Young adult type 1 diabetes care in the West of Ireland: an audit of hospital practice.

BACKGROUND: It is well recognised that management of young adults with type 1 diabetes (T1DM) poses difficult challenges for physicians and health care organisations as a whole. In Ireland and in particular the west of Ireland there has been little audit or research on young adults with T1DM and the services available to them. DESIGN: In 2011 a retrospective review of this patient population in our territory referral centre was carried out. RESULTS: The average glycaemic control in this population was poor at 81mmols/mol and diabetes related complications were present in 32%. Engagement by this population with services was poor with an average of 3 missed clinic appointments over a 24 month period. CONCLUSION: These results have prompted a re think of how health care professionals can deliver a service that better suits the needs of this challenging patient group.

Restructuring of the Diabetes Day Centre: a pilot lean project in a tertiary referral centre in the West of Ireland.

INTRODUCTION: Diabetes is a chronic disease amenable to management in the community and outpatient setting. The increasing incidence of diabetes places outpatient endocrinology services under pressure to provide a quality service in a timely manner. Our aim was to apply lean thinking to the diabetes clinic in a tertiary referral centre in the West of Ireland to improve flow, as reflected in reduced patient journey times. METHODS: The project lasted 6 months, from January to June 2011. An introductory seminar on lean thinking was arranged to inform and motivate the Diabetes Day Centre staff. Two 'rapid improvement events' took place. Value stream mapping (VSM) was the predominant lean tool employed. Patient journeys were mapped and quantified (minutes) using timesheets allocated to each step in the process at baseline, and following intervention. Data were analysed using Minitab V.16.0. RESULTS: VSM allowed the value-adding and problem-causing steps in the patient journey through the diabetes clinic process to be identified and addressed. Total patient journey time through the clinic was significantly reduced from 118 (± 38.02) min to 58 (± 18.30) min (p<0.001). CONCLUSIONS: This project reflects the successful application of VSM as a lean tool in a pilot study at our institution as evidenced by improved patient flow and a significant reduction in patient journey time through the clinic. Through the incorporation of Lean into the ethos of the hospital, we have the potential to deliver excellent care in a safe environment and in an efficient manner, while benefiting the patient, employees and tax-payer.

In vitro and in vivo responses to interleukin 12 are maintained until the late SIV infection stage but lost during AIDS.

The in vitro proliferative responses of macaque peripheral blood mononuclear cells (PBMCs) to IL-12 appeared similar before and early after SIV infection, whereas macaque PBMCs sampled during symptomatic stages of SIV infection showed markedly decreased responses. IL-12 was administered to SIVmac239-infected rhesus macaques either during the asymptomatic or the AIDS stage of infection in efforts to evaluate the effect of this cytokine on immune responses, viral loads, and hematopoietic functions in vivo. IFN-gamma secretion levels induced during the asymptomatic or early symptomatic phase were similar to preinfection induced levels, whereas in later AIDS stages this response was lost. The constitutive levels of other measured cytokines were not affected by IL-12 administration in vivo. The frequency and activity of circulating NK cells were markedly enhanced at early stages but not at symptomatic stages of SIV infection. pCTL frequencies were enhanced at early symptomatic stages but not at late AIDS stages. Despite its immunomodulatory effect, IL-12 did not seem to exacerbate or inhibit the replication of SIV in vivo, or the frequency of circulating infected lymphocytes. IL-12 administration was associated with a significant yet subclinical and transient decrease in hematocrit and hemoglobin levels without evidence of hemolysis, hemodilution, or reduction in the frequency of colony-forming unit potential of bone marrow CD34+ cells. This phenomenon may be explained by a functional inhibition of differentiation rather than an altered generation of bone marrow precursors. Thus, these results suggest that IL-12 may benefit HIV-1-infected patients only as long as their immune system retains its capability to respond to cytokine stimulation.

Down-regulation of the pharmacokinetic-pharmacodynamic response to interleukin-12 during long-term administration to patients with renal cell carcinoma and evaluation of the mechanism of this "adaptive response" in mice.

BACKGROUND: Interleukin-12 (IL-12) is a cytokine that promotes type-1 helper T-cell responses and may have therapeutic utility in the treatment of cancer, asthma, and a variety of infectious diseases. METHODS: In a phase I trial, recombinant human IL-12 (rHuIL-12) was administered subcutaneously once a week at a fixed dose of 0.1 to 1.0 microg/kg to 24 patients with renal cell carcinoma. A similar study was later performed in mice to evaluate the mechanism of down-regulation of pharmacokinetic-pharmacodynamic response observed in patients with cancer. RESULTS: Adverse events, serum IL-12 levels, and serum levels of interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) produced in response to IL- 12 were all maximum in the week after the first dose of rHuIL-12 and decreased after long-term administration. Similar to these results, repetitive subcutaneous administration of recombinant mouse IL-12 (rMoIL-12) to normal mice led to down-regulation of serum levels of IL-12 and IFN-gamma measured 5 hours after rMoIL-12 injection. Down-regulation of IL-12 serum levels was inversely correlated with the up-regulation of IL-12 receptor expression and may be the result of increased clearance of rMoIL-12 from serum by binding to lymphoid cells expressing increased amounts of IL-12 receptor. The down-regulation of serum IFN-gamma levels correlated with decreased IFN-gamma messenger ribonucleic acid expression and may result from feedback inhibition of IL-12 signaling or from a more specific inhibition of IFN-gamma synthesis. CONCLUSION: Administration of rHuIL-12 in fixed weekly doses resulted in decreased serum levels of IL-12 and of IFN-gamma, a secondary cytokine believed to be critical to response of IL-12. A better understanding of the complex regulation of the pharmacokinetic-pharmacodynamic response to IL-12 should facilitate the development of more effective dosing regimens for its use in the clinic.

IL-12 is dispensable for innate and adaptive immunity against low doses of Listeria monocytogenes.

We have studied IL-12p35-deficient (IL-12p35(-/-)) mice to evaluate the role of IL-12 in resistance against Listeria monocytogenes. In the absence of bioactive IL-12p75, mutant mice acquired higher bacterial organ burden than wild-type mice and died during the first week following infection with normally sublethal doses of Listeria. Moreover, blood IFN-gamma levels were strikingly reduced in mutant mice at day 2 post-infection. These results suggest that in IL-12p35-deficient mice impaired production of IFN-gamma which is crucial for activation of listericidal effector functions of macrophages leads to defective innate immunity against Listeria. In contrast to mice deficient for IFN-gamma or IFN-gamma receptor which are unable to resist very low infection doses of Listeria, IL-12p35(-/-) mice resisted up to 1000 c.f.u. and were able to eliminate Listeria. Spleen cells from mutant mice re-stimulated with heat-killed Listeria produced considerable amounts of IFN-gamma, suggesting that at low dose infection sufficient IFN-gamma is produced independently of IL-12. Subsequent challenge of these immunized mice with high doses of L. monocytogenes resulted in sterile elimination demonstrating efficient memory responses. These results demonstrate for the first time that at low doses of Listeria IL-12 is neither critical for innate immunity nor for the development of protective T cell-dependent acquired immunity.

Interleukin-12 is essential for a protective Th1 response in mice infected with Cryptococcus neoformans.

To analyze the roles of interleukin-12 (IL-12) and the IL-12-dependent Th1 response in resistance to Cryptococcus neoformans, we have established a chronic infection model in wild-type mice and in mice with targeted disruptions of the genes for the IL-12p35 and IL-12p40 subunits (IL-12p35(-/-) and IL-12p40(-/-) mice, respectively) as well as in mice with a targeted disruption of the IL-4 gene. Long-term application of exogenous IL-12 prevented death of infected wild-type mice for the entire period of the experiment (up to 180 days) but did not resolve the infection. Infected IL-12p35(-/-) and IL-12p40(-/-) mice died significantly earlier than infected wild-type mice, whereas infection of IL-4-deficient mice led to prolonged survival. Interestingly, infected IL-12p40(-/-) mice died earlier and developed higher organ burdens than IL-12p35(-/-) mice, which, for the first time in an infection model, suggests a protective role of the IL-12p40 subunit independent of the IL-12 heterodimer. The fungal organ burdens of IL-4-deficient mice and IL-12-treated wild-type mice were significantly reduced compared to those of untreated wild-type mice and IL-12-deficient mice. Histopathological analysis revealed reduction of the number of granulomatous lesions following treatment with IL-12. Susceptibility of both IL-12p35(-/-) and IL-12p40(-/-) mice was associated with marginal production of gamma interferon and elevated levels of IL-4 from CD4(+) T cells, which indicates Th2 polarization in the absence of IL-12, whereas wild-type mice developed a Th1 response. Taken together, our data emphasize the essential role of IL-12 for protective Th1 responses against C. neoformans.

The interleukin-12/interleukin-12-receptor system: role in normal and pathologic immune responses.

Interleukin-12 (IL-12) is a heterodimeric cytokine that plays a central role in promoting type 1 T helper cell (Th1) responses and, hence, cell-mediated immunity. Its activities are mediated through a high-affinity receptor composed of two subunits, designated beta 1 and beta 2. Of these two subunits, beta 2 is more restricted in its distribution, and regulation of its expression is likely a central mechanism by which IL-12 responsiveness is controlled. Studies with neutralizing anti-IL-12 antibodies and IL-12-deficient mice have suggested that endogenous IL-12 plays an important role in the normal host defense against infection by a variety of intracellular pathogens. However, IL-12 appears also to play a central role in the genesis of some forms of immunopathology. Inhibition of IL-12 synthesis or activity may be beneficial in diseases associated with pathologic Th1 responses, such as multiple sclerosis or Crohn's disease. On the other hand, administration of recombinant IL-12 may have utility in the treatment of diseases associated with pathologic Th2 responses such as allergic disorders and asthma.

Analysis of the multiple interactions between IL-12 and the high affinity IL-12 receptor complex.

IL-12 is a heterodimeric cytokine, composed of a p40 and a p35 subunit, that exerts its biological effects by binding to specific cell surface receptors. Two IL-12R proteins, designated human IL-12 (huIL-12) receptor beta1 (huIL-12Rbeta1) and huIL-12Rbeta2, have been previously identified. These IL-12R individually bind huIL-12 with low affinity and in combination bind huIL-12 with high affinity and confer IL-12 responsiveness. In this study the interactions of hulL-12 with these two identified human IL-12R protein subunits are examined. The heterodimer-specific anti-huIL-12 mAb 20C2, which neutralizes huIL-12 bioactivity but does not block 125I-huIL-12 binding to huIL-12Rbeta1, blocked binding of huIL-12 to huIL-12Rbeta2. In contrast, anti-huIL-12Rbeta1 mAb 2B10 and mouse IL-12 p40 subunit homodimer (mo(p40)2) blocked 125I-huIL-12 binding to huIL-12Rbeta1, but not to huIL-12Rbeta2. Therefore, two classes of IL-12 inhibitors can be identified that differ in their ability to block huIL-12 interaction with either huIL-12Rbeta1 or huIL-12Rbeta2. Both mo(p40)2 and 20C2 blocked high affinity binding to huIL-12Rbeta1/beta2-cotransfected COS-7 cells, although, as previously reported, mo(p40)2 does not block high affinity binding to IL-12R on PHA-activated human lymphoblasts. Furthermore, these two classes of IL-12 inhibitors synergistically decreased hulL-12-stimulated proliferation and IFN-gamma production. Therefore, IL-12, in binding to the high affinity IL-12R, interacts with IL-12Rbeta1 primarily via regions on the IL-12 p40 subunit and with IL-12Rbeta2 via 20C2-reactive, heterodimer-specific regions of IL-12 to which the p35 and p40 subunits both contribute.

Treatment with homodimeric interleukin-12 (IL-12) p40 protects mice from IL-12-dependent shock but not from tumor necrosis factor alpha-dependent shock.

The role of interleukin-12 (IL-12) was investigated in different shock models using anti-IL-12 reagents. IL-12 is composed of two disulfide-bonded subunits, p35 and p40. The IL-12 p40 homodimer (p40)2 has been shown to be a potent IL-12 antagonist in vitro. We investigated its in vivo inhibitory capacity in different shock models of mice. We could demonstrate that (p40)2 is able to protect mice from septic shock in primarily IL-12-dependent models such as the Shwartzman reaction and lipopolysaccharide (LPS)-induced shock, whereas (p40)2 has no effect in the tumor necrosis factor alpha-dependent LPS/D-GalN shock model. In IL-12-dependent shock models, (p40)2 inhibits IL-12-induced gamma interferon production and thereby interferes with the cascade of cytokine release, finally leading to death.

Deviation of pancreas-infiltrating cells to Th2 by interleukin-12 antagonist administration inhibits autoimmune diabetes.

Nonobese diabetic (NOD) mice develop spontaneous insulin-dependent diabetes mellitus (IDDM), and the pancreas-infiltrating T cells invariably show a Th1 phenotype. We demonstrated here that the interleukin (IL)-12 antagonist (p40)2 can deviate the default Th1 development of naive T cell receptor (TCR)-transgenic CD4+ cells to the Th2 pathway in vitro. Although (p40)2 does not modify the cytokine profile of polarized Th1 cells, it prevents further recruitment of CD4- cells into the Th1 subset. To study the involvement of Th1 and Th2 cells in the initiation and progression of IDDM, we targeted endogenous IL-12 by administration of (p40)2 in NOD mice. (p40)2 administration to NOD mice inhibits interferon-gamma but not IL-10 production in response to lipopolysaccharide (LPS) or to the putative autoantigen IA-2. Serum immunoglobulin isotypes determined after (p40)2 treatment indicate an increase in Th2 and a decrease in Th1 helper activity. Administration of (p40)2 from 3 weeks of age onwards, before the onset of insulitis, results in the deviation of pancreas-infiltrating CD4+ but not CD8+ cells to the Th2 phenotype as well as in the reduction of spontaneous and cyclophosphamide-accelerated IDDM. After treating NOD mice with (p40)2 from 9 weeks of age, when insulitis is well established, few Th2 and a reduced percentage of Th1 cells are found in the pancreas. This is associated with a slightly decreased incidence of spontaneous IDDM, but no protection from cyclophosphamide-accelerated IDDM. In conclusion, deviation of pancreas-infiltrating CD4+ cells to Th2 is associated with protection from IDDM. However, targeting IL-12 after the onset of insulitis, when the pancreas contains polarized Th1 cells, is not sufficient to induce an effective immune deviation able to significantly modify the course of disease.

Characterization of IL-12 receptor beta1 chain (IL-12Rbeta1)-deficient mice: IL-12Rbeta1 is an essential component of the functional mouse IL-12 receptor.

Two chains of the IL-12R, IL-12Rbeta1 and IL-12Rbeta2, have recently been cloned. To determine the role of IL-12Rbeta1 in mediating the biologic functions of IL-12 in mice, we have generated IL-12Rbeta1-deficient (IL-12Rbeta1(-/-)) mice by targeted mutation in ES cells. Con A-activated splenocytes from IL-12Rbeta1(+/+) mice displayed both high and low affinity IL-12-binding sites, whereas Con A-activated splenocytes from IL-12Rbeta1(-/-) mice expressed only low affinity IL-12-binding sites. Consistent with the expression of low affinity IL-12-binding sites on IL-12Rbeta1(-/-) lymphoblasts, these cells expressed normal amounts of IL-12Rbeta2 mRNA. Unlike those from IL-12Rbeta1(+/+) mice, Con A-activated splenocytes from IL-12Rbeta1(-/-) mice failed to proliferate or produce IFN-gamma in response to IL-12, even at very high concentrations (67 nM). In contrast, lymphoblasts from both types of mice proliferated equally well to IL-2 or IL-7. Splenocytes from IL-12Rbeta1(-/-) mice also failed to display enhanced NK lytic activity when cultured with IL-12 but responded normally to IL-2. Similar to IL-12 p40-deficient mice, IL-12Rbeta1(-/-) mice were impaired in their ability to produce IFN-gamma in response to endotoxin administration in vivo, and IL-12Rbeta1(-/-) splenocytes were deficient in IFN-gamma secretion when stimulated with either Con A or soluble anti-CD3 mAb in vitro. These results demonstrate that IL-12Rbeta1 is required for mouse T and NK cells to respond to IL-12 and that expression of low affinity IL-12-binding sites, presumably reflecting expression of IL-12Rbeta2, is by itself insufficient to mediate IL-12 responsiveness, even in the presence of very high concentrations of IL-12.

Bioactive murine and human interleukin-12 fusion proteins which retain antitumor activity in vivo.

Interleukin-12 (IL-12) is unique amongst cytokines in being a disulfide-linked heterodimer of two separately encoded subunits (p35 and p40). We expressed single chain IL-12 proteins from retroviral constructs in which the two IL-12 subunits were linked by a 6-15 amino acid polypeptide linker, with deletion of the 22 amino acid leader sequence of the trailing subunit. The murine fusion protein IL-12.p40.L.delta p35 containing a (Gly4Ser)3 linker was stably expressed, bioactive in vitro, and had an apparent specific activity comparable to that of native and recombinant IL-12. Western blotting confirmed that murine IL-12.p40.L.delta p35 retained the linking polypeptide sequences. The analogous human IL-12.p40.L.delta p35 fusion protein containing a Gly6Ser linker was bioactive with an apparent specific activity comparable to recombinant human IL-12. In a preexisting CMS-5 tumor model, CMS-5 cells secreting either native or fusion protein forms of IL-12 prolonged survival and led to complete tumor regression.

Regulation of interleukin-12 receptor beta1 chain expression and interleukin-12 binding by human peripheral blood mononuclear cells.

The interleukin-12 receptor (IL-12R)beta1 chain is an essential component of the functional IL-12R on both human T and natural killer cells. In this report it is shown that activation of human peripheral blood mononuclear cells (PBMC) with anti-CD3 monoclonal antibody (mAb) or phytohemagglutinin resulted in the up-regulation of IL-12Rbeta1 expression and IL-12 binding. Kinetic studies revealed that maximum expression of IL-12Rbeta1 and IL-12 binding occurred on days 3-4. Anti-CD3-induced expression of IL-12Rbeta1 chain and IL-12 binding by PBMC was augmented by anti-CD28 mAb, indicating that the potentiating effect of anti-CD28 on T cell responses to IL-12 could be mediated, at least in part, by the enhancement of IL-12R expression. Among 16 cytokines tested, IL-2, IL-7 and IL-15 markedly induced IL-12Rbeta1 expression and IL-12 binding on resting PBMC, whereas IL-1alpha and tumor necrosis factor-alpha had a minimal enhancing effect. In contrast, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, interferon (IFN)-alpha, IFN-gamma, granulocyte/macrophage colony-stimulating factor and transforming growth factor (TGF)-beta2 had no detectable enhancing effect. Anti-CD3-induced expression of IL-12Rbeta1 and of low-affinity IL-12 binding sites was partially inhibited by TGF-beta2, IL-10 and IL-4; however, TGF-beta2 and IL-10 completely abolished anti-CD3-induced expression of high-affinity IL-12 binding sites. Consistent with the reduction of high affinity IL-12 binding sites, PBMC activated with anti-CD3 mAb in the presence of TGF-beta2 or IL-10 failed to produce IFN-gamma or to proliferate in response to IL-12. These results suggest that Th2 cell-derived cytokines can inhibit IL-12-induced biological functions by inhibiting IL-12R expression and that expression of a second subunit of the IL-12R (IL-12Rbeta2), required for the formation of high-affinity IL-12 binding sites, may be more highly regulated by TGF-beta2 and IL-10 than is expression of IL-12Rbeta1.

Sterile protection of monkeys against malaria after administration of interleukin-12.

An estimated 300-500 million new infections and 1.5-2.7 million deaths attributed to malaria occur annually in the developing world, and every year tens of millions of travelers from countries where malaria is not transmitted visit countries with malaria. Because the parasites that cause malaria have developed resistance to many antimalarial drugs, new methods for prevention are required. Intraperitoneal injection into mice of one dose of 150 ng (approximately 7.5 micrograms per kg body weight) recombinant mouse interleukin-12 (rmIL-12) 2 days before challenge with Plasmodium yoelii sporozoites protects 100% of mice against malaria. We report that one subcutaneous injection of 10 micrograms/kg recombinant human IL-12 (rhIL-12) 2 days before challenge with P. cynomolgi sporozoites protected seven of seven rhesus monkeys. Protection was associated with marked increases in plasma levels of interferon-gamma (IFN-gamma), and relative increases of lymphoid cell messenger RNA coding for IFN-gamma and several other cytokines. We speculate that rIL-12 protects monkeys through IFN-gamma-dependent elimination of P. cynomolgi-infected hepatocytes. This first report of rIL-12-induced protection of primates against an infectious agent supports assessment of rhIL-12 for immunoprophylaxis of human malaria.

Interleukin-12: potential clinical applications in the treatment of chronic viral hepatitis.

Interleukin-12 (IL-12) is a heterodimeric cytokine that promotes cell-mediated immunity by facilitating type 1 helper T-lymphocyte responses, inducing the secretion of interferon-gamma from both T and natural killer cells, enhancing the lytic activity of natural killer cells, and augmenting specific cytolytic T-lymphocyte responses. In addition, IL-12 can increase the production of some subclasses of IgG antibodies. IL-12 has been shown to have potent therapeutic effects in a number of animal models of tumours and infectious diseases, including several viral infections. These results have led to the initiation of clinical trials to evaluate the therapeutic potential of IL-12 in human cancer patients and in patients infected with the human immunodeficiency virus. The biological activities of IL-12 suggest that it may also have clinical utility in the treatment of patients suffering from chronic hepatitis B or C virus infections. Because of the lack of suitable animal models for evaluating the efficacy of IL-12 in the treatment of infections with these viruses, only a clinical trial in patients with chronic viral hepatitis can address the potential role of IL-12 as an effective treatment for these disorders.

Reduced incidence and severity of collagen-induced arthritis in interleukin-12-deficient mice.

Collagen-induced arthritis (CIA) is an animal model for rheumatoid arthritis. The disease is elicited by immunization of genetically susceptible DBA/1 mice with type II collagen, resulting in a debilitating arthritis characterized by inflammation and involvement of multiple joints. We investigated the role of endogenous interleukin (IL)-12 in the pathogenesis of this disease by undertaking an analysis of IL-12-deficient mice on the DBA/1 genetic background after immunization with type II collagen. Both the incidence and severity of disease were significantly reduced in mice unable to produce biologically active IL-12. Concomitant decreases were observed in serum levels of pathogenic, collagen-specific IgG2a antibodies and collagen-induced secretion of interferon-gamma by immune splenocytes in vitro, consistent with an impaired T helper-1 response. There were, however, a few animals which developed severe disease in a single paw in spite of this highly diminished Th1 response. Taken together, these results demonstrate an important role for IL-12 in the pathogenesis of CIA, although it is not absolutely required for disease development.

A functional interleukin 12 receptor complex is composed of two beta-type cytokine receptor subunits.

We have identified a cDNA from a human phytohemagglutinin-activated lymphoblast library encoding a protein that binds 125I-labeled human interleukin 12 (125I-huIL-12) with a Kd of about 5 nM when expressed in COS-7 cells. When coexpressed in COS-7 cells with the previously identified IL-12 beta receptor (IL-12R beta) protein, two classes of 125I-huIL-12 binding sites were measured with Kds of about 55 pM and 8 nM, corresponding to the high- and low-affinity binding sites seen on phytohemagglutinin-activated lymphoblasts. This newly identified huIL-12R subunit is a member of the cytokine receptor superfamily, with closest resemblance to the beta-type cytokine receptor gp130 and the receptors for leukemia inhibitory factor and granulocyte colony-stimulating factor. Consequently, we have reclassified the previously identified IL-12R beta subunit as huIL-12R beta 1 and designated the newly identified subunit as huIL-12R beta 2. huIL-12R beta 2 is an 862-amino acid type I transmembrane protein with a 595-amino-acid-long extracellular domain and a cytoplasmic tail of 216 amino acids that contains three tyrosine residues. A cDNA encoding the mouse homolog of the huIL12R beta 2 protein has also been isolated. Human and mouse IL-12R beta 2 proteins show a 68% amino acid sequence identity. When expressed in COS-7 cells, huIL-12R beta 2 exists as a disulfide-linked oligomer with an apparent monomeric molecular weight of 130 kDa. Coexpression of the two identified IL-12R subunits in Ba/F3 cells conferred IL-12 responsiveness, and clones of these cotransfected Ba/F3 cells that grew continuously in the presence of IL-12 were isolated and designated LJM-1 cells. LJM-1 cells exhibited dose-dependent proliferation in response to huIL-12, with an ED50 of about 1 pM huIL-12. Interestingly, Ba/F3 cells transfected with IL-12R beta 2 alone proliferated in response to huIL-12 with an ED50 of about 50 pM, although a role for endogenous mouse IL-12R beta 1 in IL-12 signal transduction in these transfectants cannot be ruled out. These results demonstrate that the functional high-affinity IL-12R is composed of at least two beta-type cytokine receptor subunits, each independently exhibiting a low affinity for IL-12.

Interleukin-12 antagonist activity of mouse interleukin-12 p40 homodimer in vitro and in vivo.

Mo(p40)2 is a potent IL-12 antagonist that interacts strongly with the beta 1 subunit of the IL-12R to block binding of moIL-12 to the high-affinity mouse IL-12R. Mo(p40)2, alone or in synergy with the 2B5 mAb specific for the moIL-12 heterodimer, blocked IL-12-induced responses in vitro, Mo(p40)2 was thus used alone or with 2B5 mAb to examine the role of IL-12 in vivo, Mo(p40)2 caused a dose-dependent inhibition of both the rise in serum IFN-gamma levels in mice injected with endotoxin and the Th1-like response to immunization with KLH. Treatment with mo(p40)2 plus 2B5 anti-moIL-12 mAb also suppressed DTH responses to methylated bovine serum albumin but not specific allogeneic CTL responses in vivo. In each of these models, responses seen in mice treated with mo(p40)2 +/- 2B5 anti-moIL-12 mAb were similar to those observed in IL-12 knockout mice. Thus, mo(p40)2 can act as a potent IL-12 antagonist in vivo, as well as in vitro, and is currently being used to investigate the role of IL-12 in the pathogenesis of some Th1-associated autoimmune disorders in mice.

IL-12-deficient mice are defective but not devoid of type 1 cytokine responses.

Interleukin-12 (IL-12) has been described as a pivotal molecule in the immune response based in part on its ability to influence the differentiation of T helper (Th) cells into a type 1 (Th1) phenotype. This event is crucial in that appropriate differentiation of naive T cells can determine susceptibility or resistance to given pathogens by influencing the balance between cellular and humoral immunity. In order to further delineate the role of IL-12 in the immune response, we generated mice deficient for this cytokine. IL-12 knockout mice were viable, fully fertile, and displayed no obvious developmental abnormalities. Upon immunological analysis, these mice demonstrated an impaired ability to effect a Th1 response as well as an impaired ability to produce interferon-gamma in response to endotoxin in vivo. These data establish an essential role for IL-12 in the generation of optimal Th1 responses in vivo, but weak responses can occur independently of IL-12.