Estimate and needs of the transgender adult population: the SPoT study.

BACKGROUND: Despite the increasing interest in transgender health research, to date little is known about the size of the transgender and gender diverse (TGD) population. METHODS: A web-based questionnaire survey was developed, including a collection of socio-demographic characteristics and disseminated online through social media. Gender incongruence was evaluated by using a 2-item approach assessing gender recorded at birth and gender identity. The primary objective of the present population-based study was to estimate the proportion of TGD people across ages among a large sample of people who answered a web-based survey. The secondary endpoints were to identify gender-affirming needs and possible barriers to healthcare access. RESULTS: A total of 19,572 individuals participated in the survey, of whom 7.7% reported a gender identity different from the sex recorded at birth. A significantly higher proportion of TGD people was observed in the youngest group of participants compared with older ones. Among TGD people who participated in the study, 58.4% were nonbinary, and 49.1% experienced discrimination in accessing health care services. Nonbinary TGD participants reported both the need for legal name and gender change, along with hormonal and surgical interventions less frequently compared to binary persons. CONCLUSIONS: Being TGD is not a marginal condition In Italy. A large proportion of TGD persons may not need medical and surgical treatments. TGD people often experience barriers to healthcare access relating to gender identity.

Sociodemographic profile, health-related behaviours and experiences of healthcare access in Italian transgender and gender diverse adult population.

PURPOSE: Information on the general health of transgender and gender diverse (TGD) individuals continues to be lacking. To bridge this gap, the National Institute of Health in Italy together with the National Office against Racial Discriminations, clinical centres, and TGD organizations carried out a cross-sectional study to define the sociodemographic profile, health-related behaviours, and experiences of healthcare access in Italian TGD adult population. METHODS: A national survey was conducted by Computer-Assisted Web Interviewing (CAWI) technique. Collected data were compared within the TGD subgroups and between TGD people and the Italian general population (IGP). RESULTS: TGD respondents were 959: 65% assigned female at birth (AFAB) and 35% assigned male at birth (AMAB). 91.8% and 8.2% were binary and non-binary TGD respondents, respectively. More than 20% of the TGD population reported to be unemployed with the highest rate detectable in AMAB and non-binary people. Cigarette smoking and binge drinking were higher in the TGD population compared with IGP (p < 0.05), affecting TGD subgroups differently. A significant lower percentage of AFAB TGD people reported having had screening for cervical and breast cancer in comparison with AFAB IGP (p < 0.0001, in both cases). Over 40% was the percentage of AFAB and non-binary TGD people accessing healthcare who felt discriminated against because of their gender identity. CONCLUSIONS: Our results are a first step towards a better understanding of the health needs of TGD people in Italy in order to plan the best policy choices for a more inclusive public health.

The HIV-1 vpr protein acts as a negative regulator of apoptosis in a human lymphoblastoid T cell line: possible implications for the pathogenesis of AIDS.

Although apoptosis is considered one of the major mechanisms of CD4(+) T cell depletion in HIV-infected patients, the virus-infected cells somehow appear to be protected from apoptosis, which generally occurs in bystander cells. Vpr is an auxiliary HIV-1 protein, which, unlike the other regulatory gene products, is present at high copy number in virus particles. We established stable transfectants of CD4+ T Jurkat cells constitutively expressing low levels of vpr. These clones exhibited cell cycle characteristics similar to those of control-transfected cells. Treatment of control clones with apoptotic stimuli (i.e., cycloheximide/tumor necrosis factor alpha (TNF-alpha), anti-Fas antibody, or serum starvation) resulted in a massive cell death by apoptosis. In contrast, all the vpr-expressing clones showed an impressive protection from apoptosis independently of the inducer. Notably, vpr antisense phosphorothioate oligodeoxynucleotides render vpr-expressing cells as susceptible to apoptosis induced by cycloheximide and TNF-alpha as the control clones. Moreover, the constitutive expression of HIV-1 vpr resulted in the upregulation of bcl-2, an oncogene endowed with antiapoptotic activities, and in the downmodulation of bax, a proapoptotic factor of the bcl-2 family. Altogether, these results suggest that low levels of the endogenous vpr protein can interfere with the physiological turnover of T lymphocytes at early stages of virus infection, thus facilitating HIV persistence and, subsequently, viral spread. This might explain why apoptosis mostly occurs in bystander uninfected cells in AIDS patients.

Death receptor ligation triggers membrane scrambling between Golgi and mitochondria.

Subcellular organelles such as mitochondria, endoplasmic reticulum (ER) and the Golgi complex are involved in the progression of the cell death programme. We report here that soon after ligation of Fas (CD95/Apo1) in type II cells, elements of the Golgi complex intermix with mitochondria. This mixing follows centrifugal dispersal of secretory membranes and reflects a global alteration of membrane traffic. Activation of apical caspases is instrumental for promoting the dispersal of secretory organelles, since caspase inhibition blocks the outward movement of Golgi-related endomembranes and reduces their mixing with mitochondria. Caspase inhibition also blocks the FasL-induced secretion of intracellular proteases from lysosomal compartments, outlining a novel aspect of death receptor signalling via apical caspases. Thus, our work unveils that Fas ligand-mediated apoptosis induces scrambling of mitochondrial and secretory organelles via a global alteration of membrane traffic that is modulated by apical caspases.

Human immunodeficiency virus (HIV)-1 proteins and cytoskeleton: partners in viral life and host cell death.

Cytoskeletal components play a major role in the human immunodeficiency virus-1 (HIV-1) infection. A wide variety of molecules belonging to the microfilament system, including actin filaments and actin binding proteins, as well as microtubules have a key role in regulating both cell life and death. Cell shape maintenance, cell polarity and cell movements as well as cytoplasmic trafficking of molecules determining cell fate, including apoptosis, are in fact instructed by the cytoskeleton components. HIV infection and viral particle production seem to be controlled by cytoskeleton as well. Furthermore, HIV-associated apoptosis failure can also be regulated by the actin network function. In fact, HIV protein gp120 is able to induce cytoskeleton-driven polarization, thus sensitizing T cells to CD95/Fas-mediated apoptosis. The microfilament system seems thus to be a sort of cytoplasmic supervisor of the viral particle, the host cell and the bystander cell's very fate.

Interferon-beta induces S phase slowing via up-regulated expression of PML in squamous carcinoma cells.

Type I Interferon (IFN) and all-trans retinoic acid (RA) inhibit cell proliferation of squamous carcinoma cell lines (SCC). Examinations of growth-affected cell populations show that SCC lines ME-180 and SiHa treated with IFN-beta undergo a specific slower progression through the S phase that seems to trigger cellular death. In combination treatment RA potentiates IFN-beta effect in SCC ME-180 but not in SiHa cell line, partially resistant to RA antiproliferative action. RA added as single agent affects cell proliferation differently by inducing a slight G1 accumulation. The IFN-beta-induced S phase lengthening parallels the increased expression of PML, a nuclear phosphoprotein specifically up-regulated at transcriptional level by IFN, whose overexpression induces cell growth inhibition and tumor suppression. We report that PML up-regulation may account for the alteration of cell cycle progression induced by IFN-beta in SCC by infecting cells with PML-PINCO recombinant retrovirus carrying the PML-3 cDNA under the control of the 5' LTR. In fact PML overexpression reproduces the IFN-beta-induced S phase lengthening. These findings provide important insight into the mechanism of tumor suppressing function of PML and could allow PML to be included in the pathways responsible for IFN-induced cell growth suppression.

The HIV-1 vpr protein induces anoikis-resistance by modulating cell adhesion process and microfilament system assembly.

We have previously shown that CD4+ T Jurkat cells constitutively expressing low levels of the human immunodeficiency virus 1 (HIV-1) vpr protein were less susceptible to undergo apoptosis than control cells.1 In this study we have investigated the role of vpr in affecting mechanisms of importance in the control of apoptosis. Vpr-expressing clones consistently aggregated in clusters with time in culture, whereas mock-transfected cells grew as dispersed cultures. The analysis of adhesion molecules involved in cell-to-cell as well as in cell-substrate interactions showed a higher expression of cadherin and integrins alpha5 and alpha6 in vpr-transfected clones with respect to mock-transfected cells. This up-modulation was specifically blocked by cell exposure to antisense oligonucleotides targeted at the vpr. In addition, F-actin microfilament cytoskeletal organization, known to be involved in cell-cell interaction pathways and in the modulation of cell surface molecule expression, was significantly improved in vpr-expressing clones, in which filament polymerization was increased. We thus envisage that vpr viral protein can maintain cell survival via a specific activity on cytoskeleton-dependent cell adhesion pathways, i.e. by inducing anoikis-resistance. These particular effects of vpr might enhance the homing, spreading and survival of the infected lymphocytes, thus contributing to virus persistence in the course of acute HIV-1 infection.

Rho-dependent cell spreading activated by E.coli cytotoxic necrotizing factor 1 hinders apoptosis in epithelial cells.

Cell-cell and cell-matrix interactions play a pivotal role in numerous cell functions including cell survival and death. In this work, we report evidence that the Rho-dependent cell spreading activated by a protein toxin from E. coli, the cytotoxic necrotizing factor 1 (CNF1), is capable of hindering apoptosis in HEp-2 cells. In addition to the promotion of cell spreading, CNF1 protects cells from the experimentally-induced rounding up and detachment and improves the ability of cells to adhere to each other and to the extracellular matrix by modulating the expression of proteins related to cell adhesion. In particular, the expression of integrins such as alpha 5, alpha 6 and alpha v, as well as of some heterotypic and homotypic adhesion-related proteins such as the Focal Adhesion Kinase, E-cadherin, alpha and beta catenins were significantly increased in cells exposed to CNF1. Our results suggest, however, that the promotion of Rho-dependent cell spreading is the key mechanism in protecting cells against apoptosis rather than cell adhesion per se. A toxin inducing cell spreading without activating Rho, such as Cytochalasin B, was in fact ineffective in favouring cell survival. These data are of relevance (i) for the understanding of the role of the actin-dependent and especially Rho-dependent cellular activities involved in apoptosis regulation and (ii) in providing some clues to understanding the mechanisms by which bacteria, by controlling cell fate, might exert their pathogenic activity.

CD95/phosphorylated ezrin association underlies HIV-1 GP120/IL-2-induced susceptibility to CD95(APO-1/Fas)-mediated apoptosis of human resting CD4(+)T lymphocytes.

CD95(APO-1/Fas)-mediated apoptosis of bystander uninfected T cells exerts a major role in the HIV-1-mediated CD4+ T-cell depletion. HIV-1 gp120 has a key role in the induction of sensitivity of human lymphocytes to CD95-mediated apoptosis through its interaction with the CD4 receptor. Recently, we have shown the importance of CD95/ezrin/actin association in CD95-mediated apoptosis. In this study, we explored the hypothesis that the gp120-mediated CD4 engagement could be involved in the induction of susceptibility of primary human T lymphocytes to CD95-mediated apoptosis through ezrin phosphorylation and ezrin-to-CD95 association. Here, we show that gp120/IL-2 combined stimuli, as well as the direct CD4 triggering, on human primary CD4(+)T lymphocytes induced an early and stable ezrin activation through phosphorylation, consistent with the induction of ezrin/CD95 association and susceptibility to CD95-mediated apoptosis. Our results provide a new mechanism through which HIV-1-gp120 may predispose resting CD4(+)T cell to bystander CD95-mediated apoptosis and support the key role of ezrin/CD95 linkage in regulating susceptibility to CD95-mediated apoptosis.

TM9SF4 is a novel V-ATPase-interacting protein that modulates tumor pH alterations associated with drug resistance and invasiveness of colon cancer cells.

An inverted pH gradient across the cell membranes is a typical feature of malignant cancer cells that are characterized by extracellular acidosis and cytosol alkalization. These dysregulations are able to create a unique milieu that favors tumor progression, metastasis and chemo/immune-resistance traits of solid tumors. A key event mediating tumor cell pH alterations is an aberrant activation of ion channels and proton pumps such as (H+)-vacuolar-ATPase (V-ATPase). TM9SF4 is a poorly characterized transmembrane protein that we have recently shown to be related to cannibal behavior of metastatic melanoma cells. Here, we demonstrate that TM9SF4 represents a novel V-ATPase-associated protein involved in V-ATPase activation. We have observed in HCT116 and SW480 colon cancer cell lines that TM9SF4 interacts with the ATP6V1H subunit of the V-ATPase V1 sector. Suppression of TM9SF4 with small interfering RNAs strongly reduces assembly of V-ATPase V0/V1 sectors, thus reversing tumor pH gradient with a decrease of cytosolic pH, alkalization of intracellular vesicles and a reduction of extracellular acidity. Such effects are associated with a significant inhibition of the invasive behavior of colon cancer cells and with an increased sensitivity to the cytotoxic effects of 5-fluorouracil. Our study shows for the first time the important role of TM9SF4 in the aberrant constitutive activation of the V-ATPase, and the development of a malignant phenotype, supporting the potential use of TM9SF4 as a target for future anticancer therapies.

Changes in cell volume and internal sodium concentration in HeLa cells during exponential growth and following lonidamine treatment.

Cell volumes decreased in HeLa cells as a function of time after seeding during exponential growth. Cell volume distributions revealed the presence of two cell populations in all stages of growth. When cells approached confluence, the ratio of the two populations abruptly shifted towards that characterised by the smallest volume. Percentages of G1-, S- and G2 + M-phase cells were also measured and it was found that G1 frequency increased as a function of cell density during exponential growth. Intracellular sodium concentration, [Na]i was monitored by 23Na NMR in the presence of 5 mM dysprosium (III) tripolyphosphate. [Na]i increased from 22.8 to 59.0 mM in cells from the second to the seventh day after seeding. Treatment with lonidamine, an antitumoral drug that it is known to slow down cell growth by affecting aerobic glycolysis, produced a complete block of cell progression after a few days of treatment. The progression of cell volume distributions towards smaller volumes and the increase in internal sodium concentration as a function of time after seeding were also affected by the drug. These phenomena were related to the existence of a subpopulation of mitotically inactive G1-phase cells during exponential growth, pointing out that a density-dependent cellular mechanism regulates the cell cycling in HeLa cells.

Galectin-3 overexpression protects from cell damage and death by influencing mitochondrial homeostasis.

Galectins are a family of proteins involved in several cell processes, including their survival and death. Galectin-3 has in particular been described as an anti-apoptotic molecule entangled with a number of subcellular activities including anoikis resistance. In this work we partially address the mechanisms underlying this activity pointing at two key factors in injury progression: the alteration of mitochondrial membrane potential and the formation of reactive oxygen species. Overexpression of galectin-3 appears in fact to exert a protective effect towards both these events. On the basis of these data, we propose a reappraisal of the role of galectin-3 as a regulator of mitochondrial homeostasis.

Protection against apoptosis by monoamine oxidase A inhibitors.

Several lines of evidence have been accumulating indicating that an important role may be played by mitochondrial homeostasis in the initiation phase, the first stage of apoptosis. This work describes the results obtained by using different inhibitors of monoamine oxidases (MAO), i.e. pargyline, clorgyline and deprenyl, on mitochondrial integrity and apoptosis. Both pargyline and clorgyline are capable of protecting cells from apoptosis induced by serum starvation while deprenyl is ineffective. These data represent the first demonstration that MAO-A inhibitors may protect cells from apoptosis through a mechanism involving the maintenance of mitochondrial homeostasis.

Antioxidant N-acetyl-cysteine increasing cell adhesion capability could facilitate the biocompatibility processes.

Cell adhesion plays an important role in several cell processes and functions, including differentiation, proliferation and death. An important role for cell attachment to medical devices in biocompatibility studies has also been hypothesized. In this paper we report that the use of the antioxidant drug N-acetyl-cysteine is capable of increasing the adhesion properties of epithelial cells in culture. This is associated with a modification of specific cytoskeletal element assembly, such as microfilament system molecules. In contrast, no quantitative alterations in the expression of certain surface receptors for extracellular matrix molecules, such as VLA2, VLA3 and VLA6, are found. These data seem to indicate that intracellular oxidative balance, in particular of thiol groups, could play a key role in the cell adhesion properties and that N-acetyl-cysteine treatment, acting as 'thiol supply', could be of importance in several circumstances, including biocompatibility of medical devices.

The effect of oxygen on the growth and cell morphology of Helicobacter pylori.

The in vitro effect of progressive oxygen decrease on the growth and morphology of Helicobacter pylori was studied. H. pylori ATCC 43,504 was used for the experiments. The strain inoculated in Brucella broth plus fetal calf serum was incubated under a controlled atmosphere with oxygen concentration from 5 to 0%. CFU ml-1 and bacterial morphology were detected at the time of spreading and at 24 h, 72 h, 7 days and 14 days. A detailed ultrastructural investigation of the bacterial cells, grown in different experimental conditions, was performed by scanning electron microscopy. Oxygen deprivation produced a rapid reduction of CFU ml-1. In particular, a significant reduction of viable bacteria was recorded at 72 h of incubation in the presence of 1% oxygen and anaerobiosis, and 0 CFU ml-1 was found after 7 days of incubation at the above mentioned oxygen concentrations. The coccoid phenotype was already prevalent after 24 h of incubation with a progressive tendency to aggregate in clusters. These clusters were progressively larger, depending on the reduction of oxygen concentration, since the aggregation phenomenon can be the expression of a hypothesized mechanism of protection among bacterial cells.

Apoptosis and growth inhibition of squamous carcinoma cells treated with interferon-alpha, IFN-beta and retinoic acid are associated with induction of the cyclin-dependent kinase inhibitor p21.

Recent studies have revealed promising leads on the potential of interferons (IFNs) in combination with retinoids in solid tumor therapy. The role of IFN-alpha and retinoic acid (RA) in cervical cancer is currently under active study. Because preclinical and clinical data on IFN-beta in combination with retinoids show promising results against breast carcinoma, we analysed the anti-proliferative effect of human recombinant IFN-beta alone or in combination with all-trans RA on two human squamous cervical carcinoma cell (SCC) lines (ME180 and SiHa). The two cell lines differ in their sensitivity to the anti-proliferative effects of the different agents and their combination: i) both cell lines were more responsive to IFN-beta than to IFN-alpha2b; ii) combined treatment with RA increases the growth inhibitory effect of the single agents in ME180, but not in SiHa; iii) the antiproliferative effect correlates with the induction of apoptosis. We suggest as a possible mechanisms of action that interferon regulatory factor-1 (IRF-1), a transcription factor which belongs to the IFN machinery, and the cyclin-dependent kinase inhibitor (CDKi) p21 can be involved in cellular growth inhibition and in the induction of apoptosis. These results support the use of IFN-beta in further clinical investigation possibly in combination with retinoids.

Okadaic acid induces changes in the organization of F-actin in intestinal cells.

Okadaic acid, a polyether fatty acid associated with diarrhetic seafood poisoning, is capable of inhibiting protein phosphatases 1 and 2A which are considered among the major protein phosphatases in the cytosol of mammalian cells. One of the substrates for these phosphatases has been reported to be the cytoskeleton. In this paper, we focused on the effects of okadaic acid in intestinal cells, the more physiological target for this toxin. By fluorescence and scanning electron microscopy, we evidenced a dose- and time-dependent effect on F-actin which preceded any detectable change of tubulin and vimentin network. By a flow cytometric approach, we observed that plasma membrane permeability and transmembrane potential, two indicators of early cell damage or activation, respectively, remained unaffected in intoxicated cells. The present data strongly support the theory that actin is one of the main cytosolic targets for the phosphatases inhibited by okadaic acid in intestinal cells.

The mode of action of the antitumor agent lonidamine involves calcium-associated actin filament rounding.

The action of Lonidamine (LND), a dichlorinated derivative of indazole-3-carboxylic acid, on the membrane and cytoskeleton of Ehrlich tumor cells was investigated. A remarkable alteration in the molecular organization of the plasma membrane was observed. In particular, changes of plasma membrane and mitochondrial membrane protein distribution were induced by the drug. These membrane alterations were positively related to a rearrangement of microfilaments. In particular, characteristic ring-like structures of actin filaments were observed after 8 h of LND treatment. Intracellular calcium imbalance appeared-to be necessary to produce such peculiar structures. In fact, the administration of a calcium ionophore prevented the actin modifications induced by LND treatment while the simultaneous exposure to the antineoplastic drug verapamil, also considered a calcium channel blocker, was ineffective. The results reported herein suggest that the cytoskeleton as well as cell membranes might be involved in the cytotoxic action of LND and that they could share a common mechanism related to the calcium homeostasis. Moreover, Ehrlich tumor cells display a specific, peculiar rearrangement of F-actin filaments which can be considered as a marker effect of LND.

The cytoskeleton as a subcellular target of the antineoplastic drug lonidamine.

Lonidamine (LND), a dichlorinated derivative of indazole-3-carboxylic acid, has proved to exert a powerful antiproliferative effect and to impair the energy metabolism of normal and neoplastic cells. A target effect of the drug on the cell membrane structure was hypothesized. Thus, in order to elucidate better the mechanism of action of LND, the drug effects on the cell surface as well as on main cytoskeletal elements, i.e. actin microfilaments, microtubules and intermediate filaments, were investigated. In particular, an immunocytochemical and ultrastructural study was performed using two different cell lines: epithelial squamous carcinoma (A431) and melanoma (M14) cells. Treatment with 0.8 mM LND for 8 hr induced a remarkable rearrangement of the F-actin molecules with the disappearance of the stress fibers. As far as microtubules are concerned, formation of perinuclear patches of tubulin were detected after LND treatment. Intermediate filaments appeared to be differently affected by LND in the two cell types. Such changes were detected as an early phenomenon and the extent of the effects observed was positively related to the cell surface alterations and to the loss of cell viability, suggesting that the cytoskeletal elements might represent an additional target in the mechanisms of cytotoxic action of LND.

Modulation of the effects of the hyperthermic treatment by N-methylformamide on a human melanoma cell line.

The modulatory activity of the polar solvent N-methylformamide (NMF) on the effects of hyperhermic treatment was investigated on a human melanoma cell line (M14). Cells treated with NMF alone (1% for 20 h), hyperthermia (Hyp) alone (42.5 degrees C for 2 h) and with the two different sequences of treatment (NMF-->Hyp and Hyp-->NMF) were analysed by scanning electron microscopy and fluorescence microscopy. Moreover, their clonogenic efficiency and adherence properties were assessed. The results obtained can be summarized as follows. (a) The sequence Hyp-->NMF appeared to be more cytotoxic than the reverse sequence or NMF and Hyp given alone. (b) Heat induced cell swelling and detachment from the substrate. The pretreatment with the polar solvent was capable of preventing such alterations. (c) Fluorescence microscopy revealed remarkable changes induced by hyperthermia on actin network, vimentin distribution and vinculin expression. NMF administration proved to be capable of modulating these changes. In particular, the actin and vimentin networks showed a quite normal arrangement in NMF-->Hyp treated cells and very altered patterns in cells treated with the reverse sequence. Concerning the effects on the adhesion plaques, revealed by vinculin labeling, a considerable increase in the expression of these structures was observed after NMF treatment. (d) A remarkable increase of the attachment to collagen I and laminin molecules was revealed in NMF treated cells, whereas heat exposure reduced the number of adherent cells. Considered all together, the results obtained indicate that the administration of NMF after hyperthermia enhances the cytotoxic effect and modifies cell adherence properties, responsible for dissemination and metastasis.