Evolution of Graves' Disease during Immune Reconstitution following Nonmyeloablative Haploidentical Peripheral Blood Stem Cell Transplantation in a Boy Carrying Germline SAMD9L and FLT3 Variants.

Graves' disease, characterized by hyperthyroidism resulting from loss of immune tolerance to thyroid autoantigens, may be attributable to both genetic and environmental factors. Allogeneic hematopoietic stem cell transplantation (HSCT) represents a means to induce immunotolerance via an artificial immune environment. We present a male patient with severe aplastic anemia arising from a germline SAMD9L missense mutation who successfully underwent HSCT from his HLA-haploidentical SAMD9L non-mutated father together with nonmyeloablative conditioning and post-transplant cyclophosphamide at 8 years of age. He did not suffer graft-versus-host disease, but Graves' disease evolved 10 months post-transplant when cyclosporine was discontinued for one month. Reconstitution of peripheral lymphocyte subsets was found to be transiently downregulated shortly after Graves' disease onset but recovered upon antithyroid treatment. Our investigation revealed the presence of genetic factors associated with Graves' disease, including HLA-B*46:01 and HLA-DRB1*09:01 haplotypes carried by the asymptomatic donor and germline FLT3 c.2500C>T mutation carried by both the patient and the donor. Given his current euthyroid state with normal hematopoiesis, the patient has returned to normal school life. This rare event of Graves' disease in a young boy arising from special HSCT circumstances indicates that both the genetic background and the HSCT environment can prompt the evolution of Graves' disease.

Soluble epoxide hydrolase activity regulates inflammatory responses and seizure generation in two mouse models of temporal lobe epilepsy.

Neuroinflammation is known to be involved in epileptogenesis with unclear mechanisms. Inhibition of soluble epoxide hydrolase (sEH) seems to offer anti-inflammatory protection to ischemic brain injury in rodents. Thus, it is hypothesized that sEH inhibition might also affect the neuroinflammatory responses caused by epileptic seizures. In the present study, we investigated the involvement of sEH in neuroinflammation, seizure generation and subsequent epileptogenesis using two mouse models of temporal lobe epilepsy. Experimental epileptic seizures were induced by either pilocarpine or electrical amygdala kindling in both wild-type (WT) C57BL/6 mice and sEH knockout (sEH KO) mice. The sEH expression in the hippocampus was detected by immunohistochemistry and Western blot analysis. The effects of the sEH hydrolase inhibitors, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) and N-[1-(1-oxopropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy) phenyl)-urea (TPPU), and of the genetic deletion of sEH on seizure-induced neuroinflammatory responses and the development of epilepsy were evaluated. In the hippocampus of WT mice, sEH was mainly expressed in astrocytes (GFAP(+)), neurons (NeuN(+)) and scattered microglia (Iba-1(+)) in the regions of CA1, CA3 and dentate gyrus. Expression of sEH was significantly increased on day 7, 14, 21 and 28 after pilocarpine-induced status epilepticus (SE). Administration with sEH inhibitors attenuated the SE-induced up-regulation of interleukin-1ß (IL-1ß) and interleukin-6 (IL-6), the degradation of EETs, as well as IκB phosphorylation. Following treatment with AUDA, the frequency and duration of spontaneous motor seizures in the pilocarpine-SE mice were decreased and the seizure-induction threshold of the fully kindled mice was increased. Up-regulation of hippocampal IL-1ß and IL-6 was found in both WT and sEH KO mice after successful induction of SE. Notably, sEH KO mice were more susceptible to seizures than WT mice. Seizure related neuroinflammation and ictogenesis were attenuated by pharmacological inhibition of sEH enzymatic activity but not by sEH genetic deletion. Therefore, sEH may play an important role in the generation of epilepsy. Furthermore, the effectiveness of AUDA in terms of anti-inflammatory and anti-ictogenesis properties suggests that it may have clinical therapeutic implication for epilepsy in the future, particularly when treating temporal lobe epilepsy.

Monocyte chemoattractant protein-1 affects migration of hippocampal neural progenitors following status epilepticus in rats.

BACKGROUND: Epilepsy is a common brain disorder characterized by a chronic predisposition to generate spontaneous seizures. The mechanisms for epilepsy formation remain unknown. A growing body of evidence suggests the involvement of inflammatory processes in epileptogenesis. In the present study, we investigated the involvement of monocyte chemoattractant protein-1 (MCP-1) in aberrant migration of hippocampal progenitors in rats after the insult of status epilepticus (SE). METHODS: SE was induced with pilocarpine in Sprague-Dawley rats. Transcriptional expression of MCP-1 in the dentate gyrus (DG) was measured using quantitative real-time PCR. From 1 to 28 days after SE, the temporal profiles of MCP-1 protein expression in DG were evaluated using enzyme-linked immunosorbent assay. Chemokine (C-C motif) receptor 2 (CCR2) expression in doublecortin-positive neuronal progenitors was examined using double-labeling immunohistochemistry. The involvement of MCP-1/CCR2 signaling in aberrant neuronal progenitor migration in the epileptic hippocampus was assessed in the SE rats using a CCR2 antagonist, RS102895, and the ectopic migration of neuronal progenitors was determined using Prox1/doublecortin double immunostaining. RESULTS: After SE, MCP-1 gene was significantly upregulated and its corresponding protein expression in the DG was significantly increased on days 1 and 3. Some hilar ectopic progenitor cells of SE rats expressed the MCP-1 receptor, CCR2. Notably, the ectopic migration of neuronal progenitors into hilus was attenuated by a blockade of the MCP-1/CCR2 interaction with a selective CCR2 inhibitor, RS102895. CONCLUSIONS: An increase in dentate MCP-1 is associated with seizure-induced aberrant migration of neuronal progenitors through the interaction with CCR2. The upregulation of MCP-1 after an insult of SE may play a role in the generation of epilepsy.

Association analysis of polymorphisms in lumican gene and systemic lupus erythematosus in a Taiwan Chinese Han population.

OBJECTIVE: Lumican (LUM) is predominantly localized in areas of pathological fibrosis. To determine whether polymorphisms in LUM gene are associated with development of systemic lupus erythematosus (SLE), we analyzed 2 single-nucleotide polymorphisms (SNP) of LUM in a Taiwan Chinese Han population. METHODS: Participants included 168 patients with SLE and 192 age-matched controls in whom examinations had excluded SLE. Genotyping of -628 A/-(rs17018757) and c.1567 T/C polymorphisms in LUM were carried out in each patient and control using the polymerase chain reaction-restriction fragment-length polymorphism method, and validated by Taqman SNP genotyping assay. Data were correlated with the development of SLE and various clinical symptoms by chi-square analysis. RESULTS: Frequencies of C/C genotype and the C allele at c.1567 T/C were significantly higher in patients than controls. Polymorphism at c.1567 C/T was found to be associated with arthritis and photosensitivity in patients with SLE, which are both connective tissue-related symptoms. CONCLUSION: The c.1567 T/C polymorphism of LUM is related to the development and clinical symptoms of SLE.

Overexpression of a chromatin remodeling factor, RSF-1/HBXAP, correlates with aggressive oral squamous cell carcinoma.

RSF-1, also known as hepatitis B X-antigen associated protein (HBXAP), is a subunit of an ISWI chromatin remodeling complex, remodeling and spacing factor (RSF). Recent studies have provided new evidence that chromatin remodeling participates in the pathogenesis of neoplastic diseases by altering cell cycle regulation and gene expression. In this report, we studied the biological roles of RSF-1 in oral squamous cell carcinoma (OSCC), a highly invasive neoplastic disease. Based on IHC and quantitative real-time PCR, we demonstrated that RSF-1 expression could be detected in the majority of OSCC cases, and the levels were significantly higher in OSCC cells than in their normal counterparts. Moreover, expression levels of RSF-1 significantly correlated with the presence of angiolymphatic invasion, abnormal mitoses, metastasis, tumor recurrence, and advanced stage disease at presentation. Univariate and multivariate analyses showed a significant association of RSF-1 overexpression and worse overall survival in OSCC patients. RSF-1 knockdown remarkably decreased cellular proliferation and induced apoptosis in OSCC cells with high RSF-1 expression levels, but not in those without. Taken together, our results suggest that RSF-1 up-regulation is associated with several clinicopathological features of disease aggressiveness in OSCC patients, and RSF-1 plays an important role in maintaining cellular growth and survival in OSCC.

Prophylaxis of experimentally induced calcium oxalate nephrolithiasis in rats by Zhulingtang, a traditional Chinese herbal formula.

BACKGROUND: Zhulingtang (ZLT), a traditional Chinese medicine formula, was used to evaluate the antilithic effects of experimentally induced calcium oxalate (CaOx) nephrolithiasis in ethylene glycol (EG)-fed rats. MATERIALS AND METHODS: A total of 35 male Sprague-Dawley rats were randomly divided into 4 groups. Rats in group 1 (n = 8) served as the normal control. Rats in group 2 (n = 11) were treated with gastric gavages of starch as placebo and 0.75% EG as a stone inducer. Rats in group 3 (n = 8) were given 0.75% EG and a low dose (305 mg/kg) of ZLT. Rats in group 4 (n = 8) were treated with EG and a high dose (915 mg/kg) of ZLT. Twenty-four-hour urine and blood samples were collected at the beginning and at the end of the experiment for biochemical analysis. The histological appearances of the kidneys were observed under a polarized light microscope, and the crystal deposits were evaluated by a semiquantitative scoring method, computer assisted with ImageScoring software. RESULTS: Our results revealed that rats fed with 0.75% EG for 4 weeks successfully produced renal deposition of CaOx. The severities of crystal deposition were significantly reduced in the 2 ZLT-fed groups compared with the placebo group (p = 0.025 and 0.047, respectively). Rats in the low-dose ZLT and placebo groups exhibited significantly lower serum phosphorus in comparison with the control rats (p = 0.005 and 0.03, respectively). Rats of the placebo group (EG + starch) encountered growth retardation, with their body weights slowly increasing, expressed as 160.63 +/- 23.06 g, compared with 179.63 +/- 13.41 g in normal rats (p < 0.001). CONCLUSION: ZLT reduced the severity of CaOx crystallization and slowed down the body weight loss effects. Therefore, the traditional Chinese medicine herbal formula ZLT may be an effective reagent for renal stone prophylaxis. Although the mechanism of ZLT in crystal inhibition remains unclear, macromolecules may be involved.

Global gene expression profiling of wild type and lysC knockout Escherichia coli W3110.

Aspartokinase III, encoded by lysC, is responsible for the first step of lysine biosynthesis in Escherichia coli. In this study, a lysC knockout E. coli W3110 strain was generated to study the differential gene expression profiles of wild type and lysC knockout strains. Several significant changes were observed, including biosynthesis of lysine, oxaloacetate, alpha-ketoglutarate and glutamate genes. Genes related to transporters and heat shock proteins were also affected by lysC knockout. The results indicated that the lysC knockout strain exhibited some phenomena similar to lysine starvation. The data generated by this study further clarify the systematic role of lysC in lysine biosynthesis.

Cell growth restoration and high level protein expression by the promoter of hexose transporter, HXT7, from Saccharomyces cerevisiae.

The promoters of high-affinity hexose transporter, HXT6 and HXT7, are sufficient for complementary expression of invertase to restore the growth of Saccharomyces cerevisiae in raffinose medium. The HXT7 promoter produced higher invertase activity at 139- and 30-fold of the basal activity in strains GN 3C.2 and W303-1, respectively. In addition, the HXT7 promoter expressed 3- to 9-fold more of enhanced green fluorescent protein than that of the constitutive ADH1 in three different S. cerevisiae strains, even during short-term incubation in glucose medium. Therefore, HXT7 promoter could be used for heterologous protein expression in S. cerevisiae.

Reidentification of cellulolytic enzyme-producing Trichoderma strains W-10 and G-39.

Strain W-10, originally identified as Trichoderma koningii, and its supposed mutant G-39, published for production and gene expression of cellulase and xylanase, demonstrated morphological characteristics distinct from those of T. koningii, respectively. To clarify the identification derived from morphological characteristics, several methods were used, including electrophoretic karyotyping, internal transcribed spacer (ITS) analysis of rDNA, and polymerase chain reaction (PCR) fingerprinting using the universal primer L45. All the molecular characteristics showed that strains G-39 and W-10 were identical to T. reesei and T. longibrachiatum, respectively. The results strongly supported that T. koningii G-39 and W-10 should be reassigned as T. reesei and T. longibrachiatum, respectively. Strain G-39 should be considered a mutant from T. reesei QM9414 whose spores were contaminated with those of strain W-10 during a laboratory operation. According to this, we declare that T. koningii G-39 and W-10 must be renamed as T. reesei and T. longibrachiatum, respectively.

Geldanamycin induction of grp78 requires activation of reactive oxygen species via ER stress responsive elements in 9L rat brain tumour cells.

The molecular mechanism whereby anticancer agent geldanamycin (GA) impacts endoplasmic reticulum (ER) stress pathway is largely unknown. Here, we investigate the effect of GA on the expression of grp78 coding for ER stress protein and the mechanistic relationship of GA signalling to ER stress. GA induces the expression of mRNA and protein of grp78 by Northern blot analysis and metabolic labelling experiment in cultured rat brain tumour 9L cells. The induced grp78 expression is sensitive to antioxidant N-acetylcysteine (NAC) addition, indicating the involvement of reactive oxygen species (ROS) in GA-induced ER stress. Results from direct determination of oxidation status using dichlorodihydrofluorescein diacetate (H(2)DCFDA) showed that accumulation of ROS elicited GA was quenched by addition of NAC. Reporter genes harbouring deletions of transcription elements from grp78 promoter demonstrated that controlling elements of ERSE1, ERSE2 and CRE are required in GA treatment. The critical ROS-dependent elements in grp78 promoter can be confined within ER stress responsive element (ERSE) region, since reporter constructs loss of ERSE elements that lost the susceptibility to be modulated by NAC after GA treatment. Hence, ER stress elements correlate well with ROS-mediated elements in grp78 promoter. Reporter construct loss of ERSE element retains the susceptibility by NAC after GA treatment, indicating that CRE element might represent a ROS-independent, GA-inductive element. Conclusively, we show that ROS is required for GA to launch the transactivation of grp78, and a firm link was established between the ROS signalling pathway to specific promoter elements-ERSE1 and ERSE2 elements in ER stress marker gene grp78 promoter.

Mitochondrial calcium-mediated reactive oxygen species are essential for the rapid induction of the grp78 gene in 9L rat brain tumour cells.

The glucose-regulated protein grp78 gene is rapidly transactivated in 9L rat brain tumour (RBT) cells treated with okadaic acid (OA) followed by heat shock (HS) (termed OA-->HS treatment). By Northern blotting analyses and transient transfection assays, we herein show that transactivation of grp78 by OA-->HS is abolished by an intracellular calcium chelator, bis(aminophenoxy)ethane N,N'-tetraacetic acid (BAPTA), and an inhibitor of mitochondrial Ca(2+) uniporter, ruthenium red (RR), while unaffected by cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore (MTP). The inhibitory effects of BAPTA and RR also present in OA-->HS induction of transient elevation of intracellular hydrogen peroxide. The requirement of reactive oxygen intermediates (ROIs) is confirmed by substitutional addition of antioxidants, N-acetyl cysteine (NAC) and pyrrolidinedithiocarbamate (PDTC) during OA-->HS treatment, mimicking these inhibitory effects of BAPTA and RR. Western blotting analyses show that phosphorylation of transcription factor CREB is diminished only by BAPTA but not by RR, while phosphorylation of ATF-2 is unaffected by either agent. Conclusively, we present that both the disturbances of mitochondrial calcium homeostasis and reactive oxygen intermediates are essential for rapid transactivation of grp78, and this pathway is separate from protein kinase A (PKA)-dependent CREB activation or p38 mitogen-activated protein kinase (p38(MAPK))-dependent ATF-2 activation and signalling.