Inflammatory cell burden and phenotype in endomyocardial biopsies with antibody-mediated rejection (AMR): a multicenter pilot study from the AECVP.

This multicenter case-controlled pilot study evaluated myocardial inflammatory burden (IB) and phenotype in endomyocardial biopsies (EMBs) with and without pathologic antibody-mediated rejection (pAMR). Sixty-five EMBs from five European heart transplant centers were centrally reviewed as positive (grade 2, n = 28), suspicious (grade 1, n = 7) or negative (n = 30) for pAMR. Absolute counts of total, intravascular (IV) and extravascular (EV) immunophenotyped mononuclear cells were correlated with pAMR grade, capillary C4d deposition, donor specific antibody (DSA) status and acute cellular rejection (ACR). In pAMR+ biopsies, equivalent number of IV CD3+ T lymphocytes (23 ± 4/0.225 mm(2) ) and CD68+ macrophages (21 ± 4/0.225 mm(2) ) were seen. IB and cell phenotype correlated with pAMR grade, C4d positivity and DSA positivity (p < 0.0001). High numbers of IV T lymphocytes were associated with low grade ACR (p = 0.002). In late-occurring AMR EV plasma cells occurring in 34% of pAMR+ EMBs were associated with higher IB. The IB in AMR correlated with pAMR+, C4d positivity and DSA positivity. In pAMR+ equivalent numbers of IV T lymphocytes and macrophages were found. The presence of plasma cells was associated with a higher IB and occurrence of pAMR late after transplantation.

Intensification of GVHD prophylaxis with low-dose ATG-F before allogeneic PBSC transplantation from HLA-identical siblings in adult patients with hematological malignancies: results from a retrospective analysis.

Several studies have shown that chronic GVHD (cGVHD) is more frequent in patients receiving transplants from PBSC than in those receiving BM. In the setting of PBSC-unrelated transplants, the addition of anti-T-cell globulin (ATG) has shown a significant decrease in incidence/severity of cGVHD, without an increase in relapses or infections. However, no prospective data are yet available in the sibling setting. We retrospectively analyzed the effects of intensification of standard GVHD prophylaxis (CsA+MTX) by the addition of low-dose ATG in 245 patients receiving a transplant from HLA-identical sibling. From 1996 to 2001, patients received PBSC as the preferred source (group 2), and then ATG was added before transplant (group 3) because of a high cGVHD rate. Patients receiving BM in the same time period were analyzed as a control group (group 1). The incidence of grade III-IV acute GVHD and cGVHD was not significantly different in the three groups, but extensive cGVHD was highest in group 2 (38%) compared with group 3 (21%) or group 1 (28%; P=0.03). OS, TRM and time to relapse/progression were similar in the three groups. Our analysis shows that adding ATG to PBSC sibling allogeneic transplants can lower cGVHD, without an increase of relapse. Further prospective studies are needed to confirm these findings.

Reduced incidence of GVHD without increase in relapse with low-dose rabbit ATG in the preparative regimen for unrelated bone marrow transplants in CML.

SUMMARY: Antithymocyte globulin (ATG) treatment prevents graft failure and results in a low incidence of GVHD, but an increased risk of relapse could be expected as a consequence of reduced GVHD. From September 1995 to June 2001, 28 consecutive chronic myeloid leukemia (CML) patients underwent unrelated bone marrow transplants: 21 were in chronic phase (CP) and seven in advanced phase (AP). Median age was 35.5 years (range 20-50). HLA typing was based on high-resolution molecular techniques; in eight cases there were one or more allele mismatches. The preparative regimen consisted of TBI, EDX 120 mg/kg and rabbit ATG 15 mg/kg. All patients engrafted and no rejection occurred. Acute GVHD grade III-IV occurred in six patients (21%). Chronic GVHD occurred in 10 (40%) and it was extensive in one. Four out of seven patients transplanted in AP had a hematological relapse. Of 21 in CP, there was one cytogenetic and one molecular relapse: these two patients are now in complete remission with imatinib mesylate. With a median follow-up of 45.7 months, the 5-year survival is 76.2% for those transplanted in CP. These data demonstrate that transplants performed in CP, with low-dose ATG, are associated with a good outcome, low incidence of GVHD and no increase of relapse.

Bone marrow transplantation from unrelated donors: the impact of mismatches with substitutions at position 116 of the human leukocyte antigen class I heavy chain.

The hypothesis was tested that amino acid substitutions in specific positions within human leukocyte antigen class I heavy chain would have different impacts on transplant-related mortality (TRM) in patients receiving transplanted bone marrow from unrelated donors. One hundred patients and their unrelated donors were typed by sequence-based typing for the human leukocyte antigen (HLA)-A, -B, and -C loci. All pairs were matched for DRB1, DRB3, DRB4, DRB5, DQA1, and DQB1 loci. Forty pairs were also matched at class I, and 60 pairs had one or more mismatches at class I loci. It was found that substitutions at positions 116 and 114 of class I heavy chain significantly increased the risk for TRM in univariate and bivariate Cox analyses. Conversely, no association between number of multiple mismatches or number of amino acid substitutions and TRM was seen when positions 116 and 114 were adjusted for. Variables predictive of TRM in multivariate Cox analysis were number of cells infused, diagnosis (chronic myeloid leukemia [CML] or non-CML), and amino acid substitution at position 116 or 152. The only variable predictive of severe acute graft-versus-host disease (GVHD) in multivariate Cox analysis was substitution at position 116. Actuarial risk for acute GVHD grade III-IV, TRM, and relapse in pairs with substitutions at position 116 (n = 37) compared to other pairs (n = 63) was, respectively, 36% versus 14% (P =.01), 59% versus 28% (P =.001), and 25% versus 31% (P =.4). In conclusion these data suggest that substitutions at position 116 of class I heavy chain increase the risk for acute GVHD and TRM in patients who receive transplanted bone marrow from unrelated donors.

White cell apoptosis in platelet concentrates.

BACKGROUND: The aim of the present study was the evaluation of the apoptosis in residual white cells (WBCs) contained in platelet concentrates (PCs) and of the relationship of this apoptosis with the concentration of inflammatory cytokines in the medium and with platelet activation. STUDY DESIGN AND METHODS: Three independent methods were used to evaluated apoptosis in WBCs present in 9 PCs, either from single donors by apheresis (SD-PCs) or from pooled buffy coats (BC-PCs). All PCs were divided in two parts, one of which was irradiated. PCs were stored up to 4 days at room temperature, and samples were withdrawn daily for analysis of apoptosis, of platelet activation (surface and soluble CD62P), and of cytokine concentration (interleukin [IL]-1alpha, IL-1beta, IL-6, IL-8, and tumor necrosis factor alpha). RESULTS: Apoptosis was found to occur with storage in both irradiated and nonirradiated units. Platelet activation increased with storage time and was higher in BC-PCs. The amount of released cytokines was rather variable among PC units. Only IL-8 was consistently found to increase with storage time. CONCLUSIONS: Apoptosis of residual WBCs occurred in PC units as a function of storage time. The amount and the time course of apoptosis seem to correlate with IL-8 release rather than with platelet activation or with the occurrence of febrile nonhemolytic transfusion reactions.

Synergism between gamma interferon and doxorubicin in a human MDR colon adenocarcinoma cell line.

Recent interest in cancer therapy derives from the ability of interferons to synergistically increase the activity of chemotherapeutic agents. To understand the biological basis of this synergism we evaluated the effects of human recombinant IFN-gamma on the expression of the mdr1 gene and on the cellular growth of a human colon adenocarcinoma cell line (LoVo) and its MDR subline (LoVo/Dx) after coincubation with doxorubicin. Treatment with IFN-gamma showed unchanged levels of MDR1-glycoprotein, no perturbation on cell cycle distribution and a significant reduction of colony formation in both lines (P < 0.05) starting from 100 U/ml. A synergistic effect was observed in the LoVo/Dx cell line when doxorubicin was added after exposure to 0.1-10 U/ml of IFN-gamma. Our data indicate that the effects of IFN-gamma, independent from action on cell proliferation and from modulation of p-glycoprotein expression, are a cause of the synergistic activity between this lymphokine and conventional chemotherapeutic agents such as doxorubicin.

Immunological and non-immunological influence of H-2Kb gene transfection on the metastatic ability of B16 melanoma cells.

The H-2b-negative B78HI clone (derived from B16 melanoma) was transfected with the H-2Kb gene; 4 cell clones expressing membrane H-2Kb antigens and 2 control clones (transfected with pSV2neo alone) were used for studies of metastatic ability, immunogenicity, NK sensitivity and homotypic adhesion. The experimental metastatic capacity of H-2Kb transfectants in syngenic mice was greatly diminished in comparison with control and parent cells. Both immune-mediated and intrinsic properties of transfectants correlated with their lower metastatic ability. A cell-mediated cytotoxic response was induced by repeated in vivo immunizations of syngeneic mice followed by in vitro restimulation of effectors when transfectants (but not controls) were used as immunizers and as targets. Moreover, homotypic adhesion of H-2Kb transfectants was significantly lower than that of controls. Sensitivity to NK cells of transfectants was not decreased in comparison to H-2-negative controls. It is known that in vitro treatment with IFN-gamma of H-2-positive B16 melanoma cells induces a simultaneous increase in H-2 expression and in experimental metastasis; treatment of H-2Kb transfectants with IFN-gamma induced a higher Kb expression, but no increase in metastatic ability, thus suggesting that the IFN-sensitive component that mediates enhancement of metastasis is not H-2Kb.

CD34 or S313 positive cells selection by avidin-biotin immunoadsorption.

A column immunoadsorption method, based on the high affinity between the protein Avidin and the vitamin Biotin has been used to obtain positive CD34+ or S313+ marrow cells selection. Cell suspensions from marrow samples of six healthy subjects were incubated either the monoclonal antibody S313 or HPCA 1 (My 10-like) and a biotinilated goat anti-mouse Immunoglobulins antiserum, passed over a column containing an Avidin coated Polyacrylamide matrix, at the flow rate of 1 ml/min. The phenotype and the clonogenic efficiency of the recovered adherent cell fraction were studied by cytofluorimetric analysis and haemopoietic progenitors short term cultures. The results obtained show a mean CD34+ and S313+ cells recovery greater than 50% with a lower stem cells enrichment. Although these data could not considered optimal for clinical application in haematologic neoplasias, these preliminary studies demonstrate the possible use of the method for autologous bone marrow transplantation.

Evaluation of LAK-mediated tumor cell killing in a plasma clot clonogenic assay.

An assay based on the inhibition of the cloning capacity in a plasma clot semisolid medium assay has been used to test the sensitivity of the Raji cell line to lymphokine-activated killer (LAK) cells. This method overcomes some limitations intrinsic to the widely employed 51Cr release assay and always shows a higher degree of sensitivity. No inhibition of colony growth was found when the effector cells were plated without prior pre-incubation with interleukin 2 or with the addition of the medium derived from the LAK cells. Though more time-consuming than the classic 51Cr release assay, this technique does not require radioactive material. This test may be suitable for a more precise evaluation of LAK activity and for the study of the mechanisms involved in cell killing.

In vitro bone marrow purging of multidrug-resistant cells with a mouse monoclonal antibody directed against Mr 170,000 glycoprotein and a saporin-conjugated anti-mouse antibody.

Selective elimination of multidrug resistance-positive cells (LoVo/Dx) was obtained by using the monoclonal antibody MRK 16, which recognizes a surface epitope of the Mr 170,000 glycoprotein, and a sheep anti-mouse immunoglobulin antibody, conjugated to the ribosome-inactivating protein saporin 6. The killing was greatly decreased or even abolished by adding the monoclonal antibody at a 100-fold concentration. Both the MRK 16 and anti-mouse saporin 6 conjugate did not show any killing activity when they were used separately. In cell suspensions composed of 90% normal bone marrow cells and 10% multidrug resistance-positive cells, the monoclonal antibody MRK 16 followed by the anti-mouse immunotoxin caused the elimination of 99% multidrug resistance-positive cells, as revealed by immunofluorescence and immunocytochemistry as well as by a clonal assay. Human normal hematopoietic precursors (granulomonocytic colony-forming units, erythroid burst-forming units, and multipotent granulomonocytic, erythroid, and megakaryocytic-forming units) were not affected by the MRK 16 plus immunotoxin treatment. This technique might be suitable for ex vivo bone purging in an appropriate clinical setting, such as autologous bone marrow transplantation.

Enhancement of experimental metastatic ability by tumor necrosis factor-alpha alone or in combination with interferon-gamma.

The effects of recombinant tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on B16 mouse melanoma experimental metastatic ability and major histocompatibility complex (H-2b) antigens expression were studied. B16 cells exposed in vitro to TNF-alpha had an increased H-2 expression and were more metastatic than untreated cells. The simultaneous treatment with TNF-alpha and IFN-gamma amplified the enhancement of experimental metastasis and all other effects obtained with TNF-alpha alone. The B16 clone B78H1, selectively resistant to H-2 induction and to enhancement of metastatic ability by IFN-gamma, was not affected by treatment with TNF-alpha and with TNF-alpha + IFN-gamma. These findings contribute to a better understanding of the pleiotropic effects of TNF, some of which can have opposing actions in the complex tumor-host relationships.

Improved methods for the determination of 5-bromo-2-deoxyuridine labelling index in clones and colonies growing in plasma clots.

Bromodeoxyuridine (BrdUrd), an analogue of thymidine is one of the most employed marker to detect the S-phase of the cell cycle. Difficulties are described for the in situ detection of S-phase cells in normal and neoplastic growing clones. In this paper we propose new methods for the detection of BrdUrd in neoplastic clones, growing in plasma clots. In particular, these methods are based on the immunocytochemical staining with horseradish peroxidase and alkaline phosphatase, as well as on a new immunofluorescent streptavidin technique. They allow easy detection of S-phase cells with an inverted light microscope.

Comparison of the DNA Content, Bromodeoxyuridine Incorporation and Ki-67 Antigen Expression in Human Acute Myeloid Leukemia.

The cell kinetics of twenty-two acute myeloid leukemias (AML) were investigated by means of flow cytometry evaluating the S-phase DNA content, bromodeoxyuridine labelling index (BrdUrd L.I.) and Ki-67 antigen expression. Eight patients showed a good correlation between the DNA content and BrdUrd L.I., while nine gave rise to divergent results. In the remaining five patients the S-phase DNA content could not be evaluated due to the presence of an additional aneuploid population. The Ki-67 antigen expression defined the extent of the growth fraction in all cases and allowed for better characterization of the cell cycle. These results suggest that the three methods explore only partly overlapping events; thus, it seems that a reliable picture of the cell kinetics in leukemic populations can only be achieved by combining all these methods.

S-phase evaluation with bromodeoxyuridine in lymphocytes from cattle infected with bovine leukemia virus (BLV).

Bromodeoxyuridine (BrdUrd), an analogue of thymidine, can be detected by means of monoclonal antibodies and utilized as a marker of the S-phase. In this paper a determination of the S-phase in BLV+ cattle with lymphocytosis has been performed by incorporating bromodeoxyuridine in the DNA. This evaluation was compared to the DNA content, demonstrating that i) bromodeoxyuridine incorporation is a reliable marker of S-phase in BLV+ cattle with lymphocytosis and ii) cytofluorimetry is the method of choice, together with immunocytochemistry, to demonstrate bromodeoxyuridine incorporation.

An immunotoxin containing momordin suitable for bone marrow purging in multiple myeloma patients.

Attempts have been made by a number of methods to eliminate minimal residual disease from bone marrow to be reinfused in autologous transplantation. In this paper we describe a conjugate containing a monoclonal antibody, named 8A, recognising a plasma cell-associated antigen, and momordin, a ribosome-inactivating protein similar to the ricin A-chain. This immunotoxin is active on target cell lines and on neoplastic plasma cells, while myeloid progenitors are fairly resistant. The conjugate is shown to be acceptable for ex vivo purging in autologous bone marrow transplantation in multiple myeloma patients.

Immunotoxins containing saporin 6 and monoclonal antibodies recognizing plasma cell-associated antigens: effects on target cells and on normal myeloid precursors (CFU-GM).

Monoclonal antibodies 8A and 62B1, recognizing plasma cell-associated antigens, were covalently linked to saporin 6, a ribosome-inactivating protein similar to the A-chain of ricin. Both immunotoxins were tested on target human cell lines U266 and Raji, on non-target K562 cell line and on myeloid CFU-GM progenitors. The cloning efficiency and viability of target cells were strongly reduced by 8A-saporin 6 and 62B1-saporin 6 immunotoxins, with an ID50 up to 200,000-fold lower than free saporin 6, whilst the K562 non-target cell line was unaffected. Normal human myeloid precursors (CFU-GM) were inhibited by immunotoxins only to a limited extent. An application of this model for autologous bone marrow transplantation in multiple myeloma patients is proposed. Since no eradication of cloning target cells was achieved by a single immunotoxin, mixtures made with different antibodies could help to reach this goal.

Autologous bone marrow transplantation with immunotoxin-purged marrow for advanced multiple myeloma.

A system to purge the bone marrow of myeloma cells has been developed in our laboratories with the aim of treating with myeloablative radiochemotherapy patients suffering from advanced multiple myeloma. This system is based on the ex vivo incubation of the marrow with an immunotoxin composed of the 8A monoclonal antibody--that recognizes plasma cells and B-cell precursors--and the ribosome-inactivating protein momordin. 8 patients have so far been treated. 4 are surviving from 4 to 18 months after ABMT, whereas 4 died after 1 to 6 months, 2 from infections, 1 from relapsing disease and 1 from veno-occlusive disease. A marked tumour reduction was observed in all evaluable patients; however, none has achieved complete disappearance of the disease. The haemopoietic reconstitution was significantly delayed in 3 patients. These preliminary results show the feasibility of this approach in advanced MM patients with heavily infiltrated marrow. The place of ABMT in the treatment of MM remains to be determined; the selection of patients with still responding and less advanced disease would probably produce better results.

A sensitive test for the detection of NK activity. Plasma clot clonogenic assay.

Natural killer (NK) activity, which has been implicated in the immune response against viral infections and neoplasias, is currently evaluated by means of a chromium (51Cr) release assay. However, criticisms have been raised with regard to the reliability and reproducibility of the test. We have developed a different in vitro method for measuring NK activity, based on the inhibition of the target clone growth in plasma clot semisolid medium. This method overcomes the limitations inherent to the 51Cr release test and more closely mimics the in vivo situation. The inhibitory activity revealed by the cloning assay was always greater than the lytic activity in the 51Cr release assay. Moreover, effector/target ratios of 3:1 and 1.5:1 still produced clonal inhibition. B-CLL cells, used as effectors, showed no inhibitory activity and the Raji cell line employed as target was resistant in both techniques. Thus, the clonogenic assay appears to be more sensitive for the evaluation of low levels of NK activity, for basic studies on the effector/target interactions, for the evaluation of LAK cell activity, and in diseases in which an involvement of the NK compartment has been hypothesized.