Combining germline, tissue and liquid biopsy analysis by comprehensive genomic profiling to improve the yield of actionable variants in a real-world cancer cohort.

BACKGROUND: Comprehensive next-generation sequencing is widely used for precision oncology and precision prevention approaches. We aimed to determine the yield of actionable gene variants, the capacity to uncover hereditary predisposition and liquid biopsy appropriateness instead of, or in addition to, tumor tissue analysis, in a real-world cohort of cancer patients, who may benefit the most from comprehensive genomic profiling. METHODS: Seventy-eight matched germline/tumor tissue/liquid biopsy DNA and RNA samples were profiled using the Hereditary Cancer Panel (germline) and the TruSight Oncology 500 panel (tumor tissue/cfDNA) from 23 patients consecutively enrolled at our center according to at least one of the following criteria: no available therapeutic options; long responding patients potentially fit for other therapies; rare tumor; suspected hereditary cancer; primary cancer with high metastatic potential; tumor of unknown primary origin. Variants were annotated for OncoKB and AMP/ASCO/CAP classification. RESULTS: The overall yield of actionable somatic and germline variants was 57% (13/23 patients), and 43.5%, excluding variants previously identified by somatic or germline routine testing. The accuracy of tumor/cfDNA germline-focused analysis was demonstrated by overlapping results of germline testing. Five germline variants in BRCA1, VHL, CHEK1, ATM genes would have been missed without extended genomic profiling. A previously undetected BRAF p.V600E mutation was emblematic of the clinical utility of this approach in a patient with a liver undifferentiated embryonal sarcoma responsive to BRAF/MEK inhibition. CONCLUSIONS: Our study confirms the clinical relevance of performing extended parallel tumor DNA and cfDNA testing to broaden therapeutic options, to longitudinally monitor cfDNA during patient treatment, and to uncover possible hereditary predisposition following tumor sequencing in patient care.

Chediak-Higashi syndrome: Lessons from a single-centre case series.

BACKGROUND: Chediak-Higashi syndrome (CHS) is a rare and potentially fatal autosomal recessive disease characterized by frequent bacterial infections, bleeding tendency, oculocutaneous albinism, photosensitivity and progressive neurologic dysfunction. Owing to the rarity of this condition, the objective of this study was to describe patients with CHS. METHODS: Retrospective evaluation of patients followed in a paediatric tertiary centre of Allergy and Immunology of São Paulo, Brazil, between 1986 and 2018 with a confirmed diagnosis of CHS. Data were obtained from medical records. Demographic aspects, family history, clinical findings, laboratory data, diagnosis, treatment and outcome were described. RESULTS: A total of 14 patients (five male) were included. Clinical manifestations were first recognized at a median age of two months (at birth-20 months). Median age at diagnosis was 1.7 years (0-5 years). All patients had recurrent infections. Albinism was present in 13 patients and silvery or light hair was present in 14. Seven patients developed hemophagocytic lymphohistiocytosis (HLH); the median age at the diagnosis of HLH was 5.7 years (2.6-6.7 years) and the median interval between the diagnosis of CHS and HLH was 3.3 years (0-5 years). Four of the most recently diagnosed patients underwent bone marrow transplantation (BMT). Nine patients are deceased, and one was lost to follow-up. The median age of death was 6.7 years (3.8-22 years). Five patients died of HLH, one of lymphoma, and three of infection. All the patients who had HLH before the year of 2000 died of HLH. The two most recently diagnosed patients with HLH were able to cure the HLH, although they died of other causes. Four patients are alive, three of them after successful BMT. CONCLUSION: Thirty years of follow up showed an improvement in the prognosis in patients with CHS. The better understanding of the underlying biological mechanisms of HLH allowed the standardization of management protocols, resulting in survival improvement. BMT is the only treatment that can change CHS prognosis, which emphasizes the need for early identification of the disease.

Baked milk tolerant patient: Is there any special feature?

BACKGROUND: Determining whether patients with cow's milk allergy (CMA) can tolerate foods produced with baked milk could provide a better quality of life, a better prognosis, and an option for desensitization. OBJECTIVES: The aim of this study was to identify which patients over four years of age with persistent CMA could tolerate baked milk, to compare the clinical and laboratory characteristics of reactive and non-reactive groups and to describe their clinical evolution. MATERIALS AND METHODS: A cross-sectional study was conducted (January/13 to November/14) that included all the patients followed at a food allergy center who met the inclusion criteria. The patients underwent an oral food challenge (OFC) with a muffin (2.8g of cow's milk protein). To exclude cow's milk (CM) tolerance, the patients were subsequently challenged with unheated CM. RESULTS: Thirty patients met all the inclusion criteria. Fourteen patients (46.7%) were considered non-reactive to baked milk and reactive to unheated CM. When the groups that were reactive and non-reactive to baked milk were compared, no statistically significant differences in clinical features were found. The prick test for α-lactalbumin (p=0.01) and casein (p=0.004) and the serum specific IgE for casein (p=0.05) presented statistical differences. After one year, none of the patients who were reactive to baked milk were ingesting CM, while 28% of the tolerant patients were consuming fresh CM (p=0.037). CONCLUSIONS: Baked milk can be tolerated by patients with CMA, especially those with lower levels of casein and α-lactalbumin. This option can improve quality of life and accelerate tolerance.

Thyroid nodules with indeterminate cytology: prospective comparison between 18F-FDG-PET/CT, multiparametric neck ultrasonography, 99mTc-MIBI scintigraphy and histology.

PURPOSE: To evaluate the role of (18)F-flurodeoxiglucose positron emission tomography/computed tomography ((18)F-FDG-PET/CT) in predicting malignancy of thyroid nodules with indeterminate cytology. PATIENTS AND METHODS: We analysed 87 patients who have been scheduled to undergo surgery for thyroid nodule with indeterminate cytology. All patients underwent (18)F-FDG-PET/CT, multiparametric neck ultrasonography (MPUS), and (99m)Tc-methoxyisobutylisonitrile scintigraphy ((99m)Tc-MIBI-scan). Histopathology was the standard of reference. We compared the sensitivity (SE), specificity (SP), accuracy (AC), positive (PPV) and negative predictive (NPV) values of (18)F-FDG-PET/CT with those of (99m)Tc-MIBI-scan and MPUS in detecting cancer. Univariate and multivariate analyses evaluated the association between each diagnostic tool and histopathology. RESULTS: On histopathology, 69 out of 87 nodules were found to be benign and 18 to be malignant. The SE, SP, AC, PPV and NPV of (18)F-FDG-PET/CT were 94, 58, 66, 37 and 98% respectively. The SE, AC and NPV of (18)F-FDG-PET/CT were significantly higher than those of MPUS and (99m)Tc-MIBI-scan. The association of both positive (18)F-FDG-PET/CT and MPUS (FDG+/MPUS+) showed significantly lower SE (61% vs 94%) and NPV (88% vs 98%) than (18)F-FDG-PET/CT alone, but significantly higher SP (77% vs 58%). On univariate analysis, (18)F-FDG-PET/CT and the combination of FDG+/MPUS+ and of FDG+/MIBI- were all significantly associated with histopathology. On multivariate analysis, only FDG+/MIBI- was significantly associated with histopathology. CONCLUSION: The AC of (18)F-FDG-PET /CT in detecting thyroid malignancy is higher than that of (99m)Tc-MIBI-scan and MPUS. A negative (18)F-FDG-PET/CT correctly predicts benign findings on histopathology. The association of FDG+/MPS+ is significantly more specific than (18)F-FDG-PET/CT alone in identifying differentiated thyroid cancer. A positive (18)F-FDG-PET/CT is significantly associated with malignancy when qualitative (99m)Tc-MIBI-scan is rated as negative.

Establishing a cut-off for the serum levels of specific IgE to milk and its components for cow's milk allergy: results from a specific population.

BACKGROUND: Cow's milk allergy diagnosis many times requires double-blind placebo-controlled food challenge (DBPCFC), which presents high accuracy but involves risks, specifically in infants and anaphylactic patients. The identification of the cut-off values for specific IgE to milk or its components would contribute to cow's milk allergy (CMA) diagnosis. The aim of this study was to compare discriminating concentration of a cow's milk specific IgE and its fractions (α-lactoalbumin, ß-lactoglobulin, casein) in children for the CMA diagnosis. METHODS: this study included 123 patients (M:F=1.3:1) median age at diagnosis=1.91 years, (3.5m to 13.21y) with CMA diagnosis via DBPCFC (n=26), proven anaphylaxis due to cow's milk (n=46) or a suggestive clinical history associated with a positive skin prick test (n=51) and open oral food challenge. The control group included 61 patients (1 male:1.1 female) ages ranging from 0.66 to 16.7 years (median=6.83 years). Receiver operator characteristics (ROC) curves were constructed to determine the best cut-offs that guarantees high specificity (>95%) for cow's milk and its components. RESULTS: considering 98% specificity, cut-off points were: 3.06 kU/L for cow's milk, 2.06 kU/L for α-lactalbumin, 1.85 kU/L for ß-lactoglobulin and 1.47kU/L for casein. The best ROC curve (area under the curve=0.929) was obtained evaluating cow's milk. CONCLUSION: this study showed that the cut-off point detected for whole cow's milk revealed a better discriminatory capacity for CMA diagnosis without the necessity of the milk components testing.

Double-blind, placebo-controlled food challenges in Brazilian children: adaptation to clinical practice.

BACKGROUND: A double-blind, placebo-controlled food challenge (DBPCFC) is considered the gold standard for diagnosing food allergy, but because of methodological difficulties it is rarely conducted in clinical practice, especially in paediatric patients. The purpose of the study was to propose a DBPCFC protocol that is adapted to our conditions for the diagnosis of an IgE-mediated cow's milk allergy (CMA) in a Brazilian reference centre for paediatric allergies. METHODS: This study includes the experimental phase (choice of materials, adjustments made to protocols described in the literature) and the test execution phase. DBPCFCs were performed in 58 patients aged 1-15 years who were separated into two groups: Group 1 (n=39), sex 1.6 M:F, 5.3 years median age, suggestive history of IgE-mediated CMA; and Group 2 (n=19), sex 1.4 M:F, 8.3 years median age with symptoms not associated with milk ingestion and laboratory data not compatible with IgE-mediated CMA. RESULTS: The materials were standardised for testing: containers and disposable products, low-lactose cow's milk (CM) and vehicles, such as natural fruit juice, vegetable soup and soybean-based beverages. Each DBPCFC was performed in a single day with two blind, randomised phases with a 2-h interval between them. The milk doses were gradually increased and offered in regular intervals of 15-30 min. Following negative or inconclusive results, patients underwent an open oral challenge test with 200 mL of low-lactose CM. CONCLUSIONS: The proposed adaptation for the DBPCFC allowed to implement this important test for the diagnosis of IgE-mediated CMA in a reference centre for paediatric allergies. It was considered feasible and safe if performed in an appropriate setting with physician supervision.

Common variable immunodeficiency associated with hepatosplenic T-cell lymphoma mimicking juvenile systemic lupus erythematosus.

Common variable immunodeficiency (CVID) is a heterogeneous disorder with susceptibility to infections, autoimmune manifestations, and cancer. To our knowledge, CIVD with T-cell lymphoma mimicking juvenile systemic lupus erythematosus (JSLE) was not described in the literature, and one case was reported herein. An 8-year-old female was admitted in our Pediatric Immunology Unit with a clinical history of hypogammaglobulinemia, recurrent upper respiratory infections, and pneumonias. She had a marked decrease of three serum immunoglobulin isotypes, and the diagnosis of CVID was established. At the age of 17 years, she presented with oral ulceration, nonerosive arthritis, nephritis, serositis, cytopenia, positive antiphospholipid antibodies, and positive antinuclear antibody fulfilling the American College of Rheumatology (ACR) criteria for SLE. She was treated with intravenous methylprednisolone for three consecutive days, and intravenous immunoglobulin, and maintenance therapy of chloroquine, azathioprine and prednisone 40 mg/day. Two months later, she died of septic shock secondary to acute pneumonia. The necropsy showed hepatosplenic T-cell lymphoma with diffuse involvement of bone marrow, spleen, liver, and lungs. The lymphoma cells were positive for CD3 immunostaining and negative for CD20 and lysozyme. In conclusion, the association of CVID and hepatosplenic T-cell lymphoma may simulate JSLE diagnosis.

Molecular characterization of patients with X-linked Hyper-IgM syndrome: description of two novel CD40L mutations.

Type 1, X-linked Hyper-IgM syndrome (HIGM1) is caused by mutations in the gene encoding the CD154 protein, also known as CD40 ligand (CD40LG). CD40L is expressed in activated T cells and interacts with CD40 receptor expressed on B lymphocytes and dendritic cells. Affected patients present cellular and humoral immune defects, with infections by intracellular, opportunistic and extracellular pathogens. In the present study we investigated the molecular defects underlying disease in four patients with HIGM1. We identified four distinct CD40L mutations, two of them which have not been previously described. P1 harboured the novel p.G227X mutation which abolished CD40L expression. P2 had a previously described frame shift deletion in exon 2 (p.I53fsX65) which also prevented protein expression. P3 demonstrated the previously known p.V126D change in exon 4, affecting the TNF homology (TNFH) domain. Finally, P4 evidenced the novel p.F229L mutation also located in the TNFH domain. In silico analysis of F229L predicted the change to be pathological, affecting the many hydrophobic interactions of this residue. Precise molecular diagnosis in HIGM syndrome allows reliable detection of carriers, making genetic counselling and prenatal diagnosis possible.

Sensitisation to aeroallergens in Brazilian adolescents living at the periphery of large subtropical urban centres.

OBJECTIVES: To evaluate the sensitization to aeroallergens determined by skin prick test (SPT) in Brazilian adolescents, and to correlate its positivity with the diagnosis of asthma and/or rhinitis based on the written questionnaire (WQ) of ISAAC phase III study. PATIENTS AND METHODS: A total of 996 adolescents (387 boys) were selected by systematic samples. A standard allergen extracts panel (positive/negative control, D pteronyssinus [Dpt], P americana [Pa], B germanica [Bg], dog, cat, fungal and grass mix) was used and its positivity compared with positive responses to asthma, rhinitis or both. RESULTS: Positive SPT to at least one allergen was observed in 466 adolescents (46.8 %), with sensitisation to Dpt in 79.1 %. Positivity to more than one allergen occurred in 232 students (49.8 %). The frequency of positive SPTs was significantly higher among adolescents with asthma (OR = 2.16), rhinitis (OR = 1.69), and asthma and rhinitis (OR = 2.03). Positive SPT to four or more allergens were higher among asthmatics (OR = 2.6) and among adolescents with asthma and rhinitis (OR = 3). CONCLUSIONS: A high sensitisation rate to aeroallergens was observed, significantly higher among those with asthma, rhinitis or a combination of both, especially in multiple sensitisations.

Prevalence of asthma, rhinitis and eczema in 6 - 7 years old students from the western districts of São Paulo City, using the standardized questionnaire of the "International Study of Asthma and Allergies in Childhood" (ISAAC)-phase IIIB.

UNLABELLED: The aims of the present work were the evaluation of allergic disease prevalence among 6 and 7 year-old students from the western districts of São Paulo city and the comparison of these data with those obtained in the International Study of Asthma and Allergies in Childhood (ISAAC) phase I, performed in the central-southern districts of São Paulo, using the ISAAC standardized written questionnaire. METHODS: 5,040 questionnaires were distributed and 3,312 were returned. Proportional differences were estimated by Chi square or Fisher exact tests. Odds Ratio and 95% confidence intervals between genders and allergic diseases were calculated. Values of p<0.05 were considered statistically significant. RESULTS: The corrected prevalences found were: asthma 24.4%, medical diagnosis of asthma 5.7%, rhinitis 25.7%, rhinoconjunctivitis 11.3%, medical diagnosis of rhinitis 20.0%, atopic eczema 9.2%. Significant associations between asthma and rhinitis (OR=3.3), asthma and eczema (OR=2.2), and rhinitis and eczema (OR=2.8) occurred. The male gender was prevalent regarding asthma and rhinitis. Compared to data from ISAAC phase I, higher asthma prevalence and severity, and lower values for rhinitis and eczema were observed in this study. CONCLUSIONS: The present study evidenced high prevalences for asthma and rhinitis compared to the children's medical diagnosis. The male gender predominated in all positive responses regarding asthma and rhinitis. The most frequent associations observed were between asthma and rhinitis and asthma and eczema. In the western districts of São Paulo, a higher prevalence of asthma symptoms and severity and lower prevalences for rhinitis and eczema occurred compared to the central-southern districts of the city.

Prevalence of symptoms of asthma, rhinitis, and atopic eczema in Brazilian adolescents related to exposure to gaseous air pollutants and socioeconomic status.

OBJECTIVES: To evaluate the relationship between exposure to gaseous air pollutants (ozone [O3], carbon monoxide [CO], nitrogen dioxide [NO2], and sulfur dioxide [SO2]) socioeconomic status and the prevalence of symptoms of asthma, rhinitis and atopic eczema in adolescents. SUBJECTS AND METHODS: A sample of 16 209 adolescents from São Paulo West (SPW), São Paulo South (SPS), Santo André (SA), Curitiba (CR), and Porto Alegre (PoA) were enrolled. Data on air pollutants and socioeconomic status were compared to prevalence of symptoms with the Spearman correlation coefficient. RESULTS: Socioeconomic status was quite similar in all cities. The levels of O3 in SPW, SPS, and SA, and of CO in SA were higher than the acceptable ones. In relation to O3 and CO exposures, adolescents from SPW and SA had a significant risk of current wheezing, whereas living in SPW was associated with a high risk of rhinoconjunctivitis, eczema, and flexural eczema and living in CR to rhinitis. Exposure to NO2 was associated with a high risk of current wheezing in SPW and SA, and of severe asthma in SPW and PoA. Exposure to SO2 was associated with a high risk of current wheezing in SPW and SA, severe asthma in SPW and PoA, and nighttime cough, eczema, flexural eczema and severe eczema in SPW. Living in SPW, CR, or PoA was associated with a high risk of rhinitis, rhinoconjunctivitis, and severe rhinitis. CONCLUSIONS: Although we did not detect a characteristic pattern for all symptoms evaluated or a specific air pollutant, our data suggest a relationship between higher exposure to photochemical pollutants and high prevalence or risk of symptoms of asthma, rhinitis, and atopic eczema.

Prevalence of asthma, rhinitis and eczema in 6 - 7 years old students from the western districts of São Paulo City, using the standardized questionnaire of the "International Study of Asthma and Allergies in Childhood" (ISAAC)-phase IIIB

The aims of the present work were the evaluation of allergic disease prevalence among 6 and 7 year-old students from the western districts of São Paulo city and the comparison of these data with those obtained in the International Study of Asthma and Allergies in Childhood (ISAAC) phase I, performed in the central-southern districts of São Paulo, using the ISAAC standardized written questionnaire. METHODS: 5,040 questionnaires were distributed and 3,312 were returned. Proportional differences were estimated by Chi square or Fisher exact tests. Odds Ratio and 95 percent confidence intervals between genders and allergic diseases were calculated. Values of p<0.05 were considered statistically significant. RESULTS: The corrected prevalences found were: asthma 24.4 percent, medical diagnosis of asthma 5.7 percent, rhinitis 25.7 percent, rhinoconjunctivitis 11.3 percent, medical diagnosis of rhinitis 20.0 percent, atopic eczema 9.2 percent. Significant associations between asthma and rhinitis (OR=3.3), asthma and eczema (OR=2.2), and rhinitis and eczema (OR=2.8) occurred. The male gender was prevalent regarding asthma and rhinitis. Compared to data from ISAAC phase I, higher asthma prevalence and severity, and lower values for rhinitis and eczema were observed in this study. CONCLUSIONS: The present study evidenced high prevalences for asthma and rhinitis compared to the children's medical diagnosis. The male gender predominated in all positive responses regarding asthma and rhinitis. The most frequent associations observed were between asthma and rhinitis and asthma and eczema. In the western districts of São Paulo, a higher prevalence of asthma symptoms and severity and lower prevalences for rhinitis and eczema occurred compared to the central-southern districts of the city.

OBJETIVOS: Avaliar a prevalência das doenças alérgicas na região oeste de São Paulo entre escolares de 6 a 7 anos e comparar os dados obtidos com aqueles da fase I da região centro-sul, através do questionário padronizado do International Study of Asthma and Allergies in Childhood. MÉTODOS: Foram enviados 5040 questionários escritos com resposta de 3.312 alunos. As diferenças entre proporções foram avaliadas pelo Teste do Qui-quadrado ou Teste Exato de Fisher, se calculado a Razão das Chances, intervalo de confiança 95 por cento entre os sexos e doenças alérgicas. Os valores de p < 0,05 foram considerados como significantes. RESULTADOS: As prevalências corrigidas encontradas foram: asma 24,4 por cento, diagnóstico médico de asma 5,7 por cento, rinite 25,7 por cento, rinoconjuntivite 11,3 por cento, diagnóstico médico de rinite 20 por cento, eczema atópico 9,2 por cento. Houve associação significativa entre asma e rinite (OR=3,3), asma e eczema (OR=2,2) e rinite e eczema atópico (OR=2,8). O sexo masculino foi predominante para asma e rinite. Comparando-se os dados da fase I, observou-se prevalência mais elevada dos sintomas e gravidade de asma e valores menores para rinite e eczema. CONCLUSÕES: As prevalências de asma e rinite neste estudo mostraram valores elevados em comparação ao diagnóstico médico. Houve predomínio do sexo masculino para asma e rinite. As associações mais freqüentes foram entre asma e rinite e asma e eczema. Em relação à região centro-sul de São Paulo, observou-se que na região oeste houve maior prevalência dos sintomas e gravidade da asma e menor prevalência de rinite e eczema.

Effect of pH on the chemical composition and structure-function relationships of cheddar cheese.

The objectives of this study were to determine the effect of pH on chemical, structural, and functional properties of Cheddar cheese, and to relate changes in structure to changes in cheese functionality. Cheddar cheese was obtained from a cheese-production facility and stored at 4 degrees C. Ten days after manufacture, the cheese was cut into blocks that were vacuum-packaged and stored for 4 d at 4 degrees C. Cheese blocks were then high-pressure injected one, three, or five times with a 20% (wt/wt) glucono-delta-lactone solution. Successive injections were performed 24 h apart. Cheese blocks were then analyzed after 40 d of storage at 4 degrees C. Acidulant injection decreased cheese pH from 5.3 in the uninjected cheese to 4.7 after five injections. Decreased pH increased the content of soluble calcium and slightly decreased the total calcium content of cheese. At the highest level, injection of acidulant promoted syneresis. Thus, after five injections, the moisture content of cheese decreased from 34 to 31%, which resulted in decreased cheese weight. Lowered cheese pH, 4.7 compared with 5.3, also resulted in contraction of the protein matrix. Acidulant injection decreased cheese hardness and cohesiveness, and the cheese became more crumbly. The initial rate of cheese flow increased when pH decreased from 5.3 to 5.0, but it decreased when cheese pH was further lowered to 4.7. The final extent of cheese flow also decreased at pH 4.7. In conclusion, lowering the pH of Cheddar cheese alters protein interactions, which then affects cheese functionality. At pH greater than 5.0, calcium solubilization decreases protein-to-protein interactions. In contrast, at pH lower than 5.0, the acid precipitation of proteins overcomes the opposing effect caused by increased calcium solubilization and decreased calcium content of cheese, and protein-to-protein interactions increase.

Effect of salt on structure-function relationships of cheese.

Our objective was to determine the effect of salt on structural and functional properties of cheese. Unsalted Muenster cheese was obtained on 1 d, vacuum packaged, and stored for 10 d at 4 degrees C. The cheese was then cut into blocks that were vacuum packaged. After 4 d of storage at 4 degrees C, cheese blocks were high-pressure injected one, three, or five times, with a 20% (wt/wt) sodium chloride solution. Successive injections were performed 24 h apart. After 40 d of storage at 4 degrees C, cheese blocks were analyzed for chemical, structural, and functional attributes. Injecting sodium chloride increased the salt content of cheese, from 0.1% in the control, uninjected cheese to 2.7% after five injections. At the highest levels, salt injection promoted syneresis, and, after five injections, the moisture content of cheese decreased from 41 to 38%. However, the increased salt content caused a net weight gain. Cheese pH, soluble nitrogen, and total and soluble calcium content were unaffected. Cheese injected five times had a 4% increased area of cheese occupied by protein matrix compared with uninjected cheese. Hardness, adhesiveness, and initial rate of cheese flow increased, and cohesiveness decreased upon salt injection. However, the final extent of cheese flow, or melting was unaffected. We concluded that adding salt to cheese alters protein interactions, such that the protein matrix becomes more hydrated and expands. However, increasing the salt content of cheese did not cause an exchange of calcium with sodium. Therefore, calcium-mediated protein interactions remain a major factor controlling cheese functionality.

Effect of calcium and water injection on structure-function relationships of cheese.

Our objectives were to determine the effect of calcium and water injection on cheese structure and to relate changes in structure to changes in functional properties of cheese. Cheese with fat and moisture content similar to that of low-moisture part-skim Mozzarella was made according to a direct-acid, stirred/pressed-curd procedure. The cheese was then cut into blocks that were high-pressure-injected from one to five times, with either water or a 40% calcium chloride solution. Successive injections were performed 24 h apart. After 42 d of refrigerated storage, cheese microstructure and functionality were analyzed. When injected three or more times, water tended to increase cheese weight. The control, uninjected cheese, had the typical structure of a stirred/pressed-curd cheese: protein matrix interspersed with areas that originally contained fat and/or serum. Injecting water increased the area of cheese matrix occupied by protein, but it did not affect textural properties or melting of cheese. In contrast, when calcium was injected, a decrease in cheese weight was observed that was manifested through syneresis. The moisture content and pH of the cheese decreased as well. Calcium injection also decreased the area of cheese matrix occupied by protein. Cheese hardness increased, and cohesiveness and melting of cheese decreased upon calcium injection. We concluded that adding calcium to cheese alters how the proteins interact, which is manifested as changes in cheese microstructure. Such changes in cheese structure provide an understanding of changes in functional attributes of the cheese.

Temperature effect on structure-opacity relationships of nonfat mozzarella cheese.

Our objective was to determine the effect of heating on the structure of nonfat Mozzarella cheese and then to relate changes in structure to changes in cheese opacity. Cheese was made according to a direct-acid, stirred-curd procedure. Cheese samples, at 4 degrees C, were taken on d 1 and placed into glass bottles, which were sealed and heated. Once the cheese reached 10 degrees C or 50 degrees C, the bottles were placed on a scanner and color values measured. Samples were also taken on d 1 for chemical, micro, and ultrastructural analyses. Applying heat increased cheese opacity. At 50 degrees C the cheese was more opaque than at 10 degrees C. The increase in temperature induced changes in cheese structure. Larger high-density protein aggregates and increased protein concentration in the protein matrix were observed in cheese at 50 degrees C. Applied heat would favor hydrophobic interactions, and possibly, re-association of beta-casein and calcium with the protein matrix, promoting protein-to-protein interactions. Thus, the protein matrix contracts, occupying less cheese matrix area, and microphase separation occurs, causing serum pockets to grow in size, and microstructural heterogeneity to increase. It is proposed that the increased size of aggregates and heterogeneity of the cheese at 50 degrees C promote light reflection, thus increasing cheese opacity. We concluded that applying heat alters protein interactions in the cheese matrix, manifested as changes in cheese structure. Such changes in structure help provide an understanding of changes in cheese opacity.

Can voltammetry measure nitrogen monoxide (NO) and/or nitrites?

Recently, voltammetry with carbon fibre electrodes (CFE) has been implemented for real time measurement of nitrogen monoxide (NO) indicating that it is oxidised at the potential value of nitrites, approximately +700 mV. In contrast, here we show that modified CFE can monitor NO at oxidation potentials different than that of nitrites, i.e. +550 mV. Indeed, at +550 mV a significant increase of amperometric current levels was obtained when NO but not nitrites, were added to a phosphate buffer saline solution (PBS). Differential pulse voltammetry (DPV) supports these findings as two oxidation peaks were obtained when examining air preserved NO; peak 1 at +550 mV and peak 2 at +700 mV, respectively. In contrast, only peak 2 was monitored when nitrites or a solution of NO oxidised in air was added to PBS. Biological support to these in vitro data comes from the observation that the relaxation of an adrenaline-contracted aortic ring produced via addition of NO is concomitant with peak 1 at +550 mV. The relaxation is almost completed before the appearance of peak 2 at +700 mV. Furthermore, in vivo experiments performed in the striatum of rats show that the amperometric signal monitored at +550 mV is responsive to glutamatergic stimulation or inhibition of NO synthase.

The binding of oxidized low density lipoprotein (ox-LDL) to ox-LDL receptor-1 reduces the intracellular concentration of nitric oxide in endothelial cells through an increased production of superoxide.

Oxidized low density lipoprotein (ox-LDL) has been suggested to affect endothelium-dependent vascular tone through a decreased biological activity of endothelium-derived nitric oxide (NO). Oxidative inactivation of NO is regarded as an important cause of its decreased biological activity, and in this context superoxide (O(2)) is known to inactivate NO in a chemical reaction during which peroxynitrite is formed. In this study we examined the effect of ox-LDL on the intracellular NO concentration in bovine aortic endothelial cells and whether this effect is influenced by ox-LDL binding to the endothelial receptor lectin-like ox-LDL receptor-1 (LOX-1) through the formation of reactive oxygen species and in particular of O(2). ox-LDL induced a significant dose-dependent decrease in intracellular NO concentration both in basal and stimulated conditions after less than 1 min of incubation with bovine aortic endothelial cells (p < 0.01). In the same experimental conditions ox-LDL also induced O(2) generation (p < 0.001). In the presence of radical scavengers and anti-LOX-1 monoclonal antibody, O(2) formation induced by ox-LDL was reduced (p < 0.001) with a contemporary rise in intracellular NO concentration (p < 0.001). ox-LDL did not significantly modify the ability of endothelial nitric oxide synthase to metabolize l-arginine to l-citrulline. The results of this study show that one of the pathophysiological consequences of ox-LDL binding to LOX-1 may be the inactivation of NO through an increased cellular production of O(2).

Secondary hypogammaglobilinemia after use of carbamazepine: case report and review.

Immunologic disorders related to anticonvulsant therapy have been described in the last three decades, including cellular and humoral alterations that result in recurrent infections; however, the physiopathologic mechanisms are not completely understood. This report describes a patient with complex partial epilepsy and hypogammaglobulinemia while in treatment with carbamazepine, with significant improvement in clinical signs and laboratory tests after substitution to sodium valproate. The authors stress the importance of clinical and laboratory evaluation of patients in continuous anticonvulsant therapy, including immunoglobulins levels and peripheral blood evaluations.