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1.  RIM, Munc13, and Rab3A Interplay in Acrosomal Exocytosis 
Experimental Cell Research  2012;318(5):478-488.
Exocytosis is a highly regulated, multistage process consisting of multiple functionally definable stages, including recruitment, targeting, tethering, priming, and docking of secretory vesicles with the plasma membrane, followed by calcium-triggered membrane fusion. The acrosome reaction of spermatozoa is a complex, calcium-dependent regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. Not much is known about the molecules that mediate membrane docking in this particular fusion model. In neurons, the formation of the ternary RIM/Munc13/Rab3A complex has been suggested as a critical component of synaptic vesicles docking. Previously, we demonstrated that Rab3A localizes to the acrosomal region in human sperm, stimulates acrosomal exocytosis, and participates in an early stage during membrane fusion. Here, we report that RIM and Munc13 are also present in human sperm and localize to the acrosomal region. Like Rab3A, RIM and Munc13 participate in a prefusion step before the efflux of intra-acrosomal calcium. By means of a functional assay using antibodies and recombinant proteins, we show that RIM, Munc13 and Rab3A interplay during acrosomal exocytosis. Finally, we report by electron transmission microscopy that sequestering RIM and Rab3A alters the docking of the acrosomal membrane to the plasma membrane during calcium-activated acrosomal exocytosis. Our results suggest that the RIM/Munc13/Rab3 A complex participates in acrosomal exocytosis and that RIM and Rab3A have a central role in membrane docking.
doi:10.1016/j.yexcr.2012.01.002
PMCID: PMC3288645  PMID: 22248876
RIM; Munc13; Rab3A; acrosome reaction; membrane docking; human sperm
2.  Early postoperative MRI in detecting hematoma and dural compression after lumbar spinal decompression: prospective study of asymptomatic patients in comparison to patients requiring surgical revision 
European Spine Journal  2010;19(12):2216-2222.
Early postoperative MRI after spinal surgery is difficult to interpret because of confounding postoperative mass effects and frequent occurrence of epidural hematomas. Purpose of this prospective study is to evaluate prevalence, extent and significance of hematoma in the first postoperative week in asymptomatic patients after decompression for lumbar stenosis and to determine the degree of clinically significant dura compression by comparing with the patients with postoperative symptoms. MRI was performed in 30 asymptomatic patients (47 levels) in the first week after lumbar spine decompression for degenerative stenosis. Eleven patients requiring surgical revision (16 levels) for symptomatic early postoperative hematoma were used for comparison. In both groups the cross-sectional area of the maximum dural compression (bony stenosis and dural sac expansion) was measured preoperatively and postoperatively by an experienced radiologist. Epidural hematoma was seen in 42.5% in asymptomatic patients (20/47 levels). The median area of postoperative hematoma at the operated level was 176 mm2 in asymptomatic patients and 365 mm2 in symptomatic patients. The median cross-sectional area of the dural sac at the operated level was 128.5 and 0 mm2 in asymptomatic and symptomatic patients, respectively, at the site of maximal compression. In the symptomatic group 75% of the patients had a maximal postoperative dural sac area of 58.5 mm2 or less, whereas in the asymptomatic group 75% of patients with epidural hematoma had an area of 75 mm2 or more. The size of hematoma and the degree of dural sac compression were significantly larger in patients with symptoms needing surgical revision. Dural sac area of less than 75 mm2 in early postoperative MRI was found to be the threshold for clinical significance.
doi:10.1007/s00586-010-1483-x
PMCID: PMC2997206  PMID: 20556438
Epidural hematoma; Early postoperative MRI; Spinal stenosis; Neural compression
3.  MR/CT image fusion of the spine after spondylodesis: a feasibility study 
European Spine Journal  2010;19(10):1771-1775.
The objective of this study is to evaluate feasibility, accuracy and time requirements of MR/CT image fusion of the lumbar spine after spondylodesis. Sagittal MR and CT images derived from standard imaging protocols (sagittal T2-weighted MR/sagittal reformatted multi-planar-reformation of the CT) of the lumbar spine with correct (n = 5) and incorrect (n = 5) implant position were fused by two readers (R1, R2) using OsiriX in two sessions placing one (session 1) or two (session 2) reference point(s) on the dorsal tip(s) of the cranial and caudal endplates from the second lumbar to the first sacral vertebra. R1 was an experienced musculoskeletal radiologist; R2 a spine surgeon, both had received a short training on the software tool. Fusion times and fusion accuracy, defined as the largest deviation between MR and CT in the median sagittal plane on the ventral tip of the cranial end plate of the most cranial vertebra visible on the CT, were measured in both sessions. Correct or incorrect implant position was evaluated upon the fused images for all patients by an experienced senior staff musculoskeletal radiologist. Mean fusion time (session 1/session 2; in seconds) was 100.4/95 (R1) and 104.2/119.8 (R2). Mean fusion deviation (session 1/session 2; in mm) was 1.24/2.20 (R1) and 0.79/1.62 (R2). The correct/incorrect implant position was identified correctly in all cases. In conclusion, MR/CT image fusion of the spine with metallic implants is feasible, fast, accurate and easy to implement in daily routine work.
doi:10.1007/s00586-010-1430-x
PMCID: PMC2989216  PMID: 20473623
Image fusion; MRI; CT; Lumbar spine; Spondylodesis
4.  Femoral component rotation and arthrofibrosis following mobile-bearing total knee arthroplasty 
International Orthopaedics  2006;30(5):420-425.
The purpose of this study was to evaluate the femoral component rotation in a small subset of patients who had developed arthrofibrosis after mobile-bearing total knee arthroplasty (TKA). Arthrofibrosis was defined as flexion less than 90° or a flexion contracture greater than 10° following TKA. From a consecutive cohort of 3,058 mobile-bearing TKAs, 49 (1.6%) patients were diagnosed as having arthrofibrosis, of which 38 (86%) could be recruited for clinical assessment. Femoral rotation of a control group of 38 asymptomatic TKA patients matched for age, gender, and body mass index was also evaluated. The surgical epicondylar axis was compared with the posterior condylar axis for the femoral prosthesis. Femoral components in the arthrofibrosis group were significantly internally rotated by a mean of 4.7° (SD 2.2°, range 10° internal to 1° external). In the control group, the femoral component had a mean 0.3° internal rotation (SD 2.3°, range 4° internal to 6° external). Following mobile-bearing TKA, there is a significant correlation between internal femoral component rotation and chronic arthrofibrosis.
doi:10.1007/s00264-006-0085-z
PMCID: PMC3172765  PMID: 16521009
5.  Surveillance programme of cirrhotic patients for early diagnosis and treatment of hepatocellular carcinoma: a cost effectiveness analysis 
Gut  2001;48(2):251-259.
BACKGROUND—Hepatocellular carcinoma (HCC) is a major cause of death in cirrhotic patients. This neoplasm is associated with liver cirrhosis (LC) in more than 90% of cases. Early diagnosis and treatment of HCC are expected to improve survival of patients.
AIMS—To assess the cost effectiveness of a surveillance programme of patients with LC for the early diagnosis and treatment of HCC.
PATIENTS—A cohort of 313 Italian patients with LC were enrolled in the surveillance programme between March 1989 and November 1991. In the same period, 104 consecutive patients with incidentally detected HCC were referred to our centre and served as a control group.
METHODS—Surveillance was based on ultrasonography (US) and α fetoprotein (AFP) determinations repeated at six month intervals. Risk factors for HCC were assessed by multivariate analysis (Cox model). Outcome measures analysed were: (1) number and size of tumours; (2) eligibility for treatment; and (3) survival of patients. Economic issues were: (1) overall cost of surveillance programme; (2) cost per treatable HCC; and (3) cost per year of life saved (if any). Costs were assessed according to charges for procedures at our university hospital.
RESULTS—Surveillance lasted a mean of 56 (31) months (range 6-100). During the follow up, 61 patients (19.5%) developed HCC (unifocal at US in 49 cases), with an incidence of 4.1% per year of follow up. AFP, Child-Pugh classes B and C, and male sex were detected as independent risk factors for developing HCC. Only 42 (68.9%) of 61 liver tumours were treated by surgical resection, orthotopic liver transplantation, or local therapy. The cumulative survival rate of the 61 patients with liver tumours detected in the surveillance programme was significantly longer than that of controls (p=0.02) and multivariate analysis showed an association between surveillance and survival. The overall cost of the surveillance programme was US$753 226, the cost per treatable HCC was US$17 934, and the cost for year of life saved was US$112 993.
CONCLUSION—Our surveillance policy of patients with LC requires a large number of resources and offers little benefit in terms of patient survival. The decision whether to adopt a surveillance policy towards HCC should rely on the prevalence of the disease in the population and on the resources of a particular country.


Keywords: hepatocellular carcinoma; surveillance programme; cost effectiveness
doi:10.1136/gut.48.2.251
PMCID: PMC1728189  PMID: 11156649
6.  Kidney, splanchnic, and leg protein turnover in humans. Insight from leucine and phenylalanine kinetics. 
Journal of Clinical Investigation  1996;98(6):1481-1492.
The rate of kidney protein turnover in humans is not known. To this aim, we have measured kidney protein synthesis and degradation in postabsorptive humans using the arterio-venous catheterization technique combined with 14C-leucine, 15N-leucine, and 3H-phenylalanine tracer infusions. These measurements were compared with those obtained across the splanchnic bed, the legs (approximately muscle) and in the whole body. In the kidneys, protein balance was negative, as the rate of leucine release from protein degradation (16.8 +/- 5.1 mumol/min.1.73 m2) was greater (P < 0.02) than its uptake into protein synthesis (11.6 +/- 5.1 mumol/min. 1.73 m2). Splanchnic net protein balance was approximately 0 since leucine from protein degradation (32.1 +/- 9.9 mumol/min. 1.73 m2) and leucine into protein synthesis (30.8 +/- 11.5 mumol/min. 1.73 m2) were not different. In the legs, degradation exceeded synthesis (27.4 +/- 6.6 vs. 20.3 +/- 6.5 mumol/min. 1.73 m2, P < 0.02). The kidneys extracted alpha-ketoisocaproic acid, accounting for approximately 70% of net splanchnic alpha-ketoisocaproic acid release. The contributions by the kidneys to whole-body leucine rate of appearance, utilization for protein synthesis, and oxidation were approximately 11%, approximately 10%, and approximately 26%, respectively; those by the splanchnic area approximately 22%, approximately 27%, and approximately 18%; those from estimated total skeletal muscle approximately 37%, approximately 34%, and approximately 48%. Estimated fractional protein synthetic rates were approximately 42%/d in the kidneys, approximately 12% in the splanchnic area, and approximately 1.5% in muscle. This study reports the first estimates of kidney protein synthesis and degradation in humans, also in comparison with those measured in the splanchnic area, the legs, and the whole-body.
PMCID: PMC507576  PMID: 8823315
7.  Mechanisms of postprandial protein accretion in human skeletal muscle. Insight from leucine and phenylalanine forearm kinetics. 
Journal of Clinical Investigation  1996;98(6):1361-1372.
The relative role of protein synthesis and degradation in determining postprandial net protein deposition in human muscle is not known. To this aim, we studied forearm leucine and phenylalanine turnover by combining the arteriovenous catheterization with tracer infusions, before and following a 4 h administration of a mixed meal in normal volunteers. Forearm amino acid kinetics were assessed in both whole blood and plasma. Fasting forearm protein degradation exceeded synthesis (P < 0.01) using either tracer, indicating net muscle protein loss. The net negative forearm protein balance was quantitatively similar in whole blood and in plasma. After the meal, forearm proteolysis was suppressed (P < 0.05- < 0.03), while forearm protein synthesis was stimulated (P < 0.05- < 0.01). However, stimulation of protein synthesis was greater (P < 0.05- < 0.01) in whole blood (leucine data: +50.4 +/- 7.8 nmol/min x 100 ml of forearm; phenylalanine data: +30.4 +/- 11.6) than in plasma (leucine data: +17.8 +/- 5.6 nmol/min x 100 ml of forearm; phenylalanine data: +5.7 +/- 2.1). Consequently, the increment of net amino acid balance was approximately two to fourfold greater (P < 0.04- < 0.03) in whole blood than in plasma. In conclusion, meal ingestion stimulates forearm protein deposition through both enhanced protein synthesis and inhibited proteolysis. Plasma data underestimate net postprandial forearm protein synthesis, suggesting a key role of red blood cells and/or of blood mass in mediating mealenhanced protein accretion.
PMCID: PMC507562  PMID: 8823301
8.  Analysis of human antitopoisomerase-I idiotypes. 
Journal of Clinical Investigation  1993;92(3):1302-1313.
Antibodies to topoisomerase-I are present in approximately 26% of patients with scleroderma and are rarely found in patients with other diseases. In the current study, the expression of the antitopoisomerase-I (antitopo-I) idiotype from two scleroderma patients (E.M. and S.G.) and from a healthy individual (N.M.) were studied. Idiotype EM-SCL was restricted to the three classes of antitopo-I, whereas idiotypes SG-SCL and NM were found in all classes of antitopo-I as well as in their non-antitopo-I Igs. Sera from 9 of 10 antitopo-I-positive unrelated scleroderma patients expressed idiotype SG-SCL and some also expressed idiotype NM. Sera from N.M.'s 3 daughters and from 7 of 18 nonrelated normals expressed idiotype NM in the three immunoglobulin classes of non-antitopo-I. Two of the antitopo-I antibodies expressed a cross-reacting idiotype (CRI) that is present in non-antitopo-I antibodies from the same donor. Contrary to the natural CRI, SG-SCL's CRI is closely associated with the antigen binding site. Antitopo-I idiotypes are on the heavy chains. Like many other autoantibodies, Id-SG-SCL use VH4.2-1, DXP1, and JH4 in germline configuration.
Images
PMCID: PMC288272  PMID: 8397223
9.  Shared idiotypes and restricted immunoglobulin variable region heavy chain genes characterize murine autoantibodies of various specificities. 
Journal of Clinical Investigation  1986;78(3):753-759.
The study of the Ig variable region heavy chain (VH) genes used to encode antibodies specific for self-epitopes from murine hybridomas showed that three VH families are primarily utilized: VH J558, the largest family, and VH QPC52 and VH 7183, the families most proximal to the Ig joining region heavy chain genes. These monoclonal autoantibodies express cross-reactive idiotopes shared by rheumatoid factors and antibodies specific for Sm. The expression of these idiotypes is independent of major histocompatibility complex and Ig constant region heavy chain haplotypes, self-antigen specificity, and even the VH gene family utilized. Though the experiments described here are limited to murine autoantibodies, similarities exist between murine and human autoimmune diseases. Studies that aim to investigate the relationship between VH gene expression and the presence of cross-reactive idiotypes among human autoantibodies should enable us to better understand the mechanisms of autoimmunity and self-tolerance.
Images
PMCID: PMC423668  PMID: 2427543

Results 1-9 (9)