Isolation, characterisation and phagocytic function of human macrophages from human peripheral blood.

Macrophages are among the most important cells of the immune system. Among other functions, they take part in almost all defense actions against foreign bodies and bacteria, being particularly important in infections, wound healing, and foreign body reactions. Considering their importance for the health of the human body, as well as their important role in several diseases, the in vitro studies based on these cells, are a crucial research field. Taking all mentioned into account, this study describes a simple isolation method of human macrophages (MFUM-HMP-001 and MFUM-HMP-002 cell lines) from peripheral blood. For this purpose, the morphology, the viability, and the phagocytotic activity of the isolated cells were tested. The Immunostaining of MFUM-HMP-001 and MFUM-HMP-002 cells confirmed the macrophage cell markers CD68, CD80, and CD163/M130. The phagocytotic activity was marked in both MFUM-HMP-001 and MFUM-HMP-002 cells, as was the phagocytosis of the pHrodo green Escherichia coli bioparticles conjugates, which was enhanced with the addition of lipopolysaccharide. The cells were stable and exhibited good growth. According to our results, both cell lines are useful for the development of novel macrophage cell-based in vitro models.

High-density and targeted glycoproteomic profiling of serum proteins in pancreatic cancer and intraductal papillary mucinous neoplasm.

OBJECTIVE: Glycoproteomics is an emerging subfield of proteomics. Tumor-specific variations in protein glycosylation might be potential targets for the development of new cancer diagnostics. Here, we performed high-throughput screening and targeted verification of glycome alterations in serum samples from patients with pancreatic cancer and the precancerous lesion intraductal papillary mucinous neoplasm (IPMN). MATERIAL AND METHODS: The glycosylation profile of 1000 proteins was mapped in a discovery cohort comprising serum samples from 16 individuals, including 8 patients with pancreatic cancer and 8 healthy controls. The top 10 glycoprotein biomarker candidates with the highest signal intensity difference in glycosylation levels were evaluated in a cohort consisting of 109 serum samples, including 49 patients with resectable pancreatic cancer, 13 patients with resectable noninvasive IPMN and 47 healthy controls, using a targeted assay. RESULTS: Multivariable analysis defined sets of panels comprising CA19-9 and distinctively glycosylated proteins for discrimination between pancreatic cancer, IPMN and healthy controls. A panel including CA 19-9, IL.17E, B7.1 and DR6 gave an AUC of 0.988 at 100% sensitivity at 90% specificity for the discrimination of stage 1 pancreatic cancer and healthy controls. B7.1 was found to be a valuable biomarker for differentiating between IPMN and healthy controls, with better performance alone than CA 19-9. CONCLUSIONS: Measurement of protein glycosylation profiles in serum may aid in the early detection of pancreatic cancer and precursor lesions.

Toll-like receptor 3 (TLR-3), TLR-4 and CD80 expression in peripheral blood mononuclear cells and urinary CD80 levels in children with idiopathic nephrotic syndrome.

BACKGROUND: The aims of this study were (1) to detect toll-like receptor (TLR)-3, TLR-4 and CD80 expression in peripheral blood mononuclear cells (PBMCs) and estimate urinary CD80 levels in children with idiopathic nephrotic syndrome and (2) to investigate the utility of these markers to differentiate between biopsy-proven minimal change disease (MCD) and focal segmental glomeruloscelerosis (FSGS). METHODS: The study included 70 patients with idiopathic nephrotic syndrome (NS), of whom 40 had steroid-sensitive NS (SSNS; 25 with active NS, 15 in remission) and 30 had steroid-resistant NS (SRNS) patients, and 23 healthy controls. TLR-3, TLR- 4 and CD80 mRNA expression levels in PBMCs were determined and the urinary CD80 level estimated. RESULTS: Median TLR-3, TLR-4 and CD80 mRNA expression levels were higher in patients with active SSNS than in those with SRNS, and the latter patient group also had significantly lower expression levels than the controls. The expression levels of these markers were associated with reductions in remission. Patients with biopsy-proven MCD had higher median expression levels of these markers than those with FSGS, but the differences were not statistically significant. Median urinary CD80/creatinine values were significantly higher in patients with SSNS and SRNS than in the controls and steroid-sensitive patients in remission (p < 0.001). CD80 levels were also significantly higher in patients with MCD than in those with FSGS (p = 0.002). A cut-off level of >914.5 ng/g had a sensitivity of 86.6%, specificity 71.4% and area under the curve of 0.828 (95% confidence interval 0.678-0.978, p = 0.002) for the diagnosis of MCD. CONCLUSIONS: Increased expressions of TLR-3, TLR-4 and CD80 mRNA and the level of urinary CD80/creatinine could be useful markers to differentiate patients of SSNS in relapse from those with SRNS. Further these markers can also distinguish biopsy proven MCD from FSGS in SRNS patients.

Ouabain affects the expression of activation markers, cytokine production, and endocytosis of human monocytes.

Ouabain is a steroid capable of binding to and inhibiting Na(+),-K(+)-ATPase. Studies have demonstrated some actions of ouabain on immune cells, which indicated both pro- and anti-inflammatory properties of this molecule. Nevertheless, its effects on human monocytes are still poorly understood. Thus, the present work investigated effects of ouabain in the activation and function of human adherent monocytes. Our results show that there is an increase in intracellular calcium levels already 5 minutes following monocyte treatment with 10(-7) M of ouabain. Furthermore, monocytes expressed increased amounts of surface activation markers such as CD69, HLA-DR, CD86, and CD80 and also presented an augmented endocytic activity of dextran-FITC particles after 24 hours of culture in the presence of ouabain. However, monocytes treated with ouabain did not have an increased stimulatory capacity in allogeneic mixed leukocyte reaction. Ouabain-treated monocytes produced higher levels of IL-1 ß and TNF- α as reported before. A novel observation was the fact that ouabain induced IL-10 and VEGF as well. Collectively, these results suggest that ouabain impacts monocyte activation and modulates monocyte functions, implying that this steroid could act as an immunomodulator of these cells.

Suppressed pro-inflammatory properties of circulating B cells in patients with multiple sclerosis treated with fingolimod, based on altered proportions of B-cell subpopulations.

The chief therapeutic mechanism of fingolimod in multiple sclerosis (MS) is considered to be sequestration of pathogenic lymphocytes into secondary lymphoid tissues. B cells have recently been recognized as important immune regulators in MS. In this study, the effects of fingolimod on B cells in MS patients were analyzed. MS patients treated with fingolimod (MS-F) had a significantly lower number of B cells in the circulation. The remaining B cells in the blood of MS-F had a reduced proportion of memory B cells and an increased proportion of naïve B cells, expressed lower levels of the costimulatory molecule CD80, and produced less tumor necrosis factor-α and more interleukin-10. These observations in MS-F were based on an increased proportion of the transitional B-cell subpopulation within the naïve B-cell compartment. The observed findings in B cells of MS-F might be related to the therapeutic effect of this drug in MS.

CD80, suPAR and nephrotic syndrome in a case of NPHS2 mutation.

BACKGROUND: Podocin mutations are characterized by progression to end stage renal disease and histologic findings of Focal Segmental Glomerulosclerosis (FSGS). CD80 is a podocytes protein that may play a role in proteinuria, particularly in Minimal Change Disease whereas the soluble urokinase receptor (suPAR) is characteristically elevated in the serum of FSGS patients. METHODS: In a patient with nephrotic syndrome and podocin mutation, urinary and serum CD80 as well as suPAR were measured using commercially available kits. Urinary CD80 molecular size was determined by western blot analysis. Glomerular staining for CD80 and podocin was performed. RESULTS: Patient displayed marked elevated CD80 and mildly increased suPAR urinary levels compared to controls. Serum CD80 level was within the range observed in normal controls. Serum suPAR level was elevated, albeit in the lower range reported for patients with primary FSGS. Immunofluorescence examination of kidney biopsy revealed glomerular CD80 expression. CONCLUSION: The combination of serum and urinary biomarkers can help differentiate various forms of FSGS. High urinary CD80 and elevated serum and urinary suPAR might represent a profile to differentiate this genetic form of FSGS from primary FSGS.

Immunoregulatory profile of monocytes from cutaneous leishmaniasis patients and association with lesion size.

Leishmaniasis is an important tropical disease composed of several clinical forms that adversely affect millions of people globally. Critical cells involved in the host-Leishmania interaction are monocytes and macrophages, which act to protect against infections due to their ability to both control intracellular infections and regulate the subsequent adaptive immune response. Both soluble factors and cell surface receptors are keys in directing the immune response following interaction with pathogens such as Leishmania. Toll-like receptors (TLRs) have an essential role in immune responses against infections, but little is known about their role in human infection with Leishmania braziliensis. In this work, we evaluated peripheral blood CD14+ monocytes for the expression of immunoregulatory cytokines, co-stimulatory molecules and TLR9 from cutaneous leishmaniasis patients infected with L. braziliensis and noninfected individuals. Our results showed that patients present decreased expression of co-stimulatory molecules such as CD80 and CD86 following culture with media alone or after stimulus with soluble Leishmania antigen. Interestingly, TLR9 expression was higher after culture with soluble Leishmania antigen (SLA), suggesting a role of this molecule in immunoregulation of active disease. Lastly, higher frequencies of TLR9+ monocytes were correlated with greater lesion size. These findings demonstrate a peripheral monocytes profile compatible with important immunoregulatory potential.

[Clinical significance of determination of serum B7-H4 in patients with malignant hematologic diseases].

AIM: To study the clinical significance of determination of serum B7-H4 in patients with malignant hematologic diseases. METHODS: Serum B7-H4 levels were determined in 65 patients with leucemia, 34 patients with lymphoma, 12 patients with multiple myeloma as well as in 50 healthy controls. RESULTS: The serum B7-H4 levels in patients with lymphoma [(38.81+/-10.34) kappag/L] were significantly higher than healthy controls [(31.62+/-9.850) kappag/L] (P<0.01). But there are no significant difference of B7-H4 levels in serum among patients with leucemia, patients with multiple myeloma and healthy controls. CONCLUSION: These results suggest that the B7-H4 may correlated with lymphoma, but uncorrelated with leucemia and multiple myeloma. Measurement of serum B7-H4 level provide useful information for distinctive diagnosis of different kinds of malignant hematologic diseases.

Expression of B7-H4 in blood of patients with gastric cancer predicts tumor progression and prognosis.

BACKGROUND AND OBJECTIVES: B7-H4 is a novel molecular B7 ligand that plays an important role as a negative regulator of the T cell-mediated immune response. However, the clinical significance of B7-H4 expression in gastric cancer remains uncertain. Here, we assessed B7-H4 expression in blood of patients with gastric cancer to determine whether or not it can predict tumor progression and prognosis. METHODS: We measured B7-H4 mRNA expression by quantitative RT-PCR in five gastric cell lines as well as in blood specimens from 94 patients with gastric cancer and from 22 healthy volunteers. RESULTS: Significantly more B7-H4 mRNA copies were found in gastric cell lines and in blood from patients with gastric cancer than in blood from healthy volunteers (P < 0.0001 and P < 0.0001, respectively). B7-H4 expressed in 71 (75.5%) of 94 patients with gastric cancer significantly correlated with depth of tumor invasion, lymph node metastasis, and overall stage (P = 0.006, P = 0.001, and P < 0.001, respectively). The 5-year survival rate was significantly lower in patients with than without B7-H4 expression (P = 0.04). CONCLUSIONS: The evaluation of B7-H4 expression in blood is a useful tool for predicting the progression of gastric cancer and prognosis.

Peripheral blood gene expression of B7 and CD28 family members associated with tumor progression and microscopic lymphovascular invasion in colon cancer patients.

PURPOSE: To associate the global gene expression of B7/CD28 family transcripts with pathologic features of colon cancer, we determined the B7/CD28 family transcripts in peripheral blood mononuclear cells (PBMCs) from normal subjects and patients with adenomatous polyps and colon cancer, and correlated the results with pathologic features of colon cancer. METHODS: PBMCs from age-matched normal subjects and patients with adenomatous polyps and colon cancer were analyzed for peripheral blood transcripts (PBTs) of B7/CD28 family using real-time PCR. Differences in expression levels of B7/CD28 PBTs across all cancer stages and between colon cancer patients with or without microscopic lymphovascular invasion (LVI) were analyzed. RESULTS: The results showed a significant upregulation of PBTs of co-inhibitory molecules such as B7-H3 and PD-1 and a significant PBT downregulation of co-stimulatory molecules including CD28 and ICOS in colon cancer patients. Furthermore, the increase of B7-H3 PBT was strongly associated with tumor invasion (P = 0.025) and advanced TNM stages (P = 0.019), whereas the decline of co-stimulatory ligand B7-H2 PBT was related to regional lymph node metastasis (P = 0.028) and aggressive tumor invasion (P = 0.031). In addition, the ratios of PBT expression of CD28 family to B7 family such as CTLA-4 to B7-H2 and PD-1 to B7-H2 were significantly higher in colon cancer patients with microscopic LVI than in those without LVI (P = 0.001 and P = 0.016, respectively). CONCLUSIONS: Our results suggest that B7/CD28 family PBTs may serve as valuable markers reflecting the pathological features of colon cancer.

Assessing lead time of selected ovarian cancer biomarkers: a nested case-control study.

BACKGROUND: CA125, human epididymis protein 4 (HE4), mesothelin, B7-H4, decoy receptor 3 (DcR3), and spondin-2 have been identified as potential ovarian cancer biomarkers. Except for CA125, their behavior in the prediagnostic period has not been evaluated. METHODS: Immunoassays were used to determine concentrations of CA125, HE4, mesothelin, B7-H4, DcR3, and spondin-2 proteins in prediagnostic serum specimens (1-11 samples per participant) that were contributed 0-18 years before ovarian cancer diagnosis from 34 patients with ovarian cancer (15 with advanced-stage serous carcinoma) and during a comparable time interval before the reference date from 70 matched control subjects who were participating in the Carotene and Retinol Efficacy Trial. Lowess curves were fit to biomarker levels in cancer patients and control subjects separately to summarize mean levels over time. Receiver operating characteristic curves were plotted, and area-under-the curve (AUC) statistics were computed to summarize the discrimination ability of these biomarkers by time before diagnosis. RESULTS: Smoothed mean concentrations of CA125, HE4, and mesothelin (but not of B7-H4, DcR3, and spondin-2) began to increase (visually) in cancer patients relative to control subjects approximately 3 years before diagnosis but reached detectable elevations only within the final year before diagnosis. In descriptive receiver operating characteristic analyses, the discriminatory power of these biomarkers was limited (AUC statistics range = 0.56-0.75) but showed increasing accuracy with time approaching diagnosis (eg, AUC statistics for CA125 were 0.57, 0.68, and 0.74 for > or = 4, 2-4, and <2 years before diagnosis, respectively). CONCLUSION: Serum concentrations of CA125, HE4, and mesothelin may provide evidence of ovarian cancer 3 years before clinical diagnosis, but the likely lead time associated with these markers appears to be less than 1 year.

Immunoglobulin-like transcript 3, an inhibitor of T cell activation, is reduced on blood monocytes during multiple sclerosis relapses and is induced by interferon beta-1b.

Immunoglobulin-like transcripts (ILTs) are immunoregulatory proteins that either activate or inhibit immune responses. ILT3 is inhibitory and is expressed preferentially by antigen-presenting cells. When its extracellular domain binds to an unidentified ligand of activated T cells, the T cell is silenced. Our objective was to study the expression of ILT3 on circulating monocytes in RRMS. Freshly isolated peripheral blood mononuclear cells were analyzed by multicolored flow cytometry. The proportion of ILT3(+)CD14(+) monocytes in blood, and ILT3 levels expressed by them, is lower in untreated multiple sclerosis in relapse than in: (1) untreated multiple sclerosis in remission (p < 0.009); (2) stable interferon beta-treated relapsing-remitting multiple sclerosis (p < 0.001) and; (3) healthy controls (p < 0.009). Glatiramer acetate-stimulated CD4( +) T cells, co-cultured with freshly isolated monocytes, proliferate significantly better (p = 0.0017 for multiple sclerosis; p = 0.0015 for controls) when T cell interaction with monocyte-expressed ILT3 is blocked by anti-ILT3 antibody. Interferon beta is beneficial in multiple sclerosis; why so remains unclear. Interferon beta-1b markedly increases ILT3 expression in vitro by monocytes from multiple sclerosis patients and controls. These findings identify a putative novel mechanism for the therapeutic benefit bestowed by Interferon beta and a new target for therapeutic intervention in relapsing-remitting multiple sclerosis.

A double-negative (IgD-CD27-) B cell population is increased in the peripheral blood of elderly people.

The T cell branch of the immune system has been extensively studied in the elderly and it is known that the elderly have impaired immune function, mainly due to the chronic antigenic load that ultimately causes shrinkage of the T cell repertoire and filling of the immunologic space with memory T cells. In the present paper, we describe the IgD(-)CD27(-) double-negative B cell population which (as we have recently described) is higher in the elderly. Most of these cells were IgG(+). Evaluation of the telomere length and expression of the ABCB1 transporter and anti-apoptotic molecule, Bcl2, shows that they have the markers of memory B cells. We also show that these cells do not act as antigen presenting cells, as indicated by the low levels of CD80 and DR, nor do they express significant levels of the CD40 molecule necessary to interact with T lymphocytes through the ligand, CD154. Hence, we hypothesize that these expanded cells are late memory or exhausted cells that have down-modulated the expression of CD27 and filled the immunologic space in the elderly. These cells might be the age-related manifestation of time-enduring stimulation or dysregulation of the immune system.

[Expression of co-stimulatory molecules in peripheral blood of patients with idiopathic thrombocytopenic purpura in relation with platelet antibodies].

This study was aimed to detect the expression of co-stimulatory molecules CD80, CD86 and CD137 in peripheral blood of patients with idiopathic thrombocytopenic purpura (ITP), the content of platelet antibodies in serum (PAIgG), and to analyze the relationship between them and their correlation with the number of platelet in peripheral blood, so as to clarify the roles of co-stimulatory molecules in pathogenesis of idiopathic thrombocytopenic purpura and evaluation of disease status. The co-stimulatory molecules CD80, CD86 and CD137 in peripheral blood mononuclear cells (PBMNCs) of 48 ITP patients and 40 normal persons were detected by immunofluorescence and flow cytometry (FACS), PAIgG content in serum was detected by enzyme-linked immunosorbent assay (ELISA). The results showed that CD80, CD86 and CD137 expression levels in ITP patients were (4.92 +/- 2.02)%, (8.68 +/- 4.25)%, (5.32 +/- 2.67)% respectively, PAIgG content was 210 +/- 3.02 ng/10(7) PA, all these of which were significantly higher than these in normal control group (2.01 +/- 0.75)%, (4.56 +/- 2.06)%, (1.37 +/- 1.25)%, PAIgG 20 +/- 1.13 ng/10(7) PA (p < 0.01). Correlation of co-stimulatory molecule expression with PAIgG content were positive (r = 0.302, p < 0.05), but correlation of co-stimulatory molecule expression with platelet number was negative (r = -0.369, p < 0.05). It is concluded that the co-stimulatory molecules CD80, CD86 and CD137 are involved in immune response and the incidence of ITP. Their over-expression closely associates with the pathogenesis of ITP and clinical status, so that correcting the abnormal expression and regulating the immune status may be one therapeutic strategy and have important clinical significance.

Immunoglobulin subclass levels in patients with non-Hodgkin lymphoma.

Allergy/atopy has been suggested to protect against non-Hodgkin lymphoma (NHL) and specific IgE levels are decreased in patients with NHL. We speculated that all immunoglobulin subclass levels might be downregulated in NHL and examined levels of IgM, IgD, IgA, IgE, IgG and IgG(4) in 200 NHL patients and 200 age- and sex-matched controls. Patients with B-cell NHL of many types had consistently lower median immunoglobulin subclass levels than controls. In every subclass except IgD, about 10-15% of B-cell NHL patients had absolute levels below the 2.5 percentile of controls. Subclass levels correlated with each other and many patients had more than one significantly low level. Levels were lowest for IgG(4) and IgE. Patients with chronic lymphocytic leukemia/small lymphocytic lymphoma had especially low total IgE levels. In other B-cell NHL types, total IgE levels were decreased to a similar extent as other immunoglobulin subclasses. In conclusion, low IgE levels are only part of a more generalized loss of immunoglobulins of all subtypes in a wide variety of B-cell NHL types. Low immunoglobulin levels appear to be a consequence of B-cell NHL presence, and we speculate about molecular mechanisms that could reduce all immunoglobulin subclasses in B-cell NHL.

Therapeutic influence on circulating and monocyte-derived dendritic cells in laryngeal squamous cell carcinoma patients.

CONCLUSION: Surgical resection of laryngeal squamous cell carcinoma (LSCC) could be associated with improved circulating myeloid dendritic cell (mDC) number and monocyte-derived dentritic cell (MoDC) function. Although adjunctive radiotherapy after surgery did not effect the normalization of mDC number, it may have an impact on MoDC function. OBJECTIVE: To investigate the effects of surgery and adjunctive radiotherapy on both circulating dendritic cells (DCs) and MoDCs of LSCC patients. SUBJECTS AND METHODS: Forty-six patients with LSCC and 15 age-matched healthy control subjects were enrolled in this study. Blood samples were taken before, during, and after conventional treatment from both patients who underwent surgery only (n=18) and those who had adjunctive radiotherapy after tumor removal (n=28). Three-color flow cytometry was used for determination of circulating DC subsets. Moreover, MoDCs were generated utilizing granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), then the phenotype of MoDCs was measured by flow cytometry and the ability to stimulate autologous T cells was tested in a mixed leukocyte reaction (MLR). RESULTS: The preoperative mDC counts, MoDC surface molecular expression and stimulatory capacity were impaired in patients in comparison with controls. The number of mDCs and the expression of CD80, CD83, and HLA-DR on MoDCs were significantly increased as compared with those pretreatment in patients who underwent surgery only and in those who had surgery followed by adjunctive radiotherapy. However, the recovery of CD86 expression and allostimulatory activity was only observed in patients who underwent surgery alone.

Serum-soluble B7x is elevated in renal cell carcinoma patients and is associated with advanced stage.

B7x is the newest member of the B7-CD28 family and is thought to dampen immune responses via negative costimulation. Tumor expression of B7x was recently described in renal cell carcinoma (RCC) and was associated with poor outcome. We developed an assay to detect serum-soluble B7x (sB7x) and investigated 101 patients with clear cell RCC who underwent nephrectomy between 2003 and 2007. For controls, we obtained serum from 101 sex-matched blood donors within the same age range. Following an ELISA for sB7x, detectable levels (>0.1 ng/mL) of sB7x were observed in 53 RCC patients compared with 18 controls (P < 0.001). Median (range) concentrations of sB7x for RCC patients and controls were 14.4 ng/mL (0.1-56.9) and 2.7 ng/mL (0.2-37.1), respectively. For RCC patients with detectable sB7x, median levels were significantly higher for patients with a tumor thrombus (19.2 versus 6.6 ng/mL; P = 0.007), positive lymph nodes (41.3 versus 10.3 ng/mL; P = 0.018), and distant metastases at nephrectomy (43.3 versus 8.5 ng/mL; P = 0.002) and tended to be higher in patients with high-grade tumors (18.8 versus 8.5; P = 0.090). Additionally, median sB7x levels for tumor-node-metastasis stage I to IV RCC were 6.6, 10.3, 14.5, and 43.3 ng/mL, respectively (P = 0.012). In this first evaluation of sB7x in RCC, we show that RCC patients are more likely to have detectable sB7x compared with controls and higher sB7x levels correlate with advanced tumor stage. These early results merit further investigation of this serum marker for potential diagnostic and prognostic purposes.

Identification of two subpopulations of purified human blood B cells, CD27- CD23+ and CD27high CD80+, that strongly express cell surface Toll-like receptor 9 and secrete high levels of interleukin-6.

B-cell expression of certain Toll-like receptors (TLRs) is important in linking innate and adaptive immune responses in normal and pathological conditions. The expression of TLR9 plays a role in the recognition of conserved pathogen motifs in a manner that is dependent on B-cell localization, deduced from B-cell phenotype. The nature of TLR9 function is unclear. A first step in unravelling the function of this pattern recognition receptor is to discover the precise nature of the cell types that express TLR9. This study used three-colour flow cytometry to characterize the B lymphocytes from human peripheral blood mononuclear cells (PBMCs) that express TLR9 on the surface. We sorted TLR9-positive B and non-B cells from the PBMC population and detected TLR9 expression on naïve and memory B cells. Moreover, we identified two discrete subpopulations of B cells: CD19(+) CD27(-) CD23(+) cells and CD19(+) CD27(high) CD80(+) cells. These subpopulations expressed high levels of membrane TLR9 and exhibited a strong in vitro response to binding a relevant CpG motif by secreting high levels of interleukin-6 (compared to controls). Our finding that this pattern recognition receptor is expressed on a variety of cell subsets adds to the current understanding of the functional complexity of B-cell membrane TLR9.

Reduction of dendritic cells by granulocyte and monocyte adsorption apheresis in patients with ulcerative colitis.

The influence of the granulocyte/monocyte apheresis (GMCAP) on cell populations participating in mechanisms of tolerance, e.g. dendritic cells (DCs), is still not very clear. In a first step, we aimed to investigate changes in the DC population of patients suffering from ulcerative colitis (UC) (n = 13) compared to healthy subjects (n = 9). In a second step, we studied the changes in peripheral DCs in a small group of patients with active UC before and after Adacolumn apheresis (n = 7). For this purpose, plasmacytoid and myeloid DCs and their maturation markers CD40, CD80, and CD86 were measured using four-color flow cytometry in the peripheral blood. After apheresis, and in acute flare-ups, we identified a significantly lower number of lymphocytes, plasmacytoid, and myeloid DCs. In conclusion, the additional removal of peripheral DCs by GMCAP, which otherwise would contribute to the inflammatory process in the gut, may lead to a higher tolerogeneic status towards luminal antigens.

Aberrant expression of soluble co-stimulatory molecules and adhesion molecules in type 2 diabetic patients with nephropathy.

Co-stimulatory molecules together with leukocyte adhesion molecules are important for T lymphocyte and leukocyte-mediated inflammatory responses. We investigated the soluble costimulatory molecules CD80, CD86, CD28, and CTLA-4 and soluble adhesion molecules in plasma of 94 type 2 diabetic patients with or without nephropathy (DN and NDN) and 20 healthy controls. Plasma concentration of sCTLA-4 was significantly lower, whereas sCD28 was significantly higher in DN patients than that in control subjects (all P < 0.05). sCD28 and sCD80 were found to be positively correlated with fasting urine albumin: creatinine ratio in DN patients but not in NDN patients. Elevated soluble adhesion molecule vascular cell adhesion molecule-1 and P-selectin could be related with the disease severity of DN (all P < 0.05). Therefore, the aberrant expression of soluble co-stimulatory molecules and adhesion molecules can be related to the activation of T cells and leukocytes in the progression of inflammation in diabetic nephropathy.