Pilot study: placental biomarker predictive capability (sFlt-1, PlGF and their ratio) of postpartum maternal outcome.

BACKGROUND: Prenatal measurement of placental biomarkers was able to improve screening and diagnosis of preeclampsia. Little is known about the clinical role of placental biomarkers in the postpartum period. METHODS: This study is a prospective monocentric trial that included a total of 30 women with preeclamptic pregnancies. Serum placental biomarkers including soluble fms-like tyrosine kinase 1 (sFlt-1) and placental growth factor (PlGF) were measured before and 2 h after delivery by Enzyme-Linked Immunosorbent Assay (ELISA) using commercially available kits according to manufacturer's instructions and correlated with the postpartum outcome. RESULTS: Postpartum higher serum PlGF level was associated with postpartum elevation of the systolic blood pressure. Yet, the placental biomarkers were not able to predict general worsening of postpartum preeclampsia or other individual clinical or laboratory parameters. CONCLUSION: Serum concentrations of sFlt-1 and PlGF or their ratio in our study cohort did not completely predict the occurrence of postpartum preeclampsia. Yet, postpartum higher serum PlGF level was associated with postpartum elevation of the systolic blood pressure.

[Epidemiology, diagnostics, therapy, prevention and management of uncomplicated bacterial outpatient acquired urinary tract infections in adult patients : Update 2017 of the interdisciplinary AWMF S3 guideline]. / Epidemiologie, Diagnostik, Therapie, Prävention und Management unkomplizierter, bakterieller, ambulant erworbener Harnwegsinfektionen bei erwachsenen Patienten : Aktualisierung 2017 der interdisziplinären AWMF S3­Leitlinie.

BACKGROUND: Update of the 2010 published evidence-based S3 guideline on epidemiology, diagnostics, therapy and management of uncomplicated, bacterial, outpatient-acquired urinary tract infections in adult patients. The guideline contains current evidence for the rational use of antimicrobial substances, avoidance of inappropriate use of certain antibiotic classes and development of resistance. METHODOLOGY: The update was created under the leadership of the German Association of Urology (DGU). A systematic literature search was conducted for the period 01 January 2008 to 31 December 2015. International guidelines have also been taken into account. Evidence level and risk of bias were used for quality review. RESULTS: Updated information on bacterial susceptibility, success, collateral damage and safety of first- and second-line antibiotics was given. For the treatment of uncomplicated cystitis the first line antibiotics are fosfomycin trometamol, nitrofurantoin, nitroxoline, pivmecillinam, trimethoprim (with consideration of the local resistance rates). Fluoroquinolones and cephalosporins should not be used as first choice antibiotics. In the case of uncomplicated pyelonephritis of mild to moderate forms, preferably cefpodoxime, ceftibuten, ciprofloxacin or levofloxacin should be used as oral antibiotics. CONCLUSION: The updated German S3 guideline provides comprehensive evidence- and consensus-based recommendations on epidemiology, diagnostics, therapy, prevention and management of uncomplicated bacterial outpatient acquired urinary tract infections in adult patients. Antibiotic stewardship aspects have significantly influenced the therapeutic recommendations. A broad implementation in all clinical practice settings is necessary to ensure a foresighted antibiotic policy and thus t improve clinical care.

Alloreactive T Cells to Identify Risk HLA Alleles for Retransplantation After Acute Accelerated Steroid-Resistant Rejection.

The risk of rejection by cellular alloreactivity to the transplant donor is not routinely assessed. Here we analyzed alloreactive T cells in kidney transplant recipients and report how their detection may have helped to prevent rejection of a second kidney graft in a patient with a history of acute accelerated steroid-resistant nonhumoral rejection. Alloreactive CD4 and CD8 T cells were quantified using a flow-cytometric mixed lymphocyte reaction assay based on interferon-γ induction. A group of 16 nonrejecting transplant recipients did not show any alloreactive T-cell immunity to their respective donors, whereas alloreactivity to third-party controls was detectable. In the patient with rejection, HLA-specific antibodies were not detectable before and shortly after rejection, but after transplantation the patient showed exceptionally high frequencies of alloreactive T cells against 2 of 11 HLA-typed controls (0.604% and 0.791% alloreactive CD4 T cells and 0.792% and 0.978% alloreactive CD8 T cells) who shared HLA alleles (HLA-A*24, -B*44, -C*02, -DQB1*5) with the kidney donor. These HLA alleles were subsequently excluded for allocation of a second graft. No alloreactive T cells were observed toward the second kidney donor, and this transplantation was performed successfully. Thus, shared HLA alleles between the donor and third-party controls may suggest that alloreactive T cells had contributed to rejection of the first graft. The rejecting patient highlights that determination of cellular alloreactivity before transplantation may be applied to identify unacceptable mismatches and to reduce the risk for acute cellular rejection episodes.

Superior Sensitivity of Ex Vivo IFN-γ Release Assays as Compared to Skin Testing in Immunocompromised Patients.

Comparative assessment of the tuberculin skin testing (TST) and commercial IFN-γ release-assays (IGRAs) is hampered by the use of different antigens (tuberculin PPD in TST vs. ESAT-6/CFP-10 in IGRAs). Thus, PPD was used as a common stimulus to compare performance of the TST and three IGRAs in 72 controls, 101 hemodialysis patients and 100 renal transplant recipients. Results of the TST were compared with PPD-induced IFN-γ induction in vitro detected by ELISPOT, ELISA or a flow-cytometric FACS assay. Percentages of positive tests were significantly lower in TST (9.2%) compared to ELISA (55.3%), ELISPOT (45.3%) and FACS (44.9%, p < 0.0001). Agreement between TST and IGRAs was highest for controls (κ = 0.19-0.32) and poor in immunocompromised patients (κ = 0 for transplant patients, κ = 0.06-0.13 for hemodialysis patients). Discrepant results were largely TST negative and IGRA positive. Among IGRAs, agreement was highest between ELISPOT and FACS (κ = 0.61). Unlike TST, all IGRAs were associated with variables of mycobacterial exposure. Among IGRAs, the FACS assay was least affected by the level of immunosuppression. In conclusion, both the percentage of positive results and between-test-agreement were higher with IGRAs as compared to TST. This indicates superiority of IGRAs in detecting a PPD-specific immune response which may also apply for immunity toward Mycobacterium tuberculosis-specific antigens.

Comparative analysis of assays for detection of cell-mediated immunity toward cytomegalovirus and M. tuberculosis in samples from deceased organ donors.

Cell-mediated immunity assays could be valuable for risk assessment of organ donors, but no data exist on their feasibility in deceased donors. In this study, 105 deceased donors (52.3 ± 16.9 years) were screened at the time of organ procurement. Pathogen-specific stimulation was performed using a cytomegalovirus (CMV) lysate, tuberculin (purified protein derivative [PPD]) and soluble Mycobacterium tuberculosis-specific ESAT-6/CFP-10 proteins in combination with an in-house fluorescence-activated cell sorting (FACS) assay or commercial assay formats (QuantiFERON-CMV/TB for ELISA, T-SPOT.TB for ELISPOT). CMV-IgG antibody titers were determined as gold standard for CMV infection; 51.4% of samples were CMV seropositive. Indeterminate results were observed in 47.6% of ELISA, 12.5% of FACS and 0% of ELISPOT assays. Agreement with serology was highest for FACS (95.6%, κ = 0.91), followed by ELISPOT (84.0%, κ = 0.68) and ELISA (80.0%, κ = 0.60). Agreement between ELISA and serology increased if the CMV lysate was used as stimulus (96.7%, κ = 0.92). Among the T cell assays, agreement between ELISPOT and FACS was highest (κ = 0.70). PPD-positive results among valid samples differed between assays (26.5% for ELISA, 23.1% for FACS and 50.5% for ELISPOT); 2.0% were QuantiFERON-TB positive, 3.3% were ESAT-6/CFP-10-positive in FACS and 13.4% were positive in the T-SPOT.TB assay. In conclusion, cellular immunity may be analyzed from samples of deceased donors, although the assays differ in the rate of positivity and indeterminate results.

BK polyomavirus-specific cellular immune responses are age-dependent and strongly correlate with phases of virus replication.

BK polyomavirus (BKPyV) infection is widespread and typically asymptomatic during childhood, but may cause nephropathy in kidney transplant recipients. However, there is only limited knowledge on BKPyV-specific immunity in children and adults, and its role in BKPyV-replication and disease posttransplant. We therefore characterized BKPyV-specific immunity from 122 immunocompetent individuals (1-84 years), 38 adult kidney recipients with (n = 14) and without BKPyV-associated complications (n = 24), and 25 hemodialysis (HD) patients. Blood samples were stimulated with overlapping peptides of BKPyV large-T antigen and VP1 followed by flow-cytometric analysis of activated CD4 T cells expressing interferon-γ, IL-2 and tumor necrosis factor-α. Antibody-levels were determined using enzyme-linked immunosorbent assay. Both BKPyV-IgG levels and BKPyV-specific CD4 T cell frequencies were age-dependent (p = 0.0059) with maximum levels between 20 and 30 years (0.042%, interquartile range 0.05%). Transplant recipients showed a significantly higher BKPyV-specific T cell prevalence (57.9%) compared to age-matched controls (21.7%) or HD patients (28%, p = 0.017). Clinically relevant BKPyV-replication was associated with elevated frequencies of BKPyV-specific T cells (p = 0.0002), but decreased percentage of cells expressing multiple cytokines (p = 0.009). In conclusion, BKPyV-specific cellular immunity reflects phases of active BKPyV-replication either after primary infection in childhood or during reactivation after transplantation. Combined analysis of BKPyV-specific T cell functionality and viral loads may improve individual risk assessment.

PD-1 analysis on CD28(-) CD27(-) CD4 T cells allows stimulation-independent assessment of CMV viremic episodes in transplant recipients.

Expression of the inhibitory receptor programmed death 1 (PD-1) on cytomegalovirus (CMV)-specific CD4 T cells defines a phenotype associated with CMV viremia in transplant recipients. Moreover, CD28(-) CD27(-) double negativity is known as a typical phenotype of CMV-specific CD4 T cells. Therefore, the co-expression of inhibitory receptors on CD28(-) CD27(-) CD4 T cells was assessed as a rapid, stimulation-independent parameter for monitoring CMV complications after transplantation. Ninety-three controls, 67 hemodialysis patients and 81 renal transplant recipients were recruited in a cross-sectional and longitudinal manner. CMV-specific CD4 T cell levels quantified after stimulation were compared to levels of CD28(-) CD27(-) CD4 T cells. PD-1 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expression on CD28(-) CD27(-) CD4 T cells were related to viremia. A percentage of ≥0.44% CD28(-) CD27(-) CD4 T cells defined CMV seropositivity (93.3% sensitivity, 97.1% specificity), and their frequencies correlated strongly with CMV-specific CD4 T cell levels after stimulation (r = 0.73, p < 0.0001). Highest PD-1 expression levels on CD28(-) CD27(-) CD4 T cells were observed in patients with primary CMV viremia and reactivation (p < 0.0001), whereas CTLA-4 expression was only elevated during primary CMV viremia (p < 0.05). Longitudinal analysis showed a significant increase in PD-1 expression in relation to viremia (p < 0.001), whereas changes in nonviremic patients were nonsignificant. In conclusion, increased PD-1 expression on CD28(-) CD27(-) CD4 T cells correlates with CMV viremia in transplant recipients and may serve as a specific, stimulation-independent parameter to guide duration of antiviral therapy.

Blockade of programmed death receptor-1 signaling restores expression of mostly proinflammatory cytokines in anergic cytomegalovirus-specific T cells.

BACKGROUND: Programmed death receptor-1 (PD-1) compromises cytomegalovirus (CMV)-specific T-cell responses and has been linked to CMV viremia after transplantation. An impaired functional and proliferative capacity of PD-1-positive CMV-specific T cells may be reversed by the antibody-mediated blockade of PD-1 signaling. However, knowledge is limited on changes in "cytokinome" expression profiles associated with reversal of functional exhaustion. METHODS: The "cytokinome" was analyzed by 27-plex Luminex technology comparing renal transplant recipients with low (n = 5) and high (n = 5) PD-1 expression on CMV-specific T cells. The effect of blocking PD-1 by PD-ligand (PD-L) antibodies on restoration of cytokine expression was examined. RESULTS: CMV-specific cytokine release and proliferation was lower in patients with high PD-1 expression on CMV-specific T cells. Antibody-mediated blockade of PD-L in CMV-stimulated samples restored expression levels of interleukin (IL)-1ß, IL-2, IL-6, IL-9, IL-10, granulocyte colony-stimulating factor, interferon-γ, macrophage inflammatory protein-1α, and tumor necrosis factor-α. By contrast, no profound effect was observed for controls or patients with low PD-1 expression, or in staphylococcal enterotoxin B-stimulated cells. CONCLUSION: Taken together, this pilot study provides evidence that a high PD-1 expression on CMV-specific T cells actively impairs proliferation and "cytokinome" responses in an antigen-specific manner. Importantly, blockade of PD-L restores CMV-specific T-cell proliferation and expression of a panel of different proinflammatory and/or type 1 cytokines, suggesting a common but as yet unknown regulatory principle. We conclude that PD-1 exhaustion is reversible and potentially amenable to therapeutic ex vivo and possibly in vivo manipulation. However, detailed knowledge of the differential effects on the "cytokinome" will be necessary to increase the safety and the efficacy of such manipulations.

[National S3 guideline on uncomplicated urinary tract infection: recommendations for treatment and management of uncomplicated community-acquired bacterial urinary tract infections in adult patients]. / Nationale S3-Leitlinie "Unkomplizierte Harnwegsinfektionen" : Empfehlungen zu Therapie und Management unkomplizierter bakterieller ambulant erworbener Harnwegsinfektionen bei erwachsenen Patienten.

BACKGROUND: Urinary tract infections (UTI) belong to the most frequent bacterial infections in outpatients. Increasing antibiotic resistance rates and a new appreciation of the epidemiological side effects of antibiotics ("collateral damage") have warranted an update of the guidelines on uncomplicated UTI as an S3 clinical guideline. METHODS: The guideline was developed by the Deutsche Gesellschaft für Urologie (DGU) in collaboration with the Deutsche Gesellschaft für Allgemein- und Familienmedizin (DEGAM), Deutsche Gesellschaft für Gynäkologie und Geburtshilfe (DGGG), Deutsche Gesellschaft für Hygiene und Mikrobiologie (DGHM), Deutsche Gesellschaft für Infektiologie (DGI), Deutsche Gesellschaft für Nephrologie (DGfN), Paul-Ehrlich-Gesellschaft für Chemotherapie (PEG) and a patient representative. The systematic review of the literature on the topics of the guideline was performed for the time period of 1 January 1998 to 30 April 2008 in the databases of the Cochrane Library and MEDLINE. International guidelines of the years 1999-2007 were included. RESULTS: Uncomplicated UTI comprise uncomplicated cystitis and uncomplicated pyelonephritis. The leading uropathogen is Escherichia coli. The choice of the antibiotic substance follows the five primary aspects: (1) individual patient risk and antibiotic pretreatment; (2) bacterial spectrum and antibiotic susceptibility; (3) effectivity of the antimicrobial substance demonstrated in clinical studies; (4) epidemiological effects ("collateral damage"); and (5) adverse effects. If antibiotics such as trimethoprim/sulfamethoxazole or fluoroquinolones have previously been given, the risk for pathogens to become resistant against these substances is increased. Because of increasing resistance rates of E. coli against trimethoprim/sulfamethoxazole also in uncomplicated UTI, trimethoprim alone or in combination with sulfamethoxazole is no longer regarded as the first-line agent in the empiric treatment of uncomplicated cystitis, unless the regional resistance rate is below 20%. The antibiotic resistance rates of fluoroquinolones in uncomplicated UTI are still below 10% in Germany, but there is a significant emergence of resistance compared to earlier years. Moreover, fluoroquinolones and group 3 cephalosporins exhibit negative epidemiological effects resulting in selection of multi-resistant pathogens. Because these antibiotic classes are needed in therapy of life-threatening infections, such effects should be taken seriously. For substances like fosfomycin, nitrofurantoin or mecillinam"collateral damage" has not been documented or only to a lesser degree. Therefore, for empiric therapy of frequent uncomplicated cystitis fosfomycin-trometamol, nitrofurantoin or pivmecillinam (not listed in Germany) are recommended as first-line antibiotics. For oral first-line treatment of uncomplicated pyelonephritis, fluoroquinolones are still recommended in sufficiently high dosage due to the resistance rates of E. coli still being below 10% and the superior effectivity compared to other antibiotics. Asymptomatic bacteriuria (ASB) should only be treated in exceptional cases such as pregnant women or prior to expected mucocutaneous traumatising interventions of the urinary tract. CONCLUSION: The S3 guideline on uncomplicated urinary tract infections is a comprehensive set of evidence- and consensus-based recommendations dealing with epidemiology, diagnosis, therapy and management of uncomplicated bacterial UTI of adult outpatients. A broad implementation in all disciplines taking care of patients with UTI is necessary in order to ensure a prudent antibiotic policy in these frequent infections and thus improve patient care.

Impaired detection of Mycobacterium tuberculosis immunity in patients using high levels of immunosuppressive drugs.

We have previously shown, in renal transplant recipients on maintenance immunosuppression, that a whole-blood assay was superior in detecting immunity towards purified protein derivative (PPD) compared with skin testing. As blood tests may have limitations during high-dose immunosuppression therapy, the present study was aimed at characterising the effect of high immunosuppressive drug levels on PPD-specific T-cell immunity. PPD-reactive CD4 T-cells from 13 renal transplant recipients were longitudinally quantified by the induction of cytokines using flow cytometry. To further address the effect of high and low maintenance immunosuppression, drug effects were studied in vitro and in 49 age-matched lung transplant recipients and 49 renal transplant recipients. Maintenance immunosuppression after renal transplantation did not affect PPD-specific T-cell detection (median T-cell frequencies 0.55% before and 0.46% >12 months after transplantation), whereas specific T-cell frequencies were significantly lower 3 months after transplantation (0.15%; p = 0.0002). Likewise, high-level maintenance immunosuppression after lung transplantation was associated with a significantly lower prevalence in PPD-specific T-cell reactivity compared with renal transplant recipients (16.7% versus 52.1%; p = 0.0005). In line with the observations made in vivo, calcineurin inhibitors analysed in vitro led to a dose-dependent decrease in antigen-specific T-cell reactivity. The flow cytometric assay is not adversely affected by low drug doses. In contrast, decreased levels of PPD-specific T-cells early after transplantation and low prevalence of PPD-reactivity in lung transplant recipients suggest a reduced sensitivity of in vitro testing during high-level immunosuppression.

PD-1 expression and IL-2 loss of cytomegalovirus- specific T cells correlates with viremia and reversible functional anergy.

Cytomegalovirus (CMV) represents a major cause of infectious complications after transplantation. Recently, chronic infections with lymphocyte choriomeningitis virus (LCMV), HIV or HCV were shown to be associated with functionally exhausted T cells characterized by high expression of the programmed death (PD)-1 molecule and altered cytokine expression patterns. We therefore hypothesized that functional exhaustion of CMV-specific CD4 T cells may determine impaired CMV control in patients after renal transplantation. In viremic transplant recipients, a significantly higher proportion of CMV-specific CD4 T cells was PD-1 positive (median 40.9%, 17.0-88.7%) as compared to nonviremic transplant patients (8.8%, 0.8-80.5%), dialysis patients (8.8%, 0-36.7%) or controls (3.2%, 0.3-15.4%, p < 0.0001). In line with functional impairment, PD-1-positive T cells produced significantly less IFNgamma as compared to PD-1-negative T cells (p < 0.0001). Moreover, unlike controls or nonviremic patients, CMV-specific T cells from viremic patients showed a significant loss of IL-2 production (p < 0.0001). Interestingly, functional anergy of CMV-specific CD4 T cells was reversible in that antibody-mediated blockade of PD-1 signaling with its ligands PD-L1/-L2 led to an up to 10-fold increase in CMV-specific proliferation. In conclusion, expression of PD-1 defines a reversible defect of CMV-specific CD4 T cells that is associated with viremia, and blocking PD-1 signaling may provide a potential target for enhancing the function of exhausted T cells in chronic CMV infection.

Evaluation of latent tuberculosis infection in patients with inflammatory arthropathies before treatment with TNF-alpha blocking drugs using a novel flow-cytometric interferon-gamma release assay.

OBJECTIVE: To compare the efficacy of the conventional skin test and a novel flow cytometric whole blood assay in the diagnosis of latent tuberculosis infection (LTBI) in patients with rheumatological diseases evaluated for treatment with TNF-alpha-blocking agents. METHODS: Prospective study of 97 consecutively enrolled patients, who were assessed for the presence of LTBI through clinical history, Mendel-Mantoux skin testing and chest X-ray. In addition, T-cell reactivity towards tuberculin (PPD, purified protein derivative) and the Mycobacterium tuberculosis-specific proteins ESAT-6 and CFP-10 was determined ex vivo using a flow cytometric whole blood assay. RESULTS: After standard screening, 15% of patients receiving TNF-alpha-blocking therapy were pretreated with isoniazide (INH), another 5% of patients did not receive TNF-alpha-blocking therapy because of LTBI. PPD-reactivity in the skin was observed in 14% of patients compared with 39% with the whole blood test. Analysis of the M. tuberculosis-specific response to ESAT-6 and CFP-10 revealed positive results in 16% of patients. Using a decision tree incorporating history, chest X-ray and either skin-test or ESAT-6/CFP-10 results, 18 or 22% of the patients, respectively, were classified as latently infected with M. tuberculosis. Four patients treated with INH because of a positive skin reaction did not show reactivity to ESAT-6/CFP-10 in the whole blood assays. Another six patients not pretreated with INH because of negative skin tests would have received INH, had the results of the whole blood assay been taken into account. CONCLUSION: The Mendel-Mantoux skin test has a low sensitivity and specificity for the diagnosis of LTBI in this cohort of patients, potentially resulting in both over- and under-treatment with prophylactic INH when compared with the flow cytometric analysis of whole blood T-cell reactivity to proteins specific to M. tuberculosis. Use of T-cell based in vitro tests may help to refine diagnostic testing for LTBI.

Human cytomegalovirus protein pp65: an efficient protein carrier system into human dendritic cells.

Protein-based immunogens are usually poor inducers of CD8(+) T cells. To enhance the induction of CD8(+) T cells, one approach is the use of protein immunogens coupled to protein transduction domains (PTDs). These are small cationic peptide sequences that significantly enhance the uptake of fused proteins into dendritic cells (DC) and then mediate their presentation in the context of major histocompatibility complex class I (MHC-I) and MHC-II molecules. One drawback of this system is the high concentrations of PTD-fusion proteins required. Here, we show that proteins fused to the human cytomegalovirus tegument protein pp65 were bound with higher efficiency to DCs than those fused to the described PTDs TatPTD and Penetratin. Furthermore, the fusion of pp65 to proteins led to an enhanced uptake of these proteins by DCs. Once taken up, CD4(+) and CD8(+) memory T cells were strongly stimulated ex vivo demonstrating that pp65 was efficiently processed and presented in the context of both MHC-I and MHC-II. These data make pp65 a promising delivery system to induce cellular immune responses by fused protein vaccines.

[Volume retention in heart failure, nephrotic syndrome, and liver cirrhosis]. / Volumenüberladung bei Herzinsuffizienz, nephrotischem Syndrom und Leberzirrhose.

Volume retention in heart failure, nephrotic syndrome, and liver cirrhosis reflects pathological changes in homeostatic mechanisms that regulate the extracellular volume (sympathetic activity, renin-angiotensin-aldosterone system [RAAS], natriuretic peptides) and plasma osmolality (antidiuretic hormone [ADH]). In heart failure and liver cirrhosis, these changes are induced by a reduction of the effective circulating volume, which is the part of the extracellular fluid that is within the arterial system and effectively perfusing the tissues. This reduction in the effective circulating volume is caused by reduced cardiac output (heart failure), or by splanchnic vasodilatation with arterial underfilling (liver cirrhosis). In both cases, baroreceptors in both the carotid sinuses and in the glomerular afferent arterioles upregulate RAAS- and sympathetic activity, resulting in systemic vasoconstriction and renal sodium (and volume) retention. More severe reductions in the effective circulating volume may additionally stimulate ADH release, thus increasing the reabsorption of free water with subsequent hyponatriemia. In nephrotic syndrome, volume retention results either directly from the primary renal disease, which induces renal sodium and volume retention ("overfilling"), or indirectly from the reduced plasma oncotic pressure due to hypoalbuminemia, which induces a fluid shift from the intravascular to the interstitial space ("underfilling") with subsequent acitivation of baroreceptors and secondary sodium and volume retention.

A novel flow-cytometry-based assay for quantification of affinity and avidity changes of integrins.

Cell adhesion plays an important role in cell-cell contact formation and cell migration. Thus, the assessment of cellular adhesiveness is one important feature when studying cell-mediated immune responses. The interaction of lymphocytes with other cell types such as antigen-presenting cells or vascular-endothelial cells occurs via adhesion molecules including L-selectin, VCAM-1 or ICAM-1. There are principally two mechanisms by which cell adhesion can be enhanced: namely changes in the affinity or avidity of receptor interactions. Conventional plate-based adhesion assays detect both forms. However, they do not permit discrimination between affinity- and avidity-mediated changes in the adhesiveness. Moreover, analysis of cell subpopulations requires cell separation prior to performance of the adhesion assay. Conventional flow-cytometry-based tests make it possible to determine changes in the affinity of integrins at the single cell level. However, they fail to quantify avidity-mediated adhesiveness. Here we describe a novel flow-cytometry-based assay, which allows the detection of both integrin-mediated affinity as well as avidity changes at the single cell level. This opens up the possibility of precisely characterizing the adhesive capacity of subpopulations in heterogeneous cell populations.

The interleukin-10 promoter genotype determines clinical immune function in hemodialysis patients.

BACKGROUND: Immune dysfunction and the impaired hepatitis B vaccination response are complications of chronic renal failure that are tightly associated with inflammation induced by uremia and blood-membrane contacts. Proinflammatory cytokines, such as interleukin (IL)-6, are counter-regulated by IL-10 with a large interindividual variability. Part of the variability of cytokine production is genetically determined since polymorphisms in the cytokine gene promoters lead to high or low production. The aim of this study was to detect the genetic influence of the IL-10 promoter on immune function of chronic hemodialysis patients. METHODS: The IL-10 genotype (polymorphic bases at positions -1082 and -819) was determined in 272 chronic hemodialysis patients using highly specific PCR and related to the patients' response to a triple vaccination against hepatitis B. Secretion of IL-10 and IL-6 by peripheral blood leucocytes in vitro was determined by ELISA. RESULTS: The prevalence of the IL-10 genotypes in dialysis patients with well-preserved immune function (vaccination responders) was similar to the general population. In contrast, prevalence of the -1082G* allele (associated with high production of IL-10) was low in the nonresponders. The relative risk of vaccination nonresponse in patients homozygous for the -1082A* allele was 1.394 (95% CI, 1.091 to 1.781, P < 0.05) compared to those homozygous for -1082G*. There was no relationship between the IL-10 genotype and the type of renal disease. CONCLUSIONS: The IL-10 genotype determines IL-10 production in dialysis patients, which down-regulates uremia- and dialysis-induced chronic inflammation and helps to preserve immune defense functions.

Levels of virus-specific CD4 T cells correlate with cytomegalovirus control and predict virus-induced disease after renal transplantation.

BACKGROUND: Immunosuppressive treatment in transplant patients frequently causes infectious complications with cytomegalovirus (CMV). The extent of CMV replication can be followed by a number of diagnostic methods. There is, however, no simple diagnostic tool to assess the quality of the cellular antiviral immune response of an individual patient. This would be of particular importance for therapy decisions, as patients with detectable virus load do not necessarily develop CMV-related disease. Using a rapid whole blood assay, the frequencies of CMV-reactive CD4 and CD8 T cells were followed after renal transplantation to characterize their relative contribution in the containment of CMV infection. METHODS: T cells from transplant patients ands healthy control persons were stimulated with CMV antigen in vitro. Based on specific cellular activation and induction of intracellular cytokines, the frequency of CMV-reactive CD4 and CD8 T cells was determined using flow cytometry. Viral load quantified using the "hybrid-capture" assay. RESULTS: The absence of CMV complications in long-term transplant recipients is reflected by stable virus-specific T-cell frequencies, which do not differ from healthy CMV-positive controls. In contrast, during the first months after transplantation, clinical symptoms are preceded by a decrease in CMV-reactive CD4 T-cell frequencies and an increase in CMV load. CONCLUSIONS: The individual immune response and CMV replication are critically balanced and can be characterized by assesing both viral load and antiviral T cells. Our experimental design allows the identification of patients with sufficient, insufficient, or absent T-cell activity and can serve as diagnostic tool to facilitate decisions on antiviral therapy.