Strong and frequent T-cell responses to the minor allergen Phl p 12 in Spanish patients IgE-sensitized to Profilins.

BACKGROUND: Profilins are dominant pan-allergens known to cause cross-sensitization, leading to clinical symptoms such as pollen-food syndrome. This study aimed to determine the T-cell response to Phl p 12 in profilin-sensitized patients, by measuring the prevalence, strength and cross-reactivity to clinically relevant profilins. METHODS: The release of Phl p allergens from pollen was determined by mass spectrometry and immunochemistry. T-cell responses, epitope mapping and cross-reactivity to profilins (Phl p 12, Ole e 2, Bet v 2 and Mal d 4) were measured in vitro using PBMCs from 26 Spanish grass-allergic donors IgE-sensitized to profilin. Cross-reactivity was addressed in vivo using 2 different mouse strains (BALB/c and C3H). RESULTS: Phl p 12 and Phl p 1 are released from pollen simultaneously and in similar amounts. Both T-cell response frequency (17/26 donors) and strength were comparable between Phl p 12 and Phl p 1. T-cell cross-reactivity to other profilins correlated with overall sequence homology, and 2 immunodominant epitope regions of Phl p 12 were identified. Data from mice immunized with Phl p 12 showed that cross-reactivity to Bet v 2 was mediated by conserved epitopes and further influenced by additional genetic factors, likely to be MHC II. CONCLUSION: The strength, prevalence and cross-reactivity of T-cell responses towards Phl p 12 are comparable to the major allergen Phl p 1, which supports the hypothesis that T cells to Phl p 12 can play an important role in development of allergic symptoms, such as those associated with pollen-food syndrome.

Profilin as a severe food allergen in allergic patients overexposed to grass pollen.

BACKGROUND: Profilins are ubiquitous proteins that act as panallergens in sensitized patients, considered to be mild or incomplete food allergens. The aim of the study was to evaluate the role of profilins as severe food allergens in allergic patients overexposed to grass who were referred for severe food reactions and were sensitized to profilins. METHODS: After a careful in vitro screening, 26 patients were included, classified into two groups, mild (17) and severe reactors (9), based on clinical history and subsequently provoked orally with purified profilin in a double-blind placebo-controlled food challenge setup. RESULTS: A significant number of patients presented severe positive food challenge test reactions at low doses of the allergen profilin. Patients prone to suffer from severe reactions had lower IgG4/IgE ratio to major grass allergens than those who did not. CONCLUSION: Profilins are complete food allergens in food-allergic patient populations that are exposed to high levels of grass pollen. This type of patient constitutes an optimal model to understand the link between respiratory and food allergies. The nature of the observed reactions and the low level of allergen eliciting the reactions suggest that intake through the oral mucosa might constitute a relevant route of exposure to food allergens.

Pulse treatment with the proteasome inhibitor bortezomib inhibits osteoclast resorptive activity in clinically relevant conditions.

Myeloma bone disease is due to bone degradation by osteoclasts, and absence of repair by bone forming osteoblasts. Recent observations suggest that the anti-myeloma drug bortezomib, a proteasome inhibitor, stimulates bone formation and may inhibit bone resorption. Here, we tested bortezomib on cultured osteoclasts in conditions mimicking the pulse treatment used in the clinic, thereby avoiding continuous proteasome inhibition and unselective toxicity. A 3 h pulse with 25 nM bortezomib followed by a 3-day culture in its absence markedly inhibited osteoclast activity as evaluated through bone resorption, TRAcP release, and RANKL-induced NF-kappaB translocation into nuclei, an event dependent on proteasomes and critical for osteoclast function. The effect on TRAcP was maximal during the first 24 h post-pulse, and then tended to subside. Importantly, applying this pulse treatment to cultured myeloma cells drastically reduced their survival. We measured next the levels of two bone resorption markers in patients during the 3 days following five and seven therapeutic bortezomib administrations, respectively. These levels decreased significantly already 1-2 days after injection, and then increased, showing temporary inhibition of osteoclast activity and paralleling the in vitro effect on TRAcP. Our study demonstrates a direct inhibition of osteoclasts by bortezomib in conditions relevant to treatment of myeloma.

Post-translational protein modifications in type 1 diabetes: a role for the repair enzyme protein-L-isoaspartate (D-aspartate) O-methyltransferase?

AIMS/HYPOTHESIS: Post-translational modifications, such as isomerisation of native proteins, may create new antigenic epitopes and play a role in the development of the autoimmune response. Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PIMT), encoded by the gene PCMT1, is an enzyme that recognises and repairs isomerised Asn and Asp residues in proteins. The aim of this study was to assess the role of PIMT in the development of type 1 diabetes. MATERIALS AND METHODS: Immunohistochemical analysis of 59 normal human tissues was performed with a monoclonal PIMT antibody. CGP3466B, which induces expression of Pcmt1, was tested on MIN6 and INS1 cells, to assess its effect on Pcmt1 mRNA and PIMT levels (RT-PCR and western blot) and apoptosis. Forty-five diabetes-prone BioBreeding (BB) Ottawa Karlsburg (OK) rats were randomised to receive 0, 14 or 500 microg/kg (denoted as the control, low-dose and high-dose group, respectively) of CGP3466B from week 5 to week 20. RESULTS: A high level of PIMT protein was detected in beta cells. CGP3466B induced a two- to threefold increase in Pcmt1 mRNA levels and reduced apoptosis by 10% in MIN6 cells. No significant effect was seen on cytokine-induced apoptosis or PIMT protein levels in INS1 cells. The onset of diabetes in the BB/OK rats was significantly delayed (85.6+/-9.0 vs 84.3+/-6.8 vs 106.6+/-13.5 days, respectively; p<0.01 for high-dose vs low-dose and control groups), the severity of the disease was reduced (glucose 22.2+/-3.2 vs 16.9+/-2.6 vs 15.8+/-2.7 mmol; p<0.01 for high- and low-dose groups vs control group) and residual beta cells were more frequently identified (43% vs 71% vs 86%; p<0.05 for high-dose vs control group) in the treated animals. CONCLUSIONS/INTERPRETATION: The results support a role for post-translational modifications and PIMT in the development of type 1 diabetes in the diabetes-prone BB rat, and perhaps also in humans.

Osteoclast nuclei of myeloma patients show chromosome translocations specific for the myeloma cell clone: a new type of cancer-host partnership?

A major clinical manifestation of bone cancers is bone destruction. It is widely accepted that this destruction is not caused by the malignant cells themselves, but by osteoclasts, multinucleated cells of monocytic origin that are considered to be the only cells able to degrade bone. The present study demonstrates that bone-resorbing osteoclasts from myeloma patients contain nuclei with translocated chromosomes of myeloma B-cell clone origin, in addition to nuclei without these translocations, by using combined FISH and immunohistochemistry on bone sections. These nuclei of malignant origin are transcriptionally active and appear fully integrated amongst the other nuclei. The contribution of malignant nuclei to the osteoclast population analysed in this study was greater than 30%. Osteoclast-myeloma clone hybrids contained more nuclei than normal osteoclasts and their occurrence correlated with the proximity of myeloma cells. Similar hybrid cells were generated in myeloma cell-osteoclast co-cultures, as revealed by tracing myeloma nuclei using translocations, bromo-deoxyuridine, or the Y chromosome of male myeloma cells in female osteoclasts. These observations indicate that hybrid cells can originate through fusion between myeloma cells and osteoclasts. In conclusion, malignant cells contribute significantly to the formation of bone-resorbing osteoclasts in multiple myeloma. Osteoclast-myeloma clone hybrids reflect a previously unrecognized mechanism of bone destruction in which malignant cells participate directly. The possibility that malignant cells corrupt host cells by the transfer of malignant DNA may have been underestimated to date in cancer research.

In home telerehabilitation for older adults after discharge from an acute hospital or rehabilitation unit: A proof-of-concept study and costs estimation.

PURPOSE: The purpose of this study is to investigate rehabilitation through teletreatment as an alternative to a physical homecare visit to deliver services to individuals at home following discharge from an acute hospital or rehabilitation unit. METHOD: Four community-living elderly people were recruited for telerehabilitation services prior being discharged from an acute-care hospital and a geriatric rehabilitation unit. Once the patient returned home, an appointment was made for the assessing therapist to take the clinical measurements (T1) in a face-to-face session. Four clinical variables were used (functional autonomy, balance, locomotor performance in walking and lower-body strength). Telerehabilitation sessions with the participants were conducted with trained personnel in the individual's home. The system used to support telerehabilitation services for this proof of concept was built around network-attached remotely-controlled pan/tilt/zoom cameras with MJPEG compression, media displays and hands-free phones. Before the patient was discharged from the physiotherapy program, the same assessing therapist visited the subject again to take the T2 measurements in a face-to-face session. The satisfaction of the health-care professional was determined for each session with the homemade questionnaire. Costs related to telerehabilitation were compared to theoretically home visits. RESULTS: All four subjects improved on the four clinical variables. Mean costs for the telerehabilitation program, comprising 12 sessions over 4 weeks was $487. CONCLUSION: Telerehabilitation seems to be a practical alternative for dispensing rehabilitation services after patients are discharged from an acute hospital or rehabilitation unit.

RANKL induces formation of avian osteoclasts from macrophages but not from macrophage polykaryons.

We have shown that chick macrophages express RANK at their surface and human RANKL (hRANKL) triggers the formation of osteoclasts able to degrade dentine. As described for mammalian osteoclasts, hRANKL also stimulates the resorbing activity of chick bone-derived osteoclasts. In other hands, in culture, chick macrophages spontaneously form polykaryons sharing most of the osteoclast markers but unable to resorb bone. Since both bone-resorbing osteoclasts and macrophage polykaryons found in inflammatory tissues are multinucleated cells deriving from the fusion of macrophages, we examined whether macrophage polykaryons could be induced toward bone-resorbing osteoclasts. Long-term exposure of macrophage polykaryons to hRANKL failed to activate any resorbing activity, indicating that although deriving from the same precursors macrophage polykaryons and osteoclasts are independent cell types and polykaryons are not immature osteoclasts.

Effects of upper and lower limb static exertions on global synkineses in hemiparetic subjects.

OBJECTIVES: Global synkineses are nonpurposive pathological involuntary muscle activities or movements elicited at several or all of the joints of the affected limb or limbs during voluntary forceful resisted contractions. The purpose of this study was to assess the effect of upper and lower limb exertions on manifestations of upper limb global synkineses in hemiparetic subjects. DESIGN: Involuntary muscle activities on the affected upper limb of 11 hemiparetic subjects and on the left or right upper limb of 10 control subjects were recorded using surface electromyography during successive bilateral maximal ankle exertions and during contralateral grips. RESULTS: Significant differences in the level of involuntary electromyography (EMG) activities were observed between experimental conditions (ANOVAs, p < 0.05). EMG levels in hemiparetic subjects were significantly higher during contralateral grip tasks than during the ankle exertions. CONCLUSION: These results suggest that upper limb global synkinases are more prevalent in specific tasks and that this task specificity may reflect the neurophysiological mechanisms involved in the generation of global synkinases.

Maximal grip force in chronic stroke subjects and its relationship to global upper extremity function.

OBJECTIVES: Previous studies have shown that recovery of recordable grip strength in acute stroke subjects is one of the most sensitive assessments of initial upper limb recovery and a good prognostic factor for latter recovery. The objectives of this study were to test the reliability of maximal voluntary grip force (MVGF) measures and evaluate the relationship between paretic grip strength deficit and paretic upper extremity function in chronic stroke subjects. DESIGN: Over a three-week period, bilateral MVGF was assessed three times with a modified strain gauge dynamometer in 15 chronic stroke subjects and 10 control subjects. The paretic MVGF deficit was expressed in relation to the MVGF of the nonaffected hand. OUTCOME MEASURES: Upper extremity function in stroke subjects was measured using the Fugl-Meyer, the upper extremity performance test for the elderly (TEMPA), Box and Block and finger-to-nose tests. RESULTS: MVGF measures in both groups of subjects demonstrated good reliability (intraclass correlation, ICC >0.86) and low standard error measurements (SEM). The paretic MVGF of the stroke subjects was greatly impaired in comparison to the control subjects. Results of linear and quadratic regressions analyses show that this impairment was significantly correlated (p <0.01) with the performance of the stroke subjects on the four upper extremity function tests. The percentages of variances explained by the MVGF deficit on all four upper extremity tests varied from 62% to 78% for the linear regressions and from 72% to 93% for the quadratic regressions. CONCLUSIONS: These results suggest that the paretic maximal grip strength, normalized with the maximal grip strength on the nonaffected side, appears to be a valuable outcome measure of upper extremity function in chronic stroke subjects.

A static dynamometer measuring simultaneous torques exerted at the upper limb.

The majority of available dynamometers are designed to measure force or torque in one specific direction, one joint at a time. For the quantification of motor incoordination in neurological patient populations, these dynamometers provide limited information about the global behavior of the limb under investigation. This report describes the potential use and function of a static dynamometer measuring torques exerted simultaneously at the shoulder (flexion-extension, abduction-adduction, internal-external rotation), elbow (flexion-extension), and forearm (pronation-supination). Orthogonal forces were measured at the arm and wrist using strain gauge transducers interfaced with a laboratory computer. The lever arms were specified to a software program and the joint torques were calculated in real time according to static equilibrium equations. The use of the dynamometer is illustrated by characterizing for one hemiparetic subject, the joints torques recorded at the shoulder, elbow, and forearm during isolated submaximal grip exertions at different force levels on both sides. The torques generated at the shoulder, elbow and forearm during the hand grip tasks on the affected side were significantly higher than those obtained on the nonaffected side and increased with the grip force level. These differences probably reflect the loss of movement selectivity observed following a lesion in the central nervous system. Further studies are currently being undertaken in neurological patient populations to characterize and quantify motor deficits using this dynamometer. As a long term goal, we hope that the method and technologies described here will contribute to the evaluation and rehabilitation of these populations.

Aggregation of mononucleated precursors triggers cell surface expression of alphavbeta3 integrin, essential to formation of osteoclast-like multinucleated cells.

Alphavbeta3 is a key integrin mediating adhesion of multinucleated osteoclasts during bone resorption. 1, 25-dihydroxyvitamin D3 upregulates alphavbeta3 integrin expression in mononucleated osteoclast precursors and concomitantly stimulates their differentiation into osteoclasts. This suggests that this integrin could play a major role during osteoclast differentiation. We have developed an in vitro model, in which 1, 25-dihydroxyvitamin D3 sequentially modifies the behavior of macrophages: It first induces rounding up of these cells, then their subsequent aggregation and spreading, which finally leads to cell fusion and the formation of osteoclast-like multinucleated giant cells. We show that, while 1,25-dihydroxyvitamin D3 stimulates the de novo synthesis of alphavbeta3 in macrophages early in this process, its accumulation on the surface is triggered by cell aggregation. A high level of integrin alphavbeta3 cell surface expression correlates with macrophage spreading preceding fusion. This was confirmed by means of novel cell permeable peptides containing the C-terminal sequence of the integrin beta3 tail to specifically block (alphavbeta3 function. Although this peptide has no effect on the aggregation step, it disrupts the spreading of osteoclast precursors and consequently inhibits their fusion. These findings suggest a novel role of the integrin alphavbeta3 in a discrete step of osteoclast differentiation.

Characterization of global synkineses during hand grip in hemiparetic patients.

OBJECTIVE: Global synkineses are defined as nonpurposive associated movements on the involved side of hemiparetic subjects that are triggered during a voluntary movement. The purpose of this study was to characterize the intensity and pattern of upper limb global synkineses in hemiparetic subjects with a static biarticular dynamometer and electromyography during maximal progressive hand grip on the unaffected side. DESIGN: Survey, convenience sample. SETTINGS: University secondary care rehabilitation center. DATA SET: Global synkineses (ie, torques and electromyographic activities) in patients with severe (n = 8) and moderate (n = 7) deficits in motor performance, as evaluated by the Fugl-Meyer assessment, were compared with those obtained in a group of healthy subjects (n = 11). Clinically the subjects from the severe deficit group were more spastic and showed less strength at the elbow than the subjects from the moderate deficit group. RESULTS: Results of analyses of variance showed significant increases of shoulder torque in flexion and internal rotation, and elbow torque in flexion, with increasing force exertion during contralateral hand grip in subjects with severe deficits (p < .05). Furthermore, in these subjects increases of electromyographic activity were also observed in biceps brachii, brachioradialis, and triceps brachii muscles with increasing hand grip force levels. In contrast, no significant torques or electromyographic increases were observed in subjects with moderate deficits and in control subjects during contralateral hand grip exertions. CONCLUSION: These results provide a quantitative assessment of the kinematic and electromyographic patterns of global synkineses and their correlates with clinical observations. Within the limits of the experimental results presented in this study, it is suggested that global synkineses result from contralateral overflow of the voluntary command to hyperexcitable motoneuron pools.