COVID-19 and tuberculosis in South Africa: A dangerous combination.

There has been much speculation during the past week about the catastrophe that awaits once coronavirus disease 2019 (COVID-19) establishes itself in the poorest communities of South Africa (SA) and, importantly, in informal settlements. Evidence to date suggests that COVID-19 is efficiently passed from infected individuals via large droplets and hard-surface fomites.

MRI findings in calcific deposits in and around shoulder: atypical locations beyond supraspinatus.

Hydroxyapatite deposition disease is a spectrum of disorders including calcific tendinitis, calcific periarthritis, and hydroxyapatite-induced arthritis. These usually present with acute onset of pain, and clinically, may be confused with acute infection or adhesive capsulitis. Supraspinatus calcific tendinitis and its imaging appearance is well known; however, calcific deposits may occur less commonly in other structures in and around the shoulder joint. We describe nine cases of relatively uncommon locations of calcific deposits around the shoulder with their imaging appearances.

Pilot evaluation of a second-generation electronic pill box for adherence to Bedaquiline and antiretroviral therapy in drug-resistant TB/HIV co-infected patients in KwaZulu-Natal, South Africa.

BACKGROUND: The introduction of Bedaquiline, the first new antimycobacterial drug in over 40 years, has highlighted the critical importance of medication adherence in drug-resistant tuberculosis (DR-TB) treatment to prevent amplified drug-resistance and derive sustained benefit. Real-time electronic dose monitoring (EDM) accurately measures adherence and allows for titration of adherence support for anti-retroviral therapy (ART). The goal of this study was to evaluate the accuracy and acceptability of a next-generation electronic pillbox (Wisepill RT2000) for Bedaquiline-containing TB regimens. METHODS: Eligible patients were DR-TB/HIV co-infected adults hospitalized for the initiation of Bedaquiline-containing treatment regimens in KwaZulu-Natal, South Africa. A one-way crossover design was used to evaluate levels of adherence and patient acceptance of EDM. Each patient was given a Wisepill device which was filled with ART, Levofloxacin or Bedaquiline over three consecutive weeks. Medication adherence was measured using Wisepill counts, patient-reported seven-day recall, and weekly pill count. An open-ended qualitative questionnaire at the end of the study evaluated participant acceptability of the Wisepill device. RESULTS: We enrolled 21 DR-TB/HIV co-infected inpatients admitted for the initiation of Bedaquiline from August through September 2016. In aggregate patients were similarly adherent to Bedaquiline (100%) compared to Levofloxacin (100%) and ART (98.9%) by pill count. Wisepill was more sensitive (100%) compared to seven-day recall (0%) in detecting non-adherence events (p = 0.02). Patients reported positive experiences with Wisepill and expressed willingness to use the device during a full course of DR-TB treatment. There were no concerns about stigma, confidentiality, or remote monitoring. CONCLUSION: In this pilot study patients were highly adherent to Bedaquiline by all adherence measures. However, there was lower adherence to ART by pill count and Wisepill suggesting a possible challenge for adherence with ART. The use of EDM identified significantly more missed doses than seven-day recall. Wisepill was highly acceptable to DR-TB/HIV patients in South Africa, and is a promising modality to support and monitor medication adherence in complex treatment regimens.

Training social workers to enhance patient-centered care for drug-resistant TB-HIV in South Africa.

KwaZulu-Natal, South Africa, is the epicenter of an epidemic of drug-resistant tuberculosis (DR-TB) and human immunodeficiency virus (HIV) co-infection, characterized by low rates of medication adherence and retention in care. Social workers may have a unique role to play in improving DR-TB-HIV outcomes. We designed, implemented and evaluated a model-based pilot training course on patient-centered care, treatment literacy in DR-TB and HIV coinfection, patient support group facilitation, and self-care. Ten social workers participated in a 1-day training course. Post-training questionnaire scores showed significant overall gains (P = 0.003). A brief training intervention may be a useful and feasible way to engage social workers in patient-centered care for DR-TB and HIV coinfection.

Le KwaZulu-Natal, en Afrique du Sud, est l'épicentre d'une épidémie de coïnfection par la tuberculose pharmacorésistante (TB-DR) et le virus de l'immunodéficience humaine caractérisée par des taux faibles d'adhérence aux médicaments et de rétention en soins. Les travailleurs sociaux pourraient avoir un rôle unique dans l'amélioration des résultats de la coïnfection TB-DR et VIH. Nous avons conçu, mis en œuvre et évalué une formation pilote basée sur un modèle de soins centré sur le patient, de connaissance du traitement de la coïnfection TB-DR et VIH, de facilitation des groupes de soutien aux patients et de soins auto-administrés. Dix travailleurs sociaux ont participé à une formation d'un jour. Les scores des questionnaires après la formation ont montré des gains d'ensemble significatifs (P = 0,003). Une brève intervention de formation pourrait être une façon utile et faisable d'engager les travailleurs sociaux dans la prise en charge centrée sur le patient coïnfecté par la TB-DR et le VIH.

KwaZulu-Natal, en Suráfrica, es el epicentro de una epidemia de coinfección por el virus de la inmunodeficiencia humana (VIH) y la tuberculosis farmacorresistente (TB-DR), que se caracteriza por bajas tasas de cumplimiento terapéutico y una deficiente retención en la atención. Los trabajadores sociales pueden cumplir una función muy útil en el mejoramiento de los desenlaces clínicos de estos casos. En el presente artículo se describe el diseño, la ejecución y la evaluación de un curso experimental de capacitación a partir de un modelo, sobre la atención centrada en el paciente, la divulgación terapéutica relacionada con la coinfección por el VIH y la TB-DR, la facilitación en grupos de apoyo de pacientes y la autoasistencia. Diez trabajadores sociales participaron en un curso de capacitación de un día de duración. La puntuación de los cuestionarios posteriores a la capacitación reveló progresos notables en general (P = 0,003). Una intervención breve de capacitación puede representar un medio útil y viable para fomentar la participación de los trabajadores sociales en la atención centrada en el paciente de los casos de coinfección por el VIH y la TB-DR.

Household context and psychosocial impact of childhood multidrug-resistant tuberculosis in KwaZulu-Natal, South Africa.

SETTING: Referral hospital for drug-resistant tuberculosis (TB) in KwaZulu-Natal, South Africa. OBJECTIVES: We conducted interviews with primary care givers of children admitted with multidrug-resistant TB (MDR-TB) during a 3-month period in 2015 to identify broader household challenges. RESULTS: We interviewed 26 care givers, most of whom were women (85%). Most households had been decimated by TB/MDR-TB and human immunodeficiency virus (HIV) infection, and were dependent upon government grants. In 54% of cases, parents were absent due to illness or death, or their whereabouts were not known. The median age of the children treated for MDR-TB was 8 years (range 2-14); 72% were HIV-co-infected. Four themes emerged in the interviews: 1) the psychosocial impact of hospitalisation and separation on the child and the household, 2) the psychosocial impact of MDR-TB on children and 3) on care givers, and 4) the economic hardship of affected households. Children had to contend with multiple diseases and medications, and personal family losses; they faced behavioural, emotional and cognitive difficulties. Care givers were often anxious and concerned about the child's longer-term prospects, while the cost of hospital visits exacerbated the pre-existing economic vulnerability of affected households. CONCLUSION: The socio-economic impact of childhood MDR-TB reverberates beyond diseased children to their affected households. Enhanced social protection, psychosocial support and treatment literacy would create the foundations for family-centred care.

Defining the research agenda to measure and reduce tuberculosis stigmas.

Crucial to finding and treating the 4 million tuberculosis (TB) patients currently missed by national TB programmes, TB stigma is receiving well-deserved and long-delayed attention at the global level. However, the ability to measure and evaluate the success of TB stigma-reduction efforts is limited by the need for additional tools. At a 2016 TB stigma-measurement meeting held in The Hague, The Netherlands, stigma experts discussed and proposed a research agenda around four themes: 1) drivers: what are the main drivers and domains of TB stigma(s)?; 2) consequences: how consequential are TB stigmas and how are negative impacts most felt?; 3) burden: what is the global prevalence and distribution of TB stigma(s) and what explains any variation? 4): intervention: what can be done to reduce the extent and impact of TB stigma(s)? Each theme was further subdivided into research topics to be addressed to move the agenda forward. These include greater clarity on what causes TB stigmas to emerge and thrive, the difficulty of measuring the complexity of stigma, and the improbability of a universal stigma 'cure'. Nevertheless, these challenges should not hinder investments in the measurement and reduction of TB stigma. We believe it is time to focus on how, and not whether, the global community should measure and reduce TB stigma.

Tuberculosis stigma as a social determinant of health: a systematic mapping review of research in low incidence countries.

Tuberculosis (TB)-related stigma is an important social determinant of health. Research generally highlights how stigma can have a considerable impact on individuals and communities, including delays in seeking health care and adherence to treatment. There is scant research into the assessment of TB-related stigma in low incidence countries. This study aimed to systematically map out the research into stigma. A particular emphasis was placed on the methods employed to measure stigma, the conceptual frameworks used to understand stigma, and whether structural factors were theorized. Twenty-two studies were identified; the majority adopted a qualitative approach and aimed to assess knowledge, attitudes, and beliefs about TB. Few studies included stigma as a substantive topic. Only one study aimed to reduce stigma. A number of studies suggested that TB control measures and representations of migrants in the media reporting of TB were implicated in the production of stigma. The paucity of conceptual models and theories about how the social and structural determinants intersect with stigma was apparent. Future interventions to reduce stigma, and measurements of effectiveness, would benefit from a stronger theoretical underpinning in relation to TB stigma and the intersection between the social and structural determinants of health.

Provider perspectives on drug-resistant tuberculosis and human immunodeficiency virus care in South Africa: a qualitative case study.

OBJECTIVE: To examine influences on health care workers' (HCWs') capacity to deliver health care for multi- and/or extensively drug-resistant tuberculosis (MDR/XDR-TB) and human immunodeficiency virus (HIV) infection in South Africa. DESIGN: Qualitative data were collected via group and individual interviews with a purposive sample of 17 HCWs at a centralised, tertiary TB facility and analysed using grounded theory. RESULTS: Four themes were identified: 1) personal infection control practices among HCWs may be weakened by a workplace culture comprising low motivation, disparate risk perceptions and practices across workforce hierarchies, physical discomfort, and problems managing patients with treatment-induced hearing loss. 2) Patient-provider interactions are likely stronger among nurses, and in HIV vs. MDR/XDR-TB service delivery, due to greater attention to patient empowerment and support. Stigma associated with MDR/XDR-TB, considered worse than HIV, may be perpetuated within non-specialised facilities less familiar with MDR/XDR-TB. 3) HCWs who struggle with the daily tedium of MDR/XDR-TB treatment supervision are becoming increasingly supportive of treatment literacy and self-administration. 4) Effective integration of HIV and MDR/XDR-TB services may be impeded by administrative restrictions, workplace norms and provider mindsets. CONCLUSION: Comprehensive, decentralised management of MDR/XDR-TB and HIV coinfection requires the creation of patient-provider trust and treatment literacy in MDR/XDR-TB programmes, and defying workplace norms that could provoke nosocomial TB exposure and fragmented service provision.

Re-inventing adherence: toward a patient-centered model of care for drug-resistant tuberculosis and HIV.

BACKGROUND: Despite renewed focus on molecular tuberculosis (TB) diagnostics and new antimycobacterial agents, treatment outcomes for patients co-infected with drug-resistant TB and human immunodeficiency virus (HIV) remain dismal, in part due to lack of focus on medication adherence as part of a patient-centered continuum of care. OBJECTIVE: To review current barriers to drug-resistant TB-HIV treatment and propose an alternative model to conventional approaches to treatment support. DISCUSSION: Current national TB control programs rely heavily on directly observed therapy (DOT) as the centerpiece of treatment delivery and adherence support. Medication adherence and care for drug-resistant TB-HIV could be improved by fully implementing team-based patient-centered care, empowering patients through counseling and support, maintaining a rights-based approach while acknowledging the responsibility of health care systems in providing comprehensive care, and prioritizing critical research gaps. CONCLUSION: It is time to re-invent our understanding of adherence in drug-resistant TB and HIV by focusing attention on the complex clinical, behavioral, social, and structural needs of affected patients and communities.

Integrating patients' perspectives into integrated tuberculosis-human immunodeficiency virus health care.

BACKGROUND: Escalating rates of tuberculosis-human immunodeficiency virus (TB-HIV) co-infection call for improved coordination of TB and HIV health care services in high-burden countries such as South Africa. Patient perspectives, however, are poorly understood in the context of current integration efforts. METHOD: Under a qualitative research framework, we interviewed 40 HIV-positive adult TB patients and eight key-informant health care workers across three clinics in KwaZulu-Natal Province to explore non-clinical and non-operational aspects of TB-HIV health care. FINDINGS: Qualitative analysis highlighted critical social and ethical considerations for the concurrent delivery of TB and HIV care. Co-infected patients navigating between TB and HIV programs are exposed to missed opportunities for TB and HIV service integration, fragmented or vertical care for their dual infections and contrasting experiences within TB and HIV clinics. These intersecting issues appear to affect patients' health-related decisions, particularly nondisclosure of HIV status to non-HIV health care workers and their preferences for integrated health care. CONCLUSION: Our study highlights the imperative to address service fragmentation, HIV medical confidentiality and provider mistrust within the health care system, and the cultural differences associated with TB and HIV disease control.

Case series of the long-term psychosocial impact of drug-resistant tuberculosis in HIV-negative medical doctors.

BACKGROUND: Health care workers (HCWs) are at greater risk for tuberculosis (TB), including multidrug-resistant TB (MDR-TB), compared to the general population. The psychosocial impact of nosocomial TB on HCWs has received little attention in the literature. METHODS: A retrospective medical record review from 1999 to 2003 found 15 HCWs who were treated for drug-resistant TB at a specialist hospital in South Africa. Five human immunodeficiency virus (HIV) negative doctors with no predisposing factors for drug resistance are included in this case series. We collectively present their clinical case histories based on medical records from 2000 to 2005, and explore the long-term psychosocial impact of TB from interviews conducted in 2009. RESULTS: Four doctors had primary MDR-TB and one had primary resistance to multiple first-line drugs. Time from symptom onset to commencement of effective treatment ranged from 8 to 39 weeks. Time for bacteriological confirmation of drug-resistant TB ranged from 6 to 24 weeks. All were cured within 3 years of initial presentation. Content analysis of follow-up interviews revealed five main themes: 1) prolonged morbidity, 2) psychological impact, 3) poor infection control, 4) weak support structures and 5) attrition from the field. CONCLUSION: Themes emergent from this case series encourage prioritisation of TB infection control education and practice to minimise HCW morbidity and prevent HCW attrition from high-burden resource-constrained settings.

Dynamic postural stability during advancing pregnancy.

UNLABELLED: Pregnant women are at an increased risk of experiencing a fall. Numerous anatomical, physiological, and hormonal alterations occur during pregnancy, but the influence of these factors on dynamic postural stability has not been explored. The purpose of this study was to examine dynamic postural stability in pregnant women during their second and third trimesters as well as in a group of non-pregnant control women. METHODS: Eighty-one women (41 pregnant, 40 controls) participated stood on a force plate that translated anteroposteriorly at small, medium, and large magnitudes. Reaction time and center of pressure (COP) movement during the translations were analyzed. Trimester, perturbation direction, and perturbation magnitude were the independent variables in a mixed-model analysis of variance on each of the following dependent variables: reaction time, initial sway, total sway, and sway velocity. RESULTS: Reaction time to the perturbation was not significantly different between the groups. Initial sway, total sway, and sway velocity were significantly less during the third trimester than during the second trimester and when compared to the non-pregnant controls (P<0.05). No differences were found in any of the measures between the pregnant women in their second trimesters and the control group. CONCLUSION: Alterations in sway responses to perturbations are seen in the third trimester in healthy women with uncomplicated pregnancies. Further study is needed to examine the biomechanical and physiological reasons behind this altered dynamic postural stability.

Dynamic postural stability in pregnant fallers and non-fallers.

OBJECTIVE: To compare dynamic postural stability in pregnant women who have fallen during their pregnancies with those who have not, and with a group of non-pregnant women. DESIGN: The study was both longitudinal and cross-sectional. A cohort of pregnant women were followed through their second and third trimesters. A non-pregnant control group was used for comparison. SETTING: University-based laboratory. POPULATION: A total of 81 women (41 pregnant and 40 controls) participated. Twenty-nine pregnant women completed the protocol. METHODS: Data were collected on the pregnant women in the middle of their second and third trimesters. Pregnant women were surveyed about their daily activities, exercise participation, and fall history. Postural reaction time and centre of pressure (COP) movement data, in response to translational perturbations, were collected using a force plate. A mixed-model analysis of variants (ANOVA) was performed on each of the dependent variables (alpha = 0.05). Chi-square analysis was performed to determine if exercise participation altered the likelihood of a subject experiencing a fall (alpha = 0.05). MAIN OUTCOME MEASURES: Reaction time, initial sway, total sway, and sway velocity. RESULTS: Fifty-two percent of our pregnant subjects experienced a fall. Initial sway response, total sway, and sway velocity were smaller in the pregnant fallers than in the non-fallers and control participants (P < 0.05). Thirty-one of the pregnant subjects participated in regular exercise. Sedentary pregnant women were more likely to experience a fall than those who exercised. CONCLUSIONS: Dynamic balance is altered in pregnant women who have fallen compared with non-fallers and controls. Exercise may play a role in fall prevention in pregnant women.

Adherence to treatment for latent tuberculosis infection: systematic review of studies in the US and Canada.

BACKGROUND: There is renewed attention to the critical role of successfully treating latent tuberculosis infection (LTBI) in reducing the overall impact of tuberculosis (TB). However, levels of treatment adherence are consistently low in industrialized countries such as the United States and Canada. OBJECTIVE: A systematic review of studies in the US and Canada was undertaken to analyze measurement of adherence to treatment of LTBI (TLTBI), TLTBI completion rates, predictors of TLTBI adherence and TLTBI adherence interventions. METHODS: PUBMED, MEDLINE and PsycINFO electronic databases were searched for quantitative studies published between 1997 and 2007. Full texts of articles were reviewed for data abstraction and studies were critically examined for their methodology and rigor. The present review presents outcomes from 78 studies. RESULTS: Adherence and completion rates of TLTBI are suboptimal across high-risk groups, regardless of regimen. Associations between adherence and patient factors, clinic facilities or treatment characteristics were found to be inconsistent across studies. Several adherence interventions have been developed to improve TLTBI adherence in the US and Canada; however, no single intervention has shown consistent effectiveness. CONCLUSION: LTBI must be effectively treated if the goal of TB elimination is to be realized. Consistently employing tools for measuring and improving adherence is fundamental. Identifying barriers to adherence and treatment completion will facilitate the development of effective, appropriate interventions. A 'one-size-fits-all' approach to treatment for TLTBI adherence is not likely to succeed across all settings. Innovative approaches can inspire future interventions and suggest solutions for the current problems facing LTBI programs and their patients.

Proteasomal activity in placentas from women with preeclampsia and intrauterine growth restriction: implications for expression of HIF-alpha proteins.

Hypoxia-inducible transcription factor-1alpha and -2alpha (HIF-alpha) proteins and regulated genes are increased in preeclamptic (PE) placentas. Although placental hypoxia likely stabilizes HIF-alpha proteins, we previously reported that there is also a defect in oxygen-dependent reduction of HIF-alpha proteins in PE relative to normal pregnant (NP) placentas that could contribute to their over-expression. After a 4-h exposure to 2% oxygen, placental villous explants were exposed to 21% oxygen over 90 min. As assessed by Western analysis, the defective oxygen-dependent reduction of HIF-1alpha protein in villous explants from PE placenta was unaffected by the protein synthesis inhibitor, cycloheximide. However, after incubation with the proteasomal inhibitor, clasto-lactacystin, oxygen-dependent reduction of HIF-1alpha protein was markedly and similarly impaired in the villous explants from both normal and PE placentas. Thus, impairment of protein degradation rather than increased synthesis causes inadequate oxygen-dependent reduction of HIF-1alpha protein in PE placentas. Immunoprecipitation studies revealed comparable association of HIF-1alpha with von Hippel Lindau (VHL) protein in placentas from NP and PE women. Furthermore, prolyl hydroxylase-3 protein was appropriately upregulated in the PE placentas as determined by Western analysis paralleling the increases of HIF-alpha proteins. These results suggest that molecular events leading to the formation of the HIF-1alpha:VHL:ubiquitin ligase complex are most likely not impaired in PE placentas. Finally, proteasomal trypsin, chymotrypsin, and peptidyl glutamyl-like activities were significantly reduced by approximately 1/3 in PE placentas by using specific peptide substrates coupled to a fluorescent tag. Unexpectedly, however, they were even further decreased in placentas from normotensive women delivering growth restricted babies >37 weeks gestation-placentas which do not have elevated HIF-alpha proteins. In conclusion, accumulation of HIF-alpha proteins in PE placentas may occur as a consequence of both increased formation secondary to relative ischemia/hypoxia and reduced degradation after reperfusion/oxygenation consequent to proteasomal dysfunction. In contrast, in placentas from normotensive women delivering growth restricted babies >37 weeks gestation, proteasomal activity, albeit markedly reduced, is adequate to cope with degradation of HIF-alpha proteins, which have not been increased by an hypoxic environment.

HIV testing and disclosure: a qualitative analysis of TB patients in South Africa.

In South Africa, more than 60% of TB patients have HIV co-infection. Voluntary counseling and testing (VCT) is critical to effective HIV prevention, and TB facilities are optimal venues for delivery of these services. This study employed qualitative research methods to explore the decision-making processes for HIV testing and serostatus disclosure by 21 patients hospitalized with multi/extensively-drug resistant TB (M/XDR-TB) in Durban, KwaZulu Natal. Data collected from in-depth interviews characterized 3 broad themes: HIV testing history, experiences and perceptions of stigma and disclosure, and the relationship between TB and HIV/AIDS. Fear of AIDS-related stigma, the singular stress of TB infection, the absence of partner's consent, asymptomatic or incurable disease, and uncertainty about subsequent eligibility for antiretroviral treatment while still receiving TB treatment were identified as potential barriers to the uptake of VCT. HIV serostatus disclosure was impeded by the felt stigma of a 'discreditable' infection, manifested by social rejection and discrimination. The public disclosure of TB illness helped relieve some co-infected patients' overall burden of stigma through a process of 'covering'. HIV prevention [corrected] measures such as VCTare likely to be more effective within TB facilities if greater sensitivity is paid to TB patients' specific social issues and perceptions. These patients are not only at greater risk for HIV co-infection but also for experiencing the double stigma of TB and HIV/AIDS.

Expression of von Hippel Lindau (pVHL) protein in placentae from normal pregnant women and women with preeclampsia.

The hypoxia inducible transcription factors, HIF-1alpha and -2alpha proteins, are overexpressed in placentae from women with preeclampsia (Biol Reprod 2001;64:499-506; Biol Reprod 2001;64:1019-1020). Normally, these proteins are regulated in an oxygen-dependent manner being rapidly degraded by the ubiquitin-mediated proteasomal pathway. Recent studies have shown that the tumor suppressor protein, von Hippel Lindau (VHL), targets HIF for ubiquitinylation under nonhypoxic conditions. The objectives of the present work were: (1) to investigate VHL protein expression in normal pregnant (NP), preeclamptic (PE), and preterm (without PE) placentae, (2) to test whether VHL protein is hypoxia inducible in term and first trimester placental villous explants, and (3) to analyze the ontogeny of VHL protein expression in the human placenta. To begin evaluating the potential contribution of VHL to HIF overexpression in preeclamptic placentae, we analyzed the levels of the VHL protein in both normal and preeclamptic placentae (n=7 each). We hypothesized a deficiency of VHL protein in preeclamptic placentae. Eight biopsy sites were tested in each placenta and protein extracts were made. Western analysis was performed using VHL specific antibodies. Human renal adenocarcinoma (ACHN) cell extracts and extracts from COS-7 cells transfected with a VHL expression vector were used as positive controls. In a total of 112 biopsy sites that were analyzed (56 each for normal and preeclamptic placentae), the composite densitometry ratios (PE/NP) for the long (28 kDa) and short (19 kDa) forms of VHL were 1.09+/-0.2 and 1.16+/-0.11, respectively (both p=NS vs 1.0). A ratio of 1.0 indicates equal expression by preeclamptic and normal placentae. The same placentae exhibited composite densitometry (PE/NP) ratios of 1.97+/-0.23 and 1.68+/-0.20 for HIF-1alpha and -2alpha proteins, respectively (both p<0.05 vs 1.0). In a separate analysis, the protein expression of the short form of VHL was also comparable among NP, PE and preterm (n=6) placentae. VHL immunoreactivity was localized to cells within the basal plate and the syncytiotrophoblast. Despite induction of HIF proteins by hypoxia in first and term villous explants, there was no significant upregulation of VHL proteins. Finally, the expression of both the short and long forms of VHL protein decreased with gestational age (both p<0.05 by ANOVA), and in villous tissue from first trimester placentae VHL immunoreactivity was predominantly localized to the cytotrophoblast. These results suggest that (1) deficiency of VHL protein does not account for HIF-alpha overexpression in preeclamptic placentae, (2) VHL protein is not regulated by hypoxia in either first trimester or term placental villous explants, and (3) VHL protein expression in the placenta decreases as a function of gestational age.

Evidence for the functional activity of hypoxia-inducible transcription factors overexpressed in preeclamptic placentae.

Placentas from women with preeclampsia overexpress the hypoxia-inducible transcription factor proteins, HIF-1alpha and -2alpha (Rajakumar 2001, Biol Reprod 64; p499-506 and p1019-1020). As a first step in evaluating whether HIF-alpha overexpressed in preeclamptic placentae is capable of transactivation, we tested its ability to bind to the DNA hypoxia response element (HRE). Six pairs of normal and preeclamptic placentae obtained by cesarean section were investigated. Three biopsy sites per placenta were analyzed. We first confirmed HIF-1alpha protein overexpression in the preeclamptic placentae using Western analysis. The ratios of the arbitrary densitometry units for HIF-1alpha protein from the preeclamptic and normal placentae (PE/NP) in the three biopsy sites were: 1.9 +/- 0.3, 1.7 +/- 0.2 and 1.8 +/- 0.2, each p < 0.05 vs 1.0. (A ratio of >1.0 indicates that HIF-1alpha protein expression in placentas of women with PE exceeds that in placentas of NP women.) Conventional methods for extracting nuclear proteins and subsequent analysis by electrophoretic mobility shift assay were not suited for the frozen, archived samples (data not shown). Therefore, we employed DNA affinity chromatography using a biotinylated oligonucleotide representing the HRE of the erythropoietin gene coupled to streptavidin-coated Dynabeads. The HRE-bound proteins were then characterized by Western blot analysis. The PE/NP ratios of HRE-bound HIF-1alpha in the three biopsy sites from the six pairs of normal and preeclamptic placentae were 1.7 +/- 0.2, 2.1 +/- 0.4 and 2.4 +/- 0.5, each p < 0.05 vs 1.0. Having established DNA-binding potential at least in vitro, we subsequently analyzed three proteins that have been shown to be regulated by HIF-alpha as downstream, molecular markers of HIF-1alpha activity in vivo. VEGF receptor Flt-1 and Flk-1 play key roles in angiogenesis. Tyrosine hydroxylase is the rate-limiting enzyme in catecholamine synthesis. All three genes contain functional HRE in their promoter sequences. Total proteins were extracted from the same biopsy samples that were used for total and HRE-bound HIF-1alpha. Using specific antibodies we performed Western analysis and the levels of these three proteins were quantitated. The Flt-1 and tyrosine hydroxylase proteins were significantly higher, and Flk-1 significantly lower in placentae from preeclamptic compared to normal pregnancies. In summary, HIF-1alpha protein overexpressed in preeclamptic placentae is capable of binding to its DNA recognition sequence in vitro, and modulates gene expression in vivo.