Single-cell multiomic dissection of response and resistance to chimeric antigen receptor T cells against BCMA in relapsed multiple myeloma.

Markers that predict response and resistance to chimeric antigen receptor (CAR) T cells in relapsed/refractory multiple myeloma are currently missing. We subjected mononuclear cells isolated from peripheral blood and bone marrow before and after the application of approved B cell maturation antigen-directed CAR T cells to single-cell multiomic analyses to identify markers associated with resistance and early relapse. Differences between responders and nonresponders were identified at the time of leukapheresis. Nonresponders showed an immunosuppressive microenvironment characterized by increased numbers of monocytes expressing the immune checkpoint molecule CD39 and suppressed CD8+ T cell and natural killer cell function. Analysis of CAR T cells showed cytotoxic and exhausted phenotypes in hyperexpanded clones compared to low/intermediate expanded clones. We identified potential immunotherapy targets on CAR T cells, like PD1, to improve their functionality and durability. Our work provides evidence that an immunosuppressive microenvironment causes resistance to CAR T cell therapies in multiple myeloma.

Primary Sjögren's syndrome independently promotes premature subclinical atherosclerosis.

OBJECTIVES: Cardiovascular comorbidities are common in patients with autoimmune diseases. This study investigates the extent of subclinical atherosclerosis in patients with primary Sjögren's syndrome (pSS). Correlations with clinical factors such as organ involvement (OI) or disease activity were analysed and oxLDL antibodies (oxLDL ab) were measured as potential biomarkers of vascular damage. METHODS: Patients with pSS were consecutively included from the rheumatology outpatient clinic. Age- and sex-matched controls were recruited (2:1 ratio). Data collection was performed by a standardised questionnaire and Doppler ultrasound to evaluate the plaque extent and carotid intima-media thickness (cIMT). Propensity score matching included all cardiovascular risk (CVR) factors and corresponding laboratory markers. RESULTS: Data were available for 299 participants (199 pSS/100 controls), aged 59.4 years (50.6-65.0), 19.1% male. After matching, the pSS cohort had greater cIMT (p<0.001) and plaque extent (OR=1.82; 95% CI 1.14 to 2.95). Subgroup analyses of patients with pSS revealed that OI was associated with increased cIMT (p=0.025) and increased plaque occurrence compared with patients without OI (OR=1.74; 95% CI 1.02 to 3.01). OxLDL ab tended to be lower in patients with plaque (p=0.052). Correlations of higher Oxidized Low Density Lipoprotein (oxLDL) ab with EULAR Sjögren's Syndrome Disease Activity Index (p<0.001) and anti-Sjögren's-syndrome-related antigen A autoantibodies (SSA/Ro antibodies) (p=0.026) were observed. CONCLUSIONS: Subclinical atherosclerosis occurs earlier and more severely in patients with pSS. The difference in cIMT between pSS and controls seems mainly driven by patients with OI, suggesting that this subgroup is particularly at risk. OxLDL ab might protect against atherosclerotic progression in patients with pSS. CVR stratification and preventive medications such as Hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors should be discussed and further longitudinal studies are needed.

Cellular dynamics following CAR T cell therapy are associated with response and toxicity in relapsed/refractory myeloma.

B-cell maturation antigen (BCMA)-targeting chimeric antigen receptor (CAR) T cells revolutionized the treatment of relapsed/refractory multiple myeloma (RRMM). However, data on cellular (CAR) T cell dynamics and the association with response, resistance or the occurrence of cytokine release syndrome (CRS) are limited. Therefore, we performed a comprehensive flow cytometry analysis of 27 RRMM patients treated with Idecabtagene vicleucel (Ide-cel) to assess the expansion capacity, persistence and effects on bystander cells of BCMA-targeting CAR T cells. Additionally, we addressed side effects, like cytokine release syndrome (CRS) and cytopenia. Our results show that in vivo expansion of CD8+ CAR T cells is correlated to response, however persistence is not essential for durable remission in RRMM patients. In addition, our data provide evidence, that an increased fraction of CD8+ T cells at day of leukapheresis in combination with successful lymphodepletion positively influence the outcome. We show that patients at risk for higher-grade CRS can be identified already prior to lymphodepletion. Our extensive characterization contributes to a better understanding of the dynamics and effects of BCMA-targeting CAR T cells, in order to predict the response of individual patients as well as side effects, which can be counteracted at an early stage or even prevented.

Accelerating development of engineered T cell therapies in the EU: current regulatory framework for studying multiple product versions and T2EVOLVE recommendations.

To accelerate the development of Advanced Therapy Medicinal Products (ATMPs) for patients suffering from life-threatening cancer with limited therapeutic options, regulatory approaches need to be constantly reviewed, evaluated and adjusted, as necessary. This includes utilizing science and risk-based approaches to mitigate and balance potential risks associated with early clinical research and a more flexible manufacturing paradigm. In this paper, T2EVOLVE an Innovative Medicine Initiative (IMI) consortium explores opportunities to expedite the development of CAR and TCR engineered T cell therapies in the EU by leveraging tools within the existing EU regulatory framework to facilitate an iterative and adaptive learning approach across different product versions with similar design elements or based on the same platform technology. As understanding of the linkage between product quality attributes, manufacturing processes, clinical efficacy and safety evolves through development and post licensure, opportunities are emerging to streamline regulatory submissions, optimize clinical studies and extrapolate data across product versions reducing the need to perform duplicative studies. It is worth noting that this paper is focusing on CAR- and TCR-engineered T cell therapies but the concepts may be applied more broadly to engineered cell therapy products (e.g., CAR NK cell therapy products).

Deciphering the human antibody response against Burkholderia pseudomallei during melioidosis using a comprehensive immunoproteome approach.

Introduction: The environmental bacterium Burkholderia pseudomallei causes the often fatal and massively underreported infectious disease melioidosis. Antigens inducing protective immunity in experimental models have recently been identified and serodiagnostic tools have been improved. However, further elucidation of the antigenic repertoire of B. pseudomallei during human infection for diagnostic and vaccine purposes is required. The adaptation of B. pseudomallei to very different habitats is reflected by a huge genome and a selective transcriptional response to a variety of conditions. We, therefore, hypothesized that exposure of B. pseudomallei to culture conditions mimicking habitats encountered in the human host might unravel novel antigens that are recognized by melioidosis patients. Methods and results: In this study, B. pseudomallei was exposed to various stress and growth conditions, including anaerobiosis, acid stress, oxidative stress, iron starvation and osmotic stress. Immunogenic proteins were identified by probing two-dimensional Western blots of B. pseudomallei intracellular and extracellular protein extracts with sera from melioidosis patients and controls and subsequent MALDI-TOF MS. Among B. pseudomallei specific immunogenic signals, 90 % (55/61) of extracellular immunogenic proteins were identified by acid, osmotic or oxidative stress. A total of 84 % (44/52) of intracellular antigens originated from the stationary growth phase, acidic, oxidative and anaerobic conditions. The majority of the extracellular and intracellular protein antigens were identified in only one of the various stress conditions. Sixty-three immunoreactive proteins and an additional 38 candidates from a literature screening were heterologously expressed and subjected to dot blot analysis using melioidosis sera and controls. Our experiments confirmed melioidosis-specific signals in 58 of our immunoproteome candidates. These include 15 antigens with average signal ratios (melioidosis:controls) greater than 10 and another 26 with average ratios greater than 5, including new promising serodiagnostic candidates with a very high signal-to-noise ratio. Conclusion: Our study shows that a comprehensive B. pseudomallei immunoproteomics approach, using conditions which are likely to be encountered during infection, can identify novel antibody targets previously unrecognized in human melioidosis.

Comparison of two lab-scale protocols for enhanced mRNA-based CAR-T cell generation and functionality.

Process development for transferring lab-scale research workflows to automated manufacturing procedures is critical for chimeric antigen receptor (CAR)-T cell therapies. Therefore, the key factor for cell viability, expansion, modification, and functionality is the optimal combination of medium and T cell activator as well as their regulatory compliance for later manufacturing under Good Manufacturing Practice (GMP). In this study, we compared two protocols for CAR-mRNA-modified T cell generation using our current lab-scale process, analyzed all mentioned parameters, and evaluated the protocols' potential for upscaling and process development of mRNA-based CAR-T cell therapies.

Bone Sialoprotein Immobilized in Collagen Type I Enhances Angiogenesis In Vitro and In Ovo.

Bone fracture healing is a multistep process, including early immunological reactions, osteogenesis, and as a key factor, angiogenesis. Molecules inducing osteogenesis as well as angiogenesis are rare, but hold promise to be employed in bone tissue engineering. It has been demonstrated that the bone sialoprotein (BSP) can induce bone formation when immobilized in collagen type I, but its effect on angiogenesis still has to be characterized in detail. Therefore, the aim of this study was to analyse the effects of BSP immobilized in a collagen type I gel on angiogenesis. First, in vitro analyses with endothelial cells (HUVECs) were performed detecting enhancing effects of BSP on proliferation and gene expression of endothelial markers. A spheroid model was employed confirming these results. Finally, the inducing impact of BSP-collagen on vascular density was proved in a yolk sac membrane assay. Our results demonstrate that BSP is capable of inducing angiogenesis and confirm that collagen type I is the optimal carrier for this protein. Taking into account former results, and literature showing that BSP also induces osteogenesis, one can hypothesize that BSP couples angiogenesis and osteogenesis, making it a promising molecule to be used in bone tissue regeneration.

Bone Sialoprotein Immobilized in Collagen Type I Enhances Bone Regeneration In vitro and In vivo.

The use of bioactive molecules is a promising approach to enhance the bone healing properties of biomaterials. The aim of this study was to define the role of bone sialoprotein (BSP) immobilized in collagen type I in various settings. In vitro studies with human primary osteoblasts in mono- or in co-culture with endothelial cells demonstrated a slightly increased gene expression of osteogenic markers as well as an increased proliferation rate in osteoblasts after application of BSP immobilized in collagen type I. Two critical size bone defect models were used to analyze bone regeneration. BSP incorporated in collagen type I increased bone regeneration only marginally at one concentration in a calvarial defect model. To induce the mechanical stability, three-dimensional printing was used to produce a stable porous cylinder of polylactide. The cylinder was filled with collagen type I and immobilized BSP and implanted into a femoral defect of critical size in rats. This hybrid material was able to significantly induce bone regeneration. Our study clearly shows the osteogenic effect of BSP when combined with collagen type I as carrier and thereby offers various approaches and options for its use as bioactive molecule in bone substitute materials.

Identificación de riesgos ergonómicos en personal administrativo que realizó teletrabajo / Identificación of ergonomic risks un administrative personnel that performed teleworking

INTRODUCCIÓN. El riesgo ergonómico es la situación adversa que surge de una inadecuada interacción entre el trabajador y su puesto de trabajo. Su identificación es el punto de partida para la corrección y prevención de daños a la salud. OBJETIVO. Describir los riesgos ergonómicos del personal administrativo que realizó teletrabajo. MATERIALES Y MÉTODOS. Estudio observacional, descriptivo, retrospectivo, con una población de 66 empleados y una muestra de 25 encuestas a personal administrativo de una empresa importadora de la ciudad de Quito, que realizó teletrabajo desde marzo de 2020. Criterios de inclusión: mayores de 18 años de ambos sexos; que realizaron teletrabajo al menos 60 días. Para el análisis de datos se utilizó el programa Microsoft Excel 2013. RESULTADOS. Se observaron y registraron riesgos ergonómicos en la conformación del puesto de trabajo por mobiliario inadecuado; 20% (5; 25) del personal tuvo una silla ergonómica; 48% (12; 25) un computador tipo PC; 72% (18; 25) utilizó mouse. Se identificó disconfort en zonas corporales, resaltó lumbar 48% (12; 25), codo o antebrazo derecho 36% (9; 25) y cervical 28% (7; 25). CONCLUSIÓN. Se pudo identificar riesgos ergonómicos a los que estuvo expuesto el personal administrativo que realizó teletrabajo.

INTRODUCTION. Ergonomic risk is the adverse situation that arises from an inappropriate interaction between the worker and his job. Their identification is the starting point for the correction and prevention of damage to health. OBJECTIVE. To describe the ergonomic risks of administrative personnel who performed telework. MATERIALS AND METHODS. Observational, descriptive, retrospective study, with a population of 66 employees and a sample of 25 surveys to administrative staff of an importing company in the city of Quito, who performed telework since March 2020. Inclusion criteria: over 18 years of age of both sexes; who teleworked for at least 60 days. For data analysis, the Microsoft Excel 2013 program was used. RESULTS. Ergonomic risks were observed and recorded in the conformation of the workplace due to inadequate furniture; 20% (5; 25) of the staff had an ergonomic chair; 48% (12; 25) a PC type computer; 72% (18; 25) used a mouse. Discomfort was identified in body areas, 48% of the lumbar (12; 25), the right elbow or forearm 36% (9; 25) and the cervical 28% (7; 25). CONCLUSION. It was possible to identify ergonomic risks to which the administrative personnel who performed teleworking were exposed.

Greater hippocampal gray matter volume in subjective hyperosmia: a voxel-based morphometry study.

Subjective hyperosmia refers to a self-reported olfactory ability that is superior to a normal, intact sense of smell (normosmia), and is associated with olfactory emotional experience. The current study used voxel-based morphometry to investigate the gray matter volume (GMV) in people with self-rated hyperosmia (subjective hyperosmia, SH, N = 18) in comparison to people with self-rated normal olfaction (subjective normosmia, SN, N = 14). Participants' olfactory function were assessed by the extensive olfactory test battery, the "Sniffin' Sticks" test. Within the predicted brain regions (regions-of-interest analyses), the SH participants showed larger GMV of the left hippocampus as compared to SN participants (FWE corrected p < 0.05). Further, the whole-brain search indicated that SH had larger GMV of the bilateral hippocampus, the right hypothalamus, the left precuneus, and the left superior frontal gyrus as compared to the SN group. ROI analyses showed positive correlations between the left hippocampal GMV and odor threshold or discrimination scores across all participants. In addition, the whole-brain analysis suggested that the self-rated olfactory ability was positively associated with GMV in the cerebellum, superior frontal gyrus and the precentral gyrus among SH participants. In conclusion, the current results suggest that SH was associated with increased GMV in several brain regions that were previously shown to be involved in the processing of cognitive aspects of odors.

Serum Parathyroid Hormone Predicts Mortality in Coronary Angiography Patients with Type 2 Diabetes.

BACKGROUND: Elevated serum levels of parathyroid hormone (PTH), one of the main regulators of calcium homeostasis and vitamin D metabolism, have been proposed as predictors of mortality. The impact of type 2 diabetes mellitus (T2DM) on the putative association between PTH and mortality has not been investigated thus far. AIM: The aim of our study was to investigate the impact of T2DM on the power of PTH to predict mortality risk. METHODS: Serum PTH levels were determined in 904 consecutive Caucasian patients referred to coronary angiography for the evaluation of established or suspected stable coronary artery disease (CAD), including 235 patients with T2DM. Prospectively, deaths were recorded over a mean follow-up period of 6.3 years. RESULTS: PTH at baseline did not differ significantly between patients with and without T2DM (P = .307). Cox regression analysis revealed that the serum PTH level strongly predicted all-cause mortality in patients with T2DM (hazard ratio [HR] = 2.35 [1.37-4.03]; P = .002), whereas PTH did not predict all-cause mortality in patients without T2DM (HR = 1.04 [0.81-1.32]; P = .766). The interaction term PTH × T2DM was significant (P = .006), indicating a significantly stronger impact of PTH on mortality risk in patients with T2DM than in individuals without diabetes. The impact of PTH on mortality risk in patients with T2DM remained significant after adjustment for glycated hemoglobin A1c, diabetes duration, classical cardiovascular risk factors, serum levels of vitamin D, and kidney function (HR = 2.10 [1.10-4.10]; P = .030). CONCLUSION: We conclude that PTH is a significantly stronger predictor of all-cause mortality in patients with T2DM than in those without T2DM.

Measuring Vitamin D Status in Chronic Inflammatory Disorders: How does Chronic Inflammation Affect the Reliability of Vitamin D Metabolites in Patients with IBD?

Evidence gained from recent studies has generated increasing interest in the role of vitamin D in extraskeletal functions such as inflammation and immunoregulation. Although vitamin D deficiency has been implicated in the pathophysiology of inflammatory diseases including inflammatory bowel disease (IBD), evidence as to whether vitamin D supplementation may cure or prevent chronic disease is inconsistent. Since 25OH-vitamin D (25OHD) has been suggested to be an acute-phase protein, its utility as a vitamin D status marker is therefore questionable. In this study, possible interactions of vitamin D and inflammation were studied in 188 patients with IBD, with high-sensitivity C-reactive protein (hsCRP) levels ≥ 5 mg/dL and/or fecal calprotectin ≥ 250 µg/g defined as biochemical evidence of inflammatory activity. Levels of 25OHD and vitamin D-binding protein (VDBP) were determined by ELISA, and 1,25-dihydroxyvitamin D (1,25OHD) and dihydroxycholecalciferol (24,25OHD) by LC-MS/MS. Free and bioavailable vitamin D levels were calculated with the validated formula of Bikle. Serum 1,25OH2D and vitamin D binding protein (VDBP) levels were shown to differ between the inflammatory and noninflammatory groups: patients with inflammatory disease activity had significantly higher serum concentrations of 1,25OH2D (35.0 (16.4-67.3) vs. 18.5 (1.2-51.0) pg/mL, p < 0.001) and VDBP (351.2 (252.2-530.6) vs. 330.8 (183.5-560.3) mg/dL, p < 0.05) than patients without active inflammation. Serum 24,25OH2D levels were negatively correlated with erythrocyte sedimentation rate (ESR) (-0.155, p = 0.049) while concentrations of serum 1,25OH2D correlated positively with hsCRP (0.157, p = 0.036). Correlations with serum VDBP levels were found for ESR (0.150, p = 0.049), transferrin (0.160, p = 0.037) and hsCRP (0.261, p < 0.001). Levels of serum free and bioavailable 25OHD showed a negative correlation with ESR (-0.165, p = 0.031, -0.205, p < 0.001, respectively) and hsCRP (-0.164, p = 0.032, -0.208, p < 0.001 respectively), and a moderate negative correlation with fecal calprotectin (-0.377, p = 0.028, -0.409, p < 0.016, respectively). Serum total 25OHD concentration was the only vitamin D parameter found to have no specific correlation with any of the inflammatory markers. According to these results, the traditional parameter, total 25OHD, still appears to be the best marker of vitamin D status in patients with inflammatory bowel disease regardless of the presence of inflammation.

Divergent Evolution of Eukaryotic CC- and A-Adding Enzymes.

Synthesis of the CCA end of essential tRNAs is performed either by CCA-adding enzymes or as a collaboration between enzymes restricted to CC- and A-incorporation. While the occurrence of such tRNA nucleotidyltransferases with partial activities seemed to be restricted to Bacteria, the first example of such split CCA-adding activities was reported in Schizosaccharomyces pombe. Here, we demonstrate that the choanoflagellate Salpingoeca rosetta also carries CC- and A-adding enzymes. However, these enzymes have distinct evolutionary origins. Furthermore, the restricted activity of the eukaryotic CC-adding enzymes has evolved in a different way compared to their bacterial counterparts. Yet, the molecular basis is very similar, as highly conserved positions within a catalytically important flexible loop region are missing in the CC-adding enzymes. For both the CC-adding enzymes from S. rosetta as well as S. pombe, introduction of the loop elements from closely related enzymes with full activity was able to restore CCA-addition, corroborating the significance of this loop in the evolution of bacterial as well as eukaryotic tRNA nucleotidyltransferases. Our data demonstrate that partial CC- and A-adding activities in Bacteria and Eukaryotes are based on the same mechanistic principles but, surprisingly, originate from different evolutionary events.

Plasma Kynurenine Predicts Severity and Complications of Heart Failure and Associates with Established Biochemical and Clinical Markers of Disease.

BACKGROUND: Kynurenine, a metabolite of the L-tryptophan pathway, plays a pivotal role in neuro-inflammation, cancer immunology, and cardiovascular inflammation, and has been shown to predict cardiovascular events. OBJECTIVES: It was our objective to increase the body of data regarding the value of kynurenine as a biomarker in chronic heart failure (CHF). METHODS: We investigated the predictive value of plasma kynurenine in a CHF cohort (CHF, n = 114); in a second cohort of defibrillator carriers with CHF (AICD, n = 156), we determined clinical and biochemical determinants of the marker which was measured by enzyme immunoassay. RESULTS: In the CHF cohort, both kynurenine and NT-proBNP increased with NYHA class. Univariate binary logistic regression showed kynurenine to predict death within a 6-month follow-up (OR 1.43, 95% CI 1.03-2.00, p = 0.033) whereas NT-proBNP did not contribute significantly. Kynurenine, like NT-proBNP, was able to discriminate at a 30% threshold of left ventricular ejection fraction (LVEF; AUC-ROC, both 0.74). Kynurenine correlated inversely with LVEF (ϱ = -0.394), glomerular filtration fraction (GFR; ϱ = -0.615), and peak VO2 (ϱ = -0.626). Moreover, there was a strong correlation of kynurenine with NT-proBNP (ϱ = 0.615). In the AICD cohort, multiple linear regression analysis demonstrated highly significant associations of kynurenine with GFR, hsCRP, and tryptophan, as well as a significant impact of age. CONCLUSIONS: This work speaks in favor of kynurenine being a new and valuable biomarker of CHF, with particular attention placed on its ability to predict mortality and reflect exercise capacity.

Genotyping bacterial and fungal pathogens using sequence variation in the gene for the CCA-adding enzyme.

BACKGROUND: To allow an immediate treatment of an infection with suitable antibiotics and bactericides or fungicides, there is an urgent need for fast and precise identification of the causative human pathogens. Methods based on DNA sequence comparison like 16S rRNA analysis have become standard tools for pathogen verification. However, the distinction of closely related organisms remains a challenging task. To overcome such limitations, we identified a new genomic target sequence located in the single copy gene for tRNA nucleotidyltransferase fulfilling the requirements for a ubiquitous, yet highly specific DNA marker. In the present study, we demonstrate that this sequence marker has a higher discriminating potential than commonly used genotyping markers in pro- as well as eukaryotes, underscoring its applicability as an excellent diagnostic tool in infectology. RESULTS: Based on phylogenetic analyses, a region within the gene for tRNA nucleotidyltransferase (CCA-adding enzyme) was identified as highly heterogeneous. As prominent examples for pro- and eukaryotic pathogens, several Vibrio and Aspergillus species were used for genotyping and identification in a multiplex PCR approach followed by gel electrophoresis and fluorescence-based product detection. Compared to rRNA analysis, the selected gene region of the tRNA nucleotidyltransferase revealed a seven to 30-fold higher distinction potential between closely related Vibrio or Aspergillus species, respectively. The obtained data exhibit a superb genome specificity in the diagnostic analysis. Even in the presence of a 1,000-fold excess of human genomic DNA, no unspecific amplicons were produced. CONCLUSIONS: These results indicate that a relatively short segment of the coding region for tRNA nucleotidyltransferase has a higher discriminatory potential than most established diagnostic DNA markers. Besides identifying microbial pathogens in infections, further possible applications of this new marker are food hygiene controls or metagenome analyses.

Comparison of a Point-Of-Care Test for High-Sensitivity C-Reactive Protein with an Established Immuno-Nephelometric Method.

BACKGROUND: High-sensitivity C-reactive protein (hsCRP) is an established marker of cardiovascular risk particularly in primary prevention. For years, it was exclusively measured using automated methods in clinical laboratories, but point-of-care tests (POCT) are urgently needed to simplify and hasten the determination of hsCRP. METHODS: This study compared a novel hsCRP POCT with an established nephelometric method in 104 patients showing a broad spectrum of cardiovascular risk. RESULTS: The results indicated a moderate agreement of the POCT with the standard method, with a sensitivity of 87% and a specificity of 80% to detect elevated hsCRP (> 1 mg/L). CONCLUSIONS: The minimization of sample volume appears to be the most promising strategy for future test improvement.

Biochemical Identification of Vitamin B12 Deficiency in a Medical Office.

BACKGROUND: A vitamin B12 deficiency can be an underlying cause or a deteriorating factor in several diseases. Nevertheless, early identification of such a deficiency remains a problem. Holotranscobalamin (HTC) is presently considered to be the gold standard. We tested the predictive power of other B12 parameters by comparing them with HTC. METHODS: The blood of 77 patients from a medical office was tested for HTC, total B12 (CLIA [chemiluminescent immunoassay] and MTP [microbiological test with microtitre plates]), MMA (methylmalonic acid), HCY (homocysteine), and MCV (mean cell volume). The parameters were correlated and sensitivity, specificity, PPV (positive predictive value), NPV (negative predictive value), LR+ (positive likelihood ratio), and LR- (negative likelihood ratio) in comparison to HTC were determined. A ROC analysis was also performed. RESULTS: At a cutoff value of 35 pmol/L for HTC, the total B12 CLIA (cutoff 211 ng/L) qualified 53% of individuals as having a B12 deficiency. The total B12 MTP (cutoff 288 ng/L) classified 71% as having a B12 deficiency. Specificity was similar in both cases (CLIA, 93%; MTP, 95%). With a cutoff value of 10 µmol/L for homocysteine, the best negative prediction was achieved. MVA has a low sensitivity (41%) and a high specificity (90%). Based on the ROC analysis, which indicated superiority of the B12-MTP, the reference levels of B12-CLIA and B12-MTP were raised to 304 and 368 ng/L, respectively. Thus, a probable B12 deficiency was identified in 94% of cases with either method and with a comparable specificity. CONCLUSIONS: If total B12 is applied to identify B12 deficiency, the cutoff values should be elevated to 304 (B12-CLIA) and 368 ng/L (B12-MTP) to improve the predictive power. The negative-predictive power of HCY can be useful in daily routine. HTC has a broad grey area of uncertainty and MMA should only be applied as a confirmatory test.

Relaxin-2 does not ameliorate nephropathy in an experimental model of type-1 diabetes.

BACKGROUND/AIMS: In diabetic nephropathy (DN), the current angiotensin-II-blocking pharmacotherapy is frequently failing. For diabetic cardiomyopathy (DC), there is no specific remedy available. Relaxin-2 (Rlx) - an anti-fibrotic, anti-inflammatory, and vasoprotecting peptide ­ is a candidate drug for both. METHODS: Low-dose (32 µg/kg/day) and high-dose (320 µg/kg/day) Rlx were tested against vehicle (n = 20 each) and non-diabetic controls (n = 14) for 12 weeks in a model of type-1 diabetes induced in endothelial nitric oxide synthase knock-out (eNOS-KO) mice by intraperitoneal injection of streptozotocin. RESULTS: Diabetic animals showed normal plasma creatinine, markedly increased albuminuria and urinary malonyldialdehyde, elevated relative kidney weight, glomerulosclerosis, and increased glomerular size, but no relevant interstitial fibrosis. Neither dose of Rlx affected these changes although the drug was active and targeted plasma levels were achieved. Of note, we found no activation of the renal TGF-ß pathway in this model. In the hearts of diabetic animals, no fibrotic alterations indicative of DC could be determined which precluded testing of the initial hypothesis. CONCLUSIONS: We investigated a model showing early DN without overt tubulointerstitial fibrosis and activation of the TGF-ß-Smad-2/3 pathway. In this model, Rlx proved ineffective; however, the same may not apply to other models and types of diabetes.