Phenotypic switching of Cryptococcus neoformans can influence the outcome of the human immune response

The human pathogenic fungus Cryptococcus neoformans exhibits the phenomenon of phenotypic switching, a process that generates variant colonies that can differ in morphology, virulence and other characteristics such as capsular glucuronoxylomannan (GXM) size and structure. A previous study established that mucoid colony (MC) variants of C. neoformans were more virulent and elicited a different inflammatory response than smooth colony (SM) variants. In this study, we investigated the interaction of cells from MC and SM variants and their respective GXMs with human T cells and monocytes. Specifically, we measured CD40, CD80 and CD86 expression, lymphoproliferation and interleukin (IL)-4, IL-10, interferon (IFN)-gamma and IL-12Rbeta2 expression in the presence and absence of variant cells and their GXMs. For some immune parameters, both MC and SM strains produced similar results, in particular no differences were observed in IL-4 induction. However, for other critical parameters, including CD86 expression, lymphoproliferation and IL-10 production, the MC variant had effects that can be expected to impair the immune response. Hence, a single C. neoformans strain can elicit several different immune responses depending on the colony type expressed, and this is unlikely to be accounted for by differences in phagocytosis only. The results provide a potential explanation for the higher virulence of the MC variant based on the concept that these cells inhibit the development of a vigorous immune response. Furthermore, the results suggest a mechanism by which phenotypic switching can generate variants able to evade the immune response.     

Synthesis and characterization of new copper(I) complexes containing 4-(diphenylphosphane)benzoic acid and "scorpionate" ligands with "in vitro" superoxide scavenging activity

New copper(I) complexes have been synthesised from the reaction of CuCl with 4-(diphenylphosphane)benzoic acid and lithium tris(1H-pyrazol-1-yl)methanesulfonate, Li(SO(3))C(pz)(3), sodium hydrotris(3-trifluoromethyl-1H-pyrazol-1-yl)borate, NaHB[3-(CF(3))pz](3), potassium dihydrobis(1H-1,2,4-triazol-1-yl)borate, KH(2)B(tz)(2), hydrotris(1H-1,2,4-triazol-1-yl)borate, KHB(tz)(3), sodium hydrotris(1H-pyrazol-1-yl)borate, NaHB(pz)(3), potassium hydrotris(3,5-dimethyl-1H-pyrazol-1-yl)borate KHB(3,5-Me(2)Pz)(3) or potassium hydrotris(4-bromo-1H-pyrazol-1-yl)borate KHB(4-Brpz)(3). The complexes obtained have been characterized by elemental analyses and FT-IR in the solid state, and by NMR (1H and 31P[(1)H]) spectroscopy and conductivity measurements in solution. The solution data are consistent with partial dissociation of the sterically hindered complexes by way of breaking of Cu-P and Cu-N bonds. Electrospray mass spectrometry has been used to investigate the relative properties of the 4-(diphenylphosphane)benzoic acid and of the "scorpionate" ligands towards copper(I) ions. Chemiluminescence technique was used to evaluate the superoxide scavenging activity of these new copper complexes.     

Prolin-rich tyrosine kinase 2 (PYK2) expression and localization in mouse testis

Prolin-rich kinase 2 (PYK2) is a nonreceptor tyrosine kinase related to the focal adhesion kinase (FAK) p125(FAK). PYK2 is rapidly phosphorylated on tyrosine residues in response to various stimuli, such as tumor necrosis factor-alpha (TNF-alpha), changes in osmolarity, elevation in intracellular calcium concentration, angiotensin, and UV irradiation. PYK2 has ligand sequences for Src homology 2 and 3 (SH-2 and SH-3), and has binding sites for paxillin and p130(cas). Activation of PYK2 leads to modulation of ion channel function, phosphorylation of tyrosine residues, and activation of the MAP kinase signaling pathways. Immunocytochemistry shows that PYK2 is present in mouse germinal and Sertoli cells (ser). Northern blot and immunoprecipitation analysis demonstrate that, among germinal cells, PYK2 is more abundant in spermatocytes (spc) and spermatids (spt); in addition, immunofluorescence analysis clearly shows that the diffuse cytoplasmic localization of PYK2 changes in a specific cellular compartment in spt and spermatozoa.     

Improved pulmonary artery buffering function during phenylephrine-induced pulmonary hypertension

The goal of this study was to determine the in vivo pulmonary arterial buffering function (BF) during acute and moderate pulmonary hypertension achieved by phenylephrine-induced smooth muscle activation.Pulmonary pressure (Konigsberg P7) and diameter (sonomicrometry) were measured in nine anesthetized sheep. Transit pulmonary arterial hypertension was induced by mechanical occlusion of the pulmonary artery (HP) and by phenylephrine infusion (5 g/kg/min) (PHE). A viscoelastic Kelvin-Voigt model was used. By increasing the values of the viscous modulus, the pressure-diameter hysteresis area was reduced to a minimum in order to obtain the purely elastic pressure-diameter relationship. The elastic index (E) was calculated as the first derivative of the exponential model of the purely elastic pressure-diameter relationship at the mean pressure point.Systolic, diastolic, mean and pulse pressures were similar during HP and PHE, but significantly higher with regard to control steady state. In HP, E and arterial diameter (both its minimum and maximum values) increased significantly. In contrast, when pulmonary hypertension was induced by VSM activation, E was maintained concomitantly with pulmonary artery vasoconstriction.Pulmonary hypertension produced by occlusion of the pulmonary artery increases elasticity. Smooth muscle activation may offset the deleterious effect of pulmonary hypertension on arterial wall elasticity by reducing E and impeding arterial dilatation and collagen recruitment, maintaining BF during pulmonary hypertension.     

Targeting a recombinant adenovirus vector to HCC cells using a bifunctional Fab-antibody conjugate

We developed a specific adenoviral gene delivery system with monoclonal antibody (mAb) AF-20 that binds to a 180 kDa antigen highly expressed on human hepatocellular carcinoma (HCC) cells. A bifunctional Fab-antibody conjugate (2Hx-2-AF-20) was generated through AF-20 mAb crosslinkage to an anti-hexon antibody Fab fragment. Uptake of adenoviral particles and gene expression was examined in FOCUS HCC and NIH 3T3 cells by immunofluorescence; beta-galactosidase expression levels were determined following competitive inhibition of adenoviral CAR receptor by excess fibre knob protein. The chimeric complex was rapidly internalized at 37 degrees C, and enhanced levels of reporter gene expression was observed in AF-20 antigen positive HCC cells, but not in AF-20 antigen negative NIH 3T3 control cells. Targeting of recombinant adenoviral vectors to a tumor associated antigen by a bifunctional Fab-antibody conjugate is a promising approach to enhance specificity and efficiency of gene delivery to HCC.     

Expression Study on D123 Gene Product: Evidence for High Positivity in Testis

A novel 44-kDa gene product (D123) has been proposed as necessary for S-phase entry of the cell cycle: a point mutation resulted in a temperature-sensitive arrest in G1-phase. From human fibrosarcoma cDNA library, we have isolated an identical gene and studied its sequence and mRNA and protein expression. Compared withD123,three nucleotide differences within the human coding sequence, plus others, result in a change of two amino acids. A partial sequence similarity has been found with a yeast gene of unknown function. The protein has several potential phosphorylation sites, is highly hydrophilic, and may be highly structured in α-helix. The mRNA is abundantly expressed by a variety of normal and transformed cells and by all tissues examined, being most highly expressed in testis. Specific antibodies, raised against a rhD123 polypeptide, recognize a major 42- to 44-kDa molecule in crude extract of various human cell lines. Immunohistochemistry reveals that D123 protein is not homogeneously expressed: it is detected, often in granular vescicles, in the cytoplasm of some epithelial, stromal, and sperm cells and in varicosities lining nervous fibers, while it appears to be absent in nuclei, endothelial, and smooth muscle cells. The precise link between cytoplasmic occurrence of D123 and cell cycle progression still remains to be clarified.     

Autologous and Heterologous Neutralization Analyses of Primary Feline Immunodeficiency Virus Isolates

Feline immunodeficiency virus (FIV) provides a model system with which the significance of neutralizing antibody (NA) in immunosuppressive lentivirus infections may be studied. To date, no detailed analysis of the neutralization properties of primary FIV isolates has been reported. In this study, we have conducted the first comprehensive study of the sensitivity to autologous and heterologous neutralization in a lymphoid cell-based assay of 15 primary FIV isolates and, for comparison, of one tissue culture-adapted strain. Primary isolates in general proved highly NA resistant, although there was considerable individual variation. Variation was also observed in the capacity of immune sera to neutralize heterologous FIV isolates. The ability of sera to neutralize isolates or for isolates to be neutralized by sera did not correlate with epidemiological and genetic relatedness or with the quasispecies complexity of the isolates. From the study of specific-pathogen-free cats experimentally infected with viral isolates associated with NA of different breadths, it appears that the development of FIV vaccines cannot rely on the existence of viral strains inherently capable of inducing especially broad NA responses.Feline immunodeficiency virus (FIV) is a lentivirus that is regarded as the feline counterpart of human immunodeficiency virus (HIV) because it produces persistent infections of domestic cats which, after an incubation period of several years, progress to clinical manifestations of immunodeficiency and neurological damage that closely resemble those observed in HIV-infected humans. FIV is therefore a valuable model for investigating many aspects of AIDS pathobiology and control, including vaccination (4, 11, 39, 56).Based on DNA phylogenesis, FIV isolates worldwide have been classified into at least five distinct genetic subtypes, designated A to E, with uneven geographical distributions (2). While there is little hope of developing a monovalent vaccine capable of protecting across different FIV subtypes, a more reasonable goal is the development of one or several protective immunogens for each subtype and subsequent selection of the immunogens on the basis of the subtypes prevalent in the area where the vaccine is to be used (56). However, because genetic diversity is also high within a subtype, especially in the env region (2, 42), successful vaccines will have to induce immune responses effective against a wide range of antigenically diverse strains. Mapping the immunological relatedness of FIV strains belonging to the same genetic subtype therefore represents a prerequisite for identifying shared critical protective epitopes and an essential step for ongoing vaccine development efforts. Similar problems exist for HIV vaccine development (33).Although the humoral and cell-mediated immune responses that will eventually prove important for vaccine-induced protection against lentiviruses are unresolved (3, 7, 17), the ability to evoke a broadly reactive neutralizing-antibody (NA) response would seem to be an advantageous feature of candidate immunogens because it would at least contrast the dissemination of the initial viral inoculum from the site of entry (8, 9). In previous studies, we found that cats immunized with a fixed-cell vaccine were protected against FIV challenge in the apparent absence of NA (27, 28), but it is possible that a detectable NA response could be elicited with improved vaccines, adjuvants, and immunization regimens.FIV vaccines must be designed to protect against strains of FIV as they circulate in nature. For this reason, it is important to learn more about the immunobiological properties of fresh clinical isolates, including their ability to evoke and interact with NA and their neutralizing determinant(s). Here we report on the sensitivity of 15 FIV isolates subjected to minimal passage in culture to neutralization by autologous and heterologous immune sera. Primary FIV isolates proved only slightly prone to inhibition by immune sera. However, certain isolates were more neutralizable by heterologous sera than others and certain infected cat sera neutralized fairly large numbers of primary isolates. A relationship was also sought between neutralization properties of the isolates and immune sera and a number of factors that theoretically might influence the induction or the activity of cross-reactive NA, including epidemiological and genetic relatedness and quasispecies complexity of the isolates. Finally, to ascertain whether the cross-neutralizing potency of anti-FIV antibody was dependent on properties of the viruses that had induced their formation, we studied the NA response of specific-pathogen-free (SPF) cats inoculated with selected FIV isolates.     

Age-Related Differences in the Accuracy of Web Query-Based Predictions of Influenza-Like Illness

BackgroundWeb queries are now widely used for modeling, nowcasting and forecasting influenza-like illness (ILI). However, given that ILI attack rates vary significantly across ages, in terms of both magnitude and timing, little is known about whether the association between ILI morbidity and ILI-related queries is comparable across different age-groups. The present study aimed to investigate features of the association between ILI morbidity and ILI-related query volume from the perspective of age.MethodsSince Google Flu Trends is unavailable in Italy, Google Trends was used to identify entry terms that correlated highly with official ILI surveillance data. All-age and age-class-specific modeling was performed by means of linear models with generalized least-square estimation. Hold-out validation was used to quantify prediction accuracy. For purposes of comparison, predictions generated by exponential smoothing were computed.ResultsFive search terms showed high correlation coefficients of > .6. In comparison with exponential smoothing, the all-age query-based model correctly predicted the peak time and yielded a higher correlation coefficient with observed ILI morbidity (.978 vs. .929). However, query-based prediction of ILI morbidity was associated with a greater error. Age-class-specific query-based models varied significantly in terms of prediction accuracy. In the 0–4 and 25–44-year age-groups, these did well and outperformed exponential smoothing predictions; in the 15–24 and ≥ 65-year age-classes, however, the query-based models were inaccurate and highly overestimated peak height. In all but one age-class, peak timing predicted by the query-based models coincided with observed timing.ConclusionsThe accuracy of web query-based models in predicting ILI morbidity rates could differ among ages. Greater age-specific detail may be useful in flu query-based studies in order to account for age-specific features of the epidemiology of ILI.     

Cerebral Circulation Time is Prolonged and Not Correlated with EDSS in Multiple Sclerosis Patients: A Study Using Digital Subtracted Angiography

Literature has suggested that changes in brain flow circulation occur in patients with multiple sclerosis. In this study, digital subtraction angiography (DSA) was used to measure the absolute CCT value in MS patients and to correlate its value to age at disease onset and duration, and to expand disability status scale (EDSS). DSA assessment was performed on eighty MS patients and on a control group of forty-four age-matched patients. CCT in MS and control groups was calculated by analyzing the angiographic images. Lesion and brain volumes were calculated in a representative group of MS patients. Statistical correlations among CCT and disease duration, age at disease onset, lesion load, brain volumes and EDSS were considered. A significant difference between CCT in MS patients (mean = 4.9s; sd = 1.27s) and control group (mean = 2.8s; sd = 0.51s) was demonstrated. No significant statistical correlation was found between CCT and the other parameters in all MS patients. Significantly increased CCT value in MS patients suggests the presence of microvascular dysfunctions, which do not depend on clinical and MRI findings. Hemodynamic changes may not be exclusively the result of a late chronic inflammatory process.     

Identification and characterization of a novel salt‐tolerant esterase from a Tibetan glacier metagenomic library

A salt‐tolerant esterase, designated H9Est, was identified from a metagenomic library of the Karuola glacier. H9Est gene comprised 1071 bp and encoded a polypeptide of 357 amino acids with a molecular mass of 40 kDa. Sequence analysis revealed that H9Est belonged to the family IV of bacterial lypolitic enzyme. H9Est was overexpressed in Escherichia coli and the purified enzyme showed hydrolytic activity towards p‐nitrophenyl esters with carbon chain from 2 to 8. The optimal esterase activity was at 40°C and pH 8.0 and the enzyme retained its activity towards some miscible organic solvents such as polyethylene glycol. A three‐dimensional model of H9Est revealed that S200, D294, and H324 formed the H9Est catalytic triad. Circular Dichroism spectra and molecular dynamic simulation indicated that the esterase had a wide denaturation temperature range and flexible loops that would be beneficial for H9Est performance at low temperatures while retaining heat‐resistant features. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:890–899, 2015