Estimates of protection levels against SARS-CoV-2 infection and severe COVID-19 in Germany before the 2022/2023 winter season: the IMMUNEBRIDGE project.

PURPOSE: Despite the need to generate valid and reliable estimates of protection levels against SARS-CoV-2 infection and severe course of COVID-19 for the German population in summer 2022, there was a lack of systematically collected population-based data allowing for the assessment of the protection level in real time. METHODS: In the IMMUNEBRIDGE project, we harmonised data and biosamples for nine population-/hospital-based studies (total number of participants n = 33,637) to provide estimates for protection levels against SARS-CoV-2 infection and severe COVID-19 between June and November 2022. Based on evidence synthesis, we formed a combined endpoint of protection levels based on the number of self-reported infections/vaccinations in combination with nucleocapsid/spike antibody responses ("confirmed exposures"). Four confirmed exposures represented the highest protection level, and no exposure represented the lowest. RESULTS: Most participants were seropositive against the spike antigen; 37% of the participants ≥ 79 years had less than four confirmed exposures (highest level of protection) and 5% less than three. In the subgroup of participants with comorbidities, 46-56% had less than four confirmed exposures. We found major heterogeneity across federal states, with 4-28% of participants having less than three confirmed exposures. CONCLUSION: Using serological analyses, literature synthesis and infection dynamics during the survey period, we observed moderate to high levels of protection against severe COVID-19, whereas the protection against SARS-CoV-2 infection was low across all age groups. We found relevant protection gaps in the oldest age group and amongst individuals with comorbidities, indicating a need for additional protective measures in these groups.

Mutations in the NOD2 gene are associated with a specific phenotype and lower anti-tumor necrosis factor trough levels in Crohn's disease.

OBJECTIVE: Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) gene mutations are known to be an important risk factor in the pathogenesis of Crohn's disease (CD). Specific disease phenotypes are associated with the presence of NOD2 gene mutation. One treatment option is to use an anti-tumor necrosis factor (TNF)-α agent. Therapeutic drug monitoring (TDM) is usually performed in cases of a loss of response. Our aim was to explore whether NOD2 gene mutations have an effect on the disease phenotype, vitamin D levels, and on TDM in CD patients. METHODS: This was a retrospective genotype-phenotype association study on NOD2 gene mutations in 161 patients with CD. RESULTS: Altogether 55 (34.2%) patients carried at least one mutant allele of NOD2. NOD2 gene mutations were associated with ileocecal disease, ileocecal resection, stricturing and perianal disease, and patients with NOD2 gene mutation had significantly less frequent colonic disease and received an ostomy less frequently. TDM in patients with NOD2 gene mutation showed more frequent anti-TNF trough levels in the subtherapeutic range and lower anti-TNF trough levels than in NOD2 wild-type (WT) patients. CONCLUSIONS: CD patients with NOD2 gene mutation have a specific clinical phenotype and they may require higher doses of anti-TNF agents to achieve sufficient anti-TNF trough levels. They may therefore benefit from a proactive TDM than a reactive approach. This could be another step in the direction of personalized medicine.

CT-P13 (Inflectra™, Remsima™) monitoring in patients with inflammatory bowel disease.

The approval of infliximab biosimilars Remsima™ and Inflectra™ (CT-P13) for patients with inflammatory bowel disease (IBD) is a promising step to reduce treatment costs. Since monitoring of Remicade™ serum trough levels and anti-Remicade™ immunogenicity hold an important significance in treatment modalities, no data about monitoring of drug serum trough levels or anti-drug antibody levels in IBD patients treated with Remsima™ or Inflectra™ are present to date. Therefore, in this study we applied a Remicade™-validated ELISA to determine drug serum levels of Remsima™ or Inflectra™. Serum concentrations were measured at identical levels compared to Remicade™ at multiple time points over 38 weeks, suggesting that the monitoring of serum trough levels is equally feasible for patients receiving Remsima™ or Inflectra™ and Remicade™. Additionally, anti-drug antibody levels were not significantly different in patients treated with Remsima™ or Inflectra™ compared to patients treated with Remicade™. To our knowledge this is the first real-life experience demonstrating the feasibility of drug monitoring in IBD patients treated with the infliximab biosimilars Remsima™ and Inflectra™.

Pharmacokinetic Analysis of Mycophenolate Mofetil and Enteric-Coated Mycophenolate Sodium in Calcineurin Inhibitor-Free Renal Transplant Recipients.

BACKGROUND: Considerable interest exists in identifying calcineurin inhibitor (CNI)-free and thus, less-toxic immunosuppressive regimens, with mycophenolic acid (MPA)-based treatments being a suitable approach. Because pharmacokinetic analyses of MPA treatments in stable CNI-free renal transplant recipients are lacking, the authors aimed at comparing the steady-state pharmacokinetic characteristics of MPA in patients on stable treatment with mycophenolate mofetil (MMF) or enteric-coated mycophenolate sodium (EC-MPS) plus prednisone (≤5 mg/d). METHODS: In the prospective, nonrandomized, open-label study, patients with stable transplant function since ≥6 months received their routine single dose of either MMF (n = 12) or EC-MPS (n = 11). The MPA plasma concentration was recorded over 12 hours. Parameters assessed were predose MPA concentration (C0), postdose minimum and maximum concentration (Cmin and Cmax), time to maximum concentration (Tmax), and area under the concentration-time curve (AUC) for the 12-hours of exposure (AUC0-12). RESULTS: Baseline characteristics were comparable between both the groups. Consistent with enteric coating, the mean Tmax was significantly longer after the intake of EC-MPS compared with MMF (2.2 versus 0.8 hours; P = 0.0002). The exposure measures Cmin, Cmax, and AUC0-12 were not significantly different despite the higher mean MPA equivalent dose in patients receiving MMF compared with those receiving EC-MPS (85% versus 64% of the recommended single dose, respectively). Exposures as reflected by the median AUC0-12 values were 50.7 and 58.7 mg·h·L with MMF and EC-MPS, respectively (P = 0.340). All patients achieved a target AUC of >30 mg·h·L, and 61% had an AUC of >50 mg·h·L. CONCLUSIONS: The study provides first results on the steady-state pharmacokinetics of the 2 MPA drugs in CNI-free immunosuppressant regimens. Pharmacokinetic parameters measured in this study under real-life conditions were comparable in patients receiving MMF or EC-MPS.

Mycophenolic acid reverses TGF beta-induced cell motility, collagen matrix contraction and cell morphology in vitro.

The aim of this study was to elucidate functional and molecular effects of mycophenolic acid (MPA) on non-lymphatic, kidney epithelial cells treated with transforming growth factor (TGF). MPA effects were studied using HK2 cells incubated with EGF and TGF. The reversibility of these effects was verified using guanosine and 8-aminoguanosine. The following assays were applied: cell proliferation, viability, collagen matrix contraction, scratch wound closure, spindle index, FACS with anti-CD29 and anti-CD326, promoter demethylation of RAS protein activator like 1 (RASAL1), as well as gene expression of RASAL1, integrin 1ß (ITGB1) (CD29) and epithelial cell adhesion molecule (EpCam) (CD326). Cell proliferation was inhibited by increasing concentrations of MPA, whereas neither apoptosis nor cytotoxicity was detected. Stimulation with EGF and/or TGF led to a significant collagen matrix contraction that was successfully inhibited by MPA. In addition, scratch wound closure was inhibited by incubation with TGF alone or with EGF. Under the same conditions, cell morphology (spindle shape) and molecular phenotype (ITGB1(High)EpCam(Low)/ITGB1(Low)EpCam(High)) were both significantly changed, suggesting an epithelial to mesenchymal transformation. Cell morphology and motility, as well as molecular phenotype, were reversible after MPA treatment with TGF transformation in both presence/absence of EGF, thereby suggesting a correlation with the previously described antifibrotic effects of MPA. Dysregulation of TGF signal transduction appears to be related to progression of fibrosis. A TGF-transformed kidney epithelial cell line derived from human proximal tubules was used to study whether the immunosuppressive drug: MPA possesses any functional or molecular antifibrotic effects. Functional and morphological in vitro changes induced by both the TGF and epithelial-growth-factor were reversible by treatment with MPA. An inhibitory effect of MPA on the TGF pathway appears to be responsible for the previously described antifibrotic effects of the MPA in the COL4A3-deficient mouse model of renal fibrosis.

Lack of correlation between Treg quantification assays in inflammatory bowel disease patients.

AIM: To compare the number of regulatory T-cells (Tregs) measured by flow cytometry with those obtained using a real-time quantitative PCR (qPCR) method in patients suffering from inflammatory bowel disease (IBD). METHODS: Tregs percentages obtained by both flow cytometry and qPCR methods in 35 adult IBD patients, 18 out of them with Crohn´s disease (CD) and 17 with ulcerative colitis (UC) were compared to each other as well as to scores on two IBD activity questionnaires using the Harvey Bradshaw Index (HBI) for CD patients and the Simple Colitis Clinical Activity Index (SCCAI) for UC patients. The Treg percentages by flow cytometry were defined as CD4(+)CD25(high)CD127(low)FOXP3(+) cells in peripheral blood mononuclear cells, whereas the Treg percentages by qPCR method were determined as FOXP3 promoter demethylation in genomic DNA. RESULTS: We found an average of 1.56% ± 0.78% Tregs by using flow cytometry, compared to 1.07% ± 0.53% Tregs by using qPCR in adult IBD patients. There were no significant correlations between either the percentages of Tregs measured by flow cytometry or qPCR and the HBI or SCCAI questionnaire scores in CD or UC patients, respectively. In addition, there was no correlation between Treg percentages measured by qPCR and those measured by flow cytometry (r = -0.06, P = 0.73; Spearman Rho). These data suggest that, either Treg-related immune function or the clinical scores in these IBD patients did not accurately reflect actual disease activity. Until the cause(s) for these differences are more clearly defined, the results suggest caution in interpreting studies of Tregs in various inflammatory disorders. CONCLUSION: The two methods did not produce equivalent measures of the percentage of total Tregs in the IBD patients studied which is consistent with the conclusion that Tregs subtypes are not equally detected by these two assays.

Effects of mycophenolate mofetil on kidney function and phosphorylation status of renal proteins in Alport COL4A3-deficient mice.

BACKGROUND: We investigated the effects of mycophenolate mofetil (MMF) on kidney function and on protein phosphorylation in a mouse model for the human Alport syndrome. METHODS: COL4A3-deficient (COL4A3-/-) mice were randomly allocated to receive a placebo (PLC COL4A3-/-) or MMF treatment (MMF COL4A3-/-). Wild type mice (WT) were used as controls. Changes in serum creatinine, total protein and blood urea nitrogen (BUN), concentrations of mycophenolic acid (MPA) and its glucuronide metabolite (MPAG), serum protein electrophoresis, urine dipstick chemistry and sediment were measured. Changes in the phosphorylation status of renal proteins and histology were analyzed. RESULTS: MMF influenced kidney function and protein phosphorylation. Serum creatinine and BUN were lower in MMF treated compared to PLC treated COL4A3-/- mice. Serum albumin and alpha-1 globulins were significantly decreased while serum creatinine, alpha-2 globulins, urine dipstick protein, leukocyte esterase, hemoglobin and red blood cells were all increased in both COL4A3-/- groups compared to WT. Differential 2DE-gel analysis identified six phosphorylated kidney protein spots that were significantly altered by MMF. CONCLUSIONS: These data suggest that the MMF treatment in this murine model moderately improved kidney function and reversed the phosphorylation status of six renal phosphoprotein spots to that seen in WT mice.

CD4+ lymphocyte adenosine triphosphate--a new marker in sepsis with acute kidney injury?

BACKGROUND: AKI frequently develops in sepsis patients, significantly decreasing the overall prognosis. There are currently no diagnostic markers available which reliably predict the prognosis of sepsis-associated AKI. Recently, ATP content of CD4+ T cells (ATP_CD4) has been shown to correlate with survival in sepsis. The aim of the study was to determine ATP_CD4 in sepsis-associated AKI. METHODS: Thirty-three patients with sepsis were prospectively analyzed for ATP_CD4 at three different time points. Results were related to survival, renal recovery, and further clinical/laboratory findings. RESULTS: ATP_CD4 tended to lower in concentration at 48 h after onset of sepsis in those patients with complete renal recovery. There were no differences between patients with no AKI and those with AKI of different severity (AKIN 1-3). Urinary NGAL did not correlate with renal prognosis. CONCLUSION: ATP_CD4 may serve as risk predictor in sepsis-associated AKI. Lower concentrations may indicate a higher chance of complete renal recovery in sepsis.

Comparability and imprecision of 8 frequently used commercially available immunoassays for therapeutic drug monitoring.

BACKGROUND: The development of new immunoassays for therapeutic drug monitoring and the adaptation of current assays on new analytical platforms has led to a plethora of different tests. This variability may influence comparability between the methods and affect interpretation of test results used for guiding drug treatment. METHODS: Comparability and imprecision of 8 immunoassays for therapeutic drug monitoring were evaluated using 6 different analyzers from 3 manufacturers. Current and previous generation analytical systems used were Architect and AxSym (Abbott Laboratories), cobas c 501 module and COBAS INTEGRA 800 analyzer (Roche Diagnostics GmbH), and Dimension Vista and Dimension Xpand (Siemens Healthcare Diagnostics Inc). Carbamazepine, digoxin, phenobarbital, phenytoin, theophylline, tobramycin, vancomycin, and valproic acid were measured using leftover routine samples. RESULTS: Good performance and comparability of the drug assays was seen on all systems for carbamazepine, phenytoin, and valproic acid except for phenytoin on the Dimension Vista. Deviations from the acceptance criteria were found with digoxin, phenobarbital, theophylline, tobramycin, and vancomycin. Despite a change of the analytical principle on the Roche systems for most drugs, the results demonstrated a very good between-system comparability of the concentration measured. Systematic deviations were found between AxSYM and ARCHITECT for digoxin, phenobarbital, and tobramycin, and between Dimension Xpand and Dimension Vista for digoxin and vancomycin. CONCLUSIONS: The study revealed differences between assays, platforms, and assay principles. Care should be taken if methods or instruments are replaced in a laboratory. Laboratories are advised to perform their own method comparison studies and to inform their customers about the effect on the therapeutic ranges in case of observed clinically significant deviations.

FoxP3 demethylation is increased in human colorectal cancer and rat cholangiocarcinoma tissue.

OBJECTIVES: FoxP3 expression is a marker for Tregs which are known to be involved in tumor immunity. We aimed to evaluate FoxP3 promoter demethylation in human colorectal cancer (CRC) and rat intrahepatic cholangiocarcinoma (ICC). DESIGN AND METHODS: Bisulfite-treated genomic DNA templates of shock frozen paired samples were studied from 13 anonymous CRC patients and from 10 male rats (n=6 ICC induced by thioacetamide and n=4 age-matched controls). Real-time PCR was carried out using a LightCycler 480 system. Human FoxP3 and CD3 promoter demethylations were estimated using previously described assays; and rat FoxP3 promoter demethylation using a newly developed assay. RESULTS: A significant 3.5-fold increase of the demethylation in FoxP3 promoter region was found in human CRC and rat ICC (P<0.05). The average frequency of cells with FoxP3 demethylation in patients suffering from CRC was 0.26% in normal tissue and 0.92% in tumor tissue (n=11 paired samples). Although, no significant difference was found between the mean frequency of CD3 demethylation in normal tissue (4.80%, n=6) and in tumor tissue (4.14%, n=6) from CRC patients, the ratio of demethylated CD3/FoxP3 promoter areas was significantly lower in tumor specimens (P<0.05). Using our novel assay, we found a significant increase in mean frequencies of cells with FoxP3 demethylation in rats with ICC (7.42%, n=6) in comparison to controls (2.14%, n=4). CONCLUSION: FoxP3 seems to be an interesting biomarker for immune response to epithelial tumors. Functional consequences from the increase of Tregs remain to be demonstrated. Further studies with outcome data are necessary.

Can the Roche hemolysis index be used for automated determination of cell-free hemoglobin? A comparison to photometric assays.

OBJECTIVES: The aim of this study was to develop a novel method for automated quantification of cell-free hemoglobin (fHb) based on the HI (Roche Diagnostics). METHODS: The novel fHb method based on the HI was correlated with fHb measured using the triple wavelength methods of both Harboe [fHb, g/L = (0.915 * HI + 2.634)/100] and Fairbanks et al. [fHb, g/L = (0.917 * HI + 2.131)/100]. fHb concentrations were estimated from the HI using the Roche Modular automated platform in self-made and commercially available quality controls, as well as samples from a proficiency testing scheme (INSTAND). The fHb using Roche automated HI results were then compared to results obtained using the traditional spectrophotometric assays for one hundred plasma samples with varying degrees of hemolysis, lipemia and/or bilirubinemia. RESULTS: The novel method using automated HI quantification on the Roche Modular clinical chemistry platform correlated well with results using the classical methods in the 100 patient samples (Harboe: r = 0.9284; Fairbanks et al.: r = 0.9689) and recovery was good for self-made controls. However, commercially available quality controls showed poor recovery due to an unidentified matrix problem. CONCLUSIONS: The novel method produced reliable determination of fHb in samples without interferences. However, poor recovery using commercially available fHb quality control samples currently greatly limits its usefulness.

Circulating regulatory T cells are reduced in obesity and may identify subjects at increased metabolic and cardiovascular risk.

OBJECTIVE: Reduced numbers of regulatory T (Treg ) cells have been observed in visceral adipose tissue of obese mice and humans. However, it is unknown whether human obesity affects circulating Treg cells and whether their number is associated with markers of systemic inflammation or glucose intolerance. DESIGN AND METHODS: Peripheral blood mononuclear cells were isolated from venous blood of obese (BMI ≥ 27 kg/m(2) ; n = 30) and nonobese (BMI ≥ 27 kg/m(2) ; n = 13) individuals and analyzed using flow cytometry for the expression of CD4, CD25, and Foxp3. RESULTS: Reduced circulating Treg -cell numbers were detected in obese compared with nonobese study participants (P = 0.038). Circulating CD4(+) CD25(+) CD127(-) Foxp3 Treg cells inversely correlated with body weight (P = 0.009), BMI (P = 0.004) and plasma leptin levels (P = 0.004) and were reduced in subjects with hsCRP ≥ 3.0 mg/L (P = 0.034) or HbA1c ≥ 5.5% (P < 0.005). Receiver operating characteristic curve analysis revealed a cutoff of circulating Treg cells < 1.06% to be predictive for hsCRP levels ≥ 3.0 mg/L, and logistic regression showed that the risk of having hsCRP levels ≥ 3.0 mg/L was increased 9.6-fold (P = 0.008), if Treg cells were below this threshold. The Treg cutoff for HbA1c levels ≥ 5.5% was 0.73%, and this cutoff also predicted an increased risk of having elevated levels of both hsCRP and HbA1c, if only obese subjects were examined. CONCLUSION: Our findings thus reveal an association between circulating Treg cells and measures of adiposity, inflammation, and glucose intolerance. Although further prospective studies are needed, we present data suggesting that the determination of Treg cells might be useful to identify obese subjects at increased risk of developing cardiovascular and/or metabolic complications.

Serotonergic overstimulation in a preterm infant after sertraline intake via breastmilk.

The selective serotonin reuptake inhibitor (SSRI) sertraline is widely used as an antidepressant agent during pregnancy and lactation because of its low placental transfer and low level of excretion into breastmilk. Symptoms such as neonatal abstinence syndrome and serotonergic overstimulation have been reported after in utero exposure to SSRIs. These symptoms are self-limiting and usually peak within the first 48 hours after birth. In our case, a preterm infant was exposed to sertraline and its main metabolite desmethylsertraline in utero and via breastmilk. Beyond the first 48 hours after birth, the infant developed increasing clinical signs of serotonergic overstimulation associated with substance intake via breastmilk, until breastfeeding was discontinued on postnatal Day 9. In spite of a low calculated daily substance intake via breastmilk, the serum substance levels of the preterm infant were within the therapeutic range of adults. The serotonergic overstimulation may be explained by the limited metabolic capacity of the infant and the immaturity of the blood-brain barrier.

Confronting the challenge: individualized immune monitoring after organ transplantation using the cellular immune function assay.

The survival of a transplanted organ is dependent on avoidance of rejection, achieved through continuous immuno-suppression. Management of the transplant recipient confronts the clinician with a key challenge of post-transplant immune monitoring. Early detection of an activated allo-immune response is a harbinger of incipient rejection. Thus, timely intervention may prevent acute and chronic injury to the transplanted organ. Similarly, over immune-suppression can lead to infections or malignancies, hence the importance of early detection of the precarious suppression. The need for non-invasive systemic immune monitoring of the transplant recipient is therefore imperative. This review describes the cellular immune function assay--a non-invasive diagnostic method for evaluation of the net state of the recipient's cellular immune function. We describe the background that brought about the need for a reliable diagnostic tool for serial immune monitoring, and we overview the main mile-stones in the assimilation of the assay and its implementation in the clinic. The arising conclusion presents a novel non-invasive diagnostic bio-marker for post-transplant immune monitoring which enables the clinician to intervene prior to manifestation of clinical complications. The usefulness of the assay in detecting a state of over-suppression has been consensually described in multiple publications while its contribution in detection and management of under-suppression conditions remains to be determined by means of prospective interventional studies. The cellular immune function assay can be useful and beneficial for patient care only if used for longitudinal monitoring through serial testing.

Intracellular ATP production in CD4+ T cells as a predictor for infection and allograft rejection in trough-level guided pediatric liver transplant recipients under calcineurin-inhibitor therapy.

BACKGROUND: The assessment of cell-mediated immune responses through the measurement of intracellular adenosine-tri-phosphate (iATP) production (Cylex ImmuKnow) as a pharmacodynamic biomarker of immune function represents a potential tool to optimize individual immunosuppressive therapy independent of drug dosage or trough levels. This study aims to investigate the correlations between iATP and adverse events, immunosuppression, calcineurin-inhibitor-trough levels, and age. METHODS: In this prospective trial, 31 nontransplant pediatric subjects and 50 consecutive children were included after they underwent liver transplantation (LTX). During the study period, 4 allograft rejections and 3 acute infections occurred. The patients were treated with cyclosporine, tacrolimus, mycophenolate mofetil, and everolimus either as monotherapy or in combinations. The reactivity of the immune system was measured as iATP concentration in CD4+ T-cells after in vitro stimulation by phytohemagglutinin. RESULTS: The iATP concentrations in patients with intercurrent, clinically significant infections were in the low immune response range (median iATP 181 versus 251 ng/mL, P = 0.308), whereas the patients with incidental allograft rejection had significantly higher iATP concentrations as compared with the event-free group (median iATP 444 versus 251 ng/mL, P = 0.017). However, there was a wide range of iATP concentrations in both nontransplant and LTX patient groups, and no clear iATP cut-off values for an increased risk of infection or rejection could be defined. Post LTX, stable-phase patients showed a significantly lower iATP compared with respective controls (median iATP 297 versus 384 ng/mL, P = 0.013). No significant correlation between calcineurin-inhibitor-trough concentrations and iATP was found. iATP was not correlated with age, but was inversely correlated with time after transplantation. CONCLUSIONS: The observed correlation between clinical events and iATP concentrations is similar to the findings previously reported in adult patients who underwent transplantation. The lack of correlation of iATP with trough drug concentrations suggests that the ImmuKnow assay provides independent information that may be useful to guide immunosuppressive therapy in pediatric (liver) transplant patients. However, the wide range of iATP levels in event-free patients suggests that serial iATP measurements will be necessary to assess and guide the individual immunosuppressive therapy. Further investigations are needed to evaluate and extend these findings.

Use of total and unbound imatinib and metabolite LC-MS/MS assay to understand individual responses in CML and GIST patients.

OBJECTIVES: Trough total imatinib (t-IM) concentrations have been reported to be associated with therapeutic and toxic responses in patients with chronic myelogenous leukemia (CML) and gastrointestinal stromal tumor (GIST). Little is known about the relationships between effects and concentrations of either unbound imatinib (f-IM) or imatinib's major metabolite, N-desmethyl imatinib (NDI). In part, this is because of the lack of a single, validated, well-described clinically useful assay for these measurements. The authors report the development and application of such an assay. MATERIALS AND METHODS: A single liquid-chromatography tandem-mass-spectrometry assay was used to monitor t-IM, f-IM, and t-NDI concentrations in CML and GIST patients treated at a tertiary German teaching hospital. The assay was also validated for measuring other kinase inhibitors, including t-nilotinib, sunitinib, and erlotinib. Ultrafiltration assays were validated and used to measure f-IM and to compare free fractions to plasma α1-acid glycoprotein concentrations (AGP). RESULTS: The assays were linear over a working range (in micrograms per liter) of 8.4-8370, 8.3-4165, and 1.0-250 and had within- and between-run coefficient of variance of <7%, <12%, and <9% for t-IM, t-NDI, and f-IM, respectively. The f-IM assay was reproducible despite high (25.2%-31.6%) but concentration-independent binding to ultrafiltration devices. Clinically relevant results, such as nondetectable (ND) t-IM (<8.4 µg/L) in non-responders and >1500 µg/L in patients with major toxicity, were found. Of 156 total samples from 68 adult CML patients and 127 total samples from 42 adult GIST, only 48 samples from 22 CML patients and 40 samples from 20 GIST patients were trough samples with adequate dosing and collection information. More than half (27 of 48 CML and 24 of 40 GIST) had t-IM concentrations ≥10% below recommended target concentrations (1002 µg/L for CML and 1100 µg/L for GIST). Concentrations >50% over targets were also found in 6 of 48 CML and 4 of 40 GIST samples. Wide variations in concentrations of t-IM (range, ND to 2973 µg/L), t-NDI (range, ND to 659 µg/L), f-IM (range, 8.3-262 µg/L), and t-IM:f-IM ratios (range, 2.6%-14%) were found both between and within patients. A statistically significant association (Spearman correlation coefficient and P value for all samples, r = 0.290 and P = 0.023; for trough only, r = -0.585 and P = 0.028) was found between AGP and f-IM concentrations but wide interpatient and intrapatient variations made individual predictions unreliable. CONCLUSIONS: The liquid-chromatography tandem-mass-spectrometry methods developed provided information useful to understand individual responses to therapy even though necessary sampling and dosing information was often not available. Wide unpredictable variations in t-IM, t-NDI, and f-IM were found. Clinical outcome trials are needed to examine whether f-IM or NDI monitoring can improve the ability to predict individual responses.

Differential kidney proteome profiling in a murine model of renal fibrosis under treatment with mycophenolate mofetil.

AIM: The aim of this study was to investigate the effect of mycophenolate mofetil (MMF) using differential kidney proteome profiling of COL4A3-deficient mice as a model of progressive renal disease. METHODS: Histological evaluation of kidney sections was performed. Total protein lysate from kidneys of placebo- and MMF-treated COL4A3-deficient mice was studied for significant differences in protein abundance using 2-dimensional electrophoresis and mass spectrometry. RESULTS: While tubulointerstitial fibrosis in COL4A3-deficient mice was inhibited by MMF, 19 proteins in the kidneys were regulated: 12 with lower (ATPO, TAGL2, CAH1, TPD52, VA0D1, SERPH, GNAL, PSB6, EF1D, OTUB1, NDUS8, and NAPSA) and 7 with higher (ACADM, ACY3, CK054, ACTB/G, ACTB, UBP5, and ACY1) spot intensity. Nine differentially expressed proteins showed interaction potential (ATPO, TPD52, PSB6, EF1D, OTUB1, NAPSA, ACTB, ACTG, and UBP5). CONCLUSIONS: The identified proteins take part in different signaling pathways. With the highest probability, the VEGF signaling pathway (TAGL2, EF1D, and ACTB) and hypoxia (CAH1, PSB6, and ACTG) were influenced by MMF in fibrotic conditions. Moreover, MMF may modulate antifibrotic and apoptotic pathways as well as epithelial-mesenchymal transition (EMT). Different signaling pathways may be influenced by MMF therapy. These results suggest an inhibitory effect of MMF on renal EMT in COL4A3-deficient mice. Further studies are necessary to validate these findings.

Pre-analytical effects of different lithium heparin plasma separation tubes in the routine clinical chemistry laboratory.

BACKGROUND: In this study the pre-analytical effects of sample storage on frequently used routine clinical chemistry assays were evaluated by comparing four different lithium heparin plasma separation tubes to a reference collection procedure. METHODS: Blood was collected from 20 healthy volunteers using plasma separation tubes from four different manufacturers together with manually separated plasma as reference. In total, 15 clinical chemistry parameters were determined at 0 h, 24 h, and 72 h. Samples were stored at 4°C. Statistical differences were evaluated using a generalized estimating equation regression model. RESULTS: Significant differences could be demonstrated for almost every parameter when comparing the separation tubes to the reference collection system. The estimated maximum allowable storage time in the primary tube was considerably reduced using separation tubes, e.g., for glucose the maximum storage time was reduced from >72 h to 7-15 h, and for potassium from 60 h to 10-13 h, respectively. CONCLUSIONS: These data indicate that sample storage in the primary tube using plasma separation tubes is associated with clinically relevant changes for certain parameters. Therefore, storing samples for retesting should be avoided when using plasma separation tubes, in particular for parameters susceptible to interference by erythrocyte or platelet contamination.