An Exploration of New Zealand Crown Prosecutors' Experiences of Working with Potentially Traumatic Material and Emotions in the Courtroom

The occupational exposure to trauma and its potential impacts among legal  professionals working in the criminal justice system is an area that  has historically been neglected and has only gained traction in recent  years. Crown prosecutors, as a subset of practising criminal lawyers in  New Zealand working with potentially traumatic material (PTM), are  arguably at heightened risk of vicarious trauma (VT) and the need to  engage in emotional labour (EL). The current study qualitatively  investigated New Zealand Crown prosecutors’ experiences of working with  PTM and emotions in their role through three key research questions: 1)  What are New Zealand Crown prosecutors’ experiences of working with PTM?  2) What sort of EL do Crown prosecutors experience, if any, through  working in the criminal courts? 3) What factors in their personal and  professional lives might exacerbate or alleviate VT symptoms? Nineteen  Crown prosecutors from four Crown Solicitor firms across New Zealand  participated in the study. The data was analysed using thematic analysis  to identify recurring themes across datasets. Crown prosecutors  outlined the various negative symptoms they experienced from their  work-related exposure to trauma (VT), as well as the different workplace  and personal factors that both protected and exacerbated such symptoms.  Further, they described routinely and mandatorily engaging in EL to  mask their emotions as a function of their professional duties. EL also  doubled as a protective measure for Crown prosecutors in the  difficulties they faced in their role. These findings add to the growing  body of literature on legal professionals which has preliminarily  indicated they are an at-risk population for the negative impacts of VT  and EL, which can be significant and enduring. More research must be  dedicated to this population to understand the unique etiological  pathways for both consequences of working with PTM and ultimately,  provide empirically-sound recommendations that proactively address these  occupational risks. </p

Determination of the CD148-Interacting Region in Thrombospondin-1

<div><p>CD148 is a transmembrane protein tyrosine phosphatase that is expressed in multiple cell types, including vascular endothelial cells and duct epithelial cells. Previous studies have shown a prominent role of CD148 to reduce growth factor signals and suppress cell proliferation and transformation. Further, we have recently shown that thrombospondin-1 (TSP1) serves as a functionally important ligand for CD148. TSP1 has multiple structural elements and interacts with various cell surface receptors that exhibit differing effects. In order to create the CD148-specific TSP1 fragment, here we investigated the CD148-interacting region in TSP1 using a series of TSP1 fragments and biochemical and biological assays. Our results demonstrate that: 1) CD148 binds to the 1^st type 1 repeat in TSP1; 2) Trimeric TSP1 fragments that contain the 1^st type repeat inhibit cell proliferation in A431D cells that stably express wild-type CD148 (A431D/CD148wt cells), while they show no effects in A431D cells that lack CD148 or express a catalytically inactive form of CD148. The anti-proliferative effect of the TSP1 fragment in A431D/CD148wt cells was largely abolished by CD148 knockdown and antagonized by the 1^st, but not the 2^nd and 3^rd, type 1 repeat fragment. Furthermore, the trimeric TSP1 fragments containing the 1^st type repeat increased the catalytic activity of CD148 and reduced phospho-tyrosine contents of EGFR and ERK1/2, defined CD148 substrates. These effects were not observed in the TSP1 fragments that lack the 1^st type 1 repeat. Last, we demonstrate that the trimeric TSP1 fragment containing the 1^st type 1 repeat inhibits endothelial cell proliferation in culture and angiogenesis <i>in vivo</i>. These effects were largely abolished by CD148 knockdown or deficiency. Collectively, these findings indicate that the 1^st type 1 repeat interacts with CD148, reducing growth factor signals and inhibiting epithelial or endothelial cell proliferation and angiogenesis.</p></div

A CD148-interacting trimeric TSP1 fragment inhibits endothelial cell proliferation and angiogenesis.

<p><b>(A)</b> HRMEC cells were treated with the trimeric TSP1 fragment (1.5, 3.0, 6.0, 12.0 nM) containing the procollagen domain and the 1^st type 1 repeat or whole TSP1 (12 nM) and its effects on cell proliferation were assessed (left panel). The effects of CD148 knockdown were also assessed with 12 nM of the TSP1 fragment (right panel). The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. ** <i>P</i> < 0.05 Note: CD148 knockdown largely attenuates the activity of the TSP1 fragment to inhibit cell proliferation of HRMEC cells. <b>(B)</b> Upper panels: Gelfoam sponges loaded with vehicle or 100 ng VEGF plus or minus 100 pmol of the trimeric TSP1 fragment containing the procollagen domain and the 1^st type 1 repeat were subcutaneously implanted into the dorsal flank of either wild-type or CD148 knockout mice. At day 7, the mice were injected intravenously with 2% TRITC-dextran to label vessels, then the sponges were excised for analysis. Left panels show representative results of whole sponges under a fluorescence microscope. Right panel shows the TRITC-based quantification of vessel density in whole sponges. TRITC-positive pixel area was measured. Data show mean ± SEM of six sponges from independent mice. ** <i>P</i> < 0.05, *** <i>P</i> < 0.01 Note: A CD148-interacting trimeric TSP1 fragment inhibits VEGF-induced angiogenesis in wild-type, but not CD148 knockout, mice. Lower panels: Paraffin sections were processed from each sponge. Vessel density was assessed by vWF immunostaining. The sections were counterstained with DAPI. Left panels show representative results of vWF immunostaining in each condition. Right panel shows FITC-based quantification of vessel density in each group. Data show mean ± SEM of six sponges from independent mice. ** <i>P</i> < 0.05, *** <i>P</i> < 0.01</p

The 1^st type 1 repeat is required for TSP1/CD148-mediated cell growth inhibition.

<p><b>(A)</b> The trimeric TSP1 fragment (ΔType1-R1) that contains the procollagen domain and the 2^nd and 3^rd, but not the 1^st, type 1 repeat was prepared using HEK293E cells. Upper panel shows a schematic representation of the trimeric TSP1 fragment that lacks the 1^st type 1 repeat. Amino acid residues (aa 374–429) of the 1^st type 1 repeat were deleted. Lower panel shows colloidal blue staining of the purified ΔType1-R1 fragment. Five micrograms of protein were separated on a 10% polyacrylamide gel in reducing (+DTT) and non-reducing (-DTT) conditions and stained with colloidal blue to assess size, purity, and trimerization. The expected size of protein is also shown. <b>(B)</b> A431D/CD148wt or A431D/CD36 (stably expressing CD36) cells were treated with 12 nM of trimeric TSP1 fragments that lack or contain the 1^st type 1 repeat or whole TSP1 protein. Cell density was measured at the indicated time points. The data show mean ± SEM of quadruplicate determinations. Representative data of four independent experiments is shown. Note: The ΔType1-R1 fragment shows no growth inhibitory activity in A431D/CD148wt cells, while it inhibits cell proliferation in A431D/CD36 cells. <b>(C)</b> A431D/CD36 cells were treated with trimeric TSP1 fragments (12 nM) that lacked or contained the 1^st type 1 repeat or whole TSP1 protein (12 nM) for 18 h. Tyrosine phosphorylation of p38 and cleaved caspase 3 was assessed by immunoblotting using the phopho-specific p38 (pThr180+Tyr182) or cleaved caspase 3 antibodies. The membranes were reprobed with antibodies to total p38 or γ-tubulin. Representative data of four independent experiments is shown. <b>(D)</b> A series of monomeric TSP1 fragments were prepared from the regions containing the procollagen domain and type 1 repeats as shown in a schema on right side. Each fragment (17 nM) was incubated with either 44 pmol of CD148-Fc or control Fc (Fc alone), and Fc-proteins were pulled down with protein-G beads. Bound TSP1 fragments were assessed by anti-Myc immunoblotting (upper panel). Half of each sample was subjected to anti-CD148 immunoblotting to confirm the pull down of CD148-Fc (lower panel). Representative data of five independent experiments is shown. Note: TSP1 fragments that contain the 1^st type 1 repeat bind to CD148-Fc. <b>(E)</b> A431D/CD148wt cells were treated with or without indicated TSP1 fragments (36 nM) for 1 h, then a trimeric TSP1 fragment (12nM) containing the procollagen domain and type 1 repeats was added to the medium. Cell proliferation was assessed at day 2. The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. ** <i>P</i> < 0.05 Note: Only the 1^st type 1 repeat blocks cell growth inhibition induced by the trimeric TSP1 fragment.</p

Trimeric TSP1 fragments that contain the 1^st type 1 repeat increase CD148 catalytic activity and reduce tyrosine phosphorylation of EGFR and ERK1/2 in A431D/CD148wt cells.

<p><b>(A)</b> Left: A431D/CD148wt cells were treated with the indicated trimeric TSP1 fragments (12 nM) or whole TSP1 protein (12 nM) for 15 min. CD148 was immunoprecipitated using anti-CD148 antibody or class-matched control IgG. The washed immunocomplexes were subjected to a PTP activity assay with or without 1 mM sodium orthovanadate (VO₄). The amount of CD148 in the immunocomplexes was evaluated by immunoblotting using anti-CD148 antibody (lower panel). The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. ** <i>P</i> < 0.05 vs. vehicle-treated cells. Right: To assess the specificity of the effect, a trimeric TSP1 fragment containing the procollagen domain and the 1^st type 1 repeat was added to A431D/CD148wt cells with 11.3 nM of CD148-Fc or control Fc (Fc alone), then CD148 catalytic activity was assessed as in left panel. The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. ** <i>P</i> < 0.05 vs. vehicle-treated cells. Note: CD148-Fc, but not control Fc, abolishes the activity of the TSP1 fragment to increase CD148 catalytic activity. <b>(B)</b> Left: A431D/CD148wt cells were treated with the indicated trimeric TSP1 fragments (12 nM) or whole TSP1 protein (12 nM) for 15 min. Tyrosine phosphorylation of EGFR (immunoprecipitated) and ERK1/2 was assessed by immunoblotting using the phopho-specific EGFR (Y1173) or ERK1/2 (T202/Y204) antibodies. The membranes were reprobed with antibodies to total EGFR or ERK1/2. Representative data of four independent experiments is shown. Right: A431D and A431D/CD148cs cells were treated with a trimeric TSP1 fragment (12 nM) that contains the procollagen domain and the 1^st type 1 repeat or whole TSP1 protein (12 nM), and tyrosine phosphorylation of EGFR (immunoprecipitated) and ERK1/2 was assessed as in left panel. Representative data of four independent experiments is shown. Note: No effects are observed in A431D and A431D/CD148cs cells.</p

Type 1 repeats are required for TSP1/CD148-mediated cell growth inhibition.

<p><b>(A)</b> Trimeric TSP1 fragments containing the procollagen domain and either all three, two, one, or none of type 1 repeats were prepared using HEK293E cells. Left panel shows a schematic representation of the trimeric TSP1 fragments used in this study. The number of amino acid residues includes the signal peptide sequence. Right panel shows colloidal blue stain of the purified TSP1 fragments. Two micrograms of protein were separated on a 4–20% gradient polyacrylamide gel in reducing (+DTT) and non-reducing (-DTT) conditions, then stained with colloidal blue to assess its size, purity, and trimerization. The expected size of protein is also shown. <b>(B)</b> A431D cells stably expressing wild-type CD148 (A431D/CD148wt cells) were plated in 96-well plates, starved, and treated with 12 nM of trimeric TSP1 fragments or whole TSP1 protein. Cell density was measured at the indicated time points (left panel). The dose dependency of the effects was also evaluated at day 2 (right panel). The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. <b>(C)</b> The effects of a trimeric TSP1 fragment (6.0 nM) containing the procollagen domain and the 1^st type 1 repeat on cell proliferation of A431D (lacking CD148) and A431D/CD148cs (stably expressing a catalytically inactive form of CD148) cells are shown. Cell proliferation was assessed as in (B). The data show mean ± SEM of quadruplicate determinations. Representative data of four independent experiments is shown. ** <i>P</i> < 0.05 <b>(D)</b> CD148 was knocked down in A431D/CD148wt cells using the lentivirus encoding CD148-targeting shRNA (shRNA #1, shRNA #2). The lentivirus encoding scrambled shRNA was used as a control. The cells were subjected to a cell proliferation assay and the effects of CD148 knockdown on growth inhibition of a trimeric TSP1 fragment (12 nM) containing the procollagen domain and the 1^st type 1 repeat were assessed. The data show mean ± SEM of quadruplicate determinations. Representative data of four independent experiments is shown. ** <i>P</i> < 0.05 Note: CD148 knockdown largely attenuates the TSP1 fragment’s growth inhibitory activity in A431D/CD148wt cells.</p

Assessment of CD148-interacting region in TSP1.

<p><b>(A)</b> Recombinant TSP1 fragments that correspond to the structural elements were prepared using HEK293E cells. Left panel shows a schematic representation of the TSP1 fragments. The number of amino acid residues includes the signal peptide sequence. Right panel shows colloidal blue stain of the purified TSP1 fragments. Twelve micrograms of protein were separated on a 10% polyacrylamide gel and stained with colloidal blue to assess size and purity. The expected size of protein is also shown. <b>(B)</b> TSP1 fragments (17 nM) were incubated with either 44 pmol of CD148-Fc or control Fc (Fc alone). Fc-proteins were pulled down with Protein-G beads and the binding of TSP1 fragments was assessed by immunoblotting using anti-Myc antibody (upper panel). The membrane was reprobed with anti-CD148 antibody to confirm the pull down of CD148-Fc (lower panel). Representative data of five independent experiments is shown. Note: The TSP1 fragment containing the procollagen domain and type 1 repeats binds to CD148-Fc. <b>(C)</b> Protein-A plates conjugated with CD148-Fc (11.3 nM) or equal molar of control Fc were incubated with AP-TSP1 or AP (12 nM) in the presence or absence of TSP1 fragments (25 nM) or whole TSP1 protein (25 nM). The bound AP-TSP1 was assessed by an AP activity assay. The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. ** <i>P</i> < 0.05 Note: The binding of AP-TSP1 to CD148-Fc is blocked with either a TSP1 fragment containing the procollagen domain and type 1 repeats or whole TSP1 protein.</p

VE-PTP promoter activity in adult mouse kidney.

<p><b>(A)</b> Whole mount X-gal staining of adult VE-PTP^tlacZ/+ mouse kidney. Evident β-galactosidase activity is observed in renal cortex (C), inner stripe (IS) of outer medulla, inner medulla (IM), and papilla (P). Right panel shows vascular distribution of β-galactosidase activity in renal cortex. C, cortex; OS, outer stripe; IS, inner stripe; IM, inner medulla; P, papilla. <b>(B)</b> β-galactosidase activity in adult VE-PTP^tlacZ/+ mouse kidney sections. In medulla, β-galactosidase activity is observed in vascular bundle (VB), subpopulations of medullary tubules (red arrows in panels c and d), and papillary cells (panel e). VB, vascular bundle. <b>(C)</b> β-galactosidase activity in cortical renal vasculature. RA, renal artery: RV, renal vein; AA, arcuate artery; AV, arcuate vein; IA, interlobular artery; IV, interlobular vein; Art, arteriole; G, glomerulus; af, afferent arteriole; ef, efferent arteriole. Note: VE-PTP promoter activity is limited in efferent arterioles, peritubular capillaries, and venous circulations. Scale bar, 200 μm in B-a and B-b; 100 μm in B-c, B-d, B-e, and C; 50 μm in an insert of C.</p

Expression of receptor-type protein tyrosine phosphatase in developing and adult renal vasculature

<div><p>Renal vascular development is a coordinated process that requires ordered endothelial cell proliferation, migration, intercellular adhesion, and morphogenesis. In recent decades, studies have defined the pivotal role of endothelial receptor tyrosine kinases (RPTKs) in the development and maintenance of renal vasculature. However, the expression and the role of receptor tyrosine phosphatases (RPTPs) in renal endothelium are poorly understood, though coupled and counterbalancing roles of RPTKs and RPTPs are well defined in other systems. In this study, we evaluated the promoter activity and immunolocalization of two endothelial RPTPs, VE-PTP and PTPμ, in developing and adult renal vasculature using the heterozygous LacZ knock-in mice and specific antibodies. In adult kidneys, both VE-PTP and PTPμ were expressed in the endothelium of arterial, glomerular, and medullary vessels, while their expression was highly limited in peritubular capillaries and venous endothelium. VE-PTP and PTPμ promoter activity was also observed in medullary tubular segments in adult kidneys. In embryonic (E12.5, E13.5, E15.5, E17.5) and postnatal (P0, P3, P7) kidneys, these RPTPs were expressed in ingrowing renal arteries, developing glomerular microvasculature (as early as the S-shaped stage), and medullary vessels. Their expression became more evident as the vasculatures matured. Peritubular capillary expression of VE-PTP was also noted in embryonic and postnatal kidneys. Compared to VE-PTP, PTPμ immunoreactivity was relatively limited in embryonic and neonatal renal vasculature and evident immunoreactivity was observed from the P3 stage. These findings indicate 1) VE-PTP and PTPμ are expressed in endothelium of arterial, glomerular, and medullary renal vasculature, 2) their expression increases as renal vascular development proceeds, suggesting that these RPTPs play a role in maturation and maintenance of these vasculatures, and 3) peritubular capillary VE-PTP expression is down-regulated in adult kidneys, suggesting a role of VE-PTP in the development of peritubular capillaries.</p></div

Co-immunostaining of VE-PTP and CD31 in developing and adult mice kidneys.

<p>Kidney sections from E16.5, P0 and adult mice were double immunolabeled for VE-PTP (red) and CD31 (green) as described in the “Materials & Methods”. In developing kidneys (E16.5 and P0), VE-PTP is expressed in endothelial cells in ingrowing arteries (A) and developing glomeruli (G), which are labeled with CD31. VE-PTP is also expressed in endothelial cells (yellow arrows) that distribute around the developing nephrons. In adult kidney, VE-PTP is expressed in endothelial cells in arterial and glomerular vasculature, while its expression is limited in peritubular capillaries. A, arterial vessel; IA, interlobular artery; G, glomerulus. Scale bar, 50 μm in E16.5 and P0 kidneys; 25 μm in adult kidney.</p