[Unexplained reduction in the general condition of a female lung cancer patient]. / Unklare Minderung des Allgemeinzustandes bei Patientin mit Lungenkarzinom.

A 63-year-old female patient with lung cancer presented to our emergency room for the first time with a sudden reduction in general condition, vomiting and severe weakness. She stated that she was receiving chemotherapy for the lung cancer and reported that she had no other relevant previous illnesses. Our initial suspected diagnosis was cytostatic-induced nausea and vomiting. Contrary to this suspected diagnosis, diagnostics carried out in the emergency room revealed the findings of ketoacidosis on the basis of an initial manifestation of diabetes mellitus with hyperglycemic decompensation as well as severe, manifest hypothyroidism. After obtaining the preliminary findings, it became evident that the patient was not receiving chemotherapy, but rather immune checkpoint therapy using durvalumab. The initial manifestations described were therefore to be viewed as immune reactions associated with durvalumab. After initiating diabetic recompensation therapy and substitution with L­thyroxine, a rapid improvement in the patient's general condition was achieved.

One-sided ejaculation of echidna sperm bundles.

We report for the first time an unusual ejaculatory mechanism in the short-beaked echidna in which each side of the bilaterally symmetrical, rosettelike glans penis is used alternately, with the other being shut down. This is unparalleled in mammals but is reminiscent of the use of hemipenes in squamate reptiles, providing further reproductive evidence of a sauropsidian lineage in the Monotremata. Further, we describe the occurrence of motile sperm bundles in ejaculated echidna semen and provide scanning electron micrographs of their morphology. Sperm bundling appears to confer increased sperm motility, which may provide the potential for sperm competition between males.

Prevalence of environmental Aeromonas in South East Queensland, Australia: a study of their interactions with human monolayer Caco-2 cells.

AIMS: To investigate the prevalence of Aeromonas in a major waterway in South East Queensland, Australia, and their interactions with a gut epithelial model using Caco-2 cells. METHODS AND RESULTS: A total of 81 Aeromonas isolates, collected from a major waterway in South East Queensland, Australia, were typed using a metabolic fingerprinting method, and tested for their adhesion to HEp-2 and Caco-2 cells and for cytotoxin production on Vero cells and Caco-2 cells. Aeromonas hydrophila had the highest (43%) and Aeromonas veronii biovar sobria had the lowest (25%) prevalence. Four patterns of adhesion were observed on both HEp-2 and Caco-2 cell lines. Representative isolates having different phenopathotypes (nine strains) together with two clinical isolates were tested for their translocation ability and for the presence of virulence genes associated with pathogenic Escherichia coli. The rate and degree of translocation across Caco-2 monolayers varied among strains and was more pronounced with LogA pattern. Translocation was associated with the adherence of strains to Caco-2 cells microvilli, followed by internalization into Caco-2 cells. Two Aer. veronii biovar sobria strains were positive for the presence of heat-labile toxin genes, with one strain also positive for Shiga-like toxin gene. CONCLUSIONS: Pathogenic strains of Aeromonas carrying one or more virulence characteristics are highly prevalent in the waterways studied and are capable of translocating across a human enterocyte cell model. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that Aeromonas strains carrying one or more virulence properties are prevalent in local waterways and are capable of translocating in a human enterocyte cell culture model. However, their importance in human gastrointestinal disease has yet to be verified under competitive conditions of the gut.

A survey of the intestinal transcriptomes of the hookworms, Necator americanus and Ancylostoma caninum, using tissues isolated by laser microdissection microscopy.

The gastrointestinal tracts of multi-cellular blood-feeding parasites are targets for vaccines and drugs. Recently, recombinant vaccines that interrupt the digestion of blood in the hookworm gut have shown efficacy, so we explored the intestinal transcriptomes of the human and canine hookworms, Necator americanus and Ancylostoma caninum, respectively. We used Laser Microdissection Microscopy to dissect gut tissue from the parasites, extracted the RNA and generated cDNA libraries. A total of 480 expressed sequence tags were sequenced from each library and assembled into contigs, accounting for 268 N. americanus genes and 276 A. caninum genes. Only 17% of N. americanus and 36% of A. caninum contigs were assigned Gene Ontology classifications. Twenty-six (9.8%) N. americanus and 18 (6.5%) A. caninum contigs did not have homologues in any databases including dbEST-of these novel clones, seven N. americanus and three A. caninum contigs had Open Reading Frames with predicted secretory signal peptides. The most abundant transcripts corresponded to mRNAs encoding cholesterol-and fatty acid-binding proteins, C-type lectins, Activation-Associated Secretory Proteins, and proteases of different mechanistic classes, particularly astacin-like metallopeptidases. Expressed sequence tags corresponding to known and potential recombinant vaccines were identified and these included homologues of proteases, anti-clotting factors, defensins and integral membrane proteins involved in cell adhesion.

Ultrastructural study of Chlamydia pneumoniae in a continuous-infection model.

We have established an in vitro model of long-term continuous Chlamydia pneumoniae infection in HEp-2 cells. Using transmission electron microscopy, we demonstrated the presence of spontaneous abnormal chlamydial inclusions similar in appearance to the persistent chlamydial forms induced in vitro by treatment with cytokines or antibiotics or by nutrient deprivation.

Differential expression of ompA, ompB, pyk, nlpD and Cpn0585 genes between normal and interferon-gamma treated cultures of Chlamydia pneumoniae.

A common feature of many chlamydial infections is that they are often asymptomatic and may persist for long periods of time if left untreated. In addition to the well recognized lytic stage of the chlamydial developmental cycle, evidence is now emerging to support a persistent phase in the cycle in which the reticulate bodies are morphologically abnormal, viable but non-infectious and presumably also have altered gene expression patterns. We used an RT-PCR approach to study the differential levels of gene transcription for 14 genes (16SrRNA, ompA, ompB, omcB, 76 kDa, gseA, pmp1, gltX, hsp60, yaeT, pyk, nlpD, Cpn0585, Cpn1046) between normal and IFN-gamma treated Chlamydia pneumoniae cell cultures. Even though the level of morphologically abnormal reticulate bodies in our IFN-gamma treated cultures was low (approximately 10% morphologically discernible, although presumably a larger percentage were in the persistent state but not yet morphologically altered) we identified five genes (ompA, ompB, pyk, nlpD, Cpn0585) that were clearly upregulated when compared to normal cultures. This gene transcript profile may be characteristic of a general stress state in Chlamydia, induced by IFN-gamma treatment in this case, but perhaps more widely induced in other in vitro and in vivo situations.

Plesiomonas shigelloides enters polarized human intestinal Caco-2 cells in an in vitro model system.

This study provides the first definitive evidence that the gram-negative bacterium Plesiomonas shigelloides adheres to and enters eukaryotic intestinal host cells in vitro. P. shigelloides is increasingly regarded as an emerging enteric pathogen and has been implicated in intestinal and extraintestinal infections in humans. However, the establishment of its true role in enteric disease has been hindered by inadequacies in experimental design, deficiencies in clinical diagnosis, and the lack of an appropriate animal model. In this investigation, an in vitro system was used to evaluate plesiomonad pathogenesis. Differentiated epithelium-derived Caco-2 cell monolayers inoculated apically with 12 isolates of P. shigelloides from clinical (intestinal) origins were examined at high resolution using transmission electron microscopy. Bacterial cells were observed adhering to intact microvilli and to the plasma membrane on both the apical and the basal surfaces of the monolayer. The bacteria entered the Caco-2 cells and were observed enclosed in single and multiple membrane-bound vacuoles within the host cell cytoplasm. This observation suggests that initial uptake may occur through a phagocytic-like process, as has been documented for many other enteropathogens. P. shigelloides also was noted free in the cytosol of Caco-2 cells, suggesting escape from cytoplasmic vacuoles. Differences in invasion phenotypes were revealed, suggesting the possibility that, like Escherichia coli, P. shigelloides comprises different pathogenic phenotypes.

Bacterial contamination of cracks in symptomatic vital teeth.

As part of an ongoing study on the initiation of cracks in teeth, 20 teeth exhibiting symptoms consistent with the presence of dentinal cracks were examined. The presence of a cracked cusp was confirmed by the selective application of pressure either with a mirror handle or Fracfinder (Svenoka, Dental Instruments, Vasby, Sweden). Cracked cusps were fractured from the teeth after the removal of all existing restorations and were immediately placed into ten percent formalin. Subsequently, specimens were dehydrated, sputter-coated and examined under the scanning electron microscope (SEM). All the cracked cusps exhibited complete fracture of the dentine to the level of the dentino-enamel junction. No partial fractures were seen. Numerous bacteria of many morphological forms were present on the dentinal surfaces, of all fractured cusps, in all teeth. Cocci, bacilli and filamentous forms were consistently found. Many bacteria were in the process of division. While bacterial contamination of dentinal cracks has been described in histological studies, the nature and distribution of these bacterial and fungal forms has not been shown previously in any detail. Prior SEM studies investigating the nature and mechanisms of fracture have not revealed bacterial contamination of the fractured surface. This paper draws attention to the fact that all symptomatic cracks in teeth appear to 1. extend right through the dentine to the dentino-enamel junction, and 2. appear to be extensively contaminated by bacteria.

A survey of Blastocystis in domestic chickens.

The prevalence and morphology of Blastocystis in fresh faecal material from 227 domestic chickens was investigated. A very high prevalence of infection (approximately 95%) was found in chickens from four of the five commercial farms studied. Extremely high numbers of Blastocystis were found in the majority of samples. Blastocystis cells showed considerable variation in size, ranging from approx. 3 microm to approx. 120 microm in diameter. This size range is more extreme than those previously recognised for the organism from chickens. All chickens from one farm appeared free of Blastocystis infection. Most Blastocystis cells appeared to be the vacuolar form, although the shape of the cells and the appearance of the central vacuole contents varied considerably within and among faecal samples. Nuclei showed "spots" of electron-opaque material, generally arranged as a band within the nuclei. Multiple individual cysts within a single outer fibrillar layer were found in addition to single cysts without an encompassing fibrillar layer.

Reappraisal of vesicular types in the syncytial tegument of the Echinococcus granulosus protoscolex.

The structure, abundance, and distribution of tegumentary vesicles was compared among Echinococcus granulosus protoscoleces that had been prepared for electron microscopy using four processing schedules: a conventional method, alternative fixations using uranyl acetate or osmium tetroxide-potassium ferricyanide, and a freeze-substitution method. Four morphologically distinct types of vesicles were found in the somal region. The morphology of the first form, with moderately electron-opaque contents, and the second form, with similar size and shape but containing an electron-opaque core, varied little among the preparation methods. Two additional forms of vesicles, with characteristic intensely electron-opaque contents, were revealed only after freeze-substitution. These elongate vesicles were also found in the scolex tegument where they were most conspicuous, and appeared markedly increased in number after freeze-substitution. Large, spherical vesicles with an electron-lucent core embedded in a dense matrix of fibrillar strands were the dominant vesicle forms in the scolex region after all methods of preparation. Fixation by osmium tetroxide-potassium ferricyanide revealed the presence of spherical vesicles with amorphous electron-opaque contents and a few inclusions. This form of vesicle was also observed after freeze-substitution, but the inclusions in the vesicular lumen were more numerous. The variation in the distribution of vesicle forms among the body regions strongly implies a variety of vesicle functions. In addition, our observations suggest that comparative studies of different fixative methods are necessary to demonstrate the detailed vesicular morphology of the tegument of E. granulosus and other cestodes.

Incidental finding of Myxobolus spores (protozoa: myxozoa) in stool samples from patients with gastrointestinal symptoms.

Myxozoan spores were detected in fecal samples from three patients presenting with abdominal pain and/or diarrhea. The spores were identical to those of Myxobolus plectroplites, a previously described pathogen from the freshwater fish Plectroplites ambiguus. All patients had recently eaten fish caught from local waters, and frozen fillets of such fish were found to be infected with M. plectroplites cysts. The passage of spores unchanged through the alimentary tract suggests they were incidental findings unrelated to clinical symptoms, especially since other enteric pathogens were present in two patients.

Immunolocalisation of the glutathione S-transferases, GST-26 and GST-28, within adult Schistosoma japonicum.

This paper describes the first ultrastructural immunolocalisation study of the 26-kDa and 28-kDa glutathione S-transferases within adult Schistosoma japonicum (GST-26 and GST-28). Polyclonal antibodies raised against GST-28 (in mice) and against GST-26 (in rabbits) were used to examine the distribution of the proteins within adult parasites. Both proteins were localised within the parenchymal region of the male parasite. Additionally, both proteins were present within parenchymal cells located between the vitelline glands of female parasites. There were no detectable levels of GST-26 or GST-28 on the surface or within the tegument matrix of either the male or female worms. Possible functions for GST-26 and GST-28 within S. japonicum and their significance as vaccine target molecules are addressed.

Ultrastructural analysis of the adult Schistosoma japonicum by lectin cytochemistry.

Eight lectin probes were used to detect a range of carbohydrate residues in the tegument matrix of Schistosoma japonicum. In addition, other areas of the parasite, such as the gut, vitelline glands and flame cells, were examined for carbohydrate residues. Some minor differences in the carbohydrate residue composition between tegument orientations and between the sexes were identified. Differences between the distribution of carbohydrate residues of S. japonicum examined in this study and previous reports of Schistosoma mansoni were also noted. This study further illustrates the high level of complexity within the tegument of the adult S. japonicum and has demonstrated differences between this species and the widely studied S. mansoni.

Detection of Blastocystis sp. in domestic dogs and cats.

Blastocystis sp. was detected in faecal samples from domestic dogs and cats in Brisbane, Australia. The prevalence rates were high, with 70.8% of the dogs and 67.3% of the cats infected with this organism. Blastocystis sp. from faecal material from two dogs was successfully cultured on inspissated egg slant medium for several months, but could not be maintained for longer periods. Blastocystis sp. from feline faecal samples failed to grow in culture. The parasites found in dogs and cats were generally smaller than Blastocystis hominis from human faecal material, and were the vacuolar form rather than the multivacuolar form. Otherwise, the general morphology of these organisms appeared similar to B. hominis when examined by light and transmission electron microscopy.

Immunolocalization of the fatty acid-binding protein Sj-FABPc within adult Schistosoma japonicum.

This paper describes the first localization study of the 14.7 kDa fatty acid-binding protein in Schistosoma japonicum (SjFABPc) using transmission electron microscopy. A polyclonal antibody raised against recombinant Sj-FABPc was used in combination with a colloidal gold marker to determine the distribution of the protein within adult parasites. Sj-FABPc was localized within lipid droplets below the subtegumental region of the male parasite. Additionally, Sj-FABPc was present in the vitelline droplets of the vitelline glands of female parasites. There were no detectable levels of Sj-FABPc on the surface or within the tegument of male or female parasites. Possible functions of Sj-FABPc within S. japonicum and the relevance of these immunolocalization findings in light of the recent reports that the homologue Sm-FABPc is an important anti-S. mansoni vaccine target molecule are also discussed.

Cellular localisation of the ataxia-telangiectasia (ATM) gene product and discrimination between mutated and normal forms.

The recently cloned gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) is involved in DNA damage response at different cell cycle checkpoints and also appears to have a wider role in signal transduction. Antibodies prepared against peptides from the predicted protein sequence detected a approximately 350 kDa protein corresponding to the open reading frame, which was absent in 13/23 A-T homozygotes. Subcellular fractionation, immunoelectronmicroscopy and immunofluorescence showed that the ATM protein is present in the nucleus and cytoplasmic vesicles. This distribution did not change after irradiation. We also provide evidence that ATM protein binds to p53 and this association is defective in A-T cells compatible with the defective p53 response in these cells. These results provide further support for a role for the ATM protein as a sensor of DNA damage and in a more general role in cell signalling, compatible with the broader phenotype of the syndrome.

Virus-like particles in Blastocystis sp. from simian faecal material.

Virus-like particles were observed in the cytoplasm of Blastocystis sp. found in fresh faecal material from 2 monkeys (Macaca fascicularis). Two morphological types of particles were found during examination by transmission electron microscopy: 1 was approximately 55-60 nm diameter, with an electron-opaque core of approximately 30 nm in diameter; and the other was approximately 200 nm in diameter with a core of approximately 100 nm in diameter. This is the first report of virus-like particles identified in situ in Blastocystis.

Macromolecular transport in malaria--does the duct exist?

The proposal that a parasitophorous 'duct' traverses the malaria-infected erythrocyte cytoplasm and is responsible for the unusual molecular uptake kinetics observed in malaria, has created considerable debate on the nature of macromolecular transport in this parasite. The existence of a 'duct' has important implications for the immunobiology of this parasite, particularly the possibility that antibodies may have access to 'internal' antigens in malaria. The most compelling evidence that there is a direct connection between the parasite and the surrounding media comes from the experiment of Pouvelle et al. (Nature 353, 73-75 (1991)) using small highly fluorescent latex spheres. However, we have found that fluorescent labeling of the parasite and tubular structures that extend from the parasite is due to the release of dye from the latex spheres during the incubation and is not due to the uptake of the spheres themselves. The inability of malaria-infected erythrocytes to take up latex beads down to 14 nm diameter establishes that an 'open' channel connecting the parasite with the surrounding media does not exist. This finding has important implications for establishing the unusual nature of macromolecular transport across the infected erythrocyte cytoplasm in malaria.

Morphological differences in Blastocystis cysts-an indication of different species?

Cyst forms of Blastocystis that show disparate morphology in relation to the previously described cysts were detected in faecal material from animal hosts. Transmission electron microscopy was performed without attempts to isolate or concentrate Blastocystis from the faecal material. Large, multinucleate cyst forms were found in faecal material from Macaca monkeys. These cyst forms measured up to approximately 15 microm in diameter and were often larger than vacuolar forms present in the same samples. Four or more nuclei were frequently seen in the cysts. Multiple individual cysts enclosed by a single fibrillar layer were found in faecal material from domestic chickens. Each individual cyst within the multiple cyst form measured approximately 3-4 microm in diameter and appeared to be uninucleate.

Schistosoma japonicum: immunolocalization of paramyosin during development.

This paper describes the localization of paramyosin immunoreactivity in Schistosoma japonicum and represents the first comparative immunolocalization study among schistosome adult, cercariae and lung schistosomula by electron microscopy. A polyclonal antibody was utilized to immunolabel paramyosin or paramyosin-like proteins. Paramyosin was localized within the muscle layer of all 3 developmental stages. Furthermore, paramyosin was localized within granules of the post-acetabular glands of cercariae, and within the tegument matrix and surface of lung schistosomules. Adults and cercariae did not display any detectable paramyosin on the surface or within the tegument. The possible functions of paramyosin within S. japonicum and the relevance of these findings in relation to the reported protective properties of paramyosin as an anti-schistosome vaccine target molecule are discussed.