Role of alanine aminotransferase in the effects of urinary caffeine concentration and its primary metabolite concentration on cognitive function in older adults: Bayesian Kernel Machine regression analysis and mediation analysis.

OBJECTIVE: This study aimed to explore the role of alanine aminotransferase (ALT) in the effects of urinary caffeine and its primary metabolites on cognitive function in elderly people. MATERIALS AND METHODS: In this investigation, we meticulously curated a cohort from the 2011-2014 National Health and Nutrition Examination Survey (NHANES) database. Animal fluency emerged as the pivotal metric for assessing cognitive function within our study population. In order to navigate the intricacies of mixture analysis and circumvent potential complexities, we harnessed the power of Bayesian kernel machine regression (BKMR) models. This method allowed us to dissect the nuanced impacts of caffeine and its primary urinary metabolites on cognitive function. While accounting for caffeine and its metabolites, we analyzed the relationship between ALT and cognitive function through non-linear dynamics. Lastly, employing structural equation modeling, we probed the intriguing question of whether ALT mediates the influence of 3,7-dimethylxanthine on cognitive function. This comprehensive approach has unveiled a deeper understanding of the multifaceted interplay among these variables, offering invaluable insights into the determinants of cognitive function within our cohort. RESULTS: After meticulous adjustment for various covariates, our linear regression analysis unveiled a noteworthy finding: 3,7-dimethylxanthine demonstrated a significant positive correlation with cognitive function (p < 0.05). Importantly, within the BKMR model employed, 3,7-dimethylxanthine emerged as the most influential factor within the compound, with posterior inclusion probabilities of 0.995 and 0.939. Furthermore, our single-exposure effect model confirmed its presence at the 25th, 50th, and 75th percentile concentrations of other components within the compound. Interestingly, bivariate concentration curves indicated no interaction within the compound, underscoring the prominent impact of 3,7-dimethylxanthine on cognitive function. Subsequently, through a test of Restricted Cubic Splines (RCS), we revealed a non-linear relationship between ALT and cognitive function at the 10th, 50th, and 90th percentiles (p < 0.05), indicating a heightened risk of diminished cognitive function in the low ALT group. Employing structural equation modeling, we meticulously examined the mediating role of ALT in relation to 3,7-dimethylxanthine and cognitive function. However, our study results did not yield significant evidence of a mediating effect. This comprehensive analysis elucidates the intricate interplay between these variables, unveiling the subtle mechanisms governing cognitive function. CONCLUSIONS: In this study, a noteworthy positive correlation was observed between 3,7-dimethylxanthine and cognitive function. Additionally, a non-linear relationship was identified between ALT and cognitive function, with lower levels of ALT associated with a decline in cognitive function. The RCS trend suggested that higher levels of ALT may similarly lead to diminished cognitive performance. However, in our pursuit to ascertain potential mediation, we regrettably found no significant evidence supporting mediation among these factors involving ALT. This underscores the need for more comprehensive investigations and expanded clinical explorations into the intricate associations among these three pivotal elements.

A nomogram predicting the outcome of gallbladder cancer patients with different target organ metastases.

OBJECTIVE: Gallbladder cancer (GBC) is a highly aggressive malignancy that is associated with a high mortality rate globally. Unfortunately, distant metastases are often detected at the time of diagnosis. Therefore, we investigated the survival outcomes of gallbladder cancer patients with different metastases targeting organs, analyzed their prognosis, and explored their hidden clinical value. PATIENTS AND METHODS: Through data screening, a total of 398 patients with GBC with different target organ metastases were analyzed retrospectively, including patients with solitary bone metastasis, solitary liver metastasis, solitary lung metastasis, and multiple organ metastases. The survival results of different variables were plotted as Kaplan-Meier survival curves. Univariate and multivariate Cox regression models were used to screen study variables and identify independent prognostic factors. Finally, a nomogram was established to systematically evaluate the prognosis of patients with multiple organ metastasis. RESULTS: In the patient cohort, thirteen (3.3%) had solitary bone metastasis, 290 (72.9%) had solitary liver metastasis, 22 (5.5%) had solitary lung metastasis, and 73 (18.3%) had multiple organ metastases (including liver, lung, bone and brain metastases). Multivariate Cox analysis showed that the overall survival (OS) of patients with solitary lung metastasis was significantly better than that of patients with other organ metastasis (p = 0.038), while the difference in tumor cancer-specific survival (CSS) of this factor was not statistically significant (p > 0.05). Surgery and chemotherapy were independent prognostic protective factors for OS and CSS. The OS-related models exhibited a C-index of 0.74 (95% CI: 0.71-0.77), while the CSS-related models showed a slightly lower C-index of 0.73 (0.70-0.76). Both the OS- and CSS-related clinical prediction models had good accuracy. CONCLUSIONS: This study shows that different target organ metastases may affect the OS of patients with distant metastatic GBC. Patients receiving palliative surgery, primary site resection, radical surgery, and chemotherapy have significant survival benefits in terms of OS and CSS.

[Preliminary evaluation and mechanism of adipose-derived stem cell transplantation from allogenic diabetic rats in the treatment of diabetic rat wounds].

Objective: To investigate whether adipose-derived stem cells (ASCs) from allogeneic diabetic rats can promote wound healing in diabetic rats or not and the mechanism. Methods: (1) Fifty-six male Wistar rats aged 12-16 weeks were divided into diabetic group and healthy group according to the random number table (the same grouping method below), with 28 rats in each group. Rats in healthy group were not treated with any treatment. Rats in diabetic group were injected with 10 g/L streptozotocin 60 mg/kg intraperitoneally in one time to establish the diabetic model. Four rats in diabetic group and 4 rats in healthy group were selected according to the random number table, and the adipose tissue in the inguinal region was taken to culture and purify ASCs, so as to obtain healthy rat-derived ASCs (hereinafter referred to as nASCs) and diabetic rat-derived ASCs (hereinafter referred to as dASCs). The third passage of nASCs (n=3) and dASCs (n=3) were taken, and the positive expression rates of cell surface differentiation antigens CD105, CD31, CD34, and CD44 were detected with flow cytometer for defining ASCs purity. (2) The rest 24 rats in healthy group and 24 rats in diabetic group were used to make three round full-thickness skin defect wounds with a diameter of 12 mm on the back of each rat. Immediately after injury, phosphate buffer saline (PBS), nASCs of 2×10(7)/mL, and dASCs of 2×10(7)/mL each in the volume of 0.5 mL were subcutaneously injected into three wounds and their margins of each rat, respectively. On post injury day (PID) 1, 3, 7, and 12, 6 rats in each group were selected according to the random number table to calculate the wound area, and the wound tissue was stained with hematoxylin-eosin to observe the histological morphology of the wound. (3) Human ASCs (hASCs) were subcultured, and the 4th to 7th passage of cells were used for the subsequent experiments. The hASCs were divided into 7 groups, with 12 samples in each group. Cells in blank control group were cultured with mesenchymal stem cell culture medium, and cells in simple advanced glycation end products (AGEs) group, simple protein group, simple high glucose group, simple high osmotic pressure group, AGEs-high glucose combination group, and protein-high osmotic pressure combination group were cultured with mesenchymal stem cell culture medium containing a final mass concentration of 100 mg/L AGEs, 100 mg/L bovine serum albumin (BSA), 28 mmol/L D-glucose, 28 mmol/L mannitol, 100 mg/L AGEs+ 28 mmol/L D-glucose, 100 mg/L BSA+ 28 mmol/L mannitol, respectively. Cell proliferation was detected by cell counting kit 8 at post culture hour (PCH) 2 and on post culture day (PCD) 2, 4 and 6. (4) The hASCs were divided into blank control group, simple AGE group, simple high glucose group, and AGE-high glucose combination group, with 12 samples in each group, which were treated the same as corresponding groups in experiment (3). On PCD 0, 2, 4, and 6, the positive expression rates of cell surface differentiation antigens CD105, CD44, and CD45 were detected by flow cytometer to estimate their homeostasis. (5) The hASCs were divided into AGE-high glucose combination group and protein-high osmotic pressure combination group, with 9 samples in each group, which were treated the same as corresponding groups in experiment (3). On PCD 2, 4, and 6, the expression of intracellular protein was detected by cyanine 3-streptavidin double-antibody sandwich technique. Data were processed with analysis of variance for factorial design, least significant difference test, and Bonferroni correction. Results: (1) The positive expression rates of CD44 in nASCs and dASCs were both higher than 96%, the positive expression rates of CD31 and CD34 were low, and the positive expression rates of CD105 were about 40%, which basically met the purity requirements. (2) The areas of wounds treated by three methods in rats of healthy group and diabetic group were similar on PID 1 (P>0.05). In healthy group, compared with (0.682 1±0.078 9), (0.314 3±0.113 7), and (0.064 3±0.002 1) cm(2) of the PBS-treated wounds in rats, the area of nASCs-treated wounds in rats decreased significantly on PID 3, 7, and 12 [(0.464 1±0.092 6), (0.223 9±0.072 7), and (0.034 3±0.012 5) cm(2), P<0.05], the area of dASCs-treated wounds in rats decreased significantly on PID 3 and 12 [(0.514 1±0.124 1) and (0.043 7±0.032 8) cm(2), P<0.05] but was not obviously changed on PID 7 [(0.274 2±0.062 5) cm(2), P>0.05]. Compared with those of the dASCs-treated wounds of rats within the same group, the area of the nASCs-treated wounds of rats in healthy group decreased significantly on PID 3 and 7 (P<0.05) but was not obviously changed on PID 12 (P>0.05). In diabetic group, compared with (0.853 5±0.204 8), (0.670 5±0.164 8), and (0.131 4±0.074 4) cm(2) of the PBS-treated wounds in rats, the area of nASCs-treated wounds in rats decreased significantly on PID 3, 7, and 12 [(0.633 4±0.132 5), (0.331 8±0.023 5), and (0.074 2±0.003 8) cm(2), P<0.05], the area of dASCs-treated wounds in rats decreased significantly on PID 3 [(0.773 6±0.182 2) cm(2), P<0.05] but was not obviously changed on PID 7 and 12 [(0.510 6±0.192 2) and (0.114 4±0.003 1) cm(2), P>0.05]. Compared with the dASCs-treated wounds of rats within the same group, the area of the nASCs-treated wounds of rats in diabetic group was not obviously changed on PID 3 and 7 (P>0.05) but decreased significantly on PID 12 (P<0.05). There was no obvious difference in histological morphology of the wounds treated with three methods in rats of each group on PID 1. On PID 3, a small amount of microvessels were formed in the wounds treated with nASCs and dASCs of rats in both groups, but microvessel formation was almost undetected in the PBS-treated wounds. On PID 7, more small blood vessels and fibroblasts (Fbs) were observed in the wounds treated with nASCs and dASCs of rats in both groups, but the small blood vessels and Fbs were slightly less in the PBS-treated wounds. On PID 12, the wounds treated with nASCs and dASCs of rats in the two groups were covered by epithelial tissue, the granulation tissue in the PBS-treated wounds of rats in healthy group was not obvious, and the PBS-treated wounds of rats in diabetic group were not completely epithelialized. (3) Compared with those of blank control group, the cell number of hASCs in simple AGEs group decreased significantly on PCD 2, 4, and 6 (P<0.05), which increased significantly on PCD 2 and 4 in simple high glucose group (P<0.05), and that in AGEs-high glucose combination group decreased significantly on PCD 4 and 6 (P<0.05). (4) Compared with that on PCD 4 within the same group, the positive expression rate of CD105 in hASCs decreased significantly in blank control group, simple AGEs group, and AGEs-high glucose combination group on PCD 6 (P<0.05). The positive expression rate of CD44 was higher than 95%, and that of CD45 was less than 2% in hASCs of each group at each time point. (5) Detection values of 7 proteins were located in the confidence interval. The expression levels of basic fibroblast growth factor and tissue inhibitor of metalloproteinase-1 in hASCs of AGEs-high glucose combination group and protein-high osmotic pressure combination group showed increasing trend with the prolongation of culture time. The expression level of human monocyte chemoattractant protein 1 (MCP-1) in hASCs of AGEs-high glucose combination group showed increasing trend with the prolongation of culture time, while the expression level of growth-regulated oncogene (GRO) on PCD 6 was significantly higher than that on PCD 4 within the same group (P<0.05); the expression levels of MCP-1 and GRO in hASCs of protein-high osmotic pressure combination group showed decreasing trend with the prolongation of culture time. The expression level of follistatin in hASCs of protein-high osmotic pressure combination group decreased obviously on PCD 4, while that in hASCs of AGEs-high glucose combination group was significantly lower on PCD 6 than that on PCD 4 (P<0.05). The expression level of vascular endothelial growth factor (VEGF) in hASCs of protein-high osmotic pressure combination group decreased gradually with the prolongation of culture time, while that in hASCs of AGEs-high glucose combination group on PCD 4 decreased significantly as compared with that on PCD 2 (P<0.05). The expression level of urokinase-type plasminogen activator receptor in hASCs of protein-high osmotic pressure combination group on PCD 6 was significantly higher than that on PCD 4 within the same group (P<0.05) and that of AGEs-high glucose combination group on PCD 6 (P<0.05). Conclusions: Both nASCs and dASCs can promote wound healing in rats with simple defect injury, but dASCs have no significant effect on wound healing in rats with diabetes mellitus, which may be related to the inhibition of ASCs proliferation and the influence of high glucose and AGEs intervention on their homeostasis and secretory function.

Development of Integrated Device of Trace Bloodstains Imaging and Age Analysis.

ABSTRACT: Objective To develop a device of trace bloodstains imaging and age analysis, so as to provide a non-destructive, simple and objective method for age estimation of bloodstains at the crime scene. Methods Based on the principle of digital imaging and color pattern analysis, the mobile terminal of the device was used to collect images of bloodstains of different ages. The time-dependent pattern of 6 parameters （R, G, B, C, Y, M） reflecting the changes of color of images of different ages was obtained by computer image analysis. A multiparameter comprehensive inference equation of bloodstains age was established and embedded into the device software to realize the intelligent inference of the bloodstains age. Then the capability and reliability of the device was verified. Results This integrated device of bloodstains imaging and age analysis could quickly collect bloodstains at the crime scene and automatically analyze and infer the age of bloodstains combined with related intelligence software. In the blind test, the detection accuracy of this device was 95% in both natural light airtight group and dark airtight group, and 80% in the natural light ventilation group. Conclusion The integrated device of trace bloodstains imaging and age analysis can be used in a simple manner, which provides a new objective method for bloodstains age estimation.

Symbiotic fungal endophyte Phomopsis liquidambari-rice system promotes nitrogen transformation by influencing below-ground straw decomposition in paddy soil.

AIMS: To explore if and how symbiotic Phomopsis liquidambari-rice system influences below-ground straw decomposition and then nitrogen(N) transformation in response to environmental N levels. METHODS AND RESULTS: Litter bag experiments were utilized to trace the decay process during rice growth phases (seedling (T1), tillering (T2), heading (T3) and maturing (T4) stage), with (E+) and without endophyte (E-), under low (LN), medium (MN) and high nitrogen (HN) supply. Litter, soil and plant samples were collected to evaluate the decay process, N transformations, plant quality and relative abundance of soil ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB) and P. liquidambari. The results showed that straw decomposition increased by 19·76% (LN, T2 stage), 14·05% (MN, T3 stage) and 16·88% (MN, T4 stage) in E+ pots when compared with E- pots. Further analysis revealed that no significant endophyte × N interaction was found for straw decay rate and that the decay rate was reduced by a higher N supply (LN, 37·16 ± 0·65%; MN, 32·27 ± 1·72%; HN, 29·44 ± 1·22%) at the T1 stage, whereas straw decay rate and N release increased by 9·38 and 11·16%, respectively, mainly by endophyte colonization at the T4 stage. The abundance of AOA and AOB were altered, corresponding with the decay rate. Soil mineral N, straw mineral N and plant quality were shown to increase in E+ pots, depending on environmental N conditions and growth phase. The yield increased by 2·98% for E+ plants under MN level. CONCLUSIONS: Symbiotic P. liquidambari-rice system promoted below-ground straw decomposition and N transformation, depending on environmental N levels and plant growth phase. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides evidence that fungal endophyte-plant systems are able to promote N transformation by increasing straw decomposition. A reasonable combination of N inputs could enhance its advantage in agriculture ecosystems.

Methylene blue attenuates renal ischemia-reperfusion injury by negative regulation of NLRP3 signaling pathway.

OBJECTIVE: To investigate the effect of methylene blue (MB) on renal ischemia-reperfusion (IR) injury in mice and its possible relevant mechanisms. MATERIALS AND METHODS: A total of 30 male C57/BL6 mice aged 4 months old were randomly divided into the following three groups: Sham group (n=10), IR group (n=10), and MB group (n=10). Mice in MB group were treated with gavage continuously using methylene blue solution (dosage: 25 mg·kg-1·d-1) until they were 7 months old. Mice in the other two groups were administrated with the same amount of normal saline for gavage. After that, the abdomen of mice in Sham group was opened and closed, and bilateral renal pedicles of mice in IR group and MB group were occluded using a micro-artery clamp for 45 min for modeling. After modeling, the renal tissues and blood samples of mice were taken for detection. The levels of serum creatinine (Scr), blood urea nitrogen (BUN), and malondialdehyde (MDA), and activity of superoxide dismutase (SOD) in mice in each experimental group were detected and statistically analyzed, respectively. The degree of renal tubular necrosis in renal tissues of mice was observed under an optical microscope. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors [interleukin-1ß (IL-1ß), IL-18, IL-10 and transforming growth factor-ß1 (TGF-ß1)] in renal tissues of mice in each experimental group, followed by relevant statistical analyses. The expressions of relevant proteins [NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), nuclear factor-κB (NF-κB), caspase-1, pro-IL-1ß, and IL-1ß] in renal tissues of mice in each experimental group were detected via Western blotting, and the gray value of each band was detected for statistical analysis. RESULTS: It was found in this experimental study that Scr and BUN levels in MB group were significantly lower than those in IR group, and the differences were statistically significant (p<0.05). Compared with that in IR group, the degree of renal tubular necrosis in MB group was significantly alleviated, and the difference was statistically significant (p<0.05). Compared with those in IR group, the levels of inflammatory factors (IL-1ß and IL-18) in MB group were significantly decreased, but the levels of IL-10 and TGF-ß1 in MB group were significantly increased, and the differences were statistically significant (p<0.05). Compared with those in IR group, the activity of SOD in MB group was increased significantly, but the level of MDA was decreased, and the differences were statistically significant (p<0.05). The protein expressions of NLRP3, NF-κB, caspase-1, and pro-IL-1ß in MB group were decreased compared with those in IR group, but the expression of IL-1ß in MB group was increased, and the differences were statistically significant (p<0.05). CONCLUSIONS: We showed that methylene blue can alleviate the apoptosis and inflammatory response induced by renal IR injury in mice, and its relevant mechanism may be related to the fact that methylene blue can negatively regulate NLRP3 signaling pathway.

Comparative analysis of bilirubin in correlation to albumin between nephrotic syndrome patients and postoperative gastroparesis syndrome patients.

This study aimed to disclose the potential causality of low bilirubin in patients with nephrotic syndrome (NS). Correlation analysis was carried out on total bilirubin (TBIL) to serum albumin (ALB), urine protein (Upr), and urinary microalbumin/creatinine (Umalb/cr) for three groups in a case-control study. P < 0.001 was observed for TBIL, ALB, Umalb/cr, and Upr between the NS and chronic nephritis (CN) groups, and P values of 0.0001, 1.000, 0.0001, and 0.0001 were observed for TBIL, ALB, Umalb/cr, and Upr, respectively, between the postoperative gastroparesis (PGS) and CN groups. The values of r and P in correlation to TBIL were 0.549 and 0.000 for ALB, -0.405 and 0.000 for Umalb/cr, and -0.448 and 0.000 for Upr in the NS group; -0.007 and 0.959 for ALB, 0.213 and 0.091 for Umalb/cr, and -0.082 and 0.519 for Upr in the PGS group; and 0.509 and 0.000 for ALB, -0.431 and 0.000 for Umalb/cr, and -0.362 and 0.002 for Upr in the CN group. A probable causality is implied between the low level of blood bilirubin and its loss in urine in NS patients. This conclusion may provide a theoretical basis for the feasibility of therapies against oxidative stress in NS patients.

Post-onset inhibition of murine arthritis using combined chemokine antagonist therapy.

OBJECTIVE: To investigate the effect of targeting the chemotaxis of monocytes and polymorphonuclear monocytes (PMNs) in situ in MRL-Faslpr arthritis. METHODS: MRL-Faslpr mice were injected intradermally with complete Freund's adjuvant and cellular infiltration into the joint was monitored. Once clinical disease developed, the animals received one of three treatments: MCP-1(9-76); MCP-1(9-76) plus Gro-alpha(8-73); or control peptide, MCP-1 Ala. The bimalleolar ankle width was measured for 11 days and histological examination of the joints was then assessed. RESULTS: Cellular infiltration started after the onset of ankle swelling, and increased progressively. The incidence of swelling and the histopathology was reduced after day 6 of treatment in the MCP-1(9-76)-treated mice. Mice treated with the two antagonists MCP-1(9-76) and Gro-alpha(8-73) displayed a further significant reduction in disease parameters. CONCLUSION: Treatment after disease onset with chemotactic antagonists for monocytes and PMNs significantly alleviated both the swelling and the histopathology seen in arthritis, suggesting that chemokine antagonists are an effective anti-inflammatory therapy.

Matrix metalloproteinase activity inactivates the CXC chemokine stromal cell-derived factor-1.

Chemokines provide directional cues for leukocyte migration and activation that are essential for normal leukocytic trafficking and for host responses during processes such as inflammation, infection, and cancer. Recently we reported that matrix metalloproteinases (MMPs) modulate the activity of the CC chemokine monocyte chemoattractant protein-3 by selective proteolysis to release the N-terminal tetrapeptide. Here we report the N-terminal processing, also at position 4-5, of the CXC chemokines stromal cell-derived factor (SDF)-1alpha and beta by MMP-2 (gelatinase A). Robustness of the MMP family for chemokine cleavage was revealed from identical cleavage site specificity of MMPs 1, 3, 9, 13, and 14 (MT1-MMP) toward SDF-1; selectivity was indicated by absence of cleavage by MMPs 7 and 8. Efficient cleavage of SDF-1alpha by MMP-2 is the result of a strong interaction with the MMP hemopexin C domain at an exosite that overlaps the monocyte chemoattractant protein-3 binding site. The association of SDF-1alpha with different glycosaminoglycans did not inhibit cleavage. MMP cleavage of SDF-1alpha resulted in loss of binding to its cognate receptor CXCR-4. This was reflected in a loss of chemoattractant activity for CD34(+) hematopoietic progenitor stem cells and pre-B cells, and unlike full-length SDF-1alpha, the MMP-cleaved chemokine was unable to block CXCR-4-dependent human immunodeficiency virus-1 infection of CD4(+) cells. These data suggest that MMPs may be important regulatory proteases in attenuating SDF-1 function and point to a deep convergence of two important networks, chemokines and MMPs, to regulate leukocytic activity in vivo.

The ligands of CXC chemokine receptor 3, I-TAC, Mig, and IP10, are natural antagonists for CCR3.

Th1 and Th2 lymphocytes express a different repertoire of chemokine receptors (CCRs). CXCR3, the receptor for I-TAC (interferon-inducible T cell alpha-chemoattractant), Mig (monokine induced by gamma-interferon), and IP10 (interferon-inducible protein 10), is expressed preferentially on Th1 cells, whereas CCR3, the receptor for eotaxin and several other CC chemokines, is characteristic of Th2 cells. While studying responses that are mediated by these two receptors, we found that the agonists for CXCR3 act as antagonists for CCR3. I-TAC, Mig, and IP10 compete for the binding of eotaxin to CCR3-bearing cells and inhibit migration and Ca(2+) changes induced in such cells by stimulation with eotaxin, eotaxin-2, MCP-2 (monocyte chemottractant protein-2), MCP-3, MCP-4, and RANTES (regulated on activation normal T cell expressed and secreted). A hybrid chemokine generated by substituting the first eight NH(2)-terminal residues of eotaxin with those of I-TAC bound CCR3 with higher affinity than eotaxin or I-TAC (3- and 10-fold, respectively). The hybrid was 5-fold more potent than I-TAC as an inhibitor of eotaxin activity and was effective at concentrations as low as 5 nm. None of the antagonists described induced the internalization of CCR3, indicating that they lack agonistic effects and thus qualify as pure antagonists. These results suggest that chemokines that attract Th1 cells via CXCR3 can concomitantly block the migration of Th2 cells in response to CCR3 ligands, thus enhancing the polarization of T cell recruitment.

[High performance liquid chromatographic determination of domoic acid in shellfish samples].

Domoic acid in shellfish samples was determined by HPLC with DAD/UV detector at 242 nm. Samples were extracted with methanol-water followed by clean-up of the extracts with strong anion exchange solid phase extraction cartridge(3 mL LC-SAX). Zorbax SB-C18 column, 150 mm x 4.6 mm i.d., and mobile phase of acetonitrile-0.1% aqueous trifluoroacetic acid(13:87, V/V) were used for the assay. The quantitative analysis was performed with external standard. The calibration curve of domoic acid was linear in the range of 1.0 m/L-25.0 mg/L and the detection limit was ca. 0.2 microgram/g.

Inflammation dampened by gelatinase A cleavage of monocyte chemoattractant protein-3.

Tissue degradation by the matrix metalloproteinase gelatinase A is pivotal to inflammation and metastases. Recognizing the catalytic importance of substrate-binding exosites outside the catalytic domain, we screened for extracellular substrates using the gelatinase A hemopexin domain as bait in the yeast two-hybrid system. Monocyte chemoattractant protein-3 (MCP-3) was identified as a physiological substrate of gelatinase A. Cleaved MCP-3 binds to CC-chemokine receptors-1, -2, and -3, but no longer induces calcium fluxes or promotes chemotaxis, and instead acts as a general chemokine antagonist that dampens inflammation. This suggests that matrix metalloproteinases are both effectors and regulators of the inflammatory response.

N-terminal peptides of stromal cell-derived factor-1 with CXC chemokine receptor 4 agonist and antagonist activities.

Peptides corresponding to the N-terminal 9 residues of stromal cell-derived factor-1 (SDF-1) have SDF-1 activity. SDF-1, 1-8, 1-9, 1-9 dimer, and 1-17 induced intracellular calcium and chemotaxis in T lymphocytes and CEM cells and bound to CXC chemokine receptor 4 (CXCR4). The peptides had similar activities to SDF-1 but were less potent. Whereas native SDF-1 had half-maximal chemoattractant activity at 5 nM, the 1-9 dimer required 500 nM and was therefore 100-fold less potent. The 1-17 and a 1-9 monomer analog were 4- and 36-fold, respectively, less potent than the 1-9 dimer. Both the chemotactic and calcium response of the 1-9 dimer was inhibited by an antibody to CXCR4. The basis for the enhanced activity of the dimer form of SDF-1, 1-9 is uncertain, but it could involve an additional fortuitous binding site on the 1-9 peptide in addition to the normal SDF-1, 1-9 site. A 1-9 analog, 1-9[P2G] dimer, was found to be a CXCR4 antagonist. Overall this study shows that the N-terminal peptides are CXCR4 agonists or antagonists, and these could be leads for high affinity ligands.

An antagonist of monocyte chemoattractant protein 1 (MCP-1) inhibits arthritis in the MRL-lpr mouse model.

An antagonist of human monocyte chemoattractant protein (MCP)-1, which consists of MCP-1(9-76), had previously been characterized and shown to inhibit MCP-1 activity in vitro. To test the hypothesis that, by inhibiting endogenous MCP-1, the antagonist has antiinflammatory activity in vivo, we examined its effect in the MRL-lpr mouse model of arthritis. This strain spontaneously develops a chronic inflammatory arthritis that is similar to human rheumatoid arthritis. Daily injection of the antagonist, MCP-1(9-76), prevented the onset of arthritis as monitored by measuring joint swelling and by histopathological evaluation of the joints. In contrast, controls treated with native MCP-1 had enhanced arthritis symptoms, indicating that the inhibitory effect is specific to the antagonist. In experiments where the antagonist was given only after the disease had already developed, there was a marked reduction in symptoms and histopathology, although individuals varied in the magnitude of the response. The mechanism of inhibition of disease is not known, although the results suggest that it could be more complex than the competitive inhibition of ligand binding that is observed in vitro. The demonstration of the beneficial effects of an MCP-1 antagonist in arthritis suggests that chemokine receptor antagonists could have therapeutic application in inflammatory diseases.

Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1.

The three-dimensional structure of stromal cell-derived factor-1 (SDF-1) was determined by NMR spectroscopy. SDF-1 is a monomer with a disordered N-terminal region (residues 1-8), and differs from other chemokines in the packing of the hydrophobic core and surface charge distribution. Results with analogs showed that the N-terminal eight residues formed an important receptor binding site; however, only Lys-1 and Pro-2 were directly involved in receptor activation. Modification to Lys-1 and/or Pro-2 resulted in loss of activity, but generated potent SDF-1 antagonists. Residues 12-17 of the loop region, which we term the RFFESH motif, unlike the N-terminal region, were well defined in the SDF-1 structure. The RFFESH formed a receptor binding site, which we propose to be an important initial docking site of SDF-1 with its receptor. The ability of the SDF-1 analogs to block HIV-1 entry via CXCR4, which is a HIV-1 coreceptor for the virus in addition to being the receptor for SDF-1, correlated with their affinity for CXCR4. Activation of the receptor is not required for HIV-1 inhibition.

Immune response to recombinant mycobacterial proteins in patients with tuberculosis infection and disease.

The capacity of four Mycobacterium tuberculosis recombinant antigens to elicit proliferation and cytokine production by human T cells was evaluated. Proliferative responses of peripheral blood mononuclear cells (PBMC) to all antigens were greater in healthy tuberculin reactors than in pulmonary tuberculosis patients, and proliferative responses of pleural fluid cells were greater than those of PBMC from patients with tuberculous pleuritis. The proliferative responses to the four recombinant antigens were similar in all patient groups, and there was no selective unresponsiveness to any antigen in pulmonary tuberculosis patients. The 38-kDa antigen induced less interferon-gamma than did the 10-, 30-, and 65-kDa antigens, and all four antigens induced similar amounts of interleukin-10. These results suggest that none of the four recombinant antigens are immunodominant, and that the 10-, 30-, and 65-kDa antigens are similar in their capacity to induce a potentially protective Th1-like response.

RANTES and MCP-3 antagonists bind multiple chemokine receptors.

Antagonists of multiple chemokines could be more effective than inhibitors of specific chemokines for controlling cell migration and inflammation. To attempt to identify such antagonists we characterized a number of truncated analogs of regulated on activation normal T cell expressed protein (RANTES), monocyte chemoattractant protein (MCP)-3, and MCP-1. On the basis of their ability to compete for binding of their parent chemokines, three analogs were selected for cross-reactivity studies: RANTES (9-68), MCP-3 (10-76), and MCP-1 (9-76). These analogs bound to THP-1 monocytic cells with dissociation constants that were within 4-6-fold of their native counterparts, but they did not promote detectable chemotaxis of THP-1 cells or enzyme release from purified human monocytes. The RANTES (9-68) analog competed for the binding and inhibited the activities of all three chemokines. In contrast, native RANTES was specific for RANTES binding sites. However, truncation of either MCP-1 or MCP-3 did not change their respective binding specificity. MCP-3 and MCP-3 (10-76) competed for binding of all three labeled chemokines. MCP-1 (9-76) competed strongly for binding of labeled MCP-1, but only weakly for the other two labeled ligands and inhibited the activities induced by MCP-1 and MCP-3 but not RANTES. Although RANTES (9-68) and MCP-3 (10-76) inhibited all three chemokines, the RANTES analog was significantly more potent for RANTES-induced activity. The results indicate that NH2-terminal residues partly determine the receptor specificity of RANTES, and deletions within this region permit binding to multiple chemokine receptors. The findings suggest the feasibility of design of high affinity multi-specific CC chemokine antagonists.

Interleukin-10 downregulates Mycobacterium tuberculosis-induced Th1 responses and CTLA-4 expression.

To characterize the mechanism by which interleukin 10 (IL-10) inhibits Th1 responses to intracellular pathogens, we evaluated the interaction between IL-10 and Mycobacterium tuberculosis-induced gamma interferon (IFN-gamma) production by peripheral blood mononuclear cells from persons across the spectrum of tuberculous infection. M. tuberculosis-induced IFN-gamma production was highest in healthy tuberculin reactors, intermediate in human immunodeficiency virus (HIV)-negative tuberculosis patients, and lowest in HIV-infected tuberculosis patients. Neutralizing antibodies to IL-10 increased IFN-gamma production in HIV-infected and HIV-negative tuberculosis patients by enhancing monocyte IL-12 production. Expression of the T-cell-costimulatory molecule CTLA-4 was depressed in M. tuberculosis-stimulated peripheral blood mononuclear cells from tuberculosis patients, and anti-IL-10 and Il-12 upregulated expression of CTLA-4. These findings provide evidence that intracellular pathogens can inhibit Th1 responses and downregulate expression of specific costimulatory molecules.

Structure-activity relationships of chemokines.

Structural analysis of chemokines has revealed that the alpha/beta structural-fold is highly conserved among both the CXC and CC chemokine classes. Although dimerization and aggregation is often observed, the chemokines function as monomers. The critical receptor binding regions are in the NH2-terminal 20 residues of the protein and are the least ordered in solution. The flexible NH2-terminal region is the most critical receptor binding site and a second site also exists in the loop that follows the two disulfides. The well-ordered regions are not directly involved in receptor binding but, along with the disulfides, they provide a scaffold that determines the conformation of the sites that are critical for receptor binding. These general requirements for function are common to all the chemokines. For the CC chemokines, receptor activation and receptor binding regions are separate within the 10 residue NH2-terminal region. This has allowed identification of high affinity analogs that do not activate the receptor and are potent antagonists.

Antagonists of monocyte chemoattractant protein 1 identified by modification of functionally critical NH2-terminal residues.

Monocyte chemoattractant protein (MCP)-1 analogues were designed to determine the role of the NH2-terminal region in structure and function. The NH2-terminal residue was important for function and receptor binding, as it could not be deleted or extended. However the NH2-terminal pyroglutamate residue of the wild type was not essential as it could be replaced by several other noncyclic amino acids without loss of activity. Residues 7-10 were essential for receptor desensitization, but were not sufficient for function, and the integrity of residues 1-6 were required for functional activity. A peptide corresponding to MCP-1, 1-10 lacked detectable receptor-binding activities, indicating that residues 1-10 are essential for MCP-1 function, but that other residues are also involved. Several truncated analogues, including 8-76, 9-76, and 10-76, desensitized MCP-1-induced Ca2+ induction, but were not significantly active. These analogues were antagonists of MCP-1 activity with the most potent being the 9-76 analogue (IC50 = 20 nM) The 9-76 specifically bound to MCP-1 receptors with a Kd of 8.3 nM, which was three-fold higher than MCP-1 (Kd 2.8 nM). The 9-76 analogue desensitized the Ca2+ response to MCP-1 and MCP-3, but not to other CC chemokines, suggesting that it is MCP receptor specific. The availability of these compounds will be helpful in evaluating MCP receptor antagonists as anti-inflammatory therapeutics.