Anders Bredberg

Increased subpopulations of CD16+ and CD56+ blood monocytes in patients with active Crohn's disease

Background Circulating monocytes may be subdivided according to the presence or absence of the Fc&ggr; receptor CD16 and the neural cell adhesion molecule CD56. Monocytes classified into these subpopulations are characterized by distinct phenotypic and functional features. We hypothesized that patients with active Crohns disease differ in their peripheral monocyte subpopulations. Methods Using flow cytometry we investigated the expression of CD16 and CD56 on circulating monocytes in 11 patients with active Crohns disease and 11 controls. These monocyte subpopulations were then analyzed for expression of the chemokine receptor fractalkine, CX3CR1, and the monocyte chemoattractant protein‐1, CCR2. Results We found a median 3.7‐fold increase in the number of CD16+ monocytes related to the population with high expression of the pattern recognition receptor CD14 compared to that in the controls (P < 0.001). By studying the percentage of monocytes expressing CX3CR1, and their relative fluorescence intensity (RFI), we found significant differences, with both the highest percentage and the highest RFI in the CD14lowCD16+ subpopulation, whereas the CD14highCD16+ subgroup represented an intermediate population. Inversely, CCR2 expression was highest in the populations with high expression of CD14, whereas the CD14lowCD16+ subpopulation showed the lowest percentage and the lowest RFI for CCR2. We found the percentage of CD14+CD56+ monocytes in patients with active Crohns disease to be increased 2.7 times compared to the controls (P = 0.011). Conclusions These results show that subsets of peripheral monocytes with a more mature phenotype are expanded in patients with active Crohns disease. (Inflamm Bowel Dis 2006)

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.

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‐DRlow Mo‐MDSCs in all sepsis patients, whereas the typical PMN‐MDSCs, as well as a prominent CD14low PMN‐MDSC‐like population, appeared preferentially in gram‐positive cases. The CD14low 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.

Intestinal Barrier Dysfunction Develops at the Onset of Experimental Autoimmune Encephalomyelitis, and Can Be Induced by Adoptive Transfer of Auto-Reactive T Cells

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system with a pathogenesis involving a dysfunctional blood-brain barrier and myelin-specific, autoreactive T cells. Although the commensal microbiota seems to affect its pathogenesis, regulation of the interactions between luminal antigens and mucosal immune elements remains unclear. Herein, we investigated whether the intestinal mucosal barrier is also targeted in this disease. Experimental autoimmune encephalomyelitis (EAE), the prototypic animal model of MS, was induced either by active immunization or by adoptive transfer of autoreactive T cells isolated from these mice. We show increased intestinal permeability, overexpression of the tight junction protein zonulin and alterations in intestinal morphology (increased crypt depth and thickness of the submucosa and muscularis layers). These intestinal manifestations were seen at 7 days (i.e., preceding the onset of neurological symptoms) and at 14 days (i.e., at the stage of paralysis) after immunization. We also demonstrate an increased infiltration of proinflammatory Th1/Th17 cells and a reduced regulatory T cell number in the gut lamina propria, Peyers patches and mesenteric lymph nodes. Adoptive transfer to healthy mice of encephalitogenic T cells, isolated from EAE-diseased animals, led to intestinal changes similar to those resulting from the immunization procedure. Our findings show that disruption of intestinal homeostasis is an early and immune-mediated event in EAE. We propose that this intestinal dysfunction may act to support disease progression, and thus represent a potential therapeutic target in MS. In particular, an increased understanding of the regulation of tight junctions at the blood-brain barrier and in the intestinal wall may be crucial for design of future innovative therapies.

Ciprofloxacin does not inhibit mitochondrial functions but other antibiotics do.

At clinical concentrations, ciprofloxacin did not inhibit mitochondrial DNA replication, oxidative phosphorylation, protein synthesis, or mitochondrial mass (transmembrane potential). No difference in supercoiled forms of DNA was observed. The tetracyclines and chloramphenicol inhibited protein synthesis at clinically achievable concentrations, while rifampin, fusidic acid, and clindamycin did not.

4-quinolone antibiotics: positive genotoxic screening tests despite an apparent lack of mutation induction

The effects of different 4-quinolone antibiotic derivatives (4-Qs) in a number of short-term tests commonly employed for the evaluation of genetic toxicity were studied. Incorporation of [3H]thymidine into mitogen-stimulated peripheral blood lymphocytes was strongly enhanced at a low concentration (1.56 micrograms/ml) for most of the tested 4-Qs, whereas DNA strand breakage in lymphoblastoid cells was evident only for ciprofloxacin (10 micrograms/ml and upwards), ofloxacin (80 micrograms/ml) and norfloxacin (160 micrograms/ml). Ciprofloxacin induced a significant amount of unscheduled DNA synthesis, but was found to be negative in a shuttle vector plasmid mutation test. Ciprofloxacin (80 micrograms/ml) did not inhibit enzymes involved in the early steps of pyrimidine biosynthesis. Cell growth was slightly depressed at a concentration of 20 micrograms/ml, becoming marked at 80 micrograms/ml. In conclusion, this study seeks to contribute to an improved evaluation of genotoxic screening test data, by focusing attention on the conflicting effects imposed by the 4-Qs on a battery of such tests.

Cancer: More of polygenic disease and less of multiple mutations? A quantitative viewpoint.

The focus of cancer research is on cancer‐specific mutations, with most clinical trials involving targeted drugs. Huge numbers of DNA lesions and tumor resistance events, in each of the >1013 cells of a human individual, form a striking contrast to the low, and also very narrow, cancer incidence window (10−1‐100). A detailed consideration of these quantitative observations seems to question the present paradigm, while suggesting that a systemic regulatory network mechanism is a stronger determinant for overt cancer disease, as compared with cancer‐specific gene products. If we shall ever achieve major improvements in survival, we must gain understanding of this systemic network, rather than targeting therapy to a limited set of molecules or mutations. This may give us new opportunities for development of highly potent therapeutic tools. Cancer 2011.

Use of atorvastatin as an anti-inflammatory treatment in Crohn's disease.

Experimental and clinical investigations have revealed that statins can downregulate both acute and chronic inflammatory processes. Whether statins express anti‐inflammatory activities in the treatment of Crohns disease is unknown.

Triple helix directed psoralen adducts induce a low frequency of recombination in an SV40 shuttle vector

Triple helix forming oligonucleotide directed psoralen adducts in a mammalian shuttle vector have been reported to be repaired efficiently in human cells. In this study we examined the role of intermolecular homologous recombination in triple helix targeted psoralen adduct repair. A simian virus 40 (SV40) shuttle vector carrying a mutated supF gene was treated with a triplex forming oligonucleotide psoralen conjugate and cotransfected into human cells with a second plasmid bearing the wild type supF gene. Recombinants with a reactivated marker gene were detected by an X-gal assay in indicator bacteria. We could observe a low frequency of psoralen adduct induced recombination indicating that recombination does not play a major role in triplex directed psoralen adduct repair. The implications for targeted mutagenesis by triple helix forming oligonucleotides are discussed.

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-DRlow/-CD86low/-CD80low/-CD163low/-) 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.