Reanalysis of unclear CMV status results from buccal swab samples of potential stem cell donors is an efficient donor registry strategy.

Many stem cell donor registries determine the cytomegalovirus (CMV) IgG serostatus at donor recruitment as it is an important marker for donor selection in the context of hematopoietic stem cell transplantation. To make sample collection less uncomfortable for the donor, we have developed a method that allows CMV status determination from buccal swab samples, thus avoiding blood drawing. However, the determination fails in some cases which leads to new donors being listed for donor search without CMV status, thus hindering donor searches. In this work, we evaluated the success rate of repeating CMV status analysis from a new swab. Our results show that about 90% of the samples could be successfully determined. Due to the great importance of the CMV status in donor search, we consider the retesting approach to be highly recommended for stem cell donor registries.

A vaccine targeting mutant IDH1 in newly diagnosed glioma.

Mutated isocitrate dehydrogenase 1 (IDH1) defines a molecularly distinct subtype of diffuse glioma1-3. The most common IDH1 mutation in gliomas affects codon 132 and encodes IDH1(R132H), which harbours a shared clonal neoepitope that is presented on major histocompatibility complex (MHC) class II4,5. An IDH1(R132H)-specific peptide vaccine (IDH1-vac) induces specific therapeutic T helper cell responses that are effective against IDH1(R132H)+ tumours in syngeneic MHC-humanized mice4,6-8. Here we describe a multicentre, single-arm, open-label, first-in-humans phase I trial that we carried out in 33 patients with newly diagnosed World Health Organization grade 3 and 4 IDH1(R132H)+ astrocytomas (Neurooncology Working Group of the German Cancer Society trial 16 (NOA16), ClinicalTrials.gov identifier NCT02454634). The trial met its primary safety endpoint, with vaccine-related adverse events restricted to grade 1. Vaccine-induced immune responses were observed in 93.3% of patients across multiple MHC alleles. Three-year progression-free and death-free rates were 0.63 and 0.84, respectively. Patients with immune responses showed a two-year progression-free rate of 0.82. Two patients without an immune response showed tumour progression within two years of first diagnosis. A mutation-specificity score that incorporates the duration and level of vaccine-induced IDH1(R132H)-specific T cell responses was associated with intratumoral presentation of the IDH1(R132H) neoantigen in pre-treatment tumour tissue. There was a high frequency of pseudoprogression, which indicates intratumoral inflammatory reactions. Pseudoprogression was associated with increased vaccine-induced peripheral T cell responses. Combined single-cell RNA and T cell receptor sequencing showed that tumour-infiltrating CD40LG+ and CXCL13+ T helper cell clusters in a patient with pseudoprogression were dominated by a single IDH1(R132H)-reactive T cell receptor.

Noninvasive Determination of CMV Serostatus From Dried Buccal Swab Samples: Assay Development, Validation, and Application to 1.2 Million Samples.

BACKGROUND: Buccal swab sampling constitutes an attractive noninvasive alternative to blood drawings for antibody serostatus assays. Here we describe a method to determine the cytomegalovirus immunoglobulin G (CMV IgG) serostatus from dried buccal swab samples. METHODS: Upon solubilization, CMV IgG is determined by an ELISA assay specifically adapted to cope with low IgG concentrations. The derived CMV titer is normalized against the total protein concentration to adjust for incorrectly or less efficiently sampled buccal swabs. Assay parameters were optimized on a set of 713 samples. RESULTS: Validation with 1784 samples revealed distinct results for > 80% of samples with 98.6% specificity and 99.1% sensitivity. Based on the analysis of 1.2 million samples we derived age- and sex-stratified CMV prevalence statistics for Germany, Poland, United Kingdom, and Chile. To confirm accuracy of the assay in routine operation, the CMV status of 6518 donors was reassessed by independent laboratories based on conventional blood samples revealing 96.9% specificity and 97.4% sensitivity. CONCLUSIONS: The assay accurately delivers the CMV IgG serostatus from dried buccal swab samples for > 80% of the participants. Thereby it provides a noninvasive alternative to plasma-based CMV monitoring for nondiagnostic purposes such as hematopoietic stem cell transplantation donor screening or population studies.

Etablierung der PCR-basierten SARS-CoV-2-Testung im Hochdurchsatz.

Timely identification and isolation of affected individuals is one of the most important factors to control spreading of the newly emerged SARS-CoV-2. Consequently, it is crucial to maintain sufficient sample capacities in diagnostic laboratories. Here, we present a high-through-put approach for PCR-based SARS-CoV-2 testing. The implementation of sample pooling reduces costs and workload, especially in times with low population prevalence.

6-Locus HLA allele and haplotype frequencies in a population of 1075 Russians from Karelia.

A total of 1075 Russians from the Russian part of Karelia were genotyped at high-resolution for the human leukocyte antigen loci HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 using next generation sequencing methods. The haplotypic and allelic profiles as well as Hardy-Weinberg proportions of this population sample were evaluated. As the most frequent 6-locus haplotype, A*03:01 g ∼ B*07:02 g ∼ C*07:02 g ∼ DRB1*15:01 g ∼ DQB1*06:02 g ∼ DPB1*04:01 g was identified with an estimated frequency of 3.5%. No deviation from Hardy-Weinberg Equilibrium was detected at any of the loci studied. The HLA genotypic data of the population sample reported here are available publicly in the Allele Frequencies Net Database under the population name "Russia Karelia" and the identifier AFN3430.

Pseudomonas putida esterase contains a GGG(A)X-motif confering activity for the kinetic resolution of tertiary alcohols.

An esterase from Pseudomonas putida JD1 (PPE) was successfully cloned, actively expressed in Escherichia coli, and characterized. It was discovered that PPE is more active towards short-chain esters, hydrolyzed δ-valerolactone, and ε-caprolactone and was most active at 37°C and pH 8. After purification to homogeneity by Ni-NTA-assisted affinity chromatography, the kinetic parameters K(M) and k(cat) were determined for p-nitrophenyl acetate and butyrate, respectively, showing better catalytic efficiency for hydrolysis of the acetate residue. Investigation of the protein sequence revealed not only the classical catalytic triad for carboxylesterases, additionally the interesting GGG(A)X-motif, which is associated to activity towards tertiary alcohols, was found. Indeed, enzymatic activity was shown for a set of different tertiary alcohols with enantioselectivities up to E = 20, suggesting PPE to be a promising biocatalyst. In addition, PPE also hydrolyzed 4-hydroxyphenyl acetate, the product of a Baeyer-Villiger monooxygenase-catalyzed oxidation of 4-hydroxyacetophenone with a specific activity of 34.36 U/mg suggesting a physiological role in P. putida JD1.