Human Outbreak of Trichinellosis Caused by Trichinella papuae Nematodes, Central Kampong Thom Province, Cambodia.

In September 2017, a severe trichinellosis outbreak occurred in Cambodia after persons consumed raw wild pig meat; 33 persons were infected and 8 died. We collected and analyzed the medical records for 25 patients. Clinical signs and symptoms included myalgia, facial or peripheral edema, asthenia, and fever. We observed increased levels of creatine phosphokinase and aspartate aminotransferase-, as well as eosinophilia. Histopathologic examination of muscle biopsy specimens showed nonencapsulated Trichinella larvae. A Trichinella excretory/secretory antigen ELISA identified Trichinella IgM and IgG. Biopsy samples were digested and larvae were isolated and counted. PCR for the 5S rDNA intergenic spacer region and a multiplex PCR, followed by sequencing identified the parasite as Trichinella papuae. This species was identified in Papua New Guinea during 1999 and in several outbreaks in humans in Thailand. Thus, we identified T. papuae nematodes in humans in Cambodia.

Multiphoton imaging of tumor biomarkers with conjugates of single-domain antibodies and quantum dots.

An ideal multiphoton fluorescent nanoprobe should combine a nanocrystal with the largest possible two-photon absorption cross section (TPACS) and the smallest highly specific recognition molecules bound in an oriented manner. CdSe/ZnS quantum dots (QDs) conjugated to 13-kDa single-domain antibodies (sdAbs) derived from camelid IgG or streptavidin have been used as efficient two-photon excitation (TPE) probes for carcinoembryonic antigen (CEA) imaging on normal human appendix and colon carcinoma tissue. The TPACS for some conjugates was higher than 49,000 GM (Goeppert-Mayer units), considerably exceeding that of organic dyes being close to the theoretical value of 50,000 GM calculated for CdSe QDs. The ratio of sdAb-QD emission to the autofluorescence for 800 nm TPE was 40 times higher than that for 457.9 nm one-photon excitation. TPE ensures a clear discrimination of CEA-overexpressing tumor areas from normal tissue. Oriented sdAb-QD conjugates are bright specific labels for detecting low concentrations of antigens using multiphoton microscopy. FROM THE CLINICAL EDITOR: This study demonstrates carcinoembryonic antigen imaging on normal human appendix and colon carcinoma tissue utilizing CdSe/ZnS quantum dots conjugated to streptavidin or to 13-kDa single-domain antibodies as efficient two-photon excitation probes.

Oriented conjugates of single-domain antibodies and quantum dots: toward a new generation of ultrasmall diagnostic nanoprobes.

Common strategy for diagnostics with quantum dots (QDs) utilizes the specificity of monoclonal antibodies (mAbs) for targeting. However QD-mAbs conjugates are not always well-suited for this purpose because of their large size. Here, we engineered ultrasmall nanoprobes through oriented conjugation of QDs with 13-kDa single-domain antibodies (sdAbs) derived from llama IgG. Monomeric sdAbs are 12 times smaller than mAbs and demonstrate excellent capacity for refolding. sdAbs were tagged with QDs through an additional cysteine residue integrated within the C terminal of the sdAb. This approach allowed us to develop sdAbs-QD nanoprobes comprising four copies of sdAbs coupled with a QD in a highly oriented manner. sdAbs-QD conjugates specific to carcinoembryonic antigen (CEA) demonstrated excellent specificity of flow cytometry quantitative discrimination of CEA-positive and CEA-negative tumor cells. Moreover, the immunohistochemical labeling of biopsy samples was found to be comparable or even superior to the quality obtained with gold standard protocols of anatomopathology practice. sdAbs-QD-oriented conjugates as developed represent a new generation of ultrasmall diagnostic probes for applications in high-throughput diagnostic platforms. FROM THE CLINICAL EDITOR: The authors report the development of sdAbs-QD-oriented conjugates, comprised of single domain antibodies that are 12 times smaller than regular mAb-s and quantum dots. These ultrasmall diagnostic probes represent a new generation of functionalized ODs for applications in high-throughput diagnostic platforms.

Efficacy of ω-3 polyunsaturated fatty acids for the treatment of refractory hydroa vacciniforme.

Hydroa vacciniforme (HV) is a rare photodermatosis. Several therapies, with sometimes severe side effects, have been used in isolated cases. We report a case of refractory HV successfully treated with dietary fish oil rich in ω-3 polyunsaturated fatty acids.

Enterovirus-related activation of the cardiomyocyte mitochondrial apoptotic pathway in patients with acute myocarditis.

AIMS: We examined the impact of enterovirus (EV) cardiac replication activity on the endomyocardial mitochondrial pathway in patients with acute myocarditis. METHODS AND RESULTS: Levels of apoptotic cardiomyocytes were determined by TUNEL and ligation-mediated polymerase chain reaction (PCR) assays and EV replication activity was assessed by immunostaining of EV VP1 capsid protein in ventricular myocytes of patients with acute myocarditis (n = 25), and healthy heart controls (n = 15). Ratio of cytosolic/mitochondrial cytochrome c concentrations was determined by ELISA assay, levels of active caspase-9 were determined by western blot analysis and Bax/Bcl2 mRNA ratio was assessed by real-time reverse transcription-polymerase chain reaction (RT-PCR) in the same cardiac tissues. Patients with EV-associated acute myocarditis (n = 15) exhibited a significantly higher number of apoptotic cardiomyocytes than those with non-EV-associated acute myocarditis (n = 10) and controls (n = 15) (P < 0.001). Endomyocardial ratio of cytosolic/mitochondrial cytochrome c concentrations and levels of active caspase-9 protein were significantly increased in EV than in non-EV-related myocarditis patients (P < 0.001). Moreover, Bax/Bcl2 mRNA ratio was significantly increased in EV than in non-EV-related myocarditis patients (P < 0.001). CONCLUSION: Our findings evidence an EV-related activation of the cardiomyocyte mitochondrial apoptotic pathway in patients with acute myocarditis. Moreover, our results indicate that this EV-induced pro-apoptotic mechanism could be partly related to an up-regulation of Bax expression, and suggest that inhibition of this cell death process may constitute the basis for novel therapies.

Emerging applications of fluorescent nanocrystals quantum dots for micrometastases detection.

The occurrence of metastases is one of the main causes of death in many cancers and the main cause of death for breast cancer patients. Micrometastases of disseminated tumour cells and circulating tumour cells are present in more than 30% of breast cancer patients without any clinical or even histopathological signs of metastasis. Low abundance of these cell types in clinical diagnostic material dictates the necessity of their enrichment prior to reliable detection. Current micrometastases detection techniques are based on immunocytochemical and molecular methods suffering from low efficiency of tumour cells enrichment and observer-dependent interpretation. The use of highly fluorescent semiconductor nanocrystals, also known as "quantum dots" and nanocrystal-encoded microbeads tagged with a wide panel of antibodies against specific tumour markers offers unique possibilities for ultra-sensitive micrometastases detection in patients' serum and tissues. The nanoparticle-based diagnostics provides an opportunity for highly sensitive parallel quantification of specific proteins in a rapid and low-cost method, thereby providing a link between the primary tumour and the micrometastases for early diagnosis.

Discriminating healthy from tumor and necrosis tissue in rat brain tissue samples by Raman spectral imaging.

The purpose of this study was to investigate molecular changes associated with glioma tissues by Raman microspectroscopy in order to develop its use in clinical practice. Spectroscopic markers obtained from C6 glioma tissues were compared to conventional histological and histochemical techniques. Cholesterol and phospholipid contents were highest in corpus callosum and decreased gradually towards the cortex surface as well as in the tumor. Two different necrotic areas have been identified: a fully necrotic zone characterized by the presence of plasma proteins and a peri-necrotic area with a high lipid content. This result was confirmed by Nile Red staining. Additionally, one structure was detected in the periphery of the tumor. Invisible with histopathological hematoxylin and eosin staining, it was revealed by immunohistochemical Ki-67 and MT1-MMP staining used to visualize the proliferative and invasive activities of glioma, respectively. Hierarchical cluster analysis on the only cluster averaged spectra showed a clear distinction between normal, tumoral, necrotic and edematous tissues. Raman microspectroscopy can discriminate between healthy and tumoral brain tissue and yield spectroscopic markers associated with the proliferative and invasive properties of glioblastoma. Development of in vivo Raman spectroscopy could thus accurately define tumor margins, identify tumor remnants, and help in the development of novel therapies for glioblastoma.

Modeling and quantifying biochemical changes in C6 tumor gliomas by Fourier transform infrared imaging.

The purpose of the study was to investigate molecular changes associated with glioma tissues using FT-IR microspectroscopic imaging (FT-IRM). A multivariate statistical analysis allowed one to successfully discriminate between normal, tumoral, peri-tumoral, and necrotic tissue structures. Structural changes were mainly related to qualitative and quantitative changes in lipid content, proteins, and nucleic acids that can be used as spectroscopic markers for this pathology. We have developed a spectroscopic model of glioma to quantify these chemical changes. The model constructed includes individual FT-IR spectra of normal and glioma brain constituents such as lipids, DNA, and proteins (measured on delipidized tissue). Modeling of FT-IR spectra yielded fit coefficients reflecting the chemical changes associated with a tumor. Our results demonstrate the ability of FT-IRM to assess the importance and distribution of each individual constituent and its variation in normal brain structures as well as in the different pathological states of glioma. We demonstrated that (i) cholesterol and phosphatidylethanolamine contributions are highest in corpus callosum and anterior commissure but decrease gradually towards the cortex surface as well as in the tumor, (ii) phosphatidylcholine contribution is highest in the cortex and decreases in the tumor, (iii) galactocerebroside is localized only in white, but not in gray matter, and decreases in the vital tumor region while the necrosis area shows a higher concentration of this cerebroside, (iv) DNA and oleic acid increase in the tumor as compared to gray matter. This approach could, in the future, contribute to enhance diagnostic accuracy, improve the grading, prognosis, and play a vital role in therapeutic strategy and monitoring.

The NC1 domain of type XIX collagen inhibits in vivo melanoma growth.

Type XIX collagen is a minor collagen that localizes to basement membrane zones, together with types IV, XV, and XVIII collagens. Because several NC1 COOH-terminal domains of other chains from basement membrane collagens were reported to exhibit antitumor activity, we decided to study the effects of the NC1(XIX) collagen domain on tumor progression using an experimental in vivo model of mouse melanoma. We observed a 70% reduction in tumor volume in NC1(XIX)-treated mice compared with the corresponding controls. Histologic examination of the tumors showed a strong decrease in tumor vascularization in treated mice. In vitro, NC1(XIX) inhibited the migrating capacity of tumor cells and their capacity to invade Matrigel. It also inhibited the capacity of human microvascular endothelial cells to form pseudotubes in Matrigel. This effect was accompanied by a strong inhibition of membrane type-1 matrix metalloproteinase (matrix metalloproteinase-14) and vascular endothelial growth factor expression. Collectively, our data indicate that the NC1 domain of type XIX collagen exerts antitumor activity. This effect is mediated by a strong inhibition of the invasive capacities of tumor cells and antiangiogenic effects. NC1(XIX) should now be considered as a new member of the basement membrane collagen-derived matrikine family with antitumor and antiangiogenic activity.

Brain tissue characterisation by infrared imaging in a rat glioma model.

Pathological changes associated with the development of brain tumor were investigated by Fourier transform infrared microspectroscopy (FT-IRM) with high spatial resolution. Using multivariate statistical analysis and imaging, all normal brain structures were discriminated from tumor and surrounding tumor tissues. These structural changes were mainly related to qualitative and quantitative changes in lipids (tumors contain little fat) and were correlated to the degree of myelination, an important factor in several neurodegenerative disorders. Lipid concentration and composition may thus be used as spectroscopic markers to discriminate between healthy and tumor tissues. Additionally, we have identified one peculiar structure all around the tumor. This structure could be attributed to infiltrative events, such as peritumoral oedema observed during tumor development. Our results highlight the ability of FT-IRM to identify the molecular origin that gave rise to the specific changes between healthy and diseased states. Comparison between pseudo-FT-IRM maps and histological examinations (Luxol fast blue, Luxol fast blue-cresyl violet staining) showed the complementarities of both techniques for early detection of tissue abnormalities.

Indolent CD8-positive lymphoid proliferation of the ear: a distinct primary cutaneous T-cell lymphoma?

The authors report 4 cases of cutaneous lymphoproliferation unusual by their histology and their clinical presentation. Each presented with a history of a slow growing nodule on the ear. Despite the indolent clinical evolution, the histology suggested a high-grade lymphoma. All lesions consisted of a dense, diffuse proliferation of monomorphous medium-sized T cells throughout the dermis and subcutis. There was no epidermotropism and a grenz zone was clearly present in each case. The tumor cells displayed irregular blastlike nuclei, with small nucleoli and clear chromatin and had a CD3+, CD8+, CD4+, TIA1+, granzyme B(-)immunophenotype with a loss of other T-cell antigens. The 3 cases with available material for polymerase chain reaction studies displayed a monoclonal T-cell rearrangement of the T-cell receptor-gamma chain. These cases do not correspond to a recognized cutaneous T-cell lymphoma as described in the recent WHO/EORTC classification. The apparent striking propensity for the ear suggests that they might represent a specific entity. Further cases are needed to confirm this hypothesis. It is important for such indolent lesions to be known to avoid over treatment.

Prevalence of Abnormal Cervical Cytology in HIVNegative Women Participating in a Cervical Cancer Screening Program in Calmette Hospital, Cambodia.

Prevalence of Abnormal Cervical Cytology in HIVNegative Women Participating in a Cervical Cancer Screening Program in Calmette Hospital, Cambodia.

[Autopsy room and the July 30th 2004 memorandum]. / Salle d'autopsie et circulaire du 30 juillet 2004.

With the July 30th 2004 memorandum, for the first time a text is specifically dedicated to the architecture of the autopsy room. This memorandum reaffirms certain technical specifications stated in the May 7th 2001 decree applicable to hospital mortuaries. It supplements or modifies certain elements, particularly liquid waste processing, which will require new arrangements in death chambers and new expenditures for hospital administrations. It includes the principle of precaution and requires a new approach to handling human corpses in the autopsy room.

Immunohistochemical study of DNA topoisomerase I, DNA topoisomerase II alpha, p53, and Ki-67 in oral preneoplastic lesions and oral squamous cell carcinomas.

Human DNA topoisomerase I (topo I) is the molecular target of the camptothecin group of anticancer drugs. Laboratory studies have shown that the cellular response to topo I-targeted drugs depends on the topo I expression and DNA replication rate and the apoptotic pathway activity. In this study, we tested potential indicators of the sensitivity of topo I-targeted drugs in 36 cases of oral squamous cell carcinoma (OSCC). Formalin-fixed, paraffin-embedded tissue sections were immunostained with monoclonal antibodies against Ki-67, p53, and topo I, and with polyclonal antibodies against DNA topoisomerase II-alpha (topo II-alpha). These markers were also tested in 18 epithelial hyperplastic lesions and 18 mild dysplasias. Immunostaining was quantified by the percentage of stained nuclei in each sample (the labeling index); 200 immunoreactive epithelial nuclei were counted per case for each antibody. The results support the possibility of using topo II-alpha staining for assessing the proliferative activity. High expression of topo II-alpha and topo I in OSCCs suggests that they may serve as potential indicators of sensitivity to topo I inhibitors. However, the apoptotic pathway assessed by p53 immunostaining was found to be uninformative. Analysis of the relationship between immunohistochemical results and clinical and pathologic parameters (the T and N stages and differentiation) showed that only the differentiation parameter correlated with the topo I expression rate. Thus, significant increase in the topo I expression in the poorly differentiated OSCCs suggests their higher sensitivity to drug treatment.

Topotecan-induced alterations in the amount and stability of human DNA topoisomerase I in solid tumor cell lines.

BACKGROUND: Human DNA topoisomerase I (topo 1) is an essential nuclear enzyme involved in vital cellular processes and the sole target of antitumor drugs of the camptothecin (CPT) family. The CPT derivative topotecan (Tpt, Hycamtin) is currently used in clinic, its effectiveness varying considerably for different types of cancer. The purpose of this study was to compare time- and dose-dependent cellular responses to Tpt in terms of alterations in the amount and stability of topo 1 in lung adenocarcinoma (A-549), ovarian adenocarcinoma (CaOv-3), colorectal adenocarcinoma (HT-29) and breast adenocarcinoma (MCF-7) cell lines. MATERIALS AND METHODS: Western blot analysis of the time-dependent redistribution of a full-size topo 1 and its proteolytical fragments was performed after Tpt treatment for 1 h at concentrations 10-fold or 100-fold higher than the Tpt IC50 for the respective cell lines. RESULTS: Tpt treatment of the CaOv-3 cell line produced a substantial time-dependent decrease in the amount of topo 1 immunoprotein. Conversely, the MCF7 cell line did not exhibit a topo 1-associated response to the Tpt treatment. Strong but different time- and dose-dependent topo 1 down-regulation effects were observed in the HT-29 and A-549 cell lines. CONCLUSION: The data obtained indicate that Tpt-induced time- and dose-dependent effects on the amount and stability of topo 1 are involved in the mechanisms of Tpt activity against different solid tumor cell lines.

Camptothecin conjugated with DNA minor-groove binder netropsin: enhanced lactone stability, inhibition of human DNA topoisomerase I and antiproliferative activity.

BACKGROUND: The conjugates of camptothecin (CPT) with ligands possessing different DNA selectivity could be promising agents in cancer therapy affecting expression of specific genes by trapping DNA topoisomerase I (top I)-DNA complexes in a sequence-selective manner. Our recent data show that minor-groove binder netropsin (Nt) and its derivatives modulate the CPT-induced pattern of top I-mediated DNA cleavage. In an effort to develop a new molecule with good biological activity we have linked CPT with Nt and report here the first results of in vitro examination of the new compound. MATERIALS AND METHODS: CPT-Nt conjugate linked with flexible spacer through position 7 of CPT chromophore was synthesized and analyzed for lactone stability, the ability to modulate a top I-mediated DNA cleavage and antiproliferative activity within a panel of six tumor cell lines. RESULTS: CPT-Nt conjugate demonstrates enhanced lactone stability and concentration-dependent top I poisoning or suppression in vitro. The rate of conjugate hydrolysis in a water solution displays a 20-fold enhancement of stability compared with CPT. The cytotoxicity of the conjugate against acute promyelocytic leukaemia (HL60), chronic myelogenous leukaemia (K562), breast adenocarcinoma (MCF7), colorectal adenocarcinoma (HT29), lung carcinoma(A549) and ovarian adenocarcinoma (CaOV3) tumor cell lines was evaluated. The lowest IC50 value (0.08 microM) indicated its selective toxicity towards the ovarian adenocarcinoma cell line. CONCLUSION: The enhanced stability of CPT-Nt conjugate and its selective toxicity against the CaOV3 cell line may indicate its utility as an antitumor agent against ovarian adenocarcinoma.

[Cutaneous and bone metastases revealing hepatocarcinoma]. / Métastases mixtes cutanéo-osseuses révélatrices d'un carcinome hépatocellulaire.

We report the case of a 55-year-old patient who was admitted to our hospital for a frontal tumor. He had a left forehead nodule that had appeared several weeks before, measuring 6 cm in diameter. On palpation, it was painless and there was a pulse, and the skin above was non ulcerated. Radiography and computed tomographic scan of the skull suggested a subcutaneous metastasis. Computed tomographic scans of the lung and the abdomen showed a heterogeneous liver with irregular outlines and secondary adrenal infiltrations. A biopsy of the frontal tumor confirmed a metastatic hepatocellular carcinoma.

Juvenile frontotemporal dementia with parkinsonism associated with tau mutation G389R.

Frontotemporal lobe degeneration includes a large spectrum of neurodegenerative disorders. Patients with frontotemporal dementia with parkinsonism linked to chromosome 17 exhibit heterogeneity in both clinical and neuropathological features. Here, we report the case of a young patient with a G389R mutation. This teenager girl was 17 years old when she progressively developed severe behavioral disturbances. First, she was considered to be suffering from atypical depression. After 2 years, she was referred to the department of neurology. By this time, the patient exhibited typical frontotemporal dementia with mild extrapyramidal disorders. The main behavioral features included apathy and reduced speech output. MRI and SPECT showed a frontotemporal atrophy and hypofixation, respectively. She died 7 years after onset. Three relatives on her father side had also died after early onset dementia. Genetic testing revealed a heterozygous guanine to cytosine mutation at the first base of codon 389 (Exon 13) of MAPT, the tau gene, resulting in a glycine to arginine substitution, in the patient and her non-affected father. Postmortem neuropathological and biochemical data indicate a Pick-like tau pathology but with phosphoserine 262-positive immunoreactivity. This case is remarkable because of the extremely early onset of the disease.

Preliminary investigation of a mice model of Klebsiella pneumoniae subsp. ozaenae induced pneumonia.

In the present study, we comparatively assessed the pathophysiological mechanisms developed during lung infection of BALB/C female mice infected by an original wild type Klebsiella pneumoniae subsp. ozaenae strain (CH137) or by a referent subspecies K. pneumoniae. subsp. pneumoniae strain (ATCC10031). The mice infected with 2.106 CFU K. p. subsp. pneumoniae (n = 10) showed transient signs of infection and all of them recovered. All of those infected with 1.106 CFU K. p. subsp. ozaenae (n = 10) developed pneumonia within 24 h and died between 48 and 72 h. Few macrophages, numerous polymorphonuclear cells and lymphocytes were observed in their lungs in opposite to K. p. subsp. pneumoniae. In bronchoalveolar lavage, a significant increase in MIP-2, IL-6, KC and MCP-1 levels was only observed in K. p. subsp. ozaenae infected mice whereas high levels of TNF-α were evidenced with the two subspecies. Our findings indicated a lethal effect of a wild type K. p. subsp. ozaenae strain by acute pneumonia reflecting an insufficient alveolar macrophage response. This model might be of a major interest to comparatively explore the pathogenicity of K. p. subsp ozaenae strains and to further explore the physiopathological mechanisms of gram-negative bacteria induced human pneumonia.

Semiconductor quantum dots for multiplexed bio-detection on solid-state microarrays.

Understanding cellular systems requires identification and analysis of their multiple components and determination of how they act together and are regulated. Microarray technology is one of the few tools that is able to solve such problems. It is based on high-throughput recognition of a target to the probe and has the potential to simultaneously measure the presence of numerous molecules in multiplexed tests, all contained in a small drop of test fluid. Microarrays allow the parallel analysis of genomic or proteomic content in healthy versus disease-affected or altered tissues or cells. The signal read-out from the microarrays is done with organic dyes which often suffer of photobleaching, low brightness and background fluorescence. Recent data show that the use of fluorescent nanocrystals named "quantum dots" (QDs) allows to push these limits away. QDs are sufficiently bright to be detected as individual particles, extremely resistant against photobleaching and provide unique possibilities for multiplexing, thus supplying the microarray technology with a novel read-out option enabling the sensitivity of detection to reach the single-molecule level. This paper reviews QDs applications to microarray-based detection and demonstrates how the combination of microarray and QDs technologies may increase sensitivity and highly parallel capacities of multiplexed microarrays. Such a combination should provide the breakthrough results in drug discovery, cancer diagnosis and establish new therapeutic approaches through the identification of binding target molecules and better understanding of cell signalling pathways.