DNA vaccine encoding the moonlighting protein Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) leads to partial protection in a mouse model of human filariasis

River blindness, caused by the filarial parasite Onchocerca volvulus, is a major socio-economic and public health problem in Sub-Saharan Africa. In January 2015, The Onchocerciasis Vaccine for Africa (TOVA) Initiative has been launched with the aim of providing new tools to complement mass drug administration (MDA) of ivermectin, thereby promoting elimination of onchocerciasis in Africa. In this context we here present Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) as a possible DNA vaccine candidate. We report that in a laboratory model for filariasis, immunization with Ov-GAPDH led to a significant reduction of adult worm load and microfilaraemia in BALB/c mice after challenge infection with the filarial parasite Litomosoides sigmodontis. Mice were either vaccinated with Ov-GAPDH.DNA plasmid (Ov-pGAPDH.DNA) alone or in combination with recombinantly expressed Ov-GAPDH protein (Ov-rGAPDH). During the following challenge infection of immunized and control mice with L. sigmodontis, those formulations which included the DNA plasmid, led to a significant reduction of adult worm loads (up to 57% median reduction) and microfilaraemia (up to 94% reduction) in immunized animals. In a further experiment, immunization with a mixture of four overlapping, synthetic Ov-GAPDH peptides (Ov-GAPDHpept), with alum as adjuvant, did not significantly reduce worm loads. Our results indicate that DNA vaccination with Ov-GAPDH has protective potential against filarial challenge infection in the mouse model. This suggests a transfer of the approach into the cattle Onchocerca ochengi model, where it is possible to investigate the effects of this vaccination in the context of a natural host–parasite relationship.     

Bacteroides forsythus ATCC43037菌体蛋白与人类唾液酸性富脯蛋白的相互作用

目的：探讨福赛类杆菌与人类唾液富脯蛋白相互作用的蛋白分子。方法：Western—blot方法。将人工合成唾液富脯蛋白用生物素标记，福赛类杆菌全菌蛋白凝胶电泳，半干转移至纤维膜上，观察二者的相互作用。结果：富脯蛋白能与分子量为85KD、65KD、60KD、以及49KD的福赛类杆菌蛋白发生结合。结论：福赛类杆菌存在与人类唾液富脯蛋白相互结合的粘附素。     

PTEN及Bcl-2蛋白在膀胱移行细胞癌中的表达及其意义

目的：探讨PTEN及Bcl-2蛋白在膀胱移行细胞癌的表达规律及其临床意义。方法：应用免疫组织化学链亲和素-生物素-霉复合物（SP）方法检测43例膀胱移行细胞癌和7例正常黏膜组织中PTEN及Bcl-2蛋白的表达．分析二者的表达与膀胱癌病理参数的关系。结果：（1）G1、G2、G3肿瘤PTEN表达阳性率分别为85．7％、80．4％、73．3％，浸润性肿瘤和表浅性肿瘤表达阳性率分别为70．4％和93．8％，说明PTEN表达阳性率与肿瘤病理分级、临床分期有关。（2）G。、G2、G3肿瘤Bcl-2表达阳性率分别为14．2％、57．14％、66．67％，浸润性肿瘤和表浅性肿瘤表达阳性率分别为59．3％和43．8％，说明Bcl-2表达阳性率与肿瘤病理分级、临床分期有关。（3）随着肿瘤恶性程度的增高和临床分期进展，PTEN蛋白阳性率呈下降，Bcl-2表达呈增高趋势，二者表达呈负相关关系。结论：PTEN的抑癌作用可能与Bcl-2有关．二者的异常表达在膀胱移行细胞癌的发生、发展过程中起重要作用。二者同时检测有助于判断预后。     

Clinical safety of enamel matrix derivative (EMDOGAIN®) in the treatment of periodontal defects

Abstract The aim of the present clinical trial was to test tolerability during 2 treatments with EMDOGAIN® in a large number of patients. An open, controlled study design in 10 Swedish specialist clinics was chosen, with a test group of 107 patients treated with EMDOGAIN® in connection with periodontal surgery at 2 surgical test sites per patient. The procedures were performed 2 to 6 weeks apart on one-rooted teeth with at least 4 mm deep intraosseous lesions. A control group of 33 patients underwent flap surgery without EMDOGAIN® at I comparable site. In total 214 test and 33 control surgeries were performed. Serum samples were obtained from test patients for analysis of total and specific antibody levels. 10 of the patients had samples taken before and after the first surgery. 56 other samples were taken after one treatment with EMDOGAIN®, and 63 after 2 treatments. None of the samples, not even from allergy-prone patients after 2 treatments, indicated deviations from established baseline ranges. This indicates that the immunogenic potential of EMDOGAIN® is extremely low when applied in conjunction with periodontal surgery. Comparison between the test and control groups demonstrated the same type and frequency of post-surgical experiences, i.e., reactions caused by the surgical procedure itself. Clinical probing and radiographic evaluation was performed at baseline and 8 months postsurgery. About half of the patients (44 test and 21 control) were also evaluated after 3 years. There was a significant difference between the test and control results at 8 months post surgery. and this difference had increased further at the 3 year follow-up. The 2.5–3 mm increase in attachment and bone level after treatment with EMDOGAIN® was of the same magnitude as seen in the studies with split-mouth design aiming for lest of effectiveness of EMDOGAIN®.     

Detergent fractionation with subsequent subtractive suppression hybridization as a tool for identifying genes coding for plasma membrane proteins

Abstract:  The identification of tumor-specific proteins located at the plasma membrane is hampered by numerous methodological pitfalls many of which are associated with the post-translational modification of such proteins. Here, we present a new combination of detergent fractionation of cells and of subtractive suppression hybridization (SSH) to gain overexpressed genes coding for membrane-associated or secreted proteins. Fractionation of subcellular components by digitonin allowed sequestering mRNA of the rough Endoplasmatic reticulum and thereby increasing the percentage of sequences coding for membrane-bound proteins. Fractionated mRNAs from the cutaneous T-cell lymphoma (CTCL) cell line HuT78 and from normal peripheral blood monocytes were used for SSH leading to the enrichment of sequences overexpressed in the tumor cells. We identified some 21 overexpressed genes, among them are GPR137B, FAM62A, NOMO1, HSP90, SLIT1, IBP2, CLIF, IRAK and ARC. mRNA expression was tested for selected genes in CTCL cell lines, skin specimens and peripheral blood samples from CTCL patients and healthy donors. Several of the detected sequences are clearly related to cancer, but have not yet been associated with CTCL. qPCR confirmed an enrichment of these mRNAs in the rough endoplasmic reticulum fraction. RT-PCR confirmed the expression of these genes in skin specimens and peripheral blood of CTCL patients. Western blotting verified protein expression of HSP90 and IBP2 in HuT78. GPR137B could be detected by immunohistology in HuT78 and in keratinocytes of dysplastic epidermis, but also in sweat glands of healthy skin. In summary, we developed a new technique, which allows identifying overexpressed genes coding preferentially for membrane-associated proteins.     

Immunogenicity and safety of a novel yeast Hansenula polymorpha-derived recombinant Hepatitis B candidate vaccine in healthy adolescents and adults aged 10–45 years

The aim was to determine whether the immunogenicity of an investigational hepatitis B vaccine (spHB) is at least as high as that of a licensed control vaccine, Engerix B^®, and to evaluate its safety before inclusion in new pediatric combination vaccines. Two randomized, controlled, blind-observer, Phase 3 trials were performed: one in Argentina (344 participants aged 10–15 years, 10 μg HBsAg/dose) and one in Uruguay (344 participants aged 16–45 years, 20 μg HBsAg/dose). Both vaccines were given in a 0, 1, 6 month schedule to all participants with a baseline anti-Hep B antibody titer <0.6 mIU/mL. Antibody titers were measured pre-dose 1, 1 month after dose 2, pre-dose 3, and 1 month after dose 3. Statistical non-inferiority analyses were performed on seroprotection rates (SP) post-dose 3 (% with anti-Hep B titers ≥10 mIU/mL; delta non-inferiority limit of −10%). In both studies, SP for the spHB vaccine was 100% and the spHB vaccine was non-inferior in terms of SP to the licensed control vaccine. GMTs post-dose 3 were approximately 1.8- and 4.1-fold higher for spHB in the 10–15 year and 16–45 year age groups, respectively. Reactogenicity was low for each vaccine, after each dose. This highly immunogenic hepatitis B candidate vaccine was selected for further investigation as a component of new pediatric combination vaccines.     

Recombinant glycoprotein based vaccine for Chandipura virus infection

Chandipura virus (CHPV) has emerged as an important pediatric encephalitis-causing pathogen with very high mortality in India. No specific vaccine or treatment is available till date. We attempted to prepare a candidate vaccine employing recombinant CHPV Glycoprotein (rGp). The Glycoprotein gene (G-gene) of CHPV was expressed using Baculovirus expression system. The rGp was purified by HPLC and used for mice immunization, 3 doses, and 4 weeks apart. One microgram rGp was found to be optimum. Sero-conversion was observed as early as 2nd week by detecting anti-CHPV IgG antibodies. Antibody titres were immunogen-concentration dependent. Intracerebral challenge of the immunized mice with 100 LD₅₀ of the homologous strain demonstrated 90% protection. In in vitro neutralization, antibodies from the immunized mice were able to neutralize heterologous viruses. There was 60% T cell proliferation observed against rGp in immunized mice. The study shows that rGp induces both arms of immune response and represents an ideal vaccine candidate for further evaluations.     

Purification of antigenically intact Ro ribonucleoproteins; biochemical and immunological evidence that the 52-kD protein is not a Ro protein.

Anti-Ro sera immunoprecipitate Ro ribonucleoproteins (RNPs) from human cell extracts. Ro RNPs are biochemically heterogeneous particles whose functions are unknown and whose exact composition remains controversial. In addition to 60-kD Ro and to La proteins, a 52-kD polypeptide (p52) has been proposed to be a stable component of the Ro RNPs. To confirm the immunological studies supporting this hypothesis, we have biochemically purified Ro RNPs from HeLa cells using non-denaturing conditions. Ro RNPs segregated into three distinct populations, one of which only contained hY5 RNA (RohY5 RNPs). No p52 co-purified with Ro RNPs. Despite the absence of p52, purified Ro RNPs had biochemical and immunological properties identical to those of unfractionated Ro RNPs. Many anti-Ro sera only recognize p52 in immunoblots, and are said to be monospecific anti-p52. Preincubation with purified RohY5 RNPs (free of p52) of all human anti-Ro (including so-called monospecific anti-p52) sera abolished their capacity to immunoprecipitate Ro RNPs from unfractionated HeLa cell extracts. Conversely, preincubation of anti-Ro sera with purified p52 protein specifically inhibited recognition of p52 in immunoblots, but did not interfere with immunoprecipitation of Ro RNPs. Our data demonstrate that anti-p52 antibodies do not target intact Ro RNPs, nor do they target the native 60-kD Ro protein. Contrary to previous reports, p52 protein is not a stable component of antigenically intact Ro RNPs.     

Tau protein induces bundling of microtubules in vitro: comparison of different tau isoforms and a tau protein fragment.

Expression of tau protein in non-neuronal cells can result in a redistribution of the microtubule cytoskeleton into thick bundles of tau-containing microtubules (Lewis et al.: Nature 342:498-505, 1989; Kanai et al.: J Cell Biol 109:1173-1184, 1989). We reconstituted microtubule bundles using purified tubulin and tau in order to study the assembly of these structures. Taxol-stabilized tubulin polymers were incubated with various concentrations of recombinant human tau and examined by electron microscopy. With increasing concentrations of tau 3 (tau isoform containing three microtubule binding domains) or tau 4 (isoform containing four microtubule binding domains) the microtubules changed orientation from a random distribution to loosely and tightly packed parallel arrays and then to thick cables. In contrast, tau 4L, the tau isoform containing four microtubule binding domains plus a 58-amino acid insert near the N-terminus, showed minimal bundling activity. tau 4-induced bundling could be inhibited by the addition of 0.5 M NaCl or 0.4 mM estramustine phosphate, conditions which are known to inhibit tau binding to microtubules. A tau construct that contained only the microtubule binding domains plus 19 amino acids to the C-terminus was fully capable of bundling microtubules. Phosphorylation of tau 3 with cAMP-dependent protein kinase had no effect on its ability to induce microtubule bundling. These results indicate that tau protein is directly capable of bundling microtubules in vitro, and suggests that different tau isoforms differ in their ability to bundle microtubule filaments.