Women's Perceptions of HIV- and Sexuality-Related Stigma in Relation to PrEP: Qualitative Findings from the Masibambane Study, Durban, South Africa.

Daily oral pre-exposure prophylaxis (PrEP) offers effective HIV prevention. In South Africa, PrEP is publicly available, but use among young women remains low. We explored young women's perceptions of PrEP to inform a gender-focused intervention to promote PrEP uptake. Six focus group discussions and eight in-depth interviews exploring perceptions of PrEP were conducted with forty-six women not using PrEP, ages 18-25, from central Durban. Data were thematically analyzed using a team-based consensus approach. The study was conducted among likely PrEP users: women were highly-educated, with 84.8% enrolled in post-secondary education. Qualitative data revealed intersecting social stigmas related to HIV and women's sexuality. Women feared that daily PrEP pills would be confused with anti-retroviral treatment, creating vulnerability to misplaced HIV stigma. Women also anticipated that taking PrEP could expose them to assumptions of promiscuity from the community. To address these anticipated community-level reactions, women suggested community-facing interventions to reduce the burden on young women considering PrEP. Concerns around PrEP use in this group of urban, educated women reflects layered stigmas that may inhibit future PrEP use. Stigma-reducing strategies, such as media campaigns and educational interventions directed at communities who could benefit from PrEP, should re-frame PrEP as an empowering and responsible choice for young women.

Setting the stage for next-generation risk assessment with non-animal approaches: the EU-ToxRisk project experience.

In 2016, the European Commission launched the EU-ToxRisk research project to develop and promote animal-free approaches in toxicology. The 36 partners of this consortium used in vitro and in silico methods in the context of case studies (CSs). These CSs included both compounds with a highly defined target (e.g. mitochondrial respiratory chain inhibitors) as well as compounds with poorly defined molecular initiation events (e.g. short-chain branched carboxylic acids). The initial project focus was on developing a science-based strategy for read-across (RAx) as an animal-free approach in chemical risk assessment. Moreover, seamless incorporation of new approach method (NAM) data into this process (= NAM-enhanced RAx) was explored. Here, the EU-ToxRisk consortium has collated its scientific and regulatory learnings from this particular project objective. For all CSs, a mechanistic hypothesis (in the form of an adverse outcome pathway) guided the safety evaluation. ADME data were generated from NAMs and used for comprehensive physiological-based kinetic modelling. Quality assurance and data management were optimized in parallel. Scientific and Regulatory Advisory Boards played a vital role in assessing the practical applicability of the new approaches. In a next step, external stakeholders evaluated the usefulness of NAMs in the context of RAx CSs for regulatory acceptance. For instance, the CSs were included in the OECD CS portfolio for the Integrated Approach to Testing and Assessment project. Feedback from regulators and other stakeholders was collected at several stages. Future chemical safety science projects can draw from this experience to implement systems toxicology-guided, animal-free next-generation risk assessment.

Emtonjeni-A Structural Intervention to Integrate Sexual and Reproductive Health into Public Sector HIV Care in Cape Town, South Africa: Results of a Phase II Study.

Integration of sexual and reproductive health within HIV care services is a promising strategy for increasing access to family planning and STI services and reducing unwanted pregnancies, perinatal HIV transmission and maternal and infant mortality among people living with HIV and their partners. We conducted a Phase II randomized futility trial of a multi-level intervention to increase adherence to safer sex guidelines among those wishing to avoid pregnancy and adherence to safer conception guidelines among those seeking conception in newly-diagnosed HIV-positive persons in four public-sector HIV clinics in Cape Town. Clinics were pair-matched and the two clinics within each pair were randomized to either a three-session provider-delivered enhanced intervention (EI) (onsite contraceptive services and brief milieu intervention for staff) or standard-of-care (SOC) provider-delivered intervention. The futility analysis showed that we cannot rule out the possibility that the EI intervention has a 10 % point or greater success rate in improving adherence to safer sex/safer conception guidelines than does SOC (p = 0.573), indicating that the intervention holds merit, and a larger-scale confirmatory study showing whether the EI is superior to SOC has merit.

In vitro effects of detergent sclerosants on clot formation and fibrinolysis.

OBJECTIVE: To investigate the in vitro effects of detergent sclerosants sodium tetradecyl sulphate (STS) and polidocanol (POL) on clot formation and lysis. MATERIALS AND METHODS: clot kinetics were assessed in whole blood by thromboelastography (TEG®) and rotational thromboelastometry (ROTEM®). Fibrinogen was measured by the Clauss method in plasma and factor XIII (FXIII) by enzyme-linked immunosorbent assay (ELISA). Turbidity measurements were used to assess clot lysis in plasma, and fibrinolysis in non-cross-linked and cross-linked fibrin. D-dimer was measured by VIDAS®, STA®Liatest® and AxSYM® assays. RESULTS: Strong clots were formed at low sclerosant concentrations (0.075-0.1%). At midrange concentrations (0.15% STS, 0.15-0.3% POL), both agents inhibited the contribution of platelets to clot firmness and formed weak clots prone to lysis. At higher concentrations (STS ≥ 0.3% and POL ≥ 0.6%), clot formation was inhibited. STS destroyed FXIII at ≥ 0.15% and fibrinogen at ≥ 0.6%. Neither sclerosant had a significant effect on cross-linked fibrin, but STS had a lytic effect on non-cross-linked fibrin. STS caused an artefactual elevation of D-dimer in the VIDAS® assay when fibrinogen was present. CONCLUSION: Detergent sclerosants demonstrated a trimodal effect on clot formation, initiating strong clots at low concentrations, weak clots at midrange concentrations and preventing clot formation at higher concentrations. Neither agent had fibrinolytic activity.

In vitro effects of detergent sclerosants on antithrombotic mechanisms.

OBJECTIVES: To investigate the in vitro effects of detergent sclerosants on antithrombotic pathways. MATERIALS AND METHODS: Proteins C, S and antithrombin (AT) were assayed in normal plasma treated with increasing concentrations of sodium tetradecyl sulphate (STS) and polidocanol (POL). Activated protein C (APC) was investigated by mixing normal plasmas with sclerosants and testing with the activated partial thromboplastin time (APTT) and dilute Russell's viper venom time in the presence and absence of APC. The effect on factor Xa (FXa), heparin and enoxaparin was investigated using chromogenic anti-FXa and APTT methods. RESULTS: High concentration (>0.6%) STS significantly destroyed proteins C, S and AT whereas POL only caused a mild reduction in PC and AT and a moderate (60%) reduction in PS levels. STS potentiated the anticoagulant effect of APC while POL increased APC resistance. STS mimicked AT and demonstrated significant anti-Xa and anti-IIa activity. STS demonstrated a similar anticoagulant profile to heparin but was 1000x weaker. It also significantly potentiated the anticoagulant effect of heparin while POL had less effect. CONCLUSION: STS and POL demonstrated quite distinct and sometimes opposite effects on the antithrombotic mechanisms assayed. These effects were concentration-dependent and in general, STS had the greatest effect on antithrombotic proteins.

Clotting test results correlate better with DOAC concentrations when expressed as a "Correction Ratio"; results before/after extraction with the DOAC Stop reagent.

INTRODUCTION: Clotting test results are currently not useful for estimating direct oral anti-coagulant (DOAC) concentrations because baseline results vary. DOAC Stop is a DOAC extracting agent with no effect on clotting factors. We investigated if aPTT (activated partial thromboplastin time) and dRVVT (dilute Russells viper venom time) results might correlate better with DOAC concentrations if results after DOAC extraction were used to estimate a "before/after" value (Correction Ratio). MATERIALS AND METHODS: We used activated partial thromboplastin time (aPTT, PTT-LA) and dilute Russells viper venom time clotting test (dRVVT) results previously recorded on DOAC patient plasmas (25 dabigatran, 15 apixaban, 19 rivaroxaban) without known thrombotic risk factors before and after DOAC extraction. DOAC concentrations had been determined by standard chromogenic assays. RESULTS: Correlations between aPTT and dabigatran, apixaban, and rivaroxaban concentrations were initially poor (0.64, 0.15 and 0.39 respectively). However, they improved significantly to 0.94, 0.89 and 0.80 when the ratios of initial aPTT to the aPTT obtained after DOAC extraction were plotted against DOAC concentration. Still better correlations (0.99, 0.97, 0.95) and much higher sensitivities to the DOACs were obtained when dRVVT (LA Confirm) tests were used following this procedure on the same samples. CONCLUSIONS: The correlations of aPTT and dRVVT tests with DOAC concentrations were significantly improved by using the ratio of result "before" to those "after" DOAC extraction. The results indicate that dRVVT (especially LA Confirm) and similar tests might be useful for determining DOAC concentrations more reliably and with better sensitivity than currently possible with clotting tests.

The lytic effects of detergent sclerosants on erythrocytes, platelets, endothelial cells and microparticles are attenuated by albumin and other plasma components in vitro.

OBJECTIVE: To investigate the lytic effects of sodium tetradecyl sulphate (STS) and polidocanol (POL) on erythrocytes, platelets, endothelial cells and platelet-derived microparticle (PDMP) formation in vitro and the potential protective effects of serum albumin and agents such as procaine. MATERIALS AND METHODS: The effects of sclerosants were studied in blood samples obtained from normal individuals. Absorbance densitometry was used to assess the lytic effects of sclerosants on blood cells and cultured human microvascular endothelial cells (HMEC) in plasma and in saline. PDMP were quantified by flow cytometry. RESULTS: Haemolysis occurred in whole blood at sclerosant concentrations greater than 0.25% for STS and above 0.45% for POL. Similar concentrations of both agents caused platelet and endothelial cell lysis. Both sclerosants released PDMP at low concentrations but destroyed PDMP at higher concentrations. Albumin significantly reduced the lytic effect of both sclerosants on all cells but had a greater inhibitory effect on POL. Protamine at 0.01% had a neutralising effect on STS, whereas procaine and lignocaine showed no such activity. CONCLUSIONS: Sclerosants at therapeutic concentrations lyse blood cells and endothelial cells in vitro. This effect is strongly reduced by serum albumin possibly contributing towards the low incidence of thromboembolic complications of sclerotherapy.

Women's anal sex practices: implications for formulation and promotion of a rectal microbicide.

To gain insight into practices that may inform formulation and use of rectal microbicides, in-depth interviews were conducted with an ethnically diverse sample of 28 women who engage in anal intercourse. Microbicides are compounds under development to decrease sexually transmitted infections. Most women practiced anal sex in conjunction with vaginal intercourse. Anal sex typically was not preplanned, and few women reported preparation. Condom use was rare. Most women relied on saliva, vaginal fluids, prelubricated condoms, or used no lubrication at last intercourse. Women were uncertain about the amount of lubricant used during sex, with typical estimates of 1 to 2 teaspoons. This may prove challenging to the formulation and promotion of rectal microbicides, as substantially higher amounts may be required. Additional challenges include infrequent use of packaged lubricants, and typical male lubricant application, which may make women's control of rectal microbicides more difficult. Women overwhelmingly expressed interest in rectal microbicides.

Simple method for removing DOACs from plasma samples.

AIM: To evaluate a simple method using an adsorbent product (DOAC Stop) for extracting direct oral anti-coagulants (DOACs) from plasmas. METHOD: DOAC Stop was tested on normal and a range of abnormal plasmas initially using activated partial thromboplastin time (APTT) tests and a more DOAC-sensitive Russells viper venom-based clotting test (DOAC Test). Further tests for prothrombin time/International Normalized Ratio (PT/INR), lupus anticoagulants, activated protein C (APC) resistance, antithrombin, plasminogen, protein C and S were carried out on various patient samples. RESULTS: DOAC Stop was found to remove all types of DOACs including dabigatran, apixaban, rivaroxaban and edoxaban from test plasmas with minimal effect on any of the (mainly clotting) tests considered in this study. SUMMARY: DOAC Stop can be used to identify plasmas containing DOAcs using simple clotting tests. It reduces the false positivity for lupus anticoagulants observed in dilute Russells viper venom time (dRVVT) tests on DOAC-containing plasmas and could be useful for eliminating unwanted effects of DOACs on routine coagulation testing.

In vitro effects of detergent sclerosants on coagulation, platelets and microparticles.

OBJECTIVES: To investigate the in vitro effects of Sodium Tetradecyl Sulphate (STS) and Polidocanol (POL) on clotting tests, clotting factors, platelets and microparticles. MATERIALS AND METHODS: Platelet rich (PRP) and platelet poor (PPP) plasmas were incubated with varying concentrations of STS and POL. Clotting tests, platelet/plasma turbidity, and microparticle studies were performed. Specimens were mixed with individual factor deficient plasmas and clotting factor activities were studied. RESULTS: STS at high concentrations (>0.3%) destroyed platelets, microparticles and the clotting factors V, VII and X. It prolonged all clotting tests including prothrombin time (PT), activated partial thromboplastin time (APTT), non-activated partial thromboplastin time (NAPTT), thrombin time (TT), factor Xa clotting time (XACT) and surface activated clotting time (SACT). Higher concentrations of POL were required to achieve some anticoagulant activity. Low sclerosant concentrations shortened XACT and SACT, and induced release of procoagulant platelet derived microparticles. Increased exposure time resulted in increased procoagulant activity. STS at concentrations higher than 0.5% precipitated a complex containing apolipoprotein b and fibrinogen. CONCLUSIONS: Detergent sclerosants affect the clotting mechanism by interfering with clotting factor activities, procoagulant phospholipids and platelet derived microparticles. STS has more anticoagulant activity than POL in high concentrations. Low concentration sclerosants demonstrate procoagulant activity.

Detection of specific proenzyme activators in snake venoms by a new immunoabsorbant-chromogenic substrate method.

In separate experiments, antibodies to plasminogen, factor X and protein C were applied to microtitre trays as commonly used in enzyme-linked immunoassays. After incubation with dilute normal human plasma as a source of the corresponding proenzyme antigen, the wells were exposed to dilutions of various snake venoms. After thorough washing, the microtitre tray wells were tested overnight with chromogenic tripeptide substrates known to be relatively specific for the activated forms of the above factors, i.e., plasmin, factor Xa and activated protein C. The immunochromometric assay described detected two new activators of protein C in Agkistrodon piscivorus and Agkistrodon contortrix venoms and a new factor X activator in Agkistrodon rhodostoma venom. Gel filtration of the latter venom indicated that the factor X activator eluted with high molecular weight, was clearly distinct from the peak fibrinogen clotting activity (Ancrod) and appeared to have no procoagulant activity. Although several Bothrops venoms appeared to contain plasminogen activator by this technique, the observed strong chromogenic activity was observed in microtitre wells independently of plasminogen and represented nonspecific amidase activity.

Drop weight variation in an automated collection procedure and its relationship to apparent surface tension.

A drop counter-regulated fraction collector yields samples containing equal numbers of drops. Such fractions vary slightly in weight depending on experimental conditions such as surface tension. Provided that variables such as flow rate and eluate density remain constant, apparent surface tension may be estimated directly from the weights of eluate fractions obtained from gel filtration experiments. The detergents sodium cholate and sodium lauryl sulphate significantly decreased drop weights in this system. Following gel filtration on Sepharose 4B, sodium cholate eluted in the fractions containing low molecular weight material. It eluted in the same position when pre-mixed with human plasma. Normal plasma was found to contain two surface tension-reducing components with apparent molecular weights of 3-10(6) and 1-10(5). The apparent surface tension of whole human plasma was found to be time dependent and decreased as the flow rate was reduced.

Haemoglobin Camperdown beta104(G6) arginine leads to serine.

Routine investigation of ante natal patients revealed a subtle change in the electrophoretic pattern on cellulose acetate of the proposita. Further investigations by isoelectric focussing in polyacrylamide gel suggested the presence of two major haemoglobin components. Using a modified cellulose acetate technique globin chain separation revealed an abnormal beta-chain. Chain separation on a carboxymethyl-cellulose column provided a pure sample of the abnormal beta-chain. After amino-ethylation, tryptic digestion and peptide mapping, amino acid analysis of relevant peptides showed the abnormality in the beta-chain to be a substitution of arginine by serine at the 104 position. The presence of a positively charged residue at this position would appear to be necessary for the stabilization of the haemoglobin central cavity. The replacement by serine in this haemoglobin leads to slightly decreased stability but does not appear to affect the oxygen affinity.

Prevention of sexual risk behavior for HIV infection with women.

This paper defines the components of gender-specific interventions for HIV infections for women, i.e. negotiation skills with male partners for condom use, integration of strategies against HIV and other STD infections and for contraception, the urgent need for female controlled methods, the importance of the inclusion of heterosexual men and an expansion to couples in prevention programs. This paper also presents a critical update in HIV prevention articles for women since the beginning of the AIDS epidemic through March 1996. All reviewed interventions were conducted in the U.S., Canada or Puerto Rico and described a psychological, behavioral, or educational component that addressed sexual risk reduction and included a behavioral evaluation. Manual and computer searches identified 47 studies that targeted women and provided a female-specific analysis of intervention effects. Overall, the findings demonstrate that HIV prevention programs can be effective in reducing risky sexual behavior among women. Program effectiveness varied by intervention type, session duration, and whether studies included women alone or both men and women. The most efficacious HIV prevention programs were specifically directed toward women, focused on relationship and negotiation skills, and involved multiple, sustained contacts. Evidence also indicated that community-level interventions hold promise. It is recommended that outcomes for women be expanded to include strategies beyond the male condom, such as refusing or avoiding unsafe sex or using the female condom.

Reliability of sexual risk behavior interviews with psychiatric patients.

Test-retest interviews examining recent sexual activity were administered to 27 severely ill psychiatric patients after stabilization. Three reports were judged to be questionable. For the 16 sexually active patients among the remaining 24, high test-retest reliability was found for number of sexual partners, frequency of episodes, and proportions of episodes involving vaginal intercourse and use of condoms. The interviews did not exacerbate psychiatric symptoms.

Alkyl-substituted amino acid amides and analogous di- and triamines: new non-peptide G protein activators.

Synthesis and pharmacological properties of new potent direct activators of heterotrimeric G proteins are described. Compounds were synthesized from protected amino acids with alkylamines using coupling reagents (CDI, DCC, and EDC). Alkyl-substituted amino acid amides and their corresponding di- and triamines were subjected to structure-activity analysis. All compounds activated membrane-bound HL-60 GTPases in a pertussis toxin-sensitive fashion. This suggests a specific effect of compounds on the carboxy terminus of a defined subclass of heterotrimeric G proteins, i.e., members of the G alpha i subfamily. Elongation of the alkyl chain and increasing the number of amino groups enhanced the potency of compounds on HL-60 membrane-bound GTPase. N-(2,5-Diaminopentyl)dodecylamine (21) was selected to study its mode of action employing purified pertussis toxin-sensitive G proteins. It stimulated G alpha subunits by inducing the release of bound GDP. In contrast to receptors G beta gamma complexes were not required for 21-mediated activation of G alpha. Moderate isoform selectivity of its action was observed within a group of highly homologous members of the Gi subfamily with G alpha o1 being activated at lowest concentrations, whereas higher concentrations were necessary for the stimulation of G alpha i1 or transducin. We conclude that these compounds represent important tools for studying G protein-dependent cellular functions.

Similar mechanism of various lupus anticoagulants.

Potent lupus inhibitors from various patients were mixed with platelet free normal plasma and were compared in activated partial thromboplastin time (APTT), dilute prothrombin time (dil. PT), kaolin clotting time (KCT), contact product clotting time (CPCT), and Russell viper venom clotting time (RVVCT) tests. In the last three tests platelets and platelet lipid substitutes were avoided to enhance the sensitivities of these tests for the lupus anticoagulant. Correlations between the KCT and the other tests were mostly good, indicating that different lupus inhibitors functioned by a similar mechanism. There was no significant trend between particular clinical symptoms and individual coagulation test combinations. The KCT was found to be the most sensitive test for the lupus inhibitor, followed by the CPCT, RVVCT, dil. PT and APTT tests. Activated platelets tended to correct the APTT lupus inhibitor defect in all except the strongest inhibitor cases.

Diagnostic methodologies for circulating anticoagulants.

Various types of circulating anticoagulant are encountered in coagulation testing laboratories. Those associated with bleeding often cause problems in diagnosis. The most common type of acquired coagulation inhibitor not associated with bleeding is the so-called lupus anticoagulant (LA). Differing from SLE which occurs predominantly in women, primary LA occurs both in females and males. LA are now frequently sought in patients with recurrent foetal losses and acquired thrombotic problems as a causative factor, whereas in the past they were regarded as a laboratory nuisance. Due to the complicating effect of inhibitors on clotting tests, diagnosis of various coagulation inhibitors remains difficult. There may also be significant overlap between different types of inhibitors. With the recent interest shown in LA, almost all non-specific inhibitors tend to be classed as LA. LA are defined as phospholipid-interfering antibodies. Current criteria have recently been confirmed and include screening with phospholipid-responsive tests, abnormal mixing studies and correction with phospholipids. However it is becoming clear that even LA as defined may be heterogeneous. Most LA are not directed at negatively-charged phospholipids alone as originally suggested, but rather at complexes of either beta-2-glycoprotein 1 or prothrombin with such phospholipids. There may also be other lipid-associated antigens involved. Although earlier work suggested that all LA functioned through a similar mechanism, there is now some preliminary evidence suggesting that various subclasses of LA may account for discrepant results sometimes obtained with different clotting tests. A variety of improvements to the basic screening tests for LA (APTT, KCT, DTTI and DRVVT) have recently been suggested.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterisation and some properties of the protein C activator from Agkistrodon Contortrix Contortrix venom.

The protein C activator (PCA) detectable in the venom of Agkistrodon Contortrix Contortrix (ACCV, Southern Copperhead) by specific immunochromometric assay and anticoagulant activity has been isolated and partially characterized. Chromatography of the crude venom on SP-Sephadex followed by Con A Sepharose and finally on hydroxylapatite was necessary to achieve an electrophoretically - pure product. The isolated PCA is a single chain glycoprotein with strong positive charge and an apparent molecular weight of 36,000. It had an immediate-inhibiting effect on the activated partial thromboplastin time (APTT) of normal plasma with no noticeable effect on the prothrombin time. Its prolonging effect on the APTT was dependent on protein C and it appeared to interfere with the contact mechanism rather than with factors V and VIII. It had enzymatic activity on some tripeptide chromogenic substrates sensitive to thrombin and kallikrein. When mixed with normal plasma it generated activity on substrates sensitive to activated protein C and should be useful for studies of protein C.

Solvent extraction of test plasmas for improved recovery of lupus anticoagulant activity.

Phospholipid procoagulant material mainly derived from platelets interferes or "bypasses" the more specific tests for lupus anticoagulants. Such material in test plasmas can be inactivated with recovery of lupus anticoagulant activity by simple extraction with chloroform. This solvent treatment damages mainly factors VIII, V, VII and IX. Ether and various other solvents were less damaging to these clotting factors but not quite as effective as chloroform in the recovery of lupus anticoagulant activity.