KIR2DL3 and the KIR ligand groups HLA-A-Bw4 and HLA-C2 predict the outcome of hepatitis B virus infection.

Killer immunoglobulin-like receptors (KIRs) regulate the activation of natural killer cells through their interaction with human leucocyte antigens (HLA). KIR and HLA loci are highly polymorphic, and certain HLA-KIR combinations have been found to protect against viral infections. In this study, we analysed whether the KIR/HLA repertoire may influence the course of hepatitis B virus (HBV) infection. Fifty-seven subjects with chronic hepatitis B (CHB), 44 subjects with resolved HBV infection and 60 healthy uninfected controls (HC) were genotyped for KIR and their HLA ligands. The frequency of the HLA-A-Bw4 ligand group was higher in CHB (58%) than subjects with resolved infection (23%) (crude OR, 4.67; P<.001) and HC (10%) (crude OR, 12.38; P<.001). Similar results were obtained for the HLA-C2 ligand group, more frequent in CHB (84%), than subjects with resolved infection (70%) (crude OR, 2.24; P<.10) and HC (60%) (crude OR, 3.56; P<.01). Conversely, the frequency of KIR2DL3 was lower in CHB (81%) than in subjects with resolved infection (98%) (crude OR, 0.10; P<.05). These results suggest a detrimental role of HLA-A-Bw4 and HLA-C2 groups, which are associated with the development of CHB, and a protective role of KIR2DL3. A stepwise variable selection procedure, based on multiple logistic regression analysis, identified these three predictive variables as the most relevant, featuring high specificity (90.9%) and positive predictive value (87.5%) for the development of CHB. Our results suggest that a combination of KIR/HLA gene/alleles is able to predict the outcome of HBV infection.

Misdiagnosis of familial Mediterranean fever in patients with Anderson-Fabry disease.

Fabry disease (FD) is an underdiagnosed pathology due to its symptomatology that overlaps with various systemic and rheumatic disorders, including familial Mediterranean fever (FMF). We examined the Mediterranean fever (MEFV) and α-galactosidase A (GLA) genes, whose mutations are responsible for FMF and FD, respectively, in 42 unrelated patients diagnosed with FMF, which revealed significant ambiguity regarding some of the symptoms which are also present in FD. The objective of this study was to determine the spectrum of mutations present in these genes, in order to identify cases of mistaken diagnosis of FMF and/or missed diagnosis of FD. Ten out of 42 patients had one mutation in homozygosis or two different mutations in heterozygosis in the MEFV gene; 20/42 had a single heterozygous mutation, and 12/42 did not have genetic alterations in MEFV. The analysis of the GLA gene conducted on all the samples revealed that three subjects, and some members of their families, had two different exonic mutations associated with FD. Family studies allowed us to identify eight other cases of FD, bringing the total undiagnosed subjects to 11/53. Analyzing the MEFV and GLA genes in patients with clinical diagnoses of FMF proved to be fundamentally important for the reduction of diagnostic errors.

Production of an egg yolk antibody against Parietaria judaica 2 allergen.

Specific antibodies are essential tools for studying proteins as well as for diagnostic research in biomedicine. The egg yolk of immunized chicken is an inexpensive source of high-quality polyclonal antibodies. The 12-kDa Parietaria judaica 2 allergen was expressed as a fusion protein and was used to immunize Leghorn chickens. In this paper, we show, using 2-dimensional gel electrophoresis and immunoblotting, that chicken antibodies raised against a recombinant allergen can be used to recognize similar proteins from a pollen raw extract. Allergen identity was confirmed by nanoLC-nanospray-tandem mass spectrometry analysis. Our data demonstrate for the first time that a synergistic combination of molecular biology, 2-dimensional PAGE, and use of nonmammalian antibodies represents a powerful tool for reliable identification of allergens.

cDNA cloning, sequence analysis and allergological characterization of Par j 2.0101, a new major allergen of the Parietaria judaica pollen.

A clone (P2) coding for an allergen of Parietaria judaica (Pj) pollen has been isolated and sequenced from a cDNA library in lambda ZAP using a pool of 23 sera from Pj-allergic patients. The clone contained an insert of 622 nucleotides with an open reading frame of 133 amino acids (aa) and a putative signal peptide of 31 aa giving a deduced mature processed protein of 102 aa with a molecular mass of 11344 Da. The expressed recombinant protein, named rPar j 2.0101, was a major allergen since it reacted with IgE of 82% (23/28) of the sera of Pj-allergic subjects analyzed. It was shown to be a new allergen since (i) the amino acid sequence homology with the already reported recombinant allergen Par j 1.0101 was 45% and (ii) there was no cross-inhibition between rPar j 2.0101 and rPar j 1.0101. In addition, rPar j 2.0101 inhibited 35% of the specific IgE for 10-14 kDa native allergens and preincubation of sera from Pj-allergic patients with both rPar j 2.0101 and rPar j 1.0101 fully abolished the IgE recognition of the 10-14 kDa native allergen region, suggesting that these two allergens contributed to the region.

Utility of tumor marker CA 72.4 in patients with chronic renal failure.

The tumor marker CA 72.4 is composed of two monoclonal antibodies, B 72.3 and cc49, which detect the glycoprotein TAG 72 present in tumor cells. The levels of CA 72.4 may be modified depending on the route of excretion of the antigen TAG 72. The objective of this study was to evaluate the behavior of CA 72.4 in healthy subjects and to assess the influence of chronic renal failure (CRF) on the levels of this tumor marker. Random serum samples were collected in 181 individuals (148 healthy volunteers and 33 patients with CRF) and 214 determinations of CA 72.4 were performed. We also performed 66 determinations of plasma creatinine. In healthy subjects the cutoff value of CA 72.4 was established at 3 U/mL, with a sensitivity of 53% and a specificity of 85.8%. In the CRF patients we found no statistically significant differences when we compared the values of CA 72.4 predialysis and postdialysis (p = 0.197). However, a statistically significant difference was found in the plasma creatinine levels (p < 0.001). Chronic renal failure does not affect the result of CA 72.4 determinations; this tumor marker may therefore be useful in the monitoring of patients with cancer, independent of their renal function.

Prognostic value of the glycoprotein TAG-72 in patients with gastric cancer.

The specificity of the tumor markers used to date in patients with gastric cancer has not been satisfactory. For this reason we decided to evaluate the utility of TAG-72 in this disease. Between 1993 and 1998 we determined the levels of TAG-72 in 638 subjects (148 healthy volunteers, 33 patients with chronic renal failure (CRF), 149 patients with benign diseases of the liver, 95 patients with benign gastrointestinal diseases and 213 patients with gastric cancer). TAG-72 was measured using an IRMA method. Statistical analysis of the data was performed with the BMDP package. We established a cutoff for TAG-72 of 3 U/mL, corresponding to the 92.6th percentile of the healthy controls. We observed that neither CRF nor benign liver diseases affected TAG-72 levels, while certain benign gastrointestinal diseases did cause alterations of the marker. Using Cox multivariate analysis we discovered that the preoperative TAG-72 level was an independent prognostic variable associated with both disease-free and overall survival. We conclude that, although TAG-72 is not useful for the diagnosis of gastric cancer, it is a suitable tool for disease monitoring and prognostic assessment.

[The clinical usefulness of routine biopsy of the transition zone in the diagnosis of prostate cancer]. / Utilidad clínica de la biopsia rutinaria de la zona transicional en el diagnóstico del cáncer de próstata.

OBJECTIVE: To evaluate the effectiveness of routine biopsy of the transitional area in patients undergoing early systematic sextant biopsy. PATIENTS AND METHODS: Two biopsies were taken from the transitional area further to a sextant biopsy in 164 consecutive patients. 98 cases had serum PSA levels higher than 4 ng/mL and 66 suspicious rectal digital examination. RESULTS: Cancer was detected in 77 patients (46.9%). In 28 (36.4%) cases cancer was found only in the peripheral area, in 2 (2.6%) in the transitional area and in 47 (61%) in both areas. CONCLUSION: Routine biopsy of the transitional area in early systematic prostate biopsy appears to be little effective. This would probably be indicated for patients undergoing rebiopsy.

[The incidence of lesions associated with prostatic biopsy and its effect on the serum concentration of the prostate-specific antigen]. / Incidencia de lesiones asociadas en la biopsia prostática y su influencia en la concentración sérica del antígeno prostático específico.

OBJECTIVE: To analyze the incidence of pathoanatomical lesions seen in systematic prostate biopsies and to evaluate their influence on PSA serum levels. MATERIAL AND METHODS: 495 consecutive prostate biopsies, indicated by a suspicion digital rectal examination in 194 patients (39.2%) and raised serum PSA in 301 (60.8%), were evaluated. Biopsy was performed by sextant and hypoechoic nodes, and PSA serum measurements by dual monoclonal antibody radioimmunoassay, Tandem PSA. RESULTS: Cancer was diagnosed in 42.6% biopsies and BPH in 67.4%; additionally, other associated lesions were detected in 74.6% cases, the most frequent ones being chronic prostatitis (47.3%), glandular atrophy (35.9%) and acute prostatitis (23%). All lesions were significantly related to the primary BPH diagnosis in 74.2% to 85% cases. High grade PIN (14.1%) was related to primary cancer diagnosis in 87.1% cases. The multivariate analysis showed that the main diagnosis (BPH vs cancer) was the only variable that had a significant influence on PSA serum levels. When BPH patients were considered separately, the only variable with significant influence on PSA serum levels was the prostatic volume. The univariate analysis showed a nonsignificant increase in association with acute prostatitis and high grade PIN, and a decrease in association with chronic prostatitis. CONCLUSIONS: BPH or cancer associated damage is very frequent in prostatic biopsies. However, the only factors showing a significant contribution to PSA serum levels appear to be the presence or absence of cancer, or the prostatic volume when the main diagnosis is BPH.

[Analysis of free PSA percentage predicting potential surgical failure in patients treated with radical prostatectomy]. / Análisis del porcentaje de PSA libre en la predicción del fallo quirúrgico potencial en pacientes sometidos a prostatectomía radical.

OBJECTIVE: To analyze if free PSA percentage can help to predict a potential surgical failure (PSF) in patients undergoing radical prostatectomy. MATERIAL AND METHODS: Analysis of serum PSA concentration and free PSA percentage in 92 patients undergoing retropubic radical prostatectomy. In 38 cases, the carcinoma was organ-confined, 26 had capsule penetration, 20 had positive margins, 6 seminal vesicle invasion and 2 lymph nodes. PSF was demonstrated in 28 patients (30.4%) and in 64 (69.6%) the carcinoma was organ-confined. RESULTS: No significant relationship was found between PSA serum concentration or free PSA percentage to the pathological stage. The logistic regression analysis where the clinical status, Gleason sum, and free PSA percentage were included as predictive variables, showed that the latter was the only factor with capacity for PSF prediction. Over all, the probability of a carcinoma being confined in the surgical specimen when percentage of free PSA was greater than 10 was 83.8% and 60% when it was lower or equal, p < 0.03. However, the distribution was only significant when PSA concentration ranged between 4.1 and 10 ng/mL, p < 0.008. In this range of PSA, the relative risk of PSF was 5.5 (95% CI 1.4-21.8) when free PSA percentage was equal or lower than 10, the probability being 50% versus 9.1% when it was greater than 10. CONCLUSIONS: Free PSA percentage can help to predict PSF. PSA serum concentration lower than 10 ng/mL and free PSA percentage greater than 10 allows to detect a subgroup of patients with good prognosis and with less than 10% probability of having positive margins, seminal vesicles invasion or lymph nodes.

Age-related inflammation: the contribution of different organs, tissues and systems. How to face it for therapeutic approaches.

A typical feature of ageing is a chronic, low-grade inflammation characterized by a general increase in the production of pro-inflammatory cytokines and inflammatory markers ("inflamm-ageing"). This status may slowly damage one or several organs, especially when unfavorable genetic polymorphisms and epigenetic alterations are concomitant, leading to an increased risk of frailty together with the onset of age-related chronic diseases. The contribution of different tissues (adipose tissue, muscle), organs (brain, liver), immune system and ecosystems (gut microbiota) to age-related inflammation ("inflamm-ageing") will be discussed in this review in the context of its onset/progression leading to site-restricted and systemic effects. Moreover, some of the possible strategies and therapies to counteract the different sources of molecular mediators which lead to the age-related inflammatory phenotype will be presented.

Immune-inflammatory responses and oxidative stress in Alzheimer's disease: therapeutic implications.

Alzheimer's disease (AD) is a heterogeneous and progressive neurodegenerative disease which in Western society mainly accounts for clinical dementia. AD has been linked to inflammation and oxidative stress. Neuro-pathological hallmarks are senile plaques, resulting from the accumulation of several proteins and an inflammatory reaction around deposits of amyloid, a fibrillar protein, Abeta, product of cleavage of a much larger protein, the beta-amyloid precursor protein (APP) and neurofibrillary tangles. Inflammation clearly occurs in pathologically vulnerable regions of AD and several inflammatory factors influencing AD development, i.e. environmental factors (pro-inflammatory phenotype) and/or genetic factors (pro-inflammatory genotype) have been described. Irrespective of the source and mechanisms that lead to the generation of reactive oxygen species, mammalian cells have developed highly regulated inducible defence systems, whose cytoprotective functions are essential in terms of cell survival. When appropriately activated, each one of these systems has the possibility to restore cellular homeostasis and rebalance redox equilibrium. Increasing evidence, support the notion that reduction of cellular expression and activity of antioxidant proteins and consequent augment of oxidative stress are fundamental causes for ageing processes and neurodegenerative diseases., including AD. The better understanding of different molecular and cellular inflammatory mechanisms is crucial for complete knowledge of AD pathophysiology, hence for its prevention and drug therapy. Accordingly, two lines of preventive therapeutics can be outlined, the first based on anti-inflammatory drugs, the second one on anti-oxidative properties.

Hypoallergenic fragment of Par j 2 increases functional expression of Toll-like receptors in atopic children.

BACKGROUND: Parietaria judaica (Par j) is one of the main causes of allergy in the Mediterranean countries. The activation of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) inhibits nasal inflammation of atopic children. OBJECTIVE: To examine, in vivo and in vitro, the effect of recombinant Par j 2 (rPar j 2) and of its fragments (1-55 and 52-102) on atopic children. METHODS: We used skin prick test for in vivo evaluations. We assessed, in vitro, in peripheral blood mononuclear cells (PBMC), the effect of rPar j 2 and of the two fragments on neutrophil chemotaxis, on CD45RO, on TLR2 and TLR4 expression, on LPS binding and on interferon (IFN)-gamma release, by a microchemotaxis chamber, by flow cytometry and by enzyme-linked immunosorbent assay, respectively. RESULTS: In vivo while rPar j 2 induced a positive skin reaction, 1-55 and 52-102 fragments did not. In vitro, while rPar j 2 increased both CD45RO expression and neutrophils chemotaxis in PBMC, both Par j 2 fragments did not. 1-55 fragment of Par j 2 upregulated both TLR2 and TLR4 expression and LPS binding, while the rPar j 2 and 52-102 fragment did not. Finally, 1-55 fragment of Par j 2 induced IFNgamma release, while the rPar j 2 and 52-102 fragment did not. CONCLUSIONS: Hypoallergenic 1-55 fragment, upregulating innate immunity receptors and increasing IFNgamma, might re-orientate, in atopics, the immune system toward a physiologic balance between Th1 and Th2 responses.

Methods for recovering nucleic acid fragments from agarose gels.

Agarose gel electrophoresis is a powerful technique for the separation of nucleic acids on the basis of their size and conformation. The development of methods to recover size-fractionated nucleic acids molecules from agarose gels has greatly facilitated recombinant DNA technologies. Although several methods for recovering DNA and RNA molecules have been developed during the past fifteen years, none of them has been universally accepted. In this review we describe, discuss and evaluate the most common procedures with which we have had experience. Our evaluation is based on the criteria of yield, purity, speed, simplicity and low cost. We have considered three different approaches to the problem of recovering nucleic acids: chemical gel dissolution, physical gel disruption and physical extrusion from intact gels.

Enhanced hybridization labeling signals in Southern blotted DNAs fractionated with voltage gradient gel electrophoresis.

An enhancement of hybridization labeling signals is demonstrated in Southern blotted DNAs, fractionated by voltage gradient gel electrophoresis. This enhancement is due to a reduced thickness of each single nucleic acid band in the gel as a consequence of the gradient effect, corresponding to an increased concentration of DNA per unit area.

cDNA sequence analysis and expression of the expression of the ribosomal protein S24 during oogenesis and embryonic development of the sea urchin Paracentrotus lividus.

A gene for the Paracentrotus lividus ribosomal protein S24, called P1-S24, has been isolated and sequenced. Ribosomal protein P1-S24 consists 130 amino acids and has a molecular weight of 14869 Da. Sequence analysis shows a high percentage (90%) identity with the corresponding gene of Strongylocentrotus purpuratus. Hybridization of the cDNA to digested sperm DNA suggests that P1-S24 is represented in no more than two copies. Studies of the temporal expression of P1-S24 gene indicate a good correlation between this and the expression of the rRNA genes both during oogenesis and embryonic development.

Sequence analysis of the rDNA spacer of Paracentrotus lividus and observations about pre-rRNA processing. NTS sequence of Paracentrotus lividus rDNA.

We have isolated and sequenced one intergenic region and a small part of the flanking regions (18S and 26S rRNA coding regions) of the rRNA-encoding genes (rDNA) from the sea urchin Paracentrotus lividus. This region is about 3.8 Kb long. Northern blot hybridizations and S1 mapping experiments demonstrated the presence of a partially processed 21S rRNA precursor while has the same 5' terminus as the 32S primary precursor, also in developmental stages characterized by a low rate of rRNA synthesis.

A method for eluting DNA in a wide range of molecular weights from agarose gels.

We have developed a simple and rapid method for recovering DNAs of a wide range of molecular weights from agarose gels. A DNA-containing gel slice is placed on a Parafilm sheet in the center of a circular (positive) electrode and covered with a drop of buffer, while a linear (negative) electrode is placed on the top of the gel and driven about 1 mm into the gel itself. When a continuous current is applied, the DNA migrates into the buffer toward the circular electrode. We have obtained almost total recovery of DNAs up to 10 kb in size. Our method may also be used, under appropriate conditions, for higher molecular weight DNAs. The yield and all the biological assays performed on the DNAs obtained by our method recommend it for routine laboratory use.