ABSTRACT
OBJECTIVES: Tick-borne encephalitis virus and louping ill virus are neurotropic flaviviruses transmitted by ticks. Epidemiologically, tick-borne encephalitis is endemic in Europe whereas louping ill's predominant geographical distribution is the UK. Rarely, these flaviviruses affect dogs causing neurological signs. This case series aimed to describe the clinical, clinicopathological, and imaging findings, as well as the outcomes in six dogs with meningoencephalitis and/or meningomyelitis caused by a flavivirus in the UK in 2021. MATERIALS AND METHODS: Observational retrospective case-series study. Clinical data were retrieved from medical records of dogs with positive serological or immunohistochemical results from three different institutions from spring to winter 2021. RESULTS: Six dogs were included in the study. All dogs presented an initial phase of pyrexia and/or lethargy followed by progressive signs of spinal cord and/or intracranial disease. Magnetic resonance imaging showed bilateral and symmetrical lesions affecting the grey matter of the thalamus, pons, medulla oblongata, and thoracic or lumbar intumescences with none or mild parenchymal and meningeal contrast enhancement. Serology for tick-borne encephalitis virus was positive in five dogs with the presence of seroconversion in two dogs. The viral distinction between flaviviruses was not achieved. One dog with negative serology presented positive immunohistochemistry at post-mortem examination. Three dogs survived but presented neurological sequelae. Three dogs were euthanased due to the rapid progression of the clinical signs or static neurological signs. CLINICAL SIGNIFICANCE: These cases raise awareness of the presence of tick-borne encephalitis as an emergent disease or the increased prevalence of louping ill virus affecting dogs in the UK.
Subject(s)
Dog Diseases , Encephalitis Viruses, Tick-Borne , Encephalitis, Tick-Borne , Ticks , Dogs , Animals , Encephalitis, Tick-Borne/diagnosis , Encephalitis, Tick-Borne/epidemiology , Encephalitis, Tick-Borne/veterinary , Retrospective Studies , United Kingdom/epidemiology , Dog Diseases/diagnosisABSTRACT
Seed amplification assays (SAA) are becoming commonly used in synucleinopathies to detect α-synuclein aggregates. Studies in Parkinson's disease (PD) and isolated REM-sleep behavior disorder (iRBD) have shown a considerably lower sensitivity in the olfactory epithelium than in CSF or skin. To get an insight into α-synuclein (α-syn) distribution within the nervous system and reasons for low sensitivity, we compared SAA assessment of nasal brushings and skin biopsies in PD (n = 27) and iRBD patients (n = 18) and unaffected controls (n = 30). α-syn misfolding was overall found less commonly in the olfactory epithelium than in the skin, which could be partially explained by the nasal brushing matrix exerting an inhibitory effect on aggregation. Importantly, the α-syn distribution was not uniform: there was a higher deposition of misfolded α-syn across all sampled tissues in the iRBD cohort compared to PD (supporting the notion of RBD as a marker of a more malignant subtype of synucleinopathy) and in a subgroup of PD patients, misfolded α-syn was detectable only in the olfactory epithelium, suggestive of the recently proposed brain-first PD subtype. Assaying α-syn of diverse origins, such as olfactory (part of the central nervous system) and skin (peripheral nervous system), could increase diagnostic accuracy and allow better stratification of patients.
ABSTRACT
Gene silencing using RNA interference (RNAi) has become a widely used genetic technique to study gene function in many organisms. In insects, this technique is often applied through the delivery of dsRNA. In the adult female Aedes aegypti, a main vector of human-infecting arboviruses, efficiency of gene silencing following dsRNA injection varies greatly according to targeted genes. Difficult knockdowns using dsRNA can thus hamper gene function analysis. Here, by analysing silencing of three different genes in female Ae. aegypti (p400, ago2 and E75), we show that gene silencing can indeed be dsRNA sequence dependent but different efficiencies do not correlate with dsRNA length. Our findings suggest that silencing is likely also gene dependent, probably due to gene-specific tissue expression and/or feedback mechanisms. We demonstrate that use of high doses of dsRNA can improve knockdown efficiency, and injection of a transfection reagent along with dsRNA reduces the variability in efficiency between replicates. Finally, we show that gene silencing cannot be achieved using siRNA injection in Ae. aegypti adult females. Overall, this work should help future gene function analyses using RNAi in adult females Ae. aegypti, leading toward a better understanding of physiological and infectious processes.
Subject(s)
Aedes/genetics , Gene Knockdown Techniques/methods , RNA Interference , RNA, Double-Stranded/genetics , Animals , Base Sequence , FemaleABSTRACT
The GloPID-R (Global Research Collaboration for Infectious Disease Preparedness) chikungunya (CHIKV), o'nyong-nyong (ONNV) and Mayaro virus (MAYV) Working Group has been established to investigate natural history, epidemiology and clinical aspects of infection by these viruses. Here, we present a report dedicated to entomological aspects of CHIKV, ONNV and MAYV. Recent global expansion of chikungunya virus has been possible because CHIKV established a transmission cycle in urban settings using anthropophilic vectors such as Aedes albopictus and Aedes aegypti. MAYV and ONNV have a more limited geographic distribution, being confined to Africa (ONNV) and central-southern America (MAYV). ONNV is probably maintained through an enzootic cycle that has not been characterized yet, with Anopheles species as main vectors and humans as amplification hosts during epidemics. MAYV is transmitted by Haemagogus species in an enzootic cycle using non-human primates as the main amplification and maintenance hosts, and humans becoming sporadically infected when venturing in or nearby forest habitats. Here, we focused on the transmission cycle and natural vectors that sustain circulation of these viruses in their respective locations. The knowledge of the natural ecology of transmission and the capacity of different vectors to transmit these viruses is crucial to understand CHIKV emergence, and to assess the risk that MAYV and ONNV will expand on wide scale using anthropophilic mosquito species not normally considered primary vectors. Finally, the experts identified knowledge gaps and provided adapted recommendations, in order to address future entomological investigations in the right direction.
Subject(s)
Alphavirus Infections/transmission , Chikungunya Fever/transmission , Mosquito Vectors/virology , Aedes/virology , Africa , Animals , Anopheles/virology , Central America , Chikungunya virus/pathogenicity , Humans , O'nyong-nyong Virus/pathogenicity , Primates/virology , Research ReportABSTRACT
The GloPID-R (Global Research Collaboration for Infectious Disease Preparedness) Chikungunya (CHIKV), O'nyong-nyong (ONNV) and Mayaro virus (MAYV) Working Group is investigating the natural history, epidemiology and medical management of infection by these viruses, to identify knowledge gaps and to propose recommendations for direct future investigations and rectification measures. Here, we present the first report dedicated to diagnostic aspects of CHIKV, ONNV and MAYV. Regarding diagnosis of the disease at the acute phase, molecular assays previously described for the three viruses require further evaluation, standardized protocols and the availability of international standards representing the genetic diversity of the viruses. Detection of specific IgM would benefit from further investigations to clarify the extent of cross-reactivity among the three viruses, the sensitivity of the assays, and the possible interfering role of cryoglobulinaemia. Implementation of reference panels and external quality assessments for both molecular and serological assays is necessary. Regarding sero-epidemiological studies, there is no reported high-throughput assay that can distinguish among these different viruses in areas of potential co-circulation. New specific tools and/or improved standardized protocols are needed to enable large-scale epidemiological studies of public health relevance to be performed. Considering the high risk of future CHIKV, MAYV and ONNV outbreaks, the Working Group recommends that a major investigation should be initiated to fill the existing diagnostic gaps.
Subject(s)
Alphavirus Infections/diagnosis , Chikungunya Fever/diagnosis , Communicable Diseases, Emerging/diagnosis , Alphavirus/genetics , Alphavirus/immunology , Alphavirus/isolation & purification , Alphavirus Infections/epidemiology , Animals , Antibodies, Viral , Chikungunya virus/genetics , Chikungunya virus/immunology , Chikungunya virus/isolation & purification , Communicable Diseases, Emerging/epidemiology , Cross Reactions , Cryoglobulinemia/virology , Genes, Viral , Humans , Mosquito Vectors/virology , O'nyong-nyong Virus/genetics , O'nyong-nyong Virus/immunology , O'nyong-nyong Virus/isolation & purification , Pathology, Molecular , Phylogeny , Seroepidemiologic StudiesABSTRACT
BACKGROUND: Traditionally, perforated diverticulitis with purulent peritonitis was treated with resection and colostomy (Hartmann's procedure), with inherent complications and risk of a permanent stoma. The DILALA (DIverticulitis - LAparoscopic LAvage versus resection (Hartmann's procedure) for acute diverticulitis with peritonitis) and other randomized trials found laparoscopic lavage to be a feasible and safe alternative. The medium-term follow-up results of DILALA are reported here. METHODS: Patients were randomized during surgery after being diagnosed with Hinchey grade III perforated diverticulitis at diagnostic laparoscopy. The primary outcome was the proportion of patients with one or more secondary operations from 0 to 24 months after the index procedure in the laparoscopic lavage versus Hartmann's procedure groups. The trial was registered as ISRCTN82208287. RESULTS: Forty-three patients were randomized to laparoscopic lavage and 40 to Hartmann's procedure. Patients in the lavage group had a 45 per cent reduced risk of undergoing one or more operations within 24 months (relative risk 0·55, 95 per cent c.i. 0·36 to 0·84; P = 0·012) and had fewer operations (ratio 0·51, 95 per cent c.i. 0·31 to 0·87; P = 0·024) compared with those in the Hartmann's group. No difference was found in mean number of readmissions (1·37 versus 1·50; P = 0·221) or mortality between patients randomized to laparoscopic lavage or Hartmann's procedure. Three patients in the lavage group and nine in the Hartmann's group had a colostomy at 24 months. CONCLUSION: Laparoscopic lavage is a better option for perforated diverticulitis with purulent peritonitis than open resection and colostomy.
Subject(s)
Colon/surgery , Diverticulitis, Colonic/surgery , Intestinal Perforation/surgery , Laparoscopy/methods , Peritoneal Lavage/methods , Peritonitis/therapy , Adult , Aged , Diverticulitis, Colonic/complications , Feasibility Studies , Female , Follow-Up Studies , Humans , Intestinal Perforation/complications , Length of Stay/trends , Male , Middle Aged , Peritonitis/etiology , Prospective Studies , Rupture, Spontaneous , Time Factors , Treatment OutcomeABSTRACT
Ocean data assimilation brings together observations with known dynamics encapsulated in a circulation model to describe the time-varying ocean circulation. Its applications are manifold, ranging from marine and ecosystem forecasting to climate prediction and studies of the carbon cycle. Here, we address only climate applications, which range from improving our understanding of ocean circulation to estimating initial or boundary conditions and model parameters for ocean and climate forecasts. Because of differences in underlying methodologies, data assimilation products must be used judiciously and selected according to the specific purpose, as not all related inferences would be equally reliable. Further advances are expected from improved models and methods for estimating and representing error information in data assimilation systems. Ultimately, data assimilation into coupled climate system components is needed to support ocean and climate services. However, maintaining the infrastructure and expertise for sustained data assimilation remains challenging.
Subject(s)
Climate Change , Oceanography/trends , Seawater/chemistry , Ecosystem , Models, TheoreticalABSTRACT
OBJECTIVE: The objective of this in vitro study was an initial screening of the cytotoxic potential of widely used smooth enamel surface sealants. MATERIALS AND METHODS: A total of 20 products were allocated to four groups based on their chemical composition: (1) filled resin-based sealants, (2) unfilled resin-based sealants, (3) a resin-modified, glass ionomer-based sealant, and (4) silicone-based sealants. All materials were applied to human enamel slices both in accordance with manufacturers' instructions and in additional experiments applying 50% undercuring and 50% overcuring. An agar overlay assay was then used to test the specimens following ISO 10933. The cytotoxic potential of each material was interpreted based on a reaction index that summarized the decolorization and lysis scores obtained. RESULTS: The cytotoxic potential decreased as follows: unfilled resin-based sealants > filled resin-based sealants > resin-modified, glass ionomer-based sealant > silicone-based sealants. In 75% of the resin-based products, deliberate undercuring was associated with more extensive decolorization zones, leading to higher rates of cytotoxic potential in two of those products. Overcuring, by contrast, was associated with a tendency for smaller decolorization zones in 50% of the resin-based products. CONCLUSION: Surface sealants derived from resin monomers exhibited cytotoxic potential in the agar overlay assay. There is also evidence of a possible association with curing, as undercuring can increase the cytotoxic potential, whereas normal curing (as per manufacturers' instructions) or overcuring may help minimize such effects. More research into the biological implications of these materials is needed, especially with regard to their potential impact on the adjacent gingiva.
Subject(s)
Dental Materials/toxicity , Fibroblasts/drug effects , Fibroblasts/pathology , Pit and Fissure Sealants/toxicity , Animals , Cell Line , Cell Survival/drug effects , Materials Testing/methods , Mice , Pit and Fissure Sealants/chemistry , Toxicity TestsABSTRACT
Ultrasound and photoacoustics can be utilized as complementary imaging techniques to improve clinical diagnoses. Photoacoustics provides optical contrast and functional information while ultrasound provides structural and anatomical information. As of yet, photoacoustic imaging uses large and expensive systems, which limits their clinical application and makes the combination costly and impracticable. In this work we present and evaluate a compact and ergonomically designed handheld probe, connected to a portable ultrasound system for inexpensive, real-time dual-modality ultrasound/photoacoustic imaging. The probe integrates an ultrasound transducer array and a highly efficient diode stack laser emitting 130 ns pulses at 805 nm wavelength and a pulse energy of 0.56 mJ, with a high pulse repetition frequency of up to 10 kHz. The diodes are driven by a customized laser driver, which can be triggered externally with a high temporal stability necessary to synchronize the ultrasound detection and laser pulsing. The emitted beam is collimated with cylindrical micro-lenses and shaped using a diffractive optical element, delivering a homogenized rectangular light intensity distribution. The system performance was tested in vitro and in vivo by imaging a human finger joint.
Subject(s)
Diagnostic Imaging/methods , Lasers, Semiconductor , Light , Optics and Photonics , Photoacoustic Techniques/instrumentation , Spectrum Analysis/instrumentation , Transducers , Equipment Design , HumansABSTRACT
Toxicity tests evaluated chronic and sublethal effects of fog oil (FO) on a freshwater endangered fish. FO is released during military training as an obscurant smoke that can drift into aquatic habitats. Fountain darters, Etheostoma fonticola, of four distinct life stages were exposed under laboratory conditions to three forms of FO. FO was vaporized into smoke and allowed to settle onto water, violently agitated with water, and dosed onto water followed by photo-oxidization by ultraviolet irradiation. Single smoke exposures of spawning adult fish did not affect egg production, egg viability, or adult fish survival in 21-day tests. Multiple daily smoke exposures induced mortality after 5 days for larvae fish. Larvae and juvenile fish were more sensitive than eggs in 96-h lethal concentration (LC50) tests with FOwater mixtures and photo-oxidized FO. Water-soluble FO components photo-modified by ultraviolet radiation were the most toxic, thus indicating the value of examining weathering and aging of chemicals for the best determination of environmental impact.
Subject(s)
Oils/toxicity , Water Pollutants, Chemical/toxicity , Animals , Endangered Species , Military Personnel/education , Perches , Risk Assessment , WeatherSubject(s)
Calculi/diagnostic imaging , Foreign Bodies/diagnostic imaging , Nose/diagnostic imaging , Tomography, X-Ray Computed , Turbinates/diagnostic imaging , Aged , Calculi/surgery , Diagnosis, Differential , Endoscopy , Foreign Bodies/surgery , Humans , Incidental Findings , Male , Nose/surgery , Turbinates/surgeryABSTRACT
The emitters at the edges of high-power laser bars tend to produce less power than emitters that are near the center of the bar. We suggest that shear strain, which owes to strain induced by bonding, creates through a photoelastic effect a weak birefringence that rotates the plane of polarization of the light. A rotation of the plane of polarization reduces the net gain for the lasing modes and hence leads to a lower output power for the emitters at the edges of the bars, where the shear strain is dominant.
Subject(s)
Foreign Bodies/diagnosis , Foreign Bodies/therapy , Pistacia , Respiratory Aspiration/diagnosis , Respiratory Aspiration/therapy , Vocal Cords , Bronchoscopy , Critical Care , Heart Arrest/etiology , Humans , Infant , Intraoperative Complications/etiology , Intraoperative Complications/therapy , Intubation, Intratracheal , Laryngoscopy , Male , Respiratory Sounds/etiologyABSTRACT
Cementoblasts are cells that produce, secrete and direct the production of cementum. Resorption lacunae occur in over 90% of teeth at the compression side of the periodontal ligament and might result in an irreversible loss of the original root length. We isolated and cultivated human primary cementoblasts and investigated their expression pattern concerning markers of the osteoblastogenic differentiation (RUNX2, OCN, ALP, and BSP) and CEMP-1. Compared to osteoblasts, cementoblasts displayed an expression pattern comparable to osteoblasts in an early stage of osteoblastogenic differentiation. Next, the human primary cementoblasts were stimulated with IL-1ß (1 and 10ng/ml) for 24 and 96h and subsequently subjected to compressive forces (30.3g/cm(2)) for 1 and 6h. Our in vitro data demonstrated that BSP and CEMP-1 expression significantly decreased when stimulation was accompanied by compression, while compression or stimulation alone led to increased levels of BSP and decreased levels of CEMP-1. We concluded that human primary cementoblasts subjected to compression and IL-1ß stimulation impeded BSP and CEMP-1 expression, proteins that are associated with cementogenesis.
Subject(s)
Dental Cementum/metabolism , Gene Expression Regulation/physiology , Integrin-Binding Sialoprotein/biosynthesis , Interleukin-1beta/metabolism , Proteins/metabolism , Stress, Physiological/physiology , Adolescent , Adult , Antigens, Differentiation/biosynthesis , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cells, Cultured , Child , Compressive Strength , Dental Cementum/cytology , Female , Gene Expression Regulation/drug effects , Humans , Interleukin-1beta/pharmacology , Male , Stress, Physiological/drug effects , Time FactorsABSTRACT
Members of the ephrin/Eph family have recently been shown to be involved in the regulation of bone homeostasis in a murine model. The activation of the EphB4 receptor on osteoblasts by its ligand ephrin-B2 led to stimulation of osteoblastogenesis and therefore to bone formation. The activation of ephrin-A2-EphA2 signaling on osteoblasts inhibited the activation of osteoblast-specific gene expression, leading to bone resorption. Fibroblasts within the periodontal ligament periodontal ligament may be one of the first responders to orthodontic forces. Periodontal ligament fibroblasts (PDLF) are mechanoresponsive. Members of the ephrin/Eph family might link mechanical forces received by PDLF with the regulation of osteoblastogenesis on osteoblasts of the alveolar bone. To study whether ephrin-A2 is modulated upon compression, we subjected human primary PDLF to static compressive forces (30.3 g/cm(2)). Static compressive forces significantly induced the expression of ephrin-A2, while the expression of ephrin-B2 was significantly down-regulated. Moreover, osteoblasts of the alveolar bone stimulated with ephrin-A2 in vitro significantly suppressed their osteoblastogenic gene expression (RUNX2, ALPL) and decreased signs of osteoblastic differentiation, as demonstrated by a significantly reduced ALP activity. Together, these findings establish a role for this ligand/receptor system linking mechanical forces with the regulation of osteogenesis during orthodontic tooth movement.
Subject(s)
Alveolar Process/metabolism , Bone Remodeling/physiology , Dental Stress Analysis , Ephrin-A2/biosynthesis , Periodontal Ligament/metabolism , Tooth Movement Techniques , Adolescent , Alveolar Process/cytology , Analysis of Variance , Cells, Cultured , Child , Compressive Strength , Core Binding Factor Alpha 1 Subunit/biosynthesis , Ephrin-B2/biosynthesis , Female , Fibroblasts/metabolism , Gene Expression Regulation , Humans , MAP Kinase Signaling System , Osteoblasts/metabolism , Periodontal Ligament/cytology , Receptor, EphA2/biosynthesis , Statistics, Nonparametric , Up-Regulation , Young AdultABSTRACT
There is growing evidence that the glycaemic index (GI) of the diet is important with respect to body weight and metabolic disease risk. However, research is limited by the paucity of GI values for commonly consumed carbohydrate-rich foods in European countries. A new methodology has been developed for consistent assignment of GI values to foods across five European databases used in the Diogenes intervention study. GI values were assigned according to five decreasing levels of confidence (1) Measured values for specific foods; (2) Published values from published sources; (3) Equivalent values where published values for similar foods existed; (4) Estimated values assigned as one of three values representing low/medium/high GI ranges and (5) Nominal values assigned as 70, where no other value could be assigned with sufficient confidence. GI values were assigned to 5105 foods. In food records collected at baseline, the contribution to carbohydrate intake of foods assigned levels 1-2 ranged from 16% to 43% depending on country, and this increased to 53-81% including level 3 foods. The degree of confidence to assigned GI values differed across Europe. This standardized approach of assigning GI values will be made available to other researchers to facilitate further investigation into the effects of dietary GI on health.
Subject(s)
Blood Glucose/metabolism , Dietary Carbohydrates/classification , Food Analysis/methods , Food/classification , Glycemic Index , Blood Glucose/analysis , Dietary Carbohydrates/administration & dosage , Dietary Carbohydrates/metabolism , Europe , Glycemic Index/physiology , HumansABSTRACT
The aim of this study was to describe the development and implementation of a multifaceted, low-fat, weight-loss strategy for a Pan-European randomized controlled dietary intervention study, Diogenes. There were 891 families with at least one overweight/obese parent who underwent screening. Eligible, overweight/obese adults followed an 8-week weight-loss phase with a fixed low-energy diet (800 kcal). On attaining weight loss of > or = 8%, families were randomized to a 6- or 12-month low-fat (25-30%E) diet either based on national dietary guidelines or one of four interventions: low protein (LP)/low glycaemic index (LGI), LP/high GI (HGI), high protein (HP)/LGI and HP/HGI. The impact of each diet in preventing weight (re)gain was tested. A points-based system was used to manipulate dietary protein and carbohydrate. Manipulating carbohydrate composition involved substituting foods with a relatively high or low GI. A questionnaire was designed and completed by study investigators, providing feedback on the dietary intervention methods used to inform future interventions. The points system allowed macronutrient manipulations without compromising dietary flexibility or enforcing energy restrictions. Reported centre/participant differences in the ease of implementing the intervention may reflect dietary diversity and personal preferences for specific weight-management strategies. The points system provides a useful starting point for designing improved experimental paradigms for the manipulation of dietary intake in future trials.
Subject(s)
Diet, Reducing , Glycemic Index , Obesity/diet therapy , Weight Loss , Adolescent , Adult , Aged , Child , Child, Preschool , Cross-Cultural Comparison , Diet, Carbohydrate-Restricted , Diet, Fat-Restricted , Diet, Protein-Restricted , Dietary Carbohydrates/administration & dosage , Dietary Carbohydrates/classification , Dietary Carbohydrates/metabolism , Dietary Fats/administration & dosage , Dietary Proteins/administration & dosage , Europe , Female , Humans , Male , Middle Aged , Young AdultABSTRACT
PRIMARY OBJECTIVE: The present study used fMRI (functional magnetic resonance imaging) to objectively assess cognitive fatigue in persons with traumatic brain injury (TBI). It was hypothesized that while performing a cognitive task, TBI participants would show increased brain activity over time, indicative of increased cerebral 'effort' which might manifest as the subjective feeling of cognitive fatigue. METHODS AND PROCEDURES: Functional MRI was used to track brain activity across time while 11 TBI patients with moderate-severe injury and 11 age-matched healthy controls (HCs) performed a modified Symbol Digit Modalities Task (mSDMT). Cognitive fatigue was operationally defined as a relative increase in cerebral activation across time compared to that seen in HCs. ROIs were derived from the Chauduri and Behan model of cognitive fatigue. MAIN OUTCOMES AND RESULTS: While performing the mSDMT, participants with a TBI showed increased activity, while HCs subsequently showed decreased activity in several regions including the middle frontal gyrus, superior parietal cortex, basal ganglia and anterior cingulate. CONCLUSIONS: Increased brain activity exhibited by participants with a TBI might represent increased cerebral effort which may be manifested as cognitive fatigue. Functional MRI appears to be a potentially useful tool for understanding the neural mechanisms associated with cognitive fatigue in TBI.
Subject(s)
Brain Injuries/physiopathology , Cognition Disorders/diagnosis , Mental Fatigue/physiopathology , Adult , Basal Ganglia/physiopathology , Brain Mapping/methods , Case-Control Studies , Female , Frontal Lobe/physiopathology , Gyrus Cinguli/physiopathology , Humans , Magnetic Resonance Imaging/methods , Male , Middle Aged , Neuropsychological Tests , Thalamus/physiopathology , Young AdultABSTRACT
BACKGROUND AND OBJECTIVE: Collagen type I elevation in cyclosporin A-induced gingival overgrowth supports evidence that gingival fibroblasts play a decisive role in the manifestation of the phenotype. To analyze the role of gingival fibroblasts under more in vivo-like conditions, we evaluated the effect of cyclosporin A on collagen type I gene and protein expression in gingival overgrowth-derived gingival fibroblasts established as cocultures with gingival keratinocytes as well as in matched gingival fibroblast monolayers. MATERIAL AND METHODS: Monolayers and cocultures of primary gingival fibroblasts were treated with cyclosporin A for 6 and 72 h. The expression of collagen type I mRNA was analyzed by quantitative real time polymerase chain reaction, while expression and secretion of collagen type I protein was analyzed by indirect immunofluorescence and western blotting. RESULTS: Compared with controls, significant elevation of collagen type I mRNA was restricted to cocultures after 6 and 72 h of treatment with cyclosporin A. In keratinocytes, collagen type I remained undetectable. In monolayers and cocultures, indirect immunofluorescence showed a slightly higher level of collagen type I protein in gingival fibroblasts in response to stimulation with cyclosporin A. Semiquantitative detection of collagen type I by western blotting demonstrated a nonsignificant increase for cell extracts in monolayers and cocultures. For secreted collagen type I, western blot analysis of the supernatants revealed elevated protein levels in cultures stimulated with cyclosporin A. Compared with the corresponding monolayers, the stimulatory effect of cyclosporin A on protein secretion was significant only in coculture. CONCLUSION: Our results indicate that collagen type I is a target of cyclosporin A and that gingival fibroblasts are decisive for the manifestation of the gingival overgrowth-phenotype. Furthermore, the results suggest that cocultures of gingival overgrowth-derived gingival fibroblasts and gingival keratinocytes permit analysis of cyclosporin A-induced effects under more in vivo-like conditions.
Subject(s)
Collagen Type I/analysis , Cyclosporine/adverse effects , Fibroblasts/pathology , Gingiva/pathology , Gingival Overgrowth/chemically induced , Keratinocytes/pathology , Adult , Blotting, Western , Cell Line, Transformed , Cells, Cultured , Coculture Techniques , Collagen Type I/drug effects , Collagen Type I/genetics , Connective Tissue Cells/drug effects , Connective Tissue Cells/pathology , Female , Fibroblasts/drug effects , Fluorescent Antibody Technique, Indirect , Gingiva/drug effects , Gingival Overgrowth/pathology , Humans , Keratinocytes/drug effects , Male , Middle Aged , Phenotype , Polymerase Chain Reaction , RNA, Messenger/analysis , Time FactorsABSTRACT
The family Bunyaviridae contains over 350 named isolates, classified into five genera: Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus and Tospovirus. The Orthobunyavirus genus contains some 170 isolates that are mainly transmitted by mosquitoes and are responsible for a range of disease syndromes in humans including self-limiting febrile illness, encephalitis and haemorrhagic fever. The viruses have a tripartite, negative-sense RNA genome. Analyses of viruses in four serogroups (Bunyamwera, California, Group C and Simbu) showed that the smallest (S) RNA segment encodes the nucleocapsid protein (N) and a non-structural protein called (NSs). The NSs protein of Bunyamwera virus (BUNV) has been shown to play a role in shut-off of host cell protein synthesis in mammalian cells, but no protein shut-off is observed in BUNVinfected mosquito cells (Aedes albopictus C6/36 cells). Protein shut-off in infected mammalian cells is achieved by global inhibition of RNA polymerase II-mediated transcription and enables the virus to overcome the host innate immune response. As innate defence mechanisms constitute a significant barrier to virus infection of different hosts, NSs would appear to play a key role in determining the zoonotic capacity of orthobunyaviruses.