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"Antiretroviral therapy (ART) given to HIV patients to improve their immune response that damaged by HIV infection. Some patients with ART experience Immune Restoration Disease (IRD) as worsening of clinical symptoms from certain pathogens infection. The incidence of IRD concided with an increased number of CD4+ T cells. Hepatitis C virus can also infect HIV patients and may also lead to HCV IRD. The immunopathogenecity of IRD has not known yet. This study aims to look at the function of dendritic cells producing IL-12 and IFNα, and IFNγ-producing T cell responses in incidence of HCV IRD. Research subjects were 50 patients with HIV/HCV who were initiating antiretroviral therapy (ART) for up to 6 months of therapy. There are 9 people with HCV IRD who compared with non HCV IRD patients. Blood specimens were collected from study subjects at months 0, 1, 3 and 6 after ART. Then PBMC isolation was done and used for flowsitometri and ELISpot analysis.The results showed that the percentage of myeloid (mDC) and plasmacytoid dendritic cells (pDC) did not differ between HCV IRD patients and non-HCV IRD patients. It appears that the percentage of IL-12-producing mDC did not correlate significantly with IFNγ- producing T cells both in HCV IRD and non-IRD HCV patients. The percentage of IL-12-producing mDC in HCV IRD patients were lower than in non-IRD patients (p=0.003). While percentage of IFNα-producing pDC and IFNγ- producing T cells did not differ significantly between the two groups of patients. Antibody response to HCV proteins (core, NS3, NS4, and NS5) did not differ between HCV IRD and non-HCV IRD patients. The role of dendritic cells and T cell responses in HCV IRD incidence have not clearly seen.

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Terapi antiretroviral (ART) diberikan kepada pasien HIV akan memperbaiki respon imun tubuh yang rusak karena infeksi HIV. Beberapa pasien dengan ART mengalami sindrom pulih imun atau Immune Restoration Disease (IRD) berupa perburukan gejala klinis dari infeksi patogen tertentu. Kejadian sindrom pulih imun ini terjadi bersamaan dengan peningkatan jumlah sel T CD4+. Virus Hepatitis C yang menjadi patogen penyerta pada pasien HIV juga menjadi penyebab sindrom pulih imun. Belum diketahui dengan jelas imunopatogenesitas dari sindrom pulih imun ini. Penelitian ini bertujuan untuk melihat fungsi sel dendritik penghasil IL-12 dan IFNα, serta respon sel T penghasil IFNγ pada kejadian sindrom pulih imun HCV. Subyek penelitian adalah 50 pasien HIV/HCV yang sedang memulai terapi antiretroviral (ART). Terdapat 9 orang pasien dengan sindrom pulih imun HCV yang dibandingkan dengan pasien tanpa sindrom pulih imun HCV. Spesimen darah lengkap dikumpulkan dari subyek penelitian pada bulan ke-0, 1, 3 dan 6 setelah ART. Kemudian dilakukan isolasi PBMC dan analisis flowsitometri dan ELISpot.Hasil penelitian menunjukkan bahwa persentase sel dendritik mieloid (mDC) dan plasmasitoid (pDC) tidak berbeda antara pasien dengan dan tanpa sindrom pulih imun HCV. Persentase sel mDC penghasil IL-12 tidak berkorelasi secara signifikan dengan jumlah sel T penghasil IFNγ baik pada pasien dengan maupun tanpa sindrom pulih imun HCV. Pasien dengan sindrom pulih imun HCV memiliki persentase sel mDC penghasil IL-12 yang lebih rendah dibandingkan pasien tanpa sindrom pulih imun HCV (p=0,003). Sedangkan persentase sel pDC penghasil IFNα dan jumlah sel T penghasil IFNγ tidak berbeda secara signifikan antara kedua kelompok pasien. Respon antibodi terhadap protein HCV (core, NS3, NS4 dan NS5) pun tidak berbeda antara kedua kelompok pasien. Disimpulkan bahwa belum terlihat adanya peran dari sel dendritik dan respon sel T terhadap kejadian sindrom pulih imun HCV."

"Racun duri Acanthaster planci memiliki beragam aktifitas biologi yaitu aktifitas lethal, aktifitas hemolitik, aktifitas myonecrotic, aktifitas pendarahan, peningkatan aktifitas permeabilitas kapiler, aktifitas edema, aktifitas phospholipase-A2 (PLA2), aktifitas pelepasan histamin dari mast cell dan aktifitas kardio vaskular. Racun duri Acanthaster planci mengandung phospholipase A2 (PLA2), plancitoxin yang homolog dengan deoxyribonuklease II pada mamalia dan plancinin peptida antikoagulan. Berbagai penelitian terdahulu membuktikan bahwa racun yang berasal dari berbagai hewan mengandung senyawa yang potensial dikembangkan sebagai bahan antibiotik dan terapeutik untuk mengobati suatu penyakit. Dengan potensi aktivitas biologi tersebut racun Acanthaster planci dapat berkontribusi di bidang medis yang bisa menjadi masukan bagi pendapatan negara. Efek antimikrobial hasil aktifitas hidrolisis komponen fosfolipid membran sel mikroba oleh enzim PLA2 dapat bermanfaat bagi pengembangan bahan antibiotik. PLA2 yang dimurnikan dari racun ular memiliki aktifitas antibakteri terhadap Staphylococcus aureus, Proteus vulgaris, Proteus mirabilis, and Burkholderia pseudomallei. Selain itu, PLA2 memiliki aktifitas antiHIV melalui mekanisme penghambatan pelepasan intraseluler protein capsid virus dan diasumsikan PLA2 memblok virus masuk ke dalam sel inang sebelum virus tersebut membuka selaputnya dan secara independent memanfaatkan koreseptornya. PLA2 melindungi sel limfosit T manusia dengan memblok virus yang memiliki selubung luar mengandung fosfolipid. Acanthaster planci merupakan predator yang mengancam populasi karang terutama ketika terjadi peledakan populasi. Pemanfaatan Acanthaster planci untuk produksi PLA2 dapat menjadi alternatif produktif upaya pengendalian populasinya sekaligus membuatnya menjadi lebih berguna. Purifikasi PLA2 racun duri Acanthaster planci telah dilakukan oleh Shiomi dan koleganya menggunakan rangkaian kolom kromatografi bertingkat, memerlukan biaya yang relative mahal dan membutuhkan waktu beberapa hari, sehingga dalam penelitian ini dikembangkan metode purifikasi yang sederhana dan cepat dengan biaya yang relatif murah. Hasil penelitian ini diharapkan dapat menjadi masukan bagi upaya pemanfaatan Acanthaster planci untuk menghasilkan PLA2 yang berpeluang dikembangkan sebagai bahan antibakteri dan antiHIV. Penerapan metode percobaan yang dilakukan dalam penelitian ini memberikan hasil sebagai berikut : - Proses ekstraksi racun dari jaringan duri Acanthaster planci berlangsung efektif melalui proses sonikasi pada 20 kHz selama 2x8 menit (intensitas 80% dan output 10). Racun yang terekstraksi tertampung dalam larutan 0,01 M bufer fosfat pH 7,0 mengandung 0,001 M CaCl2 yang digunakan sebagai media ekstraksi disebut crude venom. Pengujian secara kualitatif menggunakan darah manusia yang diberi perlakuan crude venom (1:1) memperlihatkan antikoagulasi darah oleh plancinin yang terkandung dalam racun membuktikan keberhasilan proses ekstraksi. Pada awalnya dilakukan pula metode ekstraksi dengan cara duri diblender terlebih dahulu dan dilanjutkan dengan disonikasi. Untuk meminimalisir protein kontaminan yang berasal dari jaringan duri dan mempertimbangkan efisiensi maka metode ini kemudian tidak diterapkan. Purifikasi phospholipase A2 racun duri Acanthaster placi dari Ambon-Maluku melalui pengendapan amonium sulfat bertahap pada tingkat kejenuhan 20% terhadap crude venom yang telah dipanaskan efektif memurnikan PLA2. Hasil elektroforesis SDSPAGE memperlihatkan isolat PLA2 memiliki satu pita protein sedangkan crude venom memiliki empat pita protein. Isolat PLA2 yang dihasilkan memiliki aktifitas spesifik 20 kali aktifitas spesifik crude venom. Pemanasan crude venom pada 60oC selama 30 menit yang diikuti dengan sentrifugasi selama 30 menit pada 15.000xg dan 4oC memisahkan protein tidak tahan panas dari PLA2. Metode purifikasi ini juga diterapkan pada racun duri Acanthaster planci dari Sorong-Papua namun belum berhasil. Sedangkan purifikasi PLA2 melalui pengendapan menggunakan etanol dengan tingkat kejenuhan 80% tidak efektif memurnikan PLA2 namun dapat meningkatkan aktifitasnya menjadi lima kali aktifitas crude venom. Hasil eksperimen ini dipublikasikan di International Journal of Pharma and Bio Science Vol 2/issue 2/Apr-Jun 2011 and International Journal of Pharma and Bio Science 2012 Oct; 3(4):(B) 603-608 - Pengujian aktifitas antibakteri menggunakan metode difusi cakram memperlihatkan terbentuknya zona bening disekitar cakram PLA2 pada kultur Staphylococcus aureus yang mengindikasikan bahwa PLA2 racun duri Acanthaster planci memiliki aktifitas antibakteri terhadap Staphylococcus aureus pada dosis 2, 98 mg/ml. Hasil eksperimen ini dipublikasikan pada International journal of Pharma and Bio Sciene 2013 Apr; 4(2) : (B)1-5 - Pengujian aktifitas antiHIV secara kualitatif menggunakan PBMC pasien HIV (ODHA) memperlihatkan terjadinya penurunan intensitas pita protein DNA pada hasil elektroforesis RT-PCR RNA sampel kultur HIV yang diberi perlakuan PLA2. Selanjutnya analisis kuantitatif hasil Green Fluoresence Particle memperlihatkan terjadinya penurunan jumlah sel yang terinfeksi HIV secara signifikan oleh perlakuan PLA2 dari 9,72% menjadi 0,29% yang mengindikasikan PLA2 racun duri Acanthaster planci memiliki aktifitas antiHIV. Hasil eksperimen ini dipublikasikan pada Asian Pacific Journal of Tropical Medicine (2014) 412-420 - Biaya purifikasi PLA2 merupakan pembiayaan yang dibayarkan untuk 1) bahan kimia dan peralatan habis pakai, 2) listrik untuk operasional alat, 3) sewa peralatan dan 4) tenaga kerja. Hasil perhitungan biaya isolasi-purifikasi PLA2 menghasilkan nilai Rp. 446.192,- per 50 gram duri dengan hasil yang diperoleh adalah 4,622 mg PLA2. Biaya purifikasi PLA2 miniscale yang dilakukan dalam penelitian ini efisien untuk diterapkan dimana harga komersial PLA2 racun ular Crotalus amandetus (Worthington, USA) adalah Rp. 590.000 per mg (59.00 US Dolar). Hasil pengolahan data citra satelit tahun (2006) yang diunduh dari website NASA pada Juni 2013 memperlihatkan luas areal terumbu karang yang merupakan habitan Acanthaster planci adalah 94,83 hektar. Diperkirakan pada luas areal tersebut terdapat 550 individu dewasa dan jumlah yang dapat dimanfaatkan untuk menghasilkan PLA2 adalah 20% dari ketersediaannya per bulan. Berdasarkan hasil percobaan tersebut dapat disimpulkan bahwa : - Metode sederhana dan cepat dengan biaya operasionil relatif murah melalui pengendapan 20% amonium sulfat terhadap crude venom yang dipanaskan terlebih dahulu efektif memurnikan PLA2 dari racun duri Acanthaster planci dengan tingkat kemurnian dan aktifitas spesifik yang tinggi. Sedangkan metode pengendapan menggunakan etanol 80% tidak efektif memurnikan PLA2 dari racun duri Acanthaster planci namun dapat meningkatkan aktivitasnya menjadi 5 kali crude venom. PLA2 racun duri Acanthaster planci memiliki aktifitas antibakteri terhadap Staphylococcus aureus dan aktifitas antiHIV. - Biaya miniscale operasional purifikasi PLA2 efisien untuk diterapkan dan ketersediaan Acanthaster planci di perairan Liang dan pulau Pombo yang dapat dimanfaatkan untuk menghasilkan PLA2 adalah sebesar 20% per bulan.

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Spines venom of Acanthaster planci have various biological activities: lethal activity, hemolytic, myonecrotic, bleeding, increased capillary permeability, edema, phospholipase A2 (PLA2), the activity of histamine release from mast cells and cardio vascular activity. Spines venom of Acanthaster planci containing phospholipase A2 (PLA2), plancitoxin which is homologous with mammals deoxyribonuklease II and plancinin anticoagulant peptide. Previous studies prove that the venoms derived from animals contain various compounds that are potential to be developed as antibiotic and therapeutic agents to treat a disease. Acanthaster planci spines venom with various potential biological activity may contribute in the medical field that can be input for the state revenue. Antimicrobial effect results by hydrolysis activity of PLA2 on microbial cell membrane phospholipids can be beneficial to the development of antibiotic agent. PLA2 purified from snake venom have antibacterial activity against Staphylococcus aureus, Proteus vulgaris, Proteus mirabilis, and Burkholderia pseudomallei. In addition, PLA2 has antiHIV activity through inhibition of the release mechanism of intracellular viral capsid proteins and assumed PLA2 blocking viral entry into host cells before the virus opens membranes and independently utilize koreseptornya. PLA2 protect human T lymphocytes by blocking viruses that have outer sheath containing phospholipids. Acanthaster planci is a predator threatens coral populations, especially when there is a outbreak population. Acanthaster planci utilization for the production of PLA2 can be an effort population control productively and make it more useful. Purification of Acanthaster planci spines venom PLA2 has been done by Shiomi and colleagues by using a series of chromatography columns which is relatively expensive and takes a few days, so a simple and fast method with a relatively low cost was developed in this study.

The results of this study are expected to be input for utilaization of Acanthaster planci to produce PLA2 that can be developed as antibacterial and antiHIV agents. Experiments method were conducted in this study gave the following results:

- Venom extraction from the spines of Acanthaster planci was effective through the process of sonication at 20 kHz for 2x8 minutes (intensity 80% and 10 outputs). Venom was accumulated in extraction medium solution of 0.01 M phosphate buffer pH 7.0 containing 0.001 M CaCl2 called crude venom. Qualitative tested by using human blood treated with crude venom (1: 1) showed the blood anticoagulation by plancinin contained in the venom, proves the extraction process successfully. At the previous conducted on a method of extraction, the spines were blended first and followed by sonicated. To minimize contaminant proteins derived from spines tissue and consider the efficiency, this method was not implemented. Purification of phospholipase A2 from spines venom of Ambon-Maluku Acanthaster placi by using fractionated ammonium sulfate precipitation at 20% saturation of the heated crude venomwas done effectively. SDS-PAGE electrophoresis showed PLA2 isolates has one protein band while the crude venom has four protein bands. PLA2 isolates has a specific activity 20 times the specific activity of crude venom. Heated the crude venom at 60°C for 30 minutes followed by centrifugation for 30 minutes at 15.000xg and 4°C separated PLA2 from the other heat sensitive proteins. This method was also implemented to purify PLA2 spines venom of Acanthaster planci from Sorong-Papua, but have not been successful. While PLA2 purification by using ethanol precipitation at a level of 80% saturation was not effective but increased the specific activity into five times crude venom specific activity. This Experimental results were published in the International Journal of Pharma and Bio Science Vol 2 / issue 2 / Apr-June 2011 and the International Journal of Pharma and Bio Science 2012 Oct; 3 (4) :( B) 603-608.

- Investigated of antibacterial activity by using disc diffusion method exhibited clear zone around the disc pre-added PLA2 on Staphylococcus aureus culture, indicated PLA2 of Acanthaster planci spines venom has antibacterial activity against Staphylococcus aureus. This experimental result was published in International journal of Pharma and Bio Sciene 2013 Apr; 4(2) : (B)1-5

- Qualitative investigated of antiHIV activity by using PBMCs of HIV patient showed a decrease of the DNA protein band intensity in electrophoresis result of RT-PCR RNA sample of the HIV cultured treated with PLA2. Furthermore, quantitative analysis of the Green Fluorescence Particle results showed the decline significantly from 9.72% into 0.29% in the number of HIV-infected cells by PLA2 treatment, indicated PLA2 of Acanthaster planci spines venom has antiHIV activity.This experimental result was published in Asian Pacific Journal of Tropical Medicine(2014) 412-420

- The cost of PLA2 purification was paid for : 1) chemicals and equipment consumables, 2) electricity for the operation of the tools, 3) tools rental and 4) labor. The cost of PLA2 purification was Rp. 446.192,- per 50 grams spines with the results obtained was 4.622 mg PLA2. Miniscale purification costs performed in this study was efficiently implemented which is the commercial prices PLA2 is ± 590,000 rupiahs per mg (59,00 US dolar) (Worthington USA product of snake venom Crotalus amandetus PLA2). Thus purification of PLA2 from Acanthaster planci spines venom might be have a good prospect to be developed.

- Acanthaster planci survay was done on March 2013 in Eastern part Ambon water, especially in Liang (dusun Tanjung and dusun Batu Dua) and Pombo island obtained the average density value of 5.8 adult individuals per hectare. Satellite images (2006) downloaded from NASA website in June 2013 shown coral reefs area as the habitat of Acanthaster planci is 94.83 acres. Total estimated of adult Acanthaster planci in those area was 550 and the availablelity number that can be used to produce PLA2 was 20% per month. "

"Measles immunization has been introduced since 1960, thereby markedly reducing the number of cases in developed countries. However, measles epidemics still occur even in developed countries. In the United States, in 1988-1992 an increase in the number of measles cases reaching 50,000 cases was reported. Some of these cases occurred in previously immunized patients. This was thought to be caused by genetic mutation of the measles virus, aside from weaknesses of the vaccine and low immunization coverage.Since measles immunization was employed in Indonesia, the number of measles patients has decreased. However, epidemics are still frequently reported. About 15-30% of reported cases occurred in those previously immunized, raising the question of whether a genetic difference exists between the wild-type measles virus circulating in Indonesia and the vaccine virus being used. Such a difference may lead to the differences in the antigenicity of the wild-type and vaccine viruses, rendering the resulting antibody incapable of neutralizing the wild-type viruses. Based on the above, this study is aimed to demonstrate the extent of genetic and antigenic differences between the wild-type and vaccine measles viruses.We conducted an experimental laboratory study to sequence the N, H, and F genes of the wild-type measles viruses (G2, G3, and D9) and the CAM-70 vaccine virus. To show antigenic differences, the wild-type viruses (G2, G3, and D9) and the CAM-70 and Schwarz viruses were injected to BALB/c mice. Serum antibodies of the mice were analyzed using ELISA, cross-neutralization test, and immunoblotting using antigens from the respective viruses.Results of this study showed that the wild-type and the vaccine viruses differ in the sequence of the N gene by 73-79 nucleotides, resulting in amino acid substitution of 17-24 residues; the H gene by 60-99 nucleotides, resulting in amino acid substitution of 13-29 residues; the F gene by 71-88 nucleotides, resulting in amino acid substitution of 4-3 I residues. Differences between the wild-type and the CAM-70 and Schwarz vaccine viruses were also found in the epitope site of the CTL and antibodies, which are important to virus antigenicity.We conclude that a significant difference in antigenicity exists between the wild-type measles viruses circulating in Indonesia with the CAM-70 measles virus. We also found the immunogenicity of the CAM-70 and Schwarz vaccine viruses to be lower than that of the wild-type viruses."

"Latar Belakang: Pseudomonas aeruginosa, resisten terhadap obat, menyebabkan infeksi kesehatan. Resistensi terhadap terapi pilihan meropenem merupakan ancaman serius. Penelitian ini bertujuan untuk menganalisis perubahan konsentrasi hambat minimum meropenem (KHM), perubahan ekspresi gen ampC, mexA, dan oprD, serta korelasi antara KHM dengan ekspresi gen ampC, mexA, dan oprD sesudah paparan meropenem.Metode: Digunakan sepuluh isolat P. aeruginosa dari Departemen Mikrobiologi Klinik Fakultas Kedokteran Universitas Indonesia. Sesudah bakteri terbukti peka terhadap meropenem secara fenotip, gen resistensi intrinsik dideteksi menggunakan PCR. Sesudah paparan meropenem pada hari ke 5 dan 12 dilakukan uji kepekaan dengan metode gradien konsentrasi dan deteksi RNA menggunakan real-time RT-PCR.Hasil: Semua isolat P. aeruginosa yang peka secara fenotip terhadap meropenem mempunyai gen ampC, mexA, dan oprD. Peningkatan KHM, peningkatan ekspresi gen ampC dan mexA, dan penurunan ekspresi gen oprD diamati sesudah paparan meropenem. Terdapat korelasi yang sangat kuat dan signifikan (p ≤ 0,05) antara KHM dan ekspresi gen oprD sesudah hari ke-12 paparan meropenem.Kesimpulan: Meskipun tidak terdapat perbedaan yang signifikan pada ekspresi gen KHM dan ampC, mexA, dan oprD antara hari ke-5 dan hari ke-12, namun terdapat korelasi yang sangat kuat dan signifikan antara ekspresi gen KHM dan oprD pada hari ke-12 (p≤ 0,05). Hal ini menunjukkan bahwa penurunan ekspresi gen oprD berpotensi meningkatkan resistensi meropenem pada P. aeruginosa.

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Background: Pseudomonas aeruginosa, drug-resistant, causes health infections. Resistance to the preferred therapy meropenem is a serious threat. This study aimed to analyze changes in meropenem minimum inhibitory concentration (MIC), changes in ampC, mexA, and oprD gene expression, and the correlation between MIC and ampC, mexA, and oprD gene expression after meropenem exposure.

Methods: Ten isolates of P. aeruginosa from the Clinical Microbiology Department, Faculty of Medicine, Universitas Indonesia were used. After the bacteria were shown to be sensitive to meropenem phenotypically, intrinsic resistance genes were detected using PCR. After meropenem exposure on Days 5 and 12, sensitivity testing was carried out with the concentration gradient method and RNA was detected using real-time RT-PCR.

Results: All P. aeruginosa isolates that were phenotypically sensitive to meropenem had the ampC, mexA, and oprD genes. An increase in MIC, an increase in ampC and mexA gene expression, and a decrease in oprD gene expression were observed after meropenem exposure. There was a very strong and significant correlation (p ≤ 0.05) between MIC and oprD gene expression after Day 12 of meropenem exposure.

Conclusion: Although there were no significant differences in MIC and ampC, mexA, and oprD gene expression between Day 5 and Day 12, there was a very strong and significant correlation between MIC and oprD gene expression on Day 12 (p≤ 0.05). This indicates that decreasing oprD gene expression has the potential to increase meropenem resistance in Pseudomonas aeruginosa."

"Pendahuluan: Barier alamiah berupa membrane sel, kompartemen endosom, dan membrane inti sel yang menghalangi DNA ekstraseluler untuk mencapai target kerja dalam inti sel dalam mengawali proses pembentukan protein transgen. Sistem penghantar DNA, dalam hal ini protein penghantar DNA (PPD) dikembangkan untuk menghindari penghalang seluler tersebut. Karena sel tubuh berada dalam keadaan tidak membelah, maka diharapkan PPD tersebut dapat berfungsi pada sel tidak membelah. Metodologi: Metodologi yang dipakai adalah telaah literature, pengkloningan, dan uji in vitro. Dilakukan telaah literature untuk mencari peptida yang memiliki 1) kemampuan untuk masuk ke dalam sel/ berfusi dengan membran sel hospes, 2) kemampuan mengikat DNA, 3) kemampuan menghantarkan DNA melalui nuclear localization signal (NLS). Sekuen protein diubah menjadi DNA dengan menggunakan perangkat lunak. Setelah menjadi fragmen DNA, maka langkah selanjutnya DNA di klon ke dalam plasmid pQE80L untuk menghasilkan plasmid rekombian pengekspresi PPD. PPD diperoleh dengan cara mengekspresikannya ke dalam sistem prokariota. Setelah PPD dimurnikan, dilakukan uji fungsi kemampuan PPD menghantarkan DNA pada sel kultur membelah dan tidak membelah. Hasil: Berdasarkan telaah literature dihasilkan 5 rancangan PPD ALMR, SIMR, VPMR, THB2 dan THB4. Studi bioinformatika menunjukkan THB4 tidak dapat mengikat DNA dan bergerak ke inti sel. Proses pengkloningan menghasilkan 4 klon dan salah satu dari 4 kandidat PPD, THB2, tidak dapat diekspresikan dalam prokariota. ALMR, SIMR dan VPMR memiliki kemampuan untuk mengikat DNA dan melindungi DNA dari efek nuclease. Uji in vitro menunjukkan hanya ALMR yang dapat menghantarkan pcDNA3.1eGFP ke inti yang ditandai oleh ekspresi transgen oleh CHOK1. Jika dibandingkan dengan Lipofectamine, efektivitas penghantaran DNA oleh ALMR pada sel membelah kurang efektif,namun penghantaran DNA oleh ALMR pada sel tidak membelah lebih efektif dibandingkan Lipofectamine. Penambahan ALMR ke dalam komplek Lipofectamine:DNA dapat meningkatkan efisiensi penghantaran DNA dan viabilitas sel. Kesimpulan: PPD yang dapat mengikat, melindungi, dan menghantarkan DNA ke dalam sel membelah dan tidak membelah telah berhasil diperoleh. Penggabungan PPD dengan lipofectamine dapat meningkatkan efisiensi dan efektivitas penghantaran DNA kedalam sel tidak membelah serta meningkatkan viabilitas sel tidak membelah pasca transfeksi.

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Background: The journey of extracellular DNA to reach its active site, nucleus, is dependent on its capability to overcome cellular barriers such as cell membrane, endosomal compartment and nuclear membran. To overcome such natural barriers, we developed peptide-mediated DNA delivery system that could deliver DNA especially into non-dividing cells as the majority of human cells are found in nondividing state.

Methodology: Based on literature studies we found peptides that have capability 1) to translocate/fuse with cellular membrane, 2) to bind DNA and protect DNA from nuclease 3) as nuclear localization signal (NLS). We convert peptides into DNA sequences by using software. DNA coding peptide-mediated DNA delivery systems were synthesized by commercially and manually using conventional PCR. The DNA fragments were cloned into prokaryote-expression systems. After expression and purification, peptide-mediated DNA delivery systems were tested their capability to increase the efficiency and effectiveness of the transformation of extracellular DNA in dividing and non-dividing cells.

Result: We constructs 5 candidates of peptide-mediated delivery system. One of them has no capability to bind DNA and located using nucleus. One of the rest peptidemediated delivery system could not be expressed in prokaryote system. Furthermore, three candidates have capability to binds DNA and protect DNA from nuclease degradation. Based on in vitro study, only one candidate has the capability to deliver DNA pcDNA3.1 eGFP into CHOK1 nucleus that marked by the expression of transgen. Compared to the Lipofectamine, a commercial delivery system, the capability of ALMR to deliver DNA into dividing cell is less efficient, but the capability of ALMR to deliver DNA into non-dividing cells is more efficient compare to Lipofectamine. The addition of ALMR into lipofectamine-DNA complex could increasing both the percentage of transgeneexpressing cells and viability of cell.

Conclusions: We have gained one peptide-mediated delivery system that could bind, protect and deliver DNA from extracellular into intracellular environment of dividing and non dividing cells. The addition of peptide-mediated delivery into Lipofectamine could increase the efectivity of DNA transformation and increase the cell viability."

"Dalam upaya menginduksi kekebalan berspektrum luas yang responsif terhadap subtipe-subtipe HIV-1 yang berbeda, telah diteliti imunisasi vaksin DNA menggunakan vektor plasmid DNA dan virus fowlpox rekombinan dengan memanfaatkan gen-gen HIV-1 yang dirancang dari runutan konsensus turunan subtipe-subtipe HIV-1 di dunia yang mengekspresikan semua protein dari genom HIV-1 dengan peptida berukuran 30 asam amino yang overlapping dan tersusun secara acak (scrambled antigen vaccines, atau SAVINE).Tiga grup hewan coba yang terdiri dari masing-masing tujuh beruk (Macaca nemestrina) diimunisasi dengan regimen vaksin DNA standar dengan veklor plasmid DNA pHIS-64 dan vektor virus fowlpox rekombinan (rFPV) berbasis gen gag dan pol dan HIV-1 subtipe B, regimen vaksin DNA SAVINE dengan vektor pHIS-64 dan vektor rFPV berbasis genom HIV-1 yang diacak, serta vektor plasmid pHIS-64 dan FPV yang tidak mengandung gen sebagai grup kontrol. Respon kebal selular diamati dengan teknik ELiSpot dan pewamaan silokin intraselular, sedangkan respon kebal humoral diamati dengan teknik ELISA. Pada ketujuh hewan coba yang diimunisasi dengan vaksin DNA HIV-1 standar, secara umum hasil penelitian menunjukkan terinduksinya respon kebal selular terhadap protein Gag HIV-1 serta respon kebal humoral yang ditunjukkan dengan terdeteksinya antibodi terhadap protein p24 HIV-1. Respon kebal selular silang terhadap protein Gag HIV-1 dari subtipe yang berbeda juga ditunjukkan pada grup yang sama. Namun upaya melakukan imunisasi boosting ke-dua dengan vektor rFPV tidak menunjukkan perbaikan induksi respon kebal. Berbeda dari grup hewan coba yang menerima regimen vaksin DNA HIV-1 standar, pada grup yang menerima regimen vaksin DNA HIV-1 SAVINE secara umum tidak menunjukkan adanya induksi respon kebal, kecuali pada satu ekor hewan yang menunjukkan respon kebal selular yang lebih luas terhadap protein Pol dan protein-protein lain HIV-1 meski pada tingkat induksi yang amat rendah. Pengembangan teknologi vaksin SAVINE terus diperbaiki dan disempumakan dengan kemungkinan melibatkan vektor virus aktif yang lain sehingga induksi respon kebal yang diharapkan bisa tercapai.Specific Immune Responses to the Human Immunodeficiency Virus Type-1 (HIV-1) Proteins In Pigtail Macaque (Macaca nemestrina) Immunized with Whole Gene and Whole Virus Scrambled Antigen Vaccines

T cell immunity plays a critical role in controlling HIV-1 viremia, and encoding a limited set of HIV-1 genes within DNA and poxvirus vectors can, when used sequentially, induce high levels of T cell immunity in primates. However, a limited breadth of T cell immunity exposes the host to potential infection with either genetically diverse HIV-1 strains or T cell escape variants of HIV-1. In an attempt to induce maximally broad immunity, we examined DNA (prime) and recombinant Fowlpox virus (rFPV, boost) vaccines encoding all HIV-1 genes derived from a global HIV-1 consensus sequence, but expressed as multiple overlapping scrambled 30 amino acid segments (scrambled antigen vaccines, or SAVINEs).

Three groups of 7 pigtail macaques (Macaca nemestrina) were immunized with sets of DNA and rFPV expressing Gag/Pol antigens only, the whole genome SAVINE antigens, or no HIV-1 antigens. T cell immunity was monitored by ELISpot and intracellular cytokine staining, while the humoral immune response was monitored by p24 antibody capture ELISA. High levels of cross-subtype HIV-specific T cell immunity to Gag were consistently induced in the 7 macaques primed with DNA and rFPV vaccines expressing Gag/Poi as intact proteins. The humoral immunity was also induced in the animals from the same group. It was however, difficult to repeatedly boost immunity with further rFPV immunizations, presumably reflecting high levels of anti-FPV immunity. Unfortunately, this vaccine study did not consistently achieve a broadened level of T cell immunity to multiple HIV genes utilizing the novel whole-virus SAVINE approach, with only one of 7 immunized animals generating broad T cell immunity to multiple HIV-1 proteins. Further refinements are planned with alternate vector strategies to evaluate the potential of the SAVINE technology."

"Measles immunization has been introduced since 1960, thereby markedly reducing the number of cases in developed countries. However, measles epidemics still occur even in developed countries. In the United States, in 1988-1992 an increase in the number of measles cases reaching 50,000 cases was reported. Some of these cases occurred in previously immunized patients. This was thought to be caused by genetic mutation of the measles virus, aside from weaknesses of the vaccine and low immunization coverage. Since measles immunization was employed in Indonesia, the number of measles patients has decreased. However, epidemics are still frequently reported. About 15-30% of reported cases occurred in those previously immunized, raising the question of whether a genetic difference exists between the wild-type measles virus circulating in Indonesia and the vaccine virus being used. Such a difference may lead to the differences in the antigenicity of the wild-type and vaccine viruses, rendering the resulting antibody incapable of neutralizing the wild-type viruses. Based on the above, this study is aimed to demonstrate the extent of genetic and antigenic differences between the wild-type and vaccine measles viruses. We conducted an experimental laboratory study to sequence the N, H, and F genes of the wild-type measles viruses (G2, G3, and D9) and the CAM-70 vaccine virus. To show antigenic differences, the wild-type viruses (G2, G3, and D9) and the CAM-70 and Schwarz viruses were injected to BALB/c mice. Serum antibodies of the mice were analyzed using ELISA, cross-neutralization test, and immunoblotting using antigens from the respective viruses. Results of this study showed that the wild-type and the vaccine viruses differ in the sequence of the N gene by 73-79 nucleotides, resulting in amino acid substitution of 17-24 residues; the H gene by 60-99 nucleotides, resulting in amino acid substitution of 13-29 residues; the F gene by 71-88 nucleotides, resulting in amino acid substitution of 4-3 I residues. Differences between the wild-type and the CAM-70 and Schwarz vaccine viruses were also found in the epitope site of the CTL and antibodies, which are important to virus antigenicity. We conclude that a significant difference in antigenicity exists between the wild-type measles viruses circulating in Indonesia with the CAM-70 measles virus. We also found the immunogenicity of the CAM-70 and Schwarz vaccine viruses to be lower than that of the wild-type viruses."

"ABSTRAKDiagnosis HIV pada bayi masih sulit ditentukan pada daerah dengan sumber terbatas dan tidak memiliki fasilitas pemeriksaan PCR. Keterlambatan menentukan diagnosis pada bayi tertular HIV yang lahir dari ibu HIV positif akan menimbulkan angka morbiditas dan mortalitas yang tinggi. Penelitian ini bertujuan menemukan model prediksi risiko bayi tertular HIV yang efektif yaitu yang memiliki nilai sensitivitas dan spesifisitas cukup baik dan praktis penggunaannya di lapangan. Penelitian terdiri dari dua tahapan yaitu tahap pertama pembuatan model dari faktor risiko pada ibu, bayi, dan persalinan serta tahap kedua validasi skoring model. Subjek tahap pertama berasal dari data rekam medis pasangan ibu HIV positif dan bayi yang dilahirkannya di 5 rumah sakit di Jakarta dan Kepulauan Riau dan 1 puskesmas di Jakarta sebanyak 100 subjek. Didapatkan 2 model skor yang efektif sebagai model prediksi risiko bayi tertular HIV yaitu Model 1 (terdiri dari usia ibu, ARV pada ibu, infeksi TB paru, usia gestasi, cara persalinan dan jenis kelamin bayi) dan Model 2 (ARV pada ibu, infeksi TB paru ibu, dan cara persalinan). Model 2 selain efektif juga praktis untuk penggunaan di lapangan. Validasi eksterna terhadap 20 subjek bayi yang lahir dari ibu dengan HIV positif di 3 rumah sakit di Jakarta menunjukkan tidak terdapat perbedaan hasil antara Model 2 dan pemeriksaan PCR RNA HIV bayi usia 6 minggu. Model 2 adalah model prediksi yang efektif dan praktis untuk prediksi risiko bayi tertular HIV yang lahir dari ibu HIV positif di daerah dengan sumber dan fasilitas terbatas.

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ABSTRACTHIV diagnosis in infants is still difficult to determine in areas with limited resources and no PCR examination facilities. Delay in diagnosing HIV infected infants born to HIV positive mothers will lead to high morbidity and mortality. The aim of this study is to find an effective and practical model to be used in the field to predict risk of HIV transmission in infants born to HIV positive mothers, with relatively well sensitivity and specificity. This study consisted of two stages. The first stage was to develop a risk factor model consisting of maternal, infant and obstetric risk factors, and the second stage was to validate the scoring model. Data for the first stage was obtained using medical records of 100 infants born to HIV positive mothers in 5 hospitals in Jakarta and Riau Islands, as well as 1 community health center in Jakarta. Two effective models were generated in this study, namely: Model 1 (consisting of maternal age, maternal ARV therapy, maternal tuberculosis infection, gestational age, method of delivery, sex of the infant) and Model 2 (consisting of maternal ARV treatment, maternal tuberculosis infection, and mode of delivery). Model 2 is more effective and practical to be used in the field. External validation performed on 20 infants born to HIV positive mothers in three hospitals in Jakarta showed that there were no differences between the scoring model and PCR RNA HIV results. Model 2 can be used on infants born to HIV positive mothers as an effective and practical transmission risk prediction tool for in areas with limited resources and facilities"