bluuebunny/crossref_metadata_2025 huggingface dataset:
Down-regulation of MHC class II molecules and inability to up-regulate class I molecules in murine macrophages after infection withToxoplasma gondii
Authors: Lüder C G K , Lang T , Beuerle B , Gross U
Published: March 2003

Toxoplasma gondii is able to invade phagocytic cells of the monocyte-macrophage lineage and replicates within a parasitophorous vacuole. Since macrophages may activate specific T lymphocytes by presenting pathogen-derived antigens in association with molecules of the MHC, we investigated the in vitro expression of host cell molecules involved in antigen processing and presentation before and during infection of murine bone marrow-derived macrophages (BMM) with T. gondii. Fifty-one hours after addition of T. gondii tachyzoites at different parasite-to-host ratios, up-regulation of total MHC class II molecules by interferon-gamma (IFN-γ) was dose-dependently abrogated in up to 50% of macrophages compared with uninfected control cultures. Quantitative analyses by flow cytometry revealed that the IFN-γ-induced surface expression of class II antigens as well as the IFN-γ-induced up-regulation of class I molecules was significantly decreased in T. gondii-infected macrophage cultures compared with uninfected controls. However, the constitutive expression of MHC class I antigens was not altered after parasitic infection, and infected BMM remained clearly positive for these molecules. After infection of macrophages preactivated with IFN-γ for 48 h, T. gondii also actively down-regulated an already established expression of MHC class II molecules. Furthermore, kinetic analysis revealed that the reduction in intracellular and plasma membrane-bound class II molecules started ≈ 20 h after infection. While MHC class II antigens were most prominently reduced in parasite-positive host cells, culture supernatant from T. gondii-infected BMM cultures also significantly inhibited expression of these molecules in uninfected macrophages. However, down-regulation of MHC class II molecules was not mediated by an increased production of prostaglandin E2, IL-10, transforming growth factor-beta or nitric oxide by infected BMM compared with uninfected controls. Our data indicate that intracellular T. gondii interferes with the MHC class I and class II antigen presentation pathway of murine macrophages and this may be an important strategy for evasion from the host's immune response and for intracellular survival of the parasite.
Selective depletion of CD14+ CD16+ monocytes by glucocorticoid therapy
Authors: Fingerle-Rowson G , Angstwurm M , Andreesen R , Ziegler-Heitbrock H W L
Published: March 2003

Glucocorticoids (GC) are potent anti-inflammatory and immunosuppressive agents that act on many cells of the body, including monocytes. Here we show that a 5-day course of high dose GC therapy differentially affected the CD14++ and the CD14+ CD16+ monocyte subpopulations in 10 patients treated for multiple sclerosis. While the classical (CD14++) monocytes exhibited a substantial increase from 495 ± 132 to 755 ± 337 cells/μl, the CD14+ CD16+ monocytes responded with a pronounced decrease from 36 ± 15 to 2 ± 3 cells/μl (P < 0.001). In 4/10 patients the CD14+ CD16+ monocytes fell below detection limits (< 0.2 cells/μl). This observation was confirmed when the CD14+ CD16+ monocytes were identified by virtue of their low CD33 expression as these cells decreased as well. After discontinuation of GC therapy the CD14+ CD16+ monocytes reappeared and reached normal levels after 1 week. The profound depletion of CD14+ CD16+ monocytes by GC as described here is a novel effect of GC action in vivo and may contribute to GC-mediated immunosuppression. Determination of the number of this monocyte subset may also serve to monitor the effectiveness of GC therapy in patients requiring immunosuppressive treatment.
Induction of thyroiditis in mice with thyrotropin receptor lacking serologically dominant regions
Authors: Wang S H , Carayanniotis G , Zhang Y , Gupta M , Mcgregor A M , Banga J P
Published: March 2003

Grave's disease (GD) is characterized by pathogenic autoantibodies to the human thyrotropin receptor (hTSH-R), and is frequently associated with a lymphocytic infiltrate of the thyroid gland. In attempts to establish a murine model of GD, we and others have previously shown that immunization of mice with recombinant preparations of the hTSH-R ectodomain induces high titres of specific antibodies, which, however, are not pathogenic, nor is the response accompanied by the development of thyroiditis. Since earlier reports identified the serological immunodominant determinants within the N- and C-terminal regions of hTSH-R ectodomain, we reasoned that immunization of mice with truncated fragments of ectodomain lacking these dominant regions might result in skewing of the response to other determinants of the molecule, with consequent induction of immunopathological features present in GD. We show here that multiple challenge of BALB/c mice with an amino acid fragment of residues 43–282 generates antibodies directed at hTSH-R peptides 37–56, 157–176, 217–236 and 232–251. This reactivity pattern is distinct from that induced previously with the whole ectodomain of hTSH-R in BALB/c animals. Thyroid function remained unaffected in these mice, suggesting that pathogenic antibodies were not being induced. Interestingly, some animals developed lymphocytic infiltration of the thyroid gland, clearly indicating the presence of pathogenic T cell determinants within the 43–282 fragment. Challenge with the related fragment 43–316 produced the same pattern of serological response to the synthetic peptides as fragment 43–282, but was not accompanied by thyroiditis. The results demonstrate: (i) the presence of thyroiditogenic determinants within hTSH-R, and (ii) that these pathogenic determinants are likely to be cryptic, as their effect is exhibited only when the hierarchy of immunodominance within hTSH-R is drastically altered.
Cytokine-stimulated release of decay-accelerating factor (DAF; CD55) from HT-29 human intestinal epithelial cells
Authors: Nasu J , Mizuno M , Uesu T , Takeuchi K , Inaba T , Ohya S , Kawada M , Shimo K , Okada H , Fujita T , Tsuji T
Published: March 2003

Expression of DAF (CD55) is enhanced on colonic epithelial cells of patients with ulcerative colitis (UC), and stool DAF concentrations are increased in patients with active disease. Cytokines are known to modulate DAF expression in various human cells, and lesions of UC reveal altered profiles of cytokine production. In this study, we evaluate the effects of various cytokines, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, and interferon-gamma (IFN-γ), on the synthesis and kinetics of DAF protein in HT-29 human intestinal epithelial cells. Using flow cytometry and an ELISA, we found that HT-29 cells constitutively express DAF on the cell surface and spontaneously release DAF into the culture supernatant under standard culture conditions. When the culture supernatant was centrifuged at 100 000 g, nearly a half of DAF was precipitated, indicating that one half of the released DAF was present as a membrane-bound form and the other half as a soluble form. Analysis of the culture supernatant of biotin surface-labelled HT-29 cells suggested that the soluble form DAF was derived by secretion from within the cell or by cleavage from the cell surface. Among the cytokines, IL-4 markedly, and IL-1β moderately, enhanced the expression and the release of DAF. Actinomycin D, cycloheximide, and brefeldin A inhibited the increase in DAF release induced by IL-4 and IL-1β stimulation. These results suggest that DAF is released from intestinal epithelial cells in response to cytokine stimulation and that IL-4 and IL-1β are possible cytokines involved in DAF release into the colonic lumen of patients with UC.
Cytokine production by CD4 and CD8 T cells during the growth ofMycobacterium tuberculosisin mice
Authors: Howard A D , Zwilling B S
Published: March 2003

Changes in the pattern of cytokines found in CD4 and CD8 T cells during the growth of Mycobacterium tuberculosis that resulted in the establishment of a latent infection were monitored. Subsets of T cells were identified based on their differential expression of CD45 and CD44 which allowed them to be classified as naive, activated or memory. We found that the T cells in the lung produced a predominantly type 1 cytokine response. The appearance of large numbers of Th1 cells coincided with the establishment of latency. In contrast, the predominant response in the mediastinal lymph node and spleen was a Th2-type response.
Evidence of idiotypic modulation in the immune response to gp43, the major antigenic component ofParacoccidioides brasiliensisin both mice and humans
Authors: Souza A R , Gesztesi J-L , Moraes J Z , Cruz C R B , Sato J , Mariano M , Lopes J D
Published: March 2003

Paracoccidioidomycosis (PCM) is a systemic mycosis endemic in Latin America, with a high prevalence in Brazil, Argentina, Colombia and Venezuela. The aetiologic agent of disease is a thermal dimorphic fungus, Paracoccidioides brasiliensis. A glycoprotein of 43 000 D (gp43) is the major antigen of P. brasiliensis. Antibodies directed to this antigen are detected in the sera of all patients with PCM. Gp43 binds to laminin, thus participating in adhesion, invasion and pathogenesis of the fungus. As the role of antibodies in PCM is not fully understood, we decided to investigate the outcome of mice immunization with three distinct anti-gp43 MoAbs (17c, 8a and 24a) coupled with keyhole limpet haemocyanin (KLH). Results show not only the expected presence of anti-Id (AB2) antibodies in the sera of these animals but also a spontaneous and increasing amount of anti-anti-Id (AB3) antibodies after the third course of immunization. Hybridomas producing both AB2 and AB3 MoAbs were obtained using spleen cells from mice immunized with MoAb 17c. AB3 MoAbs were also obtained with spleen cells of mice immunized with MoAbs 8a and 24a. It was also shown that human PCM patients' sera with high titres of anti-gp43 antibodies generate anti-Id antibodies. These data suggest that the immune response to P. brasiliensis can be spontaneously modulated by the idiotypic network.
Differential production of IL-10 by T cells and monocytes of HIV-infected individuals: association of IL-10 production with CD28-mediated immune responsiveness
Authors: Kumar A , Angel J B , Daftarian M P , Parato K , Cameron W D , Filion L , Diaz-Mitoma F
Published: March 2003

Immune unresponsiveness in HIV-1 infection can result from impaired signals delivered by the costimulatory CD28-B7 pathway and the altered production of immunoregulatory cytokines, in particular IL-10, whose production is altered in HIV-1 infection. In this study we investigate IL-10 regulation in T cells and monocytes from HIV+ individuals, and its association with CD28-mediated T cell proliferation. IL-10 production as analysed in T cell- and monocyte-depleted peripheral blood mononuclear cells (PBMC), and by intracellular staining at the single-cell level, reveals a defect in IL-10 production by CD4+ and CD8+ T cells, whereas monocytes constitute the major IL-10-producing cell type. To investigate the impact of IL-10 on immune responsiveness, CD28-mediated proliferative responses in HIV+ individuals were correlated with PHA-induced IL-10 production. CD4+ T cells expressed CD28, yet exhibited markedly reduced CD28-mediated cell proliferation. This CD28-mediated CD4+ T cell proliferation was found to be inversely associated with the levels of PHA-induced IL-10 production and could be restored, at least in part, by anti-IL-10 antibodies. These results suggest that IL-10 production is differentially regulated in T cells and monocytes of HIV+ individuals, and that IL-10 may have a role in inducing immune unresponsiveness by modulating the CD28-B7 pathway.
T lymphocytes are not the target for estradiol-mediated suppression of DTH in reconstituted female severe combined immunodeficient (SCID) mice
Authors: Taube M , Svensson L , Carlsten H
Published: March 2003

Oestrogen has the capacity to suppress T cell-dependent DTH. To explore the mechanisms whereby oestrogen exerts its effects on the immune system we have used SCID mice which are largely devoid of functional T and B lymphocytes, hence being unable to raise DTH, but display intact antigen-presenting capacity. Transfer of lymphocytes to SCID mice restores the DTH capacity. In order to analyse if oestrogen down-regulates DTH by a direct action on T cells we reconstituted SCID mice with either splenocytes or thymocytes from congenic C.B-17 or allogeneic B6 donor mice. Either donor or recipient mice were exposed to estradiol before cell transfer. DTH response was registered in recipient SCID mice 1 and 3 weeks after challenge with oxazolone (OXA). SCID mice receiving estradiol-exposed spleen cells from congenic or allogeneic donor mice displayed lower DTH responses compared with control mice. In contrast, SCID mice receiving estradiol-exposed thymocytes from congenic donor mice showed no significant difference in DTH response compared with control mice. Estradiol-treated SCID mice, transferred with either spleen cells or thymocytes from congenic, hormonally non-treated donors, displayed a significantly lower DTH response compared with control mice. In contrast, estradiol-treated SCID mice receiving hormonally non-treated allogeneic spleen cells showed no difference in DTH response compared with control mice. The results show that T lymphocytes are not the target cell population for estradiol-mediated suppression of DTH in reconstituted female SCID mice.
Sequence analysis of V4–34-encoded antibodies from single B cells of two patients with systemic lupus erythematosus (SLE)
Authors: Mockridge C I , Chapman C J , Spellerberg M B , Sheth B , Fleming T P , Isenberg D A , Stevenson F K
Published: March 2003

SLE is an autoimmune disease characterized by the presence of autoantibodies against double-stranded (ds)DNA. A large proportion (approx. 40%) of patients with lupus also have increased levels of serum immunoglobulin encoded by the V4–34 heavy chain gene, which often fluctuate with disease activity, and this gene is utilized by a subset of anti-dsDNA antibodies. In order to probe the nature of the V4–34-encoded immunoglobulin, B cells were isolated from the blood of two patients with active disease, using the 9G4 MoAb specific for the immunoglobulin gene product. Following cell picking, single-cell polymerase chain reaction (PCR) amplification of cDNA was used to investigate both VH and VL genes. Sequences were obtained from B cells synthesizing IgM (n = 10), IgG (n = 4) and IgA (n = 1). For VH, all were derived from V4–34 as expected, and the isotype-switched sequences and 2/6 IgM sequences were somatically mutated. In contrast, VL (12 κ and 3 λ) showed a low level of mutation, possibly indicating secondary rearrangements. The three most highly mutated VH sequences were associated with unmutated VL sequences. Analysis of the distribution of mutations revealed only minor clustering in complementarity-determining regions (CDRs) characteristic of antigen selection. The CDR3 lengths of VH ranged from five to 19 amino acids, and in 3/15 there was evidence of an excess of positively charged amino acids, compared with the normal expressed repertoire. Basic amino acids were also found at the VL–JL junctions in 4/15. These findings provide insight into the V4–34–VL gene combinations used by B cells in patients with SLE which might have clinical relevance.
Mercury-induced autoimmunity in the absence of IL-4
Authors: Bagenstose L M , Salgame P , Monestier M
Published: March 2003

In susceptible H-2s mice, mercuric chloride (HgCl2) induces an autoimmune syndrome characterized by production of anti-nucleolar antibodies (ANoA) and increased serum levels of IgG1 and IgE antibodies. The increase in serum IgG1 and IgE, which are under IL-4 control, suggests a role for the Th2 subset in the induction of this syndrome. We have previously shown that administration of IL-12, a potent Th1-promoting cytokine, resulted in a dramatic reduction of the HgCl2-induced anti-nucleolar antibody titres and inhibited serum IgG1 increase. These results suggest that Th1 T cells can down-regulate ANoA, and support a role for the Th2 subset in ANoA production, possibly via IL-4. To examine the role of IL-4 in this syndrome, C57Bl/6 mice (H-2b) with a targeted deletion of the IL-4 gene were mated with A.SW mice (H-2s) to yield H-2s mice lacking IL-4. We then analysed ANoA and serum immunoglobulin levels in these mice after HgCl2 treatment. While mercury-treated IL-4−/− H-2s mice had virtually no detectable serum IgG1 or IgE, and very low levels of IgG1 ANoA, these mice had levels of IgG2a and IgG2b class ANoA comparable to mercury-treated IL-4+ H-2s mice, indicating that IL-4 is not required for the ANoA response in mercury-induced autoimmunity.
Whole virus influenza vaccine activates dendritic cells (DC) and stimulates cytokine production by peripheral blood mononuclear cells (PBMC) while subunit vaccines support T cell proliferation
Authors: Saurwein-Teissl M , Zisterer K , Schmitt T L , GLÜck R , Cryz S , Grubeck-Loebenstein B
Published: March 2003

Three types of trivalent influenza vaccines were analysed for their in vitro stimulatory properties on immune cells from young healthy volunteers. A whole inactivated virus (WV) vaccine, a conventional subunit (c-SU) preparation and a new virosomal subunit (v-SU) vaccine were used. Blood-derived DC up-regulated MHC class II, CD54, CD80 and CD86 after exposure to WV vaccine, indicating their functional maturation, but were only moderately affected by subunit (SU) vaccines. In addition, IL-12 and tumour necrosis factor-alpha (TNF-α) secretion by DC were markedly enhanced by WV, but not by SU vaccines. The production of IL-2 and interferon-gamma (IFN-γ) by PBMC was also strongly stimulated by WV, but much less by SU vaccines, among which the v-SU vaccine was a better stimulator of IL-2 secretion. In contrast to WV vaccine both SU vaccines were powerful stimulators of PBMC proliferation. Our results suggest that the presence of influenza core components leads to the activation of DC and triggers the production of cytokines by PBMC. SU vaccines are in contrast excellent stimulators of T cell growth. A combination of WV and SU vaccines in immunization regimes might allow optimal T cell priming as well as the efficient generation and maintenance of memory cells.
The immunosuppressive activity ofAscaris suumis due to high molecular weight components
Authors: Faquim-Mauro E L , Macedo M S
Published: March 2003

The role of isolated components obtained by gel filtration chromatography of Ascaris suum body extract (Asc) on the modulation of the immune response to ovalbumin (OvA) was evaluated and correlated with the immunogenic properties of such components. We showed that high (PI), but not low (PIII), molecular weight components have the ability to inhibit OvA-induced immediate and DTH reactions, lymph node (LN) cell proliferation, cytokine (IL-2, interferon-gamma (IFN-γ), IL-4 and IL-10) and antibody (IgG1, IgG2a, IgM and IgE) production in mice concomitantly immunized with OvA and these high mol. wt components. The pattern of cytokines synthesized in response to PI or PIII was totally different: the former induced more IL-4 and IL-10 and the latter more IL-2 and IFN-γ. The levels of Asc-specific IgG1 antibodies were higher in mice immunized with OvA plus PI and IgG2a anti-Asc antibodies predominated in those immunized with PIII. IgE antibody production, however, was low in the former group of mice. These results indicate that the high mol. wt components present in the body extract from the helminth A. suum are responsible for its suppressive effect upon Th1- and Th2-dependent immune responses to an unrelated antigen. The suppression of the Th1-dependent parameters could be related to high-level expression of IL-4 and IL-10 induced by such components.
Ro/SS-A-reactive B lymphocytes in salivary glands and peripheral blood of patients with Sjögren's syndrome
Authors: HALSE A-K , HARLEY J B , KRONELD U , JONSSON R
Published: March 2003

The aim of this study was to investigate the production of anti-Ro/SS-A antibodies in labial salivary glands (LSG) and peripheral blood (PB) of Sjögren's syndrome (SS) patients. The ELISPOT method was performed to quantify the frequency of LSG lymphocytes and PB lymphocytes spontaneously secreting anti-Ro/SS-A antibodies. The total number of IgG-, IgA- and IgM-producing cells was also quantified. The bovine Ro 60-kD protein was used as target antigen. Six of six primary SS patients had LSG B cells producing anti-bovine Ro 60 kD of the IgG isotype, and two of two primary SS patients had in addition PB lymphocytes producing anti-bovine Ro 60 kD of the IgG isotype. The six patients who had IgG antibodies against the Ro/SS-A antigen in LSG all had focus scores of ≥ 7 in biopsies of LSG. The results indicate that SS patients with a high degree of local inflammation in LSG have B cells producing anti-Ro/SS-A antibodies in both LSG and PB. Thus, the anti-Ro/SS-A antibodies may have pathogenic importance in the progression of the exocrinopathy of SS.
Molecular chaperones are targets of autoimmunity in Ro(SS-A) immune mice
Authors: KINOSHITA G , PURCELL A W , KEECH C L , FARRIS A D , McCLUSKEY J , GORDON T P
Published: March 2003

We have used a murine model of experimental anti-Ro(SS-A) autoimmunity to dissect additional intermolecular interactions between the 52-kD Ro (Ro52) and 60-kD Ro (Ro60) autoantigens and molecular chaperones. Immune responses to members of the heat shock protein hsp70 and hsp90 families were measured by immunoblotting and ELISA in sera from mice immunized and boosted with purified recombinant Ro52, Ro60 and La (SS-B). All Ro52 and Ro60 immune sera immunoblotted the inducible glucose-regulated protein grp78 and hsp70 species but not constitutive hsc70 or hsp90. The kinetics of antibody production and reciprocal affinity purification experiments indicated that the grp78 and hsp70 responses were cross-reactive but distinct from immune responses to the primary Ro52 and Ro60 immunogens and the endoplasmic reticulum (ER)-resident chaperone calreticulin. No responses to molecular chaperones were detected in the La-immunized mice. Control immunizations indicated that the recruited grp78 and hsp70 responses were specific for the Ro proteins and not due to immunization with denatured protein. The rapid spreading of immunity to the inducible grp78 and hsp70 in Ro52- and Ro60-immunized mice suggests that these components may co-localize and physically associate under certain physiological conditions which may promote autoimmunization. The potential importance of the ER-resident chaperones grp78 and calreticulin is further supported by their co-localization with Ro in small apoptotic membrane blebs and the finding of a novel putative grp78 binding motif in the carboxyl-terminal region of Ro52.
HLA-DMB expression by thyrocytes: indication of the antigen-processing and possible presenting capability of thyroid cells
Authors: WU Z , BIRO P A , MIRAKIAN R , HAMMOND L , CURCIO F , AMBESI-IMPIOMBATO F S , BOTTAZZO G F
Published: March 2003

Expression of HLA class II molecules on thyrocytes is a characteristic feature of autoimmune thyroid disease and may lead the thyroid cells to present autoantigens to CD4+ T lymphocytes. Since HLA-DM is a critical molecule in class II-restricted antigen processing and presentation, we assessed the expression of HLA-DMB, -invariant chain (Ii), class II transactivator (CIITA) and DRA in an untransformed, pure thyrocyte strain HTV-59A. Here we report that both HLA-DMB mRNA and the protein are expressed in thyrocytes and that CIITA expression is enhanced by interferon-gamma (IFN-γ) treatment and occurs before DMB, Ii and DRA up-regulation, suggesting CIITA expression is a requirement for antigen processing in thyrocytes. These results indicate that thyrocytes are capable of antigen processing and possibly antigen presentation to T cells.
Active suppression in orally tolerized rats coincides within situtransforming growth factor-beta (TGF-β) expression in the draining lymph nodes
Authors: LUNDIN B S , KARLSSON M R , SVENSSON L A , HANSON L Å , DAHLGREN U I H , TELEMO E
Published: March 2003

Adult rats were fed pellets containing ovalbumin (OvA) during 4 weeks, and were 2 weeks thereafter immunized subcutaneously with a mixture of OvA and human serum albumin (HSA) in Freund's complete adjuvant (day 0). As a result of the immunization, the draining lymph nodes of the non-tolerized (control) rats were heavily enlarged from day 10 to day 18; however, this size increase was absent in the OvA-fed rats. This manifestation of active suppression in the tolerized rats was preceded by the appearance of scattered CD4+ TGF-β-expressing T cells in the T cell area of their lymph nodes (days 5–8); correspondingly, the levels of TGF-β mRNA in the nodes were elevated in the tolerant rats compared with the control rats. The anti-OvA antibody levels in sera from the rats revealed that there was an initial B cell priming in the OvA-fed group, with levels higher than in the control group during the first week. Thereafter, suppression governed the response, and from day 10 onwards the anti-OvA levels were considerably lower than in the controls. When other groups of animals were pretreated with neutralizing anti-TGF-β antibodies 1 day before the immunization, the anti-OvA response of the OvA-fed rats was restored to the levels of the control group, demonstrating the importance of TGF-β in the maintenance of suppression. In conclusion, we demonstrate that TGF-β-producing cells appear in the draining lymph nodes shortly after immunization in rats made orally tolerant using a relatively high-dose feeding regime; these cells are probably responsible for the down-regulation of the immune response observed in the OvA-fed rats.
AsymptomaticBorrelia-seropositive individuals display the same incidence ofBorrelia-specific interferon-gamma (IFN-γ)-secreting cells in blood as patients with clinicalBorreliainfection
Authors: EKERFELT C , FORSBERG P , SVENVIK M , ROBERG M , BERGSTRÖM S , ERNERUDH J
Published: March 2003

Borrelia Lyme disease is a complex disorder that sometimes becomes chronic. There are contradictory reports of experimental Borrelia infections regarding which type of T cell cytokine responses, i.e. Th1 or Th2, are needed to eradicate the Borrelia spirochaetes. In human borreliosis a predominance of Borrelia-specific Th1-like responses has been shown. In this study, spontaneous, as well as Borrelia-specific, secretion of IFN-γ (Th1) and IL-4 (Th2) in Borrelia-seropositive healthy asymptomatic individuals (n = 17) was investigated in peripheral blood by a sensitive ELISPOT assay, and compared with previously reported responses in patients with clinical Borrelia infection (n = 25). The seropositive asymptomatic individuals displayed the same predominance of Borrelia-specific IFN-γ-secreting cells as the patients with clinical Borrelia infection. Interestingly, the proportion of spontaneously IL-4-secreting cells, reflecting the unstimulated in vivo secretion, was lower in the seropositive asymptomatic individuals compared with patients with chronic Borrelia infections (n = 13, P = 0.02), whereas no such difference was found compared with subacute Borrelia infections (n = 12). These findings indicate that IFN-γ secretion alone is not sufficient to eliminate Borrelia spirochaetes in humans, although IFN-γ may still have a beneficial role in borreliosis acting in concert with other mechanisms.
Serum cytokine detection in the clinical follow up of patients with cystic echinococcosis
Authors: RIGANÒ R , PROFUMO E , IOPPOLO S , NOTARGIACOMO S , TEGGI A , SIRACUSANO A
Published: March 2003

The relation of serum cytokine levels and outcome of chemotherapy was evaluated in 15 patients with cystic echinococcosis. Serum IL-4, IL-10 and interferon-gamma (IFN-γ) concentrations were determined by ELISA before and after a 3-month course of albendazole treatment: at least one serum sample per patient from 13 patients (87%) contained measurable amounts of IL-4; samples from five patients (33%) measurable amounts of IL-10 and samples from only two patients (13%) measurable amounts of IFN-γ. Clinical assessment at 1 year after the end of therapy showed that 11 of the 15 patients had responded clinically. Seven of these patients had lower IL-4 serum concentrations, two had unchanged and two undetectable amounts (pre- versus post-therapy, n = 11, P = 0.008). Conversely, of the patients who did not respond, three had higher and one patient unchanged serum IL-4 concentrations. Serum IL-10 levels also decreased in all patients who responded (3/5) and increased in all patients who did not (2/5). No association was found between cytokine concentrations and cyst characteristics or antibody levels. Overall these data suggest that serum IL-4 detection may be useful in the follow up of patients with cystic echinococcosis.
Leishmanin skin test lymphoproliferative responses and cytokine production after symptomatic or asymptomaticLeishmania majorinfection in Tunisia
Authors: SASSI A , LOUZIR H , BEN SALAH A , MOKNI M , BEN OSMAN A , DELLAGI K
Published: March 2003

Resistance to Leishmania parasite infection requires the development of a cellular immune response that activates macrophage leishmanicidal activity. In this study we have investigated the lymphoproliferative responses and in vitro cytokine production of peripheral blood mononuclear cells (PBMC) from individuals living in an endemic area for L. major infection in Tunisia. The results were compared with the DTH reaction of the leishmanin skin test (LST). Sixty-seven individuals were included in the study: 22 persons (age range 9–60 years) who developed, 2 years before the present study, a parasitologically confirmed localized cutaneous leishmaniasis (LCL) that healed spontaneously, and 45 individuals (age range 18–20 years) born and living in the same area, with no previous history of LCL. LST was positive (skin induration ≥ 5 mm) in 20/22 cured cases of LCL and in 75% of healthy individuals without history of LCL. LST+ individuals expressed vigorous Leishmania-specific lymphoproliferative responses associated with in vitro production of interferon-gamma (IFN-γ) but not IL-4. Interestingly, IL-10 was detected in parallel with the highest levels of IFN-γ in PBMC supernatants from 3/20 cured LCL and 8/25 individuals without history of LCL. Our results showed a 98% concordance between the DTH reaction assessed by LST and the in vitro proliferative assay induced by soluble leishmanial antigens. Moreover, proliferative assays as well as cytokine analysis did not show any significant difference of the immune memory to parasite antigens developed by patients who had overt cutaneous leishmaniasis and those who had apparently asymptomatic infection.
Anti-endothelial cell antibodies in systemic vasculitis and systemic lupus erythematosus (SLE): effects of heat inactivation on binding and specificity
Authors: D'CRUZ D P , KESER G , DIRESKENELI H , KHAMASHTA M A , HUGHES G R V
Published: March 2003

Heating sera is used to inactivate complement but may affect the binding characteristics of autoantibodies. We studied the effect of heating sera from patients with systemic vasculitides and SLE on antibody binding to cultured human umbilical vein endothelial cells. Sera from 32 patients with systemic vasculitides, eight with SLE and 10 healthy controls were studied for anti-endothelial cell antibodies (AECA) using an ELISA before and after heating sera to 56°C for 30 min. The median (range) AECA binding index in the patient group increased from 20% (0–153%) to 71.5% (10–259%) (P < 0.0001). The AECA binding index in the control group also increased from 14% (0–52%) to 90% (42–154%) (P < 0.0001). The increased binding was unaffected by the addition of fresh complement or removal of immune complexes and the increased binding after heating persisted even after cooling to 4°C. Specificity experiments showed that after heating, the binding specificity of sera was lost. Removal of immunoglobulin with Protein A abolished the increased binding seen after heating. Heating sera increases AECA binding in both patient and control sera. The mechanism is probably non-specific damage to the immunoglobulin molecule, and heating sera should thus be avoided.
Apoptosis ofMycobacterium avium-infected macrophages is mediated by both tumour necrosis factor (TNF) and Fas, and involves the activation of caspases
Authors: BERMUDEZ L E , PARKER A , PETROFSKY M
Published: March 2003

Mycobacterium avium causes disseminated infection in AIDS patients and several forms of infection in immunocompetent hosts. Recent studies have shown that M. avium infection of macrophages in vitro leads to apoptosis of significant numbers of infected cells. Several strains of M. avium used to infect human macrophages for 5 days (multiplicity of infection of 10) triggered 28–46% higher levels of apoptosis than observed with uninfected macrophages at the same time points. Mycobacterium avium strains unable to replicate intracellularly (rep−) resulted in a 15% rate of apoptosis, while M. smegmatis-infected monolayers showed the same percentage of apoptotic cells as the uninfected macrophage control. The presence of anti-TNF-α antibody reduced apoptosis to 17% and the presence of anti-Fas antibody reduced apoptosis to 10%. When both antibodies were used together, the apoptosis level was 5% above the control. Treatment with TGF-β also reduced the number of apoptotic cells in infected monolayers. If intracellular growth was inhibited, apoptosis of macrophages decreased significantly. It was also shown that apoptosis was associated with IL-1β-converting enzyme (ICE) activation and was significantly reduced by a caspase inhibitor. Gaining understanding of the mechanisms of M. avium-associated apoptosis of macrophages will provide important insight into M. avium pathogenesis.
Disease-associated autoantibodies and HLA-DQB1 genotypes in children with newly diagnosed insulin-dependent diabetes mellitus (IDDM)
Authors:   THE CHILDHOOD DIABETES IN FINLAND STUDY GROUP, SABBAH E , SAVOLA K , KULMALA P , REIJONEN H , VEIJOLA R , VÄHÄSALO P , KARJALAINEN J , ILONEN J , ÅKERBLOM H K , KNIP M
Published: March 2003

The possible relation between HLA-DQ genotypes and both frequencies and levels of autoantibodies associated with IDDM was assessed by examining HLA-DQB1 alleles and antibodies to islet cells (ICA), insulin (IAA), glutamic acid decarboxylase (GADA) and the protein tyrosine phosphatase-related IA-2 molecule (IA-2A) in 631 newly diagnosed diabetic children under the age of 15 years. ICA were found in 530 children (84.0%), while close to half of the subjects (n = 307; 48.7%) tested positive for IAA. GADA were detected in 461 index cases (73.1%), with a higher frequency in those older than 10 years (78.9% versus 69.2% in the younger ones; P = 0.006). More than 85% of the children (n = 541; 85.7%) tested positive for IA-2A. Altogether there were only 11 children (1.7%) who had no detectable autoantibodies at diagnosis. There were no differences in the prevalence of ICA or GADA between four groups formed according to their HLA-DQB1 genotype (DQB1*0302/02, *0302/X (X = other than *02), *02/Y (Y = other than *0302) and other DQB1 genotypes). The children with the *0302/X genotype had a higher frequency of IA-2A and IAA than those carrying the *02/Y genotype (93.8% versus 67.3%, P < 0.001; and 49.0% versus 33.6%, P = 0.002, respectively). The children with the *02/Y genotype had the highest GADA levels (median 36.2 relative units (RU) versus 14.9 RU in those with *0302/X; P = 0.005). Serum levels of IA-2A and IAA were increased among subjects carrying the *0302/X genotype (median 76.1 RU versus 1.6 RU, P = 0.001; and 50 nU/ml versus 36 nU/ml, P = 0.004) compared with those positive for *02/Y. Only three out of 11 subjects homozygous for *02 (27.3%) tested positive for IA-2A, and they had particularly low IA-2A (median 0.23 RU versus 47.6 RU in the other subjects; P < 0.001). The distribution of HLA-DQB1 genotypes among autoantibody-negative children was similar to that in the other patients. These results show that DQB1*0302, the most important single IDDM susceptibility allele, is associated with a strong antibody response to IA-2 and insulin, while GAD-specific humoral autoimmunity is linked to the *02 allele, in common with a series of other autoimmune diseases as well as IDDM. We suggest that IA-2A may represent β cell-specific autoimmunity, while GADA may represent a propensity to general autoimmunity.
B and T cell immunity in patients with lysinuric protein intolerance
Authors: LUKKARINEN M , PARTO K , RUUSKANEN O , VAINIO O , KÄYHTY H , ÖLANDER R M , SIMELL O
Published: March 2003

Lysinuric protein intolerance (LPI) is characterized by defective cellular transport of the dibasic amino acids, secondary dysfunction of the urea cycle, aversion to dietary protein, failure to thrive, hepatosplenomegaly and osteoporosis. Because several patients have suffered from recurrent respiratory infections and/or severe generalized varicella, and a few have developed systemic lupus, vasculitis or other autoimmune diseases, we have now evaluated the function of patients' immune systems. Serum concentrations of one to three IgG subclasses were decreased in 10 of the 12 patients studied. Antibody titres against diphtheria, tetanus and Haemophilus influenzae (Hib) were below the detection limit of the assay in four, three and eight of the 11 patients examined, respectively. (Re)vaccination of these 11 patients led to satisfactory responses against tetanus, but two patients still failed to develop measurable antibodies against diphtheria, two against Hib and six against one or more of the three serotypes of 23-valent pneumococcus vaccine. The proportions of T cells of all lymphocytes and the proliferative responses of the peripheral blood mononuclear cells were normal. In conclusion, humoral immune responses in some patients with LPI are defective and these patients may benefit from intravenous immunoglobulin therapy.
Chemokine expression by subchondral bone marrow stromal cells isolated from osteoarthritis (OA) and rheumatoid arthritis (RA) patients
Authors: LISIGNOLI G , TONEGUZZI S , POZZI C , PIACENTINI A , GRASSI F , FERRUZZI A , GUALTIERI G , FACCHINI A
Published: March 2003

We analysed the spontaneous and cytokine-stimulated production and expression in vitro of IL-8, GROα, MCP-1, RANTES, MIP-1α, MIP-1β, by subchondral bone marrow stromal cells (BMSC) isolated from RA, OA, post-traumatic (PT) patients and normal donors (ND). BMSC were cultured in vitro in the presence or absence of IL-1β and tumour necrosis factor-alpha (TNF-α), and assessed for chemokine production, expression and immunolocalization. BMSC from different sources constitutively released MCP-1, GROα and IL-8, but not MIP-1α or MIP-1β, while BMSC from ND constitutively released only IL-8 and MCP-1. IL-8, GROα and RANTES production in basal conditions was significantly higher in RA patients than in ND. RANTES production was also higher in OA and RA than in PT patients. The combination of TNF-α and IL-1β synergistically increased the production of all chemokines tested except for RANTES. Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that all chemokines not detectable in the supernatants were expressed at the mRNA level. Chemokine immunostaining was localized around the nuclei. This work demonstrates that BMSC from subchondral bone produce chemokines and indicates that these cells could actively participate in the mechanisms directly or indirectly causing cartilage destruction and bone remodelling.
Diminished IL-2 responses and alteration of CD2 expression on CD8+ T cells are associated with a lack of cytotoxic T cell responses during Theileria annulata infection
Authors: NICHANI A K , CRAIGMILE S C , SPOONER R L , CAMPBELL J D M
Published: March 2003

Theileria annulata is a tick-borne protozoan parasite which causes the disease bovine tropical theileriosis. In immunized or drug-treated animals, the pathogenic macroschizont stage of the parasite is destroyed by MHC class I-restricted cytotoxic T lymphocytes (CTL). Here we show that although CD8+ T cells increase greatly in number and display activation markers during an acute infection, they exhibit no killing of infected cells. During the ineffectual response, efferent lymph cells' ability to proliferate to IL-2 drops, coinciding with loss of MoAb binding to CD2 by CD8+ cells. When animals were treated with the anti-parasite drug ‘Butalex’, IL-2 responses, anti-CD2 antibody binding by CD8+ cells and strong CTL activity were restored within 24 h. The initial activation of CD4+ T cells by parasite-infected cells altering the IL-2 production in the draining lymph node is the likely cause of the failure of CTL responses.
Decreased interferon-gamma (IFN-γ)-producing T cells in patients with acute Kawasaki disease
Authors: MATSUBARA T , KATAYAMA K , MATSUOKA T , FUJIWARA M , KOGA M , FURUKAWA S
Published: March 2003

Kawasaki disease (KD) is an acute febrile illness of early childhood, in which the activation of monocytes/macrophages plays a central role in the development of vasculitis during the acute stage of disease. In this study we investigated peripheral blood T cells of 10 patients with KD, focusing on the Th1 and Th2 imbalance, using intracellular cytokine staining and analysis of the cytokine-producing T cells by flow cytometry. We observed a decrease in the numbers of IFN-γ-producing, but not IL-4-producing, CD3+ T cells, during the acute stage. Our results suggest that there is an imbalance of Th1 and Th2 subsets during the acute stage of KD.
Soluble tumour necrosis factor (TNF)-receptor levels in serum as markers of anti-viral host reactivity
Authors: BARTHOLDY C , NANSEN A , MARKER O , THOMSEN A R
Published: March 2003

The role of soluble receptors for TNF-α (sTNF-Rs) as markers of virus-induced host responses was studied by the use of murine model infections. A marked elevation in serum levels of sTNF-R75, but not sTNF-R55, was found 1 day after infection with vesicular stomatitis virus (VSV). In mice infected with lymphocytic choriomeningitis virus (LCMV), an early increase was also revealed, but peak levels of sTNF-R75 were observed later temporally related to maximal T cell-mediated anti-viral activity. Analysing different well characterized knockout mice, it was found that elevated release of sTNF-R75 into serum early after VSV infection was independent of T cells, whereas interferon (IFN)-α/β seemed to be a major mediator. In contrast, increased release of sTNF-R75 into serum 8 days post-LCMV infection was mediated via T cells but independently of both CD40 ligand and IFN-γ. A simple correlation between release of sTNF-Rs in vivo and macrophage activation in vitro was not present. These findings indicate that sTNF-R75 is indeed a sensitive marker of both innate and specific cell-mediated host reactivity during viral infection, but it is not correlated to a single immunological parameter.
Sex-limited protein:in vitroandin vivofunctions
Authors: BEURSKENS F J M , KUENEN J D M , HOFHUIS F , FLUIT A C , ROBINS D M , VAN DIJK H
Published: March 2003

Mouse complement component C4 exists in two isoforms, C4 and a protein with expression restricted to male animals called sex-limited protein (Slp). Although Slp is about 95% homologous to C4, it is generally believed to be non-functional, at least in conventional haemolytic complement assays. In a previous study, however, we showed that Slp is haemolytically active in a C1-inhibitor (C1INH)-regulated, EDTA-resistant mouse complement activation pathway. To study other possible implications of this finding, we generated constitutively expressing Slp-transgenic mice. The transgene was crossed into otherwise Slp-deficient C57Bl/6J and NZB mice. Members of the third backcross generation of C57Bl/6J mice were tested for functional Slp and classical and alternative complement pathway activities (CH50 and AP50 levels, respectively). Slp-transgenic C57Bl/6J mice showed enhanced CH50, but normal AP50 levels when compared with non-transgenic littermates. To discover a possible protective role for Slp in spontaneous systemic lupus erythematosus (SLE) in NZB×NZW (NZB×W) mice, the third backcross generation of Slp-transgenic NZB mice was mated with NZW mice and the development of SLE in the female offspring was followed. In these introductory experiments, Slp-transgenic NZB×W animals presented with a significantly extended life span. Our results imply that Slp is a mouse complement component with functions which partially resemble some of those of human C4A.
Impaired expression of MHC class II molecules in response to interferon-gamma (IFN-γ) on human thymoma neoplastic epithelial cells
Authors: INOUE M , OKUMURA M , MIYOSHI S , SHIONO H , FUKUHARA K , KADOTA Y , SHIRAKURA R , MATSUDA H
Published: March 2003

A human thymoma is a neoplasm derived from the thymic epithelial cell, and is well known for its association with autoimmune diseases, especially myasthenia gravis. The neoplastic epithelial cells of thymoma clearly retain thymic epithelial functions, but the development of T cells in thymoma is somewhat impaired. In this study, we quantified by flow cytometry the in vitro expression of MHC molecules on neoplastic epithelial cells precultured with IFN-γ. While MHC class I expression was comparable with that on normal thymic epithelial cells, the level of MHC class II molecules on neoplastic epithelial cells was lower than in controls, and also varied greatly from case to case. Additionally, there was a significant positive correlation between the expression level of MHC class II and the proportion of mature CD3+ cells in the CD4+CD8− subset. Thus, accumulation of CD3−CD4+CD8− cells in thymoma may result from impaired expression of the MHC class II molecules, suggesting that the function of the neoplastic epithelial cells might determine the maturation and the positively selected repertoire of T cells in thymomas.
Analysis of human C4A and C4B binding to an immune complex in serum
Authors: REILLY B D
Published: March 2003

Previous studies using isolated complement proteins have shown that more C4A than C4B binds to certain types of immune complexes. However, the in vivo binding of the C4 isoforms to an immune complex has not been investigated in detail and may differ from events when measured with the isolated proteins. We report here the binding of C4A and C4B to an immune complex of bovine serum albumin (BSA) anti-BSA as it occurs in serum. We found that when using the isolated C4 proteins more C4A than C4B bound to the complex, but in serum similar amounts of C4A and C4B were found to bind. Furthermore, these results were not explainable by a difference in activity between isoforms. In an attempt to explain these results a number of unexpected observations were noted. First C4A, but not C4B, bound specifically to a yet unidentified 38-kD serum protein. Second, when both covalent and non-covalent binding was assessed, we found that as serum concentration increased there followed a concomitant decrease in covalent binding and C4B was more affected than C4A. The potential biological significance of these findings is discussed.