Macrophage activating factor(s) secreted by mitogen stimulated goldfish kidney leukocytes synergize with bacterial lipopolysaccharide to induce nitric oxide production in teleost macrophages

Recent studies in our laboratory demonstrated that fish macrophages produce nitric oxide. To elucidate the mechanisms which regulate nitric oxide production in teleosts, we examined whether macrophage activating factors (MAFs) secreted by mitogen stimulated leukocytes, induced nitric oxide production in a long-term cultured macrophage cell line and in primary cultures of kidney macrophages from the goldfish. The results indicate that both primary and long term cultured goldfish macrophages produce nitric oxide in response to MAF or bacterial lipopolysaccharide (LPS), and co-stimulation with both factors results in a synergistic induction of nitric oxide production. MAF that induced nitric oxide production were present in leukocyte supernatants as early as 24 h after addition of mitogens to cell cultures. The production of MAF was dependent upon the incubation temperature, presence of serum in the culture medium and duration of incubation: maximal MAF activity was detected in 72–96 h supernatants raised in media with serum at 30 °C. MAF-induced nitric oxide production by long term cultured macrophages was inhibited by 1000 μM N^G-monomethyl- -arginine or amino-guanidine, indicating an -arginine-dependent metabolic pathway for the production of the reactive nitrogen intermediates in teleosts. The biochemical events of cytokine induced nitric oxide production by teleost macrophages appear to be similar to those of mammalian macrophages.     

Tumor necrosis factor-alpha: central regulatory cytokine in the induction of macrophage antimicrobial activities

Expression of activated macrophage resistance to infection requires the cooperative interaction of interferon-gamma (IFN-gamma) and either interleukin-2 (IL-2), interleukin-4 or granulocyte/macrophage-colony-stimulating factor: no single cytokine is effective. For IFN-gamma and IL-2, the effector activity can be suppressed by the presence of anti-tumor necrosis factor-alpha (TNF-alpha) antibodies in the reaction mixture. IFN-gamma and IL-2, only in combination, induce TNF-alpha-specific mRNA and secretion of this cytokine by macrophages. Development of intracellular killing activity by activated macrophages also requires the autocrine effects of TNF-alpha. IFN-gamma provides the first signal for the production of nitric oxide (NO), the effector molecule for intracellular destruction of parasites. When IFN-gamma-treated cells are infected with pathogens, they are stimulated to make TNF-alpha. Expression of intracellular killing, as well as production of NO, is inhibited by anti-TNF-alpha antibody.     

Dissolution rate enhancement and physicochemical characterization of carbamazepine-poloxamer solid dispersions

This study investigates the potential of poloxamers as solid dispersions (SDs) carriers in improving the dissolution rate of a poorly soluble drug, carbamazepine (CBZ). Solid dispersions were prepared with poloxamer 188 (P188) and poloxamer 407 (P407) by melting method in different drug:carrier ratios (1:1, 1:2 and 1:3). Prepared samples were characterized using differential scanning calorimetry (DSC), hot-stage polarized light microscopy (HSM), powder X-ray diffraction (PXRD) and Fourier transform infrared spectroscopy (FT-IR) to investigate drug physical state within the SDs matrix, possible polymorphic transitions and drug-polymer interactions. The interactions between CBZ molecules and polymeric chains were also evaluated using molecular dynamics simulation (MDS) technique. The most thermodynamically stable polymorphic form III of CBZ was present in all SDs, regardless of the type of poloxamer and drug-to-carrier ratio. The absence of drug-polymer interactions was observed by FT-IR analysis and additionally confirmed by MDS. Formation of persistent hydrogen bond between two CBZ molecules, observed by MDS indicate high tendency of CBZ molecules to aggregate and form crystalline phase within dispersion. P188 exhibit higher efficiency in increasing CBZ dissolution rate due to its more pronounced hydrophilic properties, while increasing poloxamers concentration resulted in decreasing drug release rate, as a consequence of their thermoreversible gelation.     

The diagnostic value of Doppler echocardiographic indexes of diastolic filling for detecting demand ischemia in anesthetized patients

In awake patients, Doppler indexes of transmitral flow velocities have been shown to be more sensitive for detection of myocardial ischemia than echocardiographic evaluation of systolic wall-motion. The diagnostic value of these indexes in anesthetized patients is unknown. It might differ from that in awake patients because anesthetics and sympathomimetic drugs, which are commonly used in surgical patients with coronary artery disease (CAD), independently affect transmitral flow velocities. Methods: Several previously published transmitral Doppler echocardiographic indicators of ischemia (marked decreases in the ratio of peak early [E] to peak atrial [A] filling velocity [E/A], in the ratio of early to atrial time–velocity integral [E_I/A_I], in E, in E acceleration, and in total diastolic time–velocity integral) were compared with standard wall-motion analysis and ST-segment analysis during dobutamine stress echocardiography (DSE) in 17 anesthetized patients with CAD and 7 age-matched control patients at low risk of CAD. Results: All patients with CAD but no control patients showed new systolic wall-motion abnormalities and/or ST-segment changes. Decreases of >10% in E/A and E_I/A_I were found in 88% and 71% of the patients with CAD and in 71% and 100% of the control patients, respectively. These decreases were found during dobutamine infusion at as low as 10 mcg/kg/min in several control patients. None of the other studied Doppler indexes were found useful to detect ischemia. Conclusions: Doppler echocardiography of diastolic transmitral flow velocities is of no appreciable diagnostic value for detecting ischemia in anesthetized patients who receive dobutamine at 10–40 mcg/kg/min.     

Early influx of macrophages determines susceptibility to experimental allergic encephalomyelitis in Dark Agouti (DA) rats

Experimental allergic encephalomyelitis (EAE) is characterized by inflammatory infiltrates of myelin antigen(s) specific T cells and consecutive demyelination. Injection of encephalitogen into the footpads induces disease in genetically susceptible Dark Agouti rats (DA) but not in Albino Oxford (AO) rats although mild inflammatory infiltrates are observed in both strains early after disease induction. In addition, only DA rats develop disease when cells from (AO×DA) F(1) hybrids are passively transferred into sub-lethally radiated AO and DA parent hosts. The aim of the study was therefore to examine the participation of accessory cells, macrophages, dendritic cells and microglia in EAE development at the level of the target tissue in these two strains using specific membrane markers. We demonstrate here that in the induction phase of EAE in DA rats, macrophages (CD68(+); CD45(hi)CD11b(+)) are the first detectable infiltrating cells in the subpial regions of the spinal cord but were not found in AO rats. During the same period, resident microglial cells which are of the ramified variety are observed in both DA and AO rats. In DA rats at the peak of disease, when profuse influx of T cells is seen, macrophages and dendritic cells appear in the parenchyma of the CNS. In addition, at that time, microglial cells are activated. FACS analyses also reveal a significant increase in CD45(hi)CD11c(+) dendritic cells and CD45(hi)D11b(+) macrophages compared with levels in na?ve and immunized AO rats. During resolution of disease in DA rats, the expression of microglia and macrophage markers is comparable with those in na?ve non-immunized DA and immunized AO rats. We conclude that an initial influx of macrophages is indispensible for the development of EAE in DA rats. The presence of dendritic cells and myeloid dendritic cells at the peak of disease supports the role of these cells in EAE especially in relapses and chronicity. The activation pattern of microglia in DA rats does not indicate their role as antigen presenting cells in disease induction since they are ramified at the induction phase and only become activated after the overwhelming influx of T cells.     

Serum CRP and IL-6, genetic variants and risk of colorectal adenoma in a multiethnic population

Chronic inflammation, which is suspected to play a role in the development of colorectal cancer (CRC), has rarely been studied in colorectal adenoma. We investigated the inter-relationships of serum levels of the inflammatory proteins CRP and in IL-6, single nucleotide polymorphisms (SNPs) in the CRP (rs1205, rs1130864, rs1800947) and IL6 (rs1800795) genes, and lifestyle factors with colorectal adenoma in a sigmoidoscopy-based case–control study of 271 adenoma cases and 539 age-, sex-, and race/ethnicity-matched controls in Hawaii. We found no association of serum CRP or IL-6 levels with the risk of adenoma. A multiple regression with stepwise selection identified elevated BMI, Caucasian and Native Hawaiian versus Japanese race/ethnicity, and current smoking as being associated with significantly higher serum CRP and IL-6 levels. Female versus male gender was also associated with higher CRP levels and older age with higher IL-6 levels. The C allele of rs1205 and the A allele of rs1130864 were significantly associated with higher serum CRP levels (p _trend: 0.0002 and 0.01, respectively), as well as with a decreased adenoma risk [rs1205: OR for CT and CC vs. TT = 0.69 (95% CI: 0.48–0.98) and 0.53 (0.34–0.83), respectively, p _trend = 0.008; rs1130864: OR for GA and AA versus GG = 0.65 (0.45–0.93) and 0.74 (0.31–1.76), respectively, p _trend = 0.04]. The findings of lower serum CRP and IL-6 levels in Japanese (a group with a high CRC risk) and of a decreased adenoma risk observed for alleles associated with higher circulating CRP levels suggest a protective effect for CRP in early colorectal neoplasia that warrants further study.