Miriam Stela Maris de Oliveira Costa

Analysis of Leishmania chagasi by 2-D difference gel electrophoresis (2-D DIGE) and immunoproteomic: identification of novel candidate antigens for diagnostic tests and vaccine.

Identification of novel antigens is essential for developing new diagnostic tests and vaccines. We used DIGE to compare protein expression in amastigote and promastigote forms of Leishmania chagasi. Nine hundred amastigote and promastigote spots were visualized. Five amastigote-specific, 25 promastigote-specific, and 10 proteins shared by the two parasite stages were identified. Furthermore, 41 proteins were identified in the Western blot employing 2-DE and sera from infected dogs. From these proteins, 3 and 38 were reactive with IgM and total IgG, respectively. The proteins recognized by total IgG presented different patterns in terms of their recognition by IgG1 and/or IgG2 isotypes. All the proteins selected by Western blot were mapped for B-cell epitopes. One hundred and eighty peptides were submitted to SPOT synthesis and immunoassay. A total of 25 peptides were shown of interest for serodiagnosis to visceral leishmaniasis. In addition, all proteins identified in this study were mapped for T cell epitopes by using the NetCTL software, and candidates for vaccine development were selected. Therefore, a large-scale screening of L. chagasi proteome was performed to identify new B and T cell epitopes with potential use for developing diagnostic tests and vaccines.

Retinohypothalamic projections in the common marmoset (Callithrix jacchus): A study using cholera toxin subunit B.

Retinal projections in vertebrates reach the primary visual, accessory optic, and circadian timing structures. The central feature of the circadian timing system is the principal circadian pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus. The direct projections from the retina to the SCN are considered the entrainment pathway of the circadian timing system. In this study, unilateral intravitreal injections of cholera toxin subunit B were used to trace the retinal projections to the marmoset hypothalamus. The retinohypothalamic tract reaches the ventral suprachiasmatic nucleus bilaterally, as anticipated from previous studies. However, labeled fibers were found in several other hypothalamic regions, such as the medial and lateral preoptic areas, supraoptic nucleus, anterior and lateral hypothalamic areas, retrochiasmatic area, and subparaventricular zone. These results reveal new aspects of retinohypothalamic projection in primates and are discussed in terms of their implications for circadian as well as noncircadian control systems. J. Comp. Neurol. 415:393–403, 1999.

Calbindin immunoreactivity delineates the circadian visual centers of the brain of the common marmoset (Callithrix jacchus)

The hypothalamic suprachiasmatic nucleus and the thalamic pregeniculate nucleus (which includes the intergeniculate leaflet) comprise the circadian visual system in the primate brain. In this study, we used intraocular injections of cholera toxin subunit B to identify those nuclei in the common marmoset brain, and demonstrated that calbindin D-28k immunoreactivity apparently labels most neurons in both the suprachiasmatic and pregeniculate nuclei. These data suggest that calbindin D-28k could represent a reliable neuronal marker for structures of the circadian visual system in marmosets and provide anatomical information on the primate equivalent of the rodent intergeniculate leaflet.

Differential distribution of afferents containing serotonin and neuropeptide Y within the marmoset suprachiasmatic nucleus.

Neuropeptide Y-containing fibers/terminals were immunohistochemically detected in the ventral portion of the marmoset suprachiasmatic nucleus, approximately matching the distribution of its retinal afferents. On the other hand, serotonergic fibers/terminals were found mostly in central and dorsal areas of the suprachiasmatic nucleus, almost completely sparing its ventral portion. These data may represent a morphological substrate for differential actions of serotonin and neuropeptide Y in the control of circadian rhythmicity in marmosets.

Retinal projections to the midline and intralaminar thalamic nuclei in the common marmoset (Callithrix jacchus)

In this study, we report the identification of a hitherto not reported direct retinal projection to midline and intralaminar thalamic nuclei in the marmoset brain. After unilateral intravitreal injections of cholera toxin subunit B (CTb), anterogradely transported CTb-immunoreactive fibers and presumptive terminals were seen in the following thalamic midline nuclei: paraventricular, rhomboid, interanteromedial, and reuniens, and thalamic intralaminar nuclei: central medial, central lateral, central dorsal, and parafascicular. Studies employing sensitive tracers in other primate species are needed in order to verify the possible universality of these projections. Some of the possible functional correlates of the present data are briefly discussed. The present results may contribute to the elucidation of the anatomical substrate of the functionally demonstrated involvement of this midline/intralaminar thalamic nuclear complex in several domains that include arousal and awareness, besides specific cognitive, sensory, and motor functions.

Sciatic nerve grafting and inoculation of FGF-2 promotes improvement of motor behavior and fiber regrowth in rats with spinal cord transection

PURPOSE Failure of severed adult central nervous system (CNS) axons to regenerate could be attributed with a reduced intrinsic growing capacity. Severe spinal cord injury is frequently associated with a permanent loss of function because the surviving neurons are impaired to regrow their fibers and to reestablish functional contacts. Peripheral nerves are known as good substrate for bridging CNS trauma with neurotrophic factor addition. We evaluated whether fibroblastic growth factor 2 (FGF-2) placed in a gap promoted by complete transection of the spinal cord may increase the ability of sciatic nerve graft to enhance motor recovery and fibers regrow. METHODS We used a complete spinal cord transection model. Rats received a 4 mm-long gap at low thoracic level and were repaired with saline (control) or fragment of the sciatic nerve (Nerve) or FGF-2 was added to nerve fragment (Nerve+FGF-2) to the grafts immediately after complete transection. The hind limbs performance was evaluated weekly for 8 weeks by using motor behavior score (BBB) and sensorimotor tests-linked to the combined behavior score (CBS), which indicate the degree of the motor improvement and the percentage of functional deficit, respectively. Neuronal plasticity were evaluated at the epicenter of the injury using MAP-2 and GAP-43 expression. RESULTS Spinal cord treatment with sciatic nerve and sciatic nerve plus FGF-2 allowed recovery of hind limb movements compared to control, manifested by significantly higher behavioral scores. Higher amounts of MAP-2 and GAP-43 immunoreactive fibers were found in the epicenter of the graft when FGF-2 was added. CONCLUSIONS FGF-2 added to the nerve graft favored the motor recovery and fiber regrowth. Thus, these results encourage us to explore autologous transplantation as a novel and promising cell therapy for treatment of spinal cord lesion.

Dopaminergic cell populations of the rat substantia nigra are differentially affected by essential fatty acid dietary restriction over two generations.

Essential fatty acids play a crucial role in the activity of several neurotransmission systems, especially in the monoaminergic systems involved in cognitive and motor aspects of behavior. The present study investigated whether essential fatty acid dietary restriction over two generations could differentially affect dopaminergic cell populations located in the substantia nigra rostro-dorso-medial (SNrm) or caudo-ventro-lateral (SNcv) regions which display distinct neurochemical profile and vulnerability to lesions under selected pathological conditions. Wistar rats were raised from conception on control or experimental diets containing adequate or reduced levels of linoleic and α-linolenic fatty acids, respectively. Stereological methods were used to estimate both the number and soma size of tyrosine hydroxylase (TH)-immunoreactive neurons in the SNrm and SNcv. TH protein levels were assessed with Western blots. Long-term treatment with the experimental diet modified the fatty acid profile of midbrain phospholipids and significantly decreased TH protein levels in the ventral midbrain (3 fold), the number of TH-positive cells in the SNrm (∼20%) and the soma size of these neurons in both SNrm (∼20%) and SNcv (∼10%). The results demonstrate for the first time a differential sensitivity of two substantia nigra dopaminergic cell populations to unbalanced levels of essential fatty acids, indicating a higher vulnerability of SNrm to the harmful effects induced by docosahexaenoic acid brain deficiency.

The suprachiasmatic nucleus and the intergeniculate leaflet in the rock cavy (Kerodon rupestris): Retinal projections and immunohistochemical characterization

In this study, two circadian related centers, the suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL) were evaluated in respect to their cytoarchitecture, retinal afferents and chemical content of major cells and axon terminals in the rock cavy (Kerodon rupestris), a Brazilian rodent species. The rock cavy SCN is innervated in its ventral portion by terminals from the predominantly contralateral retina. It also contains vasopressin, vasoactive intestinal polypeptide and glutamic acid decarboxilase immunoreactive cell bodies and neuropeptide Y, serotonin and enkephalin immunopositive fibers and terminals and is marked by intense glial fibrillary acidic protein immunoreactivity. The IGL receives a predominantly contralateral retinal projection, contains neuropeptide Y and nitric oxide synthase-producing neurons and enkephalin immunopositive terminals and is characterized by dense GFAP immunoreactivity. This is the first report examining the neural circadian system in a crepuscular rodent species for which circadian properties have been described. The results are discussed comparing with what has been described for other species and in the context of the functional significance of these centers.

Retinal projections to the thalamic paraventricular nucleus in the rock cavy (Kerodon rupestris)

The thalamic paraventricular nucleus (PVT) receives afferents from numerous brain areas, including the hypothalamic suprachiasmatic nucleus (SCN), considered to be the major circadian pacemaker. The PVT also sends projections to the SCN, limbic system centers and some nuclei involved in the control of the Sleep-Wake cycle. In this study, we report the identification of a hitherto not reported direct retinal projection to the PVT of the rock cavy, a typical rodent species of the northeast region of Brazil. After unilateral intravitreal injections of cholera toxin subunit B (CTb), anterogradely transported CTb-immunoreactive fibers and presumptive terminals were seen in the PVT. Some possible functional correlates of the present data are briefly discussed, including the role of the PVT in the modulation of the circadian rhythms by considering the reciprocal connections between the PVT and the SCN. The present work is the first to show a direct retinal projection to the PVT of a rodent and may contribute to elucidate the anatomical substrate of the functionally demonstrated involvement of this midline thalamic nucleus in the modulation of the circadian timing system.

Retinal afferents to the thalamic mediodorsal nucleus in the rock cavy (Kerodon rupestris).

The MD has reciprocal connections with the ventromedial prefrontal cortex (PFC) and with limbic cortices and appears to participate in learning and memory-related processes. In this study, we report the identification of a hitherto not reported direct retinal projection to the MD of the rock cavy, a typical rodent species of the Northeast region of Brazil. After unilateral intravitreal injections of cholera toxin subunit B (CTb), anterogradely transported CTb-imunoreactive fibers and presumptive terminals were seen in the MD. A few labeled retinal fibers/terminals detected in the MD of the rock cavy brain show clear varicosities, suggesting terminal fields. The present work is the first to show a direct retinal projection to the MD of rodents and may contribute for elucidating the anatomical substrate of the functional involvement of this thalamic nucleus in the modulation of the visual recognition, emotional learning and object-reward association memory.