A highly conserved interspecies V(H) in the human genome.

Idiotype conservation between human and mouse antibodies has been observed in association with various infectious and autoimmune diseases. We have isolated a human anti-idiotypic antibody to a mouse monoclonal anti-IgE antibody (BSW17) suggesting a conserved interspecies idiotype associated with an anti-IgE response. To find the homologue of BSW17 in the human genome we applied the guided selection strategy. Combining V(H) of BSW17 with a human V(L) repertoire resulted in three light chains. The three V(L) chains were then combined with a human V(H) repertoire resulting in three clones specific for human IgE. Surprisingly, one clone, Hu41, had the same epitope specificity and functional in vitro activity as BSW17 and V(H) complementarity-determining regions identical with that of BSW17. Real-time PCR analysis confirmed the presence of the Hu41 V(H) sequence in the human genome. These data document the first example of the isolation of a human antibody where high sequence similarity to the original murine V(H) sequence is associated with common antigen and epitope specificity.

Depletion of regulatory T cells augments a vaccine-induced T effector cell response against the liver-stage of malaria but fails to increase memory

Regulatory T cells (T(reg)) have been shown to restrict vaccine-induced T cell responses in different experimental models. In these studies CD4(+)CD25(+) T(reg) were depleted using monoclonal antibodies against CD25, which might also interfere with CD25 on non-regulatory T cell populations and would have no effect on Foxp3(+)CD25(-) T(reg). To obtain more insights in the specific function of T(reg) during vaccination we used mice that are transgenic for a bacterial artificial chromosome expressing a diphtheria toxin (DT) receptor-eGFP fusion protein under the control of the foxp3 gene locus (depletion of regulatory T cell mice; DEREG). As an experimental vaccine-carrier recombinant Bordetella adenylate cyclase toxoid fused with a MHC-class I-restricted epitope of the circumsporozoite protein (ACT-CSP) of Plasmodium berghei (Pb) was used. ACT-CSP was shown by us previously to introduce the CD8+ epitope of Pb-CSP into the MHC class I presentation pathway of professional antigen-presenting cells (APC). Using this system we demonstrate here that the number of CSP-specific T cells increases when T(reg) are depleted during prime but also during boost immunization. Importantly, despite this increase of T effector cells no difference in the number of antigen-specific memory cells was observed.

Autonomic angiotensinergic fibres in the human heart with an efferent sympathetic cophenotype

AIM The autonomic innervation of the heart consists of sympathetic and parasympathetic nerve fibres, and fibres of the intrinsic ganglionated plexus with noradrenaline and acytylcholine as principal neurotransmitters. The fibres co-release neuropeptides to modulate intracardiac neurotransmission by specific presynaptic and postsynaptic receptors. The coexpression of angiotensin II in sympathetic fibres of the human heart and its role are not known so far. METHODS Autopsy specimens of human hearts were studied (n=3; ventricles). Using immunocytological methods, cryostat sections were stained by a murine monoclonal antibody (4B3) directed against angiotensin II and co-stained by polyclonal antibodies against tyrosine hydroxylase, a catecholaminergic marker. Visualisation of the antibodies was by confocal light microscopy or laser scanning microscopy. RESULTS Angiotensin II-positive autonomic fibres with and without a catecholaminergic cophenotype (hydroxylase-positive) were found in all parts of the human ventricles. In the epicardium, the fibres were grouped in larger bundles of up to 100 and more fibres. They followed the preformed anatomic septa and epicardial vessels towards the myocardium and endocardium where the bundles dissolved and the individual fibres spread between myocytes and within the endocardium. Generally, angiotensinergic fibres showed no synaptic enlargements or only a few if they were also catecholaminergic. The exclusively catechalominergic fibres were characterised by multiple beaded synapses. CONCLUSION The autonomic innervation of the human heart contains angiotensinergic fibres with a sympathetic efferent phenotype and exclusively angiotensinergic fibers representing probably afferents. Angiotensinergic neurotransmission may modulate intracardiac sympathetic and parasympathetic activity and thereby influence cardiac and circulatory function.

Molecular profiling of lung adenosquamous carcinoma: hybrid or genuine type?

Lung adenosquamous carcinoma is a particular subtype of non-small cell lung carcinoma that is defined by the coexistence of adenocarcinoma and squamous cell carcinoma components. The aim of this study was to assess the mutational profile in each component of 16 adenosquamous carcinoma samples from a Caucasian population by a combination of next generation sequencing using the cancer hotspot panel as well as the colon and lung cancer panel and FISH. Identified mutations were confirmed by Sanger sequencing of DNA from cancer cells of each component collected by Laser Capture microdissection. Mutations typical for adenocarcinoma as well as squamous cell carcinoma were identified. Driver mutations were predominantly in the trunk suggesting a monoclonal origin of adenosquamous carcinoma. Most remarkably, EGFR mutations and mutations in the PI3K signaling pathway, which accounted for 30% and 25% of tumors respectively, were more prevalent while KRAS mutations were less prevalent than expected for a Caucasian population. Surprisingly, expression of classifier miR-205 was intermediate between that of classical adenocarcinoma and squamous cell carcinoma suggesting that adenosquamous carcinoma is a transitional stage between these tumor types. The high prevalence of therapy-relevant targets opens new options of therapeutic intervention for adenosquamous carcinoma patients.

VE1 immunohistochemistry predicts BRAF V600E mutation status and clinical outcome in colorectal cancer

AIM VE1 is a monoclonal antibody detecting mutant BRAFV600E protein by immunohistochemistry. Here we aim to determine the inter-observer agreement and concordance of VE1 with mutational status, investigate heterogeneity in colorectal cancers and metastases and determine the prognostic effect of VE1 in colorectal cancer patients. METHODS Concordance of VE1 with mutational status and inter-observer agreement were tested on a pilot cohort of colorectal cancers (n = 34), melanomas (n = 23) and thyroid cancers (n = 8). Two prognostic cohorts were evaluated (n = 259, Cohort 1 and n = 226, Cohort 2) by multiple-punch tissue microarrays. VE1 staining on preoperative biopsies (n = 118 patients) was compared to expression in resections. Primary tumors and metastases from 13 patients were tested for VE1 heterogeneity using a tissue microarray generated from all available blocks (n = 100 blocks). RESULTS Inter-observer agreement was 100% (kappa = 1.0). Concordance between VE1 and V600E mutation was 98.5%. Cohort 1: VE1 positivity (seen in 13.5%) was associated with older age (p = 0.0175) and MLH1 deficiency (p < 0.0001). Cohort 2: VE1 positivity (seen in 12.8%) was associated with female gender (p = 0.0016), right-sided tumor location (p < 0.0001), higher tumor grade (p < 0.0001) and mismatch repair (MMR)-deficiency (p < 0.0001). In survival analysis, MMR status and postoperative therapy were identified as possible confounding factors. Adjusting for these features, VE1 was an unfavorable prognostic factor. Preoperative biopsy staining matched resections in all cases except one. No heterogeneity was found across any primary/metastatic tumor blocks. CONCLUSION VE1 is highly concordant for V600E and homogeneously expressed suggesting staining can be analysed on resection specimens, preoperative biopsies, metastatic lesions and tissue microarrays.

Circulating oxidized low-density lipoprotein (LDL) is associated with risk factors of the metabolic syndrome and LDL size in clinically healthy 58-year-old men (AIR study).

OBJECTIVES Hypothetically the atherogenic effect of the metabolic syndrome may be mediated through the increased occurrence of small LDL-particles which are easily modified to atherogenic oxidized LDL (ox-LDL). The aim of this study was to test this concept by examining the association between circulating ox-LDL, LDL-particle size, and the metabolic syndrome. DESIGN AND RESULTS A population-based sample of clinically healthy 58-year-old men (n = 391) was recruited. Ox-LDL was measured by ELISA (specific monoclonal antibody, mAb-4E6) and LDL-particle size by gradient gel electrophoresis. The results showed that ox-LDL significantly correlated to factors constituting the metabolic syndrome; triglycerides (r = 0.43), plasma insulin (r = 0.20), body mass index (r = 0.20), waist-to-hip ratio (r = 0.21) and HDL (r = -0.24); (P < 0.001). Ox-LDL correlated also to LDL-particle size (r = -0.42), Apo-B (r = 0.70), LDL (r = 0.65); (P < 0.001) and, furthermore, with Apo A-1 (r = -0.13) and heart rate (r = 0.13); (P < 0.01). CONCLUSION The metabolic syndrome was accompanied by high plasma ox-LDL concentrations compared with those without the syndrome. Ox-LDL levels were associated with most of the risk factors constituting the metabolic syndrome and was, in addition related to small LDL-particle size. To our knowledge the present study is the first one to demonstrate that circulating ox-LDL levels are associated with small LDL-particle size in a population representative sample of clinically healthy middle-aged men. The high degree of intercorrelation amongst several factors makes it difficult to clarify the independent role of any specific factor.

Circulating oxidized LDL is associated with the occurrence of echolucent plaques in the carotid artery in 61-year-old men.

OBJECTIVE The aim of this study was to elucidate the relationship between the echogenicity of carotid artery plaques and the following risk factors: circulating oxLDL, hsCRP, the metabolic syndrome (MetS), and several of the traditional cardiovascular (CV) risk factors. MATERIAL AND METHODS A cross-sectional population-based study of 513 sixty-one-year-old men. The levels of circulating oxLDL were determined in plasma samples by sandwich ELISA utilizing a specific murine monoclonal antibody (mAb-4E6). High-sensitivity CRP was measured in plasma by ELISA. Plaque occurrence, size and echogenicity were evaluated from B-mode ultrasound registrations in the carotid arteries. Plaque echogenicity was assessed based on a four-graded classification scale. RESULTS A higher frequency of echolucent carotid plaques was observed with increasing levels of oxLDL and systolic blood pressure (p = 0.008 and p = 0.041, respectively). Subjects with the MetS had a significantly higher frequency of echogenic plaques than subjects without the MetS (p = 0.009). In a multiple logistic regression analysis, oxLDL turned out to be independently associated with echolucent carotid plaques. CONCLUSIONS The occurrence of echolucent carotid plaques was associated with oxLDL and systolic blood pressure, and oxLDL was associated with echolucent carotid plaques independently of systolic blood pressure.