Exosomes: novel regulators of bone remodelling and potential therapeutic agents for orthodontics.

Recent studies suggest that exosomes are involved in intercellular communication required for the maintenance of healthy bone. Exosomes are small (30-150 nm in diameter) extracellular vesicles that are formed in multivesicular bodies and are released from cells as the multivesicular bodies fuse with the plasma membrane. Regulatory exosomes have the capacity to exert profound control over target cells. They can stimulate plasma membrane receptors and are also internalized by the target cell delivering proteins, lipids, small molecules and functional RNAs from the cell of origin. We and others have recently reported on regulatory exosomes from osteoclasts and osteoblasts. Key candidate molecules identified in exosome-based regulation of bone remodelling include receptor activator of nuclear factor kappa B (RANK), RANK-ligand (RANKL), ephrinA2, semaphorin 4D, microRNA-146a and microRNA- 214-3p. Exosomes will likely prove to be crucial elements in the communication networks integrating bone cells (osteoclasts, osteoblasts, osteocytes) and linking bone to other tissue. Exosomes collected from bone cells grown in culture may prove useful to augment bone remodelling associated with orthodontic force application or required for the repair of craniofacial bone. Various technologies allow exosomes to be engineered to improve their targeting and efficacy for therapeutic purposes. In summary, exosomes have emerged as important elements of the machinery for intercellular communication between bone cells. They hold great promise as therapeutic targets, biomarkers and therapeutic agents for orthodontists.

Effects of cocoa-enriched diet on orofacial pain in a murine model.

OBJECTIVES: To investigate and discuss the effects of cocoa on orofacial pain. SETTING AND SAMPLE POPULATION: The Department of Orthodontics at the University of Florida (UF). Male and female hairless rats (N=20/group) were tested. MATERIALS AND METHODS: Rats were tested using the Orofacial Pain Assessment Device (OPAD) before and after changing their food from the standard chow to a cocoa-enriched or control-equivalent diet. RESULTS: Male rats fed the cocoa diet had a significantly higher operant pain index when tested at 37°C as compared to control diet-fed animals. Female rats on the cocoa diet had a significantly higher pain index when tested at 18°C and 44°C, as compared to animals fed the control diet. Capsaicin-induced pain was inhibited, with cocoa-diet male rats having a significantly higher pain index than control-diet male rats and cocoa-diet female rats at both 37°C and 44°C. Cocoa-diet female rats had a significantly higher pain index at 44°C than control-diet females. Mechanical sensitivity was affected following capsaicin cream, with a significantly decreased tolerated bottle distance in both cocoa- and control-diet animals, but there was no difference between cocoa- and control-diet groups. CONCLUSION: Using the OPAD operant system, we demonstrated that a diet rich in cocoa was effective in inhibiting neurogenic inflammatory pain in rats. This has implications for the use of novel alternative therapies such as diet modification for pain control.

[Juvenile systemic lupus erythematosus with unusual manifestation of lupus-associated panniculitis]. / Juveniler systemischer Lupus erythematodes mit der ungewöhnlichen Manifestation einer Lupus-assoziierten Pannikulitis.

Juvenile systemic lupus erythematosus (JSLE) is a rare multisystem autoimmune disease with broad heterogeneity of clinical manifestations. Diagnosing JSLE is often very challenging. This life-threatening, unpredictable, and relapsing disease, which may affect various organ systems, requires interdisciplinary, lifelong care. Here, we report the case of a 13-year-old patient with JSLE suffering from recurrent arthralgia, lupus panniculitis, and rashes that were successfully treated with hydroxychloroquine and prednisolone.

[Guideline for antiretroviral therapy of HIV-infected children and adolescents]. / Leitlinie der Pädiatrische Arbeitsgemeinschaft AIDS (PAAD) e.V. zur antiretroviralen Therapie bei HIV-infizierten Kindern und Jugendlichen (2011).

The HIV-infection in adults or children and adolescent differs substantially. Differences include the mode of infection, viral dynamics facing a developing immune system and the clinical course of the infection. In addition to the virological, immunological and epidemiological aspects the psychosocial situation is also very different. The above aspects and the decreased number of antiretroviral substances underline the need for specific guidelines for HIV-therapy in children and adolescents. The German Pediatric Working group AIDS (PAAD) has formulated this guideline in 2011 based on new study results, changes in international recommendations and newly available drugs.

Intradermal injection of Botulinum toxin type A alleviates infraorbital nerve constriction-induced thermal hyperalgesia in an operant assay.

Recent studies have shown that infraorbital nerve constriction (IoNC)-induced mechanical allodynia has been attenuated by administration of highly purified 150-kDa Botulinum neurotoxin type A (BoNT/A). Here, we extend these studies to determine whether BoNT/A could attenuate IoNC-induced symptoms of thermal hyperalgesia. Instead of testing head withdrawal thresholds, a thermal operant assay was used to evaluate cortical processing of sensory input following IoNC. In this assay, a fasted rat's desire to obtain a food reward (sweetened condensed milk) is coupled to its ability to tolerate facial contact with a warm (45 °C) thermode. Bilateral IoNC decreased the ratio of thermode contact duration/event, which is an indicative of thermal hyperalgesia. BoNT/A injection intradermally in the area of infraorbital nerve (IoN) innervation 7 days after IoNC resulted in decreased number of facial contacts and increased the ratio of contact duration/event (measured at 14 days after IoNC). The BoNT/A (2-200 pg) effects were dose dependent and statistically significant at 100 and 200 pg (P < 0·05). Complete reversal of thermal hyperalgesia symptoms was obtained with a 200-pg dose, without affecting sham rat behaviour. Off-site (neck) injection of BoNT/A did not relieve thermal hyperalgesia, while co-injection of BoNT/A with a neutralising antibody in the area of IoN innervation prevented relief of thermal hyperalgesia. Neither IoNC nor BoNT/A injection affected operant assay parameters with a 24 °C thermode, indicating selectivity of thermal hyperalgesia measurements. These results strongly suggest that intradermal injection of BoNT/A in the area of IoN innervation alleviates IoNC-induced thermal hyperalgesia in an operant assay.

Induction maintenance concept for HAART as initial treatment in HIV infected infants.

BACKGROUND: Early initiated antiretroviral therapy (ART) in HIV infected infants leads to improved long-term viral suppression and survival. Guidelines recommend initiating therapy with a triple ART consisting of two nucleoside reverse transcriptase inhibitors (NRTIs) and either one additional non-nucleoside reverse transcriptase inhibitor (NNRTI) or a protease inhibitor (PI). Compared to older children and adults, viral relapse is seen more frequently in infants receiving triple ART. We now address the possibility of a more potent ART with a quadruple induction and triple maintenance therapy. METHODS: We examine the longitudinal course in four HIV infected infants, who were referred from other centers and could not be recruited to multicentre trials. We introduced ART initially consisting of two NRTIs, one NNRTI and one PI and later discontinued the PI at the age of 12 months maintaining a triple regime consisting of two NRTIs and one NNRTI. RESULTS: Provided that therapy adherence was maintained we observed an effective sustained decline of viral load and significant CD4 cell reconstitution even after switching to a triple regime. No drug associated toxicity was seen. CONCLUSION: We suggest that a four drug therapy might be a possible initial therapy option in HIV infected infants, at least in those with a high viral load, followed by a maintenance triple regime after 12 months of therapy.

Disease progression in HIV-1 infected children and adolescents--results of a German-Austrian cohort study.

OBJECTIVE: The Paediatric Working Group AIDS (PAAD) initiated a prospective cohort study in order to investigate disease progression in HIV- infected children and adolescents and the effect of antiretroviral treatment regimes. PATIENTS AND METHODS: Between 1998 and 2003, paediatric centres documented HIV-infected patients under clinical care using a questionnaire for basic data and annual follow up. Main outcome measures were: use of antiretroviral therapy, adverse events, disease progression and change of therapeutic regimes. RESULTS: 174 HIV- infected paediatric patients were followed up in 12 centres in Germany and Austria between 1998 and 2003. Initially 54 (31%) patients had no antiretroviral therapy, 35 (20%) received a two-drug regimen (ART) and 85 patients (49%) a highly active antiretroviral therapy (HAART>or=3 drugs). After an observation period of 5 years, 8 patients (4%) had no therapy, 17 (10%) were on ART and 134 patients on HAART (77%). The number of patients with salvage therapy (>or=4 drugs) increased from 5 (3%) to 15 patients (9%). 72 of 166 treated patients (43%) had no change of their drug regimes, 68 patients (41%) had one change and 26 patients (16%)>or=2 changes. Main reasons for changes were increased viral load (49%), immunologic deterioration (21%) and adverse events (14%). During the follow up period no patient died. According to the CDC classification, disease progression was seen in 48 of 174 patients (28%), of whom 20 had deteriorations of clinical categories (A, B, C) and 28 of immunologic categories. Using Kaplan-Meier curves, the mean time from study onset until change of clinical categories was 61 months for patients on HAART, 26 months for patients on ART and 14 months for patients without ART. CONCLUSION: In paediatric patients with HIV infection, disease progression has declined substantially by introduction of HAART. Superiority of HAART compared with ART was demonstrated. Non-adherence as well as other reasons for treatment failure have to be studied more carefully.

Effects of High-Dose Capsaicin on TMD Subjects: A Randomized Clinical Study.

Temporomandibular joint disorder (TMD) is a complex musculoskeletal disorder that presents with pain, limited jaw opening, and abnormal noises in the temporomandibular joint. Despite the significant impact that TMD has in terms of suffering and financial burden, relatively few new treatments have emerged; therefore, development of novel treatments to treat TMD pain remains a high priority. The rationale of this study was to use a double-blind, vehicle-controlled clinical trial to evaluate the effects of a high-concentration (8%) capsaicin cream on TMD. This is based on the hypothesis that targeting TRP vanilloid subfamily member 1 (TRPV1) for pain control may provide a novel method for pain relief in TMD patients. TRPV1 is primarily expressed on a population of nociceptive-specific neurons and provides a candidate target for the development of pain treatments. Capsaicin is the primary agonist for TRPV1 and has been used previously in relatively low doses (0.025% to 0.075%) as a therapeutic for a variety of pain disorders, including postherpetic neuralgia and osteoarthritis; however, analgesic efficacy remains equivocal. TMD and healthy control subjects were assigned to either an active capsaicin or vehicle control group. The treatments were applied for 2 h and then removed. Quantitative sensory testing (QST) was completed prior to drug application (baseline), 2 h after drug application, and 1 wk later. Perceived pain intensity was measured using a visual analog scale (VAS) following capsaicin or vehicle cream application. Significantly lower pain was reported in the week after application in the capsaicin-treated TMD subjects. For QST measures, there was a decreased thermal pain threshold 2 h after capsaicin application for both the control and TMD groups, but this resolved within a week. Capsaicin had no effect on pressure pain threshold or mechanical sensitivity in both TMD and healthy individuals. This study demonstrates that 8% topical capsaicin therapy is a relatively safe, simple, and effective treatment for patients with TMD. Knowledge Transfer Statement: This study evaluated a novel topical capsaicin therapy for reducing orofacial pain. The results of this study can be used to provide another treatment option for patients with TMD.

Response to thyrotropin of normal thyroid follicular cell strain FRTL5 in hypergravity.

Thyroid hormones control every cell in the organisms and, as indicated by many hormonal changes in astronauts during and shortly after space missions, its complex regulation may be influenced by gravity. To test in vitro the effects of gravity environment on thyroid, we selected a unique cultured cell system: the FRTL5, a normal follicular thyroid cell strain in continuous culture, originally derived from adult rat thyroids. To establish if modifications of the gravitational environment may interfere with post-receptorial signal transduction mechanisms in normal mammalian cultured cells, following our previous microgravity experiments, we exposed thyrotropin-stimulated and unstimulated FRTL5 cells to hypergravity (5 g and 9 g) in a special low-speed centrifuge. At all thyrotropin doses tested, we found significant increases in terms of cyclic AMP production in FRTL5 thyroid cells. The data here reported correlate well with our previous microgravity data, showing that the FRTL5 cells functionally respond to the variable gravity force in a dose-dependent manner in terms of cAMP production following TSH-stimulation.

Relationship between capsaicin-evoked substance P release and neurokinin 1 receptor internalization in the rat spinal cord.

The relationship between substance P release and the activation of its receptor in the spinal cord remains unclear. Substance P release is usually measured by radioimmunoassay, whereas the internalization of the neurokinin 1 (NK1) receptor has been used to assess its activation by noxious stimuli. Our objective was to compare substance P release and NK1 receptor internalization produced by capsaicin in rat spinal cord slices. Superfusion of the slices with capsaicin for 3 min produced a gradual increase in substance P release that peaked 3-7 min afterward, and then decreased to baseline levels. The concentration-response curve for capsaicin was biphasic, with concentrations above 10 microM producing significantly less release. The effective concentration for 50% of response (EC(50)) for capsaicin, calculated from its stimulatory phase, was 2.3 microM. However, the potency of capsaicin to elicit NK1 receptor internalization in the same slices was one order of magnitude higher (EC(50)=0.37 microM) in lamina I, probably because NK1 receptors become saturated at relatively low concentrations of substance P. The potency of capsaicin to produce internalization was progressively lower in lamina III (EC(50)=1.9 microM) and lamina IV (EC(50)=14.5 microM), suggesting that neurokinins released in laminae I-II become diluted as they diffuse to the inner dorsal horn. To study the correlation between these two measures, we plotted substance P release against NK1 receptor internalization and fitted a saturation binding function to the points. The correlation was good for laminae I (R(2)=0.82) and III (R(2)=0.78), but it was poor (R(2)=0.35) for lamina IV because NK1 receptor internalization kept on increasing at high concentrations of capsaicin, whereas substance P release decreased. In conclusion, amounts of substance P able to activate NK1 receptors may fall under the threshold of detection of radioimmunoassay. Conversely, radioimmunoassay often detects levels of substance P release well over those required to saturate NK1 receptors in the superficial dorsal horn, but that may be able to activate these receptors in nearby regions of the spinal cord.

Altered gravity affects succinate dehydrogenase reactivity in specific nuclei of fish brain.

The effect of long-term (10 days) altered gravitational conditions upon succinate dehydrogenase (SDH) reactivity in total brain as well as in individual brain nuclei of developing cichlid fish larvae has been investigated by means of semiquantitative histochemical methods (densitometric grey value analysis). Increasing acceleration from near weightlessness (spaceflight) via 1 g controls to 3 g hypergravity (centrifuge) resulted in slightly increased total brain SDH reactivity. When focusing on distinct neuronal integration centres within the same brains in order to find the anatomical substratum of the gross histochemical data, significant effects of altered gravity on vestibulum-related brain parts were obtained. The total brain results may therefore represent the sum of such particular indirect effects but may also comprise in addition a non vestibular-related general and therefore direct influence of altered gravitational conditions, possibly on all cells.

Concurrent release of ATP and substance P within guinea pig trigeminal ganglia in vivo.

Neurons within sensory ganglia have been proposed to communicate via non-synaptic release of a diffusible chemical messenger, but the identity of the chemical mediator(s) remains unknown [J. Neurosci. 16 (1996) 4733-4741]. The present study addressed the possibility of co-released ATP and substance P (SP) within sensory ganglia to further advance the hypothesis of non-synaptic communication between sensory neurons. Microdialysis probes inserted into trigeminal ganglia (TRGs) of anesthetized guinea pigs were perfused with artificial cerebrospinal fluid and the collected perfusate analyzed for ATP and SP content using the firefly luciferin-luciferase (L/L) assay and radioimmunoassay, respectively. Significant reversible increases in ATP and SP levels were observed after infusion of 100 mM KCl or 1 mM capsaicin. Ca(2+)-free ACSF produced an eightfold increase in ATP levels, interpreted as a decrease in activity of Ca(2+)-dependent ecto-nucleotidases that degrade ATP. In contrast, KCl-induced release of ATP in the presence of normal Ca(2+) was blocked by Cd(2+), a voltage-gated Ca(2+) channel blocker, illustrating Ca(2+)-dependence of evoked ATP release. Since ganglionic release of ATP could arise from several neuronal and non-neuronal sources we directly tested acutely dissociated TRG neuron somata for ATP release. Neuron-enriched dissociated TRG cells were plated onto glass tubes and tested for ATP release using the L/L assay. Robust ATP release was evoked with 5 microM capsaicin. These data suggest that ATP is released concurrently with SP from the somata of neurons within sensory ganglia.

Inflammation-induced changes in primary afferent-evoked release of substance P within trigeminal ganglia in vivo.

Substance P (SP) is synthesized in a subset of nociceptive sensory neurons and is released from their peripheral and central terminals. Here we demonstrate with the use of in vivo microdialysis and radioimmunoassay techniques that SP is also released within trigeminal ganglia following intraganglionic application of KCl, veratridine or capsaicin, and after electrical stimulation of peripheral afferent fibers. Both the basal and KCl-evoked release of SP are shown to be dependent on extracellular calcium. Using the turpentine-induced model of unilateral orofacial inflammation we also show that both the basal and KCl-evoked release of SP within trigeminal ganglia are greatly increased on the inflamed side 48 h after induction of inflammation. Coupled with previous demonstrations of excitatory effects of SP on sensory neurons, these results suggest that SP fulfils the role of a non-synaptically released diffusible chemical messenger that may modulate the somatic excitability of neurons within sensory ganglia in inflammatory pain states.

Refined intraoperative repositioning of the osteotomized maxilla in relation to the skull and TMJ.

In dysgnathic patients, who have to be treated by surgical repositioning of the jaws, the necessary bone movements are predicted preoperatively with the help of sophisticated analysis. A high degree of accuracy, could not however be transferred to the patient, because of lack of an operative procedure as accurate as the preplanning. In this paper a face-bow is described, provided with an interocclusal splint that has been adjusted beforehand on an articulator, in a model operation taking into account the skull and TMJ relationship. Using this face-bow-splint combination the possibility of greater accuracy in transferring the preplanned movements to the patient is facilitated. The computerized preplanning, the model operation and the technical procedure as well as the operation itself are described, step by step.

Changes in the microstructure of the vestibular apparatus of tadpoles (Rana temporaria) developed in simulated weightlessness.

The vestibular apparatus of tadpoles (Rana temporaria) exposed to simulated weightlessness was examined by electron microscopy. Extended exposure to simulated weightlessness is followed by significant alterations in the sensory epithelia and also in the otolith membrane. Large vacuoles, filled with necrobiotic mitochondria and fragments of endoplasmic reticulum, were concentrated in the region where an otolith membrane covers the hair cells but were mostly absent in zones of the epithelia with undifferentiated cells. The number of otoconia in the otolith membrane was diminished. The results were compared with data from space flight experiments and some concordance was noted. The possible connection between some unusual behavior of the tadpoles after weightlessness simulation and the structural alterations in the gravitational sensors was discussed.

Light microscopic analysis of the gravireceptor in Xenopus larvae developed in hypogravity.

The paper describes an investigation of the influence of gravity on the early differentiation of gravity receptors in Xenopus embryos and larvae. There is evidence that the expression of crystals in the saccus endolymphaticus was statistically greater when the embryos developed in near weightlessness (hypogravity) than on earth. The function of these crystals is unknown but they may contribute to the functioning of the vestibular apparatus.

The reaction of Xenopus laevis Daudin (South African toad) to linear accelerations.

Preparing the German Spacelab Mission D-2 project "Gravity Perception and Neuronal Plasticity"--STATEX II--ground based experiments have been performed with larvae of the amphibian vertebrate Xenopus laevis Daud. to study the reactions to different levels of acceleration forces and profiles. The larvae have been exposed to accelerations of up to 5 g for different time periods using a modified laboratory centrifuge and the NIZEMI (Niedergeschwindigkeits-Zentrifugen-Mikroskop) which allows direct observation and video documentation. The results will be discussed and compared with those of the D1-Mission, parabolic flights, and simulated weightlessness.

Survey of the vestibulum, and behavior of Xenopus laevis larvae developed during a 7-days space flight.

Aquatic animals have almost no body weight related proprioception for spatial orientation. Xenopus larvae, like fish, maintain their attitude in water by continuous correction with their fin(s). For these reasons a special performance of the equilibrium system compared to terrestrial animals is necessary. Evidently fish therefore have more compact (dense) otoliths; Xenopus larvae have less dense otolith (membranes) similar to land vertebrates; but their sacculus-otoliths are vertically positioned, which also may lead to a higher g-sensitivity. For plausibility reasons gravity should influence the embryonic development of gravity receptors. Yet, evaluations of photographs taken from the surface of cut deep-frozen objects by incident light show no aberration of the shape of the whole vestibulum and of the shape, density, size and position of the otolith membrane in larvae developed under near-zero g (NEXPA-BW-STATEX in D-1-Mission). The further evaluation of the "weightless-larvae" revealed a probably not yet described statolith-like formation in the dorsal wall of the vestibulum. In the weightless larvae this formation outnumbers, also qualitatively, strongly the l-g controls. An extra result is the lack of striking effects of cosmic radiation on the embryonic development of the flown Xenopus eggs. The swimming behavior of the larvae which was observed about one hour after landing of the Space Shuttle showed a typical anomaly (loop swimming), which is known from larvae developed on the clinostat or from fish flown aboard Apollo capsules.

Histochemical investigations on the influence of long-term altered gravity on the CNS of developing cichlid fish: results from the 2nd German Spacelab Mission D-2.

The effect of long-term (10 days) altered gravitational conditions upon succinate dehydrogenase (SDH) reactivity in total brains as well as in individual brain nuclei of developing cichlid fish larvae had been investigated by means of semiquantitative histochemical methods (densitometric grey value analysis). Increasing accelerations from near weightlessness (spaceflight) via 1g controls to 3g hyper gravity (centrifuge) resulted in slightly increasing "all over the brain" (total brain) SDH reactivity. When focusing on distinct neuronal integration centers within the same brains in order to find the anatomical substratum of the gross histochemical data, significant effects of altered gravity only within vestibulum related brain parts were obtained.

Effects of gravity on early development.

The development of embryonic and larval stages of the South African Toad Xenopus laevis D, was investigated in hyper-g up to 5 g (centrifuge), in simulated 0 g (fast-rotating clinostat), in alternating low g, hyper-g (parabolic flights) and in microgravity (Spacelab missions D1, D-2). The selected developmental stages are assumed to be very sensitive to environmental stimuli. The results showed that the developmental reaction processes run normal also in environments different to 1 g and that aberrations in behavior and morphology normalize after return to 1 g. Development, differentiation, and morphology of the gravity perceiving parts of the vestibular system (macula-organs) had not been affected by exposure to different g-levels.