Alcoholism and Intimate Partner Violence: Effects on Children’s Psychosocial Adjustment

It is widely recognized that alcoholism and relationship violence often have serious consequences for adults; however, children living with alcoholic parents are susceptible to the deleterious familial environments these caregivers frequently create. Given the prevalence of IPV among patients entering substance abuse treatment, coupled with the negative familial consequences associated with these types of behavior, this review explores what have been, to this point, two divergent lines of research: (a) the effects of parental alcoholism on children, and (b) the effects of children’s exposure to intimate partner violence. In this article, the interrelationship between alcoholism and IPV is examined, with an emphasis on the developmental impact of these behaviors (individually and together) on children living in the home and offers recommendations for future research directions

Elucidation of Gene Essentiality and Genetic Determinants of Intrinsic p-Aminosalicylic Acid Resistance in Mycobacterium Kansasii

Mycobacterium kansasii (Mk) is an opportunistic pathogen capable of causing tuberculosis-like pulmonary disease in immunocompromised individuals and those with other risk factors including chronic obstructive pulmonary disease or malignancy. Mk is frequently isolated from man-made water sources where it forms resilient biofilms, posing a health risk to these susceptible individuals. Despite its medical relevance as an environmental pathogen, and a close phylogenetic relationship with the obligate pathogen Mycobacterium tuberculosis (Mtb), few studies to date have probed the molecular biology and genetics of Mk. Here, we sought to apply a transposon (Tn) mutagenesis tool to dissect gene function in Mk using both deep sequencing and forward genetics approaches. Through deep sequencing of 2.5 million Mk Tn mutants, we defined the in vitro essential gene set of Mk and then compared these results to essentiality data available for several other species of mycobacteria. Importantly, this analysis shows that several orthologs of essential Mtb genes encoding drug target candidates were identified as nonessential in Mk, suggesting they may not be viable drug targets in Mk. Next, we carried out a forward genetic screen of 10,000 individually arrayed Mk Tn mutants and identified those displaying increased susceptibility to the antitubercular agent p-aminosalicylic acid (PAS), to which Mk is naturally resistant. Interestingly, most of the PAS-susceptible Mk Tn mutants analyzed carried insertions in genes sharing orthologs in Mtb. This finding suggests that the natural resistance of Mk to PAS may not be due to the presence of dedicated Mk-specific PAS resistance mechanisms, but rather it is a result of fundamental differences in physiology between Mk and Mtb. Finally, we performed preliminary characterization and complementation analysis of select PAS-susceptible Mk Tn mutants to further explore these genotype-phenotype associations. Altogether, our findings from both approaches represent valuable resources to assist in the process of identifying and prioritizing potential Mk drug target candidates and to guide future studies on Mk biology

Genetic Underpinnings of Carotenogenesis and Light-Induced Transcriptome Remodeling in the Opportunistic Pathogen <i>Mycobacterium kansasii</i>

Mycobacterium kansasii (Mk) causes opportunistic pulmonary infections with tuberculosis-like features. The bacterium is well known for its photochromogenicity, i.e., the production of carotenoid pigments in response to light. The genetics defining the photochromogenic phenotype of Mk has not been investigated and defined pigmentation mutants to facilitate studies on the role of carotenes in the bacterium’s biology are not available thus far. In this study, we set out to identify genetic determinants involved in Mk photochromogenicity. We screened a library of ~150,000 transposon mutants for colonies with pigmentation abnormalities. The screen rendered a collection of ~200 mutants. Each of these mutants could be assigned to one of four distinct phenotypic groups. The insertion sites in the mutant collection clustered in three chromosomal regions. A combination of phenotypic analysis, sequence bioinformatics, and gene expression studies linked these regions to carotene biosynthesis, carotene degradation, and monounsaturated fatty acid biosynthesis. Furthermore, introduction of the identified carotenoid biosynthetic gene cluster into non-pigmented Mycobacterium smegmatis endowed the bacterium with photochromogenicity. The studies also led to identification of MarR-type and TetR/AcrR-type regulators controlling photochromogenicity and carotenoid breakdown, respectively. Lastly, the work presented also provides a first insight into the Mk transcriptome changes in response to light

<i>Mycobacterium abscessus</i> Mutants with a Compromised Functional Link between the Type VII ESX-3 System and an Iron Uptake Mechanism Reliant on an Unusual Mycobactin Siderophore

The opportunistic pathogen Mycobacterium abscessus subsp. abscessus (Mab) has become an emerging public health threat due to the increasing number of Mab-associated chronic pulmonary disease cases. Treatment requires multiple drug courses and is often combined with surgical resection. Cure rates are only ~50% due to treatment failure and comorbidities. Deeper understanding of the biology of Mab is required to illuminate potential avenues for the development of better therapeutics against Mab infections. The ESX-3 type VII protein secretion system of Mab has an important role in host inflammatory and pathological responses during infection. In this work, we demonstrate a functional link between ESX-3 and an iron uptake system based on an unusual mycobactin-type siderophore (designated MBT Ab) and exploit this link to implement a large screen for transposon mutants with an impaired ESX-3. Most mutants we identified carry insertions in genes encoding predicted ESX-3 secretion machinery components or potential ESX-3 substrates. The mutants overproduce MBT Ab, a trait consistent with an iron uptake defect. Our characterization of MBT Ab revealed structural features reminiscent of nocardial mycobactin-like compounds with cytotoxicity. This finding raises the possibility that MBT Ab may play roles in pathogenesis unlinked to iron homeostasis. The mutants generated herein will facilitate research to better understand the role of ESX-3 and its interplay with the siderophore system

Development of a vaginal fast-dissolving insert combining griffithsin and carrageenan for potential use against sexually transmitted infections

Precoital, on-demand topical microbicides to reduce a woman’s risk of sexually transmitted infections have been in development for nearly three decades, but no product has been approved due to acceptability issues and poor adherence in clinical trials. We set out to develop a self-administered vaginal fast-dissolving insert (FDI) produced by freeze-drying that would deliver safe and effective amounts of the antiviral agents griffithsin (GRFT) and carrageenan (CG) and would have properties women and their partners find acceptable. We evaluated FDI physical criteria, attributes of the gel produced upon dissolving, and GRFT stability. The lead formulation, FDI-024, was selected from 13 candidates and contains 4 mg of GRFT, 15 mg of CG, and excipients (the cryoprotectant sucrose and bulking agents dextran 40 and mannitol). The FDI exhibits good friability and hardness and is stable for at least 6 months at up to 40°C/75% relative humidity. It disintegrates in less than 60 seconds in a physiologically relevant volume (∼1 mL) of simulated vaginal fluid, forming a viscous semi-solid gel with favorable mucoadhesive and spreading properties. The formulation retains the antiviral activity of GRFT and CG against human immunodeficiency virus type 1 and human papillomavirus, respectively, in cell-based assays

MIV-150 and zinc acetate combination provides potent and broad activity against HIV-1

We previously showed that the combination of the non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 with zinc acetate (ZA) formulated in a carrageenan (CG; MZC) gel provided macaques significant protection against vaginal simian-human immunodeficiency virus-RT (SHIV-RT) challenge, better than either MIV-150/CG or ZA/CG. The MZC gel was shown to be safe in a phase 1 clinical trial. Herein, we used in vitro approaches to study the antiviral properties of ZA and the MIV-150/ZA combination, compared to other NNRTIs. Like other NNRTIs, MIV-150 has EC_50 values in the subnanomolar to nanomolar range against wild type and NNRTI or RT-resistant HIVs. While less potent than NNRTIs, ZA was shown to be active in primary cells against laboratory-adapted and primary HIV-1 isolates and HIV-1 isolates/clones with NNRTI and RT resistance mutations, with EC_50 values between 20 and 110 μM. The MIV-150/ZA combination had a potent and broad antiviral activity in primary cells. In vitro resistance selection studies revealed that previously described NNRTI-resistant mutations were selected by MIV-150. ZA-resistant virus retained susceptibility to MIV-150 (and other RTIs) and MIV-150-selected virus remained sensitive to ZA. Notably, resistant virus was not selected when cultured in the presence of both ZA and MIV-150. This underscores the potency and breadth of the MIV-150/ZA combination, supporting preclinical macaque studies and the advancement of MZC microbicides into clinical testing

First-in-human safety and pharmacokinetics (PK) of a MIV-150/zinc acetate/carrageenan gel (PC-1005)

Background: The candidate microbicide, PC-1005, completely protects Depo-Provera-treated macaques from a single vaginal SHIV-RT challenge 8 h post dose, and significantly reduces HPV and HSV-2 infection in murine models. PC-1005 contains 50 μM MIV-150 (NNRTI) and 14 mM zinc acetate dihydrate in a carrageenan gel. Methods: In preparation for a Phase 1 trial, an open-label, safety run-in was conducted at the University of Alabama at Birmingham to assess the safety and pharmacokinetics (PK) of PC-1005. Healthy, sexually-abstinent, HIV and Hepatitis B/C negative, STI-free, non-pregnant women aged 19–49 on effective contraception, were eligible. Under clinical supervision, women inserted 4 ml of PC-1005 once daily for 3 days. Evaluations included physical exam, pelvic exam with colposcopy, EKG, vitals, and safety labs. Blood was drawn for PK assessment at 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 20, and 24 h post-doses 1 and 3; and 48 h and 72 h post-dose 3. Results: From June-July 2014, 5 women (2 Black, 2 White, 1 Native American) completed the study (3 doses). Median age was 29 (range 29–38). Three women reported 4 possibly-related AEs; 3 were DAIDS Grade 1: vaginal discharge, intermenstrual bleeding, lower abdominal pressure; 1 was DAIDS Grade 2: vaginal itching. Safety labs, physical exams, vital signs, and colposcopy were all normal or not considered clinically significant by investigators. All subjects had detectable MIV-150 blood levels after dosing; no accumulation was noted. On Day 3, the median (and range) of MIV-150 PK parameters were: T_1/2 of 4.98 h (3.08–6.55); C_max of 77.4 pg/ml (55.25–166.85); T_max of 4 h (2–6); AUC_last of 774.38 pg h/ml (622.14–1188.66); AUC_inf of 803.23 pg h/ml (684.82–1252). There was no increase in zinc blood levels from baseline; median C_max of zinc was 79 μg/ml (58–94) on Day 3. Conclusions: PC-1005 was well tolerated after 3 days of dosing in 5 healthy women. MIV-150 was absorbed with low levels observed systemically; no accumulation was noted. Zinc levels were unchanged from baseline

MIV-150/zinc acetate gel inhibits cell-associated simian-human immunodeficiency virus reverse transcriptase infection in a macaque vaginal explant model

The transmission of both cell-free and cell-associated immunodeficiency viruses has been demonstrated directly in multiple animal species and possibly occurs in humans, as suggested by genotyping of the infecting human immunodeficiency virus (HIV) in acutely infected women and in semen from their partners. Therefore, a microbicide may need to block both mechanisms of HIV transmission to achieve maximum efficacy. To date, most of the preclinical evaluation of candidate microbicides has been performed using cell-free HIV. New models of mucosal transmission of cell-associated HIV are needed to evaluate candidate microbicide performance. The MIV-150/zinc acetate/carrageenan (MZC) gel protects Depo-Provera-treated macaques against cell-free simian-human immunodeficiency virus reverse transcriptase (SHIV-RT) infection when applied vaginally up to 8 h before challenge. We recently demonstrated the potent activity of MZC gel against cell-free SHIV-RT in macaque vaginal explants. In the current study, we established a cell-associated SHIV-RT infection model of macaque vaginal tissues and tested the activity of MZC gel in this model. MZC gel protected tissues against cell-associated SHIV-RT infection when present at the time of viral exposure or when applied up to 4 days prior to viral challenge. These data support clinical testing of the MZC gel. Overall, our ex vivo model of cell-associated SHIV-RT infection in macaque vaginal mucosa complements the cell-free infection models, providing tools for prioritization of products that block both modes of HIV transmission

MZC gel inhibits SHIV-RT and HSV-2 in macaque vaginal mucosa and SHIV-RT in rectal mucosa

The Population Council\u27s microbicide gel MZC (also known as PC-1005) containing MIV-150 and zinc acetate dihydrate (ZA) in carrageenan (CG) has shown promise as a broad spectrum microbicide against HIV, HSV and HPV. Previous data show antiviral activity against these viruses in cell-based assays, prevention of vaginal and rectal SHIV-RT infection and reduction of vaginal HSV shedding in rhesus macaques and also excellent antiviral activity against HSV and HPV in murine models. Recently we demonstrated that MZC is safe and effective against SHIV-RT in macaque vaginal explants. Here we established models of ex vivo SHIV-RT/HSV-2 co-infection of vaginal mucosa and SHIV-RT infection of rectal mucosa in macaques (challenge of rectal mucosa with HSV-2 did not result in reproducible tissue infection), evaluated antiviral activity of MZC and compared qPCR and ELISA readouts for monitoring SHIV-RT infection. MZC (at non-toxic dilutions) significantly inhibited SHIV-RT in vaginal and rectal mucosa and HSV-2 in vaginal mucosa when present during viral challenge. Analysis of SHIV-RT infection and MZC activity by one-step SIV gag qRT-PCR and p27 ELISA demonstrated similar virus growth dynamics and MZC activity by both methods and higher sensitivity of qRT-PCR. Our data provide more evidence that MZC is a promising dual compartment multipurpose prevention technology candidate

Antiviral activity and mode of action of Griffithsin against HSV-2 and HPV: Preliminary studies of a potential non-ARV combination microbicide

Background: Griffithsin (GRFT) is a promising HIV microbicide candidate. Nixon et al. have shown that GRFT blocks herpes simplex 2 (HSV-2) infection in a mouse model, proposing inhibition of cell-to-cell spread as the mode of action (MOA). Using in vitro studies we further investigated the MOA of GRFT against HSV-2 and studied its antiviral activity against human papillomavirus (HPV). We also combined GRFT with zinc acetate (ZA) and/or carrageenan (CG) to render a more potent microbicide. Methods: We used XTT assay to define non-cytotoxic concentrations of GRFT, ZA, CG or their combinations. Assays for anti-HIV, anti-HPV and anti-HSV-2 activities were performed in TZM-bl cells or PBMCs using MAGI and p24 ELISA; in HeLa cells using a luciferase assay; and in Vero cells using plaque forming units (pfu) assay. We performed time-of-addition and temperature dependence experiments to differentiate inhibition of viral adsorption from entry. Surface plasmon resonance (SPR) was used to assess GRFT binding to viral glycoproteins and immunohistochemistry was used to determine the specific glycoprotein involved. Antiviral activities of prototype GRFT/CG (GC) and GRFT/ZA/CG (GZC) gels in a vaginal HSV-2 mouse model were evaluated. Results: GRFT shows modest in vitro antiviral activity against HSV-2 G (IC_50 = 5.8μg/ml) and HPV 6, 16, 18, 45 PsVs (IC_50 = 10.8-26.3μg/ml), compared to potent anti-HIV activity (IC_50 = 0.7-1.4ng/ml). As with HIV, GRFT blocks the entry but not the adsorption of HSV-2 and HPV to target cells. The combined analyses of SPR and immunohistochemistry for HSV-2 gD, suggest that GRFT binds to HSV-2 gD. GC and GZ had synergistic in vitro antiviral activity against HIV and HPV (CI \u3c 1). GC and GZC gels significantly reduced (p \u3c 0.05) HSV-2 vaginal infection in vivo when administered up to 2h before challenge with 10^6pfu/mouse. Conclusions: GRFT blocks HSV-2 and HPV entry to target cells and combination with CG and/or ZA may result in a potent/broad-spectrum non-ARV microbicide