Bruton's Tyrosine Kinase Inhibitors with Distinct Binding Modes Reveal Differential Functional Impact on B-Cell Receptor Signaling.

Small molecule inhibitors of Bruton's tyrosine kinase (BTK) have been approved for the treatment of multiple B-cell malignancies and are being evaluated for autoimmune and inflammatory diseases. Various BTK inhibitors (BTKi) have distinct potencies, selectivity profiles, and binding modes within the ATP-binding site. On the basis of the latter feature, BTKis can be classified into those that occupy the back-pocket, H3 pocket, and the hinge region only. Hypothesizing that differing binding modes may have differential impact on the B-cell receptor (BCR) signaling pathway, we evaluated the activities of multiple BTKis in B-cell lymphoma models in vitro and in vivo. We demonstrated that, although all three types of BTKis potently inhibited BTK-Y223 autophosphorylation and phospholipase C gamma 2 (PLCγ2)-Y1217 transphosphorylation, hinge-only binders were defective in inhibiting BTK-mediated calcium mobilization upon BCR activation. In addition, PLCγ2 activation was effectively blocked by back-pocket and H3 pocket binders but not by hinge-only binders. Further investigation using TMD8 cells deficient in Rac family small GTPase 2 (RAC2) revealed that RAC2 functioned as a bypass mechanism, allowing for residual BCR signaling and PLCγ2 activation when BTK kinase activity was fully inhibited by the hinge-only binders. These data reveal a kinase activity-independent function of BTK, involving RAC2 in transducing BCR signaling events, and provide mechanistic rationale for the selection of clinical candidates for B-cell lymphoma indications.

Clinical Treatment Options for Carbapenem-Resistant Gram-Negative Infections in China: a Single Center Real-World Experience.

BACKGROUND: Carbapenem-resistant gram-negative bacteria pose a serious threat worldwide, and some patients even have rapidly aggravated life-threatening infection. However, as a result of the complexities of clinical therapy, antibiotic options against carbapenem-resistant pathogens have not yet been fully standardized. It should be individualized to control carbapenem-resistant pathogens in accordance with the different region. METHODS: In this study, we conducted a retrospective study in 65,000 inpatients over a 2-year period that involved a total of 86 patients from whom carbapenem-resistant gram-negative bacteria were isolated. RESULTS: Monotherapy using trimethoprim/sulfamethoxazole, amikacin, meropenem, and/or doxycycline in our hospital exhibited a clinical success rate of 83.3% for carbapenem-resistant Klebsiella pneumoniae, monotherapy using moxifloxacin, piperacillin/tazobactam, cefepime, and/or ceftazidime for carbapenem-resistant Pseudomonas aeruginosa exhibited a clinical success rate of 77.7%, and monotherapy using cefoperazone/sulbactam or combination therapy with tigecycline and cefoperazone/sulbactam for carbapenem-resistant Acinetobacter baumannii exhibited a clinical success rate of 62.1%. CONCLUSIONS: Taken together, our findings highlight the clinical strategies used in our hospital to successfully treat carbapenem-resistant gram-negative bacterial infections.

Human Insulin Growth Factor 2 mRNA Binding Protein 2 Increases MicroRNA 33a/b Inhibition of Liver ABCA1 Expression and Alters Low-Density Apolipoprotein Levels in Mice.

Genome-wide association studies (GWAS) have linked IGF2BP2 single-nucleotide polymorphisms (SNPs) with type 2 diabetes (T2D). Mice overexpressing mIGF2BP2 have elevated cholesterol levels when fed a diet that induces hepatic steatosis. These and other studies suggest an important role for insulin growth factor 2 mRNA binding protein 2 (IGF2BP2) in the initiation and progression of several metabolic disorders. The ATPase binding cassette protein ABCA1 initiates nascent high-density apolipoprotein (HDL) biogenesis by transferring phospholipid and cholesterol to delipidated apolipoprotein AI (ApoAI). Individuals with mutational ablation of ABCA1 have Tangier disease, which is characterized by a complete loss of HDL. MicroRNA 33a and 33b (miR-33a/b) bind to the 3' untranslated region (UTR) of ABCA1 and repress its posttranscriptional gene expression. Here, we show that IGF2BP2 works together with miR-33a/b in repressing ABCA1 expression. Our data suggest that IGF2BP2 is an accessory protein of the argonaute (AGO2)-miR-33a/b-RISC complex, as it directly binds to miR-33a/b, AGO2, and the 3' UTR of ABCA1 Finally, we show that mice overexpressing human IGF2BP2 have decreased ABCA1 expression, increased low-density lipoprotein-cholesterol (LDL-C) and cholesterol blood levels, and elevated SREBP-dependent signaling. Our data support the hypothesis that IGF2BP2 has an important role in maintaining lipid homeostasis through its modulation of ABCA1 expression, as its overexpression or loss leads to dyslipidemia.

First record of the New Guinea flatworm Platydemus manokwari (Platyhelminthes, Geoplanidae) as an alien species in Hong Kong Island, China.

The New Guinea flatworm (Platydemus manokwari) caused extinction of the native land snails on several Pacific island in past decades, and therefore it has been listed among the top 100 of the world's worst invasive alien species. Using morphological and molecular methods, New Guinea flatworms were discovered and identified for the first time in Hong Kong Island during a field investigation in July and August 2018. The flatworms were 32-60 mm long, 3-5 mm wide, and 1-2 mm thick. The dorsal side of the flatworm was dark brown with a thin yellow central line, and its ventral side appeared pale grey. To further verify this species, both 18S rDNA and mitochondrial cytochrome c oxidase subunit I gene (COX1) obtained from three specimens of P. manokwari were sequenced and analysed. While comparing these sequences with those previously deposited in GenBank, these 18S rDNA sequences shared 100% identity with the single available 18S rDNA sequence of P. manokwari; and the obtained COX1 sequences were identical to those of P. manokwari world genotype. Two native snails, Criptosoma imperator and Bradybaena similaris, have been found to be the prey of this predator during this investigation. Therefore, the invasive New Guinea flatworm certainly will cause a serious impact on the biodiversity of native snail populations, and an economic and environmental risk assessment for P. manokwari need to be completed in the near future in Hong Kong.

MOGAT2: A New Therapeutic Target for Metabolic Syndrome.

Metabolic syndrome is an ever-increasing health problem among the world's population. It is a group of intertwined maladies that includes obesity, hypertriglyceridemia, hypertension, nonalcoholic fatty liver disease (NAFLD), and diabetes mellitus type II (T2D). There is a direct correlation between high triacylglycerol (triglyceride; TAG) level and severity of metabolic syndrome. Thus, controlling the synthesis of TAG will have a great impact on overall systemic lipid metabolism and thus metabolic syndrome progression. The Acyl-CoA: monoacylglycerolacyltransferase (MGAT) family has three members (MGAT1, -2, and -3) that catalyze the first step in TAG production, conversion of monoacylglycerol (MAG) to diacylglycerol (DAG). TAG is then directly synthesized from DAG by a Acyl-CoA: diacylglycerolacyltransferase (DGAT). The conversion of MAG → DAG → TAG is the major pathway for the production of TAG in the small intestine, and produces TAG to a lesser extent in the liver. Transgenic and pharmacological studies in mice have demonstrated the beneficial effects of MGAT inhibition as a therapy for treating several metabolic diseases, including obesity, insulin resistance, T2D, and NAFLD. In this review, the significance of several properties of MGAT physiology, including tissue expression pattern and its relationship to overall TAG metabolism, enzymatic biochemical properties and their effects on drug discovery, and finally what is the current knowledge about MGAT small molecule inhibitors and their efficacy will be discussed. Overall, this review highlights the therapeutic potential of inhibiting MGAT for lowering TAG synthesis and whether this avenue of drug discovery warrants further clinical investigation.

Protein phosphatase 2A (PP2A) regulates low density lipoprotein uptake through regulating sterol response element-binding protein-2 (SREBP-2) DNA binding.

LDL-cholesterol (LDL-C) uptake by Ldlr is regulated at the transcriptional level by the cleavage-dependent activation of membrane-associated sterol response element-binding protein (SREBP-2). Activated SREBP-2 translocates to the nucleus, where it binds to an LDLR promoter sterol response element (SRE), increasing LDLR gene expression and LDL-C uptake. SREBP-2 cleavage and translocation steps are well established. Several SREBP-2 phosphorylation sites have been mapped and functionally characterized. The phosphatases dephosphorylating these sites remain elusive. The phosphatase(s) regulating SREBP-2 represents a novel pharmacological target for treating hypercholesterolemia. Here we show that protein phosphatase 2A (PP2A) promotes SREBP-2 LDLR promoter binding in response to cholesterol depletion. No binding to an LDLR SRE was observed in the presence of the HMG-CoA reductase inhibitor, lovastatin, when PP2A activity was inhibited by okadaic acid or depleted by siRNA methods. SREBP-2 cleavage and nuclear translocation were not affected by loss of PP2A. PP2A activity was required for SREBP-2 DNA binding. In response to cholesterol depletion, PP2A directly interacted with SREBP-2 and altered its phosphorylation state, causing an increase in SREBP-2 binding to an LDLR SRE site. Increased binding resulted in induced LDLR gene expression and increased LDL uptake. We conclude that PP2A activity regulates cholesterol homeostasis and LDL-C uptake.

Evaluating the potential of cubosomal nanoparticles for oral delivery of amphotericin B in treating fungal infection.

The oral administration of amphotericin B (AmB) has a major drawback of poor bioavailability. The aim of this study was to investigate the potential of glyceryl monoolein (GMO) cubosomes as lipid nanocarriers to improve the oral efficacy of AmB. Antifungal efficacy was determined in vivo in rats after oral administration, to investigate its therapeutic use. The human colon adenocarcinoma cell line (Caco-2) was used in vitro to evaluate transport across a model of the intestinal barrier. In vivo antifungal results showed that AmB, loaded in GMO cubosomes, could significantly enhance oral efficacy, compared against Fungizone, and that during a 2 day course of dosage 10 mg/kg the drug reached effective therapeutic concentrations in renal tissue for treating fungal infections. In the Caco-2 transport studies, GMO cubosomes resulted in a significantly larger amount of AmB being transported into Caco-2 cells, via both clathrin- and caveolae-mediated endocytosis, but not macropinocytosis. These results suggest that GMO cubosomes, as lipid nanovectors, could facilitate the oral delivery of AmB.

Identification of miR-185 as a regulator of de novo cholesterol biosynthesis and low density lipoprotein uptake.

Dysregulation of cholesterol homeostasis is associated with various metabolic diseases, including atherosclerosis and type 2 diabetes. The sterol response element binding protein (SREBP)-2 transcription factor induces the expression of genes involved in de novo cholesterol biosynthesis and low density lipoprotein (LDL) uptake, thus it plays a crucial role in maintaining cholesterol homeostasis. Here, we found that overexpressing microRNA (miR)-185 in HepG2 cells repressed SREBP-2 expression and protein level. miR-185-directed inhibition caused decreased SREBP-2-dependent gene expression, LDL uptake, and HMG-CoA reductase activity. In addition, we found that miR-185 expression was tightly regulated by SREBP-1c, through its binding to a single sterol response element in the miR-185 promoter. Moreover, we found that miR-185 expression levels were elevated in mice fed a high-fat diet, and this increase correlated with an increase in total cholesterol level and a decrease in SREBP-2 expression and protein. Finally, we found that individuals with high cholesterol had a 5-fold increase in serum miR-185 expression compared with control individuals. Thus, miR-185 controls cholesterol homeostasis through regulating SREBP-2 expression and activity. In turn, SREBP-1c regulates miR-185 expression through a complex cholesterol-responsive feedback loop. Thus, a novel axis regulating cholesterol homeostasis exists that exploits miR-185-dependent regulation of SREBP-2 and requires SREBP-1c for function.

miR-200a regulates Nrf2 activation by targeting Keap1 mRNA in breast cancer cells.

NF-E2-related factor 2 (Nrf2) is an important transcription factor that activates the expression of cellular detoxifying enzymes. Nrf2 expression is largely regulated through the association of Nrf2 with Kelch-like ECH-associated protein 1 (Keap1), which results in cytoplasmic Nrf2 degradation. Conversely, little is known concerning the regulation of Keap1 expression. Until now, a regulatory role for microRNAs (miRs) in controlling Keap1 gene expression had not been characterized. By using miR array-based screening, we observed miR-200a silencing in breast cancer cells and demonstrated that upon re-expression, miR-200a targets the Keap1 3'-untranslated region (3'-UTR), leading to Keap1 mRNA degradation. Loss of this regulatory mechanism may contribute to the dysregulation of Nrf2 activity in breast cancer. Previously, we have identified epigenetic repression of miR-200a in breast cancer cells. Here, we find that treatment with epigenetic therapy, the histone deacetylase inhibitor suberoylanilide hydroxamic acid, restored miR-200a expression and reduced Keap1 levels. This reduction in Keap1 levels corresponded with Nrf2 nuclear translocation and activation of Nrf2-dependent NAD(P)H-quinone oxidoreductase 1 (NQO1) gene transcription. Moreover, we found that Nrf2 activation inhibited the anchorage-independent growth of breast cancer cells. Finally, our in vitro observations were confirmed in a model of carcinogen-induced mammary hyperplasia in vivo. In conclusion, our study demonstrates that miR-200a regulates the Keap1/Nrf2 pathway in mammary epithelium, and we find that epigenetic therapy can restore miR-200a regulation of Keap1 expression, therefore reactivating the Nrf2-dependent antioxidant pathway in breast cancer.

MiR-28 regulates Nrf2 expression through a Keap1-independent mechanism.

NF-E2-related factor 2 (Nrf2) is an important transcription factor involved in antioxidant response. Nrf2 binds antioxidant response elements (ARE) within promoters of genes encoding detoxification enzymes (e.g., NAD (P) H-quinone oxidoreductase 1 (NQO1)) leading to their transcriptional activation. Nrf2 function is regulated post-translationally by its negative regulator Kelch-like ECH-associated protein 1 (Keap1) that binds Nrf2 and induces cytoplasmic Nrf2 degradation. Our present studies provide new evidence that Nrf2 expression can be regulated by a Keap1-independent mechanism. Here, we utilized breast epithelial cells to explore the impact of microRNA (miRNA) on Nrf2 expression. We found that Nrf2 mRNA levels are reversibly correlated with miR-28 expression and that ectopic expression of miR-28 alone reduces Nrf2 mRNA and protein levels. We further investigated the molecular mechanisms by which miR-28 inhibits Nrf2 mRNA expression. Initially, the ability of miR-28 to regulate the 3' untranslated region (3'UTR) of Nrf2 mRNA was evaluated via luciferase reporter assay. We observed that miR-28 reduces wild-type Nrf2 3'UTR luciferase reporter activity and this repression is eliminated upon mutation of the miR-28 targeting seed sequence within the Nrf2 3'UTR. Moreover, over-expression of miR-28 decreased endogenous Nrf2 mRNA and protein expression. We also explored the impact of miR-28 on Keap1-Nrf2 interactions and found that miR-28 over-expression does not alter Keap1 protein levels and has no effect on the interaction of Keap1 and Nrf2. Our findings, that miR-28 targets the 3'UTR of Nrf2 mRNA and decreases Nrf2 expression, suggest that this miRNA is involved in the regulation of Nrf2 expression in breast epithelial cells.

miR-200a regulates SIRT1 expression and epithelial to mesenchymal transition (EMT)-like transformation in mammary epithelial cells.

Evidence supports a critical role for microRNAs (miRNAs) in regulation of tissue-specific differentiation and development. Signifying a disruption of these programs, expression profiling has revealed extensive miRNA dysregulation in tumors compared with healthy tissue. The miR-200 family has been established as a key regulator of epithelial phenotype and, as such, is deeply involved in epithelial to mesenchymal transition (EMT) processes in breast cancer. However, the effects of the miR-200 family on transformation of normal mammary epithelial cells have yet to be fully characterized. By examining a TGF-ß driven model of transformation of normal mammary epithelium, we demonstrate that the class III histone deacetylase silent information regulator 1 (SIRT1), a proposed oncogene in breast cancer, is overexpressed upon EMT-like transformation and that epigenetic silencing of miR-200a contributes at least in part to the overexpression of SIRT1. We have established the SIRT1 transcript as subject to regulation by miR-200a, through miR-200a targeting of SIRT1 3'-UTR. We also observed SIRT1 and miR-200a participation in a negative feedback regulatory loop. Restoration of miR-200a or the knockdown of SIRT1 prevented transformation of normal mammary epithelial cells evidenced by decreased anchorage-independent growth and decreased cell migration. Finally, we observed SIRT1 overexpression in association with decreased miR-200a in breast cancer patient samples. These observations provide further evidence for a critical tumor suppressive role of the miR-200 family in breast epithelium in addition to identifying a novel regulatory mechanism, which may contribute to SIRT1 up-regulation in breast cancer.

L1 stimulation of human glioma cell motility correlates with FAK activation.

The neural adhesion/recognition protein L1 (L1CAM; CD171) has been shown or implicated to function in stimulation of cell motility in several cancer types, including high-grade gliomas. Our previous work demonstrated the expression and function of L1 protein in stimulation of cell motility in rat glioma cells. However, the mechanism of this stimulation is still unclear. This study further investigated the function of L1 and L1 proteolysis in human glioblastoma multiforme (GBM) cell migration and invasion, as well as the mechanism of this stimulation. L1 mRNA was found to be present in human T98G GBM cell line but not in U-118 MG grade III human glioma cell line. L1 protein expression, proteolysis, and release were found in T98G cells and human surgical GBM cells by Western blotting. Exosome-like vesicles released by T98G cells were purified and contained full-length L1. In a scratch assay, T98G cells that migrated into the denuded scratch area exhibited upregulation of ADAM10 protease expression coincident with loss of surface L1. GBM surgical specimen cells exhibited a similar loss of cell surface L1 when xenografted into the chick embryo brain. When lentivirally introduced shRNA was used to attenuate L1 expression, such T98G/shL1 cells exhibited significantly decreased cell motility by time lapse microscopy in our quantitative Super Scratch assay. These cells also showed a decrease in FAK activity and exhibited increased focal complexes. L1 binding integrins which activate FAK were found in T98G and U-118 MG cells. Addition of L1 ectodomain-containing media (1) rescued the decreased cell motility of T98G/shL1 cells and (2) increased cell motility of U-118 MG cells but (3) did not further increase T98G cell motility. Injection of L1-attenuated T98G/shL1 cells into embryonic chick brains resulted in the absence of detectable invasion compared to control cells which invaded brain tissue. These studies support a mechanism where glioma cells at the edge of a cell mass upregulate ADAM10 to proteolyze surface L1 and the resultant ectodomain increases human glioma cell migration and invasion by binding to integrin receptors, activating FAK, and increasing turnover of focal complexes.

Preparation and characterization of zedoary turmeric oil-loaded insulin-modified sterically stabilized liposomes.

The poor selectivity of anticancer drugs often leads to their multiplicate dose-limiting toxicities in humans, which severely restricts their clinical application. In this study, a novel liposomal formulation of zedoary turmeric oil (ZTO) targeting the insulin receptor (IR) was prepared by covalently conjugating insulin to the terminal of the polyethylene glycol (PEG) chain of sterically stabilized liposomes. In vitro assays indicated that a higher uptake of insulin-modified sterically stabilized liposomes (ISSLs) was observed in SMMC-7721 hepatocarcinoma cells overexpressing insulin receptors. IC(50) values of ISSLs, NTLs (nontargeted liposomes), and ZTO injection (free ZTO) against SMMC-7721, determined by MTT assays, were 157.2, 256.7, and 43.3 microg x ml(-1), respectively. Plasma-clearance profiles of ZTO in the liposomal formulations were then compared with that of ZTO injection. The liposomal formulations showed much longer terminal half-lives (11.24 and 14.73 hours for ISSLs and NTLs, respectively) than that of ZTO injection (1.45 hours). All results above indicated the ISSLs were potentially useful for the treatment of IR (+) tumors and are worthy of further investigation.

Stimulation of glioma cell motility by expression, proteolysis, and release of the L1 neural cell recognition molecule.

BACKGROUND: Malignant glioma cells are particularly motile and can travel diffusely through the brain parenchyma, apparently without following anatomical structures to guide their migration. The neural adhesion/recognition protein L1 (L1CAM; CD171) has been implicated in contributing to stimulation of motility and metastasis of several non-neural cancer types. We explored the expression and function of L1 protein as a stimulator of glioma cell motility using human high-grade glioma surgical specimens and established rat and human glioma cell lines. RESULTS: L1 protein expression was found in 17 out of 18 human high-grade glioma surgical specimens by western blotting. L1 mRNA was found to be present in human U-87/LacZ and rat C6 and 9L glioma cell lines. The glioma cell lines were negative for surface full length L1 by flow cytometry and high resolution immunocytochemistry of live cells. However, fixed and permeablized cells exhibited positive staining as numerous intracellular puncta. Western blots of cell line extracts revealed L1 proteolysis into a large soluble ectodomain (~180 kDa) and a smaller transmembrane proteolytic fragment (~32 kDa). Exosomal vesicles released by the glioma cell lines were purified and contained both full-length L1 and the proteolyzed transmembrane fragment. Glioma cell lines expressed L1-binding alphavbeta5 integrin cell surface receptors. Quantitative time-lapse analyses showed that motility was reduced significantly in glioma cell lines by 1) infection with an antisense-L1 retroviral vector and 2) L1 ectodomain-binding antibodies. CONCLUSION: Our novel results support a model of autocrine/paracrine stimulation of cell motility in glioma cells by a cleaved L1 ectodomain and/or released exosomal vesicles containing L1. This mechanism could explain the diffuse migratory behavior of high-grade glioma cancer cells within the brain.

Targeted delivery of insulin-modified immunoliposomes in vivo.

The purpose of this study was to investigate the effect of liposomes conjugated with insulin to the surface on circulation time, biodistribution, and antitumor activity after intravenous injection in tumor-bearing mice. Immunoliposomes were constructed with insulin, which was covalently linked to liposomes containing anticancer drugs. In order to investigate the targeting performance of insulin-modified immunoliposomes (SILs) in vivo, plasma pharmacokinetics, biodistribution, and antitumor activity were tested. In comparison with nontargeted liposomes (SLs), SILs were cleared faster from circulation as a result of greater liver and tumor uptake. In addition, SILs retarded the growth of the tumor effectively, compared with the ZTO injection or SL. This is the first time for selective in vivo targeting of tumor vessels using insulin-modified immunoliposomes. SILs are candidate drug-delivery systems for therapeutic anticancer approaches.

Evaluation of arsenic trioxide-loaded albumin nanoparticles as carriers: preparation and antitumor efficacy.

Bovine serum albumin (BSA) nanoparticles containing arsenic trioxide (As(2)O(3)) were prepared by a pH-coacervation method. To investigate the properties of the As(2)O(3)-loaded BSA nanoparticles, a study on drug-to-polymer ratio was done to determine the drug loading (DL), and a H-600 transmission electron microscope (TEM) was used to examine the particle sizes. The results showed that the DL was 27.8% and the average particle size was about 734 nm. The drug release in vitro test was done, which revealed that the drug release was found to provide a slow release after an initial burst release and the cumulative percentage release reached close to 95%. In vitro cytotoxicity test was carried out using APL NB4 cell lines (acute promyelocytic leukemia), and the anticancer efficacy in vivo against mouse H22 hepatoma cells was evaluated on kungming mice. The results indicated that the anticancer efficacy of the As(2)O(3)-loaded BSA nanoparticles was very obvious.

Outcome of colorectal carcinoma in patients under 40 years of age.

AIMS: Colorectal carcinoma in patients under 40 years of age usually has a poor prognosis. Controversies still exist regarding the features and the prognosis of colorectal cancer in young patients. METHODS: The records of 45 patients with histologically confirmed colorectal carcinoma treated between 1992 and 2002 at the Division of Oncology at Taipei Veterans General Hospital were reviewed. The relevance of sex, duration of symptoms, tumor site, histological type, lymph node involvement, Karnofsky performance status (KPS), carcinoembryonic antigen (CEA) and lactate dehydrogenase (LDH) levels at the diagnosis and tumor stage to overall survival (OS) were determined by univariate analysis, and their independent significance were tested by multivariate analysis. RESULTS: Most patients presented with an advanced tumor stage (24% Dukes' C and 66% Dukes' D). Colon carcinoma constituted 76% of the colorectal tumors. Family history was present in two patients and did not affect the OS. Two patients were found to have colon carcinoma during pregnancy. The 5-year survival rate in patients with Stage B, C, and D were 25, 16 and 0%, respectively. With aggressive treatment, patients with early stage carcinoma achieved longer survival. Eleven patients received resection of metastatic carcinoma of the liver, lung and ovary. Adjuvant chemotherapy with irinotecan/5-fluorouracil-based chemotherapy seemed to improve the OS in such patients, though the OS was still poorer than in patients with early stage tumors. In univariate analysis, KPS (P = 0.0001), lymph node involvement (P = 0.0024), CEA (P = 0.0423) and LDH levels (P = 0.0126) at the diagnosis and tumor stage (P = 0.0122) proved to be significant predictors of overall survival. Multivariate analyses revealed that KPS > or =70% (P = 0.007) and normal LDH levels at diagnosis (P = 0.004) were predictive of overall survival in this population. CONCLUSIONS: The present study shows that performance status and preoperative LDH levels were the major determinants for survival in patients with colorectal carcinoma under 40 years of age and the present series also suggests that surgical resection of metastatic colorectal carcinoma followed by adjuvant chemotherapy might be beneficial in certain patients. The data also suggests that current treatment modalities for young patients with advanced colorectal cancer might not be effective and more effective therapeutic regimens might be needed. Thus, it is important for surgeons to recognize the potential for colorectal cancer in young patients and to take an aggressive approach to the diagnosis and early treatment of the disease.

[Screening and analysis of genes encoding hepatocellular carcinoma associated tumor antigens].

OBJECTIVES: To screen and clone the genes encoding hepatocellular carcinoma associated tumor antigens. METHODS: A hepatocellular carcinoma cDNA express library was constructed with ZAP vector and analyzed by serological analysis of recombinant cDNA expression library (SEREX) with sera from autologous and allogenous patients. Monoclonalized positive phage clones were converted into pBK-CMV phagemid forms by in vivo excision. The cDNA inserts were determined by restriction endonuclease digestion with EcoR I and Xho I. The cDNA inserts were sequenced and analyzed with bioinformatics. LIMS1 insert was cut from the clone HCL5-70 and constructed into pQE 31 express vector. The recombinant LIMS1 was expressed in M15 and analyzed with SDS-PAGE and Western blot. RESULTS: Fourteen genes were cloned from autologous screening and eleven genes were obtained with allogeneous analysis. One gene, kinectin, was identified in both autologous and allogeneous screening. Eight of the total twenty-four genes were unknown for their functions; the other sixteen genes can be classified into eight groups according to their established or putative function. Recombinant LIMS1 was expressed in M15. CONCLUSION: The identification of hepatocellular carcinoma associated tumor antigens provides potential targets for immunotherapy of hepatocellular carcinoma patients and will help in the understanding of the carcinogenesis of hepatocellular carcinoma.

Pulmonary nocardiosis in a patient with CML relapse undergoing imatinib therapy after bone marrow transplantation.

We describe a case of pulmonary nocardiosis in a female patient with graft-versus-host disease (GVHD) underwent therapy with imatinib mesylate for a relapse of chronic myeloid leukemia (CML) after allogeneic bone marrow transplantation (BMT). The patient developed chronic GVHD 8 months after the use of imatinib and was on corticosteroid therapy. Three months after the development of chronic GVHD, she acquired pulmonary nocardiosis and a computed tomography (CT) scan of the chest showed multiple nodular lesions with cavitations over both lungs. She was successfully treated with single-agent trimethoprim-sulfamethoxazole (TMP/SMX) and the infection did not recur. Our case indicated that pulmonary nocardiosis could occur in patients with GVHD undergoing imatinib and corticosteroid therapy and might be treated by single-agent TMP/SMX.

All-trans retinoid acid increases Notch1 transcript expression in acute promyelocytic leukemia.

The effect of all-trans retinoid acid (ATRA) on the expression of Notch1 gene by real-time reverse transcriptase polymerase chain reaction (RT-PCR) in acute promyelocytic leukemia cells (APL), NB4, and HL-60 lacking t(15;17) was studied. The cells were treated with ATRA 0.5 microM for up to 96 hours. The increased transcript level of Notch1 was in concert with that of CD11b in NB4 cells, but not in HL-60 cells. The expression of Notch1 gene might be specific for APL cells. As Notch1 gene is involved in the differentiation and leukemogenesis in lymphoid neoplasm, observations suggest that Notch1 is involved in ATRA-modulated differentiation process in APL.