Utility of novel T-cell-specific extracellular vesicles in monitoring and evaluation of acute GVHD.

We have recently reported a new method for detecting T-cell-derived extracellular vesicles (EVs), CD3+CD4+EVs,CD3+CD8+EVs, and CD3+HLA-DR+EVs. In our previous study, CD3+HLA-DR+EVs were released profusely by CD8+T cells, only moderately by T helper1 (Th1) CD4+T cells, and very little from Th2 CD4+T cells in vitro. EVs were measured sequentially in patients undergoing hematopoietic stem cell transplantation (HSCT), and their relationship to GVHD was investigated in comparison with other conventional biomarkers. We analyzed peripheral blood samples from 20 patients (13 children and 7 adults) who underwent HSCT at Tokyo Medical and Dental University Hospital. CD3+CD4+EV and CD3+CD8+EV levels specifically correlated with the CD4+ and CD8+T lymphocyte counts, respectively. CD3+CD8+EVs and CD3+HLA-DR+EVs increased in GVHD and reflected the persistence of GVHD more specifically than soluble IL-2 receptor (sIL-2R). In engraftment syndrome, sIL-2R was markedly elevated, but CD3+HLA-DR+EVs were not. Furthermore, ferritin and sIL-2R markedly increased in hemophagocytic syndrome (HPS) that developed before engraftment; however, the change in CD3+HLA-DR+EVs was marginal. CD3+CD4+, CD3+CD8+, and CD3+HLA-DR+EVs efficiently reflect the cell-mediated immune response, and CD3+CD8+EVs and CD3+HLA-DR+EVs are more useful than other conventional biomarkers, such as sIL-2R, for monitoring and evaluation of acute GVHD.

Circulating CD3+HLA-DR+ Extracellular Vesicles as a Marker for Th1/Tc1-Type Immune Responses.

Extracellular vesicles (EVs) are known to contain unique proteins that reflect the cells of origins. Activated T cells are reported to secrete various EVs. To establish T cell subset-specific biomarkers, we performed proteomic analysis with Th1- and Th2-derived EVs and identified HLA-DR as a Th1-dominated EV membrane protein. We designed a measurement system for CD3+CD4+, CD3+CD8+, and CD3+HLA-DR+ EVs to specifically determine EV subpopulations derived from CD4+, CD8+, and Th1-type T cells, respectively. In vitro analysis showed that CD3+CD4+ EVs and CD3+CD8+ EVs were selectively secreted from activated CD4+ and CD8+ T cells, respectively, and that CD3+HLA-DR+ EVs were actively secreted from not only Th1 but also activated CD8+ T (probably mostly Tc1) cells. To evaluate the clinical usefulness of these EVs, we measured the serum levels in patients with inflammatory diseases, including Epstein-Barr virus (EBV, n = 13) infection, atopic dermatitis (AD, n = 10), rheumatoid arthritis (RA, n = 20), and osteoarthritis (OA, n = 20) and compared the levels with those of healthy adults (n = 20). CD3 + CD4 + EVs were significantly higher in all of EBV infection, AD, RA, and OA while CD3+CD8+ EVs were higher in EBV infection, lower in RA, and not different in AD and OA relative to the control. The levels of CD3+HLA-DR+ EVs were markedly higher in EBV infection and significantly lower in AD. The results suggest that these EV subpopulations reflect in vivo activation status of total CD4+, total CD8+, and Th1/Tc1-type T cells, respectively, and may be helpful in T cell-related clinical settings, such as cancer immunotherapy and treatment of chronic infection, autoimmune diseases, and graft-versus-host disease.

Reevaluation of quantitative flow cytometric analysis for TLR2 on monocytes using F(ab')2 fragments of monoclonal antibodies.

In patients with refractory infections, reliable markers that monitor the severity and healing process are needed. The expression level of toll-like receptor 2 (TLR2) on monocytes is such candidate. In the conventional assay system, the whole IgG (wIgG) form of anti-TLR2 mAb has been used with control IgG, which blocks nonantigen-specific bindings. However, the competitive reactions against Fcγ receptors (FcγRs) between labeled anti-TLR2 mAbs and control IgG should be considered. Our goal was to precisely quantify TLR2 expression level on monocytes by flow cytometry (FCM). In this study, we prepared anti-TLR2 mAbs, D45 (IgG2a), and D29 (IgG1), as well as their fragment antigen-binding [F(ab')(2) ] fragments to avoid nonantigen-specific binding to FcγRs. And then, we determined TLR2 expression levels on monocytes by using these mAbs/fragments and our calibration system using recombinant TLR2 beads. The binding of PE-labeled D45 wIgG to monocytes was completely blocked with unlabeled D45 wIgG, but not with unlabeled D45 F(ab')(2) fragment. Although the nonantigen-specific binding of D29 wIgG to nonstimulated monocytes was negligible, it was enhanced in interleukin-10-stimulated monocytes. It proved difficult to completely block nonantigen-specific binding of D45 and D29 wIgGs by treatment with control IgG. It was demonstrated that the use of fluorescent-labeled antigen-binding region lacking the fragment crystallizable portion of anti-TLR2 mAb [such as the PE-labeled F(ab')(2) fragment] is indispensible for quantification of TLR2 levels on monocytes in flow cytometry. .

High expression level of Toll-like receptor 2 on monocytes is an important risk factor for arteriosclerotic disease.

BACKGROUND: Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns to initiate an innate immune response. We previously reported upregulation of TLR2 expression level on monocytes of stable angina pectoris patients with significant coronary artery disease (CAD) relative to control patients without significant CAD. In this study we aimed to determine whether high level of Toll-like receptor 2 (TLR2) is a risk factor for atherogenesis, independent of established risk factors including smoking, diabetes mellitus (DM), hypertension (HT), and hyperlipidemia (HL). METHODS: TLR2 expression level on circulating monocyte surfaces was measured by using our developed flow cytometry assay. Patients were classified into two groups: "Arteriosclerotic disease" group (n=108) and "Control" group (n=70). Patients of the first group had arteriosclerotic disease such as CAD, aortic aneurysm, or peripheral arterial disease (PAD). The "Control" group was sex- and age-matched to the "Arteriosclerotic disease" group. RESULTS: TLR2 expression was significantly higher in the "Arteriosclerotic disease" group than in the "Control" group (p<0.001). Multivariate ordinal logistic regression analysis was performed; other known risk factors, which were represented to two nominal score points, 0 or 1, for patients with and without it, respectively, and TLR2 level, which was treated as a metric variable. DM (p=0.002), HT (p=0.001), HL (p<0.001), and TLR2 level (p<0.001) were identified as significant contributors for arteriosclerotic disease. CONCLUSIONS: High TLR2 expression level on monocytes may be an independent risk factor for atherogenesis.

Abeta42-to-Abeta40- and angiotensin-converting activities in different domains of angiotensin-converting enzyme.

Amyloid beta-protein 1-42 (Abeta42) is believed to play a causative role in the development of Alzheimer disease (AD), although it is a minor part of Abeta. In contrast, Abeta40 is the predominant secreted form of Abeta and recent studies have suggested that Abeta40 has neuroprotective effects and inhibits amyloid deposition. We have reported that angiotensin-converting enzyme (ACE) converts Abeta42 to Abeta40, and its inhibition enhances brain Abeta42 deposition (Zou, K., Yamaguchi, H., Akatsu, H., Sakamoto, T., Ko, M., Mizoguchi, K., Gong, J. S., Yu, W., Yamamoto, T., Kosaka, K., Yanagisawa, K., and Michikawa, M. (2007) J. Neurosci. 27, 8628-8635). ACE has two homologous domains, each having a functional active site. In the present study, we identified the domain of ACE, which is responsible for converting Abeta42 to Abeta40. Interestingly, Abeta42-to-Abeta40-converting activity is solely found in the N-domain of ACE and the angiotensin-converting activity is found predominantly in the C-domain of ACE. We also found that the N-linked glycosylation is essential for both Abeta42-to-Abeta40- and angiotensin-converting activities and that unglycosylated ACE rapidly degraded. The domain-specific converting activity of ACE suggests that ACE inhibitors could be designed to specifically target the angiotensin-converting C-domain, without inhibiting the Abeta42-to-Abeta40-converting activity of ACE or increasing neurotoxic Abeta42.

The role of infection in the development of non-valvular atrial fibrillation: up-regulation of Toll-like receptor 2 expression levels on monocytes.

Many studies have suggested that inflammation may participate in the pathogenesis of non-valvular atrial fibrillation (AF). However, it has been unknown by exposure to what the inflammation is caused. Recently, we reported that Toll-like receptor 2 (TLR2) level on monocytes was significantly up-regulated in viral and bacterial infections, but not in non-infectious inflammatory states. Our purpose was to test the hypothesis that expression of TLR2 levels may be up-regulated in patients with non-valvular AF. A total of 48 consecutive patients with non-valvular AF who were hospitalized for catheter ablation were enrolled in this study. TLR2 levels were assayed by using flow-cytometric analysis and compared with volunteers in sinus rhythm (control group, n = 24). Additionally, C-reactive protein (CRP) and interleukin-6 (IL-6) levels were assayed, and the left atrial volume indexes (LAVI) in the non-valvular AF group were measured. The results demonstrated that TLR2 levels in the non-valvular AF group were significantly higher than in the control group (median, 4682 vs. 3866 sites/cell; P < 0.01). Moreover, non-valvular AF patients had significantly higher IL-6 levels than controls. However, there was no significant difference in CRP levels between the two groups. It was observed in 44 AF patients, in whom pulmonary vein isolation was confirmed to be successful, that the LAVI significantly diminished 1 month after ablation (median, 33.6 vs. 29.5 ml/m²; P < 0.001), but not the TLR2 and IL-6 levels. Our results implied that an infectious inflammation may participate in the pathogenesis of non-valvular AF.

Toll-like receptor 2 expression level on monocytes in patients with viral infections: monitoring infection severity.

For viral infectious diseases, reliable biomarkers capable of monitoring recovery and therapeutic effects and that simultaneously discriminate between viral and bacterial infection are necessary. In this study, by using flow-cytometric quantification system, Toll-like receptor 2 (TLR2) expression levels on monocytes of influenza patients (n=47) were compared with those of healthy volunteers (n=50). Subsequently, throughout their acute, convalescent and healed phases, TLR2, C-reactive protein (CRP), serum amyroid A (SAA), and neopterin levels were followed. Additionally, TLR2 levels in other viral infectious diseases were assayed. The results showed that TLR2 level in influenza patients was remarkably up-regulated in acute phase compared to healthy volunteers (p<0.001). Thereafter, TLR2 levels normalized in good accordance with their recovery processes. CRP and neopterin levels were relatively widely distributed from normal to abnormally high levels in acute phase in spite of similar disease severity among the patients. SAA levels did not necessarily reflect the patients' clinical course during their recovery. Clinical observations of other viral infections also indicated that TLR2 levels were compatible with infection severity. TLR2 expression level on monocytes might serve as a unique biomarker useful in viral infectious diseases.

The N-terminal active centre of human angiotensin-converting enzyme degrades Alzheimer amyloid beta-peptide.

We reported recently that angiotensin-converting enzyme (ACE) significantly degraded amyloid beta-peptide (A beta) to inhibit aggregation and cytotoxicity of A beta in PC12h cells in vitro. On the other hand, others reported that ACE had two domains with highly homologous active centres, the N-domain and C-domain, but that they differed in their characteristics such as optimum chloride ion concentration, inhibition kinetics for various ACE inhibitors and rate of hydrolysis for many substrates. The aim of this study was to determine the specific ACE domain primarily responsible for degradation of A beta. For this purpose, a series of ACE recombinant proteins, each containing only one intact domain, was constructed and expressed in COS7. Our results showed that all ACE recombinant proteins obtained were enzymatically active in terms of angiotensin I cleavage. However, inhibition of A beta aggregation and cytotoxicity of the N-domain were higher than those of the C-domain. Reverse-phase high-performance liquid chromatography analyses confirmed that the N domain degraded A beta. Our results indicate that the N domain of ACE is primarily responsible for the degradation of A beta.