Platelets Induce Apoptosis during Sepsis in a Contact-Dependent Manner That Is Inhibited by GPIIb/IIIa Blockade

Purpose

End-organ apoptosis is well-described in progressive sepsis and Multiple Organ Dysfunction Syndrome (MODS), especially where platelets accumulate (e.g. spleen and lung). We previously reported an acute sepsis-induced cytotoxic platelet phenotype expressing serine protease granzyme B. We now aim to define the site(s) of and mechanism(s) by which platelet granzyme B induces end-organ apoptosis in sepsis.

Methods

End-organ apoptosis in murine sepsis (i.e. polymicrobial peritonitis) was analyzed by immunohistochemistry. Platelet cytotoxicity was measured by flow cytometry following 90 minute ex vivo co-incubation with healthy murine splenocytes. Sepsis progression was measured via validated preclinical murine sepsis score.

Measurements and Main Results

There was evident apoptosis in spleen, lung, and kidney sections from septic wild type mice. In contrast, there was a lack of TUNEL staining in spleens and lungs from septic granzyme B null mice and these mice survived longer following induction of sepsis than wild type mice. In co-incubation experiments, physical separation of septic platelets from splenocytes by a semi-permeable membrane reduced splenocyte apoptosis to a rate indistinguishable from negative controls. Chemical separation by the platelet GPIIb/IIIa receptor inhibitor eptifibatide decreased apoptosis by 66.6±10.6% (p = 0.008). Mice treated with eptifibatide in vivo survived longer following induction of sepsis than vehicle control mice.

Conclusions

In sepsis, platelet granzyme B-mediated apoptosis occurs in spleen and lung, and absence of granzyme B slows sepsis progression. This process proceeds in a contact-dependent manner that is inhibited ex vivo and in vivo by the platelet GPIIb/IIIa receptor inhibitor eptifibatide. The GPIIb/IIIa inhibitors and other classes of anti-platelet drugs may be protective in sepsis.     

Blubber morphology in wild bottlenose dolphins (Tursiops truncatus) from the Southeastern United States: influence of geographic location, age class, and reproductive state

This study investigated blubber morphology and correlations of histological measurements with ontogeny, geography, and reproductive state in live, wild bottlenose dolphins (Tursiops truncatus) from the southeastern United States. Surgical skin-blubber biopsies (N=74) were collected from dolphins during capture-release studies conducted in two geographic locations: Charleston, SC (N=38) and Indian River Lagoon, FL (N=36). Histological analysis of blubber revealed stratification into superficial, middle, and deep layers. Adipocytes of the middle blubber were 1.6x larger in Charleston subadults than in Indian River Lagoon subadults (4,590+/-340 compared to 2,833+/-335 microm2 per cell). Charleston subadult dolphins contained higher levels of total blubber lipids than Charleston adult animals (49.3%+/-1.9% compared to 34.2%+/-1.7%), and this difference was manifested in more adipocytes in the middle blubber layer (19.2+/-0.9 compared to 14.9+/-0.5 cells per field). However, dolphins from Indian River Lagoon did not exhibit this pattern, and the adipocyte cell counts of subadults were approximately equal to those of the adults (16.0+/-1.4 compared to 13.4+/-0.8 cells per field). The colder year-round water temperatures in Charleston compared to Indian River Lagoon may explain these differences. Adipocytes in the deep blubber layer were significantly smaller in lactating and simultaneously pregnant and lactating animals compared to pregnant dolphins (840+/-179, 627+/-333, and 2,776+/-586 microm2 per cell, respectively). Total blubber lipid content and adipocyte size in the deep blubber of mothers with calves decreased linearly with calf length. Lactating females may utilize lipids from the deep blubber during periods of increased energetic demands associated with offspring care. This study demonstrates that ontogeny, geography, and reproductive state may influence morphological parameters such as structural fiber densities and adipocyte numbers and sizes, measured in bottlenose dolphin blubber.     

Do male ornaments signal immunity in the common yellowthroat?

Male ornaments may advertise genetic benefits to females choosingmates. These benefits may come in the form of genes for resistanceto parasites and disease. Thus, females that prefer more ornamentedmales as mates may receive genes for enhanced immune systemfunction for their offspring. The common yellowthroat (Geothlypistrichas) is the only species to date in which a male plumageornament (size of the black facial mask) is known to be relatedto extrapair mating, and extrapair young are more immunocompetentthan their within-pair half-sibs (at least in colder years).To investigate whether male mask size signals superior immunefunction, we examined male ornaments (mask and bib size andcolor) in relation to measures of overall health (hematocrit)and immune system function (plasma immunoglobulin G [IgG] concentrationand cutaneous immune activity). We also investigated the rolethat testosterone may play in mediating the relationship betweenornaments and immunity. Male mask size was correlated positivelywith IgG level, suggesting that male mask size may indicatehumoral immunity. However, mask size was correlated negativelywith hematocrit and cutaneous immune activity (our measure ofnestling immunocompetence). Bib size and color were not relatedto these indices of immune function or health. Plasma testosteronelevel was neither related to immune function or health nor tothe expression of male ornaments. These results suggest thatthere might be a trade-off between immune system components,as well as between immunity and the production or advertisementof male ornaments.     

Physiological and molecular characterization of aluminum resistance in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

Background  

Aluminum (Al) toxicity is an important factor limiting crop production on acid soils. However, little is known about the mechanisms by which legumes respond to and resist Al stress. To explore the mechanisms of Al toxicity and resistance in legumes, we compared the impact of Al stress in Al-resistant and Al-sensitive lines of the model legume, Medicago truncatula Gaertn.     

Mechanisms of metal resistance in plants: aluminum and heavy metals

Plants have evolved sophisticated mechanisms to deal with toxic levels of metals in the soil. In this paper, an overview of recent progress with regards to understanding fundamental molecular and physiological mechanisms underlying plant resistance to both aluminum (Al) and heavy metals is presented. The discussion of plant Al resistance will focus on recent advances in our understanding of a mechanism based on Al exclusion from the root apex, which is facilitated by Al-activated exudation of organic acid anions. The consideration of heavy metal resistance will focus on research into a metal hyperaccumulating plant species, the Zn/Cd hyperaccumulator, Thlaspi caerulescens, as an example for plant heavy metal research. Based on the specific cases considered in this paper, it appears that quite different strategies are used for Al and heavy metal resistance. For Al, our current understanding of a resistance mechanism based on excluding soil-borne Al from the root apex is presented. For heavy metals, a totally different strategy based on extreme tolerance and metal hyperaccumulation is described for a hyperaccumulator plant species that has evolved on naturally metalliferous soils. The reason these two strategies are the focus of this paper is that, currently, they are the best understood mechanisms of metal resistance in terrestrial plants. However, it is likely that other mechanisms of Al and/or heavy metal resistance are also operating in certain plant species, and there may be common features shared for dealing with Al and heavy resistance. Future research may uncover a number of novel metal resistance mechanisms in plants. Certainly the complex genetics of Al resistance in some crop plant species, such as rice and maize, suggests that a number of presently unidentified mechanisms are part of an overall strategy of metal resistance in crop plants.     

Initial verification of the resistance management strategy for Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) in Australia

Shortly after the initial detection of western flower thrips (WFT), Frankiniella occidentalis (Pergande), in Australia during 1993 a resistance management strategy based on the alternation of chemical groups was implemented. This study aimed to verify this strategy by field testing α-cypermethrin against WFT with and without chemical alternation. Up to 114 times α-cypermethrin resistance (at LC50) was detected and resistance increased with and without chemical alternation; however, chemical alternation did significantly reduce numbers of thrips compared with a nonalternation strategy. Resistance has the potential to undermine the sustainable use of those chemicals to which there is no current detectable resistance. Consequently, chemicals with a high frequency and level of resistance against WFT need to be identified through monitoring and quickly eliminated from WFT chemical control recommendations.     

A simple method for studying whole sections of rice grain

We developed a new and simple method to collect sections of a whole brown rice kernel for investigation of histological properties. A single kernel of rice was dehydrated through a graded ethanol series, transferred to xylene, and embedded in paraffin. During sectioning of the blocks using a rotary microtome, we used a special adhesive tape to collect and place the sections on slides so they remained flat. The use of the adhesive tape technique combined with autofluorescence characteristics allowed us to visualize cell walls throughout an entire longitudinal or transverse section of a whole rice kernel. We obtained scanning electron microscopy images of the sections to determine section quality.     

Phylogenetic Studies in the Monocot Subclass Alismatidae: Evidence for a Reappraisal of the Aquatic Order Najadales

Within the angiosperm subclass Alismatidae (= superorder Alismatiflorae), contemporary taxonomists have often assigned the families Hydrocharitaceae and Najadaceae to different orders. The Najadaceae are presumably allied to a variety of aquatic families in the order Najadales, whereas the Hydrocharitaceae have been segregated as the order Hydrocharitales or placed within the order Alismatales. Analyses of DNA sequence data from the chloroplast gene rbcL, however, indicate that Najadaceae have a much closer phylogenetic relationship to Hydrocharitaceae than to families of the "Najadales" (Cymodoceaceae, Potamogetonaceae, Ruppiaceae, Scheuchzeriaceae, Zannichelliaceae, Zosteraceae). This association supports previous studies based upon examination of floral structure and seed coat anatomy. The rbcL sequence data also indicate that the Zosteraceae and Potamogetonaceae are closely related families. The rbcL sequence of Zostera is actually more similar to that of Potamogeton richardsonii than is the sequence of the latter to a congener, Potamogeton amplifolius. The marine, dioecious, hydrophilous genus Zostera has acquired a number of distinctive adaptations, but probably diverged relatively rapidly from freshwater Potamogetonaceae. Molecular data place Ruppiaceae as a sister group to the marine Cymodoceaceae and do not support the commonly accepted merger of Ruppiaceae and Potamogetonaceae.     

Growth factor binding and bioactivity in human kidney epithelial cell cultures

Summary Insulinlike growth factors (IGF) and epidermal growth factor (EGF) are produced in renal tissue, as are specific receptors for these hormones. To evaluate the significance of these observations to regulation of renal tubular cell proliferation, we have examined the interaction of IGF and EGF with cultured human proximal tubular epithelial cells (HPT). HPT cells showed specific binding of IGF-1, insulin, and EGF. IGF-1 binding was inhibited by antibody to the type 1 IGF receptor (α-IR3). Insulin receptors and type 1 IGF receptors were identified by bifunctional cross-linking. IGF-1, insulin, and EGF stimulated [³H]thymidine incorporation by 77, 73, and 87%, respectively. Haft maximal stimulation by IGF-1, insulin, and EGF was produced with 4×10⁻⁹ M, 2.5×10⁻⁸ M, and 8×10⁻¹⁰ M concentrations of these hormones. α-IR3 inhibited stimulation of thymidine incorporation by IGF-1 and insulin but had no effect in EGF-stimulated thymidine incorporation. EGF and high concentrations of insulin both stimulated cell proliferation by 83 and 79%, respectively. These data are consistent with regulation of tubular epithelial proliferation by IGF-1, insulin, and EGF and suggest that the mitogenic activity of both insulin and IGF-1 is mediated by the type 1 IGF receptor. Supported by grants CA37887 and DK32889 from the National Institutes of Health, Bethesda, MD, and by a Medical University of South Carolina institutional grant.     

The phenotype of mes-2, mes-3, mes-4 and mes-6, maternal-effect genes required for survival of the germline in Caenorhabditis elegans,is sensitive to chromosome dosage.

Mutations in mes-2, mes-3, mes-4, and mes-6 result in maternal-effect sterility: hermaphrodite offspring of mes/mes mothers are sterile because of underproliferation and death of the germ cells, as well as an absence of gametes. Mutant germ cells do not undergo programmed cell death, but instead undergo a necrotic-type death, and their general poor health apparently prevents surviving germ cells from forming gametes. Male offspring of mes mothers display a significantly less severe germline phenotype than their hermaphrodite siblings, and males are often fertile. This differential response of hermaphrodite and male offspring to the absence of mes+ product is a result of their different X chromosome compositions; regardless of their sexual phenotype, XX worms display a more severe germline phenotype than XO worms, and XXX worms display the most severe phenotype. The sensitivity of the mutant phenotype to chromosome dosage, along with the similarity of two MES proteins to chromatin-associated regulators of gene expression in Drosophila, suggest that the essential role of the mes genes is in control of gene expression in the germline. An additional, nonessential role of the mes genes in the soma is suggested by the surprising finding that mutations in the mes genes, like mutations in dosage compensation genes, feminize animals whose male sexual identity is somewhat ambiguous. We hypothesize that the mes genes encode maternally supplied regulators of chromatin structure and gene expression in the germline and perhaps in somatic cells of the early embryo, and that at least some of their targets are on the X chromosomes.