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.

Selectively induced apoptosis in human neutrophils in the presence of oxidative phenoxazines, 2-amino-4,4α-dihydryo-4α-7H-phenoxazine-3-one and 2-aminophenoxazine-3-one, preceded by decrease of intracellular pH, depolarization of the mitochondria, and inhibition of superoxide generation.

The present research investigated the effect of the oxidative phenoxazines, 2-amino-4,4α-dihydryo-4α-7H-phenoxazine-3-one (Phx-1) and 2-amino-phenoxazine-3-one (Phx-3) on apoptosis induction and apoptosis-related early events in human neutrophils. When Phx-1 or Phx-3 was administered to freshly drawn human blood for 18 h, these phenoxazines caused apoptotic cell death morphologically characterized by condensation of the nucleus in neutrophils, without causing it in lymphocytes and monocytes. Apoptosis, which was detectable by microscopic analysis and by using flow-cytometry, occurred significantly in human neutrophils isolated from freshly drawn blood, 6 h after the administration of 50 µM Phx-1 and Phx-3. After 24 h, every isolated neutrophil treated with Phx-1 or Phx-3 fell into apoptosis or lost its morphology, while many of the neutrophils without these phenoxazines remained alive, with normal morphology. Apoptosis-related early events including a decrease in intracellular pH (pHi) and depolarization of the mitochondria occurred in the isolated neutrophils, 30 min and 6 h after the administration of Phx-1 or Phx-3, respectively. Superoxide generation from the isolated neutrophils mimicked by phorbol myristate acetate (PMA) was very markedly inhibited by 100 µM Phx-1 or Phx-3. This result could be explained, in part, by the fact that the insufficient supply of NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) was caused by pHi decrease in neutrophils treated with Phx-1 or Phx, because NADPH is necessary for NADPH oxidase responsible for generating superoxide in the cells. The present results suggest that Phx-1 and Phx-3 have the capacity of selectively inducing apoptosis in human neutrophils and that these phenoxazines may be useful as specific drugs to induce apoptotic cell death of human neutrophils and thereby prevent inflammation caused by these phagocytic cells.

MK615 decreases RAGE expression and inhibits TAGE-induced proliferation in hepatocellular carcinoma cells.

AIM: To investigate the proliferative effect of advanced glycation end-products (AGEs) and the role of their cellular receptor (RAGE) on hepatocellular carcinoma (HCC) cells, and the inhibitory effects of MK615, an extract from Japanese apricot, against AGEs were also evaluated. METHODS: Two HCC cell lines, HuH7 and HepG2, were used. Expression of RAGE was investigated by polymerase chain reaction, Western blotting, and flow cytometry (FACS). The effect of MK615 on RAGE expression was also evaluated by FACS. The proliferative effects of a control (unglycated bovine serum albumin), glucose-derived AGEs (Glc-AGE), and glyceraldehyde-derived AGEs (Glycer-AGE), and the anti-proliferative effect of MK615 against AGEs, were evaluated using MTT assays. RESULTS: Expression of RAGE was confirmed at both the mRNA and protein levels in both HuH7 and HepG2. FACS revealed that the level of RAGE expression was higher in HuH7 than in HepG2. Treatment with 0.1 µg/mL MK615 decreased the expression level of RAGE from 24.3% to 3.7% in HuH7 and from 6.2% to 4.8% in HepG2. The growth indices for the control, Glc-AGE, and Glycer-AGE were 1.06 ± 0.08, 0.99 ± 0.04, and 1.38 ± 0.05, respectively, in HuH7 (P = 0.037), and were 1.03 ± 0.04, 1.04 ± 0.03, and 1.07 ± 0.05, respectively, in HepG2 (P > 0.05). When the cells were cultured simultaneously with Glycer-AGE and MK615, MK615 abrogated the proliferative effect of Glycer-AGE in HuH7. CONCLUSION: Only Glycer-AGE has a proliferative effect on HuH7, which expresses a higher level of RAGE. MK615 suppresses the proliferative effect of Glycer-AGE on HuH7 by decreasing the expression of RAGE.

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.

Assessment of rabbit spleen size using ultrasonography.

Assessment of spleen size using the ultrasonography has become a standard practice in human. However, the assessment is not established method in experimental animals. To establish the index to assess the spleen size using ultrasonography, we measured the cross-section image of rabbit spleen during endotoxin shock. The image of the cross-section was appeared as triangle, and the height of the triangular image was defined as the spleen index. This spleen index showed strong correlation with the spleen weight. In conclusion, this method is suitable for observation of changes in rabbit spleen size and may reduce the number of rabbit in the longitudinal studies.

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 Deltaclinical activity index (DeltaCAI=CAI at entry - CAI at post)>or=4, while clinical remission was defined as CAI

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.

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.

AIDS vaccine: Intranasal immunization using inactivated HIV-1-capturing core-corona type polymeric nanospheres.

Polymeric nanospheres have been widely used in biomedical applications, such as drug, gene and vaccine delivery systems. Nanospheres with entrapped antigens have recently been shown to possess significant potential as vaccine delivery systems and adjuvants. We previously reported that concanavalin A-immobilized polystyrene nanospheres (Con A-NS) could efficiently capture HIV-1 particles and intranasal immunization with inactivated HIV-1-capturing nanospheres (HIV-NS) induced vaginal anti-HIV-1 IgA antibody responses in mice. In addition, vaginal washes from intranasally immunized mice were capable of neutralizing HIV-1. Moreover, simian/human immunodeficiency virus KU-2-capturing nanospheres (SHIV-NS) immunized macaques exhibited partial protection when vaginally and systemically challenged with pathogenic viruses. HIV-NS is suggested to be particularly suitable to enhance antigen delivery to dendritic cells (DCs). In this study, we investigated the mucosal antibody response in mice after the intravaginal or intranasal immunization in detail with using different sized (360, 660, 940 and 1230 nm) HIV-NS. The amount of immobilized Con A to NS was dependent on the surface area of the particle. Moreover, Con A-NS with different sizes could equally capture inactivated HIV-1. Intravaginal or intranasal immunization by HIV-NS with diameters ranging 360 to 1230 nm significantly induced vaginal antibody responses. However, significant differences on vaginal anti-HIV-1 gp120 IgA and IgG antibodies were not found after intravaginal or intranasal immunization with different sized HIV-NS. These results suggest that HIV-NS provides an efficient vaccine delivery system for the induction of a mucosal immune response and the development of a mucosal vaccine.

Removal of hepatitis C virus by G-1 beads in sera from patients with chronic hepatitis C.

UNLABELLED: Recently, a new method of extracorporeal granulocyte depletion apheresis has been developed to treat inflammatory systemic diseases using an Adacolumn (Japan Immunoresearch Laboratories, Takasaki, Japan) that is filled with acetate cellulose beads (G-1 beads) to adsorb the granulocytes. We examined whether hepatitis C virus (HCV) is adsorbed after incubation of the Adacolumn with the sera from patients with HCV-RNA-positive chronic hepatitis C. PATIENTS AND METHODS: A total of 10 patients with chronic hepatitis C, whose levels of HCV RNA were greater than 800 kIU/ml were examined. The serum was incubated with 500 G-1 beads in a syringe at 37 degrees C for 1 h. After removal of the serum, the beads were washed with RNase-free water. The G-1 beads were removed from the syringe after centrifugation. RNA was extracted from 200 microl of the wash waste and from 10, 50, 100 and 200 beads, respectively, using TRIZol regent. Detection of HCV RNA was performed using the nested PCR method. RESULTS: HCV RNA was detected from as few as 10 G-1 beads. HCV RNA was not detected from waste fluid collected after the last wash from any of the patients. Further, HCV RNA was detected in the initial waste fluid after the 37 degrees C incubation with serum in all of the patients. Since HCV RNA was detected on the G-1 beads, but not from the last washing solution in the current examination, these results suggest that the G-1 beads adsorbed HCV RNA. CONCLUSIONS: Our in vitro study confirmed that G-1 beads adsorbed HCV; therefore, apheresis using a column filled with G-1 beads may reduce the HCV RNA load in the blood of patients with chronic hepatitis C.

Induction of dendritic cell-mediated immune responses against HIV-1 by antigen-capturing nanospheres in mice.

Prophylactic vaccines, designed to elicit potent humoral and cellular immune responses to human immunodeficiency virus type 1 (HIV-1) antigens in mucosa, are the important approach to the protection of individuals against HIV-1 infection, since HIV-1 transmission is largely a result of sexual contact. In this study, a novel strategy has been developed to induce HIV-1-specific immune responses, which involves inactivated HIV-1-caputring concanavalin A (Con A)-immobilized nanospheres (HIV-NS) and their interaction with bone marrow (BM)-derived dendritic cells. HIV-NS were taken up by dendritic cells via cytoskeleton-dependent but mannose-binding site-independent phagocytosis. Serial stimulations to unprimed T-cells with HIV-1 gp120-capturing NS-pulsed dendritic cells could induce antigen-specific T-cell response. Intranasal administration of fluorescein isothiocyanate-labeled nanospheres (NS) in mice proved that the particles were taken up into pulmonary dendritic cells. Analysis of mice receiving intranasal immunizations with HIV-NS revealed that the mice efficiently induced the antibodies against HIV-1 in the genital tract and specific cytotoxic T-cells in the spleen. These results suggest that the use of HIV-1-NS may provide a novel and promising approach for the induction of humoral and cellular immune responses to HIV-1.

Induction of HIV-specific antibody response and protection against vaginal SHIV transmission by intranasal immunization with inactivated SHIV-capturing nanospheres in macaques.

We have previously reported that concanavalin A-immobilized polystyrene nanospheres (Con A-NS) could efficiently capture HIV-1 particles and that intranasal immunization with inactivated HIV-1-capturing nanospheres (HIV-NS) induced vaginal anti-HIV-1 IgA antibody response in mice. In this study, to evaluate the protective effect of immunization, each three macaques was intranasally immunized with Con A-NS or inactivated simian/human immunodeficiency virus KU-2-capturing nanospheres (SHIV-NS) and then intravaginally challenged with a pathogenic virus, SHIV KU-2. After a series of six immunizations, vaginal anti-HIV-1 gp120 IgA and IgG antibodies were detected in all SHIV-NS-immunized macaques. After intravaginal challenge, one of the three macaques in each of the Con A-NS- and SHIV-NS-immunized groups was infected. Plasma viral RNA load of infected macaque in SHIV-NS-immunized macaques was substantially less than that in unimmunized control macaque and reached below the detectable level. However, it could not be determined whether intranasal immunization with SHIV-NS is effective in giving complete protection against intravaginal challenge. To explore the effect of the SHIV-NS vaccine, the remaining non-infected macaques were rechallenged intravenously with SHIV KU-2. After intravenous challenge, all macaques became infected. However, SHIV-NS-immunized macaques had lower viral RNA loads and higher CD4(+) T cell counts than unimmunized control macaques. Plasma anti-HIV-1 gp120 IgA and IgG antibodies were induced more rapidly in the SHIV-NS-immunized macaques than in the controls. The rapid antibody responses having neutralizing activity might contribute to the clearance of the challenge virus. Thus, SHIV-NS-immunized macaques exhibited partial protection to vaginal and systemic challenges with SHIV KU-2.

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.

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 degrees 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 FcgammaR 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.

Mucosal immunization with inactivated HIV-1-capturing nanospheres induces a significant HIV-1-specific vaginal antibody response in mice.

Mucosal secretory IgA is considered to have an important role in the prevention of human immunodeficiency virus type 1 (HIV-1) transmission through sexual intercourse. Therefore, substances that induce HIV-1-specific IgA antibody in the genital tract may become promising candidates for prophylactic vaccine against HIV-1 infection. We have previously reported that concanavalin A-immobilized polystyrene nanospheres (Con A-NS) could efficiently capture HIV-1 particles and gp120 antigens on their surface and that intravaginal immunization with inactivated HIV-1-capturing nanospheres (HIV-NS) induced vaginal anti-HIV-1 IgA antibody in mice. In this study, various strategies for immunization with HIV-NS were undertaken to induce HIV-1-specific IgA response in the mouse genital tract. HIV-NS were administered intravaginally, orally, intranasally or intraperitoneally to mice. Progesterone treatment enhanced the anti-HIV-1 IgA response to intravaginal immunization significantly, but intranasal immunization with HIV-NS was more effective compared with other immunization routes in terms of vaginal IgA response. In addition, vaginal washes from intranasally immunized mice were capable of neutralizing HIV-1(IIIB). Thus, application of HIV-NS is a practical approach to promote HIV-1-specific IgA response by the vaginal mucosa in the mouse and intranasal appears to be an effective immunization route in this animal model. Intranasal immunization with HIV-NS should be further pursued for its potential as an HIV-1 prophylactic vaccine.

Induction of mucosal IgA following intravaginal administration of inactivated HIV-1-capturing nanospheres in mice.

Concanavalin A-immobilized polystyrene nanospheres (Con A-NS) were developed for the HIV-1 vaccine capable of preventing sexual transmission. Con A-NS could capture efficiently HIV-1 irrespective of their cell tropism (R5 or X4). Furthermore, Con A-NS captured equally infectious and heat-inactivated HIV-1. Inactivated HIV-1-capturing Con A-NS (HIV-NS) were intravaginally administered to mice. Heat-inactivated HIV-1 alone and Con A-NS alone were also administered as control immunogens. Vaginal fluids were collected during and after immunization and analyzed for their anti-HIV-1 antibody levels. Although the anti-HIV-1 IgG antibody was undetectable in any groups, increased anti-HIV-1 IgA antibody response was identified in the vaginal fluids of immunized mice with HIV-NS. The vaginal fluids obtained from the HIV-NS-administered mice showed neutralizing activity against the immunizing HIV-1 strain. A marked difference in vaginal distribution was observed between HIV-NS and other immunogens, and the toxicity of Con A was reduced by conjugation with nanospheres. Thus, HIV-NS may have great potential as a prophylactic HIV-1 vaccine and should be examined further for its efficacy in non-human primates.