The prevalence of TTR cardiac amyloidosis among patients undergoing bone scintigraphy.

BACKGROUND: Radiolabeled bisphosphonates bone scintigraphy is highly sensitive in detecting transthyretin (TTR) cardiac amyloidosis; data on the true prevalence of cardiac involvement in TTR amyloidosis are lacking. METHODS AND RESULTS: This retrospective observational, monocentric study aims to estimate the prevalence of positive bone scan suspect for TTR cardiac amyloidosis among an all-comers population who underwent a bone scintigraphy. ECG, echocardiography and clinical status of patients with unexpected cardiac uptake (Perugini score 2-3) who underwent bone scintigraphy with [99mTc]-HDP or [99mTc]-DPD at San Luigi Gonzaga University Hospital between January 2015 and May 2020 have been collected. The prevalence of bone scintigraphy suspect for cardiac involvement was 0.54% (23/4,228). The bone scintigraphy was mainly performed using [99mTc]-HDP (82.9%) and the dominant indication for the test was oncology in the 47.9% of cases. 8 Subjects had a history of neuropathy (34.8%) and 5 of carpal tunnel syndrome (21.7%). 11 Patients suffered a previous episode of heart failure (48%) while 5 patients (21.7%) were totally asymptomatic, without any sign or symptom before the bone scintigraphy making the nuclear examination crucial for an early diagnosis of TTR amyloidosis. CONCLUSION: Bone scintigraphy allows suspecting TTR amyloidosis in a pre-clinical stage of the disease in an all-comers population of patients undergoing bone scintigraphy mainly for oncology reasons.

COVID-19 therapies and their impact on QT interval prolongation: A multicentre retrospective study on 196 patients.

BACKGROUND: SARS-CoV-2 infection has caused a global pandemic. Many of the medications identified to treat COVID-19 could be connected with QTc prolongation and its consequences. METHODS: Non-ICU hospitalized patients of the three centres involved in the study from the 19th of March to the 1st of May were included in this retrospective multicentre study. Relevant clinical data were digitally collected. The primary outcome was the incidence of QTc prolongation ≥ 500 ms, the main secondary outcomes were the Tisdale score ability to predict QTc prolongation and the incidence of ventricular arrhythmias and sudden deaths. RESULTS: 196 patients were analysed. 20 patients (10.2%) reached a QTc ≥ 500 ms. Patients with QTc ≥ 500 ms were significantly older (66.7 ± 14.65 vs 76.6 ± 8.77 years p: 0.004), with higher Tisdale score (low 56 (31.8%) vs 0; intermediate 95 (54.0%) vs 14 (70.0%); high 25 (14.2%) vs 6 (30.0%); p: 0.007) and with higher prognostic lab values (d-dimer 1819 ± 2815 vs 11486 ± 38554 ng/ml p: 0.010; BNP 212.5 ± 288.4 vs 951.3 ± 816.7 pg/ml p < 0.001; procalcitonin 0.27 ± 0.74 vs 1.33 ± 4.04 ng/ml p: 0.003). After a multivariate analysis the Tisdale score was able to predict a QTc prolongation ≥ 500 ms (OR 1,358 95% CI 1,076-1,714p: 0,010). 27 patients died because of COVID-19 (13.7%), none experienced ventricular arrhythmias, and 2 (1.02%) patients with concomitant cardiovascular condition died of sudden death. CONCLUSIONS: In our population, a QTc prolongation ≥ 500 ms was observed in a minority of patients, no suspected fatal arrhythmias have been observed. Tisdale score can help in predicting QTc prolongation.

Bi-layered collagen nano-structured membrane prototype collagen matrix CM-10826 for oral soft tissue regeneration: an in vivo ultrastructural study on 13 patients.

A new developed collagen matrix CM-10826 (CM) of porcine origin designed to be used as oral soft tissue substitute was investigated before and after implantation by light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). In a case series biopsy specimens were harvested from thirteen patients at 10, 20, 30, 43 days after abutment surgery for uncovering dental implants. The in vivo histological evaluations of each patient were performed via micro-coring of newly formed oral mucosa in the area covered by CM (test side) or left uncovered (control). Results showed that CM can be integrated in connective and epithelial tissues within 10 days, can be completely resorbed within 20 days and it is able to reduce inflammatory infiltrates and to stimulate both fibroblast/epithelial cell proliferation and neo-angiogenesis. Generally it seems to be superior in promoting soft tissue healing compared to that induced by secondary intention healing. Furthermore, it is able to act as a scaffold for soft-tissue regeneration, allowing the proliferation of keratinocytes from the wound edges and favoring neovascularization and growth of connective tissue in the mesh of porous layer. It appears that a CM might function in oral surgery as a substitute for autologous grafts and to avoid secondary intention healing in soft tissue defects.

Evaluation of the effects of a probiotic supplementation with respect to placebo on intestinal microflora and secretory IgA production, during antibiotic therapy, in children affected by recurrent airway infections and skin symptoms.

Antibiotic therapy, especially in pediatric patients, is often associated with significant modifications of the gut microflora, which can lead to intestinal dysbiosis and influence intestinal physiology and immune system functionality. Herein we report the results from a double blind controlled clinical trial in 77 pediatric patients affected by recurrent airway infections, receiving antibiotic therapy with amoxicillin and clavulanic acid. A group was treated with an oral probiotic preparation composed of Lactobacillus paracasei ssp.paracasei CRL-431, Bifidobacterium BB-12, Streptococcus thermophilus TH-4 and a fructooligosaccharide (FOS) during and after antibiotic therapy for seven days, while the other group received placebo. The study revealed a reduction in the Clostridia population, with a contemporary increase in Bifidobacteria and Lactobacilli in fecal samples in the probiotic group and an increase in the Enterobacteria population in the placebo group. Moreover, there was a decreasing trend in secretory IgA production in the probiotic group. Some relevant, but not statistically significant probiotic supplementation effects were identified.

PDX-1 (pancreatic/duodenal homeobox-1 protein 1).

The homeodomain-containing transcription factor pancreatic duodenal homeobox 1 (PDX-1) plays a key role in pancreatic development and ß-cell function. It is a major regulator of transcription in pancreatic cells, and transactivates the insulin gene by binding to a specific DNA motif in its promoter region. Glucose also regulates insulin gene transcription through PDX-1. It has been shown that PDX-1 is required for maintaining pancreatic islet functions by activating gene expression and has a dual role in pancreatic development. It initially contributes to pancreatic formation during embryogenesis and subsequently regulates the pancreatic islet cell physiology in mature islet cells. Because of this key role in the embryologic development of the pancreas, PDX-1 expression has been investigated in pancreatic cancer cell lines and human tumors. Moreover, a few reports have described expression of PDX-1 in other human neoplasms and have investigated its potential role in differential diagnosis, but data on normal human tissues are lacking. Understanding the molecular mechanisms of pancreas formation, and especially the function of PDX-1, may contribute to the improved treatment and prevention of debilitating diseases such as diabetes, insulinomas and pancreatic carcinomas. Nevertheless, further studies are needed concerning its possible application in routine practice.

Growth hormone and disease severity in early stage of multiple sclerosis.

Evidence suggests that neurohormones such as GH and IGF-I are involved in the neuroreparative processes in multiple sclerosis (MS). GH and IGF-I blood levels in naïve MS patients with different disease courses were investigated in this study. Serum GH and IGF-I in untreated MS patients (n = 64), healthy controls (HC, n = 62), and patients affected by other neurological diseases (OND, n = 46) were evaluated with a solid-phase-enzyme-labeled-chemiluminescent-immunometric assay. No differences were detected in GH across MS, OND, and HC (MS = 0.87 ± 1.32 ng/mL; OND = 1.66 ± 3.7; and HC = 1.69 ± 3.35; P = 0.858) when considering gender, disease duration, and disease course. However, GH was lower (P = 0.007) in patients with more severe disease (expanded disability scale score, EDSS ≥ 4.0) compared with milder forms (EDSS < 4). IGF-I l did not differ across the 3 groups (P = 0.160), as far as concern disease course, disability, and gender were. Lower IGF-I levels were detected in subjects older than 50 years compared to younger ones for all 3 groups. Lower GH was detected in patients with more severe MS, and age was confirmed as the main factor driving IGF-I levels in all subjects. These findings, relying on the natural course of the disease, could help in shedding lights on the mechanisms involved in autoreparative failure associated with poorer prognosis in MS.

Epidemiology of dental professional liability.

The aim of this article is to collect data relating to dental professional liability in Italy and provide a common platform for discussions among clinicians, legal medicine practitioners, and experts in law. On the basis of two different dental-legal statistical samples (1,670 reports of legal dental experts and 320 civil court decisions) we analyzed the dental professional liability lawsuit in the areas of distribution of lawsuits among the different dental specialties, recurrence and type of errors, outcome of civil suits, parameters of compensation. Some ideas are also proposed for possible strategies in the management of clinical risk (prevention of errors) and court proceedings.

Cathepsin-K is a sensitive immunohistochemical marker for detection of micro-granulomas in hypersensitivity pneumonitis.

UNLABELLED: Hypersensitivity Pneumonitis (HP) is an interstitial lung disease that occurs upon exposure to a variety of inhaled organic antigens. The presence of small non-caseating granulomas and isolated giant cells is not specific, but is considered a relevant histological feature for HP. The detection of granulomas is widely considered as easy on standard histological stains, but microgranuloma detection can be difficult and/or time consuming, especially in chronic HP cases. Cathepsin K (Cath-K) is a potent cysteine protease expressed at high levels in activated macrophages (osteoclasts, and epithelioid cells in granulomas), but is not expressed in resident macrophages thus representing a promising marker to rapidly detect and quantitatively evaluate microgranulomas in interstitial lung diseases. We analyzed the expression of Cath-K by immunohistochemistry in 22 subacute and chronic HP cases, using semi-quantitative scores. Control samples included normal lung tissue, and a variety of interstitial lung diseases: 3 Wegener's granulomatosis, 3 sarcoidosis, 3 tuberculosis, 1 berylliosis, 20 idiopathic pulmonary fibrosis (IPF), 2 Langerhans' cell histiocytosis, 5 nonspecific-interstitial pneumonia (NSIP), 5 cryptogenic organising-pneumonia (COP), 2 Airway-Centered Interstitial Fibrosis (ACIF), 5 desquamative interstitial pneumonia (DIP), 3 respiratory bronchiolitis interstitial lung disease (RB-ILD). Intense expression of Cath-K was demonstrated in epithelioid and giant cells in all cases containing granulomas (HP, sarcoidosis, Wegener's granulomatosis, berylliosis, tuberculosis). Among HP cases 19/22 (86.3%) contained granulomas that could be semiquantitatively evaluated. In all HP and control cases alveolar macrophages did not express Cath-K, including cases characterised by large collections of alveolar macrophages such as DIP and RB-ILD. CONCLUSIONS: Cath-K represents a sensitive and specific marker to detect and quantitate granulomatous reactions in interstitial lung diseases, and is particularly useful in chronic HP cases.

Cathepsin-k as a diagnostic marker in the identification of micro-granulomas in Crohn's disease.

Crohn's disease is a chronic inflammatory bowel disease, whose aetiology and pathogenesis are still unknown. The occurrence of epithelioid granulomas is one characteristic feature of the disease since these lesions are found in the bowel wall in 50-87% of colectomy specimens. Although granulomas are not pathognomonic, their identification is considered a relevant element for diagnosis. Cathepsin-k, a papain-like cysteine protease, is involved in bone remodelling, and has been widely used as a immunohistochemical marker for the in situ detection of osteoclasts. Interestingly, the expression of this potent protease is also significantly increased in stimulated tissue macrophages, epithelioid cells and granulomas, but is not expressed in resident tissue macrophages. In the present study, we evaluated Cathepsin-k expression as a diagnostic tool in the identification of small granulomas in Crohn's disease. Formalin-fixed and paraffin-embedded samples of 10 cases of Crohn's disease were collected from surgical ileo-colic resections followed by comparison of Cathepsin-k and CD68 immunoreactivity. Granulomas were identified in 4 of 10 cases examined in haematoxylin & eosin preparations. Cathepsin-k enabled the identification of small granulomas (with a diameter between 100 and 200 microm) in 6 of 10 cases, mainly localized within the submucosa and muscular layers. When compared to CD68, Cathepsin-k immunoreactivity was generally absent or only weakly expressed in resting tissue macrophages, thus allowing better identification of activated epithelioid cells. Based on these results, Cathepsin-k appears to be a reliable tool for the precise and rapid identification of small epithelioid granulomas in Crohn's disease.

[Probiotics and children: is an integration useful in allergic diseases?]. / Probiotici e bambini: è utile un'integrazione nei casi di allergia?

Recent studies report that some probiotic strains are able to improve allergic diseases. For this reason, we would verify tolerability and efficacy of a industrial preparation of Lactobacillus paracasei (11,688; Proge Farm, Italy) and Lactobacillus salivarius (11,794; Proge Farm, Italy) and value their "in vitro" immunomodulatory effect. We know that, after birth, there's a persistence of Th2 immune response that predisposes to atopy, whereas commensal bacteria are able to induce a Th1 immune response that counter-balances the original response. The "in vivo" study was set up with the recruitment of 20 atopic pediatric patients treated 30 days with 2 doses of Fiorilac (Sharper, Italy), a preparation of the two strains in the proportion of 1:12. Only one patient referred significant improvements of atopic disease, 19 patients reported a good tolerability to the product and 3 patients had a regularization of intestinal function. Immunological tests showed an increase of Th1 immune response as in CD4+ lymphocytes percentage as of IL-12 and IL-10 cytokines production and a significant increase of natural killer (NK) activity, which predisposes to an active response to viral infections and neoplastic transformations.

In vitro activation of mononuclear cells by two probiotics: Lactobacillus paracasei I 1688, Lactobacillus salivarius I 1794, and their mixture (PSMIX).

BACKGROUND: Most studies on probiotics have described their effects on the human immune system after ingestion of LAB, but little is known about their effect on in vitro stimulation of human immune cells. AIM OF THE STUDY: Evaluate the "in vitro" activity of Lactobacillus paracasei (I 1688), Lactobacillus salivarius (I 1794), and a commercial mix of the two (PSMIX, Proge Farm), on immune cells from healthy individuals. MATERIALS: Two probiotic strains, Lactobacillus salivarius (I 1794; Proge Farm, Italy) and Lactobacillus paracasei (I 1688; Proge Farm, Italy), which are contained in the functional food ENTEROBACILLI, were evaluated for their ability to stimulate peripheral blood mononuclear cells and modulate surface phenotype and cytokine production. RESULTS: All subjects responded to the bacteria, with different levels of response. The cell populations that showed a significant percent increase were CD4+/CD25+ cells (T-helper activated regulatory cells), CD8+/CD25+ (T-suppressor/cytotoxic activated cells), and CD16+/CD56+ (NK cells) (p<0.05). IL-12 and IFN-gamma in vitro production significantly increased with exposure to probiotics (p<0.05 for both). CONCLUSIONS: This study provides the first evidence that Lactobacillus paracasei and Lactobacillus salivarius are capable of inducing a specific immune response that may be useful in the clinical setting for improving innate and adaptive immune responses.

Metabolic effects of pioglitazone and rosiglitazone in patients with diabetes and metabolic syndrome treated with metformin.

BACKGROUND: Metformin is considered the gold standard for type 2 diabetes treatment as monotherapy and in combination with sulphonylureas and insulin, whereas the combination of metformin with thiazolidinediones is relatively less studied. The aim of the present study was to assess the differential effect on glycaemic metabolism and lipid variables of the combination of metformin plus pioglitazone or metformin plus rosiglitazone in diabetic patients with metabolic syndrome. METHODS: All patients began metformin and were randomized to receive pioglitazone or rosiglitazone for 12 months. We assessed body mass index, glycated haemoglobin, fasting plasma glucose, postprandial plasma glucose, fasting plasma insulin, postprandial plasma insulin, homeostasis model assessment index, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, apolipoprotein A-I, and apolipoprotein B. RESULTS: Significant decreases in glycated haemoglobin, fasting plasma glucose, postprandial plasma glucose, fasting plasma insulin, and postprandial plasma insulin were seen after 9 and 12 months in both groups. Homeostasis model assessment index improved at 12 months in both groups. Significant total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, apolipoprotein A-I, and apolipoprotein B improvement was observed in pioglitazone group after 12 months, but not in the rosiglitazone group. These variations were significant between groups. CONCLUSION: The combination of metformin plus thiazolidinediones was able to improve glycaemic control compared with previous therapy. Pioglitazone was associated with a significant improvement in lipid and lipoprotein variables.

Effects of rosiglitazone and pioglitazone combined with metformin on the prothrombotic state of patients with type 2 diabetes mellitus and metabolic syndrome.

In this multicentre, randomized, double-blind, controlled, parallel-group trial, 103 patients with type 2 diabetes mellitus and metabolic syndrome were randomized to receive one of two thiazolidinediones--pioglitazone or rosiglitazone--in combination with 1500 mg/day of metformin, increasing up to 3000 mg/day, for 12 months. Anthropometric, metabolic, coagulation and fibrinolysis parameters were assessed at baseline and after 3, 6, 9 and 12 months. Significant decreases in glycosylated haemoglobin, fasting plasma glucose and post-prandial plasma glucose levels were seen after 9 and 12 months in both groups, and significant decreases in fasting plasma insulin and post-prandial plasma insulin levels were seen after 12 months in both groups. In both groups, improvement in the homeostasis model assessment index compared with baseline was obtained only after 12 months. Plasminogen activator inhibitor-1 levels were significantly lower in both groups after 12 months compared with baseline values. In patients with type 2 diabetes mellitus and metabolic syndrome, the combination of metformin plus thiazolidinediones improved glycaemic control and produced a slight but significant reduction in plasminogen activator inhibitor-1 levels.

Metformin-pioglitazone and metformin-rosiglitazone effects on non-conventional cardiovascular risk factors plasma level in type 2 diabetic patients with metabolic syndrome.

BACKGROUND AND OBJECTIVE: Metformin is considered the gold standard for type 2 diabetes treatment as monotherapy and in combination with sulphonylureas and insulin. The combination of metformin with thiazolidinediones is less well studied. The aim of the present study was to assess the differential effect, and tolerability, of metformin combined with pioglitazone or rosiglitazone on glucose, coagulation and fibrinolysis parameters in patients with type 2 diabetes mellitus and metabolic syndrome. METHODS: This 12-month, multicentre, double-blind, randomized, controlled, parallel-group trial was conducted at three study sites in Italy. We assessed patients with type 2 diabetes mellitus (duration >or=6 months) and with metabolic syndrome. All patients were required to have poor glycaemic control with diet, or experienced adverse effects with diet and metformin, administered up to the maximum tolerated dose. Patients were randomized to receive either pioglitazone or rosiglitazone self-administered for 12 months. We assessed body mass index (BMI), glycaemic control [glycosylated haemoglobin (HbA(1c)), fasting and postprandial plasma glucose and insulin levels (FPG, PPG, FPI, and PPI respectively), homeostasis model assessment (HOMA) index], lipid profile [total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C) and triglycerides (TG)], lipoprotein (a) [Lp(a)] and homocysteine (HCT) at baseline and at 3, 6, 9 and 12 months of treatment. RESULTS AND DISCUSSION: No BMI change was observed at 3, 6, 9 and 12 months in either group. Significant HbA(1c) decreases were observed at 9 and 12 months in both groups. After 9 and 12 months, mean FPG and PPG levels decreased in both groups. Decreases in FPI and PPI were observed at 9 and 12 months compared with the baseline in both groups. Furthermore, in both groups, the HOMA index improved but only at 12 months. Significant TC, LDL-C, HDL-C, TG improvement was present in the pioglitazone group at 12 months compared with the baseline values, and these variations were significantly different between groups. No TC, LDL-C, TG improvement was present in the rosiglitazone group after 12 months. Significant Lp(a) and HCT improvement was present in the pioglitazone group at 12 months compared with the baseline values, and Lp(a) change was significant compared with the rosiglitazone group. Significant HCT decrease was observed in the rosiglitazone group at the end of the study. In our type 2 diabetic patients, both drugs were safe and effective for glycaemic control and improving HCT plasma levels. However, long-term treatment with metformin plus pioglitazone significantly reduced Lp(a) plasma levels, whereas metformin + rosiglitazone did not. CONCLUSION: For patients with type 2 diabetes mellitus and metabolic syndrome, combined treatment with metformin and rosiglitazone or pioglitazone is safe and effective, However, the pioglitazone combination also reduced the plasma Lp(a) levels whereas the rosiglitazone combination did not.

Successful treatment of end-stage hypertrophic cardiomyopathy with biventricular cardiac pacing.

The beneficial use of biventricular pacing is reported in a patient with end-stage hypertrophic cardiomyopathy, intraventricular conduction delay and echocardiographic evidence of intraventricular dyssyncrony. Marked improvement in clinical status, left ventricular ejection fraction and peak VO2 were observed. As far as we know, this is the first report of a beneficial effect of a biventricular device in this subset of patients, and may be worth further investigation.

Apo(a) size in ischemic stroke: relation with subtype and severity on hospital admission.

OBJECTIVE: To determine the distribution of apolipoprotein (a) (apo[a]) isoforms and their relation to the clinical severity of different ischemic stroke subtypes. METHODS: Ninety-four hospital cases with a first-ever ischemic stroke and 188 randomly selected control subjects matched for age, gender, and ethnicity were enrolled. Stroke etiology was defined according to Trial of Org 10172 in Acute Stroke Treatment criteria. NIH Stroke Scale (NIHSS) was used to assess the severity of stroke on admission. RESULTS: In univariate analysis, the presence of at least one small apo(a) isoform was associated with ischemic stroke in men (p = 0.02) but not in women (p = 0.33). After allowance for age, gender and traditional vascular risk factors, subjects carrying at least one small apo(a) isoform were at increased risk of atherothrombotic stroke (odds ratio [OR] 7.1, 95% CI 2.8 to 17.5, p = 0.00001) but not of lacunar infarction (OR 1.1, 95% CI 0.5 to 2.7, p = 0.78). Multivariate logistic regression analysis revealed that in the atherothrombotic stroke group, the presence of at least one small-sized apo(a) phenotype was associated with an NIHSS score > or =6 (OR 13.6, 95% CI 1.6 to 111.9, p = 0.015). CONCLUSION: Small apolipoprotein (a) isoforms distinguish atherothrombotic stroke from lacunar infarction and are associated with the severity of atherothrombotic stroke.

Apolipoprotein(a) size polymorphism is associated with coronary heart disease in polygenic hypercholesterolemia.

BACKGROUND AND AIM: In addition to high serum cholesterol levels, various cardiovascular risk factors may be involved in the development of coronary heart disease (CHD) in hypercholesterolemic subjects. As the levels of lipoprotein(a) [Lp(a)], an important and independent cardiovascular risk factor, are high in polygenic hypercholesterolemia (PH), we investigated plasma Lp(a) levels and apolipoprotein(a) [apo(a)] phenotypes in relation to occurrence of CHD events in PH patients. METHODS AND RESULTS: Lp(a) levels and apo(a) isoforms were determined in 191 PH patients, 83 normocholesterolemic subjects with CHD, and 94 normocholesterolemic controls without CHD. Lp(a) levels were similar in the hypercholesterolemic subjects with (n=100) or without CHD (n=91): 21.4 (range 6.6-59.23) vs 18.5 (range 5.25-57.25) mg/dL (p=NS). Low molecular weight apo(a) isoforms were more prevalent (55%) in the PH patients with CHD, whereas high molecular weight apo(a) isoforms were more prevalent (62.6%) in those without CHD: this difference was significant (p<0.05). A stepwise multiple-discriminant analysis made in order to determine the independence of common cardiovascular risk factors, Lp(a) levels and low molecular weight apo(a) isoforms in predicting CHD among hypercholesterolemic subjects showed that the presence of a positive family history of CHD, smoking, age, and the presence of at least one apo(a) isoform of low molecular weight were independently associated with CHD. CONCLUSIONS: Despite high Lp(a) levels, our findings do not support the hypothesis that Lp(a) plays an independent role in determining clinical CHD in PH subjects. However, the presence of at least one low molecular weight apo(a) isoform is an independent genetic predictor of CHD in hypercholesterolemic subjects. Together with other cardiovascular risk factors, apo(a) phenotypes should be assessed to evaluate the overall CHD risk status of all subjects with high serum cholesterol levels.

Molecular analysis of CDKN1C and TP53 in sporadic adrenal tumors.

OBJECTIVE: To evaluate the roles of the CDKN1C (P57KIP2) gene, which encodes for the cyclin-dependent kinase inhibitor CDNC, and the TP53 tumor suppressor gene in adrenal tumorigenesis, as a means of investigating the molecular basis of sporadic adrenal tumors, which is unknown. DESIGN: Screening for the presence CDKN1C and TP53 mutations and analyzing the expression pattern of CDNC, P53 and its downstream effector CDN1 (P21WAF1/CIP1) in a series of 79 sporadic adrenal tumors. METHODS: Single-strand conformation polymorphism and sequencing were used for mutation analysis of CDKN1C and TP53 in blood and adrenal tissue samples. In a subgroup of 48 tissues, CDKN1C expression was evaluated by RT-PCR and immunohistochemistry. Immunohistochemical analysis of P53 and CDN1 was performed. RESULTS: No somatic mutations of CDKN1C were found in the tumors analyzed, in spite of low/absent CDNC expression in adrenocortical adenomas and carcinomas. Mutations in the TP53 gene were present in 70% of adrenocortical carcinomas, associated with abnormal P53 and CDN1 expression, but not in benign neoplasms. In the normal adrenal cortex, CDNC expression was strictly nuclear and confined to the cortical zone (i.e. zona glomerulosa and reticularis), with no staining in the medulla. CONCLUSIONS: Mutations in the TP53 gene are frequent in adrenocortical carcinomas and might be used as a marker of malignancy. In the normal adrenal cortex, the zone-specific pattern of expression of CDNC suggests a role in adrenal differentiation.

Survivin is expressed on CD40 stimulation and interfaces proliferation and apoptosis in B-cell chronic lymphocytic leukemia.

In B-cell chronic lymphocytic leukemia (B-CLL), defective apoptosis causes the accumulation of mature CD5(+) B cells in lymphoid organs, bone marrow (BM), and peripheral blood (PB). These cells are the progeny of a proliferating pool that feeds the accumulating compartment. The authors sought to determine which molecular mechanisms govern the proliferating pool, how they relate to apoptosis, and what the role is of the microenvironment. To begin to resolve these problems, the expression and modulation of the family of inhibitor of apoptosis proteins (IAPs) were investigated, with consideration given to the possibility that physiological stimuli, such as CD40 ligand (CD40L), available to B cells in the microenvironment, might modulate IAP expression. The in vitro data on mononuclear cells from PB or BM of 30 patients demonstrate that B-CLL cells on CD40 stimulation express Survivin and that Survivin is the only IAP whose expression is induced by CD40L. Through immunohistochemistry, in vivo Survivin expression in lymph node (LN) and BM biopsies was evaluated. In reactive LN, Survivin was detected only in highly proliferating germinal center cells. In LN from patients with B-CLL, Survivin was detected only in pseudofollicles. Pseudofollicle Survivin(+) cells were actively proliferating and, in contrast to Survivin(+) B cells found in normal GC, were Bcl-2(+). In B-CLL BM biopsies, CD5(+), Survivin(+) cells were observed in clusters interspersed with T cells. These findings establish that Survivin controls the B-CLL proliferative pool interfacing apoptosis and that its expression may be modulated by microenvironmental stimuli.

Identification of residues in the N-terminal domain of the Yersinia tyrosine phosphatase that are critical for substrate recognition.

YopH is a 468-amino acid protein-tyrosine phosphatase that is produced by pathogenic Yersinia species. YopH is translocated into host mammalian cells via a type III protein secretion system. Translocation of YopH into human epithelial cells results in dephosphorylation of p130(Cas) and paxillin, disruption of focal adhesions, and inhibition of integrin-mediated bacterial phagocytosis. Previous studies have shown that the N-terminal 129 amino acids of YopH comprise a bifunctional domain. This domain binds to the SycH chaperone in Yersinia to orchestrate translocation and to tyrosine-phosphorylated target proteins in host cells to mediate substrate recognition. We used random mutagenesis in combination with the yeast two-hybrid system to identify residues in the YopH N-terminal domain that are involved in substrate-binding activity. Four single codon changes (Q11R, V31G, A33D, and N34D) were identified that interfered with binding of the YopH N-terminal domain to tyrosine-phosphorylated p130(Cas) but not to SycH. These mutations did not impair YopH translocation into HeLa cells infected with Yersinia pseudotuberculosis. Introduction of the V31G substitution into catalytically inactive (substrate-trapping) forms of YopH interfered with the ability of these proteins to bind to p130(Cas) and to localize to focal adhesions in HeLa cells. In addition, the V31G substitution reduced the ability of catalytically active YopH to dephosphorylate target proteins in HeLa cells. These data indicate that the substrate- and SycH-binding activities of the YopH N-terminal domain can be separated and that the former activity is important for recognition and dephosphorylation of substrates by YopH in vivo.