Background/Objectives: Pneumocystis jirovecii pneumonia (PJP) is the most frequently diagnosed AIDS-defining illness in Europe, with especially high mortality in HIV-negative patients caused by delayed diagnosis and low awareness. This study aims to evaluate cytokine release assays (CRA) to facilitate a less invasive and resource-efficient PJP specific diagnostic test. We focus on the P. jirovecii antigens Kexin 1 (KEX1), MSG1, and MSG2, which were identified in prior studies as immunologically relevant. Methods: Whole blood samples from 50 participants—22 healthy individuals and 28 immunocompromised individuals, including 8 with proven PJP—were stimulated in vitro with full-length and partial KEX1, MSG1, MSG2, and a combination of all three antigens (PJ-MIX). Following 24 h incubation at 37 °C, cytokine levels of IL-2, IFN-γ, IL-17A, and IL-17F were measured. Results: Stimulation with full-length KEX1, MSG1, MSG2, and PJ-MIX antigens induced higher IL-2 concentrations in the healthy control group compared to the groups IL-2 baseline levels and to the group of proven PJP cases. Similarly, stimulation with full-length KEX1, MSG1, and PJ-MIX elevated IFN-γ levels in the healthy control group compared to baseline IFN-γ levels. Conclusions: Our findings highlight the potential of IL-2 and IFN-γ release following stimulation with PJ antigens, with PJ-MIX eliciting the strongest and most significant responses, suggesting a cumulative antigen effect. This pilot study establishes a foundation for a PJP-specific CRA, deepening our knowledge of T-cell immunity against PJP. Clinically, such a test could, among other applications, evaluate at-risk patients who should receive prophylaxis and may consequently reduce PJP-related morbidity and mortality.

Despite powerful DNA repair systems, oxidative damage/modification to DNA is an inevitable side effect of metabolism, ionizing radiation, lifestyle habits, inflammatory pathologies such as type-2 diabetes or metabolic syndrome, cancer and natural aging.One of the most common oxidative DNA modifications is 8-OHdG (8-hydroxy-2′-deoxyguanosine), which is the most widely used marker in research and clinical diagnostics. 8-OHdG is easily and specifically detectable in various samples such as urine, plasma, cells and tissues via a large variety of methods like ELISA, HPLC, chromatographic methods, and immunochemistry.Formed by oxidation of guanine and being representative for the degree of DNA damage, 8-OHdG can be also used as biomarker for risk assessment of various cancers as well as degenerative diseases.Here, we present a highly specific, self-developed 8-OHdG antibody in successful comparison to a commercially one, tested in cells (FF95, HCT116, and HT22) and intestinal tissue, focusing on automatized evaluation via fluorescence/confocal microscopy.

Background: Improving precision medicine in chemotherapy requires highly sensitive and easily applicable diagnostic tools. In addition, non-invasive molecular real-time monitoring of cytotoxic response is highly desirable. Here, we employed the kinetics of DNA double-strand breaks (DSB) and cell-free DNA (cfDNA) in a cell model of topoisomerase II-inhibitors in T cell leukemia (Jurkat cells) compared to normal cells (peripheral blood mononuclear cells, PBMCs). Methods: We applied automated microscopy to quantify immuno-stained phosphorylated H2AX (γH2AX) as a marker for either DNA damage response (DDR) or cell death and quantitative PCR-based analysis of nuclear and mitochondrial cfDNA concentrations. Results: Jurkat cells displayed a DDR to cytotoxic drug treatment significantly earlier than PBMCs, and etoposide (ETP) induced DSB formation faster than doxorubicin (DOX) in both Jurkat and PBMCs. Jurkat cells exhibited an earlier cytotoxic response compared to PBMC, with a significantly increased mitochondrial cfDNA formation after 2 h of DOX application. In PBMCs, increased cell death was detected after 4 h of incubation with ETP, whereas DOX treatment was less effective. Conclusions: Both automated microscopy and mitochondrial cfDNA quantification analysis indicate that (malignant) Jurkat cells are more sensitive to DOX than (healthy) PBMC. Our real-time approach can improve DDR inducing drug selection and adaptation in cancer therapy and aids in decisions for optimal patient biosampling.

OBJECTIVES:Due to disease- or therapy-associated immunosuppression, oncological patients suffer from significantly higher morbidity and mortality due to infections transmitted by respiratory pathogens such as Streptococcus pneumoniae and influenza virus. Although the German Standing Committee on Vaccination (STIKO) provides specific recommendations for vaccination against these pathogens, there is no data on vaccination rates in this high-risk population. METHODS:Data from the interventional EVO study were analyzed to provide information on vaccination rates against Streptococcus pneumoniae and influenza virus in oncological patients. Numbers presented in this publication summarize baseline and follow-up data of the control group; thus, data were not influenced by the intervention. RESULTS:Data of 370 patients were analyzed; 20.5% of patients were treated for hematological malignancies and 79.5% for solid cancer. 28.1% of patients had received vaccination against influenza and 32.2% against Streptococcus pneumoniae; for the latter only 7.3% according recommendations. While vaccination rates where even lower for patients with thoracic carcinoma (influenza 26.7% and Streptococcus pneumoniae 6.0% according to STIKO recommendations), rates in patients with multiple myeloma were remarkably higher (39.0% and 14.6%). CONCLUSIONS:Despite strong recommendations to vaccinate and the clear clinical need to prevent infections in the vulnerable group of oncological patients, only the minority was vaccinated against Streptococcus pneumoniae or influenza, underlining the urgent need for better vaccination strategies in this high-risk population.

Background: To unravel how the integrity of nuclear and mitochondrial circulating cell-free DNA (cfDNA) contributes to its plasma quantity in colorectal cancer (CRC) patients. Methods: CfDNA from plasma samples of 80 CRC patients stratified by tumour stage and 50 healthy individuals were extracted. Total cfDNA concentration was determined and equal template concentrations (ETC) were analyzed by quantitative real-time PCR (qPCR) resulting in small and long fragments of KRAS, Alu and MTCO3. The obtained data was also examined relative to the total cfDNA concentration (NTC) and diagnostic accuracy was estimated using receiver operating characteristics. Results: Total cfDNA levels were significantly higher in CRC group compared to healthy control and increased with tumour stage. Long nuclear fragment levels were significantly lower in CRC patients in ETC but not NTC condition. The integrity indices of nuclear cfDNA decreased from controls to patients with highly malignant tumor. Mitochondrial cfDNA fragment quantities were strongly reduced in early and late stages of tumor patients and prognostic value was higher in ETC. Predictive models based on either ETC or NTC predictor set showed comparable classification performance. Conclusion : Increased blood cfDNA concentration in late UICC stages inversely correlate with nuclear cfDNA integrity index and suggest that necrotic degradation is not a major cause for higher total cfDNA quantity. The diagnostic and prognostic value of MTCO3 is highly significant in early stages of CRC and can be evaluated more comprehensively, using ETC for qPCR analysis. Trial Registration: The study was registered retrospectively on DRKS, the german register for clinical trials (DRKS00030257, 29/09/2022).

Whether a patient receives general or specialized outpatient palliative cancer care rarely follows clear criteria, leading to undertreatment or overtreatment. Detailed scores exist to predict prognosis, but not treatment requirements, leaving caregivers to follow their intuition. As a phenomenological indicator incorporating possibly important subjective information, intuition may in fact be a helpful tool. In this prospective observational study, a score to estimate three global dimensions of patients' resources was applied: Medical prognosis, feeling of strength and feeling of support. The score results were correlated with the actual amount and effort of care required during the subsequent palliative care time. This phenomenological score correlated well with the performance index and the Hospice and Palliative care Evaluation score. Whilst various individual items correlated significantly with the score or its constituent parameters, there was no uniform coherent pattern, reflecting the complexity of palliative care and the potential value of this predictive tool.

Standard operating procedures for autologous stem cell transplantation (SCT) aim to guarantee best possible engraftment. Three procedures are routinely used for transplant infusion: regular bag infusion (Procedure 1), injection via syringe (Procedure 2), and combination of regular bag infusion and syringe (Procedure 3). We conducted a retrospective analysis of all autologous stem cell transplants done in the hematology department of the Vivantes Clinic Neukoelln in Berlin, Germany, between January 1, 2016 and March 4, 2017. Of the total of 69 patients, 17 underwent Procedure 1, 32 Procedure 2, and 20 Procedure 3. Although speed of transplant reinfusion differed significantly between procedure types, these differences had no effect on duration of leukopenia. However, duration of leukopenia did correlate with need for blood transfusion and use of antibiotics. Our findings contradict the general perception that very rapid reinfusion is necessary. Nevertheless, considering the limitations of this study (retrospective, single center, small sample size) and that longer duration of aplasia is associated with greater need for intervention, efficient transplant reinfusion is advisable. More research is needed regarding timeliness and type of procedure used.

BACKGROUND:DNA double-strand breaks can be counted as discrete foci by imaging techniques. In personalized medicine and pharmacology, the analysis of counting data is relevant for numerous applications, e.g., for cancer and aging research and the evaluation of drug efficacy. By default, it is assumed to follow the Poisson distribution. This assumption, however, may lead to biased results and faulty conclusions in datasets with excess zero values (zero-inflation), a variance larger than the mean (overdispersion), or both. In such cases, the assumption of a Poisson distribution would skew the estimation of mean and variance, and other models like the negative binomial (NB), zero-inflated Poisson or zero-inflated NB distributions should be employed. The model chosen has an influence on the parameter estimation (mean value and confidence interval). Yet the choice of the suitable distribution model is not trivial.METHODS:To support, simplify and objectify this process, we have developed the countfitteR software as an R package. We used a Bayesian approach for distribution model selection and the shiny web application framework for interactive data analysis.RESULTS:We show the application of our software based on examples of DNA double-strand break count data from phenotypic imaging by multiplex fluorescence microscopy. In analyzing numerous datasets of molecular pharmacological markers (phosphorylated histone H2AX and p53 binding protein), countfitteR demonstrated an equal or superior statistical performance compared to the usually employed two-step procedure, with an overall power of up to 98%. In addition, it still gave information in cases with no result at all from the two-step procedure. In our data sample we found that the NB distribution was the most frequent, with the Poisson distribution taking second place.CONCLUSIONS:countfitteR can perform an automated distribution model selection and thus support the data analysis and lead to objective statistically verifiable estimated values. Originally designed for the analysis of foci in biomedical image data, countfitteR can be used in a variety of areas where non-Poisson distributed counting data is prevalent.

(1) Background: Healthcare workers (HCWs) are prone to intensified exposure to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in the ongoing pandemic. We prospectively analyzed the prevalence of antibodies against SARS-CoV-2 in HCWs at baseline and follow up with regard to clinical signs and symptoms in two university hospitals in Brandenburg, Germany. (2) Methods: Screening for anti-SARS-CoV-2 IgA and IgG antibodies was offered to HCWs at baseline and follow up two months thereafter in two hospitals of Brandenburg Medical School during the first wave of the COVID-19 pandemic in Germany in an ongoing observational cohort study. Medical history and signs and symptoms were recorded by questionnaires and analyzed. (3) Results: Baseline seroprevalence of anti-SARS-CoV-2 IgA was 11.7% and increased to 15% at follow up, whereas IgG seropositivity was 2.1% at baseline and 2.2% at follow up. The rate of asymptomatic seropositive cases was 39.5%. Symptoms were not associated with general seropositivity for anti-SARS-CoV-2; however, class switch from IgA to IgG was associated with increased symptom burden. (4) Conclusions: The seroprevalence of antibodies against SARS-CoV-2 was low in HCWs but higher compared to population data and increased over time. Screening for antibodies detected a significant proportion of seropositive participants cases without symptoms.

Seit in Deutschland der Anspruch auf spezialisierte ambulante Palliativversorgung (SAPV) eingefuhrt wurde, mussen Arzte und Arztinnen entscheiden, wer diese Versorgungsform benotigt. Offenkundig treten auch hier Unter- wie uberversorgung auf. Die Autoren untersuchen deshalb, welche Erwartungen an die Versorgung am Lebensende gestellt werden, wie gut die verschiedenen Versorgungsformen dies leisten konnen und welches Potenzial sich daraus ergibt.

Seit in Deutschland der Anspruch auf spezialisierte ambulante Palliativversorgung (SAPV) eingeführt wurde, müssen Ärzte und Ärztinnen entscheiden, wer diese Versorgungsform benötigt. Offenkundig treten auch hier Unter- wie Überversorgung auf. Die Autoren untersuchen deshalb, welche Erwartungen an die Versorgung am Lebensende gestellt werden, wie gut die verschiedenen Versorgungsformen dies leisten können und welches Potenzial sich daraus ergibt.

Background In eukaryotic cells treated with chemotherapeutic drugs DNA double strand breaks (DSB) are the most severe form of DNA damage which usually leads to apoptotic cell death. Cytotoxic chemotherapy is still a mainstay of cancer therapy, but there are few established methods to individually predict efficacy and tolerability. In most regimens dosage is based on the body surface area, a dimension of little biological precision, and individual dose adaptions are usually performed by clinical guessing or in response to adverse events. This project sets out to examine biological parameters of cell damage. Methods In particular, we measure DNA double strand breaks by an automated yH2AX assay applying the Aklides NUK® system. In preliminary work, PBMCs were stimulated with bendamustine or with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone, the components of the R-CHOP protocol, to induce DSB. Recruitment of lymphoma patients under R-CHOP therapy and its cytotoxic effects on DSB is currently ongoing. Results There was no inter-individual correlation between drug dose and number of foci, whereas does titrations on the cells of each individual donor demonstrated a clear correlation between dose and yH2AX assay result. Measurements of DSB in vivo using PBMCs of lymphoma patients is currently ongoing. First results show a rising number of DSBs in leucocytes under chemotherapy. Conclusions The lack of correlation across individuals between yH2AX foci and drug dosage based on standard calculation by body surface area suggests that standardized dosing of chemotherapeutic drugs based on gross physical determinants such as weight or body surface area does not correspond to the individual’s biological response and clinical effect. To confirm our hypothesis, different biomarkers should also to be involved in this project, like microRNAs and telomere length. The future plan of this project is the replacement of standard dosage regimens by biologically based, real-time-adapted individual dosage in lymphoma to reduce toxicity while increasing therapeutic efficacy. Legal entity responsible for the study The authors. Funding Brandenburgian Ministry of Science, Research and Culture (MWFK) Federal Ministry of Education and Research. Disclosure All authors have declared no conflicts of interest.

Chemotherapy and radiation therapy are used in malignant oncological diseases to increase the level of DNA double strand breaks (DSBs) in tumor cells. Unrepaired DSBs may either kill a cell or induce terminal arrest. Those DNA damages can be detected by fluorescence imaging of phosphorylated histon e protein H2AX (γH2AX). Peripheral blood mononuclear cells (PBMCs) are easily available human material, which are usually stored by cryopreservation for repeated experiments or postponed analysis. As DNA DSBs can be introduced through several different stressors, it was of interest to investigate the potential impact of cryopreservation, on the formation of DSBs in human PBMCs. The PBMCs were cryopreserved in four different media, containing different proportions of Dulbecco’s Modified Eagle Medium (DMEM), fetal calf serum (FCS) and dimethyl sulfoxide (DMSO) as taken from the literature. Immunofluorescence staining of γH2AX was performed on freshly isolated and cryopreserved PBMCs. A significant reduction on relative γH2AX level, after treatment with 100 μM etoposide (ETP), was observed for cryopreserved cells in medium containing DMEM, which seemed to be the limiting factor on DNA DSB formation. No change was observed for untreated PBMCs. Therefore, DSBs of cryopreserved human PBMCs could be used to investigate the influence of chemotherapeutic drugs or radiation therapy, assuming that the cryopreservation follows a standardized protocol with a tight control of potential DSB inducing stressors.

Diffuse large B-cell lymphoma (DLBCL) is a highly aggressive form of non-Hodgkin's lymphoma with a long-term survival rate of around 60% upon standard treatment with rituximab and chemotherapy. While this marks a great improvement over the pre-rituximab/pre-biologicals era, DLBCLs remain a heterogeneous disease with marked variability in clinical outcome. Efforts have been undertaken to predict its course based on characteristics such as the activated B-cell (ABC) versus germinal centre B-cell (GCB) types, but so far none of these has had much impact on treatment and outcome, and the international prognostic index, based on clinical and basic laboratory features, remains the best risk predictor. This review focuses on 13 biomarkers that have been described in the context of diagnostic tests, clinical studies or basic research related to solid and haematological malignancies in general or DLBCL in particular. These include biomarkers of the DNA repair response such as the phosphorylated histone variant 2AX (γH2AX), p53 binding protein (53BP1), Ataxia telangiectasia mutated protein (ATM), Ataxia telangiectasia and Rad3-related protein (ATR), the DNA-dependent protein kinase (DNA-PK) and Rad51 as well as proto-oncogene products like Myc, B-cell lymphoma 2 (Bcl-2), breast cancer 1 (BRCA1) and signal transducer and activator of transcription 3 (STAT3). We have also included 8-Hydroxy-2'-desoxyguanosine (8-OHdG), a marker for oxidative stress, and discuss a link between two proteins involved in apoptosis, the proteasome activator 28γ (PA28γ) and tumour suppressor p53. Our investigation revealed a connection between almost all biomarkers, which are based on methods such as indirect immunofluorescence, fluorescence in-situ hybridization or immunohistochemistry.