A usability study on the inGAIT-VSO: effects of a variable-stiffness ankle-foot orthosis on the walking performance of children with cerebral palsy.

BACKGROUND: Ankle-foot orthoses (AFOs) are commonly used by children with cerebral palsy (CP), but traditional solutions are unable to address the heterogeneity and evolving needs amongst children with CP. One key limitation lies in the inability of current passive devices to customize the torque-angle relationship, which is essential to adapt the support to the specific individual needs. Powered alternatives can provide customized behavior, but often face challenges with reliability, weight, and cost. Overall, clinicians find certain barriers that hinder their prescription. In recent work, the Variable Stiffness Orthosis (VSO) was developed, enabling stiffness customization without the need for motors or sophisticated control. METHODS: This work evaluates a pediatric version of the VSO (inGAIT-VSO) by investigating its impact on the walking performance of children with CP and its potential to be used as a tool for assessing the effect of variable stiffness on pathological gait. Data was collected for three typical developing (TD) children and six pediatric participants with CP over two sessions involving walking/balance tasks and questionnaires. RESULTS: The sensors of the inGAIT-VSO provided useful information to assess the impact of the device. Increasing the stiffness of the inGAIT-VSO significantly reduced participants' dorsiflexion and plantarflexion. Despite reduced range of motion, the peak restoring torque increased with stiffness. Overall the participants' gait pattern was altered by reducing crouch gait, preventing drop-foot and supporting body weight. Participants with CP exhibited significantly lower (p < 0.05) physiological cost when walking with the inGAIT-VSO compared to normal condition (own AFO or shoes only). Generally, the device did not impair walking and balance of the participants compared to normal conditions. According to the questionnaire results, the inGAIT-VSO was easy to use and participants reported positive experiences. CONCLUSION: The inGAIT-VSO stiffnesses significantly affected participants' plantarflexion and dorsiflexion and yielded objective data regarding walking performance in pathological gait (e.g. ankle angle, exerted torque and restored assistive energy). These effects were captured by the sensors integrated in the device without using external equipment. The inGAIT-VSO shows promise for customizing AFO stiffness and aiding clinicians in selecting a personalized stiffness based on objective metrics.

Physeal Migration During Knee-guided Growth With Tension Band Plates: Influence of Implant Position.

OBJECTIVE: Physeal migration during guided growth with tension band plates (TBPs) has been poorly described. The positioning factors associated with this phenomenon and its clinical implications are unknown. Our aim is to determine the influence of implant position on the risk of physeal migration during knee-guided growth with TBP. METHODS: Retrospective study of 491 patients who underwent temporary hemi or epiphysiodesis with TBP around the knee between 2007 and 2019. We identified 29 patients who presented physeal migration during follow-up. Demographic and clinical data were collected, and the following measures were obtained from the immediate postoperative radiographs: epiphyseal screw base-physis distance/epiphyseal screw tip-physis distance, interscrew angle, epiphyseal screw-physis angle(ES-PHa)/metaphyseal screw-physis angle, plate-physis angle, epiphyseal screw-plate angle/metaphyseal screw-plate angle, and epiphyseal screw-physis length ratio. Using follow-up radiographs, the type of physeal migration of the epiphyseal screw (touch, occupy, or traverse) and the status of the physis after implant removal (unaltered, physeal bar, and skeletal maturity) were also recorded. A descriptive analysis of the cases and a case-control comparison of imaging studies were performed. RESULTS: The median patient age at intervention was 12.2 years (interquartile range: 11.3 to 14.1), and 76% were males. A statistically significant difference between cases and controls was obtained for epiphyseal screw base-physis distance (3.7 vs 6.3; P = 0.029), epiphyseal screw tip-physis distance (3.6 vs 7.85; P = 0.002), ES-PHa (-0.1 vs 7.45; P = 0.007), and plate-physis angle (85.45 vs 88.60; P = 0.012). In a categorical analysis, a significant difference was found for the ES-PHa categories ( P = 0.002) and for the ES-PHa/metaphyseal screw-physis angle categorical pair ( P = 0.018). In 16, 17, and 12 cases the physis was touched, occupied, or traversed, respectively, although we found no physeal alterations after plate removal. CONCLUSIONS: In our study, physeal migration of TBP is not an uncommon phenomenon, although no physeal abnormalities were detected. Convergent placement of the epiphyseal screw with the base or tip close to the physis should be avoided as this position is associated with a higher risk of physeal migration. LEVEL OF EVIDENCE: Level III-case-control study.

Outcomes of the "Calcaneo-stop" procedure for treating symptomatic flexible flatfoot in children: A systematic review and meta-analysis of 2394 feet.

BACKGROUND: This study evaluates the efficacy of the calcaneo-stop (C-Stop) procedure's effectiveness in treating symptomatic flexible flatfoot (FFF) in children. METHODS: A systematic review and meta-analysis were conducted using PubMed, Embase, and Cochrane databases to identify studies until 2023 on the outcomes of the C-Stop procedure in children with FFF. The risk of bias was assessed using MINORS criteria. RESULTS: Of 85 studies screened, 20 involving 2394 feet from 1415 patients (mean age 11.2 ± 1.3 years) were included. Post-procedure, significant improvements were noted in pain reduction (93.5%), heel alignment (95.21%), and radiological measures, including reductions in Kite (7.32º), Meary (11.65º), Costa-Bartani angles (17.11º), talar declination (12.63º) and increase in Calcaneal Pitch Angle (5.92º). AOFAS scores increased by 22.32 points on average, with 94.83% reporting high satisfaction. Complication rate was low (7.8%). CONCLUSIONS: The C-Stop procedure is effective for treating FFF in children, offering significant clinical, radiological, and functional improvements with high patient satisfaction and a low complication rate. LEVEL OF EVIDENCE: Level IV, Systematic review of Level-IV studies.

Is reduction necessary in overriding metaphyseal distal radius fractures in children under 11 years: a systematic review and meta-analysis of comparative studies.

OBJECTIVE: To determine the necessity of reduction in the treatment of overriding metaphyseal distal radius fractures (DRF) in children under 11 years. METHODS: In this systematic review and meta-analysis, PubMed, Embase, and Cochrane databases were searched to retrieve studies published from inception to 2023. Two reviewers independently screened for studies with observational or randomized control design comparing two treatments for overriding metaphyseal DRF in patients under 11 years: simple casting without reduction (SC group) versus closed reduction plus casting or pin fixation (CRC/F group); with varying outcomes reported (CRD471761). The risk of bias was assessed using the ROBINS-I tool. RESULTS: Out of 3,024 screened studies, three met the inclusion criteria, 180 children (mean age 7.1 ± 0.9 years) with overriding metaphyseal DRF: SC-group (n = 79) versus CRC/F-group (n = 101). Both treatment groups achieved 100% fracture consolidation without requiring further manipulation. The SC-group showed significantly fewer complications (mean difference [MD] 0.08; 95% CI [0.01, 0.53]; I2 = 22%; P < 0.009) and trends towards better sagittal alignment (MD 5.11; 95% CI [11.92, 1.71]; I2 = 94%; P < 0.14), less reinterventions (MD 0.31; 95% CI [0.01, 8.31]; P < 0.48), and fewer patients with motion limitation at the end of follow-up (MD 0.23; 95% CI [0.03,  1.98]; P < 0.18), although these findings were not statistically significant. CONCLUSIONS: Despite a limited number of studies comparing SC versus CRC/F in overriding DRF in children under 11 years, this study suggests that anatomical reduction is not necessary. Treating these fractures with SC, even when presenting with an overriding position, leads to reduced complications, shows a trend towards fewer reinterventions, improved sagittal alignment, and less limitation in patient motion. LEVEL OF EVIDENCE: Level III, Systematic review of Level-III studies.

Long-term Impact of Planovalgus Foot on Activities of Daily Living in Patients With Down Syndrome.

INTRODUCTION: Planovalgus foot (PVF) is the most common orthopaedic abnormality in children with Down syndrome (DS), and as a result these patients rarely develop an adequate plantar arch in adulthood. The present study aims to evaluate the impact of PVF on activities of daily living and participation in sports among young adults with DS and determine whether this impact is related to the degree of foot deformity based on clinical and imaging studies. METHODS: Observational analytical study examining a database of 649 patients with DS from a pediatric referral center, identifying those individuals over age 20 years at the time of the study with a childhood diagnosis of PVF. Finally, 51 patients (102 feet) were evaluated based on clinical and imaging studies, and function was assessed using the The Foot and Ankle Outcome Score (FAOS) and the Visual Analogue Scale (VAS) pain scale. A correlation analysis was performed to determine the clinical and radiographic variables associated with functional outcomes. Linear regression models were obtained to quantify the impact of these variables on function. RESULTS: Patients had a mean age of 26.14±3.88 years and body mass index of 24.51±4.57. Clinically, 63.65% presented grade 3 or 4 PVF, and most were flexible. Radiographically, midfoot flattening was mild-moderate in 92.16%, 58.82% had medial talo-navicular uncoverage, and 30.39% had an increased hallux valgus (HV) angle. Mean scores for all FAOS subscales were between 65 and 71% and the mean VAS score was 1.45±1.96. An association analysis revealed a tendency toward lower scores on all FAOS subscales and greater pain according to the VAS scale in more severe PVF and in cases of moderate HV with asymmetry between feet. Linear regression models showed that major contributors to functional scores were radiographic evidence of hindfoot valgus, midfoot abduction, and flattening, and HV. CONCLUSIONS: Young adults with DS who are diagnosed with PVF in childhood have acceptable functional scores and low pain. Alteration of radiographic parameters toward flatter, more valgus and abducted feet and greater and asymmetric HV tend to be associated with worse long-term functional scores in activities of daily living and sports participation and increased pain. Therefore, non-operative management of these patients is justified, although individualized treatment is recommended. LEVEL OF EVIDENCE: Level IV, Case series.

Is There a Relationship Between the Functional Level of Juvenile and Adolescent Patients With Down Syndrome and Hip Dysplasia?

BACKGROUND: The prevalence of hip dysplasia among patients with Down syndrome (DS) is higher than in the general population. We hypothesize that a relationship may exist between functional level and hip dysplasia in DS, but this has not been studied to date. The aim of this study is to evaluate whether there is a relationship between functional level and radiographic parameters of hip dysplasia or other measures. METHODS: Retrospective cross-sectional comparative study of 652 patients with DS from a pediatric referral center database. Patients over 8 years of age with an anteroposterior pelvis radiograph and with no exclusion criteria were selected, totaling 132 patients (264 hips; 54.55% females; mean age 12.96 ± 2.87 y). Several radiographic parameters of the acetabulum [Sharp angle (SA), Tönnis angle (TA), Wiberg center-edge angle (W-CEA), extrusion index (EI), and acetabular retroversion signs], the proximal femur [neck shaft angle (NSA)], and joint congruence [Shenton line (SL)] were assessed. Patients were classified into 2 levels based on functional skills. A multivariate association analysis was performed between radiographic parameters and functional level. RESULTS: Sixty-one patients were compatible with a functional level I and 71 with a level II. Forty-six hips were dysplastic and 60 were borderline according to the W-CEA. A statistically significant relationship was found between the categorical distribution of certain radiographic measurements of hip dysplasia (EI, SA, TA, W-CEA, SL, and classification by functional level ( P < 0.0005). A significant receiver operating characteristic curve was obtained for W-CEA with a cutt-off point at 26.4 degrees for level I (area under the curve = 0.763; P < 0.005; sensitivity = 0.800 and specificity = 0.644). There was a fairly high correlation between EI and TA (0.749; P < 0.0005), EI and W-CEA (-0.817; P < 0.0005), and TA and W-CEA (-0.748; P < 0.0005). Numerous hips showed signs of acetabular retroversion, with no significant differences found between functional levels or association with hip dysplasia measures. CONCLUSIONS: The present study reveals a relationship between an increased risk of hip dysplasia and reduced functional levels in DS children older than 8 years. These findings may guide individualized clinical follow-up of hip development in DS children considering their functional level. LEVEL OF EVIDENCE: Level III, retrospective comparative study.

Novel dynamics of human mucociliary differentiation revealed by single-cell RNA sequencing of nasal epithelial cultures.

The upper airway epithelium, which is mainly composed of multiciliated, goblet, club and basal cells, ensures proper mucociliary function and can regenerate in response to assaults. In chronic airway diseases, defective repair leads to tissue remodeling. Delineating key drivers of differentiation dynamics can help understand how normal or pathological regeneration occurs. Using single-cell transcriptomics and lineage inference, we have unraveled trajectories from basal to luminal cells, providing novel markers for specific populations. We report that: (1) a precursor subgroup of multiciliated cells, which we have entitled deuterosomal cells, is defined by specific markers, such as DEUP1, FOXN4, YPEL1, HES6 and CDC20B; (2) goblet cells can be precursors of multiciliated cells, thus explaining the presence of hybrid cells that co-express markers of goblet and multiciliated cells; and (3) a repertoire of molecules involved in the regeneration process, such as keratins or components of the Notch, Wnt or BMP/TGFß pathways, can be identified. Confirmation of our results on fresh human and pig airway samples, and on mouse tracheal cells, extend and confirm our conclusions regarding the molecular and cellular choreography at work during mucociliary epithelial differentiation.

Long-term Outcomes of Talonavicular Arthrodesis for the Treatment of Planovalgus Foot in Children With Cerebral Palsy.

BACKGROUND: Planovalgus foot (PVF) in cerebral palsy (CP) tends toward progression and rigidity in adolescence, especially in patients with greater functional impairment. Deformity at the talonavicular joint justifies the use of talonavicular arthrodesis as a corrective surgical technique. This study aims to assess patient or caregiver functional satisfaction and radiographic outcomes of talonavicular arthrodesis for PVF in CP patients with assisted ambulation in the long-term. METHODS: Retrospective comparative study of level III and level IV pediatric CP patients who underwent talonavicular arthrodesis for PVF between 1999 and 2010 as part of multilevel surgery and with a minimum follow-up of 10 years. Radiologic correction at 10 years was compared with preintervention values, and functional impact at 10 years was measured by the foot function index (FFI); correlation between radiologic measurements and FFI were obtained, and complications were recorded. RESULTS: Forty-nine PVFs in 25 patients with CP (72% level III and 28% level IV) were included; 52% were male. The patients had a median age of 12 years at the time of surgery (range: 11 to 15) and 23 years at the time of the study (21 to 26). Significant (P<0.01) pre-post radiologic improvements were obtained in Meary angle (37.86±10.7/8.37±5.5 degrees), calcaneal pitch (3.20±8.1/13.22±5.6 degrees), lateral talocalcaneus angle (42.18±12.1/25.29±2.9 degrees), Moreau-Costa-Bartani angle (167.04±8/146.12±7.1 degrees), talus-first metatarsal angle (27.92±13.9/9.69±4.4 degrees), anteroposterior talocalcaneal angle (37.61±7.4/22.61±2.4 degrees), and talus coverage angle (37.04±11.11/2.45±2.5 degrees). At 10 years postoperatively, functional outcome measured with the FFI was satisfactory (33.9±15.2%) and the mean maximum pain was 3.04; 56% of cases had mild pain. All patients were able to wear an ankle-foot orthosis and 8 no longer needed the device. A significant correlation was found between the talofirst metatarsal angle and the FFI (P=0.024). There were 8% of cases with screw protrusion and 14% presented pseudarthrosis, most of them asymptomatic. CONCLUSIONS: The adequate functional outcome, as well as the persistence of long-term radiologic correction and acceptable number of complications, enables us to recommend talonavicular arthrodesis as an alternative treatment to consider in level III and level IV CP patients with PVF. LEVEL OF EVIDENCE: Level III-retrospective comparative study.

Heterogeneity in Human Induced Pluripotent Stem Cell-derived Alveolar Epithelial Type II Cells Revealed with ABCA3/SFTPC Reporters.

Alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, are typically identified through the use of the canonical markers, SFTPC and ABCA3. Self-renewing AEC2-like cells have been generated from human induced pluripotent stem cells (iPSCs) through the use of knock-in SFTPC fluorochrome reporters. However, developmentally, SFTPC expression onset begins in the fetal distal lung bud tip and thus is not specific to mature AEC2s. Furthermore, SFTPC reporters appear to identify only those iPSC-derived AEC2s (iAEC2s) expressing the highest SFTPC levels. Here, we generate an ABCA3 knock-in GFP fusion reporter (ABCA3:GFP) that enables the purification of iAEC2s while allowing visualization of lamellar bodies, organelles associated with AEC2 maturation. Using an SFTPCtdTomato and ABCA3:GFP bifluorescent line for in vitro distal lung-directed differentiation, we observe later onset of ABCA3:GFP expression and broader identification of the subsequently emerging iAEC2 population based on ABCA3:GFP expression compared with SFTPCtdTomato expression. Comparing ABCA3:GFP/SFTPCtdTomato double-positive with ABCA3:GFP single-positive (SP) cells by RNA sequencing and functional studies reveals iAEC2 cellular heterogeneity with both populations functionally processing surfactant proteins but the SP cells exhibiting faster growth kinetics, increased clonogenicity, increased expression of progenitor markers, lower levels of SFTPC expression, and lower levels of AEC2 maturation markers. Over time, we observe that each population (double-positive and SP) gives rise to the other and each can serve as the parents of indefinitely self-renewing iAEC2 progeny. Our results indicate that iAEC2s are a heterogeneous population of cells with differing proliferation versus maturation properties, the majority of which can be tracked and purified using the ABCA3:GFP reporter or surrogate cell surface proteins, such as SLC34A2 and CPM.

Airway Administration of Flagellin Regulates the Inflammatory Response to Pseudomonas aeruginosa.

Excessive lung inflammation and airway epithelial damage are hallmarks of human inflammatory lung diseases, such as cystic fibrosis (CF). Enhancement of innate immunity provides protection against pathogens while reducing lung-damaging inflammation. However, the mechanisms underlying innate immunity-mediated protection in the lung remain mysterious, in part because of the lack of appropriate animal models for these human diseases. TLR5 (Toll-like receptor 5) stimulation by its specific ligand, the bacterial protein flagellin, has been proposed to enhance protection against several respiratory infectious diseases, although other cellular events, such as calcium signaling, may also control the intensity of the innate immune response. Here, we investigated the molecular events prompted by stimulation with flagellin and its role in regulating innate immunity in the lung of the pig, which is anatomically and genetically more similar to humans than rodent models. We found that flagellin treatment modulated NF-κB signaling and intracellular calcium homeostasis in airway epithelial cells. Flagellin pretreatment reduced the NF-κB nuclear translocation and the expression of proinflammatory cytokines to a second flagellin stimulus as well as to Pseudomonas aeruginosa infection. Moreover, in vivo administration of flagellin decreased the severity of P. aeruginosa-induced pneumonia. Then we confirmed these beneficial effects of flagellin in a pathological model of CF by using ex vivo precision-cut lung slices from a CF pigz model. These results provide evidence that flagellin treatment contributes to a better regulation of the inflammatory response in inflammatory lung diseases such as CF.

Autoimmune neutropenia associated with influenza virus infection in childhood: a case report.

BACKGROUND: Although neutropenia is relatively frequent in infants and children and is mostly a benign condition with a self-limited course, it can lead to life-threatening severe infections. Autoimmune neutropenia is a relatively uncommon hematological disorder characterized by the autoantibody-induced destruction of neutrophils. It is usually triggered by viral infections with very few documented cases after influenza virus. CASE PRESENTATION: An 8-month-old male infant presented at the emergency room with a 5-days history of fever up to 39.7 °C, cough and runny nose. In the blood test performed, severe neutropenia was diagnosed (neutrophils 109/µL). A nasopharyngeal aspirate revealed a positive rapid test for Influenza A. Serum antineutrophil antibodies were determined with positive results. Neutropenia targeted panel showed no mutations. Despite maintenance of severe neutropenia for 9 months the course was uneventful without treatment. CONCLUSIONS: When severe neutropenia is diagnosed and confirmed, it is essential to rule out some potential etiologies and underlying conditions, since the appropriate subsequent management will depend on it. Although autoimmune neutropenia triggered by viral infections has been widely reported, it has seldom been reported after influenza infection. The benign course of the disease allows a conservative management in most cases.

Pluripotent stem cell differentiation reveals distinct developmental pathways regulating lung- versus thyroid-lineage specification.

The in vitro-directed differentiation of pluripotent stem cells (PSCs) through stimulation of developmental signaling pathways can generate mature somatic cell types for basic laboratory studies or regenerative therapies. However, there has been significant uncertainty regarding a method to separately derive lung versus thyroid epithelial lineages, as these two cell types each originate from Nkx2-1+ foregut progenitors and the minimal pathways claimed to regulate their distinct lineage specification in vivo or in vitro have varied in previous reports. Here, we employ PSCs to identify the key minimal signaling pathways (Wnt+BMP versus BMP+FGF) that regulate distinct lung- versus thyroid-lineage specification, respectively, from foregut endoderm. In contrast to most previous reports, these minimal pathways appear to be evolutionarily conserved between mice and humans, and FGF signaling, although required for thyroid specification, unexpectedly appears to be dispensable for lung specification. Once specified, distinct Nkx2-1+ lung or thyroid progenitor pools can now be independently derived for functional 3D culture maturation, basic developmental studies or future regenerative therapies.

Laquinimod arrests experimental autoimmune encephalomyelitis by activating the aryl hydrocarbon receptor.

Laquinimod is an oral drug currently being evaluated for the treatment of relapsing, remitting, and primary progressive multiple sclerosis and Huntington's disease. Laquinimod exerts beneficial activities on both the peripheral immune system and the CNS with distinctive changes in CNS resident cell populations, especially astrocytes and microglia. Analysis of genome-wide expression data revealed activation of the aryl hydrocarbon receptor (AhR) pathway in laquinimod-treated mice. The AhR pathway modulates the differentiation and function of several cell populations, many of which play an important role in neuroinflammation. We therefore tested the consequences of AhR activation in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) using AhR knockout mice. We demonstrate that the pronounced effect of laquinimod on clinical score, CNS inflammation, and demyelination in EAE was abolished in AhR-/- mice. Furthermore, using bone marrow chimeras we show that deletion of AhR in the immune system fully abrogates, whereas deletion within the CNS partially abrogates the effect of laquinimod in EAE. These data strongly support the idea that AhR is necessary for the efficacy of laquinimod in EAE and that laquinimod may represent a first-in-class drug targeting AhR for the treatment of multiple sclerosis and other neurodegenerative diseases.

In vivo Ebola virus infection leads to a strong innate response in circulating immune cells.

BACKGROUND: Ebola virus is the causative agent of a severe syndrome in humans with a fatality rate that can approach 90 %. During infection, the host immune response is thought to become dysregulated, but the mechanisms through which this happens are not entirely understood. In this study, we analyze RNA sequencing data to determine the host response to Ebola virus infection in circulating immune cells. RESULTS: Approximately half of the 100 genes with the strongest early increases in expression were interferon-stimulated genes, such as ISG15, OAS1, IFIT2, HERC5, MX1 and DHX58. Other highly upregulated genes included cytokines CXCL11, CCL7, IL2RA, IL2R1, IL15RA, and CSF2RB, which have not been previously reported to change during Ebola virus infection. Comparing this response in two different models of exposure (intramuscular and aerosol) revealed a similar signature of infection. The strong innate response in the aerosol model was seen not only in circulating cells, but also in primary and secondary target tissues. Conversely, the innate immune response of vaccinated macaques was almost non-existent. This suggests that the innate response is a major aspect of the cellular response to Ebola virus infection in multiple tissues. CONCLUSIONS: Ebola virus causes a severe infection in humans that is associated with high mortality. The host immune response to virus infection is thought to be an important aspect leading to severe pathology, but the components of this overactive response are not well characterized. Here, we analyzed how circulating immune cells respond to the virus and found that there is a strong innate response dependent on active virus replication. This finding is in stark contrast to in vitro evidence showing a suppression of innate immune signaling, and it suggests that the strong innate response we observe in infected animals may be an important contributor to pathogenesis.

Transcriptional Profiling of the Immune Response to Marburg Virus Infection.

UNLABELLED: Marburg virus is a genetically simple RNA virus that causes a severe hemorrhagic fever in humans and nonhuman primates. The mechanism of pathogenesis of the infection is not well understood, but it is well accepted that pathogenesis is appreciably driven by a hyperactive immune response. To better understand the overall response to Marburg virus challenge, we undertook a transcriptomic analysis of immune cells circulating in the blood following aerosol exposure of rhesus macaques to a lethal dose of Marburg virus. Using two-color microarrays, we analyzed the transcriptomes of peripheral blood mononuclear cells that were collected throughout the course of infection from 1 to 9 days postexposure, representing the full course of the infection. The response followed a 3-stage induction (early infection, 1 to 3 days postexposure; midinfection, 5 days postexposure; late infection, 7 to 9 days postexposure) that was led by a robust innate immune response. The host response to aerosolized Marburg virus was evident at 1 day postexposure. Analysis of cytokine transcripts that were overexpressed during infection indicated that previously unanalyzed cytokines are likely induced in response to exposure to Marburg virus and further suggested that the early immune response is skewed toward a Th2 response that would hamper the development of an effective antiviral immune response early in disease. Late infection events included the upregulation of coagulation-associated factors. These findings demonstrate very early host responses to Marburg virus infection and provide a rich data set for identification of factors expressed throughout the course of infection that can be investigated as markers of infection and targets for therapy. IMPORTANCE: Marburg virus causes a severe infection that is associated with high mortality and hemorrhage. The disease is associated with an immune response that contributes to the lethality of the disease. In this study, we investigated how the immune cells circulating in the blood of infected primates respond following exposure to Marburg virus. Our results show that there are three discernible stages of response to infection that correlate with presymptomatic, early, and late symptomatic stages of infection, a response format similar to that seen following challenge with other hemorrhagic fever viruses. In contrast to the ability of the virus to block innate immune signaling in vitro, the earliest and most sustained response is an interferon-like response. Our analysis also identifies a number of cytokines that are transcriptionally upregulated during late stages of infection and suggest that there is a Th2-skewed response to infection. When correlated with companion data describing the animal model from which our samples were collected, our results suggest that the innate immune response may contribute to overall pathogenesis.

The master regulator of the cellular stress response (HSF1) is critical for orthopoxvirus infection.

The genus Orthopoxviridae contains a diverse group of human pathogens including monkeypox, smallpox and vaccinia. These viruses are presumed to be less dependent on host functions than other DNA viruses because they have large genomes and replicate in the cytoplasm, but a detailed understanding of the host factors required by orthopoxviruses is lacking. To address this topic, we performed an unbiased, genome-wide pooled RNAi screen targeting over 17,000 human genes to identify the host factors that support orthopoxvirus infection. We used secondary and tertiary assays to validate our screen results. One of the strongest hits was heat shock factor 1 (HSF1), the ancient master regulator of the cytoprotective heat-shock response. In investigating the behavior of HSF1 during vaccinia infection, we found that HSF1 was phosphorylated, translocated to the nucleus, and increased transcription of HSF1 target genes. Activation of HSF1 was supportive for virus replication, as RNAi knockdown and HSF1 small molecule inhibition prevented orthopoxvirus infection. Consistent with its role as a transcriptional activator, inhibition of several HSF1 targets also blocked vaccinia virus replication. These data show that orthopoxviruses co-opt host transcriptional responses for their own benefit, thereby effectively extending their functional genome to include genes residing within the host DNA. The dependence on HSF1 and its chaperone network offers multiple opportunities for antiviral drug development.

Lassa and Marburg viruses elicit distinct host transcriptional responses early after infection.

BACKGROUND: Lassa virus and Marburg virus are two causative agents of viral hemorrhagic fever. Their diagnosis is difficult because patients infected with either pathogen present similar nonspecific symptoms early after infection. Current diagnostic tests are based on detecting viral proteins or nucleic acids in the blood, but these cannot be found during the early stages of disease, before the virus starts replicating in the blood. Using the transcriptional response of the host during infection can lead to earlier diagnoses compared to those of traditional methods. RESULTS: In this study, we use RNA sequencing to obtain a high-resolution view of the in vivo transcriptional dynamics of peripheral blood mononuclear cells (PBMCs) throughout both types of infection. We report a subset of host mRNAs, including heat-shock proteins like HSPA1B, immunoglobulins like IGJ, and cell adhesion molecules like SIGLEC1, whose differences in expression are strong enough to distinguish Lassa infection from Marburg infection in non-human primates. We have validated these infection-specific expression differences by using microarrays on a larger set of samples, and by quantifying the expression of individual genes using RT-PCR. CONCLUSIONS: These results suggest that host transcriptional signatures are correlated with specific viral infections, and that they can be used to identify highly pathogenic viruses during the early stages of disease, before standard detection methods become effective.

The battle of the sexes starts in the oviduct: modulation of oviductal transcriptome by X and Y-bearing spermatozoa.

BACKGROUND: Sex allocation of offspring in mammals is usually considered as a matter of chance, being dependent on whether an X- or a Y-chromosome-bearing spermatozoon reaches the oocyte first. Here we investigated the alternative possibility, namely that the oviducts can recognise X- and Y- spermatozoa, and may thus be able to bias the offspring sex ratio. RESULTS: By introducing X- or Y-sperm populations into the two separate oviducts of single female pigs using bilateral laparoscopic insemination we found that the spermatozoa did indeed elicit sex-specific transcriptomic responses. Microarray analysis revealed that 501 were consistently altered (P-value < 0.05) in the oviduct in the presence of Y-chromosome-bearing spermatozoa compared to the presence of X-chromosome-bearing spermatozoa. From these 501 transcripts, 271 transcripts (54.1%) were down-regulated and 230 transcripts (45.9%) were up-regulated when the Y- chromosome-bearing spermatozoa was present in the oviduct. Our data showed that local immune responses specific to each sperm type were elicited within the oviduct. In addition, either type of spermatozoa elicits sex-specific signal transduction signalling by oviductal cells. CONCLUSIONS: Our data suggest that the oviduct functions as a biological sensor that screens the spermatozoon, and then responds by modifying the oviductal environment. We hypothesize that there might exist a gender biasing mechanism controlled by the female.

Improving miRNA-mRNA interaction predictions.

BACKGROUND: MicroRNAs are short RNA molecules that post-transcriptionally regulate gene expression. Today, microRNA target prediction remains challenging since very few have been experimentally validated and sequence-based predictions have large numbers of false positives. Furthermore, due to the different measuring rules used in each database of predicted interactions, the selection of the most reliable ones requires extensive knowledge about each algorithm. RESULTS: Here we propose two methods to measure the confidence of predicted interactions based on experimentally validated information. The output of the methods is a combined database where new scores and statistical confidences are re-assigned to each predicted interaction. The new scores allow the robust combination of several databases without the effect of low-performing algorithms dragging down good-performing ones. The combined databases obtained using both algorithms described in this paper outperform each of the existing predictive algorithms that were considered for the combination. CONCLUSIONS: Our approaches are a useful way to integrate predicted interactions from different databases. They reduce the selection of interactions to a unique database based on an intuitive score and allow comparing databases between them.