Biomedical subjects

Jean T Jacob

Publications and source records attributed to Jean T Jacob.

9 recordsLinked to original sources

PubMed2007

Single eye analysis and contralateral eye comparison of tear proteins in normal and dry eye model rabbits by MALDI-ToF mass spectrometry using wax-coated target plates.

A study of rabbit tear protein expression in a dry eye rabbit model was performed to determine if a pattern in expressed proteins could be identified. The uniqueness of the model allows the comparison of normal (control) eye tear protein expression with surgically induced dry eye tear protein expression in individual animals. The sensitivity of the method allows for single eye analysis. One-dimensional mini-gel electrophoresis of the tear proteins did not show substantial differences between band patterns of the normal versus the dry eye, but was used to assess the molecular weight ranges of the major proteins. Specific assignments of some of the predominant proteins were obtained by tandem mass spectrometry (MS) which showed that the lower molecular weight lipid-binding proteins (approximately 10 kDa to 36 kDa) constitute a considerable amount of the observed protein, followed in lesser quantities by the transferrins which have higher molecular weights ranging from 70 kDa to 85 kDa. Enhancement of matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS linear mode analysis of intact proteins in tear fluid was demonstrated through the use of wax-coated MALDI plates and spot washing. MALDI-ToF MS analysis of the expressed tear proteins illustrates that differences between normal eye tear and dry eye tear protein content are manifested in changes in the lower molecular weight lipid-binding proteins such as lipophilin which exhibits an increase in concentration in the dry eye, and beta-2 microglobulin which undergoes a decrease.

Animals↗

PubMed2007

Tissue-engineered tear secretory system: functional lacrimal gland acinar cells cultured on matrix protein-coated substrata.

Dry eye is a general term that refers to a myriad of ophthalmic disorders resulting in the inadequate wetting of the corneal surface by the tear film. Dry eyes are typically treated by the application of artificial tears. However, patients with lacrimal insufficiencies such as Stevens-Johnson syndrome, chemical and thermal injuries, or ocular cicatricial pemphigoid have very limited options because of the short duration and action of lubricating agents. As a therapeutic strategy, we are working to develop a bioengineered tear secretory system for such patients. This article describes the growth and physiological properties of purified rabbit lacrimal gland acinar cells (pLGACs) on several matrix protein-coated polymers such as silicone, collagen I, copolymers of poly-D,L-lactide-co-glycolide (PLGA; 85:15 and 50:50), poly-L-lactic acid (PLLA), and Thermanox plastic cell culture coverslips. Monolayers of acinar cells were established on all of the polymeric substrata. An assay of beta-hexosaminidase activity in the supernatant medium showed significant increases in protein secretion, following stimulation with 100 microM carbachol on matrix protein-coated and uncoated polymers such as silicone, PLGA 85:15, and PLLA. Our study demonstrates that PLLA supported the morphological and physiological properties of purified rabbit lacrimal gland epithelial cells more successfully than the others.

Animals↗

PubMed2006

Identification and comparison of the polar phospholipids in normal and dry eye rabbit tears by MALDI-TOF mass spectrometry.

PURPOSE: To identify and compare the phosphorylated lipids in normal and dry eye rabbit tears using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). METHODS: MALDI-TOF MS studies were performed on tear samples from normal and dry eyes of female New Zealand White rabbits. Experimental dry eye was induced by complete removal of the main and accessory lacrimal glands and nictitating membranes. A solid ionic crystal MALDI matrix of paranitroaniline and butyric acid was used to enhance the mass spectral responses of the phospholipids. In addition, a novel lipid isolation, preconcentration, and clean-up method using pipettes containing immobilized metal ion affinity chromatography (IMAC) medium was used. RESULTS: The polar phospholipids present in the normal and dry eye rabbit tears showed both similarities and differences. Species related to platelet-activating factor (PAF) and/or lyso-phosphatidylcholine (lyso-PC), phosphatidylcholine (PC), and sphingomyelin (SM) were found in both the normal and dry eye rabbit tears. However, the number of types and the concentrations of SM molecules were markedly greater in the dry eye tears than in the normal tears. In addition, phosphatidylserine (PS) species that were readily detectable in dry eye tears were not found in normal tears. CONCLUSIONS: The combination of immobilized metal ion affinity chromatography and the solid ionic crystal matrix for MALDI enabled the detection and study of phosphorylated lipids in the tears. Specific differences between phospholipid levels in normal and dry eye tears were observable with this methodology. The appearance of various SM species only in the dry eye tears may provide markers for this disease state in the future.

Animals↗

PubMed2005

MALDI-TOF MS of phosphorylated lipids in biological fluids using immobilized metal affinity chromatography and a solid ionic crystal matrix.

When targeting a certain class of analytes, such as the phosphorylated lipids in complex biological extracts, interfering species can pose challenges to qualitative and quantitative analyses. Two aspects of lipid analysis were optimized to simplify the isolation and characterization of phosphorylated lipids in biological extracts. A new solid ionic crystal MALDI matrix was synthesized which combined the lipid response enhancing UV-absorber p-nitroaniline with the protonating agent butyric acid. Mass spectra of the extracts containing phosphorylated lipids were simplified by revealing only protonated molecules [M + H]+ of the zwitterionic phosphatidylcholine (PC) headgroup-containing lipids, such as lyso-PC, PC, and platelet-activating factor. For the anionic phosphorylated lipids, such as phosphatidylglycerol, phosphatidic acid, and phosphatidylserine, further spectrum simplification is obtained by the appearance of only the monosodium adducts [M + Na]+ as the major molecular ions, in preference to the double sodium adducts [M + 2Na - H]+. In addition, a new extraction, isolation, and cleanup procedure has been developed to prepare the phosphorylated lipids for MALDI-TOF analysis by the use of immobilized metal ion affinity chromatography media (i.e., ZipTip). The latter procedure was successfully applied to a complex biological tear film lipid layer extract in preparation for MALDI-TOF analysis and phospholipid characterization.

Chromatography, Affinity↗

PubMed2005

Corneal epithelial cell growth over tethered-protein/peptide surface-modified hydrogels.

In this study, we investigated the corneal epithelial cell growth rate and adhesion to novel hydrogels with (1) extracellular matrix proteins [fibronectin, laminin, substance P, and insulin-like growth factor-1 (IGF-1)] and (2) peptide sequences [RGD and fibronectin adhesion-promoting peptide (FAP)] tethered to their surface on poly(ethylene glycol) (PEG) chains. The growth rate to confluence of primary rabbit cornea epithelial cells was compared for plain polymethacrylic acid-co-hydroxyethyl methacrylate (PHEMA/MAA) hydrogels, PHEMA/MAA hydrogels coated with extracellular matrix proteins or peptides, and PHEMA/MAA hydrogels with tethered extracellular matrix proteins or peptides on the surface. The development of focal adhesions by the epithelial cells grown on the surfaces was determined by F-actin staining. Little to no epithelial cell growth occurred on the plain hydrogel surfaces throughout the 15-day culture period. Of the coated hydrogels, only the fibronectin-coated surfaces showed a significant increase in cell growth compared to plain hydrogels (p < 0.009). However, even these surfaces reached a maximum of only 20% confluence. Laminin, fibronectin adhesion-promoting peptide (FAP), and fibronectin/laminin (1:1) tether-modified hydrogels all achieved 100% confluence by the end of the culture period, although the rates at which confluence was reached differed. F-actin staining showed that focal adhesions were formed for the laminin, FAP, and fibronectin/laminin tether-modified surfaces. The results support the hypothesis that tethering certain extracellular matrix proteins and/or peptides to the hydrogel surface enhances epithelial cell growth and adhesion, compared with that seen for protein-coated or plain hydrogel surfaces.

Animals↗

PubMed2005

Corneal epithelial adhesion strength to tethered-protein/peptide modified hydrogel surfaces.

In this study, we investigated the suitability of microjet impingement for use on hydrogel materials to determine the cellular adhesion strength of corneal epithelial cells grown on novel hydrogels with extracellular matrix proteins (laminin and/or fibronectin) or a peptide sequence (fibronectin adhesion promoting peptide, FAP) tethered to their surface with poly(ethylene glycol) chains. The deformation of the hydrogel surface in response to the force of the microjet was analyzed both visually and mathematically. After the results of these experiments and calculations determined that no deformation occurred and that the pressure required for indentation (1.25 x 10(6) Pa) was three factors of 10 greater than the maximum pressure of the microjet, the relative mean adhesion strength of primary rabbit corneal epithelial cells grown on the novel poly(2-hydroxyethyl methacrylate-co-methacrylic acid) hydrogels was determined and compared with that of the same type of cells grown on control glass surfaces. Only confluent cell layers were tested. Cells grown on control glass surfaces adhered with a mean relative adhesion strength of 488 +/- 28 dynes/cm2. Under identical conditions, cells grown on laminin- and FAP-tethered hydrogel surfaces were unable to be removed, indicating an adhesion strength greater than 516 dynes/cm2. Cells grown on fibronectin- and fibronectin/laminin (1:1)-tethered surfaces showed significantly lower relative adhesion strengths (201 +/- 50 and 189 +/- 11 dynes/cm2, respectively), compared with laminin- and FAP-tethered surfaces (p = 0.001). Our results demonstrate that the microjet impingement method of cell adhesion analysis is applicable to hydrogel substrates. Additionally, analysis of our test surfaces indicates that fibronectin tethered to this hydrogel in the quantity and by the method used here does not induce stable ligand/receptor bonding to the epithelial cell membrane to the same degree as does laminin or FAP.

Animals↗

PubMed2004

Identification, quantification and comparison of major non-polar lipids in normal and dry eye tear lipidomes by electrospray tandem mass spectrometry.

Millions of individuals suffer from a health condition known as keratoconjunctivitis sicca (KCS, also known as 'dry eye'). Studies have indicated that the lipids in the tear film layer, which covers the outer portion of the eye, may be directly correlated with the existence of dry eye syndrome. By identifying and comparing the major, non-polar lipids in normal eye tears with a dry eye model, it may be possible to identify a symptom of, or a contributing factor to, dry eye. Electrospray tandem mass spectrometry (ES-MS/MS) was used to identify and compare the non-polar lipids, detected as lithium adducts, from normal and dry eye tear samples obtained from rabbits. A limited number of normal human tear samples were also examined for lipid content, and a close resemblance to rabbit was observed. Three distinct regions were delineated in the ES mass spectra of the non polar lipids, m/z 20-500, 500-800 and 800-1100. A common feature noted among identified lipid components was a glycerol backbone with fatty acyl substituents attached. Product ion spectra were obtained for lithiated monoacyl-, 1,2- and 1,3-diacyl- and triacylglyceride standards. Newly proposed structures and fragmentation pathways for the major product ions are presented for the 1,2- and 1,3-diglycerides, and also for the monoglyceride. New approaches to distinguishing asymmetric 1,2-diglycerides and 1,2- from 1,3-diglycerides are proposed. For the rabbit tear samples, the m/z 20-500 range contains monoester diols with empirical formulas C(n)H(2n)O(4), the m/z 500-800 range includes diesters with empirical formulas C(n)H(2n-2)O(5) and the m/z 800-1100 range contains triesters with empirical formulas C(n)H(2n-4)O(6). Also found in the extracts were three isoprene acetals (terpenoids).

Animals↗

PubMed2004

Tethered protein/peptide-surface-modified hydrogels.

We investigated a wet chemistry method to covalently bond polyethylene glycol (PEG)-tethered extracellular matrix (ECM) proteins (laminin and fibronectin) or peptide (fibronectin-adhesion-peptide sequence) onto the surface of a poly(2-hydroxylethyl methacrylate-co-methylacrylic acid) (PHEMA/MAA) hydrogel that could potentially be used as a replacement for corneal tissue in the eye. An essential requirement for the success of such a surface in the biological environment is its ability to support the growth and attachment of corneal epithelial cells; ECM proteins are known to promote cellular attachment and growth. We hypothesized that the use of tethers or long hydrophilic chains would allow the attached ECM protein/peptide molecules to move two-dimensionally in space, thereby increasing their ability to bind with epithelial cell membranes. Additionally, the tethers would prevent the specifically added growth-enhancing factors from being obscured by any non-specific protein binding occurring on the hydrogel surface. In this surface-modification study, carbodiimidazole (CDI) was used to activate the carboxylic groups (-COOH) on the hydrogel surface in anhydrous dimethylsulfoxide (DMSO) before addition of the PEGylated proteins/peptide. The resulting tethered protein/peptide surface-modified hydrogels were analyzed in terms of percent grafting efficiency, biological activity and mechanical properties. X-ray photoelectron spectroscopy (XPS) and 125I radioactive labeling demonstrated the successful covalent bonding between the moieties and the hydrogel surface. Radiolabeling and enzyme-linked immunosorbent assay (ELISA) studies indicated that the tethered proteins attached to the hydrogel surface at a concentration of approx. 0.1 microg/cm2. ELISA testing further showed that tethered proteins remained biologically active. However, mechanical tensile testing indicated that the mechanical properties of these chemically modified hydrogels were somewhat altered in comparison with the unmodified hydrogel. Future studies will evaluate the cellular response to these surface-modified materials in vitro and in vivo.

Biocompatible Materials↗

PubMed2002

Differences in contact lens-induced responses in the corneas of Asian and non-Asian subjects.

PURPOSE: To investigate ethnic differences in corneal endothelia bleb formation and corneal swelling during contact lens wear under closed-eye conditions, with the use of lenses of varying oxygen transmissibilities (Dk/L) in Asian and non-Asian subjects METHODS: Asian and non-Asian contact-lens naive subjects were fitted with one of four different types of contact lenses at each 20-minute session. During lens wear, the eyelid was closed Endothelial bleb formation was determined by specular microscopy or confocal microscopy immediately on eye opening. Some Asian contact-lens naive subjects were asked to wear one of two types of lenses (Dk/L values of 24 and 175) on each eye for 1 hour Corneal swelling was determined immediately after opening of the eyes. RESULTS: Asian subjects had a significantly higher degree of endothelial bleb formation than the non-Asian population for all closed eye conditions, p=0.0001. There was no significant difference in endothelial bleb formation between closed eves with or without lenses in the non-Asian population. There was a significantly higher degree of bleb formation with low Dk/L lenses, < or = 40, compared to high Dk/L lenses, > or = 110, in Asian subjects, p=0.0001. CONCLUSIONS: There is a significant difference between Asian and non-Asian subjects in the corneal response to stress. Under a closed eyelid only contact lenses with very high levels of Dk/L do not cause cornea stress in Asian patients.

Black or African American↗