IgG used in the therapy of such conditions.
A new approach to cytochrome CYP2D6 antibody detection in autoimmune hepatitis type-2 (AIH-2) and chronic hepatitis C virus (HCV) infection: a sensitive and quantitative radioligand assay
Authors: YAMAMOTO A M , JOHANET C , DUCLOS-VALLEE J-C , BUSTARRET F A , ALVAREZ F , HOMBERG J-C , BACH J-F
Published: November 2003

Antibodies specific for cytochrome CYP2D6, formally known as liver-kidney-microsome type-1 antibodies (LKM-1), are characteristically found in a subgroup of patients presenting autoimmune hepatitis. They are also found in some patients with chronic HCV infection. These autoantibodies are usually detected by indirect immunofluorescence, immunoblotting and ELISA tests. In an attempt to set up a more sensitive detection assay we developed a quantitative immunoprecipitation radioligand assay using a 35S-methionine-labelled CYP2D6 antigen obtained by in vitro transcription and translation synthesis. All 16 sera from AIH-2 patients strongly bound to this CYP2D6 antigen. Two of the nine sera (22%) from AIH-2 patients that presented only liver cytosol-1 antibodies also bound to CYP2D6. All 24 sera from HCV patients that were positive for LKM-1 antibodies by indirect immunofluorescence were also positive using this CYP2D6 radioligand assay. Lastly, all 15 sera from HCV patients negative for LKM-1 antibodies were negative by this test. The present results support the view that this quantitative radioligand assay is more sensitive than immunoblotting and ELISA CYP2D6 assays, and that it could be used in combination with indirect immunofluorescence assay.
Isolation of human anti-c-erbB-2 Fabs from a lymph node-derived phage display library
Authors: CLARK M A , HAWKINS N J , PAPAIOANNOU A , FIDDES R J , WARD R L
Published: November 2003

An immunoglobulin phage display library constructed from a tumour-associated pericolic lymph node was panned against the extracellular domain of the oncoprotein c-erbB-2. Sixteen independent clones were confirmed as positive binders based on ELISA analysis of soluble Fabs. Nucleotide sequencing demonstrated that the VH region of 12 clones belonged to four different V gene families, and the clones demonstrated varying degrees of somatic mutation compared with germ-line sequences. Fab fragments were examined for cross-reactivity by ELISA and shown to be negative against a panel of irrelevant self and non-self antigens, including bovine serum albumin (BSA), mouse immunoglobulin, tetanus toxoid, heregulin-PE40-FLAG and insulin. Reactivity of Fabs in vitro was verified by immunocytochemistry, which showed binding to the c-erbB-2 over-expressing breast cancer cell line SKBR3 but not to the low-expressing cell line MDA-MB-231. We conclude that a single lymph node library of moderate diversity (2 × 107κ light chain and γ heavy chain clones), when derived from an individual whose colorectal tumour over-expressed c-erbB-2, can be successfully panned to isolate a number of unique Fabs specific for this antigen. The nature of the anti-c-erbB-2 Fabs recovered from this library suggests that they may have resulted from a humoral immune response in the individual, and that in vivo antibody responses to tumour-associated antigens may be exploited in vitro for the production of tumour-specific recombinant antibodies.
Protective effect of granulocyte colony-stimulating factor (G-CSF) in a granulocytopenic mouse model of Pseudomonas aeruginosa lung infection through enhanced phagocytosis and killing by alveolar macrophages through priming tumour necrosis factor-alpha (TNF-α) production
Authors: SHIRAI R , KADOTA J , TOMONO K , OGAWA K , IIDA K , KAWAKAMI K , KOHNO S
Published: November 2003

We investigated the effects of G-CSF in a granulocytopenic mouse model of Pseudomonas aeruginosa lung infection. The model was prepared by intratracheal instillation of the bacteria, while granulocytopenia was induced by intraperitoneal injection of 4.0 mg of cyclophosphamide (CPA). There was no difference in the survival rate between G-CSF-treated animals and the normal group, and the number of neutrophils in the blood and lung recovered to normal in the former group. However, the phagocytic and killing activities of neutrophils were lower in G-CSF-treated mice than in controls. Interestingly, the mortality rate increased significantly when anti-TNF-α antibody was combined with G-CSF, although it was intermediate between CPA alone and CPA–G-CSF-treated mice. However, the improved mortality was not associated with a change in the number of neutrophils in the circulation and lung. Administration of anti-TNF-α antibody resulted in a significant suppression of TNF-α in bronchoalveolar lavage fluid and of enhanced alveolar macrophage function (phagocytic and bactericidal activity) against P. aeruginosa in G-CSF-treated granulocytopenic mice. We showed also increased TNF-α mRNA expression and TNF-α production in vitro using G-CSF-pretreated alveolar macrophages compared with control untreated macrophages. Our results are the first evidence to suggest that G-CSF provides a synergistic protective effect against lethal P. aeruginosa lung infection in the granulocytopenic host. This effect is probably due to enhancement of alveolar macrophage function through endogenous TNF-α production, in addition to increasing the number of circulating neutrophils.
Stimulation of peripheral blood lymphocytes with Campylobacter jejuni generates a γδ T cell response in patients with Guillain–Barré syndrome
Authors: BEN-SMITH A , GOODALL J C , GASTON J S H , WINER J B
Published: November 2003

In three patients whose Guillain–Barré syndrome (GBS) was preceded by gastrointestinal infection due to Campylobacter jejuni, γδ T cells were generated from peripheral blood in response to in vitro stimulation with C. jejuni. In one of the patients, where a diagnostic sural nerve biopsy was performed, γδ T cells were also isolated following culture of the nerve tissue. Studies with healthy volunteers and C. jejuni gastroenteritis patients also showed preferential enrichment for γδ T cells in peripheral blood cells stimulated with C. jejuni, although the response was significantly lower than that seen in GBS patients. In two out of three GBS patients and all of the controls, γδ T cell receptor (TCR) gene usage was shown to be Vγ9/Vδ2+. In the GBS patient where nerve-infiltrating γδ T cells were isolated, these and C. jejuni-specific peripheral blood cells had similar TCR gene usage, predominantly consisting of Vγ5/Vδ1+ cells. Sequencing the Vδ1 products from nerve and peripheral blood showed similarities in CDR3 length, but the single Vδ1 sequence obtained from nerve was not identified in peripheral blood. These results suggest that the generation of γδ T cells is part of a normal immune response to C. jejuni, which, in patients with GBS, may contribute to the pathogenesis of their inflammatory neuropathy.
Quantification of IgA and IgG and specificities of antibodies to viral proteins in parotid saliva at different stages of HIV-1 infection
Authors: CARTRY O , MOJA P , QUESNEL A , POZZETTO B , LUCHT F R , GENIN C
Published: November 2003

Paired sera and parotid saliva from 75 HIV-1-infected patients, divided in three equal groups with CD4+ cell counts > 500, 200–500 and < 200/mm3, respectively, were analysed for IgG, IgA and secretory IgA (sIgA) concentrations and for IgG and IgA antibody directed to HIV-1. Twenty-nine age-matched HIV− subjects were used as controls. In serum the concentrations of immunoglobulins were significantly increased in HIV-infected subjects compared with controls, and a progressive increase of IgA and sIgA was noticed while the CD4+ cell count decreased. In contrast, concentrations of IgA and sIgA were not different in parotid saliva between the four subject groups. By an ELISA test directed towards HIV-1 proteins, 73 of the 75 serum specimens from the HIV-infected subjects (97%) and 43 of the corresponding saliva (57%) were found positive for specific IgA antibodies to HIV-1, with an even distribution among the three groups of patients. By Western blotting multiple specificities of IgA to HIV-1 proteins were not frequently found in patients. By contrast, in spite of an IgG concentration in saliva about 100 times lower than that of IgA, reactivities were significantly higher for IgG than for IgA antibodies, especially to env and to pol HIV-1 products. Altogether, these data suggest that the regulation of IgA production in HIV-infected subjects is independent in serum and in parotid saliva. This imbalance of IgA/IgG antibodies to HIV-1 at the mucosal level appears to be a specific feature of HIV-1 infection, and may raise important issues in terms of local protection after immunization.
Gene expression of CC chemokines in experimental crescentic glomerulonephritis (CGN)
Authors: NATORI Yu , SEKIGUCHI M , OU Z , NATORI Ya
Published: November 2003

CGN is a rapidly progressive glomerular disease. Monocytes/macrophages are frequently observed in glomeruli in cases of CGN and they are considered to play a crucial role in the pathogenesis of this disease. We described previously the glomerular expression of monocyte chemoattractant protein-1 (MCP-1), which is a potent chemoattractant for monocytes and a member of CC chemokine family, in an experimental model of CGN. In the present study we investigated the expression of mRNAs for other CC chemokines, namely, MCP-3, macrophage inflammatory protein-1α (MIP-1α), MIP-1β, RANTES and TCA3, all of which are chemotactic for monocytes, in the CGN model. First, we established a reverse transcriptase-polymerase chain reaction (RT-PCR) method by which mRNA for each of the CC chemokines could be amplified separately, and then we measured the levels of the expression of mRNAs for the chemokines in diseased glomeruli at several time points after induction of CGN. The mRNAs for all CC chemokines examined were expressed in glomeruli of rats with CGN. Moreover, induction of the gene expression of MIP-1α and MIP-1β seemed to occur earlier than that of the others. CC chemokines may contribute to the recruitment and activation of monocytes in CGN, and each individual CC chemokine may play an overlapping but distinct role in the pathogenesis of this disease.
Surface-related triggering of the neutrophil respiratory burst. Characterization of the response induced by IgG adsorbed to hydrophilic and hydrophobic glass surfaces
Authors: LIU L , ELWING H , KARLSSON A , NIMERI G , DAHLGREN C
Published: November 2003

Hydrophilic and hydrophobic glass surfaces precoated with human albumin, fibrinogen, or IgG were investigated with respect to their ability to activate the neutrophil NADPH-oxidase. We found that IgG-coated surfaces induced a substantial and prolonged neutrophil production of reactive oxygen species (ROS). When a hydrophilic surface was used to support protein binding, a somewhat lower neutrophil response (around 35%) was obtained, compared with the response induced by IgG on a hydrophobic surface. The production of ROS was completely eliminated when cytochalasin B was added to the measuring system, suggesting the involvement of the cell cytoskeleton in the activation process. The relation between the intra- and extracellular generation of ROS was further assessed, and we found that most of the ROS produced were released from the cells, in agreement with a model in which the activating surfaces induce a ‘frustrated’ phagocytic response. Serum totally inhibited ‘frustrated’ phagocytosis provided that the IgG molecules were sticking to a hydrophilic surface.
Reduced protein tyrosine phosphatase (PTPase) activity of CD45 on peripheral blood lymphocytes in patients with systemic lupus erythematosus (SLE)
Authors: TAKEUCHI T , PANG M , AMANO K , KOIDE J , ABE T
Published: November 2003

To disclose the mechanism of aberrant function of peripheral blood lymphocytes (PBL) in SLE, we focused on the catalytic function of CD45, and determined the CD45 PTPase activity in SLE patients. The sample population consisted of 32 SLE patients with different disease activity. PTPase activity of cell lysates immunoprecipitated by anti-CD45 MoAb was assayed against phosphotyrosine analogue PNPP, followed by measuring the release of para-nitro phenol at 410 nm. CD45 PTPase activity of PBL was significantly decreased in SLE patients, compared with that of normal controls and patients with systemic sclerosis (964 ± 265, 1202 ± 172, 1210 ± 125, respectively; SLE versus normal, P < 0.05). It was correlated with SLE Disease Activity Index (SLEDAI) score (r = 0.597, P = 0.0006), but not with the dose of prednisolone (r = 0.214, P = 0.2657), indicating that CD45 PTPase activity became reduced when the disease was active, but it was not affected by prednisolone. Moreover, it was not corrected by in vitro culture with or without stimulation. The expression of CD45 on PBL was comparable between normal and SLE, raising a possibility that it may be due to aberrant regulation of catalytic function of CD45 in SLE. Given the evidence that tyrosine phosphorylation of cellular proteins by tyrosine kinases and phosphatases is one of the key biochemical events in the signal transduction pathway, the decreased CD45 PTPase activity in SLE may account for the defective signal transduction via TCR/CD3, leading to dysregulated effector function of the lymphocytes.
Elevated plasma levels of IgE in Plasmodium falciparum-primed individuals reflect an increased ratio of IL-4 to interferon-gamma (IFN-γ)-producing cells
Authors: ELGHAZALI G , PERLMANN H , RUTTA A S M , PERLMANN P , TROYE-BLOMBERG M
Published: November 2003

People living in Plasmodium falciparum-endemic areas frequently have elevated levels of total as well as P. falciparum-specific serum IgE. This study aimed at investigating whether the elevated serum IgE levels reflect a shift in the balance between CD4+ T helper 1 (Th1) and T helper 2 (Th2) cells in individuals naturally exposed to the P. falciparum parasite. To investigate the role of Th1 and Th2 cells in the human P. falciparum system we used the ELISPOT assay to determine the ratio of IFN-γ- and IL-4-producing cells after specific antigen or mitogen activation in vitro. The donors were individuals who had acquired immunity through natural exposure to the parasite. In response to the specific malaria antigens, very few IL-4-producing cells were seen. However, in the response of individual donors to the polyclonal T cell activator, leucoagglutinin (La), the anti-malarial IgE levels in plasma were correlated with an increased ratio of IL-4/IFN-γ producing cells. Thus, donors with ratios of IL-4/IFN-γ > 1 exhibited mean plasma anti-malarial IgE levels significantly greater than those with ratios < 1. In individuals not living in P. falciparum-endemic areas the ratio of IL-4/IFN-γ was always < 1. Taken together, our data suggest a shift in the balance between Th1 and Th2 cells in naturally P. falciparum-primed individuals, associated with elevated anti-P. falciparum plasma IgE levels. The role and biological significance of IgE (Th2-type immune response) for protection against P. falciparum and/or pathogenesis of malaria require further study.
Comparison of nucleic acid and protein immunization for induction of antibodies specific for HIV-1 gp120
Authors: PEET N M , McKEATING J A , RAMOS B , KLONISCH T , DE SOUZA J B , DELVES P J , LUND T
Published: November 2003

We have compared the antibody response to HIV-1 gp120 type LAI in mice immunized with either a gp120 expression plasmid or with baculovirus-derived recombinant gp120 (rgp120) formulated with Freund's complete adjuvant, TiterMax, Alum, Ribi R-700, AF-A or QuilA. DNA immunization resulted in variable levels of antibody, with endpoint titres ranging from 104 to 105, whereas mice immunized with rgp120 mixed with Ribi R-700, AF-A or QuilA produced antibody levels with endpoint titres > 105. Both types of immunization failed to elicit antibodies able to recognize denatured rgp120. The V3 region was immunogenic in animals immunized with nucleic acid, whereas only a few animals immunized with recombinant protein produced antibodies specific for V3 or other linear epitopes, irrespective of the adjuvant used. These data suggest that the immunogenicity of gp120 is dependent upon the mode of antigen delivery, and that in vivo expressed gp120 following nucleic acid immunization elicits, at least with respect to V3, an antibody response which more closely reflects that seen following natural infection in man.
Elevation of CD8+ CD11b+ Leu-8− T cells is associated with the humoral immunodeficiency in myeloma patients
Authors: WALCHNER M , WICK M
Published: November 2003

Recurrent bacterial infections due to humoral immunodeficiency are an important cause of death in myeloma patients. Recent data indicate that CD8+ T lymphocytes and a reduction of T helper type 1 cells with disease progression may be involved in the regulation of polyclonal immunoglobulin secretion. In mixed lymphocyte cultures derived from peripheral blood mononuclear cells (PBMC) of 24 myeloma patients with reduced immunoglobulin serum levels we investigated the association of CD4+ and CD8+ T cell subsets and immunoglobulin-secreting B cells (ISC) upon mitogenic stimulation with pokeweed mitogen (PWM) and concanavalin A (Con A). In supernatants of cultured PBMC of myeloma patients the spontaneous secretion of the type 1 cytokine interferon-gamma was reduced. After PWM stimulation reduced numbers of polyclonal ISC were found in 79% of patients, and monoclonal ISC were observed in 12% of patients. After Con A stimulation, again formation of polyclonal ISC was reduced, but monoclonal ISC were found in 41% of patients. Elevation of monoclonal and reduction of polyclonal ISC after stimulation with Con A were associated with an increase of CD8+ CD11b+ Leu-8− T cells (P < 0.05). We conclude that the elevated numbers of CD8+ CD11b+ Leu-8− T cells play a role in the stimulation of monoclonal and suppression of polyclonal immunoglobulin secretion in myeloma patients.
Hyperinducible expression of the interferon-gamma (IFN-γ) gene and its suppression in systemic lupus erythematosus (SLE)
Authors: GEREZ L , SHKOLNIK T , HIRSCHMANN O , LORBER M , ARAD G , KAEMPFER R
Published: November 2003

Transient expression of IFN-γ and IL-2 mRNA and its control by post-transcriptional and suppressive mechanisms were analysed in phytohaemagglutinin-induced peripheral blood mononuclear cells (PBMC) from 47 patients with SLE and 31 age-matched normal donors, using quantitative hybridization with antisense RNA probes. In SLE, basal levels of gene expression did not deviate from those of normal donors, but strongly aberrant patterns were obtained upon induction. The ratio of subjects exhibiting highly inducible IFN-γ gene expression in their PBMC to those showing moderate or low inducibility was increased five-fold in SLE (P = 0.003). High inducibility was observed for 43% of SLE patients and was equally pronounced in partial remission, mild or active disease. Inducibility of IL-2 mRNA, by contrast, remained similar to that for normal donors. However, regulation of IFN-γ gene expression differed for mild SLE. Patients with mild disease showing high inducibility of IFN-γ mRNA in their PBMC not only had the highest frequency of responders, but also the highest extent of an individual response, defined by superinduction of mRNA, to agents that relieve suppression (γ-irradiation) or post-transcriptional down-regulation (cycloheximide). By contrast, patients with active SLE showing high IFN-γ mRNA inducibility had normal suppressive capacity as well as post-transcriptional control. Hence, both high inducibility of the IFN-γ gene and its suppression are relevant to disease. Hyperactivation of the IFN-γ gene may be alleviated in mild SLE by a vigorous, concomitant activation of post-transcriptional control and of cell-mediated suppression.
Fractionation of mycobacterial integral membrane proteins by continuous elution SDS–PAGE reveals the immunodominance of low molecular weight subunits for human T cells
Authors: MEHROTRA J , MITTAL A , DHINDSA M S , SINHA S
Published: November 2003

Integral membrane proteins (IMP) represent a serologically distinct class of mycobacterial antigens which are potent stimulators of human T cells (Mehrotra et al., Clin Exp Immunol 1995; 102:626). The range of IMP from Mycobacterium fortuitum was resolved by continuous elution SDS–PAGE to recover 31 discrete fractions covering bands up to ≈ 58 kD. The fractions, after removal of SDS, were subjected to human T cell proliferation assays for the identification of immunodominant molecule(s). A low molecular weight (<20kD) fraction was able to stimulate T cells from 11 out of 12 donors comprising mainly tuberculoid leprosy patients. The described protocol is well suited to situations where large quantities of antigenic protein mixtures must be processed in order to get the purified molecules/fractions in amounts required for immunoepidemiological studies.
Characterization of complement C6 deficiency in a PVG/c rat strain
Authors: VAN DIXHOORN M G A , TIMMERMAN J J , VAN GIJLSWIJK-JANSSEN D J , MUIZERT Y , VERWEIJ C , DISCIPIO R G , DAHA M R
Published: November 2003

Complement C6 plays an important role in the effector phase of complement-mediated cell lysis. Recently, a PVG/c rat strain deficient in haemolytic C6 activity was discovered. In the present study we show that these rats lack both antigenic and functional C6, and that repetitive immunization of these rats with PVG/c+ serum results in generation of specific anti-rat C6 antibodies. The observed absence of rat C6 was further investigated at the genomic and transcriptional level using a 492-bp cDNA of rat C6, cloned from a rat liver cDNA library using full length human C6 as a probe. Northern blot analysis revealed the presence of C6 mRNA in livers of both PVG/c− and PVG/c+ rats, corresponding to a size of ≈ 3.3 kb, although the level of C6 mRNA expression was ≈ 100-fold less in PVG/c− rats. In addition, using rat C6-specific primers, positive signals were obtained in kidneys of both rat strains by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Southern blot analysis of digested genomic DNA did not reveal evidence for large C6 gene deletions. We conclude that the lack of C6 protein in the PVG/c− rat strain is not due to a (large) C6 gene deletion, but presumably is caused by an unstable mRNA or a point mutation in the C6 gene resulting in an aberrant transcription of the C6 gene. Alternatively, a gene coding for a product involved in C6 biosynthesis that acts in trans may carry a mutation.
Increased plasma levels of soluble CD23 in haemorrhagic fever with renal syndrome; relation to virus-specific IgE
Authors: ALEXEYEV O A , LINDERHOLM M , ELGH F , WADELL G , JUTO P , TÄRNVIK A
Published: November 2003

In 15 consecutive patients hospitalized with nephropathia epidemica, a European form of haemorrhagic fever with renal syndrome (HFRS) caused by Puumala virus, plasma concentrations of soluble CD23 (sCD23) and Puumala virus-specific IgE were determined. In the acute phase of illness, 11/15 patients had increased sCD23 levels (> 91 U/ml), whereas in convalescence, values of 8/10 patients were normalized. Maximal sCD23 values were correlated to maximal concentrations of Puumala virus-specific serum IgE (r = 0.597; P = 0.025). The results are compatible with a known ability of sCD23 to augment IgE production.
High incidence of autoimmune dacryoadenitis in male non-obese diabetic (NOD) mice depending on sex steroid
Authors: TAKAHASHI M , ISHIMARU N , YANAGI K , HANEJI N , SAITO I , HAYASHI Y
Published: November 2003

The NOD mouse develops spontaneous autoimmune lesions in the lacrimal and salivary glands, besides a well characterized T cell-mediated autoimmune pancreatic β cell lesion. We report unique pathological findings developed in the lacrimal glands as an autoimmune dacryoadenitis of NOD mice in contrast to those found in the salivary glands and pancreas. A high incidence of autoimmune lesions in the lacrimal glands was observed exclusively in male NOD mice at any age. Histology of autoimmune dacryoadenitis in male NOD mice showed severe destructive changes compared with those observed previously as an autoimmune lesion in the lacrimal glands. Castration in male NOD mice significantly decreased the incidence of autoimmune dacryoadenitis, and testosterone treatment with castration also increased the incidence of autoimmune lesions. Oestrogen treatment with castration did not increase the incidence, but tamoxifen treatment without castration significantly increased the incidence of autoimmune dacryoadenitis in NOD mice. In addition, we detected up-regulation of local cytokine genes (IL-1β, tumour necrosis factor-alpha (TNF-α), IL-2, interferon-gamma (IFN-γ), IL-6, IL-10, and IL-12 p40) during the course of autoimmune dacryoadenitis. These data suggest that in spontaneous autoimmune dacryoadenitis of male NOD mice there may be an intimate relationship with sex steroids, particularly testosterone, in the development and progression of autoimmune lesions, and autoreactive Th1 cells secrete up-regulated cytokine genes, including IL-10 and IL-12.
Elevated serum levels of soluble CD30 in patients with atopic dermatitis (AD)
Authors: BENGTSSON Å , HOLM L , BÄCK O , FRANSSON J , SCHEYNIUS A
Published: November 2003

The immunopathology of AD is still unclear, but evidence for an immune response polarized towards Th2 activity has been provided. The CD30 molecule belongs to the tumour necrosis factor (TNF) receptor family and is expressed on activated T cells with a sustained expression in Th2 cells. This molecule also exists in a soluble form (sCD30). Elevated serum levels of sCD30 have been found in patients with Hodgkin's disease, chronic hepatitis B infection and HIV infection. Studies were undertaken to compare the serum levels of sCD30 in patients with AD (n=49) and healthy non-atopic controls (n=94). The presence of sCD30 was analysed with ELISA. A significantly higher concentration of sCD30 was noted in AD patients, median sCD30 level 29 U/ml (range 1–708 U/ml), compared with healthy non-atopic controls (P < 0.001), where the median level was 11 U/ml with a range of 1–1042 U/ml. No correlation was found between sCD30 levels and total serum IgE, or between the AD patients' SCORAD values and concentration of sCD30. sCD30 levels were also analysed in 20 AD patients, which during ketoconazole treatment had improved their clinical scores and reduced their serum IgE and eosinophil cationic protein levels. However, no significant decrease in sCD30 levels was noted after treatment. The results show that patients with AD have elevated levels of sCD30, but without correlation to total serum IgE or disease activity.
C1q, a subunit of the first component of complement, enhances antibody-mediated apoptosis of cultured rat glomerular mesangial cells
Authors: SATO T , VAN DIXHOORN M G A , HEEMSKERK E , VAN ES L A , DAHA M R
Published: November 2003

We have shown previously that IgG2a anti-Thy-1 MoAb (ER4G) induces apoptosis of rat mesangial cells (GMC) in vitro. Since the classical complement pathway plays an essential role in Thy-1 nephritis, we analysed whether C1q, a subunit of the first component of complement, enhances the ER4G-mediated apoptosis of rat GMC. Two different subclasses of anti-Thy-1 MoAb, ER4G (IgG2a) and ER14 (IgG1), were used. It was established that ER4G binds C1q efficiently, while ER14 reacts poorly with C1q. For the experiments of apoptosis, quiescent rat GMC were exposed for 1 h at 37°C to a fixed concentration of anti-Thy-1 MoAb and incubated further for 16 h at 37°C in the presence or absence of C1q. GMC exposed to medium (M-GMC) followed by incubation of the cells with medium alone was used as controls. Apoptosis was assessed by morphological studies and quantitative analysis on FACS using FITC-annexin V (the annexin V methods) or bicolour FACS analysis using FITC-annexin V and propidium iodide (the annexin V/PI method). With the annexin V method, M-GMC revealed 9.4 ± 1.4% apoptosis. C1q had only marginal effects on apoptosis of M-GMC. GMC exposed to ER4G (ER4G-GMC) and further incubated with medium in the absence of C1q resulted in 25.7 ± 5.7% apoptosis (P < 0.01 relative to control). Incubation of ER4G-GMC together with 100 μg/ml of C1q significantly increased GMC-apoptosis up to 39.4 ± 4.9% (P < 0.01 relative to ER4G-GMC incubated in the absence of C1q). This enhancing effect of C1q on apoptosis of ER4G-GMC was time- and dose-dependent. In contrast, C1q did not significantly alter the apoptosis of either GMC exposed to ER14 (ER14-GMC) or to F(ab′)2-ER4G (F(ab′)2-ER4G-GMC), while ER14-GMC or F(ab′)2-ER4G-GMC incubated with medium resulted in significant apoptosis compared with control. These results were supported by morphological studies and bicolour FACS analyses in time course experiments using the annexin V/PI method. The effect of C1q is dependent on the presence of intact C1q-containing globular heads and does not occur with collagen-like fragments of C1q. Furthermore, incubation of ER4G-GMC with anti-mouse κ-chain antibodies also increased ER4G-mediated GMC-apoptosis. These results indicate for the first time that C1q enhances antibody-mediated apoptosis of rat GMC in vitro, presumably by its binding to ER4G and probably by additional cross-linking of Thy-1 on the surface of GMC.
Phenotype, cytokine production and cytolytic capacity of fresh (uncultured) tumour-infiltrating T lymphocytes in human renal cell carcinoma
Authors: VAN DEN HOVE L E , VAN GOOL S W , VAN POPPEL H , BAERT L , COOREVITS L , VAN DAMME B , CEUPPENS J L
Published: November 2003

We investigated the phenotype and functional capacities of tumour-infiltrating lymphocytes (TIL), freshly isolated from primary renal cell carcinoma (RCC) specimens (n = 20). Three-colour flow cytometry immunophenotyping revealed that RCC TIL consist mainly of CD3+ T cells, with a clear predominance of CD4−CD8+ over CD4+ CD8− T cells, and a marked population of CD4+ CD8+ T cells. Natural killer (NK) cells were also strongly represented (> 25% in 15 of 20 tumour samples), while B cells constituted a minor TIL subset (< 5% in 18 of 20 tumour samples). More importantly, the T and NK cells within the tumour displayed a significantly higher expression of the early activation marker CD69 than their counterparts in adjacent normal renal tissue and in peripheral blood. Expression of CD54 and of HLA-DR was also elevated on CD3+ TIL, and HLA-DR expression was further vigorously up-regulated following ex vivo stimulation with anti-CD3, all suggesting enhanced immune activity within the tumour microenvironment. CD3+ CD4+ TIL displayed a normal capacity to up-regulate CD25 expression and to secrete both Th1-type (IL-2, tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ)) and Th2-type (IL-4, IL-5 and IL-10) cytokines upon triggering with anti-CD3. Furthermore, cytokine production was susceptible to modulation by CD28 costimulation. CD3+ CD8+ TIL, on the other hand, consistently demonstrated a poor up-regulation of CD25 upon triggering with anti-CD3, and displayed poor ex vivo cytolytic activity in an anti-CD3-redirected 4-h cytotoxicity assay against murine P815 cells. Collectively, our findings indicate that the CD3+ CD4+ TIL in RCC have normal functional capacities, whereas the proportionally major CD3+ CD8+ TIL are functionally impaired. The relevance of these findings to the in vivo local immune response in RCC is discussed.
Immunoprecipitation of melanogenic enzyme autoantigens with vitiligo sera: evidence for cross-reactive autoantibodies to tyrosinase and tyrosinase-related protein-2 (TRP-2)
Authors: KEMP E H , GAWKRODGER D J , WATSON P F , WEETMAN A P
Published: November 2003

In the present study we describe the detection of TRP-2 antibodies in vitiligo patients using in vitro35S-labelled human TRP-2 in a radioimmunoassay. Of 53 vitiligo sera examined in the assay, three (5.9%) were found to be positive for TRP-2 antibodies. In contrast, 20 control sera, sera from 10 patients with Hashimoto's thyroiditis and sera from 10 patients with Graves' disease were all negative. All three patients positive for TRP-2 antibodies (mean age 54 years, age range 50–63 years) had had vitiligo of the symmetrical type for more than 1 year and all of them also had an associated autoimmune disorder: Graves' disease in one and autoimmune hypothyroidism in two. In addition, antibodies to the melanogenic enzyme tyrosinase were present in their serum. To examine any immunological cross-reactivity between TRP-2 and tyrosinase, the three vitiligo sera positive for TRP-2 antibodies were preabsorbed with COS-7 cell extract containing either expressed TRP-2 or tyrosinase, and subsequently used in the radioimmunoassay. These absorption studies indicated that preincubation with both proteins inhibited the immunoreactivity of the positive sera in the immunoassay using in vitro translated 35S-TRP-2. This antibody cross-reactivity suggests the humoral response to the two melanogenic enzymes in these patients may not be entirely independent.
Characterization of fertilization-blocking monoclonal antibody 1G12 with human sperm-immobilizing activity
Authors: KOMORI S , KAMEDA K , SAKATA K , HASEGAWA A , TOJI H , TSUJI Y , SHIBAHARA H , KOYAMA K , ISOJIMA S
Published: November 2003

A mouse hybridoma (1G12) producing sperm-immobilizing MoAb to human sperm was established and characterized in order to study the antigens relevant to sperm immobilization by antibodies. MoAb 1G12 had strong sperm-immobilizing and agglutinating activities and also showed a fertilization-blocking activity on in vitro fertilization tests. The antibody absorption experiments showed that MoAb 1G12 reacted not only to ejaculated sperm but also human seminal plasma, suggesting that the corresponding antigen might be a sperm coating antigen. The MoAb also reacted with peripheral blood lymphocytes. In histochemical studies, the epithelia of corpus epididymis were most strongly stained. Ejaculated sperm were stained with a granular pattern for their entire surface by immunofluorescence. MoAb 1G12 recognized polymorphic glycoproteins of 15–25 kD in the ejaculated sperm extract in Western blot analysis. After deglycosilation of the sperm extract, only a single staining band of under 15 kD was detected by MoAb 1G12. This suggests that the antigen epitope recognized by MoAb 1G12 might be a peptide of the core portion of the glycoprotein. MoAb 1G12 might be a useful tool for studying the mechanism of egg–sperm interaction, and also be applied to identifying the corresponding antigen by using gene technology.
Sex and parity modulate cytokine production during murine ageing
Authors: BARRAT F , LESOURD B , BOULOUIS H-J , THIBAULT D , VINCENT-NAULLEAU S , GJATA B , LOUISE A , NEWAY T , PILET Ch
Published: November 2003

We have previously shown that physiological hormone differences related to pregnancy or sex affect the age-related distribution of mononuclear cell populations during murine ageing. To determine whether such changes are involved in the age-related changes in functions of T cells, we examined the secretion of major T cell immunoregulatory cytokines (IL-2, IL-4, interferon-gamma (IFN-γ), IL-3, IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF)) of in vitro concanavalin A-activated spleen cells of C57Bl/6 mice. The study included multiparous and virgin females and males at 2, 8, 15 and 23 months of age. Short-term effects of parity (8 months) were evidenced by the decrease of IFN-γ and the preserved IL-2 production in multiparous females (8 months), while IFN-γ was unchanged and IL-2 decreased in virgin mice. The increase in IL-4 production appeared earlier in multiparous females (15 months) than in virgin mice (23 months). The increase in IL-4/IFN-γ and IL-4/IL-2 ratios at 8 and 15 months, respectively, in multiparous females, suggests that pregnancy modifies the Th1/Th2 equilibrium. In late adulthood (15 months), IL-6 and GM-CSF production was higher in multiparous females than in virgin males or females. Sex differences were also noticed: IFN-γ secretion capacity was lower in males than in females during ageing. This study underlines that the onset, magnitude and kinetics of the age-related changes in cytokine production are parity- and sex-dependent. These changes probably influence the incidence of age-related diseases and may explain the greater longevity of females.
Fine specificity of autoantibodies to La/SSB: epitope mapping, and characterization
Authors: TZIOUFAS A G , YIANNAKI E , SAKARELLOS-DAITSIOTIS M , ROUTSIAS J G , SAKARELLOS C , MOUTSOPOULOS H M
Published: November 2003

The B cell epitope mapping of La/SSB was performed using 20 mer synthetic peptides overlapping by eight amino acids covering the whole sequence of the protein. IgG, purified from sera of five patients with systemic lupus erythematosus (SLE) and four sera from patients with primary Sjögren's syndrome (pSS) were tested against the overlapping synthetic peptides. Peptides highly reactive with purified IgG were those spanning the regions 145–164, 289–308, 301–320 and 349–368 of the La protein. Determination of the minimum required length of the antigenic determinants disclosed the following epitopes:147HKAFKGSI154, 291NGNLQLRNKEVT302, 301VTWEVLEGEVEKEALKKI318 and 349GSGKGKVQFQGKKTKF364. Predicted features and molecular similarities of the defined epitopes were investigated using protein databases. The La epitope 147HKAFKGSI154 presented 83.3% similarity with the 139HKGFKGVD146 region of human myelin basic protein (MBP) and 72% similarity with the fragment YKNFKGTI of human DNA topoisomerase II. Peptides corresponding to these sequences cross-reacted with anti-La/SSB antibodies. Sixty-three sera with anti-La/SSB antibodies from patients with pSS or SLE, 35 sera without anti-La/SSB antibodies from patients with SS or SLE and 41 sera from age/sex-matched healthy blood donors were tested against biotinylated synthetic epitope analogues in order to determine their sensitivity and specificity for the detection of anti-La/SSB antibodies. Anti-La/SSB were detected with various frequencies ranging from 20% to epitope 147HKAFKGSI154 to 100% to epitope 349GSGKGKVQGKKTKF364. The overall sensitivity and specificity using all assays with the synthetic peptides were found to be 93.6% and 85.6%, respectively. In conclusion, antibodies to La/SSB constitute a heterogeneous population, directed against different linear B cell epitopes of the molecule. The epitope 147HKAFKGSI154 presents molecular similarity with fragments of two other autoantigens, i.e. human MBP and DNA topoisomerase II. Finally, synthetic epitope analogues exhibit high sensitivity and specificity for the detection of anti-La/SSB antibodies.
Expansion of restricted cellular immune responses to HIV-1 envelope by vaccination: IL-7 and IL-12 differentially augment cellular proliferative responses to HIV-1
Authors: KIM J H , LOVELAND J E , SITZ K V , RATTO KIM S , MCLINDEN R J , TENCER K , DAVIS K , BURKE D S , BOSWELL R N , REDFIELD R R , BIRX D L
Published: November 2003

The failure of immune effector mechanisms to control HIV-1 infection has important consequences for the human host. In a randomized cohort of HIV-infected patients, there was striking in vitro restriction of the proliferative response to HIV-1 envelope protein (Env), gp160; only 34% of patients recognized Env. Therapeutic vaccination with recombinant gp160 or gp120 (rgp160, rgp120) reversed the restriction in vitro, with Env recognition rising to 81%. Peripheral blood mononuclear cells (PBMC) from HIV-infected vaccine recipients, placebo recipients, and seronegative volunteers were cultured with exogenous IL-7 or IL-12 and either tetanus toxoid (TT) or gp160. IL-7 significantly augmented proliferative responses to TT and gp160, whereas IL-12 only affected proliferation to gp160. IL-7, but not IL-12, increased the number of HIV-infected placebo recipients who recognized rgp160. IL-12 had its greatest effect in the induction of rgp160-specific responses from seronegative individuals. The data suggest that these two cytokines have differential activity in the relief of restricted cellular immunity to Env; the predominant effect of IL-7 is in individuals who have been primed by exposure to antigen, while the effect of IL-12 is most evident in seronegative, unprimed individuals. Modification of restricted proliferative responses to Env by vaccination or cytokines in vitro suggests that strategies incorporating IL-7 or IL-12 as adjuvants may selectively boost cellular reactivity to HIV-1.