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Necrotizing fasciitis is one of the most common soft tissue infections, with a high risk of major amputation and a mortality ranging from 6 to 33% which has not changed in the past 20 years. Early surgical resection of necrotic tissue plays a key role in determining the prognosis. Nawijn et al. identified an optimal 6 hours window from presentation to surgery. Symptoms of necrotizing fasciitis mimic those of common skin infections, such as erysipelas and cellulitis, making rapid surgical management difficult. In this context, the aid of point-of-care-ultrasound is a valuable tool for early diagnosis, detecting the presence of subcutaneous thickening, gas and perifascial liquid. Other characteristic ultrasound findings include the "cobblestone" appearance of the subcutaneous soft tissues and reverberation artifacts due to hyperechoic outbreaks, defined as "snow globes" due to the presence of heterogeneous swirling material, and "dirty shadowing" due to the foggy shadow created by the gas., (© 2023. Società Italiana di Ultrasonologia in Medicina e Biologia (SIUMB).)

Background: The vagus nerve (VN), also called the pneumogastric nerve, connects the brainstem to organs contained in the chest and abdomen. Physiologically, VN stimulation can rapidly affect cardiac activity and heart rate (HR). VN neuropathy can increase the risk of arrhythmias and sudden death. Therefore, a selective test of VN function may be very useful. Since peripheral neurodynamic tests (NDT) are reliable for the assessment of neuropathies in somatic nerves, we aimed to validate a novel NDT to assess VN activity, namely, the VN-NTD., Methods: In this cross-sectional double-blind, sex-balanced study, 30 participants (15 females) completed a checklist of autonomic dysfunction symptoms. During the VN-NDT administration, HR and symptoms (i.e., mechanical allodynia) were monitored in parallel to a real-time ultrasonography imaging (USI) and motion capture analysis of the neck. The VN-NDT impact on HR and its accuracy for autonomic symptoms reported in the last 7 days were tested., Results: The VN-NDT induced a significant HR reduction of about 12 and 8 bpm in males and females [ t (1, 119) = 2.425; p < 0.017; η p = 0.047, 95% confidence interval (CI): 0.93-9.18], respectively. No adverse events were observed during VN-NDT. A substantial interexaminer agreement between the evaluators in symptoms induction by VN-NDT was detected [ 2 = 0.047, 95% confidence interval (CI): 0.93-9.18], respectively. No adverse events were observed during VN-NDT. A substantial interexaminer agreement between the evaluators in symptoms induction by VN-NDT was detected [ F (1, 119) = 0.540; p = 0.464; η p 2 = 0.005, low effect]. Notably, mechanical allodynia accuracy for gastrointestinal dysfunctions was excellent ( p < 0.05; 95% CI: 0.52-0.73; p < 0.001; 95% CI: 0.81-0.96)., Conclusions: The novel VN-NDT is a valid and accurate test capable of detecting VN activation with high sensitivity. Data provided are suitable for both sexes as a hallmark of HR variation due to VN normal response. The proposed VN-NDT may be reliable as daily routine neurological examination tests for the evaluation of neuropathic signs related to neuroinflammation of the VN., Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT04192877., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Carta, Seregni, Casamassima, Galli, Geuna, Pagliaro and Zago.)

Purpose: Increasing pressure pushes towards the objective competence assessment of clinical operators. Hand motion analysis (HMA) was introduced to measure surgical and clinical procedures; its recent application to FAST examinations leaves unsolved issues. This study aimed at determining optimal HMA parameters to discriminate between operators' skill levels, and which FAST tasks are experience-dependent., Methods: Ten experienced (EG) and 13 beginner (BG) sonographers performed a FAST examination on one female and one male model. A motion capture system returned the duration, working volume, number of movements (absolute and time normalized), and hand path length (absolute and time normalized) of each view., Results: BG took more time in completing specific views, with a higher working volume (p = 0.003) and longer hands path (p < 0.001). The number of movements was lower in the EG (p < 0.001) and differed between views (p = 0.014). No significant Group/Model differences were found for the normalized number of movements. The LUQ view required a higher number of movements (p < 0.001)., Conclusions: HMA identified kinematic parameters discriminating between proficiency level and critical subtasks in the FAST examination. These findings could be the base for a focused HMA-based evaluation of performances following a proctored training period. There is room to incorporate HMA into simulation metrics and evidence-based credentialing standards for clinical ultrasound applications.

Background: Acute cholecystitis (AC), frequently responsible for presentation to the emergency department, requires expedient diagnosis and definitive treatment by a general surgeon. Ultrasonography, usually performed by radiology technicians and reported by radiologists, is the first-line imaging study for the assessment of AC. Targeted point-of-care ultrasound (POCUS), particularly in the hands of the treating surgeon, may represent an evolution in surgical decision-making and may expedite care, reducing morbidity and cost., Methods: This consensus guideline was written under the auspices of the European Society of Trauma and Emergency Surgery (ESTES) by the POCUS working group. A systematic literature search identified relevant papers on the diagnosis and treatment of AC. Literature was critically-appraised according to the GRADE evidence-based guideline development method. Following a consensus conference at the European Congress of Trauma & Emergency Surgery (Valencia, Spain, May 2018), final recommendations were approved by the working group, using a modified e-Delphi process, and taking into account the level of evidence of the conclusion., Recommendations: We strongly recommend the use of ultrasound as the first-line imaging investigation for the diagnosis of AC; specifically, we recommend that POCUS may be adopted as the primary imaging adjunct to surgeon-performed assessment of the patient with suspected AC. In line with the Tokyo guidelines, we strongly recommend Murphy's sign, in conjunction with the presence of gallstones and/or wall thickening as diagnostic of AC in the correct clinical context. We conditionally recommend US as a preoperative predictor of difficulty of cholecystectomy. There is insufficient evidence to recommend contrast-enhanced ultrasound or Doppler ultrasonography in the diagnosis of AC. We conditionally recommend the use of ultrasound to guide percutaneous cholecystostomy placement by appropriately-trained practitioners., Conclusions: Surgeons have recently embraced POCUS to expedite diagnosis of AC and provide rapid decision-making and early treatment, streamlining the patient pathway and thereby reducing costs and morbidity.

For a long time surgeons have been discussing the need to improve their skills in the use of ultrasound (US). However in the recent years it has become evident the importancxe for general aklnd trauma surgeons treating critically-ill patients to learn basic and advanced US. The two last editions (9th and 10th) of the ATLS manual have officially included FAST and e-FAST in the primary assessment of trauma patients, making this tool an essential skill for surgeons. In the acute care setting FAST, e-FAST and other applications have gained a pivotal, evidence-based role in this fields. Nevertheless, surgeons are rarely performing US exams by themselves, losing a major decision-making tool. The Modular Ultrasound ESTES Course (MUSEC®) was developed to provide both fundamental and advanced US training for surgeons in trauma and acute care settings. We are strongly convinced, in the light of the results from both the surveys carried out and the customer satisfaction tests administered to all the participants in the MUSEC courses, that US courses such as these should be part of the general surgery residency programs. KEY WORDS: e-FAST, MUSEC Ultrasound in Emergency Department, Ultrasound Training Trauma Patients.

Background/aim: Colorectal Cancer is the fourth most frequent cause of cancer death worldwide and its incidence increases from 50 years of age. It is often associated with protein-caloric malnutrition and 20% of cancer deaths occur due to this event. The aim of this study was to assess the prevalence of malnutrition and inflammatory status in 78 patients undergoing surgery for colorectal carcinoma., Patients and Methods: Nutritional Status was assessed by Mini Nutritional Assessment (MNA). Serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by ELISA, while albumin, C-reactive protein (CRP) and transferrin (TRF) were tested using an immunometric assay., Results: The mean MNA score in colorectal patients was 20.4±8.4, while 23/78 patients (29.4%) were well nourished, 36/78 (46.1%) were at risk of malnutrition and 19/78 (24.3%) were malnourished, reporting in the previous six months from the date of diagnosis a significant weight loss (>10 kg), muscle mass loss and severe reduction of food intake due to loss of appetite and altered taste perception. The serum means of IL-6, TNF-α and CRP, were significantly higher in colorectal patients compared to the control group (p<0.001, p<0.0001, p<0.0001, respectively) while lower TRF, albumin and HCT serum levels in cancer patients vs. healthy subjects (p<0.0001; p<0.0001 and p<0.0001) were found., Conclusion: more than 50% of colorectal cancer patients were malnourished or at risk of malnutrition and reported an imbalance between nutritional and inflammatory status. They, therefore, require a nutritional intervention before treatment in order to have a more effective response and improve quality of life., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Matrix metalloproteinases are a family of zinc endopeptidases with proteolytic activity against the extracellular matrix components. In particular, two members of this family named Gelatinase A and B, as amply documented in the literature, play a key role in the process of tumor growth/metastasis in breast and hepatocellular carcinoma. Their activity is regulated by Tissue Inhibitor of metalloproteinases-1 and -2, which are the physiological inhibitor of Gelatinases A and B respectively. The aim of this review is to determine the current understanding of the clinical and prognostic role of Metalloproteinases-2 and -9 and their inhibitors in the course of breast cancer and liver diseases. Forty-one articles were selected from PubMed by entering the following keywords: liver diseases, breast cancer, MMP-2, TIMP-2; all articles were read and notes were made regarding the number of enrolled patients, pathology, measures, results and these data were used to write this review. Over-expression of both gelatinases is associated with the relapse of disease, metastasis, shorter overall survival in breast cancer and hepatocellular carcinoma and invasion and progression to tumors in chronic liver diseases, and MMPs/TIMPs ratio could be useful in the follow-up of these patients., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Purpose: The European Society for Trauma and Emergency Surgery (ESTES) identified the need for general and trauma surgeons involved in the management of critically ill surgical patients to embrace and learn both basic and advanced US skills. A steering group was created to address this deficit., Methods: Modular UltraSound ESTES Course (MUSEC) is a modular blended-learning course. It incorporates pre-test/post-test examinations, pre-course online materials, didactic and interactive lectures, interactive case scenarios discussion with pathological US clips, hands-on practice on healthy volunteer models, and on original phantoms for simulating both pathological US findings and practicing US-guided interventional maneuvers. Four independent modules were provided. Surgical decision-making didactics were also included in the course curriculum. Learning gain (Δ of the rating of pre-test and post-test) was calculated for each module. An anonymous post-course satisfaction survey was also administered (16 questions with a Likert's 5-point scale of evaluation)., Results: Twenty-three MUSEC Courses were run in a 30 months period, training 416 doctors from 29 countries. A total of 52 modules were delivered. The mean pre-test and post-test grades were 8.3/12 and 10.7/12, respectively, yielding a significant mean learning gain of 28.9 % (p = 0.001). Post-course satisfaction survey got an overall ranking of 4.5/5., Conclusions: MUSEC is an effective and original educational format, enjoyed by candidates, that fills an educational gap for tailored US education as a procedural skill to acute care surgeons. Ongoing revisions should reduce the current limitations and increase the educational value, in terms of number of modules and post-course credentialing.

Purpose: Acute mesenteric ischaemia (AMI) accounts for about 1:1000 acute hospital admissions. Untreated, AMI will cause mesenteric infarction, intestinal necrosis, an overwhelming inflammatory response and death. Early intervention can halt and reverse this process leading to a full recovery, but the diagnosis of AMI is difficult and failure to recognize AMI before intestinal necrosis has developed is responsible for the high mortality of the disease. Early diagnosis and prompt treatment are the goals of modern therapy, but there are no randomized controlled trials to guide treatment and the published literature contains a high ratio of reviews to original data. Much of that data comes from case reports and often small, retrospective series with no clearly defined treatment criteria., Methods: A study group of the European Society for Trauma and Emergency Surgery (ESTES) was formed in 2013 with the aim of developing guidelines for the management of AMI. A comprehensive literature search was performed using the Medical Subject Heading (MeSH) thesaurus keywords "mesenteric ischaemia", "bowel ischaemia" and "bowel infarction". The bibliographies of relevant articles were screened for additional publications. After an initial systematic review of the literature by the whole group, a steering group formulated questions using a modified Delphi process. The evidence was then reviewed to answer these questions, and recommendations formulated and agreed by the whole group., Results: The resultant recommendations are presented in this paper., Conclusions: The aim of these guidelines is to provide recommendations for practice that will lead to improved outcomes for patients.

In animals, the protein kinase C (PKC) family has expanded into diversely regulated subgroups, including the Rho family-responsive PKN kinases. Here, we describe knockouts of all three mouse PKN isoforms and reveal that PKN2 loss results in lethality at embryonic day 10 (E10), with associated cardiovascular and morphogenetic defects. The cardiovascular phenotype was not recapitulated by conditional deletion of PKN2 in endothelial cells or the developing heart. In contrast, inducible systemic deletion of PKN2 after E7 provoked collapse of the embryonic mesoderm. Furthermore, mouse embryonic fibroblasts, which arise from the embryonic mesoderm, depend on PKN2 for proliferation and motility. These cellular defects are reflected in vivo as dependence on PKN2 for mesoderm proliferation and neural crest migration. We conclude that failure of the mesoderm to expand in the absence of PKN2 compromises cardiovascular integrity and development, resulting in lethality., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The mammalian protein kinase N (PKN) family of Serine/Threonine kinases comprises three isoforms, which are targets for Rho family GTPases. Small GTPases are major regulators of the cellular cytoskeleton, generating interest in the role(s) of specific PKN isoforms in processes such as cell migration and invasion. It has been reported that PKN3 is required for prostate tumour cell invasion but not PKN1 or 2. Here we employ a cell model, the 5637 bladder tumour cell line where PKN2 is relatively highly expressed, to assess the potential redundancy of these isoforms in migratory responses. It is established that PKN2 has a critical role in the migration and invasion of these cells. Furthermore, using a PKN wild-type and chimera rescue strategy, it is shown that PKN isoforms are not simply redundant in supporting migration, but appear to be linked through isoform specific regulatory domain properties to selective upstream signals. It is concluded that intervention in PKNs may need to be directed at multiple isoforms to be effective in different cell types.

Background and Purpose: Metastatic renal cell carcinoma (MRCC) has a poor prognosis with a median overall survival of about one year. Since only a minority of patients experienced therapeutic benefit to current treatments, several studies have attempted to identify factors that may have an impact on response and survival. Cytokines play a crucial role in the host's immune response by regulating the development and function of a lot of biological compartments. Nevertheless, available data on basal cytokine levels in MRCC are very few and no clear profile of serum cytokines has been identified yet in these patients population. Thus, determining the levels of cytokines in MRCC could not only help in understanding the biological mechanisms of the tumor growth, but also in evaluating if different cytokine profiles are correlated with particular clinical behaviors., Materials and Methods: In 144 healthy donors and 55 MRCC treated with subcutaneous IL-2-based regimens, we analysed a panel of basal cytokines particularly involved in the neoplastic progression (IL-1beta, IL-6, IL-8, IL-10, IL-12, alpha-TNF) and C-reactive protein (CRP) in order to compare their levels in the two groups, and to verify their impact on patient response and survival. We first compared cytokines levels in patients population and healthy donors. Than, in definite patients group, univariate and multivariate analyses were performed to evaluate the correlation existing between each factor considered and clinical outcomes. For these analyses, baseline values were included as dichotomous variables using the median values (above and below) of control group., Results: In general, higher levels of cytokines were found in patients with respect to those of healthy donors, both in term of percentage of undetectable levels or median values. The impact on response was insignificant, except for higher levels of CRP that were strongly correlated with a worse response (p < 0.001). Within the patients groups, a worse survival was associated with higher values of CRP (8 vs 31 months, p = 0.0000), IL-6 (9 vs 25 months, p = 0.0295), and IL-8 (9 vs 17 months, p = 0.0371). Conversely, higher levels of IL-12 were associated with a better survival (25 vs 15 months, months p = 0.0882). A correlation was found between CRP and IL-6 (p = 0.009) and between CRP and IL-10 (p = 0.038). After multivariate analysis only CRP (p = 0.0035) and IL-12 (p = 0.0371) maintained an independent impact on survival, while IL-6 showed a borderline value (p = 0.0792)., Conclusion: Higher cytokines levels characterize patients population with respect to healthy donors. Moreover, higher basal level of some immunosuppressive cytokines (CRP, IL-6, IL-8) result correlated with a poorer survival, whereas higher levels of IL-12, a cytokine with a potent antineoplastic activity, was associated with a better survival. A wider sample of patients is needed to better clarify if our findings are intrinsically related to patients population or if they are simply an epiphenomenon of disease progression.

Cytokines play a crucial role in the host's immune response. In melanoma patients, cytokine profiles seems to be related to the clinical course and their imbalance could be associated to tumour progression. Thus, we studied a panel of baseline cytokines and their behaviour during treatment in order to verify their correlation with clinical outcomes. Interleukin-6, interleukin-8, interleukin-10, interleukin-12 and soluble receptor of interleukin-2 were evaluated in 90 out of 176 metastatic melanoma patients enrolled in a phase III study comparing chemotherapy and biochemotherapy. We divided patients into three different groups according to their own cytokine levels (low, intermediate and high) and then we correlated these groups with some clinical features. We also monitored the cytokines during the treatment in a subgroup of 37 patients. In univariate analysis, higher values of interleukin-6 (P = 0.005), soluble receptor of interleukin-2 (P = 0.001) and interleukin-12 (P = 0.010) were correlated with a worse survival. Conversely, interleukin-8 was unable to discriminate patients with different prognoses, and interleukin-10 was undetectable in the majority of patients. In multivariate analysis, only soluble receptor of interleukin-2 maintained its independent role in survival. The impact of baseline cytokines on response was insignificant. Regarding the behaviours of cytokines during treatment, the most remarkable aspect was a progressive increase of interleukin-12 and soluble receptor of interleukin-2 in patients with a better survival. In our metastatic melanoma patients, higher basal levels of interleukin-6, interleukin-12 and soluble receptor of interleukin-2 were associated with a worse survival. In contrast, a progressive increase of interleukin-12 and soluble receptor of interleukin-2 was observed during treatment in patients with a better survival.

Purpose: In patients with metastatic renal cell carcinoma treated with low dose subcutaneous interleukin-2 based immunotherapy we evaluated a panel of biohumoral and clinical parameters before treatment to verify their correlation with clinical outcome., Materials and Methods: We analyzed the records of 110 patients treated at our institution. Before treatment total lymphocytes, lactate dehydrogenase, the erythrocyte sedimentation rate, albumin, C-reactive protein (CRP) and fibrinogen were analyzed and correlated with clinical parameters, namely performance status, patient age, sex, prior nephrectomy, number and sites of disease, and disease-free interval (DFI) from nephrectomy to metastatic disease., Results: Median survival was 12 months (partial and complete response 33, stable disease 14 and progression 7). The overall response was 24% for a partial and complete response, 37% for stable disease and 39% for progression. On univariate analysis good performance status (p = 0.0000), prior nephrectomy (p = 0.0001), DFI longer than 12 months (p = 0.0003), bone disease site (p = 0.0013), a low number of metastatic sites (p = 0.0449), normal albumin (p = 0.0001), low/normal fibrinogen (p = 0.0140), low/normal lactate dehydrogenase (p = 0.0430) and low/normal CRP (p = 0.0000) were related to better survival. On final multivariate analysis only CRP (p = 0.002) and DFI (p = 0.0497) were found to have an independent role in survival. When we correlated clinical and biohumoral factors, only CRP correlated with DFI (p = 0.021) and prior nephrectomy (p = 0.041)., Conclusions: Our data confirm that some clinical and biohumoral factors may be strongly related to survival in metastatic renal cell carcinoma. The interesting new aspect emerging from this study is the prognostic value of CRP and fibrinogen, which are able to discriminate a good from a poor prognosis.

PURPOSE: To evaluate a panel of pretreatment clinical and laboratory parameters in metastatic melanoma (MM) in order to verify their impact on response and survival in a single prospective multi-institutional phase III study comparing out-patient chemotherapy (CT) vs bioCT. METHODS: A total of 176 patients were randomised to receive CT (cisplatin, dacarbazine, optional carmustine) or bioCT (the same CT followed by subcutaneous IL-2 plus intramuscular alpha-IFN-2b). Pretreatment total leucocytes, lymphocytes, eosinophyls, C-reactive protein (CRP), lactate dehydrogenase (LDH), erytrosedimentation rate (ESR), and fibrinogen were analyzed. Some clinical parameters (performance status, age, sex, and disease site) were also considered. As we found a positive trend for bio-CT with no statistical significance in OR (25.3% vs 20.2%) and OS (11 Mo vs 9.5 Mo), all analyses are stratified by treatment arm. RESULTS: In univariate analysis, higher value of lymphocytes percentage (P <.0001), lower value of total leucocytes (P=.005), CRP (P=.003), LHD (P <.0001), ESR (P <.027), fibrinogen (P <.0001), and no liver disease were strongly related to a better survival. In a multivariate analysis, using the Cox proportional hazards model, only fibrinogen (P=.004), LDH (P=.009) and liver disease (P=.04) were found to have an independent role on clinical outcome in metastatic melanoma patients. CONCLUSION: Liver disease and higher LDH and fibrinogen levels had an important impact on survival in MM patients. In particular, fibrinogen has been recently reconsidered both for its determinant role in the host hemostatic system, and for its capability to provide protection against NK and LAK-cell-induced lysis. These observations could have some important implications for therapeutic approaches, in particular when immunological strategies are used.

Protein kinase N 1 (PKN1), which in part resembles yeast protein kinase C, has been shown to be under the control of Rho GTPases and 3-phosphoinositide-dependent kinase 1 (PDK1). We show here that green fluorescent protein-tagged PKN1 has the ability to translocate in a reversible manner to a vesicular compartment following hyperosmotic stress. PKN1 kinase activity is not necessary for this translocation, and in fact the PKN inhibitor HA1077 is also shown to induce PKN1 vesicle accumulation. PKN1 translocation is dependent on Rac1 activation, although the GTPase binding HR1abc domain is not sufficient for this recruitment. The PKN1 kinase domain, however, localizes constitutively to this compartment, and we demonstrate that this behavior is selective for PKNs. Associated with vesicle recruitment, PKN1 is shown to undergo activation loop phosphorylation and activation. It is established that this activation pathway involves PDK1, which is shown to be recruited to this PKN1-positive compartment upon hyperosmotic stress. Taken together, our findings present a pathway for the selective hyperosmotic-induced Rac1-dependent PKN1 translocation and PDK1-dependent activation.

Cell adhesion to extracellular matrix regulates proliferation and survival of several cell types including epithelial thyroid cells. Activation of integrin receptors by binding to extracellular matrix generates a complex cell type-dependent signaling. Adhesion to extracellular matrix induces proliferation and survival in primary cultures of thyroid cells and induces survival in immortalized human thyrocytes. In this study we demonstrate that in immortalized human thyrocyte cells, adhesion to immobilized fibronectin (FN) stimulates DNA synthesis and proliferation through the p21Ras/MAPK pathway, whereas cell survival is mediated by phosphatidylinositol 3-kinase (PI3K) signal pathway. Integrin activation by immobilized FN induced phosphorylation of pp125 focal adhesion kinase and paxillin and induced the formation of focal adhesion kinase/Grb-2/Sos complex. Western blot and in vitro kinase assay demonstrated the activation of Ras and the p44/p42 MAPK/ERK1/2. Inhibition of p21Ras activity and inhibition of MAPK enzymatic activity completely arrested cell growth but did not induce cell death. Integrin activation by cell adhesion to FN also induced activation of PI3K. Inhibition of PI3K enzymatic activity induced apoptosis demonstrated by annexin V-binding assay and loss of cellular DNA content. These results demonstrate that in thyroid cells adhesion to FN regulates proliferation through the p21Ras/MAPK signal pathway, whereas integrin-mediated cell survival is mediated by PI3K.

Background: Interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) are the main immuno-biological agents used in the therapy of metastatic renal cell carcinoma (RCC). Unfortunately the promising results obtained in biological studies have not yet been confirmed in clinical studies. One reason is linked to the immunosuppression of metastatic patients which is caused by macrophage products. IL-6 in particular is considered a growth factor for RCC. Medroxyprogesterone acetate (MPA) may interfere with IL-6 macrophage production, possibly causing a synergistic effect in association with IL-2 and IFN-alpha. Therefore the purpose of our study was to evaluate the toxicity and the efficacy of the association between IL-2, IFN-alpha and MPA., Patients and Methods: Forty-two consecutive patients with metastatic RCC were enrolled. IL-2 was administered subcutaneously at doses of 4.5 million UI on days 1-5, 8-12, 15-19 and 22-26; IFN-alpha was administered s.c. at a dose of 3 million t.t.w; MPA was administered orally at a dose of 1000 mg daily. This schedule was repeated after a rest of 2 weeks., Results: Toxicity was mild: the main symptoms observed were fatigue and fever. Six CR (14%), five PR (12%), thirteen SD (31%) and seventeen PD (41%) were observed for an overall response rate of 26%. Patients with good PS and low levels of CRP had a better prognosis., Conclusion: Considering both the good activity and the low toxicity of this scheme, we think that it could be carried out in normal clinical practice.

The protein kinase C-related protein kinases (PRKs) have been shown to be under the control of the Rho GTPases and influenced by autophosphorylation. In analyzing the relationship between these inputs, it is shown that activation in vitro and in vivo involves the activation loop phosphorylation of PRK1/2 by 3-phosphoinositide-dependent protein kinase-1 (PDK1). Rho overexpression in cultured cells is shown to increase the activation loop phosphorylation of endogenous PRKs and is demonstrated to influence this process by controlling the ability of PRKs to bind to PDK1. The interaction of PRK1/2 with PDK1 is shown to be dependent upon Rho. Direct demonstration of ternary (Rho.PRK.PDK1) complex formation in situ is provided by the observation that PDK1 is recruited to RhoB-containing endosomes only if PRK is coexpressed. Furthermore, this in vivo complex is maintained after phosphoinositide 3-kinase inhibition. The control of PRKs by PDK1 thus evidences a novel strategy of substrate-directed control involving GTPases.

In normal epithelial cells, impaired cell-matrix contact leads to induction of programmed cell death, a process that has been termed 'anoikis'. We investigated the role of p53 and other apoptotic proteins in anoikis in thyroid epithelial cells. Western blot analysis demonstrated that neither p53 nor Bcl-2, Bcl-XL and Bax protein expression changed during anoikis. However, loss of endogenous p53 activity in cells transfected with a dominant-negative mutated p53 inhibited anoikis demonstrating the involvement of p53-dependent processes. The phosphatase inhibitor sodium orthovanadate opposed anoikis when added to the cells within 6 h, suggesting a role for phosphorylated proteins.

The expression of the beta1 family of integrins was determined in thyroid follicular cells from patients with Graves' disease (GD). Integrin expression was quantitated by flow fluorocytometry of single cell suspensions with antibodies against the common beta1 chain and the alpha1-alpha6 subunits. Results indicated that also in thyroid glands of GD, as previously observed in nodular goiters, two follicular cell populations with different patterns of beta1 integrin expression coexist (VLAalpha3beta1 and VLAalpha1,3,5,6beta1). The VLAalpha1,3,5,6beta1 thyrocyte population in GD was more abundant than in nodular goiters, ranging from 40 to 70% of the total follicular cells and the overall expression of the beta1 integrins was a two-fold higher. In thyrocytes from patients with GD cultured in vitro, alpha3 and alpha2 expression was regulated by cell-to-cell contact as previously described in normal thyroid cells, while the expression of alpha1, alpha5 and alpha6 was quickly lost during the culture. Our data suggest that the integrin profile of the VLAalpha1,3,5,6beta1 thyrocyte population in GD is induced by micro-environmental conditions rather than being the expression of a constitutive phenotype.

Addition of insulin growth factor-I (IGF-I) to quiescent Swiss 3T3 cells rapidly induced tyrosine phosphorylation of the p130Crk-associated substrate (p130(Cas)), a novel adaptor protein localized at focal adhesions. Half-maximal effect was obtained at 0. 6 nM. IGF-I also promoted the formation of a complex between p130(Cas) and c-Crk and elicited a parallel increase in the tyrosine phosphorylation of p125(Fak) and paxillin. IGF-I-induced p130(Cas), p125(Fak), and paxillin tyrosine phosphorylation could be dissociated from mitogen-activated protein kinase kinase, p70(S6K), and protein kinase C activation. In contrast, the structurally unrelated phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 markedly attenuated the increase in tyrosine phosphorylation of p130(Cas), p125(Fak), and paxillin induced by IGF-I. Cytochalasin D, which disrupts the network of actin microfilaments, completely prevented tyrosine phosphorylation of p130(Cas), p125(Fak), and paxillin and the formation of a p130(Cas). Crk complex in response to IGF-I. Thus, our results identified a phosphatidylinositol 3-kinase-dependent pathway that requires the integrity of the actin cytoskeleton to induce tyrosine phosphorylation of p130(Cas), p125(Fak), and paxillin in response to IGF-I and suggest that tyrosine phosphorylation of these focal adhesion proteins, together with the recruitment of c-Crk into a complex with p130(Cas), may play a novel role in IGF-I signal transduction.

Treatment of Swiss 3T3 cells with bombesin rapidly induced tyrosine phosphorylation of the p130 Crk-associated substrate (p130(cas)). Vasopressin, endothelin, bradykinin, lysophosphatidic acid, sphingosylphosphorylcholine, and phorbol 12,13-dibutyrate also stimulated p130(cas) tyrosine phosphorylation. Bombesin-induced p130(cas) tyrosine phosphorylation could be dissociated from both protein kinase C activation and Ca2+ mobilization from intracellular stores. In contrast, cytochalasin D, which disrupts the network of actin microfilaments, completely prevented tyrosine phosphorylation of p130(cas) by bombesin. Platelet-derived growth factor, at low concentrations (1-5 ng/ml), also induced tyrosine phosphorylation of p130(cas) via a pathway that depended on the integrity of the actin cytoskeleton. The phosphatidylinositol 3'-kinase inhibitors wortmannin and LY294002 prevented tyrosine phosphorylation of p130(cas) in response to platelet-derived growth factor but not in response to neuropeptides, lysophosphatidic acid, sphingosylphosphorylcholine, or phorbol 12,13-dibutyrate. All agonists that induced p130(cas) tyrosine phosphorylation also promoted the formation of a p130(cas).Crk complex in intact Swiss 3T3 cells. Thus, our results identified distinct signal transduction pathways that lead to tyrosine phosphorylation of p130(cas) in the same cells and suggest that p130(cas) could play a role in mitogen-mediated signal transduction.

The expression of integrins of the beta1 family and their possible biological effects were investigated in normal human thyroid cells in monolayer culture. The expression of beta1 and alpha(1-6) integrin subunits was determined by flow cytofluorometry with specific antibodies. Follicular cells of subconfluent monolayer cultures expressed alpha2beta1 and alpha3beta1 at high levels, while alpha1beta1 was only slightly expressed, and alpha4beta1, alpha5beta1, and alpha6beta1 were never detected. Cell attachment assays were performed in fibronectin-, type I collagen-, and laminin-coated microtiter plates. Thyroid cells, while adherent to collagen and fibronectin, showed poor attachment to laminin despite the abundance of their putative receptors alpha2beta1 and alpha3beta1. In serum-free medium, collagen and fibronectin induced cytoskeletal organization, change of cell shape from round to flat, and cell spreading. [3H]Thymidine incorporation and proliferation assays were used to evaluate the effects of collagen and fibronectin on DNA synthesis and cell growth in the absence of a change in spreading or cell shape. Both substrates, in low serum-containing medium, induced a concentration-dependent increase in [3H]thymidine incorporation partially inhibited by RGD-containing peptides that blocked the cell attachment. Thyrocytes cultured in low serum-containing medium on immobilized fibronectin or collagen showed a dose-dependent stimulation of proliferation. These data indicate that fibronectin and collagen can regulate the cytoskeletal organization and cell shape and stimulate the proliferation of normal human thyroid cells in culture and that integrins mediate these effects of extracellular matrix proteins.

In 39 women affected by cervical intraepithelial neoplasia (CIN) concentration of the cervical mucus secretory IgA (sIgA) was significantly higher than in 414 controls matched for age, reproductive status and smoking habit (196 healthy women; 109 with "specific" and "aspecific" cervicitis or cervico-vaginitis; 51 with benign epithelial disorders of the cervix; 40 with adenomatous cervical polyp and 18 with early invasive exocervical squamous carcinoma). CIN carried the highest proportion of cases with sIgA detectable or beyond the cut-off value and sIgA concentration was inversely related to CIN grade. Present data confirm our preliminary investigation on the cervical total IgA and explain that any increased levels of these IgA are induced by the secretory and not serum fraction of this immunoglobulin. We still don't know the exact meaning and value of the present results. It is only possible to suppose that they are the expression of a vigorous local immune activation adverse to CIN, which is elevated for as long as the architecture of cervical glandular epithelia is preserved.

Background: In a previous study [Psychother Psychosom 1994;61:199-204] we investigated the relationship between alexithymia, carcinogenesis and immunity in a group of women who were unconscious sufferers from precancerous lesions of the cervix (CIN). The results of this study showed a high level of association between alexithymia and CIN and, an even more interesting fact, between alexithymia and reduced levels of immunity., Methods: The aim of the present study is to check the results of the previous one by testing a larger group (43 women affected by cervical dysplasia and 67 healthy women) and by the use of a self-administered test for detection of alexithymia, the well-validated Twenty-Item Toronto Alexithymia Scale (TAS-20)., Results: The results confirm that women suffering from CIN have higher average TAS-20 ratings (55) than normal women (47.32) and that the level of alexithymia detected in the group of women suffering from dysplasia (42.5%) is higher than that of normal women (12.85%). Moreover, the present study confirms that alexithymic women have lower rates of a number of lymphocyte subsets than non-alexithymic women., Conclusions: This study fully confirms the results of our previous work and those of a number of other studies: (1) personality might be one of the factors jointly responsible for the outbreak of cancer; (2) the immune system appears to play an important part as a mediator between personality and cancer.

Several trials have evaluated the therapeutic efficacy of rIL-2 combined with more traditional treatments such as chemotherapy and radiotherapy, but the use of IL-2 as adjuvant therapy for minimal residual disease or to maintain clinical response obtained with other standard treatments has yet to be investigated. The aim of the present trial was to study the biological effects of maintenance long-term treatment (6 months) with subcutaneous low-dose IL-2 in 16 patients with different neoplasms previously treated with chemo-immuno therapeutic regimens or with surgery (7 metastatic renal cancers, 5 locally advanced renal cancers previously subjected to radical nephrectomy, 2 metastatic breast cancers, 1 small cell lung cancer, and 1 metastatic melanoma). Clinical tolerability, feasibility and therapeutic implications are also discussed. The IL-2 schedule was as follows: 4.5 million IU/day, 3 times weekly for 6 months. A total of 14 patients completed therapy without requiring dose modifications and are free of progression after a median duration of 8+ months (range: 7+ to 34+) while two patients progressed during therapy (one inflammatory breast cancer and one renal cancer). Important and persistent hemato-immunostimulating effects in both soluble (IL-2, sIL-2R, IL-6) and cellular (lymphocyte subsets, monocytes, eosinophils) parameters were noted during the entire treatment. The IL-2 related toxicity was quite low. Moreover, this long-term IL-2 therapy could control neoplastic growth and thus prolong clinical response obtained with standard treatments. Prospective randomized studies regarding the clinical efficacy have been initiated.

The expression of the beta 1 family of integrins was studied in normal thyroid tissue cultures and monolayer cell cultures. The expression of the various subunits was measured by flow cytofluorometry with specific monoclonal antibodies and by Northern analysis. In monolayer cell cultures but not in tissue cultures, the expression of the alpha 3 subunit on the cell membrane progressively increased soon after plating, reaching a 30-fold higher intensity. The alpha 2 subunit, not detectable in native follicular cells, was expressed de novo and reached a remarkable high level. Up-regulation of alpha 2 and alpha 3 in monolayer cell cultures was serum-independent and preceded the expression of proliferating cell nuclear antigen, [3H]thymidine incorporation, and cell replication. Northern analysis demonstrated an increased level of beta 1 integrin mRNA. The increase of alpha 2 and alpha 3 was readily reversible since the expression of these molecules returned to a lower level when cultures reached a high cell density. Down-regulation did not occur until cell cultures were confluent. When cells from high cell density and low integrin expression were harvested and sparsely seeded in culture, up-regulation of integrins was observed again, while rapid reaggregation of isolated cells inhibited this phenomenon. Altogether these data suggest that cell-to-cell contact may regulate the expression of beta 1 integrins in thyroid primary cultures.

Background: The relationship between solitary plasmacytoma and multiple myeloma is still unclear, but they can be distinguished by their different clinical course. Indicators of disease activity and extension, and of a possible evolution to multiple myeloma, have not been identified as yet., Methods: Two cases of solitary plasmacytoma are described: one of the mandible and one extramedullary plasmacytoma (EMP) of the rhinopharynx. Pathologic data included immunohistochemical staining for heavy and light Ig chains, and for the proliferating cell nuclear antigen (PCNA). Analysis of the peripheral immunological status and serum parameters (beta 2 microglobulin, thymidine kinase, IL-2, IL-6 and soluble IL-2 receptor) was performed and correlation was made with the clinical status. Flow cytometry analysis of nuclear DNA content and S-phase cell fraction were also studied in both neoplasms., Results: Solitary plasmacytoma of bone (SPB) showed important basal immunologic alterations and a marked increase in all serum parameters considered with respect to EMP. Ploidy analysis demonstrated an almost complete aneuploidy cell population for the SPB patient (80%), whereas in the EMP patient only 2% of the cells were aneuploid. The S-phase cells were 16% and 4%, respectively. PCNA index was 60% in SPB and 10% in EMP., Conclusions: Solitary plasmacytoma of the bone appeared to be a more aggressive form of plasmacellular neoplasia, distinct from EMP and similar to multiple myeloma. The study of serum parameters, together with analysis of PCNA, ploidy and S-phase fraction, can aid in better understanding disease activity, and in the choice of more adequate treatment. Moreover, serial analysis of some serum factors might be useful markers for monitoring the disease.

The expression and cell-membrane distribution of the beta 1 family of integrins (very-late-activation antigens, VLA) were investigated in benign and malignant human thyroid tumors. We compared tissue samples of normal glands, nodular goiters, adenomas and carcinomas. We also examined 3 thyroid-carcinoma cell lines cultured in vitro. The expression of subunits of the beta 1 family of integrins was assessed by flow cytometry and specific antibodies in dispersed single-cell suspensions and by immunofluorescence on frozen tissue sections. In contrast to the heterogeneity of the expression of beta 1 integrins observed in other tumors, thyroid neoplastic lesions showed a remarkably constant VLA profile. In all tumors, benign as well as malignant, and in carcinoma cell lines, all sub-units of beta 1 integrins were expressed at high levels. While sub-units alpha 1, alpha 3, alpha 5, alpha 6 and occasionally alpha 2 were also present in a cell sub-set of normal glands and nodular goiters, expression of alpha 4 was restricted to neoplastic lesions; this integrin can be therefore considered an antigen associated with thyroid tumors. It has been reported that in normal glands and in nodular goiters, the expression of beta 1 integrins is restricted to the basal-cell membrane. Immunofluorescence on tissue sections showed instead that, in adenomas and carcinomas, the polarized distribution of these integrins on the cell membrane is lost.

Alexithymia and circulating lymphocyte subsets were studied in 62 women [36 healthy women and 26 women affected by cervical intraepithelial neoplasia (CIN I, II, III) who were not aware of their status] in order to assess a possible relationship between alexithymia, CIN and immunological lymphocytic functions. Alexithymia was estimated by the 20-item Schalling-Sifneos Personality Scale and then correlated with peripheral blood lymphocyte subsets. The results of our study report an association between alexithymia and CIN. Alexithymic women show lower rates of almost all lymphocytic subsets compared to nonalexithymic ones. The difference was also found between alexithymic women affected by CIN and alexithymic women with an unsuspicious Pap smear. On the whole, these preliminary results seem to confirm data reported by other authors who hypothesized that a certain personality trait characterized by emotional inhibition is related to a greater cancer vulnerability. Such relationships might be mediated by certain lymphocytic functions as the result of the alexithymic status. Results reported here need more extensive surveys, in order to control potentially confounding factors related to the personality assessment of the subjects in this study.

Immunostimulating and immunosuppresive events have been noted during rIL-2 immunotherapy and cell-mediated immune deficiencies have been reported in the neutrophil and monocyte functions, determining a high incidence of infections in patients during intravenous rIL-2 treatment. The phagocytic activity of monocytes/macrophages and granulocytes was evaluated in 10 advanced solid tumor patients treated with subcutaneous rIL-2. Several indirect parameters of phagocytosis were also considered, such as Neopterin, Beta2 microglobulin, IL-2 soluble receptor, and the C3 and C4 fractions of the complement system. A decreased phagocytosis was demonstrated after subcutaneous rIL-2 administration together with an increase of indirect parameters of granulocyte/macrophage activity. Therefore, cellular activation does not seem to correspond to actual phagocytosis which is probably due to IL-2-induced secondary cytokines or to rIL-2 inhibitory effects.

Immunotherapy represents a promising biological approach to cancer treatment. Important haemato-immunological modifications during rIL-2 treatment have been demonstrated in all patients treated, independently of the schedule and mode of rIL-2 administration. Generally, there is a lack of correlation between treatment-related modifications and clinical response. rIL-2 cell-mediated responses have been extensively studied but the role of humoral immunity is still unclear. We evaluated the peripheral blood CD4, CD8, CD19 and CD25 lymphocyte subset modifications and serum levels of immunoglobulin, IL-6 and IL-2 in 19 patients with malignant melanoma and renal cell carcinoma undergoing subcutaneous administration of IL-2. Our results suggest that humoral immunity is only marginally involved; however, the parameter variations observed suggest that further studies are necessary.

A flow-cytometric analysis of ascitic fluid (AF) and peripheral blood lymphocyte subpopulations (LS) was performed on 16 patients with ovarian malignancy (OM) and 5 with metastatic peritoneal carcinomatosis (MPC). AF lymphocytes are 58% of total leukocytes in OM patients, 37% in MPC patients, while blood lymphocytes are 14% and 17%, respectively. AF absolute lymphocyte count (total and individual LS) is higher in OM patients. OM patients AF lymphocytes are: T = 70%, CD4+ = -37%, CD8+ = 32% (CD4/CD8 = 1.39), B = 5%, NK cells = 4-10%, CD25+ = 20%, CD69+ = 15%, CD71+ = 3%. In MPC patients the values are generally similar, though CD4+ cells are +7%, CD8+ cells = -14% (CD4/CD8 = 3.17), CD69+ cells = -8%. Untreated OM patients have a AF total and percent lymphocyte count higher than treated ones. Among the latter, however, the CD4/CD8 ratio as well as the number of CD4+, inducer, CD25+ and CD71+ cells are higher. In terms of percent values, the most striking differences involve total T and B lymphocytes (81-87% vs 56-62%, and 10% vs 2%, respectively). With the only exception of absolute NK cell count, OM patients show no correlation between AF and peripheral blood LS pattern. These results agree only partially with data from the literature. Pathophysiologic and clinical considerations support the practical usefulness of LS analysis in AF from OM patients.

The presence of 7 human papillomavirus (HPV) types in genital condylomata acuminata (GCA) from 94 otherwise healthy women was detected by in situ hybridization, employing 3 different DNA probe cocktails (6/11, 16/18 and 31/35/51). HPV+ and HPV- GCA did not differ significantly as to the multiplicity and site of lesions, nor the prevalence of younger age, previous pregnancies or abortions, oral contraception, concurrent vaginal infection, aspecific PAP-test abnormalities and cutaneous hypo-anergy. Of note is that 13/18 HPV+ GCA (72.2%) lodged HPV types (16, 18, 31, 35, 51) which are more commonly related to dysplastic and neoplastic lesions of the female genital tract.

The aim of the present study was to verify the efficacy and tolerability of a treatment protocol using subcutaneous administration of rIL-2. Eighteen patients with metastatic disease were treated: 10 with renal carcinoma and 8 with malignant melanoma. The drug was administered as follows: 9 million UI/mq twice daily for two days followed by 1.8 million UI/mq twice daily for five days/week for six consecutive weeks. Of the 15 patients evaluated for clinical response, all completed treatment without rIL-2 dose modifications. No complete response was obtained. One patient with malignant melanoma and 1 with renal carcinoma (13%) had partial response (soft tissue and lymph nodes respectively) which lasted 3 and 4 months. Six patients with renal carcinoma and 3 melanoma patients had stable disease (60%) which lasted 6 months (median). Toxicity was moderate and spontaneously reversing after stopping treatment. Haemato-immunological modifications and pharmacokinetic study of this modality of rIL-2 treatment were also examined.

Cervical smear total IgA (IgAc) concentration was determined in cervicitis and proliferative disorders of the cervical epithelium. All disorders except adenomatous polyp showed increased median IgAc levels as compared with healthy controls, in both the reproductive and the postmenopausal ages. In the former age group dysplasia exhibited the highest median IgAc level, while in the latter it ranked, second following specific cervicitis. Though differences between disease groups and controls were statistically non significant, they attained percent values as large as +277% (dysplasia, postmenopausal) and +596.9% (specific cervicitis, postmenopausal). Separate quantitation of the dimeric secretory and monomeric serum-derived components of total IgAc will provide more meaningful information.

The serum levels of Ceruloplasmin (CER), Properdin Factor B (PFB) and Copper (CU) were evaluated in a series of 40 patients with Hodgkin's Disease and 46 patients with non-Hodgkin's lymphoma. Concentrations of CER and PFB were determined by rate nephelometry and CU concentrations by the bathocuproine colorimetric method. The results obtained demonstrated that CER, together with the well documented CU, can be used for monitoring Hodgkin's Disease.

The authors describe a case of primary central nervous system lymphoma in a 43-year-old male. Interest in this rare form of B-lymphocyte non-Hodgkin's lymphoma is due to its increasing incidence in the last decade, especially in immunodeficient patients. Extraneural involvement was excluded by staging examinations: bilateral bone marrow biopsy from the posterior iliac crest, bipedal lymphography, abdominal CT scan, skeletal, thoracic and gastrointestinal X-rays, spinal puncture, ORL and clinical examination. The peripheral immunologic state was particularly interesting: there was a substantial decrease in total circulating B-lymphocytes at diagnosis (4.4% = 80/mmc; nv 13 +/- 4% = 287 +/- 130/mmc) and a further decrease after 2 and 4 months of therapy (0.16% = 2/mmc). Three months after completion of therapy, the B-lymphocyte level returned to the base level at diagnosis (7% = 88/mmc). At 10 months after diagnosis and 3 months after completion of chemotherapy, the patient is alive and in good health except for the after-effects of a left hemiparesis. The etiologic and possible pathogenic factors are considered.

CA 125 serum levels were assessed in 23 patients undergoing chemotherapy for advanced epithelial ovarian carcinoma (EOC). Rising, falling and unchanged levels correlated with disease in 21 out of 23 (95%) cases. Ten out of 11 patients who showed objective response to chemotherapy had a decrease in antigen levels. Two out of 3 patients with stable disease had unchanged values. Progression was always associated with rising levels. CA 125 is a reliable marker for monitoring response to chemotherapy in advanced epithelial ovarian carcinoma.