Immunohistochemical and histopathological analyses of cutaneous innervation to improve the diagnostic efficacy in hansen disease skin lesion.

BACKGROUND: The diagnosis of Hansen disease (HD) can be difficult when acid-fast bacilli are not detected in the patient's skin sample. OBJECTIVE: To demonstrate that detailed morphological analysis of nonspecific inflammatory and/or noninflammatory alterations in dermal nerves as well as skin adnexa in leprosy-suspected biopsy samples could improve the efficacy of histopathological diagnosis. METHODS: Patients with one to five skin lesions were enrolled in the study and classified into three groups by skin histopathology findings: Hansen disease (HD, n = 13), other diseases (OD, n = 11), and inconclusive cases (INC, n = 11). We quantified dermal nerve damage via the nerve lesion index (NLI) and PGP9.5-immunoreactive axon quantitative index in dermal nerves (AQI). We also measured inflammatory involvement of adnexa in cutaneous samples as indirect evidence of HD. RESULTS: We observed a higher median endoneurial inflammatory infiltrate NLI (HD = 0.5; INC = 0; OD = 0; p < 0.001) and more frequent inflammatory involvement of skin adnexa in samples of the HD group compared with those of the INC and OD groups (HD = 7; INC = 1; OD = 0). However, samples from the INC and OD groups also showed inflammatory and noninflammatory damage of dermal nerves, with 2 or more kinds of alterations in nerves in the same sample (respectively: INC = in 1 and 2 samples; OD = in 3 and 5 respectively). The quantification of PGP9.5-immunoreactive axons in dermal nerves revealed no difference between the groups. CONCLUSION: A detailed morphological analysis of cutaneous nerves in lesions with a suspicion of HD enabled us to select patients with nonspecific inflammatory or non-inflammatory lesions in the dermal nerves in the INC and OD groups, so they may be clinically monitored aiming at a possible future diagnosis of the disease. These INC and OD patients cannot have the HD diagnosis definitely excluded, and HD may coexist with another disease as a comorbidity.

ANTECEDENTES: A hanseníase pode ter o seu diagnóstico histopatológico dificultado quando bacilos álcool-ácido resistentes não são encontrados nas amostras de pele dos pacientes. OBJETIVO: Demonstrar que uma análise morfológica detalhada de alterações histopatológicas dos nervos dérmicos pode aumentar a eficácia diagnóstica. MéTODOS: Foram selecionadas amostras de pele de pacientes com uma a cinco lesões suspeitas de hanseníase. Os casos selecionados foram classificados conforme achados histopatológicos: hanseníase (HD, n = 13), casos inconclusivos (INC, n = 11), e outras doenças (OD, n = 11). Quantificamos as lesões dos nervos cutâneos por meio do índice de lesão de nervos (nerve lesion index, NLI, em inglês) e do índice quantitativo de axônios (axon quantitative index, AQI, em inglês) imunorreativos a PGP9.5 nos nervos cutâneos. Também medimos o envolvimento inflamatório dos anexos em amostras de pele como evidência indireta de hanseníase. RESULTADOS: Foram observadas no grupo HD medianas mais altas do NLI com relação a infiltrados inflamatórios endoneurais (HD = 0,5; INC = 0; OD = 0; p < 0,001) e mais alta frequência de acometimento inflamatório de anexos cutâneos (HD = 7; INC = 1; OD = 0). Entretanto, as amostras dos grupos INC e OD também mostraram comprometimento inflamatório e não inflamatório dos nervos cutâneos, com 2 ou mais tipos de alterações de nervos na mesma amostra (respectivamente: INC = 1 e 2; OD = 3 e 5). Não houve diferença significativa na quantidade de axônios endoneurais imunorreativos a PGP9.5 entre os grupos. CONCLUSãO: A análise morfológica detalhada dos nervos cutâneos em lesões suspeitas de hanseníase permitiu selecionar pacientes com lesões inespecíficas inflamatórias ou não inflamatórias nos nervos dérmicos nos grupos INC e OD, para que sejam monitorados clinicamente visando um possível diagnóstico futuro da doença. Esses pacientes INC e OD não podem ter o diagnóstico de HD definitivamente excluído, e a hanseníase pode coexistir com outra doença como uma comorbidade.

Immunohistochemical and histopathological analyses of cutaneous innervation to improve the diagnostic efficacy in hansen disease skin lesion / Análises imuno-histoquímica e histopatológica da inervação cutânea para aumento da eficácia diagnóstica nas lesões de pele da hanseníase

Abstract Background The diagnosis of Hansen disease (HD) can be difficult when acid-fast bacilli are not detected in the patient's skin sample. Objective To demonstrate that detailed morphological analysis of nonspecific inflammatory and/or noninflammatory alterations in dermal nerves as well as skin adnexa in leprosy-suspected biopsy samples could improve the efficacy of histopathological diagnosis. Methods Patients with one to five skin lesions were enrolled in the study and classified into three groups by skin histopathology findings: Hansen disease (HD, n = 13), other diseases (OD, n = 11), and inconclusive cases (INC, n = 11). We quantified dermal nerve damage via the nerve lesion index (NLI) and PGP9.5-immunoreactive axon quantitative index in dermal nerves (AQI). We also measured inflammatory involvement of adnexa in cutaneous samples as indirect evidence of HD. Results We observed a higher median endoneurial inflammatory infiltrate NLI (HD = 0.5; INC = 0; OD = 0; p < 0.001) and more frequent inflammatory involvement of skin adnexa in samples of the HD group compared with those of the INC and OD groups (HD = 7; INC = 1; OD = 0). However, samples from the INC and OD groups also showed inflammatory and noninflammatory damage of dermal nerves, with 2 or more kinds of alterations in nerves in the same sample (respectively: INC = in 1 and 2 samples; OD = in 3 and 5 respectively). The quantification of PGP9.5-immunoreactive axons in dermal nerves revealed no difference between the groups. Conclusion A detailed morphological analysis of cutaneous nerves in lesions with a suspicion of HD enabled us to select patients with nonspecific inflammatory or non-inflammatory lesions in the dermal nerves in the INC and OD groups, so they may be clinically monitored aiming at a possible future diagnosis of the disease. These INC and OD patients cannot have the HD diagnosis definitely excluded, and HD may coexist with another disease as a comorbidity.

Resumo Antecedentes A hanseníase pode ter o seu diagnóstico histopatológico dificultado quando bacilos álcool-ácido resistentes não são encontrados nas amostras de pele dos pacientes. Objetivo Demonstrar que uma análise morfológica detalhada de alterações histopatológicas dos nervos dérmicos pode aumentar a eficácia diagnóstica. Métodos Foram selecionadas amostras de pele de pacientes com uma a cinco lesões suspeitas de hanseníase. Os casos selecionados foram classificados conforme achados histopatológicos: hanseníase (HD, n = 13), casos inconclusivos (INC, n = 11), e outras doenças (OD, n = 11). Quantificamos as lesões dos nervos cutâneos por meio do índice de lesão de nervos (nerve lesion index, NLI, em inglês) e do índice quantitativo de axônios (axon quantitative index, AQI, em inglês) imunorreativos a PGP9.5 nos nervos cutâneos. Também medimos o envolvimento inflamatório dos anexos em amostras de pele como evidência indireta de hanseníase. Resultados Foram observadas no grupo HD medianas mais altas do NLI com relação a infiltrados inflamatórios endoneurais (HD = 0,5; INC = 0; OD = 0; p < 0,001) e mais alta frequência de acometimento inflamatório de anexos cutâneos (HD = 7; INC = 1; OD = 0). Entretanto, as amostras dos grupos INC e OD também mostraram comprometimento inflamatório e não inflamatório dos nervos cutâneos, com 2 ou mais tipos de alterações de nervos na mesma amostra (respectivamente: INC = 1 e 2; OD = 3 e 5). Não houve diferença significativa na quantidade de axônios endoneurais imunorreativos a PGP9.5 entre os grupos. Conclusão A análise morfológica detalhada dos nervos cutâneos em lesões suspeitas de hanseníase permitiu selecionar pacientes com lesões inespecíficas inflamatórias ou não inflamatórias nos nervos dérmicos nos grupos INC e OD, para que sejam monitorados clinicamente visando um possível diagnóstico futuro da doença. Esses pacientes INC e OD não podem ter o diagnóstico de HD definitivamente excluído, e a hanseníase pode coexistir com outra doença como uma comorbidade.

Morphometric analysis of nerve fibers in neural leprosy.

BACKGROUND: The present study assesses the contributions of axonal degeneration and demyelination in leprosy nerve damage. New clinical strategies can emerge from an in-depth understanding of the pathogenesis of neural leprosy (NL). METHODS: Morphometric analysis of myelinated nerve fibers was performed on 44 nerve biopsy samples collected from leprosy patients. Measures of density, diameter distribution, g-ratios, and the counting of axonal ovoids on the myelinated fibers were taken and compared to those in the control group. RESULTS: The proportion of small myelinated fibers increased in the leprosy group while large fiber frequency decreased. Indicative of axonal atrophy, the g-ratio was lower in the leprosy group. The frequency of axonal ovoids was identical to that found in the non-leprosy neuropathies. CONCLUSIONS: Axonal atrophy, Wallerian degeneration, and demyelination coexist in NL. Axonal degeneration predominates over demyelination in the chronic course of the disease; however, this may change during leprosy reactive episodes. This study regards demyelination and axon degeneration as concurrent mechanisms of damage to nerve fibers in leprosy. It also calls into question the view that demyelination is the primary and predominant mechanism in the complex pathogeny of NL.

Fibrosis: a distinguishing feature in the pathology of neural leprosy.

BACKGROUND: Fibrosis in the peripheral nerve is the end stage of leprous neuropathy and the cause of the resulting permanent neural function impairments. Preventive measures to avoid this irreversible pathological state are a relief strategy for leprosy sufferers. OBJECTIVES: The present study describes the frequency of fibrosis along with its characterisation and pathogenic development. METHODS: Six-hundred-and-thirteen nerve samples were sorted from 278 neural leprosy (NL) and 335 non-leprosy neuropathy patients (ON). The total number of samples was histologically examined by routine staining methods (haematoxylin-eosin, Wade staining and Gomori's trichrome) and fibrosis was evaluated via semi-quantitative estimation. FINDINGS: Fibrosis was most frequent in the NL group (33% against 0.4% in ON) while fibrosis in association with endoneurial microfasciculation was found in 38 (41.3%) of the NL samples in the examination of semithin sections. Pericytic activation in the perivascular environment was confirmed to be the source of the fibroblasts and perineurial cells delimiting microfascicles. End-stage fibrosis in leprosy displays an arrangement of microfascicles devoid of neural components (i.e., Schwann cells and axons) lined by an intermediate phenotype of fibroblastic-perineurial cells filled with bundles of collagen fibres. MAIN CONCLUSIONS: The present study underscores that fibrosis is frequently the severe end stage of neural leprosy NL pathogeny after analysing the notably distinct development of fibrosis within the neural environment.

Fibrosis: a distinguishing feature in the pathology of neural leprosy

BACKGROUND Fibrosis in the peripheral nerve is the end stage of leprous neuropathy and the cause of the resulting permanent neural function impairments. Preventive measures to avoid this irreversible pathological state are a relief strategy for leprosy sufferers. OBJECTIVES The present study describes the frequency of fibrosis along with its characterisation and pathogenic development. METHODS Six-hundred-and-thirteen nerve samples were sorted from 278 neural leprosy (NL) and 335 non-leprosy neuropathy patients (ON). The total number of samples was histologically examined by routine staining methods (haematoxylin-eosin, Wade staining and Gomori's trichrome) and fibrosis was evaluated via semi-quantitative estimation. FINDINGS Fibrosis was most frequent in the NL group (33% against 0.4% in ON) while fibrosis in association with endoneurial microfasciculation was found in 38 (41.3%) of the NL samples in the examination of semithin sections. Pericytic activation in the perivascular environment was confirmed to be the source of the fibroblasts and perineurial cells delimiting microfascicles. End-stage fibrosis in leprosy displays an arrangement of microfascicles devoid of neural components (i.e., Schwann cells and axons) lined by an intermediate phenotype of fibroblastic-perineurial cells filled with bundles of collagen fibres. MAIN CONCLUSIONS The present study underscores that fibrosis is frequently the severe end stage of neural leprosy NL pathogeny after analysing the notably distinct development of fibrosis within the neural environment.

The toxic nature of murine amylin and the immune responsivity of pancreatic islet to conformational antibody in mice.

The human amylin is a pancreatic peptide hormone found in hyperhormonemic state along with insulin in subclinical diabetes. Amylin has been associated with the pathology of type 2 diabetes, particularly due to its ability to assembly into toxic oligomers and amyloid specimens. On the other hand, some variants such as murine amylin has been described as non-amyloidogenic, either in vitro or in vivo. Recent data have demonstrated the amyloid propensity of murine amylin and the therapeutic analogue pramlintide, suggesting a universality for amylin amyloidosis. Here, we report the amyloidogenesis of murine amylin, which showed lower responsivity to the fluorescent probe thioflavin T compared to human amylin, but presented highly organized fibrilar amyloid material. The aggregation of murine amylin also resulted in the formation of cytotoxic specimens, as evaluated in vitro in INS-1 cells. The aggregation product from murine amylin was responsive to a specific antibody raised against amyloid oligomers, the A11 oligomer antibody. Pancreatic islets of wild-type Swiss male mice have also shown responsivity for the anti-oligomer, indicating the natural abundance of such specimen in rodents. These data provide for the first time evidences for the toxic nature of oligomeric assemblies of murine amylin and its existence in wild-type, non-transgenic mice.

MALDI imaging reveals lipid changes in the skin of leprosy patients before and after multidrug therapy (MDT).

Leprosy still represents a health problem in several countries. Affecting skin and peripheral nerves, it may lead to permanent disabilities. Disturbances on skin lipid metabolism in leprosy were already observed; however, the localization and distribution of lipids could not be accessed. The role of lipids on infectious disease has been fully addressed only recently, as they directly influence immune response. Matrix-assisted laser desorption/ionization imaging mass spectrometry provides a powerful tool to localize and identify lipids in tissues. The aim of this work was to study and compare the changes in lipid distribution of skin biopsies taken from leprosy patients before and after multidrug therapy (MDT). Different species of phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin and phosphatidylcholine were detected. Differences in skin lipid signal intensities, as well as in their localization, were observed before and after MDT in every patient. In general, lipid distribution in the skin after MDT had a pattern similar to control skin samples, where most of the lipids were located in the upper part of the dermis and epidermis. This study opens paths to a better understanding of lipid functions in leprosy pathogenesis and immune response.

Bone marrow-derived mononuclear cells promote improvement in glomerular function in rats with early diabetic nephropathy.

BACKGROUND/AIMS: Diabetic nephropathy is one of the main causes of end-stage renal disease. The present study investigated the effect of mononuclear cell (MC) therapy in rats subjected to diabetic nephropathy. METHODS: Male Wistar rats were divided into control (CTRL), diabetic (DM), CTRL+MC and DM+MC groups. Diabetes was induced by a single injection of streptozotocin (45 mg/kg, i.p.) and, 4 weeks later, 2×10(7) MCs were injected via the jugular vein. RESULTS: The rats in the DM and DM+MC groups showed increased glycemia, glomerular filtration rate and glomerular tuff area versus control groups. The glomerular filtration rate and glomerular tuff area were normalized in the DM+MC group. No alterations were observed in the fractional excretion of electrolytes and proteinuria between the DM and DM+MC groups. TGF-ß1 protein levels in the DM group were significantly increased versus control animals and normalized in the DM+MC group. An increase in ED1(+)/arginase I(+) macrophages and IL-10 renal expression was observed in the DM+MC group versus DM group. CONCLUSIONS: Bone marrow-derived MC therapy was able to prevent glomerular alterations and TGF-ß1 protein overexpression and modulated glomerular arginase I(+) macrophage infiltration in rats subjected to early diabetic nephropathy.

Transforming growth factor-ß1 may be a key mediator of the fibrogenic properties of neural cells in leprosy.

Fibrosis is the main cause of irreversible nerve damage in leprosy. Phenotypic changes in Mycobacterium leprae (ML)-infected Schwann cells (SCs) have been suggested to mediate this process. We found that SC line cultures stimulated with ML upregulated transforming growth factor-ß1 (TGF-ß1), and that TGF-ß1 or ML induced increased numbers of α-smooth muscle actin (α-SMA)-positive cells with characteristic stress fibers. Mycobacterium leprae and TGF-ß1 also induced increased type I collagen and fibronectin mRNA and secretion and augmented mRNA levels of SOX9 and ZEB1, which are involved in the epithelial-mesenchymal transition. These effects could be inhibited by the TGF-ß1 type I receptor (ALK5) inhibitor, SB-431542. In nerve biopsies from leprosy-infected patients with varying grades of fibrosis (n = 11), type I and III collagen and fibronectin were found in the endoneurium and perineurium, α-SMA-positive cells filled the fibrotic perineurium but not the endoneurium, and CD34-positive fibroblasts predominated in the endoneurium. Results of transcriptional studies of 3 leprosy nerves and 5 controls were consistent with these data, but α-SMA and other mRNA levels were not different from those in the control samples. Our findings suggest that TGF-ß1 may orchestrate events, including reprogramming of the SC phenotype, leading to transdifferentiation, connective tissue cell expansion, and fibrogenesis in the evolution of leprosy nerve lesions during some evolutionary stages.

Differential expression of cruzipain- and gp63-like molecules in the phytoflagellate trypanosomatid Phytomonas serpens induced by exogenous proteins.

Phytomonas serpens synthesizes metallo- and cysteine-proteases that are related to gp63 and cruzipain, respectively, two virulence factors produced by pathogenic trypanosomatids. Here, we described the cellular distribution of gp63- and cruzipain-like molecules in P. serpens through immunocytochemistry and confocal fluorescence microscopy. Both proteases were detected in distinct cellular compartments, presenting co-localization in membrane domains and intracellular regions. Subsequently, we showed that exogenous proteins modulated the production of both protease classes, but in different ways. Regarding the metalloprotease, only fetal bovine serum (FBS) influenced the gp63 expression, reducing its surface exposition (≈30%). Conversely, the cruzipain-like molecule was differentially modulated according to the proteins: human and bovine albumins reduced its expression around 50% and 35%, respectively; mucin and FBS did not alter its production, while IgG and hemoglobin drastically enhanced its surface exposition around 7- and 11-fold, respectively. Additionally, hemoglobin induced an augmentation in the cell-associated cruzipain-like activity in a dose-dependent manner. A twofold increase of the secreted cruzipain-like protein was detected after parasite incubation with 1% hemoglobin compared to the parasites incubated in PBS-glucose. The results showed the ability of P. serpens in modulating the expression and the activity of proteolytic enzymes after exposition to exogenous proteins, with emphasis in its cruzipain-like molecules.

Pulmonary abnormalities in mice with paracoccidioidomycosis: a sequential study comparing high resolution computed tomography and pathologic findings.

BACKGROUND: Human paracoccidioidomycosis (PCM) is an endemic fungal disease of pulmonary origin. Follow-up of pulmonary lesions by image studies in an experimental model of PCM has not been previously attempted. This study focuses on defining patterns, topography and intensity of lung lesions in experimentally infected PCM mice by means of a comparative analysis between High Resolution Computed Tomography (HRCT) and histopathologic parameters. METHODOLOGY: Male BALB/c mice were intranasally inoculated with 3 x 10(6) Paracoccidioides brasiliensis (Pb) conidia (n = 50) or PBS (n = 50). HRCT was done every four weeks to determine pulmonary lesions, quantify lung density, reconstruct and quantify lung air structure. Lungs were also analyzed by histopathology and histomorphometry. RESULTS: Three different patterns of lesions were evidenced by hrct and histopathology, as follows: nodular-diffuse, confluent and pseudo-tumoral. The lesions were mainly located around the hilus and affected more frequently the left lung. At the 4th week post-challenge HRCT showed that 80% of the Pb-infected mice had peri-bronchial consolidations associated with a significant increase in upper lung density when compared with controls, (-263+/-25 vs. -422+/-10 HU, p<0.001). After the 8th and 12th weeks, consolidation had progressed involving also the middle regions. Histopathology revealed that consolidation as assessed by HRCT was equivalent histologically to a confluent granulomatous reaction, while nodules corresponded to individual compact granulomas. At the 16th week of infection, confluent granulomas formed pseudotumoral masses that obstructed large bronchi. Discrete focal fibrosis was visible gradually around granulomas, but this finding was only evident by histopathology. CONCLUSIONS/SIGNIFICANCE: This study demonstrated that conventional HRCT is a useful tool for evaluation and quantification of pulmonary damage occurring in experimental mouse PCM. The experimental design used decreases the need to sacrifice a large number of animals, and serves to monitor treatment efficacy by means of a more rational approach to the study of human lung disease.

Bone marrow mononuclear cells attenuate interstitial fibrosis and stimulate the repair of tubular epithelial cells after unilateral ureteral obstruction.

The growing number of patients suffering from chronic renal disease is a challenge for the development of innovative therapies. Benefits of cell therapy in acute renal diseases in animal models have been reported but seldom for chronic lesions. We present evidence for the improvement of renal morphology in a model of tubulointerstitial fibrosis. Wistar rats were submitted to unilateral ureteral obstruction (UUO), treated with bone-marrow mononuclear cells (UUO+BMMC) infused via the cava vein, and killed on day 14. Labeled BMMC were seen in renal tissue after 7 days in the group UUO+BMMC. UUO+BMMC also showed a reduction in ED1(+) cells and tubular apoptotic cells together with enhanced tubular proliferation. Myofibroblasts were also reduced after BMMC which is consistent with a decrease in collagen deposition (picro Sirius staining) and RT-PCR data showing lower levels of procollagen-I mRNA. Simultaneously, nestin+ cells increased in the interstitium and decreased in the tubules. Double stained nestin(+)/alpha-SMA(+) cells were present only in the interstitium, and their levels did not change after BMMC infusion. These data indicate a renoprotective effect of BMMC through increased tubular cell regeneration, inhibition of tubular cell apoptosis and partially blocking of the inflammatory and fibrotic events that occur after unilateral ureteral obstruction.

Cysteine proteinases from promastigotes of Leishmania (Viannia) braziliensis.

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.

Synthesis and mobilization of glycogen and trehalose in adult male Rhodnius prolixus.

The vector of Chagas' disease, Rhodnius prolixus, feeds exclusively on blood. The blood meals are slowly digested, and these insects wait some weeks before the next meal. During the life of an insect, energy-requiring processes such as moulting, adult gonadal and reproductive growth, vitellogenesis, muscular activity, and fasting, lead to increased metabolism. Carbohydrates are a major source of energy and their mobilization is important. We determined the amounts of glycogen, trehalose, and glucose present in the fat body and/or hemolymph of adult males of R. prolixus and recorded the processes of accumulation and mobilization of these carbohydrates. We also tested our hypothesis that these processes are under endocrine control. The amount of glycogen in the fat body progressively increased until the fourth day after feeding (from 9.3+/-2.2 to 77. 3+/-7.5 microg/fat body), then declined to values around 36.3+/-4.9 microg/fat body on the fifteenth day after the blood meal. Glycogen synthesis was eliminated in decapitated insects and head-transplanted insects synthesized glycogen. The amount of trehalose in the fat body increased until the sixth day after feeding (from 16. 6+/-1.7 to 40. 6+/-5.3 nmol/fat body), decreased abruptly, and stabilized between days 7 and 15 at values ranging around 15-19 nmol/fat body. Decapitated insects did not synthesize trehalose after feeding, and this effect was reversed in head-transplanted insects. The concentration of trehalose in the hemolymph increased after the blood meal until the third day (from 0.07+/-0.01 to 0.75+/-0.05 mM) and at the fourth day it decreased until the ninth day (0.21+/-0.01 mM), when it increased again until the fourteenth day (0.79+/-0.06 mM) after the blood meal, and then declined again. In decapitated insects, trehalose concentrations did not increase soon after the blood meal and at the third day it was very low, but on the fourteenth day it was close to the control values. The concentration of glucose in the hemolymph of untreated insects remained low and constant (0.18+/-0.01 mM) during the 15 days after feeding, but in decapitated insects it progressively increased until the fifteenth day (2.00+/-0.10 mM). We recorded the highest trehalase activity in midgut, which was maximal at the eighth day after feeding (2,830+/-320 nmol of glucose/organ/h). We infer that in Rhodnius prolixus, the metabolism of glycogen, glucose, and trehalose are controlled by factors from the brain, according to physiological demands at different days after the blood meal.

The embryonic development of Schistosoma mansoni eggs: proposal for a new staging system.

Schistosomiasis is a water-borne parasitic illness caused by neoophoran trematodes of the genus Schistosoma. Using classical histological techniques and whole-mount preparations, the present work describes the embryonic development of Schistosoma mansoni eggs in the murine host and compares it with eggs maintained under in vitro conditions. Two pre-embryonic stages occur inside the female worm: the prezygotic stage is characterized by the release of mature oocytes from the female ovary until its fertilization. The zygotic stage encompasses the migration of the zygote through the ootype, where the eggshell is formed, to the uterus. Fully formed eggs are laid still undeveloped, without having suffered any cleavage. In the outside environment, eight embryonic stages can be defined: stage 1 refers to early cleavages and the beginning of yolk fusion. Stage 2 represents late cleavage, with the formation of a stereoblastula and the onset of outer envelope differentiation. Stage 3 is defined by the elongation of the embryonic primordium and the onset of inner envelope formation. At stage 4, the first organ primordia arise. During stages 5 to 7, tissue and organ differentiation occurs (neural mass, epidermis, terebratorium, musculature, and miracidial glands). Stage 7 is characterized by the nuclear condensation of neurons of the central neural mass. Stage 8 refers to the fully formed larva, presenting muscular contraction, cilia, and flame-cell beating. This staging system was compared to a previous classification and could underlie further studies on egg histoproteomics (morphological localizome). The differentiation of embryonic structures and their probable roles in granulomatogenesis are discussed herein.

Immediate dental implant failure associated with nasopalatine duct cyst.

This case report presents an analysis of the clinical, radiographic, and histological features of a peri-implant lesion around an implant placed immediately after extraction of a tooth with a periapical lesion. A 52-year-old man received an immediate implant (3.75 x 11.5 mm2) placed in the anterior region of the maxilla. Three years after implant placement, the patient presented with swelling in the anterior portion of the maxilla. Radiographic examination showed a well-circumscribed radiolucency around the implant. The implant and the lesion were removed and fixed in 10% buffered formalin and processed. Histological analysis showed 3 types of epithelium: respiratory, cuboidal, and non-keratinized stratified squamous. In the cyst wall peripheral nerves, arteries, veins, and chronic inflammation were present. The diagnosis was nasopalatine duct cyst. We concluded that the nasopalatine duct cyst can develop in association with dental implants. Clinically, the lesion is similar to the classical nasopalatine duct cyst. Histological analysis should be mandatory in all cases of peri-implant lesions and in all dental periapical lesions before immediate implant placement.

Trematode embryology: a new method for whole-egg analysis by confocal microscopy.

Trematode worms have the neoophoran mode of development in which several specialized vitelline cells surround the zygote. This vitelline cell mass appears just before the zygote passes through the ootype, a thickening of the oviduct, where the egg shell is formed. The great amount of vitelline material blurs the visualization of embryo development in whole egg seen by brightfield microscopy. The eggshell is difficult to cut into thin or ultrathin sections and acts as a barrier to fixation and infiltration with embedding media. The egg shell is also brightly fluorescent when analyzed by fluorescence microscopy. To overcome these technical disadvantages a simple staining protocol widely used in adult helminth morphological analysis was adapted for the study of the embryonic development of two different trematode species. The effects of potassium hydroxide as bleach and ethylene glycol as mounting medium were also evaluated. Confocal microscopy allowed virtual sectioning of whole-mounted eggs and made possible internal morphological detailed analysis of different embryonic stages. This method could contribute to the study of helminth egg embryology.

Aplicação de substituto de pele em oncologia cutânea: estudo experimental com derme acelular e ceratinócitos cultivados / Application of skin substitutes in skin oncology: experimental study using acellular dermis and cultured keratinocytes

FUNDAMENTOS: As neoplasias malignas da pele de grandes dimensões apresentam dificuldades de reconstrução após a excisão. OBJETIVO: O objetivo deste estudo foi avaliar a exeqüibilidade de uma nova proposta de cobertura para feridas cirúrgicas criadas após a ressecção de grandes tumores cutâneos, a combinação da derme acelular humana com epitélio autólogo cultivado. MÉTODOS: A aplicação dos substitutos de pele foi feita em quatro pacientes com área de implante variando de 33 a 120 cm2. Além da observação dos resultados clínicos, realizou-se estudo morfológico para avaliação da integração dos implantes. RESULTADOS: Ceratinócitos autólogos cultivados foram enxertados em dois pacientes e não demonstraram integração. A derme acelular foi aplicada em quatro pacientes, sendo que em um deles foram feitas duas aplicações. Dos cinco implantes de derme acelular realizados, dois não apresentaram integração, em dois a integração foi de 70 por cento, e de 50 por cento no último. CONCLUSÃO: A cobertura imediata e definitiva de defeitos cirúrgicos através da aplicação de derme acelular humana combinada com epitélio autólogo cultivado é exeqüível. Em oncologia cutânea apenas em situações especiais o uso de substitutos de pele pode ser conveniente no sentido de evitar reconstruções mais complexas.

BACKGROUND: Reconstruction difficulties may arise after excision of large malignant skin neoplasms.a OBJECTIVE: The objective of this study was to assess the feasibility of a new coverage for surgical wounds following resection of large skin tumors: a combination of human acellular dermis with cultured autologous epithelium. METHODS: The skin substitute was implanted in four patients, one of them received two implants and the area ranged from 33 to 120 cm2. Clinical results and morphologic studies were assessed as to implant integration. RESULTS: Cultured autologous epithelium was grafted in two patients and no integration was observed. The acellular dermis was applied to four patients. Out of five acellular dermis implants, two did not present integration, two presented 70 percent integration and the remaining, 50 percent integration. CONCLUSION: The immediate and definite coverage of surgical defects by means of application of human acellular dermis combined with cultured autologous epithelium is feasible. In skin oncology, the use of skin substitutes might be convenient only in special situations to avoid more complex reconstructions.

Carbohydrate accumulation and utilization by oocytes of Rhodnius prolixus.

The processes of accumulation and mobilization of carbohydrate stores in eggs of Rhodnius prolixus were analyzed. During oogenesis, the total amounts of glycogen, glucose, and trehalose increased with an accumulation of proteins, especially when oocytes grew from 1.0 to 1.5 mm in length. At 2.0 mm length, when oocytes were ready for oviposition, nutrient reserves did not increase appreciably and trehalose content decreased. Mating did not affect the final content of carbohydrates or proteins in oocytes of mated and virgin females. A trehalase activity was detected in follicles containing vitellogenic oocytes, 1.0 and 1.5 mm length, in both mated and virgin females. This activity was extremely low in chorionated, 2.0-mm oocytes. After oviposition, glycogen content decreased in fertilized eggs, but not in unfertilized ones, and some was present in newly hatched nymphs. Glucose content remained constant in unfertilized eggs, but increased in fertilized ones, while total protein amount was constant in both groups after egg laying.

Increase in dermal collagen fibril diameter and elastogenesis with UVB exposure: an optical and ultrastructural study in albino Balb/c mice.

Cutaneous aging is a complex biological phenomenon, dependent not only on the innate or intrinsic process ("biological clock"), but also on extrinsic elements, primarily chronic sun exposure (photoaging). In order to verify dermal morphological changes in the elastic fiber system and collagen associated with aged skin, we performed a light and electron microscopic study on exposed-shaved albino mice, which were exposed to UVB radiation. The experimental group consisted of 48 exposed animals, randomly distributed in three groups and submitted to different radiation doses (A, 28800 J/m2; B, 57600 J/m2; and C, 86400 J/m2) and studied 0, 30, 60 and 90 days of exposure discontinuation. Nonexposed-shaved and nonexposed-nonshaved animals were included as controls. From the day of exposure discontinuation and subsequently, the elastic system and collagen network were progressively modified. The increase in collagen fibril diameter was prominent in the 60 and 90 day groups (p<0.05), as noticed on electron microscopy. Elastic fiber density also increased after irradiation (p<0.05). On electron microscopy, elastogenesis was seen in the deep dermis. The comparative study among the groups disclosed clear relationship between doses and "elastotic changes". It also showed that chronological aging of mice skin was apparently intensified after UVB exposure. Skin elastogenesis seems to be a major consequence of UVB exposure, apart from elastolysis, and occurs not only in humans but also in hairless mice submitted to continuous, long-term UVB exposure.