Evaluation of the stability of treatment with Herbst appliance associated with Hyrax expander and fixed orthodontics through counterpart analysis: A longitudinal follow-up study.

OBJECTIVE: The aim of this study was to evaluate the stability of treatment with a Herbst appliance associated with Hyrax expander (Stage I), followed by fixed appliances (Stage II) and follow-up for an average of 4 years after Stage II, on dentoskeletal facial structures. METHODS: This study involved 50 adolescents with Angle Class II division 1 malocclusion associated with mandibular retrognathism: Treated Group (TG-25) and Control Group (CG-25). Lateral cephalometric radiographs were taken: T1, immediately before Stage I (TG) or at the beginning of the follow-up period (CG); T2, at the end of Stage I (TG) or the follow-up period (CG); T3, at the end of Stage II (TG); and T4, on average, 4 years after Stage II (TG). Enlow's counterpart analysis and some cephalometric measurements were evaluated. Parametric and non-parametric tests were used (P ≤ 0.05). RESULTS: The ramus alignment variables (P < 0.001), SNB (0.040), ANB (<0.001), 1.PP (P = 0.015), 1.MP (P < 0.001), ms/RLp (P < 0.001), mi/RLp (P < 0.001) and S-LS (P = 0.005) showed differences between TG and CG from T1 to T2. Longitudinally, there were differences in ramus alignment, P = 0.003, T1 > T2 < T3 = T4; SNB, P = 0.016, T1 < T2 = T3 = T4; ANB, P < 0.001, T1 > T2 = T3 = T4; 1.MP, P < 0.001, T1 < T2 = T3 = T4; ms/RLp, P = 0.002, T1 = T2 < T3 = T4; mi/RLp, P < 0.001, T1 < T2 = T3 = T4; S-LS, P < 0.001, T1 > T2 = T3 = T4 and S-LI, P = 0.003, T1 = T2 = T3 > T4. CONCLUSION: The nasomaxillary complex (MCF/PM alignment) tended to a retrusive effect to compensate the degree of mandibular retrusion. The protrusive effect of the lower facial third was evident after the Herbst stage and did not remain stable in the follow-up. The dentoalveolar compensation and improvement in facial profile remained stable.

A novel desmoplakin mutation causes dilated cardiomyopathy with palmoplantar keratoderma as an early clinical sign.

BACKGROUND: PPKs represent a heterogeneous group of disorders with hyperkeratosis of palmar and/or plantar skin. PPK, hair shaft abnormalities, cardiomyopathy and arrhythmias can be caused by mutations in desmosomal genes, e.g. desmoplakin (DSP). PPK should trigger genetic testing to reveal mutations with possible related cardiac disease. OBJECTIVES: To report a large multigenerational family with a novel DSP mutation associated with early-onset PPK and adult-onset cardiomyopathy and arrhythmias. METHODS: A custom-designed in-house panel of 35 PPK related genes was used to screen mutations in the index patient with focal PPK. The identified DSP mutation was verified by Sanger sequencing. DNA samples from 20 members of the large multigenerational family were sequenced for the DSP mutation. Medical records were reviewed. Clinical dermatological evaluation was performed, including light microscopy of hair samples. Cardiac evaluation included clinical examination, echocardiography, cardiac magnetic resonance imaging (CMR), electrocardiogram (ECG), Holter monitoring and laboratory tests. RESULTS: We identified a novel autosomal dominant truncating DSP c.2493delA p.(Glu831Aspfs*33) mutation associated with dilated cardiomyopathy (DCM) with arrhythmia susceptibility and focal PPK as an early cutaneous sign. The mutation was found in nine affected family members, but not in any unaffected members. Onset of dermatological findings preceded cardiac symptoms which were variable and occurred at adult age. CONCLUSIONS: We report a novel truncating DSP mutation causing focal PPK with varying severity and left ventricular dilatation and ventricular extrasystoles. This finding emphasizes the importance of genetic diagnosis in patients with PPK for clinical counselling and management of cardiomyopathies and arrhythmias.

Computed microtomography evaluation of calcium hydroxide-based root canal dressing removal from oval root canals by different methods of irrigation.

The aim of this study was to evaluate the capacity of conventional irrigation, passive ultrasonic irrigation (PUI), and Easy Clean for removing calcium hydroxide-based root canal dressing from oval root canals. Thirty mandibular uniradicular incisors with oval canals were used, and subjected to chemical-mechanical preparation with Reciproc R40 instruments. The main canal was filled with a paste based on Ca(OH)2 P.A., iodoform and propylene glycol in the ratio of 3:1:1. The teeth were stored in 100% humidity at a temperature of 37°C for 14 days. Afterwards, the teeth were divided into three groups (n = 10) according to the method of irrigation used (conventional irrigation, PUI, and Easy Clean). The specimens were analyzed by computed microtomography at three time intervals: before placing the root canal dressing, with the root canal dressing in place, and after application of the irrigation methods for removing it. The data were submitted to Kruskal-Wallis and Dunn tests for analyzing the canal as a whole, and Friedman and Dunn for analyzing the root thirds. The results showed that conventional irrigation was less efficient for removing the root canal dressing in comparison with the methods that agitated the irrigant solution (p < .05). When the root canal was analyzed as a whole, Easy Clean, and PUI were similar (p > .05). In analysis of the thirds, Easy Clean was more efficient than conventional irrigation in all the thirds, while PUI showed this behavior only in the cervical third (p < .05). The authors concluded that in oval canals, none of the irrigation methods were capable of removing all the root canal dressing, however, the methods that agitated the irrigant solution were more efficient than conventional irrigation.

Leishmania (Viannia) braziliensis Inositol Phosphorylceramide: Distinctive Sphingoid Base Composition.

Inositol phosphorylceramide (IPC), the major sphingolipid in the genus Leishmania but not found in mammals, is considered a potentially useful target for chemotherapy against leishmaniasis. Leishmania (Viannia) braziliensis is endemic in Latin America and causes American tegumentary leishmaniasis. We demonstrated that IPCs are localized internally in parasites, using a specific monoclonal antibody. Treatment with 5 µM myriocin (a serine palmitoyltransferase inhibitor) rendered promastigotes 8-fold less infective than controls in experimental hamster infection, as determined by number of parasites per inguinal lymph node after 8 weeks infection, suggesting the importance of parasite IPC or sphingolipid derivatives in parasite infectivity or survival in the host. IPC was isolated from promastigotes of three L. (V.) braziliensis strains and analyzed by positive- and negative-ion ESI-MS. The major IPC ions were characterized as eicosasphinganine and eicosasphingosine. Negative-ion ESI-MS revealed IPC ion species at m/z 778.6 (d20:1/14:0), 780.6 (d20:0/14:0), 796.6 (t20:0/14:0), 806.6 (d20:1/16:0), and 808.6 (d20:0/16:0). IPCs isolated from L. (V.) braziliensis and L. (L.) major showed significant differences in IPC ceramide composition. The major IPC ion from L. (L.) major, detected in negative-ion ESI-MS at m/z 780.6, was composed of ceramide d16:1/18:0. Our results suggest that sphingosine synthase (also known as serine palmitoyltransferase; SPT) in L. (V.) braziliensis is responsible for synthesis of a long-chain base of 20 carbons (d20), whereas SPT in L. (L.) major synthesizes a 16-carbon long-chain base (d16). A phylogenetic tree based on SPT proteins was constructed by analysis of sequence homologies in species of the Leishmania and Viannia subgenera. Results indicate that SPT gene position in L. (V.) braziliensis is completely separated from that of members of subgenus Leishmania, including L. (L.) major, L. (L.) infantum, and L. (L.) mexicana. Our findings clearly demonstrate sphingoid base differences between L. (V.) braziliensis and members of subgenus Leishmania, and are relevant to future development of more effective targeted anti-leishmaniasis drugs.

Magnetic resonance imaging in the evaluation of clinical treatment of otospongiosis: a pilot study.

OBJECTIVES: To evaluate the applicability of magnetic resonance imaging (MRI) as a method for monitoring the activity of otospongiotic lesions before and after clinical treatment. STUDY DESIGN: Prospective, randomized, controlled, double-blind study. SETTING: One single tertiary care institution in a large, cosmopolitan city. METHODS: Twenty-six patients (n = 42 ears) with clinical, audiometric, and tomographic diagnosis of otosclerosis were enrolled. If computed tomography (CT) demonstrated active lesions, these patients underwent MRI to detect otospongiotic foci, seen as areas of gadolinium enhancement. Patients were divided into 3 groups and received treatment with placebo, sodium alendronate, or sodium fluoride for 6 months. After this period, clinical and audiometric evaluations and a second MRI were performed. Each MRI was evaluated by both a neuroradiologist and an otolaryngologist in a subjective (visual) and objective (using specific eFilm Workstation software) manner. RESULTS: Otospongiosis was most predominantly identified in the region anterior to the oval window, and this site was reliable for comparing pre- and posttreatment scans. The patients in the alendronate and sodium fluoride groups had MRI findings that suggested a decrease in activity of otospongiotic lesions, more relevant in the alendronate group. These findings were statistically significant for both subjective and objective MRI evaluations. CONCLUSIONS: MRI shows higher sensitivity than clinical or audiometric assessment for detecting reduction in activity of otospongiosis. The objective MRI evaluation based on software analysis was the most accurate method of monitoring clinical treatment response in otospongiosis.

Structural diversity and biological significance of glycosphingolipids in pathogenic and opportunistic fungi.

Glycosphingolipids (GSLs) are ubiquitous membrane components and have key roles in biological systems, acting as second messengers or modulators of signal transduction by affecting several events, ranging from cell adhesion, cell growth, cell motility, regulation of apoptosis and cell cycle. Over the last 20 years our laboratory and other research groups determined the glycan and ceramide structures of more than 20 GSLs from several pathogenic/opportunistic fungi, using a combination of gas chromatography, mass spectrometry, nuclear magnetic resonance as well as other immunochemical and biochemical techniques. Fungal GSLs can be divided in two major classes: neutral GSLs, galactosyl- and glucosylceramide (GlcCer), and acidic GSLs, the glycosylinositol-phosphorylceramides (GIPCs). Glycosyl structures in fungal GIPCs exhibited significant structural diversity and distinct composition when compared to mammalian GSLs, e.g., the expression of inositol-mannose and inositol-glucosamine cores and the terminal residue of ß-D-galactofuranose which are absent in mammalian cells. Studies performed by our group demonstrated that GIPC (Galfß 6[Manα3]Manα2InsPCer) elicited in patients with paracoccidioidomycosis an immune response with production of antibodies directed to the terminal residue of ß-D-galactofuranose. Further studies also showed that inhibition of GlcCer biosynthetic pathways affects fungal colony formation, spore germination and hyphal growth, indicating that enzymes involved in GlcCer biosynthesis may represent promising targets for the therapy of fungal infections. Recently, it was shown that GlcCer and GIPCs are preferentially localized in membrane microdomains and monoclonal antibodies directed to these GSLs interfere in several fungal biological processes such as growth and morphological transition. This review focuses on glycan structures carried on sphingolipids of pathogenic/opportunistic fungi, and aspects of their biological significance are discussed.

Glycolipid sensing and innate immunity in paracoccidioidomycosis.

Distinct glycolipid profiles are described in microorganisms, which have been shown to modulate the innate immune system. We tested the hypothesis that glycosphingolipids from Paracoccidioides brasiliensis have immunomodulatory properties on monocytes and dendritic cells of two groups of healthy individuals, one cured of paracoccidioidomycosis in the past (CUR-I) and the other nonexposed to P. brasiliensis (HNE-I). Two classes of glycosphingolipids purified from yeast cells were evaluated: a neutral glycosphingolipid, monohexosylceramide (CMH), and acidic glycosylinositolphosphorylceramides (GIPCs). Both glycosphingolipids affected the functioning of innate immunity cells, interfering with the antigen presenting process: P. brasiliensis yeast cells phagocytosis, IL-10 secretion, and costimulatory molecules and recognition receptors expression by monocytes were altered, while dendritic cell antigen presentation to autologous T cells was markedly down-modulated as shown by reduced T-cell proliferative responses. The mechanisms by which CMH and GIPCs exert their effects differ since the target cells did not always respond similarly to the challenge with the glycosphingolipids. Moreover, CUR-I and HNE-I presented different responses to the glycosphingolipids. Differences not only in the glycosphingolipid structure (such as the polar head group or the ceramide moiety), but also in the innate immunity properties of CUR-I and HNE-I, may underlie these differences and contribute to individual's susceptibility or resistance to develop paracoccidioidomycosis.

Morphological changes of condyles and Helkimo clinical dysfunction index in patients treated with Herbst--orthodontic appliance.

This study evaluated the morphological changes in the temporomandibular joint (TMJ) condyles and calculated the Helkimo clinical dysfunction index (CDI) in adolescents with Class II Division 1 malocclusion and mandibular retrognathism treated with the Herbst appliance (phase I) and fixed orthodontic appliances (phase II). Thirty-two consecutive adolescents underwent phase I, and 23 completed phase II. The TMJs were evaluated qualitatively using magnetic resonance imaging (MRI) at the beginning of treatment (T1), during phase I (T2), at the end of phase I (T3) and at the end of phase II (T4). The CDI was calculated at T1, T3 and T4. From T1 to T3 (p=0.326), there were no changes in condyle morphology in 86.0% of the TMJs. From T3 to T4 (p<0.05) and T1 to T4 (p<0.05), changes occurred in 39.1% and 43.4% of the condyles. No significant changes in CDI occurred from T1 to T3, T3 to T4 and T1 to T4 (p=1.000; 86.6%, 76.2% and 76.2% concordance). After phase I, there were practically no changes in condyle morphology. At the end of phase II, a mild flattening was observed in some condyles. It may be concluded that no significant changes occurred in CDI after both treatment phases.

Morphological Changes of Condyles and Helkimo Clinical Dysfunction Index in Patients Treated with Herbst - Orthodontic Appliance

This study evaluated the morphological changes in the temporomandibular joint (TMJ) condyles and calculated the Helkimo clinical dysfunction index (CDI) in adolescents with Class II Division 1 malocclusion and mandibular retrognathism treated with the Herbst appliance (phase I) and fixed orthodontic appliances (phase II). Thirty-two consecutive adolescents underwent phase I, and 23 completed phase II. The TMJs were evaluated qualitatively using magnetic resonance imaging (MRI) at the beginning of treatment (T1), during phase I (T2), at the end of phase I (T3) and at the end of phase II (T4). The CDI was calculated at T1, T3 and T4. From T1 to T3 (p=0.326), there were no changes in condyle morphology in 86.0% of the TMJs. From T3 to T4 (p<0.05) and T1 to T4 (p<0.05), changes occurred in 39.1% and 43.4% of the condyles. No significant changes in CDI occurred from T1 to T3, T3 to T4 and T1 to T4 (p=1.000; 86.6%, 76.2% and 76.2% concordance). After phase I, there were practically no changes in condyle morphology. At the end of phase II, a mild flattening was observed in some condyles. It may be concluded that no significant changes occurred in CDI after both treatment phases.

Este estudo avaliou as mudanças morfológicas nas cabeças da mandíbula das articulações temporo mandibulares (ATMs) e calculou o índex de disfunção clínico de Helkimo (IDC) em adolescentes com má oclusão de Classe II Divisão1 e retrognatismo mandibular, tratados com aparelho de Herbst (fase I) e aparelho ortodôntico fixo (fase II). Trinta e dois adolescentes consecutivos passaram pela fase I e 23 completaram a fase II. As ATMs foram avaliadas qualitativamente por meio de imagem da resonância magnética (IRM) ao início do tratamento (T1), durante a fase I (T2), no final da fase I (T3) e no final da fase II (T4). O IDC foi calculado em T1, T3 e T4. De T1 a T3 (p=0,326) não ocorreram mudanças na morfologia da cabeça da mandíbula em 86,0% das ATMs. De T3 a T4 (p<0,05) e T1 a T4 (p<0,05) ocorreram mudanças em 39,1% e 43,4% das cabeças das mandíbulas. Não ocorreram mudanças significantes no IDC de T1 a T3, T3 a T4 e T1 a T4 (p=1,000; 86,6%, 76,2% e 76,2% concordância). Após a fase I, não houve praticamente mudanças na morfologia da cabeça da mandíbula. Ao final da fase II, um leve aplainamento foi observado em algumas cabeças das mandíbulas. Pode ser concluído que não ocorreram mudanças significantes no IDC após ambas as fases de tratamento.

Myriocin, a serine palmitoyltransferase inhibitor, blocks cytokinesis in Leishmania (Viannia) braziliensis promastigotes.

We studied the effect of myriocin, an inhibitor of serine palmitoyltransferase, on cultured Leishmania (Viannia) braziliensis promastigotes. Myriocin significantly reduced synthesis of inositol phosphorylceramide, the major sphingolipid expressed in promastigotes as characterized by thin layer chromatography and electrospray ionization mass spectrometry. Log-phase promastigotes treated with 1 µM myriocin showed a 52% reduction in growth rate and morphological alterations such as more rounded shape and shorter flagellum. Promastigotes treated with myriocin also displayed a variety of aberrant cell phenotypes. The percentage of cells with one nucleus and one kinetoplast (1N1K), following treatment with 1 or 5 µM myriocin, decreased from 89% (control value) to 27% or 3%, respectively. The percentage of cells with two nuclei (2N2K) varied from 7% (control value) to 19% and 6% for 1 or 5 µM myriocin-treated parasites, respectively. High percentage of myriocin-treated parasites exhibited large atypical cells presenting three or more nucleus (32% and 89% for 1 or 5 µM myriocin, respectively). Transmission electron microscopy following treatment with 1 µM myriocin showed the presence of 4N parasites possibly as a result of an incomplete cytokinesis. Addition of 3-ketodihidrosphingosine to myriocin-treated promastigotes rescue parasite growth and morphology. Addition of ethanolamine did not rescue the myriocin effect on parasite. Our findings indicate that sphingolipids are essential for the completion of cytokinesis, and may play a major role in cell proliferation in L. (V.) braziliensis, thus, differing from data described for Leishmania major sphingolipid-free mutant, where addition of ethanolamine rescue wild-type parasite characteristics.

Paracoccidioides brasiliensis induces secretion of IL-6 and IL-8 by lung epithelial cells. Modulation of host cytokine levels by fungal proteases.

Paracoccidioides brasiliensis is a pathogenic, dimorphic fungus that causes paracoccidioidomycosis, a systemic human mycosis that is highly prevalent in Latin America. In this study, we demonstrated that P. brasiliensis yeasts induced interleukin (IL)-8 and IL-6 secretion by human lung epithelial A549 cells. However, tumor necrosis factor-α and interferon-γ were undetectable in these cultures. Moreover, P. brasiliensis yeasts induced activation of p38 mitogen-activated protein kinase (MAPK), c-Jun NH(2)-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in A549 cells, and IL-8 and IL-6 secretion promoted by this fungus was dependent on activation of p38 MAPK and ERK 1/2. In addition, IL-8 and IL-6 levels were significantly higher in culture supernatants of A549 cells that were incubated with formaldehyde-fixed P. brasiliensis compared to cultures of cells that were infected with live yeasts. Our results indicate that the observed cytokine level differences were due to protease expression, in live yeasts, that degraded these cytokines. Degradation of human recombinant IL-8 and IL-6 by live P. brasiliensis was inhibited by AEBSF and aprotinin, suggesting that these proteases belong to a family of serine proteases. This is the first report showing that P. brasiliensis may modulate host inflammation by expressing proteases that degrade proinflammatory cytokines.

The effectiveness of audiometric evaluation in drug treatment for otospongiosis.

UNLABELLED: Otospongiosis is a primary osteodystrophy of the otic capsule that affects genetically predisposed individuals and leads to a progressive hearing loss. AIM: To evaluate the applicability of audiometric evaluation during drug treatment for otospongiosis. MATERIALS AND METHODS: A prospective, randomized, controlled, double-blind study involving 26 patients with clinical, audiometric and CT scan image of otosclerosis. Patients eligible for the study were divided into three groups (A, B and C) and received treatment with alendronate sodium (B), sodium fluoride (C) and placebo (A) for 6 months. After this period they were submitted to new tests. RESULTS: There were not statistically significant differences between air and bone conduction (gap). We also found no differences in the speech recognition threshold (SRT) and speech discrimination (IRF) between before and after treatment. CONCLUSION: After six months of drug treatment the audiometric evaluation kept the same hearing thresholds, suggesting stabilization of the otospongiotic lesions.

Eficácia da avaliação audiométrica no tratamento medicamentoso da otospongiose / The effectiveness of audiometric evaluation in drug treatment for otospongiosis

A otospongiose é uma osteodistrofia focal primária da cápsula ótica que acomete indivíduos geneticamente predispostos e promove perda auditiva progressiva. OBJETIVO: Verificar a aplicabilidade da avaliação audiométrica no tratamento medicamentoso da otospongiose. MATERIAL E MÉTODO: Estudo prospectivo, randomizado, controlado, duplo-cego, envolvendo 26 pacientes com diagnóstico clínico, audiométrico e tomográfico de otospongiose. Os pacientes elegíveis para o estudo foram alocados em três grupos (A, B e C) e receberam o tratamento com alendronato de sódio (B), fluoreto de sódio (C) e placebo (A) por 6 meses. Após este período, os mesmos realizaram nova avaliação audiométrica. RESULTADOS: Na análise das diferenças entre as vias aérea e óssea (gap), não houve diferença estatisticamente significante. Também não foram encontradas diferenças em relação ao limiar de reconhecimento da fala (SRT) e a discriminação vocal (IRF) entre os períodos pré e pós-tratamento. CONCLUSÃO: Após seis meses de tratamento medicamentoso, a avaliação audiométrica evidenciou manutenção dos limiares auditivos, sugerindo estabilização da atividade da lesão otospongiótica.

Otospongiosis is a primary osteodystrophy of the otic capsule that affects genetically predisposed individuals and leads to a progressive hearing loss. AIM: To evaluate the applicability of audiometric evaluation during drug treatment for otospongiosis. MATERIALS AND METHODS: A prospective, randomized, controlled, double-blind study involving 26 patients with clinical, audiometric and CT scan image of otosclerosis. Patients eligible for the study were divided into three groups (A, B and C) and received treatment with alendronate sodium (B), sodium fluoride (C) and placebo (A) for 6 months. After this period they were submitted to new tests. RESULTS: There were not statistically significant differences between air and bone conduction (gap). We also found no differences in the speech recognition threshold (SRT) and speech discrimination (IRF) between before and after treatment. CONCLUSION: After six months of drug treatment the audiometric evaluation kept the same hearing thresholds, suggesting stabilization of the otospongiotic lesions.

Membrane microdomain components of Histoplasma capsulatum yeast forms, and their role in alveolar macrophage infectivity.

Analysis of membrane lipids of Histoplasma capsulatum showed that ~40% of fungal ergosterol is present in membrane microdomain fractions resistant to treatment with non-ionic detergent at 4°C. Specific proteins were also enriched in these fractions, particularly Pma1p a yeast microdomain protein marker (a plasma membrane proton ATPase), a 30kDa laminin-binding protein, and a 50kDa protein recognized by anti-α5-integrin antibody. To better understand the role of ergosterol-dependent microdomains in fungal biology and pathogenicity, H. capsulatum yeast forms were treated with a sterol chelator, methyl-beta-cyclodextrin (mßCD). Removal of ergosterol by mßCD incubation led to disorganization of ergosterol-enriched microdomains containing Pma1p and the 30kDa protein, resulting in displacement of these proteins from detergent-insoluble to -soluble fractions in sucrose density gradient ultracentrifugation. mßCD treatment did not displace/remove the 50kDa α5-integrin-like protein nor had effect on the organization of glycosphingolipids present in the detergent-resistant fractions. Ergosterol-enriched membrane microdomains were also shown to be important for infectivity of alveolar macrophages; after treatment of yeasts with mßCD, macrophage infectivity was reduced by 45%. These findings suggest the existence of two populations of detergent-resistant membrane microdomains in H. capsulatum yeast forms: (i) ergosterol-independent microdomains rich in integrin-like proteins and glycosphingolipids, possibly involved in signal transduction; (ii) ergosterol-enriched microdomains containing Pma1p and the 30kDa laminin-binding protein; ergosterol and/or the 30kDa protein may be involved in macrophage infectivity.

Changes in cell wall synthesis and ultrastructure during paradoxical growth effect of caspofungin on four different Candida species.

Paradoxical growth (PG) has been described for echinocandins and is characterized by cell growth at drug concentrations above the MIC. In this study, two isolates each of Candida albicans, C. tropicalis, C. orthopsilosis, and C. parapsilosis, all of which displaying PG in response to caspofungin, were subjected to MIC, minimal fungicidal concentration (MFC), and time-kill curve assays to evaluate the levels of PG. Cell wall components and ultrastructural modifications of the PG cells were also investigated. The results showed that when cell growth and survival were evaluated by MFC or time-kill curve assays, high concentrations of caspofungin did not show fungicidal activity against PG cells. Furthermore, for C. parapsilosis and C. orthopsilosis, time-kill curves were more discriminatory than MFCs in detecting the PG effect. The four different Candida species studied demonstrated similar alterations in cell wall components and ultrastructure associated with PG. In PG cells, ß-1,3-glucan content decreased from 2.7- to 7.8-fold, whereas chitin content increased from 4.0- to 6.6-fold. An electron microscopy study of the PG cells revealed morphological alterations, clumping of cells, enlarged cells, the absence of filamentation, abnormal septa, and accumulation of chitin in the cell wall. Also, PG cells basically exhibited a single dark high-density layer in the cell wall, indicating the loss of the ß-1,3-glucan layer. Our results present novel details about the ultrastructural alterations that occur in C. albicans, C. parapsilosis, C. orthopsilosis, and C. tropicalis during PG and show that chitin is the major component of the cell walls of PG cells. Stimulation of chitin synthesis may represent a rescue mechanism against caspofungin activity.

Role of host glycosphingolipids on Paracoccidioides brasiliensis adhesion.

Binding of yeast forms to human lung fibroblast cultures was analyzed, aiming to better understand the initial steps of Paracoccidioides brasiliensis infection in humans. A significant P. brasiliensis adhesion was observed either to fibroblasts or to their Triton X-100 insoluble fraction, which contains extracellular matrix and membrane microdomains enriched in glycosphingolipids. Since human lung fibroblasts express at cell-surface gangliosides, such as GM1, GM2, and GM3, the role of these glycosphingolipids on P. brasiliensis adhesion was analyzed by different procedures. Anti-GM3 monoclonal antibody or cholera toxin subunit B (which binds specifically to GM1) reduced significantly fungal adhesion to fibroblast cells, by 35% and 33%, respectively. Direct binding of GM1 to yeast forms of P. brasiliensis was confirmed using cholera toxin subunit B conjugated to AlexaFluor(®)488. It was also demonstrated that P. brasiliensis binds to polystyrene plates coated with galactosylceramide, lactosylceramide, trihexosylceramide, GD3, GM1, GM3, and GD1a, suggesting that glycosphingolipids presenting residues of beta-galactose or neuraminic acid at non-reducing end may act as adhesion molecules for P. brasiliensis. Conversely, no binding was detected when plates were adsorbed with glycosphingolipids that contain terminal residue of beta-N-acetylgalactosamine, such as globoside (Gb4), GM2, and asialo-GM2. In human fibroblast (WI-38 cells), GM3 and GM1 are associated with membrane rafts, which remain insoluble after treatment with Triton X-100 at 4°C. Taken together, these results strongly suggest that lung fibroblast gangliosides, GM3 and GM1, are involved in binding and/or infection by P. brasiliensis.

Phospholipase-D activity and inflammatory response induced by brown spider dermonecrotic toxin: endothelial cell membrane phospholipids as targets for toxicity.

Brown spider dermonecrotic toxins (phospholipases-D) are the most well-characterized biochemical constituents of Loxosceles spp. venom. Recombinant forms are capable of reproducing most cutaneous and systemic manifestations such as dermonecrotic lesions, hematological disorders, and renal failure. There is currently no direct confirmation for a relationship between dermonecrosis and inflammation induced by dermonecrotic toxins and their enzymatic activity. We modified a toxin isoform by site-directed mutagenesis to determine if phospholipase-D activity is directly related to these biological effects. The mutated toxin contains an alanine substitution for a histidine residue at position 12 (in the conserved catalytic domain of Loxosceles intermedia Recombinant Dermonecrotic Toxin - LiRecDT1). LiRecDT1H12A sphingomyelinase activity was drastically reduced, despite the fact that circular dichroism analysis demonstrated similar spectra for both toxin isoforms, confirming that the mutation did not change general secondary structures of the molecule or its stability. Antisera against whole venom and LiRecDT1 showed cross-reactivity to both recombinant toxins by ELISA and immunoblotting. Dermonecrosis was abolished by the mutation, and rabbit skin revealed a decreased inflammatory response to LiRecDT1H12A compared to LiRecDT1. Residual phospholipase activity was observed with increasing concentrations of LiRecDT1H12A by dermonecrosis and fluorometric measurement in vitro. Lipid arrays showed that the mutated toxin has an affinity for the same lipids LiRecDT1, and both toxins were detected on RAEC cell surfaces. Data from in vitro choline release and HPTLC analyses of LiRecDT1-treated purified phospholipids and RAEC membrane detergent-extracts corroborate with the morphological changes. These data suggest a phospholipase-D dependent mechanism of toxicity, which has no substrate specificity and thus utilizes a broad range of bioactive lipids.

Changes in temporomandibular joint disc position and form following Herbst and fixed orthodontic treatment.

OBJECTIVE: To determine the changes in the position and form of the temporomandibular joint articular disc in adolescents with Class II division 1 malocclusion and mandibular retrognathism treated with the Herbst appliance (phase I) and fixed orthodontic appliance (phase II). MATERIALS AND METHODS: Thirty-two consecutive adolescents went through phase I of treatment and 23 completed phase II. The temporomandibular joints were evaluated qualitatively by means of magnetic resonance images at the beginning of treatment (T1), during phase I (T2), at the end of phase I (T3), and at the end of phase II (T4). RESULTS: Significant changes in disc position were not observed with the mouth closed between T1 x T3 (P = .317), T3 x T4 (P = .287), or T1 x T4 (P = .261). At T2, on average, the disc was positioned regressively. With the mouth open, no difference was observed between T1 x T3 (P = .223) or T1 x T4 (P = .082). We did observe a significant difference between T3 x T4 (P < .05). Significant changes in the disc form were found with the mouth closed between T1 x T2 (P < .001) and T2 x T3 (P < .001). CONCLUSIONS: At the end of the two-phase treatment, in general terms, the position and form of the initial articular discs were maintained; however, in some temporomandibular joints some seemingly adverse effects were observed at T4.

Effect of anti-glycosphingolipid monoclonal antibodies in pathogenic fungal growth and differentiation. Characterization of monoclonal antibody MEST-3 directed to Manpalpha1-->3Manpalpha1-->2IPC.

BACKGROUND: Studies carried out during the 1990's demonstrated the presence of fungal glycoinositol phosphorylceramides (GIPCs) with unique structures, some of them showed reactivity with sera of patients with histoplasmosis, paracoccidioidomycosis or aspergillosis. It was also observed that fungal GIPCs were able to inhibit T lymphocyte proliferation "in vitro", and studies regarding the importance of these molecules to fungal survival showed that many species of fungi are vulnerable to inhibitors of sphingolipid biosynthesis. RESULTS: In this paper, we describe a detailed characterization of an IgG2a monoclonal antibody (mAb), termed MEST-3, directed to the Paracoccidioides brasiliensis glycolipid antigen Pb-2 (Manpalpha1-->3Manpalpha1-->2IPC). mAb MEST-3 also recognizes GIPCs bearing the same structure in other fungi. Studies performed on fungal cultures clearly showed the strong inhibitory activity of MEST-3 on differentiation and colony formation of Paracoccidioides brasiliensis, Histoplasma capsulatum and Sporothrix schenckii. Similar inhibitory results were observed when these fungi where incubated with a different mAb, which recognizes GIPCs bearing terminal residues of beta-D-galactofuranose linked to mannose (mAb MEST-1). On the other hand, mAb MEST-2 specifically directed to fungal glucosylceramide (GlcCer) was able to promote only a weak inhibition on fungal differentiation and colony formation. CONCLUSIONS: These results strongly suggest that mAbs directed to specific glycosphingolipids are able to interfere on fungal growth and differentiation. Thus, studies on surface distribution of GIPCs in yeast and mycelium forms of fungi may yield valuable information regarding the relevance of glycosphingolipids in processes of fungal growth, morphological transition and infectivity.