The HIV-1 late domain-2 S40A polymorphism in antiretroviral (or ART)-exposed individuals influences protease inhibitor susceptibility.

BACKGROUND: The p6 region of the HIV-1 structural precursor polyprotein, Gag, contains two motifs, P7TAP11 and L35YPLXSL41, designated as late (L) domain-1 and -2, respectively. These motifs bind the ESCRT-I factor Tsg101 and the ESCRT adaptor Alix, respectively, and are critical for efficient budding of virus particles from the plasma membrane. L domain-2 is thought to be functionally redundant to PTAP. To identify possible other functions of L domain-2, we examined this motif in dominant viruses that emerged in a group of 14 women who had detectable levels of HIV-1 in both plasma and genital tract despite a history of current or previous antiretroviral therapy. RESULTS: Remarkably, variants possessing mutations or rare polymorphisms in the highly conserved L domain-2 were identified in seven of these women. A mutation in a conserved residue (S40A) that does not reduce Gag interaction with Alix and therefore did not reduce budding efficiency was further investigated. This mutation causes a simultaneous change in the Pol reading frame but exhibits little deficiency in Gag processing and virion maturation. Whether introduced into the HIV-1 NL4-3 strain genome or a model protease (PR) precursor, S40A reduced production of mature PR. This same mutation also led to high level detection of two extended forms of PR that were fairly stable compared to the WT in the presence of IDV at various concentrations; one of the extended forms was effective in trans processing even at micromolar IDV. CONCLUSIONS: Our results indicate that L domain-2, considered redundant in vitro, can undergo mutations in vivo that significantly alter PR function. These may contribute fitness benefits in both the absence and presence of PR inhibitor.

Determination of HIV-1 coreceptor tropism using proviral DNA in women before and after viral suppression.

BACKGROUND: An HIV-1 tropism test is recommended prior to CCR5 antagonist administration to exclude patients harboring non-R5 virus from treatment with this class of antiretrovirals. HIV-1 tropism determination based on proviral DNA (pvDNA) may be useful in individuals with plasma viral RNA suppression. We developed a genotypic tropism assay for pvDNA and assessed its performance in a retrospective analysis of samples collected longitudinally. RESULTS: We randomly selected paired plasma/PBMC samples from the Women's Interagency HIV Study with plasma viral load ≥5,000 cp/mL at time 1 (T1), undetectable viral load maintained for ≥1 year and CD4+ >200 cells/µL at time 2 (T2). pvDNA was isolated from cryopreserved PBMCs. Sequences were analyzed in triplicate from 49/50 women, with tropism assigned using the geno2pheno (g2p) algorithm. The median time between T1 and T2 was 4.1 years. CXCR4-using virus was detected in 24% of the RNA samples and 33% of the pvDNA samples at T1, compared to 37% of the pvDNA samples at T2. Concordance between plasma RNA and pvDNA tropism was 88% at T1 and 80% at T2. The g2p scores for RNA (T1) vs DNA (T1, T2) were strongly correlated (Spearman rho: 0.85 (T1); 0.78 (T2)). In women with evidence of tropism switch at T2 (either R5 to non-R5 or non-R5 to R5), there was a correlation between change in tropism and time. Mean pvDNA viral load decreased by 0.4 log10 copies/106 cells between T1 and T2 (p < 0.0001), but this decrease was not significantly associated with tropism status. CONCLUSIONS: We demonstrated that pvDNA tropism in women with HIV-1 suppression is concordant with prior RNA tropism results, even after a median time of >4 years. pvDNA tropism testing may be useful to determine eligibility of patients with viral suppression to switch to a CCR5-antagonist based regimen as well as for research purposes.

A genotypic HIV-1 proviral DNA coreceptor tropism assay: characterization in viremic subjects.

BACKGROUND: HIV-1 coreceptor tropism testing is used to evaluate eligibility for CCR5 antagonist therapy. However, HIV-1 RNA-based tests are not suitable for virologically suppressed patients, therefore the use of proviral DNA tropism testing has been investigated. We describe a novel proviral DNA-based genotypic tropism assay and compare its performance to that of a sensitive HIV-1 RNA-based genotypic test. METHODS: Tropism was determined using HIV-1 plasma RNA and proviral DNA from 42 paired samples from patients with plasma viral loads ≥1000 HIV-1 RNA copies/mL. Proviral DNA sample types included whole blood, separated peripheral blood mononuclear cells resuspended in phosphate-buffered saline and peripheral blood mononuclear cells resuspended in spun plasma. The HIV-1 envelope V3 region was PCR-amplified, sequenced in triplicate, and analyzed for tropism with the geno2pheno algorithm using a 10% false-positive rate (FPR). RESULTS: Amplicons were obtained from proviral DNA and plasma RNA in 41/42 samples. Tropism predictions were highly concordant (93%-98%) between proviral DNA and plasma RNA, regardless of the proviral DNA isolation method. Non-R5 proviral DNA results were obtained for 100% of patients with detectable non-R5 plasma HIV-1 RNA results. Geno2pheno FPRs for proviral DNA and plasma RNA were highly correlated (Spearman rho = 0.86). CONCLUSIONS: Our findings demonstrate that proviral DNA tropism determinations from whole blood or peripheral blood mononuclear cells were highly concordant with plasma HIV-1 RNA tropism determinations. This assay may be useful for screening virologically suppressed patients for CCR5-antagonist eligibility and for research purposes.

The S40 residue in HIV-1 Gag p6 impacts local and distal budding determinants, revealing additional late domain activities.

BACKGROUND: HIV-1 budding is directed primarily by two motifs in Gag p6 designated as late domain-1 and -2 that recruit ESCRT machinery by binding Tsg101 and Alix, respectively, and by poorly characterized determinants in the capsid (CA) domain. Here, we report that a conserved Gag p6 residue, S40, impacts budding mediated by all of these determinants. RESULTS: Whereas budding normally results in formation of single spherical particles ~100 nm in diameter and containing a characteristic electron-dense conical core, the substitution of Phe for S40, a change that does not alter the amino acids encoded in the overlapping pol reading frame, resulted in defective CA-SP1 cleavage, formation of strings of tethered particles or filopodia-like membrane protrusions containing Gag, and diminished infectious particle formation. The S40F-mediated release defects were exacerbated when the viral-encoded protease (PR) was inactivated or when L domain-1 function was disrupted or when budding was almost completely obliterated by the disruption of both L domain-1 and -2. S40F mutation also resulted in stronger Gag-Alix interaction, as detected by yeast 2-hybrid assay. Reducing Alix binding by mutational disruption of contact residues restored single particle release, implicating the perturbed Gag-Alix interaction in the aberrant budding events. Interestingly, introduction of S40F partially rescued the negative effects on budding of CA NTD mutations EE75,76AA and P99A, which both prevent membrane curvature and therefore block budding at an early stage. CONCLUSIONS: The results indicate that the S40 residue is a novel determinant of HIV-1 egress that is most likely involved in regulation of a critical assembly event required for budding in the Tsg101-, Alix-, Nedd4- and CA N-terminal domain affected pathways.

Long-term antiretroviral therapy mitigates mortality and morbidity independent of HIV tropism: 18 years follow-up in a women's cohort.

OBJECTIVE: CXCR4 (X4)-tropic HIV-1 was found previously to herald CD4 + cell depletion and disease progression in individuals who were antiretroviral-naive or took combination antiretroviral therapy (cART) for less than 5 years. We updated this finding by investigating whether the deleterious effect of X4-tropic strains is mitigated by long-term cART. DESIGN: We examined morbidity and mortality in relation to HIV-1 tropism and cART in 529 participants followed up to 18 years in the Women's Interagency HIV Study; 91% were women of color. METHODS: Plasma-derived HIV-1 tropism was determined genotypically. RESULTS: We categorized participants according to the number of visits reported on cART after initiation. Group 1: three or less visits, 74% of these participants reporting no cART; group 2: at least four visits and less than 70% of visits on cART; group 3: at least 70% of visits on cART. AIDS mortality rates for participants in each group with X4 virus compared with those with R5 virus exclusively were, respectively: 62 vs. 40% ( P  = 0.0088); 23% vs. 22% [nonsignificant (NS)]; 7% vs. 14% (NS). Kaplan-Meier curves showed accelerated progression to AIDS death or AIDS-defining illness in participants with three or less cART visits and X4 viruses ( P  = 0.0028) but no difference in progression rates stratified by tropism in other groups. Logistic regression found that HIV-1 suppression for at least 10 semiannual visits (≥5 years total) mitigated X4 tropism's deleterious effect on mortality, controlling for maximal viral load, and CD4 + nadir. CONCLUSION: Long-term cART markedly mitigated the deleterious effect of X4 viruses on AIDS morbidity and mortality. Mitigation was correlated with duration of viral suppression, supporting HIV-1 suppression as a crucial goal.

Successful surgical intervention for the management of endocarditis due to multidrug resistant Candida parapsilosis: case report and literature review.

Candida parapsilosis is an uncommon cause of invasive endocarditis. This pathogen induces severe complications and carries a high mortality rate. We describe a case of C. parapsilosis endocarditis in a 54-year-old man with a history of HIV and Hepatitis C infection who previously underwent prosthetic valve replacement due to bacterial endocarditis. The patient presented with prolonged febrile episodes and fungemia with repeat blood cultures positive for C. parapsilosis. The patient failed multiple regimens of antifungal therapy and the C. parapsilosis isolate progressively acquired resistance to a number of drugs. Due to the multidrug resistant nature of the isolate, replacement of the infected valve was required to resolve his fungemia, and the patient remained asymptomatic for two years. This case is unusual due to the multidrug resistant nature of the isolate requiring both combined medical and surgical intervention. A review of published reports indicates that endocarditis due to C. parapsilosis responds well to a combination of medical and surgical interventions; the latter is particularly suitable for immunocompromised hosts.

SARS-CoV-2 worldwide replication drives rapid rise and selection of mutations across the viral genome: a time-course study - potential challenge for vaccines and therapies.

Scientists and the public were alarmed at the first large viral variant of SARS-CoV-2 reported in December 2020. We have followed the time course of emerging viral mutants and variants during the SARS-CoV-2 pandemic in ten countries on four continents. We examined > 383,500 complete SARS-CoV-2 nucleotide sequences in GISAID (Global Initiative of Sharing All Influenza Data) with sampling dates extending until April 05, 2021. These sequences originated from ten different countries: United Kingdom, South Africa, Brazil, United States, India, Russia, France, Spain, Germany, and China. Among the 77 to 100 novel mutations, some previously reported mutations waned and some of them increased in prevalence over time. VUI2012/01 (B.1.1.7) and 501Y.V2 (B.1.351), the so-called UK and South Africa variants, respectively, and two variants from Brazil, 484K.V2, now called P.1 and P.2, increased in prevalence. Despite lockdowns, worldwide active replication in genetically and socio-economically diverse populations facilitated selection of new mutations. The data on mutant and variant SARS-CoV-2 strains provided here comprise a global resource for easy access to the myriad mutations and variants detected to date globally. Rapidly evolving new variant and mutant strains might give rise to escape variants, capable of limiting the efficacy of vaccines, therapies, and diagnostic tests.

FACS as useful tool to study distinct hyalocyte populations.

Hyalocytes, the cells of the vitreous body, are assumed to be involved in physiological as well as patho-physiological processes within the eye. However, current knowledge about the cells is still limited. As different morphological types of hyalocytes are described in the literature, it seems reasonable to try to isolate individual populations prior to characterization of single cell types. To achieve this, the present study investigated the utility of fluorescence activated cell sorting (FACS) for hyalocyte separation. Subsequent to digestion of vitreous bodies using collagenase, the resulting cell suspension was analyzed and separated using FACS without any additional staining. Two-parameter dot plots of forward scatter (indicating size) against sideward scatter (indicating granularity) showed two distinct cell populations; staining with propidium iodide confirmed that both populations represent living cells. After sorting, cells of both populations were seeded on tissue culture plastic (tissue culture treated polystyrene). Only one population attached and proliferated, whereas the other population was non-adherent. Even when seeding the native cell mix, only one population of cells was observed after two passages, as indicated by FACS. Furthermore, ascorbic acid increased proliferation of these cells similarly to the proliferation of the separated cell population. These data point out that only one of the two populations adheres and proliferates on tissue culture plastic. To conclude, the established isolation technique allows for separation of clearly defined hyalocyte populations. Moreover, clear hints were obtained that only one of the two populations adheres and proliferates under the commonly applied culture conditions.

Ascorbic acid enhances adipogenesis of bone marrow-derived mesenchymal stromal cells.

A prerequisite to successfully engineer cell-based adipose tissue surrogates is the evaluation of in vitro culture conditions that facilitate expansion of primary precursor cells under retention of their adipogenic potential and that enable a large fraction of the heterogeneous cell pool to undergo adipogenesis upon respective stimuli. Ascorbic acid (AA) was reported to enhance differentiation of precursor cells into various mesenchymal cell types. Thus, the aim of the current study was to evaluate the influence of AA on hormonally induced adipogenesis of bone marrow-derived mesenchymal stromal cells (BMSCs) in vitro when supplemented during cell propagation and/or adipogenic differentiation. BMSCs were isolated from rat bone marrow, propagated, and hormonally induced to undergo adipogenesis. Supplementation of AA from the time of induction increased the fraction of BMSCs differentiating into adipocytes and glycerol-3-phosphate dehydrogenase activity up to 2-fold. Furthermore, administration of AA already during propagation had an even larger effect with an up to 8-fold increase in adipogenic markers. Assessment of collagen accumulation suggested that the observed effects might be attributed to an enhanced collagen synthesis during propagation. The presented results demonstrate AA as a potent medium component able to enhance adipogenic conversion of BMSCs, especially when administered during cell propagation.

Methods for viral RNA isolation and PCR amplification for sequencing of near full-length HIV-1 genomes.

HIV-1 in plasma represents the viral quasispecies replicating in the patient at any given time. Studies of HIV-1 viral RNA from plasma or other body fluids therefore reflect the virus present in real time. To obtain near full-length genomic sequences derived from virion RNA it is first necessary to carefully isolate and amplify the RNA.The procedure described below, involves viral RNA extraction, reverse transcription (RT) of the extracted RNA to produce cDNA copies, and PCR amplification of long HIV-1 gene fragments using site-specific, overlapping primers. The primers are based on subtype B HIV-1 strains, and plasma specimens are used in the procedures. However, the protocol can easily be adapted to other HIV-1 subtypes by modifying the primers to match the subtype of interest.

Oral Microbiome in HIV-Infected Women: Shifts in the Abundance of Pathogenic and Beneficial Bacteria Are Associated with Aging, HIV Load, CD4 Count, and Antiretroviral Therapy.

Human immunodeficiency virus (HIV)-associated nonacquired immunodeficiency syndrome (AIDS) conditions, such as cardiovascular disease, diabetes, osteoporosis, and dementia are more prevalent in older than in young adult HIV-infected subjects. Although the oral microbiome has been studied as a window into pathogenesis in aging populations, its relationship to HIV disease progression, opportunistic infections, and HIV-associated non-AIDS conditions is not well understood. We utilized 16S rDNA-based pyrosequencing to compare the salivary microbiome in three groups: (1) Chronically HIV-infected women >50 years of age (aging); (2) HIV-infected women <35 years of age (young adult); and (3) HIV-uninfected age-matched women. We also examined correlations between salivary dysbiosis, plasma HIV RNA, CD4+ T cell depletion, and opportunistic oral infections. In both aging and young adult women, HIV infection was associated with salivary dysbiosis characterized by increased abundance of Prevotella melaninogenica and Rothia mucilaginosa. Aging was associated with increased bacterial diversity in both uninfected and HIV-infected women. In HIV-infected women with oral coinfections, aging was also associated with reduced abundance of the common commensal Veillonella parvula. Patients taking antiretroviral therapy showed increased numbers of Neisseria and Haemophilus. High plasma HIV RNA levels correlated positively with the presence of Prevotella and Veillonella, and negatively with the abundance of potentially beneficial Streptococcus and Lactobacillus. Circulating CD4+ T cell numbers correlated positively with the abundance of Streptococcus and Lactobacillus. Our findings extend previous studies of the role of the microbiome in HIV pathogenesis, providing new evidence that HIV infection is associated with a shift toward an increased pathogenic footprint of the salivary microbiome. Taken together, the data suggest a complex relationship, worthy of additional study, between chronic dysbiosis in the oral cavity, aging, viral burden, CD4+ T cell depletion, and long-term antiretroviral therapy.

Structural basis for coreceptor selectivity by the HIV type 1 V3 loop.

The third variable region (V3) of the HIV-1 surface glycoprotein, gp120, plays a central role in the interaction of the virus envelope with the cell surface chemokine receptors, triggering membrane fusion and virus entry into human lymphocytes and macrophages. The CXCR4 and CCR5 chemokine receptors are used by "X4-tropic" and "R5-tropic" viruses, respectively. Recently, the crown of the V3 loop was shown to bear a close structural homology to the beta2-beta3 loop in the CXC and CC chemokines, the natural ligands of CXCR4 and CCR5, respectively. This homology can serve as the foundation for 3D molecular modeling of the V3 loops from primary isolates whose coreceptor usage was experimentally defined. The modeling revealed a charged "patch" on the surface of V3 that correlates with coreceptor usage. This V3 surface patch is positively charged in X4-tropic viruses and negatively charged or neutral in R5-tropic viruses, and is formed by two amino acids, at position 11 and at position 24 or 25; amino acids 11 and 24 or 11 and 25 contact each other in 3D space. Residues at positions 11 and 25 were known previously to influence coreceptor usage, and the charge of the residues at these two positions is often used to predict viral tropism. However, we found that the predictive value of using the charge of residues 11, 24, and 25 to identify X4 or R5 tropism was improved over using only the charge of residues 11 and 25. Thus, the data suggest a new " 11/24/25 rule" : a positively charged amino acid at position 11, 24, or 25 defines X4; otherwise R5. This rule gave an overall predictive value of 94% for 217 viruses whose tropism had been determined experimentally as either X4 or R5. The results have additional implications for the design of HIV therapeutics, vaccines, and strategies for monitoring disease progression.

Genomic analysis of HIV type 1 strains derived from a mother and child pair of long-term nonprogressors.

To investigate the viral features of long-term nonprogressive HIV-1 infection and the selection of viral genomes, we studied serial complete HIV-1 sequences obtained from a mother-child pair, both long-term nonprogressors. Analysis of four genomic sequences demonstrated that all viral genes were intact, lacking major deletions or premature stop codons to easily explain the slow disease progression. These data suggest that viral attenuation, if present, was caused by subtle sequence variations or virus-host interactions. Serial sequences from an HIV-1-infected mother-child pair afforded us the opportunity to examine the immune selection of HIV-1 sequences years after transmission between individuals. We demonstrated that the daughter's strains were most likely subjected to immunoselection or immunoediting according to the presence of novel MHC class I alleles that differed between mother and daughter. An analysis of nef-specific cytotoxic T-lymphocyte responses in the child, whose HIV-1 nef sequence differed from the maternal nef, supported this interpretation. This study highlights the potential of full genome analysis in the investigation of pathogenesis and immune selection during HIV-1 evolution.

Ascorbic acid modulates proliferation and extracellular matrix accumulation of hyalocytes.

Ascorbic acid is known to influence proliferation and functional properties of several cell types and is therefore widely used in tissue engineering. In this study, the effect of ascorbic acid on the proliferation and functional properties of hyalocytes was evaluated. Hyalocytes were cultured with different amounts of ascorbic acid in classical two-dimensional (2-D) cultures and a three-dimensional (3-D) pellet culture system. Ascorbic acid enhanced hyalocyte proliferation dose-dependently at concentrations between 0.1 and 3 microg/mL; proliferation was constant over a wide concentration range up to 150 microg/mL, concentrations of 500 microg/mL showed toxic effects. In 2-D hyalocyte culture, the accumulation of glycosaminoglycans (GAG) and collagens increased in response to ascorbic acid supplementation of 10 or 200 microg/mL. Normalized to the cell number, GAG production was not influenced, whereas collagen production increased. These results could be verified in a pellet-like 3-D culture system. Ascorbic acid also influenced hyalocytes on the mRNA level; the expression of COL11A1 was clearly enhanced by ascorbic acid. To conclude, ascorbic acid modulates proliferation and collagen accumulation of hyalocytes; it also influences mRNA expression of the cells. Taken together with the fact that ascorbic acid is present in high concentrations in the vitreous body, this vitamin seems to be an important factor for in vitro hyalocyte culture.

Measuring cell adhesion on RGD-modified, self-assembled PEG monolayers using the quartz crystal microbalance technique.

In this study, the suitability of a flow-through quartz crystal microbalance system for the detection of the adhesion of rMSCs and 3T3-L1 fibroblasts on different surfaces is demonstrated. Frequency shifts for rMSCs of -6.7 mHz x cell(-1) and -2.0 mHz x cell(-1) for 3T3-L1 cells could be detected on non-modified gold sensors, revealing that the frequency shift per cell is comparable to that of a static setup. Modifying the sensor surface with SAMs of thioalkylated omega-amine-terminated PEG derivatives led to cell-adhesion-resistant surfaces. Total frequency shifts of only -20 +/- 7 Hz showed that protein adsorption was also significantly reduced. Attaching 35 pmol x mm(-2) of the GRGDS cell adhesion motif to the SAMs induced specific cell adhesion due to RGD-integrin interactions; the resonance frequency dropped by 3.4 mHz x cell(-1). Furthermore, the kinetics of cell detachment could be determined. The corresponding processes were completed after 10 min for trypsin, and not before 90 min with GRGDS. Moreover, the detectability of cell adhesion was shown to increase after the addition of manganese cations. The total decrease in the resonance frequency was almost 80 Hz in the presence of Mn(2+) (6.4 mHz x cell(-1)). [image: see text] Staining the cytoskeleton of the rMSCs shows that the GRGDS-modified surfaces are almost completely covered with well-spread cells.

RG7386, a Novel Tetravalent FAP-DR5 Antibody, Effectively Triggers FAP-Dependent, Avidity-Driven DR5 Hyperclustering and Tumor Cell Apoptosis.

Dysregulated cellular apoptosis and resistance to cell death are hallmarks of neoplastic initiation and disease progression. Therefore, the development of agents that overcome apoptosis dysregulation in tumor cells is an attractive therapeutic approach. Activation of the extrinsic apoptotic pathway is strongly dependent on death receptor (DR) hyperclustering on the cell surface. However, strategies to activate DR5 or DR4 through agonistic antibodies have had only limited clinical success. To pursue an alternative approach for tumor-targeted induction of apoptosis, we engineered a bispecific antibody (BsAb), which simultaneously targets fibroblast-activation protein (FAP) on cancer-associated fibroblasts in tumor stroma and DR5 on tumor cells. We hypothesized that bivalent binding to both FAP and DR5 leads to avidity-driven hyperclustering of DR5 and subsequently strong induction of apoptosis in tumor cells but not in normal cells. Here, we show that RG7386, an optimized FAP-DR5 BsAb, triggers potent tumor cell apoptosis in vitro and in vivo in preclinical tumor models with FAP-positive stroma. RG7386 antitumor efficacy was strictly FAP dependent, was independent of FcR cross-linking, and was superior to conventional DR5 antibodies. In combination with irinotecan or doxorubicin, FAP-DR5 treatment resulted in substantial tumor regression in patient-derived xenograft models. FAP-DR5 also demonstrated single-agent activity against FAP-expressing malignant cells, due to cross-binding of FAP and DR5 across tumor cells. Taken together, these data demonstrate that RG7386, a novel and potent antitumor agent in both mono- and combination therapies, overcomes limitations of previous DR5 antibodies and represents a promising approach to conquer tumor-associated resistance to apoptosis. Mol Cancer Ther; 15(5); 946-57. ©2016 AACR.

Characterization of intersubtype recombinant HIV type 1 genomes using a nonradioactive heteroduplex tracking assay.

The HIV-1 epidemic is characterized by the dominance of distinct viral subtypes in different regions of the world, and intersubtype recombinants are common. Traditional subtyping methods analyze only a small fragment of the HIV-1 genome, so the true extent of diversity and recombination has been difficult to examine. We developed a heteroduplex tracking assay (HTA) to identify viral subtypes and rapidly detect recombinant HIV-1 genomes. By using probes that target seven regions across the HIV-1 genome, HTAs can identify intersubtype recombinants on the basis of the heteroduplex mobility pattern. We used this method to analyze HIV-1 strains from 12 patients from the United States and Kenya, comparing the results with those obtained by sequencing. HTA analysis correctly identified the subtype of each region of the genome, revealing that several isolates were recombinants. This method is suitable for studies of HIV-1 diversity and recombination in areas of the world where multiple subtypes are found.

Adipose tissue engineering based on mesenchymal stem cells and basic fibroblast growth factor in vitro.

Despite the clinical need for reconstructive and plastic surgery, the supply of engineered adipose tissue equivalents still remains a challenge. As yet, only preadipocytes have been applied as a cell material for the in vitro tissue engineering of fat. Herein, we report the establishment of a three-dimensional (3-D) long-term cell culture, using bone marrow-derived mesenchymal stem cells (MSCs) as an alternative cell source and custom-made poly(lactic-co-glycolic acid) (PLGA) scaffolds as a cell carrier. Cell-polymer constructs were cultivated for 4 weeks in both the absence and presence of basic fibroblast growth factor (bFGF), which was previously shown to strongly enhance the adipogenesis of MSCs in conventional 2-D short-term culture. A striking enhancement of the adipogenic differentiation of MSCs and tissue development caused by bFGF in the 3-D culture was observed by osmium tetroxide histology and scanning electron microscopy. At the molecular level, reflecting the increased accumulation of lipids, bFGF increased the enzymatic activity of glycerol-3-phosphate dehydrogenase, a late marker of adipogenesis, and the expression of adipocyte-specific genes peroxisome proliferator activated receptor-gamma2 (PPARgamma2) and glucose transporter-4 (GLUT4), as assessed by reverse transcription-polymerase chain reaction. This study demonstrates that the use of bone marrow-derived MSCs, especially in combination with bFGF, may represent a promising approach to adipose tissue engineering.

Risk of progression to AIDS and death in women infected with HIV-1 initiating highly active antiretroviral treatment at different stages of disease.

BACKGROUND: The optimal virologic and immunologic stage at which to initiate antiretroviral therapy in individuals infected with human immunodeficiency virus type 1 (HIV-1) is undefined. METHODS: Among 1054 HIV-1-infected women in a prospective cohort study, we determined the time from initiation of highly active antiretroviral treatment (HAART) to acquired immunodeficiency syndrome (AIDS) and death. RESULTS: Median follow-up was 3.4 years. Of 553 women without AIDS at HAART initiation, 62 (11%) developed AIDS. Compared with women with CD4(+) cell counts greater than 350/microL at HAART initiation, women with cell counts of 200 to 350/microL and less than 200/microL had relative hazards (RHs) for progression to AIDS of 0.93 (95% confidence interval [CI], 0.46-1.86) and 2.48 (95% CI, 1.39-4.42), respectively. Compared with those with HIV-1 RNA values less than 5000 copies/mL, women with 5000 to 50,000 copies/mL and greater than 50,000 copies/mL had RHs of 1.39 (95% CI, 0.74-2.64) and 2.09 (95% CI, 1.09-3.99), respectively. Among women with AIDS at HAART initiation (n = 501), RHs of death were 1.97 (95% CI, 0.84-4.66) and 3.35 (95% CI, 1.59-7.08) with CD4(+) cell counts of 200 to 350/microL and less than 200/microL, respectively, relative to those with greater than 350/microL, and 1.90 (95% CI, 0.84-4.30) and 3.70 (95% CI, 1.81-7.54) for those with HIV-1 RNA values of 5000 to 50,000 and greater than 50,000 copies/mL, respectively, relative to those with less than 5000 copies/mL. CONCLUSIONS: Progression to AIDS and death was predicted by pre-HAART values of less than 200/microL for CD4(+) cells and greater than 50,000 HIV-1 RNA copies/mL, indicating that deferral of HAART until the CD4(+) cell count is between 350 and 200/microL is a valid strategy in the clinical management of HIV-1 infection.

Recombination following superinfection by HIV-1.

BACKGROUND: There is increasing recognition of recombinant HIV-1 strains globally, but it has been unclear whether recombination results from superinfection during untreated, chronic infection. OBJECTIVE: To search for evidence of recombination and superinfection in Africa, where multiple HIV-1 subtypes facilitate identification of strains. METHODS: Serial blood samples from highly exposed, chronically infected women in Nairobi's Pumwani sex workers cohort were examined. Serial, complete HIV-1 RNA sequence analyses were performed for seven untreated long-term survivors. Sequences were subjected to computational analysis. RESULTS: One woman had evidence of both superinfection and recombination. Complete HIV-1 RNA sequences were first derived from plasma obtained in 1986, when the woman had been HIV seropositive for at least 21 months; this sequence was entirely subtype A. The sequences obtained from plasma in 1995 and 1997, however, were subtype A/C recombinants with a SimPlot demonstrating that the subtype A fragment in 1995 and 1997 was derived from the original 1986 A sequence. Heteroduplex tracking assays demonstrated that the subtype C sequences were not detectable as minor species in 1986. CONCLUSION: Intersubtype recombination took place between the original non-recombinant subtype A strain and the superinfecting subtype C strain in an untreated, chronically infected woman. This finding helps to explain the rising prevalence of recombinant HIV-1 worldwide. Recombination resulting from superinfection with diverse strains may pose problems for eliciting broad immune responses necessary for an effective vaccine.