Mark J. McVey

Interleukin-7 receptor expression on CD8 T-cells is downregulated by the HIV Tat protein.

We have previously shown decreased expression of the interleukin (IL)-7 receptor α-chain (CD127) on CD8 T-cells in HIV-infected patients and an apparent recovery of this receptor in those receiving antiretroviral therapy with sustained viral suppression. Here, we demonstrate that the HIV Tat protein specifically downregulates cell surface expression of CD127 on human CD8 T-cells in a dose- and time-dependent manner. The effects of Tat on CD127 expression could be blocked with anti-Tat monoclonal antibodies or by preincubating Tat with heparin. Tat had no effect on the expression of other cell surface proteins examined, including CD132, or on cell viability over 72 hours. Further, CD127 expression was not altered by other HIV proteins, including gp160 or Nef. Preincubation of purified CD8 T-cells with Tat protein inhibited CD8 T-cell proliferation and perforin synthesis after stimulation with IL-7. Because IL-7 signaling is essential for optimal CD8 T-cell proliferation and function, the downregulation of CD127 and apparent inhibition of cytotoxic activity by Tat may play an important role in HIV-induced immune dysregulation and impaired cell-mediated immunity.

Intravenous immunoglobulin prevents murine antibody-mediated acute lung injury at the level of neutrophil reactive oxygen species (ROS) production

Transfusion-related acute lung injury (TRALI) is a leading cause of transfusion-associated mortality that can occur with any type of transfusion and is thought to be primarily due to donor antibodies activating pulmonary neutrophils in recipients. Recently, a large prospective case controlled clinical study of cardiac surgery patients demonstrated that despite implementation of male donors, a high incidence of TRALI still occurred and suggested a need for additional interventions in susceptible patient populations. To examine if intravenous immunoglobulin (IVIg) may be effective, a murine model of antibody-mediated acute lung injury that approximates human TRALI was examined. When BALB/c mice were injected with the anti-major histocompatibility complex class I antibody 34-1-2s, mild shock (reduced rectal temperature) and respiratory distress (dyspnea) were observed and pre-treatment of the mice with 2 g/kg IVIg completely prevented these symptoms. To determine IVIgs usefulness to affect severe lung damage, SCID mice, previously shown to be hypersensitive to 34-1-2s were used. SCID mice treated with 34-1-2s underwent severe shock, lung damage (increased wet/dry ratios) and 40% mortality within 2 hours. Treatment with 2 g/kg IVIg 18 hours before 34-1-2s administration completely protected the mice from all adverse events. Treatment with IVIg after symptoms began also reduced lung damage and mortality. While the prophylactic IVIg administration did not affect 34-1-2s-induced pulmonary neutrophil accumulation, bone marrow-derived neutrophils from the IVIg-treated mice displayed no spontaneous ROS production nor could they be stimulated in vitro with fMLP or 34-1-2s. These results suggest that IVIg prevents murine antibody-mediated acute lung injury at the level of neutrophil ROS production and thus, alleviating tissue damage.

T regulatory cells and dendritic cells protect against transfusion-related acute lung injury via IL-10

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related fatalities and is characterized by acute respiratory distress following blood transfusion. Donor antibodies are frequently involved; however, the pathogenesis and protective mechanisms in the recipient are poorly understood, and specific therapies are lacking. Using newly developed murine TRALI models based on injection of anti-major histocompatibility complex class I antibodies, we found CD4+CD25+FoxP3+ T regulatory cells (Tregs) and CD11c+ dendritic cells (DCs) to be critical effectors that protect against TRALI. Treg or DC depletion in vivo resulted in aggravated antibody-mediated acute lung injury within 90 minutes with 60% mortality upon DC depletion. In addition, resistance to antibody-mediated TRALI was associated with increased interleukin-10 (IL-10) levels, and IL-10 levels were found to be decreased in mice suffering from TRALI. Importantly, IL-10 injection completely prevented and rescued the development of TRALI in mice and may prove to be a promising new therapeutic approach for alleviating lung injury in this serious complication of transfusion.

When infections collide—gummatous syphilis in an HIV-infected individual

Syphilis and HIV are both transmitted sexually and have emerged as important co-pathogens with reciprocal augmentation in transmission and disease progression. HIV-positive patients tend to experience more aggressive symptomatology due to syphilis and are at greater risk of developing neurological disease. Similarly, standard therapy for syphilis may be inadequate in HIV-positive individual suggesting intensified treatment regimens may be required along with close follow-up. We report here the case of a 50-year-old HIV-positive male presenting with an unusual constellation of neurological findings. Although he had been treated appropriately 10 years previously for primary syphilis, investigations revealed multiple current intracranial gummas. Treatment with high-dose intravenous penicillin G resulted in clinical and radiographic resolution. Given the broad differential for HIV-positive patients presenting with neurological symptoms, the clinician must maintain a high index of suspicion for syphilis known for its varied and at times unusual manifestations. Further, prior treatment of syphilis does not ensure cure and so syphilis must be considered irrespective of treatment history.

Microparticles as biomarkers of lung disease: enumeration in biological fluids using lipid bilayer microspheres.

Extracellular vesicles, specifically microparticles (MPs), are rapidly gaining attention for their capacity to act as biomarkers for diagnosis, prognosis, or responsiveness to therapy in lung disease, in keeping with the concept of precision medicine. However, MP analysis by high-sensitivity flow cytometry (FCM) is complicated by a lack of accurate means for MP enumeration. To address this gap, we report here an enhanced FCM MP gating and enumeration technique based on the use of novel engineered lipid bilayer microspheres (LBMs). By comparison of LBM-based MP enumeration with conventional bead- or fluorescent-based FCM enumeration techniques and a gravimetric consumption gold standard, we found LBMs to be superior to commercial bead preparations, showing the smallest fixed bias and limits of agreement in Bland Altman analyses. LBMs had simultaneous capacity to aid FCM enumeration of MPs in plasma, BAL, and cell culture supernatants. LBM enumeration detected differences in MP counts in mice exposed to intraperitoneal lipopolysaccharide or saline. LBMs provided for 1) higher sensitivity for gating MPs populations, 2) reduced background within MP gates, 3) more appropriate size, and 4) an inexpensive alternative amenable to different fluorescent tags. LBM-based MP enumeration was useful for a series of different FCM systems assessed, whereas LBM gating benefited high- but not low-sensitivity FCM systems compared with fluorescence gating. By offering exclusive advantages over current means of gating and enumerating MPs, LBMs are uniquely suited to realizing the potential of MPs as biomarkers in biological lung fluids and facilitating precision medicine in lung disease.

Intravenous immunoglobulin treatment of spleen cells from patients with immune thrombocytopenia significantly increases the percentage of myeloid-derived suppressor cells

The response of myeloid-derived suppressor cells (MDSCs) to intravenous immunoglobulin (IVIG) treatment was evaluated in the spleens of patients with immune thrombocytopenia (ITP) and compared with spleens removed for trauma. Exposure to IVIG significantly increased the percentage of MDSCs in cultures of splenocytes from patients with ITP. IVIG also significantly increased MDSCs in the spleen cells from trauma patients, confirming the effects of IVIG. The response to IVIG was similar to that seen in patients with ITP treated with dexamethasone. This finding suggests that IVIG may also exert its effects and contribute to its salutary effects in ITP by increasing splenic MDSCs. We had the opportunity to study frozen spleen cell specimens from seven ITP and seven trauma patients. Although the exact therapies of each patient with ITP were unknown, we could at least assume that all patients that underwent splenectomy had received all of the standard treatments. MDSC phenotype was analysed by flow cytometry for the cell surface markers CD11b, CD33 and HLA-DR. Human spleen cells (5 9 10 cells/well) from ITP and trauma control patients were cultured in complete medium for 90 h in the presence or absence of IVIG. Titrations of IVIG were added to respective wells and the plates were incubated in humidified air with 5% CO2 at 37°C in HEPES buffer at a pH of 6 5. The concentration of IVIG ranged from 5 to 70 mg/ml and encompasses the approximate 25 mg/ml level in plasma that is clinically used. Cells cultured in medium alone without IVIG were run in parallel as non-treated controls. At 90 h, ITP and trauma human spleen cells were surface stained with HLA-DR, CD33 and CD11b. A mixture of 50 ll of diluted stains [HLA-DR (allophycocyanin), CD11b (phycoerythrin), CD33 (fluorescein isothiocyanate)] was added to the respective wells. Plates were re-incubated for 30 min at room temperature, centrifuged, and the pellets re-suspended in 200 ll of staining buffer. Cell viability and number were evaluated using Vi-CELL cell viability analyser (Beckman Coulter, Brea, CA, USA). At 90 h incubation 60–70% cells were viable. MDSCs in the HLA-DR negative gate were used for analysis (Fig 1) using a BD FACSort analyser (BD Biosciences, Mississauga, ON, Canada). A minimum of 10 000 events was collected, and the percentage of HLADR CD33 CD11b expressing MDSCs was evaluated. Results are expressed as the percentage of the total HLA-DR negative populations. In view of the fact that IVIG is an important agent in the treatment of ITP, we explored the effect of increasing concentrations of IVIG on the expression of MDSCs in spleen cells from patients with ITP and control trauma patients. The isolation sequence is shown in Fig 1 and described in its legend. The baseline MDSCs in the spleen cells from patients with ITP (0 28%) was 40% of that in the spleen cells from trauma patients (0 7%) (P < 0 02). The percentage of MDSCs in ITP spleen cells increased significantly with

Improved resolution in extracellular vesicle populations using 405 instead of 488 nm side scatter

ABSTRACT Improvements in identification and assessment of extracellular vesicles (EVs) have fuelled a recent surge in EV publications investigating their roles as biomarkers and mediators of disease. Meaningful scientific comparisons are, however, hampered by difficulties in accurate, reproducible enumeration and characterization of EVs in biological fluids. High-sensitivity flow cytometry (FCM) is presently the most commonly applied strategy to assess EVs, yet its utility is limited by variant ability to resolve smaller EVs. Here, we propose the use of 405 nm (violet) wavelength lasers in place of 488 nm (blue) for side scatter (SSC) detection to obtain greater resolution of EVs using high-sensitivity FCM. To test this hypothesis, we modelled EV resolution by violet versus blue SSC in silico and compared resolution of reference beads and biological EVs from plasma and bronchoalveolar lavage (BAL) fluid using either violet or blue wavelength SSC EV detection. Mie scatter modelling predicted that violet as compared to blue SSC increases resolution of small (100–500 nm) spherical particles with refractive indices (1.34–1.46) similar to EVs by approximately twofold in terms of light intensity and by nearly 20% in SSC signal quantum efficiency. Resolution of reference beads was improved by violet instead of blue SSC with two- and fivefold decreases in coefficients of variation for particles of 300–500 nm and 180–240 nm size, respectively. Resolution was similarly improved for detection of EVs from plasma or BAL fluid. Violet SSC detection for high-sensitivity FCM allows for significantly greater resolution of EVs in plasma and BAL compared to conventional blue SSC and particularly improves resolution of smaller EVs. Notably, the proposed strategy is readily implementable and inexpensive for machines already equipped with 405 nm SSC or the ability to accommodate 405/10 nm bandpass filters in their violet detector arrays.

Targeting Transfusion-Related Acute Lung Injury: The Journey From Basic Science to Novel Therapies

Objectives: Transfusion-related acute lung injury is characterized by the onset of respiratory distress and acute lung injury following blood transfusion, but its pathogenesis remains poorly understood. Generally, a two-hit model is presumed to underlie transfusion-related acute lung injury with the first hit being risk factors present in the transfused patient (such as inflammation), whereas the second hit is conveyed by factors in the transfused donor blood (such as antileukocyte antibodies). At least 80% of transfusion-related acute lung injury cases are related to the presence of donor antibodies such as antihuman leukocyte or antihuman neutrophil antibodies. The remaining cases may be related to nonantibody-mediated factors such as biolipids or components related to storage and ageing of the transfused blood cells. At present, transfusion-related acute lung injury is the leading cause of transfusion-related fatalities and no specific therapy is clinically available. In this article, we critically appraise and discuss recent preclinical (bench) insights related to transfusion-related acute lung injury pathogenesis and their therapeutic potential for future use at the patients’ bedside in order to combat this devastating and possibly fatal complication of transfusion. Data Sources: We searched the PubMed database (until August 22, 2017). Study Selection: Using terms: “Transfusion-related acute lung injury,” “TRALI,” “TRALI and therapy,” “TRALI pathogenesis.” Data Extraction: English-written articles focusing on transfusion-related acute lung injury pathogenesis, with potential therapeutic implications, were extracted. Data Synthesis: We have identified potential therapeutic approaches based on the literature. Conclusions: We propose that the most promising therapeutic strategies to explore are interleukin-10 therapy, down-modulating C-reactive protein levels, targeting reactive oxygen species, or blocking the interleukin-8 receptors; all focused on the transfused recipient. In the long-run, it may perhaps also be advantageous to explore other strategies aimed at the transfused recipient or aimed toward the blood product, but these will require more validation and confirmation first.

Gastrointestinal microbiota contributes to the development of murine transfusion-related acute lung injury

Transfusion-related acute lung injury (TRALI) is a syndrome of respiratory distress upon blood transfusion and is the leading cause of transfusion-related fatalities. Whether the gut microbiota plays any role in the development of TRALI is currently unknown. We observed that untreated barrier-free (BF) mice suffered from severe antibody-mediated acute lung injury, whereas the more sterile housed specific pathogen-free (SPF) mice and gut flora-depleted BF mice were both protected from lung injury. The prevention of TRALI in the SPF mice and gut flora-depleted BF mice was associated with decreased plasma macrophage inflammatory protein-2 levels as well as decreased pulmonary neutrophil accumulation. DNA sequencing of amplicons of the 16S ribosomal RNA gene revealed a varying gastrointestinal bacterial composition between BF and SPF mice. BF fecal matter transferred into SPF mice significantly restored TRALI susceptibility in SPF mice. These data reveal a link between the gut flora composition and the development of antibody-mediated TRALI in mice. Assessment of gut microbial composition may help in TRALI risk assessment before transfusion.

Mature murine megakaryocytes present antigen-MHC class I molecules to T cells and transfer them to platelets

Megakaryocytes (MKs) are bone marrow-derived cells that are primarily responsible for generating platelets for the maintenance of hemostasis. Although MK can variably express major histocompatibility complex (MHC) class I and II molecules during their differentiation, little is known whether they can elicit nonhemostatic immune functions such as T-cell activation. Here, we demonstrate that mature CD34- MHC class II- CD41+ MKs can endocytose exogenous ovalbumin (OVA) and proteolytically generate its immunogenic peptide ligand, which is crosspresented on their surface in association with MHC class I molecules. This crosspresentation triggered in vitro and in vivo OVA-specific CD8+ T-cell activation and proliferation. In addition, the OVA-MHC class I complexes were transferred from MK to pro-platelets upon thrombopoiesis in vitro. MK could also present endogenous MK-associated (CD61) peptides to activate CD61-specific CD8+ T cells and mediate immune thrombocytopenia in vivo. These results suggest that, in addition to their hemostatic role, mature MKs can significantly affect antigen-specific CD8+ T-cell responses via antigen presentation and are able to spread this immunogenic information through platelets.