Biomedical subjects

Massimiliano Manganini

Publications and source records attributed to Massimiliano Manganini.

5 recordsLinked to original sources

PubMed2006

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.

Animals↗

PubMed2006

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.

Adolescent↗

PubMed2004

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.

Aged↗

PubMed2003

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.

Antibodies, Monoclonal↗

PubMed2002

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.

B-Lymphocytes↗