Immune responses against SARS-coronavirus nucleocapsid protein induced by DNA vaccine

The nucleocapsid （N） protein of SARS-coronavirus （SARS-CoV） is the key protein for the formation of the helical nucleocapsid during virion assembly. This protein is believed to be more conserved than other proteins of the virus, such as spike and membrane glycoprotein. In this study, the N protein of SARS-CoV was expressed in Escherichia coli DHSalpha and identified with pooled sera from patients in the convalescence phase of SARS. A plasmid pCI-N, encoding the full-length N gene of SARS-CoV, was constructed. Expression of the N protein was observed in COS1 cells following transfection with pCI-N. The immune responses induced by intramuscular immunization with pCI-N were evaluated in a murine model. Serum anti-N immunoglobutins and splenocytes proliferative responses against N protein were observed in immunized BALB/c mice. The major immunoglobulin G subclass recognizing N protein was immunoglobulin G2a, and stimulated splenocytes secreted high levels of gamma interferon and IL-2 in response to N protein. More importantly, the immunized mice produced strong delayed-type hypersensitivity （DTH） and CD^8＋ CTL responses to N protein.     

The epidemiology of hyperuricaemia and gout in Taiwan aborigines

To determine the prevalence of hyperuricaemia, gout and gout-related factors in Central Taiwan Atayal aborigines, 342 subjects over 18 yr old were interviewed and examined. A questionnaire was designed to screen for signs and symptoms of gout and gout-related risk factors. Serum uric acid, triglyceride and creatinine were measured in all subjects. The prevalence of hyperuricaemia was 41.4% and that of gout 11.7% in aborigines. The uric acid level was 7.9+/-1.7 mg/dl in males and 5.7+/-1.5 in females, and differed significantly under age 70 yr (P < 0.001). Significantly increased triglyceride, creatinine and alcoholism was found in gouty patients compared with non-gouty patients. In 40 cases with gout, 54% had tophi and 35% of their first- degree relatives had gout. The high prevalence of hyperuricaemia and gout in Taiwan Atayal aborigines, a significant family predisposition, increased creatinine level and alcoholism suggest multiple factors affecting the hyperuricaemia.      

Intestinal pseudo-obstruction as an initial presentation of systemic sclerosis in two children

Two children are reported in whom intestinal pseudo-obstruction was the initial manifestation of systemic sclerosis. Gastrointestinal symptoms and skin changes resolved or improved in both children following treatment with prednisone and penicillamine (case 1) or methotrexate (case 2), although radiological changes of the gastrointestinal tract persisted at 3 and 2 yr of follow-up, respectively.      

Interleukin-10 promoter polymorphisms in rheumatoid arthritis and Felty's syndrome

OBJECTIVE: To examine whether promoter polymorphisms associated with variation in interleukin-10 (IL-10) production are relevant to the development of rheumatoid arthritis (RA) or Felty's syndrome (FS). METHODS: DNA was obtained from 44 FS patients, 117 RA patients and 295 controls. The promoter region between -533 and - 1120 was amplified by polymerase chain reaction, and polymorphisms detected by restriction enzyme digest or sequence-specific oligonucleotide probing. RESULTS: We found no significant difference in allele or haplotype frequencies between the groups. CONCLUSION: There is no association between FS or RA and these recently identified IL-10 promoter polymorphisms. Other genetic or environmental factors could explain the alterations in IL-10 levels seen in these conditions.      

Time course of the interaction between tadalafil and nitrates

OBJECTIVES: This study was designed to determine the time course of nitrate interaction with tadalafil, a phosphodiesterase 5 (PDE5) inhibitor with a half-life (t(1/2)) of 17.5 h. BACKGROUND: The PDE5 inhibitors augment the blood pressure (BP)-lowering effects of nitrates, yet the time course of this interaction is unclear. Recent guidelines from the American College of Cardiology/American Heart Association recommend that nitrates be withheld for 24 h after taking sildenafil (t(1/2) = 4 h). METHODS: Male subjects (n = 150) received seven consecutive daily doses of placebo or tadalafil (20 mg). On day 7 and beyond, subjects received repeated doses of sublingual nitroglycerin (0.4 mg) after the last dose of placebo or tadalafil. After a 10- to 21-day washout period, subjects crossed over to either placebo or tadalafil, and nitrate dosing was repeated. RESULTS: In response to nitroglycerin at 4, 8, and 24 h, standing systolic BP fell below 85 mm Hg in more subjects on tadalafil compared with placebo (p < 0.05), with no difference in the response to nitroglycerin at 48, 72, and 96 h (p > 0.2). Similar observations were made for standing diastolic BP <45 mm Hg, decrease in systolic BP >30 mm Hg, and decrease in diastolic BP >20 mm Hg. Nitroglycerin also evoked greater mean maximal decreases in standing systolic BP at 8 and 24 h after taking tadalafil versus placebo (p < 0.02), with no significant difference at 48, 72, or 96 h (p > 0.49). CONCLUSIONS: The hemodynamic interaction between tadalafil and sublingual nitroglycerin lasted 24 h, but was not seen at 48 h and beyond. Similar to other PDE5 inhibitors, tadalafil should not be administered in combination with organic nitrates.     

Ultra‐deep T cell receptor sequencing reveals the complexity and intratumour heterogeneity of T cell clones in renal cell carcinomas

The recognition of cancer cells by T cells can impact upon prognosis and be exploited for immunotherapeutic approaches. This recognition depends on the specific interaction between antigens displayed on the surface of cancer cells and the T cell receptor (TCR), which is generated by somatic rearrangements of TCR α‐ and β‐chains (TCRb). Our aim was to assess whether ultra‐deep sequencing of the rearranged TCRb in DNA extracted from unfractionated clear cell renal cell carcinoma (ccRCC) samples can provide insights into the clonality and heterogeneity of intratumoural T cells in ccRCCs, a tumour type that can display extensive genetic intratumour heterogeneity (ITH). For this purpose, DNA was extracted from two to four tumour regions from each of four primary ccRCCs and was analysed by ultra‐deep TCR sequencing. In parallel, tumour infiltration by CD4, CD8 and Foxp3 regulatory T cells was evaluated by immunohistochemistry and correlated with TCR‐sequencing data. A polyclonal T cell repertoire with 367–16 289 (median 2394) unique TCRb sequences was identified per tumour region. The frequencies of the 100 most abundant T cell clones/tumour were poorly correlated between most regions (Pearson correlation coefficient, –0.218 to 0.465). 3–93% of these T cell clones were not detectable across all regions. Thus, the clonal composition of T cell populations can be heterogeneous across different regions of the same ccRCC. T cell ITH was higher in tumours pretreated with an mTOR inhibitor, which could suggest that therapy can influence adaptive tumour immunity. These data show that ultra‐deep TCR‐sequencing technology can be applied directly to DNA extracted from unfractionated tumour samples, allowing novel insights into the clonality of T cell populations in cancers. These were polyclonal and displayed ITH in ccRCC. TCRb sequencing may shed light on mechanisms of cancer immunity and the efficacy of immunotherapy approaches. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Activated protein C resistance: molecular mechanisms based on studies using purified Gln506-factor V

Gln506-factor V (FV) was purified from plasma of an individual homozygous for an Arg506Gln mutation in FV that is associated with activated protein C (APC) resistance. Purified Gln506-FV, as well as Gln506-FVa generated by either thrombin or FXa, conveyed APC resistance to FV-deficient plasma in coagulation assays. Clotting assay studies also suggested that APC resistance does not involve any abnormality in FV-APC-cofactor activity. In purified reaction mixtures, Gln506-FVa in comparison to normal FVa showed reduced susceptibility to APC, because it was inactivated approximately 10-fold slower than normal Arg506-FVa. It was previously reported that inactivation of normal FVa by APC involves an initial cleavage at Arg506 followed by phospholipid- dependent cleavage at Arg306. Immunoblot and amino acid sequence analyses showed that the 102-kD heavy chain of Gln506-FVa was cleaved at Arg306 during inactivation by APC in a phospholipid-dependent reaction. This reduced but measurable susceptibility of Gln506-FVa to APC inactivation may help explain why APC resistance is a mild risk factor for thrombosis because APC can inactivate both normal FVa and variant Gln506-FVa. In summary, this study shows that purified Gln506- FV can account for APC resistance of plasma because Gln506-FVa, whether generated by thrombin or FXa, is relatively resistant to APC.     

Exploiting PI3K/mTOR signaling to accelerate epithelial wound healing

The molecular circuitries controlling the process of skin wound healing have gained new significant insights in recent years. This knowledge is built on landmark studies on skin embryogenesis, maturation, and differentiation. Furthermore, the identification, characterization, and elucidation of the biological roles of adult skin epithelial stem cells and their influence in tissue homeostasis have provided the foundation for the overall understanding of the process of skin wound healing and tissue repair. Among numerous signaling pathways associated with epithelial functions, the PI3K/Akt/mTOR signaling route has gained substantial attention with the generation of animal models capable of dissecting individual components of the pathway, thereby providing a novel insight into the molecular framework underlying skin homeostasis and tissue regeneration. In this review, we focus on recent findings regarding the mechanisms involved in wound healing associated with the upregulation of the activity of the PI3K/Akt/mTOR circuitry. This review highlights critical findings on the molecular mechanisms controlling the activation of mTOR, a downstream component of the PI3K–PTEN pathway, which is directly involved in epithelial migration and proliferation. We discuss how this emerging information can be exploited for the development of novel pharmacological intervention strategies to accelerate the healing of critical size wounds.