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[ABSTRAK
Pendahuluan: Infeksi dengue merupakan salah satu penyakit endemik di daerah tropis dan subtropis yang disebabkan oleh virus dengue (DENV). Hingga saat ini belum ada antiviral yang efektif untuk infeksi dengue. Penyebaran dan sirkulasi serotipe DENV berfariasi di setiap lokasi geografi, hal ini menyulitkan dalam melakukan evaluasi vaksin DENV. Oleh karena itu perlu dikembangkan kandidat vaksin DENV menggunakan strain Indonesia supaya dapat memberikan proteksi maksimal. Pada peneltian ini dikembangkan kandidat vaksin DNA tetravalen DENV berbasis gen prM-E DENV strain Indonesia. Metode: Konstruksi plasmid rekombinan kandidat vaksi dilakukan dengan cara menyisipkan gen prM-E setiap serotipe DENV ke dalam vektor pUMVC4a. Gen prM-E DENV merupakan strain Indonesia, yang diamplifikasi dari serum pasien yang terinfeksi dengan virus ini. Kemampuan plasmid rekombinan mengekspresikan protein prM-E DENV diuji di sel mamalia. Kemampuan kandidat vaksin menginduksi respon imun humoral dievaluasi secara monovalen dan tetravalen di mencit jenis ddY. Titer IgG anti dengue diperiksa menggunakan teknik ELISA, sedangkan titer antibodi netralisasi di tentukan dengan uji FRNT. Proteksi vaksin terhadap mencit yang diimunisasi dievaluasi dengan melakukan uji tantang menggunakan sel K562 yang diinfeksi DENV-2. Viremi virus di tentukan dengan menggunakan teknik foccus assay. Hasil: Konstruksi plasmid rekombinan kandidat vaksin DENV-1 dan DENV-3 sudah berhasil dilakukan. Plasmid dapat mengekspresikan protein prM-E DENV di sel mamalia, namun karakteristik dan kinetik protein masih belum dapat diketahui dengan jelas. Keempat kandidat vaksin DNA yang sedang dikembangkan dapat menginduksi respon imun, baik secara monovalen maupun tetravalen. Imunisasi secara tetravalen dapat memberikan proteksi pada mencit yang diuji tantang dengan sel K562 yang diinfeksi dengan DENV-2.;

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ABSTRACT
Introduction: Dengue infections are caused by dengue viruses (DENV) and are endemic in tropical and subtropical regions. At present, there is no effective antiviral treatment for dengue infection. Distribution and circulation of DENV serotypes varies by geographic location, it is difficult to evaluate DENV vaccine. Therefore, it is necessary to develop a vaccine candidate DENV using Indonesian strains in order to provide maximum protection. However, in this study, we constructed a recombinant plasmid-based prM-E gene from the Indonesia strain as a DENV DNA vaccine candidate. Methode: The recombinant plasmid was prepared by inserting the prM-E gene from each DENV serotypes into the plasmid backbone pUMVC4a. prM-E gene an Indonesia strain, which was amplified from patient sera infected with DENV. The ability of the recombinant plasmid expressing the prM-E DENV protein tested in mammalian cells. The ability of candidate vaccines induce humoral immune responses were evaluated monovalent and tetravalent in ddY mice. IgG titers of anti-dengue examined using ELISA technique, while neutralizing antibody titers determined with FRNT test. Vaccine protection against the immunized mice was evaluated by conducting challenge test using K562 cells infected by DENV-2. Viremia was determined by using the foccus assay. Result: Construction of recombinant plasmid vaccine candidate DENV-1 and DENV-3 was successfully performed. Plasmids can express prM-E DENV proteins in mammalian cells, but the characteristics and kinetics of protein still can not clearly known. Fourth DNA vaccine candidate that is being developed to induce an immune response, either monovalent or tetravalent. Tetravalent immunization may provide protection in mice challenged tested with K562 cells infected with DENV-2.;Introduction: Dengue infections are caused by dengue viruses (DENV) and are endemic in tropical and subtropical regions. At present, there is no effective antiviral treatment for dengue infection. Distribution and circulation of DENV serotypes varies by geographic location, it is difficult to evaluate DENV vaccine. Therefore, it is necessary to develop a vaccine candidate DENV using Indonesian strains in order to provide maximum protection. However, in this study, we constructed a recombinant plasmid-based prM-E gene from the Indonesia strain as a DENV DNA vaccine candidate. Methode: The recombinant plasmid was prepared by inserting the prM-E gene from each DENV serotypes into the plasmid backbone pUMVC4a. prM-E gene an Indonesia strain, which was amplified from patient sera infected with DENV. The ability of the recombinant plasmid expressing the prM-E DENV protein tested in mammalian cells. The ability of candidate vaccines induce humoral immune responses were evaluated monovalent and tetravalent in ddY mice. IgG titers of anti-dengue examined using ELISA technique, while neutralizing antibody titers determined with FRNT test. Vaccine protection against the immunized mice was evaluated by conducting challenge test using K562 cells infected by DENV-2. Viremia was determined by using the foccus assay. Result: Construction of recombinant plasmid vaccine candidate DENV-1 and DENV-3 was successfully performed. Plasmids can express prM-E DENV proteins in mammalian cells, but the characteristics and kinetics of protein still can not clearly known. Fourth DNA vaccine candidate that is being developed to induce an immune response, either monovalent or tetravalent. Tetravalent immunization may provide protection in mice challenged tested with K562 cells infected with DENV-2., Introduction: Dengue infections are caused by dengue viruses (DENV) and are endemic in tropical and subtropical regions. At present, there is no effective antiviral treatment for dengue infection. Distribution and circulation of DENV serotypes varies by geographic location, it is difficult to evaluate DENV vaccine. Therefore, it is necessary to develop a vaccine candidate DENV using Indonesian strains in order to provide maximum protection. However, in this study, we constructed a recombinant plasmid-based prM-E gene from the Indonesia strain as a DENV DNA vaccine candidate. Methode: The recombinant plasmid was prepared by inserting the prM-E gene from each DENV serotypes into the plasmid backbone pUMVC4a. prM-E gene an Indonesia strain, which was amplified from patient sera infected with DENV. The ability of the recombinant plasmid expressing the prM-E DENV protein tested in mammalian cells. The ability of candidate vaccines induce humoral immune responses were evaluated monovalent and tetravalent in ddY mice. IgG titers of anti-dengue examined using ELISA technique, while neutralizing antibody titers determined with FRNT test. Vaccine protection against the immunized mice was evaluated by conducting challenge test using K562 cells infected by DENV-2. Viremia was determined by using the foccus assay. Result: Construction of recombinant plasmid vaccine candidate DENV-1 and DENV-3 was successfully performed. Plasmids can express prM-E DENV proteins in mammalian cells, but the characteristics and kinetics of protein still can not clearly known. Fourth DNA vaccine candidate that is being developed to induce an immune response, either monovalent or tetravalent. Tetravalent immunization may provide protection in mice challenged tested with K562 cells infected with DENV-2.]

Abstrak :
ABSTRAK
Latar belakang: Zoonosis malaria telah menjadi perhatian komunitas kesehatan dunia setelah adanya laporan kasus di Sarawak pada tahun 2004. Penyakit ini disebabkan oleh parasit malaria satwa primata Plasmodium knowlesi dengan inang alami Macaca fascicularis dan M. nemestrina. Baku emas diagnosis parasit malaria masih berdasarkan pada identifikasi mikroskopik. Selain membutuhkan keahlian yang tinggi, teknik ini terkadang sulit menentukan spesies parasit bila terjadi infeksi campuran dan parasitemia yang sangat rendah. Belakangan diusulkan DNA barcoding, suatu metode identifikasi menggunakan penanda gen sitokrom c oksidase subunit I COI DNA mitokondria untuk spesiasi. Penelitian yang dilakukan bertujuan untuk mengembangkan metode identifikasi spesies parasit menggunakan gen COI sebagai penanda molekul dan mengungkap dasar molekul transmisi zoonosis parasit malaria dengan mempelajari peran gen penyandi protein DARC Duffy Antigen Receptor for Chemokines dan DBP Duffy Binding Protein yang berhubungan dengan invasi sel darah merah.Metode: Verifikasi potensi barcode COI sebagai penanda identifikasi spesies parasit malaria dilakukan dengan studi in-silico, sedangkan validasi penggunaan barcode COI dilakukan dengan analisis sensitivitas dan spesifisitas. Teknologi molekuler PCR-Sequencing dilakukan untuk mengaplikasikan barcode COI pada penapisan parasit malaria di populasi manusia dan satwa primata, serta identifikasi variasi genetik gen penyandi protein DARC dan DBP terutama pada daerah pengikatan ligan parasit dan reseptor inang.Hasil: Studi in-silico menunjukkan bahwa DNA barcoding berpotensi sebagai penanda identifikasi parasit malaria. Primer yang dirancang mengamplifikasi daerah COI sepanjang 670 pb berhasil mengidentifikasi parasit malaria dengan sensitivitas 1 ndash; 3 parasit/ l. Pada penapisan parasit malaria di populasi manusia di Kalimantan Tengah ditemukan 3,34 78/2309 kasus malaria, di mana dua diantaranya adalah kasus malaria knowlesi, yang secara statistik berbeda bermakna bila dibandingkan dengan mikroskopik 2,82 dan 18S rRNA 1,82 . Pada daerah yang sama, penapisan parasit malaria di populasi satwa primata, ditemukan 52,01 168/323 sampel orangutan dan 23,25 10/43 sampel monyet Macaca positif malaria. Spesies parasit yang ditemukan pada orangutan adalah P. species tipe parasit ovale, P. species tipe vivax-cynomolgi, P. species tipe vivax-hylobati dan P. species tipe malariae-inui, sedangkan pada monyet Macaca meliputi P. knowlesi, P. coatneyi, P. inui, juga P. spesies tipe malariae-inui, spesies parasit yang sama ditemukan di orangutan. Studi ini juga menemukan keanekaragaman genetik pada gen penyandi protein Duffy Antigen Receptor for Chemokines manusia maupun satwa primata dan Duffy Binding Protein parasit malaria yang memainkan peran penting dalam invasi parasit malaria.Kesimpulan: Barcode COI dapat secara spesifik dan sensitif mengidentifikasi spesies parasit malaria dan dapat diaplikasikan sebagai alat identifikasi zoonosis malaria. Terdapat variasi genetik gen penyandi protein Duffy Antigen Receptor for Chemokines dan Duffy Binding Protein yang berhubungan dengan invasi sel darah merah.

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ABSTRACT
Background Zoonotic case of malaria had just come to the attention of public health communities after the Sarawak study in 2004. Zoonotic malaria is caused by Plasmodium knowlesi, primarily a simian malaria parasite in wild long tail macaque Macaca fascicularis and pig tail macaque M. nemestrina as the reservoir hosts. The diagnosis of malaria parasites has mainly relied on the microscopic examination. However, this method is labor intensive, requires an experienced microscopist and difficult in identifying mixed infections in very low parasitemia cases. Recently, DNA barcoding system, which is based on the PCR amplification of a short and highly conserved region of mitochondrial cytochrome c oxidase sub unit I COI has shown to be an invaluable tool for diagnosing and differentiating the species of wide range of organisms. This study was aimed to develop identification tools of malaria parasite by using mtDNA COI gene as a molecular marker and reveal the molecular basis of zoonotic malaria by identifying the genetic variation of protein coding gene of DARC Duffy Antigen Receptor for Chemokines and DBP Duffy Binding Protein that are related to receptor ligand interaction in red blood cell invasion.Methods In silico study was carried out for verifying the potential of DNA barcoding based on the mtDNA COI gene sequence as a marker identification. Sensitivity and specificity analyses were carried out to validate the use of DNA barcoding for medical diagnosis of parasitic infection. Molecular technology of PCR Sequencing was carried out for screening malaria parasit in human and non human primate population and identifying the genetic variation within protein coding gene of DARC and DBP. Results We have initiated a study to explore the use of DNA barcoding for malaria parasite diagnosis through in silico study. We have thus designed primers spanning a 670 bp fragment of the 5 rsquo region of COI gene that could detect parasite isolates as low as 1 3 parasite per l. DNA barcode was used to detect malaria parasite in human population in Central Kalimantan. Of the 2309 subjects, 78 3.34 subjects were malaria positive of which two samples were determined as P. knowlesi infection. The detection rate of COI barcode was significantly higher as compared to microscopic 2.82 and 18S rRNA 1.82 analyses. Of the 366 non human primate samples that include 323 orangutan and 43 macaque 168 orangutan were found to be positive for either P. species ovale type, P. species vivax cynomolgi type, P. species vivax hylobati type and P. species malariae inui type. In macaque, 10 samples were positive for P. knowlesi, P. coatneyi, P. inui and P. species malariae inui type similar to that found in orangutan. The study has also found genetic variation in both human and non human primates Duffy Antigen Receptor for Chemokines and malaria parasite Duffy Binding Protein.Conclusions The study showed that mtDNA COI can be used to diagnose malaria parasites at very low parasitemia level and applied as a diagnosis tool for identification of zoonotic malaria. There is genetic variation in both human and non human primates Duffy Antigen Receptor for Chemokines and malaria parasite Duffy Binding Protein as major determinants for the invasion of malaria parasite.

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Latar belakang. Meningkatnya kasus HIV-AIDS human immunodeficiency virus-acquired immunodeficiency syndrome secara global memicu kewaspadaan akan peningkatan infeksi oportunistik, salah satunya infeksi Pneumocystis jirovecii yang mengakibatkan pneumonia PjP. Infeksi PjP merupakan kasus yang sulit ditangani terkait rendahnya sensitivitas uji diagnostik diiringi dengan peningkatan kasus resistensi terhadap antibiotik. Di Indonesia belum terdapat data demografis, epidemiologi molekuler maupun data resistensi mengenai kasus infeksi PjP. Mengantisipasi masalah tersebut, dalam penelitian ini dikembangkan uji diagnostik PjP pada ODHA Orang Dengan HIV-AIDS terduga pneumonia melalui pendekatan molekular terhadap gen MSG Major Surface Glycoprotein disertai dengan karakterisasi gen DHPS dihidropteroat sintase dan gen mtLSU mitochondrial large subunit yang berkorelasi dengan genotipe resisten dan virulensi P. jirovecii. Tujuan penelitian. Memperoleh suatu uji deteksi infeksi PjP, data genotipe resistensi dan virulensi PjP melalui pendekatan secara molekuler yang dapat dimanfaatkan sebagai dasar data demografi dan epidemiologi molekuler PjP di Indonesia. Metode penelitian. Pengembangan uji diagnosis molekuler PjP terhadap gen MSG dilakukan dengan metode real- time PCR yang diujikan terhadap 100 sampel sputum. Pola genotipe resistensi dilakukan melalui amplifikasi gen DHPS dilanjutkan dengan restriction fragment length polymorphism RFLP . Virulensi daerah hot spot gen mtLSU dianalisis dengan metode PCR dan sekuensing DNA. Hasil. Secara demografi, diketahui prevalensi PjP pada ODHA terduga pneumonia di Jakarta mencapai 20,0, laki-laki 75, rentang usia terbanyak 31-40 tahun 35, dominan 80 pada kisaran sel limfosit T CD4 200-349 sel/L. Sebanyak 12 pasien menunjukkan gen DHPS positif, lima pasien 41,66 merupakan genotipe wild type WT dan 7 pasien lainnya 58,32 merupakan genotipe resisten, terdiri dari 16,67 genotipe-3 dan 41,66 genotipe campuran WT dan genotipe 1. Analisis virulensi berdasarkan gen mtLSU diperoleh 30 strain PjP positif yang didominasi oleh variasi-3. Status imun pasien lebih berkaitan dengan genotipe resistensi dibandingkan dengan jenis varian. Kesimpulan. Uji real-time PCR yang dikembangkan mampu memberikan nilai diagnostik yang lebih baik dibandingkan pewarnaan Giemsa. Terdapat 3 genotipe gen resistensi WT, genotipe 1 dan 3 dan 7 varian P. jirovecii yang bersirkulasi di Jakarta. Genotipe resistensi lebih berkaitan terhadap kondisi klinis pasien dibandingkan dengan jenis varian.

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Background. The global rise of HIV-AIDS cases increase the alertness against oportunistic infections, one of them is Pneumocystic jirovecii pneumonia PjP. PjP infection is a one of a tough infection to be cured due to low sensitivity of its diagnostic method following the escalation of PjP resistance against antibiotics. There is no demografic, molecular epidemiology nor antibiotics resistance data were available related to PjP infection in Indonesia. Thus, this study was conducted to develop a molecular test to diagnose PjP infection in HIV-AIDS suspected pneumonia patients based on MSG Major Surface Glycoprotein gene detection, followed by characterization of DHPS dihydropteroat syinthetase and mtLSU mitochondrial large subunit genes represent genoype resistance and P. jirovecii virulence. Research objective. To obtain a molecular test in diagnosing PjP infection and information of P. jirovecii genotype resistance and virulence based on molecular characteristics, which can be used further as demographic and molecular epidemiology basis data of PjP in Indonesia. Research methods. Molecular diagnostic test aimed for MSG gene of P. jirovecii detection was done through real-time PCR against 100 sputum samples. Genotype resistance and P. jirovecii polymorphism patterns was done through DHPS and mtLSU genes amplification followed by restriction fragment length polymorphism RFLP and DNA sequencing analysis. Virulence of the hot spot area are of the mtLSU gene was analyzed by PCR method and DNA sequencing. Results. The prevalence of PjP infection in HIV-AIDS suspected pneumonia patients in Jakarta was 20.0, male 75 within 31-40 y.o 35, dominant 80 from patients with CD4 T-lymphocytes of 200-349 cells/L. Molecular real-time PCR methods give five times sensitivity higher than Giemsa stain. Twelve patients showed positive DHPS gene, five patients 41.67 were wild type WT genotypes and 7 other patients 58.32 were resistant genotypes, with 16.66 was genotype-3 and other 41.66 was mixed genotypes WT and genotype 1. Virulence analysis based on mtLSU gene show 30 positive strains which dominated by variant-3. The patients immune status is more related to the resistance genotype compared to the variant type. Conclusion. The developed real-time PCR method is proven to able to give better diagnostic value than Giemsa stain. There are 3 genotypes of resistance genes WT, genotypes 1 and 3 and 7 variants of P. jirovecii circulating in Jakarta. Resistance genotypes are more related to the clinical condition of patients compared to variant types.

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Latar Belakang: Pendekatan biologis rekayasa jaringan gigi bertujuan meregenerasi jaringan gigi secara histologis, morfologis dan fungsional. Keterbatasan DPSC gigi manusia, memberikan ide untuk menggunakan jaringan lemak sebagai penghasil sel odontoblas. Tujuan: Menganalisis potensi jaringan lemak sebagai sumber MSCs alternatif untuk menjadi sel odontoblas dengan teknik rekayasa jaringan. Material dan Metode : Kelompok perlakuan ADMSC+rhBMP-2, ADMSC+rhBMP-2+Proterin Pulpa, dan DPSC+rhBMP-2, kontrol ADMSC dan DPSC. Analisis: Stro-1, DMP-1 dan Col-1 untuk karakterisasi odontoblastik, Adhesion Assay, dan Col-1 setelah grafting dengan PRP, PRF, FG. Hasil: Ekspresi seluruh parameter menunjukkan potensi ADMSC dan DPSC yang sama untuk berdiferensiasi ke arah odontoblas. Kesimpulan: Jaringan lemak berpotensi sebagai sumber sel odontoblas dalam proses regenerasi jaringan pulpa. ......Background: Biological approach of dental tissue engineering aims to regenerate tooth structure in histological, morphological, and functional aspect. DPSC limitation of human teeth giving the idea of using adipose tissue to produce odontoblast. Objective: to analyze the potency of adipose tissue as an alternative source of MSCs to produce odontoblast cells by tissue engineering. Materials and Methods: Treatment groups were ADMSC+rhBMP-2, ADMSC+rhBMP- 2+Pulp Protein, and DPSC+rhBMP-2, and control groups of ADMSC and DPSC. Analyzed: Stro-1, DMP-1 and Col-1 for odontoblastic characterization, Adhession Assay and Col-1 after grafted with PRP, PRF, FG. Result: The expression of all markers showed the same potention of ADMSC and DPSC to differentiate towards odontoblast cells. Conclussion: Adipose tissues have the potency as a source of odontoblast cells in the process of pulp tissue regeneration.