Characterization of immune cell migration using microfabrication

The immune system provides our defense against pathogens and aberrant cells, including tumorigenic and infected cells. Motility is one of the fundamental characteristics that enable immune cells to find invading pathogens, control tissue damage, and eliminate primary developing tumors, even in the absence of external treatments. These processes are termed “immune surveillance.” Migration disorders of immune cells are related to autoimmune diseases, chronic inflammation, and tumor evasion. It is therefore essential to characterize immune cell motility in different physiologically and pathologically relevant scenarios to understand the regulatory mechanisms of functionality of immune responses. This review is focused on immune cell migration, to define the underlying mechanisms and the corresponding investigative approaches. We highlight the challenges that immune cells encounter in vivo, and the microfabrication methods to mimic particular aspects of their microenvironment. We discuss the advantages and disadvantages of the proposed tools, and provide information on how to access them. Furthermore, we summarize the directional cues that regulate individual immune cell migration, and discuss the behavior of immune cells in a complex environment composed of multiple directional cues.     

Effects of a fire retardant on the Near Eastern Fire Salamander Salamandra infraimmaculata and aquatic community structure: an experimental approach

Hydrobiologia - Fire retardants are commonly used for fighting wildfires. Retardant chemicals washed via runoff into aquatic systems may be concentrated, thus exposing aquatic species to high...     

Expression of hepatic 3β-hydroxysteroid dehydrogenase and sulfotransferase 2A1 in entire and castrated male pigs

The present study investigated the effect of surgical (SC) and immunological castration on the steroid metabolizing enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and sulfotransferase 2A1 (SULT2A1) in male pigs. Thirty-two male pigs were divided in four groups; in one group the pigs were SC before the age of 7 days, two groups were injected with Improvac(?) a vaccine against gonadotropin releasing hormone (immunological castration), while the pigs in the last group remained entire males (EMs). Immunological castration was in one group performed by vaccine injection at ages 11 and 14 weeks, while the other group received injections at ages 17 and 21 weeks. Plasma, adipose and liver tissue were collected at the time of slaughter. Plasma was analyzed for concentrations of testosterone and oestradiol. The adipose tissue was analyzed for the concentration of androstenone, while the liver tissue was analyzed for mRNA and protein expression of 3β-HSD and SULT2A1. Independent of method, all castrated pigs showed greater mRNA and protein expression of 3β-HSD and lower levels of all steroids in plasma compared with EMs. Moreover, there was a strong correlation between mRNA and protein expression of 3β-HSD and steroid levels. The same was not valid for expression of SULT2A1. It is concluded that steroid levels can increase expression of the steroid metabolizing enzyme 3β-HSD and thereby influence steroid metabolism, e.g. of androstenone.     

Cell elongation and branching are regulated by differential phosphorylation states of the nuclear Dbf2-related kinase COT1 in Neurospora crassa

Dysfunction of the Neurospora crassa nuclear Dbf2-related kinase COT1 leads to cessation of tip extension and massive induction of new sites of growth. To determine the role phosphorylation plays in COT1 function, we mutated COT1 residues corresponding to positions of highly conserved nuclear Dbf2-related phosphorylation sites. Analyses of the point-mutation cot-1 strains (mimicking non- and constitutively phosphorylated states) indicate the involvement of COT1 phosphorylation in the regulation of hyphal elongation and branching as well as asexual development by altering cell wall integrity and actin organization. Phosphorylation of COT1's activation segment (at Ser417) is required for proper in vitro kinase activity, but has only a limited effect on hyphal growth. In marked contrast, even though phosphorylation of the C-terminal hydrophobic motif (at Thr589) is crucial for all COT1 functions in vivo , the lack of Thr589 phosphorylation did not significantly affect in vitro COT1 kinase activity. Nevertheless, its regulatory role has been made evident by the significant increase observed in COT1 kinase activity when this residue was substituted in a manner mimicking constitutive phosphorylation. We conclude that COT1 regulates elongation and branching in an independent manner, which is determined by its phosphorylation state.     

Molecular features of heterogeneous vancomycin-intermediate Staphylococcus aureus strains isolated from bacteremic patients

Background  

Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) bacteremia is an emerging infection. Our objective was to determine the molecular features of hVISA strains isolated from bacteremic patients and to compare them to methicillin resistant S. aureus (MRSA) and methicillin sensitive S. aureus (MSSA) blood isolates.     

Postnatal intermittent hypoxia alters baroreflex function in adult rats

Chronic perinatal intermittent hypoxia (IH) could have long-term cardiovascular effects by altering baroreflex function. To examine this hypothesis, we exposed rats (n = 6/group) for postnatal days 1-30 or prenatal embryonic days 5-21 to IH (8% ambient O2 for 90 s after 90 s of 21% of O2, 12 h/day) or to normoxia (control). Baroreflex sensitivity (BRS) and cardiac chronotropic responses were examined in anesthetized animals 3.5-5 mo later by infusing phenylephrine or sodium nitroprusside (6-12 microg/min iv, 1-2 min) during normoxia and after 18 min of acute IH (IHA). In controls after IHA, baroreflex gain was 42% (P < 0.05) less than during normoxia. BRS in the postnatal IH group during normoxia was approximately 50% less than in control rats and similar to controls after IHA. The heart rate response to phenylephrine in the IH group was also less than in controls (P < 0.05) and was not changed by IHA. BRS and heart rate responses in the prenatal IH group were similar to the normoxic control group. Vagal efferent projections to atrial ganglia neurons in rats after postnatal IH (n = 4) were examined by injecting tracer into the left nucleus ambiguous. After 35 days of postnatal IH, basket ending density was reduced by 17% (P < 0.001) and vagal axon varicose contacts by 56% (P < 0.001) compared with controls. We conclude that reduction of vagal efferent projections in cardiac ganglia could be a cause of long-term modifications in baroreflex function.     

Antiangiogenic and Antitumor Effects of Trypanosoma cruzi Calreticulin

Background

In Latin America, 18 million people are infected with Trypanosoma cruzi, the agent of Chagas'' disease, with the greatest economic burden. Vertebrate calreticulins (CRT) are multifunctional, intra- and extracellular proteins. In the endoplasmic reticulum (ER) they bind calcium and act as chaperones. Since human CRT (HuCRT) is antiangiogenic and suppresses tumor growth, the presence of these functions in the parasite orthologue may have consequences in the host/parasite interaction. Previously, we have cloned and expressed T. cruzi calreticulin (TcCRT) and shown that TcCRT, translocated from the ER to the area of trypomastigote flagellum emergence, promotes infectivity, inactivates the complement system and inhibits angiogenesis in the chorioallantoid chicken egg membrane. Most likely, derived from these properties, TcCRT displays in vivo inhibitory effects against an experimental mammary tumor.

Methodology and Principal Findings

TcCRT (or its N-terminal vasostatin-like domain, N-TcCRT) a) Abrogates capillary growth in the ex vivo rat aortic ring assay, b) Inhibits capillary morphogenesis in a human umbilical vein endothelial cell (HUVEC) assay, c) Inhibits migration and proliferation of HUVECs and the human endothelial cell line Eahy926. In these assays TcCRT was more effective, in molar terms, than HuCRT: d) In confocal microscopy, live HUVECs and EAhy926 cells, are recognized by FITC-TcCRT, followed by its internalization and accumulation around the host cell nuclei, a phenomenon that is abrogated by Fucoidin, a specific scavenger receptor ligand and, e) Inhibits in vivo the growth of the murine mammary TA3 MTXR tumor cell line.

Conclusions/Significance

We describe herein antiangiogenic and antitumor properties of a parasite chaperone molecule, specifically TcCRT. Perhaps, by virtue of its capacity to inhibit angiogenesis (and the complement system), TcCRT is anti-inflammatory, thus impairing the antiparasite immune response. The TcCRT antiangiogenic effect could also explain, at least partially, the in vivo antitumor effects reported herein and the reports proposing antitumor properties for T. cruzi infection.     

Immunodetection of the bacteriocin lacticin RM: analysis of the influence of temperature and Tween 80 on its expression and activity

Immunoassays with specific antibodies offer higher sensitivity than do bioassays with indicator strains in the detection and quantification of several bacteriocins. Here we present the purification of lacticin RM and the production of specific polyclonal antibodies to a synthetic peptide resembling an internal fragment of the mature bacteriocin. The specificity and sensitivity of the generated polyclonal antibodies were evaluated in various immunoassays. The detection limits of lacticin RM were found to be 1.9, 0.16, and 0.18 micro g ml(-1) for Western blot, immuno-dot blot, and noncompetitive indirect enzyme-linked immunosorbent assays, respectively. Immunoassay sensitivities were 12.5-fold higher than that of the agar diffusion test (ADT). The production of lacticin RM showed temperature dependency, with 3, 4.2, 12.7, 28.9, 37.8, and 12 micro g ml(-1) at 37, 30, 20, 15, 10, and 4 degrees C, respectively. Temperature-stability analysis demonstrated that lacticin RM is sensitive to mild temperature, but the loss of activity does not seem to result from protein degradation. Tween 80 increased the concentration of lacticin RM eightfold and probably affected the results of the ADT either by enhancing the activity of lacticin RM or by increasing the sensitivity of the indicator strain. The use of antibodies for the specific detection and quantification of lacticin RM can expand our knowledge of its production and stability, with important implications for further investigation and future application.     

Carbonic anhydrase activity and photosynthetic rate in the tree species Paulownia tomentosa Steud. Effect of dimethylsulfoxide treatment and zinc accumulation in leaves

The enzyme carbonic anhydrase (CA) (EC 4.2.1.1) catalyzes the reversible conversion of CO2 to HCO3- and has been shown to be involved in photosynthesis. The enzyme has been shown in animals, plants, eubacteria and viruses, but similar reports on the evidence for CA activity in tree plants does not be appear to be available. In the preliminary analyses of the work, the CA activity in leaf extracts from the tree species Paulownia tomentosa Steud. (introduced in Bulgaria) is described. A connection between CA activity and the rate of photosynthetic CO2 fixation is shown. In the second portion of the work, the effect of 10(-4) mol/L and 10(-2) mol/L dimethylsulfoxide (DMSO) on the zinc accumulation in leaves is demonstrated. It is suggested that CA activity is an indicator of the level of physiologically active zinc in leaves of P. tomentosa Steud. A connection between the process of zinc accumulation in leaves and the activity of the enzymes CA and glycolate oxidase (GO) (EC 1.1.3.1) is established.     

Development of new insulin-like growth factor-1 receptor kinase inhibitors using catechol mimics

Because insulin-like growth factor-1 (IGF-1) and its receptor play a pivotal role in many cancers, it is an attractive target for the design of inhibitors. Here we present a new family of bioisostere inhibitors, based on the structure of AG 538. AG 538 is a substrate-competitive inhibitor of the IGF-1 receptor (IGF-1R), with an IC50 = 61 nM in a cell-free kinase assay (Blum, G., Gazit, A., and Levitzki, A. (2000) Biochemistry 39, 15705-15712). AG 538 is a low molecular weight compound containing two catechol rings, which are sensitive to oxidation in cells. We have therefore prepared and examined catechol bioisosteres of AG 538. These AG 538 bioisosteres possess similar biological properties to AG 538; they inhibit IGF-1R by a substrate-competitive mechanism and are non-competitive vis à vis ATP. They inhibit IGF-1R kinase activity in the sub-micromolar concentration range in cell-free assays. IGF-1 induced IGF-1R autophosphorylation; IRS-1 phosphorylation and protein kinase B activation are inhibited at a low micromolar concentration range when applied to intact cells. These inhibitors also block the formation of colonies in soft agar by prostate and breast cancer cells. The ability to replace catechol groups with a moiety that is more stable in cells may aid in developing non-catechol-containing substrate-competitive inhibitors targeted toward IGF-1R and possibly against other protein-tyrosine kinases.