Relapse rate and renal prognosis in ANCA-associated vasculitis according to long-term ANCA patterns.

Long-term observation of patients with ANCA-associated vasculitis (AAV) allows the identification of different longitudinal patterns of ANCA levels during follow-up. This study aimed to characterize these patterns and to determine their prognostic significance. All ANCA determinations performed in two university hospitals during a 2-year period were retrospectively reviewed. Patients were included in the analysis if they had high titers of anti-myeloperoxidase (anti-MPO) or anti-proteinase 3 (anti-PR3) antibodies at least once, ≥ 5 serial ANCA determinations and AAV diagnosed by biopsy or American College of Rheumatology (ACR) classification criteria. Patients' time-course ANCA patterns were classified as monophasic, remitting, recurrent or persistent. Associations between ANCA patterns and prognostic variables (relapse rate and renal outcome) were analysed by univariate and multivariate statistics. A total of 99 patients [55 with microscopic polyangiitis (MPA), 36 with granulomatosis with polyangiitis (GPA) and eight with eosinophilic granulomatosis with polyangiitis (EGPA)] were included. Median follow-up was 9 years. Among patients diagnosed with MPA or GPA, recurrent or persistent ANCA patterns were associated with a higher risk of clinical relapse [hazard ratio (HR) = 3·7, 95% confidence interval (CI) = 1·5-9·1 and HR = 2·9, 95% CI = 1·1-8·0, respectively], independently of clinical diagnosis or ANCA specificity. In patients with anti-MPO antibodies, the recurrent ANCA pattern was associated with worsening renal function [odds ratio (OR) = 5·7, 95% CI = 1·2-26·0]. Recurrent or persistent ANCA patterns are associated with a higher risk of clinical relapse. A recurrent ANCA pattern was associated with worsening renal function in anti-MPO-associated vasculitis.

Identification of Pen m 4 as a potential cause of occupational asthma to Gammarus shrimp.

We present the case of a 34-year-old male patient employed for 8 years in a company manufacturing and packaging animal feed. The patient developed occupational asthma to dry Gammarus powder. The diagnosis was confirmed by specific bronchial provocation test. The determination of specific IgE antibodies was positive for Pen m 4, a sarcoplasmic calcium binding protein, with a level of 6.7 ISU-E. The sensitization to Pen m 4 described here may identify a new allergen causing occupational asthma in these workers.

Sperm telomere length in motile sperm selection techniques: A qFISH approach.

Several studies have associated telomere shortening with alterations in reproductive function. The objective of the present study was to determine telomere length (TL) in spermatozoa selected by either density-gradient centrifugation (DGC) or swim-up. The analysis of TL was performed using quantitative fluorescent in situ hybridisation (qFISH) using PNA probes in combination with a chromatin decompaction protocol in sperm cells. Results of TL were 24.64 ± 5.00 Kb and 24.95 ± 4.60 Kb before and after DGC, respectively, and 19.59 ± 8.02 Kb and 20.22 ± 5.18 Kb before and after swim-up respectively. Sperm selected by DGC or swim-up did not show any significant differences in TL as compared to nonselected sperm (p > .05). Negative correlations between TL and sperm motility (r = -.308; p = .049) and concentration (r = -.353; p = .028) were found. Furthermore, exposure of sperm to increasing concentrations of hydrogen peroxide during incubation resulted in a reduction in TL. These data indicate that oxidative stress may be one of the main factors involved in the reduction of TL in sperm. Preliminary clinical results from patients included in this study indicate that TL was shorter in spermatozoa from couples who never achieved a pregnancy compared to couples who did achieve at least one natural pregnancy (p < .05); however, the clinical utility of this biomarker still needs to be confirmed in further studies.

Nuclear degraded sperm subpopulation is affected by poor chromatin compaction and nuclease activity.

There is an interest in the nuclear degraded sperm subpopulation because, although it is present in a low percentage in all semen samples, patient groups such as varicocele and rearranged genome carriers show high levels of these degraded spermatozoa. This study is designed with two objectives in mind: first, incubations of H2 O2 and nuclease on DTT-treated and untreated samples to show the aetiology of this subpopulation and second, assessment of the correlation between the protamine ratio and nuclear degraded spermatozoa. A very high increase in the nuclear degraded subpopulation has been found with nuclease incubation, and it is even higher when it has been merged with nuclear decompaction using DTT. Alternatively, incubation with H2 O2 with and without DTT did not show such a significant increase in nuclear degraded spermatozoa. The protamine ratio correlated with this subpopulation, showing, in patients, that poor nuclear compaction would turn the sperm susceptible to degradation. Then, the assessment of nuclear degraded spermatozoa might not be only a measure of DNA degradation but also an indicator of chromatin compaction in the spermatozoa. Different patient groups would fit this model for sperm nuclear degradation, such as varicocele patients, who show a high percentage of immature spermatozoa and nuclear degraded spermatozoa, and reorganised genome carriers, where reorganisation might also cause poor chromatin compaction on the sperm nucleus.

Human semen cryopreservation: a sperm DNA fragmentation study with alkaline and neutral Comet assay.

Sperm cryopreservation is widely used for both research and reproduction purposes, but its effect on sperm DNA damage remains controversial. Sperm DNA fragmentation (SDF) has become an important biomarker to assess male infertility. In particular, the differentiation between single- and double-stranded DNA fragmentation (ssSDF and dsSDF) has clinical implications for male infertility where ssSDF is associated with reduced fertility, whereas dsSDF is associated with increased risk of miscarriage. In this study, semen samples from 30 human males have been analysed in both fresh and cryopreserved using the alkaline and neutral Comet assays. Results show an increase of about 10% of ssSDF, assessed by the alkaline Comet assay, regardless of the male fertility status. Neutral Comet analysis of dsSDF does not show any statistical increase when comparing fresh and cryopreserved samples in any of the patient groups. Results support previous reports that oxidative stress is the major effector in DNA damage during sample cryopreservation, as, on one hand, ssSDF has previously been related to oxidative damage and, on the other hand, we have not found any effect on dsSDF. Therefore, there might be a slight risk of decreased fertility after using a freezed sample, but no evidence for increased miscarriage risk from cryopreserved spermatozoa should be expected.

Comprehensive analysis of sperm DNA fragmentation by five different assays: TUNEL assay, SCSA, SCD test and alkaline and neutral Comet assay.

Sperm DNA fragmentation (SDF) is becoming an important test to assess male infertility. Several different tests are available, but no consensus has yet been reached as to which tests are most predictive of infertility. Few publications have reported a comprehensive analysis comparing these methods within the same population. The objective of this study was to analyze the differences between the five most common methodologies, to study their correlations and to establish their cut-off values, sensitivity and specificity in predicting male infertility. We found differences in SDF between fertile donors and infertile patients in TUNEL, SCSA, SCD and alkaline Comet assays, but none with the neutral Comet assay. The alkaline COMET assay was the best in predicting male infertility followed by TUNEL, SCD and SCSA, whereas the neutral COMET assay had no predictive power. For our patient population, threshold values for infertility were 20.05% for TUNEL assay, 18.90% for SCSA, 22.75% for the SCD test, 45.37% for alkaline Comet and 34.37% for neutral Comet. This work establishes in a comprehensive study that the all techniques except neutral Comet are useful to distinguish fertile and infertile men.

Effectiveness of omalizumab in non-allergic severe asthma.

Omalizumab is an effective drug for allergic asthma. The purpose of this study was to evaluate the effectiveness and tolerance of this drug in non-allergic GINA step V asthma patients. This study was single-centre, prospective, open-label, observational, naturalistic. Non-allergic asthma patients requiring a mean dose of oral prednisolone of at least 5 mg/day during greater than or equal to 1 year or an accumulated oral corticosteroid dose/year greater than or equal to 1500 mg were enrolled. At entry and the end of the 12-month follow-up we measured blood eosinophilia and IgE concentration; at every monthly visit a forced spirometry and exhaled fraction of nitric oxide (NO) were carried out. The subjects were seven adult patients (5 female), age range 37-63 years, with the following mean values: IgE: 226.7+/-176 IU/mL; FVC 74+/-18 percent; FEV1 57+/-11 percent; NO: 21.2+/-7 ppb. The study was approved by the IRB of the hospital. One patient decided to stop treatment after 12 weeks and was excluded from the evaluation. We did not observe changes in eosinophil count, spirometry or NO values. Three patients considered responders did not need prednisolone during the follow-up. The mean daily dose of prednisolone fell from 6.6+/-8.1 mg/day at entry to 1.5+/-2.3 mg/day (p less than 0.16) at the end of follow-up. The mean monthly accumulated dose fell from 92+/-112 to 12+/-26 mg/month (p=0.26). Total blood IgE increased 1.93-fold. Side effects were only local: treatment tolerance was excellent; three out of six patients seemed to slightly benefit from anti-IgE treatment; to date there is no evidence strong enough to systematically prescribe omalizumab in non-allergic asthma patients.

Effects of oral antioxidant treatment upon the dynamics of human sperm DNA fragmentation and subpopulations of sperm with highly degraded DNA.

The primary aim of this study was to determine the effect of oral antioxidant treatment (1500 mg of l-Carnitine; 60 mg of vitamin C; 20 mg of coenzyme Q10; 10 mg of vitamin E; 10 mg of zinc; 200 µg of vitamin B9; 50 µg of selenium; 1 µg of vitamin B12) during a time period of 3 months upon the dynamics of sperm DNA fragmentation following varying periods of sperm storage (0 h, 2 h, 6 h, 8 h and 24 h) at 37 °C in a cohort of 20 infertile patients diagnosed with asthenoteratozoospermia. A secondary objective was to use the sperm chromatin dispersion test (SCD) to study antioxidant effects upon a specific subpopulation of highly DNA degraded sperm (DDS). Semen parameters and pregnancy rate (PR) were also determined. Results showed a significant improvement of DNA integrity at all incubation points (P < 0.01). The proportion of DDS was also significantly reduced (P < 0.05). Semen analysis data showed a significant increase in concentration, motility, vitality and morphology parameters. Our results suggest that antioxidant treatment improves sperm quality not only in terms of key seminal parameters and basal DNA damage, but also helps to maintain DNA integrity. Prior administration of antioxidants could therefore promote better outcomes following assisted reproductive techniques.

Alkaline and neutral Comet assay profiles of sperm DNA damage in clinical groups.

BACKGROUND: The analysis of sperm DNA fragmentation has become a new marker to predict male infertility, and many techniques have been developed. The sperm Comet assay offers the possibility of differentiating single- and double-stranded DNA (ssDNA and dsDNA) breaks, which could have different effects on fertility. The objective of this study was to perform a descriptive characterization of different groups of patients, such as those with asthenoteratozoospermic (ATZ) with or without varicocele, oligoasthenoteratozoospermic (OATZ) or balanced chromosome rearrangements, as compared with fertile donors. The Comet assay was used to investigate sperm samples for ssDNA and dsDNA breaks. METHODS AND RESULTS: The analysis of alkaline and neutral Comet assays in different groups of patients showed different sperm DNA damage profiles. Most fertile donors presented low values for ssDNA and dsDNA fragmentation (low-equivalent Comet profile), which would be the best prognosis for achieving a pregnancy. OATZ, ATZ and ATZ with varicocele presented high percentages of ssDNA and dsDNA fragmentation (high-equivalent Comet assay profile), ATZ with varicocele being associated with the worst prognosis, due to higher levels of DNA fragmentation. Rearranged chromosome carriers display a very high variability and, interestingly, two different profiles were seen: a high-equivalent Comet assay profile, which could be compatible with a bad prognosis, and a non-equivalent Comet assay profile, which has also been found in three fertile donors. CONCLUSIONS: Comet assay profiles, applied to different clinical groups, may be useful for determining prognosis in cases of male infertility.

Dynamics of sperm DNA fragmentation in patients carrying structurally rearranged chromosomes.

This investigation was conducted to assess the baseline level of sperm DNA fragmentation (SDF) in a cohort of patients presenting chromosomal rearrangements (nine reciprocal translocations and two inversions). In a separate experiment, a dynamic analysis to calculate the rate of SDF (rSDF), after a varying period of sperm storage (0 h, 1 h, 4 h, 8 h and 24 h) at 37 °C, was performed. Results were compared with eight fertile donors. Different experimental approaches to assess SDF, such as terminal transferase dUTP nick-end labelling (TUNEL), sperm chromatin structure assay (SCSA) and sperm chromatin dispersion test (SCDt), were used. No differences for the baseline level of SDF were found. Carriers of reorganized genomes showed statistically higher levels of SDF than did control donors (p = 0.025 for TUNEL; p = 0.022 for SCSA; p = 0.014 for SCDt). However, 54.5% (6/11) of the patients presented values similar to those of control donors. There was no significant difference in rSDF (p = 0.34). Nevertheless, the results suggest that a high variability for SDF and rSDF exists in these patients. Routine analysis of SDF and rSDF should be considered in patients presenting rearranged genomes to determine fertility status for assisted reproductive techniques (ART) purposes.

Sperm DNA integrity and meiotic behavior assessment in an infertile male carrier of a 9qh+++ polymorphism.

Although several reports on male infertility suggest a relationship between chromosome 9 polymorphisms and infertility, the effects on the phenotype have not been extensively reported. In this study, an infertile patient was found to carry a 9qh+++ chromosome. The flow cytometric TUNEL assay and SCD test have been applied to characterize sperm DNA integrity. In order to assess its meiotic behaviour, synapsis, recombination, and aneuploidy, analyses have been also performed. Sperm DNA fragmentation (SDF) was 77.81% and 87% for the TUNEL and SCD tests, respectively. Ninety-two percent of pachytene cells analyzed showed meiotic abnormalities. The mean number of MLH1 foci per pachytene in the control group was higher (49) than the mean found in the 9qh+++ patient (38) (P < .0001). In spermatozoa, significant increases of disomy rates were observed for chromosome 18 and for the sex chromosomes (P < .0001). These disturbances could be present in other male carriers of a less marked 9qh+.

Afectación renal en la crioglobulinemia mixta tipo II / Type II essential mixed cryoglobulinemia and renal disease. Hepatitis C virus association

La relación entre crioglobulinemia mixta tipo II y la infección por el virus C es un hecho bien establecido. Algunos autores han responsabilizado a dicha infección la práctica totalidad de los casos de crioglobulinemia antes denominada esencial.Sin embargo su prevalencia y significado clínico no está totalmente esclarecido. Revisamos nuestra experiencia en cuanto a las características clínicas, biológicas y evolutivas de los pacientes diagnosticados de crioglobulinemia mixta tipo II.Métodos: Estudio descriptivo y protocolizado de todos los casos de crioglobulinemia mixta tipo II diagnosticados durante un período de 8 años consecutivos. La definición de 'nefropatía crioglobulinémica' ha sido restrictiva, exigiéndose una creatinina > 1,5 mg/dl y/o proteinuria > 500 mg/24 h y/o hematuria (> 15 hematíes por campo), en ausencia de otras patologías que pudieran explicar estas alteraciones.Asimismo, se consideró la información de la biopsia renal en los casos disponibles.Resultados: Se han detectado 62 pacientes, 44 de los cuales (72 por ciento) estaban infectados por el virus C. El 52 por ciento han presentado clínica en relación con la crioglobulinemia. El 56 por ciento han presentado alteración renal, cumpliendo criterios de 'nefropatía crioglobulinémica' 17 pacientes (27 por ciento): creatinina 2,8 ñ 1,8 mg/dl, proteinuria 4,2 ñ 3,9 g/24 h. En 9 pacientes se ha obtenido material histológico, confirmándose un patrón de glomerulonefritis membrano-proliferativa en 6 casos, un caso con vasculitis extraglomerular asociada, dos casos con patrón proliferativo mesangial y una glomerulonefritis membranosa.Las diferencias más destacables entre los pacientes con nefropatía crioglobulinémica y el resto de pacientes ha sido un valor de crioglobulinas más elevado (49 ñ 28 vs 20 ñ 22), mayor frecuencia de hipocomplementemia, especialmente de C4 (93 por ciento vs 59 por ciento) y púrpura recurrente (75 por ciento vs 32 por ciento). No ha habido diferencias en la infección por el virus C (75 por ciento vs 71 por ciento).En conclusión: Se evidencia infección por el virus C en el 72 por ciento de los casos (por lo que el 28 por ciento siguen siendo 'esenciales'); el 52 por ciento presentan síntomas relacionados con la crioglobulinemia, la mitad de los cuales tienen nefropatía crioglobulinémica (AU)

[Type II essential mixed cryoglobulinemia and renal disease. Hepatitis C virus association]. / Afectación renal en la crioglobulinemia mixta tipo II.

UNLABELLED: Recently a number of studies have implicated C virus as a major cause of mixed cryoglobulinemia. Several authors described that up to 95% of "essential" mixed cryoglobulinemia could be attributed to this viral infection. Nevertheless, its prevalence and clinical significance are not well known. We review our experience in relation with the clinical, biological and evolutive characteristics of patients diagnosed of type II mixed cryoglobulinemia. METHODS: Descriptive and protocolized study of all cases found to have type II mixed cryoglobulinemia over a period of 8 years. Secondary cryoglobulinemic nephropathy was defined in a restrictive way: a plasma creatinine > 1.5 mg/dl and/or proteinuria > 500 mg/24 h and/or hematuria (> 15 red blood cells) need to be present in the absence of any other pathological conditions that could justify these alterations. Furthermore, the information obtained from available kidney biopsies was considered. RESULTS: 62 patients have been detected. C virus infection was demonstrated in 44 (72%). 52% had clinical symptoms related with cryoglobulinemia. 56% had alteration of renal tests, and 17 (27%) fulfil the conditions for the diagnosis of cryoglobulinemic nephropathy (nearly all with persistent microhematuria, median proteinuria 4.2 +/- 3.9 g/24 h; median plasma creatinine 2.8 +/- 1.8 mg/dl). Nine patients had been histologically examined, showing 6 cases a membranoproliferative glomerulonephritis pattern, one with associated extraglomerular vasculitis; two with mesangial proliferative pattern and one with membranous glomerulonephritis. The most striking differences between cryoglobulinemic nephropathy patients and the rest has been: higher amount of cryoglobulins (49 +/- 28 vs 20 +/- 22); more frequent hypocomplementemia, especially C4 (93% vs 59%) and recurrent purpura (75% vs 32%). No differences in the presence of C virus infection could be observed (75% vs 71%). IN CONCLUSION: 72% of patients with type II mixed cryoglobulinemia are infected by C virus (so 28% in our serie are "essential"); 52% have symptoms related with the presence of cryoglobulins, half of them with cryoglobulinemic nephropathy.

Plasma levels of tumor necrosis factor alpha in patients with Mediterranean spotted fever: clinical and analytical correlations.

Twenty consecutive patients with serologically confirmed Mediterranean spotted fever were analyzed for determination of plasma levels of tumor necrosis factor alpha (TNF-alpha) by means of an ELISA. Increased levels of TNF-alpha in plasma were found during the acute phase of the disease (52.3 +/- 49.8 pg/mL) compared to levels in the convalescent phase (9.0 +/- 9.3 pg/mL; P < .001) or in healthy controls (6.5 +/- 3.2 pg/mL; P < .001). Plasma TNF-alpha levels were significantly higher in patients with severe Mediterranean spotted fever. Levels of TNF-alpha correlated significantly with serum levels of C-reactive protein and triglycerides and inversely with serum levels of sodium.

Autoantibodies against a novel DNA-binding protein: DNA-protein interaction as a requisite for expression of antigenic reactivity.

A novel DNA-binding protein complex, the HB complex, has been characterized by means of autoantibodies. Three proteins of 9000, 7500, and 7000 Da constitute the HB complex. The 9000- and 7500-Da proteins are phosphorylated. Autoantibodies recognize the 7000-Da protein when it is bound to DNA. No reactivity against any protein was observed when the complex was dissociated from DNA. The three proteins are acidic (pI 5-6.2), and the complex was able to bind to synthetic double-stranded DNAs of different composition.

Serological profile of tissue autoantibodies during acute and chronic delta hepatitis.

In order to assess the serological profile in relation to other serological and histological markers of hepatitis delta virus (HDV) infection we have investigated the presence of autoantibodies during acute and chronic delta infection in 353 serum samples from different patients with acute and chronic hepatitis and autoimmune diseases. Basal cell layer antibodies (BCLA) were found in 58% acute hepatitis B, in 73% chronic hepatitis D and in 4% primary biliary cirrhosis. Stellate thymic epithelial cell antibodies (SECA) were detected in 40% acute D hepatitis and in 49% chronic D hepatitis. No tissue autoantibodies were detected in 50 acute B hepatitis, 35 autoimmune chronic liver diseases, 24 connective tissue diseases or 25 controls. In addition, two previously unreported specificities of anti-thymic antibodies reacting with reticular (TRA) and perithymocytic cells (PTA) were identified in 33% and 9% acute D hepatitis, respectively, and in 13% and 6% chronic D hepatitis cases. Among patients with acute HBV-HDV coinfection these antibodies were detected transiently (less than 4 weeks) and at low titer, whereas in those with chronic infection autoantibodies levels tend to be high and persistent throughout the follow-up. Among patients with chronic HDV infection no correlation was found between the presence of tissue autoantibodies and hepatic delta antigen expression and serum HDV-RNA which suggest that autoimmune phenomena observed during chronic delta infection are not related to the level of viral replication.

Autoantibodies in chronic delta virus infection recognize a common protein of 46 kD in rat forestomach basal cell layer and stellate thymic epithelial cells.

Chronic hepatitis delta virus infection is associated with the presence of autoantibodies to rat forestomach and thymus in approximately 60% of patients' sera. We have characterized the antigen against which these autoantibodies are directed as a protein of 46 kD by immunoblotting studies on rat forestomach and thymus extracts. Normal human sera or sera from patients with other hepatic or non-hepatic autoimmune disorders did not bind to this protein. The immunoblot assay was more sensitive than immunofluorescence. Maximal titre was 1:10,000 versus 1:5120. By techniques of elution of specific antibodies from immunoblots, our results showed that the same antigen was present in both tissues. This antigen did not share common epitopes with hepatitis delta virus (HDV). Patients' sera depleted of basal cell layer and thymic stellate epithelial cell antibodies by absorption with the corresponding tissue extract maintained the HDV antibody titres. The autoimmune phenomena observed in patients with HDV infection seems to be a colateral process induced by the replication of delta virus in the host.

Characterization of the CB antigen, a DNA-binding protein recognized by autoantibodies and which has a differential expression in lymphoid cells.

We have analyzed the characteristics of the CB Ag, a nuclear protein recognized by autoantibodies. Approximately 4% (12 out of 280) of the antinuclear-positive sera examined contained anti-CB antibodies. By immunofluorescence, these sera brightly stained the nuclei of most cells analyzed, including peripheral lymphocytes, but only dull or no staining was observed in thymocytes or B cells of the bursa of Fabricius. The CB Ag has been characterized as a DNA-binding protein, dissociable from DNA at 1.5 M NaCl, and with a Mr of 40,000 Da. Moreover, the ability of the extracted Ag to bind back to DNA has enabled us to design an ELISA system for its detection.