[Quality of life of the elderly patient on dialysis]. / La qualita' della vita nell'anziano in dialisi.

When elderly patients with end-stage renal disease start dialysis their quality of life, and particularly the emotional aspects of it, are very similar to those of age-matched controls. However, as the treatment becomes chronic the quality of life will decline not only with regard to the physical aspects (due to comorbidities) but also the emotional aspects. Dialysis-related stress episodes and the peculiar interrelationships in the dialysis facility setting may cause psychological discomfort which on the one hand reduces the patient's quality of life and on the other may unfavorably impact on the family and the health-care personnel. An integrated psychological approach involving the patient from the beginning of dialysis throughout the treatment process as well as the healthcare personnel and the family can reduce the patient's psychological discomfort, thereby improving quality of life.

[Dialysis, adaptation, quality of life, and family support]. / Rapporti tra adattamento, qualità di vita e supporto familiare, sociale nel paziente in trattamento dialitico.

Chronic dialysis treatment is characterized by a series of complex interdependent objective problems, such as the dialysis experience, the individual way to assess it, and some "protective" factors such as social and family support. Progresses in dialysis research show that dialysis patients have important alternatives to passively accept their condition: thanks to adequate psychological and relational aid, they can reach rather advanced adaptation levels, which allow them to modify both their behaviour and way of life, to keep a satisfactory compliance, and to improve their quality of life (QoL). In this adaptation process, both family and social support play an important role, although controversy still exists on it. The results of our study confirm the complexity of this role and show that either haemodialysis or peritoneal dialysis patients' adaptation process and QoL may be directly related to the extent of family member's ("caregiver") support. It is of particular interest the fact that patients, especially those undergoing haemodialysis, provided with a caregiver's assistance but who choose to "act by themselves", do have better adaptation levels and QoL than those who rely only on their caregiver. This fact reassesses the widely accepted point of view that continuous caregiver's support is always a positive and necessary factor in order to improve both the adaptation and the QoL of dialysis patients.

Decrease of HCVRNA after three days of daily interferon treatment is predictive of the virological response at one month.

Early monitoring of HCVRNA during interferon treatment may allow clinicians to obtain important information that could help them to adopt therapeutic decisions in individual cases. The hepatitis C virus infection is highly dynamic and a daily high dose of IFN may induce a decline of viremia of 95+/-10% of baseline value after 24 to 48 hours of treatment. The importance of this early antiviral efficacy has not been understood. We have measured HCVRNA levels in 47 patients with chronic hepatitis C infection during interferon treatment to study HCVRNA kinetics and evaluate the predictive value of the early decay of viremia on the virological response after one month of treatment. Sixty percent of treated patients showed early virological response (EVR) and it was significantly associated with low HCVRNA levels and a genotype other than 1b. Finally, the absence of an 85% decline in HCVRNA levels after 3 days of treatment observed in 11 out of 45 patients (24%) was an absolute and very early predictor of the absence of EVR in the study population.

Clonality of B-cells in portal lymphoid infiltrates of HCV-infected livers.

Evidence has been accumulating in favour of a role for hepatitis C virus (HCV) in the pathogenesis of human lymphoproliferative disorders. HCV infection has been documented in the majority of patients with essential mixed cryoglobulinaemia type II (MC-II); in patients with HCV infection, B-cell clonal expansion have been detected in peripheral blood and bone marrow, and a high prevalence of B-cell non-Hodgkin's lymphomas has been documented. Liver biopsies in chronic hepatitis C frequently show portal lymphoid infiltrates with features of B follicles, whose clonality has not yet been investigated. This study has analysed the B-cell clonality of portal lymphoid infiltrates from 16 patients with chronic HCV hepatitis. Portal tracts showing obvious lymphoid infiltrates were microdissected from the paraffin-embedded liver tissue sections and the clonality of lymphoid B-cells was tested using a polymerase chain reaction (PCR) approach designed to identify immunoglobulin heavy chain gene (IgH) rearrangements. A successful IgH-PCR analysis was achieved in 35 lymphoid infiltrates from 11 patients (seven with the four without MC-II) and yielded a single band in 21 cases, two bands in ten cases, and three bands in four cases. Comparison of the IgH-PCR amplification bands obtained from the different lymphoid aggregates of the same biopsy revealed that they differed in size. This finding indicates that each aggregate derives from the proliferation of one or a few founder B-cells, which are not related to each other. The results obtained in patients with and without MC-II were similar, suggesting that the presence of B-cell clonal proliferations in liver biopsies is independent of the occurrence of B-cells producing monoclonal IgMk cryoglobulins.

APC gene mutations and allelic losses in sporadic ampullary tumours: evidence of genetic difference from tumours associated with familial adenomatous polyposis.

We explored APC gene mutations and chromosome 5q21 allelic losses (5qLOH) in 18 neoplasms of the papilla of Vater, including 6 early-stage tumours (3 adenomas, 3 carcinomas) and 12 advanced-stage cancers. Eleven PCR-amplified polymorphic sequences were used to analyse 5qLOH. APC mutations were investigated both by an in vitro APC-protein truncation test and by single-strand conformation polymorphism analysis. Mutations in the Ki-ras, N-ras and p53 genes were also assessed. We found: 5qLOH in 8 of 16 cases (50%), including 1 adenoma, 3 early- and 4 advanced-stage cancers; APC mutations in 2 adenomas and 1 advanced-stage carcinoma; Ki- or N-ras mutations in 3 adenomas and 3 advanced-stage cancers; p53 mutations in 2 early-stage and 7 advanced-stage adenocarcinomas. Our results suggest that 5qLOH, APC mutations and ras mutations are present at early stages, whereas p53 inactivation is associated with progression of malignancy in a large proportion of cases. These data indicate that sporadic ampullary tumours differ from those occurring in familial adenomatous polyposis in the frequency (17% vs. 64%) as well as in the site of APC somatic mutations, suggesting a different molecular pathogenesis in the 2 conditions.

p21/WAF1 cyclin-kinase inhibitor expression in non-Hodgkin's lymphomas: a potential marker of p53 tumor-suppressor gene function.

p21WAF1 (wild-type p53-activated fragment 1) is involved in the control of mammalian cell cycle through the binding and inhibition of cyclin-dependent kinases (Cdk). Because the product of WAF1 gene is a potent downstream effector of the p53 tumor-suppressor gene function, its pattern of cellular expression might correlate with nuclear accumulation of p53-encoded protein and/or p53 gene mutations occurring in malignant lymphomas. To investigate this issue, we analyzed immunohistochemically the expression of p53 and p21WAF1 proteins in tissue involved by non-Hodgkin's lymphomas (NHLs;253 cases) of various histologic types. In a proportion of them (80 cases), we also investigated the possible presence of p53 gene mutations using single-strand conformation polymorphism analysis and direct DNA sequencing. The absence of both p21WAF1 and p53 proteins was observed in 147 of 217 cases (67.7%) among CD30-NHL and in only 8 of 36 (22.2%) CD30+cases, which were mostly anaplastic large-cell lymphomas. A consistent number (> 10%) of p21WAF1-expressing cells was shown in 48 of 253 (18.9%) NHL cases, with a higher incidence in CD30+cases (25/36 [69.4%]), which mostly (21/36) coexpressed p53. These latter cases were characterized by a germline configuration of the p53 gene. In 50 of 253 NHL samples (19.7%), 47 of which (21.6%) belong to the CD30-group, neoplastic cells were p53+/p21-. In all of these cases, the p53+cells accounted for more than 50% of neoplastic cells, up to 100%. Point mutations of p53 gene were solely observed in all investigated cases with this latter phenotype. Our findings strongly suggest that the combined immunohistochemical evaluation of p53 and p21WAF1 is a valuable means of assessing the functional status of the p53 tumor-suppressor gene product in NHL with potential application in the monitorage and prognostication of individual cases.

Chromosome 7q allelic losses in pancreatic carcinoma.

During our DNA fingerprinting studies of paired normal and pancreatic cancer tissues using arbitrarily primed PCR, we noticed a band showing an apparent homozygous deletion in a pancreatic cancer cell line and a decreased intensity in a number of primary cancers. That band was assigned to chromosome 7. Such information led us to analyze chromosome 7 loss of heterozygosity (LOH) in a panel of 12 cryostat-enriched primary pancreatic cancers and 2 pancreatic cancer cell lines, despite the reportedly low frequency of chromosome 7 LOH in xenograft-enriched pancreatic cancers. Seventeen PCR-amplified CA-microsatellite polymorphic sites were analyzed. One of the two cell lines and eight common-type cancers (including all five poorly differentiated and three of five moderately differentiated cancers) showed chromosome 7q LOH, whereas the two uncommon types of ductal cancer (one adenosquamous and one mucinous noncystic) scored negative. Our data suggest that chromosome 7q LOH is a frequent event (80%) in cryostat-enrichable common pancreatic ductal carcinomas, that is, those primarily of high cellularity. The chromosome 7q smallest common deleted region described by our cases was between 7q31.1 and 7q32.

Role of IL-1 beta and corticosteroids in the regulation of the C/EBP-alpha, beta and delta genes in vivo.

We have examined the regulatory effects of interleukin 1 beta (IL-1 beta) on the activation of three different isoforms of the C/EBP family of transcription factors (alpha, beta and delta), in hepatocytes of normal and adrenalectomized (ADX) rats. C/EBP-beta and delta mRNA levels were enhanced by IL-1 beta, whereas that of C/EBP-alpha was not affected by treatment with this interleukin in both normal and adrenalectomized rats. The magnitude of the induction was strikingly higher for C/EBP delta in adrenalectomized animals, indicating a suppressive effect of corticosteroids in the IL-1 beta regulatory pathway. The pattern of C/EBP protein synthesis did not always reflect the mRNA findings. For C/EBP-alpha the protein synthesis was higher than expected in IL-1 beta treated ADX animals compared to normal rats. The pattern of C/EBP synthesis was the one that better reflected the pattern of the mRNA transcription. Differently, the induction of C/EBP-delta was not as pronounced as that of the corresponding mRNA in IL-1 beta treated ADX rats. Hormonal modulation of C/EBP transcription factors was studied in parallel with the hormonal induction of the Alpha-1-Acid Glycoprotein (AGP) gene, which is known to be highly induced in rat liver during the acute phase response. This short report also indicates an important role of corticosteroids in the regulation of transcription factors involved in IL-1 beta signalling during the acute phase response.

Specificity of action of a herpes virus VP16/tetracycline-dependent trans-activator in mammalian cell cultures.

In this work, we have studied the activity of a tetracycline modulatable trans-activator (tTA) generated by fusing the DNA binding domain of the tetracycline repressor to the trans-activation domain of the Herpes simplex virus protein 16 (HSV VP16) (plasmid pUHD15-1Neo). In the three different cell lines studied (HTC, rat hepatoma; T47D, human breast cancer; SK-N-BE, human neuroblastoma), the expression of the luciferase gene under the control of a tetracycline operator sequence (plasmid pUHC13-3) was used as a control of the incorporation and the functionality of the trans-activator. Clones selected from these cells responded in a time and dose-dependent manner to the withdrawal of tetracycline. In all these clones, the tTA trans-activator not only modulates the activity of the luciferase gene, but also modulates the activity of a number of endogenous proteins, including C/EBP beta, the glucocorticoid receptor (GR), and SP1. In the transfected cells, the level of these transcription factors was strongly inhibited in the presence of tetracycline and was highly increased after tetracycline removal. Electrophoresis mobility shift assay (EMSA) and footprint experiments proved that the induced proteins are perfectly efficient in binding the DNA. Their transcriptional activity was also determined. In HTC/A9 cells, the level of the chloramphenicol acetyltransferase (CAT) expression driven by the promoter of the alpha 1-glycoprotein (AGP) gene was strongly enhanced at 72-84 hr following removal of tetracycline from the growth media. The accumulation of the endogenous AGP mRNA also increased at 84 hr. In the T47D/TA11 and SK-N-BE/C2.6 cells, a general activation of protein synthesis was also evidenced.

The complex interplay of the DQB1 and DQA1 loci in the generation of the susceptible and protective phenotype for insulin-dependent diabetes mellitus.

IDDM patients of North East Italian region were molecularly typed for their HLA-DQB1 and DQA1 loci by using allele specific oligonucleotide probes and PCR amplified genomic DNA. IDDM status strongly correlated with DQB1 alleles carrying a non-aspartic acid residue in position 57 of DQ beta chain and DQA1 alleles with an arginine residue in position 52 of DQ alpha chain. Genotype analysis revealed that individuals with two DQB1 alleles having a non-aspartic residue in position 57 and two DQA1 alleles with an arginine residue in position 52 had the highest relative risk of disease: they constituted 41% of IDDM patients as compared to 0% of controls. Heterozygosity either at residue 57 of DQB1 or residue 52 of DQA1 was sufficient to abrogate statistical significance for disease association, although 43.6% of IDDM patients were included in these two groups as compared to 21.6% of normal controls. On the other hand the presence of two DQB1 alleles with aspartic acid in position 57 was sufficient to confer resistance to disease irrespective of the DQA1 genotype. Based on the number of possible susceptible heterodimers an individual can form, it was found that 85% of IDDM cases could form two or more heterodimers (two in cis and two in trans), but no IDDM case was found to form one susceptible heterodimer in cis. These results demonstrate that the complete HLA-DQ genotype, more than single DQB1 or DQA1 alleles or DQB1-DQA1 haplotypes, is associated with the highest risk of disease. Screening of the population for preventive purposes and/or early signs of IDDM should then take advantage of this result and "susceptible homozygous" individuals should be followed very closely and considered the first group of choice for possible new therapeutical trials.

Detection of BCR/ABL transcripts in chronic myeloid leukaemia by polymerase chain reaction and DNA enzyme immunoassay: a DNA probe assay without DNA labelling.

The detection of the t(9;22) translocation, typical of chronic myeloid leukaemia (CML), can be accomplished by cytogenetical detection of Philadelphia (Ph1) chromosome or by molecular analysis of the bcr/abl fusion gene with nucleic acid probes after amplification by polymerase chain reaction (PCR). PCR-based approaches are now widely used for follow up of CML patients during therapy or after bone marrow transplantation (BMT). We describe here a microtitre, colorimetric assay (DNA Enzyme Immunoassay, DEIA) for analysis of t(9;22) translocation after enzymatical amplification of RNA from CML patients. This assay is based on the use of a monoclonal antibody specifically reacting with double stranded DNA, i.e. with hybridized DNA. The assay represents a nonisotopic alternative to other current hybridization assays and requires no modifications of primers, probe or target DNA.

HLA-DQB1 typing of north east Italian IDDM patients using amplified DNA, oligonucleotide probes and a rapid DNA-enzyme immunoassay (DEIA).

We report on HLA-DQB1 typing in IDDM patients of north east Italian region using an enzymatic method based on the detection of hybridization reaction between PCR amplified DNA from whole blood and allele specific oligonucleotides by an antibody directed against double stranded DNA (DNA-enzyme immunoassay or DEIA). The method is reliable, simple and sensitive as the classical radioactive method with the advantage of using a universal non radioactive detection reagent. Nineteen families, each including one subject with juvenile insulin-dependent diabetes mellitus (IDDM) were analyzed. A strong association between absence of an aspartic acid (Asp) in position 57 of DQB1 beta chain in homozygous conditions and susceptibility to IDDM was found. In contrast with some previous observations, however, no significant association was found between Asp/non-Asp heterozygous genotype and IDDM. No patients were found with an homozygous Asp/Asp genotype, known to be protective in caucasoid population. Of particular interest was the DQB1 allelic distribution in our population sample. The non-Asp allele most frequently found in IDDM subjects was the DQB1 0201 allele and this finding was statistically significant (Pc value < 0.05, relative risk = 5.01). No significant association was found for any other allele including the DQB1 0302 (Pc value = not significant although with relative risk = 3.28) previously reported as the most frequent allele in IDDM caucasoid patients.