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Effects of antimicrobial agents on spontaneous and endotoxin-induced cytokine release of human peripheral blood mononuclear cells
Abstract
Because the immunomodulatory effects of antibiotics could possibly influence the degree of the systemic and local response to infection, knowledge of their intrinsic influence on the host's inflammatory response appears to be essential. Therefore, this study investigated the effects of frequently used antimicrobial agents (beta-lactams, quinolones gentamicin, vancomycin and metronidazole) on the in-vitro tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 production of isolated human peripheral blood mononuclear cells (PBMNC), cultured with or without endotoxin, in comparison with those effects obtained in a whole-blood assay system. In the presence of ciprofloxacin, ofloxacin, gentamicin, vancomycin, and metronidazole, a significant inhibition of the endotoxin-stimulated TNF-alpha production of human peripheral blood mononuclear cells (PBMNC) was found at therapeutic levels. Only ofloxacin showed a significant inhibitory influence on the endotoxin-induced IL-6 production of PBMNC. In the whole-blood assay, significant effects were not detectable. None of the antibiotics showed cytotoxicity. It is concluded that, at present, the direct immunological effects of antibiotics should be interpreted carefully with regard to the experimental conditions, and regardless of the therapeutic implications. To assess the potential direct immunomodulatory effect of antimicrobial agents, different cell culture procedures should be used.
... Some of its metabolites have been shown to be mutagenic in certain bacterial test systems. In addition, it has been observed that MTZ induces DNA single-strand breaks in the lymphocytes of patients on standard doses of the drug678. Toxicological investigations following long-term administration of high doses of some 5-nitroimidazole derivatives to rats and mice showed induction of various tumors. ...
... The inhibitory effect of MTZ on macrophage phagocytic activity shown in the present study is supported by the data showing inhibition of macrophage TNF-α production (Fig. 13). Other researchers have found that the production of TNF-α by human peripheral blood mononuclear cells (PBMC) was also suppressed by MTZ [6], thus lending further support to the present work. It has been shown that MTZ is intracellularly activated by reduction and the toxic effect of reduced intermediates bind to DNA leading to loss of helical structure, strand breakage, and impairment of DNA function [7]. ...
The immunomodulatory effect of metronidazole (MTZ), a nitroimidazole drug used as an antiprotozoal and antibacterial agent, was investigated using Balb/c mice and human peripheral blood lymphocytes. For in vivo studies, mice were divided into six groups, six animals per group, group I received vehicle alone while the other groups (II-VI) received intraperitoneal injections of MTZ (14, 28, 42, 57, and 114 mg/kg) respectively. For in vitro studies different concentrations of MTZ (5, 10, 50, and 200 microg/ml) were used. MTZ showed a significant decrease in the percentage of circulating neutrophils and monocytes and an increase in the percentage of circulating lymphocytes. The relative weights of spleen as well as the relative body weight gain also decreased. Detectable changes were seen in the histology of spleen and thymus. Splenic plaque-forming cells (PFC), hemagglutination (HA) titer to sheep red blood cells (SRBC), spleenocytes and human peripheral blood lymphocytes proliferation (MLR) were markedly suppressed by MTZ treatment as compared to control group. MTZ also induced a significant decrease in delayed-type hypersensitivity (DTH) reaction, phagocytic activity (assessed by phagocytic capacity and phagocytic index) as well as TNF-alpha secretion by peritoneal macrophages. These observations indicate that MTZ significantly induced immunosuppression in mice and in human peripheral blood lymphocytes.
... It inverts leukocytic attachment and emigration responses by reducing rolling velocity [56]. When it contacts the monocytes, it decreases microbial endotoxin stimulated α-TNF, and thus the immunosuppression of the macrophage function [57]. The filers/excipients such as chitosan and Carbopol used in the formulation of the TAOP are organic compounds that have been employed in numerous drug delivery systems in the field of pharmaceutics and modern medicine. ...
Background and objective
Any drug or medicinal agent, when implanted into the body, gets biotransformed by various organ systems and the toxic byproducts of this process alter the normal physiological process. In this experimental study, we aimed to quantify the safety of newly formulated primary root canal obturating material by investigating the hematological and biochemical parameters related to liver function.
Methodology
Forty-eight Wistar rats (weighing 250-350 grams) were classified into three groups (n=16) through random allocation. Preoperative blood samples were collected by puncturing the orbital venous plexus, the values of which were used as control. Zinc oxide eugenol (ZOE), calcium hydroxide iodoform paste (Metapex), and newly formulated triple antibiotic obturating paste (TAOP) were implanted (100 µg) into dorsal connective tissues. Blood samples on the seventh, 15th, and 30th postoperative days were evaluated respectively by analyzing hematological, hepatic, and, renal function tests for acute and chronic inflammatory responses.
Results
The intra-group and inter-group comparisons among all the test materials after seven days exhibited high significance in terms of hemoglobin (Hb), mean corpuscular volume (MCV), neutrophils, and serum glutamic-oxaloacetic transaminase (SGOT) (p<0.001), while others showed mixed responses (p<0.05 to p>0.05). After 15 days, the comparisons showed high significance with respect to packed cell volume (PCV), mean cell volume (MCV), and serum creatinine (p<0.001), while others showed significant to nonsignificant differences (p<0.05 to p>0.05). At the end of 30 days, all the parameters showed mixed responses (p<0.001 to p>0.05).
Conclusion
The newly formulated obturating material TAOP showed lower adverse hematological, hepatic, and renal effects in experimental animals compared to other test materials, with most parameters reverting to normal after 30 days.
... Studies in animal models have demonstrated that metronidazole also has a therapeutic effect on liver injury in PSC[60]. For example, Karvonen et al [98] found that treating patients with PSC with both UDCA and metronidazole significantly reduces the serum glutamyl transpeptidase and ALP levels, and significantly improves the MRS and pathological staging compared with those treated with only UDCA. Furthermore, Krehmeier et al [99] reported that metronidazole reduced intestinal permeability, decreased bacterial endotoxin entry into the blood, inhibited endotoxin-induced TNF-α production, inhibited hepatic Kupffer cells and macrophage activation, reduced chemokine and cytokine secretion by biliary epithelial cells, attenuated liver inflammation, and prevented PSC-like bead-like liver injury. ...
Primary sclerosing cholangitis (PSC) is an autoimmune disease characterized by chronic cholestasis, a persistent inflammation of the bile ducts that leads to sclerotic occlusion and cholestasis. Gut microbes, consisting of microorganisms colonized in the human gut, play an important role in nutrient intake, metabolic homeostasis, immune regulation, and immune regulation; however, their presence might aid PSC development. Studies have found that gut-liver axis interactions also play an important role in the pathogenesis of PSC. Patients with PSC have considerably reduced intestinal flora diversity and increased abundance of potentially pathogenic bacteria. Dysbiosis of the intestinal flora leads to increased intestinal permeability, homing of intestinal lymphocytes, entry of bacteria and their associated metabolites, such as bile acids, into the liver, stimulation of hepatic immune activation, and promotion of PSC. Currently, PSC effective treatment is lacking. However, a number of studies have recently investigated the targeted modulation of gut microbes for the treatment of various liver diseases (alcoholic liver disease, metabolic fatty liver, cirrhosis, and autoimmune liver disease). In addition, antibiotics, fecal microbiota transplantation, and probiotics have been reported as successful PSC therapies as well as for the treatment of gut dysbiosis, suggesting their effectiveness for PSC treatment. Therefore, this review briefly summarizes the role of intestinal flora in PSC with the aim of providing new insights into PSC treatment.
... 24 Metronidazole is considered to exert its efficacy against the inflammatory lesions (papules/pustules) associated with rosacea via anti-inflammatory and immunomodulatory pathways. [25][26][27] Reactive oxygen species (ROS) and oxidative stress are known to be strongly associated with a range of skin conditions, 28 and it is thought that topical metronidazole can both decrease the production of ROS and also act as a scavenger. 29 Although metronidazole has been evaluated in multiple studies in rosacea patients outside of Japan, there is no clinical evidence from randomized controlled trials conducted within Japan. ...
Topical metronidazole is not currently approved in Japan as a treatment for the indication of rosacea, although 0.75% metronidazole gel was authorized in 2014 for the management of cancerous skin ulcers. We conducted a randomized, double‐blind, vehicle‐controlled study to evaluate the efficacy and safety of 0.75% metronidazole gel in Japanese patients with inflammatory lesions (papules/pustules) and erythema associated with moderate to severe rosacea. Overall, 130 patients were randomly assigned to receive 0.75% metronidazole gel (n = 65) or vehicle (n = 65), and 120 patients completed 12 weeks of treatment. The primary efficacy outcome was the proportion of patients who achieved both of the following at week 12: an improvement of >50% in the number of inflammatory lesions (papules/pustules) and a positive change of at least one degree in erythema severity. This composite outcome was achieved by 72.3% of metronidazole‐treated patients versus 36.9% of vehicle‐treated patients, with the between‐group difference demonstrating significant improvement with 0.75% metronidazole gel (p < 0.0001). All secondary efficacy endpoints (patients achieving a score of ≥3 for percent change in the number of inflammatory lesions at week 12; patients achieving a score of ≥3 for change in erythema severity at week 12; patients achieving an Investigator’s Global Assessment score of 0 or 1 at week 12; percent change over time in the number of inflammatory lesions; change over time in erythema severity) also showed improvement in the 0.75% metronidazole gel group. The incidence of adverse events was higher with metronidazole (40.0%) than with vehicle (29.2%). Of these, treatment‐related, treatment‐emergent adverse events occurred in 9.2% and 6.2% in the metronidazole and the vehicle group, respectively, but there were no new safety concerns. Overall, the results of this study have confirmed the efficacy and safety of 0.75% metronidazole gel in Japanese patients with rosacea.
... In addition, some antibiotics have been found to inhibit not only bacterial viability but also toxin release as well as to have direct immunomodulatory properties. For example, VAN at therapeutic concentrations inhibits the production of tumor necrosis factor-␣ from human peripheral blood mononuclear cells stimulated by bacterial lipopolysaccharide (37,38). It is thus possible that such an immunomodulatory action of VAN ameliorated the inflammatory response in VRE2-infected eyes. ...
Endophthalmitis due to infection with Enterococcus spp. progresses rapidly and often results in substantial and irreversible vision loss. Given that the frequency of this condition caused by vancomycin-resistant Enterococcus faecalis has been increasing, the development of novel therapeutics is urgently required. We have now demonstrated the therapeutic potential of bacteriophage ΦEF24C-P2 in a mouse model of endophthalmitis caused by vancomycin-sensitive (EF24) or vancomycin-resistant (VRE2) strains of E. faecalis . Phage ΦEF24C-P2 induced rapid and pronounced bacterial lysis in turbidity reduction assays with EF24, VRE2, and clinical isolates derived from patients with E. faecalis –related postoperative endophthalmitis. Endophthalmitis was induced in mice by injection of EF24 or VRE2 (1 × 10 ⁴ cells) into the vitreous. The number of viable bacteria in the eye increased to >1 × 10 ⁷ colony forming units and neutrophil infiltration into the eye was detected as an increase in myeloperoxidase activity at 24 h after infection. A clinical score based on loss of visibility of the fundus as well as the number of viable bacteria and the level of myeloperoxidase activity in the eye were all significantly decreased by intravitreous injection of ΦEF24C-P2 6 h after injection of EF24 or VRE2. Whereas histopathologic analysis revealed massive infiltration of inflammatory cells and retinal detachment in vehicle-treated eyes, the number of these cells was greatly reduced and retinal structural integrity was preserved in phage-treated eyes. Our results thus suggest that intravitreous phage therapy is a potential treatment for endophthalmitis caused by vancomycin-sensitive or -resistant strains of E. faecalis.
... Concurrent administration of subtherapeutic doses of amphotericin B and a pentoxifylline analog led to increased survival times in experimental candidiasis in mice (41), whereas amphotericin B increased the expression of IL-1 in human mononuclear cells in vitro (42). However, findings on the immunological properties of conventional antimicrobial agents remain inconclusive to date, with variations in findings in part depending on the experimental design (43). We did not observe any effects on cytokine production when testing VAN, GEN, and AMB on TLR2 and TLR4 agonist-stimulated cord blood samples in the absence of live microorganisms. ...
Introduction: Neonatal sepsis and its accompanying inflammatory response contribute to substantial morbidity and mortality. Pentoxifylline (PTX), a phosphodiesterase inhibitor which suppresses transcription and production of pro-inflammatory cytokines, is a candidate adjunctive therapy for newborn sepsis. We hypothesized that PTX decreases live microbe-induced inflammatory cytokine production in newborn blood.
Methods: Cord blood was stimulated with live microorganisms commonly encountered in newborn sepsis ( Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis , or Candida albicans ), and simultaneously treated with antimicrobial agents (gentamicin, vancomycin, or amphotericin B) and/or clinically relevant concentrations of PTX. Microbial colony counts were enumerated by plating, supernatant cytokines measured by multiplex assay, intracellular cytokines and signaling molecules by flow cytometry, and mRNA by qRT PCR.
Results: PTX inhibited concentration-dependent E. coli -, S. aureus-, S. epidermidis- , and C. albicans -induced TNF and E. coli -induced IL-1β production in whole blood, with greater suppression of pro-inflammatory cytokines in combination with antimicrobial agents. Likewise, PTX suppressed E. coli -induced monocytic TNF and IL-1β, whereby combined PTX and gentamicin led to significantly greater reduction of TNF and IL-1β. The anti-inflammatory effect of PTX on microbe-induced pro-inflammatory cytokine production was accompanied by inhibition of TNF mRNA expression, and was achieved without suppressing the production of the anti-inflammatory IL-10. Of note, microbial colony counts in newborn blood were not increased by PTX.
Conclusion: Our findings demonstrated that PTX inhibited microbe-induced pro-inflammatory cytokine production, especially when combined with antimicrobial agents, without enhancing microbial proliferation in human cord blood in vitro , thus supporting its utility as candidate adjunctive agent for newborn sepsis.
... For instance, both in vitro and in vivo studies show that fluoroquinolones suppress the release of inflammatory cytokines (particularly TNF-α) from lymphocytes and monocytes while increasing IL-10. [39][40][41] The β- lactam antibiotics similarly modulate the production of inflammatory cytokines. [42][43][44][45][46][47] And both ceftiofur and enrofloxacin appear to decrease B cell maturation. ...
Infections are common after stroke and associated with worse outcome. Clinical trials evaluating the benefit of prophylactic antibiotics have produced mixed results. This study explores the possibility that antibiotics of different classes may differentially affect stroke outcome.
Lewis rats were subjected to transient cerebral ischemia (2 hours) and survived for 1 month. The day after stroke they were randomized to therapy with ceftiofur (a β-lactam antibiotic), enrofloxacin (a fluoroquinolone antibiotic), or vehicle (as controls) and underwent the equivalent of 7 days of treatment. Behavioral tests were performed weekly until euthanization. In a subset of animals, histology was done.
There were no differences in outcomes at 24 hours or 1 week after stroke among the different groups. At 1 month after stroke, however, performance on the rotarod was worse in enrofloxacin-treated animals when compared with control animals.
Independent of infection, the antibiotic enrofloxacin was associated with worse stroke outcome. These data echo the clinical observations to date and suggest that the secondary effects of antibiotics on stroke outcome should be considered when treating infection in subjects with stroke. The mechanism by which this antibiotic affects outcome needs to be elucidated.
© 2015 American Heart Association, Inc.
... Antibiotic including L and V are known to have immunomodulatory effects during S. aureus infection [20,23,[38][39][40][41][42][43][44][45]. Consistent with previously reported findings [45,[50][51][52][53][54], treatment with both L and V reduced proinflammatory cytokine production in the serum of S. aureus infected mice, albeit by different mechanisms (Figure 3). Evidence from previous studies suggests that the network of inflammatory cytokines and chemokines play a major role in mediating, amplifying, and perpetuating inflammation [55,56]. ...
Linezolid (L), a potent antibiotic for Methicillin Resistant Staphylococcus aureus (MRSA), inhibits bacterial protein synthesis. By contrast, vancomycin (V) is a cell wall active agent. Here, we used a murine sepsis model to test the hypothesis that L treatment is associated with differences in bacterial and host characteristics as compared to V. Mice were injected with S. aureus USA300, and then intravenously treated with 25 mg/kg of either L or V at 2 hours post infection (hpi). In vivo alpha-hemolysin production was reduced in both L and V-treated mice compared to untreated mice but the reduction did not reach the statistical significance [P = 0.12 for L; P = 0.70 for V). PVL was significantly reduced in L-treated mice compared to untreated mice (P = 0.02). However the reduction of in vivo PVL did not reach the statistical significance in V- treated mice compared to untreated mice (P = 0.27). Both antibiotics significantly reduced IL-1β production [P = 0.001 for L; P = 0.006 for V]. IL-6 was significantly reduced with L but not V antibiotic treatment [P<0.001 for L; P = 0.11 for V]. Neither treatment significantly reduced production of TNF-α. Whole-blood gene expression profiling showed no significant effect of L and V on uninfected mice. In S. aureus-infected mice, L altered the expression of a greater number of genes than V (95 vs. 42; P = 0.001). Pathway analysis for the differentially expressed genes identified toll-like receptor signaling pathway to be common to each S. aureus-infected comparison. Expression of immunomodulatory genes like Cxcl9, Cxcl10, Il1r2, Cd14 and Nfkbia was different among the treatment groups. Glycerolipid metabolism pathway was uniquely associated with L treatment in S. aureus infection. This study demonstrates that, as compared to V, treatment with L is associated with reduced levels of toxin production, differences in host inflammatory response, and distinct host gene expression characteristics in MRSA sepsis.
... The direct impact of resveratrol on various types of inflammation induced by pro-inflammatory stimuli in different organs has been studied in recent years [34,35]. Up to now, most of the research work concerning the direct immunological action of antimicrobial agents has been performed using isolated immunocytes ex vivo [36] , but data on the potential immunomodulating capacities of medicinal phytochemicals in a natural environment have been explored less. Resveratrol is known as a regulatory agent in cells and recent research has shown it to act as a pleiotropic and multistage biological effecter, but the effects on cell viability, NO production and anti-inflammatory properties in vitro vary with the cell lines used. ...
Periodontitis is a multifactorial polymicrobial infection characterized by a destructive inflammatory process. Porphyromonas gingivalis, a Gram-negative anaerobic black-pigmented rod, which produces several virulence factors that stimulate human periodontal ligament cells (HPLCs) to produce various inflammatory mediators, has been implicated as a crucial etiologic agent in the initiation and progression of periodontitis. Since natural polyphenols such as resveratrol have growth-inhibitory effects on some bacterial pathogens and have shown chemo-preventive, anti-inflammatory and antioxidant activity, in the present study we used an HPLC model stimulated with lipopolysaccharide (LPS) of P. gingivalis to simulate the in vivo conditions such as those found in diseased periodontal sites. To determine whether resveratrol interferes with P. gingivalis LPS-activity and reduces the production of pro-inflammatory molecules, we investigated its effect on the cytokines IL-1β, IL-6, IL-8, IL-12 and TNF-α and NO production of HPLCs. The results showed that resveratrol treatment decreased in a dose- and time-dependent manner the NO expression induced by P. gingivalis LPS, correlated to an increased viability of infected HPLCs, and decreased the production of pro-inflammatory cytokines in HPLCs stimulated by P. gingivalis LPS. These results suggest that the ability of resveratrol to determine immunomodulatory effects could provide possible therapeutic applications for the treatment of periodontitis.
... In separate assays, with other stimuli, neither direct addition of MTZ nor of its metabolite influenced mononuclear cell proliferation (Bell et al. 2004). In an in vitro study with human peripheral blood mononuclear cells, MTZ displayed considerable inhibition of endotoxin-stimulated TNF-␣ generation, at therapeutic levels than in a whole-blood assay system (Krehmeier et al. 2002). Based on these findings, immunosuppression by MTZ is evident in the macrophage function. ...
Metronidazole (MTZ) is a nitroimidazole antibiotic used mainly for the treatment of infections caused by susceptible organisms, particularly anaerobic bacteria and protozoa. Distinct from its antibiotic, amoebicidal, and antiprotozoal effects, MTZ displays immunopharmacological behaviour. This review outlines multiple effects of MTZ on different aspects of immunity, including innate and acquired immunity, and also highlights the immunopharmacological behaviour of MTZ in terms of its relevance to inflammation, delayed type hypersensitivity (DTH) and graft versus host disease (GVHD).
... 9 In humans, metronidazole has been shown to induce significant inhibition of the endotoxin-stimulated tumor necrosis factor alpha production of human peripheral blood mononuclear cells at therapeutic levels. 10 Metronidazole also has beneficial effects in healing intestinal damage, reduces intestinal permeability, and has a direct protective effect on the uncoupling of mitochondrial oxidative phosphorylation caused by nonsteroidal anti-inflammatory drugs. 11 At this time no effective medical therapy is available for treatment of PSC. ...
No effective medical therapy is currently available for primary sclerosing cholangitis (PSC). Ursodeoxycholic acid (UDCA) improves liver enzymes, but its effect on liver histology is controversial. Metronidazole (MTZ) prevents PSC-like liver damage in animal models and reduces intestinal permeability. We recruited 80 patients with PSC into a randomized placebo-controlled study to evaluate the effect of UDCA and MTZ (UDCA/MTZ) compared with UDCA/placebo on the progression of PSC. Patients (41 UDCA/placebo and 39 UDCA/MTZ) were followed every third month. Assessment of liver function test, histological stage and grade, and cholangiography (via ERCP) at baseline showed no differences between the groups. After 36 months, serum aminotransferases gamma-glutamyltransferase, and alkaline phosphatase (ALP) decreased markedly in both groups, serum ALP more significantly in the UDCA/MTZ group (-337 +/- 54 U/L, P < .05) compared with the UDCA/placebo group. The New Mayo Risk Score decreased markedly only in the UDCA/MTZ group (-0.50 +/- 0.13, P < .01). The number of patients with improvement of stage (P < .05) and grade (P < .05) was higher in the combination group. ERCP findings showed no progression or improvement in 77% and 68% of patients on UDCA/MTZ and UDCA/placebo, respectively. In conclusion, combining MTZ with UDCA in PSC improved serum ALP levels and New Mayo Risk Score, but no statistically significant effect on disease progression as assessed via liver histology or ERCP was seen. Long-term studies using a higher dose of UDCA combined with MTZ in larger patient populations are indicated.
Aim: Metronidazole (MTZ) is an antimicrobial agent used to treat anaerobic infections. It has been hypothesized that MTZ may also have anti-inflammatory properties, but the evidence is limited and has not been previously reviewed. Thus, this scoping review aimed to answer the following question: “What is the evidence supporting anti-inflammatory properties of metronidazole that are not mediated by its antimicrobial effects?”
Methods: A scoping review was conducted according to the PRISMA-ScR statement. Five databases were searched up to January 2023 for studies evaluating the anti-inflammatory properties of MTZ used as monotherapy for treating infectious and inflammatory diseases.
Results: A total of 719 records were identified, and 27 studies (21 in vivo and 6 in vitro) were included. The studies reported experimental evidence of MTZ anti-inflammatory effects on (1) innate immunity (barrier permeability, leukocyte adhesion, immune cell populations), (2) acquired immunity (lymphocyte proliferation, T-cell function, cytokine profile), and (3) wound healing/resolution of inflammation.
Conclusion: Taken together, this scoping review supported a potential anti-inflammatory effect of MTZ in periodontitis treatment. We recommend that future clinical studies should be conducted to evaluate specific MTZ anti-inflammatory pathways in the treatment of periodontitis.
Introduction:
Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease associated with inflammation, fibrosis, and destruction of intra- and extrahepatic bile ducts. Despite substantial recent advances in our understanding of PSC, the only proven treatment of PSC is liver transplantation. There is an urgent unmet need to find medical therapies for this disorder.
Areas covered:
Multiple drugs are currently under evaluation as therapeutic options for this disease. This article summarizes the literature on the various novel therapeutic options that have been investigated and are currently under development for the treatment of PSC.
Expert opinion:
In the next decade, more than one drug will likely be approved for the treatment of the disease, and we will be looking at combination therapies for the optimal management of the disease.
The host defense response to microbial challenge emerging from the root canal system leads to apical periodontitis. The aim of this study was to evaluate the expression of inflammatory cytokines and Nitric Oxide (NO) by macrophages after interaction with Enterococcus faecalis in the: plankton and dislodged biofilm mode; intact biofilm mode stimulated by calcium hydroxide (CH), CH and chlorhexidine (CHX) or Triple Antibiotic Paste (TAP). For this purpose, culture of macrophages from monocytes in human peripheral blood (N=8) were exposed to the different modes of bacteria for 24 hours. Subsequently, the cytokines, such as, Tumor Necrotic Factor- alfa (TNF-α), interleukin (IL)-1β, IL-6, IL-10; and NO were quantified by Luminex xMAP and Greiss reaction, respectively. In addition to the potential therapeutic effects of the intracanal medication, their antimicrobial activity against Enterococcus faecalis biofilm were also tested in vitro by confocal microscopy. The experiments` data were analyzed by the Kruskal-Wallis test with the Dunn post hoc test (α < 0.05). Bacteria in dislodged biofilm mode were shown to be more aggressive to the immune system than bacteria in plankton mode and negative control, inducing greater expression of NO and TNF-α. Relative to bacteria in intact biofilm mode, the weakest antimicrobial activity occurred in Group CH. In Groups CH/CHX and TAP the percentage of dead bacteria was significantly increased to the same extent. Interestingly, the biofilm itself did not induce the release of pro-inflammatory cytokines - except for NO - while the biofilm treated with TAP and CH based pastes enhanced the levels of IL-6 and TNF-α; and IL-1 β, respectively. In contrast, the levels of a potent anti-inflammatory (IL-10) were increased in Group TAP.
Introduction:
Ciprofloxacin, amoxicillin, and metronidazole are antibiotics used in regenerative endodontic therapy (RET). Although their antimicrobial properties are well-documented, there is a lack of information on the effects of these antibiotics on the immune response by host macrophages and periapical healing. Thus, this study had 2 objectives: (1) to determine the immune response of macrophages to bacterial infection in response to the combination of ciprofloxacin or amoxicillin and metronidazole and (2) using conditioned media produced by these macrophages to simulate the periapical microenvironment, to determine the impact on the expression of extracellular matrix (ECM) components by periodontal fibroblasts.
Methods:
Macrophages were treated with ciprofloxacin and metronidazole or amoxicillin and metronidazole at 10-1000 μg/mL. The treated macrophages were exposed to lipopolysaccharide, and the pro- and anti-inflammatory cytokines produced were quantified with enzyme-linked immunosorbent assay. Periodontal fibroblasts were treated with conditioned media from these treated macrophages, and the expression of ECM genes was determined by quantitative polymerase chain reaction.
Results:
Lipopolysaccharides elicited the production of proinflammatory cytokines interleukin 1 beta and tumor necrosis factor alpha by macrophages, but this was suppressed by ciprofloxacin and metronidazole. Moreover, only conditioned media from macrophages treated with ciprofloxacin and metronidazole rescued microbial-induced down-regulation of ECM genes by periodontal fibroblasts. Specifically, ciprofloxacin was the antibiotic responsible for these observations. In contrast, these effects were not observed with amoxicillin and metronidazole.
Conclusions:
Apart from disinfection of the root canal system, the combination of ciprofloxacin and metronidazole also exerts an immunomodulatory effect, which may aid in periapical healing.
The effects levofloxacin (fluoroquinolone) and vaccinal BCG strain on cytokine production by blood mononuclear leukocytes was studied in patients with infiltrative pulmonary tuberculosis. Combined treatment with levofloxacin and vaccinal BCG strain suppressed the production of TNFα in drug-resistant pulmonary tuberculosis and production of IL-12 and IFNγ in drug-sensitive tuberculosis.
Alarming increase of death due to S. aureus sepsis demands newer treatment strategies. Enhancement of antibiotic resistant S. aureus strains caused increased mortality. Only antibiotic treatment for Staphylococcal sepsis has been found insufficient to improve outcomes. In the innate immune response, phagocytosis mediated killing of pathogen and further triggering of intracellular signaling cascades by the PRRs culminates in the release of a variety of pro inflammatory cytokines, which orchestrate together in the early host response to infection. Increased production of inflammatory cytokines not only delineate pathogen burden but also affects host cell by triggering inflammation. Therefore, combinational therapy of Ascorbic acid is used along with antibiotics Ofloxacin (OFX) or Chloramphenicol (CHL) to kill S. aureus by mouse peritoneal macrophages. For this ROS like H2O2, superoxide anion and NO production was accessed, TLR2 and COX2 expression was monitored. Pro-inflammatory cytokines along with antioxidant levels were also analyzed. Ascorbic acid along with antibiotics OFX or CHL promoted bacterial clearance at early infection by increasing H2O2 and O2-.NO production has been found to decrease, providing protection against harmful per-oxynitril ion. Increase in TLR-2 expression resulted in enhanced phagocytosis and subsequently more killing. Treatment with Ascorbic acid decreased proinflammatory cytokines and inflammatory markers like iNOS and COX2. This combination increased antioxidant enzymes like SOD, Catalase, GSH as well as decreased LPO, thus balancing ROS and antioxidant status inside the cell. Thus in-vitro augmentation of bacterial clearance along with regulated inflammation as found by decrease in proinflammatory cytokines like TNF-α IFN-γ,IL-6 and inflammatory markers like COX2may be considered as a novel and important therapeutic strategy.
Unlabelled:
Periodontitis represents a chronic bacterial infection that induces immuno-inflammatory conditions affecting gingiva and tooth-supporting tissues. The role of some biological mediators in periodontal disease was widely investigated, especially that of MMP-8 and MMP-9. Recently, MMP-2 was also considered to be an appropriate therapeutic target for prevention of periodontal disease progression. However, effects of the combination of metronidazole with amoxicillin or spiramycin on the release and activation of MMP-2 and the balance MMP-2÷TIMP-2 were rarely studied. This study was designed to assess the influence of two combinations of antibiotics used for treatment of periodontitis on the balance MMP-2÷TIMP-2. Gingival samples obtained from patients with no pharmacological treated chronic periodontitis and those receiving either the association between amoxicillin-metronidazole and spyramicin-metronidazole were processed for paraffin embedding and then used to perform immunohistochemical reactions in order to detect MMP-2 and TIMP-2. All subjects were evaluated clinically and radiographic at the first visit and after treatment completed, the Loe & Silnees gingival index at six sites per tooth for the whole mouth being recorded. Statistical analysis was performed using non-parametrical techniques. Gingiva samples from untreated chronic periodontitis patients revealed a diffuse positive reaction for MMP-2 in the epithelium and also in fibroblasts and macrophages from the lamina propria. For gingiva samples from patients treated with antibiotics, MMP-2 positive reaction was restricted to deep epithelial layers and few cells of the connective tissue. No significant difference was observed for TIMP-2 expression. The clinical indexes were in accordance with immunohistochemical results. After treatment, gingival index values were significantly lower then before (p<0.001) in both groups treated with antibiotics.
Conclusions:
The two combinations of antibiotics tested in our study seem to have a dual ability to reduce inflammation as well as to inhibit MMP-2 activity.
Chronic periodontitis is initiated by subgingival biofilm depots that induce a release of a cascade of inflammatory molecules in periodontal structures which finally lead to the destruction of the supporting tissues and alveolar bone. One of these cytokines is the transforming growth factor-beta 1 (TGF-beta 1) associated with inflammatory host response, but also involved in healing and fibrosis. Correlations between periodontal tissues degradation and TGF-beta 1 expression have been intensively investigated, but the studies regarding modulation of TGF-beta 1 expression under systemic antibiotic therapy are scarce. In this study we assessed the effect of two combinations of antibiotics used to treat chronic periodontitis on the relation between TGF-beta 1 expression and the indexes used for clinical evaluation of periodontium destruction. Gingival samples collected from patients with untreated chronic periodontitis and those receiving either the combination amoxicillin-metronidazole or spiramycin-metronidazole were processed for paraffin embedding and then stained for routine histological examination and immunohistochemistry in order to assess the morphological changes and to detect the localization of TGF-beta 1. TGF-beta 1 displayed different patterns of expression between the groups included in the study, being weaker and scarce in samples from patients treated with antibiotics in contrast with untreated subjects. The clinical index was in accordance with immunohistochemical results. As a conclusion, TGF-beta 1 may have a contribution not only to the pathogenesis of periodontal disease but also in the healing mechanism by means of antibiotic treatment.
The ability to monitor and confirm adequate treatment of latent TB infection (LTBI) would be a major advance. The potential immunomodulatory effects of anti-tuberculous drugs and steroids need to be considered in assessing the utility of cytokine-based assays for this purpose.
We determined whether anti-tuberculous antibiotics or dexamethasone affect the production of IFN-γ and other potential cytokine biomarkers (TNF-α, IL-1ra, IL-2, IL-10, IL-13, IP-10, MIP-1β) in the QuantiFERON-TB Gold In-Tube (QFT-IT) assay. Blood from ten adults with LTBI was added to one standard set of QFT-IT tubes and five further sets containing therapeutic concentrations of either isoniazid, rifampicin, isoniazid and rifampicin, ciprofloxacin or dexamethasone. Resulting supernatants were analysed by ELISA (QFT-IT assay IFN-γ) and xMAP-Luminex assays (all cytokines).
Anti-tuberculous antibiotics had only a limited effect on categorical QFT-IT assay results and the production of cytokines. In contrast, dexamethasone resulted in a change in categorical results from positive to negative in four of ten patients, and caused a marked reduction in IL-13 and IL-1ra responses.
Substantial changes in TB-antigen-induced IFN-γ and other cytokine responses during treatment likely primarily reflect host immunological changes rather than immunomodulatory effects of anti-tuberculous antibiotics. Results from cytokine-based assays in patients on corticosteroids should be interpreted with caution.
Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.
Periodontitis is a multifactorial polymicrobial infection characterized by a destructive inflammatory process affecting tooth-supporting tissues and resulting in periodontal pocket formation, alveolar bone resorption and, eventually, tooth loss. The continuous challenge of host immune and resident cells by periodontopathogens and their virulence factors, such as lipopolysaccharide (LPS), results in enhanced and uncontrolled secretion of cytokines. The latter directly or indirectly participate in tissue destruction and bone resorption. Metronidazole (MTZ) is a widely used antimicrobial agent. The immunomodulatory effects of antibiotics might influence the degree of the local response to infection on the human periodontal ligament cell (HPLC). HPLCs play a role in the immune response of the oral cavity. In addition, HPLC can produce cytokines that increase the inflammatory response and that supply for normal communication. MTZ has also been proposed in the field of periodontal therapy either with a systemic administration or with local biodegradable sustained-release agents. The local administration of MTZ in the form of gel significantly reduces the systemic side effects. The aim of the present study, was to simulate the in vivo conditions occurring in diseased periodontal sites, and to evaluate the effects of MTZ on the viability of isolated HPLCs. The ability of MTZ to modulate the release of interleukin (IL)-1beta, IL-6, IL-8, IL-12 and tumor necrosis factor alpha (TNF-alpha) in HPLC, treated or not with LPS of Porphyromonas gingivalis was also evaluated. The results obtained showed that MTZ had no cytotoxic effect on HPLC and was able to inhibit the production of pro-inflammatory cytokines analyzed. The ability of MTZ to determine immunomodulatory effects could provide possible therapeutic applications in the field of periodontal research.
Clostridium difficile infection is an increasing burden to the health care system, totaling more than $1 billion/year in the United States. Treatment of patients with C difficile infection with metronidazole or vancomycin reduces morbidity and mortality, although the number of patients that do not respond to metronidazole is increasing. Despite initial response rates of greater than 90%, 15%-30% of patients have a relapse in symptoms after successful initial therapy, usually in the first few weeks after treatment is discontinued. Failure to develop specific antibody response has recently been identified as a critical factor in recurrence. The review discusses the different management strategies for initial and recurrent symptomatic C difficile infections.
Evaluation of anti-adhesive gels and bioresorbable films in animal models of intra-abdominal infection has shown that a product of the cross-linking reaction between hyaluronic acid (HA) and CM-cellulose, 1-ethyl-3-(3-dimethyl aminopropyl)urea dihydrochloride (EDU), has immunomodulatory properties. The effects of EDU were evaluated by using an endotoxin-induced shock mouse model. Pre-treatment of mice with EDU (50 mg kg(-1)) in DMSO resulted in a significant reduction in mortality following injection of LPS, compared to vehicle (DMSO) pre-treatment alone. Serum levels of TNF-alpha, IL1beta and IFN-gamma in EDU-treated mice were significantly lower than those in vehicle-treated mice. Nitric oxide (NO) concentrations in the sera of mice after inoculation with LPS were significantly lower in the EDU-treated group than in the vehicle-treated group at various time-points. In contrast, EDU pre-treatment was associated with an enhanced IL10 response after LPS injection, compared to vehicle pre-treatment alone. In vitro studies revealed that IL10 production by RAW 264.7 macrophages, elicited by LPS, was increased significantly when EDU was added to the culture medium. These results suggest that the protective effect of EDU during LPS-induced shock in mice is the result of inhibition of proinflammatory cytokines and NO production and an enhanced IL10 response.
The pathogenesis of sepsis begins with the proliferation of micro-organisms at a site of infection, followed by invasion of the bloodstream and other organs. Gram-negative bacteria account for a large part of sepsis cases. The structural component of Gram-negative bacteria, endotoxin or lipopolysaccharide (LPS), induces the synthesis and release of endogenous mediators of sepsis. A growing number of investigations of the molecular mechanisms occurring in sepsis, point to endotoxin as a central mediator leading to multi-organ failure and death. In numerous clinical trials, attempts to target molecules downstream of endotoxin have been made, but have not been associated with improved survival. We describe an affinity-based system for the selective removal of endotoxin from plasma. The small-scale device, a 1.5 ml cartridge, contains beads that bind endotoxin with high specificity and efficiency. In addition, evidence is presented that this device does not affect plasma hemostasis, nor does it activate the complement system. Taken together, these results represent a proof of principle for endotoxin removal from plasma, which may be of clinical value to treat sepsis by extracorporeal circulation of the blood through a scaled-up version of this endotoxin-removing device.
Lymphomatoid granulomatosis (LG) is a rare T cell rich, B cell non-Hodgkin's lymphoma which is difficult to diagnose. We present a patient with LG who demonstrated many of the difficulties in diagnosis and highlighted the importance of reviewing the diagnosis if treatment does not have the anticipated effect.
In clinical sepsis research nearly all immune-modulators have demonstrated no benefit in regard to the 28-day mortality rate. Other endpoints such as quality of life have become more attractive, but clinically relevant animal models analyzing an equivalent to quality of life by measurement of sickness behavior are extremely rare. The concept of clinic modeling randomized trials was used in an animal trial to model clinical complexity and conditions of a randomized clinical trial.
80 adult male Wistar rats were randomly assigned to (1) control: anesthesia and sham operation, (2) sepsis: laparotomy and peritoneal infection with human stool bacteria, (3) sepsis with antibiotic prophylaxis: cefuroxime/metronidazole and (4) sepsis with antibiotic plus a cytokine prophylaxis with granulocyte-colony stimulating factor (GCSF). Endpoints were physiological and behavioral parameters.
The combination of antibiotics plus G-CSF was most effective in reducing mortality. All infected animals showed reduced open field activity acutely after infection, and recovery was improved during the 9 day follow-up in rats with prophylactic treatments. In the social interaction test, but not in the elevated plus-maze anxiety test, prophylaxis was also efficient, especially with antibiotics and G-CSF.
The results show that improving sickness behavior in septic rats with G-CSF plus antibiotics may be a promising approach.
Two assays--isolated peripheral blood mononuclear cells (PBMC) and a whole blood assay (WBA)--are commonly used to study TNF-alpha production by an individual in order to distinguish between high and low cytokine producers. We assessed the reliability and reproducibility of these assays.
The PBMC assays (n=5) were performed weekly over a period of 6 weeks and the WBAs (n=4) weekly over a 4-week period. Polymethylmethacrylate particles (approx. 6 x 10(2) particles/cell) and optimal concentrations of endotoxin (6.25 and 12.5 ng/ml) were used as the stimulatory agents in PBMC and WBAs, respectively. TNF-alpha production was measured by ELISA.
There was a high degree of both intra- and inter-individual variability of TNF-alpha secretion, with unpredictable changes in the amount of the cytokine produced by cells from the same donor. This variability could not be eliminated by correcting for cell numbers.
The PBMC and WBA models of TNF-alpha production by human peripheral blood cells cannot be used for the evaluation of inter-individual variability in cytokine secretion due to the high intra-individual variability observed. In the case of PBMC this is partly due to differences in the confluency of the cells between individuals.
Macrolide antibiotics have been licensed since the 1950s and have an important role in the treatment of a diverse range of infectious diseases. Macrolide antibiotics have antibacterial activity against gram-positive bacteria, some gram-negative bacteria and intracellular pathogens. The spectrum of antibacterial activity combined with excellent intracellular and tissue penetration has led to the extensive use of this class of drugs in respiratory disease. Macrolide antibiotics also have demonstrated anti-inflammatory properties in various in vitro and in vivo model systems. Novel antimicrobial and anti-inflammatory properties of macrolide may result in clinical benefits, particularly in conditions where the infectious agent is inherently resistant to macrolides. Three randomized control trials have demonstrated improved lung function in patients treated with the macrolide antibiotic, azithromycin. Azithromycin was generally well tolerated and resulted in reduction in the inflammatory response which may be due to an immunomodulatory role. Short term studies (three to six months) have not demonstrated the development of increased bacterial resistance or the emergence of new pathogens following azithromycin.
A review of published data on the in vitro, ex vivo, in vivo and clinical effects of fluoroquinolones on the synthesis of cytokines is provided. Fluoroquinolones (FQs) were found to affect both cellular and humoral immunity. In general, FQs exert their modulating effects only when used together with a co-stimulant. The in vitro studies generated heterogeneous data because of inhomogeneous effects triggered by different types of co-stimulants and differing responses of various cell lines on the stimuli. However, there is the general trend that FQs decrease the synthesis of pro-inflammatory cytokines. Studies in experimental animals generated homogenous data. All the FQs studied exerted significant clinical effects by attenuating cytokine responses in vivo. The FQs were found to be effective in vivo either in infections caused by organisms against which these are inactive or when dosed suboptimally, so that serum levels were lower than the susceptibilities of the causative pathogens. These in vivo effects were correlated with a significant decrease in pro-inflammatory cytokines like Il-1 and TNF. In addition, FQs were found to upregulate hematopoiesis. These immunomodulatory effects can be attributed in particular to those FQs with a cyclopropyl-moiety at the position N1 of the quinolone core structure, i. e. ciprofloxacin, moxifloxacin, grepafloxacin, sparfloxacin. The immunomodulatory effects of the FQs are due to their effects on intracellular cyclic AMP and phosphodiesterases, on transcription factors such as NF-kappa B, activator protein 1 and a triggering effect on the eucaryotic equivalent of bacterial SOS response. All these studies indicate that FQs exert immunomodulatory activities in particular in latent or chronic infections.
To explore the effects of metronidazole (Me) on intestinal microcirculation in septic rats, intravital microscopy (IVM) following 16 hours of colon ascendens stent peritonitis (CASP model) was used. Four groups of animals were studied: control group (sham operation) and CASP group, each with and without Me treatment (10 mg/kg i.v.). In order to investigate the substance-specific effects of Me independently of the antibacterial effects within a pathologically altered microcirculation, a second experimental series with lipopolysaccharide challenge (LPS model) was carried out. The LPS model consisted of the four groups (control animals and LPS animals (15 mg/kg i.v. LPS from E. coli) with and without Me). IVM in the LPS experiments was performed following a two hour observation period. Me treated CASP or LPS animals, as compared with untreated, demonstrated significant improvement of functional capillary density (FCD) of the intestinal wall. The increase in the number of leukocytes firmly adhered to the endothelium (leukocyte sticking) in the untreated CASP or LPS animals within the V1 venules of the intestinal submucosal layer, was significantly reduced in the Me treated animals. In conclusion, Me exerts beneficial anti-bacterial and anti-inflammatory effects within the septic microcirculation.
This study was performed to examine the ability of ciprofloxacin (CPFX) to suppress the inflammation associated with lipopolysaccharide (LPS) and an inflammatory cytokine in gram-negative bacterial pneumonia. For this purpose we measured viable cell counts in bronchoalveolar lavage fluid (BALF), LPS concentrations in BALF, and BALF levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) in mice exposed to Klebsiella pneumoniae. We used three groups of mice: controls, treated with physiological saline; mice treated with CPFX; and mice treated with ceftadizime (CAZ). The viable cell count in BALF was low in both the CAZ and CPFX groups. LPS values in BALF were significantly lower in the CPFX group than in the CAZ group at 48 and 72 h after K. pneumoniae exposure (48 h, P < 0.001; 72 h, P < 0.05). The BALF TNF-alpha level was significantly higher in the CAZ group at 24, 48, and 72 h compared to levels in the control and CPFX groups (P < 0.05). In conclusion, these results suggest that CPFX inhibited increases of LPS and TNF-alpha in gram-negative bacterial pneumonia, thereby suppressing the lung inflammation that accompanies pneumonia.
The fluoroquinolone ciprofloxacin is a broad-spectrum antibiotic that has been used in the treatment of inflammatory bowel diseases. There is evidence that quinolones have immunomodulating activities via the regulation of cytokine production.
We investigated the effect of ciprofloxacin on the nitric oxide (NO) production by colonic epithelium. HT-29 cells and colonic biopsies from patients (n = 4) with ulcerative colitis (UC) and normal controls (n = 4) were cultured with various concentrations of ciprofloxacin (10-100 microg mL(-1)) in the presence and absence of pro-inflammatory cytokines. The production of NO was measured in culture supernatants with a spectrophotometric method and inducible nitric oxide synthase (iNOS) mRNA expression was examined by reverse transcription-polymerase chain reaction (RT-PCR).
Ciprofloxacin did not have any effect on the basal NO production by HT-29 cells. In contrast, ciprofloxacin significantly (P < 0.001) inhibited the pro-inflammatory cytokines (interleukin-1alpha + tumour necrosis factor-alpha + interferon-gamma)-induced NO production in HT-29, in a concentration-dependent manner, via the inhibition of the cytokine-induced iNOS mRNA expression. Wortmannin produced a concentration related reversal of the inhibitory effect of ciprofloxacin at both iNOS mRNA expression and NO production in HT-29 cells. A similar inhibitory effect of ciprofloxacin on the cytokine-induced NO production and iNOS mRNA expression was detected in vitro in cultures of normal colonic tissue. In addition, ciprofloxacin significantly inhibited the NO production and iNOS mRNA expression in cultures of colonic tissue from ulcerative colitis patients, in a concentration-dependent manner.
These data suggest that ciprofloxacin, in addition to its antimicrobial role, might have an immunoregulatory effect on intestinal inflammation, via the modulation of inflammatory mediators.
Hepatic encephalopathy (HE) is a major complication for acute and chronic liver failure. Despite several decades of intensive clinical and basic research, the pathogenesis of HE is still incompletely understood, and the precise mechanisms causing brain dysfunction in liver failure are still not fully established. Several theories concerning the pathogenesis of HE have been previously suggested, including the ammonia theory, which received the most attention. These theories are not mutually exclusive and the validity of none of them has been definitely proved experimentally. In this review article, an attractive theory concerning the pathogenesis of HE, the tumour necrosis factor-alpha (TNF) theory, is presented and comprehensively discussed after accumulation of sufficient data which indicate that the pro-inflammatory cytokine, TNF, is strongly involved in the pathogenesis of HE associated with both acute and chronic liver failure. This theory seems to be superior to all other previous theories in the pathogenesis of HE, and may induce development of other beneficial therapeutical modalities for HE directed towards inhibition of TNF production and/or action, and towards enhancement of its degradation.
KKP723 (KKP), a derivative of ampicillin, is a newly developed beta-lactam antibiotic. Using an experimental endotoxemia model, the intestinal microcirculation in four groups of animals were evaluated using intravital microscopy (IVM). The groups included were a control group, an endotoxemic group (15 mg/kg i.v. LPS from E. coli), an ampicillin (50 mg/kg i.v.) treated endotoxemic group and an endotoxemic group treated with KKP (67.4 mg/kg i.v.). Ampicillin treatment resulted in a significant reduced number of firmly adhering leukocytes in intestinal submucosal venules. KKP treatment did not show this effect on leukocyte activation. We found no changes of the functional capillary density (FCD) of the intestinal wall by treatment with ampicillin or its derivative KKP. The increased leukocyte adherence in the KKP treated LPS animals may be explained by a loss of a possible ampicillin-related anti-inflammatory effect by the biotransformation process. The endotoxemia IVM model is useful to detect effects of antibiotics in an impaired microcirculation.
Alveolar macrophages from New Zealand white rabbits were incubated with twice the MIC of amikacin, ciprofloxacin, aztreonam,
ceftazidime and imipenem and exposed to either 10(4), 10(5) or 10(6) cfu/mL live Pseudomonas aeruginosa ATCC 27853 or 0.1,
1 or 10 mg/L purified lipopolysaccharide (LPS) derived from P. aeruginosa to determine the effects of different classes of
antimicrobial agent on production of tumour necrosis factor (TNF). Incubation of macrophages with ciprofloxacin and amikacin
resulted in less TNF activity after exposure to live P. aeruginosa than was found for saline, aztreonam, ceftazidime or imipenem
(P < 0.05). However, no significant differences were found between any of the agents after macrophages had been exposed to
purified LPS. Different antimicrobial agents therefore appear to exert different effects in vitro on the TNF response of macrophages
to bacterial stimulation.
Because fluoroquinolones have an immunomodulatory effect on cytokine production by lipopolysaccharide (LPS)-treated human monocytes, we examined the effect of fluoroquinolones on the survival of mice injected with a lethal dose of LPS. Trovafloxacin (100 mg/kg), ciprofloxacin (250 mg/kg), and tosufloxacin (100 mg/kg) protected 75% (P = 0.0001), 25% (P = 0.002), and 50% (P = 0.002), respectively, of mice against death. The fluoroquinolones significantly reduced serum levels of interleukin-6 and tumor necrosis factor alpha in LPS-treated mice. The protective effects of fluoroquinolones in LPS-induced shock in mice may also occur in humans.
Topoisomerase II (TOP2) poisons interfere with the breakage/reunion reaction of TOP2 resulting in DNA cleavage. In the current studies, we show that two different classes (ATP-sensitive and -insensitive) of TOP2 poisons can be identified based on their differential sensitivity to the ATP-bound conformation of TOP2. First, in the presence of 1 mm ATP or the nonhydrolyzable analog adenosine 5'-(beta,gamma-imino)triphosphate, TOP2-mediated DNA cleavage induced by ATP-sensitive TOP2 poisons (e.g. doxorubicin, etoposide, mitoxantrone, and 4'-(9-acridinylamino)methanesulfon-m-anisidide) was 30-100-fold stimulated, whereas DNA cleavage induced by ATP-insensitive TOP2 poisons (e.g. amonafide, batracylin, and menadione) was only slightly (less than 3-fold) affected. In addition, ADP was shown to strongly antagonize TOP2-mediated DNA cleavage induced by ATP-sensitive but not ATP-insensitive TOP2 poisons. Second, C427A mutant human TOP2alpha, which exhibits reduced ATPase activity, was shown to exhibit cross-resistance to all ATP-sensitive but not ATP-insensitive TOP2 poisons. Third, using ciprofloxacin competition assay, TOP2-mediated DNA cleavage induced by ATP-sensitive but not ATP-insensitive poisons was shown to be antagonized by ciprofloxacin. These results suggest that ATP-bound TOP2 may be the specific target of ATP-sensitive TOP2 poisons. Using Lac repressor-operator complexes as roadblocks, we show that ATP-bound TOP2 acts as a circular clamp capable of entering DNA ends and sliding on unobstructed duplex DNA.
We previously reported that ciprofloxacin (Cip), a quinoline-derivative antibiotic, decreases the biological activity of IL-1 released by LPS-stimulated monocytes after 24 hr of culture without affecting cell-associated IL-1 activity. To analyze further the effects of Cip on LPS-induced IL-1α and IL-1β synthesis, each species was measured in the supernatants and cell lysates of monocyte cultures over a 4-day period using IL-1α and IL-1β-specific ELISA methods. Cip had a post-transcriptional differential effect on the production of IL-1α and IL-1β, reducing the total amount of IL-1β produced by LPS-stimulated monocytes, while that of IL-1α was unaffected. In addition, the production of both species was delayed. These findings explain the discrepancy between the Cip-induced alteration of extracellular IL-1 activity and the preservation of cellassociated activity. Cip is, to our knowledge, the first pharmacological agent found to have a differential effect on the synthesis of IL-1α and IL-1β. It may form the basis for new pharmacological agents capable of selectively reducing the systemic effects of IL-1 without affecting local activity.
The mechanism of clinical effectiveness of low-dose and long-term erythromycin (EM) treatment for diffuse panbronchiolitis, sinobronchial syndrome, and associated otitis media with effusion was investigated by studying the effects of EM on tumor necrosis factor alpha (TNF-alpha) production by cultured human monocytes stimulated with lipopolysaccharide. At concentrations of 0.1 microgram/mL or more, EM inhibited TNF-alpha release from human monocytes stimulated by lipopolysaccharide in a dose-dependent manner. Of the other macrolides tested, roxithromycin, an EM derivative, also showed significant inhibition of TNF-alpha production, whereas josamycin failed to inhibit TNF-alpha release from monocytes. Nonmacrolidic drugs such as minocycline hydrochloride, ofloxacin, or penicillin G had no significant effect on TNF-alpha production. These results suggest that the clinical improvement of chronic respiratory diseases by EM may depend on the suppression of production of inflammatory cytokines such as TNF-alpha.
The antibacterial activities of the fluorinated 4-quinolones (e.g., ciprofloxacin) have been ascribed to a marked inhibition
of bacterial DNA gyrase. In contrast, the influence on purified mammalian DNA enzymes, including topoisomerases, has been
reported to be several orders of magnitude weaker, occurring at concentrations higher than 100 micrograms of ciprofloxacin
per ml. In this study, using a nondenaturing filter elution method, a marked induction of double-strand DNA breaks in human
lymphoblastoid cells exposed to 80 micrograms of ciprofloxacin per ml was seen. The proportion of single-strand versus double-strand
DNA breaks was similar to that seen with the topoisomerase II inhibitory antitumor agent VP-16. The cellular recovery was
more rapid after treatment with ciprofloxacin than after treatment with VP-16, displaying a normal elution profile within
15 min at 37 degrees C (60 min for VP-16). These data indicate that ciprofloxacin has an effect on intracellularly located
topoisomerase II in humans.
Adherence is an important regulatory signal for several monokines and the proto-oncogenes c-fms and c-fos in human peripheral blood monocytes. Although there is little if any constitutive expression of the IL-1 beta, TNF-alpha and CSF-1 genes in freshly isolated monocytes, adherence is sufficient to induce high steady-state levels of mRNA for TNF and c-fos and more slowly that of CSF-1. Expression of mRNA for the CSF-1R gene, c-fms, was transiently down-regulated by 4 h. In contrast, the induction of high levels of IL-1 beta mRNA were achieved independent of culture conditions. Although all of these genes could be induced by adherence, actual secretion of the mediators required the exposure to a second signal derived from LPS. Thus adherence rapidly primes monocytes for a variety of inflammatory responses, the magnitude of which depends on the nature of a second "activating" signal. It is likely that some of these products act locally as paracrine or autocrine factors to further regulate the phenotype of the differentiating macrophage.
A tetrazolium salt has been used to develop a quantitative colorimetric assay for mammalian cell survival and proliferation. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. We have used the assay to measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis.
To test the hypothesis that antibiotics leading to greater endotoxin release are associated with greater mortality in septic trauma patients.
Post hoc analysis of data from a previously conducted prospective, randomized, multicenter study designed to evaluate the efficacy of interferon gamma in preventing infection and death in trauma patients.
Nine level I trauma centers.
Severely injured trauma patients at high risk for sepsis. Eighty percent (N = 334) of the enrolled patients developed some manifestation of gram-negative sepsis, defined by the administration of gram-negative specific antibiotics.
The in-hospital mortality rate of patients who received penicillin-binding protein 3/tumor necrosis factor (PBP3/TNF)-specific antibiotics associated with the greatest degree of endotoxin release and TNF production (PBP3/TNF group, n = 78: aztreonam, ceftazidime, and cefotaxime sodium) was compared with that of patients not receiving these agents (non-PBP3/TNF group, n = 256).
Mortality in the PBP3/TNF group (17%) was higher than in the non-PBP3/TNF group (8%, P = .02). The two groups were similar in their mean (+/- SD) Injury Severity Scores (34 +/- 9), ages (31 +/- 12 years), and initial degree of bacterial contamination.
Antibiotics that are associated with greater release of endotoxin and production of TNF are also associated with greater mortality in septic trauma patients. Decisions regarding antibiotic administration may need to consider the endotoxin-releasing properties of antibiotics in addition to their antibacterial sensitivity spectrum. Prospective studies of the effect of endotoxin-releasing properties of antibiotics on mortality are warranted.
The efficacy of an antibiotic in human or experimental infection is presumed to be proportional to its in vitro antimicrobial
activity, yet antibiotics having comparable in vitro activity may have markedly different efficacies in vivo. For example,
we have reported that clindamycin is more efficacious than penicillin in experimental gas gangrene caused by Clostridium perfringens in animals. To explain these differences, we compared the dynamics of bacterial killing and suppression of toxin synthesis.
In addition, we investigated the ability of clindamycin and penicillin to modulate lipopolysaccharide-induced cytokine production
in human peripheral blood mononuclear cells. Our results suggest that clindamycin affects protein synthesis in both prokaryotic
and eukaryotic cells. These data may, in part, explain why the efficacy of clindamycin is greater than that of penicillin
and demonstrate that clindamycin may be an important immune modulator.
Among third-generation cephalosporins, cefodizime (CFDZ) has shown to modulate many functions of the host defense system against infections. The aim of the present study was to assess the in vitro CFDZ-dependent modulation of interleukin (IL)-6, tumor necrosis factor-alpha (TNF-alpha) and IL-8 release from lipopolysaccharide (LPS)-stimulated human peripheral mononuclear cells (MNCs). Two other third-generation cephalosporins: ceftriaxone (CFX) and ceftazidime (CFT), were also tested under the same experimental conditions. At concentrations ranging from 200 to 50 micrograms/ml, CFDZ significantly decreased TNF-alpha and IL-6 release from maximally (LPS 1 microgram/ml) stimulated MNCs (42% inhibition of TNF-alpha release with 100 micrograms/ml of CFDZ). On the other hand, CFDZ revealed a marked stimulatory effect on IL-8 release (200 micrograms/ml of CFDZ induced 51.5% enhancement of IL-8 release). On the contrary, both CFX and CFT failed to exert any significant effect on TNF-alpha, IL-6 or IL-8 release.
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.
Antibiotics may inhibit bacterial growth or may kill bacteria by inhibiting cell wall synthesis or protein synthesis. The amount of endotoxin released during antibiotic action has been found to be clinically important. Nine antibiotics, representing seven classes, were studied for the amounts of endotoxin released during their action on susceptible strains of Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa. Staphylococcus aureus, which produces no endotoxin, was used as a control organism. Aztreonam induced the highest release of endotoxin, whereas other antibiotics such as imipenem and the quinolones induced the lowest release of endotoxin. Although the quantities of endotoxin released are not easily explained from the established mechanisms of antibiotic action, our findings may have implications for therapy of the acutely ill, septic patient in whom release of large quantities of endotoxin may be catastrophic.
Levofloxacin (LVFX), the bacteriologically active isomer of ofloxacin, is a fluorinated quinolone. LVFX suppressed the proliferative activity of peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin (PHA). LVFX increased interleukin-2 (IL-2) production by PBMC stimulated with PHA in a dose-dependent manner, with more than 10 micrograms/ml of LVFX causing a significant increase. The granulocyte-macrophage colony-stimulating factor and soluble IL-2 receptor production by PHA-stimulated PBMC was suppressed at high concentrations of LVFX. Interleukin-1 beta production by lipopolysaccharide-stimulated PBMC was suppressed in a concentration-dependent manner by LVFX, and tumor necrosis factor-alpha production was suppressed at only the highest concentration. In contrast, interleukin-8 production was little affected by LVFX. These results show that LVFX has an immunomodulatory action on cytokines production by PBMC independent of its antimicrobial activity.
The cytokines play an important role in the cascade of the pathological events leading to septic shock. The TNF alpha produced by monocytes/macrophages upon stimulation with bacterial fragments may contribute to induction of this cytokine cascade. Moreover, the antibiotics used for antimicrobial therapy may cause the increase of TNF alpha production due to massive bacterial killing and exposure of monocytes/macrophages to bacterial cell constituents. To investigate the effect of Vancomycin on TNF alpha production, an in vitro model of LPS-stimulated monocytes was used. The level of TNF alpha protein or TNF biological activity were tested in the culture supernatants of monocytes with LPS. Vancomycin down-regulated, in dose-dependent manner, the TNF alpha production. Vancomycin also inhibited TNF alpha-mRNA accumulation in LPS-stimulated monocytes, as assessed by fluorescence in situ hybridization (FISH) in cell suspension. The down-regulation of TNF alpha production in LPS-stimulated monocytes may indicate that inhibition of this cytokine release is one of the important therapeutic effects of Vancomycin in sepsis.
Treatment of septicemia caused by Escherichia coli with ceftazidime (CAZ) may be associated with the development of septic shock due to the release of bacterial lipopolysaccharide. We examined the suppressive effect of clindamycin (CLDM) on CAZ-induced release of endotoxin by cultured E. coli and the subsequent production of inflammatory cytokines (tumor necrosis factor alpha [TNF-alpha] and interleukin-1 beta [IL-1 beta]). E. coli ATCC 12014 was incubated in inactivated horse serum with or without CLDM for 1, 4, or 18 h, followed by the addition of CAZ and collection of the culture supernatant at 0, 1, and 2 h. The concentration of endotoxin in each sample was measured by a chromogenic Limulus test. Another portion of the culture supernatant was added to THP-1 cell culture and incubated for 4 h, and the concentrations of TNF-alpha and IL-1 beta in the supernatant were measured by an enzyme-linked immunosorbent assay. In the control group (no CLDM), CAZ administration resulted in significant increases in endotoxin, TNF-alpha, and IL-1 beta concentrations. Pretreatment of E. coli with CLDM for 4 or 18 h before the addition of CAZ significantly suppressed the concentrations of endotoxin, TNF-alpha, and IL-1 beta in a time-dependent manner. In addition, CAZ treatment transformed E. coli from rodshaped bacteria to filament-like structures, as determined by electron microscopy, while pretreatment with CLDM prevented these morphological changes. Our in vitro studies showed that CAZ-induced release of large quantities of endotoxin by E. coli could be suppressed by prior administration of CLDM.
To study whether the endotoxin responsiveness of peripheral blood mononuclear cells correlates with the severity of injury in trauma patients.
Prospective, observational study.
University trauma center.
Fifty-nine patients with blunt trauma (Injury Severity Score [ISS] 4 to 57 points).
Standard emergency department care, surgical care, and postoperative intensive care unit treatment.
Whole blood and serum were obtained 94+/-89 (SD) mins post trauma (day 0) and during a 14-day period postinjury. Endotoxin-induced tumor necrosis factor-alpha (TNF-alpha) synthesis of peripheral blood mononuclear cells ex vivo was tested using a whole blood assay. Serum samples were assayed for TNF-alpha concentrations. A reduced capacity of whole blood to produce TNF-alpha ex vivo with endotoxin treatment was found to be closely correlated with the ISS. The capacity to produce TNF-alpha on endotoxin stimulation of whole blood from patients with an ISS > or =16 points was depressed immediately after trauma and did not reach normal values during the observation period. In patients with an ISS >22 points, maximum depression of the capacity of whole blood to produce TNF-alpha occurs within 100 mins post injury. In contrast, in patients with an ISS <22 points, maximal depression of whole blood TNF-alpha production occurs with a delay of 24 to 48 hrs after trauma. Based on pre- and postoperative values, primary surgical intervention caused a decrease of the endotoxin-stimulated TNF-alpha production of whole blood in the latter patient subgroup, as well as in the entire patient population (ISS 4 to 57) when secondary surgical treatment was necessary 5 to 13 days after trauma.
The extent of traumatic tissue damage leads to a graded depression of immunocyte function and appears to be amplified by surgical treatment. The endotoxin responsiveness of peripheral blood mononuclear cells displays a functional marker of the anatomically defined severity of injury and gives insights into the regulation of immunocyte function after severe blunt trauma.
The effect of trovafloxacin, ciprofloxacin and ceftriaxone on cytokine production of human peripheral blood mononuclear cells (PBMCs) was examined. PBMC responses were measured after stimulation with lipopolysaccharide (LPS), lipoteichoic acid (LTA) or killed or viable Streptococcus pneumoniae and Haemophilus influenzae. Trovafloxacin inhibited the production of tumour necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6 and IL-8 by PBMCs after stimulation with either LPS or LTA by 83%. Similar inhibition occurred in PBMCs incubated with killed or live bacteria and trovafloxacin, but not with ciprofloxacin or ceftriaxone. The relevance of this in vitro observation was explored by examining TNF-alpha and IL-6 responses in trovafloxacin-treated mice. Serum concentrations of both cytokines 1 h after LPS challenge were 95% less than serum concentrations in mice that were not given trovafloxacin. Reverse transcription- polymerase chain reaction studies of the mechanisms determining cytokine down-regulation demonstrated that trovafloxacin reduced TNF-alpha, IL-1beta and IL-6 mRNA to levels similar to those of unstimulated cells. These observations indicate that trovafloxacin can consistently and significantly reduce production of cytokines that play an important role in sepsis. In vitro, this effect can occur in the presence of bacteriolysis and is associated with inhibition of transcription of cytokine genes.