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Biomedical subjects

E Schrama

Publications and source records attributed to E Schrama.

15 recordsLinked to original sources
PubMed1999

Feasibility of immunotherapy of relapsed leukemia with ex vivo-generated cytotoxic T lymphocytes specific for hematopoietic system-restricted minor histocompatibility antigens.

Allogeneic bone marrow transplantation (BMT) is a common treatment of hematologic malignancies. Recurrence of the underlying malignancy is a major cause of treatment failure. Donor-derived cytotoxic T lymphocytes (CTLs) specific for patients' minor histocompatibility antigens (mHags) play an important role in both graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) reactivities. mHags HA-1 and HA-2 induce HLA-A*0201-restricted CTLs in vivo and are exclusively expressed on hematopoietic cells, including leukemic cells and leukemic precursors, but not on fibroblasts, keratinocytes, or liver cells. The chemical nature of the mHags HA-1 and HA-2 is known. We investigated the feasibility of ex vivo generation of mHag HA-1- and HA-2-specific CTLs from unprimed mHag HA-1- and/or HA-2-negative healthy blood donors. HA-1 and HA-2 synthetic peptide-pulsed dendritic cells (DCs) were used as antigen-presenting cells (APC) to stimulate autologous unprimed CD8(+) T cells. The ex vivo-generated HA-1- and HA-2-specific CTLs efficiently lyse leukemic cells derived from acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) patients. No lytic reactivity was detected against nonhematopoietic cells. Sufficient numbers of the CTLs can be obtained for the adoptive immunotherapy purposes. In conclusion, we present a feasible, novel therapy for the treatment for relapsed leukemia after BMT with a low risk of GVHD.

Antigens, Neoplasm↗
PubMed1999

Tetrameric HLA class I-minor histocompatibility antigen peptide complexes demonstrate minor histocompatibility antigen-specific cytotoxic T lymphocytes in patients with graft-versus-host disease.

Graft-versus-host disease (GvHD) is a chief complication of allogeneic bone marrow transplantation. In HLA-identical bone marrow transplantation, GvHD may be induced by disparities in minor histocompatibility antigens (mHags) between the donor and the recipient, with the antigen being present in the recipient and not in the donor. Cytotoxic T lymphocytes (CTLs) specific for mHags of the recipients can be isolated from the blood of recipients with severe GvHD (ref. 3). A retrospective study demonstrated an association between mismatch for mHags HA-1, -2, -4 and -5 and the occurrence of GvHD in adult recipients of bone marrow from HLA genotypically identical donors. Tetrameric HLA-peptide complexes have been used to visualize and quantitate antigen-specific CTLs in HIV-infected individuals and during Epstein-Barr virus and lymphocytic choriomeningitis virus infections. Here we show the direct ex vivo visualization of mHag-specific CTLs during GvHD using tetrameric HLA-class and I-mHag HA-1 and HY peptide complexes. In the peripheral blood of 17 HA-1 or HY mismatched marrow recipients, HA-1- and HY-specific CTLs were detected as early as 14 days after bone marrow transplantation. The tetrameric complexes demonstrated a significant increase in HA-1- and HY-specific CTLs during acute and chronic GvHD, which decreased after successful GvHD treatment. HLA class I-mHag peptide tetramers may serve as clinical tools for the diagnosis and monitoring of GvHD patients.

Adult↗
PubMed1998

CD80-Transfected acute myeloid leukemia cells induce primary allogeneic T-cell responses directed at patient specific minor histocompatibility antigens and leukemia-associated antigens.

Despite sufficient levels of HLA class I and class II expression, acute myeloid leukemia (AML) cells usually fail to induce a significant T-cell response in vitro. Therefore, we investigated whether in vitro modifications could enhance the T-cell stimulatory properties of AML cells. AML cells were either cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and tumor necrosis factor-alpha (TNF-alpha), or transfected with the CD80 (B7.1) gene and used as stimulator cells for primed and unprimed allogeneic T cells. Cytokine treatment increased HLA class I and II expression, but did not induce CD80 on AML cells. Cytokine-treated AML cells efficiently presented nominal and allo-antigens to primed T-cell clones, induced strong T-cell proliferation in HLA mismatched mixed lymphocyte reactions (MLR), but failed to induce primary T-cell responses from an HLA identical bone marrow donor in MLR. In contrast, CD80-transfected AML cells induced T-cell proliferation of HLA-identical bone marrow donor peripheral blood mononuclear cell (PBMC) in primary MLR, allowing the generation of leukemia reactive CD4(+) T-cell lines and clones. The majority of the generated oligoclonal (25 of 35) T-cell cultures showed patient specific reactivity that did not discriminate between patient's leukemic cells and Epstein-Barr virus (EBV)-transformed B cells (EBV-LCL). The remaining 10 oligoclonal T-cell cultures recognized only leukemic cells. One of these latter leukemia reactive oligoclonal T cells was cloned. The majority of the clones (25 of 29) reacted against both leukemic cells and patient's EBV-LCL. A minority of the T-cell clones with the CD4 phenotype (four of 29) showed strong HLA-DP restricted reactivity against leukemic cells, but not against patient's EBV-LCL or against HLA-matched nonleukemic cells, indicating that their target antigens are preferentially expressed by leukemic cells. In conclusion, our study shows that the in vitro allogeneic T-cell response induced by CD80-transfected AML cells is mainly directed against patient's specific minor histocompatibility antigens, while antigens preferentially expressed by leukemic cells can also trigger T-cell responses.

Antigens, Neoplasm↗
PubMed1997

HLA class II restricted T-cell reactivity to a developmentally regulated antigen shared by leukemic cells and CD34+ early progenitor cells.

After allogeneic bone marrow transplantation (BMT), the beneficial graft-versus-leukemia (GVL) effect but also the life-threatening graft-versus-host disease (GVHD) are mediated by T cells of the grafted marrow. The identification of leukemia cell-reactive T cells and their ligands are, therefore, crucial for the development of new anti leukemia strategies. Here we describe a leukemia-reactive allo-HLA class II restricted CD4+ T-cell clone, 6.2, isolated from a healthy individual after stimulation with allogeneic leukemic cells. Clone 6.2 recognizes leukemic cells from several AML patients without showing reactivity to unfractioned peripheral blood mononuclear cells, monocytes, B cells, T-cell blasts, and proximal tubulus epithelial cells. Interestingly, clone 6.2 also recognizes BM cells derived from healthy individuals and inhibits the colony formation of myeloid and erythroid cell lineages. In the BM, clone 6.2 recognizes only CD34+ early precursor cells but not CD34-, more differentiated cells. Thus, the target antigen of clone 6.2 is developmentally regulated and expressed only by leukemic cells and CD34+ early progenitor cells in the hematopoietic system. We suggest that targeting the T-cell immune response to leukemia-associated, developmentally regulated antigens of the hematopoietic system can provide a basis for the separation of GVL from GVHD, and may lead to new therapeutic approaches for residual and relapsed leukemia.

Acute Disease↗
PubMed1996

Interstitial rejection, vascular rejection, and diffuse thrombosis of renal allografts. Predisposing factors, histology, immunohistochemistry, and relation to outcome.

Histological and immunohistochemical analyses were made of biopsy specimens from 50 consecutive patients who experienced putative graft rejection. The mean age of the patients was 44.5 years (range, 17-69 years) and 26 were men. There were 67 evaluable allograft specimens, which were grouped according to the histological diagnosis: group 1, acute tubulointerstitial rejection (n = 42); group 2, acute vascular rejection (n = 18); and group 3, diffuse thrombosis (n = 7). Over a follow-up period of 21-57 months, the mean number of rejection episodes was 1.7, 2.8, and 3.3 in groups 1, 2, and 3, respectively. Allograft loss occurred in 7 out of 30, 10 out of 16, and 4 out of 4 patients in groups 1, 2, and 3, respectively. The following histological parameters differed significantly (P < 0.05) among the groups: interstitial edema, congestion of peritubular capillaries, glomerular thrombosis, and glomerular ischemia (group 3 > group 2 > group 1). Interstitial bleeding was seen more often in group 2 and 3 tissues than in group 1 specimens (P < 0.01). Immunohistochemical analyses showed that vascular rejection was associated with WT14 staining for monocytes and macrophages around the tubuli and with interstitial deposition of complement factor 3. With regard to serology, positive anti-endothelial cell antibody-dependent cellular cytotoxicity was associated with vascular rejection and thrombosis of the graft in all patients tested, and with graft loss in 75%. Pre-existent positive anti-IgG immunofluorescence on peritubular capillaries in pretransplant biopsy specimens incubated with patient serum was found in only 3 of the 50 patients, but was associated with graft loss in 2 of the 3. Cytomegalovirus infection was associated with a higher percentage of graft loss. There were significant intergroup differences in panel reactive antibodies before transplantation (P < 0.001), with higher titers in groups 2 and 3. The findings in relation to interstitial rejection are compatible with cellular rejection, while the data on vascular rejection support a humorally mediated pathogenesis.

Adolescent↗
PubMed1995

Evaluation by histology, immunohistology and PCR of protocollized renal biopsies 1 week post-transplant in relation to subsequent rejection episodes.

Renal biopsies were performed 1 week following renal transplantation at a time without clinical evidence of rejection in 43 patients (13 females, mean age 48 years range 18-60 and 30 males, mean age 43 years range 17-59 years). Thirty-six biopsies were available for histological or immunohistochemical analysis. Immunohistochemical analyses were performed with monoclonal antibodies against leukocytes (CD45), monocytes (WT14), complement factor 3 (C3), T-cells (Leu4), T-cell receptor alpha beta and gamma delta, tumour necrosis factor alpha (TNF alpha), IL-2 receptor (IL2-R, TAC), intercellular adhesion molecule-1 (ICAM1) and HLA-DR. The slides were scored semiquantitatively with the observers having no knowledge of clinical or patient data. TNF alpha and IL-2R were also measured by quantative PCR. None of the studied parameters correlated to delayed graft function or graft loss. Histological analysis showed that both focal interstitial infiltrate (18/35) and tubular basement membrane disruption (11/35) were followed by a higher incidence of subsequent rejection (P = 0.03 and 0.02 respectively). Also positivity for WT14 around tubuli (P = 0.02) was associated with subsequent occurrence of rejection. The intensity of staining of ICAM-1 on PTC as well as TAC on proximal tubular cells was associated with the number of subsequent rejection episodes. The association between the IL-2 receptor and subsequent rejection was also found applying PCR to the tissue specimens. We conclude that the presence of focal interstitial infiltrates and tubulitis in 1-week biopsies from well-functioning grafts carries an increased risk of subsequent rejection.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Isopropylmalate Synthase↗
PubMed1995

Tissue antigens in tubulointerstitial and vascular rejection.

We propose that tissue-specific alloantigens are of importance in interstitial and vascular rejection. To study this hypothesis we took the following approaches: multivariate analysis on our database (N = 482) was performed, the specificity of T cells cultured from kidneys with rejection was analyzed, and non-anti-HLA antibodies reactive with endothelium were studied. First we observed that in a cohort study of 482 patients receiving a cadaveric renal allograft 76 (15.8%) patients developed vascular rejection and 115 (23.9%) developed interstitial rejection. The incidence of vascular rejection was increased in patients with delayed graft function, HLA-DR mismatches, a prolonged cold ischemia period, and previous transplantations. Next we examined 40 graft infiltrating cell (GIC) lines cultured from renal biopsies taken during rejection episodes. Thirteen GIC lines reacted in a donor-specific fashion to proximal tubular cells (PTEC) but not to donor splenocytes. These GIC recognize polymorphic tissue-specific peptides in the context of allo-MHC Class I. Finally, we studied if non-conventional allo-antigen systems on endothelial cells could be the target of the humoral immune response during vascular rejection. We found the endothelial monocyte (EM) system, and another system that is present on endothelial cells and platelets, which can be tested in an antibody-dependent cellular cytotoxicity assay (ADCC).

Graft Rejection↗
PubMed1994

Expression of glomerular extracellular matrix components in human diabetic nephropathy: decrease of heparan sulphate in the glomerular basement membrane.

Diabetic nephropathy is characterized by albuminuria which proceeds to overt proteinuria. The highly negatively stained HS side chain of heparan sulphate proteoglycan (HSPG) is a major determinant of the charge-dependent permeability of the GBM. We set out to study the presence of HS and HSPG in the GBM of patients with diabetic nephropathy using newly developed monoclonal antibodies, and to compare HSPG expression to the expression of other previously investigated glomerular extracellular matrix compounds. Immunohistochemically, glomerular extracellular matrix components were analysed in 14 renal biopsies of patients with diabetic nephropathy and compared with those of normal control subjects. Monoclonal antibodies used were: JM403 against the HS side chain of GBM HSPG and JM72 against the HSPG-core protein. Also, a polyclonal antiserum (B31) against human GBM-HSPG-core protein was used. Additionally, antibodies were used against collagen types I, III, IV and against alpha 1 (IV)NC, alpha 3(IV)NC and fibronectin. Staining was scored for intensity and for staining pattern by four independent observers who had no previous knowledge of the sample origin. No glomerular staining was seen for collagen type I. Collagen type III was present in some diabetic nodules. Anti-collagen type IV showed a decreased GBM staining in patients with diabetic nephropathy (p = 0.04). With anti-alpha 1 (IV)NC no changes in GBM staining intensity were observed; with anti-alpha 3 (IV)NC brilliant GBM staining was seen in both groups. Increased mesangial staining (p = 0.003) was seen with anti-collagen type IV in biopsies with nodular lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1994

The role of unconventional alloantigens in interstitial and vascular rejection after renal transplantation.

Lymphocytes have been used as the classic target cells to measure donor specific alloreactivity before and after renal transplantation. This was based on the concept that during rejection after kidney transplantation graft cells were recognized only because of sharing of alloantigens with lymphoid cells. There is, however, sufficient evidence to suggest that tissue specific recognition of proximal tubular cells and graft endothelial cells can occur by allospecific T cells and IgG antibodies, respectively. The antigens involved have not yet been well characterized, but seem to be of relevance in the pathogenesis of both interstitial and vascular rejection. Identification of the antigens involved may lead to more appropriate matching strategies.

Animals↗
PubMed1994

Interleukin 6 production by human proximal tubular epithelial cells in vitro: analysis of the effects of interleukin-1 alpha (IL-1 alpha) and other cytokines.

Proximal tubular epithelial cells (PTEC) from human renal tissue obtained from biopsy or nephrectomy were grown in monoculture and evaluated in vitro at passage 2-4 for interleukin 6 (IL-6) production in response to medium alone or to interleukin 1 alpha (IL-1 alpha), tumour necrosis factor alpha (TNF alpha), interleukin 2 (IL-2), interferon gamma (INF gamma) or lipopolysaccharide (LPS). IL-6 bioactivity was quantitated using the IL-6-dependent murine hybridoma cell line (B9) and expressed as IL-6 units/ml/10(5) PTEC. PTEC cell lines exposed to medium alone produced intermediate amounts of IL-6 with substantial variability between cell lines. Introduction of IL-1 alpha resulted in a dose- and time-dependent increase in IL-6 production by PTEC that was maximal at 1 ng/ml IL-1 alpha at 24 h. All PTEC cell lines showed an increased IL-6 production on exposure to IL-1 alpha varying from 1.3- to 24-fold increase over baseline production. This response was completely blocked by anti-rIL-1 alpha. No significant IL-6 production by PTEC could be induced by TNF alpha, IL-2, IFN gamma, or LPS over a broad dosage range. Cycloheximide inhibited IL-6 production without irreversible cell toxicity, indicating de-novo synthesis. IL-6 produced by PTEC had a molecular weight of 26-29 kDa as demonstrated by Western blot analysis. Using PCR analysis we could demonstrate upregulation by IL-1 alpha of IL-6 mRNA in a dose-response fashion, indicating that IL-1 alpha regulates IL-6 production at a pretranslational value of protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1992

IL-1 alpha stimulated TNF alpha production by cultured human proximal tubular epithelial cells.

Tumor necrosis factor alpha (TNF alpha) production by proximal tubuli was studied by immunoperoxidase staining of 20 renal biopsies from transplant patients. A positive staining for TNF alpha on proximal tubuli was seen in nine out of 15 patients with interstitial infiltrate, five without clinical significant rejection and four with moderate to severe interstitial rejection. TNF alpha was only expressed on tubuli within areas of interstitial infiltrate. Expression of TNF alpha in the mononuclear cell infiltrate was seen only in three patients with interstitial rejection. Absence of TNF alpha could be seen in biopsies with no renal abnormalities. To obtain more information on the regulation of TNF alpha production, proximal tubular epithelial cells (PTEC) cultures were established and assessed for production of TNF alpha. Heterogenicity in production of TNF alpha was found in 14 tested PTEC lines cultured under serum free conditions. The presence of IL-1 alpha in the cultures induced a time- and dose-dependent enhancement of TNF alpha production by PTEC. Enhanced production of TNF alpha was not seen after stimulation with other cytokines such as IL-2 or IFN gamma. Inhibition studies with cycloheximide indicated de novo synthesis of TNF alpha. Western blot analysis of supernatants of unstimulated and IL-1 alpha stimulated PTEC indicated a 17 kd product, a size similar to that of recombinant TNF alpha. Northern blot analysis revealed the presence of a 2.0 kb hybridization signal in total RNA of PTEC cultures and up regulation upon treatment of PTEC with 1 ng/ml of IL-1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1991

T-cell receptors and ICAM-1 expression in renal allografts during rejection.

Sixty-two biopsies taken from 38 kidney grafts were studied for 15 histological and 10 immunohistological parameters. The biopsies were divided into three groups, according to the clinical diagnosis at the time they were performed: group 1, rejection (n = 43); group 2, other causes of dysfunction (n = 10); and group 3, stable function (n = 9). Histological signs of acute rejection included diffuse interstitial infiltrate, tubular basement membrane damage, mononuclear leukocyte infiltration, and congestion of the peritubular capillaries. Immunoperoxidase staining with monoclonal antibodies to ten markers showed a statistically significant association between detection of T-cell receptor subunits alpha-beta (TcR2) and gamma-delta (TcR1) on infiltrating lymphocytes and of intercellular adhesion molecule-1 (ICAM-1) in tubular cells and acute rejection. These results suggest that T-cell receptors and ICAM-1 may be useful markers to differentiate acute rejection from renal graft dysfunctions due to other abnormalities.

Adult↗
PubMed1990

Value of anti-neutrophil cytoplasmic autoantibodies and other laboratory parameters in follow-up of vasculitis.

The presence of anti-neutrophil cytoplasmic autoantibodies (ANCA), detected by indirect immunofluorescence, is of high sensitivity and specificity in the diagnosis of Wegener's granulomatosis and related diseases, associated with vasculitis. Titres of immunofluorescence are thought to closely reflect disease activity. In a retrospective series of 266 sera of 23 patients with at least one positive test for ANCA, disease activity was correlated with ANCA, assayed by immunofluorescence and by enzyme-linked immunosorbent assay; with rheumatoid factor and with erythrocyte sedimentation rate. All tests were of limited value in predicting disease activity or relapse. A normal sedimentation rate and, to a lesser extent, a negative result of ANCA-immunofluorescence, were useful in excluding active disease.

Autoantibodies↗