A Therapeutic Oral Vaccine Candidate against Propionibacterium acnes: Preparation and Immunological Evaluation of Nanoparticles Encapsulated Sialidase-CAMP Fusion Protein

Abstract Acne Vulgaris is a common skin disease caused by Propionibacterium acnes, an anaerobic microbiota of human skin that plays a vital role in the pathology of acne. The aim of this study was to prepare nanoparticles containing an acne recombinant protein and determine its ability as an oral acne vaccine in mice. The recombinant Sialidase-CAMP gene was expressed and purified in a prokaryotic host. The chitosan nanoparticles containing the recombinant protein were prepared, encapsulated, and administered by both oral and subcutaneous routes to Balb/c mice. Sera IgA and IgG and stool IgA titers were measured by ELISA, and the immunized mice were challenged against P. acnes. A 65 kDa recombinant protein was confirmed by SDS-PAGE and western blot. The size and zeta potential of nanoparticles were 80 nm and +18 mV, respectively. After oral immunization, the serum IgG and IgA titers were 1:3200 and 1:16, respectively, and the stool IgA titer was 1:8. In the subcutaneous route, the serum IgG titer was 1:51200. Immunized mice showed no inflammation in the ear of challenged mice. It is the first study that examines a chitosan-nanoparticulated acne fusion protein as an applicable acne vaccine candidate with appropriate immunogenicity potential. Further studies are required to validate the clinical usefulness of this vaccine candidate.

Reconhecimento molecular na doença de chagas do ponto de vista do parasita e do hospedeiro / Molecular recognition in Chagas disease from the point of view of the parasite and the host

A doença de Chagas, causada pelo parasita protozoário Trypanosoma cruzi, afeta milhões de pessoas, a maioria delas vivendo na América latina. Apesar dos avanços da medicina e da biotecnologia, ainda existem poucas opções de tratamento para indivíduos com a doença. Assim, é importante compreendermos os detalhes moleculares da infecção parasitária, para que novas alternativas terapêuticas e de diagnóstico possam ser desenvolvidas para esses pacientes. Neste trabalho estudamos esta doença em duas frentes, uma do ponto de vista do parasita, e a outra, da resposta do hospedeiro. Utilizando bioinformática, identifcamos um peptídeo conservado (denominado TS9) presente nas proteínas de superfície gp85/transsialidases do parasita. Este peptídeo é capaz de promover adesão celular e, na sua forma sintética, inibe a entrada do T. cruzi na célula hospedeira. Análise da estrutura proteica revelou que o peptídeo TS9 encontra-se num domínio do tipo laminina-G, lado-a-lado com o peptídeo FLY, outro peptídeo conservado desta grande família, previamente descrito pelo nosso grupo. Juntos, eles formam um sítio de adesão a citoqueratinas e proteínas de flamento intermediário. Na segunda parte, investigamos os antígenos e epítopos reconhecidos pelas imunoglobulinas de pacientes portadores da doença nas suas diferentes formas clínicas: assintomática e cardiomiopatias, leve ou grave. Criamos uma biblioteca de phage display contendo, virtualmente, todos os fragmentos proteicos existentes no T. cruzi, que foi varrida contra imunoglobulinas para a construção de um mapa da resposta humoral dos pacientes com a doença de Chagas. Nossos resultados mostram que a resposta dos pacientes é complexa, e mais de dois mil epítopos foram mapeados. Muitos deles, como os antígenos B13, SAPA e FRA já foram previamente descritos, validando nosso método. Porém, um grande número de novos epítopos, inclusive contra proteína descritas como hipotéticas ou sem função conhecida, também foram encontrados. Seus papéis na infecção e resposta imune da doença merecem, portanto, atenção. Em resumo, as abordagens e técnicas utilizadas nesta tese são inovadoras, e permitiram a identificação de peptídeos e moléculas que poderão ser úteis para o desenvolvimento de novos métodos diagnósticos e terapêuticos para a doença de Chagas

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, afects millions of people, most of them living in Latin America. Despite advances in medicine and biotechnology, there are still few treatment options for individuals with the disease. Thus, it is important to understand the molecular details of the parasitic infection, so that new therapeutic and diagnostic alternatives can be developed for these patients. In this work, we study this disease in two fronts, one from the point of view of the parasite, and the other, of the response of the host. Using bioinformatics, we identifed a conserved peptide (called TS9) present in the surface proteins gp85 / trans-sialidases of the parasite. This peptide is capable of promoting cell adhesion and, in its synthetic form, inhibits the entry of T. cruzi into the host cell. Analysis of the protein structure revealed that the TS9 peptide is in a laminin-G-like domain, side-by-side with the peptide FLY, another conserved peptide of this large family, previously described by our group. Together, they form an adhesion site to cytokeratins and intermediate flament proteins. In the second part, we investigated the antigens and epitopes recognized by the immunoglobulins of patients with the disease in their diferent clinical forms: asymptomatic and cardiomyopathies, mild or severe. We created a phage display library containing virtually all existing protein fragments in T. cruzi. This library was screened against immunoglobulins for the construction of a humoral response map of patients with Chagas disease. Our results show that the response of the patients is complex, and more than 2,000 epitopes have been mapped. Many of them, such as the B13, SAPA and FRA antigens have been previously described, validating our method. However, a large number of new epitopes, including many against proteins described as hypothetical or with no known function, were also found. Their roles in infection and immune response of the disease deserve, therefore, attention. In summary, the approaches and techniques used in this thesis are innovative and have allowed the identifcation of new peptides and molecules that may be useful for the development of new diagnostic and therapeutic methods for Chagas disease

An update on Gardneralla vaginalis associated bacterial vaginosis in Malaysia

Objeetive:To update the status of Gardnerella vaginalis (G.vaginalis) as a causative agent of bacterial vaginosis (BV) in Malaysia and to define its epidemiology,metronidazole resistance and virulence properties.Methods:It is a single-centre (Gynaecology clinic at the Hospital Kuala Lumpur,Malaysia) prospective study with laboratory-based microbiological follow up and analyses.Vaginal swabs collected from the patients suspected for BV were subjected to clinical BV diagnosis,isolation and identification of G.vaginalis,metronidazole susceptibility testing,vaginolysin and sialidase gene PCR,Piot's biotyping and amplified ribosomal DNA restriction analysis genotyping.Results:Among the 207 patients suspected for BV,G.vaginalis was isolated from 47 subjects.G.vaginalis coexisted with Trichomonas vaginalis and Candida albicans in 26 samples.Three G.vaginalis isolates were resistant to metronidazole.Biotyping revealed 1 and 7 as the common types.Amplified ribosomal DNA restriction analysis genotype Ⅱ was found to be more common (n =22;46％) than Ⅰ (n =12;25.53％) and Ⅲ (n =13;27.6％).All genotype Ⅰ and Ⅲ isolates carried the sialidase gene,while 91.6％ and 84.6％ contained the vaginolysin gene.Genotype Ⅰ was significantly associated with postgynaecological surgical complications and abortions (P =0.002).Conclusions:The existence of pathogenic G.vaginalis clones in Malaysia including drug resistant strains should not be taken lightly and needs to be monitored as these may bring more complications especially among women of child bearing age and pregnant women.

An update on Gardneralla vaginalis associated bacterial vaginosis in Malaysia

Objective To update the status of Gardnerella vaginalis (G. vaginalis) as a causative agent of bacterial vaginosis (BV) in Malaysia and to define its epidemiology, metronidazole resistance and virulence properties. Methods It is a single-centre (Gynaecology clinic at the Hospital Kuala Lumpur, Malaysia) prospective study with laboratory-based microbiological follow up and analyses. Vaginal swabs collected from the patients suspected for BV were subjected to clinical BV diagnosis, isolation and identification of G. vaginalis, metronidazole susceptibility testing, vaginolysin and sialidase gene PCR, Piot's biotyping and amplified ribosomal DNA restriction analysis (ARDRA) genotyping. Results Among the 207 patients suspected for BV, G. vaginalis was isolated from 47 subjects. G. vaginalis coexisted with Trichomonas vaginalis and Candida albicans in 26 samples. Three G. vaginalis isolates were resistant to metronidazole. Biotyping revealed 1 and 7 as the common types. ARDRA genotype II was found to be more common (n = 22; 46%) than I (n = 12; 25.53%) and III (n = 13; 27.6%). All genotype I and III isolates carried the sialidase gene, while 91.6% and 84.6% contained the vaginolysin gene. Genotype I was significantly associated with post-gynaecological surgical complications and abortions (P = 0.002). Conclusions The existence of pathogenic G. vaginalis clones in Malaysia including drug resistant strains should not be taken lightly and needs to be monitored as these may bring more complications especially among women of child bearing age and pregnant women.

A mouse model of bacterial vaginosis established by infecting estrogen-treated mice with Prevotella bivia / 中华微生物学和免疫学杂志

Objective To establish a mouse model of bacterial vaginosis ( BV) by infecting estro-gen-treated mice with Prevotella bivia ( P. bivia) . Methods The mice were intraperitoneally injected with beta-estradiol 17 valerate which was suspended in sesame oil and then inoculated with different doses of P. bivia strains at the logarithmic phase. Samples of vaginal flushing fluid were collected at different time points after inoculation and used for the isolation of P. bivia strains and the detection of sialidase activities. Altogether 30 mice treated with estrogen and high dose of P. bivia were killed on days 2, 7 and 21 (n=10). Samples of cornua uteri, bladder and kidney were collected from those mice for P. bivia strains isolation. Re-sults Injection the estrogen-treated mice with P. bivia via vagina could cause the P. bivia infection for more than 14 days. The numbers of P. bivia strains isolated on day 21 decreased significantly. Enhanced sialidase activities and clue cells were observed in vaginal secretions of mice with P. bivia infection. Injection of mice with the high dose of P. bivia could spread the infection to cornua uteri. Conclusion Estrogen-treated mice could be used as an animal model for researches on BV.

Quantum Chemical Investigation of C12 and C6 Position of Oseltamivir Sialidase Antiviral Inhibitor.

The ab initio and DFT investigation of C12 & C6 position of oseltamivir sialidase inhibitor reveals that the absence of pyranose oxygen ring in the inhibitor structure drastically increases binding affinity of the inhibitor in relation to the pyranose based inhibitors. The investigation further reveals that the methyl and ethyl group at the C12 position have substantial binding affinity due to their inherent hyperconjugative and charge transfer effects between C4 and C13 bond. The analysis at C6 position of oseltamivir inhibitor discloses that the methyl amine group increases the binding affinity due to their strong hydrogen bonding tendency with the vicinity receptors. Hence, the investigation validates that the 12-methyl-oseltamivir, 12-ethyl-oseltamivir and 6-methylamine-oseltamivir inhibitor become the potential candidate for the development effective sialidase antiviral inhibitors.

Trans Sialidase Genes Allow Clustering of TcI Trypanosoma cruzi Mexican Isolates.

Aims: Polymorphism of the trans sialidase (TS) family of genes is common in Trypanosoma cruzi. Our goal was to cluster Mexican TcI DTU (Discrete Typing Unit) using a set of primers specific for TS genes. Methodology: The DNA of 12 Mexican T. cruzi stocks (TcI) and reference strain were amplified using the CRP-1 primer, which anneals to the conserved 5' ends of CRP (Complement Regulatory Protein), TS, and FL-160 genes, and the CRP-2 primer, which anneals to conserved region within the GPI (Glycosil Phosphatidil Inositol) anchor sequence. Amplicons were analysed using PCR-SSCP (Single Strand Conformation Polymorphims) followed by construction of a nominal matrix data (presence/absence bands) to calculated the Jaccard and Dice similarity coefficient, and clustering with UPGMA. Results: Mexican TcI stocks produced a common pattern of amplification products and cluster in a separate group to CL-Brener strain (TcVI). The PCR-SSCP revealed that within the TcI Mexican stocks there were a complex pattern, but T. cruzi from the Yucatan peninsula clustered in special and separate group. Conclusion: The CRP-1 and CRP-2 primers were helpful for the analysis of genetic traits in T. cruzi DTU I and revealed the existence of special group in Yucatan Mexico.

Relationship between human membrane associated sialidase ( Neu3 ) and multidrug resistance (MDR) in K562 cell line / 基础医学与临床

Objective To investigate the role of human membrane associated sialidase (Neu3) in multidrug resistance ( MDR) of K562 cells. Methods K562 cells and K562/ADM cells were treated with DNR alone or with combination of DNR and NeuAC2en. The cell survival rate was measured by MTT assay; the expression of MDR related factors and apoptosis related factors were determined by Western blot and RT-PCR; Neu3 activity was detected by TBA reaction. Results The survival rate of K562/ADM cells was higher than that of K562 cells; NeuAC2en showed synergistic effect with DNR(P <0. 01) ; Neu3 activity of K562 and K562/ADM cells decreased after DNR or NeuAC2en induction and the decrease was most significant in the combination treatment group (P <0. 01). Under the identical condition, the protein expression of P-gp and Neu3 and the mRNA expression of MDR1, Neu3, BCL-xl and BCL-2 increased comparing with K562 cells. The mRNA level of BAX in K562 and K562/ADM groupsdid not changed significantly. MDR1, Neu3,BCL-xl and BCL-2 decreased after DNR induction and down-regulated most obviously in the groups treated by DNR combined with NeuAC2en. Conclusion Neu3 may be related with multidrug resistance of K562/ADM cell through regulating apoptosis and the expression of MDR1.

Application of fast detection reagent kit to diagnosis of bacterial vaginosis / 医疗卫生装备

Objective To investigate the feasibility of reagent kits of sialidase test in the diagnosis of bacterial vaginosis(BV).Methods Vaginal secretion of women was detected by applying BV reagent kits which was produced by Li Tuo Co.,Ltd.in Zhuhai.Amsel test was used as gold standard.Results In BV test,sensitivity was 93.81%,specificity was 98.29%,false positive rate was 1.14%,and false negative rate was 6.19%.Conclusion The detection by reagent kit can be used as a clinical diagnostic target.It is suitable to routine detection,and worthwhile popularized.