The complement system is old, yet it may still have something new to teach us. For many years, research has existed which shows that C3d, in addition to its established role as an adjuvant, could have an immunosuppressive activity. Being true, it suggests that a common mechanism may be used both by organisms and by their pathogens to prevent unwanted immune responses.
The objective was to investigate the frequency of human leucocyte antigen (HLA)-E alleles in Egyptian women with and without recurrent miscarriage (RM) to evaluate their role on the maintenance of pregnancy. A case-control study was adopted. HLA-E gene polymorphism typing was carried out by restriction fragment length polymorphism for 108 women with RM and 120 fertile female controls. The frequency of HLA-E *0101 allele was higher in patients with RM and HLA-E*0103 allele was higher in fertile controls, and the difference was statistically significant (P=0.003, P(c)=0.006). HLA-E*0101/0101 genotype was the most frequent genotype in patients (45.4%), followed by HLA-E*0101/0103 (44.4%) and finally HLA-E*0103/0103 genotype (10.2%). The difference in the frequency of HLA-E*0101/0101 homozygous genotype in patients with RM compared with that in the fertile controls was statistically significant (OR=2.02, 95% CI=1.13-3.62, P=0.011, P(c)=0.033). We found an increased frequency of homozygosity for HLA-E*0101 in Egyptian women with RM. HLA-E*0101 homozygosity may thus be a risk factor for RM.
Mutated oncogene peptides may be presented to T cells by HLA molecules. To be able to design the optimal peptides for stimulation of T cells in individuals with different HLA molecules, it is important to analyse the binding characteristics of oncogene peptides to HLA. HLA-DQ6 (DQ(alpha 1*0102,beta 1*0602)) and HLA-DR1 (DR(alpha,beta 1*0101)) molecules were purified from lysates of homozygous EBV-transformed cell lines. Purified HLA molecules were then tested for their ability to bind synthetic peptides in gel filtration assays. A p21 ras oncogene peptide (previously found to stimulate DQ6-restricted T-cell clones) and an influenza matrix peptide were labelled with 125I and served as indicator peptides for binding to DQ6 and DR1 respectively. Binding of homologous truncated and mutated p21 ras peptides and unrelated peptides was then evaluated by their capacity to inhibit binding of the indicator peptides. p21 ras-derived peptides were found to bind to both DQ6 and DR1 molecules indicating the existence of a promiscuous binding motif in these peptides. The binding affinities seemed to vary between the different peptides, but the amino acid substitutions resulting from natural mutations were not critical for binding. Notably, the results obtained for DQ6 in the biochemical peptide binding assay correlated well with results obtained in a functional assay using T-cell clones as probes.
Levels of nonantigen-induced pro-inflammatory cytokines and prostaglandin in macrophages isolated from human leucocyte antigen (HLA)-matched type 1 diabetes mellitus patients, first-degree relatives and healthy controls were determined. We hypothesize that monocytes isolated from patients are sensitized or preactivated and therefore, have an altered response to in vitro stimulus compared with control groups as measured by levels of pro- and anti-inflammatory mediators. In this study, peripheral blood monocytes were differentiated to macrophages with macrophage-colony stimulating factor (M-CSF) to determine lipopolysaccharide (LPS)-stimulated tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-12 and prostaglandin E-2 (PGE-2) secretion from hetero- or homozygous HLA DQB1*0201 and *0302 type 1 diabetes mellitus patients, first-degree relatives and homozygous HLA DQB1*0602 healthy controls. LPS-stimulated secretion of TNF-alpha, IL-1beta and IL-6 was immediate and markedly higher in the HLA-DQB1*0201/*0302 type 1 diabetes patients compared with all other groups including HLA-matched healthy first-degree relatives. In DQB1*0201/*0302 diabetes patients PGE-2 secretion was delayed but increased by LPS stimulation compared with HLA-matched healthy relatives. IL-12 was not detected at any condition. These data suggest that macrophages from DQB1*0201/*0302 type 1 diabetes patients are sensitized to secrete both cytokines and PGE-2 following nonantigenic stimulation. Sensitized macrophages may be important to high-risk DQB1*0201/*0302-associated type 1 diabetes.
CD8+ T cells are thought to play an important role in protective immunity against tuberculosis. We report the identification of three peptides derived from Rv1818c, Rv3812 and Rv3018c proteins of Mycobacterium tuberculosis that bound to HLA-A*0201 molecules and their ability to induce in vitro T-cell response in peripheral blood lymphocytes from HLA-A*0201-positive healthy individuals (PPD+) and patients with TB. The peptide-specific cytotoxic T lymphocytes (CTL) generated were capable of recognizing peptide pulsed targets. Three 9-mer peptides bound with high affinity to HLA-A*0201 and displayed low dissociation rates of the bound peptide from HLA. Epitope-specific recognition was demonstrated by the release of perforin and gamma-interferon. Overall, our results demonstrate the presence of HLA class I-restricted CD8+ CTL against proteins from PE and PPE proteins of M. tuberculosis and identify epitopes that are strongly recognized by HLA-A*0201-restricted CD8+ T cells in humans. These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.
CMVpp65, a candidate component of human cytomegalovirus (CMV) vaccines, has phosphokinase (PK) activity that could affect vaccine safety. A mutated form of CMVpp65 substituting asparagine for lysine at the adenosine triphosphate (ATP)-binding site (CMVpp65mII) is kinase-deficient. Using DNA immunizations in a transgenic human leucocyte antigen (HLA)A*0201.Kb mouse model, the mutated CMVpp65 induced cytotoxic T lymphocytes (CTL) immunity similarly to native CMVpp65. Murine CTL lines generated from these immunizations killed human cells either after sensitization with CMVpp65-specific peptides or after infection with either CMV-Towne strain or rvac-pp65. It is proposed that CMVpp65mII be evaluated in candidate vaccines for CMV.
The objective of this study was to investigate human leucocyte antigen (HLA) genes in patients chronically infected with hepatitis C virus (HCV) and to analyse the possible role of these genes in the progression of chronic hepatitis C. One hundred and forty-five (145) Brazilian patients infected only with HCV genotype 1 were evaluated. HLA class I (A*, B*, C*) and class II (DRB1*, DQA1*, DQB1*) typing were carried out by PCR-SSO, through Luminex technology. Associations were found with protection against development of liver damage by both DRB1*11 (5.0% versus 18.2%, P = 0.0016, OR = 0.23, CI 95% = 0.09-0.58; Pc=0.0208) and DRB1*11-DQA1*05-DQB1*03 haplotype (4.2% versus 15.3%, P = 0.0032; OR = 0.24, CI 95% = 0.08-0.64). Liver damage was associated with HLA-C*04 in patients with <20 years of infection (38.4% versus 9.1%, P = 0.002, OR = 6.25, CI 95% = 1.97-19.7; Pc=0.0238). It is concluded that HLA alleles can influence the development of liver damage in HCV type-1 chronically infected Brazilian patients.
The kinetic profile of cytokine gene expression in normal human peripheral mononuclear cells (MNC) activated by an anti-CD3 monoclonal antibody was studied. The presence or absence of 10 different cytokine mRNA were measured in a polymerase chain reaction (PCR) assisted mRNA amplification assay. After 2 h of stimulation the mRNA for interleukin-1 alpha (IL-1 alpha), interleukin-2 (IL-2), interleukin-3 (IL-3), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were detectable and remained present during the whole time period studied (22 h). Interleukin-6 (IL-6) and granulocyte macrophage colony stimulating factor (GM-CSF) were detected after 4 h, while interleukin-10 (IL-10) mRNA did not appear until after 7 h; they all remained expressed at 22 h. A transient expression of interleukin-4 (IL-4) mRNA was observed between 4 and 7 h of stimulation. No gene expression of granulocyte colony stimulating factor (G-CSF) was detected at any time. These results show that anti-CD3 stimulation of MNC leads to a rapid sequential induction of different cytokine mRNA, some with a very transient expression.
Myasthenia gravis (MG) and its animal model experimental autoimmune MG (EAMG), are T-cell dependent, antibody-mediated autoimmune disorders. A dual altered peptide ligand (APL) composed of the tandemly arranged two single amino acids analogs of two myasthenogenic peptides, p195-212 and p259-271, was demonstrated to downregulate, in vitro and in vivo, MG-associated autoimmune responses. Upregulation of regulatory CD4(+)CD25(+) cells plays a key role in the mechanism of action of the dual APL. The objectives of the present study were to address the involvement of extracellular-regulated kinase (ERK)1,2 in the mechanisms by which the dual APL-induced CD4(+)CD25(+) cells suppress MG-associated autoimmune responses. We demonstrate here that administration of the dual APL increased activated ERK1,2 in the CD4(+)CD25(+)-enriched population. Further, inhibition of ERK1,2 by its inhibitor, U0126, in dual APL-induced CD4(+)CD25(+) cells, abrogated their ability to suppress interferon (IFN)-gamma secretion by lymph node (LN) cells of mice that were immunized with the myasthenogenic peptide. Moreover, inhibition of ERK1,2 in the dual APL-induced regulatory CD4(+)CD25(+) cells, resulted in downregulation of the forkhead box p3 (Foxp3) gene and protein expression levels, as well as in the downregulation of CD4(+)CD25(+) development, suggesting that the active suppression exerted by the dual APL via CD4(+)CD25(+) cells depends on ERK1,2 activity.
The active metabolite of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3), was tested for the ability to differentiate promyelocytic leukaemia cells (HL-60) and histiocytic lymphoma cells (U937) into cytotoxic effector cells against K562 leukaemia cells. A concentration of 10(-6) M 1,25(OH)2D3 differentiated HL-60 cells into substrate-adherent monocyte-like cells with cytolytic activity against antibody-coated K562 cells. These differentiated HL-60 cells were not able to lyse uncoated K562 cells in an 18-h Cr-release assay. Similar treatment of the U937 cells with 1,25(OH)2D3 did not make them cytolytic towards K562 cells. These data indicate that 1,25(OH)2D3 can differentiate HL-60 cells to mature monocytes with cytolytic activity against antibody-coated leukaemia cells.
Excessive activation of nuclear transcription factor-κB (NF-κB) is involved in human airway smooth muscle cells (HASMCs) activities in asthma. We investigated the effects of 1,25 - dihydroxyvitamin D3 [1,25 - (OH) 2D3] on the NF- κB signaling pathway in passively sensitized HASMCs and the molecular mechanisms involved. HASMCs were treated with either healthy controls' serum, asthma patients' serum, or pre-treated with 1,25 - (OH) 2D3 prior to treatment with asthmatics' serum. At 1 h after serum treatment: electrophoretic mobility shift assay (EMSA) was used to detect NF-κB DNA binding activity; immunocytochemical staining was used to observe the nuclear translocation of NF-κB p65; Western blots were used for NF-κB p65, IκBα, and phospho-IκBα protein levels and the nuclear translocation of NF-κB p65; real-time quantitative PCR was used for NF-κB p65 and IκBα mRNA expressions; and actinomycin D treatment was used to determine IκBα mRNA stability. Our major findings were: (1) 1,25 - (OH) 2D3 significantly reduced asthma serum passively sensitized HASMCs NF-κB DNA binding activity and inhibited the nuclear translocation of NF-κB p65; (2) 1,25 - (OH) 2D3 increased the stability of IκBα mRNA with reduced IκBα phosphorylation in asthma serum passively sensitized HASMCs and significantly increased IκBα expression in these HASMCs. Inhibiting NF-κB signaling with 1,25 - dihydroxyvitamin D3 may be a therapeutic approach for controlling HASMC-related remodeling in asthma.
The influences of pretreatment with beta-1,3-D-polyglucose derivatives on levels of cytokines and arachidonic acid metabolites in body fluids in experimental peritonitis in mice are reported. Peritonitis was induced by an intraperitoneal injection of 10(8) live Escherichia coli. Pretreated animals survived the infection, untreated animals died about 12 h after inoculation with E. coli. Levels of IL-1 in plasma and peritoneal fluid, measured by cytotoxicity assay of the HT-2 cell line, increased significantly during the first 48 h after intraperitoneal treatment with beta-1,3-D-polyglucose-derivatized microbeads (GDM) or soluble, aminated beta-1,3-D-polyglucose (AG). After subsequent challenge with E. coli, the levels of IL-1 were significantly lower than in untreated animals. There was no increase in levels of TNF after treatment with GDM or AG, measured by cytotoxicity assay of the WEHI clone 13 cell line. After challenge with E. coli, TNF in plasma and peritoneal fluid was significantly lower compared with untreated animals. Both PGE2 and LTB4, measured by radioimmunoassay kits, were increased in peritoneal fluid after treatment with GDM and AG. After challenge with E. coli, PGE2 and LTB4 in peritoneal fluid increased to about half the concentration of infected control animals. Intraperitoneal injection of indomethacin to pretreated animals resulted in increased levels of IL-1 and TNF and decreased levels of PGE2 following challenge with E. coli. The levels of IL-1 and TNF remained elevated until the animals died after about 12 h. These studies demonstrate that the raised levels of arachidonic acid metabolites after pretreatment with GDM or AG seem to inhibit the otherwise lethal elevation of IL-1 and TNF in body fluids which is seen in untreated animals.
Intraperitoneal injection of β-1,3-D-glucan coupled to the surface of monodisperse methacrylate microbeads improves the resistence against bacterial infections in mice, while methacrylate microbeads alone do not. The effect of the glucan-derivatized microbeads (GDM) is considered lo be mediated through peritoneal macrophages [34, 40].
We show that both GDM and (he underivatized methacrylate microbeads (UDM) treated with normal serum were rapidly bound and phagocytozed by mouse peritoneal macrophages in vitro. We found that both complement and fibronectin opsonized the bead.-; and were responsible for the uptake.
Treatment of microbeads with serum lacking fibronectin and complement activity still gave BOOK uptake of GDM, but not uptake of UDM. The uptake of GDM was similar to the uptake of untreated GDM and was inhibited by pretreatment of macrophages with soluble β-1,3-D-gluean.
Our conclusion is that GDM and UDM intraperitoneally bind fibronectin and C3 through activation of the alternative pathway of complement. This leads to their phagocytosis by macrophages through fibronectin and complement receptors. GDM are also internalized via β-glucan receptors. We present the hypothesis that the β-glucan receptors on peritoneal macrophages account for the protective effect of GDM in intraperitoneal bacterial infections.
We have previously shown that soluble animated beta-1,3-D-glucan (AG) and glucan-derivatized microbeads (GDM) bind to the specific beta-glucan receptor on mouse peritoneal macrophages. Phagocytosis of GDM by macrophages is mediated through the beta-glucan receptor. IFN-gamma which increases macrophage phagocytic capacity, also increased the phagocytosis of GDM. In the present study we show that IFN-gamma inhibits internalization of AG in macrophages in a dose- and time-dependent manner. The inhibitory effect of IFN-gamma was neutralized by treatment of the macrophages with cycloheximide. These results were confirmed by confocal laser scanning microscopy which showed that IFN-gamma treated cells incorporated less fluorescein-labelled AG than did untreated cells. IFN-gamma did not change the macrophage-binding capacity for AG showing that the inhibitory effect of IFN-gamma is not caused by decreased number of beta-glucan receptors on the cells. The stimulatory effect of AG on IL-1 beta and TNF-alpha release from macrophages was reduced by pretreatment of the cells with IFN-gamma. We conclude that the uptake of AG and GDM in macrophages, both mediated through the beta-glucan receptor, are differently regulated by IFN-gamma. The reduced internalization of AG after IFN-gamma treatment of macrophages, is probably responsible for the down-regulation of IL-1 and TNF-alpha secretion.
The light (L) and heavy (H) chain and the idiotypic (Id) composition of the antibody (Ab) plaque-forming cells (PFC) and serum Ab specific for alpha-1,3 dextran have been characterized in murine strains exhibiting the CH-Ig-lb to e allotypes and in their F1 hybrids with Ig-la1, BALB/c prototypes. The Ab response of the Ig-lb to e mice to the alpha-1,3 dextran was low in the kappa (kappa) L chain class with only a minor, sporadic Ab in the lambda (lambda) L chain class discernible after prolonged immunization. Two of a total of sixty-eight C57Bl/6 Ig-lb mice, in a total of 318 Ig-lb to e mice tested, exhibited, in late responses, a significantly elevated Ab in the lambda L class at both serum and PFC levels, equalling at the PFC level the total non-specific lambda PFC values. An Id analysis showed this lambda Ab and the kappa Ab to lack the Id relatedness to the three BALB/c alpha-1,3 dextran-binding myeloma proteins (MP) Ab prototypes, J-558, 104 E, and UPC-102, exhibited by the lambda Id+ Ab of the Ig-la1 BALB/c prototypes and their F1 hybrids with the Ig-lb to e prototypes. Furthermore, affinity differences could be detected by alpha-1,3 nigerodextrans, PFC inhibition analysis, between the late C57Bl/6 anti-alpha-1,3 dextran lambda Id--Ab PFC and the lambda Id+ Ab PFC of the BALB/c and their F1 progeny.
The binding of a 34-kDa (mol. wt.) acylpoly(1,3)galactoside (APG) extracted from a membrane proteoglycan of Klebsiella pneumoniae to human blood leucocytes was investigated. APG is made of a long poly(1,3)galactose chain, a core-like region and a lipid moiety which comprises two glucosamine residues bound to a phosphate group and two beta OH myristic acids. Fluoresceinated APG was shown to bind preferentially to monocytes and to a lesser extent to polymorphonuclear neutrophils, as determined by flow cytometry. Binding of fluoresceinated APG was inhibited by unlabelled APG; it was concentration dependent, but not saturable, with rapid kinetics. It occurred at +4 degrees C but was markedly increased at 37 degrees C. It involved trypsin-sensitive molecules on the membrane of monocytes. Neither the parent proteoglycan nor lipopolysaccharide from K. pneumoniae or Salmonella minnesota competed for APG binding. A minor non-specific binding to lymphocytes, occurring predominantly on B cells, was observed. Unlike that of lipopolysaccharide, the APG binding was not blocked by polymyxin B sulphate. Interaction between the galactose chain of APG and the galactose receptor does not account for the binding of APG to monocytes because the galactose receptor (Mac-2) is expressed at high density on activated macrophages but not on monocytes. Despite its strong binding to human blood monocytes, APG displayed a much weaker activity than K. pneumoniae membrane proteoglycan with respect to induction of monocyte cytokine synthesis. When administered as a Technetium 99 conjugate, APG was shown to label inflammatory foci in experimental animals, and its property as a marker of macrophages is currently being evaluated in clinical trials.
Macrophages stimulated by an insoluble beta-1,3-D-glucan from yeast cell walls were able to destroy tumour cells as measured by the release of radioactive label from prelabelled 14C-thymidine cells. Target cells were B-16 melanoma, P-815 mastocytoma, and the L-929 cell line. A significant target cell killing by macrophages stimulated by glucan was observed after 72-96 h. The cytolysis of L-929 cells was investigated in some detail. No stable soluble cytolytic factor appeared to be released into the medium during the stimulation of macrophages by glucan, since cell-free spent medium had no cytotoxic effect on L-929 cells. The densities of the macrophage monolayers were critical for an effective target cell killing; dense cultures showed more cytotoxicity than less dense cultures. The kinetics of the development of macrophage-mediated cytotoxicity suggests a minimum stimulation period of 4 days for maximal cytolysis.
Human peritoneal macrophages were stimulated in vitro with beta-1,3-D-polyglucose-derivatized microbeads (GDM) or soluble aminated beta-1,3-D-polyglucose (AG) in combination with lipoproteins. The release of interleukin 1 (IL-1) was analysed in cell supernatants in a thymocyte proliferation assay. We report that the release of IL-1 is markedly enhanced in macrophages stimulated with polyglucose in either form in combination with native low-density lipoprotein (LDL) or acetyl LDL at a concentration of 100 micrograms/ml. By increasing the amount of lipoproteins up to 10-fold, the IL-1 release decreased sharply. There was only a slight increase in activity when high-density lipoprotein (HDL) or very low-density lipoprotein (VLDL) were added. Other stimulatory agents, such as gamma interferon (IFN-gamma) and lipopolysaccharide (LPS) showed about half the activity of polyglucose. There was no significant difference between native LDL and acetyl LDL in potentiating effect. Our observations also suggest that the potentiating effect of LDL or acetyl LDL is not dependent on binding to their specific receptors. These findings provide a connection between macrophages, lipoproteins, and cytokines with regard to their role in the inflammatory response.
The light (L) and heavy (H) chain and the antigenic, idiotypic (Id) composition of the antibody (Ab) plaque-forming cells (PFC) and serum Ab specific for alpha-1,3 dextran have been characterized in Ig-lal BALB/c prototype mice, in Ig-la-  mice, and in BALB/c congenic and recombinant strains. Four distinct Ab Id specificities were identified by using criteria of Id relatedness to three Id-distinct, alpha-1,3 dextran-binding BALB/c myeloma proteins (MP), all associated, in the Ig-lal mice with the lambda (lambda) chains: a major, common IdX in 40--90% of the molecules; three minor, individual IdI in 1--49% of the molecules; and a fifth, Id-undefined one. These specificities were expressed at the PFC and serum level in the mu and gamma isotypes. A minor, kappa (kappa), Id-negative Ab was discerned only at the PFC level. The Ig-la- CBA and C3H mice responded with anti-alpha-1,3 dextran, kappa Id-negative Ab. The BALB/c, congenic strain CB20 (BALB/c Ig-lb, carrying the C57Bl/Ka, Ig-lb variable H (VH) gene complement and CH phenotype, made anti-alpha-1,3 dextran kappaId-negative Ab of the Ig-lb prototype. The recombinant BAB/14, carrying in the C57Bl/Ka CH-Ig-lb phenotype the BALB/c Ig-lal VH gene(s) controlling anti-alpha-1,3 dextran lambda Ab responsiveness and Id specificity (VH-DEX+), express the BALB/c lambdaId repertoire.
Macrophages obtained from animals treated with beta-1,3-D-glucan-derivatized plastic beads were greatly stimulated, as judged by morphology, esterase release, and cytostatic effect on L-929 tumour cells in vitro. The pretreatment of mice with such beads conferred an apparent absolute local resistance to an otherwise lethal pneumococcal infection but had no effect on the growth of intraperitoneal AA ascites sarcoma. Moreover, peritoneal cells from animals pretreated with glucan beads did not protect the animals in a Winn assay.
Cramoll 1,4 is a lectin with specific glucose/mannose binding, which is extracted from seeds of Cratylia mollis Mart. Many assays have shown the cytokine expression activity and anti-inflammatory profile of this lectin. The aim of this study was to evaluate the immunostimulatory response, in vitro, of splenocytes in mice previously inoculated, in vivo, with C. mollis (Cramoll 1,4) and Canavalia ensiformis (Con A) lectins. Results demonstrated higher proliferation indexes induced by Cramoll 1,4 than Con A lectin in relation to all experimental groups. Cramoll 1,4 and Con A also induced high levels of IL-2, IL-6, IFN-γ and nitric oxide production. Moreover, Cramoll 1,4 did not induce apoptosis and stimulated a significant number of cells in the S phase of the cell cycle. Results showed that Cramoll 1,4 lectin induces proliferative response and suggested that this lectin can be used as a mitogenic agent in immunostimulatory assays.
Cockroaches have been identified as one of the major indoor allergens inducing perennial rhinitis and asthma. Per a 1s are a group of the major allergens from American cockroach. Although Per a 1s are major allergens from American cockroach, factors contributing to the allergenicity of Per a 1s are still poorly defined. To investigate the effects of Per a 1s on the expression of PARs and the release of proinflammatory cytokines from mast cells. Per a 1.0101 and Per a 1.0104 were cloned from American cockroach and then expressed in Eschericia coli. The purified allergens were used to stimulate P815 mast cells, and the expression of protease-activated receptors (PARs) was determined by real-time RT-PCR and flow cytometry. The levels of IL-4 and IL-13 in culture media were detected with ELISA. Sera from 80 and 77.3% of cockroach allergy patients reacted to recombinant Per a (rPer a) 1.0101 and rPer a 1.0104, confirming they are major allergens. Both rPer a 1.0101 and rPer a 1.0104 had no enzymatic activity, but rPer a 1.0101 upregulated the expression of PAR-1 and PAR-2, and rPer a 1.0104 enhanced the expression of PAR-1 and PAR-4 proteins. Both recombinant allergens were able to increase the release of IL-4 and IL-13 from P815 mast cells. This is the first study aiming to investigate functions of group 1 allergens of American cockroach. rPer a 1.0101 and rPer a 1.0104 have the capacity to upregulate the expression of PARs and to enhance Th2 cytokine production in mast cells.
The aim of this study was to investigate the frequency of the -1082 polymorphism of the interleukin-10 (IL-10) gene and the soluble IL-10 levels in Hungarian primary Sjögren's syndrome (SS) patients. Ninety-nine SS patients and 135 healthy volunteers were examined. Samples were analysed by the PCR restriction fragment length polymorphism method, and IL-10 plasma levels were assessed by a commercial enzyme-linked immunosorbent assay. IL-10 plasma levels were higher in the primary SS patients (36.4 +/- 57.5 pg/ml, n = 99) compared with the healthy subjects (9.9 +/- 20.3 pg/ml, n = 135, P = 10(-6)). The elevated IL-10 phenotype of SS patients was not associated with increased G allele frequency as reported earlier, while in the control group, we found higher IL-10 levels among the subjects who were carriers of the GG genotype (17.7 +/- 23.2 pg/ml) as compared with the other two genotype carriers (AA 8.98 +/- 16.5 and GA 8.5 +/- 21.1 pg/ml, P = 0.01). Our data do not support previous observations indicating an association between deregulated IL-10 secretion in SS and higher G allele frequency. However, the results clearly demonstrate that GG homozygosity is associated with elevated IL-10 levels in apparently healthy subjects, but this cannot account for the IL-10-related specific disease features observed in SS. Thus, other genetic factors contribute to the clinical spectrum of this heterogeneous disease at least in the Hungarian population.
The aim was to investigate the association of interferon-gamma (IFN-γ) +874 T/A and interleukin-10 (IL-10)-1082 A/G single nucleotide polymorphisms with tuberculous infection and post-BCG lymphadenitis in Egyptian children. IFN-γ +874 T/A and IL-10 -1082 A/G polymorphism detection by amplification refractory mutation system technique was carried out for 110 patients with TB, 40 patients with post-BCG lymphadenitis and 118 healthy controls. IFN-γ +874 A allele was higher in TB and post-BCG patients than those in healthy controls (Pc=0.006 and 0.002, respectively). IFN-γ +874 genotype AA was significantly higher in patients with TB than that in control (Pc=0.015), in extrapulmonary than patients with pulmonary TB (PTB) (Pc=0.009), and young children with TB below 5 years (Pc=0.024). No statistically significant differences were observed between patients with TB and controls for the frequency of IL-10(-1082) alleles or genotypes (P>0.05); however, a statistically significant difference in the frequency of IL-10 (-1082) GG genotype was found between patients with pulmonary and extrapulmonary TB (Pc=0.003). Low producer IFN-γ +874 A/A genotype is associated with post-BCG lymphadenitis and TB disease especially in younger children below 5 years. IL-10-1082 G/G genotype did not exhibit significant association except for increased GG frequency in PTB. Both cytokine polymorphisms have no relation to tuberculin reaction in patients with TB.
Tuberculosis (TB) constitutes the major cause of death due to infectious diseases. Cytokines play a major role in defense against Mycobacterium tuberculosis infection. Polymorphisms in the genes encoding various cytokines have been associated with tuberculosis susceptibility. Household contacts are at increased risk of developing the disease. In this study we examined association of IL-1β & IL-10 cytokine gene polymorphisms with risk of developing tuberculosis in TB patients, their Household Contacts (HHC) and Healthy Controls (HC) using JAVASTAT & SPSS. Multifactor Dimensionality Reduction (MDR) analyses were performed to explore the potential gene-gene interactions. The genotype and allele frequencies of IL-1β +3954C/T polymorphism did not vary significantly between TB patients and HC. GG (P<0.005, O.R=0.219 & 95% C.I 0.059-0.735) & GA (p<0.0001, O.R=2.938 & 95% C.I 1.526-5.696) genotypes of IL-10 -1082 G/A polymorphism were found to be significantly associated in patients vs HC. HHC with CC (p<0.03, O.R=1.833 & 95% C.I 1.1-3.35) genotype in IL-1β & GA (p<0.0001, O.R=4.612 & 95% C.I 2.225-9.702) genotype in IL-10 were at increased risk of developing tuberculosis. MDR tests revealed high risk genotypes in IL-1β & IL-10 based on the association model. Our results demonstrate that the polymorphisms of IL-1β & IL-10 genes may be valuable markers to predict the risk for the development of TB in household contacts. This article is protected by copyright. All rights reserved.
Around 20 susceptibility loci for type 1 diabetes mellitus (T1DM) have been mapped. One of these loci, IDDM10, was found on chromosome 10p11-q11. Here, we investigated whether the IDDM10 locus contributes in the susceptibility to T1DM in a Russian family dataset. One hundred and fourteen simplex Russian families, each containing two siblings (one affected with T1DM diagnosed and one nondiabetic sibling), and 97 multiplex families, containing 106 affected full sibling pairs, were studied. Genomic DNA from the venous blood of the patients was genotyped by PCR using 12 microsatellites (D10S193, D10S548, D10S565, D10S586, D10S588, D10S675, D10S1243, D10S1426, D10S1733, D10S1772, D10S1780 and D10S1783) located on chromosome 10p11-q11. Using the multipoint linkage analysis, the region of suggestive linkage, with a multipoint logarithm of odds (LOD) ratio (MLS) value of more than 2.2, was found between markers D10S1733 and D10S1780, an area of 9.0 cM on the genetic map. The maximum linkage peak (MLS = 2.85 and nonparametric logarithm = 2.68) was observed between markers D11S565 and D11S1243. Using the transmission disequilibrium test, an association of these markers, D10S565 (P overall = 0.0082) and D10S1243 (P overall = 0.017), with T1DM was shown. These results suggest the evidence for the IDDM10 susceptibility locus on chromosome 10p11-q11.
CBA/N and C57BL/10ScCr mice are low responders to the antigen dextran B512. This is due to the Xid gene in CBA/N mice and to unknown genes in C57BL/10ScCr mice, although this strain is unresponsive to lipopolysaccharide (LPS) due to a defective gene in the fourth chromosome. The female F1 hybrids (C57BL/10ScCr X CBA/N) and (CBA/N X C57BL/10ScCr) were low responders to dextran, although the Xid gene is not expressed in these hybrids, indicating lack of genetic complementation. In contrast, female F1 hybrids between the dextran high-responder strains CBA or C57BL/10 as one parental strain and the low-responder strains CBA/N or C57BL/10ScCr as the other parental strain, respectively, were responders to dextran. The C57BL/10ScCr mice did not appear to have an X-linked gene determining low responsiveness to dextran. The findings suggest that the only defect in CBA/N mice cannot be the Xid gene and the only defect in C57BL/10ScCr mice cannot be the gene determining unresponsiveness to LPS.
Macrophages altered by various Th2-associated and anti-inflammatory mediators--including IL-4 and IL-13 [inducing alternatively activated macrophages (AAMs)], IL-10 and TGF-β--were generically termed M2. However, markers that discriminate between AAMs and other M2 remain scarce. We previously described E-cadherin as a marker for AAMs, permitting these macrophages to fuse upon IL-4 stimulation. To identify novel potential contributors to macrophage fusion, we assessed the effect of IL-4 on other adherens and tight junction-associated components. We observed an induction of claudin-1 (Cldn1), Cldn2 and Cldn11 genes by IL-4 in different mouse macrophage populations. Extending our findings to other stimuli revealed Cldn1 as a mainly TGF-β-induced gene and showed that Cldn11 is predominantly associated with IL-4-induced AAMs. Cldn2 is upregulated by diverse stimuli and is not associated with a specific macrophage activation state in vitro. Interestingly, different claudin genes preferentially associate with M2 from distinct diseases. While Cldn11 is predominantly expressed in AAMs from helminth-infected mice, Cldn1 is the major macrophage claudin during chronic trypanosomiasis and Cldn2 dominates in tumour-associated macrophages. Overall, we identified Cldn1, Cldn2 and Cldn11 as genes that discriminate between diverse types of M2.
Native kinetoplastid membrane protein-11 (KMP-11), purified from crude extracts of Leishmania donovani parasites, activates T cells from individuals who have recovered from visceral leishmaniasis. In this work we used three 38-mer peptides spanning the amino acid sequence of the L. donovani KMP-11 as solid-phase ligands in enzyme-linked immunosorbent assays (ELISAs) and as stimulating antigens in lymphoproliferative assays in order to evaluate humoral and cellular immune responses to well-defined sequences of the protein. Antibody reactivity against the three peptides was measured in plasma from 63 Sudanese visceral leishmaniasis patients (VL) and the percentage of patients with anti-KMP-11 antibodies in ELISA were 37% (KMP-11-1), 30% (KMP-11-2) and 58% (KMP-11-3). The fraction of VL patients with measurable antibody reactivity in one or more of the three ELISAs was 79%. Cross-reactivity to the KMP-11 peptides was detected in plasma from Sudanese patients suffering from Leishmania major infections and in plasma from Sudanese and Danish patients infected with Plasmodium falciparum. In lymphoproliferative assays, 10 of 17 PBMC isolates from donors previously infected with L. donovani showed a response to one or more of the three KMP-11 peptides.
After binding lo the CD4 receptor, the human Immunodeficiency virus I HIV-I)may enter the T cell and induce the formation of multinucleated giant cells (syncytia). As well as the CD4 molecule, other molecules, such as the lymphocyte function-associated antigen 1(LFA-1. CD11a/CD 18) have been shown to be involved in HIV-l-mediated cell fusion.
This study was designed to define regions on the human CD1la/CD18 molecule important for the HIV-l-induced syncytium formation A CD11a/CD18 MoAb panel discriminating at least five distinct and spatially distant domains of the LFA-l molecule was used. Comparison of the functional activity of different MoAbs demonstrated that all epitopes of the LFA-1 molecule were not of equal importance m HIV-1-induced syncytium formation between H9.III cells chronically infected with HIV-1 and uninlecledCD4+ Sup T1 cells. We also demonstrated that CD11a/CD18 MoAbs inhibit syncytia formation only at the level of the uninfected Sup T1 cells, suggesting that the LPA-1 molecule expressed on Sup T1 ceils interacts with ligand(s) expressed on the infected H9. III cells. Two potential LFA-1 receptors on the H9.III cells were tested: t he ICAM-1 molecule (intercellular adhesion molecule 1.CD54)and the HIV-1 transmembrance glycoprotein41 (gp4l), A CD54 MoAb (84H10) partially inhibited syncytia formation, thus demonstrating the involvement of the ICAM-1 molecule in the HIV-l-mediated cell fusion. However, the CD11a/CD18 MoAbs do not inhibit binding of the viral envelope glycoprotein gp41 to the cell surface, irrespective of the MoAb concentration used.
Although we have not been successful in identifying all candidate fusion receptors for the LFA-1 molecule, these data suggest that some LFA.-1 regions are important for syncytium formation and. therefore, in the cell-to-cell transmission of virus and in the spread of infection.
Leu-M2 (Mac-120) antigen is a cell-surface marker present on a subpopulation of human blood monocytes and platelets. Its expression on monocytes and macrophages is necessary for their ability to present antigen to T lymphocytes. Only Leu-M2+ cells display the human Ia-like determinant, HLA-DS, which seems important in antigen presentation and stimulation in the autologous MLR. Expression of the Leu-M2/Mac-120 antigen was studied by indirect immunofluorescence analysis and flow cytofluorometry in mouse-human somatic cell hybrids segregating human chromosomes. In the mouse-human hybrid clones, expression of the Leu-M2/Mac-120 antigen was dependent on the presence of human chromosome 11. This was verified by fluorescence-activated cell sorting of heterogeneous hybrid cell populations into antigen-positive and-negative fractions. The Leu-M2 antigen co-segregated with chromosome 11. The gene controlling the expression of the Leu-M2/Mac-120 antigen thus is assigned to human chromosome 11.
In a well-established murine abortion model, stress is thought to trigger fetal rejection by inducing a proinflammatory immune response via substance P (SP), being tumour necrosis factor (TNF)-alpha-producing CD8+ T cells involved. Interestingly, the SP metabolite SP5-11 also binds to SP receptors and mediates SP-like effects on immune cells at sites of inflammation. No data were available regarding the effects of SP5-11 on pregnancy outcome in the CBA/J x DBA/2J abortion-prone combination. We investigated the influence of SP5-11 in contrast to stress or SP on the abortion rate and the cytokine production by lymphocytes as well as on the levels of CD8+ T cells. Stress and SP boosted the abortion rate and increased the percentage of type 1 [TNF-alpha, interferon-gamma, interleukin (IL)-12] and type 2 (IL-4 and IL-10) cytokine-producing lymphocytes in blood and decidua, predominantly CD8+ T cells. Interestingly, SP5-11 did not significantly affect the abortion rate or cytokine production in the decidua, while increasing the Th1 and Th2 cytokine production systemically. Our data suggest that stress and SP induce abortion by augmenting the local levels of TNF-alpha, which seems therefore to be a potent trigger of miscarriage. On the contrary, the SP metabolite SP5-11 only affects the systemic cytokine production without boosting the abortion rate in this experimental model.
This paper shows that the seven HA306-320 specific T-cell clones isolated from one individual recognize the peptide complexed to both autologous HLA-DRB1*1101 and allogeneic HLA-DRB1*1601 (or DRB5*0201) molecules. For each T-cell clone, a single T-cell receptor (TCR) is involved in the recognition of these two different peptide-DR complexes as evidenced by cold target competition experiments. Yet, the seven T-cell clones express several different TCRs as judged by V beta-J beta usage and fine specificities. Furthermore, one representative clone has the same fine specificity for HA306-320 analogues mutated at epitopic residues irrespective of the use of DR1101 or DR1601 APC. These results suggest that structural differences between DRB1*1101 and DRB1*1601 (or DRB5*0201) do not dramatically influence the orientation of HA306-320 in the grooves such that most residues interacting with TCRs are conserved. In another individual, the same pattern of restriction, i.e. DR1101 + DR1601, was found for several HA306-320 specific clones. Two additional patterns, DR1101 + DR0801 and DR1101 + DR0801 + DR1601, were identified. By comparing DR sequences the authors found that DRB1*1101 and DRB1*1601 share four important motifs, i.e. beta 85-86, beta 67-71, beta 57 and beta 28-31 supposed to line three distinct HLA-DR pockets. Three of these motifs are also shared with DRB1*0801. All the results further support that the motif similarities allow the peptide to adopt very similar orientations in the cross-reacting DR molecules.
We have previously demonstrated an association between secondary AA type amyloid fibrils and glycosaminoglycans (GAGs) in human liver. The present study was aimed ill investigating whether a similar association could be demonstrated in isolated cardiac amyloid fibrils from a unique Danish family with amyloid cardiomyopathy related to variant transthyretin (TTR) with a single amino acid substitution of a methionin for leucine at position 111 (TTR Met 111).
Using gel nitration and ion exchange chromatography. significant amounts of GAGs were detected in close association with purified myocardial amyloid fibrils, whereas only truce amounts of polysaecharides were present in the corresponding normal preparation. The GAGs were identified as 50% chondroitin sulfate, 33% heparin/heparan sulfate, and 17% hyaluronan. With the methods used the amyloid associated GAGs appeared as high molecular weight free polysaccharidc chains, and not as part of intact proteoglycans (PGs) in the fibril extracts.
We conclude that the association between purified amyloid fibrils and GAC is may be a general feature of amyloid deposits. Also, we suggest that the proportion of different GAGs in the amyloid deposits may depend both on the organ or tissues affected and the type of protein making up the fibrils.
Natural killer (NK) cell activity and related markers were analysed in childhood acute lymphoblastic leukaemia (ALL). Children with untreated ALL, children with active disease, and children in remission for less than 1 month and undergoing induction therapy had significantly lower NK cell activity in peripheral blood than the control group (P less than 0.05, P = 0.0005, and P less than 0.0025). Patients in remission for 1-3 months and undergoing consolidation chemotherapy had normal NK activity (P greater than 0.05). Children in complete remission for more than 3 months and undergoing maintenance therapy also had a normal NK activity in their peripheral blood (P greater than 0.05). However, their bone marrow cells showed an increased NK cell activity (P less than 0.0005). Cells positive for the Leu-7 marker were reduced in the peripheral blood from untreated children (P less than 0.025) and children in remission for less than 1 month (P = 0.025). The percentage of cells from peripheral blood expressing the marker Leu-11b (CD 16) did not differ significantly from that of the controls (P greater than 0.05). However, children in complete remission for more than 3 months had a higher number of bone marrow cells expressing the Leu-7 (P = 0.005) and the Leu-11b (CD 16) markers (P = 0.05) than controls. Stimulation of mononuclear cell suspensions with recombinant alpha interferon and recombinant interleukin 2 were shown to cause a normalization of the NK cell activity in peripheral blood and bone marrow.
Peripheral blood and bone marrow mononuclear cells from 25 children with acute non-lymphoid leukaemia were analysed for natural killer cell activity and for cells with the Leu-7 and Leu-11b (CD 16) markers. Significantly reduced spontaneous cytotoxicity was detected in peripheral blood from children with untreated and active acute non-lymphoid leukaemia compared with that of the controls (P = 0.01 and P less than 0.05). Patients in remission, however, had normal natural cytotoxicity and normal numbers of Leu-7 and Leu-11b (CD 16)-positive cells. The natural killer cell activity in bone marrow from patients with untreated acute non-lymphoid leukaemia was also significantly reduced (P = 0.025). On the other hand, patients in remission had both an increased percentage of Leu-7 and Leu-11b (CD 16)-positive cells (P less than 0.05) and an increased natural killer cell activity (P less than 0.0005) in their bone marrow cells in comparison with the control group. This augmented natural killer cell activity is most probably a result of anti-leukaemic treatment. Stimulation with recombinant alpha interferon and recombinant interleukin 2 caused an increase in natural killer cell activity that was both significant and normal in both peripheral blood and bone marrow from children with acute non-lymphoid leukaemia.
CD11b (Leu15) epitope is expressed on 20-30% of peripheral blood lymphocytes, including CD16+ large granular lytnphocytes and CD8+ cells. This study confirms that 30% of CD8+ lymphocytes and virtually all CD16+ NK cells from healthy subjects express this determinant. In parallel, our data show that various proportions of CD3+ 4-8-, TCR-δ cytotoxic T lymphoeytes and occasionally CD4+ lymphocytes subsets could also express this epitope.
The CD8+ 11b+ phenotype is associated with suppression of T-cell proliferative response and has been extensively used to characterize suppressor T lymphocytes. Since about 25% of CD8 lymphocytes are non-T (CD3) and express the CD16 NK antigen (CD8+ 16+ 3 -), the expression of CD11b was also studied on CD8+ 3+ T-cell and CD8+ 16+ NK-cell subsets. To this end, we developed three methods using a flow cytometer equipped with a single laser and two fluorescence detectors. Results showed that T CD8+ 3+ 11b+ and NK CD8+ 16+ 11b+ lymphocytes account for 3O% and 70% of CD8+ 11b+ cells respectively. Consequently, the CD8+ 3+ 11b+ phenotype would be more specific for suppressor T lymphocytes than the total CD8+ 11b+ phenotype which includes high proportions of CD16+ NK cells.
The calcium ionophore ionomycin and the phorbol ester phorbol-12,13-dibutyrate (PDBu) are shown to have a synergistic effect upon interleukin 2 (IL-2) production, interleukin 2 receptor expression, and T-lymphocyte proliferation.
The proliferative response was inhibited by addition of a monoclonal antibody directed against the IL-2R (Tac antigen) demonstrating that PDBu and ionomycin induce T-cell growth through an IL-2-dependent autocrine pathway.
Sequential stimulation with PDBu and ionomycin failed to induce IL-2 production, IL-2R expression, and consequently proliferation of the T cells, indicating that T-cell activation requires simultaneous activation of protein kinase C (PKC) and elevation of cytosolic calcium.
Exposure of T cells to both agents for different times resulted in IL-2 production, IL-2R expression, and proliferation in proportion to the duration of incubation with at least 4 h required for maximal T-cell activation. Further, in the presence of PDBu maximal T-cell activation was found to require stimulation with ionomycin for 4 h, indicating that a sustained increase in free cytoplasmic calcium of several hours' duration is essential for T-cell activation. In contrast T cells incubated with ionomycin were induced to produce IL-2 and express IL-2Rs upon brief exposure to PDBu with a 2-h incubation period being sufficient for maximal T-cell activation. Thus transient activation of PKC seems to be sufficient for activation of the IL-2 gene and IL-2R gene. However, maximal T-cell activation requires activation of PKC for at least 2 h.
Monoclonal antibody K20 recognizes a human glycoprotein complex that is not restricted to haematopoietic lineages but is preferentially expressed on early haematopoietic cells, T cells, and monocytes. This glycoprotein complex is made of a constant 120,000-140,000 Mr subunit noncovalently associated at the cell surface with subunits of higher Mr ranging from 150,000 to 200,000 on different cell types. Internal labelling with [35S]methionine and pulse-chase experiments revealed that in the cell the 120,000 Mr glycoprotein of this complex is also noncovalently associated with a 100,000 Mr glycoprotein, and that both glycoproteins are independently biosynthesized. This glycoprotein complex is shown by immunoprecipitation by lectin plus antilectin antibodies and by sequential immunoprecipitations to be one of the cell surface structures bound by phytohaemagglutinin on the surface of normal T cells.
Amyloid fibril material was extracted from autopsy material of a patient who died from progressive cardiac failure at age 64. He had enlarged heart on routine X-ray at age 47 and the first symptoms of cardiac failure at age 62. Fractionation of the fibril material resolved peptide fragments immunoreactive with anti-human transthyretin (TTR). One of the peptides was further purified by high-performance liquid chromatography (HPLC) and subjected to tryptic peptide mapping along with TTR isolated from the patient's serum. In both instances, sequencing procedures revealed, in addition to the normal peptide 12 (residues 105-126), an abnormal peptide with an isoleucine for valine substitution at position 122. This substitution has been described previously in a patient with systemic senile amyloidosis (SSA) homozygous for this variant. The results question whether SSA is a clinical entity related to TTR Ile 122 with phenotypic expression in the homozygous condition.
The degree of uptake of DNP 125I-HSA (= DNP *HSA) conjugates by mouse peritoneal exudate (PE) Cells in vivo and their retention in The- spleen and liver depended on the density of hapten per carrier molecule. Forty to 400 times more DNP29*HSA and DNP39*HSA were ingested by PE cells as compared to HSA alone. Significantly more DNP29*HSA and DNP39*HSA persisted in the spleen and liver after intraperitoneal injection than *HSA, DNP6*HSA or DNP11*HSA. *HSA, DNP6*HSA, and DNP11*HSA were rapidly catabolized after uptake in macrophages in vitro, but a small amount of these antigens was associated with the cells. About 50% of the *HSA was membrane-bound. *HSA conjugated with DNP29 or DNP39 was more rapidly catabolized by PE cells than when it was present in low-density hapten-carrier conjugates. More of the high-density conjugates were associated with the PE cells than was the case with the other conjugates, and again about 50% of the radioactivity was msembrane-bound. Thus, the degree of metabolism and persistence of *HSA in vitro was also related to the density of hapten on the carrier.
In a new procedure, rocket immunoelectrophoresis is performed at pH 8.7 with rabbit antibodies and 5% polyethyleneglycol 6000 in the agarose gel. After the pH in the gel has been changed to 5 the nonprecipitated immunoglobulins are electrophorsed out of the gel simultaneously with the electrophoresis of 125I-labeled swine antibodies against rabbit IgG into the gel. The latter antibodies tag the rabbit IgG, which is not present only in the precipitates. The radioactive precipitates are visualized by autoradiography. The method permits quantification of antigens down to an amount of approximately 0.5 ng; well-defined rockets are not formed below this limit. Compared to conventional protein staining with Coomassie brilliant blue, this represents an increase in sensitivity of up to 20 times.
Radioiodinated protein A (SpA) from Staphylococcus aureus was used to quantitate specific antibodies of the IgG class at the nanogram level in rabbit serum by a standardized radioimmunoassay with the antigen covalently bound to paper discs. Quantitation of IgG in micrograms of IgG per millilitre of serum was done by using a standard curve with purified anti-bovine serum albumin (BSA) IgG or by means of the calculated molar SpA to rabbit IgG ratio of 1:1, thus converting counts per minute of 125I-SpA to nanograms of IgG. The molar ratio for SpA to human IgG was also 1:1. The unspecific binding of IgG to the paper discs was studied and could be depressed by hen serum or by adsorption of the serum sample to cellulose before the assay. The affinity constants of the extracellular SpA and human, rabbit and guinea-pig IgG, respectively, were determined and found to be close to the values previously calculated for cell-wall-bound SpA. The applicability of the assay was demonstrated by quantitation of the specific IgG response on different days after immunization of rabbits with BSA and 3,4-dinitrobenzene (DNP)-BSA.
Visualization by autoradiography of specific IgE binding in crossed radioimmunoelectrophoresis (CRIE) and other 125I-immunoautoradiography (IAR) techniques is done in two different ways; either by traditional direct autoradiography (D-ARG), where the film is exposed to the 125I-anti-IgE incubated sample at room temperature, or by indirect autoradiography (ID-ARG), applying intensifying screen, low-temperature exposure and, eventually, pre-exposure. This study confirmed that D-ARG provided the benefits of simplicity and better image resolution with the disadvantage of prolonged exposure periods. ID-ARG reduced the exposures needed to produce film image densities of 0.01 and 0.1 A540 nm (i.e. autoradiographic sensitivity (AR sigma) and autoradiographic speed (ARs] to 1/18 and 1/55 respectively of the corresponding exposures in D-ARG. The lowest detection limits for 125I in 24 h were 1.2 cpm mm-2 with the indirect and 6.8 cpm mm-2 with the direct systems investigated. The major drawbacks of ID-ARG were inferior image resolution and higher background levels, especially when pre-exposure was included.
Polymorphisms in genes that encode crucial signalling molecules have been proposed as factors that influence susceptibility to, and outcome of malaria. We studied the role of a mutation, c.1264 T>G, that causes CD36 deficiency on IgG responses to MSP-1₁₉ antigen and malaria incidence. Children were genotyped for the c.1264 T>G mutation at the beginning of the study using PCR-RFLP. IgG levels [optical density (OD) readings] and per cent seropositivity to MSP-1₁₉ were determined at baseline by ELISA. Children were followed for 12 months for acquisition of anti-MSP-1₁₉ IgG antibody and malaria incidence. We observed a significant increase in the production of anti-MSP-1₁₉ IgG antibody in normal and heterozygous children during the 12 months of follow-up, but not in homozygous mutants. Normal children had a significantly lower malaria incidence rate compared to other genotypes (χ² = 115.59; P < 0.01). We conclude that the presence of the c.1264 T>G mutation that leads to CD36 deficiency is closely associated with reduced IgG production and higher malaria incidence. It is most likely that deficiency of CD36 which is known to modulate dendritic cell function suppresses the production of protective IgG antibodies directed to Plasmodium falciparum MSP-1₁₉ antigen, which predisposes to the acquisition of clinical malaria in children.
Abnormalities in CD4+CD25+ regulatory T cells (Treg) may contribute to type 1 diabetes (T1D) development. First-degree relatives of T1D patients are at increased risk especially when they carry certain HLA II haplotypes. Using two novel markers of CD4+CD25+ Treg (CD127- and FoxP3+ respectively), we evaluated number and function of Treg after specific stimulation with diabetogeneic autoantigens in 11 high-risk (according to HLA-linked risk) relatives of T1D patients and 14 age-matched healthy controls using a cytokine secretion assay based on interferon-gamma (IFN-gamma) production. High-risk relatives of T1D patients had significantly lower pre- and post-stimulatory number of CD127- Treg than that of healthy controls (P < 0.05). Labelling Treg with FoxP3+ demonstrated similar trend but did not reach statistical significance. Although the stimulation with diabetogenic autoantigens did not lead to a significant change in number of Treg in both groups, the defective function of Treg was performed by significantly higher activation of diabetogeneic T cells in high-risk relatives of T1D patients compared to healthy controls (P < or = 0.02). Individuals at increased HLA-associated genetic risk for T1D showed defects in Treg.
Trypanosoma brucei subspecies invade the brain parenchyma at late stages of human and experimental rodent infections. In this study, we compared the outcome of infection with T. b. brucei in MHC-matched (H-2b) C57BL/6 (B6) and 129Sv/Ev (Sv-129). Sv-129 showed higher parasitaemia and lower specific IgM (but not IgG) antibody levels than B6 mice. The number of trypanosomes, CD4+ and CD8+ T cells in the brain parenchyma was higher in B6 mice. B6 mice lost weight and showed higher cumulative mortality when compared with Sv-129 mice. Higher levels of IL-1beta, IL-6, IL-10, TNF-alpha, IFN-gamma, ICAM-1 and E-selectin, but low levels of TGF-beta mRNA were present in brains of B6 when compared with Sv-129-infected mice. Thus, host genetics differentially determine the invasion of T. b. brucei into the brain parenchyma, which is paralleled by the severity of inflammation in the brain and course of the disease, but not by parasitaemia nor by antibody titres.
We analysed the association of a single nucleotide polymorphism (SNP) in the gene encoding the IL-12 subunit p40 (IL12B, rs3212227, A>C) with breast cancer. The SNPs allelic and genotypic frequencies were compared between patients (n = 191) and healthy (n = 194) women in a case-control study from Croatia. The major allele (A) was associated with susceptibility to breast cancer (P = 0.003; OR = 1.67; 95% CI: 1.17-2.38). Likewise, the minor allele (C) was significantly correlated with protection (P = 0.003; OR = 0.60; 95% CI: 0.42-0.86). At the genotype level, AA homozygosity was significantly associated with predisposition to disease (P = 0.013; OR = 1.68, 95% CI: 1.09-2.59), whereas the minor allele homozygosity (CC) was correlated with protection to disease (P = 0.020, OR = 0.28, 95% CI: 0.09-0.91). The heterozygous genotype showed no significant correlation with disease. The product of the IL12B gene (IL-12 p40) can either form a homodimeric cytokine or be part of two pro-inflammatory (IL-12 and IL-23) cytokines. It is presently unclear whether the major allele is associated with higher or lower protein levels of IL-12 p40 and IL-12 p70, which are critical in inflammation and adaptive immune responses. However, as the A allele is high producer of IL12B (p40) mRNA, these results might imply that higher levels of IL-12 p40 (either as homodimers or joined with one or both of the other two subunits) predispose to breast cancer.
RATIONALE: Brucellosis remains a major zoonosis worldwide. Brucella antigens induce the production of T-helper 1 (Th1) cytokines such as interleukin- 12 (IL-12) in humans. OBJECTIVES: We aimed to investigate the association of two single nucleotide polymorphisms (SNPs) in the gene encoding the IL-12p40 cytokine (IL-12B) with brucellosis, and to examine the functionality of these SNPs through measuring serum levels of IL-12p40. We genotyped IL-12B gene rs3212227, A>C; rs6887695 G>C polymorphisms in a case-control study on a totally of 281 subjects including 153 patients with active brucellosis and 128 healthy controls, using RFLP, and serum IL-12p40 levels were assessed by ELISA. FINDINGS: The rs3212227 minor allele (C) and homozygote genotype (CC) were more frequent in controls compared with brucellosis patients (p=0.006, OR=0.608, 95%CI=0.429-0.861for the C allele; p= 0.024, OR = 0.443, 95% CI: 0.218-0.900 for the CC genotype). Comparison of IL-12B genotypes and serum levels of the IL-12p40 revealed that rs3212227 AA genotype, with higher frequency in patients than in controls, was associated with increased levels of the cytokine (p=0.0001). Furthermore, the distribution of haplotype and genotype combinations in our study suggested that rs3212227C/rs6887695C haplotype or CC/GC or CC/CC genotype combinations may protect controls against Brucella infection by contributing to a functional downregulation of the serum IL-12p40 production in vivo, as shown by ELISA (p<0.05). CONCLUSIONS: Overall, our study demonstrated that rs3212227 A variant was associated with higher levels of serum IL-12p40, and could possibly contribute to an inherited predisposition to brucellosis. This article is protected by copyright. All rights reserved.