Tuberculosis in HIV-infected South African children with complicated severe acute malnutrition.

SETTING: Academic tertiary referral hospital in Durban, South Africa. OBJECTIVE: To describe the incidence and diagnostic challenges of tuberculosis (TB) in human immunodeficiency virus (HIV) infected children with severe acute malnutrition (SAM). DESIGN: Post-hoc analysis of a randomised controlled trial that enrolled antiretroviral therapy naïve, HIV-infected children with SAM. Trial records and hospital laboratory results were explored for clinical diagnoses and bacteriologically confirmed cases of TB. Negative binomial regression was used to explore associations with confirmed cases of TB, excluding cases where the clinical diagnosis was not supported by microbiological confirmation. RESULTS: Of 82 children enrolled in the study, 21 (25.6%) were diagnosed with TB, with bacteriological confirmation in 8 cases. Sputum sampling (as opposed to gastric washings) was associated with an increased risk of subsequent diagnosis of TB (adjusted relative risk [aRR] 1.134, 95%CI 1.02-1.26). Culture-proven bacterial infection during admission was associated with a reduced risk of TB (aRR 0.856, 95%CI 0.748-0.979), which may reflect false-negative microbiological tests secondary to empiric broad-spectrum antibiotics. CONCLUSION: TB is common in HIV-infected children with SAM. While microbiological confirmation of the diagnosis is feasible, empiric treatment remains common, possibly influenced by suboptimal testing and false-negative TB diagnostics. Rigorous microbiological TB investigation should be integrated into the programmatic management of HIV and SAM.

Prevention of Tumor Formation by Latent Gammaherpesvirus Infection.

Recent reports suggested that chronic herpesvirus infection, as a constituent of the so-called virome, may not only exert harmful effects but may also be beneficial to the host, for example mediating increased resistance to secondary infections or to tumors. To further challenge this concept, specifically regarding increased resistance to tumors, we infected chimeric HLA-DR4-H2-E (DR4) mice, a mouse strain which spontaneously develops hematological tumors, with the rodent herpesvirus murine gammaherpesvirus 68 (MHV-68). Using this model, we observed that infection with wildtype MHV-68 completely prevented tumor formation. This happened, however, at the cost of hyposplenism. In contrast to wildtype infection, infection with a latency-deficient mutant of MHV-68 neither prevented tumor formation nor induced hyposplenism. The underlying mechanisms are not known but might be related to an infection-mediated priming of the immune response, resulting in the suppression of a tumor promoting endogenous retrovirus. Thus, under certain circumstances, chronic herpesvirus infection may prevent the development of tumors.

Pancreatic metastasectomy: experience of the Irish National Surgical Centre for Pancreatic Cancer.

BACKGROUND: Metastatic tumours of the pancreas are rare and the optimal management of these tumours remains unclear, given the paucity of data existing in the literature. We report our experience of pancreatic metastasectomy. METHODS: Data were reviewed on all patients who underwent pancreatic resection for pathologically confirmed metastatic lesions over a consecutive 7-year period. RESULTS: Seven patients (two men and five women) underwent a pancreatectomy for a metastatic pancreatic tumour. The primary tumours were renal cell carcinoma (n = 3), colorectal carcinoma (n = 2) and leiomyosarcoma (n = 2). There was no operative mortality. Postoperative morbidities occurred in two patients. The median follow-up was 49 months (range 17-76). Overall 1- and 2-year survivals were 100 and 86 %, respectively, with a 2-year disease-free survival of 72 %. CONCLUSIONS: Our series further supports that pancreatic metastasectomy can be performed safely and achieves acceptable survival outcomes.

Pancreatectomy for metastatic disease: a systematic review.

AIM: Tumours rarely metastasise to the pancreas. While surgical resection of such metastases is believed to confer a survival benefit, there is limited data to support such management. We present a systematic review of case series of pancreatic metastasectomy and analysis of survival outcomes. METHODS: A literature search was performed using the PubMed and Cochrane databases and the reference lists of relevant articles, searching for sizeable case series of pancreatic metastasectomy with curative intent. Data extracted included basic demographics, histological primary tumour, presentation, operative management, complications and survival, while the MINORS index was used to assess study quality. RESULTS: 18 studies were found which met our inclusion criteria, involving 399 patients. Renal cell carcinoma (RCC) was the commonest malignancy metastasising to the pancreas, responsible for 62.6% of cases, followed by sarcoma (7.2%) and colorectal carcinoma (6.2%). While survival data was not uniformly reported, the median survival post-metastasectomy was 50.2 months, with a one-year survival of 86.81% and five-year survival of 50.02%. Median survival for RCC was 71.7 months with 70.4% five-year survival. Median survival was similar in patients with synchronous and metachronous pancreatic metastases, but patients with additional extrapancreatic metastases had a significantly shorter survival than patients with isolated pancreatic metastases (26 versus 45 months). Study quality was poor, with a median MINORS score of 10/16. CONCLUSIONS: Within the limitations of a review of non-randomised case series, it would appear that pancreatic metastasectomy confers a survival benefit in selected patients. Better evidence is required, but may prove difficult to acquire.

ORF23 of murine gammaherpesvirus 68 is non-essential for in vitro and in vivo infection.

Although ORF23 is conserved among gammaherpesviruses, its role during infection is unknown. Here, we studied the expression of ORF23 of murine gammaherpesvirus 68 (MHV-68) and its role during infection. ORF23 mRNA was detected in infected cells as a late transcript. The ORF23 protein product could be expressed and detected as an N-terminally FLAG-tagged protein by Western blot and indirect immunofluorescence. To investigate the role of ORF23 in the infection cycle of a gammaherpesvirus, we constructed an ORF23 deletion mutant of MHV-68. The analysis of the ORF23 deletion mutant suggested that ORF23 of MHV-68 is neither essential for replication in cell culture nor for lytic or latent infection in vivo. A phenotype of the ORF23 deletion mutant, reflected by a moderate reduction in lytic replication and latency amplification, was only detectable in the face of direct competition to the parental virus.

The bacterial redox signaller pyocyanin as an antiplasmodial agent: comparisons with its thioanalog methylene blue.

The quorum sensor and signalling molecule pyocyanin (PYO) contributes significantly to the pathophysiology of Pseudomonas aeruginosa infections. Comparison to phenothiazine drugs suggests that the antimalarial compound methylene blue (MB) can be regarded as a sulfur analog of PYO. This working hypothesis would explain why the synthetic drug MB behaves as a compound shaped in biological evolution. Here we report on redox-associated biological and biochemical properties of PYO in direct comparison to its synthetic analog MB. We quantitatively describe the reactivity of both compounds toward cellular reductants, the reactivity of their reduced leuco-forms towards O2, and their interactions with FAD-containing disulfide reductases. Furthermore, the interaction of PYO with human glutathione reductase was studied in structural detail by x-ray crystallography, showing that a single PYO molecule binds to the intersubunit cavity of the enzyme. Like MB, also PYO was also found to be active against blood schizonts of the malaria parasite P. falciparum in vitro. Furthermore, both compounds were active against the disease transmitting gametocyte forms of the parasites, which was systematically studied in vitro. As shown for mice, PYO is too toxic to be used as a drug. It may, however, have antimalarial activity in numerous human patients with concomitant Pseudomonas infections. MB, in contrast to PYO, is well tolerated and represents a promising agent for MB-based combination therapies against malaria. Current and future clinical studies can be guided by the comparisons between MB and PYO reported here. Additionally, it is of interest to study if and to what extent the protection from malaria in patients with cystic fibrosis or with severe wound infections is based on PYO produced by Pseudomonas species.

Canine embryonic stem cells: state of the art.

Embryonic stem cells (ESCs) are permanent cell lines that can be maintained in a pluripotent, undifferentiated state. Appropriate environmental stimuli can cause them to differentiate into cell types of all three germ layers both in vitro and in vivo. Embryonic stem cells bear many opportunities for clinical applications in tissue engineering and regenerative medicine. Whereas most of our knowledge on the biology and technology of ESCs is derived from studies with mouse cells, large animal models mimicking important aspects of human anatomy, physiology, and pathology more closely than mouse models are urgently needed for studies evaluating the safety and efficacy of cell therapies. The dog is an excellent model for studying human diseases, and the availability of canine ESCs would open new possibilities for this model in biomedical research. In addition, canine ESCs could be useful for the development of cell-based approaches for the treatment of dogs. Here, we discuss the features of recently reported canine embryo-derived cells and their potential applications in basic and translational biomedical research.

The M10 locus of murine gammaherpesvirus 68 contributes to both the lytic and the latent phases of infection.

Murine gammaherpesvirus 68 (MHV-68) is closely related to Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus (KSHV) and provides a small-animal model to study the pathogenesis of gammaherpesvirus (gammaHV) infections. According to the colinear organization of the gammaHV genomes, the M10 locus is situated at a position equivalent to the K12 locus of KSHV, which codes for proteins of the kaposin family. The M10 locus of MHV-68 has been predicted to code for three overlapping open reading frames (M10a, M10b, and M10c [M10a-c]) with unknown function. In addition, the M10 locus contains a lytic origin of replication (oriLyt). To elucidate the function of the M10 locus during lytic and latent infections, we investigated, both in vitro and in vivo, the following four recombinant viruses which were generated using MHV-68 cloned as a bacterial artificial chromosome: (i) a mutant virus with a deletion which affects both the coding region for M10a-c and the oriLyt; (ii) a revertant virus in which both the M10a-c coding region and the oriLyt were reverted to those of the wild type; (iii) a virus with an ectopic insertion of the oriLyt, which restores the function of the oriLyt but not the M10a-c coding region; and (iv) a mutant virus with a deletion in the oriLyt only. While the mutants were slightly attenuated with regard to lytic replication in cell culture, they showed severe growth defects in vivo. Both lytic replication and latency amplification were strongly reduced. In contrast, both the revertant virus and the virus with the ectopic oriLyt insertion grew very similarly to the parental wild-type virus both in vitro and in vivo. Thus, we provide genetic evidence that mutation of the oriLyt, and not of putative protein coding sequences within the M10a-c region, is responsible for the observed phenotype. We conclude that the oriLyt in the M10 locus plays an important role during infection of mice with MHV-68.

Emergence of SCCmec type IV as the most common type of methicillin-resistant Staphylococcus aureus in a university hospital.

BACKGROUND: The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically changed over the last decade by the emergence of community-associated MRSA (CA-MRSA). Recent studies indicate that these strains have already spread to hospitals. To evaluate if SCCmec type IV and Panton-Valentine leukocidin (PVL) are unambiguous markers of CA-MRSA, we analyzed 77 sporadic MRSA strains isolated, in our low MRSA incidence university hospital, from inpatients between 2000 and 2004. METHODS: MRSA strains were analyzed by staphylococcal cassette chromosome mmecec (SCCmec) typing, PCR for PVL genes and pulsed-field gel electrophoresis (PFGE). MRSA was classified in HA-MRSA or CA-MRSA according to Centers for Disease Control and Prevention (CDC) criteria. Antimicrobial susceptibility testing was performed using microbroth dilution method following CLSI recommendations. RESULTS: Among 77 sporadic single-patient strains, SCCmec types I-IV and four subtypes were identified. Type IV/IVA was most common (42.9%).The distribution of SCCmec types changed over the years. Type IV/IVA strains increased from 33.3% in 2000 to 57.9% in 2004. Type IV strains were resistant to ciprofloxacin in 81.8%, and in 9.1% to tobramycin while type IVA strains were 100% resistant to both antimicrobials. In contrast, non-type IV/IVA strains were resistant to ciprofloxacin in 86.4%, and in 75.0% to tobramycin. Only one strain was PVL positive and harbored SCCmec type III variant. By PFGE analysis, the 33 SCCmec type IV/IVA strains comprised 12 distinct genotypes. 36.4% of 11 CA-MRSA and 43.9% of 66 HA-MRSA harbored SCCmec type IV/IVA. CONCLUSION: Type IV/IVA has become the most common SCCmec type in inpatients of our university hospital. The SCCmec type IV/IVA is present in both CA-MRSA and HA-MRSA limiting its use as a marker for CA-MRSA.

Proteins of the secretory pathway govern virus productivity during lytic gammaherpesvirus infection.

BACKGROUND: Diseases caused by gammaherpesviruses continue to be a challenge for human health and antiviral treatment. Most of the commonly used antiviral drugs are directed against viral gene products. However, the emergence of drug-resistant mutations ma limit the effectiveness of these drugs. Since viruses require a host cell to propagate, the search for host cell targets is an interesting alternative. METHODS: In this study, we infected three different cell types (fibroblasts, endothelial precursor cells and macrophages with a murine gammaherpesvirus and analysed the host cell response for changes either common to all or unique to a particular cell type using oligonucleotide microarrays. RESULTS: The analysis revealed a number of genes whose transcription was significantly up- or down-regulated in either one or two of the cell types tested. After infection, only two genes, Lman1 (also known as ERGIC53) an synaptobrevin-like 1 (sybl1) were significantly up-regulated in all three cell types, suggestive for a general role for the virus life cycl independent of the cell type. Both proteins have been implicated in cellular exocytosis and transport of glycoproteins through the secretory pathway. To test the significance of the observed up-regulation, the functionality of these proteins was modulated, and the effect on virus replication was monitored. Inhibition of either Lman1 or sybl1 resulted in a significant reduction in virus production. CONCLUSIONS: This suggests that proteins of the secretory pathway which appear to be rate limiting for virus production may represent new targets for intervention.

Emergence of a teicoplanin-resistant small colony variant of Staphylococcus epidermidis during vancomycin therapy.

Small colony variants of Staphylococcus aureus can cause persistent and recurrent infections. There are only a few reports of small colony variants of coagulase-negative staphylococci. Herein a case of infection with a teicoplanin-resistant small colony variant of Staphylococcus epidermidis is presented. The small colony variant was isolated from blood cultures of a patient with acute leukaemia and therapy-induced neutropenia who was treated with vancomycin for catheter-associated bloodstream infection. Despite removal of the catheter and adequate antibiotic therapy, the infection did not clear and the patient died 20 days after continuous antibiotic therapy.

Invasive pulmonary aspergillosis: effects of early resection in a neutropenic rat model.

OBJECTIVE: Invasive pulmonary aspergillosis is frequent in neutropenic patients. Usually localized in the beginning, the disease spreads and mortality is high despite antifungal treatment. The role of early adjuvant surgery is not clear. Surgery may help to confirm fungal disease, may control fungal disease locally and may prevent systemic spreading. This study examines effects of early resection on survival and dissemination in a rat model of localized invasive pulmonary aspergillosis. METHODS: Forty persistently neutropenic male albino rats were challenged with standardized conidial aspergillus inoculum injected into peripheral lung tissue of the right upper lobe under direct vision. Animals were divided into four groups. Twenty animals were treated with amphotericin B at 1 mg/kg per day beginning 48 h after inoculation, 20 animals were left untreated. In each group half the animals underwent early resection of localized invasive aspergillosis by lobectomy. Animals were checked daily and mortality was recorded up to 28 days after which surviving animals were sacrificed. RESULTS: Significantly higher survival was observed in resected animals in the non-Am B groups (survival: 10 +/- 19% without early resection and 50 +/- 32% with early resection; P = 0.044). However, early resection did not lead to improved survival in animals treated with amphotericin B (survival 70 +/- 29% without early resection and 50 +/- 32% with early resection; P = 0.316). CONCLUSIONS: In this rat model of localized invasive pulmonary aspergillosis effects of early resection on survival could be demonstrated only in animals not receiving amphotericin B treatment.

K3-mediated evasion of CD8(+) T cells aids amplification of a latent gamma-herpesvirus.

The murine gamma-herpesvirus-68 (MHV-68) K3 protein, like that of the Kaposi's sarcoma associated herpesvirus, down-regulates major histocompatibility complex (MHC) class I expression. However, how this contributes to viral replication in vivo is unclear. After intranasal MHV-68 infection, K3 was transcribed both during acute lytic infection in the lung and during latency establishment in lymphoid tissue. K3-deficient viruses were not cleared more rapidly from the lung, but the number of latently infected spleen cells was reduced and the frequency of virus-specific CD8(+) cytotoxic T lymphocytes (CTLs) was increased. CTL depletion reversed the viral latency deficit. Thus, a major function of K3 appears to be CTL evasion during viral latency expansion.

Prevalence of Leptospira spp. in various species of small mammals caught in an inner-city area in Switzerland.

In order to establish the leptospira carrier rate of small animals in an urban environment, small rodents and shrews were captured in the city of Zurich, Switzerland. Kidney specimens of 190 animals were examined using a leptospira specific PCR assay. Leptospiral DNA was amplified in kidneys of 12.6% of the animals.

Prostate-specific antigen response and systemic T cell activation after in situ gene therapy in prostate cancer patients failing radiotherapy.

In an extended phase I/II study we evaluated 36 prostate cancer patients with local recurrence after radiotherapy who received single or repeated cycles of replication-deficient adenoviral vector (ADV)-mediated herpes simplex virus-thymidine kinase (HSV-tk) plus ganciclovir (GCV) in situ gene therapy with respect to serum PSA levels, alterations in immune cells, and numbers of apoptotic cells in needle biopsies. An initial cycle of HSV-tk plus GCV gene therapy caused a significant prolongation of the mean serum PSA-doubling time (PSADT) from 15.9 to 42.5 months (p = 0.0271) and in 28 of the injected patients (77.8%) there was a mean PSA reduction (PSAR) of 28%. It took a mean of 8.5 months for the PSA to return to the initial PSA (TR-PSA) value. A repeated cycle of gene therapy failed to significantly extend PSADT but did result in significant increases in PSAR (29.4%) and TR-PSA (10.5 months). Moderately increased serum adenovirus antibody titers were generally observed 2 weeks after initial vector injection. Also at this time there was a statistically significant increase in the mean percent of CD8(+) T cells positive for the HLA-DR marker of activation in peripheral blood (p = 0.0088). Studies using prostate biopsies obtained at the same time point demonstrated that vector DNA was detectable by PCR in most samples yet all patients remained positive for prostate cancer in at least one biopsy core. Further analysis demonstrated a correlation between the level of CD8(+) cells and the number of apoptotic cells in biopsies containing cancer cells (p = 0.042). We conclude that repeated cycles of in situ HSV-tk plus GCV gene therapy can be administered to prostate cancer patients who failed radiotherapy and have a localized recurrence. Biological responses to this experimental therapy including increases in PSADT, PSAR, and TR-PSA, and activated CD8(+) T cells present in the peripheral blood, were demonstrated. Interestingly, the density of CD8(+) cells in posttreatment biopsies correlated with the number of apoptotic cells.

Enhanced resolution of membranes in cultured cells by cryoimmobilization and freeze-substitution.

Investigations of cellular processes demand immediate arresting of the process at any given time and excellent retention of cellular material and excellent visibility of membranes. To achieve this goal we used cryofixation to arrest cellular processes instantly and tested diverse freeze-substitution protocols. Madin-Darby kidney cells and Vero cells were grown on carbon-coated sapphire disks. For cryofixation the sapphire disks covered with a cell monolayer were injected with the aid of a guillotine into liquid propane or ethane or a mixture of both cooled by liquid nitrogen. Freezing of the cryogen was prevented by using a partially insulated cylinder and by vigorous stirring that results in a substantial decrement of the freezing point of the cryogen. Cell monolayers can be cryofixed successfully using the guillotine in a safety hood at ambient temperature and humidity or at 37 degrees C and 45% humidity. The freezing unit can also be placed in a laminar flow for working under biohazard conditions. For visualizing cell membranes at high contrast and high resolution, cells were substituted in the presence of various concentrations of glutaraldehyde and osmium tetroxide and the temperature was raised to diverse final temperatures. Substitution for 4 hours at -90 degrees C in anhydrous acetone containing 0.25% anhydrous glutaraldehyde and 0.5% osmium tetroxide followed by a temperature rise of 5 degrees C/hour to 0 degrees C and final incubation for 1 hour at 0 degrees C resulted in high contrast and excellent visibility of subcellular components at the level of the membrane bilayer. The high spatial and temporal resolution makes this methodology an excellent tool for studying cell membrane-bound processes, such as virus-cell interactions.