β-Lactamase–Producing Bacteria in Upper Respiratory Tract Infections

β-Lactamase–producing bacteria (BLPB) can play an important role in respiratory infections. They can have a direct pathogenic impact in causing the infection as well as an indirect effect through their ability to produce the enzyme β-lactamase. BLPB not only may survive penicillin therapy, but as demonstrated by in vitro and in vivo studies, can also protect other penicillin-susceptible bacteria from penicillin by releasing the free enzyme into their environment. The clinical in vitro and in vivo evidence supporting the role of these organisms in the increased failure rate of penicillin in eradication of these infections and the implication of that increased rate on the management of infections is discussed.     

Antibiotic resistance of oral anaerobic bacteria and their effect on the management of upper respiratory tract and head and neck infections

Anaerobes of oral origin are common in chronic upper respiratory tract and other head and neck infections. Anaerobes are the predominant components of the normal human oropharyngeal flora, and are therefore a common cause of bacterial infections of the upper respiratory tract that are of endogenous origin. These bacteria can be isolated in chronic otitis media, sinusitis, and tonsillitis, and their complications. Anaerobes also predominate in deep oral and neck infections and abscesses. Their isolation requires appropriate methods of collection, transportation, and cultivation of specimens. In addition to their active pathogenic role in these infections, many anaerobes express an indirect effect through their ability to produce the enzyme beta-lactamase. This enables these organisms to shield non-beta-lactamase-producing bacteria (BLPB) from penicillins. Inadequate therapy against BLPB may lead to clinical failures. Treatment of anaerobic infection is complicated by their slow growth, their polymicrobial nature, and the growing resistance of anaerobic bacteria to antimicrobials. Antimicrobial therapy is often the only form of therapy needed, whereas in other instances it is an important adjunct to a surgical approach. Because anaerobes generally are isolated mixed with aerobic organisms, therapy should provide for adequate coverage of both types of pathogens.     

Penicillin Failure in the Treatment of Streptococcal Pharyngo-Tonsillitis

The inadequate penetration of penicillins into the tonsillar tissues and tonsillar surface fluid and microbiologic interactions between Group A beta-hemolytic streptococci (GABHS) and other pharyngo-tonsillar bacterial flora can account for their failure in eradicating GABHS pharyngo-tonsillitis (PT). These interactions include the presence of beta-lactamase producing bacteria (BLPB) that “shield” GABHS from penicillins, the absence of bacteria that interfere with the growth of GABHS, and the coaggregation between GABHS and Moraxella catarrhalis. In the treatment of acute tonsillitis, the use of cephalosporins can overcome these interactions by eradicating aerobic BLPB, while preserving the potentially interfering organisms and eliminating GABHS. In treatment of recurrent and chronic PT, the administration of clindamycin or amoxicillin-clavulanatecan eradicates both aerobic and anaerobic BLPB, as well as GABHS.     

A study of metabolites as intermediate effectors in angiogenesis

Metabolites released from hypoxic tissues have been reported as angiogenic factors in circumstances of reduced tissue oxygenation or an increased rate of metabolism. However, in more recent reports their possible role in angiogenesis prior to the induction of hypoxia-inducible genes appears to have been neglected. In a systematic attempt to evaluate their role, metabolites common to the glycolytic and oxidative pathways (nicotinamide adenine dinucleotide, adenosine diphosphate and adenosine triphosphate), exclusively glycolytic metabolites (pyruvate and lactic acid) and exclusively oxidative metabolites (malate, succinate, fumarate and citrate) were tested to assess their effects upon in vivo angiogenesis and in vitro endothelial cell migration and proliferation. In addition, adenosine was tested due to its proposed role in hypoxia-induced angiogenesis. The angiogenic effects in vivo were examined using the chick chorioallantoic membrane assay and in vitro on chick embryonic capillary endothelial cells using a phagokinetic track/migration assay and crystal violet dye binding/proliferation assay. Metabolites common to the glycolytic and oxidative metabolic pathways and exclusively glycolytic metabolites produced an angiogenic response in vivo and in vitro on endothelial cell proliferation and migration, whereas exclusively oxidative metabolites, with the exception of malate, did not. Adenosine caused an increased proliferation of blood vessels in vivo and stimulated endothelial cell migration and proliferation. Overall, these results implicate metabolites as effectors in angiogenesis and it is proposed that they have a role which is possibly independent of the upregulation of hypoxia-inducible genes. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro and in vivo effect of immunoglobulin G on the integrity of bacterial membranes

Summary The interaction between a modified 7S immunoglobulin (MISG) and bacterial membranes was studied by adoptingin vitro as well asin vivo techniques. Preincubation ofEscherichia coli andPseudomonas aeruginosa with MISG resulted in a release of enzymatic markers from the periplasmic space, whereas no cytoplasmic or membrane-bound enzymes were liberated. Due to the interaction of MISG with the outer membrane of gram-negative rods, the bacteria became more susceptible to the antibacterial action of poorly penetrating penicillins because of a significantly increased rate of uptake. Thesein vitro effects were corroborated underin vivo conditions by adopting the granuloma pouch model. A single intravenous injection of MISG enhanced the therapeutic efficacy of mezlocillin againstE. coli; similarly, the antibacterial activity of penicillin G, oxacillin, cephalothin and cefamandole againstStaphylococcus aureus was augmented by MISG. Thesein vivo effects of MISG were not due to an increased rate of phagocytosis or complement activity. Thus, MISG sensitized bacteria to several -lactam antibiotics by disorganizing their outer membrane.

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In vitro- und In vivo-Effekte von Immunglobulin G auf die Integrität bakterieller Membranen

Zusammenfassung Die Interaktion zwischen einem modifizierten 7S Immunserumglobulin (MISG) und bakteriellen Membranen wurde sowohl unterIn vitro- als auchIn vivo-Bedingungen geprüft. Eine Präinkubation vonEscherichia coli undPseudomonas aeruginosa mit MISG resultierte in der Freisetzung von Enzymmarkern aus dem periplasmatischen Raum, wohingegen keine zytoplasmatischen oder zellwandgebundenen Enzyme freigesetzt wurden. Aufgrund der Interaktion von MISG mit der äußeren Membran gram-negativer Stäbchen wurden diese Bakterien empfindlicher gegenüber der antibakteriellen Wirkung von nur geringradig penetrierenden Penicillinen, da ihre Aufnahmerate signifikant erhöht wurde. DieseIn vitro-Effekte wurden auch unterIn vivo-Bedingungen unter Verwendung des Granuloma-Pouch Modells bestätigt. Eine einzige intravenöse MISG-Injektion erhöhte die therapeutische Effektivität von Mezlocillin gegenüberE. coli; in ähnlicher Weise wurde die antibakterielle Effektivität von Penicillin G, Oxacillin, Cephalothin und Cefamandol gegenüberStaphylococcus aureus durch MISG gesteigert. DieseIn vivo-Effekte sind nicht auf eine gesteigerte Phagozytoserate oder erhöhte Komplementaktivität zurückzuführen. Somit sensitiviert MISG Bakterien gegenüber mehreren -Laktamantibiotika aufgrund einer Desintegration der äußeren Membran.

     

Immunological cross-reactivity of mycobacterial topoisomerase I and divergence from other bacteria

Mycobacterium smegmatis topoisomerase I exhibits several distinctive characteristics among all topoisomerases. The enzyme is devoid of Zn²⁺fingers found typically in other bacterial type I topoisomerases and binds DNA in a site-specific manner. Using polyclonal antibodies, we demonstrate the high degree of relatedness of the enzyme across mycobacteria but not other bacteria. This absence of cross-reactivity from other bacteria indicates that mycobacterial topoisomerase I has diverged from Escherichia coli and other bacteria. We have investigated further the immunological properties of the enzyme by raising a panel of monoclonal antibodies that recognises different antigenically active regions of the enzyme and binds it with widely varied affinity. Inhibition of a C-terminal domain-specific antibody binding by enzyme-specific and non-specific oligonucleotides suggests the possibility of using these monoclonal antibodies to probe the structure, function and in vivo role of the enzyme.     

Intestinal IgA as a modulator of the gut microbiota

Accumulating evidence suggests that dysbiosis plays a role in the pathogenesis of intestinal diseases including inflammatory bowel disease (IBD) as well as extra-intestinal disorders. As a modulator of the intestinal microbiota, we isolated a mouse monoclonal IgA antibody (clone W27) with high affinities for multiple commensal bacteria, but not for beneficial bacteria such as Lactobacillus casei (L. casei). Via specific recognition of an epitope in serine hydroxymethyltransferase (SHMT), a bacterial metabolic enzyme, W27 IgA selectively inhibited the in vitro growth of bound bacteria, including Escherichia coli (E. coli), while having no effect on unbound beneficial bacteria such as L. casei. By modulating the gut microbiota in vivo, oral administration of W27 IgA effectively prevented development of colitis in several mouse models. Here we discuss how intestinal IgA modulates the gut microbiota through recognition of SHMT.     

The nonenzymatic glycosylation of DNA by reducing sugars in vivo may contribute to DNA damage associated with aging

Glucose and other reducing sugars have been shown to react nonenzymatically with DNA in vitro in a time and sugar concentration dependent manner. The nonenzymatic glycosylation of DNA in vitro affects both physical and biological properties of the DNA. To further study the nonenzymatic glycosylation of DNA, in vitro and in vivo models have been developed to monitor and analyze DNA modifications as a function of glucose 6-phosphate concentration and the potential effects DNA damage by reducing sugars may play in the biological aging process. In vitro studies have shown that incubation of glucose 6-phosphate and lysine alone is sufficient to form an intermediate which can rapidly react with DNA to form stable adducts. These intermediates, which accumulate over time, can not be inhibited by aminoguanidine or reduced by sodium borohydride. To study the modifications of DNA in vivo by reducing sugars, glycolytic mutant bacteria were used in a model system to monitor plasmid mutations as a function of increased intracellular glucose 6-phosphate levels. These studies have shown that increases in intracellular glucose 6-phosphate levels result in increases in plasmid mutation rates. Recipient of the Walter R. Nicolai Student Award for 1987     

Human platelet gel supernatant inactivates opportunistic wound pathogens on skin

Activation of human platelets produces a gel-like substance referred to as platelet rich plasma or platelet gel. Platelet gel is used clinically to promote wound healing; it also exhibits antimicrobial properties that may aid in the healing of infected wounds. The purpose of this study was to quantify the efficacy of human platelet gel against the opportunistic bacterial wound pathogens Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus on skin. These opportunistic pathogens may exhibit extensive antibiotic resistance, necessitating the development of alternative treatment options. The antimicrobial efficacy of platelet gel supernatants was quantified using an in vitro broth dilution assay, an ex vivo inoculated skin assay, and in an in vivo skin decontamination assay. Human platelet gel supernatants were highly bactericidal against A. baumannii and moderately but significantly bactericidal against S. aureus in vitro and in the ex vivo skin model. P. aeruginosa was not inactivated in vitro; a low but significant inactivation level was observed ex vivo. These supernatants were quite effective at inactivating a model organism on skin in vivo. These results suggest application of platelet gel has potential clinical applicability, not only in the acceleration of wound healing, but also against relevant bacteria causing wound infections.     

Targeting colorectal cancer-associated bacteria: A new area of research for personalized treatments

Most cases of colorectal cancer (CRC) are sporadic, and numerous studies have suggested that gut microbiota may play a crucial role in CRC development. Escherichia coli is a member of the gut microbiota frequently associated with colorectal tumors. CRC-associated E. coli strains frequently harbor the pks genomic island. This genomic island is responsible for the synthesis of colibactin genotoxin, which increases tumor numbers in CRC mouse models. We recently showed that targeting ClbP, a key enzyme involved in colibactin synthesis, blocks the deleterious effect of this toxin in vitro and leads to a significant decrease in tumor numbers in vivo. Altogether, our results suggest that the personalized treatment of CRC should also take into consideration the bacteria associated with the tumor in order to limit their deleterious effects.     

Contribution of Alveolar Phagocytes to Antibiotic Efficacy in an Experimental Lung Infection with Streptococcus pneumoniae

Objectives: The effect of cyclophosphamide-induced leukocytopenia on the cellular defence and on the efficacy of penicillin treatment in a Streptococcus pneumoniae pneumonia model in mice was studied.Methods: The number of alveolar phagocytes was determined in broncho-alveolar lavage (BAL) fluid as well as the number of bacteria in both BAL fluid and homogenized lung tissue.Results: Eighteen and 21 h after infection, leukocytopenic animals had significantly lower numbers of alveolar phagocytes than controls, while the numbers of bacteria in both BAL fluid and lungs were significantly higher. The number of bacteria was inversely related to the dose of penicillin and the number of alveolar macrophages. The number of alveolar granulocytes was inversely related to the dose of penicillin.Conclusions: Leukocytopenia due to cyclophosphamide impairs the cellular defence in the lung againstStreptococcus pneumoniae and the dose of penicillin must be increased to compensate for the higher outgrowth of bacteria in these leukocytopenic mice, compared to normal animals.     

Roxithromycin in the treatment of lyme disease-update and perspectives

Spirochaetal infections have been successfully treated with penicillin; more recently, erythromycin has been used in cases with known penicillin allergy. The discovery of the spirochaeteBorrelia burgdorferi and the elaboration of a new generation of macrolides with properties that differ from older macrolides have led to new ways of treating spirochaetal disease with these compounds. This paper presents data on thein vitro andin vivo efficacy of a combination of roxithromycin and co-trimoxazole againstB. burgdorferi. In vitro (checkerboard technique;B. burgdorferi strain B31; modified BSK II medium) it was found that while roxithromycin showed excellent efficacy againstB. burgdoferi (MIC 0.031 mg/l), co-trimoxazole had no effect. However, the combination of both chemotherapeutics led to a minor synergistic effect, decreasing the MIC for roxithromycin by one dilution step at concentrations of co-trimoxazole from 256 to 8 mg/l. In addition, a clearly reduced growth of microorganisms was seen at concentrations of roxithromycin as low as 0.015 mg/l in combination with 256 to 4 mg/l co-trimoxazole, when compared to the positive controls. Most interestingly, however, the motility ofB. burgdorferi was markedly reduced even when the two drugs were combined at very low concentrations. In anin vivo, non-randomised, open, prospective pilot study it was found that of 17 patients, with confirmed late Lyme borreliosis (stage II/III), treated with combined roxithromycin (300 mg b.i.d.) and co-trimoxazole for 5 weeks, 13 (76%) recovered completely by the end of treatment, and four continued to have symptoms on follow-up at 6 and 12 months. This success rate is similar to that seen with i.v. penicillin and ceftriaxone. It appears that the reduced motility ofB. burgdorferi makes the pathogen mor accessibile to the immune system. Es wird über diein vitro- undin vivo- Wirksamkeit einer Kombination von Roxithromycin und Co-trimoxazol gegenBorrelia burgdorferi berichtet.In vitro zeigte sich, da? Roxithromycin über eine ausgezeichnete Wirksamkeit gegenüberB. burgdorferi verfügt, Co-trimoxazol war jedoch unwirksam. Die Kombination beider Chemotherapeutika führt zu einem geringen synergistischen Effekt u.z., um eine Verdünnungsstufe bei einer Co-trimoxazol-Konzentration von 256/8mg/1. Bei einer 0.015 mg/l Roxithromycin-Konzentration in Kombination mit 256/4 mg/l zeigte sich in Vergleich zu positiven Kontrollen ein eindeutig reduziertes Wachstum der Mikroorganismen. Die Motilit?t war sogar eindeutig reduziert, wenn beide Substanzen nur sehr niedrige Konzentrationen aufwiesen. Bei einer offenen prospektiven Pilot-Studie, bei 17 Patienten mit einer gesicherten Borreliose in Stadium II/III, die mit einer Kombination von Roxithromycin (300 mg b.i.d.) 5 Wochen behandelt wurden, waren danach 13 ausgeheilt, vier hatten weiterhin Symptome nach 6 bis 12 Monaten. Das Ergebnis entspricht der einer i.v. Penizillin-oder Ceftriaxon-Behandlung.     

Multiresistente Erreger in der Gastroenterologie

The discovery of penicillin was one of the most important milestones in medicine. This achievement is jeopardized due to the increase of multiresistant bacteria. Whereas the increase of multiresistant gram-positive pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) has been slowed down, the rate of multiresistant gram-negative bacteria has significantly increased. Because gram-negative pathogens play an important role in gastrointestinal infections, the selection of the correct empirical antibiotic therapy is becoming increasingly more difficult. Despite the fact that access to test sample material is often difficult and complicated, microbiological analysis is becoming even more important. Risk factors for infections with multiresistant bacteria are healthcare-associated or nosocomial infections and the prior use of antibiotics. These factors have to be taken into account when considering empirical antibiotic treatment.     

NON CAMPYLOBACTER PYLORI SPIRAL ORGANISMS IN THE GASTRIC ANTRUM

Colonisation of the human gastric antrum with non Campylobacter pylori spiral organisms is described. A moderately severe histological gastritis was present despite the fact that these organisms, in contrast to C. pylori, were not closely associated with the epithelial cell surface. Although in vitro culture of the organisms was unsuccessful, in vivo culture was achieved in the mouse stomach. Further study of these spiral organisms should provide important clues to the role of bacteria in upper gastrointestinal disease.     

Impaired cAMP and Rac 1 Signaling Contribute to TNF‐α‐induced Endothelial Barrier Breakdown in Microvascular Endothelium

Objective: In sepsis, tumor necrosis factor‐alpha (TNF‐α) contributes to endothelial barrier breakdown. The involvement of Rho A/rho kinase signaling has recently been challenged. Here, we tested the role of cAMP and Rac 1 signaling. Materials and Methods: For this study, we took in vivo measurements of hydraulic conductivity in postcapillary mesenteric venules of adult rats. Measurements of transendothelial electrical resistence (TER), fluorescein isothiocyanate–dextran flux, Western blotting, immunostaining, and enzyme‐linked immunosorbent assay–based measurements of cAMP levels and Rho‐GTPase activity in human microvascular endothelial cells. Results: TNF‐α disrupted endothelial barrier functions in vivo and in vitro. Under these conditions, Rho A activity was significantly increased, whereas Rac 1 activity was decreased and Cdc42 was unaltered. Moreover, cAMP levels were reduced. Rho kinase inhibition, using Y27632, did not prevent TNF‐α‐induced barrier breakdown. In contrast, preincubation with forskolin and rolipram (F/R) to increase cAMP and cytotoxic necrotizing factor 1 to activate Rac 1 and Rho A abolished TNF‐α‐induced barrier breakdown in vivo and in vitro. Moreover, inactivation of Rac 1 was blocked by F/R‐mediated increase of cAMP, whereas Rho A activation was only partially inhibited. Conclusion: Our data indicate that decrease of cAMP and Rac 1 inactivation, rather than Rho A activation, contribute to TNF‐α‐induced endothelial barrier breakdown in vivo and in vitro.     

Pharmacological and Clinical Studies with Temocapril,an Angiotensin Converting Enzyme Inhibitor that is Excreted in the Bile

Temocapril is an angiotensin converting enzyme inhibitor (ACEI), a prodrug with a thiazepine ring. Its active form, temocaprilat, is slightly more potent than enalaprilat in inhibiting ACE isolated from rabbit lung. The inhibitory potency of temocaprilat on isolated rat aorta is 3 times that of enalaprilat. Temocapril is excreted in the bile and urine and can be used in patients with renal insufficiency. It reduces blood pressure without causing any significant change in heart rate or cardiac output. Temocapril has been reported to improve endothelial dysfunction in vitro by suppressing increased oxidative stress. In vivo it improves reactive hyperemia in patients with essential hypertension. It has been reported to prevent coronary vascular remodeling in vivo by suppressing local ACE and increased oxidative stress. In humans temocapril has been found to improve insulin resistance partly by increasing adiponectin levels. Cardiac remodeling was improved by temocapril not only in experiment animals but also in humans. It improves renal function and decreases urinary albumin excretion in diabetics as well as in hypertensive patients. Temocapril is currently marketed only in Japan. Considering its beneficial effects and unique pharmaco‐kinetics, temocapril, is likely to be introduced in other countries as well.     

Epithelial induction of serum amyloid A in experimental mucosal inflammation

We previously demonstrated epithelial induction of serum amyloid A in germ-free mice inoculated with luminal bacteria. The aims of the present study were to investigate the role of luminal bacteria and mucosal inflammation in epithelial expression of this acute-phase protein using germ-free and dextran sulfate sodium-treated mice in vivo and HT29 cells in vitro. Immunoreactivity for serum amyloid A was detected in the epithelium of esophagus, stomach, duodenum and rectum regardless of the presence or absence of luminal bacteria. Administration of dextran sulfate sodium resulted in colonic epithelial induction of serum amyloid A at the mRNA and protein levels in parallel with the progression of mucosal inflammation. Epithelial induction of serum amyloid A is possibly relevant to mucosal inflammation because that was observed in bacteria-reconstituted and dextran sulfate sodium-induced colitis in vivo and because interleukin-1 and lipopolysaccharide induced its mRNA in vitro.     

Clinical Potential of Lomerizine,a Ca2+ Channel Blocker as an Anti‐Glaucoma Drug: Effects on Ocular Circulation and Retinal Neuronal Damage

Glaucoma is defined as an optic neuropathy with characteristic changes in the optic nerve head and ultimate loss of visual field. Previous studies have suggested that (a) mechanical damage due to raised intraocular pressure and (b) a compromised tissue circulation in the optic nerve head play significant roles in the development of glaucomatous damage in the optic nerve head. Recently, we found that lomerizine, a new Ca²⁺ channel blocker, increased ocular circulation and protected neuronal cells against retinal neurotoxicity both in vitro and in vivo with minimal cardiovascular side effects. We examined the effect of lomerizine on the ocular circulation and compared it with those of other Ca²⁺ channel blockers in normal rabbits and in rabbits with an endothelin‐1‐disturbed circulation in the optic nerve head. In anesthetized rabbits, lomerizine and the other Ca²⁺ channel blockers increased the ocular circulation and also inhibited the hypoperfusion induced in optic nerve head tissue by an intravitreous injection of endothelin‐1. Whereas the other Ca²⁺ channel blockers produced changes in blood pressure and heart rate, the effects of lomerizine on these parameters were slight. In healthy humans, lomerizine increased blood velocity in the optic nerve head, without significantly altering blood pressure or heart rate. Moreover, lomerizine reduced retinal damage in rats both in vitro and in vivo, presumably through a Ca²⁺ channel blocking effect via an action that may involve a direct protection of retinal neurons as well as an improvement in the ocular circulation. These results indicate that lomerizine may be useful as a therapeutic drug against ischemic retinal diseases (such as glaucoma and retinal vascular occlusive diseases) that involve a disturbance of the ocular circulation.     

Neutralization of the activity of a Fasciola hepatica cathepsin L proteinase by anti-cathepsin L antibodies

Fasciola hepatica secretes a cathepsin L proteinase that is suggested to play an in vivo role in immunoprotection since the enzyme can cleave host immunoglobulin. In the present report, rabbit anti-cathepsin L IgG was shown to bind to the cathepsin L enzyme and inhibit its ability to cleave IgG molecules. Cathepsin L can prevent the antibody-mediated attachment of eosinophils to newly excysted juveniles in in vitro assays; however, if anti-cathepsin L IgG are mixed with the cathepsin L prior to the addition of the enzyme to the assay, eosinophils attach to the newly excysted juveniles. Thus it is possible to prepare antibodies that can bind and disrupt the biological activity of the F. hepatica cathepsin L.     

Neutrophil respiratory burst following liver transplantation: in vitro effects of granulocyte colony-stimulating factor

Early postoperative infections and septic complications are predominant causes of morbidity and mortality in patients following orthotopic liver transplantation (OLTx). Prophylactic granulocyte colony-stimulating factor (G-CSF) administration after OLTx was found to decrease the number of sepsis episodes and sepsis-related mortality. Since polymorphonuclear neutrophils (PMNs) are one of the major determinants of antimicrobial defense, alteration of their functions may influence the development of sepsis in these patients. Therefore, we investigated in vitro whether or not priming with G-CSF affects the neutrophils’ respiratory burst (RB) in immunosuppressed liver-transplanted patients. Venous blood was drawn from liver allograft recipients (n=12) between the 5th and 15th day postoperatively. Patients without clinical signs of infection or rejection were included in this study. Leukocytes were obtained as supernatant following sedimentation and incubated with 1000 IE ml^?1 G-CSF. The RB was measured by the intracellular oxidation of non-fluorescent dihydrorhodamine to the fluorescent rhodamine by flow cytometry. The results were expressed as a percentage of increasing stimulation compared to the control responses, which are made up of the percentage of cells with RB reaction after stimulation with phorbol ester (PMA), bacteria (E. coli), or the combination of a cytokine (TNF-α) and a bacterial peptide (FMLP) in the absence of G-CSF. In vitro priming with G-CSF resulted in significantly increased activity of the RB after PMA (from 71.7% to 85.6%) and TNF-α/FMLP (from 58.4% to 72.7%) stimulation. These data demonstrate that G-CSF in vitro augments the RB of PMNs, thereby suggesting a possible therapeutic role for G-CSF as immunomodulating agent during bacterial and fungal infections following OLTx.