Screening of In-Vitro Anti-Inflammatory and Antioxidant Activity of Sargassum ilicifolium Crude Lipid Extracts from Different Coastal Areas in Indonesia.

Sargassum brown seaweed is reported to exhibit several biological activities which promote human health, such as anticancer, antimicrobial, antidiabetic, anti-inflammatory, and antioxidant activity. This study aimed to investigate the anti-inflammatory and antioxidant activity of crude lipid extracts of Sargassum ilicifolium obtained from four different coastal areas in Indonesia, namely Awur Bay-Jepara (AB), Pari Island-Seribu Islands (PI), Sayang Heulang Beach-Garut (SHB), and Ujung Genteng Beach-Sukabumi (UGB). Results showed that treatment of RAW 264.7 macrophage cells with UGB and AB crude lipid extracts (12.5-50 µg/mL) significantly suppressed the nitric oxide production after lipopolysaccharide stimulation, both in pre-incubated and co-incubated cell culture model. The anti-inflammatory effect was most marked in the pre-incubated cell culture model. Both two crude lipid extracts showed 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and high ferric reducing antioxidant power, which were amounted to 36.93-37.87 µmol Trolox equivalent/g lipid extract and 681.58-969.81 µmol FeSO4/g lipid extract, respectively. From this study, we can conclude that crude lipid extract of tropical S. ilicifolium can be further developed as a source of anti-inflammatory and antioxidant agent.

Bone Growth Capacity of Human Umbilical Cord Mesenchymal Stem Cells and BMP-2 Seeded Into Hydroxyapatite/Chitosan/Gelatin Scaffold in Alveolar Cleft Defects: An Experimental Study in Goat.

OBJECTIVE: To evaluate bone regeneration in alveolar defects treated with human umbilical cord-derived mesenchymal stem cells (hUCMSCs), hydroxyapatite/chitosan/gelatin (HA/CS/Gel) scaffold, and bone morphogenic protein-2 (BMP-2) in Capra hircus models. DESIGN: Randomized posttest-only control group design. SETTING: Animal Hospital at Bogor Agricultural Institute. PARTICIPANTS: Healthy and equally treated 24 female Capra hircus/goats. INTERVENTION: Animals were randomly assigned to 3 experimental group design (iliac crest alveolar bone graft/ICABG [control], HA/Cs/Gel+BMP-2 [Novosys], and HA/Cs/Gel+BMP-2+UCMSCs). Graft materials were implanted in surgically made alveolar defects. MAIN OUTCOME MEASURES: Postoperative functional score and operating time were assessed. New bone growth, bone density, inflammatory cells recruitment, and neoangiogenesis were evaluated based on radiological and histological approach at 2 time points, week 4 and 12. Statistical analysis was done between treatment groups. RESULTS: Operating time was 34% faster and functional score 94.5% more superior in HA/Cs/Gel+BMP-2+hUCMSC group. Bone growth capacity in HA/Cs/Gel+BMP-2+UCMSCs mimicked ICABG, but ICABG showed possibility of bone loss between week 4 and 12. The HA/Cs/Gel+BMP-2+UCMSCs showed early bone repopulation and unseen inflammatory cells and angiogenesis on week 12. DISCUSSION AND CONCLUSION: The HA/Cs/Gel+BMP-2+hUCMSCs were superior in enhancing new bone growth without donor site morbidity compared to ICABG. The presence of hUCMSCs in tissue-engineered alveolar bone graft (ABG), supported with paracrine activity of the resident stem cells, initiated earlier new bone repopulation, and completed faster bone regeneration. The HA/Cs/Gel scaffold seeded with UCMSCs+BMP-2 is a safe substitute of ICABG to close alveolar bone defects suitable for patients with cleft lip, alveolus, and palate.

Sargassum Seaweed as a Source of Anti-Inflammatory Substances and the Potential Insight of the Tropical Species: A Review.

Sargassum is recognized both empirically and scientifically as a potential anti-inflammatory agent. Inflammation is an important response in the body that helps to overcome various challenges to body homeostasis such as microbial infections, tissue stress, and certain injuries. Excessive and uncontrolled inflammatory conditions can affect the pathogenesis of various diseases. This review aims to explore the potential of Sargassum's anti-inflammatory activity, not only in crude extracts but also in sulfated polysaccharides and purified compounds. The tropical region has a promising availability of Sargassum biomass because its climate allows for the optimal growth of seaweed throughout the year. This is important for its commercial utilization as functional ingredients for both food and non-food applications. To the best of our knowledge, studies related to Sargassum's anti-inflammatory activity are still dominated by subtropical species. Studies on tropical Sargassum are mainly focused on the polysaccharides group, though there are some other potentially bioactive compounds such as polyphenols, terpenoids, fucoxanthin, fatty acids and their derivatives, typical polar lipids, and other groups. Information on the modulation mechanism of Sargassum's bioactive compounds on the inflammatory response is also discussed here, but specific mechanisms related to the interaction between bioactive compounds and targets in cells still need to be further studied.

Non-invasive surveillance for Plasmodium in reservoir macaque species.

BACKGROUND: Primates are important reservoirs for human diseases, but their infection status and disease dynamics are difficult to track in the wild. Within the last decade, a macaque malaria, Plasmodium knowlesi, has caused disease in hundreds of humans in Southeast Asia. In order to track cases and understand zoonotic risk, it is imperative to be able to quantify infection status in reservoir macaque species. In this study, protocols for the collection of non-invasive samples and isolation of malaria parasites from naturally infected macaques are optimized. METHODS: Paired faecal and blood samples from 60 Macaca fascicularis and four Macaca nemestrina were collected. All animals came from Sumatra or Java and were housed in semi-captive breeding colonies around West Java. DNA was extracted from samples using a modified protocol. Nested polymerase chain reactions (PCR) were run to detect Plasmodium using primers targeting mitochondrial DNA. Sensitivity of screening faecal samples for Plasmodium was compared to other studies using Kruskal Wallis tests and logistic regression models. RESULTS: The best primer set was 96.7 % (95 % confidence intervals (CI): 83.3-99.4 %) sensitive for detecting Plasmodium in faecal samples of naturally infected macaques (n = 30). This is the first study to produce definitive estimates of Plasmodium sensitivity and specificity in faecal samples from naturally infected hosts. The sensitivity was significantly higher than some other studies involving wild primates. CONCLUSIONS: Faecal samples can be used for detection of malaria infection in field surveys of macaques, even when there are no parasites visible in thin blood smears. Repeating samples from individuals will improve inferences of the epidemiology of malaria in wild primates.

Transplantation of mesenchymal stem cells, recombinant human BMP-2,and their combination in accelerating the union after osteotomy and increasing, the mechanical strength of extracorporeally irradiated femoral autograft in rat models.

BACKGROUND: Delayed union, nonunion, and mechanical failure is still problems encountered in limb salvage surgery (LSS) using extracorporeal irradiation (ECI). This study aimed to determine whether bone marrow mesenchymal stem cells (MSC) and recombinant human bone morphogenetic protein-2 (rhBMP-2) improve hostgraft union after osteotomy and also increase its mechanical strength. METHODS: Thirty Sprague Dawley rats were randomly divided into five groups. Group I (control) underwent LSS using ECI method with 150 Gy single doses. Similar procedures were applied to other groups. Group II received hydroxyapatite (HA) scaffold. Group III received HA scaffold and MSC. Group IV received HA scaffold and rhBMP-2. Group V received HA scaffolds, MSC, and rhBMP-2. Radiograph were taken at week-2, 4, 6, and 8; serum alkaline phosphatase and osteocalcin were measured at week-2 and 4. Histopathological evaluation and biomechanical study was done at week-8. RESULTS: The highest radiological score was found in group IV and V Similar result was obtained in histological score and ultimate bending force. These results were found to be statistically significant. There was no significant difference among groups in serum alkaline phosphatase and osteocalcin level. CONCLUSION: Combination of MSC and rhBMP-2 was proven to accelerate union and improve mechanical strength of ECI autograft.

Identification and molecular characterization of simian endogenous retrovirus in Macaca fascicularis and Macaca nemestrina from captive breeding facilities in Bogor, Indonesia.

Background and Aim: Endogenous retroviruses (ERVs) found in all vertebrates, including non-human primates (NHPs), are known to be genetically inherited. Thus, recent studies have explored ERVs for human immunodeficiency virus vaccine development using human ERV (HERV) due to the hypervariability of exogenous retroviruses which cause conventional vaccines to be ineffective. HERV was also found to be able to induce an immune response in cancer patients. This study aimed to identify and molecularly characterize ERVs from Indonesian NHPs: Macaca fascicularis and Macaca nemestrina. Then, we described the phylogenetic relationship of these isolates with those of the simian ERVs (SERVs) characterized in other species and countries. Materials and Methods: First, 5 mL of whole blood samples was taken from 131 long-tailed macaques and 58 pig-tailed macaques in captive breeding facilities at Bogor, Indonesia, for DNA extraction. Next, the DNA samples were screened using the SYBR Green real-time polymerase chain reaction (PCR) technique with specific primers for env (simian retroviruses [SRV]1-5 7585U19 and SRV1-5 7695L21). Positive SERV results were those with cycle threshold (CT) values < 24 (CT < 24) and melting temperature (TM) ranges of 80°C-82°C. Then, whole-genome nucleotide sequences from two pig-tailed macaques samples detected as positive SERV were generated using a nucleic acid sequencing technique which utilized the walking primer method. Subsequently, the sequences were analyzed using bioinformatics programs, such as 4Peaks, Clustal Omega, and BLAST (NCBI). Subsequently, a phylogenetic tree was constructed using the neighbor-joining method in MEGA X. Results: SYBR Green real-time PCR amplification results indicated that SERV (Mn B1 and Mn B140910)-positive samples had CT values of 22.37-22.54 and TM of 82°C. Moreover, whole-genome sequences resulted in 7991 nucleotide sequences, comprising long terminal repeat, gag, pro, pol, and env genes identical between the sequenced samples. Furthermore, the phylogenetic tree results indicated that both samples from M. nemestrina had 99%-100% nucleotide identities to the Mn 92227 sample identified at the National Primate Center University of Washington (NaPRC UW) which was imported from Indonesia in 1998, confirmed as a novel SERV strain. The phylogenetic tree results also indicated that although SERV whole-genome nucleotide and env amino acid sequences were clustered with SRV-2 (identity values of 82% and 79%, respectively), they had a 99%-100% nucleotide identity to Mn 92227. Meanwhile, the gag, pro, and pol amino acids were clustered with SRV-1, SRV-3, SRV-4, SRV-5, SRV-8, and SERV/1997, with 82% and 88% identity values. Conclusion: Based on the SYBR Green real-time PCR profiles generated, similarities with Mn 92227 were observed. Subsequent phylogenetic analysis confirmed that both samples (Mn B1 and Mn B140919) from pig-tailed macaques in the country of origin were novel SERV strains at NaPRC UW. Therefore, it could be used in biomedical research on ERVs.

Exposure of Wild Sumatran Tiger (Panthera tigris sumatrae) to Canine Distemper Virus.

Canine distemper virus (CDV) is recognized as a conservation threat to Amur tigers (Panthera tigris altaica) in Russia, but the risk to other subspecies remains unknown. We detected CDV neutralizing antibodies in nine of 21 wild-caught Sumatran tigers (42.9%), including one sampled on the day of capture, confirming exposure in the wild.

In-vitro anti-inflammatory activity, free radical (DPPH) scavenging, and ferric reducing ability (FRAP) of Sargassum cristaefolium lipid-soluble fraction and putative identification of bioactive compounds using UHPLC-ESI-ORBITRAP-MS/MS.

Sargassum brown seaweed is well-known to contain several bioactive compounds which exhibit various biological activities, including anti-inflammatory and antioxidant activity. Lipophilic extracts and fractions of Sargassum were reported to possess promising anti-inflammatory activity. This study, therefore, aims to evaluate the anti-inflammatory and antioxidant activity of Sargassum cristaefolium crude lipid extract and its fractions. The brown seaweed was obtained from Awur Bay, Jepara - Indonesia. Crude lipid fractionation was performed using normal phase column chromatography, and three different fractions (dichloromethane, acetone, methanol) were produced. The results showed that treatment of acetone fraction exerted strongest nitric oxide inhibition in lipopolysaccharide-induced RAW 264.7 cells, both in pre-incubated and co-incubated cell culture models. This outcome was in accordance with its 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity and ferric reducing antioxidant power (FRAP). Metabolite profiling of lipid fractions was performed by ultra-high-performance liquid chromatography electrospray ionization orbitrap tandem mass spectrometry, while the orthogonal projection to latent structures analysis was conducted to determine some features with significant correlation to the bioactivity. There were 14 feature candidates considered from both positive and negative ionization mode datasets. Seven out of them were putatively identified as pheophytin a (1), all-trans fucoxanthin (2), 132-hydroxy-pheophytin a (3), pheophorbide a (4), 1-hexadecanoyl-2-(9Z-octadecenoyl)-3-O-ß-D-galactosyl-sn-glycerol (6), 1-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-2-(9Z,12Z,15Z-octadecatrienoyl)-3-O-ß-D-galactosyl-sn-glycerol (10), and 1-(9Z,12Z,15Z-octadecatrienoyl)-2-(6Z,9Z,12Z,15Z-octadecatetraenoyl)-3-O-ß-D-galactosyl-sn glycerol (12).

Construction of A Preliminary Three-Dimensional Structure Simian betaretrovirus Serotype-2 (SRV-2) Reverse Transcriptase Isolated from Indonesian Cynomolgus Monkey.

Simian betaretrovirus serotype-2 (SRV-2) is an important pathogenic agent in Asian macaques. It is a potential confounding variable in biomedical research. SRV-2 also provides a valuable viral model compared to other retroviruses which can be used for understanding many aspects of retroviral-host interactions and immunosuppression, infection mechanism, retroviral structure, antiretroviral and vaccine development. In this study, we isolated the gene encoding reverse transcriptase enzyme (RT) of SRV-2 that infected Indonesian cynomolgus monkey (Mf ET1006) and predicted the three dimensional structure model using the iterative threading assembly refinement (I-TASSER) computational programme. This SRV-2 RT Mf ET1006 consisted of 547 amino acids at nucleotide position 3284-4925 of whole genome SRV-2. The polymerase active site located in the finger/palm subdomain characterised by three conserved catalytic aspartates (Asp90, Asp165, Asp166), and has a highly conserved YMDD motif as Tyr163, Met164, Asp165 and Asp166. We estimated that this SRV-2 RT Mf ET1006 structure has the accuracy of template modelling score (TM-score 0.90 ± 0.06) and root mean square deviation (RMSD) 4.7 ± 3.1Å, indicating that this model can be trusted and the accuracy can be seen from the appearance of protein folding in tertiary structure. The superpositionings between SRV-2 RT Mf ET1006 and Human Immunodeficiency Virus-1 (HIV-1) RT were performed to predict the structural in details and to optimise the best fits for illustrations. This SRV-2 RT Mf ET1006 structure model has the highest homology to HIV-1 RT (2B6A.pdb) with estimated accuracy at TM-score 0.911, RMSD 1.85 Å, and coverage of 0.953. This preliminary study of SRV-2 RT Mf ET1006 structure modelling is intriguing and provide some information to explore the molecular characteristic and biochemical mechanism of this enzyme.

Water and Lipid-Soluble Component Profile of Sargassum cristaefolium from Different Coastal Areas in Indonesia with Potential for Developing Functional Ingredient.

Sargassum brown seaweed is known to have many health benefits and therapeutic effects. Preliminary chemical characterization of this seaweed is important as a bioprospecting strategy for seaweed industry development. This study aimed to evaluate chemical composition differences, both water and lipidsoluble component, of Sargassum cristaefolium from four different coastal areas in Indonesia, namely Pari Island/PI, Awur Bay/AB, Ujung Genteng Beach/UGB, and Sayang Heulang Beach/SHB. Principal component analysis (PCA) on water-soluble component made samples from different origins to be clearly distinguished (variance: 80.37%). SHB and UGB samples were characterized by a high content of ash, alginate, fucose-containing sulfated polysaccharides (FCSPs), and fucose content of FCSPs, while samples of AB and PI had a high amount of total sugar and crude fiber. PCA result on lipid-soluble components showed a different tendency that SHB and AB samples were located at close proximity and characterized by larger blade size, higher content of chlorophyll, fucoxanthin, carotenoid, PUFA, total n-3 fatty acids, total n-6 fatty acids, and also a lower ratio of n-6 to n-3 (variance: 75.42%). The overview of each samples' chemical characteristics can be valuable knowledge for further development, especially for developing functional ingredients.

Humoral Immune Responses to Burkholderia pseudomallei Antigens in Captive and Wild Macaques in the Western Part of Java, Indonesia.

Burkholderia pseudomallei, the Gram-negative bacterium which causes melioidosis, is a threat to human and a wide range of animal species. There is an increased concern of melioidosis in Indonesian primate facilities, especially following case reports of fatal melioidosis in captive macaques and orangutans. Our preliminary serosurveillance of immunoglobulin G (IgG) to B. pseudomallei lipopolysaccharide showed that a significant number of captive and wild macaques in the western part of Java, Indonesia, have been exposed to B. pseudomallei. To better characterize the humoral immune response in those animals, a panel of assays were conducted on the same blood plasma specimens that were taken from 182 cynomolgus macaques (M. fascicularis) and 88 pig-tailed macaques (M. nemestrina) reared in captive enclosures and wild habitats in the western part of Java, Indonesia. The enzyme-linked immunosorbent assays (ELISAs) in this study were conducted to detect IgG against B. pseudomallei proteins; alkyl hydroperoxide reductase subunit C (AhpC), hemolysin-coregulated protein (Hcp1), and putative outer membrane porin protein (OmpH). The performances of those immunoassays were compared to ELISA against B. pseudomallei LPS, which has been conducted previously. Seropositivity to at least one assay was 76.4% (139/182) and 13.6% (12/88) in cynomolgus macaques and pig-tailed macaques, respectively. Analysis of demographic factors showed that species and primate facility were significant factors. Cynomolgus macaques had higher probability of exposure to B. pseudomallei. Moreover, macaques in Jonggol facility also had higher probability, compared to macaques in other facilities. There were no statistical associations between seropositivity with other demographic factors such as sex, age group, and habitat type. There were strong positive correlations between the absorbance results of AhpC, HcpI, and OmpH assays, but not with LPS assay. Our analysis suggested that Hcp1 assay would complement LPS assay in melioidosis serosurveillance in macaques.

Characterization of Burkholderia pseudomallei from spontaneous melioidosis in a Bornean orangutan.

BACKGROUND AND AIM: Melioidosis is a potentially fatal disease affecting humans and a wide range of animal species; it is often underdiagnosed and underreported in veterinary medicine in Indonesia. This study aimed to characterize morphological and molecular features of Burkholderia pseudomallei, the causative agent of melioidosis which caused the death of a Bornean orangutan. MATERIALS AND METHODS: Pulmonary abscess samples were cultured on several types of media, including Ashdown agar, Ashdown broth, and MacConkey agar. Type three secretion system orf 2 real-time polymerase chain reaction (PCR) and latex agglutination tests were performed to identify the bacteria. Morphological characteristics were compared to all previously published morphotypes. Subsequently, the bacteria were characterized by multilocus sequence typing (MLST) and Yersinia-like flagellum/Burkholderia thailandensis-like flagellum and chemotaxis PCR. The results of the genotyping were afterward compared to all genotypes from Southeast Asia. RESULTS: Multiple morphotypes of B. pseudomallei were perceived during the growth on Ashdown agar. Furthermore, it was identified by MLST that the Type I and Type II morphotypes observed in this study were clones of a single ST, ST54, which is predominantly found in humans and the environment in Malaysia and Thailand, although a very limited number of reports was published in association with animals. Moreover, the E-BURST analysis showed that the ST is grouped together with isolates from Southeast Asian countries, including Malaysia, Thailand, Singapore, and Cambodia. ST54 was predicted to be the founding genotype of several STs from those regions. CONCLUSION: B. pseudomallei ST54 that caused the death of a Bornean orangutan has a distant genetic relationship with other STs which were previously reported in Indonesia, implying a vast genetic diversity in Indonesia that has not been discovered yet.

Mammosphere Culture of Mammary Cells from Cynomolgus Macaques (Macaca fascicularis).

The mammary gland contains adult stem cells that are capable of self-renewal. Although these cells hold an important role in the biology and pathology of the breast, the studies of mammary stem cells are few due to the difficulty of acquiring and expanding undifferentiated adult stem cell populations. In this study, we developed mammosphere cultures from frozen mammary cells of nulliparous cynomolgus macaques (Macaca fascicularis) as a culture system to enrich mammary stem cells. Small samples of mammary tissues were collected by surgical biopsy; cells were cultured in epithelial cell growth medium and cryopreserved. Cryopreserved cells were cultured into mammospheres, and the expression of markers for stemness was evaluated by using quantitative PCR analysis. Cells were further differentiated by using 2D and 3D approaches to evaluate morphology and organoid budding, respectively. The study showed that mammosphere culture resulted in an increase in the expression of mammary stem cell markers with each passage. In contrast, markers for epithelial cells and pluripotency decreased across multiple passages. The 2D differentiation of the cells showed heterogeneous morphology, whereas 3D differentiation allowed for organoid formation. The results indicate that mammospheres can be successfully developed from frozen mammary cells derived from breast tissue collected from nulliparous cynomolgus macaques through surgical biopsy. Because mammosphere cultures allow for the enrichment of a mammary stem cell population, this refined method provides a model for the in vitro or ex vivo study of mammary stem cells.

Replication deficient human adenovirus vector serotype 19a/64: Immunogenicity in mice and female cynomolgus macaques.

The human adenovirus type 19a/64 (hAd19a) is a rare serotype in the human population that transduces human dendritic cells (DCs) and human muscle cells more efficiently than the well-characterized human adenovirus type 5 (hAd5). To further characterize the potential of this vector as a vaccine we designed replication deficient hAd19a, hAd5 and MVA vectors expressing a papillomavirus (PV) antigen fused to the human MHC class II associated invariant chain T cell adjuvant (hIi) and investigated their immunogenicity in vivo in mice and cynomolgus macaques. We initially showed that the hIi encoded in the hAd5 enhanced PV specific CD8+ T cell responses in mice. The T cell responses induced after hAd19a vaccination was similar to those induced by hAd5 vaccination. The hAd19a induced responses were not reduced in presence of preexisting Ad5 immunity in mice. In macaques both vaccines were equally potent at inducing CD8+ T cells after MVA boost, while the level of CD4+ T cell responses were found to be broader in hAd19a primed animals. These data demonstrate the potential of hAd19a as an alternative vector to hAd5 to elicit potent T cell responses to PV.

Emergence of Melioidosis in Indonesia and Today's Challenges.

A recent modeling study estimated that there could be as many as 20,000 human melioidosis cases per year in Indonesia, with around 10,000 potential deaths annually. Nonetheless, the true burden of melioidosis in Indonesia is still unknown. The Indonesia Melioidosis Network was formed during the first melioidosis workshop in 2017. Here, we reviewed 101 melioidosis cases (99 human and two animal cases) previously reported and described an additional 45 human melioidosis cases. All 146 culture-confirmed cases were found in Sumatra (n = 15), Java (n = 104), Kalimantan (n = 15), Sulawesi (n = 11) and Nusa Tenggara (n = 1). Misidentification of Burkholderia pseudomallei was not uncommon, and most cases were only recently identified. We also evaluated clinical manifestations and outcome of recent culture-confirmed cases between 2012 and 2017 (n = 42). Overall, 15 (36%) cases were children (age <15 years) and 27 (64%) were adults (age ≥15 years). The overall mortality was 43% (18/42). We conducted a survey and found that 57% (327/548) of healthcare workers had never heard of melioidosis. In conclusion, melioidosis is endemic throughout Indonesia and associated with high mortality. We propose that top priorities are increasing awareness of melioidosis amongst all healthcare workers, increasing the use of bacterial culture, and ensuring accurate identification of B. pseudomalleiand diagnosis of melioidosis.

Therapeutic Vaccine Against Primate Papillomavirus Infections of the Cervix.

Currently available prophylactic vaccines have no therapeutic efficacy for preexisting human papillomavirus (HPVs) infections, do not target all oncogenic HPVs and are insufficient to eliminate the burden of HPV induced cancer. We aim to develop an alternative HPV vaccine which is broadly effective and capable of clearing preexisting infection. In an initial attempt to develop a broadly reactive therapeutic vaccine, we designed a putative papillomavirus (PV) ancestor antigen (circulating sequence derived antigenic sequences E1E2-CDSE1E2) based on the conserved E1 and E2 protein sequences from existing oncogenic HPV strains. This antigen was found to be as related to circulating oncogenic Macaca fascicularis papillomaviruses (MfPVs) as to oncogenic HPVs. The CDSE1E2 antigen was fused to a T-cell adjuvant and encoded in chimpanzee 3 and 63 adenoviral vectors. We first showed that the combination of these 2 vaccines induced long-lasting potent CDSE1E2 specific T cell responses in outbred mice. This prime-boost regimen was then tested in female macaques naturally infected with MfPVs. All immunized animals (16/16) responded to the vaccine antigen but with reduced cross-reactivity against existing PVs. Preexisting MfPV infections did not prime vaccine inducible immune responses. Importantly, immunized oncogenic MfPV type 3 (MfPV3) infected animals that responded toward MfPV3 were able to diminish cervical MfPV3 DNA content. Although insufficient breadth was achieved, our results suggest that a relevant level of E1E2 specific T cell immunity is achievable and might be sufficient for the elimination of PV infection. Importantly, naturally infected macaques, offer a relevant model for testing vaccines aimed at eliminating mucosal PV infections.

New Bone Formation in Tuberculous-Infected Vertebral Body Defect after Administration of Bone Marrow Stromal Cells in Rabbit Model.

STUDY DESIGN: Preliminary experimental study using a rabbit spondylitis model. PURPOSE: To observe the ossification in a micro-environment containing live Mycobacterium tuberculosis transplanted with bone marrow stromal cells (BMSCs) in rabbits. OVERVIEW OF LITERATURE: BMSCs differentiate to osteoblasts and then osteocytes during ossification. Mycobacterium tuberculosis does not affect BMSC growth in vitro. METHODS: Six rabbits were divided into two groups of three rabbits. One group was positive for spondylitis tuberculosis by culture, polymerase chain reaction (PCR), and histopathologically. The other group was positive by PCR and histopathologically. Both groups were treated using BMSC transplantation and anti-tuberculosis drugs. After 6 weeks, ossification was evaluated by enumerating the number of osteoblasts, osteocytes, and lesion level of calcium. RESULTS: Mean number of osteoblasts was 207.00±31.00 in the first group and 220.33±73.46 in the second group. Mean number of intra-lesions osteocytes was in the first and second group was 18.33±30.04 and 31.00±26.87, respectively. Mean calcium level in the first group and second group was 2.94%±0.89% and 2.51%±0.13%, respectively. Total ossification score in the first and second group was 31.00 and 25.67, respectively. CONCLUSIONS: Mycobacterium tuberculosis provides support for new bone formation by stimulating intra-lesion calcium metabolism. The microscopic environment containing live Mycobacterium tuberculosis enhances ossification.

Comparison of whole gene and whole virus scrambled antigen approaches for DNA prime and fowlpox virus boost HIV type 1 vaccine regimens in macaques.

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 and recombinant fowlpox virus (rFPV) 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 seven pigtail macaques were immunized with sets of DNA and rFPV expressing Gag/Pol antigens only, the whole genome SAVINE antigens, or no HIV-1 antigens and T cell immunity was monitored by ELISpot and intracellular cytokine staining. High levels of cross-subtype HIV-specific T cell immunity to Gag were consistently induced in the seven macaques primed with DNA and rFPV vaccines expressing Gag/Pol as intact proteins. 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 seven immunized animals generating broad T cell immunity to multiple HIV-1 proteins. Further refinements are planned with alternative vector strategies to evaluate the potential of the SAVINE technology.

A whole-arm translocation (WAT8/9) separating Sumatran and Bornean agile gibbons, and its evolutionary features.

Gibbons, like orangutans, are a group of threatened Asian apes, so that genetic monitoring of each species or subspecies is a pressing need for conservation programmes. We conducted a project to take, as far as possible, samples of known origin from wild-born animals from Sumatra and Borneo (Central Kalimantan) for genetic monitoring of agile gibbons. As a result, we found a whole arm translocation between chromosomes 8 and 9 (WAT8/9) specific to Sumatran agile gibbons. Furthermore, population surveys suggested that the form with the WAT8/9 seems to be incompatible with an ancestral form, suggesting that the former might have extinguished the latter from Sumatran populations by competition. In any case, this translocation is a useful chromosomal marker for identifying Sumatran agile gibbons. Population genetic analyses with DNA showed that the molecular genetic distance between Sumatran and Bornean agile gibbons is the smallest, although the chromosomal difference is the largest. Thus, it is postulated that WAT8/9 occurred and fixed in a small population of Sumatra after migration and geographical isolation at the last glacial period, and afterwards dispersed rapidly to other populations in Sumatra as a result of the bottleneck effect and a chromosomal isolating mechanism.