Search

Background and Aim: The intricate healing process involves distinct sequential and overlapping phases in thermal injury. To maintain the zone of stasis in Jackson’s burn wound model, proper wound intervention is essential. The extent of research on the histoarchitecture of thermal wound healing and the application of mesenchymal stem cell (MSC)-free-based therapy is limited. This study aimed to assess the efficacy of MSC-secretome-based hydrogel for treating partial-thickness cutaneous thermal burn wounds. Materials and Methods: Eighteen male Wistar rats were divided into three groups, namely the hydrogel base (10 mg), hydrogel secretome (10 mg) and Bioplacenton™ (10 mg) treatment groups. All groups were treated twice a day (morning and evening) for 7 days. Skin tissue samples from the animals were processed for histological evaluation using the formalin-fixed paraffin-embedded method on days 3 and 7. Results: This study’s findings showed that secretome hydrogel expedited thermal burn wound healing, decreasing residual burn area, boosting collagen deposition and angiogenesis, guiding scar formation, and influencing the inflammation response facilitated by polymorphonuclear leukocytes and macrophages. Conclusion: The secretome hydrogel significantly improves healing outcomes in partial-thickness cutaneous thermal burn wounds. The administration of secretome hydrogel accelerates the reduction of the residual burn area and promotes fibroblast proliferation and collagen density. The repairment of histo-architecture of the damaged tissue was also observed such as the reduction of burn depth, increased angiogenesis and epidermal scar index while the decreased dermal scar index. Furthermore, the secretome hydrogel can modulate the immunocompetent cells by decreasing the polymorphonuclear and increasing the mononuclear cells. Thus, it effectively and safely substitutes for thermal injury stem cell-free therapeutic approaches. The study focuses on the microscopical evaluation of secretome hydrogel; further research to investigate at the molecular level may be useful in predicting the beneficial effect of secretome hydrogel in accelerating wound healing.

Vero cell culture as a platform for producing viral vaccines is an established and standardized process in vaccine manufacture. Generally, Vero cell culture requires media as a source of nutrition with serum supplementation to provide growth factors. However, the serum has several disadvantages including batch to batch variation and adventitious agent. Therefore, chemically defined serum-free media (SFM) are formulated by using standardized growth factors. Epidermal Growth Factor (EGF) is one of growth factors that showed adequate mitogenic support in serum-free medium system, especially in Vero cells. In this study, SFM-EGF media was compared with serum supplementation media, namely MEM 10% and MEM 5% FBS supplementation. The cell morphology was observed using an inverted microscope and their proliferation was evaluated using a MTT colorimetric-based assay. Vero E6 and Vero CCL-81 cells morphology did not show any morphological changes. Vero E6 and Vero CCL-81 proliferation in SFM-EGF media on day one to four did not show a significant difference compared to MEM 10% or MEM 5% serum supplementation media. However, the OD values of both Vero E6 and Vero CCL-81 cells given SFM-EGF media produced an average value below MEM 10% but higher than MEM 5% FBS supplementation. As such, this study proved that utilizing SFM-EGF could support the proliferation of Vero E6 and Vero CCL-81 cells.

Background: SARS-CoV-2 vaccine was proven to be an effective and efficient measure for mitigating pandemic. COVID-19 infection and mortality subsided along with the increaseing COVID-19 vaccination coverage. Vaccine and health resource equity are predominant factors in COVID-19 pandemic management. Vaccine development for Indonesia, aims to ensure a sustainable pandemic control and steady national stability restoration. A decent vaccine must induce immunity against COVID-19 with minimum adverse reaction. Immunogenicity and ability to induce neutralizing antibody evaluation needs to be performed as part of the SARS-CoV-2 inactivated vaccine development from East Java, Indonesia isolate (Vaksin Merah Putih-INAVAC). Objective: This research demonstrated INAVAC performance in inducing the production neutralizing antibody along with its effects on CD4+ and CD8+ cells response in Macaca fascicularis (non-human primate). Methods: Two dosages of 3 μg and 5 μg were tested, compared to sham (NaCl 0.9%) in 10 Macaca fascicularis (2 injection intramuscular with 14 days interval). All animals were monitored daily for clinical signs. Nasopharyngeal samples were analyzed using qRT-PCR while the serum were tested using ELISA and neutralization assay, whereas PBMCs were flowcytrometrically analyzed to measure CD4+ and CD8+ population. Results: It is observed that both vaccine doses could stimulate relatively similar immune response and neutralizing antibody (end GMT post challenge = 905,1), whereas higher CD8+ cells response were reported in the 5 μg group after the 3rd day post-challenge. The dose of vaccine that produce adequate immune cell stimulation with neutralizing antibody induction can be adopted to clinical study, as favorable result of these parameters could predict minimum adverse reaction from inflammation response with balanced immune response. Conclusions: Therefore, it is concluded that Vaksin Merah Putih-INAVAC with 3 μg dose showed a favorable potential to be developed and tested as human vaccine.

Context: The SARS-CoV-2 virus is the cause of the COVID-19 pandemic, which is a severe public health crisis worldwide.Aims: To analyze the SARS-CoV-2 isolates of Surabaya and predict ORF1ab polyprotein epitopes through the bioinformatics approach for vaccine candidate development. Methods: Three genomic sequences of Surabaya isolates were obtained from the GISAID, NCBI and PDB Gen-bank databases and MEGA-11 software were used to understand the transformations in the isolates. The IEDB and VaxiJen, AllerTop, and ToxinPred web servers were used to predict B-cell epitopes and analyze their antigenicity, non-allergenicity, non-toxicity, respectively. Moreover, these epitopes were linked by EAAAK for 3D modeling, refinement, and validation through Swiss-Model, Galaxy Refine, and RAMPAGE web tools. Results: The Surabaya isolates, RSDS-RCVTD-UNAIR-49-A, 54-A, and 42-A, had 10, 20, and 16 mutations in nucleotides and depicted a phylogenetically close relationship to isolates of Egypt, Pakistan, and Bangladesh, respectively. A total of 71 sequential Orf1ab B-cell epitopes were predicted, and only three peptides were found to be antigenic, non-allergenic, and non-toxic. These epitopes were linked with the EAAAK linker to develop a 3D refined and validated structure. This construct was docked with TLR-3 receptor by the Cluspro webserver and found a high affinity of ORF1ab+TLR3 due to 15 hydrogen bonds. The construct demonstrated good humoral and cellular immune responses in the C-ImmSim server, and cloning in the expression vector pET28a (+) yielded a colon of 846bp. Conclusions: ORF1ab B-cell epitopes could be useful for developing effective vaccines to combat SARS-CoV-2 infection.

Context: The current COVID-19 pandemic has significantly impacted health and socio-economic status worldwide. The only way to combat this situation is to develop an effective vaccine and immunize people around the globe. Aims: To construct a multi-epitope spike glycoprotein-based vaccine from the SARS-CoV-2 Surabaya isolate using a bioinformatics approach. Methods: The spike protein was submitted to IEDB, VaxiJen, AllerTOP, and ToxinPred webservers to predict antigenic, non-allergic, non-toxic, B- and T-cell epitopes. To develop a multi-epitope vaccine, an adjuvant cholera toxin B subunit was linked to B-cell and B-cell with T-cell through EAAAK and GPGPG linkers, respectively. The designed vaccine 3D structure development, refinement, and validation were done through PHYRE2, Galaxy Refine, and RAMPAGE webservers. Moreover, the Cluspro-2.0 webserver was used for the molecular docking of the vaccine designed with TLR3. The vaccine+TLR3 complex was docked with Surfactant protein A as a control to validate the docking results. Finally, immune-simulation and in silico cloning of the vaccine were carried out by C-ImmSim webserver and SnapGene software, respectively. Results: A multi-epitopic vaccine containing B and T-cell was developed using 392 amino acids with a molecular weight of 40825.59 Da. The docking and immunogenicity results of the vaccine met all established parameters for constructing a quality vaccine. Furthermore, the optimized sequence of the vaccine was successfully cloned in expression vector pET 28 a (+) that yielded a colon of 2724 bp. Conclusions: The vaccine’s immunogenicity demonstrates its effectiveness against SARS-CoV-2 infection. Further confirmatory testing may therefore be performed as soon as possible in the public interest.

Purpose: This study aims to evaluate the role of high-sensitivity troponin T (hsTnT) as a complementary tool for determining cardiotoxicity in non-Hodgkin lymphoma (NHL) patients receiving cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) regimen chemotherapy. Methods: We included 35 patients diagnosed with NHL who received CHOP chemotherapy. Left ventricular ejection fraction (LVEF) and hsTnT were measured at two time points: before the first cycle (pre-test) and after the fourth cycle (post-test). The LVEF and hsTnT were analysed using IBM SPSS version 24 through the paired-sample T-test, Wilcoxon signed-rank test, Pearson’s correlation and Spearman’s correlation. Results: There was a significant difference in both LVEF and hsTnT between pre-chemotherapy and post-4th chemotherapy cycles (p = 0.001). However, more contrast difference from the baseline value of hsTnT compared to LVEF could be observed. LVEF did not detect any deterioration in myocardial function. However, 10 out of 35 subjects exhibit hsTnT higher than the 99th percentile of the population (>14 pg/ml), suggesting that myocardial injury (MI) could be detected. There was no correlation between LVEF and hsTnT (p > 0.05). Conclusion: HsTnT, together with LVEF, could complement each other and offer better coverage for detecting cardiotoxicity during the administration of CHOP in NHL patients. An insignificant correlation between hsTnT and LVEF showed that cardiotoxicity existed in a broad spectrum including cellular damage and functional impairment, as hsTnT represents cellular damage, and LVEF reflects heart functional capacity.

Background: Myelosuppressive effects of chemotherapy for breast cancer treatment may trigger chemotherapy-induced neutropenia (CIN) and febrile neutropenia (FN). Filgrastim has been widely used as prophylaxis against CIN and FN. However despite filgrastim administration, some study showed FN still occur and cause patient vulnerability to infection. This study aims to evaluate factors associated with Absolute Neutrophil Count (ANC) dynamics and Docetaxel-Adryamicin-Cyclophosphamide (TAC) CIN during extended filgrastim administration in breast cancer patients. Methods: Patients were selected among breast cancer in-patients who fulfilled the eligibility criteria. Patient characteristics data and ANC were collected. The entire patients received 5μg/kg/day filgrastim by subcutaneous injection 24 hours post-chemotherapy. ANC was monitored daily and filgrastim administration was stopped when ANC reached >10000/mm3 or 14 days of administration. Kruskall-Wallis test and Spearman Correlation test was performed to analyze ANC dynamics and CIN-related factors. Results: This study included 42 breast cancer patients. Patient age median was 52 (31-70) years old. ANC nadir could be observed around 5-7 days after chemotherapy and FN occurred in two out of 38 grade 4 neutropenia patients (4.8%). Critical ANC lasted for 1 day, 2 days, and 3 days respectively in 9 (23.7%), 25 (65.8%) and 4 (10.5%) patients. There was no correlation between neutropenia and age. ANC slope and recovery duration did not show a significant difference. However, depth of nadir is inversely correlated with the duration of ANC recovery (>10000/mm3) and the duration during the peak on the 2nd day until reaching nadir both with fair strength, r = −0.489 and r = −0.438 (p

BACKGROUND: Cancer stem cells (CSCs) eradication might serve as a robust approach for cancer eradication. MCF-7 as breast cancer continuous cell line is known to contain breast CSCs (BCSCs) for its capability to maintain its original tumor population. CSCs enriched culture is a fundamental tool for CSCs targeted therapy development. Effective and unsophisticated CSCs dedifferentiation protocol for producing CSCs enriched culture is needed. METHODS: MCF-7 cells were cultured initially in Dulbecco's Modified Eagle Medium (DMEM) low glucose medium then changed to DMEM:F12. Serum starvation was performed during each medium refreshment gradually with fetal bovine serum (FBS) concentration of 10%, 5%, 2.5% until reaching 1% FBS concentration. Stable MCF-7 culture was then adapted to serum free culture system, containing DMEM:F12, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and B27 supplement as dedifferentiation protocol for 18 days. Cluster of differentiation (CD)44 and CD24 double staining immunocytochemistry was performed to evaluate cell stemness. RESULTS: The population of cells expressing BCSCs markers (CD44+/CD24low) in non-adherent single cells subpopulation was significantly increased after the dedifferentiation procedure (70.39%) compared to control groups (0.71%) (p

BACKGROUND: Carbohydrate antigens 15-3 (CA 15-3) is a conventional tumor marker in breast cancer, with low sensitivity and specificity. MicroRNA (miRNA)-21 showed its stability in circulation and could serve as powerful biomarker. The aim of this study was to evaluate miRNA-21 as breast cancer biomarker compared to CA 15-3 in Indonesian population. METHODS: Circulating plasma miRNA-21 expression was measured using qRT-PCR in 49 patients at various stages of breast cancer and 16 healthy controls. The relative expression value of miRNA-21 was calculated using 2-ΔΔCt. Meanwhile, CA 15-3 was quantified using electrochemiluminescence immunoassay (ECLIA) methods. The results of miRNA-21 and CA 15-3 plasma circulating expression were compared with controls at each stage and between stages of breast cancer. RESULTS: CA 15-3 median level in breast cancer group was 1.60 times higher compared to control group (p=0.019), 21.00 m/mL and 13.05 m/mL, respectively. Median miRNA-21 expression in breast cancer group was elevated 4.92 folds compared to control group (p=0.001), 4.43 and 0.90, respectively. There was no significant difference of CA 15-3 level between controls and all stages of breast cancer group. CA 15-3 cut-off value was 15.05 m/mL (p=0.016) with 59.2% sensitivity and 62.5% specificity. Meanwhile, there was a significant difference of miRNA-21 expression between controls and most stages of breast cancer group. Circulating miRNA-21 expression cut-off value was 2.07 (p=0.000) with 91.8% sensitivity and 87.5% specificity. CONCLUSION: Circulating miRNA-21 expression and CA 15-3 levels were significantly increased in breast cancer group compared to control group. The miRNA-21 expression increased consistently with breast cancer stage progression. miRNA-21 could serve as superior biomarker compared to CA 15-3. KEYWORDS: biomarker, breast cancer, circulating plasma, liquid biopsy, miRNA-21

Background: An immunoinformatic approach may be useful to investigate the conserved region in the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Indonesia isolates. The aim of this study was to investigate Indonesian SARS-CoV-2 isolates based on B cell epitopes by targeting the conserved regions in the spike glycoprotein to trigger increased multi-variant virus neutralization and memory response for the development of vaccine seed candidates. Methods: SARS-CoV-2 spike glycoprotein gene sequences originating from Indonesia were compared with Wuhan (China), the United Kingdom, South Africa, India, the United States, and Brazil isolates obtained from the NCBI and GISAID databases. The recognition of antigens was carried out directly using B cells through the B cell receptor (BCR). An indirect B cell activation by Cluster of Differentiation (CD)4+ T cells and major histocompatibility complex (MHC)-II was predicted through the binding with human leukocyte antigen (HLA) based on IC50 value. In addition, vaccine allergenicity and toxicity were investigated. During the molecular complex examination, the 3D peptide structure was investigated and the lowest amount of energy formed when the vaccine candidate peptide bound to BCR and MHC-II was calculated. Results: As a result, the spike glycoprotein sequences of Indonesian SARS-CoV-2 isolates had conserved regions which were very similar to reference countries such as China, the United Kingdom, South Africa, India, the United States, and Brazil. Conclusion: It was predicted that the conserved regions could be identified as the epitope of B and T CD4+ cells that produced the peptides for vaccine candidate with antigenic, non-allergen, and non-toxic properties.

Background: Incidents of SARS-CoV-2 in East Java increased steadily, and it became the second epicenter in Indonesia. The COVID-19 pandemic caused a dire multisectoral crisis all around the world. This study investigates and characterizes local isolates from East Java, Indonesia. Methods: There were 54 patients suspected with SARS-COV-2 infection and 27 patients were COVID-19 positive. Virus isolates were obtained from COVID-19 inpatients’ nasopharyngeal swabs at the Dr Soetomo Teaching Hospital, Surabaya. There were only three isolates (#6, #11, #35) with good growth characteristics. Serial blind passage and cytopathic effect observation in the Vero E6 cell line were performed for virus isolation. Confirmation of the SARS-CoV-2 infection was proven by means of reverse transcriptase-polymerase chain reactions using SARS-CoV-2 specific primers, scanning electron microscopy, and scanning transmission electron microscopy examination. Whole genome sequencing was performed using ARTIC protocol. Furthermore, SARS-CoV-2 characterization was identified through a western blot using rabbit serum immunized with inactive SARS-CoV-2 vaccine and human natural COVID-19 infection serum. Results: Spike gene analysis of three samples (#6, #11, #35) found that the D614G mutation was detected in all isolates, although one isolate exhibited the D215Y and E484D mutation. Based on whole genome analysis, those three isolates were included in clade 20A, and two isolates were included in lineage B.1.6 with one isolate belongs to lineage B.1.4.7. Conclusion: Based on molecular characterization and immunogenicity of SARS-CoV-2 East Java, Indonesia showed high titer and it has mutation in some regions.