Kinetics of IL-8, MIP-1 alpha, TNF alpha, IL-1 beta, IL-1ra and IL-10 in human whole blood samples triggered by smooth and rough LPS.

An in vitro model for the study of sepsis mediators was used to investigate the effects of two different lipopolysaccharides (LPS), a smooth (LPS-S) and a rough (LPS-R) type, on the release of chemokines (IL-8 and MIP-1 alpha) and cytokines (TNF alpha, IL-1 beta, IL-1ra and IL-10) from human whole blood samples. TNF alpha level increased significantly vs. control, at 4 h and 8 h after the challenge with smooth and rough type of LPS respectively. Concentrations of the two chemokines studied, IL-8 and MIP-1 alpha, were significantly elevated following stimulation by both LPS, and reached concentrations significantly different from controls at 4, 8 and 24 h. After 24 h of incubation both LPS produced a significant IL-10 increase, although such change was more substantial with the rough type. Present data suggest an early and maintained release of chemokines regardless of the type of LPS used and often in absence of a significant increase in primary pro-inflammatory cytokines.

Microscopic observation of progressive immobilization of leishmania promastigotes in acridine orange stain.

To rapidly isolate Leishmania donovani promastigotes in samples from Novy-MacNeal-Nicolle (NNN) cultures, a method of staining with acridine orange was developed. Such vital staining combines the advantages of direct microscopic examination (e.g., observation of motility) with more accurate cytological and structural imaging of the stained parasites (usually obtained by Giemsa staining). Progressive immobilization of Leishmania promastigotes associated with a change in fluorescence color was also studied. Our findings may be useful for the early confirmation of a positive culture inoculated with a clinical sample.

Effect of RO 23-9424, cefotaxime and fleroxacin on functions of human polymorphonuclear cells and cytokine production by human monocytes.

The way in which an antibiotic interacts with host defences could influence the clinical outcome of many infectious diseases. The impact of RO 23-9424, a novel dual-action and extended-spectrum antibiotic, was studied on several functions of human polymorphonuclear neutrophils (PMNs). A significant (P < 0.05) increase of the superoxide (O2-) released by phorbol-myristate acetate (PMA) -stimulated PMN (10-100 mg/L) can be observed in the RO 23-9424 pre-treated cells. RO 23-9424, particularly at low dosages, showed an interesting but not statistically significant effect on PMN phagocytosis. Higher dosages of RO 23-9424 (50-200 mg/L) and fleroxacin (20-200 mg/L) significantly reduced PMN chemotaxis. Cytokine production by human monocytes were also evaluated after incubation with the antibiotic (100-200 mg/L) in both basal conditions and in response to endotoxin (lipopolysaccharide, LPS). In the LPS-treated cells, RO 23-9424 (100 mg/L) significantly (P < 0.05) enhanced the tumour necrosis factor-alpha (TNF-alpha) levels, compared with LPS controls after 4 h of incubation. RO 23-9424 (200 mg/L) was able to reduce in a dose-dependent way LPS-induced interleukin-1 beta (IL-1 beta) after 4 and 24 h of incubation. Interleukin-8 (IL-8) release was not significantly changed by RO 23-9424. Cefotaxime (200 mg/L) significantly (P < 0.05) increased the basal levels of IL-1 beta and reduced basal IL-8 concentration after 24 h of incubation. The lower concentration of cefotaxime reduced the LPS-stimulated IL-8 levels. Fleroxacin (100 mg/L) enhanced basal levels of IL-8. The potentiated PMN phagocytosis, the significantly enhanced O2- release by PMA-stimulated PMN and the dimetric changes of TNF-alpha and IL-1 beta appeared peculiar for RO 23-9424 and may have useful therapeutical implications.

Impairment of immunological functions in genetically epilepsy-prone rats.

1. In genetically epilepsy-prone rats (GEPR-9s), which represent a natural genetic model of epilepsy, we observed that the number of peritoneal macrophages was significantly lower with respect to normal rats, and that some functional parameters (i.e. phagocytosis and intracellular killing) of these macrophages were impaired. 2. The count of lymphocyte populations showed a predominance of T-helper over T-cytotoxic/suppressor both in the spleen and lymph nodes. Moreover, an increased T-cell/B-cell ratio was observed in GEPR-9s. Flow cytometry revealed that GEPR-9s spleens possessed a large percentage of T-helper cells in comparison to normal rats. 3. By using concanavalin A-induced proliferation of GEPR-9s cultured lymphocytes, we have shown increased functional activation. 4. We suggest that the alterations in T-cell functions in GEPR-9s could be due to the involvement of the neuroendocrine system in the modulation of immunity, in the shift between Th1 and Th2, and in the activation of stress response.

In vitro effects of novel glycopeptide antibiotics on the reactivity of the lipopolysaccharide (LPS) of S. minnesota R595.

Four novel glycopeptide antibiotics, namely MDL 62708, MDL 63155, MDL 62827, MDL 62873 (mideplanin), plus teicoplanin, which differ in their chemical structure, were used to examine the relationship between the structure of glycopeptides and their LPS neutralization activity. Compound MDL 62708 (100 micrograms/ml) significantly reduced (P < 0.01 vs. antibiotic-free LPS, by Fisher's PLSD Test) metachromatic reactivity of S. minnesota R595 LPS (500 micrograms/ml) as evaluated by the DMB test. The remaining glycopeptides showed a significant reduction of the metachromatic reactivity, although at concentrations (333 and 1000 micrograms/ml) higher than MDL 62708. Data obtained with LAL test appeared in accordance with those of the other techniques: all the glycopeptides used (100 and 1000 micrograms/ml) significantly (P < 0.05) reduced the reactivity of S. minnesota R595 LPS (50 pg/ml), and the lowest concentration of MDL 62708 (10 pg/ml) used produced a substantial, although not significant, reduction of the LPS reactivity with LAL. The antibiotic/LPS ratios associated to a significant reduction of LPS reactivity were 3.3/5 (wt/wt) and 2/1 (wt/wt) for DMB and LAL tests respectively. Such ratio appeared to be even lower for MDL 62708. In conclusion, the four new glycopeptides, when tested at an antibiotic/LPS ratio about 1000 times lower than that which can be found in vivo, were able to reduce the reactivity of LPS in the in vitro models used. Teicoplanin aglycone MDL 62708, which also lacks the teicoplanin fatty acid, seems to have the same anti-LPS activity as the parental antibiotic, thus suggesting an important role for the glycopeptide backbone and NH2 groups in LPS-neutralizing effect.

Meropenem: effects on human leukocyte functions and interleukin release.

Modifications of the immune response have been reported for a number of antimicrobial agents following both in vivo and in vitro experiments. Present results were obtained from in vitro incubated human polymorphonuclear cells (PMN) and monocytes treated with meropenem, a novel carbapenem antibiotic. Only the highest concentrations of meropenem (50-200 microg/ml) significantly reduced the phagocytic activity and unstimulated superoxide release of neutrophils after 1 h of incubation. Moreover meropenem (100 and 200 microg/ml) reduced PMA-stimulated superoxide release by PMN after 1 h of incubation. Only the highest concentration used (200 microg/ml) was found to reduce significantly superoxide release by PMA-unstimulated and -stimulated PMN incubated for 2 h. Meropenem did not affect some of the PMN functions studied (killing of Candida albicans, chemotaxis and glucose consumption) over a broad range of concentrations (5, 10, 20, 50, 100, 200 microg/ml). The leukocyte viability did not change even at the highest antibiotic concentration used, as showed by the trypan blue exclusion test. The LPS-induced release of IL-1alpha, IL-6 and IL-8 from isolated monocytes was not impaired by meropenem (100 and 200 microg/ml), that significantly reduced TNFalpha stimulated by LPS, after 4 h of incubation. In conclusion our data suggest that therapeutically relevant concentrations (5-20 microg/ml) of meropenem did not modify substantially the viability and the functions of human leukocytes.

Inhibition of endotoxin-induced interleukin 8 release by teicoplanin in human whole blood.

Antibiotic-induced endotoxin (lipopolysaccharide; LPS) release may precipitate septic shock. In the present study the effect of teicoplanin, which has been reported to neutralize LPS in experimental models, on LPS neutralization was investigated in human whole blood samples. Levels of interleukin 8, a preinflammatory cytokine which was stimulated by Salmonella minnesota R595 LPS (12.6 micrograms/ml), were monitored over time. Interleukin 8 concentrations increased over time up to 24 h. When LPS was preincubated with teicoplanin (antibiotic: LPS ratio 20:1, w/w), interleukin 8 concentrations were found significantly (p < 0.05) reduced at 4, 8 and 24 h after LPS challenge. Interleukin 1 beta (at 4, 8 and 24 h) and tumor necrosis factor alpha (at 8 and 24 h) levels were also significantly decreased by teicoplanin. In this experiment model, a teicoplanin:LPS ratio 100-fold less than the ratio achievable in plasma of septic shock patients was able to reduce interleukin 8, which has been correlated with the severity of septic disease.

Teicoplanin reduces in-vitro reactivity and murine lethality of Salmonella minnesota R595 lipopolysaccharide.

Three different tests were performed to investigate the effect of teicoplanin on lipopolysaccharide (LPS). After incubation for 3 h with teicoplanin, LPS from Salmonella minnesota R595 showed reduced reactivity in the metachromatic dimethyl-methylene blue assay and the limulus amoebocyte lysate test. In addition, galactosamine-sensitized mice had an increased survival rate, from 29% to 72%, when teicoplanin was pre-incubated for 3 h with the LPS to be injected intraperitoneally. The results suggest that teicoplanin may have a neutralizing effect on LPS.

Aminoglycosides modify the in vitro metachromatic reaction and murine generalized Shwartzman phenomenon induced by Salmonella minnesota R595 lipopolysaccharide.

Endotoxin-neutralizing activity may be an important property for antibiotics to be used in severe sepsis. Several antibiotics, belonging to different classes, were evaluated as to their endotoxin-neutralizing ability, using the inhibition of an in vitro metachromatic assay for lipopolysaccharides and a murine generalized Shwartzman reaction model. Gentamicin, amikacin, and sisomicin have been found to share significant in vitro antiendotoxin activity at an antibiotic/endotoxin ratio as low as 1.0/5 (by weight) and to reduce the murine generalized Shwartzman reaction at an antibiotic/endotoxin ratio of 3.3/5.

Biological effects of Veillonella parvula and Bacteroides intermedius lipopolysaccharides.

A comparative study on the endotoxic effects of lipopolysaccharide (LPS) from Veillonella parvula ATCC 10790 and from Bacteroides intermedius BMH was performed using an in vivo approach in the C57BL/6 mouse. Phenol-water extracted LPS of such anaerobes was purified by ultracentrifugation and DNase/RNase digestion, and characterized by a metachromatic assay for endotoxins and by electrophoresis on SDS-polyacrylamide gel and silver staining. Mouse LD50 for V. parvula LPS was 1.479 mg and for B. intermedius greater than 3.160 mg. Sublethal amounts of the LPS from anaerobes as well as from facultative aerobes decreased daily water intake and body weight in the mouse. Endotoxin from Salmonella typhimurium SL1102, Escherichia coli 0128:B12 and V. parvula had a strong effect on water intake and body weight, whereas Bacteroides intermedius LPS activity was very weak. The results of the present report suggest that V. parvula LPS has a toxic in vivo activity on mouse, which is comparable to LPS from classic enteric organisms and stronger than B. intermedius LPS.

Cefixime shows good effects on group A and group B beta-haemolytic streptococci.

There is continued interest in the development of oral beta-lactam compounds, which can be used clinically to treat various bacterial infections, particularly those caused by beta-haemolytic streptococci. Cefixime is a new orally active cephalosporin, with a broad spectrum of antibacterial activity, including Enterobacteriaceae, Haemophilus influenzae, Branhamella catarrhalis, Streptococcus pneumoniae and Streptococcus pyogenes. Cefixime is highly resistant to hydrolysis by most beta-lactamases. In this study the authors examined the effects of this molecule on Group A and Group B beta-haemolytic streptococci, recently isolated from clinical specimens in the authors' laboratory. MICs and the growth curves of 36 strains of Group A streptococci and the effects of sub-MICs on buccal cell adhesion were evaluated. The results show that concerning the sub-MIC cefixime effect on streptococci adherence, the treatment led to a decrease in adherence to the cells of the strains studied. Moreover cefixime showed good activity with 86.1% of the strains with MIC less than or equal to 0.5 microgram/ml, and the growth curves demonstrated that the molecule possesses a bactericidal effect after 3 h. Concerning Group B streptococci, 70.3% of the strains showed a MIC less than or equal to 2 micrograms/ml. In conclusion cefixime demonstrates good activity on beta-haemolytic streptococci, particularly those of Group A.

Cefixime does not affect polymorphonuclear cell and monocyte functions from chronic lymphoid leukemia patients and from healthy donors.

It is well documented that to evaluate the efficacy of an antibiotic treatment it is important to know the relationships between the drug and the cells belonging to the immune system, by studying the possible effects on some cellular functions, particularly in immunosuppressed and immunodeficient patients. We describe the influence of cefixime, a new orally administered cephalosporin, on some polymorphonuclear cell (PMN) and monocyte functions from healthy donors and from patients affected by chronic lymphoid leukemia (CLL).

In vitro activity of cefixime: correlation index between MIC and disc diffusion test.

In this study the in vitro antibacterial effects of cefixime are presented. Its activity was studied by evaluation of MICs, MBCs and the disc diffusion susceptibility method on Enterobacteriaceae, Staphylococcus aureus and Streptococcus pneumoniae. Moreover, the coefficient of correlation between MIC values and the disc diffusion susceptibility test was evaluated. The results support the wide antimicrobial activity of cefixime, which appears to be particularly effective against enterobacteria.

Different effects of bacterial lipopolysaccharide on superoxide anion production by macrophages from normal and tumor-bearing rats.

Bacterial endotoxins or lipopolysaccharides (LPS) exhibit a wide range of modulatory activities on immunocompetent cells. Among the numerous effects of LPS on macrophages, an enhancement of superoxide anion (O2-) release has been reported. In previous studies carried out on tumor-bearing rats, it was found that several functions of peritoneal macrophages such as phagocytic, microbicidal and antiviral activities were depressed. In this paper we evaluated the spontaneous or phorbol myristate acetate (PMA)-induced production of superoxide anion by macrophages from tumor-bearing rats with respect to controls. Moreover, the effect of in vitro priming with LPS on O2- production by the same cells was studied. It was found that the pattern of superoxide release by macrophages from tumor-bearing rats is significantly different from controls. Preincubation of macrophages from normal rats with LPS enhanced the spontaneous and PMA-induced production of O2-. In contrast, the same concentrations of LPS did not prime macrophages from tumor-bearing rats.

Splanchnic artery occlusion shock in the rat: effects of the calcium entry blockers nimodipine and verapamil.

Splanchnic artery occlusion (SAO) shock made by clamping splanchnic arteries for 45 min was performed in female rats infused for 30 min with vehicle, nimodipine, and verapamil before, during, or after SAO. Survival time, macrophage phagocytosis and killing, and white cell count were evaluated in conscious rats. Shocked animals pretreated with vehicle exhibited 24.6 +/- 0.9% phagocytic activity, 5.1 +/- 0.7% killing activity, and survived 177 +/- 1.7 min. Leukopenia was also present. Sham animals survived more than 300 min, and showed the following values: phagocytosis = 52.8 +/- 0.6%, killing = 18.8 +/- 0.6%. Pretreatment with nimodipine (1 microgram/kg/min x 30 min, intravenously [IV]) before SAO significantly prolonged survival time (274 +/- 4.7 min) and improved phagocytosis (38.1 +/- 0.6%) and killing (16.1 +/- 1.1%), but did not change leukopenia. Lower doses of nimodipine (0.25 and 0.5 microgram/kg/min x 30 min, IV), and all the doses of verapamil (100 and 200 micrograms/kg/min x 30 min, IV), when infused before SAO, were ineffective. Neither nimodipine nor verapamil, when infused for 30 min either during or immediately after SAO, were able to influence survival, macrophage functions, or white cell count. Moreover, nimodipine (5, 10, and 25 microM), when added "in vitro" to macrophages collected from SAO-shocked rats, significantly enhanced their phagocytic activity, while verapamil (100 and 200 microM) did not change it. Finally, in anaesthetized rats nimodipine pretreatment had a beneficial effect on the cardiovascular changes occurring during SAO shock. These data suggest that nimodipine has a beneficial effect in SAO-shocked rats only when given before SAO.

Effect of bacterial endotoxin on lipoxygenase and cyclo-oxygenase metabolites by rat neutrophils and correlation with cellular functional parameters.

The effect of Salmonella enteritidis endotoxin on in vitro rat neutrophil cyclo-oxygenase and lipoxygenase metabolism, phagocytic activity, superoxide (O2-) generation, and microbicidal activity was investigated. Incubation of polymorphonuclear leukocytes (PMN) with 5, 25, and 50 micrograms of endotoxin significantly enhanced synthesis of immunoreactive (i) leukotriene (LT)C4/D4 and thromboxane (Tx)B2 (P less than 0.001) as compared to control cells. Endotoxin 5 micrograms/ml produced optimal stimulation of the arachidonic acid metabolites. Calcium ionophore, A23187, significantly enhanced iLTC4/D4 and iTxB2 synthesis more than that elicited with endotoxin. Although phagocytic function was not significantly altered by endotoxin, intracellular killing of C. albicans demonstrated enhanced microbicidal activity at 5 micrograms/ml of endotoxin. Superoxide generation was significantly enhanced in neutrophils stimulated with phorbol myristate acetate (PMA). Endotoxin (5 micrograms/ml) further potentiated superoxide generation by these cells when stimulated by PMA. These findings demonstrate that endotoxin directly enhances neutrophil iLTC4/D4 and iTxB2 synthesis. The enhanced arachidonic acid metabolism elicited by endotoxin in these cells parallels increased microbicidal activity and superoxide generation.

Influence of ceftizoxime on the immune system.

In the last few years the interest about the influence on host/parasite relations exerted by antibiotics has increased. In this study we have studied the effect of ceftizoxime, a third generation cephalosporin, on some functional parameters of human macrophages and granulocytes. Ceftizoxime does not seem to exert any stimulatory effect on phagocytosis and chemotaxis, but at the same time it allows these cells to explicate their functions during an infective process. A separate series of experiments was designed in order to investigate the immunogenicity of ceftizoxime and its immunological cross-reactivity versus other beta-lactam antibiotics. The low ELISA title of ceftizoxime indicates its weak immunogenic power. Cross-reactivity was also very low (title 1:25) when ceftizoxime was tested against the other antibiotics.