Molecular alterations of Ras‐Raf‐mitogen‐activated protein kinase and phosphatidylinositol 3‐kinase‐Akt signaling pathways in colorectal cancers from a tertiary hospital at Kuala Lumpur,Malaysia

Molecular alterations in KRAS, BRAF, PIK3CA, and PTEN have been implicated in designing targeted therapy for colorectal cancer (CRC). The present study aimed to determine the status of these molecular alterations in Malaysian CRCs as such data are not available in the literature. We investigated the mutations of KRAS, BRAF, and PTEN, the gene amplification of PIK3CA, and the protein expression of PTEN and phosphatidylinositol 3‐kinase (PI3K) catalytic subunit (p110α) by direct DNA sequencing, quantitative real‐time PCR, and immunohistochemistry, respectively, in 49 CRC samples. The frequency of KRAS (codons 12, 13, and 61), BRAF (V600E), and PTEN mutations, and PIK3CA amplification was 25.0% (11/44), 2.3% (1/43), 0.0% (0/43), and 76.7% (33/43), respectively. Immunohistochemical staining demonstrated loss of PTEN protein in 54.5% (24/44) of CRCs and no significant difference in PI3K p110α expression between CRCs and the adjacent normal colonic mucosa (p = 0.380). PIK3CA amplification was not associated with PI3K p110α expression level, but associated with male cases (100% of male cases vs 56% of female cases harbored amplified PIK3CA, p = 0.002). PI3K p110α expression was significantly higher (p = 0.041) in poorly/moderately differentiated carcinoma compared with well‐differentiated carcinoma. KRAS mutation, PIK3CA amplification, PTEN loss, and PI3K p110α expression did not correlate with Akt phosphorylation or Ki‐67 expression. KRAS mutation, PIK3CA amplification, and PTEN loss were not mutually exclusive. This is the first report on CRC in Malaysia showing comparable frequency of KRAS mutation and PTEN loss, lower BRAF mutation rate, higher PIK3CA amplification frequency, and rare PTEN mutation, as compared with published reports.     

Expression of miR‐155 and miR‐126 in situ in cutaneous T‐cell lymphoma

Recently, miR‐155 has been implicated in cutaneous T‐cell lymphoma (CTCL). Thus, elevated levels of miR‐155 were observed in skin lesions from CTCL patients as judged from qPCR and micro‐array analysis and aberrant, high miR‐155 expression was associated with severe disease. Moreover, miR‐155 promoted proliferation of malignant T cells in vitro. Little is, however, known about which cell types express miR‐155 in vivo in CTCL skin lesions. Here, we study miR‐155 expression using in situ hybridization (ISH) with a miR‐155 probe, a negative control (scrambled), and a miR‐126 probe as a positive control in nine patients with mycosis fungoides, the most frequent subtype of CTCL. We provide evidence that both malignant and non‐malignant T cells stain weakly to moderately positive with the miR‐155 probe, but generally negative with the miR‐126 and negative control probes. Reversely, endothelial cells stain positive for miR‐126 and negative for miR‐155 and the control probe. Solitary T cells with a malignant morphology display brighter staining with the miR‐155 probe. Taken together, our findings suggest that both malignant and non‐malignant T cells express miR‐155 in situ in CTCL. Moreover, they indicate heterogeneity in miR‐155 expression among malignant T cells.     

Tandem pore domain K+‐channel TASK‐3 (KCNK9) and idiopathic absence epilepsies

Recently, the gene coding for the tandem pore domain K⁺‐channel TASK‐3 (KCNK9) has been localized to the chromosomal region 8q24. Because mutations in ion channel genes have been recognized as an important factor in the etiology of abnormal neuronal excitability, TASK‐3 is an interesting candidate gene for epilepsies linked to 8q24. We therefore performed a mutation analysis of the TASK‐3 gene in 65 patients with childhood and juvenile absence epilepsy. Only one silent nucleotide exchange (636C/T) was detected in exon 2 of the TASK‐3 coding region. No evidence for an allelic association was found between the exon 2 polymorphism and absence epilepsy. Accordingly, genetic variation of the TASK‐3 coding region does not play a major role in the etiology of idiopathic absence epilepsies. © 2002 Wiley‐Liss, Inc.     

Amomum tsao‐ko fruit extract suppresses lipopolysaccharide‐induced inducible nitric oxide synthase by inducing heme oxygenase‐1 in macrophages and in septic mice

Amomum tsao‐ko Crevost et Lemarié (Zingiberaceae) has traditionally been used to treat inflammatory and infectious diseases, such as throat infections, malaria, abdominal pain and diarrhoea. This study was designed to assess the anti‐inflammatory effects and the molecular mechanisms of the methanol extract of A. tsao‐ko (AOM) in lipopolysaccharide (LPS)‐induced RAW 264.7 macrophages and in a murine model of sepsis. In LPS‐induced RAW 264.7 macrophages, AOM reduced the production of nitric oxide (NO) by inhibiting inducible nitric oxide synthase (iNOS) expression, and increased heme oxygenase‐1 (HO‐1) expression at the protein and mRNA levels. Pretreatment with SnPP (a selective inhibitor of HO‐1) and silencing HO‐1 using siRNA prevented the AOM‐mediated inhibition of NO production and iNOS expression. Furthermore, AOM increased the expression and nuclear accumulation of NF‐E2‐related factor 2 (Nrf2), which enhanced Nrf2 binding to antioxidant response element (ARE). In addition, AOM induced the phosphorylation of extracellular regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) and generated reactive oxygen species (ROS). Furthermore, pretreatment with N‐acetyl‐l ‐cysteine (NAC; a ROS scavenger) diminished the AOM‐induced phosphorylation of ERK and JNK and AOM‐induced HO‐1 expression, suggesting that ERK and JNK are downstream mediators of ROS during the AOM‐induced signalling of HO‐1 expression. In LPS‐induced endotoxaemic mice, pretreatment with AOM reduced NO serum levels and liver iNOS expression and increased HO‐1 expression and survival rates. These results indicate that AOM strongly inhibits LPS‐induced NO production by activating the ROS/MAPKs/Nrf2‐mediated HO‐1 signalling pathway, and supports its pharmacological effects on inflammatory diseases.     

A randomized,double‐blind,placebo‐controlled,dose‐finding trial with Lolium perenne peptide immunotherapy

Background

A novel subcutaneous allergen immunotherapy formulation (gpASIT+?) containing Lolium perenne peptides (LPP) and having a short up‐dosing phase has been developed to treat grass pollen–induced seasonal allergic rhinoconjunctivitis. We investigated peptide immunotherapy containing the hydrolysate from perennial ryegrass allergens for the optimum dose in terms of clinical efficacy, immunogenicity and safety.

Methods

This prospective, double‐blind, placebo‐controlled, phase IIb, parallel, four‐arm, dose‐finding study randomized 198 grass pollen–allergic adults to receive placebo or cumulative doses of 70, 170 or 370 μg LPP. All patients received weekly subcutaneous injections, with the active treatment groups reaching assigned doses within 2, 3 and 4 weeks, respectively. Efficacy was assessed by comparing conjunctival provocation test (CPT) reactions at baseline, after 4 weeks and after completion. Grass pollen–specific immunoglobulins were analysed before and after treatment.

Results

Conjunctival provocation test (CPT) response thresholds improved from baseline to V7 by at least one concentration step in 51.2% (170 μg; P = .023), 46.3% (370 μg), and 38.6% (70 μg) of patients receiving LPP vs 25.6% of patients receiving placebo (modified per‐protocol set). Also, 39% of patients in the 170‐μg group became nonreactive to CPT vs 18% in the placebo group. Facilitated allergen‐binding assays revealed a highly significant (P < .001) dose‐dependent reduction in IgE allergen binding across all treatment groups (70 μg: 17.1%; 170 μg: 18.8%; 370 μg: 26.4%). Specific IgG₄ levels increased to 1.6‐fold (70 μg), 3.1‐fold (170 μg) and 3.9‐fold (370 μg) (mPP).

Conclusion

Three‐week immunotherapy with 170 μg LPP reduced CPT reactivity significantly and increased protective specific antibodies.

     

Characterization of CTX‐M‐producing Escherichia coli by repetitive sequence‐based PCR and real‐time PCR‐based replicon typing of CTX‐M‐15 plasmids

The emergence of extended‐spectrum β‐lactamase (ESBL)‐producing Enterobacteriaceae is a major global concern. CTX‐M is the dominating ESBL type worldwide, and CTX‐M‐15 is the most widespread CTX‐M type. The dissemination of CTX‐M appears to be in part due to global spread of the Escherichia coli clone O25b‐ST131. However, the gene‐encoding CTX‐M is mainly located on mobile genetic elements, such as plasmids, that also promote the horizontal dissemination of the CTX‐M genes. In this study, 152 CTX‐M‐producing E. coli isolated in 1999–2008 in Örebro County, Sweden, were typed using a commercial repetitive sequence‐based PCR (the DiversiLab system), and the prevalence of ST131 was investigated by pabB PCR. Real‐time PCR‐based plasmid replicon typing was performed on 82 CTX‐M‐15‐producing E. coli isolates. In general, the CTX‐M‐producing E. coli population was genetically diverse; however, ST131 was highly prevalent (27%), and the dominating clone in our area. The bla_CTX‐M‐15 gene was mainly located on IncF plasmids (69%), but a relatively high proportion of IncI1 plasmids (29%) were also detected among E. coli with diverse rep‐PCR patterns, indicating that horizontal transmission of IncI1 plasmids carrying bla_CTX‐M‐15 may have occurred between different E. coli strains.     

SPINK5 is associated with early‐onset and CHI3L1 with late‐onset atopic dermatitis

We have recently showed that filaggrin (FLG) mutations are associated only with early‐onset of AD, but not with late‐onset of AD. Consequently, other susceptibility genes should receive attention, especially in patients with late‐onset of AD. Our aim was to assess the associations between development of AD and the polymorphisms rs2303067 in SPINK5 and rs490928 in CHI3L1. A study population of 241 AD patients and 164 healthy controls was genotyped for two polymorphisms (rs2303067 in SPINK5 and rs490928 in CHI3L1). Rs2303067 in SPINK5 was significantly associated with early‐onset AD (≤8 years: p = .003; OR = 2.57) and was characterized by the need for hospitalization (p = .006; OR = 2.76), prolonged duration (≥10 years; p = .008; OR = 2.32) and more body parts affected (p = .015; OR = 2.01). In contrast, rs490928 in CHI3L1 was associated with late‐onset AD (>8 years: p = .048; OR = 1.65) and was characterized by no need for hospitalization (p = .049; OR = 1.59), shorter duration (<10 years; p = .017; OR = 1.94) and fewer body parts affected (p = .049; OR = 1.75). Our results confirmed that different AD phenotypes, specifically early‐ and late‐onset AD, have different genetic backgrounds. Early‐onset AD was associated with rs2303067 in SPINK5, which is involved in skin barrier functioning, and late‐onset was associated with rs4950928 in CHI3L1, which is involved in the immune response. Future studies should examine the early‐ versus late‐onset subgrouping more closely.     

Posttraumatic Stress Disorder in DSM‐5: New Criteria and Controversies

The fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM‐5; American Psychiatric Association, 2013) featured extensive changes to the posttraumatic stress disorder (PTSD) diagnosis. PTSD was moved out of the anxiety disorders into a new class of “trauma‐ and stressor‐related disorders,” and the definition of what constitutes a traumatic experience was revised. Three new symptoms were added, existing ones were modified, and a new four‐cluster organization and diagnostic algorithm were introduced. Finally, a new dissociative subtype was added to the diagnosis. We review these changes, discuss some of the controversies surrounding them, and then introduce a new debate involving a radically different conceptualization of PTSD proposed for International Classification of Diseases, 11th edition.