NF‐κB‐inducing kinase is a key regulator of inflammation‐induced and tumour‐associated angiogenesis

Angiogenesis is essential during development and in pathological conditions such as chronic inflammation and cancer progression. Inhibition of angiogenesis by targeting vascular endothelial growth factor (VEGF) blocks disease progression, but most patients eventually develop resistance which may result from compensatory signalling pathways. In endothelial cells (ECs), expression of the pro‐angiogenic chemokine CXCL12 is regulated by non‐canonical nuclear factor (NF)‐κB signalling. Here, we report that NF‐κB‐inducing kinase (NIK) and subsequent non‐canonical NF‐κB signalling regulate both inflammation‐induced and tumour‐associated angiogenesis. NIK is highly expressed in endothelial cells (ECs) in tumour tissues and inflamed rheumatoid arthritis synovial tissue. Furthermore, non‐canonical NF‐κB signalling in human microvascular ECs significantly enhanced vascular tube formation, which was completely blocked by siRNA targeting NIK. Interestingly, Nik^?/? mice exhibited normal angiogenesis during development and unaltered TNFα‐ or VEGF‐induced angiogenic responses, whereas angiogenesis induced by non‐canonical NF‐κB stimuli was significantly reduced. In addition, angiogenesis in experimental arthritis and a murine tumour model was severely impaired in these mice. These studies provide evidence for a role of non‐canonical NF‐κB signalling in pathological angiogenesis, and identify NIK as a potential therapeutic target in chronic inflammatory diseases and tumour neoangiogenesis. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

In vitro responses to electrosprayed alkaline phosphatase/calcium phosphate composite coatings

Surface modification of titanium implants to improve their fixation in bone tissue is of great interest. We present a novel approach to enhance implant performance by applying important principles of bone mineralization to biomedical coatings. As an attempt to mimic the biphasic biomineralization process, both the enzyme alkaline phosphatase (ALP) and calcium phosphate (CaP) were immobilized onto Ti discs, thereby triggering enzymatically and physicochemically controlled biomineralization pathways. ALP, CaP and ALP–CaP composite coatings with preserved functionality of ALP were successfully deposited using electrospray deposition. In vitro soaking studies in cell culture medium revealed that crystal growth initially proceeded at a faster rate on CaP-coated Ti than on ALP-containing coatings, but mineral deposition onto ALP-coated Ti caught up with the calcification behaviour of CaP coatings upon long-term soaking. Cell culture experiments with osteoblast-like cells, however, demonstrated the opposite effect in mineral deposition on the electrosprayed CaP and ALP coatings. The ALP–CaP composite coatings showed delayed proliferation as well as accelerated mineralization in comparison to cells cultured on the CaP-coated and uncoated Ti. In conclusion, these in vitro results showed that the osteogenic potential of Ti can be stimulated by ALP-containing coatings.     

Linkage and association analysis of CACNG3 in childhood absence epilepsy

Childhood absence epilepsy (CAE) is an idiopathic generalised epilepsy characterised by absence seizures manifested by transitory loss of awareness with 2.5-4 Hz spike-wave complexes on ictal EEG. A genetic component to aetiology is established but the mechanism of inheritance and the genes involved are not fully defined. Available evidence suggests that genes encoding brain expressed voltage-gated calcium channels, including CACNG3 on chromosome 16p12-p13.1, may represent susceptibility loci for CAE. The aim of this work was to further evaluate CACNG3 as a susceptibility locus by linkage and association analysis. Assuming locus heterogeneity, a significant HLOD score (HLOD = 3.54, alpha = 0.62) was obtained for markers encompassing CACNG3 in 65 nuclear families with a proband with CAE. The maximum non-parametric linkage score was 2.87 (P < 0.002). Re-sequencing of the coding exons in 59 patients did not identify any putative causal variants. A linkage disequilibrium (LD) map of CACNG3 was constructed using 23 single nucleotide polymorphisms (SNPs). Transmission disequilibrium was sought using individual SNPs and SNP-based haplotypes with the pedigree disequilibrium test in 217 CAE trios and the 65 nuclear pedigrees. Evidence for transmission disequilibrium (P < or = 0.01) was found for SNPs within a approximately 35 kb region of high LD encompassing the 5'UTR, exon 1 and part of intron 1 of CACNG3. Re-sequencing of this interval was undertaken in 24 affected individuals. Seventy-two variants were identified: 45 upstream; two 5'UTR; and 25 intronic SNPs. No coding sequence variants were identified, although four variants are predicted to affect exonic splicing. This evidence supports CACNG3 as a susceptibility locus in a subset of CAE patients.     

An improved protocol for generation of immuno-potent dendritic cells through direct electroporation of CD14+ monocytes

In this study we demonstrate a novel protocol showing that electroporation of CD14+ monocytes directly isolated from blood with green fluorescent protein (GFP) RNA results in a 3-fold higher yield of antigen presenting dendritic cells (DCs) when compared to conventional methods employing immature DCs for electroporation. We further show a stable electroporation efficacy resulting in 60% of GFP positive cells. Expression of co-stimulatory molecules and maturation markers such as CD80, CD86, CD83 as well of the chemokine receptor 7 (CCR7) was found in 90% of the mature DCs. Importantly, production of IL-12p70 was 10 times higher in cells electroporated at the monocyte stage compared to cells electroporated at the immature DC stage. Stimulation of autologous na?ve lymphocytes by DCs electroporated at monocytes stage elicited proliferation of CD8+ T-cell with 7-fold increase in IFN-gamma release. Blocking of the MHC-Class I molecules significantly inhibited the IFN-gamma release, indicating that antigen presentation was MHC-Class I mediated. In summary, electroporation of CD14+ monocytes with RNA results in a high yield of antigen presenting DCs with high immuno-stimulatory capacity and antigen presentation on MHC-Class I molecules. This improved method may represent an attractive approach for RNA-based DC immunotherapy.     

Pregnant couples at increased risk for common aneuploidies choose maximal information from invasive genetic testing

Genomic array detects more pathogenic chromosome aberrations than conventional karyotyping (CK), including genetic variants associated with a susceptibility for neurodevelopmental disorders; susceptibility loci (SL). Consensus regarding the scope of invasive prenatal diagnosis (PND) pregnant couples should be offered is lacking. This study examined pregnant couples' preferences, doubts and satisfaction regarding the scope of invasive PND. Eighty‐two couples choosing prenatal screening (PNS) and 59 couples choosing invasive PND were offered a choice between 5 (comparable to CK) and 0.5 Mb resolution array analysis outcomes, the latter with or without reporting SL. A pre‐test self‐report questionnaire and post‐test telephone interview assessed their choices in‐depth. Actual (PND) and hypothetical (PNS) choices differed significantly (p < 0.001). Ninety‐five percent of the couples in the PND group chose 0.5 Mb array, vs 69% in the PNS group. Seven percent of the PND group wished not to be informed of SL. Ninety percent was satisfied with their choice and wished to decide about the scope themselves. Pregnant couples wish to make their own choices regarding the scope of invasive PND. It therefore seems justified to offer them a choice in both the resolution of array and disclosure of SL.     

Differences in complexity of isolated brachydactyly type C cannot be attributed to locus heterogeneity alone

Hereditary isolated brachydactyly type C (OMIM 113100) mostly follows an autosomal dominant pattern of inheritance with a marked variability in expression. This phenotype has been mapped to two different loci on chromosomes 12q24 and 20q11.2. The latter locus contains the cartilage-derived morphogenetic protein (CDMP)1 gene, in which a null mutation has been found in patients with malformations restricted to the upper limbs. A more complex brachydactyly type C phenotype has been mapped to chromosome 12q24. Differences in complexity of these phenotypes have been attributed to locus heterogeneity. Clinical subclassification based on the degree of complexity of the phenotype has therefore been suggested. We present patients with a complex brachydactyly type C phenotype in whom there is considerable intra- and interfamilial variability in expression. We show that clinical subclassification based on the complexity of the brachydactyly type C phenotype related to the genetic defect is not feasible. We present evidence that differences in complexity are not only due to locus heterogeneity, but that genetic modifiers and/or environmental factors must also play a role.     

Expression of killer cell inhibitory receptors is restricted to true NK cell lymphomas and a subset of intestinal enteropathy-type T cell lymphomas with a cytotoxic phenotype

BACKGROUND/AIMS: Killer inhibitory receptors (KIR) have a modulating effect on the cytotoxic functions of natural killer (NK) cells and T cells. Because lymphoma cells often have the same receptors as their non-neoplastic counterparts, this study investigated the expression of KIR on well defined groups of NK and T cell lymphomas, with and without a cytotoxic phenotype, from different sites of origin. METHODS: Nine CD56+/CD3- NK cell lymphomas, 29 CD3+/CD56- T cell lymphomas with a cytotoxic phenotype, and 19 T cell lymphomas without a cytotoxic phenotype were stained for KIR using monoclonal antibodies specific for CD94, CD158a, and CD158b. In addition, the expression of KIR was studied on normal lymphoid tissues. RESULTS: KIR expression was seen in five of nine true NK cell lymphomas including three of four nasal, one of four cutaneous, and one of one intestinal lymphoma nasal type. Double staining for CD56 and CD94 in normal lymphoid tissues revealed that KIR was predominantly expressed by CD56+ NK cells and sporadically on CD8+ T cells. Moreover, enteropathy-type T cell lymphomas with a cytotoxic phenotype showed KIR expression (three cases expressing CD94 and one case expressing CD158a). All nodal and extranodal nonintestinal T cell lymphomas with or without a cytotoxic phenotype lacked expression of KIR. CONCLUSIONS: These results show that KIR expression is restricted to CD56+/CD3- true NK cell lymphomas originating from the nose, gut, and skin, as well as in a subset of extranodal T cell lymphomas originating from the small intestine, which possessed a cytotoxic phenotype. Thus, the presence of KIR on NK/T cell lymphomas seems to mimic the distribution of KIR found on NK and T cells in normal lymphoid tissue.     

Identification of wheat flour allergens by means of 2-dimensional immunoblotting

BACKGROUND: Wheat flour proteins are allergens for 60% to 70% of bakers with workplace-related respiratory symptoms. OBJECTIVE: The aim of the study was to investigate the variability of IgE antibody patterns of wheat flour-sensitized bakers and to identify the most frequently recognized allergens. METHODS: Water/salt-soluble wheat flour proteins from the cultivar Bussard were separated by using 2-dimensional gel electrophoresis with immobilized pH gradients. IgE-reactive proteins were identified by means of immunoblotting with sera of 10 subjects with baker's asthma. Mass spectrometric fingerprinting was used to identify the proteins most frequently recognized by IgE. RESULTS: The IgE immunoblots obtained with 10 different sera exhibited a remarkable heterogeneity. Each patient showed an individual IgE-binding pattern with 4 to 50 different allergen spots. Altogether, more than 100 IgE-binding protein spots were detected. Nine of the predominant IgE-binding protein spots were identified by using mass spectrometric fingerprinting. The obtained masses matched 2 different isoforms of glycerinaldehyde-3-phosphate dehydrogenase from Hordeum vulgare, triosephosphate isomerase from H vulgare, and serpin, a serine proteinase inhibitor from Triticum aestivum. CONCLUSIONS: The results show a great interindividual variation of IgE-binding patterns of wheat flour proteins in baker's asthma. The clinical relevance of the identified 4 new allergens will be further investigated in the near future.     

Mycobacterium bovis BCG recA deletion mutant shows increased susceptibility to DNA-damaging agents but wild-type survival in a mouse infection model

Pathogenic microorganisms possess antioxidant defense mechanisms for protection from reactive oxygen metabolites which are generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxifies reactive oxygen species, and DNA repair systems, which repair damage resulting from oxidative stress. To (i) determine the relative importance of the DNA repair system when oxidative stress is encountered by the Mycobacterium tuberculosis complex during infection of the host and to (ii) provide improved mycobacterial hosts as live carriers to express foreign antigens, the recA locus was inactivated by allelic exchange in Mycobacterium bovis BCG. The recA mutants are sensitive to DNA-damaging agents and show increased susceptibility to metronidazole, the first lead compound active against the dormant M. tuberculosis complex. Surprisingly, the recA genotype does not affect the in vitro dormancy response, nor does the defect in the DNA repair system lead to attenuation as determined in a mouse infection model. The recA mutants will be a valuable tool for further development of BCG as an antigen delivery system to express foreign antigens and as a source of a genetically stable vaccine against tuberculosis.     

Molecular analysis of T-cell clonality with concomitant specific T-cell proliferation in vitro in nickel-allergic individuals

BACKGROUND: The peripheral blood mononuclear cells (PBMC) of individuals with nickel contact allergy are reported to proliferate to a varying degree upon nickel stimulation in vitro. Different phenotypes of the T cells involved are described. With regard to preferential use of the T-cell receptor (TCR), analysis of the several families of the TCR-gamma gene allows rearrangement evaluation of all T cells regardless of predominant surface expression of TCR alpha/beta. METHODS: The PBMC of 10 nickel-allergic and five nonallergic individuals were cultured for 4 days in the presence of either medium, PHA, NiSO4, or tetanus toxoid (TT). Proliferation was measured by radioactive thymidine uptake and expressed as stimulation index (SI). T-cell clonality was assessed by analysis of the TCR-gamma chain gene, including the use of PCR with a primer combination covering the four main groups (Vgamma1-8, Vgamma9, Vgamma10, and Vgamma11) of the variable region of the TCR-beta chain gene. RESULTS: In the allergic individuals, proliferation to NiSO4 was significantly (P<0.05) higher than in nonallergics (mean SI: 18.05/17.87 vs 0.67/2.27). In unstimulated and PHA-stimulated cultures, there was a random TCR spectrum in both groups. In contrast, in nickel-allergic individuals or individuals with recent TT-booster, oligoclonality could be observed in the correspondingly stimulated cultures. CONCLUSION: In addition to proliferation assay, analysis of T-cell clonality may be a further means to characterize clinical hypersensitivity reactions on the basis of antigen-dependent oligoclonal T-cell expansion, as in the case of tissue-infiltrating lymphocytes.