Harold Kochounian

A simple and inexpensive on-column frit fabrication method for fused-silica capillaries for increased capacity and versatility in LC-MS/MS applications

A modified sol–gel method for a one‐step on‐column frit preparation for fused‐silica capillaries and its utility for peptide separation in LC‐MS/MS is described. This method is inexpensive, reproducible, and does not require specialized equipments. Because the frit fabrication process does not damage polyimide coating, the frit‐fabricated column can be tightly connected on‐line for high pressure LC. These columns can replace any capillary liquid transfer tubing without any specialized connections up‐stream of a spray tip column. Therefore multiple columns with different phases can be connected in series for one‐ or multiple‐dimensional chromatography.

The ability of peptides to induce cytotoxic T cells in vitro does not strongly correlate with their affinity for the H-2Ld molecule: implications for vaccine design and immunotherapy.

The hypothesis that the ability of a peptide to bind to a class I molecule correlates with its immunogenicity is controversial. In this paper we have measured the affinity constants of nine synthetic peptides, which have been previously identified as binding to H-2L(d) molecules, and have determined their immunogenicity in an in vitro cytotoxic T lymphocyte (CTL) induction assay. We find that six peptides bind with high affinity (K(a) > 10(7)/M); of these, four are of viral origin but only two elicit potent CTLs, one is a self peptide which is not immunogenic, while the sixth is of bacterial origin and also does not generate effective CTLs. Two peptides bind with intermediate affinity (K(a) > 10(6)/M); one of these elicits a moderate CTL response, while the other, a tumor-derived epitope, is highly immunogenic. Intriguingly, the peptide with lowest affinity (p2Ca) is exceedingly effective at eliciting CTLs. The efficacy of peptides with modest affinity for their restriction elements appears to correlate well with the CTL precursor frequency. We have also examined intrinsic parameters of some of the peptides such as solubility and stability. Taken together, our results underscore the relevance of factors other than affinity which affect immunogenicity and which may be critical in the design of peptide-based vaccines as well as tumor immunotherapy approaches.

Gene therapy in a murine model for clinical application to multiple sclerosis.

Female SJL/J mice, suffering from experimental autoimmune encephalomyelitis (EAE), were injected with 1 × 107 cells from a syngeneic fibroblast line transduced with a retroviral vector designed to encode proteolipid protein (101‐157) targeted for secretion. A striking abrogation of both clinical and histological signs of disease resulted. The treatment was efficacious when given after the first or the third relapses, protected naive mice from challenge with spinal cord homogenate, and was dose dependent. This strategy was devised to provide a systemic, antigen‐specific signal to pathogenic T cells in the absence of costimulation and, hence, render them anergic. Cytokine analyses of brain and spinal cord lymphocytes demonstrate that the treatment induces an antiinflammatory Th2 profile, indicating that this antigen‐specific therapy acts by a cytokine‐induced pathway. This study was designed for translation to the clinic. We envision using allogeneic transduced fibroblasts, encapsulated in a chamber, to deliver the antigen‐specific signal. This will enable us to use one therapeutic cell line for all patients and to remove the device should the therapy exacerbate disease.

Accumulation of extracellular RGR-d in Bruch's membrane and close association with drusen at intercapillary regions.

Human retinal pigment epithelial (RPE) cells synthesize an extraneous splice isoform of retinal G protein-coupled receptor (RGR). In this study, we analyzed the exon-skipping variant of RGR (RGR-d) that is found in extracellular deposits. RPE-choroid tissue sections were prepared from postmortem human eyes from donors of various ages. RGR-d was analyzed in drusen and Bruchs membrane by immunohistochemical localization. Extracellular RGR-d is present in most drusen, including hard, soft, confluent and early-stage. Initial drusen formation is known to be preferentially associated with the intercapillary regions of Bruchs membrane. We corroborated this significant association of drusen, including early-stage drusen, with the intercapillary regions. The distribution of extracellular RGR-d in Bruchs membrane differs in old and young donors. In older persons, nodes of concentrated RGR-d accumulate at intercapillary loci, predominantly at the lateral edges of the capillaries of the choriocapillaris. RGR-d loci at the lateral capillary wall appear numerous in old, but not young, donors. Intensely immunostained RGR-d loci can be found at the base of early-stage drusen mounds in the older donors and may precede the formation of these drusen.