Quantitative and functional evaluation of innate immune responses in patients with common variable immunodeficiency.

OBJECTIVES: We evaluate the frequency and functional response of innate immune cells in peripheral blood (PB) from patients with common variable immunodeficiency (CVID) and healthy controls upon activation with agonists of the Toll-like receptors (TLR) TLR2, TLR4, and TLR9. In addition, several nonsynonymous single nucleotide polymorphisms (SNPs) within these TLR genes were examined. METHODS: Flow cytometry was used to perform immunophenotyping and evaluate the expression of cell surface markers. Levels of cytokines in the culture supernatants were evaluated using cytometric bead array technology. SNPs in the TLR genes were evaluated from genomic DNA using different sequencing techniques. RESULTS: Our results demonstrate that the frequency of CD1d-restricted TCR invariant natural killer T cells in PB was significantly reduced in the patients with CVID. A marked, though not significant, reduction in absolute numbers of plasmacytoid dendritic cells and natural killer cells was also observed in these patients. Interestingly, CD80 and CD86 expression on innate cells upon stimulation with TLR ligands was not altered in the patients although 3 of them exhibited low baseline levels of these surface molecules on monocytes compared to healthy controls. We also observed a significant increase in TNF-alpha levels in supernatants of PB mononuclear cells from CVID patients after stimulation with lipopolysaccharide. Finally, no association was found between the presence of nonsynonymous SNPs within the TLR genes and the clinical presentation of CVID. CONCLUSIONS: Taken together, our study demonstrates than innate immune responses are disturbed in some CVID patients and prompts the evaluation of innate immunity genes as candidates to explain the CVID clinical phenotype.

Study of adult neurogenesis in the Gallotia galloti lizard during different seasons.

In a previous study we found a seasonal distribution of cell proliferation (the first stage of adult neurogenesis) in the telencephalic ventricular walls of the adult Gallotia galloti lizard. The aim of the present work was to determine the influence of seasonality on the subsequent migration of the resulting immature neurons. We used wild animals injected with bromodeoxyuridine and kept in captivity within 30 days. To confirm the neuronal identity of these cells, we used double immunohistochemical 5-bromo-2'-deoxyuridine (BrdU) and doublecortin (DCX, an early neuronal marker) labeling, as well as autoradiography after the administration of methyl-[³H]thymidine ([³H]T). We found that: (1) the rate of cell division and/or migration from the ventricular walls varied with the season, especially in regions related with olfaction. (2) Immature neuron-like cells appeared to migrate in an apparently radial and tangential way towards different parts of the telencephalic parenchyma. (3) We did not observe ultrastructurally mature neurons until at least 90 days later, a period considerably greater than that reported for other species of vertebrates in similar studies.

Seasonal differences in ventricular proliferation of adult Gallotia galloti lizards.

Lizards present neuronal production throughout the telencephalon in their adult state, both naturally and after experimentally induced brain lesions. As in birds, lizards present seasonal behavioural variations. In birds, such variations have been shown to alter neuronal production. In birds and mammals, lack of stimuli or exposure to stress interferes with adult neurogenetic capacity. The effect of this type of study has not been performed with lizards. In the present study we used bromodeoxyuridine to label dividing cells in the ventricular walls of Gallotia galloti lizards during all four seasons and we investigated the effect of captivity on such proliferation. We found that G. galloti presented a particular distribution that differed from that previously described in other reptiles with respect to regions of greater or lesser proliferative rate. In addition, proliferative rate varied seasonally, with greater production of cells in Spring and low production in Autumn and Winter. Proliferative rate was significantly lower throughout the telencephalon and during all seasons in those lizards kept in captivity as compared with wild animals, even though photoperiod and temperature were similar to natural conditions. Our results indicate that cell production in lizards is species-dependent, varies with seasons and is significantly reduced in captive animals.

Endemically exposed asymptomatic individuals show no increase in the specific Leishmania (Viannia) panamensis-Th1 immune response in comparison to patients with localized cutaneous leishmaniasis.

In Colombia, most cases of human cutaneous leishmaniasis are caused by Leishmania (Viannia) panamensis. Interestingly, up to 30% of the exposed population do not suffer from clinical leishmaniasis although it is likely that they are continuously infected with Leishmania parasites. Since it is believed that the induction of efficient Th1 immune responses protects against Leishmania infections both in humans and in animal models, we determined if endemically exposed asymptomatics showed stronger Leishmania-specific Th1 immune responses than patients with active localized cutaneous leishmaniasis (LCL). We found that Montenegro skin test responses were slightly higher among asymptomatic individuals compared to patients suffering from LCL. However, PBMC from patients with LCL showed similar Leishmania-specific proliferative responses compared to PBMC from asymptomatic individuals. Furthermore, PBMC from both groups also secreted similar amounts of IFN-gamma, IL-12p40 and IL-10 after in vitro exposure to L. panamensis. No IL-4 was detected in the supernatants. Taken together our results suggest that lack of LCL development in endemically exposed asymptomatics cannot be explained by stronger systemic anti-Leishmania Th1 immune responses or decreased Th2 responses in these individuals in comparison to individuals who develop LCL. It may be possible that other mechanisms are responsible for resistance to cutaneous leishmaniasis in Colombia in endemically exposed asymptomatics.

Cytoarchitectonic subdivisions in the subtectal midbrain of the lizard Gallotia galloti.

Contemporary study of molecular patterning in the vertebrate midbrain is handicapped by the lack of a complete topological map of the diverse neuronal complexes differentiated in this domain. The relatively less deformed reptilian midbrain was chosen for resolving this fundamental issue in a way that can be extrapolated to other tetrapods. The organization of midbrain centers was mapped topologically in terms of longitudinal columns and cellular strata on transverse, Nissl-stained sections in the lizard Gallotia galloti. Four columns extend along the whole length of the midbrain. In dorsoventral order: 1) the dorsal band contains the optic tectum, surrounded by three ventricularly prominent subdivisions, named griseum tectale, intermediate area and torus semicircularis, in rostrocaudal order; 2) a subjacent region is named here the lateral band, which forms the ventral margin of the alar plate and also shows three rostrocaudal divisions; 3) the basal band forms the basal plate or tegmentum proper; it appears subdivided into medial and lateral parts: the medial part contains the oculomotor and accessory efferent neurons and the medial basal part of the reticular formation, which includes the red nucleus rostrally; the lateral part contains the lateral basal reticular formation, and includes the substantia nigra caudally; 4) the median band contains the ventral tegmental area, representing the mesencephalic floor plate. The alar regions (dorsal and lateral) show an overall cellular stratification into periventricular, central and superficial strata, with characteristic cytoarchitecture for each part. The lateral band contains two well developed superficial nuclei, one of which is commonly misidentified as an isthmic formation. The basal longitudinal subdivisions are simpler and basically consist of periventricular and central strata.

The lacertidian reticular thalamic nucleus topographically upon the dorsal thalamus: experimental study in Gallotia galloti.

The projection pattern of the ventral thalamic reticular nucleus onto the dorsal thalamus was studied in the lizard Gallotia galloti using in vitro horseradish peroxidase and fluorescent carbocyanine labelling techniques. Localized label deposits at three dorsoventrally spaced sites in the dorsal thalamus elicited retrograde transport into separate, though partly overlapping, medial, dorsolateral and ventrolateral sectors within an extended cytoarchitectonic complex which may be globally identifiable as the reticular nucleus. Neurons found in the dorsolateral and ventrolateral sectors mainly corresponded to the cell group named nucleus ventromedialis (or nucleus of the dorsal supraoptic decussation) in the literature, whereas neurons labelled in the medial sector corresponded to the so-called dorsal hypothalamic nucleus. Sparser cells appear labelled in the superficially placed nucleus suprapeduncularis. Thalamotelencephalic fibers arising from the injected dorsal thalamic nuclei also project to the corresponding retrogradely labeled sectors within the reticular nucleus. These findings reveal a rough topographic organization in the connections of the extended reticular nucleus complex with the whole dorsal thalamus. This supports the hypothesis of hodological homology between this ventral thalamic formation in Gallotia and the mammalian thalamic reticular nucleus.

Golgi study of the anterior dorsal ventricular ridge in a lizard. II. Neuronal cytodifferentiation.

The development of the morphologic features of neurons in the anterior dorsal ventricular ridge (ADVR) has been followed in Golgi preparations from the lizard Gallotia galloti between embryonic stage 32 and post-eclosion stages of specimens 3.6-4.5 cm in length. The differentiation sequence of multipolar and bitufted neurons was established. Dendritic growth cones are present after stage 34. Filiform dendritic processes are replaced later on by spines. Clusters of neurons first appear at stage 39 in the periventricular zone, the cells becoming Golgi-impregnated in pairs. After hatching, the number of impregnated cells per cluster increases.

Golgi study of the anterior dorsal ventricular ridge in a lizard. I. neuronal typology in the adult.

Using Golgi techniques we have studied neuronal cell types in the anterior dorsal ventricular ridge (ADVR) of the adult lizard Gallotia galloti. Multipolar, bitufted, and juxtaependymal neuronal forms were found. The multipolar and bitufted neurons are present in both the periventricular and central ADVR zones. Multipolar neurons can be subdivided into multipolar neurons with polygonal somata and four to six main dendritic trunks and multipolar neurons with pyramidal somata and three or more dendritic trunks. The former are the cells most frequently impregnated in the ADVR. In the population of bitufted neurons, we distinguish subtypes I, II, and III according to the number of dendritic trunks that emerge from the somata. Juxtaependymal neurons are restricted to a cell-poor zone, adjacent to ependymal cells. Their dendrites either are orientated parallel to the ventricular surface or extend into the periventricular zone. The dendrites of ADVR neurons have pedunculated spines with knob-like tips. However, such spines do not appear on the somata or on the primary dendritic trunks. The number of spines is scarce or moderate. The periventricular neuronal clusters contain two to five cells. The morphology of these neurons is mainly multipolar, but we also found some bitufted neurons.

Cell death in the lizard cerebellar anlage at early embryonic stages.

Three types of cell degeneration were observed at early embryonic stages of the development of cerebellar cortex of Gallotia galloti (Reptilia: Lacertidae). One is characterized by overall cell shrinkage whereas in the other two the degenerative process is recognized by a differential appearance of the cellular nucleus. We have also studied the further phagocytosis of the resultant cellular debris.

Cell death in the embryonic brain of Gallotia galloti (Reptilia; Lacertidae): a structural and ultrastructural study.

In the striatum, thalamus and cerebellum of a Lacertid reptile, we have found three types of cellular death during embryonic development, both at the light and electron microscopic level. The first affects the undifferentiated neuro-epithelial cells and is commonest during the early stages (E. 32-E. 36). The second corresponds to the type of 'nuclear' death described in the bibliography and reaches a maximum in the middle embryonic period (E. 37-E. 39); nevertheless important variations were observed in different zones. The third is the same as the 'cytoplasmic' death type and appears in the perinatal stages. Phagocytosis involved in the elimination of dead cells is of two types. One is associated with early death and is carried out by undifferentiated neuro-epithelial cells. The other is carried out by microglial cells which appear around Stage 37. Much cellular debris was observed in the intermediate zone and this was associated with the second type of phagocytosis. In both cases lipid production was associated with the degenerative process. Comparison of the temporal cellular death pattern with synaptogenesis, gliogenesis and maturation of neuronal processes is consistent with the view that the various types of cellular death found by us had different causes.

Cell death in the normal development of Gallotia galloti mesencephalon (Reptilia Lacertidae). An ultrastructural study.

We have studied the development of the mesencephalon of Gallotia galloti and we have observed two classes of cellular death: 'nuclear' or 'Type I' and 'cytoplasmatic' or 'Type II'. The first appears in stages around E-34 and the second one is observed from stages around hatching to the adult lizard. The degenerative cells have been observed in the profundus, torus semicircularis, the 5th pair of the trigeminal nerve nuclei as well as the 4th and 5th layers of the optic tectum where this last nucleus mentioned is situated.

Neuronal cilia in the embryonic thalamus and striatum of Gallotia galloti (Reptilia, Lacertidae).

Cilia are a common characteristic of the embryonic neurons of the thalamus and striated centers of Gallotia galloti from E.35. The ciliogenesis process occurs around the stages E.33 and E.34. The ciliary morphology shows no substantial variations between the different stages and the ciliary structure in the embryonic period is 8 + 1. On the other hand, the adult cilia has a 9 + 2 arrangement of microtubules. We do not rule out the possibility of other axonemes.

A Nissl, Golgi-Kopsch and ultrastructural study of the Gallotia galloti red nucleus.

The object of this study was to understand the different types of morphological neurons of the Gallotia galloti red nucleus using quantitative and qualitative analysis, under the aspects of optical (Nissl and Golgi-Kopsch) and electronic microscopy. The results from this study and considering the size, the distribution of NISSL bodies and the dendritic arborization, we have identified three neuronal types: ellipsoidal or ovoid (15 microns), triangular (15-35 microns) and polygonal (20-50 microns). In general the polygonal neurons contain a cytoplasm with abundant organelles, well developed Nissl bodies, perinuclear Golgi complex, numerous mitochondria, an ovoid nucleus and multiple dendrites. The triangular neurons have a similar structure, although the dendritic model is less ramified than that of the polygonal and the ellipsoidal neurons contain a smaller cytoplasm with less dendritic ramification. The contact with spines is not very frequent but can be observed in somas and dendrites. The neuronal population is heterogeneous and therefore neither the magnocellular part nor the other parvicellular were observed separately, but a mixture of both.