Aequorin as Intracellular Ca2+ Indicator Incorporated in Follicular Lymphoma Cells by Hypoosmotic Shock Treatment.

Natural proteins can be used in measuring intracellular Ca(2+) concentration. As one of the Ca(2+)- regulated photoproteins, aequorin has several advantages in comparison to widely used Ca(2+) fluorescence indicators (e.g., fura-2, indo-1 and fluo-3), including high dynamic range and resistance to motion artefacts. However, incorporation of aequorin into cells remains a challenge. Hypoosmotic shock treatment was optimized and used as a method for loading aequorin into the cytoplasm of follicular lymphoma cells. Measurement of aequorin luminescence in the cells was performed using a luminometer with a sensitive photomultiplier tube and the luminescence intensity was recalculated into intracellular [Ca(2+)]. The value of (0.85 ± 0.52)·10-6 M was found. We show that the optimized method of incorporation was effective for loading aequorin into follicular lymphoma cells in vitro. The cell viability remains high immediately after the procedure. This method can also be used for measuring intracellular Ca(2+) concentration in other types of non-adherent cells.

[The Role of Regulatory T-cells in Antitumor Immune Response]. / Úloha regulacních T bunek v protinádorové imunitní odpovedi.

Regulatory T-lymphocytes (Treg) are essential for regulation of immune homeostasis and prevention of autoimmune disease development. Regulatory T-cells prevent the onset of autoimmune diseases; they keep immune homeostasis and modulate immune response during infection. Their activity is precisely controlled. Regulatory T-cells belong to one group of immune cells, which can support tumor survival and growth. They realize their function through inhibition of effector T-cells and by regulation of tumor microenvironment through production of various soluble factors. Many publications have proven that the amount of Treg cells is elevated in both solid tumors and in hematologic malignancies. Nevertheless, little is known about mechanisms, which allow increase and maintenance of elevated Treg cells in cancer patients. In this review, we will focus, among others, on the description of function and phenotype of Treg cells, their modulation of humoral immune response and interaction with cancer stem cells. Current development of modern tumor immunotherapy allows new possibilities of influencing Treg cells function.

Thiazolidinediones Regulate the Level of ABC Transporters Expression on Lung Cancer Cells.

ATP binding cassette (ABC) transporters related to multidrug resistance (MDR) actively efflux various xenobio-tics from the cells across the cell membrane and decrease a drugs efficiency. Lung cancer is the leading cause of death among all types of cancer in the Czech Republic, and its incidence is still rising. Ciglitazone, rosiglitazone and troglitazone belonging to PPARγ agonist family (formerly used in diabetes mellitus treatment) were selected to investigate their capability to influence expression of ABC transporters on lung cancer cells. Therefore, the effect of PPARγ of agonists on transcription of following ABC transporters was investigated: multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein (BCRP). We have investigated if these PPARγ agonists are substrates of ABC transporters using HL60 and HL60 derived cell lines (HL60-MDR1, HL60-MRP1, PLB-BCRP) by cytotoxicity test WST-1. We have mapped the changes in mRNA expression level of those transporters in A549 and HEK293 cells after PPARγ agonists treatment using quantitative reverse transcription real-time PCR (qRT-PCR). All three PPARγ agonists serve as substrates to at least one ABC transporter under study. PPARγ activation correlates with up-regulation of PTEN which may modulate the expression of ABC transporters through PI3K/ Akt signaling pathway. We have shown that rosiglitazone and troglitazone inhibit mRNA expression of MDR1 transporter in both cell lines whereas the expression of MRP1 in HEK293 cell was up-regulated after rosiglitazone treatment and the expression of MDR1 was upregulated after ciglitazone treatment.

Tebentafusp in the treatment of metastatic uveal melanoma - the first patient treated in the Czech Republic.

BACKGROUND: Uveal melanoma is a rare cancer, in which metastases occur in approximately one half of cases. In metastatic disease, the prognosis is unfavorable and the median of survival does not exceed 6 months. Effective treatment options were very limited up to date. Tebentafusp is a bispecific fusion protein, which as the first drug proved efficacy in uveal melanoma. CASE: The patient was referred for suspected uveal melanoma of the left eye. She was treated for Hodgkin's disease in the past. Primarily, the tumor was treated by radiosurgery with radiotherapy of a small lesion of the vertebral body. However, later the patient had to undergo bulbus enucleation with confirmation of a large tumor category pT4b. PET/CT revealed metastases of the bones and the liver; simultaneously, haplotype A*02: 01 was confirmed. The patient underwent radiotherapy of the sternum and later, after confirmation of payment from the health insurance company, she started treatment with tebentafusp. The first three doses were administered during admission to the hospital, with a need to treat cytokine release syndrome by corticosteroids. Later, the administration was performed in an out-patient regimen, without complications, except for a transient elevation of transaminases. The first CT restaging confirmed stable disease; however, the second restaging confirmed a new osteolytic lesion in the processus of Th11. Because of progression, the treatment with tebentafusp was withdrawn after 6 months. Unfortunately, the lesion could not be treated by radiotherapy. Two months later, the patient was urgently admitted to the hospital because of right-sided hemiplegia; MRI revealed bleeding metastatic lesion in the brain stem. CONCLUSION: In this case report, we present the case of the first patient treated with this drug in the Czech Republic.

Relative expression of γδ T-cell receptor gene families detected by RT-PCR and capillary electrophoresis.

γδ T cells are intensively studied because their function in infection, allergy, autoimmune disease, cancer and post-transplant period is not yet fully understood. PCR-based techniques were established to study the γ variable (Vγ) and δ variable (Vδ) gene families. PCR product evaluation is routinely carried out by Southern blot analysis or the third complementarity-determining region spectratyping, but a fast and simple assessment of Vγ and Vδ gene family expression is missing. The aim of our study was to test capillary electrophoresis as a potential method for evaluating the composition of the γδ T-cell population. This report provides optimized PCR conditions for γδ T-cell receptor amplification. Further, it describes the utilization of capillary electrophoresis in the Agilent 2100 Bioanalyzer to evaluate the relative expression of Vγ and Vδ gene families after their amplification. An application of the methodology to peripheral blood mononuclear cell samples from patients during haemato-oncological treatment is shown. The described methodology is fast and simple to operate and is therefore suitable as a first screening of the γδ T-cell population composition in tissues of interest.

[Schnitzler syndrome: case report, the experience with glucocorticoid and anakinra (Kineret) therapies and monitoring of systemic cytokine response]. / Schnitzler-syndrom: popis prípadu, zkusenosti s lécbou glukokortikoidy a preparátem anakinra (Kineret) a sledování cytokinové odpovedi organizmu.

Schnitzler syndrome is a rare idiopathic disease characterized by chronic urtica, presence of monoclonal IgM immunoglobuline and further, less common symptoms. This case report describes another case of this disease affecting a male adult born in 1963. The first symptoms, eruptions of non-pruritic urticarial rash, appeared in this patient at the age of 43. In addition, bone pains (mainly tibias) and joint pains (mainly knees) were present. Later on however, severe attacks of fever, chills and shaking together with bone and joint pains were added to during which new urticarial eruptions appeared. Primarily, the man was followed up without any substantial therapeutic results at a department of dermatovenerology, subsequently, due to a finding of monoclonal IgM kappa immunoglobulin (serum concentration 1.9 g/l) he was referred to our department for the reason of gammopathy being a differential diagnosis. On a CT scan hyperostosis in claviculae and pelvic bones was identified. Also on the CT, an increase in cortical thickness was described in the long bones of the lower extremities, where areas of technetium pyrophosphate accumulation were identified on a bone scintigraphy. These areas were found in the chest and sacral regions as well. From the blood exams, the proinflammatory status of the organism was apparent (CRP 35.9 mg/l, erythrocyte sedimentation rate 92 mm/h, leukocytes 12.4 x 10(9)/l). After excluding other differential diagnoses, the patient was diagnosed with Schnitzler syndrome. As regards therapy, we made initial use of the effect of corticoids which abated the symptoms, however, these were causing serious adverse reactions in the form of iatrogenous Cushing's syndrome. The therapy took a turn only after biologic therapy with anakinra (interleukin-1 receptor antagonist) had started, which minimized the Schnitzler symptoms with very good drug tolerance. In the work we measured serum levels of interleukins for disease activity monitoring. The most sensitive were interleukins IL-6 and especially IL-18 the levels of which were the highest at the time of clinical exacerbation of the disease, whereas the levels of IL-1beta and TNF-alpha (tumour necrosis factor) were during all measurements below the limit of detection. Concerning the growing numbers of the reports on successful biological therapy with anakinra and our positive experience, we propose that the therapeutic response to anakinra should be included within the diagnostic criteria of Schnitzler syndrome, which is significant above all in differential diagnosis thereof.

Microarray analysis using a limited amount of cells.

cDNA microarray technology is widely used in various biological and medical disciplines to determine gene expression profiles. Unfortunately, this technology requires a large quantity of input RNA. Since there is an increasing need for more precise analyses of defined cell subpopulations with low cell counts, working protocols using a minimal number of input cells are required. Optimal RNA isolation and its accurate amplification are crucial to the success of these protocols. The HL-60 cell line was used in the search for a suitable protocol that can be used for clinical samples of CD34+ haematopoietic cells obtained from bone marrow. The goal was to discover the best method for isolating and amplifying RNA from a small number of cells. Our evaluation of various methods and kits available in the market revealed that the combination of RNAqueous Kit for RNA isolation and the SenseAmp Plus Kit for one-round RNA amplification produced the best results. This article presents a verified protocol describing a reliable and reproducible method for obtaining enough input RNA for microarray experiments when the number of cells is limited.

[Effect of growth factor on the phenotype of subpopulations and on the kinetics of CD34+ cells in the peripheral blood and in grafts of peripheral stem cells in patients with non-Hodgkin's lymphoma indicated for autologous peripheral blood stem cell transplantation]. / Vliv typu rustového faktoru na fenotyp subpopulací a kinetiku CD34+ bunek v periferní krvi a stepech periferních kmenových bunek pacientu s non-hodgkinskými lymfomy indikovaných k autologní transplantaci krvetvorby.

BACKGROUND: Peripheral blood stem cells are the preferred source for transplantation of hematopoiesis in patients with non-Hodgkin's lymphoma. Application of hematopoietic growth factors is a part of the mobilization chemotherapy regimen. Time overlap of the highest leukocyte and CD34+ cell count is required for optimal graft collection. Authors analyzed the effect of two growth factors (leridistim and filgrastim) on the kinetics and phenotype of CD34+ cells in patients with non-Hodgkin's lymphoma indicated for autologous peripheral blood stem cell transplantation. METHODS AND RESULTS: Authors analyzed phenotype of CD34+ cell subpopulations and their kinetics in peripheral blood and leukapheresis products by flow cytometry during mobilization and graft collection. Statistically significant differences in expression of lineage-committed antigens between growth factors were found (CD3, CD5--T-lineage, CD56 NK-lineage, CD20 for B-lineage, p < 0.05), as well as for lineage non-specific antigens (CD38, p < 0.05 and CD54, p < 0.01). The most significant divergence was observed between CD34+CD19+ subpopulations of leridistim and filgrastim stimulated blood and graft (p < 0.001). CONCLUSIONS: Expression of lineage-committed antigens on CD34+ subpopulations between two growth factors was statistically different. Kinetics of CD34+ cells during mobilization regimen with leridistim was not superior to filgrastim concerning the quality of graft.

[Rare diagnosis of CD4+56+ leukemia from dendritic cells type DC2]. / Vzácná diagnóza: CD4+56+ leukémie z dendritických bunek typu DC2.

CD4+56+ hematodermic neoplasm or leukemia from early plasmocytoid dendritic cells type DC2 was recognized by WHO-EORTC classification of cutaneous lymphomas as a separate entity related to the plasmacytoid precursor dendritic cell (pDC). This diagnosis is based on expression of CD4 and CD56 antigens and absence of B, T or myeloid lineage markers. Immunohistochemistry and flow cytometry are the only methods, which allow identification of this disease, either in isolated skin lesions or in a leukemic form. Although the co-expression of CD4 and CD56 is rare and the number of described cases is low, this group bears similar characteristics in a clinical course of disease. It is a very aggressive leukemia/lymphoma, usually with primary skin involvement, in half of the cases infiltrating bone marrow or lymph nodes. Despite high rate of initial response to treatment, early and widespread relapses occur and patients die of disease progression. Although the physiological counterpart of tumour cells was identified, the origin of the disease is still discussed because of aberrant expression of cell markers. Optimal treatment is not known. However, this aggressive disease requires radical approach with intensive chemotherapy regimens, prophylaxis of CNS involvement and early indication of allogeneic bone marrow transplantation. Two case reports are described.

The importance of immune system disorders related to the Glasgow Outcome Score in patients after severe brain injury.

BACKGROUND: It is assumed that changes in different parts of the host defence mechanisms could influence the outcome by making way for extracranial complications. MATERIAL AND METHODS: For our study we enrolled 121 patients who had sustained various types of severe head injury (GCS 3-8). We investigated several laboratory parameters. Furthermore we observed all extracranial complications that occurred within first ten days of hospitalization. Six months after the discharge we assessed GOS and we put all these parameters in correlation. RESULTS: 55.37 % of the patients ended our treatment with an unfavourable outcome; mortality of the group was 40.5 %. We noted 117 cases of extracranial inflammatory complications, occurring in 94 patients (77.6 %). We confirmed a significant correlation between lower GCS and higher incidence of infection. Concerning the relationship between immune system disorders and outcome, we disclosed a significant difference, especially when comparing the group with good recovery with the patients who died. The difference was seen in cellular parameters, whereas humoral parameters showed no significant changes. CONCLUSIONS: While GCS changes didn't relate to the intensity of immunity disorders, GOS value shows a strong relationship, especially to its cellular part. However, both GCS and GOS values showed an important correlation with the occurrence of infectious complications. Patients ending our treatment with bad outcome have more extracranial complications when compared with the patients that have ended our treatment successfully (Tab. 3, Fig. 6, Ref. 15). Full Text (Free, PDF) www.bmj.sk.

The effect of head injury upon the immune system.

Severe head injuries are characterized by high mortality and morbidity. In spite of guidelines based therapy the treatment is frequently unsuccessful. Extracranial infectious complications are considered to be an important problem during the course of recovery, and possibly immunological changes could explain their occurrence. Head injuries cause an imbalance within the helper cell community, resulting in a T(H)2 dominance. This development is influenced by the soluable agents of the sympathic nervous system and the hypothalamic-pituitary-adrenal axis. The crucial research of damaged cellular immunity concluded Quattrocchi in 1991. Both the activation of microglial cells and the accumulation of T-cells after crossing the BBB indicate production of pro-inflammatory mediators in the CNS after injury. The leaking of pro-inflammatory mediators to the circulation develops to a systemic inflammatory response syndrome (SIRS). On the contrary, an overwhelming of anti-inflammatory substances leads to an anti-inflammatory response syndrome (CARS). It is suggested that an imbalance between these two immune responses is responsible for organ dysfunction and increased susceptibility to infections in polytrauma victims. Concerning mediators, IL-6 draws attention because of its high marker ability. Finally, post-traumatic infections have also been correlated with an altered function of antigenpresenting cells (APC). Concerning the quantity, the humoral part of immune system seems to be stimulated, but its function and phagocyte activity shows several defects. Finally, T(H)2 dominance induces IgE levels accumulation. All these changes are strongly under effect of stress based release of endogenous glucocorticoids and catecholamine, which influence the complex network of cytokines and cell mediators (Fig. 3, Ref 18).

[The subpopulation of CD34+ cells and their importance in the healing of grafts in family-related allogenic transplants of peripheral stem cells]. / Subpopulace CD34+ bunek a jejich vyznam pro prihojeni stepu u príbuzenských alogenních transplantací periferních kmenových bunek.

CURRENT STATUS: The determination of concentration of CD34+ cells is the standard method for evaluation of the quality of a bone marrow graft and of peripheral stem cells. Although the relationship between the dose of CD34+ cells and the speed of graft healing in autologous transplants is a proven fact, it may not always be the case in allogenic transplants. PATIENTS AND METHOD: The correlation between the dose of CD34+ cell subpopulations and the speed of healing was monitored in patients indicated for allogenic transplantation of haematopoietic stem cells. The patients were divided according to the type of preparatory regimen they underwent for the purpose of analysis; one group contained those under a myeloablative regimen; a second group contained those under a non-myeloablative regimen. The data was subject to analysis of variance in regression models and non-parametric tests. RESULTS: From among the monitored subpopulations, CD34+36+ cells had the greatest effect on the healing process and were the most significant predictor of the speed of healing in patients under a myeloablative regimen. Nevertheless, a dose ofCD34+ cells continued to be the best healing predictor in patients under a non-myeloablative regimen. Also subpopulations of CD34+38+ and CD34+61+ cells had a significant effect on the speed of healing in both groups. CONCLUSION: Haematopoietic stem cells and progenitor cells defined by co-expression of specific antigens are likely to play a role, through different mechanisms of action, in the process of healing in patients in different pre-transplant regimens. While the dose of CD34+ cells is still the one which correlates best with the speed of healing in patients who underwent transplantation after non-myeloablative regimen, the dose of CD34+36+ cells appears to be a better predictor for the speed of healing after myeloablative regimens.

The use of controlled mild hypothermia and immune system status in patients with severe brain injury.

INTRODUCTION: Severe brain injuries pose one of the most important problems on our health care because of their high morbidity and mortality. MATERIAL AND METHODS: A group of 89 patients after severe brain injury (Glasgow Coma Scale< or =8) was included into our research of detecting the changes of immune system parameters and their relation to the application of mild hypothermia during the early period after the insult. RESULTS: In both of the groups CD3+ and CD4+ lymphocytic levels decreased significantly after the insult and gradually got to normal (p<0.01). The NK cells levels have changed in correlation with the course of infection. Immunoglobulin (IgA, IgG) levels were normal or slightly increased. IgM levels changes had a close relation to the occurrence of inflammatory complications, especially that of pneumonia (p<0.01). The most surprising moment in our research was the level of IgE antibodies. They had been high and got even higher. They achieved the values typical for atopic reactions or parasitic diseases. 77.52 % of the patients with decreased parameters of immune system developed extra cranial complications. Immune system disorders appeared more frequently in the patients with lower Glasgow Coma Scale after admission (p<0.01). The application of mild hypothermia caused an unimportant increase in extra cranial complications (p>0.05) having no relation to immunity disorders. CONCLUSION: Intensive treatment of intracranial hypertension fundamentally affects results of our treatment (Glasgow Outcome Score). The application of controlled mild hypothermia doesn't escalate the occurrence of extra cranial inflammatory complications after severe brain injury (Tab. 2, Fig. 11, Ref. 15).

[Density expression of the CD20 antigen on population of tumor cells in patients with chronic B-lymphocyte lymphoproliferative diseases]. / Denzita exprese antigenu CD20 na populaci nádorových bunek u pacientu s chronickými lymfoproliferacemi B-lymfocytární rady.

BACKGROUND: Rituximab is being used successfully in the treatment of patients with chronic B-cell lymphoproliferative diseases. The success of treatment by rituximab is influenced, among other factors, by the antigen density on tumor cells. Therefore, the authors analyzed and compared the densities of the CD20 antigen in patients with chronic lymphoproliferative diseases. METHODS AND RESULTS: Previously untreated patients with B-chronic lymphocytic leukemia (B-CLL), mantle-cell lymphoma (MCL), and small-cell lymphocytic lymphoma (SCLL) were evaluated by flow cytometry. The control group consisted of blood donors. The CD20 density was measured on tumor cell populations in patients and on the B-lymphocytes of the control group. The density was expressed in MESE In the patients with B-CLL and SCLL, the CD20 density was low (25,300 vs. 36,100 MESF) and it was significantly lower than in donors (172,800 MESF; p<0.001). The difference between B-CLL and SCLL patients was not statistically significant. The density in MCL patients (196,300 MESF) was comparable to that of donors. CONCLUSIONS: We did not prove statistical different density of CD20 antigen in patients with SCLL when compared with B-CLL patients. High density in MCL patients may be helpful in differential diagnosis against B-CLL and

Immune system status in the patients after severe brain injury.

INTRODUCTION: Extracranial complications occuring after severe brain injury definitely aggravate clinical status and debase Glasgow Outcome Score (GOS). The immune system disorders could cause for example pneumonia and other inflammatory complications - urinary infection, coagulopathy, etc. MATERIAL AND METHODS: We have admitted and observed a group of 8 patients with various degree of brain injury and we have demonstrated some changes of immune system parametres after the insult. RESULTS: Most of the patients had a significant loss in cell mediated immunity parametres, especially T-lymphocytes (CD3+, helper cells) levels were decreased, whereas B-lymphocytes levels were increased. Humoral parametres and acute phase proteins levels were also changed. C-reatcive protein level increased in all cases. However the levels of C3 and C4 were lower. The level of IgE antibodies were high and they even got higher. They achieved the values typical for atopic reaction or parasitic diseases. CONCLUSION: Patients with immune system disorders have more extracranial complications. Patients with lower Glasgow Coma Scale after admission have often immune system disorders. However the prognostic value of monitoring of immue system disorders seems to be low. (Tab. 2, Fig. 3, Ref: 9.)

Some commonly overlooked properties of calcium buffer systems: a simple method for detecting and correcting stoichiometric imbalance in CaEGTA stock solutions.

When the two-solution approach is used to make CaEGTA buffers, the most critical requirement is that the CaEGTA stock solution contain equimolar quantities of calcium and chelator. The impact on [Ca2+] of errors in the molar ratio is magnified at high buffer ratios, so stoichiometric imbalance is most readily detected there. In this paper we examine some of the properties of calcium buffer systems that illustrate these principles, and describe a simple and sensitive method for detecting and correcting stoichiometric imbalance in the CaEGTA stock solution. The method derives its high sensitivity from the fact that the CaEGTA stock solution itself represents a calcium buffer system with a very high buffer ratio.

[Detection of myeloma cells in peripheral blood using multi-parameter flow cytometry: monitoring residual disease]. / Detekce myelomových bunek v periferní krvi vícerozmernou prutokovou cytometrií: monitorování zbytkové nemoci.

BACKGROUND: The aim of this study was to evaluate significance of CD 38(+2)45-54+, CD 38(+2)45-56+ and CD 38(+2)45-138+ cells counts in peripheral blood of patients with multiple myeloma for monitoring of the minimal residual disease. METHODS AND RESULTS: A triple-color flow cytometric analysis was used for this purpose. Peripheral blood of 29 patients with multiple myeloma who underwent high dose chemotherapy and autologous stem cells transplantation was repeatedly analysed. Counts of myeloma cells in peripheral blood were compared to serum monoclonal immunoglobulin concentration, serum calcium level, serum C-reactive protein, serum beta 2 microglobulin, and number of myeloma cells in bone marrow (morphology). From 29 patients in this study, 5 patients have relapsed. Patients in relapse had significantly higher counts of CD 38(+2)45-54+, CD 38(+2)45-56+ and CD 38(+2)45-138+ cells in peripheral blood than patients in remission (geometrical average: 12.41; 6.20; 14.45 cells/microliter versus 4.08; 2.87; 2.58 cells/microliter). The number of these cells correlated well with serum monoclonal immunoglobulin level and counts of myeloma cells in bone marrow. CONCLUSION: The authors conclude that the longitudinal multi-color flow cytometric analysis of CD 38(+2)45-54+, CD 38(+2)45-56+ and CD 38(+2)45-138+ cells in peripheral blood of patients with multiple myeloma is a useful method for evaluation of the disease activity. Significance of peripheral myeloma cells count for prediction of the relapse of the multiple myeloma remains to be evaluated.

[Analysis of phenotypes and kinetics of CD34+ cells and lymphocytes in donors of hematopoietic stem cells for allogenic transplantation]. / Analýza fenotypu a kinetiky subpopulací CD34+ bunek a lymfocytu dárcu periferních hematopoetických kmenových bunek pro alogenní transplantace.

BACKGROUND: Amount of CD34+ cells is a critical parameter for quality assessment and successful engraftment of peripheral blood hematopoietic stem cells (PBSC) during the transplantation of haemopoisis. CD34+ cells are routinely analysed by immunophenotyping in PBSC and in peripheral blood during mobilization. Other leukocyte subpopulations are not usually assessed. METHODS AND RESULTS: The authors present results of immunophenotyping of subpopulations of CD34+ cells and leucocytes in samples from donors of PBSC for allogeneic transplantations, who were stimulated with the growth factor G-CSF at dose 16 micrograms/kg/day. The amount of CD34+ cells was not significantly different between days 4 and 5; however, there was a significant drop at day 6. CD34+90+ and CD34+61+ subpopulations reached their maximum at the day 4; partially differentiated CD34+ cells with co-expression of CD33, CD19, and CD7 reached maximum at the day 6. CD4+ Th-lymphocytes were concentrated in the grafts during leukapheresis, CD4/CD8 ratio in the grafts was increased to average 3.06. CONCLUSIONS: The knowledge of kinetics of CD34+ subpopulations, together with stem cell selection and ex vivo manipulation, may have an impact on the speed of engraftment or GvHD prevention in transplanted patients.

[Evaluation of the clinical effectivity and toxicity of the FDN regimen (fludarabin, mitoxantron, dexamethason) in patients with follicular lymphoma]. / Hodnocení klinické ucinnosti a toxicity rezimu FND (fludarabin, mitoxantron, dexamethason) u pacientu s folikulárním lymfomem.

BACKGROUND: One of the perspective therapeutic possibilities in follicular lymphomas (FL) is the fludarabine-based regimen FND (fludarabine, mitoxantron, dexamethason). However serious signs of myelotoxicity and significant immunosuppression with appearance of the opportunistic infections were described after the fludarabine treatment. METHODS AND RESULTS: Follicular lymphoma patients with advanced disease grade I-II were treated with FND (6-8 cycles). The immunotoxicity was evaluated by measuring of immunoglobuline levels (IgA, IgG, IgM) and that of lymphocytes subpopulations (CD3+, CD4+, CD8+, CD20+, CD56+) in peripheral blood. The myelotoxicity was evaluated by cultures of progenitor cells (CFC and LTC-IC). Totally 34 patients (median age 55.5 years) were evaluated, the overall response was 72% (CR 61%, PR 11%, progression 28%). 73% patients of 11 after 6-8 FND show persisting CR (27% relapsed) with median follow-up about 15 months. The dominating toxicity was myelotoxicity. The leucopenia grade III-IV occurred in about 30% cycles. Because of toxicity it was necessary to reduce doses of FND in 10% cycles and this treatment had to be finished ahead of schedule in 29% patients. The significant immunotoxicity was found only in the decrease of whole lymphocyte population (p < 0.05) and of IgG level (p < 0.05). The decrease of lymphocyte subpopulations did not reach any statistical significance. The long-term myelotoxicity caused the decrease of LTC-IC that had a clinical correlation with the very difficult mobilization of PBSC. CONCLUSIONS: FND is efficient in treatment of follicular lymphoma. However myelotoxicity seems to be limiting. Myelotoxicity doesn't allow administering scheduled dose of FND in substantial amount of patients, long-term myelotoxicity complicates PBSC-mobilization. Lymphotoxicity is apparent, but seems not to be clinically important.