Serine synthesis and catabolism in starved lung cancer and primary bronchial epithelial cells.

Serine and glycine give rise to important building blocks in proliferating cells. Both amino acids are either synthesized de novo or taken up from the extracellular space. In lung cancer, serine synthesis gene expression is variable, yet, expression of the initial enzyme, phosphoglycerate dehydrogenase (PHGDH), was found to be associated with poor prognosis. While the contribution of de novo synthesis to serine pools has been shown to be enhanced by serine starvation, the impact of glucose deprivation, a commonly found condition in solid cancers is poorly understood. Here, we utilized a stable isotopic tracing approach to assess serine and glycine de novo synthesis and uptake in different lung cancer cell lines and normal bronchial epithelial cells in variable serine, glycine, and glucose conditions. Under low glucose supplementation (0.2 mM, 3-5% of normal plasma levels), serine de novo synthesis was maintained or even activated. As previously reported, also gluconeogenesis supplied carbons from glutamine to serine and glycine under these conditions. Unexpectedly, low glucose treatment consistently enhanced serine to glycine conversion, along with an up-regulation of the mitochondrial one-carbon metabolism enzymes, serine hydroxymethyltransferase (SHMT2) and methylenetetrahydrofolate dehydrogenase (MTHFD2). The relative contribution of de novo synthesis greatly increased in low serine/glycine conditions. In bronchial epithelial cells, adaptations occurred in a similar fashion as in cancer cells, but serine synthesis and serine to glycine conversion, as assessed by label enrichments and gene expression levels, were generally lower than in (PHGDH positive) cancer cells. In summary, we found a variable contribution of glucose or non-glucose carbon sources to serine and glycine and a high adaptability of the downstream one-carbon metabolism pathway to variable glucose supply.

Genetic Modulation: Future Trends Toward Graft Optimization During Machine Perfusion.

Dynamic preservation methods such as normothermic, subnormothermic, and hypothermic machine perfusion circuits have emerged as viable alternatives to conventional static cold storage. These organ perfusion technologies serve as preservation methods and enable organ assessment, reconditioning, and repair before transplantation. Gene therapy is a novel strategy with the potential to transform the field of graft optimization and treatment. Thereby specific pathways involved in the transplantation process can be targeted and modified. This review aims to provide an overview of gene delivery methods during ex vivo machine perfusion of kidney and liver grafts. Recent literature on state-of-the-art gene therapy approaches during ex situ organ preservation, especially with respect to ischemia-reperfusion injury, as well as acute and chronic graft rejection have been analyzed. Additionally, potential challenges that could affect further refinement of this therapeutic modality are outlined.

Pharmacokinetics of immunosuppressive agents during hemoperfusion in a sheep model.

Introduction: Hemoadsorption shows promising signals in organ preservation and post lung transplantation. However, its potential impact on the pharmacokinetics of immunosuppressant drugs (ID) is still unknown. Methods: In this interventional study, CytoSorb® hemoperfusion was tested in healthy sheep (n = 5) against a sham extracorporeal circuit (n = 3). Seven different ID (tacrolimus (TAC), cyclosporin A (CYA), mycophenolate mofetil (MMF), everolimus (EVER), basiliximab (BAS), methylprednisolone (MP) and prednisolone (PRED)) were administered in clinically relevant doses and combinations. Their levels were measured repeatedly in blood samples from the extracorporeal circulation over 6 h following administration. Population pharmacokinetic modeling analysis (NONMEM® 7.5) was performed. Results: Negligible clearance was observed for PRED and BAS. For all other substances, a saturable adsorption sub-model with linear decrease of the adsorption effect over the adsorbed amount best described the measured concentrations. The maximum absolute adsorbed amounts (95% CI) for TAC, CYA, MMF, EVER, and MP were 0.040 (0.028-0.053), 1.15 (0.39-1.91), 4.17 (2.00-6.35), 0.0163 (0.007-0.026), and 53.4 mg (20.9-85.9), respectively, indicating an adsorption of less than 5% of the daily administered dosages for all investigated substances. Discussion: In this large animal model, CytoSorb® hemoperfusion appears to have a limited effect on the clearance of tested ID.

Different RONS Generation in MTC-SK and NSCL Cells Lead to Varying Antitumoral Effects of Alpha-Ketoglutarate + 5-HMF.

BACKGROUND: Carbonylated proteins (CPs) serve as specific indicators of increased reactive oxygen and nitrogen species (RONS) production in cancer cells, attributed to the dysregulated mitochondrial energy metabolism known as the Warburg effect. The aim of this study was to investigate the potential of alpha-ketoglutarate (aKG), 5-hydroxymethylfurfural (5-HMF), and their combination as mitochondrial-targeting antioxidants in MTC-SK or NCI-H23 cancer cells. METHODS: MTC-SK and NCI-H23 cells were cultured in the absence or presence of varying concentrations (0-500 µg/mL) of aKG, 5-HMF, and the combined aKG + 5-HMF solutions. After 0, 24, 48, and 72 h, mitochondrial activity, cancer cell membrane CP levels, cell growth, and caspase-3 activity were assessed in aliquots of MTC-SK and NCI-H23 cells. RESULTS: The mitochondrial activity of MTC-SK cells exhibited a concentration- and time-dependent reduction upon treatment with aKG, 5-HMF, or the combined aKG + 5-HMF. The half-maximal inhibitory concentration (IC50%) for mitochondrial activity was achieved at 500 µg/mL aKG, 200 µg/mL 5-HMF, and 200 µg/mL aKG + 66.7 µg/mL 5-HMF after 72 h. In contrast, NCI-H23 cells showed a minimal reduction (10%) in mitochondrial activity even at the highest combined concentration of aKG + 5-HMF. The CP levels in MTC-SK cells were measured at 8.7 nmol/mg protein, while NCI-H23 cells exhibited CP levels of 1.4 nmol/mg protein. The combination of aKG + 5-HMF led to a decrease in CP levels specifically in MTC-SK cells. The correlation between mitochondrial activity and CP levels in the presence of different concentrations of combined aKG + 5-HMF in MTC-SK cells demonstrated a linear and concentration-dependent decline in CP levels and mitochondrial activity. Conversely, the effect was less pronounced in NCI-H23 cells. Cell growth of MTC-CK cells was reduced to 60% after 48 h and maintained at 50% after 72 h incubation when treated with 500 µg/mL aKG (IC50%). Addition of 500 µg/mL 5-HMF inhibited cell growth completely regardless of the incubation time. The IC50% for 5-HMF on MTC-CK cell growth was calculated at 375 µg/mL after 24 h incubation and 200 µg/mL 5-HMF after 72 h. MTC-SK cells treated with 500 µg/mL aKG + 167 µg/mL 5-HMF showed no cell growth. The calculated IC50% for the combined substances was 250 µg/mL aKG + 83.3 µg/mL 5-HMF (48 h incubation) and 200 µg/mL aKG + 66.7 µg/mL 5-HMF (72 h incubation). None of the tested concentrations of aKG, 5-HMF, or the combined solution had any effect on NCI-H23 cell growth at any incubation time. Caspase-3 activity increased to 21% in MTC-CK cells in the presence of 500 µg/mL aKG, while an increase to 59.6% was observed using 500 µg/mL 5-HMF. The combination of 500 µg/mL aKG + 167.7 µg/mL 5-HMF resulted in a caspase-3 activity of 55.2%. No caspase-3 activation was observed in NCI-H23 cells when treated with aKG, 5-HMF, or the combined solutions. CONCLUSION: CPs may serve as potential markers for distinguishing between cancer cells regulated by RONS. The combination of aKG + 5-HMF showed induced cell death in high-RONS-generating cancer cells compared to low-RONS-generating cancer cells.

Transplanting a left or right donor kidney into the left or right iliac fossa: importance of laterality and site of venous anastomosis.

Data on the impact of donor-to-recipient laterality on kidney transplantation are lacking. This study evaluated the impact of donor-to-iliac fossa laterality and the site of venous anastomosis on operating time and surgical outcome. This retrospective single-center study analyzed 1262 deceased donor adult kidney transplants into pristine iliac fossa. Multivariable linear and logistic regression analyses were used to identify variables with an impact on operating time and surgical complications. Operating time was shorter by 11 min in median for transplantations into the right iliac fossa compared to the left iliac fossa (p < 0.001). Operating time in left-to-right donor-to-recipient combination was shorter by 17 min in median if venous anastomoses were performed on the caval vein or common iliac vein as compared to anastomoses to the external iliac vein (p < 0.001). Overall, the shortest operating times (median 112.5 min) were achieved in left-to-right donor-to-recipient combinations with venous anastomosis to the caval or common iliac vein, without an increase in surgical complications. Kidney transplantation into the right iliac fossa with anastomosis to the caval vein or the common iliac vein saves operating time and reduces thrombotic complications. Acceptance of a left donor kidney is likely to further reduce operating time.

A combined computational and functional approach identifies IGF2BP2 as a driver of chemoresistance in a wide array of pre-clinical models of colorectal cancer.

AIM: Chemoresistance is a major cause of treatment failure in colorectal cancer (CRC) therapy. In this study, the impact of the IGF2BP family of RNA-binding proteins on CRC chemoresistance was investigated using in silico, in vitro, and in vivo approaches. METHODS: Gene expression data from a well-characterized cohort and publicly available cross-linking immunoprecipitation sequencing (CLIP-Seq) data were collected. Resistance to chemotherapeutics was assessed in patient-derived xenografts (PDXs) and patient-derived organoids (PDOs). Functional studies were performed in 2D and 3D cell culture models, including proliferation, spheroid growth, and mitochondrial respiration analyses. RESULTS: We identified IGF2BP2 as the most abundant IGF2BP in primary and metastastatic CRC, correlating with tumor stage in patient samples and tumor growth in PDXs. IGF2BP2 expression in primary tumor tissue was significantly associated with resistance to selumetinib, gefitinib, and regorafenib in PDOs and to 5-fluorouracil and oxaliplatin in PDX in vivo. IGF2BP2 knockout (KO) HCT116 cells were more susceptible to regorafenib in 2D and to oxaliplatin, selumitinib, and nintedanib in 3D cell culture. Further, a bioinformatic analysis using CLIP data suggested stabilization of target transcripts in primary and metastatic tumors. Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) revealed a decreased basal OCR and an increase in glycolytic ATP production rate in IGF2BP2 KO. In addition, real-time reverse transcriptase polymerase chain reaction (qPCR) analysis confirmed decreased expression of genes of the respiratory chain complex I, complex IV, and the outer mitochondrial membrane in IGF2BP2 KO cells. CONCLUSIONS: IGF2BP2 correlates with CRC tumor growth in vivo and promotes chemoresistance by altering mitochondrial respiratory chain metabolism. As a druggable target, IGF2BP2 could be used in future CRC therapy to overcome CRC chemoresistance.

Probiotic Supplementation Attenuates Chemotherapy-Induced Intestinal Mucositis in an Experimental Colorectal Cancer Liver Metastasis Rat Model.

The use of chemotherapeutic agents is of paramount importance when treating colorectal cancer (CRC). Unfortunately, one of the most frequent chemotherapy (CTx) side effects is intestinal mucositis (IM), which may present with several clinical symptoms such as nausea, bloating, vomiting, pain, and diarrhea and even can result in life-threatening complications. There is a focused scientific effort towards developing new therapies to prevent and treat IM. The aim of this study was to assess the outcomes of probiotic supplementation on CTx-induced IM in a CRC liver metastasis rat model. Six-week-old male Wistar rats received either a multispecies probiotic or placebo mixture. On the 28th experiment day, rats received FOLFOX CTx, and afterwards, the severity of diarrhea was evaluated twice daily. Stool samples were collected for further microbiome analysis. Additionally, immunohistochemical stainings of ileum and colon samples with were performed with MPO, Ki67, and Caspase-3 antibodies. Probiotic supplementation alleviates the severity and length of CTx-induced diarrhea. Additionally, probiotics significantly reduced FOLFOX-induced weight and blood albumin loss. Furthermore, probiotic supplementation mitigated CTx-induced histological changes in the gut and promoted intestinal cell regeneration. This study shows that multispecies probiotic supplementation attenuates FOLFOX-induced IM symptoms by inhibiting apoptosis and promoting intestinal cell proliferation.

Experimental Static Cold Storage of the Rat Uterus: Protective Effects of Relaxin- or Erythropoietin-Supplemented HTK-N Solutions.

Uterus transplantation (UTx) is the only treatment method for women with absolute uterine infertility. Currently, the number of grafts retrieved from deceased donors is increasing; hence, prolonged cold ischemia time is inevitable. Thus, this study was designed to assess the effect of the novel relaxin (RLN)- or erythropoietin (EPO)-supplemented Custodiol-N (HTK-N) solutions in an experimental uterus static cold storage (SCS) model. A total of 15 Sprague Dawley rats were used. Uterus horns were randomly assigned into three groups (n = 10/group). SCS was performed by keeping samples at 4 °C in HTK-N solution without or with different additives: 10 IU/mL EPO or 20 nM RLN. Tissue samples were taken after 8 and 24 h of preservation. Uterine tissue histology, and biochemical and immunohistochemical markers were analyzed. No significant differences in SCS-induced tissue damage were observed between groups after 8 h of preservation. Uterine tissue histology, MDA, SOD levels and the TUNEL-positive cell number showed severe damage in HTK-N without additives after 24 h of preservation. This damage was significantly attenuated by adding RLN to the preservation solution. EPO showed no favorable effect. Our study shows that RLN as an additive to an HTK-N solution can serve as an effective uterine tissue preservative in the uterus SCS setting.

Machine Perfusion in Liver Transplantation: A Systematic Review and Meta-Analysis.

Background: Liver transplantation (LTx) is the only treatment option for patients with end-stage liver disease. Novel organ preservation techniques such as hypothermic machine perfusion (HMP) or normothermic machine perfusion (NMP) are under investigation in order to improve organ quality from extended criteria donors and donors after circulatory death. The aim of this study was to systematically review the literature reporting LTx outcomes using NMP or HMP compared to static cold storage (SCS). Methods: The following data were retrieved: graft primary nonfunction rate, early allograft dysfunction (EAD) rate, biliary complication rate, and 12-month graft and patient survival. A total of 15 studies were included (6 NMP and 9 HMP studies), and meta-analysis was performed only for HMP studies because NMP had considerable differences. Results: The systematic review showed the potential of NMP to reduce graft injury and lower the liver graft discard rate. The performed quantitative analyses showed that the use of HMP reduces the rate of EAD (odds ratio [OR] 0.51; 95% confidence interval [CI] 0.34-0.76; p = 0.001; I 2 = 0%) and non-anastomotic biliary strictures (OR 0.34; 95% CI 0.17-0.67; p = 0.002; I 2 = 0%) compared to SCS. Conclusion: Our systematic review and meta-analysis revealed that the use of HMP reduces the rate of EAD and non-anastomotic biliary strictures compared to SCS.

Alpha-Ketoglutarate or 5-HMF: Single Compounds Effectively Eliminate Leukemia Cells via Caspase-3 Apoptosis and Antioxidative Pathways.

BACKGROUND: We recently showed that a combined solution containing alpha-ketoglutarate (aKG) and 5-hydroxymethyl-furfural (5-HMF) has a solid antitumoral effect on the Jurkat cell line due to the fact of its antioxidative, caspase-3 and apoptosis activities, but no negative effect on human fibroblasts was obtained. The question arises how the single compounds, aKG and 5-HMF, affect peroxynitrite (ONOO-) and nitration of tyrosine residues, Jurkat cell proliferation and caspase-activated apoptosis. METHODS: The ONOO- luminol-induced chemiluminescence reaction was used to measure the ONOO- scavenging function of aKG or 5-HMF, and their protection against nitration of tyrosine residues on bovine serum albumin was estimated with the ELISA technique. The Jurkat cell line was cultivated in the absence or presence of aKG or 5-HMF solutions between 0 and 3.5 µM aKG or 0 and 4 µM 5-HMF. Jurkat cells were tested for cell proliferation, mitochondrial activity and caspase-activated apoptosis. RESULTS: aKG showed a concentration-dependent reduction in ONOO-, resulting in a 90% elimination of ONOO- using 200 mM aKG. In addition, 20 and 200 mM 5-HMF were able to reduce ONOO- only by 20%, while lower concentrations of 5-HMF remained stable in the presence of ONOO-. Nitration of tyrosine residues was inhibited 4 fold more effectively with 5-HMF compared to aKG measuring the IC50%. Both substances, aKG and 5-HMF, were shown to cause a reduction in Jurkat cell growth that was dependent on the dose and incubation time. The aKG effectively reduced Jurkat cell growth down to 50% after 48 and 72 h of incubation using the highest concentration of 3.5 µM, and 1, 1.6, 2, 3 and 4 µM 5-HMF inhibited any cell growth within (i) 24 h; 1.6, 2, 3 and 4 µM 5-HMF within 48 h (ii); 2, 3 and 4 µM 5-HMF within 72 h (iii). Furthermore, 4 µM was able to eliminate the starting cell number of 20,000 cells after 48 and 72 h down to 11,233 cells. The mitochondrial activity measurements supported the data on aKG or 5-HMF regarding cell growth in Jurkat cells, in both a dose- and incubation-time-dependent manner: the highest concentration of 3.5 µM aKG reduced the mitochondrial activity over 24 h (67.7%), 48 h (57.9%) and 72 h (46.8%) of incubation with Jurkat cells compared to the control incubation without aKG (100%). 5-HMF was more effective compared to aKG; the mitochondrial activity in the presence of 4 µM 5-HMF decreased after 24 h down to 68.4%, after 48 h to 42.9% and after 72 h to 32.0%. Moreover, 1.7 and 3.4 µM aKG had no effect on caspase-3-activated apoptosis (0.58% and 0.56%) in the Jurkat cell line. However, 2 and 4 µM 5-HMF increased the caspase-3-activated apoptosis up to 22.1% and 42.5% compared to the control (2.9%). A combined solution of 1.7 µM aKG + 0.7 µM 5-HMF showed a higher caspase-3-activated apoptosis (15.7%) compared to 1.7 µM aKG or 2 µM 5-HMF alone. In addition, 3.5 µM µg/mL aKG + 1.7 µM 5-HMF induced caspase-activated apoptosis up to 55.6% compared to 4.5% or 35.6% caspase-3 activity using 3.5 µM aKG or 4 µM 5-HMF. CONCLUSION: Both substances showed high antioxidative potential in eliminating either peroxynitrite or nitration of tyrosine residues, which results in a better inhibition of cell growth and mitochondrial activity of 5-HMF compared to aKG. However, caspase-3-activated apoptosis measurements revealed that the combination of both substances synergistically is the most effective compared to single compounds.

Probiotic Supplementation Suppresses Tumor Growth in an Experimental Colorectal Cancer Liver Metastasis Model.

Colorectal cancer (CRC) ranks third in incidence and second in mortality of all cancers worldwide. At the time of primary diagnosis, around 20% of patients already have metastatic CRC and only around 20% are candidates for radical resection. Thus, most of the patients have to undergo chemotherapy (CTx). Due to chemoresistance and side effects, novel treatment additives are crucial for controlling the disease and prolonging patient survival. The aim of this study was to evaluate probiotic supplementation and its antitumorigenic effects in an experimental CRC liver metastasis model. Six-week-old male Wistar rats received either a multispecies probiotic (1.2 × 109 CFU/daily) or placebo mixture. On day 14 of the experiment, rat CRC cells (CC531) were implanted under the liver capsule later treated by FOLFOX CTx. Change in tumor volume was measured by performing micro computed tomography (micro-CT) scanning on experimental days 28 and 34. Additionally, immunohistochemical staining with anti-MPO, anti-Ki67, and anti-CD31 were performed. Tumor apoptosis was evaluated using TUNEL staining. Micro-CT image analysis indicates that probiotic supplementation significantly inhibits tumor growth. No synergistic effects between probiotic supplementation and FOLFOX CTx was observed. Reduced tumor volume was achieved by inhibiting angiogenesis, as tumor microvascular density was significantly lower in rats receiving probiotic supplementation. This study shows that a multispecies probiotic mixture significantly reduces angiogenesis and inhibits CRC liver metastasis growth in an experimental rat model.

Relaxin and Erythropoietin Significantly Reduce Uterine Tissue Damage during Experimental Ischemia-Reperfusion Injury.

Successful uterus transplantation, a potential treatment method for women suffering from absolute uterine infertility, is negatively affected by ischemia-reperfusion injury (IRI). The aim of this study is to investigate the protective effect of relaxin (RLX) or/and erythropoietin (EPO) on experimental uterus IRI. Eighty rats, randomly assigned into eight groups (n = 10/group), were pretreated with either saline, 5 µg/kg human relaxin-2, 4000 IU/kg recombinant human erythropoietin or their combination. Ischemia was achieved by clamping the aorta and ovarian arteries for 60 min, following 120 min of reperfusion and tissue sampling. For sham animals, clamping was omitted during surgery. There were no differences in tissue histological score, malondialdehyde (MDA) and superoxide dismutase (SOD) levels, myeloperoxidase (MPO) and TUNEL-positive cell count between all sham-operated rats. Pretreatment with RLX preserved normal tissue morphology, reduced MDA levels, MPO and TUNEL-positive cell count, preserved SOD activity and upregulated NICD and HES1 gene expression when compared to the control group. Pretreatment with EPO reduced MDA levels. In conclusion, pretreatment with RLX, EPO or a combination of both EPO and RLX significantly alleviates uterine tissue damage caused by IRI.

Circulating microRNAs as molecular biomarkers for lung adenocarcinoma.

BACKGROUND: The potential of microRNAs (miRNAs) as molecular tumor biomarkers for early diagnosis and prognosis in lung cancer is still unclear. OBJECTIVE: To analyze expression of miRNAs in A549 lung adenocarcinoma (LUAD) cells and in primary, non-malignant bronchial epithelial (BE) cells from healthy donors. To analyze the most prominently deregulated miRNAs in plasma samples of LUAD patients and healthy donors. MATERIALS AND METHODS: The expression of 752 miRNAs in LUAD and BE cells was assessed by RT-qPCR with mean-centering restricted normalization. The relative plasma levels of 18 miRNAs in LUAD patients and healthy donors were analyzed using RT-qPCR and normalized to miR-191-5p and miR-16-3p. Putative interactions between miRNAs and their target genes were investigated in silico. RESULTS: Out of 752 miRNAs, 37 miRNAs were significantly deregulated in A549 cells compared to BE cells. MiR-15b-3p, miR-148a-3p, miR-193b-3p, and miR-195-5p were significantly deregulated in plasma samples of LUAD patients compared to donors. The target genes of those four miRNAs are involved in essential mechanisms in cancer development and progression. CONCLUSIONS: There are substantial differences between cancer and control miRNA expression in vitro and in plasma samples of LUAD patients compared to healthy donors. Four deregulated miRNAs are promising as a diagnostic biomarker for adenocarcinoma of the lung.

Necroptosis in Solid Organ Transplantation: A Literature Overview.

Ischemia-reperfusion injury (IRI) is encountered in various stages during solid organ transplantation (SOT). IRI is known to be a multifactorial inflammatory condition involving hypoxia, metabolic stress, leukocyte extravasation, cellular death (including apoptosis, necrosis and necroptosis) and an activation of immune response. Although the cycle of sterile inflammation during IRI is consistent among different organs, the underlying mechanisms are poorly understood. Receptor-interacting protein kinase 3 (RIPK3) and mixed-lineage kinase domain-like pseudokinase (MLKL) are thought to be crucial in the implementation of necroptosis. Moreover, apart from "silent" apoptotic death, necrosis also causes sterile inflammation-necroinflammation, which is triggered by various damage-associated molecular patterns (DAMPs). Those DAMPs activate the innate immune system, causing local and systemic inflammatory responses, which can result in graft failure. In this overview we summarize knowledge on mechanisms of sterile inflammation processes during SOT with special focus on necroptosis and IRI and discuss protective strategies.

B Cell Composition Is Altered After Kidney Transplantation and Transitional B Cells Correlate With SARS-CoV-2 Vaccination Response.

BACKGROUND: The COVID-19 pandemic has major implications on kidney transplant recipients (KTRs) since they show increased mortality due to impaired immune responses to SARS-CoV-2 infection and a reduced efficacy of SARS-CoV-2 vaccination. Surprisingly, dialysis patients have shown superior seroconversion rates after vaccination compared to KTRs. Therefore, we investigated peripheral blood B cell (BC) composition before and after kidney transplantation (KT) and aimed to screen the BC compartment to explain impaired antibody generation. METHODS: A total of 105 patients were recruited, and multicolor flow cytometric phenotyping of peripheral venous blood BC subpopulations was performed before and 1 year after KT. Complete follow-up was available for 71 individuals. Anti-SARS-CoV-2 antibodies were collected retrospectively and were available for 40 subjects, who had received two doses of an mRNA-based vaccine (BNT162b2 or mRNA-1273). RESULTS: Overall, relative BC frequencies within lymphocytes decreased, and their absolute counts trended in the same direction 1 year after KT as compared to CKD G5 patients. Frequencies and absolute numbers of naïve BCs remained stable. Frequencies of double negative BCs, a heterogeneous subpopulation of antigen experienced BCs lacking CD27 expression, were increased after KT, yet their absolute counts were similar at both time points. Transitional BCs (TrBCs) and plasmablasts were significantly reduced after KT in absolute and relative terms. Memory BCs were affected differently since class-switched and IgM-only subsets decreased after KT, but unswitched and IgD-only memory BCs remained unchanged. CD86+ and CD5+ expression on BCs was downregulated after KT. Correlational analysis revealed that TrBCs were the only subset to correlate with titer levels after SARS-CoV-2 vaccination. Responders showed higher TrBCs, both absolute and relative, than non-responders. CONCLUSION: Together, after 1 year, KTRs showed persistent and profound compositional changes within the BC compartment. Low TrBCs, 1 year after KT, may account for the low serological response to SARS-CoV-2 vaccination in KTRs compared to dialysis patients. Our findings need confirmation in further studies as they may guide vaccination strategies.

Quantification of increased MUC5AC expression in airway mucus of smoker using an automated image-based approach.

Microscopic analysis of mucus quantity and composition is crucial in research and diagnostics on muco-obstructive diseases. Currently used image-based methods are unable to extract concrete numeric values of mucosal proteins, especially on the expression of the key mucosal proteins MUC5AC and MUC5B. Since their levels increase under pathologic conditions such as extensive exposure to cigarette smoke, it is imperative to quantify them to improve treatment strategies of pulmonary diseases. This study presents a simple, image-based, and high-processing computational method that allows determining the ratio of MUC5AC and MUC5B within the overall airway mucus while providing information on their spatial distribution. The presented pipeline was optimized for automated downstream analysis using a combination of bright field and immunofluorescence imaging suitable for tracheal and bronchial tissue samples, and air-liquid interface (ALI) cell cultures. To validate our approach, we compared tracheal tissue and ALI cell cultures of isolated primary normal human bronchial epithelial cells derived from smokers and nonsmokers. Our data indicated 18-fold higher levels of MUC5AC in submucosal glands of smokers covering about 8% of mucosal areas compared to <1% in nonsmoking individuals, confirming results of previous studies. We further identified a subpopulation of nonsmokers with slightly elevated glandular MUC5AC levels suggesting moderate exposure to second-hand smoke or fine particulate air pollution. Overall, this study demonstrates a novel, user-friendly and freely available tool for digital pathology and the analysis of therapeutic interventions tested in ALI cell cultures.

Custodiol® Supplemented with Synthetic Human Relaxin Decreases Ischemia-Reperfusion Injury after Porcine Kidney Transplantation.

Objective. Ischemia-reperfusion injury (IRI) is inevitable after kidney transplantation (KT), impairing outcomes. Relaxin-2 (RLX) is a promising insulin-related peptide hormone that protects against renal IRI in rodents, although large animal models are needed before RLX can be tested in a human setting. Methods. In this blinded, randomized, and placebo-controlled experimental study kidneys from 19 donor pigs were retrieved after perfusion with Custodiol® ± RLX (5 or 20 nmol/L) and underwent static cold storage (SCS) for 24 and 48 h, respectively. Subsequently, KT was performed after unilateral right nephrectomy. Study outcomes included markers for kidney function, oxidative stress, lipid peroxidation, and endothelial cell damage. PCR analysis for oxidative stress and apoptosis-related gene panels as well as immunohistochemistry were performed. Results. RLX upregulated SOD2 and NFKB expression to 135% (p = 0.042) and 125% (p = 0.019), respectively, while RIPK1 expression was downregulated to 82% (p = 0.016) of corresponding controls. Further RLX significantly downregulated RIPK1 and MLKL expression and decreased the number of Caspase 3- and MPO-positive cells in grafts after SCS. Conclusions. RLX supplemented Custodiol® significantly decreased IRI via both antioxidant and anti-apoptotic mechanisms. Clinical trials are warranted to implement synthetic human RLX as a novel additive to preservation solutions against IRI.

Alpha-Ketoglutarate and 5-HMF: A Potential Anti-Tumoral Combination against Leukemia Cells.

We have recently shown that a combined solution containing alpha-ketoglutarate (aKG) and 5-hydroxymethyl-furfural (5-HMF) might have anti-tumoral potential due to its antioxidative activities. The question arises if these substances have caspase-3- and apoptosis-activating effects on the cell proliferation in Jurkat and HF-SAR cells. Antioxidative capacity of several combined aKG + 5-HMF solution was estimated by cigarette smoke radical oxidized proteins of fetal calf serum (FCS) using the estimation of carbonylated proteins. The usage of 500 µg/mL aKG + 166.7 µg/mL 5-HMF showed the best antioxidative capacity to inhibit protein modification of more than 50% compared to control measurement. A Jurkat cell line and human fibroblasts (HF-SAR) were cultivated in the absence or presence of combined AKG + 5-HMF solutions between 0 µg/mL aKG + 0 µg/mL 5-HMF and different concentrations of 500 µg/mL aKG + 166.7 µg/mL 5-HMF. Aliquots of Jurkat cells were tested for cell proliferation, mitochondrial activity, caspase activity, apoptotic cells and of the carbonylated protein content as marker of oxidized proteins in cell lysates after 24, 48, and 72 h of incubation. The combined solutions of aKG + 5-HMF were shown to cause a reduction in Jurkat cell growth that was dependent on the dose and incubation time, with the greatest reductions using 500 µg/mL aKG + 166.7 µg/mL 5-HMF after 24 h of incubation compared to 24 h with the control (22,832 cells vs. 32,537 cells), as well as after 48 h (21,243 vs. 52,123 cells) and after 72 h (23,224 cells). Cell growth was totally inhibited by the 500 µg/mL AKG + 166.7 µg/mL solution between 0 and 72 h of incubation compared to 0 h of incubation for the control. The mitochondrial activity measurements supported the data on cell growth in Jurkat cells: The highest concentration of 500 µg/mL aKG + 166.7 µg/mL 5-HMF was able to reduce the mitochondrial activity over 24 h (58.9%), 48 h (28.7%), and 72 h (9.9%) of incubation with Jurkat cells compared not only to the control incubation, but also to the concentrations of 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 375 µg/mL aKG 125 µg/mL 5-HMF, which were able to significantly reduce the mitochondrial activity after 48 h (28.7% or 35.1%) and 72 h (9.9% or 18.2%) compared to 24 h with the control (100%). A slight increase in cell proliferation was found in HF-SAR using the highest concentration (500 µg/mL aKG + 166.7 µg/mL 5-HMF) between 0 h and 72 h incubation of 140%, while no significant differences were found in the mitochondrial activity of HF-SAR in the absence or presence of several combined aKG + 5-HMF solutions. The solutions with 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 250 µg/mL aKG + 83.3 µg/mL 5-HMF showed a significantly higher caspase activity (51.6% or 13.5%) compared to the control (2.9%) in addition to a higher apoptosis rate (63.2% or 31.4% vs. control: 14.9%). Cell lysate carbonylated proteins were significantly higher in Jurkat cells compared to HF-SAR cells (11.10 vs. 2.2 nmol/mg). About 72 h incubation of Jurkat cells with 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 250 µg/mL aKG + 83.3 µg/mL 5-HMF reduced significantly the carbonylated protein content down to 5.55 or 7.44 nmol/mg whereas only the 500 µg/mL aKG + 166.7 µg/mL 5-HMF solution showed a significant reduction of carbonylated proteins of HF-SAR (1.73 nmol/mg).

Ischemic Preconditioning for Liver Transplantation: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

BACKGROUND: In recent decades, liver transplantation (LTx) has increased the survival and quality of life of patients with end-stage organ failure. Unfortunately, LTx is limited due to the shortage of donors. A lot of effort is put into finding new ways to reduce ischemia-reperfusion injury (IRI) in liver grafts to increase the number of suitable organs procured from expanded-criteria donors (ECD). The aim of this study was to systematically review the literature reporting LTx outcomes when using ischemic preconditioning (IPC) or remote ischemic preconditioning (RIPC) to reduce IRI in liver grafts. METHODS: A literature search was performed in the MEDLINE, Web of Science, and EMBASE databases. The following combination was used: "Liver" OR "Liver Transplantation" AND "Ischemic preconditioning" OR "occlusion" OR "clamping" OR "Pringle." The following outcome data were retrieved: the rates of graft primary nonfunction (PNF), retransplantation, graft loss, and mortality; stay in hospital and the intensive care unit; and postoperative serum liver damage parameters. RESULTS: The initial search retrieved 4,522 potentially relevant studies. After evaluating 17 full-text articles, a total of 9 randomized controlled trials (RCTs) were included (7 IPC and 2 RIPC studies) in the qualitative synthesis; the meta-analysis was only performed on the data from the IPC studies. RIPC studies had considerable methodological differences. The meta-analysis revealed the beneficial effect of IPC when comparing postoperative aspartate aminotransferase (AST) corresponding to a statistically lower mortality rate in the IPC group (odds ratio [OR] 0.51; 95% confidence interval [CI] 0.27-0.98; p = 0.04). CONCLUSION: IPC lowers postoperative AST levels and reduces the mortality rate; however, data on the benefits of RIPC are lacking.