Validation of the Sysmex XN analyser and Blood Bank mode for the quality and safety of donor blood and transfusion products.

OBJECTIVES: Our objective was to compare the measurement of residual white blood cell (rWBC) and residual red blood cell (rRBC) counts in blood products using the XN Blood Bank mode and the laboratory standard operating procedures for manual counts. In addition, to compare the whole blood complete blood count (CBC) values of blood donors and the quality of blood products using the Sysmex XN analyser versus the XS-1000i analyser. MATERIALS AND METHODS: For blood donors, 190 samples from blood or apheresis donors were analysed on both the Sysmex XS-1000i and XN-1000 analysers and the mean values of six CBC parameters were compared: the white blood cell count (WBC), the red blood cell count (RBC), haemoglobin (HGB), haematocrit (HCT), the mean corpuscular volume (MCV), the platelet count (PLT). For blood products, 164 samples were collected: 13 Plasma products - whole blood, 9 Plasma products - apheresis, 36 RBC concentrates - whole blood, 30 PLT concentrates - buffy coats, 36 PLT concentrates - buffy coats - pooled and 55 PLT concentrates - apheresis. RESULTS: All CBC parameters of the blood donors tested showed similar performance, with excellent correlation coefficients (r) ranging from 0.821 to 0.995. The majority of the blood products did not have a quantifiable number of residual cells, meaning the number of rWBC and rRBC, if present, was below the limit of quantitation (LoQ) of the different methods. rWBC were detected by Blood Bank mode in Plasma products - whole blood with a mean rWBC of 0.012 × 109 /L and in PLT concentrates - buffy coats with a mean rWBC of 0.19 × 109 /L. The correlation coefficient in both analysers for all three parameters (HGB, HCT, RBC) in RBC concentrates - whole blood was excellent, ranging from 0.95 to 0.99. For platelet count, r ranged from 0.98 to 0.99. CONCLUSION: The XN-Series analyser, equipped with a Blood Bank mode, demonstrated reliable performance when used for blood donor evaluation, rWBC enumeration and measurement of end blood products.

The world apheresis association registry, 2023 update.

The WAA apheresis registry contains data on more than 140,000 apheresis procedures conducted in 12 different countries. The aim is to give an update of indications, type and number of procedures and adverse events (AEs). MATERIAL AND METHODS: The WAA-registry is used for registration of apheresis procedures and is free of charge. The responsible person for a center can apply at the site www.waa-registry.org RESULTS: Data includes reported AEs from 2012 and various procedures and diagnoses during the years 2018-2022; the latter in total from 27 centers registered a total of 9500 patients (41% women) that began therapeutic apheresis (TA) during the period. A total of 58,355 apheresis procedures were performed. The mean age was 50 years (range 0-94). The most common apheresis procedure was stem cell collection for which multiple myeloma was the most frequent diagnosis (51%). Donor cell collection was done in 14% and plasma exchange (PEX) in 28% of patients; In relation to all performed procedures PEX, using a centrifuge (35%) and LDL-apheresis (20%) were the most common. The main indication for PEX was TTP (17%). Peripheral veins were used in 56% as the vascular access. The preferred anticoagulant was ACD. AEs occurred in 2.7% of all procedures and were mostly mild (1%) and moderate 1.5% (needed supportive medication) and, only rarely, severe (0.15%). CONCLUSION: The data showed a wide range of indications and variability in apheresis procedures with low AE frequency.

NPM1 and DNMT3A mutations are associated with distinct blast immunophenotype in acute myeloid leukemia.

The immune system is important for elimination of residual leukemic cells during acute myeloid leukemia (AML) therapy. Anti-leukemia immune response can be inhibited by various mechanisms leading to immune evasion and disease relapse. Selected markers of immune escape were analyzed on AML cells from leukapheresis at diagnosis (N = 53). Hierarchical clustering of AML immunophenotypes yielded distinct genetic clusters. In the absence of DNMT3A mutation, NPM1 mutation was associated with decreased HLA expression and low levels of other markers (CLIP, PD-L1, TIM-3). Analysis of an independent cohort confirmed decreased levels of HLA transcripts in patients with NPM1 mutation. Samples with combined NPM1 and DNMT3A mutations had high CLIP surface amount suggesting reduced antigen presentation. TIM-3 transcript correlated not only with TIM-3 surface protein but also with CLIP and PD-L1. In our cohort, high levels of TIM-3/PD-L1/CLIP were associated with lower survival. Our results suggest that AML genotype is related to blast immunophenotype, and that high TIM-3 transcript levels in AML blasts could be a marker of immune escape. Cellular pathways regulating resistance to the immune system might contribute to the predicted response to standard therapy of patients in specific AML subgroups and should be targeted to improve AML treatment.

Mass spectrometry, data re-analysis, and homology modelling predict posttranslational modifications of leucine-rich alpha-2-glycoprotein as a marker of myelodysplastic syndrome.

BACKGROUND: Leucine-rich alpha-2-glycoprotein (LRG) has been repeatedly proposed as a potential plasma biomarker for myelodysplastic syndrome (MDS). OBJECTIVE: The goal of our work was to establish the total LRG plasma level and LRG posttranslational modifications (PTMs) as a suitable MDS biomarker. METHODS: The total plasma LRG concentration was determined with ELISA, whilst the LRG-specific PTMs and their locations, were established using mass spectrometry and public mass spectrometry data re-analysis. Homology modelling and sequence analysis were used to establish the potential impact of PTMs on LRG functions via their impact on the LRG structure. RESULTS: While the results showed that the total LRG plasma concentration is not a suitable MDS marker, alterations within two LRG sites correlated with MDS diagnosis (p= 0.0011). Sequence analysis and the homology model suggest the influence of PTMs within the two LRG sites on the function of this protein. CONCLUSIONS: We report the presence of LRG proteoforms that correlate with diagnosis in the plasma of MDS patients. The combination of mass spectrometry, re-analysis of publicly available data, and homology modelling, represents an approach that can be used for any protein to predict clinically relevant protein sites for biomarker research despite the character of the PTMs being unknown.

Proteome changes of plasma-derived extracellular vesicles in patients with myelodysplastic syndrome.

BACKGROUND: Extracellular vesicles are released into body fluids from the majority of, if not all, cell types. Because their secretion and specific cargo (e.g., proteins) varies according to pathology, extracellular vesicles may prove a rich source of biomarkers. However, their biological and pathophysiological functions are poorly understood in hematological malignancies. OBJECTIVE: Here, we investigated proteome changes in the exosome-rich fraction of the plasma of myelodysplastic syndrome patients and healthy donors. METHODS: Exosome-rich fraction of the plasma was isolated using ExoQuick™: proteomes were compared and statistically processed; proteins were identified by nanoLC-MS/MS and verified using the ExoCarta and QuickGO databases. Mann-Whitney and Spearman analyses were used to statistically analyze the data. 2D western blot was used to monitor clusterin proteoforms. RESULTS: Statistical analyses of the data highlighted clusterin alterations as the most significant. 2D western blot showed that the clusterin changes were caused by posttranslational modifications. Moreover, there was a notable increase in the clusterin proteoform in the exosome-rich fraction of plasma of patients with more severe myelodysplastic syndrome; this corresponded with a simultaneous decrease in their plasma. CONCLUSIONS: This specific clusterin proteoform seems to be a promising biomarker for myelodysplastic syndrome progression.

Using the World Apheresis Association Registry Helps to Improve the Treatment Quality of Therapeutic Apheresis.

Therapeutic apheresis (TA) is prescribed to patients that suffer from a severe progressive disease that is not sufficiently treated by conventional medications. A way to gain more knowledge about this treatment is usually by the local analysis of data. However, the use of large quality assessment registries enables analyses of even rare findings. Here, we report some of the recent data from the World Apheresis Association (WAA) registry. Data from >104,000 procedures were documented, and TA was performed on >15,000 patients. The main indication for TA was the collection of autologous stem cells (45% of patients) as part of therapy for therapy. Collection of stem cells from donors for allogeneic transplantation was performed in 11% of patients. Patients with indications such as neurological diseases underwent plasma exchange (28%). Extracorporeal photochemotherapy, lipid apheresis, and antibody removal were other indications. Side effects recorded in the registry have decreased significantly over the years, with approximately only 10/10,000 procedures being interrupted for medical reasons. CONCLUSION: Collection of data from TA procedures within a multinational and multicenter concept facilitates the improvement of treatment by enabling the analysis of and feedback on indications, procedures, effects, and side effects.

Integrin expression and adhesivity to fibronectin in primary acute myeloid leukemia cells: Impact of NPM1 and FLT3 mutations.

OBJECTIVES: Interaction of leukemia cells with the bone marrow extracellular matrix promotes cell survival and resistance to chemotherapy. In this work, we analyzed integrin expression and adhesivity to fibronectin in primary cells from patients with acute myeloid leukemia. METHODS: Surface expression of integrins ß1 and αVß3 on primary leukemia cells (N = 46) was correlated with the stem cell marker CD34, as well as with cell adhesivity to fibronectin. The results were analyzed with regard to the mutational status of NPM1 and FLT3 genes. RESULTS: The integrin ß1 was omnipresent, whereas αVß3 was often more expressed on CD34-positive cells. In particular, higher αVß3 expression on CD34+ cells was associated with NPM1 mutation (P = .0018). Monocytic leukemias had significantly higher αVß3 expression compared to less maturated cases (P = .0008). Cells from patients with internal tandem duplications in FLT3 (FLT3-ITD) had lower adhesivity to fibronectin compared to cells with wild-type FLT3 (P = .031), specifically in less differentiated myeloblasts. Inhibition of a putative FLT3-ITD target, EZH2, increased cell adhesivity in MV4-11 cell line (P = .024). CONCLUSIONS: The integrin αVß3 is expressed in particular on CD34+ cells with NPM1 mutation and might have a prognostic value in patients with mutated NPM1. FLT3-ITD is associated with lower cell adhesivity, especially in patients with less differentiated leukemias.

High PD-L1 Expression Predicts for Worse Outcome of Leukemia Patients with Concomitant NPM1 and FLT3 Mutations.

Compared to solid tumors, the role of PD-L1 in hematological malignancies is less explored, and the knowledge in this area is mostly limited to lymphomas. However, several studies indicated that PD-L1 is also overexpressed in myeloid malignancies. Successful treatment of the acute myeloid leukemia (AML) is likely associated with elimination of the residual disease by the immune system, and possible involvement of PD-L1 in this process remains to be elucidated. We analyzed PD-L1 expression on AML primary cells by flow cytometry and, in parallel, transcript levels were determined for the transcription variants v1 and v2. The ratio of v1/v2 cDNA correlated with the surface protein amount, and high v1/v2 levels were associated with worse overall survival (p = 0.0045). The prognostic impact of PD-L1 was limited to AML with mutated nucleophosmin and concomitant internal tandem duplications in the FLT3 gene (p less than 0.0001 for this particular AML subgroup).

Phase I/II trial of dendritic cell-based active cellular immunotherapy with DCVAC/PCa in patients with rising PSA after primary prostatectomy or salvage radiotherapy for the treatment of prostate cancer.

OBJECTIVE: Immunotherapy of cancer has the potential to be effective mostly in patients with a low tumour burden. Rising PSA (prostate-specific antigen) levels in patients with prostate cancer represents such a situation. We performed the present clinical study with dendritic cell (DC)-based immunotherapy in this patient population. MATERIALS AND METHODS: The single-arm phase I/II trial registered as EudraCT 2009-017259-91 involved 27 patients with rising PSA levels. The study medication consisted of autologous DCs pulsed with the killed LNCaP cell line (DCVAC/PCa). Twelve patients with a favourable PSA response continued with the second cycle of immunotherapy. The primary and secondary objectives of the study were to assess the safety and determine the PSA doubling time (PSADT), respectively. RESULTS: No significant side effects were recorded. The median PSADT in all treated patients increased from 5.67 months prior to immunotherapy to 18.85 months after 12 doses (p < 0.0018). Twelve patients who continued immunotherapy with the second cycle had a median PSADT of 58 months that remained stable after the second cycle. In the peripheral blood, specific PSA-reacting T lymphocytes were increased significantly already after the fourth dose, and a stable frequency was detected throughout the remainder of DCVAC/PCa treatment. Long-term immunotherapy of prostate cancer patients experiencing early signs of PSA recurrence using DCVAC/PCa was safe, induced an immune response and led to the significant prolongation of PSADT. Long-term follow-up may show whether the changes in PSADT might improve the clinical outcome in patients with biochemical recurrence of the prostate cancer.

Alpha-2-HS-glycoprotein plasma level decrease correlates with age in patients with myelodysplastic syndromes.

BACKGOUND: It has been indicated in plasma proteomic studies on different myelodysplastic syndrome (MDS) cohorts that alpha-2-HS-glycoprotein could be a promising MDS biomarker candidate. OBJECTIVE: The goal of this work was to estimate alpha-2-HS-glycoprotein (AHSG) plasma levels and its biomarker value in the low- and high-risk subgroups of MDS patients. METHODS: The level of AHSG was estimated for 115 plasma samples using ELISA. RESULTS: The AHSG plasma level was found to be decreased significantly (p= 2.59 × 10-7) in MDS patients (515 ± 58 µg/ml) when compared to healthy controls (579 ± 64 µg/ml). Pearson and Spearman correlation analyses showed that age is the principal factor affecting the AHSG plasma level, rather than risk/diagnosis in MDS. CONCLUSIONS: In this work we demonstrate that although the total plasma level of AHSG is decreased in myelodysplastic syndrome patients, in particular in advanced MDS, that decrease correlates more strongly with age than with diagnosis within our studied cohort. Thus, according to the AHSG data gathered so far, AHSG total plasma level does not seem to be a suitable MDS biomarker, but its particular proteoforms should be considered for the next steps in MDS research.

Phase I/II clinical trial of dendritic-cell based immunotherapy (DCVAC/PCa) combined with chemotherapy in patients with metastatic, castration-resistant prostate cancer.

PURPOSE: We conducted an open-label, single-arm Phase I/II clinical trial in metastatic CRPC (mCRPC) patients eligible for docetaxel combined with treatment with autologous mature dendritic cells (DCs) pulsed with killed LNCaP prostate cancer cells (DCVAC/PCa). The primary and secondary endpoints were safety and immune responses, respectively. Overall survival (OS), followed as a part of the safety evaluation, was compared to the predicted OS according to the Halabi and MSKCC nomograms. EXPERIMENTAL DESIGN: Twenty-five patients with progressive mCRPC were enrolled. Treatment comprised of initial 7 days administration of metronomic cyclophosphamide 50 mg p.o. DCVAC/PCa treatment consisted of a median twelve doses of 1 × 107 dendritic cells per dose injected s.c. (Aldara creme was applied at the site of injection) during a one-year period. The initial 2 doses of DCVAC/PCa were administered at a 2-week interval, followed by the administration of docetaxel (75 mg/m2) and prednisone (5 mg twice daily) given every 3 weeks until toxicity or intolerance was observed. The DCVAC/PCa was then injected every 6 weeks up to the maximum number of doses manufactured from one leukapheresis. RESULTS: No serious DCVAC/PCa-related adverse events have been reported. The median OS was 19 months, whereas the predicted median OS was 11.8 months with the Halabi nomogram and 13 months with the MSKCC nomogram. Kaplan-Meier analyses showed that patients had a lower risk of death compared with both MSKCC (Hazard Ratio 0.26, 95% CI: 0.13-0.51) and Halabi (Hazard Ratio 0.33, 95% CI: 0.17-0.63) predictions. We observed a significant decrease in Tregs in the peripheral blood. The long-term administration of DCVAC/PCa led to the induction and maintenance of PSA specific T cells. We did not identify any immunological parameter that significantly correlated with better OS. CONCLUSIONS: In patients with mCRPC, the combined chemoimmunotherapy with DCVAC/PCa and docetaxel was safe and resulted in longer than expected survival. Concomitant chemotherapy did not preclude the induction of specific anti-tumor cytotoxic T cells.

Roscovitine sensitizes leukemia and lymphoma cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand with selective antitumor activity. However, many primary tumors are TRAIL resistant. Previous studies reported that roscovitine, a cyclin-dependent kinase inhibitor, sensitized various solid cancer cells to TRAIL. We show that roscovitine and TRAIL demonstrate synergistic cytotoxicity in hematologic malignant cell lines and primary cells. Pretreatment of TRAIL-resistant leukemia cells with roscovitine induced enhanced cleavage of death-inducing signaling complex-bound proximal caspases after exposure to TRAIL. We observed increased levels of both pro- and antiapoptotic BCL-2 proteins at the mitochondria following exposure to roscovitine. These results suggest that roscovitine induces priming of cancer cells for death by binding antiapoptotic BCL-2 proteins to proapoptotic BH3-only proteins at the mitochondria, thereby decreasing the threshold for diverse proapoptotic stimuli. We propose that the mitochondrial priming and enhanced processing of apical caspases represent major molecular mechanisms of roscovitine-induced sensitization to TRAIL in leukemia/lymphoma cells.

Efficacy and safety of peripheral blood stem cell collection in elderly donors; does age interfere?

Elderly patients with hematological malignancies are often reliant on allogeneic transplantations. Older family relatives are increasingly involved in utilization as PBSC donors. We analyzed the mobilization results from 103 donors of age ≥55 years in comparison with 121 younger donors of age <55 years. The median CD34+ count in peripheral blood on day +5 of the mobilization was higher in younger than in older donor group (72.0 vs. 37.0 cells/µL, P < 0.0001). Linear regression showed a negative correlation between the age and CD34+ count in peripheral blood (P < 0.0001) and apheresis product (P < 0.0001). Based on multivariate analysis, the amount of circulating CD34+ cells appeared to be negatively influenced by age (P < 0.001) and positively by the preapheresis WBC count (P < 0.001). The precollection CD34+ (P < 0.0001), PLT (P = 0.0144) counts, and age (P = 0.0392) were confirmed as independent factors determining the collection yield. The side effects of G-CSF administration were similar in both the groups. Apheresis complications were more frequently recorded in elderly donors (29 vs. 15%, P = 0.0096). Higher age represents a risk factor for poorer mobilization results. A requirement for more than one apheresis in older donors occurs more frequently to obtain the adequate amount of CD34+ cells. Mobilization and collection procedures are associated with acceptable risks and complication rates in elderly donors.

PBPC collections: Management, techniques and risks.

We evaluated the efficiency, safety and risks of three techniques which were used for autologous PBPC collections: (a) large-volume leukapheresis (LVL), (b) standard collections, and (c) a new modified technique which was named as "Mixed" collections. In spite of the fact that the standard and LVL collection techniques are used routinely, there may occur special conditions in which the procedures cannot be recommended. Some patients may suffer from serious clinical complications and they cannot tolerate either standard procedures with administration of higher doses of ACD-A, or the high extent of procedure in the course of LVL. We tried to find the safe and efficient collection technique which could help this group of patients to overcome their problems. The "Mixed" collection technique could be such a choice. The numbers of 136 autologous PBPC collections were performed in 98 patients who suffered from hemato-oncological diseases. We evaluated the results of (a) 93 LVL (more than 3 TBV, total blood volumes of the patients were processed; anticoagulation: ACD-A and Heparin), (b) 16 Standard procedures (less than 3 TBV were processed; anticoagulation: ACD-A), and (c) 27 "Mixed" collections (less than 3 TBV of patients were processed; anticoagulation: ACD-A+ Heparin). Collections were performed by the use of separator Cobe Spectra, Caridian. In patients (a) with a good effect of mobilization (precollection CD 34+ cells in blood higher than 20×10(3)/mL) we prepared almost the same median dose of CD 34+ cells from the standard and "Mixed" collections, 3.8 and 4×10(6)/kg, respectively. In LVL the median yield of CD 34+ cells was 8.2×10(6)/kg. In patients (b) who were mobilized weakly (precollection CD 34+ cells in blood lower than 20×10(3)/mL), LVL enabled to prepare 1.5×10(6) of CD 34+/kg from one collection, while the median yield of CD 34+ cells from the standard and "Mixed" collections was 0.9 and 1.2×10(6)/kg. All the standard, LVL and "Mixed" procedures were tolerated well without any serious adverse reactions. We detected 22 adverse reactions, but only three reactions were associated directly with the procedure. Mild hypocalcemia (2) and hypotensive reaction (1) were transient and treated efficiently. Procedures could continue and were finished according to the planned programme. Other reactions were related either to the insufficient function of central venous catheter or to the poor clinical condition of the patients. LVL enabled to get a higher yield of CD 34+ cells than the Standard and "Mixed" collections in well mobilized patients as well as in weakly mobilized patients. We observed the similar efficiency in standard and "Mixed" collections in well mobilized and weakly mobilized patients. We can recommend LVL in all patients who can tolerate it due to a greater chance of collecting higher yields of progenitor cells. In the weakly mobilized patients LVL offers a greater chance of collecting at least a minimum amount of CD 34+ cells needed for transplantation. "Mixed" collections may be used as an alternative technique under the circumstances in which standard or LVL cannot be recommended - like in patients who do not tolerate a high amount of citrate or a high extent of the procedure, e.g. patients with cardiac arrhytmia, impaired liver or renal function or unstable vital signs.

The influence of RhD phenotype on toxoplasmosis- and age-associated changes in personality profile of blood donors.

Toxoplasma gondii, a parasite infecting 20-60% of humans in various countries, influences the behaviour of infected animal and human hosts. Infected human subjects have changes in several of Cattell's and Cloninger's personality factors. Recently, three independent studies have shown that Rh-positive subjects are protected against the T. gondii-induced changes of reaction times and increased risk of traffic accidents. Here we searched for evidence of similar effects of RhD phenotype on toxoplasmosis- or aging-associated changes in the personality profile of about 302 blood donors. We found that Rh-positive and Rh-negative subjects responded differently to toxoplasmosis. In addition to the already known effects of toxoplasmosis on novelty seeking, self transcendence, superego strength and protension, we also found effects of RhD phenotype on ego strength, protension, and praxernia, as well as opposite effects of toxoplasmosis on ego strength, praxernia, ergic tension and cooperativeness in Rh-positive and Rh-negative subjects. Moreover, our results indicate that RhD phenotype might influence not only the effect of toxoplasmosis but also the effect of aging on specific personality traits.

TRAIL-induced apoptosis of HL60 leukemia cells: two distinct phenotypes of acquired TRAIL resistance that are accompanied with resistance to TNFalpha but not to idarubicin and cytarabine.

TNF-related apoptosis-inducing ligand (TRAIL) is a proapoptotic cytokine implicated in cancer cell surveillance. A potential of TRAIL as a cancer-specific therapeutic agent has been proposed, either as a single agent or in combination with chemotherapy. Prolonged exposure of TRAIL-sensitive leukemia cell line, wild-type (WT) HL60 cells to recombinant soluble TRAIL or to cytostatic agents, cytarabine and idarubicin, resulted in the establishment of resistant subclones with distinct phenotypic features. The TRAIL resistant HL60 subclones were characterized by decreased expression of TRAIL and TNFalpha death receptors. These resistant subclones had impaired activation of caspases 8 and 10 in response to TRAIL and TNFalpha, decreased TRAIL-induced nuclear translocation of NFkappaB RelA/p65, and dysregulation of the expression of several apoptosis regulators. Among the TRAIL resistant HL60 subclones we identified two separate phenotypes that differed in the expression of CD14, osteoprotegerin, and several apoptosis regulators. Both these TRAIL resistant HL60 subclones were resistant to TNFalpha, suggesting disruption of the extrinsic apoptotic pathway, but not to cytostatic agents, cytarabine and idarubicin. The concurrently derived HL60 subclones were cytarabine and idarubicin-resistant but remained sensitive to TRAIL-induced apoptosis. We identified distinct pathways for the development of HL60 leukemia cell resistance to apoptosis induction. These findings are relevant for the design of more effective strategies for leukemia therapy.

Extracorporeal photochemotherapy (ECP) in treatment of patients with c-GVHD and CTCL.

We evaluated the immunomodulatory and clinical effect of 279 extracorporeal photochemotherapy (ECP) procedures which were performed in six patients with chronic extensive GVHD and in two patients with CTCL (cutaneous T-cell lymphoma)/Mycosis fungoides. ECP was performed using the off line regimen. In five of six patients with c-GVHD the improvement of sclerodermatous skin changes, joint mobility, and the reduction of joint pain was observed. Two patients with CTCL responded to ECP with rapid improvement of the skin changes. In patients with c-GVHD and CTCL who responded to ECP efficiently, we found the similar tendency to increase in the number of CD 3/8+ T-lymphocytes and the decrease of CD 4/8 IRI. In patients with CTCL we observed also the decrease in levels of CD 3/4+ T-lymphocytes and in the number of leukocytes. The influence of ECP on T-cell subsets and on the dendritic cells function, which we observed in our previous study, leads to the suggestion that interactions between T-cell subsets and dendritic cells may participate in the process of ECP. ECP did not cause any significant changes in levels of IgG, parameters of liver and renal functions in patients with c-GVHD and with CTCL. No increased incidence of infections and no serious adverse reactions in patients have been observed so far.

UVA-activated 8-methoxypsoralen (PUVA) causes G2/M cell cycle arrest in Karpas 299 T-lymphoma cells.

We investigated the effect of UVA-activated 8-methoxypsoralen (PUVA) on the cell line Karpas 299 derived from anaplastic large-cell lymphoma (ALCL) expressing chimeric fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM/ALK). NPM/ALK activates phosphatidylinositol 3 kinase (PI3K)/Akt pathway responsible for the cell protection from apoptosis. We found that PUVA treatment first induced G2/M cell cycle arrest resulting in a decrease in the cell proliferation rate. The mitochondrial apoptosis was triggered immediately following PUVA treatment, as we judged from the unmasking of mitochondrial membrane antigen 7A6. However, the mitochondrial membrane depolarization was not observed and caspase-3 was only slightly activated. The late apoptotic events were lacking: neither translocation of phosphatidylserine to the outer side of plasma membrane nor DNA fragmentation occurred. We revealed that PUVA enhanced the expression of peroxiredoxin, stress protein endoplasmin and galectin-3. Galectin-3 has been shown to protect mitochondrial membrane integrity and prevent cytochrome c release thereby blocking the effector stage of apoptosis. We suggest that the elevated level of this protein following PUVA treatment acts in synergy with the constitutively expressed chimeric kinase NPM/ALK to block the apoptosis.

Decreased level of novelty seeking in blood donors infected with Toxoplasma.

OBJECTIVES: Toxoplasma gondii, a parasitic protozoan, infects about 30 - 60% of people worldwide. Toxoplasma is known to induce behavioral changes and an increase of dopamine in mice. The presence of anti-Toxoplasma antibodies (latent toxoplasmosis) is also a risk factor for schizophrenia. Latent toxoplasmosis in men (male soldiers) is associated with lower novelty seeking. As the novelty seeking is supposed to negatively correlate with level of dopamine, the observed effect was interpreted as indirect evidence of increased dopamine levels in subjects with toxoplasmosis. However, it is also possible that the observed effect was caused by association of both novelty seeking and Toxoplasma infection with a third factor, e.g. size of place of residence. METHODS: Personality profile of 290 blood donors (205 men and 85 women) were measured by Cloninger's TCI (Temperament and Character Inventory) and their blood samples were assayed for the presence of anti-Toxoplasma antibodies. Difference between Toxoplasma-infected and Toxoplasma-free subjects was tested with ANCOVA method with gender, size of place of residence, and age as covariates. RESULTS: The present analysis revealed that lower novelty seeking was associated with latent toxoplasmosis both in men and women. The effect of infection on novelty seeking remained significant even after adjustment for size of place of residence (p<0.01). CONCLUSION: Decreased novelty seeking in Toxoplasma-infected subjects have been already confirmed in three independent populations (male soldiers and male and female blood donors). These findings suggest that the local inflammation-induced increase in dopamine in the brain of infected subjects can represent a missing link between toxoplasmosis and schizophrenia.

PBPC collection techniques: standard versus large volume leukapheresis (LVL) in donors and in patients.

Transplantations of autologous and allogeneic peripheral blood progenitor cells (PBPC) are able to assure a complete hematopoietic and immunologic reconstitution in patients. PBPC are collected by leukapheresis technique after prior mobilization therapy, but procedures and results remain still highly variable and are poorly characterized. An optimum regimen for PBPC collections has not yet been recommended, but 2-3 total blood volumes (TBV) of the donor or patient are regarded as a standard. Another promising technique is large volume leukapheresis (LVL) with processing of 3-6 TBV of donor or patient. The aim of this paper is to find the most efficient and safe collection technique for an individual donor or patient and, consequently minimize the number of procedures required. Finding the optimal collection procedure would be helpful while considering which method would be preferred in an individual donor or patient with respect to the result of mobilization, health state and required yield of CD 34+ cells for transplantation. We evaluated the results in a total of 134 standard and LVL procedures, which were performed in 21 well mobilized donors (Group I), in 65 well mobilized patients (Group II), and in 14 weakly mobilized patients (Group III) with hemato-oncological diseases. A precollection concentration of CD 34+ cells in peripheral blood higher than 20 x 10(3)/mL was considered to be the criterion for efficient mobilization. Such levels of concentration indicating the start of PBPC collections could be easily reached in Group I of donors and Group II of well mobilized patients. Heavily pretreated patients at advanced stages of disease (Group III) did not respond to mobilization sufficiently and had a concentration of CD 34+ cells lower than 20x10(3)/mL. LVL technique made it possible to obtain higher numbers of CD 34+ cells than in the standard collection in well mobilized donors (Group I), well mobilized patients (Group II), and even in weakly mobilized patients in Group III. In donors and well mobilized patients (Group I and Group II) it was possible to collect sufficient amounts of CD 34+ cells for allogeneic or for autologous transplantation from one LVL collection. The median yield of CD 34+ cells from one LVL collection was 5.5 x 10(6)/kg b.w. in donors, and 6.0 x 10(6)/kg b.w. in well mobilized patients. Due to the linear dependence of the yield of collected CD 34+ cells on the concentration of CD 34+ cells in blood, it can be used as a simple prediction of the success of collection in Group II (correlation coefficient 0.93 for standard procedures, and correlation coefficient 0.88 for LVL). In Group III of weakly mobilized patients the standard collections were usually ineffective and the relationship between the yield of CD 34+ cells/kg in the product and the precollection concentration of CD 34+ cells was much less significant (correlation coefficient 0.56 for standard procedures and correlation coefficient 0.66 for LVL). The median of CD 34+ cells collected from one standard procedure was only 0.7 x 10(6)/kg but using LVL the median increased to 1.4 x 10(6)/kg. Our results prove that the yield of CD 34+ cells in the product can be enhanced by large volume leukapheresis (LVL). Based on the results obtained, we recommend LVL in all donors and patients who can tolerate it due to a greater chance of collecting higher yields of progenitor cells while minimizing adverse reactions. LVL procedures should also be preferred in weakly mobilized patients where it is not possible to collect sufficient amounts of CD 34+ cells for transplantation using the standard regime. In weakly mobilized patients LVL provides a greater chance to at least collect a minimum amount of CD 34+ cells necessary. LVL should be used in circumstances where extremely high doses of CD 34+ cells has to be prepared, e.g. planned "tandem" transplantations or manipulations with a graft in which a significant loss of cells is expected.