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.

The elevation of plasma galectin-9 levels in patients with psoriasis and its associations with inflammatory and immune checkpoint molecules in skin tissues.

Psoriasis is a chronic, immune-mediated disorder that mainly affects the skin, with an estimated global prevalence of 2-3%. Galectin-9 (Gal-9) is a ß-galactoside-binding lectin capable of promoting or suppressing the progression of infectious and immune-mediated diseases. Here, we determined if the expression of Gal-9 is observed in psoriasis. Gal-9 levels were measured in plasma of psoriasis (n = 62) and healthy control (HC) (n = 31) using an enzyme-linked immunosorbent assay. In addition, skin samples from seven patients were screened for RNA transcriptomes and the expression of Gal-9 was compared with inflammatory, immune checkpoint molecules (ICMs) and Foxp3. The plasma Gal-9 levels in patients with psoriasis were significantly higher (841 pg/mL) than in HCs (617 pg/mL) (P < 0.0001) and were associated with white blood cell numbers, eosinophils (%) and alanine transaminase. The levels of inflammatory molecules IL-36B, IL-17RA, IL-6R, IL-10, IRF8, TGFb1, and IL-37, and those of ICMs of Tim-3, CTLA-4, CD86, CD80, PD-1LG2, CLEC4G, and Foxp3 were significantly correlated with Gal-9 (LGALS9) in skin. However, HMGB1, CD44, CEACAM1 and PDL1-known to be associated with a variety of Gal-9 biological functions were not correlated with LGALS9. Thus, it is likely that Gal-9 expression affects the disease state of PS.

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.

Galectins: Their Network and Roles in Infection/Immunity/Tumor Growth Control 2021.

Galectins constitute a protein family of soluble and non-glycosylated animal lectins that show a ß-galactoside-binding activity via a conserved sequence of approximately 130-140 amino acids located in the carbohydrate recognition domain (CRD) [...].

Development of Indole Alkaloid-Type Dual Immune Checkpoint Inhibitors Against CTLA-4 and PD-L1 Based on Diversity-Enhanced Extracts.

Cancer immunotherapy involves the use of the immune system for cancer treatment. Recently, immune checkpoint-blocking antibodies have become integral for the treatment of some cancers. However, small molecules exhibit advantages over monoclonal antibody drugs, such as cell penetration, long half-life, and low manufacturing costs, and the possibility of oral administration. Thus, it is imperative to develop small-molecule immune checkpoint inhibitors. Previously, we have screened a library of synthetic indole-alkaloid-type compounds, which are produced by diversity-enhanced extracts of Japanese cornelian cherry, and reported that an unnatural pentacyclic compound inhibits CTLA-4 gene expression. In this study, immune checkpoint inhibitors with increased potency were developed by introducing substituents and conversion of functional groups based on the unnatural pentacyclic compound. The developed compounds suppressed not only CTLA-4 and PD-L1 gene expression but also protein expression on the cell surface. Their efficacy was not as potent as that of the existing small-molecule immune checkpoint inhibitors, but, to the best of our knowledge, the developed compounds are the first reported dual small-molecule inhibitors of CTLA-4 and PD-L1.

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.

Blood Levels of Galectin-9, an Immuno-Regulating Molecule, Reflect the Severity for the Acute and Chronic Infectious Diseases.

Galectin-9 (Gal-9) is a ß-galactoside-binding lectin capable of promoting or suppressing the progression of infectious diseases. This protein is susceptible to cleavage of its linker-peptides by several proteases, and the resulting cleaved forms, N-terminal carbohydrate recognition domain (CRD) and C-terminal CRD, bind to various glycans. It has been suggested that full-length (FL)-Gal-9 and the truncated (Tr)-Gal-9s could exert different functions from one another via their different glycan-binding activities. We propose that FL-Gal-9 regulates the pathogenesis of infectious diseases, including human immunodeficiency virus (HIV) infection, HIV co-infected with opportunistic infection (HIV/OI), dengue, malaria, leptospirosis, and tuberculosis (TB). We also suggest that the blood levels of FL-Gal-9 reflect the severity of dengue, malaria, and HIV/OI, and those of Tr-Gal-9 markedly reflect the severity of HIV/OI. Recently, matrix metallopeptidase-9 (MMP-9) was suggested to be an indicator of respiratory failure from coronavirus disease 2019 (COVID-19) as well as useful for differentiating pulmonary from extrapulmonary TB. The protease cleavage of FL-Gal-9 may lead to uncontrolled hyper-immune activation, including a cytokine storm. In summary, Gal-9 has potential to reflect the disease severity for the acute and chronic infectious diseases.

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.

Possible role of plasma Galectin-9 levels as a surrogate marker of viremia in HIV infected patients on antiretroviral therapy in resource-limited settings.

BACKGROUND: Early detection of viremia in HIV infected patients on anti-retroviral therapy (ART) is important to prevent disease progression as well as accumulation of drug resistance mutations. This makes HIV viral load (VL) monitoring indispensable in HIV infected patients on ART. However VL, being an expensive test, results in heavy financial burden on health services. Hence, cheaper surrogate markers of viremia are desired to reduce overall cost of management of HIV infected patients. METHODS: We enrolled aviremic (n = 63, M:F = 31:32) and viremic (n = 43, M:F = 21:22) HIV infected patients at 1 year after ART initiation. Viremic individuals were identified as those having a plasma VL of more than 1000 copies/µl and aviremic individuals as less than 40 copies/µl. The study participants also included immuno-virologically discordant patients as they demonstrate differential degrees of immune-reconstitution and are likely to harbour concomitant infections influencing levels of immune-activation markers screened as the surrogate markers. Immune activation markers viz. plasma hs-CRP, soluble-CD14 and Galectin-9 levels were estimated by ELISA, IL-6 by luminex assay and percentages of CD38+ CD8+ cells were determined by flow cytometry. The levels were compared between viremic and aviremic patients and correlated with plasma viral load. Receiver operated curve (ROC) analysis was done for plasma Galectin-9 levels. RESULTS: Viremic patients had significantly higher levels of Galectin-9 and %CD38+ CD8+ cells (p values < 0.0001) than aviremic patients. Levels of the other activation markers did not differ between viremic and aviremic individuals. Galectin-9 levels (r = 0.76) and %CD38+ CD8+ cells (r = 0.39) correlated positively with VL. Area under curve for Galectin-9 levels for distinguishing between viremic and aviremic individuals was 0.98. Youden index, sensitivity, specificity, positive predictive value and negative predictive value for Galectin-9 levels were 0.87, 0.97, 0.90, 0.87 and 0.98, respectively, at the cut-off value of 5.79 ng/ml. CONCLUSIONS: Plasma Galectin-9 levels could identify viremic individuals with sensitivity and specificity of more than 90%. Thus, they showed a potential to serve as a surrogate marker of viremia in HIV infected patients on ART and would have cost implications on HIV management especially in resource-limited settings. However, the findings need to be confirmed in the patients on ART for different durations of time.

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.

Downregulation of PD-L1 via amide analogues of brefelamide: Alternatives to antibody-based cancer immunotherapy.

The therapeutic blockade of immune checkpoint has emerged as an effective treatment option for a broad range of tumors. However, the objective tumor response is still limited to a small number of cases and tumor types. The full utility of monoclonal antibody (mAb)-based treatment is hindered by several inherent limitations. Thus, there is an urgent requirement to explore alternative modalities targeting the same pathways. In the present study, two amide analogues of brefelamide, TPFS-201 and TPFS-202, were identified as small molecular immune checkpoint inhibitors, as they downregulated PD-L1 expression in tumor cells. PD-L1 was suppressed in cancer cells treated with TPFD compounds at both mRNA and protein levels, as detected by reverse transcription quantitative PCR and flow cytometric analysis, respectively. Reporter assays using a PD-L1 promoter luciferase construct confirmed the transcriptional inhibition of PD-L1 by TPFS compunds. TPFS compound-mediated PD-L1 downregulation in cancer cells consequently restored T cell activity, as identified by the reduction of apoptosis and an increase in interleukin-2 promoter activity in Jurkat T cells, which were co-cultured with TPFS compound-treated A549 cells. TPFS compound-mediated PD-L1 inhibition was partially abolished by the disruption of the putative transcriptional co-activator with PDZ (TAZ)/TEA domain (TEAD)-binding motif in the PD-L1 promoter. The inhibitory effect of TPFS compounds on PD-L1 was markedly inhibited in mouse cell lines, which is consistent with previous research demonstrating that PD-L1 regulation by TAZ is not conserved in mice due to distinct promoter sequences flanking the TAZ/TEAD-binding motif. Together, the data of the current study indicated the potential utility of the brefelamide amide analogues as small molecule immune checkpoint inhibitors, thereby providing therapeutic alternatives, which could be used as monotherapy or in combination with mAbs-based treatment.

Mongolian Mind-Body Interactive Psychotherapy enhances the quality of life of patients with esophageal cancer: A pilot study.

Esophageal cancer is a major public health issue in China. Mongolian Mind-Body Interactive Psychotherapy (MMIP) is a new psychotherapy that combines modern psychology with traditional Mongolian medicine. Previous cases have shown better quality of life (QoL) after MMIP in patients with cancer and other diseases. This study aimed to shed light on the effect of MMIP on the quality of life of patients in Inner Mongolia. A total of 21 patients diagnosed with esophageal cancer were studied. QoL assessment was performed using the two questionnaires of EORTC QLQ-OES 18 and QLICP-OES. The results showed that MMIP had statistical significant improvement on body function, psychological function, common symptoms, and side effects, such as reflux. As alternative and complementary medicine, MMIP could help esophageal cancer patients experience better QoL. Further large-scale studies are required to determine the impact of MMIP for QoL in patients undergoing surgery or chemotherapy for esophageal cancer.

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.

Plasma Galectin-9 Concentrations in Normal and Diseased Condition.

BACKGROUND/AIMS: Galectin-9 is a soluble immune modulator with versatile functions, including a role as an immune checkpoint molecule. Therefore, the amount of galectin-9 in the blood may reflect an individual's immunological balance. Many studies have conducted galectin-9 measurements; however, the reported galectin-9 concentration in the blood varies greatly, even within healthy controls. This study investigates the variation between the reported and actual concentrations of galectin-9. METHODS: A GalPharma ELISA and an R&D Systems ELISA kit were directly compared using the same set of plasma and a series of recombinant galectins, including degraded galectin-9. Furthermore, galectin-9 in plasma was concentrated using anti-galectin-9 antibody-conjugated beads, and subjected to western blotting to estimate the quantity and integrity of galectin-9 and assess the consistency of ELISA measurements. RESULTS: The R&D Systems' ELISA indicated a 50-fold higher median concentration of plasma galectin-9 than that indicated by the GalPharma ELISA. This variation is due to aberrantly enhanced reactivity of the R&D Systems' ELISA to degraded galectin-9 present in small quantities in the plasma. The GalPharma ELISA could detect only intact galectin-9 and its results correlated well with the plasma galectin-9 level obtained by western blotting. CONCLUSION: ELISA kits from R&D Systems reacts aberrantly higher against degraded galectin-9 than the intact galectin-9. Therefore, the existence of a small amount of degraded galectin-9 in a test sample hinders the quantification. As galectin-9 is a fragile protein, this is a serious concern when using this kit. Based on quantifications from the GalPharma ELISA, the median (25th-75th percentiles) galectin-9 concentration in healthy subjects in the current study cohort was calculated as 110 pg/mL (67 -154 pg/mL).

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.

Secretion of Galectin-9 as a DAMP during Dengue Virus Infection in THP-1 Cells.

Damage-associated molecular patterns (DAMPs) are endogenous cellular molecules released to the extracellular environment in response to stress conditions such as virus infection. Galectins are ß-galactoside-binding proteins that are widely expressed in cells and tissues of the immune system, are localized in the cell cytoplasm, and have roles in inflammatory responses and immune responses against infection. Elevated levels of galectin-9 (Gal-9) in natural human infections have been documented in numerous reports. To investigate the effect of dengue virus (DENV) infection on expression of endogenous Gal-9, monocytic THP-1 cells were infected with varying doses of DENV-3 (multiplicity of infection (MOI) 0.01, 0.03 and 0.1) and incubated at varying time points (Day 1, Day 2, Day 3). Results showed augmentation of Gal-9 levels in the supernatant, reduction of Gal-9 levels in the cells and decreased expression of LGALS9 mRNA, while DENV-3 mRNA copies for all three doses remained stable through time. Dengue virus induced the secretion of Gal-9 as a danger response; in turn, Gal-9 and other inflammatory factors, and stimulated effector responses may have limited further viral replication. The results in this pilot experiment add to the evidence of Gal-9 as a potential DAMP.