A qualitative exploration of forensic pathology service staff perceptions of the implementation barriers and facilitators of manual- and electronic injury mortality surveillance system methods in South Africa.

BACKGROUND: Injury mortality surveillance systems are critical to monitor changes in a population's injury outcomes so that relevant injury prevention responses may be adopted. This is particularly the case in South Africa, where the injury burden is nearly twice the global rate. Regular evaluations of surveillance systems are pivotal to strengthening surveillance capacity, performance, and cost effectiveness. The National Injury Mortality Surveillance System (NIMSS) is an injury mortality surveillance system that is currently focused in Mpumalanga and utilises manual and electronic web-based systems for data collection. This study explored Forensic Pathology Service (FPS) staff perceptions of the implementation barriers and facilitators of manual- and electronic injury mortality surveillance system methods. METHODS: A qualitative study was employed using purposive sampling. Forty-seven participants, aged 29 to 59 years comprising 31 males and 16 females were recruited across 21 FPS facilities that serve the province. The formative evaluation occurred over the November 2019 to November 2022 period. Twelve focus group discussions were thematically analysed to determine emerging themes and patterns related to the use of the system using the WHO surveillance system guidelines as a framework. RESULTS: The key themes concerning the barriers and facilitators were located along WHO attributes of simplicity, acceptability, timeliness, flexibility, data quality and stability. Distinctions between the manual and e-surveillance systems were drawn upon across the attributes highlighting their experience with the system, user preference, and its contextual relevance. With Mpumalanga predominantly rural, internet connectivity was a common issue, with most participants consequently showing a preference for the manual system, even though the electronic system's automated internal validation process was of benefit. The data quality however remained similar for both methods. With program stability and flexibility, the manual system proved more beneficial as the dataset was reported to be easily transferrable across computer devices. CONCLUSION: Obtaining FPS perceptions of their experiences with the system methodologies are pertinent for the enhancement of injury surveillance systems so to improve prospective engagements with the systems. This will facilitate timely and accurate injury mortality information which is vital to inform public policy, and injury control and prevention responses.

Provision of Organ at Risk Contouring Guidance in UK Radiotherapy Clinical Trials.

AIMS: Accurate delineation of organs at risk (OAR) is vital to the radiotherapy planning process. Inaccuracies in OAR delineation arising from imprecise anatomical definitions may affect plan optimisation and risk inappropriate dose delivery to normal tissues. The aim of this study was to review the provision of OAR contouring guidance in National Institute of Health Research Clinical Research Network (NIHR CRN) portfolio clinical trials. MATERIALS AND METHODS: The National Radiotherapy Quality Trials Assurance (RTTQA) Group carried out a two-round Delphi assessment to determine which OAR descriptions provided optimal guidance. RESULTS: Eighty-four clinical trials involving radiotherapy quality assurance were identified as either in recruitment or in setup within the NIHR CRN portfolio. Fifty-nine trials mandated OAR contouring. In total there were 412 OAR; 171 were uniquely named; 159 OAR had more than one name associated with a single structure, with the greatest nomenclature variation seen for the femoral head ± neck, the parotid gland, and bowel. The two-round Delphi assessment determined 42 OAR descriptions as providing optimal contouring guidance. CONCLUSIONS: This study identified the need for OAR nomenclature and contouring guidance consistency across clinical trials. In response to this study and in conjunction with the Global Quality Assurance of Radiation Therapy Clinical Trials Harmonisation Group, the RTTQA Group is in collaboration with international partners to provide consensus recommendations for OAR delineation in clinical trials.

Vitamin E delivery to human skin by a rinse-off product: penetration of alpha-tocopherol versus wash-out effects of skin surface lipids.

alpha-Tocopherol, the major biologically active form of vitamin E, represents a frequently added lipophilic compound of skin care products. Despite its emerging use in rinse-off formulations, little is known on its efficacy with respect to its deposition or its antioxidant potential in human skin. The objective of this study was to investigate whether the single use of an alpha-tocopherol-enriched rinse-off product provides effective deposition of alpha-tocopherol on human stratum corneum. To test this, forearm skin of 13 volunteers was washed either with an alpha-tocopherol-enriched rinse-off product (test product, TP) or with an alpha-tocopherol-free vehicle control (control product, CP) (contralateral arm) using a standardized wash protocol. Thereafter, skin surface lipids were extracted with pure ethanol after the wash procedure as well as after 24 h. Additionally, one group of volunteers was subjected to irradiation of their forearms with low-dose UVA (8 J/cm(2)) prior to lipid extraction. Skin lipid extracts were analyzed by high performance liquid chromatography using electrochemical detection for vitamin E and UV detection for squalene (SQ) and squalene monohydroperoxide. The results of this in vivo study demonstrated that (1) while CP treatment lowers, TP treatment strongly increases alpha-tocopherol levels of skin barrier lipids; (2) increased vitamin E deposition levels were maintained for a period of at least 24 h, and (3) TP treatment significantly inhibited photooxidation of SQ. In conclusion, the use of alpha-tocopherol-enriched rinse-off products may help to maintain the integrity of the skin barrier by providing protection against photooxidative stress at the level of skin surface lipids.

Phorbol ester-induced loss of membrane sialic acid: implications for tumor cytolysis by natural killer cells.

Freeze-fracture analysis has shown that treatment of cells with phorbol myristate acetate (PMA) results in a loss of intramembranous particles (IMP) associated with the external leaflet of their plasma membranes. It has also been demonstrated that phorbol esters markedly enhance the sensitivity of tumor targets to natural killer (NK) cells, although the mechanism underlying this phenomenon has remained unexplained. Since the ability of NK cells to recognize neoplasms appears to be inversely related to the concentration of sialic acid on the target cell surface, it seemed possible that phorbols affect membrane glycoproteins which have terminal carbohydrates bearing sialic acid residues. To investigate whether phorbol treatment could be responsible for the loss of sialic acid, four tumor cell lines were examined before and after exposure to PMA. A reduction in surface sialic acid was established by four different methods: 1) standard thiobarbituric acid analysis of cell hydrolysates, 2) metabolic labelling of cells with [3H]-mannosamine followed by treatment with neuraminidase, 3) chromatography of membrane extracts, and 4) freeze-fracture analysis of lectin-labelled intact cells. These observations suggest a mechanism whereby phorbols may facilitate NK-cell-mediated cytolysis. In addition, an entirely novel effect of these tumor-producing agents may have been uncovered.

Unmasking of cryptic natural killer (NK) cell recognition sites on chronic lymphocytic leukemia lymphocytes.

The sensitivity of chronic lymphocytic leukemia (CLL) lymphocytes to attack by natural killer (NK) cells has remained questionable. To clarify this issue, freshly isolated lymphocytes of 37 patients with B-CLL, five with WDLL and two with HCL, were tested with a standard cytotoxicity assay with NK cells from normal donors. All these targets were resistant to cytolysis by the effectors. Freeze-fracture analysis of CLL cell plasma membranes revealed that they have a larger number of intramembranous particles (IMP) associated with the external leaflet (E-face) than have normal lymphocytes. Unlike other neoplastic cells, exposure of CLL lymphocytes to phorbol esters or treatment with neuraminidase did not render them vulnerable to attack by NK cells, nor did 5 days of culture have an effect. Incubation of CLL lymphocytes with anti-Ig-mu (24-72 hr) or with 0.1% pepsin (15 min) resulted in 15% and 27% cytolysis, respectively. B-lymphocytes from the blood of healthy donors were not killed when treated similarly: These data establish that freshly isolated B-CLL lymphocytes are resistant to NK cytolysis but that in contrast to normal B-cells, they possess cryptic NK-recognition structures, which may be uncovered by surface modulation.

Susceptibility to NK cell lysis is abolished in tumor cells by a factor which restores their contact inhibited growth.

It is well recognized that physical contact between natural killer (NK) cells and tumor targets is necessary for cell lysis. Therefore, any modulation of the tumor cell surface that alters intercellular contact could affect NK cell cytotoxicity. To examine this hypothesis, a contact inhibitory factor (CIF), which had been shown to restore contact inhibition of growth to several malignant cell lines was tested for its ability to render such cells immune to recognition by NK cells. When three NK-sensitive melanoma and two NK-sensitive colon carcinoma targets were cultured with CIF, they did not only change morphologically, but also showed a 70% to 95% reduction in their sensitivity to lysis by NK cells. In addition, K562 cells, which grow in suspension and do not permit a morphologic evaluation of the CIF effect, also became resistant to lysis by NK cells after culture with CIF. CIF did not reduce the viability nor the cytotoxicity of NK cells. CIF did not contain interferon nor did the CIF-treated targets induce the production of interferon during the cytotoxicity assay. It is concluded that restoration of contact inhibition of growth and resistance to NK cell lysis are cell surface phenomena that may run in parallel.

Loss of intercalated membrane particles by treatment with phorbols.

Because brief exposure to phorbol esters renders normal cells vulnerable to deformation and cytolysis by lymphocytes, it was postulated that these tumor promoters might cause a hitherto unrecognized physical alteration in membrane architecture. To investigate this possibility, four tissue culture cell lines (K-562 erythroleukemia cells, melanoma cells, N1121 adult fibroblasts, and normal fetal fibroblasts) and three blood cell types (lymphocytes, monocytes, and platelets) were subjected to freeze-fracture analysis before and after brief treatment with phorbol myristate acetate. Phorbol myristate acetate caused a 50% reduction of intramembranous particles associated with the external leaflet (E face) of the plasma membrane of every cell except platelets. In contrast, no change in size or number of intramembranous particles associated with the protoplasmic membrane leaflet (P face) was evident. Since the platelet membrane is known to be turned "inside out," as regards the partition coefficient of the intramembranous particles, the disparity between the results obtained with platelets and other cells may serve to determine the nature of intramembranous particles affected by phorbols. Also, since phorbols affect primarily glycolipids and/or glycoproteins anchored in the external membrane leaflet, these findings may provide a useful tool for future exploration of membrane structure.

Phorbol-induced recruitment of lymphocytes for spontaneous cytolysis of natural killer-insensitive tumor targets.

Natural killer (NK) cells lyse a variety of tumor cells in vitro whereas NK-depleted unsensitized lymphocytes do not have this effect. In studies designed to elucidate the NK phenomenon, a series of experiments was carried out to identify properties of NK-sensitive targets and compare these with those of NK-insensitive targets and with targets rendered sensitive by treatment with phorbol esters. Following brief exposure to phorbol-12-myristate-13-acetate (PMA), the targets were thoroughly washed, and then incubated with lymphocyte preparations which were either enriched for or depleted of NK cells. PMA treatment increased the susceptibility of sensitive targets to NK-enriched fractions by only 20-30%, but made the NK-cell-insensitive targets markedly vulnerable to these effectors (80% lysis). Unexpectedly, brief PMA exposure also rendered cells susceptible to lysis by NK-cell-depleted lymphocytes. Yet, such targets were not killed by monocytes or B lymphocytes. Elimination of T8 lymphocytes from the NK-depleted fractions abolished lysis. To explore whether PMA had induced membrane changes not detectable on electron microscopy of thin sections, freeze-fracture studies were carried out on target cells before and after treatment with PMA. Freeze-fracture replicas of target cells which had been exposed to PMA exhibited a 50% reduction of the intramembranous particles (IMP) on the external leaflet of the plasma membrane but no changes in the number or size of the IMP associated with the protoplasmic leaflet face. The exact relationship of the structural changes and enhanced susceptibility to cytolysis has not yet been established. However, the observation that normal and tumor cells can be rendered vulnerable to lysis by lymphocytes which have not been sensitized immunologically may have practical applications.

A substrate analog inhibitor for arylsulfatase reduces NK cell cytotoxicity.

A synthetic aromatic sulfonate (I), which has been found to be an effective inhibitor of arylsulfatase, reduces NK-cell mediated cytotoxicity by ca. 60% at 10 microM concentration. At lower concentrations the effect is concentration dependent, but no further reduction of cytotoxicity is observed at concentrations above 10 microM.

Inhibition by superoxide dismutase and catalase of the damage of isolated Leishmania mexicana amazonensis by phenazine methosulfate.

Phenazine methosulfate, a cationic electron carrier, inhibits the extracellular growth of promastigotes and the conversion of amastigotes into promastigote forms of Leishmania mexicana amazonensis. Growth inhibition and damage of extracellular parasites by PMS was counteracted by superoxide dismutase, a scavenger of the superoxide anion (O2-), and to a lesser extent, by catalase, a scavenger of hydrogen peroxide (H2O2). Inactivated dismutase and catalase were ineffective. Thus, damage of isolated L.m. amazonensis by phenazine methosulfate, involves the participation of O2- and H2O2. The role of the oxygen metabolites in the toxicity of phenazine methosulfate remains unknown. That O2- can damage the parasites is supported by the finding that superoxide dismutase also protected promastigotes from damage induced by oxygen intermediates generated by a xanthine-xanthine oxidase system. Killing of the parasites by crystal violet, a triphenylmethane, or basic blue 24, a phenothiazine, was not inhibited by superoxide dismutase.

Release of the membrane-calcium and its relation to the superoxide formation by polymorphonuclear leukocytes.

The relationship between the intracellular translocation of calcium from the storage pool and the oxidative metabolism was studied. An intracellular calcium-antagonist, TMB-8, inhibited the release of superoxide induced by a calcium ionophore A23187 and the inhibition was relieved by the addition of calcium ions. The release induced by cytochalasin D or by the ingestion of bacteria was similarly inhibited by TMB-8. The mobilization of intracellular divalent cations of leukocytes was monitored by a fluorescent probe, CTC. When the CTC-loaded cells were stimulated with cytochalasin D or E. coli, a fluorescence change ascribable to the release of calcium from the intracellular hydrophobic environment was observed. The dose-response curve of the fluorescence change and that of the superoxide release of th cells were very similar. TMB-8 inhibited both metabolic and fluorescence changes in parallel. The results support the hypothesis that an intracellular translocation of calcium is stimulated the oxidative metabolism of leukocytes.