STRAIN ANALYSIS OF MASSETER MUSCLE BY ULTRASOUND.

The masseter muscle represents an area of important functional interest. The present study aims to verify the feasibility of ultrasound imaging for quantifying the muscular deformation pattern in the masseter. Fifteen consecutive subjects were enrolled and underwent masseter ultrasound according to a repeatable protocol. Ultrasound was carried out during teeth clenching in natural conditions and after the insertion of a medical device that alters the distance between the dental arches, and was repeated on 3 different days. Results showed that masseter deformation is not uniform within the muscle. The same strain patterns were repeated in the different ultrasounds of the same patient and were modified after the introduction of a medical device. This was supported by quantitative comparisons in the deep portion of the muscle (standard deviation on the three measures: 3% normal conditions, 2% with medical device) showing a systematic reduction with the prosthesis (30% on average). This study demonstrated that masseter strain analysis is a repeatable and sensitive tool for the study of functional analysis of the masticatory organ. This opens new technical perspectives for the diagnosis and therapy of dysfunctional pathologies of the masticatory organ.

Cardiac mesoangioblasts are committed, self-renewable progenitors, associated with small vessels of juvenile mouse ventricle.

Different cardiac stem/progenitor cells have been recently identified in the post-natal heart. We describe here the identification, clonal expansion and characterization of self-renewing progenitors that differ from those previously described for high spontaneous cardiac differentiation. Unique coexpression of endothelial and pericyte markers identify these cells as cardiac mesoangioblasts and allow prospective isolation and clonal expansion from the juvenile mouse ventricle. Cardiac mesoangioblasts express many cardiac transcription factors and spontaneously differentiate into beating cardiomyocytes that assemble mature sarcomeres and express typical cardiac ion channels. Cells similarly isolated from the atrium do not spontaneously differentiate. When injected into the ventricle after coronary artery ligation, cardiac mesoangioblasts efficiently generate new myocardium in the peripheral area of the necrotic zone, as they do when grafted in the embryonic chick heart. These data identify cardiac mesoangioblasts as committed progenitors, downstream of earlier stem/progenitor cells and suitable for the cell therapy of a subset of juvenile cardiac diseases.

Fibrinogen induces adhesion, spreading, and microfilament organization of human endothelial cells in vitro.

Human umbilical vein endothelial cells (ECs) have been shown to attach to a substratum of fibrinogen (fg). Later, ECs undergo spreading, organization of thick microfilament bundles of the stress fiber type, and formation of focal contacts (adhesion plaques) that correspond to accumulation of vinculin at the cytoplasmic aspect of the ventral membrane. The rate of attachment to fg and the type of spreading is virtually identical to that obtained on substrata coated with fibronectin (FN). Antibodies to fg, but not to FN, prevent EC adhesion to fg; conversely, antibodies to FN, but not to fg, prevent adhesion of ECs to a FN-coated substratum. The removal of residual FN contamination from fg preparations by means of DEAE-cellulose chromatography does not result in any difference in EC adhesion on fg. Moreover, pretreatment of cells with inhibitors of synthesis and release of proteins does not impair their adhesion capacity on an fg-coated substratum. In contrast, human arterial smooth muscle cells do not adhere and spread on fg substrata but do so on FN. The synthetic peptides (Gly-Arg-Gly-Asp[GRGD] and Gly-Arg-Gly-Asp-Ser-Pro[GRGDSP]) containing the tripeptide Arg-Gly-Asp (RGD), originally found to be responsible for the cell binding activity of FN, have been found to inhibit EC spreading and the redistribution of their cytoskeleton, including the formation of stress fibers and the localization of vinculin either on fg or on FN. Conversely, the synthetic peptide Arg-Gly-Gly (RGG) was completely uneffective in inhibiting the adhesion and the sequence of events leading to spreading and cytoskeletal organization. These results indicate that ECs, but not smooth muscle cells, specifically adhere and spread on an fg substratum and this occurs by recognition mechanisms similar to those reported for FN.

Cultured human endothelial cells generate tissue factor in response to endotoxin.

Bacterial infection is associated with disseminated intravascular coagulation and fibrin deposition in the microcirculation; the mechanism of these effects in humans is still unclear. We have studied the generation of procoagulant activity (PCA) by cultured human endothelial cells (EC) in response to endotoxin. Cells from umbilical cord veins were grown in Eagle's minimum essential medium with 20% fetal calf serum till confluence. Absence of fibroblasts and macrophages was carefully checked. Endotoxin (Salmonella enteritidis lipopolysaccharide (LPS) W or Escherichia coli 0111:B4 LPS W, 0.01-1.0 micrograms/ml) was added to culture dishes for 4-6 h. PCA of EC was measured by a one-stage clotting assay and/or a two-stage amidolytic assay with the chromogenic substrate S-2222. In the absence of endotoxin, EC generated little, if any PCA (2-5 units/10(5) cells). In contrast, the addition of endotoxin resulted in generation of strong PCA that reached a maximum within 4-6 h (185-241 units/10(5) cells) and was dose-dependent between 1 and 0.01 microgram endotoxin/ml of culture medium. The generation of PCA required RNA and protein synthesis but did not require the presence of serum. No activity was found in the culture medium. The activity was of tissue thromboplastin type, as indicated by biological and immunological criteria. These endotoxin effects were observed in the absence of endothelial damage, as shown by phase-contrast microscopy and lack of 51Cr release. These data could contribute to elucidate the pathogenesis of vascular complications associated with endotoxemia in man.

Interaction between fibrinogen and cultured endothelial cells. Induction of migration and specific binding.

It has been suggested that fibrinogen (fg) or its physiological derivatives influence the motility and growth of endothelial cells (ECs), but direct support for this concept is still lacking. In the present study, the capacity of fg to interact with ECs and induce the migration of ECs was examined. The capacity of fg to induce EC migration was studied by means of a modification of the Boyden chamber technique. fg in the lower compartment of the chamber caused a time- and concentration-dependent migration of ECs across filters. fg present in equal concentrations above and below the filter increased EC migration, but the maximal effect invariably occurred in the presence of a gradient between the lower and the upper compartments. Trypsin or plasmin digestion of fg and preincubation of fg with Fab fragments from specific antibody completely abolished fg-induced EC migration. Dialysis of fg to eliminate small peptides that might contaminate the preparation did not modify fg-induced migration. Plasma obtained from healthy donors induced EC migration, but plasma from an afibrinogenemic patient was completely ineffective. The addition of purified fg to afibrinogenemic plasma restored plasma-induced EC migration. Plasmin degradation fragments D and E, of 100,000 and 50,000 mol wt, respectively, did not induce EC migration. However, fragment E caused dose-related inhibition of fg-induced EC migration Direct interaction of highly purified radioiodinated human fg with cultured human and bovine Ecs was observed. The binding was time dependent and plateaued at 10 min. Nonlabeled fg in a large molar excess inhibited the interaction, but unrelated proteins, including fibronectin, ovalbumin, and myoglobin, did not. Monospecific Fab fragments directed to fg inhibited binding by 38% at a 50 to 1 molar ratio whereas nonimmune Fab caused only 2% inhibition at a similar concentration. The binding of 125I-fg with ECs was saturable, and an apparent dissociation constant of 0.23 x 10(-6) M was estimated from binding isotherms. After 30 min of incubation the interaction between 125I-fg and the cells was completely reversible and displaceable by a large molar excess of unlabeled fg. Autoradiography of the display of EC-bound 125I on polyacrylamide gel showed the constitutive B beta- and gamma-chains of the fg molecule, with a partial loss of the A alpha-chain. Purified fragment E and E were tested for their capacity to inhibit fg binding. At a 1 to 400 125I-fg-to-fragment molar ratio, fragment E, which also inhibited migration, competed for binding by 44%, but fragment D was completely ineffective. These data show that fg may specifically associate with ECs and induce migration of these cells; it also appears that the structural requirement of this activity is located in the N-terminal part of the molecule.

Ultrasound and analysis of the deformation patterns of the masseter muscle: comparing surgical anatomy, ultrasound and functional anatomy.

PURPOSE: We have tried to demonstrate whether the analysis of the muscle strain allows us to identify the three distinct functional areas of the architecture of the masseter, as one would see them by performing or viewing an anatomical dissection of said muscle, and whether these sections have behave differently in terms of origin and coping of the strain they face (quantitative analysis). MATERIALS AND METHODS: This work has been elaborated by the use of an ultrasound machine (MicrUs ext-1H Telemed Medical Systems Milano) and a linear probe (L12-5l40S-3 5-12 MHz 40 mm) which allowed us to record a 45 frame per second video (DCM). Videos has been elaborated by use of an ultrasound machine (MicrUs ext-1H Telemed Medical Systems Milano) and a linear probe (L12-5l40S-3 5-12 MHz 40 mm) which allowed us to record a 45 frame per second video (DCM). We applied to the resulting video a software (Mudy 1.7.7.2 AMID Sulmona Italy) for the analysis of muscle deformation patters (contraction, dilatation, cross-plane, vertical strain, horizontal strain, vertical shear, horizontal shear, horizontal displacement, vertical displacement). The number of videos of masseter muscles in contraction at maximum exertion due to dental clenching made during this research is around 12,000. Out of these we chose 1,200 videos which examine 200 patients (100 females, 100 males). RESULTS: The deformation pattern analysis of the skeletal muscle on ultrasound basis seems to be an adequate instrument to use during the investigation of the functional structure of the masseter muscle given its ability to highlight the distinct activity of each separate part of the muscle. CONCLUSIONS: Moreover the strain does not apply to the muscle uniformly; instead it varies according to the observed area.

Management and control of isotonic contraction generated stress: evaluation of masseter muscle deformation pattern by means of ecography.

PURPOSE: The objective of the following study is to observe the behavior of the six layers of the masseter during an isometric contraction at maximum exertion with the deformation pattern analysis method. MATERIALS AND METHODS: This study has been conducted by use of an ultrasound machine (MicrUs ext-1H Telemed Medical Systems Milano) and a linear probe (L12-5l40S-3 5-12 MHz 40 mm) which allowed us to record a video (DCM) comprised of 45 frames per second. The probe was fixed to a brace and the patient was asked to clench their teeth as hard as possible, obtain the muscle's maximum exertion, for 5 seconds three times, with 30 seconds intervals in between. Both right and left masseter muscles were analyzed. Then we applied to the resulting video a software (Mudy 1.7.7.2 AMID Sulmona Italy) for the analysis of muscle deformation patterns (contraction, dilatation, cross-plane, vertical strain, horizontal strain, vertical shear, horizontal shear, horizontal displacement, vertical displacement). The number of videos of masseter muscles in contraction at maximum exertion due to dental clenching made during this research is around 12,000. Out of these we chose 1,200 videos which examine 200 patients (100 females, 100 males). RESULTS: The analysis of the deformation patterns of the masseter allows us to observe how the six layers of the muscle have different and specific functions each, which vary depending on the applied force (application point, magnitude and direction) so that we find it impossible to assign to one of the three sections of the muscle a mechanical predominance. Therefore it appears that the three parts of the muscle have specific and synergistic tasks.

Analysis of masseter deformation patterns during a maximum exertion clenching in patients with unilateral chewing.

PURPOSE: The aim of the following study is to examine both masseter muscles (left/right) in a group of patients suffering from unilateral chewing during a maximum exertion isometric contraction using the deformation pattern analysis of ultrasound videos and compare them with the results obtained by studying patients with alternate bilateral chewing patterns. MATERIALS AND METHODS: This study has been conducted by use of an ultrasound machine and a linear probe which allowed us to record a video (DCM) comprised of 45 frames per second (MicrUs ext-1H Telemed Medical Systems Milano) and a linear probe (L12-5l40S-3 5-12 MHz 40 mm). The probe was fixed to a brace and the patients were asked to clench their teeth as hard as possible, obtain the muscle's maximum exertion, for 5 seconds three times, with 30 seconds intervals in between. Both right and left masseter muscles were analyzed. We applied to the ultrasound video a dedicated software (Mudy 1.7.7.2 AMID Sulmona Italy) for the analysis of muscle deformation patterns. The total number of patients for this study is 150. Out of this number, 50 belong to Group A, mono lateral chewing on the left side arch, and 50 to Group B, mono lateral chewing on the right side arch. The remains patients belong to Group C, bilateral alternate chewing. The deformation pattern analysis of the skeletal muscles on ultrasound videos allows us to highlight with ease the clear difference in the clenching capabilities and strain management between the dominant masseter and the subordinate masseter in a unilaterally chewing patient. RESULTS: In the sample investigated both in Group A and Group B the unilateral chewing is associated with a series of parameters (number, shape, volume, position and orientation of the teeth) and is also associated with the extension of the cutting surface really available.

Prostacyclin production by human endothelial and bovine smooth muscle cells in culture. Effect of repeated stimulation with arachidonic acid, thrombin and ionophore A23187.

Prostacyclin (prostaglandin I2) is the major product of arachidonic acid metabolism in vascular cells. Its physiological role may be linked to the ability of the cells to respond continuously with prostaglandin I2 production to a variety of stimuli. We report that human endothelial cells or bovine smooth muscle cells in culture respond with prostaglandin I2 synthesis to a first but not to a second stimulation with arachidonic acid. The development of this refractoriness was independent of the arachidonic acid concentration used (6.6-25 microM) and lasted for about 6 h. The same time was required for the cells to recover completely after inhibition of cyclooxygenase activity by aspirin. Neither cis-polyunsaturated fatty acids (linoleic or oleic acids) nor stearic acid (a long-chain saturated fatty acid) prevented the generation of prostaglandin I2 by arachidonic acid. Similarly to arachidonic acid, thrombin and ionophore A23187 could elicit vascular prostaglandin I2 synthesis only once. Pretreatment of the cells with arachidonic acid rendered the cells unresponsive to any other stimulus. These results indicate that the mechanism of the refractoriness induced by arachidonic acid was different from that induced by the other stimuli. It is proposed that vascular cells cannot be stimulated continuously to produce prostaglandin I2, but this process is regulated by different feedback mechanisms.

Further studies on the mechanism of action of human plasma in stimulating prostacyclin production by rat smooth muscle cells.

Human normal plasma stimulates prostacyclin (PGI2) production by vascular cells. This plasma activity may be greatly modified in different pathological conditions. The purpose of this study was to characterize some aspects of the mechanism of action of plasma in modulating PGI2 release. Cultured rat aortic smooth muscle cells were used. Citrated plasma from healthy donors stimulated PGI2 production in a concentration-dependent way. Plasma-derived serum containing increasing concentrations of platelets had the same PGI2 stimulating activity as citrated plasma. Plasma stimulation of PGI2 production was accompanied by release of endogenously incorporated arachidonic acid (AA) from the cell membrane. Similarly to AA, plasma induced PGI2 synthesis only once, a second or third challenge producing a reduced response from the cells. Cells stimulated twice with plasma responded to AA like unstimulated cells while cells stimulated twice with AA were poorly responsive to subsequent stimulation with plasma. When the cells were repeatedly stimulated with AA in the presence of plasma no refractoriness was apparent. This study suggests that plasma increases PGI2 synthesis by the release of endogenous substrate from the cell membrane and by protecting the cells from self-inactivation during AA conversion to prostaglandins.

Medullary-thyroid carcinoma - clinical-evaluation of 19 cases.

Medullary thyroid carcinoma (MTC) accounts for 5-10% of thyroid malignancies and occurs either as a sporadic or as a familial form. The familial form is inherited in an autosomal dominant pattern, and it is clinically expressed as multiple endocrine neoplasia (MEN), types IIa and IIb or as familial MTC alone. It is possible to make an early diagnosis in patients who have the familial form of the disease as well as to perform an organ specific localisation regarding possible spread of the disease. Calcitonin is a major product of MTC cells and represent the most used tumour marker for diagnosis and evaluation of prognosis. The purpose of this investigation is to analyse our experience with patients treated for MTC in the period 1980-1993.

Primary HIV infection: molecular approaches in diagnosis and monitoring.

OBJECTIVE: The aim of this study was to evaluate, in patients with primary HIV infection (PHI), the modification of HIV molecular parameters (HIV, RNA, and DNA) induced by highly active antiretroviral therapy (HAART) in peripheral blood mononuclear cells (PBMC) and in lymphoid tissue (LNMC). METHODS: Nineteen patients with primary HIV infection, 4 women and 15 men with an average age of 35 years (range 27-62), were included in this study. Ten patients received 4 drugs: zidovudine plus lamivudine plus saquinavir plus ritonavir, 7 patients received 3 drugs: zidovudine plus lamivudine plus saquinavir and 2 patients received a different combination of 3 drugs: zidovudine plus lamivudine plus indinavir. As control group we included 8 patients who had been enrolled in a placebo-controlled trial of zidovudine between 1991 and 1995: four received placebo and 4 were treated with zidovudine alone. Peripheral blood samples and lymphoid tissue obtained by echo-driven fine needle biopsies were drawn to monitor molecular HIV parameters. A quantitative in house PCR method in the HIV gag region was used to monitor viral DNA burden and the NASBA system for viremia. RESULTS: A certain heterogeneity in the baseline values of HIV, DNA, and RNA was observed. Early HAART determined a rapid recovery of the CD4 cell number with normalisation of the CD4/CD8 ratio in most patients. HIV-RNA levels dropped to undetectable levels after a few months of therapy and HIV-DNA was consistently reduced although it never reached undetectable levels. Lymph-node biopsies were well tolerated due to the non-invasive sampling, however an optimisation of the method is needed to improve cell recovery. In the valuable samples the amount of HIV DNA recovered is comparable to that from peripheral blood samples, both at baseline and at follow-up.

Studies on the sensitivity of anticancer agents of normal human cells in culture.

Two biopsies of human periosteal tissue have been tested for sensitivity to several antitumoral agents. The tests were performed during the lifespan of the cultured cells in order to reveal possible variations in sensitivity. The results indicate that the age of the donor affects drug sensitivity; cells obtained from the young donor show a higher growth potential, together with a variable sensitivity and morphology during their life in vitro, while cells from the adult donor have a lower growth potential, a constant sensitivity to chemotherapy and a constant morphology.

Complications of muscle injuries.

Muscle injuries can be classified into strain injuries and contusions. Depending on the type of injury, different complications may occur, which in turn can be divided into early, intermediate and delayed complications. A prompt diagnosis of complications allows early treatment and permits to avoid harmful sequelae. Imaging studies, ultrasonography in particular, allow (recognizing) the assessment of complications whenever clinically suspected. In this article the most frequent complications of muscle injuries are presented.

Interventional ultrasonography of the chest: Techniques and indications.

Thoracic ultrasonography can be used for diagnostic purposes as well as a guide for diagnostic and therapeutic interventions.When the lesion or fluid collection has been located and the patient properly positioned, the angle of the needle is identified with respect to the transducer. The insertion tract should transgress the smallest possible area of aerated parenchyma. The needle can be introduced with a free-hand technique or with the aid of a needle guide. Correct planning of the procedure reduces intervention time and decreases the risk of complications.The main indications are superficial masses that require biopsy, pleural and parenchymal lesions formerly biopsied with CT or fluoroscopic guidance, and fluid collections that need to be drained.Ultrasound, thanks to its widespread use, simple execution, and low costs, represents a safe, manageable guide for thoracic interventions.