Saliva suppresses osteoclastogenesis in murine bone marrow cultures.

Saliva can reach mineralized surfaces in the oral cavity; however, the relationship between saliva and bone resorption is unclear. Herein, we examined whether saliva affects the process of osteoclastogenesis in vitro. We used murine bone marrow cultures to study osteoclast formation. The addition of fresh sterile saliva eliminated the formation of multinucleated cells that stained positive for tartrate-resistant acid phosphatase (TRAP). In line with the histochemical staining, saliva substantially reduced gene expression of cathepsin K, calcitonin receptor, and TRAP. Addition of saliva led to considerably decreased gene expression of receptor activator of nuclear factor kappa-B (RANK) and, to a lesser extent, that of c-fms. The respective master regulators of osteoclastogenesis (c-fos and NFATc1) and the downstream cell fusion genes (DC-STAMP and Atp6v0d2) showed decreased expression after the addition of saliva. Among the costimulatory molecules for osteoclastogenesis, only OSCAR showed decreased expression. In contrast, CD40, CD80, and CD86-all costimulatory molecules of phagocytic cells-were increasingly expressed with saliva. The phagocytic capacity of the cells was confirmed by latex bead ingestion. Based on these in vitro results, it can be concluded that saliva suppresses osteoclastogenesis and leads to the development of a phagocytic cell phenotype.

Incorporation of RANKL promotes osteoclast formation and osteoclast activity on β-TCP ceramics.

β-Tricalcium phosphate (β-TCP) ceramics are approved for the repair of osseous defects. In large defects, however, the substitution of the material by authentic bone is inadequate to provide sufficient long-term mechanical stability. We aimed to develop composites of β-TCP ceramics and receptor activator of nuclear factor κ-B ligand (RANKL) to enhance the formation of osteoclasts and promote cell mediated calcium phosphate resorption. RANKL was adsorbed superficially onto β-TCP ceramics or incorporated into a crystalline layer of calcium phosphate by the use of a co-precipitation technique. Murine osteoclast precursors were seeded onto the ceramics. After 15 days, the formation of osteoclasts was quantified cytologically and colorimetrically with tartrate-resistant acidic phosphatase (TRAP) staining and TRAP activity measurements, respectively. Additionally, the expression of transcripts encoding the osteoclast gene products cathepsin K, calcitonin receptor, and of the sodium/hydrogen exchanger NHA2 were quantified by real-time PCR. The activity of newly formed osteoclasts was evaluated by means of a calcium phosphate resorption assay. Superficially adsorbed RANKL did not induce the formation of osteoclasts on β-TCP ceramics. When co-precipitated onto β-TCP ceramics RANKL supported the formation of mature osteoclasts. The development of osteoclast lineage cells was further confirmed by the increased expression of cathepsin K, calcitonin receptor, and NHA2. Incorporated RANKL stimulated the cells to resorb crystalline calcium phosphate. Our in vitro study shows that RANKL incorporated into β-TCP ceramics induces the formation of active, resorbing osteoclasts on the material surface. Once formed, osteoclasts mediate the release of RANKL thereby perpetuating their differentiation and activation. In vivo, the stimulation of osteoclast-mediated resorption may contribute to a coordinated sequence of material resorption and bone formation. Further in vivo studies are needed to confirm the current in vitro findings.

Disruption of estrogen signaling does not prevent progesterone action in the estrogen receptor α knockout mouse uterus

Estrogen is known to increase progesterone receptor (PR) levels in the wild-type mouse uterus, and this estrogen induction was thought to be important for progesterone action through the PR. The estrogen receptor α knockout (ERKO) mouse uterus was observed to express PR mRNA that cannot be induced by estrogen. Progesterone action was characterized to determine whether it was diminished in ERKO mice. The PR protein is present in the ERKO uterus at 60% of the level measured in a wild-type uterus. The PR-A and PR-B isoforms are both detected on Western blot, and the ratio of isoforms is the same in both genotypes. Although the level of PR is reduced in the ERKO uterus, the receptor level is sufficient to induce genomic responses, since both calcitonin and amphiregulin mRNAs were increased after progesterone treatment. Finally, the ERKO uterus can be induced to undergo a progesterone-dependent decidual response. Surprisingly, the decidual response is estrogen independent in the ERKO, although it remains estrogen dependent in a wild type. These results indicate that estrogen receptor α modulation of PR levels is not necessary for expression of the PR or genomic and physiologic responses to progesterone in the ERKO uterus.

Neuropeptides, by direct interaction with T cells, induce cytokine secretion and break the commitment to a distinct T helper phenotype

Searching for nervous system candidates that could directly induce T cell cytokine secretion, I tested four neuropeptides (NPs): somatostatin, calcitonin gene-related peptide, neuropeptide Y, and substance P. Comparing neuropeptide-driven versus classical antigen-driven cytokine secretion from T helper cells Th0, Th1, and Th2 autoimmune-related T cell populations, I show that the tested NPs, in the absence of any additional factors, directly induce a marked secretion of cytokines [interleukin 2 (IL-2), interferon-γ, IL-4, and IL-10) from T cells. Furthermore, NPs drive distinct Th1 and Th2 populations to a “forbidden” cytokine secretion: secretion of Th2 cytokines from a Th1 T cell line and vice versa. Such a phenomenon cannot be induced by classical antigenic stimulation. My study suggests that the nervous system, through NPs interacting with their specific T cell-expressed receptors, can lead to the secretion of both typical and atypical cytokines, to the breakdown of the commitment to a distinct Th phenotype, and a potentially altered function and destiny of T cells in vivo.

Epigenetic phenotypes distinguish microsatellite-stable and -unstable colorectal cancers

Aberrant DNA methylation is a common phenomenon in human cancer, but its patterns, causes, and consequences are poorly defined. Promoter methylation of the DNA mismatch repair gene MutL homologue (MLH1) has been implicated in the subset of colorectal cancers that shows microsatellite instability (MSI). The present analysis of four MspI/HpaII sites at the MLH1 promoter region in a series of 89 sporadic colorectal cancers revealed two main methylation patterns that closely correlated with the MSI status of the tumors. These sites were hypermethylated in tumor tissue relative to normal mucosa in most MSI(+) cases (31/51, 61%). By contrast, in the majority of MSI(−) cases (20/38, 53%) the same sites showed methylation in normal mucosa and hypomethylation in tumor tissue. Hypermethylation displayed a direct correlation with increasing age and proximal location in the bowel and was accompanied by immunohistochemically documented loss of MLH1 protein both in tumors and in normal tissue. Similar patterns of methylation were observed in the promoter region of the calcitonin gene that does not have a known functional role in tumorigenesis. We propose a model of carcinogenesis where different epigenetic phenotypes distinguish the colonic mucosa in individuals who develop MSI(+) and MSI(−) tumors. These phenotypes may underlie the different developmental pathways that are known to occur in these tumors.

Control of alternative pre-mRNA splicing by distributed pentameric repeats

Multiple copies of the hexamer TGCATG have been shown to regulate fibronectin pre-mRNA alternative splicing. GCATG repeats also are clustered near the regulated calcitonin-specific 3′ splice site in the rat calcitonin/CGRP gene. Specific mutagenesis of these repeats in calcitonin/CGRP pre-mRNA resulted in the loss of calcitonin-specific splicing, suggesting that the native repeats act to enhance alternative exon inclusion. Mutation of subsets of these elements implies that alternative splicing requires a minimum of two repeats, and that the combination of one intronic and one exonic repeat is necessary for optimal cell-specific splicing. However, multimerized intronic repeats inhibited calcitonin-specific splicing in both the wild-type context and in a transcript lacking endogenous repeats. These results suggest that both the number and distribution of repeats may be important features for the regulation of tissue-specific alternative splicing. Further, RNA containing a single repeat bound cell-specific protein complexes, but tissue-specific differences in protein binding were not detected by using multimerized repeats. Together, these data support a novel model for alternative splicing regulation that requires the cell-specific recognition of multiple, distributed sequence elements.

Human blood-mobilized hematopoietic precursors differentiate into osteoclasts in the absence of stromal cells.

Osteoclastogenesis is a complex process that is facilitated by bone marrow stromal cells (SCs). To determine if SCs are an absolute requirement for the differentiation of human hematopoietic precursors into fully mature, osteoclasts (OCs), CD34+ cells were mobilized into the peripheral circulation with granulocyte colony-stimulating factor, harvested by leukapheresis, and purified by magnetic-activated cell sorting. This procedure yields a population of CD34+ cells that does not contain SC precursors, as assessed by the lack of expression of the SC antigen Stro-1, and that differentiates only into hematopoietic cells. We found that CD34+, Stro-1- cells cultured with a combination of granulocyte/macrophage colony-stimulating factor, interleukin 1, and interleukin 3 generated cells that fulfill current criteria for the characterization of OCs, including multinucleation, presence of tartrate-resistant acid phosphatase, and expression of the calcitonin and vitronectin receptors and of pp60c-src tyrosine kinase. These OCs also expressed mRNA for the noninserted isoform of the calcitonin receptor and excavated characteristic resorption pits in devitalized bone slices. These data demonstrate that accessory SCs are not essential for human osteoclastogenesis and that granulocyte colony-stimulating factor treatment mobilizes OC precursors into the peripheral circulation.

A complex of nuclear proteins mediates SR protein binding to a purine-rich splicing enhancer.

A purine-rich splicing enhancer from a constitutive exon has been shown to shift the alternative splicing of calcitonin/CGRP pre-mRNA in vivo. Here, we demonstrate that the native repetitive GAA sequence comprises the optimal enhancer element and specifically binds a saturable complex of proteins required for general splicing in vitro. This complex contains a 37-kDa protein that directly binds the repetitive GAA sequence and SRp40, a member of the SR family of non-snRNP splicing factors. While purified SR proteins do not stably bind the repetitive GAA element, exogenous SR proteins become associated with the GAA element in the presence of nuclear extracts and stimulate GAA-dependent splicing. These results suggest that repetitive GAA sequences enhance splicing by binding a protein complex containing a sequence-specific RNA binding protein and a general splicing activator that, in turn, recruit additional SR proteins. This type of mechanism resembles the tra/tra-2-dependent recruitment of SR proteins to the Drosophila doublesex alternative splicing regulatory element.

Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage.

Focal brain ischemia is the most common event leading to stroke in humans. To understand the molecular mechanisms associated with brain ischemia, we applied the technique of mRNA differential display and isolated a gene that encodes a recently discovered peptide, adrenomedullin (AM), which is a member of the calcitonin gene-related peptide (CGRP) family. Using the rat focal stroke model of middle cerebral artery occlusion (MCAO), we determined that AM mRNA expression was significantly increased in the ischemic cortex up to 17.4-fold at 3 h post-MCAO (P < 0.05) and 21.7-fold at 6 h post-MCAO (P < 0.05) and remained elevated for up to 15 days (9.6-fold increase; P < 0.05). Immunohistochemical studies localized AM to ischemic neuronal processes, and radioligand (125I-labeled CGRP) displacement revealed high-affinity (IC50 = 80.3 nmol) binding of AM to CGRP receptors in brain cortex. The cerebrovascular function of AM was studied using synthetic AM microinjected onto rat pial vessels using a cranial window or applied to canine basilar arteries in vitro. AM, applied abluminally, produced dose-dependent relaxation of preconstricted pial vessels (P < 0.05). Intracerebroventricular (but not systemic) AM administration at a high dose (8 nmol), prior to and after MCAO, increased the degree of focal ischemic injury (P < 0.05). The ischemia-induced expression of both AM mRNA and peptide in ischemic cortical neurons, the demonstration of the direct vasodilating effects of the peptide on cerebral vessels, and the ability of AM to exacerbate ischemic brain damage suggests that AM plays a significant role in focal ischemic brain injury.

Localization of choline acetyltransferase in rat peripheral sympathetic neurons and its coexistence with nitric oxide synthase and neuropeptides.

Indirect immunofluorescence methods using a mouse monoclonal antibody raised to rat choline acetyltransferase (ChAT) revealed dense networks of ChAT-immunoreactive fibers in the superior cervical ganglion, the stellate ganglion, and the celiac superior mesenteric ganglion of the rat. Numerous and single ChAT-immunoreactive cell bodies were observed in the stellate and superior cervical ganglia, respectively. The majority of ChAT-immunoreactive fibers in the stellate and superior cervical ganglia were nitric oxide synthase (NOS) positive. Some ChAT-immunoreactive fibers contained enkephalin-like immunoreactivity. Virtually all ChAT-positive cell bodies in the stellate ganglion were vasoactive intestinal polypeptide (VIP)-positive, and some were calcitonin gene-related peptide (CGRP)-positive. After transection of the cervical sympathetic trunk almost all ChAT- and NOS-positive fibers and most enkephalin- and CGRP-positive fibers disappeared in the superior cervical ganglion. The results suggest that most preganglionic fibers are cholinergic and that the majority of these in addition can release nitric oxide, some enkephalin, and a few CGRP. Acetylcholine, VIP, and CGRP are coexisting messenger molecules in some postganglionic sympathetic neurons.

Lineage specification of neuronal precursors in the mouse spinal cord.

We have investigated the differentiation potential of precursor cells within the developing spinal cord of mice and have shown that spinal cord cells from embryonic day 10 specifically give rise to neurons when plated onto an astrocytic monolayer, Ast-1. These neurons had the morphology of motor neurons and > 83% expressed the motor neuron markers choline acetyltransferase, peripherin, calcitonin gene-related peptide, and L-14. By comparison, < 10% of the neurons arising on monolayers of other neural cell lines or 3T3 fibroblasts had motor neuron characteristics. Cells derived from dorsal, intermediate, and ventral regions of the spinal cord all behaved similarly and gave rise to motor neuron-like cells when plated onto Ast-1. By using cells that expressed the lacZ reporter gene, it was shown that > 93% of cells present on the Ast-1 monolayers were motor neuron-like. Time-lapse analysis revealed that the precursors on the Ast-1 monolayers gave rise to neurons either directly or following a single cell division. Together, these results indicate that precursors in the murine spinal cord can be induced to differentiate into the motor neuron phenotype by factors produced by Ast-1 cells, suggesting that a similar factor(s) produced by cells akin to Ast-1 may regulate motor neuron differentiation in vivo.

Avaliação da inervação entérica e análise proteômica do intestino delgado de ratos expostos à dose aguda ou crônica de fluoreto

O trato gastrointestinal (TGI) é a principal rota de exposição ao fluoreto (F) e o seu mais importante sítio de absorção. Acredita-se que a toxicidade do F comprometa a fisiologia do intestino, devido à relevante sintomatologia gastrointestinal relatada em consequência da exposição excessiva ao F. A função intestinal é controlada por uma complexa rede neuronal interligada e incorporada à parede deste órgão, denominada Sistema Nervoso Entérico (SNE). Embora os efeitos tóxicos do F sobre o Sistema Nervoso Central sejam descritos na literatura, não há estudos relacionados à sua toxicidade sobre o SNE. Neste estudo realizado em ratos, foi avaliado o efeito da exposição aguda ou crônica ao F, sobre a população geral de neurônios entéricos e sobre as subpopulações que expressam os principais neurotransmissores entéricos: Acetilcolina (ACh), Óxido Nítrico (NO), Peptídeo Vasoativo Intestinal (VIP), Peptídeo Relacionado ao Gene da Calcitonina (CGRP) e Substância P (SP). Os animais foram divididos em 5 grupos: 3 destinados à exposição crônica (0 ppm, 10 ppm ou 50 ppm de F na água de beber) e 2 à exposição aguda (0 ou 25 mgF/Kg por gavagem gástrica). Foram coletados os 3 segmentos do intestino delgado (duodeno, jejuno e íleo) e processados para a detecção da HuC/D, ChAT, nNOS, VIP, CGRP e SP, através de técnicas de imunofluorescência, no plexo mioentérico. Foram obtidas imagens para a realização da análise quantitativa dos neurônios da população geral (HuC/D) e nitrérgicos (imunorreativos à nNOS); e morfométrica dos neurônios imunorreativos à HuC/D ou nNOS; e das varicosidades imunorreativas à ChAT, VIP, CGRP ou SP. Amostras dos 3 segmentos intestinais foram preparadas e coradas em Hematoxilina e Eosina para análise histológica da morfologia básica. O segmento intestinal considerado mais afetado na análise morfométrica da população geral de neurônios, o duodeno, foi selecionado para a realização da análise proteômica, com o objetivo de oferecer o seu perfil proteico e determinar diferenças na expressão proteica em decorrência da exposição crônica ou aguda ao F. A análise da concentração de F no plasma sanguíneo foi realizada para a confirmação da exposição. Na análise quantitativa, o grupo de 50 ppm F, apresentou uma diminuição significativa na densidade da população geral de neurônios do jejuno e do íleo e na densidade dos neurônios imunorreativos à nNOS no duodeno e no jejuno. Quanto à análise morfométrica, a população geral e as subpopulações neuronais entéricas avaliadas apresentaram alterações morfológicas significativas, tanto após a exposição crônica quanto a aguda. Para a análise proteômica do duodeno, verificou-se que da associação de seus genes a um termo, e assim classificadas de acordo com diferentes processos biológicos. No caso do grupo da dose aguda, o processo biológico com a maior porcentagem de genes associados foi a geração de metabólitos precursores e energia (27% das proteínas); enquanto para os grupos de 10 e 50 ppm F foram o processo metabólico da piridina (41%) e a polimerização proteica (33%), respectivamente.

Structural basis for control of secondary vessels in the long-finned eel, Anguilla reinhardtii

Histological sections of primary segmental arteries and associated interarterial anastomoses and secondary vessels from the long-finned eel Anguilla reinhardtii were examined by light and transmission electron microscopy. Interarterial anastomoses were found to originate from the primary vasculature as depressions through the tunica intima and media, from where they ran perpendicularly to the adventitial layer, before coiling extensively. From here the anastomoses travelled a relatively linear path in the outer margin of the adventitia to anastomose with a secondary vessel running in parallel with the primary counterpart. In contrast to findings from other species, secondary vessels had a structure quite similar to that of primary vessels; they were lined by endothelial cells on a continuous basement membrane, with a single layer of smooth muscle cells surrounding the vessel. Smooth muscle cells were also found in the vicinity of interarterial anastomoses in the adventitia, but these appeared more longitudinally orientated. The presence of smooth muscle cells on all aspects of the secondary circulation suggests that this vascular system is regulated in a similar manner as the primary vascular system. Because interarterial anastomoses are structurally integrated with the primary vessel from which they originate, it is anticipated that flow through secondary vessels to some extent is affected by the vascular tone of the primary vessel. Immunohistochemical studies showed that primary segmental arteries displayed moderate immunoreactivity to antibodies against 5-hydroxytryptamine and substance P, while interarterial anastomoses and secondary vessels showed dense immunoreactivity. No immunoreactivity was observed on primary or secondary arteries against neuropeptide Y or calcitonin gene-related peptide.

Serum procalcitonin and C-reactive protein as markers of sepsis and outcome in patients with neurotrauma and subarachnoid haemorrhage

This prospective study evaluated serum procalcitonin (PCT) and C-reactive protein (CRP) as markers for systemic inflammatory response syndrome (SIRS)/sepsis and mortality in patients with traumatic brain injury and subarachnoid haemorrhage. Sixty-two patients were followed for 7 days. Serum PCT and CRP were measured on days 0, 1, 4, 5, 6 and 7. Seventy-seven per cent of patients with traumatic brain injury and 83% with subarachnoid haemorrhage developed SIRS or sepsis (P= 0.75). Baseline PCT and CRP were elevated in 35% and 55% ofpatients respectively (P=0.03). There was a statistically non-significant step-wise increase in serum PCT levels from no SIRS (0.4 +/- 0.6 ng/ml) to SIRS (3.05 +/- 9.3 ng/ml) to sepsis (5.5 +/- 12.5 ng/ml). A similar trend was noted in baseline PCT in patients with mild (0.06 +/- 0.9 ng/ml), moderate (0.8 +/- 0.7 ng/ml) and severe head injury (1.2 +/- 1.9 ng/ml). Such a gradation was not observed with serum CRP There was a non-significant trend towards baseline PCT being a better marker of hospital mortality compared with baseline CRP (ROC-AUC 0.56 vs 0.31 respectively). This is the first prospective study to document the high incidence of SIRS in neurosurgical patients. In our study, serum PCT appeared to correlate with severity of traumatic brain injury and mortality. However, it could not reliably distinguish between SIRS and sepsis in this cohort. This is in pan because baseline PCT elevation seemed to correlate with severity of injury. Only a small proportion ofpatients developed sepsis, thus necessitating a larger sample size to demonstrate the diagnostic usefulness of serum PCT as a marker of sepsis. Further clinical trials with larger sample sizes are required to confirm any potential role of PCT as a sepsis and outcome indicator in patients with head injuries or subarachnoid haemorrhage.

Protease-activated receptor-2 peptides activate neurokinin-1 receptors in the mouse isolated trachea

Protective roles for protease-activated receptor-2 (PAR2) in the airways including activation of epithelial chloride (Cl-) secretion are based on the use of presumably PAR(2)-selective peptide agonists. To determine whether PAR(2) peptide-activated Cl- secretion from mouse tracheal epithelium is dependent on PAR(2), changes in ion conductance across the epithelium [short-circuit current (I-SC)] to PAR(2) peptides were measured in Ussing chambers under voltage clamp. In addition, epithelium and endothelium-dependent relaxations to these peptides were measured in two established PAR(2) bioassays, isolated ring segments of mouse trachea and rat thoracic aorta, respectively. Apical application of the PAR(2) peptide SLIGRL caused increases in I-SC, which were inhibited by three structurally different neurokinin receptor-1 (NK1R) antagonists and inhibitors of Cl- channels but not by capsaicin, the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8-37), or the nonselective cyclooxygenase inhibitor indomethacin. Only high concentrations of trypsin caused an increase in I-SC but did not affect the responses to SLIGRL. Relaxations to SLIGRL in the trachea and aorta were unaffected by the NK1R antagonist nolpitantium (SR 140333) but were abolished by trypsin desensitization. The rank order of potency for a range of peptides in the trachea I-SC assay was 2-furoyl-LIGRL > SLCGRL > SLIGRL > SLIGRT > LSIGRL compared with 2-furoyl-LIGRL > SLIGRL > SLIGRT > SLCGRL (LSIGRL inactive) in the aorta relaxation assay. In the mouse trachea, PAR(2) peptides activate both epithelial NK1R coupled to Cl- secretion and PAR(2) coupled to prostaglandin E-2-mediated smooth muscle relaxation. Such a potential lack of specificity of these commonly used peptides needs to be considered when roles for PAR(2) in airway function in health and disease are determined.