[Utility of denaturing high performance liquid chromatography (DHPLC) for the diagnosis of mevalonate kinase deficiency in periodic disease]. / Utilità della tecnica denaturing high performance liquid chromatography (DHPLC) per la diagnosi di deficit di mevalonato chinasi nelle febbri periodiche di sospetta natura autoinfiammatoria.

OBJECTIVES: We developed a genetic investigation using denaturing high performance liquid chromatography (DHPLC), in order to identify polymorphisms of the gene MVK in patients with autoinflammatory syndrome suspicion. METHODS: We evaluated 19 patients affected by recurrent fevers and other clinical manifestations usually found in autoinflammatory syndromes and not correlated with infections or autoimmune disease and 10 healthy controls. IgD level was measured in all patients. Molecular testing was performed in DNA extracted from PBMC and MVK gene was analysed either with DHPLC or with automatic sequencer. Primers for PCR amplifications, amplicon lengths and PCR conditions were designed in our laboratory. RESULTS: IgD level was normal in 14 patients. Healthy controls did not show any alteration of the DHPLC-profiles and of the DNA sequences. Twelve patients had at least one altered DHPLC-profile and these data have been confirmed by sequencing. In particular we detected the polymorphisms c.78+61A>G, S52N, S135S, D170D, c.632-18A>G, c.885+24G>A already described in the database INFEVERS. With DHPLC we got the results in shorter time (10 hours/patient) and with lower cost (40 euro/patient) in comparison to direct sequencing (25 hours and 150 euro/patient). CONCLUSIONS: High IgD levels do not represent an essential marker for diagnosis of MKD, as already reported in literature. DHPLC is a rapid low cost technique in order to screen mutations in patients with MKD suspicion. Twelve patients carried at the same time D170D and c.632-18A>G: such event suggests that these SNPs could be in linkage disequilibrium and that such polymorphisms could predispose to MKD.

An open population screening study for HFE gene major mutations proves the low prevalence of C282Y mutation in Central Italy.

BACKGROUND: The C282Y mutation in the HFE gene is responsible for most cases of hereditary haemochromatosis. AIM: To investigate the allele frequency of HFE mutations and the associations between mutations and cases of iron overload or liver diseases in an open population of Central Italy. METHODS: A total of 502 individuals over 8 years of age, comprising 203 males and 299 females, who were residents in Arsita (a small town in Central Italy), were assayed for: C282Y, H63D and S65C mutations of the HFE gene by TaqMan probes; body mass index, serum ferritin, transferrin saturation, transaminases, GGT, glucose, insulin, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, HBV and HCV serum markers. Information was obtained on alcohol intake. Liver ultrasound was performed in 334 (67%) subjects. RESULTS: The allele frequencies for C282Y, H63D and S65C were 2%, 15%, and 0.01%, respectively. C282Y/wt was found in 19 subjects (4%), H63D/wt in 127 (25%), H63D/H63D in 11 (2%) and S65C/wt in one (2.0 per thousand). No homozygosity for C282Y or compound mutation (C282Y/H63D) was found in the study population, but 27 subjects (5%) had TfSat >45% (including 10 subjects with high serum ferritin). Overall, 49 subjects (9.8%) were HCV-RNA-positive. Logistic regression analysis indicated that male gender (P = 0.000) and hepatic steatosis (P = 0.017) were independent variables correlating to a high serum ferritin. CONCLUSION: C282Y HFE mutation is less frequent in Central Italy than in Northern Italy.

Pancreatic cancer cells invasiveness is mainly affected by interleukin-1beta not by transforming growth factor-beta1.

BACKGROUND: We investigated in vitro whether IL-1beta and TGF-beta1 affect pancreatic cancer cell growth, adhesion to the extracellular matrix and Matrigel invasion. MATERIALS AND METHODS: Adhesion to fibronectin, laminin and type I collagen, and Matrigel invasion after stimulation with saline, IL-1beta and TGF-beta1 were evaluated using three primary and three metastatic pancreatic cancer cell lines. RESULTS: Extracellular matrix adhesion of control cells varied independently of the metastatic characteristics of the studied cell lines, whereas Matrigel invasion of control cells was partly correlated with the in vivo metastatic potential. IL-1beta did not influence extracellular matrix adhesion, whereas it significantly enhanced the invasiveness of three of the six cell lines. TGF-beta1 affected the adhesion of one cell line, and exerted contrasting effects on Matrigel invasion of different cell lines. CONCLUSIONS: IL-1beta enhances the invasive capacity of pancreatic cancer cells, whereas TGF-beta1 has paradoxical effects on pancreatic cancer cells; this makes it difficult to interfere with TGF-beta1 signaling in pancreatic cancer treatment.

Killer genes in pancreatic cancer therapy.

This review describes: 1. The main genetic alterations found in pancreatic cancer (EGF-R overexpression, SST-2 somatostatin receptor loss of expression, k-ras, p53 mutations and DPC4 mutations) and the effect of their replacements by gene therapy on tumor growth; 2. The use of suicide genes (HSV-TK and CD) for pancreatic cancer gene therapy in vitro and in vivo; 3. The implications for pancreatic cancer treatment when using cytotoxic bacterial toxins; 4. Viral and non-viral delivery systems for the transfer of therapeutical genes into pancreatic cancer cells. Overall both the correction of pancreatic cancer cells main genetic alterations and the use of suicide genes allow only partial tumor regression in vitro and in vivo. The lack of a 100% effect for any studied strategy considered alone, indicates the need for combined therapies to achieve a satisfactory treatment of this tumor.

Helicobacter pylori babA2, cagA, and s1 vacA genes work synergistically in causing intestinal metaplasia.

AIMS: To determine any associations between the Helicobacter pylori genes babA2, oipA, cagA and the s and m alleles of vacA. In addition, to verify whether these genes work synergistically or independently in causing gastritis, peptic ulcer, and intestinal metaplasia. METHODS: One hundred and sixty seven H pylori positive patients were studied (52 antral gastritis, 41 diffuse gastritis, 41 peptic ulcer, and 33 duodenitis). Helicobacter pylori virulence genes were amplified by means of the polymerase chain reaction. RESULTS: Significant associations were found between babA2 and the other H pylori genes studied. When considered singly, all the genes were associated with disease diagnosis, inflammation, and intestinal metaplasia. Four H pylori groups were defined. Group A: cagA-, s2m2, babA2-; group B: cagA+, s1m1, babA2+; group C: cagA+, s1m2, babA2+; group D: cagA+, s1m2, babA2-. Group A infecting strains were associated with less severe endoscopic and inflammatory conditions, whereas group B strains were associated with the worst endoscopic and inflammatory findings. Intestinal metaplasia was a rare finding in group A infected patients (< 10%), whereas it was frequent in those infected with group B strains (48%). CONCLUSIONS: The H pylori genes cagA, oipA "on", s1 and m1 vacA, and babA2 are associated with each other, possibly as a result of shared selective pressure. When coexpressed by the same H pylori strain, cagA, s1 and m1 vacA, and babA2 work synergistically in worsening inflammation. Infections caused by strains coexpressing cagA, s1m1 vacA, and babA2 are those at higher risk for intestinal metaplasia.

Helicobacter pylori non-cytotoxic genotype enhances mucosal gastrin and mast cell tryptase.

AIMS: To determine the association, if any, between H pylori genotype and the gastric mucosal variations in the levels of gastrin, somatostatin, tryptase, and histamine. METHODS: 49 patients affected by duodenal ulcer and 48 by non-ulcer dyspepsia were studied. To identify the H pylori genotype, the presence of the cagA gene and vacA alleles m1, m2, s1, and s2 were analysed by polymerase chain reaction. Gastrin, somatostatin, tryptase, and histamine were measured in antral mucosal biopsies. RESULTS: 57 patients were infected with H pylori (30 with duodenal ulcer and 27 with non-ulcer dyspepsia). Gastrin and tryptase were increased in patients with H pylori infection, although the variations were statistically significant only for gastrin; somatostatin and histamine were not influenced by H pylori infection. In patients with non-ulcer dyspepsia the absence of the cagA gene and the presence of vacA alleles s2 and m2 were associated with higher values of tryptase and to a lesser extent of gastrin. These associations were not found in patients with duodenal ulcer. CONCLUSIONS: The cagA negative s2m2 strain of H pylori may be less dangerous for the gastric mucosa than other H pylori strains since it enhances tryptase production by gastric mucosal mast cells; this enzyme is thought to stimulate tissue turnover and favour wound healing.

Total PSA, free PSA/total PSA ratio, and molecular PSA detection in prostate cancer: which is clinically effective and when?

OBJECTIVES: To ascertain when the serum determination of the free prostate-specific antigen (PSA)/total PSA (fPSA/tPSA) ratio is clinically useful, and whether the identification of PSA or prostate-specific membrane antigen (PSM) mRNA in circulating cells has diagnostic advantages over the determination of their protein product. METHODS: fPSA, tPSA, and the fPSA/tPSA ratio were determined in the sera of 50 men with benign nonprostatic urologic diseases (EPD), 112 patients with prostate cancer (PCa), and 218 with benign prostatic hyperplasia (BPH). mRNA was extracted from the circulating mononuclear cells of 13 EPD samples, 25 PCa samples, and 38 BPH samples. PSA and PSM mRNA signals were identified in these samples by means of reverse transcriptase-polymerase chain reaction. RESULTS: Overall, at a fixed specificity of 95%, the sensitivity of tPSA was 19% and that of the fPSA/tPSA ratio was 40% in distinguishing PCa from BPH. The fPSA/tPSA ratio allowed the discrimination of PCa from BPH with satisfactory sensitivity and specificity when considering patients less than 60 years of age (100% and 95%, respectively). PSA and PSM mRNA were positive in 1 and 7 of 13 EPD samples, 6 and 13 of 25 PCa samples, and 6 and 17 of 38 BPH samples. The Gleason score did not correlate with tPSA, the fPSA/tPSA ratio, PSA mRNA, or PSM mRNA. CONCLUSIONS: The serum determination of the fPSA/tPSA ratio is an excellent index of PCa for subjects younger than 60 years of age; the clinical utility of PSA mRNA identification in circulating cells needs to be validated by large follow-up studies, and the analysis of PSM mRNA seems to be of no clinical interest.

ME-PCR for the identification of mutated K-ras in serum and bile of pancreatic cancer patients: an unsatisfactory technique for clinical applications.

Our aim was to assess the clinical reliability of mutated K-ras detection in serum or bile for the diagnosis of pancreatic cancer using ME-PCR. DNA was extracted from 1 ml serum obtained from 29 patients with pancreatic cancer and 12 control subjects. ME-PCR was optimized using a mixture of normal DNA added with different amounts of mutated DNA. The analysis of sera obtained from the 29 patients and of bile obtained from 11 pancreatic cancer patients demonstrated the presence of mutated K-ras in two (6.9%) and four cases (36%). By contrast K-ras was not amplifiable in any of the 12 serum samples obtained from healthy controls. In conclusion the DNA obtained from pancreatic cancer patients' sera is suitable for K-ras amplification and for the identification of codon 12 point mutations. However ME-PCR alone has an unsatisfactory sensitivity for the detection of pancreatic cancer using serum DNA as starting template.

Suicide gene therapy with HSV-TK in pancreatic cancer has no effect in vivo in a mouse model.

AIM: To study in vivo whether pancreatic cancer tumour growth and metastasis can be modified by a gene construct with HSV-TK suicide gene and IL2 co-expression. METHODS: Seventy-eight female SCID mice were i.p. inoculated with retrovirally transduced or control MIA PaCa 2, CAPAN-1 and PANC-1 cell lines. The animals were then randomly selected for saline or ganciclovir (GCV) treatment from the second week, for a total of two weeks. RESULTS: Most inoculated mice developed tumour nodules and spleen metastases. The liver was colonized by control CAPAN-1 and MIA PaCa 2, but not by PANC-1. Tumours in transduced MIA PaCa 2 cell injected mice were smaller, and in transduced CAPAN-1 injected mice larger, than in control-inoculated mice. There were increased pancreatic and decreased spleen metastases from transduced CAPAN-1, and diminished liver involvement from transduced MIA PaCa 2. No differences were found between mice inoculated with transduced and control PANC-1 cell lines. GCV treatment had no effect on tumour's size or metastases. CONCLUSIONS: The HSV-TK suicide gene does not confer GCV sensitivity to pancreatic cancer in this in vivo model. Different pancreatic cancer cell lines cause different growth and metastasis patterns after inoculation in SCID mice, possibly because of variations in their inherent characteristics. The different effects of our vector on cell growth and metastasis may be attributable to the effects of the immunostimulatory cytokine IL2.

Metastatic colorectal cancer stimulates collagen synthesis by fibroblasts.

BACKGROUND: We ascertained in vitro whether there were any differences in the growth of fibroblasts and the production of collagen (PIIIP), in relation to the presence of conditioning sera or tumor tissues from colorectal cancer (CRC) patients with disease progression or regression after surgery. PATIENTS AND METHODS: Fibroblasts were conditioned with: 1) 10% of control (n=20) or CRC patients' (n=57) sera; 2) 10% of tumor tissue homogenates obtained from CRC patients without (group A, n=6) or with (group B, n=5) liver metastases. After surgery, 29/57 patients (group 1) developed while 28/57 (group 2) did not develop a recurrent disease. RESULTS: The ratio between cell growth and PIIIP production increases when fibroblasts were conditioned by group I (p<0.05), but not by controls or group 2 sera. Tumor homogenates from group B inhibited cell growth (p<0.001), while they induced PIIIP production (p<0.001) in comparison with results from group A. CONCLUSION: CRC non-metastatic cancer cells induce mainly fibroblasts growth, while metastatic cells mainly induce collagen synthesis. This effect is probably caused by soluble mediators, which partly pass into the systemic circulation.

Combined use of urinary UGP and serum CA 125 in the diagnosis of gynecological cancers.

UGP, the beta-core fragment of human chorionic gonadotropin has been proposed as a tumor marker for gynecological malignancies. This fragment may be detected in a single morning-specimen of urine using an enzyme immunoassay. In this study, the diagnostic usefulness of urine UGP and serum CA 125 measurement for gynecological neoplasias (149 cases) was evaluated using a control group of patients with benign gynecological diseases (69 cases) and healthy females (99 cases). Considering the neoplastic patients in comparison to patients with benign diseases, the best diagnostic efficiency (78%) was found to correspond to a cut-off level of 120 pmol/mol creatinine the sensitivity being 73% and the specificity 90%. With this cut-off, an efficiency of 82% for healthy controls was obtained. Since the menopausal condition increases UGP levels, and though no significant difference for UGP was found between healthy subjects and patients with benign diseases, we decided to consider the reference populations as a single group. Thus, we evaluated the UGP performance on the basis of menopausal status. When a specificity of 95% was fixed, the cut-off values were 120 and 180 pmol/mol creatinine for pre- and postmenopausal women respectively, the sensitivity being 73% and 64%. Finally the combined evaluation of UGP and CA 125 improved their individual clinical efficiency for the diagnosis of ovarian serous cystadenocarcinomas, assuring a sensitivity of 86% and a specificity of 89%.

CD44v10: an antimetastatic membrane glycoprotein for pancreatic cancer.

AIMS: The aims of this study were 1) to investigate the mRNA pattern of CD44 variants in three primary (MIA PaCa 2, PANC-1, PSN-1) and two metastatic (CAPAN-1, SUIT-2) pancreatic cancer (PC) cell lines; 2) to ascertain whether the genetic transfer of CD44s and CD44v10 modifies the adhesion of PC cells to the extracellular matrix (ECM) in vitro and their metastatic behavior in vivo. METHODS: CD44 mRNA analysis was done by means of RT-PCR. Adhesion to ECM the was assessed using coated microtiter plates. For the study of CD44v10 insertion in the CAPAN-1 line, liposome-mediated DNA transfer was used. SCID mice were employed for in vivo experiments. RESULTS: CD44v10 mRNA was not expressed by the CAPAN-1 nor by four of the six SUIT-2-derived clones. The stable expression of CD44v10 by modified CAPAN-1 significantly enhanced fibronectin adhesion. Mice without either liver or pancreatic metastases were more frequently found among the animals injected with modified (CD44v10 expressing) than with non-modified CAPAN-1. CONCLUSIONS: 1) It is possible to differentiate between metastatic and non-metastatic PC cells on the basis of CD44v10 expression; 2) CD44v10 seems to be involved in mediating fibronectin adhesion in vitro and in counteracting metastases in vivo.

[The role of the laboratory in the diagnosis and monitoring of Helicobacter pylori infection]. / Ruolo del laboratorio nella diagnosi e nel monitoraggio dell'infezione da Helicobacter pylori.

The diagnosis of Helicobacter pylori infection represents a fundamental step for a correct clinical approach to the patient with gastritis, peptic ulcer or gastric adenocarcinoma or lymphoma. Upper gastrointestinal endoscopy has first to be made in order to obtain gastric juice and mucosal biopsies, where Hp can be found. Two different procedures are recommended to diagnose Hp infection (e.g. urease test and histology or culture). The identification of specific DNA sequences with the polymerase chain reaction represents a sensitive and specific method to diagnose Hp infection. Serum anti Hp antibodies, pepsinogen A and C determination is recommended at diagnosis and during the follow-up to assess the success of therapy.

[Diagnosis of Helicobacter pylori infection: comparison of techniques]. / Diagnostica dell'infezione da Helicobacter pylori: tecniche a confronto.

UNLABELLED: Several diagnostic assays are available for evaluating Helicobacter pylori infection: histological examination, culture of gastric biopsies, urea breath test and serology. Recently a new enzyme immunoassay has been introduced for the detection of H. pylori antigens in stool samples (HpSA). The aim of our study was to evaluate and compare diagnostic efficacy of HpSA with histological examination, culture, urea breath test and serology in a group of 95 patients. Patients were classified H. pylori positive (43) or negative (52) on the basis of histology, culture and urea breath test. HpSA optical densities were significantly higher in infected patients compared to those obtained in H. pylori-negative patients (t = 5.47, p < 0.001). Overall, with a fixed cut-off of 0.1 unit of optical density, the sensitivity was 79% and the specificity 100%. In the H. pylori positive patients, HpSA optical density correlated with bacterial load histologically evaluated in the gastric antrum (r = 0.405, p < 0.05) and was inverse correlated with levels of serum IgG elicited against H. pylori (r = -0.315, p < 0.05). Considering patients with a positive HpSA finding and/or levels of anti-H. pylori antibodies upper than 30 U/mL, sensitivity in detecting infected patients was 98%. IN CONCLUSION: (1) immunodetection of H. pylori antigens in stools is a good alternative of breath test; (2) a reduction in H. pylori density grade might be accompanied by low HpSA optical density, leading to a false negative result and (3) combining the HpSA determination with the serum detection of anti-H. pylori antibodies a better clinical sensitivity is obtained.

[Biochemical markers of gastric functioning]. / Marcatori biochimici di funzionalità gastrica.

The serum determination of pepsinogen A (PGA) and pepsinogen C (PGC) might indicate gastric mucosal inflammation and atrophy. Body gastric mucosa produces both PGA and PGC, while antral mucosa produces only PGC. Therefore, diseases involving mainly the antrum, such as H. pylori infection, are mainly indicated by the variations in serum PGC than in serum PGA. In agreement, when the antral mucosa is infected by the more virulent cagA positive H. pylori strains, which cause severe inflammation, serum PGC significantly increases. Another indirect indicator of gastric inflammation is polymorphonuclear (PMN) oxidative burst after the stimulation with water extracts from H. pylori culture: this parameter is significantly increased in infected if compared to non-infected subjects. The higher oxidative burst response of peripheral PMN in infected patients, possibly consequent to the release of specific cytokines able to prime PMN towards H. pylori products, is unable to eliminate the infection, but it might concur in damaging the gastric mucosa.

Heat-induced transcription of diphtheria toxin A or its variants, CRM176 and CRM197: implications for pancreatic cancer gene therapy.

Vectors combining the heat shock proteins (HSPs) promoter with the catalytic subunit A of the diphtheria toxin (DTA) or its variants, cross-reacting material (CRM) 176 and 197, were engineered to investigate the effect of bacterial toxins on pancreatic cancer (PC) cells. Three heat-inducible enhanced green fluorescent protein (eGFP)-expression vectors were obtained: V1 (91% homology to HSPA6), V2 (five heat shock elements upstream the minimal HSPA6 promoter) and V3 (V1 and V2 combined). The highest eGFP transcription and translation levels were found in V3 transfected PC cells. The V3 promoter was used to control DTA, CRM176 and CRM197 expression, treatment response being investigated in four PC cell lines. DTAwt or CRM176 transfected cell growth was completely arrested after heat shock. CRM197 toxin presumed to be inactive, caused mild distress at 37 degrees C and induced a 25-50% reduction in cell growth after heat shock. Preliminary in vivo findings showed that heat treatment arrests tumor growth in DTA197 stably transfected PSN1 cells. In conclusion, the efficient HSP promoter identified in this study may be extremely useful in controlling the transcription of toxins such as CRM197, which have lethal dose-related effects, and may thus be a promising tool in PC gene therapy in vivo.

Insights in the laboratory diagnosis of celiac disease.

The present report focuses on the diagnosis of celiac disease and its pathogenesis, which depends on a genetic predisposition (HLA DQ2 or DQ8 haplotypes), gluten ingestion and T cell activation, type II transglutaminase (TG2), the autoantigen recognized by the antiendomysial antibody playing a key role. IgA class antibody anti-environmental (gliadin) and endogenous (TG2) antigens are present in the sera of patients with celiac disease. The anti-TG2 antibody has the best available diagnostic accuracy, especially when measured employing second generation ELISA tests, which use the human TG2 antigen, or immunochemiluminescent assay, which is highly sensitive. A diagnosis of celiac disease must always be confirmed by the histological evaluation of multiple duodenal mucosa specimens, and serology is recommended for follow-up controls.

Plasma chromogranin A in patients with prostate cancer improves the diagnostic efficacy of free/total prostate-specific antigen determination.

INTRODUCTION: We ascertained whether plasma chromogranin A enhances the power of serology assessing prostate cancer (PC). MATERIALS AND METHODS: We studied 56 PC and 83 benign prostatic hyperplasia (BPH) patients. In the sera we measured total prostate-specific antigen (tPSA) and free PSA (fPSA) and calculated the ratio between fPSA and tPSA (f/tPSA). In plasma samples the levels of chromogranin A (CgA) were also assayed. RESULTS: PC patients had higher CgA (p < 0.005) and tPSA (p < 0.05) levels, and a lower f/tPSA ratio (p < 0.001), than BPH patients. When f/tPSA and CgA were combined, the diagnostic sensitivity was enhanced (57-73%), while the specificity had only an 8% reduction (from 89 to 80%). CgA was only correlated to the Gleason PC score (p < 0.05). CONCLUSIONS: CgA determination in PC may enhance the diagnostic accuracy of the f/tPSA assay and provides useful information on the tumor grade.