Olfactory Heterogeneity in LRRK2 Related Parkinsonism

LRRK2 mutations can cause familial and sporadic Parkinson`s disease (PD) with Lewy-body pathology at post-mortem. Studies of olfaction in LRRK2 are sparse and incongruent. We applied a previously validated translation of the 16 item smell identification test from Sniffin` Sticks (SS-16) to 14 parkinsonian carriers of heterozygous G2019S LRRK2 mutation and compared with 106 PD patients and 118 healthy controls. The mean SS-16 score in LRRK2 was higher than in PD (p < 0.001, 95% CI for beta = -4.7 to -1.7) and lower than in controls (p = 0.007, 95% CI for beta = +0.6 to +3.6). In the LRRK2 group, subjects with low scores had significantly more dyskinesia. They also had younger age of onset, longer disease duration, and reported less frequently a family history of PD, but none of these other differences reached significance. Odor identification is diminished in LRRK2 parkinsonism but not to the same extent as in idiopathic PD. (C) 2010 Movement Disorder Society

Identification of the alpha(6) integrin as a candidate receptor for papillomaviruses

Papillomaviruses (PVs) bind in a specific and saturable fashion to a range of epithelial and other cell lines. Treatment of cells with trypsin markedly reduces their ability to bind virus particles, suggesting that binding is mediated via a cell membrane protein. We have investigated the interaction bf human PV type 6b L1 virus-like particles (VLPs) with two epithelial cell lines, CV-1 and HaCaT, which bind VLPs, and a B-cell line (DG75) previously shown not to bind VLPs. Immunoprecipitation of a mixture of PV VLPs with [S-35]methionine-labeled cell extracts and with biotin-labeled cell surface proteins identified four proteins from CV-1 and HaCaT cells of 220, 120, 87, and 35 kDa that reacted with VLPs and were not present in DG75 cells. The alpha(6) beta(4) integrin complex has subunits corresponding to the VLP precipitated proteins, and the tissue distribution of this complex suggested that it was a candidate human PV receptor. Monoclonal antibodies (MAbs) to the alpha(6) or beta(4) integrin subunits precipitated VLPs from a mixture of CV-1 cell proteins and VLPs, whereas MAbs to other integrin subunits did not. An alpha(6) integrin-specific MAb (GoH3) inhibited VLP binding to CV-1 and HaCaT cells, whereas an anti-beta(4) integrin MAb and a range of integrin-specific and other MAbs did not. Furthermore, human laminin, the natural ligand for the alpha(6) beta(4) integrin, was able to block VLP binding. By use of sections of monkey esophagus, the distribution of alpha(6), integrin expression in the basal epithelium was shown to coincide with the distribution of bound VLPs. Taken together, these data suggest that VLPs bind specifically to the alpha(6) integrin subunit and that integrin complexes containing alpha(6) integrin complexed with either beta(1) or beta(4) integrins may act as a receptor for PV binding and entry into epithelial cells.

High numbers of human skin cancers express MMP2 and several integrin genes

Background: Basal cell carcinomas (BCC), squamous cell carcinomas (SCC) and cutaneous malignant melanoma (MM) are solid skin cancers derived from different cell types, with different ability to metastasize. Several subtypes of integrins and matrix metalloproteinases (MMP) have been related to malignization and metastasis processes. This work aimed at a quantitative evaluation of skin cancers expressing eight integrins and MMP2 genes. Methods: Expression of integrins and MMP2 genes was evaluated on fresh tumor biopsies from BCC, SCC and MM, and respective controls, by the reverse transcriptase polychain reaction (RT-PCR) technique. Results: More than 90% tumors expressed alpha 6a, beta 1, beta 3 and beta 6 (non-melanoma), and alpha 5a, alpha 6a and MMP2 (MM). Up to 100% controls also expressed beta 1 and beta 3. The results were significant for alpha 6a in BCC (p = 0.026), alpha 6b in SCC (p = 0.035), alpha 2a in BCC (p = 0.003), beta 5 and beta 6 in BCC (p = 0.005). MMP2 was expressed in 100% MM (p = 0.0004). Conclusion: Integrin subunits alpha 2a and alpha 6a would be interesting targets for BCC anti-tumor therapy, as well as alpha 6b in case of SCC. The elevated number of BCC expressing alpha 2 and alpha 6, and of MM expressing alpha v and MMP2, corroborate literature data.

Both alpha 1 and alpha 2-adrenoceptors mediate the cardiovascular responses to noradrenaline microinjected into the bed nucleus of the stria terminal of rats

Background and purpose: We have previously shown that noradrenaline microinjected into the bed nucleus of stria terminalis (BST) elicited pressor and bradycardiac responses in unanaesthetized rats. In the present study, we investigated the subtype of adrenoceptors that mediates the cardiovascular response to noradrenaline microinjection into the BST. Experimental approach: Cardiovascular responses following noradrenaline microinjection into the BST of male Wistar rats were studied before and after BST pretreatment with different doses of the selective alpha(1)-adrenoceptor antagonist WB4101, the alpha(2)-adrenoceptor antagonist RX821002, the combination of WB4101 and RX821002, the non-selective beta-adrenoceptor antagonist propranolol, the selective beta(1)-adrenoceptor antagonist CGP20712 or the selective beta(2)-adrenoceptor antagonist ICI118,551. Key results: Noradrenaline microinjected into the BST of unanaesthetized rats caused pressor and bradycardiac responses. Pretreatment of the BST with different doses of either WB4101 or RX821002 only partially reduced the response to noradrenaline. However, the response to noradrenaline was blocked when WB4101 and RX821002 were combined. Pretreatment with this combination also shifted the resulting dose-effect curve to the left, clearly showing a potentiating effect of this antagonist combination. Pretreatment with different doses of either propranolol or CGP20712 increased the cardiovascular responses to noradrenaline microinjected into the BST. Pretreatment with ICI118,551 did not affect cardiovascular responses to noradrenaline. Conclusion and implications: The present results indicate that alpha(1) and alpha(2)-adrenoceptors mediate the cardiovascular responses to noradrenaline microinjected into the BST. In addition, they point to an inhibitory role played by the activation of local beta(1)-adrenoceptors in the cardiovascular response to noradrenaline microinjected into the BST.

Effect of acute restraint stress on the tachycardiac and bradycardiac responses of the baroreflex in rats

In the present study, we evaluated cardiac baroreflex responses of rats submitted to acute restraint stress. The baroreflex was tested: immediately before, during a 30 min exposure to restraint stress, as well as 30 and 60 min after ending the stress session (recovery period). Restraint increased both mean arterial pressure (MAP) and heart rate (HR). The magnitude of tachycardiac responses evoked by intravenous infusion of sodium nitroprusside was higher during restraint stress, whereas that of bradycardiac responses evoked by intravenous infusion of phenylephrine was decreased. Restraint-evoked baroreflex changes were still observed at 30 min into the recovery period, although MAP and HR values had already returned to control values. The baroreflex was back to control values at 60 min of the recovery period. Intravenous administration of the selective beta(1)-adrenoceptor antagonist atenolol blocked the restraint-evoked increase in the tachycardiac baroreflex response, but did not affect the effects on the bradycardiac response. In conclusion, the present results suggest that psychological stresses, such as those resulting from acute restraint, affect the baroreflex. Restraint facilitated the tachycardiac baroreflex response and reduced the bradycardiac response. Restraint-related effects on baroreflex persisted for at least 30 min after ending restraint, although MAP and HR had already returned to control levels. The cardiac baroreflex returned to control values 60 min after the end of restraint, indicating non-persistent effects of acute restraint on the baroreflex. Results also indicate that the influence of restraint stress on the baroreflex tachycardiac response is mainly dependent on cardiac sympathetic activity, whereas the action on the bradycardiac response is mediated by the cardiac parasympathetic component.

Evaluating budesonide efficacy in nasal polyposis and predicting the resistance to treatment

Cell resistance to glucocorticoids is a major problem in the treatment of nasal polyposis (NP). The objectives of this study were to observe the effect of budesonide on the expression of IL-1 beta, TNF-alpha, granulocyte macrophage-colony stimulating factor, intercellular adhesion molecule (ICAM)-1, basic fibroblast growth factor, eotaxin-2, glucocorticoid receptor (GR)-alpha, GR-beta, c-Fos and p65 in nasal polyps and to correlate their expression to clinical response. Biopsies from nasal polyps were obtained from 20 patients before and after treatment with topical budesonide. Clinical response to treatment was monitored by a questionnaire and nasal endoscopy. The mRNA levels of the studied genes were measured by real-time quantitative (RQ)-PCR. There was a significant decrease in the expression of TNF-alpha (P < 0.05), eotaxin-2 (P < 0.05) and p65 (P < 0.05) in NP after treatment. Poor responders to glucocorticoids showed higher expression of IL-1 beta (3.74 vs. 0.14; P < 0.005), ICAM-1 (1.91 vs. 0.29; P < 0.05) and p65 (0.70 vs. 0.16; P < 0.05) before treatment. Following treatment, IL-1 beta (4.18 vs. 0.42; P < 0.005) and GR-beta (0.95 vs. 0.28; P < 0.05) mRNA expression was higher in this group. Topical budesonide reduced the expression of TNF-alpha, eotaxin-2 and p65. Poor responders to topical budesonide exhibit higher levels of IL-1 beta, ICAM-1 and nuclear factor (NF)-kappa B at diagnosis and higher expression of both IL-1 beta and GR-beta after treatment. These results emphasize the anti-inflammatory action of topical budesonide at the molecular level and its importance in the treatment of NP. Nevertheless, IL-1 beta, ICAM-1 and NF-kappa B may be associated with primary resistance to glucocorticoids in NP, whereas higher expression of GR-beta in poor responders only after glucocorticoid treatment may represent a secondary drug resistance mechanism in this disease.

Dental caries status and salivary properties of asthmatic children and adolescents

Aims. This study aimed to investigate the dental caries status and salivary properties in 3- to 15-year-old children/adolescents. Methods. The sample was split in two groups: asthma group (AG), composed of 65 patients who attended Public Health Service; asthma-free group (AFG), composed of 65 nonasthmatic children/adolescents recruited in two public schools. Stimulated salivary samples were collected for 3 min. Buffering capacity and pH were ascertained in each salivary sample. A single trained and calibrated examiner (kappa = 0.98) performed the dental caries examination according to WHO criteria. Results. The AFG showed salivary flow rate (1.10 +/- 0.63 mL/min) higher (P = 0.002) than AG (0.80 +/- 0.50 mL/min). An inverse relationship was observed between asthma severity and salivary flow rate (Phi coefficient, r phi: 0.79, P = 0.0001). Children with moderate or severe asthma showed an increased risk for reduced salivary flow rate (OR: 17.15, P < 0.001). No association was observed between drug use frequency (P > 0.05) and drug type (P > 0.05) with salivary flow rate. Buffering capacity was similar in both groups. No significant differences were encountered in dental caries experience between AFG and AG groups. Conclusions. Although asthma can cause reduction in flow rate, the illness did not seem to influence dental caries experience in children with access to proper dental care.

Human papillomavirus type 6b virus-like particles are able to activate the Ras-MAP kinase pathway and induce cell proliferation

The initial step in viral infection is the attachment of the virus to the host cell via an interaction with its receptor. We have previously shown that a receptor for human papillomavirus is the alpha6 integrin. The alpha6 integrin is involved in the attachment of epithelial cells with the basement membrane, but recent evidence suggests that ligation of many integrins results in intracellular signaling events that influence cell proliferation. sere we present evidence that exposure of A431 human epithelial cells to human papillomavirus type 6b L1 virus-like particles (VLPs) results in a dose-dependent increase in cell proliferation, as measured by bromodeoxyuridine incorporation. This proliferation is Lost if VLPs are first denatured or incubated with a monoclonal antibody against L1 protein. The MEK1 inhibitor PB98059 inhibits the VLP-mediated increase in fell proliferation, suggesting involvement of the Ras-MAP kinase pathway, Indeed, VLP binding results in rapid phosphorylation of the beta4 integrin upon tyrosine residues and subsequent recruitment of the adapter protein She to beta4, Within 30 min, the activation of Ras, Raf, and Erk2 was observed. Finally, the upregulation of c-myc mRNA was observed at 60 min, These data indicate that human papillomavirus type 6b is able to signal cells via the Ras-MAP kinase pathway to induce cell proliferation. We hypothesize that such a mechanism would allow papillomaviruses to infect hosts more successfully by increasing the potential pool of cells they are able to infect via the initiation of proliferation in resting keratinocyte stem and suprabasal cells.

The oral medicine of tooth wear

This review illustrates, through a series of case histories, how oral medicine insights aid the diagnosis and management of patients with excessive tooth wear. The cases reviewed are drawn from the records of 500 southeast Queensland patients referred to the author over a 12 year period. Patients most at risk of dental erosion have work and sports dehydration, caffeine addiction, gastro-oesophageal reflux, asthma, diabetes mellitus, hypertension or other systemic diseases or syndromes that predispose to xerostomia. Saliva protects the teeth from the extrinsic and intrinsic acids which cause dental erosion. Erosion, exacerbated by attrition and abrasion, is the main cause of tooth wear. These cases illustrate that teeth, oral mucosa, salivary glands, skin and eyes should be examined for evidence of salivary hypofunction and attendant medical conditions. Based on comprehensive oral medicine, dietary analyses and advice, it would seem patients need self-management plans to deal with incipient chronic tooth wear. The alternative is the expensive treatment of pain, occlusal damage and pulp death required to repair the effects of acute severe tooth wear.

Laminin 10/11: an alternative adhesive ligand for epidermal keratinocytes with a functional role in promoting proliferation and migration

We have investigated the expression and function of the isoforms of laminin bearing the alpha(5) chain, i.e. laminin-10/11 in neonatal and adult human skin. By immunostaining human skin derived from a variety of anatomic sites, we found that the laminin-alpha(5) chain is expressed abundantly in the basement membrane underlying the interfollicular epidermis and the blood vessels in the dermis. Interestingly, while the expression level of the well-studied laminin-5 isoform did not change significantly with age, laminin-10/11 (a5 chain) appeared to decrease in the basement membrane underlying the epidermis, in adult skin. In contrast, the levels of laminin-10/11 in the basement membrane underlying blood vessels remained unchanged in neonatal vs. adult skin. Importantly, in vitro cell adhesion assays demonstrated that laminin-10/11 is a potent adhesive substrate for both neonatal and adult keratinocytes and that this adhesion is mediated by the alpha(3)beta(1), and alpha(6)beta(4) integrins. Adhesion assays performed with fractionated basal keratinocytes showed that stem cells, transit amplifying cells and early differentiating cells all adhere to purified laminin-10/11 via these receptors. Further, laminin-10/11 provided a proliferative signal for neonatal foreskin keratinocytes, adult breast skin keratinocytes, and even a human papillomavirus type-18 transformed tumorigenic keratinocyte cell line in vitro. Finally, laminin-10/11 was shown to stimulate keratinocyte migration in an in vitro wound healing assay. These results provide strong evidence for a functional role for laminin-10/11 in epidermal proliferation during homeostasis, wound healing and neoplasia.

Dental erosion in asthma: A case-control study from south east Queensland

Background: Asthma medication places patients at risk of dental erosion by reducing salivary protection against extrinsic or intrinsic acids. But patterns of lesions in asthmatics may differ from patterns in non-asthmatics, because gastro-oesophageal reflux (GOR) is found in 60 per cent of asthmatics. Methods: The lesions in 44 asthma cases were compared to those of age and sex match controls with no history of asthma or medications drawn from the dental records of 423 patients referred concerning excessive tooth wear. The subjects were 70 males age range 15 to 55 years and 18 females age range 18 to 45. Anamnestic clinical data were compared between the two groups. Models of all 88 subjects were examined by light microscopy, and wear patterns were recorded on permanent central incisor, canine, premolar and first molar teeth. Results: Clinical differences were a higher incidence of tooth hypersensitivity; xerostomia, salivary gland abnormalities, gastric complaints, and self induced vomiting in the cases. No differences were found between the cases and controls on citrus fruit and acid soft drink consumption. More occlusal erosion sites were found in cases, whereas more attrition sites were found in the controls. There were no significant differences in palatal erosion on maxillary anterior teeth found between cases and controls. Lingual erosion of the mandibular incisors, found only in GOR patients, was not observed. Conclusions: A higher incidence of erosion was found in asthmatics. Gastro-oesophageal reflux symptoms were not associated with the sign of lingual mandibular incisor erosion. The clinical significance is that asthmatics are at risk of dental erosion from extrinsic acid, but GOR does not appear to contribute in a site-specific manner.

Anti-leishmania effector functions of CD4+ Th1 cells and early events instructing Th2 cell development and susceptibility to Leishmania major in BALB/c mice.

Resistance and susceptibility to infection with the intracellular parasite, Leishmania major, are mediated by parasite-specific CD4+ Th1 and Th2 cells, respectively. It is well established that the protective effect of parasite-specific CD4+ Th1 cells is largely dependent upon the IFN-gamma produced. However, recent results indicate that the effect of Th1 cells on resolution of lesions induced by L. major in genetically resistant mice also requires a functional Fas-FasL pathway of cytotoxicity. In contrast to resistant mice, susceptible BALB/c mice develop aberrant Th2 responses following infection with L. major and consequently suffer progressive disease. These outcomes clearly depends upon the production of interleukin 4 (IL-4) early after infection. We have shown that a burst of IL-4 mRNA, peaking in draining lymph nodes of BALB/c mice 16 hrs after infection, occurs within CD4+ T cells that express V beta 4-V alpha 8 T cell receptors. In contrast to control and V beta 6-deficient mice, V beta 4-deficient BALB/c mice were resistant to infection, demonstrating the role of these cells in Th2 development. The early IL-4 response was absent in these mice, and Th1 responses occurred following infection. The LACK antigen of L. major induced comparable IL-4 production in V beta 4-V alpha 8 CD4+ T cells. Thus, the IL-4 required for Th2 development and susceptibility to L. major is produced by a restricted population of V beta 4-V alpha 8 CD4+ T cells after cognate interaction with a single antigen from this complex parasite. The IL-4 produced rapidly by these CD4+ T cells induces within 48 hours a state of unresponsiveness to IL-12 among parasite-specific CD4+ T cell precursors by downregulating the IL-12 receptor beta 2 chain expression.

Synthesis of a radioiodinated photoreactive MAGE-1 peptide derivative and photoaffinity labeling of cell-associated human leukocyte antigen-A1 molecules.

The synthesis of a photoreactive derivative of the human leukocyte antigen-A1 (HLA-A1)-restricted MAGE-1 peptide 161-169 (EADPTGHSY) is described. Using conventional automated solid-phase peptide synthesis, a photoreactive derivative of this peptide was synthesized by replacing histidine-167 with photo-reactive N-beta-4-azidosalicyloyl-L-2,3-diaminopropionic acid. The C-terminal tyrosine was incorporated as phosphotyrosine. This peptide derivative was radioiodinated in the presence of chloramine T. This iodination took place selectively at the photoreactive group, because the phosphate ester prevented tyrosine iodination. Following dephosphorylation with alkaline phosphatase and chromatographic purification, the radiolabeled peptide derivative was incubated with cells expressing HLA-A1 or other HLA molecules. Photoactivation resulted in efficient photoaffinity labeling of HLA-A1. Other HLA molecules or other cellular components were not detectably labeled. This labeling was inhibited by HLA-A1 but not by HLA-A2-binding peptides. This synthesis is generally applicable and can also be adapted to the synthesis of well-defined radiolabeled nonphotoreactive peptide derivatives.

Induction of CTL in vivo by major histocompatibility complex class I-peptide complexes covalently associated on the cell surface.

The identification of endogenously produced antigenic peptides presented by MHC class I molecules has opened the way to peptide-based strategies for CTL induction in vivo. Here we demonstrate that the induction in vivo of CTL directed against naturally processed antigens can be triggered by injection of syngeneic cells expressing covalent major histocompatibility complex class I-peptide complexes. In the model system used, the induction of HLA-Cw3 specific cytotoxic T lymphocytes (CTL) in mice by cell surface-associated, covalent H-2Kd (Kd)-Cw3 peptide complexes was investigated. The Kd-restricted Cw3 peptide 170-179 (RYLKNGKETL), which mimics the major natural epitope recognized by Cw3-specific CTL in H-2d mice, was converted to a photoreactive derivative by replacing Arg-170 with N-beta-(4-azidosalicyloyl)-L-2,3-diaminopropionic acid. This peptide derivative was equivalent to the parental Cw3 peptide in terms of binding to Kd molecules and recognition by Cw3-specific CTL clones and could be cross-linked efficiently and selectively to Kd molecules on the surface of Con A-stimulated spleen cells from H-2d mice. Photocross-linking prevented the rapid dissociation of Kd-peptide derivative complexes that takes place under physiological conditions. Cultures of spleen cells or peritoneal exudate cells from mice inoculated i.p. with peptide-pulsed and photocross-linked cells developed a strong CTL response following antigenic stimulation in vitro. The cultured cells efficiently lysed not only target cells sensitized with the Cw3 170-179 peptide but also target cells transfected with the Cw3 gene. Moreover, their TCR preferentially expressed V beta 10 and J alpha pHDS58 segments as well as conserved junctional sequences, as has been observed previously in Cw3-specific CTL responses. In contrast, no Cw3-specific CTL response could be obtained in cultures derived from mice injected with Con A-stimulated spleen cells pulsed with the peptide derivative without photocross-linking.

Vascular Integrins: Therapeutic and Imaging Targets of Tumor Angiogenesis

Cells, including endothelial cells, continuously sense their surrounding environment and rapidly adapt to changes in order to assure tissues and organs homeostasis. The extracellular matrix (ECM) provides a physical scaffold for cell positioning and represents an instructive interface allowing cells to communicate over short distances. Cell surface receptors of the integrin family emerged through evolution as essential mediators and integrators of ECM-dependent communication. In preclinical studies, pharmacological inhibition of vascular integrins suppressed angiogenesis and inhibited tumor progression. alpha(V)beta(3) and alpha(V)beta(5) were the first integrins targeted to suppress tumor angiogenesis. Subsequently, additional integrins, in particular alpha(1)beta(1), alpha(2)beta(1), alpha(5)beta(1), and alpha(6)beta(4), emerged as potential therapeutic targets. Integrin inhibitors are currently tested in clinical trials for their safety and antiangiogenic/antitumor activity. In this chapter, we review the role of integrins in angiogenesis and present recent advances in the use of integrin antagonists as potential therapeutics in cancer and discuss future perspectives.