Lysosomal enzymes and the oxygen burst capability of monocyte-derived macrophages in active drug-resistant tuberculosis patients in relation to cell attachment.

Drug resistance to tuberculosis (TB) has become an obstacle in eliminating tuberculosis. The transmission of drug-resistant TB from patients increases the incidence of primary drug-resistant (DR) TB in individuals who are in close contact. Therefore, it is necessary to incorporate an immunological approach into preventive therapy. This study focuses on the activity of lysosomal enzymes, oxygen bursts, and the attachment ability of macrophages among individuals diagnosed with active drug-resistant TB compared with close contacts with latent TB or healthy cases. We measured macrophage oxygen burst ability (Water-soluble tetrazolium salt (WST) test, Nitric Oxide production, and myeloperoxidase activity) and the degradative ability of lysosomes (activity of the ß-glucuronidase and acid phosphatase enzymes). Six active DR-TB patients and 18 close-contact cases (8 Latent Tuberculosis Infection (LTBI); 10 healthy) were recruited at Universitas Indonesia Hospital. The macrophage attachment of the LTBI group was higher than in the other groups. NO production, myeloperoxidase activity, ß-glucuronidase, and acid phosphatase were higher in the active DR-TB group. A negative correlation was uncovered between phagocytosis and NO production, myeloperoxidase activity, and lysosomal enzymes. The difference in macrophage function is expected to be a further reference in active DR-TB treatment or preventive therapy.

Activities and concentration of alpha-1 antitrypsin and cystatin C in serum from patients with house dust mite asthma.

Background: The proteolytic activities of house dust mite (HDM) allergens are involved in the pathogenesis of asthma by cleaving T-junction protein complexes, increasing the permeability of airway epithelial cells, and enabling the allergens to reach the interstitial tissue. The human body contains natural protease inhibitors such as alpha-1 antitrypsin (AAT) with antiserine protease activity and cystatin C with anticysteine protease activity. Objective: This study aimed to investigate the behavior of serum AAT and cystatin C levels in patients with HDM-allergic asthma. Methods: Ten individuals with HDM-allergic asthma and 10 healthy volunteers participated in a cross-sectional study. The serum AAT and cystatin C inhibitory activities were measured using enzymatic assays. ELISA was used to determine the serum AAT and cystatin C concentrations. Results: Serum AAT inhibitory activity (P = 0.445; P > 0.05), AAT concentration (P = 0.290; P > 0.05), and cystatin C concentration (P = 0.419; P > 0.05) did not significantly differ between the patient and control groups. However, serum cystatin C inhibitory activity in the asthmatic patient group was significantly higher than in the healthy subjects (P = 0.001; P < 0.05). There was no correlation between AAT inhibitory activity and AAT concentration or between cystatin C inhibitory activity and cystatin C concentration. Conclusion: These findings suggest that serum cystatin C activity is involved in asthma pathogenesis. Additional research is required to address this issue.

Inhibition of ALA dehydratase activity in heme biosynthesis reduces cytoglobin expression which is related to the proliferation and viability of keloid fibroblasts.

The aim of this study was to analyze the effect of heme synthesis inhibition on cytoglobin expression and its correlation with keloid fibroblast viability and proliferation. The study was conducted on primary culture of keloid fibroblasts. Heme synthesis in keloid fibroblasts was inhibited using succinyl acetone. We measured amino levulinic acid dehydratase (ALAD) enzyme activity using a colorimetric method; cytoglobin mRNA expression using qRT-PCR, cytoglobin protein expression using ELISA and immunocytochemistry, fibroblast viability using the MTT test; and fibroblast proliferation using BrdU test. The results showed that the ALAD enzyme activity level was lower in the keloid fibroblasts treated with succinyl-acetone (SA, 1, 2.5, and 5 mM) than in the control. The cytoglobin mRNA and protein expressions level were significantly lower in the keloid fibroblasts cultured with 2.5 mM and 5 mM SA than in the control and 1 mM SA. The viability and proliferation of the keloid fibroblasts decreased when the SA concentration was increased. In conclusion, the use of succinyl acetone at a concentration of 1; 2.5; and 5 mM caused decrease ALAD enzyme activity which indicated the inhibition of the heme synthesis. Inhibition of heme synthesis can affect cytoglobin expression, which correlates with the viability and proliferation of keloid fibroblasts.

The Use of Chitosan Nanoparticles for Delivery of CpG ODN in Treatment of Allergic Balb/C Mice.

Background: This study aims to prepare high stability chitosan nanoparticles (CNP) and examine the ability of CNP in CpG-ODN delivery when treating allergic mice model. Methods: Preparation and characterization of CNP were performed by ionic gelation, dynamic light scattering, and zeta sizer. The CNP cytotoxicity and activation ability of CpG ODN delivered with CNP were tested using a cell counting kit-8 and Quanti blue method. Allergic mice were injected intraperitoneal with 10 ug ovalbumin on day 0 and 7, and then treated with intranasal CpG ODN/CpG ODN, delivered with CNP/CNP, on the third week three times per week for three weeks. The ELISA method measured cytokine and IgE profiles in the allergic mice's plasma and spleen. Results: CNP results have sizes 27.73 nm±3.67 dan 188.23 nm±53.47, spherical in shape and non-toxic, and did not alter the NF-κB activation of CpG ODN in RAW-blue cells. The application of CpG ODN delivered by chitosan nanoparticles shows no statistical difference between groups of IFN-γ, IL-10, and IL-13 in Balb/c mice's plasma and spleen, in contrast with IgE level. Conclusions: The results showed that using chitosan nanoparticles as a delivery system for CpG ODN has the potency to safely CpG ODN efficacy.

High concentration of γ­H2AX correlates with a marker of apoptotic suppression and PI3K/Akt pathway upregulation in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a very aggressive type of primary brain tumor in adults with a poor prognosis. DNA double-strand breaks are known to be associated with the development of numerous cancer types due to their ability to generate genomic instabilities. In GBM, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is a common pathway that can be activated by exogenous and endogenous factors. Genomic instability may be an endogenous stimulating factor for activation of the PI3K/Akt pathway, which may inhibit the apoptosis of GBM cells. Spontaneous DNA double-strand breaks play an essential role in the survival of GBM cells, and apoptosis levels may reflect survival ability. However, no study has yet been conducted to analyse the association between spontaneous DNA double-strand breaks and apoptosis in patients with GBM prior to treatment. Therefore, the present study examined the concentrations of γ-histone 2AX (γ-H2AX), a sensitive marker of spontaneous DNA double-strand breaks, and cleaved caspase-3, a marker of apoptosis, in patients with GBM. The correlation of γ-H2AX with cleaved caspase-3, PI3K and Akt was also investigated. A total of 26 pre-treatment tumor tissue specimens from patient with GBM were analyzed to determine the concentrations of γ-H2AX, PI3K, Akt and cleaved caspase-3 using sandwich enzyme-linked immunosorbent assays. The results showed a moderate positive correlation between γ-H2AX and PI3K (r=0.52; P=0.007), a moderate positive correlation between γ-H2AX and Akt (r=0.4; P=0.041) and a strong negative correlation between γ-H2AX and cleaved caspase-3 (r=-0.61; P=0.0009). These analyses were also performed in seven tumor tissue specimens from patients with grade I glioma as controls, but no significant correlations were detected. The findings of the present study suggest that a high level of γ-H2AX may affect GBM cell apoptosis via the PI3K/Akt pathway.

Plasma Protein Profile of Lactating Women from Two Primary Health Centers in Jakarta, Indonesia.

Background: Plasma protein profile test is a potential laboratory method to assess nutritional status especially albumin and globulins levels which reflects protein adequacy. The purpose of this study is to evaluate plasma protein profile of lactating women from two primary health centers in Jakarta. Methods: A cross-sectional study was conducted involving lactating women attending routine maternal examinations in two public primary health centers in Jakarta, Indonesia. The mother's plasma total protein, albumin, globulin, and immunoglobulin levels were measured. Results: Sixty lactating women were recruited, mostly were 28 years old, slightly overweight, bearing two children, and their recent children were 2 months old. The mean total protein level was 8.13 g/dl, albumin 5.00 g/dl, globulin 3.18 g/dl, albumin: globulin ratio 1.558, mean total IgG level of 1255.98 and mean total IgM level of 135.819. All the measured plasma protein parameters were shown to be not correlated with maternal age, maternal BMI, or maternal parity. Discussion: The plasma total protein, albumin, globulin, as well as total IgG and IgM level of lactating women in Jakarta were within normal range. These biochemical parameters were shown to be not correlated with anthropometrical data such as maternal age and BMI. The small and relatively homogenous samples were supposed to be the cause of such findings. Conclusion: The plasma protein profile of lactating women in Jakarta was adequate. Further studies are required to evaluate the eligibility of plasma protein profile as biochemical parameter of nutritional status in lactating women.

Isolated Diaphorase From Bovine Erythrocyte Cannot Reduce Oxidized Cytoglobin (Metcygb).

Background: Cytoglobin (Cygb) is a relatively newly identified globin protein that acts as an oxygen transporter in tissues like hemoglobin (Hb) in erythrocytes and myoglobin (Mb) in muscles. The natural oxidation of the Fe2+ ion in its heme group into metglobin (globin-Fe3+) made the loses of oxygen binding functions. It is known metHb and metMb can be reduced enzymatically using diaphorase or cyb5r3. However, metCygb reductase had not been previously identified. This study aims to analyze the reducing activity of bovine diaphorase on metCygb. Methods: Diaphorase was isolated from bovine erythrocyte and purified using gel filtration and cationic-exchanger chromatography. Its purity was verified by SDS-PAGE and western blot (WB). The metCygb was obtained from Cygb oxidation with potassium ferrocyanide and its reducing activity was determined by spectroscopy. Results: The diaphorase (MW=30.09 kDa) was purified 10.77-fold from crude enzyme with specific activity against metHb 8.479 U/mg. The purity was confirmed by WB using primary antibody anti-cyb5r3. The purified enzyme reduced metCygb at 0.785 µgmin-1, which was 13.7 times less than the Vmax of metHb. Discussion: In conclusion, the purified diaphorase from bovine erythrocytes did not significantly reduce metCygb rather than metHb, a natural substrate in cells.

Fibropreventive and Antifibrotic Effects of Uncaria gambir on Rats with Pulmonary Fibrosis.

Pulmonary fibrosis causes scar tissue formation that disrupts the functioning of the lungs. Uncaria gambir (Hunter) Roxb (hereafter gambir)-a plant native to West Sumatra in Indonesia-contains flavonoid (+)-catechin, which has strong antioxidant activity and can be used to combat pulmonary fibrosis. This random in vivo experimental study analyzed the antifibrotic effect of gambir on the lungs of rats with bleomycin-induced fibrosis. The subjects were 10 groups of 10-week-old male rats weighing around 200-250 g. All groups were terminated at the end of the seventh week or on day 50. The lungs were cleaned, and tissues were taken to analyze inflammatory cell counts and TGF-ß1 levels using bronchoalveolar lavage (BAL) with ELISA; type I collagen and tissue inhibitor of metalloproteinase 1 (TIMP-1) levels using immunohistochemistry (IHC); and activation of NF-κB using ELISA and Western blot assays. The most severe histopathological characteristic based on the modified Ashcroft score was in the bleomycin group (BG), whereas the mildest was in the 262 mg/kg of the bodyweight antifibrotic gambir-dosed group (AF G262). The results showed a significant difference in the BAL inflammatory cell count (p=0.017; p < 0.05). AF G262 differed most from the other antifibrotic groups in terms of the number of inflammatory cells (0.63), TGF-ß1 levels (3.80), and NF-κB levels (0.48), followed by the 131 mg/kg of the bodyweight antifibrotic gambir-dosed group (AF G131), which also differed most from other antifibrotic groups in terms of NF-κB (0.48), TIMP-1 (11.74), and collagen I (14.50) levels. Western blot analysis showed that the fibropreventive and antifibrotic groups had a specific band size of p65, whereas no specific band binding existed in the control group. This study concluded that the administration of AF G262 could improve fibrosis by lysing the extracellular matrix (ECM) in rat lungs.

The Effect of Avidin on Viability and Proliferation of Colorectal Cancer Cells HT-29.

OBJECTIVE: The aim of this study was to analyze the effect of avidin treatment on cell viability, proliferation and cyclin D1 expression in colorectal cancer cells HT-29. METHODS: Colorectal cancer cell line HT-29 incubated with 50, 100, 150, and 200 µg/mL of avidin concentration during 24, 48, and 72 hours, then the cell viability and proliferation   were analyzed. Each avidin concentration was conducted together with HT-29 cell line without avidin treatment as a control group. The cell viability was measured by MTS assay and the proliferation was measured by BrdU (5-bromo-2'-deoxyuridine) cell proliferation assay. According to cell viability and proliferation result, we determined the 100 µg/mL avidin concentration for analyzing mRNA and protein of cyclin D1. RESULTS: We demonstrated that the viability and proliferation of HT-29 cells were significantly decreased in all concentration of avidin treatment compared to control.   The cell proliferation showed larger reduction in avidin treatment rather than cell viability. This proves avidin could inhibit proliferation of colorectal cancer cell HT-29 quite well. The expression of cyclin D1, both mRNA and protein, was also significantly decreased after the avidin treatment group compared to control group, it supports the suppression of proliferation result. CONCLUSION: We concluded that avidin treatment could decrease cell viability and proliferation, accompanied by suppression of cyclin D1 expression in colorectal cells HT-29.

Conjugation of Cetuximab - Puromycin and Its Target-Specific Effect on Triple Negative Breast Cancer Cell Lines.

Cancer is life-threatening disease and being global health problems. Chemotherapy is one of the most used therapy for cancer since many years ago. Chemotherapy is also toxic for normal cell, not specific to the target cells. Consequently, chemotherapy has various side effects. Monoclonal antibody (MAb) has been developed for specific therapy which only has killing effect in cancer cells, but the survival rate of most MAbs around 20%. Therefore, in clinical practice, MAbs administration should combine with chemotherapeutic agents. For effectiveness of therapy and to minimalize adverse effects, anticancer agent with selective cytotoxic effect on target cells is needed, the immunotoxin. OBJECTIVE: This study introduces a novel approach to conjugate monoclonal antibody (Cetuximab) and toxin (Puromycin), in order to selectively inhibit proliferation of triple negative breast cancer (TNBC) and to enhance the efficacy of MAb in target cells killing. METHODS: Cetuximab was conjugated with Puromycin using a linker, i.e SATP (Succinimidyl-acetylthiopropionate) and tested on triple negative breast cancer cell lines (MDA-MB-231) which expressed EGFR (epidermal growth factor receptor). Cetuximab is MAb which targets EGFR. MCF-7 was used as control cells since it has low or no EGFR expression. Cell counting were conducted as viability assay at 24 hours, 48 hours, and 72 hours after treatment. RESULTS: The results showed significant reduction of live cells number in Conjugate 20 µg/mL cultured in MDA-MB-231 compared to MCF-7 after 24 hours, 48 hours, and 72 hours incubation. In all time period of incubation, significant reduction of MDA-MB-231 live cells number was also observed in Conjugate 20 µg/mL compared to Cetuximab 20 µg/mL. CONCLUSION: Synthesized conjugate showed its target-specific effect in TNBC and improved the efficacy of Cetuximab on TNBC. In the future, this conjugate can be a potential anticancer therapy in treating triple-negative breast cancer.

Is the Mitochondrial Function of Keloid Fibroblasts Affected by Cytoglobin?

BACKGROUND: A keloid is a benign skin tumour characterised by excessive proliferation of fibroblasts, a process that requires a sufficient amount of energy. The energy needs are associated with adequate oxygen (O2) flow and well-functioning mitochondria. It is known that cytoglobin (CYGB) has a function in O2 distribution. The aim of the present study was to explore whether the inhibition of CYGB expression caused impaired mitochondrial function of keloid fibroblasts. METHODS: An in vitro study was conducted on a keloid fibroblast derived from our previous study. The study was carried out in the laboratory of the Biochemistry & Molecular Biology Department, Faculty of Medicine, Universitas Indonesia (FMUI), from July to December 2018. CYGB expression was inhibited by small interfering ribonucleic acid (siRNA) and CYGB. Analysis of mitochondrial function was observed through peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), a mitochondrial biogenesis marker and the activity of the succinate dehydrogenase (SDH) enzyme in mitochondria. RESULTS: The CYGB gene and protein were downregulated after treatment with CYGB siRNA. Inhibition of CYGB expression with siRNA also tended to decrease the levels of PGC-1α messenger ribonucleic acid (mRNA) and protein, as well as SDH enzyme activity. CONCLUSION: Inhibition of CYGB expression with siRNA tended to decrease mitochondrial biogenesis and function. This may be useful for understanding the excessive proliferation of fibroblasts in keloids and for development of treatment for keloids.

Effects of paternal lymphocyte immunization in women with unexplained infertility.

OBJECTIVE: Infertility in women could be a result of an excessive production of antisperm antibody (ASA). Paternal lymphocyte immunization (PLI) could decrease the ASA levels, but the mechanism is still unclear. The aim of this study was to analyse the impact of a PLI-induced ASA decline on regulatory T-cell populations and serum interleukin-10 (IL-10) levels in women with unexplained infertility. METHODS: Samples were obtained from patients who came to Sayyidah Mother and Child Hospital in Jakarta from July 2018 to April 2019 with infertility problems. The inclusion criterion for this study was unexplained infertility. Each patient was examined for ASA titres using husband's sperm auto-agglutination test (HSAaT) method, and patients with ASA titres >1:128 were given PLI subcutaneously every 3 weeks. ASA titres were evaluated again 2 weeks after PLI with HSAaT. A total of 12 samples were analysed. Regulatory T-cell populations were evaluated using flow cytometry and human forkhead box P3 FoxP3 staining kit of Biotech and Device, and serum IL-10 was determined using an Abcam ELISA kit. The data were analysed using Wilcoxon and Spearman tests. RESULTS: PLI decreased serum ASA and percentage of regulatory T cells (p = 0.023). The decrease in ASA and subsequent decrease in regulatory T cell population was due to the strong negative correlation between regulatory T cells and IL-10 (r = -0.817, p = 0.004). CONCLUSIONS: The decline in ASA was associated with a decrease in regulatory T cells due to a negative correlation with IL-10levels.

Phagocytosis and the antigen-processing abilities of macrophages derived from monocytes in spinal tuberculosis patients.

This study examined the hypothesis that there is an impairment of macrophageal function in spinal TB. We examined macrophageal functions in spinal TB patients. Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of five spinal TB patients and five healthy persons as control. The isolated monocytes were cultured with stimulation of macrophage colony-stimulating factor (M-CSF) for seven days for maturation. The phagocytic ability of the macrophages derived from monocytes was measured. Also, nitric oxide (NO), myeloperoxidase (MPO), beta-glucuronide, and acid phosphatase activity was investigated. We found that the monocytes collected from patient PBMCs were significantly fewer than those of the control group (2992.103 vs. 6474.103 (cells/mL)). There were also fewer macrophages that had adhered to sheep red blood cells (SRBC) (598.103 vs. 264.103 (cells/mL)). However, NO production (2346 vs. 325.17 (µmol/gram of protein)), and the MPO (570.7 vs. 17.4 (unit/mg), beta-glucuronide (0.149 vs. 0.123 (µmol/hour/100 mg of protein)), and acid phosphatase activities (1776.9 vs. 287.9 (µmol/hour/100 mg of protein)) of the macrophages in the spinal TB group were markedly higher than in the healthy group. Despite the low adhesion to foreign bodies, the intracellular processing of TB macrophages, including oxidative activity and lysosome function, was significantly high. These results suggested the impairment of macrophageal function in spinal TB. Possibly, there is a dominance of innate non-specific immunity in spinal TB infection.

Difference of regulatory T cells, IL10, IL6, IFNγ, and IDO levels in female with high ASA and virgin: A research article.

OBJECTIVE: The maternal immune system requires tolerance for conception to occur. It is not only the balance of Th 1/Th2 that plays a role in pregnancy, but also the regulatory T cells (Tregs) that regulate the important role in pregnancy. One cause of failure in pregnancy is due to immunological factors, including antisperm antibodies (ASA). About 10-30% of infertile couples are caused by ASA. Th1 secretes Interferon γ (IFNγ). IFNγ is also an inducer indoleamin 2,3 dioksigenase (IDO). Cooperation between Tregs and IDO will induce tolerance for pregnancy.Th2 secretes most ofinterleukin (IL)10. Increased IL10 and decreased IL6 occur during pregnancy. The aim of this study was to analyse difference of Tregs, IL10, IL6, IFNγ, and IDO levels in female with high ASA and virgin. METHODS: Samples with high ASA were examined ASA titres using the husband's sperm auto-agglutination test (HSAaT) method.49 samples were analysed. Tregs were evaluated using flowcytometry with the human forkhead box P3 (FoxP3) staining kit of Biotech and Device.Level of IL10, IL6, and IFNγ was determined using an Abcam ELISA kit. Level of IDO was determined using an RnD ELISA kit. The data were analysed using the Mann-whitney tests. RESULTS: There are differences in the Tregs population (p = 0.000<0.05) but there is no difference IL10, IL6, IFNγ, and IDO levels in female with high ASA and virgin (p 0.140 > 0.05, p 0.680 > 0.05, p 0.204 > 0.05, and p 0.362 > 0.05). CONCLUSION: High ASA affects of the Tregs population but has no effect on cytokines IL10, IL6, IFNγ, and IDO.

High expressions of the cytoglobin and PGC-1α genes during the tissue regeneration of house gecko (Hemidactylus platyurus) tails.

BACKGROUND: The tissue regeneration process requires high oxygen and energy levels. Cytoglobin (Cygb) is a member of the globin family, which has the ability to bind oxygen, plays a role in dealing with oxidative stress, and carries oxygen into the mitochondria. Energy production for tissue regeneration is associated with mitochondria-especially mitochondrial biogenesis. The peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha protein helps to regulate mitochondrial biogenesis. House geckos (Hemidactylus platyurus) are reptiles that have the ability to regenerate the tissue in their tails. House geckos were selected as the animal models for this study in order to analyze the association of Cygb with oxygen supply and the association of PGC-1α with energy production for tissue regeneration. RESULTS: The growth of house gecko tails showed a slow growth at the wound healing phase, then followed by a fast growth after wound healing phase of the regeneration process. While Cygb mRNA expression reached its peak at the wound healing phase and slowly decreased until the end of the observation. PGC-1α mRNA was expressed and reached its peak earlier than Cygb. CONCLUSIONS: The expressions of both the Cygb and PGC-1α genes were relatively high compared to the control group. We therefore suggest that Cygb and PGC-1α play an important role during the tissue regeneration process.

Hypoxia and autophagic response of obese adult rat adipocytes which differ in nutritional state during childhood.

The prevalence of obesity in adults is increasing worldwide, which is problematic since obesity is associated with degenerative diseases. Nowadays Indonesia is facing an interesting phenomenon since there are adults who have been obese since childhood and others who conversely were undernourished while young. The biological differences of these two types of obesities are not well understood. This study aims to analyse the difference in the size and number of visceral adipocytes, HIF-1α, HIF-2α and MAP1LC3A/LC3 in obese adult rat groups that were undernourished at a young age compared to groups who were normal or even already fat from childhood. We analyzed Hif-1α, Hif-2α, Lc3 mRNA by RT-qPCR; HIF-1α, HIF-2α, MAP1LC3A/LC3 protein level by ELISA. The HIF-1α and HIF-2α protein level of visceral adipocytes derived from the group of rat which were undernourished while young increased significantly compared to the group which was overnourished. The visceral adipocytes of the group which was overnourished since childhood showed an increase in Hif-2α mRNA level. The Lc3 mRNA of the rat group which were undernourished since young increased significantly compared to rat group which was obese since childhood.

Expression and role of HIF-1α and HIF-2α in tissue regeneration: a study of hypoxia in house gecko tail regeneration.

The house gecko (Hemidactylus platyurus) has evolved the ability to autotomize its tail when threatened. The lost part is then regrown via epimorphic regeneration in a process that requires high energy and oxygen levels. Oxygen demand is therefore likely to outstrip supply and this can result in relative hypoxia in the tissues of the regenerating tail. The hypoxic state is stabilized by the Hypoxia Inducible Factor-1α (HIF-1α) and HIF-2α proteins. We induced tail autotomy in 30 mal H. platyurus adults using a standard procedure and then collected samples of the regenerated tail tissue on days 1, 3, 5, 8, 10, 13, 17, 21, 25, and 30 post autotomy. For each sample, mRNA expression was analyzed by qPCR, proteins were analyzed using Western Blot tests and immunohistochemistry, and the histological structure was analyzed using Hematoxylin and Eosin staining. On day 1, HIF-1α mRNA expression increased and the tissue was dominated by leucocyte and erythrocyte cells. HIF-1α mRNA expression peaked on day 3, at which time some cells were actively proliferating, migrating, and differentiating. At the same time as HIF-1α expression decreased, HIF-2α mRNA expression increased, as did overall cellular activity. HIF-2α expression increased more gradually but was present over a longer period of time than HIF-1α. We hypothesize that HIF-1α helps to initially stimulate the tissue regeneration process while HIF-2α functionally takes over the role of HIF-1α after HIF-1α succumbs to the oxygen conditions, but we suspect that both HIF-1α and HIF-2α play a role in overcoming the tissue's hypoxic state.

Role of Hypoxia Inducible Factor-1 Alpha (HIF-1α) in Cytoglobin Expression and Fibroblast Proliferation of Keloids.

BACKGROUND: Keloids are characterized by an overabundance of collagen deposition due to elevated activity and proliferation of fibroblasts, which lead to hypoxic conditions. Adaptation to these conditions is regulated by the transcription factor hypoxia inducible factor-1α (HIF-1α). Cytoglobin (Cygb), a reactive oxygen species scavenger, is a target gene of HIF-1α. In our previous study, we showed that Cygb expression in keloid tissue was correlated with HIF-1α expression. However, whether HIF-1α regulates Cygb expression and the proliferation of keloid fibroblasts remained unclear. Therefore, this study aimed to determine the role of HIF-1α in Cygb expression and fibroblast proliferation of keloids. METHODS: This was an in vitro study using a primary culture of keloid fibroblasts in which ibuprofen was used to inhibit HIF-1α expression. The expression of HIF-1α and Cygb mRNA were analyzed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) methods, and their protein levels were analyzed using an enzyme-linked immunosorbent assay (ELISA). Fibroblast proliferation was analyzed using a Trypan blue exclusion assay. RESULTS: Inhibition of HIF-1α by ibuprofen decreased Cygb mRNA expression but not in all the samples, followed by a decrease in the protein level of Cygb. There was a positive correlation between the HIF-1α protein and Cygb mRNA, probably due to the regulation of Cygb by HIF-1α at the mRNA level, but not the protein level. The proliferation of keloid fibroblasts was significantly decreased and positively correlated with the HIF-1α protein. CONCLUSION: HIF-1α regulates Cygb expression and fibroblast proliferation in keloids.

Intact Glycocalyx of Intestinal Mucosa in Intraabdominal Infection: an Investigation Using Blood Group Antigen.

BACKGROUND: intestinal glycocalyx plays a role in bacterial translocation as the pathogenesis sepsis derived from intra-abdominal infections that vulnerable in certain blood types. However, the link between intestinal glycocalyx in specific types of blood groups and abdominal infections remains unknown. This study aims to find out the condition of intestinal glycocalyx in certain blood types with intraabdominal sepsis. METHODS: descriptive study involved subjects with intraabdominal infections who underwent laparotomy. Samples are in the form of intestinal specimens. The measurement of intestinal glycocalyx proceeded by the ELISA method using blood group antigens (A and B). Expression data on the secretors were analyzed using the Kolmogorov - Smirnov test followed by parametric comparisons using ANOVA and t-tests. RESULTS: there were 32 subjects with intra-abdominal infections studied in this study. All of them are secretors and express A and B antigens strongly. We found no difference between intraabdominal infections in those with complications or without complications. Blood type O is a predominant blood type found (43.8%). Escherichia coli is the most commonly found microbe in the culture (61.3%). CONCLUSION: this study shows there is no disrupted intestinal glycocalyx of sepsis patients caused by intraabdominal infection.

Biochemical studies of membrane bound Plasmodium falciparum mitochondrial L-malate:quinone oxidoreductase, a potential drug target.

Plasmodium falciparum is an apicomplexan parasite that causes the most severe malaria in humans. Due to a lack of effective vaccines and emerging of drug resistance parasites, development of drugs with novel mechanisms of action and few side effects are imperative. To this end, ideal drug targets are those essential to parasite viability as well as absent in their mammalian hosts. The mitochondrial electron transport chain (ETC) of P. falciparum is one source of such potential targets because enzymes, such as L-malate:quinone oxidoreductase (PfMQO), in this pathway are absent humans. PfMQO catalyzes the oxidation of L-malate to oxaloacetate and the simultaneous reduction of ubiquinone to ubiquinol. It is a membrane protein, involved in three pathways (ETC, the tricarboxylic acid cycle and the fumarate cycle) and has been shown to be essential for parasite survival, at least, in the intra-erythrocytic asexual stage. These findings indicate that PfMQO would be a valuable drug target for development of antimalarial with novel mechanism of action. Up to this point in time, difficulty in producing active recombinant mitochondrial MQO has hampered biochemical characterization and targeted drug discovery with MQO. Here we report for the first time recombinant PfMQO overexpressed in bacterial membrane and the first biochemical study. Furthermore, about 113 compounds, consisting of ubiquinone binding site inhibitors and antiparasitic agents, were screened resulting in the discovery of ferulenol as a potent PfMQO inhibitor. Finally, ferulenol was shown to inhibit parasite growth and showed strong synergism in combination with atovaquone, a well-described anti-malarial and bc1 complex inhibitor.