Ontogenesis and functional aspects of oxytocin and vasopressin gene expression in the thymus network.

Ontogenesis of oxytocin (OT) and vasopressin (VP) gene expression and function were investigated in murine thymus. OT and VP transcripts were detected in the thymus on embryonic days 13 and 15, respectively. Corresponding messenger RNAs were evidenced in thymic epithelial cells by in situ hybridization with a neurophysin probe. From all OT and VP receptors, only OTR was expressed by all T-cell subsets, while V1bR was found in double positive and single positive CD8 cells. In fetal thymic organ cultures, OTR antagonist d[D-Tyr(Et)2, Thr4]OVT increased early apoptosis of CD8 cells, while V1bR antagonist (Sanofi SSR149415) inhibited T-cell differentiation, and favored CD8 T-cell commitment.

Neurohypophysial receptor gene expression by thymic T cell subsets and thymic T cell lymphoma cell lines.

Neurohypophysial oxytocin (OT) and vasopressin (VP) genes are transcribed in thymic epithelium, while immature T lymphocytes express functional neurohypophysial receptors. Neurohypophysial receptors belong to the G protein-linked seven-transmembrane receptor superfamily and are encoded by four distinct genes, OTR, V1R, V2R and V3R. The objective of this study was to identify the nature of neurohypophysial receptor in thymic T cell subsets purified by immunomagnetic selection, as well as in murine thymic lymphoma cell lines RL12-NP and BW5147. OTR is transcribed in all thymic T cell subsets and T cell lines, while V3R transcription is restricted to CD4+CD8+ and CD8+ thymic cells. Neither V1R nor V2R transcripts are detected in any kind of T cells. The OTR protein was identified by immunocytochemistry on thymocytes freshly isolated from C57BL/6 mice. In murine fetal thymic organ cultures, a specific OTR antagonist does not modify the percentage of T cell subsets, but increases late T cell apoptosis further evidencing the involvement of OT/OTR signaling in the control of T cell proliferation and survival. According to these data, OTR and V3R are differentially expressed during T cell ontogeny. Moreover, the restriction of OTR transcription to T cell lines derived from thymic lymphomas may be important in the context of T cell leukemia pathogenesis and treatment.

Involvement of insulin-like growth factors in early T cell development: a study using fetal thymic organ cultures.

The expression of insulin-like growth factor (IGF) and IGF receptor genes was investigated by RT-PCR during ontogeny of the murine thymus. IGF-1, IGF-1R, M6P/IGF-2R genes are expressed in the thymus both in fetal and postnatal life, whereas IGF-2 messenger RNAs (mRNAs) decline after birth but are still detectable on the seventh week. By in situ hybridization, IGF-2 transcripts were located in the outer cortex and medulla of the postnatal thymus, and on the whole surface ofthe epithelial-like network in the fetal thymus. The effects of anti-IGFs and IGF-receptors neutralizing Abs on the generation of pre-T cell subpopulations were then investigated using fetal thymic organ cultures (FTOC). FTOC treatment with an anti-IGF-2 mAb, an anti-IGF-1R mAb, or an anti-M6P/IGF-2R polyclonal Ab induced a blockade of T cell differentiation at the CD4-CD8- stage, as shown by a significant increase in the percentage of CD4-CD8- cells and a decrease in the percentage of CD4+CD8+ cells. Moreover, anti-IGF-2 Ab treatment induced an increase in CD8+ cells suggesting that thymic IGF-2 might have a role in determining differentiation into the CD4 or CD8 lineage. Anti-IGF-1 Ab treatment decreased the proportion in CD4-CD8- cells and increased the frequency in CD4+CD8+. FTOC treatment with anti-(pro)insulin did not exert any significant effect on T cell development. These data indicate that the intrathymic IGF-mediated signaling plays an active role in the early steps of T cell differentiation during fetal development.

[Bacterial resistance during anti-acne antibiotic therapy. How to limit the risk]. / Résistances bactériennes au cours de l'antibiothérapie anti-acnéique. Comment en limiter le risque.

The treatment of moderate to severe acne often relies on antibiotherapy in order to eradicate as much as possible microorganisms such as Propionibacterium spp colonizing the sebaceous follicles. In recent years, bacterial resistances against specific antibiotics have emerged. Both the antibiotic and its administration modalities must be considered in order to control the risk. With regard to this conundrum, minocycline is a medication of choice among the diverse anti-acneic therapies.

Reactive oxygen intermediate-dependent NF-kappaB activation by interleukin-1beta requires 5-lipoxygenase or NADPH oxidase activity.

We previously reported that the role of reactive oxygen intermediates (ROIs) in NF-kappaB activation by proinflammatory cytokines was cell specific. However, the sources for ROIs in various cell types are yet to be determined and might include 5-lipoxygenase (5-LOX) and NADPH oxidase. 5-LOX and 5-LOX activating protein (FLAP) are coexpressed in lymphoid cells but not in monocytic or epithelial cells. Stimulation of lymphoid cells with interleukin-1beta (IL-1beta) led to ROI production and NF-kappaB activation, which could both be blocked by antioxidants or FLAP inhibitors, confirming that 5-LOX was the source of ROIs and was required for NF-kappaB activation in these cells. IL-1beta stimulation of epithelial cells did not generate any ROIs and NF-kappaB induction was not influenced by 5-LOX inhibitors. However, reintroduction of a functional 5-LOX system in these cells allowed ROI production and 5-LOX-dependent NF-kappaB activation. In monocytic cells, IL-1beta treatment led to a production of ROIs which is independent of the 5-LOX enzyme but requires the NADPH oxidase activity. This pathway involves the Rac1 and Cdc42 GTPases, two enzymes which are not required for NF-kappaB activation by IL-1beta in epithelial cells. In conclusion, three different cell-specific pathways lead to NF-kappaB activation by IL-1beta: a pathway dependent on ROI production by 5-LOX in lymphoid cells, an ROI- and 5-LOX-independent pathway in epithelial cells, and a pathway requiring ROI production by NADPH oxidase in monocytic cells.

Apoptosis during the development of radiogenic thymic lymphomas: effects of treatments inhibiting lymphoma development.

INTRODUCTION: Whole body fractionated irradiation induces thymic lymphomas in C57BL/Ka mice after a latent period during which preleukemic cells progressively transform into leukemic cells within an abnormal thymic microenvironment. A bone marrow graft or repeated cytokine injections prevent lymphoma development. We think that these treatments restore altered mechanisms controlling apoptosis. MATERIALS AND METHODS: Apoptosis was analyzed by flow cytometry in thymocytes from different groups of mice (control, preleukemic, prevented mice). RESULTS: The apoptotic rates did not change in freshly isolated thymocytes from different experimental groups. However, after culture, the level of apoptosis increased in preleukemic thymuses; and returned to normal value in cultured thymocytes from irradiated mice after lymphoma preventing treatments. Furthermore, thymic microenvironmental factors can control thymocyte apoptosis. CONCLUSION: We propose that after leukemogenic irradiation, there is an increase of cells with an activated suicide program, but that alterations of thymic environmental factors rescue them from apoptosis, allowing their further neoplastic transformation.

Reciprocal regulation of protein tyrosine kinases p56lck and p59fyn, and altered tyrosine phosphorylation in murine AIDS.

Murine AIDS (MAIDS), caused by a defective murine leukemia virus, is a severe lymphoproliferative disease associated with profound immunodeficiency and increased susceptibility to opportunistic infections. Most subsets of lymphocytes, including CD4+ and CD8+ T cells, are refractory to mitogen stimulation. As a first step to examine proximal signal transduction in the infected mice, Western and Northern blot analyses were performed, and showed that p56lck is dramatically decreased at the protein as well as the mRNA level in the lymph nodes (LN). In contrast, p59(fyn) and its mRNA were slightly increased in the LN of the same mice. Similar results were obtained with purified T cells. Interestingly, the thymus of the infected animals did not show any abnormality regarding p56(lck) or p59(fyn). Tyrosine phosphorylation was constitutively increased in the infected mice and was barely amplified by anti-CD3 mAb stimulation. A similar pattern was observed when tyrosine phosphorylation was selectively examined at the level of ZAP-70. Our results suggest that a reciprocal regulation of p56(lck) and p59(fyn) protein tyrosine kinases, previously described in various models of anergy, could also be involved in the pathogenesis of MAIDS.

In vitro propagated dendritic cells from patients with human-papilloma virus-associated preneoplastic lesions of the uterine cervix: use of Flt3 ligand.

Dendritic cells (DC) are the most efficient antigen presenting cells. The clinical use of DC as vectors for antitumor and anti-infectious disease immunotherapy has been limited by their low level and accessibility in normal tissue. Substantial numbers of DC can be generated from peripheral blood cultured in the presence of interleukin-4 (IL-4) and granulocyte/macrophage-colony-stimulating factor (GM-CSF). We showed in this study that substantial numbers of DC can be obtained from the peripheral blood of patients with (pre)neoplastic lesions of the uterine cervix. The procedure required relatively small blood samples (10 ml) and the presence of 100 U/ml IL-4 and 800 U/ml GM-CSF in the culture medium. There was no significant difference in the morphology, yield, phenotype and function of generated DC between patients with cervical (pre)neoplastic lesions and healthy individuals. When the hematopoietic factor Flt3 ligand (Flt3L, 40 ng;ml) was added, there was an average increase in the DC population of 26% compared to cultures with GM-CSF and IL-4 alone. Approximately 1.2 x 10(6) cells with the characteristics of dendritic cells could be obtained when Flt3L was included in the medium. The addition of Flt3L did not modify the phenotypic profile of DC (HLA-DR+, CD1a+, CD4+, CD54+, CD80+, CD86+. CD40+, CD3- and CD14-). In addition, Flt3L generated functional DC capable of stimulating the proliferation of alloreactive T cells. These results suggest that Flt3L, in association with GM-CSF and IL-4, provides an advantageous tool for the large-scale generation of DC and that an immunotherapy based on the use of DC generated in vitro is possible in patients with (pre)neoplastic lesions of the uterine cervix.

A pilot study on bacterial viability in acne. Assessment using dual flow cytometry on microbials present in follicular casts and comedones.

BACKGROUND: Antibiotic therapy is one of the main methods of acne treatment, however, bacterial resistance is on the rise and can affect the treatment outcome. Quantitative bacteriologic cultures are the gold standard methodology for the assessment of such a problem; however, certain important biological aspects remain uncovered. OBJECTIVE: The purpose of this study was to compare the antibacterial activity of minocycline and lymecycline in sebaceous follicle infundibula and comedones of acne patients. METHOD: We used a recently introduced flow cytometric method, allowing a distinction to be made between viable, injured (presumably resistant), and dead microorganisms. RESULTS: Minocycline (100 mg) proved to be superior to lymecycline (600 mg) in abating the microflora harboring in the sebaceous follicles of acne patients. CONCLUSIONS: The dissimilar bioavailability and antimicrobial efficacy between the two bacteriostatic agents may impart different clinical efficacy.

Regulation of major histocompatibility complex class I expression by NF-kappaB-related proteins in breast cancer cells.

Downregulation of MHC Class I antigens has been observed in many cancers and usually results from a decreased gene transcription. A reporter CAT gene dependent on the MHC Class I kappaB site or on a longer promoter is transactivated by NF-kappaB complexes containing p65 or RelB. p100 as well as IkappaB-alpha are potent inhibitors of this transcription and p100 sequesters RelB and p65 complexes in the cytoplasm of breast cancer cells. However, although p100 is highly expressed in a number of breast cancer cell lines, MHC Class I antigen expression was observed on all the cell lines we analysed and could be further induced by stimulation with the cytokines IFN-gamma or TNF-alpha. Stable transfection of a unresponsive mutated IkappaB-alpha Ser 32-36 expression vector showed that TNF-alpha induced MHC Cl I expression in an NF-kappaB-dependent way while IFN-gamma did it independently of any NF-kappaB activation.

CD28-B7 costimulatory blockade by CTLA4Ig delays the development of retrovirus-induced murine AIDS.

Mouse AIDS (MAIDS) induced in C57BL/6 mice by infection with a replication-defective retrovirus (Du5H) combines extensive lymphoproliferation and profound immunodeficiency. Although B cells are the main target of viral infection, recent research has focused on CD4(+) T cells, the activation of which is a key event in MAIDS induction and progression. A preliminary observation of increased expression of B7 molecules on B cells in MAIDS prompted us to address the possible involvement of the CD28/B7 costimulatory pathway in MAIDS. Mice infected with the MAIDS-inducing viral preparation were treated with murine fusion protein CTLA4Ig (3 x 50 microg/week given intraperitoneally), a competitive inhibitor of physiological CD28-B7 interactions. In CTLA4Ig-treated animals, the onset of the disease was delayed, lymphoproliferation progressed at a much slower rate than in untreated mice, and the loss of in vitro responsiveness to mitogens was reduced. Relative expression of Du5H did not differ between treated and untreated animals. These results suggest that the CD28/B7 costimulatory pathway contributes to MAIDS development.

The DNA intercalating alkaloid cryptolepine interferes with topoisomerase II and inhibits primarily DNA synthesis in B16 melanoma cells.

Cryptolepine hydrochloride is an indoloquinoline alkaloid isolated from the roots of Cryptolepis sanguinolenta. It is characterized by a multiplicity of host-mediated biological activities, including antibacterial, antiviral, and antimalarial properties. To date, the molecular basis for its diverse biological effects remains largely uncertain. Several lines of evidence strongly suggest that DNA might correspond to its principal cellular target. Consequently, we studied the strength and mode of binding to DNA of cryptolepine by means of absorption, fluorescence, circular, and linear dichroism, as well as by a relaxation assay using DNA topoisomerases. The results of various optical and gel electrophoresis techniques converge to reveal that the alkaloid binds tightly to DNA and behaves as a typical intercalating agent. In DNAase I footprinting experiments it was found that the drug interacts preferentially with GC-rich sequences and discriminates against homo-oligomeric runs of A and T. This study has also led to the discovery that cryptolepine is a potent topoisomerase II inhibitor and a promising antitumor agent. It stabilizes topoisomerase II-DNA covalent complexes and stimulates the cutting of DNA at a subset of preexisting topoisomerase II cleavage sites. Taking advantage of the fluorescence of the indoloquinoline chromophore, fluorescence microscopy was used to map cellular uptake of the drug. Cryptolepine easily crosses the cell membranes and accumulates selectively into the nuclei rather than in the cytoplasm of B16 melanoma cells. Quantitative analyses of DNA in cells after Feulgen reaction and image cytometry reveal that the drug blocks the cell cycle in G2/M phases. It is also shown that the alkaloid is more potent at inhibiting DNA synthesis rather than RNA and protein synthesis. Altogether, the results provide direct evidence that DNA is the primary target of cryptolepine and suggest that this alkaloid is a valid candidate for the development of tumor active compounds.

Stages in the development of radiation-induced thymic lymphomas in C57 BL/Ka mice: preleukemic cells become progressively resistant to the tumor preventing effects of a bone marrow graft.

Fractionated whole body irradiation induces thymic lymphomas in C57 BL/Ka mice after 6-12 months. A graft of normal congenic bone marrow cells immediately after the last irradiation prevents the development of lymphomas by inducing the disappearance of preleukemic cells. When such a graft is performed one month later, it does not inhibit the emergence of tumors. It could be because, one month after irradiation, preleukemic cells become insensitive to the effects of the grafted bone marrow on their leukemogenic potential. To check this hypothesis, we have investigated the capacity of grafted bone marrow cells to prevent the development of lymphomas in mice inoculated with radiation-induced preleukemic cells collected at several time intervals after the completion of the radiation regimen. It was found that the bone marrow graft reduced the incidence of thymic lymphoma at day 2 (10 vs. 43%; p < 0.01) and 10 (39 vs. 86%; p < 0.01) but not at day 15 (64 vs. 80%; NS) or 30 (93 vs. 82%; NS). The inefficacy of the marrow graft was not associated with proliferation of the inoculate in the recipient thymus nor with inhibition by preleukemic cells of thymic repopulation by bone marrow precursors. The data provide evidence that preleukemic cells undergo intrinsic changes which are reflected by the acquisition of resistance to bone marrow grafts.

Subset-specific analysis of calcium fluxes in murine AIDS.

Infection of susceptible strains of mice with the Duplan strain of murine leukemia viruses induces a syndrome called MAIDS (murine acquired immunodeficiency syndrome) characterized by immunodeficiency and lymphoproliferation. In addition to a complete refractoriness of most subsets of lymphocytes to mitogen stimulation, the development of phenotypic abnormalities occurs such as the appearance of an abnormal CD4+ T cell subset lacking membranes Thy-1. This study was performed to compare the calcium responses during the early stages of MAIDS (week 9 or earlier) between T cells and B cells and between CD4+Thy-1- and CD4+Thy-1+ T cells. B cells were strikingly less affected than T cells: their baseline [Ca2+]i did not significantly increase, and their calcium response to anti-IgM antibody and concanavalin A (Con A) was partially maintained. In contrast, the response to Con A was completely abolished in T cells. Interestingly, calcium mobilization in response to membrane receptor-independent stimuli such as ionophores and thapsigargin was strongly inhibited in T cells, while no such inhibition was found in B cells. In comparison with their CD4+Thy-1+ counterparts, CD4+Thy-1- T cells had blunted calcium responses in controls, as well as in infected mice. However, CD4+Thy-1+ T cells were also strikingly altered, suggesting that the loss of membrane Thy-1 could be associated with, but not directly responsible for abnormalities of calcium responses in CD4+ T cells from RadLV-Rs-infected mice.

Further characterization of cytotoxic T cells generated by short-term culture of human peripheral blood lymphocytes with interleukin-2 and anti-CD3 mAb.

In this study we have specifically investigated the participation of T cells in the cytotoxic activity of peripheral blood lymphocytes (PBL) activated by interleukin-2 (IL-2, 50 U/ml) alone or in combination with an anti-CD3 mAb (BMA030, 10 ng/ml, IgG2a). Purified CD3+ T cells, incubated in the presence of the anti-CD3 mAb for 4 days, mediated a cytotoxic activity against HL60 and U937 tumor cell lines. Several findings suggested the involvement of a redirected-cytotoxicity phenomenon, since the lytic process was restricted to target cell lines bearing the high-affinity Fc gamma receptor (Fc gamma RI) and T lymphocytes stimulated by IL-2 alone did not lyse these cell lines. Furthermore, anti-CD3 mAb F(ab')2, anti-CD3 IgG1 (UCHT1), phytohemagglutinin or staphylococcal enterotoxin A did not induce a similar cytotoxic activity in T lymphocytes. The cytotoxic process occurred in the presence of a very low level of anti-CD3 antibodies (in the nanomolar range). The cytotoxic activity of T cells stimulated by IL-2 or by IL-2 + BMA030, against OVCAR-3 cells (MOv18+ ovarian tumor cell line), was also compared in the presence of a bispecific antibody OC/TR, anti-CD3 x MOv18). The stimulation by IL-2 + BMA030 induced approximately a twofold higher cytotoxic activity than IL-2-activated T cells. This could be related to the state of activation of effector cells stimulated by IL-2 + BMA030, since the phenotypic analysis showed an increased proportion of T cells expressing several activation/differentiation markers (CD25, HLA-DR, CD45R0, adhesion molecules). These findings could be applied to the design of therapeutic protocols using anti-CD3 x antitumoral bispecific antibodies.

Prevention of murine radiogenic thymic lymphomas by tumor necrosis factor or by marrow grafting.

BACKGROUND: Split-dose irradiation (1.75 Gy given weekly for 4 weeks) of C57BL/Ka mice induces the emergence of preleukemic cells (PLCs). These cells develop into leukemic cells after a latency period of 3-6 months. The survival and transformation of PLCs are dependent on radiation-induced alterations of the thymic epithelium and of resident lymphocyte (i.e., thymocyte) subpopulations in the thymus. PLCs can be eliminated, concomitantly with the restoration of the thymus, by grafting bone marrow cells immediately after the last irradiation. Our hypothesis was that any agent able to restore the thymus after leukemogenic irradiation would exert the same effects as a bone marrow graft. Tumor necrosis factor-alpha (TNF-alpha) is one such possible agent, since it has been shown to modulate some functions of the thymic epithelium and thymocyte subpopulations. PURPOSE: The goal of this study was to assess the ability of repeated intraperitoneal injections of TNF-alpha to functionally replace bone marrow transplantation in the restoration of normal intrathymic lymphopoiesis and in the prevention of thymic lymphomas in split-dose-irradiated mice. METHODS: We replaced the bone marrow graft with repeated injections of TNF-alpha (25 000 U/injection) in the split-dose-irradiated (4 x 1.75 Gy) C57BL/Ka mouse model. We analyzed the expression of the cell differentiation markers CD4 and CD8 on thymocytes by flow cytometry. We also studied the thymic environment by isolating thymic nurse cells, the bone marrow prothymocyte activity by analyzing thymic repopulation, and the evolution of PLCs by an in vivo transplantation assay. Local production of TNF-alpha after bone marrow grafting was examined by in situ hybridization. Injections of anti-TNF-alpha antibodies were given to split-dose-irradiated mice to test the effect of neutralizing TNF-alpha in vivo. One-way analysis of variance and Newman-Keuls two-tailed tests were used to test statistical significance. RESULTS: Multiple injections of TNF-alpha into split-dose-irradiated mice did not influence bone marrow prothymocyte activity but restored thymocyte subpopulations and thymic epithelium, induced the disappearance of PLCs, and prevented the development of lymphomas. Moreover, a bone marrow graft significantly stimulated intrathymic production of TNF-alpha messenger RNA (P<.01), and anti-TNF-alpha antibodies partially inhibited the antilymphomatous effects of bone marrow graft in split-dose-irradiated mice (P<.05). CONCLUSION: These data strongly suggest that TNF-alpha is a mediator that is involved in the mechanisms by which bone marrow transplantation functions to prevent thymic lymphomas in split-dose-irradiated mice. IMPLICATIONS: Cytokines might be used in some biological systems, particularly in the hemopoietic system, as a therapeutic agent for the secondary prevention of cancer.

Improved four-color flow cytometry method using fluo-3 and triple immunofluorescence for analysis of intracellular calcium ion ([Ca2+]i) fluxes among mouse lymph node B- and T-lymphocyte subsets.

A visible-light, dual-laser, flow cytometric method was developed for the simultaneous analysis of intracellular ionized calcium concentration ([Ca2+]i) and three cell-surface markers (CD4, CD8, and Thy-1.2 antigens) by using the calcium probe fluo-3 and using R-phycoerythrin (PE), peridinin chlorophyll-alpha protein (PerCP), and allophycocyanin (APC) conjugated monoclonal antibodies (MoAbs). This improved method was used in the analysis of [Ca2+]i mobilization upon in vitro stimulation with mitogenic lectins [phytohaemagglutinin (PHA) or concanavalin A (ConA)], anti-CD3 MoAbs, or A23187 calcium ionophore in the heterogeneous lymph node cell populations from healthy C57BL/Ka mice. The present results show that the calcium responses were heterogeneous and dependent on the cellular immunophenotype, not only on lectins or anti-CD3 MoAbs stimulation, but also on the receptor-independent A23187 ionophore stimulation. An in situ fluo-3 calibration method (using A23187 and metabolic poisons in Ca2+ /EGTA buffers with known free calcium concentrations) indicated a resting [Ca2+]i in lymphocytes of 103 +/- 23 nM (mean +/- S.D.) but with significant differences between the [Ca2+]i in B cells and in all of the T-cell subsets (CD4+Thy-1+, CD4+Thy-1-, and CD8+T cells). Both the B cells and the T-cell subsets showed an increase of fluo-3 fluorescence upon in vitro stimulation with ConA or PHA, but the calcium mobilization following lectin stimulation was time delayed in all T-cell subsets. Only the T cells, including the CD4+Thy-1- subset, responded to anti-CD3 MoAbs. The percentage of responding cells upon stimulation with ConA was higher in T cells than in B cells. By contrast, PHA gave a higher response in B cells. After stimulation with different mitogens, [Ca2+]i increased in both CD4+ and CD8+ T-cell subsets. However, the percentage of responding cells was far higher in the CD4+Thy-1+ subset than in the CD4+Thy-1- or the CD8+T-cell subsets. The stimulation with A23187 ionophore induced a higher calcium response in B cells than in T cells. Interestingly, it also induced greater Ca2+ mobilization in CD4+ than in CD8+T cells. These results demonstrate the potential use of fluo-3 simultaneously with three fluorescein (FITC)-compatible fluorochromes. This technique may be useful for investigating the role of the CD4+Thy-1-T cells, a rare subset that is abnormally expanded in a murine acquired immunodeficiency syndrome (murine AIDS).

Assessment of Ki-67 antigen immunostaining in squamous intraepithelial lesions of the uterine cervix. Correlation with the histologic grade and human papillomavirus type.

A formalin-fixation and paraffin-processing resistant epitope of Ki-67 cell proliferation-associated antigen was immunohistochemically detected by the MIB-1 monoclonal antibody (Immunotech, Marseille, France) in 25 routinely processed cervical biopsies showing normal squamous epithelium or squamous metaplasia and in 65 cervical intraepithelial lesions (SILs) (44 low grade and 21 high grade SILs) with human papillomavirus (HPV) infection. Expression of Ki-67 antigen was exclusively confined to the parabasal and basal layers of normal and metaplastic epithelium. There was no significant difference of Ki-67 antigen immunostaining between normal cervical biopsies and cases of squamous metaplasia. In SIL specimens, the staining was markedly increased in the parabasal and basal layers and Ki-67-positive cells were also distributed in the intermediate (low grade SIL) or all layers of epithelium (high grade SIL). Statistically significant differences for the density of Ki-67 antigen-labeled cells, which were assessed with an image analysis system, were found in comparisons between normal or metaplastic epithelium and SILs (P < .001) and between low grade SILs and high grade SILs (P < .001). In our series of SILs, HPV 16/18 and 31/33/35/novel types, which were found in both low grade and high grade SILs, were significantly associated with higher densities of Ki-67 antigen-positive cells than HPV 6/11 types that were found exclusively in low grade SILs. There was no significant difference found between the densities of Ki-67 antigen-labeled cells in HPV 16/18-positive and HPV 31/33/35/novel types-positive tissues in our series of SILs taken as a whole or when segregating SILs into low grade and high grade.

Spontaneous and induced apoptosis after whole body radiation exposure: experimental approaches. Observations in radio-induced thymic lymphomagenesis.

Radio-induced thymic lymphomagenesis is associated with alterations in the balance between thymocyte subsets and cytokinetic perturbations. The objectives of this work were to investigate whether these alterations are associated with alterations in the basic levels of thymocyte apoptosis. For this purpose, we tested DNA fragmentation by gel electrophoresis, analyzed DNA content by propidium iodide staining of ethanol fixed cells and looked for DNA strand breaks on tissue sections by in situ end labeling. We described an increase of the levels of apoptosis in cultured thymocytes during the preleukemic period, while the basic levels of apoptosis observed in situ are similar in normal and in preleukemic thymuses. We propose that after leukemogenic irradiations, there is an increase of cells wherein the cell suicide program is activated but that environmental thymic factors rescue them from apoptosis. Preleukemic cells could belong to this abnormally surviving population of cells "programmed to die," wherein additional genomic abnormalities would lead to fully neoplastic transformation.

A RadLV-induced gamma delta T cell lymphoma displaying an antitumoral cytotoxicity.

We described previously the induction by RadLV infection of a lymphoma (NS8) expressing a cytolytic activity against an MCA-induced fibrosarcoma. We report here that the cytolytic activity of these immortalized CD3+, CD8+ T cells is non-MHC-restricted. We then determined the structure and expression of the TCR chains expressed by these cells. Only partial rearrangement of the beta chain associated to an abnormally short transcript was detected in NS8 cells, whereas the gamma chain is rearranged and normally transcribed. On the opposite, rearrangement and expression of these genes were found in the other RadLV-induced lymphomas analysed. Moreover, gamma delta TCR proteins were detected on the cell surface of NS8 cells only, whereas the alpha beta complex, presents on the other T cell lines, was not expressed by NS8 cells. The ability of NS8 cells or of cells obtained from activated lymph nodes (harvested from mice grafted with the T2 sarcoma used to induce the NS8 line) to lyse the T2 sarcoma cell line was analysed. With both types of lymphocytes, the cytotoxicity was partially inhibited by a preincubation of the effector cells with anti-gamma delta antibodies. These results demonstrate that gamma delta lymphocytes can mediate anti-tumour cytotoxicity and NS8 lymphoma line may be representative of the TCR gamma delta CD8+ T cell subpopulation expressing non MHC-restricted cytotoxicity and displaying antitumoral activity.