177Lu radiolabeling and preclinical theranostic study of 1C1m-Fc: an anti-TEM-1 scFv-Fc fusion protein in soft tissue sarcoma.

PURPOSE: TEM-1 (tumor endothelial marker-1) is a single-pass transmembrane cell surface glycoprotein expressed at high levels by tumor vasculature and malignant cells. We aimed to perform a preclinical investigation of a novel anti-TEM-1 scFv-Fc fusion antibody, 1C1m-Fc, which was radiolabeled with 177Lu for use in soft tissue sarcomas models. METHODS: 1C1m-Fc was first conjugated to p-SCN-Bn-DOTA using different excess molar ratios and labeled with 177Lu. To determine radiolabeled antibody immunoreactivity, Lindmo assays were performed. The in vivo behavior of [177Lu]Lu-1C1m-Fc was characterized in mice bearing TEM-1 positive (SK-N-AS) and negative (HT-1080) tumors by biodistribution and single-photon emission SPECT/CT imaging studies. Estimated organ absorbed doses were obtained based on biodistribution results. RESULTS: The DOTA conjugation and the labeling with 177Lu were successful with a radiochemical purity of up to 95%. Immunoreactivity after radiolabeling was 86% ± 4%. Biodistribution showed a specific uptake in TEM-1 positive tumor versus liver as critical non-specific healthy organ, and this specificity is correlated to the number of chelates per antibody. A 1.9-fold higher signal at 72 h was observed in SPECT/CT imaging in TEM-1 positive tumors versus control tumors. CONCLUSION: TEM-1 is a promising target that could allow a theranostic approach to soft-tissue sarcoma, and 1C1m-Fc appears to be a suitable targeting candidate. In this study, we observed the influence of the ratio DOTA/antibody on the biodistribution. The next step will be to investigate the best conjugation to achieve an optimal tumor-to-organ radioactivity ratio and to perform therapy in murine xenograft models as a prelude to future translation in patients.

Reduced-intensity and non-myeloablative allogeneic stem cell transplantation from alternative HLA-mismatched donors for Hodgkin lymphoma: a study by the French Society of Bone Marrow Transplantation and Cellular Therapy.

Allogeneic stem cell transplantation (allo-SCT) following a non-myeloablative (NMA) or reduced-intensity conditioning (RIC) is considered a valid approach to treat patients with refractory/relapsed Hodgkin lymphoma (HL). When an HLA-matched donor is lacking a graft from a familial haploidentical (HAPLO) donor, a mismatched unrelated donor (MMUD) or cord blood (CB) might be considered. In this retrospective study, we compared the outcome of patients with HL undergoing a RIC or NMA allo-SCT from HAPLO, MMUD or CB. Ninety-eight patients were included. Median follow-up was 31 months for the whole cohort. All patients in the HAPLO group (N=34) received a T-cell replete allo-SCT after a NMA (FLU-CY-TBI, N=31, 91%) or a RIC (N=3, 9%) followed by post-transplant cyclophosphamide. After adjustment for significant covariates, MMUD and CB were associated with significantly lower GvHD-free relapse-free survival (GRFS; hazard ratio (HR)=2.02, P=0.03 and HR=2.43, P=0.009, respectively) compared with HAPLO donors. In conclusion, higher GRFS was observed in Hodgkin lymphoma patients receiving a RIC or NMA allo-SCT with post-transplant cyclophosphamide from HAPLO donors. Our findings suggest they should be favoured over MMUD and CB in this setting.

[Selective and non-invasive monitoring of the posterior columns and pyramidal tract during surgery of the spine and spinal cord]. / Monitorage non invasif et sélectif des cordons postérieurs et de la voie pyramidale pendant les chirurgies du rachis et de la moelle épinière.

For intra-operative neurophysiological monitoring during spine and spinal cord surgery, the challenge is to detect, in real-time, the occurrence of neurological impairment at onset in order to remedy the problem as quickly as possible before it becomes definitely acquired and irreversible. The past three decades were marked by considerable technical progress. Feasibility and reliability have now reached a very high level. A multimodal approach combining simultaneous monitoring of somatosensory and motor pathways has enabled a considerable decline in the rate of false negatives reported in the 80s when somatosensory evoked potentials (SSEP) were monitored alone. Several methods of monitoring are described in the literature. Combining transcranial electric stimulation of muscle motor evoked potentials (mMEP) and cortical SSEP allows non-invasive and selective monitoring of the posterior columns and pyramidal tract. One of the most widely used techniques internationally backed by a broad consensus within the International Society of Intra-operative Neurophysiology (ISIN), this technique remains exceptional in French-speaking countries. We expose here the methodological aspects for the readers of La Revue Neurologique.

[The multifibrillar network of the tendon sliding system]. / Les tendons et le système de glissement multifibrillaire.

After carrying out 215 in vivo dissections, 65 of which were video-recorded, the authors propose that the current representation of the notion of the tendon sliding is incorrect. It is suggested that tendon sliding is explained by the existence of a mechanical adaptable multimicrovacuolar and fibrillar tissue. This tissue enables complete sliding without any dynamic influence on the surrounding tissues. The new theory is based on a polyhedric fibrillar framework, apparently chaotic and complex, subtending the microvacuolar gel, a concept that is to be found everywhere in the human body.

[Muscles and connective tissue: histology]. / Muscles et tissu conjonctif: données histologiques.

Here, we give some comments about the DVD movies "Muscle Attitudes" from Endovivo productions, the movies up lighting some loss in the attention given to studies on the connective tissue, and especially them into muscles. The main characteristics of the different components in the intra-muscular connective tissue (perimysium, endomysium, epimysium) are shown here with special references to their ordered architecture and special references to their spatial distributions. This connective tissue is abundant into the muscles and is in continuity with the muscles in vicinity, with their tendons and their sheath, sticking the whole on skin. This connective tissue has also very abundant connections on the muscles fibres. It is then assumed that the connective tissue sticks every organs or cells of the locomotion system. Considering the elastic properties of the collagen fibres which are the most abundant component of connective tissue, it is possible to up light a panel of connective tissue associated functions such as the transmission of muscle contractions or the regulation of protein and energetic muscles metabolism.

The role and mechanical behavior of the connective tissue in tendon sliding.

After carrying out 215 in-vivo dissections, 65 of which were video-recorded, the authors propose that the current representation of the notion of the tendon sliding is incorrect. It is suggested that tendon sliding is explained by the existence of a mechanical adaptable multimicrovacuolar and fibrillar tissue. This tissue enables complete sliding without any dynamic influence on the surrounding tissues. The new theory is based on a polyhedric fibrillar framework, apparently chaotic and complex, subtending the microvacuolar gel, a concept that is to be found everywhere in the human body.

The microvacuolar system: how connective tissue sliding works.

The term 'fascia' has been applied to a large number of very different tissues within the hand. These range from aligned ligamentous formations such as the longitudinal bands of the palmar fascia or Grayson's and Cleland's ligaments, to the loose packing tissues that surround all of the moving structures within the hand. In other parts of the body the terms 'superficial' and 'deep fascia' are often used but these have little application in the hand and fingers. Fascia can be divided into tissues that restrain motion, act as anchors for the skin, or provide lubrication and gliding. Whereas the deep fascia is preserved and easily characterized in anatomical dissection, the remaining fascial tissue is poorly described. Understanding its structure and dynamic anatomy may help improve outcomes after hand injury and disease. This review describes the sliding tissue of the hand or the 'microvacuolar system' and demonstrates how movement of tissues can occur with minimal distortion of the overlying skin while maintaining tissue continuity.

[New views about the skin]. / Faire peau neuve.

As the follow up article to "Introduction to the knowledge of subcutaneous sliding system in humans" published in the "Annales de chirurgie plastique" we further investigate the architecture of the skin and comment on the subcutaneous multifibrillar and microvacuolar arrangements that provide form, mobility, adaptability and resistance to force of gravity. The study aimed to highlight the direct link between the skin and subcutaneous environment in dynamic living tissue. Through high resolution endoscopic observations made during live surgery it is revealed how microvacuoles and microspaces can provide dynamic structure and form during movement between the epidermis, dermis and hypodermis. The study reveals intriguing morphodynamics which are necessary to maintain mobility and continuity to neighboring tissues. The polyhedric design of the skin surface directly relates to multifibrillar pillars beneath the skin which dictate their patterning and movement. The concept of tissue continuity is realised by the chaotic and fractal organisation of multifibrils interlaced with cellular components which characteristics alter depending on the state of hydration. Understanding the integral arrangement that provides continuity of all the structures below the skin provides an appreciation to how skin behaves in relation to movement of the rest of the body.

Bioenergetics of lung tumors: alteration of mitochondrial biogenesis and respiratory capacity.

Little is known on the metabolic profile of lung tumors and the reminiscence of embryonic features. Herein, we determined the bioenergetic profiles of human fibroblasts taken from lung epidermoid carcinoma (HLF-a) and fetal lung (MRC5). We also analysed human lung tumors and their surrounding healthy tissue from four patients with adenocarcinoma. On these different models, we measured functional parameters (cell growth rates in oxidative and glycolytic media, respiration, ATP synthesis and PDH activity) as well as compositional features (expression level of various energy proteins and upstream transcription factors). The results demonstrate that both the lung fetal and cancer cell lines produced their ATP predominantly by glycolysis, while oxidative phosphorylation was only capable of poor ATP delivery. This was explained by a decreased mitochondrial biogenesis caused by a lowered expression of PGC1alpha (as shown by RT-PCR and Western blot) and mtTFA. Consequently, the relative expression of glycolytic versus OXPHOS markers was high in these cells. Moreover, the re-activation of mitochondrial biogenesis with resveratrol induced cell death specifically in cancer cells. A consistent reduction of mitochondrial biogenesis and the subsequent alteration of respiratory capacity was also observed in lung tumors, associated with a lower expression level of bcl2. Our data give a better characterization of lung cancer cells' metabolic alterations which are essential for growth and survival. They designate mitochondrial biogenesis as a possible target for anti-cancer therapy.

Physical continuity of the perimysium from myofibers to tendons: involvement in lateral force transmission in skeletal muscle.

Advances in muscle physiology suggest that the perimysium plays a role in the transmission of lateral contractile forces. This hypothesis is strongly supported by our recent demonstration of the existence of "Perimysial Junctional Plates" in bovine Flexor carpi radialis muscle [Passerieux, E., Rossignol, R., Chopard, A., Carnino, A., Marini, J.F., Letellier, T., Delage, J.P. 2006. Structural organization of the perimysium in bovine skeletal muscle: junctional plates and associated intracellular subdomains. J. Struct. Biol. 154 (2), 206-216] However, the overall organization of the perimysium collagen network, as well as its continuity and heterogeneity, have still not been described in detail throughout the entire muscle. We used an extension of the standard NaOH digestion technique and scanning electron microscopy to analyze perimysium architecture in bovine Flexor carpi radialis muscle. First, we observed that the perimysium is made of a highly ordered network of collagen fibers, binding the myofibers from tendon to tendon. We identified basic collagen cable structures, characterized by a straight portion (3 cm long) in the direction of the myofibers and a curved terminal portion at 60 degrees. These cables reach the myofiber surface at the level of the previously described "Perimysial Junctional Plates". At a higher level of organization, these cables stick together to form the walls of numerous tubes arranged in a overlapping honeycomb pattern around the myofibers. At the ends of these tubes, the straight portions of the collagen cables ramify in large bundles that merge with the tendons. Taken together, these observations identify four levels of organization in the perimysium: (i) Perimysial Junctional Plates that constitute the focal attachment between the perimysium and the myofibers, (ii) collagen plexi attaching adjacent myofibers, (iii) a loose lattice of large interwoven fibers, and (iv) honeycomb tubes connecting two tendons. This spatial arrangement of the perimysium supports the view of a complex pattern of lateral force transmission from myofibers to tendons and adjacent muscles.

Physiological diversity of mitochondrial oxidative phosphorylation.

To investigate the physiological diversity in the regulation and control of mitochondrial oxidative phosphorylation, we determined the composition and functional features of the respiratory chain in muscle, heart, liver, kidney, and brain. First, we observed important variations in mitochondrial content and infrastructure via electron micrographs of the different tissue sections. Analyses of respiratory chain enzyme content by Western blot also showed large differences between tissues, in good correlation with the expression level of mitochondrial transcription factor A and the activity of citrate synthase. On the isolated mitochondria, we observed a conserved molar ratio between the respiratory chain complexes and a variable stoichiometry for coenzyme Q and cytochrome c, with typical values of [1-1.5]:[30-135]:[3]:[9-35]:[6.5-7.5] for complex II:coenzyme Q:complex III:cytochrome c:complex IV in the different tissues. The functional analysis revealed important differences in maximal velocities of respiratory chain complexes, with higher values in heart. However, calculation of the catalytic constants showed that brain contained the more active enzyme complexes. Hence, our study demonstrates that, in tissues, oxidative phosphorylation capacity is highly variable and diverse, as determined by different combinations of 1) the mitochondrial content, 2) the amount of respiratory chain complexes, and 3) their intrinsic activity. In all tissues, there was a large excess of enzyme capacity and intermediate substrate concentration, compared with what is required for state 3 respiration. To conclude, we submitted our data to a principal component analysis that revealed three groups of tissues: muscle and heart, brain, and liver and kidney.

Structural organization of the perimysium in bovine skeletal muscle: Junctional plates and associated intracellular subdomains.

We analyzed the structural features of the perimysium collagen network in bovine Flexor carpi radialis muscle using various sample preparation methods and microscopy techniques. We first observed by scanning electron microscopy that perimysium formed a regular network of collagen fibers with three hierarchical levels including (i) a loose lattice of large interwoven fibers ramified in (ii) numerous collagen plexi attaching together adjacent myofibers at the level of (iii) specific structures that we call perimysial junctional plates. Second, we looked more closely at the intracellular organization underneath each plate using transmission electron microscopy, immunohistochemistry, and a three-dimensional reconstruction from serial sections. We observed the accumulation of myonuclei arranged in clusters surrounded by a high density of subsarcolemmal mitochondria and the proximity of capillary branches. Third, we analyzed the distribution of these perimysial junctional plates, subsarcolemmal mitochondria, and myonuclei clusters along the myofibers using a statistical analysis of the distances between these structures. This revealed a global colocalization and the existence of adhesion domains between endomysium and perimysium. Taken together, our observations give a better description of the perimysium organization in skeletal muscle, and provide evidence that perimysial junctional plates with associated intracellular subdomains may participate in the lateral transmission of contractile forces as well as mechanosensing.

Modification of mitochondrial metabolism in fibroblasts from mice with a skeletal muscle mutation (muscular dysgenesis). Evidence of embryonic communication between myoblasts and fibroblasts.

Muscle development during embryogenesis is a complex process involving many mechanisms. It requires a close communication among the different cellular types of the muscle, especially the fibroblasts and myoblasts. Indeed, any abnormality in one cell type might influence the differentiation of the other. Thus, any disturbance altering the metabolism of the myoblasts might lead to modifications in the fibroblasts. To study this phenomenon, we used the dysgenic mouse (mdg-"muscular dysgenesis") carrying a homozygous recessive lethal mutation expressed only in skeletal muscle cells. First, we found that fibroblasts isolated from such mutant muscle (and not from mutant skin tissue) and grown in culture exhibited an altered metabolism. Secondly, muscle fibroblasts showed a lower capacity for proliferation. We also observed that respiration and ATP synthesis of dysgenic muscle fibroblasts were deficient, while respiratory chain enzymatic activities were normal. Finally, intracellular [Ca2+] levels of dysgenic fibroblasts are 50% of those of normal fibroblasts. These results support the hypothesis that certain characteristics of fibroblasts are determined by the surrounding cellular environment during embryonic organogenesis, and that such modifications are stable when the fibroblasts are isolated in vitro. Since fibroblast differentiation was disrupted permanently, this suggests, in the case of myopathies, that the modified cells, surrounding the muscle tissue, could contribute to the muscle pathology. Synergistic activities of this type should be considered when studying the course of pathologies in different types of muscle diseases.

M-calpain levels increase during fusion of myoblasts in the mutant muscular dysgenesis (mdg) mouse.

Previous studies have led to the hypothesis of a possible role for the calcium-dependent neutral protease m-calpain in myoblast fusion in culture. To evaluate this hypothesis, we chose as our model, the "muscular dysgenesis" mouse (mdg), which presents in vivo and in vitro characteristics of an elevated process of fusion (Yao and Essien, 1975; Dussartre, 1993; Ashby et al., 1993, Joffroy et al., 1999). The aim of this study was to demonstrate using myoblast cell lines and muscle biopsies from this mdg mutant, that the amount of m-calpain increases significantly as multinucleated myotubes are formed. Using immunoblot analysis, it was shown that the m-calpain concentration in a dysgenic cell line (GLT) increased 3-fold compared to what it was upon the introduction of the differentiation medium. On the other hand, in a normal cell line (NLT), the concentration of m-calpain did not vary significantly. Thus, when the transition from myoblasts to myotubes was slow, and the absolute level of fusion was reduced, as in the NLT cell line, the level of m-calpain was stable. In contrast, when the process of fusion was precocious and fast, and the level of fusion was elevated, such as in the GLT cell line, the concentration of m-calpain increased during fusion. Moreover, when myoblast fusion was prevented by the addition of calpain inhibitor II, the process was reduced by approximately 93%. Taking into account these observations, it is clear from our data that the muscular dysgenesis mouse provides a relevant model to study myoblast fusion and that m-calpain is involved in this process.

Fluorescence study of some terbium-oligopeptide complexes in methanolic solution.

This study concerned the use of lanthanide chelates to detect glycyl-leucyl-phenylalanine (GLF) and its homologues. Spectroscopic analysis of peptides without or with terbium complexation revealed the formation of (LF)(3)(Tb)(2), (GF)(3)(Tb)(2), (GLF)(3)(Tb)(2) and (FL)(4)Tb, (FG)(4)Tb complexes with high stability constants in methanolic solutions (pK(d)>13). Lanthanide chelate emission displayed a large Stokes shift (>270 nm), which allowed Tb chelates of GLF and its derivatives to be used for detection purposes. However, this preliminary study indicated some important limitations associated with lanthanide chelation, such as high methanolic content.

Multiple bcl-2/Ig gene rearrangements in persistent polyclonal B-cell lymphocytosis.

Persistent polyclonal B-cell lymphocytosis is a benign lymphoproliferative disorder of unknown aetiology occurring exclusively in women, characterized by typical binucleated lymphocytes, polyclonal expansion of B cells and elevated serum IgM. Owing to the role of Bcl-2 oncogene in inhibition of apoptosis, we have investigated the presence of the bcl-2/Ig gene rearrangement. Bcl-2/Ig gene rearrangement was determined by polymerase chain reaction targeting the usual breakpoint regions of the t(14;18). Bcl-2/Ig gene rearrangement was identified in all six patients and, more importantly, multiple rearrangements were present in five patients. The frequency of the bcl-2/Ig gene rearrangement is estimated to be of one translocation in 1 x 10(2) to 1 x 10(3) peripheral blood mononuclear cells. We conclude that persistent polyclonal B-cell lymphocytosis is associated with bcl-2/Ig gene rearrangement. These findings are of clinical importance because these patients may be misdiagnosed as having a leukaemic expression of non-Hodgkin's lymphoma.

Familial hemolytic-uremic syndrome and homozygous factor H deficiency.

Inherited hemolytic-uremic syndrome (HUS) is unusual. We report the occurrence of HUS in two siblings; one died at an early age while the other (the proband) has presented with three episodes of HUS since the age of 19 years. The finding of a persistently low serum C3 level in this patient led to a thorough evaluation of her complement cascade and a family investigation. The proband and her asymptomatic younger sister were found to have very low serum levels (5% of normal) of factor H, a regulatory protein of the alternative complement pathway. Both patients had low levels of serum C3, factor B, CH50 and VAH50, reflecting persistent alternative pathway activation. The father and mother both had half-normal serum factor H levels but an otherwise normal complement profile. Other members of the extended pedigree were also found to have half-normal serum factor H levels. In conclusion, in this family, factor H deficiency appears to be associated with HUS and is transmitted as an autosomal recessive trait. Persistent C3 hypocomplementemia in the setting of familial and/or recurrent HUS should be a clue to a possible inherited complement deficiency.