Immune response induced by 14-3-3.3 recombinant protein vaccine of Taenia solium in mice / 中华地方病学杂志

Objective:To explore the potential of Taenia solium (Ts) 14-3-3.3 protein as a candidate molecule for cysticercosis vaccine. Methods:Sixty Kunming mice with the body weight of 18 - 22 g were selected and divided into 3 groups according to their body weight via the random number table method, including normal saline control group (control group), Ts14-3-3.3 recombinant protein vaccine group (vaccine group), and Ts14-3-3.3 recombinant protein vaccine + adjuvant group (vaccine + adjuvant group), with 20 mice in each group. The multi-point subcutaneous injection method was adopted. After the first immunization at 0 week, the booster immunization was carried out twice, a total of 3 times, with an interval of 2 weeks. Four mice in the three groups were killed at 0, 2, 4, 6 and 8 weeks after the first immunization, and the blood of eyeballs and spleen were collected aseptically for serum separation and preparation of spleen lymphocytes suspension [treatment: cell suspension, antigen-stimulate and concanavalin (Con) A-stimulate], respectively. The levels of mouse serum specific immunoglobulin (Ig) G, IgG2a, IgG1 and IgE were detected by indirect enzyme-linked immunosorbent assay (ELISA). The proliferation level of mouse spleen lymphocytes was detected via the CCK-8 method. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-12, IL-13 and IL-10 in culture supernatant of mouse spleen lymphocytes were determined by double-antibody sandwich ELISA.Results:The IgG, IgG2a, and IgG1 levels of the vaccine and vaccine + adjuvant groups immunized for 2 to 8 weeks were higher than those of the control group, and the above indicators of the vaccine + adjuvant group were higher than those of the vaccine group ( P < 0.05). With the same treatment between the groups, the proliferation levels of spleen lymphocytes and the levels of TNF-α, IL-12, IL-13, and IL-10 in the culture supernatant after 2 - 8 weeks of immunization were statistically significantly different ( P < 0.05); the proliferation levels of spleen lymphocytes and the levels of TNF-α, IL-12, IL-13 and IL-10 in the culture supernatant of the vaccine and vaccine + adjuvant groups immunized for 2 to 8 weeks were higher than those of the control group, and the above indicators of the vaccine + adjuvant group were higher than those of the vaccine group ( P < 0.05). When treatment was different in the group, the proliferation levels of spleen lymphocytes and the levels of TNF-α, IL-12, IL-13 and IL-10 in the culture supernatant of the antigen-stimulate and ConA-stimulate were higher than those of the cell suspension, and the above indicators of the ConA-stimulate were higher than those of the antigen-stimulate ( P < 0.05). Conclusion:The recombinant protein vaccine of Ts14-3-3.3 can induce an effective immune response in mice.

Cutaneous Cysticercosis - A Rare Case Report.

Cysticercosis is caused by Cysticercus Cellulosae, which is the larval stage of a tapeworm, Taenia Solium in humans. This larva commonly affects the central nervous system, but it can involve eyes, muscles, lungs, liver, subcutaneous tissues and heart with variable clinical manifestations. Here we report a rare case of cutaneous Cysticercosis of the left scapular region without any associated neurological or eye involvement in a 50 year old male patient. He was managed with albendazole and steroids with complete recovery.

Construction and identification of recombinant Mycobacterium smegmatis vaccine expressing Cysticercus cellulosae cC1 antigen / 中国血吸虫病防治杂志

Objective To construct recombinant Mycobacterium smegmatis vaccine expressing Cysticercus cellulosae cC1 anti-gen. Methods The recombinant pET28a-cC1 plasmid was extracted and double digested by Xho I and BamH I restriction en-zymes,and shuttle plasmid pMV261 was extracted and double digested by Hind III and BamH I restriction enzymes. Both frag-ments were modified by Klenow fragment to form blunt end,then the large fragments of cC1 and pMV261 plasmid were purified and ligated by T4 ligase enzyme. The recombinant pMV261-cC1 plasmid was constructed and sequenced. Then the pMV261-cC1 plasmid was transformed into Mycobacterium smegmatis by the electrotransformation method. The recombinant cC1-Mycobacterium smegmatis was induced by heat and identified by the Western blotting method with the sera of cysticercosis patients. In addition, the growth states of the Mycobacterium smegmatis and the recombinant cC1-Mycobacterium smegmatis were compared and the growth curves were drawn. Results The restriction enzyme and sequencing results showed that the recombinant pMV261-cC1 plasmid was successfully constructed. After heat induction,a 40 kD band was showed by PAGE analysis of cC1-Mycobacterium smegmatis. The Western blotting results showed that the sera of cysticercosis patients could recognize the 40 kDa band,which sug-gested that cC1 protein was expressed in Mycobacterium smegmatis. Compared with the Mycobacterium smegmatis,the recombi-nant cC1-Mycobacterium smegmatis showed no significant difference in proliferation characteristics. Conclusion The recombi-nant cC1-Mycobacterium smegmatis vaccine has been successfully constructed.

Diagnóstico laboratorial da neurocisticercose: revisão e perspectivas / Laboratory diagnosis of neurocysticercosis: review and perspectives

A neurocisticercose é causada por Cysticercus cellulose, a forma larval de Taenia solium, quando este se aloja no sistema nervoso central. O seu diagnóstico é realizado com base em dados clínicos, epidemiológicos, demonstração do agente etiológico pelas técnicas de imagem e testes laboratoriais. No presente estudo, apresentamos uma revisão do diagnóstico laboratorial, com ênfase no desempenho dos testes para pesquisa de anticorpos específicos e detecção de antígenos circulantes, utilização de antígeno homólogo ou heterólogo, nativo e recombinante, bem como a aplicação de métodos moleculares.

Neurocysticercosis is caused by Cysticercus cellulosae, the larval form of Taenia solium, when it lodges in the central nervous system. The diagnosis of neurocysticercosis is based on clinical and epidemiological data, neuroimaging findings of etiological agent and serologic test results. Herein we present a review of clinical diagnosis, emphasizing test performance for specific antibody and antigen detection, the use of homologous or heterologous antigen, native and recombinant antigens as well as the application of molecular methods.

Cysticercosis of the masseter.

Cysticercosis of the oral cavity is a very rare soft tissue lesion and very few cases have been reported worldwide. Here we report a case of a cysticercous cellulosae within the masseter muscle which was diagnosed with the help of high resolution ultrasonography (USG) and ultrasound guided fine needle aspiration cytology (FNAC) and managed conservatively using oral antiparasitic medication. Cysticercosis is not commonly considered in the diagnosis of swellings of the head and neck and this is the reason why they are of utmost interest to the practitioner and have to be studied.

Comparação de técnicas imunológicas in-house empregadas na rotina diagnóstica de cisticercose humana / Comparative analysis on in-house immunologic assays used for performing the routine laboratorial diagnosis of human cysticercosis

Neste estudo foram analisados os resultados obtidos do diagnóstico de cisticercose no Centro de Imunologia do Instituto Adolfo Lutz (IAL), no período de março/2007 a julho/2010. A detecção de anticorpos específicos em 522 amostras de soro e líquido cefalorraquidiano (LCR)foi realizada pelas técnicas de imunofluorescência indireta (IFI) e hemaglutinação indireta (HAI). A frequência de amostras reagentes foi de 11,0% no LCR e 8,2% no soro. Em 50% das amostras não houve informações sobre suspeita clínica de neurocisticercose dos pacientes, sendo disponíveis nos18,3% e 16,4%, em amostras, respectivamente, de LCR e soro. Nas amostras de paciente com suspeita de neurocisticercose, a positividade foi de 22,6% (LCR) e de 18,4% (soro). Houve associação entre a suspeita clínica e a positividade dos testes (p>0.05). A maioria das amostras testadas foi proveniente do Estado de São Paulo, e 16,9% de amostras de LCR e 35,9% de amostras séricas foram enviados de outros Estados do país. Os ensaios de IFI e HAI apresentaram teste de concordância Kappa de 86%. Pela indisponibilidade de kits de reagentes diagnósticos de cisticercose em amostras de LCR no mercado, os testes in-house produzidos no IAL têm sido de grande relevância para os serviços de saúde pública.

Evaluation of an enzyme immunoassay for clinical diagnosis of neurocysticercosis in symptomatic patients / Avaliação de um teste imunoenzimático para o diagnóstico clínico de neurocisticercose em pacientes sintomáticos

INTRODUCTION: Neurocysticercosis is an infection of the human central nervous system caused by the metacestode larvae of Taenia solium. Neurocysticercosis is the most common parasitic disease in developing countries. Epilepsy is the most common clinical manifestation. Difficulties in confirming the diagnosis motivated the evaluation of the enzyme-linked immunosorbent assay on cerebral spinal fluid (CSF). METHODS: Twenty-two patients with NCC and 44 control patients were studied. CSF was analyzed using a commercial ELISA kit developed for NCC. Sensitivity and specificity were measured and a multivariate logistic regression was performed. RESULTS: Sensitivity and specificity of ELISA were 31.8 percent and 100 percent, respectively, with accuracy of 77.3 percent. Only the size of the lesions proved to be important for performance of the test. CONCLUSIONS: The results showed that ELISA contributes to the diagnosis of neurocysticercosis if the result is negative or if the patient has a lesion of 2 cm or more.

INTRODUÇÃO: Neurocisticercose é a infecção do sistema nervoso central causada pela larva metacestódea da Taenia solium. Neurocisticercose é a parasitose mais comum nos países em desenvolvimento. Epilepsia é a sua manifestação clínica mais comum. A dificuldade para confirmar o diagnóstico motivou a avaliação do ensaio imunoenzimático ligado à enzima no líquido cérebro-espinhal. MÉTODOS: Vinte e dois pacientes com NCC e 44 pacientes controles foram estudados. Líquido cérebro-espinhal foi analisado por um kit ELISA comercial desenvolvido para NCC. A sensibilidade e especificidade foram medidas e uma análise multivariada de regressão logística foi realizada. RESULTADOS: A sensibilidade e a especificidade de ELISA foram, respectivamente, 31,8 por cento e 100 por cento, com acurácia de 77,3 por cento. Apenas o tamanho das lesões mostrou-se importante para o desempenho do teste. CONCLUSÕES: Este estudo concluiu que ELISA contribui para o diagnóstico de NCC, caso o teste seja negativo ou caso o paciente seja portador de lesão cuja dimensão seja igual ou maior que dois centímetros.

Epidemiologic pattern of patients with neurocysticercosis diagnosed by computed tomography in Curitiba, Brazil / Perfil epidemiológico de pacientes com neurocisticercose diagnosticados por tomografia computadorizada em Curitiba, Brazil

The aim of this study was to drawn an epidemiological pattern of neurocystisticercosis (NCC) patients diagnosed by computed tomography at the major private diagnostic center in Curitiba, Brazil. A total of 1,009 medical files of consecutive patients presenting neurological indications were diagnosed by computed tomography from July 2007 to April 2008. Patient data included sex, age, municipality and tomography findings were analysed by Epi-info version 6.0.1. software. Most patients (81.10 percent) were living in Curitiba. A total of 91/1,009 cases (9.02 percent) were considered positive to NCC; 88 (96.7 percent) patients had inactive form of NCC and only 3 (3.2 percent) patients had cysts in granulomatous process. No patients had both forms. The prevalence of NCC cases in studied group was greater in patients between 51 to 60 years old, however, difference between sex was not significant. This epidemiological pattern of NCC was similar to the first NCC study in Curitiba, performed in 1995-1996 with 9.24 percent of positive cases.

Determinou-se o perfil epidemiológico da neurocisticercose (NCC) em pacientes diagnosticados por tomografia computadorizada (TC) no maior centro privado de diagnósticos de Curitiba, Brasil. Foram analisados 1009 registros médicos de pacientes consecutivos com indicações neurológicas submetidos a TC entre julho de 2007 a abril de 2008. Os dados dos pacientes que incluíram sexo, idade, município de residência e achados tomográficos foram analisados pelo software Epi-info versão 6.01. A maioria dos pacientes (80,10 por cento) era procedente de Curitiba; 91/1.009 casos (9,02 por cento) foram positivos para NCC; 88 (96,7 por cento) apresentaram a forma inativa e apenas em 3 (3,2 por cento) cistos em processo granulomatoso; não houve formas mistas. A prevalência de casos de NCC foi maior entre 51 e 60 anos. Não houve diferença significativa para o sexo entre os casos. O perfil dos pacientes diagnosticados para NCC por TC neste estudo é semelhante ao estudo anterior realizado em Curitiba entre 1995-1996, com 9,24 por cento de casos de NCC.

Determination of Circulating Antigen in Neurocysticercosis Patients by Monoclonal Antibody / 热带医学杂志

Objective To detect circulating antigen (Cag) for diagnosing neurocysticercosis. Method ELISA was performed with monoclonal antibody 4B6 against the cyst fluid antigen of Cyticercus cellulosae for detecting Cag in serum and/or cerebrospinal fluid (CSF) of patients with neurocysticercosis or with other diseases. Results In the group of 82 cases of neurocysticercosis, the positive rate of serum Cag was 79.2% (65/82) and the positive rate of CSF Cag was 100% (26/26). After chemotherapy for 20 cases with positive serum Cag, the titer of serum Cag in 17 cases dropped to zero(85% ). Cag could not be detected in specimens from patients with other diseases. Conclusion These results indicate that the determination of Cag, especially of the CSF Cag, is useful for the diagnosis of neurocysticercosis and the drop in serum Cag is a good parameter for the evaluation of the effectiveness of chemotherapy.

Molecular Cloning of a Protein Antigen Gene of Cysticercus cellulosae / 中国寄生虫学与寄生虫病杂志

Objective To immunoscreen one protein antigen gene from a cDNA library of Cysticercus cellulosae . Methods A cDNA library of C. cellulosae was constructed after cDNA was synthesized, and immunoscreened using rabbit anti C. cellulosae polyclone antibody. The gene structure and its possible function were analyzed by comparing with the sequences available in the GenBank, after the insert of positive clone was subcloned to pBluescript SK plasmid and the nucleotide sequence of the cDNA was determined by dideoxynucleotide chain termination method using a Taq DyeDeoxy Terminator Cycle Sequencing Kit. The amino acid sequence was deduced from nucleotide sequence using GENETYX software. Homological search of the nucleotide sequences was done using BLAST in GenBank. Results and Conclusion A cDNA clone of 1 320 bp was isolated. The clone contained one open reading frame composed of 1 260 bp encoding 420 amino acids, in which two potential glycosylation sites were found. The partial nucleotide sequence of the gene fragment showed high homology with the essential spectrin gene of Caenorhabditis elegans and the erythrocyte surface antigen gene of Plasmodium falciparum , when the gene fragment was homologically analyzed in GenBank.

Immunostimulatory Activity Elicited by CpG Sequences in Cysticercus Cellulosae Paramyosin cDNA / 中国寄生虫学与寄生虫病杂志

Objective To analyse the immunostimulatory activity of CpG sequences in cysticercus cellulosae paramyosin (also named Antigen B,AgB)cDNA. Methods C57BL/6 mice were immunized with pcDNA3 AgB plasmid,pcDNA3 AgB′(CpG sequences were mutated),pcDNA3 or AgB protein and two weeks later,immune response was assayed by ELISA. Results IgG and IgG 2a were detectable at week 2 after immunization and continually increased until week 4.The antibody levels elicited by pcDNA3 AgB were significantly higher( P

Intravitreal cysticercosis

Examination of a 36-year-old man with naked visual acuity of 20/20 revealed a floating, conspicuous cyst of Cysticercus cellulosae in the vitreous cavity of the right eye. A vitreous traction band from the vitreous base and the optic disc was connected to the lodging bulb of the cyst. In the superonasal area, an ovoid retinal break surrounded by a white retinal lesion with two elliptical retinal hemorrhages was found, and this seems to be the previous lodging site of the cyst. A pars plana vitrectomy was performed to remove the parasite, and laser photocoagulation was carried out around the retinal break. Four months after the operation, the patient was satisfied with naked visual acuity of 25/20 without any complication in the affected eye.

MOLECULAR CLONING OF A cDNA ENCODING AN ANTIGENIC POLYPEPTIDE OF CYSTICERCUS CELLULOSAE / 中国血吸虫病防治杂志

Objective To immunoscreen one protein antigen gene from a cDNA library of cysticercus cellulosae. Methods The cDNA library of cysticercus cellulosae was immunoscreened by using the serum of patient with cerebral cysticercosis. After the insert of positive clone was amplified by PCR and subcloned into pUC18, the nucleotide sequence of the cDNA was determined by the dideoxynucleotide chain termination method using a Taq DyeDeoxy Terminator Cycle Sequencing Kit. The homological search of the nucleotide sequence was done by using BLASTN in NCBI. Results A cDNA clone (named cY1) with a length of 546 bp was isolated. The clone contained one open reading frame composed of 474 bp encoding 158 amino acids. The nucleotide sequence of cY1 showed 99% homology with one immunogenic protein gene named NC-9 of cysticercus cellulosae. Conclusion A gene encoding protein antigen of cysticercus cellulosae is cloned.

Screening of cDNA Clone for Putative RNA Polymerase Subunit of Cysticercus cellulosae / 中国寄生虫学与寄生虫病杂志

Objective To obtain related genes of Cysticercus cellulosae from spliced leader (SL) cDNA library. Methods Spliced leader library of Cysticercus cellulosae was constructed using SL specific primer and oligo (dT)15 with M13M4 primer, and positive clones were then screened randomly, identified with enzyme restriction, followed by sequencing and homologous analysis. Results The amino acid sequence, encoded by the positive clone with a poly (A) 22 tail and a complete open reading frame (ORF), was with homology of RNA polymerase subunit genes of human, B. napus, fission yeast, A. thaliana, C. elegans and fruit fly up to 71.6%. Conclusion The protein, RNA polymerase subunit encoded putatively by the clone, is high conservative in different species.

Cloning and Characterization of Mitochondrial NADH Dehydrogenase Gene of Cysticercus cellulosae / 中国寄生虫学与寄生虫病杂志

Objective To clone and characterize the NADH1 gene of Cysticercus cellulosae. \ Methods\ A \{cDNA\} library was constructed from Cysticercus cellulosae and was immunoscreened by using rabbit anti\|Cysticercus cellulosae \{polyclonal\} antibody. The gene structure and its possible function were analyzed by comparing with sequences available in the GenBank, after the insert of positive clone was subcloned and the nucleotide sequence of the insert was \{determined\} by dideoxynucleotide chain termination method. \ Results and Conclusion \ A cDNA clone (named TS5) with a length of \{1 082\} bp was isolated. The 5′ terminal of cloned gene contained one open reading frame of 1-578 bp encoding 192 amino acid residues of mitochondrial NADH dehydrogenase subunit 1 and the 3′ terminal contained three kinds of tRNA genes.

Protective immunity induced by DNA vaccine of Cysticercus cellulosae antigen / 第二军医大学学报

Objective: To investigate the immune response induced by Cysticercus cellulosae protective antigen cC1 DNA vaccine in mice and the protective immunity induced by immunized newborn pigs. Methods: Recombined plasmid p3 cC1 was constructed by inserting full length cC1 cDNA into an eukaryotic expression vector pcDNA3. Mice were injected intramuscularly with the recombined construct. Anti cC1 antibody and IgG 2a in serum were screened by ELISA. Then the protective immunization in pigs was done. Results: The immune response of specific antibody was induced during the 3r week. The highest level was during the 8th week. IgG 2a response was detected during the 2nd week. The higher duration of IgG 2a response induced by DNA vaccine was longlived. The protective rate induced in immunized newborn pigs was 73%. Conclusion: The cC1 DNA vaccine can effectively induce protective immunity in newborn pigs.

Frequência de anticorpos anti-Cysticercus cellulosae em população de risco para cisticercose e em segmento de população considerado supostamente normal, em regiões do Estado de São Paulo, Brasil / Frequency of anti-cysticercus cellulosae antibodies in high risk population for cysticercosis and in a segment considered as normal population, in areas of Säo Paulo state, Brazil

Examinaram-se, através de reação de fixação de complemento, os soros de 234 pacientes internados em hospital psiquiátrico localizado no município de Presidente Prudente, considerados de risco para infecção cisticercótica, além de 454 soros de gestantes procedentes da Região Administrativa de Santos e 397 soros de indivíduos considerados supostamente normais, procedentes da Região Administrativa de Presidente Prudente. O antígeno utilizado na reação de fixação de complemento foi obtido através de extração metílica, à temperatura ambiente, dos císticercos tratados com acetona. Consideraram-se positivas as reações em que ocorreu fixação de complemento a partir da diluição 1 :2. Dos 1.085 soros testados, 27 apresentaram atividade anticomplementar e 17 (1,6%) mostraram-se reagentes. Todavia, quando se consideraram, separadamente, os grupos procedentes de Santos, Presidente Prudente e os doentes mentais, percebe-se diferença significativa nos resultados: assim, os índices de freqüência foram, respectivamente, 0,88% e 1,00% para os indivíduos procedentes de Santos e Presidente Prudente e considerados supostamente normais e 3,8% para os doentes mentais. Os resultados indicam que não é desprezível a ocorrência de anticorpos anti-Cysticercus cellulosae em nosso meio, especialmente entre pacientes de hospitais psiquiátricos (AU).