Mutations in ABCR (ABCA4) in patients with Stargardt macular degeneration or cone-rod degeneration.

PURPOSE: To determine the spectrum of ABCR mutations associated with Stargardt macular degeneration and cone-rod degeneration (CRD). METHODS: One hundred eighteen unrelated patients with recessive Stargardt macular degeneration and eight with recessive CRD were screened for mutations in ABCR (ABCA4) by single-strand conformation polymorphism analysis. Variants were characterized by direct genomic sequencing. Segregation analysis was performed on the families of 20 patients in whom at least two or more likely pathogenic sequence changes were identified. RESULTS: The authors found 77 sequence changes likely to be pathogenic: 21 null mutations (15 novel), 55 missense changes (26 novel), and one deletion of a consensus glycosylation site (also novel). Fifty-two patients with Stargardt macular degeneration (44% of those screened) and five with CRD each had two of these sequence changes or were homozygous for one of them. Segregation analyses in the families of 19 of these patients were informative and revealed that the index cases and all available affected siblings were compound heterozygotes or homozygotes. The authors found one instance of an apparently de novo mutation, Ile824Thr, in a patient. Thirty-seven (31%) of the 118 patients with Stargardt disease and one with CRD had only one likely pathogenic sequence change. Twenty-nine patients with Stargardt disease (25%) and two with CRD had no identified sequence changes. CONCLUSIONS: This report of 42 novel mutations brings the growing number of identified likely pathogenic sequence changes in ABCR to approximately 250.

Frequency of mutations in the gene encoding the alpha subunit of rod cGMP-phosphodiesterase in autosomal recessive retinitis pigmentosa.

PURPOSE: To determine the mutation spectrum of the PDE6A gene encoding the alpha subunit of rod cyclic guanosine monophosphate (cGMP)phosphodiesterase and the proportion of patients with recessive retinitis pigmentosa (RP) due to mutations in this gene. METHODS: The single-strand conformation polymorphism (SSCP) technique and a direct genomic sequencing technique were used to screen all 22 exons of this gene for mutations in 164 unrelated patients with recessive or isolate RP. Variant DNA fragments revealed by SSCP analysis were subsequently sequenced. Selected alleles that altered the coding region or intron splice sites were evaluated further through segregation analysis in the families of the index cases. RESULTS: Four new families were identified with five novel mutations in this gene that cosegregated with disease. Combining the data presented here with those published earlier by the authors, eight different mutations in six families have been discovered to be pathogenic. Two of the mutations are nonsense, five are missense, and one affects a canonical splice-donor site. CONCLUSIONS: The PDE6A gene appears to account for roughly 3% to 4% of families with recessive RP in North America. A compilation of the pathogenic mutations in PDE6A and those reported in the homologous gene PDE6B encoding the beta subunit of rod cGMP-phosphodiesterase shows that the cGMP-binding and catalytic domains are frequently affected.

Further characterization of histamine releasing chemokines present in fractionated supernatants derived from human mononuclear cells.

BACKGROUND: Histamine releasing factors (HRF) are members of the beta chemokine family of cytokines and have been characterized using recombinant proteins. Mononuclear cell and/or platelet supernatants have been shown to contain HRF and the initial void peak obtained using Mono Q anion exchange chromatography possesses such activity, as do two later peaks eluted from the column. OBJECTIVE: We wished to further characterize the activity present in the void peak and determine which of the chemokines present are responsible for the activity measured. METHODS: We fractionated the void peak obtained from Mono Q chromatography on Mono S. The elution profile of individual chemokines was determined and the fractions were assayed for histamine releasing capability. We used monospecific antisera to inhibit the activity and quantitate the contribution of each protein. RESULTS: The fractions contained MCP-1/MCAF, CTAPIII/NAP-2, IL8, and a small quantity of RANTES. About 90-95% of the total histamine containing capability was attributable to MCP-1/MCAF. There was a small contribution by CTAPIII/NAP-2, and RANTES, and no activity associated with IL8. CONCLUSION: MCP-1/MCAF is the critical HRF present in the initial void peak obtained by anion exchange chromatography of supernatants derived from human mononuclear cells and platelets. The alpha chemokine CTAPIII/NAP-2 has relatively weak activity and IL8 has none although they are prominent in this fraction and overlap with MCP-1/MCAF. RANTES makes a minor contribution but most of it is eluted in a later peak.

Chemokines of the alpha, beta-subclass inhibit human basophils' responsiveness to monocyte chemotactic and activating factor/monocyte chemoattractant protein-1.

Monocyte chemotactic and activating factor (MCAF) is the most potent cytokine that activates basophils to release histamine. The response of human basophils to either simultaneous or sequential addition of the chemokines RANTES, macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, platelet factor (PF)4, connective tissue activating peptide III (CTAP-III), interleukin (IL)-8, and inflammatory protein (IP)-10 on MCAF-induced histamine release was studied. Simultaneous addition of MCAF and any of the chemokines studied evoked an augmented response as measured by histamine release, whereas preincubation of leukocytes or purified basophils (80%) with these chemokines decreased MCAF-induced histamine release in a dose-dependent manner. Histamine release by anti-IgE remained unchanged. When tested at 5 x 10(-9) mol/L, the decrease in histamine release by RANTES was 69.2% +/- 3.5%, by MIP-1 alpha 48.8% +/- 3.1%, by MIP-1 beta 42.9% +/- 3.1%, by PF4 56.5% +/- 2.9%, by IL-8 41.2% +/- 2.2, by CTAP III 27% +/- 4.4%, and by IP-10 15.3% +/- 2.6%. The peak inhibition of histamine release by the chemokines was reached within 10 minutes of preincubation with basophils and remained unchanged thereafter. Washing basophils after preincubation with chemokines abolished the inhibition, with the exception of desensitization by low concentrations of MCAF. With the exclusion of MCAF and RANTES, none of the chemokines (at the concentration range of 5 x 10(-8) to 5 x 10(-11)) induced significant (> 10% above spontaneous) histamine release from basophils. Preincubation of basophils with C5a (5 x 10(-10) mol/L) did not affect histamine release, whereas preincubation with granulocyte-macrophage colony-stimulating factor (10 ng/ml) or IL-5 (10 ng/ml) enhanced MCAF-induced histamine release by 121.8% +/- 10.1% and 108% +/- 10.8%, respectively. We have therefore characterized RANTES, MIP-1 alpha, MIP-1 beta, CTAP III, PF4, IL-8, and IP-10 as inhibitors of MCAF-induced histamine release. Although the results are consistent with receptor blockade, the alpha and beta chemokines appear to interact with separate receptors linked to G proteins; thus, a mechanism of receptor class desensitization is proposed. Interaction of this group of cytokines at the site of allergic inflammation may modulate a function of basophils to initiate, augment, or inhibit histamine release.

Characterization of the human basophil response to cytokines, growth factors, and histamine releasing factors of the intercrine/chemokine family.

We have tested the histamine releasing properties and priming abilities of a wide range of recombinant or purified cytokines and growth factors on the basophils of 20 subjects (10 atopic and 10 nonatopic). We found that monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 (MCAF/MCP-1), RANTES, human macrophage inflammatory protein-1 alpha and human inflammatory protein-1 beta, Connective tissue activating peptide III and Neutrophil Activating Peptide-2 (NAP-2) cause histamine release from basophils and are all members of the intercrine/chemokine family. MCAF/MCP-1 was as potent as anti-IgE or C5a and it is clearly the major contributor to histamine releasing factor activity. RANTES was the second major histamine releasing factor among the positive cytokines. Both MCAF/MCP-1 and RANTES are present in conditioned mononuclear cell media and can be separated using Mono Q anion exchange chromatography. We also demonstrated that RANTES has unusual chromatographic properties in spite of its isoelectric point of > 9.0 because it is largely found in peak-2 of the Mono Q column rather than peak-1 in which intercrines such as MCAF/MCP-1, IL-8, and connective tissue activating peptide III are found. All other cytokines and growth factors tested were negative, with the exception of IL-3, which caused histamine release in a subpopulation of subjects, and also primed basophils for release by anti-IgE. Other basophil primers for anti-IgE-dependent histamine release were IL-5, mast cell growth factor (c-kit ligand), and insulin-like growth factor II. Using specific neutralizing antibodies we have shown that MCAF/MCP-1, RANTES, and IL-3 contribute significantly to the activity found in mononuclear cell culture supernatants. Granulocyte-macrophage-CSF, IP-10, I-309, IL-7, IL-8, IL-9, IL-10, IL-11, IgE-binding factor, TNF-alpha, TGF-beta 1, fibroblast growth factor, epidermal growth factor, and endothelial cell growth factor were negative for direct histamine release and as primers of basophils. Our results indicate that cytokines belonging to the intercrine/chemokine family are major constituents of the activity known as "histamine releasing factor" found in MNC supernatants.

RANTES, a monocyte and T lymphocyte chemotactic cytokine releases histamine from human basophils.

Chemotaxis of different populations of cells and release of proinflammatory mediators in response to antigenic stimulation are important processes in allergic diseases. These lead to the late phase response, a hallmark of chronic allergic diseases. Recombinant RANTES, a member of the "intercrine/chemokine" family of cytokines, has been previously shown to be chemotactic for monocytes and T cells of memory/helper phenotype. In this manuscript, we show that it is capable of inducing histamine release from human basophils at concentrations as low as 10(-10) M and compare its activity with that of monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 (MCAF/MCP-1), another intercrine/chemokine. RANTES (10(-7) M) caused histamine release from the leukocytes of 26 of 33 donors tested (mean 21.8 +/- 3.1%). In the same group of donors, MCAF/MCP-1, goat anti-human IgE (anti-IgE; 1 microgram/ml), and FMLP (10(-5) M) released 41.1 +/- 2.9%, 40.5 +/- 4.6%, and 44 +/- 3.1% histamine, respectively. The percent histamine release by RANTES in atopic vs nonatopics was 30.3 +/- 6.7 and 16.5 +/- 2.4, respectively (p less than 0.05), and histamine release by RANTES correlated significantly with histamine release by MCAF (r = 0.69; p less than 0.001) but not with histamine release by anti-IgE (r = 0.29; p greater than 0.05). Histamine release by RANTES and MCAF/MCP-1 was extremely rapid, reaching a maximum within 1 min. RANTES was also shown to activate highly purified basophils (80% pure), and its activity was inhibited by a polyclonal anti-RANTES antibody. At a suboptimal concentration (6 x 10(-9) M), RANTES did not prime basophils to enhance histamine release by secretagogues such as anti-IgE, C5a, or FMLP. On the other hand, preincubation of basophils with RANTES or MCAF/MCP-1 desensitized basophils to either factor but not to anti-IgE, C5a, or FMLP. Preincubation of basophils with pertussis toxin markedly diminished the basophil response to either RANTES or MCAF/MCP-1. These results suggest that RANTES and MCAF/MCP-1: 1) are potent activators of basophils; 2) may function via the same, or a closely related, receptor system in basophils; and 3) may represent a link between activation of monocytes, lymphocytes, and basophils in inflammatory disorders such as the late phase allergic reaction.

Monocyte chemotactic and activating factor is a potent histamine-releasing factor for human basophils.

Recombinant monocyte chemotactic-activating factor (MCAF) has been shown to induce histamine release from human basophils with a dose response between 10(-9) and 10(-6) M. The peak of activity was reached at 10(-7) M. Histamine release by MCAF was rapid with an initial rate comparable with histamine release by an optimal dose of anti-IgE. MCAF led to peak histamine release within 1 min. 80% of the subjects tested were responsive to MCAF or anti-IgE, while all were responsive to FMLP. The percentage histamine release by MCAF was, however, less than that seen with anti-IgE or FMLP, but this was attributable to a lesser percent release in nonatopic subjects; atopic subjects responded similarly to all three agonists. MCAF was also shown to activate highly purified human basophils more readily than mixed leukocytes, and its activity was inhibited by a polyclonal rabbit antibody. At a suboptimal concentration (2.5 x 10(-9) M), MCAF was unable to prime the basophil to histamine release by other secretagogues. However, interleukin 3 (IL-3) and IL-5 could each prime basophils for MCAF-induced secretion. Therefore, our results suggest that MCAF may be a major contributor to the histamine-releasing activity seen in peripheral blood mononuclear cell supernatants that has been designated histamine releasing factor(s).