J774 macrophage-like cell line cytokine and chemokine patterns are modulated by Francisella tularensis LVS strain infection.

Mutual interactions were investigated between intracellular parasitic bacterium Francisella tularensis (F.t.; highly virulent bacterium responsible for tularemia, replicating within the host macrophages) and murine macrophage-like cell line J774. Recombinant murine lymphokine INF-gamma and/or LPS derived from E. coli were determined to stimulate in vitro antimicrobial activity of macrophage-like J774 cell line against the live vaccine strain (LVS) of F.t. through their ability to produce proinflammatory cytokines and chemokines. F.t. infection up-regulated IL-12 p40 production and down-regulated TNF-alpha production by stimulated macrophages; on the other hand, F.t. infection did not affect the production of IL-8, IL-6, MCP-5, and RANTES by stimulated macrophages. This showed that F.t. infection modulates the cytokine synthesis by J774 macrophage cell line.

[Significance of confirmatory testing of HLA system in unrelated haematopoietic stem cell donors]. / Význam konfirmacních testu HLA systému nepríbuzných dárcu hematopoetických kmenových bunek.

BACKGROUND: Haematopoietic stem cell transplantation is a standard curative therapy for some acquired haematological diseases and inherited metabolic and immunological disorders. The HLA compatibility in five loci (HLA class I -A, -B and -C and HLA class II -DRB1 and -DQB 1) of the donor/recipient pair is a prerequisite for the success of haematopoietic stem cell transplantation which represents a process of adoption donors immunity. METHODS AND RESULTS: HLA is the most polymorphic system in the human genome and this polymorphism is exactly detected by molecular genetics methods on DNA level only. In period of 2001-2004 we performed confirmatory testing of 366 unrelated haematopoietic stem cells donors from Czech and foreign registers for 256 patients. Only 16% of the donors completely matched the patients in all HLA loci. We detected HLA mismatches in the samples of 81% patient/donor pairs but these results were consonant with previous results from registers. 3% of confirmatory samples were discrepant with previous registry data. CONCLUSIONS: Despite of increasing number of available unrelated haematopoietic stem cell donors and the quality of registry HLA typing the possibility of finding the completely match donor is still limited.

[HLA-DRB1/DQA1/DQB1 alleles and haplotypes in Czech children with celiac sprue]. / HLA-DRB1/DQA1/DQB1 alely a haplotypy ceských deti s celiakální sprue.

BACKGROUND: The celiac disease (CD) is a multifactor disease resulting from a life time abnormal immune response to gluten accompanied by autoimmune characteristics, which can in sensitive individuals evoke small bowel mucosa morphologic changes. The genetically sensitive individual to CD has not been defined yet, it is obvious, however, that this illness is closely linked to the HLA class II genes. The objective of our study was to detect associations of HLA class II alleles and haplotypes DRB1/DQA1/DQB1 in Czech CD children. METHODS AND RESULTS: A group of Czech CD children diagnosed according to ESPGHAN criteria was genotyped HLA for alleles of DRB1/DQA1/DQB1 loci. Genotyping of the HLA-DRB1/DQB1 haplotypes proved statistically significant association CD with haplotypes and alleles of this genetic system. 92.9% of patients have in their HLA phenotype allele DQA1*0501 in either cis or trans configuration with the DQB1 allele *0201/*0202. The extended HLA haplotype DRB*0301/DQA1*0501/DRB1*0201 as well as the haplotype DRB1*0701/DQA1*0201/DQB1*0202, are presented in 63.6% or in 61.0% CD patients respectively. The individual HLA class II alleles DRB*0301, *0701, DQA1*0201, *0501, DQB1*0201, *0202 and the above mentioned HLA haplotypes inclusively provide genotypic frequencies significantly different from healthy Czech individuals (P < or = 0.06 +/- 0.001). These results support the opinion that the HLA molecule expressed on the cellular surface as a alpha beta heterodimer encoded by the DQA1*0501 and DQB1*0201/02 alleles in either cis or trans configuration is responsible for the primary sensitivity to this disease. We were, however, not able to find an association of various clinical forms of the CD with a certain HLA haplotype in the followed group. CONCLUSION: The CD patients have in comparison with healthy population significantly different frequency of HLA class II haplotypes. Though the finding of these alleles is not sufficient for an explicit confirmation of this diagnosis, the proof of this risky haplotype/s may notably contribute to it, namely in case of potential or latent forms of this disease.

[Immunofluorescence determination using flow cytometry of platelet surface antigens in peripheral blood of healthy individuals (pilot study)]. / Imunofluorescencní stanovení zastoupení vybraných povrchových antigenu na trombocytech z periferní zilní krve u zdravých dospelých osob prutokovou cytometrií (pilotní práce).

The authors present normal values of selected constitutive and activating platelet surface antigens expression. The results were obtained by flow cytometry analysis after immunofluorescent staining of the peripheral whole blood specimens from 25 healthy adults. Data are presented as a mean value and standard deviation (SD) of percentage of positive labelled platelets: constitutively expressed molecules CD9: 94.97% (+/- 2.95), CD31: 92.78% (+/- 2.97), CD36: 90.98% (+/- 5.25), CD41: 95.62% (+/- 2.23), CD42a: 94.98% (+/- 2.62), CD42b: 94.13% (+/- 2.58), activation molecules CD62P: 0.45% (+/- 0.33), CD63: 0.32% (+/- 0.22). This study is the first report in Czech literature.

Cytotoxic T lymphocyte precursor frequency analysis in the selection of HLA matched unrelated donors for hematopoietic stem cell transplantation: the correlation of CTLp frequency with HLA class I genotyping and aGVHD development.

The selection of human leukocyte antigen (HLA) compatible unrelated donors for hematopoietic stem cell transplantation (HSCT) is based on the direct genotyping of HLA class I and class II alleles (HLA-A, -B, -C, -DRB1, -DQB1 loci). The cellular test estimating the frequency of cytotoxic T lymphocyte precursors (CTLp) has been included into the selection procedure of unrelated donors to detect the class I alloreactivity and to predict acute graft versus host disease (aGVHD) occurrence and severity. The relationship between HLA-A, -B, -C high/medium resolution genotyping and CTLp activation was analysed in the cohort of 78 unrelated donor/patient pairs indicated for HSCT. The high frequency of CTLp (> 1:100,000) correlated significantly (p < or = 0.0002) with the incompatibilities in alleles of HLA-A, -B, -C loci. Nevertheless, the results of HLA-A, -B, -C genotyping and CTLp assay are not fully alternative, suggesting that the CTLp test gives its specific information. The high CTLp frequency (CTLpf) in 14/35 pairs fully matched by HLA class-I alleles genotyping could reflect the influence of another factors upon the CTLp activation. On the contrary, the low CTLp frequency values (< or = 1:100,000) found in 8/43 pairs with existing HLA class-I alleles incompatibilities could indicate the immunological permissivity of these particular mismatches. The clinical relevance of the CTLp test for aGVHD prediction has been also analysed. The relationship between CTLp activation in vitro and the incidence and severity of aGVHD was evaluated in 37 patients who underwent allogeneic HSCT. The severe form of aGVHD (grade III-IV) developed in 9 of 18 cases (50%) with the high pretransplant CTLpf value. The patients with the low CTLpf (n = 19) suffered from the severe form of aGVHD in 2 cases (10%) only, the remaining 17 patients from this group were without aGVHD symptoms or developed only the mild form of aGVHD (I-II). The relationship between CTLp results and the incidence and severity of aGVHD was found statistically significant (p < or = 0.01).

The relationship between HA-1 compatibility and the activation of helper T lymphocyte precursors.

The relationship between the compatibility in minor histocompatibility HA-1 antigen and the activation of helper (IL-2 producing) T lymphocyte precursors in vitro was studied in the group of 17 HLA-A2 positive HLA identical siblings. Although the number of pairs studied is still small, no correlation has been found between HA-1 compatibility and helper T lymphocyte precursors activation. The results presented here could suggest the possibility that the HTLp assay does not have to be a relevant parameter for the detection of HA-1 mismatches in HLA identical siblings.

[Cytoimunofluorometry and its use in the detection of blood platelet activation]. / Cytoimunofluorometrie a její vyuzití v detekci aktivované desticky.

Blood platelet activation is a complex process involved in the physiological hemostasis and also in a number of disorders. Platelets change their morphology, modify their surfaces glycoprotein receptors with adhesive functions. Procoagulant microparticles are shedding and the aggregates of platelets with leucocytes are appearing. The activated platelet can be detected by flow cytometry after imunofluorescent staining.

Frequencies of HLA-DRB1, -DQB1 and -DPB1 alleles in Czech population.

The frequencies of phenotypes, alleles and allelic subtypes of DRB1 and DQB1 HLA loci in 420 unrelated individuals from the Czech population were determined. The frequencies of DPB1 alleles of the HLA locus were determined in 92 individuals. The assays were performed using the polymerase chain reaction (PCR) method or the restriction fragment length polymorphism (RFLP) analysis. The most frequent DRB1 allele was *07, the most frequent DQB1 allele was *03 and the most frequent DPB1 allele detected was *04. These assays define the extent of polymorphism of the HLA system and are useful for determining the selection strategy of HLA-identical donor-recipient pair suitable for bone marrow transplantation.

The frequency of HLA-DRB1 and -DQB1 alleles in cervical cancer cases in the Czech Republic.

The distribution of HLA class II DRB1 and DQB1 alleles in cervical carcinoma patients was compared with the frequency of these alleles found in healthy population living in the Czech Republic. The RFLP analysis and PCR-SSP were used for DNA typing. Although the differences in the frequency of DRB1*03, DQB1*02 and DQB1*0303 alleles between the cases and the controls were rather large, corrected P values did not reach significance.

[Allogenic transplantation of hematopoietic stem cells in the treatment of children with high-risk acute leukemia]. / Alogenní transplantace kmenových bunek krvetvorby v lécbe detí s vysoce rizikovou akutní leukémií.

BACKGROUND: Most children with acute lymphoblastic leukemia (ALL) and increasing number of children with acute myelogenous leukemia (AML) are currently cured with conventional chemotherapy. Despite of this success there is a subset of patients with high-risk features at diagnosis who are predisposed to a very high risk of relapse. Relapse of AML and early bone marrow relapse of ALL can not be cured by conventional chemotherapy. Allogeneic hematopoietic stem cell transplantation (HSCT) is therapeutic option in these children with very high-risk acute leukemia. METHODS AND RESULTS: Between XI/1989-XII/1996 33 children with acute leukemia (ALL: 22, AML: 11) underwent an allogeneic HSCT from HLA identical related donors (HLA-identical sibling: 30, twin: 1, other HLA-identical relative: 2) at the 2nd Dept. of Pediatrics, University Hospital Motol. Median age of our group was 9 years (1.5-19 y.), boys (n = 23) clearly dominated over the girls (n = 10). The resource of stem cells was bone marrow in 31 children, bone marrow and peripheral blood progenitor cells (PBPC) and PBPC in one child respectively. Myeloablative conditioning regimen varied, consisting of total body irradiation and chemotherapy in 21 children and chemotherapy in 12 children. HSCT was performed in first complete remission of acute leukemia in 9 children (AML: 7, ALL: 2), in second remission in 14 children (AML: 2, ALL: 12), in third remission in 4 children (ALL: 4). Six children underwent HSCT in first partial remission (n = 1) and in second (n = 4) or third (n = 1) chemoresistant relapse. Seven (21%) children died due to post-transplant complications. Nine (28%) children suffered from clinically significant acute graft-versus-host reaction (GVH) and 15% (4/27) children who survived 100 days post-transplant suffered from chronic GVH disease. Relapse of leukemia was diagnosed in 39% (12/31) children. Fourteen (42%) children are alive and well in continuous remission with median follow-up 42 months. CONCLUSIONS: Allogeneic HSCT can cure children with very high-risk acute leukemia in the situations where conventional chemotherapy fails. Relapse of leukemia and GVH reaction are most important causes of post-transplant morbidity and mortality.

Helper T lymphocyte precursor frequency analysis in alloreactivity detection.

The utility of IL-2 secreting helper T lymphocyte precursors (HTLp) frequency testing has been evaluated for detecting alloreactivity. The frequency of HTLp was approached by limiting dilution assay. High HTLp frequency was detected in 20 out of 30 HLA matched unrelated pairs (67%). The comparison of HTLp and CTLp (cytotoxic T lymphocyte precursors) frequencies in HLA matched unrelated pairs showed that the two examinations are not fully alternative in detecting alloreactivity. This could suggest the utility of combined testing of both HTLp and CTLp frequencies for alloreactivity assessment. In contrast, five positive HTLp values were only found among 28 HLA genotypic identical siblings (18%). Previous CTLp limiting dilution studies showed very low or undetectable CTLp frequency results in that group. For that, HTLp assay remains to be the only cellular in vitro technique detecting alloreactivity in these combinations.

Predictive value of HLA class II PCR typing for the outcome of mixed lymphocyte reaction.

The use of HLA-DRB1 and -DQB1 polymerase chain reaction-sequence-specific primer (PCR-SSP) typing at different levels of resolution for MLR prediction was assessed in 54 HLA-A and -B matched donor/recipient unrelated pairs and 89 HLA-A and -B identical siblings. Graft-versus-host (GvH) direction one-way MLR was evaluated unless stated otherwise. The typing of DRB1 alleles satisfactory for MLR prediction in HLA identical siblings (P = 0.0015) does not appear to be sufficient in matched unrelated pairs (P = 0.2407). Using more discriminatory PCR-SSP typing, the disparity in DRB1 allelic subtypes was predominantly found in the category of DRB1 allele compatible, MLR positive unrelated pairs. Besides, DRB1 allelic subtype mismatches were revealed in five of the forty-one DRB1 allele compatible, MLR negative unrelated pairs. More discriminatory typing made the correlation between DRB1 compatibility and MLR negativity extremely significant (P = 0.0001). As for these five exceptional cases, the reciprocal host-versus-graft (HvG) direction MLR was considered, too. This allowed HLA-D disparity to be disclosed in two of them. An uninterpretable result reflecting defective MLR reactivity occurred in one case. Negative reciprocal MLR in the last two DRB1 allelic subtype incompatible pairs is hardly to explain without postulation of MLR silent DRB1 allelic subtype mismatches. An analysis in unrelated pairs showed a role of some DQB1 gene products in the proliferative response too. GvH direction positive MLR was found in two HLA identical siblings among the 89 tested. The DPB1 incompatibility detected in one of them could be a potential cause of proliferative response but MLR reactivity in the other, DPB1 identical, pair cannot be interpreted easily.

Transplantation of allogeneic peripheral blood progenitor cells--a single centre experience.

13 patients have been transplanted at Institute of Hematology and Blood Transfusion since 1995 using allogeneic PBPC either alone or with bone marrow as a source of progenitor cells. All donors were G-CSF mobilised HLA identical family members. PBPC harvests were performed on D 4,5, (6) of G-CSF administration. The medium content of TNC, CD34+, CD3+, CD4+and CD8+cells/kg b.w. of the recipients in the grafts were: 13,1x10(8)(TNC), 11,4x10(6)(CD34+), 393x10(6)(CD3+) 243x10(6)(CD4+), 125x10(6)(CD8+) The patients received either BuCy2 or CyTBI preparative regimen and Cyclosporin A + short course of Methotrexate for GVHD prophylaxis. Engraftment of ANC >500 was achieved by D+16 and PLT >20.000 by D+19. Three of ten evaluable patients developed acute and three of nine chronic GVHD. 8 patients survive with the longest follow up 776 days.

[Frequency of cytotoxic and helper T-lymphocyte precursors in donors with alloreactivity to recipient histocompatibility antigens. Possible use in predicting acute graft vs host disease in bone marrow transplantation]. / Frekvence prekurzoru cytotoxických a pomocných T lymfocytu dárce aloreaktivních vuci histokompatibilitním antigenum príjemce. Moznosti vyuzití pro predpoved aGVHD pri transplantaci kostní drene.

BACKGROUND: The utility of cytotoxic T lymphocyte precursor (CTLp) and helper T lymphocyte precursor (HTLp) frequencies estimation for detecting alloreactivity and for the prediction of acute graft versus host disease (aGVHD) has been evaluated. METHODS AND RESULTS: The limiting dilution assay and a maximum likelihood statistical programme were used for CTLp and HTLp frequency estimation. A high CTLp frequency suggesting the presence of hidden class I mismatches was detected in 41.2% of unrelated pairs. HLA-A and -B matched by serological typing and DRB1 and DQB1 matched by DNA analysis. Severe aGVHD (grade III-IV) occurred in all patients of this group who underwent bone marrow transplantation (BMT). In two patients of the three evaluated with low pretransplant CTLp frequency a mild form (grade I) or no aGVHD developed after unrelated BMT. Positive frequency of alloreactive HTLp was found in 50% of HLA matched unrelated pairs. The comparison of CTLp and HTLp values in the same individuals showed that these two methods are not fully alternative in detecting alloreactivity. In the group of HLA identical siblings, 18.7% of positive HTLp results were only found. Besides HLA-DP incompatibilities, the differences in non-HLA genes could cause this alloreactivity. CONCLUSIONS: CTLp assay has a potential for the prediction for aGVHD development following BMT from HLA matched unrelated donors. CTLp results suggest the necessity of more accurate class I typing in these cases. The comparison of CTLp and HTLp frequencies showed that the results can differ in some unrelated donor-recipient BMT pairs suggesting the convenience of simultaneous performing of both assays for the alloreactivity assessment. More cases have to be considered to determine the relationship between pretransplant HTLp frequency and posttransplant aGVHD development in HLA identical siblings.

[Cell-mediated immunity in patients with Sjogren's syndrome]. / Bunkami zprostredkovaná imunita u nemocných se Sjögrenovým syndromem.

BACKGROUND: Sjögren's syndrome (SS) is considered an autoimmune immunopathological disease, which is characterized by disorders of humoral and cell-mediated immunity. The objective of the work was to assess the ratio of T and B lymphocytes, subpopulations of T lymphocytes and NK cells in the peripheral blood of patients suffering from SS. METHODS AND RESULTS: The investigated group was formed by 22 patients with the diagnosis of primary SS (mean age 55.7 years, range 39-83 years), 17 patients with the diagnosis of secondary SS (mean age 60.5 years, range 45-79 years). The ration of CD3-, CD4-, CD8-, CD19-, CD5-, CD25- HLA DR- cells in full blood was assessed by the monoclonal antibody technique of direct immunofluorescence, using evaluation by means of a flow cytometer. The results were compared by statistical methods with a control group of blood donors using Student's t-test, A relative increase of the ratio of CD3- T lymphocytes was revealed in patients with primary SS (75.3 +/- 17.9 %, p = 0.380) and secondary SS (73.7 +/- 11.8, p = 0.326), as compared with controls (65.3 +/- 10.3%). The absolute number of the subpopulation of suppressor helper inducer CD4+ T lymphocytes was markedly lower in patients with primary SS (0.056 +/- 0.27 - 10(9)/l, p = 0.0154), as compared with controls (0.81 +/- 0.27, 10(9)/l. The relative number of suppressor cytotoxic CD8- T lymphocytes was significantly higher in patients with primary SS (33.7 +/- 15.0%, p = 0.0001 and secondary SS (30.5 +/- 11.0%, p = 0.0002), as compared with controls (17.2 +/- 6.0%). The absolute number of inhibitory cytotoxic T lymphocytes was significantly higher in patients with primary SS (0.47 +/- 0.3. 10(9)/1, p = 0.0084) and patients with secondary SS (0.48 +/- 0.3, 10(9)/l, p = 0.0072). The relative number of CD57+ NK cells was significantly higher in patients with primary SS (21.1 +/- 11.0%, p = 0.0001) and secondary SS (19.3 +/- 11.7%, p = 0.0023). The absolute number of NK cells was significantly higher in patients with primary SS (0.28 +/- 0.20, 10(9)/l, p = 0.0025 and in patients with secondary SS (0.31 +/- 0.23, 10(9)/l, p = 0.01). CONCLUSIONS: The revealed statistically significant increases in the number of T lymphocytes in patients with Sjögren's syndrome is probably caused by a significant increase of the subpopulation of CD8+ suppressor cytotoxic T lymphocytes. The latter probably overlap with the population of NK cells. These immunological abnormalities are even more marked in patients with primary Sjögren's syndrome due to the decrease of the absolute number of CD4+ helper inducer T lymphocytes.

[Blood platelet adhesion molecules]. / Adhezní molekuly krevních desticek.

Homeostasis is mediated through interactions of adhesion molecules on the surface of platelets which belong into the families of selectins, integrins and immunoglobulins with soluble plasma proteins, surface molecules of activated endothelial cells and molecules of extracellular matrix. The membrane expression of some adhesion molecules (P-selectin) which are the part of granular system of resting platelet identify activated platelet. Adhesion molecules of platelets could be detected by immunochemistry through flow cytometry. By this approach the inherited defects of adhesion molecules on platelets and the activated platelet could be detected. The detection of activated platelets is very useful in many clinical conditions.

[Immunologic profile of patients with a transplanted kidney in the postoperative period]. / Imunologický profil pacienta s transplantovanou ledvinou v pooperacním období.

In our study we follow up a response of specific and a part of nonspecific cellular immunity in recipient organism with renal transplantation of cadaveric kidney to a standard immunosuppressive treatment used in the Department of Urology in Faculty hospital in Hradec Králové.

[Determination of biological paternity yesterday and today]. / Prukaz biologického otcovství vcera a dnes.

The paper deals with methodical development of expertise in cases of disputed paternity. Presently used approaches to paternity testing were introduced following deeper understanding of heredity of human features as well as development of new methods of their studying. While the genetic characterization of individuals tested was till recent time limited to the level of gene products, in the present also direct molecular genetic analysis of their genome is possible. The most frequently used molecular genetic technique is Polymerase Chain Reaction (PCR) which leads to multiplication of chosen hypervariable regions of DNA followed with direct with following direct detection of polymorphic alleles. Examination of several DNA polymorphisms provides an exclusion of all falsely accused men and in opposite cases a practical proof of parenthood. That is the reason why the world-wide trends of forensic medicine tend to consecutive replacement of all procedures use until now with methods of molecular genetics.

Sub-milliMorgan map of the proximal part of mouse Chromosome 17 including the hybrid sterility 1 gene.

We have generated a high-resolution genetic map, 0.071 cM per backcross animal, of the 13 cM T-H2 region of the mouse Chromosome (Chr) 17. The map contains two phenotypic loci, T and Hst1, 12 RFLP markers, and 24 microsatellite loci. The Hst1 gene was mapped to a chromosomal interval contained within a single 580-kb YAC clone. The FFEH11 YAC is 0.44 cM long and carries, besides the Hst1 gene, five polymorphic DNA markers and recombination breakpoints of six backcross animals. Two candidate genes for Hst1 were identified based on their location and testicular expression. These are Tbp and D17Ph4e. The submilliMorgan map of the T-H2 region revealed significant clustering of (CA)n loci. The clustering, if shown to be a common feature in the mouse genome, may cause gaps in the physical map of the mouse genome.

The search for HLA-compatible bone marrow donors. Procedure currently used in the Institute of Hematology and Blood Transfusion, Prague.

The search for compatible donors is based on the HLA types of donors and recipients. HLA-A, -B and -DRB1 antigens or alleles must be unequivocally typed in donors and recipients. The typing of HLA-DQB, -DPB and -C gene products is also useful to characterize the HLA phenotype, but it is not absolutely necessary in the donor selection. The standard serological methods using alloantisera and one-dimensional isoelectric focusing are used for the typing of HLA class I antigens. Recently, DNA analysis of class I alleles has been introduced. In HLA class II typing the serological analysis was generally replaced by DNA analysis. In addition to typing techniques that determine the individual HLA alleles or HLA gene products, the cellular matching techniques are used in the selection procedure (mixed lymphocyte culture, cytotoxic T lymphocyte precursor frequency assay, and IL-2-producing helper T lymphocyte precursor frequency assay). The cellular matching techniques determine the compatibility in the regions of HLA comprising several HLA loci; some of them may detect minor histocompatibility (non-HLA) gene disparities as well.