Hepatitis C virus NS3/4A quasispecies diversity in acute hepatitis C infection in HIV-1 co-infected patients.

The growing number of cases of acute hepatitis C (AHC) infections among human immunodeficiency virus type 1 (HIV-1)-positive men who have sex with men (MSM) in the last 10 years has promoted the search for predictors of AHC clearance as well as for epidemiological networks of viral transmission. We characterized the diversity and catalytic efficiency of HCV NS3/4A protease quasispecies in AHC patients coinfected with HIV-1. Plasma samples obtained at HCV diagnosis from 18 MSM HIV-coinfected patients with AHC were studied. Five HCV monoinfected patient samples with AHC were also investigated. An average of 39 clones from each sample was analysed. The catalytic efficiency of the dominant quasispecies (i.e. the most abundant) from each quasispecies was also assayed for mitochondrial antiviral signalling protein (MAVS) cleavage. Phylogenetic analysis identified two clusters of patients with highly related viruses, suggesting a common source of HCV infection. None of the 18 MSM HIV-coinfected patients spontaneously cleared HCV, although 78% of the treated patients achieved a sustained virological response after early treatment with pegylated interferon (pegIFN) plus ribavirin (RBV). The synonymous-nonsynonymous (ds/dn) mutation ratio, a marker of selective pressure, was higher in AHC compared to 26 HIV-1-infected men with genotype 1a chronic hepatitis C (CHC) (P < 0.0001). NS3/4A proteases from AHC patients also exhibited higher catalytic efficiency compared to CHC patients (P < 0.0001). No differences were found when ds/dn mutation ratios and NS3/4A protease catalytic efficiencies from AHC HIV-coinfected patients were compared with AHC monoinfected patients. The presence of epidemiological networks of HCV transmission was confirmed among HIV-1-positive MSM. In addition, substantial genetic diversity was demonstrated in AHC. NS3/4A protease efficiency cleaving MAVS may be associated with virus transmission and response to pegIFN/RBV treatment.

Balearic archipelago: three islands, three beta-thalassemia population patterns.

The mutation spectrum of 175 ß-thalassemia (ß-thal) carriers, identified in pilot carrier screening on 22,713 individuals from Balearic Islands (Spain), is reported. The ß(0) CD39 (C>T) mutation is the most frequent (61.1%), followed by ß(+) IVS-I-110 (G>A) (12.0%), ß(+) IVS-I-6 (T>C) and ß(0) IVS-1-1 (G>A) (3.4% both) and eight other rare mutations (2.9-0.6%); with a distinct prevalence and distribution between islands. Minorca shows the highest prevalence in Iberian populations, with a single mutation, CD39 (C>T), present in most ß-thal carriers. Ibiza is the only Western Mediterranean population where the most frequent ß-thal mutation is IVS-I-110 (G>A). These results can be explained by a combination of historical-demographic characteristics together with evolutionary forces such as founder effect, genetic drift and probably selection by malaria. Knowledge of the mutational spectrum in the Balearic Islands will enable to optimize mutation detection strategy for genetic diagnosis of ß-thal in these islands.

Rubeosis iridis as a sign of underlying carotid stenosis.

Severe carotid stenosis may be associated with uncommon clinical symptoms. We report a case of ocular ischemic syndrome and subsequent rubeosis iridis due to a high-grade carotid stenosis. The patient recovered visual acuity and his normal iris coloring after carotid endarterectomy. Rubeosis iridis may be the only clinical sign associated with severe carotid stenosis, making it mandatory to rule out the presence of carotid narrowing when it is detected. Establishing an early diagnosis is essential to improve quality of life, prognosis, and patients' outcome.

Risk of recurrence during follow-up for optimally treated advanced epithelial ovarian cancer (EOC) with a low-level increase of serum CA-125 levels.

BACKGROUND: Our group evaluated the risk of recurrence for optimally treated advanced epithelial ovarian cancer (adEOC) in patients with a low-level rising serum CA-125 concentration within the normal range (0-35 kU/l). In addition, we tested the new proposed early CA-125 signal of progressive disease (EPD) criterion in the same study population. PATIENTS AND METHODS: Patients treated from 1998 to 2006 for adEOC were identified at our institution. Inclusion criteria were as follows: CA-125 at time of diagnosis (>35 kU/l); International Federation of Gynecology and Obstetrics stages III-IV treated with optimal primary treatment; and complete response (CR) to primary treatment with normalization of CA-125. RESULTS: Median progression-free survival and overall survival for the recurrence group (n = 60) were 17.7 and 38.2 months, respectively. The median follow-up time from CR to last contact was 40.2 months for patients in the nonrecurrence group (n = 36). An absolute increase in serum CA-125 levels of >or=5 kU/l compared with baseline CA-125 nadir values was significantly predictive of recurrence (odds ratio for recurrence = 402.98, P < 0.0001). The progression date was predated by the EPD criterion in 77% of patients with known progressive disease (median, 58 days early) with a sensitivity of 90%, a positive predictive value of 96.4%, and a false-positive rate of 5.6%. CONCLUSIONS: Among patients with optimally treated adEOC in complete remission, a low-level increase in serum CA-125 concentration within the normal range is a strong independent predictive factor for disease recurrence. In this patient population, future prospective randomized trials should consider the evaluation of the EPD criterion.

Nadir CA-125 concentration in the normal range as an independent prognostic factor for optimally treated advanced epithelial ovarian cancer.

BACKGROUND: The amount of residual disease after surgery is considered the most important factor influencing the survival of patients with advanced epithelial ovarian cancer (adEOC). In optimally treated patients with adEOC, there are no well-established prognostic factors [excluding International Federation of Gynecology and Obstetrics (FIGO) stage]. The aim of this retrospective study is to analyze the prognostic value of the CA-125 nadir after the completion of an optimal primary treatment. PATIENTS AND METHODS: Patients treated for adEOC were identified from January 1998 to December 2006. INCLUSION CRITERIA: elevated CA-125 at time of diagnosis (>35 kU/l); FIGO stage III-IV treated with optimal primary treatment (residual tumor <1 cm and carboplatin/taxane-based combination chemotherapy); and complete response to optimal primary treatment with normalization of CA-125. RESULTS: Patients, n = 96: 44 group A (< or =10 kU/l); 52 group B (11-35 kU/l). Median progression-free survival (PFS) was 42 and 20 months for groups A and B, respectively (P = 0.0087). Median overall survival (OS) was 84 and 43 months for groups A and B, respectively (P < 0.0001). The Cox model showed a highly significant impact on PFS and OS in relation to CA-125 nadir levels. CONCLUSIONS: The CA-125 nadir value is a strong independent prognostic factor for optimally treated adEOC after achieving a complete response.

Prevalence of HFE C282Y and H63D in Jewish populations and clinical implications of H63D homozygosity.

The frequencies of C282Y and H63D mutations of the HFE gene vary between different populations. A previous study showed an unexpectedly high H63D frequency in Chuetas (a population of Jewish descent). The present study addressed the question of the distribution of these mutations in Jewish populations from different origins and studied the possible causes of the high H63D frequency in Chuetas. Moreover, to improve the understanding of the controversial relationship between H63D homozygosity and iron overload, a group of patients with altered iron metabolism were studied. The high frequency of H63D mutation in Chuetas is not due to a high prevalence of this mutation in Sephardic Jews. Jewish populations have low C282Y and moderate H63D frequencies, suggesting slight gene flow from their surrounding populations. In accordance with historical and demographic data, genetic drift is the most probable cause for the singular H63D frequency in Chuetas. Clinically, this study of H63D homozygotes supports the conclusion that this genotype must be taken into account, because it confers an increased risk of iron overload and therefore genetic susceptibility to developing hereditary hemochromatosis or to aggravating other diseases.

In hereditary hemochromatosis, red cell apheresis removes excess iron twice as fast as manual whole blood phlebotomy.

The current treatment of hereditary hemochromatosis (HH) consists of performing periodic manual whole blood phlebotomies. Erythroapheresis (EPH) is considered to be an alternative procedure if the classic treatment is contra-indicated. A prospective study of 13 consecutive cases of HH were included in a periodic EPH program with the aim of assessing the efficacy, feasibility, and tolerability of EPH in the treatment of HH by induction and maintenance. Iron depletion (ferritin <20 microg/l) was achieved in all patients after a mean of 6.7 +/- 2.9 months of treatment and a mean of 13.5 +/- 7.2 EPH sessions. The procedure was well tolerated and there were no complications. After a follow-up period of 10.5 +/- 6.6 months, only four patients have required further maintenance sessions beyond 6 months after completing the induction therapy. The efficacy, speed, tolerability, and more favorable schedule of an EPH program facilitate treatment of HH.

Distribution of HFE C282Y and H63D mutations in the Balearic Islands (NE Spain).

The HFE gene contains two main missense mutations: C282Y and H63D. Individuals with these mutations carry a risk of developing hereditary haemochromatosis (HH). The common form of this disease is due to homozygosity for the C282Y mutation. Population studies have shown the variation of the prevalence of these mutations in different countries and ethnic groups. The purposes of this current study were to determine the prevalence of the C282Y and H63D mutations in the Balearic Islands and the genotypic characterization of patients diagnosed with HH, as well as those with iron overload and liver diseases. A total of 1330 Balearic chromosomes were analyzed. The results showed that the populations of the Balearic Islands were not homogeneous. No C282Y carriers were observed in a group of descendants of Majorcan Jews (Chuetas) and the frequency was very low in Minorca (1.2%) in comparison with the other islands of Majorca (4.7%) and Ibiza (6.5%). The carrier frequency of the H63D mutation was similar in the three islands and very high (43.1%) in the descendants of Majorcan Jews. The study of patients was carried out in 129 individuals. The homozygous C282Y genotype was the principal one involved in hereditary haemochromatosis (90%), whereas the other HH patients were C282Y/H63D compound heterozygous and H63D homozygous.

Role of a dipeptide insertion between codons 69 and 70 of HIV-1 reverse transcriptase in the mechanism of AZT resistance.

The 3'-azido-3'-deoxythymidine (AZT)-resistant pheno type of a heavily mutated human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) carrying a dipeptide (Ser-Ser) insertion between codons 69 and 70 as well as other mutations related to resistance to RT inhibitors has been studied. Recombinant virus carrying this variant RT (termed SS RT) showed reduced susceptibility to all nucleoside RT inhibitors in clinical use, particularly to AZT. In the presence of ATP, recombinant SS RT had an increased ability to remove the 3'-terminal nucleotide from AZT- terminated primers and extend the unblocked primer, compared with wild-type HIV-1 RT (BH10 isolate). Insertion of two serines in the sequence context of BH10 RT did not affect the ATP-dependent phosphorolytic activity of the enzyme, and had no influence in resistance to RT inhibitors. However, SS RT mutants lacking the dipeptide insertion or bearing a four-serine insertion showed reduced ATP-dependent phosphorolytic activity that correlated with increased AZT sensitivity, as determined using a recombinant virus assay. Therefore, the insertion appears to be critical to enhance AZT resistance in the sequence context of multidrug-resistant HIV-1 RT.

Prevalence of the C282Y mutation for haemochromatosis on the Island of Majorca.

The C282Y mutation of the HFE gene has been reported to be present in most of the patients with hereditary haemochromatosis (HH) of Northern European ancestry. HH affects approximately 1/300 individuals, but it is not evenly distributed in the different European countries. In the present study, polymerase chain reaction (PCR) and restriction-enzyme digestion were used to analyse the frequency of the most important mutation in haemochromatosis (C282Y) in subjects from Majorca (Balearic Islands, Spain) and patients with haemochromatosis. The results were compared with other studies from Spain and Europe. A total of 420 Majorcan chromosomes were analysed and the C282Y mutation was observed at a frequency of 2.62%+/-0.8 (11 heterozygotes: eight men and three women). In the group of hereditary haemochromatosis probands, 13 out of 14 were homozygous for the C282Y mutation. In the distribution of the C282Y mutation, a north-west to south-east cline was detected, supporting the Celtic origin of this mutation.

A bacteriophage lambda-based genetic screen for characterization of the activity and phenotype of the human immunodeficiency virus type 1 protease.

Human immunodeficiency virus type 1 (HIV-1) resistance to antiretroviral drugs is the main cause of patient treatment failure. Despite the problems associated with interpretation of HIV-1 resistance testing, resistance monitoring should help in the rational design of initial or rescue antiretroviral therapies. It has previously been shown that the activity of the HIV-1 protease can be monitored by using a bacteriophage lambda-based genetic assay. This genetic screening system is based on the bacteriophage lambda regulatory circuit in which the viral repressor cI is specifically cleaved to initiate the lysogenic to lytic switch. We have adapted this simple lambda-based genetic assay for the analysis of the activities and phenotypes of different HIV-1 proteases. Lambda phages that encode HIV-1 proteases either from laboratory strains (strain HXB2) or from clinical samples are inhibited in a dose-dependent manner by the HIV-1 protease inhibitors indinavir, ritonavir, saquinavir, and nelfinavir. Distinct susceptibilities to different drugs were also detected among phages that encode HIV-1 proteases carrying different resistance mutations, further demonstrating the specificity of this assay. Differences in proteolytic processing activity can also be directly monitored with this genetic screen system since two phage populations compete in culture with each other until one phage outgrows the other. In summary, we present here a simple, safe, and rapid genetic screening system that may be used to predict the activities and phenotypes of HIV-1 proteases in the course of viral infection and antiretroviral therapy. This assay responds appropriately to well-known HIV-1 protease inhibitors and can be used to search for new protease inhibitors.

[Comparative study of the analyzers L-9100 (Merck-Hitachi) and HA-8121 (Menarini) for the determination of glycosylated hemoglobin]. / Estudio comparativo de los analizadores L-9100 (Merck-Hitachi) y HA-8121 (Menarini) para la determinación de hemoglobina glucosilada.

A comparison study about the determination of HbA1c by L-9100 (Merck Hitachi) and HA-8121 (Menarini) was made. HbA1c was measured by high pressure liquid chromatography (HPLC). The within-run imprecision was CV < 3% (for HA-8121) and CV < 4.9% (for L-9100). The between-run imprecision was CV < 1% (for L-9100) and CV < 2% (for HA-121). Analysis of means (Student test) showed that both instruments were statistically different for HbA1Ac measurements (p < 0.001). The results of regression analysis (Passsing-Bablock test) were as follows: y = 0.88x + 0.58 (y = L-9100, r = 0.982, p = 0.005). In conclusion, the results showed that there are statistical differences between both instruments. Regression and correlation was good, but the data also showed that there is a constant and proportional error. We must make a new reference range values.

The role of interleukin 2 (IL-2) and serum-soluble IL-2 receptor cells in idiopathic IgA nephropathy.

Interleukin 2 (IL-2) plays a central role in the immune response and may be involved in the derangement of cellular immunoregulation of idiopathic IgA nephropathy (IgAN). The aim of this study was to investigate the serum levels and production of IL-2 from peripheral blood mononuclear cells (PBMC) and the distribution of IL-2 receptor cells and serum-soluble IL-2 receptor cells (sIL-2R) in patients with IgAN. Twenty-four patients with IgA nephropathy and 11 healthy controls (age and sex matched) were studied during an infection-free period without signs of clinical activity at the moment of the study. Serum IL-2 concentrations did not differ between patients and controls. The supernatant levels of IL-2 taken from 24-hr cultures of PBMC stimulated with phytohemagglutinin or tumor necrosis factor increased significantly in the patients but not in the controls. The percentage of IL-2R positive cells (CD25+) was increased in patients compared with controls. Moreover, IgAN patients had increased activated CD4+ lymphocytes when compared with the controls. Serum levels of sIL-2R were significantly higher in patients than in controls. There were no correlations among renal function, serum IgA levels, and urinary findings with cellular subsets or with IL-2 levels. However, sIL-2R was higher in the subgroup of patients with episodic macrohematuria and was closely related with the presence of red blood cells in the urinary sediment. We conclude that PBMC of IgA nephropathy patients have an overproduction of IL-2 after mitogenic stimulation, an increased helper T cell activity, increased IL-2R+ cells, and elevated serum levels of sIL-2R. These alterations are present in periods of apparent clinical inactivity. Finally, sIL-2R is closely related with hematuria, providing a good marker for disease activity. Our results suggest a pivotal role of IL-2 in cellular immune responses with regard to T cell activation in patients with IgAN.

T-cell alterations in immunoglobulin A nephropathy.

In the present paper are reported alterations of the cellular immunity found in 24 patients with immunoglobulin A nephropathy (IgAN). CD4+ and CD25+ cells were increased in patients in comparison with controls. The mean of interleukin-2 (IL-2) and interleukin-4 (IL-4) levels in sera were similar in patients and controls, but the levels of IL-2 in supernatants of stimulated peripheral blood mononuclear cells from patients were higher than those of controls. There were no correlations between renal function, serum IgA levels, urinary findings, cellular subsets, and IL-2 or IL-4 sera levels. These immunological data were also unrelated to the mode of clinical presentation. The results suggest a pivotal role of IL-2 in cellular immune response with regard to T-cell activation in patients with IgAN.

[Metabolic disorders in the gastrectomized patient]. / Estudio de los trastornos metabólicos en el enfermo gastrectomizado.

The present study attempts on one hand to study the metabolic disorders which may present themselves in the gastrectomized patient, such as the malabsorption of fat, vitamin B12, folic acid and iron as well as the possible correlation between steatorrhea and the presence of exocrine pancreatic insufficiency. For this purpose a group of 71 patients have been studied who have undergone a subtotal gastrectomy (70.42%) or total (29.58%) in the General Surgery Services of Elche. The results obtained show the presence of ferropenic or megaloblastic anemia in 61.97% of the group, serious steatorrhea in only 3 patients (4.22%) and calciumphosphorous metabolism alterations appeared in 21.13%. With this we conclude that anemia is the most frequent ferropenic alteration in the gastrectomy patient; steatorrhea does not seem to be produced exclusively by the presence of exocrine pancreatic insufficiency and bone alterations in the gastrectomized patient appear in an insidious manner, being more a question of biochemical alterations than actual clinical lesions.