Dino Collavo

An improved method for the detection of DNA fragmentation

An application of the Southern blot technique is described which permits the detection of DNA fragmentation due to cell death by apoptosis. DNA fragments were isolated from cell suspensions and tissues, separated on agarose gel, transferred by Southern blot and hybridized with a radiolabeled total cellular DNA probe. The application of this procedure to thymus cell samples, revealed the distinct ladder pattern of DNA fragments in multiples of about 180-200 base pairs, a characteristic feature of DNA fragmentation. In comparison to conventional DNA visualization with ethidium bromide staining, the radiolabeled probe improved the detection of DNA fragments at least eight-fold. This method detects low levels of DNA fragments, as well as physiological tissue DNA fragmentation, while avoiding cell damage due to DNA radiolabeling.

HIV-mediated immunodepression: in vitro inhibition of T-lymphocyte proliferative response by ultraviolet-inactivated virus

In order to assess whether the human retrovirus HIV, like other animal retroviruses, is endowed with intrinsic immunosuppressive activity, we studied the effects of noninfectious, uv-irradiated virus on in vitro lymphocyte function. uvHIV preparations inhibited T-cell proliferation to mitogens and alloantigens, as well as mitogen-driven IL-2 production. The inhibitory effect, which was not exerted by uv-irradiated HTLV-I, was apparently not due to a decrease in cell viability and was likely associated with thermoresistant viral component(s). The suppression proved to be selective for T-cell responses, while sparing other lymphocyte functions, such as the B-cell proliferative response to a selective B-cell mitogen. The inhibitory effect of uvHIV was not counteracted by a substantial reduction in the number of monocytes or by indomethacin. Moreover, IL-1 production by monocytes was not affected upon virus incubation. On the other hand, the proliferative response of both CD4+ and CD8+ T-cell clones was inhibited by uvHIV, suggesting that T cells represent the actual target for the inhibitory effect. Although a sizeable decrease in IL-2 production was observed following uvHIV incubation, exogenous IL-2 was not capable of reversing the virus-induced suppression of the proliferation. The possibility that the immunosuppressive activity of noninfectious HIV contributes to the T-cell defect in infected patients by mechanisms other than the cytopathic effect on CD4+ T lymphocytes is discussed.

Immunologic Unresponsiveness to Murine Leukemia Virus Antigens: Mechanisms and Role in Tumor Development

Publisher Summary This chapter discusses the immunological aspects of retrovirus oncogenesis, with particular emphasis on the mouse system and specific immune unresponsiveness. Members of the retrovirus family have been causally associated with the tumor induction in host animals. Several hypotheses have been advanced to explain the ways oncogene-devoid retroviruses manage to subvert the proliferative machinery of cells. In the experimental systems, murine leukemia virus (MuLV) expression on lymphoreticular cells during the perinatal period must be considered as the initial step of multiple events eventually leading to lymphoma development. Endogenous M-MuLV activation during embryogenesis in Mov-13 mice or in the few days following birth in BALB/Mo mice, as well as exogenous M-MuLV infection and expression in newborn mice, render the still immature immune system tolerant to viral antigens, thus, enabling the establishment of a lifelong virus carrier state. However, it is commonly held that the MuLV system is a misleading model for the studies of retrovirus-induced human diseases and that better human analogs are the feline and bovine leukemia virus systems.

Therapeutic effect of interleukin 12 on mouse haemangiosarcomas is not associated with an increased anti-tumour cytotoxic T-lymphocyte activity

In syngeneic mice, the H5V polyoma middle-T oncogene-transformed endothelioma cell line induces Kaposis sarcoma-like cavernous haemangiomas that regress transiently, probably because of an anti-tumour immune response, but eventually grow progressively and kill the host. To evaluate the generation of tumour-specific cytotoxic T lymphocytes (CTLs), spleen cells of tumour-bearing mice were restimulated with irradiated H5V cells in mixed leucocyte-tumour cell cultures. Tumour-specific CTLs were demonstrable only when low numbers of H5V stimulator cells were used (<1 H5V cell per 50 splenocytes). We found that H5V cells secrete immunosuppressive mediators because CTL generation was blocked when H5V cells culture supernatants were added to allogeneic mixed leucocyte cultures. As numerous tumour-derived immunosuppressive mediators may interfere with interleukin 12 (IL-12) production, we tested whether IL-12 treatment of the tumour-bearing mice would augment their immune response and thus suppress tumour growth. Indeed, IL-12 inhibited tumour growth and prevented mortality, but did not increase anti-H5V CTL generation either in vitro or in vivo. Moreover, the anti-tumour activity in IL-12-treated mice was abrogated by anti-interferon (IFN)-gamma monoclonal antibody (MAb) co-administration. These results strongly suggest that the anti-tumour effect of IL-12 is principally mediated by IFN-gamma release that in turn blocks H5V cell proliferation and induces the release of factors that suppress angiogenesis.

In vitro and in vivo evaluation of T and B lymphocyte functions in AKR mice.

To investigate whether AKR spontaneous leukaemogenesis is associated with a reduction in functional activity of T lymphocytes, the PHA response of AKR blood cells at different ages up to and including the preleukaemic period was studied. No significant differences were observed among young, adult and preleukaemic donors. In addition, the in vitro and in vivo AKR lymphocyte functions were compared with those of CBA lymphocytes by means of their response to stimulation with T and B lymphocyte selective mitogens (PHA, Con A and LSP respectively), and their response to immunization with thymus dependent (SRBC) or independent (LPS) antigens. We observed in vitro that while the B lymphocytes responded normally to mitogen, an intrinsic hyporeactivity to mitogens characterizes the T lymphocytes. Moreover, AKR mice exhibited a reduced in vivo response to both thymus dependent and independent antigens.

Resistance of lymphokine-activated T lymphocytes to cell-mediated cytotoxicity☆

We observed that lymphokine-activated T lymphocytes, obtained in short- and long-term cultures following stimulation with recombinant interleukin-2 (rIL-2), are resistant to cell-mediated cytotoxicity. In particular, lymphokine-activated killer (LAK) cells do not undergo self-lysis or lysis by alloreactive cytotoxic T lymphocytes (CTL), in line with recent reports concerning CTL clones. Similar findings were further confirmed in a lectin-dependent cell cytotoxicity assay. LAK cell lysis resistance was not due to an inability to recognize itself, since inactivated LAK cells used as cold competitors inhibited tumor cell lysis in a dose-dependent manner. In contrast, the addition on Day 0 of irradiated LAK cells or alloreactive CTL, as well as a CTL clone having LAK-like activity to rIL-2-stimulated cultures abrogated or strongly reduced LAK cell generation. Therefore, LAK cell precursors were most likely susceptible to the lytic activity of differentiated cytotoxic cells, as no inhibition was detected when cell to cell contact was prevented by using a diffusible chamber culture system. These findings, on the whole, suggest that the emergence of the lysis-resistant phenotype is most likely the result of a selective process occurring in vitro that leads to the elimination of lysis-susceptible lymphocytes present in culture.

Prevention of Murine Sarcoma Virus Oncogenesis in Offspring of Immunized Female Mice

BALB/c mice born to and nursed by females immunized against MSV-M showed a reduced tumour incidence and a high tumour regression rate following MSV-M injection at 7-14 days of age. Females immunized long before mating could also confer protection to their offspring whereas females immunized after parturition could not. A reduced number of tumours was observed in 3 out of 14 MSV-M injected litters whose mothers had been previously exposed to the virus while nursing infected offspring. Sera from suckling mice born to and nursed by immunized mothers contained MSV-M neutralizing antibody as shown by an in vitro focus reduction assay. Cell-free extracts from mice which developed leukaemia after MSV-M inoculation were tested for oncogenic activity in 1-week old mice. Out of 6 extracts, 4 induced typical MSV-M tumours and 2 caused leukaemias.

Delivery of methoxymorpholinyl doxorubicin by interleukin 2-activated NK cells: effect in mice bearing hepatic metastases

SummaryThe possibility of using interleukin 2 (IL-2)-activated natural killer cells (A-NK) to carry methoxymorpholinyl doxorubicin (MMDX; PNU 152243) to liver-infiltrating tumours was explored in mice bearing 2-day established M5076 reticulum cell sarcoma hepatic metastases. In vitro, MMDX was 5.5-fold more potent than doxorubicin against M5076 tumour cells. MMDX uptake by A-NK cells correlated linearly with drug concentration in the incubation medium [correlation coefficient (r) = 0.999]; furthermore, as MMDX incorporation was readily reproducible in different experiments, the amount of drug delivered by A-NK cells could be modulated. In vivo experiments showed that intravenous (i.v.) injection of MMDX-loaded A-NK cells exerted a greater therapeutic effect than equivalent or even higher doses of free drug. The increase in lifespan (ILS) following A-NK cell delivery of 53 μg kg–1 MMDX, a dosage that is ineffective when administered in free form, was similar to that observed in response to 92 μg kg–1 free drug, a dosage close to the 10% lethal dose (ILS 42% vs. 38% respectively). These results correlated with pharmacokinetic studies showing that MMDX encapsulation in A-NK cells strongly modifies its organ distribution and targets it to tissues in which IL-2 activated lymphocytes are preferentially entrapped after i.v. injection.

In Vivo Interactions between Murine Leukemia and Sarcoma Viruses

Experiments have been performed with the aim of elucidating the nature and the extent of the in vivo interactions between murine leukemia viruses (MuLVs) and murine sarcoma virus (MSV). BALB/c and CBA mice, injected neonatally with Graffi or passage A Gross viruses (MuLV-Gi, MuLV-G), have been inoculated as young adults with murine sarcoma virus, Moloney strain (MSV-M). A higher percentage of nonregressing sarcomas appeared in these animals, sometimes accompanied simultaneously by leukemia. The immune reactivity of mice receiving MuLV-Gi at birth was found to be significantly depressed when evaluated by the hemolytic palque-forming cell (PFC) technique. However, in mice infected with MuLV-Gi and MSV-M the number of PFC ranged within the control values or slightly increased. The potentiation of MSV-M oncogenicity following infection with MuLV was studied in a more natural situation. Adult AKR mice, known to release endogenous MuLV continuously, were injected with MSV-M. The incidence of induced sarcomas was similar to that observed in control BALB/c mice inoculated with MSV-M. Moreover, tumors developed with a very long latent period. On the other hand, the great majority of tumors showed no regression and ultimately killed the host. Additional experiments, making use of immunologic manipulation of the host and Fl hybrids, suggest that the relative resistance to MSV-M oncogenesis in AKR mice is influenced by genetic and immunologic factors. MSV recovered from MSV-M-induced tumors in AKR and C58 mice was typed by highly specific mouse antisera. The results clearly showed that formation of a new MSV pseudotype occurred in vivo, the endogenous Gross virus acting as helper.

Chieco-Bianchi L,+CHIECOAABIANCHI L: [Cytoxic activity in vitro of normal and leukemic mouse antileukocytic serum].

These experiments were designed to evaluate the specificity of cytotoxic antibodies for normal and leukemic leukocytes from mice with different genotypes. C57BL and C3Hf/Gs mice were immunized with allogeneic splenic cells from C3Hf/Gs, C57BL and (C3Hf/Gs x CBA T6T6) F1 donors. The animals were injected intraperitoneally once a week for 6–7 weeks and serum was obtained 7 days after the last injection. The cytotoxic effect of the sera on leukocytes was evaluated using the Terasaki test, which is based on direct phase contrast observation of refraction modification in damaged cellular elements. By using donors and recipients with dissimilar H-2 locus, an increase in the cytotoxic index was observed which was related to the number of immunizing injections. Values were fairly uniform for the different combinations tested. The maximum antibody titer was obtained 7 days after the last injection with notable decrease after 14 and 30 days. Cross tests between antisera and leukocytes with the same or different H-2 allele have demonstrated a high specificity of the isoantisera for the different H-2 locus alleles (k, b). In another group of experiments, the effect of isoantisera on leukocytes from animals with generalized leukemia was tested. The leukemia was induced by inoculation at birth with either Graffi or Gross virus. In the 12 cases studied, the cytotoxic effect on leukemic cells was always less than on normal cells of the same strain. Finally, a group of experiments was carried out using leukemic anti-leukocyte sera obtained by immunizing C57BL animals with cells derived from 2 Graffi virus-induced leukemias grafted into (C3Hf/Gs x CBA T6T6) F1 mice. These sera presented maximum cytotoxic index values on leukocytes of (C3Hf/Gs x CBA T6T6) F1 animals with Graffi virus-induced leukemia. Lower values were observed for normal leukocytes of the same strain. (C3Hf/Gs x CBA T6T6) F1 cells originating from Gross virus-induced leukemia were less sensitive to these sera, not only in comparison with Graffi virus-induced leukemia leukocytes but with normal (C3Hf/Gs x CBA T6T6) F1 leukocytes as well. It was also possible to demonstrate positivity in the cytotoxic response with C57BL leukemic leukocytes (Graffi virus-induced). These data seem to indicate that a reduction in the histocompatibility antigens of the leukemic leukocyte occurs contemporaneously with the appearance of tumor-specific antigens, and both phenomena are most likely related to neoplastic transformation. The following hypotheses are discussed as possible explanations of these phenomena: the possibility of true antigen deletion; the spreading out of membrane antigenic receptors as a consequence of increased cellular volume; the appearance of the TL antigen in the leukemic cells causing a partial loss of H-2 antigens.These experiments were designed to evaluate the specificity of cytotoxic antibodies for normal and leukemic leukocytes from mice with different genotypes. C57BL and C3Hf/Gs mice were immunized with allogeneic splenic cells from C3Hf/Gs, C57BL and (C3Hf/Gs x CBA T6T6) F1 donors. The animals were injected intraperitoneally once a week for 6–7 weeks and serum was obtained 7 days after the last injection. The cytotoxic effect of the sera on leukocytes was evaluated using the Terasaki test, which is based on direct phase contrast observation of refraction modification in damaged cellular elements. By using donors and recipients with dissimilar H-2 locus, an increase in the cytotoxic index was observed which was related to the number of immunizing injections. Values were fairly uniform for the different combinations tested. The maximum antibody titer was obtained 7 days after the last injection with notable decrease after 14 and 30 days. Cross tests between antisera and leukocytes with the same or different H-2...