AAV8 Induces Tolerance in Murine Muscle as a Result of Poor APC Transduction,T Cell Exhaustion,and Minimal MHCI Upregulation on Target Cells

Following gene transfer of adeno-associated virus 2/8 (AAV2/8) to the muscle, C57BL/6 mice show long-term expression of a nuclear-targeted LacZ (nLacZ) transgene with minimal immune activation. Here, we show that pre-exposure to AAV2/8 can also induce tolerance to the more immunogenic AAV2/rh32.33 vector, preventing otherwise robust T-cell activation and allowing stable transgene expression. Depletion and adoptive transfer studies showed that a suppressive factor was not sufficient to account for AAV2/8-induced tolerance, whereas further characterization of the T-cell population showed upregulation of the exhaustion markers PD1, 2B4, and LAG3. Furthermore, systemic administration of Toll-like receptor (TLR) ligands at the time of AAV2/rh32.33-administration broke AAV2/8-induced tolerance, restoring T-cell activation and β-gal clearance. As such, AAV2/8 transduction appears to lack the inflammatory signals necessary to prime a functional cytotoxic T-cell response. Inadequate T-cell priming could be explained upstream by AAV2/8''s poor transduction and activation of antigen-presenting cells (APCs). Immunohistochemical analysis indicates that AAV2/8 transduction also fails to upregulate major histocompatibility complex class I (MHCI) expression on the surface of myocytes, rendering transduced cells poor targets for T-cell–mediated destruction. Overall, AAV2/8-induced tolerance in the muscle is multifactorial, spanning from poor APC transduction and activation to the subsequent priming of functionally exhausted T-cells, while simultaneously avoiding upregulation of MHCI on potential targets.     

Intravenous azathioprine in severe ulcerative colitis: a pilot study

OBJECTIVE: Azathioprine use in acute ulcerative colitis has been limited by its perceived long onset of action. The aim of this study was to determine the safety and clinical effect of an i.v. loading dose of azathioprine in the setting of severe steroid refractory ulcerative colitis. METHODS: Nine hospitalized patients with severe steroid refractory ulcerative colitis were enrolled. Patients 1-3 received 20 mg/kg i.v. azathioprine over 36 h. Patients 4-6 received 40 mg/kg i.v. azathioprine over 36 h. Patients 7-9 received 40 mg/kg i.v. azathioprine as three 8-h infusions over 3 days. Clinical remission was defined as steroid withdrawal and an Ulcerative Colitis Disease Activity Index score of 0. The Inflammatory Bowel Disease Questionnaire was obtained at each visit. White blood cell concentrations and erythrocyte concentrations of 6-thioguanine were obtained. RESULTS: Five of nine patients (56%) had a response and avoided colectomy. Three of nine patients (33%) met the definition for clinical remission. Response was seen within 4 wk. The mean 6-thioguanine concentration for those five patients at 12 wk after infusion was 148.2 pmol/8 x 10(8). Two patients had transient leukopenia and one had transient hepatotoxicity. CONCLUSIONS: Intravenous azathioprine appears to be safe and of clinical benefit in inducing response and avoiding colectomy in severe steroid refractory ulcerative colitis. Data from an i.v. azathioprine trial in Crohn's disease suggests oral dosing alone may obtain the same results. The role of oral dosing alone in severe ulcerative colitis and the role of azathioprine metabolite levels in monitoring efficacy should be investigated further.     

Prion disease tempo determined by host-dependent substrate reduction

The symptoms of prion infection can take years or decades to manifest following the initial exposure. Molecular markers of prion disease include accumulation of the misfolded prion protein (PrP^Sc), which is derived from its cellular precursor (PrP^C), as well as downregulation of the PrP-like Shadoo (Sho) glycoprotein. Given the overlapping cellular environments for PrP^C and Sho, we inferred that PrP^C levels might also be altered as part of a host response during prion infection. Using rodent models, we found that, in addition to changes in PrP^C glycosylation and proteolytic processing, net reductions in PrP^C occur in a wide range of prion diseases, including sheep scrapie, human Creutzfeldt-Jakob disease, and cervid chronic wasting disease. The reduction in PrP^C results in decreased prion replication, as measured by the protein misfolding cyclic amplification technique for generating PrP^Sc in vitro. While PrP^C downregulation is not discernible in animals with unusually short incubation periods and high PrP^C expression, slowly evolving prion infections exhibit downregulation of the PrP^C substrate required for new PrP^Sc synthesis and as a receptor for pathogenic signaling. Our data reveal PrP^C downregulation as a previously unappreciated element of disease pathogenesis that defines the extensive, presymptomatic period for many prion strains.     

Update on current approaches to diagnosis and treatment of onychomycosis

ABSTRACT

Introduction: Onychomycosis is a chronic fungal infection of the nail bed, matrix or plate. It accounts for roughly 50% of all nail disease. As the prevalence of onychomycosis is increasing, a critical review of diagnostic techniques and treatment options is required.

Areas covered: This review discusses the current diagnostic techniques associated with diagnosing onychomycosis, such as microscopy, culture, periodic acid Schiff stain (PAS) and polymerase chain reaction (PCR). Oral and topical therapies are also discussed, as well as, the utility of device-based treatments and combination therapy.

Expert commentary: Culture for the diagnosis of onychomycosis is the gold standard; however, PCR is more sensitive and should be considered. In general, topical treatments are recommended for mild to moderate disease and oral treatments should be considered for moderate to severe disease. Combination therapy and device-based treatments may enhance cure rates, further study is required.     

Measuring morbidity for resource allocation.

The RAWP (Resource Allocation Working Party) report used population weightings based on standardised mortality ratios (SMRs) as a proxy measure of differences in morbidity (and therefore in the need for health care resources) that existed between geographical areas after allowing for the age and sex structure of their populations. The adequacy of SMRs as a proxy for morbidity has aroused controversy, particularly from RAWP losers in London, and is one of the main themes of the National Health Service Management Board's current review of RAWP. Critics have argued, firstly, that the nature of the relation between morbidity and mortality is unknown; and, secondly, that SMRs are incomplete because they fail to take account of the effect of social deprivation on the need for health care. As a result several alternative proxies for morbidity based on social indicators have been proposed. One of their principal drawbacks is that their use is justified by their relation to measures of use of services known to be affected by the prevailing level of supply. Furthermore, the evidence suggests that mortality data actually correlate quite well with the available measures of both morbidity and social deprivation. But without access to comprehensive morbidity data the SMR debate is bound to remain inconclusive. As measures of health need, however, SMRs have the twin merits of being (a) independent of supply, and (b) more direct measures of health state than social indicators.     

氯化四乙基铵及维拉帕米对家兔缺血心肌不应期的影响

实验比较了K⁺通道阻断剂氯化四乙基铵(TEA)及Ca²⁺通道阻断剂维拉帕米(verapamil,Ver)对家兔缺血性心室肌不应期(ERP)的影响。结果表明,阻断冠脉10min及30min后,缺血中心区(CIZ)和缺血边缘区(BIZ)心肌ERP比缺血前均明显缩短,而TEA和Ver均能延长急性缺血性心肌的ERP,可能对心肌缺血引起的心律失常有预防作用。     

Small diameter versus conventional laparoscopy: a prospective, self- controlled study

The objective of this study was to determine visual quality, diagnostic accuracy, and surgical merits of small diameter laparoscopy (SDL). Thirty-seven patients were randomly selected. The indications for laparoscopy were infertility, desire for tubal sterilization or chronic pelvic pain. Patients underwent SDL, followed by conventional laparoscopy (CL) as a control under general anaesthesia. Findings at operation were compared. The mean time for diagnostic work-up was longer with SDL than CL, 11.7 +/- 5.6 versus 7.6 +/- 3.2 min respectively (P < 0.04). Visual quality was scored from 4 to 1 by the operator; mean visual quality, mean endometriosis score and mean adnexal adhesions score were slightly lower with SDL than CL. Sensitivity of SDL in diagnosing endometriosis, adhesions, ovarian, uterine and pouch of Douglas lesions were 71, 58, 81, 89 and 73% respectively; specificity was 100, 96, 100, 100, 100% in the same order. Suction irrigation, cyst aspiration, tissue biopsies, simple adhesiolysis, tubal ligation and cauterization were easily performed with SDL. We conclude that SDL seems a good alternative to CL in diagnosing macro-pelvic anatomy and coarse pelvic pathologies and may also be good in performing surgical procedures such as: tubal ligation, biopsies and differential diagnosis of pelvic fluids. But SDL must be used cautiously in micro-oriented, functional conditions such as infertility, pelvic pain, endometriosis and adhesion scoring or treatment. SDL may be regarded as a less invasive but less sensitive tool with limited surgical merits.