Utility of Botulinum Injections in Stiff-Person Syndrome.

Stiff-person syndrome (SPS) is an uncommon neurological disorder characterized by significant rigidity and muscle spasms primarily affecting the truncal and proximal musculature. Furthermore, a wide-based gait with functional impairment is generally seen. High-dose benzodiazepines or baclofen are widely considered the optimal initial therapy; however, major adverse effects often preclude adequate dosing. Refractory cases may be treated with intravenous immunoglobulins (IVIG), plasma exchange, or B-cell depletion with rituximab, although these are also associated with major, sometimes fatal, adverse reactions. Several reports have validated the safety and utility of botulinum injections in this setting, yet botulinum remains markedly underutilized in this cohort. Below, a case report and review of the literature show botulinum can decrease pain and stiffness, improve gait and balance, and decrease dependence on powerful systemic treatments in this group.

Preclinical evaluation of the penciclovir analog 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl)guanine for in vivo measurement of suicide gene expression with PET.

UNLABELLED: The gene for herpes simplex virus thymidine kinase (HSV-tk) is widely used as a suicide gene in experimental gene therapy of cancer. 9-(4-Fluoro-3-hydroxymethylbutyl)guanine (FHBG) is an antiviral nucleoside analog that is rapidly phosphorylated by viral thymidine kinase but is a poor substrate for mammalian thymidine kinase. Recently, FHBG labeled in the 4-fluoro position with (18)F has shown promise relative to other similar compounds for imaging in vivo expression of HSV-tk using PET. In this study, we evaluated the uptake of [(18)F]FHBG in vitro and in vivo using transduced and wild-type human colon cancer cells (HT-29). We also imaged [(18)F]FHBG and measured the radioactivity concentrations of circulating [(18)F]FHBG and its metabolites in monkeys. METHODS: Sterile, pyrogen-free [(18)F]FHBG was produced routinely in good yields. Cells were transduced with the retroviral vector G1Tk1SvNa containing HSV-tk gene. In vitro uptake studies were performed by incubating cells with [(18)F]FHBG at 37 degrees C for 1 and 5 h. Biodistribution studies were performed at 2 and 5 h after injection in nude mice bearing tumors grown from wild-type or transduced cells. Sequential, whole-body PET scans of cynomolgus monkeys were obtained over a period of >2 h after intravenous injection of [(18)F]FHBG. Arterial plasma samples obtained from monkeys 15-120 min after intravenous injection were subjected to acid extraction, and the acid-soluble fractions were analyzed by high-performance liquid chromatography. RESULTS: In vitro studies showed 31 and 71 (P < 0.001) times higher uptake of the probe at 1 and 5 h, respectively, in transduced cells compared with nontransduced cells. In vivo studies in mice showed that tumor uptake of the radiotracer was 4-fold (P < 0.05) and 13-fold (P < 0.001) higher at 2 and 5 h, respectively, in tumors grown from transduced cells compared with control cells. Transduced tumor-to-normal tissue ratios ranged from 2 to 25 at 2 h and from 2 to 22 at 5 h. Recirculating labeled metabolites had only a minor effect on the biodistribution of radiolabel from [(18)F]FHBG in monkeys. CONCLUSION: These results indicate that [(18)F]FHBG may yield high-contrast PET images of HSV-tk expression in tumors and, therefore, it is a very promising radiotracer for monitoring of gene therapy of cancer with PET.

ICOS is essential for effective T-helper-cell responses.

The outcome of T-cell responses after T-cell encounter with specific antigens is modulated by co-stimulatory signals, which are required for both lymphocyte activation and development of adaptive immunity. ICOS, an inducible co-stimulator with homology to CD28, is expressed on activated, but not resting T cells, and shows T-cell co-stimulatory function in vitro. ICOS binds specifically to its counter-receptor B7RP-1 (refs 5,6,7), but not to B7-1 or B7-2. Here we provide in vivo genetic evidence that ICOS delivers a co-stimulatory signal that is essential both for efficient interaction between T and B cells and for normal antibody responses to T-cell-dependent antigens. To determine the physiological function of ICOS, we generated and characterized gene-targeted ICOS-deficient mice. In vivo, a lack of ICOS results in severely deficient T-cell-dependent B-cell responses. Germinal centre formation is impaired and immunoglobulin class switching, including production of allergy-mediating IgE, is defective. ICOS-deficient T cells primed in in vivo and restimulated in vitro with specific antigen produce only low levels of interleukin-4, but remain fully competent to produce interferon-gamma.

Blocking catabolism with eniluracil enhances PET studies of 5-[18F]fluorouracil pharmacokinetics.

UNLABELLED: Noninvasive methods for measuring the pharmacokinetics of chemotherapeutic drugs such as 5-fluorouracil (FU) are needed for individualized optimization of treatment regimens. PET imaging of [18F]FU (PET/[18F]FU) is potentially useful in this context, but PET/[18F]FU is severely hampered by low tumor uptake of radiolabel and rapid catabolism of FU in vivo. Pretreatment with eniluracil (5-ethynyluracil) prevents catabolism of FU. Hypothesizing that suppression of catabolism would enhance PET/[18F]FU, we examined the effects of eniluracil on the short-term pharmacokinetics of the radiotracer. METHODS: Anesthetized rats bearing a subcutaneous rat colorectal tumor were given eniluracil or placebo and injected intravenously 1 h later with [18F]FU or [3H]FU. In the 18F studies, dynamic PET image sequences were obtained 0-2 h after injection. Tumors were excised and frozen at 2 h and then analyzed for labeled metabolites by high-performance liquid chromatography. Biodistribution of radiolabel was determined by direct tissue assay. RESULTS: Eniluracil improved tumor visualization in PET images. With eniluracil, tumor standardized uptake values ([activity/g]/[injected activity/g body weight]) increased from 0.72 +/- 0.06 (mean +/- SEM; n = 6) to 1.57 +/- 0.20 (n = 12; P < 0.01), and tumor uptake increased by factors of 2 or more relative to plasma (P < 0.05) and bone, liver, and kidney (P < 0.01). Without eniluracil (n = 5), 57% +/- 4% of recovered radiolabel in tumor at 2 h was on catabolites, with the rest divided among FU (2% +/- 1%), anabolites of FU (38% +/- 7%), and unidentified peaks (4% +/- 2%). With eniluracil (n = 8), catabolites, FU, and anabolites comprised 2% +/- 1%, 41% +/- 5%, and 57% +/- 4%, respectively, of the recovered radiolabel in tumors. CONCLUSION: Eniluracil increased tumor accumulation of 18F relative to host tissues and fundamentally changed the biochemical significance of that accumulation. With catabolism suppressed, tumor radioactivity reflected the therapeutically relevant aspect of FU pharmacokinetics--namely, uptake and anabolic activation of the drug. With this approach, it may be feasible to measure the transport and anabolism of [18F]FU in tumors by kinetic modeling and PET. Such information may be useful in predicting and increasing tumor response to FU.

Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-kappaB-dependent gene transcription.

Induction of NF-kappaB-dependent transcription requires phosphorylation and subsequent degradation of I-kappaB, an inhibitor of NF-kappaB, followed by nuclear translocation and DNA binding of NF-kappaB. Tumor necrosis factor receptor-associated factor 2 (TRAF2) plays a role in NF-kappaB activation in response to cytokines such as tumor necrosis factor alpha (TNFalpha). In this study, we purified and characterized a novel kinase (T2K, also known as TBK1 or NAK), which associates with TRAF2 and exhibits kinase activity towards I-kappaBalpha in vitro. The physiological function of T2K was investigated using T2K-deficient mice. Heterozygotes appear normal, but t2k(-/-) animals die at approximately E14.5 of massive liver degeneration and apoptosis. Never theless, hematopoietic progenitors from T2K-deficient fetal liver support normal lymphocyte development. Furthermore, t2k(-/-) embryonic fibroblasts and thymocytes do not display increased sensitivity to TNFalpha-induced apoptosis. In response to either TNFalpha or IL-1 induction, t2k(-/-) embryonic fibroblasts exhibit normal degradation of I-kappaB and kappaB-binding activity. However, NF-kappaB-directed transcription is dramatically reduced. These results demonstrate that, like I-kappaB kinase beta and the RelA subunit of NF-kappaB, T2K is critical in protecting embryonic liver from apoptosis. However, T2K has a unique role in the activation of NF-kappaB-directed transcription, apparently independent of I-kappaB degradation and NF-kappaB DNA binding.

Validation of fluorouracil metabolite analysis in excised tumor. Lability of anabolites.

Rapid excision and freezing of tissue commonly is assumed to preserve the molecular composition of the tissue just prior to its removal from the host. We examined the lability of radiolabeled 5-fluorouracil (FUra) and its anabolites during excision and freeze-clamping in a rat tumor model. Acid-soluble metabolites were identified by HPLC. Two rats, each bearing multiple, subcutaneously-implanted colon tumors, were treated with eniluracil (an inactivator of dihydropyrimidine dehydrogenase) to prevent catabolism of FUra and then injected intravenously with [(3)H]FUra. After 2 hr, tumors were harvested sequentially and segmented. The tumor pieces were kept at room temperature for various times up to 4 min prior to freezing. These specimens showed a decrease (P < 0.01) in labeled nucleoside triphosphate content of 13 +/- 2%/min and commensurate increases (P < 0.005) in labeled nucleoside monophosphates and nucleosides with increasing time-to-freeze. The amounts of labeled macromolecules, nucleoside diphosphates, and FUra each remained approximately constant. The study indicates that substantial errors may occur in measured tissue concentrations of pyrimidine nucleosides and nucleotides due to lability during tissue excision and freeze-clamping. Such errors can be corrected using data of the type obtained in this study.

Pharmacokinetics of the thymidine analog 2'-fluoro-5-[(14)C]-methyl-1-beta-D-arabinofuranosyluracil ([(14)C]FMAU) in rat prostate tumor cells.

2'-Fluoro-5-[(14)C]-methyl-1-beta-D-arabinofuranosyluracil (FMAU) is an analog of thymidine (TdR) that is resistant to catabolism, is incorporated into DNA, and has been labeled with (11)C for use with positron emission tomography. We compared the uptake and metabolism of [(14)C]FMAU with that of [(3)H]TdR in fast- and slow-growing cell lines of a rat prostate tumor. Although FMAU was incorporated much less rapidly than TdR, FMAU behaved very similarly to TdR with respect to correlation between uptake velocity and cell growth rate, saturability of cellular incorporation, and intracellular metabolite pools. Thus, FMAU warrants further evaluation as an in vivo indicator of tumor cell division.

TRAF2 deficiency results in hyperactivity of certain TNFR1 signals and impairment of CD40-mediated responses.

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) can interact with various members of the TNF receptor family. Previously, we reported that TRAF2-deficient mice die prematurely and have elevated serum TNF levels. In this study, we demonstrate that TRAF2-deficient macrophages produce increased amounts of nitric oxide (NO) and TNF in response to TNF stimulation. Furthermore, we could enhance the survival of TRAF2-deficient mice by eliminating either TNF or TNFR1. Using these double-knockout mice, we show that in the absence of TRAF2, the T helper-dependent antibody response, CD40-mediated proliferation, and NF-kappaB activation are defective. These data demonstrate two important roles of TRAF2, one as a negative regulator of certain TNFR1 signals and the other as a positive mediator of CD40 signaling.

Evaluation of 9-[(3-18F-fluoro-1-hydroxy-2-propoxy)methyl]guanine ([18F]-FHPG) in vitro and in vivo as a probe for PET imaging of gene incorporation and expression in tumors.

Preparation of 9-[(3-18F-fluoro-1-hydroxy-2-propoxy)methyl]-guanine ([18F]-FHPG) for clinical use, and its evaluation as a positron emission tomography (PET) imaging agent for gene incorporation and expression in tumors are reported. In vitro studies in human colon cancer cells, HT-29, transduced with the retroviral vector G1Tk1SvNa and nontransduced (wild type) showed 4, 8, 12, and 15 times higher uptake of the probe in 1, 3, 5, and 7 h, respectively, in transduced cells compared with the controls. In vivo studies in tumor-bearing nude mice demonstrated that the tumor uptake of the radiotracer is three and six-fold higher in 2 and 5 h, respectively, in transduced cells compared with the control cells. These results suggest that [18F]-FHPG is a potential in vivo PET imaging agent for monitoring gene incorporation and expression in gene therapy of cancer.

T-cell co-stimulation through B7RP-1 and ICOS.

T-cell activation requires co-stimulation through receptors such as CD28 and antigen-specific signalling through the T-cell antigen receptor. Here we describe a new murine costimulatory receptor-ligand pair. The receptor, which is related to CD28 and is the homologue of the human protein ICOS, is expressed on activated T cells and resting memory T cells. The ligand, which has homology to B7 molecules and is called B7-related protein-1 (B7RP-1), is expressed on B cells and macrophages. ICOS and B7RP-I do not interact with proteins in the CD28-B7 pathway, and B7RP-1 co-stimulates T cells in vitro independently of CD28. Transgenic mice expressing a B7RP-1-Fc fusion protein show lymphoid hyperplasia in the spleen, lymph nodes and Peyer's patches. Presensitized mice treated with B7RP-1-Fc during antigen challenge show enhanced hypersensitivity. Therefore, B7RP-1 exhibits co-stimulatory activities in vitro and in vivo. ICOS and B7RP-1 define a new and distinct receptor-ligand pair that is structurally related to CD28-B7 and is involved in the adaptive immune response.

Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes.

Caspases are fundamental components of the mammalian apoptotic machinery, but the precise contribution of individual caspases is controversial. CPP32 (caspase 3) is a prototypical caspase that becomes activated during apoptosis. In this study, we took a comprehensive approach to examining the role of CPP32 in apoptosis using mice, embryonic stem (ES) cells, and mouse embryonic fibroblasts (MEFs) deficient for CPP32. CPP32(ex3-/-) mice have reduced viability and, consistent with an earlier report, display defective neuronal apoptosis and neurological defects. Inactivation of CPP32 dramatically reduces apoptosis in diverse settings, including activation-induced cell death (AICD) of peripheral T cells, as well as chemotherapy-induced apoptosis of oncogenically transformed CPP32(-/-) MEFs. As well, the requirement for CPP32 can be remarkably stimulus-dependent: In ES cells, CPP32 is necessary for efficient apoptosis following UV- but not gamma-irradiation. Conversely, the same stimulus can show a tissue-specific dependence on CPP32: Hence, TNFalpha treatment induces normal levels of apoptosis in CPP32 deficient thymocytes, but defective apoptosis in oncogenically transformed MEFs. Finally, in some settings, CPP32 is required for certain apoptotic events but not others: Select CPP32(ex3-/-) cell types undergoing cell death are incapable of chromatin condensation and DNA degradation, but display other hallmarks of apoptosis. Together, these results indicate that CPP32 is an essential component in apoptotic events that is remarkably system- and stimulus-dependent. Consequently, drugs that inhibit CPP32 may preferentially disrupt specific forms of cell death.

FADD: essential for embryo development and signaling from some, but not all, inducers of apoptosis.

FADD (also known as Mort-1) is a signal transducer downstream of cell death receptor CD95 (also called Fas). CD95, tumor necrosis factor receptor type 1 (TNFR-1), and death receptor 3 (DR3) did not induce apoptosis in FADD-deficient embryonic fibroblasts, whereas DR4, oncogenes E1A and c-myc, and chemotherapeutic agent adriamycin did. Mice with a deletion in the FADD gene did not survive beyond day 11.5 of embryogenesis; these mice showed signs of cardiac failure and abdominal hemorrhage. Chimeric embryos showing a high contribution of FADD null mutant cells to the heart reproduce the phenotype of FADD-deficient mutants. Thus, not only death receptors, but also receptors that couple to developmental programs, may use FADD for signaling.

The transcription factor NF-ATc1 regulates lymphocyte proliferation and Th2 cytokine production.

NF-ATc1 is a member of a family of genes that encodes the cytoplasmic component of the nuclear factor of activated T cells (NF-AT). In activated T cells, nuclear NF-AT binds to the promoter regions of multiple cytokine genes and induces their transcription. The role of NF-ATc1 was investigated in recombination activating gene-1 (RAG-1)-deficient blastocyst complementation assays using homozygous NF-ATc1-/- mutant ES cell lines. NF-ATc1-/-/RAG-1-/- chimeric mice showed reduced numbers of thymocytes and impaired proliferation of peripheral lymphocytes, but normal production of IL-2. Induction in vitro of Th2 responses, as demonstrated by a decrease in IL-4 and IL-6 production, was impaired in mutant T cells. These data indicate that NF-ATc1 plays roles in the development of T lymphocytes and in the differentiation of the Th2 response.

Early lethality, functional NF-kappaB activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice.

TRAF2 is an intracellular signal-transducing protein recruited to the TNFR1 and TNFR2 receptors following TNF stimulation. To investigate the physiological role of TRAF2, we generated TRAF2-deficient mice. traf2-/- mice appeared normal at birth but became progressively runted and died prematurely. Atrophy of the thymus and spleen and depletion of B cell precursors also were observed. Thymocytes and other hematopoietic progenitors were highly sensitive to TNF-induced cell death and serum TNF levels were elevated in these TRAF2-deficient animals. Examination of traf2-/- cells revealed a severe reduction in TNF-mediated JNK/SAPK activation but a mild effect on NF-kappaB activation. These results suggest that TRAF2-independent pathways of NF-kappaB activation exist and that TRAF2 is required for an NF-kappaB-independent signal that protects against TNF-induced apoptosis.

CD44 regulates hematopoietic progenitor distribution, granuloma formation, and tumorigenicity.

CD44 is expressed in various isoforms on numerous cell types and tissues during embryogenesis and in the mature organism. CD44 may also be involved in tumor growth. To study the multiple roles of CD44, we abolished expression of all known isoforms of CD44 in mice by targeting exons encoding the invariant N-terminus region of the molecule. Surprisingly, mice were born in Mendelian ratio without any obvious developmental or neurological deficits. Hematological impairment was evidenced by altered tissue distribution of myeloid progenitors with increased levels of colony-forming unit-granulocyte-macrophage (CFU-GM) in bone marrow and reduced numbers of CFU-GM in spleen. Fetal liver colony-forming unit-spleen and granulocyte colony-stimulating factor mobilization assays, together with reduced CFU-GM in peripheral blood, suggested that progenitor egress from bone marrow was defective. In what was either a compensatory response to CD44 deficiency or an immunoregulatory defect, mice also developed exaggerated granuloma responses to Cryotosporidium parvum infection. Finally, tumor studies showed that SV40-transformed CD44-deficient fibroblasts were highly tumorigenic in nude mice, whereas reintroduction of CD44s expression into these fibroblasts resulted in a dramatic inhibition of tumor growth.

Impaired CD28-mediated interleukin 2 production and proliferation in stress kinase SAPK/ERK1 kinase (SEK1)/mitogen-activated protein kinase kinase 4 (MKK4)-deficient T lymphocytes.

The dual specific kinase SAPK/ERK1 kinase (SEK1; mitogen-activated protein kinase kinase 4/Jun NH2 terminal kinase [ JNK] kinase) is a direct activator of stress-activated protein kinases ([SAPKs]/JNKs) in response to CD28 costimulation, CD40 signaling, or activation of the germinal center kinase. Here we show that SEK1(-/-) recombination-activating gene (RAG)2(-/-) chimeric mice have a partial block in B cell maturation. However, peripheral B cells displayed normal responses to IL-4, IgM, and CD40 cross-linking. SEK1(-/-) peripheral T cells showed decreased proliferation and IL-2 production after CD28 costimulation and PMA/Ca2+ ionophore activation. Although CD28 expression was absolutely crucial to generate vesicular stomatitis virus (VSV)-specific germinal centers, SEK1(-/-)RAG2(-/-) chimeras mounted a protective antiviral B cell response, exhibited normal IgG class switching, and made germinal centers in response to VSV. Interestingly, PMA/Ca2+ ionophore stimulation, which mimics TCR-CD3 and CD28-mediated signal transduction, induced SAPK/JNK activation in peripheral T cells, but not in thymocytes, from SEK1(-/-) mice. These results show that signaling pathways for SAPK activation are developmentally regulated in T cells. Although SEK1(-/-) thymocytes failed to induce SAPK/JNK in response to PMA/Ca2+ ionophore, SEK1(-/-)RAG2(-/-) thymocytes proliferated and made IL-2 after PMA/Ca2+ ionophore and CD3/CD28 stimulation, albeit at significantly lower levels compared to SEK1(+/+)RAG2(-/-) thymocytes, implying that CD28 costimulation and PMA/Ca2+ ionophore-triggered signaling pathways exist that can mediate proliferation and IL-2 production independently of SAPK activation. Our data provide the first genetic evidence that SEK1 is an important effector molecule that relays CD28 signaling to IL-2 production and T cell proliferation.

CD28 is not required for rejection of unmanipulated syngeneic and autologous tumors.

To gain a better understanding of the requirement of CD28 co-stimulation in different types of T cell-dependent tumor rejection responses, we performed a series of syngeneic and autologous tumor rejection experiments on CD28 knockout mice. In a preimmunization-challenge model, virally-induced ALC lymphoma and methylcholanthrene-induced MC57X fibrosarcoma transplants were rejected similarly by syngeneic CD28 knockout and immunocompetent controls. ALC-specific cytotoxic T lymphocytes (CTL) and MC57X-specific tumor necrosis factor (TNF) release were induced in CD28 knockouts, although at a reduced level in the latter case. Secondly, the spontaneous regression of Moloney murine sarcoma virus (MMSV)-induced primary tumors in the autologous hosts occurred equally in CD28 knockouts and in immunocompetent control mice. A comparable virus-specific CTL response was generated in both, as revealed in cytolytic assays against RBL-5 targets. Thirdly, the spontaneous rejection of the B7-transfected EL-4 lymphoma by immunocompetent hosts was abrogated in CD28 knockout mice, since more than 82% CD28 knockouts developed tumors after inoculation with B7-transfected EL-4 cells. Our results therefore show that CD28 co-stimulatory molecules are not required for the rejection of unmanipulated syngeneic tumors in hyperimmunized hosts and the regression of MMSV-induced sarcoma in autochthonous hosts.

Acute graft-versus-host disease without costimulation via CD28.

T lymphocyte activity is enhanced by costimulatory signals mediated through CD28 binding to B7-1/B7-2 on antigen-presenting cells. Several recent studies have shown that graft-versus-host disease (GVHD) can be inhibited by in vivo treatment with CTLA4Ig, which blocks CD28-B7 interactions. These findings prompted us to investigate the role of CD28 in acute GVHD, using gene-targeted mice. We performed the experiments in the context of strong allogeneic MHC stimulation (H2(b) anti-H2(d)) and weak stimulation (H2(d) anti-H2(b)). In both directions, efficient in vitro T-cell cytotoxicity and acute lethal GVHD were induced by CD28-deficient lymphocytes, which was only partially delayed when compared with wild-type mice. We conclude that lethal GVHD can develop without costimulation via CD28.

Alterations in the level of CD45 surface expression affect the outcome of thymic selection.

CD45 is a receptor protein tyrosine phosphatase whose activity is required for thymocyte development and TCR-mediated signal transduction. Here we show that positive selection of TCR-alphabeta transgenic thymocytes is completely blocked in CD45 exon 6 -/- gene-deficient (CD45 -/-) mice that express the P14 TCR specific for the lymphocytic choriomeningitis virus. Thymocytes from mice heterozygous for the targeted disruption of the CD45 gene (CD45 +/-) displayed a reduction in both CD45 surface intensity and enzymatic CD45 protein tyrosine phosphatase activity. Surprisingly, positive thymocyte selection was enhanced in CD45 +/- mice as characterized by an up-regulation of the P14 TCR on thymocytes and increased numbers of transgenic T cells. Using a variant of lymphocytic choriomeningitis virus that is impaired in the induction of negative selection of P14 thymocytes, we also show that the reduction of CD45 surface expression in CD45 +/- mice rendered P14 transgene thymocytes susceptible to negative selection. These data demonstrate that changes in the expression level of the receptor protein tyrosine phosphatase CD45 can alter thymic selection.

Stress-signalling kinase Sek1 protects thymocytes from apoptosis mediated by CD95 and CD3.

Distinct and evolutionarily conserved signal transduction cascades mediate survival or death in response to developmental and environmental cues. The stress-activated protein kinases, or Jun N-terminal kinases (SAPKs/JNKs), are activated in response to a variety of cellular stresses such as changes in osmolarity and metabolism, DNA damage, heat shock, ischaemia, or inflammatory cytokines. Sek1 (JNKK/MKK4) is a direct activator of SAPKs/JNKs in response to environmental stresses or mitogenic factors. Here we investigate the role of Sek1 in development and apoptosis by deleting sek1 in embryonic stem (ES) cells by homologous recombination. We provide genetic evidence that different stresses utilize distinct signalling pathways for SAPK/JNK activation. sek1(-/-) rag2(-/-) chimaeric mice have normal numbers of mature T cells but fewer immature CD4+CD8+ thymocytes. The sek1 mutation did not affect the induction of apoptosis in response to environmental stresses in ES and T cells: instead, sek1 protected thymocytes from CD95 (Fas)- and CD3-mediated apoptosis. These data indicate that SEK1 mediates survival signals in T-cell development.