1,8-cineole and castor oil in sodium lauryl ether sulphate disrupt reproduction and ovarian tissue of Rhipicephalus (Boophilus) microplus.

Essential and fixed oils have been researched as alternatives to chemical acaricides. The activity of volatile compounds from essential oils (1,8-cineole, citral and eugenol) at 1.0% (w/v) and fixed oil (castor oil) at 0.3% (w/v) dissolved in 2.0% (v/v) dimethyl sulfoxide (DMSO) + 0.2% (w/v) Tween 80® was assessed against Rhipicephalus microplus using immersion tests. 1,8-cineole (29.0%) and castor oil (30.2%) had the highest reproductive inhibition rate. A second experiment was performed to verify the effect of the 1,8-cineole (10.0% w/v) and, or castor oil (0.3% w/v) on tick reproduction using different solubilizing agents. The highest reproductive inhibition was observed for the combination of 1,8-cineole/castor oil (94.1%) and 1,8-cineole in 2.0% (w/v) sodium lauryl ether sulphate (SLES) (92.8%). A third experiment showed morphological changes in R. microplus oocytes at different stages of development, as well as in pedicel cells. The most intense effects were observed when ticks were immersed in the formulation containing 1,8-cineole (10.0% w/v) and castor oil (0.3% w/v) dissolved in 2% (w/v) SLES. These findings highlight the potential of this formulation as an alternative for managing cattle ticks as their cytotoxic effects can reduce R. microplus reproductive success.

On the Person-Place Interaction and Its Relationship with the Responses/Outcomes of Listeners of Urban Soundscape (Compared Cases of Lisbon and Bogotá): Contextual and Semiotic Aspects.

Design, planning, and management of the urban soundscape require various interacting fields of knowledge given the fact that it is the human person that experiences and provides meaning to the urban places and their acoustic environments. The process of environmental perception involves contextual information that conditions people's responses and outcomes through the relationship between the variables Person, Activity, and Place. This research focuses on the interaction between Person and Place and its impact on responses and outcomes from listeners with different geographical origin and background. Laboratory studies were conducted in the cities of Lisbon (Portugal) and Bogotá (Colombia), where local listeners were introduced to known and unknown acoustic environments. Sound data recorded in the two cities allowed comparison of responses and outcomes of the listeners according to the Person-Place Interaction, leading to different meanings depending on the contextual variables. The results clearly show a relationship between site, acoustic environment, soundscape, Person-Place Interaction, and meaning of the place. This information can be useful for urban technicians and designers dealing with planning and management of urban soundscapes.

Prospective, randomized and controlled study of mesh displacement after laparoscopic inguinal repair: fixation versus no fixation of mesh.

INTRODUCTION: Repair of inguinal hernia is one of the most common elective operations performed in general surgery practice. Mesh hernia repair became the gold standard because of its low recurrence rate in comparison with non-tension-free repair. Laparoscopic approach seems to have potential advantages over open techniques, including faster recovery and reduced acute and chronic pain rate. Laparoscopic mesh fixation is usually performed using staples, which is associated with higher cost and risk for chronic pain. Recently, the role of mesh fixation has been questioned by several surgeons. AIM: To evaluate mesh displacement in patients undergoing laparoscopic inguinal hernia repair comparing mesh fixation with no fixation. METHODS: From January 2012 to May 2014, 60 consecutive patients with unilateral inguinal hernia were randomized into two groups: control group--10 patients underwent totally extraperitoneal (TEP) repair with mesh fixation; NO FIX group-50 patients underwent TEP repair with no mesh fixation. Mesh was marked with three 3-mm surgical clips at its medial inferior, medial superior and lateral inferior corners. Mesh displacement was measured by comparing an initial X-ray, performed in the immediate postoperative period, with a second X-ray obtained 30 days later. RESULTS: The mean displacement of all three clips in control group was 0.1-0.35 cm (range 0-1.2 cm), while in NO FIX group was 0.1-0.3 cm (range 0-1.3 cm). The overall displacement of control and NO FIX group did not show any difference (p = 0.50). CONCLUSION: Fixation of the mesh for TEP repair is unnecessary. TEP repair with no mesh fixation is safe and is not associated with increased risk of mesh displacement.

Consanguinity and founder effect for Gaucher disease mutation G377S in a population from Tabuleiro do Norte, Northeastern Brazil.

Gaucher's disease (GD) is caused by a ß-glucocerebrosidase deficiency, leading to the accumulation of glucocerebroside in the reticuloendothelial system. The prevalence of GD in Tabuleiro do Norte (TN) (1:4000) is the highest in Brazil. The purpose of this study was to present evidence of consanguinity and founder effect for the G377S mutation (c.1246G>A) among GD patients in TN based on enzyme, molecular and genealogical studies. Between March 2009 and December 2010, 131 subjects at risk for GD (GC in dried blood ≤2.19 nmol/h/ml) and 5 confirmed GD patients from the same community were submitted for molecular analysis to characterize the genetic profile of the population. Based on the enzymatic and molecular analysis, the subjects were classified into three categories: affected (n = 5), carrier (n = 20) and non-carrier (n = 111). All carriers were (G377S/wt). Affected subjects were homozygous (G377S/G377S). The identification of a single mutation in carriers and homozygotes from different generations, the history of the community and the genealogy study suggest that the high prevalence of GD in this population may be due to a combination of consanguinity and founder effect for the G377S mutation.

Detecção de coinfecções por Leishmania (l) Chagasi, Trypanosoma evansi, Toxoplasma gondii e neospora caninum em cães / Detection of co-infections by Leishmania (l) Chagasi, Trypanosoma evansi, Toxoplasma gondii and neospora caninum in dogs

Foram investigadas coinfecções por Leishmania (L.) chagasi, Trypanosoma evansi, Toxoplasma gondii e Neospora caninum em cães. Formas amastigotas de Leishmania spp. foram detectadas pela análise citopatológica de linfonodos em 46.42% (39/84) dos cães. Em um cão macho, adulto, sem raça definida, proveniente de área rural e positivo para Leishmania, foram observadas formas flageladas de T. evansi em esfregaço sanguíneo. Pela imunofluorescência indireta (RIFI), 5.95% (5/84) dos cães foram considerados reagentes para T. gondii, com titulação igual a 64, enquanto que 3.57% (3/84) foram reagentes para N. caninum, com título 50. Entre os animais com leishmaniose visceral, um apresentou resposta sorológica positiva para T. gondii e dois para N. caninum. Todos os cães reagentes para N. caninum eram de área rural e, o predomínio da infecção pelo T. gondii ocorreu em cães da área urbana. Um cão macho, jovem, da zona rural e soropositivo para T. gondii, apresentou mórulas de Ehrlichia spp. na citologia e reação positiva para o vírus da cinomose. Deste modo, mais estudos são necessários para avaliar a epidemiologia dessas infecções na população canina, principalmente com relação aos reservatórios de Trypanosoma spp. nas zonas rurais.(AU)

Co-infections by Leishmania (L.) chagasi, Trypanosoma evansi, Toxoplasma gondii and Neospora caninum in dogs were investigated. Amastigotes forms of Leishmania spp. were detected by cytopathological analysis of lymph nodes in 46,42% (39/84) of dogs. In a male dog, adult, without defined breed, from rural area and positive for Leishmania, were observed flagellated forms of T. evansi in blood smear. By immunofluorescence antibody test, 5,95% (5/84) of dogs were considered reactive to T. gondii, with titer equal to or higher than 1:64, while 3,57% (3/84) were reactive to N. caninum, with titer ≥1:50. Among the animals with visceral leishmaniasis, one showed positive serological response to T. gondii and two for N. caninum. All dogs reactive to N. caninum were from rural area and the predominance of infection by T. gondii was in dogs from urban area. A young male dog from the rural area and seropositive for T. gondii showed Ehrlichia spp. morulae in the cytology and positive reaction for canine distemper virus. Thus, further studies are needed to assess the epidemiology of these infections in canine population, especially with respect to the reservoirs of Trypanosoma spp. in rural areas.(AU)

Osteoporosis/osteopenia as an independent factor associated with periodontitis in postmenopausal women: a case-control study.

UNLABELLED: This study investigated whether osteoporosis/osteopenia has an influence on the progression of periodontitis in postmenopausal women. The findings highlight that postmenopausal women with osteoporosis/osteopenia had a greater chance of presenting periodontitis than those with normal bone mineral density, particularly among nonusers of osteoporosis medications and women with a greater number of remaining teeth, showing that osteoporosis/osteopenia has had an influence on the progression of periodontitis. INTRODUCTION: This study investigated whether osteoporosis/osteopenia has an influence on the progression of periodontitis in postmenopausal women and explored the effects of use of osteoporosis medication and tooth loss on this association. METHODS: This case-control study involved 521 postmenopausal women, with minimum age of 50 years, in Feira de Santana, Bahia, Brazil. Sociodemographic characteristics, health conditions/medications, and lifestyle habits were recorded. A complete periodontal examination was performed and periodontitis was diagnosed. Bone mineral density was evaluated through lumbar spine and femoral bone densitometry, obtained using dual-energy X-ray absorptiometry. Logistic regression was used to calculate the strength of association between the occurrences of osteoporosis/osteopenia and periodontitis. RESULTS: Women with osteoporosis/osteopenia were twice as likely to present periodontitis, as were those with normal bone mineral density, even after adjusting for smoking, age, family income, and last visit to dentist (odds ratios (OR)adjusted=2.24, 95% CI [1.24-4.06], p=0.008). Among nonusers of osteoporosis medication (ORadjusted=2.51, 95% CI [1.33-4.73], p=0.004) and women with at least 10 remaining teeth (ORadjusted=2.50 95% CI [1.18-5.27], p=0.02), the odds ratio was higher and statistically significant. CONCLUSIONS: These findings highlight that postmenopausal women with osteoporosis/osteopenia had a greater chance of presenting periodontitis than those with normal bone mineral density, particularly among nonusers of osteoporosis medications and women with a greater number of remaining teeth.

Detecção de coinfecções por Leishmania (l) Chagasi, Trypanosoma evansi, Toxoplasma gondii e neospora caninum em cães / Detection of co-infections by Leishmania (l) Chagasi, Trypanosoma evansi, Toxoplasma gondii and neospora caninum in dogs

Foram investigadas coinfecções por Leishmania (L.) chagasi, Trypanosoma evansi, Toxoplasma gondii e Neospora caninum em cães. Formas amastigotas de Leishmania spp. foram detectadas pela análise citopatológica de linfonodos em 46.42% (39/84) dos cães. Em um cão macho, adulto, sem raça definida, proveniente de área rural e positivo para Leishmania, foram observadas formas flageladas de T. evansi em esfregaço sanguíneo. Pela imunofluorescência indireta (RIFI), 5.95% (5/84) dos cães foram considerados reagentes para T. gondii, com titulação igual a 64, enquanto que 3.57% (3/84) foram reagentes para N. caninum, com título 50. Entre os animais com leishmaniose visceral, um apresentou resposta sorológica positiva para T. gondii e dois para N. caninum. Todos os cães reagentes para N. caninum eram de área rural e, o predomínio da infecção pelo T. gondii ocorreu em cães da área urbana. Um cão macho, jovem, da zona rural e soropositivo para T. gondii, apresentou mórulas de Ehrlichia spp. na citologia e reação positiva para o vírus da cinomose. Deste modo, mais estudos são necessários para avaliar a epidemiologia dessas infecções na população canina, principalmente com relação aos reservatórios de Trypanosoma spp. nas zonas rurais.

Co-infections by Leishmania (L.) chagasi, Trypanosoma evansi, Toxoplasma gondii and Neospora caninum in dogs were investigated. Amastigotes forms of Leishmania spp. were detected by cytopathological analysis of lymph nodes in 46,42% (39/84) of dogs. In a male dog, adult, without defined breed, from rural area and positive for Leishmania, were observed flagellated forms of T. evansi in blood smear. By immunofluorescence antibody test, 5,95% (5/84) of dogs were considered reactive to T. gondii, with titer equal to or higher than 1:64, while 3,57% (3/84) were reactive to N. caninum, with titer ≥1:50. Among the animals with visceral leishmaniasis, one showed positive serological response to T. gondii and two for N. caninum. All dogs reactive to N. caninum were from rural area and the predominance of infection by T. gondii was in dogs from urban area. A young male dog from the rural area and seropositive for T. gondii showed Ehrlichia spp. morulae in the cytology and positive reaction for canine distemper virus. Thus, further studies are needed to assess the epidemiology of these infections in canine population, especially with respect to the reservoirs of Trypanosoma spp. in rural areas.

DETECTION OF CO-INFECTIONS BY Leishmania (l.) chagasi, Trypanosoma evansi, Toxoplasma gondii AND Neospora caninum IN DOGS / Detecção de co-infecções por Leishmania (l.) chagasi, Trypanosoma evansi, Toxoplasma gondii e Neospora caninum em cães

Foram investigadas coinfecções por Leishmania (L.) chagasi, Trypanosoma evansi, Toxoplasma gondii e Neospora caninum em cães. Formas amastigotas de Leishmania spp. foram detectadas pela análise citopatológica de linfonodos em 46.42% (39/84) dos cães. Em um cão macho, adulto, sem raça definida, proveniente de área rural e positivo para Leishmania, foram observadas formas flageladas de T. evansi em esfregaço sanguíneo. Pela imunofluorescência indireta (RIFI), 5.95% (5/84) dos cães foram considerados reagentes para T. gondii, com titulação igual a 64, enquanto que 3.57% (3/84) foram reagentes para N. caninum, com título 50. Entre os animais com leishmaniose visceral, um apresentou resposta sorológica positiva para T. gondii e dois para N. caninum. Todos os cães reagentes para N. caninum eram de área rural e, o predomínio da infecção pelo T. gondii ocorreu em cães da área urbana. Um cão macho, jovem, da zona rural e soropositivo para T. gondii, apresentou mórulas de Ehrlichia spp. na citologia e reação positiva para o vírus da cinomose. Deste modo, mais estudos s&at

DETECTION OF CO-INFECTIONS BY Leishmania (l.) chagasi, Trypanosoma evansi, Toxoplasma gondii AND Neospora caninum IN DOGS / Detecção de co-infecções por Leishmania (l.) chagasi, Trypanosoma evansi, Toxoplasma gondii e Neospora caninum em cães.

Foram investigadas coinfecções por Leishmania (L.) chagasi, Trypanosoma evansi, Toxoplasma gondii e Neospora caninum em cães. Formas amastigotas de Leishmania spp. foram detectadas pela análise citopatológica de linfonodos em 46.42% (39/84) dos cães. Em um cão macho, adulto, sem raça definida, proveniente de área rural e positivo para Leishmania, foram observadas formas flageladas de T. evansi em esfregaço sanguíneo. Pela imunofluorescência indireta (RIFI), 5.95% (5/84) dos cães foram considerados reagentes para T. gondii, com titulação igual a 64, enquanto que 3.57% (3/84) foram reagentes para N. caninum, com título 50. Entre os animais com leishmaniose visceral, um apresentou resposta sorológica positiva para T. gondii e dois para N. caninum. Todos os cães reagentes para N. caninum eram de área rural e, o predomínio da infecção pelo T. gondii ocorreu em cães da área urbana. Um cão macho, jovem, da zona rural e soropositivo para T. gondii, apresentou mórulas de Ehrlichia spp. na citologia e reação positiva para o vírus da cinomose. Deste modo, mais estudos s&at

Cervical cytological abnormalities and factors associated with high-grade squamous intraepithelial lesions among HIV-infected women from Rio de Janeiro, Brazil.

Although cervical cancer remains a major public health problem in Brazil, knowledge of cervical cytological abnormalities among HIV-infected women remains scarce. At baseline evaluation of a cohort followed in Rio de Janeiro, Brazil, 703 HIV-infected women underwent cytology-based cervical cancer screening and human papillomavirus (HPV) DNA testing. Poisson regression analysis was used to evaluate the association of factors with the presence of high-grade squamous intraepithelial lesions (HSIL). Cervical cytology was abnormal in 24.3% of the women; 4.1% had HSIL. Beyond HPV infection, factors independently associated with the presence of HSIL was age (≥25 and ≤40 years, prevalence ratio [PR] 2.60, 95% confidence interval [CI] 1.11-6.10), and more than three pregnancies was protective (PR 0.33, 95% CI 0.11-0.94). High coverage of cervical cancer screening is warranted to prevent morbidity and mortality from cervical cancer in this population.

Effect of one year of cryopreservation on the activity of lysosomal hydrolases from EBV-transformed lymphocytes.

BACKGROUND: The Epstein-Barr virus (EBV) was used as an agent of B lymphocyte proliferation for subsequent diagnosis of lysosomal storage disease. Due to the constant handling of long-preserved samples in our cell bank, we decided to observe the behavior and then compare cultured and frozen samples for at least one year's cryopreservation. METHODS: Twenty-five samples from healthy individuals were used to assess the possible changes in activity of enzymes ß-galactosidase, ß-glucosidase, α-iduronidase, α-galactosidase, and α-glucosidase. Transmission electron microscopy was used to confirm cell transformation of B lymphocytes into EBV-infected cells, generating lymphoblastoid cell lines. RESULTS: Transmission electron microscopy findings confirmed previous reports in the literature that is, significant and evident morphological changes in the nucleus occur after day 12 and the consequent cell transformation into EBV-infected cells. After thawing and subsequent treatment with the five enzymes utilized, we observed no significant changes in samples cryopreserved for more than one year, as compared to samples cultured for 12 days.

The effect of Mycoplasma and mycoplasma removal agent on the hydrolase activity in fibroblasts of patients with lysosomal diseases.

This study was designed to evaluate the effect of mycoplasma contamination on acid hydrolase activity and the action of the mycoplasma removal agent (MRA), in cultures of human fibroblasts from individuals with lysosomal diseases. For this purpose, we measured the activity of the b-galactosidase, arylsulphatase B (ASB), hexosaminidase A and a-glucosidase enzymes. The activity of the above mentioned enzymes in fibroblasts contaminated by mycoplasma was measured before and after the addition of the MRA. The results were then compared to the enzymatic activity in contamination-free cultures. Only the ASB enzyme showed significant alteration in activity both in the presence of mycoplasma and MRA. The remaining enzymes did not suffer significant interference by the presence of the two agents. Of the four enzymes tested, three did not suffer significant alterations by the presence of the mycoplasma nor from the MRA. However, the activity measured in the ASB enzyme increased significantly in the presence of mycoplasma and MRA and could lead to a doubtful diagnosis. Therefore, we suggest that contamination should be prevented by using aseptic techniques as well as the MRA in those fibroblast cultures that cannot be discarded.

Feasibility of using cryopreserved lymphoblastoid cells to diagnose some lysosomal storage diseases.

OBJECTIVE: The Epstein-Barr virus (EBV) is utilized as a tool in the study of cellular biology because of its capacity to transform B-lymphocytes. For this reason, EBV is used in conservation of human B-lymphocytes for long periods for subsequent evaluation of lysosomal hydrolase activity. Lymphoblastoid cell lines have several advantages for use over other cell types, such as prompt availability and possibility to develop, characterize and standardize cell banks, to test effects of promising pharmaceutical reagents. The study below presents biochemical data that demonstrate validity of lymphoblastoid cell lines for diagnosis of GM1-gangliosidosis, Gaucher, Fabry and Pompe diseases and mucopolysaccharidosis type I. MATERIALS AND METHODS: Cultures were prepared from peripheral blood, collected from 25 normal subjects and 13 affected individuals. Enzyme activities and immunohistochemistry (IHC) were measured. Activities of enzymes beta-galactosidase, beta-glucosidase, alpha-iduronidase, alpha-galactosidase and alpha-glucosidase were measured before and after cryopreservation for 180 days. Enzymatic activity was measured when transformation was confirmed by IHC. RESULTS: We observed some significant alterations in enzymatic activity of non-cultured cells when compared to others that had been cultured for 12 days and kept frozen for 180 days. CONCLUSIONS: However, these alterations did not invalidate use of the technology of transformation of lymphoblastoid cell lines with EBV, to diagnose the diseases mentioned above, in view of the fact that the cultured cells, before and after freezing, demonstrated similar enzymatic activities.

Screening filamentous tropical fungi for their nutritional potential as sources of crude proteins, lipids and minerals.

The use of fungal and yeast biomass in foodstuff, either as supplements or as major ingredients in formulations is an area of growing interest for the modern food industry. The aim of this study was to evaluate the nutritional potential of biomasses obtained from filamentous fungi Penicillium sclerotiorum, Penicillium janthinellum, Rhizopus stolonifer and Syncephalastrum racemosum. Biomasses presented 26-37% of total proteins, 1.7-3.5% of lipids and 4.6-9.1% of ashes. The humidity level reached 75-83%. Ashes were screened for minerals contents with a special outcome for S. racemosum biomass that presented 3438 mg/100 g (dw) of magnesium. Fatty acids present in the biomasses were screened and the palmitic (C16:0), estearic (C18:0), elaidic (18:1n9-t), oleic (18:1n9-c), linolelaidic (C18:2n6-t), linoleic (C18:2n6-c) and γ-linolenic (C18:3n6) acids were found to be the most abundant, from a total of 88-90% of identified fatty acids. Overall data indicate that the filamentous fungi studied have good nutritional properties, possessing a combination of good level of proteins, low level of fat, and presence of essential fatty acids, including omega-3 derivatives, along with the presence of Ca, Mg, Zn and Fe.

Acute hepatitis due to dengue virus in a chronic hepatitis patient

We present a case of acute hepatitis caused by dengue virus, with a significant increase in aspartate transferase and alanine transferase levels in a chronic hepatitis patient attended at the Cane Sugar Planters Hospital of Campos dos Goytacazes, RJ.

Apolipoprotein CIII polymorphism and triglyceride levels of a Japanese population living in Southern Brazil.

Apolipoprotein CIII (apo-CIII) participates in the regulation of triglyceride-rich lipoprotein metabolism. Several polymorphic sites have been detected within and around the apo-CIII gene. Here, we examined the relationship between apo-CIII SstI polymorphism (CC, CG, GG genotypes) and plasma triglyceride (TG) levels in a group of 159 Japanese individuals living in Southern Brazil. The sample was divided into a group of Japanese descendants (N = 51) with high TG (HTG; >200 mg/dL) and a group of Japanese descendants (N = 108) with normal TG (NTG; <200 mg/dL). TG and total cholesterol levels were analyzed by an enzymatic method using the Labtest-Diagnostic kit and high- and low-density lipoproteins by a direct method using the Labtest-Diagnostic kit and DiaSys Diagnostic System International kit, respectively. A 428-bp sequence of apo-CIII gene was amplified using oligonucleotide primers 5' GGT GAC CGA TGG CTT CAG TTC CCT GA 3' and 5' CAG AAG GTG GAT AGA GCG CTG GCC T 3'. The PCR products were digested with a restriction endonuclease SstI. Rare G allele was highly prevalent in our study population (0.416) compared to Caucasians (0.00-0.11). G allele was almost two times more prevalent in the HTG group compared to the NTG group (P < 0.001). The genotype distribution was consistent with the Hardy-Weinberg equilibrium. There was a significant association between rare G allele and HTG in Japanese individuals living in Southern Brazil as indicated by one-way ANOVA, P < 0.05.

SOCS in situ expression in tuberculous lymphadenitis in an endemic area.

BACKGROUND: The immune response to Mycobacterium tuberculosis is complex and multifactorial, the cytokine system being a major factor in M. tuberculosis immunity. AIM: To analyze the immunohistochemical aspects of tuberculous lymph nodes in immunocompetent patients and search for associations between SOCS and cytokine expression in human tuberculous lymphadenitis. METHODS: Thirteen lymph nodes were assayed by immunohistochemistry for SOCS-1 and 3, STAT-3, RANTES, MIP-1-alpha, ICAM-1, IFN-gamma as well as CD45RO, CD20, CD34, CD68, trypsin and lysozyme. Additionally, the RT in situ PCR was performed for SOCS-1 and 3 mRNA detection. RESULTS: Decreased MIP-1 alpha expression together with reduced SOCS-3 (p=0.042), lysozyme (p=0.024) and CD45RO (p=0.05) was observed in the TB lymph nodes compared to the control lymph nodes. In conclusion, the lymphadenitis due to M. tuberculosis was associated with a downregulation of memory T cells (CD45RO), activated lysozymes and SOCS-3 compared to controls, which may play a role in the long-term bacterial replication and altered immune modulation characteristic of the disease.

Apolipoprotein CIII polymorphism and triglyceride levels of a Japanese population living in Southern Brazil

Apolipoprotein CIII (apo-CIII) participates in the regulation of triglyceride-rich lipoprotein metabolism. Several polymorphic sites have been detected within and around the apo-CIII gene. Here, we examined the relationship between apo-CIII SstI polymorphism (CC, CG, GG genotypes) and plasma triglyceride (TG) levels in a group of 159 Japanese individuals living in Southern Brazil. The sample was divided into a group of Japanese descendants (N = 51) with high TG (HTG; >200 mg/dL) and a group of Japanese descendants (N = 108) with normal TG (NTG; <200 mg/dL). TG and total cholesterol levels were analyzed by an enzymatic method using the Labtest-Diagnostic kit and high- and low-density lipoproteins by a direct method using the Labtest-Diagnostic kit and DiaSys Diagnostic System International kit, respectively. A 428-bp sequence of apo-CIII gene was amplified using oligonucleotide primers 5' GGT GAC CGA TGG CTT CAG TTC CCT GA 3' and 5' CAG AAG GTG GAT AGA GCG CTG GCC T 3'. The PCR products were digested with a restriction endonuclease SstI. Rare G allele was highly prevalent in our study population (0.416) compared to Caucasians (0.00-0.11). G allele was almost two times more prevalent in the HTG group compared to the NTG group (P < 0.001). The genotype distribution was consistent with the Hardy-Weinberg equilibrium. There was a significant association between rare G allele and HTG in Japanese individuals living in Southern Brazil as indicated by one-way ANOVA, P < 0.05.

Transient high-level expression of beta-galactosidase after transfection of fibroblasts from GM1 gangliosidosis patients with plasmid DNA.

GM1 gangliosidosis is an autosomal recessive disorder caused by the deficiency of lysosomal acid hydrolase beta-galactosidase (beta-Gal). It is one of the most frequent lysosomal storage disorders in Brazil, with an estimated frequency of 1:17,000. The enzyme is secreted and can be captured by deficient cells and targeted to the lysosomes. There is no effective treatment for GM1 gangliosidosis. To determine the efficiency of an expression vector for correcting the genetic defect of GM1 gangliosidosis, we tested transfer of the beta-Gal gene (Glb1) to fibroblasts in culture using liposomes. Beta-Gal cDNA was cloned into the expression vectors pSCTOP and pREP9. Transfection was performed using 4 microL lipofectamine 2000 and 1.5-2.0 microg DNA. Cells (2 x 10(5)/well) were harvested 24 h, 48 h, and 7 days after transfection. Enzyme specific activity was measured in cell lysate and supernatant by fluorometric assay. Twenty-four hours after transfection, treated cells showed a higher enzyme specific activity (pREP9-beta-Gal: 621.5 +/- 323.0, pSCTOP-beta-Gal: 714.5 +/- 349.5, pREP9-beta-Gal + pSCTOP-beta-Gal: 1859.0 +/- 182.4, and pREP9-ss-Gal + pTRACER: 979.5 +/- 254.9 nmol x h-1 x mg-1 protein) compared to untreated cells (18.0 +/- 3.1 for cell and 32.2 +/- 22.2 nmol x h-1 x mg-1 protein for supernatant). However, cells maintained in culture for 7 days showed values similar to those of untreated patients. In the present study, we were able to transfect primary patients' skin fibroblasts in culture using a non-viral vector which overexpresses the beta-Gal gene for 24 h. This is the first attempt to correct fibroblasts from patients with GM1 gangliosidosis by gene therapy using a non-viral vector.

Transient high-level expression of ß-galactosidase after transfection of fibroblasts from GM1 gangliosidosis patients with plasmid DNA

GM1 gangliosidosis is an autosomal recessive disorder caused by the deficiency of lysosomal acid hydrolase ß-galactosidase (ß-Gal). It is one of the most frequent lysosomal storage disorders in Brazil, with an estimated frequency of 1:17,000. The enzyme is secreted and can be captured by deficient cells and targeted to the lysosomes. There is no effective treatment for GM1 gangliosidosis. To determine the efficiency of an expression vector for correcting the genetic defect of GM1 gangliosidosis, we tested transfer of the ß-Gal gene (Glb1) to fibroblasts in culture using liposomes. ß-Gal cDNA was cloned into the expression vectors pSCTOP and pREP9. Transfection was performed using 4 µL lipofectamine 2000 and 1.5-2.0 µg DNA. Cells (2 x 10(5)/well) were harvested 24 h, 48 h, and 7 days after transfection. Enzyme specific activity was measured in cell lysate and supernatant by fluorometric assay. Twenty-four hours after transfection, treated cells showed a higher enzyme specific activity (pREP9-ß-Gal: 621.5 ± 323.0, pSCTOP-ß-Gal: 714.5 ± 349.5, pREP9-ß-Gal + pSCTOP-ß-Gal: 1859.0 ± 182.4, and pREP9-ß-Gal + pTRACER: 979.5 ± 254.9 nmol·h-1·mg-1 protein) compared to untreated cells (18.0 ± 3.1 for cell and 32.2 ± 22.2 nmol·h-1·mg-1 protein for supernatant). However, cells maintained in culture for 7 days showed values similar to those of untreated patients. In the present study, we were able to transfect primary patients' skin fibroblasts in culture using a non-viral vector which overexpresses the ß-Gal gene for 24 h. This is the first attempt to correct fibroblasts from patients with GM1 gangliosidosis by gene therapy using a non-viral vector.