Correlation between compartmental tenofovir concentrations and an ex vivo rectal biopsy model of tissue infectibility in the RMP-02/MTN-006 phase 1 study

Design: Phase 1, randomized, two-site (US), double-blind, placebo-controlled study of sexually-abstinent males and females

Rectal Transmission of Transmitted/Founder HIV-1 Is Efficiently Prevented by Topical 1% Tenofovir in BLT Humanized Mice

Rectal microbicides are being developed to prevent new HIV infections in both men and women. We focused our in vivo preclinical efficacy study on rectally-applied tenofovir. BLT humanized mice (n = 43) were rectally inoculated with either the primary isolate HIV-1(JRCSF) or the MSM-derived transmitted/founder (T/F) virus HIV-1(THRO) within 30 minutes following treatment with topical 1% tenofovir or vehicle. Under our experimental conditions, in the absence of drug treatment we observed 50% and 60% rectal transmission by HIV-1(JRCSF) and HIV-1(THRO), respectively. Topical tenofovir reduced rectal transmission to 8% (1/12; log rank p = 0.03) for HIV-1(JRCSF) and 0% (0/6; log rank p = 0.02) for HIV-1(THRO). This is the first demonstration that any human T/F HIV-1 rectally infects humanized mice and that transmission of the T/F virus can be efficiently blocked by rectally applied 1% tenofovir. These results obtained in BLT mice, along with recent ex vivo, Phase 1 trial and non-human primate reports, provide a critically important step forward in the development of tenofovir-based rectal microbicides

Selective Enhancement of Donor Hematopoietic Cell Engraftment by the CXCR4 Antagonist AMD3100 in a Mouse Transplantation Model

The interaction between stromal cell-derived factor-1 (SDF-1) with CXCR4 chemokine receptors plays an important role in hematopoiesis following hematopoietic stem cell transplantation. We examined the efficacy of post transplant administration of a specific CXCR4 antagonist (AMD3100) in improving animal survival and in enhancing donor hematopoietic cell engraftment using a congeneic mouse transplantation model. AMD3100 was administered subcutaneously at 5 mg/kg body weight 3 times a week beginning at day +2 post-transplant. Post-transplant administration of AMD3100 significantly improves animal survival. AMD3100 reduces pro-inflammatory cytokine/chemokine production. Furthermore, post transplant administration of AMD3100 selectively enhances donor cell engraftment and promotes recovery of all donor cell lineages (myeloid cells, T and B lymphocytes, erythrocytes and platelets). This enhancement results from a combined effect of increased marrow niche availability and greater cell division induced by AMD3100. Our studies shed new lights into the biological roles of SDF-1/CXCR4 interaction in hematopoietic stem cell engraftment following transplantation and in transplant-related mortality. Our results indicate that AMD3100 provides a novel approach for enhancing hematological recovery following transplantation, and will likely benefit patients undergoing transplantation

Diffuse idiopathic skeletal hyperostosis in ancient clergymen

Diffuse idiopathic skeletal hyperostosis (DISH) is a common but often unrecognized systemic disorder observed mainly in the elderly. DISH is diagnosed when the anterior longitudinal ligament of the spine is ossified on at least four contiguous spinal levels or when multiple peripheral enthesopathies are present. The etiology of DISH is unknown but previous studies have shown a strong association with obesity and insulin-independent diabetes mellitus. DISH can lead to back pain, dysphagia, myelopathy, musculoskeletal impairment and grossly unstable spine fractures after minor trauma. In archeological studies a high prevalence of DISH has been demonstrated in ancient clergymen. The present study describes the pathological changes of human remains excavated from the abbey court (Pandhof) in the city of Maastricht, The Netherlands. Human remains of 51 individuals buried between 275 and 1795 ce were excavated and examined. The remains were investigated according to a standardized physical anthropological report and individuals demonstrating ossification of spinal ligaments and/or multiple peripheral enthesopathies were included in the study group. The authors reviewed all available material and after reaching consensus, each abnormality found was given a diagnosis and subsequently recorded. After examination, 28 individuals were considered to be adult males; 11 adult females; three adults of indeterminate sex and nine individuals were of sub adult age. The mean age at death for adults was 36.8 years. Seventeen adult individuals (40.4% of all adults), displayed ossifications of at least four contiguous spinal levels and/or multiple enthesopathies of the appendicular skeleton and were therefore, assigned the diagnosis DISH. The mean age of these individuals was 49.5 ± 13.0 years. In at least three of these individuals, DISH had led to extensive ossification and subsequent ankylosis of axial and peripheral skeletal structures. In this population of (presumably) clergymen and high-ranking citizens, DISH was observed in unusual high numbers at a relatively young age. Some of the examined cases suggest that DISH may be a seriously incapacitating disorder when the more advanced stages of the disease have been reached. It is hypothesized that “a monastic way of life” can predispose to DISH. Present demographic trends in obesity and diabetes mellitus as potential co-factors for the development of DISH warrant further study to investigate its future prevalence

A phase 1 randomized, open label, rectal safety, acceptability, pharmacokinetic, and pharmacodynamic study of three formulations of tenofovir 1% Gel (the CHARM-01 study)

Objectives: The CHARM-01 study characterized the safety, acceptability, pharmacokinetics (PK), and pharmacodynamics (PD) of three tenofovir (TFV) gels for rectal application. The vaginal formulation (VF) gel was previously used in the CAPRISA 004 and VOICE vaginal microbicide Phase 2B trials and the RMP-02/MTN-006 Phase 1 rectal safety study. The reduced glycerin VF (RGVF) gel was used in the MTN-007 Phase 1 rectal microbicide trial and is currently being evaluated in the MTN-017 Phase 2 rectal microbicide trial. A third rectal specific formulation (RF) gel was also evaluated in the CHARM-01 study. Methods: Participants received 4 mL of the three TFV gels in a blinded, crossover design: seven daily doses of RGVF, seven daily doses of RF, and six daily doses of placebo followed by one dose of VF, in a randomized sequence. Safety, acceptability, compartmental PK, and explant PD were monitored throughout the trial. Results: All three gels were found to be safe and acceptable. RF and RGVF PK were not significantly different. Median mucosal mononuclear cell (MMC) TFV-DP trended toward higher values for RF compared to RGVF (1136 and 320 fmol/106 cells respectively). Use of each gel in vivo was associated with significant inhibition of ex vivo colorectal tissue HIV infection. There was also a significant negative correlation between the tissue levels of TFV, tissue TFV-DP, MMC TFV-DP, rectal fluid TFV, and explant HIV-1 infection. Conclusions: All three formulations were found to be safe and acceptable. However, the safety profile of the VF gel was only based on exposure to one dose whereas participants received seven doses of the RGVF and RF gels. There was a trend towards higher tissue MMC levels of TFV-DP associated with use of the RF gel. Use of all gels was associated with significant inhibition of ex vivo tissue HIV infection. Trial Registration: ClinicalTrials.gov NCT01575405

Engineering HIV-Resistant Human CD4+ T Cells with CXCR4-Specific Zinc-Finger Nucleases

HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5Δ32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5) virus strains. As an approach to inactivating CCR5, we introduced CCR5-specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 (X4) in place of or in addition to CCR5 (R5X4) remains. Here we describe engineering a pair of zinc finger nucleases that, when introduced into human T cells, efficiently disrupt cxcr4 by cleavage and error-prone non-homologous DNA end-joining. The resulting cells proliferated normally and were resistant to infection by X4-tropic HIV-1 strains. CXCR4 could also be inactivated in ccr5Δ32 CD4+ T cells, and we show that such cells were resistant to all strains of HIV-1 tested. Loss of CXCR4 also provided protection from X4 HIV-1 in a humanized mouse model, though this protection was lost over time due to the emergence of R5-tropic viral mutants. These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals

Sialoadhesin Expressed on IFN-Induced Monocytes Binds HIV-1 and Enhances Infectivity

Background: HIV-1 infection dysregulates the immune system and alters gene expression in circulating monocytes. Differential gene expression analysis of CD14 + monocytes from subjects infected with HIV-1 revealed increased expression of sialoadhesin (Sn, CD169, Siglec 1), a cell adhesion molecule first described in a subset of macrophages activated in chronic inflammatory diseases. Methodology/Principal Findings: We analyzed sialoadhesin expression on CD14 + monocytes by flow cytometry and found significantly higher expression in subjects with elevated viral loads compared to subjects with undetectable viral loads. In cultured CD14 + monocytes isolated from healthy individuals, sialoadhesin expression was induced by interferon-a and interferon-c but not tumor necrosis factor-a. Using a stringent binding assay, sialoadhesin-expressing monocytes adsorbed HIV-1 through interaction with the sialic acid residues on the viral envelope glycoprotein gp120. Furthermore, monocytes expressing sialoadhesin facilitated HIV-1 trans infection of permissive cells, which occurred in the absence of monocyte selfinfection. Conclusions/Significance: Increased sialoadhesin expression on CD14 + monocytes occurred in response to HIV-1 infection with maximum expression associated with high viral load. We show that interferons induce sialoadhesin in primary CD14 + monocytes, which is consistent with an antiviral response during viremia. Our findings suggest that circulating sialoadhesinexpressing monocytes are capable of binding HIV-1 and effectively delivering virus to target cells thereby enhancing th

Breast adenocarcinoma liver metastases, in contrast to colorectal cancer liver metastases, display a non-angiogenic growth pattern that preserves the stroma and lacks hypoxia

Although angiogenesis is a prerequisite for the growth of most human solid tumours, alternative mechanisms of vascularisation can be adopted. We have previously described a non-angiogenic growth pattern in liver metastases of colorectal adenocarcinomas (CRC) in which tumour cells replace hepatocytes at the tumour-liver interface, preserving the liver architecture and co-opting the sinusoidal blood vessels. The aim of this study was to determine whether this replacement pattern occurs during liver metastasis of breast adenocarcinomas (BC) and whether the lack of an angiogenic switch in such metastases is due to the absence of hypoxia and subsequent vascular fibrinogen leakage. The growth pattern of 45 BC liver metastases and 28 CRC liver metastases (73 consecutive patients) was assessed on haematoxylin- and eosin-stained tissue sections. The majority of the BC liver metastases had a replacement growth pattern (96%), in contrast to only 32% of the CRC metastases (P<0.0001). The median carbonic anhydrase 9 (CA9) expression (M75 antibody), as a marker of hypoxia, (intensity x % of stained tumour cells) was 0 in the BC metastases and 53 in the CRC metastases (P<0.0001). There was CA9 expression at the tumour-liver interface in only 16% of the BC liver metastases vs 54% of the CRC metastases (P=0.002). There was fibrin (T2G1 antibody) at the tumour-liver interface in only 21% of the BC metastases vs 56% of the CRC metastases (P=0.04). The median macrophage count (Chalkley morphometry; KP-1 anti-CD68 antibody) at the interface was 4.3 and 7.5, respectively (P<0.0001). Carbonic anhydrase 9 score and macrophage count were positively correlated (r=0.42; P=0.002) in all metastases. Glandular differentiation was less in the BC liver metastases: 80% had less than 10% gland formation vs only 7% of the CRC metastases (P<0.0001). The liver is a densely vascularised organ and can host metastases that exploit this environment by replacing the hepatocytes and co-opting the vasculature. Our findings confirm that a non-angiogenic pattern of liver metastasis indeed occurs in BC, that this pattern of replacement growth is even more prevalent than in CRC, and that the process induces neither hypoxia nor vascular leakage

Increasing the Effectiveness of Vaginal Microbicides: A Biophysical Framework to Rethink Behavioral Acceptability

Microbicide candidates delivered via gel vehicles are intended to coat the vaginal epithelium after application. The coating process depends on intrinsic biophysical properties of the gel texture, which restricts the potential choices for an effective product: the gel first must be physically synthesizable, then acceptable to the user, and finally applied in a manner promoting timely adequate coating, so that the user adherence is optimized. We present a conceptual framework anchoring microbicide behavioral acceptability within the fulfillment of the product biophysical requirements.We conducted a semi-qualitative/quantitative study targeting women aged 18-55 in Northern California to assess user preferences for microbicide gel attributes. Attributes included: (i) the wait time between application and intercourse, (ii) the gel texture and (iii) the trade-off between wait time and gel texture. Wait times were assessed using a mathematical model determining coating rates depending upon the gel's physical attributes.71 women participated. Results suggest that women would independently prefer a gel spreading rapidly, in 2 to 15 minutes (P<0.0001), as well as one that is thick or slippery (P<0.02). Clearly, thick gels do not spread rapidly; hence the motivation to study the trade-off. When asked the same question 'constrained' by the biophysical reality, women indicated no significant preference for a particular gel thickness (and therefore waiting time) (P>0.10) for use with a steady partner, a preference for a watery gel spreading rapidly rather than one having intermediate properties for use with a casual partner (P = 0.024).Biophysical constraints alter women's preferences regarding acceptable microbicide attributes. Product developers should offer a range of formulations in order to address all preferences. We designed a conceptual framework to rethink behavioral acceptability in terms of biophysical requirements that can help improve adherence in microbicide use ultimately enhancing microbicide effectiveness

Requirement of the CXXC Motif of Novel Francisella Infectivity Potentiator Protein B FipB, and FipA in Virulence of F. tularensis subsp. tularensis

The lipoprotein encoded by the Francisella tularensis subsp. tularensis locus FTT1103 is essential for virulence; an FTT1103 deletion mutant is defective in uptake and intracellular survival, and mice survive high dose challenges of greater than 108 bacteria. This protein has two conserved domains; one is found in a class of virulence proteins called macrophage infectivity potentiator (Mip) proteins, and the other in oxidoreductase Disulfide Bond formation protein A (DsbA)-related proteins. We have designated the protein encoded by FTT1103 as FipB for Francisella infectivity potentiator protein B. The locus FTT1102 (fipA), which is upstream of fipB, also has similarity to same conserved Mip domain. Deletion and site-specific mutants of fipA and fipB were constructed in the Schu S4 strain, and characterized with respect to intracellular replication and in vivo virulence. A nonpolar fipA mutant demonstrated reduced survival in host cells, but was only slightly attenuated in vivo. Although FipB protein was present in a fipA mutant, the abundance of the three isoforms of FipB was altered, suggesting that FipA has a role in post-translational modification of FipB. Similar to many DsbA homologues, FipB contains a cysteine-any amino acid-any amino acid-cysteine (CXXC) motif. This motif was found to be important for FipB's role in virulence; a deletion mutant complemented with a gene encoding a FipB protein in which the first cysteine was changed to an alanine residue (AXXC) failed to restore intracellular survival or in vivo virulence. Complementation with a gene that encoded a CXXA containing FipB protein was significantly defective in intracellular growth; however, only slightly attenuated in vivo