CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia.

Patients with chemo-refractory acute myeloid leukemia (AML) have a dismal prognosis. Chimeric antigen receptor T (CART) cell therapy has produced exciting results in CD19+ malignancies and may overcome many of the limitations of conventional leukemia therapies. We developed CART cells to target CD33 (CART33) using the anti-CD33 single chain variable fragment used in gemtuzumab ozogamicin (clone My96) and tested the activity and toxicity of these cells. CART33 exhibited significant effector functions in vitro and resulted in eradication of leukemia and prolonged survival in AML xenografts. CART33 also resulted in human lineage cytopenias and reduction of myeloid progenitors in xenograft models of hematopoietic toxicity, suggesting that permanently expressed CD33-specific CART cells would have unacceptable toxicity. To enhance the viability of CART33 as an option for AML, we designed a transiently expressed mRNA anti-CD33 CAR. Gene transfer was carried out by electroporation into T cells and resulted in high-level expression with potent but self-limited activity against AML. Thus our preclinical studies show potent activity of CART33 and indicate that transient expression of anti-CD33 CAR by RNA modification could be used in patients to avoid long-term myelosuppression. CART33 therapy could be used alone or as part of a preparative regimen prior to allogeneic transplantation in refractory AML.

Jagged1 (JAG1) mutations in Alagille syndrome: increasing the mutation detection rate.

Alagille syndrome (AGS) is caused by heterozygous mutations in JAG1, and mutations have been previously reported in about 70% of patients who meet clinical diagnostic criteria. We studied a cohort of 247 clinically well-defined patients, and using an aggressive and sequential screening approach we identified JAG1 mutations in 94% of individuals. Mutations were found in 232 out of 247 patients studied and 83 of the mutations were novel. This increase in the mutation rate was accomplished by combining rigorous clinical phenotyping, with a combination of mutation detection techniques, including fluorescence in situ hybridization (FISH), genomic and cDNA sequencing, and quantitative PCR. This higher rate of mutation identification has implications for clinical practice, facilitating genetic counseling, prenatal diagnosis, and evaluation of living-related liver transplant donors. Our results suggest that more aggressive screening may similarly increase the rate of mutation detection in other dominant and recessive disorders.

Defective intracellular transport and processing of JAG1 missense mutations in Alagille syndrome.

Jagged1 (JAG1) is a cell surface ligand in the Notch signaling pathway and mutations in this gene cause Alagille syndrome (AGS). JAG1 mutations have been identified in 60-70% of AGS patients studied, and these include total gene deletions ( approximately 6%), protein-truncating mutations (insertions, deletions and nonsense mutations) (82%) and missense mutations (12%). Based on the finding that total JAG1 deletions cause AGS, haploinsufficiency has been hypothesized to be a mechanism for disease causation; however, the mechanism by which missense mutations cause disease is not understood. To date, 25 unique missense mutations have been observed in AGS patients. Missense mutations are non-randomly distributed across the protein with clusters at the 5' end of the protein, in the conserved DSL domain, and two clusters within the EGF repeats. To understand the effect of the missense mutations on protein localization and function, we have studied four missense mutations (R184H, L37S, P163L and P871R). In two assays of JAG1 function, R184H and L37S are associated with loss of Notch signaling activity relative to wild-type JAG1. Neither R184H or L37S is present on the cell surface and both are abnormally glycosylated. Furthermore, these mutations lead to abnormal accumulation of the protein, possibly in the endoplasmic reticulum. Both P163L and P871R are associated with normal levels of Notch signaling activity and are present on the cell surface, consistent with these changes being polymorphisms rather than disease-causing mutations.

Boy with bilateral retinoblastoma due to an unusual ring chromosome 13 with activation of a latent centromere.

We present a patient with bilateral retinoblastoma and developmental delay who has an abnormal male karyotype containing 47 chromosomes, including an acentric derivative chromosome 13. We postulate that the derivative 13 occurred after a break at 13q14, with the proximal portion of the chromosome forming a ring and the distal portion undergoing duplication. Thus, this patient is trisomic for 13q14-->qter. The derivative chromosome with duplicated distal portion (13q14-->qter) lacked the 13 centromere and was negative for chromosome 13 alpha-satellite DNA by low stringency FISH. Nevertheless, this chromosome is stably transmitted in lymphocytes and fibroblasts. A single primary constriction was observed at band 13q21, consistent with activation of a latent centromere (neocentromere) at this band. The neocentromere on der(13) was positive for multiple centromeric proteins, suggesting that it acts as the functional centromere. By FISH, the Rb gene was present on the normal 13, the proximally derived ring chromosome, but not on the derivative chromosome. Although there was no evidence for disruption of the Rb gene, this chromosome rearrangement most likely results in abnormal expression of the Rb gene product.

Characterization of RyR1-slow, a ryanodine receptor specific to slow-twitch skeletal muscle.

Two distinct skeletal muscle ryanodine receptors (RyR1s) are expressed in a fiber type-specific manner in fish skeletal muscle (11). In this study, we compare [(3)H]ryanodine binding and single channel activity of RyR1-slow from fish slow-twitch skeletal muscle with RyR1-fast and RyR3 isolated from fast-twitch skeletal muscle. Scatchard plots indicate that RyR1-slow has a lower affinity for [(3)H]ryanodine when compared with RyR1-fast. In single channel recordings, RyR1-slow and RyR1-fast had similar slope conductances. However, the maximum open probability (P(o)) of RyR1-slow was threefold less than the maximum P(o) of RyR1-fast. Single channel studies also revealed the presence of two populations of RyRs in tuna fast-twitch muscle (RyR1-fast and RyR3). RyR3 had the highest P(o) of all the RyR channels and displayed less inhibition at millimolar Ca(2+). The addition of 5 mM Mg-ATP or 2.5 mM beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP) to the channels increased the P(o) and [(3)H]ryanodine binding of both RyR1s but also caused a shift in the Ca(2+) dependency curve of RyR1-slow such that Ca(2+)-dependent inactivation was attenuated. [(3)H]ryanodine binding data also showed that Mg(2+)-dependent inhibition of RyR1-slow was reduced in the presence of AMP-PCP. These results indicate differences in the physiological properties of RyRs in fish slow- and fast-twitch skeletal muscle, which may contribute to differences in the way intracellular Ca(2+) is regulated in these muscle types.

Two primary osteosarcomas in a patient with Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome is an autosomal recessive disorder characterized by poikilodermatous skin changes that develop in infancy. Associated manifestations include juvenile cataracts, sparse hair, short stature, skeletal defects, dystrophic nails and teeth, and hypogonadism. An increased incidence of malignancy, including osteosarcoma, has been reported in patients with Rothmund-Thomson syndrome. The molecular basis of the disorder is not known. This report describes a patient with Rothmund-Thomson syndrome in whom two primary osteosarcomas developed 12 years apart. The presentation, diagnosis, and treatment of osteosarcoma in this patient with Rothmund-Thomson syndrome are described. Cytogenetic and molecular analysis of peripheral blood and skin fibroblasts had low level mosaicism for trisomy of chromosomes 2 and 8. Although several patients have been described with mosaic trisomy 8 and i(2q) (mosaic isochromosome for the long arm of chromosome 2), the patient described here is the first to have mosaic trisomy for the entire chromosomes 2 and 8. The cytogenetic findings in this patient are consistent with an underlying defect in chromosomal stability.

Molecular cytogenetic analysis of eight inversion duplications of human chromosome 13q that each contain a neocentromere.

Neocentromeres are fully functional centromeres that have arisen in previously noncentromeric chromosomal locations on rearranged chromosomes. The formation of neocentromeres results in the mitotic stability of chromosomal fragments that do not contain endogenous centromeres and that would normally be lost. Here we describe a unique collection of eight independent patient-derived cell lines, each of which contains a neocentromere on a supernumerary inversion duplication of a portion of human chromosome 13q. Findings in these patients reveal insight into the clinical manifestations associated with polysomy for portions of chromosome 13q. The results of FISH and immunofluorescent analysis of the neocentromeres in these chromosomes confirm the lack of alpha-satellite DNA and the presence of CENtromere proteins (CENP)-C, -E, and hMAD2. The positions of the inversion breakpoints in these chromosomes have been placed onto the physical map of chromosome 13, by means of FISH mapping with cosmid probes. These cell lines define, within chromosome 13q, at least three distinct locations where neocentromeres have formed, with five independent neocentromeres in band 13q32, two in band 13q21, and one in band 13q31. The results of examination of the set of 40 neocentromere-containing chromosomes that have thus far been described, including the 8 neocentromere-containing chromosomes from chromosome 13q that are described in the present study, suggest that chromosome 13q has an increased propensity for neocentromere formation, relative to some other human chromosomes. These neocentromeres will provide the means for testing hypotheses about sequence requirements for human centromere formation.

Chromosomal localization, genomic characterization, and mapping to the Noonan syndrome critical region of the human Deltex (DTX1) gene.

The human Deltex (DTX1) gene encodes a cytoplasmic protein that functions as a positive regulator of the Notch signaling pathway. We have determined the genomic organization and map location of the human gene. DTX1 encodes a 2.5-kb cDNA that is composed of nine exons. The DTX1 gene maps to chromosomal region 12q24 in the vicinity of the Noonan syndrome critical region. We have fine-mapped DTX1 to within this critical region and evaluate it as a candidate gene for this disorder.

Cloning and characterization of fiber type-specific ryanodine receptor isoforms in skeletal muscles of fish.

We have cloned a group of cDNAs that encodes the skeletal ryanodine receptor isoform (RyR1) of fish from a blue marlin extraocular muscle library. The cDNAs encode a protein of 5,081 amino acids with a calculated molecular mass of 576,302 Da. The deduced amino acid sequence shows strong sequence identity to previously characterized RyR1 isoforms. An RNA probe derived from a clone of the full-length marlin RyR1 isoform hybridizes to RNA preparations from extraocular muscle and slow-twitch skeletal muscle but not to RNA preparations from fast-twitch skeletal or cardiac muscle. We have also isolated a partial RyR clone from marlin and toadfish fast-twitch muscles that shares 80% sequence identity with the corresponding region of the full-length RyR1 isoform, and a RNA probe derived from this clone hybridizes to RNA preparations from fast-twitch muscle but not to slow-twitch muscle preparations. Western blot analysis of slow-twitch muscles in fish indicates the presence of only a single high-molecular-mass RyR protein corresponding to RyR1. [3H]ryanodine binding assays revealed the fish slow-twitch muscle RyR1 had a greater sensitivity for Ca2+ than the fast-twitch muscle RyR1. The results indicate that, in fish muscle, fiber type-specific RyR1 isoforms are expressed and the two proteins are physiologically distinct.

Purification and characterization of ryanotoxin, a peptide with actions similar to those of ryanodine.

We purified and characterized ryanotoxin, an approximately 11.4-kDa peptide from the venom of the scorpion Buthotus judiacus that induces changes in ryanodine receptors of rabbit skeletal muscle sarcoplasmic reticulum analogous to those induced by the alkaloid ryanodine. Ryanotoxin stimulated Ca2+ release from sarcoplasmic reticulum vesicles and induced a state of reduce unit conductance with a mean duration longer than that of unmodified ryanodine receptor channels. With Cs+ as the current carrier, the slope conductance of the state induced by 1 microM ryanotoxin was 163 +/- 12 pS, that of the state induced by 1 microM ryanodine was 173 +/- 26 pS, and that of control channels was 2.3-fold larger (396 +/- 25 pS). The distribution of substate events induced by 1 microM RyTx was biexponential and was fitted with time constants approximately 10 times shorter than those fitted to the distribution of substates induced by 1 microM ryanodine. Bath-applied 5 microM ryanotoxin had no effect on the excitability of mouse myotubes in culture. When 5 microM ryanotoxin was dialyzed into the cell through the patch pipette in the whole-cell configuration, there was a voltage-dependent increase in the amplitude of intracellular Ca2+ transients elicited by depolarizing potentials in the range of -30 to +50 mV. Ryanotoxin increased the binding affinity of [3H]ryanodine in a reversible manner with a 50% effective dose (ED50) of 0.16 microM without altering the maximum number (Bmax) of [3H]ryanodine-binding sites. This result suggested that binding sites for ryanotoxin and ryanodine were different. Ryanotoxin should prove useful in identifying domains coupling the ryanodine receptor to the voltage sensor, or domains affecting the gating and conductance of the ryanodine receptor channel.

Primary structure and properties of helothermine, a peptide toxin that blocks ryanodine receptors.

Helothermine, a protein from the venom of the Mexican beaded lizard (Heloderma horridum horridum), was found to inhibit [3H]ryanodine binding to cardiac and skeletal sarcoplasmic reticulum, to block cardiac and skeletal ryanodine receptor channels incorporated into planar bilayers, and to block Ca(2+)-induced Ca2+ release triggered by photolysis of nitr-5 in saponin-permeabilized trabeculae from rat ventricle. Cloning of the helothermine cDNA revealed that the protein is composed of 223 amino acids with a molecular mass of 25,376 daltons, and apparently is stabilized by eight disulfide bridges. The peptide sequence showed significant homology with a family of cysteine-rich secretory proteins found in the male genital tract and in salivary glands. The interaction of helothermine and ryanodine receptors should serve to define functional domains within the channel structure involved in the control of Ca2+ release from sarcoplasmic reticulum.

Mechanism of chloride-dependent release of Ca2+ in the sarcoplasmic reticulum of rabbit skeletal muscle.

We investigated the effect of Cl- on the Ca2+ permeability of rabbit skeletal muscle junctional sarcoplasmic reticulum (SR) using 45Ca2+ fluxes and single channel recordings. In 45Ca2+ efflux experiments, the lumen of the SR was passively loaded with solutions of 150 mM univalent salt containing 5 mM 45Ca2+. Release of 45Ca2+ was measured by rapid filtration in the presence of extravesicular 0.4-0.8 microM free Ca2+ and 150 mM of the same univalent salt loaded into the SR lumen. The rate of release was 5-10 times higher when the univalent salt equilibrated across the SR-contained Cl- (Tris-Cl, choline-Cl, KCl) instead of an organic anion or other halides (gluconate-, methanesulfonate-, acetate-, HEPES-, Br-, I-). Cations (K+, Tris+) could be interchanged without a significant effect on the release rate. To determine whether Cl- stimulated ryanodine receptors, we measured the stimulation of release by ATP (5 mM total) and caffeine (20 mM total) and the inhibition by Mg2+ (0.8 mM estimated free) in Cl(-)-free and Cl(-)-containing solutions. The effects of ATP, caffeine, and Mg2+ were the largest in K-gluconate and Tris-gluconate, intermediate in KCl, and notably poor or absent in choline-Cl and Tris-Cl. Procaine (10 mM) inhibited the caffeine-stimulated release measured in K-gluconate, whereas the Cl- channel blocker clofibric acid (10 mM) but not procaine inhibited the caffeine-insensitive release measured in choline-Cl. Ruthenium red (20 microM) inhibited release in all solutions. In SR fused to planar bilayers we identified a nonselective Cl- channel (PCl: PTris: PCa = 1:0.5:0.3) blocked by ruthenium red and clofibric acid but not by procaine. These conductive and pharmacological properties suggested the channel was likely to mediate Cl(-)-dependent SR Ca2+ release. The absence of a contribution of ryanodine receptors to the Cl(-)-dependent release were indicated by the lack of an effect of Cl- on the open probability of this channel, a complete block by procaine, and a stimulation rather than inhibition by clofibric acid. A plug model of Cl(-)-dependent release, whereby Cl- removed the inhibition of the nonselective channel by large anions, was formulated under the assumption that nonselective channels and ryanodine receptor channels operated separately from each other in the terminal cisternae. The remarkably large contribution of Cl- to the SR Ca2+ permeability suggested that nonselective Cl- channels may control the Ca2+ permeability of the SR in the resting muscle cell.

Structure and function of ryanodine receptors.

Membrane depolarization, neurotransmitters, and hormones evoke a release of Ca2+ from intracellular Ca(2+)-storing organelles like the endoplasmic reticulum and, in muscle, the sarcoplasmic reticulum (SR). In turn, the released Ca2+ serves to trigger a variety of cellular responses. The presence of Ca2+ pumps to replenish intracellular stores was described more than 20 years ago. The presence of Ca2+ channels, like the ryanodine receptor, which suddenly release the organelle-stored Ca2+, is a more recent finding. This review describes the progress made in the last five years on the structure, function, and regulation of the ryanodine receptor. Numerous reports have described the response of ryanodine receptors to cellular ions and metabolites, kinases and other proteins, and pharmacological agents. In many cases, comparative measurements have been made using Ca2+ fluxes in SR vesicles, single-channel recordings in planar bilayers, and radioligand binding assays using [3H]ryanodine. These techniques have helped to relate the activity of single ryanodine receptors to global changes in the SR Ca2+ permeability. Molecular information on functional domains within the primary structure of the ryanodine receptor is also available. There are at least three ryanodine receptor isoforms in various tissues. Some cells, such as amphibian muscle cells, express more than a single isoform. The diversity of ligands known to modulate gating and the diversity of tissues known to express the protein suggest that the ryanodine receptor has the potential to participate in many types of cell stimulus-Ca(2+)-release coupling mechanisms.

Cyclic ADP-ribose induced Ca2+ release in rabbit skeletal muscle sarcoplasmic reticulum.

The Ca(2+)-mobilizing metabolite cyclic ADP-ribose (cADPR) has been shown to release Ca2+ from ryanodine-sensitive stores in many cells. We show that this metabolite at a concentration of 17 microM, but not its precursor beta-NAD+ nor non-cyclic ADPR at the same concentration, is active in releasing Ca2+ from rabbit skeletal muscle sarcoplasmic reticulum. The release was not sensitive to Ruthenium red (1 microM) nor to the ryanodine receptor-specific scorpion toxin Buthotus1-1 (10 microM). In planar bilayer single channel recordings, concentrations up to 50 microM cADPR did not increase the open probability of Ruthenium red and toxin-sensitive Ca2+ release channels. Thus Ca2+ release induced by cADPR in skeletal muscle sarcoplasmic reticulum may not involve opening of ryanodine receptors.

Acute, but not chronic, exercise lowers the body weight set-point in male rats.

The influence of muscular training on overall energy balance and body weight is not clear. A group of male rats was trained to feed every day from 1000 to 1200. Then the intersect of regression line of food hoarded during meal time vs. body weight with the X-axis was measured. Finally, the rats were trained to run 1 h every day on a motor-driven treadmill. When the training took place in the morning, just before the hoarding session, the mean intersect was significantly lowered from control (497 +/- 18 g to 433 +/- 9 g). When the training took place in the afternoon, after the hoarding session, the mean intersect was not significantly different (504 +/- 21 g) from control. Food intake during the hoarding sessions was affected neither by body weight changes nor by muscular exercise. These results suggest that the set-point for body weight regulation is acutely lowered just after muscular exercise, but is not influenced by chronic training.

Activation of the ryanodine receptor Ca2+ release channel of sarcoplasmic reticulum by a novel scorpion venom.

We identified a peptide fraction from the venom of the scorpion Buthotus hottentota that stimulated binding of [3H]ryanodine to ryanodine receptors of skeletal and cardiac sarcoplasmic reticulum and brain microsomes in a highly specific manner. Activity was concentrated in a peptide fraction of Mr 5,000-8,000. Assuming a single active peptide in this fraction, we estimated a dissociation constant of 20-30 nM for the interaction of the peptide with the ryanodine receptor. The whole venom and the purified fraction activated skeletal ryanodine receptor Ca2+ release channels incorporated into planar lipid bilayers. The venom produced a 10-fold increase in the mean open time and induced the appearance of a long lasting subconductance state not seen in controls. Changes were reversible and could be induced by the partially purified venom fraction. This novel scorpion venom should be helpful in establishing the role of ryanodine receptors in the initiation of intracellular Ca2+ release in striated muscle and in nonmuscle cells containing functional ryanodine receptors such as neurons and secretory cells.

Transport of N1-methylnicotinamide by organic cation-proton exchange in rat liver membrane vesicles.

The characteristics of hepatic organic cation transport were examined in basolateral (blLPM) and canalicular (cLPM) rat liver plasma membrane vesicles, using the naturally occurring organic cation, N1-methylnicotinamide (NMN). In blLPM vesicles, an outwardly directed H+ gradient (pHin 5.9/pHout 7.9) stimulated [3H]NMN uptake compared with [3H]NMN uptake under pH-equilibrated conditions. The time course of [3H]NMN uptake exhibited a transient "over-shoot" phenomenon, consistent with active transport. The proton ionophore, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, had no effect on [3H]NMN uptake, demonstrating that pH-dependent [3H]NMN uptake was not the result of a H+ diffusion potential. An outwardly directed H+ gradient also stimulated [3H]NMN uptake under voltage-clamped conditions, consistent with electroneutral NMN-H+ exchange. Under conditions that effectively dissipated the H+ gradient, no active transport of [3H]NMN was observed. In the absence of a pH gradient, the intravesicular presence of NMN trans-stimulated the uptake of [3H]NMN. NMN-H+ exchange was differentiated from sinusoidal Na(+)-H+ exchange by determining sensitivity to amiloride. The substrate specificity of NMN-H+ exchange in blLPM vesicles was examined by determining the cis-inhibitory effects of typical endogenous and exogenous substrates of other epithelial organic cation-H+ exchangers. Kinetic analysis of initial rates of carrier-mediated [3H]NMN uptake over a NMN concentration range of 0.05-15 mM demonstrated that uptake occurred via two saturable transport systems, one a high-affinity low-capacity process and the other a low-affinity high-capacity type. In contrast, in cLPM vesicles, no pH gradient-dependent [3H]NMN uptake was demonstrated. These findings are consistent with the presence of an organic cation-H+ antiport on the sinusoidal membrane, with features distinct from the renal antiport, such as substrate specificity and membrane localization, that may account for differences in drug disposition by these two organs.

Inhibition of bile acid transport by cyclosporine A in rat liver plasma membrane vesicles.

In selectively isolated basolateral (bILPM) and canalicular (cLPM) rat liver plasma membrane vesicles, the in vitro effect of cyclosporine A (CsA) on specific hepatic membrane transport processes was examined. CsA (0.1-200 microM) caused a concentration-dependent inhibition of initial rates of Na(+)-dependent taurocholate uptake in bILPM and cLPM vesicles and Na(+)-independent taurocholate efflux from cLPM vesicles. In contrast, CsA had no effect on Na(+)-dependent L-alanine uptake in bILPM and in cLPM vesicles. In addition, electroneutral pH gradient-driven Na+ uptake in bILPM vesicles was unaffected by CsA treatment. CsA-induced inhibition of taurocholate transport in bILPM and cLPM vesicles was competitive in nature. A hydroxylated (OL-17) and a N-demethylated (OL-21) metabolite of CsA had no effect on taurocholate transport in either membrane vesicle population. These findings suggest that the mechanism of CsA-induced cholestasis is, in part, the result of selective inhibition of bile acid transport by the parent compound at both domains of the hepatocyte plasma membrane.