Clinical presentation and pathogenesis of cold-induced autoinflammatory disease in a family with recurrence of an NLRP12 mutation.

OBJECTIVE: NLRP12 mutations have been described in patients affected with peculiar autoinflammatory symptoms. This study was undertaken to characterize NLRP12 mutations in patients with autoinflammatory syndromes, particularly a novel missense mutation, p.D294E, affecting a protein sequence crucial for ATP binding, which was identified in a Caucasian family with familial cold-induced autoinflammatory syndrome in some family members. METHODS: Fifty patients were tested for NLRP12 mutations. A Caucasian family with the p.D294E missense mutation of NLRP12 in some family members was clinically characterized. In vitro analysis of the effects of the mutation on NF-κB activity was performed in HEK 293 cells after cotransfection of the cells with a luciferase NF-κB-responsive element and mutant or wild-type (WT) NLRP12 expression plasmids. NF-κB activity was also evaluated 24 hours after stimulation with tumor necrosis factor α in monocytes from individual family members carrying the mutation. Furthermore, secretion of interleukin-1ß (IL-1ß), production of reactive oxygen species (ROS), and activation of antioxidant systems in patient and healthy donor monocytes, under resting conditions and after stimulation with pathogen-associated molecular patterns (PAMPs), were also assessed. RESULTS: In the family assessed, the p.D294E mutation segregated in association with a particular sensitivity to cold exposure (especially arthralgias and myalgia), but not always with an inflammatory phenotype (e.g., urticarial rash or fever). In vitro, the mutant protein maintained the same inhibitory activity as that shown by WT NLRP12. Consistently, NLRP12-mutated monocytes showed neither increased levels of p65-induced NF-κB activity nor higher secretion of IL-1ß. However, the kinetics of PAMP-induced IL-1ß secretion were significantly accelerated, and high production of ROS and up-regulation of antioxidant systems were demonstrated. CONCLUSION: Even with a variable range of associated manifestations, the extreme sensitivity to cold represents the main clinical hallmark in an individual carrying the p.D294E mutation of the NLRP12 gene. Although regulation of NF-κB activity is not affected in patients, redox alterations and accelerated secretion of IL-1ß are associated with this mild autoinflammatory phenotype.

Central nervous system tuberculosis in non-HIV-positive children: a single-center, 6 year experience.

PURPOSE: The aim of this paper is to describe the imaging features of central nervous system (CNS) tuberculosis on computed tomography (CT) and magnetic resonance imaging (MRI) studies in non-HIV-positive children. MATERIALS AND METHODS: A retrospective descriptive evaluation was conducted on imaging studies obtained from ten children admitted to our hospital over a 6-year period who fulfilled criteria for a diagnosis of CNS tuberculosis. Data were collected with regard to patients' clinical, laboratory and demographic characteristics, as well as results of radiological investigation. RESULTS: We studied ten children, of whom five were boys and five were girls and whose mean age was 4 (range 7 months to 16) years. Neuroradiological findings on the first imaging study were basal meningeal enhancement (100%), hydrocephalus (70%), infarcts (90%), tuberculomas (40%) and cranial nerve involvement (20%). Follow-up studies revealed basal meningeal enhancement, hydrocephalus, and infarcts in all patients, tuberculomas in 70% and cranial nerve involvement in 50%. Only one patient showed a pattern of miliary tuberculosis. CONCLUSIONS: CNS tuberculosis is still an important cause of childhood morbidity and mortality even in nonimmunosuppressed children. Because prompt diagnosis results in earlier treatment, it is crucial to be aware of tuberculous meningitis and its complications at imaging, especially because of the impact on patients' prognosis.

Nearly automated analysis of coronary Doppler flow velocity from transthoracic ultrasound images: validation with manual tracings.

Coronary flow velocity reserve is obtained by manual tracings of transthoracic coronary Doppler flow velocity profiles as the ratio of stress versus baseline diastolic peak velocities. This approach introduces subjectivity in the measurements and limits the information which could be exploited from the Doppler velocity profile. Accordingly, our goals were to develop a technique for nearly automated detection of Doppler coronary flow velocity profile, and automatically compute both conventional and additional amplitude, derivative and temporal parameters, and validate it with manual tracings. A total of 100 patients (17 normals, 15 patients with severe coronary stenosis, 41 with connective tissue disease and 27 with diabetes mellitus) were studied. Linear correlation and Bland-Altman analyses showed that the proposed method was highly accurate and repeatable compared to the manual measurements. Comparison between groups evidenced significant differences in some of the automated parameters, thus representing potentially additional indices useful for the noninvasive diagnosis of microcirculatory or coronary artery disease.

Low prevalence of selective human leukocyte antigen (HLA)-A and HLA-B epitope losses in early-passage tumor cell lines.

The down-regulation of human leukocyte antigen (HLA) class I molecules, especially the selective down-regulation of certain allelic products, is believed to represent a major mechanism of tumor escape from immune surveillance. In the present report, an original approach is described to precisely evaluate and classify HLA class I epitope losses in 30 cancer patients with malignant melanoma and lung, breast, endometrium, ovary, and colon carcinoma tumors. Early-passage tumor cell lines were established in culture from the corresponding metastatic tumor lesions obtained in each patient. Both the cell lines and the tumor lesions were compared, in their HLA-A and -B expression, to the peripheral blood mononuclear cells (PBMCs) obtained from the same patient (autologous PBMCs). On the basis of HLA-genotyping data, the appropriate monoclonal antibodies identifying mono- and poly-morphic HLA-A and HLA-B epitopes were selected from a panel of 34 antibodies for a total of 24 testable alleles. The selected antibodies were used not only in immunohistochemical assays on cryostatic tumor sections and cytospins of PBMCs but also in quantitative, sensitive flow cytometry assays on early-passage tumor cells and PBMC suspensions. With this latter method, a low overall HLA expression was detected in 26 tumor cell explants and a complete, generalized HLA-A, HLA-B, HLA-C loss in the remaining 4 cases. However, no complete, selective loss of any of the 45 tested HLA-A and HLA-B allomorphs was observed. Sequences from all of the HLA class I alleles could be detected at the genomic DNA level in tumor cells and tissues. At variance from the literature and the results of immunohistochemical experiments performed in parallel on the corresponding tumor lesions, the relative proportions of the various HLA epitopes were relatively preserved in each early-passage cell line/PBMC pair, and selective increases, rather than decreases, in the expression of polymorphic HLA epitopes had the highest prevalence and greatest magnitude. Our data suggest an alternative tumor stealth strategy in which up- and down-regulation are equally important. This alternative model of tumor-host interaction better fits the available models of tumor cell recognition by CTLs and natural killer cells bearing activatory and inhibitory receptors for HLA-A, HLA-B, HLA-C molecules.

Stromal damage as consequence of high-dose chemo/radiotherapy in bone marrow transplant recipients.

Bone marrow transplant (BMT) relies on the engraftment of donor hemopoietic precursors in the host marrow space. Colony forming units-fibroblasts (CFU-f), the precursor compartment for the osteogenic lineage, are essential to hemopoietic stem cell survival, proliferation and differentiation. We have studied CFU-f in donors (aged 5 months to 62 years) and in patients who had received allogeneic BMT (aged 2 months to 63 years). In donor marrows we found an inverse correlation between CFU-f frequency and age. In BMT recipients CFU-f frequencies were reduced by 60%-90% (p < 0.05) and the numbers did not recover up to 12 years after transplant. Stromal reconstitution to normal levels was found only in patients < 5 years old. In all patients studied CFU-f post-BMT were of host origin. Patients with low CFU-f levels displayed also a decreased bone mineral density (p < 0.05) and significantly reduced levels of long-term culture-initiating cells (LTC-IC) (p < 0.05). Our study demonstrates that the marrow stromal microenvironment is seriously and irreversibly damaged after BMT. Donor cells do not contribute to reconstitute the marrow microenvironment, whose residual CFU-fs remain of host origin.

HLA polymorphism in a Mataco South American Indian tribe: serology of class I and II antigens. Molecular analysis of class II polymorphic variants.

In the present study, HLA-A, B, C, DR, DQ, and DP loci were analyzed in a group of Mataco Amerindians of Argentina. Using reagents from the 11th International Histocompatibility Workshop (11th IHW), class I specifities such as Bw70, Bw75, and Bw48 were found in this population, other than the HLA determinants commonly described in South American Indians. The class II antigens found were DR4, DRw14, and DRw8 at the DR locus, and DQw4 and DQw7 at the DQ locus. The analysis of DRB1-DR4 related alleles, performed by PCR amplification and oligonucleotide probe hybridization, showed the presence of DRB1*0403, *0404, *0405, and *0411 in individuals from this ethnic group. By the analysis of DRB1-DRw14 related alleles, two variants were found: DRB1*1402 and DRB1*1406, the latter provisionally called DRB1 14.6 in 11th IHW. The DRw8-related allele present was DRB1*0802. The analysis of DRB3 gene revealed only the presence of DRB3*0101 allele in DRw14 individuals. DPB1 locus was also analyzed in unrelated individuals of the same population. Only five DPB1 alleles were found: DPB1*0201, *0301, *0402, *0501, and *1301 over the 19 previously described in the literature. These findings emphasize the restricted HLA class I and II variation observed in this ethnic group as it has been previously shown in other American groups. Some particular haplotypes in this Mataco tribe are described in this work.

HLA-DP typing by DNA amplification and hybridization with specific oligonucleotides.

HLA-DP genotyping was performed using dot-blot analysis with synthetic oligonucleotide probes. Fourteen probes were designed based on the known sequence variations in the polymorphic segments of the DP beta second exon. Each probe was tested against genomic DNA amplified by the polymerase chain reaction, using DP beta-specific primers. A total of 45 HLA homozygous B-cell lines, selected from the Tenth International Histocompatability Workshop and pretyped for the known DP omega specificities, were analyzed. Different hybridization patterns were found for each DP omega specificity. The oligonucleotide hybridization performed on DP omega-negative B-cell lines gave a pattern distinct from those of known DP omega specificities, indicating the presence of novel DP allelic sequences. The use of sequence-specific oligonucleotides combined with DNA amplification allows a simple and reliable genotyping of DP antigens.

Analysis of HLA DP, DQ, and DR alleles in adult Italian rheumatoid arthritis patients.

We analyzed the distribution of DRB1, DQA1, DQB1, and DPB1 allelic variants in 48 rheumatoid arthritis (RA) patients, compared with 109 Italian random controls, using PCR amplification and hybridization with specific oligonucleotides. We confirm the previously reported increase of DR4 specificity, in comparison with healthy Italian individuals. In particular, we find a statistically significant positive association of DRB1*0401 and DRB1*0404 alleles with RA. However, when we compare the DR4+ groups, none of the DRB1*04 alleles is increased in the RA group. By sequence analysis, performed on 10 patients, we demonstrate that the DRB1*04 genes of RA show no difference from the DRB1*04 sequences previously published. From the molecular analysis of the other DRB1 polymorphic variants, we find a trend of positive association of DRB1*0101 in DR4-negative patients versus DR4-negative healthy controls and, in the group of DR4-negative and/or DR1-negative patients, a similar increase of DRB1*06. Also, we observe in RA patients a statistically significant increase of DQA1*0301 and DQB1*0302 accompanied by a significant decrease of DQA1*0201, DQA1*0501 and DQB1*0201. Finally, from the analysis of DPB1 gene, it can be assessed that the distribution of DPB1 alleles does not differ significantly between RA patients and healthy controls.

Bone marrow transplantation for chronic myeloid leukemia (CML) from unrelated and sibling donors: single center experience.

This is a report on 60 consecutive patients with chronic myeloid leukemia (CML) who received an allogeneic bone marrow transplant (BMT) in this Unit. Donors were HLA-identical siblings (SIB) (n = 36) or unrelated donors (MUD) (n = 24) matched by serology for HLA A and B and by molecular biology for HLA DR. All patients were prepared with cyclophosphamide 120 mg/kg and fractionated total body irradiation 10-12 Gy. GVHD prophylaxis consisted of cyclosporin A (CsA) starting on day -7 and short-course methotrexate. Bone marrow was unmanipulated in all cases. Cytomegalovirus prophylaxis consisted of acyclovir for SIBs and foscarnet for MUDs. When compared to SIB transplants, MUD patients were younger (29 vs 36 years; P = 0.002), had younger donors (31 vs 39; P = 0.001), had a longer interval between diagnosis and BMT (1459 vs 263 days; P < 0.001) and received a smaller number of nucleated cells at transplant (3.3 vs 4.4 x 10(8)/kg; P = 0.003). More MUDs had advanced disease (50 vs 17%, P = 0.005). The median day to 0.5 x 10(9)/l neutrophils was similar in both groups (18 days for SIBs vs 17 days for MUDs; P = 0.06); the median platelet count on days +30, +50, +100 was significantly (P < 0.01) higher in SIB than in MUD patients (122 vs 38, 113 vs 50 and 97 vs 45 x 10(9)/l, respectively). Acute GVHD was scored as absent-mild, moderate, or severe, in 36, 58 and 6% of SIBs vs 25, 42 and 33% in MUD patients (P = 0.01). Chronic GVHD was comparable (P = 0.1). The actuarial risk of CMV antigenemia at 1 year was 60% in both groups. There were six deaths in SIB patients (two leukemia, two infections, one GVHD, one pneumonitis) and four deaths in MUD patients (three acute GVHD and one infection). Fifty patients survive with a median follow-up of 656 days for SIBs and 485 for MUDs. The actuarial 3-year transplant-related mortality is 12% in SIBs and 17% in MUDs (P = 0.5); the actuarial relapse is 18% in SIBs vs 6% in MUDs (P = 0.4) and 3-year survival 78% in SIBs vs 82% in MUDs (P = 0.7). This study suggests that survival of CML patients after marrow transplantation from unrelated or sibling donors is currently similar, provided the former are well matched. The increased incidence of GVHD in MUD patients is possibly compensated by a lower risk of relapse.

HLA-DQA1 allele and genotype frequencies in a northern Italian population.

HLA-DQA1 typing of 227 randomly selected Northern Italian people by the use of polymerase chain reaction are reported. The combined use of commercial Amplitype HLA-DQalpha system and four sequence-specific oligonucleotide probes allows the definition of 8 alleles and 36 genotypes, arranged according to World Health Organisation nomenclature. Seven of these genotypes are not observed among the analyzed samples. Allele frequencies range from 1.5 to 35.7% and genotype observations do not deviate significantly from Hardy-Weinberg equilibrium; observed heterozygosity is 0.8238 with an allelic diversity value of 0.79 and the power of discrimination is 0.925. Our Italian population sample shows differences from other Caucasian samples both for allele and genotype frequencies. This locus typing for the 8 defined alleles provides a rapid and sensitive method in individual identification and paternity investigation.

Noninvasive prediction of sudden death and sustained ventricular tachycardia after acute myocardial infarction using a neural network algorithm.

BACKGROUND: The early and accurate noninvasive identification of postinfarction patients at risk of sudden death and sustained ventricular tachycardia (arrhythmic events) still remains an unsolved problem. The aim of the present study was to identify the combination of clinical and laboratory noninvasive variables, easy to obtain in most patients, that best predicts the occurrence of arrhythmic events after an acute myocardial infarction. METHODS: Four hundred and four consecutive patients with acute myocardial infarction were enrolled and followed for a median period of 21.4 months. In each patient, 61 clinical and laboratory noninvasive variables were collected before hospital discharge and used for the prediction of arrhythmic events using an artificial neural network. RESULTS: During follow-up, 13 (3.2%) patients died suddenly and 11(2.5%) had sustained ventricular tachycardia. The neural network showed that the combination best predicting arrhythmic events included: left ventricular failure during coronary care stay, ventricular dyskinesis, late potentials, number of ventricular premature depolarizations/hour, nonsustained ventricular tachycardia, left ventricular ejection fraction, bundle branch block and digoxin therapy at discharge. The neural network algorithm allowed identification of a small high-risk patient subgroup (12% of the study population) with an arrhythmic event rate of 46%. The sensitivity and specificity of the test were 96 and 93% respectively. CONCLUSIONS: These results suggest that, in postinfarction patients, it is possible to predict early and accurately arrhythmic events by noninvasive variables easily obtainable in most patients. Patients identified as being at risk are candidates for prophylactic antiarrhythmic therapy.

[Allelic variations of DPB1, DQA1, DQB1 and DRB1 and rheumatoid arthritis: further genetic and statistical considerations]. / Varianti alleliche dei DPB1, DQA1, DQB1, DRB1 e artrite reumatoide: ulteriori considerazioni genetiche e statistiche.

We used polymerase chain reaction amplification and hybridization with specific oligonucleotides to analyze the distribution of DPB1, DQA1, DQB1, and DRB1 allelic variants in 48 patients with rheumatoid arthritis (RA) and compared our results with those from 109 randomly chosen, healthy control subjects. Our work confirms a previously reported increase in DR4 specificity in RA: in particular, we found a statistically significant positive association of the DRB1*0401 and DRB1*0404 alleles with RA. When we compared the DR4 groups, however, none of the DRB1*04 alleles were increased in the RA group. Molecular analysis of the other DRB1 polymorphic variants disclosed the trend of a positive association of DRB1*0101 (DR1) in DR4 negative patients vs DR4 negative healthy control subjects, and an increase in DRw6 (DRB1*13,*14) in the DR4 and/or DR1 negative patient group. Moreover, analysis of the association between RA and a heptapeptide motif (positions 67-74) in the third hypervariable region confirmed that this epitope confers enhanced risk for the development of RA with respect to the allele DRB1*0404 (etiologic fraction = 0.53 vs 0.12). We also observed a statistically significant increase in DQA1*0301 and DQB1*0302 accompanied by a significant decrease in DQA1*0202, DQA1*0501 and DQB1*0201 in RA patients. Analysis of DPB1 alleles disclosed no significant differences between RA patients and healthy control subjects.

Magnetic resonance spectroscopy of ischemic heart disease.

An overview of the basic knowledge necessary to understand the procedure of Magnetic Resonance Spectroscopy of the myocardium and its most significant applications in the study of ischemic heart disease, is presented, with reference to the personal experience. The chemical shift phenomenon, the main techniques of spectroscopic localization and the general aspects of myocardial 31P and 1H Magnetic Resonance Spectroscopy, including proton decoupling and magnetization transfer, are illustrated. Postprocessing techniques before and after Fourier transform are mentioned. 31P Magnetic Resonance Spectroscopy allows the noninvasive assessment of the metabolism of high energy phosphates, PCr/ATP ratio in particular, in the in vivo myocardial tissue with significant applications in the diagnostic approach to ischemic patients with the support of provocative tests (dobutamine). 1H Magnetic Resonance Spectroscopy allows similar evaluations based on the peak of total creatinine.

Oftalmopatía de Graves y tratamiento con I131 / Graves´ Ophthalmopathy and Radioiodine Treatment

La oftalmopatía de Graves (OG) es un desorden autoinmune que representa la manifestación extratiroidea más frecuente de la Enfermedad de Graves. Se describen múltiples factores que pueden influir en el desarrollo/progresión de la enfermedad, entre ellos el tratamiento con I131. El objetivo de esta monografía es analizar la bibliografía existente sobre la relación entre OG y tratamiento con I131, teniendo en cuenta su fisiopatología así como los factores de riesgo asociados y su profilaxis.

Graves´ ophthalmopathy (GO) is an autoimmune disorder representing the most common extrathyroidal manifestation of Graves' disease. Multiple factors can influence the development / progression of the disease, including treatment with 131I. The aim of this paper is to analyze the existing literature on the relationship between OG and treatment with I131, considering the pathophysiology and associated risk factors and prophylaxis.

Dorsal brain stem syndrome: MR imaging location of brain stem tegmental lesions in neonates with oral motor dysfunction.

The anatomic extent of brain stem damage may provide information about clinical outcome and prognosis in children with hypoxic-ischemic encephalopathy and oral motor dysfunction. The aim of this study was to retrospectively characterize the location and extent of brain stem lesions in children with oral motor dysfunction. From January 2005 to August 2009, 43 infants hospitalized at our institution were included in the study because of a history of hypoxic-ischemic events. Of this group, 14 patients showed oral motor dysfunction and brain stem tegmental lesions detected at MR imaging. MR imaging showed hypoxic-ischemic lesions in supra- and infratentorial areas. Six of 14 patients revealed only infratentorial lesions. Focal symmetric lesions of the tegmental brain stem were always present. The lesions appeared hyperintense on T2-weighted images and hypointense on IR images. We found a strong association (P < .0001) between oral motor dysfunction and infratentorial lesions on MR imaging. Oral motor dysfunction was associated with brain stem tegmental lesions in posthypoxic-ischemic infants. The MR imaging examination should be directed to the brain stem, especially when a condition of prolonged gavage feeding is necessary in infants.

Parapharyngeal neuroglial heterotopia in Pierre Robin sequence: MR imaging findings.

Heterotopic neuroglial tissue is a rare lesion, occurring more frequently in the nasal cavities. Other rare locations are the orbit, the scalp, the palate, the pharynx, the parapharyngeal space and the lungs. They are usually detected occasionally because they are often asymptomatic, but sometimes they might present with dyspnoea, feeding difficulty, snorting and nasal flaring. Respiratory symptoms occur when heterotopic neuroglial tissue is located in the parapharyngeal space. We report a case of an infant affected by Pierre Robin sequence (PRS) who was admitted to our Institution for a worsening respiratory distress that was not explainable only by PRS.

Semi-automated analysis of coronary flow Doppler images: validation with manual tracings.

Coronary flow velocity reserve (CFVR) is conventionally obtained by manual tracings of Doppler profiles, as ratio of stress vs baseline diastolic peak velocity. When <1.9, this parameter evidences reduced coronary flow and possible microcirculatory disease. Our goals were: 1) to develop a novel technique for semi-automated detection of Doppler flow velocity profile, allowing the automated computation of CFVR and other parameters; 2) to validate this technique in comparison with conventional measurements obtained by manual tracing; 3) to test for differences between normal (N) subjects and patients with rheumatoid arthritis (RA). Linear correlation and Bland-Altman analyses showed that the proposed method was highly accurate and repeatable compared to the manual measurements. Comparison between N and RA groups evidenced significant differences in some of the automated parameters.

Continuous renal replacement therapy and charcoal plasmaperfusion in treatment of amanita mushroom poisoning.

Hemoperfusion has been used in the treatment of mushroom poisoning for many years. The aim of this study was to study the efficacy of charcoal plasmaperfusion (CPP) and continuous renal replacement therapy (CRRT) in 2 patients severely poisoned by the amanita mushroom. Both patients arrived at the ICU from another hospital with a diagnosis of amanita phalloides mushroom poisoning. The patients were precociously treated with CRRT for 20 h and CPP for 3 h every day. The treatments were effected for 3 and 5 days, respectively. Both patients recovered completely and were discharged asyntomatic after 7 and 10 days.