Leukoreduced whole blood-derived platelets treated with antimicrobial peptides maintain in vitro properties during storage.

BACKGROUND: Bacterial sepsis is still a complication in patients transfused with stored platelets (PLTs). We have recently demonstrated that selected antimicrobial peptides (AMPs) have bactericidal activity in bacteria-spiked PLTs. In a subsequent preclinical study, we have also shown that these AMPs do not elicit antibody response in rabbits and treatment of PLTs before transfusion does not affect their in vivo recovery and survival in severe combined immunodeficient mice. Here we have selected two such AMPs, Arg-Trp (RW) repeats of tri- and tetra-peptides (RW3 and RW4) in combination (i.e., cocktail), and evaluated their effect on the in vitro properties of PLTs. STUDY DESIGN AND METHODS: Leukoreduced ABO- and D-identical whole blood-derived PLT concentrates were pooled and divided into two 60-mL aliquots in CLX storage bags. On Day 0, one bag received a peptide cocktail of RW3 plus RW4 at 0.01 mmol/L final concentration (test) and the other bag received only phosphate-buffered saline (PBS), the AMP solvent (control). The treated PLTs were stored for 7 days at 20 to 24°C. Samples were collected on Days 1, 5, and 7 to evaluate the in vitro properties of PLTs with standard assays. RESULTS: In vitro properties of the RW3 plus RW4 cocktail-treated PLTs were similar to those incubated with PBS only. There were no significant differences between the control and test PLTs during the 7-day storage. CONCLUSION: Leukoreduced whole blood-derived PLTs treated with a mixture of RW3 and RW4 peptides maintain their in vitro properties during 7 days of storage.

Preclinical safety evaluation of human platelets treated with antimicrobial peptides in severe combined immunodeficient mice.

BACKGROUND: Bacterial sepsis is a complication attributed to room temperature (RT)-stored platelets (PLTs) in transfusion medicine. Antimicrobial peptides (AMPs) are emerging as new therapeutic agents against microbes. We had previously demonstrated bactericidal activity of select synthetic AMPs against six types of bacteria in stored PLTs. In this report, we tested these AMPs for their potential antibody response and interference with the recovery and survival of human PLTs in an animal model. STUDY DESIGN AND METHODS: Two separate studies were conducted to evaluate the safety of the synthetic AMPs. 1) Two AMPs (PD3 and PD4), derived from thrombin-induced human PLT microbicidal protein, and four repeats of arginine-tryptophan (RW), containing two to five repeats (RW2-RW5), were tested in rabbits for potential antibody response. 2) RT-stored human PLTs treated for 2 hours with each of the six AMPs individually or with phosphate-buffered saline (PBS) alone were infused into severe combined immunodeficient (SCID) mice to evaluate their in vivo recovery and survival by flow cytometry. RESULTS: Except for PD3, which showed a weak immune response, all other peptides did not induce any detectable antibodies in rabbits. Furthermore, all six AMPs tested did not significantly affect the in vivo recovery and survival of human PLTs in SCID mice compared to PBS alone-treated PLTs. CONCLUSION: Preclinical evaluation studies reported here demonstrate that the selected AMPs used in the study did not adversely affect the human PLT recovery and survival in the SCID mouse model, suggesting further study of AMPs toward addressing the bacterial contamination of PLTs.

A peptide derived from phage display library exhibits antibacterial activity against E. coli and Pseudomonas aeruginosa.

Emergence of drug resistant strains to currently available antibiotics has resulted in the quest for novel antimicrobial agents. Antimicrobial peptides (AMPs) are receiving attention as alternatives to antibiotics. In this study, we used phage-display random peptide library to identify peptides binding to the cell surface of E. coli. The peptide with sequence RLLFRKIRRLKR (EC5) bound to the cell surface of E. coli and exhibited certain features common to AMPs and was rich in Arginine and Lysine residues. Antimicrobial activity of the peptide was tested in vitro by growth inhibition assays and the bacterial membrane permeabilization assay. The peptide was highly active against gram-negative organisms and showed significant bactericidal activity against E. coli and P. aeruginosa resulting in a reduction of 5 log(10) CFU/ml. In homologous plasma and platelets, incubation of EC5 with the bacteria resulted in significant reduction of E. coli and P. aeruginosa, compared to the peptide-free controls. The peptide was non-hemolytic and non-cytotoxic when tested on eukaryotic cells in culture. EC5 was able to permeabilize the outer membrane of E. coli and P. aeruginosa causing rapid depolarization of cytoplasmic membrane resulting in killing of the cells at 5 minutes of exposure. The secondary structure of the peptide showed a α-helical conformation in the presence of aqueous environment. The bacterial lipid interaction with the peptide was also investigated using Molecular Dynamic Simulations. Thus this study demonstrates that peptides identified to bind to bacterial cell surface through phage-display screening may additionally aid in identifying and developing novel antimicrobial peptides.

Identification and evaluation of a novel peptide binding to the cell surface of Staphylococcus aureus.

Identification of short peptides that serve as specific ligands to biological materials such as microbial cell surfaces has major implications in better understanding the molecular recognition of cell surfaces. In this study we screened a commercially available random phage-display library against Staphylococcus aureus cells and identified peptides specifically binding to the bacteria. A synthetic peptide (SA5-1) representing the consensus sequence (VPHNPGLISLQG) of the bacteria-binding peptide was evaluated for its binding potential against S. aureus. Dot-blot, immunoblot assay and ELISA results revealed the SA5-1 peptide to be highly specific to S. aureus. The SA5-1 peptide binding was optimal between pH 6.0 and 8.0. Nanogold Transmission Electron Microscopy demonstrated that the SA5-1 binds to the outer membrane surface of S. aureus. Diagnostic potential of the SA5-1 peptide was evaluated in human platelet samples spiked with S. aureus and specific detection of the bacteria by biotinylated-SA5-1 and streptavidin-conjugated fluorescent quantum dots. Fluorometry results indicated that the peptide was able to detect ∼100 organisms per ml in a spiked biological sample providing a proof-of-concept towards potential of this peptide as a S. aureus diagnostic tool that can be of use in different detection platforms.

Identification of XMRV infection-associated microRNAs in four cell types in culture.

INTRODUCTION: XMRV is a gammaretrovirus that was thought to be associated with prostate cancer (PC) and chronic fatigue syndrome (CFS) in humans until recently. The virus is culturable in various cells of human origin like the lymphocytes, NK cells, neuronal cells, and prostate cell lines. MicroRNAs (miRNA), which regulate gene expression, were so far not identified in cells infected with XMRV in culture. METHODS: Two prostate cell lines (LNCaP and DU145) and two primary cells, Peripheral Blood Lymphocytes [PBL] and Monocyte-derived Macrophages [MDM] were infected with XMRV. Total mRNA was extracted from mock- and virus-infected cells at 6, 24 and 48 hours post infection and evaluated for microRNA profile in a microarray. RESULTS: MicroRNA expression profiles of XMRV-infected continuous prostate cancer cell lines differ from that of virus-infected primary cells (PBL and MDMs). miR-193a-3p and miRPlus-E1245 observed to be specific to XMRV infection in all 4 cell types. While miR-193a-3p levels were down regulated miRPlus-E1245 on the other hand exhibited varied expression profile between the 4 cell types. DISCUSSION: The present study clearly demonstrates that cellular microRNAs are expressed during XMRV infection of human cells and this is the first report demonstrating the regulation of miR193a-3p and miRPlus-E1245 during XMRV infection in four different human cell types.

Detection technologies for Bacillus anthracis: prospects and challenges.

Bacillus anthracis is a Gram-positive, spore-forming bacterium representing the etiological agent of acute infectious disease anthrax, a lethal but rare disease of animals and humans in nature. With recent use of anthrax as a bioweapon, a number of techniques have been recently developed and evaluated to facilitate its rapid detection of B. anthracis in the environment as well as in point-of-care settings for humans suspected of exposure to the pathogen. Complex laboratory methods for B. anthracis identification are required since B. anthracis has similarities with other Bacillus species and its existence in both spore and vegetative forms. This review discusses current challenges and various improvements associated with anthrax agent detection.

Peptides panned from a phage-displayed random peptide library are useful for the detection of Bacillus anthracis surrogates B. cereus 4342 and B. anthracis Sterne.

Recent use of Bacillus anthracis as a bioweapon has highlighted the need for a sensitive monitoring system. Current bacterial detection tests use antibodies as bio-molecular recognition elements which have limitations with regard to time, specificity and sensitivity, creating the need for new and improved cost-effective high-affinity detection probes. In this study, we screened a commercially available bacteriophage-displayed random peptide library using Bacillus cereus 4342 cells as bait to identify peptides that could be used for detection of Bacillus. The method enabled us to identify two 12-amino acid consensus peptide sequences that specifically bind to B. cereus 4342 and B. anthracis Sterne, the nonpathogenic surrogates of B. anthracis strain. The two Bacillus-binding peptides (named BBP-1 and BBP-2) were synthesized with biotin tag to confirm their binding by four independent detection assays. Dot-blot analysis revealed that the peptides bind specifically to B. cereus 4342 and B. anthracis Sterne. Quantitative analysis of this interaction by ELISA and fluorometry demonstrated a detection sensitivity of 10(2) colony forming U/ml (CFU/ml) by both assays. When the peptides were used in combination with Qdots, the sensitivity was enhanced further by enabling detection of even a single bacterium by fluorescence microscopy. Immunoblot analysis and protein sequencing showed that BBP-1 and BBP-2 bound to the S-layer protein of B. anthracis Sterne. Overall, our findings validate the usefulness of synthetic versions of phage-derived peptides in combination with Qdot-liquid nanocrystals as high sensitivity bioprobes for various microbial detection platforms.

Antiviral activity of selected antimicrobial peptides against vaccinia virus.

Antimicrobial peptides (AMPs) are gaining importance as effective therapeutic alternatives to conventional antibiotics. Recently we have shown that a set of nine synthetic antimicrobial peptides, four originating from thrombin-induced human platelet-derived antimicrobial proteins named PD1-PD4 and five synthetic repeats of arginine-tryptophan (RW) repeats (RW1-5) demonstrate antibacterial activity in plasma and platelets. Using WR strain of vaccinia virus (VV) as a model virus for enveloped virus in the present study, we tested the same nine synthetic peptides for their antiviral activity. A cell culture-based standard plaque reduction assay was utilized to estimate antiviral effectiveness of the peptides. Our analysis revealed that peptides PD3, PD4, and RW3 were virucidal against VV with PD3 demonstrating the highest antiviral activity of 100-fold reduction in viral titers, whereas, PD4 and RW3 peptide treatments resulted in 10-30-fold reduction. The EC(50) values of PD3, PD4 and RW3 were found to be 40 microg/ml, 50 microg/ml and 6.5 microM, respectively. In VV-spiked plasma samples, the virucidal activity of PD3, PD4 and RW3 was close to 100% (90-100-fold reduction). Overall, the present study constitutes a new proof-of-concept in developing peptide therapeutics for vaccinia virus infections in biothreat scenarios and as in vitro viral reduction agents.

Evaluation of antimicrobial peptides as novel bactericidal agents for room temperature-stored platelets.

BACKGROUND: A single cost-effective pathogen inactivation approach would help to improve the safety of our nation's blood supply. Several methods and technologies are currently being studied to help reduce bacterial contamination of blood components. There is clearly need for simple and easy-to-use pathogen inactivation techniques specific to plasma, platelets (PLTs), and red blood cells. STUDY DESIGN AND METHODS: In this report, we introduce a novel proof of concept: using known therapeutic antimicrobial peptides (AMPs) as bactericidal agents for room temperature-stored PLT concentrates (PCs). Nine synthetic AMPs, four from PLT microbicidal protein-derived peptides (PD1-4) and five Arg-Trp (RW) repeat peptides containing one to five repeats, were tested for bactericidal activity in plasma and PC samples spiked with Staphylococcus aureus, S. epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Bacillus cereus. A 3-log reduction of viable bacteria was considered as the bactericidal activity of a given peptide. RESULTS: In both plasma alone and PCs, RW3 peptide demonstrated bactericidal activity against S. aureus, S. epidermidis, E. coli, P. aeruginosa, and K. pneumoniae; PD4 and RW2 against P. aeruginosa; and RW4 against K. pneumoniae. The activity of each of these four peptides against the remaining bacterial species in the test panel resulted in less than a 3-log reduction in the number of viable bacteria and hence considered ineffective. CONCLUSIONS: These findings suggest a new approach to improving the safety of blood components, demonstrating the potential usefulness of screening therapeutic AMPs against selected bacteria to identify suitable bactericidal agents for stored plasma, PCs, and other blood products.

Gamma-phage lysin PlyG sequence-based synthetic peptides coupled with Qdot-nanocrystals are useful for developing detection methods for Bacillus anthracis by using its surrogates, B. anthracis-Sterne and B. cereus-4342.

BACKGROUND: Previous reports of site-directed deletion analysis on gamma (gamma)-phage lysin protein (PlyG) have demonstrated that removal of a short amino acid sequence in the C-terminal region encompassing a 10-amino acid motif (190LKMTADFILQ199) abrogates its binding activity specific to the cell wall of Bacillus anthracis. Whether short synthetic peptides representing the10-amino acid PlyG putative binding motif flanked by surrounding N- and C-terminal residues also selectively bind to the bacterial cell wall has not been evaluated. If such peptides do demonstrate selective binding to the cell wall, they could serve as bio-probes towards developing detection technologies for B. anthracis. Furthermore, by using B. anthracis (Sterne, 34F2), an animal vaccine and B. cereus-4342, a gamma-phage susceptible rare strain as surrogates of B. anthracis, development of proof-of-concepts for B. anthracis are feasible. RESULTS: Using four different methods, we evaluated six synthetic peptides representing the putative binding motif including flanking sequences (PlyG-P1 through P6) for the bacterial cell wall binding capacity. Our analysis identified PlyG-P1, PlyG-P3 and PlyG-P5 to have binding capability to both B. anthracis (Sterne, 34F2) and B. cereus-4342. The peptides however did not bind to B. cereus-11778, B. thuringiensis, and B. cereus-10876 suggesting their specificity for B. anthracis-Sterne and B. cereus-4342. PlyG-P3 in combination with fluorescent light microscopy detected even a single bacterium in plasma spiked with the bacteria. CONCLUSION: Overall, these studies illustrate that the short 10-amino acid sequence 'LKMTADFILQ' in fact is a stand-alone bacterial cell wall-binding motif of PlyG. In principle, synthetic peptides PlyG-P1, PlyG-P3 and PlyG-P5, especially PlyG-P3 coupled with Qdot-nanocrystals are useful as high-sensitivity bio-probes in developing detection technologies for B. anthracis.

The proteoglycan bamacan is a host cellular ligand of vaccinia virus neurovirulence factor N1L.

Neurovirulence is one of the pathological complications associated with vaccinia virus (VV) infection/vaccination. Although the viral N1L protein has been identified as the neurovirulence factor, none of the host N1L-interacting factors have been identified so far. In the present study, we identified N1L-interacting proteins by screening a human brain cDNA expression library with N1L as a bait protein in a yeast two-hybrid analysis. The analysis revealed that N1L interacts with human brain-originated cellular basement membrane-associated chondroitin sulfate proteoglycan (bamacan). The N1L-binding domain of bamacan was mapped to its C-terminal 227 amino acids. The N1L-bamacan interaction was further confirmed in both VV-infected and N1L-transfected mammalian cells. Following the confirmation of the protein interactions by coimmunoprecipitation experiments, confocal microscopic analysis revealed that N1L colocalizes with bamacan both in VV-infected B-SC-1 cells as well as in mice neuronal tissue. Furthermore, a human neural cell line, which expresses bamacan to moderately elevated levels relative to a non-neural cell line, supported enhanced viral growth. Overall, these studies clearly suggest that bamacan interacts with the VV-N1L and such interactions seem to play a positive role in promoting the viral growth and perhaps contribute to the virulence of VV in neural cells.

Membrane array-based differential profiling of platelets during storage for 52 miRNAs associated with apoptosis.

BACKGROUND: Enucleated platelets (PLTs) utilize posttranscriptional gene (mRNA) regulation (PTGR) for their normal morphologic and physiologic functions, which are altered in their ex vivo storage, also collectively referred to as storage lesions. While cellular micro-RNAs (miRNAs) play a significant role in posttranscriptional gene (mRNA) regulation by binding to their target mRNAs, comprehensive analysis of apoptosis-associated miRNAs and global changes in their profiles during PLT storage have not been evaluated to date. STUDY DESIGN AND METHODS: In this report room temperature-stored PLTs of Days 0, 2, and 9 were analyzed by differential profiling for 52 apoptosis-associated human miRNAs. After total RNA extraction from the samples, a membrane array-based miRNA analysis was carried out. Prediction of target genes was performed by bioinformatics-based approaches. RESULTS: Our analysis revealed that during storage, Let-7a, -7c, -7e, -7f, -7g, and -7i miRNA profiles of the PLTs were barely detectable, while levels of miR-150, -151, -152, -184, -188, -196a, -197, and -202 remained at high levels in PLTs. The rest of the miRNA levels were in between. However, two miRNAs, Let-7b and miR-16, distinctly demonstrated an increasing trend while miR-7 and miR-145 showed a decreasing profile during PLT storage. For these four miRNAs, we also identified their potential target mRNAs. CONCLUSIONS: Overall, these results confirm the fact that miRNAs do exist in PLTs, and among 52 apoptosis-specific miRNAs studied, only a few selected miRNAs did perturb during PLT storage. Future experimental evaluation of these miRNA-target mRNA interactions will provide new insights into the molecular mechanisms of PLT storage-associated lesions.

Defective rotavirus particle assembly in lovastatin-treated MA104 cells.

Rotavirus is a non-enveloped virus that depends on cellular lipids for cell entry and associates with lipid rafts during assembly. However, the effects of cellular lipids on rotavirus assembly are still not fully understood. The present study analyzes the effects of lovastatin, an inhibitor of cholesterol biosynthesis, during rotavirus infection in MA104 cells with regard to viral growth and particle assembly. Following viral infection, a 2-log relative reduction of viral titers was observed in drug-treated cells, while viral mRNA levels in infected cells remained unaltered in both groups. Furthermore, the levels of some viral proteins in drug-treated cells were elevated. The observed discordance between the viral RNA and protein levels and the decrease in infectivity titers of viral progeny in the drug-treated cells suggested that the drug affects viral assembly, the viral proteins not being properly incorporated into virions. Transmission electron microscopic (TEM) analysis revealed that in drug-treated cells there was an increase in "empty-looking" rotavirus particles devoid of an electron-dense core as compared to the normal, electron-dense particles seen in untreated infected cells. The present study thus provides visual evidence of defective rotavirus particle assembly as a result of cholesterol depletion.

A human vaccine strain of lamb rotavirus (Chinese) NSP4 gene: complete nucleotide sequence and phylogenetic analyses.

A lamb strain of rotavirus has recently been licensed for use in China as a live vaccine to prevent rotavirus diarrhea in children. As rotavirus NSP4, especially the cytotoxic domain alone is considered to be associated with diarrhea, we sequenced gene segment 10, which encodes NSP4, of lamb rotavirus. Comparative analyses was performed to identify differences from human rotavirus strains, that might be associated with attenuation, and to ascertain whether the lamb rotavirus gene fits among the NSP4 of other sequenced rotavirus strains. Our comparative nucleotide sequence analysis suggests its close identity (91.17% homology) with that of group-A equine rotavirus (strain HI23). Multiple alignment of the deduced amino acid sequence of lamb NSP4 with that of other group A rotaviruses demonstrated homology ranging from 63.42% with that of porcine YM strain to 93.71% with equine HI23 strain of rotavirus. A group A-specific NSP4 monoclonal antibody recognized the glycosylated and unglycosylated forms of the protein from virus-infected lysates, suggesting a well-conserved group-specificity of the lamb NSP4. Phylogenetic analysis of the lamb rotavirus gene, with 60 other NSP4 gene sequences of human and animal rotavirus strains, demonstrated that the lamb rotavirus strain belongs to genotype A. Comparative analysis also revealed that although it is a vaccine strain, the NSP4 cytotoxic domain of lamb strain demonstrated an overall amino acid conservation similar to that of other strains, whose NSP4 alone causes diarrhea in animal models. These results taken together with our previous observations clearly reaffirm the idea that the attenuation phenotype of rotaviruses does not involve NSP4 cytotoxic domain, perhaps due to the suppression of NSP4 cytotoxic activity by other rotaviral proteins.

An atypical protein disulfide isomerase from the protozoan parasite Leishmania containing a single thioredoxin-like domain.

In higher eukaryotes, secretory proteins are under the quality control of the endoplasmic reticulum for their proper folding and release into the secretory pathway. One of the proteins involved in the quality control is protein disulfide isomerase, which catalyzes the formation of protein disulfide bonds. As a first step toward understanding the endoplasmic reticulum quality control of secretory proteins in lower eukaryotes, we have isolated a protein disulfide isomerase gene from the protozoan parasite Leishmania donovani. The parasite enzyme shows high sequence homology with homologs from other organisms. However, unlike the four thioredoxin-like domains found in most protein disulfide isomerases, of which two contain an active site, the leishmanial enzyme possesses only one active site present in a single thioredoxin-like domain. When expressed in Escherichia coli, the recombinant parasite enzyme shows both oxidase and isomerase activities. Replacement of the two cysteins with alanines in its active site results in loss of both enzymatic activities. Further, overexpression of the mutated/inactive form of the parasite enzyme in L. donovani significantly reduced their release of secretory acid phosphatases, suggesting that this single thioredoxin-like domain protein disulfide isomerase could play a critical role in the Leishmania secretory pathway.

The N-terminal conserved domain of rubella virus capsid interacts with the C-terminal region of cellular p32 and overexpression of p32 enhances the viral infectivity.

Cellular 'defense collagens' are produced to launch virus-specific responses to clear the invading viruses. Cellular p32, the C1q binding protein is one such protein. In this report, we identified the interaction of p32 derived from a human lung diploid cell line (WI-38) with rubella virus capsid (RVCP from Therien strain) N-terminal 28-amino acid domain, which is conserved among several RV strains including the vaccine strains. We further identified that the C-terminal 69 aa of the mature p32 is sufficient to interact with the CP. In addition, we observed that in three independent Vero 76-derived cell lines constitutively overexpressing p32, the RV infectivity was enhanced. Our results suggest that RV has evolved a strategy whereby one of its proteins is recruited to interact with, and exploit the cellular defense machinery to its advantage.