Cellular Targeting of Engineered Heterologous Antigens Is a Determinant Factor for Bovine Herpesvirus 4-Based Vaccine Vector Development

In a previous study, an apathogenic strain of bovine herpesvirus 4 (BoHV-4) cloned as a bacterial artificial chromosome and expressing a chimeric peptide (gE2/gD) as a secreted form was described. Recombinant virus-inoculated animals produced antibodies against bovine viral diarrhea virus (BVDV) gE2 and BoHV-1 gD. However, neutralizing antibodies were produced only against BVDV, not against BoHV-1. In the present work a recombinant BoHV-4 expressing a membrane-linked form of gE2/gD chimeric peptide was constructed, and inoculated rabbits produced serum-neutralizing antibodies against both BVDV and BoHV-1. Protein cell sorting and targeting are a very important issue when immunodominant antigens are engineered for recombinant virus vaccine development.Bovine viral diarrhea virus (BVDV) is a member of the family Flaviviridae and is one of the three viruses in the Pestivirus genus of importance in veterinary medicine, including hog cholera virus and border disease virus. BVDV is a virus frequently associated with several manifestations in cattle ranging from unapparent infections to disease of high mortality (4). Bovine herpesvirus 1 (BoHV-1) is another very important herpesvirus pathogen for the cattle industry and causes significant economic losses worldwide (2). Infection is accompanied by various clinical manifestations, such as infectious bovine rhinotracheitis, infectious pustular vulvovaginitis, balanoposthitis, abortion, and generalized systemic infection. BoHV-1 is known to play an important role in the bovine respiratory disease complex, commonly referred to as shipping fever (2). Inflammation and necrosis of respiratory epithelia and immunosuppression often lead to increased susceptibility to secondary viral and bacterial infections, resulting in severe clinical disease. For both pathogens, vaccination remains the most important tool in terms of prevention, and novel vaccines are desired. The use of recombinant viral vaccines, although still far from reality, seems to be the most promising in terms of their safety and efficacy, and bovine herpesvirus 4 (BoHV-4), due to its biological characteristics, has been suggested to be a good candidate (8, 16).In a previous work (8), an apathogenic Movar-like strain of BoHV-4 was isolated from the cell milk fraction of a healthy cow and its genome was cloned as a bacterial artificial chromosome (BAC) and manipulated to express as a secreted form a chimeric peptide (gE2/gD) made by the fusion of BVDV gE2 and the BoHV-1 gD immunodominant ectodomain. Infected rabbits produced antibodies against both BVDV and BoHV-1, but the serum-neutralizing fraction of such antibodies was detected only for BVDV. Because the cellular location of antigens expressed by DNA-based vaccines has been shown to modulate the immune response (17), in the present work a membrane-linked version of the chimeric peptide (gE2/gD-TM) expressed by a BoHV-4-based vector was constructed and compared with the secreted one. Inoculated rabbits successfully produced serum-neutralizing antibodies against BoHV-1 and BVDV.     

Two Spectrophotometric Assays for Dopamine Derivatives in Pharmaceutical Products and in Biological Samples of Schizophrenic Patients Using Copper Tetramine Complex and Tri-iodide Reagent

Two simple, rapid, and sensitive spectrophotometric methods areproposed for the determination of levodopa (LD). The first methodis based on coupling of 4-aminoantipyrine (4-AAP) with one of thedopamine derivatives (LD, CD) to give a new ligand that reactswith copper tetramine complex to give intenselycolored chelates. The colored products are quantifiedspectrophotometrically at 525 and 520nm for LD and CD,respectively. The optimization of the experimental conditions isdescribed. The method has been used for the determination of19.7–69.0 and 18.1–54.3μg mL⁻¹ of LDand CD, respectively. The accuracy of the method is achieved bythe values of recovery (100 ± 0.2%) and the precision issupported by the low standard deviation (SD = 0.17–0.59) andrelative standard deviation (CV = 0.4%–1.54%) values. Thesecond method is based on the formation of ion-pair iodinatedinner sphere or outer sphere colored complexes between the LDand triiodide ions at pH 5 and room temperature (23 ± 3^°C). This method has been used for the determination of LDwithin the concentration range 39.44–78.88μgmL⁻¹ with SD = 0.22–0.24 and recovery percent = 100 ± 0.3%.The sensitivity of the two methods is indicated bySandell's sensitivity of 0.014–0.019g cm⁻². Theresults of the two methods are compared with those of theofficial method. The interference of common drug additives,degradation products, and excipients was also studied. Theproposed methods were applied successfully to the determinationof the LD-CD synthetic mixture and Levocare drug. Thedetermination of LD in urine of some schizophrenic patients wasapplied with good precision and accuracy. The reliability of themethods was established by parallel determinations against theofficial British pharmacopoeia method.     

Electrocardiographic abnormalities of left ventricular repolarization: Prognostic implications in hypertensive post-menopausal women

Background

Although repolarization abnormalities on ECG are frequent in post-menopausal hypertensive women, their prognostic value in these women is uncertain.

Methods

We analyzed 908 hypertensive post-menopausal women consecutively included in the PIUMA (Progetto Ipertensione Umbria Monitoraggio Ambulatoriale) study. The median duration of follow-up was 8.6 years (range: 1–21). All women were untreated at entry. Drug treatment during follow-up was adjusted to single individuals. Standard 12-lead ECG was carried out at entry. The Minnesota Coding was used to define minor and major (“typical strain”) repolarization abnormalities.

Design

prospective observational study in essential hypertension.

Results

Mean age at entry was 60 years. At baseline, ECG was normal in 707 women, minor ST-T changes were noted in 152 women, and a typical strain pattern was present in 49 subjects. Predictors of typical strain were age, diabetes and systolic blood pressure (BP). During follow-up there were 119 new cardiovascular (CV) events and 75 all-cause deaths. Typical strain was associated with a threefold higher risk of CV disease (HR: 3.16; 95% CI: 1.59–6.31; p = 0.001) after adjustment for the significant influence of age, diabetes, serum creatinine, systolic BP and HDL-cholesterol. Women with minor LV repolarization abnormalities showed a non-significant excess risk of CV disease when compared with women with normal LV repolarization (HR: 1.25; 95% CI: 0.69–2.26; p = 0.467). Similar results were obtained for all-cause mortality.

Conclusions

Typical strain pattern, an easily detectable marker of altered LV repolarization, identifies post-menopausal hypertensive women at increased risk of CV disease and all-cause mortality.     

Blocking of Histamine Release and IgE Binding to FcεRI on Human Basophils by Antibodies Produced in Camels

Purpose

The production of camel heavy-chain antihuman IgE (huIgE) that has the potential to block IgE-FcεRI interaction and histamine release by basophils.

Methods

Camels were immunized with a synthetic loop peptide (SLP) designed in a multiple antigen peptide system (MAPS) forming SLP-MAPS immunogen. Camel polyclonal antibodies (PCAs) were produced, purified, characterized using Protein A & G, ELISA, and SDS-PAGE, and tested for their potency to block passive sensitization and histamine release of human basophils using flow cytometry (FCM) and ELISA, respectively.

Results

FCM data indicated that camel conventional (IgG1) and heavy chain antibodies (HCAbs; IgG2, and IgG3) had blocking activities of 43.9%, 72%, and 96.6%, respectively. Moreover, both IgG2 and IgG3 achieved remarkable inhibition rates of 93.98% and 97.05% in histamine release, respectively, whereas the IgG1inhibiting activity was 60.05%.

Conclusions

Camel PCAs produced against SLP-MAPS were capable of blocking the IgE-receptor interaction and the release of histamine by basophils with superiority to HCAbs. These findings may pave the way toward the possible use of camel anti-huIgE HCAbs as blocking antibodies in the treatment of IgE-mediated allergy and asthma.     

Does parvovirus infection have a role in systemic lupus erythematosus?

We sought to evaluate a possible link between parvovirus B19 infection and the clinical and laboratory expression of systemic lupus erythematosus (SLE). SLE patients were examined to evaluate their clinical status and disease activity. A complete Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score was obtained for each patient. In addition, we determined the level of systemic involvement throughout the course of the disease. Blood levels of IgM and IgG antibodies to parvovirus B19, levels of anti-dsDNA, C3, and C4 were measured. A PCR real-time assay was used to determine the presence of parvovirus B19 genetic material. The viral genome was found in sera of 2 of 51(3.9%) patients with SLE. There was no correlation between viral serology and the clinical and serological parameters of the disease. More SLE patients with secondary antiphospholipid syndrome (APS) had IgG and IgM antibodies to the virus (p < 0.029 and p < 0.018, respectively). These patients also had a higher titer of IgG antibodies to parvovirus B19 compared to SLE patients without APS. In this group of SLE patients, no association was found between parvovirus infection and the presence or activity of SLE. The results of the study suggest an association between parvovirus infection and antibody production directed against phospholipids.

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Systemic sclerosis and cryoglobulinemia: Our experience with overlapping syndrome of scleroderma and severe cryoglobulinemic vasculitis and review of the literature

Objective

Systemic sclerosis (SSc) is an immune-mediated disorder characterized by multiple organ fibrotic alterations and diffuse microangiopathy. The SSc can be associated with other connective tissue diseases and less frequently with systemic vasculitides, including cryoglobulinemic vasculitis (CV). The aim of the present study was to investigate the prevalence of CV in a large series of SSc patients.

Methods

The presence of serum cryoglobulins was detected in 246 SSc patients (24 M and 222 F, age 61 ± 13.5 SD years, disease duration 9.3 ± 6.7 SD years); the observed clinico-serological findings, in particular the presence of SSc–CV overlapping syndrome, were carefully analyzed and compared with previous data reported in the literature.

Results

The presence of circulating cryoglobulins was found in 7/246 (2.8%) of SSc patients; namely, 2 subjects only trace amounts of cryoglobulins, while 5 (2%) showed mixed cryoglobulinemia (type II, IgG-IgMk), low C4, rheumatoid factor seropositivity, and hepatitis C virus infection. Among SSc patients with serum mixed cryoglobulins, 4 (1.6%) developed a clinically overt CV, while the other one was totally asymptomatic with regard to typical vasculitic manifestations. Patients with SSc–CV overlapping syndrome had limited cutaneous SSc with serum anticentromere antibodies, pulmonary hypertension, clinico-serological features of HCV-related CV, and non-healing skin ulcers of the lower limbs. In all cases, the diagnosis of SSc preceded the clinical onset of CV, from 3 to 17 years. The treatment with rituximab was useful on skin ulcers of lower limb in 2/3 patients; however, the overall clinical outcome of the four SSc–CV patients was unusually severe: one with very severe skin ulcers complicated by gangrene required bilateral through-the knee amputation, the other three subjects died because of severe heart failure, and in two cases because of untreatable pulmonary hypertension.In the literature, the prevalence of mixed cryoglobulinemia in scleroderma patients is quite rare (range 0.3–2%); while, the association of SSc with clinically overt CV is only anecdotally described, always in the absence of HCV infection.

Conclusion

The SSc–CV overlapping syndrome described here is characterized by markedly severe vascular manifestations responsible for very poor prognosis; these peculiar clinical manifestations suggest a synergic activity of typical scleroderma microangiopathy and cryoglobulinemic vasculitis.     

Histogram Analysis of Hepatobiliary Phase MR Imaging as a Quantitative Value for Liver Cirrhosis: Preliminary Observations

Purpose

To investigate whether histogram analysis of the hepatobiliary phase on gadoxetate enhanced-MRI could be used as a quantitative index for determination of liver cirrhosis.

Materials and Methods

A total of 63 patients [26 in a normal liver function (NLF) group and 37 in a cirrhotic group] underwent gadoxetate-enhanced MRI, and hepatobiliary phase images were obtained at 20 minutes after contrast injection. The signal intensity of the hepatic parenchyma was measured at four different regions of interest (ROI) of the liver, avoiding vessels and bile ducts. Standard deviation (SD), coefficient of variation (CV), and corrected CV were calculated on the histograms at the ROIs. The distributions of CVs calculated from the ROI histogram were examined and statistical analysis was carried out.

Results

The CV value was 0.041±0.009 (mean CV±SD) in the NLF group, while that of cirrhotic group was 0.071±0.020. There were statistically significant differences in the CVs and corrected CV values between the NLF and cirrhotic groups (p<0.001). The most accurate cut-off value among CVs for distinguishing normal from cirrhotic group was 0.052 (sensitivity 83.8% and specificity 88.5%). There was no statistically significant differences in SD between NLF and cirrhotic groups (p=0.307).

Conclusion

The CV of histograms of the hepatobiliary phase on gadoxetate-enhanced MRI may be useful as a quantitative value for determining the presence of liver cirrhosis.     

Detection of Circulating Anti-Mucin 1 (MUC1) Antibodies in Breast Tumor Patients by Indirect Enzyme-Linked Immunosorbent Assay Using a Recombinant MUC1 Protein Containing Six Tandem Repeats and Expressed in Escherichia coli

Mucin 1 (MUC1), a tumor-associated antigen, is a transmembrane glycoprotein expressed by normal epithelial cells and overexpressed by carcinomas of epithelial origin. Autoantibodies against MUC1 are often found in circulation, either free or bound to immune complexes, which might contribute to limit tumor outgrowth and dissemination by antibody-dependent cell-mediated cytotoxicity, and were found favorably predictive of survival in early breast cancer patients. There is no commercial enzyme-linked immunosorbent assay (ELISA) kit for detecting the anti-MUC1 antibodies in human serum thus far. To detect circulating anti-MUC1 antibodies, we established an indirect ELISA (I-ELISA) using a recombinant MUC1 protein containing six tandem repeat sequences of MUC1 after the antigenicity and specificity of the protein were confirmed. The I-ELISA had a sensitivity of 91.3% and a specificity of 94.1% when a competitive I-ELISA was used as a reference test. The results showed that more patients with benign breast tumors (P = 0.001) and breast cancer patients before primary treatment (P = 0.010) were found to have anti-MUC1 IgG than healthy women; anti-MUC1 IgG before primary treatment was found more than after primary treatment (P = 0.016) in breast cancer patients. Interestingly, the anti-MUC1 IgG serum level was reversely correlated to that of CA15-3 antigen in advanced-stage patients (r = −0.4294, P = 0.046). Our study has demonstrated the suitability of the established I-ELISA for detecting circulating anti-MUC1 antibodies in human serum. Furthermore, we found that circulating anti-MUC1 antibodies may still bind MUC1 shed into blood in stage IV breast cancer, which can support the use of MUC1-target immune therapy strategies.Mucin 1 (MUC1), also called cancer antigen 15-3 (CA15-3) or polymorphic epithelial mucin, is a transmembrane glycoprotein with variable number tandem repeats (VNTR) of a 20-amino-acid motif as its large extracellular fragment. The repeat units contain potential O glycosylation sites represented by serine and threonine residues, which act as a scaffold for the attachment of O-glycans, resulting in the formation of a highly glycosylated extended repetitive structure (22). CA15-3 is defined as the glycoprotein that binds with two monoclonal antibodies (MAbs): DF3 and 115D8. The DF3 antibody recognizes the VNTR of MUC1 (sequence DTRPAPGS), which corresponds to amino acids Asp-Thr-Arg-Pro-Ala-Pro-Gly-Ser. The 115D8 MAb is the solid-phase capture antibody, which binds to a peptide-carbohydrate epitope on the same repeat (11). As a tumor-associated antigen, MUC1 is overexpressed on various carcinomas of epithelial origin, including breast cancer, pancreatic cancer, ovarian cancer, and multiple myeloma, etc. Because of its deficient glycosylation with exposed VNTR in cancer cells, MUC1 can behave as a self-antigen to stimulate an immune response, which provides evidence for vaccine immunotherapy of targeting MUC1 (6, 19, 29).Free and compound autoantibodies against MUC1 can be detected both in patients with malignant tumors and in healthy people (2, 17, 24). Studies have demonstrated that circulating anti-MUC1 antibodies may be used as a favorable prognostic factor for patients with early breast cancer and pancreatic cancer (7, 25). In addition, previous studies have shown that the antibodies might contribute to limit tumor outgrowth and dissemination by antibody-dependent cellular cytotoxicity (1, 8, 28). It is believed that free anti-MUC1 antibodies can bind MUC1 and form MUC1 circulating immune complexes (MUC1-CIC) in blood circulation (3); however, patients with stage IV of breast cancer present low MUC1-CIC, although more common anti-MUC1 antibodies and MUC1 exist in their sera (4, 26). A contradictory result indicated that anti-MUC1 antibodies in stage IV of breast cancer could not bind or neutralize MUC1 antigen, and they were of low affinity (4).Thus far, there is no commercial enzyme-linked immunosorbent assay (ELISA) kit for detecting the anti-MUC1 antibodies in human serum. Mostly, synthetic MUC1 VNTR peptides were used as coating antigens in ELISA for detecting anti-MUC1 antibodies in human sera (13, 27). Alternatively, recombinant MUC1 VNTR containing peptide was also used as antigen for detecting circulating anti-MUC1 antibodies by Western blotting (9). Although the recombinant MUC1 VNTR containing peptide expressed in Escherichia coli cannot be glycosylated as in eukaryotic cells, it has been demonstrated to be efficient in detecting anti-MUC1 antibody because MUC1 is less or not glycosylated when expressed in tumor cells.In the present study, we constructed a recombinant MUC1 protein, 8R-MUCPT, which contained six MUC1 VNTRs. After the antigenicity and specificity of the 8R-MUCPT were verified, we established an indirect ELISA (I-ELISA) using 8R-MUCPT as a coating antigen to detect anti-MUC1 antibodies in the sera of patients with benign breast tumors and breast cancer. The results have demonstrated the potential of this recombinant MUC1 protein as detecting antigen and the suitability of the established I-ELISA for detecting circulating anti-MUC1 antibodies. In addition, the results suggested that anti-MUC1 antibodies in serum may play a role in neutralizing MUC1 VNTR core peptides and forming MUC1-CIC. By analyzing the relationship between circulating MUC1 and anti-MUC1 antibodies in advanced-stage patients, we were able to deduce the same neutralizing role for the antibodies in stage IV breast cancer.     

Abnormal vaginal microbioma is associated with severity of localized provoked vulvodynia. Role of aerobic vaginitis and <Emphasis Type="Italic">Candida</Emphasis> in the pathogenesis of vulvodynia

Localized provoked vulvodynia (LPV) causes introital dyspareunia in up to 14% of premenopausal women. Vaginal infections like candidosis may play a initiating role. The aim of this study was to test a possible association of vaginal microbiota alternations such as Candida vaginitis (CV), aerobic vaginitis (AV) and bacterial vaginosis (BV) with severity of vulvodynia and painful intercourse. In an observational study, Q-tip touch test (score 1 (no pain) to 10 (worst possible pain)) was performed on seven vestibular locations in 231 LPV patients presenting in the Vulvovaginal Disease Clinics in Tienen, Leuven and Antwerp, Belgium. Severity of pain upon attempting sexual intercourse was recorded in a similar scale. Both scales were compared to results from fresh wet mount phase contrast microscopy on vaginal fluid smears tested for abnormal vaginal flora (AVF), BV, AV and CV according the standardized microscopy method (Femicare). Fisher’s exact test was used. Average age was 31.3?±?11.6 years, and 58.8% (n?=?132) had secondary vestibulodynia. There was an inverse relation between the presence of Candida in the vaginal smears and pain score (p?=?0.03). There was no relation of pain score, nor Q-tip score with BV. LPV patients with Q-tip score above 7 at 5 and/or 7 o’clock or at 1 and/or 11 o’clock had more often AV than women with lower pain scores (30 vs 14.5%, p?=?0.01, and 39 vs 14.7%, p?<?0.005, respectively). Detailed study of the vaginal microflora in patients demonstrates that the most severe patients suffer more from AV and less from Candida. These abnormalities need to be actively looked for and corrected before considering surgery or other therapies.     

Serological study in Tunisian cervical cancer patients

Objective

The aim of this study is to use a novel ELISA, based on five recombinant HPV-16 and HPV-18 proteins, for detection HPV-specific antibodies in a case-control study.

Patients and methods

L1, E6 and E7 genes have been over expressed in Escherichia coli as double fused proteins. These recombinant proteins were used in a GST-capture ELISA as coating antigens. Human sera were collected from women with cervical cancer. Negative human sera were collected from patients apparently healthy and may be affected by other infectious agents.

Results

Most of the sera showed a positive reactivity to at least one of the HPV-16 or HPV-18 proteins (52/71). A percentage of 39.50% of the sera from HPV-16 infected women and 21.12% of the sera from women infected by HPV-18 genotype recognised at least one of the HPV-16 or HPV-18 proteins. Sera showed different reactivity to L1, E6 and E7 antigens, and only a few serum samples reacted to L1, E6 and E7 HPV-16, E6 and E7 HPV-18 (co-infection). Differences of reactivity between cases and controls were significant (P < 0.0001).

Conclusion

This novel ELISA, based on recombinant HPV-16 and HPV-18 antigens, is able to detect antibodies in women infected by HPV genotypes. The assay is easy to perform and has low cost, making it suitable for monitoring the natural history of HPV infections as well as for detecting pre-existing HPV antibodies in women who receive vaccination.     

RAPD and SSR Based Genetic Diversity Analysis of Elite-2 Set of Synthetic Hexaploid Wheats

Background

Synthetic hexaploid wheats are artificially reconstituted hexaploid wheats that possess high genetic variation which could be utilized for the development of new improved wheat varieties. One such group of synthetic wheats is called the Elite-II set of synthetic wheats that are derived from crossing durum wheat with different Aegilops tauschii wheats.

Materials and Methods

In the current study genetic diversity was investigated among 18 Elite-II synthetic hexaploid wheat lines at DNA level. Two types of molecular markers i.e. RAPD and SSR were used for this purpose.

Results

Both types of markers proved useful in estimating the overall genetic diversity among these lines. Based on RAPD data range of genetic distances in these lines was from 0 to 100 percent. Seven D-genome specific SSRs were also used to get further estimation of the genetic diversity contributed by Aegilops tauschii parent. On the basis of results obtained it is inferred that the Aegilops tauschi accessions used in the production of these synthetic lines were genetically different and they contributed to the enhancement of genetic variation in the synthetic lines. These results could be helpful for future genome mapping programs.

Conclusion

The overall extensive genitive diversity indicates that these lines are good candidates for development of improved wheat varieties by crossing with cultivated wheat varieties.     

Application of histone modification-specific interaction domains as an alternative to antibodies

Post-translational modifications (PTMs) of histones constitute a major chromatin indexing mechanism, and their proper characterization is of highest biological importance. So far, PTM-specific antibodies have been the standard reagent for studying histone PTMs despite caveats such as lot-to-lot variability of specificity and binding affinity. Herein, we successfully employed naturally occurring and engineered histone modification interacting domains for detection and identification of histone PTMs and ChIP-like enrichment of different types of chromatin. Our results demonstrate that histone interacting domains are robust and highly specific reagents that can replace or complement histone modification antibodies. These domains can be produced recombinantly in Escherichia coli at low cost and constant quality. Protein design of reading domains allows for generation of novel specificities, addition of affinity tags, and preparation of PTM binding pocket variants as matching negative controls, which is not possible with antibodies.The unstructured N-terminal tails of histones protrude from the core nucleosome and harbor complex patterns of post-translational modifications (PTMs) (Kouzarides 2007; Margueron and Reinberg 2010; Bannister and Kouzarides 2011; Tan et al. 2011). These PTMs regulate a multitude of chromatin-templated transactions, play a central role in development, and are implicated in many diseases, such as cancer (Suva et al. 2013). Therefore, understanding the role of histone marks in chromatin-dependent processes is of paramount importance. So far, techniques based on the specific binding of antibodies to modified histone proteins have been the only method available for genome-wide analyses of histone modifications with locus-specific resolution. The central role of antibodies for the characterization of histone PTMs in chromatin research makes the quality and reliability of these reagents a very important scientific issue. In general, antibodies are very powerful and important reagents in biomolecular research, but the validation of commercial antibodies is not always sufficiently rigorous (Bordeaux et al. 2010). This is particularly important in the chromatin field, where specific recognition and discrimination of subtle epitopes defined only by the presence of distinct PTMs is required. Moreover, several important modifications occur in very similar amino acid sequence motifs, like the methylation of H3K9 and H3K27, which are both placed in the context of an ARKS sequence. In addition, histone tails are hypermodified, as exemplified by the H3 tail, where the adjacent R8, K9, and S10 amino acid side chains are known to be methylated, acetylated, or phosphorylated. This implies that secondary modifications often occur on the peptide segment contacted by the antibody in the immediate vicinity of the target PTM and sometimes prevent the binding of antibodies in spite of the presence of the target modification, yielding false negative results. When undocumented, the cross-reactivity with related or unrelated marks and the combinatorial effect of neighboring marks compromise the application of antibodies, as illustrated in Figure 1. Additionally, different antibodies show distinct profiles of false positive and false negative signals, and even antibodies with the same catalog numbers regularly show lot-to-lot fluctuations of properties (also illustrated in Fig. 1). This variability is not unexpected for polyclonal antibodies, where new batches are produced by immunization of a new animal, but changes of purification procedure may cause variance of properties of monoclonal antibodies as well. Occasionally some lots of commercial antibodies even prefer to bind to secondary targets (see Fig. 1 and H3K36me3 antibodies documented in Bock et al. 2011a). This necessitates a detailed quality control and documentation of each antibody and each lot in order to give the user all relevant information for correct data interpretation, which is often not sufficiently provided. The urgency for better quality assessment and documentation of antibodies used in chromatin research has been widely recognized in the field (Bock et al. 2011a; Egelhofer et al. 2011; Fuchs et al. 2011; Nishikori et al. 2012; Peach et al. 2012; Hattori et al. 2013; Heubach et al. 2013), and the ENCODE Project Consortium has set up quality criteria for histone PTM antibodies (Egelhofer et al. 2011; Landt et al. 2012). According to these guidelines, antibodies must specifically detect modified histones in Western blots and fulfill one or more of the following secondary criteria: (1) specific binding to modified peptides in dot blot assays; (2) mass spectrometric detection of the modification in precipitated chromatin; (3) loss of signal upon knockdown of the corresponding histone modifying enzyme; (4) reproducibility of ChIP-seq; (5) similarity of ChIP-seq results of two different antibodies directed against the same modification; or (6) overlap of ChIP-seq peaks with expected genomic annotations.Open in a separate windowFigure 1.Peptide array analyses showing lot-to-lot fluctuations, cross-reactivity, and effects of proximal marks on the binding of popular histone tail antibodies. Peptide spots are annotated on the left side of the glass slide. The color-coded boxes denote the presence of the designated modifications. (A) Anti-H3K36me3 antibodies showed significant cross-reactivity to H4K20me1, H4K20me3, H3K27me3, H3K9me3, and H3K36me2. All lots of the H3K36me3 antibody had a weak binding specificity, and they showed very different properties. (B) Anti-H3K9me3 antibodies displayed cross-reactivity to H3K27me3 (Lot 3 even prefers H3K27me3 peptides) and H4K20me3 (very overt in the case of Lot 1 and Lot 4). H3K9me3 peptide spots that were not bound by these antibodies generally also contained H3S10ph or H3T11ph, indicating that this secondary mark prevents binding. For a more detailed annotation of the modifications at each spot, refer to Supplemental File S2. Among the H3K9me3 antibodies, only Lot 2 showed a binding specificity of acceptable quality. Anti-H3K36me3 (Lot 1) and anti-H3K9me3 (Lot 1) data were taken from Bock et al. (2011a) and reprocessed. Additional examples of commercial H3K36me3 antibodies with insufficient specificity are shown in Bock et al. (2011a). Examples of the fundamental differences in the specificity profiles of different antibodies directed against H3K27me3 are given in Supplemental Figure S1.To develop an alternative to antibodies for chromatin research, we assessed the applicative potential and utility of naturally occurring and engineered histone modification interacting domains (HMIDs). This approach has several distinct advantages over antibodies, such as the ease and cost-effectiveness of recombinant production of HMIDs in Escherichia coli, the amenability of HMIDs to protein engineering, and the possibility of producing them at constant quality, eliminating lot-to-lot variability. In support of this concept, affinity methods based on protein domains have been successfully employed for the enrichment of methylated or unmethylated CpG islands in the analysis of DNA methylation (Cross et al. 1994; Blackledge et al. 2012) or in proteome-wide analyses of non-histone lysine methylation (Liu et al. 2013; Moore et al. 2013). In this proof-of-principle study, we started by characterizing the specificity of several HMIDs and compared them with ENCODE-validated antibodies for the same PTM using peptide arrays. Specificities were further validated in Western blots by detecting histone tail PTMs using unmodified histones and histones specifically depleted with the target PTM as controls. Then, we investigated the applicative potential of HMIDs in ChIP-like experiments to enrich for chromatin with particular modifications, which represents one of the most important and commonly used applications of histone tail PTM antibodies.     

Relationship of critical velocity to marathon running performance

The purpose of this investigation was to evaluate the critical velocity (CV) test for prediction of marathon running performance. Twelve subjects [mean age (SD) = 29 (4) years; mean body mass = 63 (13) kg] were tested for CV and completed the 1994 New York City Marathon. The CV (m?·?s^?1) was determined from times to exhaustion at four treadmill running velocities. In addition, peak oxygen consumption ( O _{2 peak}; ml?·?kg^?1?·?min^?1) and ventilatory threshold (Th_vent) were determined from an incremental treadmill test. The Th_vent was calculated using bi-segmental linear regression and was expressed as the velocity (m?·?s^?1) at Th_vent. Separate simple linear regression analyses showed that marathon time [MT; mean (SD) = 231.9 (27.4) min] correlated more highly with CV [MT = 445.3 – 50.3 (CV); r ² = 0.76, SEE = 14.1 min] than either O_2peak [MT = 390.7 – 2.7 ( O_2peak); r ² = 0.51, SEE = 20.1 min] or Th_vent [MT = 353.5 – 30.1 (Th_vent) r ² = 0.28, SEE = 27.4 min]. A stepwise regression analysis resulted in CV (entered first) and Th_vent being included in the prediction equation [MT = 443.5 – 78.9 (CV) + 34.3 (Th_vent), R ² = 0.88, SEE = 10.7 min], while O_2peak was not included. These preliminary data indicate that the CV test may be an attractive field test for assessing marathon performance capabilities.     

Peptide Mimic Isolated by Autoantibody Reveals Human Arrest Defective 1 Overexpression Is Associated with Poor Prognosis for Colon Cancer Patients

Tumor-associated antigens, which induce the generation of autoantibodies, are useful as cancer biomarkers in early detection and prognostic prediction of cancer. To isolate a novel cancer marker, we used serum antibodies from colon cancer patients to screen a phage display peptide library. A positive peptide 249C (VPLYSNTLRYGF) that could specifically react with serum from colon cancer patients was isolated, and the corresponding antigen–human arrest defective 1 (ARD1A), which shares an identical LYSNTL motif with 249C, was identified. Both immunological assays and three-dimensional structure analysis showed that the LYSNTL region is an epitope of ARD1A. Using ELISA and immunohistochemistry, we found anti-ARD1A antibody levels in serum from patients with colon cancer were significantly higher than those in healthy volunteers (P < 0.001), and ARD1A expression was detected in 84.1% (227/270) of colon cancer tissues compared with 22.7% (55/242) of matched noncancerous tissues (P < 0.001) and 4.8% (2/42) of benign lesions (P < 0.001). Furthermore, multivariate analysis with Cox proportional hazards regression models revealed that ARD1A-positive patients had significantly shortened overall survival (OS) (HR, 1.91, P = 0.039) and borderline significantly shortened disease-free survival (DFS) (HR, 1.70; P = 0.068). Kaplan–Meier survival curves also showed that ARD1A expression was associated significantly with shortened DFS (P = 0.037) and OS (P = 0.019). These results indicate that ARD1A is a novel tumor-associated antigen and a potential prognostic factor for colon cancer.Colon cancer is one of the most common cancers worldwide and is the second leading cause of cancer death in developed countries.¹ The 5-year survival rate of patients with colon cancer has improved during the past decade because of advances in screening and systemic treatment, but the survival rate remains relatively poor in patients with stage III and IV cancer.² Colon cancer is treated mainly by surgery, and it would be beneficial if the prognosis after initial surgery could be predicted. Although the tumor-node-metastasis (TNM) system is helpful for this purpose,² it is not always correlated with outcome of the cancer patients. To predict the prognosis more exactly, new molecular markers are needed to be exploited.Considerable evidence has shown that an immune response in the form of autoantibodies to various tumor antigens was developed in patients with cancer.^3,4,5,6,7 Thus, autoantibodies in the serum of patients with colon cancer could be used to isolate specific tumor-associated antigens. More sensitive and specific molecular markers have the potential to improve the preclinical diagnosis of primary and recurrent colon cancer, as well as hold the promise of prognostic value. Phage display libraries have proved to be a useful tool for identifying autoantigens or epitopes recognized by antibodies.^8,9,10,11,12 Selection of peptide libraries with mAbs and mixed serum from patients with disease has led to the isolation of immunoreactive peptide epitopes.^4,12,13,14 The strategy has also led to the identification of some tumor-associated antigens from phage display libraries using patient serum.^4,12In the present study, by screening a phage display peptide library with serum antibodies from patients with colon cancer, together with bioinformatic analysis and a series of immune experiments, we first revealed that human arrest defective 1 (ARD1A) may serve as an indicator of unfavorable prognosis in colon cancer.     

Posttranslational modifications of α-tubulin of Toxoplasma gondii

The posttranslational modifications of -tubulin of Toxoplasma gondii were characterized by antibodies and biochemical analysis of the carboxy-terminal peptide. -Tubulin is acetylated and glutamylated. Side chains with up to three glutamate residues are linked to Glu445 of T. gondii -tubulin. The data suggest that the site of glutamylation on -tubulin is conserved over a broad range of species.     

Inexpensive Designer Antigen for Anti-HIV Antibody Detection with High Sensitivity and Specificity

A novel recombinant multiepitope protein (MEP) has been designed that consists of four linear, immunodominant, and phylogenetically conserved epitopes, taken from human immunodeficiency virus (HIV)-encoded antigens that are used in many third-generation immunoassay kits. This HIV-MEP has been evaluated for its diagnostic potential in the detection of anti-HIV antibodies in human sera. A synthetic MEP gene encoding these epitopes, joined by flexible peptide linkers in a single open reading frame, was designed and overexpressed in Escherichia coli. The recombinant HIV-MEP was purified using a single affinity step, yielding >20 mg pure protein/liter culture, and used as the coating antigen in an in-house immunoassay. Bound anti-HIV antibodies were detected by highly sensitive time-resolved fluorometry, using europium(III) chelate-labeled anti-human antibody. The sensitivity and specificity of the HIV-MEP were evaluated using Boston Biomedica worldwide HIV performance, HIV seroconversion, and viral coinfection panels and were found to be comparable with those of commercially available anti-HIV enzyme immunoassay (EIA) kits. The careful choice of epitopes, high epitope density, and an E. coli-based expression system, coupled with a simple purification protocol and the use of europium(III) chelate-labeled tracer, provide the capability for the development of an inexpensive diagnostic test with high degrees of sensitivity and specificity.Human immunodeficiency virus (HIV) is a lentivirus of the family Retroviridae, whose members characteristically have an RNA genome within a capsid and a lipid envelope. HIV infection induces a profound immune dysfunction, with abnormalities in every arm of the immune system, resulting in AIDS (5). In 2007, there were 2.7 million new HIV infections and 2 million HIV-related deaths. Globally, there were an estimated 33 million people living with HIV in 2007. India is one of the largest and most populated countries in the world, with a population of over 1 billion. Of this number, it is estimated that around 2.4 million Indians were living with HIV in 2007 (26). The genes of HIV are located in the central region of the proviral DNA and encode at least nine proteins. These proteins are divided into three classes: the major structural proteins (Gag, Pol, and Env), the regulatory proteins (Tat and Rev), and the accessory proteins (Vpu, Vpr, Vif, and Nef) (11). The gag gene of HIV type 1 (HIV-1) encodes a polyprotein precursor, p55, which is cleaved by the virus-encoded protease into three proteins, p24, p17, and p15. Linear B-cell epitopes have already been identified within p24 (14). The antigen p24 is of special significance because of its ability to be expressed first in body fluids after HIV-1 infection. The linear immunodominant epitope of p24 serves as an important diagnostic intermediate to detect antibodies to HIV-1 in human sera (23). The envelope glycoproteins (gp), gp41 of HIV-1 and gp36 of the closely related HIV-2, are highly immunogenic and are important diagnostic intermediates for the detection of antibodies to these viruses in human sera (17, 24). HIV-1 comprises three lineages, denoted M, N, and O (22). HIV-2 and divergent forms have been detected in West African or West Africa-related patients with AIDS (7-9). Several enzyme immunoassay (EIA)-based diagnostic kits are available on the market for the detection of antibodies to HIV in human sera. These anti-HIV EIA kits use synthetic peptides and/or recombinant proteins mainly from the envelope gp of HIV-1 group M, HIV-1 group O, and HIV-2. The fourth-generation kits also have antibodies to p24 antigen. The requirement of multiple peptides and/or multiple recombinant proteins for reliable diagnosis of HIV infections adds to the cost of these EIA kits. The high cost of anti-HIV EIA kits becomes prohibitive for routine use in many developing countries, precluding early detection and prevention of new infections (18, 25, 27). We have designed a single recombinant multiepitope protein (MEP) antigen, consisting of several immunodominant, linear, and conserved virus-specific epitopes from structural proteins of HIV-1 and HIV-2. DNAs encoding these epitopes have been assembled in tandem in a single open reading frame, with intervening sequences encoding flexible linkers, and expressed in Escherichia coli. A polyhistidine tag has also been included which allows for facile purification of recombinant MEP by Ni-NTA chromatography. The purified protein has been used as the coating antigen for developing an anti-HIV indirect immunoassay. We have evaluated the performance of this assay with that of other multiple-antigen-based immunoassay kits currently available on the market, using well-characterized commercially available serum panels.     

Complex Serology and Immune Response of Mice to Variant High-Molecular-Weight O Polysaccharides Isolated from Pseudomonas aeruginosa Serogroup O2 Strains

The O antigen of the Pseudomonas aeruginosa lipopolysaccharide is the optimal target for protective antibodies, but the unusual and complex nature of their sugar substituents has made it difficult to define the range of these structures needed in an effective vaccine. Most clinical isolates of P. aeruginosa can be classified into 10 O-antigen serogroups, but slight chemical differences among O polysaccharides within a serogroup give rise to subtype epitopes. These epitopes could impact the reactivity of O-antigen-specific antibodies, as well as the susceptibility of a target strain to protective, opsonic antibodies. To define parameters of serogroup and subtype-epitope immunogenicity, antigenicity, and surface expression on P. aeruginosa cells, we prepared high-molecular-weight O-polysaccharide vaccines from strains of P. aeruginosa serogroup O2, for which eight structurally variant O antigens expressing six defined subtype epitopes (O2a to O2f) have been identified. A complex pattern of immune responses to these antigens was observed following vaccination of mice. The high-molecular-weight O polysaccharides were generally more immunogenic at low doses (1 and 10 μg) than at a high dose (50 μg) and usually elicited antibodies that opsonized the homologous strain for phagocytic killing. Some of the individual polysaccharides elicited cross-opsonic antibodies to a variable number of strains that express all of the defined serogroup O2 subtype epitopes. Combination into one vaccine of two antigens that individually elicited cross-reactive opsonic antibodies to most members of the O2 serogroup inhibited, instead of enhanced, the production of antibodies broadly reactive with most serogroup O2 subtype strains. Thus, immune responses to P. aeruginosa O antigens may be restricted to a limited range of epitopes on structurally complex O antigens, and combining multiple related antigens into a single vaccine formulation may inhibit the production of those antibodies best able to protect against most P. aeruginosa strains within a given O-antigen serogroup.It has been established through animal and human experimentation that the lipopolysaccharide (LPS) O antigen of Pseudomonas aeruginosa is a target for protective antibodies (3, 36, 38). The studies of Knirel and colleagues (17, 19) on the chemical composition and structure of the major O-side-chain polysaccharides have provided important insights into the immunochemical properties of these antigens, but our understanding of their antigenic and immunogenic properties is incomplete. This point is highlighted by the inability to date to develop effective, LPS-specific immunotherapies for human P. aeruginosa infection (7).Results obtained with animals by using immunogens and antibodies specific to the O polysaccharides have indicated that slight chemical differences among bacterial strains with otherwise closely related O-side-chain structures can produce a complex pattern of reactions between antibodies and related antigens (13). With standard serologic methods using whole-cell agglutinations, strains of P. aeruginosa can be classified as members of one serogroup (serotype); members of each serogroup share a group-specific antigen. Further subdivision into subtypes, which correlate with structural variants determined by Knirel and colleagues (17), can be accomplished with appropriate antisera (22).To develop safe and effective O-antigen-specific P. aeruginosa vaccines, we have utilized the high-molecular-mass (>100,000-Da) fraction of O polysaccharides. These antigens are safe and immunogenic in humans and animals (13, 27, 37) and elicit protective antibodies to the strains from which they are isolated. However, in recent studies of animals immunized with a heptavalent high-molecular-weight O-polysaccharide vaccine whose individual components were isolated from single strains representative of the major serogroups causing P. aeruginosa infection, opsonic antibody responses to the group-specific antigens were not commonly elicited (13). Thus, in spite of chemical and serologic relatedness among subtype strains within a P. aeruginosa serogroup, single antigens isolated from one subtype strain do not always elicit opsonic antibodies to all of the strains within the serogroup (13). Previous results showed that a particular O antigen from a given serogroup may elicit group-specific immunity, while an O antigen from another serogroup may elicit only immunity specific to the subtype epitopes expressed on that particular O antigen.To explore this situation further and gain additional insight into the serologic diversity among P. aeruginosa LPS O antigens, we prepared high-molecular-weight O-polysaccharide immunogens from five strains of P. aeruginosa serogroup O2 that, together, express all six of the identified subtype antigens (Table ​(Table1).1). These polysaccharides were used to immunize mice, and the resultant sera were assessed by enzyme-linked immunosorbent assay (ELISA) and for opsonic killing activity. The results showed a complex interaction among the strains with regard to high-molecular-weight O-polysaccharide immunogenicity, antigenicity, serogroup and subtype epitope density, and susceptibility to opsonic killing. These findings indicate that the current serogroup classifications of P. aeruginosa are probably inadequate to define the full range of LPS antigens needed to elicit comprehensive immunity to a wide range of clinical isolates.

TABLE 1

Strains used for immunogen production, their serologic classification by subtype epitope, and chemical structures of the associated O antigens Open in a separate window^aBoldface type indicates a feature of a structure that distinguishes it from a related structure of the same serogroup. Abbreviations: FucNAc, 2-acetamido-2,6-dideoxygalactose (N-acetylfucosamine); Man(NAc)₂A, 2,3-diacetamido-2,3-dideoxymannuronic acid; Man(2NAc3N)A, 2-acetamido-3-acetamidino-2,3-dideoxymannuronic acid; Gul(NAc)₂A, 2,3-diacetamido-2,3-dideoxyguluronic acid. ^bThe lower structure is also part of the O antigen of strain 170007; there is about a 2:1 ratio of the upper and lower structures.      

Production of monoclonal antibodies to thyroglobulin by in vitro immunization with a free synthetic peptide

A synthetic peptide corresponding to amino acids 1-19 of thyroglobulin was used to test the possibility of generating protein-reactive monoclonal antibodies by immunization in vitro with a synthetic peptide as antigen. Splenocytes from non-immunized Balb/c mice were cultured in serum-free medium for 3 days in the presence of thymocyte-conditioned medium and the synthetic peptide prior to fusion with SP2/0 murine myeloma cells. The synthetic peptide was used in its free form, i.e. not coupled to a protein carrier. Hybridomas secreting monoclonal antibodies reactive with the synthetic peptide were obtained after immunization in vitro with as little as 10 ng/ml of the synthetic peptide. Between 50 and 70% of the primary clones obtained in different experiments produced monoclonal antibodies also reactive with the intact protein. Six stable hybridomas were isolated; all produced antibodies of the IgM class. We conclude that immunization in vitro with a free synthetic peptide is an efficient method for the generation of monoclonal antibodies reactive with the intact protein.     

Immune Abnormalities Induced by Human Endogenous Retroviral Peptides: With Reference to the Pathogenesis of Systemic Lupus Erythematosus

P15E is a specific sequence among the envelope gene (env)-encoded transmembrane proteins of exogenous and endogenous retroviruses. A synthetic peptide (CKS-17) that shows homology to this p15E region in several species of retrovirus is known to induce immune abnormalities. In this study, we examined the effect of a synthetic peptide derived from a region of human endogenous retrovirus (HERV) clone 4-1 ( 4-1) similar to sequences of CKS-17 on the induction of systemic lupus erythematosus (SLE)-related immune abnormalities. Our results indicated that this peptide could induce T-cell activation and anergy in normal peripheral blood mononuclear cells, and the peptide could also promote the production of interleukins IL-6 and IL-16. These phenomena are representative immune abnormalities observed in SLE patients. Thus, our findings support the possibility that HERV acts as a pathogen in human SLE.     

Effect of Synthetic Truncated Apolipoprotein C-I Peptide on Plasma Lipoprotein Cholesterol in Nonhuman Primates

The present studies were conducted to determinewhether a synthetic truncated apoC-I peptide thatinhibits CETP activity in baboons would raise plasmaHDL cholesterol levels in nonhuman primates with lowHDL levels. We used 2 cynomolgus monkeys and 3baboons fed a cholesterol- and fat-enriched diet. Incynomolgus monkeys, we injected synthetic truncatedapoC-I inhibitor peptide at a dose of 20mg/kgand, in baboons, at doses of 10, 15, and 20mg/kgat weekly intervals. Blood samples were collected 3times a week and VLDL + LDL and HDL cholesterolconcentrations were measured. In cynomolgus monkeys,administration of the inhibitor peptide caused arapid decrease in VLDL + LDL cholesterolconcentrations (30%–60%) and an increase in HDLcholesterol concentrations (10%–20%). VLDL + LDLcholesterol concentrations returned to baselinelevels in approximately 15days. In baboons,administration of the synthetic inhibitor peptidecaused a decrease in VLDL + LDL cholesterol (20%–60%)and an increase in HDL cholesterol (10%–20%). VLDL+ LDL cholesterol returned to baseline levels byday 21, whereas HDL cholesterol concentrationsremained elevated for up to 26days. ApoA-Iconcentrations increased, whereas apoE andtriglyceride concentrations decreased. Subcutaneousand intravenous administrations of the inhibitorpeptide had similar effects on LDL and HDLcholesterol concentrations. There was no change inbody weight, food consumption, or plasma IgGlevels of any baboon during the study. Thesestudies suggest that the truncated apoC-I peptide canbe used to raise HDL in humans.