A Melting Method for RNA Extraction from the Mucosal Membrane of the Mouse Middle Ear

PurposeThere is much confusion surrounding the methods of RNA extraction from the middle ear mucosa of mice. In this study, we worked to develop a "melting method," which is faster, purer, and more reliable than other methods in common use.ResultsEach resected half bulla contained a well distributed mucosal membrane. After a TRIzol melting duration of 10-30 minutes, only mucosal marker (MUC5AC) was expressed without bony marker (total osteocalcin). The same results were determined from SEM.ConclusionThis melting method, compared with stripping and irrigation methods, is effective and offers an easier, more robust approach to extracting RNA from the middle ear mucosal membranes of mice.     

Comparison of the Astigmatic Power of Toric Intraocular Lenses Using Three Toric Calculators

Purpose

To compare the astigmatic power of toric intraocular lenses (IOLs) obtained from the AcrySof, TECNIS, and iTrace toric calculator in patients with preoperative with-the-rule (WTR) or against-the-rule (ATR) corneal astigmatism.

Materials and Methods

Fifty eyes with cataract and corneal astigmatism greater than 0.75 diopters were enrolled in each group (WTR and ATR). Keratometric values were measured using autokeratometry, an IOLMaster, and an iTrace, which incorporated corneal topography and ray-tracing aberrometry. Based on measured keratometric values, the astigmatic power of each toric IOL was calculated using three toric calculators.

Results

Bland-Altman plots showed good agreement between six pairwise corneal astigmatism values in both groups. The TECNIS calculator tended to suggest a higher astigmatic power of the toric IOL than the AcrySof calculator. With the higher astigmatism and keratometric values from the IOLMaster, in both groups, calculations from the AcrySof and TECNIS calculators resulted in higher calculated astigmatic powers than those from same calculators with autokeratometry-measured values, demonstrating good agreement. With the higher calculated astigmatic power values, the values from the iTrace toric calculator using keratometric values obtained from iTrace ray tracing wavefront aberrometry or iTrace simulated keratometry showed fair to moderate agreement with those from the other calculator-keratometry pairs in both groups.

Conclusion

To achieve the best refractive outcome after toric IOL implantation, understanding the differences in keratometric values between instruments and in calculated astigmatic power among toric calculator programs is necessary. Moreover, systemic analysis of each toric calculator in conjunction with postoperative data is required.     

High-Dose Vitamin C Promotes Regression of Multiple Pulmonary Metastases Originating from Hepatocellular Carcinoma

We report a case of regression of multiple pulmonary metastases, which originated from hepatocellular carcinoma after treatment with intravenous administration of high-dose vitamin C. A 74-year-old woman presented to the clinic for her cancer-related symptoms such as general weakness and anorexia. After undergoing initial transarterial chemoembolization (TACE), local recurrence with multiple pulmonary metastases was found. She refused further conventional therapy, including sorafenib tosylate (Nexavar). She did receive high doses of vitamin C (70 g), which were administered into a peripheral vein twice a week for 10 months, and multiple pulmonary metastases were observed to have completely regressed. She then underwent subsequent TACE, resulting in remission of her primary hepatocellular carcinoma.     

The prognostic factors of pancreatic cancer can be different according to clinical stages]

BACKGROUND/AIMS: The prognosis of patients with pancreatic cancer remains very poor. Although many studies have evaluated the prognostic factors of pancreatic cancer, their results are inconclusive because of different inclusion criteria, tumor stages, and treatment modalities. This large scale retrospective analysis was performed to assess whether active treatment of pancreatic cancer, even in its advanced stage, could improve patients' survival. In addition, we sought to identify factors associated with favorable prognosis of pancreatic cancer. METHODS: Between 1994 and 2004, a total of 971 patients with pancreatic cancer were treated at Asan Medical Center. The patients were classified into three groups according to clinical stages: resectable (RE, n=226), locally advanced (LA, n=409), and far advanced (FA, n=336). Treatment response and prognostic factors for survival were analyzed in each group. RESULTS: Compared to supportive care, active treatment significantly increased the median survival time in all groups (RE: 18.0 vs. 9.0 months; LA: 10.0 vs. 7.0 months; FA: 5.0 vs. 3.0 months). Multivariate analysis showed that prognostic factors for survival differed according to clinical stages. In the RE group, unfavorable prognostic factors were high CA 19-9, poor histologic differentiation, large tumor size, and regional lymph node involvement. In the FA group, however, poor outcomes were associated with old age, poor performance status, and hypoalbuminemia. CONCLUSIONS: More active treatment of pancreatic cancer, even in advanced stage, can make a significant difference in terms of patient's survival. The prognosis of resectable pancreatic cancer is dependent on tumor-related factors, while the prognosis of patients with far advanced pancreatic cancer is dependent on patient-related factors.     

The siRNA cocktail targeting interleukin 10 receptor and transforming growth factor‐β receptor on dendritic cells potentiates tumour antigen‐specific CD8+ T cell immunity

Dendritic cells (DCs) are promising therapeutic agents in the field of cancer immunotherapy due to their intrinsic immune‐priming capacity. The potency of DCs, however, is readily attenuated immediately after their administration in patients as tumours and various immune cells, including DCs, produce various immunosuppressive factors such as interleukin (IL)‐10 and transforming growth factor (TGF)‐β that hamper the function of DCs. In this study, we used small interfering RNA (siRNA) to silence the expression of endogenous molecules in DCs, which can sense immunosuppressive factors. Among the siRNAs targeting various immunosuppressive molecules, we observed that DCs transfected with siRNA targeting IL‐10 receptor alpha (siIL‐10RA) initiated the strongest antigen‐specific CD8⁺ T cell immune responses. The potency of siIL‐10RA was enhanced further by combining it with siRNA targeting TGF‐β receptor (siTGF‐βR), which was the next best option during the screening of this study, or the previously selected immunoadjuvant siRNA targeting phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or Bcl‐2‐like protein 11 (BIM). In the midst of sorting out the siRNA cocktails, the cocktail of siIL‐10RA and siTGF‐βR generated the strongest antigen‐specific CD8⁺ T cell immunity. Concordantly, the knock‐down of both IL‐10RA and TGF‐βR in DCs induced the strongest anti‐tumour effects in the TC‐1 P0 tumour model, a cervical cancer model expressing the human papillomavirus (HPV)‐16 E7 antigen, and even in the immune‐resistant TC‐1 (P3) tumour model that secretes more IL‐10 and TGF‐β than the parental tumour cells (TC‐1 P0). These results provide the groundwork for future clinical development of the siRNA cocktail‐mediated strategy by co‐targeting immunosuppressive molecules to enhance the potency of DC‐based vaccines.     

The Role of Spleen Stiffness in Determining the Severity and Bleeding Risk of Esophageal Varices in Cirrhotic Patients

Esophageal varix and its hemorrhage are serious complications of liver cirrhosis. Recent studies have focused on noninvasive prediction of esophageal varices. We attempted to evaluate the association of liver and spleen stiffness (LS and SS) as measured by acoustic radiation force impulse imaging, with the presence and severity of esophageal varices and variceal hemorrhage in cirrhotic patients.We measured LS and SS, along with endoscopic examination of esophageal varices for a total of 125 cirrhotic patients at a single referral hospital in this prospective observational study. The diagnostic utility of noninvasive methods for identifying varices and their bleeding risk was compared, including LS, SS, spleen length, Child-Pugh score, and various serum fibrosis indices.Esophageal varices were present in 77 patients (61.6%). SS was significantly higher in patients with varices than in those without varices (3.58 ± 0.47 vs 3.02 ± 0.49; P < 0.001). A tendency toward increasing SS levels was observed with increasing severity of varices (no varix, 3.02 ± 0.49; F1, 3.39 ± 0.51; F2, 3.60 ± 0.42; F3, 3.85 ± 0.37; P < 0.001)_. SS was significantly higher in patients who experienced variceal hemorrhage than in those who did not (3.80 ± 0.36 vs 3.20 ± 0.51; P = 0.002). An optimal cut-off value of SS for high-risk varices (≥F2) or variceal hemorrhage was 3.40 m/s.SS was significantly correlated with the presence, severity, and bleeding risk of esophageal varices. Prompt endoscopic evaluation of variceal status and prophylactic measures based on the SS may be warranted for cirrhotic patients.     

Basolateral amygdala bidirectionally modulates stress-induced hippocampal learning and memory deficits through a p25/Cdk5-dependent pathway

Repeated stress has been suggested to underlie learning and memory deficits via the basolateral amygdala (BLA) and the hippocampus; however, the functional contribution of BLA inputs to the hippocampus and their molecular repercussions are not well understood. Here we show that repeated stress is accompanied by generation of the Cdk5 (cyclin-dependent kinase 5)-activator p25, up-regulation and phosphorylation of glucocorticoid receptors, increased HDAC2 expression, and reduced expression of memory-related genes in the hippocampus. A combination of optogenetic and pharmacosynthetic approaches shows that BLA activation is both necessary and sufficient for stress-associated molecular changes and memory impairments. Furthermore, we show that this effect relies on direct glutamatergic projections from the BLA to the dorsal hippocampus. Finally, we show that p25 generation is necessary for the stress-induced memory dysfunction. Taken together, our data provide a neural circuit model for stress-induced hippocampal memory deficits through BLA activity-dependent p25 generation.Chronic stress can have devastating psychological consequences that include depression and cognitive impairment (1–3). Decades of research suggest that the hippocampus, a structure important for learning and memory and implicated in depression, is particularly sensitive to the effects of chronic stress. In animal models, for example, chronic stress impairs hippocampus-dependent forms of learning and memory (2). This sensitivity is partially conferred by a dense concentration of glucocorticoid receptor (GR) in the hippocampus (4), as well as through hippocampal connectivity to important stress response coordinators, such as the amygdala, from which the hippocampus receives abundant glutamatergic inputs (5–7). Following chronic stress, the hippocampus shows marked reductions in dendritic arborization and neurogenesis, along with impaired plasticity (2). Many of these effects have been attributed to connections between the hippocampus and a specific amygdalar subregion, the basolateral amygdala (BLA) (8–10).Abundant evidence suggests that these BLA inputs have a major impact on hippocampus function; for example, the hippocampus and BLA synchronize their activity during fear memory retrieval and fear extinction (11, 12), whereas electrical stimulation of the BLA disrupts the induction of long-term potentiation (LTP), a measure of synaptic plasticity, in the hippocampal CA1 subregion (13). Lesions of the BLA have been shown to block the detrimental effects of repeated stress, a model of chronic stress in rodents, on LTP and spatial memory (8, 10), as well as the deleterious effect of hippocampal GR activation on hippocampus-dependent memory (9). Although the BLA sends abundant projections to the hippocampus (5–7), this region also projects diffusely throughout the brain and thereby regulates a myriad of behaviors, including valence or social interaction (14), as well as hormonal cascades (15). Because of this complexity, whether BLA activity affects hippocampus-dependent learning and memory directly or indirectly through distinct relay brain regions or other downstream mediators, such as stress hormones, remains unclear.Cdk5 (cyclin-dependent kinase 5) plays a pleiotropic role in the nervous system (16). This enzyme is essential for proper brain development and regulates synaptic plasticity and cognitive function. Activation of Cdk5 requires association with a regulatory subunit known as p35. p35 is subjected to calpain-mediated cleavage into p25 in a process dependent on the activation of glutamate receptors, specifically NR2B-containing NMDA receptors, following neurotoxic stimulation, such as exposure to β-amyloid peptides, oxidative stress, or excitotoxicity, as well as in response to physiological neuronal activity (16).A number of studies have implicated p25 production in Alzheimer’s disease (AD)-like phenotypes, including learning and memory impairments (16), and long-term overexpression of p25 in the forebrain is known to lead to cognitive deficits (16). Furthermore, stress and the heightened sensitivity to stress are known risk factors for the development of AD (1). The role of p25 production after repeated stress remains undetermined, however.One pathway through which p25/Cdk5 might be implicated in stress-induced cognitive dysfunction is stress hormone receptor-mediated epigenetic signaling in the hippocampus. Indeed, it was previously shown that GR is activated by p25/Cdk5-dependent phosphorylation on Ser211 (17, 18), and that increased GR phosphorylation leads to increased expression of histone deacetylase 2 (HDAC2) in a mouse model of AD (18). HDAC2 in turn suppresses the expression of genes important for learning and memory (18, 19), suggesting a mechanism by which elevated p25 generation leads to cognitive impairment. Although GR activation has been shown to be required for stress-induced hippocampal dysfunction and is dependent upon its phosphorylation (20–22), and HDAC2 has been shown to be up-regulated in the ventral striatum of mice following chronic stress (23), the possible up-regulation of HDAC2 in the hippocampus after repeated stress, and the role of p25/Cdk5 signaling in this process, are unknown. We tested the hypothesis that p25 is generated in the hippocampus after repeated stress in an amygdala-dependent manner and contributes to stress-associated learning and memory deficits. Blockade of p25 generation would then protect the hippocampus from the detrimental effects of repeated stress.Here we identify that the activity of a specific BLA to dorsal hippocampus neural circuit mediates the detrimental effects of repeated stress on hippocampal learning and memory via a molecular pathway dependent on p25 generation.     

Damage caused by Wegener's granulomatosis and its treatment: prospective data from the Wegener's Granulomatosis Etanercept Trial (WGET)

OBJECTIVE: To analyze damage occurring in patients with Wegener's granulomatosis (WG) enrolled in the WG Etanercept Trial (WGET) and to correlate that damage with disease activity, adverse events, and quality of life. METHODS: The Vasculitis Damage Index (VDI) was applied to all 180 patients at trial entry and every 6 months throughout the trial. Items of damage were analyzed by presumed etiology (i.e., secondary to WG, to therapy, or both) and time of occurrence. Spearman's rank correlation coefficients were calculated between VDI scores and the Birmingham Vasculitis Activity Score for WG (BVAS/WG), frequency of flares, number of adverse events, and the patients' quality-of-life assessments. RESULTS: The mean VDI score was 1.3 at the study enrollment and 1.8 at the end of the trial. This increase was due to damage that occurred despite (or because of) therapy, including visual impairment, hearing loss, nasal blockade, pulmonary fibrosis, hypertension, renal insufficiency, peripheral neuropathy, gonadal failure, and diabetes mellitus. Only 11% of the enrolled patients had not sustained a single VDI item after 1 year of enrollment. When adjusted for baseline VDI, the baseline BVAS/WG correlated moderately well with the VDI score at 1 year (r = 0.20, P = 0.015). Increases in adjusted VDI scores also correlated with the number of adverse events, particularly among patients with limited WG (P = 0.06). CONCLUSION: Damage from both active disease and its treatment remain important problems for patients with WG. Despite the dramatic improvements in patient survival achieved over the last several decades, only a few patients with WG emerge from a period of active disease without sustaining some damage from the disease itself, its treatment, or both. An important measure of future therapeutic approaches will be their ability to reduce the damage accrued over time.