Anti-SARS-CoV-2 antibody-containing plasma improves outcome in patients with hematologic or solid cancer and severe COVID-19: a randomized clinical trial.

Patients with cancer are at high risk of severe coronavirus disease 2019 (COVID-19), with high morbidity and mortality. Furthermore, impaired humoral response renders severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines less effective and treatment options are scarce. Randomized trials using convalescent plasma are missing for high-risk patients. Here, we performed a randomized, open-label, multicenter trial ( https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-001632-10/DE ) in hospitalized patients with severe COVID-19 (n = 134) within four risk groups ((1) cancer (n = 56); (2) immunosuppression (n = 16); (3) laboratory-based risk factors (n = 36); and (4) advanced age (n = 26)) randomized to standard of care (control arm) or standard of care plus convalescent/vaccinated anti-SARS-CoV-2 plasma (plasma arm). No serious adverse events were observed related to the plasma treatment. Clinical improvement as the primary outcome was assessed using a seven-point ordinal scale. Secondary outcomes were time to discharge and overall survival. For the four groups combined, those receiving plasma did not improve clinically compared with those in the control arm (hazard ratio (HR) = 1.29; P = 0.205). However, patients with cancer experienced a shortened median time to improvement (HR = 2.50; P = 0.003) and superior survival with plasma treatment versus the control arm (HR = 0.28; P = 0.042). Neutralizing antibody activity increased in the plasma cohort but not in the control cohort of patients with cancer (P = 0.001). Taken together, convalescent/vaccinated plasma may improve COVID-19 outcomes in patients with cancer who are unable to intrinsically generate an adequate immune response.

Inflammation induces pro-NETotic neutrophils via TNFR2 signaling.

Cytokines released during chronic inflammatory diseases induce pro-inflammatory properties in polymorphonuclear neutrophils (PMNs). Here, we describe the development of a subgroup of human PMNs expressing CCR5, termed CCR5+ PMNs. Auto- and paracrine tumor necrosis factor (TNF) signaling increases intracellular neutrophil elastase (ELANE) abundance and induces neutrophil extracellular traps formation (NETosis) in CCR5+ PMNs, and triggering of CCR5 amplifies NETosis. Membranous TNF (mTNF) outside-in signaling induces the formation of reactive oxygen species, known activators of NETosis. In vivo, we find an increased number of CCR5+ PMNs in the peripheral blood and inflamed lamina propria of patients with ulcerative colitis (UC). Notably, failure of anti-TNF therapy is associated with higher frequencies of CCR5+ PMNs. In conclusion, we identify a phenotype of pro-NETotic, CCR5+ PMNs present in inflamed tissue in vivo and inducible in vitro. These cells may reflect an important component of tissue damage during chronic inflammation and could be of diagnostic value.

A Randomized Open label Phase-II Clinical Trial with or without Infusion of Plasma from Subjects after Convalescence of SARS-CoV-2 Infection in High-Risk Patients with Confirmed Severe SARS-CoV-2 Disease (RECOVER): A structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: Primary objectives • To assess the time from randomisation until an improvement within 84 days defined as two points on a seven point ordinal scale or live discharge from the hospital in high-risk patients (group 1 to group 4) with SARS-CoV-2 infection requiring hospital admission by infusion of plasma from subjects after convalescence of SARS-CoV-2 infection or standard of care. Secondary objectives • To assess overall survival, and the overall survival rate at 28 56 and 84 days. • To assess SARS-CoV-2 viral clearance and load as well as antibody titres. • To assess the percentage of patients that required mechanical ventilation. • To assess time from randomisation until discharge. TRIAL DESIGN: Randomised, open-label, multicenter phase II trial, designed to assess the clinical outcome of SARS-CoV-2 disease in high-risk patients (group 1 to group 4) following treatment with anti-SARS-CoV-2 convalescent plasma or standard of care. PARTICIPANTS: High-risk patients >18 years of age hospitalized with SARS-CoV-2 infection in 10-15 university medical centres will be included. High-risk is defined as SARS-CoV-2 positive infection with Oxygen saturation at ≤ 94% at ambient air with additional risk features as categorised in 4 groups: • Group 1, pre-existing or concurrent hematological malignancy and/or active cancer therapy (incl. chemotherapy, radiotherapy, surgery) within the last 24 months or less. • Group 2, chronic immunosuppression not meeting the criteria of group 1. • Group 3, age ≥ 50 - 75 years meeting neither the criteria of group 1 nor group 2 and at least one of these criteria: Lymphopenia < 0.8 x G/l and/or D-dimer > 1µg/mL. • Group 4, age ≥ 75 years meeting neither the criteria of group 1 nor group 2. Observation time for all patients is expected to be at least 3 months after entry into the study. Patients receive convalescent plasma for two days (day 1 and day 2) or standard of care. For patients in the standard arm, cross over is allowed from day 10 in case of not improving or worsening clinical condition. Nose/throat swabs for determination of viral load are collected at day 0 and day 1 (before first CP administration) and subsequently at day 2, 3, 5, 7, 10, 14, 28 or until discharge. Serum for SARS-Cov-2 diagnostic is collected at baseline and subsequently at day 3, 7, 14 and once during the follow-up period (between day 35 and day 84). There is a regular follow-up of 3 months. All discharged patients are followed by regular phone calls. All visits, time points and study assessments are summarized in the Trial Schedule (see full protocol Table 1). All participating trial sites will be supplied with study specific visit worksheets that list all assessments and procedures to be completed at each visit. All findings including clinical and laboratory data are documented by the investigator or an authorized member of the study team in the patient's medical record and in the electronic case report forms (eCRFs). INTERVENTION AND COMPARATOR: This trial will analyze the effects of convalescent plasma from recovered subjects with SARS-CoV-2 antibodies in high-risk patients with SARS-CoV-2 infection. Patients at high risk for a poor outcome due to underlying disease, age or condition as listed above are eligible for enrollment. In addition, eligible patients have a confirmed SARS-CoV-2 infection and O2 saturation ≤ 94% while breathing ambient air. Patients are randomised to receive (experimental arm) or not receive (standard arm) convalescent plasma in two bags (238 - 337 ml plasma each) from different donors (day 1, day 2). A cross over from the standard arm into the experimental arm is possible after day 10 in case of not improving or worsening clinical condition. MAIN OUTCOMES: Primary endpoints: The main purpose of the study is to assess the time from randomisation until an improvement within 84 days defined as two points on a seven-point ordinal scale or live discharge from the hospital in high-risk patients (group 1 to group 4) with SARS-CoV-2 infection requiring hospital admission by infusion of plasma from subjects after convalescence of a SARS-CoV-2 infection or standard of care. Secondary endpoints: • Overall survival, defined as the time from randomisation until death from any cause 28-day, 56-day and 84-day overall survival rates. • SARS-CoV-2 viral clearance and load as well as antibody titres. • Requirement mechanical ventilation at any time during hospital stay (yes/no). • Time until discharge from randomisation. • Viral load, changes in antibody titers and cytokine profiles are analysed in an exploratory manner using paired non-parametric tests (before - after treatment). RANDOMISATION: Upon confirmation of eligibility (patients must meet all inclusion criteria and must not meet exclusion criteria described in section 5.3 and 5.4 of the full protocol), the clinical site must contact a centralized internet randomization system ( https://randomizer.at/ ). Patients are randomized using block randomisation to one of the two arms, experimental arm or standard arm, in a 1:1 ratio considering a stratification according to the 4 risk groups (see Participants). BLINDING (MASKING): The study is open-label, no blinding will be performed. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): A total number of 174 patients is required for the entire trial, n=87 per group. TRIAL STATUS: Protocol version 1.2 dated 09/07/2020. A recruitment period of approximately 9 months and an overall study duration of approximately 12 months is anticipated. Recruitment of patients starts in the third quarter of 2020. The study duration of an individual patient is planned to be 3 months. After finishing all study-relevant procedures, therapy, and follow-up period, the patient is followed in terms of routine care and treated if necessary. Total trial duration: 18 months Duration of the clinical phase: 12 months First patient first visit (FPFV): 3rd Quarter 2020 Last patient first visit (LPFV): 2nd Quarter 2021 Last patient last visit (LPLV): 3rd Quarter 2021 Trial Report completed: 4th Quarter 2021 TRIAL REGISTRATION: EudraCT Number: 2020-001632-10, https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-001632-10/DE , registered on 04/04/2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2). The eCRF is attached (Additional file 3).

Spatial oxidation of L-plastin downmodulates actin-based functions of tumor cells.

Several antitumor therapies work by increasing reactive oxygen species (ROS) within the tumor micromilieu. Here, we reveal that L-plastin (LPL), an established tumor marker, is reversibly regulated by ROS-induced thiol oxidation on Cys101, which forms a disulfide bridge with Cys42. LPL reduction is mediated by the Thioredoxin1 (TRX1) system, as shown by TRX1 trapping, TRX1 knockdown and blockade of Thioredoxin1 reductase (TRXR1) with auranofin. LPL oxidation diminishes its actin-bundling capacity. Ratiometric imaging using an LPL-roGFP-Orp1 fusion protein and a dimedone-based proximity ligation assay (PLA) reveal that LPL oxidation occurs primarily in actin-based cellular extrusions and strongly inhibits cell spreading and filopodial extension formation in tumor cells. This effect is accompanied by decreased tumor cell migration, invasion and extracellular matrix (ECM) degradation. Since LPL oxidation occurs following treatment of tumors with auranofin or γ-irradiation, it may be a molecular mechanism contributing to the effectiveness of tumor treatment with redox-altering therapies.

Human monocytes downregulate innate response receptors following exposure to the microbial metabolite n-butyrate.

INTRODUCTION: Hyporesponsiveness of human lamina propria immune cells to microbial and nutritional antigens represents one important feature of intestinal homeostasis. It is at least partially mediated by low expression of the innate response receptors CD11b, CD14, CD16 as well as the cystine-glutamate transporter xCT on these cells. Milieu-specific mechanisms leading to the down-regulation of these receptors on circulating monocytes, the precursor cells of resident macrophages, are mostly unknown. METHODS: Here, we addressed the question whether the short chain fatty acid n-butyrate, a fermentation product of the mammalian gut microbiota exhibiting histone deacetylase inhibitory activity, is able to modulate expression of these receptors in human circulating monocytes. RESULTS: Exposure to n-butyrate resulted in the downregulation of CD11b, CD14, as well as CD16 surface expression on circulating monocytes. XCT transcript levels in circulating monocytes were also reduced following exposure to n-butyrate. Importantly, treatment resulted in the downregulation of protein and gene expression of the transcription factor PU.1, which was shown to be at least partially required for the expression of CD16 in circulating monocytes. PU.1 expression in resident macrophages in situ was observed to be substantially lower in healthy when compared to inflamed colonic mucosa. CONCLUSIONS: In summary, the intestinal microbiota may support symbiosis with the human host organism by n-butyrate mediated downregulation of protein and gene expression of innate response receptors as well as xCT on circulating monocytes following recruitment to the lamina propria. Downregulation of CD16 gene expression may at least partially be caused at the transcriptional level by the n-butyrate mediated decrease in expression of the transcription factor PU.1 in circulating monocytes.

Monitoring of calcineurin inhibitors by NFAT-regulated gene expression in de novo renal allograft recipients on cyclosporine A.

INTRODUCTION: Calcineurin inhibitors are critical-dose drugs with a narrow therapeutic range and optimal monitoring strategies are discussed in terms of safety and efficacy. A new pharmacodynamic monitoring tool - assessing the expression of nuclear factor of activated T-cells (NFAT)-regulated genes - has been established to directly measure the functional effect of cyclosporine A (CsA) in an individual patient. Until now, only sparse data on NFAT-regulated gene expression within the early post-transplant period have been available. METHOD: Altogether 80 de novo renal transplant patients were enrolled in this non-interventional cohort-study. Immunosuppression consisted of interleukin (IL)-2 receptor antagonist induction, CsA, mycophenolic acid and steroids. Expression of NFAT-regulated genes (IL-2, granulocyte-macrophage colony stimulating-factor (GM-CSF), interferon-γ (IFN-γ)) was determined by qRT-PCR (real-time reverse transcription-PCR) at CsA C0 (prior to CsA intake) and C2 (2 hours after CsA intake) at regular follow-up visits within 6 months after transplantation. RESULTS: The median age of all patients was 47.9 ± 13.7 years (54 male). Residual NFAT-regulated gene expression showed a high interindividual variability. Inversely to reduction of CsA doses, NFAT-regulated genes increased from 1.78 ± 1.33% to 8.04 ± 7.36% in month 1 to month 6. Despite comparable CsA C0 levels, NFAT-regulated gene expression was significantly less inhibited in patients with treated biopsy-proven acute rejections (2.9 ± 2.2% vs. 2.0 ± 1.7%, p = 0.047). Patients with very low residual expression of NFAT-regulated genes were at an increased risk for early infectious episodes. Residual expression of IFN-γ and GM-CSF genes correlated significantly with clinical outcomes. CONCLUSION: NFAT-regulated gene expression is highly inhibited in the early post-transplant period in renal allograft recipients on CsA treatment. High residual NFAT-regulated gene expression was related to acute rejection episodes and low residual expression with infectious complications. Thus, NFAT-monitoring has the potential to support pharmacokinetic monitoring during the early post-transplant period.

Standardization of a human organ culture model of intestinal inflammation and its application for drug testing.

Targeting early molecular events in intestinal inflammation may represent a useful therapeutic strategy for maintaining remission in inflammatory bowel disease. Recently, we established an intestinal organ culture model (LEL model), which allows to study the initiation of an intestinal inflammatory response in human tissue. In this model, EDTA-mediated depletion of epithelial cells of colonic mucosa results in an instantaneous inflammatory response in resident lamina propria cells, which shows features of intestinal inflammation in vivo. Furthermore, activated immune cells emigrate from the lamina propria onto the luminal side of the basement membrane. Here, we standardize the LEL model and explore its suitability for drug testing. To this end, human mucosal punches of defined surface area were prepared, depleted of epithelial cells, and cultured at an optimized ratio of medium volume/punch area. The intra-assay variability of measurements of inflammatory parameters ranged from 13% for cell migration to 19% for secretion and 30% for tissue gene expression, respectively, of the inflammatory mediators IL-8 and IL-6. Importantly, known suppressive effects of dexamethasone, a drug employed for the treatment of inflammatory bowel diseases, on leucocyte migration, IL8, IL6, and TNF-α production as well as CD86 surface expression by myeloid cells were observed in this model. In conclusion, the present results suggest that the LEL model may represent a useful human experimental system not only for studying initial activation mechanisms in intestinal inflammation but also for evaluating drug compounds for the treatment of mucosal inflammation.

The extent of HLA-DR expression on HLA-DR(+) Tregs allows the identification of patients with clinically relevant borderline rejection.

Regulatory T cells (Tregs) were shown to be involved into the pathogenesis of acute rejection after transplantation. The suppressive activity of the total regulatory T cell pool depends on its percentage of highly suppressive HLA-DR(+) -Treg cells. Therefore, both the suppressive activity of the total Treg pool and the extent of HLA-DR expression of HLA-DR(+) -Tregs (MFI HLA-DR) were estimated in non transplanted volunteers, patients with end-stage renal failure (ESRF), healthy renal transplant patients with suspicion on rejection, due to sole histological Bord-R or sole acute renal failure (ARF), and patients with clinically relevant borderline rejection (Bord-R and ARF). Compared to patients with only Bord-R or only ARF, the suppressive activity of the total Treg cell pool was exclusively reduced in patients with clinically relevant Bord-R. In parallel, the HLA-DR MFI of the DR(+) -Treg subset was significantly decreased in these patients, due to a significantly lower proportion of DR(high+) -Tregs, which were shown to have the highest suppressive capacity within the total Treg pool. Our findings clearly demonstrate that the determination of the HLA-DR MFI of the HLA-DR(+) -Treg subset allows a highly sensitive, specific and non-invasive discrimination between patients with clinically relevant Bord-R (Bord and ARF) and patients with subclinical rejection or other causes of transplant failure.

Increased cyclosporin a sensitivity in vivo in pediatric renal transplant recipients compared with adults.

BACKGROUND: Developmental regulation of the pharmacodynamics of cyclosporin A (CsA) has been suggested by in vitro studies. However, these results have not yet been reproduced in the complexity of an in vivo immune system, because reliable biomarkers of CsA effects have not been available. METHODS: Gene expression of interleukin-2 (IL-2), interferon (IFN)-γ, and granulocyte macrophage colony stimulating factor (GM-CSF) in peripheral blood from stable pediatric (N = 31) and adult renal transplant recipients (N = 153) (age range 6.5-78 years) was measured by quantitative real-time polymerase chain reaction before (C0) and 2 hours (C2) after oral CsA intake. To control for the effect of varying CsA concentrations, an index was calculated as a measure of individual CsA sensitivity. RESULTS: The CsA sensitivity of IL-2 gene expression in pediatric patients was 3.9% higher than in middle-aged adults and 5.2% higher than in seniors, indicating stronger immunosuppression at a given CsA blood concentration in younger patients. For the entire patient cohort, there was a statistically significant inverse correlation between the CsA sensitivity of IL-2 and chronological age (r = 0.142, P < 0.0001). Also, the CsA sensitivity of IFN-γ (r = 0.131, P < 0.0001) and GM-CSF (r = 0.036, P < 0.01) were inversely correlated with chronological age. Multiple linear regression analysis revealed that age was a highly significant (P = 0.0027) independent predictor for residual gene expression of IL-2, but not of IFN-γ and GM-CSF. CONCLUSIONS: An increased sensitivity of IL-2 to suppression by CsA was found in pediatric renal transplant recipients in vivo compared with adults. Hence, there seems to be an effect of human development on CsA pharmacodynamics, which, besides the effect of age on pharmacokinetics, should also be considered for the design of treatment regimens of CsA and potentially other calcineurin inhibitors in the pediatric patient population.

DR(high+)CD45RA(-)-Tregs potentially affect the suppressive activity of the total Treg pool in renal transplant patients.

Recent studies show that regulatory T cells (Tregs) play an essential role in tolerance induction after organ transplantation. In order to examine whether there are differences in the composition of the total CD4(+)CD127(low+/-)FoxP3(+)- Treg cell pool between stable transplant patients and patients with biopsy proven rejection (BPR), we compared the percentages and the functional activity of the different Treg cell subsets (DR(high+)CD45RA(-)-Tregs, DR(low+)CD45RA(-)-Tregs, DR(-)CD45RA(-)-Tregs, DR(-)CD45RA(+)-Tregs). All parameters were determined during the three different periods of time after transplantation (0-30 days, 31-1,000 days, >1,000 days). Among 156 transplant patients, 37 patients suffered from BPR. The most prominent differences between rejecting and non-rejecting patients were observed regarding the DR(high+)CD45RA(-)-Treg cell subset. Our data demonstrate that the suppressive activity of the total Treg pool strongly depends on the presence of these Treg cells. Their percentage within the total Treg pool strongly decreased after transplantation and remained relatively low during the first year after transplantation in all patients. Subsequently, the proportion of this Treg subset increased again in patients who accepted the transplant and reached a value of healthy non-transplanted subjects. By contrast, in patients with acute kidney rejection, the DR(high+)CD45RA(-)-Treg subset disappeared excessively, causing a reduction in the suppressive activity of the total Treg pool. Therefore, both the monitoring of its percentage within the total Treg pool and the monitoring of the HLA-DR MFI of the DR(+)CD45RA(-)-Treg subset may be useful tools for the prediction of graft rejection.

Pharmacodynamic monitoring of cyclosporin A reveals risk of opportunistic infections and malignancies in renal transplant recipients 65 years and older.

BACKGROUND: The cohort of senior renal allograft recipients is increasing. Age-related physiologic changes are believed to influence the pharmacokinetics and pharmacodynamics of immunosuppression. Measuring the residual nuclear factor of activated T-cell (NFAT)-regulated gene expression (RGE) is a promising pharmacodynamic tool to individually monitor cyclosporin A (CsA) therapy. PATIENTS AND METHODS: In stable senior renal allograft recipients (≥65 years), the expression of 3 calcineurin-dependent NFAT-regulated genes (interleukin-2, interferon-γ, and granulocyte-macrophage colony-stimulating factor) was measured in whole-blood samples before (C0) and 2 hours (C2) after oral drug intake. Clinical data on opportunistic infections were collected in a clinical observational period of 12 months. RESULTS: Thirty-six senior patients [22 male, median age 70 years (65-77)] were enrolled in this clinical study. Median daily CsA dosage was 150 mg (50-250), CsA C0 concentration 102 mcg/L (range 33-157), and CsA C2 concentration 551 mcg/L (range 254-1228). The NFAT RGE varied between 3% and 37% (median 10%). CsA peak concentrations and inhibition of gene expression correlated significantly (r = -0.737, P < 0.001). NFAT RGE in patients with opportunistic infections including atypical pneumonia, cytomegalovirus and herpes viral infections was lower compared with that in patients without infections [4% (3-13) versus 11% (3-37), P = 0.05], whereas the daily CsA dosage, CsA C0, and CsA C2 concentrations were comparable. Renal allograft function correlated inversely with NFAT RGE. CONCLUSIONS: A higher degree of immunosuppression correlated with more infectious complications in a considerable proportion of senior renal allograft recipients treated with standard CsA therapy. Pharmacodynamic monitoring is an approach to individualize immunosuppression and could provide the opportunity to reduce complications caused by infections.

Selective downregulation of retinoic acid-inducible gene I within the intestinal epithelial compartment in Crohn's disease.

BACKGROUND: A defective innate immune response may contribute to the pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC). Employing a global gene expression analysis, this study was aimed at identifying specifically regulated genes within the epithelial compartment in inflammatory bowel disease (IBD). METHODS: The epithelial fraction of human ileal mucosa samples from surgical specimens was obtained by laser microdissection. Gene expression was examined by global expression profiling (n = 18, Affymetrix), quantitative reverse-transcription polymerase chain reaction (RT-PCR) (n = 35), immunoblot analysis (n = 9), and immunohistochemistry (n = 25). RESULTS: Global expression profiling revealed a pronounced downregulation of the retinoic acid-inducible gene I (RIG-I) within the epithelial layer of the ileum in patients with CD but not with UC. The downregulation of RIG-I was confirmed by quantitative RT-PCR, immunoblot analysis, and immunohistochemistry. CONCLUSIONS: Epithelial downregulation of RIG-I, a known pattern recognition receptor for viral components, might contribute to alterations of the innate mucosal immune response, particularly in CD.

Molecular detection of breast cancer metastasis in sentinel lymph nodes by reverse transcriptase polymerase chain reaction (RT-PCR): identifying, evaluating and establishing multi-marker panels.

The potential advantage of using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) methodology to detect metastasis in sentinel lymph nodes (SLNs) of breast cancer (BC) patients was evaluated in this prospective study. We measured the expression of relevant gene transcripts in SLNs using an innovative algorithm and compared the results of single-marker assays versus multi-marker assays with conventional histological detection methods. SLNs from women aged ≥ 18 years diagnosed with unilateral BC were examined by haematoxylin-eosin staining and immunohistochemistry and analysed for transcripts of several relevant genes using qRT-PCR (learning group). Four candidate panels of expressed transcript combinations with high sensitivity and specificity were selected for further investigation. The candidate panels were then validated using SLNs from a second group of BC patients (validation group). In the learning group, 74/314 SLN sections from 150 patients were positive for metastasis by histology. The transcripts analysed showed the following individual sensitivities/specificities: cytokeratin 19 (CK19) 94.6%/97.9%; mammaglobin 1 (MGB1) 82.4%/91.7%; mammaglobin 2 (MGB2) 82.4%/96.7%; carcinoembryonic antigen (CEA) 71.6%/97.5%; EPCAM (epithelial cell adhesion molecule) 91.9%/97.1%; and NY-BR-1 82.4%/93.8%. The optimal panel based on the predefined criteria comprised four markers: CK19, MGB1, EPCAM, and NY-BR-1, of which ≥ 2 had to be positive (95.9% sensitivity, 95.0% specificity, 85.5% positive predictive value (PPV), and 98.7% negative predictive value (NPV)). Overall concordance with histology was 95.2%. In the validation group, 84/315 SLN sections from 235 patients were histologically positive, and panel sensitivity, specificity and overall accuracy were 88.1, 95.2 and 93.3%, respectively, at the SLN section level. In conclusion, molecular staging using expression patterns of relevant transcripts in SLNs could serve as a useful complement to standard diagnostic work-up in BC patients. The proposed flexible multi-parametric approach does not improve the overall accuracy compared with the single-marker approach. However, it overcomes several limitations of the previously reported molecular assays for SLN diagnosis.

Pharmacodynamic disparities in tacrolimus-treated patients developing cytomegalus virus viremia.

BACKGROUND: The optimal balance between efficacy and toxicity of tacrolimus (Tac) treatment remains unsolved. The quantification of nuclear factor of activated T cell (NFAT)-regulated gene expression may provide a tool to monitor the individual susceptibility to Tac. METHODS: Expression of NFAT-regulated genes (interleukin-2, interferon-gamma, and granulocyte-macrophage colony stimulating factor) in peripheral blood from renal transplant patients (N = 73) was measured by quantitative real-time polymerase chain reaction (at C0, C1.5, and C4) and correlated to clinical endpoints in a 1-year observation period. In a subgroup (n = 10), NFAT expression was quantified over a 12-hour dose interval. RESULTS: Median daily Tac dose of 73 stable renal transplant patients [median age 47 years (range 19-69 years)] was 5 mg (1-13), Tac trough (C0), 1.5-hour (C1.5) and 4-hour (C4) concentrations were 8.5 mcg/L (3-20), 20 mcg/L (4.7-50.4), and 14.5 mcg/L (4.5-37.5), respectively. The mean residual expression of all 3 NFAT-regulated genes was 21% at C1.5 (1-84) and 35% at C4 (2-88). The relative reduction of gene transcripts was inversely correlated with the individual Tac blood concentrations. Seven patients had cytomegalus virus viremia during the observation period, and their residual NFAT-regulated gene expression at C1.5 was significantly lower [13% (1-21) versus 26% (1-84), P = 0.02] compared with those without viremia despite comparable Tac blood concentrations (6.3 versus 8.6 mcg/L). CONCLUSIONS: Monitoring of NFAT-regulated gene expression in Tac-treated transplant recipients provides a tool to assess the individual response to Tac, identify patients at the risk of developing cytomegalus virus viremia, and may, thus, help to select the optimal Tac dose with respect to safety and toxicity.

WF10 stimulates NK cell cytotoxicity by increasing LFA-1-mediated adhesion to tumor cells.

The redox-active chlorite-based drug WF10 (Immunokine) was shown to have modulatory effects on both the innate and adaptive immune system in vitro and in vivo. Animal studies suggest that WF10 enhances immunity against tumors. One possible explanation for such an effect is that WF10 stimulates natural killer cell cytotoxicity against malignant cells. Here, we show that WF10 regulates human NK cell cytotoxicity in a time-dependent manner, following an S-shaped kinetic with an initial stimulation of activity followed by a decrease in activity relative to the untreated controls. WF10 does not activate NK cells on its own but co-stimulates NK cell activation mediated by different activating receptors. This is mediated by enhancing NK cell adhesion to target cells through promoting the activation of the integrin LFA-1. These data demonstrate a direct effect of WF10 on the cytotoxicity of human NK cells.

Individualized monitoring of nuclear factor of activated T cells-regulated gene expression in FK506-treated kidney transplant recipients.

BACKGROUND: The suggested key mechanism of both cyclosporine A (CsA) and FK506 is the inhibition of calcineurin phosphatase activity, preventing nuclear factor of activated T cells (NFAT)-translocation into the nucleus of T cells, with a subsequent transcriptional block of crucial cytokine genes. However, the two drugs exert different clinical activities as exemplified by the ability of FK506 to treat acute rejections. Inhibition of calcineurin activity by FK506 occurs in vitro at the same or even higher dose as for CsA; however, the magnitude of clinical and experimental immunosuppression is higher, indicating that FK506 may act in a calcineurin-independent way. METHODS: To test this hypothesis, we measured the inhibition of NFAT-regulated gene expression in 262 stable kidney transplanted patients after FK506 intake. RESULTS: Previously, we showed that the optimal degree of NFAT inhibition in patients treated with CsA is between 15% and 30% residual gene expression. A considerable number of patients treated with FK506 do not achieve this level of immunosuppression despite therapeutic drug concentrations. Importantly, FK506 does inhibit protein translation. This insufficient degree of NFAT inhibition was associated with a higher rate of biopsy-proven acute rejection but also with a lower incidence of recurrent infections. Conversion of CsA to FK506 causes immediately reduced inhibition of NFAT-regulated gene expression. CONCLUSION: We could demonstrate that a considerable number of FK506-treated patients benefit from the drug, irrespective of the potency of NFAT inhibition in T cells by a yet unknown mechanism. Nevertheless, residual expression of NFAT-regulated genes seems to be a useful pharmacodynamic method to monitor FK506 therapy in renal transplant patients.

Approaches towards individualized immune intervention.

Long-term immunosuppression causes a significantly increased risk for the development of malignancies in transplanted patients. A link between immunosuppression and incidence of cancer is well documented and involves the effect of immunosuppression on antitumor surveillance and antiviral adaptive immune responses. Using a recently described pharmacodynamic assay, a strong correlation between the incidence of malignancies and the individual degree of immunosuppression after cyclosporin A treatment in patients with kidney transplants was observed. The availability of a quantitative and quick laboratory test for the assessment of the individual functional activity of immunocompetent cells crucial for transplant rejection, defense against viral infection and tumor surveillance, along with the ability to adjust doses of immunosuppressive agents such that patients are largely protected against malignant disease and/or viral infection while maintaining a stable allograft function, represents an enormous breakthrough in transplantation medicine and advances our attempts to individualize treatment in transplanted patients.

Exposure of NK cells to intravenous immunoglobulin induces IFN gamma release and degranulation but inhibits their cytotoxic activity.

The mechanisms underlying the modulation of Natural Killer (NK) cell functions by intravenous immunoglobulin (IVIg) are poorly understood. Using an ex vivo whole blood assay system we demonstrate that IVIg suppresses NK cell cytotoxicity. This was paralleled by IVIg-induced degranulation of CD56(bright), CD16(positive) NK cells, reduced expression of CD16 and elevated IFN gamma release. To assess whether these findings also occur in vivo we analyzed whole blood before and after IVIg therapy of patients. Following IVIg treatment the number of NK cells in peripheral blood dropped significantly. We observed reduced CD16 expression, elevated IFN gamma-amounts in plasma, reduced NK cell cytotoxicity, and granzyme B release into the plasma, confirming our in vitro data. These effects on the functions of NK cells describe a novel immunomodulatory effect of IVIg. The in vitro assays employed here could represent informative test systems to monitor effects of in vivo IVIg treatment at an individual level.

Heme oxygenase-1 and its metabolites affect pancreatic tumor growth in vivo.

BACKGROUND: Pancreatic cancer (PaCa) is a fatal human cancer due to its exceptional resistance to all current anticancer therapies. The cytoprotective enzyme heme oxygenase-1 (HO-1) is significantly overexpressed in PaCa and seems to play an important role in cancer resistance to anticancer treatment. The inhibition of HO-1 sensitized PaCa cells to chemo- and radiotherapy in vitro. Therefore, we investigated the effects of HO-1 and its metabolites biliverdin, carbon monoxide and iron on PaCa cells. PaCa cell lines with divergent HO-1 expression patterns were used in a murine orthotopic cancer model. HO-1 expression and activity was regulated by zinc (inhibition) and cobalt (induction) protoporphyrin. Furthermore, the influence of cellular HO-1 levels and its metabolites on effects of standard chemotherapy with gemcitabine was tested in vivo and in vitro. RESULTS: High HO-1 expression in PaCa cell lines was associated with increased chemoresistance in vitro. Chemoresistance to gemcitabine was increased during HO-1 induction in PaCa cells expressing low levels of HO-1. The inhibition of HO-1 activity in pancreatic tumors with high HO-1 boosted chemotherapeutic effects in vivo significantly. Furthermore, biliverdin and iron promoted PaCa resistance to chemotherapy. Consequently, specific iron chelation by desferrioxamine revealed profound anticancerous effects. CONCLUSION: In summary, the inhibition of HO-1 and the chelation of iron in PaCa cells were associated with increased sensitivity and susceptibility of pancreatic tumors to chemotherapy in vivo. The metabolites biliverdin and iron seem to be involved in HO-1-mediated resistance to anticancer treatment. Therefore, HO-1 inhibition or direct interference with its metabolites may evolve new PaCa treatment strategies.