Safety assessment of intraportal liver cell application in New Zealand white rabbits under GLP conditions.

Liver cell transplantation (LCT) is considered a new therapeutic strategy for the treatment of acute liver failure and inborn metabolic defects of the liver. Although minimally invasive, known safety risks of the method include portal vein thrombosis and pulmonary embolism. Since no systematic data on these potential side effects exist, we investigated the toxicological profile of repeated intraportal infusion of allogeneic liver cells in 30 rabbits under GLP conditions. Rabbit liver cells were administered once daily for 6 consecutive days at 3 different dose levels, followed by a 2-week recovery period. No test item-related mortality was observed. During cell infusion, clinical findings such as signs of apathy and hyperventilation, moderate elevations of liver enzymes ALT and AST and a slight decrease in AP were observed, all fully reversible. Cell therapy-related macroscopic and histological findings, especially in liver and lungs, were observed in animals of all dose groups. In conclusion, the liver and lungs were identified as potential toxicological target organs of intraportal allogeneic liver cell infusion. A NOAEL (no observed adverse effect level) was not defined because of findings observed also in the low-dose group. No unexpected reactions became apparent in this GLP study. Overall, LCT at total doses up to 12 % (2 % daily over 6 days) of the total liver cell count were tolerated in rabbits. Observed adverse effects are not considered critical for treatment in the intended patient populations provided that a thorough monitoring of safety relevant parameters is in place during the application procedure.

Regional transient portal ischemia and irradiation as preparative regimen for hepatocyte transplantation.

Hepatocyte transplantation is regarded as a promising option to correct hereditary metabolic liver disease. This study describes a novel method involving regional transient portal ischemia (RTPI) in combination with hepatic irradiation (IR) as a preparative regimen for hepatocyte transplantation. The right lobules of rat livers (45% of liver mass) were subjected to RTPI of 30-120 min. Liver specimens and serum samples were analyzed for transaminase levels, DNA damage, apoptosis, and proliferation. Repopulation experiments involved livers of dipeptidylpeptidase IV (DPPIV)-deficient rats preconditioned with RTPI (60-90 min) either with or without prior partial hepatic IR (25 Gy). After reperfusion intervals of 1 and 24 h, 12 million wild-type (DPPIV positive) hepatocytes were transplanted into recipient livers via the spleen. RTPI of 60-90 min caused limited hepatic injury through necrosis and induced a distinct regenerative response in the host liver. Twelve weeks following transplantation, small clusters of donor hepatocytes were detected within the portal areas. Quantitative analysis revealed limited engraftment of 0.79% to 2.95%, whereas control animals (sham OP) exhibited 4.16% (determined as relative activity of DPPIV when compared to wild-type liver). Repopulation was significantly enhanced (21.43%) when IR was performed prior to RTPI, optimum preconditioning settings being 90 min of ischemia and 1 h of reperfusion before transplantation. We demonstrate that RTPI alone is disadvantageous to donor cell engraftment, whereas the combination of IR with RTPI comprises an effective preparative regimen for liver repopulation. The method described clearly has potential for clinical application.

Three monthly intravenous injections of ibandronate in the treatment of postmenopausal osteoporosis.

PURPOSE: Oral treatment of osteoporosis with bisphosphonates relies on compliance, the absorption being low and suppressed by simultaneous food intake. Intravenous (IV) treatment with an aminobisphosphonate, pamidronate (once every 3 months) was effective, but required infusions. Ibandronate, a new very potent aminobisphosphonate, can be administered safely as an IV bolus injection, and therefore offers an interesting alternative suitable for outpatient treatment. PATIENTS AND METHODS: To test the efficacy of this bolus IV treatment in postmenopausal osteoporosis in randomized partly double-blind, placebo controlled study, 125 postmenopausal women (mean age, 64 years) with osteoporosis (bone mineral density [BMD] < -2.5 SD T score) received a placebo or ibandronate (0.25, 0.5, 1, or 2 mg) every 3 months. All patients received 1 g calcium/day. BMD, in g/cm2, was measured by dual-energy x-ray absorptiometry at all standard sites. RESULTS: Lumbar spine BMD (L2 to L4) did not change (0.85%) in the placebo group, but increased by 2.4%, 3.5%, 3.7%, and 5.2% at 12 months for dose-ranging groups (no significant differences among ibandronate groups). The increase was statistically significantly different from placebo for the 0.5 mg (P < 0.006), 1 mg (P < 0.004), and 2 mg (P < 0.001) group, whereas with 0.25 mg no significant differences occured. After 1 year there were no significant changes in BMD compared with placebo at the femoral neck, Ward's triangle, and distal forearm. Total hip and trochanter BMD increased significantly, by 1.8% and 2.9% for total hip and by 2.7% and 4.2% for trochanter in the 1 and 2 mg group, respectively. Urinary excretion of C-telopeptide and N-telopeptide decreased after 1 month in all ibandronate groups, with a clear dose dependency. Three months after the first injection of 2 mg ibandronate there was still a significant reduction in these markers of bone resorption. Osteocalcin decreased progressively and dose dependently over time. There was a correlation between the decrease in C-telopeptide measured after 1 month and the increase in lumbar spine BMD after 1 year (n = 115, r = -0.26, P < 0.012). Ibandronate therapy proved to be safe. There was no significant difference in the overall number of adverse events in the ibandronate groups compared with the placebo group. Considering specific adverse events, no dose dependency and difference to placebo could be observed apart from acute reactions that occurred in 7% of the patients. CONCLUSION: Treatment of postmenopausal osteoporosis by interval IV bolus injections of the bisphosphonate ibandronate was safe and effective in increasing BMD through a dose-dependent inhibition of bone resorption. The high potency of ibandronate allows 3-month interval bolus IV injections as a new therapeutic approach with optimal compliance.

Immunomodulatory action of eicosanoids and other small molecular weight products of macrophages.

There is an increasing body of evidence that T cell-mediated immune response is regulated by a variety of small molecular weight products of macrophages. One of the best known immunoregulatory products in this context is prostaglandin E2 (PGE2) which is known to regulate both T cell and macrophage functions. Recently, ornithine, cysteine, and lactate have also been recognized as immunoregulatory mediators. In analogy to the hormone-like cytokines and lymphokines, all these substances are produced by immunologically relevant cells (macrophages) at a variable and regulated rate; and they have been shown to regulate the functional activities of other cells (T cells and macrophages). This brief review describes the key observations that underscore the important regulatory role of these metabolites in physiological and pathological conditions.

Correlation of immunogenicity and production of ornithine by peritoneal macrophages.

The release of ornithine by macrophages and its correlation with their immunogenicity after treatment with various macrophage-stimulating substances were analyzed. Pristane-elicited peritoneal macrophages (PM) were found to express strong arginase activity and to release L-ornithine into the extracellular space. This activity is strongly reduced within 3 hr after treatment with tetradecanoylphorbol acetate (TPA) but not with lipopolysaccharide (LPS). Resident PM usually express little arginase activity, but this activity is markedly augmented within 24 or 48 hr after treatment with LPS. The release of ornithine by peritoneal cells (PC) (60 to 90% macrophages) was found to be correlated with their immunogenicity as determined by the in vivo immunization for a subsequent in vitro secondary cytotoxic response against minor H antigens. The immunogenicity of pristane-elicited PC is markedly stronger than that of resident PC or TPA-treated, pristane-elicited PC. Moreover, the immunogenicity of the resident PC and TPA-treated elicited PC is substantially augmented by the simultaneous injection of ornithine, whereas the immunogenicity of the untreated elicited PC is not further augmented by exogenous ornithine, indicating that the endogenous production of ornithine by the stimulating cells had a strong influence on the resulting immune response. Injection of glutathione into pristane-treated mice also reduces the ornithine production and immunogenicity of the resulting peritoneal exudate cells. The immunogenicity in this case is at least partly reconstituted by application of exogenous ornithine. Our experiments revealed no correlation between the production of ornithine and prostaglandin E2. Prostaglandin E2 production of resident and pristane-elicited PC is not markedly different and is in either case strongly augmented by TPA. Elicited or resident PM which have been incubated for several days in culture release practically no ornithine; but ornithine production can be induced again by incubation for 24 hr with LPS and to some extent also with interferon-gamma.

Release of L-alanine by tumor cells.

Culture supernatants from the weakly immunogenic T cell lymphoma L5178Y ESb were found to contain substantial amounts of alanine and lactate at a ratio of about 1:10. Supernatants from cells of the highly immunogenic mutant line ESb-D also contained lactate but only minute amounts of alanine. Moreover, ESb cells converted 14C-labeled glucose or pyruvate into labeled alanine and lactate at a ratio of about 1:10, whereas ESb-D cells yielded only labeled lactate and no detectable alanine. The injection of L-alanine in combination with L-lactate into mice strongly suppressed the capacity of their spleen cells to generate cytotoxic responses. The injection of L-alanine also suppressed the immunogenicity of ESb-D cells, as demonstrated by the generation of cytotoxic activity in vivo and by the in vivo immunization (priming) for secondary cytotoxic responses against ESb-D cells in vitro. Taken together, these experiments suggest the possibility i) that the ESb cells prevent the induction of cytotoxic responses by releasing immunosuppressive alanine, and ii) that the immunogenic mutant ESb-D may have gained immunogenicity by losing this immunosuppressive property.