Study of proteolysis in river buffalo mozzarella cheese using a proteomics approach.

The guarantee of the origin and quality of raw material is essential for the protection and valorization of Campana buffalo mozzarella cheese. The risk of utilization of semifinished products and stored milk in substitution for fresh milk is increasing, due to the continuous desire to reduce production costs. A proteomics approach and electrophoresis survey of retail mozzarella cheeses indicated different rates of proteolysis in the production of dairy industries. The use of fresh milk and correct cheesemaking protocol yielded only γ-caseins, which are derived from ß-casein by plasmin, and para-κ-casein, which is derived from κ-casein by chymosin. The detection of abnormal hydrolysis resulting in ß- and αS1-casein fragments, identified by mass spectrometry, indicates the use of stored milk or stored and pressed curd, or the reuse of unsold mozzarella cheese, to produce mozzarella. The formation of γ-caseins and other fragments during a long storage of raw materials at room or refrigeration temperature was ascribed to plasmin (endogenous milk enzyme), whereas formation of αS1-casein fragments, mainly αS1-I(6P)- and αS1-I(7P)-casein during the storage of curd was ascribed to the action of chymosin (exogenous enzyme) from rennet. Sodium dodecyl sulfate-PAGE and alkaline urea-PAGE permitted us to evaluate the freshness of the raw materials used in the manufacturing of buffalo mozzarella cheese and to reveal possible inappropriate preservation.

Physico-chemical features of the environment affect the protein conformation and the immunoglobulin E reactivity of kiwellin (Act d 5).

BACKGROUND: Allergy diagnostic systems sometimes give false positive or negative results. In this respect, the influence of protein conformational changes on the allergen-IgE interaction sites is worthy to be investigated. OBJECTIVE: To investigate the influence of different experimental conditions on the structural properties and IgE reactivity of kiwellin (Act d 5) as a model system. METHODS: Act d 5 was purified from the natural source. To study its conformational features, experiments of circular dichroism (CD) in different media were performed. The IgE reactivity was investigated by skin testing, immunoblotting and ISAC microarray system, in a population of kiwifruit allergic subjects. RESULTS: CD experiments indicated that Act d 5 has a mainly helical structure and the conformation is strongly affected by the experimental conditions. The protein is more structured in low polarity media and at acidic pH values, similar to those of the natural source. Eleven subjects of 29 (38%) allergic to kiwifruit were positive to purified natural Act d 5 by skin test. Among them, three patients (10%) showed a reaction only to Act d 5 at pH 4.5, and three (10%) showed a reaction only to the allergen in standard neutral conditions. No one of the 11 subjects with positive skin test recognized Act d 5 immobilized on the ISAC system. Eight of nine subjects detected Act d 5 by IgE immunoblotting. One subject did not recognize the sequence epitopes of Act d 5 in IgE immunoblotting experiments and reacted to the skin test only when the allergen was in acidic conditions. CONCLUSIONS AND CLINICAL RELEVANCE: The conformation and IgE reactivity of Act d 5 are affected by the physico-chemical characteristics of the solvent. These findings suggest that the assay conditions influence the results of the diagnostic systems by modulating the pattern of exposed antigenic epitopes.

Cortical metabolic changes and clinical outcome in normal pressure hydrocephalus after ventriculoperitoneal shunt: Our preliminary results. / Cambios metabólicos corticales y resultado clínico en la hidrocefalia normotensiva después de la derivación ventrículo-peritoneal: nuestros resultados preliminares.

INTRODUCTION: Our objective was to evaluate the cortical metabolic changes and clinical outcome in patients affected by idiopathic normal pressure hydrocephalus (iNPH) after a placement of ventriculoperitoneal (VP) shunt. MATERIALS AND METHODS: 10 patients affected by suspected iNPH underwent a CSF hydrodynamics evaluation based on a lumbar infusion test (LIT). The main selection criterion for surgery was based on intracranial elasticity (IE)>0.30. All subjects with an IE>0.30 underwent a PET scan with 18 fluorodeoxiglucose (18F-FDG) at baseline (PET1) and 1 month after surgery (PET2). Furthermore, the same patients were submitted to clinical evaluation before and 1 month after surgery through neuropsychological tests and gait analysis. RESULTS: An overall number of 20 18F-FDG PET scans were performed in all the enrolled patients. As compared to PET1, PET2 showed an increase in glucose consumption in the left frontal and left parietal lobe in PET2 as compared to PET1 (P<.001). All the enrolled patients presented a significant increase in neuropsychological scores (i.e Frontal Assessment Battery and Montreal Cognitive Assessment) and have clinically improved at gait analysis. A significant correlation was found between the increase of cortical glucose consumption in the left parietal area and the cognitive improvement as detectable by neuropsychological assessment. CONCLUSIONS: Improvement in 18F FDG PET glucose metabolism could be considered a useful imaging marker for the assessment of iNPH response to VP shunting.

Pathological classification of DCIS and planning of therapeutic management.

BACKGROUND: Ductal intraepitelial neoplasia (DIN) represents a spectrum of disease that may progress from usual hyperplasia to ductal carcinoma in situ (DCIS) grade 3. The aim of the study was to asses the correlation between the DIN classification and the surgical treatment including sentinel lymph node biopsy (SLNB). PATIENTS AND METHODS: In this retrospective study, 229 patients with DIN had undergone conservative or radical surgical treatment and SLNB in cases of DIN1C-DIN3. RESULTS: Breast conservative surgery was the definitive treatment in 80% of the cases. The H&E evaluation of excised sentinel nodes was negative for metastatic disease; nevertheless the immunohistochemical (IHC) evaluation revealed the presence of metastatic cells in 6 patients (3.7%). CONCLUSION: In cases of DIN lesions SLNB is not indicated. The only reason SLNB should be considered is when there is an evidence of invasive foci at definitive histology or when radical mastectomy is proposed.

In vivo assessment of hydrocephalus shunt.

OBJECTIVES: Over a 3-year period, we have performed 312 tests in 197 shunted patients. The data have been analyzed retrospectively to: (1) investigate the parameters describing CSF dynamics that correlate with shunt under-drainage and (2) estimate accuracy of this method. METHODS: Constant rate infusion tests into shunt prechamber were performed. RESULTS: In 161 of the 312 infusion tests, results indicated under-draining shunts. Patients in the under-draining group had higher baseline and plateau CSF pressures, higher resistance to CSF outflow and higher levels of baseline pulse amplitude waveform. During the test, a significantly greater vasogenic waves and lower compensatory reserve was noticed in patients with blocked shunts. In 21 patients with suggestion of shunt blockage and who subsequently underwent operative revision of the shunt, reports of intraoperative shunt patency were available. Shunt blockage was confirmed intra-operatively during surgery in 19 cases. CONCLUSIONS: In vivo shunt testing is easy, safe and clinically useful, aiding decision in difficult clinical situations, where shunt malfunction is suspected but not certain. It also has satisfactory positive predictive power.

Cerebral blood flow autoregulation during intracranial hypertension: a simple, purely hydraulic mechanism?

OBJECTIVE: In this paper, we re-propose the role of a hydraulic mechanism, acting where the bridging veins enter the dural sinuses in cerebral blood flow (CBF) autoregulation. MATERIALS AND METHODS: We carried out an intraventricular infusion in ten albino rabbits and increased intracranial pressure (ICP) up to arterial blood pressure (ABP) levels. We measured CBF velocity by an ultrasound probe applied to a by-pass inserted in a carotid artery and recorded ICP by an intraventricular needle. Diastolic and pulsatile ICP and ABP values were analyzed from basal conditions up to brain tamponade and vice versa. CONCLUSIONS: A biphasic pattern of pulsatile intracranial pressure (pICP) was observed in all trials. Initially, until the CBF velocity remained constant, pICP increased (from 1.2 to 5.4 mmHg) following a rise in diastolic intracranial pressure (dICP); thereafter, in spite of a further rise in dICP, pICP decreased (2.87 mmHg) following CBF velocity reduction until intracranial circulation arrest (pICP=1.2 mmHg). A specular pattern was observed when the intraventricular infusion was stopped and CBF velocity returned to basal levels. These findings can be interpreted as indicating a hydraulic mechanism. Initially, when CBF is still constant, pICP rise is due to an increase in venous outflow resistance; subsequently, when CBF decreases following a further increase in venous outflow resistance, the vascular engorgement produces an arteriolar vasodilation. This vasodilation determines an increase in vascular wall stiffness, thus reducing pulse transmission to surrounding subarachnoid spaces.

How does CSF dynamics change after shunting?

OBJECTIVE: Hydrocephalus is much more complex than a simple disorder of cerebrospinal fluid (CSF) circulation. Shunting primarily corrects disturbed fluid flow which may have an impact on cerebral blood flow and metabolism. We studied hydrocephalic patients before and after shunting to characterize changes in their CSF compensatory parameters. MATERIAL AND METHODS: We selected 25 patients and studied them retrospectively. All patients had ventriculomegaly and clinical symptoms of normal pressure hydrocephalus. After shunting, they were still presenting with some adverse symptoms, mainly headaches, slow improvement or no improvement of ventriculomegaly. Therefore, they underwent further infusion studies to assess shunt function. In all cases, the shunts were confirmed to be draining CSF adequately. Parameters of CSF dynamics: baseline intracranial pressure (ICP), resistance to CSF outflow, cerebrospinal elasticity, content of vasogenic pressure waves (pulse, respiratory and B waves) and compensatory reserve assessed as moving correlation coefficient between mean CSF pressure and pulse amplitude (RAP), were compared before and after shunting. RESULTS: Mean ICP and resistance to CSF outflow decreased (P < 0.003) after shunting. All vasogenic pressure waves decreased (P < 0.005). Compensatory reserve (RAP) significantly improved (P < 0.005). CONCLUSION: A functioning shunt has an important impact on CSF circulation and pressure-volume compensation. Infusion studies can demonstrate the return of disturbed CSF dynamics to normal values even if clinical or radiological changes are not dramatic.

Anesthesia and analgesia in breast surgery: the benefits of peripheral nerve block.

Breast surgery is frequently associated with postoperative pain, nausea and vomiting, that result in increased patient's suffering, prolongation of hospital stays and related costs. Thoracic paravertebral nerve block (TPVB) is a viable option to the classic multimodal analgesia in breast surgery as it enhances surgical anesthesia and postoperative analgesia. In this review, we report the results of a number of studies on the role of TPVB in breast surgery. This technique is associated with a superior control of the pain, a reduction in opioids consumption after surgery, a decrease in postoperative nausea and vomiting, and an overall decrease in length of hospital stay. In particular, TPVB seems to provide the most benefits in patients undergoing an unilateral or bilateral mastectomy followed by immediate reconstruction. Some studies also suggest that the use of regional anesthesia-analgesia could attenuate perioperative immunosuppression and minimize metastases in breast cancer patients. TPVB can be also coupled with other regional anesthetic techniques such as pectoral nerve block (PNB), thus increasing the reduction in postsurgical pain, opioids consumption and length of hospital stays.

Stage IV Kienböck's disease: Proximal row carpectomy and application of RCPI implant.

Kienböck disease is an aseptic osteonecrosis of the lunate, which gradually leads to total carpal collapse. Lichtman's classification relates radiographic evidence of wrist damage to four different stages and supports surgical decision-making. This study pertains to six patients (2 males and 4 females) affected by stage IV Kienböck's disease who underwent proximal row carpectomy and received an RCPI implant. The clinical outcomes consisted of wrist range of motion (ROM), pain on a VAS scale (0-10), the DASH score and the patient's level of satisfaction. The mean follow-up was 27.6 months (16-36). Pain relief and improvements in wrist flexion - extension ROM, radial - ulnar deviation and strength were achieved in every patient. There were no cases of implant failure or dislocation. Considering the good results obtained, we believe that proximal row carpectomy associated with the use of a pyrocarbon RCPI implant is a valid surgical technique for the treatment of stage IV Kienböck's disease. It is a good alternative to carpal fusion, which leads to wrist immobility, and to total wrist joint replacement, which has a high incidence of dislocation and fracture.

An arthroscopic-assisted minimal invasive method for the reconstruction of the scapho-lunate ligament using a bone-ligament-bone graft.

UNLABELLED: We report the outcomes of an arthroscopic-assisted minimally invasive technique to reconstruct the scapho-lunate ligament using a bone-ligament-bone graft in 11 patients (11 wrists). The mean follow-up time was 29 months (range 20 to 38). The preoperative mean wrist flexion, extension, grip strength and patient-rated wrist evaluation score values were 61°, 54°, 115 N and 54, respectively. The postoperative mean values were 64°, 58°, 142 N and 15, respectively. There were no statistical differences between the pre- and postoperative wrist flexion and extension, whereas changes in grip strength and patient-rated wrist evaluation score were significant. Scapho-lunate angles decreased significantly from 69° to 60°. Based on our clinical outcomes, this method provides a reliable alterative for the reconstruction of the scapho-lunate ligament in patients with persistent Geissler type 3 and 4 lesions in the short-term. LEVEL OF EVIDENCE: IV.

Comparative study of oncoplastic versus non-oncoplastic breast conserving surgery in a group of 211 breast cancer patients.

OBJECTIVE: Breast conserving surgery (BCS) followed by radiotherapy is the standard of care for most patients with early breast cancers; however, the aesthetic outcomes of this procedure is not always desirable. Oncoplastic breast surgery is an innovative approach that aims at the safe and effective treatment of the cancerous lesion while achieving the best possible aesthetic outcome. The aim of this study was to evaluate and compare oncoplastic and non-oncoplastic procedures. PATIENTS AND METHODS: A retrospective observational study was conducted on a group of patients who underwent oncoplastic or non-oncoplastic breast surgery at the Breast Unit of the University of Rome Tor Vergata. RESULTS: Out of 211 patients, 154 (73%) underwent non-oncoplastic surgery, while 61 (27%) underwent an oncoplastic procedure. The percentage of patients requiring re-excision was twice greater for women in the non-oncoplastic group: 12.9% vs. 6.5% in the oncoplastic group. The rate of complications was higher in the oncoplastic group compared to the non-oncoplastic one (4.9% vs. 1.3%). The size of the tumor, the number of the patients receiving post-operative radiation, the interval between surgery and radiotherapy and follow-up were similar between the two groups. CONCLUSIONS: Oncoplastic surgery represents a valid alternative to breast conserving surgery for patients with breast cancer, with only minimal differences in long-term complications and similar results in terms of local recurrence and number of metastasis.

Microsatellite instability is correlated with lymph node-positive breast cancer.

We analyzed 81 cases of primary breast carcinoma and 7 cases of fibroadenoma for microsatellite instability at eight loci. Twenty-seven cases (33.3%) manifested aberrant microsatellite alleles: 7 (8.6%) at one locus and 20 (24.7%) at two or more loci [tumors with replication error-positive (RER+) phenotype]. No evidence of microsatellite instability was observed in fibroadenomas. We investigated correlations between RER+ phenotype and clinicopathological characteristics of the carcinomas. The RER+ phenotype was statistically associated with large tumor diameter; of 19 RER+ tumors with measured size, 16 were > 2 cm, compared to 28 of 58 tumors with no evidence of microsatellite instability or with shifts in allele sizes limited to one locus (P

Measurement of neovascularization is an independent prognosticator of survival in node-negative breast cancer patients with long-term follow-up.

We measured neovascularization, epidermal growth factor receptor, and c-erbB-2 expression in a consecutive series of 233 surgically resected axillary lymph node-negative breast cancer patients with a long-term follow-up to define the usefulness of these parameters as independent prognostic indicators of overall survival (OAS). Microvessel count (MVC), as a measure of neovascularization, was determined using a monoclonal antibody against human factor VIII-related antigen. The median MVC of 20 (range, 4-76) was used as a cutoff value for discriminating between low and high vascularized tumors. Epidermal growth factor receptor and c-erbB-2 expression were evaluated by immunohistochemistry. Tumors were considered positive if >10% of the cells showed specific membrane staining. OAS curves were estimated by the Kaplan-Meier method. The independent prognostic effect of each variable was determined with the Cox proportional hazards model. High MVC (P = 0.04), high nuclear grade (P = 0.005), and high S-phase (P = 0.02) significantly affected OAS at univariate analysis. In a Cox multivariate analysis, the characteristics with an independent prognostic effect on OAS were: MVC (relative hazard, 2.12; 95% confidence interval, 1.18-3.81; P = 0.01) and nuclear grade (relative hazard, 2.83; 95% confidence interval, 1.12-7.17; P = 0.01). These results demonstrate that quantification of neovascularization adds useful independent prognostic information on survival in node-negative breast cancer patients with long-term follow-up.

Intraperitoneal insulin absorption after long-term intraperitoneal insulin therapy.

OBJECTIVE: To evaluate insulin absorption through the peritoneal membrane after long-term intraperitoneal insulin therapy using an implanted programmable device. RESEARCH DESIGN AND METHODS: Seven insulin-dependent diabetes mellitus (IDDM) patients implanted with a programmable pump were studied after 3 and 30 months of intraperitoneal insulin therapy. A 20-min square wave infusion of 15 IU of insulin was administered in the peritoneal space, and plasma glucose and plasma free insulin levels were monitored for 180 min. Hypoglycemia was prevented by intravenous glucose infusion. RESULTS: After 30 months of intraperitoneal insulin therapy, plasma free insulin profiles following the administration of insulin in the peritoneal space were similar to those observed at the beginning of this mode of therapy. CONCLUSIONS: In IDDM patients, intraperitoneal insulin absorption does not change after long-term intraperitoneal insulin therapy using an implanted device.

Free-insulin profiles after intraperitoneal, intramuscular, and subcutaneous insulin administration.

The aim of our study was to compare the time course of plasma free-insulin appearance after injection of equal amounts of insulin into the peritoneal cavity above and below the transverse mesocolon, intramuscularly, and subcutaneously. Seven nondiabetic subjects undergoing cholecystectomy received in random sequence 0.2 IU/kg of insulin into the peritoneal cavity above or below the transverse mesocolon. Concentrations of plasma free insulin were compared with those obtained from seven other nondiabetic subjects after repeated injections of equal amounts of insulin intramuscularly and subcutaneously. Intraperitoneal insulin above the transverse mesocolon yielded a faster rise of free insulin, peaking at 15 min, whereas intraperitoneal insulin below the transverse mesocolon produced a somewhat slower rise, peaking at 30 min. The area under the curves between 0 and 15 min was greater after the injection above than below the transverse mesocolon (P less than .05). Intramuscular and subcutaneous insulin injections resulted in a slower rise of plasma free insulin, peaking at 60 and 90 min, respectively. We conclude that the pattern of insulin appearance in the plasma resembles more closely physiologic events after intraperitoneal than after subcutaneous or intramuscular insulin administration.

Peritoneal and subcutaneous absorption of insulin in type I diabetic subjects.

We and others have shown that in type I diabetes, ip insulin delivery results in lower free insulin levels than sc delivery. The aim of this study was to compare the rate of appearance of insulin in the peripheral circulation during ip and sc insulin administration in type I diabetes, in steady state and nonsteady state. To do this, we determined free insulin levels during ip or sc infusion as well as the impulse response of the insulin system after iv injection of a 6-nmol bolus of insulin. Twelve hours after a constant basal insulin infusion (5.5 +/- 1.4 nmol/h) was started, five C-peptide-negative type I diabetic subjects showed a lower systemic rate of appearance of insulin (expressed as a percentage of the administered dose) with ip than sc administration (27 +/- 6% vs. 40 +/- 10%; P < 0.001). In nonsteady state, when the infusion rate was increased from basal to 15 nmol/h (0-150 min) and subsequently to 42 nmol/h (150-300 min), the percent increase in insulin's systemic rate of appearance was higher with ip than sc infusion (P < 0.05 from 60-150 min; P < 0.01 from 150-300 min), indicating faster absorption. Thus, we conclude that insulin is more rapidly absorbed from the peritoneal cavity than from sc tissue. However, with ip administration, a sizable amount of insulin, once absorbed, is extracted before reaching the peripheral circulation, most likely by the liver. This is indirect evidence that ip insulin delivery results in a portal-peripheral insulin gradient in humans.

Expression of epitopes of the tumour-associated glycoprotein 72 and clinicopathological correlations in mammary carcinomas.

We analysed the immunohistochemical expression pattern of the distinct carbohydrate epitopes of the TAG-72 molecule, defined by the monoclonal antibodies (MAbs) B72.3, CC-49 and CC-83, in 92 breast carcinomas of different histological type, and in other histological components identified in the mammary tissue samples studied. The results were correlated with the clinico-pathological characteristics of the tumours, and with their proliferative activity, assessed by thymidine labelling index (TLI). Expression of the TAG-72 epitopes was detected in all the tumour histotypes analysed, but patterns of immunoreactivity tended to vary in relation to type and level of differentiation. The comparative analysis of the reactivities of the three anti-TAG-72 MAbs revealed differences in their ability to recognise neoplastic lesions. MAb CC-49 reacted with the highest percentage of tumours (82%), and also tended to yield the highest percentages of immunoreactive cancer cells, while B72.3 and CC-83 reacted with lower percentages of tumours (respectively, 55 and 51%), and identified lower percentages of immunoreactive cells. High levels of expression of the three TAG-72 epitopes were detected in areas of in situ ductal carcinoma. Comparatively lower levels of immunohistochemical positivity were found in atypical epithelial hyperplasia, normal mammary epithelium and epithelium with cystic disease. TAG-72 epitope expression was correlated with prognostic parameters. The synchronous expression of the three epitopes significantly correlated with large tumour size (> 2 cm), and with high histological grade. No correlations could be demonstrated between TAG-72 phenotypes and nuclear grade, lymph node status and proliferative activity (high versus low).

Selecting high-risk early breast cancer patients: what to add to the number of metastatic nodes?

High-risk early breast cancer patients are usually identified by the number of metastatic axillary nodes. To study whether other easily and inexpensively detectable morphological factors are able to detect high-risk patients, we performed a retrospective analysis of tumor size, and skin/fascia and nipple invasion. The data consisted of 941 node-positive cases registered between 1978 and 1991. Tumour size, and skin/fascia and nipple invasion were closely associated with the number of metastatic nodes (chi 2 test). The number of metastatic nodes, tumour size, skin/fascia and nipple invasion significantly affected disease free survival (DFS) and overall survival (OS) at univariate analysis. These results were confirmed by multivariate analysis with a model containing the number of metastatic nodes, tumour diameter categories, skin/fascia invasion, nipple invasion and adjuvant therapy as covariates: all variables significantly and independently affected risk of relapse and of death. All the variables studied were prognostic, within individual nodal categories, for both DFS and OS. In conclusion, the number of metastatic nodes is not the only prognostic tool with which to select high-risk patients for new intensive adjuvant programmes. Tumour size, and skin/fascia invasion or nipple invasion, taken singly or combined, are valuable prognostic factors that can identify patients with few metastatic nodes and poor outcome. On the basis of our data, we believe that a reconsideration of the pT4 category within the pTNM classification is in order, that is, chest wall invasion should be substituted by fascia invasion, and combined skin/fascia invasion could be a subcategory of each class defined by tumour size.