Brittle cornea syndrome: recognition, molecular diagnosis and management.

Brittle cornea syndrome (BCS) is an autosomal recessive disorder characterised by extreme corneal thinning and fragility. Corneal rupture can therefore occur either spontaneously or following minimal trauma in affected patients. Two genes, ZNF469 and PRDM5, have now been identified, in which causative pathogenic mutations collectively account for the condition in nearly all patients with BCS ascertained to date. Therefore, effective molecular diagnosis is now available for affected patients, and those at risk of being heterozygous carriers for BCS. We have previously identified mutations in ZNF469 in 14 families (in addition to 6 reported by others in the literature), and in PRDM5 in 8 families (with 1 further family now published by others). Clinical features include extreme corneal thinning with rupture, high myopia, blue sclerae, deafness of mixed aetiology with hypercompliant tympanic membranes, and variable skeletal manifestations. Corneal rupture may be the presenting feature of BCS, and it is possible that this may be incorrectly attributed to non-accidental injury. Mainstays of management include the prevention of ocular rupture by provision of protective polycarbonate spectacles, careful monitoring of visual and auditory function, and assessment for skeletal complications such as developmental dysplasia of the hip. Effective management depends upon appropriate identification of affected individuals, which may be challenging given the phenotypic overlap of BCS with other connective tissue disorders.

Freeze-fracture study of Trichomonas vaginalis

The freeze-fracture technique was used to analyse the organization of the plasma membrane, as well as membranes of cytoplasmic organelles, of the pathogenic protozoan Trichomonas vaginalis. Rosettes formed by 4 to 14 intramembranous particles were seen on the fracture faces of the membrane lining the anterior flagella as well as in fracture faces of the plasma membrane enclosing the anterior region of the protozoan and in cytoplasmic organelles. Special organization of the membrane particles were also seen in the region of association of the recurrent flagellum to the cell body.

Predators promote defence of rhizosphere bacterial populations by selective feeding on non-toxic cheaters.

Soil pseudomonads increase their competitiveness by producing toxic secondary metabolites, which inhibit competitors and repel predators. Toxin production is regulated by cell-cell signalling and efficiently protects the bacterial population. However, cell communication is unstable, and natural populations often contain signal blind mutants displaying an altered phenotype defective in exoproduct synthesis. Such mutants are weak competitors, and we hypothesized that their fitness depends on natural communities on the exoproducts of wild-type bacteria, especially defence toxins. We established mixed populations of wild-type and signal blind, non-toxic gacS-deficient mutants of Pseudomonas fluorescens CHA0 in batch and rhizosphere systems. Bacteria were grazed by representatives of the most important bacterial predators in soil, nematodes (Caenorhabditis elegans) and protozoa (Acanthamoeba castellanii). The gacS mutants showed a negative frequency-dependent fitness and could reach up to one-third of the population, suggesting that they rely on the exoproducts of the wild-type bacteria. Both predators preferentially consumed the mutant strain, but populations with a low mutant load were resistant to predation, allowing the mutant to remain competitive at low relative density. The results suggest that signal blind Pseudomonas increase their fitness by exploiting the toxins produced by wild-type bacteria, and that predation promotes the production of bacterial defence compounds by selectively eliminating non-toxic mutants. Therefore, predators not only regulate population dynamics of soil bacteria but also structure the genetic and phenotypic constitution of bacterial communities.

Viral envelope glycoprotein processing by proprotein convertases.

The proprotein convertases (PCs) are a family of nine mammalian enzymes that play key roles in the maintenance of cell homeostasis by activating or inactivating proteins via limited proteolysis under temporal and spatial control. A wide range of pathogens, including major human pathogenic viruses can hijack cellular PCs for their own purposes. In particular, productive infection with many enveloped viruses critically depends on the processing of their fusion-active viral envelope glycoproteins by cellular PCs. Based on their crucial role in virus-host interaction, PCs can be important determinants for viral pathogenesis and represent promising targets of therapeutic antiviral intervention. In the present review we will cover basic aspects and recent developments of PC-mediated maturation of viral envelope glycoproteins of selected medically important viruses. The molecular mechanisms underlying the recognition of PCs by viral glycoproteins will be described, including recent findings demonstrating differential PC-recognition of viral and cellular substrates. We will further discuss a possible scenario how viruses during co-evolution with their hosts adapted their glycoproteins to modulate the activity of cellular PCs for their own benefit and discuss the consequences for virus-host interaction and pathogenesis. Particular attention will be given to past and current efforts to evaluate cellular PCs as targets for antiviral therapeutic intervention, with emphasis on emerging highly pathogenic viruses for which no efficacious drugs or vaccines are currently available.

Characterisation of microbial communities colonising the hyphal surfaces of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) are symbiotic soil fungi that are intimately associated with the roots of the majority of land plants. They colonise the interior of the roots and the hyphae extend into the soil. It is well known that bacterial colonisation of the rhizosphere can be crucial for many pathogenic as well as symbiotic plant-microbe interactions. However, although bacteria colonising the extraradical AMF hyphae (the hyphosphere) might be equally important for AMF symbiosis, little is known regarding which bacterial species would colonise AMF hyphae. In this study, we investigated which bacterial communities might be associated with AMF hyphae. As bacterial-hyphal attachment is extremely difficult to study in situ, we designed a system to grow AMF hyphae of Glomus intraradices and Glomus proliferum and studied which bacteria separated from an agricultural soil specifically attach to the hyphae. Characterisation of attached and non-attached bacterial communities was performed using terminal restriction fragment length polymorphism and clone library sequencing of 16S ribosomal RNA (rRNA) gene fragments. For all experiments, the composition of hyphal attached bacterial communities was different from the non-attached communities, and was also different from bacterial communities that had attached to glass wool (a non-living substratum). Analysis of amplified 16S rRNA genes indicated that in particular bacteria from the family of Oxalobacteraceae were highly abundant on AMF hyphae, suggesting that they may have developed specific interactions with the fungi.

Cured by tonsillectomy: was it really a PFAPA syndrome?

We show how an ultrafast pump-pump excitation induces strong fluorescence depletion in biological samples, such as bacteria-containing droplets, in contrast with fluorescent interferents, such as polycyclic aromatic compounds, despite similar spectroscopic properties. Application to the optical remote discrimination of biotic versus non-biotic particles is proposed. Further improvement is required to allow the discrimination of one pathogenic among other non-pathogenic micro-organisms. This improved selectivity may be reached with optimal coherent control experiments, as discussed in the paper.

Description of Leishmania equatorensis sp. n. (Kinetoplastida: Trypanosomatidae), a new parasite infecting arboreal mammals in Ecuador

Characterization is given of a new parasite, Leishmania equatoriensis sp.n. wich was isolated from the viscera of a sloth (Choloepus hoffmanni) and a squirrel (Sciurus granatensis), captured in humid tropical forest onthe Pacific Coast of Ecuador. Data based on biological and molecular criteria, as well as numerical zymotaxonomical analysis, indicate that this parasite is a new species of the L. brasiliensis complex. L. equatoriensis is cleary distinguishable form all other known species within this complex, using the following molecular criteria: reactivity patterns with specific monoclonal antibodies, isoenzyme electrophoresis, and restriction-endonuclease fragment patterns of kinetoplast DNA (k-DNA).

Antigenic differences among Leishmania amazonensis isolates and their relationship with distinct clinical forms of the disease

Immunoblot analysis was used to investigate antigenic differences among clinical isolates of Leishmania amazonensis and their role in the etiology of the diseases. Western blots of promastigote homogenates were analyzed with either monoclonal antibodies (MAbs) specific for the L. mexicana complex (M-4, M-6, M-9 and M-11) or polyclonal sera from L. amazonensis infected patients with the various forms of clinical disease. In the case of the MAbs, no significant variation was observed among the strains of L. amazonensis, isolated from cases of cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), diffuse cutaneous leishmaniasis (DCL), visceral leishmaniasis (VL) or post kala-azar dermal leishmaniasis (PKDL), in either the relative morbility (Mr) or the quantitative amount (intensity) of the antigenic determinats. In the case of the sera of the infected patients, the patterns of antigenic reactivity of these strains revealed that, despite showing the presence of shared antigens, differences were observed between some of the antigenic components of the various isolates of L. amazonensis that were recognized by a single serum. Differences were also demonstrated between the antigenic determinants of a single isolate of L. amazonensis that were recognized by the different patient's sera. No apparent association was consistently found, however, between the Mr components identified in these isolates and clinical form of the disease or the geographical area of isolation. In addition, the spectrum of antigens recognized by the sera from patients with the same clinical form were not identical; although in some instances, similar Mr antigens were shared. These results indicate that isolates of L. amazonensis are not antigenically identical (homogeneous) and that the immune responses (antibodies) observed among infected patients are heterogeneous.

Predator-prey chemical warfare determines the expression of biocontrol genes by rhizosphere-associated Pseudomonas fluorescens.

Soil bacteria are heavily consumed by protozoan predators, and many bacteria have evolved defense strategies such as the production of toxic exometabolites. However, the production of toxins is energetically costly and therefore is likely to be adjusted according to the predation risk to balance the costs and benefits of predator defense. We investigated the response of the biocontrol bacterium Pseudomonas fluorescens CHA0 to a common predator, the free-living amoeba Acanthamoeba castellanii. We monitored the effect of the exposure to predator cues or direct contact with the predators on the expression of the phlA, prnA, hcnA, and pltA genes, which are involved in the synthesis of the toxins, 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin, hydrogen cyanide, and pyoluteorin, respectively. Predator chemical cues led to 2.2-, 2.0-, and 1.2-fold increases in prnA, phlA, and hcnA expression, respectively, and to a 25% increase in bacterial toxicity. The upregulation of the tested genes was related to the antiprotozoan toxicity of the corresponding toxins. Pyrrolnitrin and DAPG had the highest toxicity, suggesting that bacteria secrete a predator-specific toxin cocktail. The response of the bacteria was elicited by supernatants of amoeba cultures, indicating that water-soluble chemical compounds were responsible for induction of the bacterial defense response. In contrast, direct contact of bacteria with living amoebae reduced the expression of the four bacterial toxin genes by up to 50%, suggesting that protozoa can repress bacterial toxicity. The results indicate that predator-prey interactions are a determinant of toxin production by rhizosphere P. fluorescens and may have an impact on its biocontrol potential.

Genes associated with retinitis pigmentosa and allied diseases are frequently mutated in the general population.

BACKGROUND: Retinitis pigmentosa and other hereditary retinal degenerations (HRD) are rare genetic diseases leading to progressive blindness. Recessive HRD are caused by mutations in more than 100 different genes. Laws of population genetics predict that, on a purely theoretical ground, such a high number of genes should translate into an extremely elevated frequency of unaffected carriers of mutations. In this study we estimate the proportion of these individuals within the general population, via the analyses of data from whole-genome sequencing. METHODOLOGY/PRINCIPAL FINDINGS: We screened complete and high-quality genome sequences from 46 control individuals from various world populations for HRD mutations, using bioinformatic tools developed in-house. All mutations detected in silico were validated by Sanger sequencing. We identified clear-cut, null recessive HRD mutations in 10 out of the 46 unaffected individuals analyzed (∼22%). CONCLUSIONS/SIGNIFICANCE: Based on our data, approximately one in 4-5 individuals from the general population may be a carrier of null mutations that are responsible for HRD. This would be the highest mutation carrier frequency so far measured for a class of Mendelian disorders, especially considering that missenses and other forms of pathogenic changes were not included in our assessment. Among other things, our results indicate that the risk for a consanguineous couple of generating a child with a blinding disease is particularly high, compared to other genetic conditions.

Impact of Babesia bovis and Babesia bigemina on the production of beef cattle in Uruguay

Uruguay is situated in a marginal area for the development of Boophilus microplus (30- 35- South Lat.) with important areas of enzootic instability for Babesia bovis and B. bigemina. The livestock products represent 70% of our exports, for wich reason it is fundamental to evaluate the losses in the production that these haemoparasites cause as basic information to take future decisions. In the period 1988-1990, several works were carried out by our laboratory to know the incidence of babesiosis in the reduction of liveweight gains. The results are shown and discussed in the work. Experiment I: the weight increase of the control group (x = 0,248kg/day), was 23% higher than that of the infected group with Babesia spp (from Uruguay), but significant statistical differences were not found (P < 0,05). These animals were kept in boxes and the food was controlled for 76 days. Experiment II: the incidence of Babesis spp (same strain ) was studied for 140 on Hereford heifers (n = 14) on natural pastures. The control group obtained x = 25,29kg of liveweight gain and it was 45% higher than that of the infected group, significant statistical difference were found (P < 0,05). Experiments with attenuated strains III: four studies were carried out inoculating B. bovis and B. bigemina in bovines about one year old, in different growth systems, searching for the limit of application. Significant statistical differences between those groups were found during the experiment (about 180 days) (P < 0,05). Experiment combining and pathogenic strains IV: the liveweight gain, in immune and challanged group (n = 14) was the same than that of the unchallenged group and did not show significant statistical differences (P < 0,05). However the control challenged group had less weight gain and statistical differences were found (P < 0,05). Although this is a preliminary information, it shows that: (a) the incidence of babesiosis on the reduction of weight gains is important; (b) the decrease in weight gain was not observed when attenuated strains were used; when the challenge was done in immunized animals, losses in liveweight gain were not observed. These results are discussed in order to plan future studis in different real systems of production.

Insights into the regulation of two caspase-activating platforms, the inflammasome and the PIDDosome

Résumé: Les organismes multicellulaires ont adopté diverses stratégies pour répondre aux stress auxquels ils sont exposés. Cette étude a exploré deux de ces stratégies l'inflammation en réponse à une invasion par un pathogène, et l'apoptose ou la survie en réponse aux dommages à l'ADN. L'interleukine-lß (IL-lß) est une importante cytokine inflammatoire. Elle est synthétisée sous forme d'un précurseur inactif et nécessite un clivage par la caspase-1 pour être activée. La caspase-1 elle-même est activée dans un complexe appelé inflammasome. Certains NLRs (Nod-like receptors), IPAF et les NALPs, sont capables de former des inflammasomes fonctionnels. Cette étude s'est intéressée au rôle d'un autre NLR structurellement proche, la protéine NAIP, dans la régulation de la caspase-1 et la maturation de l'IL-1 ß. NAIP est incorporé à l'inflammasome contenant NALP3 et est capable d'inhiber l'activation de la caspase-1 et la maturation de l'IL-lß. Cette fonction inhibitrice dépend des ses domaines BIR et est inhibée par ses LRRs. Le mécanisme exact d'inhibition reste à définir et la régulation de l'activation de NAIP est discutée. La deuxième partie de cette étude concerne la protéine PIDD. Cette protéine est impliquée avec RAIDD dans l'activation de la caspase-2, et est aussi capable, avec l'aide de RIP et de NEMO, d'activer NF-κB en réponse aux dommages à l'ADN. Deux isoformes de PIDD ont déjà été décrites dans la littérature, PIDD (isoforme 1) et LRDD (isoforme 2) et une troisième isoforme est rapportée ici. L'étude de l'expression de ces isoformes a montré qu'elles sont exprimées différemment dans les tissus et dans les lignées cellulaires, et que l'isoforme 3 est induite en réponse à un stress génotoxique. La caractérisation fonctionnelle a établi que les trois isoformes sont capables d'activer NF-κB, donc la survie, mais que seule l'isoforme 1 peut interagir avec RAIDD pour activer la caspase-2 et sensibiliser les cellules à la mort induite par un stress génotoxique. Le domaine intermédiaire de PIDD, situé entre le deuxième ZU5 et le DD est essentiel pour l'interaction entre PIDD et RAIDD et l'activation de la caspase-2 qui en découle. En conclusion, l'épissage différentiel de l'ARNm de PIDD permet la production d'au moins trois protéines possédant des fonctions agonistes ou antagonistes et qui peuvent participer au choix cellulaire entre survie et apoptose en réponse aux dommages à l'ADN. Summary: Multicellular organisms have evolved several strategies to cope with the stresses they encounter. The present study has explored two of these strategies: inflammation in response to a pathogenic invasion, and apoptosis or repair/survival in response to DNA damage. Interleukin-lß (IL-lß) is a key mediator of inflammation. It is synthesized as an inactive precursor and requires cleavage by caspase-1 to be activated. caspase-1 itself is activated in molecular platforms called inflammasomes, which can be formed by members of the NOD-like receptors (NLR) family, like IPAF and NALPs. This study has investigated the role of another NLR, the structurally related protein NAIP, in the regulation of caspase-1 activation and IL-lß maturation. An inhibitory role of NAIP on caspase-1 activation and IL-lß maturation was demonstrated, as well as NAIP incorporation in the NALP3 inflammasome. This inhibitory property relies on NAIP BIR domains and is inhibited by NAIP LRRs. The exact mechanism of NAIP-mediated caspase-1 activation remains to be elucidated and the regulation of NAIP activation is discussed. The second part of this study focused on the caspase-2 activating protein PIDD. This protein is known to mediate caspase-2 activation via RAIDD and to signal NF-κB via RIP and NEMO in response to DNA damage. Two isoforms of PIDD, PIDD (isoform 1) and LRDD (isoform 2), have already been reported and a third isoform is described here. Investigation of the expressional regulation of these isoforms indicated that they are differentially expressed in tissues and cell lines, and that isoform 3 mRNA levels are upregulated in response to genotoxic stress. Functional studies demonstrated that all three isoforms can activate NF-κB in response to DNA damage, but only isoform 1 is able to interact with RAIDD and activate caspase-2, sensitizing cells to genotoxic stress-induced cell death. The intermediate domain located between the second ZUS and the DD is essential for the interaction of PIDD and RAIDD and the subsequent caspase-2 activation. Thus the differential splicing of PIDD mRNA leads to the formation of at least thrée proteins with antagonizing/agonizing functions that could participate in determining cell fate in response to DNA damage.

Interrelationship between Schistosomiasis and concomitant diseases

The biological literature contains many examples of mutual influences between different species of parasites, especially with respect to concomitant helminth infections. Several situations are known in wich the association of infection by Shistosoma mansoni with other pathogens in the same host results in a type of disease wich differs from the simple summation of the individual effects of each infection. The present study concerns concomitant infections involving S. mansoni and enterobacteriaceae; S. mansoni and other helmints such as Ascaris lumbricoides, Ancylostomids, Toxocara canis and species of the genus Hymenolepis; S. mansoni and different protozoa such as Trypanosoma cruzi, T. brucei, Toxoplasma gondii and Plasmodium berghei. The interaction between hepatitis B virus and S. mansoni, leading to prolonged viremia and worsening of liver damage, is also discussed. The paper also treats the simultaneous occurrence of schistosomiasis and other aggravating factors such as malnutrition and neoplasias wich may alter the host's response to the trematode.

Coevolution of hosts and microorganisms: an analysis of the involvment of cytokines in host-parasite interactions

Parasites may employ particular strategies of eluding an immune response by taking advantage of those mechanisms that normally guarantee immunological self-tolerance. Much in the way as it occurs during the establishment of self-tolerance, live pathogens may induce clonal deletion, functional inactivation(anergy) and immunosupression. At this latter level, it appears that certain pathogens produce immunosupresive cytokine-like mediators or provoke like host the secrete cytokines that will compromise the anti-parasite immune response. It appears that immune responses that preferentially involve T helper l cells (secretors of interleukin-2-and interferon-y) tend to be protective, whereas T helper 2 cells (secretors of IL-4, IL5, IL-6, and IL-10), a population that antagonizes T helper cells, mediate disease susceptibility and are immunopathological reactions. Cytokines produced by T helper 2 cells mediate many symptoms of infection, including eosinophilia, mastocytosis, hyperimmunoglobulinemia, and elevated IgE levels. Administration of IL-2 and IFN-y has beneficial effects in many infections mediated by viruses, bacteria, and protozoa. The use of live vaccinia virus might be an avenue for the treatment of or vaccination against infection. We have found that a vaccinia virus expressing the gene for human IL-2, though attenuated, precipitates autoimmune disease in immunodeficient athymic mice. Thus, although T helper l cytokines may have desired immunostimulatory properties, they also may lead to unwarranted autoaggressive responses.

Host tissue destruction by Entamoeba histolytica: molecules mediating adhesion, cytolysis, and proteolysis

Entamoeba histolytica, the protozoan parasite causing human amoebisis, has recently been found to comprise two genetically distinct forms, potentially pathogenic and constitutively nonpathogenic ones. Host tissue destruction by pathogenic forms is belived to result from cell functions mediaed by a lectin-type adherence receptor, a pore-forming peptide involved in host cell lysis, and abundant expression of cysteine proteinase(s). Isolation and molecular cloning of these amoeba products have provided the tools for structural analyses and manipulations of cell functions including comparisons between pathogenic and nonpathogenic forms.