Functional relevance of NLRP3 inflammasome-mediated interleukin (IL)-1ß during acute allergic airway inflammation.

Overall asthmatic symptoms can be controlled with diverse therapeutic agents. However, certain symptomatic individuals remain at risk for serious morbidity and mortality, which prompts the identification of novel therapeutic targets and treatment strategies. Thus, using an adjuvant-free T helper type 2 (Th2) murine model, we have deciphered the role of interleukin (IL)-1 signalling during allergic airway inflammation (AAI). Because functional IL-1ß depends on inflammasome activation we first studied asthmatic manifestations in specific inflammasome-deficient [NACHT, LRR and PYD domains-containing protein 3 (NLRP3(-/-) ) and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC(-/-) )] and IL-1 receptor type 1(-/-) (IL-1R1(-/-) ) mice on the BALB/c background. To verify the onset of disease we assessed cellular infiltration in the bronchial regions, lung pathology, airway hyperresponsiveness and ovalbumin (OVA)-specific immune responses. In the absence of NLRP3 inflammasome-mediated IL-1ß release all symptoms of AAI were reduced, except OVA-specific immunoglobulin levels. To address whether manipulating IL-1 signalling reduced asthmatic development, we administered the IL-1R antagonist anakinra (Kineret®) during critical immunological time-points: sensitization or challenge. Amelioration of asthmatic symptoms was only observed when anakinra was administered during OVA challenge. Our findings indicate that blocking IL-1 signalling could be a potential complementary therapy for allergic airway inflammation.

Farm-derived Gram-positive bacterium Staphylococcus sciuri W620 prevents asthma phenotype in HDM- and OVA-exposed mice.

BACKGROUND: Farm-derived dust samples have been screened for bacteria with potential allergo-protective properties. Among those was Staphylococcus sciuri W620 (S. sciuri W620), which we tested with regard to its protective capacities in murine models of allergic airway inflammation. METHODS: We employed two protocols of acute airway inflammation in mice administering either ovalbumin (OVA) or house dust mite extract (HDM) for sensitization. Mechanistic studies on the activation of innate immune responses to S. sciuri W620 were carried out using human primary monocytic dendritic cells (moDC) and co-culture with autologous T cells. RESULTS: The allergo-protective properties of S. sciuri W620 were proven in a T(H)2-driven OVA model as well as in a mixed T(H)1/T(H)2 phenotype HDM model as demonstrated by abrogation of eosinophils and neutrophils in the airways after intranasal treatment. In the HDM model, lymph node cell T(H)1/T(H)2 signature cytokines were decreased in parallel. Studies on human moDC revealed an activation of TLR2 and NOD2 receptors and initiation of DC maturation following incubation with S. sciuri W620. Cytokine expression analyses after exposure to S. sciuri W620 showed a lack of IL-12 production in moDC due to missing transcription of the IL-12p35 mRNA. However, such DC selectively supported T(H)1 cytokine release by co-cultured T cells. CONCLUSION AND CLINICAL RELEVANCE: Our proof-of-concept experiments verify the screening system of farm-derived dust samples as suitable to elucidate new candidates for allergo-protection. S. sciuri W620 was shown to possess preventive properties on airway inflammation providing the basis for further mechanistic studies and potential clinical implication.

Suppression of adrenomedullin contributes to vascular leakage and altered epithelial repair during asthma.

BACKGROUND: The anti-inflammatory peptide, adrenomedullin (AM), and its cognate receptor are expressed in lung tissue, but its pathophysiological significance in airway inflammation is unknown. OBJECTIVES: This study investigated whether allergen-induced airway inflammation involves an impaired local AM response. METHODS: Airway AM expression was measured in acute and chronically sensitized mice following allergen inhalation and in airway epithelial cells of asthmatic and nonasthmatic patients. The effects of AM on experimental allergen-induced airway inflammation and of AM on lung epithelial repair in vitro were investigated. RESULTS: Adrenomedullin mRNA levels were significantly (P < 0.05) reduced in acute ovalbumin (OVA)-sensitized mice after OVA challenge, by over 60% at 24 h and for up to 6 days. Similarly, reduced AM expression was observed in two models of chronic allergen-induced inflammation, OVA- and house dust mite-sensitized mice. The reduced AM expression was restricted to airway epithelial and endothelial cells, while AM expression in alveolar macrophages was unaltered. Intranasal AM completely attenuated the OVA-induced airway hyperresponsiveness and mucosal plasma leakage but had no effect on inflammatory cells or cytokines. The effects of inhaled AM were reversed by pre-inhalation of the putative AM receptor antagonist, AM ((22-52)) . AM mRNA levels were significantly (P < 0.05) lower in human asthmatic airway epithelial samples than in nonasthmatic controls. In vitro, AM dose-dependently (10(-11) -10(-7) M) accelerated experimental wound healing in human and mouse lung epithelial cell monolayers and stimulated epithelial cell migration. CONCLUSION: Adrenomedullin suppression in T(H) 2-related inflammation is of pathophysiological significance and represents loss of a factor that maintains tissue integrity during inflammation.

Bayesian analysis of new and old malaria parasite DNA sequence data demonstrates the need for more phylogenetic signal to clarify the descent of Plasmodium falciparum.

Molecular systematic studies published during the last 15 years to clarify the phylogenetic relationships among the malaria parasites have led to two major hypotheses on the descent of Plasmodium falciparum: One supports an avian origin as a result of a relatively recent host switch, and another one favours the evolutionary development of P. falciparum together with its human host from primate ancestors. In this paper, we present phylogenetic analyses of three different Plasmodium genes, the nuclear 18 small sub-unit (SSU) ribosomal ribonucleic acid (rRNA), the mitochondrial cytochrome b (cyt b) and the plastid caseinolytic protease C (ClpC) gene, using numerous haemosporidian parasite DNA sequences obtained from the GenBank as well as several new sequences for major malaria parasites including the avian one Plasmodium cathemerium, which has never been considered in molecular phylogenetic analyses before. Most modern and sophisticated DNA substitution models based on Bayesian inference analysis were applied to estimate the cyt b and ClpC phylogenetic trees, whereas the 18 SSU rRNA gene was examined with regards to its secondary structure using PHASE software. Our results indicate that the data presently available are generally neither sufficient in number nor in information to solve the problem of the phylogenetic origin of P. falciparum.

Expression of calcitonin receptor-like receptor in human vascular tumours.

BACKGROUND: Vascular tumours such as Kaposi's sarcoma and capillary haemangioma are characterised by abnormal vascularisation and proliferation of endothelial cells or neoplastic cells. Adrenomedullin, a potent vasodilative peptide, and its receptor, calcitonin receptor-like receptor (CRLR), play an important part in angiogenesis. AIM: To establish whether this system also plays a part in vascular diseases, showing abnormal proliferation such as vascular tumours. METHODS: CRLR expression was investigated in several specimens of Kaposi's sarcoma and other vascular tumours, using immunohistochemical analysis with a previously described CRLR-specific polyclonal antibody and reverse transcriptase-polymerase chain reaction. RESULTS: Intense and specific CRLR-immunoreactive staining of neoplastic cells was observed in all specimens, which was of greater intensity than similar staining of adjacent normal endothelium. CONCLUSIONS: CRLR is expressed in vascular tumours and, with adrenomedullin, may have a role in neoplastic vascular growth.

Two novel cell specific receptor proteins, CRLR and CD 117 in human glial tumors.

OBJECTIVE: CRLR (calcitonin receptor-like receptor) and CD 117, the gene product of c-kit have been shown to be expressed in cells of glial tumors, especially in those with higher malignancy. Here we report the distribution of these peptides in various cellular compartments within those tumors. MATERIAL: Both receptor proteins have been investigated in 95 glial tumor biopsies of different grades. METHODS: Both proteins were visualized by immunohistochemistry with antibodies either commercially available or raised for this purpose. RESULTS: Both receptor peptides can be identified in or around tumor blood vessels. CRLR occurs in some endothelial cells, especially in the microvascular proliferations of glioblastoma multiforme, whereas CD 117 preferentially occurs in cells of the thickened vascular wall within cells of pericyte or fibroblast morphology. Both antigens are found in addition in few neoplastic cells of overt astrocyte morphology. CONCLUSIONS: The occurrence of identical antigens in glial tumor blood vessels and in neighboring tumor cells underlines the common origin of "mesenchymal" and "neuroepithelial" components of such (malignant) glial neoplasms.

The immunohistochemical expression of calcitonin receptor-like receptor (CRLR) in human gliomas.

BACKGROUND: Gliomas are the most common primary tumours of the central nervous system and exhibit rapid growth that is associated with neovascularisation. Adrenomedullin is an important tumour survival factor in human carcinogenesis. It has growth promoting effects on gliomas, and blockade of its actions has been experimentally shown to reduce the growth of glioma tissues and cell lines. There is some evidence that the calcitonin receptor-like receptor (CRLR) mediates the tumorigenic actions of adrenomedullin. AIM: To determine whether CRLR is expressed in human gliomas and the probable cellular targets of adrenomedullin. METHODS: Biopsies from 95 human gliomas of varying grade were processed for immunohistochemical analysis using a previously developed and characterised antibody to CRLR. RESULTS: All tumour specimens were positive for CRLR. As previously found in normal peripheral tissues, CRLR immunostaining was particularly intense in the endothelial cells. This was evident in all the various vascular conformations that were observed, and which are typical of gliomas. In addition, clear immunostaining of tumour cells with astrocyte morphology was observed. These were preferentially localised around vessels. CONCLUSIONS: This study has shown for the first time that the CRLR protein is present in human glioma tissue. The expression of the receptor in endothelial cells and in astrocytic tumour cells is consistent with the evidence that its endogenous ligand, adrenomedullin, may influence glioma growth by means of both direct mitogenic and indirect angiogenic effects. CRLR may be a valuable target for effective therapeutic intervention in these malignant tumours.

Expression and distribution of calcitonin receptor-like receptor in human hairy skin.

Calcitonin gene-related peptide and adrenomedullin exert potent effects in skin but their cellular targets are unknown. This study aimed to identify the cellular location of calcitonin receptor-like receptor (CRLR) which is pharmacologically identical to CGRP receptor-1, a putative molecular target of CGRP and adrenomedullin. RT-PCR analysis of human hairy skin revealed the presence of CRLR mRNA and immunohistochemical analysis, employing a previously characterized polyclonal antibody raised to CRLR, provided novel evidence of the cellular distribution of CRLR. Extensive and specific CRLR-immunostaining was detected in arteriolar smooth muscle and venular endothelium and is consistent with CGRP's putative role in neurogenic inflammation. Novel targets for CGRP and/or adrenomedullin were identified, including capillary endothelium, hair follicles and sweat glands.

Immunohistochemical detection of calcitonin gene-related peptide receptor (CGRPR)-1 in the endothelium of human coronary artery and bronchial blood vessels.

The potent vasodilatory peptide, calcitonin gene-related peptide (CGRP) is present in the innervation of vascular tissue. The actions of CGRP occur via a receptor, CGRP receptor(R)-1, which is also a target for the cardioprotective peptide adrenomedullin. The human version of the pharmacologically-defined CGRPR-1 has been cloned but its distribution and cellular location is unknown. A rabbit antibody was generated to a synthetic peptide that corresponds to the C-terminus of human CGRPR-1 Immunochemical analysis of the human cell-line, SK-N-MC, which exhibits functional expression of the CGRPR-1 confirmed the antibody's specificity. The antiserum revealed specific staining in the endothelium of human coronary arteries. The vascular smooth muscle and ventricular myocardium were not immunoreactive. In bronchial blood vessels CGRPR-1-immunoreactivity was detected in the endothelium of the venules and not in the arterioles, which is particularly relevant for elucidating the putative role of CGRP in inflammation in this tissue.

Stroke prevention trial in sickle cell anemia.

Stroke occurs in 7-8% of children with Sickle Cell Disease (Hb SS) and is a major cause of morbidity. Rates of recurrence have been reduced from 46-90% to less than 10% through chronic blood transfusions. Prevention of first stroke, however, would be preferable because even one stroke can cause irreversible brain injury. Transcranial Doppler (TCD) ultrasound can detect arterial blood flow rates associated with subsequent stroke risk. By combining TCD screening and a potentially effective treatment, first stroke may be prevented. The Stroke Prevention Trial in Sickle Cell Anemia (STOP) is the first stroke prevention trial in Hb SS and the first randomized, controlled use of transfusion in Hb SS. This multi-center trial is designed to test whether reducing sickle hemoglobin to 30% or less with periodic blood transfusions will reduce first-time stroke by at least 70% compared to standard care. Primary endpoints will be clinically evident symptoms of cerebral infarction with consistent findings on Magnetic Resonance Imaging and Angiography (MRI/MRA) or symptomatic intracranial hemorrhage. Secondary endpoints will be asymptomatic brain lesions detected by MRI in brain areas not involved in primary endpoints. The design calls for a 6-month start-up interval, 18 months of TCD screening and randomization, and observation for stroke from entry through month 54. Key features of the trial are standardized TCD and MRI/MRA protocols interpreted blindly, and blinded adjudication of endpoints. The sample size (60 per treatment group) is based on prospective data relating TCD velocity to risk of stroke. A time-averaged mean velocity of > or = 200 cm/sec is associated with a 46% risk of cerebral infarction over 39 months. The sample size is sufficient to detect 70% reduction in the primary endpoint at 90% power. This trial will determine if transfusion is effective in the primary prevention of stroke. Secondary aims may further the understanding of the effects of transfusion on the brain and guide future research into cerebrovascular disease in Hb SS.

Enhanced cAMP accumulation by the human thyrotropin receptor variant with the Pro52Thr substitution in the extracellular domain.

Recently, a naturally occurring variant of the human thyrotropin receptor with a Pro52Thr substitution in the N-terminal extracellular domain of the receptor has been identified. To determine the functional significance of this substitution, cDNAs of wild-type and variant thyrotropin receptors were stably expressed in Chinese hamster ovary cells. The Pro52Thr substitution did not affect synthesis and membrane localization of the receptor, as evidenced by 125I-thyrotropin binding analysis to intact cells. The variant receptor and the wild-type receptor were expressed in equivalent numbers and displayed identical binding affinity for thyrotropin. Strikingly, thyrotropin increased cAMP accumulation to a much greater extent in cells expressing the variant receptor as compared to the wild-type receptor-expressing cells. Basal and cholera toxin-stimulated or forskolin-stimulated cAMP levels were not different. It is concluded that the Pro52Thr substitution in the N-terminal region of the human thyrotropin receptor produces a receptor protein with enhanced coupling to cAMP production. This naturally occurring hyperactive thyrotropin receptor may participate in hyperthyroidism of patients with Graves' disease.