Nutritional management of hyperlipaemia in a jenny: a brief report.

An adult jenny (5-years-old, non-pregnant) was presented to the Veterinary Teaching Hospital (VTH) of the University of Sassari, with a recent history of appetite loss, extreme underweight condition and reluctance to move. On physical inspection, emaciation [body condition score, BCS: 3/9], muscular waste [muscular condition score, MCS: 1/5], loose/running faeces [faecal score, FS: 2/8], and a general state of mild dehydration were found. Blood analyses outlined a general undernourishment condition [circulating albumins, ALB: 17.6 g/L (21.6-31.6 g/L)] with underlying systemic inflammatory profile and moderate increase in circulating enzymes to explore liver function [aspartate amino-transferase, AST: 657 u/L (279-430 u/L); alanine amino-transferase ALT: 60 u/L (5-14 u/L); gamma-glutamyl-transferase, γ-GT: 87 IU/L (14-69 IU/L); total bilirubin close to the upper limit, TB: 0.20 mg/dL(0.07-0.21 mg/dL)]and hyperlipaemia [TG: 8.70 mmol/L (0.60-2.87 mmol/L)], following fat depots mobilisation, with total cholesterol closed to the lower limit of the physiological range. Hyper-phosphataemia was linked to haemolytic anaemia [P:1.81 mmol/L (0.77-1.39 mmol/L) and red blood cells, RBC: 4.14 1012/L (4.40-7.10 1012)] aligned with the TB to the upper limit. On ultrasound abdominal imaging, enlarged and hyper-echogenic liver was observed. Based on the clinical evaluation, a condition of hepatic lipidosis was diagnosed, requiring dedicated nutritional treatment to solve the extreme emaciation along with the metabolic disorder in support of medical therapy. A two-step feeding protocol was planned to support treatments aiming at immediate re-hydration (Ringer lactate solution 2 ml/kg/8 h). The nutritional objectives were meant at first to restart the voluntary feed intake. Gradual increasing energy provision through a palatable hay-based diet was planned to cover one fourth of daily metabolizable energy requirement calculated on the expected metabolic weight, adjusted according to the daily intake of feed and clinical condition. At the conclusion of this first 7-day phase, circulating blood parameters were closer to the reference values and the BCS moved from 3 to 4 out of 9. Bowel motility was restored, and faecal score improved (4/8). In the second phase, allowance to pasture and a combination diet with compound mixed feed were designed. Within four weeks of starting the nutritional plan, blood parameters were re-established to reference values. The gradual feed provision calculated in this two-phase approach proved successful in support of the overall clinical improvement observed after four weeks of treatment, in a severely undernourished jenny with compromised liver functions.

Meta-analysis on the utility of radiotherapy for the treatment of Ocular Melanoma

Introduction: Uveal melanoma is the most common intraocular tumor in the adult population. It can affect any part of the uveal tract: the iris, ciliary body, and choroid. Historically, enucleation has been the mainstay of treatment for primary melanoma. In the last decade, however, radiotherapy has acquired an increasingly important role and has now become our first-line modality. However, it is still widely debated what is the most effective radiotherapy technique for this tumor. Purpose to perform a literature review on the utility of radiotherapy for primary ocular melanoma and determine the most effective radiotherapy technique Materials and Methods: We included all systematic and narrative reviews on the topic, published between September 2007 and November 2017 on PubMed and SCOPUS. Two independent reviewers assessed the eligibility criteria for each article using the PRISMA checklist. The methodological quality of narrative and systematic reviews was evaluated with the INSA and AMSTAR checklists, respectively Results: Our study analyzed a total of 23 studies, including 18 narrative reviews and 5 systematic reviews. Radiotherapy with Brachytherapy, Proton Therapy, SRS/SRT with gamma knife and cyber knife, are the most common choices for the treatment of primary ocular melanoma. These techniques allow for excellent lesion spread control, eye, and vision conservation, and improve overall patients' quality of life. Among the narrative reviews, the highest INSA score was 5/7, the lowest 2/7, the mean was 3.83/7 and median was 4/7. Among the systematic reviews, the highest AMSTAR score was 9/12, the lowest 4/12, the mean 5.6/7 and median 4/7 Conclusion: The number of studies available on this topic is scarce. Among those published, the methodological quality is modest, as assessed with the INSA and AMSTAR checklists. As a result, we are not able to determine what the most effective radiotherapy technique is

Ultrasound delivery of Surface Enhanced InfraRed Absorption active gold-nanoprobes into fibroblast cells: a biological study via Synchrotron-based InfraRed microanalysis at single cell level.

Ultrasound (US) induced transient membrane permeabilisation has emerged as a hugely promising tool for the delivery of exogenous vectors through the cytoplasmic membrane, paving the way to the design of novel anticancer strategies by targeting functional nanomaterials to specific biological sites. An essential step towards this end is the detailed recognition of suitably marked nanoparticles in sonoporated cells and the investigation of the potential related biological effects. By taking advantage of Synchrotron Radiation Fourier Transform Infrared micro-spectroscopy (SR-microFTIR) in providing highly sensitive analysis at the single cell level, we studied the internalisation of a nanoprobe within fibroblasts (NIH-3T3) promoted by low-intensity US. To this aim we employed 20 nm gold nanoparticles conjugated with the IR marker 4-aminothiophenol. The significant Surface Enhanced Infrared Absorption provided by the nanoprobes, with an absorbance increase up to two orders of magnitude, allowed us to efficiently recognise their inclusion within cells. Notably, the selective and stable SR-microFTIR detection from single cells that have internalised the nanoprobe exhibited clear changes in both shape and intensity of the spectral profile, highlighting the occurrence of biological effects. Flow cytometry, immunofluorescence and murine cytokinesis-block micronucleus assays confirmed the presence of slight but significant cytotoxic and genotoxic events associated with the US-nanoprobe combined treatments. Our results can provide novel hints towards US and nanomedicine combined strategies for cell spectral imaging as well as drug delivery-based therapies.

Reduction of autophagy and increase in apoptosis correlates with a favorable clinical outcome in patients with rheumatoid arthritis treated with anti-TNF drugs.

BACKGROUND: Autophagy has emerged as a key mechanism in the survival and function of T and B lymphocytes, and its activation was involved in apoptosis resistance in rheumatoid arthritis (RA). To investigate whether the relationship between autophagy and apoptosis may impact the response to the therapy, we analyzed ex vivo spontaneous autophagy and apoptosis in patients with RA subjected to treatment with anti-tumor necrosis factor (TNF) drugs and in vitro the effects of TNFα and anti-TNF drugs on cell fate. METHODS: Peripheral blood mononuclear cells (PBMCs) from 25 RA patients treated with anti-TNF drugs were analyzed for levels of autophagy marker LC3-II by western blot and for the percentage of annexin V-positive apoptotic cells by flow cytometry. The same techniques were used to assess autophagy and apoptosis after in vitro treatment with TNFα and etanercept in both PBMCs and fibroblast-like synoviocytes (FLS) from patients with RA. RESULTS: PBMCs from patients with RA responsive to treatment showed a significant reduction in LC3-II levels, associated with an increased apoptotic activation after 4 months of therapy with anti-TNF drugs. Additionally, the expression of LC3-II correlated with DAS28. TNFα was able to induce autophagy in a dose-dependent manner after 24 h of culture in RA PBMCs and FLS. Moreover, etanercept caused a significant reduction of autophagy and of levels of citrullinated proteins. CONCLUSIONS: Our results show how the crosstalk between autophagy and apoptosis can sustain the survival of immune cells, thus influencing RA progression. This suggests that inhibition of autophagy represents a possible therapeutic target in RA.

Differential microRNA expression between decidual and peripheral blood natural killer cells in early pregnancy.

STUDY QUESTION: Have decidual natural killer (dNK) cells a different microRNA (miRNA or miR) expression pattern compared to NK cells circulating in the peripheral blood (pb) of healthy pregnant women in the first trimester of gestation? SUMMARY ANSWER: dNK cells have a unique miRNA profile, showing exclusive expression of a set of miRNAs and significant up- or down-regulation of most of the miRNAs shared with pbNK cells. WHAT IS KNOWN ALREADY: dNK cells differ from pbNK cells both phenotypically and functionally, and their origin is still debated. Many studies have indicated that miRNAs regulate several important aspects of NK cell biology, such as development, activation and effector functions. STUDY DESIGN, SIZE, DURATION: Decidua basalis and peripheral blood specimens were collected from women (n = 7) undergoing voluntary termination of gestation in the first trimester of pregnancy. dNK and pbNK cells were then highly purified by cell sorting. PARTICIPANTS/MATERIALS, SETTING, METHODS: miRNAs expression was analysed by quantitative RT-PCR (qRT-PCR)-based arrays using RNA purified from freshly isolated and highly purified pbNK and dNK cells. Results from arrays were validated by qRT-PCR assays. The bioinformatics tool ingenuity pathway analysis (IPA) was applied to determine the cellular network targeted by validated miRNAs and the correlated biological functions. MAIN RESULTS AND THE ROLE OF CHANCE: Herein, we identified the most differentially expressed miRNAs in NK cells isolated from peripheral blood and uterine decidua of pregnant women. We found that 36 miRNAs were expressed only in dNK cells and two miRNAs only in pbNK cells. Moreover, 48 miRNAs were commonly expressed by both NK cell preparations although at different levels: 28 were upregulated in dNK cells, while 15 were downregulated compared to pbNK cells. Validation of a selected set (n = 11) of these miRNAs confirmed the differential expression of nine miRNAs: miR-10b and miR-214 expressed only in dNK cells and miR-200a-3p expressed only in pbNK cells; miR-130b-3p, miR-125a-5p, miR-212-3p and miR-454 were upregulated while miR-210-3p and miR-132 were downregulated in dNK cells compared to pbNK cells. IPA network analysis identified a single network connecting all the miRNAs as well as their significant involvement in several classes of functions: 'Organismal injury, Reproductive system disease, Inflammatory disease' and 'Cellular development'. These miRNAs target molecules such as argonaute 2, tumour protein p53, insulin and other genes that belong to the same network and significantly influence cell differentiation and pregnancy. LIMITATIONS, REASONS FOR CAUTION: In the present study, the cellular network and biological functions modulated by miRNAs differentially expressed in dNK and pbNK cells were identified by IPA considering only molecules and relationships that were with confidence 'experimentally observed' in leucocytes. The decidual and pbNK cells that were analysed here are a heterogeneous population and further study will help to disentangle whether there are differences in miRNA production by the different subsets of NK cells. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study describing a different miRNA expression profile in dNK cells compared to matched pbNK cells during the first trimester of pregnancy. Our findings improved the body of knowledge on dNK cell biology and strongly suggest further investigation into the roles of miRNAs that are differentially expressed in human dNK compared to pbNK cells. Our results suggest that specific miRNAs can modulate dNK cell origin and functions, highlighting a potential role of this miRNA signature in human development and diseases. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from the Istituto Pasteur, Fondazione Cenci Bolognetti, the European NoE EMBIC within FP6 (Contract number LSHN-CT-2004-512040), Istituto Italiano di Tecnologia, and Ministero dell'Istruzione, dell'Università e della Ricerca (Ricerche Universitarie), and from Università Politecnica delle Marche. There are no conflicts of interest to declare.

Selective inhibitors of aurora kinases inhibit proliferation, reduce cell viability and impair cell cycle progression in papillary thyroid carcinoma cells.

The three members of the Aurora kinase family, Aurora-A, -B and -C, regulate several aspects of the mitotic process, and their aberrant expression and/or function causes mitotic abnormalities leading either to cell death or aneuploidy. They are found overexpressed in several human malignancies, including the papillary thyroid carcinoma (PTC). In the present study, we sought to establish whether Aurora kinase inhibition could be of any therapeutic value in the treatment of aggressive forms of PTC, enduring to radioactive iodide (RAI) ablation. To this end, the effects of selective inhibitors of Aurora-A (MLN8237) and Aurora-B (AZD1152) were analyzed on 3 human PTC cell lines expressing either wild-type (K1 and TPC1) or mutant p53 (BCPAP). The two inhibitors were capable of reducing cell proliferation in a time- and dose-dependent manner, with IC50 comprised between 65.4 and 114.9 nM for MLN8237, and between 26.6 and 484.6 nM for AZD1152. Immunofluorescence experiments confirmed that AZD1152 inhibited Aurora-B phosphorylation of histone H3 on Ser10, however, it did not affect Aurora-A autophosphorylation. MLN8237 inhibited Aurora-A autophosphorylation as expected, but at concentrations required to achieve the maximum antiproliferative effects it also abolished H3 (Ser10) phosphorylation. Time-lapse videomicroscopy evidenced that both inhibitors prevented the completion of cytokinesis, and cytofluorimetric analysis showed accumulation of cells in G2/M phase and/or polyploidy. Apoptosis was induced in all the cells by both inhibitors independently from the p53 status. In conclusion, in the present preclinical study MLN8237 and AZD1152 have emerged as promising drug candidates for RAI-insensitive PTC.

The dual Aurora kinase inhibitor ZM447439 prevents anaplastic thyroid cancer cell growth and tumorigenicity.

The anaplastic thyroid cancer (ATC) is among the most aggressive human tumors which fail to respond to all the currently available therapeutic approaches. As a consequence most patients die within a few months from diagnosis. In the present preclinical study, the effects of the ZM447439, a functional inhibitor of Aurora kinases, on the growth and tumorigenicity of a panel of ATC derived cell lines (CAL-62, 8305C, 8505C and BHT-101) were evaluated. The treatment of the different ATC cells with ZM447439 inhibited proliferation in a time- and dose-dependent manner, with IC50 comprised between 0.5 mM and 5 mM. Moreover, the drug remarkably impaired the formation of colonies in soft agar of all the cell lines. Consistently with Aurora inhibition, immunofluorescence and immunoblotting experiments demonstrated that Aurora auto-phosphorylation following drug treatment was completely abrogated, and treated cells were characterized by the presence of multiple spindles with short microtubules. In the same experiments we observed the loss of histone H3 phosphorylation on Ser10, specifically due to Aurora-B, after ZM447439 treatment. Time-lapse videomicroscopy and flow cytometric analysis demonstrated that in presence of ZM447439 the cells were able to enter mitosis but not to complete it, becoming polyploid. Almost all the ATC cell lines studied showed increased apoptosis after only 48 h of treatment. In conclusion, our data demonstrate that ZM447439 is effective in reducing cell growth and tumorigenicity of different ATC derived cell lines, and further investigations are needed to exploit its potential therapeutic value for ATC treatment.

Effects of the Aurora kinases pan-inhibitor SNS-314 mesylate on anaplastic thyroid cancer derived cell lines.

OBJECTIVES: Anaplastic thyroid carcinomas (ATC) are highly aggressive tumours unresponsive to any available radio- or chemotherapeutic protocol, with a median survival rate of 4-5 months from the time of diagnosis. We previously demonstrated that ATC are characterized by increased expression of the kinases Aurora-A, -B and -C, involved in the regulation of multiple steps of the mitotic phase. In this study, the in vitro effects of SNS-314 mesylate, a pan-inhibitor of the Aurora kinases, on growth and tumorigenicity of ATC cells were evaluated. MATERIALS AND METHODS: The effects of SNS-314 mesylate were assessed on the ATC derived cell lines CAL-62, 8305C, 8505C and BHT-101 by means of cell proliferation assay, immunofluorescence, cytofluorimetry, time lapse microscopy, and colony formation in soft agar. RESULTS: Treatment of the different ATC cells with SNS-314 mesylate inhibited proliferation in a time- and dose-dependent manner, with IC(50) comprised between 2.6 nM and 26.6 nM. CAL-62 cells exposed for 24 h to SNS-314 mesylate 100 nM evidenced a significant augmentation of the apoptotic index. Time-lapse video-microscopy of CAL-62 cells showed that SNS-314 mesylate prevents the completion of mitosis leading to polyploidy. Western blot experiments demonstrated that the auto-phosphorylation of the Aurora kinases as well as histone H3 phosphorylation in CAL-62 treated cells was inhibited. Finally, the drug inhibited colony formation in soft agar of all cell lines. CONCLUSIONS: Our results demonstrated that SNS-314 mesylate is capable to efficiently reduce cell growth and tumorigenicity of different ATC derived cell lines suggesting its potential therapeutic value for ATC treatment.

UVB radiation induced effects on cells studied by FTIR spectroscopy.

We have made a preliminary analysis of the results about the effects on tumoral cell line (lymphoid T cell line Jurkat) induced by UVB radiation (dose of 310 mJ/cm(2)) with and without a vegetable mixture. In the present study, we have used two techniques: Fourier transform infrared spectroscopy (FTIR) and flow cytometry. FTIR spectroscopy has the potential to provide the identification of the vibrational modes of some of the major compounds (lipid, proteins and nucleic acids) without being invasive in the biomaterials. The second technique has allowed us to perform measurements of cytotoxicity and to assess the percentage of apoptosis. We already studied the induction of apoptotic process in the same cell line by UVB radiation; in particular, we looked for correspondences and correlations between FTIR spectroscopy and flow cytometry data finding three highly probable spectroscopic markers of apoptosis (Pozzi et al. in Radiat Res 168:698-705, 2007). In the present work, the results have shown significant changes in the absorbance and spectral pattern in the wavenumber protein and nucleic acids regions after the treatments.

Chlamydia pneumoniae induces T cell apoptosis through glutathione redox imbalance and secretion of TNF-alpha.

Chlamydia pneumoniae persistent infection has been implicated in the pathogenesis of several chronic inflammatory diseases including atherosclerosis, and we hypothesized that modulation of the apoptosis of macrophages and/or T cells by C. pneumoniae infection may contribute to the development of such diseases. We therefore evaluated apoptosis, cytokine response, and redox status in human primary T cells and macrophages infected with C. pneumoniae. In addition, co-cultures of T cells and macrophages infected with C. pneumoniae were also carried out. Apoptosis, and levels of glutathione (GSH), glutathione disulfide (GSSG), and tumour necrosis factor (TNF)-alpha were measured by flow cytometry, high performance liquid chromatography and enzyme-linked immunosorbent assay. C. pneumoniae induced apoptosis in T cells as well as in co-cultures of T cells and infected macrophages by marked decrease in GSH/GSSG ratio and increased production of TNF-alpha, respectively. The results demonstrate that interaction of C. pneumoniae with T cells and/or macrophages characterized by interference with redox status, and secretion of tumour necrosis factor-alpha culminates in the induction of T cell apoptosis and survival of infected macrophages. In conclusion, the inappropriate T cell response against C. pneumoniae and survival of infected macrophages could explain the persistence of this intracellular obligate pathogen in the host-organism; it may contribute to the development of chronic inflammatory diseases, although further studies are needed to clarify such a complex mechanism.

The adaptor protein shc is involved in the negative regulation of NK cell-mediated cytotoxicity.

The activation of protein tyrosine kinase(s) (PTK) is a critical event required for the development of NK cell-mediated cytotoxicity. Here we demonstrate that the adaptor protein shc undergoes tyrosine phosphorylation during the generation of antibody-dependent cellular cytotoxicity (ADCC) and natural killing. In addition, we report that, upon direct or antibody-dependent target cell interaction, shc coprecipitates with the Src homology 2 (SH2)-containing inositol phosphatase, SHIP. To gain information on the functional role of shc in NK cytotoxicity, we overexpressed wild-type or dominant negative shc constructs in the human NKL cell line. Our findings show a consistent shc-mediated down-regulation of ADCC and natural killing. Such functional effect correlates with a perturbation of the phosphoinositide (PI) metabolism by means of a shc-mediated negative regulation of inositol 1,4,5 triphosphate (IP3) generation and intracellular calcium flux upon CD16 ligation. Furthermore, our data show that dominant-negative shc-mediated perturbation of shc/SHIP interaction leads to inhibition of ligand-dependent SHIP recruitment to CD16 zeta chain. We suggest that shc plays a role of negative adaptor by modulating SHIP recruitment to activation receptors involved in the generation of NK cytotoxic function.

Effects of triptorelin, a gonadotropin-releasing hormone agonist, on the human prostatic cell lines PC3 and LNCaP.

Some analogues of gonadotropin-releasing hormone (GnRH) influence the in vitro proliferation of cultured human cells by complex interactions that are only partially understood. This study explored the effect of Triptorelin, a GnRH agonist, on the LNCaP and PC3 prostatic cell lines, which are, respectively, responsive and unresponsive to androgen stimulation. The toxicity and cell cycle modifications induced by the drug were investigated by FACScan analysis; the effect on cell proliferation in different culture conditions was determined by counting in a Burker chamber; and the expression of binding sites for 125I-Triptorelin was revealed by displacement experiments. PC3 cell growth was completely unaffected by Triptorelin. The drug caused a double stimulatory-inhibitory action on the growth of actively proliferating LNCaP cells, depending upon the dose and environment. A significant inhibitory effect on proliferation, ranging from 25% to 65% compared with controls, was observed at a high dose (10(-4) M) according to the culture conditions; and a proliferative effect (42% compared with controls) was observed at a lower dose (10(-7) M) only in fetal bovine serum-supplemented medium. Displacement experiments revealed the expression of moderately high affinity and low affinity binding sites in LNCaP cells (Kd = 2.6 x 10(-8) and 7.7 x 10(-6) M) but only low affinity binding sites in PC3 cells (Kd = 2.7 x 10(-6) M), which suggests that the expression of binding sites with different affinity could be associated with a biological response to the drug. Proliferation studies in the presence of Cetrorelix, a GnRH antagonist, confirmed the different sensitivity of the 2 cell lines to GnRH analogues and showed that the proliferative effect of Triptorelin on LNCaP cells can be inhibited by the antagonist. Data confirm the cell specificity of Triptorelin's action and the peculiarity of its effects on prostatic cell proliferation in our experimental conditions.

Expression of neurotrophin receptors in normal and malignant B lymphocytes.

In order to define a cellular model suitable for studying, in vitro, the molecular properties and functions of neurotrophin receptors in human lymphocytes, TrkA, TrkB, TrkC and p75(NTR) expression was investigated in a panel of EBV immortalized lymphoblastoid (LCL) and Burkitt lymphoma-derived cell lines (BLs) compared to primary B lymphocytes by RT-PCR and flow cytometric analysis. Our data show that trkA and trkB are transcribed in most B cell lines of normal and malignant origin. For several of them, we also gained first evidence of trkC expression in B cells. All cell lines and primary B cells lack p75(NTR) expression. These data suggest that neurotrophin receptors expression in the B cell lines correlates to some extent with the phenotypic maturation stage and endogenous viral activity levels. Our data suggest that TrkA and TrkB, once activated, provide a partial rescue from apoptosis, whereas TrkC stimulates the progression through the cell cycle without affecting cell survival. Finally, the identification of a number of cell lines showing single expression of one of the Trk receptors has disclosed the availability of a cellular tool for further studies on their function, and mechanisms of signal transduction in the B cell moiety in the absence of p75(NTR).

Augmentation of leukocyte infiltration in murine tumors expressing B-cell derived but not nasopharyngeal carcinoma derived EBV membrane protein LMP1.

The Epstein-Barr virus (EBV) encoded latent membrane protein of B cell origin, B-LMP1 (B95-8 prototype) and nasopharyngeal carcinoma (NPC) derived C-LMP1 (CAO prototype) were transfected individually in S6C adenocarcinoma cells of ACA (H-2f) origin. We have shown previously that inoculation of B-LMP1 expressing S6C cells led to tumor rejection in pre-immunized, immunocompetent syngeneic ACA mice, whereas the C-LMP1 transfectants were not immunogenic. Furthermore, B-LMP1 but not C-LMP1 expressing S6C cells grew with necrosis and extensive skin damage in non-immunized mice. A study was carried out to determine whether the in vivo growth pattern of S6C cells expressing two different LMP1 isolates could be correlated to any immunomodulatory mechanism. An increased infiltration of CD45+ leukocytes was found in B-LMP1 expressing S6C tumors originating in non-immunized, syngeneic ACA mice. The C-LMP1 expressors, vector transfectants and untransfected parental tumors had significantly lower number of infiltrating leukocytes. The immunoaccessory molecules ICAM-1, B7-1 and MHC Class I and II expression was unaltered in both B- and C-LMP1 transfectants. The data suggest that B-LMP1 but not C-LMP1 induce anti-tumor immune response.

In vitro infection of CD4+ T lymphocytes with HTLV-I generates immortalized cell lines coexpressing lymphoid and myeloid cell markers.

The human T cell leukemia/lymphoma virus (HTLV-I) is the etiologic agent of adult T cell leukemia (ATL). CD4+ lymphocytes are the preferential targets of infection, even though other cell types can be infected in vitro by the virus. Although ATL cells show CD3 and CD4 surface markers, some ATL-derived cell lines were reported to express also myeloid antigens. In order to analyze possible phenotypic changes induced by HTLV-I after infection of human lymphocytes, CD4+ cells were isolated from peripheral blood of three healthy donors, by separation through immunomagnetic beads. CD4+ lymphocytes were then infected by coculture with irradiated HTLV-I producing MT-2 cells. The phenotypic profile of infected cells was studied by flow cytometric analysis using monoclonal antibodies against lymphoid (CD3, CD4, TCR alpha/beta) and myelomonocitic markers (CD13, CD14, CD15, CD33, CD34). The results show that HTLV-I immortalized cell lines coexpressed CD13, CD33 and lymphoid markers. No expression of CD14, CD15 and CD34 was observed. These data suggest that the presence of both myeloid and lymphoid phenotype in HTLV-I infected T cells is the results of an induction rather than a selection mechanism.

Overexpression of transforming growth factor beta-type II receptor reduces tumorigenicity and metastastic potential of K-ras-transformed thyroid cells.

Expression of type II receptor of transforming growth factor beta (TbetaRII) is necessary for this factor to inhibit the growth of thyroid epithelial cells. In rat thyroid transformed cells, the resistance to transforming growth factor beta (TGFbeta) is associated with a decreased expression of TbetaRII mRNA and protein. Reduced TbetaRII expression has also been found in human thyroid differentiated and undifferentiated carcinomas. To investigate the role of TbetaRII in modulating the tumorigenic potential of k-ras-transformed thyroid cells, we transfected these cells with an expression vector carrying the human TbetaRII gene, regulated by an inducible promoter. Isolated clones, overexpressing TbetaRII, showed a reduction in the anchorage-dependent and -independent cell growth, compared with control k-ras-transformed cells. When transplanted in athymic nude mice, the transfected clones presented a decrease in tumorigenicity with respect to the highly malignant parental cells. Moreover, the diminished tumorigenic ability of the clones studied was accompanied by a statistically significant reduction in spontaneous and lung artificial metastases. Taken together, our data demonstrate that TbetaRII acts as a potent tumor suppressor gene when overexpressed in malignant thyroid cells.

Defective Th1 and Th2 cytokine synthesis in the T-T cell presentation model for lack of CD40/CD40 ligand interaction.

In this study, T or NK cell clones used as antigen-presenting cells (T- or NK-APC) were shown to be significantly less efficient than professional APC in inducing Th1 and Th2 cytokines by antigen-specific T cell clones. This phenomenon was not related to a limited engagement of TCR by T-APC, since comparable thresholds of TCR down-regulation were shown when antigen was presented by either T-APC or professional APC. Rather, the stimulatory T-APC weakness was due to their inability, because they are CD40-, to provide the appropriate co-stimuli to responder T cells both indirectly via IL-12, and partially via direct CD40L triggering on T cells. Indeed, the simultaneous addition of IL-12 and reagents directly engaging CD40L on responder T cells restored T cell cytokine synthesis when antigen was presented by T-APC. In addition, either IL-12 production or blocking of T cell cytokine synthesis by anti-IL-12 p75 antibodies was evident only when professional APC were used in our antigen-specific system. The down-regulation of cytokine synthesis in the system of T-T cell presentation could represent a novel mechanism of immune regulation, which may intervene to switch off detrimental Th1- or Th2-mediated responses induced by antigen presentation among activated T cells infiltrating inflamed tissues.

The lymphoepithelial organization of the tonsil: an immunohistochemical study in chronic recurrent tonsillitis.

Interactions between leukocytes and crypt epithelium were extensively investigated in 12 cases of chronic recurrent tonsillitis, using immunohistochemistry and cytofluorimetric analysis of cell suspensions. Intraepithelial leukocytes are a mixed cell population composed of 50 per cent CD20-positive B lymphocytes, 40 per cent T lymphocytes with a 2.7 CD4/CD8 ratio, and 10 per cent CD68-positive macrophages. About 4 per cent of intraepithelial leukocytes are proliferating cells, as indicated by Ki-67 staining. Leukocyte infiltration is associated with expression on epithelial cells of the adhesion molecules ICAM-1 and VCAM-1. Crypt epithelium is supported by a basement membrane showing frequent interruptions and connected with the reticular stroma of the lymphoid tissue, which was stained for fibronectin, tenascin, collagen, and laminin. Extracellular matrix (ECM) distribution was correlated with integrin expression on B and T lymphocytes. It was found that the ECM was arranged differently in the follicles and in the extrafollicular area and that B and T lymphocytes exhibited different patterns of integrin expression.

Effects of sodium fusidate in animal models of insulin-dependent diabetes mellitus and septic shock.

We have evaluated the effects of the novel immunosuppressant sodium fusidate (fusidin) in the non-obese diabetic (NOD) mouse and in D-galactosamine (D-Gal)-presensitized BALB/c mice challenged with the bacterial superantigen, Staphylococcus aureus enterotoxin B (SEB) or with the endotoxin, Escherichia coli lipopolysaccharide (LPS). The NOD mouse model has clinical and histoimmunological features similar to those of human insulin-dependent diabetes mellitus (IDDM). The SEB- and LPS-treated BALB/c mouse models exhibit pathogenic similarities with human septic shock conditions. In the NOD mouse, fusidin suppressed the spontaneous development of insulitis (mean inhibition 73%) and hyperglycaemia (IDDM incidence 25% versus 0%) when administered at 40 mg/kg five times weekly for 8 consecutive weeks from the fourth week of age; concurrently treated animals exhibited reduced percentages of splenic T lymphocytes. This anti-diabetogenic effect was confirmed in the accelerated model of diabetes induced in the NOD mouse with cyclophosphamide (CY) (IDDM incidence 55% versus 21-6% using dosages of fusidin from 40 to 80 mg/kg five times weekly); protection from IDDM development was achieved even when the drug (80 mg/kg/day) was first administered 7 days after CY challenge. In contrast, fusidin did not reverse hyperglycaemia when administered to CY-treated animals within 3 days of IDDM development. In the two models of septic shock, prophylactic treatment with fusidin, 80 mg/kg given three times for 2 days prior to D-Gal/SEB or D-Gal/LPS challenge, drastically reduced the lethality compared with D-Gal/buffer-treated mice. This effect may depend on the inhibitory action of fusidin on the secretion of cytokines such as interferon-gamma and tumour necrosis factor-alpha, the serum levels of which were greatly diminished in the fusidin-treated mice (mean inhibition 50-90%). These results demonstrate that fusidin may have a role in the treatment of cell-mediated autoimmune diseases and cytokine-mediated infectious diseases in humans.

Generation and characterization of two human alpha/beta T cell clones. Recognizing autologous breast tumor cells through an HLA- and TCR/CD3-independent pathway.

Cell-mediated immune response to breast tumor has only been marginally investigated. To gain insight into this issue we have developed two clones of distinct phenotype, CD3+ alpha/beta, CD4+, CD8-, CD16-, and CD3+ alpha/beta, CD4-, CD8+, CD16-, respectively, from peripheral blood lymphocytes (PBL) of a breast cancer patient. These effectors, selected on the basis of their cytolytic activity against autologous tumor cells and lack of lysis on NK-sensitive cell lines, preferentially recognize autologous tumor cells. The two clones' cytotoxic activity, while inhibited by anti-LFA-1 mAb, could not be abolished by mAbs to CD3, to class I and class II MHC molecules, and by mAbs to molecules involved in T cell function (i.e., CD4, CD8, CD2). The molecular structure of the alpha and beta T cell receptor chains of the two effector cells, confirmed their clonality and showed that, despite an overlapping killing pattern, they possess distinct TCR alpha and beta chains. These findings demonstrate that breast tumor-specific CTL clones can be generated through current technology and that a alpha/beta effector cell population operating through a HLA-unrestricted and TCR/CD3-independent pathway may be involved in the identification and killing of this tumor.