Unraveling the mystery of Gaucher bone density pathophysiology.

Gaucher disease (GD) is caused by pathogenic mutations in GBA1, the gene that encodes the lysosomal enzyme ß-glucocerebrosidase. Despite the existence of a variety of specific treatments for GD, they cannot completely reverse bone complications. Many studies have evidenced the impairment in bone tissue of GD, and molecular mechanisms of bone density alterations in GD are being studied during the last years and different reports emphasized its efforts trying to unravel why and how bone tissue is affected. The cause of skeletal density affection in GD is a matter of debates between research groups. and there are two opposing hypotheses trying to explain reduced bone mineral density in GD: increased bone resorption versus impaired bone formation. In this review, we discuss the diverse mechanisms of bone alterations implicated in GD revealed until the present, along with a presentation of normal bone physiology and its regulation. With this information in mind, we discuss effectiveness of specific therapies, introduce possible adjunctive therapies and present a novel model for GD-associated bone density pathogenesis. Under the exposed evidence, we may conclude that both sides of the balance of remodeling process are altered. In GD the observed osteopenia/osteoporosis may be the result of contribution of both reduced bone formation and increased bone resorption.

Gaucher disease-associated alterations in mesenchymal stem cells reduce osteogenesis and favour adipogenesis processes with concomitant increased osteoclastogenesis.

Gaucher disease (GD) is caused by pathogenic mutations in GBA1, the gene that encodes the lysosomal enzyme ß-glucocerebrosidase. Until now, treatments for GD cannot completely reverse bone problems. The aim of this work was to evaluate the potential of MSCs from GD patients (GD MSCs) to differentiate towards the osteoblast (GD Ob) and adipocyte (GD Ad) lineages, and their role in osteoclastogenesis. We observed that GD Ob exhibited reduced mineralization, collagen deposition and alkaline phosphatase activity (ALP), as well as decreased gene expression of RUNX2, COLA1 and ALP. We also evaluated the process of osteoclastogenesis and observed that conditioned media from GD MSCs supernatants induced an increase in the number of osteoclasts. In this model, osteoclastogenesis was induced by RANKL and IL-1ß. Furthermore, results showed that in GD MSCs there was a promotion in NLRP3 and PPAR-γ gene expression. Adipogenic differentiation revealed that GD Ad had an increase in PPAR-γ and a reduced RUNX2 gene expression, promoting adipocyte differentiation. In conclusion, our results show that GD MSCs exhibited deficient GD Ob differentiation and increased adipogenesis. In addition, we show that GD MSCs promoted increased osteoclastogenesis through RANKL and IL-1ß. These changes in GD MSCs are likely to contribute to skeletal imbalance observed in GD patients.

The stimulated innate resistance event in Bordetella pertussis infection is dependent on reactive oxygen species production.

The exacerbated induction of innate immune responses in airways can abrogate diverse lung infections by a phenomenon known as stimulated innate resistance (StIR). We recently demonstrated that the enhancement of innate response activation can efficiently impair Bordetella pertussis colonization in a Toll-like receptor 4 (TLR4)-dependent manner. The aim of this work was to further characterize the effect of lipopolysaccharide (LPS) on StIR and to identify the mechanisms that mediate this process. Our results showed that bacterial infection was completely abrogated in treated mice when the LPS of B. pertussis (1 µg) was added before (48 h or 24 h), after (24 h), or simultaneously with the B. pertussis challenge (10(7) CFU). Moreover, we detected that LPS completely cleared bacterial infection as soon as 2 h posttreatment. This timing suggests that the observed StIR phenomenon should be mediated by fast-acting antimicrobial mechanisms. Although neutrophil recruitment was already evident at this time point, depletion assays using an anti-GR1 antibody showed that B. pertussis clearance was achieved even in the absence of neutrophils. To evaluate the possible role of free radicals in StIR, we performed animal assays using the antioxidant N-acetyl cysteine (NAC), which is known to inactivate oxidant species. NAC administration blocked the B. pertussis clearance induced by LPS. Nitrite concentrations were also increased in the LPS-treated mice; however, the inhibition of nitric oxide synthetases did not suppress the LPS-induced bacterial clearance. Taken together, our results show that reactive oxygen species (ROS) play an essential role in the TLR4-dependent innate clearance of B. pertussis.

Modelling pertussis transmission to evaluate the effectiveness of an adolescent booster in Argentina.

Due to the current epidemiological situation of pertussis, several countries have implemented vaccination strategies that include a booster dose for adolescents. Since there is still no evidence showing that the adolescent booster has a positive effect on the most vulnerable group represented by infants, it is difficult to universalize the recommendation to include such reinforcement. In this work we present an age-structured compartmental deterministic model that considers the outstanding epidemiological features of the disease in order to assess the impact of the booster dose at age 11 years (Tdap booster) to infants. To this end, we performed different parameterizations of the model that represent distinct possible epidemiological scenarios. The results obtained show that the inclusion of a single Tdap dose at age 11 years significantly reduces the incidence of the disease within this age group, but has a very low impact on the risk group (0-1 year). An effort to improve the coverage of the first dose would have a much greater impact on infants. These results hold in the 18 scenarios considered, which demonstrates the robustness of these conclusions.

Bone marrow adipocytes alteration in an in vitro model of Gaucher Disease.

Gaucher disease (GD) is caused by biallelic pathogenic variants in GBA1 gene that encodes the lysosomal enzyme glucocerebrosidase. Up to now, specific treatment for GD cannot completely reverse bone complications. Bone is composed of different cell types; including osteoblasts, osteocytes and osteoclasts. Osteoblasts are present on bone surfaces and are derived from local mesenchymal stem cells (MSCs). Depending on environment conditions, MSCs could differentiate into osteoblasts and adipocytes. Mature adipocytes-secreted adipokines and free fatty acids affect both osteoblasts and osteoclasts formation/activity and therefore mediate skeletal homeostasis. The aim of this study was to evaluate possible alterations in GD adipocyte (GD Ad) that could contribute to bone complications. MSCs were grown in adipogenic media in order to evaluate expression of differentiation markers as PPAR-γ. PPAR-γ was observed into the nucleus of GD Ad, indicating that these cells are properly stimulated. However, these cells accumulate lesser lipid droplets (LDs) than Control Ad. In order to study lipid droplet metabolism, we evaluated the lipolysis of these structures by the measurement of free glycerol in culture supernatant. Our results indicated that GD Ad had an alteration in this process, evidenced by an increase in glycerol release. We have also evaluated two enzymes involved in LDs synthesis: fatty acid synthase (FASN) and stearoyl-coenzyme A desaturase 1 (SCD1). The transcription of these genes was decreased in GD Ad, suggesting a dysfunction in the synthesis of LDs. In conclusion, our results show an alteration in LDs metabolism of GD Ad, independent of adipocyte differentiation process. This alteration would be caused by an increase in lipolysis in early stages of differentiation and also by a reduction of lipid synthesis, which could contribute with the skeletal imbalance in GD.

Effect of build direction dependent grain structure on fatigue crack growth of biomedical Co-29Cr-6Mo alloy processed by laser powder bed fusion.

Biomedical Co-29Cr-6Mo alloy is one of the alloys that are suitable for laser powder bed fusion (LPBF) additive manufacturing and as an implant material is often used in situations of critical and cyclic loading. Thus, fatigue crack growth (FCG) behaviour and resistance of the alloy processed by LPBF are an important consideration for dental and orthopaedic applications. In this study, FCG testing has been conducted to evaluate how build direction (BD) dependent grain/cell structure in relation to crack growth direction (CD), either CD⊥BD or CD//BD, affects FCG behaviour. It has been found that the threshold stress intensity factor (ΔKTh) value is significantly higher and the values of c and m in Paris equation are slightly lower for CD//BD samples than the values for CD⊥BD samples, respectively. Failure analysis has revealed that the effects of the commonly known defect, lack of fusion, on both ΔKTh and FCG rate are weak. It has been identified that crack has mainly propagated in a transgranular and transcellular manner, consistent with the observation of the crack path being more torturous and with the higher crack growth resistance determined in CD//BD samples than in CD⊥BD samples. This will be further discussed linking the difference in the size of crack segment, which is BD and thus grain/cell length dependent, to the roughness-induced crack closure mechanism.

In vitro osteoclastogenesis from Gaucher patients' cells correlates with bone mineral density but not with Chitotriosidase.

Gaucher disease (GD) is caused by mutations on the gene encoding for the lysosomal enzyme glucocerebrosidase. Type I GD (GD1) patients present anemia, hepatosplenomegaly and bone alterations. In spite of treatment, bone alterations in GD patients persist, including poor bone mineral density (BMD). Mechanisms leading to bone damage are not completely understood, but previous reports suggest that osteoclasts are involved. Chitotriosidase (CHIT) is the most reliable biomarker used in the follow up of patients, although its correlation with bone status is unknown. The aim of this work was to study the pro-osteoclastogenic potential in patients and to evaluate its correlation with CHIT activity levels and clinical parameters. PBMCs from treated patients and healthy controls were cultured in the presence of M-CSF, and mature osteoclasts were counted. BMD, blood CHIT activity and serum levels of CTX, BAP, and cytokines were evaluated in patients. We found that blood CHIT activity and osteoclast differentiation were significantly increased in patients, but no correlation between them was observed. Interestingly, osteoclast numbers but not CHIT, presented a negative correlation with BMD expressed as Z-score. CTX, BAP and serum cytokines involved in bone remodeling were found altered in GD1 patients. These results show for the first time a correlation between osteoclast differentiation and BMD in GD1 patients, supporting the involvement of osteoclasts in the bone pathology of GD1. Our results also suggest that an altered immune response may play an important role in bone damage.

Use of extracorporeal membrane oxygenation for respiratory failure in term infants.

Eight infants with intractable respiratory failure were treated with extracorporeal membrane oxygenation. Intractable respiratory failure was defined as alveolar-arterial oxygen gradient of more than 620 torr for six to 12 hours that did not respond to hyperventilation and the use of tolazoline. Infants with overt sepsis, CNS damage, or other debilitating conditions were not considered for extracorporeal membrane oxygenation. Six of the eight infants survived after a mean extracorporeal membrane oxygenation time of 164 hours. Five of the six survivors were normal neurologically and developmentally when examined at 1 year of age.

Influence of pertussis toxin on the calcitonin-opioid interaction in isolated tissues.

1. In order to clarify one of the mechanisms involved in the analgesic effect of calcitonin, we have tested the in vitro modifications induced by calcitonin on the effect of opioids. 2. The inhibition of the contractions induced by opioids or clonidine, in guinea-pig ileum or in mouse vas deferens, were significantly reduced in tissues incubated with pertussis toxin (PTX). When tissues were incubated with PTX and calcitonin, the inhibitory effect was restored. 3. These results suggest that calcitonin is able to potentiate a non-PTX-sensitive mechanism of transduction and support the possibility of involvement of similar G-proteins in the effects of opioid and alpha 2-adrenoceptor agonists.

Effects of nimodipine and nifedipine upon behavior and regional brain monoamines in the rat.

The effects of single and repeated (9 times) administration of two dihydropyridines (DHPs), nimodipine (NIM) and nifedipine (NIF) (5 mg/kg per 12 h and 2.5 mg/kg per 12 h, IP), on the behavior of male adult rats in the holeboard and in the plus-maze, were investigated. Besides, the effects of repeated administration of the drugs on the levels of dopamine (DA), serotonin (5-HT), and their respective major metabolites in several regions of the central nervous system (CNS) were also assessed. The effects of single and repeated administration of the drugs were similar. Both DHPs caused a significant decrease in general motor activity which was evident in both tests and more marked, with the higher doses. The two exploratory parameters measured in the holeboard, i.e. head-dipping frequency and duration, were dissociated under pharmacological treatment. The drug-treated animals did not show an increased emotionality in the holeboard. However, in the plus-maze, NIF (5 mg/kg) and to a lesser extent NIM, appeared to induce some anxiety-related responses which may be secondary, at least in part, to the depressing effect on activity and exploration. Repeated administration of NIM and NIF caused an increase in striatal DA and DOPAC levels, whilst no effects were found on serotonergic system in any of the regions of the CNS analyzed.

Study of mechanisms of calcitonin analgesia in mice. Involvement of 5-HT3 receptors.

The analgesic effect of calcitonin when serotonin (5-HT) concentration is increased and the involvement of some 5-HT receptors were studied using the writhing test in mice. 5-hydroxytryptophan (5-HTP) administration increased both 5-HT levels in the central nervous system (CNS) and calcitonin analgesia. The 5-HT(1A) agonist (+/-)-8-hydroxy-2-dipropylaminotetralin hydrobromide (8-OH-DPAT) diminished calcitonin analgesia, this effect being antagonised by the 5-HT(1A) antagonist (WAY 100, 135). As the stimulation of 5-HT(1A) autoreceptors reduces the turnover of 5-HT, the effect of 8-OH-DPAT on calcitonin analgesia may be attributed to this decrease. The 5-HT(2A-2C) agonist (+/-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride (DOI) diminished calcitonin analgesia. A sub-analgesic dose of the 5-HT(2A) antagonist ketanserin failed to prevent this effect. The 5-HT(3) agonist (+/-)-2-methyl-5-hydroxytryptamine maleate (2-methyl-5-HT) potentiated calcitonin analgesia, whereas it was significantly reduced by the 5-HT(3) antagonist tropisetron. The effect of 2-methyl-5-HT on calcitonin analgesia was also reversed by tropisetron, This result suggests that the 5-HT(3) receptor may play an important role in the relationship between calcitonin and the serotonergic system. Tropisetron also reversed the analgesia induced by calcitonin plus 5-HTP corroborating importance of the 5-HT(3) receptors.

Serotonergic mechanisms involved in calcitonin potentiation of kappa-opioid receptor-mediated effects on adrenal secretion.

Calcitonin can selectively modulate the effects of opioids on the rat hypothalamic-pituitary-adrenal axis and increase the release of corticosterone induced by a kappa-opioid receptor agonist. Considerable evidence supports the involvement of opioid and serotonergic systems in the analgesic effect of calcitonin. In this study, the involvement of hypothalamic serotonergic pathways in the calcitonin potentiation of the effect of (trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]-benzeneacetamide methane sulphonate (U-50,488H) on the secretion of corticosterone was examined. The correlation between the calcitonin-induced potentiation of the pituitary adrenal response to U-50,488H and changes in serotonin turnover was evaluated. Our results show that the increase in the release of corticosterone induced by treatment with calcitonin + U-50,488H was not evident when the turnover of serotonin was decreased by inhibition of its synthesis with m-hydroxybenzylhydrazine (NSD 1015) or by blockade of its metabolism with trans-2-phenylcyclopropylamine (tranylcypromine). Although other factors can not be discarded, from the present data it can be suggested that the serotonergic system plays an important role in the interaction calcitonin-kappa-opioid receptor agonist in the hypothalamic-pituitary-adrenal axis.

Involvement of central serotonergic pathways in analgesia elicited by salmon calcitonin in the mouse.

The contribution of central serotonergic pathways to the analgesic activity induced by salmon calcitonin in the writhing test was investigated. Salmon calcitonin was administered to mice after lesioning of the ascending and descending serotonergic pathways by means of i.p. administration of p-chloroamphetamine (40 mg/kg, for 2 days) or p-chlorophenylalanine (300 mg/kg, for 3 days). The analgesic effect induced by salmon calcitonin at the doses of 10 and 20 IU/kg was not evident in mice previously treated with p-chloroamphetamine or p-chlorophenylalanine. However, the analgesic effect of salmon calcitonin 40 IU/kg was not significantly modified by p-chloroamphetamine or p-chlorophenylalanine pretreatment. Salmon calcitonin did not alter the depletion of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid after p-chloroamphetamine or p-chlorophenylalanine administration. Similarly, this hormone did not change the NSD 1015-induced accumulation of 5-hydroxytryptophan or the tranylcypromine-induced accumulation of 5-HT. These results indicate that although salmon calcitonin does not influence the synthesis and metabolism of 5-HT, it does require the integrity of the serotonergic system in order to cause analgesia.

Effect of salmon-calcitonin on the analgesic effect of selective mu, delta and kappa opioid agonists in mice.

The analgesic effect of three different opioid agonists, DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin), U-50,488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidynyl)cyclohexyl] benzene-aceramide methane sulphonate), and [D,Pen2-D,Pen5]-enkephalin, which act upon mu, delta and kappa opioid receptors, respectively, was compared in the presence and absence of salmon-calcitonin (s-CT). The analgesic test used was the writhing test in mice. The analgesic effect of the opioids was significantly enhanced by pretreatment of the animals with s-CT intraperitoneally (i.p.) administered. This effect was more evident for the delta and kappa-agonists. The present result suggests that the joint administration of s-CT and opioids may be a useful and interesting alternative in the treatment of painful diseases resistant to other treatments.

Calcitonin reverts pertussis toxin blockade of the opioid analgesia in mice.

The aim of this paper is to study the influence of salmon calcitonin (SCT) on opioid analgesia when opioid transduction pathways are functionally uncoupled from Gi/o proteins by treatment with pertussis toxin (PTX). The antinociceptive effect of morphine and three selective opioid agonists, [D-Ala2,N-Me-Phe2,Gly5-ol]enkephalin (DAMGO) (OP(3-mu receptor agonist), [D-Pen2.5]-enkephalin (OP-1-delta receptor agonist) and trans-( +/- )-3,4-dichloro-N-methyl-N-[2-1(-pyrrolidinyl)-cyclohexyl]-benzene-acetam ide methane sulfonate (U-50, 488H) (OP1-kappareceptor agonist) was evaluated, using the tail flick test, in mice treated with PTX or with PTX and SCT. PTX blocked the antinociceptive effect of the opioids, being the antinociception similar in control animals and in mice treated with PTX and SCT. Thus, SCT prevents the effect of the blockade of Gi/o-proteins. From this it could be suggested that calcitonin activates alternative antinociceptive mechanisms that are not dependent on Gi/o-proteins.

Salmon calcitonin potentiates the analgesia induced by antidepressants.

Antidepressants are used in the treatment of a variety of pain syndromes. Most of them act by blocking noradrenaline (NA) and serotonin (5-HT) reuptake. It is also well known that the serotonergic system is also involved in calcitonin (CT) analgesia. Taking these two evidences into account, the modification of the analgesic effect of nortriptyline, amitriptyline, and paroxetine in the presence of salmon CT (s-CT) was examined in mice. The forced-swimming test was carried out in order to choose doses of each drug that did not induce an antidepressant effect under our experimental conditions (nortriptyline: 0.2-5 mg/kg ip, amitriptyline: 2.5-20 mg/kg ip, and paroxetine: 5-30 mg/kg ip). The analgesic effect of each antidepressant was then evaluated using the acetic acid test. At the doses tested, the antidepressants induced a dose-dependent analgesic effect. When mice were pre-treated with a subanalgesic dose of s-CT (2.5 IU/kg), the analgesic effect of amitriptyline and paroxetine was significantly increased though no modification was found for nortriptyline. In summary, s-CT was able to increase the analgesic effect of the antidepressant drugs that reduce the uptake of 5-HT, suggesting that the joint administration of antidepressants and CT may be an interesting alternative in pain management.

Effect of Bay K 8644 on the synthesis and metabolism of dopamine and 5-hydroxytryptamine in various brain areas of the rat.

The effects of Bay K 8644 (1, 2 and 4 mg kg-1, i.p.) on the synthesis and metabolism of dopamine and 5-hydroxytryptamine (5-HT) in rat brain after m-hydroxybenzylhydrazine administration were studied. Bay K 8644 (2 and 4 mg kg-1, i.p.) caused an increase in the synthesis of both dopamine in the striatum and 5-HT in the midbrain and striatum, measured as the accumulation of 3,4-dihydroxyphenylalanine (dopa) and 5-hydroxytryptophan, respectively. Moreover, Bay K 8644 at the dose of 4 mg kg-1 increased the turnover of dopamine in the striatum and of 5-HT in midbrain and striatum. These neurochemical changes were antagonized by the calcium channel antagonist nimodipine (10 mg kg-1, i.p.). It is concluded that dihydropyridine receptors may mediate the brain region-specific changes in the dopaminergic and 5-HT-ergic neurotransmission which occur following activation of neuronal calcium channels.

Cholesterol-fed rabbits: study of the response of the vas deferens to adrenergic and non-adrenergic stimulus and to a kappa-opioid agonist.

The aim of the present study was to analyse the contractility of the isolated vas deferens from hypercholesterolaemic rabbits; for this purpose we evaluated the contractile response induced by noradrenaline and by electrical stimulation. A significant increase in the amplitude of adrenergic and non-adrenergic responses was observed in vas deferens from hypercholesterolaemic rabbits. These data suggest an increase in the contractility of the smooth muscle in these animals.

Alveolar-arterial oxygen gradients versus the Neonatal Pulmonary Insufficiency Index for prediction of mortality in ECMO candidates.

Current selection criteria necessary for intelligent application of extracorporeal membrane oxygenation (ECMO) in hypoxic neonates remains controversial. Both the Neonatal Pulmonary Insufficiency Index (NPII) and serial alveolar-arterial oxygenation gradient measurements (A-a)Do2 have been recommended. Accordingly, an analysis of 50 consecutive severely hypoxic neonates was undertaken to assess the predictive value of (A-a)Do2 determinations and NPII in discriminating survivors from non-survivors. These infants with meconium aspiration syndrome (MAS), congenital diaphragmatic hernia (CDH), or persistent pulmonary hypertension of the newborn (PPHN) required maximum mechanical ventilation for hypoxia. Pharmacologic manipulation of pulmonary vascular resistance was attempted in 83%. If postductal (A-a)Do2 remained greater than or equal to 620 torr despite 12 hours of maximum medical therapy, mortality was 100%; however, 35% of nonsurvivors were unfortunately excluded. (A-a)Do2 greater than or equal to 600 torr for 12 hours demonstrated 93.8% mortality, and only 12% of all mortalities were thus excluded. Among nonsurvivors successfully hyperventilated, the NPII could not predict mortality. Ideal selection criteria must exclude those who would otherwise survive without ECMO, yet allow early accurate identification of the neonate certain to die. It would appear that serial (A-a)Do2 determinations best permit this identification and thus orderly application of ECMO.