Deregulation of HOX B13 expression in urinary bladder cancer progression.

Urinary bladder cancer is a common malignancy in industrialized countries. More than 90% of bladder cancer originates in the transitional cells. Bladder transitional cancer prognosis is, according to the most recent definition related to the level of tumor infiltration, characterized by two main phenotypes, Non Muscle Invasive Bladder Transitional Cancer (NMIBC) and Muscle Invasive Bladder Transitional Cancer (MIBC). The genetic profile and the clinical course of the two subtypes are completely different, however among NMIBC the prognosis is not completely predictable, since 20% of the cases experience a relapse, even in the form of MIBC. It has recently been reported that the chromosomal region 12q13-15, containing crucial cancer genes such as MDM2, CDK4, GLI and an entire cluster of HOX genes, is amplified in bladder cancer. HOX genes codify for transcriptionl factor, involved in embryonal development and cancer progression, with main nuclear expression. Particularly it was also described the strong involvement of HOX B13 in several tumors of urogenital system. In this study we have been investigated, by immunohistochemisty and quantitative Real Time PCR, the HOX B13 expression in bladder cancer evolution and progression, evaluating its ability to discriminate between NMIBC and MBCI phenotypes. Cytoplasmic HOX B13 delocalization significantly relates with muscle invasion (p 0.004). In addition in the series of NMIBC nuclear HOX B13 expression loss is significantly associated to shorter disease free survival (p-value=0.038) defining a potential prognostic role. Overexpression of HOX B13 in more aggressive phenotype is also demonstrate at gene level by quantitative RT-PCR. The de-regulation and delocalization of HOX B13 in urinary bladder cancer supports again the important role of HOX genes in tumor evolution and represents a starting point to establish an integrated analysis, in which HOX genes represent important prognostic and predictive markers for bladder cancer.

Experimental therapy of genetic arrhythmias: disease-specific pharmacology.

The integration between molecular biology and clinical practice requires the achievement of fundamental steps to link basic science to diagnosis and management of patients. In the last decade, the study of genetic bases of human diseases has achieved several milestones, and it is now possible to apply the knowledge that stems from the identification of the genetic substrate of diseases to clinical practice. The first step along the process of linking molecular biology to clinical medicine is the identification of the genetic bases of inherited diseases. After this important goal is achieved, it becomes possible to extend research to understand the functional impairments of mutant protein(s) and to link them to clinical manifestations (genotype-phenotype correlation). In genetically heterogeneous diseases, it may be possible to identify locus-specific risk stratification and management algorithms. Finally, the most ambitious step in the study of genetic disease is to discover a novel pharmacological therapy targeted at correcting the inborn defect (locus-specific therapy) or even to "cure" the DNA abnormality by replacing the defective gene with gene therapy. At present, this curative goal has been successful only for very few diseases. In the field of inherited arrhythmogenic diseases, several genes have been discovered, and genetics is now emerging as a source of information contributing not only to a better diagnosis but also to risk stratification and management of patients. The functional characterization of mutant proteins has opened new perspectives about the possibility of performing gene-specific or mutation-specific therapy. In this chapter, we will briefly summarize the genetic bases of inherited arrhythmogenic conditions and we will point out how the information derived from molecular genetics has influenced the "optimal use of traditional therapies" and has paved the way to the development of gene-specific therapy.

The short QT syndrome as a paradigm to understand the role of potassium channels in ventricular fibrillation.

The recently discovered hereditary channelopathy, the Short QT Syndrome (SQTS), is an important advance in clinical and molecular cardiology that has opened new doors for investigating the manner in which alterations in excitability and action potential morphology may facilitate the occurrence of ventricular fibrillation. In this brief review we address the molecular and genetic features of SQTS in which specific mutations in one of three different potassium channels involved in cardiac repolarization substantially increase the risk of life-threatening tachyarrhythmias. We then summarize new knowledge on the mechanism of wavebreak, which is the hallmark of reentry initiation, and on the role of potassium channels in the ionic mechanisms underlying cardiac excitation and its frequency dependence. The article argues for a detailed understanding of the ionic mechanisms that promote wavebreaks and stable rotors as an essential tool for successful anti-arrhythmic therapy in SQTS and other diseases leading to sudden cardiac death.

[Long QT syndrome and Brugada syndrome: 2 aspects of the same disease?]. / Sindrome del QT lungo e sindrome di Brugada: due aspetti della stessa malattia?

In clinical cardiology, resort has recently been made to molecular genetics in order to explain some mechanisms that underlie sudden cardiac death in young people with structurally normal hearts. It has become evident that genetic mutations regarding cardiac ion channels may disrupt the delicate balance of currents in the action potential, thus inducing malignant ventricular tachyarrhythmias. The cardiac sodium channel gene, SCN5A, is involved in two of such arrhythmogenic diseases, the Brugada syndrome and one form of the long QT syndrome (LQT3). It is believed that these syndromes result from opposite molecular effects: Brugada syndrome mutations cause a reduced sodium current, while LQT3 mutations are associated with a gain of function. The effects of class I antiarrhythmic drugs have been used to differentiate these diseases. Intravenous flecainide is used as a highly specific test to unmask the electrocardiographic phenotype of the Brugada syndrome. On the other hand, on the basis of experimental and clinical studies, the possibility that the same drugs act as a gene-specific therapy in this disorder by contrasting the effect of mutations in LQT3 has been explored. Recent evidence shows that phenotypic overlap may exist between the Brugada syndrome and LQT3. One large family with a SCN5A mutation and a "mixed" electrocardiographic pattern (prolonged QT interval and ST-segment elevation) has been reported. Moreover, our recent data showed that flecainide challenge may elicit ST-segment elevation in some LQT3 patients. The presence of "intermediate" phenotypes highlights a remarkable heterogeneity suggesting that clinical features may depend upon the single mutation. Only deepened understanding of the genotype-phenotype correlation will allow the definition of the individual patient's risk and the development of guidelines for clinical management.

[The role of transvaginal ultrasonography in the screening of ovarian tumors]. / Ruolo dell'ecografia transvaginale nello screening delle tumefazioni ovariche.

BACKGROUND: From January 1992 to March 1996, 1987 women underwent vaginal sonography screening, at the Department of Obstetrics and Gynaecology of the II Faculty of Medicine and Surgery "Federico II" in Naples. This mass screening aimed at early diagnosis of ovarian cancer. METHODS: Patients included in this investigations were all asymptomatic and had no pelvic abnormalities. Each ovary was measured in three planes and ovarian volume was calculated using the prolate ellipsoid formula. In premenopausal women, ovaries were normal if their volume was of > or = 18 cm3 and if they were hypoechogenic or anechogenic. In postmenopausal women a normal ovary was defined as having a volume of > or = 8 cm3 and a uniformly hypogenic internal structure. RESULTS AND CONCLUSIONS: In thirty-five premenopausal women was detected an abnormal volume of the ovaries. Forty-six postmenopausal women had abnormal vaginal sonograms. In this investigations vaginal sonography has permitted the detection of about 4.88% ovarian tumors in asymptomatic women, so that, it can be considered a more accurate and direment screening method for ovarian cancer than abdominal sonography.

[Needle aspiration of ovarian cysts. Our experience]. / Agoaspirazione delle cisti ovariche. Nostra esperienza.

80 patients underwent ultrasound guided cyst puncture of ovarian cysts. Six patients were pregnant and in four of them needle aspiration was executed through the transvaginal tract. The sediment aspirated was examined by a cytologic method and when possible it was also correlated to a histological test. Technique and results have been dealt with. No relevant complication was found. Finally, the echoguided (induced) needle aspiration is a simple and safe method to treat benign ovarian cysts in fertile women. It is particularly advisable in pregnancy and in patients for whom both laparascopy and traditional surgery are not practicable.

Effect of antigen-specific T helper cells or interleukin-2 on suppressive ability of macrophage subsets detected in spleens of Trypanosoma cruzi-infected mice as determined by limiting dilution-partition analysis.

Trypanosoma cruzi, a protozoan parasite and the causative agent of Chagas' disease, induces a state of lymphocyte hyporesponsiveness to both mitogenic and antigenic stimuli in mice during the acute phase of infection. Addition of spleen cells from T. cruzi-infected mice (SCinf) to microcultures of spleen cells from noninfected mice (SCn) suppresses the responsiveness of such cultures to antigenic challenge and to mitogenic stimulation. We analyzed the regulatory cell populations in SCinf by limiting dilution-partition analysis and found a complex regulatory circuit in T. cruzi-infected mice consisting of two suppressive macrophage subsets and an enhancing T-cell population. This T-cell population was able to abrogate or escape the suppressive ability of one suppressor macrophage subset, yet was suppressed by the other macrophage subset. To further study the cellular interactions of this regulatory circuit and analyze the suppressive abilities of the two suppressor macrophage subsets, we examined the effect of adding either primed T helper cells of known specificity or interleukin-2 to the limiting dilution-partition analysis microcultures. The results of these experiments suggest that one suppressor macrophage subset, which is abundant and, therefore, detected with low doses of SCinf, is able to suppress both mitogen- and primary antigen-specific responses but is unable to inhibit cells once they are already activated or primed. The other macrophage subset, which is presumably a less abundant or less active population (since high doses of SCinf are required to detect it), is able to suppress the response of activated or primed T cells by the inhibition of interleukin-2 production.

Macrophage regulation of immune responses of spleen cells from mice infected with Trypanosoma cruzi.

Mice infected with the protozoan parasite, Trypanosoma cruzi, are known to be immunosuppressed in responsiveness to heterologous antigens and parasite-specific antigens. This suppression is mediated by suppressor macrophages and is exemplified by deficient T cell activity and abnormal cytokine production. Neither the mechanism by which suppressor macrophages effect suppression nor the characteristics of these suppressor macrophages is known. In the present study, we analyzed the regulatory cell populations in splenocytes of infected mice (SCinf) and their interactions by limiting dilution-partition analysis, an approach which allows the functional separation of multiple regulatory cell subpopulations within cell mixtures. Our results demonstrate the presence of a complex immunoregulatory circuit in SCinf affecting the generation of anti-sheep erythrocyte antibody responses in vitro. Titration of SCinf (but not peritoneal exudate or lymph node cells) into Mishell-Dutton microcultures of normal spleen cells generated complex dose-response curves with two zones of suppressed responses following the addition of either low or high doses of SCinf to the cultures. Addition of intermediate doses of SCinf to the microcultures restored responsiveness. Both the low- and high-dose zones of suppression were shown to be mediated by macrophages, whereas T cells were responsible for the restored responsiveness at intermediate doses of SCinf. Examination of the development of this complex regulatory pattern during the course of the acute phase of infection indicated the sequential development of one suppressor macrophage population, followed by the development of the beneficial T cell population, and finally the expression of the second suppressive macrophage population.

Description of a urease-based microELISA for the analysis of limiting dilution microcultures.

Limiting dilution analysis has been a valuable approach for both determining the frequency of cell subpopulations elicited during immune responses, as well as for the analysis of immunoregulatory circuits. We describe a simple, visually scored spot test for evaluating the response of Mishell-Dutton microcultures used in limiting dilution analysis. This spot test is based on a microELISA using immunoreagents conjugated to the enzyme, urease, as an alternative to the hemolytic spot test. The assay as performed in Terasaki trays requires minute quantities (less than 10 microliters) of culture supernatant, yet the ELISA yields a distinct color difference between tray wells containing culture supernatants derived from responding (purple) and nonresponding (yellow) microcultures. Although designed to be scored rapidly by visual inspection, the assay can be quantified by manual alignment of the Terasaki tray wells on commercially available ELISA plate readers with an accuracy and reproducibility comparable to assays performed in 96-well ELISA plates. Determination of anti-sheep RBC responses in limiting dilution Mishell-Dutton microcultures with both the hemolytic spot test and the urease-microELISA spot test showed a very close correlation between the results of the two assays. However, the urease-microELISA should be amenable for use with antigens not readily conjugated to an indicator RBC, and should be useful in those situations where determination of the antibody subclass(es) produced by responding microcultures is desired.

Cloning and sequence of the gene for heat shock protein 60 from Chlamydia trachomatis and immunological reactivity of the protein.

We isolated and sequenced the gene for the chlamydial heat shock protein 60 (HSP-60) from a Chlamydia trachomatis genomic library by molecular genetic methods. The DNA sequence derived revealed an operon-like gene structure with two open reading frames encoding an 11,122- and a 57,956-Da protein. The translated amino acid sequence of the larger open reading frame showed a high degree of homology with known sequences for HSP-60 from several bacterial species as well as with plant and human sequences. By using the determined nucleotide sequence, fragments of the gene were cloned into the plasmid vector pGEX for expression as fusion proteins consisting of glutathione S-transferase and peptide portions of the chlamydial HSP-60. HSP-60 antigenic identity was confirmed by an immunoblot with anti-HSP-60 rabbit serum. Sera from patients that exhibited both high antichlamydial titers and reactivity to chlamydial HSP-60 showed reactivity on immunoblots to two fusion proteins that represented portions of the carboxyl-terminal half of the molecule, whereas fusion proteins defining the amino-terminal half were nonreactive. No reactivity with the fusion proteins was seen with sera from patients that had been previously screened as nonreactive to native chlamydial HSP-60 but which had high antichlamydial titers. Sera from noninfected control subjects also exhibited no reactivity. Definition of recognized HSP-60 epitopes may provide a predictive screen for those patients with C. trachomatis infections who may develop damaging sequelae, as well as providing tools for the study of immunopathogenic mechanisms of Chlamydia-induced disease.

Cysteine-rich outer membrane proteins of Chlamydia trachomatis display compensatory sequence changes between biovariants.

Two cysteine-rich proteins of Chlamydia trachomatis are essential structural components of the unique outer membrane of the infectious elementary body. These 58,000 (outer membrane protein 2; OMP2) and 15,000 (OMP3) proteins also differ structurally and chemically between biovariants that differ in invasive capability. We have identified the gene for OMP3 and sequenced both trachoma and lymphogranuloma venereum (LGV) omp3 genes. We have previously sequenced omp2 from the LGV biovar and now describe the omp2 sequence for a trachoma biovariant. Amino acid sequence differences between biovariants were few but, significantly, these changes have altered the charge of both OMP2 and OMP3 such that the net charge of each protein differs between biovariants. These compensatory charge alterations have implications for the outer membrane organization of these proteins. In addition, examination of the OMP3 sequence suggests that OMP3 may be a lipoprotein.

Increased natural killer cell activity in experimental American trypanosomiasis.

These studies showed that increased natural killer (NK) cell activity develops during experimental American Trypanosomiasis in mice. Increased cytotoxicity against YAC target cells by peritoneal cavity cells (PEC) was detected as early as 1 day into infection in both C3H/He and C57BL/6 mice. Peak activity occurred 2 days into infection, with significant but variable increases detected in the spleen cells (SC) from these mice. The response subsided in PEC and to a lesser extent in SC by 4 days post-infection. Similar responses were not detected in infected C57BL/6 bgJ/bgJ (beige mutant) mice until the fourth day of infection and peaked at significantly lower levels than the peak on day 2 in beige hybrid mice ([C57BL/6 +/+ X C57BL/6 bgJ/bgJ]F1). The effector cell from infected mice was sensitive to pretreatment with anti-NK 1.2 serum plus complement (C), partially sensitive to anti-Thy 1.2 + C, and insensitive to polyvalent anti-mouse immunoglobulin + C. The cytotoxic activity induced in cells from mice infected for 2 days was recovered in subpopulations nonadherent to plastic or Sephadex G-10. Thus, the anti-YAC effector cell found very early in mice infected with T. cruzi possessed many of the characteristics of natural killer (NK) cells found in normal mice. Other experiments demonstrated that injection of heat-killed preparations of blood-form trypomastigotes (BFT) induced increases in NK activity. The presence of augmented NK activity against YAC tumor cells in both resistant and susceptible strains of mice, together with the presentation of the resistance phenotype in beige mutant mice, which had a lower NK response to infection, is not indicative of a direct role for host protection during infection with T. cruzi by NK cells.