The role of calcineurin/NFAT in SFRP2 induced angiogenesis--a rationale for breast cancer treatment with the calcineurin inhibitor tacrolimus

Tacrolimus (FK506) is an immunosuppressive drug that binds to the immunophilin FKBPB12. The FK506-FKBP12 complex associates with calcineurin and inhibits its phosphatase activity, resulting in inhibition of nuclear translocation of nuclear factor of activated T-cells (NFAT). There is increasing data supporting a critical role of NFAT in mediating angiogenic responses stimulated by both vascular endothelial growth factor (VEGF) and a novel angiogenesis factor, secreted frizzled-related protein 2 (SFRP2). Since both VEGF and SFRP2 are expressed in breast carcinomas, we hypothesized that tacrolimus would inhibit breast carcinoma growth. Using IHC (IHC) with antibodies to FKBP12 on breast carcinomas we found that FKBP12 localizes to breast tumor vasculature. Treatment of MMTV-neu transgenic mice with tacrolimus (3 mg/kg i.p. daily) (n?=?19) resulted in a 73% reduction in the growth rate for tacrolimus treated mice compared to control (n?=?15), p?=?0.003; which was associated with an 82% reduction in tumor microvascular density (p<0.001) by IHC. Tacrolimus (1 μM) inhibited SFRP2 induced endothelial tube formation by 71% (p?=?0.005) and inhibited VEGF induced endothelial tube formation by 67% (p?=?0.004). To show that NFATc3 is required for SFRP2 stimulated angiogenesis, NFATc3 was silenced with shRNA in endothelial cells. Sham transfected cells responded to SFRP2 stimulation in a tube formation assay with an increase in the number of branch points (p<0.003), however, cells transfected with shRNA to NFATc3 showed no increase in tube formation in response to SFRP2. This demonstrates that NFATc3 is required for SFRP2 induced tube formation, and tacrolimus inhibits angiogenesis in vitro and breast carcinoma growth in vivo. This provides a rationale for examining the therapeutic potential of tacrolimus at inhibiting breast carcinoma growth in humans.     

Staphylococcus aureus Alpha Toxin Suppresses Effective Innate and Adaptive Immune Responses in a Murine Dermonecrosis Model

An optimal host response against Staphylococcus aureus skin and soft tissue infections (SSTI) is dependent on IL-1β and IL-17 mediated abscess formation. Alpha toxin (AT), an essential virulence factor for SSTI, has been reported to damage tissue integrity; however its effect on the immune response has not been investigated. Here, we demonstrate that infection with USA300 AT isogenic mutant (Δhla), or passive immunization with an AT neutralizing mAb, 2A3, 24 h prior to infection with wild type USA300 (WT), resulted in dermonecrotic lesion size reduction, and robust neutrophil infiltration. Infiltration correlates with increase in proinflammatory cytokines and chemokines, as well as enhanced bacterial clearance relative to immunization with a negative control mAb. In addition, infection with Δhla, or with WT +2A3, resulted in an early influx of innate IL-17⁺γδT cells and a more rapid induction of an adaptive immune response as measured by Th1 and Th17 cell recruitment at the site of infection. These results are the first direct evidence of a role for AT in subverting the innate and adaptive immune responses during a S. aureus SSTI. Further, these effects of AT can be overcome with a high affinity anti-AT mAb resulting in a reduction in disease severity.     

Cuticular wax biosynthesis in petunia petals: cloning and characterization of an alcohol-acyltransferase that synthesizes wax-esters

The surface of plants is covered by cuticular wax, which contains a mixture of very long-chain fatty acid (VLCFA) derivatives. This wax surface provides a hydrophobic barrier which reduces non-stomatal water loss. One component of the cuticular wax is the alkyl esters, which typically contain a VLCFA esterified to an alcohol of a similar length. As part of an EST project, we recently identified an acyltransferase with 19% sequence identity (amino acid) to a bacterial ‘bifunctional’ wax-ester synthase/diacylglycerol acyltransferase (WS/DGAT). Northern analysis revealed that this petunia homologue was expressed predominantly within the petals. The cDNA encoding the WS/DGAT homologue was introduced into a yeast strain deficient in triacylglycerol biosynthesis. The expressed protein failed to restore triacylglycerol biosynthesis, indicating that it lacked DGAT activity. However, isoamyl esters of fatty acids were detected, which suggested that the petunia cDNA encoded a wax-synthase. Waxes were extracted from petunia petals and leaves. The petal wax extract was rich in VLCFA esters of methyl, isoamyl, and short-to-medium straight chain alcohols (C4–C12). These low molecular weight wax-esters were not present in leaf wax. In-vitro enzymes assays were performed using the heterologously expressed protein and ¹⁴C-labelled substrates. The expressed protein was membrane bound, and displayed a preference for medium chain alcohols and saturated very long-chain acyl-CoAs. In fact, the activity would be sufficient to produce most of the low molecular wax-esters present in petals, with methyl-esters being the exception. This work is the first characterization of a eukaryotic protein from the WS/DGAT family. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Proteome cataloging and relative quantification of Francisella tularensis tularensis strain Schu4 in 2D PAGE using preparative isoelectric focusing

The protein complement of whole cell extract of the bacterium Francisella tularensis tularensis was analyzed using two-dimensional electrophoresis with preparative isoelectric focusing in the first dimension. The format allows the quantification of relative protein abundance by linear densitometry and extends the potential dynamic range of protein detection by as much as an order of magnitude. The relative abundance and rank order of 136 unique proteins identified in F. tularensis tularensis were established. It is estimated that 16% of the moderately to highly expressed proteins and 8% of all predicted non-pseudogenes were identified by comparing this proteome information with the relative abundance of mRNA as measured by microarray. This rank-ordered proteome list provides an important resource for understanding the pathogenesis of F. tularensis and is a tool for the selection and design of synthetic vaccines. This method represents a useful additional technique to improve whole proteome analyses of simple organisms.     

Chromosome aberrations in Chinese hamster and human cells: a comparison using compounds with various genotoxicity profiles

Chromosome aberrations (Cabs) can be induced in vitro by non-DNA damaging compounds, often associated with cytotoxicity and DNA synthesis inhibition, and under conditions that would not be relevant in vivo. Such misleading positive results are reported both in Chinese hamster cell lines and in human peripheral blood lymphocytes (HL). We assessed the response of HL to compounds with varied genetic toxicity profiles, all of which induced Cabs in CHO cells Seven of 10 compounds were negative or equivocal in HL. Results in purified lymphocytes for four verified that the difference was not due to the presence of blood in cultures. Two compounds that were weakly positive in the Ames test and one that induced DNA adducts were negative or equivocal in the HL assay; their overall mutagenic potential in vivo is not clear. Of four Ames-negative compounds, three of which inhibited DNA synthesis in CHO cells, three were negative and one was equivocal in the HL assay. A potent Cab inducer, which also induced micronuclei in vivo (but was negative in the Ames test) was clearly positive in the HL assay. Two compounds were clearly positive in HL only when the mitotic indices (MI) were below 50% of control. These are genotoxic in other assays but our evidence suggests that Cab induction is related more to toxicity than to primary DNA damage. For this limited set of 10 compounds, HL were more likely than CHO cells to give negative or equivocal results. It is likely that more stringent checkpoint controls in human cells prevent damaged cells reaching mitosis, and may also influence the reported greater sensitivity to induction of aneuploidy and polyploidy of normal rodent compared with human cells. In the studies reported here, two strong inducers of polyploidy in CHO cells gave weaker increases in HL. Human lymphocytes have disadvantages as a routine screening assay (finding donors, known individual variability, increased time required and the inadequacy of the MI as a toxicity measure), but may be useful in follow-up testing to assess weight of evidence about genotoxic risk to humans, for compounds that are positive in the Chinese hamster cell Cabs assays.     

Effects of social reorganization on cellular immunity in male cynomolgus monkeys

Exposure to acute stressors has been shown to impair cellular immunity in human beings and other animal species. Comparatively little is known, however, about the effects of long-term stressors on immune function and how individual behavioral characteristics may mediate differences in immune function and clinical disease susceptibility. To determine the effects of social stress on cellular immunity and reactivation of a latent herpesvirus, 20 Herpes B virus-positive male cynomolgus monkeys were exposed to four periodic reorganizations of social group memberships over 5 months. Observations were made to categorize individuals as high or low in expression of aggressive, fearful, and affiliative behaviors. Complete blood counts, lymphocyte proliferation tests, and natural killer cell cytotoxicity assays were performed immediately before and 4 days after reorganizations. Herpesvirus-specific immunoglobulin G antibody levels were measured, and oral and conjunctival swabs were cultured for virus. Reorganization was associated with increased lymphocyte counts (P = 0.0009) and decreased lymphocyte proliferation in response to phytohemagglutinin (P < 0.005), particularly among monkeys showing high levels of fear (P = 0.0137). High-aggressive monkeys showed lower baseline natural killer cell activity (P = 0.0013) and higher lymphocyte counts (P = 0.013) than low-aggressive monkeys. Herpesvirus antibody titers decreased over time (P < 0.004) and no positive virus cultures were obtained. Measures of cellular immunity and behavior were unrelated to virus-specific antibody titers. These results suggest that repeated exposure to a social stressor alters several measures of cellular immunity, and that some of these changes may be predicted by individual differences in agonistic behavior. In contrast to human studies, the results suggest that some psychological stressors may not cause reactivation of a common herpesvirus in this species. © 1996 Wiley-Liss, Inc.     

Ultrastructure of human labial salivary glands. I. Acinar secretory cells

The structure of human labial salivary gland acini was studied by light and electron microscopy. Contrary to previous reports, these glands were pure mucous in nature; no serous elements were present. The acinar cells were found in all stages of maturation. Immature cells were characterized by an extensive and highly organized rough-surfaced endoplasmic reticulum. The Golgi complex was extremely prominent, consisting of stacks of flattened cisternae and swarms of small vesicles. Mucous droplets were almost completely absent. As secretory activity progressed, the endoplasmic reticulum involuted, while the Golgi cisternae became distended and formed many vacuoles. In mature mucous cells, the apical cytoplasm was filled with membrane-bounded mucous droplets, and the nucleus was displaced basally. The droplets frequently showed great variation in density from cell to cell, and even within the same cell they sometimes were quite heterogeneous. They were liberated from the acinar cells by an apocrine process, so that droplets with intact limiting membranes were often observed in the acinar lumen. These droplets soon lysed, their contents fusing into streams of mucus. Occasionally during apocrine secretion a mucous cell failed to reconstitute its apical surface, and its entire contents spilled into the acinar lumen. Unusual cytoplasmic inclusions were present in many of the acinar cells. These inclusions, which were surrounded by a single membrane, consisted of lipid droplets closely associated with bundles of fine filaments.     

Eine Elektrode zur kontinuierlichen Messung des Gelöstsauerstoffs (pO2) in Fermenterkulturen

Zusammenfassung Die Arbeit beschreibt Aufbau und Eigenschaften einer Elektrode zur kontinuierlichen Messung des Sauerstoffpartialdruckes in Fermenterkulturen. Die nach dem polarographischen Prinzip arbeitende Elektrode leitet sich von der Clarkschen Meßanordnung ab. Ihr wesentliches Merkmal ist die Auftrennung in zwei selbständige Bauelemente, einen Membran-tragenden Glasmantel und den Elektroden-tragenden Meßgeber. Der Elektrodenmantel wird gemeinsam mit dem Fermentergefäß autoklaviert, der thermolabile Meßfühler dagegen erst nachträglich eingesetzt. Die Eichung der Elektrode kann vor dem Autoklavieren in einem Spezialgefäß oder danach im Fermentergefäß vorgenommen werden. Der sterilisierbare, in die Kulturlösung hineinragende Elektrodenmantel wird von der Lösung durch eine O₂-permeable, 250 starke Siliconglasgewebemembran abgeschlossen. Zwischen Siliconschicht und der blanken Platinoberfläche der Kathode befindet sich eine weitere, stabilisierende Membran von 12 Stärke (Cellophanfolie). Die Empfindlichkeit der Elektrode beträgt 10^–9 A/mm Hg pO₂ bei 37°C, die Einstellzeit auf 95% des Endwertes 50 sec bei 37°C. Die Abhängigkeit der Messungen von der Turbulenz des Mediums ist zwischen 400 und 1200 UpM Rührergeschwindigkeit zu vernachlässigen. Wiederholtes Autoklavieren der Siliconmembran hat keinen Einfluß auf die Einstellzeiten.

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An electrode for continuous measurement of oxygen tension in fermenter cultures

Summary The paper describes the construction and properties of an electrode for continuous measurement of oxygen tension in fermenter cultures. The equipment is a modified Clark electrode with a cathode of 99,99% pure Pt and a silver anode. It works polarographically. Its most outstanding feature is the subdivision into two separate construction elements: an outer glass-envelope containing a membrane, and a membrane-carrying electrode. The glass-envelope is autoclavable together with the fermenter vessel, whereas the thermolabile electrode must be inserted into the sterilized glass-envelope. The electrode can be calibrated either in a special vessel before autoclaving the glass envelope, or after sterilization in the air-saturated sterile culture medium, before inoculation.The steam resistent glass-envelope, which is immersed in the culture medium, is covered on the lower part with a silicone membrane of 250 thickness which is stabilized by glass fibers. The silicone membrane is separated from the polished Pt cathode by a second stabilizing membrane (cellophane) of 12 thickness, which is part of the electrode. The sensitivity of the electrode is 10^–9 A/mm Hg pO₂ at 37° C. When the oxygen tension is changed from 0 to 100%, the electrode follows this change up to 95% within 50 sec at 37° C. The reading is nearly independent of the agitation of the medium between 400 and 1,200 rpm of the impeller turbine in the culture vessel. Repeated steam-sterilization has no influence on the response time of the electrode.

     

Interspecies exchange of β 2-microglobulin and associated MHC and differentiation antigens

Radiolabeled human ₂-microglobulin (₂m) can bind to mouse histocompatibility (H-2) antigens on the cell surface or to partially purified H-2 antigens in solution. The complexes containing human ₂m and H-2 antigens from C3H (H-2^k) mice could be immunoprecipitated specifically with alloantisera, rabbit anti-H-2 xenoantisera, and with monoclonal H-2-specific antibodies. Specific association with H-2 antigens was also observed with other haplotypes. The only exception was B10.D2 (H-2 ^d ) from which complexes containing human ₂M could only be precipitated with anti-H-2 xenosera. Thus radiolabeled human ₂M can be used as a specific label for mouse H-2 antigens in precipitation and radioimmunoassays. The application of this finding extends to major histocompatibility complex antigens of other species, and to differentiation antigens with primary association with ₂m.Abbreviations used in this paper MHC major histocompatibility complex - ₂m ₂-microglobulin - LcH Lens culinaris hemagglutinin