Shotgun metagenomic screening to improve the retrospective diagnosis of undocumented intrauterine infections on embedded placenta samples: Lessons from a brucellosis case.

BACKGROUND: Microbiological diagnosis of intrauterine infections (IIU) still relies on bacteriological cultures or targeted DNA amplification lacking in sensitivity. Shotgun metagenomics (SMg) is an emerging unbiased molecular approach that makes it possible to sequence all the nucleic acids from any sample. It had never previously been used for IIU. METHODS: We here report the case of a patient with an unexplained IIU and fetal loss that could be documented by a combined SMg/microbiological approach, leading to the diagnosis of maternal brucellosis. RESULTS: A 31-year-old woman presented with an undocumented IIU with fetal loss at 24 weeks of gestation. Culture-based work-up failed to identify the pathogen involved. Paraffin-embedded placenta sample was retrospectively analyzed by SMg. Brucella spp nucleic acids were detected, and subacute maternal brucellosis was confirmed by targeted PCR and serological testing. CONCLUSION: This case provides grounds for further utilization of SMg for the microbiological diagnosis of unexplained obstetrical infections.

Social network analysis of cattle movement in Kampong Cham, Kampong Speu and Takeo, Cambodia.

The aim of this study is to provide insight the network of cattle movement in Kampong Cham, Kampong Speu and Takeo, Cambodia. A cross-sectional study was carried out from July 2014 to August 2014, using questionnaires. It was implemented with 435 interviewees (24.4%, 24.6% and 51.0% from Kampong Cham, Kampong Speu and Takeo, respectively) using one-step snowball sampling. The findings suggest that the key players in all three provinces are producers who raise their cattle as backyard animals. In all three provinces the key players in spreading disease are probably the middlemen, collectors, brokers or traders. The network of cattle movement is presented as a strong component of varying size in each location. In this network we found three cut-points in both Kampong Cham and Kampong Speu. The network in each province indicates a random pattern of node distribution. The results of our study are useful to relevant authorities and researchers to understand the spread of infectious diseases into different areas. The middlemen, collectors, brokers and traders need to be controlled as first priority in order to reduce the magnitude of the spread of disease.

Caught on camera: hairy polyp of the posterior tonsillar pillar.

BACKGROUND: Hairy polyps are rare congenital growths of the head and neck, mainly found in the nasopharynx and oropharynx. They are made up of two germ cell layers: the ectoderm and mesoderm. METHODS: This paper reports a four-month-old who presented with breathing and feeding difficulties. Clinical examination was unremarkable, but a video taken by the patient's mother on her smartphone showed a mass protruding from the infant's mouth. Laryngoscopy performed in the operating theatre showed that the mass emanated from the left posterior tonsillar pillar. RESULTS: The mass was removed transorally with no complications. Pathological examination showed a skin-covered pedunculated structure characteristic of a hairy polyp. The patient's follow up was unremarkable. CONCLUSION: To the best of our knowledge, this is the second English-language case report of a patient with a hairy polyp emanating from a posterior tonsillar pillar. This paper also highlights the growing usage of smartphones by patients to help physicians with their diagnosis and management.

Inhibition of fatty acid amide hydrolase activates Nrf2 signalling and induces heme oxygenase 1 transcription in breast cancer cells.

BACKGROUND AND PURPOSE: Endocannabinoids such as anandamide (AEA) are important lipid ligands regulating cell proliferation, differentiation and apoptosis. Their levels are regulated by hydrolase enzymes, the fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL). Here, we investigated whether FAAH or AEA are involved in NF (erythroid-derived 2)-like 2 (Nrf2)/antioxidant responsive element (ARE) pathway. EXPERIMENTAL APPROACH: The aim of this study was to analyse the effects of AEA or FAAH inhibition by the URB597 inhibitor or FAAH/siRNA on the activation of Nrf2-ARE signalling pathway and heme oxygenase-1 (HO-1) induction and transcription. KEY RESULTS: Endogenous AEA was detected in the immortalized human mammary epithelial MCF-10A cells (0.034 ng per 10(6) cells) but not in MCF-7 or MDA-MB-231 breast cancer cells. Because breast tumour cells express FAAH abundantly, we examined the effects of FAAH on Nrf2/antioxidant pathway. We found that inhibition of FAAH by the URB597 inhibitor induced antioxidant HO-1 in breast cancer cells and MCF-10A cells. RNAi-mediated knockdown of FAAH or treatment with AEA-activated ARE-containing reporter induced HO-1 mRNA and protein expression, independent of the cannabinoid receptors, CB1, CB2 or TRPV1. Furthermore, URB597, AEA and siRNA-FAAH treatments induced the nuclear translocation of Nrf2, while siRNA-Nrf2 treatment and Keap1 expression blocked AEA, URB597 and si-FAAH from activation of ARE reporter and HO-1 induction. siRNA-HO-1 treatment decreased the viability of breast cancer cells and MCF-10A cells. CONCLUSIONS AND IMPLICATIONS: These data uncovered a novel mechanism by which inhibition of FAAH or exposure to AEA induced HO-1 transcripts and implicating AEA and FAAH as direct modifiers in signalling mediated activation of Nrf2-HO-1 pathway, independent of cannabinoid receptors.

NRP/B mutations impair Nrf2-dependent NQO1 induction in human primary brain tumors.

Brain tumors are associated with genetic alterations of oncogenes and tumor suppressor genes. Accumulation of reactive oxygen species (ROS) in cells leads to oxidative stress-induced damage, resulting in tumorigenesis. Here, we showed that the nuclear matrix protein nuclear restricted protein in brain (NRP/B) was colocalized and interacted with NF-E2-related factor 2 (Nrf2). During oxidative stress response, NRP/B expression and its interaction with Nrf2 were upregulated in SH-SY5Y cells. Association of NRP/B with Nrf2 was crucial for NAD(P)H:quinone oxidoreductase 1 (NQO1) expression. NRP/B was localized predominantly in the nucleus of normal brain cells, whereas in primary brain tumors NRP/B was almost exclusively contained in the cytoplasm. In addition, unlike wild-type NRP/B, the expression of NRP/B mutants isolated from primary brain tumors was found in the cytoplasm, and these mutants failed to induce Nrf2-dependent NQO1 transcription. Thus, NRP/B mutations and their altered localization resulted in changes in NRP/B function and deregulation of Nrf2-dependent NQO1 activation in brain tumors. This study provides insights into the mechanism by which the NRP/B modulates Nrf2-dependent NQO1 induction in cellular protection against ROS in brain tumors.

In-vitro fertilisation in women aged 40 years and above.

INTRODUCTION: To compare the results of in-vitro fertilisation (IVF) in women aged less than 40 years with those aged 40 years and above with baseline follicle-stimulating hormone (FSH) levels less than 15iu and using their own oocytes. METHODS: A total of 2179 fresh IVF cycles were started in KK Women's and Children's Hospital IVF Centre from 1997 to 2002, of which 247 cycles were done in women 40 to 45 years with FSH levels less than 15iu. The remaining 1932 cycles were performed in another group of women aged less than 40 years old. All couples were treated using our hospital's IVF protocol, and the same clinical and embryological team was involved in all treatments. The medical records of patient outcomes were retrospectively reviewed. The main outcomes measured were clinical pregnancy, miscarriage and delivery rates. RESULTS: The total number of fresh cycles performed in women over 40 years was 247 cycles. Of these, 186 (75.3 percent) cycles reached oocyte collection, and 179 (72.5 percent) cycles reached embryo transfer. The total number of pregnancies was 22 (12.3 percent). The number of cancelled cycles was 61 (24.7 percent). Women less than 40 years of age demonstrated higher rates in cycles reaching oocyte collection (89.2 percent), embryo transfer (84.6 percent), pregnancy rates (32.9 percent) and live-birth rates (24.0 percent). They also reported a lower miscarriage (36.1 percent) and cancellation rate (10.9 percent) as compared to the group of older women. CONCLUSION: As older women seek IVF treatment, it is necessary for them to understand that chances of pregnancy decrease with increasing age. Our results show that as women exceed 40 years old, pregnancy and live-birth rates fall with concurrent rising miscarriage and cycle cancellation rates.

A study on SARS awareness and health-seeking behaviour - findings from a sampled population attending National Healthcare Group Polyclinics.

INTRODUCTION: The study aimed to assess the effectiveness of massive SARS public education effort on SARS awareness and the conduct of those suspected of having SARS. MATERIALS AND METHODS: Five hundred and ninety-three respondents attending the National Healthcare Group Polyclinics (NHGP) participated in the survey from 9 to 13 June 2003. Associations between awareness of SARS symptoms and (i) first action to be taken and (ii) mode of transportation used, if the respondent was suspected of having SARS, were analysed using Chi-square or Fisher's exact tests. Logistic regression was performed to adjust for relevant covariates. RESULTS: The majority (92.7%) of the respondents were aware of SARS symptoms. Television (91.6%), newspaper (65.2%) and radio (30.4%) formed the top 3 sources of information on SARS. Slightly more than half (51.6%) of those who suspect themselves of having SARS would choose to visit their primary health care doctors, while 22.7% of the respondents would go to Tan Tock Seng Hospital (TTSH). If they suspected themselves to have SARS, most (84.9%) of the 578 respondents would react appropriately by taking the SARS ambulance or driving themselves to TTSH. However, 60 respondents would nonetheless take public transport to TTSH [by taxi 8.5%, mass rapid transit (MRT) or bus 1.9%]. In particular, the retired with lower educational levels were likely to be oblivious both to the symptoms of SARS and the possible consequences of travelling by inappropriate transport. CONCLUSION: Despite more than 2 months of intensive SARS public education in Singapore, there remain important gaps in knowledge and appropriate behaviour that have to be bridged.

SAG1 is a host-targeted antigen for protection against toxoplasma gondii infection.

We previously reported that SAG1 transgenic (tg) mice have an elevated susceptibility resulting from their inability to elicit strong Th1-based protection against Toxoplasma gondii infection. Here, we demonstrate that SAG1 tg mice were protected against T. gondii infection, characterized by a decline in IFN-gamma levels, following administration of a lethal dose of T. gondii. Moreover, immunization with T. gondii homogenate conferred protection and induced production of IgG, with IgG1 and IgG2a subclasses driven by Th2 and Th1 responses, respectively, in both SAG1 tg and wild-type (wt) mice. IgG titers were significantly higher from day 10 after immunization in wt mice compared to those in SAG1 tg mice. There were no significant differences observed in levels of IgG1 in both groups. However, significantly lower IgG2a titers were measured in the sera from SAG1 tg mice on days 10, 15 and 20. IFN-gamma levels in sera were significantly lower in SAG1 tg mice compared to those in wt mice on day 20 after immunization. When challenged with a lethal dose of the Beverley strain of T. gondii, 80 and 100% survival rates were observed in SAG1 tg and wt mice, respectively, indicating that SAG1 tg mice were protected to a lesser extent from challenge due to the decrease in protective immunity. These results suggest that SAG1 plays a critical role in eliciting protection, hence a target antigen for the development of protective Th1-based responses against T. gondii infection in mice.

Visualization of the uptake of high-density lipoprotein by rat aortic endothelial cells and smooth muscle cells in vitro.

High-density lipoproteins (HDL) were conjugated to Fluorescein 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) or colloidal gold for the investigation of ultrastructural aspects of binding and uptake of HDL by cholesterol-loaded cultured endothelial and smooth muscle cells from rat aorta. When cells were incubated for 2 h at 4 degrees C, HDL-DiI and HDL-gold conjugates were seen only on the cell surface. When cells were returned to incubation at 37 degrees C for 5 min, HDL-DiI appeared in the cytoplasm and colocalized with the fluorescent cholesteryl ester tag BODIPY-FL-C12. HDL-gold conjugates appeared in the plasmalemmal invaginations and plasmalemmal vesicles. After incubation for 15 min, most of the HDL-gold conjugates reappeared on the cell surface. After incubation for 30 min, only a few conjugates were observed and they localized in lysosomal-like bodies. Quantitative data indicated that when the cholesterol-loaded cells were incubated at 4 degrees C for 2 h, the numbers of HDL-gold associated in clusters on the endothelial cell surface was 1.18 clusters/microm. When cells were returned to incubation at 37 degrees C for 5 min, this value decreased to 0.7, increased again to 1.13 at 15 min, and decreased to 0.29 at 30 min. The numbers of clusters in the plasmalemmal invaginations were 0.06 clusters/microm at 4 degrees C for 2 h, increased to 0.34 at 37 degrees C for 5 min and decreased gradually to 0.19 and 0.04 at 15 and 30 min, respectively. The incidence of clusters in the plasmalemmal vesicles per non-nuclear cytoplasm was 0.01 clusters/microm2 at 4 degrees C for 2 h, increased significantly to 1.08 at 37 degrees C for 5 min, and decreased to 0.43 and 0.14 at 15 and 30 min, respectively. This work supports that the plasmalemmal invaginations and plasmalemmal vesicles are linked to the HDL uptake in cholesterol-loaded aortic endothelial cells and smooth muscle cells.

Unresponsiveness to surface antigen 1 modifies cytokine profiles in acute Toxoplasma gondii infection.

Resistance to Toxoplasma gondii involves the development of a highly polarized Th1-type cytokine expression. SAG1 transgenic mice are highly susceptible to T. gondii infection due to their non-reactivity to SAG1 of the protozoan parasite. Here we describe cytokine profiles during the acute phase of T. gondii infection, which are associated with the susceptibility of SAG1 transgenic mice. SAG1 transgenic mice showed a 4.5-fold increase in susceptibility upon inoculation with a sublethal dose of the Beverley strain of T. gondii compared to their wild-type counterparts (mortality: 81 vs. 18%, respectively). When analysis of the most important cytokines involved in the mediation of resistance to infection was carried out, SAG1 transgenic mice exhibited low production levels of IL-12, IFN-gamma and TNF-alpha in sera during the acute phase of T. gondii infection. Antibody and T cells specific for SAG1 were not mounted upon SAG1 stimulation in SAG1 transgenic mice. Moreover, in vitro studies indicated that in SAG1 transgenic mice IFN-gamma and IL-12 production was lower than in their wild-type counterparts, although levels of TNF-alpha increased in SAG1 transgenic mice on day 9 after infection. Low IgG2a levels were detected in SAG1 transgenic mouse sera. Unresponsiveness to SAG1 of T. gondii renders SAG1 transgenic mice unable to develop a strong Th1-based protection against T. gondii infection. These results provide evidence that SAG1 is a pivotal antigen involved in the induction of immune responses towards the development of Th1-protective immunity during T. gondii infection.

[Adenocarcinoma of the ethmoid sinuses: results of a retrospective study in Rouen]. / Les adénocarcinomes de l'ethmoïde.

Ethmoid adenocarcinoma is a rare tumour of the ethmoidal sinuses. The authors report on the clinical features, treatment and follow-up results in 19 cases. Risk factors were those regularly encountered. Delay to diagnosis was long due to the nonspecific clinical features and course. Nasal endoscopy was essential for follow-up. Computed tomography and magnetic resonance imaging were also required to assess tumour spread. Our results suggest that radiotherapy following surgery should be preferred. Survival rate is generally low for this type of tumor. We had 77% survival at 5 years.

The WD motif-containing protein receptor for activated protein kinase C (RACK1) is required for recruitment and activation of signal transducer and activator of transcription 1 through the type I interferon receptor.

An obligatory step in the activation of Signal Transducers and Activators of Transcription (STATs) by cytokines is their docking to specific receptors via phosphotyrosines. However, this model does not address whether STATs pre-associate with their corresponding receptor or exist free in the cytoplasm before receptor activation. In this report, we demonstrate that pre-association of STAT1 with the receptor is required for type I interferon (IFN) signaling. Interestingly, the interaction between the human type I IFN receptor and STAT1 is not direct but mediated by the adapter protein receptor for activated protein kinase C (RACK1). Disruption of the IFNalpha receptor-RACK1 interaction abolishes not only IFNalpha-induced tyrosine phosphorylation of STAT1 but also activation of STAT2, indicating that RACK1 plays a central role in early signaling through the Jak-STAT pathway. These findings demonstrate the involvement of RACK1 in STAT1 activation and raise the possibility that other STATs may pre-associate with cytokine receptors through similar adapter-STAT-mediated interactions.

Magnetic resonance cholangiographic assessment of a delayed radiation-induced bile duct stricture.

Radiation-induced bile duct strictures are rare since bile ducts are considered to be resistant in radiation injury. We report a case of bile duct stenosis where evidence is presented that bile duct stricture was the result of radiation injury and which illustrates the major contribution of magnetic res-onance cholangiography in biliary tract disease evaluation.

Sarcoma after radiation therapy: retrospective multiinstitutional study of 80 histologically confirmed cases. Radiation Therapist and Pathologist Groups of the Fédération Nationale des Centres de Lutte Contre le Cancer.

PURPOSE: To evaluate the best strategy for treatment of sarcoma that occurs after radiation therapy. MATERIALS AND METHODS: Records were retrospectively reviewed for 80 patients with a confirmed histologic diagnosis of sarcoma that occurred after radiation therapy performed during 1975-1995. The patients were treated for breast cancer (n = 33, 42%), non-Hodgkin lymphoma (n = 9, 11%), cervical cancer (n = 9, 11%), benign lesions (n = 4, 5%), or other tumors (n = 25, 31%). Sarcoma occurred after a mean latency of 12 years (range, 3-64 years), with most (70%) developing in the soft tissue. Treatment included surgery (28 patients), surgery and chemotherapy (18 patients), chemotherapy only (15 patients), and radiation therapy (14 patients). RESULTS: By the end of the study, 51 patients were dead, including 46 due to sarcoma. Median survival was 23 months. Overall survival rates at 2 and 5 years, respectively, were 69% and 39% for patients treated with surgery, 10% and 0% for those treated with chemotherapy, and 52% and 35% for those treated with surgery and chemotherapy (P =.001). The 2- and 5-year rates for survival without recurrence were 54% and 32%, respectively. CONCLUSION: The results confirm the beneficial effect of surgery. Further study is needed to explore the roles of combined treatments.

Expression of SAG-1 of Toxoplasma gondii in transgenic mice.

We describe the expression of SAG-1 cDNA in B6C3F1 mice by microinjecting a 3.3 kbp DNA fragment, consisting of the cytomegalovirus enhancer-chicken beta-actin hybrid promoter and SAG-1 into the pronucleus of a fertilized egg at the one-cell stage. Offspring derived from this microinjection were analyzed for the integration and functional expression of the SAG-1 transgene. Steady-state expressions of both the mRNA for SAG-1 and SAG-1 protein product were detected in the brain, thymus, spleen and liver. Approximately 50% of F1 and F2 progeny inherited the SAG-1 transgene from SAG-1 transgenic mice in Mendelian fashion. These results indicated that SAG-1 transgenic lines were established. Transgenic mice harboring the SAG-1 gene will contribute a critical tool of defining the molecular mechanisms of SAG-1 in pathogenesis and host immune response.

Increased susceptibility to Toxoplasma gondii infection in SAG-1 transgenic mice.

SAG-1, one of the major surface proteins of Toxoplasma gondii, has been reported to play an important role in immune and pathogenic mechanisms of the parasites but its exact function is still unclear. We investigated the time courses of T. gondii infection in B6C3F1 transgenic mice carrying the SAG-1 gene. SAG-1 transgenic mice were infected intraperitoneally with a high virulent RH strain or a low virulent Beverley strain of T. gondii. When infected with RH strain tachyzoites, no significant differences in time courses of survivals between SAG-1 transgenic and wild-type mice were observed. Both groups succumbed to an acute infection within 8 days after infection. However, a lower survival rate (20%) was observed in SAG-1 transgenic mice than in wild-type (80%), when infected with Beverley strain cysts. This result indicates that SAG-1 transgenic mice are more susceptible to T. gondii infection as compared with their wild-type counterpart. ELISA using recombinant SAG-1 protein indicates that SAG-1 transgenic mice do not produce antibodies to the SAG-1 molecule. These findings may provide a critical tool for analysing the molecular mechanisms of pathogenesis and host immune responses during toxoplasmosis.

Roles of CD4(+) T cells and gamma interferon in protective immunity against Babesia microti infection in mice.

Babesia microti produces a self-limiting infection in mice, and recovered mice are resistant to reinfection. In the present study, the role of T cells in protective immunity against challenge infection was examined. BALB/c mice which recovered from primary infection showed strong protective immunity against challenge infection. In contrast, nude mice which failed to control the primary infection and were cured with an antibabesial drug did not show protection against challenge infection. Treatment of immune mice with anti-CD4 monoclonal antibody (MAb) diminished the protective immunity against challenge infection, but treatment with anti-CD8 MAb had no effect on the protection. Transfer of CD4(+) T-cell-depleted spleen cells resulted in higher parasitemia than transfer of CD8(+) T-cell-depleted spleen cells. A high level of gamma interferon (IFN-gamma), which was produced by CD4(+) T cells, was observed for the culture supernatant of spleen cells from immune mice, and treatment of immune mice with anti-IFN-gamma MAb partially reduced the protection. Moreover, no protection against challenge infection was found in IFN-gamma-deficient mice. On the other hand, treatment of immune mice with MAbs against interleukin-2 (IL-2), IL-4, or tumor necrosis factor alpha did not affect protective immunity. These results suggest essential requirements for CD4(+) T cells and IFN-gamma in protective immunity against challenge infection with B. microti.

Effect of radiation therapy on the potential doubling time of tumours in colorectal cancers.

OBJECTIVE: The aim of the present study was to investigate the effects of standard fractionated radiation therapy on the kinetic parameters of colorectal adenocarcinomas. METHODS: The study of tumour kinetics involved in vivo injection of bromodeoxyuridine. Endoscopic biopsies were obtained from the tumour and analysed with flow cytometry. This procedure provides a rapid calculation of qualitative parameters such as ploidy and quantitative parameters such as the in vivo S-phase fraction labelling index which indicates the percentage of cells that have entered into the cycle, the duration of S-phase (Ts) and the potential tumour doubling time (Tpot). RESULTS: Thirty-eight colorectal carcinomas were studied without prior chemotherapy or radiation therapy (group 1) and ten rectal carcinomas were studied following radiation therapy (group 2). In diploid tumours, the labelling index was significantly lower in the post-radiotherapy group than in the pre-radiotherapy group (2.7 +/- 1.1% versus 6.4 +/- 4.2%, respectively; P= 0.01), and the Tpot was significantly longer after radiotherapy (group 2) (22.0 +/- 7.0 days versus 8.6 +/- 6.0 days, P = 0.002). Standard fractionated radiation therapy also appears to result in a longer Tpot in diploid adenocarcinomas of the colon and rectum. This effect was not observed in aneuploid tumours. CONCLUSIONS: The effectiveness of hyperfractionated schedules of radiation therapy for aneuploid rectal tumours with short Tpot warrants further investigation in a larger patient population.

Pasteurella multocida infection in Singapore.

A case of Pasteurella multocida infection in Singapore is presented. The patient was a 21-year-old Chinese male who developed fever and cellulitis with abscess formation of his right index finger after it was bitten by a stray cat. The organism was isolated in pure culture and identified as Pasteurella multocida subspecies septica. The patient responded to antibiotic therapy and had an uneventful recovery.

Quality control of radiotherapeutic treatment of medulloblastoma in a multicentric study: the contribution of radiotherapy technique to tumour relapse. The French Medulloblastoma Group.

Between 06.86 and 11.89, 88 medulloblastoma or primitive neuroectodermic tumour (PNET) localised in the posterior fossa have been included in the M7 multicentric protocol, 82 received the totality of the radiotherapy treatment and were evaluable for this study. Twenty-two of these 82 patients relapsed: their radiotherapy treatment is analysed in the present study. In 10 cases out of the 22 relapses treatment failure was probably due to a radiotherapeutic imperfection. This study confirms the necessity of a strict radiotherapy control, particularly in multicentric study.