Altered expression of integrins and basement membrane proteins in malignant and pre-malignant lesions of oral mucosa

Integrins are transmembrane receptors that regulate cell-cell and cell-extracellular matrix (ECM) contact. In epithelial tissues, they interact with ECM components of the basement membrane (BM) to maintain the homeostasis and the architecture of the tissue. This interaction controls several cell functions such as adhesion, migration, proliferation, differentiation, and therefore has a key role in cancer development and metastasis. We studied the expression of integrins and ECM components of the BM by immunohistochemistry in frozen specimens of malignant squamous cell carcinoma (SCC), pre-malignant lesions of the oral mucosa (leucoplakia) and oral lichen planus. In invasive SCC, we observed altered polarity and distribution of alpha2beta1, alpha6beta4 and alpha3beta1 integrins, whereas in the in situ carcinoma alpha6beta4 and alpha3beta1 patterns only were altered. Immunostaining for ECM components such as Laminin-1 (Ln-1), Ln-5, and Collagen IV (Coll IV) was discontinuous and interrupted in invasive SCC, whereas it was normal in the in situ carcinoma. In both pre-malignant lesions and lichen planus specimens, integrins were expressed in a polarized manner in the presence of a normal BM, whereas were abnormally distributed in those tissues with altered staining patterns of the ECM components. In conclusion, we suggest that abnormal re-distribution of alpha3beta1 and alpha6beta4 integrins and expression of ECM components such as Ln-5 could play an important role in SCC invasion and metastasis.     

Morphological variability and genetic identity in Rhinoestrus spp. causing horse nasal myiasis

Larvae of Rhinoestrus purpureus (Brauer) and Rhinoestrus usbekistanicus Gan (Diptera: Oestridae) cause nasal myiases of equids. During a recent epidemiological survey in southern Italy some morphological and taxonomical doubts arose concerning the identification of Rhinoestrus third stage larvae on the basis of the features of the posterior spiracles and the distribution of dorsal spines on the third segment. Four different morphotypes were retrieved: R. usbekistanicus-like, R. purpureus-like and two morphotypes with shared features. The genes encoding for the mitochondrial cytochrome oxidase I (COI) and for the ribosomal subunits 16S and 28S of the four morphotypes of Rhinoestrus were investigated to determine whether they belonged to a single taxon or they displayed genetic differences indicative of more than one species. The three genes showed a very low level of sequence variation (COI 0-0.43%, 16S 0-1.45%, 28S 0-0.23%) falling within the intraspecific ranges previously described for Oestridae species. Finally, the peritreme features and the spinulation of the third segment of the four morphotypes examined could not be used to differentiate the two species.     

A novel heterozygous mutation of the AIRE gene in a patient with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED)

Background

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED) is an autosomal recessive disease due to mutations of the autoimmune regulator (AIRE) gene. Typical manifestations include candidiasis, Addison's disease, and hypoparathyroidism. Type 1 diabetes, alopecia, vitiligo, ectodermal dystrophy, celiac disease and other intestinal dysfunctions, chronic atrophic gastritis, chronic active hepatitis, autoimmune thyroid disorders, pernicious anemia and premature ovarian failure are other rare associated diseases although other conditions have been associated with APECED.

Case presentation

What follows is the clinical, endocrinological and molecular data of a female APECED patient coming from Lithuania. The patient was affected by chronic mucocutaneous candidiasis, hypoparathyroidism and pre-clinical Addison's disease. Using direct sequencing of all the 14 exons of the AIRE gene in the patient's DNA, we identified in exon 6 the known mutation c.769 C>T (p.Arg257X) in compound heterozygosity with the newly discovered mutation c.1214delC (p.Pro405fs) in exon 10. The novel mutation results in a frameshift that is predicted to alter the sequence of the protein starting from amino acid 405 as well as to cause its premature truncation, therefore a non-functional Aire protein.

Conclusions

A novel mutation has been described in a patient with APECED with classical clinical components, found in compound heterozygosity with the c.769 C>T variation. Expanded epidemiological investigations based on AIRE gene sequencing are necessary to verify the relevancy of the novel mutation to APECED etiopathogenesis in the Lithuanian population and to prove its diagnostic efficacy in association with clinical and immunological findings.     

Gas-chromatographic determination of levamisole residues in sheep flesh

The Authors carried out a research on the residues of Levamisole, a well known antihelmintic drug, in the muscles, livers, kidneys and blood from sheep treated with therapeutical doses. The results suggest that highest levamisole concentrations in the organs have been detected between the second and the third day (3.25 ppm in muscles; 1.40 ppm in livers; 0.22 ppm in kidneys). Remarkable residues have already been verified in blood during the first determination (4 h after the treatment) but these have been rapidly eliminated. Besides, a gradual diminution of the residues was observed until the seventh day from the treatment.     

Cross-sectional survey of ticks (Acari: Ixodidae) in sheep from an area of the southern Italian Apennines

A cross-sectional survey of ticks was conducted on 197 ovine farms with animals pasturing in an area (3971 km2) of the southern Italian Apennines. The farms were selected to be uniformly distributed throughout the study area using Geographical Information System (GIS). Ticks were collected from 309 (31.4%) out of the 985 sheep sampled, belonging to 92 (46.7%) out of the 197 farms included in the study. The following tick species were found (farm prevalence): Dermacentor marginatus (37.6%), Haemaphysalis punctata (29.4%), H. sulcata (2.5%), H. parva (2.0%). H. inermis (0.5%), Ixodes gibbosus (2.0%), I. ricinus (0.5%), Rhipicephalus sanguineus group (1.0%), and R. bursa (0.5%). A point distribution map (PDM) was drawn by GIS in order to display the distribution of each tick genus in the study area. The general trends of the PDM show that Dermacentor marginatus and Haemaphysalis spp. were widely and homogeneously spread throughout the study area, whereas Rhipicephalus spp. and Ixodes spp. were present only in a few concentrated zones of the study area in accordance to their biological and ecological characteristics.     

Dual Orientation of the Outer Membrane Lipoprotein P6 of Nontypeable Haemophilus influenzae

The majority of outer membrane (OM) lipoproteins in Gram-negative bacteria are tethered to the membrane via an attached lipid moiety and oriented facing in toward the periplasmic space; a few lipoproteins have been shown to be surface exposed. The outer membrane lipoprotein P6 from the Gram-negative pathogenic bacterium nontypeable Haemophilus influenzae (NTHi) is surface exposed and a leading vaccine candidate for prevention of NTHi infections. However, we recently found that P6 is not a transmembrane protein as previously thought (L. V. Michel, B. Kalmeta, M. McCreary, J. Snyder, P. Craig, M. E. Pichichero, Vaccine 29:1624–1627, 2011). Here we pursued studies to show that P6 has a dual orientation, existing infrequently as surface exposed and predominantly as internally oriented toward the periplasmic space. Flow cytometry using three monoclonal antibodies with specificity for P6 showed surface staining of whole NTHi cells. Confocal microscopy imaging confirmed that antibodies targeted surface-exposed P6 of intact NTHi cells and not internal P6 in membrane-compromised or dead cells. Western blots of two wild-type NTHi strains and a mutant NTHi strain that does not express P6 showed that P6 antibodies do not detect a promiscuous epitope on NTHi. Depletion of targets to nonlipidated P6 significantly decreased bactericidal activity of human serum. Protease digestion of surface-exposed P6 demonstrated that P6 is predominantly internally localized in a manner similar to its homologue Pal in Escherichia coli. We conclude that P6 of NTHi is likely inserted into the OM in two distinct orientations, with the predominant orientation facing in toward the periplasm.     

Otodectes cynotis (Acari: Psoroptidae): examination of survival off-the-host under natural and laboratory conditions

The biological and environmental factors affecting survival off-the-host of Otodectes cynotis (Acari: Psoroptidae) ear mites were investigated under natural and laboratory conditions. From November 2000 to November 2002 mites were collected monthly from cats and divided into four groups according to sex and stage. In laboratory conditions, the mites were placed in an incubator with a steady 95% relative humidity (r.h.), at 10 °C. All the plates were examined by stereomicroscopy every 24 h until all the mites had died. The data were analysed statistically by multiple linear regression and survival analysis. At 10 °C, the maximum survival time of mites was between 15 and 17 days, while at 34 °C, it was between 5 and 6 days. The maximum survival time of adult females was significantly longer than that of other stages. No differences were observed in maximum survival times of mites that had been offered food and those that had not, or in the time (in days) to reach 50% mortality (LT50). When exposed to environmental conditions, the maximum survival time (12 days) was observed at temperatures ranging from 12.3 to 14.2 °C and r.h.s between 57.6 and 82.9%. Multiple regression analysis showed that temperature alone influenced the maximum survival time and LT50 of mites, and that the rate of survival declined linearly with increasing mean temperature. This basic understanding of off-host survival suggests that, places which have been inhabited by infected animals may need to be disinfected or remain vacated for at least 12 days before occupancy by clean cats or dogs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Musca domestica is not a vector of Thelazia callipaeda in experimental or natural conditions

Thelazia callipaeda Railliet and Henry (Spirurida: Thelaziidae), commonly called oriental eyeworm for its widespread presence in the Far East, has been recently found to affect dogs, cats and foxes in northern and southern Italy. Although the biology of T. callipaeda in the definitive hosts has been recently investigated, many doubts still remain about its biology in insect vectors. It has been suggested that more than one species of Diptera, namely Musca domestica Linnaeus (Diptera: Muscidae) and Amiota okadai Maca (Diptera: Drosophilidae), may be involved in the transmission of T. callipaeda in China. The aim of the work described here was to verify the role of M. domestica as a vector of T. callipaeda both in experimental and natural conditions. A total of 310 m. domestica (Group 1) were put in a cage and allowed to feed for 14 days around the eyes of a dog naturally infected by T. callipaeda. Ten flies were collected daily for 14 days. A total of 149 houseflies (Group 2) were fed with T. callipaeda first stage larvae (L1) and dissected at 1, 2 and 7 days post-infection. From June to August 2003, flies were netted (Group 3) in two different sites every 10 days both from the environment and directly from the periocular region of dogs affected by thelaziosis. Musca flies were examined for eyeworms by dissection and visual inspection of house flies (Groups 1 and 2) and using a molecular approach (Groups 1-3) via a specific amplification of the ribosomal internal transcribed spacer 1 (ITS1) sequence of T. callipaeda. On the whole, 180 pools of M. domestica flies were processed molecularly and all the experimentally infected flies (Groups 1 and 2) were found to be negative both at the visual dissection and at the molecular assay. Similarly, the 234 M. domestica collected from Group 3 were negative for T. callipaeda. The results clearly suggest that M. domestica is unlikely to act as a vector of T. callipaeda in southern Europe, in contrast with a single previous report.     

Otodectes cynotis (Acari: Psoroptidae): examination of survival off-the-host under natural and laboratory conditions

The biological and environmental factors affecting survival off-the-host of Otodectes cynotis (Acari: Psoroptidae) ear mites were investigated under natural and laboratory conditions. From November 2000 to November 2002 mites were collected monthly from cats and divided into four groups according to sex and stage. In laboratory conditions, the mites were placed in an incubator with a steady 95% relative humidity (r.h.), a 10 degrees C. All the plates were examined by stereomicroscopy every 24 h until all the mites had died. The data were analysed statistically by multiple linear regression and survival analysis. At 10 degrees C, the maximum survival time of mites was between 15 and 17 days, while at 34 degrees C, it was between 5 and 6 days. The maximum survival time of adult females was significantly longer than that of other stages. No differences were observed in maximum survival times of mites that had been offered food and those that had not, or in the time (in days) to reach 50% mortality (LT50). When exposed to environmental conditions, the maximum survival time (12 days) was observed at temperatures ranging from 12.3 to 14.2 degrees C and r.h.s between 57.6 and 82.9%. Multiple regression analysis showed that temperature alone influenced the maximum survival time and LT50 of mites, and that the rate of survival declined linearly with increasing mean temperature. This basic understanding of off-host survival suggests that, places which have been inhabited by infected animals may need to be disinfected or remain vacated for at least 12 days before occupancy by clean cats or dogs.     

Utility of mitochondrial and ribosomal genes for differentiation and phylogenesis of species of gastrointestinal bot flies

Larvae of Gasterophilus spp. (Diptera: Oestridae) cause gastrointestinal myiasis of equids. However, their identification may be problematic due to morphological similarities between species infesting identical regions of the digestive tract. In this study, genes encoding for mitochondrial cytochrome oxidase I (COI) and for the 16S and 28S ribosomal subunits of the most commonly encountered Gasterophilinae subfamily species [i.e., Gasterophilus haemorrhoidalis (L.), Gasterophilus inermis (Brauer), Gasterophilus intestinalis (De Geer), Gasterophilus nasalis (L.), and Gasterophilus pecorum (F.)] were studied, together with Gyrostigma pavesii (Corti), a rhinoceros parasite, and Hypoderma lineatum (De Villers), as outgroup taxa. Analysis identified interspecific differences that allowed their unequivocal identification. The high genetic homology among the sequences of G. haemorrhoidalis and G. intestinalis (i.e., 100, 99.86, and 99.46% in the 28S, COI, and 16S genes, respectively) strongly support the hypothesis that they are morphotypes of the same species. Phylogenetic analyses (maximum-likelihood and parsimony) were performed using PAUP; all analyses supported monophyly of subfamily Gasterophilinae. This study confirms the utility of the COI and 16S and 28S rRNA genes to address diagnostic and phylogenetic questions in Gasterophilus species.