Safety and efficacy of allogeneic adipose tissue-derived mesenchymal stem cells for treatment of dogs with inflammatory bowel disease: Endoscopic and histological outcomes.

Systemic administration of mesenchymal stem cells (MSCs) has been shown to be safe and efficacious in humans with Crohn's disease. The aim of this study was to evaluate the safety of an intravenous (IV) infusion of adipose tissue-derived mesenchymal stem cells (ASCs) and to assess macroscopic and histological effects in the digestive tract of dogs with inflammatory bowel disease (IBD). Eleven dogs with confirmed IBD received a single ASC infusion (2 × 10(6) cells/kg bodyweight). Full digestive endoscopic evaluation was performed pre-treatment and between 90 and 120 days post-treatment with mucosal changes being assessed using a fit-for-purpose endoscopic scale. Endoscopic biopsies from each digestive section were evaluated histologically according to the World Small Animal Veterinary Association (WSAVA) Gastrointestinal Standardization Group criteria. The pre- and post-treatment canine IBD endoscopic index (CIBDEI) and histological score (HS) were calculated and compared using the Wilcoxon test. Remission was defined as a reduction of >75% of the CIBDEI and HS compared with pre-treatment. No acute reactions to ASC infusion or side effects were reported in any dog. Significant differences between pre- and post-treatment were found in both the CIBDEI (P = 0.004) and HS (P = 0.004). Endoscopic remission occurred in 4/11 dogs with the remaining dogs showing decreased CIBDEI (44.8% to 73.3%). Histological remission was not achieved in any dog, with an average reduction of the pre-treatment HS of 27.2%. In conclusion, a single IV infusion of allogeneic ASCs improved gastrointestinal lesions as assessed macroscopically and slightly reduced gastrointestinal inflammation as evaluated by histopathology in dogs with IBD.

Safety and efficacy of allogeneic adipose tissue-derived mesenchymal stem cells for treatment of dogs with inflammatory bowel disease: Clinical and laboratory outcomes.

Mesenchymal stem cells (MSCs) have shown immunomodulatory and anti-inflammatory effects in experimental colitis, and promising clinical results have been obtained in humans with Crohn's disease and ulcerative colitis. The aim of this study was to determine the safety and feasibility of adipose tissue-derived MSC (ASC) therapy in dogs with inflammatory bowel disease (IBD). Eleven dogs with confirmed IBD received one ASC intravascular (IV) infusion (2 × 10(6) cells/kg bodyweight). The outcome measures were clinical response based on percentage reduction of the validated Clinical Inflammatory Bowel Disease Activity Index (CIBDAI) and Canine Chronic Enteropathy Clinical Activity Index (CCECAI), as well as normalisation of C-reactive protein (CRP), albumin, folate and cobalamin serum concentrations at day 42 post-treatment. The Wilcoxon test was used to compare variables before and after treatment. No acute reaction to ASC infusion and no side effects were reported during follow-up in any dog. Six weeks post-treatment, the CIBDAI and CCECAI decreased significantly and albumin, cobalamin and folate concentrations increased substantially. Differences in CRP concentrations pre- and post-treatment were not significant (P = 0.050). Clinical remission (defined by a reduction of initial CIBDAI and CCECAI >75%) occurred in 9/11 dogs at day 42. The two remaining dogs showed a partial response with reduction percentages of 69.2% and 71.4%. In conclusion, a single IV infusion of allogeneic ASCs was well tolerated and appeared to produce clinical benefits in dogs with severe IBD.

Assay of total homocysteine and other thiols by capillary electrophoresis and laser-induced fluorescence detection. II. Pre-analytical and analytical conditions.

In recent papers, we presented a new analytical method for thiol quantification in serum. This method was developed with capillary electrophoresis (CE) and laser-induced fluorescence (LIF) to analyze thiol-iodoacetamidofluoresceine (IAF) derivatives. Quantitative results for homocysteine, glutathione, cysteinylglycine, and cysteine were presented (Caussé E., et al., Clin. Chem. 45 (1999) 412). An exhaustive comparison of the quantitation of homocysteine in plasma, using high-performance liquid chromatography with either conventional fluorescence detection or fluorescence polarization immunoassay was also reported (Caussé E., et al., Electrophoresis 21 (2000) 2074). Sample preparation prior to derivatization with IAF had never been investigated. Recently we studied protein precipitation in serum with different organic agents (Caussé E., et al., J. Chromatogr. A 895 (2000) 173). In this work, we evaluated the conditions of protein precipitation in function of the amounts of acetonitrile and their influence on quantitation and quality of the electropherograms. Then, we looked at the variation of thiol concentrations in the haemolysis states and studied the thiol stability of blood samples cooled on ice.

In vitro generation of cytotoxic effectors activated by interleukin 2 (IL-2): comparison of autologous peripheral blood stem cells (PBSC) from adults and children.

Previous studies demonstrated that ex vivo IL-2- activated PBSC could generate cytotoxic effectors without impairing haematopoietic reconstitution. Clinical experience and previous studies indicated that children with solid tumours could benefit from high-dose chemotherapy with immune modulation. We studied the generation of cytotoxic effectors from growth-factor +/- chemotherapy-mobilised PBSC from 10 patients (five adults and five children) with different solid tumours. Cells were placed in culture in serum-free culture medium supplemented with IL-2 1000 U/ml +/- IL-12 for 1, 2, 4 or 7 days. Anti-tumour cytotoxicity was tested against K562, Daudi and two neuroblastoma cell lines (Gau, NB91). Cultured adult PBSC in the presence of IL-2 (1000 U/ml) showed marked cytotoxicity against all the cell lines tested from day 1. At day 2, with an E:T ratio of 25:1, cytotoxicity was 53% +/- 10.4, 63.2% +/- 23.8, 38% +/- 9.1, and 39% +/- 15.7 against K562, Daudi, Gau and NB91, respectively. Cytotoxic activity of child PBSC was significantly lower (P < 0.05) and was displayed after longer culture times (day 4). No difference was found in the phenotype analysis of lymphoid subsets before and after IL-2 activation between adult and child PBSC. Haematological properties of the graft were not significantly impaired by IL-2 activation.

Assays for total homocysteine and other thiols by capillary electrophoresis-laser-induced fluorescence detection. I. Preanalytical condition studies.

In recent papers, we presented a new analytical method for thiol quantification in serum. It is based on the use of capillary electrophoresis and laser-induced fluorescence to analyze thiol 6-iodoacetamidofluoresceine (IAF) derivatives. Quantitative results of homocysteine, glutathione, cysteine-glycin, and cysteine were shown (Clin. Chem. 45 (1999) 412). A comprehensive comparison of the quantitation of homocysteine in serum, using high-performance liquid chromatography/conventional fluorescence detection and fluorescence polarization immunoassay was also used (E. Caussé et al., Electrophoresis 21 (2000) 2074). Sample preparation prior to derivatization with IAF had never been investigated. In this work we present the results of quantitation of thiols in serum and plasma with three different anticoagulants widely used: ethylenediaminetetraacetic acid (EDTA), heparin, and sodium citrate. We show that serum and EDTA plasma gave the same results. Then serum protein precipitations by acetonitrile, acetone, sulfosalicylic acid, perchloric acid and trichloracetic acid, prior to derivatization by IAF, were also investigated. Their influence on the concentrations of the thiols were determined. Sulfosalicylic acid and acetonitrile precipitations are well adapted, whereas acetone cannot be used.

Plasma total homocysteine and other thiols analyzed by capillary electrophoresis/laser-induced fluorescence detection: comparison with two other methods.

We present a new analytical method for thiol quantification in plasma, based on the use of capillary electrophoresis (CE) and laser-induced fluorescence (LIF) to analyze 6-iodoacetamidofluorescein derivatives. Quantitative results of homocysteine, glutathione, cysteinylglycine, and cystationine are presented. A comparison of the quantitation of homocysteine in plasma, using high performance liquid chromatography/fluorescence detection and fluorescence polarization immunoassay is proposed. The results indicate that these techniques for plasma total homocysteine (tHcy) determination can be used interchangeably. The major advantage of CE-LIF is that it can quantitate the thiols in one run while keeping the price of consumables reasonable.

Remote tumor cells in the case of breast cancers: significance of their presence for prognosis [corrected].

Through two clinical studies, tumor cells were searched for in the bone biopsies and cytapherisis of patients suffering from inflammatory tumors and who had undergone intensive therapy and autografts (Pegase 2 program). In these studies we used immunocytochemical test with two monoclonal antibodies. The results have shown the presence of tumor cells in 14% of the patients (respectively 18%), with no correlation to the appearance of metastases after 4 years in the first study. Nevertheless, the presence of these tumor cells seems to be an important factor in the number of relapses. It seems important to develop research into tumor contamination especially in the selection of grafts over the next few years.

Leukaemic presentation of small cell variant anaplastic large cell lymphoma: report of four cases.

We report four cases of a rare subtype of CD30-positive anaplastic large cell lymphoma (ALCL) with a predominant small cell component (small cell variant of ALCL) presenting with a leukaemic feature. Lymph node biopsy showed malignant cells of varying size with a predominant population of small to medium-sized malignant cells associated with large anaplastic cells strongly positive for CD30 and epithelial membrane antigen (EMA). Both large and small cells were reactive with antibody ALK1, which recognizes the chimaeric NPM-ALK protein associated with the t(2;5)(p23;q35). All patients presented with hyperleucocytosis with atypical small lymphocytes. Bone marrow involvement was detected on both aspirate and bone marrow trephine where scattered malignant cells were only demonstrated by immunostaining for CD30 and ALK protein. Atypical cells in peripheral blood, lymph node and skin biopsies showed a T or null cell phenotype. Cytogenetic analysis of blood, bone marrow and/or lymph node revealed the t(2:5)(p23;q35) characteristic of ALCL. The patients responded to chemotherapy but showed early relapse without abnormal cells in peripheral blood. This report shows that the small cell variant of ALCL may have a leukaemic presentation with peripheral blood involvement by atypical lymphocytes and provides evidence that, in the small cell variant of ALCL, the small cell component is a part of the malignant clone.

Unfractionated peripheral blood stem cell autografts and CD(34+)-enriched autografts have similar long-term culture initiating capacity in multiple myeloma.

CD(34+)-enriched peripheral blood stem cells (PBSC) are increasingly being used as an autograft in patients with multiple myeloma (MM). The rationale for the use of the CD34+-enriched fraction in MM is the ability to obtain a graft with a significant reduction of contamination by plasma cells. However, the effect of such a manipulation on the proliferating potential of the engrafted cells is not known. We wished to study, as part of a randomized trial comparing the outcome in MM patients transplanted with either CD(34+)-enriched cells or unfractionated PBSC, the primitive hematopoietic cell content of the autografts using long-term culture initiating cell (LTC-IC) assays in 7 MM patients. In 3 patients CD(34+)cell-enriched fraction was compared to unfractionated PBSC whereas in the remaining 4 patients the LTC-IC assay was performed on total PBSC. The mean percentage of CD34+ cells of the CD34+ selected fraction in three patients was 82% (range 71%-96%) whereas the same percentage in PBSC varied from 0.6% to 10% in 4 patients (mean: 4.2%). Out of three patients transplanted with CD34+ cell fraction, two patients were found to have a very similar LTC-IC generating potential in their CD34+ versus PBSC fractions as this was assessed by the clonogenic cell output at week+5 per 10(4) CD34+ cells initiating the culture (PBSC: 92 and 168 and CD34+ fraction: 102 and 16, respectively) whereas one patient had a slightly different values (PBSC: 51 and CD34+ fraction: 103). When the PBSC fraction was compared in all 7 patients, the LTC-IC generation potential was very heterogenous, varying from 1.4 to 168. To determine if the selection procedure influences the numbers of LTC-IC's in both fractions, we have performed limiting dilution assays to determine both the frequency of distribution of hematopoietic colonies and the frequency of LTC-IC's in two patients. The frequency of distribution of hematopoietic colonies was linear in both CD34+ and PBSC fractions as was the frequency of LTC-IC when the corrections were made with regard to the CD34+ cell-content of the cultures (1/20). Our results indicate that the CD34+ selection procedure used in all three patients (Ceprate) is not deleterious for the generation of LTC-IC's and these findings support the rationale for the use of this procedure in multiple for the purposes of tumor depletion.

Burkitt-like lymphomas in AIDS patients: characterization within a series of 103 human immunodeficiency virus-associated non-Hodgkin's lymphomas. Burkitt's Lymphoma Study Group.

PURPOSE: Burkitt-like lymphoma (BLL) is a tumor with morphologic features intermediate between Burkitt's lymphoma (BL) and large-cell lymphoma, but its relationship with these lymphomas is currently unclear. We have therefore analyzed its characteristics within a large series of human immunodeficiency virus (HIV)-associated lymphomas. MATERIALS AND METHODS: Clinical, histologic, immunophenotypic, and molecular analyses were performed on 103 patients with AIDS lymphomas. RESULTS: Nineteen cases (18.4%) were identified as BLL. They were monoclonal B-cell proliferations, as evaluated by immunoglobulin (Ig) gene rearrangement analyses, and had rearrangement of the c-myc oncogene in 68% of cases but not the bcl-2 gene, in contrast to a previous study on non-HIV-associated BLL. This molecular pattern was therefore identical to that of typical BL, suggesting that they represented tumors of similar origin. However, some features could clearly differentiate BLL from BL and were similar to those seen in the diffuse large-cell immunoblastic lymphomas (DLC-IBL) group. These included a greater frequency of Epstein-Barr Virus (EBV) infection (79% v 48%, P = .04), an upregulation of CD39 (50% v 0%, P = .0007) and CD70 (75% v 15%, P = .003) activation antigens and of the CD11a/LFA-1 adhesion molecule (83% v30%, P = .05), and, finally, a lower CD4 count (mean, 119/microL v 270/microL, P = .04). CONCLUSION: BLL is a frequent entity among AIDS lymphomas and should be considered as a morphologic variant of BL in the context of severe immunodepression that occurs in HIV-infected patients.

Phase II trial of fludarabine monophosphate in patients with mantle-cell lymphomas.

PURPOSE: The aim of this phase II trial was to assess the efficacy of fludarabine monophosphate in untreated and pretreated mantle-cell lymphomas (MCL). PATIENTS AND METHODS: Fifteen patients with MCL were included in the study. In two cases, fludarabine was the first-line therapy, the second in four cases, the third in five cases, and the fourth in four cases. The diagnosis of MCL was based on the criteria of the European Lymphoma Task Force (ELTF), with morphologic, immunologic, and cytogenetic data. Patients were treated with intravenous fludarabine 25 mg/m2/d for 5 days every 4 weeks. RESULTS: Toxicity of fludarabine was mild: World Health Organization (WHO) grade 3 and 4 granulocytopenia occurred in 15 of 56 assessable cycles (cy) (27%), there was no grade 3 or 4 thrombocytopenia, one grade 3 bacterial lung infection, and no treatment-related death. There were five partial responses (33%) but no complete response. The duration of these responses was short and ranged from 4 to 8 months. CONCLUSION: These results suggest that fludarabine can be moderately effective in the treatment of MCL. Fludarabine appears to be far less effective than in chronic lymphocytic leukemia (CLL) and follicular non-Hodgkin's lymphoma (NHL). Therefore, fludarabine should be evaluated in association with other chemotherapeutic agents in MCL.

Successful cryopreservation of purified autologous CD34+ cells: influence of freezing parameters on cell recovery and engraftment.

Conventional hematopoietic stem cell cryopreservation methods use a DMSO concentration of 10%. However, cells manipulated ex vivo may require more refined freezing protocols adapted to the specific cell suspension. In this retrospective study, we evaluated the results obtained with CD34+ cells purified from peripheral blood of 39 patients on the CEPRATE SC System and frozen in 7.5% DMSO with a view to transplantation. The post-freezing recovery of progenitor cells was 89.4 +/- 27.87% for CD34+ cells, 59.13 +/- 36.93% for CFU-GM, and 53.49 +/- 40.71 for BFU-E. Neither the purity of the suspension nor the nucleated cell density during freezing was predictive of cell recovery. No difference was observed between cells stored in vials and bags. Thirty-seven patients transplanted with the concentrated CD34+ fraction received 4.46 x 10(6) CD34+ cells/kg and 33.04 x 10(4) CFU-GM/kg. The median time to granulocyte (>0.5 x 10(9)/l) and platelet (>50 x 10(9)/l) engraftment was 11 and 13 days, respectively. Only cell density and the infused number of CD34+ cells and CFU-GM were significantly related to hematological recovery. Our data suggest that purified CD34+ cells can be successfully cryopreserved in 7.5% DMSO and may represent a first step in establishing freezing parameters for selected CD34+ cells.

Mantle cell lymphomas: characteristics, natural history and prognostic factors of 45 cases.

We reviewed 77 cases considered as lymphocytic lymphomas of intermediate differentiation or diffuse centrocytic lymphomas. Forty-five cases were diagnosed as mantle cell lymphoma (MCL). The architectural pattern was diffuse in 95%, 8 cases presented large blastoid cells and CD5 positivity was observed in 28/34 cases. Of 20 cases studied, 8 presented a t(11;14)(q13;q32). Patient characteristics were: median age 59 years, B symptoms in 38%, 87% stages III-IV, bone marrow involvement in 67% with peripheral leukemic cells in 24%. Forty-four patients were treated with chemotherapy and 7 received radiotherapy. The complete response (CR) rate was 58%. Of the 26 CR, 19 relapsed at a median of 15 months. Disease-free survival was 42% and overall survival was 73% at 3 years. In a univariate analysis, overall survival was related to liver and bone marrow involvement, the presence of peripheral lymphomatous cells and achieving a complete response.

Multicentric evaluation of the MDR phenotype in leukemia. French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris.

The wide discrepancies in the frequency of 'positive' samples for multidrug resistance (MDR) phenotype within the same type of tumor observed in the literature justified the need for the definition of consensus recommendations. To define standard techniques of MDR phenotype measurement, we ran a large multicentric evaluation of the different methods available. Thirty-six French centers participated in the study, and 742 samples of 2-10 x 10(6) viable cells were sent by overnight express mail between December 1993 and February 1996. The same batches of MRK16, 4E3 and UIC2 were used. Nineteen samples of leukemia (12 AML, 1 ALL, 6 lymphoproliferative syndromes) and six leukemic cell lines with different levels of MDR expression were tested. Five meetings reached agreement concerning the guidelines for each technique, except immunocytochemistry. The 19 fresh samples were tested by each center using one to four techniques among cytofluorometry, immunocytochemistry, functional tests and RT-PCR. Five samples were diagnosed as 'negative' according to local criteria, with few discordant results (0 to 16% of 'positive' results). For all the 14 remaining samples, large discrepancies were observed from center to center, and from one technique to another. No correlations could be found between techniques. Flow cytometric analysis of cells already exposed to MRK16 or control IgG2A, fixed in paraformaldehyde and sent to centers did not reduce the discrepancies between centers in two of the four samples with moderate expression, emphasizing the role of histogram interpretation. The use of alternative monoclonal antibodies (4E3 and UIC2) did not reduce the discrepancies observed. In a second step, the K562 parental cell line, a low resistant subline (K562/HHT100, x7 resistance index to DNR) and a high resistant subline (K562/HHT300, x125 resistance index to DNR) were sent blindly three times, with an increasing level of recommendations for flow cytometry. Dramatic improvements were observed in cytometric results when the result was expressed as the ratio of arithmetic mean of fluorescence of antibody (10 microg of MRK16)/arithmetic mean of fluorescence of control (10 microg IgG2A): the proportion of expected results increased from 61 to 100% for K562, and from 37 to 85% for K562/HHT100. For uptake and drug efflux measurements, the use of 1 h uptake of 0.1 microM of rhodamine, followed by 1 h efflux +/-10 microM of verapamil, permitted an increased reproducibility of the technique from 71 to 100% for K562 and K562/HHT100. Whatever the technique used, concordant results were obtained for K562/HHT300. The immunocytochemistry, using several antibodies (MRK16, JSB1 and C219) gave many non-interpretable results (44%), due to a frequent high background and discordant results between antibodies in the same centers, and discordant conclusions between centers. The group does not recommend this technique for circulating tumoral cells.

French multicentric evaluation of mdr1 gene expression by RT-PCR in leukemia and solid tumours. Standardization of RT-PCR and preliminary comparisons between RT-PCR and immunohistochemistry in solid tumours. French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris.

Since there is no consensus on the techniques for multidrug resistance (MDR) phenotype evaluation, many discrepancies concerning the importance and frequency of mdr1 gene expression in leukemias and solid tumors are observed in the literature. In order to establish an inter-laboratory consensus in France, a multicenter study was carried out to propose further guidelines for MDR phenotype evaluation. The techniques used by the 38 laboratories participating in the trial were: immunodetection (immunohisto and/or cytochemistry, flow cytometry), functional tests, reverse transcription-polymerase chain reaction (RT-PCR) or Northern blot. We present the results obtained by 19 laboratories concerning the measurement of mdr1 gene expression assessed by RT-PCR or Northern blot in: (1)19 samples of tumor cells obtained from leukemic patients; (2) six solid tumor samples obtained at surgery; (3) eight cell lines exhibiting variable levels of resistance, and; (4)10 preparations of RNA and of cDNA obtained from solid tumors. Standardization of the RT-PCR technique and preliminary results comparing RT-PCR with immunohistochemistry in solid tumors are also reported.