Nutritional value and antioxidant compounds during the ripening and after domestic cooking of bananas and plantains.

Genotypes of bananas and plantains have been studied for biofortification purposes, mainly due to content of resistant starch (RS) and polyphenols. This study aims to identify banana and plantain genotypes with a high content of resistant starch, phenolic compounds and minerals, and to evaluate the impact of the ripening stage and domestic thermal processing to select superior genotypes with high levels of functional compounds. In this study, it was used bunches of bananas and plantain genotypes. The phenolic compounds profiles were determined by HPLC-DAD in pulps and peels. The resistant starch and the minerals (K, Na, Zn, Cu and Fe) were evaluated in pulps and peels of unripe fruit. The results of phenolic compounds were studied in three ripening stages, and after thermal processing (ripe stage) of two genotypes, which were most promising for biofortification studies. Resistant starch and minerals were analysed in the unripe fruits. The peel biomass showed the highest values of phenolic compounds and minerals. The total starch content in the pulp varied from 42.3% ('FC06-02') to 80.6% ('Pelipita'). Plantains and cooking bananas presented the highest contents of starch and resistant starch (stage 2 - green with yellow traces). The pulps of the dessert genotypes 'Khai' and 'Ouro da Mata', and cooking genotype 'Pacha Nadam' stood out due to their minerals high contents (P, K and Fe; Zn and Fe; Ca, Mg and Zn, respectively). The dessert bananas (e.g., 'Ney Poovan') and cooking bananas (e.g., 'Tiparot') had the highest concentrations of phenolic compounds, mainly in ripe fruit (stage 5 - yellow with green). In addition, the thermal processing of Musa spp. fruit led to increasing these secondary metabolites, mainly the cooking of fruit with peel by boiling, which should be preferred in domestic preparations.

Keratoconus: The ABCD Grading System.

PURPOSE: To propose a new keratoconus classification/staging system that utilises current tomographic data and better reflects the anatomical and functional changes seen in keratoconus. METHOD: A previously published normative database was reanalysed to generate both anterior and posterior average radii of curvature (ARC and PRC) taken from a 3.0 mm optical zone centred on the thinnest point of the cornea. Mean and standard deviations were recorded and anterior data were compared to the existing Amsler-Krumeich (AK) Classification. ARC, PRC, thinnest pachymetry and distance visual acuity were then used to construct a keratoconus classification. RESULTS: 672 eyes of 336 patients were analysed. Anterior and posterior values were 7.65 ± 0.236 mm and 6.26 ± 0.214 mm, respectively, and thinnest pachymetry values were 534.2 ± 30.36 µm. The ARC values were 2.63, 5.47 and 6.44 standard deviations from the mean values of stages 1-3 in the AK classification, respectively. PRC staging uses the same standard deviation gates. The pachymetric values differed by 4.42 and 7.72 standard deviations for stages 2 and 3, respectively. CONCLUSION: A new keratoconus staging incorporates anterior and posterior curvature, thinnest pachymetric values, and distance visual acuity and consists of stages 0-4 (5 stages). The proposed system closely matches the existing AK classification stages 1-4 on anterior curvature. As it incorporates posterior curvature and thickness measurements based on the thinnest point, rather than apical measurements, the new staging system better reflects the anatomical changes seen in keratoconus.

Comparison of subgluteal sciatic nerve block duration in type 2 diabetic and non-diabetic patients.

BACKGROUND: Although animal studies demonstrated delayed recovery after nerve block in laboratory models of diabetes, the duration of the action of sciatic nerve blocks clinically in patients with diabetes remains to be determined. We studied the duration of a sciatic nerve block in type 2 diabetic patients compared with non-diabetic patients. METHODS: We prospectively included consecutive patients aged 50-80 yr, with type 2 diabetes with minor nerve injury (confirmed with 5.07 at 10 g monofilament test, n=23) and non-diabetic patients (n=49) scheduled for distal lower limb surgery. Before surgery, a subgluteal sciatic nerve block (20 ml of ropivacaine 4.75 mg ml(-1)) was performed with an ultrasound approach coupled with nerve stimulation. The primary endpoint was the sensory block duration. RESULTS: There was no significant difference between groups for age, but haemoglobin A1c and creatinine values were significantly higher in the diabetic group. There was no difference in 5.07 (10 g) monofilament testing, but the diabetic group had lower scores for the 0.4 and 0.07 g tests (P<0.01). There was no significant difference in the median onset time for the sensory block (25 vs 25 min, NS), but the median duration of the sensory block (21 vs 17 h, P<0.01) and the motor block (16 vs 12 h, P<0.01) were higher in the diabetic group. No complication occurred in either group. CONCLUSIONS: These findings demonstrate that diabetic patients with pre-existing incipient neuropathy exhibit delayed recovery from the block with ropivacaine, confirming animal studies. Clinical trial registration ClinicalTrials.gov, NCT01704612.

[The IC3D classification of the corneal dystrophies]. / IC3D-Klassifikation von Hornhautdystrophien.

BACKGROUND: The recent availability of genetic analyses has demonstrated the shortcomings of the current phenotypic method of corneal dystrophy classification. Abnormalities in different genes can cause a single phenotype, whereas different defects in a single gene can cause different phenotypes. Some disorders termed corneal dystrophies do not appear to have a genetic basis. PURPOSE: The purpose of this study was to develop a new classification system for corneal dystrophies, integrating up-to-date information on phenotypic description, pathologic examination, and genetic analysis. METHODS: The International Committee for Classification of Corneal Dystrophies (IC3D) was created to devise a current and accurate nomenclature. RESULTS: This anatomic classification continues to organize dystrophies according to the level chiefly affected. Each dystrophy has a template summarizing genetic, clinical, and pathologic information. A category number from 1 through 4 is assigned, reflecting the level of evidence supporting the existence of a given dystrophy. The most defined dystrophies belong to category 1 (a well-defined corneal dystrophy in which a gene has been mapped and identified and specific mutations are known) and the least defined belong to category 4 (a suspected dystrophy where the clinical and genetic evidence is not yet convincing). The nomenclature may be updated over time as new information regarding the dystrophies becomes available. CONCLUSIONS: The IC3D Classification of Corneal Dystrophies is a new classification system that incorporates many aspects of the traditional definitions of corneal dystrophies with new genetic, clinical, and pathologic information. Standardized templates provide key information that includes a level of evidence for there being a corneal dystrophy. The system is user-friendly and upgradeable and can be retrieved on the website www.corneasociety.org/ic3d .

Disease severity and family history in keratoconus.

BACKGROUND/AIMS: To determine if disease severity is associated with a family history of keratoconus. METHODS: Markers of disease severity in the CLEK Study cohort were assessed to determine if they could discriminate individuals with and without family history. Logistic regression was used to examine association between corneal scarring, average corneal power, flat and steep keratometry readings, and higher-order root mean square (RMS) wavefront error with family history. RESULTS: In univariate analyses, none of the severity indices had any significant associations with family history; however, contact lens use, gender, and Caucasian race were found to be significant predictors. After controlling for these confounders, there were no significant associations between any severity indices and family history. CONCLUSIONS: Presence or absence of family history is not associated with more severe clinical disease, at least when each marker for severity is considered independently. The results of this analysis are important for genetic studies of keratoconus in that it will allow recruitment of keratoconus patients across all stages of disease severity because it does not influence familial aggregation.

Identification of Urop11, a novel leptin-modulated gene that is upregulated in the hypothalamus of mice with virus-induced obesity.

Obesity results from disturbances of tightly regulated interactions between the nervous, endocrine, and metabolic systems that can be caused by external factors, such as viral infections. A mouse model of obesity induced by brain infection with a morbillivirus, canine distemper virus, allowed us to identify obesity-related genes. Using a subtractive library for the hypothalamus, the main brain structure regulating energy homeostasis, we identified a new gene on mouse chromosome 19 which we named upregulated obese product (Urop) 11 and, which has no homology with any known mRNA. A step-by-step molecular approach allowed us to isolate the full-length mRNA, predict the protein sequence, and identify consensus sites. Urop11 was mainly detected in the hypothalamus and adipocytes, and was dramatically upregulated in these central and peripheral structures in obese mice. Urop11 was also expressed in human neural and lymphoid samples and its expression seemed to be regulated by the state of lymphocyte activation. Interestingly, Urop11 expression was strongly upregulated both in vivo in mouse hypothalamus and in vitro in mouse neural cell lines, after leptin treatment. Taken together, our data show that Urop11 is a target of leptin, the satiety factor produced by adipocytes, in physiological and pathological conditions, including obesity. This new gene can be considered a key molecule in the hypothalamic integration pathway and demonstrates the importance of Urop11 as a target of leptin action.

Bone marrow CD34+/B220+ progenitors target the inflamed brain and display in vitro differentiation potential toward microglia.

Recent evidence indicates that microglial cells may not derive from blood circulating mature monocytes as they express features of myeloid progenitors. Here, we observed that a subpopulation of microglial cells expressed CD34 and B220 antigens during brain development. We thus hypothesized that microglia, or a subset of microglial cells, originate from blood circulating CD34+/B220+ myeloid progenitors, which could target the brain under developmental or neuroinflammatory conditions. Using experimental allergic encephalomyelitis (EAE) as a model of chronic neuroinflammation, we found that a discrete population of CD34+/B220+ cells expands in both blood and brain of diseased animals. In EAE mice, intravenous transfer experiments showed that macrophage-colony stimulating factor (M-CSF) -expanded CD34+ myeloid progenitors target the inflamed central nervous system (CNS) while keeping their immature phenotype. Based on these results, we then assessed whether CD34+/B220+ cells display in vitro differentiation potential toward microglia. For this purpose, CD34+/B220+ cells were sorted from M-CSF-stimulated bone marrow (BM) cultures and exposed to a glial cell conditioned medium. Under these experimental conditions, CD34+/B220+ cells were able to differentiate into microglial-like cells showing the morphological and phenotypic features of native microglia. Overall, our data suggest that under developmental or neuroinflammatory conditions, a subpopulation of microglial cells derive from CNS-invading CD34+/B220+ myeloid progenitors.

Dendritic cells are essential for priming but inefficient for boosting antitumour immune response in an orthotopic murine glioma model.

The prognosis of malignant gliomas remains dismal and alternative therapeutic strategies are required. Immunotherapy with dendritic cells (DCs) pulsed with tumour antigens emerges as a promising approach. Many parameters influence the efficacy of DC-based vaccines and need to be optimised in preclinical models. The present study compares different vaccine schedules using DCs loaded with tumour cell lysate (DC-Lysate) for increasing long-term survival in the GL26 orthotopic murine glioma model, focusing on the number of injections and an optimal way to recall antitumour immune response. Double vaccination with DC-Lysate strongly prolonged median survival compared to unvaccinated animals (mean survival 87.5 days vs. 25 days; p < 0.0001). In vitro data showed specific cytotoxic activity against GL26. However, late tumour relapses frequently occurred after 3 months and only 20% of mice were finally cured at 7 months. While one, two or three DC injections gave identical survival, a boost using only tumour lysate after initial DC-Lysate priming dramatically improved long-term survival in vaccinated mice, compared to the double DC-Lysate group, with 67.5% of animals cured at 7 months (p < 0.0001). In vitro data showed better specific CTL response and also the induction of specific anti-GL26 antibodies in the DC-Lysate/Lysate group, which mediated Complement Dependent Cytotoxicity. These experimental data may be of importance for the design of clinical trials that currently use multiple DC injections.

[Dendritic cells and gliomas: a hope in immunotherapy?]. / Cellules dendritiques et gliomes: un espoir en immunothérapie?

Immunotherapy has been explored for several decades to try to improve the prognosis of gliomas, but until recently no therapeutic benefit has been achieved. The discovery of dendritic cells, the most potent professional antigen presenting cells to initiate specific immune response, and the possibility of producing them ex vivo gave rise to new protocols of active immunotherapy. In oncology, promising experimental and clinical therapeutic results were obtained using these dendritic cells loaded with tumor antigen. Patients bearing gliomas have deficit antigen presentation making this approach rational. In several experimental glioma models, independent research teams have showed specific antitumor responses using these dendritic cells. Phase I/II clinical trials have demonstrated the feasibility and the tolerance of this immunotherapeutic approach. In neuro-oncology, the efficiency of such an approach remains to be established, similarly in oncology where positive phase III studies are missing. Nevertheless, dendritic cells comprise a complex network which is only partially understood and capable of generating either immunotolerance or immune response. Numerous parameters remain to be explored before any definitive conclusion about their utility as an anticancer weapon can be drawn. It seems however logical that immunotherapy with dendritic cells could prevent or delay tumor recurrence in patients with minor active disease. A review on glioma and dendritic cells is presented.

Differential MAP kinases activation during semaphorin3A-induced repulsion or apoptosis of neural progenitor cells.

Semaphorins are multifunctional factors implicated in various developmental processes. Little is known about the intracellular pathways ensuring appropriate signal transduction that encode the diverse functions observed. In this study, we investigated whether mitogen-activated protein kinases (MAPK), which are key elements of signal transduction in eukaryotic cells, were activated during semaphorin 3A (Sema3A)-induced repulsion or apoptosis of neural progenitor cells. We found that selective recruitment of the ERK1/2 pathway occurred during Sema3A-induced neural progenitor cell repulsion, whereas p38 MAPK activation was necessary for induction of apoptosis. Moreover, we provide evidence for the involvement of vascular endothelial growth factor receptor 1 (VEGFR1) in the activation of ERK1/2. Additional experiments performed with native cerebellar progenitors confirmed such a selective recruitment of MAPK during Sema3A-dependent migration or apoptosis. Altogether, our results suggest a model to explain how a single factor can exert different functions for a given cell type by the selective recruitment of intracellular pathways.

Extracellular matrix protein expression in cerebrospinal fluid from patients with tropical spastic paraparesis associated with HTLV-I and Creutzfeldt-Jakob disease.

The cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the CNS, thus biochemical processes in the CNS could potentially be reflected in the CSF. Changes in extracellular matrix (ECM) proteins can be studied through their analysis in the CSF. ECM plays an essential role in CNS homeostasis and several proteins such as laminin (LN), fibronectin (FN), thrombospondin (TS) and heparan sulphate proteoglycan (HS, perlecan) form part of its structure. Possible changes in the levels of these proteins were investigated in two different pathologies--tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM) (n=25) and Creutzfeldt-Jakob disease (CJD) (n=19)--and compared with those in a control group with or without neurological disease (n=25). CSF analyses were carried out using monoclonal or monospecific polyclonal antibodies. In comparison with the control group, it was found that TSP/HAM patients presented significantly higher levels of LN, TS and HS, while in CJD patients the levels of FN, TS and HS were increased. In CJD patients the HS level was almost double that of the TSP/HAM patients. These results suggest a distinct pattern of ECM proteins in CSF in relation to the type of neurological disease. TSP/HAM is a chronic motor disease that affects the white matter of the spinal cord, while CJD is a subacute dementia that affects cerebral neurons and their synapsis.

Establishment of human tumoral ependymal cell lines and coculture with tubular-like human endothelial cells.

Ependymomas, rare neoplasms of the central nervous system, occur predominantly in children. They are highly vascularized, and histological findings show many perivascular rosettes of tumoral cells radially organized around capillaries. Treatment of ependymomas relies on surgery combined with radio- or chemotherapy, but the efficiency of chemotherapy is limited, probably because of their multidrug resistance (MDR) phenotype. Progress in the therapy of these neoplasms is dramatically limited by the absence of cell line models. We established conditions for the long-term culture of human tumoral ependymocytes and their 3D coculture in Matrigel with endothelial cells. Histological, immunological, and ultrastructural studies showed that the morphological features (microvilli, cilia, and caveolae) of these cultured cells were similar to those of the tumor in vivo. The cells expressed potential oncological markers related to the immature state of tumoral cells (nestin and Notch-1), their tumorigenicity [caveolae and epidermal growth factor-receptor (EGF-R)], or the MDR phenotype [P-glycoprotein (P-gp)]. The expression of P-gp, EGF-R, and caveolin-1 by these tumoral ependymocytes could be useful in studies on new drugs. This coculture model might represent a new powerful tool to study new therapeutic delivery strategies in tumoral cells.

Isolation and expression pattern of human Unc-33-like phosphoprotein 6/collapsin response mediator protein 5 (Ulip6/CRMP5): coexistence with Ulip2/CRMP2 in Sema3a- sensitive oligodendrocytes.

The Unc-33-like phosphoprotein/collapsin response mediator protein (Ulip/CRMP) family consists of four homologous phosphoproteins considered crucial for brain development. Autoantibodies produced against member(s) of this family by patients with paraneoplastic neurological diseases have made it possible to clone a fifth human Ulip/CRMP and characterize its cellular and anatomical distribution in developing brain. This protein, referred to as Ulip6/CRMP5, is highly expressed during rat brain development in postmitotic neural precursors and in the fasciculi of fibers, suggesting its involvement in neuronal migration/differentiation and axonal growth. In the adult, Ulip6/CRMP5 is still expressed in some neurons, namely in areas that retain neurogenesis and in oligodendrocytes in the midbrain, hindbrain, and spinal cord. Ulip2/CRMP2 and Ulip6/CRMP5 are coexpressed in postmitotic neural precursors at certain times during development and in oligodendrocytes in the adult. Because Ulip2/CRMP2 has been reported to mediate semaphorin-3A (Sema3A) signal in developing neurons, in studies to understand the function of Ulip6/CRMP5 and Ulip2/CRMP2 in the adult, purified adult rat brain oligodendrocytes were cultured in a Sema3A-conditioned medium. Oligodendrocytes were found to have Sema3A binding sites and to express neuropilin-1, the major Sema3A receptor component. In the presence of Sema3A, these oligodendrocytes displayed a dramatic reduction in process extension, which was reversed by removal of Sema3A and prevented by anti-neuropilin-1, anti-Ulip6/CRMP5, anti-Ulip2/CRMP2 antibodies, or VEGF-165, another neuropilin-1 ligand. These results indicate the existence in the adult brain of a Sema3A signaling pathway that modulates oligodendrocyte process extension mediated by neuropilin-1, Ulip6/CRMP5, and Ulip2/CRMP2, and they open new fields of investigation of neuron/oligodendrocyte interactions in the normal and pathological brain.

Functional changes in astrocytes by human T-lymphotropic virus type-1 T-lymphocytes.

The human T-lymphotropic virus type-1 (HTLV-1) is the causative agent of a chronic progressive myelopathy (TSP/HAM) in which lesions of the central nervous system (CNS) are associated with infiltration of HTLV-1-infected T-cells. In a model that mimics the interaction between glial and T-cells, we show that transient contact with T-lymphocytes chronically infected with HTLV-1 induce profound metabolic alterations in astrocytes. Within the first week post-contact, an overall activation of astrocyte metabolism was observed as assessed by enhanced uptake of glutamate and glucose, and lactate release. In contrast, longer examination showed a reduced astrocytic accumulation of glutamate. The time course of the change in glutamate uptake was in fact biphasic. Previous observations indicated that HTLV-1 protein Tax-1 was involved in this delayed decrease, via the induction of TNF-alpha. The expression of the glial glutamate transporters, GLAST and GLT-1 decreased in parallel. These decreases in glutamate uptake and transporters' expression were associated with an imbalance in the expression of the catabolic enzymes of glutamate, GS and GDH, presumably due to Tax-1. Given the fact that impairment of glutamate management in astrocytes is able to compromise the functional integrity of neurons and oligodendrocytes, our results altogether give new insights into the physiopathology of TSP/HAM.

Morbillivirus infection of the mouse central nervous system induces region-specific upregulation of MMPs and TIMPs correlated to inflammatory cytokine expression.

Viral infection of the central nervous system (CNS) can result in perturbation of cell-to-cell communication involving the extracellular matrix (ECM). ECM integrity is maintained by a dynamic balance between the synthesis and proteolysis of its components, mainly as a result of the action of matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs). An MMP/TIMP imbalance may be critical in triggering neurological disorders, in particular in virally induced neural disorders. In the present study, a mouse model of brain infection using a neurotropic strain of canine distemper virus (CDV) was used to study the effect of CNS infection on the MMP/TIMP balance and cytokine expression. CDV replicates almost exclusively in neurons and has a unique pattern of expression (cortex, hypothalamus, monoaminergic nuclei, hippocampus, and spinal cord). Here we show that although several mouse brain structures were infected, they exhibited a differential pattern in terms of MMP, TIMP, and cytokine expression, exemplified by (i) a large increase in pro-MMP9 levels, in particular in the hippocampus, which occurred mainly in neurons and was associated with in situ gelatinolytic activity, (ii) specific and significant upregulation of MT1-MMP mRNA expression in the cortex and hypothalamus, (iii) an MMP/TIMP imbalance, suggested by the upregulation of TIMP-1 mRNA in the cortex, hippocampus, and hypothalamus and of TIMP-3 mRNA in the cortex, and (iv) a concomitant region-specific large increase in expression of Th1-like cytokines, such as gamma interferon, tumor necrosis factor alpha, and interleukin 6 (IL-6), contrasting with weaker induction of Th2-like cytokines, such as IL-4 and IL-10. These data indicate that an MMP/TIMP imbalance in specific brain structures, which is tightly associated with a local inflammatory process as shown by the presence of immune infiltrating cells, differentially impairs CNS integrity and may contribute to the multiplicity of late neurological disorders observed in this viral mouse model.

Down regulation of melanin concentrating hormone in virally induced obesity.

Obesity is a complex disease involving genetic components and environmental factors and probably associated with the dysregulation of central homeostasis normally maintained by the hypothalamic neuroendocrine/neurotransmitter network. We previously reported that canine distemper virus (CDV), which is closely related to human measles virus, can target hypothalamic nuclei, and lead to obesity syndrome in the late stages of infection. Here, using differential display PCR, we demonstrate specific down-regulation of melanin-concentrating hormone precursor mRNA (ppMCH) in infected-obese mice. Although ppMCH was down-regulated in all infected mice during the acute stage of infection, this was only seen during the late stage of infection in infected-obese mice. In addition, ppMCH mRNA and protein expression in the lateral hypothalamus was decreased in the absence of neuronal death. These results show the importance of ppMCH in the establishment and maintenance of obesity and the involvement of a virus as an environmental factor.

Human medulloblastoma cell line DEV is a potent tool to screen for factors influencing differentiation of neural stem cells.

The aim of our study was to investigate whether a human neural cell line could be used as a reliable screening tool to examine the functional conservation, in humans, of transcription factors involved in neuronal or glial specification in other species. Gain-of-function experiments were performed on DEV cells, a cell line derived from a human medulloblastoma. Genes encoding nine different transcription factors were tested for their influence on the process of specification of human DEV cells towards a neuronal or glial fate. In a first series of experiments, DEV cells were transfected with murine genes encoding transcription factors known to be involved in the neuronal differentiation cascade. Neurogenins-1, -2, and -3; Mash-1; and NeuroD increased the differentiation of DEV cells towards a neuronal phenotype by a factor of 2-3.5. In a second series of experiments, we tested transcription factors involved in invertebrate glial specification. In the embryonic Drosophila CNS, the development of most glial cells depends on the master regulatory gene glial cell missing (gcm). Expression of gcm in DEV cells induced a twofold increase of astrocytic and a sixfold increase of oligodendroglial cell types. Interestingly, expression of tramtrack69, which is required in all Drosophila glial cells, resulted in a fourfold increase of only the oligodendrocyte phenotype. Expression of the related tramtrack88 protein, which is not expressed in the fly glia, or the C. elegans lin26 protein showed no effect. These results show that the Drosophila transcription factor genes tested can conserve their function upon transfection into the human DEV cells, qualifying this cell line as a screening tool to analyze the mechanisms of neuronal and glial specification.