In Situ Tissue Labeling of Cerebral Amyloid Using HIV-Related Tat Peptide.

Delivering peptide-based drugs to the brain is a major challenge because of the existence of the blood-brain barrier (BBB). To overcome this problem, cell-penetrating peptides derived from proteins that are able to cross biological membranes have been used as cell-permeable and brain-penetrant compounds. An example is the transactivator of transcription protein transduction domain (Tat) of the human immunodeficiency virus. The basic domain of Tat is formed of arginine and lysine amino acid residues. Tat has been used as brain-penetrant carrier also in therapies for Alzheimer disease (AD), the most common form of dementia characterized by extracellular cerebral deposits of amyloid made up of Aß peptide. The aim of our study was to assess whether Tat bind to amyloid deposits of AD and other amyloidoses. An in situ labeling using biotinylated Tat 48-57 peptide was employed in the brain tissue with amyloid deposits made up of Aß (patients with AD and transgenic AD mice), of prion protein (patients with Gerstmann-Straussler-Scheinker disease), and other amyloidosis, processed by different fixations and pretreatments of histological sections. Our results showed that Tat peptide binds amyloid deposits made up of Aß, PrP, and immunoglobulin lambda chains in the brain and other tissues processed by alcoholic fixatives but not in formalin-fixed tissue. The fact that biotinylated Tat peptide stains amyloid of different biochemical composition and the specific charge characteristics of the molecules suggests that Tat may bind to heparan sulfate glicosaminoglicans, that are present in amyloid deposits. Inhibition of the binding by Tat pre-incubation with protamine reinforces this hypothesis. Binding of Tat to amyloid deposits should be kept in mind in interpreting the results of studies employing this molecule as brain-penetrating compound for the treatment of cerebral amyloidoses. Our results also suggest that Tat may be helpful for the analysis of the mechanisms of amyloidogenesis, and in particular, the interactions between specific amyloid peptides and glicosaminoglicans.

A key role for abdominal ultrasound examination in "difficult" diagnoses of celiac disease.

PURPOSE: To evaluate the usefulness of abdominal ultrasound examination (US) for the diagnostic workup of cases of suspected CD involving negative serum antibodies and difficult diagnosis. MATERIALS AND METHODS: 524 consecutive patients with symptoms of suspected CD underwent an extensive diagnostic workup. 76 (14 %) were excluded since they were positive for serum anti-tTG and/or EmA antibodies. 377 were excluded since they were diagnosed with something other than CD or did not have the alleles encoding for HLA DQ 2 or DQ 8. A diagnosis of CD with negative serum antibodies was probable in 71 patients who underwent abdominal US and duodenal biopsy for histology evaluation. RESULTS: Intestinal histology and subsequent clinical and histological follow-up confirmed the CD diagnosis in 12 patients (GROUP 1) and excluded it in 59 subjects (GROUP 2). Abdominal US showed that the presence of dilated bowel loops and a thickened small bowel wall had a sensitivity of 83 % and a negative predictive value (NPV) of 95 % in CD diagnosis. Furthermore, in 11 of the 12 CD seronegative patients there was at least one of these two abdominal US signs. Therefore, considering the presence of one of these two signs, abdominal US sensitivity increased to 92 % and NPV to 98 %. CONCLUSION: Abdominal US is useful in the diagnostic workup of patients with a high clinical suspicion of CD but with negative serology.

Haematological deficiencies in patients with recurrent aphthosis.

BACKGROUND: Recurrent aphthosis is a common oral ulcerative condition consisting also of a subset of similar ulcers, properly named 'aphthous-like' ulcers (ALU), linked to systemic diseases and among these, to iron, folic acid and vitamin B(12) deficiencies. OBJECTIVES: The main objectives of this study were: (i) to evaluate the association between recurrent aphthosis and the most common predisposing factors; (ii) to assess the frequency of ALU in recurrent aphthosis; (iii) to verify the efficacy of a replacement therapy in all ALU patients. METHODS: Thirty-two adults with recurrent aphthosis and 29 otherwise healthy controls were consecutively recruited, interviewed and subjected to haematological investigations. RESULTS: Family history of recurrent aphthosis was significantly associated (P < 0.01). The overall frequency of haematinic deficiencies was 56.2% in recurrent aphthosis patients vs. 7% in controls (P < 0.0001). All ALU patients with a negative family history showed a complete remission of the ulcerative episodes after replacement therapy, while those with a positive family history only had a reduction in frequency and severity. In the logistic regression model, only family history was associated with recurrent aphthosis (P = 0.0137). CONCLUSION: The strong association with familiarity, the unexpected higher frequency of ALU (compared with the idiopathic variant) and the good response to replacement therapy means that familiarity should always be investigated. Furthermore, routine haematological screening and tests for serum iron, folic acid and vitamin B(12) deficiencies should be assessed in all patients with recurrent aphthosis to treat any nutritional deficiency and to prevent more important related systemic manifestations.

"The Linosa Study": epidemiological and heritability data of the metabolic syndrome in a Caucasian genetic isolate.

BACKGROUND AND AIMS: Growing evidence suggests that the metabolic syndrome (MetS) has both a genetic and environmental basis. To evaluate the possibility of a further genetic analysis, we estimated prevalence rates and heritabilities for the MetS and its individual traits in the adult population of Linosa, a small and isolated Italian Island in the southern-central part of the Mediterranean Sea. METHODS AND RESULTS: The Linosa Study (LiS) group consisted of 293 Caucasian native subjects from 51 families (123 parents; 170 offsprings). The MetS was defined according to NCEP/ATP III criteria and the following prevalence rates were calculated: hyperglycaemia 20.3%; central obesity 34.9%; hypertension 43.4%; hypertriglyceridaemia 29.9%; "low HDL" 56.6%; MetS 29.9%. Waist circumference was significantly related to all the quantitative parameters included in the NCEP/ATP III MetS definition. The MetS showed a heritability of 27% (p=0.0012) and among its individual components, treated as continuous and discrete traits, heritability ranged from 10% for blood glucose to 54% for HDL-cholesterol. Among MetS subtypes, the clustering of central obesity, hypertriglyceridaemia and "Iow HDL" had the highest heritability (31%; p<0.001). CONCLUSION: These data showed high prevalence rates for the MetS and its related traits in an isolated and small Caucasian population. The appreciable heritability estimates for the MetS and some of its components/clusters in the LiS population might support the observation of genetic factors underlying the pathogenesis of the MetS and encourage further analysis to identify new susceptibility genes.

Atherogenic forms of dyslipidaemia in women with polycystic ovary syndrome.

OBJECTIVE: Dyslipidaemia is very common in patients with polycystic ovary syndrome (PCOS) but, beyond plasma lipids, atherogenic lipoprotein (Lp) and apolipoprotein (apo) alterations are still ill defined. DESIGN: We measured concentrations of apoB, Lp(a) and small, dense low-density lipoprotein (LDL) in 42 patients with PCOS [age: 28 +/- 7 years, body mass index (BMI): 27 +/- 5 kg/m(2)] vs. 37 age- and BMI-matched healthy controls. METHODS: Elevated Lp(a) levels considered were those > 30 mg/dl while elevated apoB concentrations were those > 100 g/l. RESULTS: Polycystic ovary syndrome showed increased triglycerides levels (p = 0.0011) and lower high-density lipoprotein (HDL)-cholesterol concentrations (p = 0.0131) while total- and LDL cholesterol were similar. PCOS also showed smaller LDL size (p = 0.0005), higher levels of total small, dense LDL (p < 0.0001), higher concentrations of Lp(a), as considered as absolute values (p = 0.0143) and log-transformed (p = 0.0014), while no differences were found in apoB levels. Elevated Lp(a) concentrations were found in 24% of PCOS, while elevated apoB levels were relatively uncommon (14%). Spearman correlation analysis revealed that Lp(a) concentrations were weakly correlated only with HDL-cholesterol levels (r = -0.378, p = 0.0431). In addition, 36% of patients with PCOS with normal plasma lipid profile showed elevated levels of Lp(a), apoB or small, dense LDL. CONCLUSIONS: Atherogenic Lp abnormalities may be found in one-third of women with PCOS who have a normal lipid pattern. Future prospective studies are needed to test to which extent such atherogenic forms of dyslipidaemia may contribute to the increased cardiovascular risk in young women with PCOS.

Histomorphology of healthy oral mucosa in untreated celiac patients: unexpected association with spongiosis.

BACKGROUND: The jejunal mucosa is the major site involved in celiac disease, but modifications have also been found in the gastric, rectal and esophageal mucosa. Few studies have focused on the histomorphological features of the oral mucosa in celiac disease patients. Our objectives were: (i) to assess the presence, quality and intensity of lymphocytic infiltrate in clinically healthy oral mucosa and its relation to celiac disease severity (villous height to crypt depth ratio); and (ii) to detect any other histological features connected to celiac disease. METHODS: Twenty-one untreated celiac disease patients (age range 13-68 years) with clinically healthy oral mucosa were enrolled and compared with 14 controls. Intestinal and oral biopsies were carried out and specimens were evaluated after staining with hematoxylin and eosin. RESULTS: Intra-epithelial lymphocyte B and T infiltrates of the oral mucosa were found to be similar in both groups; likewise, intensity of the lymphocytic infiltrate in the lamina propria was similar in both groups and was not related to intestinal damage; important signs of spongiosis were found to be more significantly present in celiac disease patients compared with controls (P = 0.0002). CONCLUSIONS: Our study showed that the healthy oral mucosa of untreated patients does not reflect the intestinal damage by celiac disease, but it is unexpectedly affected by spongiosis, as being detected for the first time in the literature. This latter feature could be related to gliadin ingestion and could contribute to explain the higher susceptibility of celiac disease patients to suffering from oral mucosa lesions.

Clinical importance of eosinophil count in nasal fluid in patients with allergic and non-allergic rhinitis.

Eosinophil count in nasal fluid (ECNF) was used to differentiate nasal pathologies. Receiver operating characteristic (ROC) curve analysis and the area under the curve (AUC) were performed to evaluate the ECNF's accuracy in distinguishing allergic rhinitis (AR) from non-allergic rhinitis (NAR). We also evaluated the accuracy of ECNF in recognizing patients with mild and severe symptoms of rhinitis and patients with ineffective and effective clinical responses to antihistamines. 1,170 consecutive adult patients with a clinical history of rhinitis were studied. ECNF's median in AR was 6.0 and 2.0 in NAR and the best cut-off value was > 3.0, AUC = 0.75. ECNF's median in AR with mild nasal symptoms was 3.0 and 7.0 with severe symptoms, and the best cut-off value was 4.0, AUC = 0.90. ECNF's median in NAR with mild nasal symptoms was 2.0 and 8.5 with severe symptoms, and the best cut-off value was > 4.0, AUC = 0.86. ECNF's median in AR with effective clinical response to antihistamines was 4.0 and 8.0 with ineffective response, the best cut-off value was < or = 5.0, AUC = 0.94. ECNF's median in NAR with an effective clinical response to antihistamines was 1.0 and 2.0 with ineffective response, and the best cut-off value was < or = 3.0, AUC = 0.64. Our results suggest an interesting practical use of ECNF data as evaluator of the clinical severity both AR and NAR. As predictor of the clinical response to antihistamines, ECNF is accurate only in patients with AR. The ECNF's performance was moderately accurate in distinguish patients with AR and NAR.

A novel insertional mutation in the prion protein gene: clinical and bio-molecular findings.

A young man, presenting with early onset of personality and behavioural changes followed by slowly progressive cognitive impairment associated with marked bi-parietal cerebral atrophy, was found to carry a novel seven extra-repeat insertional mutation in the prion protein gene (PRNP). In vitro, the mutated recombinant prion protein (PrP) showed biochemical properties that were consistent with pathological PrP variants. Our results further underline the heterogeneity of neurological pictures associated with insertional mutations of PRNP, indicating the diagnostic difficulties of sporadic cases with early-onset atypical dementia.

Atypical presentation of Creutzfeldt-Jakob disease: the first Italian case associated with E196K mutation in the PRNP gene.

Genetic Creutzfeldt-Jakob disease (gCJD) is caused by a range of mutations in the prion protein gene (PRNP). We describe the first Italian case of gCJD associated with the rare PRNP E196K mutation. The disease showed an atypical presentation featuring dementia without motor signs in a 75-year-old woman. The case lacked both a known family history of a similar neurological disease and the typical EEG pattern; it was misdiagnosed as frontotemporal dementia. The present case emphasizes that vigilance must be kept high to avoid missing gCJD cases falling outside a typical phenotypical presentation and a known family history, especially in the elderly, in whom an alternative, more common, but incorrect diagnosis may be made.

Coeliac disease: oral ulcer prevalence, assessment of risk and association with gluten-free diet in children.

AIMS: Oral mucosal lesions may be markers of chronic gastrointestinal disorders, such as those causing malabsorption. Our objectives were to assess the prevalence of recurrent oral aphthous-like ulcers in coeliac disease patients living in the Mediterranean area, and to evaluate the impact of a gluten-free diet. METHODS: A test group of 269 patients (age range 3-17 years) with coeliac disease confirmed both serologically and histologically was compared with a control group of 575 otherwise clinically healthy subjects for the presence, or a positive history of aphthous-like ulcers. Coeliac disease patients with aphthous-like ulcers were re-evaluated 1-year after starting a gluten-free diet. RESULTS: Aphthous-like ulcers were found significantly more frequently in coeliac disease, in 22.7% (61/269) of patients with coeliac disease versus 7.1% (41/575) of controls (p=<0.0001; chi-square=41.687; odds ratio=4.3123; 95% confidence interval=2.7664:6.722). Most coeliac disease patients with aphthous-like ulcers and adhering strictly to gluten-free diet (71.7%; 33/46) reported significant improvement on gluten-free diet, with no or reduced episodes of aphthous-like ulcers (p=0.0003; chi-square=13.101; odds ratio=24.67; 95% confidence interval=2.63:231.441). CONCLUSIONS: The epidemiological association found between coeliac disease and aphthous-like ulcers suggests that recurrent aphthous-like ulcers should be considered a risk indicator for coeliac disease, and that gluten-free diet leads to ulcer amelioration.

Utilization of extremely low frequency (ELF) magnetic fields in chronic disease; five years experience: three case reports.

We present three examples of the use of ELF magnetic therapy, two cases of multiple sclerosis and one of chronic pulmonary disease. In each of the two MS cases the Seqex device was applied as an adjunct to antioxidant medication two times a week for six weeks. Radiological and MRI examination indicated improvement in the two MS patients and stabilization in the patient with obstructive pulmonary disease following merely five treatments.

A novel phenotype of sporadic Creutzfeldt-Jakob disease.

An atypical case of sporadic Creutzfeldt-Jakob disease (CJD) is described in a 78-year-old woman homozygous for methionine at codon 129 of the prion protein (PrP) gene. The neuropathological signature was the presence of PrP immunoreactive plaque-like deposits in the cerebral cortex, striatum and thalamus. Western blot analysis showed a profile of the pathological form of PrP (PrP(Sc)) previously unrecognised in sporadic CJD, marked by the absence of diglycosylated protease resistant species. These features define a novel neuropathological and molecular CJD phenotype.

FokI polymorphism of the vitamin D receptor gene correlates with parameters of bone mass and turnover in a female population of the Italian island of Lampedusa.

One of the most promising genetic approaches to dissecting a multifactorial disease is represented by genetically isolated population studies. We studied a genetic marker in a cohort of women living on the Mediterranean island of Lampedusa, a geographically isolated population. Lampedusa, located between the African coast and Sicily, consists of a young genetic isolate (<20 generations) with an exponential growth in the last generations. We analyzed the association between the FokI vitamin D receptor (VDR) gene polymorphism, previously proposed as a predictor of bone mass, with parameters of bone mass and turnover in a cohort of pre- and postmenopausal women living on Lampedusa. In 424 women (277 postmenopausal and 147 premenopausal), allelic frequencies were 49% for the F allele and 51% for the f allele. Using analysis of covariance, we found that subjects with ff genotype exhibited a significantly (P < 0.001) lower lumbar spine bone mass, by dual-energy X-ray absorptiometry, and lower values of bone ultrasonographic parameters (speed of sound and broadband ultrasound attenuation) relative to those with Ff and FF genotypes. Conversely, osteocalcin and serum cross-laps were significantly higher in ff and Ff compared to FF genotype. Our data suggest that FokI VDR polymorphism may contribute to the determination of bone mass and turnover in both pre- and postmenopausal women in this geographically isolated population.

Relationship between specific serum IgE to Ascaris lumbricoides and onset of respiratory symptoms in Bangladesh immigrants.

The role of helminths in asthma and/or rhinitis and in allergic sensitization is still unclear. We assessed the relationship between Ascaris-specific IgE, respiratory symptoms and allergic sensitization in Bangladesh immigrants. 246 individuals were examined from 1996 to 2001. Serum total IgE, Ascaris IgE, specific IgE to inhalant allergens, skin prick tests (SPT) and parasitological evaluation of the stool were performed. Total serum IgE were significantly higher in Ascaris-IgE positive (> 0.35 kU/L) individuals (806.5 [409.0-1436.0] kU/L vs. 207.0 [127.0-332.5] kU/L; P < 0.0001) and in subjects with respiratory symptoms (413.0 [239.0-1096.0] kU/L vs. 259.5 [147.0-387.0] kU/L), (P < 0.0001), but not in SPT positive subjects (413.0 [179.0-894.0] kU/L vs. 404.6 [305.0-1201.0] kU/L (P = 0.5). Ascaris-specific IgE were detected in 48 subjects with respiratory symptoms (40.0%) and in 46 subjects without respiratory symptoms (36.5%) (P = 0.5). The SPT positivity was similar between Ascaris-IgE seropositive (38.2%) and Ascaris-IgE seronegative (38.1%) subjects (P = 0.9). Total IgE and length of stay in Italy correlated with SPT positivity (OR 5.6 [CI 95% 1.5-19.8], P = 0.007, and OR 1.5 [CI 95% 1.3-1.7], P< 0.0001), and with respiratory symptoms (OR 13.7 [CI 95% 3.0-62.4];, P = 0.0007, and OR 2.4 [CI 95% 1.9-3.0], P < 0.0001). Ascaris-IgE were negatively associated with SPT positivity (OR 0.3 [CI 95% 0.1-0.8], P = 0.02) and with respiratory symptoms (OR 0.1 [CI 95% 0.04-0.7], P = 0.01). Our findings favour the role of environmental factors in the development of respiratory symptoms in immigrants, irrespective of Ascaris-IgE.

In vitro effects of aminobisphosphonates on Vgamma9Vdelta2 T cell activation and differentiation.

In this study we have evaluated the in vitro effects of four different aminobisphosphonates, alendronate, risedronate, neridronate and zoledronate, on Vgamma9Vdelta2 T cell activation and differentiation. All tested aminobisphosphonates induce an IL-2-dependent activation and expansion of Vgamma9Vdelta2 T lymphocytes in primary PBMC cultures of healthy donors. Most notably, they also determine a different distribution of Vgamma9Vdelta2 T cell subsets, with decrease of T(naive) and T(CM) cells and increase of T(EM) and T(EMRA) Vgamma9Vdelta2cells, indicating that in vitro treatment with aminobisphosphonates induces Vgamma9Vdelta2 T lymphocytes to differentiate towards an effector/cytotoxic phenotype. Accordingly, Vgamma9Vdelta2 T lymphocytes cultured with aminobisphosphonates and IL-2 showed a major content of IFN-gamma and acquired the ability to kill tumor target cells.

Significance of P16INK4A hypermethylation gene in primary head/neck and colorectal tumors: it is a specific tissue event? Results of a 3-year GOIM (Gruppo Oncologico dell'Italia Meridionale) prospective study.

BACKGROUND: Methylation of the p16 promoter is one of the most frequent mechanisms of gene inactivation; its incidence is extremely variable according to the type of tumor involved. Our purpose was to analyze the hypermethylation of the p16 promoter in laryngeal squamous cell carcinomas (LSCC), salivary gland (SG) tumors and in colorectal cancer (CRC), to detect any possible association with the clinicopathological features and to determine the prognostic significance of the p16 gene in the tumors analyzed. PATIENTS AND METHODS: The hypermethylation of the p16 promoter was prospectively analyzed, by MSP, in a consecutive series of 64 locally advanced LSCC patients, in a consecutive series of 33 SG tumor patients and in a consecutive series of 66 sporadic CRC patients. RESULTS: Hypermethylation was observed in 9% of the LSCC cases, in all cases of SG cancer and in 21% of the CRC cases. No significant association was observed between p16 hypermethylation and clinicopathological variables in all the tissue samples analyzed. Moreover at univariate analysis p16 mutations were not independently related at disease relapse and death in LSCC and CRC. CONCLUSIONS: The results of this study suggest that the lack of p16 function could happen in advanced stage of SG tumors.

Detection and quantification of mammaglobin in the blood of breast cancer patients: can it be useful as a potential clinical marker? Preliminary results of a GOIM (Gruppo Oncologico dell'Italia Meridionale) prospective study.

BACKGROUND: Mammaglobin is expressed mainly in mammary tissue, overexpressed in breast cancer (BC) and rarely in other tissue. The aim of this study was to assess the sensitivity and specificity of transcript MGB1 detection and to evaluate the role of MGB1 as potential clinical marker for the detection of disseminated cancer cells in the blood of BC patients. PATIENTS AND METHODS: A consecutive series of 23 BC tissues, 36 peripheral blood BC samples and 35 healthy peripheral blood samples was prospectively recruited to investigate MGB1 expression by means of a quantitative Real Time RT-PCR assay. RESULTS: MGB1 overexpression in tissue samples of BC patients is significantly associated only with high level of Ki67 (P <0.05). None of the samples from peripheral blood of 35 healthy female individuals were positive for MGB1 transcript. In contrast MGB1 mRNA expression was detected in three of 36 (8%) peripheral blood of BC patients. CONCLUSIONS: Our preliminary results demonstrate that the detection of MGB1 transcript in peripheral blood of BC patients was specific but with low sensitivity. MGB1 overexpression by itself or in combination with Ki67 might be considered an index of BC progression.

Molecular detection of TP53, Ki-Ras and p16INK4A promoter methylation in plasma of patients with colorectal cancer and its association with prognosis. Results of a 3-year GOIM (Gruppo Oncologico dell'Italia Meridionale) prospective study.

BACKGROUND: Despite the improvement in detection and surgical therapy in the last years, the outcome of patients affected by colorectal carcinoma (CRC) remains limited by metastatic relapse. The aim of this study was to investigate the presence of free tumor DNA in the plasma of CRC patients in order to understand its possible prognostic role. PATIENTS AND METHODS: Ki-Ras, TP53 mutations and p16(INK4A) methylation status were prospectively evaluated in tumor tissues and plasma of 66 CRC patients. RESULTS: In 50 of the 66 primitive tumor cases (76%) at least one significant alteration was identified in Ki-Ras and/or TP53 and/or p16(INK4A) genes. Eighteen of the 50 patients presented the same alteration both in the plasma and in the tumor tissue. At univariate analysis, Ki-Ras mutations proved to be significantly related to quicker relapse (P <0.01), whereas only a trend towards statistical significance (P = 0.083) was observed for the TP53 mutations CONCLUSIONS: Detection of Ki-Ras and TP53 mutation in plasma should be significantly related to disease recurrence. These data suggest that patients with a high risk of recurrence can be identified by means of the analysis of tumor-derived plasma DNA with the use of fairly non-invasive techniques.

Is there a role for antileukotrienes in urticaria?

In vitro and in vivo clinical and experimental data have suggested that leukotrienes play a key role in inflammatory reactions of the skin. Antileukotriene drugs, i.e. leukotriene receptor antagonists and synthesis inhibitors, are a new class of anti-inflammatory drugs that have shown clinical efficacy in the management of asthma. We searched the MedLine database and carried out a manual search on journals specializing in allergy and dermatology for the use of antileukotriene drugs in urticaria. Montelukast might be effective in chronic urticaria associated with aspirin or food additive hypersensitivity or with autoreactivity to intradermal serum injection when taken with an antihistamine but not in moderate chronic idiopathic urticaria. Evidence for the effectiveness of zafirlukast and the 5-lipoxygenase inhibitor, zileuton, in chronic urticaria is mainly anecdotal. In addition, there is anecdotal evidence of effectiveness of antileukotrienes in primary cold urticaria, delayed pressure urticaria and dermographism. No evidence exists for other physical urticarias, including cholinergic, solar and aquagenic urticarias, vibratory angio-oedema, and exercise-induced anaphylaxis.