Extended-Release Versus Immediate-Release Triamcinolone Acetonide in Patients Who Have Knee Osteoarthritis and Type 2 Diabetes Mellitus.

BACKGROUND: Intra-articular (IA) corticosteroid injections may cause hyperglycemia (glucose level > 180 mg/dL). In a phase 2 study of 33 patients who had osteoarthritis of the knee (OAK) and type 2 diabetes mellitus (T2D), triamcinolone acetonide extended-release (TA-ER) was associated with minimal glycemic control disruption compared with triamcinolone acetonide immediate-release (TA-IR). This post hoc analysis characterizes the clinical relevance of these results. METHODS: Patients who had symptomatic OAK for ≥ 6 months, T2D for ≥ 1 year, and hemoglobin A1c ≥ 6.5 and ≤ 9.0% were randomized to receive an IA injection of either TA-ER or TA-IR. Changes in continuous glucose monitor daily glucose level, percentage of time in or above the target glucose range (> 70 to 180 mg/dL), time to glucose level 250 mg/dL and maximum glucose level > 250 mg/dL, and glycemic variability were evaluated. RESULTS: Across postinjection days 1 to 3, the TA-ER group (n = 18) had a lower median change from baseline in maximum glucose level (92.3 versus 169.1 mg/dL), a reduced percentage of time with a glucose level > 250 mg/dL (12 versus 26%), a smaller proportion of patients who had a maximum glucose level > 250 mg/dL (50 versus 93%), and a greater percentage of time in the target glucose range (62 versus 48%) versus the TA-IR group (n = 15). There was less glycemic variability and lower glucose spikes in the TA-ER versus TA-IR group. Median times to glucose level 250 mg/dL (44 versus 6 hours) and maximum glucose level (34 versus 13 hours) were significantly longer in the TA-ER versus TA-IR group. CONCLUSIONS: Use of TA-ER was associated with a clinically meaningful reduction in hyperglycemia versus TA-IR.

Modeling homophily in dynamic networks with application to HIV molecular surveillance.

BACKGROUND: Efforts to control the HIV epidemic can benefit from knowledge of the relationships between the characteristics of people who have transmitted HIV and those who became infected by them. Investigation of this relationship is facilitated by the use of HIV genetic linkage analyses, which allows inference about possible transmission events among people with HIV infection. Two persons with HIV (PWH) are considered linked if the genetic distance between their HIV sequences is less than a given threshold, which implies proximity in a transmission network. The tendency of pairs of nodes (in our case PWH) that share (or differ in) certain attributes to be linked is denoted homophily. Below, we describe a novel approach to modeling homophily with application to analyses of HIV viral genetic sequences from clinical series of participants followed in San Diego. Over the 22-year period of follow-up, increases in cluster size results from HIV transmissions to new people from those already in the cluster-either directly or through intermediaries. METHODS: Our analytical approach makes use of a logistic model to describe homophily with regard to demographic, clinical, and behavioral characteristics-that is we investigate whether similarities (or differences) between PWH in these characteristics are associated with their sequences being linked. To investigate the performance of our methods, we conducted on a simulation study for which data sets were generated in a way that reproduced the structure of the observed database. RESULTS: Our results demonstrated strong positive homophily associated with hispanic ethnicity, and strong negative homophily, with birth year difference. The second result implies that the larger the difference between the age of a newly-infected PWH and the average age for an available cluster, the lower the odds of a newly infected person joining that cluster. We did not observe homophily associated with prior diagnosis of sexually transmitted diseases. Our simulation studies demonstrated the validity of our approach for modeling homophily, by showing that the estimates it produced matched the specified values of the statistical network generating model. CONCLUSIONS: Our novel methods provide a simple and flexible statistical network-based approach for modeling the growth of viral (or other microbial) genetic clusters from linkage to new infections based on genetic distance.

Sex Differences in Human Immunodeficiency Virus Persistence and Reservoir Size During Aging.

BACKGROUND: Sex differences in human immunodeficiency virus (HIV) reservoir dynamics remain underexplored. METHODS: Longitudinal samples from virally suppressed midlife women (n = 59, median age 45 years) and age-matched men (n = 31) were analyzed retrospectively. At each time point, we measured sex hormones (by means of enzyme-linked immunosorbent assay) and cellular HIV DNA and RNA (by means of digital droplet polymerase chain reaction). Number of inducible HIV RNA+ cells, which provides an upper estimate of the replication-competent reservoir, was quantified longitudinally in a different subset of 14 women, across well-defined reproductive stages. Mixed-effects models included normalized reservoir outcomes and sex, time since antiretroviral therapy (ART) initiation, and the sex-by-time interaction as predictors. RESULTS: At ART initiation, women and men had median (interquartile range [IQR]) CD4+ T-cell counts of 204/µL (83-306/µL) versus 238/µL (120-284/µL), respectively; median ages of 45 (42-48) versus 47 (43-51) years; and median follow-up times of 79.2/µL (60.5-121.1/µL) versus 66.2/µL (43.2-80.6/µL) months. We observed a significant decline of total HIV DNA over time in both men and women (P < .01). However, the rates of change differed significantly between the sexes (P < .01), with women having a significantly slower rate of decline than men, more pronounced with age. By contrast, the levels of inducible HIV RNA increased incrementally over time in women during reproductive aging (P < .01). CONCLUSIONS: In contrast to men, in whom the HIV reservoir steadily declines with aging, the HIV reservoir in women is more dynamic. Total HIV DNA (including intact and defective genomes) declines more slowly in women than in men, while the inducible HIV RNA+ reservoir, which is highly enriched in replication-competent virus, increases in women after menopause.

Effective Human Immunodeficiency Virus Molecular Surveillance Requires Identification of Incident Cases of Infection.

BACKGROUND: Ending the human immunodeficiency virus (HIV) epidemic requires knowledge of key drivers of spread of HIV infection. METHODS: Between 1996 and 2018, 1119 newly and previously diagnosed, therapy-naive persons with HIV (PWH) from San Diego were followed. A genetic distance-based network was inferred using pol sequences, and genetic clusters grew over time through linkage of sequences from newly observed infections. Cox proportional hazards models were used to identify factors associated with the rate of growth. These results were used to predict the impact of a hypothetical intervention targeting PWH with incident infection. Comparison was made to the Centers for Disease Control and Prevention (CDC) Ending the HIV Epidemic (EHE) molecular surveillance strategy, which prioritizes clusters recently linked to all new HIV diagnoses and does not incorporate data on incident infections. RESULTS: Overall, 219 genetic linkages to incident infections were identified over a median follow-up of 8.8 years. Incident cluster growth was strongly associated with proportion of PWH in the cluster who themselves had incident infection (hazard ratio, 44.09 [95% confidence interval, 17.09-113.78]). The CDC EHE molecular surveillance strategy identified 11 linkages to incident infections a genetic distance threshold of 0.5%, and 24 linkages at 1.5%. CONCLUSIONS: Over the past 2 decades, incident infections drove incident HIV cluster growth in San Diego. The current CDC EHE molecular detection and response strategy would not have identified most transmission events arising from those with incident infection in San Diego. Molecular surveillance that includes detection of incident cases will provide a more effective strategy for EHE.

Subclinical Cytomegalovirus and Epstein-Barr Virus Shedding Is Associated with Increasing HIV DNA Molecular Diversity in Peripheral Blood during Suppressive Antiretroviral Therapy.

Cytomegalovirus (CMV) almost universally infects persons with HIV (PWH), and it is a driver of persistent inflammation and HIV persistence. The mechanisms underlying the association between CMV (and possibly other herpesviruses) and HIV persistence are unclear. Serially collected blood samples were obtained from men who have sex with men (MSM) who started antiretroviral therapy (ART) within 1 year of their estimated date of HIV infection (EDI). Total CMV and Epstein-Barr virus (EBV) DNA were quantified in peripheral blood mononuclear cells by droplet digital PCR (ddPCR). Deep sequencing of the HIV DNA partial env gene was performed, and the dynamics of viral diversity over time were analyzed in relation to CMV and EBV shedding status. In total, 37 MSM PWH were included and followed for a median of 23 months (IQR, 22 to 28). Participants started ART within a median of 3.1 months (IQR, 1.5 to 6.5) after EDI and remained virally suppressed thereafter. A total of 18 participants (48.6%) were classified as high EBV shedders, while 19 (51.4%) were classified as CMV shedders. In longitudinal analyses, normalized molecular diversity levels tended to increase over time among participants with detectable CMV and high EBV DNA (0.03 ± 0.02, P = 0.08), while they significantly declined among participants with no/low viral shedding (-0.04 ± 0.02, P = 0.047, interaction P < 0.01). Subclinical CMV and EBV shedding could contribute to the dynamics of the HIV DNA reservoir during suppressive ART. Whether persistent CMV/EBV replication could be targeted as a strategy to reduce the size of the latent HIV reservoir is an avenue that should be explored.IMPORTANCE As part of this study, we evaluated the molecular characteristics of the HIV DNA reservoir over time during antiretroviral treatment (ART) in relation to those of other chronic viral infections (i.e., cytomegalovirus [CMV] and Epstein-Barr virus [EBV]). We demonstrated that the presence of CMV and high-level EBV DNA in peripheral blood cells was associated with changes in HIV DNA molecular diversity. Specifically, HIV DNA molecular diversity increased over time among participants with detectable CMV and high-level EBV DNA, while it significantly declined among participants with no/low viral shedding. Although the current study design does not allow causality to be inferred, it does support the theory that persistent CMV and EBV shedding could contribute to the dynamics of the HIV DNA reservoir during suppressive ART, even when ART is initiated during the earliest phases of HIV infection.

Effect of Cannabis Use on Human Immunodeficiency Virus DNA During Suppressive Antiretroviral Therapy.

Cannabis use is frequent among people living with human immunodeficiency virus (HIV) and is associated with reduced systemic inflammation. We observed a faster HIV DNA decay during antiretroviral therapy among cannabis users, compared to those with no drug use. No cannabis effect was observed on cellular HIV RNA transcription.

Subclinical Cytomegalovirus DNA Is Associated with CD4 T Cell Activation and Impaired CD8 T Cell CD107a Expression in People Living with HIV despite Early Antiretroviral Therapy.

Most people living with HIV (PLWH) are coinfected with cytomegalovirus (CMV). Subclinical CMV replication is associated with immune dysfunction and with increased HIV DNA in antiretroviral therapy (ART)-naive and -suppressed PLWH. To identify immunological mechanisms by which CMV could favor HIV persistence, we analyzed 181 peripheral blood mononuclear cell (PBMC) samples from 64 PLWH starting ART during early HIV infection with subsequent virologic suppression up to 58 months. In each sample, we measured levels of CMV and Epstein-Barr virus (EBV) DNA by droplet digital PCR (ddPCR). We also measured expression of immunological markers for activation (HLA-DR+ CD38+), cycling (Ki-67+), degranulation (CD107a+), and the immune checkpoint protein PD-1 on CD4+ and CD8+ T cell memory subsets. Significant differences in percentages of lymphocyte markers by CMV/EBV shedding were identified using generalized linear mixed-effects models. Overall, CMV DNA was detected at 60/181 time points. At the time of ART initiation, the presence of detectable CMV DNA was associated with increased CD4+ T cell activation and CD107a expression and with increased CD8+ T cellular cycling and reduced CD107a expression on CD8+ T cells. While some effects disappeared during ART, greater CD4+ T cell activation and reduced CD107a expression on CD8+ T cells persisted when CMV was present (P < 0.01). In contrast, EBV was not associated with any immunological differences. Among the covariates, peak HIV RNA and CD4/CD8 ratio had the most significant effect on the immune system. In conclusion, our study identified immune differences in PLWH with detectable CMV starting early ART, which may represent an additional hurdle for HIV cure efforts.IMPORTANCE Chronic viral infections such as with HIV and CMV last a lifetime and can continually antagonize the immune system. Both viruses are associated with higher expression of inflammation markers, and recent evidence suggests that CMV may complicate efforts to deplete HIV reservoirs. Our group and others have shown that CMV shedding is associated with a larger HIV reservoir. Subclinical CMV replication could favor HIV persistence via bystander effects on our immune system. In this study, we collected longitudinal PBMC samples from people starting ART and measured immune changes associated with detectable CMV. We found that when CMV was detectable, CD4+ T cell activation was higher and CD8+ T cell degranulation was lower. Both results may contribute to the slower decay of the size of the reservoir during CMV replication, since activated CD4+ T cells are more vulnerable to HIV infection, while the loss of CD8+ T cell degranulation may impede the proper killing of infected cells.

Early Antiretroviral Therapy Is Associated with Lower HIV DNA Molecular Diversity and Lower Inflammation in Cerebrospinal Fluid but Does Not Prevent the Establishment of Compartmentalized HIV DNA Populations.

Even when antiretroviral therapy (ART) is started early after infection, HIV DNA might persist in the central nervous system (CNS), possibly contributing to inflammation, brain damage and neurocognitive impairment. Paired blood and cerebrospinal fluid (CSF) were collected from 16 HIV-infected individuals on suppressive ART: 9 participants started ART <4 months of the estimated date of infection (EDI) ("early ART"), and 7 participants started ART >14 months after EDI ("late ART"). For each participant, neurocognitive functioning was measured by Global Deficit Score (GDS). HIV DNA levels were measured in peripheral blood mononuclear cells (PBMCs) and CSF cell pellets by droplet digital (dd)PCR. Soluble markers of inflammation (sCD163, IL-6, MCP-1, TNF-α) and neuronal damage (neurofilament light [NFL]) were measured in blood and CSF supernatant by immunoassays. HIV-1 partial C2V3 env deep sequencing data (Roche 454) were obtained for 8 paired PBMC and CSF specimens and used for phylogenetic and compartmentalization analysis. Median exposure to ART at the time of sampling was 2.6 years (IQR: 2.2-3.7) and did not differ between groups. We observed that early ART was significantly associated with lower molecular diversity of HIV DNA in CSF (p<0.05), and lower IL-6 levels in CSF (p = 0.02), but no difference for GDS, NFL, or HIV DNA detectability compared to late ART. Compartmentalization of HIV DNA populations between CSF and blood was detected in 6 out of 8 participants with available paired HIV DNA sequences (2 from early and 4 from late ART group). Phylogenetic analysis confirmed the presence of monophyletic HIV DNA populations within the CSF in 7 participants, and the same population was repeatedly sampled over a 5 months period in one participant with longitudinal sampling. Such compartmentalized provirus in the CNS needs to be considered for the design of future eradication strategies and might contribute to the neuropathogenesis of HIV.

No Substantial Evidence for Sexual Transmission of Minority HIV Drug Resistance Mutations in Men Who Have Sex with Men.

During primary HIV infection, the presence of minority drug resistance mutations (DRM) may be a consequence of sexual transmission, de novo mutations, or technical errors in identification. Baseline blood samples were collected from 24 HIV-infected antiretroviral-naive, genetically and epidemiologically linked source and recipient partners shortly after the recipient's estimated date of infection. An additional 32 longitudinal samples were available from 11 recipients. Deep sequencing of HIV reverse transcriptase (RT) was performed (Roche/454), and the sequences were screened for nucleoside and nonnucleoside RT inhibitor DRM. The likelihood of sexual transmission and persistence of DRM was assessed using Bayesian-based statistical modeling. While the majority of DRM (>20%) were consistently transmitted from source to recipient, the probability of detecting a minority DRM in the recipient was not increased when the same minority DRM was detected in the source (Bayes factor [BF] = 6.37). Longitudinal analyses revealed an exponential decay of DRM (BF = 0.05) while genetic diversity increased. Our analysis revealed no substantial evidence for sexual transmission of minority DRM (BF = 0.02). The presence of minority DRM during early infection, followed by a rapid decay, is consistent with the "mutation-selection balance" hypothesis, in which deleterious mutations are more efficiently purged later during HIV infection when the larger effective population size allows more efficient selection. Future studies using more recent sequencing technologies that are less prone to single-base errors should confirm these results by applying a similar Bayesian framework in other clinical settings.IMPORTANCE The advent of sensitive sequencing platforms has led to an increased identification of minority drug resistance mutations (DRM), including among antiretroviral therapy-naive HIV-infected individuals. While transmission of DRM may impact future therapy options for newly infected individuals, the clinical significance of the detection of minority DRM remains controversial. In the present study, we applied deep-sequencing techniques within a Bayesian hierarchical framework to a cohort of 24 transmission pairs to investigate whether minority DRM detected shortly after transmission were the consequence of (i) sexual transmission from the source, (ii) de novo emergence shortly after infection followed by viral selection and evolution, or (iii) technical errors/limitations of deep-sequencing methods. We found no clear evidence to support the sexual transmission of minority resistant variants, and our results suggested that minor resistant variants may emerge de novo shortly after transmission, when the small effective population size limits efficient purge by natural selection.

Asymptomatic CMV Replication During Early Human Immunodeficiency Virus (HIV) Infection Is Associated With Lower CD4/CD8 Ratio During HIV Treatment.

BACKGROUND: A low CD4/CD8 ratio in human immunodeficiency virus (HIV)-infected individuals is associated with inflammation and higher risk of non-AIDS morbidity and mortality. In this study, we investigated the effect of subclinical cytomegalovirus (CMV) and Epstein-Barr virus (EBV) replication on CD4+ and CD8+ T-cell dynamics when antiretroviral therapy (ART) is started during early infection. METHODS: We investigated 604 peripheral blood mononuclear cell samples from 108 CMV- and EBV-seropositive HIV-infected men who have sex with men, who started ART within a median of 4 months from their estimated date of infection and were followed for a median of 29.1 months thereafter. Levels of CMV and EBV DNA were measured at each timepoint. Mixed-effects asymptotic regression models were applied to characterize CD4+ and CD8+ T-cell dynamics, and Bayesian hierarchical models were used to quantify individual differences in CMV and EBV DNA replication. RESULTS: Higher levels of subclinical CMV replication were associated with lower predicted maximum levels of CD4/CD8 ratio (P < .05), which was driven by higher levels of CD8+ T-cell counts (P < .05), without affecting CD4+ T-cell counts (P > .1). Age was negatively associated with CD4/CD8 levels (P < .05), and this effect was independent of the CMV association (P < .05 for both CMV and age in a multivariate model). CONCLUSIONS: Subclinical CMV replication in blood cells during early HIV infection and younger age were associated with lower CD4/CD8 ratios during suppressive ART. These findings suggest that active CMV infection in the setting of treated HIV may represent an attractive potential target for therapeutic intervention.

Plasma acylcarnitines during insulin stimulation in humans are reflective of age-related metabolic dysfunction.

The purpose of this study was to determine if plasma acylcarnitine (AC) profiling is altered under hyperinsulinemic conditions as part of the aging process. Fifteen young, lean (19-29 years) and fifteen middle-to older-aged (57-82 years) individuals underwent a 2-hr euglycemic-hyperinsulinemic clamp. Plasma samples were obtained at baseline, 20 min, 50 min, and 120 min for analysis of AC species and amino acids. Skeletal muscle biopsies were performed after 60 min of insulin-stimulation for analysis of acetyl-CoA carboxylase (ACC) phosphorylation. Insulin infusion decreased the majority of plasma short-, medium-, and long-chain (SC, MC, and LC, respectively) AC. However, during the initial 50 min, a number of MC and LC AC species (C10, C10:1, C12:1, C14, C16, C16:1, C18) remained elevated in aged individuals compared to their younger counterparts indicating a lag in responsiveness. Additionally, the insulin-induced decline in skeletal muscle ACC phosphorylation was blunted in the aged compared to young individuals (-24% vs. -56%, P < 0.05). These data suggest that a desensitization to insulin during aging, possibly at the level of skeletal muscle ACC phosphorylation, results in a diminished ability to transition to glucose oxidation indicative of metabolic inflexibility.

The power of the mind: the cortex as a critical determinant of muscle strength/weakness.

We tested the hypothesis that the nervous system, and the cortex in particular, is a critical determinant of muscle strength/weakness and that a high level of corticospinal inhibition is an important neurophysiological factor regulating force generation. A group of healthy individuals underwent 4 wk of wrist-hand immobilization to induce weakness. Another group also underwent 4 wk of immobilization, but they also performed mental imagery of strong muscle contractions 5 days/wk. Mental imagery has been shown to activate several cortical areas that are involved with actual motor behaviors, including premotor and M1 regions. A control group, who underwent no interventions, also participated in this study. Before, immediately after, and 1 wk following immobilization, we measured wrist flexor strength, voluntary activation (VA), and the cortical silent period (SP; a measure that reflect corticospinal inhibition quantified via transcranial magnetic stimulation). Immobilization decreased strength 45.1 ± 5.0%, impaired VA 23.2 ± 5.8%, and prolonged the SP 13.5 ± 2.6%. Mental imagery training, however, attenuated the loss of strength and VA by ∼50% (23.8 ± 5.6% and 12.9 ± 3.2% reductions, respectively) and eliminated prolongation of the SP (4.8 ± 2.8% reduction). Significant associations were observed between the changes in muscle strength and VA (r = 0.56) and SP (r = -0.39). These findings suggest neurological mechanisms, most likely at the cortical level, contribute significantly to disuse-induced weakness, and that regular activation of the cortical regions via imagery attenuates weakness and VA by maintaining normal levels of inhibition.

Wnt5a, TLR2 and TLR4 are elevated in advanced human atherosclerotic lesions.

OBJECTIVE AND DESIGN: Atherosclerosis (ATH) is a chronic inflammatory disease that involves cascades of signaling events mediated by various effector proteins. Here we sought to determine if the expression of Wnt5a, a secreted glycoprotein, is altered in discrete regions of the arterial plaque. METHODS: Atherosclerotic plaque tissues from 14 human subjects undergoing elective carotid endarterectomy were used in this study. Immunohistochemistry and laser capture microdissection combined with quantitative real-time PCR were used to determine the expression of Wnt5a and Toll-like receptors (TLRs) in different sections of the arterial lesions. Atherosclerotic serum samples (n = 30) and serum from healthy subjects (n = 16) were quantified for Wnt5a using an enzyme-linked immunosorbent assay (ELISA). RESULTS: The data analysis revealed that Wnt5a transcripts and protein were elevated in advanced arterial lesions relative to less advanced arterial lesions; that Wnt5a expression correlated with the presence of TLR4 and TLR2 transcripts; and that the average amount of Wnt5a protein present in atherosclerotic patient serum was significantly higher compared to healthy controls. CONCLUSIONS: This study is the first to provide evidence that the expression of Wnt5a increases as the disease progresses to a more advanced stage, and that this expression is coincident with that of TLR2 and TLR4. In addition, we found that the average Wnt5a levels in the serum of atherosclerotic patients are elevated relative to healthy controls, which is consistent with the hypothesis that Wnt5a plays a role in ATH.

Zinc finger genes Fezf1 and Fezf2 control neuronal differentiation by repressing Hes5 expression in the forebrain.

Precise control of neuronal differentiation is necessary for generation of a variety of neurons in the forebrain. However, little is known about transcriptional cascades, which initiate forebrain neurogenesis. Here we show that zinc finger genes Fezf1 and Fezf2, which encode transcriptional repressors, are expressed in the early neural stem (progenitor) cells and control neurogenesis in mouse dorsal telencephalon. Fezf1- and Fezf2-deficient forebrains display upregulation of Hes5 and downregulation of neurogenin 2, which is known to be negatively regulated by Hes5. We show that FEZF1 and FEZF2 bind to and directly repress the promoter activity of Hes5. In Fezf1- and Fezf2-deficient telencephalon, the differentiation of neural stem cells into early-born cortical neurons and intermediate progenitors is impaired. Loss of Hes5 suppresses neurogenesis defects in Fezf1- and Fezf2-deficient telencephalon. Our findings reveal that Fezf1 and Fezf2 control differentiation of neural stem cells by repressing Hes5 and, in turn, by derepressing neurogenin 2 in the forebrain.

Compensation effects on clinical trial data collection in opioid-dependent young adults.

BACKGROUND: Attrition in studies of substance use disorder treatment is problematic, potentially introducing bias into data analysis. OBJECTIVES: This study aimed to determine the effect of participant compensation amounts on rates of missing data and observed rates of drug use. METHODS: We performed a secondary analysis of a clinical trial of buprenorphine/naloxone among 152 treatment-seeking opioid-dependent subjects aged 15-21 during participation in a randomized trial. Subjects were randomized to a 2-week detoxification with buprenorphine/naloxone (DETOX; N = 78) or 12 weeks buprenorphine/naloxone (BUP; N = 74). Participants were compensated $5 for weekly urine drug screens and self-reported drug use information and $75 for more extensive assessments at weeks 4, 8, and 12. RESULTS: Though BUP assignment decreased the likelihood of missing data, there were significantly less missing data at 4, 8, and 12 weeks than other weeks, and the effect of compensation on the probability of urine screens being positive was more pronounced in DETOX subjects. CONCLUSION: These findings suggest that variations in the amount of compensation for completing assessments can differentially affect outcome measurements, depending on treatment group assignment. SCIENTIFIC SIGNIFICANCE: Adequate financial compensation may minimize bias when treatment condition is associated with differential dropout and may be a cost-effective way to reduce attrition. Moreover, active users may be more likely than non-active users to drop out if compensation is inadequate, especially in control groups or in groups who are not receiving active treatment.

Optimization of the conditions for the immobilization of glycopolypeptides on hydrophobic silica particulates and simple purification of lectin using glycopolypeptide-immobilized particulates.

Glycopolypeptide-immobilized particulates exhibit high binding selectivities and affinities for several analytes. However, to date, the conditions for the synthesis of glycopolypeptide-immobilized particulates have not been optimized and the application of these particulates as carriers for affinity chromatography has not been reported. Accordingly, herein, as a model compound for determining the optimal conditions for the immobilization of an artificial glycopolymer on hexyl-containing hybrid silica particulates (HSPs), the glycopolypeptide poly [GlcNAcß1,4GlcNAc-ß-NHCO-(CH2)5NH-/CH3(CH2)9NH-/γ-PGA] (3) containing multivalent chitobiose moieties and multivalent decyl groups with a γ-polyglutamic acid backbone was synthesized. Immobilization of 3 on HSPs under each condition was evaluated by a lectin-binding assay using wheat germ (Triticum vulgaris) agglutinin (WGA), which is an N-acetylglucosamine-binding lectin. As a result, the optimal immobilization conditions for HSPs at 25 mg/mL were obtained at dimethyl sulfoxide (DMSO) concentration of reaction solvent in the range of 1(DMSO):9(water) to 4(DMSO):6(water) and a compound 3 concentration in the range of 125 nM-1250 nM. Furthermore, the influence of the alkyl group structure introduced into glycopolypeptide for imparting hydrophobicity to it on the immobilization of glycopolypeptide on HSPs was investigated. As a result of comparing three types, poly [GlcNAcß1,4GlcNAc-ß-NHCO-(CH2)5NH-/γ-PGA] (1) with no alkyl group, poly [GlcNAcß1,4GlcNAc-ß-NHCO-(CH2)5NH-/CH3(CH2)4NH-/γ-PGA] (2) with a pentyl group, and 3 with a decyl group, 3 showed the best immobilization efficiency on HSPs. Finally, 1 mg 3-immobilized HSPs prepared under the optimum conditions adsorbed approximately 7.5 µg WGA in a structure-specific manner. We also achieved a simple WGA purification from raw wheat germ extract as a practical example using 3-immobilized HSPs. We believe that in the future, these glycopolypeptide-immobilized particulates will be used not only for the purification of plant lectins, but also as specific adsorbents for various lectins-like substances such as in vivo lectins, pathogenic viruses, and toxin proteins.

Cloning of complementary DNAs encoding structurally related homeoproteins from preimplantation mouse embryos: their involvement in the differentiation of embryonic stem cells.

During the preimplantation development of mouse embryos between the 4-cell to 8-cell stage and the morula stage, when the first irreversible segregation of cell fates proceeds into the pluripotent inner cell mass (progenitor cells to form the fetus) and the trophectoderm (to form the placenta) of blastocysts, pluripotency-maintaining and differentiation-inducing genes are expressed to coordinately regulate cell fates. Three structurally related cDNAs (Crxos1, Crxos1 sv2, and Crxos1 tv3) that exhibited concomitant elevated expression during this critical period were identified by subtractive cDNA cloning. CRXOS1 contains two homeodomains, while CRXOS1 sv2 and CRXOS1 tv3 each contain one of the homeodomains included in CRXOS1. Crxos1, Crxos1 sv2, and Crxos1 tv3 were expressed differentially during in vitro embryonic stem (ES) cell differentiation. Even under differentiation-inducing conditions, forced expression of Crxos1 sv2 inhibited the differentiation of ES cells. In contrast, under conditions that promote self-renewal of ES cells, forced expression of Crxos1 induced differentiation. Forced expression of Crxos1 resulted in induction of Gata4 but in repression of T, probably indicating that Crxos1 promotes the differentiation of ES cells into primitive endoderm, while inhibiting differentiation into mesoderm. On the other hand, no apparent effects of forced expression of Crxos1 tv3 were observed. Taken together, it was concluded that these transcripts encoding homeoproteins are capable of regulating the maintenance and/or differentiation of mouse ES cells and likely regulate that of preimplantation embryos.

Association of Cytomegalovirus DNA and Immunologic Markers of Cardiovascular Disease.

BACKGROUND: Persons living with human immunodeficiency virus (HIV) (PLWH) with high cytomegalovirus (CMV)-specific interferon (IFN) γ response have increased numbers of endothelium homing receptor (CX3CR1)+-expressing cells that are associated with cardiovascular disease. The current study was performed to investigate the effect of cellular levels of CMV DNA on these markers. METHODS: Eighty paired peripheral blood mononuclear cell samples were collected ≥12 months apart from 40 CMV-seropositive PLWH with suppressed HIV RNA, who started antiretroviral therapy at median of 3-months of infection. The samples were assessed for CMV-specific IFN-γ response by means of enzyme-linked immunospot assay, and participants were classified as low responders (LRs) or high responders (HRs) based on IFN-γ production (≤100 or >100 spot-forming units [SFUs]/105 cells). RESULTS: Of the 40 participants, 26 (65%) were HRs and 14 (35%) LRs at baseline, which did not change over time or by CMV levels (median at first/second time points, 383/308 SFUs/106 cells for HRs vs 21/41 SFUs/106 for LRs). A decrease in IFN-γ over time was associated with higher CMV DNA levels (P < .01). High CMV response was also associated with increased CD28+CD27-CD4+ T cells expressing CX3CR1 (P < .001). Similarly, increased IFN-γ production was associated with increased CMV-specific CX3CR1+CD28+CD27-CD4+ and CD8+ T cells (P < .001). CONCLUSIONS: These findings demonstrate that levels of CMV-specific IFN-γ response in PLWH are stable over time, and that HRs have increased circulating T cells expressing CX3CR1 that may put them at increased risk of cardiovascular disease and other inflammatory diseases.

Blood Flow-restricted Exercise Does Not Induce a Cross-Transfer of Effect: A Randomized Controlled Trial.

PURPOSE: The goal of this trial was to determine whether low-load blood flow-restricted (BFR) exercise of appendicular muscles induces a cross-transfer of effect to the trunk extensor (TE) muscles, such that low-load TE exercise would enhance TE size and function to a greater extent than standard low-load exercise in people with recurrent low back pain (LBP). We also investigated the direct effects of BFR exercise in the appendicular muscles. METHODS: Thirty-two adults with recurrent, nonspecific LBP were randomized into two groups: Appendicular BFR exercise (BFR exercise) or control exercise (CON exercise). All participants trained (two times per week) for 10 wk, with a 12-wk follow-up. Participants performed three sets of leg extension (LE), plantar flexion (PF), and elbow flexion (EF) exercises followed by low-load TE exercise without BFR. Outcome measures included magnetic resonance imaging-derived muscle size (quadriceps and TE), strength (LE, PF, EF, and TE), and endurance (LE and TE). RESULTS: There was no evidence for a cross-transfer of effect to the TE. There was also no statistically significant enhancement of limb skeletal muscle size or function of BFR relative to CON exercise at any time point; though, moderate effect sizes for BFR exercise were observed for enhanced muscle size and strength in the leg extensors. CONCLUSIONS: Low-load BFR exercise of the appendicular muscles did not result in a cross-transfer of effect to the TE musculature. There was also no significant benefit of low-load BFR exercise on the appendicular muscle size and function, suggesting no benefit from low-load BFR exercise in adults with recurrent, nonspecific LBP.

Improved assays to measure and characterize the inducible HIV reservoir.

BACKGROUND: Improved assays are critical to better characterize the HIV reservoir and to reliably evaluate candidate intervention strategies. Here we describe different methods to quantify the HIV reservoir. METHODS: We developed an optimized quantitative viral outgrowth assay (QVOA) to quantify the frequency of cells harboring replication-competent HIV, which is simpler and more sensitive than classical QVOAs. We also developed new inducible RNA assays that concomitantly measure the frequency of cell-associated [ca-] (gag and tat-rev) and cell-free [cf-] HIV RNA after three days of anti-CD3/CD28 stimulation. FINDINGS: The median frequency of the infected cells measured after induction was 94 IQR[60-132], 16 IQR [9-29] and 2.9 IQR[1.9-6.8] cells/106 CD4+ T-cells for ca-RNA gag and tat-rev, and cf-RNA, respectively. There are a large proportion of transcription-competent proviruses (ca-RNA) that seemed unable to form complete virions (cf-RNA), suggesting post-transcriptional blocks or defective proviruses. Importantly, the median frequency of infected CD4+ T-cells as estimated by 3-day inducible cf-RNA assay was not statistically different from the frequency measured by the QVOA (median of 3.3 [1.9-6.2] IUPM). The latently infected cells detected by the inducible cf-RNA assay correlated highly with the QVOA ( r= 0.67, p < .001), and both assays were equivalent in 60% of the samples tested, suggesting that most cells induced to produce virions are generating replication-competent virus. INTERPRETATION: These inducible RNA assays provide more sensitivity and a greater dynamic range for the monitoring of reduction of the reservoir by eradication strategies. Such assays may serve as robust and useful tools for clinical investigations of the HIV reservoir.