Byrsonima fagifolia Niedenzu Apolar Compounds with Antitubercular Activity.

Bioassay-guided fractionation of the chloroform extract of Byrsonima fagifolia leaves led to the isolation of active antitubercular compounds alkane dotriacontane (Minimal Inhibitory Concentration-MIC, 62.5 µg mL(-1)), triterpenoids as bassic acid (MIC = 2.5 µg mL(-1)), α-amyrin acetate (MIC = 62.5 µg mL(-1)), a mixture of lupeol, α- and ß-amyrin (MIC = 31.5 µg mL(-1)) and a mixture of lupeol, and acetates of α- and ß-amyrin (MIC = 31.5 µg mL(-1)). The antimycobacterial activity was determined by the Microplate Alamar Blue Assay (MABA) and the structures of promising compounds were determined by spectroscopic analysis. This investigation constitutes the first report of a chemical and antitubercular study of apolar compounds from B. fagifolia Niedenzu (IK).

Plasma LH, ovulation and conception rates in cats mated once or three times on different days of oestrus.

Although cats are induced ovulators, the relationship between the day of breeding, the number of matings and the likelihood of ovulation and conception have not been extensively investigated. In this experiment, cats were mated either once or three times on day 1 or day 5 of oestrus to study the incidence of the LH surge, ovulation and conception rates. The percentage ovulating and the conception rates after a single mating on day 1 of oestrus were 60% (6/10) and 33.3% (2/6), respectively, and for cats mated once on day 5 of oestrus were 83.3% (10/12) and 40% (4/10), respectively. When cats were mated three times on day 1 of oestrus, the ovulation rates and conception rates were 70% (7/10) and 85.7% (6/7), respectively, and for those mated three times on day 5 of oestrus were 100% (10/10) and 100% (10/10), respectively. The concentration of LH did not increase in non-ovulating cats, and cats that were mated three times had LH concentrations that were numerically higher than those that were mated once. Litter size was neither related to the day of mating nor to the number of matings. Although an increase in the number of matings on day 1 of oestrus produced a numerically larger LH surge, it did not increase the ovulation rate, suggesting that plasma oestradiol concentrations were not sufficiently elevated to induce a high pituitary response to mating stimulation. The conception rate after a single mating was low, suggesting that the number of sperm per mating was not sufficient. These results suggest that mating more than once in the middle of oestrus is required to improve ovulation rates and conception rates in cats.

Comparison of human serum with fetal bovine serum for expansion and differentiation of human synovial MSC: potential feasibility for clinical applications.

The aim of this study was to evaluate the effect of human serum (HS) on growth and differentiation capacity of human synovium-derived mesenchymal stem cells (MSC) in comparison to cells grown in fetal bovine serum (FBS). Human MSCs were isolated from the synovium of knee joints of three donors and the cells were cultured individually in varying concentrations of allogenic HS or FBS. Bovine MSCs were isolated from synovium and cultured in the same manner. Cell proliferation was assessed by the tetrazolium assay after passage 3. The capacity for chondrogenic and osteogenic differentiation was investigated in specific media followed by 1,9-dimethylmethylene blue assay and alcian blue staining, or by alizarin red staining, respectively. Human MSCs proliferated significantly more rapidly in the presence of HS than with equivalent levels of FBS. Chondrogenic or osteogenic differentiation occurred to nearly identical levels in HS or FBS. The results of this study indicate that HS is superior for the culture of human MSCs compared with FBS in terms of cellular expandability, without losing chondrogenic or osteogenic differentiation capacity. Coupled with the advantage in eliminating the potential risk accompanied with the use of xeno-derived materials, pooled, well-characterized HS could be a useful reagent to promote cellular expansion for clinical synovial stem cell-based therapy.

Costal cartilage transplantation for treatment of growth plate injury in a rabbit model.

OBJECTIVES: The purpose of this study is to evaluate the effects of allogenic costal cartilage transplantation on preventing bony bridge formation and angular deformities for the treatment of partial growth plate injury using a rabbit model. METHODS: An experimental model of partial growth injury was created by resecting the medial part of the proximal tibial growth plate in male six-week-old New Zealand White rabbits. The rabbits were divided into four groups: no surgery; no transplantation; bone wax transplantation; and allogenic costal cartilage transplantation. The angular deformities of the tibia and bony bridge were analysed using radiographs and microcomputed tomography, and the repair of the injured growth plate cartilage and bony bridge formation rate were histologically evaluated. RESULTS: On radiographic evaluation, the varus deformities in the costal cartilage group were significantly improved compared with the no transplantation group at four and eight weeks after operation and with the bone wax group at eight weeks after operation. Micro-computed tomography showed that the bony bridge formation was prevented in the bone wax and costal cartilage groups. Histological findings showed that the bony bridge formation in the bone wax and costal cartilage group was decreased. In addition, the growth plate was continuous and stained with safranin O and immunohistochemically stained for type II collagen. CONCLUSION: Transplantation of costal cartilage improved angular deformities and decreased bony bridge formation in the partial growth plate injury. Costal cartilage might be a suitable graft for the treatment of growth plate injury.

Lymphokine-activated killer function following autologous bone marrow transplantation for refractory hematological malignancies.

Disease recurrence remains the major factor which limits the success of autologous bone marrow transplantation (ABMT) for refractory hematological malignancies. The administration of interleukin 2 (IL2) with or without ex vivo generated lymphokine-activated killer (LAK) cells represents a potential approach to eradicating residual disease after ABMT. However, since LAK precursor activity is radiosensitive, high dose chemoradiotherapy may abrogate LAK function and preclude clinical responsiveness to IL2 after ABMT. Furthermore, since lymphocyte subsets which mediate LAK activity may recover at different rates after ABMT, LAK cells may be phenotypically and/or functionally altered after ABMT. To determine whether IL2 responsive LAK precursor cells are present in the circulation after ABMT, peripheral blood mononuclear cells (PBMC) from 21 patients with acute leukemia or lymphoma were tested for IL2-inducible LAK activity 17-83 days after ABMT. Cells were cultured with IL2 (1000-2000 units/ml) for 4 or 5 days and then tested for cytolytic activity and/or cell phenotype. LAK activity against the Daudi cell line was detected in every PBMC sample from every patient at every time point tested. The Raji cell line and a fresh allogeneic ovarian carcinoma were also lysed by LAK cells generated after ABMT. In the subgroup of patients transplanted for non-Hodgkin's lymphoma, LAK precursor activity appeared comparable to that of healthy controls. Culture with IL2 resulted in increased mean IL2 receptor expression in lymphocytes from patients after ABMT (3.1-9.9%) and from healthy controls (3.1-12.0%). After culture with IL2, the percentage of cells bearing the natural killer cell-associated Leu-19 determinant was significantly higher in patient PBMC than in normal control PBMC (28.3 versus 8.7%). Positive and negative cell selection by fluorescence sorting after culture with IL2 revealed that most of the LAK activity after ABMT was mediated by the Leu-19+ cells. Although CD5+ T-cells were devoid of LAK activity, a subset LAK effectors was CD8+. Thus, LAK activity is rapidly reconstituted after ABMT and is mediated by cells phenotypically similar to those in normal controls. These results support the feasibility of IL2 +/- LAK as consolidative immunotherapy after ABMT.

Induction of lymphokine-activated killer activity by interleukin 4 in human lymphocytes preactivated by interleukin 2 in vivo or in vitro.

In an attempt to augment the generation of human cytotoxic effector cells for potential cancer therapy with interleukin 2 (IL2) and lymphokine-activated killer (LAK) cells, the effect of interleukin 4 (IL4) on LAK cell induction was studied. In normal human peripheral blood lymphocytes (PBL), IL4 does not induce LAK activity and inhibits LAK induction by IL2. However, since lymphocyte activation, such as with antigen or mitogen, can render them responsive to IL4, the ability of IL4 to induce LAK activity in lymphocytes preactivated in vivo or in vitro with IL2 was investigated. PBL obtained from 12 patients with advanced cancer 1 to 3 days after IL2 therapy and from eight healthy control subjects were cultured 4 to 5 days with or without IL4 and/or IL2 and then tested for LAK activity as assessed by lysis of Daudi in a 4-h 51Cr release assay. In normal PBL, IL4 failed to induce LAK activity and consistently inhibited LAK induction by a suboptimal concentration of IL2 (10 units/ml). By contrast, IL4 induced LAK activity in PBL from seven of twelve IL2-treated patients and augmented LAK induction by the suboptimal IL2 in PBL from five of twelve IL2-treated patients. With an optimal LAK-inducing concentration of IL2 (1000 units/ml), IL4 less consistently inhibited LAK induction in normal PBL and had a variable effect upon LAK induction in PBL from IL2-treated patients. IL4 induced LAK activity in PBL obtained from a cancer patient after, but not before, systemic IL2 therapy. Similarly, IL4 induced LAK activity in normal PBL only after they had been preincubated with IL2. Thus, IL4 induces LAK activity in lymphocytes preactivated by IL2 in vivo or in vitro. Fluorescence-activated cell sorting revealed that the LAK activity, whether induced by IL4 or by IL2, was mediated largely by non-T (CD5-) natural killer-like (CD56+) cells. The results suggest a regulatory relationship between IL2 and IL4 in the induction and/or maintenance of LAK activity, which might be exploited to augment the generation of cytotoxic cells for lymphokine-mediated immunotherapy of human cancer.

Enhancement by interleukin 4 of interleukin 2- or antibody-induced proliferation of lymphocytes from interleukin 2-treated cancer patients.

Systemic interleukin 2 (IL-2) and IL-2-activated lymphocytes have induced tumor regression in some cancer patients. The IL-2-activated cells have usually been generated by obtaining peripheral blood mononuclear cells (PBMC) from cancer patients shortly after systemic IL-2 therapy and culturing them with IL-2 in vitro. In an effort to augment the ex vivo generation of such cells preactivated in vivo, we examined the proliferative responses of PBMC from IL-2-treated cancer patients to several proliferative signals including IL-2, interleukin 4 (IL-4), and mitogenic antibodies to CD3 and CD28. Although much is known about the response of normal PBMC to these signals, the possibility was considered that the response of lymphocytes preactivated by IL-2 in vivo might differ from that of normal PBMC. Accordingly, PBMC obtained from ten normal, healthy controls and from 17 patients with advanced cancer 1 to 3 days after systemic IL-2 therapy were cultured for 4 days with IL-4 (1000 units/ml) and/or IL-2 (10 units/ml or 1000 units/ml) or with combinations of IL-4 and anti-CD3 +/- anti-CD28, and they were then tested for proliferation by [3H]thymidine incorporation. IL-4 failed to induce proliferation of normal PBMC and inhibited IL-2-induced proliferation, whereas IL-4 alone induced proliferation in PBMC from five of 11 IL-2-treated patients and did not inhibit but augmented the proliferation induced by IL-2 (10 units/ml and 1000 units/ml) in PBMC from six of nine patients and five of 11 patients, respectively. Anti-CD3 induced proliferation in PBMC from eight of nine patients, and the proliferation was consistently augmented by coculture with anti-CD28. Finally, IL-4 significantly augmented the proliferative responses of PBMC from IL-2-treated patients to anti-CD3, as well as to the combination of anti-CD3 and anti-CD28. Thus, in PBMC from IL-2-treated cancer patients, IL-4 enhanced the in vitro proliferation induced by IL-2 or by anti-CD3 +/- anti-CD28. The results suggest that IL-4 and/or mitogenic antibodies may be useful in augmenting the ex vivo generation of lymphocytes for clinical adoptive immunotherapy.

Histopathological predictors of breast cancer death among Caucasians and Japanese in Hawaii.

In the United States, Caucasian women are at higher risk of death from breast cancer than age-matched Japanese-Americans. The tumors of Japanese-Americans exhibit a greater uniformity of nuclear grade (NG), greater degrees of intratumoral lymphocytic infiltration (LI), and more conspicuous sinus histiocytosis (SH) in the regional lymph nodes. To assess the impact of histopathology upon the ethnic disparity in breast cancer mortality, we compared the survival experience of Japanese and Caucasian women with breast cancer in Hawaii. The study group consisted of 443 women, aged 45-74, whose cancers were diagnosed between 1975 and 1980. Survival status at 9 or more years after diagnosis, known for 416 of these women, was used in the analyses. Age and tumor stage at diagnosis were significant predictors of breast cancer death in the logistic regression analysis. When histopathological predictor variables (NG, LI, and SH) were included in the model, age, stage, NG, and LI were independently predictive. Although NG predicted stage among all patients, and SH predicted stage among the women with invasive disease, race was an independent predictor breast cancer stage in multivariate analyses. Finally, analysis within stage subgroups revealed that race was independently predictive of cancer death among women with localized disease (confined to the breast) but not among women with regional spread (local extension or axillary nodes involves). These results indicate that histopathological differences contribute to, but only partially explain, the disparity in breast cancer mortality between Caucasians and Japanese in Hawaii.

Correlations between mutagen sensitivity and plasma nutrient levels of healthy individuals.

Increased mutagen sensitivity and decreased intake of antioxidant-rich fruits and vegetables have been associated with an increased risk of upper aerodigestive tract cancers. The objective of this study was to investigate the intraindividual variation in mutagen sensitivity and its possible correlation with plasma nutrient levels in a group of 25 healthy individuals in Hawaii. Mutagen sensitivity, as assessed by bleomycin-induced chromosomal breaks in cultured peripheral blood lymphocytes and plasma nutrient levels were measured monthly for 11 months. The monthly numbers of chromosomal breaks/cell ranged from 0.04 to 0.80 and showed considerable intraindividual variation. Based on individual means, significant inverse correlations were found between mutagen sensitivity scores and the plasma levels of alpha-carotene (r = -0.64), total carotenoids (r = -0.41), and ascorbic acid (r = -0.40). There were also significant inverse associations between monthly mean plasma levels of alpha-carotene (r = -0.58), beta-carotene (r = -0.76) and total carotenoids (r = -0.72) and monthly mean chromosomal breaks. In contrast, there was a significant positive correlation between monthly mean plasma triglyceride level (r = 0.60) and monthly mean mutagen sensitivity. These results suggest that mutagen sensitivity as assessed by the bleomycin assay may be influenced by plasma levels of certain nutrients and could potentially be modified by dietary interventions or micronutrient supplementation.

Induction of NAD(P)H:quinone reductase by vitamins A, E and C in Colo205 colon cancer cells.

High consumption of fruits and vegetables which are abundant in dietary antioxidants has been linked to a reduced incidence of colorectal cancer. A potential mechanism of dietary anticarcinogenesis involves the induction of detoxifying phase II enzymes, including NAD(P)H:quinone reductase (QR) and glutathione-S-transferase (GST). This study therefore examined the ability of the dietary antioxidant vitamins beta-carotene, alpha-tocopherol and ascorbic acid to induce cellular expression of QR and GST activities in human colon cancer cells. Colo205 cells were cultured in the presence or absence of various concentrations (10(-10) to 10(-5) M) of each antioxidative micronutrient, then assessed for cytosolic QR and GST activities and cell growth. beta-Carotene, alpha-tocopherol and ascorbic acid each resulted in dose-dependent increases in QR activity, without adverse effects upon cell proliferation. To investigate whether the ability of beta-carotene to induce QR may be attributable to its conversion to vitamin A and/or to its antioxidant capacity as a carotenoid, retinol, retinoic acid, and lycopene were similarly tested for their capacity for enzyme induction. Although retinol and retinoic acid were both noted to be antiproliferative at higher concentrations (10(-6) to 10(-5) M), both retinoids stimulated QR at physiological concentrations. Lycopene, a carotenoid which is not converted to vitamin A, was devoid of biologic activity. By contrast with the effects upon QR, GST activity was unaffected by treatment with any of the micronutrients tested in this in vitro model. The results support a hypothesis that a high dietary consumption of vitamins A, E and C may confer partial protection against colorectal cancer by the induction of specific detoxifying enzymes. The antioxidant capacity of beta-carotene appears to have less biologic impact vis-a-vis QR induction than its function as a non-toxic reservoir of vitamin A. Measurements of QR activity within the colorectal mucosa may provide an index of cancer susceptibility, and may be an appropriate surrogate endpoint biomarker for colorectal cancer prevention studies involving diet modification or specific relevant micronutrients.

Induction of NADPH:quinone reductase by dietary phytoestrogens in colonic Colo205 cells.

Phytoestrogens are a group of naturally occurring diphenolic compounds present in legumes, whole grains, fruits, and vegetables. High consumption of phytoestrogen-rich foods has been linked to a reduced incidence of cancers at many sites. A potential mechanism of dietary anticarcinogenesis involves the induction of detoxifying phase II enzymes such as NADPH:quinone reductase (QR). This study, therefore, examined the ability of six prominent phytoestrogens to affect cellular expression of QR in colonic cells. Colo205 cells were cocultured with various concentrations (0.001 to 10.0 microM) of each phytoestrogen, and then were assessed for cytosolic QR activity, cell growth, and QR mRNA expression. A maximum of 6- to 8-fold induction of QR activity was observed for both enterolactone and genistein, although at high concentrations they showed an adverse effect upon cell growth. The concentrations required to double the specific activity of QR for enterolactone and genistein were about 0.04 and 0.14 microM, respectively. A 2- to 3-fold increase of QR specific activity was found with either biochanin A (1.1 microM) or coumestrol (12.0 microM) treatments. No significant effects were found for daidzein or formononetin treatments. QR induction was further confirmed by using reverse transcription-polymerase chain reaction (RT-PCR) techniques to measure mRNA expression. A significant correlation between the expression of QR mRNA and the corresponding QR activity was observed (r = 0.76, P < 0.001). The results demonstrated that certain dietary phytoestrogens are capable of QR induction in Colo205 cells by promoting QR mRNA expression, and suggest a novel mechanism by which dietary phytoestrogens may be implicated in colorectal cancer chemoprevention.

Accessory cells and T cell activation. The relationship between two components of macrophage accessory cell function: I-A and IL1.

Accessory cells are required for the activation of helper T cells. We have examined two characteristics of accessory cells, their expression of I-A, and their ability to release IL1. We provide evidence that these two properties are related, and postulate that membrane I-A molecules participate in the pathway leading to IL1-release. Experimental results are described relating I-A to IL1-release as follows: 1. In vitro-educated Ly1 T cells stimulate IL1-release from M phi; this process is H-2-restricted and blocked by anti-I-A antibodies. 2. H-2-restriction between T cells and M phi is overcome in the presence of ConA, but this unrestricted interaction is also blocked by anti-I-A. 3. LPS stimulation of IL1-release is blocked by anti-I-A. These findings suggested an active role for I-A molecules on IL1-producing cells. We next describe a series of experiments designed to assess the requirements for I-A versus IL1 during T cell activation. In a number of experimental systems, T cells demonstrated a requirement for I-A-recognition, but none that could not also be satisfied by IL1: 1. Generation of helper T cells in allogeneic chimeras. 2. Proliferation of KLH-primed lymph node cells. 3. Proliferation of KLH-primed lymph node cells from chronically anti-I-A-suppressed mice. 4. Proliferation of GAT-primed lymph node cells from nonresponder mice. These findings suggest that for many kinds of T cells (not necessarily all) the apparent requirement for I-A-recognition is primarily involved in stimulating IL1-release from accessory cells.

Interleukin-2 with or without lymphokine-activated killer cells as consolidative immunotherapy after autologous bone marrow transplantation for acute myelogenous leukemia.

IL-2 with or without autologous lymphokine-activated killer (LAK) cells, administered early after ABMT for AML may eradicate residual disease and reduce relapses. This paper reports 14 patients who received IL-2 or IL-2 plus LAK cells after ABMT for AML in first relapse or at a later stage, in two separate trials. Patients with AML in first relapse (n = 9), second CR (n = 3) or second relapse (n = 2) underwent ABMT after busulfan (BU), CY and total body irradiation (n = 11) or BU/CY alone (n = 3), with marrow that was (n = 6) or was not (n = 8) purged with 4-HC. In a previously-reported Phase I trial, eight patients received IL-2 (Roche) by continuous infusion at 0.3-3.0 x 10(6) U/m2/day x 5 days and, after 6 days of rest, 0.3 x 10(6) U/m2/day x 10 days. In a subsequent trial, five patients received IL-2 at 3.0 x 10(6) U/m2/day x 5 days, underwent leukapheresis for 3 days and received their LAK cells plus IL-2 (0.3 x 10(6) U/m2/day x 10 days). A sixth patient received only 2 days of IL-2, developed sepsis and died of multiorgan failure. All other patients had mild to moderate toxicity which was reversible. All patients developed neutrophilia, lymphocytosis and thrombocytopenia. IL-2 with or without LAK therapy was initiated 21-91 days (median 51 days) after ABMT. Severe thrombocytopenia (< 10 x 10(9)/l) occurred during the apheresis days.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunotherapy with interleukin 2 with or without lymphokine-activated killer cells after autologous bone marrow transplantation for malignant lymphoma: a feasibility trial.

Early relapse remains a major challenge after autologous bone marrow transplant for malignant lymphoma (ML). It is postulated that consolidative immunotherapy with interleukin 2 (IL-2) with or without lymphokine-activated killer (LAK) cells administered after autologous bone marrow (ABMT) or peripheral blood stem cell transplantation (PBSCT) for ML might eradicate residual disease and reduce relapse rates. A previous trial identified an IL-2 regimen that could be administered early after ABMT. This paper presents the clinical results of 16 patients with ML, who participated in a study to determine whether LAK cells could be administered after ABMT with this IL-2 regimen, as well as 6 patients who received IL-2 alone after ABMT or PBSCT. Seventeen patients with non-Hodgkin's lymphoma (NHL), and 5 with Hodgkin's disease (HD), underwent ABMT (20 patients) or PBSCT (2 patients). At the time of transplantation, 7 patients were in untreated or chemotherapy-sensitive first relapse, 3 were in CR2, and 12 were beyond CR2. Beginning 22-85 days (median 43) after ABMT/PBSCT, patients received IL-2 at 3.0 x 10(6) U/m2/day by continuous infusion days 1-5 of the IL-2 protocol. On protocol days 7-9 the first 16 patients underwent apheresis for LAK cell generation. The cells were cultured in IL-2 for 5 days and were infused on days 12-14. Low-dose IL-2 (0.9 x 10(6) IU/m2/day) was administered on days 12-21 in the outpatient department. Patients received a median of 148 (62-279) x 10(9) LAK cells. LAK cell infusions were associated with transient fevers, chills and dyspnea in most patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation of phosphatydilcholine-hydroperoxide in dialysis patients with diabetic nephropathy.

Plasma lipid peroxidation in noninsulin dependent diabetes mellitus (DM) patients were evaluated in DM patients undergoing hemodialysis (HD) by means of a chemiluminescence-HPLC for the specific determination of phosphatidylcholine hydroperoxide (PCOOH). Thirty-three uremic patients with DM nephropathy, undergoing 12 hours HD a week using polymethylmethacrylate membrane, were studied. Of them 22 DM patients on HD were divided into 2 age and sex matched groups treated and conventional group in order to clarify therapeutic effect of 500 mg alfa-tocopherol and 600 mg probucol daily. Fifty DM patients without end-stage renal disease (ESRD) who were age-, period of diabetes-, and sex-matched, were selected as positive control of the subjects. Plasma PCOOH levels were significantly elevated in both DM patients, while the plasma PCOOH in normal controls were 227.0 +/- 68.7 pmol/ml. Plasma PCOOH levels of DM patients undergoing HD were significantly higher than that of patients without ESRD (1,330.8 +/- 642.7 pmol/ml vs. 756.6 +/- 431.9 pmol/ml, p < 0.025). Partial correlation coefficient of plasma PCOOH level demonstrated PCOOH and period of HD in DM patients were highly significantly positively correlated (p < 0.01), although single session of HD was not found to produce significantly increased lipid-peroxidation. Plasma PCOOH roughly remained within similar levels as base lines by medication with anti-oxidant compared to that of conventional group. From these results we conclude that HD intensifies lipid peroxidation and such accumulation of hydroperoxide could account for accelerated progress of atherosclerosis in DM patients with renal insufficiency. It is worthwhile to try an administration of free radical scavenger in order to reduce PCOOH and slow down the progression of atherosclerotic vascular disease.

The role of protein kinase C activity in the proliferation of peritoneal fibroblasts.

OBJECTIVE: To clarify the effect of glucose on peritoneal sclerosis, we performed several experiments to determine how glucose influences the proliferation of, and the production of extracellular matrix proteins by, peritoneal fibroblasts. The effect of heparin on these phenomena was also studied. DESIGN: Using rat peritoneal fibroblasts, cells were cultured in four separate media: M199 with 5% fetal bovine serum (FBS) (control medium), control medium with 4% glucose (glucose medium), glucose medium with H7 [an inhibitor of protein kinase C (PKC)] 50 micromol, and glucose medium with heparin 50 microg/mL. Cell proliferation and concentrations of procollagen 3 peptide (P3P) and hyaluronic acid (HA) in the supernatants were evaluated at 24 hours, 72 hours, and 120 hours after culture. PKC activity in cytosolic and cell membrane fractions were measured 30 minutes after incubation in control medium and in glucose medium with and without heparin. RESULTS: Glucose accelerated cell proliferation 24 hours after culture, but inhibited it 120 hours after culture. Glucose stimulated the production of HA from these cells 72 hours and 120 hours after culture, but it did not stimulate the production of P3P at any time. Heparin and H7 inhibited cell proliferation by glucose 24 hours after culture. Heparin and H7 also inhibited the production of HA in the peritoneal fibroblasts after culture, but did not affect the production of P3P. Glucose accelerated PKC activity in cell membrane, but not in cytosol. Heparin inhibited the elevated PKC activity of the membrane fraction by glucose. CONCLUSION: Glucose may accelerate the proliferation of, and HA production in, peritoneal fibroblasts by stimulation of cellular PKC activity. Heparin suppresses these phenomena by inhibiting PKC activity.

The effect of glucose on the proliferation of peritoneal fibroblasts.

The purpose of this study was to clarify the pathophysiological role of glucose and glucose transporters (GLUTs) in the proliferation and production of extracellular matrix (ECM) in peritoneal fibroblasts. Peritoneal fibroblasts from rat omentum were cultured in medium M199 with 5% fetal bovine serum (FBS) with (Group B) and without (Group A) 4% glucose. The rate of cell growth at the M stage of the cell cycle in Group B was higher than that in Group A at 12 and 24 hours after culture. However, cell numbers in Group B were less than in Group A at 24, 72, and 120 hours after culture. The GLUT inhibitor suppressed the proliferation of cells 72 and 120 hours after culture. The procollagen III peptide (PIIIP) contents in the supernatant of cells cultured in a high glucose medium were higher than those of control cells at 24, 72, and 120 hours after culture. PIIIP levels of cells cultured with the GLUT inhibitor were also higher than those of cells without the GLUT inhibitor. These results suggest that initially glucose stimulates cell proliferation and thereafter inhibits its proliferation. GLUTs may accelerate the proliferation of peritoneal fibroblasts. We suggest that glucose stimulates the ECM component PIIIP, and GLUT may have an inhibitory effect on the production of the ECM component PIIIP.

[Pathophysiological mechanism of ischemic acute renal failure: protective effect of coenzyme Q10, Ca channel blocker, superoxide dismutase and protease inhibitor against ischemic acute renal failure].

Ischemic insult has been considered a cause of cellular injuries under certain circumstance, such as the disturbance of energy metabolism, the alternation of calcium homeostasis, the production of oxygen radical and the release of lysosomal protease. The present study was designed to clarify the pathophysiological effects of coenzyme Q10 (CoQ10), diltiazem, superoxide dismutase (SOD) and urinastatin on the development and progression of ischemic acute renal failure (IARF) of the rat. At 24 hours after reflow following 45 minutes ischemia, serum urea nitrogen, creatinine and fractional excretion of sodium were 99.3 mg/dl, 3.14 mg/dl, 5.95% respectively, in non-treated IARF rats. Renal ATP content was reduced to 0.91 micrograms/mg. prot. from 10.59 micrograms/mg. prot. at 10 minutes after ischemic insult, and remained at almost the same level throughout the entire 45 minutes ischemia. Although the subsequent blood reflow resulted in the recovery of ATP content, it was up to 50% of normal level at 24 hours after reflow following 45 minutes ischemia. During the ischemic period, the pathological changes were mild, whereas, after reflow, tissue involvement was mainly localized in the S3 segment of the proximal tubule. Major alteration were the loss of brush border, high amplitude swelling of mitochondria with matrical densities and fragmentation of the epithelial cell. At 24 hours after reflow, it was observed that renal function was superior in IARF rats treated with CoQ10, diltiazem, SOD and urinastatin. The treated rats also had higher ATP contents and showed less pathological changes than non-treated rats. Among these inhibitory agents, diltiazem exerted the most reliable effect. From these results, it was concluded that IARF was obviously caused by such pathophysiological mechanisms as mentioned above. Especially, Ca influx into the cells is one of the most important factors on pathogenesis of IARF.

Comparison of insulin detemir and insulin glargine on glycemic variability in patients with type 1 and type 2 diabetes.

AIMS: To compare the glycemic variability of insulin detemir and insulin glargine in type 1 and type 2 diabetic patients. METHODS: 15 type 1 and 14 type 2 diabetic patients receiving intensive insulin therapy with insulin glargine were enrolled. Before and after switching insulin glargine to insulin detemir, we assessed fasting glucose variability using the standard deviation (SD) and the coefficient of variance (CV) of self-monitored fasting blood sugar (FBS) levels. RESULTS: The SD and CV values were significantly decreased in type 1 diabetes after switching the therapy, though there was no significant difference in type 2 diabetes. The frequency of hypoglycemia was decreased in type 1 diabetes and there was no change in type 2 diabetes. The changes of the CV value also showed significant positive correlation with fasting serum CPR levels in all patients and total insulin dose in type 1 diabetes. The changes of frequency of hypoglycemia showed significant positive correlation with total and basal insulin dose adjusted for body weight in type 1 diabetes. CONCLUSION: The present study demonstrated lower within-subject variability of insulin detemir compared to insulin glargine, suggesting that the basal insulin replacement with insulin detemir may provide a useful therapeutic strategy for uncontrolled type 1 diabetes with high glucose variability.