Storage of evidence and delayed reporting after sexual assault: Rates and impact factors on subsequent reporting.

BACKGROUND: Sexual assault (SA) is alarmingly prevalent, yet reporting rates remain disproportionately low. Forensic examinations (FE) play a crucial role in both immediate medical care and evidence collection, yet many victims/survivors may not report the crime initially, leading to the loss of vital forensic evidence. The storage of evidence "Option 3″ care alternative provides post-SA care including FE without initial police involvement. METHODS: This is a cross-sectional study analysing the attendances of people who chose to store evidence at the Dublin Sexual assault Treatment Unit (SATU) between January 1, 2017 and December 31, 2023. RESULTS: There were 238 storage of evidence FEs ('Option 3') performed during the study period, which represented 12.8 % of all FEs. The majority identified as female (89.1 %), with an average age of 26.6 years. 31.9 % attended within 24 h of the incident, and 51.3 % self-referred. Most assaults occurred over weekends (64.7 %), with alcohol consumption reported in 82.2 % of cases and drug-facilitated SA concerns in 20.2 %. Genital injuries were present in 17.9 % of females and 19 % of males. Those that availed of storage of evidence (compared with those who initially reported to the police) were significantly more likely to have consumed alcohol (p < 0.001) and the assault was more likely to have occurred indoors (p = 0.002). There was no significant difference in care option choice for those 'unsure' of the assault occurrence (p = 0.353). Among storage of evidence cases, 20.2 % subsequently reported to the police, with females more likely to report (p = 0.02), while people who were uncertain whether an assault had occurred were less likely to report (p = 0.04). Genital injury (p = 0.822), victim-assailant relationship (p = 0.465), assault location (p = 0.487), and substance consumption (p = 0.332) did not significantly affect subsequent reporting rates. CONCLUSIONS: The availability of storage of evidence has afforded people the opportunity to access prompt, responsive SATU care including collection of forensic evidence which may have significant evidential value. This approach provides further opportunity for comprehensive detection of a crime, even if reporting to the police is delayed.

Mast cells, basophils and eosinophils: From allergy to cancer.

Basophils, eosinophils and mast cells were first recognized by Paul Ehrlich in the late 19th century. These cells have common, but non-redundant roles, in the pathogenesis of allergic diseases and in the protection against parasites. Nevertheless, in virtue of their shared-adeptness to produce a huge variety of immunological mediators and express membrane-bound receptors, they are able to interact with immune and non-immune components of the tissue microenvironment, contributing to the regulation of tissue homeostasis and immune response while participating to further deregulation of tissues transforming into neoplasia.

The Role of Mast Cells in Molding the Tumor Microenvironment.

Mast cells (MCs) are granulocytic immune cells that reside in tissues exposed to the external environment. MCs are best known for their activity in allergic reactions, but they have been involved in different physiological and pathological conditions. In particular, MC infiltration has been shown in several types of human tumors and in animal cancer models. Nevertheless, the role of MCs in the tumor microenvironment is still debated because they have been associated either to good or poor prognosis depending on tumor type and tissue localization. This dichotomous role relies on MC capacity to secrete a broad spectrum of molecules with modulatory functions, which may condition the final tumor outcome also promoting angiogenesis and tissue remodeling. In this review, we analyze the multifaceted role of mast cell in tumor progression and inhibition considering their ability to interact with: i) immune cells, ii) tumor cells and iii) the extracellular matrix. Eventually, the current MC targeting strategies to treat cancer patients are discussed. Deciphering the actual role of MCs in tumor onset and progression is crucial to identify MC-targeted treatments aimed at killing cancer cells or at making the tumor vulnerable to selected anti-cancer drugs.

Mast cells are critically involved in serum-mediated vascular leakage in chronic urticaria beyond high-affinity IgE receptor stimulation.

BACKGROUND: Chronic urticaria (CU) is one of the most common skin disorders whose pathogenic mechanisms are not fully clarified. Autoimmune aetiology can be ascribed to 45% of patients with CU, and basophil histamine release is positive in 40% of cases. Our aim was to use a novel approach to evaluate the serum permeabilizing effect to identify the mediators of endothelial cell (EC) leakage and to define the role of mast cells (MCs) in the process. METHODS: Permeabilizing activity of sera from 19 patients with CU and 11 healthy blood donors was evaluated by measuring serum-induced degranulation of two MC lines, expressing (LAD2) or lacking (HMC-1) the IgE receptor. Mast cell supernatant (SN) was then incubated with an EC monolayer, and endothelial permeability was evaluated by Fluorescein isothiocyanate-bovine serum albumin leakage in a transwell system. RESULTS: All 19 patient sera failed to induce direct EC leakage, but 15/19 and 17/19 promoted degranulation of HMC-1 and LAD2, respectively. Interestingly, 85% of autologous serum skin test-negative sera were able to cause MC degranulation. Also, 17/19 SNs from HMC-1 and all SNs from LAD2 incubated with CU sera increased endothelial permeability. Endothelial cell leakage remained unchanged after Ig depletion and was prevented by antihistamine, platelet-activating factor or leukotriene antagonist. CONCLUSIONS: Our study shows that CU sera are able to degranulate MCs through an IgE- and IgG-independent mechanism. The nature of histamine-releasing factors involved is still unclear, but our finding opens new ways to the understanding of the pathogenesis of CU, particularly in patients not showing circulating autoantibodies to FcεRI or IgE.

Outside inside signalling in CD40-mediated B cell activation.

CD40 is a member of the growing tumor necrosis factor receptor (TNF-R) family of molecules, and has been shown to play important roles in T cell-mediated B lymphocyte activation. Ligation of B cell CD40 by CD154 expressed on activated T cells stimulates B cell proliferation, differentiation, isotype switching, upregulation of surface molecules contributing to antigen presentation, development of the germinal center, and the humoral memory response. The present review will summarize recent literature data on the various CD40 signalling pathways, which involve both the TNF-R associated factors (TRAFs) and additional signalling proteins, and lead to activation of kinases and transcription factors.

Mitochondrial localization of APE/Ref-1 in thyroid cells.

Mutations of mitochondrial DNA (mtDNA) are associated with different human diseases, including cancer and aging. Reactive oxygen species produced during oxidative phosphorylation are a major source of mtDNA damage. It is not clear, however, whether DNA repair mechanisms, able to abolish effects due to oxidative damage, are present in mitochondria. APE/Ref-1 is a nuclear protein possessing both redox activity (by which activates, "in vitro", the DNA-binding functions of several transcription factors) and DNA repair activity over apurinic/apyrimidinic sites. Immunohistochemical evidences indicate that in follicular thyroid cells, APE/Ref-1 is located in both nucleus and cytoplasm. Electronmicroscopy immunocytochemistry performed in the rat thyroid FRTL-5 cell line, indicates that part of the cytoplasmatic APE/Ref-1 is located in mitochondria. The presence of APE/Ref-1 inside mitochondria is further demonstrated by western blot analysis after cell fractionation. In the Kimol cell line (which is derived from FRTL-5, transformed by the Ki-ras oncogene) the amount of mitochondrial APE/Ref-1 is reduced by three to fourfold with respect to the normal FRTL-5 cells. These results suggest that: (i) a machinery capable of repairing DNA damaged by oxidative stress is present in mitochondria and (ii) mtDNA repair mechanisms may be impaired during cell transformation.

Monoclonal antibody detection of naphthalene dioxygenase from Pseudomonas aeruginosa 2NR.

A monoclonal antibody, designated mAb alpha(CT), was generated against a peptide of the ISP(NAP) alpha-subunit of the naphthalene dioxygenase (NDO) enzyme of Pseudomonas aeruginosa. Since NDO expression is induced by aromatic hydrocarbons, its detection is important as a tool for environmental biomonitoring. This antibody is highly specific and works well both in an indirect ELISA assay and Western Blot analysis, allowing the detection of Pseudomonas spp. expressing the NDO inducible enzyme. The detection threshold for the ELISA assay developed in this work was 10(4) colony forming units (cfu) per ml. Thus, this mAb could represent a powerful tool to test for pollutants in soil, groundwater, and other natural environments.

Humanized antibodies as potential drugs for therapeutic use.

Antibodies have been used therapeutically to treat a variety of clinical conditions. The introduction of monoclonal antibodies (mAb) and, recently, engineered antibodies has greatly refined and expanded the therapeutic potential of this modality of treatment. Expanded use will depend on improvement in their efficacy (affinity and specificity), demonstration of their safety, and reduction of their immunogenicity depending on the size, suboptimal biodistribution and pharmacokinetics. To surmount these problems the molecules have to be redesigned and the basic issues of how monoclonal antibodies kill cells reinvestigated. The review will survey the literature for humanized antibodies in clinical trials and the perspective of the use of mAbs or engineered antibodies in clinical practice.

TSH controls Ref-1 nuclear translocation in thyroid cells.

Ref-1 (called also APE) is a bifunctional protein playing a role in a large variety of cell functions. It is a major member of the DNA base excision repair system. Moreover, through reduction of cysteine residues, Ref-1 controls the activity of several transcription factors. It has been previously demonstrated that TSH up-regulates Ref-1 gene expression in thyroid cells. By using the rat FRTL-5 cell line, we demonstrate that TSH controls Ref-1 intracellular localization. Western blot experiments indicate that addition of TSH to the culture medium increases the Ref-1 cytoplasm-to-nucleus translocation. This phenomenon occurs at early times of TSH stimulation and is not dependent on protein neosynthesis. The Ref-1 cellular compartmentalization was also investigated in human thyroid tumors. A Ref-1 nuclear/cytoplasmic ratio difference between normal and cancerous thyroid tissues was observed. These results suggest that Ref-1 localization may have a critical role in the control of thyroid cell functions.

An 'environment to nucleus' signaling system operates in B lymphocytes: redox status modulates BSAP/Pax-5 activation through Ref-1 nuclear translocation.

The Ref-1 (also called APE or HAP1) protein is a bifunctional enzyme impacting on a wide variety of important cellular functions. It acts as a major member of the DNA base excision repair pathway. Moreover, Ref-1 stimulates the DNA-binding activity of several transcription factors (TFs) through the reduction of highly reactive cysteine residues. Therefore, it represents a mechanism that regulates eukaryotic gene expression in a fast way. However, it has been demonstrated that external stimuli directly act on Ref-1 by increasing its expression levels, a time-consuming mechanism representing a paradox in terms of rapidity of TF regulation. In this paper we demonstrate that this is only an apparent paradox. Exposure of B lymphocytes to H(2)O(2)induced a rapid and sustained increase in Ref-1 protein levels in the nucleus as evaluated by both western blot analysis and by pulse-chase experiments. A time course, two color in situ immunocytochemistry indicated that the up-regulation of Ref-1 in the nucleus at <30 min was primarily the consequence of translocation of its cytoplasmic form. This early nuclear accumulation is effective in modulating the DNA-binding activity of the B cell-specific activator protein BSAP/Pax-5. In fact, EMSA experiments demonstrate that a transient interaction with Ref-1 up-regulates the DNA-binding activity of BSAP/Pax-5. Moreover, in a co-transfection experiment, Ref-1 increased the BSAP/Pax-5 activating effect on an oligomerized BSAP/Pax-5 binding site of the CD19 promoter by 5- to 8-fold. Thus, Ref-1 mediates its effect by up-regulating the DNA-binding activity of BSAP/Pax-5, accounting for a new and fast outside/inside pathway of signaling in B cells.

Comparative stability analysis of the thyroid transcription factor 1 and Antennapedia homeodomains: evidence for residue 54 in controlling the structural stability of the recognition helix.

The thyroid transcription factor 1 homeodomain (TTF-1 HD) shows a peculiar DNA-binding specificity which is partially dictated by several amino acids of the recognition helix. TTF-1 preferentially recognizes sequences containing the 5'-CAAG-3' core motif while most other homeodomains, such as Antennapedia (Antp), recognizes sites containing the 5'-TAAT-3' core motif. Since phenomena of 'induced fit' may occur during protein/DNA interaction, a primary role for high affinity binding and target discrimination has to be searched in the effect played by subtle structural determinants in these proteins. By using spectroscopic analysis in aqueous solution, we compared the structural stability of TTF-1 and Antp homeodomains. Although the three-dimensional structural architecture of homeodomains is conserved, some differences are detectable in terms of their structural stability. At 24 degrees C the TTF-1 HD is less structured than the Antp HD with 24 and 34% of the residues in the alpha-helical conformation, respectively. This poor folded structure reflects into different thermal and isothermal stability between the two homeodomains. TTF-1 HD exhibits a Tm of 39 degrees C and is stabilized by a delta GDH2O of +1487 cal/mol, calculated by Urea unfolding, while Antp HD exhibits a Tm of 48 degrees C and is stabilized by a delta GDH2O of +2742 cal/mol. By using mutants of both TTF-1 and Antp HDs we demonstrate that one of the major determinants in controlling the structural stability of the recognition helix is the residue at position 54. Since previous studies have shown that also residue at position 56 is involved in stabilization of the recognition helix, we conclude that the structure of this critical element is controlled by an interplay between residues at position 54 and 56 of the homeodomain.

Adhesion to fibronectin promotes the activation of the p125(FAK)/Zap-70complex in human T cells.

The beta1 integrins are a family of heterodimeric adhesion receptors involved in cell-to-cell contacts and cell-to-extracellular matrix interactions. Through their adhesive role, integrins participate in transduction of outside/inside signals and contribute to trigger a multitude of cellular events such as differentiation, cell activation, and motility. The fibronectin integrin receptors, alpha4beta1 and alpha5beta1, can function as costimulatory molecules in T-cell receptor (TCR)-dependent T-cell activation. In the current study the Jurkat T-cell line was used as a model system to investigate the TCR-independent role of cell adhesion to fibronectin in the activation of Zap-70, a central molecule in the signalling events in T cells. Upon adhesion to plastic immobilized fibronectin but not to bovine serum albumin (BSA) the phosphorylation of p125FAK, a protein kinase that localizes to focal adhesion sites, was induced. Moreover, clustering of fibronectin receptors led to the detection of a p125FAK/Zap-70 complex. Finally, while the complex between fak-B, another protein kinase localized to focal adhesion sites, and Zap-70 was detected in cells plated either on BSA or on fibronectin, the formation of the p125FAK/Zap-70 complex appeared specifically induced following fibronectin-mediated integrin clustering. These data suggest the existence of a high degree of specificity when the members of the beta1 integrin family mediate signalling pathways in T cells.

Structural defects of a Pax8 mutant that give rise to congenital hypothyroidism.

Pax proteins are transcriptional regulators that play important roles during embryogenesis. These proteins recognize specific DNA sequences via a conserved element: the paired domain (Prd domain). The low level of organized secondary structure, in the free state, is a general feature of Prd domains; however, these proteins undergo a dramatic gain in alpha-helical content upon interaction with DNA ('induced fit'). Pax8 is expressed in the developing thyroid, kidney and several areas of the central nervous system. In humans, mutations of the Pax8 gene, which are mapped to the coding region of the Prd domain, give rise to congenital hypothyroidism. Here, we have investigated the molecular defects caused by a mutation in which leucine at position 62 is substituted for an arginine. Leu62 is conserved among Prd domains, and contributes towards the packing together of helices 1 and 3. The binding affinity of the Leu62Arg mutant for a specific DNA sequence (the C sequence of thyroglobulin promoter) is decreased 60-fold with respect to the wild-type Pax8 Prd domain. However, the affinities with which the wild-type and the mutant proteins bind to a non-specific DNA sequence are very similar. CD spectra demonstrate that, in the absence of DNA, both wild-type Pax8 and the Leu62Arg mutant possess a low alpha-helical content; however, in the Leu62Arg mutant, the gain in alpha-helical content upon interaction with DNA is greatly reduced with respect to the wild-type protein. Thus the molecular defect of the Leu62Arg mutant causes a reduced capability for induced fit upon DNA interaction.

The overlooked "nonclassical" functions of major histocompatibility complex (MHC) class II antigens in immune and nonimmune cells.

Besides their "classical" antigenic peptide-presenting activity, major histocompatibility complex (MHC) class II antigens can activate different cellular functions in immune and nonimmune cells. However, this "nonclassical" role and its functional consequences are still substantially overlooked. In this review, we will focus on these alternative functional properties of MHC class II antigens, to reawaken attention to their present and foreseeable immunobiologic and pathogenetic implications. The main issues that will be addressed concern 1) the role of MHC class II molecules as basic components of exchangeable oligomeric protein complexes with intracellular signaling ability; 2) the nonclassical functions of MHC class II antigens in immune cells; 3) the pathogenetic role of MHC class II antigens in inflammatory/autoimmune and infectious disease; and 4) the functional role of MHC class II antigens in solid malignancies.

Ref-1 controls pax-8 DNA-binding activity.

Redox potential controls the DNA-binding activity of several transcription factors. In some cases, the regulation of DNA-binding activity by the redox state is mediated by the Ref-1 nuclear protein. In this study, we demonstrate that Ref-1 is able to induce "in vitro" the DNA-binding activity of the Pax-8 paired domain. In co-transfection experiments, Ref-1 increases the Pax-8 activating effect on thyroglobulin promoter. Moreover, immunoreactivity data suggest that, in nuclear extracts of thyroid cells, the levels of Ref-1 correlate with the amounts of reduced Pax-8. Therefore, the regulation of the Pax-8 DNA-binding activity by redox potential, that we have demonstrated occurring "in vitro", could represent a means to control "in vivo" the function of Pax proteins. Alignment of the Paired domains sequences present in the Protein Data Bank demonstrates a strong conservation of Cys residues, suggesting that the redox regulation of the Paired domain DNA-binding activity is widely conserved along phylogenesis.

Redox potential controls the structure and DNA binding activity of the paired domain.

Pax proteins are transcriptional regulators controlling a variety of cell fates during animal development. This role depends on the intact function of the paired (Prd) domain that is able to recognize specific DNA sequences. The Prd domain is composed of two distinct helix-turn-helix subdomains, PAI and RED. Molecular functions of Pax proteins are subjected to different levels of regulation involving both pre-translational and post-translational mechanisms. By using Pax-5 and Pax-8 recombinant proteins, we demonstrate that the binding activity of the Prd domain is regulated through the oxidation/reduction of conserved cysteine residues. Mass spectrometry analysis and mutagenesis experiments demonstrate that the redox regulation is accomplished through the reversible formation of an intramolecular disulfide bridge involving the cysteines present in the PAI subdomain, whereas the RED subdomain appears quite insensitive to redox potential. Circular dichroism experiments indicate that only the reduced form of the Prd domain is able to undergo the proper conformational change necessary for sequence-specific DNA binding. Nuclear extracts from different cell lines contain an activity that is able to reduce the Paired domain and, therefore, to control the DNA binding activity of this protein. Immunodepletion of nuclear extracts demonstrate that the protein Ref-1 contributes to the redox regulation of the Prd DNA binding activity. Given the modular nature of the Prd domain and the independent DNA binding specificity of the PAI and RED subdomains, we propose that this control mechanism should be involved in "switching" among different DNA sequences and therefore different target genes.

Fibronectin binding promotes a PKC-dependent modulation of NF-kappa B in human T cells.

NF-kappa B was identified as one of the transcription factors leading to antigen-independent stimulation through activation of integrin receptors. This effect was dependent upon stimulation of alpha 4 beta 1 and alpha 5 beta 1 integrins, the major fibronectin-binding integrins of Jurkat T cells, since either RGD or CS-1 peptides at 10(-4) M could prevent NF-kappa B activation. At variance with fibroblasts and smooth muscle cells, in which only p50 and p65 components of the NF-kappa B complex are induced, adhesion of T cells to fibronectin resulted in a strong upregulation of p50 and c-Rel and in a partial increase in p65 activity. The upregulation of NF-kappa B activity was abrogated by calphostin C, an inhibitor of protein kinase C. Cell adhesion determined a strong reduction in the cytoplasmic levels of the NF-kappa B inhibitor I kappa B alpha, reduction that was prevented after treatment with calphostin C, suggesting that PKC-dependent I kappa B alpha phosphorylation might be involved in the upregulation of NF-kappa B.

Structural and functional properties of the N transcriptional activation domain of thyroid transcription factor-1: similarities with the acidic activation domains.

The thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor involved in the development of thyroid and lung. TTF-1 contains two transcriptional activation domains (N and C domain). The primary amino acid sequence of the N domain does not show any typical characteristic of known transcriptional activation domains. In aqueous solution the N domain exists in a random-coil conformation. The increase of the milieu hydrophobicity, by the addition of trifluoroethanol, induces a considerable gain of alpha-helical structure. Acidic transcriptional activation domains are largely unstructured in solution, but, under hydrophobic conditions, folding into alpha-helices or beta-strands can be induced. Therefore our data indicate that the inducibility of alpha-helix by hydrophobic conditions is a property not restricted to acidic domains. Co-transfections experiments indicate that the acidic domain of herpes simplex virus protein VP16 (VP16) and the TTF-1 N domain are interchangeable and that a chimaeric protein, which combines VP16 linked to the DNA-binding domain of TTF-1, undergoes the same regulatory constraints that operate for the wild-type TTF-1. In addition, we demonstrate that the TTF-1 N domain possesses two typical properties of acidic activation domains: TBP (TATA-binding protein) binding and ability to activate transcription in yeast. Accordingly, the TTF-1 N domain is able to squelch the activity of the p65 acidic domain. Altogether, these structural and functional data suggest that a non-acidic transcriptional activation domain (TTF-1 N domain) activates transcription by using molecular mechanisms similar to those used by acidic domains. TTF-1 N domain and acidic domains define a family of proteins whose common property is to activate transcription through the use of mechanisms largely conserved during evolutionary development.

Biphasic control of NF-kappa B activation induced by the triggering of HLA-DR antigens expressed on B cells.

The regulation of NF-kappa B activation following the triggering of HLA-DR antigens by mAb L243 has been studied at various times in Raji cells. Electrophoretic mobility shift assays demonstrated a strong increase of NF-kappa B DNA binding after triggering of HLA-DR antigens. Using TNF-alpha-activity neutralizing antibodies, the authors demonstrated that the upregulation of NF-kappa B was found to depend, at later time point, on an autocrine effect of TNF-alpha secreted following triggering of HLA-DR antigens. In contrast, it was found to be TNF-alpha independent in the early time point. Moreover, the upregulation of NF-kappa B binding activity is regulated by the triggering of selected epitopes of HLA-DR antigens. In fact, mAb L243 but not the staphylococcal superantigens, staphylococcal exotoxin toxic shock syndrome toxin-I or staphylococcal enterotoxin B, regulate the NF-kappa B binding activity.