Drug resistance testing in PBMCs of HIV infected patients.

HIV infected patients are considered a sort of reservoir having different genetically distinct viral variants (quasispecies), that evolve from the starting virus inoculum. Frequently, during replication, HIV can generate nucleotide differences in the new viral population; such genetic changes may be uninfluential in viral "fitness" (replication capacity) or give the virus some advantages under a selective pressure, due to immune response or drug treatment. The use of potent combination therapy for the treatment of HIV infections has certainly improved the "quality of life" for patients, decreasing the viral load in the plasma (HIV RNA). In our study, we investigated whether detection of drug resistance-related mutations was possible in circulating PBMCs, which represent a sort of genetic archive of viral drug resistances, when the levels of viral RNA were reduced to below 400 or 50 copies/ml, since, generally, plasma samples with more than 1,000 copies/ml of HIV RNA are needed to generate some results. The study was successfully performed sequencing proviral HIV DNA in PBMCs from 32 samples belonging to 25 patients, using a new modified protocol, that showed a good reproduciblity and very interesting data, also in patients with low or without circulating HIV RNA levels.     

A beta-1,3 glucan sulfate induces resistance in grapevine against Plasmopara viticola through priming of defense responses, including HR-like cell death

Sulfated laminarin (PS3) has been shown previously to be an elicitor of plant defense reactions in tobacco and Arabidopsis and to induce protection against tobacco mosaic virus. Here, we have demonstrated the efficiency of PS3 in protecting a susceptible grapevine cultivar (Vitis vinifera cv. Marselan) against downy mildew (Plasmopara viticola) under glasshouse conditions. This induced resistance was associated with potentiated H2O2 production at the infection sites, upregulation of defense-related genes, callose and phenol depositions, and hypersensitive response-like cell death. Interestingly, similar responses were observed following P. viticola inoculation in a tolerant grapevine hybrid cultivar (Solaris). A pharmacological approach led us to conclude that both callose synthesis and jasmonic acid pathway contribute to PS3-induced resistance.     

Rapid and accurate quantification of different HCV genotypes by LightCycler Real Time PCR and direct sequencing of HCV amplicons

Follow-up of chronically infected HCV patients is the primary clinical goal in therapy administration. In the absence of an HCV vaccine, the timely monitoring of HCV viral load combined with the information of the viral genotype could contribute to patient disease management. A LightCycler Real Time RT-PCR assay was developed and optimized allowing rapid and accurate quantification of HCV RNA over an extended dynamic range using a single human reference standard. A total of 5,096 plasma samples, collected over almost 5 years, were tested and HCV RNA was quantified in 2,435 samples with levels ranging from 5.7x10(1) to 2.52x10(9) IU/ml. The precision and reproducibility of the test are documented by various inter-assay parameters of the reference standard obtained in 409 RT-PCR runs. This Real Time RT-PCR protocol uses the LightCycler cDNA amplicons for direct sequence analysis and reduces the sequencing time to approximately 3 hours. Nearly all HCV genotypes were identified. Viral sequences showed a similarity level close to 100%, independently from the viral load, while the LightCycler melting temperature analysis did not correlate with HCV genotypes. All this makes the LightCycler Real Time RT-PCR protocol a suitable tool for the diagnosis and monitoring of HCV infections.     

Serological, biological, and molecular characterization of New Zealand white rabbits infected by intraperitoneal inoculation with cell-free human immunodeficiency virus.

The availability of a small laboratory animal model suitable for the evaluation of methods for prevention and treatment of human immunodeficiency virus type 1 infection would be a valuable resource for AIDS research. Here we describe the infection of a strain of domestic rabbits by intraperitoneal inoculation with cell-free human immunodeficiency virus type 1. Evidence of infection includes the presence of an immune response that has persisted for almost 3 years and the detection of an reisolation of infectious virus from peripheral blood mononuclear cells (PBMCs) and other tissues during the first 2 years. Typical viral proteins, DNA and RNA patterns, were observed in rabbit PBMCs and in cells infected by cocultivation with rabbit PBMCs. While a number of possible pathological changes were evaluated in infected rabbits, the presence of changes in lymph node structure similar to those reported in infected humans merits further investigation.     

A Case of Cunninghamella bertholettiae Rhino-cerebral Infection in a Leukaemic Patient and Review of Recent Published Studies

Cunninghamella bertholletiae infection occurs most frequently in neutropenic patients affected by haematological malignancies, is associated with an unfavourable outcome. We report a case of rhino-mastoidal fungal infection in a leukaemic patient. Bioptical tissue cultures yield the isolation of a mould with typical properties of Cunninghamella species. Liposomal amphotericin B (L-Amb) therapy combined with surgical intervention brought the lesion to recovery. Nevertheless, the patient died 14 days after bone marrow transplantation (BMT) from bacterial sepsis. Mastoiditis was documented at CT-scan. The conditioning regimen probably caused the reactivation of the Cunninghamella infection that led to the patient's fatal outcome; fungal hyphae were detected after autopsy of brain and lung tissue.     

The interaction of peripheral proteins and membranes studied with alpha-lactalbumin and phospholipid bilayers of various compositions

To characterize the interaction of peripheral proteins and membranes at the molecular level, we studied the reversible association of bovine alpha-lactalbumin (BLA) with lipid bilayers composed of different molecular forms of phosphatidylserine or equimolar mixtures of these phosphatidylserine forms and egg yolk phosphatidylcholine. At pH 4.5, almost all BLA (>90%) associates to negatively charged small unilamellar vesicles. The conformational changes that binding to these bilayers induced on the protein were characterized by circular dichroism and fluorescence spectroscopy. Because binding of BLA to negatively charged vesicles is reverted by adjusting the pH back to >6.0, we also investigated the conformation of the membrane-bound protein by NMR-monitored H-D exchange of the backbone amide protons. The conformation adopted by BLA bound to these bilayers resembles a molten globule-like state but the negative ellipticity at 222 nm and the apparent alpha-helix content of the bound protein senses the changes in the physical properties of the membrane. Binding to bilayers in the gel state appears to correlate with an increased amount of alpha-helical structure and with a lower extent of integration into the membrane, corresponding to the adsorbed protein, while the opposite is found for BLA bound to vesicles in the liquid-crystalline phase, corresponding to the embedded conformation. A common feature for the membrane-bound conformations of BLA is that the amphipathic helix C (residues 86 to 99) is an important determinant for the adsorption and further integration of the protein into the membrane.     

In vivo genetic variability of the human immunodeficiency virus type 2 V3 region.

The principal neutralizing epitope of the human immunodeficiency virus type 1 (HIV-1) lies between two invariant cysteines in the third variable region (V3) of the viral envelope (gp120), and its amino acid sequence varies among different HIV-1 isolates. HIV-2 carries an analogous amino acid sequence between two cysteines of the V3 regions, but its functional similarity with the HIV-1 principal neutralizing epitope is uncertain. We studied the degree of genetic variation of the HIV-2 V3 region in fresh blood samples from 12 HIV-2-seropositive individuals from Guinea-Bissau. Polymerase chain reaction was used to amplify viral fragments of 465 bp containing the V3 region from cellular DNA. Nucleotide sequence analysis of the entire envelope fragment from each patient revealed that the degree of variation among field isolates of HIV-2 is comparable to that observed in the analogous region of HIV-1. Most of the HIV-2 isolates studied were highly related, suggesting the existence of a limited number of different viral strains in the cohort studied. Thus, the HIV-2 and HIV-1 V3 regions vary to a similar degree and may also have analogous functions.     

Energetic aspects of CO oxidation in desulfovibrio desulfuricans

In cell suspension of Desulfovibrio desulfuricans B-1388, oxidation of CO as the only energy source is associated with reduction of SO42-. After a 2-h incubation of cells in 8% CO, 81% of the gas is converted. Oxidation of 1 mole CO results in formation of 0.23 mole H2S. Intracellular ATP content increases from 2.5 (control) to 8.3 nmoles/mg (during CO conversion). Dinitrophenol inhibits sulfate reduction and CO oxidation. CO dehydrogenase was detected in cytoplasmic and membrane cell fractions (59 and 34%, respectively).     

Reactive complexes in myoglobin and nitric oxide synthase

In this overview some of our crystallographic and spectroscopic studies on reactive complexes in myoglobin and nitric oxide synthase are summarised. Myoglobin and nitric oxide synthase are both haemoproteins with some similar reaction intermediates. For myoglobin we have studied different intermediates generated in the reaction with hydrogen peroxide by X-ray diffraction, single-crystal microspectrophotometry, electron paramagnetic resonance spectroscopy, Mössbauer spectroscopy, resonance Raman spectroscopy and quantum refinement. Several of these myoglobin states are quite susceptible to radiation-induced changes during crystallographic data collection, and we have observed a radiation-induced change of the ferric resting myoglobin to aqua ferrous myoglobin, of myoglobin compound II to a proposed intermediate H, and of myoglobin compound III to peroxy myoglobin. For the myoglobin compound II/ intermediate H we observe a single-bonded Fe^IV-O species, which is probably protonated. The long Fe-O bond seen in the crystal structure can be supported by the observation of a new ¹⁸O-sensitive resonance Raman mode at 687 cm⁻¹. For nitric oxide synthase we detected with cryobiochemical methods in electron paramagnetic resonance spectra the first biopterin radical serving as electron donor to the ferrous-oxy complex, and that biopterin serves as a proton donor as well, in addition we could observe formation of the Fe(NO) complex with a amino-pterin cofactor capable to form a reactive radical.