Griscelli syndrome-type 2 in twin siblings: case report and update on RAB27A human mutations and gene structure

Griscelli syndrome (GS) is a rare autosomal recessive disorder caused by mutation in the MYO5A (GS1, Elejalde), RAB27A (GS2) or MLPH (GS3) genes. Typical features of all three subtypes of this disease include pigmentary dilution of the hair and skin and silvery-gray hair. Whereas the GS3 phenotype is restricted to the pigmentation dysfunction, GS1 patients also show primary neurological impairment and GS2 patients have severe immunological deficiencies that lead to recurrent infections and hemophagocytic syndrome. We report here the diagnosis of GS2 in 3-year-old twin siblings, with silvery-gray hair, immunodeficiency, hepatosplenomegaly and secondary severe neurological symptoms that culminated in multiple organ failure and death. Light microscopy examination of the hair showed large, irregular clumps of pigments characteristic of GS. A homozygous nonsense mutation, C-T transition (c.550C>T), in the coding region of the RAB27A gene, which leads to a premature stop codon and prediction of a truncated protein (R184X), was found. In patient mononuclear cells, RAB27A mRNA levels were the same as in cells from the parents, but no protein was detected. In addition to the case report, we also present an updated summary on the exon/intron organization of the human RAB27A gene, a literature review of GS2 cases, and a complete list of the human mutations currently reported in this gene. Finally, we propose a flow chart to guide the early diagnosis of the GS subtypes and Chédiak-Higashi syndrome.

Synergistic antitumor effect of TRAIL and adriamycin on the human breast cancer cell line MCF-7

The aim of the present study was to determine the effect of the combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and adriamycin (ADM) on the human breast cancer cell line MCF-7 and to identify potential mechanisms of apoptosis. Cell viability was analyzed by the MTT assay and the synergistic effect was assessed by the Webb coefficient. Apoptosis was quantified using the annexin V-FITC and propidium iodide staining flow cytometry. The mRNA expression of TRAIL receptors was measured by RT-PCR. Changes in the quantities of Bax and caspase-9 proteins were determined by Western blot. MCF-7 cells were relatively resistant to TRAIL (IC50 >10 µg/mL), while MCF-7 cells were sensitive to ADM (IC50 <10 µg/mL). A subtoxic concentration of ADM (0.5 µg/mL) combined with 0.1, 1, or 10 µg/mL TRAIL had a synergistic cytotoxic effect on MCF-7 cells, which was more marked with the combination of TRAIL (0.1 µg/mL) and ADM (0.5 µg/mL). In addition, the combined treatment with TRAIL and ADM significantly increased cell apoptosis from 9.8% (TRAIL) or 17% (ADM) to 38.7%, resulting in a synergistic apoptotic effect, which is proposed to be mediated by up-regulation of DR4 and DR5 mRNA expression and increased expression of Bax and caspase-9 proteins. These results suggest that the combination of TRAIL and ADM might be a promising therapy for breast cancer.

Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood

Lipopolysaccharide (LPS) activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R) and smooth (S) forms signal through Toll-like receptor 4 (TLR4), but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS) and nitric oxide (NO) generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30% increase followed by a 40% decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50% reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.

Challenges in the research and development of new human vaccines

The field of vaccinology was born from the observations by the fathers of vaccination, Edward Jenner and Louis Pasteur, that a permanent, positive change in the way our bodies respond to life-threatening infectious diseases can be obtained by specific challenge with the inactivated infectious agent performed in a controlled manner, avoiding the development of clinical disease upon exposure to the virulent pathogen. Many of the vaccines still in use today were developed on an empirical basis, essentially following the paradigm established by Pasteur, “isolate, inactivate, and inject” the disease-causing microorganism, and are capable of eliciting uniform, long-term immune memory responses that constitute the key to their proven efficacy. However, vaccines for pathogens considered as priority targets of public health concern are still lacking. The literature tends to focus more often on vaccine research problems associated with specific pathogens, but it is increasingly clear that there are common bottlenecks in vaccine research, which need to be solved in order to advance the development of the field as a whole. As part of a group of articles, the objective of the present report is to pinpoint these bottlenecks, exploring the literature for common problems and solutions in vaccine research applied to different situations. Our goal is to stimulate brainstorming among specialists of different fields related to vaccine research and development. Here, we briefly summarize the topics we intend to deal with in this discussion.

Myosin Va is developmentally regulated and expressed in the human cerebellum from birth to old age

Myosin Va functions as a processive, actin-based motor molecule highly enriched in the nervous system, which transports and/or tethers organelles, vesicles, and mRNA and protein translation machinery. Mutation of myosin Va leads to Griscelli disease that is associated with severe neurological deficits and a short life span. Despite playing a critical role in development, the expression of myosin Va in the central nervous system throughout the human life span has not been reported. To address this issue, the cerebellar expression of myosin Va from newborns to elderly humans was studied by immunohistochemistry using an affinity-purified anti-myosin Va antibody. Myosin Va was expressed at all ages from the 10th postnatal day to the 98th year of life, in molecular, Purkinje and granular cerebellar layers. Cerebellar myosin Va expression did not differ essentially in localization or intensity from childhood to old age, except during the postnatal developmental period. Structures resembling granules and climbing fibers in Purkinje cells were deeply stained. In dentate neurons, long processes were deeply stained by anti-myosin Va, as were punctate nuclear structures. During the first postnatal year, myosin Va was differentially expressed in the external granular layer (EGL). In the EGL, proliferating prospective granule cells were not stained by anti-myosin Va antibody. In contrast, premigratory granule cells in the EGL stained moderately. Granule cells exhibiting a migratory profile in the molecular layer were also moderately stained. In conclusion, neuronal myosin Va is developmentally regulated, and appears to be required for cerebellar function from early postnatal life to senescence.

Expression of β-catenin and c-myc during human common bile duct development: a possible role in the morphogenesis of the common bile duct

β-catenin and c-myc play important roles in the development of tissues and organs. However, little is known about their expression patterns during the development of the human common bile duct. Immunohistochemistry was used to detect β-catenin and c-myc expression in common bile duct samples from postmortem tissues of 14 premature infants and 6 spontaneously aborted fetuses. The expression of β-catenin and c-myc was also analyzed by Western blot. The samples were divided into four groups based on the stage of human fetal development: 12, 13-27, 28-37, and >37 weeks. The Image-Pro Plus v. 6.0 image analysis software was used to calculate the mean qualifying score (MQS). At fetal stages 12, 13-27, 28-37, and >37 weeks, MQS of β-catenin were 612.52±262.13, 818.38±311.73, 706.33±157.19, and 350.69±110.19, respectively. There was a significant difference in MQS among the four groups (ANOVA, P=0.0155) and between the scores at >37 and 13-27 weeks (Student-Newman-Keuls, P<0.05). At fetal stages 12, 13-27, 28-37, and >37 weeks, the MQS of c-myc were 1376.64±330.04, 1224.18±171.66, 1270.24±320.75, and 741.04±219.19, respectively. There was a significant difference in MQS among the four groups (ANOVA, P=0.0087) and between the scores at >37 and 12 weeks, >37 and 13-27 weeks, and >37 and 28-37 weeks (all P<0.05, Student-Newman-Keuls). Western blots showed that β-catenin and c-myc expression were significantly higher in fetal than in postnatal control duct tissue (P<0.05). c-myc and β-catenin are involved in the normal development of the human common bile duct.

Effect of estradiol and bisphenol A on human hepatoblastoma cell viability and telomerase activity

Sex hormones from environmental and physiological sources might play a major role in the pathogenesis of hepatoblastoma in children. This study investigated the effects of estradiol and bisphenol A on the proliferation and telomerase activity of human hepatoblastoma HepG2 cells. The cells were divided into 6 treatment groups: control, bisphenol A, estradiol, anti-estrogen ICI 182,780 (hereinafter ICI), bisphenol A+ICI, and estradiol+ICI. Cell proliferation was measured based on average absorbance using the Cell Counting-8 assay. The cell cycle distribution and apoptotic index were determined by flow cytometry. Telomerase activity was detected by polymerase chain reaction and a telomeric repeat amplification protocol assay. A higher cell density was observed in bisphenol A (P<0.01) and estradiol (P<0.05) groups compared with the control group. Cell numbers in S and G2/M phases after treatment for 48 h were higher (P<0.05), while the apoptotic index was lower (P<0.05) and telomerase activities at 48 and 72 h (P<0.05) were higher in these groups than in the control group. The cell density was also higher in bisphenol A+ICI (P<0.01) and estradiol+ICI (P<0.05) groups compared with the ICI group. Furthermore, cell numbers were increased in S and G2/M phases (P<0.05), while the apoptotic index was lower (P<0.05) and telomerase activities at 48 and 72 h were higher (P<0.05) in these groups than in the ICI group. Therefore, bisphenol A and estradiol promote HepG2 cell proliferation in vitro by inhibition of apoptosis and stimulation of telomerase activity via an estrogen receptor-dependent pathway.

Psychophysical estimation of acid intensity and determination of sweet-acid interaction in a fish paste containing glycerol

In a fish paste made with cooked Brazilian flathead (Percophis brasiliensis), glycerol (17%), sodium chloride (1.5%) and potassium sorbate (0.1%) the following acid percentages: 0.2; 0.4; 0.6; 0.8, 1 and 1.5% w/w were incorporated to determine the relationship between added acetic acid and the sensorially perceived intensity, and the effects of the combination of sweet-acid tastes. Tests for paired comparison, ranking and structured verbal scales for sweet and acid attributes and psychophysical test were carried out. There was a perceptible difference among samples for differences of 0.4 units of acid concentration. Samples indicated as sweeter by 89.47% of the judges were those containing a lesser acid concentration. A reduction in glycerol sweetness when increasing acid levels was observed. Acetic acid reduced the sweetness of glycerol and inversely glycerol reduced the acidity of acetic acid. The data obtained with the magnitude estimation test agree with Steven's law.

Human mucocutaneous leishmaniasis in Três Braços, Bahia - Brazil: an area of Leishmania braziliensis braziliensis transmission. II. Cutaneous disease. Presentation and evolution

The clinical records of 182 patients with cutaneous leishmaniasis probably due to Leishmania braziliensis braziliensis are analysed. 68% had a single lesion which was usually an ulceron the lower anterior tibial third. Many had short histories of one to two months and all age groups were represented 13% had closed lesions of a verrucose or plaque like nature. Evolution of these skin lesions after treatment was related to the regularity of antimony therapy. Although healing usually occurred in three months, the time to scarring after commencing treatment was variable and related to the size ofthe lesion (p < 0.01). Usually if sufficient antimony treatment was given the lesion closed. Seven of the ten patients with initially negative leishmanin skin tests converted to positive after treatment. A significant decline of indirect fluorescent antibody titres occurred in patients followed, during and after therapy.

Interaction of an opportunistic fungus Purpureocillium lilacinum with human macrophages and dendritic cells

IntroductionPurpureocillium lilacinum is emerging as a causal agent of hyalohyphomycosis that is refractory to antifungal drugs; however, the pathogenic mechanisms underlying P. lilacinum infection are not understood. In this study, we investigated the interaction of P. lilacinum conidia with human macrophages and dendritic cells in vitro.MethodsSpores of a P. lilacinum clinical isolate were obtained by chill-heat shock. Mononuclear cells were isolated from eight healthy individuals. Monocytes were separated by cold aggregation and differentiated into macrophages by incubation for 7 to 10 days at 37°C or into dendritic cells by the addition of the cytokines human granulocyte-macrophage colony stimulating factor and interleukin-4. Conidial suspension was added to the human cells at 1:1, 2:1, and 5:1 (conidia:cells) ratios for 1h, 6h, and 24h, and the infection was evaluated by Giemsa staining and light microscopy.ResultsAfter 1h interaction, P. lilacinum conidia were internalized by human cells and after 6h contact, some conidia became inflated. After 24h interaction, the conidia produced germ tubes and hyphae, leading to the disruption of macrophage and dendritic cell membranes. The infection rate analyzed after 6h incubation of P. lilacinumconidia with cells at 2:1 and 1:1 ratios was 76.5% and 25.5%, respectively, for macrophages and 54.3% and 19.5%, respectively, for cultured dendritic cells.ConclusionsP. lilacinum conidia are capable of infecting and destroying both macrophages and dendritic cells, clearly demonstrating the ability of this pathogenic fungus to invade human phagocytic cells.

Interaction and cystogenesis of Toxoplasma gondii within skeletal muscle cells in vitro

Infection by the protozoan parasite Toxoplasma gondii is widely prevalent in humans and animals. To prevent human infection, all meat should be well cooked before consumption, since the parasite is present in skeletal muscle. In this context, the use of skeletal muscle cells (SkMCs) as a cellular model opens up new approaches to investigate T. gondii-host cell interactions. Immunofluorescent detection of proteins that are stage-specific for bradyzoites indicated that complete cystogenesis of T. gondii in in vitro cultures of SkMCs occurs after 96 h of infection. Ultrastructural analysis showed that, after 48 h of interaction, there were alterations on the parasitophorous vacuole membrane, including greater thickness and increased electron density at the inner face of the membrane. The present study demonstrates the potential use of primary cultures of SkMCs to evaluate different molecular aspects of T. gondii invasion and cystogenesis and presents a promising in vitro model for the screening of drug activities toward tissue cysts and bradyzoites.

Interaction of Trichomonas vaginalis and Tritrichomonas foetus with keratin: an important role in parasite infection

Trichomonas vaginalis and Tritrichomonas foetus are human and bovine parasites, respectively, that provoke the sexually transmitted disease trichomoniasis. These extracellular parasites adhere to the host epithelial cell surface. Although mucinases and proteases have been described as important proteins for parasite adhesion to epithelial cells, no studies have examined the role of the keratin molecules that cornify the vaginal epithelium. Here, we investigated the interaction of T. vaginalis and T. foetus with human keratin in vitro; additionally, adherence assays were performed in cattle with T. foetus to elucidate whether trichomonads were able to interact with keratin in vivo. We demonstrated that both T. vaginalisand T. foetusinteracted directly with keratin. Additionally, the trichomonads ingested and digested keratin, shedding new light on the Trichomonas infection process.

Cognitive performance of young and elderly subjects on the free word recall memory test: effect of presentation order on recall order

The influence of aging on memory has been extensively studied, but the importance of short-term memory and recall sequence has not. The objective of the current study was to examine the recall order of words presented on lists and to determine if age affects recall sequence. Physically and psychologically healthy male subjects were divided into two groups according to age, i.e., 23 young subjects (20 to 30 years) and 50 elderly subjects (60 to 70 years) submitted to the Wechsler Adult Intelligence Scale-Revised and the free word recall test. The order of word presentation significantly affected the 3rd and 4th words recalled (P < 0.01; F = 14.6). In addition, there was interaction between the presentation order and the type of list presented (P < 0.05; F = 9.7). Also, both groups recalled the last words presented from each list (words 13-15) significantly more times 3rd and 4th than words presented in all remaining positions (P < 0.01). The order of word presentation also significantly affected the 5th and 6th words recalled (P = 0.05; F = 7.5) and there was a significant interaction between the order of presentation and the type of list presented (P < 0.01; F = 20.8). The more developed the cognitive functions, resulting mainly from formal education, the greater the cognitive reserve, helping to minimize the effects of aging on the long-term memory (episodic declarative).

Smoking, consumption of alcohol and sedentary life style in population grouping and their relationships with lipemic disorders

The study, part of the project "Atherosclerotic cardiovascular diseases, lipemic disorders, hypertension, obesity and diabetis mellitus in a population of the metropolitan area of the southeastern region of Brazil", had the following objectives: a) the characterization and distribution among typical human socio-economic groupings, of the prevalence of some particular habits which constitute aspects of life-style-the use of tobacco, the use of alcohol and sedentary activity; b) the establishment of the interrelation between the above-mentioned habits and some lipemic disorders. The prevalence of the habits cited behaved in the following manner: the use of tobacco predominated among men, distributed uniformly throughout the social strata; among the women the average percentage of smokers was 18,9%, a significant difference occurring among the highest socio-economic class, where the average was of 40.2%. The sedentary style of life presented high prevalence, among both men and women with exception of the women of the highest socio-economic level and of the skilled working class. The use of alcohol, as one would expect, is a habit basically practised by the men, without any statistically significant differences between classes. For the purpose of establishing associations between these risk fictors and lipemic conditions four situations were chosen, of the following characteristics: 1- total cholesterol > or = 220 mg/dl and triglycerides > or = 150 mg/dl; 2- HDL cholesterol <35 mg/dl for men and <45 mg/dl for women and triglycerides levels > or = 150 mg/dl; 3- HDL cholesterol <35 mg/dl for men and <45 mg/dl for women and triglycerides levels <150 mg/dl; 4- total cholesterol 220 mg/dl with triglycerides levels <150 mg/dl. Six models of multiple (backward) regression were established, with seven independent variables- age, sex, use of tobacco, consumption of alcohol, light physical activity, hypertension and obesity. Significant associations (P<0,05) were revealed with hypercholesterolemia, accompanied by triglyceride levels > or = 150 mg/dl, and the following independent variables: age, use of tobacco and the interactions between obesity and smoking, age and sedentary lifestyle, sex and obesity (R2=22%); the standardized B coefficient showed that the variables with the greatest weight in the forecasting of the variation in the levels of cholesterol were smoking and the interaction between obesity and smoking. The hypercholesterolemia accompanied by triglycerides levels <150 mg/dl showed a positive association between total cholesterol and sex and the interactions obesity/smoking and sex/obesity. As regards HDL cholesterol accompanied by triglyceride/ levels > or = 150 mg/dl was inversely associated with obesity and the interaction smoking/ age and directly with age (R=31%). The standardized B coeffients, indicated that the variables obesity and the interactions smoking/age possessed a weight three times greater than age alone in accounting for the variation in the serum levels of HDL cholesterol. When accompanied by triglycerides <150 mg/dl there was no association between and the independent variables and the set of them presented R equal to 22%. The sum of top, in the population stutied in this project, the component habits of life-style (smoking, alcohol consumption and sedentary activity) which constitute risk factors which determine morbidity from atherosclerotic cardiovascular diseases are be found distributed through all the typical social groupings of this particular form of social organization. On the other hand, the seven independent variables used in the multiple regression models for the explanation of the lipemic conditions considered presented multiple determination coefficients which varied, approximately, between 20% and 30%. Thus it is important that in the genetic epidemiology the study of the morbidities in question be emphasized.

POSSIBLE ROLE OF LUTZOMYIA MARANONENSIS AND LUTZOMYIA ROBUSTA (DIPTERA: PSYCHODIDAE) AS VECTORS OF HUMAN BARTONELLOSIS IN THREE PROVINCES OF REGION NOR ORIENTAL DEL MARAÑON, PERU

Human bartonellosis is found predominantly in Perú2, 6, 8, 12, 15, as well as in Ecuador3, 7, 10 and Colombia13, 15. In Peru, the disease is restricted to the valleys of the western-side and a few inter-andean and eastern-slopes of the andean valleys6, 15, 18 at altitudes between 1000 and 3200 masl. Most human cases are reported from the regions of Chavin, Nor Oriental del Marañon and Lima16. Lutzomyia verrucarum is presumed to be the only vector of human bartonellosis in the valleys of Peru1, 2, 8, 11, 17, 19/ Our research objetive was to detect the presence of Lu. verrucarum in various localities known to be endemic for human bartonellosis in three provinces of Region Nor Oriental del Marañon. Sandfly collections were made between 1987 and 1992 during four visits to bartonellosis-endemic provinces: San Ignacio (districts of San José de Lourdes: 1020-1260 m and La Coipa: 1200-1560 m), Jaén (districts of Santa Rosa: 1300-1680 m and Jaén: 1220-1680 m) and Utcubamba (districts of Lonya Grande: 1200 m and El Milagro: 1200-1540 m)