CONTENTS
Transplantation
- Incidence of Bone Marrow Transplantation in Europe. Report from the European Group for Blood and Marrow Transplantation
ALOIS GRATWOHLAbstract. The European Group of Blood and Marrow Transplantation (EBMT) is collecting information on incidence of bone marrow transplantation in Europe. 203 teams in 26 European countries performed in 1992 a total of 6065 bone marrow transplants (BMT). There were 2666 (44%) allogeneic transplants from 2171 HLA identical sibling donors, 170 non-identical family donors, 29 twin donors and 296 unrelated volunteer donors. There were 3399 autologous transplants (56%): 2494 autologous BMT, 644 autologous peripheral blood stem cell transplants and 261 combined autologous bone marrow peripheral blood stem cell trans- plants. Transplant indications were leukemias in 2963 patients (49%) (1987 allogeneic, 976 autologous), lymphoproliferative disorders in 1890 patients (31 %) (201 allogeneic, 1689 autologous), solid tumors in 739 patients (12%) (10 allogeneic, 739 autologous), aplastic anemia in 194 patients (3%) (193 allogeneic, 1 auto- logous), thalassemia in 128 patients (2%) (all allogeneic), inborn errors in 115 patients (2%) (all allogeneic) and miscellaneous disorders in 36 patients (32 allogeneic, 4 autologous). Main differences were observed in the number of transplanting teams, and in the number of allogeneic and autologous transplants performed in the different European countries compared to the number of inhabitants.
Keywords: bone marrow transplantation; allogeneic; autologous.logous.
- HLA and Bone Marrow Transplantation
THOMAS H. EIERMANNAbstract. Bone marrow transplantation between HLA-identical siblings is an established therapeutic strategy in leukemia and a variety of lympho-hematopoetic disorders. In recent years unrelated donors were increasin- gly used for patients lacking a family donor. Here we report on current progress in HLA class II typing by molecular methods which have influenced donor search and improved matching.
Keywords: bone marrow transplantation; mixed lymphocyte culture; polymerase chain reaction sequence-specific oligonucleotide.eotide.
- DRB Polymerase Chain Reaction Fingerprinting, DQA Oligotyping and Mixed Lymphocyte Culture Results In related Bone Marrow Donors and Recipients
EWA BROJER, MONIKA SANKOWSKA, JANUSZ J. STAŃCZAK, ANNA GRONKOWSKA, BARBARA STĘPIEŃ-SOPNIEWSKA, ANDRZEJ GÓRSKI and BARBARA ŻUPAŃSKAAbstract. Donor and recipient are usually regarded as well matched for bone marrow transplantation when they are compatible in HLA class I and II antigens and in mixed lymphocyte culture (MLC). However, results of serological typing of class II antigens may be unreliable. Hence, polymerase chain reaction fingerprinting of HLA DRB (PCR FP) was introduced for the screening of related donors for 29 patients awaiting bone marrow transplantation. In addition, the sequence-specific oligonucleotide (SSO) typing of DQA alleles was performed. In 18 pairs the results of DNA analysis methods were compared with the results of MLC. 72% of pairs were HLA DQA compatible and 59% showed compatibility in PCR FP. MLC compatibility was found in 61%. A high correlation of PCR FP and MLC results was observed.
Keywords: bone marrow transplantation; HLA; polymerase chain reaction; fingerprinting.inting.
- Analysis of Reconstituting T Cell Receptor Repertoires in Bone Marrow Transplant Recipients
JACK GORSKI, MARYAM YASSAI, CAROLYN KEEVER and NEAL FLOMENBERGAbstract. Allogeneic bone marrow transplantation (BMT) has become the treatment of choice for a variety of hematopoetic disorders. An important factor limiting the use of allogeneic BMT is delayed restoration of immune function. A quantitative understanding of immune reconstitution of the T cell compartment based on an efficient method of analysis would be of benefit. Distinct lineages of T cells which have resulted from previous and ongoing clonal expansion can be identified by their unique T cell receptors (TCR). Thus, TCR complexity can be used as a measure of repertoire complexity. Here we use a polymerase chain reaction-based approach which visualizes the size heterogeneity of the third complementarity determining region (CDR3) to study T cell reconstitution in adult bone marrow transplant recipients. We find that repertoire complexity, as determined by the number of bands of different length for each V family, reflects the general immune status of individuals tested. Contractions and gaps in repertoires are revealed in individuals suffering from recurrent infections associated with T cell impairment. This approach provides a new tool in the analysis of recons- titution of a/ß T cell repertoires and it can also be applied to B cells and y/â T cells.
Keywords: T cell receptor; polymerase chain reaction; bone marrow transplantation.tation.
- Cytokines, Adhesion Molecules (E-Selectin and VCAM-1) and Graft-Versus-Host Disease
ANDRZEJ LANGE, ALEKSANDRA KLIMCZAK, LIDIA KARABON and KRZYSZTOF SUCHNICKIAbstract. Analysis of skin biopsy specimens for the presence of adhesion molecules, composition of cellular rofiltrates, Ki-67 antigen expression, and examination of serum for interleukin 6 (IL-6) and tumor necrosis factor a (TNF-a) levels, was performed in patients after allogeneic bone marrow transplantation (alloBMT), to study the pathomechanism of acute graft-versus-host disease (aGvHD). It was found that: 1) early hemato- bgical recovery constitutes a risk factor for grade IV GvHD, 2) vascular cell adhesion molecule-1 (VCAM-1) is present in the matrix organizing the cells in the bone marrow and in aGvHD infiltrates, 3) HLA DR antigens aberrant expression in epithelial cells, as well as 4) strong expression of Ki-67 is seen in early stages of aGvHD. These immunopathomorfological lesions are cytokine-dependent. High levels of IL-6 and TNF-a were found in sera of patients affected with the aGvHD process and infectious complications. An increase of IL-6 in the course of aGvHD is a sign of poor prognosis. These data support the notion that cytokines facilitate the cell accumulation at the site of aGvHD at the beginning of this process and again, at the final stage of the disease, cytokines high levels are associated with the organ damage.
Keywords: bone marrow transplantation; cytokines; graft-versus-host disease; immunopathomorphology.hology.
- Statistical Evaluation of Bone Marrow Transplantation Data
RYSZARD M. SZYDŁOAbstract. The analysis of data generated from a transplant programme is fundamental to assessing both the etfectiveness of different treatments, and the prognostic role of disease and patient variables. Therefore, the use of life-table analyses, methods of comparing survival curves, and a brief discussion of multivariate methods will be presented.
Keywords: bone marrow transplantation; survival analysis; life-table; logrank; proportional hazards regres- sion analysis.alysis.
Molecular Immunology
- Use of Spontaneously Mutated Human DNA as Competitive Internal Standard for Nucleic Acid Quantification by Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
LIDIA RUDNICKA, ARTURO DIAZ, JOHN VARGA, ANGELO CHRISTIANO, JOUNI UITTO and SERGIO A. JIMENEZAbstract. Quantification of gene expression is of increasing interest in many medical sciences. Methods based on reverse transcription-polymerase chain reactions (RT-PCRs) are timesaving and require only very small amounts of RNA. A limiting factor, however, is the significant fluctuation in the efficacy of reverse transcrip- tion as well as in the polymerase chain reactions. Various external and internal standards have been suggested for correcting these fluctuations. We describe á novel way of creating an internal standard for assessing the expression of type VII collagen in human cells. The total RNA of a patient with hereditary epidermolysis bullosa dystrophica associated with a homozygous T to A point mutation in type VII collagen gene was reverse transcribed and a 382bp fragment of type VII collagen cDNA containing the mutation was amplified. The mutated cDNA, unlike normal type VII collagen cDNA could be cleaved by Ear1 endonuclease into 244bp and 138bp fragments. Semiquantitative PCR was performed with the mutated cDNA as internal standard and the studied cDNA sample in the same tube in the presence of a3zP-labeled dCTP. The reaction was followed by Ear1 digestion, electrophoresis on a polyacrylamide gel and exposure to a X-ray film. In conclusion, we describe a timesaving method for creating internal standards for semiquantitative RT-PCR.
Keywords: polymerase chain reaction; internal standard; transforming growth factor ß.ctor ß.
- Cell Proliferation-Associated Nuclear Antigen Defined by Antibody Ki-67: a New Kind of Cell Cycle-Maintaining Proteins
MICHAEL DUCHROW, CARSTEN SCHLÜTER, GORAN KEY, MICHAEL H. G. KUBBUTAT, CLAUDIA WOHLENBERG, HANS-DIETER FLAD and JOHANNES GERDESAbstract. A decade of studies on the human nuclear antigen defined by monoclonal antibody Ki-67 (the „Ki-67 protein”) has made it abundantly clear that this structure is strictly associated with human cell proliferation and that the expression of this protein can be used to assess the growth fraction of a given cell population. Until recently the Ki-67 protein was described as a nonhistone protein that is highly susceptible to protease treatment. We have isolated and sequenced cDNAs encoding for this antigen and found two isoforms of the full length cDNA of 11.5 and 12.5 kb, respectively, sequence and structure of which are thus far unique. The gene encoding the Ki-67 protein is organized in 15 exons and is localized on chromosome 10. The center of this gene is formed by an extraordinary 6845 bp exon containing 16 successively repeated homologous segments of 366 bp („Ki-67 repeats”), each containing a highly conserved new motif of 66 bp („Ki-67 motif’). The deduced peptide sequence of this central exon possesses 10 ProGluSerThr (PEST) motifs which are associated with high turnover proteins such as other cell cycle-related proteins, oncogenes and transcription factors, etc. Like the latter proteins the Ki-67 antigen plays a pivotal role in maintaining cell proliferation because Ki-67 protein antisense oligonucleotides significantly inhibit 3H-thymidine incorportation in per- manent human tumor cell lines in a dose-dependent manner.
Keywords: Ki-67; Mib-1; monoclonal antibody; cell proliferation; nuclear antigen; cDNA.; cDNA.
- Analysis of T Cell Receptor aß Variability in Tumor-Infiltrating Lymphocytes in Primary and Metastatic Melanoma
JESPER ZEUTHEN, ANETTE BIRCK and PER THOR STRATENAbstract. The T cell receptor (TCR) aß variable (V) gene family usage of tumor-infiltrating lymphocytes (TIL) in different primary human malignant melanomas and corresponding metastatic lesions were characterized using a recently developed method using the reverse transcription coupled polymerase chain reaction (RT-PCR). This semiquantitative RT-PCR method could be adapted to analysis of formalin-fixed, paraffin-embedded histopathological samples of primary tumor material and demonstrated to be reproducible and to be useful for the assessment of V( and Vß-gene family usage in tumor samples. The TIL in primary tumors were observed to preferentially express certain TCR V( and Vß-gene families: V(4, V(22 and Vß8 were highly expressed in several of the primary tumors analyzed using this method. With respect to V(22 and VßB, the preferential expression of these V-gene families was demonstrated to be due in situ clonal expansion of T cells by means of cloning and sequencing. of the CDR3 regions (V-J or V-D-J, respectively) corresponding to the RT-PCR products from one of the primary tumors. The observed preferential usage of certain TCR V( and Vß-genes strongly suggest the in situ clonal expansion of specific populations of T cells in accordance with recent results from others. These clonal T cell populations probably react with certain melanoma-associated peptides presen- ted by specific HLA molecules. The preferential usage of certain V(- and Vß-gene families observed in several tumors further supports the involvement of a limited number of shared melanocyte or melanoma-associated peptides. Since the HLA status of the patients is obviously important to interpret these results, some of the patients were typed for HLA-A1 and -A2, the two most well-characterized restriction elements for melano- ma-associated antigens, either serologically or by a newly developed RT-PCR method which similarly could be applied directly to the tumor material. In TIL in primary melanomas, a possible correlation was suggested between HLA-A2 and the preferential usage of the TCR V-gene families V(4, V(5, V(22 and VßB, whereas the Vß3-gene family appeared to be expressed together with HLA-A1. The V-gene families which were highly expressed in the primary tumors were generally not, or only very weakly, expressed in the corresponding metastases and vice versa, possibly reflecting a substantial change in the phenotype of the metastatic melano- ma target cells. Continued studies of larger patient materials will be necessary to extend and validate these conclusions and of obvious interest for the further analysis of the T cell response in melanoma.
Keywords: T cell receptor; tumor-infiltrating lymphocytes; melanoma.lanoma.
- HLA-B27 Subtypes Among the Chukotka Native Groups
MIKHAIL Y. KRYLOV, .IOHN D. REVEILLE, LUDMILA I. ALEXEEVA, SHANDOR ERDESZ, FRANK C. ARNETT and LIDIYA I. BENEVOLENSKAYAAbstract. The purpose of this study was to assess the relative frequency of the known HLA=B27 subtypes in HLA-B27 positive Chukotka natives, which have higher frequencies of HLA-B27 (to 40%) and spondyloarth- ropathies (to 2%) than the Russian Caucasian population. Using oligotyping of the polymerase-chain reaction amplified second and third exons of the HLA-B27 gene in 86 DNA samples from HLA-B27 positive in- dividuals were successfully typed. All had HLA-B*2705, including 4 patients with Reiter’s syndrome and 5 with ankylosing spondyloarthritis, except one Eskimo who had HLA-B*2702. None had HLA-B*2704 a frequent subtype in Orientals. With respect to HLA-B27 subtypes the indigenous populations from the eastern part of the Chukotka Peninsula are genetically more closely related to Caucasians than to Orientalis.
Keywords: HLA-B27 subtypes; Chukotka natives; Eskimos.skimos.
- Role of Hydrophobic Amino Acids at Position 74 o DRB1 Chain in Reumatoid Arthritis
MARIE ĆERNA, STANISLAV HAVELKA and EVA IVAŚKOVAAbstract. We have analyzed HLA class II alleles in a group of 153 Czech children with rheumatoid arthritis by PCR and hybridization with oligonucleotide probes. When we try to find a common sequence for all DRB1 alleles involved in juvenile and adult arthritis, we can notice hydrophobic amino acid at position 74, which is present in all these alleles, but not in nonsusceptible alleles, where is the hydrophilic amino acid at position 74. In our model, we speculate that the hydrophilic amino acid at position 74 creates a such kind of epitope which is not suitable for rheumatoid-associated peptides or T cells, and only hydrophobic amino acid can permit binding of these peptides or recognition by certain T cells. Analyses of the DPBI sequences have shown that alleles which have a negatively charged amino acid at position 69, are more frequent in pauciarticular patients while those with a positively charged amino acid are more frequent in polyarticular patients. A positively charged amino acid at position 69 might present the same rheumatoid associated peptide as susceptible DRB1 alleles. The presence of more rheumatoid-associated peptide on the cell surface may cause conversion to more severe polyarticular forms. A negatively charged amino acid at position 69 could not present this peptide and a low concentration of the peptide on the cell surface presented just by DRB1 molecules keeps disease in a relatively benign condition of pauciarticular forms.
Keywords: HLA; DRB1; amino acid sequences; peptide presentation.tation.
- Use of Specific Anti-HLA Antibodies in Clinical Nephrology
SOPHYA E. BUGLOVA, VALERY I. LEVIN, VALERY S. PILOTOVICH, GENNADY T. KOZLOV and LUDMILA N. DRAPEZOAbstract. In order to optimize the regimen of pathogenetic therapy of glomerulonephritis with nephrotic syndrome we used transfusions of plasma containing specific anti-HLA antibodies against the patient’s pheno- type as a part of the treatment in the acute stages of disease. The method was theoretically based on the conception of autoimmune genesis of glomerular lesion as well as on the assumption that the highest expres- sion of H LA-antigens are on the surface of activated immunocompetent cells. In previous experiments in vitro we demonstrated immunosuppressive activity of cytotoxic serum on lymphocytes in the absence of comp- lement. Stable remission that was associated with the recovery of serum IgG/IgM balance and normalization of cellular immunity indices was achieved in 72% of 50 treated patients after 2-4 transfusions. Immuno- therapeutic properties of anti-HLA antibodies were also confirmed with preliminary results of plasma tran- sfusions in 5 kidney graft recipients. Posttransfusion reactions of a non-haemolytic type were slight and transient. The data presented allow us to conclude that our method may be useful in clinical cases associated with undesirable immune activation induced by both auto- and alloantigens.
Keywords: anti-HLA antibodies; immunotherapy; glomerulonephritis; kidney transplantation.tation.
Cytokines
- Cytokine Regulation of Monocyte Recruitment
ALBERTO MANTOVANI, BARBARA BOTTAZZI, SILVANO SOZZANI, GIUSEPPE PERI, PAOLA ALLAVENA, QUIANG(GANG DONG, ANNUNCIATA VECCHI and FRANCESCO COLOTTAAbstract. Phagocytes infiltrating neoplastic tissues have peculiar membrane phenotype and functional proper- ties. Tumor-associated macrophages (TAM) play a complex, ambiguous role in the regulation of primary tumor grwoth and metastasis (a `macrophage balance”). Yet these cells are strategically located at the very interface between tumor and host and represent a potential target for immonomodulation. A better undrestan- ding of the regulation and function of TAM may provide a less empirical basis of or rational design of therapeutic approaches, as vividly illustrated by the antitumor activity of i.p. in IFN ovarian cancer patients with minimal residual disease resistant to chemotherapy.
Keywords: tumor-associated macrophages; cytokine; ovarian cancer.cancer.
- Cytokines in Mycobacterial Infections in vitro and ex vivo Studies
HANS-DIETER FLAD 1, JENS GERCKEN 1, LORENZ HUBNER t, CARSTEN SCHLUTER t JULIUSZ PRYJMA 2 and MARTIN ERNST 1,
Abstract. Different species of mycobacteria differ in their capacity to induce the production of tumor necrosis factor-a (TNF-alpha) by human monocytes in vitro. Whereas M. tuberculosis is a potent inducer of TNF-a, M. leprae is much less potent. TNF-a production is found to be associated with the availability of H2O2 genera- ted by activated monocytes, as superoxide enhancing H202 concentration increases and catalase degrading H2O2 decreases TNF( production. Furthermore, M. kansasii with high intrinsic catalase induce less TNF-a than mycobacteria with low intrinsic catalase. In vitro infection of monocytes with M. tuberculosis leads to an impairment of the antigen-presenting capacity, as determined by a reduction of antigen-induced T cell prolife- ration and interferon y (IFN-y) production. Of crucial importance in this impairment is the M. tuberculo- sis-induced down-modulation of MHC class II antigens. The role of TNF(alpha in vivo is reflected in patients with various forms of leprosy. In skin lesions of lepromatous leprosy patients TNF(alpha, interleukin lß (IL-lß), and IFN-(y production are found to be rare, whereas these cytokines are well expressed in skin lesions of patients with tuberculoid leprosy. After multidrug chemotherapy an increase of local cytokine production is found. Taken together, these findings suggest that components of mycobacteria may interfere with local cell-mediated immune reactions in vivo. The molecular mechanisms involved in these local responses need to be defined.
Keywords: tumor necrosis factor; cytokines; mycobacterial infections.ctions.