Levetiracetam Inhibits both ryanodine and IP3 receptor activated calcium induced calcium release in hippocampal neurons in culture

Epilepsy affects approximately 1% of the population worldwide, and there is a pressing need to develop new anti-epileptic drugs (AEDs) and understand their mechanisms of action. Levetiracetam (LEV) is a novel AED and despite its increasingly widespread clinical use, its mechanism of action is as yet undetermined. Intracellular calcium ([Ca²⁺]_i) regulation by both inositol 1,4,5-triphosphate receptors (IP3R) and ryanodine receptors (RyR) has been implicated in epileptogenesis and the maintenance of epilepsy. To this end, we investigated the effect of LEV on RyR and IP3R activated calcium-induced calcium release (CICR) in hippocampal neuronal cultures. RyR-mediated CICR was stimulated using the well-characterized RyR activator, caffeine. Caffeine (10 mM) caused a significant increase in [Ca²⁺]_i in hippocampal neurons. Treatment with LEV (33 μM) prior to stimulation of RyR-mediated CICR by caffeine led to a 61% decrease in the caffeine induced peak height of [Ca²⁺]_i when compared to the control. Bradykinin stimulates IP3R-activated CICR—to test the effect of LEV on IP3R-mediated CICR, bradykinin (1 μM) was used to stimulate cells pre-treated with LEV (100 μM). The data showed that LEV caused a 74% decrease in IP3R-mediated CICR compared to the control. In previous studies we have shown that altered Ca²⁺ homeostatic mechanisms play a role in seizure activity and the development of spontaneous recurrent epileptiform discharges (SREDs). Elevations in [Ca²⁺]_i mediated by CICR systems have been associated with neurotoxicity, changes in neuronal plasticity, and the development of AE. Thus, the ability of LEV to modulate the two major CICR systems demonstrates an important molecular effect of this agent on a major second messenger system in neurons.     

Molecular analysis of bacterial species associated with childhood caries

Although substantial epidemiologic evidence links Streptococcus mutans to caries, the pathobiology of caries may involve more complex communities of bacterial species. Molecular methods for bacterial identification and enumeration now make it possible to more precisely study the microbiota associated with dental caries. The purpose of this study was to compare the bacteria found in early childhood caries (ECC) to those found in caries-free children by using molecular identification methods. Cloning and sequencing of bacterial 16S ribosomal DNAs from a healthy subject and a subject with ECC were used for identification of novel species or uncultivated phylotypes and species not previously associated with dental caries. Ten novel phylotypes were identified. A number of species or phylotypes that may play a role in health or disease were identified and warrant further investigation. In addition, quantitative measurements for 23 previously known bacterial species or species groups were obtained by a reverse capture checkerboard assay for 30 subjects with caries and 30 healthy controls. Significant differences were observed for nine species: S. sanguinis was associated with health and, in order of decreasing cell numbers, Actinomyces gerencseriae, Bifidobacterium, S. mutans, Veillonella, S. salivarius, S. constellatus, S. parasanguinis, and Lactobacillus fermentum were associated with caries. These data suggest that A. gerencseriae and other Actinomyces species may play an important role in caries initiation and that a novel Bifidobacterium may be a major pathogen in deep caries. Further investigation could lead to the identification of targets for biological interventions in the caries process and thereby contribute to improved prevention of and treatment for this significant public health problem.     

Macrophages but not B cells from aged mice are defective in stimulating autoreactive T cells in vitro

In the present study the effect of aging on the capacity of Ia+ cells to stimulate autoreactive T cells in the syngeneic mixed lymphocyte reaction (SMLR) was investigated. Using young CD4+ T cells as responders, it was observed that unseparated whole spleen cells from aged mice had normal stimulatory activity comparable to that of young spleen cells. Interestingly, however, when purified splenic adherent cells (SAC) enriched for macrophages or splenic B cells were used as stimulators, aged SAC but not aged B cells were found to be defective in stimulating autoreactive T cells. This defect in aged SAC was not due to decreased expression of Ia antigens since the percentage of Ia+ SAC and density of Ia antigen expression was similar in both young and old mice. Also, the B cells from aged mice expressed normal levels of Ia antigens. Aged SAC, when mixed with young SAC could also actively suppress the normal SMLR. However, this suppression was not due to increased prostaglandin production but was found to be associated with interleukin-1 (IL-1) regulation, inasmuch as addition of exogenous IL-1 could completely reconstitute the defective stimulatory activity of aged SAC and also abolished the suppressor activity of the SAC. Aged mice also demonstrated an intrinsic defect in the CD4+ T cells responding in the SMLR. Together, our studies on the SMLR demonstrate an age-related defect in responder autoreactive T cells and in stimulator splenic macrophages but not in the stimulatory activity of B cells.     

Association of IL4 gene polymorphisms with asthma in North Indians

BACKGROUND: Asthma is a complex airway disorder, and a number of genetic loci have been found to be associated with asthma. The 5q31-33 region is one of the most important loci linked to asthma and atopic disorders. However, association studies with candidate genes in this region, such as IL4, were inconclusive, as both positive and negative results were obtained in several populations studied. The aim of our case-control study was to determine the association between IL4 and asthma in North Indians. PATIENTS AND METHODS: Polymorphisms in the promoter and a dinucleotide repeat in the 2nd intron in IL4 were genotyped by sequencing and GeneScan analysis, respectively, in ethnically matched, unrelated patients (n = 171) and controls (n = 128), following the guidelines of the American Thoracic Society. RESULTS: The proximal promoter region of the IL4 gene was found to be invariant. Previously reported polymorphisms, -590 C/T and +33 C/T, were found to be absent in our population. The chi2 test using only large expected cell counts (more than 5% of the sample size) showed a significant association between allele size and disease status (chi2 = 38.08, d.f. = 6, p < 0.05). In addition, a significant difference was observed for the allele and genotype frequencies (p < 0.0005 and p = 0.0009, respectively) in the patient and the control groups using the Fisher-Freeman-Halton test. CONCLUSION: Our studies indicate that the promoter of the IL4 gene is invariant in our population. The case-control studies on the CA repeat polymorphism in the 2nd intron of the IL4 gene have shown interesting results and indicate the need for further family-based studies.     

Relaxation of imprinting of IGFII gene in juvenile nasopharyngeal angiofibromas.

IGFII and H19 genes are expressed only from one allele due to genomic imprinting, biallelic expression (loss of imprinting) being associated with the tumorigenic process of different types of tumors. The mechanism responsible for genomic imprinting is not yet determined, although DNA methylation has been considered the main genetic event for an imprinted mark. In the current study, the authors analyzed the imprinting status and expression levels of the IGFII and H19 genes in 27 cases of Juvenile Nasopharyngeal Angiofibroma (JNA) using RFLPs, RT-PCR, and Southern and Northern Blots. The authors found that four out of eight informative cases (50%) for ApaI/IFGII polymorphism showed biallelic expression of IFGII whereas none of the nine informative cases for the polymorphism showed biallelic expression of the H19 gene. Overexpression of IFGII was observed in 8 out of 22 cases (36.4%), and 7 out of 19 cases (36.8%) showed H19 overexpression. Hypomethylation was found only in the H19 gene in six out of eight cases analyzed. Therefore, our results demonstrate that alterations in the IFGII/H19 imprinted region occur in JNA.     

Fas Ligand-dependent and -independent mechanisms of toxicity induced by T cell lymphomas in lymphoid organs and in the liver

In the current study, we investigated the effect of growth of FasL⁺ tumors in vivo on the functions of peripheral lymphoid organs and the liver. Injection of FasL⁺ LSA tumor cells into syngeneic C57BL/6 wild-type mice but not C57BL/6 lpr/lpr (Fas-deficient) mice caused apoptosis in splenocytes. Spleen cells expressing CD3, CD4, CD8, CD19, Mac-3, and CD44 were all susceptible to tumor-induced apoptosis. Also, activated T cells were more sensitive to apoptosis induced by LSA tumor cell lysate when compared to naïve T cells. In contrast, anti-Fas Abs (Jo2) induced apoptosis in only activated but not naïve T cells. When the LSA tumor-bearing mice were injected with a superantigen (SEA), these mice showed a significant decrease in the expansion of SEA-reactive Vβ3⁺ and Vβ11⁺ T cells. When injected into syngeneic mice, the FasL⁺ LSA tumor cells caused hepatotoxicity, as indicated by an increase in serum aspartate aminotransferase (AST) levels. Interestingly, Fas-deficient C57BL/6 lpr/lpr mice also showed significant AST levels in the serum following LSA tumor growth. Moreover, hepatocytes isolated from C57BL/6 wild-type and C57BL/6 lpr/lpr mice were equally susceptible to apoptosis induced by LSA tumor cell lysate in vitro. Using cDNA array, LSA tumor cells were found to express several cytokine genes including IL-2, IL-7, IL-11, IL-13, IL-16, lymphotoxin β, and tumor necrosis factor β. Together, these data suggested that, in mice bearing FasL⁺ LSA tumor, the immunotoxicity is FasL-based, whereas the hepatotoxicity, at least in part, may be FasL-independent.     

Mortality After Revision Total Hip Arthroplasty

BackgroundIn counseling patients about the complications of revision total hip arthroplasty (revTHA), it is imperative that mortality be considered. The actual mortality rate by indication of revision is ill-defined. The purpose of this study is to determine the mortality rate after revTHA.MethodsAn institutional database identified 596 patients who had undergone revTHA between 2012 and 2018. Medical records, national, state, and local death indexes were queried for mortality status and indication for revTHA. For survivors, the last clinical visit date was used for censoring in the mortality analysis. Mortality rates were calculated for all clinical patients and then by specific indication for revision.ResultsThe overall 2-year mortality rate following revTHA was 19.5 deaths per 1000 or 1 in 51 patients. Patients presenting with a periprosthetic fracture had a significantly higher 2-year mortality rate of 74.5 deaths per 1000 or 1 in 13 patients (P < .001), while an indication of dislocation or instability had a slightly higher 2-year mortality rate of 50.3 per 1000 (1 in 20) but this difference was not significant (P = .531). Other indications such as mechanical loosening or infection did not have a significantly different mortality rate.ConclusionThe overall 2-year mortality rate following revTHA was 19.5 deaths per 1000 which was largely attributed to patients with a periprosthetic fracture (74.5 per 1000) with other indications not significantly impacting mortality. Mortality rates and specific rates by indication for revision should be considered when counseling patients prior to revTHA.     

Soft tissue sarcomas: ultrasonographic evaluation of local recurrences

OBJECTIVE: The diagnosis of early local recurrence of soft tissue sarcomas, especially in those treated with surgery and radiotherapy, is a difficult clinical problem. Financial constraints led us to use ultrasonography instead of CT or MR imaging. The aim of this study was to evaluate the role of ultrasonography (US) in detecting local recurrence. METHODS AND RESULTS: Fifty patients with previous treatment for soft tissue sarcomas were evaluated prospectively for recurrence by US and histopathology. Seven of the 50 patients were clinically suspected to have recurrent tumour. Ultrasonography showed recurrence in 26, no recurrence in 18, benign disease in four and was indeterminate in two cases. Ultrasonography was instrumental in guiding fine needle aspiration biopsies of small local recurrences and indeterminate lesions in 17 patients. In the sonographically tumour positive patients, histopathology confirmed recurrence in 24; one case had benign disease and one patient refused surgery. Thirteen of the 18 sonographically tumour negative patients were operated upon; all were negative for tumour on histopathology. Both the indeterminate cases showed recurrence on histopathology. The benign cases were confirmed by histopathology correlation. Ultrasound guided fine needle aspiration cytology (FNAC) was positive in 14 out of 17 patients (88%). The sensitivity and specificity of US was 92.30% and 94.4% respectively. CONCLUSION: Our study concludes that US is an extremely useful and cost effective method in the detection of early local recurrences of soft tissue sarcomas and should therefore be used for initial routine follow-up and guided biopsies.     

Combined screening of thymocytes using apoptosis-specific cDNA array and promoter analysis yields novel gene targets mediating TCDD-induced toxicity.

We have used pathway-specific cDNA arrays coupled with analysis of gene promoter regions to identify novel genes that may mediate the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the thymus. C57BL/6 mice were injected ip with 50 microg/kg TCDD, and 3, 6, or 24 h later, RNA was extracted from the thymus and subjected to microarray analysis. Several members of the TNF and TNFR family were induced following TCDD exposure, including receptor/ligand pairs Ltbeta-R/LIGHT, OX40/OX40L and TNF-alpha/TNFR1. In addition, Fas and CD30 were also upregulated. Pro-apoptotic bcl-2 gene family members Bax and Hrk, among others, were also induced, as were pro-survival bcl-2 family genes Bcl-x and Bcl-w. Cell-cycle regulator p21Cip1 was also induced. In addition, we analyzed the promoter regions of genes induced by TCDD for the presence of dioxin-responsive elements (DREs). The Fas and LIGHT gene promoters were found to contain DREs as analyzed by Matinspector Web-based search algorithm. Furthermore, binding of the aryl hydrocarbon receptor (AhR) to the DREs present on these genes was confirmed by chromatin immunoprecipitation. Given that several of the genes, including Fas, LIGHT, and CD30 are involved in negative selection of T cells in the thymus, our studies suggest that TCDD-induced upregulation of these genes may enhance negative selection leading to thymic atrophy.