Effects of Hormone Therapy on Cognition and Mood in Recently Postmenopausal Women: Findings from the Randomized,Controlled KEEPS–Cognitive and Affective Study

Background

Menopausal hormone therapy (MHT) reportedly increases the risk of cognitive decline in women over age 65 y. It is unknown whether similar risks exist for recently postmenopausal women, and whether MHT affects mood in younger women. The ancillary Cognitive and Affective Study (KEEPS-Cog) of the Kronos Early Estrogen Prevention Study (KEEPS) examined the effects of up to 4 y of MHT on cognition and mood in recently postmenopausal women.

Methods and Findings

KEEPS, a randomized, double-blinded, placebo-controlled clinical trial, was conducted at nine US academic centers. Of the 727 women enrolled in KEEPS, 693 (95.3%) participated in the ancillary KEEPS-Cog, with 220 women randomized to receive 4 y of 0.45 mg/d oral conjugated equine estrogens (o-CEE) plus 200 mg/d micronized progesterone (m-P) for the first 12 d of each month, 211 women randomized to receive 50 μg/d transdermal estradiol (t-E2) plus 200 mg/d m-P for the first 12 d of each month, and 262 women randomized to receive placebo pills and patches. Primary outcomes included the Modified Mini-Mental State examination; four cognitive factors: verbal learning/memory, auditory attention/working memory, visual attention/executive function, and speeded language/mental flexibility; and a mood measure, the Profile of Mood States (POMS). MHT effects were analyzed using linear mixed-effects (LME) models, which make full use of all available data from each participant, including those with missing data. Data from those with and without full data were compared to assess for potential biases resulting from missing observations. For statistically significant results, we calculated effect sizes (ESs) to evaluate the magnitude of changes.On average, participants were 52.6 y old, and 1.4 y past their last menstrual period. By month 48, 169 (24.4%) and 158 (22.8%) of the 693 women who consented for ancillary KEEPS-Cog were lost to follow-up for cognitive assessment (3MS and cognitive factors) and mood evaluations (POMS), respectively. However, because LME models make full use all available data, including data from women with missing data, 95.5% of participants were included in the final analysis (n = 662 in cognitive analyses, and n = 661 in mood analyses). To be included in analyses, women must have provided baseline data, and data from at least one post-baseline visit. The mean length of follow-up was 2.85 y (standard deviation [SD] = 0.49) for cognitive outcomes and 2.76 (SD = 0.57) for mood outcomes. No treatment-related benefits were found on cognitive outcomes. For mood, model estimates indicated that women treated with o-CEE showed improvements in depression and anxiety symptoms over the 48 mo of treatment, compared to women on placebo. The model estimate for the depression subscale was −5.36 × 10⁻² (95% CI, −8.27 × 10⁻² to −2.44 × 10^−2; ES = 0.49, p < 0.001) and for the anxiety subscale was −3.01 × 10⁻² (95% CI, −5.09 × 10⁻² to −9.34 × 10⁻³; ES = 0.26, p < 0.001). Mood outcomes for women randomized to t-E2 were similar to those for women on placebo. Importantly, the KEEPS-Cog results cannot be extrapolated to treatment longer than 4 y.

Conclusions

The KEEPS-Cog findings suggest that for recently postmenopausal women, MHT did not alter cognition as hypothesized. However, beneficial mood effects with small to medium ESs were noted with 4 y of o-CEE, but not with 4 y of t-E2. The generalizability of these findings is limited to recently postmenopausal women with low cardiovascular risk profiles.

Trial Registration

ClinicalTrials.gov NCT00154180 and NCT00623311     

Macrophage-Tumor Cell Fusions from Peripheral Blood of Melanoma Patients

Background

While the morbidity and mortality from cancer are largely attributable to its metastatic dissemination, the integral features of the cascade are not well understood. The widely accepted hypothesis is that the primary tumor microenvironment induces the epithelial-to-mesenchymal transition in cancer cells, facilitating their escape into the bloodstream, possibly accompanied by cancer stem cells. An alternative theory for metastasis involves fusion of macrophages with tumor cells (MTFs). Here we culture and characterize apparent MTFs from blood of melanoma patients.

Methods

We isolated enriched CTC populations from peripheral blood samples from melanoma patients, and cultured them. We interrogated these cultured cells for characteristic BRAF mutations, and used confocal microscopy for immunophenotyping, motility, DNA content and chromatin texture analyses, and then conducted xenograft studies using nude mice.

Findings

Morphologically, the cultured MTFs were generally large with many pseudopod extensions and lamellipodia. Ultrastructurally, the cultured MTFs appeared to be macrophages. They were rich in mitochondria and lysosomes, as well as apparent melanosomes. The cultured MTF populations were all heterogeneous with regard to DNA content, containing aneuploid and/or high-ploidy cells, and they typically showed large sheets (and/or clumps) of cytoplasmic chromatin. This cytoplasmic DNA was found within heterogeneously-sized autophagic vacuoles, which prominently contained chromatin and micronuclei. Cultured MTFs uniformly expressed pan-macrophage markers (CD14, CD68) and macrophage markers indicative of M2 polarization (CD163, CD204, CD206). They also expressed melanocyte-specific markers (ALCAM, MLANA), epithelial biomarkers (KRT, EpCAM), as well as the pro-carcinogenic cytokine MIF along with functionally related stem cell markers (CXCR4, CD44). MTF cultures from individual patients (5 of 8) contained melanoma-specific BRAF activating mutations. Chromatin texture analysis of deconvoluted images showed condensed DNA (DAPI-intense) regions similar to focal regions described in stem cell fusions. MTFs were readily apparent in vivo in all human melanomas examined, often exhibiting even higher DNA content than the cultured MTFs. When cultured MTFs were transplanted subcutaneously in nude mice, they disseminated and produced metastatic lesions at distant sites.

Conclusions and Hypothesis

Apparent MTFs are present in peripheral blood of patients with cutaneous melanomas, and they possess the ability to form metastatic lesions when transplanted into mice. We hypothesize that these MTFs arise at the periphery of primary tumors in vivo, that they readily enter the bloodstream and invade distant tissues, secreting cytokines (such as MIF) to prepare “niches” for colonization by metastasis initiating cells.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Carvedilol Prevents Ovariectomy-Induced Myocardial Contractile Dysfunction in Female Rat

Carvedilol has beneficial effects on cardiac function in patients with heart failure but its effect on ovariectomy-induced myocardial contractile dysfunction remains unclear. Estrogen deficiency induces myocardial contractile dysfunction and increases cardiovascular disease risk in postmenopausal women. Our aim was to investigate whether carvedilol, a beta receptor blocker, would prevent ovariectomy-induced myocardial contractile dysfunction. Female rats (8 weeks old) that underwent bilateral ovariectomy were randomly assigned to receive daily treatment with carvedilol (OVX+CAR, 20 mg/kg), placebo (OVX) and SHAM for 58 days. Left ventricle papillary muscle was mounted for isometric tension recordings. The inotropic response to Ca²⁺ (0.62 to 3.75 mM) and isoproterenol (Iso 10⁻⁸ to 10⁻² M) were assessed. Expression of calcium handling proteins was measured by western blot analysis. Carvedilol treatment in the OVX animals: prevented weight gain and slight hypertrophy, restored the reduced positive inotropic responses to Ca²⁺ and isoproterenol, prevented the reduction in SERCA2a expression, abolished the increase in superoxide anion production, normalized the increase in p22^phox expression, and decreased serum angiotensin converting enzyme (ACE) activity. This study demonstrated that myocardial contractile dysfunction and SERCA2a down regulation were prevented by carvedilol treatment. Superoxide anion production and NADPH oxidase seem to be involved in this response.     

Inhibition of Histone Deacetylase Impacts Cancer Stem Cells and Induces Epithelial-Mesenchyme Transition of Head and Neck Cancer

The genome is organized and packed into the nucleus through interactions with core histone proteins. Emerging evidence suggests that tumors are highly responsive to epigenetic alterations that induce chromatin-based events and dynamically influence tumor behavior. We examined chromatin organization in head and neck squamous cell carcinoma (HNSCC) using acetylation levels of histone 3 as a marker of chromatin compaction. Compared to control oral keratinocytes, we found that HNSCC cells are hypoacetylated and that microenvironmental cues (e.g., microvasculature endothelial cells) induce tumor acetylation. Furthermore, we found that chemical inhibition of histone deacetylases (HDAC) reduces the number of cancer stem cells (CSC) and inhibits clonogenic sphere formation. Paradoxically, inhibition of HDAC also induced epithelial-mesenchymal transition (EMT) in HNSCC cells, accumulation of BMI-1, an oncogene associated with tumor aggressiveness, and expression of the vimentin mesenchymal marker. Importantly, we observed co-expression of vimentin and acetylated histone 3 at the invasion front of human HNSCC tumor tissues. Collectively, these findings suggest that environmental cues, such as endothelial cell-secreted factors, modulate tumor plasticity by limiting the population of CSC and inducing EMT. Therefore, inhibition of HDAC may constitute a novel strategy to disrupt the population of CSC in head and neck tumors to create a homogeneous population of cancer cells with biologically defined signatures and predictable behavior.     

Altered Visual and Feet Proprioceptive Feedbacks during Quiet Standing Increase Postural Sway in Patients with Severe Knee Osteoarthritis

Objective

The objective was to investigate how postural control in knee osteoarthritis (KOA) patients, with different structural severities and pain levels, is reorganized under different sensory conditions.

Methods

Forty-two obese patients (BMI range from 30.1 to 48.7 kg*m⁻², age range from 50 to 74 years) with KOA were evaluated. One minute of quiet standing was assessed on a force platform during 4 different sensory conditions, applied 3 times at random: Eyes open (EO) and eyes closed (EC) standing on firm and soft (foam) surfaces (EO-soft and EC-soft). Centre of pressure (Cop) standard deviation, speed, range and Cop mean position in both directions (anterior-posterior and medial-lateral) were extracted from the force platform data. Structural disease severity was assessed from semiflexed standing radiographs and graded by the Kellgren and Lawrence (KL) score. Pain intensity immediately before the measurements was assessed by numeric rating scale (range: 0–10).

Results

The patients were divided into “less severe” (KL 1 and 2, n = 24) and “severe” (KL>2, n = 18) group. The CoP range in the medial-lateral direction was larger in the severe group when compared with the less severe group during EC-soft condition (P<0.01). Positive correlation between pain intensity and postural sway (range in medial-lateral direction) was found during EC condition, indicating that the higher the pain intensity, the less effective is the postural control applied to restore an equilibrium position while standing without visual information.

Conclusion

The results support that: (i) the postural reorganization under manipulation of the different sensory information is worse in obese KOA patients with severe degeneration and/or high pain intensity when compared with less impaired patients, and (ii) higher pain intensity is related to worse body balance in obese KOA patients.     

Gene delivery mediated by cationic liposomes: from biophysical aspects to enhancement of transfection

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. However, the relationship between the features of the lipid-DNA complexes (`lipoplexes') and their mode of interaction with cells, the efficiency of gene transfer and gene expression remain to be clarified. To gain insights into these aspects, the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP) and its mixture with phosphatidylethanolamine (PE)), and their complexes with DNA at different (+/-) charge ratios were determined. A lipid mixing assay was used to assess the interaction of liposomes and lipoplexes with monocytic leukaemia cells. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. However, very limited transfection of these cells was achieved using the above complexes. It is possible that the topology of the cationic liposome-DNA complexes does not allow the entry of DNA into cells through a fusion process at the plasma membrane. In an attempt to enhance transfection mediated by lipoplexes composed of DOTAP and its equimolar mixture with dioleoylphosphatidylethanolamine (DOPE) two different strategies were explored: (i) association of a targeting ligand (transferrin) to the complexes to promote their internalization, presumably by receptor-mediated endocytosis; and (ii) association of synthetic fusogenic peptides (GALA or the influenza haemagglutinin Nterminal peptide HA-2) to the complexes to promote endosomal destabilization and release of the genetic material into the cytoplasm. These strategies were effective in enhancing transfection in a large variety of cells, including epithelial and lymphoid cell lines, as well as human macrophages, especially with the use of optimized lipid/ DNA (+/-) charge ratios. Besides leading to high levels of transfection, the ternary complexes of cationic liposomes, DNA, and protein or peptide, have the advantages of being active in the presence of serum and being non-toxic. Moreover, such ternary complexes present a net negative charge and, thus, are likely to alleviate the problems associated with the use of highly positively charged complexes in vivo, such as avid complexation with serum proteins. Overall, the results indicate that these complexes, and their future derivatives, may constitute viable alternatives to viral vectors for gene delivery in vivo.