Ultrasound-assisted resection of oral tongue cancer.

BACKGROUND: In surgical resection of squamous cell carcinoma of the oral tongue (SCCOT), achieving clear margins is important for prognosis. Insufficient histopathological margins are common, particularly deep margins. AIMS/OBJECTIVES: The aim of the present study was to determine whether ultrasound (US)-assisted resection could decrease the proportion of insufficient histopathological deep margins in SCCOT. MATERIAL AND METHODS: 34 patients with SCCOT undergoing US-assisted resection (study group) were compared to 76 whose resections were performed without US (conventional group). Outcome measures were insufficient deep histopathological resection margins and mean difference in deep margins. RESULTS: Insufficient deep resection margins (<5.0 mm) were seen in 8 of 34 (23.5%) in the study group, compared to 31 of 76 (40.8%) in the conventional group, unadjusted RR 0.58 [95% CI 0.30-1.12; p = .11], adjusted RR 0.82 [95% CI 0.35-1.92; p = .64]. Unadjusted mean difference was 1.4 mm (95% CI 0.1-2.7, p = .04), adjusted mean difference 1.1 mm (95% CI -2.7 to 0.5, p = .19). CONCLUSIONS: Intraoperative US can visualize the deep resection margins in T1/T2 SCCOT. US-assisted resection seems to decrease the number of insufficient histopathological deep margins, though the results are not statistically significant. Comparatively good results in the conventional group is one explanation for the lack of significance. CLINICALTRIALS.GOV ID: NCT04059861.

Ultrasound accurately assesses depth of invasion in T1-T2 oral tongue cancer.

Background: Depth of invasion (DOI) is important for the T-classification of squamous cell carcinoma of the oral tongue (SCCOT) and incorporated in the TNM 8 classification of oral cavity cancer. To determine DOI clinical palpation is performed, but the preferred radiological modality remains controversial. The aim of this study was to investigate the assessment of DOI using ultrasound (US-DOI). Methods: The DOI was assessed in 40 patients with T1-T3 SCCOT by ultrasound, palpation, computed tomography and magnetic resonance imaging (MRI). Histopathological DOI (H-DOI) was gold standard. Bland-Altman analysis was used to compare mean difference and 95% limits of agreement (LOA). Results: The mean difference of US-DOI was -0.5 mm (95% LOA -4.9-4.0) compared to H-DOI and the mean difference for MRI was 3.9 mm (95% LOA -2.3-10.2). In the subgroup analysis of cT1-T2 the US-DOI mean difference was 0.1 mm and the 95% LOA limits -2.5-2.7. Conclusions: Ultrasound seems to be the most accurate method to assess DOI in T1-T2 SCCOT. MRI overestimates DOI and cannot assess a substantial proportion of the tumors. Level of Evidence: 2c.

Protocol for RNA fluorescence in situ hybridization in mouse meningeal whole mounts.

The multilayered meninges surrounding the brain and spinal cord harbor distinct immune cell populations with prominent roles in health and diseases. Here we present an optimized protocol for RNA fluorescence in situ hybridization (RNA FISH) in meningeal whole mounts, allowing the visualization of gene expression. We also describe the combination of this protocol with immunohistochemistry for simultaneous visualization of mRNA and proteins. This protocol can be used for assessing spatial gene expression within the meninges.

Cytosolic replication in epithelial cells fuels intestinal expansion and chronic fecal shedding of Salmonella Typhimurium.

Persistent and intermittent fecal shedding, hallmarks of Salmonella infections, are important for fecal-oral transmission. In the intestine, Salmonella enterica serovar Typhimurium (STm) actively invades intestinal epithelial cells (IECs) and survives in the Salmonella-containing vacuole (SCV) and the cell cytosol. Cytosolic STm replicate rapidly, express invasion factors, and induce extrusion of infected epithelial cells into the intestinal lumen. Here, we engineered STm that self-destruct in the cytosol (STmCytoKill), but replicates normally in the SCV, to examine the role of cytosolic STm in infection. Intestinal expansion and fecal shedding of STmCytoKill are impaired in mouse models of infection. We propose a model whereby repeated rounds of invasion, cytosolic replication, and release of invasive STm from extruded IECs fuels the high luminal density required for fecal shedding.

Bacterial protein domains with a novel Ig-like fold target human CEACAM receptors.

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is the major cause of neonatal sepsis in humans. A critical step to infection is adhesion of bacteria to epithelial surfaces. GBS adhesins have been identified to bind extracellular matrix components and cellular receptors. However, several putative adhesins have no host binding partner characterised. We report here that surface-expressed ß protein of GBS binds to human CEACAM1 and CEACAM5 receptors. A crystal structure of the complex showed that an IgSF domain in ß represents a novel Ig-fold subtype called IgI3, in which unique features allow binding to CEACAM1. Bioinformatic assessment revealed that this newly identified IgI3 fold is not exclusively present in GBS but is predicted to be present in adhesins from other clinically important human pathogens. In agreement with this prediction, we found that CEACAM1 binds to an IgI3 domain found in an adhesin from a different streptococcal species. Overall, our results indicate that the IgI3 fold could provide a broadly applied mechanism for bacteria to target CEACAMs.

Foodborne infection of mice with Salmonella Typhimurium.

The bacterial pathogen Salmonella enterica serovar Typhimurium is one of the most common causes of foodborne disease in humans and is also an important model system for bacterial pathogenesis. Oral inoculation of C57Bl/6 mice, which are genetically susceptible to Salmonella, results in systemic infection but the murine intestine is not efficiently colonized unless the intestinal microbiota is disrupted. Pretreatment of C57Bl/6 mice with streptomycin, followed by oral inoculation with Salmonella Typhimurium results in colitis resembling human intestinal Salmonellosis. The predominant method of delivery of bacteria is oral gavage, during which organisms are deposited directly into the stomach via a feeding needle. Although convenient, this method can be stressful for mice, and may lead to unwanted tracheal or systemic introduction of bacteria. Here, we developed a method for oral infection of mice by voluntary consumption of regular mouse chow inoculated with bacteria. Mice readily ate chow fragments containing up to 108 CFU Salmonella, allowing for a wide range of infectious doses. In mice pretreated with streptomycin, infection with inoculated chow resulted in reproducible infections with doses as low as 103 CFU. Mice not treated with streptomycin, as well as resistant Nramp1 reconstituted C57Bl/6J mice, were also readily infected using this method. In summary, voluntary consumption of chow inoculated with Salmonella represents a natural route of infection for foodborne salmonellosis and a viable alternative to oral gavage.

Dissecting Flavivirus Biology in Salivary Gland Cultures from Fed and Unfed Ixodes scapularis (Black-Legged Tick).

The Ixodes scapularis tick transmits a number of pathogens, including tick-borne flaviviruses (TBFVs). In the United States, confirmed human infections with the Powassan virus (POWV) TBFV have a fatality rate of ∼10% and are increasing in incidence. Tick salivary glands (SGs) serve as an organ barrier to TBFV transmission, and little is known regarding the location of TBFV infection in SGs from fed ticks. Previous studies showed I. scapularis vanin (VNN) involved with TBFV infection of I. scapularis ISE6 embryonic cells, suggesting a potential role for this gene. The overall goal of this study was to use SG cultures to compare data on TBFV biology in SGs from fully engorged, replete (fed) ticks and from unfed ticks. TBFV multiplication was higher in SGs from fed ticks than in those from unfed ticks. Virus-like particles were observed only in granular acini of SGs from unfed ticks. The location of TBFV infection of SGs from fed ticks was observed in cells lining lobular ducts and trachea but not observed in acini. Transcript knockdown of VNN decreased POWV multiplication in infected SG cultures from both fed and unfed ticks. This work was the first to identify localization of TBFV multiplication in SG cultures from a fed tick and a tick transcript important for POWV multiplication in the tick SG, an organ critical for TBFV transmission. This research exemplifies the use of SG cultures in deciphering TBFV biology in the tick and as a translational tool for screening and identifying potential tick genes as potential countermeasure targets.IMPORTANCE Tick-borne flaviviruses (TBFVs) are responsible for more than 15,000 human disease cases each year, and Powassan virus lineage 2 (POWV-L2) deer tick virus has been a reemerging threat in North America over the past 20 years. Rapid transmission of TBFVs in particular emphasizes the importance of preventing tick bites, the difficulty in developing countermeasures to prevent transmission, and the importance of understanding TBFV infection in tick salivary glands (SGs). Tick blood feeding is responsible for phenomenal physiological changes and is associated with changes in TBFV multiplication within the tick and in SGs. Using SG cultures from Ixodes scapularis female ticks, the primary aims of this study were to identify cellular localization of virus-like particles in acini of infected SGs from fed and unfed ticks, localization of TBFV infection in infected SGs from fed ticks, and a tick transcript (with associated metabolic function) involved in POWV-L2 infection in SG cultures.

Legg-Calvé-perthes disease: quality of life, physical activity, and behavior pattern.

BACKGROUND: Legg-Calvé-Perthes disease (LCPD) is a disease in children leading to deformation of the femoral head and can be a promoter for early dysfunction of the hip and early osteoarthritis of the hip. The study of health-related quality of life, physical activity, and behavior patterns in patients with LCPD can reveal its consequences later in life and also contribute to a better understanding of the etiology of the disease. PATIENTS AND METHODS: We identified 145 patients with LCPD diagnosed and treated at Uppsala University Hospital between 1978 and 1995. A total of 116 patients answered questionnaires regarding health-related quality of life (EQ-5D-3L), physical activity [International Physical Activity Questionnaire (IPAQ)], and hyperactive/inattentive behavior pattern [ADHD self-reporting symptom checklist (ASRS v1.1)] by interview. Patients were asked to report on fractures or soft-tissue injuries that required medical care. Medical charts were reviewed to determine age at onset of LCPD and treatment received. RESULTS: Patients with LCPD had significantly lower EQ-5D-3L and EQ VAS scores than the Swedish general population in all age groups. A total of 28% of our patient group had ASRS scores indicating they are likely or highly likely to have an ADHD diagnosis. A lower EQ-5D-3L score was significantly correlated with a higher total ASRS v1.1 score (ρ=-0.309**). Over 90% of our patient group was physically active on a moderate or high level, despite 52% reporting either some or severe problems with pain according to the EQ-5D-3L questionnaire. Patients with high ASRS v1.1 scores (>16) had a significantly higher incidence of soft-tissue injuries than those with lower ASRS v1.1 scores. CONCLUSION: The consequence of LCPD in adulthood was expressed in a lower quality of life compared with the Swedish general population. Despite this, the patients in our study reported a higher level of physical activity than the general population. A tendency toward hyperactive behavior pattern and high physical activity level may be present even in childhood and could contribute to the etiology of LCPD. LEVEL OF EVIDENCE: A retrospective study, level II.

Legg-Calvé-Perthes disease and the risk of injuries requiring hospitalization: a register study involving 2579 patients.

BACKGROUND AND PURPOSE: Previous studies have suggested that Legg-Calvé-Perthes disease (LCPD) is associated with repetitive trauma, coagulation problems and anatomical abnormalities of the blood supply to the femoral head. The hypothesis that repetitive trauma can affect the blood supply of the femoral head, leading to LCPD, is supported by an animal model. For evidence of an increased risk of repetitive trauma, we investigated whether patients with LCPD have a higher risk for severe injuries requiring hospitalization. PATIENTS AND METHODS: We identified 2579 patients with LCPD in Sweden during the period 1964-2005. 13,748 individuals without LCPD were randomly selected from the Swedish general population, matched by year of birth, sex and region (control group). Cox proportional hazard regression estimated the risks. RESULTS: Compared to the control group, patients with LCPD had a modestly raised hazard ratio (HR) of 1.2 (95% CI 1.1-1.3) for injury requiring hospitalization. The risks were slightly higher for soft tissue injuries (HR = 1.3, 95% CI:1.1-1.4) than for fractures (HR = 1.1, 95% CI: 1.0-1.3) and more pronounced among females. Compared to the control group, the higher risk for injury only applied to the lower extremities (HR = 1.2, 95% CI: 1.0-1.4) in patients with LCPD. INTERPRETATION: Patients with LCPD are vulnerable to injuries which could be interpreted as a marker of hyperactive behavior. It could also implicate that anatomical changes in the bone formation or blood supply of the femoral head - increasing its sensibility for trauma - contribute to the etiology of LCPD.

Legg-Calve-Perthes disease and risks for cardiovascular diseases and blood diseases.

OBJECTIVE: We hypothesized that patients with Legg-Calvé-Perthes disease (LCPD) might have higher risks of cardiovascular and blood diseases. METHODS: A total of 3141 patients, 2 to 15 years of age, with LCPD diagnosed between 1965 and 2005 were identified with the Swedish Inpatient Register. A total of 15 595 individuals without LCPD were selected randomly from among the Swedish general population, with matching according to year of birth, age, gender, and region of residence. Cox proportional-hazard regression analyses, with adjustment for socioeconomic index, were used to estimate relative risks. The patients also were compared with their same-gender siblings. RESULTS: Patients with LCPD had a hazard ratio (HR) of 1.70 (95% confidence interval [CI]: 1.39-2.09) for cardiovascular diseases, compared with individuals without LCPD. The point estimate was slightly higher among subjects >30 years of age at the follow-up (HR: 2.10 [95% CI: 1.52-2.91]). There were statistically significantly higher risks for blood diseases, including anemias and coagulation defects (HR: 1.41 [95% CI: 1.07-1.86]), which were more pronounced among subjects >30 years of age at the follow-up (HR: 2.70 [95% CI: 1.50-4.84]). Patients also had statistically significantly higher risks of hypertensive disease (HR: 2.97 [95% CI: 1.87-4.72]) and nutritional anemia (HR: 2.92 [95% CI: 1.58-5.40]). Analyses using siblings as the comparison group showed consistent results for cardiovascular diseases. CONCLUSION: The results are consistent with the hypothesis that an insufficient blood supply to the femoral head, attributable to vascular pathologic conditions, is involved in the pathogenesis of LCPD.

Targeted screening of cis-regulatory variation in human haplotypes.

Regulatory cis-acting variants account for a large proportion of gene expression variability in populations. Cis-acting differences can be specifically measured by comparing relative levels of allelic transcripts within a sample. Allelic expression (AE) mapping for cis-regulatory variant discovery has been hindered by the requirements of having informative or heterozygous single nucleotide polymorphisms (SNPs) within genes in order to assign the allelic origin of each transcript. In this study we have developed an approach to systematically screen for heritable cis-variants in common human haplotypes across >1,000 genes. In order to achieve the highest level of information per haplotype studied, we carried out allelic expression measurements by using both intronic and exonic SNPs in primary transcripts. We used a novel RNA pooling strategy in immortalized lymphoblastoid cell lines (LCLs) and primary human osteoblast cell lines (HObs) to allow for high-throughput AE. Screening hits from RNA pools were further validated by performing allelic expression mapping in individual samples. Our results indicate that >10% of expressed genes in human LCLs show genotype-linked AE. In addition, we have validated cis-acting variants in over 20 genes linked with common disease susceptibility in recent genome-wide studies. More generally, our results indicate that RNA pooling coupled with AE read-out by second generation sequencing or by other methods provides a high-throughput tool for cataloging the impact of common noncoding variants in the human genome.

A risk haplotype of STAT4 for systemic lupus erythematosus is over-expressed, correlates with anti-dsDNA and shows additive effects with two risk alleles of IRF5.

Systemic lupus erythematosus (SLE) is the prototype autoimmune disease where genes regulated by type I interferon (IFN) are over-expressed and contribute to the disease pathogenesis. Because signal transducer and activator of transcription 4 (STAT4) plays a key role in the type I IFN receptor signaling, we performed a candidate gene study of a comprehensive set of single nucleotide polymorphism (SNPs) in STAT4 in Swedish patients with SLE. We found that 10 out of 53 analyzed SNPs in STAT4 were associated with SLE, with the strongest signal of association (P = 7.1 x 10(-8)) for two perfectly linked SNPs rs10181656 and rs7582694. The risk alleles of these 10 SNPs form a common risk haplotype for SLE (P = 1.7 x 10(-5)). According to conditional logistic regression analysis the SNP rs10181656 or rs7582694 accounts for all of the observed association signal. By quantitative analysis of the allelic expression of STAT4 we found that the risk allele of STAT4 was over-expressed in primary human cells of mesenchymal origin, but not in B-cells, and that the risk allele of STAT4 was over-expressed (P = 8.4 x 10(-5)) in cells carrying the risk haplotype for SLE compared with cells with a non-risk haplotype. The risk allele of the SNP rs7582694 in STAT4 correlated to production of anti-dsDNA (double-stranded DNA) antibodies and displayed a multiplicatively increased, 1.82-fold risk of SLE with two independent risk alleles of the IRF5 (interferon regulatory factor 5) gene.

Vitamin D receptor 3' haplotypes are unequally expressed in primary human bone cells and associated with increased fracture risk: the MrOS Study in Sweden and Hong Kong.

UNLABELLED: The VDR is a prime candidate gene for osteoporosis. Here, we studied three common VDR haplotypes in relation to bone phenotypes in 5014 participants of the global MrOS Study. We also studied the relative expression of the haplotypes in human bone cells. One haplotype was associated with increased fracture risk and differently expressed in primary human bone cells. INTRODUCTION: Vitamin D plays an essential role in skeletal metabolism by binding to its nuclear steroid receptor, the vitamin D receptor (VDR). The heritability of BMD is well established, and the VDR gene is considered a prime candidate suggested to partially account for genetically controlled BMD variance in the population. MATERIALS AND METHODS: Here, we reconstructed common haplotypes in the VDR 3' untranslated region (UTR) and studied the association to BMD and risk of vertebral fractures in elderly men from Sweden (n = 3014) and Hong Kong (n = 2000), all participants of the global MrOS Study. To assess any functional implications of the VDR polymorphisms, we studied allele-specific expressions of the different VDR 3' UTR haplotypes in the normal chromosomal context of 70 unrelated human trabecular bone samples. This was performed by quantitative genotyping of coding polymorphisms in RNA samples and in corresponding DNA samples isolated from the bone samples. RESULTS: Three major haplotypes were reconstructed and in agreement with the previously well-defined baT, BAt, and bAT haplotypes, herein denoted Hap1, Hap2, and Hap3. The Hap1 haplotype was independently associated with increased risk of vertebral fractures in Swedish men (OR, 1.655; 95% CI, 1.146-2.391; p < 0.01) and with lower lumbar spine BMD in elderly men from Sweden (p < 0.01) and Hong Kong (p < 0.05). The VDR gene was also shown to exhibit a 3' UTR haplotype dependent allelic imbalance, indicating that the VDR Hap1 allele was overexpressed in human trabecular bone samples. CONCLUSIONS: The results indicate that the relatively overexpressed VDR Hap1 haplotype could be considered a risk allele for osteoporosis regardless of ethnicity.

Corticosteroids peroperatively diminishes damage to the C-fibers in microscopic lumbar disc surgery.

STUDY DESIGN: A subgroup analysis of patient outcomes from a double-blind randomized controlled study comparing corticosteroid versus saline in microscopic lumbar disc surgery. OBJECTIVES: To study if the use of corticosteroids combined with surgery alleviates the damage to the nerve fibers in lumbar disc herniation. SUMMARY OF BACKGROUND DATA: The use of quantitative sensory testing can detect damage to the myelinated A-delta fibers (cold sense) and the unmyelinated C-fibers (warmth sense). Corticosteroids combined with surgery in lumbar disc surgery enhance the outcome after surgery in terms of less pain and more rapid rehabilitation. METHODS: Analyzing quantitative sensory testing before surgery and after 2 weeks and 2 years. RESULTS: In the corticosteroid group, we saw a statistically significant normalization for the warmth disturbance comparing with control group, which not was detected concerning the cold disturbance. CONCLUSIONS: The use of corticosteroids combined with surgery seems to protect some of the damage to the C-fibers in lumbar disc herniation.