Bacterial survival in whole blood depends on plasma sensitivity and resistance to neutrophil killing.

BACKGROUND: Whole blood (WB) is held at room temperature for not more than 24 hours before blood component manufacturing. The ability of several culture collection, skin-derived, and transfusion-related bacteria to survive in WB stored at 22 ± 2°C for 24 hours was investigated in this study. STUDY DESIGN AND METHODS: Twenty-one bacteria of the species Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus capitis, Streptococcus agalactiae, Serratia liquefaciens, Serratia marcescens, Klebsiella pneumoniae, Escherichia coli, and Yersinia enterocolitica were inoculated into 7-mL aliquots of WB at a concentration of 500 colony-forming units (CFU)/mL. Spiked WB was stored aerobically at 22 ± 2°C, and bacterial viability and growth were monitored at 3, 8, and 24 hours during WB storage. Bacteria that showed decreased viability during WB incubation were further characterized for their sensitivity to plasma factors and neutrophil killing. RESULTS: There were three different scenarios for bacterial behavior during the hold of WB at 22 ± 2°C. Five bacteria proliferated (p < 0.03), 11 remained viable or showed low proliferation, and a third group of five bacteria had decreased or lost viability (p < 0.01). Three of the latter five bacteria were plasma-sensitive while the other two were plasma-resistant but susceptible to neutrophil killing (p = 0.01). CONCLUSIONS: The bactericidal activity of WB can be the result of plasma sensitivity or neutrophil killing. Bacteria with a starting inoculum of 500 CFU/mL, and able to resist WB immune factors, can proliferate to clinically significant levels posing a potential safety risk to transfusion patients. Results of this pilot study should be validated under standard WB collection and storage conditions.

Differential Leukocyte MicroRNA Responses Following Pan T Cell, Allorecognition and Allosecretome-Based Therapeutic Activation.

Effective immunomodulation of T-cell responses is critical in treating both autoimmune diseases and cancer. Our previous studies have demonstrated that secretomes derived from control or methoxypolyethylene glycol mixed lymphocyte alloactivation assays exerted potent immunomodulatory activity that was mediated by microRNAs (miRNA). The immunomodulatory effects of biomanufactured miRNA-based allo-secretome therapeutics (SYN, TA1, IA1 and IA2) were compared to Pan T-cell activators (PHA and anti-CD3/CD28) and lymphocyte alloactivation. The differential effects of these activation strategies on resting peripheral blood mononuclear cells (PBMC) were assessed via T-cell proliferation, subset analysis and miRNA expression profiles. Mitogen-induced PBMC proliferation (> 85%) significantly exceeded that arising from either allostimulation (~ 30%) or the pro-inflammatory IA1 secretome product (~ 12%). Consequent to stimulation, the ratio of CD4 to CD8 cells of the resting PBMC (CD4:CD8; 1.7 ± 0.1) decreased in the Pan T cell, allrecognition and IA1 activated cells (averages of 1.1 ± 0.2; 1.2 ± 0.1 and 1.0 ± 0.1). These changes arose consequent to the expansion of both CD4+CD8+ and CD4-CD8- populations as well as the shrinkage of the CD4 subset and the expansion of the CD8 T cells. Importantly, these activation strategies induced vastly different miRNA expression profiles which were associated with significant differences in cellular differentiation and biological function. These findings support the concept that the "differential patterns of miRNA expression" regulate the biologic immune response in a "lock and key" manner. The biomanufacturing of miRNA-enriched secretome biotherapeutics may be a successful therapeutic approach for the systemic treatment of autoimmune diseases (TA1) and cancer (IA1).

Enhancing the pro-inflammatory anti-cancer T cell response via biomanufactured, secretome-based, immunotherapeutics.

T lymphocytes play a critical role in the pro-inflammatory anti-cancer response; hence, significant pharmacologic efforts have been made to enhance the endogenous T cell response. Unfortunately, significant toxicity arises consequent to pan T cell activation. In contrast, the less robust T cell alloresponse has also demonstrated an anti-cancer effect, but poses an inherent risk of GvHD. To overcome the GvHD risk, an acellular pro-inflammatory agent (IA1) has been biomanufactured from the secretome of the allorecognition response. To assess IA1's immunomodulatory activity, T cell proliferation and differentiation were determined in vitro. The pro-inflammatory properties of the IA1 therapeutic were mediated by the miRNA-enriched fractions. Moreover, cross-species efficacy was observed consequent to the evolutionary conservation of miRNA. IA1 exerted no toxicity to resting PBMC but induced significant proliferation of resting CD3+ (CD4+ and CD8+) T cells and skewed the response towards a pro-inflammatory state (i.e., increased Teff:Treg ratio). Crucially, IA1-activated PBMC demonstrated a potent inhibition of cancer cell (HeLa and SH-4 melanoma) proliferation relative to the resting PBMC. The anti-proliferation effect of IA1-activated PBMC was noted within ˜12 h versus 4-5 days for resting cells. A second biomanufactured therapeutic (IA2; produced using HeLa cells) surprisingly demonstrated direct toxicity to cancer cells but was less effective than IA1 in inducing a cell-mediated response. This study demonstrates that miRNA-enriched therapeutics can be biomanufactured from the secretome and can induce a potent pro-inflammatory, anti-cancer, effect on resting lymphocytes.

Cyclin E and survival in patients with breast cancer.

BACKGROUND: Cyclin E, a regulator of the cell cycle, affects the behavior of breast-cancer cells. We investigated whether levels of cyclin E in the tumor correlated with survival among patients with breast cancer. METHODS: Tumor tissue from 395 patients with breast cancer was assayed for cyclin E, cyclin D1, cyclin D3, and the HER-2/neu oncogene with the use of Western blot analysis. Full-length, low-molecular-weight, and total cyclin E were measured. Immunohistochemical assessments of cyclin E were also made of 256 tumors. We sought correlations between levels of these molecular markers and disease-specific and overall survival. RESULTS: The median follow-up was 6.4 years. A high level of the low-molecular-weight isoforms of cyclin E, as detected by Western blotting, correlated strongly with disease-specific survival whether axillary lymph nodes were negative or positive for metastases (P<0.001). Among 114 patients with stage I breast cancer, none of the 102 patients with low levels of cyclin E in the tumor had died of breast cancer by five years after diagnosis, whereas all 12 patients with a high level of low-molecular-weight cyclin E had died of breast cancer within that period. In multivariate analysis, a high total cyclin E level or high levels of the low-molecular-weight forms of cyclin E were significantly correlated with poor outcome. The hazard ratio for death from breast cancer for patients with high total cyclin E levels as compared with those with low total cyclin E levels was 13.3--about eight times as high as the hazard ratios associated with other independent clinical and pathological risk factors. CONCLUSIONS: Levels of total cyclin E and low-molecular-weight cyclin E in tumor tissue, as measured by Western blot assay, correlate strongly with survival in patients with breast cancer.

Immunogenicity of murine mPEG-red blood cells and the risk of anti-PEG antibodies in human blood donors.

The immunocamouflage of non-ABO blood group antigens by membrane-grafted methoxypoly(ethylene glycol) (mPEG) may attenuate the risk of red blood cell (RBC) alloimmunization. However, concerns have been raised over the immunogenic risk of PEG and PEG-RBCs. To assess this risk, murine and human studies were performed. Mice were exposed to soluble PEG prior to, or between, multiple transfusions (∼60-day intervals) of control or mPEG-RBCs, and cell survival was determined by flow cytometry. In some studies, the control and mPEG-RBC groups were reversed after one or more transfusions. Furthermore, human blood donors and commercial intravenous immunoglobulin products were examined to detect anti-PEG antibodies and to assess the risk for false positives. Naïve mice receiving chronic mPEG-RBC transfusions had normal RBC survival curves with no evidence of anti-PEG antibodies. Similarly, challenge with soluble PEG did not elicit anti-PEG antibodies in mice. Studies in humans revealed no evidence of a high prevalence of anti-PEG antibodies in either blood donors or commercial intravenous immunoglobulin. However, by use of the methods employed by studies identifying high levels of anti-PEG antibodies, a significant level (∼15%) of "false positives" were detected in commercial antibodies of known (non-PEG) specificities. These findings suggest that methodologic problems yielded a high rate of false positives in these earlier studies. These data continue to support the clinical utility of cellular PEGylation and the low immunogenic risk of grafted mPEG.

Inhibition of allogeneic cytotoxic T cell (CD8+) proliferation via polymer-induced Treg (CD4+) cells.

T cell-mediated immune rejection remains a barrier to successful transplantation. Polymer-based bioengineering of cells may provide an effective means of preventing allorecognition and the proliferation of cytotoxic (CD8+) T lymphocytes (CTL). Using MHC-disparate murine splenocytes modified with succinimidyl valerate activated methoxypoly(ethylene glycol) [SVA-mPEG] polymers, the effects of leukocyte immunocamouflage on CD8+ and CD4+ alloproliferation and T regulatory (Treg) cell induction were assessed in a mixed lymphocyte reaction (MLR) model. Polymer-grafting effectively camouflaged multiple leukocyte markers (MHC class I and II, TCR and CD3) essential for effective allorecognition. Consequent to the polymer-induced immunocamouflage of the cell membrane, both CD8+ and CD4+ T cell alloproliferation were significantly inhibited in a polymer dose-dependent manner. The loss of alloproliferation correlated with the induction of Treg cells (CD4+CD25+Foxp3+). The Tregs, surprisingly, arose primarily via differentiation of naive, non-proliferating, CD4+ cells. Of biologic importance, the polymer-induced Treg were functional and exhibited potent immunosuppressive activity on allogeneic CTL proliferation. These results suggest that immunocamouflage-mediated attenuation of alloantigen-TCR recognition can prevent the tissue destructive allogeneic CD8+ T cell response, both directly and indirectly, through the generation/differentiation of functional Tregs. Immunocamouflage induced tolerance could be clinically valuable in attenuating T cell-mediated transplant rejection and in the treatment of autoimmune diseases. STATEMENT OF SIGNIFICANCE: While our previous studies have demonstrated that polymer-grafting to MHC disparate leukocytes inhibits CD4+ cell proliferation, the effects of PEGylation on the alloproliferation of CD8+ cytotoxic T cells (CTL) was not examined. As shown here, PEGylation of allogeneic leukocytes prevents the generation of the CTL response responsible for acute rejection. The loss of CTL proliferation is consequent to the polymer-based attenuation of allorecognition and the induction of T regulatory cells (Tregs). Interestingly, the Tregs are primarily generated via the differentiation of non-proliferating naive T cells. Importantly, the Tregs are functional and effectively induce a tolerogenic environment when transferred to an alloresponsive environment. The use of polymer-modified leukocytes provides a unique approach to effectively maximize the biologic production of functional Tregs both in vitro and in vivo. By using this approach it may be possible to attenuate unwanted alloresponses (e.g., graft rejection) or to treat autoimmune diseases.

Inhibition of Autoimmune Diabetes in NOD Mice by miRNA Therapy.

Autoimmune destruction of the pancreatic islets in Type 1 diabetes is mediated by both increased proinflammatory (Teff) and decreased regulatory (Treg) T lymphocytes resulting in a significant decrease in the Treg:Teff ratio. The non-obese diabetic (NOD) mouse is an excellent in vivo model for testing potential therapeutics for attenuating the decrease in the Treg:Teff ratio and inhibiting disease pathogenesis. Here we show for the first time that a bioreactor manufactured therapeutic consisting of a complex of miRNA species (denoted as TA1) can effectively reset the NOD immune system from a proinflammatory to a tolerogenic state thus preventing or delaying autoimmune diabetes. Treatment of NOD mice with TA1 resulted in a systemic broad-spectrum upregulation of tolerogenic T cell subsets with a parallel downregulation of Teff subsets yielding a dramatic increase in the Treg:Teff ratio. Moreover, the murine-derived TA1 was highly effective in the inhibition of allorecognition of HLA-disparate human PBMC. TA1 demonstrated dose-responsiveness and exhibited equivalent or better inhibition of allorecognition driven proliferation than etanercept (a soluble TNF receptor). These findings demonstrate that miRNA-based therapeutics can effectively attenuate or arrest autoimmune disease processes and may be of significant utility in a broad range of autoimmune diseases including Type 1 diabetes.

The potential utility of methoxypoly(ethylene glycol)-mediated prevention of rhesus blood group antigen RhD recognition in transfusion medicine.

Red blood cell (RBC) transfusions are an important clinical intervention. However, RBC express hundreds of non-ABO antigens making alloimmunization a significant risk. RhD expression is the most immunologically important non-ABO antigen. Availability of RhD(-) blood, often problematic in North America and Europe, is a significant issue in Asia and Africa where RhD(-) blood is uncommon (<0.5% of supply). The immunocamouflage of RhD is readily accomplished by the covalent grafting of methoxypoly(ethylene glycol) [mPEG] to the RBC membrane. To determine if RhD immunocamouflage would inhibit its immunologic recognition, an in vitro RhD-sensitized antigen presentation assay using PBMC and dendritic cells (DC) from RhD-sensitized women was used. The immunological effects of polymer grafting to an immunodominant RhD peptide, purified RhD protein and intact RhD(+) RBC were examined via T cell proliferation and cytokine release assays. At Day 11, PEGylation significantly attenuated T cell proliferation arising from RhD peptide (~80 → 5%), protein (36 → 0.2%) and intact RBC (33 → 1.4%). Cytokine secretion was similarly blunted following PEGylation of the purified protein or intact RBC. These data support the immunomodulatory effects of PEGylation and the potential utility of this technology in transfusion medicine - especially in situations where RhD(-) blood is rare or in short supply.

Induction of immunotolerance via mPEG grafting to allogeneic leukocytes.

The induction of anergy or tolerance to prevent allorecognition is of clinical interest. To this end, the effects of methoxypoly(ethylene glycol) [mPEG] grafting to allogeneic lymphocytes on proliferation and phenotype (Th17 and Treg) was examined in vitro and in vivo. Control studies demonstrated that PEGylation did not affect cells viability or proliferation (mitogen) potential. Conditioned media (1° MLR) collected at 72 h from resting PBMC demonstrated no immunomodulatory effects whereas the control MLR demonstrated significant (p < 0.001) pro-proliferative potential and significantly increased in IL-2, TNF-α, and INF-γ. However, 1° media from either resting mPEG-PBMC or the PEGylated MLR resulted in a significant inhibitory effect (p < 0.001) in the 2° MLR and no increase in cytokines. PEGylation of donor murine splenocytes resulted in significant in vivo immunosuppressive effects in H2-disparate mice. While unmodified allogeneic splenocytes resulted in a significant in vivo decrease in Treg and increased Th17 lymphocytes, PEGylated allogeneic splenocytes yielded significantly increased Tregs and baseline levels of Th17 lymphocytes. This effect was persistent to at least 30 days post challenge and was not reversed by unmodified allogeneic cells. These studies demonstrate that PEGylation of allogeneic lymphocytes induced an immunoquiescent state both in vitro and in vivo.

Polymer-mediated immunocamouflage of red blood cells: effects of polymer size on antigenic and immunogenic recognition of allogeneic donor blood cells.

Developing a practical means of reducing alloimmunization in chronically transfused patients would be of significant clinical benefit. Immunocamouflaging red blood cells (RBCs) by membrane grafting of methoxypoly(ethylene glycol) (mPEG) may reduce the risk of allo-immunization. The results of this study showed that antibody recognition of non-ABO antigens was significantly reduced in an mPEG-dose- and polymer size-dependent manner, with higher molecular weight mPEGs providing better immunoprotection. Furthermore, in vivo immunogenicity was significantly reduced in mice serially transfused with mPEG-modified xenogeneic (sheep; sRBCs), allogeneic (C57Bl/6), or syngeneic (Balb/c) RBCs. Following a primary transfusion of sRBCs, mice receiving mPEG-sRBCs showed a >90% reduction in anti-sRBC IgG antibody levels. After two transfusions, mice receiving mPEG-sRBCs showed reductions of >80% in anti-sRBC IgG levels. Importantly, mPEG-modified autologous cells did not induce neoantigens or an immune (IgG or IgM) response. These data suggest that the global immunocamouflage of RBCs by polymer grafting may provide a safe and cost-effective means of reducing the risk of alloimmunization.

Evidence that accumulation of mutants in a biofilm reflects natural selection rather than stress-induced adaptive mutation.

The accumulation of mutant genotypes within a biofilm evokes the controversy over whether the biofilm environment induces adaptive mutation or whether the accumulation can be explained by natural selection. A comparison of the virulence of two strains of the dental pathogen Streptococcus mutans showed that rats infected with one of the strains accumulated a high proportion (average, 22%) of organisms that had undergone a deletion between two contiguous and highly homologous genes. To determine if the accumulation of deletion mutants was due to selection or to an increased mutation rate, accumulations of deletion mutants within in vitro planktonic and biofilm cultures and within rats inoculated with various proportions of deletion organisms were quantified. We report here that natural selection was the primary force behind the accumulation of the deletion mutants.