The identification of potentially pathogenic and therapeutic epitopes from common human allergens

ObjectivesTo outline the processes involved in large-scale T-cell epitope identification from common allergens and illustrate their relevance to development of allergy specific immunotherapy.Data SourcesA set of studies recently published by our laboratory illustrating high-throughput identification of allergen specific T-cell epitopes.Study SelectionT-cell responses contribute both directly and indirectly to allergy-related disease. However, the molecular targets (epitopes) recognized by allergen-specific T cells are largely undefined. We review several different studies in the last 2 years that identified novel T-cell epitopes from a panel of 32 different allergen sources.ResultsAllergen-specific T-cell responses are highly heterogeneous. Epitopes prevalently recognized in allergic patients are often capable of binding to multiple HLA class II molecules. This feature can be used to predict these promiscuous epitopes by bioinformatic predictions. This approach was validated in the Timothy grass system and then applied to a panel of 31 other allergen sources.ConclusionT-cell epitopes for common allergens have been identified, and a general method to identify epitopes from additional allergens has been validated. Characterization of epitopes for common allergens might enable new diagnostics and immunotherapy regimens. These data will also allow the study of T-cell responses in different patient populations and throughout disease progression.     

Loofah sponges as reservoirs and vehicles in the transmission of potentially pathogenic bacterial species to human skin.

Loofah sponges are natural products used as exfoliative beauty aids. As a consequence of tracing a case of Pseudomonas aeruginosa folliculitis to a contaminated loofah sponge, we assessed the role of loofah sponges in supporting the growth of a wide variety of bacterial species. Our data show growth enhancement of sterile loofah fragments for numerous gram-negative (Pseudomonas, Xanthomonas, and Klebsiella) and gram-positive (Enterococcus and group B Streptococcus) species of human and environmental origin. Furthermore, hydrated new, unused loofah sponges undergo a shift in bacterial flora from sparse colonies of Bacillus spp. and Staphylococcus epidermidis to a predominantly gram-negative flora. The growth-promoting potential of loofah sponges (and other exfoliatives) can be further augmented by desquamated epithelial cells entrapped in the loofah fibrous matrix. Therefore, as loofah sponges (and other exfoliatives) can serve as a reservoir and a vehicle for the transmission of potentially pathogenic species to the human skin, we recommend their decontamination with hypochlorite (10%) bleach at regular intervals.     

Isolation and genotyping of potentially pathogenic Acanthamoeba and Naegleria species from tap-water sources in Osaka, Japan

Here, we carried out a survey to determine the prevalence of free-living amoebae (FLA) in tap-water sources from rivers and water treatment plants located in Osaka Prefecture, Japan. A total of 374 raw water samples were collected from 113 sampling points. The samples were filtrated and transferred to non-nutrient agar plates seeded with a heat-killed suspension of Escherichia coli and incubated for 2 to 7 days at 30°C or 42°C. The plates were examined by microscopy to morphologically identify FLA families, and polymerase chain reaction and sequence analysis were then performed to define the species of the detected Naegleria and Acanthamoeba isolates. A total of 257 of 374 samples (68.7%) were positive for FLA by microscopy, and among these there were 800 FLA isolates, including Acanthamoeba and Naegleria species. Sequence analysis identified five Acanthamoeba spp. isolates of the known pathogenic T4 genotype and 43 Naegleria australiensis isolates, a reported pathogen to mice and also of concern as a potential pathogen to humans. Our results suggest a wide distribution of FLA, including potential pathogenic species, in tap-water sources of western Japan.     

Diversity of the 32-kilodalton protein gene may form a basis for species determination of potentially pathogenic mycobacterial species.

In this study, partial gene sequences of the mycobacterial 32-kDa protein gene were determined by PCR-based sequencing. A total of 50 strains representing 18 potentially pathogenic mycobacterial species were studied. In 10 cases, all three strains of the species studied were identical, and intraspecies variability was found in 6 cases. Thus, the 32-kDa protein gene may be a good target for identification of mycobacteria by PCR-based sequencing.     

Simultaneous detection of four human pathogenic microsporidian species from clinical samples by oligonucleotide microarray

Microsporidian species have been rapidly emerging as human enteric pathogens in immunocompromised and immunocompetent individuals in recent years. Routine diagnostic techniques for microsporidia in clinical laboratories are laborious and insensitive and tend to underestimate their presence. In most instances, they are unable to differentiate species of spores due to their small sizes and similar morphologies. In this study, we report the development of another protozoan oligonucleotide microarray assay for the simultaneous detection and identification to the species level of four major microsporidian species: Enterocytozoon bieneusi, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis. The 18S small-subunit rRNA gene was chosen as the amplification target, labeled with fluorescence dye, and hybridized to a series of species-specific oligonucleotide probes immobilized on a microchip. The specificity and sensitivity of the microarray were clearly demonstrated by the unique hybridization profiles exhibited by each species of microsporidian tested and its ability to detect as few as 10 spores. In order to assess the applicability of this microarray in a clinical setting, we conducted microarray assays of 20 fecal samples from AIDS patients. Twelve of these samples were positive for the presence of microsporidia and could be confidently identified; 11 of them were positive for more than one species. Our results suggested that this microarray-based approach represents an attractive diagnostic tool for high-throughput detection and identification of microsporidian species in clinical and epidemiological investigations.     

Thermophilic potentially pathogenic amoebae isolated from natural water bodies in Poland and their molecular characterization

The free-living amoebae (FLA) may live in the environment and also within other organisms as parasites and then they are called amphizoic. They are potentially pathogenic for humans and animals and are found in water that is a source of infection. The aim of this study was molecular detection and identification of these FLA in natural water bodies in North-Western Poland to evaluate the risk of the pathogenic amoebae infections. We examined surface water samples collected from 50 sites and first, the tolerance thermic test was performed in order to select thermophilic, potentially pathogenic strains. For molecular identification of FLA, regions of 18S rDNA, 16S rDNA and intergenic spacers were amplified. Acanthamoeba T4 and T16 genotypes of 18S rDNA gene and 18S rDNA of H. vermiformis were detected. We identified two variants of Acanthamoeba T4 genotype, two variants of Acanthamoeba T16 genotype and one variant of H. vermiformis. Identification of the T16 genotype and H. vermiformis in water was for the first time in Poland. Additionally, we made attempts to adapt the RLB method for detection and differentiation of FLA species and strains. PCR seems to be more sensitive than RLB hybridization, though.     

Effectiveness of selected disinfectant and antiseptic preparations on potentially pathogenic micromycetes

Using the contact test, the author investigated the antifungal efficiency of 13 disinfectants and antiseptic preparations (hydrogen peroxide, Persteril, Jodonal B, Jodisol, Chlordetal, Famosept-Super, Septonex, Ajatin, Cresolum saponatum, ethanol, Desident spray, chlorhexidine, Galli-Valerio solution) against 23 strains of eight genera of microscopic fungi (Absidia, Aspergillus, Candida, Geotrichum, Mucor, Rhizopus, Torulopsis and Trichosporon). The results revealed that filamentous fungi and among them in particular zygomycetes are more resistant against the above preparations than yeasts. The highest antifungal activity was observed in 5% Creolum saponatum and 1% and 2% Famosept Super. On the other hand, 3% H2O2, 5% Jodisol and 0.1% Chlorhexidine were almost ineffective. On investigations of the vitality of spores and fragments of the mycelium of the tested micromycetes the greatest resistance to desiccation was manifested by conidia and hyphae of aspergilli, the smallest one by blastopores of yeasts Candida albicans and Torulopsis glabrata.     

A comparative analysis of pilin genes from pathogenic and nonpathogenic Neisseria species

Pathogenic Neisseria species elaborate type IV pili, which are considered important for virulence. In this study, we examined pilin-encoding expression loci (pilE) in nonpathogenic Neisseria species. PCR based screening detected homology to a conserved N-terminal region of pilE in 12 of 15 Neisseria species, including all human commensal isolates. The three species failing to display homology were isolated from nonhuman sources. We have also characterized complete pilE loci from the human commensal species N. lactamica and N. cinerea. As anticipated, the predicted protein sequences from these species display features typical of all type IV pilins. In addition, these commensal pilins possess two highly conserved regions, SV2 and CYS2, which are shared among all neisserial pilins. However, a comparative analysis of pilE loci from pathogenic and nonpathogenic Neisseria species reveals two distinct structural groups, one composed of the pilin genes from N. lactamica, N. cinerea, and the class II pilin-producing subset of N. meningitidis isolates, the other of gonococcal and meningococcal class I pilin-encoding genes. Since both class I and class II pilin-producing meningococci can act as pathogens, structural relationships among neisserial pilin genes do not obviously reflect either species membership or ability to cause human disease.     

Corynebacterium tuberculostearicum: a potentially misidentified and multiresistant Corynebacterium species isolated from clinical specimens

Corynebacterium tuberculostearicum is a lipophilic corynebacterium validly characterized in 2004. We provide clinical information on 18 patients from whom this organism was isolated. The majority of the patients were hospitalized and had a history of prolonged treatment with broad-spectrum antimicrobials. In 7 (38.9%) of the 18 cases, the isolates were found to be clinically relevant. The present report also includes detailed data on the biochemical and molecular identification of C. tuberculostearicum, as well as its identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Our data demonstrate that routine biochemical tests do not provide reliable identification of C. tuberculostearicum. MALDI-TOF MS represents a helpful tool for the identification of this species, since all of the strains matched C. tuberculostearicum as the first choice and 58.3% (7/12) of the strains processed with the full extraction protocol generated scores of >2.000. Nevertheless, partial 16S rRNA gene sequencing still represents the gold standard for the identification of this species. Due to the challenging identification of C. tuberculostearicum, we presume that this organism is often misidentified and its clinical relevance is underestimated. The antimicrobial susceptibility profile of C. tuberculostearicum presented here reveals that 14 (87.5%) of the 16 strains analyzed exhibited multidrug resistance.     

Isolation and molecular characterization of potentially pathogenic Acanthamoeba genotypes from diverse water resources including household drinking water from Khyber Pakhtunkhwa, Pakistan

Acanthamoeba, an opportunistic protozoan pathogen, is ubiquitous in nature, and therefore plays a predatory role and helps control microbial communities in the ecosystem. These Acanthamoeba species are recognized as opportunistic human pathogens that may cause blinding keratitis and rare but fatal granulomatous encephalitis. To date, there is not a single report demonstrating Acanthamoeba isolation and identification from environmental sources in Pakistan, and that is the aim of this study. Acanthamoeba were identified by morphological characteristics of their cysts on non-nutrient agar plates seeded with Escherichia coli. Additionally, the polymerase chain reaction (PCR) was performed with genus-specific primers followed by direct sequencing of the PCR product for molecular identification. Furthermore, our PCR and sequencing results confirmed seven different pathogenic and nonpathogenic genotypes, including T2–T10, T4, T5, T7, T15, T16, and T17. To the best of our knowledge, we have identified and isolated Acanthamoeba sp., for the first time, from water resources of Khyber Pakhtunkhwa, Pakistan. There is an urgent need to address (1) the pathogenic potential of the identified genotypes and (2) explore other environmental sources from the country to examine the water quality and the current status of Acanthamoeba species in Pakistan, which may be a potential threat for public health across the country.     

Urease from a potentially pathogenic coccoid isolate: purification, characterization, and comparison to other microbial ureases.

Strain SL100 is a gram-positive coccoid isolate prototype with an adhesin specific for gastric mucin and is representative of potentially pathogenic organisms obtained at biopsy from patients with gastric disorders. The urease of this isolate constitutes a significant fraction of the total cell protein, and the outcome of the purification strategy described herein suggests that it is associated with a cell wall fraction. The urease was purified 138-fold to apparent homogeneity, as indicated by gel electrophoresis, to a specific activity of 1,120 U/mg. The urease was unstable during purification in the absence of nickel, which is present in a metallocenter in other microbial ureases. When nickel sulfate was present during growth (5 microM) and in buffers during sonication and purification (100 microM), the urease was completely stable at room temperature during the purification procedure. The native urease was approximately 260 kDa and was composed of three subunits of 65 kDa and three subunits of 21 kDa. The purified urease was relatively stable in acid and retained most of its activity after incubation for 30 min at pH 1.3. The K(m)s for urease measured from whole cells and for the purified enzyme were 0.56 and 1.7 mM, respectively, indicating that some cell wall component(s) affects the affinity of the enzyme for urea. The V(max)s for urea hydrolysis measured from whole cells and for the purified enzyme were 8.1 and 1,120 mol/min/mg of protein, respectively. The kinetic parameters, relative abundance, and subunit composition are more similar to those of the ureases of Helicobacter than to those of the ureases of other microbial species. These similarities are consistent with an adaptation of this organism to colonization of the stomach and indicate that the urease may be a virulence factor during colonization.     

Molecular ecology and pathogenic potential of Fonsecaea species.

The genus Fonsecaea is revised on the basis of ribosomal DNA internal transcribed spacer (ITS) sequence data. Two species are recognized, F. pedrosoi and the new defined F. monophora. The distinction between these species does not correspond with the classical distinction of F. pedrosoi and F. compacta. The latter appears to be no more than a morphological variant. Both species recognized in this study are agents of human chromoblastomycosis; however, in F. pedrosoi a strict association with this disease is noted, while F. monophora is a more general opportunist. Subspecific randomly amplified polymorphic DNA (RAPD) typing revealed a high degree of strain diversity, although clonal reproduction is also likely to occur. Most strains with Fonsecaea-like morphology isolated from environments to which symptomatic human patients were exposed were found to be more closely related to species of Cladophialophora than to Fonsecaea.     

Monozygotic twins discordant for epilepsy differ in the levels of potentially pathogenic autoantibodies and cytokines

Correcting errors and proofreading are crucial in a post-genomic era, when DNA sequences are already part of an effective medical screening and treatments. SNPs genotyping of complex human genes can lead to questionable associations if not properly handled. Here, we report about a spurious (reitered) association between FCGR2B genetic polymorphisms and systemic lupus erythematosus.     

Monozygotic twins discordant for epilepsy differ in the levels of potentially pathogenic autoantibodies and cytokines

Can autoantibodies (Ab's) and cytokines play a role in epilepsy?Monozygotic twins discordant for epilepsy (most probably Rasmussen's encephalitis (RE)), compared to 49 neurologically intact controls, were both found to contain in their serum (at the time of epilepsy diagnosis) significantly elevated levels of specific Ab's against peptide B (amino acids 372-395) of the ionotropic glutamate receptor of AMPA subtype 3 (i.e. GluR3B peptide). Interestingly, both twins also had clinically elevated levels of Ab's to double-stranded (ds) DNA, glutamic acid decarboxylase, nuclear antigens, beta2-glycoprotein I and cardiolipin, as in "classical" autoimmune diseases. Both twins also had significantly elevated levels of IFNgamma, TNFalpha, IL-4 and IL-10 in the serum, compared to the controls. Comparing the twins revealed that the epileptic twin had significantly higher levels of five of the above anti-self Ab's, but significantly lower levels of all four cytokines compared to her healthy sister. Importantly, the epileptic twin, alike three other RE patients tested herein, contained elevated levels of Ab's to GluR3B and dsDNA also in cerebrospinal fluid (CSF) (unavailable of the healthy twin). Our results suggest that the various autoimmune Ab's studied herein, all of which are known already to have a potential to be pathogenic in the nervous system and/or peripheral organs, may play a role in some types of epilepsy. The titer of such Ab's and of key cytokines may be crucial for either facilitating or arresting the development of epilepsy. Our findings also show that anti-GluR3B Ab's in serum are not necessarily detrimental (their presence in the CSF may be more dangerous), and that they are not a mere side effect of already existing epilepsy, as they were found herein in serum of a healthy individual. These findings and suggestions may be of clinical importance and call for further studies.     

Effects of freezing and storage temperature on MS2 viability

Monitoring human enteric virus levels in domestic wastewater effluent is crucial to protecting human health. Occasionally, during intensive sampling, wastewater samples must be stored for later viral analysis. Little data exist regarding how enteric viruses survive during storage at different temperatures in secondary treated wastewater. During a field-scale study assessing pathogen removal performance by various onsite treatment technologies, the MS2 bacteriophage, an indicator of enteric viruses, was inoculated into septic tank (STE), sand filter, peat filter and constructed wetland (CW) effluents to determine virus decay at various storage temperatures. Virus stored at temperatures > or =10 degrees C and at -20 degrees C decayed nearly twice as fast as those stored at 4 degrees C or -80 degrees C. Decreased water quality decreased viral decay rates at 4 degrees C and -80 degrees C, with slowest decay occurring in STE and the fastest in sterile PBS and low pH peat effluent. In CW effluent after 8 days, less MS2 was inactivated when stored at 4 degrees C (20%) compared to -80 degrees C (58%); however, during extended storage (approximately 300 days), less MS2 was inactivated at -80 degrees C (75%) compared to 4 degrees C (93%). We recommend that viruses in wastewater be stored in the dark at 4 degrees C unless storage for >40 days is necessary.