Endothelial cell dysfunction in homocystinuria

Abstract. This report describes the isolation and culture of venous endothelial cells from the umbilical cord of an obligate heterozygote for homocystinuria. The effect of different sulphur-containing amino acids on the viability and function of these cells was studied and compared with cultured normal endothelial cells. When endothelial cells were cultured in the presence of methionine (10 mmol/l) or homocystine (10 mmol/l), differences occurred between the viability and function of the heterozygote and normal cells in terms of ⁵¹Cr release and ability to prevent platelet adherence. The Cr release corrected for spontaneous release increases for the heterozygote cells after incubation for 21 h in the presence of methionine to 81.3% (control cells, range: 0–23.3%, n = 5) and in the presence of homocystine to 141% (control cells, range: 13.5–55.2%, n = 5). The total number of platelets that adhere to confluent monolayers increases for heterozygote cells cultured in the presence of methionine to 0.98 ± 10⁷ platelets cm^-2 (normal cells, range: 0.56–0.72 ± 10⁷ platelets cm^-2) and in the presence of homocystine to 1.41 ± 10⁷ platelets cm^-2 (normal cells, range: 0.94–1±06 ± 10⁷ platelets cm^-2). Both normal and control cells were sensitive to homocysteine. This study indicates for the first time what vascular endothelial cells, derived from an obligate heterozygote, are (partly) deficient in cysthathionine synthase and are more susceptible to methionine- and homocystine-mediated injury than normal endothelial cells. Consequently, in homocystinuria, due to dysfunction of the endothelial cells, toxic sulphur-containing amino acids may accumulate in these cells, causing injury of these cells.     

Mild elevations of plasma ornithine in homocystinuria

Hyperornithinemia was found on many occasions during monitoring of amino acids in the fasting plasma of homocystinuric patients. Ornithine concentrations were elevated 2- to 3-fold above the normal mean value. Hyperornithinemia was of much less marked degree than that seen in gyrate atrophy of the choroid and retina, and was not accompanied by overflow ornithinuria or hypolysinemia. Elevations in plasma ornithine concentrations in homocystinuric patients were generally proportional to elevations in the plasma concentration of homocystine, but not that of methionine.     

Increased plasma copper in patients with homocystinuria due to cystathionine β-synthase deficiency

We measured blood copper-containing proteins and plasma total copper in 15 patients with homocystinuria (14 with cystathionine β-synthase deficiency and one with abnormal cobalamin metabolism), in 13 heterozygotes for cystathionine β-synthase deficiency, and in 44 normal subjects. Plasma total copper was increased in patients with cystathionine β-synthase deficiency compared with age- and sexmatched controls; the ratio was 1.41 ± 0.14 for females and 1.39 ± 0.15 for males (means ± SD). This was due to corresponding increases in caeruloplasmin concentrations, but levels were unrelated to total plasma homocysteine. Erythrocyte superoxide dismutase levels were normal. The heterozygotes had normal plasma copper and caeruloplasmin levels.The increased copper and caeruloplasmin may contribute to the precocious atherogenesis occurring in homocystinuria by decreasing the adhesion of vascular endothelial cells to the intima. It is unlikely that decreased lysyl oxidase activity due to chelation of copper by homocysteine is important for the pathogenesis of the connective tissue defect in homocystinuria.     

Increase in urinary purines and pyrimidines in patients with methylmalonic aciduria combined with homocystinuria

BackgroundMethylmalonic aciduria combined with homocystinuria (MMA–HC) is the biochemical trait of a metabolic disorder resulting from impaired conversion of dietary cobalamin (cbl, or vitamin B₁₂) to its two metabolically active forms. Effects on urinary purine and pyrimidine levels have not been described for this condition.MethodsUrine samples were collected from three patients with methylmalonic aciduria combined with homocystinuria and from 70 healthy subjects. Urinary purine and pyrimidine levels were quantitated by the use of LC/UV–Vis and LC/ESI/MS.ResultsHigher urine levels of pyrimidines were detected with both methods in patients compared to controls.ConclusionMethylmalonic aciduria with homocystinuria is due to deficiency of the enzyme, cobalamin reductase. The enzyme defect leads to altered hepatic metabolism, which appears to modify circulating pyrimidine levels.     

Health-related quality of life in Brazilian pacemaker patients

Background: Most quality of life (QoL) studies of pacemaker patients have been conducted in either North America or Europe and their applicability to Latin American populations is largely unknown. Our aim is to study health-related QoL indices in Brazilian pacemaker patients and their determinants using both a generic (SF-36) and a disease-specific questionnaire (AQUAREL).
Methods: The study enrolled 139 clinically stable patients (aged 59 ± 14, 60.4% female) without any communication or cognitive impairments who went to the Pacemaker Laboratory for postimplantation follow-up. All patients were submitted to a standard protocol, which included an interview, functional class assessment, and QoL questionnaires. Additionally, 74 patients were requested to perform a 6-minute walk test.
Results: Female patients and patients without a partner displayed low QoL scores in both the SF-36 mental component summary and the AQUAREL arrhythmia domain. Chagas disease patients displayed low scores only in AQUAREL domains. All health-related QoL scores were low in patients with the worst, high-numbered functional classes, the strongest determinant of low QoL scores in multivariate analysis.
Conclusion: In this first systematic study of QoL in a Latin American pacemaker population, AQUAREL detected well-impaired health-related QoL scores in different groups of patients, particularly in those with Chagas disease. Heart failure, evaluated by functional class, was the strongest predictor of low QoL in pacemaker patients.     

Molecular identification of nontuberculous mycobacteria isolates in a Brazilian mycobacteria reference laboratory

This study utilized the hsp65 polymerase chain reaction restriction analysis (PRA) method in the identification of nontuberculous mycobacteria (NTMs) isolated in a Brazilian mycobacteria laboratory. NTM isolates from clinical specimens collected from 192 patients were characterized using the hsp65 PRA method and analyzed using both 16S rRNA and hsp65 gene sequencing. Only 30% of the NTM strains were correctly identified through PRA, though the suggested inclusion of an additional restriction enzyme could increase the resolution to roughly 90%. A total of 17 NTM strains were not identified to species level and may represent a new taxonomic entity classified as belonging to the Mycobacterium simiae complex. This study demonstrates the applicability of hsp65 PRA in the identification of several NTM strains in a reference laboratory, though the results suggest that some modifications to the original PRA method could increase its resolution substantially.     

Molecular analysis of FVIII gene in severe HA patients of Costa Rica

Haemophilia A (HA) is X-chromosome linked bleeding disorders caused by deficiency of the coagulation factor VIII (FVIII). It is caused by FVIII gene intron 22 inversion (Inv22) in approximately 45% and by intron 1 inversion (Inv1) in 5% of the patients. Both inversions occur as a result of intrachromosomal recombination between homologous regions, in intron 1 or 22 and their extragenic copy located telomeric to the FVIII gene. The aim of this study was to analyze the presence of these mutations in 25 HA Costa Rican families. Patients, methods: We studied 34 HA patients and 110 unrelated obligate members and possible carriers for the presence of Inv22or Inv1. Standard analyses of the factor VIII gene were used incl. Southern blot and long-range polymerase chain reaction for inversion analysis. Results: We found altered Inv22 restriction profiles in 21 patients and 37 carriers. It was found type 1 and type 2 of the inversion of Inv22. During the screening for Inv1 among the HA patient, who were Inv22 negative, we did not found this mutation. Discussion: Our data highlight the importance of the analysis of Inv22 for their association with development of inhibitors in the HA patients and we are continuous searching of Inv1 mutation. This knowledge represents a step for genetic counseling and prevention of the inhibitor development.     

Molecular analysis of resistance to interferon in patients with laryngeal papillomatosis.

Although interferon (IFN)-alpha has been used successfully as an adjuvant therapy in laryngeal papillomatosis, some patients are resistant to this treatment. In order to know which patients will benefit from the therapy, we have tried to find a relationship between the IFN response and the viral and host parameters in the lesion. Detection of viral type and copy numbers by polymerase chain reaction (PCR) showed that all patients infected with human papillomavirus (HPV)-11 were sensitive to the treatment, in contrast to those infected with HPV-6. These differences could be explained in part by the inability of HPV-11 E7 to inhibit the induction of an IFN-responsive element, whereas HPV-6 E7 almost completely inhibited the activity of this promoter in transient transfection experiments. Local immune status in the lesion showed that all HPV-11-infected patients had detectable levels of interleukin (IL)-15 and IFN-gamma mRNA, in contrast to HPV-6-infected patients, in whom mRNA for these cytokines was almost absent. Viral copy numbers and levels of IL-4 mRNA could not be correlated with IFN response. Only one patient resistant to recombinant IFN-alpha2b and negative for HPV DNA presented high titers of neutralizing anti-IFN-alpha2b antibodies. This patient became sensitive when natural IFN-alpha was administered. These results suggest that response to IFN may be a complex phenomenon resulting from the interaction between viral and host elements.     

Molecular analysis of ABCD1 gene in Indian patients with X-linked adrenoleukodystrophy

BackgroundX-linked Adrenoleukodystrophy (X-ALD), with an incidence of 1:14,000 is the most frequent monogenic demyelinating disorder worldwide. The principal biochemical abnormality in X-ALD is the increased levels of saturated, unbranched very long chain fatty acids (VLCFA). It is caused by mutations in ABCD1 gene. No molecular data on X-ALD is available in India and mutational spectrum in Indian patients is not known.MethodsWe standardized conformation sensitive gel electrophoresis (CSGE) method to detect mutations in ABCD1 gene in twenty Indian patients with X-ALD. The results were confirmed by sequencing. Genotype–phenotype correlation was also attempted. Prenatal diagnosis (PND) in one family was done using chorionic villi (CV) sample at 12 weeks of gestation.ResultsOut of twenty, causative mutations could be identified in twelve patients (60%). Six reported and four novel mutations were identified. Three polymorphisms were also observed. No hot spot was found. No significant genotype–phenotype correlation could be established.ConclusionsThe study identified the mutation spectrum of Indian X-ALD patients, which enabled us to offer accurate genetic counseling, carrier detection and prenatal diagnosis where needed.     

Drug interactions in Brazilian type 2 diabetes patients

The aim of this paper is to identify the prevalence of the most frequent drug interactions in patients using oral antidiabéticos and their association with capillary glucose and medication adherence. In total, 579 type 2 diabetes mellitus patients from 12 health institutions in Fortaleza, Brazil were interviewed in 2009. A form was applied, including questions on medication use, comorbidities, lifestyle, body mass index and random capillary glucose. Results revealed that 26.7% used five or more different drugs simultaneously and daily. Statistically significant drug interactions occurred between antidiabéticos and diuretics, angiotensin‐converting enzyme inhibitors, anti‐lipidaemics and corticoids. No significant association was found between polypharmacy, medication adherence and glucose. It is important for nurses, in consensus with other health professionals, to consider the possibility of other drugs that mean less risk for diabetes patients’ glucose control or of increased antidiabetics doses.     

慢性HBV感染者低浓度HBsAg相关分子调查研究

Objective To investigate the molecular characteristics and epidemiological signification of patients with low-level HBsAg. Methods PCR and gene sequencing were used to detect HBV DNA and Tyr-Met-Asp-Asp(YMDD) mutant in 136 serum samples with low-level HBsAg and 44 sernm samples with high-level HBsAg. Genotyping was performed in 47 cases with HBV DNA 105 copies/L by concentration method and 37 cases with high-level HBsAg. S gene sequences and serotypes were analyzed in 14 cases with HBV DNA 105 copies/L and 29 cases with high-level HBsAg. S gene sequences were compared with the consensus sequence of Chinese strain by BioEdit software. Results The HBV DNA-positive rate, YMDD mutation rate and HBV DNA load (logarithm) in low-level and high-level HBsAg group were 34.6% (47/136), 0% (0/136), 6.5±1.4 and 84.1% (37/44), 9.1% (4/44), 8.9±1.8, respectively. There was statistically significant differences between two groups (for concentration method,χ2 = 30.8, P < 0.05; for direct method, χ2 = 53.5, P < 0.05; for YMDD mutation ratio, P = 0.003, For HBV DNA (log), t = 6.5, P < 0.05). The genotypes in low-level HBsAg group included type B (16/47), type C (5/47) and non-classified ones(26/47). There were significant differences between two groups (χ2=21.8, P <0.01). The serotypss included adw (7/14), ayw (4/14), adr (2/14) and ayr (1/14). There were significant differences in genotypes (χ2 = 13.5, P < 0.05) but not in serotypes between two groups (χ2 = 4.7, P >0.05). S gene sequencing results showed no S gnne variation was detected, but there were 6 single nucleotide polymorphisms in 16 cases, which would not result in the alternation of amino acid. Conclusions Low-replication phenomenon of HBV DNA was present in patients with low-level HBsAg. The major genotyps and serotype was type B and adw/ayw, respectively. Polymorphic variants have been found in the S gene. The existence of low-level HBsAg might be related with its own molecular characteristics resulting in low expression of HBsAg or immune tolerance induced by low-level HBsAg after HBV infection.     

慢性HBV感染者低浓度HBsAg相关分子调查研究

目的 探讨低浓度HBsAg人群的分子生物学特征及流行病学意义.方法 采用PCR及基因测序的方法 对HBV慢性感染者136份低浓度HBsAg(低浓度HBsAg组)和44份高浓度HBsAg(高浓度HBsAg组)血清标本进行HBV DNA、酪氨酸-蛋氨酸-天冬氨酸-天冬氨酸(YMDD)变异检测,并分别对浓缩法HBV DNA105拷贝/L的47份低浓度HBsAg和37份高浓度HBsAg血清标本进行基因型检测,以及对直接法HBV DNA10~5拷贝/L的14份低浓度HBsAg和29份高浓度HBsAg血清标本进行S基因序列、血清型进行检测分析,S基因序列采用BioEdit软件与中国株参照序列进行比对.结果低浓度HBsAg组HBV DNA阳性率、YMDD变异率和HBV DNA对数值分别为34.6%(47/136)、0(0/136)和6.5±1.4,高浓度HBsAg组分别为84.1%(37/44)、9.1%(4/44)和8.9±1.8,两组之间差异有统计学意义(浓缩法χ~2=30.8,P<0.05;直接法χ~2=53.5,P<0.05;YMDD变异率精确概率法,P=0.003;HBV DNA对数值t=6.5,P<0.05);47例低浓度HBsAg病例中分别检出B基因型16例、C基因型5例、未分型26例,14例血清型分别为adw 7例、ayw4例、adr2例、ayr 1例,在两组人群中基因型的分布差异有统计学意义(χ~2=13.5,P<0.05),血清型的分布差异无统计学意义(χ~2=4.7,P>0.05),S基因测序结果未发现S基因变异,但6处16例次存在核苷酸碱基差异而氨基酸同义的多态性特征.结论 低浓度HBsAg人群HBV DNA存在低复制现象,基因型、血清型分别以B型、adw/ayw为主,S基因呈多态性特征,低浓度HBsAg存在可能与HBV S基因特殊的分子生物学特征使HBsAg表达低下有关,或与患者感染HBV后机体免疫系统的个体反应导致低浓度HBsAg诱导机体免疫耐受有关.     

2016年浙江省急性胃肠炎患者诺如病毒的分子流行病学特征分析

摘要：目的：探讨2016年浙江省急性胃肠炎患者中诺如病毒的流行病学和基因特征。 方法：收集2016年1月至12月共1 308份急性胃肠炎患者的临床流行病学资料和粪便标本,用一步法双重荧光RT-PCR技术检测诺如病毒GⅠ和GⅡ型,用分层抽样抽取阳性标本核酸进行普通RT-PCR扩增,并对扩增产物进行测序分析。 结果：诺如病毒阳性率为10.55%（138/1 308）,其中GⅠ型12例,GⅡ型118例,GⅠ/GⅡ型混合感染8例。诺如病毒在不同年龄组的阳性率随患者年龄上升而下降,以≥60岁组最低;不同性别组间的阳性率差异无统计学意义;诺如病毒感染全年分布,其中12月阳性率达到顶峰,为37.50%。测序结果显示GⅠ的基因亚型主要是GⅠ.6;GⅡ的基因亚型以GⅡ.4与GⅡ.17为主,分别占40.91%（18/44）和34.09%（15/44）。 结论：诺如病毒是引起2016年浙江省急性胃肠炎的重要病原体,且GⅡ.4和GⅡ.17是主要的基因亚型。     

Molecular networking prospection and characterization of terpenoids and C15-acetogenins in Brazilian seaweed extracts

Molecular networking (MN) can efficiently dereplicate extracts and pure compounds. Red algae of the genus Laurencia are rich in halogenated secondary metabolites, mainly sesquiterpenes and C₁₅-acetogenins. Brown algae of the genus Dictyopteris produce mainly C₁₁-hydrocarbons, sesquiterpenes and sulfur-containing compounds, while Dictyota and Canistrocarpus are reported to contain mainly diterpenes. This study performs an exploratory MN analysis of 14 extracts from algae collected in Brazil (including the oceanic islands) and characterizes the secondary metabolites from the analyzed species. The extracts and some isolated metabolites were analyzed by LC-MS using the FastDDA algorithm, and the MS/MS spectra were submitted to GNPS and displayed in Cytoscape 3.5.1. The GNPS platform generated 68 individual nodes and nine family networks. The MN exploratory analysis indicated chemical differences among species, and also in sampling sites for the same species. For some extracts, it was possible to identify mass values that could correspond to terpenoids and C₁₅-acetogenins that have already been isolated from those or related species. An interesting chemodiversity was highlighted between Laurencia catarinensis from two nearby islands, and this was revealed and was also suggested by the family networks. Many nodes in the MN could not be characterized, and these metabolites can be used as targets for isolation in future works.

Molecular networking of Brazilian marine algae.