T cells support osteoclastogenesis in an in vitro model derived from human multiple myeloma bone disease: the role of the OPG/TRAIL interaction

The development of multiple myeloma (MM) bone disease is mediated by increased number and activity of osteoclasts (OCs). Using an in vitro osteoclastogenesis model consisting of unstimulated and unfractionated peripheral blood mononuclear cells (PBMCs) from patients with MM, we showed that T cells support the formation of OCs with longer survival. Different from T-cell-depleted MM PBMC cultures, exogenous macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB ligand (RANKL) were necessary for the formation of OCs; however, they did not exhibit longer survival. We found up-regulated production of RANKL, osteoprotegerin (OPG), and TNF-related apoptosis-inducing ligand (TRAIL) by fresh MM T cells. Despite high OPG levels, the persistence of osteoclastogenesis can be related to the formation of the OPG/TRAIL complex demonstrated by immunoprecipitation experiments and the addition of anti-TRAIL antibody which decreases OC formation. OCs overexpressed TRAIL decoy receptor DcR2 in the presence of MM T cells and death receptor DR4 in T-cell-depleted cultures. In addition, increased Bcl-2/Bax (B-cell lymphoma-2/Bcl2-associated protein X) ratio, following Bcl-2 up-regulation, was detected in OCs generated in the presence of T cells. Our results highlight that MM T cells support OC formation and survival, possibly involving OPG/TRAIL interaction and unbalanced OC expression of TRAIL death and decoy receptors.     

Decreased plasma levels of orexin-A in sleep apnea

BACKGROUND: Orexin-A, also known as hypocretin, is a neuropeptide implicated in appetite and sleep regulation. Because the obstructive sleep apnea syndrome (OSAS) is characterized by obesity and excessive daytime sleepiness, we hypothesized that orexin-A levels may be abnormal in patients with OSAS. Further, since treatment with continuous positive airway pressure (CPAP) in patients with OSAS is very effective in normalizing daytime sleepiness, we also hypothesized that the chronic use of CPAP may influence plasma levels of orexin-A in these patients. OBJECTIVE: To evaluate plasma levels of orexin-A in patients with OSAS and the effect of CPAP treatment. PATIENTS AND METHODS: We compared the plasma levels of orexin-A in 13 healthy controls, 27 untreated patients with OSAS and 14 patients treated with CPAP during at least 1 year (4.5 +/- 0.5 h/night; mean +/- SEM). All patients had severe OSAS (apnea-hypopnea index, 57 +/- 4 h(-1)). Results: Orexin-A plasma levels were significantly lower in untreated (9.4 +/- 1.9 pg.ml(-1), p < 0.01) and treated patients with OSAS (4.2 +/- 1.5 pg.ml(-1), p < 0.001) than in healthy subjects (20.6 +/- 4.5 pg.ml(-1)). In untreated patients, orexin-A levels were not significantly related to daytime somnolence assessed by Epworth scale (r = -0.18, p = 0.37) or the body mass index (r = -0.13, p = 0.52). CONCLUSIONS: Orexin-A plasma levels are abnormally low in patients with OSAS, independently of the level of somnolence and/or presence of obesity. These results suggest that these low orexin-A levels may be related to the pathogenesis of OSAS.     

Highly active antiretroviral therapy and cervical intraepithelial neoplasia

Highly active antiretroviral therapy (HAART) has improved HIV prognosis, but its effect on cervical intraepithelial neoplasia (CIN), which is associated with HIV, is uncertain. Among 71 HAART-treated women the prevalence of CIN before HAART was 55%. After a median of 10 months after starting HAART the prevalence had increased to 62% (P = 0.20); 13% of patients experienced regression of a CIN lesion, and this was most strongly associated with a greater increase in CD4 cell count. Such studies will provide the basis for guidelines for monitoring CIN in HIV-positive women in the HAART era.     

Effect of gluten-free diet on preventing recurrence of gastroesophageal reflux disease-related symptoms in adult celiac patients with nonerosive reflux disease

Background and Aim:  In Celiac Disease (CD) the role of a gluten-free diet (GFD) on gastroesophageal reflux disease–related symptoms (GERD-rs) is unclear. The aim of this study was to establish the recurrence of GERD-rs, in CD patients with nonerosive reflux disease (NERD).
Methods:  From a total of 105 adult CD patients observed, 29 who presented with the NERD form were enrolled in the study. Thirty non-CD patients with NERD were studied as controls. Recurrence of GERD-rs was clinically assessed at 6, 12, 18, and 24 months follow-up (FU) after withdrawal of initial proton-pump inhibitor (PPI) treatment for 8 weeks.
Results:  GERD-rs were resolved in 25 (86.2%) CD patients and in 20 (66.7%) controls after 8 weeks of PPI treatment. In the CD group, recurrence of GERD-rs was found in five cases (20%) at 6 months but in none at 12, 18, and 24 months while in the control group recurrence was found in six of 20 controls (30%), in another six (12/20, 60%), in another three (15/20, 75%), and in another two (17/20, 85%) at 6, 12, 18, and 24 months FU respectively.
Conclusions:  The present study is the first to have evaluated the effect of a GFD in the nonerosive form of GERD in CD patients, by means of clinical long-term follow-up, suggesting that GFD could be a useful approach in reducing GERD symptoms and in the prevention of recurrence.     

Human herpesvirus-8/Kaposi's sarcoma-associated herpesvirus in organ transplant patients with immunosupression

Several recent studies have demonstrated Kaposi's sarcoma-associated herpes virus (KSHV), also known as putative human herpes virus-8 (HHV-8), DNA in various epidemiologic forms of Kaposi's sarcoma (KS), including AIDS-associated, classic, and endemic types. Risk of developing KS in non-HIV-infected immunosuppressed hosts, such as patients following solid organ transplantation, is also significantly higher compared to normal individuals. We have retrospectively evaluated 28 organ transplant patients with KS (23 cutaneous and five visceral) for the presence of KSHV genome by polymerase chain reaction (PCR) amplification of DNA isolated from formalin-fixed, paraffin-embedded archival tissue samples. 27/28 KS patients were positive for the presence of KSHV. In four KS patients, tissue samples with no histologic evidence of KS were also analysed for KSHV. No evidence of positivity in three samples was noted, but one patient had weak positive amplification products on DNA samples isolated from a gastric biopsy with chronic gastritis and lymph node with sinus histiocytosis. These data support the association of KSHV with KS developing in non-HIV-infected immunosuppressed patients, similar to other forms of KS, and suggest that KSHV may play a significant role in the development of all forms of KS.     

Detection of Chlamydiae pneumoniae but not Helicobacter pylori DNA in atherosclerosis plaques

Chronic infections have been associated with cardiovascular disease. We used bacterial culture, polymerase chain reaction (PCR), and immunohistochemical staining with anti-vacA and anticagA antibodies to search for Helicobacter pylori and Chlamydiae pneumoniae in atherosclerotic plaques obtained at endarterectomy. Serum IgG antibodies to H. pylori and C. pneumoniae were also determined. Thirty-two patients were enrolled. Anti-H. pylori and anti-C. pneumoniae IgG were present in 72% and 81%, respectively. Culture and PCR for H. pylori of vessel walls and plaques were negative. Atherosclerotic plaque and normal vessel sections from H. pylori-negative and- positive patients showed reactivity with anti-vacA and anti-cagA antibodies. C. pneumoniae DNA was amplified in three atherosclerotic lesions. These findings suggest that the association between H. pylor infection and atherosclerosis does not result from continuing direct effects of H. pylori antigens in the vessel walls. Antigens within vessel atherosclerotic plaques cross-react with H. pylori virulence factors and could act as cofactors in determining instability for the atherosclerotic plaques.     

Sural nerve biopsy and functional studies support the pathogenic role of a novel MPZ mutation

Our patient is a 65‐year‐old woman presenting with bilateral pes cavus, pronounced distal muscle wasting, weakness and areflexia. Electrophysiological findings included diffuse unrecordable motor and sensory responses. While the CMT phenotype was evident, the lack of family history and the severe, but unspecific electrophysiological impairment, was a challenge for genetic diagnosis. A sural nerve biopsy was performed, showing a severe loss of myelinated fibers with residual axons surrounded by myelin outfoldings. Whereas myelin outfoldings are a pathological hallmark of autosomal recessive CMT4B1 and CMT4B2, due to mutations in myotubularin‐related 2 (MTMR2) and 13 (MTMR13) genes respectively, they may also occur in nerve biopsies from CMT1B patients. By direct sequencing, a novel heterozygous transversion c.410G>T in MPZ gene was demonstrated, producing an amino acid change from glycine to valine in position 108 (p.G108V). In HeLa cells the fusion P0G108V‐EGFP was normally trafficked to the cell membrane, but with decreased P0 adhesion function, compared with wild‐type P0, thus supporting a pathogenic role of the new variant. In conclusion this case highlights the relevance, in selected cases, of sural nerve biopsy to orient the genetic/molecular tests, while in vitro analyses may strengthen the pathogenic role of novel mutations.     

Direct measurement of the 3-dimensional DNA lesion distribution induced by energetic charged particles in a mouse model tissue

Charged particles are increasingly used in cancer radiotherapy and contribute significantly to the natural radiation risk. The difference in the biological effects of high-energy charged particles compared with X-rays or γ-rays is determined largely by the spatial distribution of their energy deposition events. Part of the energy is deposited in a densely ionizing manner in the inner part of the track, with the remainder spread out more sparsely over the outer track region. Our knowledge about the dose distribution is derived solely from modeling approaches and physical measurements in inorganic material. Here we exploited the exceptional sensitivity of γH2AX foci technology and quantified the spatial distribution of DNA lesions induced by charged particles in a mouse model tissue. We observed that charged particles damage tissue nonhomogenously, with single cells receiving high doses and many other cells exposed to isolated damage resulting from high-energy secondary electrons. Using calibration experiments, we transformed the 3D lesion distribution into a dose distribution and compared it with predictions from modeling approaches. We obtained a radial dose distribution with sub-micrometer resolution that decreased with increasing distance to the particle path following a 1/r² dependency. The analysis further revealed the existence of a background dose at larger distances from the particle path arising from overlapping dose deposition events from independent particles. Our study provides, to our knowledge, the first quantification of the spatial dose distribution of charged particles in biologically relevant material, and will serve as a benchmark for biophysical models that predict the biological effects of these particles.Charged particles, including protons, α-particles, and heavy ions, are increasingly used in cancer radiotherapy and represent a significant component of the natural background irradiation on earth and in space (1–3). Their biological effect is often very different from that of photons (X- or γ-rays), largely because charged particles deposit their energy along a track, whereas photons produce a fairly homogeneous dose distribution. Linear energy transfer (LET; typically given in units of keV/µm) has been introduced as a parameter to describe the amount of energy that charged particles deposit along their track. Particles with high LET are densely ionizing and typically biologically more effective than photons or low-LET particles.Energy deposition along a particle track is not restricted to the path itself (the so-called “track core”), but extends laterally into an area known as the penumbra of the particle, which can reach considerable distances for high-energy particles. Energy deposition in the penumbra arises from energetic secondary electrons, so-called δ-electrons, which are generated by ionization events of the charged particles and carry energy away from the immediate path into the penumbra. According to classical track structure theory, ∼50% of the total energy is deposited in the penumbra, where it spreads out over a much greater volume than the energy in the track core (4). Thus, the penumbra represents a sparsely ionized region within the track of high-LET particles. Thus, charged particles deposit their energy in a complex 3D manner and typically comprise a spectrum of high- and low-ionization densities. The total biological effect of a charged particle is a result of this complex energy deposition pattern.Our knowledge about the energy deposition pattern of charged particles is based largely on theoretical predictions and physical measurements in inorganic material. Most information in this respect was gained from microdosimetric experiments with gas-filled detectors (5). In these devices, the dose distribution around an ion trajectory is measured and rescaled to a track structure profile in water by comparing the electron density of the gas with that of water. Such a density scaling approach is expected to fail, however, when the energies of the δ-electrons are in the same order of magnitude as the intermolecular binding energies (6, 7). These limitations call for alternative approaches for assessing the 3D energy deposition pattern of charged particles directly in biologically relevant material.Computational models, mostly Monte Carlo codes, have been developed to describe the energy deposition patterns of charged particles. Results from such approaches are in reasonable agreement with physical measurements, although track structure modeling at a nanometer or micrometer scale remains a challenge. Computational models are also used to predict the biological effects of charged particles, such as the induction of DNA damage (8–11). Unlike Monte Carlo calculations, amorphous track structure models exploit the radial dose distribution to predict the biological response (12, 13). One of these approaches, known as the local effect model (LEM), is currently applied in particle cancer therapy to predict the biological effects of heavy ions (14, 15). The LEM assumes that the same local dose, independent of the radiation type that deposits this dose, leads to the same biological effect. Essentially, LEM derives the biological effects of charged particles from the response of cells or tissues to photon radiation.Double-strand breaks (DSBs) are among the most hazardous DNA lesions induced by ionizing radiation (IR) because they can give rise to more complex lesions when occurring in clusters or associated with other lesions (16). On DSB induction, the histone variant H2AX is phosphorylated at the break site to γH2AX (17). This histone modification can be visualized by microscopy and forms so-called γH2AX foci, which arise within minutes after IR in a 1:1 ratio to isolated DSBs and are lost with time due to DSB repair (18, 19). Because this technology detects single isolated DSBs in single cells, it has exceptional sensitivity and can monitor the effect of IR doses of a few mGy (20, 21). Not surprisingly, γH2AX foci analysis has been applied to assess radiation doses encountered by humans during diagnostic medical procedures, such as computed tomography scanning (22–25). Moreover, this technology can be applied to different tissues (26, 27) and is becoming the gold standard for various biodosimetric applications (28, 29).In the present study, we combined the particular sensitivity of the γH2AX foci technology with its second major advantage, the ability to precisely determine the position of the DNA lesions, to investigate the spatial distribution of γH2AX foci induced by charged particles. Calibration experiments allowed transformation of the lesion distribution into a 3D dose distribution, which could be compared with predictions from the amorphous track structure model, the LEM. Using this procedure, we were able to quantify the lateral (radial) dose profile of titanium (Ti) ions with sub-micrometer resolution and verify the theoretically predicted 1/r² dose decline using a biological model system.