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A protein-expressing citrus tristeza virus (CTV)-based vector construct, pT36CA-V1.3, obtained from a California isolate of the T36 strain (T36CA), was retooled into a virus induced gene silencing (VIGS) system intended for use with studies of California citrus. VIGS constructs engineered with a truncated Citrus macrophylla (Cm) PHYTOENE DESATURASE (PDS) gene sequence in the sense or anti-sense orientation worked equally well to silence the endogenous CmPDS gene. In a parallel effort to optimize vector performance, two non-synonymous nucleotides in open reading frame 1a of pT36CA-V1.3 were replaced with those conserved in the reference sequences from the T36CA cDNA library. The resulting viruses, T36CA-V1.4 (with one amino acid modification: D760N) and T36CA-V1.5 (with two amino acid modifications: D760N and P1174L), along with T36CA-V1.3 were individually propagated in Nicotiana benthamiana and C. macrophylla plants. Enzyme-linked immunosorbent assay (ELISA) measurements of extracts of the newly emerged leaves suggested that all three viruses accumulated to similar levels in N. benthamiana plants at 5 week-post-inoculation. ELISA values of T36CA-V1.4- and -V1.5-infected C. macrophylla samples were significantly higher than that of T36CA-V1.3-infected samples within an 8 to 12 month-post-inoculation (mpi) window, suggesting a higher accumulation of T36CA-V1.4 and -V1.5 than T36CA-V1.3. However, at 36 mpi, the ELISA values suggested that all three viruses accumulated to similar levels. When C. macrophylla plants infected with each of the three viruses were grafted to commercial citrus varieties, a limited number of receptor plants became infected, demonstrating a weak but nonetheless (the first) successful delivery of T36CA to California-grown commercial citrus.
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Transmission of the crinivirus, lettuce infectious yellows virus (LIYV), is determined by a minor coat protein (CPm)-mediated virion retention mechanism located in the foregut of its whitefly vector. To better understand the functions of LIYV CPm, chimeric CPm mutants engineered with different lengths of the LIYV CPm amino acid sequence and that of the crinivirus, lettuce chlorosis virus (LCV), were constructed based on bioinformatics and sequence alignment data. The 485 amino acid-long chimeric CPm of LIYV mutant, CPmP-1, contains 60â% (from position 3 to 294) of LCV CPm amino acids. The chimeric CPm of mutants CPmP-2, CPmP-3 and CPmP-4 contains 46 (position 3 to 208), 51 (position 3 to 238) and 41â% (position 261 to 442) of LCV CPm amino acids, respectively. All four mutants moved systemically, expressed the chimeric CPm and formed virus particles. However, following acquisition feeding of the virus preparations, only CPmP-1 was retained in the foreguts of a significant number of vectors and transmitted. In immuno-gold labelling transmission electron microscopy (IGL-TEM) analysis, CPmP-1 particles were distinctly labelled by antibodies directed against the LCV but not LIYV CPm. In contrast, CPmP-4 particles were not labelled by antibodies directed against the LCV or LIYV CPm, while CPmP-2 and -3 particles were weakly labelled by anti-LIYV CPm but not anti-LCV CPm antibodies. The unique antibody recognition and binding pattern of CPmP-1 was also displayed in the foreguts of whitefly vectors that fed on CPmP-1 virions. These results are consistent with the hypothesis that the chimeric CPm of CPmP-1 is incorporated into functional virions, with the LCV CPm region being potentially exposed on the surface and accessible to anti-LCV CPm antibodies.
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Proteínas do Capsídeo/metabolismo , Crinivirus/fisiologia , Hemípteros/virologia , Insetos Vetores/virologia , Nicotiana/virologia , Doenças das Plantas/virologia , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Crinivirus/genética , Sistema Digestório/virologia , Engenharia Genética , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/metabolismo , Mutação , Plantas Geneticamente Modificadas/virologia , Vírion/fisiologiaRESUMO
Changing to burosumab, a monoclonal antibody targeting fibroblast growth factor 23, significantly improved phosphorus homeostasis, rickets, lower-extremity deformities, mobility, and growth versus continuing oral phosphate and active vitamin D (conventional therapy) in a randomized, open-label, phase 3 trial involving children aged 1-12 years with X-linked hypophosphatemia. Patients were randomized (1:1) to subcutaneous burosumab or to continue conventional therapy. We present patient-reported outcomes (PROs) from this trial for children aged ≥ 5 years at screening (n = 35), using a Patient-Reported Outcomes Measurement Information System (PROMIS) questionnaire and SF-10 Health Survey for Children. PROMIS pain interference, physical function mobility, and fatigue scores improved from baseline with burosumab at weeks 40 and 64, but changed little with continued conventional therapy. Pain interference scores differed significantly between groups at week 40 (- 5.02, 95% CI - 9.29 to - 0.75; p = 0.0212) but not at week 64. Between-group differences were not significant at either week for physical function mobility or fatigue. Reductions in PROMIS pain interference and fatigue scores from baseline were clinically meaningful with burosumab at weeks 40 and 64 but not with conventional therapy. SF-10 physical health scores (PHS-10) improved significantly with burosumab at week 40 (least-squares mean [standard error] + 5.98 [1.79]; p = 0.0008) and week 64 (+ 5.93 [1.88]; p = 0.0016) but not with conventional therapy (between-treatment differences were nonsignificant). In conclusion, changing to burosumab improved PRO measures, with statistically significant differences in PROMIS pain interference at week 40 versus continuing with conventional therapy and in PHS-10 at weeks 40 and 64 versus baseline.Trial registration: ClinicalTrials.gov NCT02915705.
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Raquitismo Hipofosfatêmico Familiar , Anticorpos Monoclonais , Anticorpos Monoclonais Humanizados , Criança , Raquitismo Hipofosfatêmico Familiar/tratamento farmacológico , Humanos , Medidas de Resultados Relatados pelo PacienteRESUMO
BACKGROUND AND HYPOTHESIS: Post-traumatic elbow contracture remains a common and challenging complication with often unsatisfactory outcomes. Although the etiology is unknown, elevated or abnormal post-fracture synovial fluid cytokine levels may result in the migration of fibroblasts to the capsule and contribute to capsular pathology. Thus, the purpose of this study was to characterize the cytokine composition in the synovial fluid fracture hematoma of patients with intra-articular elbow fractures. METHODS: The elbow synovial fluid fracture hematoma of 11 patients with intra-articular elbow fractures was analyzed for CTXII (C-terminal telopeptides of type II collagen [a cartilage breakdown product]) as well as 15 cytokines and matrix metalloproteinases (MMPs) including interferon γ, interleukin (IL) 1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, tumor necrosis factor α, MMP-1, MMP-2, MMP-3, MMP-9, and MMP-10. The uninjured, contralateral elbow served as a matched control. Mean concentrations of each factor were compared between the fluid from fractured elbows and the fluid from control elbows. RESULTS: The levels of 14 of 15 measured cytokines and MMPs-interferon γ, IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, tumor necrosis factor α, MMP-1, MMP-3, MMP-9, and MMP-10-were significantly higher in the fractured elbows. In addition, post hoc power analysis revealed that 10 of 14 significant differences were detected with greater than 90% power. The mean concentration of CTXII was not significantly different between groups. CONCLUSIONS: These results demonstrate a proinflammatory environment after fracture that may be the catalyst to the development of post-traumatic elbow joint contracture. The cytokines with elevated levels were similar, although not identical, to the cytokines with elevated levels in studies of other weight-bearing joints, indicating the elbow responds uniquely to trauma.
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Citocinas/metabolismo , Lesões no Cotovelo , Hematoma/metabolismo , Fraturas Intra-Articulares/metabolismo , Metaloproteinases da Matriz/metabolismo , Líquido Sinovial/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Colágeno Tipo II/metabolismo , Feminino , Hematoma/etiologia , Humanos , Inflamação/metabolismo , Fraturas Intra-Articulares/complicações , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/metabolismo , Estudos ProspectivosAssuntos
Falência Renal Crônica , Lúpus Eritematoso Sistêmico , Insuficiência Renal Crônica , Humanos , Diálise Renal , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Falência Renal Crônica/complicações , Falência Renal Crônica/diagnóstico , Falência Renal Crônica/terapia , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/terapiaRESUMO
BACKGROUND: The non-translated regions at the genome ends of RNA viruses serve diverse functions and can exhibit various levels of nucleotide (nt) heterogeneity. However, the extent of nt heterogeneity at the extreme termini of Citrus tristeza virus (CTV) genomes has not been comprehensively documented. This study aimed to characterize two widely prevalent CTV genotypes, T36-CA and T30-CA, from California that have not been sequenced or analyzed substantially. The information obtained will be used in our ongoing effort to construct the infectious complementary (c) DNA clones of these viruses. METHODS: The terminal nts of the viral genomes were identified by sequencing cDNA clones of the plus- and/or minus-strand of the viral double-stranded (ds) RNAs generated using 5' and 3' rapid amplification of cDNA ends. Cloned cDNAs corresponding to the complete genome sequences of both viruses were generated using reverse transcription-polymerase chain reactions, sequenced, and subjected to phylogenetic analysis. RESULTS: Among the predominant terminal nts identified, some were identical to the consensus sequences in GenBank, while others were different or unique. Remarkably, one of the predominant 5' nt variants of T36-CA contained the consensus nts "AATTTCAAA" in which a highly conserved cytidylate, seen in all other full-length T36 sequences, was absent. As expected, but never systematically verified before, unique variants with additional nt (s) incorporated upstream of the 5' terminal consensus nts of T36-CA and T30-CA were also identified. In contrast to the extreme 5' terminal nts, those at the extreme 3' termini of T36-CA and T30-CA were more conserved compared to the reference sequences, although nt variants were also found. Notably, an additional thymidylate at the extreme 3' end was identified in many T36-CA sequences. Finally, based on pairwise comparisons and phylogenetic analysis with multiple reference sequences, the complete sequences of both viruses were found to be highly conserved with those of the respective genotypes. CONCLUSIONS: The extreme terminal nts in the T36-CA and T30-CA genomes were identified, revealing new insights on the heterogeneity of these CTV genomic regions. T36-CA and T30-CA were the first and the second genotypes, respectively, of CTV originating from California to be completely sequenced and analyzed.
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Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Closterovirus/genética , Variação Genética , Genoma Viral , RNA Viral/genética , California , Closterovirus/classificação , Closterovirus/isolamento & purificação , Genótipo , Análise de Sequência de DNARESUMO
Numerous pathogens of humans, animals, and plants are transmitted by specific arthropod vectors. However, understanding the mechanisms governing these pathogen-vector interactions is hampered, in part, by the lack of easy-to-use analytical tools. We investigated whitefly transmission of Lettuce infectious yellows virus (LIYV) by using a unique immunofluorescent localization approach in which we fed virions or recombinant virus capsid components to whiteflies, followed by feeding them antibodies to the virions or capsid components, respectively. Fluorescent signals, indicating the retention of virions, were localized in the anterior foregut or cibarium of a whitefly vector biotype but not within those of a whitefly nonvector biotype. Retention of virions in these locations strongly corresponded with the whitefly vector transmission of LIYV. When four recombinant LIYV capsid components were individually fed to whitefly vectors, significantly more whiteflies retained the recombinant minor coat protein (CPm). As demonstrated previously and in the present study, whitefly vectors failed to transmit virions preincubated with anti-CPm antibodies but transmitted virions preincubated with antibodies recognizing the major coat protein (CP). Correspondingly, the number of insects that specifically retained virions preincubated with anti-CPm antibodies were significantly reduced compared with those that specifically retained virions preincubated with anti-CP antibodies. Notably, a transmission-defective CPm mutant was deficient in specific virion retention, whereas the CPm-restored virus showed WT levels of specific virion retention and transmission. These data provide strong evidence that transmission of LIYV is determined by a CPm-mediated virion retention mechanism in the anterior foregut or cibarium of whitefly vectors.
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Proteínas do Capsídeo/metabolismo , Crinivirus/metabolismo , Transmissão de Doença Infecciosa , Imunofluorescência/métodos , Hemípteros/virologia , Insetos Vetores/virologia , Vírion/metabolismo , Animais , Anticorpos Antivirais/metabolismo , Crinivirus/genética , DNA Complementar/genética , Ensaio de Imunoadsorção Enzimática , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: In patients with X-linked hypophosphatemia (XLH), conventional therapy with oral phosphate salts and active vitamin D has been associated with nephrocalcinosis. However, the nature of the relationships among XLH, its treatment, nephrocalcinosis, and kidney function remain poorly understood. METHODS: Renal ultrasounds were performed and glomerular filtration rates were estimated (eGFR) at baseline in burosumab-naïve patients with XLH who participated in burosumab clinical trials (NCT02181764, NCT02526160, NCT02537431, NCT02163577, NCT02750618, NCT02915705) or enrolled in the XLH Disease Monitoring Program (XLH-DMP; NCT03651505). In this cross-sectional analysis, patient, disease, and treatment characteristics were described among patients with and without nephrocalcinosis. RESULTS: The analysis included 196 children (mean [SD] age 7.6 [4.0] years) and 318 adults (40.3 [13.1] years). Mean (SD) height z-score was -1.9 (1.2) for children and -2.3 (1.7) for adults. Nearly all children (97%) and adults (94%) had previously received conventional therapy. Nephrocalcinosis was detected in 22% of children and 38% of adults. In children, reduced eGFR <90 ml/min/1.73 m2 was more prevalent in those with nephrocalcinosis (25%) than in those without (11%), a finding that was not observed in adults. Children with nephrocalcinosis had lower mean values of TmP/GFR (P<.05), serum 1,25(OH)2D (P<.05), and eGFR (P<.001) and higher mean serum calcium concentrations (P<.05) than did those without nephrocalcinosis. Adults with nephrocalcinosis had lower mean serum phosphorus (P<.01) and 1,25(OH)2D (P<.05) concentrations than those without. Exploratory logistic regression analyses revealed no significant associations between the presence of nephrocalcinosis and other described patient or disease characteristics. CONCLUSIONS: Nephrocalcinosis was observed in nearly one quarter of children and more than one-third of adults with XLH. Further study is needed to better understand the predictors and long-term consequences of nephrocalcinosis, with surveillance for nephrocalcinosis remaining important in the management of XLH.
BACKGROUND: Conventionally, patients with X-linked hypophosphatemia (XLH) were treated with phosphate and vitamin D taken by mouth. However, this therapy might lead to a buildup of calcium in the kidney, called nephrocalcinosis. Here, we tried to better understand how XLH, conventional therapy, nephrocalcinosis, and kidney function are related. METHODS: Nephrocalcinosis was detected with kidney ultrasounds. Kidney function, called the estimated glomerular filtration rate (eGFR), was determined using the blood level of creatinine. Patients had been part of burosumab clinical trials or part of the XLH Disease Monitoring Program. Data were collected from patients before they received burosumab. RESULTS: The study included 196 children and 318 adults. Almost all children and adults had received conventional therapy. 22% of children had nephrocalcinosis and 38% of adults had nephrocalcinosis. In children, low eGFR was more common in those with nephrocalcinosis (25%) than in those without (11%). In adults, levels of eGFR were similar among those with and without nephrocalcinosis.Some lab values were different among patients with versus those without nephrocalcinosis. Children with nephrocalcinosis had significantly greater loss of phosphate by the kidneys, lower blood levels of the active form of vitamin D (1,25(OH)2D), lower eGFR, and higher blood levels of calcium than those without nephrocalcinosis. Adults with nephrocalcinosis had significantly lower blood levels of phosphorus and 1,25(OH)2D concentrations than those without. CONCLUSIONS: Nearly one quarter of children and more than one-third of adults with XLH, most of whom had received conventional therapy, had nephrocalcinosis. Further study is needed to better understand what factors can predict who will get nephrocalcinosis and to understand the long-term consequences of nephrocalcinosis. It remains important to monitor patients with XLH for nephrocalcinosis.
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BACKGROUND: Little is known about the procedural characteristics, case volumes, and mortality rates for early- vs non-early-career interventional cardiologists in the United States. OBJECTIVES: This study examined operator-level data for patients who underwent percutaneous coronary intervention (PCI) between April 2018 and June 2022. METHODS: Data were collected from the National Cardiovascular Data Registry CathPCI Registry, American Board of Internal Medicine certification database, and National Plan and Provider Enumeration System database. Early-career operators were within 5 years of the end of training. Annual case volume, expected mortality and bleeding risk, and observed/predicted mortality and bleeding outcomes were evaluated. RESULTS: A total of 1,451 operators were early career; 1,011 changed their career status during the study; and 6,251 were non-early career. Overall, 514,540 patients were treated by early-career and 2,296,576 patients by non-early-career operators. The median annual case volume per operator was 59 (Q1-Q3: 31-97) for early-career and 57 (Q1-Q3: 28-100) for non-early-career operators. Early-career operators were more likely to treat patients presenting with ST-segment elevation myocardial infarction and urgent indications for PCI (both P < 0.001). The median predicted mortality risk was 2.0% (Q1-Q3: 1.5%-2.7%) for early-career and 1.8% (Q1-Q3: 1.2%-2.4%) for non-early-career operators. The median predicted bleeding risk was 4.9% (Q1-Q3: 4.2%-5.7%) for early-career and 4.4% (Q1-Q3: 3.7%-5.3%) for non-early-career operators. After adjustment, an increased risk of mortality (OR: 1.08; 95% CI: 1.05-1.17; P < 0.0001) and bleeding (OR: 1.08; 95% CI: 1.05-1.12; P < 0.0001) were associated with early-career status. CONCLUSIONS: Early-career operators are caring for patients with more acute presentations and higher predicted risk of mortality and bleeding compared with more experienced colleagues, with modestly worse outcomes. These data should inform institutional practices to support the development of early-career proceduralists.
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Cardiologistas , Intervenção Coronária Percutânea , Sistema de Registros , Humanos , Estados Unidos/epidemiologia , Intervenção Coronária Percutânea/estatística & dados numéricos , Feminino , Masculino , Pessoa de Meia-Idade , Cardiologistas/estatística & dados numéricos , Idoso , Competência ClínicaRESUMO
In a randomized, open-label phase 3 study of 61 children aged 1-12 years old with X-linked hypophosphatemia (XLH) previously treated with conventional therapy, changing to burosumab every 2 weeks (Q2W) for 64 weeks improved the phosphate metabolism, radiographic rickets, and growth compared with conventional therapy. In this open-label extension period (weeks 64-88), 21 children continued burosumab Q2W at the previous dose or crossed over from conventional therapy to burosumab starting at 0.8 mg/kg Q2W with continued clinical radiographic assessments through week 88. Efficacy endpoints and safety observations were summarized descriptively for both groups (burosumab continuation, n = 6; crossover, n = 15). At week 88 compared with baseline, improvements in the following outcomes were observed in the burosumab continuation and crossover groups, respectively: mean (SD) RGI-C rickets total score (primary outcome), +2.11 (0.27) and +1.89 (0.35); mean (SD) RGI-C lower limb deformity score, +1.61 (0.91) and +0.73 (0.82); and mean (SD) height Z-score + 0.41 (0.50) and +0.08 (0.34). Phosphate metabolism normalized rapidly in the crossover group and persisted in the continuation group. Mean (SD) serum alkaline phosphatase decreased from 169% (43%) of the upper limit of normal (ULN) at baseline to 126% (51%) at week 88 in the continuation group and from 157% (33%) of the ULN at baseline to 111% (23%) at week 88 in the crossover group. During the extension period, treatment-emergent adverse events (AEs) were reported in all 6 children in the burosumab continuation group and 14/15 children in the crossover group. The AE profiles in the randomized and extension periods were similar, with no new safety signals identified. Improvements from baseline in radiographic rickets continued in the extension period among children with XLH who remained on burosumab. Children who crossed over from conventional therapy to burosumab demonstrated a rapid improvement in phosphate metabolism and improved rickets healing over the ensuing 22 weeks.
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Regenerative therapies for tendon are falling behind other tissues due to the lack of an appropriate and potent cell therapeutic candidate. This study aimed to induce tenogenesis using stable Scleraxis (Scx) overexpression in combination with uniaxial mechanical stretch of iPSC-derived mesenchymal stromal-like cells (iMSCs). Scx is the single direct molecular regulator of tendon differentiation known to date. Bone marrow-derived (BM-)MSCs were used as reference. Scx overexpression alone resulted in significantly higher upregulation of tenogenic markers in iMSCs compared to BM-MSCs. Mechanoregulation is known to be a central element guiding tendon development and healing. Mechanical stimulation combined with Scx overexpression resulted in morphometric and cytoskeleton-related changes, upregulation of early and late tendon markers, and increased extracellular matrix deposition and alignment, and tenomodulin perinuclear localization in iMSCs. Our findings suggest that these cells can be differentiated into tenocytes and might be a better candidate for tendon cell therapy applications than BM-MSCs.
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Células-Tronco Pluripotentes Induzidas , Células-Tronco Mesenquimais , Diferenciação Celular , Tendões , Matriz ExtracelularRESUMO
During vertebrate embryogenesis, axial tendons develop from the paraxial mesoderm and differentiate through specific developmental stages to reach the syndetome stage. While the main roles of signaling pathways in the earlier stages of the differentiation have been well established, pathway nuances in syndetome specification from the sclerotome stage have yet to be explored. Here, we show stepwise differentiation of human iPSCs to the syndetome stage using chemically defined media and small molecules that were modified based on single cell RNA-sequencing and pathway analysis. We identified a significant population of branching off-target cells differentiating towards a neural phenotype overexpressing Wnt. Further transcriptomics post-addition of a WNT inhibitor at the somite stage and onwards revealed not only total removal of the neural off-target cells, but also increased syndetome induction efficiency. Fine-tuning tendon differentiation in vitro is essential to address the current challenges in developing a successful cell-based tendon therapy.
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CONTEXT: In an open-label, randomized, controlled, phase 3 trial in 61 children aged 1 to 12 years with X-linked hypophosphatemia (XLH), burosumab improved rickets vs continuing conventional therapy with active vitamin D and phosphate. OBJECTIVE: We conducted an analysis to determine whether skeletal responses differed when switching to burosumab vs continuing higher or lower doses of conventional therapy. METHODS: Conventional therapy dose groups were defined as higher-dose phosphate [greater than 40 mg/kg] (HPi), lower-dose phosphate [40 mg/kg or less] (LPi), higher-dose alfacalcidol [greater than 60 ng/kg] or calcitriol [greater than 30 ng/kg] (HD), and lower-dose alfacalcidol [60 ng/kg or less] or calcitriol [30 ng/kg or less] (LD). RESULTS: At week 64, the Radiographic Global Impression of Change (RGI-C) for rickets was higher (better) in children randomly assigned to burosumab vs conventional therapy for all prebaseline dose groups: HPi (+1.72 vs +0.67), LPi (+2.14 vs +1.08), HD (+1.90 vs +0.94), LD (+2.11 vs +1.06). At week 64, the RGI-C for rickets was also higher in children randomly assigned to burosumab (+2.06) vs conventional therapy for all on-study dose groups: HPi (+1.03), LPi (+1.05), HD (+1.45), LD (+0.72). Serum alkaline phosphatase (ALP) also decreased in the burosumab-treated patients more than in the conventional therapy group, regardless of on-study phosphate and active vitamin D doses. CONCLUSION: Prior phosphate or active vitamin D doses did not influence treatment response after switching to burosumab among children with XLH and active radiographic rickets. Switching from conventional therapy to burosumab improved rickets and serum ALP more than continuing either higher or lower doses of phosphate or active vitamin D.
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Raquitismo Hipofosfatêmico Familiar , Hipofosfatemia , Criança , Humanos , Fosfatos , Anticorpos Monoclonais/uso terapêutico , Vitamina D/uso terapêutico , Calcitriol/uso terapêutico , Vitaminas/uso terapêutico , Fatores de Crescimento de FibroblastosRESUMO
Cranial bone loss presents a major clinical challenge and new regenerative approaches to address craniofacial reconstruction are in great demand. Induced pluripotent stem cell (iPSC) differentiation is a powerful tool to generate mesenchymal stromal cells (MSCs). Prior research demonstrated the potential of bone marrow-derived MSCs (BM-MSCs) and iPSC-derived mesenchymal progenitor cells via the neural crest (NCC-MPCs) or mesodermal lineages (iMSCs) to be promising cell source for bone regeneration. Overexpression of human recombinant bone morphogenetic protein (BMP)6 efficiently stimulates bone formation. The study aimed to evaluate the potential of iPSC-derived cells via neural crest or mesoderm overexpressing BMP6 and embedded in 3D printable bio-ink to generate viable bone graft alternatives for cranial reconstruction. Cell viability, osteogenic potential of cells, and bio-ink (Ink-Bone or GelXa) combinations were investigated in vitro using bioluminescent imaging. The osteogenic potential of bio-ink-cell constructs were evaluated in osteogenic media or nucleofected with BMP6 using qRT-PCR and in vitro µCT. For in vivo testing, two 2 mm circular defects were created in the frontal and parietal bones of NOD/SCID mice and treated with Ink-Bone, Ink-Bone + BM-MSC-BMP6, Ink-Bone + iMSC-BMP6, Ink-Bone + iNCC-MPC-BMP6, or left untreated. For follow-up, µCT was performed at weeks 0, 4, and 8 weeks. At the time of sacrifice (week 8), histological and immunofluorescent analyses were performed. Both bio-inks supported cell survival and promoted osteogenic differentiation of iNCC-MPCs and BM-MSCs in vitro. At 4 weeks, cell viability of both BM-MSCs and iNCC-MPCs were increased in Ink-Bone compared to GelXA. The combination of Ink-Bone with iNCC-MPC-BMP6 resulted in an increased bone volume in the frontal bone compared to the other groups at 4 weeks post-surgery. At 8 weeks, both iNCC-MPC-BMP6 and iMSC-MSC-BMP6 resulted in an increased bone volume and partial bone bridging between the implant and host bone compared to the other groups. The results of this study show the potential of NCC-MPC-incorporated bio-ink to regenerate frontal cranial defects. Therefore, this bio-ink-cell combination should be further investigated for its therapeutic potential in large animal models with larger cranial defects, allowing for 3D printing of the cell-incorporated material.
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Células-Tronco Pluripotentes Induzidas , Humanos , Camundongos , Animais , Osteogênese , Tinta , Crista Neural , Camundongos Endogâmicos NOD , Camundongos SCID , Diferenciação CelularRESUMO
PURPOSE: In X-linked hypophosphatemia (XLH), excess fibroblast growth factor-23 causes hypophosphatemia and low calcitriol, leading to musculoskeletal disease with clinical consequences. XLH treatment options include conventional oral phosphate with active vitamin D, or monotherapy with burosumab, a monoclonal antibody approved to treat children and adults with XLH. We have previously reported outcomes up to 64 weeks, and here we report safety and efficacy follow-up results up to 160 weeks from an open-label, multicenter, randomized, dose-finding trial of burosumab for 5- to 12-year-old children with XLH. METHODS: After 1 week of conventional therapy washout, patients were randomized 1:1 to burosumab every 2 weeks (Q2W) or every 4 weeks (Q4W) for 64 weeks, with dosing titrated based on fasting serum phosphorus levels between baseline and week 16. From week 66 to week 160, all patients received Q2W burosumab. RESULTS: Twenty-six children were randomized initially into each Q2W and Q4W group and all completed treatment to week 160. In 41 children with open distal femoral and proximal tibial growth plates (from both treatment groups), total Rickets Severity Score significantly decreased by 0.9â ±â 0.1 (least squares meanâ ±â SE; Pâ <â 0.0001) from baseline to week 160. Fasting serum phosphorus increases were sustained by burosumab therapy throughout the study, with an overall population mean (SD) of 3.35 (0.39) mg/dL, within the pediatric normal range (3.2-6.1 mg/dL) at week 160 (mean change from baseline Pâ <â 0.0001). Most adverse events were mild to moderate in severity. MAIN CONCLUSIONS: In children with XLH, burosumab administration for 160 weeks improved phosphate homeostasis and rickets and was well-tolerated. Long-term safety was consistent with the reported safety profile of burosumab. CLINICALTRIALS.GOV: NCT02163577.
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Anticorpos Monoclonais Humanizados/administração & dosagem , Raquitismo Hipofosfatêmico Familiar/tratamento farmacológico , Fator de Crescimento de Fibroblastos 23/antagonistas & inibidores , Anticorpos Monoclonais Humanizados/efeitos adversos , Criança , Pré-Escolar , Relação Dose-Resposta a Droga , Raquitismo Hipofosfatêmico Familiar/sangue , Raquitismo Hipofosfatêmico Familiar/diagnóstico , Feminino , Fator de Crescimento de Fibroblastos 23/metabolismo , Humanos , Masculino , Fosfatos/sangue , Fosfatos/metabolismo , Reabsorção Renal/efeitos dos fármacos , Índice de Gravidade de DoençaRESUMO
CONTEXT: Younger age at treatment onset with conventional therapy (phosphate salts and active vitamin D; Pi/D) is associated with improved growth and skeletal outcomes in children with X-linked hypophosphatemia (XLH). The effect of age on burosumab efficacy and safety in XLH is unknown. OBJECTIVE: This work aimed to explore the efficacy and safety of burosumab vs Pi/D in younger (<â 5 years) and older (5-12 years) children with XLH. METHODS: This post hoc analysis of a 64-week, open-label, randomized controlled study took place at 16 academic centers. Sixty-one children aged 1 to 12 years with XLH (younger, nâ =â 26; older, nâ =â 35) participated. Children received burosumab starting at 0.8 mg/kg every 2 weeks (younger, nâ =â 14; older, nâ =â 15) or continued Pi/D individually titrated per recommended guidelines (younger, nâ =â 12; older, nâ =â 20). The main outcome measure included the least squares means difference (LSMD) in Radiographic Global Impression of Change (RGI-C) rickets total score from baseline to week 64. RESULTS: The LSMD in outcomes through 64 weeks on burosumab vs conventional therapy by age group were as follows: RGI-C rickets total score (younger, +0.90; older, +1.07), total Rickets Severity Score (younger, -0.86; older, -1.44), RGI-C lower limb deformity score (younger, +1.02; older, +0.91), recumbent length or standing height Z-score (younger, +0.20; older, +0.09), and serum alkaline phosphatase (ALP) (younger, -31.15% of upper normal limit [ULN]; older, -52.11% of ULN). On burosumab, dental abscesses were not reported in younger children but were in 53% of older children. CONCLUSION: Burosumab appears to improve outcomes both in younger and older children with XLH, including rickets, lower limb deformities, growth, and ALP, compared with Pi/D.
Assuntos
Raquitismo Hipofosfatêmico Familiar , Hipofosfatemia , Adolescente , Anticorpos Monoclonais , Anticorpos Monoclonais Humanizados/uso terapêutico , Criança , Raquitismo Hipofosfatêmico Familiar/tratamento farmacológico , Fatores de Crescimento de Fibroblastos , HumanosRESUMO
Lettuce infectious yellows virus is the first crinivirus for which the retention of purified virions ingested into the whitefly (Bemisia tabaci New World (NW)) vector's foregut, has been demonstrated to be a requisite for successful virus transmission. This key finding supports the hypothesis that the determinant of foregut retention and transmission is present on the virion itself. However, whether this is also true for other criniviruses has not been established. Here, we provide evidence that lettuce chlorosis virus (LCV) acquired from plants is retained in the foreguts of both the B. tabaci NW and Middle East-Asia Minor 1 (MEAM1) vector species and transmitted upon inoculation feeding. An association between foregut retention and transmission by NW vectors is also observed following the acquisition and inoculation feeding of LCV virions purified using a standard procedure involving 2% or 4% (v/v) Triton™ X-100 (TX-100). However, while virions purified with 2% or 4% TX-100 are also retained in the foreguts of MEAM1 vectors, transmission is observed with the 4% TX-100-purified virions or when more vectors are used for acquisition and inoculation feeding. These results suggest that an intrinsic difference exists between NW and MEAM1 vectors in their interactions with, and transmission of, LCV virions.
Assuntos
Crinivirus/fisiologia , Sistema Digestório/virologia , Hemípteros/fisiologia , Hemípteros/virologia , Insetos Vetores/fisiologia , Insetos Vetores/virologia , Animais , Sistema Digestório/anatomia & histologia , Doenças das Plantas/virologia , Vírion/fisiologiaRESUMO
INTRODUCTION: Lubiprostone capsules are approved for managing three different chronic constipation conditions. A "sprinkle" formulation may facilitate use in individuals with difficulty swallowing capsules. Our objective was to evaluate the bioequivalence, pharmacokinetics (PK), and bioavailability of lubiprostone sprinkles vs lubiprostone capsules, compared with placebo. METHODS: A 1-week randomized, placebo-controlled, double-blinded, bioequivalence study (study 302) and a single-dose PK and bioavailability crossover study (study 304) were conducted. In study 302, 522 subjects with chronic constipation were randomized to lubiprostone sprinkle 24 µg twice daily (BID), lubiprostone capsule 24 µg BID, or placebo. The primary efficacy endpoint was observed spontaneous bowel movement (SBM) counts (equivalence defined as showing the 90% confidence interval [CI] of the "between-group SBM ratio" to be contained within 0.8-1.25). Study 304 included two cohorts of healthy volunteers randomized to a single 48-µg lubiprostone dose, sprinkle, or capsule (n = 35) or to a single 48-µg sprinkle dose, in fed or fasted state (n = 14). RESULTS: Both lubiprostone formulations significantly improved SBM count (sprinkle, 4.82 ± 3.66, P = 0.002; capsule, 5.74 ± 3.79, P < 0.0001) vs placebo (3.68 ± 2.16), but equivalent efficacy was not demonstrated, with a 90% CI for the SBM count ratio of 0.69-0.95. Quantifiable PK data on lubiprostone were limited; however, overall exposure to the M3 metabolite was approximately 44% higher with sprinkles vs capsules under fasted conditions (geometric mean ratio 1.441 [90% CI, 1.166, 1.782]), and exposure with the sprinkle formulation was 11% lower in the fed state vs the fasted state (geometric mean ratio 0.888 [90% CI, 0.675, 1.168]). Both formulations were generally well tolerated. CONCLUSION: Despite the significant improvement in SBM counts vs placebo, the sprinkle formulation did not demonstrate bioequivalence to the capsule formulation in either pharmacodynamic or PK key parameters. TRIAL REGISTRATION: Study 302: ClinicalTrials.gov identifier, NCT03097861; https://www.clinicaltrials.gov/ct2/show/NCT03097861 ; Study 304: ClinicalTrials.gov identifier, NCT03010631; https://www.clinicaltrials.gov/ct2/show/NCT03010631 .
Assuntos
Alprostadil , Constipação Intestinal , Constipação Intestinal/tratamento farmacológico , Estudos Cross-Over , Método Duplo-Cego , Humanos , LubiprostonaRESUMO
Complementary (c)DNA clones corresponding to the full-length genome of T36CA (a Californian isolate of Citrus tristeza virus with the T36 genotype), which shares 99.1% identity with that of T36FL (a T36 isolate from Florida), were made into a vector system to express the green fluorescent protein (GFP). Agroinfiltration of two prototype T36CA-based vectors (pT36CA) to Nicotiana benthamiana plants resulted in local but not systemic GFP expression/viral infection. This contrasted with agroinfiltration of the T36FL-based vector (pT36FL), which resulted in both local and systemic GFP expression/viral infection. A prototype T36CA systemically infected RNA silencing-defective N. benthamiana lines, demonstrating that a genetic basis for its defective systemic infection was RNA silencing. We evaluated the in planta bioactivity of chimeric pT36CA-pT36FL constructs and the results suggested that nucleotide variants in several open reading frames of the prototype T36CA could be responsible for its defective systemic infection. A single amino acid substitution in each of two silencing suppressors, p20 (S107G) and p25 (G36D), of prototype T36CA facilitated its systemic infectivity in N. benthamiana (albeit with reduced titre relative to that of T36FL) but not in Citrus macrophylla plants. Enhanced virus accumulation and, remarkably, robust systemic infection of T36CA in N. benthamiana and C. macrophylla plants, respectively, required two additional amino acid substitutions engineered in p65 (N118S and S158L), a putative closterovirus movement protein. The availability of pT36CA provides a unique opportunity for comparative analysis to identify viral coding and noncoding nucleotides or sequences involved in functions that are vital for in planta infection.
Assuntos
Closterovirus/genética , Nicotiana/virologia , Doenças das Plantas/virologia , Proteínas Virais/metabolismo , Closterovirus/fisiologia , Interações Hospedeiro-Patógeno , Interferência de RNA , Nicotiana/genética , Proteínas Virais/genéticaRESUMO
PURPOSE OF REVIEW: Childhood vaccination recommendations in the United States have increased throughout the years. Many providers, patients, and families are overwhelmed and have concerns regarding the safety and efficacy of vaccines. Various barriers and challenges exist for healthcare providers to successfully implement the vaccination recommendations. This review will discuss the 2009 and newly released 2010 immunization recommendations, as well as challenges and strategies to improve vaccination in children and adolescents. RECENT FINDINGS: Seasonal influenza immunization continues to be promoted for all children, and recommendations for vaccination against novel influenza A have emerged as well. Concerns surrounding vaccine safety and necessity may cause increasing rates of vaccine refusal among some parents, but clear messages from providers and unbiased information about benefits and risks of immunization may counteract these doubts. Barriers to immunizing adolescents continue as access to healthcare in this age group changes. SUMMARY: Pediatric providers currently face numerous challenges in improving rates of immunization among children and adolescents. Promoting coverage through the influenza vaccines, counseling parents with clear information about the risks and benefits of vaccines, and taking advantage of nonpreventive visits for immunization are some strategies suggested to address these challenges.