Antonio Gallego

Horizontal and amacrine cells in the mammal's retina

Abstract The study of the retina of several mammals by both light and electron microscopy shows the existence of two types of horizontal cells: horizontal cells with a short axon, and horizontal cells without axon, which we can name “amacrine” cells of the outer plexiform layer. The horizontal cells with a short axon are related to cones. The amacrine cells of the outer plexiform layer make contacts with rods. Abundant membrane-to-membrane contacts, with total or partial fusion, can be seen between horizontal cells of both types as well as between them and the bipolar cells. In the retina of primates, only the horizontal cells with short axon have been seen. The horizontal cells of the monkeys retina show, in the soma, large processes and even in the thinnest terminals, the Kolmer “Kristaloide”. Such crystalline structures have been seen in the lateral processes of the cone “triads”. The membrane-to-membrane contacts between horizontal cells of the retina of primates adopt a structural aspect similar to that of the desmosomes. Both the diffuse and stratified type of amacrine cells have been found in all the retinae studies. A new cellular type, with processes expanding in both plexiform layers, have been seen in the cats retina. A new cellular type, probably an amacrine cell, has been seen at the upper level of the ganglion cell layer. Horizontal and amacrine cells are not typical neurons. Due to their structure, disposition in plexus and electrophysiological response, they can be classified as a new type of nerve cell.

The relation of the microglia with the pericytes in the cat cerebral cortex.

SummaryCapillaries, pericytes and microglial cells in layer I of the cerebral cortex of normal adult cats have been studied with electron microscopy. The data obtained in this study show that pericytes are cells which are able to transform themselves into microglial cells by virtue of an activation process in which the astrocytic neuroglia appears to play a decisive role. By virtue of its structure, its mesodermic origin and its function the microglia has to be distinguished clearly from the astrocytic neuroglia and the oligodendroglia.

Immunological risk factors for infection after immunosuppressive and biologic therapies

Immunosuppressive and biologic therapies are costly and can involve a considerable risk of infection. Noninvasive diagnostic tools for early prediction of infection before and after administration of these therapies are of major interest. Serial longitudinal immune monitoring would provide data on immunocompetence and complement clinical follow-up protocols. Biomarkers of immune response may be useful to identify patients at risk of developing infection and who could be candidates for immunosuppressant dose reduction. This article focuses on the potential use of biomarkers of immune response to predict development of infection after immunosuppressive and biologic therapies in selected settings of autoimmune disease (rituximab for treatment of rheumatoid arthritis) and solid organ transplantation.

Quantitative Abnormalities of Peripheral Blood Distinct T, B, and Natural Killer Cell Subsets and Clinical Findings in Obstetric Antiphospholipid Syndrome

Objetive. Few studies have assessed immunophenotypic abnormalities on lymphocyte subsets in patients with antiphospholipid syndrome (APS). We performed an extended immunological study to define alterations of distinct T, B, and natural killer (NK) cell subsets in obstetric patients with APS and their relationship with APS–associated complications. Methods. Patients and controls: 36 women with APS [Sydney criteria, Group A1 without thrombosis (n = 26), Group A2 with thrombosis (n = 10)]; and 36 age matched women with recurrent abortion without antiphospholipid antibodies (disease controls; Group B), 36 healthy parous women (healthy controls; Group C), and 36 healthy nonparous women (healthy controls; Group D). Thrombotic events occurred after history of abortions in all A2 women. Three-color whole-blood flow cytometry was used to characterize the distinct immunophenotypes. Results. A1 patients had significantly higher percentages of CD4+CD45RA–CCR7+ central memory cells (A1 vs D), higher percentages of activated CD4+CD25+ T cells (A1 vs D), and lower percentages and absolute counts of CD4+CD45RA–CCR7– effector memory cells (A1 vs D). GroupA2 patients had higher percentages and absolute numbers of CD19+CD27–IgD+ naive B cells (A2 vs A1 vs all controls), lower percentages and absolute numbers of CD3–CD56+CD16+ NK cells (A2 vs all controls), and higher percentages of activated CD4+DR+ (A2 vs all controls), CD8+DR+ (A2 vs A1 vs C vs D), CD4+CD38+DR+ (A2 vs D), and CD4+CD25+DR+ T cells (A2 vs all controls). Increased percentages of CD8+DR+ T cells [relative risk (RR) 2.43, 95% CI 1.09–5.44, p = 0.02] and of naive B cells (RR 3.05, 95% CI 1.30–7.11, p = 0.009) were associated with development of thrombosis. Conclusion. In obstetric patients with APS we documented significant changes in T, B, and NK cell homeostasis. Increased levels of CD8+DR+ and CD19+CD27–IgD+ cells might identify obstetric patients with APS at risk of having thrombosis.

Organization of the outer plexiform layer of the diurnal and nocturnal bird retinae

Cajal (1892) described the outer plexiform layer of the gallinaceous retina as composed of three distinct strata or “concentric plexuses”: the external plexus formed by the basal rod filaments and the dendritic expansions of some bipolar cells; the intermediate plexus formed by the vitreal end or synaptic bodies of the “straight” cones and the ascending dendrites of other bipolar cell types; the internal plexus formed by the basal filaments of the “oblique” cones and the dendritic expansions of the bipolar cells. No further information about the structure of the outer plexiform layer in other bird species is found in Cajal’s work. The presence of rods, single cones and double cones in the birds retina was noted by Schultze (1866, 1867) who observed in the cones red, yellow and colorless droplets. A comprehensive review on the visual cells of several bird species was made with light microscopy by Rochon-Duvigneaud (1943). Nevertheless later studies (Meyer and Cooper, 1966; Morris and Shorey, 1967; and Morris, 1970) on bird visual cells using phase contrast and electron microscopic techniques, have contributed largely to the current knowledge on this subject. Morris (1970) describes different types of visual cells in the chicken retina. namely, rods with no oil droplets, double cones whose “chief member possesses a large yellow oil droplet and whose “accessory” one has a small yellow-green droplet and three types of single cones. All types of single cones have oil droplets and can be differentiated by their affinity for lead staining and their electron density. According to Morris and Shorey (1967) the synaptic bodies of the chicken photoreceptors are located at two different levels in the outer plexiform layer: at the external or sclerad level lie the rod and double cone synaptic bodies; at the internal or v&read level lie the synaptic bodies of the single cones. In the present investigation, the retinae of three diurnal birds, GalLs domesticus, Hieratrrs fasciurus and Mihs mihs and of two nocturnal birds, Carinae noctua and Asiojammetrs were used. Histological processing started immediately after sacrificing the animals. In several instances portions of the retinas were studied in fresh or just after formaldehyde fixation. with the light microscope using Nomarsky optics. For regular light microscopy the techniques used were as follows: the Gallego technique (1953) of silver impregnation of the entire retina. the classical Golgi procedure and its Colonnier modification. Routinely all the retinas were prepared as flat-mounts and studied in toto. Samples of the retinas were selected either for embedding in paraffin and sectioning at different orientations or for plastic embedding, ultrathin sectioning and examination by the electron microscope. The electron microscope is necessary to identify the photoreceptor types and to decide upon the organization of the synaptic bodies of the photoreceptors in the outer plexiform layer.

Immune monitoring of anti cytomegalovirus antibodies and risk of cytomegalovirus disease in heart transplantation.

We sought to determine whether quantitative assessment of anti-cytomegalovirus (CMV) antibodies could be useful to identify patients at risk of cytomegalovirus (CMV) disease after heart transplantation (HT). 75 patients who underwent HT at a single health care center were prospectively studied. Induction therapy included 2 doses of daclizumab and maintenance tacrolimus (n=42) or cyclosporine (n=29), mycophenolate mofetil and prednisone. All patients received prophylaxis with gancyclovir or valganciclovir. Anti-CMV intravenous immunoglobulin (CMV-IG) was added in high risk patients (CMV D+/R- serostatus). Serial determinations of anti-CMV antibodies, immunoglobulins (IgG, IgA, IgM) and IgG-subclasses were analysed. CMV infection was based on detection of the virus by antigenemia. CMV disease consisted of detection of signs or symptoms attributable to this microorganism. Ten patients (13.3%) developed CMV disease. Mean time of development of CMV disease was 3.4+/-1.6 months. In Cox regression analysis, patients with low baseline anti-CMV titers (<4.26 natural logarithm of titer, RH: 8.1, 95%CI: 1.93-34.1, p=0.004) and recipients with 1-month post-HT IgG hypogammaglobulinemia (IgG<500 mg/dl, RH: 4.49, 95%CI: 1.26-15.94, p=0.02) were at higher risk of having CMV disease. Despite use of prophylactic CMV-IG, D+/R- patients showed significantly lower titers of anti-CMV antibodies at 7 d, 30 d and 90 d post HT as compared with HT recipients without infections. Four out of 6 of these patients developed late CMV disease. Monitoring of specific anti-CMV antibodies on the bedside warrants further evaluation as a potential tool to identify heart transplant recipients at higher risk of CMV disease.

Decreased levels of serum complement C3 and natural killer cells add to the predictive value of total immunoglobulin G for severe infection in heart transplant recipients

Infection remains a source of mortality in heart recipients. We previously reported that post‐transplant immunoglobulin G (IgG) quantification can help identify the risk for infection. We assessed whether other standardized parameters of humoral and cellular immunity could prove useful when identifying patients at risk of infection.

Short-axon horizontal cells of the monkey's retina.

Since Cajal’s early studies (1892, 1933) it has been widely accepted that the retinae of terrestrial vertebrates have two types of horizontal cells. “‘external” and “internal”, each with a short axon. Recent studies on mammalian horizontal cells (Gallego, 1964, 1965, 1971: Kolb, 1970; Boycott and Kolb. 1973) have shown that such an assumption does not hold for all mammalian species. According to Gallego ( 197 I. 1972) there is only a single type of horizontal cell with axon in mammals and the distinction between “external” and “internal” horizontal cells, based on Cajal’s idea should not be maintained. In the primate retina the existence of a single type of horizontal cell with a short axon was first stated by Polyak (1941) and later confirmed by Kolb (1970), GalIego (1971). and Boycott and Kolb ( 1973). Lower mammals with rod dominated retinae (mouse, rat. guinea-pig, cat, dog, etc.) have, as the primates, the horizontal cell with axon and also a second type of horizontal cell that differs from the former in its morphology, staining properties and its relationship with the photoreceptors. The absence of an axon and their distribution in a plexus that spreads all over the retina from the ‘ora serrata” to the “papilla” (including the central area) are the main features of this second type of horizontal cell (Gallego, 1964, 1965, 1971). In the “rhesus monkey” retina, Boycott and Kolb (1973) describe a single type of horizontal cell with axon, whose size varies from the fovea to the peripheral retina. Its dendritic terminals enter the cone triad; one horizontal cell contacts 6-9 cones in the parafoveal region and 30-40 cones in the peripheral retina. Each horizontal cell has a single axon whose origin has been stained by several investigators (Polyak, 1941; Boycott and Dowling, 1969; Kolb, 1970; Gallego, 1971; Boycott and Dowling, 1973). Complete staining of the whole length of the axon to its terminal branches has been achieved by Ogden (1974) in the peripheral retina of the owl monkey Aotes. The idea that they probably contact the rod spherules was put forward (Gallego. 1971), but the connections that these terminals might make with the photoreceptors have not been demonstrated. In whole-flat mounted retinae of rhesus, Erirrocebus patas and of Cercopithecus monkeys. stained with the Golgi technique and its Colonnier and Lasansky modifications. a complete staining of the whole axon length of the horizontal cell has not been achieved, but we have been able to follow it for long distances (Fig. I I more than 600 pm, and to obtain a complete staining of its terminal branches. The axon has no coliaterals and its progression is often straight although it bends sharply in some instances. No pattern in the axon orien~tion has been found. The dendrites of the horizontal ceils and the axonic terminals display clear morphological differences at their thin terminal branches. As described by Boycott and Kolb (1973) the dendritic endings form clusters of small “boutons”, corresponding in size to the diameter of the cone pedicle; in contrast. the terminal branches of the axon give out thin fibers which end bv isolated “boutons” directed towards the synaptic bodies of the photoreceptors (Figs. 4 and 6). The dendritic endings are the lateral elements of the cone triads as shown by Boycott and Dowling (1969) and Kolb (1970). In Golgi stained and whole-flat mounted retinas. it can be seen very often that the thin dendrites of a bipolar cell join a cluster of horizontal cell dendritic endings (Fig. I!. and also that the dendrites of two horizontal cells contribute to form one cluster of endings (Fig. 3). With the light microscope the endings of the axonic terminal are seen in contact with the rod spherules (Fig. 4). In perpendicular sections of the reembedded tissue showing one of these axonic terminats, its endings in form of small -boutons” directed towards the photoreceptors can be seen (Fig. 6): ultrathin sections .studied with electron microscopy show clearly that these terminals penetrate the rod synaptic bodies (Fig. 7). The number of cones contacting the dendrites of a singie horizontal cell has been estimated by the procedure of Boycott and Kolb (1973) and our results are in agreement with theirs. Counting the stained “boutons” of an axonic terminal and assuming that each one enters the synaptic complex of one rod. as a first approach we can estimate that one horizontal celL whose dendrites contact from 6 to 30 cones. contacts through its axonic terminals 80150 rods located at a minimal distance of 600 pm from the soma of the horizontal cell.

Horizontal cells of the avian retina

Consistent with his own general description of the retinal structure of vertebrates, Cajal (1892, 1904, 1933) described two types of horizontal cells in the avian retina: “stellate” and “brush” horizontal cells. Cajal’s distinction was based on the different location of each cell type with regard to the outer plexiform layer and on some of their morphological features. Recently, a great deal of work has been done on the structure and classification of the photoreceptor cells. as well as on the efferent centrifugal fiber system in the avian retina. However, much less information is available concerning the organization of the outer plexiform layer or the horizontal cells morphology and their relationship with the photoreceptor cells. A previous paper (Gallego, Baron and Gayoso, 1975) shows that in the diurnal bird retina the synaptic bodies of the photoreceptors are found at three different strata in the outer plexiform layer. whereas in the nocturnal species they are found at a single stratum. The present investigation attempts to establish the organization of the outer plexiform layer which includes the horizontal cell types found in the retinae of both diurnal and nocturnal birds. The retinae of three diurnal bird species, Gallus domesticus. Hieratus fasciatus and Milcus milaus, and those of two nocturnal ones, Carinae noctua and Asio jlammeus, were studied. The retinae were obtained inmediately after sacrificing the animal. In the study of the horizontal cells the most suitable procedure was found to be the Golgi silver impregnation and its Colonnier and Lasansky modifications. All retinas were prepared as flat-mounts and observed in toto. Samples of the retinas showing horizontal cells were selected and embedded in paraffin. These samples were then sectioned at different orientations. Further samples of the cell soma and/or the axonic terminal were prepared for thin sectioning and analysis by the electron microscope.

Alterations of naïve and memory B‐cell subsets are associated with risk of rejection and infection in heart recipients

Rejection and infection are relevant causes of mortality in heart recipients. We evaluated the kinetics of the maturation status of B lymphocytes and its relationship with acute cellular rejection and severe infection in heart recipients. We analyzed B‐cell subsets using 4‐color flow cytometry in a prospective follow‐up study of 46 heart recipients. Lymphocyte subsets were evaluated at specific times before and up to 1 year after transplantation. Higher percentages of pretransplant class‐switched memory B cells (CD19+CD27+IgM‐IgD‐ >14%) were associated with a 74% decrease in the risk of severe infection [Cox regression relative hazard (RH) 0.26, 95% confidence interval (CI), 0.07–0.86; P = 0.027]. Patients with higher percentages of naïve B cells at day 7 after transplantation (CD19+CD27‐IgM+IgD+ >58%) had a 91% decrease in the risk of developing acute cellular rejection (RH 0.09; 95% CI, 0.01–0.80; P = 0.02). Patients with infections showed a strong negative correlation between baseline serum B‐cell–activating factor (BAFF) concentration and absolute counts of memory class‐switched B cells (R = −0.81, P = 0.01). The evaluation of the immunophenotypic maturation status of B lymphocytes could prove to be a useful marker for identifying patients at risk of developing rejection or infection after heart transplantation.