Microsporidia - Emergent Pathogens in the Global Food Chain.

Intensification of food production has the potential to drive increased disease prevalence in food plants and animals. Microsporidia are diversely distributed, opportunistic, and density-dependent parasites infecting hosts from almost all known animal taxa. They are frequent in highly managed aquatic and terrestrial hosts, many of which are vulnerable to epizootics, and all of which are crucial for the stability of the animal-human food chain. Mass rearing and changes in global climate may exacerbate disease and more efficient transmission of parasites in stressed or immune-deficient hosts. Further, human microsporidiosis appears to be adventitious and primarily associated with an increasing community of immune-deficient individuals. Taken together, strong evidence exists for an increasing prevalence of microsporidiosis in animals and humans, and for sharing of pathogens across hosts and biomes.

Development of dual fluorescent stage specific reporter strain of Toxoplasma gondii to follow tachyzoite and bradyzoite development in vitro and in vivo.

Toxoplasma gondii is a protozoan that infects 30% of humans as intermediate hosts. T Sexual reproduction can occur only within the intestinal tract of felines, however, infection in other mammals and birds is associated with asexual replication and interconversion between the tachyzoite and bradyzoite stages. Bradyzoites are slow growing forms found in tissue cysts in latent infection. Recently, our group described the biological behavior of the EGS strain that forms thick walled cysts spontaneously in tissue culture, constituting a useful tool for examining the developmental biology of T. gondii. To further improve the usefulness of this model, we constructed genetically modified EGS parasites that express fluorescent tags under the control of stage specific promoters. The promoter regions for SAG-1 (tachyzoite specific), BAG-1 and LDH-2 (bradyzoite specific) were amplified by PCR and plasmids were constructed with mCherry (redT) and sfGFP (greenB) sequences, respectively. Strains of parasites were selected using FACS to arrive at single fluorescent and dual fluorescent strains of EGS expressing tags in a stage specific manner. In cell cultures, vacuoles labeled by immunofluorescence assay using anti-CST-1 a marker for T. gondii cyst wall contained parasites that were positive for BAG1-GFP and negative for SAG1-mCherry. Tachyzoites and bradyzoites harvested from the mice expressed stage specific mCherry and GFP proteins, respectively. These new dual fluorescent transgenic EGS strains are a promising tool to elucidate the mechanisms of T. gondii differentiation both in vitro and in vivo.

A meta-analysis of Hodgkin lymphoma reveals 19p13.3 TCF3 as a novel susceptibility locus.

Recent genome-wide association studies (GWAS) of Hodgkin lymphoma (HL) have identified associations with genetic variation at both HLA and non-HLA loci; however, much of heritable HL susceptibility remains unexplained. Here we perform a meta-analysis of three HL GWAS totaling 1,816 cases and 7,877 controls followed by replication in an independent set of 1,281 cases and 3,218 controls to find novel risk loci. We identify a novel variant at 19p13.3 associated with HL (rs1860661; odds ratio (OR)=0.81, 95% confidence interval (95% CI) = 0.76-0.86, P(combined) = 3.5 × 10(-10)), located in intron 2 of TCF3 (also known as E2A), a regulator of B- and T-cell lineage commitment known to be involved in HL pathogenesis. This meta-analysis also notes associations between previously published loci at 2p16, 5q31, 6p31, 8q24 and 10p14 and HL subtypes. We conclude that our data suggest a link between the 19p13.3 locus, including TCF3, and HL risk.

Overexpression of miR-26a-2 in human liposarcoma is correlated with poor patient survival.

Approximately 90% of well-differentiated/de-differentiated liposarcomas (WDLPS/DDLPS), the most common LPS subtype, have chromosomal amplification at 12q13-q22. Many protein-coding genes in the region, such as MDM2 and , have been studied as potential therapeutic targets for LPS treatment, with minimal success. In the amplified region near the MDM2 gene, our single nucleotide polymorphism (SNP) array analysis of 75 LPS samples identified frequent amplification of miR-26a-2. Besides being in the amplicon, miR-26a-2 was overexpressed significantly in WDLPS/DDLPS (P<0.001), as well as in myxoid/round cell LPS (MRC) (P<0.05). Furthermore, Kaplan-Meier survival analysis showed that overexpression of miR-26a-2 significantly correlated with poor patient survival in both types of LPS (P<0.05 for WDLPS/DDLPS; P<0.001 for MRC). Based on these findings, we hypothesized that miR-26a-2 has an important role in LPS tumorigenesis, regardless of LPS subtypes. Overexpression of miR-26a-2 in three LPS cell lines (SW872, LPS141 and LP6) enhanced the growth and survival of these cells, including faster cell proliferation and migration, enhanced clonogenicity, suppressed adipocyte differentiation and/or resistance to apoptosis. Inhibition of miR-26a-2 in LPS cells using anti-miR-26a-2 resulted in the opposite responses. To explain further the effect of miR-26a-2 overexpression in LPS cells, we performed in silico analysis and identified 93 candidate targets of miR-26a-2. Among these genes, RCBTB1 (regulator of chromosome condensation and BTB domain-containing protein 1) is located at 13q12.3-q14.3, a region of recurrent loss of heterozygosity (LOH) in LPS. Indeed, either overexpression or inhibition of RCBTB1 made LPS cells more susceptible or resistant to apoptosis, respectively. In conclusion, our study for the first time reveals the contribution of miR-26a-2 to LPS tumorigenesis, partly through inhibiting RCBTB1, suggesting that miR-26a-2 is a novel therapeutic target for human LPS.

Host Cell Preference of Toxoplasma gondii Cysts in Murine Brain: A Confocal Study.

Toxoplasma gondii is a protozoan parasite that is widely prevalent in humans and typically results in a chronic infection characterized by cysts located predominantly in the central nervous system. In immunosuppressed hosts, such as patients with HIV infection, the infection can be reactivated from the cysts in the brain resulting in a severe and potentially fatal encephalitis. Studies suggest that the chronic infection may also have neuropathological and behavioral effects in immune competent hosts. An improved understanding of tissue cyst behavior is of importance for understanding both the reactivation as well as the neurophysiological consequences of chronic infection. In vivo studies have identified neurons as host cells for cysts but in vitro studies have found that astrocytes can also foster development of the cysts. In this study we have addressed the question of which neural cell tissue cysts of T. gondii reside during chronic infection using a mouse model. Mice were infected with Me49 Strain T. gondii and the intracellular localization of the cysts analyzed during the development and establishment of a chronic infection at 1, 2, and 6 months post infection. Brains were fixed, cryosectioned, and stained with FITC-Dolichos biflorans to identify the Toxoplasma cysts and they were labeled with cell specific antibodies to neurons or astrocytes and then analyzed using confocal fluorescence microscopy. Cysts were found to occur almost exclusively in neurons throughout chronic infection. No cysts were identified in astrocytes, using the astrocyte marker, GFAP. Astrocyte interactions with neuronal-cysts, however, were frequently observed.

The gamma interferon (IFN-gamma)-inducible GTP-binding protein IGTP is necessary for toxoplasma vacuolar disruption and induces parasite egression in IFN-gamma-stimulated astrocytes.

Toxoplasma gondii is a common central nervous system infection in individuals with immunocompromised immune systems, such as AIDS patients. Gamma interferon (IFN-gamma) is the main cytokine mediating protection against T. gondii. Our previous studies found that IFN-gamma significantly inhibits T. gondii in astrocytes via an IFN-gamma-inducible GTP-binding protein (IGTP)-dependent mechanism. The IGTP-dependent-, IFN-gamma-stimulated inhibition is not understood, but recent studies found that IGTP induces disruption of the parasitophorous vacuole (PV) in macrophages. In the current study, we have further investigated the mechanism of IFN-gamma inhibition and the role of IGTP in the vacuolar disruption in murine astrocytes. Vacuolar disruption was found to be dependent upon IGTP, as PV disruption was not observed in IGTP-deficient (IGTP(-/-)) astrocytes and PV disruption could be induced in IGTP(-/-) astrocytes transfected with IGTP. Live-cell imaging studies using green fluorescent protein-IGTP found that IGTP is delivered to the PV via the host cell endoplasmic reticulum (ER) early after invasion and that IGTP condenses into vesicle-like structures on the vacuole just prior to PV disruption, suggesting that IGTP is involved in PV disruption. Intravacuolar movement of the parasite occurred just prior to PV disruption. In some instances, IFN-gamma induced parasite egression. Electron microscopy and immunofluorescence studies indicate that the host cell ER fuses with the PV prior to vacuolar disruption. On the basis of these results, we postulate a mechanism by which ER/PV fusion is a crucial event in PV disruption. Fusion of the ER with the PV, releasing calcium into the vacuole, may also be the mechanism by which intravacuolar parasite movement and IFN-gamma-induced parasite egression occur.

Microsporidiosis in South Africa: PCR detection in stool samples of HIV-positive and HIV-negative individuals and school children in Vhembe district, Limpopo Province.

Microsporidia were initially recognized as pathogens of insects and fish but have recently emerged as an important group of human pathogens, especially in immune-compromised individuals, such as those with HIV infection. In this study, we used a PCR-RFLP assay confirmed by quantitative real-time PCR and trichrome staining to determine the prevalence of microsporidian infections among hospital patients and school children in Vhembe region. Enterocytozoon bieneusi was the only microsporidian species detected in these stool samples. It was found in 33 (12.9%) of 255 samples from the hospitals and in 3 (4.5%) of 67 samples from primary school children and was significantly associated (P=0.039) with diarrhea in HIV-positive patients (21.6%) compared to HIV-negative individuals (9%). However, microsporidian infections were not associated with intestinal inflammation as indicated by the lactoferrin test. These results suggest that microsporidia might be a cause of secretory diarrhea in HIV-positive patients. To our knowledge, this is the first report of E. bieneusi in the Vhembe region of South Africa. Further investigations are needed in order to clarify the pathogenesis of E. bieneusi in HIV-positive patients.

The immunology of parasite infections in immunocompromised hosts.

Immune compromise can modify the severity and manifestation of some parasitic infections. More widespread use of newer immnosuppressive therapies, the growing population of individuals with immunocompromised states as well as the prolonged survival of these patients have altered the pattern of parasitic infection. This review article discusses the burden and immunology of parasitic infections in patients who are immunocompromised secondary to congenital immunodeficiency, malnutrition, malignancy, and immunosuppressive medications. This review does not address the literature on parasitic infections in the setting of HIV-1 infection.

Alpha-methylacyl-CoA racemase: a multi-institutional study of a new prostate cancer marker.

AIM: To test whether alpha-methylacyl-CoA racemase (AMACR) is a sensitive and specific marker of prostate cancer. METHODS AND RESULTS: The expression levels of AMACR mRNA were measured by real-time polymerase chain reaction. A total of 807 prostatic specimens were further examined by immunohistochemistry specific for AMACR. Quantitative immunostaining analyses were carried out by using the ChromaVision Automated Cellular Imaging System and the Ariol SL-50 Imaging System, respectively. AMACR mRNA levels measured in prostatic adenocarcinoma were 55 times higher than those in benign prostate tissue. Of 454 cases of prostatic adenocarcinoma, 441 were positive for AMACR, while 254 of 277 cases of benign prostate were negative for AMACR. The sensitivity and specificity of AMACR immunodetection of prostatic adenocarcinomas were 97% and 92%, respectively. Both positive and negative predictive values were 95%. By automatic imaging analyses, the AMACR immunostaining intensity and percentage in prostatic adenocarcinomas were also significantly higher than those in benign prostatic tissue (105.9 versus 16.1 for intensity, 45.7% versus 0.02% and 35.03% versus 4.64% for percentage, respectively). CONCLUSIONS: We have demonstrated the promising features of AMACR as a biomarker for prostate cancer in this large series and the potential to develop automated quantitative diagnostic tests.

Usefulness of immunohistochemistry in delineating renal spindle cell tumours. A retrospective study of 31 cases.

AIMS: To assess the usefulness of immunohistochemistry in delineating tumour diagnoses on a series of morphologically diagnosed renal spindle cell tumours (RSCTs). METHODS AND RESULTS: Formalin-fixed paraffin-embedded tissues from 31 morphologically diagnosed tumours were reinterpreted in light of newly obtained immunohistochemical information. By morphology, six had originally been classified as sarcomatoid carcinoma, five as spindle cell tumour (NOS), four as sarcoma (NOS), three as leiomyoma, three as leiomyosarcoma, and one each as fibrous polyp, hamartoma, neurilemmoma, mesoblastic nephroma, medullary fibroma, angiomyolipoma, haemangiopericytoma, malignant rhabdoid tumour, malignant Triton tumour, and carcinosarcoma. The application of immunohistochemistry verified the original diagnosis in 18 cases (18/31, 58%), confirming the diagnosis of sarcomatoid renal carcinoma (4/6), leiomyoma (2/3), leiomyosarcoma (3/3), sarcoma (NOS) (2/4), carcinosarcoma (1/1), malignant rhabdoid tumour (1/1), malignant Triton tumour (1/1), fibrous polyp (1/1), mesoblastic nephroma (1/1), hamartoma (1/1), and angiomyolipoma (1/1). Different tumour designations were suggested in 13 cases (13/31, 42%), including carcinosarcoma, sarcoma (NOS), leiomyosarcoma, solitary fibrous tumour, monomorphic/biphasic angiomyolipoma, endometrial stromal tumour, and congenital mesoblastic nephroma. CONCLUSIONS: Our data indicate that although morphology is most important in formulating the initial differential diagnosis, the addition of immunohistochemistry is vital in arriving at the correct classification of RSCTs.

Polyamine metabolism in the Microsporidia.

Members of the phylum Microspora are all obligate intracellular parasites. Little is known concerning metabolic pathways in these parasites, some of which pose serious problems in immunocompromised patients. We investigated polyamine metabolism in the systemic pathogen Enterocytozoon cuniculi using intact pre-emergent spores, and cell-free preparations. We found both polyamine synthetic and interconversion pathways to be operative, as evidenced by conversion of ornithine into polyamines, and production of spermidine from spermine by pre-emergent spores. Recent developments in the antitumour field have highlighted the ability of bis-ethylated polyamine analogues to reduce polyamine levels and block growth of tumour cells. In light of enhanced polyamine uptake in Enc. cuniculi, we have begun to study bis-aryl 3-7-3 and bis-ethyl oligoamine analogues as leads for chemotherapy of microsporidia.

Absence of interferon-gamma-inducible gene IGTP does not significantly alter the development of chagasic cardiomyopathy in mice infected with Trypanosoma cruzi (Brazil strain).

Interferon-gamma (IFN-gamma) contributes to host resistance during acute infection with Trypanosoma cruzi, the causative agent of Chagas' disease. Inducibly expressed guanosine triphosphatase (IGTP), a 48-kDa guanosine triphosphatase (GTPase), is a member of a family of GTPase proteins inducibly expressed by IFN-gamma. The expression pattern of IGTP suggests that it may mediate IFN-gamma-induced responses in a variety of cell types. IGTP has been demonstrated to be important for control of Toxoplasma gondii infection but not for resistance against Listeria monocytogenes. We evaluated the role of IGTP in development of chronic chagasic cardiomyopathy in IGTP null mice and C57X129sv (wild type [WT]) mice infected with the Brazil strain for 6 mo. There was no significant difference in parasitemia or cardiac histopathology between null and WT mice. Right ventricular remodeling was observed in infected IGTP null mice, suggesting that IGTP does not significantly alter the course of T. cruzi infection.

Tumours of histiocytes and accessory dendritic cells: an immunohistochemical approach to classification from the International Lymphoma Study Group based on 61 cases.

Neoplasms of histiocytes and dendritic cells are rare, and their phenotypic and biological definition is incomplete. Seeking to identify antigens detectable in paraffin-embedded sections that might allow a more complete, rational immunophenotypic classification of histiocytic/dendritic cell neoplasms, the International Lymphoma Study Group (ILSG) stained 61 tumours of suspected histiocytic/dendritic cell type with a panel of 15 antibodies including those reactive with histiocytes (CD68, lysozyme (LYS)), Langerhans cells (CD1a), follicular dendritic cells (FDC: CD21, CD35) and S100 protein. This analysis revealed that 57 cases (93%) fit into four major immunophenotypic groups (one histiocytic and three dendritic cell types) utilizing six markers: CD68, LYS, CD1a, S100, CD21, and CD35. The four (7%) unclassified cases were further classifiable into the above four groups using additional morphological and ultrastructural features. The four groups then included: (i) histiocytic sarcoma (n=18) with the following phenotype: CD68 (100%), LYS (94%), CD1a (0%), S100 (33%), CD21/35 (0%). The median age was 46 years. Presentation was predominantly extranodal (72%) with high mortality (58% dead of disease (DOD)). Three had systemic involvement consistent with 'malignant histiocytosis'; (ii) Langerhans cell tumour (LCT) (n=26) which expressed: CD68 (96%), LYS (42%), CD1a (100%), S100 (100%), CD21/35 (0%). There were two morphological variants: cytologically typical (n=17) designated LCT; and cytologically malignant (n=9) designated Langerhans cell sarcoma (LCS). The LCS were often not easily recognized morphologically as LC-derived, but were diagnosed based on CD1a staining. LCT and LCS differed in median age (33 versus 41 years), male:female ratio (3.7:1 versus 1:2), and death rate (31% versus 50% DOD). Four LCT patients had systemic involvement typical of Letterer-Siwe disease; (iii) follicular dendritic cell tumour/sarcoma (FDCT) (n=13) which expressed: CD68 (54%), LYS (8%), CD1a (0%), S100 (16%), FDC markers CD21/35 (100%), EMA (40%). These patients were adults (median age 65 years) with predominantly localized nodal disease (75%) and low mortality (9% DOD); (iv) interdigitating dendritic cell tumour/sarcoma (IDCT) (n=4) which expressed: CD68 (50%), LYS (25%), CD1a (0%), S100 (100%), CD21/35 (0%). The patients were adults (median 71 years) with localized nodal disease (75%) without mortality (0% DOD). In conclusion, definitive immunophenotypic classification of histiocytic and accessory cell neoplasms into four categories was possible in 93% of the cases using six antigens detected in paraffin-embedded sections. Exceptional cases (7%) were resolvable when added morphological and ultrastructural features were considered. We propose a classification combining immunophenotype and morphology with five categories, including Langerhans cell sarcoma. This simplified scheme is practical for everyday diagnostic use and should provide a framework for additional investigation of these unusual neoplasms.

Keratin expression in human tissues and neoplasms.

Keratin expression in human tissues and neoplasms Keratin filaments constitute type I and type II intermediate filaments (IFs), with at least 20 subtypes named keratin 1-20. Since certain keratin subtypes are only expressed in some normal human tissues but not others, and vice versa, various tissues have been subclassified according to the pattern of keratin staining. Simple epithelia generally express the simple epithelial keratins 7, 18, 19, and 20, while complex epithelia express complex epithelial keratins 5/6, 10, 14, and 15. When an epithelium undergoes malignant transformation, its keratin profile usually remains constant. The constitution and expression patterns of keratin filaments in human epithelial neoplasms are complex and often distinctive. In this article, we first briefly review the molecular and cell biology of keratin filaments. We then focus on the expression patterns of keratin filaments in various human neoplasms.

Neospora caninum in vitro: evidence that the destiny of a parasitophorous vacuole depends on the phenotype of the progenitor zoite.

We previously reported that Neospora caninum can be induced to express BAGI, a bradyzoite antigen, within 3 days of culture under stress conditions. The main goals of the present experiment were to increase the expression of BAGI in vitro (in part by extending cultures for 9 days), to observe parasitophorous vacuoles at various points of stage differentiation, and to test the ability of organisms produced in vitro to function like mature bradyzoites. Expression of BAG1 and of a tachyzoite antigen (NcSAGI) was monitored using a double-label immunofluorescence assay. For the purpose of this study, organisms expressing NcSAG1 were designated as tachyzoites, those expressing BAG1 were designated as bradyzoites, and those expressing both antigens were designated as intermediate zoites. The greatest percentage of intermediate zoites and bradyzoites (14%) occurred in bovine monocytes maintained for 9 days. These bradyzoites did not appear to be functionally mature; they did not induce patent infections in dogs. in contrast to bradyzoites that were produced in chronically infected mice. In vitro, large parasitophorous vacuoles contained either a pure population of tachyzoites or a mixture of tachyzoites and intermediate zoites, which is indicative of asynchronous stage conversion of organisms within a vacuole. Bradyzoites were first observed within small vacuoles on day 6. and bradyzoites never shared vacuoles with tachyzoites. This finding suggests that vacuoles containing bradyzoites may develop only if the cell is invaded by a zoite that has already begun bradyzoite differentiation. An alternative possibility is that cysts may develop if the establishing tachyzoite undergoes bradyzoite differentiation before multiplying. Cysts do not appear to arise from transformation of tachyzoites within large parasitophorous vacuoles.

Cytokeratin 14 immunoreactivity distinguishes oncocytic tumour from its renal mimics: an immunohistochemical study of 63 cases.

AIMS: The cytokeratin 14 (CK14) expression in oncocytomas or oncocytic tumours of various tissue origins has not been established. We have studied CK14 expression in 30 cases of oncocytic tumours of various tissue origins and 33 cases of renal cell carcinoma with overlapping features (mimics) by immunohistochemistry. METHODS AND RESULTS: Immunohistochemistry (ABC-HRP method) was performed for detection of CK14 in 30 cases of oncocytic tumour and 33 cases of renal mimics. To demonstrate CK14 specificity and sensitivity in oncocytic tumours, mES-13 (an anti-mitochondrial monoclonal antibody) immunohistochemistry was also performed in 20 of 30 cases on oncocytic tumour and all 33 cases of renal mimics. We found that all 30 cases of oncocytic tumour showed cytoplasmic CK14 positivity. All 20 cases of oncocytic tumour studied with mES-13 were positive. CK14 immunoreactivity was identified in only four cases of renal cell carcinoma (one conventional renal cell carcinoma with granular cytoplasm and three chromophobe renal cell carcinomas with eosinophilic cytoplasm). In contrast, all 33 cases of renal cell carcinoma were positive for mES-13 to varying degrees. CONCLUSION: The homogeneous, cytoplasmic, and granular CK14 immunoreactivity is sensitive and specific for oncocytic tumours, whereas CK14 immunoreactivity in renal mimics is light and sporadic with peripheral accentuation.