Wnt5a induces a tolerogenic phenotype of macrophages in sepsis and breast cancer patients.

A well-orchestrated inflammatory reaction involves the induction of effector functions and, at a later stage, an active downregulation of this potentially harmful process. In this study we show that under proinflammatory conditions the noncanonical Wnt protein, Wnt5a, induces immunosuppressive macrophages. The suppressive phenotype induced by Wnt5a is associated with induction of IL-10 and inhibition of the classical TLR4-NF-κB signaling. Interestingly, this phenotype closely resembles that observed in reprogrammed monocytes in sepsis patients. The Wnt5a-induced feedback inhibition is active both during in vitro LPS stimulation of macrophages and in patients with sepsis caused by LPS-containing, gram-negative bacteria. Furthermore, using breast cancer patient tissue microarrays, we find a strong correlation between the expression of Wnt5a in malignant epithelial cells and the frequency of CD163(+) anti-inflammatory tumor-associated macrophages. In conclusion, our data point out Wnt5a as a potential target for an efficient therapeutic modality in severe human diseases as diverse as sepsis and malignancy.

Lymphocyte and monocyte flow cytometry immunophenotyping as a diagnostic tool in uncharacteristic inflammatory disorders.

BACKGROUND: Patients with uncharacteristic inflammatory symptoms such as long-standing fatigue or pain, or a prolonged fever, constitute a diagnostic and therapeutic challenge. The aim of the present study was to determine if an extended immunophenotyping of lymphocytes and monocytes including activation markers can define disease-specific patterns, and thus provide valuable diagnostic information for these patients. METHODS: Whole blood from patients with gram-negative bacteraemia, neuroborreliosis, tuberculosis, acute mononucleosis, influenza or a mixed connective tissue disorders, as diagnosed by routine culture and serology techniques was analysed for lymphocyte and monocyte cell surface markers using a no-wash, no-lyse protocol for multi-colour flow cytometry method. The immunophenotyping included the activation markers HLA-DR and CD40. Plasma levels of soluble TNF alpha receptors were analysed by ELISA. RESULTS: An informative pattern was obtained by combining two of the analysed parameters: (i), the fractions of HLA-DR-expressing CD4+ T cells and CD8+ T cells, respectively, and (ii), the level of CD40 on CD14+ CD16- monocytes. Patients infected with gram-negative bacteria or EBV showed a marked increase in monocyte CD40, while this effect was less pronounced for tuberculosis, borrelia and influenza. The bacterial agents could be distinguished from the viral agents by the T cell result; CD4+ T cells reacting in bacterial infection, and the CD8+ T cells dominating for the viruses. Patients with mixed connective tissue disorders also showed increased activation, but with similar engagement of CD4+ and CD8+ T cells. Analysis of soluble TNF alpha receptors was less informative due to a large inter-individual variation. CONCLUSION: Immunophenotyping including the combination of the fractions of HLA-DR expressing T cell subpopulations with the level of CD40 on monocytes produces an informative pattern, differentiating between infections of bacterial and viral origin. Furthermore, a quantitative analysis of these parameters revealed the novel finding of characteristic patterns indicating a subacute bacterial infection, such as borreliosis or tuberculosis, or a mixed connective tissue disorder. The employed flow cytometric method is suitable for clinical diagnostic laboratories, and may help in the assessment of patients with uncharacteristic inflammatory symptoms.

Human G-MDSCs are neutrophils at distinct maturation stages promoting tumor growth in breast cancer.

Myeloid-derived suppressor cells (MDSCs) are known to contribute to immune evasion in cancer. However, the function of the human granulocytic (G)-MDSC subset during tumor progression is largely unknown, and there are no established markers for their identification in human tumor specimens. Using gene expression profiling, mass cytometry, and tumor microarrays, we here demonstrate that human G-MDSCs occur as neutrophils at distinct maturation stages, with a disease-specific profile. G-MDSCs derived from patients with metastatic breast cancer and malignant melanoma display a unique immature neutrophil profile, that is more similar to healthy donor neutrophils than to G-MDSCs from sepsis patients. Finally, we show that primary G-MDSCs from metastatic breast cancer patients co-transplanted with breast cancer cells, promote tumor growth, and affect vessel formation, leading to myeloid immune cell exclusion. Our findings reveal a role for human G-MDSC in tumor progression and have clinical implications also for targeted immunotherapy.

Humoral response to Clostridium difficile in inflammatory bowel disease, including correlation with immunomodulatory treatment.

BACKGROUND AND AIM: An abnormal immune response to intestinal bacteria has been observed in Crohn's disease (CD). Clostridium difficile infection incidence and severity are increased in CD, but reports on the humoral response have provided conflicting results. We aimed to shed light on the possible role of C. difficile in CD pathogenesis by paying attention to the influence of immunomodulatory treatment on the humoral response. METHODS: A total of 71 consecutive outpatients with CD, 67 with ulcerative colitis (UC), and 121 healthy controls were analyzed for serum IgA and IgG to C. difficile toxins A and B. RESULTS: IgA levels were similar in all study groups. IgG to toxin A was increased similarly in CD and UC (P = 0.02 for both). In contrast, IgG to toxin B was elevated only in CD patients not receiving disease-modifying anti-inflammatory bowel disease drugs (DMAID) (n = 16) (P = 0.0001), while the CD medication subgroup (n = 47) had a level similar to healthy controls. The UC results were not influenced by DMAID treatment. CONCLUSION: Our findings add support to the idea of a disturbed interaction between intestinal cells and the microbiota being part of the CD disease mechanism. An abnormal immune response to C. difficile toxin B may be a critical component of this interaction.

Increased sporulation rate of epidemic Clostridium difficile Type 027/NAP1.

Clostridium difficile PCR ribotype 027 comprised 0.2% of a collection of Swedish isolates in 1997-2001 (3 of 1,325 isolates). These isolates had lower moxifloxacin MICs than the epidemic type 027 isolates, but they had the same tcdC sequence and toxin yield. Type 027 produced 3- to 13-fold more toxin than did major Swedish types. One epidemic strain (027/NAP1a) sporulated more than did other type 027 isolates, a feature that should contribute to its survival and spread.

Pharmacodynamic studies of vancomycin, metronidazole and fusidic acid against Clostridium difficile.

BACKGROUND: Pharmacodynamic studies of antibiotics have attracted great interest in recent years. However, studies on the pharmacodynamics of different antibiotics against Clostridium difficile are scarce. METHODS: The postantibiotic effects (PAE) and the postantibiotic sub-minimum inhibitory concentration (MIC) effects (PA SME) of vancomycin, metronidazole and fusidic acid were investigated by viable counts against three different strains of C. difficile. The killing rate and extent of the three antibiotics against the same strains were also studied by adding 2, 4, 8, 16 and 32x MIC of the three antibiotics, respectively. RESULTS: Metronidazole exerted a very rapid bactericidal effect at concentrations of 8x MIC and above against all three strains investigated. Vancomycin gave overall less kill in comparison to metronidazole and was bacteriostatic against two of the three strains. Fusidic acid exerted a concentration-dependent killing against two of the strains. Vancomycin exerted short PAEs and PA SMEs against all three strains. Significantly longer PAEs and PA SMEs were noted for fusidic acid. Metronidazole gave similar short PAEs like vancomycin but longer PA SMEs were noted against two of the investigated strains. CONCLUSION: Metronidazole exerted the most prominent bactericidal effect greater than fusidic acid and greater than vancomycin. Fusidic acid gave overall the longest PAEs and PA SMEs greater than metronidazole and greater than vancomycin.

Systemic Monocytic-MDSCs Are Generated from Monocytes and Correlate with Disease Progression in Breast Cancer Patients.

Myeloid-derived suppressor cells (MDSCs) are highly immunosuppressive myeloid cells, which increase in cancer patients. The molecular mechanism behind their generation and function is unclear. Whereas granulocytic-MDSCs correlate with poor overall survival in breast cancer, the presence and relevance of monocytic-MDSCs (Mo-MDSCs) is unknown. Here we report for the first time an enrichment of functional blood Mo-MDSCs in breast cancer patients before they acquire a typical Mo-MDSC surface phenotype. A clear population of Mo-MDSCs with the typical cell surface phenotype (CD14(+)HLA-DR(low/-)CD86(low/-)CD80(low/-)CD163(low/-)) increased significantly first during disease progression and correlated to metastasis to lymph nodes and visceral organs. Furthermore, monocytes, comprising the Mo-MDSC population, from patients with metastatic breast cancer resemble the reprogrammed immunosuppressive monocytes in patients with severe infections, both by their surface and functional phenotype but also at their molecular gene expression profile. Our data suggest that monitoring the Mo-MDSC levels in breast cancer patients may represent a novel and simple biomarker for assessing disease progression.

Activity of three disinfectants and acidified nitrite against Clostridium difficile spores.

OBJECTIVE: To identify environmentally safe, rapidly acting agents for killing spores of Clostridium difficile in the hospital environment. DESIGN: Three classic disinfectants (2% glutaraldehyde, 1.6% peracetyl ions, and 70% isopropanol) and acidified nitrite were compared for activity against C. difficile spores. Four strains of C. difficile belonging to different serogroups were tested using a dilution-neutralization method according to preliminary European Standard prEN 14347. For peracetyl ions and acidified nitrite, the subjective cleaning effect and the sporicidal activity was also tested in the presence of organic load. RESULTS: Peracetyl ions were highly sporicidal and yielded a minimum 4 log10 reduction of germinating spores already at short exposure times, independent of organic load conditions. Isopropanol 70% showed low or no inactivation at all exposure times, whereas glutaraldehyde and acidified nitrite each resulted in an increasing inactivation factor (IF) over time, from an IF greater than 1.4 at 5 minutes of exposure time to greater than 4.1 at 30 minutes. Soiling conditions did not influence the effect of acidified nitrite. There was no difference in the IF among the 4 strains tested for any of the investigated agents. Acidified nitrite demonstrated a good subjective cleaning effect and peracetyl ions demonstrated a satisfactory effect. CONCLUSIONS: Cidal activity was shown against C. difficile spores by glutaraldehyde, peracetyl ions, and acidified nitrite. As acidified nitrite and peracetyl ions are considered to be environmentally safe chemicals, these agents seem well suited for the disinfection of C. difficile spores in the hospital environment.

A high frequency of MDSCs in sepsis patients, with the granulocytic subtype dominating in gram-positive cases.

The causative microorganisms dictate the type of MDSC generated in sepsis patients, and a large proportion of PMN-MDSCs in gram-positive sepsis includes immunosuppressive myeloid blasts. MDSCs constitute a heterogeneous population of immature myeloid cells that potently suppress immune responses. They were identified originally in cancer patients and have since been reported to occur also in chronic inflammation, autoimmunity, and even bacterial infections. Human MDSCs are commonly divided into Mo-MDSCs and granulocytic (PMN-MDSCs) subtypes. To what extent the bona fide cancer MDSCs are representative of the proposed MDSCs found in other diseases is not well known. PMN-MDSCs have been found previously to be enriched among LDGs in density gradient-centrifuged blood. In this study, we analyzed potential MDSCs in sepsis patients with different causative microorganisms, using total peripheral blood compared with density gradient-centrifuged blood. We found a high frequency of typical CD14(+)HLA-DR(low) Mo-MDSCs in all sepsis patients, whereas the typical PMN-MDSCs, as well as a prominent CD14(low) PMN-MDSC-like population, appeared preferentially in gram-positive cases. The CD14(low) PMN-MDSC variant was demonstrated to suppress T cell proliferation in vitro via a ROS-dependent mechanism, to display an increased IL-10:TNF-α ratio, and to present with signs of immaturity: blast morphology and low cytokine levels. We conclude that a spectrum of cells with MDSC features is enriched in sepsis and that the microbial origin of sepsis contributes to the substantial interindividual patient variation in the MDSC pattern.

Ribotyping of Clostridium difficile strains associated with nosocomial transmission and relapses in a Swedish County.

Clostridium difficile is an emerging threat in hospital environments. To analyse possible transmission and to distinguish between relapse and reinfection a collection of C. difficile isolates, sampled from 162 consecutive episodes of C. difficile infection, were PCR ribotyped. Two ribotypes (001 and 012) were prone to cause nosocomial acquisition. Moreover, ribotype 001 had a tendency to cause relapses as almost one in two patients with this ribotype had one or more relapses. By using PCR ribotyping strains inclined to cause relapses and strains associated with hospital transmission might be detected. This enables optimized hygiene measures and may improve the choice of treatment regimen.

Lactobacillus plantarum 299v enhances the concentrations of fecal short-chain fatty acids in patients with recurrent clostridium difficile-associated diarrhea.

Our objective was to document how intake of Lactobacillus plantarum 299v affects the concentrations of fecal organic acids during and after metronidazole treatment in 19 patients with recurrent Clostridium difficile-associated diarrhea. Fecal samples were analyzed by gas-liquid chromatography. After intake of metronidazole a significant decrease in total short-chain fatty acids was seen in the placebo group (from 77.1 to 45.5 micromol/g; P=0.028) but not in the Lactobacillus group (79.8-60.4 micromol/g). In addition, a statistically significant difference between treatment groups was noted for butyrate (5.6-1.2 micromol/g in the placebo group vs. 7.6-5.6 micromol/g in the Lactobacillus group; P=0.047). At the end of the study and after cessation of placebo or Lactobacillus, the total short-chain fatty acids rose to the same levels as before antibiotic treatment in the placebo group. Both treatment groups showed a significant decrease in concentrations of succinate at the end of the study in comparison to the time when metronidazole intake was stopped (6.3-1.5 micromol/g in the placebo group versus 9.3-0.9 micromol/g in the Lactobacillus group; P=0.028). The present study of fecal samples from a clinical trial is the first to demonstrate that administration of Lactobacillus plantarum 299v reduces the negative effects of an antibiotic on colonic fermentation. The intake of this probiotic strain may thereby provide an additional benefit for patients with recurrent Clostridium difficile-associated diarrhea.

A double-blind randomized controlled trial of fusidic acid and metronidazole for treatment of an initial episode of Clostridium difficile-associated diarrhoea.

OBJECTIVES: Few treatment options are currently available to treat patients suffering from an initial episode of Clostridium difficile-associated diarrhoea (CDAD). PATIENTS AND METHODS: A prospective, randomized controlled, double-blind trial was conducted to compare the efficacy of fusidic acid and metronidazole for treatment of patients experiencing a first episode of CDAD. The primary outcomes were clinical cure and clearance of C. difficile toxin determined on days 8-13, and secondary outcomes were clinical recurrence and reappearance of C. difficile toxin evaluated on days 35-40. RESULTS: Of the patients in the fusidic acid group, 83% were clinically cured in comparison to 93% in the metronidazole group (P=0.116) at the first follow-up visit. Clearance of C. difficile toxin did not differ between the two groups at that time. Clinical recurrence and reappearance of C. difficile toxin were noted in 27% and in 13% of the patients receiving fusidic acid, respectively and in 29% and 10% of those given metronidazole at the second follow-up on days 35-40. CONCLUSION: Since three of the four primary and secondary outcomes were almost identical for the two groups, the results indicate that fusidic acid is as effective as metronidazole in curing an initial episode of CDAD and can therefore be considered as an adequate alternative for treatment of this disease.

Lactobacillus plantarum 299v for the treatment of recurrent Clostridium difficile-associated diarrhoea: a double-blind, placebo-controlled trial.

A double-blind, placebo-controlled trial was performed to analyse the ability of Lactobacillus plantarum 299v to prevent further recurrent episodes of Clostridium difficile-associated diarrhoea (RCDAD). Recurrence of clinical symptoms (main outcome) was seen in 4 of 11 patients who received metronidazole in combination with L. plantarum 299v and in 6 of 9 treated with metronidazole in combination with placebo. The lactobacilli treatment had no side-effects. Although the small sample size does not allow any conclusion to be drawn concerning the efficacy of L. plantarum in patients with RCDAD, these results may contribute to the ongoing discussion about the benefits of probiotics in patients with RCDAD and encourage the performance of larger multicentre studies.