Molekulare Therapie in der Gastroenterologie und Hepatologie

Zusammenfassung Mittels molekularer Techniken gewonnene Erkenntnisse haben in den letzten Jahren erheblichen Einfluss auf die Behandlung gastrointestinaler und hepatologischer Erkrankungen gewonnen. Die Diagnose bestimmter Erkrankungen wird nicht mehr allein phänotypisch, sondern aufgrund des Genotyps gestellt. Dies betrifft sowohl monogene Erkrankungen als auch die Identifikation von genetischen Risikokonstellationen (z. B. NOD2/CARD15-Mutation bei M. Crohn). Auch für die Therapieplanung bei viralen Erkrankungen wird eine erweiterte molekulare Diagnostik eingesetzt. Die Versuche, die Lebertransplantation in der Behandlung hereditärer Lebererkrankungen durch gezielte genetische Eingriffe (z. B. mittels viraler Vektoren) zu ersetzen, sind noch in der experimentellen Phase, aber die verwendeten Methoden haben bereits wegweisende Verbesserungen erfahren. Mit der molekularen Identifikation neuer Zielstrukturen war die Entwicklung maßgeschneiderter Therapien möglich. Diese finden insbesondere in der Behandlung chronisch entzündlicher Darmerkrankungen und gastrointestinaler Tumorerkrankungen Anwendung.     

Clonal dysregulation of the antibody response to tetanus-toxoid after bone marrow transplantation

After bone marrow transplantation (BMT), a prolonged dysregulation of humoral immunity can be observed. In the present study, we investigated whether this is reflected in an abnormal production of specific antibodies (Ab) to the T-cell-dependent recall antigen tetanus-toxoid (TT). The study group consisted of children receiving transplants of an unmodified allogeneic graft and of adults receiving either a T-cell- depleted allogeneic or an unmodified autologous BM graft. Findings were compared with those in healthy controls. In pediatric graft recipients, who were routinely revaccinated early after BMT, the Ab response was quantitatively superior to that in adult graft recipients who did not receive early revaccination. In the majority of graft recipients, the time period after vaccination required to reach the peak level of antibodies was prolonged and the number of responding TT-specific B- cell clones was markedly decreased in comparison with controls. In controls, a low frequency of dominant B-cell clones may produce low quantities of homogeneous Ab components (H-Ab) against a heterogeneous background. However, in BM graft recipients, "overshooting" of Ab production by separate B-cell clones was observed, resulting in the development of H-Ab at a relatively high concentration. These abnormalities were present up to 10 years after BMT, irrespective of either the age of the recipient, the modulation of the graft, or the vaccination schedule used. It is hypothesized that the dysregulated Ab production is the consequence of activation of a restricted number of resting memory B cells, present in germinal centers, repopulating gradually after BMT. Our data show that routine revaccination early after BMT improves the humoral immune response. However, because of a clonally dysregulated Ab production, long-lasting qualitative defects may be present even after normalization of Ab titers.     

Angiogenic factors stimulate mast-cell migration

Mast cells accumulate at sites of angiogenesis. The factor(s) that control mast-cell recruitment at these sites have yet to be defined. We sought to determine if angiogenic factors result in mast-cell chemotaxis. In this study, we observed that platelet-derived growth factor-AB (PDGF-AB), vascular endothelial cell growth factor (VEGF), and basic fibroblast growth factor (bFGF) each cause directed migration of murine mast cells at picomolar concentrations, with a typical bell- shaped dose-response curve. Another potent angiogenic factor, platelet- derived endothelial cell growth factor (PD-ECGF), appears to promote chemokinesis of mast cells, whereas tumor necrosis factor-alpha, a weak angiogenic factor, is less robust but still functions as a mast cell chemotactic factor. Epidermal growth factor (EGF), a growth factor with minimal angiogenic properties, was ineffective as a mast cell chemotactic factor. A checkerboard analysis confirmed the directional chemotactic response of PDGF-AB, VEGF, and bFGF, while indicating the chemokinetic response induced by PD-ECGF. Cross-desensitization of growth-factor-induced directed migration was observed between PDGF-AB and bFGF, and also between PDGF-AB and PD-ECGF. Tyrosine kinase- inhibitor genistein effectively dampened the chemotactic responses, whereas pertussis toxin had no effect. In summary, our findings suggest that factors known to act on endothelial cells and stimulate neovascularization may simultaneously serve to recruit mast cells to these sites. The local accumulation of mast cells is believed to facilitate new vessel formation through complex cell:cell interactions.     

T-cell receptor delta/alpha rearrangements in lymphoid neoplasms

Rearrangements within the T-cell receptor (TCR)delta/alpha locus were analyzed in a wide variety of lymphoid neoplasms by eight DNA probes specific for TCR J delta, J alpha and C alpha segments. In all 11 T- cell malignancies, rearrangement and/or deletion of TCR delta was detected irrespective of the stage of maturation of the tumor. The organization of TCR delta correlated with the phenotype of the tumor: In "prethymic" T-cell acute lymphocytic leukemia (ALL), TCR delta was the only TCR gene to be rearranged. More mature T cell malignancies expressing CD4 together with CD3 showed deletion of both alleles of TCR delta, suggestive of TCR V alpha-J alpha rearrangement. All 43 B-cell tumors expressing surface immunoglobulin (sIg), including two cases of adult B-cell ALL, had germline configuration of TCR delta/alpha. In contrast, all 17 B-cell precursor ALLs (null, common, and pre-B-cell ALLs) had rearrangement and/or deletion of TCR delta/alpha. A single case of "histiocytic" lymphoma also showed biallelic deletion of TCR delta. Oligoclonal rearrangements of Ig and TCR genes were observed in two cases of B-cell precursor ALL and in one case of T-cell lymphoblastic lymphoma. Patterns of such "aberrant" TCR rearrangement were similar to those observed in T-lineage malignancies. In particular, seven of eight cases of B-cell precursor ALL and the histiocytic lymphoma which demonstrated biallelic TCR delta deletion, (suggestive of a V alpha-J alpha rearrangement) had clonal TCR beta rearrangement. These data support the hypothesis that supposedly aberrant rearrangements of the TCR genes may follow the same developmental controls as found in T-cell differentiation, despite the lack of evidence for further commitment to the T-cell lineage. TCR delta rearrangement is a useful marker of clonality of immature T-cell tumors which may have only this gene rearranged but is not specific to the T-cell lineage.     

Sustained, retransplantable, multilineage engraftment of highly purified adult human bone marrow stem cells in vivo

Data from many laboratory and clinical investigations indicate that CD34+ cells comprise approximately 1% of human bone marrow (BM) mononuclear cells, including the progenitor cells of all the lymphohematopoietic lineages and lymphohematopoietic stem cells (stem cells). Because stem cells are an important but rare cell type in the CD34+ cell population, investigators have subdivided the CD34+ cell population to further enrich stem cells. The CD34+/CD38- cell subset comprises less than 10% of human CD34+ adult BM cells (equivalent to < 0.1% of marrow mononuclear cells), lacks lineage (lin) antigens, contains cells with in vitro replating capacity, and is predicted to be highly enriched for stem cells. The present investigation tested whether the CD34+/CD38- subset of adult human marrow generates human hematopoiesis after transfer to preimmune fetal sheep. CD34+/ CD38- cells purified from marrow using immunomagnetic microspheres or fluorescence-activated cell sorting generated easily detectable, long- term, multilineage human hematopoiesis in the human-fetal sheep in vivo model. In contrast, transfer of CD34+/CD38+ cells to preimmune fetal sheep generated only short-term human hematopoiesis, possibly suggesting that the CD34+/CD38+ cell population contains relatively early multipotent hematopoletic progenitor cells, but not stem cells. This work extends the prior in vitro evidence that the earliest cells in fetal and adult human marrow lack CD38 expression. In summary, the CD34+/ CD38- cell population has a high capacity for long-term multilineage hematopoietic engraftment, suggesting the presence of stem cells in this minor adult human marrow cell subset.     

A phase II study of continuous infusion recombinant human granulocyte- macrophage colony-stimulating factor as an adjunct to autologous bone marrow transplantation for patients with non-Hodgkin's lymphoma in first remission

Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) clearly hastens myeloid recovery in patients with relapsed hematologic malignancies undergoing autologous bone marrow transplantation (ABMT). In efforts to further improve neutrophil engraftment and shorten hospital stay in ABMT patients, rhGM-CSF was administered by a potentially more potent route (continuous infusion) to non-Hodgkin's lymphoma (NHL) patients with better BM reserve (first remission). Time to myeloid engraftment was compared with that of NHL patients treated in first remission at our institution on a similar ABMT protocol but without growth factor support (controls). Median neutrophil engraftment (absolute neutrophil count, 500 cells/microL) in first remission patients treated with rhGM-CSF was 14 days, compared with 22 days in controls (P = .0001). Hospital stays were also significantly reduced for rhGM-CSF patients (P = .0003). Platelet engraftment did not differ between the two groups. Persistent fever and generalized serositis were the primary toxicities. rhGM-CSF, delivered by this route, was efficacious but more toxic than 2-hour rhGM-CSF infusions previously reported by other investigators. Future alterations in both dose and schedule may retain comparable efficacy yet diminish toxicity.     

Enhancement of in vivo humoral immunity by estrogen: permissive effect of a thymic factor

Physiological levels of estrogen enhance humoral immune responses. Several in vitro studies indicate the hormone to have a direct effect on immune cells, and other studies show that estrogen may affect humoral immunity indirectly through the thymus. Therefore, we have conducted experiments to investigate the requirement of the thymus in the enhancement of humoral immune responsiveness by estrogen. In Exp I, adult ovariectomized Lewis rats were thymectomized or sham thymectomized and given estradiol (E2; 0.25 microgram E2 in sesame oil, sc, once every 4 days) or the oil vehicle in a 2 x 2 factorial design, and their antifluorescein responses were followed by enzyme-linked immunosorbant assay across 21 days. Only animals that were thymus intact and given estrogen replacement showed significantly (P less than 0.05) greater serum antifluorescein titers than all other treatments. In Exp II, ovariectomized thymectomized rats were submitted to a 2 x 3 factorial design of oil vehicle or E2 replacement and saline, gelatin, or thymus replacement (thymosin fraction 5; 1 mg/kg in saline, sc). As described above, only the animals receiving both thymosin fraction 5 and E2 replacement displayed antifluorescein titers that were significantly (P less than 0.03) increased over titers of all other treatment groups. These results indicate that the enhancement of in vivo humoral immunity by estrogen requires the thymus, and that a constitutive thymic factor, found in thymosin fraction 5, exerts a permissive influence on the action of E2 outside the thymus to increase a specific humoral immune response.     

Insulin binding substances,autoimmunity and type i diabetes in kuwaiti patients and their kindred

Summary Insulin autoantibodies (IAAs) are associated with type I diabetes mellitus (DM) and have been suggested as predictive markers of the disease. Using an ELISA assay, we have studied the prevalence of binding to human insulin in sera from an Arab type I DM population and compared it with the prevalence in the family members (FMs) of the probands, in type II DM patients from the same population, and in Arab control subjects. Significant levels of binding occurred in 11/16 (69%) of type I DM patients and in 21/34 (62%) of their FMs, but in only 5/31 (16%) of type II DM patients and in 1/25 (4%) of control subjects. Within families, there was homogeneity with regard to the level of insulin binding and the mean family levels correlated with those of the proband (r=0.68, df=7, p=0.05). HLA-DR3 or -DR4 antigens occurred in 55/63 (87%) of type I DM patients and in 95/118 (81%) of their FMs. This was significantly higher (p<0.001) than in either type II DM patients (39/75, 52%) or in control subjects (34/93, 37%). ICAs were present in significantly more (25/43, 58%) of type I DM patients than their FMs (3/82, 3%) (p<0.001). They did not occur in either type II DM patients or in the control group. In conclusion, insulin binding occurred in sera from both type I diabetic patients and their kindred, and hence did not appear to be specifically associated with the development of clinical diabetes.