The association of the vitamin D status with the persistence of anti-HBs antibody at 20years after primary vaccination with recombinant hepatitis B vaccine in infancy.

BACKGROUND AND OBJECTIVE: Vitamin D has potent immunoregulatory effects due to the expression of its receptor on the majority of immune cells. The aim was to evaluate the association of the vitamin D status with the persistence of anti-HBs antibody and immune response to booster immunization at 20years after primary vaccination with hepatitis B (HB) vaccine. METHODS: Blood samples were collected from 300 adults 20years after completion of the primary HB vaccination in infancy. The serum levels of vitamin D and anti-HBs antibody were measured by ELISA. A single booster dose of a recombinant HB vaccine was administered to a total of 138 subjects, whose anti-HBs titer was<10IU/L. The sera of revaccinated subjects were re-tested for anti-HBs antibody, 4weeks after booster vaccination. RESULTS: At 20years after primary vaccination, the mean vitamin D concentrations were significantly higher in seroprotective subjects as compared to non-seroprotective individuals (P<0.01). The levels of anti-HBs were significantly increased with advanced concentrations of vitamin D (P<0.01). Overall, 125/138 (90.6%) of the revaccinated subjects showed an anamnestic response to booster vaccination. The concentrations of vitamin D were significantly higher in subjects with an anamnestic response to booster vaccination as compared with subjects without this response (P<0.01). CONCLUSION: Vitamin D status may influence the persistence of anti-HBs antibody and durability of protection after primary vaccination with a recombinant HB vaccine in infancy.

The pre-mir-27a variant rs895819 may contribute to type 2 diabetes mellitus susceptibility in an Iranian cohort.

PURPOSE: The study was aimed at investigating the association between hsa-mir-27a polymorphism rs895819 (T/C) and type 2 diabetes mellitus (T2DM) susceptibility in a large Iranian cohort. METHODS: In this case-control study, the investigated population consisted of T2DM patients (n = 204) and sex- and age-matched controls (n = 209). We used the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) for genotyping. RESULTS: We observed significant differences between T2DM patients and controls for weight (p = 0.002), BMI (p < 0.001), systolic blood pressure (p < 0.001), diastolic blood pressure (p < 0.001), fasting plasma glucose (p < 0.001), triglyceride (p = 0.004) and LDL cholesterol (p = 0.051). Moreover, we found that genotype distributions were significantly different between groups (p < 0.05) and that the rs895819-C allele is more frequent in controls (p = 0.030, OR = 0.72, 95 % CI 0.53-0.97). CONCLUSION: Our study shows that rs895819 in hsa-mir-27a is associated with T2DM susceptibility and that the C allele conveyed a protective role against T2DM. Larger multicentric and specific functional studies will be necessary to obtain a deeper comprehension of the role of rs895819 and hsa-mir-27a and how they are involved in the development of diabetes.

Leishmania tarentolae secreting the sand fly salivary antigen PpSP15 confers protection against Leishmania major infection in a susceptible BALB/c mice model.

Cutaneous leishmaniasis is a zoonotic, vector-borne disease causing a major health problem in several countries. No vaccine is available and there are limitations associated with the current therapeutic regimens. Immune responses to sand fly saliva have been shown to protect against Leishmania infection. A cellular immune response to PpSP15, a protein from the sand fly Phlebotomus papatasi, was sufficient to control Leishmania major infection in mice. This work presents data supporting the vaccine potency of recombinant live non-pathogenic Leishmania (L.) tarentolae secreting PpSP15 in mice and its potential as a new vaccine strategy against L. major. We generated a recombinant L. tarentolae-PpSP15 strain delivered in the presence of CpG ODN and evaluated its immunogenicity and protective immunity against L. major infection in BALB/c mice. In parallel, different vaccination modalities using PpSP15 as the target antigen were compared. Humoral and cellular immune responses were evaluated before and at three and eight weeks after challenge. Footpad swelling and parasite load were assessed at eight and eleven weeks post-challenge. Our results show that vaccination with L. tarentolae-PpSP15 in combination with CpG as a prime-boost modality confers strong protection against L. major infection that was superior to other vaccination modalities used in this study. This approach represents a novel and promising vaccination strategy against Old World cutaneous leishmaniasis.

Administration of naloxone in combination with recombinant Plasmodium vivax AMA-1 in BALB/c mice induces mixed Th1/Th2 immune responses.

Naloxone (NLX) has the ability to shift the immune response to a Th1 profile. Therefore, the adjuvant efficacy of NLX with recombinant P. vivax apical membrane antigen-1(rPvAMA-1) in BALB/c mice was evaluated. Mice were immunized subcutaneously with purified rPvAMA-1 formulated with NLX (doses of 5 mg/kg body weight) alone or in combination with IFA. A significant increase in anti-PvAMA-1 IgG antibody after the second boost (mean OD490  = 2·08 and 2·17, in groups received, rPvAMA-1/NLX and rPvAMA-1/NLX/IFA, respectively) was detected. IgG1 and IgG2b were the predominant isotypes in all immunized mouse groups. In immunized mice with rPvAMA-1/NLX (mean: 1036 pg/mL) and with rPvAMA-1/NLX/IFA (mean: 1024 pg/mL), IFN-γ was elicited in response to rPvAMA-1 after the second boost. No detectable IL-4 secretion was determined in all tested groups. In conclusion, the administration of NLX alone or NLX/IFA with rPvAMA-1 in BALB/c mice, which induced mixed Th1/Th2 immune responses, was comparable with that of the same recombinant antigen with CFA/IFA adjuvant. The results indicate that NLX alone may possibly not be considered as a potent Th1 adjuvant in PvAMA-1-based vaccine. However, in order to modulate immune responses from mixed Th1/Th2 to strong and protective Th1 response, further study is warranted on combination of NLX with other adjuvants such as CpG motifs or MPL in proper vaccine formulation. Additionally, dose-response study is necessary to determine the effect of different doses of antigen combined with NLX (at various doses) in Balb/c mice.

Frequency analysis of HLA class I alleles in Iranian patients with progressive and non-progressive chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a malignant disorder of B cell origin, with low incidence in Asian populations. In this study we investigated the HLA-class I A and B allele frequencies in 87 Iranian CLL patients and 64 healthy controls using sequence specific primer-polymerase chain reaction (SSP-PCR) technique. Our results showed increased frequencies of HLA-A11:01 (p=0.02) and HLA-B35:01 (p=0.002) alleles and HLA-A11:01/B35:01 haplotype (p=0.036) and decreased frequencies of HLA-A01:01 (p=0.02), HLA-A26:01 (p=0.03), HLA-B65:01 (p=0.03) and HLA-B53:01 (p<0.00001) alleles in CLL patients compared to the control group. Classification of the patients into non-progressive and progressive groups did not reveal significant differences for the frequency of any of the HLA-A and -B alleles or haplotypes between these two subtypes. Comparison between patients with immunoglobulin heavy chain variable region genes (IGHV) mutated (n=56) and unmutated (n=31) subtypes showed a significant increase in HLA-A32:01 (p=0.05) and HLA-A33:01 (p=0.05) alleles in IGHV unmutated patients compared to IGHV mutated patients. Similarly, a higher frequency of HLA-B52:01 (p=0.037) alleles was observed in CD38(+) compared with CD38(-) patients. Our results obtained from an Iranian population indicate that CLL is associated with distinct HLA class I alleles and haplotypes some of which are linked to disease prognostic factors.

Monoclonal antibodies against ROR1 induce apoptosis of chronic lymphocytic leukemia (CLL) cells.

ROR1 is a receptor tyrosine kinase (RTK) recently identified to be overexpressed at the gene and protein levels in chronic lymphocytic leukemia (CLL). Monoclonal antibodies (MAbs) against RTKs have been successfully applied for therapy of solid tumors. We generated five MAbs against the Ig (n = 1), cysteine-rich (CRD) (n = 2) and kringle (KNG) (n = 2) domains, respectively, of the extracellular part of ROR1. All CLL patients (n = 20) expressed ROR1 on the surface of the leukemic cells. A significantly higher frequency of ROR1 expression was found in patients with progressive versus non-progressive disease, and in those with unmutated versus mutated IgVH genes. All five MAbs alone induced apoptosis in the absence of complement or added effector cells (Annexin-V and MTT, as well as cleavage of poly-(ADP ribose)-polymerase, caspase-8 and caspase-9) of CLL cells but not of normal B cells. Most effective were MAbs against CRD and KNG, significantly superior to rituximab (P < 0.005). Cross-linking of anti-ROR1 MAbs using the F(ab')(2) fragments of anti-Fc antibodies significantly augmented apoptosis. Two of the MAbs induced complement-dependent cytotoxicity (CDC) similar to that of rituximab and one anti-ROR1 MAb (KNG) (IgG1) showed killing activity by antibody-dependent cellular cytotoxicity. The identified ROR1 epitopes may provide a basis for generating human ROR1 MAbs for therapy.

Differential Responses of Anopheles stephensi (Diptera: Culicidae) to Skin Emanations of a Man, a Cow, and a Guinea Pig in the Olfactometer.

BACKGROUND: Biting habit of mosquitoes plays an important role in the epidemiology of mosquito-borne diseases. Mosquitoes use a set of elaborate sensory modalities to find their preferred hosts by exploiting cues emanating from a nearby host. It has been suggested that the chemical profile of skin can provide further support for anthropophilic mosquito species to find their suitable hosts. This study aimed at revealing the value of skin emanation for a zoophilic species like Anopheles stephensi as a model. METHODS: Skin emanations of a man, a cow and a Guinea pig were collected by ethanol soaked cottons. Upwind responses of mosquitoes to 100 and 200 µl of filtered skin materials were non-competitively explored in a dual-choice olfactometer. L-lactic acid and other chemical content of the skin samples were identified by an enzymatic kit and GC-MS, respectively. RESULTS: Unexpectedly, only human skin emanation was resulted in the statistically significant activation and attraction responses of An. stephensi in the wind tunnel. L-lactic acid content of this skin sample was 10 and 29 times more than the cow and the Guinea pig, respectively. The possible role of lactic acid and a few other identified compounds have been discussed here. CONCLUSION: Anopheles stephensi showed higher and more specific upwind responses to human skin emanation in the olfactometer. Undoubtedly, the thorough explanation of this unexpected finding needs further investigation. But, if new data verify this result, then, it may be necessary to reconsider the role of skin emanation and thence the human blood index and vectorial capacity of this zoophilic mosquito.

Low dose revaccination induces robust protective anti-HBs antibody response in the majority of healthy non-responder neonates.

A sizeable proportion (1-10%) of healthy adults and to lesser extent neonates vaccinated with triple 10 microg hepatitis B (HB) vaccine fail to mount a protective antibody response. Revaccination with the same vaccine dose has proved to be effective in a significant number of primary non-responders. The influence of revaccination with lower vaccine doses however has not been studied adequately in non-responder neonates. This study was conducted to evaluate the influence of supplementary vaccination with a single low and standard dose of a recombinant hepatitis B (HB) vaccine in healthy Iranian non-responder neonates to primary vaccination. Iranian neonates unable to respond to primary vaccination with 10, 5 or 2.5 microg doses of recombinant HB vaccine were revaccinated with a single additional dose of the same concentration. Serum anti-HBs antibody titer was measured by sandwich ELISA. Administration of a single additional dose induced seroprotection (anti-HBs> or =10IU/L) in 10/12 (83%), 10/12 (83%) and 21/24 (87.5%) of non-responder neonates in 10, 5 and 2.5 microg vaccine recipients with geometric mean titers (and 95% confidence limits) of 1358 (258-7142), 401 (79-2038) and 164 (62-433) IU/L, respectively. The log-transformed antibody titer obtained for the 10 microg dose recipients was significantly higher than that of the 2.5 microg dose vaccinees (p=0.028). No significant differences in anti-HBs titer were observed between other groups of vaccinees. However, the total seroprotection rates obtained after administration of four low doses of 2.5 and 5 microg were significantly higher than that obtained after administration of the classical three 10 microg doses (p=0.029 and p=0.006, respectively). The total seroprotection rates were similar between all groups of vaccines receiving four doses of 2.5, 5 and 10 microg vaccine doses. These results indicate that a significant proportion of non-responder neonates can be induced to develop a protective anti-HBs response following revaccination with a single low dose vaccine. Thus adaptation of four low dose (2.5 or 5 microg) vaccination is expected to induce higher seroprotection rate and lower or comparable anti-HBs antibody titer in healthy neonates.

Analysis of the immunoglobulin heavy chain variable region gene expression in Iranian patients with chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) results from clonal expansion of phenotypically mature but functionally immature B-lymphocytes. The incidence of this type of leukemia is low in Asian countries, whereas it is the most frequent type of leukemia in the West. Previous investigations mainly conducted in Western populations have demonstrated non-random rearrangement of certain immunoglobulin variable region heavy (VH) and/or light (VL) chain genes in different groups of B-CLL patients. Little is known about the profile of VH gene expression in Asian patients. In the present study, we determined the frequency of VH gene family usage in 59 Iranian patients with B-CLL. VH gene family of patients was determined by reverse transcriptase-polymerase chain reaction using VH1-VH7 family specific primers. The most frequently expressed VH gene family was found to be VH3 (45.8%) followed by VH4 (32.2%), VH1 (18.6%), VH5 (1.7%) and VH6 (1.7%), with no expression of VH2 and VH7 gene families. The results indicate a lower representation of the VH1 and VH2 gene families and a higher representation of the VH4 gene family in Iranian B-CLL patients compared to Western patients, suggesting involvement of ethnic and/or environmental factors in B-CLL disease initiation.

Frequency analysis of HBsAg-specific B lymphocytes in high-responder individuals to recombinant hepatitis B vaccine: comparison of LDA and ELISPOT assays.

The determination of the frequency of antigen-specific lymphocytes may provide invaluable information for the evaluation of the immune response to different antigens and pathogens. Different methods are employed to determine the frequency of specific B lymphocytes in peripheral blood. In this study, the frequency of hepatitis B surface antigen (HBsAg)-specific B lymphocytes was determined by a limiting dilution assay (LDA) and an enzyme-linked immunospot assay (ELISPOT) in seven healthy adult high responders to recombinant HBsAg. Peripheral blood mononuclear cells isolated at different time intervals (1, 2, 4, 8 and 16 weeks) following administration of a booster dose were either transformed with Epstein-Barr virus (LDA) or stimulated with Pokeweed mitogen (ELISPOT). In an LDA, anti-HBs positive wells were screened by a sandwich ELISA and the frequency of specific B lymphocytes was estimated based on the Poisson statistical analysis. In an ELISPOT, coloured spots representing specific B lymphocytes were finally enumerated by stereomicroscope. Our results showed a significant increase in the number of specific B lymphocytes in the first week by an ELISPOT compared with an LDA (1:190 versus 1:13,462) (P < 0.001). No significant differences were observed at other time intervals. A significant correlation was observed between the serum titer of anti-HBs antibody and frequency of HBsAg-specific B cells obtained by LDA and ELISPOT methods at different time intervals. The highest correlation was found at fourth week in LDA (r = 0.83, P < 0.01) and ELISPOT (r = 0.85, P < 0.01) assays. Furthermore, a significant correlation was observed between an LDA and ELISPOT at different time intervals (highest correlation in second week, r = 0.88, P < 0.008). These findings suggest that in addition to technical advantages, such as speed and simplicity, an ELISPOT is a more sensitive assay, compared with an LDA.

Epitope mapping of recombinant hepatitis B surface antigen by murine monoclonal antibodies.

Hepatitis B surface antigen (HBsAg) induces a potent protective antibody response in immunized healthy individuals. The antibody response in humans is largely directed to a restricted conformational immunodominant region of HBsAg, identified as "a" determinant. Our aim was generation and characterization of murine monoclonal antibodies (MAbs) against recombinant HBsAg and their use for epitope mapping of the antigen. Hybridoma cells were established from Balb/c mice immunized with recombinant HBsAg of the "adw" subtype and cloned by limiting dilution. Specificity of MAbs was studied by indirect ELISA and immunoblotting. Topology of the epitopes was analyzed by competitive and inhibition ELISA. Eight hybridoma clones producing MAbs specific for the immunogen were established. Five of the MAbs recognized overlapping conformational epitopes, whereas the remaining three MAbs were found to identify linear epitopes. Cross-inhibition studies suggest recognition of mutually exclusive epitopes by these MAbs. Our data suggest that, similar to the human system, the mouse antibody response is largely directed to restricted conformational overlapping epitopes of HBsAg.

Identification of cross-reactive and restricted epitopes localized on human chorionic gonadotropin beta-subunit by monoclonal antibodies.

Human chorionic gonadotropin (hCG) belongs to the family of glycoprotein hormones. All members of the family are composed of an identical alpha subunit and structurally related beta subunit which confers biological specificity. Specific quantification and functional analysis of hCG require the use of monoclonal antibodies recognizing different epitopes of hCGbeta. This study describes the production and characterization of monoclonal antibodies (MAbs) to hCGbeta with no cross-reactivity to other glycoprotein hormones. Spleen cells from Balb/c mice immunized with hCG were fused with mouse SP2/0 myeloma cells. Fused cells were grown in hypoxanthine, aminopterine, and thymidine (HAT) selective medium and cloned by limiting dilution assay. Antibody-secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed, using a panel of highly purified and recombinant glycoprotein hormones, their subunits and peptides representing the C-terminal end of hCGbeta (hCGbeta-CTP) by ELISA and immunoblotting. The affinity constant (K(aff)) was also determined by ELISA. Three murine hybridomas designated G5M1, B12M2 and F4M3 were obtained that secrete MAbs specific for hCGbeta. The G5M1 MAb reacts only with hCGbeta, hCGbeta-CTP and intact hCG with no detectable cross-reaction with hCGalpha or any of the other glycoprotein hormones. The specificity of B12M2 MAb is very similar to G5M1, but it does not react with hCGbeta-CTP. The F4M3 MAb also has similar specificity to G5M1 and B12M2, but it strongly cross-reacts with hLH. The affinity constant (Kaff) of G5M1, B12M2 and F4M3 was found to be 4.28 x 10(9), 5.2 x 10(8), and 1.97 x 10(9) M(-1), respectively. Our results indicate that G5M1 and B12M2 MAbs are specific for hCG and recognize epitopes restricted to hCGbeta, but F4M3 recognizes a common epitope expressed both on hCGbeta and hLHbeta.

Frequency analysis of B lymphocytes specific for Rh antigens in naturally immunized Rh-negative women.

BACKGROUND AND OBJECTIVES: Despite a successful outcome of the anti-D prophylaxis programme, alloimmunization still occurs. The aim of this study was to estimate the frequency of Rh-specific B lymphocytes in the peripheral blood of nine Rh-alloimmunized individuals at different time intervals after parturition. MATERIALS AND METHODS: The donors' B lymphocytes were transformed with Epstein-Barr virus (EBV) and cultured at different cell densities over a feeder of human fetal fibroblasts. Culture supernatants were screened for human immunoglobulin by enzyme-linked immunosorbent assay (ELISA) and for anti-Rh antibody by using a direct haemagglutination technique. The percentage of CD19+ B lymphocytes in peripheral blood was determined by flow cytometry, and the frequency of Rh-specific B lymphocytes was estimated by limiting-dilution assay (LDA). RESULTS: The frequency of Rh-specific B lymphocytes varied from 1 : 150 to 1 : 27,850 in different donors. There was a decrease in this frequency and level of anti-Rh antibody with increase in time interval between bleeding and last exposure to the antigen. Furthermore, a positive correlation was observed between the titre of Rh-specific antibody and frequency of Rh-specific B cells in each of three subjects bled at multiple time-points postdelivery. CONCLUSIONS: The magnitude of the specific antibody response to Rh antigens varies greatly in Rh-alloimmunized women, which partly reflects the difference in frequency of specific B cells in these individuals.

Characterization of human hybridoma clones isolated from hemophilia patients with specificity for different domains of coagulating factor VIII.

Hemophilia A patients treated with human coagulating factor VIII (FVIII) may develop inhibitory antibodies (inhibitors). Characterization of the inhibitors at the clonal level may help exploring new therapeutic strategies. We have generated lymphoblastoid cell lines (LCLs) producing anti-FVIII antibodies from peripheral blood lymphocytes of hemophilia A patients with high inhibitor titers. We fused the anti-FVIII-positive LCLs with a heteromyeloma, to produce FVIII specific hybridomas. We determined the specificity, isotype, idiotypic and immunoglobulin (Ig) variable region heavy (VH) chain gene family profiles of the secreted antibodies (Ab) by ELISA, immunoblotting and RT-PCR. We established eight hybridomas which produced high titers of anti-FVIII Ab. All hybridomas secreted IgM Ab, associated with either kappa(5/8) or lambda(3/8) light chain. Analysis of the expressed VH genes by RT-PCR revealed that the hybridomas utilized only the VH1 (63%) or the VH3 (37%) gene families. Among the cross-reactive idiotypes (CRIs) we tested, only the VH1 and VK3b-associated CRIs were expressed by 3 hybridomas. Immunoblotting of thrombin-digested FVIII demonstrated distinct patterns of reactivity of the monoclonal Ab (MAb) secreted by the hybridomas, which recognized either the A2 domain of the Fvm heavy chain, or the light chain, or both. Our findings suggest that: a) the isotype of the anti-FVIII Ab secreted by LCLs and hybridoma clones (IgM) differs from that of anti-FVIII Ab in vivo, which are predominantly IgG4: this suggests a negative selection of the isotype-switched FVIII-specific B-cells in the periphery of these patients; b) the anti-FVIII Ab have a biased representation of the VH1 gene family, and c) somatic mutations in the VH genes coding for FVIII specificity occur in the anti-FVIII Ab response, as evidenced by lack of expression of the VH-associated CRI.

Kinetics of cytokine gene expression in human CD4+ and CD8+ T-lymphocyte subsets using quantitative real-time PCR.

The time kinetics of five cytokines [interleukin-2 (IL-2), IL-5, interferon-gamma (IFN-gamma), granulocyte macrophage-colony stimulating factor (GM-CSF) and tumour necrosis factor-alpha (TNF-alpha)] and one cytotoxic effector protein (granzyme B) was analysed by real-time quantitative polymerase chain reaction (PCR) following in vitro stimulation of human CD4 and CD8 T lymphocytes. Two stimuli were used, a mitogen [phytohemagglutinin (PHA)] and a recall antigen [purified protein derivative (PPD)]. The pattern of cytokine mRNA expression was found to be dependent on the T-cell subset and stimulus used. A wide interindividual variability in the cytokine gene expression pattern was demonstrated. Two expression patterns were observed. A bell-shaped expression profile was seen for most cytokines upon PHA activation in both subsets and PPD-activated CD4 T cells, whereas a biphasic/multiphasic expression pattern was noted in CD8 T cells upon PPD stimulation. For most cytokines, the time to induction was within 30 min of activation, and maximum accumulation seemed to be obtained after 4-8 h of activation. A sustained high level could, however, be noticed for up to 24 h. Granzyme B gene expression was also induced within 30 min of activation but showed a continuous gradual increase and late maximal accumulation (48-72 h). The findings of the present study are of importance when designing studies using the cytokine gene expression profile as a marker for antigen-specific T lymphocytes. It might be recommended that cytokine gene expression (IL-2, IL-5 and IFN-gamma) should be measured after 4-8 h of specific activation but also up to 24 h of stimulation is acceptable. Granzyme B should preferentially be measured after 48-72 h of activation.

Analysis of T-cell receptor beta chain variable gene segment usage in healthy adult responders and nonresponders to recombinant hepatitis B vaccine.

One to 10 per cent of healthy adult individuals do not produce protective levels of anti-hepatitis B surface (HBs) antibodies, following a standard vaccination protocol. Lack of an HBs antigen (Ag)-specific T-cell repertoire is amongst the possible defects, which may lead to humoral unresponsiveness and is the main objective of this study. We analysed TcR BV (T-cell receptor beta chain variable) gene usage in T lymphocytes from nine healthy adult responders and six nonresponders to recombinant HB vaccine, before and after booster vaccination. CD4+ and CD8+ T-cell populations were isolated from peripheral blood mononuclear cells by magnetic beads, and the expression of TcR BV genes in each population was investigated by reverse transcription polymerase chain reaction and hybridization with specific probe. When the usage of each TcR BV gene within CD4+ and CD8+ T cells of the responders was compared with that of nonresponders, statistically significant difference (P < 0.01) was noted for BV5S2-3 gene family in CD4+ T cells of nonresponders. Furthermore, individual vaccinees were shown to overexpress several TcR BV genes. To characterize the T-cell repertoire and determine their clonal nature, analysis of CDR3 length polymorphism was performed. Our results show that T-cell response to HBsAg is generally oligoclonal and involves multiple BV families. Furthermore, overexpressed individual TcR BV genes and CDR3 length distributions in response to HBsAg are subject-dependent. In conclusion, our results are not in line with the notion that defective TcR repertoire may be an explanation for unresponsiveness to recombinant HBsAg vaccine.

Establishment of human heterohybridoma and lymphoblastoid cell lines specific for the Rh D and C antigens.

Human monoclonal antibodies specific for the D antigen of the Rh system are valuable tools for blood group typing and prevention of erythroblastosis. In this study, peripheral blood lymphocytes obtained from an Rh-negative woman immunized with Rh-positive fetuses were immortalized with Epstein-Barr virus (EBV), and transformed lymphoblastoid cell lines (LCLs) secreting antibodies to Rh antigens were generated. The presence of specific antibody was assessed by direct haemagglutination using Rh-positive, papain-treated red blood cells (RBCs), and the production of human antibody was assayed by enzyme-linked immunosorbent assay (ELISA). Specificities of the antibodies were determined by a panel of RBCs of known Rh phenotypes. Five LCLs produced antibody specific for the D antigen, and one LCL showed specificity towards the C antigen of the Rh blood group system. High-titre anti-Rh antibody-producing LCLs were subsequently selected and fused with a human x mouse heteromyeloma cell line. A hybridoma line producing human antibody of the immunoglobulin M (IgM) isotype, which strongly reacted with the D antigen, was established. The hybridoma was cloned, and the monoclone has been stable for growth and antibody production during 8 months of continuous culture, with a mean antibody concentration of 11.5 microg mL-1 and haemagglutination titre of 1/20 480. This antibody was not able to agglutinate a sample of native weak D RBCs (Du); however, agglutination was achieved with papain-treated Du RBCs. Immunoprecipitation of the D antigen by this antibody, followed by Western blot analysis, did not reveal any immobilized D-specific polypeptide. As this human antibody readily agglutinates D+ RBCs in saline, it has the potential to be used as an efficient reagent in routine blood group typing.

Generation and characterization of a mouse monoclonal antibody with specificity similar to staphylococcal protein A (SPA).

Human IgG is comprised of four subclasses (IgG(1), IgG(2), IgG(3), and IgG(4)). Each subclass possesses different biological properties. One of the differential specificities of human IgG subclasses is binding of Fc fragment of IgG(1), 2, and 4 but, not IgG(3) to staphylococcal protein A (SPA). This study was conducted to produce, select and characterize a monoclonal antibody (MAb) recognizing human IgG subclasses with specificity similar to SPA. Splenocytes from Balb/c mice immunized with Fc fraction of a human IgG(1) myeloma protein were fused with Sp2/0 myeloma cells. Fused cells were grown in hypoxanthine, aminopterine, and thymidine (HAT) selective medium and cloned by limiting dilution assay. Antibody-secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAb was further analyzed, using a panel of purified myeloma proteins by ELISA and immunoblotting. A murine hybridoma designated 6F11E1 was obtained that secretes an MAb specific for the Fc fragment of the immunizing protein. This MAb reacts with isotypic epitope common to IgG(1), 2 and 4 subclasses. An allelic epitope linked to IgG(3) molecules is also recognized by 6F11E1. This pattern of reactivity was found to be highly similar to that of SPA. Our findings imply that similar or overlapping epitopes are recognized by 6F11E1 and SPA.

The antibody response to HBs antigen is regulated by coordinated Th1 and Th2 cytokine production in healthy neonates.

A proportion of healthy neonates fail to produce protective levels of anti-HBs antibody following vaccination with recombinant hepatitis B vaccine. This study was undertaken to investigate contribution of Th1 and Th2 responses to anti-HBs antibody production and to explore the mechanism(s) of unresponsiveness to HBsAg in human neonates. Peripheral blood manonuclear cells (PBMCs) were isolated form 28 nonresponder (anti-HBs antibody <10 IU/l) and 25 responder neonates. The cells were stimulated in vitro with recombinant HBsAg and PHA mitogen and concentrations of IL-4, IL-10 and IFN-gamma were quantified in culture supernatants by sandwich ELISA. Our results demonstrated significantly increased production of all cytokines, including IL-4 (P < 0.001), IL-10 (P < 0.002) and IFN-gamma (P < 0.01) in responder compared to nonresponder vaccinees. No significant differences, however, were observed between the two groups of neonates in the levels of cytokines induced by PHA or secreted in absence of antigen and mitogen. Our findings suggest that unresponsiveness to recombinant HBsAg in healthy neonates is linked to inadequate secretion of both Th1 and Th2 cytokines.

Morphological and cytochemical characteristics of purified murine splenic dendritic cells.

Dendritic cells function as the main cellular population responsible for professional antigen presentation and hence for induction of primary immune responses. Although they are present in virtually every tissue, nevertheless their number is usually so low that it makes their isolation for studies very difficult. In this study, we purified dendritic cells from mouse spleen by a three-step enrichment method and evaluated morphological and cytochemical characteristics of isolated cells. We showed that isolated dendritic cells from mouse spleen had all lobulated nuclei with multiple cytoplasmic projections and their morphological features changed after an overnight incubation. It was also shown that typical dendritic cells lacked both Myeloperoxidase (MPO) and Non Specific Esterase (NSE) activity. In conclusion, for reaching a reasonable purity in isolation of dendritic cells from lymphoid tissues, many enrichment steps should be taken, and for determining the purity of isolated cells, we recommend that a combination of morphological and cytochemical studies be used.