Controlled delivery of paclitaxel from stent coatings using poly(hydroxystyrene-b-isobutylene-b-hydroxystyrene) and its acetylated derivative

A poly(styrene-b-isobutylene-b-styrene) (SIBS) triblock polymer is employed as the polymer drug carrier for the TAXUS Express2 Paclitaxel-Eluting Coronary Stent system (Boston Scientific Corp.). It has been shown that the release of paclitaxel (PTx) from SIBS can be modulated by modification of either drug-loading ratio or altering the triblock morphology by blending. In the present work, results toward achieving release modulation of PTx by chemical modification of the styrenic portion (using hydroxystyrene or its acetylated version) of the SIBS polymer system are reported. The synthesis of the precursor poly[(p-tert-butyldimethylsilyloxystyrene)]-b-isobutylene-b-[(p-tert-butyldimethylsilyloxystyrene] triblock copolymers was accomplished by living sequential block copolymerization of isobutylene (IB) and p-(tert-butyldimethylsiloxy)styrene (TBDMS) utilizing the capping-tuning technique in a one-pot procedure in methylcyclohexane/CH3Cl at -80 degrees C. This procedure involved the living cationic polymerization of IB with the 5-tert-butyl-1,3-bis(1-chloro-1-methylethyl)benzene/TiCl4 initiating system and capping of living difunctional polyisobutylene (PIB) chain ends with 1,1-ditolylethylene (DTE) followed by addition of titanium(IV) isopropoxide (Ti(OIp)4) to lower the Lewis acidity before the introduction of TBDMS. Deprotection of the product with tetrabutylammonium fluoride yielded poly(hydroxystyrene-b-isobutylene-b-hydroxystyrene), which was quantitatively acetylated to obtain the acetylated derivative. The hydroxystyrene and acetoxystyrene triblock copolymers have acceptable mechanical properties for use as drug delivery coatings for coronary stent applications. It was concluded that the hydrophilic nature of the endblocks and polarity effects on the drug/polymer miscibility lead to enhanced release of PTx from these polymers. The drug-polymer miscibility was confirmed by differential scanning calorimetry and atomic force microscopy evaluations.     

Evidence for minority male mating success and minority female mating disadvantage in Drosophila ananassae

Frequency-dependent mating success was tested for three pairs of wild-type and mutant strains of Drosophila ananassae, MY and yellow body color (y), PN and claret eye color (ca), and TIR and cut wing (ct). The two strains of each pair were chosen for their approximately equal mating propensities. Multiple-choice experiments, using different experimental procedures, were employed. The tests were carried out by direct observation in Elens-Wattiaux mating chambers with five different sex ratios (4:16, 8:12, 10:10, 12:8, and 16:4). There was no assortative mating and sexual isolation between the strains, based on 2 x 2 contingency chi2 analysis and isolation estimate values. One-sided rare male mating advantages were found in two experiments, one for ca males and the other for wild-type males (TIR). However, no advantage was found for rare males in the experiment with MY and y flies. Mating disadvantages for rare females were found for sex-linked mutants (y and ct). Two different observational methods (removal or direct observation of mating pairs) imparted no overall significant effects on the outcome of the frequency-dependent mating tests.     

Cytoplasmic replication of Staphylococcus aureus upon phagosomal escape triggered by phenol‐soluble modulin α

Staphylococcus aureus is a Gram‐positive human pathogen that is readily internalized by professional phagocytes such as macrophages and neutrophils but also by non‐professional phagocytes such as epithelial or endothelial cells. Intracellular bacteria have been proposed to play a role in evasion of the innate immune system and may also lead to dissemination within migrating phagocytes. Further, S. aureus efficiently lyses host cells with a battery of cytolytic toxins. Recently, phenol‐soluble modulins (PSM) have been identified to comprise a genus‐specific family of cytolytic peptides. Of these the PSMα peptides have been implicated in killing polymorphonuclear leucocytes after phagocytosis. We questioned if the peptides were active in destroying endosomal membranes to avoid lysosomal killing of the pathogen and monitored integrity of infected host cell endosomes by measuring the acidity of the intracellular bacterial microenvironment via flow cytometry and by a reporter recruitment technique. Isogenic mutants of the methicillin‐resistant S. aureus (MRSA) strains USA300 LAC, USA400 MW2 as well as the strongly cytolytic methicillin‐sensitive strain 6850 were compared with their respective wild type strains. In all three genetic backgrounds, PSMα mutants were unable to escape from phagosomes in non‐professional (293, HeLa, EAhy.926) and professional phagocytes (THP‐1), whereas mutants in PSMβ and δ‐toxin as well as β‐toxin, phosphatidyl inositol‐dependent phospholipase C and Panton Valentine leucotoxin escaped with efficiencies of the parental strains. S. aureus replicated intracellularly only in presence of a functional PSMα operon thereby illustrating that bacteria grow in the host cell cytoplasm upon phagosomal escape.     

Dynamic changes in blood immune cell composition and function in Holstein and Jersey steers in response to heat stress

Heat stress has detrimental effects on livestock via diverse immune and physiological changes; heat-stressed animals are rendered susceptible to diverse diseases. However, there is relatively little information available regarding the altered immune responses of domestic animals in heat stress environments, particularly in cattle steers. This study aimed to determine the changes in the immune responses of Holstein and Jersey steers under heat stress. We assessed blood immune cells and their functions in the steers of two breeds under normal and heat stress conditions and found that immune cell proportions and functions were altered in response to different environmental conditions. Heat stress notably reduced the proportions of CD21⁺MHCII⁺ B cell populations in both breeds. We also observed breed-specific differences. Under heat stress, in Holstein steers, the expression of myeloperoxidase was reduced in the polymorphonuclear cells, whereas heat stress reduced the WC1⁺ γδ T cell populations in Jersey steers. Breed-specific changes were also detected based on gene expression. In response to heat stress, the expression of IL-10 and IL-17A increased in Holstein steers alone, whereas that of IL-6 increased in Jersey steers. Moreover, the mRNA expression pattern of heat shock protein genes such as Hsp70 and Hsp90 was significantly increased in only Holstein steers. Collectively, these results indicate that altered blood immunological profiles may provide a potential explanation for the enhanced susceptibility of heat-stressed steers to disease. The findings of this study provide important information that will contribute to developing new strategies to alleviate the detrimental effects of heat stress on steers.     

Proteome Analysis of Plasmodium falciparum Extracellular Secretory Antigens at Asexual Blood Stages Reveals a Cohort of Proteins with Possible Roles in Immune Modulation and Signaling

The highly co-evolved relationship of parasites and their hosts appears to include modulation of host immune signals, although the molecular mechanisms involved in the host-parasite interplay remain poorly understood. Characterization of these key genes and their cognate proteins related to the host-parasite interplay should lead to a better understanding of this intriguing biological phenomenon. The malaria agent Plasmodium falciparum is predicted to export a cohort of several hundred proteins to remodel the host erythrocyte. However, proteins actively exported by the asexual intracellular parasite beyond the host red blood cell membrane (before merozoite egress) have been poorly investigated so far. Here we used two complementary methodologies, two-dimensional gel electrophoresis/MS and LC-MS/MS, to examine the extracellular secreted antigens at asexual blood stages of P. falciparum. We identified 27 novel antigens exported by P. falciparum in the culture medium of which some showed clustering with highly polymorphic genes on chromosomes, suggesting that they may encode putative antigenic determinants of the parasite. Immunolocalization of four novel secreted proteins confirmed their export beyond the infected red blood cell membrane. Of these, preliminary functional characterization of two novel (Sel1 repeat-containing) parasite proteins, PfSEL1 and PfSEL2 revealed that they down-regulate expression of cell surface Notch signaling molecules in host cells. Also a novel protein kinase (PfEK) and a novel protein phosphatase (PfEP) were found to, respectively, phosphorylate/dephosphorylate parasite-specific proteins in the extracellular culture supernatant. Our study thus sheds new light on malaria parasite extracellular secreted antigens of which some may be essential for parasite development and could constitute promising new drug targets.Plasmodium falciparum is a wide spread protozoan parasite responsible for over a million deaths annually mainly among children in sub-Saharan Africa (1). Like other apicomplexan parasites such as Leishmania, Trypanosoma, and Toxoplasma, Plasmodia depend on a series of intricate and highly evolved adaptations that enable them to evade destruction by the host immune responses. These protozoan parasites have provided some of the best leads in elucidating the mechanisms to circumvent innate immunity and adaptive humoral and cellular immunity (2). Ingenious strategies to escape innate defenses include subversion of attack by humoral effector mechanisms such as complement lysis and lysis by other serum components (3), remodeling of phagosomal compartments in which they reside (4), modulation of host cell signaling pathways (5), and modification of the antigen-presenting and immunoregulatory functions of dendritic cells, which provide a crucial link with the adaptive immune response (6). Malaria parasites also predominantly use antigenic diversity and clonal antigenic variation to evade adaptive immunity of the host (7). Surface-associated and secreted parasite proteins are major players in host-parasite cross-talk and are advantageously used by the parasite to counter the host immune system. Proteins secreted by a wide range of parasitic pathogens into the host microenvironment result in symptomatic infections. For example, the excretory-secretory (ES)¹ products of the parasitic fluke Fasciola hepatica are key players in host-parasite interactions (8). Among the apicomplexans, proteomics analyses of rhoptry organelles of Toxoplasma gondii have revealed many novel constituents of host-parasite interactions (9).The identification and trafficking of Plasmodium proteins exported into the host erythrocyte have been subjects of recent detailed investigations. A number of studies have identified Plasmodium proteins that contain signature sequence motifs, the host cell targeting signal or the Plasmodium export element (PEXEL), that target these proteins into the infected erythrocytes (10, 11). Recent proteomics analyses have identified novel proteins in the raftlike membranes of the parasite and on the surface of infected erythrocytes (12, 13). P. falciparum translationally controlled tumor protein (PfTCTP), a homolog of the mammalian histamine-releasing factor, has been shown to be released into the culture supernatant from intact as well as ruptured infected RBCs and causes histamine release from human basophils and IL-8 secretion from eosinophils (14). However, the total spectrum of proteins actively exported by the asexual intracellular parasite beyond the host RBC membrane (before merozoite egress) has been poorly investigated so far.In the present study, we used two complementary methodologies, two-dimensional gel electrophoresis (2DE)/MS and LC-MS/MS to examine the cohort of extracellular secreted antigens (ESAs) at asexual blood stages of P. falciparum. Our findings reveal that malaria parasites secrete a number of effector molecules such as immunomodulators and signaling proteins that are potentially involved in host-parasite interactions. Prominent among these are proteins with Sel1 domain, a protein of the LCCL family, a novel protein kinase, and a novel protein phosphatase. Secreted-extracellular/iRBC surface localization of some of these proteins was validated by immunolocalization studies. We also characterized the functions of some of these proteins in the culture supernatant, thus providing an insight into the nature of some of the malaria parasite extracellular antigens.     

Hormonal regulation of apoptosis in the endometrium of common marmosets (Callithrix jacchus)

Phase-dependent apoptotic changes in the human endometrium during an ovarian cycle imply a potential role of steroids in the regulation of apoptosis. The present study was undertaken to determine the direct role of hormones in endometrial apoptosis in marmosets (Callithrix jacchus), a primate species which shows similarity to humans in terms of the cycle length and pattern. Endometrial apoptosis was detected by 3'-end labeling (TUNEL) in various phases of ovarian cycle in naturally cycling healthy marmosets (n=14) and also in ovariectomized marmosets (n=13) treated with either estradiol alone (E) or progesterone alone (P) or estradiol followed by progesterone (E+P). Expressions of apoptosis associated genes such as Bcl-2 family members (Bax and Bcl-2), proliferating cell nuclear antigen (PCNA)--a proliferation marker and steroid receptors, ERalpha and PR A were analysed by immunohistochemical methods. Apoptosis was intense in the glandular epithelial cells of endometrium during the mid-luteal phase as compared to other phases in naturally cycling animals; in the E+P group as compared to other groups of ovariectomized animals (P<0.05). Pronounced apoptosis in the mid-luteal phase was accompanied by the increased expression of Bax in glandular epithelial cells; while Bcl-2 immunoreactivity remained unchanged. PCNA expression was higher in the naturally cycling animals in the follicular phase and in the E group of the ovariectomized animals as compared those in the other groups. Immunoreactive ERalpha and PR A in glandular epithelial cells were most abundant during early follicular phase in naturally cycling animals and in both E and E+P groups among the ovariectomized animals. The present study highlights the importance of apoptosis in endometrial remodeling during the ovarian cycle and secondly, the role of both estradiol and progesterone in the regulation of apoptosis.