Cloning, production and characterization of antigen 5 like proteins from Simulium vittatum and Culicoides nubeculosus, the first cross-reactive allergen associated with equine insect bite hypersensitivity

Insect bite hypersensitivity (IBH) is an IgE-mediated seasonal dermatitis of the horses associated with bites of Simulium (black fly) and Culicoides (midge) species. Although cross-reactivity between Simulium and Culicoides salivary gland extracts has been demonstrated, the molecular nature of the allergens responsible for the observed cross-reactivity remains to be elucidated. In this report we demonstrate for the first time in veterinary medicine that a homologous allergen, present in the salivary glands of both insects, shows extended IgE cross-reactivity in vitro and in vivo. The cDNA sequences coding for both antigen 5 like allergens termed Sim v 1 and Cul n 1 were amplified by PCR, subcloned in high level expression vectors, and produced as [His](6)-tagged proteins in Escherichia coli. The highly pure recombinant proteins were used to investigate the prevalence of sensitization in IBH-affected horses by ELISA and their cross-reactive nature by Western blot analyses, inhibition ELISA and intradermal skin tests (IDT). The prevalence of sensitization to Sim v 1 and Cul n 1 among 48 IBH-affected horses was 37% and 35%, respectively. In contrast, serum IgE levels to both allergens in 24 unaffected horses did not show any value above background. Both proteins strongly bound serum IgE from IBH-affected horses in Western blot analyses, demonstrating the allergenic nature of the recombinant proteins. Extended inhibition ELISA experiments clearly showed that Sim v 1 in fluid phase is able to strongly inhibit binding of serum IgE to solid phase coated Cul n 1 in a concentration dependent manner and vice versa. This crucial experiment shows that the allergens share common IgE-binding epitopes. IDT with Sim v 1 and Cul n 1 showed clear immediate and late phase reactions to the allergen challenges IBH-affected horses, whereas unaffected control horses do not develop relevant immediate hypersensitivity reactions. In some horses, however, mild late phase reactions were observed 4h post-challenge, a phenomenon reported to occur also in challenge experiments with Simulium and Culicoides crude extracts probably related to lipopolysaccaride contaminations which are also present in E. coli-expressed recombinant proteins. In conclusion our data demonstrate that IgE-mediated cross-reactivity to homologous allergens, a well-known clinically relevant phenomenon in human allergy, also occurs in veterinary allergy.

Sequencing a new target genome: The Boophilus microplus (Acari : Ixodidae) genome project

The southern cattle tick, Boophilus microplus (Canestrini), causes annual economic losses in the hundreds of millions of dollars to cattle producers throughout the world, and ranks as the most economically important tick from a global perspective. Control failures attributable to the development of pesticide resistance have become commonplace, and novel control technologies are needed. The availability of the genome sequence will facilitate the development of these new technologies, and we are proposing sequencing to a 4-6X draft coverage. Many existing biological resources are available to facilitate a genome sequencing project, including several inbred laboratory tick strains, a database of approximate to 45,000 expressed sequence tags compiled into a B. microplus Gene Index, a bacterial artificial chromosome (BAC) library, an established B. microplus cell line, and genomic DNA suitable for library synthesis. Collaborative projects are underway to map BACs and cDNAs to specific chromosomes and to sequence selected BAC clones. When completed, the genome sequences from the cow, B. microphis, and the B. microphis-borne pathogens Babesia bovis and Anaplasma marginale will enhance studies of host-vector-pathogen systems. Genes involved in the regeneration of amputated tick limbs and transitions through developmental stages are largely unknown. Studies of these and other interesting biological questions will be advanced by tick genome sequence data. Comparative genomics offers the prospect of new insight into many, perhaps all, aspects of the biology of ticks and the pathogens they transmit to farm animals and people. The B. microplus genome sequence will fill a major gap in comparative genomics: a sequence from the Metastriata lineage of ticks. The purpose of the article is to synergize interest in and provide rationales for sequencing the genome of B. microplus and for publicizing currently available genomic resources for this tick.

Evaluation of an IgE ELISA with Culicoides spp. extracts and recombinant salivary antigens for diagnosis of insect bite hypersensitivity in Warmblood horses

Insect bite hypersensitivity (IBH) in horses represents an immunoglobulin E (IgE)-mediated hypersensitivity to salivary antigens from biting midges (Culicoides spp.). The aim of this study was to evaluate and compare the performances of IgE ELISAs using recombinant Culicoides spp. Obsoletus group salivary gland antigens or crude whole body extracts ('ObsWBE'), C. nubeculosus recombinant proteins (Culn1, 3, 4, 5, 7, 8 and 10) and Obsoletus group recombinant proteins (Culo1 and 2). IgE levels were measured in plasma of 343 Warmblood horses classified as IBH-affected (n=167) and IBH-unaffected (n=176) according to the owners' descriptions. IBH-affected horses were subdivided based on the severity of their clinical signs at sampling and whether or not their IBH history was considered to be classical. The accuracies of the tests increased when clinical signs at sampling were more pronounced or when the IBH history could be considered as classical. A combination of IgE levels against the three best performing Culicoides spp. recombinant proteins (Culn4, Culo1 and Culo2) and ObsWBE resulted in the best performing test. When IBH-affected horses showing a classical history of the disease and severe clinical signs were compared with IBH-unaffected horses, the Youden's index at the optimal cut-off for the three tests in combination was 0.67. This optimal cut-off had a sensitivity of 70%, a specificity of 97% and a total accuracy of 92%. The performance of the IgE ELISA was affected by the severity of IBH clinical signs at sampling and was improved when IgE levels against several recombinant proteins were combined.

Mandibular gland secretions of meliponine worker bees: further evidence for their role in interspecific and intraspecific defence and aggression and against their role in food source signalling

Like ants and termites some species of stingless bees (Meliponini), which are very important pollinators in the tropics, use pheromone trails to communicate the location of a food source. We present data on the communicative role of mandibular gland secretions of Meliponini that resolve a recent controversy about their importance in the laying of such trails. Volatile constituents of the mandibular glands have been erroneously thought both to elicit aggressive/defensive behaviour and to signal food source location. We studied Trigona spinipes and Scaptotrigona aff. depilis (`postica`), two sympatric species to which this hypothesis was applied. Using extracts of carefully dissected glands instead of crude cephalic extracts we analysed the substances contained in the mandibular glands of worker bees. Major components of the extracts were 2-heptanol (both species), nonanal (T. spinipes), benzaldehyde and 2-tridecanone (S. aff. depilis). The effect of mandibular gland extracts and of individual components thereof on the behaviour of worker bees near their nest and at highly profitable food sources was consistent. Independent of the amount of mandibular gland extract applied, the bees overwhelmingly reacted with defensive behaviour and were never attracted to feeders scented with mandibular gland extract or any of the synthetic chemicals tested. Both bee species are capable of using mandibular gland secretions for intra-and interspecific communication of defence and aggression and share 2-heptanol as a major pheromone compound. While confirming the role of the mandibular glands in nest defence, our experiments provide strong evidence against their role in food source signalling.

Xerostomia in Sjogren`s syndrome and lupus erythematosus: a comparative histological and immunofluorescence study of minor salivary glands alterations

Background: Xerostomia is a symptom that can be triggered by chronic diseases such as Sjogren`s syndrome (SS) and lupus erythematosus (LE). Many authors regard most cases of salivary hypofunction in LE to secondary SS. Others believe that salivary changes in patients with LE might reflect a multisystem presentation of the disease. The present study compared histopathological and direct immunofluorescence (DIF) alterations in salivary glands of patients with xerostomia and diagnosis of LE or SS. Methods: Twenty-eight salivary gland biopsies from patients with xerostomia and diagnosed with LE or SS were submitted to histopathological and DIF exams. Results: From the 28 patients, 16 had SS and 12 had LE. In SS, a moderate to intense sialadenitis was detected, with infiltration and destruction of excretory salivary ducts. In LE, mild/moderate sialadenitis with thickening and hyalinization of the ductal basement membrane was observed. DIF revealed that 50% of SS patients presented intercellular ductal IgA deposits, whereas 58% of LE patients showed deposits of IgG in the ductal basement membrane. Conclusions: Alterations in salivary glands of LE patients may be a specific manifestation of the disease (lupus sialadenitis), reflecting its multisystemic presentation, instead of an association of secondary SS and LE.

PLUNC protein expression in major salivary glands of HIV-infected patients

Objective: To analyse and compare the expression of Palate, Lung, and Nasal Epithelium Clone (PLUNC) proteins in salivary glands from patients with and without AIDS (control group) using autopsy material. Methods: We analysed the expression of PLUNCs using immunohistochemistry in parotid (n = 45), submandibular (n = 47) and sublingual gland (n = 37) samples of AIDS patients [30 with normal histology, 21 with mycobacteriosis, 14 with cytomegalovirus (CMV) infection, 30 with chronic non-specific sialadenitis, and 30 HIV-negative controls. In situ hybridization (ISH) for SPLUNC 2 in the HIV-negative group was performed. Results: SPLUNC 1 expression was detected in the mucous acini of submandibular and sublingual glands, and SPLUNC 2 were seen in the serous cells. LPLUNC 1 expression was only positive in the salivary ducts. There was a higher expression of SPLUNC 2 in AIDS patients with CMV infection and mycobacteriosis when compared with all other groups. The intensity of staining for SPLUNC 2 was greater around the lesions than the peripheral ones. ISH for SPLUNC 2 showed perinuclear positivity in the serous cells in all HIV-negative cases. Conclusions: SPLUNC 1 and LPLUNC 1 proteins were similarly expressed in the salivary glands of AIDS patients and non-HIV patients. CMV infection and mycobacteriosis increase SPLUNC 2 expression in serous cells in the salivary gland of AIDS patients.

A tick salivary protein targets cathepsin G and chymase and inhibits host inflammation and platelet aggregation

Platelet aggregation and acute inflammation are key processes in vertebrate defense to a skin injury. Recent studies uncovered the mediation of 2 serine proteases, cathepsin G and chymase, in both mechanisms. Working with a mouse model of acute inflammation, we revealed that an exogenous salivary protein of Ixodes ricinus, the vector of Lyme disease pathogens in Europe, extensively inhibits edema formation and influx of neutrophils in the inflamed tissue. We named this tick salivary gland secreted effector as I ricinus serpin-2 (IRS-2), and we show that it primarily inhibits cathepsin G and chymase, while in higher molar excess, it affects thrombin activity as well. The inhibitory specificity was explained using the crystal structure, determined at a resolution of 1.8 angstrom. Moreover, we disclosed the ability of IRS-2 to inhibit cathepsin G-induced and thrombin-induced platelet aggregation. For the first time, an ectoparasite protein is shown to exhibit such pharmacological effects and target specificity. The stringent specificity and biological activities of IRS-2 combined with the knowledge of its structure can be the basis for the development of future pharmaceutical applications. (Blood. 2011;117(2):736-744)

Expressed sequence tags (ESTs) from the salivary glands of the tick Amblyomma cajennense (Acari : Ixodidae)

The neotropical tick Amblyomma cajennense is a significant pest to domestic animals, the most frequently human-biting tick in South America and the main vector of Brazilian spotted fever (caused by Rickettsia rickettsii), a deadly human disease. The purpose of this study is to characterize the adult A. cajennense salivary gland transcriptome by expressed sequence tags (ESTs). We report the analysis of 1754 clones obtained from a cDNA library, which reveal mainly transcripts related to proteins involved in the hemostatic processes, especially proteases and their inhibitors. Remarkably, five types of possible serine protease inhibitors were found, including a molecule with a distinguished structure that contains repeats of the active motif of hirudin inhibitors. Besides, other components that may be active over the host immune system or acting as defensins against infecting microorganisms were also described, including a molecule similar to insect venom allergens. The conjunction of components from this transcriptome suggests a diverse strategy of A. cajennense tick during feeding, but emphasized in the coagulation system. (c) 2008 Published by Elsevier Ltd.

Developing human minor salivary glands: morphological parallel relation between the expression of TGF-beta isoforms and cytoskeletal markers of glandular maturation

Morphogenesis of salivary glands involves complex coordinated events. Synchronisation between cell proliferation, polarisation and differentiation, which are dependent on epithelial-mesenchymal interactions and on the microenvironment, is a requirement. Growth factors mediate many of these orchestrated biological processes and transforming growth factor-beta (TGF-beta) appear to be relevant. Using immunohistochemistry and immunofluorescence, we have mapped the distribution of TGF-beta 1, 2 and 3 and compared it with the expression of maturation markers in human salivary glands obtained from foetuses ranging from weeks 4 to 24 of gestation. TGF-beta 1 first appeared during canalisation stage in the surrounding mesenchyme and, in the more differentiated stages, was expressed in the cytoplasm of acinar cells throughout the adult gland. TGF-beta 2 was detected since the bud stage of the salivary gland. Its expression was observed in ductal cells and increased along gland differentiation, TGF-beta 3 was detected from the canalisation stage of the salivary gland, being weakly expressed on ductal cells, and it was the only factor detected on myoepithelial cells. The data suggest that TGF-beta have a role to play in salivary gland development and differentiation.

Development of human minor salivary glands: expression of mucins according to stage of morphogenesis

The formation of salivary glands entails the proliferation of epithelial cells from the stomatodeum into the underlying ectomesenchyme, culminating in a complex network of ducts and acinar bulbs. The extent to which mucins regulate this process is unknown, but they appear to mediate luminal space formation and maturation. Our aim was to examine mucin expression patterns during the morphogenesis of human salivary glands. Mucin expression - MUC1, 2, 3, 4, 5AC, 5B, 6, and 16 - was analyzed in specimens of developing human salivary glands, obtained from fetuses at 4-24 weeks` gestation, and fully developed salivary glands by immunohistochemistry. Expression patterns were analyzed qualitatively according to the development stage of the salivary glands. Mucins 1, 3, 4, 5B, and 16 were expressed during salivary gland development - being stronger in all ductal segments by the final phases of branching morphogenesis and in mature glands. Acinar cells were negative for most mucins, including MUC1 in mature salivary glands. Mucins 2, 5AC, and 6 were not expressed. Mucins MUC1, 3, 4, 5B, and 16 are expressed in developing human salivary glands and in mature glands, suggesting important roles in the maturation and maintenance of the ductal network.

Differential expression of galectin-3, beta-catenin, and cyclin D1 in adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma of salivary glands

Background: Galectin-3 has been implicated in tumor progression of some malignancies as thyroid, prostate, and salivary gland tumors. Recently, it has been suggested that this protein may be an important mediator of the beta-catenin/Wnt pathway. Moreover, nuclear galectin-3 expression has been implicated in cell proliferation, promoting cyclin D1 activation. Thus, the present study aimed to correlate galectin-3 expression with beta-catenin and cyclin D1 expressions in adenoid cystic carcinoma (ACC) and in polymorphous low-grade adenocarcinoma (PLGA). Methods: Fifteen formalin-fixed paraffin-embedded cases of each tumor were retrieved from the files of the Surgical Oral Pathology Service at the University of Sao Paulo and the proteins were analyzed by immunohistochemistry. Results: Adenoid cystic carcinoma showed galectin-3 immunostaining mainly in the nuclei, while PLGA revealed a positive mostly cytoplasmic reaction to galectin-3 in the largest part of tumor cells. Both tumors showed intense cytoplasmic/nuclear staining for beta-catenin in majority of cases. Cyclin D1 immunoreactivity was not detected in 14/15 PLGA and showed specific nuclear staining in 10/15 cases of ACC in more than 5% of the neoplastic cells. Cyclin D1 expression was correlated with cytoplasmic and nuclear galectin-3 expression in ACC (P < 0.05). Conclusions: These results suggest that in ACC galectin-3 may play a role in cellular proliferation through cyclin D1 activation. In addition, nuclear expression of galectin-3 in ACC may be related to a more aggressive behavior of this lesion. Although beta-catenin seems to play a role in carcinogenesis in both lesions, it seems that it does not bind to galectin-3 for cyclin D1 stimulation.

Comparison of the effects of pilocarpine and cevimeline on salivary flow

Objective: The aim of the present study was to compare the effect of low-dose pilocarpine and cevimeline as stimulants for salivary flow in healthy subjects. Methods: In this cross-over clinical trial with a 1-week washout period, 40 male volunteers were submitted to an oral dose of pilocarpine 1% (Salagen (TM)) -60 mu g kg(-1) body-weight (Group 1) or Cevimeline (Evoxac (TM)) -30 mg (Group 2). Saliva samples were collected and the salivary flow rate was measured (ml min(-1)) at baseline and 20, 40, 60, 80, 140 and 200 min after administration of drugs. In addition, salivary secretion was also measured under mechanical stimulation to observe salivary gland function. Results: The data were analyzed by Friedman and Wilcoxon signed-rank tests (significance level = 5%). Pilocarpine and cevimeline significantly increased salivary flow 140 min after intake. There was a significant higher secretion with cevimeline 140 and 200 min after administration. There were no differences seen among subjects in the salivary glands function by mechanical stimulation. Conclusion: Both drugs showed efficacy in increasing the salivary flow in healthy volunteers, but cevimeline was more effective than pilocarpine.

Sialolithiasis of the Submandibular Gland

Sialolithiasis of the salivary gland is a benign pathology that occurs most frequently in the submandibular gland because of its anatomic features. Depending on the sialolith size and calcification degree, it can be visible in radiographic examinations. Commonly, patients may experience pain and/or edema, when the ducts are obstructed. The authors report the case of sialolithiasis of the submandibular gland in a 42-year-old, female, white-skinned patient, noticed during routine dental examination. Following diagnosis confirmed by clinical and radiographic examinations, the treatment plan consisted of surgery for removal of the calcified mass. The prognosis is often good, and generally there is no recurrence.

Submandibular gland aplasia and progressive dental caries: A case report

When severe caries occurs in mandibular permanent incisor teeth, the clinician should consider the possibility of associated submandibular gland aplasia or salivary hypofunction. Early diagnosis of submandibular gland disease is essential, as operative problems involving restoration of mandibular incisor teeth are considerable. Furthermore, progressive severe dental caries can present a dilemma for the clinician in affected individuals, despite intensive preventive and restorative therapy. A case report describing severe progressive dental caries and enamel demineralization of the permanent mandibular incisor teeth in a young girl is presented. Further investigation revealed absence of functional bilateral submandibular salivary glands contributing to the rapid breakdown of the teeth despite intensive preventive measures.

Ablation of caterpillar labial salivary glands: technique for determining the role of saliva in insect-plant interactions.

There has been an ardent interest in herbivore saliva due to its roles in inducing plant defenses and its impact on herbivore fitness. Two techniques are described that inhibit the secretion of labial saliva from the caterpillar, Helicoverpa zea, during feeding. The methods rely on cauterizing the caterpillar's spinneret, the principal secretory structure of the labial glands, or surgically removing the labial salivary gland. Both methods successfully inhibit secretion of saliva and the principal salivary enzyme glucose oxidase. Caterpillars with inhibited saliva production feed at similar rates as the untreated caterpillars, pupate, and emerge as adults. Glucose oxidase has been suggested to increase the caterpillar's survival through the suppression of inducible anti-herbivore defenses in plants. Tobacco (Nicotiana tabacum) leaves fed on by caterpillars with ablated salivary glands had significantly higher levels of nicotine, an inducible anti-herbivore defense compound of tobacco, than leaves fed upon by caterpillars with intact labial salivary glands. Tomato (Lycopersicon esculentum) leaves fed upon by caterpillars with suppressed salivary secretions showed greatly reduced evidence of hydrogen peroxide formation compared to leaves fed upon by intact caterpillars. These two methods are useful techniques for determining the role that saliva plays in manipulating plant anti-herbivore defenses.