Pro-fibrotic phenotype of bone marrow stromal cells in Modic type 1 changes.

Modic type 1 changes (MC1) are painful vertebral bone marrow lesions frequently found in patients suffering from chronic low-back pain. Marrow fibrosis is a hallmark of MC1. Bone marrow stromal cells (BMSCs) are key players in other fibrotic bone marrow pathologies, yet their role in MC1 is unknown. The present study aimed to characterise MC1 BMSCs and hypothesised a pro-fibrotic role of BMSCs in MC1. BMSCs were isolated from patients undergoing lumbar spinal fusion from MC1 and adjacent control vertebrae. Frequency of colony-forming unit fibroblast (CFU-F), expression of stem cell surface markers, differentiation capacity, transcriptome, matrix adhesion, cell contractility as well as expression of pro-collagen type I alpha 1, α-smooth muscle actin, integrins and focal adhesion kinase (FAK) were compared. More CFU-F and increased expression of C-X-C-motif-chemokine 12 were found in MC1 BMSCs, possibly indicating overrepresentation of a perisinusoidal BMSC population. RNA sequencing analysis showed enrichment in extracellular matrix proteins and fibrosis-related signalling genes. Increases in pro-collagen type I alpha 1 expression, cell adhesion, cell contractility and phosphorylation of FAK provided further evidence for their pro-fibrotic phenotype. Moreover, a leptin receptor high expressing (LEPRhigh) BMSC population was identified that differentiated under transforming growth factor beta 1 stimulation into myofibroblasts in MC1 but not in control BMSCs. In conclusion, pro-fibrotic changes in MC1 BMSCs and a LEPRhigh MC1 BMSC subpopulation susceptible to myofibroblast differentiation were found. Fibrosis is a hallmark of MC1 and a potential therapeutic target. A causal link between the pro-fibrotic phenotype and clinical characteristics needs to be demonstrated.

Muscle fiber capillarization is associated with various indices of skeletal muscle mass in healthy, older men.

INTRODUCTION: Muscle fiber capillarization plays a fundamental role in the regulation of skeletal muscle mass maintenance. However, it remains unclear to what extent capillarization is related to various other skeletal muscle characteristics. In this study we determined whether muscle fiber capillarization is independently associated with measures of skeletal muscle mass, both on a whole-body and cellular level, and post-absorptive muscle protein synthesis rates in healthy older men. METHODS: Forty-six healthy older (70 ± 4 y) men participated in a trial during which basal muscle protein synthesis rates were assessed using stable isotope tracer methodology. Blood and muscle biopsy samples were collected to assess post-absorptive muscle protein synthesis rates over a 3-hour period. Immunohistochemistry was performed to determine various indices of muscle fiber capillarization, size, type distribution, and myonuclear content/domain size. Dual energy x-ray absorptiometry scans were performed to determine whole-body and appendicular lean tissue mass. RESULTS: Capillary-to-fiber ratio (C/Fi) and perimeter exchange (CFPE) index correlated with whole-body lean tissue mass (r = 0.43, P < 0.01 and r = 0.25, P < 0.10, respectively), appendicular lean tissue mass (r = 0.52, P < 0.001 and r = 0.37, P < 0.05, respectively) as well as appendicular lean tissue mass divided by body mass index (r = 0.65, P < 0.001 and r = 0.62, P < 0.001, respectively). Muscle fiber size correlated with C/Fi (r = 0.45, P < 0.01), but not with CFPE index. No associations were observed between different indices of muscle fiber capillarization and post-absorptive muscle protein synthesis rates in healthy, older men. CONCLUSION: The present study provides further evidence that muscle fiber capillarization may be a critical factor in the regulation of skeletal muscle maintenance in healthy older men.

Pull-out strength of patient-specific template-guided vs. free-hand fluoroscopically controlled thoracolumbar pedicle screws: a biomechanical analysis of a randomized cadaveric study.

PURPOSE: To assess the pull-out strength of thoracolumbar pedicle screws implanted via either a patient-specific template-guided or conventional free-hand fluoroscopically controlled technique in a randomized cadaveric study, and to evaluate the influence of local vertebral bone density, quantified by Hounsfield units (HU), on pedicle screw pull-out strength. METHODS: Thoracolumbar pedicles of three spine cadavers were instrumented using either a free-hand fluoroscopically controlled or a patient-specific template-guided technique. Preoperative bone density was quantified by HU measured on CT. Pedicle perforation was evaluated on postoperative CT scans by an independent and blinded radiologist. After dissected vertebrae were embedded in aluminum fixation devices, pull-out testing was initiated with a preload of 50 N and a constant displacement rate of 0.5 mm/s. Subgroup analyses were performed excluding pedicle screws with a pedicle breach (n = 47). RESULTS: Pull-out strength was significantly different with 549 ± 278 and 441 ± 289 N in the template-guided (n = 50) versus fluoroscopically controlled (n = 48) subgroups (p = 0.031), respectively. Subgroup analysis limited to screws with an intrapedicular trajectory revealed a tendency toward a higher pull-out strength in the template-guided (n = 30) versus fluoroscopically controlled screws (n = 21) with 587 ± 309 and 454 ± 269 N (p = 0.118), respectively. There was a trend toward a higher pull-out strength (709 ± 418 versus 420 ± 149 N) in vertebrae with a bone density of (>171 HU) versus (<133 HU), respectively (p = 0.061). CONCLUSIONS: There was a significantly higher pull-out strength of thoracolumbar pedicle screws when inserted via a patient-specific template-guided versus conventional free-hand fluoroscopically controlled technique, potentially associated with screw trajectory.

Review of current best practice and priorities for research in radiation oncology for elderly patients with cancer: the International Society of Geriatric Oncology (SIOG) task force.

Radiotherapy (RT) is a key component of the management of older cancer patients. Level I evidence in older patients is limited. The International Society of Geriatric Oncology (SIOG) established a task force to make recommendations for curative RT in older patients and to identify future research priorities. Evidence-based guidelines are provided for breast, lung, endometrial, prostate, rectal, pancreatic, oesophageal, head and neck, central nervous system malignancies and lymphomas. Patient selection should include comorbidity and geriatric evaluation. Advances in radiation planning and delivery improve target coverage, reduce toxicity and widen eligibility for treatment. Shorter courses of hypofractionated whole breast RT are safe and effective. Conformal RT and involved-field techniques without elective nodal irradiation have improved outcomes in non-small-cell lung cancer (NSCLC) without increasing toxicity. Where comorbidities preclude surgery, stereotactic body radiotherapy (SBRT) is an option for early-stage NSCLC and pancreatic cancer. Modern involved-field RT for lymphoma based on pre-treatment positron emission tomography data has reduced toxicity. Significant comorbidity is a relative contraindication to aggressive treatment in low-risk prostate cancer (PC). For intermediate-risk disease, 4-6 months of hormones are combined with external beam radiotherapy (EBRT). For high-risk PC, combined modality therapy (CMT) is advised. For high-intermediate risk, endometrial cancer vaginal brachytherapy is recommended. Short-course EBRT is an alternative to CMT in older patients with rectal cancer without significant comorbidities. Endorectal RT may be an option for early disease. For primary brain tumours, shorter courses of postoperative RT following maximal debulking provide equivalent survival to longer schedules. MGMT methylation status may help select older patients for temozolomide alone. Stereotactic RT provides an alternative to whole-brain RT in patients with limited brain metastases. Intensity-modulated radiation therapy provides an excellent technique to reduce dose to the carotids in head and neck cancer and improves locoregional control in oesophageal cancer. Best practice and research priorities are summarised.

The expression of glycophorin A and osteoprotegerin is locally increased in carotid atherosclerotic lesions of symptomatic compared to asymptomatic patients.

The aim of this study was to evaluate in detail the histopathological characteristics of endarterectomized carotid atherosclerotic lesions in symptomatic versus asymptomatic patients. Twenty carotid lesions, 10 from asymptomatic and 10 from symptomatic patients who underwent carotid endarterectomy were classified according to histomorphological features. Samples were analyzed for intraplaque localization and for the expression of proteins associated with inflammation, such as CD68, interleukin (IL)-1ß, tumor necrosis factor-α (TNF-α), pentraxin-3 (PTX-3), nuclear factor-κB (NF-κB), C-reactive protein (CRP) and transforming growth factor-ß (TGF-ß), as well as for proteins associated with vascular remodelling, such as matrix-metalloproteinase-9 (MMP-9), glycophorin A (GYPA), osteoprotegerin (OPG), vascular cell adhesion molecule-1 (VCAM-1), endothelin-1 (ET-1), vascular endothelial growth factor (VEGF) and vascular smooth muscle cell actin (VSMA). Corresponding expression scores were compared between the symptomatic and asymptomatic patients and evaluated statistically. The expression of all 14 evaluated markers was significantly elevated in the border zone adjacent to the mixed plaque compared with the unaffected control area of the same sample (p<0,016). The expression scores of GYPA and OPG were significantly higher in the border zones around the calcified (GYPA, p=0.035; OPG, p=0.043) and mixed (GYPA, p<0.001; OPG, p=0.007) plaque zones of symptomatic patients compared to asymptomatic patients. No difference in expression scores was observed for any of the analyzed inflammatory marker proteins between the border zones of symptomatic and asymptomatic patients. In conclusion, the increased expression of GYPA, indicating intraplaque hemorrhage, and OPG, indicating the transdifferentiation of vascular cells, in carotid atherosclerotic lesions may be associated with an increased risk of plaque instability.

Impact of intensity-modulated and image-guided radiotherapy on elderly patients undergoing chemoradiation for locally advanced head and neck cancer.

PURPOSE: In this work, the treatment tolerance of elderly patients (≥70 years) undergoing intensity-modulated radiotherapy (IMRT) and image-guided radiotherapy (IGRT) and chemotherapy for locally advanced head and neck cancer was assessed. PATIENTS AND METHODS: A retrospective review of 112 patients undergoing concurrent chemoradiation for locally advanced head and neck cancer was performed. Treatment toxicity, protocol violations, long-term complications, and survival were compared between 85 younger patients (< 70 years) and 27 older patients (≥ 70 years). RESULTS: Grade 3-4 treatment toxicity was observed in 88.2% and 88.8% for younger and older patients, respectively. Mean weight loss and treatment break were 5.9 and 3.9 kg (p = 0.03) and 7.3 and 7.8 days (p = 0.8) for younger and older patients, respectively. Seven patients (8.2%) did not complete treatment in the younger group compared to 1 patient (3.7%) in the older group (p = 0.6). No significant differences in protocol violations and survival were found between the two groups. CONCLUSION: Compared to younger patients, elderly patients with locally advanced head and neck cancer tolerated chemoradiation with IMRT and IGRT well, and should not be denied curative treatment based solely on age.

Identifying patients at risk for late radiation-induced toxicity.

The impact of curative radiotherapy depends mainly on the total dose delivered in the targeted volume. Nevertheless, the dose delivered to the surrounding healthy tissues may reduce the therapeutic ratio of many treatments. Two different side effects (acute and late) can occur during and after radiotherapy. Of particular interest are the radiation-induced late complications (LC) due to their irreversibility and the potential impact on quality of life. In one population treated with the same technique, it appears that individual radiosensitivity clearly exists. In the hypothesis that genetic is involved in this area of research, low CD4 and CD8 lymphocyte apoptosis were shown to be correlated with high grade of LC. In addition, recent data suggest that patients with severe radiation-induced LC possess 4 or more single nucleotide polymorphisms (SNPs) in candidate genes and low radiation-induced CD8 lymphocyte apoptosis in vitro. On-going studies are being analyzing the entire genome using a genome-wide association study (GWAS).

[Management of anal canal carcinoma]. / Prise en charge du carcinome du canal anal.

Although anal canal squamous cell carcinoma is rare, the general practitioner should consider this diagnosis in a patient with persistent lower abdominal symptoms. While classically observed in older women, an increased incidence is also seen in HIV-positive patients or patients with a history of human papillomavirus infection. Initial diagnosis and local work-up require assessment by a proctologist. Standard curative treatment combines radiotherapy with 5-FU- and MMC-based chemotherapy. Salvage surgery should be discussed in case of local relapse. The general practitioner, the proctologist and the radiation oncologist, all participate in post-treatment surveillance.

[Oesophageal cancer: multidisciplinary approach]. / Cancer de l'oesophage: traitement multidisciplinaire.

Despite recent progresses, the prognosis of oesophageal cancer is still bad, mostly because of frequent late diagnosis. In early cases, radical surgery alone is able to cure 60-70% of the patients. In locally-advanced cases, on the other hand, surgical results are considerably worse and combined therapies are contemplated. In these cases, neoadjuvant therapy (induction chemotherapy followed by radiotherapy and surgical resection) is often proposed, but without formal proof of superiority. These combined therapies are heavy for the patient and complex for the team. They can only be decided and managed in the frame of intensive multidisciplinary collaboration. Future progresses will come at the same time from larger studies and from the efforts of the medical community towards earlier diagnosis of this disease.

Sepsis and cardiomyopathy as rare clinical manifestations of pheochromocytoma--two case report studies.

The clinical manifestation of pheochromocytomas is highly variable and can closely resemble numerous clinical conditions. Here, we report on two cases of patients with pheochromocytoma, which manifested as sepsis or cardiomyopathy. The first patient initially presented with bacterial urosepsis due to klebsiella oxytoca. Despite effective antibiotic therapy, the patient developed recurring fever accompanied by hypertension. The inconsistency between therapy-refractory hypertension and fever indicated the possibility of excessive catecholamine production. In the second case, the patient presented with a suspected ST-segment elevation myocardial infarction accompanied by E. coli sepsis and a previously undiagnosed unilateral tumor mass of the adrenal gland. Severely impaired myocardial contraction of the apical anterior and inferior regions without significant coronary artery disease was consistent with the Takotsubo cardiomyopathy, a known transient functional myocardial complication associated with pheochromocytoma. Both patients were diagnosed with unilateral pheochromocytoma. Following pre-operative antihypertensive therapy, both patients were cured by surgery and still remain free of disease after two years of follow-up.

Insulin receptors and signal transduction proteins in the hypothalamo-hypophyseal system: a review on morphological findings and functional implications.

Receptors for insulin are widely distributed in the brain and pituitary. The current hypothesis on receptor function in these regions points to a role of insulin as a mediator in the communication of the peripheral endocrine system with the brain via various steps of the neuroendocrine axis. Recent data demonstrate that receptor-positive neurons in the brain, i.e. in the hypothalamus, and secretory cells in the anterior pituitary gland possess specific proteins that are thought to be involved in key steps of post receptor signal transduction, in particular insulin receptor substrate-1 and phosphatidylinositol 3'-kinase (PI3k). PI3k is a critical enzyme of the intracellular signaling pathway that is activated by a number of receptor tyrosine kinases, including receptors for insulin and IGF-1. This information further completes the framework indicating in vivo activity of insulin receptors in central neuroendocrine cells and their involvement in one branch of several physiological mechanisms that control body metabolism and nutritional behaviour.

Mechanism of inhibition of the human matrix metalloproteinase stromelysin-1 by TIMP-1.

Matrix metalloproteinases (MMPs) are zinc endopeptidases that are required for the degradation of extracellular matrix components during normal embryo development, morphogenesis and tissue remodelling. Their proteolytic activities are precisely regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in diseases such as arthritis, atherosclerosis, tumour growth and metastasis. Here we report the crystal structure of an MMP-TIMP complex formed between the catalytic domain of human stromelysin-1 (MMP-3) and human TIMP-1. TIMP-1, a 184-residue protein, has the shape of an elongated, contiguous wedge. With its long edge, consisting of five different chain regions, it occupies the entire length of the active-site cleft of MMP-3. The central disulphide-linked segments Cys 1-Thr 2-Cys 3-Val 4 and Ser 68-Val 69 bind to either side of the catalytic zinc. Cys 1 bidentally coordinates this zinc, and the Thr-2 side chain extends into the large specificity pocket of MMP-3. This unusual architecture of the interface between MMP-3 and TIMP-1 suggests new possibilities for designing TIMP variants and synthetic MMP inhibitors with potential therapeutic applications.

1.8-A crystal structure of the catalytic domain of human neutrophil collagenase (matrix metalloproteinase-8) complexed with a peptidomimetic hydroxamate primed-side inhibitor with a distinct selectivity profile.

Matrix metalloproteinases (MMP) are zinc endopeptidases involved in tissue remodelling. They have been implicated in a series of pathologies, including cancer, arthritis, joint destruction and Alzheimer's disease. Human neutrophil collagenase represents one of the three interstitial collagenases that cleave triple-helical collagen of type I, II and III. Its catalytic domain (residues Phe79-Gly242) has been heterologously expressed in Escherichia coli and crystallized as a non-covalent complex with the hydroxamate inhibitor BB-1909, which has distinct selectivity against different MMP, in a crystal form. The crystal structure, refined to 0.18-nm resolution, shows that BB-1909 is a right-hand-side inhibitor that binds to the S1'-S3' subsites and coordinates to the catalytic Zn2+ in a bidentate manner via the hydroxyl and carbonyl oxygen atoms of the hydroxamate group in a similar manner to batimastat. The collagenase/BB-1909 complex is described in detail and compared with the collagenase/batimastat complex. These studies provide information on MMP specificity and thus may assist the development of more-selective MMP inhibitors.

The helping hand of collagenase-3 (MMP-13): 2.7 A crystal structure of its C-terminal haemopexin-like domain.

Collagenase-3 (MMP-13) is a matrix metalloproteinase involved in human breast cancer pathology and in arthritic processes. The crystal structure of its C-terminal haemopexin-like domain has been solved by molecular replacement and refined to an R-value of 0.195 using data to 2.7 A resolution. This structure reveals a disk-like shape. The chain is folded into a beta-propeller structure of pseudo 4-fold symmetry, with the four propeller blades arranged around a funnel-like tunnel. This central tunnel tube harbours four ions assigned as two calcium and two chloride ions. The C-terminal domain of collagenase-3 has a similar structure to the equivalent domain of gelatinase A and fibroblast collagenase 1; however, its detailed structure and surface charge pattern has a somewhat greater similarity to the latter, in agreement with the subgrouping of MMP-13 with the collagenase subfamily of MMPs. It is proposed that several small structural differences may act together to confer the characteristic binding and cleavage specificities of collagenases for triple-helical substrates, probably in co-operation with a fitting interdomain linker.

Reconstitution of the core complex (alpha beta)3APCLC8.9 of the phycobilisome from Mastigocladus laminosus using the Lc8.9 linker polypeptide overexpressed in Escherichia coli.

The apcC gene from Mastigocladus laminosus encodes the linker polypeptide LC8.9 located in the phycobilisome core. A T7 RNA polymerase expression system was used to express the linker polypeptide LC8.9 from M. laminosus in Escherichia coli. The apcC gene product was expressed as an inclusion body which was solubilized in a buffer containing 8M urea. Final purification was achieved by ion exchange chromatography on Fractogel TSK CM 650 (S). In addition, a method for preparative isolation of the LC8.9 linker polypeptide from M. laminosus by reverse phase chromatography is presented. Both LC8.9 isolated from M. laminosus and overexpressed in E. coli were capable of reconstituting the complex (alpha beta)3APCLC8.9. The reconstituted complex was identical to preparations isolated from M. laminosus in terms of polypeptide composition, absorption and fluorescence emission spectroscopy.

Mortality from lung cancer and cardiovascular diseases among stainless-steel producing workers.

The mortality pattern of workers involved in the production of stainless steel (SS) was studied from 1968 to 1984 in order to investigate a possible risk of lung cancer in relation to exposure to chromium compounds, polycyclic aromatic hydrocarbons, and silica. The role of heat exposure in mortality from cardiovascular diseases also was examined. The cohort was comprised of 4,227 workers. Complete individual job histories were provided by the company (UGINE SA). The smoking habits of 24 percent of the cohort members were known from the interview of workers still active during the data collection. The observed numbers of deaths were compared with the expected ones based on national rates with adjustment for age, sex, and calendar time (standardized mortality ratio, SMR). No significant excesses of lung cancer were observed among workers employed in the manufacture of ferroalloys (SMR = 0.68) and in the melting and casting of SS (SMR = 1.04), whereas a significant excess appeared among SS foundry workers (SMR = 2.29). This excess was higher and remained significant among workers with more than 30 years since first employment in the foundry area (SMR = 3.34). Among subjects exposed to heat, no excess was observed for all cardiovascular diseases or for ischemic heart diseases.

A mortality study of cobalt production workers: an extension of the follow-up.

The follow-up of a cohort of workers employed in an electrochemical plant producing cobalt and sodium, previously studied from 1950-1980, has been extended from 1981-1988. The standardized mortality ratio (SMR) for all causes of death was 0.85 (95% confidence interval [CI] = 0.76-0.95, 309 observed) for the whole cohort, and 0.95 (95% CI = 0.83-1.08, 247 observed) for the subcohort of workers born in France. With regard to lung cancer mortality among cobalt production workers, which is the main objective of the study, the SMRs were, respectively, 0.85 (95% CI = 0.18-2.50, 3 observed) and 1.16 (95% CI = 0.24-3.40, 3 observed). Neither did any excess of mortality from diseases of the circulatory and of the respiratory systems appear among cobalt production workers. Maintenance workers, however, exhibited high SMRs for lung cancer, reaching statistical significance for duration of exposure and time since first exposure > or 30 years. This study does not support the hypothesis of a relationship between lung cancer and cobalt exposure.

Partial purification of a potassium channel with low permeability for sodium from tonoplast membranes of Hordeum vulgare cv. Gerbel.

A potassium-specific tonoplast channel was identified by reconstitution of tonoplast polypeptides into planar lipid bilayer membranes. Highly purified tonoplast membranes were solubilized in Triton X-100-containing buffer and fractionated by size-exclusion chromatography. The protein fractions were assayed for ion channel activity in a planar bilayer system, and the potassium channel was routinely recovered in specific fractions corresponding to an apparent molecular mass of 80 kDa. In symmetrical electrolyte solutions of 100 mM potassium chloride, the potassium channel had a single-channel conductance of 72 pS. Substates of the channel with conductances of 17, 33 and 52 pS were frequently observed. After identification of the channel in low or high KCl, addition of sodium acetate or sodium chloride caused only insignificant conductance changes. This result suggested that the channel was not or little permeable for sodium or chloride, whereas it had similar single-channel conductance for rubidium and caesium ions as compared with potassium ions. The channel is presumably responsible for the equilibration of potassium between the vacuole and the cytosol. The role of the channel in the physiology of the barley cell under salt stress is discussed.

Characterization of the epidermis from barley primary leaves : I. Isolation of epidermal protoplasts.

A method is described for isolating epidermal protoplasts from the primary leaves of barley (Hordeum vulgare L.). Epidermal protoplasts are lighter than mesophyll protoplasts because of their smaller ratio of cytoplasm to vacuole, and can be separated from the latter by density-gradient centrifugation after complete digestion of the leaves. We have started a basic characterization of the epidermal protoplast fraction in comparison with mesophyll protoplasts. Epidermal protoplasts had a mean diameter of 63.5 µm, whereas that of mesophyll protoplasts was 35.7 µm. Their respiratory oxygen consumption was not influenced by light. They contained acid hydrolases and cytoplasmic enzymes in relative activities different from those of mesophyll protoplasts. Their polypeptide pattern as judged from two-dimensional separations was, in principle, similar to that of mesophyll cells after elimination of the plastids from the latter by the preparation of vacuoplasts. However, in addition, a considerable number of epidermis-specific polypeptides were observed. Isolated epidermal protoplasts were viable and efficiently incorporated [(35)S]methionine into newly synthesized proteins. The results show that epidermal protoplasts are suitable for the investigation of the physiological and molecular properties of epidermal cells in leaves.