Katsuya Hiraishi

Studies on the mechanisms of leukocyte adhesion to cellulose acetate beads: an in vitro model to assess the efficacy of cellulose acetate carrier-based granulocyte and monocyte adsorptive apheresis.

Abstract: Granulocyte and monocyte adsorptive apheresis (GMA) using a column filled with cellulose acetate (CA) beads (carriers) has been associated with a significant clinical efficacy in patients with rheumatoid arthritis and ulcerative colitis. To obtain further understanding on the mechanisms of disease modification by cellulose acetate‐carrier‐based GMA, in the present study, we investigated the mechanisms of granulocyte and monocyte adhesion to CA beads following exposure of human peripheral blood to the carriers at 37°C for up to 60 min under controlled conditions. Cellulose acetate beads selectively adsorbed granulocytes, monocytes, CD19+ (B cells) and CD56+ (NK cells) lymphocyte subpopulations. The granulocyte and monocyte adsorption was inhibited by heat‐inactivated plasma and EDTA, indicating that the adsorption was plasma protein (immunoglobulin, complement) and calcium dependent. Accordingly, granulocyte and monocyte adsorption was markedly enhanced by coating the carriers with IgG. Similarly, C3b was adsorbed onto the CA beads as a marker of complement activation. The results indicated that IgG and active complement fragments mediated leukocyte adhesion to CA beads via the FcγR and/or leukocyte complement receptor like CR3. Additionally, CA beads induced loss of expression of TNF receptors on CD16+ granulocytes and CD14+ monocytes, but not on CD3+ lymphocytes. In conclusion, CA beads might be an appropriate biomaterial for inducing extracorporeal immunomodulation as a treatment for auto‐immune diseases which are associated with pathological leukocyte activity.

The first observation of O2− generation at real time in vivo from non-Kupffer sinusoidal cells in perfused rat liver during acute ethanol intoxication

Infusion of ethanol or phorbol myristate acetate (PMA) into the perfused rat liver immediately produces O2 − which was detected directly by infusion of a Cypridina luciferin analogue, MCLA as a chemiluminescence reagent. The MCLA photon emission was inhibitable by SOD. Generation of O2 − in the liver was further verified by nitroblue tetrazolium, formazan precipitate formation. Ethanol‐induced O2 − generation was unaffected by gadolinium chloride (GdCl3), an inhibitor of kupffer cells, while PMA induced O2 − generation was completely abolished by GdCl3, Since PMA is a known stimulator of phagocytic cells including Kupffer cells, the results indicate, for the first time that ethanol stimulates a non‐Kupffer cell population, probably liver sinusoid endothelial cell to produce O2 −

Granulocyte and Monocyte Adsorption Apheresis (GCAP) for Refractory Skin Diseases Caused by Activated Neutrophils and Psoriatic Arthritis: Evidence that GCAP Removes Mac-1-Expressing Neutrophils

Abstract:  In the present study, we have shown that granulocyte and monocyte adsorption apheresis (GCAP), an extracorporeal apheresis instrument whose column contains cellulose acetate (CA) beads, is useful for skin diseases attributable to activated granulocytes and psoriatic arthritis (PsA). We assessed the clinical effectiveness of GCAP and investigated the mechanisms underlying the adsorption of pathogenic granulocytes. The effect of GCAP was assessed in 14 patients with neutrophilic dermatoses and 16 with PsA. The mechanisms by which the instrument adsorbs activated granulocytes were investigated using an in vitro mini‐column system that mimics the GCAP. Skin lesions and arthropathy improved in 22 of 29 patients (75.9%) and 14 of 18 (77.8%), respectively. Mac‐1 (CD11b/CD18) expression on the peripheral neutrophils, increased compared with normal subjects, was reduced by GCAP. In the mini‐column system, CA beads adsorbed 50% neutrophils; and adsorption was inhibited significantly by treating plasma with EDTA and blood cells with antihuman CD11b monoclonal antibody. GCAP was useful for treating neutrophilic dermatoses and PsA. GCAP adsorbs Mac‐1‐expressing neutrophils to the CA beads by the binding of complement component (iC3b) on CA beads and CD11b expressed on activated neutrophils.

Clinical response is associated with elevated plasma interleukin-1 receptor antagonist during selective granulocyte and monocyte apheresis in patients with ulcerative colitis

Depletion of granulocytes and monocytes (GM) by selective apheresis (GMA) with an Adacolumn exerts an anti-inflammatory effect in patients with ulcerative colitis (UC) or rheumatoid arthritis. However, the mechanism of the anti-inflammatory effect of GMA is not fully understood yet. We investigated the effect of GMA on the plasma concentration of interleukin-1 receptor antagonist (IL-1ra), a potent anti-inflammatory cytokine. Twenty-six patients with active UC received GMA at one session per week for 5 consecutive weeks. Clinical response was defined as Δclinical activity index (ΔCAI=CAI at entry – CAI at post)≥4, while clinical remission was defined as CAI≤4. Twenty-one of twenty-six patients (80.8%) responded to GMA. In the first session, plasma from responder patients showed a significant (P < 0.01) increase in IL-1ra in the Adacolumn outflow. In contrast, there was no change in IL-1ra in nonresponders. In conclusion, release of IL-1ra during GMA might be one mechanism of clinical efficacy associated with this therapy.

The granulocyte/lymphocyte ratio as an independent predictor of tumour growth, metastasis and progression : its clinical applications

Several investigators have suggested that the granulocyte/lymphocyte (G/L) ratio is a good indicator for the evaluation of the condition of a tumour-bearing host, although its prognotic significance is unclear. To further investigate the clinical applications of the G/L ratio, we injected 1x105 and 1x106 Lewis lung carcinoma cells (3LLc) into the feet of 4-week-old C57BL/6 mice separated into groups A, B, C and D (1x105 cells) and E, F, G and H (1x106 cells). For the observation of tumour metastasis and G/L ratio, the mice in groups A-D were sacrificed on days 11, 14, 17 and 21 after inoculation with the 3LLc cells, and the mice in groups E-H on days 7, 11, 14 and 17. The results suggest that in mice the number of granulocytes increases with time after 3LLc cell injection (P<0.05). We also retrospectively investigated the correlation between G/L ratio, clinicopathologic features and prognosis in 62 patients with gastric carcinoma. There was a significant correlation between the G/L ratio and tumour weight (r=0.746, P<0.05), as well as a significant difference between the G/L ratio and the extent of metastases (P<0.05). Additionally, the G/L ratio was significantly associated with lymph node metastasis and higher tumour stage, tumour progression (P=0.017) and 5-year survival (P=0.013). In conclusion, the G/L ratio is associated with tumour progression and shorter survival. The close correlation between G/L ratio and tumour stage or lymph node status suggests that it could be used to predict tumour metastasis, prognosis and overall survival in patients with gastric carcinoma before they undergo surgical treatment.

MK615, A Compound Extract from the Japanese Apricot "Prunus mume" Inhibits In vitro Cell Growth and Interleukin-8 Expression in Non-small Cell Lung Cancer Cells

The Japanese apricot “Prunus mume,” which is also known as the Ume fruit in Japan, is a centuries-old traditional Japanese medicine, and it is a commonly consumed food. MK615, a compound extract from Ume fruits, has been shown to have anti-tumor and anti-inflammatory effects. In this study, we assessed the effects of MK615 on the in vitro growth of nine non-small cell lung cancer (NSCLC) cell lines and the HBEC4 immortalized bronchial epithelial cell line. While MK615 inhibited the in vitro cell growth of the majority of the NSCLC cell lines, the growthinhibitory effects varied among the cell lines, and some cell lines exhibited MK615 resistance. In the H1299 and H157 NSCLC cell lines that are highly sensitive to MK615, the induction of autophagy was observed after MK615 treatment. In addition, cell-cycle analysis showed that MK615 increased the proportion of cells in the G0-G1 phase in H1299 and H157 cells. In H1792 cells that overexpress IL-8, MK615 down-regulated IL-8 expression at the mRNA and protein levels in a dose-dependent manner. These results suggest that MK615 has multiple anti-tumor activities including the inhibition of cell proliferation, autophagy induction, G0-G1 cell cycle arrest and the downregulation of IL-8 expression in NSCLC cells.

Pathogenesis of Focal Cytoplasmic Necrosis of the Smooth Muscle Cells in Hypertensive Rat Arterial Media

Hypertensive rat arteries exhibited severe medial smooth muscle cell injury and necrosis. Electron microscopic observations showed the smooth muscle cells of these arteries exhibited characteristics of focal cytoplasmic necrosis forming new cytodemarcating membrane between the healthy cytoplasm and necrotic cytoplasm. When the focal necrotic cytoplasm disappeared from the injured smooth muscle cells, it left it with a moth-eaten leaf-like appearance (moth-eaten necrosis). At an advanced stage of injury, smooth muscle cells changed to islet-like cell bodies with newly formed basement membranes around them, and further islet-like cell bodies and cell debris disappeared leaving lamellar and reticular basement membranes. In hypertensive rats injected with nitroblue tetrazolium (NBT), formazan deposits were observed in the medial cells and nitrotyrosine, a biomarker of peroxynitrite, were immunohistochemically observed in the arterial media. Nick-end positive extranuclear small granular bodies, which might have derived from focal necrotic cytoplasm and nucleus, were detected in the arterial media using DNA nick-end labeling method. Based on electron microscopical and histochemical findings, we conjectured that the focal cytoplasmic necrosis of the smooth muscle cells in the arterial media depended on injury arising from mitochondria-derived oxidants.