Marta E. García

Immune response to fungal infections

The immune mechanisms of defence against fungal infections are numerous, and range from protective mechanisms that were present early in evolution (innate immunity) to sophisticated adaptive mechanisms that are induced specifically during infection and disease (adaptive immunity). The first-line innate mechanism is the presence of physical barriers in the form of skin and mucous membranes, which is complemented by cell membranes, cellular receptors and humoral factors. There has been a debate about the relative contribution of humoral and cellular immunity to host defence against fungal infections. For a long time it was considered that cell-mediated immunity (CMI) was important, but humoral immunity had little or no role. However, it is accepted now that CMI is the main mechanism of defence, but that certain types of antibody response are protective. In general, Th1-type CMI is required for clearance of a fungal infection, while Th2 immunity usually results in susceptibility to infection. Aspergillosis, which is a disease caused by the fungus Aspergillus, has been the subject of many studies, including details of the immune response. Attempts to relate aspergillosis to some form of immunosuppression in animals, as is the case with humans, have not been successful to date. The defence against Aspergillus is based on recognition of the pathogen, a rapidly deployed and highly effective innate effector phase, and a delayed but robust adaptive effector phase. Candida albicans, part of the normal microbial flora associated with mucous surfaces, can be present as congenital candidiasis or as acquired defects of cell-mediated immunity. Resistance to this yeast is associated with Th1 CMI, whereas Th2 immunity is associated with susceptibility to systemic infection. Dermatophytes produce skin alterations in humans and other animals, and the essential role of the CMI response is to destroy the fungi and produce an immunoprotective status against re-infection. The resolution of the disease is associated with a delayed hypersensitive response. There are many effective veterinary vaccines against dermatophytoses. Malassezia pachydermatis is an opportunistic yeast that needs predisposing factors to cause disease, often related to an atopic status in the animal. Two species can be differentiated within the genus Cryptococcus with immunologic consequences: C. neoformans infects predominantly immunocompromised hosts, and C. gattii infects non-immunocompromised hosts. Pneumocystis is a fungus that infects only immunosupressed individuals, inducing a host defence mechanism similar to that induced by other fungal pathogens, such as Aspergillus.

Anticoagulants Interfere with PCR Used To Diagnose Invasive Aspergillosis

Invasive aspergillosis is a disease that affects immunodepressed individuals and has become one of the leading causes of death in many transplant centers ([1][1]). Owing to the difficulties encountered in diagnosis of the disease and to the high sensitivity of the PCR technique, PCR-based methods

Prevalence of Clostridium difficile in diarrhoeic and non-diarrhoeic piglets

Clostridium difficile is considered to be an important causative agent of porcine neonatal diarrhoea, having taken over from classic bacterial pathogens. However, there are currently no clear data concerning the prevalence of this microorganism in piglets, or about its relative distributions among diarrhoeic and non-diarrhoeic animals. In the present study, we analyzed the presence of C. difficile in rectal swabs from 780 piglets from two age groups (newborn and 1-2-month-old pigs) by means of molecular and microbiological procedures. Furthermore DNA was isolated from the bacteria in order to identify toxin A and toxin B genes.C. difficile was not found in any of the 239 samples taken from 1- to 2-month-old pigs. Bacteria were, however, recovered from 140 out of 541 newborn piglets (25.9%), including both diarrhoeic and non-diarrhoeic animals, and animals from control farms (free of diarrhoeic animals). Genes for the production of both toxins A and B were identified in 132 of the 140 isolates (A(+)B(+)). Only seven isolates, all from the same farm and from non-diarrhoeic animals, lacked both toxin genes (A(-)B(-)), while one isolate from this same group of animals was A(-)B(+).This study provides the first report comparing the prevalence of C. difficile in large numbers of diarrhoeic and non-diarrhoeic animals. There was no clear link between bacterial isolation and neonatal porcine diarrhoea.

Acetylsalicylic Acid Inhibits Cell Proliferation by Involving Transforming Growth Factor-β

Background—Acetylsalicylic acid (ASA) inhibits cell proliferation. This may be mediated by transforming growth factor-&bgr; (TGF-&bgr;). TGF-&bgr; directly stops cell proliferation, restrains cells in G0, and inhibits the uptake of platelet-derived growth factor and insulin-like growth factor. These effects are identical to those observed with ASA treatment. Methods and Results—We cultured rat thoracic aorta vascular smooth muscle cells and measured cytotoxicity, cell proliferation, cell cycle, transcription of TGF-&bgr;1, and concentration of TGF-&bgr;1 in supernatant medium. ASA dose-dependently restrained cells in G0 phase with no cytotoxic effect and inhibited cell proliferation by 30.86%. Anti–TGF-&bgr;1 reversed this inhibition by 30.21%. However, ASA treatment decreased TGF-&bgr;1 transcription and had no significant effect on TGF-&bgr;1 concentration. Conclusions—TGF-&bgr; seems to play an important role in ASA-mediated inhibition of cell proliferation. Therefore, treatment with ASA prevents coronary disease not only by means of its antiplatelet properties but also by an important inhibition of plaque growth. This relationship between ASA and TGF-&bgr; explains many other effects, such as cancer chemoprevention, immunomodulation, and wound healing. The aim of this study was to demonstrate this link.

Mating type and invasiveness are significantly associated in Aspergillus fumigatus.

In recent years, several lines of evidence have questioned the asexual nature of Aspergillus fumigatus , showing that this fungus possesses a fully functional sexual reproductive cycle that leads to the production of cleistothecia and ascospores. The presence of a sexual cycle in A. fumigatus could have signifi cant medical implications, as sexual reproduction might contribute to increased virulence or resistance to antifungal agents. In the present work, we studied the relationship between mating type and invasiveness in A. fumigatus . Statistical analysis of the results showed a signifi cant association between the mating type MAT1-1 and an invasive origin of the isolates. Similarly, when the clinical or environmental origin of isolates was considered instead of their invasive or non-invasive origin, a signifi cant association between the mating type MAT1-1 and clinical origin was observed. Finally, the association between mating type MAT1-1 and pathogenicity, measured by an Elastase Activity Index � 1, was signifi cant. Our results suggest a possible association between the MAT1-1 mating type and A. fumigatus invasiveness.

Modeling normal and pathological processes through skin tissue engineering.

Skin tissue engineering emerged as an experimental regenerative therapy motivated primarily by the critical need for early permanent coverage of extensive burn injuries in patients with insufficient sources of autologous skin for grafting. With time, the approach evolved toward a wider range of applications including disease modeling. We have established a skin‐humanized mouse model system consisting in bioengineered human‐skin‐engrafted immunodeficient mice. This new model allows to performing regenerative medicine, gene therapy, genomics, and pathology studies in a human context on homogeneous samples. Starting from skin cells (keratinocytes and fibroblasts) isolated from normal donor skin or patients biopsies, we have been able to deconstruct‐reconstruct several inherited skin disorders including genodermatoses and cancer‐prone diseases in a large number of skin humanized mice. In addition, the model allows conducting studies in normal human skin to gain further insight into physiological processes such as wound healing or UV‐responses.

Invasive aspergillosis caused by cryptic Aspergillus species: a report of two consecutive episodes in a patient with leukaemia.

We report a case of two consecutive episodes of invasive aspergillosis caused by cryptic Aspergillus species in a patient with leukaemia. A first episode of pulmonary infection was caused by Aspergillus calidoustus and Aspergillus novofumigatus, and the second episode by A. novofumigatus and Aspergillus viridinutans. Fungal isolates were identified to species level using traditional and sequencing-based molecular methods.

Polyclonal Aspergillus fumigatus infection in captive penguins.

Aspergillus fumigatus is a known opportunistic pathogen of penguins. Most reports of penguin aspergillosis have focused on clinical, histopathological and microbiological findings. The molecular characterization of A. fumigatus isolates involved in invasive infections in penguins has yet to be addressed. The aim of this work was to study the possible coexistence of different A. fumigatus genotypes in five clinical cases of invasive aspergillosis in captive penguins. Differences in other relevant characteristics of the isolates, including mating type and invasiveness, were also considered. Alkaline protease and elastase production by the A. fumigatus isolates was evaluated by plate assays. Random amplified polymorphic DNA, and microsatellite analysis techniques were used for molecular typing, and mating type (MAT1-1 or MAT1-2) was determined by multiplex PCR. Although all isolates showed protease activity, differences in elastase activity were observed. The typing techniques indicated different genotypes in all the penguins, although one genotype was predominant in some cases. Fungal strains of different mating type were found in two different penguins, confirming infection polyclonality. In conclusion, captive penguins are susceptible to infection by multiple strains of A. fumigatus that differ not only in their genotype, but also in mating type and invasiveness. This finding has important consequences for the diagnosis and treatment of avian aspergillosis.

Characterization of multiple isolates of Aspergillus fumigatus from patients: genotype, mating type and invasiveness.

The possible co-existence of different genotypes of Aspergillus fumigatus in the same case was studied in five patients colonized or infected by this opportunistic mould. A total of 22 isolates were typed by random amplified polymorphic DNA (RAPD) and microsatellite analysis. Differences in the mating type and invasiveness of the isolates were also considered. The combination of four arbitrary primers used in RAPD typing differentiated all the isolates. In microsatellite analysis, at least two different genotypes were identified in four of the five patients. The 22 isolates showed elastase activity after 10 days of incubation at 37 degrees C in an elastin-containing medium. The presence of strains of compatible mating type was observed in one of the colonized patients and one of the individuals with invasive aspergillosis. Some isolates that belonged to the same genotype in microsatellite analysis were of a different mating type. Taken together, our results suggest that multiple isolates of A. fumigatus obtained from colonized or infected patients may differ not only in their genotypes, but also in their invasiveness and mating types. Furthermore, mating type determination may be of great assistance in differentiating some isolates, as two isolates of different mating type cannot be genotypically identical.

Shedding of Clostridium difficile PCR ribotype 078 by zoo animals, and report of an unstable metronidazole-resistant isolate from a zebra foal (Equus quagga burchellii)

Clostridium difficile is an emerging and potentially zoonotic pathogen, but its prevalence in most animal species, including exhibition animals, is currently unknown. In this study we assessed the prevalence of faecal shedding of C. difficile by zoo animals, and determined the ribotype, toxin profile and antimicrobial susceptibility of recovered isolates. A total of 200 samples from 40 animal species (36.5% of which came from plains zebra, Equus quagga burchellii) were analysed. C. difficile was isolated from 7 samples (3.5% of total), which came from the following animal species: chimpanzee (Pan troglodytes troglodytes), dwarf goat (Capra hircus), and Iberian ibex (Capra pyrenaica hispanica), with one positive sample each; and plains zebra, with 4 positive samples from 3 different individuals. Most recovered isolates (4/7, 57.1%) belonged to the epidemic PCR ribotype 078, produced toxins A and B, and had the genes encoding binary toxin (i.e. A(+)B(+)CDT(+) isolates). The remaining three isolates belonged to PCR ribotypes 039 (A(-)B(-)CDT(-)), 042 (A(+)B(+)CDT(-)) and 110 (A(-)B(+)CDT(-)). Regardless of their ribotype, all isolates displayed high-level resistance to the fluoroquinolones ciprofloxacin, enrofloxacin and levofloxacin. Some isolates were also resistant to meropenem and/or ertapenem. A ribotype 078 isolate recovered from a male zebra foal initially showed in vitro resistance to metronidazole (MIC ≥ 256 μg/ml), but lost that trait after subculturing on non-selective media. We conclude that zoo animals belonging to different species can carry ribotype 078 and other toxigenic strains of C. difficile showing resistance to antimicrobial compounds commonly used in veterinary and/or human medicine.