Tumorigenicity and lysis by natural killers.

Detailed analysis of the natural killer (NK) activity directed at nontumorigenic cell lines and their transformed tumorigenic derivatives has revealed a paradox. On the one hand, a correlation has been found between the tumorigenic potential of chemically transformed fibroblast cell lines and their sensitivity to NK cells in vitro. Nontransformed cells (N-type cell lines) and cells tumorigenic in normal mice (C-type cell lines) are resistant to NK-mediated lysis. In contrast, cell lines that are tumorigenic in ATxFL mice (these mice are very low in NK activity), but not in normal mice (I-type cell lines) are sensitive to NK-mediated lysis. These findings support the concept that NK activity is involved in host surveillance against tumors. On the other hand, NK-resistant fibroblasts, whether taken directly form animals or derived as tumorigenic or nontumorigenic cell lines, compete with NK-sensitive target cells to inhibit their lysis by NK effectors. Not only are both NK-sensitive and -resistant cells recognized by NK effectors but both receive lytic signals from NK effector cells. Target cell resistance is a result of a protein synthesis-dependent mechanism that prevents lysis such that in the presence of inhibitors of protein synthesis all fibroblasts tested are NK sensitive. Those fibroblasts that are normally sensitive to NK-mediated lysis must be deficient in their ability to produce or respond to this counterlytic mechanism. These findings are in contrast with the general findings when lymphoid cells are studied as NK targets where sensitivity appears to be a result of recognition by NK effectors. Because our findings show that transformed and normal cells express the same recognition determinants, in order for NK activity to play an important in vivo role in tumor surveillance, a mechanism must operate to permit NK effectors to find their targets in vivo. In the absence of a special discrimination mechanism, the killing of NK-sensitive transformants that arise autochronously would be less than optimal as a consequence of competition by the normal, NK-resistant, cells.

The synthetic liver X receptor agonist GW3965 reduces tissue factor production and inflammatory responses in human islets in vitro.

AIMS/HYPOTHESIS: Optimising islet culture conditions may be one strategy for reducing islet loss prior to, and immediately after, islet transplantation. Liver X receptor (LXR) agonism has previously been shown to increase insulin release from pancreatic islets and reduce inflammation in leucocytes. Our aim was to investigate whether the synthetic LXR agonist GW3965 could modulate the inflammatory status of human pancreatic islets. METHODS: Levels of pro-inflammatory cytokines and tissue factor in isolated human islets were determined by TaqMan low density array and/or real-time quantitative RT-PCR (mRNA levels) and enzyme immunoassay (EIA) (protein levels). Islet viability was measured using intracellular ATP content, ADP/ATP ratio, mitochondrial dehydrogenase activity (XTT assay) and insulin secretion in a dynamic glucose-challenge model. Apoptosis was determined by EIA measurement of histone-DNA complexes present in cytoplasm and by assaying caspase-3/-7 activity. RESULTS: Treatment of LPS-stimulated human islets with the synthetic LXR agonist GW3965 (1 micromol/l) for 24 h reduced mRNA and protein levels of selected pro-inflammatory cytokines (IL-8, monocyte chemotactic protein-1 and tissue factor). Moreover, GW3965 had no adverse effect on insulin secretion, islet viability or apoptosis. No excess of lipid accumulation could be detected with the dosage and exposure time used. CONCLUSIONS/INTERPRETATION: LXR activation suppresses inflammation in human islets in vitro without adverse effects on islet viability. Short-term moderate activation of LXR prior to islet transplantation may represent a possible strategy for improving post-transplant islet survival.

The human nuclear xenobiotic receptor PXR: structural determinants of directed promiscuity.

The human nuclear pregnane X receptor (hPXR) activates cytochrome P450-3A expression in response to a wide variety of xenobiotics and plays a critical role in mediating dangerous drug-drug interactions. We present the crystal structures of the ligand-binding domain of hPXR both alone and in complex with the cholesterol-lowering drug SR12813 at resolutions of 2.5 and 2.75 angstroms, respectively. The hydrophobic ligand-binding cavity of hPXR contains a small number of polar residues, permitting SR12813 to bind in three distinct orientations. The position and nature of these polar residues were found to be critical for establishing the precise pharmacologic activation profile of PXR. Our findings provide important insights into how hPXR detects xenobiotics and may prove useful in predicting and avoiding drug-drug interactions.

Autoregulation of the human liver X receptor alpha promoter.

Previous work has implicated the nuclear receptors liver X receptor alpha (LXR alpha) and LXR beta in the regulation of macrophage gene expression in response to oxidized lipids. Macrophage lipid loading leads to ligand activation of LXRs and to induction of a pathway for cholesterol efflux involving the LXR target genes ABCA1 and apoE. We demonstrate here that autoregulation of the LXR alpha gene is an important component of this lipid-inducible efflux pathway in human macrophages. Oxidized low-density lipoprotein, oxysterols, and synthetic LXR ligands induce expression of LXR alpha mRNA in human monocyte-derived macrophages and human macrophage cell lines but not in murine peritoneal macrophages or cell lines. This is in contrast to peroxisome proliferator-activated receptor gamma (PPAR gamma)-specific ligands, which stimulate LXR alpha expression in both human and murine macrophages. We further demonstrate that LXR and PPAR gamma ligands cooperate to induce LXR alpha expression in human but not murine macrophages. Analysis of the human LXR alpha promoter led to the identification of multiple LXR response elements. Interestingly, the previously identified PPAR response element (PPRE) in the murine LXR alpha gene is not conserved in humans; however, a different PPRE is present in the human LXR 5'-flanking region. These results have implications for cholesterol metabolism in human macrophages and its potential to be regulated by synthetic LXR and/or PPAR gamma ligands. The ability of LXR alpha to regulate its own promoter is likely to be an integral part of the macrophage physiologic response to lipid loading.

A novel technique for fascial fixation of laparoscopic adjustable gastric band ports.

Access port dislodgement after laparoscopic adjustable gastric banding is a recurring problem that often requires operative revision. Securing the port to the abdominal wall fascia in the traditional way with standard instruments is challenging in obese patients due to a thick abdominal wall. Therefore, we have devised a novel and simple technique for access port fixation using the EndoStitch device.

Analysis of immune surveillance of sequentially derived cell lines that differ in their tumorigenic potential.

The immune surveillance hypothesis suggests that cancer evolves as a multistage process. Further, it predicts that cells intermediate on the pathway to cancer are susceptible to host protective mechanisms, and only those variants that are able to escape the protective mechanisms are able to grow as tumors. We have isolated, as lineages, fibroblast lines that express phenotypes predicted by the surveillance hypothesis. The lineages were derived by treating nontransformed cells (N-cells) with chemical carcinogens and by isolating transformed variants in vitro. From the transformants that are tumorigenic in immune-depressed ATXFL mice but rejected by normal mice (I-cells), variants were selected in vivo that had escaped the rejection mechanism(s) and had grown as tumors in normal mice (C-cells). Thus lineages were established comprised of sequentially derived cell lines with the following phenotypes: nontransformed, transformed but susceptible to host protective mechanisms, transformed and resistant to host protective mechanisms (i.e., N----I----C). With the use of in vivo cross-protection experiments, two independently derived I-cell lines were shown to express non-cross-reactive antigens that are not expressed by the parental nontransformed N-cells (i.e., transformation-associated antigens). The transformation-associated antigens are expressed at an equivalent level on the cells that are susceptible to rejection (i.e., I-lines) and those that have escaped rejection (i.e., C-lines). In addition, although the transformation-associated antigens expressed by I-cells induce an effective immune response capable of rejecting both the I-line and C-line, the expression of these antigens on C-cells does not induce an effective immune response. The role of host defense mechanisms in the rejection of these chemically transformed I-cells and the possible mechanisms by which C-cells escape rejection are discussed.

Characterization of an antigen from the myelogenous leukemia cell line K-562.

A protein was solubilized from the myelogenous leukemia cell line K-562 WITh 3 M KCl that specifically inhibited the antibody-dependent, complement-mediated cytolysis of 51Cr-labeled K-562 cells by a monkey antiserum to K-562. When the crude 3 M KCl extract uas fractionated with ammonium sulfate, an eightfold increase in specific activity (U inhibition/mg protein) resulted. This purified fraction migrated as a single protein band after polyacrylamide gel electrophoresis (PGE) with no detectable carbohydrate or lipid. The molecular weight of the denatured protein determined by sodium dodecyl sulfate-PGE was 77,000, similar to that of the native protein (80,000) determined by Sephadex exclusion chromatography. The protein was stable at pH 6-8, with an apparent isoelectric point between pH 5 and 6. In addition to being irreversibly denatured at pH 5 or less, it was unstable at osmolarities below 0.25 M (NaCl). It was denatured at temperatures of 56 degrees C or above. Normal human peripheral blood leukocytes were extracted similarly with 3 M KCl and fractionated with ammonium sulfate. Neither the crude preparation nor any fraction purified as described for the specific antigen inhibited the cytolytic assay, which indicated at least a quantitative lack of the protein on the surfaces of normal leukocytes.

Resistance to cytolysis by tumor necrosis factor alpha in malignant gynecological cell lines is associated with the expression of protein(s) that prevent the activation of phospholipase A2 by tumor necrosis factor alpha.

Although there are a limited number of cell lines that are sensitive to cytolysis by tumor necrosis factor alpha (TNF alpha), the vast majority are resistant. The analysis of TNF alpha-sensitive cells has shown that phospholipase A2 is activated by TNF alpha in these cells and that the activity of phospholipase A2 is required for their cytolysis. Many cell lines that are resistant to TNF alpha-mediated cytolysis are dependent on the maintenance of protein synthesis for their resistance. We have recently shown that this is also true for TNF alpha-resistant cell lines derived from cervical (ME-180 and SiHa) and ovarian (SK-OV-3 and OVCAR-3) carcinomas, in that they are sensitive to cytolysis by TNF alpha only in the presence of protein synthesis inhibitors. Here we show that the TNF alpha-mediated cytolysis of these resistant cell lines in the presence of the protein synthesis inhibitor emetine is similar to that of sensitive cells, in that cytolysis is inhibited by the inhibitors of phospholipase A2. The measurement of the release of radiolabeled material from cervical and ovarian carcinoma cell lines prelabeled with [3H]arachidonic acid showed that not only was phospholipase A2 required for the cytolysis of these cells by TNF alpha in the presence of protein synthesis inhibitors, but more importantly, phospholipase A2 was not activated by TNF alpha unless protein synthesis was inhibited. These results indicate that a protein synthesis-dependent resistance mechanism expressed by these cell lines blocks TNF alpha-mediated cytolysis by preventing the activation of phospholipase A2 by TNF alpha.

Expression of a resistance mechanism in ovarian and cervical carcinoma cells prevents their lysis by gamma-interferon.

Despite extensive evidence that recombinant human gamma-interferon (IFN-gamma) exerts antiproliferative effects on a variety of cancer cell lines, IFN-gamma has not been shown to lyse cells in vitro. In order to determine whether some cancer cells might actively resist lysis by IFN-gamma, we examined eight arbitrarily selected cell lines derived from gynecological malignancies (ME-180, MS751, HT-3, SiHa, and C-33A human cervical carcinoma lines; Caov-3, SK-OV-3, and NIH:OVCAR-3 human ovarian carcinoma cell lines) for lysis by IFN-gamma. In a 24-h assay involving release of 51Cr from cells, none of these cell lines was lysed by IFN-gamma, either alone or in combination with actinomycin-D or emetine, two inhibitors of protein synthesis. However, pretreatment of cells with 100 units/ml of IFN-gamma for 24 h, followed by inhibition of protein synthesis, led to significantly increased lysis of the cell lines ME-180, MS751, and Caov-3. These results indicate that IFN-gamma induces a lytic mechanism in some cancer cells that is opposed by a protein synthesis-dependent resistance mechanism. This suggests that a combination therapy involving IFN-gamma and inhibitors of protein synthesis may be useful in the treatment of some cancers.

Simultaneous detection of multiple genetic aberrations in single cells by spectral fluorescence in situ hybridization.

Spectral fluorescence in situ hybridization (S-FISH) is a novel molecular cytogenetic approach that detects multiple disease-specific chromosomal aberrations in interphase nuclei using combinatorial fluorescence and digital imaging microscopy. A panel of six centromeric probes for chromosomes 7, 8, 9, 10, X, and Y, using a unique two-dye combination of four fluorophores, was developed to assess ploidy in breast tumors, bladder washings, and leukemia. Validation of S-FISH was performed by classic cytogenetics when metaphases were available or by standard fluorescence in situ hybridization (FISH) analyses. S-FISH identified clonal aberrations in newly diagnosed breast tumors and recurrent bladder cancer and revealed minimal residual disease in hyperdiploid acute lymphocytic leukemia, providing "proof of concept." Like standard FISH, aberrations were identified in poor growth/no growth specimen at the single cell level; however, S-FISH provided increased sensitivity over standard FISH by surveying six genetic targets instead of one or two. Disadvantages of the current assay include labor intensive screening and interpretative challenges with signal overlap in highly aneuploid samples and focal plane distortions. S-FISH appears to be a sensitive oncology assay with significant clinical application for early detection of new or reemerging clones, allowing for earlier therapeutic intervention and development of probe panels for individualized therapy.

Minimal residual disease (MRD) in remission t(8;21) AML and in vivo differentiation detected by FISH and CD34+ cell sorting.

Many patients with t(8;21) AML have residual positive cells during remission. We previously developed D-FISH probes that detect both derivative chromosomes and the normal alleles. In negative controls, only 2/44,000 (0.0045%) positive signals were observed. To investigate MRD, we examined specimens from 29 patients who had initially obtained CR. In remission patients, 61% had 1-4/2000 positive cells (0.05-0.19%). Higher frequencies were found in two patients in early relapse and in one patient in early remission. However, a negative test did not exclude relapse. Since false positives were negligible and because most t(8;21) AMLs express CD34, we asked whether cell sorting combined with FISH would increase the sensitivity. In one patient, we observed that 80% of CD34+ cells were t(8;21)+ at 2 months from initial clinical and cytogenetic remission. However, by 5 months the pre- and post-sorted populations contained 0.15% and 0.06% t(8;21) cells, respectively. Whereas essentially all t(8;21) cells in the initial specimen expressed CD34, only 0.6% were subsequently CD34+. These results are consistent with in vitro assays showing that residual t(8;21) cells undergo differentiation. Thus, FISH can identify MRD in a majority of t(8;21) patients and, combined with CD34+ selection, may provide an indirect assessment of the differentiation state of residual t(8;21) cells.

N-Phenylamidines as selective inhibitors of human neuronal nitric oxide synthase: structure-activity studies and demonstration of in vivo activity.

Selective inhibition of the neuronal isoform of nitric oxide synthase (NOS) compared to the endothelial and inducible isoforms may be required for treatment of neurological disorders caused by excessive production of nitric oxide. Recently, we described N-(3-(aminomethyl)benzyl)acetamidine (13) as a slow, tight-binding inhibitor, highly selective for human inducible nitric oxide synthase (iNOS). Removal of a single methylene bridge between the amidine nitrogen and phenyl ring to give N-(3-(aminomethyl)phenyl)acetamidine (14) dramatically altered the selectivity to give a neuronal selective nitric oxide synthase (nNOS) inhibitor. Part of this large shift in selectivity was due to 14 being a rapidly reversible inhibitor of iNOS in contrast to the essentially irreversible inhibition of iNOS observed with 13. Structure-activity studies revealed that a basic amine functionality tethered to an aromatic ring and a sterically compact amidine are key pharmacophores for this class of NOS inhibitors. Maximal nNOS inhibition potency was achieved with N-(3-(aminomethyl)phenyl)-2-furanylamidine (77) (Ki-nNOS = 0.006 microM; Ki-eNOS = 0.35 microM; Ki-iNOS = 0.16 microM). Finally, alpha-fluoro-N-(3-(aminomethyl)phenyl)acetamidine (74) (Ki-nNOS = 0. 011 microM; Ki-eNOS = 1.1 microM; Ki-iNOS = 0.48 microM) had excellent brain penetration and inhibited nNOS in a rat brain slice assay as well as in the rat brain (cerebellum) in vivo. Thus, N-phenylamidines should be useful in validating the role of nNOS in neurological disorders.

Pharmacophore analysis of the nuclear oxysterol receptor LXRalpha.

A cell-free assay was developed for the orphan nuclear receptor LXRalpha that measures the ligand-dependent recruitment of a peptide from the steroid receptor coactivator 1 (SRC1) to the nuclear receptor. Using this ligand-sensing assay (LiSA), the structural requirements for activation of the receptor by oxysterols and related compounds were studied. The minimal pharmacophore for receptor activation was shown to be a sterol with a hydrogen bond acceptor at C24. 24(S),25-Epoxycholesterol (1), which meets this criterion, is among the most efficacious of the oxysterols and is an attractive candidate as the LXRalpha natural hormone. Cholenic acid dimethylamide (14) showed increased efficacy compared to 1, whereas the unnatural oxysterol 22(S)-hydroxycholesterol (4) was shown to be an antagonist of 1 in the LiSA. The structural requirements for SRC1 recruitment in the LiSA correlated with the transcriptional activity of compounds in a cell-based reporter assay employing LXRalpha-GAL4 chimeric receptors. Site-directed mutagenesis identified Trp(443) as an amino acid critical for activation of LXRalpha by oxysterol ligands. This information was combined with the structure-activity relationship developed from the LiSA to develop a 3D homology model of LXRalpha. This model may aid the design of synthetic drugs targeted at this transcriptional regulator of cholesterol homeostasis.

N-(2-Benzoylphenyl)-L-tyrosine PPARgamma agonists. 2. Structure-activity relationship and optimization of the phenyl alkyl ether moiety.

We previously reported the identification of (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(5-methyl-2-phenyloxazol-4-y l)e thoxy]phenyl¿propanoic acid (2) (PPARgamma pKi = 8.94, PPARgamma pEC50 = 9.47) as a potent and selective PPARgamma agonist. We now report the expanded structure-activity relationship around the phenyl alkyl ether moiety by pursuing both a classical medicinal chemistry approach and a solid-phase chemistry approach for analogue synthesis. The solution-phase strategy focused on evaluating the effects of oxazole and phenyl ring replacements of the 2-(5-methyl-2-phenyloxazol-4-yl)ethyl side chain of 2 with several replacements providing potent and selective PPARgamma agonists with improved aqueous solubility. Specifically, replacement of the phenyl ring of the phenyloxazole moiety with a 4-pyridyl group to give 2(S)-((2-benzoylphenyl)amino)-3-¿4-[2-(5-methyl-2-pyridin-4-yloxazol+ ++- 4-yl)ethoxy]phenyl¿propionic acid (16) (PPARgamma pKi = 8.85, PPARgamma pEC50 = 8.74) or a 4-methylpiperazine to give 2(S)-((2-benzoylphenyl)amino)-3-(4-¿2-[5-methyl-2-(4-methylpiperazin+ ++- 1-yl)thiazol-4-yl]ethoxy¿phenyl)propionic acid (24) (PPARgamma pKi = 8.66, PPARgamma pEC50 = 8.89) provided two potent and selective PPARgamma agonists with increased solubility in pH 7.4 phosphate buffer and simulated gastric fluid as compared to 2. The second strategy took advantage of the speed and ease of parallel solid-phase analogue synthesis to generate a more diverse set of phenyl alkyl ethers which led to the identification of a number of novel, high-affinity PPARgamma ligands (PPARgamma pKi's 6.98-8.03). The combined structure-activity data derived from the two strategies provide valuable insight on the requirements for PPARgamma binding, functional activity, selectivity, and aqueous solubility.

N-(2-Benzoylphenyl)-L-tyrosine PPARgamma agonists. 3. Structure-activity relationship and optimization of the N-aryl substituent.

3-¿4-[2-(Benzoxazol-2-ylmethylamino)ethoxy]phenyl¿-(2S)-((2- benzoylph enyl)amino)propionic acid (1) and (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(5-methyl-2-phenyloxazol-4-y l)e thoxy]phenyl¿propionic acid (2) are peroxisome proliferator-activated receptor gamma (PPARgamma) agonists and have antidiabetic activity in rodent models of type 2 diabetes. As part of an effort to develop the SAR of the N-2-benzoylphenyl moiety of 1 and 2, a series of novel carboxylic acid analogues, 23-66, modified only in the N-2-benzoylphenyl moiety were synthesized from L-tyrosine and evaluated as PPARgamma agonists. In general, only modest changes in the N-2-benzoylphenyl moiety of 1 and 2 are tolerated. More specifically, the best changes involve bioisosteric replacement of one of the two phenyl rings of this moiety. Addition of substituents to this moiety generally produced compounds that are less active in the cell-based functional assays of PPARgamma activity although binding affinity to PPARgamma may be maintained. A particularly promising set of analogues is the anthranilic acid esters 63-66 in which the phenyl ring in the 2-benzoyl group of 1 and 2 has been replaced by an alkoxy group. In particular, (S)-2-(1-carboxy-2-¿4-[2-(5-methyl-2-phenyloxazol-4-yl)ethoxy]phen yl¿ ethylamino)benzoic acid methyl ester (63) has a pKi of 8.43 in the binding assay using human PPARgamma ligand binding domain and a pEC50 of 9.21 in the in vitro murine lipogenesis functional assay of PPARgamma activity. Finally, 63 was found to normalize glycemia when dosed at 3 mg/kg bid po in the Zucker diabetic fatty rat model of type 2 diabetes.

N-(2-Benzoylphenyl)-L-tyrosine PPARgamma agonists. 1. Discovery of a novel series of potent antihyperglycemic and antihyperlipidemic agents.

We have identified a novel series of antidiabetic N-(2-benzoylphenyl)-L-tyrosine derivatives which are potent, selective PPARgamma agonists. Through the use of in vitro PPARgamma binding and functional assays (2S)-3-(4-(benzyloxy)phenyl)-2-((1-methyl-3-oxo-3-phenylpropenyl)+ ++amin o)propionic acid (2) was identified as a structurally novel PPARgamma agonist. Structure-activity relationships identified the 2-aminobenzophenone moiety as a suitable isostere for the chemically labile enaminone moiety in compound 2, affording 2-((2-benzoylphenyl)amino)-3-(4-(benzyloxy)phenyl)propionic acid (9). Replacement of the benzyl group in 9 with substituents known to confer in vivo potency in the thiazolidinedione (TZD) class of antidiabetic agents provided a dramatic increase in the in vitro functional potency and affinity at PPARgamma, affording a series of potent and selective PPARgamma agonists exemplified by (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(methylpyridin-2-ylamino+ ++)ethoxy ]phenyl¿propionic acid (18), 3-¿4-[2-(benzoxazol-2-ylmethylamino)ethoxy]phenyl¿-(2S)-((2- benzoylph enyl)amino)propanoic acid (19), and (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(5-methyl-2-phenyloxazol-4-y l)e thoxy]phenyl¿propanoic acid (20). Compounds 18 and 20 show potent antihyperglycemic and antihyperlipidemic activity when given orally in two rodent models of type 2 diabetes. In addition, these analogues are readily prepared in chiral nonracemic fashion from L-tyrosine and do not show a propensity to undergo racemization in vitro. The increased potency of these PPARgamma agonists relative to troglitazone may translate into superior clinical efficacy for the treatment of type 2 diabetes.

Fragile X syndrome and neoplasia.

Among 100 males with fragile X [fra(X)] or Martin-Bell syndrome, two have developed malignancies. The first case, a 57-year-old man with fra(X) expression in 12% of peripheral blood lymphocytes, developed a seminoma of the left testis at age 45 years and in the right testis at age 50 years. The second case, a 16-year-old white boy with fra(X) expression in 23% of lymphocytes, developed a mucin-producing adenocarcinoma of the colon at age 14 years. Because of the unusual nature of the tumors observed in these patients and in 2 other patients from the literature, we suggest that individuals with the fra(X) syndrome may be at increased risk of cancer.

Surface immunoglobulin light chain-positive acute lymphoblastic leukemia of FAB L1 or L2 type: a report of 6 cases in adults.

Acute lymphoblastic leukemia (ALL) of B-cell lineage may be classified using the French-American-British (FAB) classification as L1, L2, or L3 type. L1 and L2 ALLs characteristically express terminal deoxynucleotidyl transferase (TdT) and are surface immunoglobulin (sIg)-negative. In contrast, L3 ALL is typically TdT-negative and sIg-positive. However, in a few large studies of children with ALL, sIg expression has been reported in less than 2% of L1 and L2 neoplasms. In these sIg-positive cases, IgM usually has been detected, with Ig light chain in a subset of tumors. Surface Ig expression in L1 or L2 ALL in adults is extremely rare; we found only 1 case report in the English literature. We report 6 cases of L1 or L2 ALL with an unusual immunophenotype arising in adults. In each tumor, the neoplastic cells expressed monotypic sIg light chain (4 lambda, 2 kappa) and TdT. Three tumors expressed CD34. Cytogenetic studies in 4 cases at diagnosis or relapse revealed no evidence of chromosomal translocations involving the c-myc locus, such as the t(8;14), t(2;8), or t(8;22). Three patients responded completely to chemotherapy and are alive; follow-up ranges from 18 to 57 months. Three patients died at 3, 13, and 14 months after diagnosis.

Acute lymphoblastic leukemia. Survey of immunophenotype, French-American-British classification, frequency of myeloid antigen expression, and karyotypic abnormalities in 210 pediatric and adult cases.

Immunophenotypic studies are essential to distinguish acute lymphoblastic leukemia (ALL) from minimally differentiated acute myeloid leukemia (AMLM0) and to classify ALL into immunologic subtypes. Frequently, immunophenotyping identifies myeloid antigen expression in ALL, causing a potential diagnostic problem. To evaluate the immunophenotype of ALL, we studied 210 cases of pediatric and adult ALL by flow cytometry and compared the results with the French-American-British (FAB) Cooperative Group classification and the karyotypic findings. Myeloid-associated antigens were expressed in 78 (45.6%) of precursor B-cell ALL cases. Pediatric precursor B ALLs had a higher frequency of myeloid antigen expression than did adult cases. All mature B-cell ALL cases were negative for TdT and myeloid antigens. Myeloid antigen expression was less frequent in T-cell ALL cases compared with precursor B-cell ALL cases. Of the 192 cases submitted for cytogenetic analysis, 147 were abnormal. The most common chromosomal translocation was the Philadelphia chromosome, which was more likely to have L2 blast morphology and a precursor B immunophenotype. Myeloid antigen expression was present in 70.8% of Ph-positive cases (P = .008). Chromosome rearrangements involving 11q23 also showed an increased frequency of myeloid antigen expression. Chromosome translocations involving regions of T-cell receptor genes were present in 24% of T-cell ALL cases. A high percentage of ALL cases, however, had various other cytogenetic abnormalities, many of which involved less well-studied chromosomal regions.

Receptor binding profile suggests multiple mechanisms of action are responsible for ibogaine's putative anti-addictive activity.

The indole alkaloid ibogaine (NIH 10567, Endabuse) is currently being examined for its potential utility in the treatment of cocaine and opioid addiction. However, a clearly defined molecular mechanism of action for ibogaine's putative anti-addictive properties has not been delineated. Radioligand binding assays targeting over 50 distinct neurotransmitter receptors, ion channels, and select second messenger systems were employed to establish a broad in vitro pharmacological profile for ibogaine. These studies revealed that ibogaine interacted with a wide variety of receptors at concentrations of 1-100 microM. These included the mu, delta, kappa, opiate, 5HT2, 5HT3, and muscarinic1 and 2 receptors, and the dopamine, norepinephrine, and serotonin uptake sites. In addition, ibogaine interacted with N-methyl-D-aspartic acid (NMDA) associated ion and sodium ion channels as determined by the inhibition of [3H]MK-801 and [3H]bactrachotoxin A 20-alpha-benzoate binding (BTX-B), respectively. This broad spectrum of activity may in part be responsible for ibogaine's putative anti-addictive activity.