G. Robin Barclay

Damaged intestinal epithelial integrity linked to microbial translocation in pathogenic simian immunodeficiency virus infections

The chronic phase of HIV infection is marked by pathological activation of the immune system, the extent of which better predicts disease progression than either plasma viral load or CD4+ T cell count. Recently, translocation of microbial products from the gastrointestinal tract has been proposed as an underlying cause of this immune activation, based on indirect evidence including the detection of microbial products and specific immune responses in the plasma of chronically HIV-infected humans or SIV-infected Asian macaques. We analyzed tissues from SIV-infected rhesus macaques (RMs) to provide direct in situ evidence for translocation of microbial constituents from the lumen of the intestine into the lamina propria and to draining and peripheral lymph nodes and liver, accompanied by local immune responses in affected tissues. In chronically SIV-infected RMs this translocation is associated with breakdown of the integrity of the epithelial barrier of the gastrointestinal (GI) tract and apparent inability of lamina propria macrophages to effectively phagocytose translocated microbial constituents. By contrast, in the chronic phase of SIV infection in sooty mangabeys, we found no evidence of epithelial barrier breakdown, no increased microbial translocation and no pathological immune activation. Because immune activation is characteristic of the chronic phase of progressive HIV/SIV infections, these findings suggest that increased microbial translocation from the GI tract, in excess of capacity to clear the translocated microbial constituents, helps drive pathological immune activation. Novel therapeutic approaches to inhibit microbial translocation and/or attenuate chronic immune activation in HIV-infected individuals may complement treatments aimed at direct suppression of viral replication.

Distribution of cell‐associated prion protein in normal adult blood determined by flow cytometry

Leucocyte subpopulations from normally healthy individuals were identified by recognized combinations of fluorochrome‐conjugated antibodies to CD markers and stained by different monoclonal antibodies (MAb) to normal cellular prion protein (PrPC), including the 3F4 MAb. Cell preparations were examined by three‐colour flow cytometry. All mononuclear leucocyte subpopulations and platelets expressed PrPC, but polymorphonuclear leucocytes and red blood cells expressed little or no PrPC. The amounts of PrPC expressed by the different cells were calculated by comparison to bead standards. Mononuclear leucocytes expressed 3000–4000 molecules of antibody‐reactive PrPC per cell. Resting platelets expressed around 1400 molecules of PrPC per cell, whereas activated platelets expressed around 4800 molecules of PrPC per cell. Extrapolation of these values to the amounts of the various cells in whole blood showed that platelet PrPC accounted for at least 96% of cell‐expressed PrPC in blood. The PrPC on mononuclear cells and platelets was sensitive to enzymatic treatment of cells by proteinase k and phosphatidylinositol‐specific phospholipase C. Certain anti‐PrPC MAbs which showed equivalent intensity of staining to MAb 3F4 on fresh cells showed relative reductions of staining compared to MAb 3F4 on stored cells, indicating possible structural alterations of PrPC under these conditions.

Late Outgrowth Endothelial Cells Resemble Mature Endothelial Cells and Are Not Derived from Bone Marrow

A decade of research has sought to identify circulating endothelial progenitor cells (EPC) in order to harness their potential for cardiovascular regeneration. Endothelial outgrowth cells (EOC) most closely fulfil the criteria for an EPC, but their origin remains obscure. Our aim was to identify the source and precursor of EOC and to assess their regenerative potential compared to mature endothelial cells. EOC are readily isolated from umbilical cord blood (6/6 donors) and peripheral blood mononuclear cells (4/6 donors) but not from bone marrow (0/6) or peripheral blood following mobilization with granulocyte‐colony stimulating factor (0/6 donors). Enrichment and depletion of blood mononuclear cells demonstrated that EOC are confined to the CD34+CD133−CD146+ cell fraction. EOC derived from blood mononuclear cells are indistinguishable from mature human umbilical vein endothelial cells (HUVEC) by morphology, surface antigen expression, immunohistochemistry, real‐time polymerase chain reaction, proliferation, and functional assessments. In a subcutaneous sponge model of angiogenesis, both EOC and HUVEC contribute to de novo blood vessel formation giving rise to a similar number of vessels (7.0 ± 2.7 vs. 6.6 ± 3.7 vessels, respectively, n = 9). Bone marrow‐derived outgrowth cells isolated under the same conditions expressed mesenchymal markers rather than endothelial cell markers and did not contribute to blood vessels in vivo. In this article, we confirm that EOC arise from CD34+CD133−CD146+ mononuclear cells and are similar, if not identical, to mature endothelial cells. Our findings suggest that EOC do not arise from bone marrow and challenge the concept of a bone marrow‐derived circulating precursor for endothelial cells. STEM CELLS2013;31:338–348

In Vivo Mononuclear Cell Tracking Using Superparamagnetic Particles of Iron Oxide Feasibility and Safety in Humans

Background— Cell therapy is an emerging and exciting novel treatment option for cardiovascular disease that relies on the delivery of functional cells to their target site. Monitoring and tracking cells to ensure tissue delivery and engraftment is a critical step in establishing clinical and therapeutic efficacy. The study aims were (1) to develop a Good Manufacturing Practice–compliant method of labeling competent peripheral blood mononuclear cells with superparamagnetic particles of iron oxide (SPIO), and (2) to evaluate its potential for magnetic resonance cell tracking in humans. Methods and Results— Peripheral blood mononuclear cells 1–5×109 were labeled with SPIO. SPIO-labeled cells had similar in vitro viability, migratory capacity, and pattern of cytokine release to unlabeled cells. After intramuscular administration, up to 108 SPIO-labeled cells were readily identifiable in vivo for at least 7 days using magnetic resonance imaging scanning. Using a phased-dosing study, we demonstrated that systemic delivery of up to 109 SPIO-labeled cells in humans is safe, and cells accumulating in the reticuloendothelial system were detectable on clinical magnetic resonance imaging. In a healthy volunteer model, a focus of cutaneous inflammation was induced in the thigh by intradermal injection of tuberculin. Intravenously delivered SPIO-labeled cells tracked to the inflamed skin and were detectable on magnetic resonance imaging. Prussian blue staining of skin biopsies confirmed iron-laden cells in the inflamed skin. Conclusions— Human peripheral blood mononuclear cells can be labeled with SPIO without affecting their viability or function. SPIO labeling for magnetic resonance cell tracking is a safe and feasible technique that has major potential for a range of cardiovascular applications including monitoring of cell therapies and tracking of inflammatory cells. Clinical Trial Registration— URL: http://www.clinicaltrials.gov; Unique identifier: NCT00972946, NCT01169935.

Preparation and Preclinical Evaluation of a Novel Liposomal Complete-Core Lipopolysaccharide Vaccine

ABSTRACT Our objective is to develop a prophylactic vaccine strategy that can be evaluated for surgical and other high-risk hospitalized patients. In this paper, we describe the preparation and preclinical evaluation of a liposomal complete-core lipopolysaccharide (LPS) vaccine that is nontoxic and broadly antigenic. Complete-core (Ra-chemotype) LPSs were isolated from four gram-negative bacterial strains (Escherichia coli K-12, E. coli R1,Pseudomonas aeruginosa PAC608, and Bacteroides fragilis), mixed together to form a cocktail of complete-core LPSs, and then incorporated into multilamellar liposomes consisting of dimyristoyl phosphatidyl choline, dimyristoyl phosphatidylglycerol, and cholesterol in a 4:1:4 molar ratio. The endotoxic activities of these LPS-containing liposomes were less than 0.1% of the endotoxicities of the original free LPSs as measured by the Limulusamoebocyte lysate assay. In vivo administration of liposomal complete-core LPS mixed with Al(OH)3 to rabbits resulted in no pyrogenicity or overt toxicity over a 7-day period. In immunoblots, sera from rabbits following active immunization elicited cross-reactive antibodies to a large panel of rough and smooth LPSs from numerous clinically relevant gram-negative bacteria, including E. coli (serotypes O1, O4, O6, O8, O12, O15, O18, O75, O86, O157, and O111), P. aeruginosa (Fisher-Devlin serotypes 1, 2, and 3, which correspond to International Antigenic Typing Scheme types 6, 11, and 2, respectively), Klebsiella pneumoniae (serotypes O1, O2ab, and O3), B. fragilis, and Bacteroides vulgatus. Active immunization of mice with liposomal complete-core LPS provided protection against a lethal challenge withE. coli O18 LPS. The vaccine tested was nontoxic, nonpyrogenic, and immunogenic against a wide variety of pathogens found in clinical settings.

Decreased Endotoxin Immunity Is Associated with Greater Mortality and/or Prolonged Hospitalization after Surgery

Background Patients undergoing noncardiac surgery often develop postoperative morbidity, potentially attributable to endotoxemia and the systemic inflammatory response syndrome. Endogenous antibodies to endotoxin may confer protection from endotoxin-mediated toxicity. The authors sought to determine the association of preoperative antiendotoxin immunity and death or prolonged hospitalization in a broad population of general surgical patients undergoing major surgery. Methods To test the hypothesis that low preoperative serum antiendotoxin core antibody (EndoCAb) concentration is an independent predictor of adverse outcome after general surgery, 1,056 patients undergoing routine noncardiac surgery were enrolled into a prospective, blinded, cohort study. Immunoglobulin M EndoCAb, immunoglobulin G EndoCAb, total immunoglobulin M, and immunoglobulin G concentrations were measured in serum obtained preoperatively. A physiologic risk score using the established POSSUM criteria was assigned preoperatively to each patient. The primary predefined composite end point (postoperative complication) was either in-hospital death or postoperative length of stay greater than 10 days. Multivariate logistic regression was used to test the study hypothesis. Results Overall, postoperative complication occurred in 234 of the 1,056 patients (22.1%). Lower immunoglobulin M EndoCAb concentration (P = 0.006) predicted increased risk of postoperative complication independent of POSSUM physiologic risk score (P < 0.001). In contrast, total immunoglobulin M and total immunoglobulin G concentrations did not predict adverse outcome. Complications involved multiple organ systems and were generally unrelated to the type or site of surgery, consistent with the systemic inflammatory response syndrome. Conclusions Adverse outcome after routine noncardiac surgery is common and is predicted in part by low concentrations of EndoCAb. The authors’ findings suggest that endotoxemia may be a cause of postoperative morbidity after routine noncardiac surgery.

Circulating endothelial progenitor cells are not affected by acute systemic inflammation

Vascular injury causes acute systemic inflammation and mobilizes endothelial progenitor cells (EPCs) and endothelial cell (EC) colony-forming units (EC-CFUs). Whether such mobilization occurs as part of a nonspecific acute phase response or is a phenomenon specific to vascular injury remains unclear. We aimed to determine the effect of acute systemic inflammation on EPCs and EC-CFU mobilization in the absence of vascular injury. Salmonella typhus vaccination was used as a model of acute systemic inflammation. In a double-blind randomized crossover study, 12 healthy volunteers received S. typhus vaccination or placebo. Phenotypic EPC populations enumerated by flow cytometry [CD34(+)VEGF receptor (VEGF)R-2(+)CD133(+), CD14(+)VEGFR-2(+)Tie2(+), CD45(-)CD34(+), as a surrogate for late outgrowth EPCs, and CD34(+)CXCR-4(+)], EC-CFUs, and serum cytokine concentrations (high sensitivity C-reactive protein, IL-6, and stromal-derived factor-1) were quantified during the first 7 days. Vaccination increased circulating leukocyte (9.8 + or - 0.6 vs. 5.1 + or - 0.2 x 10(9) cells/l, P < 0.0001), serum IL-6 [0.95 (0-1.7) vs. 0 (0-0) ng/l, P = 0.016], and VEGF-A [60 (45-94) vs. 43 (21-64) pg/l, P = 0.006] concentrations at 6 h and serum high sensitivity C-reactive protein at 24 h [2.7 (1.4-3.6) vs. 0.4 (0.2-0.8) mg/l, P = 0.037]. Vaccination caused a 56.7 + or - 7.6% increase in CD14(+) cells at 6 h (P < 0.001) and a 22.4 + or - 6.9% increase in CD34(+) cells at 7 days (P = 0.04). EC-CFUs, putative vascular progenitors, and the serum stromal-derived factor-1 concentration were unaffected throughout the study period (P > 0.05 for all). In conclusion, acute systemic inflammation causes nonspecific mobilization of hematopoietic progenitor cells, although it does not selectively mobilize putative vascular progenitors. We suggest that systemic inflammation is not the primary stimulus for EPC mobilization after acute vascular injury.

Comparative analysis of normal prion protein expression on human, rodent, and ruminant blood cells by using a panel of prion antibodies.

BACKGROUND: It is not known whether variant CJD can be transmitted within the human population by blood transfusion. The expression of normal cellular prion protein (PrPC) by different blood cell types may permit selective uptake and dissemination of infectivity.

Systematic assessment in an animal model of the angiogenic potential of different human cell sources for therapeutic revascularization

IntroductionEndothelial progenitor cells (EPC) capable of initiating or augmenting vascular growth were recently identified within the small population of CD34-expressing cells that circulate in human peripheral blood and which are considered hematopoietic progenitor cells (HPC). Soon thereafter human HPC began to be used in clinical trials as putative sources of EPC for therapeutic vascular regeneration, especially in myocardial and critical limb ischemias. However, unlike HPC where hematopoietic efficacy is related quantitatively to CD34+ cell numbers implanted, there has been no consensus on how to measure EPC or how to assess cellular graft potency for vascular regeneration. We employed an animal model of spontaneous neovascularization to simultaneously determine whether human cells incorporate into new vessels and to quantify the effect of different putative angiogenic cells on vascularization in terms of number of vessels generated. We systematically compared competence for therapeutic angiogenesis in different sources of human cells with putative angiogenic potential, to begin to provide some rationale for optimising cell procurement for this therapy.MethodsHuman cells employed were mononuclear cells from normal peripheral blood and HPC-rich cell sources (umbilical cord blood, mobilized peripheral blood, bone marrow), CD34+ enriched or depleted subsets of these, and outgrowth cell populations from these. An established sponge implant angiogenesis model was adapted to determine the effects of different human cells on vascularization of implants in immunodeficient mice. Angiogenesis was quantified by vessel density and species of origin by immunohistochemistry.ResultsCD34+ cells from mobilized peripheral blood or umbilical cord blood HPC were the only cells to promote new vessel growth, but did not incorporate into vessels. Only endothelial outgrowth cells (EOC) incorporated into vessels, but these did not promote vessel growth.ConclusionsThese studies indicate that, since EPC are very rare, any benefit seen in clinical trials of HPC in therapeutic vascular regeneration is predominantly mediated by indirect proangiogenic effects rather than through direct incorporation of any rare EPC contained within these sources. It should be possible to produce autologous EOC for therapeutic use, and evaluate the effect of EPC distinct from, or in synergy with, the proangiogenic effects of HPC therapies.

Absence of a relationship between immunophenotypic and colony enumeration analysis of endothelial progenitor cells in clinical haematopoietic cell sources

BackgroundThe discovery of adult endothelial progenitor cells (EPC) offers potential for vascular regenerative therapies. The expression of CD34 and VEGFR2 by EPC indicates a close relationship with haematopoietic progenitor cells (HPC), and HPC-rich sources have been used to treat cardiac and limb ischaemias with apparent clinical benefit. However, the laboratory characterisation of the vasculogenic capability of potential or actual therapeutic cell autograft sources is uncertain since the description of EPC remains elusive. Various definitions of EPC based on phenotype and more recently on colony formation (CFU-EPC) have been proposed.MethodsWe determined EPC as defined by proposed phenotype definitions (flow cytometry) and by CFU-EPC in HPC-rich sources: bone marrow (BM); cord blood (CB); and G-CSF-mobilised peripheral blood (mPB), and in HPC-poor normal peripheral blood (nPB).ResultsAs expected, the highest numbers of cells expressing the HPC markers CD34 or CD133 were found in mPB and least in nPB. The proportions of CD34+ cells co-expressing CD133 is of the order mPB>CB>BM≈nPB. CD34+ cells co-expressing VEGFR2 were also most frequent in mPB. In contrast, CFU-EPC were virtually absent in mPB and were most readily detected in nPB, the source lowest in HPC.ConclusionHPC sources differ in their content of putative EPC. Normal peripheral blood, poor in HPC and in HPC-related phenotypically defined EPC, is the richest source of CFU-EPC, suggesting no direct relationship between the proposed EPC immunophenotypes and CFU-EPC potential. It is not apparent whether either of these EPC measurements, or any, is an appropriate indicator of the therapeutic vasculogenic potential of autologous HSC sources.