5'-O-beta-D-galactosyl-5-fluorouridine is a prodrug that can be converted by the enzyme beta-D-galactosidase to the potent antineoplastic drug 5-fluorouridine. The prodrug is more than 100x less toxic than the drug to bone marrow cells in Balb/c mice. The ratio of the IC50 of the prodrug to that of the drug determined on a variety of tumor cell lines in vitro ranged from 500:1-1000:1. An antibody-enzyme conjugate (AEC) was synthesized and purified. Maleimide-substituted COL-1 anti-CEA monoclonal antibody was linked to free thiol groups of beta-D-galactosidase. The conjugate was purified by size exclusion and ion exchange chromatography. It retained full immunoreactivity and enzyme activity. After binding to antigen-positive tumor cells, the conjugate was able to activate the prodrug and specifically kill the cells. We are continuing to investigate this model for its potential use in antibody-directed enzyme prodrug therapy (ADEPT).

Osteoarthritis (OA) is a common chronic condition which involves the loss of articular cartilage, the cushion between joints that provides a smooth surface for the bones to glide. Three compounds that make up most of the articular cartilage are type II collagen, proteoglycans, and water. In OA there is an increase in collagenase activity that degrades collagens and proteoglycans necessary for healthy cartilage. The composition of the extracellular matrix changes in OA as evidenced by production of type I collagen. Previous studies in our lab have shown that primary cultures of human osteoarthritic chondrocytes (HOACs) can be reared in serum‐free alginate culture but measurable extracellular matrix endpoints of the OA profile are dampened when chondrocytes isolated from sides of greater and least pathology in the joint are pooled together in culture. In this study, we obtained HOACs from patients undergoing total knee arthroplasty. Femoral condyles and the tibial plateau were labeled greater or least pathology, determined by gross anatomical observation, the cells were isolated and plated separately in three‐dimensional, twelve‐well, alginate cultures at a density of 1.8 × 106 cells per 0.5 mL. Samples were treated for 48 hours from day of plating with either 250nM Extracellular Matrix Protective Factor‐1 (ECPF‐1) or hyaluronic acid conjugated ECPF‐1 (HA‐ECPF‐1). Collagen I degradation and sulfated proteoglycan synthesis were measured in alginate‐associated matrix of the treated cultures. HOACs isolated from the side of greatest pathology responded to treatment by reducing collagen type I degradation (8.42ng/culture Control, 7.25ng/culture ECPF‐1 and 4.25ng/culture HA‐ECPF‐1) and increasing retention of sulfated proteoglycans in the extracellular matrix (0.36ug/culture Control versus 0.56ug/culture ECPF‐1 treated). These data confirm that 48 hour treatment in serum free HOAC culture is sufficient to measure the therapeutic efficacy of an OA drug intervention and that both formulations of ECPF‐1 can slow the progression of OA by altering production of sulfated proteoglycan and collagen type I.Support or Funding InformationThis work was supported in part by a grant from the New Jersey Health Foundation and Philadelphia College of Osteopathic Medicine's Center for Chronic Disorders of Aging and the Division of Research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Osteoarthritis (OA) is a common condition involving the loss of articular cartilage which is primarily made up of type II collagen, proteoglycans, and water. In OA, there is an increase in collagenase activity that degrades collagens and proteoglycans necessary for healthy cartilage and leads to the abnormal production of type I collagen. Previous studies in our lab have shown that primary cultures of human osteoarthritic chondrocytes (HOACs) can be reared in serum‐free, three‐dimensional alginate culture and components of the extracellular matrix (ECM) can be measured. However, we had noted that the ECM phenotype of these cells seemed to be changing when cultured past 5 days. In this study, we obtained HOACs from the femoral condyles and the tibial plateau of patients undergoing total knee arthroplasty. HOACs of greater or least pathology, determined by gross observation, were isolated and plated in 12‐well cultures at a density of 1.8 × 106 cells/0.5 mL alginate. Collagens I and II degradation and proteoglycan synthesis were measured in conditioned media and alginate‐associated matrix of the cultures at days 2, 5, 8 and 11. During long term HOAC culture, collagen degradation was reduced (p<0.001 for coll I) while proteoglycans were retained in the ECM. This trend suggests that long term, three‐dimensional, serum‐free culture of HOACs may revert to a healthier phenotype. The largest changes in extracellular matrix production were demonstrated between days 2 and 5 in HOAC culture giving insight of a treatment window to test possible therapeutics. Support or Funding Information Support was provided by New Jersey Health Foundation Research Award, Cooper Foundation Research Grant and the Division of Research, PCOM.

The pathogenic process that defines osteoarthritis (OA) is the imbalance between excess degradation of the extracellular matrix (ECM) in articular cartilage and a decreased ability of chondrocytes to remodel the matrix. There are many factors that contribute to this disease including unchecked actions of matrix metalloproteinase 13 (MMP‐13), an enzyme specific for the breakdown of collagen type II which is the main component of cartilage extracellular matrix. We have developed a three‐dimensional, serum‐free culture system to rear primary, human OA chondrocytes (HOACs). However, we had noted that the ECM phenotype of these cells seemed to be changing when cultured past 5 days. Primary human articular chondrocytes were isolated from patients who underwent surgery for total knee arthroplasty (TKA). The medial and lateral aspects of the joint samples were kept separated and labeled as greatest or least pathology. The number of live cells were determined and plated at 1.8 × 106 cells/0.5ml alginate per well of a 12‐well plate. Cells were cultured in complete serum‐free media for 11 days with RNA and DNA collected at days 2, 5, 8 and 11. Total RNA and DNA were isolated via Trizol reagent protocol. Quantitation of nucleic acids was accomplished with the NanoDrop system. Over the eleven days in culture there was no statistically significant difference in the total amount of RNA isolated either in the least pathology samples (day 2=4.3ugRNA/culture versus day 11=2.8ugRNA/culture) or the greatest pathology samples (day 2=5.3ugRNA/culture versus day 11=6.2ugRNA/culture). The same was true for the total DNA isolated from the least pathology samples (day 2=1.9ugDNA/culture versus day 11=1.6ugDNA/culture) or the greatest pathology samples (day 2=8ugDNA/culture versus day 11=1.7ugDNA/culture). Future studies include quantitative, real‐time PCR to measure expression of metabolic agents including MMPs and collagens present in OA chondrocytes. This study is the first of its kind to isolate RNA and DNA from completely serum‐free, three‐dimensional cultures of primary human OA chondrocytes. Support or Funding Information This study has been supported in part by the New Jersey Health Foundation and PCOM Division of Research

Periodontal disease is present in 48% of people over the age of 30 years and 70% over the age of 65 years. It is marked by an inflammatory state that affects the gum tissue, periodontal ligaments, and the alveolar bone itself. It has several different causes, but a significant risk factor is smoking because it impairs the healthy biological pathway of repair of the tissues. In a generally healthy patient, there is a balance of broken down collagen and repair with intact collagen. The enzyme responsible for this collagen type 1 degradation is matrix metalloproteinase‐8 (MMP‐8). In this study, the metabolic activity of primary human gingival fibroblasts (HGVF) isolated from inflamed non‐smoker (IFN) and inflamed smoker (IFC) were cultured in serum‐free media over a 48 hour period. We measured production of intact and degraded collagen type I in the secreted fraction (conditioned media) and in the incorporated extracellular matrix. In the secreted and incorporated fractions at both time points (24 and 48hr), HGVF isolated from a smoker (IFC) produced more intact collagen (100% more secreted and incorporated) and more degraded collagen (97% more secreted and 95% more incorporated) than HGVF isolated from the non‐smoker (IFN) (p<0.001). These data support the hypothesis that inflamed tissue isolated from a current smoker exhibits a profile of collagen metabolism in line with more acute diseased state (fibrotic collagen profile). In addition, the culture of primary human gingival fibroblasts in serum‐free medium for at least 48 hours is a viable method to study the disease mechanism of periodontitis and potential therapeutics to treat the disease. Support or Funding Information This work is supported by the Division of Research at PCOM