Evaluation of a lateral flow assay-based IFN-γ release assay as a point-of-care test for the diagnosis of latent tuberculosis infection.

INTRODUCTION: We compared the performance of a fluorescence lateral flow assay (ichroma™ IGRA-TB) with the QuantiFERON-TB Gold PLUS (QFT-PLUS) for the diagnosis of latent tuberculosis infection (LTBI) in patients with immune-mediated inflammatory diseases (IMID) prior to receiving biologics therapy. METHOD: The comparability of the ichroma™ IGRA-TB assay with the QFT-PLUS assay for the diagnosis of LTBI was determined in prospectively enrolled patients with IMID prior to receiving biologics between August 2018 and October 2019. To determine the best cut-off value of the ichroma™ IGRA-TB, an ROC curve analysis was performed. RESULTS: Patients with IMID (n = 145) had inflammatory bowel disease (n = 83; 57.2%), rheumatoid arthritis (n = 44; 30.3%), or spondyloarthropathy (n = 18; 12.4%). The median age was 40.5 (interquartile range: 27.0-56.0), 72 (49.7%) were men, and 140 (96.6%) received BCG vaccination. With the manufacturer-recommended cut-off values, 11 (7.6%) and 20 (13.8%) patients showed positive results with the ichroma™ IGRA-TB and QFT-PLUS tests, respectively. The overall agreement between the two tests was 91.0% with a Cohen's kappa value of 0.535 (95% confidence interval: 0.317-0.754). ROC curve analysis of the QFT-PLUS results showed that a cut-off value of > 0.21 IU/mL would improve the performance of the ichroma™ IGRA-TB. Using the new cut-off value, the concordance rate was improved to 93.1% with a Cohen's kappa value of 0.668 (95% confidence interval: 0.478-0.858). CONCLUSIONS: The ichroma™ IGRA-TB could be used as a point-of-care test for LTBI screening in IMID patients before starting biologics, especially in resource-limited settings. Key Points • The ichroma™ IGRA-TB is an automated fluorescence lateral flow assay-based IGRA. • The test has advantages like short turn-around time, low-cost, and ease of use. • The ichroma™ IGRA-TB showed high agreement with the QuantiFERON-TB Gold In-Tube in patients with chronic immune-mediated inflammatory diseases before starting biologics.

Comparison of Shear Stress-Induced Thrombotic and Thrombolytic Effects Among 3 Different Antithrombotic Regimens in Patients With Acute Coronary Syndrome.

Shear stress (SS)-induced platelet activation is suggested as an essential mechanism of the acute coronary syndrome (ACS). We aimed to compare SS-induced thrombotic and thrombolytic activities among 3 treatment regimens in patients with ACS who underwent percutaneous coronary intervention (PCI). Patients were nonrandomly enrolled and treated with one of 3 regimens (TICA: ticagrelor 180 mg/d; RIVA: clopidogrel 75 mg/d and rivaroxaban 5 mg/d; CLP: clopidogrel 75 mg/d), administered in addition to aspirin (100 mg/d) for 30 days. The global thrombosis test was applied to measure SS-induced thrombotic (occlusion time [OT]) and thrombolytic activity (lysis time [LT]) at day 2 and 30. Aspirin reaction unit (ARU) and P2Y12 reaction unit (PRU) were simultaneously measured using VerifyNow. Group differences in the OT, LT, ARU, and PRU were evaluated. Seventy-five patients (25 patients in each group) finished 30 days of follow-up. Clinical and angiographic characteristics did not differ among the 3 groups, except ACS subtype and pre-PCI coronary flow. No major adverse cardiovascular events occurred in any group during follow-up. The OT and LT did not differ among the 3 groups at day 30 (OT: TICA, 447.2 ± 87.1 vs RIVA, 458.5 ± 70.3, vs CLP, 471.9 ± 90.7, LT: 1522.3 ± 426.5 vs 1734.6 ± 454.3 vs 1510.2 ± 593.9) despite significant differences in the PRU among the 3 groups. Shear stress-induced thrombotic and thrombolytic activities did not differ among the 3 investigated antithrombotic treatments.

Mycobacterium tuberculosis-Secreted Protein, ESAT-6, Inhibits Lipopolysaccharide-Induced MMP-9 Expression and Inflammation Through NF-κB and MAPK Signaling in RAW 264.7 Macrophage Cells.

-20pt?>Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium that causes contagious tuberculosis (TB). Recently, Mtb-secreted proteins have been considered virulence factors and candidates for drugs and vaccines. Among these proteins, 6-kDa early secreted antigenic target (ESAT-6) is known to be able to induce component of matrix metalloproteinase-9 (MMP-9) in epithelial cells, leading to recruitment of macrophages. However, detailed function of ESAT-6 during macrophage recruitment to inflammatory sites remains unknown. Thus, the objective of the present study was to elucidate such function of EAST-6 and mechanism(s) involved. In the present study, we have found that recombinant ESAT-6 purified in the form of ESAT-6 double-connected structure (2E6D) could inhibit lipopolysaccharide (LPS)-induced potential of cell migration and inflammation in murine macrophage cells. Interestingly, 2E6D suppressed LPS-induced MMP-9 expression at both protein and mRNA levels as well as its enzyme activity. Levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzymes as known upregulators of MMP-9 were significantly decreased when 2E6D has been treated. In addition, nitric oxide (NO) as a second messenger was also significantly decreased by treatment with the purified 2E6D. Furthermore, 2E6D inhibited LPS-induced phosphorylation of IκB and translocation of NF-κB. Moreover, 2E6D suppressed phosphorylation of MAPK signaling proteins. Taken together, these results suggest that ESAT-6 can suppress LPS-induced MMP-9 and inflammation by downregulating COX-2, iNOS, and NO through NF-κB and MAPK signaling.

Enhancement of the stability of Mycobacterium tuberculosis recombinant antigen expressed in Escherichia coli using cell lysis additives.

BACKGROUND: Mycobacteria tuberculosis (Mtb), the causative agent of tuberculosis, is a slow-growing bacterium. Expression in Escherichia coli is a widely used method for large-scale production of diagnostic antigenic recombinant proteins. Expression of Mtb antigen in E. coli offers a rapid and, inexpensive alternative to conventional protein synthesis from Mtb. The addition of stabilizing additives during cell lysis or storage of Mtb antigenic protein plays a vital role in enhancing antigen stability. In this study, we evaluated the effects of additives on the stability of Mtb antigens expressed in E. coli. METHODS: Immunodominant Mtb antigens, i.e., CFP-10, Rv3872, TB7.7, and TB9.7, were cloned, and recombinant proteins overexpressed in E. coli were gradually degraded in a time-dependent manner by incubation at 37 °C. Various stabilizing additives during storage or cell lysis before protein purification were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. RESULTS: CFP-10 and Rv3872 were mainly expressed in soluble form. The degraded form of the expressed protein after incubation at 37 °C was easily observed after 1 week. Increased stability was observed in a solution containing glycine for recombinant CFP-10 and Rv3872. TB9.7 was stable in a solution containing trehalose or mannitol. TB7.7 was stable in a solution containing sucrose, glycine, or polyethylene glycol. CONCLUSION: Recombinant Mtb antigen stabilization using chemical additives inhibited protein degradation, leading to increased antigen stability and purification efficiency.

Mitogen-Induced Interferon Gamma Production in Human Whole Blood: The Effect of Heat and Cations.

BACKGROUND: Interferon-gamma release assays (IGRAs) are blood tests used to measure the amount of interferon-γ (IFN-γ) released by T lymphocytes after stimulation by antigens specific for the diagnosis of latent tuberculosis infection. A mitogen serves as a positive control to assess the immune function in IGRAs. METHODS: This in vitro study was conducted to evaluate IFN-γ production by human whole blood stimulated with heat-treated and/or cation-supplemented phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM), using QuantiFERON-TB Gold Kit ELISA tests. RESULTS: The optimal concentrations of PWM, Con A and PHA for IGRAs were 2 µg/mL, 5 µg/mL and 10 µg/mL, respectively. The results showed that IFN-γ production in response to PWM was the highest and PHA was the lowest amount. The median values of three mitogens were in the following order: PWM≥Con A≥ positive control>>PHA-P>>negative control. PWM and PHA were heat stable, while Con A was heat sensitive. The mitogen response of lymphocytes to untreated or heat-treated PWM and heat-treated Con A was increased in 1 mM Ca2+-supplemented groups, whereas the response to heat-treated PHA was decreased. Exposure to 1 mM Mg2+ had no effect on untreated or heat-treated PWM, and a concentration of 1 mM Zn2+ inhibited the stimulation of un-treated PWM. We found that calcium supplementation improved the PWM-induced production of IFN-γ. CONCLUSION: Therefore, PWM is an appropriate mitogen for use as a positive control in IGRAs. It is a potential indicator of cytokine production in the diagnostic as well as research settings, and calcium supplementation improved stimulation.

Malaria and Japan's colonial frontier: Manchuria, 1900s-1940s.

This article examines the malaria problem among Chinese migrant laborers in Manchuria, particularly laborers on the South Manchuria Railway's mining sites, the Fushun Mines, during the first half of the twentieth century. Almost all of the malaria cases in Manchuria were caused by the parasite Plasmodium vivax, which rarely causes death but leads to debilitation and makes sufferers susceptible to other illnesses. Malaria epidemics in Manchuria during this period were the result of Japan's economic and military exploitation. The expansion of malaria mosquito habitats caused by large-scale constructions and development of mines and massive immigration for these industries led to these epidemics. Most of the malaria victims were Chinese laborers who worked for Japanese businesses and no less than two-thirds of these patients were reportedly from Fushun, where the Fushun Mines were located. The living and working conditions of the laborers made them vulnerable to various diseases, including malaria. As Japanese employers concentrated on the human-centered approach to malaria control general sanitary reforms were often ignored. After the promulgation of the Five-Year Industrial Development Plan of Manchukuo and the outbreak of the Second-Sino Japanese War, Japanese authorities' attitude to malaria among Chinese laborers changed dramatically. A steady supply of labor was essential to enable the production of more coal for the war-efforts as the Fushun Mines were designated a key industry for Japan's national defense. To achieve this manpower efficiency was crucial but malaria epidemics decreased the productivity of labor. As the coal shortage was considered a great obstacle for Japanese and Manchukuo industries, as well as for the conduct of the war, the malaria problem among Chinese laborers could no longer be ignored.

Malaria and Colonialism in Korea, c.1876-c.1945.

This article considers the problem of malaria in the Korean peninsula from 1876 to 1945, focusing particularly on the impact of Japanese colonial rule. One aspect which receives special attention is malaria in urban contexts. The relationship between malaria and urbanisation is shown to be extremely complex, fluctuating regardless of specific interventions against the disease. In rural and urban areas, Japanese antimalarial measures concentrated on military garrisons, at the expense of both civilian settlers and Koreans. However, it was Koreans who bore the brunt of the malaria problem, which was exacerbated in many areas by agricultural and industrial development and, ultimately, by the war regime introduced from 1938. The worsening of the malaria burden in the final years of Japanese rule left a legacy which lasted long after independence.

The borderline of 'empire': Japanese maritime quarantine in Busan c.1876-1910.

This paper seeks to balance the regional and thematic focus of cholera historiography by examining maritime quarantine in Busan, as it was devised and implemented by Japanese officials and doctors during the pre-colonial period. It also places the relationship between Korea and Japan in the context of relations with China, Russia and Britain. This paper shows that quarantine measures in Busan and other Korean ports reflected the rise of Japanese imperial power and the increasing desire on the part of the Japanese to establish an effective borderline for their regional empire. From 1879 Japan began to impose maritime quarantine in Busan, where Japanese influence was very strong even before the colonial period, though at that time Japan was unable to perform quarantine in its own ports independently due to the objections of Western powers, particularly Britain. Victories in the Sino-Japanese and Russo-Japanese wars established Japan as a regional power on equal terms with the West, and as the dominant power in Korea and Eastern Asia. With the acquisition of the right to impose quarantine in its homeland, Japan strengthened and extended the range of quarantine from Japan to Korea, China and Russia. Now quarantine screened Japan from potentially harmful agents ­ pathogenic and political ­ and its functions diversified further as modernisation and imperial expansion gathered pace. The reliance which Japan placed upon quarantine in maintaining its empire explains why it was increasingly out of step with other powers regarding international sanitary precautions. The Japanese maritime quarantine in Busan during this period therefore shows many aspects of Japan's 'national empire'.

Ethylacetate fraction from Korean seaside starfish, Asterias amurensis, has an inhibitory effect on MMP-9 activity and expression and on migration behavior of TNF-α induced human aortic smooth muscle cells.

Atherosclerosis is accompanied by the proliferation of human aortic smooth muscle cells (HASMC) and their movement into the intima. Many reports have indicated the involvement of gelatinases (MMP-9 and MMP-2) in this pathogenesis. The ethylacetate fraction from starfish, Asterias amurensis (EFA), harvested from the Korean seaside has an inhibitory effect on MMP-9 and MMP-2 activities, as well as on the expression of MMP-9 in TNF-α induced HASMC in a dose-dependent manner. Also, EFA inhibits the migration of TNF-α induced HASMC in transwells containing gelatin coated plugs. EFA was not cytotoxic to HASMC over the range 0-1mg/ml. By Western-blot analysis, it was revealed that the phosphorylation of extracellular signal regulated kinase (ERK) in TNF-α induced cells was inhibited and nuclear factor kappa B (NF-κB) p65 levels in nuclear extracts were decreased by EFA treatment. In addition, ERK inhibitor (U0126) treated cells exhibited decreased MMP-9 activity in the zymographic assay. From these results, it was found that the gelatinolytic activity was regulated (1) by enzymatic inhibition of both MMP-9 and MMP-2, as well as (2) by the decreased production of MMP-9 via ERK pathways in EFA treated HASMCs. Taken together, it has been shown that EFA has a putative anti-atherosclerotic effect.

Regulation of matrix metalloproteinase-9 expression between gingival fibroblast cells from old and young rats.

Gingival fibroblast cells (rGF) from aged rats have an age-related decline in proliferative capacity compared with young rats. We investigated G1 phase cell cycle regulation and MMP-9 expression in both young and aged rGF. G1 cell cycle protein levels and activity were significantly reduced in response to interleukin-1beta (IL-1beta) stimulation with increasing in vitro age. Tumor necrosis factor-alpha (TNF-alpha)-induced matrix metalloproteinase-9 (MMP-9) expression was also decreased in aged rGF in comparison with young rGF. Mutational analysis and gel shift assays demonstrated that the lower MMP-9 expression in aged rGF is associated with lower activities of transcription factors NF-kappaB and AP-1. These results suggest that cell cycle dysregulation and down-regulation of MMP-9 expression in rGF may play a role in gingival remodeling during in vitro aging.

Deoxypodophyllotoxin, flavolignan, from Anthriscus sylvestris Hoffm. inhibits migration and MMP-9 via MAPK pathways in TNF-alpha-induced HASMC.

The matrix metalloproteinases (MMP-9 and MMP-2) in aortic smooth muscle cells (SMC) play key roles in the pathogenesis atherosclerosis. The SMC migration into the vascular wall via the bloodstream is directly linked with MMP-9 expression. Deoxypodophyllotoxin (DPT), a naturally occurring flavolignan with anti-inflammatory activity, was isolated from Anthriscus sylvestris Hoffm. and has been known inhibit the expression of MMP-9 in tumor necrosis factor-alpha (TNF-alpha) stimulated human aortic smooth muscle cells (HASMC). In this study, DPT was purified and demonstrated to inhibit the MMP-9/2 activities in TNF-alpha-induced HASMC. In addition, MMP-9 expression and migration was strongly inhibited by DPT in TNF-alpha-induced HASMC. To examine whether TNF-alpha-induced MMP-9 expressions are involved with migrations of HASMC, reverse transcription-polymerase chain reaction (RT-PCR) and luciferase-tagged promoter analysis were applied. These experiments revealed that DPT inhibited the mRNA transcription of MMP-9 gene expression. Furthermore, Western blot analysis indicated that the TNF-alpha-induced phosphorylation of extracellular signal regulated kinase 1 and 2 (ERK1/2), p38 and c-Jun N-terminal kinase (JNK) were strongly inhibited by DPT. From these results, it is concluded that DPT has an inhibitory activities on migration and MMP-2/9 activities, and MMP-9 transcription in HASMC.

Regulation of type II collagen expression by cyclin-dependent kinase 6, cyclin D1, and p21 in articular chondrocytes.

This study examined whether cell cycle regulatory proteins, such as cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors, regulate type II collagen expression and mediate interlukin-1 (IL-1beta)-induced suppression of type II collagen expression in articular chondrocytes. IL-1beta inhibited type II collagen expression, but activated CDK6. Ectopic expression of CDK2 did not alter type II collagen expression. However, overexpression of CDK6 inhibited type II collagen expression, whereas inhibition of CDK6 activity blocked IL-1beta-induced suppression of type II collagen expression. IL-1beta upregulated the expression of cyclin D1, which is known to activate CDK6. In turn, overexpression of cyclin D1 suppressed type II collagen expression. In contrast to cyclin D1, IL-1beta triggered down-regulation of the CDK inhibitor, p21. Overexpression of p21 blocked IL-1beta- or CDK6-induced suppression of type II collagen expression. Our results collectively indicate that CDK6/cyclin D1/p21 complex regulates type II collagen expression in articular chondrocytes.

Protein kinase D2 contributes to either IL-2 promoter regulation or induction of cell death upon TCR stimulation depending on its activity in Jurkat cells.

Members of protein kinase D (PKD) family serine/threonine kinases (PKD1, PKD2 and PKD3) are expressed in wide range of cells and regulate various cellular responses including immune responses. We have previously shown that PKD is involved in the signaling pathways of a human CD4(+) T cell clone stimulated with its cognate antigen. Contrary to foregoing publications, PKD1 mRNA was not detected in human T cells, Jurkat cells and mouse thymocytes and splenocytes. Instead, mass-spectrometric and reverse transcription-PCR analyses revealed that PKD2 was predominant in T cells. To investigate the roles of PKD2, wild-type (WT) and constitutively active (CA) PKD2 were expressed in Jurkat cells together with IL-2 promoter-driven reporter gene. Expression of WT-PKD2 enhanced IL-2 promoter activity upon stimulation with anti-CD3 mAb, while expression of CA-PKD2 inhibited IL-2 promoter activity and induced cell death. Although the cell death was suppressed by the treatment with caspase inhibitor, the IL-2 promoter activity was rarely recovered in CA-PKD2-expressing cells upon TCR stimulation. WT-PKD2 localized mainly in the cytoplasm translocated into the nucleus after TCR stimulation, while CA-PKD2 was present in both the cytoplasm and the nuclei before and after stimulation. Proteomic analyses revealed that CA-PKD2 enhanced the amount of phosphorylated SET protein, a histone chaperon that regulates histone acetylation, in Jurkat cells and the recombinant SET protein was phosphorylated by CA-PKD2 in vitro. The data provide a renewing insight into the subset of PKD family kinases expressed in T cells and suggest that PKD2 is involved in IL-2 promoter regulation and cell death depending on its activity upon TCR stimulation.

TCR ligand avidity determines the mode of B-Raf/Raf-1/ERK activation leading to the activation of human CD4+ T cell clone.

The interactions between peptide/MHC complexes and their cognate TCR are essential for various T cell responses. However, the relationship between the avidity of TCR ligand and the subsequent intracellular signaling through the TCR is still unclear. To investigate the effects of TCR ligand avidity on TCR-mediated signaling, we established L cells expressing HLA-DR4 molecules covalently linked with agonistic peptide (high-affinity ligand) or altered peptide ligand (APL; low-affinity ligand) at various densities as APC for a cognate human CD4(+) T cell clone. Using this system, we demonstrated that the T cell clone stimulated with APL/HLA-DR4 complexes presented at an excessive density provoked the up-regulation of CD69, IL-2 production and proliferation, but no detectable phosphorylation of ZAP-70/LAT/SLP-76. Furthermore, in contrast to the high-affinity stimulation, the low-affinity stimulation evoked delayed and sustained activation of the B-Raf/extracellular signal-regulated kinase (ERK) pathway without Raf-1 activation. The strength and duration of B-Raf/ERK activations closely correlated with the density of the TCR ligand. A knockdown approach confirmed that B-Raf activation was indispensable for the APL-induced T cell responses. These observations suggest that the differences in TCR-peptide/MHC interactions reflect the strength and duration of B-Raf/Raf-1/ERK activation in the human CD4(+) T cells.

A role of kinase inactive ZAP-70 in altered peptide ligand stimulated T cell activation.

T cell activation signals induced by altered peptide ligands (APLs) are different from those induced by the original agonistic peptide. The characteristics of the former are partial phosphorylation of TCR-zeta and no tyrosine-phosphorylation of zeta-associated protein-70 (ZAP-70). To analyze further those signaling pathways, we introduced a dominant negative (DN) form of ZAP-70 into a human CD4(+) T cell clone in which fully and partially agonistic peptide ligands have been well characterized. We found that some over-expressed partially agonistic ligands (OPALs) induced T cell responses without tyrosine-phosphorylation and kinase activation of ZAP-70. However, those responses were inhibited in T cells expressing DN ZAP-70, which could associate with partially phosphorylated TCR-zeta. In OPAL-stimulated T cells, PLC-gamma1 was phosphorylated and it was suppressed by DN ZAP-70 expression, suggesting that the ZAP-70-TCR-zeta association mediates the activation of PLC-gamma1 leading to T cell responses even in the absence of kinase activation of ZAP-70.

Association of a high activity of matrix metalloproteinase-9 to low levels of tissue inhibitors of metalloproteinase-1 and -3 in human hepatitis B-viral hepatoma cells.

Matrix metalloproteinases (MMPs) play a major role in the turnover of extracellular matrix (ECM) during cancer invasion and metastasis, and tissue inhibitors of metalloproteinases (TIMPs) control MMPs, thus maintaining a balanced ECM catabolism under physiological conditions. The aim of this study was to assess the behavior of some MMPs (FASEB J., 7, 1993, 1434; Cancer Metastasis Rev., 9(4) 1990, 289) and TIMPs (Biochem. Biophys. Res. Commun., 301, 2003, 1069; FASEB J., 7, 1993, 1434; Nature, 370, 1994, 61). Competitive RT-PCR, gelatin-substrate zymography, and ELISA techniques were used for quantification. The hepatitis B virus (HBV)-DNA-containing hepatocellular carcinoma cell lines, Hep3B, HepG2-HBV and HFF-T2 contain highly activated matrix metallproteinase-9 (MMP-9), which is rarely found in normal liver cell lines such as the Chang lines. MMP-9 activities of HFH-T2, HepG2-HBV and Hep3B were significantly higher than that of non-HBV-hepatocellular carcinoma SK-Hep1 and HepG2 (HCC origin, HBV not detected), as assayed by gelatin zymography. Low levels of TIMP-1 and TIMP-3 were observed in HFH-T2, HepG2-HBV and Hep3B, while the TIMP-2 level was high, as evidenced by reverse zymography. In contrast, 3 TIMP-1, -2 and -3 were largely detected in Chang, HepG2 and SK-Hep1 cells. To investigate the nature of the quantitative regulation of MMPs and TIMPs for these cell lines at the transcriptional levels, a reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out. Not only MMP-9 mRNAs of HFH-T2, HepG2-HBV and Hep3B but also MMP-9 mRNA of SK-Hep1 and HepG2 were highly expressed with no major differences among these four cell lines. However, TIMP-1 and TIMP-3 mRNAs of HFH-T2, HepG2-HBV and Hep3B were markedly reduced, while those of SK-Hep1, HepG2 and Chang cells were maintained at high levels. Finally, an invasion assay using matrigel indicated in an increase in invasiveness in HFH-T2, HepG2-HBV and Hep3B cells, but a decrease in invasiveness of Chang, HepG2 and SK-Hep1 cells. These results indicate that the overexpression of MMP-9 mRNAs and the suppression of TIMP-1 and TIMP-3 in HFH-T2, HepG2-HBV and Hep3B were the result of HBV transfection. Based on these results, it is concluded that HBV affects the malignance of hepatocellular cancer by elevating MMP-9 activity, and suppressing TIMP-1 and TIMP-3.

Structure-related cytotoxicity and anti-hepatofibric effect of asiatic acid derivatives in rat hepatic stellate cell-line, HSC-T6.

The structural relationship of 16 asiatic acid (AA) derivatives, including AA and asiaticoside (AS) to cytotoxicity and anti-hepatofibrotic activity in HSC-T6 cells, were investigated. Cytotoxicities of AA derivatives varied from 5.5 microM to over 2000 microM of IC50 depending on AA functional group modifications. Substituting the hydroxyl group at the C(2) to N[triple bond]C and substituting bulky groups for dihydroxyl groups at (3), (23) of the A-ring increased the cytotoxicity, but keto group at C(11) and benzoyl ester at C(2) were greatly reduced it. Modification of the carboxylic acid group at C28 also reduced the cytotoxicity. The collagen synthesis determined by hydroxyproline content in the cells was inhibited from a maximum of 48% (Zlx-i-85 and 87) to 15% (AS) by AA derivatives. The anti-hepatofibrotic effect of these compounds might be due to the reduced expression of prolyl 4-hydroxylase alpha and beta subunits and TIMP2. However, the inhibition of collagen by asiaticoside derivatives did not show any structural-activity relationship.

Correlation between plasma levels of matrix metalloproteinase (MMP)-9 /MMP-2 ratio and alpha-fetoproteins in chronic hepatitis carrying hepatitis B virus.

BACKGROUND: Matrix metalloproteases (MMP) and alpha-fetoproteins (AFP) are involved in hepatitis B virus (HBV)-induced chronic hepatitis. In the present study, we have determined the correlation between the MMP-9/MMP-9 ratio and AFP levels in the serum of patients during chronic viral B hepatitis. METHODS: Twenty-eight healthy individuals (18 men and 10 women) with a mean age of 36.3 years (range 23-58 years) and 50 patients (42 men, 8 women) with a mean age of 39.7 years (range 22-61 years) participated in the study. Forty-eight participants had HBV and the remaining two were either hepatitis G virus (HGV) or hepatitis C virus (HCV) carriers. Values of patients were compared with those obtained from 12 blood donor controls (5 men, 7 women), mean age 36 years (range 21-46 years). Patients' sera were subjected to examination of hepatitis B surface (HBs) and hepatitis B early (Hbe) antigen, SGOT, SGPT, AFP, MMP-2 and MMP-9. Serum levels of MMP-2 and MMP-9 activities were measured by a zymogram protease assay and densitometric measurement. The ratios of MMP-9 to MMP-2 were calculated by dividing the densitometric results. RESULTS: Compared with the healthy controls, the mean serum concentrations of MMP-2 were slightly increased in the chronic HBV patients. In contrast, compared with the healthy controls, the mean serum concentrations of MMP-9 were significantly increased in the chronic HBV patients. When the ratios of the MMP-9/MMP-2 and amounts of the serum AFP were compared, a specific correlation between these two parameters was observed. Higher amounts of AFP were detected in the patients with a low ratio of MMP-9/MMP-2. Patients with hepatocellular carcinoma (HCC) and cirrhosis showed relatively low MMP-9/MMP-2 ratios in chronic hepatitis B. In addition, AFP levels of HCC and cirrhosis were higher than in chronic HBV patients. CONCLUSIONS: These results indicate that the AFP level and ratio of MMP-9 and MMP-2 is highly correlated in chronic HBV-induced hepatitis. Because the serum MMP activities were significantly varied between each stage of AFP production in liver disease, an individual profile of these parameters might serve as an easy accessing serum marker to monitor the progression of liver disease.