David A. Crouse

Purification of Monoclonal Antibodies Using High Performance Liquid Chromatography (HPLC)

Recent increases in the ability to detect low levels of immunofluorescence have shown the need for highly purified primary immunoreagents. There are now reports of purification of monoclonal antibodies using HPLC with reverse phase columns. In this study we have utilized standard size exclusion HPLC to purify both biotinylated and non-biotinylated monoclonal antibodies from hybridoma culture supernatants. Results indicated that both biotinylated and non-biotinylated monoclonal antibodies retained their antigen binding capacity after purification, and were not different in this capacity from commercially available, affinity purified reagents. These findings indicate that size exclusion HPLC may be used in the purification of biologically active monoclonal antibodies, and suggest that this technique may be used in the large scale production of antibodies and their fragments, in antibody purification from ascites fluid, and in antisera quality control.

An in vitro investigation of the hematopoietic micro-environment in young and aged mice

Dexter-type cultures derived from the bone marrow of young and aged mice were established as in vitro correlates to the hematopoietic microenvironment and inoculated two weeks later with fresh bone marrow-derived stem cells (colony-forming units, CFUs) from young, syngeneic mice. Such cultures allowed the observation, quantitation and evaluation of interactions between aged or young microenvironments and the young stem cells. The hematopoietic microenvironments derived from aged marrow were found to support a greater total nucleated cellularity and a significantly greater number of CFUs. Also, the production of CFUs on aged monolayers occurred at an elevated rate. Though cyclic variations in total cellularity were noted in all cultures, the granulocyte--macrophage lineage always predominated. Lymphocyte populations in all cultures were seen to decline rapidly with time as other cell types became more abundant. The number of megakaryocytes in the aged marrow-derived cultures was significantly elevated in the early time periods post-refeeding. Differences in the adherent cell population densities were noted with the aged monolayers being somewhat less dense. However, there were no differences in morphologically identifiable cell types comprising the adherent layers derived from marrow of young and old mice. From these results, we conclude that there are differences in the ability of aged versus young hematopoietic microenvironments to support normal young stem cells in vitro and that the microenvironmental influences present in the in vitro system are reflective of those seen in the in vivo marrow microenvironment.

Thymic non-lymphoid cells

SummaryIn formulating this summary of our simon-pure knowledge of the structure/function relationships in the thymus, we decided that the time may have come to introduce a suitable dose of cynicism to balance the sometimes hopeless optimism of the past.Are the non-lymphoid cells of the thymus necessary for thymic function? Probably, but not to the extent or uniqueness that some authors including ourselves have previously claimed; T cells can probably differentiate in other tissues but may acquire their preference for MHC class II in the thymus.Mouse thymic lymphoid cell traffic and surface phenotype has recently been summarized pictorally byScollay and Shortman [95]. Briefly stated, within the thymus, cells are hatched, matched and then dispatched. Minimally, the non-lymphoid cells act either as scenically varied obstacles along the way, nurseries for newborn T cells, or as tombstones for life’s disenfranchized, effete and autoaggressive thymocytes. Hassall’s corpuscles are morphological structures unique to the thymus, which are most useful to medical students for identification of this tissue. Their function remains one of life’s great mysteries. Morphologically, they are suitable companions to the more recently described strange multicellular complexes of lymphocytes and epithelial cells which might be functionally important. The thymus of the much studied inbred, environmentally mollycoddled, laboratory mouse has been often and majestically described. It is probably typical for that of man and most mammals. It may, however, be unrepresentative of the thymus of stressed and parasitized wild animals. Diseases of the thymus generally can be categorized as not having enough thymus, having a neoplastic thymus or having a thymus which does not work properly. The bottom line in our knowledge of thymic non-lymphoid cells is that if you are born without them, you get sick and die; unless, of course, you are a nude mouse in Omaha, in which case you just freeze to death.

Effect of systematic interleukin-3 administration on epithelial cell proliferation in mouse intestine

The effect of interleukin-3 (IL-3) on the crypt cell production rate (CCPR) in the intestine of mice was studied using a stathmokinetic technique combined with crypt microdissection. Interleukin-3 (0.71 micrograms/injection) was administered subcutaneously (s.c.) as two injections per day for 7 successive days and small mucosal pieces (duodenum, jejunum, ileum and colon) removed at necropsy were organ cultured in the presence of the metaphase arrest agent vincristine sulfate for two hours. The number of metaphases was enumerated in dissected crypts and CCPR calculated. The results demonstrated that the CCPR was significantly increased in all mucosal segments in the IL-3 treated animals compared to saline injected controls. These results suggest that the growth promoting properties of IL-3 are not restricted to hematopoietic cells when used in vivo and may directly or indirectly increase epithelial cell turnover in gut mucosa.

Epithelial cell proliferation and biodistribution of radiolabeled urogastrone in the gastrointestinal mucosa of young and old mice

We have evaluated epithelial cell proliferation and biodistribution of radiolabeled recombinant human urogastrone/epidermal growth factor (125I rhUG/EGF) in the gastrointestinal mucosa of young (2+ months) and old (30+ months) mice. Animals were injected intraperitoneally with the metaphase arrest agent vincristine sulfate and intravenously with 125I rhUG/EGF. Animals were killed after two hours. Crypt cell production rate and uptake of radiolabeled urogastrone were measured in the same intestinal tissues. The results demonstrated that older animals had significantly greater crypt cell production rate as compared to young. However, the uptake of 125I rhUG/EGF was not significantly different (except in duodenum, where the uptake of 125I rhUG/EGF was significantly greater in young compared to old animals) between young and old animals. This suggests that increased epithelial cell proliferation in the aging animals is not associated with increased uptake of 125I rhUG/EGF. Thus epidermal growth factor/urogastrone may not be a primary factor for the intestinal kinetic differences which exist between young and old animals.

A decrease in thymus-mediated immune responses as a result of treatment of neonatal rats with glutamate

We investigated whether administration of monosodium 1-glutamate (MSG) to neonatal rats would disrupt immune responses in intact and orchidectomized adult male rats. Neonatal male rats were treated with saline or MSG which causes severe endocrine abnormalities. Half of each group of animals were orchidectomized as adults and killed one week later along with intact rats. MSG treatment resulted in suppressed serum LH levels in intact rats. Thymus weight and spleen cellularity in intact animals were not affected by MSG treatment, but thymus weight increased within one week after orchidectomy in both saline- and MSG-treated groups. In intact rats, lymphocyte stimulation by the T cell specific mitogens (concanavalin A or phytohemagglutinin) or the B cell specific mitogen (lipopolysaccharide) was unaffected by prior treatment with MSG. However, MSG treatment blocked the decrease attributable to orchidectomy in concanavalin A and phytohemagglutinin stimulation of lymphocyte blastogenesis. The results suggest that administration of MSG to neonatal male rats can alter some immune responses in the adult animal.

Homing Receptor Expression in Grafts of Purified Thymic Epithelium

Grafts of purified thymic epithelium have been used to study the role of the thymic microenvironment in T cell differentiation1–6. The ability of such grafts to immunologically reconstitute either nude mice or thymectomized, irradiated and hematopoietically reconstituted hosts has demonstrated that they are only partially able to reconstitute the host animal, as neither lymphocyte phenotype frequencies nor functional responsiveness of the graft derived peripheral (splenic) T cell population reaches normal (intact) levels2,3,6. More recently it has been suggested that the expression of lymphocyte homing receptors may be important in the selection of thymocytes destined to emigrate from the thymus, as well as their localization in the periphery7–9. In the studies reported here, we have examined homing receptor expression in grafts of purified thymic epithelium and in the peripheral lymphoid organs of grafted nude mice. Results showed that, although incomplete, T cell reconstitution did take place in these animals. The presence of MEL-14 positive cells in the graft suggests that the presence or absence of homing receptor expression in these grafts was not solely responsible for the incomplete T cell emigration and peripheralization seen in these animals.