Induction of human immunodeficiency virus (HIV-1) envelope specific cell-mediated immunity by a non-homologous synthetic peptide

Background

Cell mediated immunity, including efficient CTL response, is required to prevent HIV-1 from cell-to-cell transmission. In previous investigations, we have shown that B1 peptide derived by Fourier transformation of HIV-1 primary structures and sharing no sequence homology with the parent proteins was able to generate antiserum which recognizes envelope and Tat proteins. Here we have investigated cellular immune response towards a novel non-homologous peptide, referred to as cA1 peptide.

Methodology/Principal Findings

The 20 amino acid sequence of cA1 peptide was predicted using the notion of peptide hydropathic properties; the peptide is encoded by the complementary anti-sense DNA strand to the sense strand of previously described non-homologous A1 peptide. In this report we demonstrate that the cA1 peptide can be a target for major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes in HIV-1-infected or envelope-immunized individuals. The cA1 peptide is recognized in association with different MHC class I allotypes and could prime in vitro CTLs, derived from gp160-immunized individuals capable to recognize virus variants.

Conclusions/Significance

For the first time a theoretically designed immunogen involved in broad-based cell-immune memory activation is described. Our findings may thus contribute to the advance in vaccine research by describing a novel strategy to develop a synthetic AIDS vaccine.     

Effect of Maraviroc Intensification on HIV-1-Specific T Cell Immunity in Recently HIV-1-Infected Individuals

Background

The effect of maraviroc on the maintenance and the function of HIV-1-specific T cell responses remains unknown.

Methods

Subjects recently infected with HIV-1 were randomized to receive anti-retroviral treatment with or without maraviroc intensification for 48 weeks, and were monitored up to week 60. PBMC and in vitro-expanded T cells were tested for responses to the entire HIV proteome by ELISpot analyses. Intracellular cytokine staining assays were conducted to monitor the (poly)-functionality of HIV-1-specific T cells. Analyses were performed at baseline and week 24 after treatment start, and at week 60 (3 months after maraviroc discontinuation).

Results

Maraviroc intensification was associated with a slower decay of virus-specific T cell responses over time compared to the non-intensified regimen in both direct ex-vivo as well as in in-vitro expanded cells. The effector function profiles of virus-specific CD8⁺ T cells were indistinguishable between the two arms and did not change over time between the groups.

Conclusions

Maraviroc did not negatively impact any of the measured parameters, but was rather associated with a prolonged maintenance of HIV-1-specific T cell responses. Maraviroc, in addition to its original effect as viral entry inhibitor, may provide an additional benefit on the maintenance of virus-specific T cells which may be especially important for future viral eradication strategies.     

The RNA editing enzyme APOBEC1 induces somatic mutations and a compatible mutational signature is present in esophageal adenocarcinomas

Background

The AID/APOBECs are deaminases that act on cytosines in a diverse set of pathways and some of them have been linked to the onset of genetic alterations in cancer. Among them, APOBEC1 is the only family member to physiologically target RNA, as the catalytic subunit in the Apolipoprotein B mRNA editing complex. APOBEC1 has been linked to cancer development in mice but its oncogenic mechanisms are not yet well understood.

Results

We analyze whether expression of APOBEC1 induces a mutator phenotype in vertebrate cells, likely through direct targeting of genomic DNA. We show its ability to increase the inactivation of a stably inserted reporter gene in a chicken cell line that lacks any other AID/APOBEC proteins, and to increase the number of imatinib-resistant clones in a human cellular model for chronic myeloid leukemia through induction of mutations in the BCR-ABL1 fusion gene. Moreover, we find the presence of an AID/APOBEC mutational signature in esophageal adenocarcinomas, a type of tumor where APOBEC1 is expressed, that mimics the one preferred by APOBEC1 in vitro.

Conclusions

Our findings suggest that the ability of APOBEC1 to trigger genetic alterations represents a major layer in its oncogenic potential. Such APOBEC1-induced mutator phenotypes could play a role in the onset of esophageal adenocarcinomas. APOBEC1 could be involved in cancer promotion at the very early stages of carcinogenesis, as it is highly expressed in Barrett''s esophagus, a condition often associated with esophageal adenocarcinoma.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0417-z) contains supplementary material, which is available to authorized users.     

Antigen-Presenting Cells Represent Targets for R5 HIV-1 Infection in the First Trimester Pregnancy Uterine Mucosa

Background

During the first trimester of pregnancy, HIV-1 mother-to-child transmission is relatively rare despite the permissivity of placental cells to cell-to-cell HIV-1 infection. The placenta interacts directly with maternal uterine cells (decidual cells) but the physiological role of the decidua in the control of HIV-1 transmission and whether decidua could be a source of infected cells is unknown.

Methodology/Principal Findings

To answer to this question, decidual mononuclear cells were exposed to HIV-1 in vitro. Decidual cells were shown to be more susceptible to infection by an R5 HIV-1, as compared to an X4 HIV-1. Infected cells were identified by flow cytometry analysis. The results showed that CD14⁺ cells were the main targets of HIV-1 infection in the decidua. These infected CD14⁺ cells expressed DC-SIGN, CD11b, CD11c, the Fc gamma receptor CD16, CD32 and CD64, classical MHC class-I and class-II and maturation and activation molecules CD83, CD80 and CD86. The permissivity of decidual tissue was also evaluated by histoculture. Decidual tissue was not infected by X4 HIV-1 but was permissive to R5 HIV-1. Different profiles of infection were observed depending on tissue localization.

Conclusions/Significance

The presence of HIV-1 target cells in the decidua in vitro and the low rate of in utero mother-to-child transmission during the first trimester of pregnancy suggest that a natural control occurs in vivo limiting cell-to-cell infection of the placenta and consequently infection of the fetus.     

Lysine residue at position 22 of the AID protein regulates its class switch activity

Background

Activation induced deaminase (AID) mediates class switch recombination and somatic hypermutation of immunoglobulin (Ig) genes in germinal centre B cells. In order to regulate its specific activity and as a means to keep off-target mutations low, several mechanisms have evolved, including binding to specific cofactors, phosphorylation and destabilization of nuclear AID protein. Although ubiquitination at lysine residues of AID is recognized as an essential step in initiating degradation of nuclear AID, any functional relevance of lysine modifications has remained elusive.

Methodology/Principal Findings

Here, we report functional implications of lysine modifications of the human AID protein by generating a panel of lysine to arginine mutants of AID and assessment of their catalytic class switch activity. We found that only mutation of Lys22 to Arg resulted in a significant reduction of class switching to IgG1 in transfected primary mouse B cells. This decrease in activity was neither reflected in reduced hypermutation of Ig genes in AID-mutant transfected DT40 B cell lines nor recapitulated in bacterial deamination assays, pointing to involvement of post-translational modification of Lys22 for AID activity in B cells.

Conclusions/Significance

Our results imply that lysine modification may represent a novel level of AID regulation and that Lys22 is important for effective AID activity.     

Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells

Background

Fatal human respiratory disease associated with influenza A subtype H5N1 has been documented in Hong Kong, and more recently in Vietnam, Thailand and Cambodia. We previously demonstrated that patients with H5N1 disease had unusually high serum levels of IP-10 (interferon-gamma-inducible protein-10). Furthermore, when compared with human influenza virus subtype H1N1, the H5N1 viruses in 1997 (A/Hong Kong/483/97) (H5N1/97) were more potent inducers of pro-inflammatory cytokines (e.g. tumor necrosis factor-a) and chemokines (e.g. IP-10) from primary human macrophages in vitro, which suggests that cytokines dysregulation may play a role in pathogenesis of H5N1 disease. Since respiratory epithelial cells are the primary target cell for replication of influenza viruses, it is pertinent to investigate the cytokine induction profile of H5N1 viruses in these cells.

Methods

We used quantitative RT-PCR and ELISA to compare the profile of cytokine and chemokine gene expression induced by H5N1 viruses A/HK/483/97 (H5N1/97), A/Vietnam/1194/04 and A/Vietnam/3046/04 (both H5N1/04) with that of human H1N1 virus in human primary alveolar and bronchial epithelial cells in vitro.

Results

We demonstrated that in comparison to human H1N1 viruses, H5N1/97 and H5N1/04 viruses were more potent inducers of IP-10, interferon beta, RANTES (regulated on activation, normal T cell expressed and secreted) and interleukin 6 (IL-6) in primary human alveolar and bronchial epithelial cells in vitro. Recent H5N1 viruses from Vietnam (H5N1/04) appeared to be even more potent at inducing IP-10 than H5N1/97 virus.

Conclusion

The H5N1/97 and H5N1/04 subtype influenza A viruses are more potent inducers of proinflammatory cytokines and chemokines in primary human respiratory epithelial cells than subtype H1N1 virus. We suggest that this hyper-induction of cytokines may be relevant to the pathogenesis of human H5N1 disease.     

Two Forms of Activation-Induced Cytidine Deaminase Differing in Their Ability to Bind Agarose

Background

Activation-induced cytidine deaminase (AID) is a B-cell-specific DNA mutator that plays a key role in the formation of the secondary antibody repertoire in germinal center B cells. In the search for binding partners, protein coimmunoprecipitation assays are often performed, generally with agarose beads.

Methodology/Principal Findings

We found that, regardless of whether cell lysates containing exogenous or endogenous AID were examined, one of two mouse AID forms bound to agarose alone.

Conclusions/Significance

These binding characteristics may be due to the known post-translational modifications of AID; they may also need to be considered in coimmunoprecipitation experiments to avoid false-positive results.     

X4 Human immunodeficiency virus type 1 gp120 promotes human hepatic stellate cell activation and collagen I expression through interactions with CXCR4

Background & Aims

Patients coinfected with HIV-1 and HCV develop more rapid liver fibrosis than patients monoinfected with HCV. HIV RNA levels correlate with fibrosis progression implicating HIV directly in the fibrotic process. While activated hepatic stellate cells (HSCs) express the 2 major HIV chemokine coreceptors, CXCR4 and CCR5, little is known about the pro-fibrogenic effects of the HIV-1 envelope protein, gp120, on HSCs. We therefore examined the in vitro impact of X4 gp120 on HSC activation, collagen I expression, and underlying signaling pathways and examined the in vivo expression of gp120 in HIV/HCV coinfected livers.

Methods

Primary human HSCs and LX-2 cells, a human HSC line, were challenged with X4 gp120 and expression of fibrogenic markers assessed by qRT-PCR and Western blot +/− either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Downstream intracellular signaling pathways were evaluated with Western blot and pre-treatment with specific pathway inhibitors. Gp120 immunostaining was performed on HIV/HCV coinfected liver biopsies.

Results

X4 gp 120 significantly increased expression of alpha-smooth muscle actin (a-SMA) and collagen I in HSCs which was blocked by pre-incubation with either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Furthermore, X4 gp120 promoted Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and pretreatment with an ERK inhibitor attenuated HSC activation and collagen I expression. Sinusoidal staining for gp120 was evident in HIV/HCV coinfected livers.

Conclusions

X4 HIV-1 gp120 is pro-fibrogenic through its interactions with CXCR4 on activated HSCs. The availability of small molecule inhibitors to CXCR4 make this a potential anti-fibrotic target in HIV/HCV coinfected patients.     

Polyclonal B Cell Differentiation and Loss of Gastrointestinal Tract Germinal Centers in the Earliest Stages of HIV-1 Infection

Background

The antibody response to HIV-1 does not appear in the plasma until approximately 2–5 weeks after transmission, and neutralizing antibodies to autologous HIV-1 generally do not become detectable until 12 weeks or more after transmission. Moreover, levels of HIV-1–specific antibodies decline on antiretroviral treatment. The mechanisms of this delay in the appearance of anti-HIV-1 antibodies and of their subsequent rapid decline are not known. While the effect of HIV-1 on depletion of gut CD4⁺ T cells in acute HIV-1 infection is well described, we studied blood and tissue B cells soon after infection to determine the effect of early HIV-1 on these cells.

Methods and Findings

In human participants, we analyzed B cells in blood as early as 17 days after HIV-1 infection, and in terminal ileum inductive and effector microenvironments beginning at 47 days after infection. We found that HIV-1 infection rapidly induced polyclonal activation and terminal differentiation of B cells in blood and in gut-associated lymphoid tissue (GALT) B cells. The specificities of antibodies produced by GALT memory B cells in acute HIV-1 infection (AHI) included not only HIV-1–specific antibodies, but also influenza-specific and autoreactive antibodies, indicating very early onset of HIV-1–induced polyclonal B cell activation. Follicular damage or germinal center loss in terminal ileum Peyer''s patches was seen with 88% of follicles exhibiting B or T cell apoptosis and follicular lysis.

Conclusions

Early induction of polyclonal B cell differentiation, coupled with follicular damage and germinal center loss soon after HIV-1 infection, may explain both the high rate of decline in HIV-1–induced antibody responses and the delay in plasma antibody responses to HIV-1. Please see later in the article for Editors'' Summary     

In-depth characterization of viral isolates from plasma and cells compared with plasma circulating quasispecies in early HIV-1 infection

Background

The use of in vitro models to unravel the phenotypic characteristics of circulating viral variants is key to understanding HIV-1 pathogenesis but limited by the availability of primary viral isolates from biological samples. However, overall in vivo genetic variability of HIV-1 within a subject may not be reflected in the viable viral population obtained after isolation. Although several studies have tried to determine whether viral populations expanded in vitro are representative of in vivo findings, the answer remains unclear due to the reduced number of clonal sequences analyzed or samples compared. In order to overcome previous experimental limitations, here we applied Deep Pyrosequencing (DPS) technology in combination with phenotypic experiments to analyze and compare with unprecedented detail the composition of viral isolates and in vivo quasispecies.

Methodology/Principal Findings

We amplified by DPS HIV-1 genomic regions covering gag, protease, integrase and env-V3 to characterize paired isolates from plasma and peripheral blood mononuclear cells and compare them with total plasma viral RNA in four recently HIV-1 infected subjects. Our study demonstrated the presence of unique haplotypes scattered between sample types with conservation of major variants. In addition, no differences in intra- and inter-population encoded protein variability were found between the different types of isolates or when these were compared to plasma viral RNA within subjects. Additionally, in vitro experiments demonstrated phenotypic similarities in terms of replicative capacity and co-receptor usage between viral isolates and plasma viral RNA.

Conclusion

This study is the first in-depth comparison and characterization of viral isolates from different sources and plasma circulating quasispecies using DPS in recently HIV-1 infected subjects. Our data supports the use of primary isolates regardless of their plasma or cellular origin to define genetic variability and biological traits of circulating HIV-1 quasispecies.     

In Vitro and Ex Vivo Testing of Tenofovir Shows It Is Effective As an HIV-1 Microbicide

Background

Tenofovir gel has entered into clinical trials for use as a topical microbicide to prevent HIV-1 infection but has no published data regarding pre-clinical testing using in vitro and ex vivo models. To validate our findings with on-going clinical trial results, we evaluated topical tenofovir gel for safety and efficacy. We also modeled systemic application of tenofovir for efficacy.

Methods and Findings

Formulation assessment of tenofovir gel included osmolality, viscosity, in vitro release, and permeability testing. Safety was evaluated by measuring the effect on the viability of vaginal flora, PBMCs, epithelial cells, and ectocervical and colorectal explant tissues. For efficacy testing, PBMCs were cultured with tenofovir or vehicle control gels and HIV-1 representing subtypes A, B, and C. Additionally, polarized ectocervical and colorectal explant cultures were treated apically with either gel. Tenofovir was added basolaterally to simulate systemic application. All tissues were challenged with HIV-1 applied apically. Infection was assessed by measuring p24 by ELISA on collected supernatants and immunohistochemistry for ectocervical explants. Formulation testing showed the tenofovir and vehicle control gels were >10 times isosmolar. Permeability through ectocervical tissue was variable but in all cases the receptor compartment drug concentration reached levels that inhibit HIV-1 infection in vitro. The gels were non-toxic toward vaginal flora, PBMCs, or epithelial cells. A transient reduction in epithelial monolayer integrity and epithelial fracture for ectocervical and colorectal explants was noted and likely due to the hyperosmolar nature of the formulation. Tenofovir gel prevented HIV-1 infection of PBMCs regardless of HIV-1 subtype. Topical and systemic tenofovir were effective at preventing HIV-1 infection of explant cultures.

Conclusions

These studies provide a mechanism for pre-clinical prediction of safety and efficacy of formulated microbicides. Tenofovir was effective against HIV-1 infection in our algorithm. These data support the use of tenofovir for pre-exposure prophylaxis.     

Antiproliferative and apoptosis-inducing activity of schisandrin B against human glioma cells

Background

Malignant glioma is the most devastating and aggressive tumour in the brain and is characterised by high morbidity, high mortality and extremely poor prognosis. The main purpose of the present study was to investigate the effects of schisandrin B (Sch B) on glioma cells both in vitro and in vivo and to explore the possible anticancer mechanism underlying Sch B-induced apoptosis and cell cycle arrest.

Methods

The anti-proliferative ability of Sch B on glioma cells were assessed by MTT and clony formation assays. Flow cytometric analysis was used to detect cell cycle changes. Apoptosis was determined by Hoechst 33342 staining and annexin V/PI double-staining assays. The mitochondrial membrane potential was detected by Rhodamine 123 staining. The in vivo efficacy of Sch B was measured using a U87 xenograft model in nude mice. The expressions of the apoptosis-related and cell cycle-related proteins were analysed by western blot. Student’s t-test was used to compare differences between treated groups and their controls.

Results

We found that Sch B inhibited growth in a dose- and time-dependent manner as assessed by MTT assay. In U87 and U251 cells, the number of clones was strongly suppressed by Sch B. Flow cytometric analysis revealed that Sch B induced cell cycle arrest in glioma cells at the G0/G1 phase. In addition, Sch B induced glioma cell apoptosis and reduced mitochondrial membrane potential (ΔΨm) in a dose-dependent manner. Mechanically, western blot analysis indicated that Sch B induced apoptosis by caspase-3, caspase-9, PARP, and Bcl-2 activation. Moreover, Sch B significantly inhibited tumour growth in vivo following the subcutaneous inoculation of U87 cells in athymic nude mice.

Coclusions

In summary, Sch B can reduce cell proliferation and induce apoptosis in glioma cells and has potential as a novel anti-tumour therapy to treat gliomas.     

A Common Carcinogen Benzo[a]pyrene Causes Neuronal Death in Mouse via Microglial Activation

Background

Benzo[a]pyrene (B[a]P) belongs to a class of polycyclic aromatic hydrocarbons that serve as micropollutants in the environment. B[a]P has been reported as a probable carcinogen in humans. Exposure to B[a]P can take place by ingestion of contaminated (especially grilled, roasted or smoked) food or water, or inhalation of polluted air. There are reports available that also suggests neurotoxicity as a result of B[a]P exposure, but the exact mechanism of action is unknown.

Methodology/Principal Findings

Using neuroblastoma cell line and primary cortical neuron culture, we demonstrated that B[a]P has no direct neurotoxic effect. We utilized both in vivo and in vitro systems to demonstrate that B[a]P causes microglial activation. Using microglial cell line and primary microglial culture, we showed for the first time that B[a]P administration results in elevation of reactive oxygen species within the microglia thereby causing depression of antioxidant protein levels; enhanced expression of inducible nitric oxide synthase, that results in increased production of NO from the cells. Synthesis and secretion of proinflammatory cytokines were also elevated within the microglia, possibly via the p38MAP kinase pathway. All these factors contributed to bystander death of neurons, in vitro. When administered to animals, B[a]P was found to cause microglial activation and astrogliosis in the brain with subsequent increase in proinflammatory cytokine levels.

Conclusions/Significance

Contrary to earlier published reports we found that B[a]P has no direct neurotoxic activity. However, it kills neurons in a bystander mechanism by activating the immune cells of the brain viz the microglia. For the first time, we have provided conclusive evidence regarding the mechanism by which the micropollutant B[a]P may actually cause damage to the central nervous system. In today''s perspective, where rising pollution levels globally are a matter of grave concern, our study throws light on other health hazards that such pollutants may exert.     

Lineage tracing of cardiac explant derived cells

Aims

Cultured cardiac explants produce a heterogeneous population of cells including a distinctive population of refractile cells described here as small round cardiac explant derived cells (EDCs). The aim of this study was to explore the source, morphology and cardiogenic potential of EDCs.

Methods

Transgenic MLC2v-Cre/ZEG, and actin-eGFP mice were used for lineage-tracing of EDCs in vitro and in vivo. C57B16 mice were used as cell transplant recipients of EDCs from transgenic hearts, as well as for the general characterisation of EDCs. The activation of cardiac-specific markers were analysed by: immunohistochemistry with bright field and immunofluorescent microscopy, electron microscopy, PCR and RT-PCR. Functional engraftment of transplanted cells was further investigated with calcium transient studies.

Results

Production of EDCs was highly dependent on the retention of blood-derived cells or factors in the cultured explants. These cells shared some characteristics of cardiac myocytes in vitro and survived engraftment in the adult heart in vivo. However, EDCs failed to differentiate into functional cardiac myocytes in vivo as demonstrated by the absence of stimulation-evoked intracellular calcium transients following transplantation into the peri-infarct zone.

Conclusions

This study highlights that positive identification based upon one parameter alone such as morphology or immunofluorescene is not adequate to identify the source, fate and function of adult cardiac explant derived cells.     

Icariin Ameliorates Cigarette Smoke Induced Inflammatory Responses via Suppression of NF-κB and Modulation of GR In Vivo and In Vitro

Purpose

To investigate the effects of icariin, a major constituent of flavonoids isolated from the herb Epimedium, on cigarette smoke (CS) induced inflammatory responses in vivo and in vitro.

Methods

In vivo, BALB/c mice were exposed to smoke of 15 cigarettes for 1 h/day, 6 days/week for 3 months and dosed with icariin (25, 50 and 100 mg/kg) or dexamethasone (1 mg/kg). In vitro, A549 cells were incubated with icariin (10, 50 and 100 µM) followed by treatments with CSE (2.5%).

Results

We found that icariin significantly protected pulmonary function and attenuated CS-induced inflammatory response by decreasing inflammatory cells and production of TNF-α, IL-8 and MMP-9 in both the serum and BALF of CS-exposed mice and decreasing production of TNF-α and IL-8 in the supernatant of CSE-exposed A549 cells. Icariin also showed properties in inhibiting the phosphorylation of NF-κB p65 protein and blocking the degradation of IΚB-α protein. Further studies revealed that icariin administration markedly restore CS-reduced GR protein and mRNA expression, which might subsequently contribute to the attenuation of CS-induced respiratory inflammatory response.

Conclusion

Together these results suggest that icariin has anti-inflammatory effects in cigarette smoke induced inflammatory models in vivo and in vitro, possibly achieved by suppressing NF-κB activation and modulating GR protein expression.     

Evidence of at least two introductions of HIV-1 in the Amerindian Warao population from Venezuela

Background

The Venezuelan Amerindians were, until recently, free of human immunodeficiency virus (HIV) infection. However, in 2007, HIV-1 infection was detected for the first time in the Warao Amerindian population living in the Eastern part of Venezuela, in the delta of the Orinoco river. The aim of this study was to analyze the genetic diversity of the HIV-1 circulating in this population.

Methodology/Principal Findings

The pol genomic region was sequenced for 16 HIV-1 isolates and for some of them, sequences from env, vif and nef genomic regions were obtained. All HIV-1 isolates were classified as subtype B, with exception of one that was classified as subtype C. The 15 subtype B isolates exhibited a high degree of genetic similarity and formed a highly supported monophyletic cluster in each genomic region analyzed. Evolutionary analyses of the pol genomic region indicated that the date of the most recent common ancestor of the Waraos subtype B clade dates back to the late 1990s.

Conclusions/Significance

At least two independent introductions of HIV-1 have occurred in the Warao Amerindians from Venezuela. The HIV-1 subtype B was successfully established and got disseminated in the community, while no evidence of local dissemination of the HIV-1 subtype C was detected in this study. These results warrant further surveys to evaluate the burden of this disease, which can be particularly devastating in this Amerindian population, with a high prevalence of tuberculosis, hepatitis B, among other infectious diseases, and with limited access to primary health care.     

A biomimetic membrane device that modulates the excessive inflammatory response to sepsis

Objective

Septic shock has a clinical mortality rate approaching fifty percent. The major clinical manifestations of sepsis are due to the dysregulation of the host''s response to infection rather than the direct consequences of the invading pathogen. Central to this initial immunologic response is the activation of leukocytes and microvascular endothelium resulting in cardiovascular instability, lung injury and renal dysfunction. Due to the primary role of leukocyte activation in the sepsis syndrome, a synthetic biomimetic membrane, called a selective cytopheretic device (SCD), was developed to bind activated leukocytes. The incorporation of the SCD along an extracorporeal blood circuit coupled with regional anticoagulation with citrate to lower blood ionized calcium was devised to modulate leukocyte activation in sepsis.

Design

Laboratory investigation.

Setting

University of Michigan Medical School.

Subjects

Pigs weighing 30-35 kg.

Interventions

To assess the effect of the SCD in septic shock, pigs were administered 30×10¹⁰ bacteria/kg body weight of Escherichia coli into the peritoneal cavity and within 1 hr were immediately placed in an extracorporeal circuit containing SCD.

Measurements and Main Results

In this animal model, the SCD with citrate compared to control groups without the SCD or with heparin anticoagulation ameliorated the cardiovascular instability and lung sequestration of activated leukocytes, reduced renal dysfunction and improved survival time compared to various control groups. This effect was associated with minimal elevations of systemic circulating neutrophil activation.

Conclusions

These preclinical studies along with two favorable exploratory clinical trials form the basis of an FDA-approved investigational device exemption for a pivotal multicenter, randomized control trial currently underway.