Microencapsulation of Renealmia alpinia (Rottb.) Maas pulp pigment and antioxidant compounds by spray-drying and its incorporation in yogurt.

Renealmia alpinia (Rottb.) Maas pulp was processed by spray drying using Maltodextrin (MDX), and Gum Arabic (GA), and the mixture of both encapsulating agents (MDX-GA). Yield, moisture, water activity (a w ), apparent and bulk densities, size and morphology of capsules, color, and antioxidant potential (antioxidant activity, total carotenoids, and phenolic compounds) were analyzed. The encapsulates were incorporated as pigments in yogurt and the stability of antioxidant compounds (1, 7, 14, 21, and 28 days of storage) and the sensory properties were evaluated. The yields of all formulations (MDX, GA, MDX-GA) were around 17.86% with low moisture and a w range values (2.62-3.29% and 0.276-0.309, respectively). The microcapsules presented multiples particle sizes (0.67-27.13 µm) with irregular and smooth surfaces. Furthermore, these capsulates preserved yellow color and the retention of carotenoids was significantly higher with MDX (34.12 mg/100 g of powder), while the phenolic compounds and antioxidant activity increased with GA (474.17 mg GAE/100 g and 552.63 mg TE/100 g of powder, respectively). The main compounds ß-carotene and gallic acid were identified and quantified in positive and negative mode respectively using LC-MS/MS. Finally, the addition of the encapsulated pigments to yogurt allowed to obtain a yellow coloration and the yogurt added with MDX-GA presented the best formulation with not significant changes in antioxidant activity and acceptable sensory attributes up 28 days of storage.

In vivo assessment of specific cytotoxic T lymphocyte killing.

The direct killing of target cells by cytotoxic T lymphocytes (CTLs) plays a fundamental role in protective immunity to viral, bacterial, protozoan and fungi infections, as well as to tumor cells. In vivo cytotoxic assays take into account the interaction of target and effector cells in the context of the proper microenvironment making the analysis biologically more relevant than in vitro cytotoxic assays. Thus, the development, improvement and validation of in vivo methods are necessary in view of the importance of the results they may provide. We describe and discuss in this manuscript a method to evaluate in vivo specific cytotoxic T lymphocyte killing. We used as model system mice immunized with human recombinant replication-deficient adenovirus 5 (HAd5) containing different transgenes as the trigger of a CTL-mediated immune response. To these mice, we adoptively transferred syngeneic cells labeled with different vital fluorescent dyes. Donor cells were pulsed (target) or not (control non-target) with distinct CD8 T-cell epitopes, mixed in a 1:1 ratio and injected i.v. into immunized or non-immunized recipient mice. After 18-24h, spleen cells are collected and analysed by flow cytometry. A deviation from the 1:1 ratio of control and target cell populations indicates antigen specific lysis of target cells.

The inhibitor of the redox activity of APE1/REF-1, APX2009, reduces the malignant phenotype of breast cancer cells.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/REF-1) is a multifunctional protein acting on cellular signaling pathways, including DNA repair and redox activities. APE1/REF-1 has emerged as a target for cancer therapy, and its role in breast cancer models would reveal new strategies for cancer therapy. APX2009 is a specific APE1/REF-1 redox inhibitor whose anticancer properties have not been described in breast cancer cells. Here, we investigated the effect of the APX2009 treatment in the breast cancer cell lines MDA-MB-231 and MCF-7. Breast cancer cell lines were cultured, and WST1 and colony formation assays were performed to evaluate cell proliferation. Annexin V-FITC/7-AAD and LDH-Glo™ assays were performed to evaluate cell death. The wound healing assay and Matrigel transwell assay were performed after APX2009 treatment to evaluate the cellular migration and invasion processes, respectively. Our findings demonstrated that APX2009 treatment decreased breast cancer cell proliferative, migratory, and invasive properties. Furthermore, it induced apoptosis in both cell lines. Our study is the first to show the effects of APX2009 treatment on apoptosis in a breast cancer cell. Therefore, this study suggested that APX2009 treatment is a promising anticancer molecule for breast cancer.

Frequency of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis and Ureaplasma species in cervical samples.

We investigated the relative frequencies of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis and Ureaplasma sp. in cervical samples. PCR analyses were performed in ectocervical and endocervical samples from 224 patients attending public health services in Belo Horizonte and Contagem, Minas Gerais Brazil. A high prevalence of colonisation of the cervix (6.3% for C. trachomatis, 4.0% for N. gonorrhoeae, 0.9% for M. genitalium, 21.9% for M. hominis, 38.4% for Ureaplasma sp.) was demonstrated not only for pathogens classically associated to cervicitis (C. trachomatis and N. gonorrhoeae), but also for M. hominis and Ureaplasma sp. These findings may be useful to guide more adequate diagnosis to interrupt transmission and to avoid negative impacts on the female reproductive tract.

Evaluation of attractive toxic sugar baits (ATSB) against Aedes aegypti (Diptera: Culicidae) in laboratory.

Toxic baits, widely used against insect pests, are being successfully used to control mosquito vectors. In the present study, basic aspects for Attractive Toxic Sugar Baits (ATSBs) use as a control tool against Aedes aegypti including insecticide dosage, bait composition and plant application under laboratory conditions were evaluated. The Lethal Concentrations (LC 50 and 90) of boric acid (insecticide) Ae. aegypti engorgement and mortality were determined using ATSBs prepared using fruits (guava, mango and cupuaçu) and offered to mosquitoes on cotton discs and also sprayed on a Kalanchoe blossfeldiana plant. LCs of Ae. aegypti males and females did not differ significantly and varied from 0.53 to 2.46%, decreasing from 24 to 48 hours. No significant difference in the proportion of engorged male mosquitoes in ATSB (0.60) and Attractive Sugar Bait (ASB) (0.65) was found, but females engorged more on ASB (control bait) (0.80) compared to ATSBs (0.67). General mortality rate of mosquitoes in ATSB and ASB were 0.81 and 0.10 for males, respectively; 0.61 and 0.12 for females, respectively. Fruit composition affected neither engorgement nor mortality. ATSB applied on plants caused the mortality of males and females ranging from 0.75-0.87 while mortality on ASB sprayed plants varied from 0.07-0.14. Different common fruit juices and a low toxic oral insecticide are readily accepted, engorged and causes a high mortality both males and females Ae. aegypti using ATSBs. Moreover, the use of a common indoor plant in the region sprayed with ATSB under laboratory conditions leads to significant mosquito mortality.

The inhibitor of the redox activity of APE1/REF-1, APX2009, reduces the malignant phenotype of breast cancer cells

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/REF-1) is a multifunctional protein acting on cellular signaling pathways, including DNA repair and redox activities. APE1/REF-1 has emerged as a target for cancer therapy, and its role in breast cancer models would reveal new strategies for cancer therapy. APX2009 is a specific APE1/REF-1 redox inhibitor whose anticancer properties have not been described in breast cancer cells. Here, we investigated the effect of the APX2009 treatment in the breast cancer cell lines MDA-MB-231 and MCF-7. Breast cancer cell lines were cultured, and WST1 and colony formation assays were performed to evaluate cell proliferation. Annexin V-FITC/7-AAD and LDH-Glo™ assays were performed to evaluate cell death. The wound healing assay and Matrigel transwell assay were performed after APX2009 treatment to evaluate the cellular migration and invasion processes, respectively. Our findings demonstrated that APX2009 treatment decreased breast cancer cell proliferative, migratory, and invasive properties. Furthermore, it induced apoptosis in both cell lines. Our study is the first to show the effects of APX2009 treatment on apoptosis in a breast cancer cell. Therefore, this study suggested that APX2009 treatment is a promising anticancer molecule for breast cancer.

Congenital diaphragmatic hernia - the neonatal period (part I).

AIM: The aim of the study was to review our experience in the management of newborns with congenital diaphragmatic hernia (CDH). METHODS: A retrospective study including all infants with CDH at the Hospital de São João, a center that does not provide ECMO support, for the period from 1997 to 2006. Since 2003, a new treatment protocol has been used. RESULTS: There were 61 newborns (30 male/31 female) with a birth weight of 2800 g (880 - 3770), and a gestational age of 38 weeks (28 - 41); 46 (75 %) were inborn and 42 (69 %) had a prenatal diagnosis of CDH. There were 2 (3 %) chromosomal anomalies, 3 (5 %) with other congenital anomalies and 1 (2 %) with nonimmune hydrops fetalis. The diaphragmatic defect was left sided in 55 (90 %) cases. Corrective surgery was performed in 43 (70 %) patients. New therapies were used: HFOV 13 % (n = 8); inhaled nitric oxide 13 % (n = 8); and sildenafil 7 % (n = 4). We found that systemic arterial hypotension (p = 0.001), the severity of pulmonary hypertension (p = 0.001), prenatal diagnosis (p = 0.006), birth weight (p = 0.022), female gender (p = 0.029), inborn birth (p = 0.030), arterial pH < 7.35 at admission (p = 0.030), right-sided defect (p = 0.033) and pneumothorax (p = 0.033) to be predictive of mortality. The overall survival rate was 43 % (n = 26), and since 2003 this rate has improved to 61 % for term neonates without other congenital or chromosomal anomalies. CONCLUSIONS: Our survival rate for infants with CDH has improved over the last ten years, and this improvement is associated with the use of new therapies such as HFOV, inhaled nitric oxide and sildenafil.

Congenital Diaphragmatic Hernia. The Post-neonatal period. Part II.

AIM: The aim of the study was to review our experience in the management of newborns with congenital diaphragmatic hernia (CDH). METHODS: A retrospective study including all infants with CDH at the Hospital de São João, a center that does not provide ECMO support, for the period from 1997 to 2006. Since 2003, a new treatment protocol has been used. RESULTS: There were 61 newborns (30 male/31 female) with a birth weight of 2800 g (880 - 3770), and a gestational age of 38 weeks (28 - 41); 46 (75 %) were inborn and 42 (69 %) had a prenatal diagnosis of CDH. There were 2 (3 %) chromosomal anomalies, 3 (5 %) with other congenital anomalies and 1 (2 %) with nonimmune hydrops fetalis. The diaphragmatic defect was left sided in 55 (90 %) cases. Corrective surgery was performed in 43 (70 %) patients. New therapies were used: HFOV 13 % (n = 8); inhaled nitric oxide 13 % (n = 8); and sildenafil 7 % (n = 4). We found that systemic arterial hypotension (p = 0.001), the severity of pulmonary hypertension (p = 0.001), prenatal diagnosis (p = 0.006), birth weight (p = 0.022), female gender (p = 0.029), inborn birth (p = 0.030), arterial pH < 7.35 at admission (p = 0.030), right-sided defect (p = 0.033) and pneumothorax (p = 0.033) to be predictive of mortality. The overall survival rate was 43 % (n = 26), and since 2003 this rate has improved to 61 % for term neonates without other congenital or chromosomal anomalies. CONCLUSIONS: Our survival rate for infants with CDH has improved over the last ten years, and this improvement is associated with the use of new therapies such as HFOV, inhaled nitric oxide and sildenafil.

Cyclosporin a. Inhibition of experimental autoimmune uveitis in Lewis rats.

Cyclosporin A (CS-A), a selective inhibitor of T lymphocytes, is reported here to prevent S antigen (S-Ag) induced uveitis in Lewis rats. The S-Ag, found in all mammalian retinas, is uveitogenic under experimental conditions and patients with certain uveitic entities demonstrate cell mediated responses to this antigen. Daily treatment with CS-A (10 mg/kg) begun on the same day as S-Ag immunization totally inhibited the development of the uveitis in this experimental autoimmune model. Moreover a greater CS-A dose (40 mg/kg) efficiently prevented the disease process when therapy was started 7 d after S-Ag immunization. Anti-S-Ag antibody titers were observed to be similar in rats either protected or not protected with CS-A. Our data support strongly the need for T cell participation in this disease model. Since ocular inflammatory disease is an important cause of visual impairment, the data further suggest that CS-A may be useful in the treatment of patients with intractable uveitis.

Nasal airway evaluation in obstructive sleep apnoea patients: volumetric tomography and endoscopic findings.

Obstructive sleep apnoea (OSA) results from the recurrent collapse of the upper airway during sleep. Nasal abnormalities influence the stability of the pharynx. The aim of this study was to evaluate the volumetric and anatomical changes of the nasal cavity in patients with OSA. The Nasal Obstruction Symptom Evaluation (NOSE) scale was used to grade nasal obstruction. Sleep-related breathing disorders were evaluated by polysomnography. The nasal airway volume was obtained from computed tomography scans through volumetric reconstruction of the nasal airway. Alterations to the nasal anatomy were identified by nasal fibre-optic endoscopy. Ninety-four patient charts were analyzed. The final sample comprised 32 patients with severe OSA, 16 with moderate OSA, 23 with mild OSA, and 20 without OSA. Three groups were established based on nasal obstruction and OSA. The groups were compared for nasal airway volume (P=0.464) and body mass index (P=0.001). The presence of nasal septum deviation and inferior turbinate hypertrophy were related to the NOSE score (P=0.05 for both), apnoea-hypopnoea index (P=0.03 and P=0.05, respectively), and nasal airway volume (P=0.71 and P=0.78, respectively). In this nasal airway evaluation of OSA patients, the presence of sites of obstruction was correlated with the severity of OSA; this was not the case for the evaluation of the nasal airway volume dimensions.

The non-palindromic adaptor-PCR method for the identification of the T-cell receptor genes of an interferon-gamma-secreting T-cell hybridomaspecific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen.

Cloning of the T-cell receptor genes is a critical step when generating T-cell receptor transgenic mice. Because T-cell receptor molecules are clonotypical, isolation of their genes requires reverse transcriptase-assisted PCR using primers specific for each different Valpha or Vbeta genes or by the screening of cDNA libraries generated from RNA obtained from each individual T-cell clone. Although feasible, these approaches are laborious and costly. The aim of the present study was to test the application of the non-palindromic adaptor-PCR method as an alternative to isolate the genes encoding the T-cell receptor of an antigen-specific T-cell hybridoma. For this purpose, we established hybridomas specific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen. These T-cell hybridomas were characterized with regard to their ability to secrete interferon-gamma, IL-4, and IL-10 after stimulation with the antigen. A CD3+, CD4+, CD8- interferon-gamma-producing hybridoma was selected for the identification of the variable regions of the T-cell receptor by the non-palindromic adaptor-PCR method. Using this methodology, we were able to rapidly and efficiently determine the variable regions of both T-cell receptor chains. The results obtained by the non-palindromic adaptor-PCR method were confirmed by the isolation and sequencing of the complete cDNA genes and by the recognition with a specific antibody against the T-cell receptor variable beta chain. We conclude that the non-palindromic adaptor-PCR method can be a valuable tool for the identification of the T-cell receptor transcripts of T-cell hybridomas and may facilitate the generation of T-cell receptor transgenic mice.

Quantification of antigen specific CD8+ T cells using an ELISPOT assay.

An ELISPOT assay to detect and determine the number of antigen specific CD8+ T cells was standardized using cloned murine CD8+ T cells specific for the epitope SYVPSAEQI of a rodent malaria antigen. This assay is based on the detection of IFN-gamma secretion by single cells after their stimulation with antigen. The interferon secretion is visualized as spots revealed by using enzyme labeled anti-IFN-gamma monoclonal antibodies. Using known numbers of cloned murine CD8+ T cells it was determined that the assay detects 80-95% of these CD8+ T cells. The optimal culture conditions for the stimulation of the CD8+ T cells were determined and the antigen concentration, number of antigen presenting cells and supplement of growth factors required to perform the assay were defined. This ELISPOT assay can be performed with spleen cells from immunized mice, and provide the precise number of antigen specific CD8+ T cells present in mixed lymphocyte populations. This method is more sensitive than the chromium-51 release assay, and much simpler than the conventional precursor frequency analysis, providing the number of antigen specific CD8+ T cells in 36-48 h.

Change in epithelial keratin expression during healing of rabbit corneal wounds.

Corneal epithelial wound healing following full-thickness trephination and transcorneal freeze injury was studied by electron microscopy and immunofluorescent microscopy using monoclonal antibodies AE1, AE2, and AE3 to human epithelial keratin. Wounds were evaluated at various time intervals between 4 hr and 2 mo after injury. By scanning and transmission electron microscopy, epithelial migration was evident 4 hr after injury and was characterized by thinning of the epithelium and extension of filopodial processes. AE1 monoclonal antibody, which stains specifically the superficial cells of normal corneal epithelium, reacted to cells at the leading edge of the migrating epithelium. By 24 hr, all cells migrating over the wound displayed positive fluorescence with AE1 while the epithelium over the undamaged cornea exhibited normal fluorescence limited to the superficial epithelial cells. In full-thickness corneal wounds, reepithelialization was complete by 1-2 wk; however, all epithelial cells covering the wound remained positive for the AE1 antikeratin antibody. By 2 mo, the AE1 fluorescence returned to normal. In transcorneal freeze injuries, reepithelialization was complete by 4 to 7 days after injury, with all cells overlying the wound reacting with the AE1 antibody. By 2 wk after freeze injury, all epithelial cells appeared to express a normal AE1 staining pattern. No change was noted in the fluorescent distribution of either AE2 antibody, which did not react with the corneal epithelium, or AE3, which reacts with all corneal epithelial cells. These results suggest that healing of corneal epithelial wounds involves changes in keratin expression of the corneal epithelium.

Corpora amylacea of the optic nerve and retina: a form of neuronal degeneration.

One hundred autopsy eyes were examined by light microscopy. Corpora amylacea (CA) occurred in 93% of the cases. Histochemical stains showed that these deposits are composed of sulfated polysaccharides. The fine structure of CA showed delicate, straight 6 to 7 nm thick filaments best demonstrated with the Thiery stain. In three cases, electron-dense vesicles resembling presynaptic vesicles were noted. These structures are characteristic of axons rather than of glial cells and could be related to protein synthesis. CA were present within myelinated and unmyelinated nerves and probably represent products of axonal degeneration.

Langerhans cells in the normal conjunctiva and peripheral cornea of selected species.

The distribution of Langerhans cells (LCs) in human corneal and conjunctival epithelial sheets was investigated by histochemical, immunofluorescence, and immuno-electron microscopic methods. The LCs stained positive with ATPase and with antibodies to HLA-DR antigen and were negative to DOPA-oxidase. Human conjunctiva showed 250 to 300 LCs/mm2 compared to 15 to 20/mm2 in the peripheral third of the corneal epithelium, approximately similar of LCs were present in Lewis rat, fewer cells in guinea pigs and mice, and no detectable cells in the chick.

Alterations in stromal glycoconjugates in macular corneal dystrophy.

Nine biotinylated lectins were used as histochemical probes to localize the carbohydrates residues of glycoconjugates in normal corneas and in corneas with macular and granular dystrophy. The lectin binding patterns of normal corneas and of corneas with granular dystrophy were indistinguishable from one another, but were distinctly different from those found in corneas with macular dystrophy. Concanavalin A reacted weakly with normal corneal stromal matrix, but stained stromal matrix of corneas with macular dystrophy intensely. Furthermore, unlike the normal corneal matrix, stromal matrix of corneas with macular dystrophy reacted positively with wheat germ agglutinin (WGA), Ricinus communis agglutinin I (RCA-I), Ulex europeus I, Dolichos biflorus, Bandeiraea simplicifolia I, Bandeiraea simplicifolia II, and soybean and peanut lectins. This study demonstrates specific alterations in glycoconjugates which occur in the corneal matrix of patients with macular dystrophy, namely the presence of oligosaccharides with terminal alpha-fucose, beta-galactose, N-acetylglucosamine and N-acetylgalactosamine residues, and oligosaccharide chains with a beta-galactose-N-acetylgalactosamine sequence.

Long-term effects of Q-switched ruby laser on monkey anterior chamber angle.

In an attempt to clarify whether pulsed lasers might be able to cause permanent fistulas from the anterior chamber to the interior of the canal of Schlemm, slightly suprathreshold, low energy, small diameter Q-switched ruby laser pulses were applied to the trabecular meshwork of nine eyes of six rhesus monkeys. Clinical examinations during the next 2 months disclosed no adverse effect on the cornea, iris, lens, or retina. There was transient mild inflammation in five eyes. Intraocular pressure was not changed significantly; the facility determined by perfusion of the anterior chambers at 2 months was normal. Light microscopy and scanning electron microscopy show localized trabecular lesions; some are slightly indented, but there is no persistent penetration to Schlemm's canal. Endothelial cells, confluent with those of the cornea, cover the inner (anterior chamber) surface of the lesions. Cross-sections through the center of a lesion show that trabecular meshwork and Schlemm's canal have been obliterated by the treatment and healing; these changes are similar to those previously seen after argon laser monkey trabecular treatment. In the untreated areas, between pulsed laser application sites, trabecular meshwork and Schlemm's canal are normal by light microscopic and scanning electron microscopic examination. Any effect on IOP from this particular type of pulsed laser treatment of the trabecular meshwork is probably not due to trabeculopuncture and flow of fluid through the fistula.

Epithelialization of the corneal endothelium in posterior polymorphous dystrophy.

The unusual cell type present on the posterior corneal surface of posterior polymorphous dystrophy patients has been characterized. In addition to microvilli and desmosomes, these cells contain abundant 10 nm filaments which by immunofluorescent staining were shown to consist of keratin proteins, a marker for epithelial cells.

Demonstration of biogenic amines and adrenergic innervation in uveal malignant melanomas by the histoflurometric method.

Clinical and histologic studies were performed on 25 patients with malignant melanomas of the ciliary body and choroid. Portions of fresh tumor were quick frozen and processed by the histofluorometric technique to demonstrate the presence of biogenic amines. Separate portions of each tumor were fixed and processed for routine light microscopy. Specific fluorescence was visible in 21 of 23 pigmented neoplasms. Catecholamine-induced fluorescence of biogenic amines was related to tumor cell type. In two amelanotic tumors no specific fluorescence was seen.

Altered antigenicity of keratan sulfate proteoglycan in selected corneal diseases.

Monoclonal antibody against keratan sulfate (KS) was used for immunofluorescent staining of sections of human corneas from 8 normal eyes, 19 with keratoconus, 4 with pellucid marginal degeneration, 5 with primary macular corneal dystrophy, and 1 with recurrent macular corneal dystrophy. The anti-KS monoclonal antibody did not stain the corneas with primary macular corneal dystrophy, but stained all other corneas to varying degrees. Staining intensity was weaker than normal in most keratoconus and pellucid marginal degeneration corneas, and was very weak in a case of macular corneal dystrophy that had recurred in a transplanted normal cornea. In several corneas with keratoconus, normal staining was seen at the periphery, and staining intensity decreased in the thinned central portion of the stroma. The decreased KS staining was not localized in stromal scar tissue found in the keratoconus and pellucid marginal degeneration corneas. Quantitation of relative staining intensity found keratoconus and pellucid marginal degeneration corneas to be 49% and 40% as intensely stained, respectively, as normal corneas, a statistically significant decrease (P less than 0.01). Distribution of staining intensities of the keratoconus corneas demonstrated a single modality. These results are in agreement with findings of previous biochemical studies, which show reduction of highly sulfated keratan sulfate epitopes in corneas from keratoconus and pellucid marginal degeneration, and absence of sulfated keratan sulfate epitopes in macular corneal dystrophy.