Gender, cholinesterase, platelet count and red cell count are main predictors of peripheral blood stem cell mobilization in healthy donors.

BACKGROUND AND OBJECTIVES: Mobilization of CD34+ cells by stimulation with G-CSF shows considerable variation across stem cell donors. Upfront prediction of CD34+ cell counts in peripheral blood based on easily available steady-state parameters would be helpful for the planning of apheresis and stem cell transplantation. Commonly accepted steady-state predictors for the mobilization are gender, body mass index and platelet count. The aim of the study was the identification of novel predictors that might influence mobilization efficacy and to create a model for the prediction of stem cell mobilization. METHODS: A total of 333 healthy stem cell donors who donated peripheral stem cells in our institution were retrospectively analysed. All available data before stem cell mobilization with G-CSF were included in the database. Primary end-point was CD34+ cell count before the first apheresis. RESULTS: In this cohort cholinesterase, differential blood cell counts including platelets, gender and body mass index were significantly correlated with CD34+ cell count. G-CSF dose per lean body weight showed a significant correlation with mobilization efficacy in women but not in men. A multivariate analysis identified gender, cholinesterase and platelet and red cell count as main predictors of mobilization. Red cell count showed a strong gender dependence, with higher predictive value in females. CONCLUSION: The counts of eosinophils, platelets, red cells, cholinesterase and gender are the most important predictors of CD34+ cell mobilization in our deduced models. The red cell count as a predictor for mobilization showed a differential gender dependence.

The Monoclonal Anti-CD157 Antibody Clone SY11B5, Used for High Sensitivity Detection of PNH Clones on WBCs, Fails to Detect a Common Polymorphic Variant Encoded by BST-1.

BACKGROUND: CD157, encoded by BST-1, has been described as a useful flow cytometric marker for the analysis of paroxysmal nocturnal hemoglobinuria (PNH) as it is a glycosylphosphatidylinositol (GPI)-linked molecule highly expressed on normal monocytes and neutrophils. We and others observed isolated CD157 signal dropouts during intended PNH analysis. We hypothesize that these negative populations occur due to an antibody failure. To investigate the reason for this finding, we compared two different anti-CD157 antibody clones for PNH analysis. METHODS: We sequenced BST-1 of CD157-negative probands that are not suffering from PNH and expressed wild type and a discovered variant form of CD157 in HEK293 cells. We compared the binding patterns of two different anti-CD157 antibody clones (SY11B5 and RF3) by flow cytometry and western blot analysis. RESULTS: When sequencing two CD157-negative probands we detected a common SNP (p.Arg145Gln) in exon 3 of BST-1. We found that only anti-CD157 antibody clone RF3 but not the more widely used clone SY11B5 was able to detect both, the wild type and the variant form of CD157 in flow cytometric experiments. CONCLUSION: The failure of anti-CD157 antibody clone SY11B5 to detect a common SNP can explain some CD157-negative cytometric data. This provides crucial knowledge for laboratories performing PNH analyses as such results can potentially lead to false-positive PNH interpretation. Our results confirm the importance of published PNH guidelines. © 2018 International Clinical Cytometry Society.

Peptide vaccination in the presence of adjuvants in patients after hematopoietic stem cell transplantation with CD4+ T cell reconstitution elicits consistent CD8+ T cell responses.

Rationale: Patients receiving an allogeneic stem cell graft from cytomegalovirus (CMV) seronegative donors are particularly prone to CMV reactivation with a high risk of disease and mortality. Therefore we developed and manufactured a novel vaccine and initiated a clinical phase I trial with a CMV phosphoprotein 65 (CMVpp65)-derived peptide. Methods: Ten patients after allogeneic stem cell transplantation received four vaccinations at a biweekly interval. All patients were monitored for CMVpp65 antigenemia. Flow cytometry for CMV-specific CD8+ and γδ T cells as well as neutralizing anti-CMV antibodies were correlated to clinical parameters. Results: The vaccination was well tolerated. Seven of nine patients cleared CMVpp65 antigenemia after four vaccinations and are still free from antigenemia to this day. Two patients with CMV reactivation showed persisting CMV antigenemia. One patient received prophylactic vaccination and did not develop antigenemia. An increase of up to six-fold in frequency of both CMV-specific CD8+ T cells and/or Vδ2negative γδ T cells was detected. Titers of neutralizing antibodies increased up to the tenfold. Humoral and cellular immune responses correlated with clearance of CMV. Conclusion: In summary, CMVpp65 peptide vaccination for patients after allogeneic stem cell transplantation at high risk for CMV reactivation was safe, well tolerated and clinically encouraging. A study in solid-organ transplant patients is ongoing.

Impact of cell number and microvascular obstruction in patients with bone-marrow derived cell therapy: final results from the randomized, double-blind, placebo controlled intracoronary Stem Cell therapy in patients with Acute Myocardial Infarction (SCAMI) trial.

BACKGROUND: In patients with acute myocardial infarction (AMI), the number of transplanted autologous bone-marrow cells (BMC) has been linked to improvement in left ventricular ejection fraction (LVEF). Complete obstruction of myocardial microvasculature is indicated by microvascular obstruction (MO) in cardiac magnetic resonance imaging (CMR). We analyzed whether the number of transplanted cells and presence of MO were associated with improved LVEF in the double-blind, placebo-controlled, randomized intracoronary Stem Cell therapy in patients with Acute Myocardial Infarction (SCAMI) trial. METHODS AND RESULTS: Patients (N = 42) received study therapy mean 7 days after AMI. Median number of transplanted BMC was 324 × 10(6). CMR was performed prior to study therapy and annually up to 3 years and revealed no difference between BMC and placebo population. Patients treated with a cell number above the median experienced a significant improvement in LVEF compared with patients with cell number below the median 3.6 ± 3.4 versus -0.5 ± 6.4 % (difference 4.1, 95 % CI 0.2 to 8.1 %, p = 0.04) at 6 months. The difference in LVEF change between the groups remained with 3.8 % (p = 0.12) at 12 months, 4.5 % (p = 0.07) at 24 months and 5.6 % (p = 0.03) at 36 months. BMC treated patients without MO experienced a better improvement in LVEF compared with patients with MO at 6, 12, 24 and 36 months with 3.5, 5.3, 6.4 and 3.2 %. CONCLUSIONS: In the randomized, placebo-controlled double-blind SCAMI trial improvement in LVEF up to 3 years was higher in BMC patients treated with a high cell number or without MO.

Results of intracoronary stem cell therapy after acute myocardial infarction.

To assess the effect of autologous bone-marrow cell (BMC) therapy in patients with acute myocardial infarction in a rigorous double-blind, randomized, placebo-controlled trial. Patients with reperfusion >6 hours after symptom onset were randomly assigned in a 2:1 ratio to receive intracoronary BMC or placebo therapy 5 to 7 days after symptom onset. The patients were stratified according to age, acute myocardial infarction localization, and left ventricular (LV) function. Rigorous double-blinding was ensured using autologous erythrocytes for the placebo preparation that was visually indistinguishable from the active treatment. Serial cardiac magnetic resonance imaging studies were performed before study therapy and after 1, 3, and 6 months. The primary end point was the difference in the LV ejection fraction from baseline to 6 months. The secondary end points included changes in the LV end-diastolic and end-systolic volume indexes and infarct size. A total of 42 patients were enrolled (29 in the BMC group and 13 in the placebo group) in the integrated pilot phase. A mean of 381 x 10(6) mononuclear BMCs were administered. The baseline clinical and cardiac magnetic resonance imaging parameters did not differ. Compared to baseline, the difference in LV ejection fraction for the placebo group versus BMC group was 1.7 +/- 6.4% versus -0.9 +/- 5.5% at 1 month, 3.1 +/- 6.0% versus 1.9 +/- 4.3% at 3 months, and 5.7 +/- 8.4% versus 1.8 +/- 5.3% at 6 months (primary end point; not significant). No difference was found in the secondary end points between the 2 groups, including changes in infarct size or LV end-diastolic and end-systolic volume indexes. In conclusion, in this rigorous double-blind, randomized, placebo-controlled trial, we did not observe an evidence for a positive effect for intracoronary BMC versus placebo therapy with respect to LV ejection fraction, LV volume indexes, or infarct size.

Cytomegalovirus vaccination of leukemia and lymphoma patients after allogeneic stem cell transplantation--validation of a peptide vaccine.

Peptide vaccination constitutes a novel immunotherapeutical approach for the treatment of patients with solid tumors, lymphoma and leukemia. Moreover it might be of use in hematooncological patients for the prevention and therapy of infections like cytomegalovirus (CMV) reactivation due to immunosuppression. To meet good manufacturing practice (GMP) criteria, we introduce here a bio-assay to validate peptide vaccines for peptide content and bio-activity. As a paradigm for peptide vaccine preparation the immunogenic CMV peptide 495-503 NLVPMVATV lyophilisate was resolubilized in dimethyl sulfoxide, phosphate buffered saline and admixed with Montanide. Addition of different amounts of peptide (10-80 microg) to a mixed lymphocyte peptide culture (MLPC) resulted in the generation of interferon (IFN) gamma and granzyme B releasing CD8(+) CMV tetramer(+) T cells in a dose dependent manner. The combination of FACS and ELISPOT results allowed the definition of the peptide amount in a vaccine preparation. Storage at +/-4 degrees C over 24 h did not result in a significant change of the immunogenicity of the vaccine. In contrast, cryopreservation of the vaccine at -20 degrees C resulted in a loss of immunogenicity. Quantitation of tumor/viral antigen peptides admixed with adjuvants, such as incomplete Freund's adjuvant (IFA), is feasible through bio-assays as the modified ELISPOT/FACS assay described here, meeting GMP criteria for multi-center trials.

Adoptive therapy of head and neck squamous cell carcinoma with antibody coated immune cells: a pilot clinical trial.

BACKGROUND: Catumaxomab is an antibody that binds with one arm epithelial cell adhesion molecule (EpCAM) positive tumors and with the other arm CD3+ T cells. Intravenous application of therapeutic antibodies may result in intravascular cytokine release. AIM: In this pilot trial we assessed whether cytokine release can be controlled by ex vivo cell opsonization and cytokine wash-out before administration of catumaxomab, preserving its anti-cancer activity. In addition, preliminary data on safety of and clinical response to catumaxomab coated autologous immune cells were acquired. METHODS: Peripheral blood mononuclear cells (PBMNC) of four patients with recurrent head and neck carcinoma were collected by leukapheresis, incubated ex vivo with catumaxomab for 24 h and cleared from released cytokines. Each patient received an escalated number of antibody-coated PBMNC equivalent to 1 x 10(4), 1 x 10(5), 1 x 10(6) and 1 x 10(7) CD3(+) cells/kgBW intravenously at bi-weekly intervals. RESULTS: After opsonization, PBMNC released substantial amounts of interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) in vitro, which were removed before administration. Catumaxomab up-regulated CD25, CD69, and CD83 on PBMNC, and catumaxomab loaded PBMNC released IFNgamma and granzyme B when coincubated with EpCAM(+) BHY cells, suggesting cell activation and target directed biological activity. During the study period, one patient died of aspiration pneumonia and one patient needed a tracheotomy. Treatment related adverse events (AE) occurred at the highest cell dose in two patients, whereas 1 x 10(6) loaded CD3(+) cells/kgBW were well tolerated by all patients. One patient showed stable disease for 6 months and one patient is in complete remission for 27 months. CONCLUSION: Ex vivo opsonization of PBMNC with catumaxomab provided biologically active, tumor targeting cells. Extracorporeal PBMNC coating may be an option to control intravascular cytokine release induced by therapeutic antibodies.

188Re or 90Y-labelled anti-CD66 antibody as part of a dose-reduced conditioning regimen for patients with acute leukaemia or myelodysplastic syndrome over the age of 55: results of a phase I-II study.

In a phase I-II study for patients aged 55-65 years, we employed radioimmunotherapy using an anti-CD-66 antibody as part of a dose-reduced conditioning regimen, which was followed by a T-cell-depleted graft. 20 patients with a median age of 63 years suffering from acute leukaemia (n=17) or myelodysplastic syndrome (n=3) received the antibody labelled either with 188Rhenium (n=8) or with 90Yttrium (n=12) during conditioning. Radioimmunotherapy provided a mean dose of 21.9 (+/-8.4) Gy to the bone marrow with a significantly higher dose when 90Yttrium was used. Additional conditioning was fludarabine-based plus anti-thymocyte globulin in matched related donor transplants (n=11), or plus melphalan in matched unrelated donor transplants (n=9). Regimen-related toxicity was low, with two patients developing three episodes of grade III organ toxicity. All patients engrafted, grade II-IV acute graft-versus-host disease (GvHD) was observed in one patient (5%) and chronic GvHD in three patients (15%). The cumulative incidence of non-relapse mortality was 25%, the cumulative incidence of relapse 55%. The probability of survival was estimated to be 70% at 1 year and 52% at 2 years post-transplant, although no plateau was reached afterwards. In conclusion, radioimmunotherapy using the anti-CD66 antibody was feasible and safe in our elderly patient group and provided a high marrow dose.

[Dyspepsia and Helicobacter pylori infection in employees of a large industry. Results of a prospective BASF Helicobacter pylori prevention campaign]. / Dyspepsie und Helicobacter-pylori-Infektion bei Beschäftigten eines grossen Industrieunternehmens. Ergebnisse der prospektiven BASF-Helicobacter-pylori-Vorsorgeaktion.

BACKGROUND: The role of Helicobacter pylori (H. pylori) infection in dyspepsia is controversial. In the course of a health initiative within a large industrial corporation, we investigated the prevalence of both dyspepsia and positive H. pylori serology and the outcome of eradication therapy in symptomatic H. pylori positive employees. TEST PERSONS AND METHODS: H. pylori serology (IgG ELISA) was determined in 6,143 employees of BASF AG Ludwigshafen/Germany who were also asked to complete a standardized health history administered by a physician. Peptic ulcer disease (PUD) and dyspepsia subgroups were defined based on past medical history and symptom profiles using the criteria of Heading. Upper GI endoscopy, abdominal ultrasound and eradication therapy (Italian Triple Therapy) was recommended for symptomatic H. pylori positive individuals. The prognostic value of antibodies against CagA and VacA was evaluated in 37 and 39 employees with PUD and non-ulcer dyspepsia (NUD) confirmed by endoscopy, respectively. RESULTS: Of 6,143 employees, 1,255 (20.4%) were classified as dyspeptic, 492 (39.2%) of whom were H. pylori positive. The seroprevalence of H. pylori in asymptomatic employees was 35.8%. There were no significant differences in H. pylori seroprevalence among dyspepsia subgroups (reflux only, dysmotility only, reflux/dysmotility, ulcer-like and non-specific). However, individuals reporting severe dyspeptic symptoms were significantly more likely to be H. pylori positive (OR 2.09, CI 1.43-3.05). The seroprevalence of CagA and VacA was not significantly different among employees with NUD compared to referents or among employees with NUD compared to those with PUD. 330 (72%) of 458 employees with dyspepsia received eradication therapy, 128 persons refused therapy. Based on a 12-month follow-up of 402 individuals (300 of whom had received therapy), eradication success was 81.5% as judged by serology. Of the successfully treated employees, 33.2% reported a total absence and 42.8% reported a decrease in symptoms. Among the employees who refused therapy, the corresponding percentages were 37.3% and 16.7%, respectively. An increase in reflux complaints was not observed among treated employees. CONCLUSION: In a large active employee population, at most a very weak association was observed between the prevalence of H. pylori seropositivity and dyspepsia. Frequent and severe dyspeptic symptoms were associated with an increased rate of H. pylori seropositivity. The analysis of the virulence factors is not particularly helpful in discriminating PUD or NUD. Eradication of H. pylori infection leads to a decrease in dyspeptic symptoms after 12 months, but not more often to their complete absence compared to untreated individuals.