Rankl genetic deficiency and functional blockade undermine skeletal stem and progenitor cell differentiation.

BACKGROUND: Skeletal Stem Cells (SSCs) are required for skeletal development, homeostasis, and repair. The perspective of their wide application in regenerative medicine approaches has supported research in this field, even though so far results in the clinic have not reached expectations, possibly due also to partial knowledge of intrinsic, potentially actionable SSC regulatory factors. Among them, the pleiotropic cytokine RANKL, with essential roles also in bone biology, is a candidate deserving deep investigation. METHODS: To dissect the role of the RANKL cytokine in SSC biology, we performed ex vivo characterization of SSCs and downstream progenitors (SSPCs) in mice lacking Rankl (Rankl-/-) by means of cytofluorimetric sorting and analysis of SSC populations from different skeletal compartments, gene expression analysis, and in vitro osteogenic differentiation. In addition, we assessed the effect of the pharmacological treatment with the anti-RANKL blocking antibody Denosumab (approved for therapy in patients with pathological bone loss) on the osteogenic potential of bone marrow-derived stromal cells from human healthy subjects (hBMSCs). RESULTS: We found that, regardless of the ossification type of bone, osteochondral SSCs had a higher frequency and impaired differentiation along the osteochondrogenic lineage in Rankl-/- mice as compared to wild-type. Rankl-/- mice also had increased frequency of committed osteochondrogenic and adipogenic progenitor cells deriving from perivascular SSCs. These changes were not due to the peculiar bone phenotype of increased density caused by lack of osteoclast resorption (defined osteopetrosis); indeed, they were not found in another osteopetrotic mouse model, i.e., the oc/oc mouse, and were therefore not due to osteopetrosis per se. In addition, Rankl-/- SSCs and primary osteoblasts showed reduced mineralization capacity. Of note, hBMSCs treated in vitro with Denosumab had reduced osteogenic capacity compared to control cultures. CONCLUSIONS: We provide for the first time the characterization of SSPCs from mouse models of severe recessive osteopetrosis. We demonstrate that Rankl genetic deficiency in murine SSCs and functional blockade in hBMSCs reduce their osteogenic potential. Therefore, we propose that RANKL is an important regulatory factor of SSC features with translational relevance.

The direct impact of pegvisomant on osteoblast functions and bone development.

PURPOSE: Acromegaly is a chronic disease characterized by growth hormone (GH) hypersecretion, usually caused by a pituitary adenoma, resulting in elevated circulating levels of insulin-like growth factor type I (IGF-I). Pegvisomant (PEG), the GH-receptor (GHR) antagonist, is used in treating acromegaly to normalize IGF-I hypersecretion. Exposure to increased levels of GH and IGF-I can cause profound alterations in bone structure that are not completely reverted by treatment of GH hypersecretion. Indeed, there is evidence that drugs used for the treatment of acromegaly might induce direct effects on skeletal health regardless of biochemical control of acromegaly. METHODS: We investigated, for the first time, the effect of PEG on cell proliferation, differentiation, and mineralization in the osteoblast cell lines MC3T3-E1 and hFOB 1.19 and its potential impact on bone development in zebrafish larvae. RESULTS: We observed that PEG did not affect osteoblast proliferation, apoptosis, alkaline phosphatase (ALP) activity, and mineralization. After PEG treatment, the analysis of genes related to osteoblast differentiation showed no difference in Alp, Runx2, or Opg mRNA levels in MC3T3-E1 cells. GH significantly decreased cell apoptosis (- 30 ± 11%, p < 0.001) and increased STAT3 phosphorylation; these effects were suppressed by the addition of PEG in MC3T3-E1 cells. GH and PEG did not affect Igf-I, Igfbp2, and Igfbp4 mRNA levels in MC3T3-E1 cells. Finally, PEG did not affect bone development in zebrafish larvae at 5 days post-fertilization. CONCLUSION: This study provides a first evidence of the impact of PEG on osteoblast functions both in vitro and in vivo. These findings may have clinically relevant implications for the management of skeletal health in subjects with acromegaly.

Direct effects of octreotide on osteoblast cell proliferation and function.

PURPOSE: Octreotide (OCT) is a first-generation somatostatin analog (SSA) used in the treatment of acromegaly and neuroendocrine tumors (NETs). In both diseases, OCT interacts with somatostatin receptors 2 and 5 (SSTR2 and SSTR5), inhibiting hormone hypersecretion and cell proliferation. Skeletal health is an important clinical concern in acromegaly and NETs, since acromegalic osteopathy and NET bone metastasis occur in a remarkable number of patients. While OCT's effect on NET and pituitary cells has been extensively investigated, its direct action on bone cells remains unknown. METHODS: Here, we investigated OCT direct effects on cell proliferation, differentiation, mineralization, and chemoattractant capacity of murine primary osteoblasts and osteoblast cell line MC3T3-E1. RESULTS: OCT inhibited osteoblasts and MC3T3-E1 cell proliferation (- 30 ± 16%, and - 22 ± 4%, both p < 0.05 vs control) and increased MC3T3-E1 cell apoptosis (+ 76 ± 32%, p < 0.05 vs control). The anti-proliferative action of OCT was mediated by SSTR2 and SSTR5 in MC3T3-E1, while its pro-apoptotic effect was abrogated in SSTR2-silenced cells. The analysis of genes related to the early and late phases of osteoblast differentiation showed that OCT did not affect Alp, Runx2, Bglap, Spp1, and Sost levels in MC3T3-E1 cells. Similarly, OCT did not affect ALP activity, mineralization, and osteoclastogenic induction. Finally, Vegfa expression decreased in OCT-treated MC3T3-E1 cells and OCT inhibited pancreatic NET cell migration toward the osteoblast-conditioned medium. CONCLUSION: This study provides the first evidence of the direct action of OCT on osteoblasts which may have clinically relevant implications for the management of skeletal health in subjects with acromegaly and metastatic NETs.

In vitro performance of ultrasound enamel preparation compared with classical bur preparation on pit and fissure sealing.

AIM: Fissure sealants are effective in preventing caries. The aim of this in vitro study was to evaluate the effects of two different enamel surface preparation techniques for pit and fissure sealing. MATERIALS AND METHODS: Sixty extracted sound third molars were used. For each tooth, the mesial half of the occlusal fissures was treated with ultrasound diamond tip T1 mounted on an ultrasonic handpiece, while the distal half with conventional diamond bur. The teeth were randomly divided into 2 groups (n = 30/each). Group 1 samples were stored in distilled water at 4 °C. For group 2 samples, sealing of occlusal fissures was performed according to standard procedures. Bucco-lingual cuts parallel to the long axis of the tooth were made in order to separate the two different types of preparations. The effects of the executed procedures were assessed with SEM. RESULTS: Surfaces prepared with ultrasound system showed the presence of residual debris and appeared more irregular than surfaces prepared with traditional bur system. Furthermore, images showed the presence of cracks on the bottom and on the walls of the ultrasound prepared fissures. CONCLUSION: Conventional bur surface treatment showed a better performance when compared to ultrasound preparation and could probably ensure superior sealant retention.

Gestational diabetes: An overview with attention for developing countries.

Gestational diabetes mellitus (GDM) is defined as a glucose intolerance that occurs for the first time or it is first identified during pregnancy. The GDM etiology is multifactorial. It has not completely been established yet and several known risk factors may contribute to its onset. To date, there are no shared guidelines on the management and follow-up, especially regarding the low-income countries. In this paper, we describe the state of art about epidemiology, physiopathology, diagnosis, and management of GDM. Moreover, we focus on the current state in low income countries trying to outline basis for further research.

Sesqui- and triterpenoids from the liverwort Lepidozia chordulifera inhibitors of bacterial biofilm and elastase activity of human pathogenic bacteria.

Five dammarane-type triterpenoids, five pentacyclic triterpenoids (three of them carrying a carboxylic acid group), and two aromadendrane-type sesquiterpenoids were isolated from an Argentinian collection of the liverwort Lepidozia chordulifera. Compounds were characterized by comparison of their spectral data with those previously reported and tested in their ability to control bacterial growth, biofilm formation, bacterial Quorum Sensing process (QS), and elastase activity of Pseudomonas aeruginosa, as well as bacterial growth and biofilm formation of Staphylococcus aureus. The key role played by biofilm and elastase activity in bacterial virulence make them a potential target for the development of antibacterial agents. The aromadendrane-type sesquiterpenoid viridiflorol was the most potent biofilm formation inhibitor, producing 60% inhibition in P. aeruginosa and 40% in S. aureus at 50µg/ml. Ursolic and betulinic acids (two of the pentacyclic triterpenoids isolated) were able to reduce 96 and 92% the elastase activity of P. aeruginosa at 50µg/ml, respectively. Among the analyzed triterpenoids, those that carry a dammarane skeleton were the most potent inhibitors of the P. aeruginosa biofilm formation and were active against both P. aeruginosa and S. aureus. Subsequently, a computer-assisted study of the triterpenoid compounds was carried out for a better understanding of the structure-activity relationships.

Nanoscale effects of caspofungin against two yeast species, Saccharomyces cerevisiae and Candida albicans.

Saccharomyces cerevisiae and Candida albicans are model yeasts for biotechnology and human health, respectively. We used atomic force microscopy (AFM) to explore the effects of caspofungin, an antifungal drug used in hospitals, on these two species. Our nanoscale investigation revealed similar, but also different, behaviors of the two yeasts in response to treatment with the drug. While administration of caspofungin induced deep cell wall remodeling in both yeast species, as evidenced by a dramatic increase in chitin and decrease in ß-glucan content, changes in cell wall composition were more pronounced with C. albicans cells. Notably, the increase of chitin was proportional to the increase in the caspofungin dose. In addition, the Young modulus of the cell was three times lower for C. albicans cells than for S. cerevisiae cells and increased proportionally with the increase of chitin, suggesting differences in the molecular organization of the cell wall between the two yeast species. Also, at a low dose of caspofungin (i.e., 0.5× MIC), the cell surface of C. albicans exhibited a morphology that was reminiscent of cells expressing adhesion proteins. Interestingly, this morphology was lost at high doses of the drug (i.e., 4× MIC). However, the treatment of S. cerevisiae cells with high doses of caspofungin resulted in impairment of cytokinesis. Altogether, the use of AFM for investigating the effects of antifungal drugs is relevant in nanomedicine, as it should help in understanding their mechanisms of action on fungal cells, as well as unraveling unexpected effects on cell division and fungal adhesion.

Apoptotic effects of a chimeric plant virus carrying a mimotope of the hepatitis C virus hypervariable region 1: role of caspases and endoplasmic reticulum-stress.

The role of apoptosis in the persistence of hepatitis C virus (HCV) infection is controversial. Moreover, conflicting data on the modulation of this process by HCV proteins have been provided. We evaluated the susceptibility of peripheral lymphocytes from patients with chronic hepatitis C to apoptosis both spontaneous and after incubation with a chimeric Cucumber mosaic virus (CMV) carrying 180 copies of the synthetic R9 mimotope obtained from more than 200 hypervariable region-1 sequences of HCV. Resting T lymphocytes were found to be sensitized to apoptosis as a result of chronic HCV infection. The plant virus-derived vector R9-CMV displayed a strong pro-apoptotic effect associated with activation of both caspase-8 and -9, indicating the involvement of both extrinsic and intrinsic apoptotic pathways. A parallel R9-CMV-mediated activation of endoplasmic reticulum-stress was suggested by the significant induction of BiP/GRP78, GADD153 and caspase-12. These data contribute to define the complex HCV/host interaction, and open new prospects for developing a plant-derived antigen-presenting system to strengthen host defences against persistent pathogens.

Emtricitabine (FTC) for the treatment of HIV infection.

Emtricitabine (FTC) is a synthetic nucleoside analogue of cytosine, which is intracellularly phosforylated to form the active form emtricitabine 5'triphosphate (E5TP). E5TP inhibits both HIV and Hepatitis B reverse transcriptase. Clinical trials have shown FTC to be active as part of highly active anti-retroviral therapy in naïve HIV-positive patients. FTC may be dosed once daily and in vitro is less associated with the M184V mutation, which is classically associated with failure of treatment with lamivudine. In clinical practice, toxicity with FTC is unusual. The most common treatment-related adverse events are diarrhoea, headache, nausea, dizziness, abdominal pain, aesthenia and rash. Skin discolouration, which is typically reported as hyperpigmentation and usually affecting either the palms of the hands or the soles of the feet, is reported on under 2% of individuals and is almost exclusive to patients of African origin.

Unconventional medicine teaching at the Universities of the European Union.

CONTEXT: The recent rapid increase in demand for and use of unconventional medicine requires an adequate medical education. In the United States, 64% of medical schools offer undergraduate courses. No information is available about similar courses at European universities. OBJECTIVES: To document the incidence of educational courses on unconventional medicine offered by the European universities and their topic content. DESIGN: Mail survey, which consisted of two questionnaires and was conducted in 1999 (January-June). The first questionnaire was sent to the universities' Rectorats, the second one to the faculties or lecturers indicated by the replies to the first questionnaire. PARTICIPANTS: The Universities listed by the Confederation of European Union Rectors' Conference. MAIN OUTCOME MEASURES: Courses offered at European Universities, both at medical faculties and at other faculties. RESULTS: Five hundred and fifty (550) universities were contacted. Replies were received from 326 (59%); 141 have a faculty of medicine and 107 (76%) of them replied. We also received answers from 29 faculties of health sciences. In addition we received 190 (50%) answers from 380 other miscellaneous universities. Courses on unconventional medicine were offered by 43 (40%) medical schools, 21 (72%) health sciences faculties, and by 15 (8%) other faculties. Topics covered encompassed a wide range of techniques (33), from homeopathy to shamanism. CONCLUSIONS: Unconventional medicine courses are widely represented at European universities. They cover a wide range of therapies. Many of them are used clinically. Research work is underway at several faculties.

Cranio-orbital missile wound and bullet migration. Case report.

An unusual case of craniocerebral missile injury, with orbital roof perforation and spontaneous bullet migration into the maxillary sinus, is reported. Emergency treatment consisted in wide craniectomy around the bullet entry point, blood and foreign bodies debridement. Subsequent procedures were necessary for abscess evacuation, transmaxillary bullet removal and later cranial vault reconstruction. Challenging aspects were the treatment of the infectious complications, following cerebrospinal fluid fistula through the wound, and the onset of post-traumatic epilepsy, scarcely responsive to common antiepileptic drugs. The treatment of the abscess by combined systemic and intracavitary antibiotic therapy and of the chronic seizures by progressive adjustment with new protocols of antiepileptic drugs under EEG and brain mapping revealed successful.

Conservative management of C5-C6 fracture-luxation in the elderly. Case report.

The case of a 86-year-old female victim of cervical trauma with C5-C6 fracture-luxation and bilateral locked facets is hereinafter described. The case was reputed representative of the complexity in deciding between conservative and surgical treatment in spine surgery. Conservative management would have been dictated by the additional risks linked to the age of the patient, whereas the surgical treatment would have been favored by the easy access to the anterior cervical spine for reduction and fixation. On the way to plan the definitive treatment, the patient died by pulmonary embolism. The controversial aspects and the indication to anticoagulant therapy in spinal surgery, frequently faced in the treatment of spine trauma in patients of this age group, are discussed in this case report.

[Endoscopic sphincterotomy in acute biliary pancreatitis. A multicenter study]. / La sfinterotomia endoscopica nella pancreatite acuta biliare. Studio multicentrico.

BACKGROUND: Severe acute pancreatitis are still dangerous, as can be seen from the high mortality rate (around 30%). It's commonly known that in the last years diagnostic ability, precise stadiation and control over the disease development were increased. Surgical therapy is directed mainly to achieve the control over septic evolution, and in biliary pancreatitis to resolve hypertension and infection inside the biliary tree (now widely accepted as the real cause of this disease). Recently, some authors demonstrated that the prognosis of a pancreatitis can be greatly improved by stopping the evolution toward a severe phase with necrosis and multiple organ failure without any surgical operation. They believe that's possible to achieve this result performing an early endoscopic sphincterotomy in all biliary pancreatitis (recognised by means of echography and CT scan), before the development of necrosis. METHODS: In our department, following this address, very good results have been obtained: that's why a multicentric trial has been carried out to appraise the results obtained by endoscopic sphincterotomy within 48 hours from the disease onset. Answers from 114 hospitals were received, with a total survey of more than 10,000 pancreatitis. Endoscopic sphincterotomy was performed in 55% of billiary pancreatitis, which were estimated at the onset, on second or third degree of Balthazar classification. RESULTS: In 86% of patients, endoscopic sphincterotomy was performed within 48 hours from symptoms onset. Complications related to this treatment are reported in less than 4% of patients. CONCLUSIONS: The procedure, avoiding the evolution toward necrosis and sepsis, allowed the reduction of surgical operations to 6.7%, and of the mortality amongst all cases gathered to 2.5%.

Efficacy of lodoxamide eye drops on mast cells and eosinophils after allergen challenge in allergic conjunctivitis.

PURPOSE: The purpose of the study is to evaluate in a double-blind, randomized study the efficacy of lodoxamide tromethamine 0.1% versus placebo. METHODS: Signs and symptoms, tear tryptase, and tear fluid cytology were evaluated in 20 asymptomatic subjects with allergic conjunctivitis. The study included three allergen challenges in skin test-positive patients. At the first visit, a threshold dose of allergen was established. At the second visit, a bilateral ocular challenge was performed without pretreatment. At the third visit, either lodoxamide or placebo eye drops were used for 1 week before ocular challenge. RESULTS: Lodoxamide significantly reduced tryptase levels (P < 0.01), neutrophils (P < 0.04), and eosinophils (P < 0.01) in the tear fluid and significantly inhibited ocular itching (P < 0.02) when compared with that of placebo. CONCLUSIONS: Lodoxamide is effective in reducing tryptase levels and the recruitment of inflammatory cells in the tear fluid after allergen challenge.

Tear tryptase levels and allergic conjunctivitis.

We measured tryptase, a neutral protease stored in the secretory granules of mast cells, by solid-phase radioimmunoassay in tears of 12 subjects with vernal keratoconjunctivitis (VKC) during remission phases, nine subjects with seasonal or perennial allergic conjunctivitis, and eight healthy controls. Mean values of tear tryptase levels were significantly (P < 0.02) increased in VKC patients (14.5 +/- 13 micrograms/l) when compared to those measured in patients with seasonal or perennial allergic conjunctivitis (0.6 +/- 0.1 microgram/l) and in controls (3.3 +/- 3.2 micrograms/l). In subjects with allergic conjunctivitis, the levels of tryptase, almost undetectable before allergen conjunctival challenge, showed a significant increase in the challenged eye 20 min-but not 6 h-after provocation in 5/9 cases. Our results indicate that VKC a severe ocular disease characterized by an increased number and abnormal distribution of mast cells in the conjunctiva, also shows elevated levels of tryptase in tears even during remission phases. Evidence of mast-cell activation, as revealed by a significant increase of tryptase levels in tears, in documented during the early-phase reaction, but not during the late-phase reaction, of allergic conjunctivitis patients challenged topically by specific allergen.

Efficacy of mipragoside ophthalmic gel in vernal keratoconjunctivitis.

To evaluate the efficacy of Mipragoside, a ganglioside derivative, in vernal keratoconjunctivitis we performed a controlled randomised clinical trial involving 24 patients (mean age 10 +/- 3.4 years, range 5-20 years). Patients received either Mipragoside 0.5% aqueous ophthalmic gel or placebo four times a day for 2 weeks after a week of treatment with placebo. Ocular signs and symptoms were evaluated and considered for statistical analysis. Results show that Mipragoside significantly reduces all symptoms, being most effective on itching (p = 0.01; p = 0.0001) and hyperaemia (p = 0.01; p = 0.0006) after 1 and 2 weeks respectively when compared with placebo. Physician judgement of drug efficacy at the end of treatment was significantly in favour of Mipragoside (p = 0.0001) compared with placebo. We conclude that Mipragoside topical treatment improves symptoms of patients with vernal keratoconjunctivitis and we postulate a possible anti-inflammatory activity of this compound.

Estrogen and progesterone receptors in vernal keratoconjunctivitis.

PURPOSE: Sex-related influences have been implicated in the pathogenesis of vernal keratoconjunctivitis (VKC), an allergic eosinophilic disease. METHODS: The authors evaluated tarsal and bulbar conjunctival biopsies from seven patients with severe and symptomatic VKC for the presence of estrogen and progesterone receptors by using monoclonal antibodies with a peroxidase-antiperoxidase technique. RESULTS: Both the epithelium and subepithelium of the tarsal and bulbar conjunctiva of patients with VKC, but not those of four nonatopic control subjects, showed intense positive staining for estrogen and progesterone receptors. Immunofluorescence colocalization of both estrogen and progesterone receptors with eosinophil cationic protein showed that approximately 70% of positive cells were eosinophils. CONCLUSIONS: Sexual hormones, through their receptors, may influence the activity of eosinophils in patients with VKC.

Vernal keratoconjunctivitis: a model of 5q cytokine gene cluster disease.

Clinical studies of vernal keratoconjunctivitis (VKC) patients show that total IgE serum levels are increased even in the absence of IgE antibodies to common allergens. Activated eosinophils are also a constant feature of VKC at both the circulation (cytofluorimetry) and tissue (tear cytology and conjunctival scrapings) levels. Moreover, allergen challenge induces a prolonged inflammatory reaction with a prevalent participation of eosinophils, lymphocytes and possibly basophils. Immunohistochemical studies of VKC biopsies show a multicellular inflammatory infiltrate with prevalence of activated eosinophils, mast cells and CD4 lymphocytes in both epithelium and subepithelium. Mediator studies indicate that eosinophil products (eosinophil peroxidase, eosinophinal cationic protein and eosinophil-derived neurotoxin/eosinophil protein X) are increased in both serum and tears, where tryptase and interleukin (IL)-5 are also detectable in higher amounts than in controls. On the basis of these findings, we postulate that VKC can represent a phenotypic model of up-regulation of the cytokine gene cluster on chromosome 5q which through its products (IL-3, IL-4, IL-5 and granulocyte/macrophage-colony-stimulating factor) regulates Th2 prevalence, IgE production as well as mast cell and eosinophil growth and function in VKC.

Hepatitis B virus infection in chronic uremia: long-term follow-up of a two-step integrated protocol of vaccination.

Active immunization is crucial for eradicating hepatitis B virus infection from dialysis units. A prospective study was performed in 63 consecutive chronic uremic patients, which included the following: (1) the intramuscular (IM) administration of 40 micrograms of a DNA-recombinant vaccine (Engerix-B, Smith Kline & French Laboratories, Milan, Italy) to all chronic uremic patients at 0, 1, 2, and 6 months; (2) the antibody titer determination at the seventh month (chronic uremic patients with a titer > 100 mIU/mL received an IM booster dose of 40 micrograms at 18 months [group A], and those with a titer < 100 mIU/mL received a further IM dose of 40 micrograms at 12 months [group B]); and (3) the intradermal inoculation of 5 micrograms of vaccine every 2 weeks until the protective titer (> or = 10 mIU/mL) was achieved, and then monthly for 6 months, in chronic uremic patients who did not have a protective titer even after 19 months (group C). Thus, 41, 17, and five chronic uremic patients were allocated to groups A, B, and C, respectively. All developed a protective titer: 79.4%, 84.0%, and 87.5% after the fourth, fifth, and sixth IM dose at 7, 13, and 19 months, respectively. Five chronic uremic patients (group C) achieved seroprotection after 3.8 +/- 0.5 (SEM) intradermal inoculations.(ABSTRACT TRUNCATED AT 250 WORDS)