Optical coherence tomography-based decision making in exudative age-related macular degeneration: comparison of time- vs spectral-domain devices.

PURPOSE: To determine whether optical coherence tomography (OCT) device-type influences clinical grading of OCT imaging in the context of exudative age-related macular degeneration (AMD). METHODS: Ninety-six paired OCT scans from 49 patients with active exudative AMD were obtained on both the time-domain Stratus OCT system and the spectral-domain Cirrus OCT system at the same visit. Three independent graders judged each scan for the presence of intraretinal fluid (IRF) or subretinal fluid (SRF). The degree of grader consensus was evaluated and the ability of the systems to detect the presence of disease activity was analysed. RESULTS: Cirrus OCT generated a higher degree of inter-grader consensus than Stratus OCT with higher intraclass correlation coefficients for all parameters analysed. A pair-wise comparison of Cirrus OCT with Stratus OCT systems revealed that Cirrus-based gradings more frequently reported the presence of SRF and IRF and detected overall neovascular activity at a higher rate (P<0.05) compared with Stratus-based gradings. CONCLUSIONS: The choice of time-domain (Stratus) vs spectra-domain (Cirrus) OCT systems has a measurable impact on clinical decision making in exudative AMD. Spectral-domain OCT systems may be able to generate more consensus in clinical interpretation and, in particular cases, detect disease activity not detected by time-domain systems. Clinical trials using OCT-based clinical evaluations of exudative AMD may need to account for these inter-system differences in planning and analysis.

Evaluation for synergistic suppression of T cell responses to minor histocompatibility antigens by chloroquine in combination with tacrolimus and a rapamycin derivative, SDZ-RAD.

The 4-aminoquinolines, chloroquine and hydroxychloroquine, can suppress chronic graft-versus-host disease (GVHD) following blood and marrow transplantation (BMT) in mice and humans, respectively. We hypothesized that chloroquine in combination with tacrolimus and the rapamycin derivative SDZ-RAD can synergistically suppress T cell responses and antigen-presenting cell (APC) function in vitro. We used the APC-dependent C57BL/6 anti-BALB.B T cell response and APC-independent anti-CD3epsilon antibody-induced response to evaluate the role of synergism between chloroquine and tacrolimus or SDZ-RAD on each component of a T cell response to minor histocompatibility antigens. We found that chloroquine with tacrolimus had a greater synergistic suppression of APC-dependent compared to the APC-independent T cell responses, with a combination index (CIx) for 50% inhibition by mean effect analysis of 0.16 and 0.50, respectively (a lower number indicates greater suppression). By contrast, chloroquine with SDZ-RAD had a similar CIx between the two responsed 0.50 vs0.45) suggesting only T cell suppression. Synergy between chloroquine and SDZ-RAD involved a direct effect on T cell cytokine production, whereas synergism between chloroquine and tacrolimus was due to an effect on both T cells and APCs. We conclude that the renal-sparing 4-aminoquinolines may be used syneristically with immunosuppressive drugs currently used for BMT.

Identification of a novel truncated isoform of trkB in the kitten primary visual cortex.

Neurotrophins have been shown to play important roles in development and plasticity of the visual cortex (VC). Since signal transduction of neurotrophins is mediated through neurotrophin receptors, we attempted to analyze neurotrophin receptors in the VC. In this study, we isolated cDNAs encoding the intracellular regions of truncated isoforms of the trkB receptor from 30-d-old kitten primary VC. Two distinct truncated isoforms of trkB were isolated and characterized by sequence analyses. One of the isoforms corresponds to the previously described truncated trkB in several mammalian species. The second isoform represents a novel truncated trkB variant form in the kitten VC. Sequence analysis revealed that this contains a sequence that has not yet been reported in any species. This novel isoform, designated trkB.T4, results from alternative splicing 189-bp (63 amino acids) downstream from the splice site giving rise to the first known truncated isoforms of trkB. In the context of recent hypotheses regarding the function of truncated trkB receptors, sequence analysis indicates that trkB.T4 may bear putative signaling/internalization sequences.

Molecular analysis of trkC in the cat visual cortex.

trkC belongs to the trk family of neurotrophin receptors. Several isoforms of trkC have been cloned to date; a full-length catalytic form containing a tyrosine kinase (TK) domain, three full-length isoforms with amino-acid insertions (14, 25, and 39 amino acids) in the TK domain, and five noncatalytic truncated forms that completely lack the TK domain. These isoforms have been studied in several mammalian species, including the pig, rat, mouse, monkey, and human. In this article we report the cloning and sequencing of five trkC isoforms isolated from 30-d postnatal cat visual cortex. The first isoform corresponded to the previously reported full-length trkC transcript containing the 14 amino-acid insert. To search for the presence of other inserts, reverse transcription polymerase chain reaction (RT-PCR) was performed on 30-d postnatal cat visual cortex mRNA using primers that flank the insertion site in the TK domain. Both the isoform containing the 14 amino-acid insert and the isoform lacking any insertion were present in abundant amounts, whereas the other two insert containing isoforms (TK25 and TK39) were much less abundant. The fifth isoform discovered corresponds to the previously reported truncated transcript. Overall, there is a high degree of identity (89-98%) and homology (97-99%) between the cat trkC nucleotide and amino-acid sequences among all mammals. The extracellular juxtamembrane domain was found to be highly divergent among all mammals that have been studied to date. This divergent region also included a proline deletion in the cat trkC sequence. This is the first report of the cloning, sequencing, and RT-PCR analysis of trkC in cat visual cortex, a system extensively studied using anatomical and physiological approaches.

Bivalency and epitope specificity of a high-affinity IgG3 monoclonal antibody to the Streptococcus group A carbohydrate antigen. Molecular modeling of a Fv fragment.

The binding of Strep 9, a mouse monoclonal antibody (mAb) of the IgG3 subclass directed against the cell-wall polysaccharide of Group A Streptococcus (GAS), has been characterized. The intact antibody and proteolytic fragments of Strep 9 bind differently to GAS: the intact mAb and F(ab)2' have greater affinity for the carbohydrate epitope than the monomeric Fab or F(ab)'. A mode of binding in which Strep 9 binds bivalently to portions of the polysaccharide on adjacent chains on GAS is proposed. A competitive ELISA protocol using a panel of carbohydrate inhibitors shows that the branched trisaccharide, beta-D-GlcpNAc-(1-->3)-[alpha-L-Rhap-(1-->2)]-alpha-L-Rhap, and an extended surface are key components of the epitope recognized by Strep 9. Microcalorimetry measurements with the mAb and two synthetic haptens, a tetrasaccharide and a hexasaccharide, show enthalpy-entropy compensation as seen in other oligosaccharide-protein interactions. Molecular modeling of the antibody variable region by homology modeling techniques indicates a groove-shaped combining site that can readily accommodate extended surfaces. Visual docking of an oligosaccharide corresponding to the cell-wall polysaccharide into the site provides a putative model for the complex, in which a heptasaccharide unit occupies the site and the GlcpNAc residues of two adjacent branched trisaccharide units occupy binding pockets within the groove-shaped binding site.

Efficient, convergent syntheses of oligosaccharide allyl glycosides corresponding to the Streptococcus group A cell-wall polysaccharide.

Convergent syntheses of di-, tri, tetra-, penta-, and hexa-saccharide allyl glycosides corresponding to the beta-hemolytic Streptococcus Group A cell-wall polysaccharide are described. The strategy relies on the preparation of related di- and tri-saccharide building blocks: beta-D-Glc pNAc-(1-3)-alpha-L-Rhap and alpha-L-Rhap-(1-2)-[(beta-D-Glc p NAc-(1-3)]-alpha-L-Rhap, which could be used either as glycosyl donors or acceptors in subsequent glycosylation reactions. The protecting groups were chosen to allow the selective removal of the allyl aglycon to access the intermediate glycosyl donors but also to allow their own removal without affecting the allyl group. The allyl group was intended for use in conjugation of the oligosaccharides to soluble protein carriers or solid supports for the preparation of antigens and immunoadsorbents, respectively.