Calcium regulation of growth and differentiation of normal human keratinocytes: modulation of differentiation competence by stages of growth and extracellular calcium

In this study we examined the different aspects of the pathway leading to the differentiation of keratinocytes as a function of time in culture and calcium concentration of the culture medium. Human neonatal foreskin keratinocytes were grown in a serum-free, defined medium containing 0.07, 1.2, or 2.4 mM calcium and assayed for the rate of growth and protein synthesis, involucrin content, transglutaminase activity, and cornified envelope formation at preconfluent, confluent, and postconfluent stages of growth. We observed that keratinocytes grown to postconfluence in all calcium concentrations showed an increased protein/DNA ratio and an increased rate of membrane-associated protein synthesis. Extracellular calcium concentrations did not have a clear influence on these parameters. However, preconfluent and confluent keratinocytes grown in 0.07 mM calcium showed markedly retarded differentiation at all steps, i.e., involucrin synthesis, transglutaminase activity, and cornified envelope formation. Within 1 week after achieving confluence, these keratinocytes began synthesizing involucrin and transglutaminase and developed the ability to form cornified envelopes. Cells grown in 1.2 and 2.4 mM calcium synthesized involucrin and transglutaminase prior to confluence and were fully competent to form cornified envelopes by confluence. Thus external calcium-regulated keratinocyte differentiation is not an all or none phenomenon, but rather it is the rate at which keratinocytes differentiate that is controlled by calcium. We conclude that either or both higher extracellular calcium concentration and the achievement of cell-cell contacts lead to a coordinate increase of at least two precursors--involucrin content and transglutaminase activity--required for cornified envelope formation. We speculate that a critical level of cytosolic calcium, achieved by increased extracellular calcium or by achievement of intercellular communication established by cell-cell contact, may trigger mechanisms required for initiation of keratinocyte differentiation.     

Occurrence of yeasts in psittacines droppings from captive birds in Italy

Three-hundred twenty five droppings from parrots raised in the premises of 4 breeders and in several private households were cultured for yeasts. One-hundred sixty droppings (49.2%) resulted positive. From these specimens 212 isolates belonging to 27 different species were obtained. Mainly Candida species such as C. albicans, C. catenulata, C. curvata, C. famata, C. glabrata, C. guilliermondi, C. holmii, C. intermedia, C. krusei, C. lambica, C. lusitaniae, C. membranaefaciens, C. parapsilosis, C. pelliculosa, C. sake and C. valida were isolated. Debaryomyces marama, D. polymorphus, Geotrichum sp., Pichia etchelsii, P. ohmeri, Rhodotorula glutinis, R. rubra, Rhodotorula sp., Saccharomyces cerevisiae, S. kluyiveri and Zygosaccharomyces sp. were also obtained. Dark colonies on Staib medium were never observed. The psittacine birds apparently serve as carriers for several Candida species or their perfect states and to a lesser extent for other opportunistic yeasts such as Rhodotorula, Trichosporon and Saccharomyces spp., which are considered part of the transient microbiota of the gastrointestinal tract. The most striking finding was the absence of Cryptococcus spp. among the isolates. The present survey confirms the role of pet birds in carrying potential zoonotic yeasts. This revised version was published online in June 2006 with corrections to the Cover Date.     

A simple duplex-PCR protocol for routine diagnosis and follow up of canine leishmaniasis

The introduction of PCR has efficiently improved diagnosis of canine leishmaniasis. In order to provide a robust, efficient and reliable diagnostic method, a duplex PCR assay targeting the Leishmania infantum kDNA minicircle and the canine GAPDH gene as inner control was designed. Sensitivity of the assay reached 0.15 parasites/ml blood. Development, testing and application of this system on a group of 10 dogs during therapy administration (60 days) are also described. Six dogs (out of eight that have been showing a positive PCR result on peripheral blood during the study) were tested negative at day 62, indicating a reduction of parasitaemia at the end of the treatment period. All the animals had a positive PCR on lymph node aspirate both at the beginning and at the end of treatment. These findings seem to suggest that, in order to test therapy efficacy, PCR on whole blood could be a useful assay in dogs that have a positive PCR at the beginning of the treatment, while PCR positivity on lymph nodes lasts longer than the observation period during therapy administration. The presence of the GAPDH inner control band efficiently contributed to prevent false negatives, by highlighting samples affected by haemoglobin inhibition or inappropriate DNA isolation.     

Survey of keratinophilic fungi isolated from city park soils of Pisa,Italy

A survey of geophilic dermatophytes and related keratinophilic fungi isolated from city park soils of Pisa is reported. Twenty-three (48%) soil samples out of 48 were positive by hair baiting. The following species were isolated: Microsporum gypseum (39%), Trichophyton ajelloi (31%), Chrysosporium keratinophilum (14%), T. terrestre (8%), M. fulvum, Ch. luteum, Ch. indicum (5% each) and M. cookei (2%). The presence of the different species is discussed in relation to the risk of fungal skin infections. This revised version was published online in June 2006 with corrections to the Cover Date.     

Extracellular enzymatic activity ofMicrosporum canis isolates

The enzymatic activity of 70 feline and canineMicrosporum canis isolates was determined by the Api-Zym^® test. The liquid phase of cultures, inoculated into Tryptic Soy Broth, was used to examine 19 enzymes. Considerable differences were observed among the extracellular enzymatic patterns. All the isolates produced alkaline phosphatase and beta-glucosidase, while lipase (C14), trypsin, chymotrypsin, beta-glucuronidase, and alpha-fucosidase activity was never revealed. Esterase (C4) activity was present in 57 samples (81%), esterase lipase (C8) in 31 (44%), leucine arylamidase in 35 (50%), valine arylamidase and cystine arylamidase in 7 (10%), acid phosphatase in 64 (91%), naphthol-AS-BI-phosphohydrolase in 60 (86%), alpha-galactosidase in 5 (7%), beta-galactosidase in 6 (8%), alpha-glucosidase in 25 (36%), N-acetyl-beta-glucosaminidase in 41 (58%), and alpha-mannosidase in 51 (73%). The beta-galactosidase activity ofM. canis has not been reported previously. Remarkable variations of intensity for each enzymatic activity were also detected. It is believed that these results could provide basic data for further investigations on the pathogenic role of enzymes secreted byM. canis.     

Aspergillosis in Larus cachinnans micaellis: Survey of Eight Cases

Avian aspergillosis is reported in several avian species, with Aspergillus fumigatus as the main aetiological agent. Predisposing factors such as starvation, thermal stress, migratory stress, primary infectious disease or toxicosis may play a role. Eight cases of disseminated aspergillosis in free ranging seagulls sheltered at C.R.U.M.A. (Centro Recupero Uccelli Marini e Acquatici, Livorno, Italy) with different clinical histories are presented. The infection was demonstrated by cultural and histological methods from lesions of all birds, and the presence of airborne A. fumigatus viable elements ranging from 450 to 525 CFU/m³ inside and outside the shelter by means of a surface air sampler (SAS) Super-90 was also assessed. The role of this fungal species as an opportunistic factor in the captivity of seagulls is considered and some control measures, such as a clean and stress free environment and the use of antifungal drugs are suggested.     

Feline leishmaniasis: what's the epidemiological role of the cat?

Feline leishmaniasis (FL) is a quite uncommon feature. Clinical disease has been described in cats since nineties begin. More than 40 reports in world literature have been referred, but the clinical cases have been only recently well defined. Most of the reports focus on infected cats living in endemic areas, even if, more recently FL due to Leishmania infantum was found in Sao Paulo State, in Brazil where autochthonous human or canine leishmaniasis cases have never reported. In Europe clinical cases of FL have been described from Portugal, France, Spain and Italy from 1996 to 2002. When a typing of the etiological agent was performed L. infantum was identified in all reported cases. In some endemic areas serological surveys have also been carried out in cats, using IHAT in Egypt, Western blot in France or IFAT in Italy. Sixty Egyptian cats had low serological antibody titers, from 1/32 to 1/128, in the endemic focus of canine leishmaniasis of Alpes Maritimes 12 out of 97 (12.5%) cats showed antibodies versus antigens 14 and/or 18 kDa of L. infantum. A previous survey by means of IFAT in Liguria and Toscana on 110 and 158 feline sera respectively reports a seroprevalence of 0.9% with low titer, while sera from Sicily seem to be positive at higher dilutions. Animals living in an endemic area can develop specific antibodies against leishmania and, in our experience, they can be evidentiated by means of IFAT. The antibody titers appear to be lower in affected cats than in dogs, even if the number of clinical cases is very scanty. PCR tests on feline blood samples are in progress, but preliminary results confirm the presence of leishmania DNA in such specimens. Cutaneous leishmaniasis is the more frequent form in cats and it was reported from several countries. Typical signs include nodular to ulcer or crusty lesions on the nose, lips, ears, eyelids, alopecia: clinical signs of cutaneous FL are unspecific and in endemic area this infection must be taken into account. Visceral leishmaniasis is not common in cats: this form shows visceral involvement: liver and spleen are interested, with lymph nodes and kidney. The cat probably has to considerate to play an active role in the disease, in contrast to goats, calves and horses who could act as accidental reservoirs of leishmania, while sheep appears to be not susceptible to experimental infection. In endemic foci for kala-azar in Sudan cows, goats and donkeys had a high prevalence of specific antibodies. Recently in Europe sporadic cases of equine leishmaniasis have been reported: L. infantum was the causative agent. Equine leishmaniasis appears as a self-healing skin-dwelling disease, with a massive accumulation of parasites. The animals do not often show detectable specific antibodies and recover without any chemotherapy. Untreated affected cats can frequently die and we also observed lymph nodes and blood involvement indicating a spread of leishmania in feline hosts. The epidemiological role of the cat has never been clarified due also to lack of xenodiagnosis trials. This species is believed to have a high degree of natural resistance, as observed following experimental infection. Some of the affected cats were FIV and/or FeLV positive and these viroses such as stress may induce an impaired cellular immune response, even if leishmania infected cat was not submitted to CD4+, CD8+ lymphocyte counts nor other immunological test. However the resistance of the cat to leishmania infection probably depends on genetic factors, not strictly related to cell mediated immunity, taking into account the high seroprevalence of FIV infections (30%) in our country versus the number of clinical cases.     

Dermatophytes isolated from symptomatic dogs and cats in Tuscany,Italy during a 15-year-period

Between January, 1, 1986 and December, 31, 2000, dermatological specimens from 10.678 animals (7.650 cats and 3.028 dogs) were examined for dermatophytes. All the animals presented clinical signs of ringworm. Two thousand-four hundred fifty-six of the 10.678 (23%) examined animals scored positive for dermatophytes, 566 out of 3.028 canine (18.7%) and 1890 out of 7.650 feline specimens (24.7%). Microsporum canis constituted 83% and 97% of the isolated dermatophytes respectively in dogs and cats, M. gypseum represented 13% and 2.6% and T. mentagrophytes 5.5% and 0.2%. A sexual predisposition for mycotic infections was not observed. The animals with less than 1 year of age were more frequently infected. Canine toy breeds showed a significantly higher (P < 0.001) prevalence of infections by M. canis. Microsporum gypseum was mostly recorded from sporting (hunting) breeds [such as T. mentagrophytes (6.7%)]. Microsporum canis was isolated from long-haired cats with a ratio of 2:1 versus short-haired cats, while M. gypseum and T. mentagrophytes were never recovered from Persian cats. The annual distribution of the infections in dogs showed a significantly higher incidence for M. gypseum in summer versus winter and spring, while the recovery rate of M. canis from cats was very significantly higher in fall and winter than in summer and spring. Trichophyton mentagrophytes did not show a similar seasonal distribution.     

Use of monoclonal antibody 225.28S in the study of nevus cells in culture

The aim of this work is to identify and purify cultured nevus cells. We used for our study monoclonal antibody 225.28S. This antibody reacts with a surface antigen which is expressed by nevus cells and melanoma cells, but it does not react with normal melanocytes. We studied 10 dermic nevi and we observed that antigenic determinant survives in cultured nevus cells. These results allowed us to employ the method of panning for purify cultured nevus cells.     

Identification and seasonal distribution of airborne fungi in three horse stables in Italy

Fungal agents are responsible for a variety of respiratory diseases both in humans and animals. The nature and seasonal variations of fungi have been investigated in many environments with wide ranging results. The aims of the present report were (i) to evaluate the quality and magnitude of exposure to airborne fungi in three differently structured equine stalls (open air, partially and completely enclosed buildings) during a one-year period, using an air sampling technique and (ii) to compare the distribution and frequency of fungal species, with regards to these different environments. Air samples were collected monthly from December 2001 to November 2002 by means of a surface air sampler (SAS) Super-90, (PBI International, Milan, Italy). Penicillium and Aspergillus spp. were cultured from all the stables in all seasons. Mucoraceae were also recovered in all seasons in stalls 1 and 2, while they were not isolated in spring and fall in stall 3. These fungi were detected in 28.4%, 72.9% and 60.5% of the total number of samples, respectively. Other fungal genera such as Alternaria, Cladosporium, Fusarium, Beauveria and Drechslera were also occasionally recovered.Viable fungal concentrations varied greatly, ranging from below the limit of detection to more than 3000 CFU/m³ for stables 1 and 2, and 1750 CFU/m³ for stable 3. The median fungal concentration was approximately 178 CFU/m³. Total fungal concentration appeared to be highest in summer, winter and spring, and lowest in the fall.