Effect of intraperitoneally administered recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) on the cytotoxic potential of murine peritoneal cells

We studied the effect of recombinant murine granulocyte–macrophage colony-stimulating factor(rmGM-CSF) on the cytotoxic potential of murine peritoneal cells. Mice received rmGM-CSF intraperitoneally using different dosages and injection schemes. At different time points after the last injection, mice were sacrificed, peritoneal cells isolated and their tumour cytotoxicity was determined by a cytotoxicity assay using syngeneic [methyl-3H]thymidine-labelled colon carcinoma cells. Also, the cytotoxic response to a subsequent in vitro stimulation with lipopolysaccharide was determined. Upon daily injection of 6000–54 000 U rmGM-CSF over a 6-day period, the number of peritoneal cells increased over ten fold with the highest rmGM-CSF dose. Increases in cell numbers was mainly due to increases in macrophage numbers. Upon injection of three doses of 3000 U rmGM-CSF per day for 3 consecutive days, the number of macrophages remained elevated for minimally 6 days. Although the peritoneal cells from rmGM-CSF-treated mice were not activated to a tumoricidal state, they could be activated to high levels of cytotoxicity with an additional in vitro stimulation of lipopolysaccharide. Resident cells isolated from control mice could be activated only to low levels of tumour cytotoxicity with lipopolysaccharide. Tumour cytotoxicity strongly correlated with nitric oxide secretion. When inhibiting nitric oxide synthase, tumour cell lysis decreased. Thus, the expanded peritoneal cell population induced by multiple injections of rmGM-CSF has a strong tumour cytotoxic potential and might provide a favourable condition for immunotherapeutic treatment of peritoneal neoplasms. © 1999 Cancer Research Campaig

MAK-4 and -5 supplemented diet inhibits liver carcinogenesis in mice

<p>Abstract</p> <p>Background</p> <p>Maharishi Amrit Kalash (MAK) is an herbal formulation composed of two herbal mixtures, MAK-4 and MAK-5. These preparations are part of a natural health care system from India, known as Maharishi Ayur-Veda. MAK-4 and MAK-5 are each composed of different herbs and are said to have maximum benefit when used in combination. This investigation evaluated the cancer inhibiting effects of MAK-4 and MAK-5, <it>in vitro </it>and <it>in vivo</it>.</p> <p>Methods</p> <p><it>In vitro </it>assays: Aqueous extracts of MAK-4 and MAK-5 were tested for effects on <it>ras </it>induced cell transformation in the Rat 6 cell line assessed by focus formation assay. <it>In vivo </it>assays: Urethane-treated mice were put on a standard pellet diet or a diet supplemented with MAK-4, MAK-5 or both. At 36 weeks, livers were examined for tumors, sera for oxygen radical absorbance capacity (ORAC), and liver homogenates for enzyme activities of glutathione peroxidase (GPX), glutathione-S-transferase (GST), and NAD(P)H: quinone reductase (QR). Liver fragments of MAK-fed mice were analyzed for connexin (cx) protein expression.</p> <p>Results</p> <p>MAK-5 and a combination of MAK-5 plus MAK-4, inhibited <it>ras</it>-induced cell transformation. In MAK-4, MAK-5 and MAK4+5-treated mice we observed a 35%, 27% and 46% reduction in the development of urethane-induced liver nodules respectively. MAK-4 and MAK4+5-treated mice had a significantly higher ORAC value (<it>P </it>< 0.05) compared to controls (200.2 ± 33.7 and 191.6 ± 32.2 <it>vs. </it>152.2 ± 15.7 ORAC units, respectively). The urethane-treated MAK-4, MAK-5 and MAK4+5-fed mice had significantly higher activities of liver cytosolic enzymes compared to the urethane-treated controls and to untreated mice: GPX(0.23 ± 0.08, 0.21 ± 0.05, 0.25 ± 0.04, 0.20 ± 0.05, 0.21 ± 0.03 U/mg protein, respectively), GST (2.0 ± 0.4, 2.0 ± 0.6, 2.1 ± 0.3, 1.7 ± 0.2, 1.7 ± 0.2 U/mg protein, respectively) and QR (0.13 ± 0.02, 0.12 ± 0.06, 0.15 ± 0.03, 0.1 ± 0.04, 0.11 ± 0.03 U/mg protein, respectively). Livers of MAK-treated mice showed a time-dependent increased expression of cx32.</p> <p>Conclusion</p> <p>Our results show that a MAK-supplemented diet inhibits liver carcinogenesis in urethane-treated mice. The prevention of excessive oxidative damage and the up-regulation of connexin expression are two of the possible effects of these products.</p

Eosinophil Cationic Protein and Histamine Production by Neutrophils from Periodontitis Patients

Periodontitis develops through an inflammatory process caused by an infection at the microbial biofilm, followed by tissue destruction mediated by leukocytes, which cause clinically significant destruction of connective tissue and bone. Several elements derived from the bacteria cause the inflammatory response and the release of mediators involved in destruction of the periodontium. There are number of inflammatory mediators released by leukocytes, mainly neutrophils, upon bacterial challenge. Neutrophils produce and release eosinophil cationic protein (ECP) and histamine, two important inflammatory mediators; however, their role has not been characterized in periodontal inflammation. Thus, the purpose of this study is to investigate whether neutrophils from patients with periodontitis can produce ECP and histamine in response to lipopolysaccharides (LPSs). ECP and histamine production in response to LPSs was analyzed by enzyme-linked immunosorbent assay. Expression of the histidine decarboxylase and ECP was also analyzed by flow cytometry and fluorescence microscopy in neutrophils from patients with periodontitis in response to LPS. It was found that neutrophils from patients with periodontitis express higher levels of histidine decarboxylase and ECP than those from healthy volunteers, and they also release higher levels of histamine. Findings described could represent new knowledge indicating neutrophils as a source of histamine and ECP in the progression of periodontitis

Dose-dependent activation of immune function in mice by ingestion of Maharishi Amrit Kalash 5

This study was carried out to evaluate the dose-effects of ingestion of Maharishi Amrit Kalash 5 (MAK 5), an Ayurvedic food supplement, on the immune function in 10 week female inbred BALB/c mice. Superoxide anion (O2 ) production of peritoneal macrophages and the response of spleen cells to concanavalin A (Con A) were examined in mice given MAK 5 by gastric intubation of an aqueous emulsion at the doses of 10, 50, 100 and 200 mg/kg once a day for 20 days. Glucose consumption of peritoneal macrophages in the MAK 5-treated mice at all doses after 24 hours of incubation, and only at the dose of 200 mg/kg after 48 hours of incubation were significantly higher than those in the control group. O2 production of peritoneal macrophages in the presence of stimulator was significantly higher in the MAK 5-treated group at the dose of 200 mg/kg than in the control group. Activities of β -glucuronidase and lactate dehydrogenase in the peritoneal macrophages were significantly increased in the MAK 5treated mice at all doses. MAK 5 did not enhance spontaneous splenic lymphocyte proliferation at any dose in mice. Stimulation indices in the MAK 5-treated groups at the doses of 50, 100 and 200 mg/kg were significantly higher than that of the control group. These results indicate that gastric intubation of MAK 5 once a day at the dose of 50 mg/kg enhances not only macrophage function but also lymphocyte responsiveness in mice

Histamine induces Toll-like receptor 2 and 4 expression in endothelial cells and enhances sensitivity to Gram-positive and Gram-negative bacterial cell wall components

Histamine is a major inflammatory molecule released from the mast cell, and is known to activate endothelial cells. However, its ability to modulate endothelial responses to bacterial products has not been evaluated. In this study we determined the ability of histamine to modulate inflammatory responses of endothelial cells to Gram-negative and Gram-positive bacterial cell wall components and assessed the role of Toll-like receptors (TLR) 2 and 4 in the co-operation between histamine and bacterial pathogens. Human umbilical vein endothelial cells (HUVEC) were incubated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or peptidoglycan (PGN) in the presence or absence of histamine, and the expression and release of interleukin-6 (IL-6), and NF-κB translocation were determined. The effect of histamine on the expression of mRNA and proteins for TLR2 and TLR4 was also evaluated. Incubation of HUVEC with LPS, LTA and PGN resulted in marked enhancement of IL-6 mRNA expression and IL-6 secretion. Histamine alone markedly enhanced IL-6 mRNA expression in HUVEC, but it did not stimulate proportional IL-6 release. When HUVEC were incubated with LPS, LTA, or PGN in the presence of histamine marked amplification of both IL-6 production and mRNA expression was noted. HUVEC constitutively expressed TLR2 and TLR4 mRNA and proteins, and these were further enhanced by histamine. The expression of mRNAs encoding MD-2 and MyD88, the accessory molecules associated with TLR signalling, were unchanged by histamine treatment. These results demonstrate that histamine up-regulates the expression of TLR2 and TLR4 and amplifies endothelial cell inflammatory responses to Gram-negative and Gram-positive bacterial components