In Vitro Activity of Gentamicin-Loaded Bioabsorbable Beads against Different Microorganisms

Abstract: Osteomyelitis is responsible for high treatment costs, long hospital stays, and results in substantial morbidity. Treatment with surgical debridement and antibiotic-impregnated Polymethylmetacrylate (PMMA) beads is the standard of care, providing high local but low serum antibiotic concentrations, thereby avoiding systemic toxicity. However, for several reasons, the beads require surgical removal. Alternative antibiotic delivery systems should improve the treatment of bone infection, actively encourage bone healing and require no additional surgery for removal. We investigated the activity of gentamicin-loaded bioabsorbable beads against different microorganisms (Staphylococcus epidermidis, S. aureus, Escherichia coli, Enterococcus faecalis, Candida albicans) commonly causing surgical site bone infection, by microcalorimetry. Calcium sulphate beads containing gentamicin were incubated in microcalorimetry ampoules containing different concentrations of the corresponding microorganism. Growth medium with each germ and unloaded beads was used as positive control, growth medium with loaded beads alone as negative control. Bacterial growth-related heat production at 37 °C was measured for 24 h. Cultures without gentamicin-loaded beads produced heat-flow peaks corresponding to the exponential growth of the corresponding microorganisms in nutrient-rich medium. In contrast, cultures with gentamicin-loaded beads completely suppressed heat production during 24 h, demonstrating their antibiotic activity. Gentamicin-loaded beads effectively inhibited growth of susceptible microorganisms, under the described in vitro conditions.

Cycle length of spermatogenesis in shrews (mammalia: soricidae) with high and low metabolic rates and different mating systems.

The aim of the present study was to establish and compare the durations of the seminiferous epithelium cycles of the common shrew Sorex araneus, which is characterized by a high metabolic rate and multiple paternity, and the greater white-toothed shrew Crocidura russula, which is characterized by a low metabolic rate and a monogamous mating system. Twelve S. araneus males and fifteen C. russula males were injected intraperitoneally with 5-bromodeoxyuridine, and the testes were collected. For cycle length determinations, we applied the classical method of estimation and linear regression as a new method. With regard to variance, and even with a relatively small sample size, the new method seems to be more precise. In addition, the regression method allows the inference of information for every animal tested, enabling comparisons of different factors with cycle lengths. Our results show that not only increased testis size leads to increased sperm production, but it also reduces the duration of spermatogenesis. The calculated cycle lengths were 8.35 days for S. araneus and 12.12 days for C. russula. The data obtained in the present study provide the basis for future investigations into the effects of metabolic rate and mating systems on the speed of spermatogenesis.

High Expression of hTERT and Stemness Genes in BORIS/CTCFL Positive Cells Isolated from Embryonic Cancer Cells.

BORIS/CTCFL is a member of cancer testis antigen family normally expressed in germ cells. In tumors, it is aberrantly expressed although its functions are not completely well-defined. To better understand the functions of BORIS in cancer, we selected the embryonic cancer cells as a model. Using a molecular beacon, which specifically targets BORIS mRNA, we demonstrated that BORIS positive cells are a small subpopulation of tumor cells (3-5% of total). The BORIS-positive cells isolated using BORIS-molecular beacon, expressed higher telomerase hTERT, stem cell (NANOG, OCT4, SOX2) and cancer stem cell marker genes (CD44 and ALDH1) compared to the BORIS-negative tumor cells. In order to define the functional role of BORIS, stable BORIS-depleted embryonic cancer cells were generated. BORIS silencing strongly down-regulated the expression of hTERT, stem cell and cancer stem cell marker genes. Moreover, the BORIS knockdown increased cellular senescence in embryonic cancer cells, revealing a putative role of BORIS in the senescence biological program. Our data indicate an association of BORIS expressing cells subpopulation with the expression of stemness genes, highlighting the critical role played by BORIS in embryonic neoplastic disease.

Edar/Eda interactions regulate enamel knot formation in tooth morphogenesis.

tabby and downless mutant mice have apparently identical defects in teeth, hair and sweat glands. Recently, genes responsible for these spontaneous mutations have been identified. downless (Dl) encodes Edar, a novel member of the tumour necrosis factor (TNF) receptor family, containing the characteristic extracellular cysteine rich fold, a single transmembrane region and a death homology domain close to the C terminus. tabby (Ta) encodes ectodysplasin-A (Eda) a type II membrane protein of the TNF ligand family containing an internal collagen-like domain. As predicted by the similarity in adult mutant phenotype and the structure of the proteins, we demonstrate that Eda and Edar specifically interact in vitro. We have compared the expression pattern of Dl and Ta in mouse development, taking the tooth as our model system, and find that they are not expressed in adjacent cells as would have been expected. Teeth develop by a well recorded series of epithelial-mesenchymal interactions, similar to those in hair follicle and sweat gland development, the structures found to be defective in tabby and downless mice. We have analysed the downless mutant teeth in detail, and have traced the defect in cusp morphology back to initial defects in the structure of the tooth enamel knot at E13. Significantly, the defect is distinct from that of the tabby mutant. In the tabby mutant, there is a recognisable but small enamel knot, whereas in the downless mutant the knot is absent, but enamel knot cells are organised into a different shape, the enamel rope, showing altered expression of signalling factors (Shh, Fgf4, Bmp4 and Wnt10b). By adding a soluble form of Edar to tooth germs, we were able to mimic the tabby enamel knot phenotype, demonstrating the involvement of endogenous Eda in tooth development. We could not, however, reproduce the downless phenotype, suggesting the existence of yet another ligand or receptor, or of ligand-independent activation mechanisms for Edar. Changes in the structure of the enamel knot signalling centre in downless tooth germs provide functional data directly linking the enamel knot with tooth cusp morphogenesis. We also show that the Lef1 pathway, thought to be involved in these mutants, functions independently in a parallel pathway.

An Essential Role for Insulin and IGF1 Receptors in Regulating Sertoli Cell Proliferation, Testis Size, and FSH Action in Mice.

Testis size and sperm production are directly correlated to the total number of adult Sertoli cells (SCs). Although the establishment of an adequate number of SCs is crucial for future male fertility, the identification and characterization of the factors regulating SC survival, proliferation, and maturation remain incomplete. To investigate whether the IGF system is required for germ cell (GC) and SC development and function, we inactivated the insulin receptor (Insr), the IGF1 receptor (Igf1r), or both receptors specifically in the GC lineage or in SCs. Whereas ablation of insulin/IGF signaling appears dispensable for GCs and spermatogenesis, adult testes of mice lacking both Insr and Igf1r in SCs (SC-Insr;Igf1r) displayed a 75% reduction in testis size and daily sperm production as a result of a reduced proliferation rate of immature SCs during the late fetal and early neonatal testicular period. In addition, in vivo analyses revealed that FSH requires the insulin/IGF signaling pathway to mediate its proliferative effects on immature SCs. Collectively, these results emphasize the essential role played by growth factors of the insulin family in regulating the final number of SCs, testis size, and daily sperm output. They also indicate that the insulin/IGF signaling pathway is required for FSH-mediated SC proliferation.

Fas (CD95/APO-1) limits the expansion of T lymphocytes in an environment of limited T-cell antigen receptor/MHC contacts.

Fas-deficient mice (Fas(lpr/lpr)) and humans have profoundly dysregulated T lymphocyte homeostasis, which manifests as an accumulation of CD4(+) and CD8(+) T cells as well as an unusual population of CD4(-)CD8(-)TCRαβ(+) T cells. To date, no unifying model has explained both the increased T-cell numbers and the origin of the CD4(-)CD8(-)TCRαβ(+) T cells. As Fas(lpr/lpr) mice raised in a germ-free environment still manifest lymphadenopathy, we considered that this process is primarily driven by recurrent low-avidity TCR signaling in response to self-peptide/MHC as occurs during homeostatic proliferation. In these studies, we developed two independent systems to decrease the number of self-peptide/MHC contacts. First, expression of MHC class I was reduced in OT-I TCR transgenic mice. Although OT-I Fas(lpr/lpr) mice did not develop lymphadenopathy characteristic of Fas(lpr/lpr) mice, in the absence of MHC class I, OT-I Fas(lpr/lpr) T cells accumulated as both CD8(+) and CD4(-)CD8(-) T cells. In the second system, re-expression of β(2)m limited to thymic cortical epithelial cells of Fas(lpr/lpr) β(2)m-deficient mice yielded a model in which polyclonal CD8(+) thymocytes entered a peripheral environment devoid of MHC class I. These mice accumulated significantly greater numbers of CD4(-)CD8(-)TCRαβ(+) T cells than conventional Fas(lpr/lpr) mice. Thus, Fas shapes the peripheral T-cell repertoire by regulating the survival of a subset of T cells proliferating in response to limited self-peptide/MHC contacts.

PPARgamma in placental angiogenesis.

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor involved in diverse biological processes including adipocyte differentiation, glucose homeostasis, and inflammatory responses. Analyses of PPARγ knockout animals have been so far preempted by the early embryonic death of PPARγ-/- embryos as a consequence of the severe alteration of their placental vasculature. Using Sox2Cre/PPARγL2/L2 mice, we obtained fully viable PPARγ-null mice through specific and total epiblastic gene deletion, thereby demonstrating that the placental defect is the unique cause of PPARγ-/- embryonic lethality. The vasculature defects observed in PPARγ-/- placentas at embryonic d 9.5 correlated with an unsettled balance of pro- and antiangiogenic factors as demonstrated by increased levels of proliferin (Prl2c2, PLF) and decreased levels of proliferin-related protein (Prl7d1, PRP), respectively. To analyze the role of PPARγ in the later stage of placental development, when its expression peaks, we treated pregnant wild-type mice with the PPARγ agonist rosiglitazone. This treatment resulted in a disorganization of the placental layers and an altered placental microvasculature, accompanied by the decreased expression of proangiogenic genes such as Prl2c2, vascular endothelial growth factor, and Pecam1. Together our data demonstrate that PPARγ plays a pivotal role in controlling placental vascular proliferation and contributes to its termination in late pregnancy.

Telomerase activation in colorectal carcinogenesis.

Telomerase activity has been detected in germ cells as well as in the developing embryo. Activity is no longer detectable in most somatic cells of the neonate, although low levels of activity persist in regenerative tissues. Telomerase has been found to be reactivated or up-regulated in the majority of cancers. The colorectal adenoma-carcinoma sequence is one of the best-characterized models of multistep tumourigenesis and is thus suitable for determining at which stage telomerase is activated. Telomerase activity was examined by telomeric repeat amplification protocol (TRAP) assay in 96 cases of colorectal tissues, including 50 carcinomas, 31 adenomas, and 15 normal colonic tissues. For each case, histological diagnosis and telomerase activity were determined on consecutive frozen sections. In order to reduce the chance of a false-negative TRAP assay due to RNA degradation, the integrity of rRNA in the tissues was verified in each case. Twenty-five carcinomas, 30 adenomas, and all of the 15 normal colorectal mucosal samples showed no or only partial rRNA degradation and only in these cases was the TRAP assay interpreted. None of the normal tissues exhibited telomerase activity. In contrast, all of the 25 cancers and 47 per cent (14/30) of the adenomas were positive. In adenomas, telomerase activation was highly significantly related to the grade of dysplasia (p< 0.0001). All adenomas which contained high-grade dysplasia revealed telomerase activity, whereas telomerase activity was detectable in only 20 per cent (4/20) of cases with exclusively low-grade dysplasia. These results indicate that telomerase activation, which may be an obligatory step in colorectal carcinogenesis, occurs in the progression from low-grade to high-grade dysplasia in adenomas. Furthermore, in the adenoma-carcinoma sequence, telomerase activation seems to occur later than K- ras mutation but earlier than p53 mutation.

High dose chemotherapy with autologous hematopoietic stem cell support for solid tumors other than breast cancer in adults.

Since the early 1980s high dose chemotherapy with autologous hematopoietic stem cell support was adopted by many oncologists as a potentially curative option for solid tumors, supported by a strong rationale from laboratory studies and apparently convincing results of early phase II studies. As a result, the number and size of randomized trials comparing this approach with conventional chemotherapy initiated (and often abandoned before completion) to prove or disprove its value was largely insufficient. In fact, with the possible exception of breast carcinoma, the benefit of a greater escalation of dose of chemotherapy with stem cell support in solid tumors is still unsettled and many oncologists believe that this approach should cease. In this article, we critically review and comment on the data from studies of high dose chemotherapy so far reported in adult patients with small cell lung cancer, ovarian cancer, germ cell tumors and sarcomas.

Role of FGF and Wnt pathways in endoderm patterning and pancreas organogenesis

Summary Between gastrulation and gut tube formation, the endoderm becomes regionally specified along the anterior-posterior axis. An early sign of patterning is the expression of organ-specific genes in restricted endoderm domains. We studied the role of the fibroblast growth factor (FGF) and Wnt pathways in the establishment of the antero-posterior (A-P) axis domains. Here we report the first evidence that graded FGF4-mediated signaling establishes gut tube domains along the A-P axis in vivo from gastrulation to somitogenesis. At gastrulation, FGF4 may act cooperatively with Wnts, since both of them affect the gut tube patterning by promoting posterior and inhibiting anterior endoderm cell fate. The activity of the Wnt pathway is however time restricted, since. it does not affect patterning at somitogenesis. Our experiments point to a global mechanism that coordinates the A-P patterning of all three primary germ layers. Soon after regionalization of the gut tube, morphogenetic evidences of organogenesis appear. We focused our attention on one of these organs, the pancreas. We report a comprehensive investigation of the activity and the role of the Wnt pathway in pancreas organogenesis. We have used two mouse reporter lines to monitor canonical Wnt-pathway activity during development and after birth and demonstrate activity in early pancreatic bud, endocrine cells and in the mesenchyme. We have specifically deleted the ß-catenin .gene, a key component of the Wnt pathway, in the epithelium of the pancreas and duodenum using Pdxl -Cre mice. In agreement with Wnt pathway activity in pancreatic endocrine cells, we find a reduction in endocrine islet numbers. Our study reveals that ß-catenin deletion also affects cells in which Wnt pathway activity is not detected. Indeed, ß-catenin mutant cells have a competitive disadvantage during development that also' affects the exocrine compartment. Moreover, the conditional KO mice develop acute edematous pancreatitis perinatally due to the disruption of the epithelial structure of acini. These effects are likely to be due to the function of ß-catenin at the membrane. Résumé Entre la gastrulation et la formation du tube digestif, l'endoderme est progressivement régionalisé le long de l'axe antéropostérieur (A-P). Un des premiers signes de cette régionalisation est l'expression de gènes spécifiques à certains organes dans une région restreinte. Nous avons étudié l'implication des voies de signalisation FGF et Wnt dans l'établissement de la régionalisation A-P. Nous rapportons les premières preuves que FGF4 établit la ségrégation des domaines de l'endoderme le long de l'axe A-P in vivo de la gastrulation à la somitogenèse. Cette activité peut être menée en collaboration avec les Wnts, puisque ceux-ci influencent aussi l'endoderme en inhibant le destin antérieur et en induisant le destin postérieur des cellules. Cette activité des Wnts est perdue à la somitogenèse. Nos expériences démontrent une régionalisation coordonnée des trois feuillets germinaux le long de l'axe A-P. Peu après la régionalisation, les premiers signes morphologiques de l'organogenèse apparaissent. Nous nous sommes intéressés au rôle des Wnts dans un des dérivés de l'endoderme : le pancréas. Nous avons utilisés deux lignés de souris rapportrices de l'activité de la voie canonique des Wnts, qui montrent une activité dans le bourgeon précoce du pancréas avant la différentiation, puis plus tard dans les cellules endocrines et le mésenchyme. Nous avons utilisé la souris transgénique Pdxl -Cre pour inactiver spécifiquement le gène de la ß-caténine, un intermédiaire de la voie des Wnts, dans la région pancréatique. En accord avec l'activité de la voie de signalisation Wnt, la perte de la ßcaténine conduit à une réduction du nombre de cellules endocrines. De plus certaines cellules qui ne montrent aucune activité de la voie Wnt sont aussi affectées. En effet, les cellules ayant perdu la ß-caténine ont un désavantage compétitif face aux cellules sauvages dans un environnement mosaïque. Cette compétition résulte en l'absence de cellules déplétées en ßcaténine chez l'adulte. De plus, vers la naissance, les animaux déficients pour la ß-caténine développent une pancréatite aiguë due à la destruction de l'architecture des acini. Ceci est probablement aux fonctions d'adhésion de la ß-caténine à la membrane.

Keeping the Vermin Out: Perceived Disease Threat and Ideological Orientations as Predictors of Exclusionary Immigration Attitudes

Integrating evolutionary and social representations theories, the current study examines the relationship between perceived disease threat and exclusionary immigration attitudes in the context of a potential avian influenza pandemic. This large-scale disease provides a realistic context for investigating the link between disease threat and immigration attitudes. The main aim of this cross-sectional study (N=412) was to explore mechanisms through which perceived chronic and contextual disease threats operate on immigration attitudes. Structural equation models show that the relationship between chronic disease threat (germ aversion) and exclusionary immigration attitudes (assimiliationist immigration criteria, health-based immigration criteria and desire to reduce the proportion of foreigners) was mediated by ideological and normative beliefs (social dominance orientation, belief in a dangerous world), but not by contextual disease threat (appraisal of avian influenza pandemic threat). Contextual disease threat only predicted support for health-based immigration criteria. The conditions under which real-life disease threats influence intergroup attitudes are scrutinized. Convergence and dissimilarity of evolutionary and social representational approaches in accounting for the link between disease threat and immigration attitudes are discussed.

Cellular source and mechanisms of high transcriptome complexity in the Mammalian testis.

Understanding the extent of genomic transcription and its functional relevance is a central goal in genomics research. However, detailed genome-wide investigations of transcriptome complexity in major mammalian organs have been scarce. Here, using extensive RNA-seq data, we show that transcription of the genome is substantially more widespread in the testis than in other organs across representative mammals. Furthermore, we reveal that meiotic spermatocytes and especially postmeiotic round spermatids have remarkably diverse transcriptomes, which explains the high transcriptome complexity of the testis as a whole. The widespread transcriptional activity in spermatocytes and spermatids encompasses protein-coding and long noncoding RNA genes but also poorly conserves intergenic sequences, suggesting that it may not be of immediate functional relevance. Rather, our analyses of genome-wide epigenetic data suggest that this prevalent transcription, which most likely promoted the birth of new genes during evolution, is facilitated by an overall permissive chromatin in these germ cells that results from extensive chromatin remodeling.

Development of projections from auditory to visual areas in the cat.

In newborn kittens, cortical auditory areas (including AI and AII) send transitory projections to ipsi- and contralateral visual areas 17 and 18. These projections originate mainly from neurons in supragranular layers but also from a few in infragranular layers (Innocenti and Clarke: Dev. Brain Res. 14:143-148, '84; Clarke and Innocenti: J. Comp. Neurol. 251:1-22, '86). The postnatal development of these projections was studied with injections of anterograde tracers (wheat germ agglutinin-horseradish peroxidase [WGA-HRP]) in AI and AII and of retrograde tracers (WGA-HRP, fast blue, diamidino yellow, rhodamine-labeled latex beads) in areas 17 and 18. It was found that the projections are nearly completely eliminated in development, this, by the end of the first postnatal month. Until then, most of the transitory axons seem to remain confined to the white matter and the depth of layer VI; a few enter it further but do not appear to form terminal arbors. As for other transitory cortical projections the disappearance of the transitory axons seems not to involve death of their neurons of origin. In kittens older than 1 month and in normal adult cats, retrograde tracer injections restricted to, or including, areas 17 and 18 label only a few neurons in areas AI and AII. Unlike the situation in the kitten, nearly all of these are restricted to layers V and VI. A similar distribution of neurons projecting from auditory to visual areas is found in adult cats bilaterally enucleated at birth, which suggests that the postnatal elimination of the auditory-to-visual projection is independent of visual experience and more generally of information coming from the retina.

Gene expression profiling of rat spermatogonia and Sertoli cells reveals signaling pathways from stem cells to niche and testicular cancer cells to surrounding stroma.

Background: Stem cells and their niches are studied in many systems, but mammalian germ stem cells (GSC) and their niches are still poorly understood. In rat testis, spermatogonia and undifferentiated Sertoli cells proliferate before puberty, but at puberty most spermatogonia enter spermatogenesis, and Sertoli cells differentiate to support this program. Thus, pre-pubertal spermatogonia might possess GSC potential and pre-pubertal Sertoli cells niche functions. We hypothesized that the different stem cell pools at pre-puberty and maturity provide a model for the identification of stem cell and niche-specific genes. We compared the transcript profiles of spermatogonia and Sertoli cells from pre-pubertal and pubertal rats and examined how these related to genes expressed in testicular cancers, which might originate from inappropriate communication between GSCs and Sertoli cells. Results: The pre-pubertal spermatogonia-specific gene set comprised known stem cell and spermatogonial stem cell (SSC) markers. Similarly, the pre-pubertal Sertoli cell-specific gene set comprised known niche gene transcripts. A large fraction of these specifically enriched transcripts encoded trans-membrane, extra-cellular, and secreted proteins highlighting stem cell to niche communication. Comparing selective gene sets established in this study with published gene expression data of testicular cancers and their stroma, we identified sets expressed genes shared between testicular tumors and pre-pubertal spermatogonia, and tumor stroma and pre-pubertal Sertoli cells with statistic significance. Conclusions: Our data suggest that SSC and their niche specifically express complementary factors for cell communication and that the same factors might be implicated in the communication between tumor cells and their micro-enviroment in testicular cancer.

Potential detrimental effects of a phytoestrogen-rich diet on male fertility in mice.

Soy and soy-based products are widely consumed by infants and adult individuals. There has been speculation that the presence of isoflavone phytoestrogens in soybean cause adverse effects on the development and function of the male reproductive system. The purpose of this study was to examine the influence of dietary soy and phytoestrogens on testicular and reproductive functions. Male mice were fed from conception to adulthood with either a high soy-containing diet or a soy-free diet. Although adult mice fed a soy-rich diet exhibited normal male behaviour and were fertile, we observed a reduced proportion of haploid germ cells in testes correlating with a 25% decrease in epididymal sperm counts and a 21% reduction in litter size. LH and androgens levels were not affected but transcripts coding for androgen-response genes in Sertoli cells and Gapd-s, a germ cell-specific gene involved in sperm glycolysis and mobility were significantly reduced. In addition, we found that dietary soy decreased the size of the seminal vesicle but without affecting its proteolytic activity. Taken together, these studies show that long-term exposure to dietary soy and phytoestrogens may affect male reproductive function resulting in a small decrease in sperm count and fertility.