Gender, migration and violence among third country nationals accessing the sexual health clinic in Malta.

BACKGROUND: In 2020, the number of displaced people worldwide reached 41.3 million (IOM, 2020). Among them, are many migrants and refugees at risk of sexual and gender-based violence (SGBV). Healthcare providers have a key role to play in identifying migrant victims/survivors of violence. OBJECTIVES: This paper seeks to assess STIs prevalence, sexual health and sexual violence among third country nationals (TCNs) attending the GUC in Malta. METHODS: This is a mixed methods study carried out at the Genitourinary Clinic (GUC), which is the only public sexual health clinic in Malta. Demographic data, sexual history and diagnoses of patients attending the GUC between January 2018 and December 2019 were collected and retrospectively analysed. A SGBV risk assessment was performed through a semi-structured questionnaire. RESULTS: In the 24-month study period, a total of 12 654 patients accessed the GUC in Malta. Demographic data were collected on age, gender, nationality, marital status and sexual orientation. 16.4% (n = 2064) of these were extra-European migrants, predominantly male. 80 different nationalities were recorded, with the 5 most common being Nigerian, Filipino, Libyan, Syrian and Brazilian. The average age was 32.6 years. Over 110 sex workers were visited at the GUC in the study period - 20 were foreign, primarily from China. The presence of a 'massage parlour owner' during consultation, lack of control over passports and other factors were identified as warning signs of trafficking. 5 cases of sexual violence and forced prostitution involving girls from Sub-Saharan Africa and, in 2 cases, boys recently arrived in Malta by boat, were encountered. 6 African women accessing the service exhibited a type of female genital mutilation (FGM). CONCLUSIONS: Migration, sexual health and SGBV overlap in important ways. Further research and training in SGBV and migration in the healthcare setting and awareness-raising about existing services among the migrant population are required.

Glypican-3 modulates BMP- and FGF-mediated effects during renal branching morphogenesis.

The kidney of the Gpc3-/ mouse, a novel model of human renal dysplasia, is characterized by selective degeneration of medullary collecting ducts preceded by enhanced cell proliferation and overgrowth during branching morphogenesis. Here, we identify cellular and molecular mechanisms underlying this renal dysplasia. Glypican-3 (GPC3) deficiency was associated with abnormal and contrasting rates of proliferation and apoptosis in cortical (CCD) and medullary collecting duct (MCD) cells. In CCD, cell proliferation was increased threefold. In MCD, apoptosis was increased 16-fold. Expression of Gpc3 mRNA in ureteric bud and collecting duct cells suggested that GPC3 can exert direct effects in these cells. Indeed, GPC3 deficiency abrogated the inhibitory activity of BMP2 on branch formation in embryonic kidney explants, converted BMP7-dependent inhibition to stimulation, and enhanced the stimulatory effects of KGF. Similar comparative differences were found in collecting duct cell lines derived from GPC3-deficient and wild type mice and induced to form tubular progenitors in vitro, suggesting that GPC3 directly controls collecting duct cell responses. We propose that GPC3 modulates the actions of stimulatory and inhibitory growth factors during branching morphogenesis.

Glypican-3-deficient mice exhibit developmental overgrowth and some of the abnormalities typical of Simpson-Golabi-Behmel syndrome.

Glypicans are a family of heparan sulfate proteoglycans that are linked to the cell surface through a glycosyl-phosphatidylinositol anchor. One member of this family, glypican-3 (Gpc3), is mutated in patients with the Simpson-Golabi-Behmel syndrome (SGBS). These patients display pre- and postnatal overgrowth, and a varying range of dysmorphisms. The clinical features of SGBS are very similar to the more extensively studied Beckwith-Wiedemann syndrome (BWS). Since BWS has been associated with biallelic expression of insulin-like growth factor II (IGF-II), it has been proposed that GPC3 is a negative regulator of IGF-II. However, there is still no biochemical evidence indicating that GPC3 plays such a role.Here, we report that GPC3-deficient mice exhibit several of the clinical features observed in SGBS patients, including developmental overgrowth, perinatal death, cystic and dyplastic kidneys, and abnormal lung development. A proportion of the mutant mice also display mandibular hypoplasia and an imperforate vagina. In the particular case of the kidney, we demonstrate that there is an early and persistent developmental abnormality of the ureteric bud/collecting system due to increased proliferation of cells in this tissue element. The degree of developmental overgrowth of the GPC3-deficient mice is similar to that of mice deficient in IGF receptor type 2 (IGF2R), a well characterized negative regulator of IGF-II. Unlike the IGF2R-deficient mice, however, the levels of IGF-II in GPC3 knockouts are similar to those of the normal littermates.

Syndecan-1 is up-regulated in ras-transformed intestinal epithelial cells.

The syndecans, a family of cell-surface heparan sulphate proteoglycans, have been proposed to mediate cellular interactions with extracellular effector molecules, such as growth factors and components of the extracellular matrix, during critical phases of development. Transcripts of all four syndecans are expressed at varying levels in the developing rat intestine and in a series of immature rat intestinal epithelial cell lines. In addition, we report the novel finding that, in the intestinal epithelial cell lines, expression of syndecan-1 transcript is up-regulated by transformation with activated H-ras. This is in contrast to other cell lines in which ras transformation is associated with a decrease in syndecan-1 levels. The observed increase in the syndecan-1 occurs as a result of increased transcription and can be correlated with the degree of transformation of the IEC-18 cells. Transformation is also associated with a decrease in apparent molecular weight and increased shedding of the proteoglycan into the culture medium. Increased shedding of syndecan-1 into the culture medium after transformation with H-ras may contribute to the disruption of proteoglycan interactions with the extracellular matrix, leading to alterations in cell adhesion and organization.

Reversible zinc exchange between metallothionein and the estrogen receptor zinc finger.

We report here the first demonstration that reversible metal exchange occurs between metallothionein (MT) and full-length estrogen receptor (ER). Specific binding of ER to estrogen response element is inhibited in the presence of 40 microM thionein and restored by 120 microM zinc. Moreover, ER in metal-depleted nuclear extracts exhibits reduced DNA binding which can be restored by 140 microM native MT. Hence, thionein inhibits DNA binding by abstracting zinc from functional ER while native MT is capable of restoring binding to metal-depleted extracts by donating metal to ER. This indicates MT may be an important physiological regulator of intracellular zinc and/or other metals.

Selection and characterization of verapamil-resistant multidrug resistant cells.

Multidrug resistant cells may become acutely sensitive to the calcium channel blocker verapamil, in spite of the fact that its accumulation by these cells is negligible. We selected verapamil-resistant mutants from multidrug resistant Chinese hamster ovary cells. Levels of P-glycoprotein expression and cross-resistance profiles remained unaltered in the verapamil-resistant multidrug resistant cells. As well, a photoactive verapamil analog specifically bound to P-glycoprotein in these cells. We had previously used a photoactive anthracycline to show that calcium antagonists and several anticancer drugs bind to P-glycoprotein at overlapping or interacting sites. Verapamil and its analogues no longer inhibit the binding of either anticancer drugs or calcium channel blockers to P-glycoprotein. Sequencing of P-glycoprotein revealed that no change had occurred in the coding sequence as a result of the selection procedure.

Mouse Paneth cell defensins: primary structures and antibacterial activities of numerous cryptdin isoforms.

Cryptdins are antimicrobial peptides of the defensin family that are produced by intestinal Paneth cells. mRNAs encoding 17 cryptdin isoforms have been characterized from a cDNA library generated from a single jejunal crypt. Six cryptdin cDNAs correspond to known peptides, and the remainder predict 11 novel Paneth cell defensins. Most cryptdin cDNAs have > or = 93% nucleotide sequence identity overall, except for cryptdin 4 and 5 cDNAs, whose respective mature peptide-encoding regions are only 74 and 78% identical to that of cryptdin 1. Cryptdin cDNAs differ at a small number of nucleotide positions: frequent substitutions were found in codons 38 and 52 of the propiece and in codons 68, 73, 76, 87, and 89 of the deduced peptides; cDNA clones with changes in codons 74, 83, and 88 were found, but there were fewer of these. The antimicrobial activities of cryptdins 1 to 6 were tested against Escherichia coli ML35 in two assays. In an agar diffusion assay, the potencies of cryptdins 1 to 3, 5, and 6 were approximately equivalent to that of rabbit neutrophil defensin NP-1 but cryptdin 4 was 30 times more active than NP-1. In a bactericidal assay system, cryptdins 1 and 3 to 6 were equally active at 10 micrograms/ml but cryptdin 2 and rabbit NP-1 were not active at this concentration. Since cryptdins 2 and 3 differ only at residue 10 (Thr and Lys, respectively), this amino acid appears to function in bactericidal interaction with E. coli. The demonstration that Paneth cells express a diverse population of microbicidal defensins further implicates cryptdins in restricting colonization or invasion of small intestinal epithelium by bacteria.

Activated ras and src induce CD44 overexpression in rat intestinal epithelial cells.

CD44 is an adhesion molecule that is involved in the progression of several tumor types, including those originating in the intestine. There are several alternatively spliced forms of CD44. Here we show that intestinal epithelial cells express the standard form of CD44 (CD44s). The same form of CD44 is found in IEC-18, a cell line derived from normal rat intestinal crypts. Upon transfection of IEC-18 cells with ras or src, two oncogenes that are frequently activated in intestinal tumors, a significant induction of CD44s is observed. A causal role for ras in this induction is shown by using IEC clones transfected with an inducible ras expression vector. The oncogene-transformed IEC clones display a high degree of hyaluronic acid-dependent cell-cell adhesion that is not observed in the parental IEC-18 cells suggesting that ras- and src-induced overexpression of CD44 can alter the adhesion properties of intestinal cells.

In vitro cDNA amplification from individual intestinal crypts: a novel approach to the study of differential gene expression along the crypt-villus axis.

The mouse small intestine is lined with a monolayer of continuously renewing epithelial cells. Cells of four distinct epithelial lineages are derived clonally from the stem cell zone, located near the crypt base, from which cells differentiate and migrate to the villus tip. The kinetics of the multilineage process are well understood. However, the molecular mechanisms underlying gene expression during lineage commitment and cell proliferation and differentiation remain obscure. A novel approach to the problem is presented here. Single intact epithelial crypts were isolated by incubation in ethylenediaminetetraacetic acid and mechanical vibration of everted mouse intestinal or colonic segments. Crypts isolated in this manner were suitable for mRNA-directed polymerase chain reaction, thus generating crypt epithelium-specific cDNA. The fidelity of transcript amplification was confirmed by Southern blot hybridization with cloned intestinal transcripts. To demonstrate the potential utility of crypt-specific cDNA, the amplified transcripts from a single jejunal crypt were used to construct a cDNA library, characterization of which revealed a high representation of cryptdin-1-related transcripts. This study presents a technique which will facilitate comprehensive analyses of gene expression in the differentiating mammalian intestine.

Fast kinetic analysis of drug transport in multidrug resistant cells using a pulsed quench-flow apparatus.

One major feature of multidrug resistance is the reduced cellular level of drugs maintained by MDR cells. Although there is now strong evidence that drugs are actively pumped out of MDR cells, transport experiments have indicated decreased initial rates of influx at the earliest times at which measurements could be made. We have used a pulsed quench-flow apparatus to study transport characteristics of colchicine resistant MDR cells on a very fast time scale. A rapid association of daunomycin with drug sensitive cells occurred within 0.11 sec. This association is virtually absent in MDR cells. In efflux experiments performed on the same rapid time scale, greater than 50% of daunomycin efflux occurred within 0.1 sec. No substantial efflux from B1, drug sensitive cells was observed. On the other hand, vinblastine accumulation by both cell types was similar for approx. 10 seconds. Thus, kinetically, not all drugs are handled in a similar fashion by MDR cells. The pulsed quench-flow apparatus was useful in making fast time measurements of drug influx and efflux and in demonstrating the differences between drug recognition patterns by MDR cells.

Equilibrium, kinetic and photoaffinity labeling studies of daunomycin binding to P-glycoprotein-containing membranes of multidrug-resistant Chinese hamster ovary cells.

The binding of daunomycin and its Bolton-Hunter derivative iodomycin to plasma membranes isolated from multidrug-resistant Chinese hamster ovary cells (CHO B30) and their drug-sensitive parents (B1) was investigated. The thermodynamics and kinetics of equilibrium binding monitored by fluorescence titrations and temperature-jump relaxation spectrometry were compared with the specificity of covalent photolabeling with [3H]daunomycin and [125I]iodomycin. The facts that the uptake of anthracycline from aqueous solution into the CHO membranes was not accompanied by any substantial increase of fluorescence anisotropy nor by any spectral shift of the fluorescence emission spectrum and that the partition ratio into the membrane was 20-30-fold higher when compared to a lecithin bilayer, provided evidence that the non-covalent drug binding sites are constituted by polar protein domains without any substantial contribution from the surrounding lipids. Photoaffinity labeling with nanomolar concentrations of anthracycline and equilibrium binding curves independently showed that a 150-170-kDa plasma membrane glycoprotein (P-glycoprotein), whose overexpression is the major difference between B1 and B30 membranes, provides the binding sites of highest affinity for daunomycin and iodomycin (K approximately equal to 4 x 10(7) M-1). Comparison of photolabeling and equilibrium data suggested that the same binding sites on P-glycoprotein were most probably being monitored. The photolabeling of P-glycoprotein by iodomycin was inhibited in a dose-dependent manner by other compounds to which multi-drug-resistant cells are either resistant or collaterally sensitive with the following orders of effectiveness: vinblastine greater than verapamil greater than nitrendipine greater than daunomycin much greater than colchicine. Temperature-jump experiments covering the time range of 1 microseconds to 1 s revealed a single concentration-dependent relaxation time of 10-30 microseconds. The association of daunomycin with its binding sites in the membranes was found to be a diffusion-controlled process with kon rates of 2-4 X 10(9) M-1 s-1. Therefore, the selectivity of drug binding was entirely reflected in the dissociation rates.

Preparation and utility of a radioiodinated analogue of daunomycin in the study of multidrug resistance.

Anthracyclines are an important class of cytotoxic drugs that are frequently used in cancer chemotherapy, especially in acute leukemia. The pharmacokinetics and disposition of these compounds in whole animals and in cells have been studied employing 3H-labeled forms. However, their usefulness is limited by their low specific activities and the low energy of 3H. Therefore, we have labeled daunomycin using 125I-Bolton-Hunter reagent. The resultant anthracycline analogue, iodomycin, has a specific activity of approximately 2000 Ci/mmol. Although this compound was 10-fold less toxic to normal cells than daunomycin, multidrug-resistant cells were cross-resistant to it. Like other drugs to which these cells are cross-resistant, its accumulation by them was greatly reduced, compared with drug-sensitive cells. We have also utilized this compound in photoaffinity labeling experiments to identify its target in multidrug-resistant cells. We observed the specific binding of iodomycin to P-glycoprotein in membrane vesicles as well as in intact cells, thereby directly demonstrating that this protein specifically binds anthracyclines as well as Vinca alkaloids.

Action of calcium antagonists on multidrug resistant cells. Specific cytotoxicity independent of increased cancer drug accumulation.

Previous studies have shown that calcium channel blockers can overcome, at least partially, multidrug resistance (MDR). This study was undertaken to attempt to determine the mechanisms whereby these agents bring about this effect. Their influence on the uptake and retention of several cancer drugs and on the toxic actions of these compounds was assessed employing MDR cell lines from several species. The wild-type drug sensitive parent cells proved to be more susceptible than the multidrug resistant variants to the effects of calcium channel blockers on cancer drug accumulation. This was shown for verapamil, nifedipine and the calmodulin inhibitor trifluoperazine acting on human, mouse and Chinese hamster ovary (CHO) cell lines. The enhancement of drug accumulation by calcium antagonists was similar to that caused by non-ionic detergents. Furthermore, verapamil was unable to alter 45Ca2+ accumulation in sensitive or resistant cells, suggesting that these agents act in a calcium-independent manner. Verapamil accumulation in multidrug resistant cells was reduced compared to sensitive cells. In spite of this reduced accumulation, however, verapamil alone was much more toxic to multidrug resistant cells than to the sensitive cells. This suggests that calcium channel blockers are specifically toxic to MDR cells by virtue of an interaction with the MDR cell surface distinct from that involved in promoting cellular accumulation.