Engineering Tolerance toward Allogeneic CAR-T Cells by Regulation of MHC Surface Expression with Human Herpes Virus-8 Proteins.

Allogeneic, off-the-shelf (OTS) chimeric antigen receptor (CAR) cell therapies have the potential to reduce manufacturing costs and variability while providing broader accessibility to cancer patients and those with other diseases. However, host-versus-graft reactivity can limit the durability and efficacy of OTS cell therapies requiring new strategies to evade adaptive and innate-immune responses. Human herpes virus-8 (HHV8) maintains infection, in part, by evading host T and natural killer (NK) cell attack. The viral K3 gene encodes a membrane-tethered E3 ubiquitin ligase that discretely targets major histocompatibility complex (MHC) class I components, whereas K5 encodes a similar E3 ligase with broader specificity, including MHC-II and the MHC-like MHC class I polypeptide-related sequence A (MIC-A)- and sequence B (MIC-B)-activating ligands of NK cells. We created γ-retroviruses encoding K3 and/or K5 transgenes that efficiently transduce primary human T cells. Expression of K3 or K5 resulted in dramatic downregulation of MHC-IA (human leukocyte antigen [HLA]-A, -B, and -C) and MHC class II (HLA-DR) cell-surface expression. K3 expression was sufficient for T cells to resist exogenously loaded peptide-MHC-specific cytotoxicity, as well as recognition in one-way allogeneic mixed lymphocyte reactions. Further, in immunodeficient mice engrafted with allogeneic T cells, K3-transduced T cells selectively expanded in vivo. Ectopic K5 expression in MHC class I-, MIC-A+/B+ K562 cells also reduced targeting by primary NK cells. Coexpression of K3 in prostate stem cell antigen (PSCA)-directed, inducible MyD88/CD40 (iMC)-enhanced CAR-T cells did not impact cytotoxicity, T cell growth, or cytokine production against HPAC pancreatic tumor target cells, whereas K5-expressing cells showed a modest reduction in interleukin (IL)-2 production without effect on cytotoxicity. Together, these results support application of these E3 ligases to advance development of OTS CAR-T cell products.

Inducible MyD88/CD40 synergizes with IL-15 to enhance antitumor efficacy of CAR-NK cells.

Natural killer (NK) cells expressing chimeric antigen receptors (CARs) are a promising anticancer immunotherapy, leveraging both innate NK cell antitumor activity and target-specific cytotoxicity. Inducible MyD88/CD40 (iMC) is a potent, rimiducid-regulated protein switch that has been deployed previously as a T-cell activator to enhance proliferation and persistence of CAR-modified T cells. In this study, iMC was extended to CAR-NK cells to enhance their growth and augment cytotoxicity against tumor cells. iMC-activated NK cells substantially increased cytokine and chemokine secretion and displayed higher levels of perforin and granzyme B degranulation. In addition, iMC activation could be coupled with ectopic interleukin-15 (IL-15) to further enhance NK cell proliferation. When coexpressed with a target-specific CAR (CD123 or BCMA), this IL-15/iMC system showed further augmented antitumor activity through enhanced CAR-NK cell expansion and cytolytic activity. To protect against potential toxicity from engineered NK cells, an orthogonal rapamycin-regulated Caspase-9 (iRC9) was included in a 4-gene, dual-switch platform. After infusion of dual-switch NK cells, pharmacologic iRC9 dimerization led to rapid elimination of a majority of expanded transduced NK cells. Thus, CAR-NK cells utilizing dual molecular switches provide an innovative and effective approach to cancer immunotherapy with controlled specificity, efficacy, and safety.

Constitutively active MyD88/CD40 costimulation enhances expansion and efficacy of chimeric antigen receptor T cells targeting hematological malignancies.

Successful adoptive chimeric antigen receptor (CAR) T-cell therapies against hematological malignancies require CAR-T expansion and durable persistence following infusion. Balancing increased CAR-T potency with safety, including severe cytokine-release syndrome (sCRS) and neurotoxicity, warrants inclusion of safety mechanisms to control in vivo CAR-T activity. Here, we describe a novel CAR-T cell platform that utilizes expression of the toll-like receptor (TLR) adaptor molecule, MyD88, and tumor-necrosis factor family member, CD40 (MC), tethered to the CAR molecule through an intentionally inefficient 2A linker system, providing a constitutive signal that drives CAR-T survival, proliferation, and antitumor activity against CD19+ and CD123+ hematological cancers. Robust activity of MC-enhanced CAR-T cells was associated with cachexia in animal models that corresponded with high levels of human cytokine production. However, toxicity could be successfully resolved by using the inducible caspase-9 (iC9) safety switch to reduce serum cytokines, by administration of a neutralizing antibody against TNF-α, or by selecting "low" cytokine-producing CD8+ T cells, without loss of antitumor activity. Interestingly, high basal activity was essential for in vivo CAR-T expansion. This study shows that co-opting novel signaling elements (i.e., MyD88 and CD40) and development of a unique CAR-T architecture can drive T-cell proliferation in vivo to enhance CAR-T therapies.

Two-Dimensional Regulation of CAR-T Cell Therapy with Orthogonal Switches.

Use of chimeric antigen receptors (CARs) as the basis of targeted adoptive T cell therapies has enabled dramatic efficacy against multiple hematopoietic malignancies, but potency against bulky and solid tumors has lagged, potentially due to insufficient CAR-T cell expansion and persistence. To improve CAR-T cell efficacy, we utilized a potent activation switch based on rimiducid-inducible MyD88 and CD40 (iMC)-signaling elements. To offset potential toxicity risks by this enhanced CAR, an orthogonally regulated, rapamycin-induced, caspase-9-based safety switch (iRC9) was developed to allow in vivo elimination of CAR-T cells. iMC costimulation induced by systemic rimiducid administration enhanced CAR-T cell proliferation, cytokine secretion, and antitumor efficacy in both in vitro assays and xenograft tumor models. Conversely, rapamycin-mediated iRC9 dimerization rapidly induced apoptosis in a dose-dependent fashion as an approach to mitigate therapy-related toxicity. This novel, regulatable dual-switch system may promote greater CAR-T cell expansion and prolonged persistence in a drug-dependent manner while providing a safety switch to mitigate toxicity concerns.

Influence of Various Factors on Circulating 25(OH) Vitamin D Concentrations in Dogs with Cancer and Healthy Dogs.

BACKGROUND: Low blood 25-hydroxyvitamin D (25(OH)D) concentrations have been associated with cancer in dogs. Little research has examined what other factors may affect 25(OH)D concentrations. OBJECTIVES: (1) To determine whether the presence of cancer (lymphoma, osteosarcoma, or mast cell tumor [MCT]) in dogs is associated with plasma 25(OH)D concentrations and (2) identify other factors related to plasma 25(OH)D concentrations in dogs. ANIMALS: Dogs newly diagnosed with osteosarcoma (n = 21), lymphoma (n = 27), and MCT (n = 21) presented to a tertiary referral oncology center, and healthy, client-owned dogs (n = 23). METHODS: An observational study design was used. Dietary vitamin D intake, sex, age, body condition score (BCS), muscle condition score (MCS), and plasma concentrations of 25(OH)D, 24,25-dihydroxyvitamin D (24,25(OH)2 D) (a marker of CYP24A1 activity), as well as ionized calcium (ICa), parathyroid hormone, and parathyroid hormone-related protein concentrations were measured. An analysis of covariance was used to model plasma 25(OH)D concentrations. RESULTS: Cancer type (P = 0.004), plasma 24,25(OH)2 D concentrations (P < 0.001), and plasma ICa concentrations (P = 0.047) had significant effects on plasma 25(OH)D concentrations. Effects of age, sex, body weight, BCS, MCS, and plasma PTH concentrations were not identified. A significant interaction between ICa and cancer was found (P = 0.005). Plasma 25(OH)D concentrations increased as ICa concentrations increased in dogs with cancer, whereas plasma 25(OH)D concentrations decreased as ICa concentrations increased in healthy dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Results support a relationship between cancer and altered vitamin D metabolism in dogs, mediated by plasma ICa concentrations. The CYP24A1 activity and plasma ICa should be measured in studies examining plasma 25(OH)D concentrations in dogs.

Regulated Expansion and Survival of Chimeric Antigen Receptor-Modified T Cells Using Small Molecule-Dependent Inducible MyD88/CD40.

Anti-tumor efficacy of T cells engineered to express chimeric antigen receptors (CARs) is dependent on their specificity, survival, and in vivo expansion following adoptive transfer. Toll-like receptor (TLR) and CD40 signaling in T cells can improve persistence and drive proliferation of antigen-specific CD4+ and CD8+ T cells following pathogen challenge or in graft-versus-host disease (GvHD) settings, suggesting that these costimulatory pathways may be co-opted to improve CAR-T cell persistence and function. Here, we present a novel strategy to activate TLR and CD40 signaling in human T cells using inducible MyD88/CD40 (iMC), which can be triggered in vivo via the synthetic dimerizing ligand, rimiducid, to provide potent costimulation to CAR-modified T cells. Importantly, the concurrent activation of iMC (with rimiducid) and CAR (by antigen recognition) is required for interleukin (IL)-2 production and robust CAR-T cell expansion and may provide a user-controlled mechanism to amplify CAR-T cell levels in vivo and augment anti-tumor efficacy.

Ligands for FKBP12 increase Ca2+ influx and protein synthesis to improve skeletal muscle function.

Rapamycin at high doses (2-10 mg/kg body weight) inhibits mammalian target of rapamycin complex 1 (mTORC1) and protein synthesis in mice. In contrast, low doses of rapamycin (10 µg/kg) increase mTORC1 activity and protein synthesis in skeletal muscle. Similar changes are found with SLF (synthetic ligand for FKBP12, which does not inhibit mTORC1) and in mice with a skeletal muscle-specific FKBP12 deficiency. These interventions also increase Ca(2+) influx to enhance refilling of sarcoplasmic reticulum Ca(2+) stores, slow muscle fatigue, and increase running endurance without negatively impacting cardiac function. FKBP12 deficiency or longer treatments with low dose rapamycin or SLF increase the percentage of type I fibers, further adding to fatigue resistance. We demonstrate that FKBP12 and its ligands impact multiple aspects of muscle function.

Sudden infant death syndrome in mice with an inherited mutation in RyR2.

BACKGROUND: Mutations in the cardiac ryanodine receptor gene (RyR2) have been recently identified in victims of sudden infant death syndrome. The aim of this study was to determine whether a gain-of-function mutation in RyR2 increases the propensity to cardiac arrhythmias and sudden death in young mice. METHODS AND RESULTS: Incidence of sudden death was monitored prospectively in heterozygous knock-in mice with mutation R176Q in RyR2 (R176Q/+). Young R176Q/+ mice exhibited a higher incidence of sudden death compared with wild-type littermates. Optical mapping of membrane potentials and intracellular calcium in 1- to 7-day-old R176Q/+ and wild-type mice revealed an increased incidence of ventricular ectopy and spontaneous calcium releases in neonatal R176Q/+ mice. Surface ECGs in 3- to 10-day-old mice showed that R176Q/+ mice developed more ventricular arrhythmias after provocation with epinephrine and caffeine. Intracardiac pacing studies in 12- to 18-day-old mice revealed the presence of an arrhythmogenic substrate in R176Q/+ compared with wild-type mice. Reverse transcription-polymerase chain reaction and Western blotting showed that expression levels of other calcium handling proteins were unaltered, suggesting that calcium leak through mutant RyR2 underlies arrhythmogenesis and sudden death in young R176Q/+ mice. CONCLUSIONS: Our findings demonstrate that a gain-of-function mutation in RyR2 confers an increased risk of cardiac arrhythmias and sudden death in young mice and that young R176Q/+ mice may be used as a model for elucidating the complex interplay between genetic and environmental risk factors associated with sudden infant death syndrome.

Cartilage oligomeric matrix protein expression in systemic sclerosis reveals heterogeneity of dermal fibroblast responses to transforming growth factor beta.

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) accumulates in systemic sclerosis (SSc) skin and is upregulated by transforming growth factor (TGF)beta. To further characterise the response to TGFbeta in SSc, we investigated TGFbeta1 and COMP expression and myofibroblast staining in SSc skin. METHODS: Skin biopsies from patients with diffuse cutaneous SSc (dSSc), limited cutaneous SSc (lSSc) and healthy controls were evaluated for COMP mRNA expression using real-time PCR. COMP, alpha-smooth muscle actin (SMA) and TGFbeta were assessed in skin sections and in cultured fibroblasts by immunohistochemistry. Clinical disease status was assessed by the modified Rodnan skin score (mRSS). RESULTS: Myofibroblasts expressing SMA and COMP were found coexpressed in many cells in dSSc dermis, but each also stained distinct cells in the dermis. Cultured SSc dermal fibroblasts also showed heterogeneity for COMP and SMA expression, with cells expressing SMA, COMP, both or neither. TGFbeta treatment increased COMP and SMA-expressing cells. COMP mRNA expression in lesional skin from patients with dSSc correlated with the mRSS and TGFbeta1 staining. CONCLUSION: These findings suggest that TGFbeta upregulation of COMP and/or SMA expression in subpopulations of fibroblasts contributes to different pathways of fibrosis and that multiple TGFbeta regulated genes may serve as biomarkers for the degree of SSc skin involvement.

[Predictors of prophylactic response to lithium]. / Prédicteurs de réponse prophylactique au lithium.

INTRODUCTION: Since their first utilization in psychiatry as mood stabilizers in the 1940s, lithium salts have been widely studied in the medical literature. The considerable amount of data available to date, supports the use of lithium salts as first-line mood stabilizing agents, with acute antimanic and antidepressant properties and proven efficacy in the long term prevention of manic and depressive relapses. LITERATURE FINDINGS: Several predictors were reported by different authors in early articles and were confirmed later on by the medical literature. All the psychopathological, environmental, biological, neurophysiologic and genetic predictors known to date are reviewed here. PSYCHOLOGICAL PREDICTORS: Psychopathological predictors of a good response to lithium prophylaxis include: the initial good response to lithium during the first 6-12 months of treatment, considered to date to be the most reliable predictor of a favourable response to lithium; the classical pattern of elated manic episodes; a positive familial history of bipolar disorders, especially those known to be responsive to lithium; the absence of comorbid personality disorders; bipolar type I disorders; melancholic features during depressive episodes; MDI pattern in the illness course and early onset of lithium treatment. In contrast, the following have been confirmed as psychopathological predictors of poor prophylactic lithium response: mixed episodes, considered to be one of the most reliable predictors of poor response to lithium since Kraeplin's description; rapid cycling bipolar disorders; comorbid alcohol and/or drug abuse; mood disorders with incongruent psychotic features; early onset bipolar disorder before the age of 18; discontinuation of lithium treatment; high number of previous affective episodes in the illness course before lithium initiation and DMI pattern. ENVIRONMENTAL PREDICTORS: Among environmental factors, being single was found to be the only predictor of a poor response to lithium treatment in prophylaxis. BIOLOGICAL PREDICTORS: Biological predictors of a good prophylactic response to lithium include a high RBC/plasma-lithium ratio, one of the most controversial predictors of a favourable response to lithium in the literature, a higher platelet serotonin-induced calcium mobilization, and a high rate of red blood cell membrane phospholipids, especially of phosphatidylcholine, and a phospholipid implicated in lithium intracellular transport. Among neurophysiologic predictors of a favourable response to lithium, the following have been reported: brain lithium concentrations above 0.2 mEq/L when measured by 7Li-MRS; decreased cerebral intracellular pH and white matter hyper intensity at (31)P-MRS and a high intensity of loudness dependence auditory-evoked potentials (LDAEP), the latter being one of the best indicators of human cerebral serotoninergic functioning. In contrast, the following have been reported as neurophysiological predictors of a poor lithium response in prophylaxis: epileptiform anomalies with diffuse theta waves on electroencephalography, a predictor of poor response to lithium known since the descriptions of Dalen in 1965 and decreased cerebral phosphocreatine levels at (31)P-MRS, the latter being an indicator of cerebral mitochondrial dysfunction. GENETIC PREDICTORS: Genetic predictors of good response to lithium in prophylaxis include a lower-inositol-monophosphatase (IMPase-2) mRNA expression, IMPase-2 being a key enzyme of the calcium-intracellular-signalling pathway and IMPase-2 gene being studied recently as a candidate gene in bipolar disorder. A higher frequency of phospholipase C isoenzyme gamma1 (PLCG1)-5 repeat allele genes has also been associated with a good response to lithium, PLCG1 being a major enzyme of the phosphatidylinositol second messenger system. Genetic predictors of negative prophylactic lithium response include the homozygotic forms of the short allele of the serotonin transporter gene (5-HTT), the presence of the A/A subtype of tryptophan hydroxylase (TPH) gene and a high frequency of human leukocyte antigens type A3 (HLA-A3), this genotype being associated with cellular membrane anomalies implicated in alteration of lithium intracellular transport. DISCUSSION: The search for new predictors of lithium prophylactic response is currently facing several methodological problems: lack of representativity of the samples of bipolar patients enrolled in research studies, poor reliability of retrospective reconstructions of the course of the bipolar disorder before initiation of lithium treatment, absence of consensus on tools used to assess response to lithium prophylaxis in study designs, difficult access and high costs of most of the laboratory and neuroimaging techniques used in recent studies such as magnetic resonance spectroscopy and LDAEP measures, and problematic evaluation of the impact of treatment on a disorder whose natural intrinsic course is often irregular.

Endocardial Brg1 represses ADAMTS1 to maintain the microenvironment for myocardial morphogenesis.

Developing myocardial cells respond to signals from the endocardial layer to form a network of trabeculae that characterize the ventricles of the vertebrate heart. Abnormal myocardial trabeculation results in specific cardiomyopathies in humans and yet trabecular development is poorly understood. We show that trabeculation requires Brg1, a chromatin remodeling protein, to repress ADAMTS1 expression in the endocardium that overlies the developing trabeculae. Repression of ADAMTS1, a secreted matrix metalloproteinase, allows the establishment of an extracellular environment in the cardiac jelly that supports trabecular growth. Later during embryogenesis, ADAMTS1 expression initiates in the endocardium to degrade the cardiac jelly and prevent excessive trabeculation. Thus, the composition of cardiac jelly essential for myocardial morphogenesis is dynamically controlled by ADAMTS1 and its chromatin-based transcriptional regulation. Modification of the intervening microenvironment provides a mechanism by which chromatin regulation within one tissue layer coordinates the morphogenesis of an adjacent layer.

Factors predicting survival following noninvasive ventilation in amyotrophic lateral sclerosis.

BACKGROUND/AIMS: The involvement of respiratory muscles is a major predicting factor for survival in amyotrophic lateral sclerosis (ALS). Recent studies show that noninvasive ventilation (NIV) can relieve symptoms of alveolar hypoventilation. However, factors predicting survival in ALS patients when treated with NIV need to be clarified. METHODS: We conducted a retrospective study of 33 consecutive ALS patients receiving NIV. Ten patients had bulbar onset. We determined the median survivals from onset, diagnosis and initiation of NIV and factors predicting survival. Statistical analysis was performed using the Kaplan-Meier test and Cox proportional hazard models. RESULTS: The median initial and maximal total uses of NIV were 10 and 14 h/24h. The overall median survival from ALS onset was 34.2 months and worsened with increasing age and bulbar onset of the disease. The median survival from initiation of NIV was 8.4 months and was significantly poorer in patients with advanced age or with airway mucus accumulation. Survival from initiation of NIV was not influenced by respiratory parameters or bulbar symptoms. CONCLUSION: Advanced age at diagnosis and airway mucus accumulation represent poorer prognostic factors of ALS patients treated with NIV. NIV is a helpful treatment of sleep-disordered breathing, including patients with bulbar involvement.

Rescue of degradation-prone mutants of the FK506-rapamycin binding (FRB) protein with chemical ligands.

We recently reported that certain mutations in the FK506-rapamycin binding (FRB) domain disrupt its stability in vitro and in vivo (Stankunas et al. Mol. Cell, 2003, 12, 1615). To determine the precise residues that cause instability, we calculated the folding free energy (Delta G) of a collection of FRB mutants by measuring their intrinsic tryptophan fluorescence during reversible chaotropic denaturation. Our results implicate the T2098L point mutation as a key determinant of instability. Further, we found that some of the mutants in this collection were destabilized by up to 6 kcal mol(-1) relative to the wild type. To investigate how these mutants behave in cells, we expressed firefly luciferase fused to FRB mutants in African green monkey kidney (COS) cell lines and mouse embryonic fibroblasts (MEFs). When unstable FRB mutants were used, we found that the protein levels and the luminescence intensities were low. However, addition of a chemical ligand for FRB, rapamycin, restored luciferase activity. Interestingly, we found a roughly linear relationship between the Delta G of the FRB mutants calculated in vitro and the relative chemical rescue in cells. Because rapamycin is capable of simultaneously binding both FRB and the chaperone, FK506-binding protein (FKBP), we next examined whether FKBP might contribute to the protection of FRB mutants. Using both in vitro experiments and a cell-based model, we found that FKBP stabilizes the mutants. These findings are consistent with recent models that suggest damage to intrinsic Delta G can be corrected by pharmacological chaperones. Further, these results provide a collection of conditionally stable fusion partners for use in controlling protein stability.

Rapamycin analogs with differential binding specificity permit orthogonal control of protein activity.

Controlling protein dimerization with small molecules has broad application to the study of protein function. Rapamycin has two binding surfaces: one that binds to FKBP12 and the other to the Frb domain of mTor/FRAP, directing their dimerization. Rapamycin is a potent cell growth inhibitor, but chemical modification of the surface contacting Frb alleviates this effect. Productive interactions with Frb-fused proteins can be restored by mutation of Frb to accommodate the rapamycin analog (a rapalog). We have quantitatively assessed the interaction between rapalogs functionalized at C16 and C20 and a panel of Frb mutants. Several drug-Frb mutant combinations have different and nonoverlapping specificities. These Frb-rapalog partners permit the selective control of different Frb fusion proteins without crossreaction. The orthogonal control of multiple target proteins broadens the capabilities of chemical induction of dimerization to regulate biologic processes.

Analytical performances of two liquid crystals and their mixture as stationary phases in capillary gas chromatography.

Comparative gas chromatographic applications of two new liquid crystals called LCa and LCb and their equimolar mixture LC(a+b) were investigated. The thermal properties of LCa, LCb and LC(a+b) were established with differential scanning calorimetry (DSC) and polarizing microscopy. Differential scanning calorimetry of LC(a+b) showed that the melting or clearing temperature was intermediate between the corresponding temperatures of the pure compounds. Polarizing microscopy showed that the liquid crystal phase of A + B was nematic. The chromatographic separation abilities LCa, LCb and LC(a+b) were studied using fused silica capillary columns. Interesting analytical performances were obtained: isomeric separation of aromatics, polyaromatics, phenols.

A field of myocardial-endocardial NFAT signaling underlies heart valve morphogenesis.

The delicate leaflets that make up vertebrate heart valves are essential for our moment-to-moment existence. Abnormalities of valve formation are the most common serious human congenital defect. Despite their importance, relatively little is known about valve development. We show that the initiation of heart valve morphogenesis in mice requires calcineurin/NFAT to repress VEGF expression in the myocardium underlying the site of prospective valve formation. This repression of VEGF at E9 is essential for endocardial cells to transform into mesenchymal cells. Later, at E11, a second wave of calcineurin/NFAT signaling is required in the endocardium, adjacent to the earlier myocardial site of NFAT action, to direct valvular elongation and refinement. Thus, NFAT signaling functions sequentially from myocardium to endocardium within a valvular morphogenetic field to initiate and perpetuate embryonic valve formation. This mechanism also operates in zebrafish, indicating a conserved role for calcineurin/NFAT signaling in vertebrate heart valve morphogenesis.

Biochemical and structural basis for partially redundant enzymatic and transcriptional functions of DCoH and DCoH2.

An inherited form of diabetes, maturity-onset diabetes of the young type 3 (MODY3), results from mutations in the transcriptional activator, hepatocyte nuclear factor-1alpha (HNF1alpha). Transcription by HNF1alpha is stimulated by the bifunctional coactivator DCoH (dimerization cofactor of HNF1). Strikingly, an HNF1alpha deletion in mice causes more severe phenotypes than a DCoH deletion. It has been hypothesized that a DCoH homolog, DCoH2, partially complements the DCoH deletion. To test this idea, we determined the biochemical properties and the 1.6-A-resolution crystal structure of DCoH2. Like DCoH, DCoH2 forms a tetramer, displays pterin-4alpha-carbinolamine dehydratase activity, and binds HNF1alpha in vivo and in vitro. DCoH and DCoH2 adopt identical folds with structural differences confined largely to the protein surfaces and the tetramer interface. In contrast to the hyperstable DCoH tetramer, DCoH2 readily disproportionates and forms a 2:2 complex with HNF1 in vitro. Phylogenetic analysis reveals six major subfamilies of DCoH proteins, including unique DCoH and DCoH2 branches in metazoans. These results suggest distinct roles for DCoH and DCoH2. Differences in conserved surface residues could mediate binding to different effectors. We propose that HNF1alpha binding kinetics may distinguish regulation by DCoH2, under thermodynamic control, from regulation by DCoH, under kinetic control.

Conditional protein alleles using knockin mice and a chemical inducer of dimerization.

We have developed a general method of making conditional alleles that allows the rapid and reversible regulation of specific proteins. A mouse line was produced in which proteins encoded by the endogenous glycogen synthase kinase-3 beta (GSK-3beta) gene are fused to an 89 amino acid tag, FRB*. FRB* causes the destabilization of GSK-3beta, producing a severe loss-of-function allele. In the presence of C20-MaRap, a highly specific, nontoxic, cell-permeable small molecule, GSK-3betaFRB* binds to the ubiquitously expressed FKBP12 protein. This interaction stabilizes GSK-3betaFRB* and restores both protein levels and activity. C20-MaRap-mediated stabilization is rapidly reversed by the addition of an FKBP12 binding competitor molecule. This technology may be applied to a wide range of FRB*-tagged mouse genes while retaining their native transcriptional control. Inducible stabilization could be valuable for many developmental and physiological studies and for drug target validation.

["Spontaneous" resolution of two severe methotrexate-induced pneumonias]. / Résolution spontanée de deux pneumopathies sévères au méthotrexate.

The beneficial role of corticosteroid therapy for the treatment of methotrexate-induced pneumonia remains controversial. We report two cases of acute severe interstitial pneumonia induced by methotrexate in patients with non-Hodgkin lymphoma given a polychemotherapy protocol (M'BACOD). The first signs appeared on the eleventh day of the first cycle in patient one and on the tenth day of the third cycle in patient two. The causal implication of methotrexate was based on the history, the clinical and radiological presentation, and the negative tests in both patients: lymphocyte alveolitis with granulomatous lesions on the transbronchial biopsy in patient one and positive leukocyte migration test in the presence of methotrexate in patient two. Early acute respiratory failure required high flow rate oxygen therapy with positive expiratory pressure ventilatory assistance. The course was rapidly favorable both for blood gases and radiographic presentation without corticosteroids. These two cases illustrate that pulmonary disease can be cured without corticosteroids despite severe respiratory failure at onset. This provides a further argument on reservations about using corticosteroids for suspected methotrexate-induced pneumonia.