Development of highly stable galectins: truncation of the linker peptide confers protease-resistance on tandem-repeat type galectins.

Galectin-9 and galectin-8, members of beta-galactoside-binding animal lectin family, are promising agents for the treatment of immune-related and neoplastic diseases. The proteins consist of two carbohydrate recognition domains joined by a linker peptide, which is highly susceptible to proteolysis. To increase protease resistance, we prepared mutant proteins by serial truncation of the linker peptide. As a result, mutant forms lacking the entire linker peptide were found to be highly stable against proteolysis and retained their biological activities. These mutant proteins might be useful tools for analyzing the biological functions and evaluating the therapeutic potential of galectin-9 and galectin-8.

Differences in systemic and central nervous system cellular immunity relevant to relapsing-remitting multiple sclerosis.

In order to elucidate the differences between systemic and central nervous system (CNS) immunity that are relevant to exacerbations of multiple sclerosis (MS), paired peripheral blood and cerebrospinal fluid (CSF) samples obtained from 36 non-treated patients with relapsing-remitting MS (RRMS) were simultaneously examined using flow cytometry to determine the percentages of functional lymphocyte subsets, as well as enzyme-linked immunosorbent assays for measuring soluble immune mediators. Active RRMS patients (n = 27) were characterized by an increase in CD4+ CXCR3+ Th1 cells in blood as compared with inactive patients (n = 9), and this parameter was inversely correlated with plasma levels of IL-10 and IL- 12p70. In contrast, an increase in the percentage of CD4+ CD25+ cells and a decrease in the percentage of CD8+ CD11a(high) cells were features of CSF samples from those with active RRMS. Further, CSF CD4+ CD25+ cells had a close association with leukocyte counts as well as albumin and CXCL10 levels in the CSF, and, thus, could be useful as a measure for inflammatory reactions in the CNS. On the other hand, CD8+ CD11a(high) cells may function as immunoregulatory cells, as their percentage in the CSF showed a positive correlation with CSF levels of the anti-inflammatory cytokine IL-4. These findings suggest that MS relapses occur in a combination with altered cell-mediated immunity that differs between the peripheral blood and CSF compartments, while measurement of lymphocyte subsets may be helpful for monitoring disease status.

Chemokine receptors associated with immunity within and outside the central nervous system in early relapsing-remitting multiple sclerosis.

Thirty-four patients with early relapsing-remitting multiple sclerosis (RRMS) were studied to clarify the differences in chemokine receptor usage by blood and cerebrospinal fluid (CSF) lymphocytes relevant to the pathogenesis of MS. A total of 45 examinations (33 active and 12 inactive stages) revealed that circulating CD4+CXCR3+ T helper 1 (Th1) cells were increased in active MS patients and correlated with the number of gadolinium-enhanced lesions on magnetic resonance (MR) images. In contrast, CSF samples obtained during active stages were characterized by a decrease in the percentage of CD8+CXCR3+ T cells, which was inversely correlated with CSF cell count and intra-blood-brain barrier (BBB) IgG production.

Immune parameters associated with early treatment effects of high-dose intravenous methylprednisolone in multiple sclerosis.

To determine the immunological effects of high-dose intravenous methylprednisolone (IVMP) and elucidate immune measurements used for evaluation of its therapeutic effect, we analyzed lymphocyte subsets and humoral immune parameters in peripheral blood and cerebrospinal fluid (CSF) samples, before and within 2 weeks of treatment during 19 acute exacerbations in 16 relapsing-remitting multiple sclerosis (MS) patients. In addition to decreases in CSF albumin and IgG levels, treatment resulted in an increase of CD8(+)CXCR3(+) cells as well as a decrease in CD4(+) subsets expressing CD25, CD29, and CCR4 in the CSF. Further, the percentage of circulating CD4(+)CXCR3(+) Th1 cells also decreased. Clinical improvement was achieved following 15 of the 19 treatment occasions. Early (<2 weeks of treatment) clinical improvement was significantly associated with a decrease in CSF CD4(+)CD29(+) helper inducer T cells, whereas they were nearly unchanged in four patients who showed no improvement. Changes in other parameters following IVMP treatment were not different between the responder and non-responder groups.

Relevance of distinct monocyte subsets to clinical course of ischemic stroke patients.

BACKGROUND AND PURPOSE: The most common strategy for treating patients with acute ischemic stroke is thrombolytic therapy, though only a few patients receive benefits because of the narrow time window. Inflammation occurring in the central nervous system (CNS) in association with ischemia is caused by immune cells including monocytes and involved in lesion expansion. If the specific roles of monocyte subsets in stroke can be revealed, they may become an effective target for new treatment strategies. METHODS: We performed immunological examinations of 36 consecutive ischemic stroke patients within 2 days of onset and compared the results with 24 age-matched patients with degenerative disorders. The stroke patients were repeatedly tested for the proportions of monocyte subsets in blood, and serum levels of pro- and anti-inflammatory cytokines immediately after admission, on days 3-7 and 12-16 after stroke onset, and on the day of discharge. In addition, immunological measurements were analyzed for relationships to stroke subtypes and complications, including progressive infarction (PI) and stroke-associated infection (SAI). RESULTS: Monocyte count was significantly increased from 0-16 days after stroke as compared to the controls (p<0.05). CD14(high)CD16(-) classical and CD14(high)CD16(+) intermediate monocytes were significantly increased from 0-7 and 3-16 days after stroke, respectively (p<0.05), whereas CD14 (dim)CD16(high) non-classical monocytes were decreased from 0-7 days (p<0.05). Cardioembolic infarction was associated with a persistent increase in intermediate monocytes. Furthermore, intermediate monocytes were significantly increased in patients with PI (p<0.05), while non-classical monocytes were decreased in those with SAI (p<0.05). IL-17A levels were positively correlated with monocyte count (r=0.485, p=0.012) as well as the percentage of non-classical monocytes (r=0.423, p=0.028), and negatively with that of classical monocytes (r=-0.51, p=0.007) during days 12-16. CONCLUSIONS: Our findings suggest that CD14(high)CD16(+) intermediate monocytes have a role in CNS tissue damage during acute and subacute phases in ischemic stroke especially in relation to cardioembolism.