Lynne P. Rutzky

Human colonic adenocarcinoma cells: I. Establishment and description of a new line

SummaryA series of human colonic epithelial cell lines have been cultured from a single patient: LS-180 the original adenocarcinoma, LS-174T a trypsinized variant, and normal colonic tissue. The malignant cells, 20 to 40 µm in diameter and oval to polygonal, exhibited characteristics of normal colonic mucosal cells, namely, abundant microvilli prominent in secretory cells, and the presence of intracytoplasmic mucin vacuoles. The cultured adenocarcinoma cells, but not normal, demonstrated neoplastic properties by producing high levels of carcinoembryonic antigen (CEA) and by the ability to be propagated in hamster cheek pouches and in immunodeprived mice. The CEA production by the newly established line LS-180 released 900 times more CEA per cell into the culture medium and bore 30 times more cell-associated material than the established line, HT-29. These cell lines may permit detection of distinctive chemical, physiological, pharmacologic, and immunologic characteristics of neoplastic colonic cells.

Challenges in cyclosporine therapy: the role of therapeutic monitoring by area under the curve monitoring.

Summary Cyclosporine has revolutionized the practice of transplantation, but its clinical application has been beclouded by a narrow therapeutic window between immunosuppressive and nephrotoxic concentrations. Marked intra-and interindividual pharmacokinetic differences preclude the use of routine dosing regimens. For example, at the intraindividual level, cyclosporine absorption improves during the first 90 days after institution of therapy. A wide range of demographic factors, namely, age, race, and concomitant drug therapy, as well as individual-specific factors produce unique pharmacokinetic behaviors in any given patient. We introduced a pharmacokinetic strategy for cyclosporine administration almost 10 years ago based on the observation that the best estimate of drug exposure was the area under the concentration-time kinetic curve (AUC) not the trough level. Early studies documented the relation between AUC and the incidence of acute rejection. Subsequent studies revealed that not only is the AUC an important predictor, but so is the consistency of drug absorption over time; namely, patients with variations >25% among AUC determinations display an increased risk of chronic rejection episodes. Therefore therapeutic drug monitoring plays an important role in the optimal care of patients under cyclosporine therapy.

Microgravity culture condition reduces immunogenicity and improves function of pancreatic islets1.

Background. The failure of pancreatic islet allotransplants observed in almost all clinical attempts is related to poor initial islet function and allograft rejection. To remedy these problems we cultured islets in microgravity conditions to improve their function and to reduce their immunogenicity. Methods. Fresh mouse islets or mouse islets cultured in stationary dishes or microgravity bioreactors were transplanted to streptozotocin-induced diabetic mouse recipients. Results. Both allogeneic dish- or bioreactor-cultured islets survived more than 100 days compared with fresh allogeneic islets, which were rejected in less than 15 days. Islet titration studies revealed that 250 fresh or dish-cultured, but only 30 to 120 bioreactor-cultured, islets were necessary to produce euglycemia. Furthermore, glucose tolerance tests showed that bioreactor-cultured islets functioned better compared with fresh and dish-cultured islets on day 30 postgrafting. Immunostaining and transmission electron microscopy (TEM) analyses showed the gradual disappearance of dendritic cells in cultured islets compared with fresh islets. TEM revealed that the ultrastructure of islets from bioreactor, but not dish, appeared healthy and closely resembled fresh islets. Interestingly, TEM and scanning electron microscopy showed that only bioreactor-cultured islets developed unique and multiple nutritional channels between arrays of islet cells. TEM with colloidal lanthanum tracer revealed that only bioreactor islet cell cultures were devoid of tight junctional complexes, which may facilitate channel formation. Conclusion. Microgravity condition decreases immunogenicity and significantly improves the function of secretory cells.

Immature syngeneic dendritic cells potentiate tolerance to pancreatic islet allografts depleted of donor dendritic cells in microgravity culture condition

Background. Previously we showed that pancreatic islets cultured for seven days in rotating bioreactors survived for >100 days in allogeneic recipients without immunosuppression. This survival coincided with almost complete elimination of “passenger” donor dendritic cells (DCs). Herein, we examined the necessity of DCs in the generation of CD4+CD25+ T regulatory (Treg) cells. Methods. Allogeneic fresh islets or islets cultured for three days in bioreactors were transplanted to streptozotocin-induced diabetic Balb/c as well as signal transducers and activators of transcription (Stat)4-deficient Balb/cStat4−/− or Balb/cStat6−/− mice. Some Balb/cStat4−/− recipients of fresh islet allografts were also treated with a tolerogenic protocol of anti-CD40 Ligand MR1 mAb and CTLA4Ig. Results. Islet allografts cultured for three days in bioreactors survived >100 days in all Balb/cStat4−/− recipients and in 56% of Balb/cStat6−/− recipients, but in none of the Balb/c recipients; the same recipients rejected fresh islet allografts. Purified T cells from long-term surviving Balb/cStat4−/− recipients failed to transfer tolerance to SCID recipients of donor-type fresh islet allografts. In contrast, MR1/CTLA4Ig therapy induced tolerance to fresh islet allografts and their T cells adoptively transferred tolerance. When Balb/c or Balb/cStat4−/− recipients of bioreactor-cultured islets were injected intravenously with immature syngeneic DCs, they became tolerant and developed potent alloantigen-specific CD4+CD25+ Treg cells expressing Foxp3. Conclusion. Allogeneic islets depleted of donor DCs by culture in bioreactors have almost twofold better acceptance in Balb/cStat4−/− than in Balb/cStat6−/− mice, but lack Treg cells. Additional injection of host immature DCs improves tolerance in Balb/c and Balb/cStat4−/− recipients by inducing potent CD4+CD25+ Treg cells.

In vitro correlates of in vivo therapy with cyclosporine to immunosuppress rejection of heterotopic rat cardiac allografts across strong (RT-1) plus weak (non-RT-1) histocompatibility differences

This study correlated different oral cyclosporine doses with in vivo graft survival, blood and tissue drug levels, and in vitro immune performances. Wistar-Furth (WFu, RT-1u) hosts engrafted with heterotopic cardiac transplants from strongly histoincompatible Buffalo (BUF, RT-lb) rats were treated postoperatively with 14-day courses of different doses of CsA delivered per gavage. There was a graded prolongation of graft survival—namely, no effect at the 1.5 mg/kg dose; a modest effect at 3 mg/kg; a therapeutic effect at 5 mg/kg; and long-term unresponsiveness at 10 mg/kg. Whole blood, serum, and tissue CsA concentrations correlated with drug dose. On day 7 posttransplantation—that is, during the peak of the immune response of untreated recipients and midway during the period of daily CsA therapy—in vitro immune performances were examined in each experimental group. On the one hand, the mixed lymphocyte reaction of WFu host splenic T cells toward donor-type BUF stimulators poorly reflected the administered CsA dose. On the other hand, there was a good correlation between drug dose and both impaired cell-mediated lympholysis and reduced frequency of alloantigen-specific T cytotoxic cell precursors f(CTL)p. Animals treated with therapeutic doses of CsA showed different patterns of T cell-mediated lympholysis: 3 mg/kg did not prevent anti-BUF Tc cell sensitization; 5 mg/kg maintained f (CTL)P levels similar to the normal controls; and 10 mg/kg significantly reduced Tc clones against donor but not third-party targets. These data demonstrate that the fate of alloantigen-specific Tc clones activated in vivo depends upon the local drug concentration. Furthermore, the present studies suggest that CML and f(CTL)p afford useful in vitro indices of in vivo immunosuppression with CsA in rat cardiac allograft recipients.

IN VITRO STIMULATION OF RAT LIVER CELLS BY HOMOLOGOUS PARTIAL HEPATECTOMY SERUM

SummaryA low passage rat liver cell line demonstrated in vitro growth stimulation when cultured in the presence of serum of homologous, partially hepatectomized rats. After 4-day incubation a 3.25-fold increase in the cell population was observed in cultures supplemented with posthepatectomy serum at a dilution of 1∶10. No response was observed with sham-operated animal serum. Continous cultures of Chang human liver and Don hamster lung cells were not responsive to the posthepatectomy serum. The limitations of tetraphenylboron as a dispersing agent for primary rat liver cells are discussed.

Novel Sphingosine-1-Phosphate Receptor Modulator KRP203 Combined With Locally Delivered Regulatory T Cells Induces Permanent Acceptance of Pancreatic Islet Allografts

Background KRP203, a structural FTY720 analogue, has 5-fold greater selectivity for binding to sphingosine-1-phosphate receptor (S1PR) 1 (S1PR1) versus S1PR3 and 100-fold greater selectivity over S1PR2 and S1PR5. Although the immunoregulatory effects of FTY720 have been tested in clinical and experimental research, the therapeutic efficacy of KRP203 in allograft models remains elusive. In this study, we investigated the potential of KRP203 alone and in combination with intragraft injection of CD4+CD25+FoxP3+ regulatory T cells (Tregs) to induce islet allograft tolerance. Methods BALB/c (H-2d) mice received transplants of fresh C57BL/10 (H-2b) islet allografts under the kidney capsule and were treated for 7 days with 0.3, 1.0, or 3.0 mg/kg KRP203 alone or in combination with intragraft-infused Tregs. Results Untreated BALB/c mice acutely rejected C57BL/10 islet allografts at a mean survival time of 13.8±2.7 days (n=5). A 7-day dosing of 0.3 or 1.0 mg/kg KRP203 produced long-term islet allograft survival (>200 days) in one of five and two of seven recipients, respectively. A 3 mg/kg KRP203 dose resulted in islet graft survival for more than 200 days in 5 of 12 recipients. Whereas recipients that received 500 allogeneic islets admixed with 5×105–7×105 Tregs survived 83.6±67.2 days, addition of transient 3 mg/kg KRP203 therapy induced prolonged drug-free graft survival (>200 days) in all recipients. Conclusions A brief treatment with KRP203 significantly prolonged islet allograft survival, whereas additional intragraft delivery of Tregs induced tolerogenic effects selective to islet alloantigens.

Purification of tumor-specific antigens. An overview of the relevance to human colon carcinoma.

Methods which dissociate intramolecular noncovalent bonds have been used to prepare soluble derivatives of cell‐surface antigens. Applications of these techniques to human colon carcinoma are underway. Continuous tissue‐culture strains derived from primary lesions were developed and shown to be composed of malignant epithelial elements. Parallel data on the preparation and activity of soluble materials in a murine model methylcholanthrene system reveal that although cultured cells are a satisfactory source for antigen extraction, they are poor targets of the immune response. The development of methods to quantitate the biologic activity of colon‐specific, soluble materials may provide indicator systems to define the antigenic determinants, to permit purification, and to serve as assays of the efficacy of immunotherapy.