Rapid Detection and Identification of Mucormycetes in Bronchoalveolar Lavage Samples from Immunocompromised Patients with Pulmonary Infiltrates by Use of High-Resolution Melt Analysis

Rapid differential diagnostics of pulmonary infiltrates suspected of invasive fungal disease in an immunocompromised host and early initiation of effective antifungal therapy are crucial for patient outcomes. There are no serological tests available to detect mucormycetes; therefore, PCR-based methods are highly suitable. We validated our previously published PCR followed by high-resolution melt analysis (PCR/HRMA) to detect Rhizopus spp., Rhizomucor pusillus, Lichtheimia corymbifera, and Mucor spp. in bronchoalveolar lavage (BAL) samples from immunocompromised patients who were at risk of invasive fungal disease. All PCR/HRMA-positive samples were retested using novel real-time quantitative PCR (RQ PCR) assays specific to the species identified. In total, between January 2009 and December 2012 we analyzed 99 BAL samples from 86 patients with pulmonary abnormalities using PCR/HRMA. Ninety (91%) BAL samples were negative, and 9 (9%) samples were positive. The sensitivity and specificity of PCR/HRMA were 100% and 93%, respectively. By combining the positive results of PCR/HRMA with positive RQ PCR results, the specificity was raised to 98%. PCR/HRMA, due to its high negative predictive value (99%), represents a fast and reliable tool for routine BAL sample screening for the differential diagnosis of pulmonary infiltrates in immunocompromised patients for the four most clinically important mucormycetes.     

Safety and Immunogenicity of a Vero Cell Culture-Derived Whole-Virus H5N1 Influenza Vaccine in Chronically Ill and Immunocompromised Patients

The development of vaccines against H5N1 influenza A viruses is a cornerstone of pandemic preparedness. Clinical trials of H5N1 vaccines have been undertaken in healthy subjects, but studies in risk groups have been lacking. In this study, the immunogenicity and safety of a nonadjuvanted cell culture-derived whole-virus H5N1 vaccine were assessed in chronically ill and immunocompromised adults. Subjects received two priming immunizations with a clade 1 A/Vietnam H5N1 influenza vaccine, and a subset also received a booster immunization with a clade 2.1 A/Indonesia H5N1 vaccine 12 to 24 months later. The antibody responses in the two populations were assessed by virus neutralization and single radial hemolysis assays. The T-cell responses in a subset of immunocompromised patients were assessed by enzyme-linked immunosorbent spot assay (ELISPOT). The priming and the booster vaccinations were safe and well tolerated in the two risk populations, and adverse reactions were predominantly mild and transient. The priming immunizations induced neutralizing antibody titers of ≥1:20 against the A/Vietnam strain in 64.2% of the chronically ill and 41.5% of the immunocompromised subjects. After the booster vaccination, neutralizing antibody titers of ≥1:20 against the A/Vietnam and A/Indonesia strains were achieved in 77.5% and 70.8%, respectively, of chronically ill subjects and in 71.6% and 67.5%, respectively, of immunocompromised subjects. The T-cell responses against the two H5N1 strains increased significantly over the baseline values. Substantial heterosubtypic T-cell responses were elicited against the 2009 pandemic H1N1 virus and seasonal A(H1N1), A(H3N2), and B subtypes. There was a significant correlation between T-cell responses and neutralizing antibody titers. These data indicate that nonadjuvanted whole-virus cell culture-derived H5N1 influenza vaccines are suitable for immunizing chronically ill and immunocompromised populations. (This study is registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT00711295","term_id":"NCT00711295"}}NCT00711295.)     

Influence of Cryo-Processing and Post-SPD Annealing on Creep Behavior of CP Titanium

The commercial purity of VT1-0 titanium was processed by the rolling process and executed at elevated, room, and cryo-temperatures. These processings led to the formation of an ultrafine-grained microstructure, with the mean grain size at a nanometer level. Some of these materials were statically annealed at a temperature of 823 K for 1 h, which led to significant subgrains and grain coarsening. The constant load creep tests in tension were carried out in argon on all states of materials, at temperatures of 648–723 K and different ranges of applied stresses. From the value of the steady-state creep rate, the control creep mechanisms were determined. The microstructure analyses were carried out via SEM and TEM. It was found that titanium prepared at elevated and room temperatures have a higher creep strength than titanium prepared at cryo-temperatures. Furthermore, the post-SPD —annealing led to a significant decrease in the creep properties. The influence of the preparation temperature on the difference of the creep behavior were discussed and explained using the microstructure analyses of the tests’ samples.     

Temperature modulation of growth rates and glucosephosphate isomerase isozyme activity in Trypanosoma cruzi

In an attempt to understand the conditions which might influence the geographical distribution of Trypanosoma cruzi isozyme profiles (zymodemes), the thermal response of different glucosephosphate isomerase (GPI) phenotypes was studied. T. cruzi clones with single- and triple-banded GPI phenotype showed a similar response to temperature with respect to both growth rates and GPI kinetic parameters. However, the relative activity of each GPI isozyme was dependent on parasite incubation temperature. In view of the similar kinetic properties of the isozymes, enzyme regulation is not a consequence of an adaptative response to thermal conditions and the suggestion of a phenotype distribution determined selectively by temperature is not supported by the present study.     

Vessel co‐option is common in human lung metastases and mediates resistance to anti‐angiogenic therapy in preclinical lung metastasis models

Anti‐angiogenic therapies have shown limited efficacy in the clinical management of metastatic disease, including lung metastases. Moreover, the mechanisms via which tumours resist anti‐angiogenic therapies are poorly understood. Importantly, rather than utilizing angiogenesis, some metastases may instead incorporate pre‐existing vessels from surrounding tissue (vessel co‐option). As anti‐angiogenic therapies were designed to target only new blood vessel growth, vessel co‐option has been proposed as a mechanism that could drive resistance to anti‐angiogenic therapy. However, vessel co‐option has not been extensively studied in lung metastases, and its potential to mediate resistance to anti‐angiogenic therapy in lung metastases is not established. Here, we examined the mechanism of tumour vascularization in 164 human lung metastasis specimens (composed of breast, colorectal and renal cancer lung metastasis cases). We identified four distinct histopathological growth patterns (HGPs) of lung metastasis (alveolar, interstitial, perivascular cuffing, and pushing), each of which vascularized via a different mechanism. In the alveolar HGP, cancer cells invaded the alveolar air spaces, facilitating the co‐option of alveolar capillaries. In the interstitial HGP, cancer cells invaded the alveolar walls to co‐opt alveolar capillaries. In the perivascular cuffing HGP, cancer cells grew by co‐opting larger vessels of the lung. Only in the pushing HGP did the tumours vascularize by angiogenesis. Importantly, vessel co‐option occurred with high frequency, being present in >80% of the cases examined. Moreover, we provide evidence that vessel co‐option mediates resistance to the anti‐angiogenic drug sunitinib in preclinical lung metastasis models. Assuming that our interpretation of the data is correct, we conclude that vessel co‐option in lung metastases occurs through at least three distinct mechanisms, that vessel co‐option occurs frequently in lung metastases, and that vessel co‐option could mediate resistance to anti‐angiogenic therapy in lung metastases. Novel therapies designed to target both angiogenesis and vessel co‐option are therefore warranted. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Trypanosoma cruzi: elemental composition heterogeneity of cloned stocks

Concentrates of the epimastigote stage of Trypanosoma cruzi stocks derived from single cell clones and cultured under identical conditions display a spectrum of 'colors' varying from dark brown to milk white. The color of the concentrate is reproducible for a parasite stock. An essential component of the culture medium for epimastigote growth is hemin, an iron-containing compound. Consequently, it seemed possible that the color spectrum of the epimastigote stocks reflected differences in the uptake, concentration or utilization of iron. This report describes the quantitative studies utilizing electron probe X-ray elemental mapping, energy dispersive X-ray microanalysis, and energy dispersive X-ray fluorescence spectrometry of the epimastigote stage of two T. cruzi stocks (CA-I/72 and HO-3/15) which display extreme color differences. Striking and statistically significant quantitative differences were found in the levels of Fe, Zn, and K between the two stocks. On the basis of atomic ratios, the CA-I/72 stock contains approximately two-fold more Fe per cell than the HO-3/15 stock. However, in the case of Zn the ratio is reversed; the HO-3/15 stock contains approximately two-fold more Zn per cell than the CA-I/72 stock. The marked inter-stock differences which exist in the levels of several elements could modulate the pathogenicity, survival, or adaptability of T. cruzi and, consequently, be important factors in our understanding of the complex problem of Chagas' disease.     

Nucleoside uptake in Trypanosoma cruzi: analysis of a mutant resistant to tubercidin

Nucleoside salvage pathways are vital to the parasitic protozoan Trypanosoma cruzi, and have become important targets in the development of new chemotherapeutic agents against this organism. We produced a mutant T. cruzi clone with a defect in the uptake of the adenosine analogue tubercidin which allowed us to hypothesize that there are at least two distinct nucleoside transport pathways in this parasite. The mutant shows a marked defect in the uptake of tubercidin and thymidine, whereas the uptake of adenosine and inosine are normal. Inhibition and metabolic studies suggest that the defect is related to transport and that there are two transport processes relatively specific for purines and pyrimidines, respectively, although tubercidin is transported via the latter. This is similar to the reported dual nucleoside transport pathways in Leishmania donovani and may be a common system in the Trypanosomatidae. These transport processes are markedly different from those which have been described for mammalian cells and may play an important role in the design of strategies for the chemotherapy of human infection with these pathogenic parasites.     

In vivo flexion/extension of the normal cervical spine

Twenty-two women (age range 25-49 years, average 30.9 years) and twenty-two men (age range 23-42 years, average 31.6 years), all healthy and asymptomatic, underwent passive flexion/extension examinations of the cervical spine. Functional x-rays were taken and analyzed using a computer-assisted method that quantified intervertebral rotations, translations, and locations of the centers of rotation for each level C1-C2-C6-C7. The aim of the study was to establish values for these parameters for a normal population as related to age and gender. In the process, a statistically significant difference was found in the average value of rotation between male and female groups at the C5-C6 level. A new parameter, the ratio between translation and rotation, was also established and may prove useful for clinical diagnoses. This parameter has a smaller error associated with it than do pure translations and may aid the clinician by helping to account for the large variation in rotatory ranges of motion within the population. This translation/rotation ratio indicated highly significant differences in the lower segments of the cervical spine between gender groups.