Interferon Induction by HIV-1-Infected Cells: A Possible Role of Sulfatides or Related Glycolipids

AbstractWe have investigated the mechanism of interferon (IFN) induction in peripheral blood mononuclear cells by HIV-1(IIIB)-infected H9 cells or by recombinant gp120. A monoclonal antibody specific for the galactosylsphingosinyl moiety in galactocerebrosides and sulfatides inhibited IFN induction in a dose-dependent manner. Furthermore, exogenous sulfatides inhibited with an ID50of approximately 1 μM,whereas galactocerebrosides were not inhibitory at 40 times higher concentrations. These studies suggest that sulfate containing galactolipids such as sulfatides on responder cells may be part of the gp120–membrane complex that initiates the induction of IFN. A partial homology of an epitope on the V3 loop of gp120 with a previously suggested binding domain for sulfated glycoconjugates supports this conclusion

Granulocytic myeloid-derived suppressor cells increased in early phases of primary HIV infection depending on TRAIL plasma level

Background It has been demonstrated that Myeloid Derived Suppressor Cells (MDSC) are expanded in HIV-1 infected individuals and correlated with disease progression. The phase of HIV infection during which MDSC expansion occurs, and the mechanisms that regulate this expansion remain to be established. In this study we evaluated the frequency of MDSC in patients during primary HIV infection, and factors involved in MDSC control. Methods Patients with primary (PHI) and chronic (CHI) HIV infection were enrolled. PHI staging was performed according to Fiebig classification, and circulating MDSC frequency and function were evaluated by flow cytometry. Cytokine levels were evaluated by Luminex technology. Results We found that granulocytic MDSC (Gr-MDSC) frequency was higher in PHI compared to healthy donors, but lower than CHI. Interestingly, Gr-MDSC expansion was observed in the early phases of HIV infection (Fiebig II/III), but it was not associated to HIV viral load and CD4 T cell count. Interestingly, in PHI Gr-MDSC frequency was inversely correlated with plasmatic level of TRAIL, while a direct correlation was observed in CHI. Further, lower level of GMCSF was observed in PHI compared with CHI. In vitro experiments demonstrated that, differently from CHI, recombinant TRAIL induced apoptosis of Gr-MDSC from PHI, can effect that can be abrogated by GM-CSF. Conclusion We found that Gr-MDSC are expanded early during primary HIV infection and may be regulated by TRAIL and GM-CSF levels. These findings shed light on the fine mechanisms regulating the immune system during HIV infection, and open new perspectives for immune-based strategies

A molecular beacon, bead-based assay for the detection of nucleic acids by flow cytometry

Molecular beacons are dual-labelled probes that are typically used in real-time PCR assays, but have also been conjugated with solid matrices for use in microarrays or biosensors. We have developed a fluid array system using microsphere-conjugated molecular beacons and the flow cytometer for the specific, multiplexed detection of unlabelled nucleic acids in solution. For this array system, molecular beacons were conjugated with microspheres using a biotin-streptavidin linkage. A bridged conjugation method using streptavidin increased the signal-to-noise ratio, allowing for further discrimination of target quantitation. Using beads of different sizes and molecular beacons in two fluorophore colours, synthetic nucleic acid control sequences were specifically detected for three respiratory pathogens, including the SARS coronavirus in proof-of-concept experiments. Considering that routine flow cytometers are able to detect up to four fluorescent channels, this novel assay may allow for the specific multiplex detection of a nucleic acid panel in a single tube

Simultaneous control of DNA and RNA processing efficiency using a nucleic acid calibration set.

PCR-based detection techniques enables reliable and sensitive nucleic acid target detection. However, quantitative determination methods often fail to control for the efficiency of nucleic acid extraction, reverse transcription, and PCR amplification. This problem is even more prominent when working with clinical samples due to target sequence loss during nucleic acid processing or the co-purification of PCR inhibitors (1,2). Handling processes are often assumed to approach 100% efficiency in the laboratory, even if practical experience shows that this efficiency can be much lower. This inability to ensure accuracy can lead to significant error in uncalibrated DNA sample quantitation. The additional need for reverse transcription of RNA may further increase the quantitative error rate, as yet another enzymatic process is involved. Nucleic acid controls have been developed based upon known sequences to calibrate either DNA or RNA handling; DNA calibrators have been used to control for the amplification of target sequences using realtime PCR methods (3–8), while RNA calibrators have been developed to test reverse transcription and amplification efficiencies (9–11). A nonpathogenic viral particle carrying a sequence for use as an external positive control of extraction and amplification has also been described (12). Unfortunately, most of the established processing controls are only suitable for limited applications (i.e., either DNA or RNA detection). Cross-contamination of biological samples or minute detection from natural sources reveals the need for completely synthetic sequences, with no homology to sequences in the nucleic acid databases. It is, therefore, beneficial to design an internal, synthetic calibration system that can control for both DNA and RNA processing steps in a single tube. This set includes both RNA and DNA targets with identical primer binding sites and, thus, primer binding efficiency, but easily distinguishable sequence characteristics, allowing for simultaneous detection, quantitation, and calibration of nucleic acid processing efficiency. A 150-bp randomly generated nucleic acid sequence was developed for use as a short control (SC). A GCrich 75-bp sequence was inserted in the middle of the 150-bp sequence to generate a 225-bp sequence, long control (LC). Besides size, the two sequences were designed to have easily distinguishable probe binding sites with a predicted product melting temperature difference of 4°C. Calibrator sequences have been published as GenBank® accession nos. EF143258 (DNA control, LC) and EF143257 (RNA control, SC). Simultaneous control of DNA and RNA processing efficiency using a nucleic acid calibration se

Viral hemorrhagic fevers: advancing the level of treatment

The management of viral hemorrhagic fevers (VHFs) has mainly focused on strict infection control measures, while standard clinical interventions that are provided to patients with other life-threatening conditions are rarely offered to patients with VHFs. Despite its complexity, a proper clinical case management of VHFs is neither futile nor is it lacking in scientific rationale. Given that patient outcomes improve when treatment is started as soon as possible, development and implementation of protocols to promptly identify and treat patients in the earliest phases of diseases are urgently needed. Different pharmacological options have been proposed to manage patients and, as for other life-threatening conditions, advanced life support has been proved effective to address multiorgan failure. In addition, high throughput screening of small molecular libraries has emerged as a novel promising way to find new candidates drugs for VHFs therapy and a relevant number of new molecules are currently under investigation. Here we discuss the current knowledge about VHF clinical management to propose a way to step up the approach to VHFs beyond the mere application of infection control measures

Orthopoxvirus Seroprevalence in Cats and Veterinary Personnel in North-Eastern Italy in 2011

Orthopoxviruses (OPV) are emerging zoonotic pathogens, and an increasing number of human infections is currently reported in Europe and in other continents, warranting heightened attention on this topic. Following two OPV infections reported in veterinarians scratched by sick cats in 2005 and 2007 in North-Eastern-Italy, involving a previously undescribed OPV, a similar strain was isolated by a sick cat from the same territory in 2011, i.e., 6 years later, raising attention on OPV circulation in this region. A surveillance program was launched to assess the OPV seroprevalence among the veterinarians working in local veterinary clinics and in the local wild and domestic cat population; seroprevalence was 33.3% in veterinarians and 19.5% in cats. Seroprevalence in cats was unevenly distributed, peaking at 40% in the area where OPV-infected cats had been observed

Performance of rapid tests in the management of dengue fever imported cases in Lazio, Italy 2014-2019

Abstract Background In Italy, dengue virus is the most frequent agent of imported viral infections. The use of rapid diagnostic tests (RDTs) may be of help as a preliminary user-friendly quick assay to facilitate dengue diagnosis, as ordinary laboratory diagnosis of dengue fever may require special efforts in terms of tools availability, interpretation of results, and skilled personnel. The performance of RDTs, however, may vary according to different epidemiological and laboratory background. Methods We reviewed five years of laboratory records of two dengue RDT results (Colorimetric SD-Bioline Dengue-Duo-RDT and Fluorimetric SD-Biosensor-STANDARD-F-Dengue-RDT), able to detect viral NS1 antigen and specific IgM and IgG. Diagnostic parameters were calculated using as reference the results of molecular (RT-PCR) and serological (immunofluorescence, IFA) tests. Overall performance, calculated considering the final case definition, was included in the accuracy assessment of RDTs. Results The combined use of NS1 and IgM/IgG RDT for the detection of acute dengue cases resulted in an overall sensitivity and specificity of 87.2% and 97.9% for Colorimetric RDT, 96.2% and 96.2% for Fluorimetric RDT. NS1 was the most reliable marker of acute infection, while IgM resulted falsely positive in nine samples, including sera derived from 2 Zika and 4 non-arbovirus infected patients. Conclusions The inclusion of RDT in the diagnostic algorithm is of undeniable help in the prompt management and surveillance of dengue infection in non-endemic areas. Confirmatory tests are, however, necessary to rule in or rule out dengue fever diagnosis