Preservation of clonal heterogeneity of the Pneumocystis carinii-specific CD4 T cell repertoire in HIV infected, asymptomatic individuals.

The loss of CD4 lymphocytes in HIV disease associates with opportunistic infections. Since diverse CD4 T cell clones respond to an opportunistic pathogen, we asked whether CD4 depletion deletes selected clones in the repertoire (vertical depletion) or it affects all clones by reducing the cell number in each progeny without affecting the overall number of clones (horizontal depletion). Understanding this point may help explain the mode of CD4 depletion and the mode of immunoreconstitution after therapy. Therefore we examined the CD4 T cell repertoire specific for Pneumocystis carinii, a relevant opportunistic pathogen in AIDS, in HIV-infected, asymptomatic individuals. We identified two patients of 36 asymptomatics for lack of proliferation to P. carinii, suggesting selective depletion of specific CD4 cells. To investigate clonal heterogeneity of P. carinii-responsive CD4 lymphocytes, specific CD4 T cell lines were generated and studied by TCR BV gene family usage and CDR3 length analysis (spectratyping). Clonal heterogeneity was similar in antigen-specific CD4 lines generated from P. carinii non-responding HIV seropositives and from controls. Thus, despite undetectable response to the pathogen, residual specific cells probably prevent overt infection and, when expanded in vitro, exhibit a clonal diversity similar to normal controls. These findings suggest a horizontal, rather than vertical, depletion in these asymptomatic patients.

PCR-restriction fragment length polymorphism analysis of a diagnostic 452-base-pair DNA fragment discriminates between Cryptosporidium parvum and C. meleagridis and between C. parvum isolates of human and animal origin.

Genomic DNAs from human Cryptosporidium isolates previously typed by analysis of the 18S ribosomal DNA locus (Cryptosporidium parvum bovine genotype, C. parvum human genotype, Cryptosporidium meleagridis, and Cryptosporidium felis) were used to amplify the diagnostic fragment described by Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg., 45:688-694, 1991). The obtained 452-bp amplified fragments were sequenced and aligned with the homologous Cryptosporidium wrairi sequence. Polymorphism was exploited to develop a restriction fragment length polymorphism method able to discriminate Cryptosporidium species and C. parvum genotypes.

Molecular characterization of Cryptosporidium isolates obtained from humans in France.

Cryptosporidium parvum is usually considered the agent of human cryptosporidiosis. However, only in the last few years, molecular biology-based methods have allowed the identification of Cryptosporidium species and genotypes, and only a few data are available from France. In the present work, we collected samples of whole feces from 57 patients from France (11 immunocompetent patients, 35 human immunodeficiency virus [HIV]-infected patients, 11 immunocompromised but non-HIV-infected patients) in whom Cryptosporidium oocysts were recognized by clinical laboratories. A fragment of the Cryptosporidium 18S rRNA gene encompassing the hypervariable region was amplified by PCR and sequenced. The results revealed that the majority of the patients were infected with cattle (29 of 57) or human (18 of 57) genotypes of Cryptosporidium parvum. However, a number of immunocompromised patients were infected with C. meleagridis (3 of 57), C. felis (6 of 57), or a new genotype of C. muris (1 of 57). This is the first report of the last three species of Cryptosporidium in humans in France. These results indicate that immunocompromised individuals are susceptible to a wide range of Cryptosporidium species and genotypes.

Immunohistochemical contribution to the study of morphine metabolism in Calliphoridae larvae and implications in forensic entomotoxicology.

Morphine was detected by immunohistochemistry on sections of third stage larvae of Calliphora vomitoria (Diptera, Calliphoridae) reared on minced beef meat previously treated with morphine hydrochloride. The detection was performed with an avidin-biotin-peroxidase-complex method. Positive specimens showed specific staining of the haemolymph and a more intense immunoreaction in an area located at the limit between exocuticle and endocuticle. These results constitute an evidence of morphine accumulation inside the cuticle of Diptera larvae during their development. During the pupariation, the larval cuticle is transformed into the sclerotized puparium. This study consequently points out the possibilities of analyzing empty pupariae when suitable tissues or living necrophagous insects are absent.

[Human nasal myiasis due to Oestrus ovis]. / Myiase nasale humaine à Oestrus ovis.

One case of human nasal myiasis caused by second and third instar larvae of Oestrus ovis was observed in Lille (North France). Oestrosis is a common myiasis of sheep and goats in Mediterranean and Tropical countries. The authors examine the oestrosis pathogenesis in the usual host (sheep) or in humans, and analyse the epidemiology, the symptomatology and the treatment of human oestrosis.

Transmission of Pneumocystis carinii disease from immunocompetent contacts of infected hosts to susceptible hosts.

Pneumocystis carinii organisms constitute a large group of heterogeneous atypical microscopic fungi that are able to infect immunocompromised mammals by an airborne route and to proliferate in their lungs, inducing Pneumocystis carinii pneumonia. This pneumonia remains a crucial epidemiological challenge, since neither the source of Pneumocystis carinii infection in humans nor the process by which humans become infected has been clearly established. Polymerase chain reaction (PCR) assays have shown that profoundly immunosuppressed patients without pneumocystosis can be subclinically infected with Pneumocystis. Other PCR-based studies have suggested that healthy immunocompetent hosts are not latent carriers of the parasite. However, recent reports have indicated that Pneumocystis carinii can persist for limited periods in the lungs of convalescent rats after recovery from corticosteroid-induced pneumocystosis, and also that immunocompetent mammals can be transiently parasitized by Pneumocystis carinii after close contact with hosts with Pneumocystis carinii pneumonia. Can transiently parasitized hosts be a source of infection for immunosuppressed hosts? In order to investigate this important clinical question, the ability of immunocompetent BALB/c mice, which were carrying subclinical levels of Pneumocystis carinii, to transmit the infection by the airborne route to highly susceptible, uninfected mice with severe combined immunodeficiency was studied. The results indicated that the immunocompetent mice, transiently parasitized by Pneumocystis carinii organisms after close contact with Pneumocystis carinii-infected mice, were able to transmit the infection to Pneumocystis carinii-free mice with severe combined immunodeficiency.

[Cryptosporidium and wildlife: a risk for humans?]. / Cryptosporidium et faune sauvage: un risque pour l'homme?

The present review underlines the knowledge of Cryptosporidium, especially its biodiversity and transmission. The presence of the parasite in different mammal host species is discussed with real, potential risk of transmission to humans. The potential role of insects in mechanical transmission of the parasite is evaluated by experimental protocols. The cost of cryptosporidiosis at health and economic levels are mentioned, which emphasises the importance of detection and identification of the parasite in the environment and in wild mammal species, using specific molecular tools. Potential measures to be accomplished in order to fight off cryptosporidiosis are also noted.

Necrophilous insect succession on rabbit carrion in sand dune habitats in northern France.

Six freshly killed rabbit carcasses were exposed in different habitats in the coastal dune massif of Ambleteuse (northern France) during the spring seasons of 1996 and 1997. In total, 66 arthropod species were collected during the decomposition of these carcasses, and particular attention was paid to taxa of necrophilic significance. The pattern of insect activity was recorded and is discussed in relation to meteorological conditions. One significant feature in this study was the delay in initial oviposition by flies during 1996, which demonstrates the need for caution in estimation of postmortem interval by entomological techniques in early spring. Although daily temperatures may be favorable for adult fly activity, flies may be absent because of small population size and low nocturnal temperatures during this period.

Effect of a killer toxin of Pichia anomala to Pneumocystis. Perspectives in the control of pneumocystosis.

Despite the development of drugs in the prophylaxis of pneumocystosis, Pneumocystis carinii remains a major opportunistic microorganism in immunosuppressed individuals, especially in human immunodeficiency virus-infected patients. Since side effects were frequently observed after administration of trimethoprim-sulfamethoxazole or pentamidine, the drugs which are mainly used in treating human P. carinii pneumonia (PCP), new therapeutic strategies should be developed. Over the last years, the inhibitory effect of a Pichia anomala killer toxin (PaKT), a molecule with a wide spectrum of antimicrobial activity, was characterized on P. carinii. The susceptibility of mouse and rat-derived Pneumocystis to PaKT has been demonstrated by in vitro attachment tests and in vivo infectivity assays. Nevertheless, PaKT is strongly antigenic, toxic and could not be used directly as a therapeutic agent. Then, a new strategy using killer toxin-like anti-idiotypic antibodies (KT-antiIds) mimicking the fungal toxin activity has been developed. Different KT-antiIds were obtained by idiotypic immunization with a monoclonal antibody (mabKT4). This mabKT4 neutralized the killer properties of the PaKT. KT-antiIds were produced by immunization against the variable domain (idiotype) of mAbKT4 (internal image of the killer toxin receptor), or they were obtained directly from vaginal fluid of patients affected by recurrent vaginal candidiosis. In this last case, such natural KT-antiIds were immunopurified by affinity-chromatography with mAbKT4 and their anti-P. carinii activity was then evaluated. Our results showed that both the in vitro attachment of rat-derived parasites and their infectivity to nude rats were inhibited by the KT-antiIds. With regard to KT-antiIds obtained by immunization, the antimicrobial activity of a monoclonal KT-antiIds (mAbK10) has been evaluated by using a PCP experimental nude rat model treated by mAbK10 administered by aerosol. The pneumocystosis extension was significantly reduced in this model. The monoclonal KT-antiIds were effective against P. carinii in reducing parasite proliferation in lungs of nude rats. Further experiments are in progress to study the in vivo anti-P. carinii activity of KT-antiIds by using recombinant single-chain of the variable fragment of KT-antiIds. Yeast killer toxin-like recombinant molecules could provide the basis for a new therapeutic strategy towards the control of pneumocystosis.

Morphological and ultrastructural methods for Pneumocystis.

Pneumocystis is a eukaryotic unicellular microorganism with marked fungal affinities. All known life cycle stages of this parasite were observed in the lung of mammals. The cystic forms of this microorganism may be observed microscopically by using stains with affinity for the components of their relatively thick cell wall. However, about 100 years ago they were observed for the first time thanks to panoptic stains which do not stain their cell wall. Methanol-Giemsa technique as well as Giemsa-like rapid stainings are often used to reveal vegetative or cystic forms of this parasite on air dried smears of clinical or experimental samples. For many years, hypotheses on its life cycle, which remains unknown, were based on transmission electron microscopy (TEM) studies. However, only for the last years progresses in the quality of fixation for TEM led to a better understanding of the Pneumocystis cell structure. In this chapter, strategies to reveal Pneumocystis organisms in clinical or experimental specimens by using light microscopy, as well as techniques allowing a good preparation of parasitic samples for TEM, are given and shortly discussed.

Different ultrastructural morphology of Pneumocystis carinii derived from mice, rats, and rabbits.

Pneumocystis carinii (PC) is a fungus present in the lungs of many mammal species. Even though studies of the genome, the isoenzymes, and the antigens have proved some host-species-linked heterogeneity, the existence of distinct Pneumocystis species or subspecies has still not been accepted. Comparative studies of the ultrastructural morphology of pneumocysts derived from several host species may support evidence of host-species-linked heterogeneity. We have compared the ultrastructural morphology of pneumocysts derived from mice, rats, and rabbits. The density of membrane-limited electron-dense cytoplasmic granules was found to be higher in mouse-derived pneumocysts than in rabbit-derived pneumocysts, and furthermore the average diameter of the granules from mouse pneumocysts was larger than that of granules from rabbit-derived pneumocysts. The average diameter of the filopodia of mouse-derived pneumocysts was smaller than that of filopodia from rat-derived pneumocysts, which was smaller than that of filopodia from rabbit-derived pneumocysts. Globular electron-dense bulbous dilatations at the tip of the filopodia were described for the first time and they were only found on filopodia of mouse-derived pneumocysts. These distinct host-species-linked morphological differences of pneumocysts from mouse, rat, and rabbit may support previous biochemical data indicating the existence of different Pneumocystis species or subspecies.

Inhibitory effect of human natural yeast killer toxin-like candidacidal antibodies on Pneumocystis carinii.

BACKGROUND: Human natural antibodies have been found that owe their candidacidal action to the mimicry of a yeast killer toxin produced by the yeast Pichia anomala (PaKT). Candidacidal human natural antibodies (KTAb) are elicited by and bind to a KT receptor (PaKTR) present on the cell surface of infectious PaKT-sensitive microorganisms. Because of the recognized susceptibility of Pneumocystis carinii organisms to PaKT upon the occurrence of specific PaKTR, we examined whether human natural KTAb could also bind to and inhibit P. carinii. MATERIALS AND METHODS: Immunoaffinity-purified KTAb from the vaginal fluid of patients affected by candidiasis were tested and compared with PaKT for their ability to inhibit rat-derived P. carinii attachment to epithelial lung cells as well as infectivity to nude rats. Immunofluorescence studies were also performed by biotinylated PaKT in competition with human KTAb to establish their specific binding to PaKTR on the surface of rat-derived and human P. carinii organisms. RESULTS: Human natural candidacidal KTAb exerted a strong, specific inhibitory activity against rat-derived P. carinii organisms that are susceptible to PaKT itself. The antimicrobial activity of human KTAb was abolished by adsorption with a specific PaKT-neutralizing mAb KT4. Immunofluorescence studies of competition with PaKT showed that human KTAb efficiently bind to the specific PaKTR on the surface of rat-derived and human P. carinii organisms. CONCLUSIONS: The results strongly suggest that human KTAb, elicited by a common transphyletic receptor of different pathogenic microorganisms during infection, may play a role in antibody-mediated cross-immunity and, if properly engineered, as functionally equivalent recombinant antibodies they could exert a therapeutic activity against pneumocystosis in vivo.

Monoclonal yeast killer toxin-like candidacidal anti-idiotypic antibodies.

Rat monoclonal yeast killer toxin (KT)-like immunoglobulin M (IgM) anti-idiotypic antibodies (KT-IdAbs) were produced by idiotypic vaccination with a mouse monoclonal antibody (MAb; MAb KT4) that neutralized a Pichia anomala KT characterized by a wide spectrum of antimicrobial activity. The characteristics of the KT-IdAbs were demonstrated by their capacity to compete with the KT to the idiotype of MAb KT4 and to interact with putative KT cell wall receptors (KTRs) of sensitive Candida albicans cells. The internal-image properties of KT-IdAbs were proven by their killer activity against KT-sensitive yeasts. This lethal effect was abolished by prior adsorption of KT-IdAbs with MAb KT4. These findings stressed the potential importance of antibody-mediated immunoprotection against candidiasis and suggested a feasible experimental approach for producing antimicrobial receptor antibodies without purifying the receptor. KT-IdAbs might represent the basis for producing engineered derivatives with a high potential for effective therapeutic antifungal activity.