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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.

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 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.

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.

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.

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.

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.

BackgroundC-reactive protein (CRP) is a well-established marker of inflammation. The level of CRP is affected by several lifestyle factors. A slightly increased CRP level, also known as low-grade inflammation (LGI), is associated with increased risk of several diseases, especially cardiovascular disease. The aim of this study was to identify predictors of increased CRP levels in healthy individuals. We therefore assessed CRP in a large cohort of blood donors.MethodsWe measured plasma CRP levels in 15,684 participants from the Danish Blood Donor Study. CRP was measured by a commercial assay. Furthermore, all participants completed a standard questionnaire on smoking status, alcohol consumption, physical activity, diet, and various body measurements. Female participants also reported the use of contraception, childbirth, and menopausal status. The relationship between LGI (defined here as a plasma CRP level between 3 mg/L and 10 mg/L) and predictors was explored by multivariable logistic regression analysis. Results were presented as odds ratios (OR) with 95% confidence intervals (CI).ResultsWe found LGI in a total of 1,561 (10.0%) participants. LGI was more frequent in women using combined oral contraception (OC) (29.9%) than in men (6.1%) and women not using OC (7.9%). Among premenopausal women, OC was the strongest predictor of LGI (odds ratio = 8.98, pConclusionHigh BMI and abdominal obesity strongly predicted LGI among healthy individuals. However, the most striking finding was the high prevalence of LGI among premenopausal women who used combined oral contraception. Although the significance of CRP as a marker of inflammation is well known, the role of CRP in pathogenesis is still uncertain. The impact of oral contraception on CRP levels should nevertheless be considered when CRP is used in risk assessment.

A total of 11 HIV-1 positive patients, with CD4+ cell counts between 200 and 500/microl, who were in stable anti-retroviral therapy, were treated with subcutaneous recombinant human IL-2 thrice weekly administered on an out-patient basis in a dose-escalating manner. Subcutaneous IL-2 was well tolerated and associated with only mild to moderate constitutional symptoms and local inflammation at the injection site. CD4+ cell count increased from 404 +/- 48/microl at baseline to 639 +/- 88/microl at week 6, with proportionate increases in naive cells and memory cells. Increased doses of IL-2 were then needed to sustain the number of CD4+ cells. After discontinuation of IL-2 treatment, CD4+ cell count returned to baseline levels. IL-2 induced a reduction in the percentage of CD8+ CD38+ and CD8+ HLA-DR+ cells, an increase in the fraction of CD8+ CD25+ and CD8+ CD122+, and an elevation in the number of NK-cells. IL-2 did not induce any clinically significant change in plasma HIV-RNA. In conclusion, IL-2 can safely be administered subcutaneously on an out-patient basis to HIV-infected individuals with CD4+ cell counts from 200/microl to 500/microl and with some improvement in immunological abnormalities. Continuous therapy, however, seems to result in the development of tachyphylaxia.

Background. Posttransplant lymphoproliferative disorder (PTLD) is a life-threatening complication of transplantation, which comprises a morphologically and clinically heterogeneous spectrum of B-lymphocyte diseases. Risk factors include primary or reactivated Epstein-Barr virus (EBV) infection, and the type and duration of immunosuppression. Interleukin-10 (IL-10) is a pleiotropic cytokine, produced primarily by T-helper 2 (Th2) lymphocytes in the later stages of T-cell activation, suggested to play a role in EBV-associated PTLD. We recently reported preliminary findings on IL-10 in relation to the development of PTLD in three kidney transplanted patients. The study now includes nine patients that could be followed before and/or after the occurrence of lymphoma. Methods. Nine patients with lymphomas (eight PTLDs and one Hodgkin's disease) were diagnosed among 268 consecutive renal transplantations (1990-1997). All were treated with cyclosporine with an initial 10-day course of antilymphocyte globulin, supplemented from 1995 with mycophenolate mofetil. Serum antibodies against EBV were detected using recombinant antigens. A double sandwich enzyme-linked immunosorbent assay using rabbit antibodies to purified human recombinant IL-10 was employed; the assay is specific for human natural and viral IL-10. Results. Three patients experienced primary EBV infection, five reactivated EBV infections, and one did not change EBV status. Three patients had a fulminant course and died with EBV-associated PTLD confirmed post mortem. The other six are alive and are apparently cured. Treatment was immediate discontinuation of immunosuppression (in all PTLDs) and long-term high-dose aciclovir in all but one. Two patients have maintained excellent graft function for 3 and 2 years, respectively, without immunosuppression and are now in a state of operational tolerance. In three of four cases with initial lymphoma, EBV infection (primary or reactivation) preceded the increase in IL-10. In all four cases, the IL-10 increase preceded the PTLD diagnosis. In six cases, IL-10 could be followed after treatment showing either immediate zero or a decrease to zero. Conclusion. IL-10 seems to play a role in EBV-associated PTLD. Moreover, IL-10 may have an important role in transplant tolerance by inducing long-lasting anergy to donor- and host-specific alloantigens, perhaps caused by down-regulation of Th1 cytokines in the graft. If substantiated, this may provide new insight into the pathogenesis of PTLD introducing new strategies for prevention and therapy of PTLD, and for the induction of tolerance in transplanted patients.

Dipeptidyl peptidase IV (DPPIV; CD26) is a 110‐kDa cell surface glycoprotein of T lymphocytes isolated from synovial fluid and peripheral blood in rheumatoid arthritis (RA) [1], and so far there ar...

OBJECTIVES: The aim of this study was to measure, in early rheumatoid arthritis (RA) patients, the concentration of CC-chemokine ligand 19 (CCL19) in plasma and the cell-surface expression of CC-chemokine receptor 7 (CCR7) on circulating monocytes and CD4+ T lymphocytes and to analyse correlations with disease activity and 5-year radiographic progression.METHOD: In disease-modifying anti-rheumatic drug (DMARD)-naïve RA patients (disease duration < 6 months), we measured plasma CCL19 by enzyme-linked immunosorbent assay (ELISA) (n = 160) and CCR7 cell-surface expression on monocytes and CD4+ T lymphocytes by flow cytometry (n = 40) at baseline and after 1 year of treatment with methotrexate (MTX) or methotrexate+cyclosporin A (MTX/CyA). Radiographic progression was scored by the van der Heijde-modified Total Sharp Score (TSS) from 0 to 5 years.RESULTS: Increased baseline CCL19 (median 85 pg/mL, range 31-1008 pg/mL, p = 0.01) decreased after 1 year (median 31 pg/mL, range 31-1030 pg/mL, p < 0.001) and 5 years (median 31 pg/mL, range 31-247 pg/mL, p < 0.001) to a level below the controls (n = 45) (median 60 pg/mL, range 31-152 pg/mL). Baseline plasma CCL19 levels [p = 0.011, 95% confidence interval (CI) 0.0030-0.0176], anti-cyclic citrullinated peptide (anti-CCP) antibody status (p = 0.002, 95% CI 0.61-2.38), and TSS > 0 at baseline (p < 0.001, 95% CI 1.21-3.16) were independent predictors of 5-year radiographic progression evaluated by multiple logistic regression in contrast to never smoked, C-reactive protein (CRP), gender, age, number of tender (NTJ) and swollen joints (NSJ), and 28-joint Disease Activity Score (DAS28). Increased CCR7 expression on monocytes (p = 0.008) correlated to CRP (p = 0.006, r = 0.52) and normalized (n = 15) after 1 year (p = 0.02).CONCLUSIONS: In DMARD-naïve RA patients, CCL19 plasma level and CCR7 surface expression on monocytes were upregulated and normalized after 1 year of treatment. Increased baseline plasma CCL19 level, anti-CCP antibody status, and TSS > 0 at baseline correlated independently with 5-year radiographic progression. OBJECTIVES: The aim of this study was to measure, in early rheumatoid arthritis (RA) patients, the concentration of CC-chemokine ligand 19 (CCL19) in plasma and the cell-surface expression of CC-chemokine receptor 7 (CCR7) on circulating monocytes and CD4+ T lymphocytes and to analyse correlations with disease activity and 5-year radiographic progression.METHOD: In disease-modifying anti-rheumatic drug (DMARD)-naïve RA patients (disease duration < 6 months), we measured plasma CCL19 by enzyme-linked immunosorbent assay (ELISA) (n = 160) and CCR7 cell-surface expression on monocytes and CD4+ T lymphocytes by flow cytometry (n = 40) at baseline and after 1 year of treatment with methotrexate (MTX) or methotrexate+cyclosporin A (MTX/CyA). Radiographic progression was scored by the van der Heijde-modified Total Sharp Score (TSS) from 0 to 5 years.RESULTS: Increased baseline CCL19 (median 85 pg/mL, range 31-1008 pg/mL, p = 0.01) decreased after 1 year (median 31 pg/mL, range 31-1030 pg/mL, p < 0.001) and 5 years (median 31 pg/mL, range 31-247 pg/mL, p < 0.001) to a level below the controls (n = 45) (median 60 pg/mL, range 31-152 pg/mL). Baseline plasma CCL19 levels [p = 0.011, 95% confidence interval (CI) 0.0030-0.0176], anti-cyclic citrullinated peptide (anti-CCP) antibody status (p = 0.002, 95% CI 0.61-2.38), and TSS > 0 at baseline (p < 0.001, 95% CI 1.21-3.16) were independent predictors of 5-year radiographic progression evaluated by multiple logistic regression in contrast to never smoked, C-reactive protein (CRP), gender, age, number of tender (NTJ) and swollen joints (NSJ), and 28-joint Disease Activity Score (DAS28). Increased CCR7 expression on monocytes (p = 0.008) correlated to CRP (p = 0.006, r = 0.52) and normalized (n = 15) after 1 year (p = 0.02).CONCLUSIONS: In DMARD-naïve RA patients, CCL19 plasma level and CCR7 surface expression on monocytes were upregulated and normalized after 1 year of treatment. Increased baseline plasma CCL19 level, anti-CCP antibody status, and TSS > 0 at baseline correlated independently with 5-year radiographic progression.

The vascular disrupting agent combretastatin A-4 disodium phosphate (CA4P) induces fluctuations in peripheral blood neutrophil concentration. Because neutrophils have the potential to induce both vascular damage and angiogenesis we analyzed neutrophil involvement in the anti-tumoral effects of CA4P in C3H mammary carcinomas in CDF1 mice and in SCCVII squamous cell carcinomas in C3H/HeN mice. Flow cytometry analyses of peripheral blood before and up to 144 h after CA4P administration (25 and 250 mg/kg) revealed a decrease 1 h after treatment, followed by an early (3-6 h) and a late (>72 h) increase in the granulocyte concentration. We suggest that the early increase (3-6 h) in granulocyte concentration was caused by the initial decrease at 1 h and found that the late increase was associated with tumor size, and hence independent of CA4P. No alterations in neutrophil infiltration into the C3H tumor after CA4P treatment (25 and 250 mg/kg) were found. Correspondingly, neutrophil depletion in vivo, using an anti-neutrophil antibody, followed by CA4P treatment (25 mg/kg) did not increase the necrotic fraction in C3H tumors significantly. However, by increasing the CA4P dose to 250 mg/kg we found a significant increase of 359% in necrotic fraction when compared to neutrophil-depleted mice; in mice with no neutrophil depletion CA4P induced an 89% change indicating that the presence of neutrophils reduced the effect of CA4P. In contrast, neither CA4P nor 1A8 affected the necrotic fraction in the SCCVII tumors significantly. Hence, we suggest that the initial decrease in granulocyte concentration was caused by non-tumor-specific recruitment of neutrophils and that neutrophils may attenuate CA4P-mediated anti-tumor effect in some tumor models.

The IGF-IR density on CD4+T-lymphocytes was studied using flow cytometry in 40 early steroid- and DMARD-naïve rheumatoid arthritis (RA) patients before and after 52 weeks of treatment with methotrexate+placebo or methotrexate+cyclosporine A and in 15 controls. RA patients had increased IGF-IR density on CD4+T-lymphocytes at week 0 and week 52, irrespective of treatment. IGF-IR-positive CD4+T-lymphocytes fraction decreased during treatment, but neither at week 0 nor at week 52 did it differ from healthy controls. No correlations were found to disease activity parameters.

Clozapine remains the drug of choice for treatment resistant schizophrenia, but is associated with potentially life threatening side effects, including agranulocytosis and myocarditis. Immunological mechanisms may be involved in the development of these side effects or in the unique antipsychotic efficacy in subgroups of schizophrenia patients. This systematic review presents the immunomodulatory effects of clozapine from human in vitro and in vivo studies and relates these findings to the developments of adverse and therapeutic effects of clozapine. Several studies confirm the immunomodulatory actions of clozapine, but only few studies investigated their relationship to the unique adverse and therapeutic effects of clozapine. During the first month of clozapine treatment, up to 50% of patients develop fever and flu like symptoms, which is seemingly driven by increased cytokines. Within the same time period, the risk of side-effects with a suspected immunological mechanism peaks. Patients developing fever during the first weeks of treatment should have a thorough physical examination, and measurements of white blood cell count, absolute neutrophil count, ECG, C-reactive protein, creatinine kinase, and troponin to exclude infection, agranulocytosis, myocarditis and neuroleptic malignant syndrome. To what degree the unique antipsychotic efficacy of clozapine in subgroups of schizophrenia patients is related to its immunomodulatory effects has not been studied. Research relating the immunomodulatory actions of clozapine and its early markers to clinically relevant adverse and therapeutic outcomes is hoped to provide new leads for the understanding of the pathophysiology of schizophrenia and aid the development of novel treatment targets.

Collection of peripheral blood stem cells (PBSC) must be performed in a safe and effective manner. Issues like automation, collection efficiency (CE), and adverse events must be considered. Auto-PBSC (COBE Spectra) is a fully automated program for PBSC collection. Changes in the protocol were made to achieve high CE, low product volume, and resulted in three groups of patients. Standard operating procedures (SOPs) were developed to reduce citrate toxicity and patients with central venous catheter. Twenty patients and 27 collections (Group 1), 88 patients and 112 collections (Group 2), and 158 patients and 194 collections (Group 3) were recorded. The protocol changes increased CE significantly from 31% (Group 1) to 57 and 59% (Group 2 and 3). Adjusting endpoint according to the preapheresis number of CD34+ cells reduced the collection time and the volume of the product significantly (median 227 min and 56 mL) without affecting CE. Mean level of ionized calcium before collection was 1.22 mmol/L, measured in 31 patients. This declined to a mean of 1.07 mmol/L after 1 h of collection and remained unchanged despite continuous calcium infusion. The number of patients with mild symptoms of citrate toxicity was reduced from 20 to 6%. A central venous catheter was used in 15%. Compared to peripheral access no differences in blood flow rate or time to perform the collection were found. Changes in the Auto-PBSC protocol resulted in an improved CE and a small product. SOPs reduced the number of patients with citrate toxicity and with central venous catheter.

Udgivelsesdato: 2010-null BACKGROUND: Real-Time quantitative PCR is an important tool in research and clinical settings. Here, we describe two new approaches that broaden the scope of real-time quantitative PCR; namely, run-internal mini standard curves (RIMS) and direct real-time relative quantitative PCR (drqPCR). RIMS are an efficient alternative to traditional standard curves and provide both run-specific and target-specific estimates of PCR parameters. The drqPCR enables direct estimation of target ratios without reference to conventional control samples. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we compared RIMS-based drqPCR with classical quantifications based on external standard curves and the "comparative Ct method". Specifically, we used a raw real-time PCR dataset as the basis for more than two-and-a-half million simulated quantifications with various user-defined conditions. Compared with classical approaches, we found that RIMS-based drqPCR provided superior precision and comparable accuracy. CONCLUSIONS/SIGNIFICANCE: The obviation of referencing to control samples is attractive whenever unpaired samples are quantified. This may be in clinical and research settings; for instance, studies on chimerism, TREC quantifications, copy number variations etc. Also, lab-to-lab comparability can be greatly simplified.

Aim Scandiatransplant is the organ exchange organization for the Nordic countries. The number of highly immunized (HI) patients on the waiting list (WL) for deceased donor kidney transplant is steadily increasing and the HI patients wait longer than non-immunized patients do. The Scandiatransplant Acceptable Mismatch Program (STAMP) started in March 2009. The aim was to improve the probability for a HI patient to receive a suitable kidney graft from a deceased donor. Methods Patients are HLA typed at split level (HLA-A, B, C, DR, DQ) using serological or genomic techniques. The patients fulfill all the following criteria: (a) On WL >1 year, (b) HI, PRA ⩾80% based on CDC and/or solid phase assay, (c) HI in two consecutive samples over a period of more than 3 months, (d) Antibody reactivity against HLA cl I and/or II antigens, (e) The last tested sample drawn less than 3 months before acceptance. Eligible patients must have acceptable mismatches defined. We regard HLA antigens which the kidney patient has not developed clinical relevant antibodies (MFI > 1000) towards as acceptable. Upon registration, a transplantability score (TS) is calculated that take HLA antigens and AB0 blood group into account. The TS gives the likelihood of finding a suitable donor by counting the number of compatible donors in 1000 recently HLA typed deceased donors within Scandiatransplant. Results In the study period (March 2009–February 2015) 94/245 patients on the STAMP WL were transplanted, the mean waiting time for the transplanted patients being 137 days (2–721). In the same period, 4338/8583 patients on the ordinary WL were transplanted, with a mean waiting time of 421 days (0–2260). The TS ranged from 0 to 87/1000. The patients with a score ⩾10/1000 ( n = 40) were transplanted, with a mean waiting time of 40 days, while the patients with a TS n = 54) had a mean waiting time of 193 days. Ninety percent of the patients remaining on STAMP for more than a year have a TS Conclusion STAMP is a feasible and immunologically safe way to transplant HI patients. Patients with a TS

Supplemental digital content is available in the text., Background Posttransplant lymphoproliferative disorder (PTLD) is a feared complication to organ transplantation, associated with substantial morbidity and inferior survival. Risk factors for PTLD include T cell–depleting induction therapy and primary infection or reactivation of Epstein-Barr virus. Possible associations between certain HLA types and the risk of developing PTLD have been reported by other investigators; however, results are conflicting. Methods We conducted a retrospective, population-based study on 4295 Danish solid organ transplant patients from the Scandiatransplant database. Having identified 93 PTLD patients in the cohort, we investigated the association of HLA types with PTLD, Epstein-Barr virus status and time to PTLD onset. The outcomes survival and PTLD were evaluated using Cox regression; mismatching, and the PTLD-specific mortality were evaluated in a competing risk analysis. Results Risk of PTLD was associated with male sex (odds ratio, 1.70; 95% confidence interval, 1.07-2.71), and, in women, HLA-DR13 conferred an increased risk (odds ratio, 3.22; 95% confidence interval, 1.41-7.31). In multivariate analysis, HLA-B45 and HLA-DR13 remained independent predictive factors of PTLD. Mismatching in the B locus was associated with a reduced risk of PTLD (P < 0.001). Overall survival was poor after a PTLD diagnosis and was significantly worse than that in the remaining transplant cohort (P < 0.001). Conclusions Our data indicate risk-modifying HLA associations, which can be clinically useful after transplantation in personalized monitoring schemes. Given the strong linkage disequilibrium in the HLA region, the associations must be interpreted carefully. The large size, virtually complete ascertainment of cases and no loss to follow-up remain important strengths of the study.

Objectives: To evaluate the effect of orally administered methotrexate (MTX) on the density of CC chemokine receptor 2 (CCR2) and CXC chemokine receptor 3 (CXCR3) on circulating monocytes, and the coexpression of CXCR3 and CCR2 on CD4 T lymphocytes in patients with active chronic rheumatoid arthritis. Methods: All 34 patients with rheumatoid arthritis fulfilled the 1987 American Rheumatism Association criteria and were followed for 16 weeks after starting MTX. Peripheral blood mononuclear cells were analysed for CCR2 and CXCR3 density by three-colour flow cytometry before initiation of MTX and at week 12. Results: 22 (65%) patients were non-responders, 12 (35%) patients responded to MTX by American College of Rheumatology (ACR)20% criteria, and 8 (24%) of these patients responded by ACR50%. In patients with active rheumatoid arthritis before starting MTX, CCR2 density on circulating monocytes, CD4+ CXCR3+ and CD4+ CXCR3− T lymphocytes was increased compared with controls. During 12 weeks of MTX treatment, the CCR2 density on monocytes decreased significantly in the ACR50% group but not in the ACR20% and non-responder groups. The increased CCR2 density on CD4+ CXCR3+ and CD4+ CXCR3− T lymphocytes was unaffected by the reduction in disease activity measured in relation to MTX treatment. The percentage of both monocytes and CD4+ CXCR3+ and CD4+ CXCR3− T lymphocytes among the peripheral circulating mononuclear cells did not change during MTX treatment. Conclusions: Active chronic rheumatoid arthritis is characterised by enhanced CCR2 density on circulating monocytes and CD4+ CXCR3+ and CD4+ CXCR3− T lymphocytes. During MTX treatment, a decrease in CCR2 density on monocytes in the ACR50% responder group was associated with decreased disease activity. The increased CCR2 density on CD4+ CXCR3+ and CD4+ CXCR3− T lymphocytes was uninfluenced by MTX and disease activity.

Objective: To evaluate in vitro migration of mononuclear cells towards synovial fluid (SF) and plasma in relation to RANTES synovial fluid levels and clinical disease activity. Methods: 31 RA patients with synovitis in one knee were included. Modified Boyden chamber technique was used to determine a migratory index defined as: 'In vitro migrating cells towards SF' divided by 'In vitro migrating cells towards plasma'. RANTES was quantified by ELISA. Disease activity was assessed by the swollen joint count, the Ritchie articular index (RAI), global assessment, pain on VAS, HAQ, ESR and CRP. Results: A positive significant correlation was found between the migratory index and the RANTES levels in SF (r=0.48, p=0.006), the RAI (r=0.56, p=0.0001) and pain on VAS (r=0.43, p=0.04). The in vitro migration could be inhibited in 3 of 4 SF samples by neutralising antibodies towards RANTES (12-18%). Conclusion: The migratory index correlate to SF levels of RANTES and parameters for joint pain.

As a complication to immunosuppressive treatment in allotrans-plantation, malignant diseases such as post-transplant lymphoproliferative disorder (PTLD) may occur. The patient in the present case is a 21-year-old man transplanted at the age of 11 with a kidney from his mother and at the age of 15 with a kidney from his father. During the immunosuppressive treatment the patient developed PTLD resulting in the withdrawal of the immunosuppressive drugs. At the time of writing, the immunosuppressive drugs have been withdrawn for more than 3 years. We report the findings of a state of donor-specific tolerance occurring after transplantation. Post-transplant cells from the patient show a non-reactive response in mixed lymphocyte cultures (MLCs) to cells from both the mother and the father. We demonstrate a reduction in the mRNA expression of the Thl cytokines LL-2 and LFN-γ in the very same MLCs. The expression of Thl cytokine mRNA was measured semi-quanritatively using competitive reverse bran-scription-polymerase chain reaction (RT-PCR). The reduction in the Thl cytokine mRNA expression is not seen in the MLCs with patient cells against cells from a paternal HLA-A, B and DR-matched individual, suggesting the influence of other allorecognition factors than HLA-A, B and DR. Detection in vitro of a lowered expression of Thl cytokine mRNA supports the notion of these mRNAs as indicators of post-transplant tolerance. Further studies will reveal whether the cytokine mRNA measurements on short time stimulated lymphocytes can be used more generally as a monitoring parameter of tolerance in kidney transplantation.