Cooling of Cells and Organs Confers Extensive DNA Strand Breaks Through Oxidative Stress and ATP Depletion

Cooling at 4 degrees C is routinely used to lower metabolism and preserve cell and tissue integrity in laboratory and clinical settings, including organ transplantation. However, cooling and rewarming produce cell damage, attributed primarily to a burst of reactive oxygen species (ROS) upon rewarming. While DNA represents a highly vulnerable target of ROS, it is unknown whether cooling and/or rewarming produces DNA damage. Here, we show that cooling alone suffices to produce extensive DNA damage in cultured primary cells and cell lines, including double-strand breaks (DSBs), as shown by comet assay and pulsed-field gel electrophoresis. Cooling-induced DSB formation is time- and temperature-dependent and coincides with an excess production of ROS, rather than a decrease in ATP levels. Immunohistochemistry confirmed that DNA damage activates the DNA damage response marked by the formation of nuclear foci of proteins involved in DSB repair, gamma-H2Ax, and 53BP1. Subsequent rewarming for 24 h fails to recover ATP levels and only marginally lowers DSB amounts and nuclear foci. Precluding ROS formation by dopamine and the hydroxychromanol, Sul-121, dose-dependently reduces DSBs. Finally, a standard clinical kidney transplant procedure, using cold static storage in UW preservation solution up to 24 h in porcine kidney, lowered ATP, increased ROS, and produced increasing amounts of DSBs with recruitment of 53BP1. Given that DNA repair is erroneous by nature, cooling-inflicted DNA damage may affect cell survival, proliferation, and genomic stability, significantly impacting cellular and organ function, with relevance in stem cell and transplantation procedures

Evidence for local dendritic cell activation in pulmonary sarcoidosis

<p>Abstract</p> <p>Background</p> <p>Sarcoidosis is a granulomatous disease characterized by a seemingly exaggerated immune response against a difficult to discern antigen. Dendritic cells (DCs) are pivotal antigen presenting cells thought to play an important role in the pathogenesis. Paradoxically, decreased DC immune reactivity was reported in blood samples from pulmonary sarcoidosis patients. However, functional data on lung DCs in sarcoidosis are lacking. We hypothesized that at the site of disease DCs are mature, immunocompetent and involved in granuloma formation.</p> <p>Methods</p> <p>We analyzed myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in broncho-alveolar lavage (BAL) and blood from newly diagnosed, untreated pulmonary sarcoidosis patients and healthy controls using 9-color flowcytometry. DCs, isolated from BAL using flowcytometric sorting (mDCs) or cultured from monocytes (mo-DCs), were functionally assessed in a mixed leukocyte reaction with naïve allogeneic CD4+ T cells. Using Immunohistochemistry, location and activation status of CD11c⁺DCs was assessed in mucosal airway biopsies.</p> <p>Results</p> <p>mDCs in BAL, but not in blood, from sarcoidosis patients were increased in number when compared with mDCs from healthy controls. mDCs purified from BAL of sarcoidosis patients induced T cell proliferation and differentiation and did not show diminished immune reactivity. Mo-DCs from patients induced increased TNFα release in co-cultures with naïve allogeneic CD4⁺T cells. Finally, immunohistochemical analyses revealed increased numbers of mature CD86⁺DCs in granuloma-containing airway mucosal biopsies from sarcoidosis patients.</p> <p>Conclusion</p> <p>Taken together, these finding implicate increased local DC activation in granuloma formation or maintenance in pulmonary sarcoidosis.</p

Hypomagnesaemia and its determinants in a contemporary primary care cohort of persons with type 2 diabetes

AIMS: Among persons with type 2 diabetes mellitus (T2DM) hypomagnesaemia has been reported in 14-48% of patients. This may be of significance given the emerging associations of hypomagnesaemia with glucometabolic disturbances and possibly even complications. We assessed the prevalence of hypomagnesaemia and its determinants, in a well-defined cohort of persons with T2DM treated in primary care. METHODS: Observational cohort study among persons with T2DM treated in primary care in the Northeast of the Netherlands. Magnesium was measured using a colorimetric endpoint assay (Roche). Hypomagnesaemia was defined as a serum magnesium level <0.70 mmol/L. Pearson correlations were performed to correlate variables with serum magnesium. Next, a stepwise backward regression model was made. RESULTS: Data of 929 persons (55% male) with a mean age of 65 (± 10) years, diabetes duration 6.5 [3.0-10.1] years, and HbA1c concentration 6.7 (± 0.7)% (50 (± 9) mmol/mol) were analysed. Serum magnesium was 0.79 (± 0.08) mmol/L. The percentage of persons with magnesium deficiency was 9.6%. Age, diabetes duration, BMI, HbA1c, use of metformin, sulfonylurea derivatives, and DPP4 inhibitors were negatively associated with magnesium concentrations. In contrast, LDL cholesterol and serum creatinine were positively associated serum magnesium. CONCLUSIONS: Hypomagnesaemia was present in 9.6% of T2DM patients treated in primary care. This percentage is remarkably lower than reported previously, possibly due to the unselected nature of our population. Concerning T2DM-related factors, only BMI, HbA1c and the use of metformin, sulfonylurea derivatives and DPP4 inhibitors correlated negatively with magnesium concentrations

Inhibition of Ferroptosis Enables Safe Rewarming of HEK293 Cells following Cooling in University of Wisconsin Cold Storage Solution

The prolonged cooling of cells results in cell death, in which both apoptosis and ferroptosis have been implicated. Preservation solutions such as the University of Wisconsin Cold Storage Solution (UW) encompass approaches addressing both. The use of UW improves survival and thus extends preservation limits, yet it remains unclear how exactly organ preservation solutions exert their cold protection. Thus, we explored cooling effects on lipid peroxidation and adenosine triphosphate (ATP) levels and the actions of blockers of apoptosis and ferroptosis, and of compounds enhancing mitochondrial function. Cooling and rewarming experiments were performed in a cellular transplantation model using Human Embryonic Kidney (HEK) 293 cells. Cell viability was assessed by neutral red assay. Lipid peroxidation levels were measured by Western blot against 4-Hydroxy-Nonenal (4HNE) and the determination of Malondialdehyde (MDA). ATP was measured by luciferase assay. Cooling beyond 5 h in Dulbecco's Modified Eagle Medium (DMEM) induced complete cell death in HEK293, whereas cooling in UW preserved ~60% of the cells, with a gradual decline afterwards. Cooling-induced cell death was not precluded by inhibiting apoptosis. In contrast, the blocking of ferroptosis by Ferrostatin-1 or maintaining of mitochondrial function by the 6-chromanol SUL150 completely inhibited cell death both in DMEM- and UW-cooled cells. Cooling for 24 h in UW followed by rewarming for 15 min induced a ~50% increase in MDA, while concomitantly lowering ATP by &gt;90%. Treatment with SUL150 of cooled and rewarmed HEK293 effectively precluded the increase in MDA and preserved normal ATP in both DMEM- and UW-cooled cells. Likewise, treatment with Ferrostatin-1 blocked the MDA increase and preserved the ATP of rewarmed UW HEK293 cells. Cooling-induced HEK293 cell death from hypothermia and/or rewarming was caused by ferroptosis rather than apoptosis. UW slowed down ferroptosis during hypothermia, but lipid peroxidation and ATP depletion rapidly ensued upon rewarming, ultimately resulting in complete cell death. Treatment throughout UW cooling with small-molecule Ferrostatin-1 or the 6-chromanol SUL150 effectively prevented ferroptosis, maintained ATP, and limited lipid peroxidation in UW-cooled cells. Counteracting ferroptosis during cooling in UW-based preservation solutions may provide a simple method to improve graft survival following cold static cooling.</p

Bispecific antibody CD73xEpCAM selectively inhibits the adenosine-mediated immunosuppressive activity of carcinoma-derived extracellular vesicles

Tumor-derived extracellular vesicles (EVs) carry potent immunosuppressive factors that affect the antitumor activities of immune cells. A significant part of the immunoinhibitory activity of EVs is attributable to CD73, a GPI-anchored ecto-5'-nucleotidase involved in the conversion of tumor-derived proinflammatory extracellular ATP (eATP) to immunosuppressive adenosine (ADO). The CD73-antagonist antibody oleclumab inhibits cell surface-exposed CD73 and is currently undergoing clinical testing for cancer immunotherapy. However, a strategy to selectively inhibit CD73 exposed on EVs is not available. Here, we present a novel bispecific antibody (bsAb) CD73xEpCAM designed to bind with high affinity the common EV surface marker EpCAM and concurrently inhibit CD73. Unlike oleclumab, bsAb CD73xEpCAM potently inhibited the immunosuppressive activity of EVs from CD73pos/EpCAMpos carcinoma cell lines and patient-derived colorectal cancer cells. Taken together, selective blockade of EV-exposed CD73 by bsAb CD73xEpCAM may be useful as an alternate or complementary targeted approach in cancer immunotherapy

Bispecific antibody CD73xEpCAM selectively inhibits the adenosine-mediated immunosuppressive activity of carcinoma-derived extracellular vesicles

Tumor-derived extracellular vesicles (EVs) carry potent immunosuppressive factors that affect the antitumor activities of immune cells. A significant part of the immunoinhibitory activity of EVs is attributable to CD73, a GPI-anchored ecto-5'-nucleotidase involved in the conversion of tumor-derived proinflammatory extracellular ATP (eATP) to immunosuppressive adenosine (ADO). The CD73-antagonist antibody oleclumab inhibits cell surface-exposed CD73 and is currently undergoing clinical testing for cancer immunotherapy. However, a strategy to selectively inhibit CD73 exposed on EVs is not available. Here, we present a novel bispecific antibody (bsAb) CD73xEpCAM designed to bind with high affinity the common EV surface marker EpCAM and concurrently inhibit CD73. Unlike oleclumab, bsAb CD73xEpCAM potently inhibited the immunosuppressive activity of EVs from CD73pos/EpCAMpos carcinoma cell lines and patient-derived colorectal cancer cells. Taken together, selective blockade of EV-exposed CD73 by bsAb CD73xEpCAM may be useful as an alternate or complementary targeted approach in cancer immunotherapy.</p

Serum free thiols in type 2 diabetes mellitus:A prospective study

Aims: Oxidative stress is a driver in the development of type 2 diabetes (T2DM) complications. As thiols (R-SH) are oxidized by reactive oxygen and sulfur species, circulating concentrations may directly reflect systemic redox status. We hypothesized that high serum R-SH concentrations are a reflection of a favourable redox status and may therefore positively associate with disease status.Methods: R-SH were measured in serum of 943 T2DM outpatients (55% males, 65 years and HbA1c of 6.7% (50 mmol/mol)) with a follow-up period of 1.2 years.Results: In the highest R-SH tertile patients were younger, more often men, had less microvascular complications, lower HbA1c and were more often treated nutritionally or with oral glucose-lowering drugs. Age-and sex adjusted hazard ratios for developing micro-, macro- or any complication plus death were 0.994, 0.992 and 0.993: even after adjustment for potential confounders. The Harrell's C statistic to predict microvascular complications or any complication plus death was higher in the models with R-SH than in those without R-SH.Conclusions: Although R-SH concentrations were associated with a favourable disease status, it did not add to the predictive capacity for long-term complications. Based on the current data R-SH seems unsuitable as a prognostic marker in T2DM.</p

Targeted Genomic Sequencing of TSC1 and TSC2 Reveals Causal Variants in Individuals for Whom Previous Genetic Testing for Tuberous Sclerosis Complex Was Normal

Tuberous sclerosis complex (TSC) is caused by inactivating variants in TSC1 and TSC2. Somatic mosaicism, as well as the size and complexity of the TSC1 and TSC2 loci, makes variant identification challenging. Indeed, in some individuals with a clinical diagnosis of TSC, diagnostic testing fails to identify an inactivating variant. To improve TSC1 and TSC2 variant detection, we screened the TSC1 and TSC2 genomic regions using targeted HaloPlex custom capture and next-generation sequencing (NGS) in genomic DNA isolated from peripheral blood of individuals with definite, possible or suspected TSC in whom no disease-associated variant had been identified by previous diagnostic genetic testing. We obtained &gt;95% target region coverage at a read depth of 20 and &gt;50% coverage at a read depth of 300 and identified inactivating TSC1 or TSC2 variants in 83/155 individuals (54%); 65/113 (58%) with clinically definite TSC and 18/42 (43%) with possible or suspected TSC. These included 19 individuals with deep intronic variants and 54 likely cases of mosaicism (variant allele frequency 1-28%; median 7%). In 13 cases (8%), we identified a variant of uncertain significance (VUS). Targeted genomic NGS of TSC1 and TSC2 increases the yield of inactivating variants found in individuals with suspected TSC.</p

Dendritic cells are crucial for maintenance of tertiary lymphoid structures in the lung of influenza virus–infected mice

Tertiary lymphoid organs (TLOs) are organized aggregates of B and T cells formed in postembryonic life in response to chronic immune responses to infectious agents or self-antigens. Although CD11c+ dendritic cells (DCs) are consistently found in regions of TLO, their contribution to TLO organization has not been studied in detail. We found that CD11chi DCs are essential for the maintenance of inducible bronchus-associated lymphoid tissue (iBALT), a form of TLO induced in the lungs after influenza virus infection. Elimination of DCs after the virus had been cleared from the lung resulted in iBALT disintegration and reduction in germinal center (GC) reactions, which led to significantly reduced numbers of class-switched plasma cells in the lung and bone marrow and reduction in protective antiviral serum immunoglobulins. Mechanistically, DCs isolated from the lungs of mice with iBALT no longer presented viral antigens to T cells but were a source of lymphotoxin (LT) β and homeostatic chemokines (CXCL-12 and -13 and CCL-19 and -21) known to contribute to TLO organization. Like depletion of DCs, blockade of LTβ receptor signaling after virus clearance led to disintegration of iBALT and GC reactions. Together, our data reveal a previously unappreciated function of lung DCs in iBALT homeostasis and humoral immunity to influenza virus

High-yield identification of pathogenic NF1 variants by skin fibroblast transcriptome screening after apparently normal diagnostic DNA testing

Neurofibromatosis type 1 (NF1) is caused by inactivating mutations in NF1. Due to the size, complexity, and high mutation rate at the NF1 locus, the identification of causative variants can be challenging. To obtain a molecular diagnosis in 15 individuals meeting diagnostic criteria for NF1, we performed transcriptome analysis (RNA-seq) on RNA obtained from cultured skin fibroblasts. In each case, routine molecular DNA diagnostics had failed to identify a disease-causing variant in NF1. A pathogenic variant or abnormal mRNA splicing was identified in 13 cases: 6 deep intronic variants and 2 transposon insertions causing noncanonical splicing, 3 postzygotic changes, 1 branch point mutation and, in 1 case, abnormal splicing for which the responsible DNA change remains to be identified. These findings helped resolve the molecular findings for an additional 17 individuals in multiple families with NF1, demonstrating the utility of skin-fibroblast-based transcriptome analysis for molecular diagnostics. RNA-seq improves mutation detection in NF1 and provides a powerful complementary approach to DNA-based methods. Importantly, our approach is applicable to other genetic disorders, particularly those caused by a wide variety of variants in a limited number of genes and specifically for individuals in whom routine molecular DNA diagnostics did not identify the causative variant.</p