Bacterial phenotype variants in group B streptococcal toxic shock syndrome

We conducted genetic and functional analyses of isolates from a patient with group B streptococcal (GBS) necrotizing fasciitis and toxic shock syndrome. Tissue cultures simultaneously showed colonies with high hemolysis (HH) and low hemolysis (LH). Conversely, the HH and LH variants exhibited low capsule (LC) and high capsule (HC) expression, respectively. Molecular analysis demonstrated that the 2 GBS variants were of the same clonal origin. Genetic analysis found a 3-bp deletion in the covR gene of the HH/LC variant. Functionally, this isolate was associated with an increased growth rate in vitro and with higher interleukin-8 induction. However, in whole blood, opsonophagocytic and intracellular killing assays, the LH/HC phenotype demonstrated higher resistance to host phagocytic killing. In a murine model, LH/HC resulted in higher levels of bacteremia and increased host mortality rate. These findings demonstrate differences in GBS isolates of the same clonal origin but varying phenotypes.

Tenascin-C and tenascin-W in whisker follicle stem cell niches: possible roles in regulating stem cell proliferation and migration

The whisker follicle has CD34-positive stem cells that migrate from their niche near the bulge along the glassy membrane to the whisker bulb, where they participate in the formation of the whisker shaft. Using immunohistochemistry we found the glycoprotein tenascin-C in the fibrous capsule of mouse whisker follicles, along the glassy membrane and in the trabecular region surrounding keratin-15-negative, CD34-positive stem cells. The related glycoprotein tenascin-W is found in the CD34-positive stem cell niche, in nearby trabeculae, and along the glassy membrane. Tenascin-W is also found in the neural stem cell niche of nearby hair follicles. The formation of stress fibers and focal adhesion complexes in CD34-positive whisker-derived stem cells cultured on fibronectin was inhibited by both tenascin-C and tenascin-W, which is consistent with a role for these glycoproteins in promoting the migration of these cells from the niche to the whisker bulb. Tenascin-C, but not tenascin-W, increased the proliferation of whisker follicle stem cells in vitro. Thus, the CD34-positive whisker follicle stem cell niche contains both tenascin-C and tenascin-W, and these glycoproteins may play a role in directing the migration and proliferation of these stem cells.

Target genes for virulence assessment of Escherichia coli isolates from water, food and the environment

The widespread species Escherichia coli includes a broad variety of different types, ranging from highly pathogenic strains causing worldwide outbreaks of severe disease to avirulent isolates which are part of the normal intestinal flora or which are well characterized and safe laboratory strains. The pathogenicity of a given E. coli strain is mainly determined by specific virulence factors which include adhesins, invasins, toxins and capsule. They are often organized in large genetic blocks either on the chromosome ('pathogenicity islands'), on large plasmids or on phages and can be transmitted horizontally between strains. In this review we summarize the current knowledge of the virulence attributes which determine the pathogenic potential of E. coli strains and the methodology available to assess the virulence of E. coli isolates. We also focus on a recently developed procedure based on a broad-range detection system for E. coli-specific virulence genes that makes it possible to determine the potential pathogenicity and its nature in E. coli strains from various sources. This makes it possible to determine the pathotype of E. coli strains in medical diagnostics, to assess the virulence and health risks of E. coli contaminating water, food and the environment and to study potential reservoirs of virulence genes which might contribute to the emergence of new forms of pathogenic E. coli.

Biomechanical evaluation of the stabilizing function of the atlantoaxial ligaments under shear loading: A canine cadaveric study

OBJECTIVES To evaluate the stabilizing function of atlanto-axial ligaments in dogs. STUDY DESIGN Cadaveric biomechanical study. ANIMALS Beagle dog cadavers (n = 10). METHODS The craniocervical region was collected from 10 Beagle cadavers, and the occipito-atlanto-axial region was prepared and freed from the surrounding muscles. Care was taken to preserve integrity of the atlantoaxial ligaments and atlantoaxial joint capsule. The atlanto-occipital joints were blocked with 2 diverging transarticular 1.8 mm positive threaded K-wires. Specimen extremities were embedded in polymethylmethacrylate (PMMA) and mounted on a simulator testing shear load at the atlantoaxial joint. Range of motion (ROM) and neutral zone (NZ) were determined with all ligaments intact, after cutting the apical ligament, both alar ligaments, the transverse ligaments and finally after cutting the dorsal atlantoaxial ligament. RESULTS ROM increased similarly and stepwise during testing. The most significant increase was observed after transection of the alar ligaments. CONCLUSION The alar ligaments seem to be the most important ligamentous structures for stabilization of the atlantoaxial joint under shear load.

Pleomorphic adenoma of the parotid: formal parotidectomy or limited surgery?

BACKGROUND Optimal surgery for pleomorphic adenoma of the parotid is controversial. In the present review, we discuss the advantages and disadvantages of the various approaches after addressing the surgical pathology of the parotid pleomorphic adenoma capsule and its influence on surgery. DATA SOURCES PubMed literature searches were performed to identify original studies. CONCLUSIONS Almost all pleomorphic adenomas can be effectively treated by formal parotidectomy, but the procedure is not mandatory. Extracapsular dissection is a minimal margin surgery; therefore, in the hands of a novice or occasional parotid surgeon, it may result in higher rates of recurrence. Partial superficial parotidectomy may be a good compromise. The tumor is removed with a greater cuff of healthy parotid tissue than in extracapsular dissection. This may minimize the recurrence rate. On the other hand, the removal of healthy parotid tissue compared with formal parotidectomy is limited, thus minimizing complications such as facial nerve dysfunction and Frey syndrome.

A novel technique, dynamic intraligamentary stabilization creates optimal conditions for primary ACL healing: A preliminary biomechanical study

BACKGROUND Anterior cruciate ligament (ACL) rupture is a common lesion. Current treatment emphasizes arthroscopic ACL reconstruction via a graft, although this approach is associated with potential drawbacks. A new method of dynamic intraligamentary stabilization (DIS) was subjected to biomechanical analysis to determine whether it provides the necessary knee stability for optimal ACL healing. METHODS Six human knees from cadavers were harvested. The patellar tendon, joint capsule and all muscular attachments to the tibia and femur were removed, leaving the collateral and the cruciate ligaments intact. The knees were stabilized and the ACL kinematics analyzed. Anterior-posterior (AP) stability measurements evaluated the knees in the following conditions: (i) intact ACL, (ii) ACL rupture, (iii) ACL rupture with primary stabilization, (iv) primary stabilization after 50 motion cycles, (v) ACL rupture with DIS, and (vi) DIS after 50 motion cycles. RESULTS After primary suture stabilization, average AP laxity was 3.2mm, which increased to an average of 11.26mm after 50 movement cycles. With primary ACL stabilization using DIS, however, average laxity values were consistently lower than those of the intact ligament, increasing from an initial AP laxity of 3.00mm to just 3.2mm after 50 movement cycles. CONCLUSIONS Dynamic intraligamentary stabilization established and maintained close contact between the two ends of the ruptured ACL, thus ensuring optimal conditions for potential healing after primary reconstruction. The present ex vivo findings show that the DIS technique is able to restore AP stability of the knee.

Synchrotron x ray induced axonal transections in the brain of rats assessed by high-field diffusion tensor imaging tractography

Since approximately two thirds of epileptic patients are non-eligible for surgery, local axonal fiber transections might be of particular interest for them. Micrometer to millimeter wide synchrotron-generated X-ray beamlets produced by spatial fractionation of the main beam could generate such fiber disruptions non-invasively. The aim of this work was to optimize irradiation parameters for the induction of fiber transections in the rat brain white matter by exposure to such beamlets. For this purpose, we irradiated cortex and external capsule of normal rats in the antero-posterior direction with a 4 mm×4 mm array of 25 to 1000 µm wide beamlets and entrance doses of 150 Gy to 500 Gy. Axonal fiber responses were assessed with diffusion tensor imaging and fiber tractography; myelin fibers were examined histopathologically. Our study suggests that high radiation doses (500 Gy) are required to interrupt axons and myelin sheaths. However, a radiation dose of 500 Gy delivered by wide minibeams (1000 µm) induced macroscopic brain damage, depicted by a massive loss of matter in fiber tractography maps. With the same radiation dose, the damage induced by thinner microbeams (50 to 100 µm) was limited to their paths. No macroscopic necrosis was observed in the irradiated target while overt transections of myelin were detected histopathologically. Diffusivity values were found to be significantly reduced. A radiation dose ≤ 500 Gy associated with a beamlet size of < 50 µm did not cause visible transections, neither on diffusion maps nor on sections stained for myelin. We conclude that a peak dose of 500 Gy combined with a microbeam width of 100 µm optimally induced axonal transections in the white matter of the brain.

Focal white matter changes in spasmodic dysphonia: a combined diffusion tensor imaging and neuropathological study.

Spasmodic dysphonia is a neurological disorder characterized by involuntary spasms in the laryngeal muscles during speech production. Although the clinical symptoms are well characterized, the pathophysiology of this voice disorder is unknown. We describe here, for the first time to our knowledge, disorder-specific brain abnormalities in these patients as determined by a combined approach of diffusion tensor imaging (DTI) and postmortem histopathology. We used DTI to identify brain changes and to target those brain regions for neuropathological examination. DTI showed right-sided decrease of fractional anisotropy in the genu of the internal capsule and bilateral increase of overall water diffusivity in the white matter along the corticobulbar/corticospinal tract in 20 spasmodic dysphonia patients compared to 20 healthy subjects. In addition, water diffusivity was bilaterally increased in the lentiform nucleus, ventral thalamus and cerebellar white and grey matter in the patients. These brain changes were substantiated with focal histopathological abnormalities presented as a loss of axonal density and myelin content in the right genu of the internal capsule and clusters of mineral depositions, containing calcium, phosphorus and iron, in the parenchyma and vessel walls of the posterior limb of the internal capsule, putamen, globus pallidus and cerebellum in the postmortem brain tissue from one patient compared to three controls. The specificity of these brain abnormalities is confirmed by their localization, limited only to the corticobulbar/corticospinal tract and its main input/output structures. We also found positive correlation between the diffusivity changes and clinical symptoms of spasmodic dysphonia (r = 0.509, P = 0.037). These brain abnormalities may alter the central control of voluntary voice production and, therefore, may underlie the pathophysiology of this disorder.

Effect of indomethacin on bile acid-phospholipid interactions: implication for small intestinal injury induced by nonsteroidal anti-inflammatory drugs.

The injurious effect of nonsteroidal anti-inflammatory drugs (NSAIDs) in the small intestine was not appreciated until the widespread use of capsule endoscopy. Animal studies found that NSAID-induced small intestinal injury depends on the ability of these drugs to be secreted into the bile. Because the individual toxicity of amphiphilic bile acids and NSAIDs directly correlates with their interactions with phospholipid membranes, we propose that the presence of both NSAIDs and bile acids alters their individual physicochemical properties and enhances the disruptive effect on cell membranes and overall cytotoxicity. We utilized in vitro gastric AGS and intestinal IEC-6 cells and found that combinations of bile acid, deoxycholic acid (DC), taurodeoxycholic acid, glycodeoxycholic acid, and the NSAID indomethacin (Indo) significantly increased cell plasma membrane permeability and became more cytotoxic than these agents alone. We confirmed this finding by measuring liposome permeability and intramembrane packing in synthetic model membranes exposed to DC, Indo, or combinations of both agents. By measuring physicochemical parameters, such as fluorescence resonance energy transfer and membrane surface charge, we found that Indo associated with phosphatidylcholine and promoted the molecular aggregation of DC and potential formation of larger and isolated bile acid complexes within either biomembranes or bile acid-lipid mixed micelles, which leads to membrane disruption. In this study, we demonstrated increased cytotoxicity of combinations of bile acid and NSAID and provided a molecular mechanism for the observed toxicity. This mechanism potentially contributes to the NSAID-induced injury in the small bowel.

Progressive multifocal leukoencephalopathy in a patient without apparent immunosuppression.

An 80-year-old man with no history of an immune-compromising disorder was diagnosed with progressive multifocal leukoencephalopathy (PML). He presented with dysphagia and left-sided weakness; magnetic resonance imaging demonstrated marked signal abnormality in the subcortical white matter of the left frontal lobe and in the posterior limb of the right internal capsule. Polymerase chain reaction (PCR) analysis of the cerebrospinal fluid (CSF) was negative for John Cunningham (JC) virus. On brain biopsy, foamy macrophages infiltrating the white matter were identified, staining positive for anti-simian virus 40 antibodies. Postoperatively, PCR for JC viral DNA in the CSF was positive, establishing the diagnosis of PML. Extensive investigation for an occult immunocompromising disorder was negative. The patient's neurologic deficits rapidly increased throughout his hospital stay, and he died 3.5 months after his diagnosis.

Brain fiber tract plasticity in experimental spinal cord injury: diffusion tensor imaging.

Diffusion tensor imaging (DTI) and immunohistochemistry were performed in spinal cord injured rats to understand the basis for activation of multiple regions in the brain observed in functional magnetic resonance imaging (fMRI) studies. The measured fractional anisotropy (FA), a scalar measure of diffusion anisotropy, along the region encompassing corticospinal tracts (CST) indicates significant differences between control and injured groups in the 3 to 4 mm area posterior to bregma that correspond to internal capsule and cerebral peduncle. Additionally, DTI-based tractography in injured animals showed increased number of fibers that extend towards the cortex terminating in the regions that were activated in fMRI. Both the internal capsule and cerebral peduncle demonstrated an increase in GFAP-immunoreactivity compared to control animals. GAP-43 expression also indicates plasticity in the internal capsule. These studies suggest that the previously observed multiple regions of activation in spinal cord injury are, at least in part, due to the formation of new fibers.

Enforced expression of Tbx1 in fetal thymic epithelial cells antagonizes thymus organogenesis

Enforced expression of Tbx1 in fetal thymic epithelial cells antagonizes thymus organogenesis Kim T. Cardenas The thymus and parathyroid glands originate from organ-specific domains of 3rd pharyngeal pouch (PP) endoderm. At embryonic day 11.5 (E11.5), the ventral thymus and dorsal parathyroid domains can be identified by Foxn1 and Gcm2 expression respectively. Neural crest cells, (NCCs) play a role in regulating patterning of 3rd PP endoderm. In addition, pharyngeal endoderm influences fate determination via secretion of Sonic hedgehog (Shh), a morphogen required for Gcm2 expression and generation of the parathyroid domain. Gcm2 is a downstream target of the transcription factor Tbx1, which in turn is positively regulated by Shh. Although initially expressed throughout pharyngeal pouch endoderm, Tbx1 expression is excluded from the thymus-specific domain of the 3rd PP by E10.5, but persists in the parathyroid domain. Based on these observations, we hypothesized that Tbx1 expression is non-permissive for thymus fate specification and that enforced expression of Tbx1 in the fetal thymus would impair thymus development. To test this hypothesis, we generated knock-in mice containing a Cre-inducible allele that allows for tissue-specific Tbx1 expression. Expression of the R26iTbx1 allele in fetal and adult thymus using Foxn1Cre resulted in severe thymus hypoplasia throughout ontogeny that persisted in the adult. Thymic epithelial cell (TEC) development was impaired as determined by immunohistochemical and FACS analysis of various differentiation markers. The relative level of Foxn1 expression in fetal TECs was significantly reduced. TECs in R26iTbx1/+ thymi assumed an almost universal expression of Plet-1, a marker associated with a TEC stem/progenitor cell fate. In addition, embryonic R26iTbx1/+ mice develop a perithymic mesechymal capsule that appears expanded compared to control littermates. Interestingly, thymi from neonatal and adult R26iTbx1/+ but not R26+/+ mice were encased in adipose tissue. This thymic phenotype also correlated with a decrease in thymocyte cellularity and aberrant thymocyte differentiation. The results to date support the conclusion that enforced expression of Tbx1 in TECs antagonizes their differentiation and prevents normal organogenesis via both direct and indirect effects.

Neural correlates of free T3 alteration after catecholamine depletion in subjects with remitted major depressive disorder and in controls.

RATIONALE: Thyroid hormones and their interactions with catecholamines play a potentially important role in alterations of mood and cognition. OBJECTIVES: This study aimed to examine the neurobiological effects of catecholamine depletion on thyroid hormones by measuring endocrine and cerebral metabolic function in unmedicated subjects with remitted major depressive disorder (RMDD) and in healthy controls. METHODS: This was a randomized, placebo-controlled, and double-blind crossover trial that included 15 unmedicated RMDD subjects and 13 healthy control subjects. The participants underwent two 3-day-long sessions at 1-week intervals; each participant was randomly administered oral α-methyl-para-tyrosine in one session (catecholamine depletion) and an identical capsule containing hydrous lactose (sham depletion) in the other session prior to a [(18)F]-fluorodeoxyglucose positron emission tomography scan. RESULTS: Serum concentrations of free T3 (FT3), free T4 (FT4), and TSH were obtained and assessed with respect to their relationship to regional cerebral glucose metabolism. Both serum FT3 (P = 0.002) and FT4 (P = 0.0009) levels were less suppressed after catecholamine depletion compared with placebo treatment in the entire study sample. There was a positive association between both FT3 (P = 0.0005) and FT4 (P = 0.002) and depressive symptoms measured using the Montgomery-Åsberg Depression Rating Scale. The relative elevation in FT3 level was correlated with a decrease in regional glucose metabolism in the right dorsolateral prefrontal cortex (rDLPFC; P < 0.05, corrected). CONCLUSIONS: This study provided evidence of an association between a thyroid-catecholamine interaction and mood regulation in the rDLPFC.

Phenotypic and molecular characterization of hyperpigmented group B Streptococci.

Group B Streptococcus (GBS) causes invasive infections in neonates, older adults and patients with comorbidities. β-hemolysin/cytolysin is an important GBS virulence factor. It is encoded by the cyl operon and confers GBS hemolytic activity. Isolates displaying hyperpigmentation are typically hyperhemolytic. Comparison of clonally identical isolates displaying different levels of pigmentation has shown transcriptional dysregulation due to mutations in components of the control of the virulence S/R (CovS/R) regulatory system. In addition, hyperpigmented isolates show decreased CAMP factor and decreased capsule thickness. In analogy to findings in group A Streptococcus, a pivotal role of CovS/R has been proposed in the host-pathogen interaction of invasive GBS infection. However, corresponding investigations on multiple clinical GBS isolates have not been performed. We prospectively collected hyperpigmented isolates found in a diagnostic laboratory and performed phenotypic, molecular and transcriptional analyses. In the period from 2008 to 2012, we found 10 isolates obtained from 10 patients. The isolates reflected both invasive pathogens and colonizers. In three cases, clonally identical but phenotypically different variants were also found. Hence, the analyses included 13 isolates. No capsular serotype was found to be significantly more frequent. Bacterial pigments were analyzed via spectrophotometry and for their hemolytic activity. Data obtained for typical absorbance spectra peaks correlated significantly with hemolytic activity. Molecular analysis of the cyl operon showed that it was conserved in all isolates. The covR sequence displayed mutations in five isolates; in one isolate, the CovR binding site to cylX was abrogated. Our results on clinical isolates support previous findings on CovR-deficient isogenic mutants, but suggest that - at least in some clinical isolates - for β-hemolysin/cytolysin and CAMP factor production, other molecular pathways may be involved.

A point mutation in cpsE renders Streptococcus pneumoniae nonencapsulated and enhances its growth, adherence and competence

BackgroundThe polysaccharide capsule is a major virulence factor of the important human pathogen Streptococcus pneumoniae. However, S. pneumoniae strains lacking capsule do occur.ResultsHere, we report a nasopharyngeal isolate of Streptococcus pneumoniae composed of a mixture of two phenotypes; one encapsulated (serotype 18C) and the other nonencapsulated, determined by serotyping, electron microscopy and fluorescence isothiocyanate dextran exclusion assay.By whole genome sequencing, we demonstrated that the phenotypes differ by a single nucleotide base pair in capsular gene cpsE (C to G change at gene position 1135) predicted to result in amino acid change from arginine to glycine at position 379, located in the cytoplasmic, enzymatically active, region of this transmembrane protein. This SNP is responsible for loss of capsule production as the phenotype is transferred with the capsule operon. The nonencapsulated variant is superior in growth in vitro and is also 117-fold more adherent to and more invasive into Detroit 562 human epithelial cells than the encapsulated variant.Expression of six competence pathway genes and one competence-associated gene was 11 to 34-fold higher in the nonencapsulated variant than the encapsulated and transformation frequency was 3.7-fold greater.ConclusionsWe identified a new single point mutation in capsule gene cpsE of a clinical S. pneumoniae serotype 18C isolate sufficient to cause loss of capsule expression resulting in the co-existence of the encapsulated and nonencapsulated phenotype. The mutation caused phenotypic changes in growth, adherence to epithelial cells and transformability. Mutation in capsule gene cpsE may be a way for S. pneumoniae to lose its capsule and increase its colonization potential.