Can unexplained infertility be evaluated by a new immunological four-biomarkers panel? A pilot study.

BACKGROUND: Inflammation and oxidative stress are known to be triggering factors for a decrease of the pregnancy rate like maternal immunosuppression. Under these circumstances our study was performed to verify four immunological biomarkers (IMMUNOX Panel) in terms of incidence in a sine-causa infertile population and the overall pregnancy rate when the Panel was showing some non-physiologic values. METHODS: Sera of 86 women affected by unexplained infertility were screened for the IMMUNOX panel of biomarkers composed by: tumor necrosis factor alpha (TNF-α,) glycodelin (GLY), total oxidative status (TOS), and complement activity toxic factor (CATF). When at least one of the biomarkers tested was showing values outside the physiologic range, the woman was considered IMMUNOX-Positive. RESULTS: The first data was indented to verify the incidence of the women with an IMMUNOX-positive panel. Results show that 19.8%, 18.6%, 25.6%, and 47.7% were IMMUNOX-positive for GLY, TNF-α, TOS and CATF respectively. The overall incidence of IMMUNOX-positive patients, with at least one biomarker positive was 70,9%. Subsequently we have analysed the correlation between IMMUNOX Panel positivity and the pregnancy rate. The pregnancy rate in a subgroup (N.=55) of the entire population tested (N.=86) was 2.9% and 36.6% for the IMMUNOX-positive and IMMUNOX-negative patients respectively. CONCLUSIONS: Further validation studies are needed to prove that there is a correlation between unexplained infertility and immunological disorders screened by the IMMUNOX Panel, nevertheless our data shows that this diagnostic approach may be helpful to predict and to identify women at higher risk of IVF cycles failure.

Fetal mesenchymal stromal cells from cryopreserved human chorionic villi: cytogenetic and molecular analysis of genome stability in long-term cultures.

BACKGROUND AIMS: First-trimester chorionic villi (CV) are an attractive source of human mesenchymal stromal cells (hMSC) for possible applications in cellular therapy and regenerative medicine. Human MSC from CV were monitored for genetic stability in long-term cultures. METHODS: We set up a good manufacturing practice cryopreservation procedure for small amounts of native CV samples. After isolation, hMSC were in vitro cultured and analyzed for biological end points. Genome stability at different passages of expansion was explored by karyotype, genome-wide array-comparative genomic hybridization and microsatellite genotyping. RESULTS: Growth curve analysis revealed a high proliferative potential of CV-derived cells. Immunophenotyping showed expression of typical MSC markers and absence of hematopoietic markers. Analysis of multilineage potential demonstrated efficient differentiation into adipocytes, osteocytes, chondrocytes and induction of neuro-glial commitment. In angiogenic experiments, differentiation in endothelial cells was detected by in vitro Matrigel assay after vascular endothelial growth factor stimulation. Data obtained from karyotyping, array-comparative genomic hybridization and microsatellite genotyping comparing early with late DNA passages did not show any genomic variation at least up to passage 10. Aneuploid clones appeared in four of 14 cases at latest passages, immediately before culture growth arrest. CONCLUSIONS: Our findings indicate that hCV-MSC are genetically stable in long-term cultures at least up to passage 10 and that it is possible to achieve clinically relevant amounts of hCV-MSC even after few stages of expansion. Genome abnormalities at higher passages can occasionally occur and are always associated with spontaneous growth arrest. Under these circumstances, hCV-MSC could be suitable for therapeutic purposes.

Mechanism of action of type II, glycoengineered, anti-CD20 monoclonal antibody GA101 in B-chronic lymphocytic leukemia whole blood assays in comparison with rituximab and alemtuzumab.

We analyzed in B-chronic lymphocytic leukemia (B-CLL) whole blood assays the activity of therapeutic mAbs alemtuzumab, rituximab, and type II glycoengineered anti-CD20 mAb GA101. Whole blood samples were treated with Abs, and death of CD19(+) B-CLL was measured by flow cytometry. Alemtuzumab efficiently lysed B-CLL targets with maximal lysis at 1-4 h (62%). In contrast, rituximab induced a more limited cell death (21%) that was maximal only at 24 h. GA101 killed B-CLL targets to a similar extent but more rapidly than rituximab, with 19.2 and 23.5% cell death at 4 and 24 h, respectively, compared with 7.9 and 21.4% for rituximab. Lysis by both rituximab and GA101 correlated directly with CD20 expression levels (r(2) = 0.88 and 0.85, respectively). Interestingly, lysis by all three Abs at high concentrations was mostly complement dependent, because it was blocked by the anti-C5 Ab eculizumab by 90% in the case of alemtuzumab and rituximab and by 64% in the case of GA101. Although GA101 caused homotypic adhesion, it induced only limited (3%) direct cell death of purified B-CLL cells. Both rituximab and GA101 showed the same efficiency in phagocytosis assays, but phagocytosis was not significant in whole blood due to excess Igs. Finally, GA101 at 1-100 µg/ml induced 2- to 3-fold more efficient NK cell degranulation than rituximab in isolated B-CLL or normal PBMCs. GA101, but not rituximab, also mediated significant NK cell degranulation in whole blood samples. Thus, complement and Ab-dependent cellular cytotoxicity are believed to be the major effector mechanisms of GA101 in whole blood assays.

The sensitivity of acute lymphoblastic leukemia cells carrying the t(12;21) translocation to campath-1H-mediated cell lysis.

BACKGROUND AND OBJECTIVES: Campath-1H is used in conditioning regimens and more recently as an anti-leukemic therapy in acute lymphoblastic leukemias (ALL). We therefore investigated CD52 expression and campath-1H-mediated lysis of ALL cells in vitro. DESIGN AND METHODS: Complement-mediated cytotoxicity assays were performed on freshly isolated neoplastic cells and cell lines using human serum. Antibody-dependent cellular cytotoxicity (ADCC) was performed by calcein-AM release assays. RESULTS: CD52 was expressed in four out of eight ALL cell lines studied. Among 61 freshly isolated ALL samples CD52 was expressed at varying levels in 87% of cases. Whereas ADCC was equivalent in different CD52+ lines, complement-dependent cytotoxicity (CDC) was variable. The REH cell line bearing the t(12;21) translocation showed 47-60% lysis when treated with 10 microg/mL campath-1H compared to 0-6% for the other cell lines expressing equivalent amounts of CD52. Furthermore all nine ALL samples with t(12;21) showed very high CDC (mean 97%) compared to the other 24 CD52+cases (mean 24%)(p<0.0001). In t(12;21) samples, efficient CDC was obtained with as little as 1 microg/mL campath-1H. CDC correlated in part with CD52 levels, suggesting that CD52 expression and other yet undefined factors contribute to the particular sensitivity of t(12;21) cells. The resistance of non t(12;21) ALL cases could be overcome to a limited extent by increasing the concentration of campath-1H, blocking the CD55 and CD59 complement inhibitors, and more effectively by combining campath-1H with fludarabine. INTERPRETATION AND CONCLUSIONS: We conclude that most ALL samples express CD52 to a variable level and that campath-1H has cytotoxic activity against CD52+ALL, alone or in combination with cytotoxic drugs.

The role of complement in the therapeutic activity of rituximab in a murine B lymphoma model homing in lymph nodes.

BACKGROUND AND OBJECTIVES: We have set up a murine B lymphoma model stably expressing human CD20 and homing in lymph nodes in immunocompetent mice to study the mechanism of action of rituximab. DESIGN AND METHODS: The B lymphoma line 38C13 was stably transduced with the human CD20 cDNA by retroviral infection and injected into syngeneic mice. RESULTS: The transduced 38C13-CD20(+) cells stably expressed human CD20 on 100% of cells. Rituximab alone did not inhibit 38C13-CD20+ cell growth but relocalized the human CD20 into lipid rafts and induced complement-mediated lysis in vitro. Inoculation of 4x10(3) 38C13-CD20(+) intravenously into syngeneic mice led to the development of tumor masses in the spleen, bone marrow and lymph nodes, detectable from day 15 by polymerase chain reaction (PCR) analysis, and with a median survival of 21-24 days. Treatment with 250 mg rituximab i.p. given 1-10 days after tumor inoculation cured 100% of animals, with disappearance of tumor documented by immunohistochemistry and PCR analysis. Depletion of both NK cells and neutrophils did not affect the therapeutic activity of rituximab in vivo. Similarly, removal of phagocytic macrophages using clodronate-liposomes did not modify the capacity of rituximab to control tumor growth. In contrast, the protective activity of the antibody was completely abolished after complement depletion with cobra venom factor. Complement was also required when cells were inoculated subcutaneously in nude mice. INTERPRETATION AND CONCLUSIONS: These data demonstrate that complement is required for the therapeutic activity of rituximab in vivo in a murine model of B-cell lymphoma, independently of its localization in lymph nodes or subcutaneously.

Effect of alemtuzumab on neoplastic B cells.

BACKGROUND AND OBJECTIVES: The therapeutic antibody alemtuzumab is directed against the CD52 molecule and is used for the treatment of B-cell lymphocytic leukemia (B-CLL). We investigated the mechanism of action of this antibody in vitro against different neoplastic B cells and compared it to the anti-CD20 antibody rituximab. DESIGN AND METHODS: Complement-mediated cytotoxicity assays were performed on freshly isolated neoplastic cells using human serum as the source of complement. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated by chromium release assays, using peripheral blood mononuclear cells as effector cells, before and after 2 days of culture with interleukin-2 (IL-2). RESULTS: Alemtuzumab lysed cells from the 23 B-CLL samples through complement activation (mean 80%) much more efficiently than rituximab did (mean 16%), presumably because of the higher expression of CD52 than of CD20. All other leukemic B cells, including 1 prolymphocytic leukemia, 2 hairy cell leukemias and 6 B-non Hodgkin's lymphomas were effective targets for both antibodies, with 88% and 85% mean lysis, respectively. Both CD52 and CD20 were highly expressed in these cells. In contrast, most neoplastic B cell samples were poorly lysed through ADCC using freshly isolated peripheral blood mononuclear cells as effectors with either monoclonal antibody and regardless of target antigen levels. ADCC was, however, significantly increased in all cases by culturing the effector cells with IL-2 for 2 days. INTERPRETATION AND CONCLUSIONS: Complement-mediated lysis is likely to be an important mechanism of action of alemtuzumab in B-CLL and combination with IL-2 may increase this antibody's efficacy through ADCC. Mature neoplastic B cells other than B-CLL express high levels of CD52 and are good targets for alemtuzumab-mediated cytotoxicity.

Rituximab-mediated antibody-dependent cellular cytotoxicity against neoplastic B cells is stimulated strongly by interleukin-2.

BACKGROUND AND OBJECTIVES: We analyzed the sensitivity of freshly isolated neoplastic B cells to rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC), using different effector cells. DESIGN AND METHODS: ADCC was performed by 51Cr release assays in vitro, using peripheral blood mononuclear cells, IL-2-activated or expanded NK cells, neutrophils or macrophages as effector cells. B lymphoma lines and freshly isolated leukemic samples were used as targets. RESULTS: NK cells, but PMN or macrophages mediated rituximab dependent cellular cytotoxicity against two B lymphoma lines. Purified NK cells (95% CD56+/CD16+) reached 70% lysis at the highest E:T ratio. By contrast, all freshly isolated B leukemia or lymphoma cases, including 5 chronic lymphocytic leukemia, 1 B-prolymphocytic leukemia, 1 mantle cell lymphoma, 2 marginal zone lymhomas and 2 follicular lymphomas were poorly lysed by ADCC in the same conditions and regardless of CD20 expression levels, reaching a mean of 4% and 27% maximal lysis with PBMC or purified NK cells, respectively. Interestingly, short term IL-2 cultured PBMC, containing 10 % activated NK cells, as well as long-term expanded NK cells, containing 80-95% activated NK cells, became strong ADCC effector cells with rituximab and lysed all leukemic samples to a mean of 57% and 67% at the highest E:T ratio, respectively. INTERPRETATION AND CONCLUSIONS: Primary leukemic cells are more resistant than cell lines to rituximab- and NK cell-mediated ADCC but short-term exposure to IL-2 or long-term expansion of NK cells in vitro may provide effective tools to improve the therapeutic activity of rituximab.

A human immunodeficiency virus type 1 pol gene-derived sequence (cPPT/CTS) increases the efficiency of transduction of human nondividing monocytes and T lymphocytes by lentiviral vectors.

We have investigated the capacity of two human immunodeficiency virus type 1-derived lentivectors, differing in the presence of a 118-bp pol fragment containing the cPPT/CTS element, to transduce human normal primary cells of different hematopoietic lineages. Infection of resting monocytes with a high multiplicity of infection (MOI > 10) revealed that the lentivirus carrying the pol fragment (cPPT) is effective, transducing 75% of cells compared with 36% for the no-cPPT vector. Even at low MOIs (< or =1) the cPPT vector still shows a better transduction efficiency than the no-cPPT vector. Moreover, transduction does not require dendritic cell differentiation. In contrast, infection of nonactivated T lymphocytes showed that both vectors, tested at high MOIs, can transduce a small, although measurable, percentage of cells (up to 10%), which may correspond to G(1a) "activated" cells as detected by simultaneous staining of DNA and RNA, in our cultures in the presence of medium alone. Furthermore, we show that the sole addition of interleukin 2 or interleukin 15 represents a full proliferative signal under our conditions and permits high transduction efficiency (up to 30% with the cPPT vector and 15% with the no-cPPT vector). Still higher transduction of T lymphocytes can be achieved after stimulation with phytohemagglutinin and interleukin 2 (up to 78% with the cPPT vector vs. 42% with the no-cPPT vector). Finally, both viruses do not transduce either resting or proliferating tonsillar B lymphocytes.