Killing of Latently HIV-Infected CD4 T Cells by Autologous CD8 T Cells Is Modulated by Nef.

The role of HIV-specific CD8 T cell activity in the course of HIV infection and the way it affects the virus that resides in the latent reservoir resting memory cells is debated. The PBMC of HIV-infected patients contain HIV-specific CD8 T cells and their potential targets, CD4 T cells latently infected by HIV. CD4 T cells and CD8 T cells procured from PBMC of HIV-infected patients were co-incubated and analyzed: Formation of CD8 T cells and HIV-infected CD4 T cell conjugates and apoptosis of these CD4 T cells were observed by fluorescence microscopy with in situ PCR of HIV LTR DNA. Furthermore, conjugation of CD8 T cells with CD4 T cells and apoptosis of CD4 T cells was observed and quantified by imaging flow cytometry using anti-human activated caspase 3 antibody and TUNEL assay. The conjugation activity and apoptosis were found to be much higher in patients with acute HIV infection or AIDS compared to patients in chronic infection on antiretroviral therapy (ART) or not. Patients on ART had low grade conjugation and apoptosis of isolated CD69, CD25, and HLA-DR-negative CD4 T cells (latent reservoir cells) by CD8 T cells. Using in situ PCR The latent reservoir CD4 T cells were shown to contain most of the HIV DNA. We demonstrate in HIV-infected patients, that CD8 T cells conjugate with and kill HIV-infected CD4 T cells, including HIV-infected resting memory CD4 T cells, throughout the course of HIV infection. We propose that in HIV-infected patients CD4 T cell annihilation is caused in part by ongoing activity of HIV-specific CD8 T cells. HIV Nef protein interacts with ASK 1 and inhibits its pro-apoptotic death signaling by Fas/FasL, thus protecting HIV-infected cells from CD8 T cells killing. A peptide that interrupts Nef-ASK1 interaction that had been delivered into CD4 T cells procured from patients on ART resulted in the increase of their apoptosis inflicted by autologous CD8 T cells. We suggest that elimination of the HIV-infected latent reservoir CD4 T cells can be achieved by Nef inhibition.

Peripheral blood mononuclear cells of HIV-infected patients contain CD8 T cells that form conjugates with and kill HIV-infected autologous CD4 T cells.

Peripheral blood mononuclear cells (PBMC) of untreated, HIV-infected patients contain HIV-specific CD8 T cells as well as their corresponding targets, HIV-infected CD4 T cells. To determine if CD4 T-cell depletion in HIV-infected patients may result from autologous CD8-CD4 T-cell interaction, CD8 and CD4 T cells procured from PBMC of acute and chronic untreated HIV-infected patients were sorted and co-incubated. Formation of CD8-CD4 T-cell conjugates was observed by fluorescence microscopy. Apoptosis of CD4 T cells in conjugation was recorded by digitized images and was further observed and measured by FACS using Annexin staining. Perforin expression in the CD8 T cells was measured using intracellular monoclonal perforin antibody staining. HIV DNA in the conjugated CD4 T cells was detected by in situ PCR. We found that 6·1 ± 0·5% of CD4 T cells from acute HIV-infected patients and 3·0 ± 0·5% from chronic HIV-infected patients formed CD8-CD4 T-cell conjugates. Annexin binding and cell morphology typical of apoptosis were observed in the conjugated CD4 T cells. The majority of CD8 T cells that had conjugated to CD4 T cells expressed perforin. The conjugated CD4 T cells exhibited nuclear HIV DNA. CD8 T cells and HIV-infected CD4 T cells, both procured from the PBMC of untreated HIV-infected patients, form conjugates. Apoptotic lytic activity has been observed in the conjugated CD4 T cells. We propose that CD4 T-cell annihilation in HIV-infected patients results, at least in part, from the interactions of perforin-rich CD8 T cells with autologous, HIV-infected CD4 T cells.

Emotional processing of personally familiar faces in the vegetative state.

BACKGROUND: The Vegetative State (VS) is a severe disorder of consciousness in which patients are awake but display no signs of awareness. Yet, recent functional magnetic resonance imaging (fMRI) studies have demonstrated evidence for covert awareness in VS patients by recording specific brain activations during a cognitive task. However, the possible existence of incommunicable subjective emotional experiences in VS patients remains largely unexplored. This study aimed to probe the question of whether VS patients retain a brain ability to selectively process external stimuli according to their emotional value and look for evidence of covert emotional awareness in patients. METHODS AND FINDINGS: In order to explore these questions we employed the emotive impact of observing personally familiar faces, known to provoke specific perceptual as well as emotional brain activations. Four VS patients and thirteen healthy controls first underwent an fMRI scan while viewing pictures of non-familiar faces, personally familiar faces and pictures of themselves. In a subsequent imagery task participants were asked to actively imagine one of their parent's faces. Analyses focused on face and familiarity selective regional brain activations and inter-regional functional connectivity. Similar to controls, all patients displayed face selective brain responses with further limbic and cortical activations elicited by familiar faces. In patients as well as controls, Connectivity was observed between emotional, visual and face specific areas, suggesting aware emotional perception. This connectivity was strongest in the two patients who later recovered. Notably, these two patients also displayed selective amygdala activation during familiar face imagery, with one further exhibiting face selective activations, indistinguishable from healthy controls. CONCLUSIONS: Taken together, these results show that selective emotional processing can be elicited in VS patients both by external emotionally salient stimuli and by internal cognitive processes, suggesting the ability for covert emotional awareness of self and the environment in VS patients.

Evaluation of a benchtop HIV ultradeep pyrosequencing drug resistance assay in the clinical laboratory.

Detection of low-abundance drug resistance mutations (DRMs) of HIV-1 is an evolving approach in clinical practice. Ultradeep pyrosequencing has shown to be effective in detecting such mutations. The lack of a standardized commercially based assay limits the wide use of this method in clinical settings. 454 Life Sciences (Roche) is developing an HIV ultradeep pyrosequencing assay for their benchtop sequencer. We assessed the prototype plate in the clinical laboratory. Plasma samples genotyped by the standardized TruGene kit were retrospectively tested by this assay. Drug-treated subjects failing therapy and drug-naive patients were included. DRM analysis was based on the International AIDS Society USA DRM list and the Stanford algorithm. The prototype assay detected all of the DRMs detected by TruGene and additional 50 low-abundance DRMs. Several patients had low-abundance D67N, K70R, and M184V reverse transcriptase inhibitor mutations that persisted long after discontinuation of the drug that elicited these mutations. Additional patient harbored low-abundance V32I major protease inhibitor mutation, which under darunavir selection evolved later to be detected by TruGene. Stanford analysis suggested that some of the low-abundance DRMs were likely to affect the resistance burden in these subjects. The prototype assay performs at least as well as TruGene and has the advantage of detecting low-abundance drug resistance mutations undetected by TruGene. Its ease of use and lab-scale platform will likely facilitate its use in the clinical laboratory. The extent to which the detection of low-abundance DRMs will affect patient management is still unknown, but it is hoped that use of such an assay in clinical practice will help resolve this important question.

Cytotoxic T lymphocyte perforin and Fas ligand working in concert even when Fas ligand lytic action is still not detectable.

The reason(s) why individual cytotoxic T lymphocytes (CTL) possess a fast-acting, perforin/granzyme-mediated, as well as a much slower, Fas ligand (FasL) -driven killing mechanism is not clear, nor is the basis for wide variations in killing activity exhibited by individual CTL, ranging from minutes to hours. We show that perforin expression among individual, conjugated CTL varies widely, which can account for the heterogeneity in killing speeds exhibited by individual CTL. Despite a 2-hr lag in FasL-based killing, CTL lytic action is enhanced when the two mechanisms operate in concert. This is explained by finding that the two pathways in fact are jump-started simultaneously with the lag in FasL lytic action reflecting pre-lytic caspase-8 activation and BH3-interacting domain (BID) cleavage. The complementary action of the two lytic pathways, co-expressed at varying levels among individual CTL, facilitates the lytic action of late-stage poor perforin-expressing CTL, ensuring optimal cytocidal action throughout the CTL response.

Switch from perforin-expressing to perforin-deficient CD8(+) T cells accounts for two distinct types of effector cytotoxic T lymphocytes in vivo.

Although CD8(+) cytotoxic T lymphocytes (CTL) exhibit both Fas ligand (FasL) -based and perforin-based lytic activities, the accepted hallmark of a fully active CTL remains its perforin killing machinery. Yet the origin, rationale for possessing both a slow-acting (FasL) and a fast-acting (perforin) killing mechanism has remained enigmatic. Here we have investigated perforin expression in CTL directly involved in acute tumour (i.e. leukaemias EL4 and L1210) allograft rejection occurring within the peritoneal cavity. We show that at the height of the immune response, the majority of conjugate-forming CD8(+) CTL express high levels of perforin messenger RNA and protein, and kill essentially via perforin. Later however, coinciding with complete rejection, fully cytocidal CTL emerge which exhibit a stark decrease in perforin and now kill preferentially via constitutively expressed FasL. Although late in emergence, and persistent, these powerful CTL are neither effector-memory nor memory CTL. This finding has implications for the monitoring of anti-transplant responses in clinical settings, based on assessing perforin expression in graft infiltrating CD8(+) T cells. The results show that as the immune response progresses in vivo, targeted cellular suicide mainly prunes high perforin-expressing CD8(+) cells, resulting in the gradual switch in effector CTL, from mostly perforin-based to largely Fas/FasL-based killers. Hence, two kinds of CD8(+) CTL have two killing strategies.

Comparison of nitinol urethral stent infections with indwelling catheter-associated urinary-tract infections.

BACKGROUND AND PURPOSE: To determine the efficacy of intraurethral metal stents in preventing or eradicating urinary-tract infections (UTI) during the management of bladder outlet obstruction (BOO) by comparing the frequency and nature of the infections with indwelling-catheter-associated UTI. PATIENTS AND METHODS: The SAS relative-risk test was used to compare the risks of UTI in 76 patients with temporary urethral stents, 60 patients with BOO who had never been catheterized nor stented, and 34 patients with a permanent indwelling urethral catheter (PIUC). Infection was assessed 1 month after placement of the devices. Scanning electron microscopy (SEM) of the proximal and distal pieces of the stents removed from five patients with and five patients without UTI was carried out in a search for predisposing changes on the surfaces. RESULTS: After insertion of the catheter, UTI developed in 79.4% of the patients who originally had sterile urine. However, after insertion of the stent, UTI developed in only 40.9% of the patients with sterile urine. In 21 (44.6%) of the catheterized patients who had infected urine, UTI was eradicated after stent insertion. The SEM analysis of the stents showed that a thick organic layer had formed only on the infected devices but with no sign of erosion. CONCLUSION: Urinary infection is a significant problem in patients with PIUC but is significantly less frequent and less severe in patients with urethral stents. This advantage of stents over the conventional urethral catheter, in addition to their obvious convenience for the patient, make them good alternatives to reduce the risk of UTI.

Agranulocytosis associated with parvovirus B19 infection in otherwise healthy patients.

In the course of 6 years, 23 otherwise healthy patients with acute febrile illness and leukopenia were diagnosed as having acute parvovirus B19 infection. Five of these patients had agranulocytosis associated with acute parvovirus B19 infection and one had chronic agranulocytosis due to persistent parvovirus B19 infection. The diagnosis was made after positive anti-parvovirus B19 IgM antibodies were found in all of the patients and viral DNA was detected by PCR in four patients. Neutropenia and agranulocytosis appear to be much more frequently associated with parvovirus B19 infection than previously reported.

Listeria monocytogenes infection in Israel and review of cases worldwide.

Listeria monocytogenes, an uncommon foodborne pathogen, is increasingly recognized as a cause of life-threatening disease. A marked increase in reported cases of listeriosis during 1998 motivated a retrospective nationwide survey of the infection in Israel. From 1995 to 1999, 161 cases were identified; 70 (43%) were perinatal infections, with a fetal mortality rate of 45%. Most (74%) of the 91 nonperinatal infections involved immunocompromised patients with malignancies, chronic liver disease, chronic renal failure, or diabetes mellitus. The common clinical syndromes in these patients were primary bacteremia (47%) and meningitis (28%). The crude case-fatality rate in this group was 38%, with a higher death rate in immunocompromised patients.