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Biomedical subjects

H Kolb

Publications and source records attributed to H Kolb.

At least 91 records · Page 5Linked to original sources
PubMed1998

Gene array and expression of mouse retina guanylate cyclase activating proteins 1 and 2.

PURPOSE: To identify gene arrangement, chromosomal localization, and expression pattern of mouse guanylate cyclase activating proteins GCAP1 and GCAP2, retina-specific Ca2+-binding proteins, and photoreceptor guanylate cyclase activators. METHODS: The GCAP1 and GCAP2 genes were cloned from genomic libraries and sequenced. The chromosomal localization of the GCAP array was determined using fluorescent in situ hybridization. The expression of GCAP1 and GCAP2 in mouse retinal tissue was determined by immunocytochemistry. RESULTS: In this study, the mouse GCAP1 and GCAP2 gene array, its chromosomal localization, RNA transcripts, and immunolocalization of the gene products were fully characterized. The GCAP tail-to-tail array is located at the D band of chromosome 17. Each gene is transcribed into a single transcript of 0.8 kb (GCAP1) and 2 kb (GCAP2). Immunocytochemistry showed that both GCAP genes are expressed in retinal photoreceptor cells, but GCAP2 was nearly undetectable in cones. GCAP2 was also found in amacrine and ganglion cells of the inner retina. Light-adapted and dark-adapted retinas showed no significant difference in the distribution of the most intense GCAP2 staining within the outer segment and outer plexiform layers. CONCLUSIONS: Identical GCAP gene structures and the existence of the tail-to-tail gene array in mouse and human suggest an ancient gene duplication-inversion event preceding mammalian diversification. Identification of both GCAPs in synaptic regions, and of GCAP2 in the inner retina suggest roles of these Ca-binding proteins in addition to regulation of phototransduction.

Adaptation, Ocular↗
PubMed1998

The localization of guanylyl cyclase-activating proteins in the mammalian retina.

PURPOSE: To explore the distribution of guanylyl cylase-activating proteins 1 and 2 (GCAP1 and GCAP2) in the mammalian retina. METHODS: Cryostat and vibratome vertical sections and wholemount retinas from mouse, rat, cat, bovine, monkey, and human eyes were prepared for immunocytochemistry and viewing by light and confocal microscopy. RESULTS: In all mammalian retinas investigated, intense GCAP1 immunoreactivity (GCAP1-IR) was seen in cone photoreceptor inner and outer segments, cell bodies, and synaptic regions. Intensity of the GCAP1-IR was strong in inner segments of rods in all species but weaker in outer segments-particularly so in primates and cats. GCAP2 immunoreactivity (GCAP2-IR) was weak in bovine, mouse, and rat cones but was intense in human and monkey cones. In all species except primates, GCAP2 staining was intense in rod inner and outer segments. In primates GCAP2-IR was intense in the rod inner segment but faint in the rod outer segment. A striking difference from the GCAP1 pattern of immunoreactivity was seen with GCAP2 antibodies as far as the inner retina was concerned. GCAP2-IR was evident in certain populations of bipolar, amacrine, and ganglion cells in all species. CONCLUSIONS: GCAP1 and GCAP2, which are involved in Ca2+-dependent stimulation and inhibition of photoreceptor guanylyl cyclase, can be detected in mammalian photoreceptor inner and outer segments, consistent with their physiological function. The occurrence of both GCAPs in the synaptic region of the photoreceptors indicates participation of these proteins in pathways other than regulation of phototransduction. The occurrence of GCAP2 in inner retinal neurons is indicative of second-messenger chemical transduction, possibly in metabotropic glutamate, gamma-aminobutyric acid (GABA) receptor, and nitric oxide-activated neural circuits.

Animals↗
PubMed1997

Uniqueness of the S-cone pedicle in the human retina and consequences for color processing.

The purpose of this study was to investigate more fully the shape and content of ribbons and synapses to second-order neurons in the short-wavelength cone (S-cone, blue cone) pedicle and to learn more concerning the uniqueness of the S-cone system in the primate retina. A piece of well-fixed peripheral human retina (10 mm, 35 degrees nasal to the fovea) was serially thick sectioned in the tangential plane from the level of the outer segments to the tops of the cone pedicles. Then serial electron microscope (EM) sections were collected through the whole depth of the pedicle-occupying region into the neuropil of the outer plexiform layer (OPL). The resultant EM micrograph montages of a large field of cone pedicles were perused, and S-cone pedicles were identified. Serial micrographs of a single S-cone pedicle, picked out of the montages, were digitized and reconstructed by computer three-dimensional methods. The S-cone pedicle arose from a slightly oblique axon and projected 0.5-1 microm more vitread in the OPL than other cone pedicles. It was bilobed in shape, with synaptic invaginations and ribbons in both lobes. No cone-contacting telodendria projected from the S-cone pedicle itself, but a small number of neighboring cones sent telodendria to its surface to make small gap junctions. Neighboring rod spherules also made small gap junctions. Four robust bipolar cell dendrites, most likely from S-cone-specific bipolar cells, made synapses at ribbons and basal (distal) junctions. A small number of other bipolar cell dendrites made narrow-cleft basal junction only. The majority of lateral elements were thought to be from HII horizontal cells, and a minority from HI horizontal cells. We conclude that the S-cone pedicle has a unique morphology and connectivity to second-order neurons that makes it quite different from the other two longer wavelength cone systems, and we speculate on the consequences for color processing in the visual system in general.

Adult↗
PubMed1997

Soluble CD44 splice variants in metastasizing human breast cancer.

The local expression of CD44 splice variants in human breast cancer tissue has been previously shown to be associated with metastasis. We show here that elevated systemic serum levels of CD44 splice variants occur in breast cancer and may represent a new tool for staging and differential diagnosis. Sera of node-negative and node-positive breast cancer patients in comparison with healthy control subjects were analyzed for serum CD44 (sCD44) and 2 different splice variants (v5 and v6). Node-positive breast cancer patients showed significantly (p < 0.01) elevated levels of sCD44-v5 and -v6 splice variants in comparison to node-negative patients and healthy controls. None of the node-negative breast cancer patients or healthy controls showed elevated levels of both sCD44-v5 and -v6. Interestingly, no differences were seen for serum levels of non-spliced sCD44-standard between the 3 groups. Soluble forms of CD44 variants may promote migration of tumor cells. This may occur through interference with tumor cell adhesion or by modulation of immune defense mechanisms.

Alternative Splicing↗
PubMed1997

Neural circuitry and light responses of the dopamine amacrine cell of the turtle retina.

PURPOSE: To understand the circuitry and electrophysiology of the dopamine cells in the turtle retina. METHODS: Preembedding immunocytochemistry for tyrosine hydroxylase (Toh) was done on vibratome sections of turtle retina. Resultant Toh-immunoreactive (Toh-IR) amacrine cells were then serially thin-sectioned for analysis by electron microscopy (EM). Some sections of Toh-IR cells also were post-embedding immunostained for glycine and GABA content. Intracellular recordings and dye markings were made from the turtle eyecup and slice preparation to determine the light responses of cells called A28, which have the same morphology as Toh-IR amacrine cells. RESULTS: Physiologically A28 cells were L-type (luminosity) and gave sustained depolarizing (ON-center) responses to light pulses. High intensity light pulses produced immediate transients and long depolarizations, lasting beyond the stimulus duration. An after-hyperpolarization and an antagonistic surround could be elicited. EM reconstruction of a Toh-IR cell revealed new circuitry over that described before (Pollard, J. & Eldred, W.D. (1990). J. Neurocytol. 19, 53-66). Bipolar ribbon synapses occurred in all three dendritic tiers. However, amacrine cell inputs dominated numerically (95% amacrine input, 5% bipolar input) many of them in a serial synaptic configuration. GABA+ inputs were seen but not glycine+ inputs. Output from Toh-IR profiles was primarily to large ganglion cell dendrites but also to bipolar cell axons, GABA-IR amacrines, unspecified amacrine cells and other Toh-IR dendrites. CONCLUSIONS: The synaptology of the dopamine cells of the turtle retina suggests that sustained inhibitory amacrine cell pathways, including GABAergic pathways, are chiefly responsible for their response characteristics at low light levels. Conversely, at higher light intensities, transient excitatory amacrine cells probably have influence.

Animals↗
PubMed1997

Active stage of autoimmune diabetes is associated with the expression of a novel cytokine, IGIF, which is located near Idd2.

Recently, interferon-gamma-inducing-factor (IGIF) has been described as a novel monokine that is a more potent interferon-gamma (IFN-gamma) inducer than IL-12. By cloning IGIF from affected tissue and studying IGIF gene expression, we describe for the first time a close association of this cytokine with an autoimmune disease. The non-obese diabetic (NOD) mouse spontaneously develops autoimmune insulitis and diabetes which can be accelerated and synchronized by a single injection of cyclophosphamide. IGIF mRNA was demonstrated by reverse transcriptase PCR in NOD mouse pancreas during early stages of insulitis. Levels of IGIF mRNA increased rapidly after cyclophosphamide treatment and preceded a rise in IFN-gamma mRNA, and subsequently diabetes. Interestingly, these kinetics mimick that of IL-12p40 mRNA, resulting in a close correlation of individual mRNA levels. Cloning of the IGIF cDNA from pancreas RNA followed by sequencing revealed identity with the IGIF sequence cloned from Kupffer cells and in vivo preactivated macrophages. When extending our study to macrophages of the spleen we observed that NOD mouse macrophages responded to cyclophosphamide with IGIF gene expression while macrophages from Balb/c mice treated in parallel did not. The IGIF gene position is located within the Idd2 interval on mouse chromosome 9 and therefore it is a candidate for the Idd2 susceptible gene. We conclude that IGIF expression is abnormally regulated in autoimmune NOD mice and closely associated with diabetes development.

Animals↗
PubMed1997

Immunostaining with antibodies against protein kinase C isoforms in the fovea of the monkey retina.

We reported previously that an antibody to the alpha isoform of protein kinase C (PKC) immunostained rod bipolar cells and bipolar cells that could be blue-cone (B-cone)-specific in postmortem human retina (Kolb et al. (1993) Vis. Neurosci. 10:341-351). In addition, we showed that antibodies to the beta isoform of PKC immunostained cone system bipolar, amacrine, and ganglion cells. Since the fixation of the human material was poor, we were unable to make positive identifications of the specific cell types that were immunoreactive, particularly in the case of PKC-beta antibodies. Thus, herein we have repeated the study on well-fixed monkey foveal retina. PKC-alpha immunoreactivity (IR) was restricted to a single type of cone bipolar cell that contacted only a minority of the cone pedicles at central invaginating contacts of ribbon triads. This bipolar type shares some morphological characteristics of B-cone-specific bipolar cells of primate retina. PKC-beta immunoreactivity was found in cone bipolar cells that made primarily basal contacts with cone pedicles and had axon terminals in sublamina alpha of the inner plexiform layer (IPL). Immunoreactivity also occurred in a type of cone bipolar that made central element contacts and had axon terminals in sublamina b of the IPL. Some ganglion cells, particularly those postsynaptic to flat midget bipolar cells also exhibited PKC-beta-IR. One type of amacrine with an 8 microns diameter cell body showed strong PKC-beta-IR. It was postsynaptic to cone bipolar cells in both sublamina a and b and presynaptic to bipolar axons, other immunoreactive amacrine cells, and ganglion cell dendrites and bodies. The other amacrine cell type showed less strong PKC-beta-IR, large-bodied (12-15 microns cell body diameter), and probably diffuse in branching pattern. The latter interacted with the intensely immunoreactive amacrines, bipolars, and ganglion cells. By comparison to cat and primate retinas where morphology and physiology of many retinal neurons are well documented, we suggest that PKC-beta may be specific to flat midget, flat diffuse, and invaginating diffuse cone bipolar cells and to at least two amacrine cells. Some of these neural types are proposed to be involved in OFF-center cone pathways in the monkey retina.

Animals↗
PubMed1997

Systemic production of interferon-gamma inducing factor (IGIF) versus local IFN-gamma expression involved in the development of Th1 insulitis in NOD mice.

We report that the onset of Th1 insulitis is preceded by a rise of interferon-gamma inducing factor (IGIF) mRNA expression in the spleen. This systemic shift towards Th1 reactivities was underlined by a close correlation of IGIF and IL-12p40 mRNA levels in the spleen, as determined by semi-quantitative RT-PCR. Cyclophosphamide-induced IGIF expression was also observed in MHC congenic NOR mice, but not in MHC class II-incompatible NON mice. The systemic rise of IGIF was followed by the development of destructive Th1-associated intra-insulitis. Interestingly, immunohistochemistry showed IL-4-positive cells evenly dispersed throughout the infiltrate, while IFN-gamma-positive cells were restricted to the vicinity of beta-cells. We conclude that cyclophosphamide induces a systemic shift in antigen presenting cells towards favouring Th1 responses, in an MHC dependent manner. Despite this general bias in immune reactivity, activation of Th1 cells in insulitis occurs only close to beta-cells, indicating a crucial role of antigen presentation by beta-cells or in their immediate vicinity.

Animals↗
PubMed1997

Suppression of cyclophosphamide induced diabetes development and pancreatic Th1 reactivity in NOD mice treated with the interleukin (IL)-12 antagonist IL-12(p40)2.

The macrophage product interleukin (IL)-12 is known to drive Th1 reactions in physiological and pathological immune responses. Here we report that treatment with the homodimeric IL-12p40 subunit, an antagonist of the bioactive IL-12p35/p40 heterodimer, suppresses diabetes development in cyclophosphamide-injected NOD mice. Female mice of 70 days old received cyclophosphamide (250 mg/kg) to accelerate and synchronize diabetes development, and daily injections of 1 microgram IL-12(p40)2. While there was no delay of the first diabetes cases, the incidence of overt diabetes was significantly decreased in treated mice (46 vs 23%, p < 0.05). Analysis of mRNA expression in the pancreas showed that administration of the IL-12 antagonist had dampened interferon-gamma gene expression, decreased the ratio of interferon-gamma/IL-10 mRNA levels and in parallel suppressed the expression of the inducible nitric oxide synthase. At the same time intra-islet infiltration was significantly decreased (p < 0.001). Interestingly, the administration of IL-12(p40)2 also affected IL-12 gene expression, by downregulation of p35 mRNA. We conclude that IL-12 p40 homodimer suppresses diabetes development in the NOD mouse by dampening islet inflammation via selective down-regulation of Th1 type responses. The naturally occurring IL-12 antagonist IL-12(p40)2 represents a new and specific Th1 directed approach to prevent autoimmune diabetes.

Animals↗
PubMed1997

Oral insulin for diabetes prevention in NOD mice: potentiation by enhancing Th2 cytokine expression in the gut through bacterial adjuvant.

Oral administration of insulin suppresses the development of diabetes in nonobese diabetic (NOD) mice and deviates the cytokine balance in the islets of Langerhans from a Th1 to a Th2 type cytokine pattern. However, the effect of oral insulin is limited and disease suppression is limited to a narrow dose range. Therefore we tried to improve the outcome of suboptimal insulin dosing by bacterial adjuvant. Mice treated with a suboptimal dose of oral insulin showed no change in diabetes incidence although a shift from Th1 towards Th2 cytokine expression occurred in inflamed islets. Significant suppression of diabetes development was only seen in NOD mice receiving both, insulin and the bacterial preparation OM-89 as adjuvant. OM-89 is a protein extract of Escherichia coli, with nonspecific immunostimulatory properties. Potentiation of the effect of oral insulin by the adjuvant was associated with upregulation of interleukin (IL)-4 Th2 cells in infiltrated islets and sustained local IL-2 gene expression. RT PCR analyses of cytokine expression in the gut showed a clear deviation to Th2 type reactivity and downregulation of inducible nitric oxide (NO) synthase (iNOS) expression by the bacterial adjuvant but not by oral insulin alone. Since macrophages are the primary target cells of adjuvant action we tested its effect on mouse macrophages in vitro. Treatment with OM-89 induced transient release of tumour necrosis factor alpha and nitrite but rendered macrophages refractory to restimulation by the potent macrophage activator lipopolysaccharide. In conclusion, the protective effect of oral insulin can be potentiated by pretreatment with the bacterial adjuvant OM-89. This effect correlates with enhanced Th2 cytokine and decreased iNOS gene expression in the gut, probably due to the downregulation of proinflammatory mediators by exposure to the adjuvant.

Adjuvants, Immunologic↗
PubMed1997

Amacrine cells of the mammalian retina: neurocircuitry and functional roles.

Since amacrine cells are important interneurons of the inner retina and their activity may be detected in certain waveforms of the electroretinogram, this paper reviews their morphologies, classification, mosaics, neurotransmitter content, neural circuitry and physiological responses to light. Nine different amacrine cell types of cat, rabbit and human retinas are presently quite well studied in terms of the aforementioned aspects and are described in detail in this paper.

Animals↗
PubMed1997

Dobesilate enhances endothelial nitric oxide synthase-activity in macro- and microvascular endothelial cells.

1. Dobesilate is used for normalizing vascular dysfunction in a number of diseases. In search for an effect on endothelial NO production, macrovascular endothelial cells from rat aorta, microvascular endothelial cells from rat exocrine pancreatic tissue, and capillary endothelial cells from rat islets, were cultured in the presence or absence of Mg-Dobesilate. The activity of constitutive nitric oxide synthase (ecNOS) in resident cells as well as of inducible nitric oxide synthase (iNOS) in cytokine-activated cells was measured indirectly by recording the citrulline concentrations in culture supernatants. 2. In each of the different endothelial cells Mg-Dobesilate incubation (0.25-1 mM) for 24 h led to a significant and concentration-dependent increase in ecNOS-activities. With cytokine-activated endothelial cell cultures only moderate effects were seen with little or no concentration-dependency. Addition of the NOS-inhibitor N(G)-monomethyl-L-arginine led to a significant suppression of citrulline formation in all cultures as an evidence for the enzyme specificity of these effects. 3. iNOS- and ecNOS-specific reverse transcription and semi-quantitative polymerase chain reaction (RT-PCR) with RNA from resident or cytokine-activated endothelial cells gave no evidence for an increase in NOS-specific mRNA after Mg-Dobesilate-treatment. Furthermore, Dobesilate-mediated enhancement of NO synthesis in resting endothelial cells was not due to iNOS induction in these cells, as no iNOS-specific signal was found by RT-PCR.

Animals↗
PubMed1997

Low stress response enhances vulnerability of islet cells in diabetes-prone BB rats.

In islet cells isolated from normal outbred Wistar rats, the known high vulnerability of islet cells toward oxygen radicals or nitric oxide can be abolished by inducing a stress response, such as by heat shock. We show here that islet cells from diabetes-prone BB rats are unable to mount such a protective response. Islet cells from diabetes-prone BB rats without recognizable insulitis were heat stressed. Subsequently, cells were exposed to nitric oxide, to oxygen radicals, or to the beta-cell toxin streptozotocin. While prior heat shock substantially increased the survival of toxin-treated Wistar rat islet cells, no protective stress response was noted for islet cells from diabetes-prone BB rats. Islet cells from diabetes-resistant BB rats were protected by heat stress to the same extent as Wistar rats. A survey of four additional major histocompatibility complex (MHC)-disparate rat strains confirmed the existence of a low and high responder type to stress. Parallel analysis of heat shock protein (hsp)70 induction by Western blot showed a low and high hsp70 response phenotype. A high hsp70 response coincided with a protective stress response. The presence (or absence) of a protective stress response correlated with the preservation (or loss) of intracellular NAD+ in toxin-treated islet cells. The lack of a protective stress response in islet cells from diabetes-prone BB rats, but not in diabetes-resistant BB rats, may promote beta-cell lysis and autoantigen release, and hence could be important for initiation or propagation of the disease process.

Animals↗
PubMed1997

Systemic bias of cytokine production toward cell-mediated immune regulation in IDDM and toward humoral immunity in Graves' disease.

Disturbed immune regulation has been postulated to be crucial in the pathogenesis of IDDM and other autoimmune or allergic diseases. We therefore tested the hypothesis of a general bias in the peripheral immune system in patients with recent-onset IDDM or Graves' disease in comparison to healthy control subjects by studying whole blood cultures stimulated with phytohemagglutinin. Cells from IDDM patients (n = 53) produced significantly higher amounts of Th1 cytokines gamma-interferon (IFN-gamma) (P = 0.028) and tumor necrosis factor alpha (TNF-alpha) (P = 0.007) than normal control subjects (n = 56), while Th2 cytokine levels (interleukin [IL]-4, IL-10) were similar. Low levels of islet cell antibodies (ICAs) in IDDM patients were associated with high levels of Th1 and Th2 cytokines. Antibodies to GAD, ICA512, or insulin did not correlate with individual cytokine profiles. Also, HLA-DQ types did not significantly correlate with either Th1 or Th2 cytokine production. Conversely, whole blood cultures from patients with Graves' disease (n = 18) produced significantly less TNF-alpha and IL-4 than normal subjects (P = 0.001-0.006). However, when the balance between Th1 and Th2 cytokine production was analyzed in individuals, the ratio between IFN-gamma or TNF-alpha and IL-4 or IL-10 was clearly biased toward Th1 reactivity in patients with IDDM (P = 0.0001), while a dominance of Th2 cytokine production was seen in Graves' disease (P = 0.0001). The ratio of counterregulatory cytokines appeared to be the most reliable marker of the individual disease process. This study provides first evidence of a systemic bias in the immune regulation of humans, which might be either toward cell-mediated immunity (Th1) in IDDM or humoral immunity (Th2) in Graves' disease.

Adolescent↗