Indira Hewlett

Identification of a Potential HIV-Induced Source of Bystander-Mediated Apoptosis in T Cells: Upregulation of TRAIL in Primary Human Macrophages by HIV-1 Tat

The induction of apoptosis in T cells by bystander cells has been repeatedly implicated as a mechanism contributing to the T cell depletion seen in HIV infection. It has been shown that apoptosis could be induced in T cells from asymptomatic HIV-infected individuals in a Fas-independent, TNF-related apoptosis-inducing ligand (TRAIL)-dependent manner if the cells were pretreated with anti-CD3. It has also been shown that T cells from HIV-infected patients were even more sensitive to TRAIL induction of apoptosis than they were to Fas induction. Recently, it has been reported that in an HIV-1 SCID-Hu model, the vast majority of the T cell apoptosis is not associated with p24 and is therefore produced by bystander effects. Furthermore, few apoptotic cells were associated with neighboring cells which were positive for either Fas ligand or TNF. However, most of the apoptotic cells were associated with TRAIL-positive cells. The nature of these TRAIL-positive cells was undetermined. Here, we report that HIV infection of primary human macrophages switches on abundant TRAIL production both at the RNA and protein levels. Furthermore, more macrophages produce TRAIL than are infected by HIV, indicating that a bystander mechanism may, at least in part, upregulate TRAIL. Exogenously supplied HIV-1 Tat protein upregulates TRAIL production by primary human macrophages to an extent indistinguishable from infection. The results suggest a model in which HIV-1-infected cells produce extracellular Tat protein, which in turn upregulates TRAIL in macrophages which then can induce apoptosis in bystander T cells.

Prevalence of Human Immunodeficiency Virus Type 1 p24 Antigen in U.S. Blood Donors — An Assessment of the Efficacy of Testing in Donor Screening

Abstract Background. We performed a multicenter study in 1989 to determine whether screening whole-blood donors for human immunodeficiency virus type 1 (HIV-1) p24 antigen would improve transfusion safety by identifying carriers of the virus who are seronegative for HIV-1 antibody. Methods. More than 500,000 donations were tested at 13 U.S. blood centers with test kits from two manufacturers. Units found repeatedly reactive were retested in a central laboratory; if the results were positive, they were confirmed by a neutralization assay. A subgroup of units was also tested for HIV-1 by the polymerase chain reaction. Selected donors confirmed or not confirmed as having p24 antigen were contacted for follow-up interviews to identify risk factors and undergo retesting for HIV-1 markers. Results. Positive tests for p24 antigen were confirmed by neutralization in five donors (0.001 percent of all donations tested), all of whom were also positive for HIV-1 antibody and HIV-1 by polymerase chain reaction. Three ...

An international collaborative study to establish the 1st international standard for HIV-1 RNA for use in nucleic acid-based techniques

Twenty-six laboratories from 10 different countries participated in a collaborative study to establish the 1st International Standard for HIV-1 RNA for use in nucleic acid-based techniques (NAT). Three candidate preparations were tested all based on genotype B viruses. The candidates were tested by each laboratory at a range of dilutions in four independent assays and the results collated and analysed statistically. All three candidates gave results that were tightly grouped, with little difference between the results from different laboratories or from the use of different assays. Studies of relative potency showed good agreement between laboratories. There were no significant differences between five commercial assay types, except that candidate XX showed a slightly lower potency compared to YY and ZZ with a single commercial assay. The reason for this was not established. Degradation studies showed that the freeze-dried preparations were stable at -20,4 and 20 degrees C for 26 weeks, the longest period studied, but that they became difficult to reconstitute after 3 weeks at 45 degrees C and 9 weeks at 37 degrees C. As a result of the study, the World Health Organisation (WHO) Expert Committee on Biological Standardisation (ECBS) established the preparation referred to as candidate YY (NIBSC Code No. 97/656) as the 1st International Standard for HIV-1 RNA for use with NAT with an assigned potency of 100000 International Units per vial.

Failure to confirm XMRV/MLVs in the blood of patients with chronic fatigue syndrome: a multi-laboratory study.

The assays used to detect XMRV in patients with chronic fatigue syndrome are unreliable. Murine leukemia viruses (MLVs), including xenotropic-MLV–related virus (XMRV), have been controversially linked to chronic fatigue syndrome (CFS). To explore this issue in greater depth, we compiled coded replicate samples of blood from 15 subjects previously reported to be XMRV/MLV–positive (14 with CFS) and from 15 healthy donors previously determined to be negative for the viruses. These samples were distributed in a blinded fashion to nine laboratories, which performed assays designed to detect XMRV/MLV nucleic acid, virus replication, and antibody. Only two laboratories reported evidence of XMRV/MLVs; however, replicate sample results showed disagreement, and reactivity was similar among CFS subjects and negative controls. These results indicate that current assays do not reproducibly detect XMRV/MLV in blood samples and that blood donor screening is not warranted.

Nanoparticle-based immunoassays for sensitive and early detection of HIV-1 capsid (p24) antigen

We evaluated the feasibility of using nanoparticle (NP)-based assays for improving detection sensitivity of human immunodeficiency virus type 1 (HIV-1) p24 antigen. One assay that was evaluated is a gold NP-based biobarcode amplification (BCA) assay, which can detect HIV-1 p24 antigen at levels as low as 0.1 pg/mL. The lower limit of detection for an enzyme-linked immunosorbent assay (ELISA) is 10-15 pg/mL. These results demonstrate that the HIV-1 p24 BCA assay offers 100-150-fold enhancement in the detection limit over the traditional colorimetric ELISA. Furthermore, the BCA assay detected HIV-1 infection 3 days earlier than did ELISA in samples from patients who had experienced seroconversion. The other assay that we tested is the europium NP-based immunoassay, which uses europium NPs to replace gold NPs in the BCA assay to further simplify the detection method and decrease the incubation time. For detection of HIV-1 p24, the lower limit of detection for the europium NP-based immunoassay was 0.5 pg/mL. These results indicate that the universal labeling technology based on NPs and its application may provide a rapid and sensitive testing platform for clinical diagnosis and laboratory research.

Detection of anthrax toxin by an ultrasensitive immunoassay using europium nanoparticles.

ABSTRACT We developed a europium nanoparticle-based immunoassay (ENIA) for the sensitive detection of anthrax protective antigen (PA). The ENIA exhibited a linear dose-dependent pattern within the detection range of 0.01 to 100 ng/ml and was approximately 100-fold more sensitive than enzyme-linked immunosorbent assay (ELISA). False-positive results were not observed with serum samples from healthy adults, mouse plasma without PA, or plasma samples collected from mice injected with anthrax lethal factor or edema factor alone. For the detection of plasma samples spiked with PA, the detection sensitivities for ENIA and ELISA were 100% (11/11 samples) and 36.4% (4/11 samples), respectively. The assay exhibited a linear but qualitative correlation between the PA injected and the PA detected in murine blood (r = 0.97731; P < 0.0001). Anthrax PA was also detected in the circulation of mice infected with spores from a toxigenic Sterne-like strain of Bacillus anthracis, but only in the later stages of infection. These results indicate that the universal labeling technology based on europium nanoparticles and its application may provide a rapid and sensitive testing platform for clinical diagnosis and laboratory research.

Nanoparticle-Based biobarcode amplification assay (BCA) for sensitive and early detection of human immunodeficiency type 1 capsid (p24) antigen.

Nanotechnology-based techniques are being widely evaluated in medical testing and could provide a new generation of diagnostic assays due to their high degrees of sensitivity, high specificity, multiplexing capabilities, and ability to operate without enzymes. In this article, we have modified a nanoparticle-based biobarcode amplification (BCA) assay for early and sensitive detection of HIV-1 capsid (p24) antigen by using antip24 antibody-coated microplates to capture viral antigen (p24) and streptavidin-coated nanoparticle-based biobarcode DNAs for signal amplification, followed by detection using a chip-based scanometric method. The modified BCA assay exhibited a linear dose-dependent pattern within the detection range of 0.1 to 500 pg/ml and was approximately 150-fold more sensitive than conventional enzyme-linked immunosorbent assay (ELISA). No false positive results were observed in 30 HIV-1-negative samples, while all 45 HIV-1 RNA positive samples were found HIV-1 p24 antigen positive by the BCA assay. In addition, the BCA assay detected HIV-1 infection 3 days earlier than ELISA in seroconversion samples. Preliminary evaluation based on testing a small number of samples indicates that the HIV-1 p24 antigen BCA may provide a new tool for sensitive and early detection of HIV-1 p24 antigen in settings where HIV-1 RNA testing is currently not routinely performed.

Window-period human immunodeficiency virus transmission to two recipients by an adolescent blood donor

BACKGROUND:  Pooled NAT and donor screening have reduced the diagnostic window period for HIV in the blood donor population to approximately 10 to 15 days. This report describes two cases of transfusion‐acquired HIV infection and verification of transmission from the donor to the recipients, and attempts to identify how the 18‐year‐old donor acquired her infection.

Monocytes-macrophages are a potential target in human infection with West Nile virus through blood transfusion

BACKGROUND:  West Nile virus (WNV) transmission by transfusion was documented in 2002. Approximately 80 percent of WNV infections are asymptomatic and 1 percent develop severe neurological illness. In animals, Langerhans‐dendritic cells support initial viral replication, followed by replication in lymphoid tissues and dissemination to organs and possibly to the CNS. The cellular tropism of WNV infection after transfusion and the particular human blood cells that sustain viral replication remain largely unknown. Whether primary monocyte‐derived macrophages (MDMs) support WNV infection‐replication and produce infectious virions, with an in vitro system, was investigated.

Ultrasensitive detection of HIV-1 p24 antigen using nanofunctionalized surfaces in a capacitive immunosensor.

The HIV-1 capsid protein, p24 antigen, is of considerable diagnostic interest because following HIV exposure it is detectable several days earlier than host-generated HIV antibodies (which are the target of almost all current tests used in the field) and can be used to design very sensitive assays without the need for PCR. Here, we present an ultrasensitive capacitive immunosensor that is capable of detecting subattogram per milliliter concentrations of p24 antigen, which to our knowledge is the lowest level of detection ever reported. Dilution studies using p24-spiked human plasma samples indicate that the immunosensor is robust against the interfering effects of a complex biological matrix. Moreover, the capacitive immunosensor assay is rapid (<20 min), label-free, and generates data in real-time, with a portable format in development. Additional optimization of the capture agents and/or surface chemistries may further improve performance, highlighting the potential of this platform to serve as a diagnostic tool for early detection of HIV in field settings.