Interferon gamma receptor deficient mice are resistant to endotoxic shock.

Antibody neutralization studies have established interferon gamma (IFN-gamma) as a critical mediator of endotoxic shock. The advent of IFN-gamma receptor negative (IFN gamma R-/-) mutant mice has enabled a more direct assessment of the role of IFN-gamma in endotoxin (lipopolysaccharide [LPS]-induced shock. We report that IFN gamma R-/- mice have an increased resistance to LPS-induced toxicity, this resistance manifesting well before the synthesis and release of LPS-induced IFN-gamma. LPS-induced lymphopenia, thrombocytopenia, and weight loss seen in wild-type mice were attenuated in IFN gamma R-/- mice. IFN gamma R-/- mice tolerated 100-1,000 times more LPS than the minimum lethal dose for wild-type mice in a D-galactosamine (D-GalN)/LPS model. Serum tumor necrosis factor (TNF) levels were 10-fold reduced in mutant mice given LPS or LPS/D-GalN. Bone marrow and splenic macrophages from IFN gamma R-/- mice had a four- to sixfold decreased LPS-binding capacity which correlated with similar reduction in CD14. Serum from mutant mice reduced macrophage LPS binding by a further 50%, although LPS binding protein was only 10% reduced. The expression of TNF receptor I (p55) and II (p75) was identical between wild-type and mutant mice. Thus, depressed TNF synthesis, diminished expression of CD14, and low plasma LPS-binding capacity, in addition to blocked IFN-gamma signaling in the mutant mice, likely to combine to manifest in the resistant phenotype of IFN gamma R-/- mice to endotoxin.

The stimulatory effects of interleukin (IL)-12 on hematopoiesis are antagonized by IL-12-induced interferon gamma in vivo.

Interleukin (IL)-12 synergizes with other cytokines to stimulate the proliferation and differentiation of early hematopoietic progenitors in vitro. However, in vivo administration of IL-12 decreases peripheral blood counts and bone marrow hematopoiesis. Here, we used interferon (IFN) gamma receptor-deficient (IFN gamma R-/-) mice to investigate whether the in vivo inhibition of hematopoiesis by IL-12 is indirectly mediated by IL-12-induced IFN-gamma. IL-12 administered for 4 d (1 microgram/mouse per day) resulted in lower peripheral blood counts and a 2-fold decrease in bone marrow cellularity in wild-type mice, but not in IFN gamma R-/- mice. Bone marrow hematopoietic progenitors were decreased after IL-12 treatment in wild-type mice, but rather increased in IFN gamma R-/- mice. Splenic cellularity was 2.3-fold higher after IL-12 administration in wild-type mice, largely due to natural killer (NK) cell and macrophage infiltration together with some extramedullary hematopoiesis. In IFN gamma R-/- mice, spleen cellularity was less increased, there were fewer infiltrating NK cells, but a strong extramedullary hematopoiesis. Thus, alterations mediated by IL-12-induced IFN-gamma include reduction in bone marrow cellularity and hematopoietic progenitors, as well as pronounced splenomegaly, largely caused by NK cell infiltration. In the absence of IFN-gamma signaling, IL-12 promotes hematopoiesis, consistent with its in vitro activities.

IkappaBalpha deficiency results in a sustained NF-kappaB response and severe widespread dermatitis in mice.

The ubiquitous transcription factor NF-kappaB is an essential component in signal transduction pathways, in inflammation, and in the immune response. NF-kappaB is maintained in an inactive state in the cytoplasm by protein-protein interaction with IkappaBalpha. Upon stimulation, rapid degradation of IkappaBalpha allows nuclear translocation of NF-kappaB. To study the importance of IkappaBalpha in signal transduction, IkappaBalpha-deficient mice were derived by gene targeting. Cultured fibroblasts derived from IkappaBalpha-deficient embryos exhibit levels of NF-kappaB1, NF-kappaB2, RelA, c-Rel, and IkappaBbeta similar to those of wild-type fibroblasts. A failure to increase nuclear levels of NF-kappaB indicates that cytoplasmic retention of NF-kappaB may be compensated for by other IkappaB proteins. Treatment of wild-type cells with tumor necrosis factor alpha (TNF-alpha) resulted in rapid, transient nuclear localization of NF-kappaB. IkappaBalpha-deficient fibroblasts are also TNF-alpha responsive, but nuclear localization of NF-kappaB is prolonged, thus demonstrating that a major irreplaceable function Of IkappaBalpha is termination of the NF-kappaB response. Consistent with these observations, and with IkappaBalpha and NF-kappaB's role in regulating inflammatory and immune responses, is the normal development Of IkappaBalpha-deficient mice. However, growth ceases 3 days after birth and death usually occurs at 7 to 10 days of age. An increased percentage of monocytes/macrophages was detected in spleen cells taken from 5-, 7-, and 9-day-old pups. Death is accompanied by severe widespread dermatitis and increased levels of TNF-alpha mRNA in the skin.

Cyclooxygenase-2 is essential for normal recovery from 5-fluorouracil-induced myelotoxicity in mice.

Cyclooxygenase (COX) plays a key regulatory role in prostaglandin synthesis. COX-2 is inducible and is the major isoform of inflammatory cells. COX-2-deficient mice were shown to have normal basal hematopoiesis and hematology. We hypothesized that COX-2 induction plays a role in the recovery phase of 5-fluorouracil (5-FU) induced bone marrow injury, because significant macrophage-driven phagocytic removal of necrotic debris and stromal cell reorganization of repopulating marrow occur after 5-FU induction of bone marrow necrosis. Hematologic recovery was markedly delayed with moderately severe leukopenia, thrombocytopenia and reticulocytopenia compared to heterozygotes on day 8 or 12 in Cox-2-/- mice. Mild anemia was present in 5-FU-treated Cox-2-/- and Cox-2+/- mice on days 8 and 12, which was more severe in Cox-2-/- mice. Cox-2-/- mice had markedly decreased bone marrow cell counts per femur and reduced numbers of erythroid and myeloid colony-forming cells compared to heterozygote mice on days 8 and 12 post 5-FU. Histologic examination of 5-FU-treated Cox-2-/- mice revealed a failure to repopulate the intact marrow stroma with hematopoietic cells. Accelerated erythropoiesis following phenylhydrazine-induced hemolytic anemia, however, was comparable between Cox-2-/- and Cox+/- mice, as were induced levels of renal erythropoietin mRNA. COX-2 induction is likely a central event in the accelerated hematopoiesis following myelotoxic injury, because recovery from 5-FU-induced myeloablation is markedly impaired in Cox-2-/- mice but is normal after phenylhydrazine induction of anemia.

Interferon-gamma enhances tumor necrosis factor-alpha production by inhibiting early phase interleukin-10 transcription.

The ability of cytokine synthesis inhibitory factor or interleukin-10 (IL-10) and interferon-gamma (IFN-gamma) to modulate the production of tumor necrosis factor (TNF-alpha) induced by lipopolysaccharide (LPS) was examined in mouse bone marrow-derived macrophages (BMDM). IFN-gamma profoundly enhances LPS-stimulated TNF-alpha production, whereas IL-10 is markedly inhibitory, demonstrating the opposing effects of IFN-gamma and IL-10 on BMDM. Early neutralization of endogenously produced, LPS-stimulated IL-10 markedly enhanced short term TNF-alpha production, an effect further amplified by the absence of IFN-gamma priming. The regulatory effects of IFN-gamma and IL-10 apparently occurred at the translational (or post-translational) level, with TNF-alpha mRNA steady-state levels remaining unchanged. Furthermore, IFN-gamma exerts its enhancing effect on TNF synthesis by the transcriptional inhibition of IL-10. This in vitro finding was also confirmed in vivo. In the absence of LPS, IFN-gamma was not capable of inducing TNF-alpha production in BMDM, indicating that LPS or other signals are necessary for transcriptional activation. Reduced but significant TNF-alpha production in LPS-injected IFN-gamma receptor -/- mice suggests that IFN-gamma is not an absolute requirement and that other cytokines or cell types contribute in a secondary fashion to the priming of LPS-induced TNF-alpha production in vivo.

Endotoxin-mediated bovine alveolar macrophage procoagulant induction is dependent on protein kinase C activation.

The induction of pulmonary alveolar macrophage (PAM) tissue factor-dependent procoagulant activity is central to the deposition of inflammatory fibrin in the pulmonary alveolus. The presence of enhanced tissue factor activity is often associated with pulmonary fibrin deposition, an important pathogenetic event that can delay resolution of pulmonary inflammation and promote the induction of pulmonary fibrosis. Since tissue factor synthesis induction and activation pathways are potential therapeutic targets for modulation of alveolar macrophage tissue factor (procoagulant) activity, we examined the pathways through which endotoxin lipopolysaccharide (LPS) induces bovine PAM tissue factor-dependent procoagulant activity. PAM procoagulant activity was markedly enhanced to 10 times the levels of freshly isolated PAM after 8 h of culture in the presence of either the protein kinase C (PKC) agonist phorbol 12-myristate 13-acetate (PMA) or LPS. Both LPS-(P less than 0.002) and PMA-induced activity (P less than 0.007) was completely ablated by the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H 7,100 microM) but was unaffected by the cyclic nucleotide-dependent protein kinase inhibitor N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004, 100 microM). The arachidonate cyclooxygenase pathway inhibitor phenylbutazone (10(-4) M) had modest effects that were not statistically significant. The unstimulated increase of procoagulant activity in 8-h cultures was unaffected by the same inhibitory modulations.(ABSTRACT TRUNCATED AT 250 WORDS)

Special considerations in the evaluation of the hematology and hemostasis of mutant mice.

The study of mutant mice with altered or deficient hematopoietic or hemostatic gene products provides a challenge to the researcher, particularly when genetic alterations lead to lethal phenotypes. The following review provides a framework for understanding murine hematopoiesis, based on work with mutant mice, and details experimental approaches used to evaluate these animals. Mice with deficiencies in hemostatic and fibrinolytic system proteins are discussed, and the investigation of their phenotypes is reviewed.

Formation of neutrophil-activating peptide 2 from platelet-derived connective-tissue-activating peptide III by different tissue proteinases.

Neutrophil-activating peptide 2 (NAP-2) is generated by cleavage of two inactive precursors, connective-tissue-activating peptide III (CTAP-III) and platelet basic protein (PBP), which are stored in the alpha-granules of blood platelets. Using highly purified CTAP-III as the substrate we studied the generation of NAP-2 by several neutral tissue proteinases. CTAP-III was rapidly cleaved by chymotrypsin, cathepsin G and trypsin, yielding products with neutrophil-stimulating activity. This activity remained unchanged for 24 h in the presence of chymotrypsin, decreased only slowly in the presence of cathepsin G, but was rapidly destroyed by trypsin. CTAP-III was also degraded by human neutrophil elastase and porcine pancreatic elastase, but no active fragments were obtained. By contrast, no degradation of CTAP-III was observed with thrombin, plasmin or 'granzymes' from cytolytic T-lymphocyte granules. Two active fragments of CTAP-III, generated by chymotrypsin or cathepsin G, were purified and partially sequenced, and were found to have the same N-terminal sequence as NAP-2. These results indicate that both proteinases cleave preferentially the bond between amino acids 15 (Tyr) and 16 (Ala) of CTAP-III. We conclude that chymotrypsin-like proteolytic activity in the vicinity of activated platelets may generate NAP-2 intravascularly. Due to its presence in the primary granules of neutrophils and monocytes cathepsin G is likely to be involved in this process.

Inhibition of kinases impairs neutrophil activation and killing of Staphylococcus aureus.

Intracellular phosphorylations polymorphonuclear neutrophils are mediated by kinases, including mitogen activated-protein (MAP) kinases and phosphatidylinositol 3-kinase. In the present study we demonstrate their effector functions upon both ligation of cell-surface seven-transmembrane-spanning receptors by bacterial peptide formylmethionyl-leucylphenylalanine as well as in the process of destruction of Staphylococcus aureus. To regulate neutrophil MAP kinases p38 and p44/42, specifically, we made use of their specific inhibitors 10 microM SK&F 86002 (for p38) and PD 098059 (for activating kinase of p44/42). SK&F 86002 was a potent inhibitor (by 70%) of induced antimicrobial oxygen-radical generation compared with PD 098059 (by 20%). SK&F 86002 and PD 098059 inhibited mobilization of a dominant neutrophil adhesion molecule, beta2 integrin, from cytoplasmic granules to the plasma membrane by 40 and 10% respectively, and the combination of the two drugs resulted in a 90% effect. The combined effect of both drugs was moderate inhibition of bacterial destruction, despite the fact that neither compound had detectable effect on bactericidal activity if applied individually. Bacterial destruction was also inhibited by wortmannin (0.1 microM), the specific inhibitor of phosphatidylinositol 3-kinase, which had previously been described to target various other activations of the neutrophil, including oxygen-radical generation. Although the relative contribution of p38 and p44/42 MAP kinases varied, the marked effects of the combined inhibition of the kinases revealed their concerted actions to be critical for normal neutrophil function.

Prevention of intraabdominal adhesions in ponies by low-dose heparin therapy.

An ischemic bowel model was used to stimulate adhesion formation in eight ponies. Heparin (40 USP u/kg) or saline was administered intravenously at surgery and was continued subcutaneously every 12 hours for 48 hours to evaluate the efficacy of heparin in preventing intraabdominal adhesions. Ponies were euthanatized after 6 weeks, and postmortem examinations were performed. A statistically significant difference was found between the heparin-treated and the control groups. Adhesions developed in three of four control ponies, and adhesions did not develop in three of four heparin-treated ponies. None of the adhesions resulted in clinical disease. In this study, heparin decreased the formation of adhesions in ponies after experimentally induced intestinal ischemia.

Expression and kinetics of induced procoagulant activity in bovine pulmonary alveolar macrophages.

Leukocytes, especially macrophages, are important cellular mediators of fibrin deposition and removal at tissue sites of inflammation. Pulmonary fibrin deposition is a prominent feature of bovine acute lung injury; therefore, we studied the resting and stimulated procoagulant responses of bovine pulmonary alveolar macrophages (PAM) and peripheral blood neutrophils (PMN). Freshly isolated normal PAM and PMN expressed negligible procoagulant activity. PAM stimulated with endotoxin lipopolysaccharide (LPS), 4 beta-phorbol 12-myristate 13-acetate (PMA) and bovine recombinant interleukin-1 beta (rBIL-1 beta) exhibited protein synthesis- and dose-dependent enhancement of procoagulant activity in 8-h cultures. Bovine recombinant granulocyte macrophage-colony stimulating factor (rBGM-CSF) and recombinant human gamma-interferon (rHIFN-gamma) did not induce procoagulant activity. The kinetics of LPS- and PMA-enhanced PAM procoagulant activity differed: LPS-induced enhancement developed earlier and more rapidly than PMA-induced enhancement. Pasteurella haemolytica LPS was more potent than Escherichia coli LPS in enhancing PAM procoagulant activity, while dexamethasone decreased both baseline and LPS- or PMA-stimulated activity by approximately 50%. PAM procoagulant activity resulted from tissue factor expression. Bovine PMN produced negligible procoagulant activity when stimulated, and are thus unlikely to be major contributors to procoagulant activity in bovine lung. Activity inhibitory to bovine tissue factor was present in both calf and adult sera, and was partly dependent on the presence of factor X for activity. Rapid induction of bovine PAM procoagulant activity by inflammatory mediators, and subsequent resistance to degradation, may thus combine to promote an alveolar microenvironment permissive to fibrin deposition in bovine acute lung injury.

The role of leukocytes in the pathogenesis of fibrin deposition in bovine acute lung injury.

The peculiarly fibrinous nature of bovine acute lung injury due to infection with Pasteurella haemolytica A1 suggests an imbalance between leukocyte-directed procoagulant and profibrinolytic influences in the inflamed bovine lung. Calves with experimental pneumonia produced by intratracheal inoculation with P. haemolytica A1 developed acute locally extensive cranioventral fibrinopurulent bronchopneumonia. Pulmonary alveolar macrophages (PAM) recovered by segmental lavage from affected lung lobes were 30 times more procoagulant than PAM obtained from unaffected lung lobes and 37-fold more procoagulant than PAM from control calf lungs. Unlike the enhancement of procoagulant activity, profibrinolytic activity (plasminogen activator amidolysis) of total lung leukocytes (PAM and plasminogen activator neutrophils [PMN]) was decreased 23 times in cells obtained from affected lung lobes and also was decreased four times in cells obtained from unaffected lobes of infected animals. This marked imbalance in cellular procoagulant and fibrinolytic activity probably contributes significantly to enhanced fibrin deposition and retarded fibrin removal. In addition, PAM from inflamed lungs were strongly positive for bovine tissue factor antigen as demonstrated by immunocytochemistry. Intensely tissue factor-positive PAM enmeshed in fibrinocellular exudates and positive alveolar walls were situated such that they were likely to have, in concert, initiated extrinsic activation of coagulation in the acutely inflamed lung. These data collectively suggest that enhanced PAM-directed procoagulant activity and diminished PAM- and PMN-directed profibrinolytic activity represent important modifications of local leukocyte function in bovine acute lung injury that are central to the pathogenesis of lesion development with extensive fibrin deposition and retarded fibrin removal.

Elevated IL-8 and MCP-1 in the bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis and pulmonary sarcoidosis.

The potential for interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) to induce neutrophil and mononuclear phagocyte accumulation in the lungs of patients with pulmonary sarcoidosis and idiopathic pulmonary fibrosis (IPF) was investigated. Bronchoalveolar lavage (BAL) fluids from 12 patients with IPF and 15 with sarcoidosis were concentrated by reversed-phase chromatography, and their IL-8 and MCP-1 concentrations assessed by enzyme-linked immunosorbent assay (ELISA), chemotaxis, and enzyme-releasing assays with monocytes and neutrophils. ELISA revealed significantly elevated concentrations of MCP-1 (20.1 ng/mg albumin) in the BAL fluids of patients with pulmonary sarcoidosis and those with IPF (41.8 ng/mg) in comparison to 11 normal individuals (4.24 ng/mg) and 15 patients with chronic bronchitis (CB) (5.16 ng/mg). Similarly, the chemotactic activity for monocytes (MCP-1 equivalent) was strongly increased in patients with sarcoidosis (86.03 ng/mg) as well as in those with IPF (54.47 ng/mg). The chemoattractant activity of normal individuals and CB patients was 7- or 3-fold lower, respectively. Patients with IPF and sarcoidosis also had elevated IL-8 levels (15.5 and 26.0 ng/mg, respectively; normals: 2.14 ng/mg; and CB patients: 4.23 ng/mg) and greater neutrophil chemotaxis (60.25 and 49.68 ng/mg, respectively; normals: 0.35 ng/mg; and CB patients: 11.06 ng/mg). These data suggest that increased levels of both MCP-1 and IL-8 may be characteristic for sarcoidosis or IPF. It appears likely that both of these chemoattractants contribute to the influx of monocytes and neutrophils into the pulmonary alveolus and interstitium in these diseases.

Interleukin-6 exacerbates glomerulonephritis in (NZB x NZW)F1 mice.

The ability of interleukin-6 (IL-6) to modulate immune parameters and mesangial cell function suggests a role for this cytokine in the development of autoimmune glomerulonephritis. This hypothesis was tested in 6-month-old female (NZB x NZW)F1 mice that were administered recombinant human IL-6 (rhIL-6) (50 and 250 micrograms/kg s.c.) for 12 weeks, resulting in an accelerated and severe form of membranoproliferative glomerulonephritis associated with marked upregulation of mesangial major histocompatibility complex class II antigen and glomerular ICAM-1 expression. To distinguish direct effects of rhIL-6 on the renal mesangium from those mediated through the immune system, (NZB x NZW)F1 mice were immunosuppressed with cyclosporin. Immunosuppression by cyclosporin inhibited the development of glomerulonephritis, decreased class II antigen expression, and abrogated IL-6-mediated effects. Administration of neutralizing anti-IL-6 antibody had no effect on the spontaneous development of glomerulonephritis in (NZB x NZW)F1 mice. This finding, together with undetectable IL-6 serum levels, makes a pathogenetic role of endogenously produced IL-6 in this disease model unlikely. In contrast to (NZB x NZW)F1 mice, parental NZW or BALB/c mice given high doses of rhIL-6 (500 micrograms/kg) or recombinant murine IL-6 (100 micrograms/kg) daily for 4 weeks failed to develop morphological or biochemical evidence of glomerulonephritis. Induction of acute phase proteins, anemia, thrombocytosis, and induction of renal class II antigen confirmed the biological activity of IL-6 in these mice. In conclusion, while non-nephritogenic in normal mice, IL-6 accelerates the development of the genetically determined glomerulonephritis of (NZB x NZW)F1 mice through effects mediated by a modulated immune system. Since neutralizing IL-6 antibody treatment did not prevent the development of glomerulonephritis, it is unlikely that increased IL-6 production plays a role in the pathogenesis of lupus nephritis.

Growth inhibition signalled through the interleukin-4/interleukin-13 receptor complex is associated with tyrosine phosphorylation of insulin receptor substrate-1.

Induction of growth inhibition in human colorectal carcinoma cell lines by interleukin (IL)-4 and IL-13 was associated with the neophosphorylation of a 170 kDa cellular protein, identified as insulin receptor substrate-1 (IRS-1) by immunoprecipitation. Tyrosine phosphorylation of IRS-I was also induced by insulin and insulin-like growth factor I. Sublines of colorectal carcinoma cells unresponsive to growth modulation by IL-4, IL-13 or insulin-like growth factor I-induced growth did not phosphorylate IRS-1. A functional, multimeric IL-4 receptor complex was present on all carcinoma cell lines with a subunit composition of 65 kDa, 75 kDa and the previously characterized 130 kDa band as demonstrated by affinity cross-link with 126I labelled IL-4. The 65 kDa subunit is novel whereas the 75 kDa band represents the common IL-2 receptor gama-chain the novel 65 kDa receptor was present as a double band and bound primarily 125I-labelled IL-13. The present study demonstrates the involvement of a novel chain other than the gama-chain in the receptor complexes of IL-4 and IL-13 and and post-receptor tyrosine phosphorylation of IRS-1. The association of IRS-1 with growth inhibitory signals in carcinoma cells suggests a novel mechanism of tumour growth control.

Bovine melanoma growth stimulatory activity: a new monocyte-macrophage-derived cytokine of the IL-8 family. Partial structure, function, and expression in acute pulmonary inflammation.

BACKGROUND: Neutrophil-chemotactic peptides are a family of small basic peptides 70 to 80 amino acids in length. They contain four conserved cysteine residues, the first two spaced by one amino acid (C-X-C). The best characterized species is human IL-8. Other prominent members are melanoma growth stimulatory activity (GRO-alpha), neutrophil-activating peptide-2, and epithelial-cell derived neutrophil-activating protein 78. EXPERIMENTAL DESIGN: Bovine monocytes and alveolar macrophages were induced by lipopolysaccaride, and a major neutrophil chemotactic activity in the supernatant was purified by cation-exchange chromatography and reversed-phase HPLC. The chemotaxin was then analyzed for biologic activity on bovine neutrophils by in vitro chemotaxis, shape change, and transient rise of intracellular-free calcium concentration. The in vivo role of bovine GRO (boGRO) was tested immunohistologically in confirmed cases of pneumonic pasteurellosis. RESULTS: We have purified and partially sequenced a bovine homologue of human GRO-alpha. The partial amino acid sequence of boGRO was: APVVNELRCQCLQTLQGIHLKNIQSVKVTTPGP. BoGRO was biologically active and induced a dose-dependent neutrophil migration in the range of 10(-7) to 10(-9) M. BoGRO also induced a dose-dependent shape change in bovine neutrophils similar to human IL-8. This effect was detectable down to 10(-10) M. Similar effects were observed on the transient rise of intracellular-free calcium concentration. In bovine pneumonic pasteurellosis and, to a lesser extent, in normal lungs, immunoreactivity to human GRO was highly positive in hypertrophic type-II epithelial cells and in mesothelial cells, whereas pleural fibroblasts and bronchial epithelial cells were negative. CONCLUSIONS: BoGRO is a prominent neutrophil chemoattractant secreted by monocytes and alveolar macrophages. It is active at similar concentrations as human IL-8. The strong immunoreactivity in type-II epithelial and mesothelial cells of bovine pneumonia strongly suggest a role for boGRO in the genesis of pulmonary inflammation.

The toxicology of interleukin-12: a review.

Recombinant murine interleukin (IL)-12 (rmIL-12) exhibits antitumor, antiviral, and antimicrobial activities and can modify allergic inflammatory reactions in animal models. Recombinant human IL-12 (rhIL-12) is currently in clinical trials for treatment of cancer, asthma, and viral hepatitis. Principally a phagocyte-derived cytokine, IL-12 targets natural killer cells and T lymphocytes, stimulating their activity and the secretion of interferon (IFN)-gamma. An understanding of the toxicology of IL-12, due in part to effects mediated by IFN-gamma, has emerged from preclinical safety and mechanistic studies and initial clinical trials. Target organs common to several animal species and humans include the lymphohematopoietic system, intestines, liver, and lung.

Effects of recombinant human interleukin 6 (rhIL-6) in marmosets (Callithrix jacchus). 1. General toxicity and hematological changes.

The physiological and toxicological properties of recombinant human interleukin 6 (rhIL-6) were assessed in marmoset monkeys (Callithrix jacchus). Two experimental series were performed with daily subcutaneous administration: (a) 5 or 1000 micrograms rhIL-6/kg per day for three weeks and (b) 25, 100 or 500 micrograms rhIL-6/kg per day for 3 months. RhIL-6 was well tolerated and did not induce fever or any other non-specific signs of toxicity. The main findings were: (1) A two- to threefold increase in platelet counts at 2-4 weeks, which decreased following further continuous rhIL-6 administration; (2) increase in total white blood cells between 1 and 4 weeks of administration, including an absolute increase in granulocytes (including band forms) and basophils. A change in the number of monocytes was not detected; (3) an increase in total red blood cells, which peaked at 4 weeks, sustained elevation of red cell distribution width and a slight decrease in hemoglobin between week 1 and 4, concurrent with a distinct decrease in mean corpuscular hemoglobin at 4 weeks. This effect persisted for 9 weeks in the 100 micrograms/kg and 500 micrograms/kg groups; (4) decrease in plasma AST activity and increase in plasma protein concentration after 2 weeks of treatment; (5) no clinical or biochemical signs of renal glomerular dysfunction; (6) RhIL-6 after s.c. administration was detectable in the plasma, peak levels (mean values +/- SD) of 9.4 +/- 6.3 and 72.4 +/- 7.7 ng/ml were measured after a single dose of 100 or 1000 micrograms/kg; (7) antibodies against rhIL-6 developed within 2 weeks, increased during administration and neutralized the biological effect of rhIL-6 progressively from 4 to 9 weeks. In conclusion, aside from a mild anemia, rhIL-6 was well tolerated in marmosets and had a profound and sustained effect on thrombopoiesis. Due to the formation of neutralizing antibodies, the chronic biological effect of rhIL-6 is lost in marmosets and studies beyond 4 weeks are rendered less meaningful. The analyses of antibody formation, induction of acute phase proteins, histological changes and alterations on lymphocyte receptors will be reported in two following publications.

Ileocolostomy. A technique for surgical management of equine cecal impaction.

Several surgical alternatives have been described for the management of cecal impaction in the horse, but none has met with consistently successful results. This study was done to evaluate a surgical bypass of the cecum by anastomosis of the ileum to the right ventral colon (ileocolostomy). A ventral midline celiotomy was performed on nine adult ponies (155-350 kg) and a mechanically stapled 10 cm side-to-side ileocolostomy was created. In five ponies a complete cecal bypass (CCB) was created by transecting the ileum distal to the anastomosis. In the other four, an incomplete cecal bypass (ICB) was created with no interruption of the ileum. Six horses with clinical cecal impaction also underwent cecal bypass procedures. Five had a CCB and one had an ICB. All the ponies maintained body weight, had no change in consistency of the feces and had no abdominal pain during the 6 month observation period. At necropsy, the lengths of the lateral cecal band, lateral free band of the colon, and the diameter of the anastomotic stoma were compared to measurements made at surgery. The lateral cecal band length decreased significantly more in the CCB ponies than in the ICB ponies (p = 0.008). The anastomotic stoma diameter was significantly larger in the ICB group than in the CCB group (p = 0.032). Five of the six clinical cases recovered and returned to their previous activity. CCB by an ileocolostomy resulted in removal of the cecum from the functional flow of ingesta without complication in the ponies, and was successful in five clinical cases of cecal impaction.