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Recurrent Clostridioides difficile infection (CDI) results in significant morbidity and mortality. We previously established that CDI in mice does not protect against reinfection and is associated with poor pathogen-specific B cell memory (Bmem), recapitulating our observations with human Bmem. Here, we demonstrate that the secreted toxin TcdB2 is responsible for subversion of Bmem responses. TcdB2 from an endemic C. difficile strain delayed immunoglobulin G (IgG) class switch following vaccination, attenuated IgG recall to a vaccine booster, and prevented germinal center formation. The mechanism of TcdB2 action included increased B cell CXCR4 expression and responsiveness to its ligand CXCL12, accounting for altered cell migration and a failure of germinal center-dependent Bmem. These results were reproduced in a C. difficile infection model, and a US Food and Drug Administration (FDA)-approved CXCR4-blocking drug rescued germinal center formation. We therefore provide mechanistic insights into C. difficile-associated pathogenesis and illuminate a target for clinical intervention to limit recurrent disease., Competing Interests: Declaration of interests K.M.N., M.L.L., and J.D.B. are listed as inventors on provisional US patent OKLA.P0023US.P1 filed on 09/13/23, which is related to the work described here., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: The aim of this study was to implement a standardized, hospital-based bedside handoff report (IPASS [Illness severity, Patient summary, Action items, Situational awareness, and Synthesis]) in prehospital pediatric critical care transport to increase patient safety by eliminating the risk of misreporting or omitting critical patient care information received before arrival at a tertiary care center. The setting for this project was a level 1 pediatric trauma center in the Southern United States., Methods: Pre- and postsurveys were used to assess staff perception of clinical handoff comprehensiveness and satisfaction with the use of a standardized IPASS handoff tool., Results: Improvement was identified in 6 of 8 survey items. Team members were better able to hear all the information provided in the handoff, the format was functional, and physical transfers of patients from the transport team to the ED went more smoothly. Overall, satisfaction of the handoff process increased by 80%., Conclusion: Many factors contribute to patient safety events and errors in health care, with communication failures contributing to the majority. Overall, findings support the use of standardized IPASS handoffs in pediatric critical care transport to promote patient safety, increase comprehension of patient information, and increase staff satisfaction., (Published by Elsevier Inc.)

Fragile X syndrome is a learning disability caused by excess of CGG repeats in the 5' untranslated region of the Fragile X gene (FMR1) silencing its transcription and translation. We used a murine model of this condition, Fmr1 knock-out mice (KO) to study acetylcholine (ACh) metabolism and compared it to that of wild-type control mice (WT). Brain endogenous ACh (D0ACh), free choline (D0Ch), their deuterated variants D4ACh and D4Ch and mole ratios (AChMR and ChMR) were measured by gas chromatography-mass spectrometry in the cerebral hemisphere, cerebral cortex, hippocampus and cerebellum, following D4Ch administration. Regression analysis indicated a significant decrease with age (negative slope) of D4ACh, AChMR, D4Ch and ChMR in WT mice. Age dependence was only present for D4ACh and AChMR in KO mice. Analysis of variance with age as covariate indicated a significant greater D4Ch in the cerebral cortex of KO females when compared to WT females. Contrasts between sexes within genotypes indicated lower D0Ch in cortex and cerebellum of female KO mice but not in WT and lower D4Ch in hippocampus of female KO and WT mice. In conclusion, after adjusting for age, D0ACh concentrations and synthesis from deuterium-labeled Ch were similar in KO and control WT mice in all brain regions. In contrast, significant changes in Ch dynamics were found in hippocampus and cerebral cortex of KO mice that might contribute to the pathogenesis of FXS., (Published by Elsevier Ltd.)

A dualistic pathway model of ovarian carcinoma (OvCA) pathogenesis has been proposed: type I OvCAs are low grade, genetically stable, and relatively more indolent than type II OvCAs, most of which are high-grade serous carcinomas. Endometrioid OvCA (EOC) is a prototypical type I tumor, often harboring mutations that affect the Wnt and phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin signaling pathways. Molecular and histopathologic analyses indicate type I and II OvCAs share overlapping features, and a subset of EOCs may undergo type I→type II progression accompanied by acquisition of somatic TP53 or PIK3CA mutations. We used a murine model of EOC initiated by conditional inactivation of the Apc and Pten tumor suppressor genes to investigate mutant Trp53 or Pik3ca alleles as key drivers of type I→type II OvCA progression. In the mouse EOC model, the presence of somatic Trp53 or Pik3ca mutations resulted in shortened survival and more widespread metastasis. Activation of mutant Pik3ca alone had no demonstrable effect on the ovarian surface epithelium but resulted in papillary hyperplasia when coupled with Pten inactivation. Our findings indicate that the adverse prognosis associated with TP53 and PIK3CA mutations in human cancers can be functionally replicated in mouse models of type I→type II OvCA progression. Moreover, the models should represent a robust platform for assessment of the contributions of Trp53 or Pik3ca defects in the response of EOCs to conventional and targeted drugs., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Cholinergic mechanisms are known to play a key role in cognitive functions that are profoundly altered in traumatic brain injury (TBI). The present investigation was designed to test the ability of continuous administration, starting at the time of injury, of physostigmine (PHY), an acetylcholinesterase (AChE) inhibitor that crosses the blood-brain barrier (BBB), to ameliorate the alterations of learning and memory induced by cerebral cortex impact injury in rats under isoflurane anesthesia. Learning and memory were assessed with the Morris water maze implemented during days 7-11 (WM1), and days 21-25 post-TBI (WM2), with four trials per day for 3 days, followed by target reversal and 2 additional days of training. These groups of Sprague-Dawley male rats were used: TBI treated with PHY at 3.2 μmol/kg/day (TBI-PHY3.2), or 6.4 μmol/kg/day (TBI-PHY6.4), by subcutaneous osmotic pumps, or TBI and no injury (Sham) treated with saline. AChE activity was measured in brain tissue samples of non-traumatized animals that received PHY at the doses used in the TBI animals. In WM1 tests, PHY3.2 improved learning within sessions, but not between sessions, in the recall of the target position, while PHY6.4 had no significant effects. In WM2 tests, PHY improved within- and between-sessions performance at both dose levels. We found that continuous AChE inhibition interacted with repeated training on the water maze task to completely reverse the deficits seen in learning and memory induced by TBI. The PHY treatment also reduced the amount of brain tissue loss as measured using cresyl violet staining.

Traumatic brain injury (TBI) induces transient or persistent dysfunction of gait and balance. Enhancement of cholinergic transmission has been reported to accelerate recovery of cognitive function after TBI, but the effects of this intervention on locomotor activity remain largely unexplored. The hypothesis that enhancement of cholinergic function by inhibition of acetylcholinesterase (AChE) improves locomotion following TBI was tested in Sprague-Dawley male rats after a unilateral controlled cortical impact (CCI) injury of the motor-sensory cortex. Locomotion was tested by time to fall on the constant speed and accelerating Rotarod, placement errors and time to cross while walking through a horizontal ladder, activity monitoring in the home cages, and rearing behavior. Assessments were performed the 1st and 2nd day and the 1st, 2nd, and 3rd week after TBI. The AChE inhibitor physostigmine hemisulfate (PHY) was administered continuously via osmotic minipumps implanted subcutaneously at the rates of 1.6-12.8 μmol/kg/day. All measures of locomotion were impaired by TBI and recovered to initial levels between 1 and 3 weeks post-TBI, with the exception of the maximum speed achievable on the accelerating Rotarod, as well as rearing in the open field. PHY improved performance in the accelerating Rotarod at 1.6 and 3.2 μmol/kg/day (AChE activity 95 and 78% of control, respectively), however, higher doses induced progressive deterioration. No effect or worsening of outcomes was observed at all PHY doses for home cage activity, rearing, and horizontal ladder walking. Potential benefits of cholinesterase inhibition on locomotor function have to be weighed against the evidence of the narrow range of useful doses.

Background: More than 1.5 million Americans have Parkinson disease (PD), and this figure is expected to rise as the population ages. However, the dental literature offers little information about the illness., Types of Studies Reviewed: The authors conducted a MEDLINE search using the key terms "Parkinson's disease," "medical management" and "dentistry." They selected contemporaneous articles published in peer-reviewed journals and gave preference to articles reporting randomized controlled trials., Results: PD is a progressive neurodegenerative disorder caused by loss of dopaminergic and nondopaminergic neurons in the brain. These deficits result in tremor, slowness of movement, rigidity, postural instability and autonomic and behavioral dysfunction. Treatment consists of administering medications that replace dopamine, stimulate dopamine receptors and modulate other neurotransmitter systems., Clinical Implications: Oral health may decline because of tremors, muscle rigidity and cognitive deficits. The dentist should consult with the patient's physician to establish the patient's competence to provide informed consent and to determine the presence of comorbid illnesses. Scheduling short morning appointments that begin 90 minutes after administration of PD medication enhances the patient's ability to cooperate with care. Inclination of the dental chair at 45 degrees, placement of a bite prop, use of a rubber dam and high-volume oral evacuation enhance airway protection. To avoid adverse drug interactions with levodopa and entacapone, the dentist should limit administration of local anesthetic agents to three cartridges of 2 percent lidocaine with 1:100,000 epinephrine per half hour, and patients receiving selegiline should not be given agents containing epinephrine or levonordefrin. The dentist should instruct the patient and the caregiver in good oral hygiene techniques.

Radiation therapy (RT) is a component of the treatment of patients with head and neck malignancies. This therapy may damage the nearby carotid arteries, thereby initiating or accelerating the atherosclerotic process (atheroma formation). Dentists treating patients who have been irradiated should examine the patient's panoramic radiograph for evidence of atheroma-like calcifications, which appear 1.5 to 2.5 cm posterior and inferior to the angle of the mandible. Patients with evidence of such lesions should be referred to their primary care physician with the suggestion that an ultrasound examination of the carotid arteries is indicated.

Background: The authors review the clinical features, epidemiology, pathophysiology, medical management, dental findings and dental treatment of patients with Alzheimer's disease (AD)., Studies Reviewed: The authors conducted MEDLINE searches for 2000 through 2005 using the terms "Alzheimer's disease," "geriatric," "epidemiology," "pathophysiology," "treatment" and "dentistry." Reports selected for further review included those published in English in peer-reviewed journals. The authors gave preference to articles reporting randomized, controlled trials., Results: AD is a progressive and fatal neurodegenerative disorder characterized by cognitive dysfunctions, particularly in learning and memory, and the emergence of behavioral abnormalities. Deficiencies in the cells responsible for storage and processing of information underlie the cognitive, functional and behavioral changes seen in patients with the disorder., Clinical Implications: As the elderly population grows, increasing numbers of Americans with AD will require dental treatment. The prevalence of dental disease likely will be extensive, because of diminished salivary flow and patients' inability to perform appropriate oral hygiene techniques. Preventive dental education for the caregiver and use of saliva substitutes and anticaries agents by the patient are indicated.

A rabbit antiserum against chicken gizzard tropomyosin reacted with two doublets of 30,000 and 40,000 mol. wt., respectively. The heat stability of these polypeptides argued in favour of their being tropomyosins. Immunocytochemical staining of cryostat sections with the antibody showed that tropomyosin was present in glial cells (Bergman glia and oligodendrocytes) and in all identified neuronal cell types in the rat cerebellum. Tropomyosin was not localized homogeneously within neurons, being highly enriched in dendrites and cell bodies in comparison with axons. Myelinated axons of the cerebellar white matter showed only faint staining by the antibody. These results give further evidence for the compartmentalization of cytoskeletal proteins within neurons resulting in major differences in axonal and dendritic cytoskeletons.

We have examined the question of whether alpha-tubulin is detyrosylated during axonal transport in retinal ganglion cell axons and axons of spinal motor neurons. The degree of tyrosylation of alpha-tubulin was estimated from immunocytochemistry and immunoblotting with two anti-alpha-tubulin monoclonals, one of which (YL1/2) recognizes only the tyrosylated form of alpha-tubulin. In the case of retinal ganglion cells, the axons were depleted of tyrosylated alpha-tubulin both in the retina and proximal region of the optic nerve. Distal regions of the axons, in the optic tract, gave a pattern of staining consistent with a reduction in the total level of alpha-tubulin at the expense of detyrosylated alpha-tubulin. Axons within the L5 ventral root, the sciatic nerve and tibial nerve were consistently unstained by YL1/2 indicating that these axons were depleted in tyrosylated alpha-tubulin in all 3 segments. The results indicate that alpha-tubulin destined for axonal microtubules is detyrosylated close to or in cell bodies and not progressively during its transport. Therefore the segregation of detyrosylated alpha-tubulin to axonal microtubules may occur at their site of assembly.

The presence of tropomyosins in adrenal chromaffin cells was demonstrated by immunoperoxidase staining of cultured chromaffin cells by an anti-tropomyosin antibody. In immunoblotting the antibody labelled polypeptides of Mr 39 000, 36 500 and 31 000 in 100 000 X g supernatants and heat-stable fractions of adrenal medulla. The adrenal medullary tropomyosins did not comigrate with smooth muscle tropomyosins. The 39-kDa and 31-kDa polypeptides comigrated with polypeptides labelled by anti-tropomyosin in brain supernatants. Chromaffin granule membranes possessed a 39-kDa polypeptide that was labelled by anti-tropomyosin.

Stimulation of adrenal chromaffin cells results in a rise in the concentration of intracellular free calcium which initiates catecholamine secretion by exocytosis. An understanding of the molecular basis of exocytosis will require knowledge of the sites of action of calcium. A role for calmodulin has been implicated in secretion from chromaffin cells, and isolated granule membranes bind both calmodulin and a series of cytosolic proteins in a calcium-dependent fashion. Here, we demonstrate that one of the cytosolic granule-binding proteins with a relative molecular mass (Mr) of 70,000 (70K) is a form of the calmodulin-regulated actin-binding protein caldesmon, first isolated from smooth muscle. Cytoplasmic gels assembled from an adrenal medullary extract in the absence of Ca2+ contained actin and the 70K protein. The association of both of these proteins with the cytoplasmic gel was inhibited by a micromolar concentration of Ca2+. In addition, we have demonstrated that the 70K protein is localized at the periphery of chromaffin cells. These results are consistent with the notion that 70K protein (caldesmon) has a role in regulating the organization of actin filaments of the cell periphery during the secretory process.

We have examined bovine adrenal chromaffin cells in culture for the presence of the microtubule-associated protein 2. Chromaffin cells could be identified in culture on the basis of their staining with antibodies directed against secretory granule proteins. Using immunoperoxidase staining microtubule-associated protein 2 immunoreactivity was demonstrated to be present in chromaffin cells but not fibroblasts in culture. Microtubule-associated protein 2 immunoreactivity was present in the cell body, processes and varicosities of the chromaffin cells. Microtubule-associated protein 2 polypeptides were shown to be present in an adrenal medullary homogenate but not chromaffin granule membranes by immunoblotting. The results indicate that the neuronal cytoskeletal polypeptide microtubule-associated protein 2 is present in adrenal chromaffin cells. The presence of microtubule-associated protein 2 in both neurons and chromaffin cells may be related to their common embryonic origin.

The expression of the microtubule-associated protein MAP1A and the synaptic vesicle protein p65 by rat cerebellar granule cells developing in vivo and in vitro was examined. MAP1A in common with a series of previously described cytoskeletal proteins was present in immature parallel fibres and began to disappear from parallel fibres around P15. From the earliest age (P6) at which the molecular layer could be identified parallel fibres throughout the molecular layer expressed p65 with no evidence of any gradient of expression from young to mature axons. Granule cells in culture did not express p65 until later than 1 day in vitro. By 7 days in vitro numerous p65 containing varicosities were present on granule cell processes though MAP1A was still expressed. The results show that transport of synaptic vesicles and formation of varicosities is an early event in granule cell differentiation and precedes a major reorganisation of the axonal cytoskeleton.

Carbamylcholine-stimulated catecholamine release from adrenal chromaffin cells was completely inhibited by pretreatment of the cells for 10 min with 1 microM calmidazolium. Catecholamine release due to 55 mM K+ and ionophore A23187 was also inhibited by calmidazolium but less effectively than release due to carbamylcholine. Inhibition of release appeared to be due to an effect of calmidazolium on a step distal to Ca2+ entry, since the carbamylcholine-stimulated rise in the concentration of intracellular free calcium, monitored using quin-2, was unaffected by calmidazolium. The possibility was considered that calmidazolium inhibited secretion through an effect on protein kinase C rather than calmodulin. However, the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), had no demonstrable effect on catecholamine release, arguing against a significant role for protein kinase C in secretion from adrenal chromaffin cells. These results give further support to the notion that calmodulin plays a role in the secretory process in chromaffin cells.

The tyrosine kinase substrate p36 (calpactin heavy chain) is a calcium-dependent membrane- and cytoskeletal-binding protein. Using an affinity-purified antiserum raised against the p36 heavy chain from bovine adrenal medulla, we have examined the cellular distribution of p36 in developing and adult cerebellum. From immunoblotting, the level of p36 in cerebellum was found to decline during development. In dissociated cell cultures of P4 cerebellum, all cell types were labeled by anti-p36. In vibratome sections from cerebella of P10 rats, anti-p36 stained Purkinje cell bodies strongly and all other cell types less strongly, with the exception of cells in the external germinal layer, which were unstained. By 18 days postnatally p36 was present at higher levels in Bergmann glia and astrocytes of the white matter. In sections of adult cerebella, anti-p36 staining was restricted to Bergmann glial processes and to the processes of a subclass of astrocytes in the granular layer and the white matter. At no developmental stage was anti-p36 staining detectable in axons or dendrites in vibratome sections. These results suggest that p36 plays a role in some aspect of cellular differentiation common to all cerebellar cell types and may have additional functions in astrocytes of the adult cerebellum.