Jesse J. Kwiek

Androgen Receptor Phosphorylation REGULATION AND IDENTIFICATION OF THE PHOSPHORYLATION SITES

Activation of signal transduction kinase cascades has been shown to alter androgen receptor (AR) activity. Although it has been suggested that changes in AR phosphorylation might be directly responsible, the basal and regulated phosphorylations of the AR have not been fully determined. We have identified the major sites of AR phosphorylation on ARs expressed in COS-1 cells using a combination of peptide mapping, Edman degradation, and mass spectrometry. We describe the identification of seven AR phosphorylation sites, show that the phosphopeptides seen with exogenously expressed ARs are highly similar to those seen with endogenous ARs in LNCaP cells and show that specific agonists differentially regulate the phosphorylation state of endogenous ARs in LNCaP prostate cancer cells. Treatment of LNCaP cells with the synthetic androgen, R1881, elevates phosphorylation of serines 16, 81, 256, 308, 424, and 650. Ser-94 appears constitutively phosphorylated. Forskolin, epidermal growth factor, and phorbol 12-myristate 13-acetate increase the phosphorylation of Ser-650. The kinetics of phosphorylation of most sites in response to hormone or forskolin is temporally delayed, reaching a maximum at 2 h post-stimulation. The exception is Ser-81, which continues to display increasing phosphorylation at 6 h. These data provide a basis for analyzing mechanisms of cross-talk between growth factor signaling and androgen in prostate development, physiology, and cancer.

Maternal Syphilis Infection is Associated With Increased Risk of Mother-to-Child Transmission of HIV in Malawi

Objective:To determine the association between maternal syphilis and HIV mother-to-child transmission (MTCT). Design:Prospective cohort study. Methods:Pregnant women admitted at Queen Elizabeth Central Hospital (Malawi) in late third trimester were screened for HIV (by HIV rapid tests) and syphilis (by rapid plasma regain test and Treponema pallidum hemagglutination assay). HIV-infected women and their infants received nevirapine, according to the HIVNET 012 protocol. They were followed up at 6 and 12 weeks postpartum. Infant HIV infection was diagnosed by DNA PCR. Findings:Of the 1155 HIV-infected women enrolled, 1147 had syphilis test results, of whom 92 (8.0%) were infected. Only 751 HIV-positive women delivered live singleton infants who were tested for HIV at birth. Of these, 65 (8.7%) were HIV-infected, suggesting in utero (IU) HIV MTCT. Of the 686 infants who were HIV-negative at birth, 507 were successfully followed up. Of these, 89 (17.6%) became HIV-infected, suggesting intrapartum/postpartum (IP/PP) HIV MTCT. Maternal syphilis was associated with IU HIV MTCT, after adjusting for maternal log10 HIV-1 viral load and low birth weight (LBW) [adjusted relative risk (ARR), 2.77; 95% CI, 1.40–5.46]. Furthermore, maternal syphilis was associated with IP/PP HIV MTCT (ARR, 2.74; 95% CI, 1.58–4.74), after adjusting for recent fever, breast infection, LBW and maternal log10 HIV-1 viral load. Conclusion:Maternal syphilis is associated with IU and IP/PP HIV MTCT. Screening and early treatment of maternal syphilis during pregnancy may reduce pediatric HIV infections.

A randomized controlled pilot trial of azithromycin or artesunate added to sulfadoxine-pyrimethamine as treatment for malaria in pregnant women.

Objective New anti-malarial regimens are urgently needed in sub-Saharan Africa because of the increase in drug resistance. We investigated the safety and efficacy of azithromycin or artesunate combined with sulfadoxine-pyrimethamine used for treatment of malaria in pregnant women in Blantyre, Malawi. Methods/Findings This was a randomized open-label clinical trial, conducted at two rural health centers in Blantyre district, Malawi. A total of 141 pregnant women with uncomplicated Plasmodium falciparum malaria were recruited and randomly allocated to 3 treatment groups: sulfadoxine-pyrimethamine (SP; 3 tablets, 500 mg sulfadoxine and 25 mg pyrimethamine per tablet); SP plus azithromycin (1 g/day×2 days); or SP plus artesunate (200 mg/day×3 days). Women received two doses administered at least 4 weeks apart. Heteroduplex tracking assays were performed to distinguish recrudescence from new infections. Main outcome measures were incidence of adverse outcomes, parasite and fever clearance times and recrudescence rates. All treatment regimens were well tolerated. Two women vomited soon after ingesting azithromycin. The parasite clearance time was significantly faster in the SP-artesunate group. Recrudescent episodes of malaria were less frequent with SP-azithromycin [Hazard Ratio 0.19 (95% confidence interval 0.06 to 0.63)] and SP-artesunate [Hazard Ratio 0.25 (95% confidence interval 0.10 to 0.65)] compared with SP monotherapy. With one exception (an abortion in the SP-azithromycin group), all adverse pregnancy outcomes could be attributed to known infectious or obstetrical causes. Because of the small sample size, the effect on birth outcomes, maternal malaria or maternal anemia could not be evaluated. Conclusions Both SP-artesunate and SP-azithromycin appeared to be safe, well tolerated and efficacious for the treatment of malaria during pregnancy. A larger study is needed to determine their safety and efficacy in preventing poor birth outcomes. Trial Registration ClinialTrials.gov NCT00287300

Microbial Contamination of Human Milk Purchased Via the Internet

OBJECTIVE: To quantify microbial contamination of human milk purchased via the Internet as an indicator of disease risk to recipient infants. METHODS: Cross-sectional sample of human milk purchased via a popular US milk-sharing Web site (2012). Individuals advertising milk were contacted to arrange purchase, and milk was shipped to a rented mailbox in Ohio. The Internet milk samples (n = 101) were compared with unpasteurized samples of milk donated to a milk bank (n = 20). RESULTS: Most (74%) Internet milk samples were colonized with Gram-negative bacteria or had >104 colony-forming units/mL total aerobic count. They exhibited higher mean total aerobic, total Gram-negative, coliform, and Staphylococcus sp counts than milk bank samples. Growth of most species was positively associated with days in transit (total aerobic count [log10 colony-forming units/mL] β = 0.71 [95% confidence interval: 0.38–1.05]), and negatively associated with number of months since the milk was expressed (β = −0.36 [95% confidence interval: −0.55 to −0.16]), per simple linear regression. No samples were HIV type 1 RNA-positive; 21% of Internet samples were cytomegalovirus DNA-positive. CONCLUSIONS: Human milk purchased via the Internet exhibited high overall bacterial growth and frequent contamination with pathogenic bacteria, reflecting poor collection, storage, or shipping practices. Infants consuming this milk are at risk for negative outcomes, particularly if born preterm or are medically compromised. Increased use of lactation support services may begin to address the milk supply gap for women who want to feed their child human milk but cannot meet his or her needs.

Umbilical Cord–Blood Infections with Plasmodium falciparum Malaria Are Acquired Antenatally in Kenya

BACKGROUND It is unknown whether the presence of Plasmodium falciparum malaria parasites in umbilical cord blood denotes infection acquired antenatally or contamination with infected maternal blood at delivery. METHODS Parasites were quantified by real-time quantitative polymerase chain reaction (RTQ-PCR) and were genotyped in paired maternal- and cord-blood samples obtained from 632 pregnant Kenyan women and their newborns. Placental alkaline phosphatase (PLAP) and polyclonal immunoglobulin E levels were also quantified in paired maternal- and cord-blood samples, as markers of admixture of maternal blood with cord blood. RESULTS Sixty-six cord-blood samples (10.4%) contained falciparum malaria, as detected by RTQ-PCR. For 25 of the infected cord-blood samples, either absence of infection was noted in paired maternal-blood samples at delivery (n=16) or amplicon levels in cord-blood samples were 10-fold higher than those in maternal-blood samples (n=9). Of the paired maternal- and cord-blood samples that were both infected, 57% showed discordant malaria parasite strains. There was no correlation between maternal parasitemia and levels of PLAP and immunoglobulin E in cord blood. PLAP levels, however, were significantly higher in cord-blood samples obtained from newborns of primigravid or secundigravid women with placental malaria, compared with cord-blood samples obtained from newborns of women without placental malaria or multigravid women. These findings indicate that parity and placental malaria are risk factors for maternofetal transfusion. CONCLUSIONS Malaria parasites identified in cord blood are acquired antenatally by transplacental transmission of infected erythrocytes. Primigravid and secundigravid women with placental malaria are at increased risk for congenital infection.

The Genetic Bottleneck in Vertical Transmission of Subtype C HIV-1 Is Not Driven by Selection of Especially Neutralization-Resistant Virus from the Maternal Viral Population

ABSTRACT Subtype C human immunodeficiency virus type 1 (HIV-1C) continues to cause the majority of new cases of mother-to-child transmission (MTCT), and yet there are limited data on HIV-1C transmission. We amplified env from plasma RNA for 19 HIV-1C MTCT pairs, 10 transmitting in utero (IU) and 9 transmitting intrapartum (IP). There was a strong genetic bottleneck between all mother-infant pairs, with a majority of transmission events involving the transmission of a single virus. env genes of viruses transmitted to infants IP, but not IU, encoded Env proteins that were shorter and had fewer putative N-linked glycosylation sites in the V1-V5 region than matched maternal sequences. Viruses pseudotyped with env clones representative of each maternal and infant population were tested for neutralization sensitivity. The 50% inhibitory concentration of autologous serum was similar against both transmitted (infant) and nontransmitted (maternal) viruses in a paired analysis. Mother and infant Env proteins were also similar in sensitivity to soluble CD4, to a panel of monoclonal antibodies, and to heterologous HIV-1C sera. In addition, there was no difference in the breadth or potency of neutralizing antibodies between sera from 50 nontransmitting and 23 IU and 23 IP transmitting HIV-1C-infected women against four Env proteins from heterologous viruses. Thus, while a strong genetic bottleneck was detected during MCTC, with viruses of shorter and fewer glycosylation sites in env present in IP transmission, our data do not support this bottleneck being driven by selective resistance to antibodies.

Efficacy of DB289 in Thai Patients with Plasmodium vivax or Acute, Uncomplicated Plasmodium falciparum Infections

BACKGROUND DB289 is the orally active prodrug of the diamidine DB75, which was developed for the treatment of human African trypanosomiasis. METHODS We tested the safety and efficacy of DB289 for the treatment of Plasmodium vivax and acute, uncomplicated P. falciparum infections in an open-label pilot study at the Hospital for Tropical Diseases in Bangkok. Nine patients with P. vivax infections and 23 patients with P. falciparum infections were admitted and treated with 100 mg of DB289 given orally twice a day for 5 days and were followed for 28 days. Patients with P. vivax infections were also treated with primaquine on days 10-23. RESULTS All patients cleared parasites by day 7, with a mean+/-SD clearance time of 43+/-41 h. One patient with a P. vivax infection had a recurrence of parasitemia on day 9. Of the 23 patients with P. falciparum infections, 3 had recurrences of parasitemia caused by P. vivax and 2 had recurrences of parasitemia caused by P. falciparum. In only 1 of 2 recurrences of parasitemia caused by P. falciparum were the parasites genotypically distinct from the infecting parasites the patient had at enrollment, which means there was a 96% cure rate. CONCLUSIONS DB289 is a promising new antimalarial compound that could become an important component of new antimalarial combinations.

Maternal-fetal microtransfusions and HIV-1 mother-to-child transmission in Malawi

Background Between 25% and 35% of infants born to HIV-infected mothers become HIV-1 infected. One potential route of mother-to-child transmission (MTCT) could be through a breakdown in the placental barrier (i.e., maternal–fetal microtransfusions). Methods and Findings Placental alkaline phosphatase (PLAP) is a 130-kD maternal enzyme that cannot cross the intact placental barrier. We measured PLAP activity in umbilical vein serum as an indicator of maternal–fetal microtransfusion, and related this to the risk of HIV-1 MTCT. A case-cohort study was conducted of 149 women randomly selected from a cohort of HIV-1-infected pregnant Malawians; these women served as a reference group for 36 cases of in utero MTCT and 43 cases of intrapartum (IP) MTCT. Cord PLAP activity was measured with an immunocatalytic assay. Infant HIV status was determined by real-time PCR. The association between cord PLAP activity and HIV-1 MTCT was measured with logistic regression using generalized estimating equations. Among vaginal deliveries, PLAP was associated with IP MTCT (risk ratio, 2.25 per log10 ng/ml PLAP; 95% confidence interval, 0.95–5.32) but not in utero MTCT. In a multivariable model adjusted for HIV-1 RNA load, chorioamnionitis, and self-reported fever, the risk of IP MTCT almost tripled for every log10 increase in cord PLAP activity (risk ratio, 2.87; 95% confidence interval, 1.05–7.83). Conclusion These results suggest that during vaginal deliveries, placental microtransfusions are a risk factor for IP HIV-1 MTCT. Future studies are needed to identify factors that increase the risk for microtransfusions in order to prevent IP HIV-1 MTCT.

Malaria during pregnancy and foetal haematological status in Blantyre, Malawi

BackgroundAlthough maternal anaemia often stems from malaria infection during pregnancy, its effects on foetal haemoglobin levels are not straightforward. Lower-than-expected cord haemoglobin values in malarious versus non-malarious regions were noted by one review, which hypothesized they resulted from foetal immune activation to maternal malaria. This study addressed this idea by examining cord haemoglobin levels in relation to maternal malaria, anaemia, and markers of foetal immune activation.MethodsCord haemoglobin levels were examined in 32 malaria-infected and 58 uninfected women in Blantyre, Malawi, in relation to maternal haemoglobin levels, malaria status, and markers of foetal haematological status, hypoxia, and inflammation, including TNF-α, TGF-β, and ferritin. All women were HIV-negative.ResultsAlthough malaria was associated with a reduction in maternal haemoglobin (10.8 g/dL vs. 12.1 g/dL, p < 0.001), no reduction in cord haemoglobin and no significant relationship between maternal and cord haemoglobin levels were found. Cord blood markers of haematological and hypoxic statuses did not differ between malaria-infected and uninfected women. Maternal malaria was associated with decreased TGF-β and increased cord ferritin, the latter of which was positively correlated with parasitaemia (r = 0.474, p = 0.009). Increased cord ferritin was associated with significantly decreased birth weight and gestational length, although maternal and cord haemoglobin levels and malaria status had no effect on birth outcome.ConclusionIn this population, cord haemoglobin levels were protected from the effect of maternal malaria. However, decreased TGF-β and elevated ferritin levels in cord blood suggest foetal immune activation to maternal malaria, which may help explain poor birth outcomes.

Estimating True Antimalarial Efficacy by Heteroduplex Tracking Assay in Patients with Complex Plasmodium falciparum Infections

ABSTRACT Heteroduplex tracking assays (HTAs) of Plasmodium falciparum merozoite surface protein 1 block-2 were used to assess complexity of infection and treatment efficacy in a trial of three antimalarial treatments in 141 Malawian pregnant women. An elevated complexity of infection (COI) was associated with anemia, parasite burden, and human immunodeficiency virus infection but was not associated with age or gravidity. Comparisons of HTA patterns before and after treatment allowed the classification of 20 of 30 (66%) recurrent episodes as either definite treatment failures or reinfections. An elevated COI was strongly associated with treatment failure (P = 0.003). An algorithm was developed to assign a probability of failure for the 10 indeterminate participants, some of whose infections shared a single variant of high prevalence (>10%). By summing these probabilities, treatment efficacy was estimated.