Treatment of chronic COVID-19 with convalescent/postvaccination plasma in patients with hematologic malignancies.

Immunocompromised patients are at high risk to fail clearance of SARS-CoV-2. Prolonged COVID-19 constitutes a health risk and a management problem as cancer treatments often have to be disrupted. As SARS-CoV-2 evolves, new variants of concern have emerged that evade available monoclonal antibodies. Moreover, antiviral therapy promotes SARS-CoV-2 escape mutations, particularly in immunocompromised patients. These patients frequently suffer from prolonged infection. No successful treatment has been established for persistent COVID-19 infection. Here, we report on a series of 21 immunocompromised patients with COVID-19-most of them hematologic malignancies-treated with plasma obtained from recently convalescent or vaccinated donors or a combination thereof. Repeated dosing of SARS-CoV-2-antibody-containing plasma could clear SARS-CoV-2 infection in 16 out of 21 immunocompromised patients even if COVID-19-specific treatments failed to induce sustained viral clearance or to improve clinical course of SARS-CoV-2 infection. Ten patients were major responders defined as an increase delta(d)Ct of > = 5 after the first administration of convalescent and/or vaccinated plasma (C/VP). On average, SARS-CoV-2 PCR Ct values increased from a median value of 22.55 (IQR = 19.10-24.25) to a median value of 29.57 (IQR = 27.55-34.63; p = <.0001) in the major response subgroup. Furthermore, when treated a second time with C/VP, even 4 out of 5 of the initial nonresponders showed an increase in Ct-values from a median value of 23.13 (IQR = 17.75-28.05) to a median value of 32.79 (IQR = 31.75-33.75; p = .013). Our results suggest that C/VP could be a feasible treatment of COVID-19 infection in patients with hematologic malignancies who did not respond to antiviral treatment.

Impact of ABO-blood group type on haemorrhagic and thromboembolic complications after resection of intracranial meningiomas.

OBJECTIVE: Recent studies have suggested an impact of the ABO-blood group type on thromboembolic and haemorrhagic events following trauma and surgical procedures. However, only limited data are available on the impact of ABO-blood group types in neurosurgical patients. The goal of the present study was to evaluate the role of the ABO-blood group type on the frequency of thromboembolic and haemorrhagic complications in patients treated surgically for intracranial meningiomas at our institution. METHODS: We retrospectively analysed the medical records of consecutive patients undergoing resection of intracranial meningiomas at our institution during a period of 12.5 years (2006-2018). Clinical characteristics, modalities of surgical treatment, histopathological results and the postoperative course of patients were analysed with specific focus on ABO-blood group typing results, need for transfusion of blood products, events of postoperative thromboembolism and intracranial re-haemorrhage requiring surgical revision, as well as in-hospital mortality. RESULTS: A total of 1,782 patients were included in this study. Based on the ABO-blood group type, patients were subdivided into four categories, corresponding to their ABO-blood group: Blood group A (n = 773; 43%); blood group B (n = 222; 12%); blood group AB (n = 88; 5%); and blood group O (n = 699; 39%). Intracranial re-haemorrhage requiring re-craniotomy and haematoma evacuation occurred in a total of 49 patients (2.7%). Thromboembolic events such as pulmonary embolism occurred in a total of 27 patients (1.5%). Statistical analysis showed no significant differences regarding the ABO-blood group type in patients suffering from re-haemorrhage or thromboembolism compared with patients with uneventful course after surgery. The overall in-hospital mortality rate was 0.17% (n = 3). CONCLUSION: Our findings suggest a lack of relevance of the ABO-blood group type regarding haemorrhagic and thromboembolic complications in patients undergoing neurosurgical meningioma resection.

Outcomes and complications following ABO-incompatible kidney transplantation performed after desensitization by semi-selective immunoadsorption - a retrospective study.

Because of the current organ shortage, ABO-incompatible (ABOi) transplantations have been increasingly performed in recent years. The results seem comparable to those of compatible transplantations, but there have also been reports of increased side effects possibly because of the desensitization therapy. To address an increase in severe infectious complications, we compared the outcomes of 48 ABOi transplant recipients to outcomes of 96 matched ABO-compatible (ABOc) controls transplanted at Heidelberg University Hospital from August 2005 to April 2018. Over a follow-up period of 8 years, ABOi transplant recipients had comparable graft and patient survival as well as graft function compared with ABOc patients. T-cell-mediated and antibody-mediated rejections were not different between groups. In ABOi transplant recipients, urosepsis (22.9% vs. 8.5%; P = 0.019) and pneumonia with opportunistic pathogens (8.3% vs. 1.0%, P = 0.025) appeared more frequently. As a consequence, a significantly higher number of deaths from infection have been observed after ABOi transplantations (6.3% vs. 0%, P = 0.010). High-titer recipients (isoagglutinin titer of ≥1:256) showed a higher incidence of BK virus replication and postoperative bleeding complications. ABO-incompatible transplantations can be performed with results that are not different from results after ABOc transplantations. However, an increased rate of serious infectious complications must be taken into account.

Induction of type I IFN is a physiological immune reaction to apoptotic cell-derived membrane microparticles.

Membrane microparticles (MMP) released from apoptotic cells deliver signals that secure the anti-inflammatory response beyond the nearest proximity of the apoptotic cell. Plasmacytoid dendritic cells (pDC) are sentinels prepared to detect cellular processes that endanger the organism. They play a key role in the regulation of both pro- and anti-inflammatory immune responses. Based on the assumption that pDC could participate in the initiation of the anti-inflammatory response to apoptotic cells, we investigated the effects of apoptotic cell-derived MMP on human pDC. The results obtained in our experiments confirmed that MMP released from apoptotic cells trigger IFN-α secretion from human pDC. They further suggest that pDC activation results from sensing of DNA contained in MMP. MMP-DNA displays a particularly strong stimulatory activity compared with MMP-RNA and other sources of DNA. Inhibition of MMP-induced IFN-α secretion by cytochalasin D, chloroquine, and an inhibitory G-rich oligodeoxynucleotide identify TLR9 as the receptor for MMP-DNA. In marked contrast to the pDC response in autoimmune patients, in healthy subjects MMP-mediated stimulation of pDC-derived IFN-α was found to be independent of FcγRIIA (CD32A). Based on our findings, we conclude that induction of pDC-derived IFN-α by MMP is a physiological event; future investigations are necessary to elucidate whether pDC activation promotes inflammation or propagates tolerance in the context of apoptotic cell clearance.

Intravascular immune hemolysis caused by the contrast medium iomeprol.

BACKGROUND: Immune-mediated hemolysis is not included in the list of adverse reactions related to contrast medium (CM). Here, we report on a patient who developed immune hemolytic anemia (IHA) related to iomeprol, a nonionic CM. STUDY DESIGN AND METHODS: A 34-year-old female patient developed massive hemolysis during infusion of 50 mL of iomeprol. Serologic studies were performed using standard techniques. RESULTS: Before hemolysis, the patient's serum was weakly positive with e+ red blood cells (RBCs; autoanti-e) and the direct antiglobulin test (DAT) was negative. After hemolysis, the patient's serum samples became significantly reactive with e- RBCs in the presence of iomeprol but not in the presence of two other similar CM. The DAT became strongly positive only with anti-C3d. CONCLUSION: Initially, an allergic reaction was suggested, and as the hemolysis became obvious, a toxic hemolysis was suspected. However, serologic reexamination revealed an iomeprol-dependent antibody. IHA related to CM has yet only been described in one patient in 1991. The hemolysis in that patient was related to Isopaque, an older ionic CM. Here, we describe an additional patient and recommend that CM should be considered as a rare risk in the development of IHA.

Anti-SARS-CoV-2 antibody-containing plasma improves outcome in patients with hematologic or solid cancer and severe COVID-19: a randomized clinical trial.

Patients with cancer are at high risk of severe coronavirus disease 2019 (COVID-19), with high morbidity and mortality. Furthermore, impaired humoral response renders severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines less effective and treatment options are scarce. Randomized trials using convalescent plasma are missing for high-risk patients. Here, we performed a randomized, open-label, multicenter trial ( https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-001632-10/DE ) in hospitalized patients with severe COVID-19 (n = 134) within four risk groups ((1) cancer (n = 56); (2) immunosuppression (n = 16); (3) laboratory-based risk factors (n = 36); and (4) advanced age (n = 26)) randomized to standard of care (control arm) or standard of care plus convalescent/vaccinated anti-SARS-CoV-2 plasma (plasma arm). No serious adverse events were observed related to the plasma treatment. Clinical improvement as the primary outcome was assessed using a seven-point ordinal scale. Secondary outcomes were time to discharge and overall survival. For the four groups combined, those receiving plasma did not improve clinically compared with those in the control arm (hazard ratio (HR) = 1.29; P = 0.205). However, patients with cancer experienced a shortened median time to improvement (HR = 2.50; P = 0.003) and superior survival with plasma treatment versus the control arm (HR = 0.28; P = 0.042). Neutralizing antibody activity increased in the plasma cohort but not in the control cohort of patients with cancer (P = 0.001). Taken together, convalescent/vaccinated plasma may improve COVID-19 outcomes in patients with cancer who are unable to intrinsically generate an adequate immune response.

AB0 blood group and prognosis in patients with pancreatic cancer.

BACKGROUND: Although blood group 0 is associated with a reduced risk of pancreatic cancer, little is known about the role of AB0 blood group antigens in disease progression. We assessed the prognostic relevance of AB0 blood status in a large cohort of patients with resected pancreatic cancer. METHODS: A total of 627 patients, who underwent resection for pancreatic ductal adenocarcinoma between October 2001 and December 2008 were enrolled. The relationship between AB0 blood group status and outcome was analyzed using univariate and multivariate Cox regression analyses. RESULTS: In patients with pancreatic cancer the incidence of blood group 0 (31%) was lower compared to 13.044 patients without pancreatic cancer (38%) (p = 0.0005). There were no significant differences in clinicopathologic characteristics among patients with different AB0 blood groups. The 3-year and 5-year overall survival rates were 29% and 14%. On univariate analysis AB0 blood group status did not correlate with survival (p = 0.39). Multivariate analysis, however, revealed a favorable and independent impact of blood group 0 on survival (Hazard ratio 0.78; 95% confidence interval 0.62 - 0.99; p = 0.037). CONCLUSION: AB0 blood group status is associated independently with the prognosis of patients with resected pancreatic cancer.

Early Development of Ubiquitous Acanthocytosis and Extravascular Hemolysis in Lung Cancer Patients Receiving Alectinib.

Alectinib is a standard initial treatment for patients with advanced anaplastic lymphoma kinase (ALK) rearranged non-small-cell lung cancer (NSCLC). The current study analyzed a prospective cohort of 24 consecutive alectinib-treated patients and controls in order to comprehensively characterize longitudinal erythrocyte changes under treatment with ALK inhibitors. Upon starting alectinib, all examined patients developed reticulocytosis and abnormal erythrocyte morphology with anisocytosis and a predominance of acanthocytes (64% of red blood cells on average, range 36−100%) in the peripheral blood smear within approximately 2 weeks. Changes were accompanied by a gradual reduction in Eosin-5-maleimide (EMA) binding, which became pathologic (<80% of cells) within 1−2 months in all cases, mimicking an abortive form of hereditary spherocytosis. The latter could be ruled out in 3/3 of analyzed cases by normal sequencing results for the ANK1, EPB42, SLC4A1, SPTA1, or SBTB genes. The direct Coombs test was also negative in 11/11 tested cases. Besides, anemia, increased LDH, and increased bilirubin were noted in a fraction of patients only, ranging between 42 and 68%. Furthermore, haptoglobin decreases were infrequent, occurring in approximately 1/3 of cases only, and mild, with an average value of 0.93 g/L within the normal range of 0.3−2 g/dL, suggesting that hemolysis occurred predominantly in the extravascular compartment, likely due to splenic trapping of the deformed erythrocytes. These changes showed no association with progression-free survival under alectinib or molecular features, i.e., ALK fusion variant or TP53 status of the disease, and resolved upon a switch to an alternative ALK inhibitor. Thus, alectinib induces mild, reversible erythrocyte changes in practically all treated patients, whose most sensitive signs are aberrant red cell morphology in the peripheral smear, a pathologic EMA test, and reactive reticulocytosis. Frank hemolytic anemia is rare, but mild subclinical hemolysis is very frequent and poses differential-diagnostic problems. Alectinib can be continued under the regular control of hemolysis parameters, but the risk of long-term complications, such as cholelithiasis due to increased serum bilirubin in most patients, remains unclear at present.

A Randomized Open label Phase-II Clinical Trial with or without Infusion of Plasma from Subjects after Convalescence of SARS-CoV-2 Infection in High-Risk Patients with Confirmed Severe SARS-CoV-2 Disease (RECOVER): A structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: Primary objectives • To assess the time from randomisation until an improvement within 84 days defined as two points on a seven point ordinal scale or live discharge from the hospital in high-risk patients (group 1 to group 4) with SARS-CoV-2 infection requiring hospital admission by infusion of plasma from subjects after convalescence of SARS-CoV-2 infection or standard of care. Secondary objectives • To assess overall survival, and the overall survival rate at 28 56 and 84 days. • To assess SARS-CoV-2 viral clearance and load as well as antibody titres. • To assess the percentage of patients that required mechanical ventilation. • To assess time from randomisation until discharge. TRIAL DESIGN: Randomised, open-label, multicenter phase II trial, designed to assess the clinical outcome of SARS-CoV-2 disease in high-risk patients (group 1 to group 4) following treatment with anti-SARS-CoV-2 convalescent plasma or standard of care. PARTICIPANTS: High-risk patients >18 years of age hospitalized with SARS-CoV-2 infection in 10-15 university medical centres will be included. High-risk is defined as SARS-CoV-2 positive infection with Oxygen saturation at ≤ 94% at ambient air with additional risk features as categorised in 4 groups: • Group 1, pre-existing or concurrent hematological malignancy and/or active cancer therapy (incl. chemotherapy, radiotherapy, surgery) within the last 24 months or less. • Group 2, chronic immunosuppression not meeting the criteria of group 1. • Group 3, age ≥ 50 - 75 years meeting neither the criteria of group 1 nor group 2 and at least one of these criteria: Lymphopenia < 0.8 x G/l and/or D-dimer > 1µg/mL. • Group 4, age ≥ 75 years meeting neither the criteria of group 1 nor group 2. Observation time for all patients is expected to be at least 3 months after entry into the study. Patients receive convalescent plasma for two days (day 1 and day 2) or standard of care. For patients in the standard arm, cross over is allowed from day 10 in case of not improving or worsening clinical condition. Nose/throat swabs for determination of viral load are collected at day 0 and day 1 (before first CP administration) and subsequently at day 2, 3, 5, 7, 10, 14, 28 or until discharge. Serum for SARS-Cov-2 diagnostic is collected at baseline and subsequently at day 3, 7, 14 and once during the follow-up period (between day 35 and day 84). There is a regular follow-up of 3 months. All discharged patients are followed by regular phone calls. All visits, time points and study assessments are summarized in the Trial Schedule (see full protocol Table 1). All participating trial sites will be supplied with study specific visit worksheets that list all assessments and procedures to be completed at each visit. All findings including clinical and laboratory data are documented by the investigator or an authorized member of the study team in the patient's medical record and in the electronic case report forms (eCRFs). INTERVENTION AND COMPARATOR: This trial will analyze the effects of convalescent plasma from recovered subjects with SARS-CoV-2 antibodies in high-risk patients with SARS-CoV-2 infection. Patients at high risk for a poor outcome due to underlying disease, age or condition as listed above are eligible for enrollment. In addition, eligible patients have a confirmed SARS-CoV-2 infection and O2 saturation ≤ 94% while breathing ambient air. Patients are randomised to receive (experimental arm) or not receive (standard arm) convalescent plasma in two bags (238 - 337 ml plasma each) from different donors (day 1, day 2). A cross over from the standard arm into the experimental arm is possible after day 10 in case of not improving or worsening clinical condition. MAIN OUTCOMES: Primary endpoints: The main purpose of the study is to assess the time from randomisation until an improvement within 84 days defined as two points on a seven-point ordinal scale or live discharge from the hospital in high-risk patients (group 1 to group 4) with SARS-CoV-2 infection requiring hospital admission by infusion of plasma from subjects after convalescence of a SARS-CoV-2 infection or standard of care. Secondary endpoints: • Overall survival, defined as the time from randomisation until death from any cause 28-day, 56-day and 84-day overall survival rates. • SARS-CoV-2 viral clearance and load as well as antibody titres. • Requirement mechanical ventilation at any time during hospital stay (yes/no). • Time until discharge from randomisation. • Viral load, changes in antibody titers and cytokine profiles are analysed in an exploratory manner using paired non-parametric tests (before - after treatment). RANDOMISATION: Upon confirmation of eligibility (patients must meet all inclusion criteria and must not meet exclusion criteria described in section 5.3 and 5.4 of the full protocol), the clinical site must contact a centralized internet randomization system ( https://randomizer.at/ ). Patients are randomized using block randomisation to one of the two arms, experimental arm or standard arm, in a 1:1 ratio considering a stratification according to the 4 risk groups (see Participants). BLINDING (MASKING): The study is open-label, no blinding will be performed. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): A total number of 174 patients is required for the entire trial, n=87 per group. TRIAL STATUS: Protocol version 1.2 dated 09/07/2020. A recruitment period of approximately 9 months and an overall study duration of approximately 12 months is anticipated. Recruitment of patients starts in the third quarter of 2020. The study duration of an individual patient is planned to be 3 months. After finishing all study-relevant procedures, therapy, and follow-up period, the patient is followed in terms of routine care and treated if necessary. Total trial duration: 18 months Duration of the clinical phase: 12 months First patient first visit (FPFV): 3rd Quarter 2020 Last patient first visit (LPFV): 2nd Quarter 2021 Last patient last visit (LPLV): 3rd Quarter 2021 Trial Report completed: 4th Quarter 2021 TRIAL REGISTRATION: EudraCT Number: 2020-001632-10, https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-001632-10/DE , registered on 04/04/2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2). The eCRF is attached (Additional file 3).

Effectiveness of different immunoadsorption columns for anti-A/B antibody depletion.

OBJECTIVE: In recent studies, semi-selective compared to antigen-specific immunoadsorption (IA) columns showed comparable effectiveness in anti-A/B antibody removal before incompatible living donor kidney transplantation. Semi-selective columns allow a greater number of IA treatments at lower costs. They are also capable of removing potentially harmful human leukocyte antigen alloantibodies. Nevertheless, additional plasma exchange treatments are often necessary to reach the preoperative target titer, most likely due to an inadequate anti-A/B IgM antibody depletion. METHODS: We compared the effectiveness of immunoglobulin and anti-A/B antibody reduction by different semi-selective (Therasorb Ig-flex, Therasorb Ig-omni5, Immunosorba) and antigen-specific (Glycosorb) IA columns during the desensitization of 63 ABO-incompatible living donor kidney transplant candidates with a total of 375 IA treatments. Fifty-three patients were eventually transplanted. RESULTS: Total IgM reduction during the first IA treatment was significantly greater with the Therasorb Ig-omni5 compared to the Therasorb Ig-flex (mean: -71.3 vs -41.6; p = 0.001) or Immunosorba columns (mean: -71.3 vs -42.8; p = 0.03). During a median of 5.5-6 pre-transplant IA treatments, Therasorb Ig-flex and Therasorb Ig-omni5 columns were equally effective in the reduction of total IgM while both showed superior IgM reduction compared to the Immunosorba columns (Therasorb Ig-flex, mean: -81.2 vs -72.2; p = 0.01; Therasorb Ig-omni5, mean: -88.2 vs -72.2; p = 0.02). IgG reduction was not significantly different between groups. Likewise, anti-A/B IgM antibody reduction (titer Saline) during the first IA treatment was significantly greater with the Therasorb Ig-omni5 compared to the Therasorb Ig-flex (mean titer reduction: -1.9 vs -1.1; p = 0.02) and tended to be greater than with Immunosorba or Glycosorb columns. During a median of 5-6 pre-transplant IA treatments, overall anti-A/B IgM antibody reduction was significantly greater when IA was performed with the Therasorb Ig-flex (mean titer reduction: -3.8 vs -1.3; p < 0.001) or Therasorb Ig-omni5 (mean titer reduction: -4.3 vs -1.3; p = 0.01) compared to the Immunosorba columns with no differences compared to the Glycosorb columns. Again, anti-A/B IgG antibody reduction (titer Coombs) was not significantly different between groups. CONCLUSIONS: The semi-selective Therasorb Ig-omni5 device offers potential advantages in reducing total IgM as well as anti-A/B IgM antibodies.

Effect of platelet-rich plasma on the in vitro proliferation and osteogenic differentiation of human mesenchymal stem cells on distinct calcium phosphate scaffolds: the specific surface area makes a difference.

The in vitro effect of platelet-rich plasma (PRP) on cell loading, proliferation, and osteogenic differentiation of human mesenchymal stem cells (MSC) is assessed on distinct resorbable and synthetic calcium phosphate scaffolds. A high specific surface area scaffold composed of calcium-deficient hydroxyapatite (CDHA; 48m2/g) is compared with one made out of beta-tricalcium phosphate (beta-TCP; surface area <0.5 m2/g). Fivefold concentrated fresh PRP is applied to scaffolds loaded with 2 x 10(5) MSC (n = 5). These constructs are kept in a medium with osteogenic supplements for 3 weeks. The addition of PRP leads to a higher cell loading efficiency of MSC on CDHA (p = 0.0001), that reaches the values of beta-TCP. Proliferation over 21 days is improved by PRP both on CDHA (p = 0.0001) and beta-TCP (p = 0.014) compared to MSC/calcium phosphate composites. Without the addition of PRP, CDHA has a lower cell loading efficiency (p= 0.0001) and proliferation (p= 0.001) than beta-TCP. The ALP activity is higher in the MSC/ceramics groups than in the monolayer controls (p<0.05). The addition of PRP does not significantly affect ALP activity. However, ALP activity varies considerably within the cell donors and different PRP-pools (p = 0.001), while the cell numbers do not vary within these two parameters. PRP generates a positive effect on the loading efficiency of MSC on the high specific surface scaffold CDHA that thereby reaches the loading efficiency of beta-TCP. PRP improved proliferation, but its osteogenic properties on both calcium phosphate scaffolds are weak.

Characterization of hospitalized cardiovascular patients with suspected heparin-induced thrombocytopenia.

BACKGROUND: Little is known about heparin-induced thrombocytopenia (HIT), a pro-thrombotic, potentially life-threatening immune-mediated reaction to heparin exposure, in conservative and interventional cardiovascular medicine. HYPOTHESIS: The 4T score, validated for prediction of HIT in surgical patients before, is also suitable for assessing HIT probability in cardiovascular patients with unclear thrombocytopenia. METHODS: A total of 403 consecutive patients from our Department of Cardiology, Angiology and Pneumology in whom a HIT screening test was performed between 2009 and 2016 were identified. All 72 patients with a positive screening test were subjected to a functional confirmation test (heparin-induced platelet activation test, HIPA), resulting in 23 patients with serologically confirmed HIT (positive screening test, positive HIPA) and 49 patients with nonconfirmed HIT (positive screening test, negative HIPA). RESULTS: The 4TScore had a sensitivity of 82.6% and a specificity of 28.6% in our patients, suggesting that it might not sufficiently predict the clinical probability of HIT in cardiovascular patients. In both confirmed and nonconfirmed HIT, intrahospital mortality was high without a significant difference (30% in confirmed HIT vs 43% in nonconfirmed HIT). Bacteremia was more often found in patients with nonconfirmed HIT, suggesting infection as a frequent differential diagnosis of thrombocytopenia in these patients (49% vs 17%, P = 0.0185). CONCLUSION: HIT screening should be initiated in cardiovascular patients with unclear thrombocytopenia despite a low 4Tscore in order to distinguish patients requiring alternative anticoagulants from those with other causes such as infections. Further research is needed to specify the risk profile for HIT in cardiovascular patients.

The bcl-2/IgH rearrangement in a population of 204 healthy individuals: occurrence, age and gender distribution, breakpoints, and detection method validity.

This study assessed prevalence, frequency, age and gender distribution and breakpoint locations, and detection method validity for the bcl-2/IgH rearrangement in 204 healthy individuals. For this purpose, both classic two-step, nested, semi-quantitative PCR as well as a newly established sequence-specific, hybridization probe-based real-time quantitative PCR (RQ-PCR) were employed and tested for their sensitivity and specificity for detecting t(14;18) positive cells in healthy blood donors. Interestingly, almost a quarter (24%; 39/204) of all healthy individuals carried the translocation, confirming data of one large prior report [Summers KE, Goff LK, Wilson AG, Gupta RK, Lister TA, Fitzgibbon J. Frequency of the Bcl-2/IgH rearrangement in normal individuals: implications for the monitoring of disease in patients with follicular lymphoma. J Clin Oncol 2001;19(2):420-4]. Regarding presence as well as frequency of the translocation, no correlation to age (mean frequency 2.0:10(4), with a median of or=40 years) nor gender was detected. Comparing the two PCR approaches, a 95.1% concordance (194/204) regarding t(14;18) detection was determined for nested and RQ-PCR, with nested PCR being slightly more sensitive (reproducible detection limit l:10(5) cells versus 1:10(4); maximum detection limit l:10(6) versus 1:10(5)). Sequence analysis confirmed individual breakpoints for all samples analyzed (29/29), indicating detection validity for both PCR approaches and ruling out contamination. The breakpoint location distribution pattern appeared to be comparable to the pattern seen with follicular lymphoma (FL) patient collectives. In conclusion, clonal bcl-2/IgH rearrangements are indeed a very frequent observation in healthy individuals, and appear to be independent of age and gender in regard to presence and frequency. This represents a conflicting finding in context of potential biological significance, and presents a potential disruptive factor for minimal residual disease (MRD) monitoring in FL patients. Prospective future trials will have to clarify the biological significance of this important observation.

Ectopic bone formation associated with mesenchymal stem cells in a resorbable calcium deficient hydroxyapatite carrier.

Bone substitute materials can induce bone formation in combination with mesenchymal stem cells (MSC). The aim of the current study was to examine ectopic in vivo bone formation with and without MSC on a new resorbable ceramic, called calcium deficient hydroxyapatite (CDHA). Ceramic blocks characterized by a large surface (48 m2/g) were compared with beta-tricalcium phosphate (beta-TCP), hydroxyapatite (HA) ceramics (both ca. 0.5 m2/g surface) and demineralized bone matrix (DBM). Before implantation in the back of SCID mice carriers were freshly loaded with 2x10(5) expanded human MSC or loaded with cells and kept under osteogenic conditions for two weeks in vitro. Culture conditions were kept free of xenogenic supplements. Deposits of osteoid at the margins of ceramic pores occurred independent of osteogenic pre-induction, contained human cells, and appeared in 416 MSC/CDHA composites compared to 216 MSC/beta-TCP composites. ALP activity was significantly higher in samples with MSC versus empty controls (p<0.001). Furthermore, ALP was significantly (p<0.05) higher for all ceramics when compared to the DBM matrix. Compared to previous studies, overall bone formation appeared to be reduced possibly due to the strict human protocol. Ectopic bone formation in the novel biomaterial CDHA varied considerably with the cell pool and was at least equal to beta-TCP blocks.

Outcomes Following ABO-Incompatible Kidney Transplantation Performed After Desensitization by Nonantigen-Specific Immunoadsorption.

BACKGROUND: For desensitization of ABO-incompatible kidney transplant recipients we recently proposed nonantigen-specific immunoadsorption (IA) and rituximab. METHODS: We now compared clinical outcomes of 34 ABO-incompatible living-donor kidney recipients who were transplanted using this protocol with that of 68 matched ABO-compatible patients. In addition, we analyzed efficacy and cost of nonantigen-specific as compared to blood group antigen-specific IA. RESULTS: Before desensitization, the median isoagglutinin titer of 34 ABO-incompatible patients was 1:64 (Coombs technique). Patients received a median of 7 preoperative IA treatments. Twenty-four patients had a median of 2 additional plasmapheresis treatments to reach the preoperative target isoagglutinin titer of 1:8 or less. After a median postoperative follow-up of 22 months, overall graft survival in the ABO-incompatible group was not significantly different from that in ABO-compatible patients (log-rank P = 0.20), whereas patient survival tended to be lower (log-rank P = 0.05). The incidence of rejection episodes was 15% in both groups. The ABO-incompatible kidney recipients had a higher incidence of BK virus replication (P = 0.04) and nephropathy (P = 0.01) and showed more often colonization with multidrug resistant bacteria (P = 0.02). In comparison to blood group antigen-specific IA, nonantigen-specific IA showed equal efficacy but was associated with reduction in cost. CONCLUSIONS: Clinical outcomes of ABO-incompatible patients desensitized with a nonantigen-specific IA device and rituximab do not differ from that of matched ABO-compatible patients although a trend toward reduced patient survival was noted. Special attention must be paid to the higher incidence of BK virus infection in recipients of ABO-incompatible grafts.

ABO-incompatible kidney transplantation enabled by non-antigen-specific immunoadsorption.

BACKGROUND: ABO-incompatible kidney transplantation performed after desensitization with antigen-specific immunoadsorption (IA) results in good outcomes. However, a unique single-use IA device is required, which creates high costs. METHODS: From August 2005 to August 2010, 19 patients were desensitized for ABO-incompatible living donor kidney transplantation. Six patients treated with a single-use antigen-specific IA device and 12 patients treated with a reusable non-antigen-specific IA device were analyzed. RESULTS: Six patients who received antigen-specific IA had a median of 5 IA treatments and 12 patients with non-antigen-specific IA had a median of 6 IA treatments preoperatively. Median average titer drop in Coombs technique was 1.2 in antigen-specific IA and 1.7 in non-antigen-specific IA. In two patients with antigen-specific IA and four patients with non-antigen-specific IA, additional plasmapheresis treatments were necessary for recipient desensitization. Despite six treatments with antigen-specific IA and 12 plasmapheresis treatments, one patient with a starting isoagglutinin titer of 1:1024 (Coombs) could not be transplanted. The 18-month graft survival rate for the 17 ABO-incompatible living donor kidney transplants was 100%. One male recipient who was desensitized with antigen-specific IA died 44 months after transplantation from sudden cardiac death with a serum creatinine of 1.2 mg/dL. At last follow-up, a median of 13 months after transplantation, median serum creatinine for 16 patients was 1.5 mg/dL, median glomerular filtration rate as estimated by the modification of diet in renal disease formula 54 mL/min/1.73 m, and median urinary protein-to-creatinine ratio 0.1, with no differences between treatments. CONCLUSIONS: A reusable non-antigen-specific IA device allows high number of treatments at reasonable cost, and at the same time might deplete human leukocyte antigen-alloantibodies.

Influence of platelet-rich plasma on osteogenic differentiation of mesenchymal stem cells and ectopic bone formation in calcium phosphate ceramics.

Platelet-rich plasma (PRP) contains a mixture of growth factors that play an important role in wound and fracture healing. While PRP enhanced bone formation by autogenous cancellous bone grafts, its influence in combination with different bone substitutes remained unknown. This study evaluated the effect of PRP on osteogenic differentiation and ectopic bone formation of human mesenchymal stem cells (MSC) in distinct resorbable calcium phosphate ceramics. Calcium-deficient hydroxyapatite (CDHA) blocks with a large specific surface area (48 m2/g) and beta-tricalcium phosphate (beta-TCP) with a low specific surface area (<0.5 m2/g) were loaded with 2 x 10(5) bone marrow-derived MSC. Half of the specimens were treated with 5-fold concentrated PRP. Biocomposites were implanted subcutaneously into SCID mice or kept under osteogenic culture conditions for 2 weeks before implantation. The addition of PRP increased the specific alkaline phosphatase (ALP) activity (p = 0.012) in undifferentiated MSC/CDHA composites but not in MSC/beta-TCP composites. Osteogenic preinduction was ineffective for CDHA and reduced ALP activity of beta-TCP composites significantly at explantation. Ectopic bone formation was stronger in MSC/CDHA (7/32) compared to MSC/beta-TCP (2/30) composites, but no influence of PRP was evident. In conclusion, the effect of PRP depended on the type of ceramic and the differentiation status of the MSC, and enhanced ALP activity of MSC on the high surface scaffold CDHA only, but PRP did not improve osteogenesis in our setting.

Differential role of glycolipid-enriched membrane domains in glycoprotein VI- and integrin-mediated phospholipase Cgamma2 regulation in platelets.

The platelet collagen receptor glycoprotein VI (GPVI) and the fibrinogen receptor integrin alphaIIbbeta3 trigger intracellular signalling cascades involving the tyrosine kinase Syk, the adapter SLP-76 and phospholipase Cgamma2 (PLCgamma2). Similar pathways are activated downstream of immune receptors in lymphocytes, where they have been localized in part to glycolipid-enriched membrane domains (GEMs). Here we provide several lines of evidence that GPVI-mediated tyrosine phosphorylation of PLCgamma2 in platelets is dependent on GEM-organized signalling and utilizes the GEM resident adapter protein LAT (linker for activation of T cells). In sharp contrast, although fibrinogen binding to platelets stimulates alphaIIbbeta3-dependent activation of Syk and tyrosine phosphorylation of SLP-76 and PLCgamma2, it does not utilize GEMs to promote these responses or to support platelet aggregation. These results establish that GPVI and alphaIIbbeta3 trigger distinct patterns of receptor signalling in platelets, leading to tyrosine phosphorylation of PLCgamma2, and they highlight the role of GEMs in compartmentalizing signalling reactions involved in haemostasis.