Dynamics of Matricellular Protein Levels in Blood Predict Recovery in Patients with Human Immunodeficiency Virus-Tuberculosis Coinfection.

Chronic immune activation in tuberculosis (TB) associated with human immunodeficiency virus (HIV) infection (HIV/TB) modifies their clinical course. We prospectively measured osteopontin (OPN), full-length galectin-9 (FL-Gal9), and total-Gal9 (T-Gal9) levels in 32 patients with HIV/TB coinfection treated with anti-tuberculosis and antiretroviral therapies over 6-18 months to determine the amelioration of inflammatory conditions in response to the therapies. We observed a significant time-dependent decrease in FL-Gal9 in both pulmonary TB (PTB, n = 20) and extrapulmonary TB (EPTB, n = 12) patients. The levels of T-Gal9, OPN, and CRP decreased significantly after treatment in only PTB patients. We calculated the inflammatory score (INS) indicating immunologic recovery based on the decline in OPN, FL-Gal9, T-Gal9, and CRP levels. Baseline levels of T-Gal9 and OPN positively correlated with INS in all TB and only PTB patients, respectively, indicating that their levels predict better recovery. In contrast, FL-Gal9 levels at the second visit negatively correlated with INS in EPTB patients. The decrease rate in OPN levels at the second visit also correlated positively with INS in PTB patients. Women showed a higher INS and lower levels of FL-Gal9 than men. The patients with moderate grade severity on chest X-ray had higher CD4 cell numbers than those with limited grade severity. Monitoring these markers will help to predict and assess the response to therapy as well as to devise strategies to reduce the complications caused by chronic immune activation in patients with HIV/TB coinfection.

Transcriptome-Based Identification of the SaR2R3-MYB Gene Family in Sophora alopecuroides and Function Analysis of SaR2R3-MYB15 in Salt Stress Tolerance.

As one of the most prominent gene families, R2R3-MYB transcription factors significantly regulate biochemical and physiological processes under salt stress. However, in Sophora alopecuroides, a perennial herb known for its exceptional saline alkali resistance, the comprehensive identification and characterization of SaR2R3-MYB genes and their potential functions in response to salt stress have yet to be determined. We investigated the expression profiles and biological functions of SaR2R3-MYB transcription factors in response to salt stress, utilizing a transcriptome-wide mining method. Our analysis identified 28 SaR2R3-MYB transcription factors, all sharing a highly conserved R2R3 domain, which were further divided into 28 subgroups through phylogenetic analysis. Some SaR2R3-MYB transcription factors showed induction under salt stress, with SaR2R3-MYB15 emerging as a potential regulator based on analysis of the protein-protein interaction network. Validation revealed the transcriptional activity and nuclear localization of SaR2R3-MYB15. Remarkably, overexpression of SaR2R3-MYB15 in transgenic plants could increase the activity of antioxidant enzymes and the accumulation of proline but decrease the content of malondialdehyde (MDA), compared with wild-type plants. Moreover, several salt stress-related genes showed higher expression levels in transgenic plants, implying their potential to enhance salt tolerance. Our findings shed light on the role of SaR2R3-MYB genes in salt tolerance in S. alopecuroides.

Plasma N-Cleaved Galectin-9 Is a Surrogate Marker for Determining the Severity of COVID-19 and Monitoring the Therapeutic Effects of Tocilizumab.

Galectin-9 (Gal-9) is known to contribute to antiviral responses in coronavirus disease 2019 (COVID-19). Increased circulating Gal-9 in COVID-19 is associated with COVID-19 severity. In a while, the linker-peptide of Gal-9 is susceptible to proteolysis that can cause the change or loss of Gal-9 activity. Here, we measured plasma levels of N-cleaved-Gal9, which is Gal9 carbohydrate-recognition domain at the N-terminus (NCRD) with attached truncated linker peptide that differs in length depending on the type of proteases, in COVID-19. We also investigated the time course of plasma N-cleaved-Gal9 levels in severe COVID-19 treated with tocilizumab (TCZ). As a result, we observed an increase in plasma N-cleaved-Gal9 levels in COVID-19 and its higher levels in COVID-19 with pneumonia compared to the mild cases (healthy: 326.1 pg/mL, mild: 698.0 pg/mL, and with pneumonia: 1570 pg/mL). N-cleaved-Gal9 levels were associated with lymphocyte counts, C-reactive protein (CRP), soluble interleukin-2 receptor (sIL-2R), D-dimer, and ferritin levels, and ratio of percutaneous oxygen saturation to fraction of inspiratory oxygen (S/F ratio) in COVID-19 with pneumonia and discriminated different severity groups with high accuracy (area under the curve (AUC): 0.9076). Both N-cleaved-Gal9 and sIL-2R levels were associated with plasma matrix metalloprotease (MMP)-9 levels in COVID-19 with pneumonia. Furthermore, a decrease in N-cleaved-Gal9 levels was associated with a decrease of sIL-2R levels during TCZ treatment. N-cleaved-Gal9 levels showed a moderate accuracy (AUC: 0.8438) for discriminating the period before TCZ from the recovery phase. These data illustrate that plasma N-cleaved-Gal9 is a potential surrogate marker for assessing COVID-19 severity and the therapeutic effects of TCZ.

Both Full-Length and Protease-Cleaved Products of Osteopontin Are Elevated in Infectious Diseases.

Circulating full-length osteopontin (FL-OPN) is elevated in plasma from patients with various infectious diseases, such as adult T-cell leukemia, Mycobacterium tuberculosis (TB), hepatitis virus infection, leptospirosis, acquired immune deficiency syndrome (AIDS), AIDS/TB, and coronavirus disease 2019 (COVID-19). Proteolysis of OPN by thrombin, matrix metalloproteases, caspase 8/3, cathepsin D, plasmin, and enterokinase generates various cleaved OPNs with a variety of bioactivities by binding to different target cells. Moreover, OPN is susceptible to gradual proteolysis. During inflammation, one of the cleaved fragments, N-terminal thrombin-cleaved OPN (trOPN or OPN-Arg168 [OPN-R]), induces dendritic cell (DC) adhesion. Further cleavage by carboxypeptidase B2 or carboxypeptidase N removes Arg168 from OPN-R to OPN-Leu167 (OPN-L). Consequently, OPN-L decreases DC adhesion. In particular, the differences in plasma level over time are observed between FL-OPN and its cleaved OPNs during inflammation. We found that the undefined OPN levels (mixture of FL-OPN and cleaved OPN) were elevated in plasma and reflected the pathology of TB and COVID-19 rather than FL-OPN. These infections are associated with elevated levels of various proteases. Inhibition of the cleavage or the activities of cleaved products may improve the outcome of the therapy. Research on the metabolism of OPN is expected to create new therapies against infectious diseases.

High Levels of the Cleaved Form of Galectin-9 and Osteopontin in the Plasma Are Associated with Inflammatory Markers That Reflect the Severity of COVID-19 Pneumonia.

Numbers of patients with coronavirus disease 2019 (COVID-19) have increased rapidly worldwide. Plasma levels of full-length galectin-9 (FL-Gal9) and osteopontin (FL-OPN) as well as their truncated forms (Tr-Gal9, Ud-OPN, respectively), are representative inflammatory biomarkers. Here, we measured FL-Gal9, FL-OPN, Tr-Gal9, and Ud-OPN in 94 plasma samples obtained from 23 COVID-19-infected patients with mild clinical symptoms (CV), 25 COVID-19 patients associated with pneumonia (CP), and 14 patients with bacterial infection (ID). The four proteins were significantly elevated in the CP group when compared with healthy individuals. ROC analysis between the CV and CP groups showed that C-reactive protein had the highest ability to differentiate, followed by Tr-Gal9 and ferritin. Spearman's correlation analysis showed that Tr-Gal9 and Ud-OPN but not FL-Gal9 and FL-OPN, had a significant association with laboratory markers for lung function, inflammation, coagulopathy, and kidney function in CP patients. CP patients treated with tocilizumab had reduced levels of FL-Gal9, Tr-Gal9, and Ud-OPN. It was suggested that OPN is cleaved by interleukin-6-dependent proteases. These findings suggest that the cleaved forms of OPN and galectin-9 can be used to monitor the severity of pathological inflammation and the therapeutic effects of tocilizumab in CP patients.

Cecum microbiota in rats fed soy, milk, meat, fish, and egg proteins with prebiotic oligosaccharides.

Diet is considered the most influential factor in modulating the gut microbiota but how dietary protein sources differ in their modulatory effects is not well understood. In this study, soy, meat (mixture of beef and pork), and fish proteins (experiment 1) and soy, milk (casein), and egg proteins (experiment 2) were fed to rats with cellulose (CEL) and raffinose (RAF); the microbiota composition and short-chain fatty acid concentration in the cecum were determined. Egg protein feeding decreased the concentration of acetic acid and the richness and diversity of the cecum microbiota. RAF feeding increased the concentrations of acetic and propionic acids and decreased the richness and diversity of the cecum microbiota. When fed with CEL, the abundance of Ruminococcaceae and Christensenellaceae, Akkermansiaceae and Tannerellaceae, and Erysipelotrichaceae enhanced with soy protein, meat and fish proteins, and egg protein, respectively. The effects of dietary proteins diminished with RAF feeding and the abundance of Bifidobacteriaceae, Erysipelotrichaceae, and Lachnospiraceae increased and that of Ruminococcaceae and Christensenellaceae decreased regardless of the protein source. These results indicate that, although the effect of prebiotics is more robust and distinctive, dietary protein sources may influence the composition and metabolic activities of the gut microbiota. The stimulatory effects of soy, meat, and egg proteins on Christensenellaceae, Akkermansiaceae, and Erysipelotrichaceae deserve further examination to better elucidate the dietary manipulation of the gut microbiota.

Stimulation of THP-1 Macrophages with LPS Increased the Production of Osteopontin-Encapsulating Exosome.

Osteopontin (OPN) mediates bone remodeling and tissue debridement. The OPN protein is cleaved, but it is unclear how full-length (FL)-OPN or its cleaved form perform their biological activities in target cells. We, therefore, performed the molecular characterization of OPN in exosomes (Exo). The Exo were isolated from lipopolysaccharide (LPS)-stimulated phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages. The Exo were also isolated from PMA-differentiated THP-1 macrophages. The Exo were identified using the qNano multiple analyzer (diameter 59-315 nm) and western blotting with a CD9 antibody. LPS-stimulated cells produced more particles than non-stimulated cells. The presence of the FL or the cleaved form of OPN was confirmed using western blot analysis. A mixture of FL and cleaved OPN was also measured using an ELISA system (Ud-OPN) and their presence in the Exo was confirmed. Ud/FL ratios became low after LPS stimulation, indicating the enhanced encapsulation of FL-OPN in the Exo by LPS. These findings suggest that LPS stimulation of human macrophages facilitates the synthesis of FL-OPN, which is cleaved in cells or the Exo after release. These findings indicate that Exo is a suitable vehicle to transfer OPN to the target cells.

Plasma Levels of a Cleaved Form of Galectin-9 Are the Most Sensitive Biomarkers of Acquired Immune Deficiency Syndrome and Tuberculosis Coinfection.

Acquired immunodeficiency syndrome (AIDS) complicated with tuberculosis (TB) is a global public issue. Due to the paucity of bacteria in AIDS/TB, blood-based biomarkers that reflect disease severity are desired. Plasma levels of matricellular proteins, such as osteopontin (OPN) and galectin-9 (Gal-9), are known to be elevated in AIDS and TB. Therefore, full-length (FL)-Gal9 and FL-OPN, and their truncated forms (Tr-Gal9, Ud-OPN), and 38 cytokines/chemokines were measured in the plasma of 24 AIDS (other than TB), 49 TB, and 33 AIDS/TB patients. Receiver-operating characteristic analysis was used to screen molecules that could distinguish either between disease and normal group, among each disease group, or between deceased patients and survivors. Selected molecules were further analyzed for significant differences. Tr-Gal9 had the highest ability to differentiate TB from AIDS or AIDS/TB, while Ud-OPN distinguished multidrug resistance (MDR)-TB from non-MDR TB, and extra-pulmonary TB from pulmonary TB. Molecules significantly elevated in deceased patients included; FL-Gal9, Tr-Gal9, interleukin (IL)-1 receptor antagonist, IL-17A and transforming growth factor-α in AIDS; IL-6, granulocyte colony-stimulating factor and monocyte chemotactic protein-1 in TB; and macrophage inflammatory protein-1ß in AIDS/TB. From the sensitivity, specificity, and significant elevation, Tr-Gal9 is the best biomarker of inflammation and severity in AIDS and AIDS/TB.

Elevated Levels of Galectin-9 but Not Osteopontin in HIV and Tuberculosis Infections Indicate Their Roles in Detecting MTB Infection in HIV Infected Individuals.

Galectin-9 (Gal-9) and osteopontin (OPN) play immunomodulatory roles in tuberculosis and HIV infections. Evaluation of their levels as well as their interplay with different pro-inflammatory cytokines is critical to understand their role in immunopathogenesis of HIV/tuberculosis co-infection considering the complexity of the disease. Plasma levels of these proteins were measured by ELISAs in HIV-negative individuals with pulmonary (n = 21), extrapulmonary (n = 33), and latent tuberculosis (n = 22) and in HIV infected patients with pulmonary (n = 14), latent tuberculosis (n = 17), and without tuberculosis (n = 41). Levels of pro-inflammatory cytokines were estimated by Luminex assay. Receiver operated characteristic curve analysis was performed to evaluate discriminatory roles of these proteins. Spearman's correlation analysis was performed with the markers of HIV and tuberculosis disease progression to evaluate their immunopathogenic roles. Gal-9 and OPN levels were higher in HIV uninfected patients with active tuberculosis than with latent tuberculosis. Gal-9 but not OPN levels were higher in HIV infected patients with active tuberculosis than with latent tuberculosis. Area under curve for Galectin-9 was >0.9 in HIV/tuberculosis co-infection and extrapulmonary tuberculosis. OPN and IL-6 levels were higher in patients with severe chest X-ray grade indicating its association with severity of the disease and positively correlated with each other. Stronger positive and negative correlations of Gal-9 levels, respectively, with viral loads and CD4 cell counts in HIV infected patients were observed than OPN levels indicating their association with HIV disease progression. Thus, significantly elevated Gal-9 levels were reported for the first time in HIV/tuberculosis co-infection and extrapulmonary tuberculosis in our study than single infections with HIV and tuberculosis. The study indicated a need for further evaluation of monitoring role of Gal-9 for detection of developing tuberculosis in HIV infected individuals. The findings also indicated differential roles of Gal-9 and OPN in the pathogenesis of tuberculosis and HIV infections.

Plasma Osteopontin Levels is Associated with Biochemical Markers of Kidney Injury in Patients with Leptospirosis.

: Leptospirosis becomes severe, with a fatality rate of >10%, and manifests as severe lung injury accompanied by acute kidney injury. Using urine and blood samples of 112 patients with leptospirosis, osteopontin (OPN), galectin-9 (Gal-9) and other kidney-related biomarkers were measured to understand the pathological and diagnostic roles of OPN and Gal-9 in leptospirosis. Plasma levels of full-length (FL)-OPN (pFL-OPN) (p < 0.0001), pFL-Gal-9(p < 0.0001) and thrombin-cleaved OPN (p < 0.01) were significantly higher in patients with leptospirosis than in healthy controls (n = 30), as were levels of several indicators of renal toxicity: serum cystatin C (p < 0.0001), urine N-acetyl-ß-glucosaminidase (NAG)/creatinine (p < 0.05), and urine clusterin/creatinine (p < 0.05). pFL-Gal-9 levels were negatively correlated with pFL-OPN levels (r = -0.24, p < 0.05). pFL-OPN levels were positively correlated with serum cystatin C (r = 0.41, p < 0.0001), urine NAG/creatinine (r = 0.35, p < 0.001), urine clusterin/creatinine (r = 0.33, p < 0.01), and urine cystatin C/creatinine (r = 0.33, p < 0.05) levels. In a group of patients with abnormally high creatinine levels, significantly higher levels of serum cystatin C (p < 0.0001) and pFL-OPN (p < 0.001) were observed. Our results demonstrate that pFL-OPN reflect kidney injury among patients with leptospirosis.

Inhibition of inflammatory-molecule synthesis in THP-1 cells stimulated with phorbol 12-myristate 13-acetate by brefelamide derivatives.

Plasma osteopontin (OPN) levels are elevated in tuberculosis patients and may involve granuloma formation. New inhibitors using brefelamide, an aromatic amide isolated from Dictyostelium cellular slime molds that may inhibit OPN transcription in A549 cells at 1 µM concentration, were synthesized as compounds C, D, and E. Their inhibitory activity against OPN synthesis in phorbol 12-myristate 13-acetate (PMA)-stimulated THP-1 cells was confirmed using enzyme-linked immunosorbent assay (ELISA), a multicolor immune-fluorescent microscope, and western blot. In the ELISA performed using full-length OPN, each compound showed significant inhibition in culture supernatants with half maximal inhibitory concentration (IC50) values of 1.6, 1.8, and 2.2 µM for C, D, and E, respectively. In another ELISA to detect the immune-related form of OPN, IC50 values were 0.6, 1.2, and 2.5 µM for compounds C, D, and E, respectively. The decreases in OPN expression and synthesis were confirmed using immunofluorescence and western blot studies using compound-treated cells or cell lysates. Luminex assay of the supernatants of PMA-treated THP-1 cells showed significant reduction in the synthesis of interleukin (IL)-1ß, galectin-9, and tumor necrosis factor (TNF)-α. Elucidation of the detailed mechanisms of the biological activities of these compounds would be necessary; however, they may be used in clinical trials for infectious diseases, inflammatory disorders, and cancer.

Evaluation of a rapid diagnostic test for detection of dengue infection using a single-tag hybridization chromatographic-printed array strip format.

A dipstick DNA chromatography assay, a single-tag hybridization-printed array strip (STH-PAS), was evaluated for its efficacy to detect dengue virus (DENV). Reverse-transcribed DNA was amplified by PCR, and the amplified DNA was detected using the STH-PAS system. The method was evaluated using stored RNA samples previously identified to carry all 4 serotypes of dengue, chikungunya, and influenza viruses. Clinical performance was also assessed in a prospective study using plasma from 269 febrile cases from the Emergency Department of St. Luke's Medical Center, Quezon City, Philippines, and 30 afebrile normal healthy volunteers. A Taqman real-time PCR (RT-PCR) assay and a rapid Dengue NS1 test, SD Bioline, were used for comparison. The STH-PAS system was more sensitive in detecting dengue infection compared to Taqman RT-PCR. For DENV serotypes 1, 2, and 3, the detection was 1 to 2 dilutions (10-fold) higher, and for DENV serotype 4, the detection was 2-4 dilutions higher. In clinical studies, the STH-PAS system showed 100% sensitivity with 88.9% and 86.6% specificities compared to Taqman RT-PCR and SD Dengue Duo NS1 test, respectively. The STH-PAS system was found to have a superior sensitivity than the Taqman system. Further evaluation of its performance in the field may provide important data to extend its usefulness for surveillance and epidemiological research in outbreak situations.

Synthesis of a Cleaved Form of Osteopontin by THP-1 Cells and Its Alteration by Phorbol 12-Myristate 13-Acetate and BCG Infection.

The protease-cleaved osteopontin (OPN) was proposed to enhance the migration of memory T cells to granulomas in tuberculosis. Various forms of OPN were identified in human monocytic THP-1 cells stimulated by phorbol 12-myristate 13-acetate (PMA). Antibodies O-17, 10A16 and 34E3, which recognize N-terminus, the C-half, and thrombin-cleaved site of OPN, respectively, all detected distinct bands on Western blots following PMA stimulation. Bands corresponding to 18 and 30 kD were detected by antibodies 34E3 and 10A16, indicating that OPN cleavage occurred by endogenous proteases in the PMA-stimulated THP-1 cells. In immune-fluorescence (IF) assay, 34E3 positive signals were detected in intracellular space of non-infected and bacillus Calmette-Guérin (BCG)-infected cells; however, 10A16 positive signals were confirmed in extracellular area in PMA-stimulated cells followed by BCG infection. Small amounts of full-length (FL) and thrombin-cleaved (Tr) OPN were detected by ELISA in the supernatants of non-PMA-stimulated cells, and increased levels of all forms, including undefined (Ud) OPN, in PMA-stimulated cells. ELISA showed a decrease in OPN synthesis during BCG infection. To our knowledge, this is the first report of OPN cleavage in THP-1 macrophages after PMA stimulation, and of enhanced cleavage induced by BCG infection.

Dietary soy, meat, and fish proteins modulate the effects of prebiotic raffinose on composition and fermentation of gut microbiota in rats.

Soy, meat (mixture of pork and beef), and fish proteins were fed to rats with and without prebiotic raffinose (RAF), and the composition and fermentation of gut microbiota were examined. Bifidobacterium spp. populations were higher, and propionic acid concentration was lower in soy protein-fed than meat protein-fed rats. Likewise, Enterobacteriaceae populations were higher in fish protein-fed rats than other rats. RAF feeding increased Bifidobacterium spp. and decreased Faecalibacterium prausnitzii populations regardless of the dietary protein source. Interactions between dietary proteins and RAF were shown for Lactobacillus spp. and Clostridium perfringens group; the increase of Lactobacillus spp. populations by RAF was seen only for soy protein-fed rats, whereas the reduction of C. perfringens group by RAF was evident in fish and meat protein-fed rats. It is concluded that dietary proteins may differentially modulate the effects of prebiotic oligosaccharides on gut fermentation and microbiota, with differences observed between plant and animal proteins.

Dietary Casein and Soy Protein Isolate Modulate the Effects of Raffinose and Fructooligosaccharides on the Composition and Fermentation of Gut Microbiota in Rats.

Although diet has an important influence on the composition of gut microbiota, the impact of dietary protein sources has only been studied to a minor extent. In this study, we examined the influence of different dietary protein sources regarding the effects of prebiotic oligosaccharides on the composition and metabolic activity of gut microbiota. Thirty female rats were fed casein and soy protein isolate with cellulose, raffinose (RAF), and fructooligosaccharides (FOS). Microbiota composition was examined by real-time qPCR and denaturing gradient gel electrophoresis. Dietary protein source affected cecum microbiota; acetic acid concentration and Lactobacillus spp. populations were greater with soy protein than with casein. Prebiotic oligosaccharides had distinctive effects on gut microbiota; RAF increased the acetic acid concentration and Bifidobacterium spp. populations, and FOS increased the butyric acid concentration regardless of the dietary protein. Likewise, Bifidobacterium sp., Collinsella sp., and Lactobacillus sp. were detected in microbiota of the rats fed RAF, and Bacteroides sp., Roseburia sp., and Blautia sp. were seen in microbiota of the rats fed FOS. Interactions between dietary proteins and prebiotic oligosaccharides were observed with Clostridium perfringens group populations and cecum IgA concentration. RAF and FOS decreased C. perfringens group populations in casein-fed rats, and the combination of soy protein and RAF substantially increased cecum IgA concentration. These results indicate that dietary proteins can differentially modulate the effects of prebiotic oligosaccharides on gut fermentation and microbiota, depending on the type of carbohydrate polymers involved.