Experiments In Knockout Mice Research Articles

Polymorphisms in DNA repair genes as risk factors for spina bifida and orofacial clefts

Repairing DNA damage is critical during embryogenesis because development involves sensitive periods of cell proliferation, and abnormal cell growth or death can result in malformations. Knockout mouse experiments have demonstrated that disruption of DNA repair genes results in embryolethality and structural defects. Studies using mid-organogenesis rat embryos showed that DNA repair genes were variably expressed. It is hypothesized that polymorphisms that alter the functionality of DNA repair enzymes may modify the risk of malformations. We conducted a case-control analysis to investigate the relationship between DNA repair gene polymorphisms and the risk of spina bifida and oral clefts. Newborn screening blood spot DNA was obtained for 250 cases (125 spina bifida, 125 oral clefts) identified by the California Birth Defects Monitoring Program, and 350 non-malformation controls identified from birth records. Six single nucleotide polymorphisms of five DNA repair genes representing three distinct repair pathways were interrogated including: XRCC1 (Arg399Gln), APE1 (Asp148Glu), XRCC3 (Thr241Met), hOGG1(Ser326Cys), XPD (Asp312Asn, Lys751Gln). Elevated or decreased odds ratios (OR, adjusted for race/ethnicity) for spina bifida were found for genotypes containing at least one copy of the variant allele for XPD [751Gln, OR = 1.62; 95% confidence interval (CI) = 1.05-2.50] and APE 148 (OR = 0.58; CI = 0.37-0.90). A decreased risk of oral clefts was found for XRCC3 (OR = 0.62; CI = 0.39-0.99) and hOGG1 (326 Cys/Cys, OR = 0.22; CI = 0.06-0.78). This study suggested that polymorphisms of DNA repair genes, representing different major repair pathways, may affect risk of two major birth defects. Future, larger studies, examining additional repair genes, birth defects, and interaction with exposures are recommended.

Cell cycle in mouse development

Mice likely represent the most-studied mammalian organism, except for humans. Genetic engineering in embryonic stem cells has allowed derivation of mouse strains lacking particular cell cycle proteins. Analyses of these mutant mice, and cells derived from them, facilitated the studies of the functions of cell cycle apparatus at the organismal and cellular levels. In this review, we give some background about the cell cycle progression during mouse development. We next discuss some insights about in vivo functions of the cell cycle proteins, gleaned from mouse knockout experiments. Our text is meant to provide examples of the recent experiments, rather than to supply an extensive and complete list.

Book review

Book review

The quantitative role of alternative pathway amplification in classical pathway induced terminal complement activation

Complement activation with formation of biologically potent mediators like C5a and the terminal C5b-9 complex (TCC) contributes essentially to development of inflammation and tissue damage in a number of autoimmune and inflammatory conditions. A particular role for complement in the ischaemia/reperfusion injury of the heart, skeletal muscle, central nervous system, intestine and kidney has been suggested from animal studies. Previous experiments in C3 and C4 knockout mice suggested an important role of the classical or lectin pathway in initiation of complement activation during intestinal ischaemia/reperfusion injury while later use of factor D knockout mice showed the alternative pathway to be critically involved. We hypothesized that alternative pathway amplification might play a more critical role in classical pathway-induced C5 activation than previously recognized and used pathway-selective inhibitory mAbs to further elucidate the role of the alternative pathway. Here we demonstrate that selective blockade of the alternative pathway by neutralizing factor D in human serum diluted 1 : 2 with mAb 166-32 inhibited more than 80% of C5a and TCC formation induced by solid phase IgM and solid- and fluid-phase human aggregated IgG via the classical pathway. The findings emphasize the influence of alternative pathway amplification on the effect of initial classical pathway activation and the therapeutic potential of inhibiting the alternative pathway in clinical conditions with excessive and uncontrolled complement activation.

Myogenic regulatory factors: Redundant or specific functions? Lessons from Xenopus

The discovery, in the late 1980s, of the MyoD gene family of muscle transcription factors has proved to be a milestone in understanding the molecular events controlling the specification and differentiation of the muscle lineage. From gene knock-out mice experiments progressively emerged the idea that each myogenic regulatory factor (MRF) has evolved a specialized as well as a redundant role in muscle differentiation. To date, MyoD serves as a paradigm for the MRF mode of function. The features of gene regulation by MyoD support a model in which subprograms of gene expression are achieved by the combination of promoter-specific regulation of MyoD binding and MyoD-mediated binding of various ancillary proteins. This binding likely includes site-specific chromatin reorganization by means of direct or indirect interaction with remodeling enzymes. In this cascade of molecular events leading to the proper and reproducible activation of muscle gene expression, the role and mode of function of other MRFs still remains largely unclear. Recent in vivo findings using the Xenopus embryo model strongly support the concept that a single MRF can specifically control a subset of muscle genes and, thus, can be substituted by other MRFs albeit with dramatically lower efficiency. The topic of this review is to summarize the molecular data accounting for a redundant and/or specific involvement of each member of the MyoD family in myogenesis in the light of recent studies on the Xenopus model.

Prolonged production of TNF-alpha exacerbates illness during respiratory syncytial virus infection.

CD8(+) CTL are the main effector cells responsible for resolving viral infections. However, the CTL response to respiratory syncytial virus (RSV) infection in mice facilitates viral clearance at the expense of significant immunopathology. Previous reports have shown a strong correlation between the mechanism of CTL activity and the severity of RSV-induced illness. Furthermore, experiments in perforin knockout mice revealed that antiviral cytokine production temporally correlated with RSV-induced illness. In the current study, we show that TNF-alpha is the dominant mediator of RSV-associated illness, and it is also important for clearance of virus-infected cells during the early stages of infection. We also demonstrate that IFN-gamma plays a protective role in conjunction with perforin/granzyme-mediated killing. Preliminary experiments in gld mice that express nonfunctional Fas ligand (FasL) revealed that RSV-induced illness is significantly reduced in the absence of FasL-mediated killing. Antiviral cytokine production was not elevated in the absence of FasL, suggesting a possible link between FasL and antiviral cytokine activity. This work shows that multiple phenotypic subsets of CD8(+) CTLs respond to RSV infection, each with varying capacities for clearance of virus-infected cells and the induction of illness. In addition, the revelation that TNF-alpha is the principal mediator of RSV-induced illness means that administration of TNF receptor antagonists, in combination with antiviral therapy, may be an effective method to treat RSV infections.

Increased expression of regulator of G protein signaling-2 (RGS-2) in Bartter's/Gitelman's syndrome. A role in the control of vascular tone and implication for hypertension.

Regulator of G protein signaling-2 (RGS-2) plays a key role in the G protein-coupled receptor (GPCR) angiotensin II (Ang II) signaling. NO and cGMP exert a vasodilating action also through activation and binding to RGS-2 of cGMP dependent protein kinase 1-alpha, which phosphorylates RGS-2 and dephosphorylates myosin light chain. In Bartter's/Gitelman's patients (BS/GS) Ang II related signaling and vasomotor tone are blunted. Experiments were planned to explore whether RGS-2 may play a role in BS/GS vascular hyporeactivity. NO metabolites and cGMP urinary excretion were also measured. Mononuclear cells (PBM) from six BS/GS patients and six healthy controls were used. PBM RGS-2 mRNA and RGS-2 protein were increased in BS/GS: 0.47 +/- 0.06 d.u. vs 0.32 +/- 0.04, (p < 0.006) (RGS-2 mRNA), and 0.692 +/- 0.02 vs 0.363 +/- 0.06 (p < 0.0001) (RGS2 protein). Incubation of PBM with Ang II increased RGS-2 protein in controls (from 0.363 +/- 0.06 d.u. to 0.602 +/- 0.05; p < 0.0001) but not in BS/GS (from 0.692 +/- 0.02 to 0.711 +/- 0.02). NO(2)(-)/NO(3)(-) and cGMP urinary excretion were increased in BS/GS (0.46 +/- 0.13 vs 0.26 +/- 0.05 micromol/micromol of urinary creatinine, p < 0.005, and 0.060 +/- 0.030 vs 0.020 +/- 0.01 p < 0.009, respectively). These results demonstrate that RGS-2 is increased and maximally stimulated in BS/GS and human RGS-2 system reacts as predicted by knockout mice experiments. This is the first report of RGS-2 level in a human clinical condition characterized by altered vascular tone, underlines the importance of RGS-2 as a key regulator element for Ang II signaling and provides insight into the links between BS/GS genetic abnormalities and abnormal vascular tone regulation.

Nonspecific antiviral immunity by formalin-fixed Coxiella burnetii is enhanced in the absence of nitric oxide

Mice treated with a single injection of formalin-fixed Coxiella burnetii showed a significant increase in resistance to vaccinia virus (VV) infection compared to untreated mice. C. burnetii stimulated dramatically high levels of nitric oxide (NO) in the serum of treated mice, suggesting that NO might play a role in resistance to virus infection. To test this hypothesis, the effect of C. burnetii treatment on VV replication was examined in NOS2 −/− and wild-type mice. C. burnetii treatment inhibited VV replication in both the knockout and wild-type mice but the effect was significantly greater in the NOS2 −/− mice. Experiments in IFNγ receptor knockout mice indicated that the nonspecific antiviral immunity induced by C. burnetii was dependent on IFNγ and not NO. In the absence of NO, indoleamine 2,3-dioxygenase (IDO) was increased in C. burnetii-treated mice and this may contribute to the accelerated virus clearance in NOS2 −/− mice.

LONGTERM CARDIAC ALLOGRAFT SURVIVAL IN VAV1 KNOCKOUT MICE

O331* Aims: T cells are key players in the immune response leading to graft rejection. The guanine nucleotide exchange factor Vav1 mediates TCR and CD28-triggered activation and proliferation of T lymphocytes by activating small GTPases such as Rac1. To assess the involvement of Vav1 in graft rejection, we performed heart transplantation experiments in Vav1 knockout (ko) mice. Moreover, immune responses in Vav1-deficient mice were characterized in vivo and in vitro. Methods: BALB/c cardiac allografts are transplanted heterotopically into Vav1 ko C57B/6 recipient mice. Graft survival is monitored by palpation of heart beat. At the end of the study graft histology is performed. The effect of Vav1 deficiency on T cell-dependent antibody formation is assessed by i.p. immunization with DNP-KLH. Ex vivo immune assays include one-way mixed lymphocyte reaction (MLR) with irradiated BALB/c splenocytes as stimulators as well as FACS-based analysis of activation marker expression on lymphocyte subsets in whole blood and spleen. Results: In the absence of immunosuppressive treatment, the median survival time of cardiac allografts in Vav1-deficient recipient mice was more than 100 days as compared to 7 days in WT recipients. Terminal immunohistological evaluation of grafts revealed scattered infiltrates of T and B cells as well as of perivascular macrophages which was associated with signs of remodeling of vessels in grafts surviving 100 days post transplantation. In the DNP-KLH mouse model the anti-DNP titer is largely suppressed by cyclosporine. In contrast Vav1-deficient mice are capable of T cell-dependent antibody formation indicating that Vav1-dependent and CsA-inhibited pathways are different. In the one-way MLR assay the proliferation was markedly decreased using various ratios of BALB/c and Vav1-deficient splenocytes. Stimulation of Vav1-deficient lymphocytes from spleen and whole blood with ConA, anti-CD3 or anti-CD3/anti-CD28 resulted in markedly reduced expression of the activation marker CD69 as compared to WT cells. This defect was observed with CD4+, CD8+ and B220+ lymphocytes. Conclusions: We show for the first time that absence of Vav1 in recipient mice prevents allograft rejection. Vav1 deficiency was associated with defects in T cell activation.

Functions of intermediate filaments in neuronal development and disease.

Five major types of intermediate filament (IF) proteins are expressed in mature neurons: the three neurofilament proteins (NF-L, NF-M, and NF-H), alpha-internexin, and peripherin. While the differential expression of IF genes during embryonic development suggests potential functions of these proteins in axogenesis, none of the IF gene knockout experiments in mice caused gross developmental defects of the nervous system. Yet, deficiencies in neuronal IF proteins are not completely innocuous. Substantial developmental loss of motor axons was detected in mice lacking NF-L and in double knockout NF-M;NF-H mice, supporting the view of a role for IFs in axon stabilization. Moreover, the absence of peripherin resulted in approximately 30% loss of small sensory axons. Mice lacking NF-L had a scarcity of IF structures and exhibited a severe axonal hypotrophy, causing up to 50% reduction in conduction velocity, a feature that would be very detrimental for large animal species. Unexpectedly, the NF-M rather than NF-H protein turned out to be required for proper radial growth of large myelinated axons. Studies with transgenic mice suggest that some types of IF accumulations, reminiscent of those found in amyotrophic lateral sclerosis (ALS), can have deleterious effects and even cause neurodegeneration. Additional evidence for the involvement of IFs in pathogenesis came from the recent discovery of neurofilament gene mutations linked to ALS and Charcot-Marie-Tooth disease (CMT2E). Conversely, we discuss how certain types of perikaryal neurofilament aggregates might confer protection in motor neuron disease.

Role of proteolysis in polyglutamine disorders.

To date, nine polyglutamine disorders have been characterised, including Huntington's disease (HD), spinobulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), and spinocerebellar ataxias 1, 2, 3, 6, 7 and 17 (SCAs). Although knockout and transgenic mouse experiments suggest that a toxic gain of function is central to neuronal death in these diseases (with the probable exception of SCA6), the exact mechanisms of neurotoxicity remain contentious. A further conundrum is the characteristic distribution of neuronal damage in each disease, despite ubiquitous expression of the abnormal proteins. One mechanism that could possibly underlie the specific distribution of neuronal toxicity is proteolytic cleavage of the full-length expanded polyglutamine tract-containing proteins. There is evidence found in vitro or in vivo (or both) of proteolytic cleavage in HD, SBMA, DRPLA, and SCAs 2, 3, and 7. In HD, cleavage has been demonstrated to be regionally specific, occurring as a result of caspase activation. These diseases are also characterised by development of intraneuronal aggregates of the abnormal protein that co-localise with components of the ubiquitin-proteasome pathway. It remains unclear whether these aggregates are pathogenic or merely disease markers; however, at least in the case of ataxin-3, cleavage promotes aggregation. Inhibition of specific proteases constitutes a potential therapeutic approach in these diseases.

Expression profiling of Epo receptor in non-hematopoietic tissues: implications for a direct effect of rHuEpo on cardiac cells

Growing experimental evidence suggests that in addition to erythropoietin's (Epo) role in stimulating erythropoiesis, Epo may function in multiple tissues by preventing cellular apoptosis under stressful conditions such as hypoxia or exposure to cytotoxins and by stimulation of proliferation. EPO and EPOR knockout experiments in mice have demonstrated that the Epo/EpoR axis is essential for normal cardiac morphogenesis. While data suggests that there is an indirect effect of Epo on cardiac myocytes through increased hematocrit or other indirect effects, it is also possible that Epo acts directly on these cells. Therefore we have investigated the expression and function of EpoR in human cardiac cells. EpoR expression profiling in multiple murine, rat, monkey and human tissues using quantitative RT-PCR and RNAse protections (RPA's) identified EpoR transcripts in hematopoietic tissues, brain, kidney, adrenal gland, pancreas, liver and heart. More specifically in heart, EpoR transcripts were detected in monkey ventricle, atrium and septum. EpoR transcripts were also detected in primary human cardiac cell cultures, demonstrating that transcript detection was not due to contaminating hematopoietic cells. To determine whether transcription of EpoR correlated with expression of protein, immunolocalization experiments using a monoclonal antibody to human EpoR detected EpoR expression in human heart, located within cardiomyocytes, and within primary cardiac myocyte cultures. Staining was specific as demonstrated by reduced staining when the antibody was pre-incubated with soluble human EpoR. These data suggest that EpoR is expressed by cardiac cells and may suggest potential new functions of Epo in the heart. This may offer new options for the treatment of heart disease with erythropietic stimulating proteins such as darbepoetin alfa. Growing experimental evidence suggests that in addition to erythropoietin's (Epo) role in stimulating erythropoiesis, Epo may function in multiple tissues by preventing cellular apoptosis under stressful conditions such as hypoxia or exposure to cytotoxins and by stimulation of proliferation. EPO and EPOR knockout experiments in mice have demonstrated that the Epo/EpoR axis is essential for normal cardiac morphogenesis. While data suggests that there is an indirect effect of Epo on cardiac myocytes through increased hematocrit or other indirect effects, it is also possible that Epo acts directly on these cells. Therefore we have investigated the expression and function of EpoR in human cardiac cells. EpoR expression profiling in multiple murine, rat, monkey and human tissues using quantitative RT-PCR and RNAse protections (RPA's) identified EpoR transcripts in hematopoietic tissues, brain, kidney, adrenal gland, pancreas, liver and heart. More specifically in heart, EpoR transcripts were detected in monkey ventricle, atrium and septum. EpoR transcripts were also detected in primary human cardiac cell cultures, demonstrating that transcript detection was not due to contaminating hematopoietic cells. To determine whether transcription of EpoR correlated with expression of protein, immunolocalization experiments using a monoclonal antibody to human EpoR detected EpoR expression in human heart, located within cardiomyocytes, and within primary cardiac myocyte cultures. Staining was specific as demonstrated by reduced staining when the antibody was pre-incubated with soluble human EpoR. These data suggest that EpoR is expressed by cardiac cells and may suggest potential new functions of Epo in the heart. This may offer new options for the treatment of heart disease with erythropietic stimulating proteins such as darbepoetin alfa.

Thyroid Hormone Action: Insight from Transgenic Mouse Models

Thyroid hormone receptors (TRs) are cellular homologues of the viral erythroblastic leukemia oncogene (v-erbA). TRs (c-crbA isoforms) are derived from two separate gene loci in mammals: α and β. Through...

Thyroid Hormone Action: Insight from Transgenic Mouse Models

Thyroid hormone receptors (TRs) are cellular homologues of the viral erythroblastic leukemia oncogene (v-erbA). TRs (c-crbA isoforms) are derived from two separate gene loci in mammals: a and p. Through a series of knockout experiments in mice in which one or several of the TR isoforms were deleted, it has been demonstrated that the TR-β isoforms control central regulation of thyroid-stimulating hormone. Of these isoforms, TR-β2 is the most important in mediating negative feedback control of the hypothalamic-pituitary-thyroid axis. Further analysis of TR knockout animals revealed, however, that they exhibited a much milder overall phenotype than hypothyroid animals, indicating that receptor loss was not equivalent to ligand loss in vivo. To understand this apparent paradox, we generated animals expressing a non-T3 binding receptor (Δ337T) from the TR-β allele. These mice displayed a complete hypothyroid phenotype, demonstrating that the unliganded TR mediates the effect of hypothyroidism. Because this mutant TR constitutively binds to nuclear coreprssors, it also suggests that this class of proteins is essential for mediating hypothyroidism in vivo.

Ca 2+ Buffer Saturation Underlies Paired Pulse Facilitation in Calbindin-D28k-Containing Terminals

Ca 2+ buffer saturation was proposed as a mechanism of paired pulse facilitation (PPF). However, whether it operates under native conditions remained unclear. Here we show that saturation of the endogenous fast Ca 2+ buffer calbindin-D28k (CB) plays a major role in PPF at CB-containing synapses. Paired recordings from synaptically connected interneurons and pyramidal neurons in the mouse neocortex revealed that dialysis increased the amplitude of the first response and decreased PPF. Loading the presynaptic terminals with BAPTA or CB rescued the effect of the CB washout. We extended the study to the CB-positive facilitating excitatory mossy fiber-CA3 pyramidal cell synapse. The effects of different extracellular Ca 2+ concentrations and of EGTA indicated that PPF in CB-containing terminals depended on Ca 2+ influx rather than on the initial release probability. Experiments in CB knockout mice confirmed that buffer saturation is a novel basic presynaptic mechanism for activity-dependent control of synaptic gain.

The angiotensin converting enzyme (ACE)

Angiotensin converting enzyme 1, found widely throughout the animal kingdom, is an integral membrane bound protein whose active sites are directed to the extracellular spaces. Two isoforms are expressed in mammals, a single domain germinal isoform required for male fertility, and a double domain somatic isoform which has a key role in the renin-angiotensin system. Both somatic domains are active with different substrate affinities. Mouse knockout experiments, and comparative work with invertebrate homologues, suggest that the two domains have clearly distinct roles. The importance of therapies involving inhibition of angiotensin converting enzyme are undisputed, but our understanding of how and why these therapies work is now being informed by the tools of genomic and comparative biology.

Tumor suppression induced by intratumor administration of adenovirus vector expressing NK4, a 4-kringle antagonist of hepatocyte growth factor, and naive dendritic cells

NK4, a 4-kringle antagonist of hepatocyte growth factor (HGF), is a potent inhibitor of tumor angiogenesis and functions independently of its HGF-antagonistic activity. We have shown previously that in vivo genetic modification of tumors with an adenovirus vector that expresses NK4 (AdNK4) restrains tumor angiogenesis and slows the rate of tumor growth in vivo. In the present study, we investigated the hypothesis that this can be made more efficient by also administering bone marrow-generated dendritic cells (DCs) to the tumor. The data show that the growth of mouse subcutaneous tumors is significantly suppressed by direct administration of DCs into established tumors that had been pretreated with AdNK4 3 days previously. The synergistic antitumor effect produced by the combination therapy of AdNK4 with DCs correlated with the in vivo priming of tumor-specific cytotoxic T lymphocytes. Analysis of mice treated with fluorescence-labeled DCs suggested that DCs injected into the flank tumor could migrate to lymphoid organs in vivo for activation of immune-relevant processes. Knockout mice experiments demonstrated that the tumor regression produced by this combination therapy depends on both major histocompatibility complex (MHC) class I antigen presentation of DCs injected into the tumors and CD8(+) T cells of the treated host. Finally, a mechanism for this synergism was suggested by the histological observation that tumor necrosis and apoptosis were induced by genetic engineering of the tumors to express NK4. These findings should be useful in designing novel strategies that use a combination of 2 monotherapies directed against the vascular and immune systems for cancer therapy.

The use of germ line-mutated mice in understanding host-pathogen interactions.

Microbial pathogenesis reflects an imbalance between parasite and host factors that favour pathogen multiplication and tissue destruction over those required for microbial elimination and preservation of the integrity of host tissues. In vivo analysis of host-pathogen interactions has been revolutionized by the ability to engineer specific genetic alterations including loss of function mutations and transgenes into the mouse germline. This brief review recapitulates what we have learned about the host response to Toxoplasma gondii infection to illustrate the usefulness of gene-altered mice in microbial pathogenesis research. A consideration of the pitfalls and limitations of experiments in knockout mice and ways of addressing these concerns are discussed. Finally, advances in inducible and tissue-restricted alterations in gene function are presented and their possible applications to microbiology research are considered.

Blockade of central neuropeptide Y (NPY) Y2 receptors reduces ethanol self-administration in rats

Activation of central neuropeptide Y (NPY) receptors is known to produce several behavioral effects, including feeding, modulation of memory and antagonism of behavioral effects of stress. In addition, experiments in knock-out and transgenic mice have suggested a possible role of NPY regulation of voluntary ethanol intake. NPY receptors involved in this action are not known. Here, we examined the effects of a selective NPY-Y2 receptor antagonist, BIIE0246, on operant responding for ethanol in a sweetened solution, or the sweetened solution without ethanol, during 30 min sessions of free choice between the two. BIIE0246 produced a robust suppression of responding for ethanol (40% reduction, P=0.013) at an intracerebroventricular dose of 1.0 nmol, but not 0.3 nmol. Responding for the saccharin solution was not significantly affected. The dose range examined was selected since preliminary experiments with doses of 3 nmol and higher indicated sedative effects, but such effects were absent up to 1.0 nmol, as shown by unaffected exploratory locomotor activity. In summary, antagonism at central NPY-Y2 receptors seems to selectively suppress operant self-administration of ethanol. This suggests that Y2 receptors might be candidate targets for developing novel pharmacological treatments of alcoholism.

Bioavailability of recombinant tumor necrosis factor determines its lethality in mice.

In mice, tumor necrosis factor (TNF) displays a selective species specificity. In contrast to murine TNF (mTNF), human TNF (hTNF) only induces lethality at extremely high doses of about 500 microg/mouse, whereas it still has a powerful antitumor activity in combination with interferon-gamma. The observation that hTNF does not interact with the p75 mTNF receptor seemed to provide a plausible explanation for these species-specific biological effects. Experiments in TNF receptor knockout mice and tests with hTNF muteins in baboons did not, however, support this hypothesis. We here show that an mTNF mutein selective for the p55 mTNF receptor induces lethality in a manner comparable to wild-type mTNF, and conclude that other differences between hTNF and mTNF must account for the reduced lethality of hTNF. Pharmacokinetics showed that hTNF is cleared much faster than mTNF or the mTNF mutein used. In contrast to the hardly lethal effect(s) of a bolus administration of hTNF, fractionated repetitive administration of the same total hTNF dose induced lethality. This suggests that prolonged exposure rather than peak levels determine the lethal effects of hTNF in mice. Experiments with receptor and ligand knockouts demonstrated that the difference in pharmacokinetics is independent of an interaction with (soluble) TNF receptor, TNF-induced effects or induction of endogenous TNF. These results show that manipulation of the clearance rate of TNF may broaden the therapeutic range of systemic treatments with TNF.