ZIKV infection regulates inflammasomes pathway for replication in monocytes.

ZIKV causes microcephaly by crossing the placental barrier, however, the mechanism of trans-placental dissemination of ZIKV remains unknown. Here, we sought to determine whether monocytes, which can cross tissue barriers, assist ZIKV dissemination to the fetus. We determined this by infecting monocytes with two strains of ZIKV: South American (PRVABC59) and Nigerian (IBH30656) and analyzing viral replication. We found that ZIKV infects and replicates in monocytes and macrophages, which results in the modulation of a large number of cellular genes. Analysis of these genes identified multiple pathways including inflammasome to be targeted by ZIKV, which was confirmed by analyzing the transcript levels of the proteins of inflammasome pathways, NLRP3, ASC, caspase 1, IL-1 and IL-18. Interestingly, IFNα and the IFN inducible gene, MxA were not enhanced, suggesting prevention of innate antiviral defense by ZIKV. Also, inhibition of inflammasome led to an increased transcriptional activity of IFNα, MxA and CXCL10. Based on these results we suggest that ZIKV transcription is regulated by inflammasomes.

Perivascular stromal cells as a potential reservoir of human cytomegalovirus.

Human cytomegalovirus (HCMV) infection is an important cause of morbidity and mortality among both solid organ and hematopoietic stem cell transplant recipients. Identification of cells throughout the body that can potentially serve as a viral reservoir is essential to dissect mechanisms of cell tropism and latency and to develop novel therapies. Here, we tested and compared the permissivity of liver-, brain-, lung (LNG)- and bone marrow (BM)-derived perivascular mesenchymal stromal cells (MSC) to HCMV infection and their ability to propagate and produce infectious virus. Perivascular MSC isolated from the different organs have in common the expression of CD146 and Stro-1. While all these cells were permissive to HCMV infection, the highest rate of HCMV infection was seen with LNG-MSC, as determined by viral copy number and production of viral particles by these cells. In addition, we showed that, although the supernatants from each of the HCMV-infected cultures contained infectious virus, the viral copy number and the quantity and timing of virus production varied among the various organ-specific MSC. Furthermore, using quantitative polymerase chain reaction, we were able to detect HCMV DNA in BM-MSC isolated from 7 out of 19 healthy, HCMV-seropositive adults, suggesting that BM-derived perivascular stromal cells may constitute an unrecognized natural HCMV reservoir.

Characterization of an antisense transcript spanning the UL81-82 locus of human cytomegalovirus.

In this study we present the characterization of a novel transcript, UL81-82ast, UL81-82 antisense transcript, and its protein product. The transcript was initially found in a cDNA library of monocytes from a seropositive donor. mRNA was obtained from monocytes isolated from a healthy donor with a high antibody titer against human cytomegalovirus (HCMV). The mRNAs were cloned into a lambda phage-derived vector to create the cDNA library. Using PCR, UL81-82ast was amplified from the library. The library was tested for the presence of numerous HCMV genes. Neither structural genes nor immediate-early genes were found. UL81-82ast was detected in five bone marrow samples from healthy antibody-positive donors. This same transcript was also found in in vitro-infected human fibroblasts early after infection but disappears at the same time that UL82 transcription begins. Not only was the transcript amplified using reverse transcription-PCR and sequenced but its protein product (UL82as protein) was detected by both Western blot and immunofluorescence. Phylogenetic studies using UL82as protein were conducted, showing a high degree of conservation in clinical isolates, laboratory strains of HCMV, and even in chimpanzee CMV. The transcript could be involved in the posttranscriptional regulation of the UL82 gene, affecting its mRNA stability or translation. Since the UL82 product, pp71, functions as an immediate-early transactivator, its posttranscriptional control could have some effect over latency reactivation and lytic replication.

Complete coding sequences of dengue-1 viruses from Paraguay and Argentina.

We have determined the complete coding sequences of six dengue-1 (DEN-1) viruses isolated from Paraguay and Argentina in 2000 from patients with dengue fever. Sequences of strains 259par00, 280par00, 295arg00, 297arg00 and 301arg00 can encode a polyprotein of 3392 amino acids. Strain 293arg00 circulated as a "wild type+deletion mutant" quasispecies, with a subpopulation characterized by a 3-nucleotide deletion in the NS4A region. This variant, which would encode a three amino acid change in the NS4A protein, was found as a minority population in one additional partially-sequenced isolate from the same outbreak. These six South American strains group into two different clades of the "American-African" DEN-1 genotype-one clade is most closely related to strains isolated from Brazil in 1997, the other to a Peruvian strain isolated in 1991 for which only partial sequence information is available. DEN-1 viruses isolated worldwide comprise at least four different genotypes according to previously defined classification criteria.

Phylogenetic relationships of dengue-1 viruses from Argentina and Paraguay.

We sequenced the Capsid-pre Membrane (C/prM) and the Envelope-Nonstructural protein 1 (E/NS1) regions of 24 recent isolates of dengue-1 (DEN-1) from South America. This included 12 Argentinean and 11 Paraguayan DEN-1 strains isolated in 2000 plus a Paraguayan strain isolated in 1988. These sequences were compared with published sequences of DEN-1 isolated worldwide to determine the origin of these isolates. Pairwise comparisons of strains from Paraguay and Argentina revealed a nucleotide divergence of 0-5% in the E/NS1 region and 0-3% in the C/prM region. Our results showed that these viruses belong to the same genotype, but can be separated into two clades. Interestingly, both clades circulated simultaneously in the same geographic area during the 2000 outbreaks. Amino acid differences were found between both clades in the C/prM region at position 100 (Lys vs. Arg) and in the E/NS1 region at positions 722 (Ala vs. Thr). Although the geographic movement of DEN-1 virus can not be unequivocally traced from the genetic relationship determined here, our results suggest that the recent epidemics in Argentina and Paraguay were due to the re-emergence of a previously circulating strain, or to the virus circulating unnoticed, rather than to the introduction of a new genotype.

Generation of a life-expanded rhesus monkey fibroblast cell line for the growth of rhesus rhadinovirus (RRV).

RRV, the rhesus macaque equivalent to HHV-8 or kaposi's sarcoma-associated herpesvirus (KSHV) was recently isolated from a simian immunodeficiency virus (SIV) infected macaque with a lymphoproliferative disorder. The growth of RRV in tissue culture requires propagation of primary rhesus monkey fibroblasts (RFs). In an effort to extend the life of these primary cells in tissue culture, the catalytic subunit of telomerase (hTERT) was introduced into RF cells using a recombinant retrovirus. This new cell line, Telo-RFs, have currently been passed in tissue culture over 80 times compared to a maximum passage number of 38 for wild type RFs, remain fully permissive for RRV DNA replication and production of infectious virus. Viral gene expression of immediate-early and early RNA transcripts was virtually identical to that observed in wild-type (wt) RFs. In addition, transfection experiments show that telo-RFs are easily and more efficiently transfected than wtRFs.

Meal replacements in weight intervention.

OBJECTIVE: To evaluate the effectiveness of meal replacements (MRs) in weight loss interventions in premenopausal women. RESEARCH METHODS AND PROCEDURES: Overweight premenopausal women (n = 113; body mass index: 25 to 35 kg/m(2); 30 to 50 years old) were randomized into three interventions: group A, a dietitian-led intervention; group B, a dietitian-led intervention incorporating MRs; and group C, a clinical office-based intervention incorporating MRs. In year 1, groups A and B attended 26 group sessions, whereas group C received the same educational materials during 26 10-minute office visits with a physician-nurse team. In year 2, participants attended monthly group seminars and drop-in visits with a dietitian. RESULTS: For the 74 subjects completing year 1, weight loss in the office-based group C was as effective as the traditional dietitian-led group A (4.3 +/- 6.5% vs. 4.1 +/- 6.4%), while group B maintained a significantly greater weight loss (9.1 +/- 8.9%; p < 0.02; mean +/- SD). For the 43 subjects completing year 2, group B showed significant differences in the percentage of weight loss (-8.5 +/- 7.0%) compared with group A (-1.5 +/- 5.0%) and group C (-3.0 +/- 7.0%; p < 0.001). DISCUSSION: Study results showed that a traditional weight loss intervention incorporating MRs was effective as a weight loss tool in the medical office practice and in the dietitian-led group setting.

Dietary protein and weight reduction: a statement for healthcare professionals from the Nutrition Committee of the Council on Nutrition, Physical Activity, and Metabolism of the American Heart Association.

High-protein diets have recently been proposed as a "new" strategy for successful weight loss. However, variations of these diets have been popular since the 1960s. High-protein diets typically offer wide latitude in protein food choices, are restrictive in other food choices (mainly carbohydrates), and provide structured eating plans. They also often promote misconceptions about carbohydrates, insulin resistance, ketosis, and fat burning as mechanisms of action for weight loss. Although these diets may not be harmful for most healthy people for a short period of time, there are no long-term scientific studies to support their overall efficacy and safety. These diets are generally associated with higher intakes of total fat, saturated fat, and cholesterol because the protein is provided mainly by animal sources. In high-protein diets, weight loss is initially high due to fluid loss related to reduced carbohydrate intake, overall caloric restriction, and ketosis-induced appetite suppression. Beneficial effects on blood lipids and insulin resistance are due to the weight loss, not to the change in caloric composition. Promoters of high-protein diets promise successful results by encouraging high-protein food choices that are usually restricted in other diets, thus providing initial palatability, an attractive alternative to other weight-reduction diets that have not worked for a variety of reasons for most individuals. High-protein diets are not recommended because they restrict healthful foods that provide essential nutrients and do not provide the variety of foods needed to adequately meet nutritional needs. Individuals who follow these diets are therefore at risk for compromised vitamin and mineral intake, as well as potential cardiac, renal, bone, and liver abnormalities overall.

Blood pressure and symptoms of depression and anxiety: a prospective study.

This study investigated whether symptoms of depression and anxiety were related to the development of elevated blood pressure in initially normotensive adults. The study's hypothesis was addressed with an existing set of prospective data gathered from an age-, sex-, and weight-stratified sample of 508 adults. Four years of follow-up data were analyzed both with logistic analysis, which used hypertension (blood pressure > or =140 mm Hg systolic or 90 mm Hg diastolic) as the dependent variable, and with multiple regression analysis, which used change in blood pressure as the dependent variable. Five physical risk factors for hypertension (age, sex, baseline body mass index, family history of hypertension, and baseline blood pressure levels) were controlled for in the regression analyses. Use of antidepressant/antianxiety and antihypertensive medications were controlled for in the study. Of the 433 normotensive participants who were eligible for our study, 15% had missing data in the logistic regression analysis focusing on depression (n = 371); similarly, 15% of the eligible sample had missing data in the logistic regression using anxiety as the psychological variable of interest (n = 370). Both logistic regression analyses showed no significant relationship for either depression or anxiety in the development of hypertension. The multiple regression analyses (n = 369 for the depression analysis; n = 361 for the anxiety analysis) similarly showed no relationship between either depression or anxiety in changes in blood pressure during the 4-year follow-up. Thus, our results do not support the role of depressive or anxiety symptoms in the development of hypertension in our sample of initially normotensive adults.

Weight control in the physician's office.

BACKGROUND: Lifestyle changes involving diet, behavior, and physical activity are the cornerstone of successful weight control. Incorporating meal replacements (1-2 per day) into traditional lifestyle interventions may offer an additional strategy for overweight patients in the primary care setting. METHODS: One hundred thirteen overweight premenopausal women (mean +/- SD age, 40.4 +/- 5.5 years; weight, 82 +/- 10 kg; and body mass index, 30 +/- 3 kg/m(2)) participated in a 1-year weight-reduction study consisting of 26 sessions. The women were randomly assigned to 3 different traditional lifestyle-based groups: (1) dietitian-led group intervention (1 hour per session), (2) dietitian-led group intervention incorporating meal replacements (1 hour per session), or (3) primary care office intervention incorporating meal replacements with individual physician and nurse visits (10-15 minutes per visit). RESULTS: For the 74 subjects (65%) completing 1 year, the primary care office intervention using meal replacements was as effective as the traditional dietitian-led group intervention not using meal replacements (mean +/- SD weight loss, 4.3% +/- 6.5% vs 4.1% +/- 6.4%, respectively). Comparison of the dietitian-led groups showed that women using meal replacements maintained a significantly greater weight loss (9.1% +/- 8.9% vs 4.1% +/- 6.4%) (P =.03). Analysis across groups showed that weight loss of 5% to 10% was associated with significant (P =.01) reduction in percentage of body fat, body mass index, waist circumference, resting energy expenditure, insulin level, total cholesterol level, and low-density lipoprotein cholesterol level. Weight loss of 10% or greater was associated with additional significant (P =.05) improvements in blood pressure and triglyceride level. CONCLUSIONS: A traditional lifestyle intervention using meal replacements can be effective for weight control and reduction in risk of chronic disease in the physician's office setting as well as in the dietitian-led group setting.

Effects of tumor necrosis factor alpha on sin nombre virus infection in vitro.

Previous data indicate that immune mechanisms may be involved in developing capillary leakage during Sin Nombre virus (SNV) infection. Therefore, we investigated production of tumor necrosis factor alpha (TNF-alpha) by human alveolar macrophages and human umbilical vein endothelial cells (HUVEC) after infection with SNV. In addition, we examined the effect of TNF-alpha on HUVEC monolayer leakage. Our results reveal that although TNF-alpha decreases accumulation of viral nucleoproteins, TNF-alpha levels do not change in SNV-infected cells. In addition, supernatants from SNV-infected human alveolar macrophages did not cause a significant increase in endothelial monolayer permeability.