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Normothermic machine perfusion (NMP) of injured kidneys offers the opportunity for interventions to metabolically active organs prior to transplantation. Mesenchymal stromal cells (MSCs) can exert regenerative and anti-inflammatory effects in ischemia-reperfusion injury. The aims of this study were to evaluate the safety and feasibility of MSC treatment of kidneys during NMP using a porcine autotransplantation model, and examine potential MSC treatment-associated kidney improvements up to 14 days posttransplant. After 75 min of kidney warm ischemia, four experimental groups of n = 7 underwent 14 h of oxygenated hypothermic machine perfusion. In three groups this was followed by 240 min of NMP with infusion of vehicle, 10 million porcine, or 10 million human adipose-derived MSCs. All kidneys were autotransplanted after contralateral nephrectomy. MSC treatment did not affect perfusion hemodynamics during NMP or cause adverse effects at reperfusion, with 100% animal survival. MSCs did not affect plasma creatinine, glomerular filtration rate, neutrophil gelatinase-associated lipocalin concentrations or kidney damage assessed by histology during the 14 days, and MSCs retention was demonstrated in renal cortex. Infusing MSCs during ex vivo NMP of porcine kidneys was safe and feasible. Within the short posttransplant follow-up period, no beneficial effects of ex vivo MSC therapy could be demonstrated.

Background.Mesenchymal stromal cell (MSC) therapy may improve renal function after ischemia-reperfusion injury in transplantation. Ex vivo renal intraarterial administration is a targeted delivery method, avoiding the lung vasculature, a known barrier for cellular therapies. In a randomized and blinded study, we tested the feasibility and effectiveness of MSC therapy in a donation after circulatory death autotransplantation model to improve posttransplant kidney function, using an ex vivo MSC delivery method similar to the clinical standard procedure of pretransplant cold graft flush.Methods.Kidneys exposed to 75 minutes of warm ischemia and 16 hours of static cold storage were intraarterially infused ex vivo with 10 million male porcine MSCs (Tx-MSC, n = 8) or vehicle (Tx-control, n = 8). Afterwards, the kidneys were autotransplanted after contralateral nephrectomy. Biopsies an hour after reperfusion confirmed the presence of MSCs in the renal cortex. Animals were observed for 14 days.Results.Postoperatively, peak plasma creatinine was 1230 and 1274 mu mol/L (Tx-controls versus Tx-MSC, P = 0.69). During follow-up, no significant differences over time were detected between groups regarding plasma creatinine, plasma neutrophil gelatinase-associated lipocalin, or urine neutrophil gelatinase-associated lipocalin/creatinine ratio. At day 14, measured glomerular filtration rates were 40 and 44 mL/min, P = 0.66. Renal collagen content and fibrosis-related mRNA expression were increased in both groups but without significant differences between the groups.Conclusions.We demonstrated intraarterial MSC infusion to transplant kidneys as a safe and effective method to deliver MSCs to the graft. However, we could not detect any positive effects of this cell treatment within 14 days of observation.

Naturally occurring antibodies are abundant in human plasma, but their importance in the defense against bacterial pathogens is unclear. We studied the role of the most abundant of such antibodies, the antibody against terminal galactose-α-1,3-galactose (anti-αGal), in the protection against pneumococcal infections (Streptococcus pneumonia). All known pneumococcal capsular polysaccharides lack terminal galactose-α-1,3-galactose, yet highly purified human anti-αGal antibody of the IgG class reacted with 48 of 91 pneumococcal serotypes. Anti-αGal was found to contain multiple antibody subsets that possess distinct specificities beyond their general reactivity with terminal galactose-α-1,3-galactose. These subsets in concert targeted a wide range of microbial polysaccharides. We found that anti-αGal constituted up to 40% of the total antibody reactivity to pneumococci in normal human plasma, that anti-αGal drives phagocytosis of pneumococci by human neutrophils, and that the anti-αGal level was 2-fold lower in patients prone to pneumococcal infections compared to controls. Moreover, during a 48-year period in Denmark, the 48 anti-αGal-reactive serotypes caused fewer invasive pneumococcal infections (n = 10,927) than the 43 non-reactive serotypes (n = 18,107), supporting protection on the population level. Our findings explain the broad-spectrum pathogen reactivity of anti-αGal and support that these naturally occurring polyreactive antibodies contribute significantly to human protective immunity.

BACKGROUND: Adipose tissue-derived stem cells are of great interest because of their properties of immune modulation, tissue regeneration, and multipotent differentiation. To advance development of stem cell-based treatments, determination of the physiologic concentration of adipose tissue-derived stem cells in human adipose tissue is relevant for proper guidance of stem cell treatment dosage, oncologic safety, and evaluation of efficacy.METHODS: A prospective comparative case-control study of 20 patients was conducted to determine the yield of adipose tissue-derived stem cells in periumbilical adipose tissue harvested by the widely used method of aspiration and in structurally intact adipose tissue harvested by excision. Stem cells were isolated using conventional enzymatic digestion and by a method combining enzymatic digestion with mechanical distortion. Stem cell yield was quantified by multicolor flow cytometry and colony-forming capacity.RESULTS: When only the conventional enzymatic digestion was used, no significant difference in adipose tissue-derived stem cell yield was observed. However, when enzymatic digestion was combined with mechanical distortion, twice as many stem cells were isolated from excised adipose tissue compared to aspirated adipose tissue. Inclusion of mechanical distortion significantly increased yield 5-fold in excised adipose tissue and 2-fold in aspirated adipose tissue. Combining enzymatic digestion and mechanical distortion, measured levels of excised adipose tissue reached 140 × 10 (95 percent CI, 62 to 220 × 10) adipose tissue-derived stem cells per gram of adipose tissue that corresponded to 26 × 10 (95 percent CI, 18 to 33 × 10) colony-forming units per gram.CONCLUSIONS: The study indicates that harvesting by aspiration halves the concentration of adipose tissue-derived stem cells in adipose tissue samples when compared to structural intact adipose tissue. Furthermore, the study presents stem cell yield higher than previously described in the current literature.CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Inflammation resulting from ischaemia/reperfusion injury can cause kidney graft dysfunction, increase the risk of delayed graft function and possibly reduce long-term graft survival. Remote ischaemic conditioning may protect against ischaemia/reperfusion injury and mitigate the immunological response to the graft. We investigated the immunological effects of remote ischaemic conditioning on kidney transplantation from deceased donors in the randomized CONTEXT study. Three circulating dendritic cell (DC) subtypes identified in peripheral blood from kidney transplant recipients [myeloid DCs, plasmacytoid DCs and immunoglobulin-like transcript (ILT)3+ DCs] were measured at baseline, days 1, 3 and 5 and 1 and 3 months after transplantation. We also quantified 21 cytokines at baseline, days 1 and 5 and 3 months after transplantation. Neither DC counts nor cytokine levels differed between patients receiving remote ischaemic conditioning and controls; however, several parameters exhibited dynamic and parallel alterations in the two groups over time, reflecting the immunological response to the kidney transplantation and immunosuppression.

Background and aims Allergic rhinitis (AR), allergic conjunctivitis (AC), and asthma are characterized by activation of the immune system. The aim of this study was to explore the long-term association between AR, AC, asthma, and specific immunoglobulin E (IgE) and blood platelet and leukocyte differential counts. Material and methods In the Danish Blood Donor Study, 14,440 participants from Central Denmark Region had platelet and leukocyte differential counts available and completed a questionnaire regarding AR, AC, and asthma. Of these participants, 8485 were tested for IgE to inhalation allergens. Results The prevalence of AR, AC, asthma, and IgE sensitization was 19%, 15%, 9%, and 29%, respectively. AR, AC, asthma, wheeze, and IgE sensitization was associated with increased blood eosinophil concentration even in IgE sensitized participants who did not report any allergy or asthma. The strongest associations were observed for participants with current disease. We found no differences in eosinophil concentration between months without symptoms and months with symptoms of AR and asthma. Conclusion AR, AC, asthma, wheezing, and IgE sensitization to inhalation allergens are associated with increased eosinophil concentration. This may reflect a persistent inflammation even in periods without symptomatic disease.

BACKGROUND: For unknown reasons, HIV-2 is less pathogenic than HIV-1, and HIV-2-induced immunodeficiency may be different from that caused by HIV-1. Previous immunological studies have hinted at possible shifts in both T-cell and B-cell subsets, which we aimed to characterize further. METHODS: From an HIV clinic in Guinea-Bissau, 63 HIV-2, 83 HIV-1, and 26 HIV-negative participants were included. All HIV-infected participants were ART-naive. The following cell subsets were analysed by flow cytometry; T cells (maturation and activation), regulatory T cells, and B cells (maturation and activation). RESULTS: After standardizing for sex, age, and CD4 T-cell count HIV-2 had 0.938 log10 copies/ml lower HIV RNA levels than the HIV-1-infected patients. Whereas T-cell maturation and regulatory T-cell profiles were similar between patients, HIV-2-infected patients had higher proportions of CD8CD28 and lower proportions of CD8PD-1+ T cells than HIV-1-infected patients. This finding was independent of HIV RNA levels. HIV-2 was also associated with a more preserved proportion of naive B cells. CONCLUSION: HIV-2 is characterized by lower viral load, and lower T-cell activation, which may account for the slower disease progression.

Background Do prescription drugs with anti-inflammatory properties such as nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and statins increase the risk of postoperative bleeding measured with RBC transfusion in elderly hip fracture surgery patients? Study design and methods Using the Danish Multidisciplinary Hip Fracture Database, 74,791 patients aged 65 years or older with surgery-treated hip fracture were identified during 2005-2016, and their use of NSAIDs, corticosteroids, and statins was ascertained. For each drug, patients were categorized as nonusers (no prescription ≤365 days prior to surgery), former users (one prescription 91-365 days), and current users (one prescription ≤90 days). Information on surgical treatment, transfusion, other medication and comorbidities were collected using Danish nationwide registries. A log-binomial model was used to estimate relative risks for RBC transfusion within 7 days of surgery and corresponding 95% confidence intervals. Adjustments were made for patient- and surgery-related factors. Results Former and current users of NSAIDs, corticosteroids, and statins accounted for 22%, 10%, and 24%, respectively. Current users of NSAIDs had an increased adjusted relative risk of transfusion (1.07; confidence interval, 1.04-1.10) compared to nonusers. There was no association between current users of corticosteroids and statins and risk of transfusion. Conclusion NSAID use within 90 days of a hip fracture surgery was associated with an increased risk of RBC transfusion. Thus, current use of NSAIDs can be associated with an increased risk of postoperative bleeding, but we cannot rule out the influence of confounding.

Serological assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to support clinical diagnosis and epidemiological investigations. Recently, assays for large-scale detection of total antibodies (Ab), immunoglobulin G (IgG), and IgM against SARS-CoV-2 antigens have been developed, but there are limited data on the diagnostic accuracy of these assays., Serological assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to support clinical diagnosis and epidemiological investigations. Recently, assays for large-scale detection of total antibodies (Ab), immunoglobulin G (IgG), and IgM against SARS-CoV-2 antigens have been developed, but there are limited data on the diagnostic accuracy of these assays. This study was a Danish national collaboration and evaluated 15 commercial and one in-house anti-SARS-CoV-2 assays in 16 laboratories. Sensitivity was evaluated using 150 samples from individuals with asymptomatic, mild, or moderate COVID-19, nonhospitalized or hospitalized, confirmed by nucleic acid amplification tests (NAAT); samples were collected 13 to 73 days either from symptom onset or from positive NAAT (patients without symptoms). Specificity and cross-reactivity were evaluated in samples collected prior to the SARS-CoV-2 epidemic from >586 blood donors and patients with autoimmune diseases, cytomegalovirus or Epstein-Barr virus infections, and acute viral infections. A specificity of ≥99% was achieved by all total-Ab and IgG assays except one, DiaSorin Liaison XL IgG (97.2%). Sensitivities in descending order were Wantai ELISA total Ab (96.7%), CUH-NOVO in-house ELISA total Ab (96.0%), Ortho Vitros total Ab (95.3%), YHLO iFlash IgG (94.0%), Ortho Vitros IgG (93.3%), Siemens Atellica total Ab (93.2%), Roche Elecsys total Ab (92.7%), Abbott Architect IgG (90.0%), Abbott Alinity IgG (median 88.0%), DiaSorin Liaison XL IgG (median 84.6%), Siemens Vista total Ab (81.0%), Euroimmun/ELISA IgG (78.0%), and Snibe Maglumi IgG (median 78.0%). However, confidence intervals overlapped for several assays. The IgM results were variable, with the Wantai IgM ELISA showing the highest sensitivity (82.7%) and specificity (99%). The rate of seropositivity increased with time from symptom onset and symptom severity.

Background Allergic rhinitis (AR), allergic conjunctivitis (AC), and asthma composing multiple phenotypes and improved understanding of these phenotypes and their respective risk factors are needed. Objectives The objective of this study was to define the prevalence of AR, AC, and asthma and their association with allergen-specific immunoglobulin E (sIgE) sensitization in a large cohort of blood donors and identify risk factors. Methods From the nationwide population-based Danish Blood Donor Study, 52,976 participants completed an electronic questionnaire including AR, AC, asthma, allergic predisposition, and childhood residence. Of these, 25,257 were additionally tested for sIgE to inhalation allergens (Phadiatop). Results The prevalence of sIgE sensitization, AR, AC, and asthma was 30%, 19%, 15%, and 9%, respectively. The youngest birth cohorts had the highest prevalence of sIgE sensitization and symptoms of asthma, AR, and AC, and for asthma, they apparently experienced symptoms at an earlier age. The sIgE sensitization was positively associated with male sex. The sIgE seroprevalence was higher in participants with both AR and AC (ARC) than in participants with either AR or AC. Allergic predisposition and sIgE sensitization increased the risk of the diseases, while farm upbringing was associated with reduced prevalence of ARC, however, only in sIgE sensitized participants. Conclusion Birth year, childhood residence, sIgE sensitization, and allergic predisposition were associated with asthma, AR, and AC prevalence. Individuals with self-reported ARC represent a primarily sIgE-positive phenotype, while those with either AR or AC represent more diverse phenotypes. Background: Allergic rhinitis (AR), allergic conjunctivitis (AC), and asthma composing multiple phenotypes and improved understanding of these phenotypes and their respective risk factors are needed. Objectives: The objective of this study was to define the prevalence of AR, AC, and asthma and their association with allergen-specific immunoglobulin E (sIgE) sensitization in a large cohort of blood donors and identify risk factors. Methods: From the nationwide population-based Danish Blood Donor Study, 52,976 participants completed an electronic questionnaire including AR, AC, asthma, allergic predisposition, and childhood residence. Of these, 25,257 were additionally tested for sIgE to inhalation allergens (Phadiatop). Results: The prevalence of sIgE sensitization, AR, AC, and asthma was 30%, 19%, 15%, and 9%, respectively. The youngest birth cohorts had the highest prevalence of sIgE sensitization and symptoms of asthma, AR, and AC, and for asthma, they apparently experienced symptoms at an earlier age. The sIgE sensitization was positively associated with male sex. The sIgE seroprevalence was higher in participants with both AR and AC (ARC) than in participants with either AR or AC. Allergic predisposition and sIgE sensitization increased the risk of the diseases, while farm upbringing was associated with reduced prevalence of ARC, however, only in sIgE sensitized participants. Conclusion: Birth year, childhood residence, sIgE sensitization, and allergic predisposition were associated with asthma, AR, and AC prevalence. Individuals with self-reported ARC represent a primarily sIgE-positive phenotype, while those with either AR or AC represent more diverse phenotypes.

Supplemental Digital Content is available in the text., Background. In an era where global kidney shortage has pushed the field of transplantation towards using more marginal donors, modified kidney preservation techniques are currently being reviewed. Some techniques require further optimization before implementation in full scale transplantation studies. Using a porcine donation after circulatory death kidney model, we investigated whether initial kidney hemodynamics improved during normothermic machine perfusion if this was preceded by a short period of oxygenated hypothermic machine perfusion (oxHMP) rather than static cold storage (SCS). Methods. Kidneys subjected to 75 minutes of warm ischemia were randomly assigned to either SCS (n = 4) or SCS + oxHMP (n = 4), with a total cold storage time of 240 minutes. Cold preservation was followed by 120 minutes of normothermic machine perfusion with continuous measurement of hemodynamic parameters and renal function. Results. oxHMP preserved kidneys maintained significantly lower renal resistance throughout the normothermic machine perfusion period compared to SCS kidneys (P

BackgroundThe pandemic due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has tremendous consequences for our societies. Knowledge of the seroprevalence of SARS-CoV-2 is needed to accurately monitor the spread of the epidemic and also to calculate the infection fatality rate (IFR). These measures may help the authorities to make informed decisions and adjust the current societal interventions. Blood donors comprise approximately 4.7% of the similarly aged population of Denmark and blood is donated in all areas of the country. The objective of this study was to perform real-time seroprevalence surveying among blood donors as a tool to estimate previous SARS-CoV-2 infections and the population based IFR.MethodsAll Danish blood donors aged 17-69 years giving blood April 6 to 17 were tested for SARS-CoV-2 immunoglobulin M and G antibodies using a commercial lateral flow test. Antibody status was compared between areas and an estimate of the IFR was calculated. The seroprevalence was adjusted for assay sensitivity and specificity taking the uncertainties of the test validation into account when reporting the 95% confidence intervals (CI).ResultsThe first 9,496 blood donors were tested and a combined adjusted seroprevalence of 1.7% (CI: 0.9-2.3) was calculated. The seroprevalence differed across areas. Using available data on fatalities and population numbers a combined IFR in patients younger than 70 is estimated at 82 per 100,000 (CI: 59-154) infections.ConclusionsThe IFR was estimated to be slightly lower than previously reported from other countries not using seroprevalence data. The IFR, including only individuals with no comorbidity, is likely several fold lower than the current estimate. This may have implications for risk mitigation. We have initiated real-time nationwide anti-SARS-CoV-2 seroprevalence surveying of blood donations as a tool in monitoring the epidemic.

Identification of risk factors for contracting and developing serious illness following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of paramount interest. Here, we performed a retrospective cohort analysis of all Danish individuals tested for SARS-CoV-2 between 27 February 2020 and 30 July 2020, with a known ABO and RhD blood group, to determine the influence of common blood groups on virus susceptibility. Distribution of blood groups was compared with data from nontested individuals. Participants (29% of whom were male) included 473 654 individuals tested for SARS-CoV-2 using real-time polymerase chain reaction (7422 positive and 466 232 negative) and 2 204 742 nontested individuals, accounting for ∼38% of the total Danish population. Hospitalization and death from COVID-19, age, cardiovascular comorbidities, and job status were also collected for confirmed infected cases. ABO blood groups varied significantly between patients and the reference group, with only 38.41% (95% confidence interval [CI], 37.30-39.50) of the patients belonging to blood group O compared with 41.70% (95% CI, 41.60-41.80) in the controls, corresponding to a relative risk of 0.87 (95% CI, 0.83-0.91) for acquiring COVID-19. This study identifies ABO blood group as a risk factor for SARS-CoV-2 infection but not for hospitalization or death from COVID-19.

Background The renal endothelium is a prime target for ischemia-reperfusion injury (IRI) during donation and transplantation procedures. Mesenchymal stromal cells (MSC) have been shown to ameliorate kidney function after IRI. However, whether this involves repair of the endothelium is not clear. Therefore, our objective is to study potential regenerative effects of MSC on injured endothelial cells and to identify the molecular mechanisms involved. Methods Human umbilical vein endothelial cells (HUVEC) were submitted to hypoxia and reoxygenation and TNF-α treatment. To determine whether physical interaction or soluble factors released by MSC were responsible for the potential regenerative effects of MSC on endothelial cells, dose-response experiments were performed in co-culture and transwell conditions and with secretome-deficient MSC. Results MSC showed increased migration and adhesion to injured HUVEC, mediated by CD29 and CD44 on the MSC membrane. MSC decreased membrane injury marker expression, oxidative stress levels, and monolayer permeability of injured HUVEC, which was observed only when allowing both physical and paracrine interaction between MSC and HUVEC. Furthermore, viable MSC in direct contact with injured HUVEC improved wound healing capacity by 45% and completely restored their angiogenic capacity. In addition, MSC exhibited an increased ability to migrate through an injured HUVEC monolayer compared to non-injured HUVEC in vitro. Conclusions These results show that MSC have regenerative effects on injured HUVEC via a mechanism which requires both physical and paracrine interaction. The identification of specific effector molecules involved in MSC-HUVEC interaction will allow targeted modification of MSC to apply and enhance the therapeutic effects of MSC in IRI.

Background Remote ischaemic conditioning (RIC) is currently being explored as a non-invasive method to attenuate ischaemia/reperfusion injuries in organs. A randomised clinical study (CONTEXT) evaluated the effects of RIC compared to non-RIC controls in human kidney transplants. Methods RIC was induced prior to kidney reperfusion by episodes of obstruction to arterial flow in the leg opposite the transplant using a tourniquet (4 × 5 min). Although RIC did not lead to clinical improvement of transplant outcomes, we explored whether RIC induced molecular changes through precision analysis of CONTEXT recipient plasma and kidney tissue samples by high-resolution tandem mass spectrometry (MS/MS). Results We observed an accumulation of muscle derived proteins and altered amino acid metabolism in kidney tissue proteomes, likely provoked by RIC, which was not reflected in plasma. In addition, MS/MS analysis demonstrated transient upregulation of several acute phase response proteins (SAA1, SAA2, CRP) in plasma, 1 and 5 days post-transplant in RIC and non-RIC conditions with a variable effect on the magnitude of acute inflammation. Conclusions Together, our results indicate sub-clinical systemic and organ-localised effects of RIC.

Objectives This study aimed to characterize the association between HLA alleles and ANCA-associated vasculitis (AAV) in a genetically homogeneous population, and to analyse the contribution of specific HLA molecule amino acid sequences to the risk of AAV. Methods We included 187 Danish patients with AAV and 1070 healthy controls. All were HLA typed at two-field resolution. The association of HLA alleles to PR3- or MPO-AAV was analysed. The contribution of the dominant molecular motifs of the HLA-DPB1 molecule to the risk of AAV was investigated by association studies that included specific amino acid sequences of the hypervariable regions in exon 2. Results Ninety-four percent of patients with PR3-AAV were carriers of HLA-DPB1*04:01 while all patients with PR3-AAV were carriers of an HLA-DPB1*04 allele, and 85% were homozygous. This was significantly more than in the control group (P < 0.0001). The association was even stronger when HLA-DPB1*04:02 and -DPB1*23:01 were included. HLA-DPB1*04:01, -DPB1*04:02 and -DPB1*23:01 share amino acids in positions 8–9, 69, 76 and 84–87 within the hypervariable regions, but only positions 69 and 84–87 contributed significantly to the disease risk. HLA-DRB1*15 was associated with an increased risk of developing PR3-AAV, while HLA-DRB1*04, -DRB1*07 and -DQB1*03 were associated with a reduced risk of kidney involvement in PR3-AAV. MPO-AAV was only weakly associated with HLA class I alleles. Conclusion PR3-AAV is strongly associated with the HLA-DPB1 alleles HLA-DPB1*04:01, -DPB1*04:02 and -DPB1*23:01, which share amino acid sequences crucial for the peptide-binding groove.

Background & Aim Renal endothelium is damaged by ischemia reperfusion injury (IRI) during transplantation as a result of the hypoxic metabolism and the resumption of oxygen supply after reperfusion. It has been shown that mesenchymal stromal cells (MSC) are effective in enhancing kidney function after IRI. However, the cellular targets and mechanisms of action behind MSC effects are still poorly understood. Therefore, our objective was to evaluate the role of MSC in repairing injured endothelium as it is the first tissue with which they interact after infusion and to determine the specific interactions involved in this process. The role of physical communication and cytokine secretion in the reparative action of MSC was studied as well as its molecular mechanisms. Methods, Results & Conclusion Human umbilical vein endothelial cells (HUVEC) were submitted to hypoxic and inflammatory insult (TNF-a). This increased the expression of activation molecules such as CD54, CD62e and HLA-II on HUVEC's cell surface. Consequently, an increase of almost 20% in the adherence of MSC to HUVEC was observed in comparison to untreated HUVEC. Moreover, an increase of around 20% in the migratory capacity of MSC towards injured HUVEC was observed. MSC showed a 15 to 20% increase in their ability to transmigrate through injured HUVEC monolayers. Once MSC were in contact with HUVEC, the oxidative stress of HUVEC decreased as observed by a decrease in reactive oxygen species levels, their proliferative status improved and MSC restored the angiogenic potential to levels close to those from healthy HUVEC. To determine whether physical interaction or soluble factors released by MSC are fundamental for MSC regenerative capacity, a Boyden chamber system experiment was performed and we observed that both were required to achieve the full reparative effect of MSC. Moreover, we observed an increase in the expression of CD44 and CD29 adhesion molecules on MSC membrane after co-culture with injured HUVEC, as well as their ligands CD62e and CD106 on HUVEC membrane. We conclude that MSC are able to repair the injury caused by ischemic and inflammatory injury on HUVEC via a mechanism that requires both physical and paracrine interaction between MSC and HUVEC. We are currently studying the specific effector molecules involved in MSC and HUVEC cell-to-cell interaction which will allow targeted modification to improve the regenerative effects of MSC therapy.

Antibodies of the IgG class to terminal Galα3Gal (IgG anti-αGal) is abundant in human plasma and are reported to bind most sepsis-causing Gram-negative bacteria. However, these seminal findings, made more than two decades ago, have not been reexamined. Our aim was to assess IgG anti-αGal´s pathogen reactivity. We affinity purified IgG anti-αGal from a therapeutic grade normal human IgG pool applying two rounds of positive selection with Galα3Gal-coupled beads and included removal of column matrix reactive antibodies. The purified antibodies were rigorously characterized in terms of specificity and purity in various solid-phase immunoassays. We used flow cytometry to study reactivity against 100 consecutive clinical isolates diagnosed as cause of sepsis in humans. We found that the purified IgG anti-αGal displays high specificity for Galα3Gal. Also, IgG anti-αGal at 5 mg/L bound 56 out of 100 pathogens with predilection for Gram-positive bacteria binding 39 out of 52 strains. We confirm that although IgG anti-αGal comprise a small fraction of the human antibody pool (~0.1%), these antibodies targets an impressively large part of pathogens causing invasive disease.

The regenerative capacities of mesenchymal stromal cells (MSCs) make them suitable for renal regenerative therapy. The most common delivery route of MSC is through intravenous infusion, which is associated with off-target distribution. Renal intra-arterial delivery offers a targeted therapy, but limited knowledge is available regarding the fate of MSCs delivered through this route. Therefore, we studied the efficiency and tissue distribution of MSCs after renal intra-arterial delivery to a porcine renal ischemia-reperfusion model. MSCs were isolated from adipose tissue of healthy male pigs, fluorescently labeled and infused into the renal artery of female pigs. Flow cytometry allowed MSC detection and quantification in tissue and blood. In addition, quantitative polymerase chain reaction was used to trace MSCs by their Y-chromosome. During infusion, a minor number of MSCs left the kidney through the renal vein, and no MSCs were identified in arterial blood. Ischemic and healthy renal tissues were analyzed 30 min and 8 h after infusion, and 1-4 × 104 MSCs per gram of tissue were detected, predominantly, in the renal cortex, with a viability >70%. Confocal microscopy demonstrated mainly glomerular localization of MSCs, but they were also observed in the capillary network around tubuli. The infusion of heat-inactivated (HI) MSCs, which are metabolically inactive, through the renal artery showed that HI-MSCs were distributed in the kidney in a similar manner to regular MSCs, suggesting a passive retention mechanism. Long-term MSC survival was analyzed by Y-chromosome tracing, and demonstrated that a low percentage of the infused MSCs were present in the kidney 14 days after administration, while HI-MSCs were completely undetectable. In conclusion, renal intra-arterial MSC infusion limited off-target engraftment, leading to efficient MSC delivery to the kidney, most of them being cleared within 14 days. MSC retention was independent of the metabolic state of MSC, indicating a passive mechanism.

Metformin is today the first choice treatment for type‐2 diabetes, but has also protective effects in several renal disease models. Previously, we have demonstrated that the protective effects in response to unilateral ureteral obstruction (UUO) are independent of organic cation transporters (OCTs), the transporters responsible for the metformin uptake into the renal cells. The mechanisms behind the renoprotective effects are incompletely understood, but our previous results indicate that the renoprotective effects at least partly could be dependent on actions of metformin outside the renal cells. In this study, we investigate whether the renoprotective effects of metformin can be mediated via systemic immunomodulatory actions. We demonstrated that metformin can affect the immune system in the kidney as well as in the peripheral blood and spleen following UUO. UUO kidneys showed infiltration of immune cells including monocytes, B cells, and T cells, but metformin limited infiltration of all cell types. UUO animals had increased spleen sizes, but this increase was attenuated by metformin. Metformin treatment surprisingly resulted in a higher proportion of monocytes with infiltratory capacity 7 days after UUO. Other studies have suggested that metformin regulates monocyte maturation through signal transducer and activator of transcription 3 (STAT3) activation, as also indicated by our results. In conclusion, our results demonstrate that metformin limits the infiltration of immune cells into the kidney, as well as modulates immune cell composition at a systemic level.

Background: This pilot study aimed to maintain acceptable animal welfare in the development of a porcine autotransplantation model with severe and incremental renal ischemic injury, a model for usage in future intervention studies. Secondary aims were to develop and test methods to collect blood and urine without the need to restrain or use sedative and avoid transportation to optimize welfare of the pig. Methods: Kidneys from 7 female pigs were subjected to incremental durations of warm ischemia (WI) 30, 45, or 75 minutes by left renal artery and vein clamping. After static cold storage, contralateral nephrectomy was performed, and the injured graft was autotransplanted and animals observed for 14 days. Animal welfare was assessed and recorded using a structured scoring sheet before and 4 days after the kidney autotransplantation. Furthermore, blood samples were drawn daily the first week and every second day the following week using a semi-central venous catheter. An ostomy bag around the genitals was tested for urine collection. Measured glomerular filtration rate was calculated using renal clearance of chromium-51-labeled ethylenediamine tetraacetic acid on day 14. Results: None of the 7 animals died during the follow-up. The animal welfare was moderately affected when applying 75 minutes of WI (n = 2), and for that reason WI was not further increased. Pigs with lower WI had no observed welfare issues. With 75 minutes of WI peak, plasma creatinine was 1486 and 1317 µmol/L, reached on day 4. Lowest glomerular filtration rate levels were observed in the pigs with 75 minutes of WI. Conclusions: WI up to 75 minutes caused the intended severely impaired renal function without significantly compromising animal welfare. Blood and urine was collected postoperatively without sedation of the pigs or use of a metabolic cage.

Ischemic preconditioning (IPC) has been protective against ischemia-reperfusion injury (IRI), but the underlying mechanism is poorly understood. We examined whether IPC modulates the early inflammatory response after IRI. Nineteen healthy males participated in a randomised crossover trial with and without IPC before IRI. IPC and IRI were performed by cuff inflation on the forearm. IPC consisted of four cycles of five minutes followed by five minutes of reperfusion. IRI consisted of twenty minutes followed by 15 min of reperfusion. Blood was collected at baseline, 0 min, 85 min and 24 h after IRI. Circulating monocytes, T-cells subsets and dendritic cells together with intracellular activation markers were quantified by flow cytometry. Luminex measured a panel of inflammation-related cytokines in plasma. IRI resulted in dynamic regulations of the measured immune cells and their intracellular activation markers, however IPC did not significantly alter these patterns. Neither IRI nor the IPC protocol significantly affected the levels of inflammatory-related cytokines. In healthy volunteers, it was not possible to detect an effect of the investigated IPC-protocol on early IRI-induced inflammatory responses. This study indicates that protective effects of IPC on IRI is not explained by direct modulation of early inflammatory events.

CD46 is an important immune regulatory receptor with dual functions, however, the CD46 isoform distribution and the effect of CD46 activation on the cytokine production in monocytes and monocyte-derived dendritic cells (moDCs) is unclear. Here, we show that CD46 activation of moDCs downregulates LPS-induced CXCL-10 expression, while the expression of CXCL-10 in monocytes is unaffected. Furthermore, the differentiation of moDCs induces a switch towards dominance of CYT-2 isoforms of CD46. These data indicate that CD46 activation exerts different functions in monocytes and moDCs and this correlates with a switch in CD46 isoform expression upon differentiation of moDCs.

Background: High dose chemotherapy (HDT) burdens patients with a high risk of serious infections while neutropenic. The role of host antibodies as risk factors for infections is unclear. Our aim was to investigate if pre-HDT levels of IgG antibodies against terminal Galα3Gal (anti-αGal) predict infections. Anti-αGal is reported to be the most abundant antibody in human plasma and is able to recognize most sepsis-causing Gram-negative bacteria. The antibody exerts poor complement activation, which promotes pathogen survival by blocking access of more effective complement activators. This may be particularly problematic in neutropenic patients. Thus, we hypothesized that patients with high anti-αGal serum concentrations are particularly prone to suffer infections following HDT. Methods: We conducted a clinical cohort study on patients ≥16 years receiving HDT with autologous stem cell transplantation for myelomatosis (MM) or non-Hodgkin lymphoma (NHL) at Department of Hematology, Aarhus University Hospital, Denmark from 25 November 2009 through 26 June 2015. Of eligible patients (N=308), we excluded previous transplanted (N=14), those without a pre-HDT serum sample available for anti-αGal quantification (N=115), and recipients of plasma transfusion/IgG substitutions within 90 days before the pre-HDT serum sample and until end of follow-up (N=9). All included received prophylactic antimicrobial therapy (levofloxacin and fluconazol) and filgrastim. Serum anti-αGal was quantified using an in-house Time-resolved Immuno-Fluoremetric Assay. We ascertained infectious episodes within 30 days following the date of autologous stem cell re-infusion through review of all medical charts and data retrieval from the laboratory information systems, blinded for anti-αGal concentrations. Infectious episodes were defined as fever (≥38.5°C) or hypothermia ( 21 mg/L preceded by at least 24 hours without any of these. We used a Cox proportional hazards model to investigate the association between anti-αGal concentrations and risk of infection, with adjustment for total plasma IgG concentrations, ABO blood group, comorbidity, and underlying disease (MM or NHL) in the adjusted hazard ratios (HR). Optimum cutoff for dichotomizing was determined from ROC analysis applying Youden´s J statistic and groups were compared by Kaplan-Meier method. Results: We included 170 patients (MM, 55%; males, 58%; median age 62 years). Severe neutropenia was transient in all, with blood neutrophils increasing to above 500/µL on median day 11 (range 8-18). We identified one infectious episode in 103 patients (61%) and two episodes in six (3.5%) patients. The infectious episodes began during severe neutropenia in 92% of the patients. The infection types were fever of unknown origin (FUO) (72%), pneumonia (19%), catheter related (4.3%), typhlitis (1.7%), C. difficile colitis (1.7%), and hypothermia of unknown origin (0.87%). None died from infections. Pre-HDT serum was collected on median day -31 (range -142- -16). Anti-αGal was detectable in all but one sample, averaging 0.51 mg/dL (range: 0.016-12). Overall, anti-αGal concentrations predicted infectious episodes (all types), crude HR 1.2 (95% confidence interval (CI) 1.1-1.4), adjusted HR 1.1 (95% CI: 1.0-1.3). Using anti-αGal at 1.3 mg/dL as cut-off, we identified that patients with higher concentrations (N=41) experienced considerably increased risk of infectious episodes (crude estimates): all types, HR 1.7 (95% CI: 1.3-3.2); FUO, HR 2.0 (95%CI: 1.5-4.5); and pneumonia HR 2.1 (95%CI: 0.83-8.7). Discussion: We found that high anti-αGal serum concentrations predict an increased risk of infection after HDT. These findings support that anti-αGal may critically enhance pathogen-survival in the neutropenic host. Studies of other cohorts and mechanistic studies are required. Our findings may pave the way for future optimized risk stratification and therapeutic measures. Disclosures No relevant conflicts of interest to declare.

BACKGROUND: HIV-2 may slow progression of a subsequently acquired HIV-1 infection through cross-neutralizing antibodies and polyfunctional CD8 T cells. We hypothesized that HIV-1/2 dually infected patients compared with HIV-1-infected patients had more preserved immune maturation subsets and less immune activation of T and B cells. METHODS: ART-naive patients with HIV-1 (n = 83) or HIV-1/2 dual (n = 27) infections were included in this cross-sectional study at an HIV clinic in Guinea-Bissau. Peripheral blood mononuclear cells (PBMCs) were analyzed by flow cytometry according to T-cell maturation and activation, regulatory T-cell fraction, and B-cell maturation and activation. RESULTS: HIV-1/2 dually infected patients had lower levels of HIV-1 RNA compared with patients with HIV-1 infection, but the levels of total HIV RNA (HIV-1 and HIV-2) were similar in the two patient groups. T-cell maturation, and proportions of regulatory T cells (FoxP3+) were also similar in the two groups. HIV-1/2 dually infected patients had higher proportions of CD4 and CD8 T cells positive for the activation marker CD38, but there was no difference in other T-cell activation markers (CD28, CTLA-4, PD-1). HIV-1/2 dually infected patients also had higher proportions of IgM-only B cells and plasmablasts. CONCLUSION: HIV-1/2 was not associated with less immune perturbations than for HIV-1 infection.

The detection of donor-specific antibodies (DSAs) is a cornerstone in the immunological risk assessment prior to organ transplantation. The detection methods have developed rapidly during the last decade, and the evidence for clinical interpretation of results obtained by solid phase immunoassays (SPI) is slowly accumulating. Nevertheless, technical limitations and theoretical concerns still mean that "expert opinions" govern clinical decision-making when results of bead-based arrays are applied in immunological risk assessment prior to transplantation. This article underlines the prognostic value of SPI in the immunized recipient of an organ transplant while cautioning uncritical clinical interpretation of mean fluorescence intensity (MFI) as a quantitative parameter in organ transplantation based on documented as well as theoretical shortcomings of the method. The role of SPI-based detection of anti-HLA antibodies in clinical transplantation diagnostics is summarized and put into perspective of the Sensitization in Transplantation: Assessment of Risk (STAR) working group report 2017.

Common variable immunodeficiency (CVID) is a heterogeneous primary immunodeficiency disease, leading to recurrent bacterial airway infections and often also autoimmune complications. To shed light on the regulatory lymphocytes from these patients, we analyzed the levels of regulatory B (pro-B10) cell and regulatory T (Treg) cell subpopulations in PBMCs from twenty-six patients diagnosed with CVID using multi-color flowcytometry. Pro-B10 cells were induced by 48h in vitro stimulation prior to analysis. Suppressor function was measured on a subset of patients with splenomegaly and autoimmune complications. The levels of regulatory B and T cells were correlated to clinical manifestations, including autoimmunity, splenomegaly and CVID EUROclass subgroups. We demonstrate a significant association between elevated levels of pro-B10 cells, decreased levels of Tregs and autoimmune phenomena in CVID patients. The finding of marked abnormalities in regulatory lymphocyte populations contribute to our understanding of the pathogenesis of CVID and potentially be valuable in the clinical management and treatment of patients.

Background: Enumeration of stem cells is essential in the management of peripheral blood stem cell (PBSC) harvest. An alternative to the gold standard flow cytometric CD34+ stem cell count is the fully automated hematopoietic stem cell (HPC) count on the Sysmex XN hematology analyzer. Materials and methods: Eighty-nine patients and healthy stem cell donors who underwent PBSC harvest were included in the study. Stem cells were enumerated in pre-harvest peripheral blood and the apheresis yield by both flow cytometric CD34+ stem cell enumeration and by the Sysmex XN HPC count. Results: The Sysmex HPC concentration overestimated the CD34+ stem cell concentration by a ratio of 1.3 in average. The agreement between the two methods was poor at concentration 43.5 HPC/μL, where PBSC harvest can be initiated. And a negative cut off

Objectives: HIV-2 is endemic in West Africa and is characterized by lower transmissibility because of lower viral load, and HIV-2-infected persons usually have a slower progression to AIDS. The mechanisms behind the slower disease progression are unknown. The main objective was to identify specific HLA class I and II alleles that may influence the disease progression of HIV-2 infection. Design: Cohort follow-up study. Methods: We used high-resolution HLA typing of DNA from 437 antiretroviral naive HIV-2-infected patients from the Bissau HIV Cohort, Guinea-Bissau, to identify HLA alleles with an influence on HIV-2 disease progression. The effect of HLA-type on viral load and CD4 + cell count was assessed initially by ranksum-test and t-test, followed by adjusted logistic regression and multivariable linear regression analysis, respectively. Results: Three alleles (HLA-B ∗ 58:01, HLA-DPB1 ∗ 10:01 and HLA-DRB1 ∗ 11:01) were associated with lower possibility of detectable baseline plasma viral load (P = 0.002, P = 0.044 and P = 0.033, respectively), and no alleles were associated with higher possibility of detectable plasma viral load. HLA-DPB1 ∗ 10:01 and HLA-DRB1 ∗ 11:01 were in linkage disequilibrium (P = 0.047). Patients with heterozygous HLA types in all their HLA class I loci or in one or two loci were not more likely to have undetectable viral load compared with patients that were homozygous in all their class I loci after adjusting for sex and CD4 + cell count (P = 0.93 and P = 0.88, respectively). Conclusion: The three alleles HLA-B ∗ 58:01, HLA-DPB1 ∗ 10:01 and HLA-DRB1 ∗ 11:01 may protect against HIV-2 disease progression towards AIDS.

Background Several frequently used prescription drugs may affect bleeding risk. We investigated use of aspirin, nonsteroidal anti-inflammatory drugs (NSAIDs), selective serotonin reuptake inhibitors (SSRIs), and statins and risk of postoperative red blood cell transfusion in breast cancer patients. Methods Using Danish population-based registries, we identified a cohort of women who underwent surgery for primary breast cancer (n = 22,238) during 2005–2012 and ascertained their use of aspirin, NSAIDs, SSRIs, and statins. For each drug, patients were categorized as users if they filled ≥1 prescription in the 60 days prior to surgery. We calculated the 14-day risk of red blood cell transfusion and relative risks (RRs) with 95% confidence intervals (CIs), comparing users with nonusers for each drug and adjusting for age, cancer stage, and Charlson Comorbidity Index score. Results In our cohort, 1385 (6.2%) women were aspirin users, 1794 (8.0%) were NSAID users, 1110 (4.9%) were SSRI users, and 2053 (9.1%) were statin users. The overall risk of red blood cell transfusion was 1.3%. The 14-day risk of RBC transfusion was 3.5% among aspirin users versus 1.1% among aspirin nonusers (adjusted RR = 1.9, 95% CI: 1.4–2.7), and 1.8% among SSRI users versus 1.2% among SSRI nonusers (adjusted RR = 1.2, 95% CI: 0.7–1.9). Red blood cell transfusion risk was increased among NSAID users, but not in a sensitivity analysis with a 30-day exposure window. Red blood cell transfusion risk was not increased among SSRI and statin users. Conclusions Primary breast cancer surgery confers a low risk of RBC transfusion. Still, use of aspirin and possibly NSAIDs, but not SSRIs and statins, is associated with increased red blood cell transfusion. This increased risk is not explained by differences in age, stage, or comorbidity level. Electronic supplementary material The online version of this article (doi:10.1186/s13058-017-0926-2) contains supplementary material, which is available to authorized users.

BACKGROUND: It is well known that obesity complicates the course of several diseases. However, it is unknown whether obesity affects the risk of infection among healthy individuals.METHODS: We included 37,808 healthy participants from the Danish Blood Donor Study, who completed a questionnaire on health-related items. Obesity was defined as a body mass index ≥ 30 kg/m. Infections among participants were identified by relevant ICD-10 codes in the Danish National Patient Register and Anatomical Therapeutic Chemical (ATC) codes in the Danish Prescription Register. Multivariable Cox proportional hazards analysis with age as the underlying timescale was used as the statistical model.RESULTS: During 113,717 person-years of observation, 1,233 participants were treated for infection at a hospital. Similarly, during 58,411 person-years of observation, 15,856 participants filled at least one prescription of antimicrobials. Obesity was associated with risk of hospital-based treatment for infection (women: hazard ratio [HR] = 1.5, 95% confidence interval [CI] = 1.1, 1.9; men: HR = 1.5, 95% CI = 1.2, 1.9). For specific infections, obesity was associated with increased risk of abscesses (both sexes), infections of the skin and subcutaneous tissue (men), and respiratory tract infections and cystitis (women). Similarly, obesity was associated with filled prescriptions of antimicrobials overall (women: HR = 1.22, 95% CI = 1.14, 1.30; men: HR = 1.23, 95% CI: 1.15, 1.33) and particularly with phenoxymethylpenicillin, macrolides, dicloxacillin and flucloxacillin, and broad-spectrum penicillins.CONCLUSIONS: In a large cohort of healthy individuals, obesity was associated with risk of infection. This result warrants further studies of metabolism and the immune response.

Influenza can cause significant morbidity and mortality in renal transplant recipients especially with a high rate of lower respiratory disease. Annual influenza vaccination is therefore recommended to renal transplant recipients. We report the first three cases of acute kidney injury in renal transplant recipients following influenza vaccination that all led to graft loss. They all had different native diseases and were all vaccinated in the same season of 2009-10. The time span from vaccination to decline of kidney function is shorter than the time to diagnosis since the three patients only had blood tests every 3 months or when symptoms became severe. These reports do not justify a change of current recommendations regarding influenza vaccination in renal transplant recipients, but they support the continued attention and registration of vaccinations to monitor side effects.

There is great demand for transplant kidneys for the treatment of end-stage kidney disease patients. To expand the donor pool, organs from older and comorbid brain death donors, so-called expanded criteria donors (ECD), as well as donation after circulatory death donors, are considered for transplantation. However, the quality of these organs may be inferior to standard donor organs. A major issue affecting graft function and survival is ischemia/reperfusion injury, which particularly affects kidneys from deceased donors. The development of hypothermic machine perfusion has been introduced in kidney transplantation as a preservation technique and has improved outcomes in ECD and marginal organs compared to static cold storage. Normothermic machine perfusion (NMP) is the most recent evolution of perfusion technology and allows assessment of the donor organ before transplantation. The possibility to control the content of the perfusion fluid offers opportunities for damage control and reparative therapies during machine perfusion. Mesenchymal stromal cells (MSC) have been demonstrated to possess potent regenerative properties via the release of paracrine effectors. The combination of NMP and MSC administration at the same time is a promising procedure in the field of transplantation. Therefore, the MePEP consortium has been created to study this novel modality of treatment in preparation for human trials. MePEP aims to assess the therapeutic effects of MSC administered ex vivo by NMP in the mechanisms of injury and repair in a porcine kidney autotransplantation model.

INTRODUCTION: Live peripheral blood mononuclear cells (PBMCs) can be frozen and thawed for later analyses by adding and removing a cryoprotectant, such as dimethyl sulfoxide (DMSO). Laboratories across the world use various procedures, but published evidence of optimal thawing procedures is scarce.MATERIALS AND METHODS: PBMCs were separated from blood collected from healthy Danish blood donors, and stored at -80°C after adding of DMSO. The essential steps in the thawing procedure were modified and performance was evaluated by flow cytometry with respect to the percentage and total yield of viable PMBCs.RESULTS: The best-performing washing medium was Roswell Park Memorial Institute (RPMI) 1640 at 37°C with 20% fetal bovine serum. When using 10 mL washing medium in a 15-mL Falcon tube, samples should be centrifuged for at least 10 minutes at 500 g. We failed to detect any differences between the tested methods of mixing PBMCs with washing medium. Likewise, neither the thawing duration nor centrifugation temperature (20°C and 37°C) had any effect. PBMCs could be incubated (rested) for up to eight hours in a 37°C 5% CO2 incubator without affecting cell counts, but incubating PBMCs for 16 hours significantly decreased viability and recovery. In general, high viability was not necessarily associated with high recovery.CONCLUSION: Changing the thawing procedure significantly impacted PBMC viability and live cell recovery. Evaluating both viability and live PBMC recovery are necessary to evaluate method performance. Investigation of differential loss of PBMC subtypes and phenotypic changes during thawing and incubation requires further evaluation.

PURPOSE: Endothelial progenitor cells (EPCs) represent a heterogeneous cell population that is believed to be involved in vasculogenesis after ischemic diseases. EPCs could have a potential for future therapies with the purpose of enhancing endothelial repair. However, due to the low amount of these cells in circulation they have to be expanded in vitro before administration into recipients. The purpose of this study was to analyse and evaluate possible changes in morphology and functionality as a result of in vitro ageing of a subtype of EPCs called endothelial outgrowth cells (EOCs), since such changes might compromise the cells' ability to participate in vasculogenesis.METHODS: EOCs were isolated and grown from human umbilical cord blood using two methodologies with varying degree of cell purification. The changes between the two culture setups and the changes occurring in EOCs over time were traced by flow cytometry and assays for growth, tube formation, and beta-galactosidase production.RESULTS: After initial expansion of EOCs from two different setups, using varying degree of cell purification, the cells showed to be indistinguishable from each other during the first weeks of culture. The cells showed a changed morphology with bigger and more granular cells and the growth rate decreased with time. The cells also showed an increased Beta-galactosidase expression and decreased tube formation ability in late passage EOCs.CONCLUSION: Our data indicates that EOCs undergo senescence during long-term expansion and therefore time for cell harvest has to be validated in order to achieve functional cells still maintaining a therapeutic potential. A possible application in large animal or humans could be local injection of EOCs into affected areas and thereby reducing the need for long-term expansion of the cells.

Background Venous thromboembolism (VTE) in patients with upper gastrointestinal (GI) cancer increases morbidity and mortality. This study aimed to determine the prevalence of VTE at diagnosis of upper GI cancer. Methods Patients admitted between February 2008 and February 2011 with upper GI cancer (pancreatic, extrahepatic biliary, lower oesophageal, gastro-oesophageal junction or gastric cancer) were investigated in a cross-sectional cohort study. At cancer diagnosis, all patients were examined for deep vein thrombosis (DVT) by means of bilateral compression ultrasonography. From February 2009 and onwards, computed tomographic pulmonary angiography (CTPA) was also performed for the diagnosis of pulmonary embolism (PE). Results Some 250 patients had ultrasonography; CTPA was performed in 143 patients on admission. DVT was detected in 13 (5·2 per cent) of the 250 patients, eight (3·2 per cent) of whom were asymptomatic. DVT was correlated with tumour location in the pancreaticobiliary tract (odds ratio (OR) 6·27, 95 per cent confidence interval 1·18 to 33·38; P = 0·031) and tumour stage IV (OR 19·34, 2·33 to 160·70; P = 0·006). PE was detected in 11 (7·7 per cent) of 143 patients, eight (5·6 per cent) of whom were asymptomatic. PE embolism was also significantly more common in patients with pancreaticobiliary tract cancer (OR 7·81, 1·28 to 47·62; P = 0·026) and in those with stage IV disease (OR 17·19, 1·83 to 161·50; P = 0·013). Conclusion The prevalence of VTE at cancer diagnosis was significantly higher in patients with pancreaticobiliary tract cancer than in those with other forms of upper GI cancer, and in patients with advanced cancer stage.

Partial defects in interferon (IFN)-γ signaling lead to susceptibility to infections with nontuberculous mycobacteria. The receptors for IFN-α and IFN-γ activate components of the Janus kinase-signal transducer and activator of transcription (STAT) signaling pathway. Some defects in STAT1 mainly affect IFN-γ signaling, thus resulting in mendelian susceptibility to mycobacterial disease (MSMD). MSMD is a severe disease but patients show a favorable response to anti-mycobacterial chemotherapy. Other defects in STAT1 affect both IFN-α and IFN-γ signaling resulting in mycobacterial and lethal viral disease. We report here a patient with two novel STAT1 alleles, which in combination results in a recessive trait with partial STAT1 deficiency and mycobacterial disease. Cells from the patient did respond to mycobacterial antigen, but both the expression of STAT1 and phosphorylation of STAT1 in response to IFN-γ treatment were reduced. This is the first report of a mutation in the N-terminal part of STAT1 involved in causing mycobacterial disease.

Mesenchymal stem cells (MSCs) are of great interest due to their properties of immune modulation, tissue regeneration, and multipotent differentiation. Future developments of clinical applications, however, require a higher yield of MSCs, lower number of passages of cells in culture, and shorter time from harvest to use. Optimization and standardization of techniques for mesenchymal adipose tissue-derived stem cell isolation offers solutions to current bottlenecks as a larger amount of MSCs can be isolated. These improvements result in shorter expansion time, fewer passages, less donor material needed, and higher MSC yield. This paper describes an MSC isolation method combining enzymatic digestion with mechanic disruption. This protocol is a standardized and easy-to-implement method for reaching significantly higher MSC yields compared to conventional enzymatic isolation protocols. Based on the results presented, we hypothesize that the combined enzymatic and mechanical method increases the surface area of the adipose tissue, facilitating digestion by enzymes. This approach reduces the amount of adipose tissue and in vitro expansion time needed to reach sufficient amounts of MSCs for clinical purposes. Importantly, the method does not require increased amounts of collagenase, nor does it impair the viability or differentiability of the MSCs. Using this protocol increases MSC yield by a factor of three. As a consequence, these results indicate that the physiological concentration of MSCs in adipose tissue is higher than previously assumed. © 2018 by John Wiley & Sons, Inc.

Interleukin-6 (IL-6) is a potent stimulator of the hepatic synthesis of acute-phase proteins. 125I-labelled IL-6 disappeared from the blood of rats with an overall half-time of about 1.5 min; 41% of the injected tracer dose was recovered in the liver by 15 min. The clearance was biphasic. The simultaneous injection of tracer and an excess of unlabelled IL-6 eliminated the initial rapid phase, and reduced the hepatic uptake to 14%. Light microscopic autoradiography showed 5% of the grains over non-hepatocytes, and 80% over hepatocytes, accumulating in areas around the bile canaliculi. Thereafter, degradation products accumulated in the bile. At 4 degrees C, isolated rat hepatocytes bound IL-6 with an apparent Kd of 39 pmol l-1 to a uniform class of 4500 receptors per cell with an apparent molar mass of 115-120 kg mol-1. The HepG2 human hepatocellular cell line bound IL-6 with an apparent Kd of 21 pmol l-1 to a uniform class of 1200 receptors per cell with an apparent molar mass of 155-160 kg mol-1. At 37 degrees C, both cell types endocytosed the bound ligand slowly, and degradation products appeared in the medium after a relatively long lag period (40 min in hepatocytes and 1 h in HepG2 cells).

The effect of low-dose methotrexate (MTX) treatment on the CD26 density on circulating monocytes and CD4(+) T lymphocytes or levels of soluble CD26 (sCD26) has not yet been described in rheumatoid arthritis (RA). While CD26 in T lymphocytes is involved in the activation and proliferation of T lymphocytes, little is known of the role of CD26 in monocytes as it has only recently been localized to monocytes. We analysed the CD26 density by flow cytometry and levels of sCD26 in plasma before initiation of MTX treatment and 12 weeks later. This was done on 34 RA patients fulfilling the 1987 American College of Rheumatology (ACR) criteria followed for 16 weeks after starting MTX treatment. CD26 density on monocytes was increased in RA patients compared with healthy controls before MTX treatment (P < 0.01). After 12 weeks of MTX treatment, the CD26 density on monocytes decreased significantly in the ACR-50% group (P = 0.03), but not in the ACR-20% and the non-responder group (P = 0.15 and 0.87). The increased CD26 density on CD4(+) T lymphocytes (P < 0.01) was unaffected by the reduction in disease activity in relation to MTX treatment. The percentage of monocytes and CD4(+) T lymphocytes among peripheral blood circulating mononuclear cells did not change during MTX treatment. No effect of MTX treatment was observed on the plasma levels of sCD26. Active chronic RA is characterized by enhanced CD26 density on circulating monocytes and CD4(+) T lymphocytes. MTX treatment decreased CD26 density on monocytes in the ACR-50% responder group and was associated with decreased disease activity. The enhanced CD26 density on CD4(+) T lymphocytes was uninfluenced by MTX treatment.

BACKGROUND AND PURPOSE: The C-C chemokine receptor 5-Δ32 deletion (CCR5-Δ32) has been associated with lower levels of C-reactive protein (CRP), but the effect on cardiovascular diseases is uncertain. This study addresses the impact of CCR5-Δ32 on the risk of low-grade inflammation and hospitalization with cardiovascular diseases in a large cohort of blood donors.METHODS: Genotyping of 15,206 healthy participants from The Danish Blood Donor Study for CCR5-Δ32 was performed and combined with CRP measurements and questionnaire data. Cardiovascular disease diagnoses were identified by ICD-10 codes in the Danish National Patient Registry.RESULTS: CCR5-Δ32-carriers had a higher risk of hospitalization for cardiovascular diseases when compared with wild-type homozygotes (hazard ratio = 1.35, 95%-confidence interval: 1.00-1.87). CRP levels were unaffected by the CCR5-Δ32 deletion.CONCLUSION: In this cohort, carriers of the CCR5-Δ32 deletion had normal CRP levels but a borderline significant increased risk of cardiovascular diseases.

INTRODUCTION: Prospective studies of chemotherapy-associated VTE in cancer patients undergoing neoadjuvant chemotherapy in combination with curative intended surgery have not been reported for upper gastrointestinal cancer. In this clinical prospective study, we sought to estimate the incidence of VTE in esophagogastric cancer (OEC) patients scheduled for a specific perioperative chemotherapy regime: oxaliplatin, capecitabine, and epirubicin, (EXE) and curative intended surgery.MATERIAL AND METHODS: A total of 129 consecutive OEC patients were examined using state-of-the-art bilateral compression ultrasound (biCUS) for deep vein thrombosis (DVT) before undergoing preoperative chemotherapy, surgery, and postoperative chemotherapy. In addition 79 were also consecutively scanned at baseline for pulmonary embolism (PE) using state-of-the-art computer tomography pulmonary angiography (CTPA).RESULTS: There were 21 VTE cases throughout the course of treatment (16%, 95% confidence interval [95% CI]: 10 - 24%) among the patients examined using both biCUS and CTPA. Fourteen of 21 VTE was incidental (68%, 95% CI: 43 -85) and 7 VTE events was symptomatic (33%, 15 - 57). The median overall survival was 18months (95% CI: 13 - 24) in patients without any VTE and 14months (95% CI: 7 -30, P = 0.820) in patients with VTE. The cancer stage (adjusted odds ratio [OR]: 5.2, 95% CI: 1 - 21, p=0.002) and gastric cancer (OR 6.4, 95% CI: 2 - 21, P = 0.002) was a significant predictor of VTE.CONCLUSION: The incidence of VTE in patients undergoing EXE neoadjuvant chemotherapy was high, particularly among patients with initial stage III and IV cancers. In addition, a substantial number of chemotherapy-related VTE cases were asymptomatic.

IL-10 is a multifunctional cytokine with known inhibitory effect on DC maturation. In the present study, we show that the presence of IL-10 during the first 6 days of maturation of Mo-DC has severe effects on the phenotype with IL-10 treated Mo-DC retaining a high expression of factors involved in antigen uptake and having low expression of costimulatory factors and MHC II. The effect of IL-10 on DC maturation is further characterized by a comprehensive investigation of the expression profile of 12500 genes using GeneArrays and comparing the data for IL-10 treated and untreated Mo-DC at day 6 and 8 with monocytes and macrophages. The data is confirmed with semi-quantitative RT-PCR. We find that the transcriptional pattern of IL-10 treated Mo-DC at day 6 and day 8 is distinct from both untreated Mo-DC and macrophages. IL-10 induces the expression of genes coding for CCL18, ILT3, ILT4 and TGFβ while inhibiting the expression of IL-12 p40 mRNA, and genes coding for CCL17, CCL19 and CCL22 suggesting a likely effect of IL-10 on Mo-DC maturation to be the generation of tolerogenic Mo-DC.

Pericellular proteolysis initiated by receptor-bound urokinase-type plasminogen activator (uPA) is considered important for directed migration of granulocytes to inflammatory sites. Using flow cytometry and whole-cell binding of radiolabelled-uPA, we found a high level of uPA-receptor (uPAR) expression in granulocytes (3.9 x 104 +/- 0.9 x 104 sites/cell). Modulation of uPAR expression was assessed in the presence of chemoattractant gradients. Our findings demonstrate that interleukin (IL)-8, leukotriene B4(LTB4) and formyl-methionyl-leucyl-phenylalanine (f MLP) caused a dose-dependent upregulation of uPAR on granulocytes in healthy controls. Modulation of uPAR expression is known to regulate chemotactic response. As determined by flow cytometry, uPAR expression by granulocytes from human immunodeficiency virus (HIV)-infected patients was distinctly lower than that of healthy control cells (P < 0.001). However, upregulation of uPAR in response to chemoattractants was similar to that observed in healthy controls. In HIV-infected patients, the uPAR expression on granulocytes correlated (P < 0.001, n = 10) with the number of CD4+ blood cells. In contrast, the expression of IL-8 receptor, CD11b, CD18 and CD62 was not significantly altered in HIV-patients compared with healthy controls.

INTRODUCTION: Endothelial progenitor cells (EPCs) has been reported to have the potential for advancing revascularization of ischemic tissue. However, the heterogeneous nature of these cells calls for specification of the angiogenic potential of each subtype. The purpose of this study was to gain additional insight on the homing capacity of the EPC subtype, endothelial outgrowth cells (EOCs) in tumours using a well-established tumour model.METHODS: (111)Indium ((111)In) - and 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) labelled EOCs derived from human umbilical cord blood were injected into mice with a C3H mammary carcinoma foot tumour. The subsequent capture of the EOCs was traced by estimation of activity in individual organs, autoradiography and fluorescence microscopy.RESULTS: (111)In activity was found in tumour and other organs. However, varying parts of the activity originated from free (111)In lost from EOCs. Autoradiography demonstrated accumulation of (111)In activity in the tumour rim. Microscopy proved that a least part of this radioactivity originated from the presence of human derived EOCs and that those EOCs were not located in the endothelial lining of vessels, in the tumour.CONCLUSION: The results demonstrated the presence of xenotransplanted EOCs in the rim of a C3H mammary carcinoma. They were, however, not located in the endothelial lining of the vessels, thus indicating that their effect in vasculogenesis might be mediated via paracrine mechanisms rather than differentiating into endothelial cells (ECs) in tumour vessels.