A majority of Brazilian patients with rheumatoid arthritis HLA-DRB1 alleles carry both the HLA-DRB1 shared epitope and anti-citrunillated peptide antibodies

The objective of the present study was to evaluate the contribution of the shared epitope (SE), the rheumatoid arthritis (RA) protection model, and the occurrence of anti-cyclic citrullinated peptide (anti-CCP) antibodies in RA patients from a genetically diverse population. One hundred and forty Brazilian RA patients and 161 matched controls were typed for HLA-DRB1 alleles using amplified DNA hybridized with sequence-specific oligonucleotide probes or primers. Patients were stratified according to the presence or absence of SE (DRB1*0401, *0404, *0405, *0101, *1001, and *1402), of the DERAA alleles (DRB1*0103, *0402, *1102, *1103, *1301, *1302, and *1304), and X (all other alleles). Anti-CCP antibodies were measured by ELISA. The combined frequency of SE-positive alleles was significantly greater (76.4 vs 23.6%, P < 0.0001) than the controls. The SE/SE and SE/X genotypes were over-represented (P < 0.0001, OR = 6.02) and DERAA/X was under-represented in RA patients (P < 0.001, OR = 0.49), whereas the frequencies of the SE/DERAA, X/X and X/DERAA genotypes were not significantly different from controls. The frequency of anti-CCP antibodies was higher in SE-positive patients than in SE-negative patients (64.6 vs 44.7%, P = 0.03; OR = 2.25). Although the Brazilian population is highly miscegenated, the results of this study support the findings observed in most genetically homogeneous populations with RA; however, they are not mutually exclusive but rather complementary. The participation of DRB1-DERAA alleles in protection against RA was also observed (OR = 0.4; 95%CI = 0.23-0.68).

Prokaryotic expression of a novel mouse pro-apoptosis protein PNAS-4 and application of its polyclonal antibodies

Mouse PNAS-4 (mPNAS-4) has 96% identity with human PNAS-4 (hPNAS-4) in primary sequence and has been reported to be involved in the apoptotic response to DNA damage. However, there have been no studies reported of the biological functions of mPNAS-4. In studies conducted by our group (unpublished data), it was interesting to note that overexpression of mPNAS-4 promoted apoptotic death in Lewis lung carcinoma cells (LL2) and colon carcinoma cells (CT26) of mice both in vitro and in vivo. In our studies, mPNAS-4 was cloned into the pGEX-6P-1 vector with GST tag at N-terminal in Escherichia coli strain BL21(DE3). The soluble and insoluble expression of recombinant protein mPNAS-4 (rmPNAS-4) was temperature-dependent. The majority of rmPNAS-4 was insoluble at 37°C, while it was almost exclusively expressed in soluble form at 20°C. The soluble rmPNAS-4 was purified by one-step affinity purification, using a glutathione Sepharose 4B column. The rmPNAS-4 protein was further identified by electrospray ionization-mass spectrometry analysis. The search parameters of the parent and fragment mass error tolerance were set at 0.1 and 0.05 kDa, respectively, and the sequence coverage of search result was 28%. The purified rmPNAS-4 was further used as immunogen to raise polyclonal antibodies in New Zealand white rabbit, which were suitable to detect both the recombinant and the endogenous mPNAS-4 in mouse brain tissue and LL2 cells after immunoblotting and/or immunostaining. The purified rmPNAS-4 and our prepared anti-mPNAS-4 polyclonal antibodies may provide useful tools for future biological function studies for mPNAS.

Anti-cyclic citrullinated peptide antibodies and rheumatoid factor in leprosy patients with articular involvement

The objective of the present research was to evaluate the usefulness of anti-cyclic citrullinated peptide (anti-CCP) antibodies and the IgM rheumatoid factor (IgM RF) test for the differential diagnosis of leprosy with articular involvement and rheumatoid arthritis (RA). Anti-CCP antibodies and IgM RF were measured in the sera of 158 leprosy patients (76 with and 82 without articular involvement), 69 RA patients and 89 healthy controls. Leprosy diagnosis was performed according to Ridley and Jopling classification criteria and clinical and demographic characteristics of leprosy patients were collected by a standard questionnaire. Leprosy patients with any concomitant rheumatic disease were excluded. Serum samples were obtained from all participants and frozen at _20°C. Measurement of anti-CCP antibodies and IgM RF were performed by ELISA, using a commercial second-generation kit, and the latex agglutination test, respectively. Anti-CCP antibodies and IgM RF were detected in low frequencies (2.6 and 1.3%, respectively) in leprosy patients and were not associated with articular involvement. Among healthy individuals both anti-CCP antibodies and IgM RF were each detected in 3.4% of the subjects. In contrast, in the RA group, anti-CCP antibodies were present in 81.2% and IgM RF in 62.3%. In the present study, both anti-CCP antibodies and IgM RF showed good positive predictive value for RA, helping to discriminate between RA and leprosy patients with articular involvement. However, anti-CCP antibodies were more specific for RA diagnosis in the population under study.

Antibodies against electronegative LDL inhibit atherosclerosis in LDLr-/- mice

In order to determine the effect of antibodies against electronegative low-density lipoprotein LDL(-) on atherogenesis, five groups of LDL low receptor-deficient (LDLr-/-) mice (6 per group) were immunized with the following antibodies (100 µg each): mouse anti-LDL(-) monoclonal IgG2b, rabbit anti-LDL(-) polyclonal IgG or its Fab fragments and mouse irrelevant monoclonal IgG and non-immunized controls. Antibodies were administered intravenously one week before starting the hypercholesterolemic diet (1.25% cholesterol) and then every week for 21 days. The passive immunization with anti-LDL(-) monoclonal IgG2b, polyclonal antibody and its derived Fab significantly reduced the cross-sectional area of atherosclerotic lesions at the aortic root of LDLr-/- mice (28.8 ± 9.7, 67.3 ± 17.02, 56.9 ± 8.02 µm² (mean ± SD), respectively) compared to control (124.9 ± 13.2 µm²). Vascular cell adhesion molecule-1 protein expression, quantified by the KS300 image-analyzing software, on endothelium and the number of macrophages in the intima was also decreased in aortas of mice treated with anti-LDL(-) monoclonal antibody (3.5 ± 0.70 per field x 10) compared to controls (21.5 ± 3.5 per field x 10). Furthermore, immunization with the monoclonal antibody decreased the concentration of LDL(-) in blood plasma (immunized: 1.0 ± 1.4; control: 20.5 ± 3.5 RLU), the amount of cholesterol oxides in plasma (immunized: 4.7 ± 2.7; control: 15.0 ± 2.0 pg COx/mg cholesterol) and liver (immunized: 2.3 ± 1.5; control: 30.0 ± 26.0 pg COx/mg cholesterol), and the hepatic content of lipid hydroperoxides (immunized: 0.30 ± 0.020; control: 0.38 ± 0.15 ng/mg protein). In conclusion, antibodies against electronegative LDL administered intravenously may play a protective role in atherosclerosis.

Use of anti-PGL-1 antibodies to monitor therapy regimes in leprosy patients

The suitability of IgM antibodies to PGL-1 for monitoring the response to multidrug therapy (MDT) was sequentially tested by ELISA in 105 leprosy patients, and bacterial indexes (BI) were also determined. Patients were divided into 3 groups: group 1, 34 multibacillary (MB) patients treated for 12 months with MDT-MB; group 2, 33 MB patients treated for 24 months with MDT-MB, and group 3, 38 paucibacillary (PB) patients treated for 6 months with MDT-PB. Untreated MB patients exhibited higher antibody levels (mean ± SEM): group 1 (6.95 ± 1.35) and group 2 (12.53 ± 2.02) than untreated PB patients (1.28 ± 0.35). There was a significant difference (P < 0.01) in anti-PGL-1 levels in group 1 patients: untreated (6.95 ± 1.35) and treated for 12 months (2.78 ± 0.69) and in group 2 patients: untreated (12.53 ± 2.02) and treated for 24 months (2.62 ± 0.79). There was no significant difference between untreated (1.28 ± 0.35) and treated (0.62 ± 0.12) PB patients. Antibody levels correlated with BI. The correlation coefficient (Pearson’s r) was 0.72 before and 0.23 (P < 0.05) after treatment in group 1 and 0.67 before and 0.96 (P < 0.05) after treatment in group 2. BI was significantly reduced (P < 0.01) after 12 and 24 months on MDT (group 1: 1.26-0.26; group 2: 1.66-0.36). Our data indicate that monitoring anti-PGL-1 levels during MDT may be a sensitive tool for evaluating treatment efficacy. These data also indicate that the control of leprosy infection can be obtained with 12 months of MDT in MB patients.

Development of two potential diagnostic monoclonal antibodies against human cytomegalovirus glycoprotein B

Human cytomegalovirus glycoprotein B (gB) represents a target for diagnosis and treatment in view of the role it plays in virus entry and spread. Nevertheless, to our knowledge, rare detection of a gB antigen has been reported in transplant patients and limited information is available about diagnostic gB monoclonal antibodies (mAbs). Our aim was to develop gB mAbs with diagnostic potential. Hydrophilic gB peptides (ST: amino acids 27-40, SH: amino acids 81-94) of favorable immunogenicity were synthesized and used to immunize BALB/c mice. Two mAbs, named ZJU-FH6 and ZJU-FE6, were generated by the hybridoma technique and limited serial dilution and then characterized by indirect ELISA, Western blotting, immunoprecipitation, and immunohistochemical staining. The mAbs displayed high titers of specific binding affinities for the ST and SH synthetic peptides at an mAb dilution of 1:60,000 and 1:240,000, respectively. Western blotting and immunoprecipitation indicated that these mAbs recognized both denatured and native gB of the Towne and AD169 strains. The mAbs, when used as the primary antibody, showed positive staining in cells infected with both Towne and AD169 strains. The mAbs were then tested on patients submitted to allogeneic hematopoietic stem cell transplantation. The gB antigen positivity rates of the patients tested using ZJU-FH6 and ZJU-FE6 were 62.0 and 63.0%, respectively. The gB antigen showed a significant correlation with the level of pp65 antigen in peripheral blood leukocytes. In conclusion, two potential diagnostic gB mAbs were developed and were shown to be capable of recognizing gB in peripheral blood leukocytes in a reliable manner.

"A Sequence in Subdomain 2 of DBL1a of Plasmodium falciparum Erythrocyte Membrane Protein 1 Induces Strain Transcending Antibodies"

Immunity to severe malaria is the first level of immunity acquired to Plasmodium falciparum. Antibodies to the variant antigen PfEMP1 (P. falciparum erythrocyte membrane protein 1) present at the surface of the parasitized red blood cell (pRBC) confer protection by blocking microvascular sequestration. Here we have generated antibodies to peptide sequences of subdomain 2 of PfEMP1-DBL1a previously identified to be associated with severe or mild malaria. A set of sera generated to the amino acid sequence KLQTLTLHQVREYWWALNRKEVWKA, containing the motif ALNRKE, stained the live pRBC. 50% of parasites tested (7/14) were positive both in flow cytometry and immunofluorescence assays with live pRBCs including both laboratory strains and in vitro adapted clinical isolates. Antibodies that reacted selectively with the sequence REYWWALNRKEVWKA in a 15-mer peptide array of DBL1a-domains were also found to react with the pRBC surface. By utilizing a peptide array to map the binding properties of the elicited anti-DBL1a antibodies, the amino acids WxxNRx were found essential for antibody binding. Complementary experiments using 135 degenerate RDSM peptide sequences obtained from 93 Ugandan patient-isolates showed that antibody binding occurred when the amino acids WxLNRKE/D were present in the peptide. The data suggests that the ALNRKE sequence motif, associated with severe malaria, induces strain-transcending antibodies that react with the pRBC surface

A sequence in subdomain 2 of DBL1 alpha of plasmodium falciparum erythrocyte membrane protein 1 induces strain transcending antibodies

Immunity to severe malaria is the first level of immunity acquired to Plasmodium falciparum. Antibodies to the variant antigen PfEMP1 (P. falciparum erythrocyte membrane protein 1) present at the surface of the parasitized red blood cell (pRBC) confer protection by blocking microvascular sequestration. Here we have generated antibodies to peptide sequences of subdomain 2 of PfEMP1-DBL1 alpha previously identified to be associated with severe or mild malaria. A set of sera generated to the amino acid sequence KLQTLTLHQVREYWWALNRKEVWKA, containing the motif ALNRKE, stained the live pRBC. 50% of parasites tested (7/14) were positive both in flow cytometry and immunofluorescence assays with live pRBCs including both laboratory strains and in vitro adapted clinical isolates. Antibodies that reacted selectively with the sequence REYWWALNRKEVWKA in a 15-mer peptide array of DBL1 alpha-domains were also found to react with the pRBC surface. By utilizing a peptide array to map the binding properties of the elicited anti-DBL1 alpha antibodies, the amino acids WxxNRx were found essential for antibody binding. Complementary experiments using 135 degenerate RDSM peptide sequences obtained from 93 Ugandan patient-isolates showed that antibody binding occurred when the amino acids WxLNRKE/D were present in the peptide. The data suggests that the ALNRKE sequence motif, associated with severe malaria, induces strain-transcending antibodies that react with the pRBC surface.

16/6-idiotype expressing antibodies induce brain inflammation and cognitive impairment in mice: the mosaic of central nervous system involvement in lupus

Background: The 16/6-idiotype (16/6-Id) of the human anti-DNA antibody was found to induce experimental lupus in naive mice, manifested by production of autoantibodies, leukopenia and elevated inflammatory markers, as well as kidney and brain involvement. We assessed behavior and brain pathology of naive mice injected intracerebra-ventricularly (ICV) with the 16/6-Id antibody. Methods: C3H female mice were injected ICV to the right hemisphere with the human 16/6-Id antibody or commercial human IgG antibodies (control). The mice were tested for depression by the forced swimming test (FST), locomotor and explorative activity by the staircase test, and cognitive functions were examined by the novel object recognition and Y-maze tests. Brain slices were stained for inflammatory processes. Results: 16/6-Id injected mice were cognitively impaired as shown by significant differences in the preference for a new object in the novel object recognition test compared to controls (P = 0.012). Similarly, the preference for spatial novelty in the Y-maze test was significantly higher in the control group compared to the 16/6-Id-injected mice (42% vs. 9%, respectively, P = 0.065). Depression-like behavior and locomotor activity were not significantly different between the16/6-Id-injected and the control mice. Immunohistochemistry analysis revealed an increase in astrocytes and microglial activation in the hippocampus and amygdala, in the 16/6-Id injected group compared to the control. Conclusions: Passive transfer of 16/6-Id antibodies directly into mice brain resulted in cognitive impairments and histological evidence for brain inflammation. These findings shed additional light on the diverse mosaic pathophysiology of neuropsychiatric lupus.

Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge

A major problem in hepatitis C virus (HCV) immunotherapy or vaccine design is the extreme variability of the virus. We identified human monoclonal antibodies (mAbs) that neutralize genetically diverse HCV isolates and protect against heterologous HCV quasispecies challenge in a human liver-chimeric mouse model. The results provide evidence that broadly neutralizing antibodies to HCV protect against heterologous viral infection and suggest that a prophylactic vaccine against HCV may be achievable.

Novel single chain antibodies to the prion protein identified by phage display

A well defined structure is available for the carboxyl half of the cellular prion protein (PrPc), while the structure of the amino terminal half of the molecule remains ill defined. The unstructured nature of the polypeptide has meant that relatively few of the many antibodies generated against PrPc recognise this region. To circumvent this problem, we have used a previously characterised and well expressed fragment derived from the amino terminus of PrPc as bait for panning a single chain antibody phage (scFv-P) library. Using this approach, we identified and characterised I predominant and 3 additional scFv-Ps that contained different V-H and V-L sequences and that bound specifically to the PrPc target. Epitope mapping revealed that all scFv-Ps recognised linear epitopes between PrPc residues 76 and 156. When compared with existing monoclonal antibodies (MAb), the binding of the scFvs was significantly different in that high level binding was evident on truncated forms of PrPc that reacted poorly or not at all with several pre-existing MAbs. These data suggest that the isolated scFv-Ps bind to novel epitopes within the aminocentral region of PrPc. In addition, the binding of MAbs to known linear epitopes within PrPc depends strongly on the endpoints of the target PrPc fragment used. (c) 2006 Elsevier Inc. All rights reserved.

Evaluation of a nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus

As an immunogen of the coronavirus, the nucleoprotein (N) is a potential antigen for the serological monitoring of infectious bronchitis virus (IBV). In this report, recombinant N protein from the Beaudette strain of IBV was produced and purified from Escherichia coli as well as Sf9 ( insect) cells, and used for the coating of enzyme-linked immunosorbent assay ( ELISA) plates. The N protein produced in Sf9 cells was phosphorylated whereas N protein from E. coli was not. Our data indicated that N protein purified from E. coli was more sensitive to anti-IBV serum than the protein from Sf9 cells. The recombinant N protein did not react with the antisera to other avian pathogens, implying that it was specific in the recognition of IBV antibodies. In addition, the data from the detection of field samples and IBV strains indicated that using the recombinant protein as coating antigen could achieve an equivalent performance to an ELISA kit based on infected material extracts as a source of antigen(s). ELISAs based on recombinant proteins are safe ( no live virus), clean ( only virus antigens are present), specific ( single proteins can be used) and rapid ( to respond to new viral strains and strains that cannot necessarily be easily cultured).

Evaluation of the time resolved fluorescence immunoassay (TRFIA) for the detection of varicella zoster virus (VZV) antibodies following vaccination of healthcare workers

Determination of varicella zoster virus (VZV) immunity in healthcare workers without a history of chickenpox is important for identifying those in need of vOka vaccination. Post immunisation, healthcare workers in the UK who work with high risk patients are tested for seroconversion. To assess the performance of the time-resolved fluorescence immunoassay (TRFIA) for the detection of antibody in vaccinated as well as unvaccinated individuals, a cut-off was first calculated. VZV-IgG specific avidity and titres six weeks after the first dose of vaccine were used to identify subjects with pre-existing immunity among a cohort of 110 healthcare workers. Those with high avidity (≥60%) were considered to have previous immunity to VZV and those with low or equivocal avidity (<60%) were considered naive. The former had antibody levels ≥400mIU/mL and latter had levels <400mIU/mL. Comparison of the baseline values of the naive and immune groups allowed the estimation of a TRFIA cut-off value of >130mIU/mL which best discriminated between the two groups and this was confirmed by ROC analysis. Using this value, the sensitivity and specificity of TRFIA cut-off were 90% (95% CI 79-96), and 78% (95% CI 61-90) respectively in this population. A subset of samples tested by the gold standard Fluorescence Antibody to Membrane Antigen (FAMA) test showed 84% (54/64) agreement with TRFIA.