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.

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.

Galectin-9 as a Predictive Marker for the Onset of Immune-Related Adverse Effects Associated with Anti-CCR4 MoAb Therapy in Patients with Adult T Cell Leukemia.

Adult T-cell leukemia/lymphoma (ATL/ATLL) is one of the most malignant lymphomas with poor prognosis. ATL/ATLL cells express CC chemokine receptor 4, and mogamulizumab (anti-CCR4 monoclonal antibody) exhibits strong cytotoxicity for ATL/ATLL cells. We analyzed plasma samples of 6 patients with ATL/ATLL treated with chemotherapy followed by mogamulizumab therapy (mogatherapy) for changes in the levels of biomarkers in relation to immune-related adverse effects. As treatment is often associated with skin eruptions, we investigated the profiles of inflammatory cytokines, including galectin-9 (Gal-9), which becomes increased in various infectious diseases and allergic patients. Gal-9, soluble interleukin (IL)-2 receptor, tumor necrosis factor-α, and IL-10 levels were increased before chemotherapy, and Gal-9 levels were associated with the sIL-2 receptor, which reflects tumor burden. Inflammatory levels decreased after chemotherapy. After mogatherapy, 5 of 6 patients attained complete remission (CR), whereas 1 patient showed no response (NR) and died. Among 5 patients with CR, the biomarkers remained low during mogatherapy, except for a 3-5-fold increment in Gal-9 (associated with skin eruptions). A skin biopsy showed infiltration by inflammatory cells and Gal-9 synthesis in areas with CD8 cell infiltration. In the patient with NR, increased levels of Gal-9 and the aforementioned biomarkers were noted 3 days after mogatherapy, followed by opportunistic infections resembling immune reconstitution inflammatory syndrome. Therefore, an increased Gal-9 plasma level in ATL/ATLL indicates tumor burden and reflects immune activation by mogatherapy. These findings may indicate that an increase in the Gal-9 level, a novel immune checkpoint molecule, can reflect immune-related adverse effects of various biotherapies.

Secretion of IFN-γ Associated with Galectin-9 Production by Pleural Fluid Cells from a Patient with Extrapulmonary Tuberculosis.

In this study, we investigated the role of a matricellular protein galectin-9 (Gal-9) in pleural effusion related to tuberculosis (TB). Plasma and pleural fluid of a patient with extrapulmonary TB were analyzed for cytokine content by ELISA and Luminex. Peripheral blood mononuclear cells (PBMCs) and pleural fluid cells (PFCs) were examined for interferon-γ (IFN-γ) secretion by the enzyme-linked immunospot (ELISPOT) assay or IFN-γ ELISA, for apoptosis and necrosis by Cell Death Detection ELISA, and also underwent cell sorting. The results indicate that compared to plasma, pleural fluid had increased levels of IFN-γ (1.6 vs. 55.5 pg/mL), IL-10, IL-12p40, vascular endothelial growth factor (VEGF), and Gal-9 (3.0 vs. 936.0 pg/mL), respectively. PFCs culture supernatant exhibited higher concentration of Gal-9 compared to PBMCs in culture, consistent with enriched Gal-9 staining in the granuloma that is in closer vicinity to PFCs compared to PBMCs. PFCS displayed higher IFN-γ secretion after stimulation with TB antigens ESAT-6/CFP-10. Furthermore, in PFCs, Gal-9 alone could stimulate IFN-γ synthesis in culture or ELISPOT, which was inhibited by a Gal-9 antagonist lactose, and which may promote apoptosis and necrosis. These findings suggest that Gal-9 could modulate immune responses and participate in immunopathology of pleural effusion during TB.

Plasma levels of Galectin-9 reflect disease severity in malaria infection.

BACKGROUND: Galectin-9 (Gal-9) is a ß-galactoside-binding lectin that interacts with sugar moieties on glycoproteins and glycolipids of cells and pathogens. Gal-9 is known as an immune modulator that induces cell death via interaction with T cell immunoglobulin and mucin domain-3 (Tim3), a co-inhibitory receptor, and it inhibits production of several pro-inflammatory cytokines (TNF, IL-6 and IL-1α) and enhances production of IL-10. To understand the immune pathology of malaria, the Gal-9 in plasma was measured. METHODS: Plasma samples and clinical parameters were obtained from 50 acute malaria cases (nine severe and 41 uncomplicated cases) from Thailand at three time points: day 0, day 7 and day 28. Gal-9 levels were determined by ELISA. A total of 38 species of cytokines and chemokines were measured using a BioPlex assay. RESULTS: Gal-9 levels were higher at day 0 compared to day 7 and day 28 (P < 0.0001). Gal-9 levels were also higher in severe malaria (SM) cases compared to uncomplicated (UM) cases at day 0 and day 7 (923 vs 617 pg/mL; P = 0.03, and 659 vs 348 pg/mL; P = 0.02 respectively). Median Gal-9 levels were higher in patients with blood urea nitrogen to creatinine ratio (BUN/creatinine) ≥20 (mg/dL) than in patients with BUN/creatinine <20 (mg/dL) at day 0 (817.3 vs 576.2 pg/mL, P = 0.007). Gal-9 was inversely significantly correlated with chloride levels in both SM and UM cases (r s = -0.73 and r s = -0.46, respectively). In both UM and SM cases, Gal-9 was significantly associated with pro- and anti-inflammatory cytokines and chemokines such as TNF, IL-6, IFN-α2, IFN-γ, IL-1Ra and IL-10. These correlations were observed at day 0 but disappeared at day 28. CONCLUSIONS: Gal-9 is released during acute malaria, and reflects its severity. This elevation of Gal-9 in acute malaria infection raises the possibility of its role in termination of the immune response by binding to Tim-3, a receptor of Gal-9.

Immunological Roles of Elevated Plasma Levels of Matricellular Proteins in Japanese Patients with Pulmonary Tuberculosis.

Elevated matricellular proteins (MCPs), including osteopontin (OPN) and galectin-9 (Gal-9), were observed in the plasma of patients with Manila-type tuberculosis (TB) previously. Here, we quantified plasma OPN, Gal-9, and soluble CD44 (sCD44) by enzyme-linked immunosorbent assay (ELISA), and another 29 cytokines by Luminex assay in 36 patients with pulmonary TB, six subjects with latent tuberculosis (LTBI), and 19 healthy controls (HCs) from Japan for a better understanding of the roles of MCPs in TB. All TB subjects showed positive results of enzyme-linked immunospot assays (ELISPOTs). Spoligotyping showed that 20 out of 36 Mycobacterium tuberculosis (MTB) strains belong to the Beijing type. The levels of OPN, Gal-9, and sCD44 were higher in TB (positivity of 61.1%, 66.7%, and 63.9%, respectively) than in the HCs. Positive correlations between OPN and Gal-9, between OPN and sCD44, and negative correlation between OPN and ESAT-6-ELISPOT response, between chest X-ray severity score of cavitary TB and ESAT-6-ELISPOT response were observed. Instead of OPN, Gal-9, and sCD44, cytokines G-CSF, GM-CSF, IFN-α, IFN-γ, IL-12p70, and IL-1RA levels were higher in Beijing MTB-infected patients. These findings suggest immunoregulatory, rather than inflammatory, effect of MCPs and can advance the understanding of the roles of MCPs in the context of TB pathology.

Urine Levels of Defensin α1 Reflect Kidney Injury in Leptospirosis Patients.

Leptospirosis is a zoonotic disease whose severe forms are often accompanied by kidney dysfunction. In the present study, urinary markers were studied for potential prediction of disease severity. Urine samples from 135 patients with or without leptospirosis at San Lazaro Hospital, the Philippines, were analyzed. Urine levels of defensin α1 (uDA1) were compared with those of neutrophil gelatinase-associated lipocalin (uNGAL) and N-acetyl-ß-d-glucosidase (uNAG). Serum creatinine (Cr) was used as a marker of kidney injury. The levels of uDA1/Cr, uNGAL/Cr, and uNAG/Cr were positive in 46%, 90%, and 80% of leptospirosis patients, and 69%, 70%, and 70% of non-leptospirosis patients, respectively. In leptospirosis patients, the correlation of uDA1/Cr, uNGAL/Cr and uNAG/Cr levels with serum Cr were r = 0.3 (p < 0.01), r = 0.29 (p < 0.01), and r = 0.02 (p = 0.81), respectively. uDA1/Cr levels were correlated with uNGAL/Cr levels (r = 0.49, p < 0.01) and uNAG/Cr levels (r = 0.47, p < 0.0001) in leptospirosis patients. These findings suggest that uDA1, uNGAL, and uNAG were elevated in leptospirosis patients and reflected various types of kidney damage. uDA1 and uNGAL can be used to track kidney injury in leptospirosis patients because of their correlation with the serum Cr level.

Leptospirosis in the Tohoku region: re-emerging infectious disease.

Leptospirosis is a zoonotic and disaster-related infectious disease. It is mainly endemic in subtropical or tropical countries and has not been reported since 2009 in the Tohoku region (northern Japan), including the Yamagata and Miyagi Prefectures. However, we experienced four patients with leptospirosis in the Tohoku region from 2012 to 2014; three patients (#1-3) live in the agricultural areas of the Yamagata Prefecture and one patient (#4) was a visitor to the Miyagi Prefecture. Patient 1 (81-year-old female) is a villager, with a rat bite, while Patient 2 (77-year-old male) and Patient 3 (84-year-old female) are farmers and were infected probably during agriculture work. Patient 4 (40-year-old male US citizen) was infected while traveling in Thailand. They had chief complaint of fever, headache, and myalgia and showed manifestations of hyperbilirubinemia (mean, 4.35 mg/dL), thrombocytopenia and acute kidney injury (AKI). All patients were diagnosed by polymerase chain reaction using blood and/or urine samples and a microscopic agglutination test for the anti-Leptospira antibody. All the patients were treated with infused antibiotics, including minocycline. The patients underwent hemodialysis due to severe AKI (mean serum creatinine, 4.44 mg/dL), except for Patient 2 with the normal serum creatinine level (1.12 mg/dL). All the patients recovered and were discharged. The presence of the three patients in the Yamagata Prefecture implies that leptospirosis does re-emerge in the Tohoku region. Therefore, careful survey of the pathogen is necessary for febrile patients with AKI who engage in agriculture or have a recent history of travelling in subtropical or tropical countries.

Elevated OPN, IP-10, and neutrophilia in loop-mediated isothermal amplification confirmed tuberculosis patients.

Tuberculosis (TB) is the second most common cause of death from infectious diseases and results in high socioeconomic losses to many countries. Proper diagnosis is the first step in TB eradication. To develop a rapid, simple, and accurate diagnostic TB test and to characterize the prevalence of Mycobacterium tuberculosis (MTB) genotypes and immune profiles of TB patients, a total of 37 TB patients and 30 healthy control (HC) from Metro Manila were enrolled. Loop-mediated isothermal amplification (LAMP) reliably detected MTB infection. Manila genotype was identified by spoligotyping method in all TB patients. Osteopontin (OPN), interferon-γ-induced protein 10 kDa (IP-10), and neutrophil counts were found to reflect the acute stage of MTB infection. The sensitivity and specificity were 94.6% and 93.3%, respectively, for both OPN and IP-10, and they were 83.8% and 78.6%, respectively, for neutrophils. The combination of OPN, IP-10, neutrophil count, IL-6, IL-8, TNF-α, MCP-1, platelets, galectin-9, and leukocyte count correctly identifies all the HC and 96.3% of TB patients. LAMP method may serve as a rapid, supportive method in addition to time-consuming culture methods. OPN, IP-10, and neutrophil counts are useful in detecting MTB infection and may have utility in monitoring the course of the disease.

Awareness of disaster reduction frameworks and risk perception of natural disaster: a questionnaire survey among Philippine and Indonesian health care personnel and public health students.

As the impacts of natural disasters have grown more severe, the importance of education for disaster medicine gains greater recognition. We launched a project to establish an international educational program for disaster medicine. In the present study, we surveyed medical personnel and medical/public health students in the Philippines (n = 45) and Indonesia (n = 67) for their awareness of the international frameworks related to disaster medicine: the Human Security (securing individual life and health), the Sphere Project (international humanitarian response), and the Hyogo Framework for Action 2005-2015 (international strategy for disaster reduction). In both countries, more than 50% responders were aware of human security, but only 2 to 12% were aware of the latter two. The survey also contained questions about the preferred subjects in prospective educational program, and risk perception on disaster and disaster-related infections. In the Philippines, significant disasters were geophysical (31.0%), hydrological (33.3%), or meteorological (24.8%), whereas in Indonesia, geophysical (63.0%) and hydrological (25.3%) were significant. Moreover, in the Philippines, leptospirosis (27.1%), dengue (18.6%), diarrhea (15.3%), and cholera (10.2%) were recognized common disaster-related infections. In Indonesia, diarrhea (22.0%) and respiratory infection (20.3%) are major disaster-related infections. Water-related infections were the major ones in both countries, but the profiles of risk perception were different (Pearson's chi-square test, p = 1.469e-05). The responders tended to overestimate the risk of low probability and high consequence such as geophysical disaster. These results are helpful for the development of a postgraduate course for disaster medicine in Asia Pacific countries.

Acyclovir reduces the duration of fever in patients with infectious mononucleosis-like illness.

Acyclovir is known for its antiviral activity against some pathogenic viruses such as the Epstein-Barr virus (EBV) that causes infectious mononucleosis (IM) and IM-like illness. Therefore, we empirically administered acyclovir to patients with suspected EBV-IM and IM like-illness, upon their admission to our hospital. We admitted 25 patients, who were hospitalized for fever and lymphadenopathy, to the Tohoku University Hospital Infectious Disease Ward. As part of treatment, 8 of these patients were given acyclovir (750 mg/day) with their consent and were assigned to the acyclovir group; the remaining 17 patients were assigned to the control group. The mean age of acyclovir patients (all men) was 42±5.2 years, and that of control patients (13 men and 4 women) was 31±3.0 years. The cause of illness was confirmed as EBV-IM in 6 patients (1, acyclovir; 5, control), and remained unknown for the other 19 IM-like illness patients (7, acyclovir; 12, control). A shorter duration of hospitalization and fever was observed in the acyclovir compared to that in the control patients (hospitalization duration: 16±3.7 vs. 27±7.7 days, P=0.36; fever duration: 4.5±1.8 vs. 18±6.5 days, P=0.04). Additionally, serum amyloid A (SAA) levels were lower in acyclovir than that in control patients (98±37 vs. 505±204 µg/mL, P=0.02). Therefore, we propose that acyclovir is a potential therapeutic agent for both EBV-IM and IM like-illnesses. Future studies should further examine its mechanism of action.

[Susceptibilities of bacteria isolated from patients with lower respiratory infectious diseases to antibiotics (2006)].

From October 2006 to September 2007, we collected the specimen from 356 patients with lower respiratory tract infections in 14 institutions in Japan, and investigated the susceptibilities of isolated bacteria to various antibacterial agents and patients' characteristics. Of 414 strains that were isolated from specimen (mainly from sputum) and assumed to be bacteria causing in infection, 407 strains were examined. The isolated bacteria were: Staphylococcus aureus 64, Streptococcus pneumoniae 96, Haemophilus influenzae 87, Pseudomonas aeruginosa (non-mucoid) 52, P. aeruginosa (mucoid) 11, Klebsiella pneumoniae 20, and Moraxella catarrhalis 44. Of 64 S. aureus strains, those with 2 microg/ml or less of MIC of oxacillin (methicillin-susceptible S. aureus: MSSA) and those with 4 microg/ml or more of MIC of oxacillin (methicillin-resistant S. aureus: MRSA) were 27 (42.2%) and 37 (57.8%) strains, respectively. Against MSSA, imipenem had the most potent antibacterial activity and inhibited the growth of all strains at 0.063 microg/ml or less. Against MRSA, vancomycin and linezolid showed the most potent activity and inhibited the growth of all the strains at 1 microg/ml. Carbapenems showed the most potent activities against S. pneumoniae and in particular, panipenem inhibited the growth of all the strains at 0.063 microg/ml or less. Imipenem and faropenem also had a preferable activity and inhibited the growth of all the strains at 0.125 and 0.5 microg/ml, respectively. In contrast, there were high-resistant strains (MIC: over 128 microg/ml) for erythromycin (45.8%) and clindamycin (20.8%). Against H. influenzae, levofloxacin showed the most potent activity and its MIC90 was 0.063 microg/ml or less. Meropenem showed the most potent activity against P. aeruginosa (mucoid) and its MIC90 was 0.5 microg/ml. Against P. aeruginosa (non-mucoid), tobramycin had the most potent activity and its MIC90 was 2 microg/ml. Against K. pneumoniae, cefozopran was the most potent activity and inhibited the growth of all the strains at 0.063 microg/ml or less. Also, all the antibacterial agents except ampicillin generally showed a potent activity against M. catarrhalis and the MIC90 of them were 2 microg/ml or less. The approximately half the number (50.6%) of the patients with respiratory infection were aged 70 years or older. Bacterial pneumonia and chronic bronchitis accounted for 49.2% and 28.1% of all the respiratory infections, respectively. The bacteria frequently isolated from the patients with bacterial pneumonia were S. pneumoniae (29.2%), S. aureus (20.8%), and H. influenzae (12.9%). H. influenzae (25.0%) and P. aeruginosa (21.7%) also were frequently isolated from the patients with chronic bronchitis. Before the antibacterial agent administration, the bacteria frequently isolated from the patients were S. pneumoniae (27.5%) and H. influenzae (22.5%). The bacteria frequently isolated from the patients treated with macrolides was P. aeruginosa, and its isolation frequently was 39.4%.

Frequent detection of anti-tubercular-glycolipid-IgG and -IgA antibodies in healthcare workers with latent tuberculosis infection in the Philippines.

Anti-tubercular-glycolipid-IgG (TBGL-IgG) and -IgA (TBGL-IgA) antibodies, and the QuantiFERON-TB Gold test (QFT) were compared in healthcare workers (HCWs, n = 31) and asymptomatic human immunodeficiency virus-carriers (HIV-AC, n = 56) in Manila. In HCWs, 48%, 51%, and 19% were positive in QFT, TBGL-IgG, and -IgA, respectively. The TBGL-IgG positivity was significantly higher (P = 0.02) in QFT-positive than QFT-negative HCWs. Both TBGL-IgG- and -IgA-positive cases were only found in QFT-positive HCWs (27%). The plasma IFN-γ levels positively correlated with TBGL-IgA titers (r = 0.74, P = 0.005), but not TBGL-IgG titers in this group, indicating that mucosal immunity is involved in LTBI in immunocompetent individuals. The QFT positivity in HIV-AC was 31% in those with CD4+ cell counts >350/µL and 12.5% in low CD4 group (<350/µL). 59 % and 29% were positive for TBGL-IgG and -IgA, respectively, in HIV-AC, but no association was found between QFT and TBGL assays. TBGL-IgG-positive rates in QFT-positive and QFT-negative HIV-AC were 61% and 58%, and those of TBGL-IgA were 23% and 30%, respectively. The titers of TBGL-IgA were associated with serum IgA (P = 0.02) in HIV-AC. Elevations of TBGL-IgG and -IgA were related to latent tuberculosis infection in HCWs, but careful interpretation is necessary in HIV-AC.

Rapid decrease of plasma galectin-9 levels in patients with acute HIV infection after therapy.

Acute HIV-1 infection is often diagnosed as infectious mononucleosis and the symptoms resolve spontaneously after varying periods of time. After the infection of HIV-1 through the mucosa, the characteristic clinical symptoms and laboratory markers of acute HIV-1 infection appear in each patient through a complicated virus-host interaction. To understand the host responses, we measured two unique proinflammatory cytokines, galectin-9 (Gal-9) and osteopontin (OPN). A ß-galactoside-binding mammalian lectin, Gal-9, reduces pro-inflammatory type-1 helper T (Th1) cells and Th17 cells and increases anti-inflammatory regulatory T cells. The plasma level of Gal-9 is known to be associated with HIV-1 viral load in chronic HIV-1 infection. On the contrary, osteopontin induces Th1/Th17 cells and promotes tissue inflammation. OPN is synthesized by variety of cells in the body, and dendritic cells are known to synthesize OPN in HIV-1 infected individuals. It was hypothesized that Gal-9 and/or OPN could be not only immune-modulators but also novel biomarkers of acute HIV-1 infection. We experienced 3 patients with acute HIV-1 and measured the levels of Gal-9 and OPN periodically before and after antiretroviral treatment. The results showed that the plasma levels of Gal-9 were extremely elevated [more than 2,300 pg/ml (normal range < 46 pg/ml)] in all three acute HIV-1 infected individuals and decreased rapidly after treatment. The changes in the OPN levels were less marked. In conclusion, the plasma levels of Gal-9 may be predictive of a severe inflammation status during the acute phase of HIV-1 infection and could be a potential biomarker during acute infection.

Distinct clinical features in nontuberculous mycobacterial disease with or without latent tuberculosis infection.

Nontuberculous mycobacteria (NTM) diseases are in the face of a progressive increase even in immune-competent subjects, and the clinical features of NTM diseases are heterogenous. The decision to institute treatment of the patients should be made after a period of follow up, because therapy is often prolonged, and frequently ineffective. The reasons why some patients develop severe NTM diseases are not clear. Here we observed the involvement of latent tuberculosis infection (LTBI) in clinical and laboratory features of NTM diseases. We evaluated various tuberculosis-related inflammatory markers including osteopontin (OPN), pentraxin-3 (PTX-3), and soluble IL-2 receptor (sIL-2R) in NTM infected patients with or without LTBI. Eight NTM and 5 tuberculosis (TB) patients, and 5 healthy subjects were enrolled. Polymerase Chain Reaction (PCR) analysis confirmed the absence of tuberculosis specific gene (RD1 region), among clinical isolates from NTM patients. Interferon-γ (IFN-γ) release assay (IGRA) using Early Secreted Antigenic Target-6 (ESAT-6) and CFP-10, the RD1-encoded protein, was employed for determining LTBI. IGRA was positive in 4/8 NTM (NTM with LTBI, 50%) and 5/5 TB patients. Only 2 of 4 NTM with LTBI were under chemotherapy among all NTM patients, and others were followed up. The plasma levels of OPN, PTX3 and sIL-2R were significantly higher in NTM patients with LTBI than in those without LTBI (P < 0.05). The two patients under therapy showed the highest OPN levels that persisted after treatment. The increased inflammatory levels in NTM patients with LTBI indicate enhanced inflammatory reaction. Extensive therapy may be necessary in such 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.

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.

The increase of plasma galectin-9 in a patient with insulin allergy: a case report.

Allergic reaction to insulin is known to be associated with eosinophilia and hyper IgE. Recent report showed that eosinophilia is related with the increased synthesis of galectin-9 (GAL-9) and osteopontin (OPN). Here, we examined plasma levels of GAL-9 and OPN first time in a case of 65-year old patient with insulin allergy. Insulin aspart & insulin aspart 30 mix were given to the patient and an elevation of the eosinophil count (8440/mul, 17.6 fold) and a moderate increase of IgE (501 U/ml, reference range: 10-350 U/ml), eotaxin-3 (168 pg/ml, 2 fold), histamine (0.95 ng/ml, 5.3 fold) were found 33 days later. The plasma levels of GAL-9 and OPN were 22.5 and 1.7 fold higher than the cut-off point, respectively. After one month cessation of insulin therapy, elevations of the eosinophil count (3,480/mul; 7.3 fold), and OPN (1.4 fold) still occurred but the GAL-9 levels became normal. Therefore, we noted the increases of GAL-9 and OPN in plasma for the first time in a patient with insulin allergy and propose that GAL-9 reflects the conditions of allergy more accurately.

High numbers of interferon-gamma-producing T cells and low titers of anti-tuberculous glycolipid antibody in individuals with latent tuberculosis.

Latent tuberculosis infection (LTBI) is defined as an infection with Mycobacterium tuberculosis (MTB) without clinical, bacteriological, or radiological findings, and its early diagnosis is essential for eradication of tuberculosis. To identify LTBI, we measured the numbers of interferon-gamma producing T cells, based on the ELISPOT assay, and the antibody titers in the sera to tuberculous glycolipid antigen (TBGL-Ab). Seventeen culture-confirmed TB patients, 13 controls from TB endemic areas (EC) and 13 controls from TB non-endemic areas (NEC) were enrolled. Peripheral blood mononuclear cells (2.5 x 10(5) per well) were cultured on plates precoated with antibody against interferon-gamma. ELISPOT response was defined as positive when the MTB-specific antigen-containing wells showed at least 6 spots and twice numbers of spots than negative control wells. ELISPOT responses were positive in 15 (88%), 8 (62%) and 4 (31%) subjects of TB, EC and NEC groups, respectively. The ELISPOT data differ between TB and NEC groups (p < 0.01) but not between TB and EC groups. In contrast, TBGL-Ab titers were elevated (> 2.0 U/ml) in 12 TB patients (71%), but only in one subject (8%) each from EC and NEC groups. These results indicate the high prevalence of LTBI in EC. In conclusion, LTBI is associated with positive ELISPOT assay and the low titer of TBGL-Ab, while positive results both in ELISPOT and TBGL-Ab assays indicate active TB. The low titer of TBGL-Ab is a helpful marker to identify LTBI in ELISPOT-positive individuals in TB endemic areas.

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.