Contribution of CD4+, CD8+CD28+, and CD8+CD28- T cells to CD3+ lymphocyte homeostasis during the natural course of HIV-1 infection.

The relationship between the number of circulating CD4+ T cells and the presence of particular CD8+ T cell subsets was analyzed by flow cytometry on PBL from asymptomatic HIV-1-infected patients whose specimens were collected every 2 mo for a total period of 32 mo. Only slight variations were detected in the absolute number of lymphocytes and percentage of CD3+ lymphocytes, whereas both CD4+ and CD8+ T cell subsets showed wide intrapatient variation. Variations in the number of CD8+CD28+ cells paralleled those of the CD4+ T cell subset in each patient tested, while the presence of CD8+CD28- T cells correlated inversely with CD4+ and CD8+CD28+ T cells. These data show that changes in the number of circulating CD4+-and CD8+CD28+ T cells are strongly related to the presence of CD8+CD28- T cells in these patients. Insight into the significance of CD8+CD28- T cell expansion will allow us to understand the mechanisms and significance of the HIV-1- driven change in CD4+CD8+ T cell homeostasis and the basic immunopathology of HIV disease.     

Skin and perivascular toxicity induced experimentally by doxorubicin

Extravasation of antitumor drugs and particularly doxorubicin (DXR) can be followed by skin ulceration and slowly evolving perivascular necrosis. DXR lesions have some characteristics in common with those induced by ionizing radiation and, with respect to gross morphology, are reminiscent of skin lesions induced by necrotizing agents. Time course and histopathology of toxic phenomena induced by intradermal or perivascular injection of various doses of either DXR or caustic chemicals have been studied in hairy outbred and hairless inbred (MF1 hr/hr) mice. The latter strain has been found to be intrinsically more sensitive to DXR induced toxic effects, particularly as far as perivascular administration is concerned. Long lasting lesions and, in a few cases, systemic involvement have been observed. On the contrary, necrotic foci induced by caustic chemicals rapidly regressed in both strains. The perivascular administration model, which has not been previously investigated, appears to be representative of what happens in clinical conditions and can be of use for assessing either skin toxicity of antitumor compound or the protective effect of candidate antidotes.     

The inhibition of lymphocyte stimulation by autologous human metastatic melanoma cells correlates with the expression of HLA-DR antigens on the tumor cells

Previous studies indicated that peripheral blood lymphocytes from patients (Pt-PBL) with lymph node metastatic melanomas proliferated in vitro and developed into tumor-restricted cytotoxic lymphocytes in response to alloantigens or interleukin 2 (IL-2). However, Pt-PBL were not stimulated by irradiated autologous metastatic melanoma (Auto-Me) cells. In the present study we report that the lack of stimulatory activity of Auto-Me cells may be due to a suppressive effect exerted by Auto-Me cells on the responder lymphocytes. In fact, we found that in 62% of cases examined, the addition of 5-10% Auto-Me cells to Pt-PBL cultures strongly inhibited both proliferation and the generation of tumor cytotoxic lymphocytes induced by alloantigens or IL-2. The inhibition was dose-dependent and tumor-restricted, and was not due either to toxicity, medium depletion or IL-2 absorption by Auto-Me cells. Normal fibroblasts, K562 cells and autologous E- lymphocytes were not suppressive. Auto-Me cells were able to inhibit Pt-PBL responses only when added during the first 24 h of culture and not later. Phenotypic analysis of Auto-Me cells using monoclonal antibodies directed against HLA-A,B,C, HLA-DR and melanoma-associated antigens revealed that the expression of high levels of DR antigens on Auto-Me cells was associated with an elevated suppressive activity. Conversely, Auto-Me cells with low or undetectable levels of DR antigens were not inhibitory. Furthermore, the increased expression of DR antigens on Auto-Me cells obtained by in vitro treatment with human interferon gamma (IFN-γ) also resulted in an increased suppressive activity. We conclude that HLA-DR+ metastatic melanoma cells can interfere with the generation of an anti-tumor immune response, thus potentially favoring the escape of the tumor from the host's control mechanism.     

Linking survival of HER2-positive breast carcinoma patients with surgical invasiveness

The early peak of relapse in patients with breast carcinomas that overexpress HER2 oncoprotein and dissemination to the axillary lymph nodes might be related to proliferation of micrometastatic lesions induced by EGF family growth factors released at the time of surgery. If the levels of these growth factors have an impact on relapse, the survival of patients with positive nodes and HER2-positive tumours should be dependent on surgery wideness. To test this hypothesis, HER2 status of primary tumours from patients included in a randomized clinical trial addressing conservative quadrantectomy versus radical mastectomy was retrospectively analyzed. In HER2-negative patients, independently of node infiltration, and in HER2-positive patients without node infiltration, no differences in survival according to the type of surgery were observed. In patients with positive nodes and HER2-positive tumours the estimation of the time-dependent log-hazard ratios showed that radical mastectomy significantly increased early death rates (P=0.037).     

Chemotactic activity for mononuclear phagocytes of culture supernatants from murine and human tumor cells: Evidence for a role in the regulation of the macrophage content of neoplastic tissues

Culture supernatants from 14 mouse solid tumors, 2 mouse lymphomas, 8 human solid tumor lines and 3 human leukemia-lymphomas were examined for chemotactic activity in blind-well chemotaxis chambers using murine peritoneal macrophages and human blood monocytes as indicator cells. Culture supernatants from various murine and human solid tumors had appreciable chemotactic activity for mononuclear phagocytes. Chemotactic activity was found in murine tumors of different histology and transplantation history, including autochthonous mammary carcinomas and chemically-induced sarcomas. Chemotactic activity was not unique to neoplastic cells because is was detected also in supernatants of two preparations of mouse embryo fibroblasts and two human lung embryo fibroblast lines. Production of tumor-derived chemo-attractants did not require serum in the culture medium, was inhibited by the protein synthesis inhibitor Cycloheximide, but was unaffected by the DNA synthesis inhibitor Mitomycin C. Murine supernatants were active on human monocytes and vice-versa. A first preliminary characterization of the chemotactic activity of the human sarcoma line 8387 revealed that it was not dialyzable, that most of the activity was retained at 56deg;C for 30 min, but not at 100deg;C, and that it was susceptible to proteolytic enzymes (trypsin and proteinase K) but unaffected by DNase and RNase. Upon fractionation on a Sephadex G-75 column, the activity eluted in a single, relatively broad peak in the cytocrome C region, corresponding to an apparent molecular weight of about 12,000. In the heterogeneous series of murine solid tumors studied, a significant, though far from absolute, correlation (r=0.71, p=0.013) was found between chemotactic activity in culture supernatants (expressed as area under the chemotaxis titration curve) and the percentage of tumor-associated macrophages. It is suggested that tumor-derived chemotactic factor(s) play a role in the regulation of the macrophage content of neoplastic tissues.     

PDGFRβ and FGFR2 mediate endothelial cell differentiation capability of triple negative breast carcinoma cells

Triple negative breast cancer (TNBC) is a very aggressive subgroup of breast carcinoma, still lacking specific markers for an effective targeted therapy and with a poorer prognosis compared to other breast cancer subtypes.In this study we investigated the possibility that TNBC cells contribute to the establishment of tumor vascular network by the process known as vasculogenic mimicry, through endothelial cell differentiation.Vascular‐like functional properties of breast cancer cell lines were investigated in vitro by tube formation assay and in vivo by confocal microscopy, immunofluorescence or immunohistochemistry on frozen tumor sections. TNBCs express endothelial markers and acquire the ability to form vascular‐like channels in vitro and in vivo, both in xenograft models and in human specimens, generating blood lacunae surrounded by tumor cells. Notably this feature is significantly associated with reduced disease free survival. The impairment of the main pathways involved in vessel formation, by treatment with inhibitors (i.e. Sunitinib and Bevacizumab) or by siRNA‐mediating silencing, allowed the identification of PDGFRβ and FGFR2 as relevant players in this phenomenon. Inhibition of these tyrosine kinase receptors negatively affects vascular lacunae formation and significantly inhibits TNBC growth in vivo.In summary, we demonstrated that TNBCs have the ability to form vascular‐like channels in vitro and to generate blood lacunae lined by tumor cells in vivo. Moreover, this feature is associated with poor outcome, probably contributing to the aggressiveness of this breast cancer subgroup. Finally, PDGFRβ and FGFR2‐mediated pathways, identified as relevant in mediating this characteristic, potentially represent valid targets for a specific therapy of this breast cancer subgroup.     

Taxanes enhance trastuzumab-mediated ADCC on tumor cells through NKG2D-mediated NK cell recognition

Recent clinical data indicate a synergistic therapeutic effect between trastuzumab and taxanes in neoadjuvantly treated HER2-positive breast cancer (BC) patients. In HER2+ BC experimental models and patients, we investigated whether this synergy depends on the ability of drug-induced stress to improve NK cell effectiveness and thus trastuzumab-mediated ADCC. HER2+ BC cell lines BT474 and MDAMB361 treated with docetaxel showed up-modulation of NK activator ligands both in vitro and in vivo, accompanied by a 15–40% increase in in vitro trastuzumab-mediated ADCC; antibodies blocking the NKG2D receptor significantly reduced this enhancement. NKG2D receptor expression was increased by docetaxel treatment in circulating and splenic NK cells from mice xenografted with tumor cells, an increase related to expansion of the CD11b⁺Ly6G⁺ cell population. Accordingly, NK cells derived from HER2+ BC patients after treatment with taxane-containing therapy expressed higher levels of NKG2D receptor than before treatment. Moreover, plasma obtained from these patients recapitulated the modulation of NKG2D on healthy donors'' NK cells, improving their trastuzumab-mediated activity in vitro. This enhancement occurred mainly using plasma from patients with low NKG2D basal expression. Our results indicate that taxanes increase tumor susceptibility to ADCC by acting on tumor and NK cells, and suggest that taxanes concomitantly administered with trastuzumab could maximize the antibody effect, especially in patients with low basal immune effector cytotoxic activity.     

TLR9 agonists oppositely modulate DNA repair genes in tumor versus immune cells and enhance chemotherapy effects

Synthetic oligodeoxynucleotides expressing CpG motifs (CpG-ODN) are a Toll-like receptor 9 (TLR9) agonist that can enhance the antitumor activity of DNA-damaging chemotherapy and radiation therapy in preclinical mouse models. We hypothesized that the success of these combinations is related to the ability of CpG-ODN to modulate genes involved in DNA repair. We conducted an in silico analysis of genes implicated in DNA repair in data sets obtained from murine colon carcinoma cells in mice injected intratumorally with CpG-ODN and from splenocytes in mice treated intraperitoneally with CpG-ODN. CpG-ODN treatment caused downregulation of DNA repair genes in tumors. Microarray analyses of human IGROV-1 ovarian carcinoma xenografts in mice treated intraperitoneally with CpG-ODN confirmed in silico findings. When combined with the DNA-damaging drug cisplatin, CpG-ODN significantly increased the life span of mice compared with individual treatments. In contrast, CpG-ODN led to an upregulation of genes involved in DNA repair in immune cells. Cisplatin-treated patients with ovarian carcinoma as well as anthracycline-treated patients with breast cancer who are classified as "CpG-like" for the level of expression of CpG-ODN modulated DNA repair genes have a better outcome than patients classified as "CpG-untreated-like," indicating the relevance of these genes in the tumor cell response to DNA-damaging drugs. Taken together, the findings provide evidence that the tumor microenvironment can sensitize cancer cells to DNA-damaging chemotherapy, thereby expanding the benefits of CpG-ODN therapy beyond induction of a strong immune response.     

Toll-like receptor agonists regulate beta-defensin 2 release in hair follicle

BACKGROUND: Skin is constantly in contact with different pathogens, which are present in the environment. The hair follicle is particularly susceptible to this microbial invasion as it offers an easy way of access for microorganisms; for this reason it is equipped with defence mechanisms to avoid frequent infections. OBJECTIVES: To analyse the expression pattern of four different members of the toll-like receptor (TLR) family in murine hair follicles and to evaluate the effects of their activation by their specific microbiota-derived agonists, in terms of production of the antimicrobial peptide beta-defensin 2 (DEFB2). METHODS: TLR and DEFB2 protein expression was investigated by immunohistochemistry on murine skin samples. RESULTS: Murine hair follicle expresses TLR2, TLR4 and TLR5; agonists of TLR2, TLR4 and TLR5 but not of TLR9 induced DEFB2 production in this compartment. The strongest DEFB2 expression was observed following TLR4 activation by lipopolysaccharide. CONCLUSIONS: Our data show that the hair follicle is equipped with TLR2, TLR4 and TLR5, and that these receptors are able to respond to microbial stimuli inducing the production of DEFB2 by epithelial cells. This immune response might be important in preserving the skin from microorganism infections.     

Tumorigenicity and dissemination of primary and metastatic human melanomas implanted into different sites in athymic nude mice

In the present study, the growth features and metastatic capacity of human primary and metastatic melanomas transplanted by different routes in nude mice was examined. Eight different human melanoma early cell cultures derived from 3 primary tumors, 1 local recurrence and 4 metastatic lesions of 6 melanoma patients were characterized for cell surface HLA (class I and II) and melanoma-associated antigens and karyotype. These tumors were then transplanted in CD-1 outbred nude mice by subcutaneous, intraperitoneal and intrasplenic routes. It was found that 7 out of 8 melanomas were tumorigenic after subcutaneous implantation without giving rise to metastases; 5 out of 7 melanomas grew when injected intraperitoneally and 3 of them disseminated to peritoneal organs and infiltrated intraperitoneal lymph nodes, liver and pancreas; of 8 melanomas implanted intrasplenically 4 grew in the spleen or invaded intraperitoneal lymph nodes, liver, pancreas, ovaries or lungs. Primary and metastatic melanomas did not differ in the pattern of dissemination. In fact, 2 out of 4 metastases and 1 of 3 primary tumors and 1 recurrence disseminated after intrasplenic or intraperitoneal inoculation. Heterogeneity in the growth pattern of different metastases of the same melanoma patient was also found. No correlation could be detected between the metastatic ability of melanoma cells studied and clinical stage of patients, tumor cell karyotype abnormalities, modal number or with the antigenic phenotype.