OBJECTIVE

Therapies targeted to the immune checkpoint mediated by PD-1 and PD-L1 show antitumor activity in a subset of patients with non-small cell lung cancer (NSCLC). We have now examined PD-L1 expression and its regulation in NSCLC positive for the EML4-ALK fusion gene.

METHODS

The expression of PD-L1 at the protein and mRNA levels in NSCLC cell lines was examined by flow cytometry and by reverse transcription and real-time PCR analysis, respectively. The expression of PD-L1 in 134 surgically resected NSCLC specimens was evaluated by immunohistochemical analysis.

RESULTS

The PD-L1 expression level was higher in NSCLC cell lines positive for EML4-ALK than in those negative for the fusion gene. Forced expression of EML4-ALK in Ba/F3 cells markedly increased PD-L1 expression, whereas endogenous PD-L1 expression in EML4-ALK-positive NSCLC cells was attenuated by treatment with the specific ALK inhibitor alectinib or by RNAi with ALK siRNAs. Furthermore, expression of PD-L1 was downregulated by inhibitors of the MEK-ERK and PI3K-AKT signaling pathways in NSCLC cells positive for either EML4-ALK or activating mutations of the EGFR. Finally, the expression level of PD-L1 was positively associated with the presence of EML4-ALK in NSCLC specimens.

CONCLUSIONS

Our findings that both EML4-ALK and mutant EGFR upregulate PD-L1 by activating PI3K-AKT and MEK-ERK signaling pathways in NSCLC reveal a direct link between oncogenic drivers and PD-L1 expression.

Bcr/Abl is a chimeric oncogene that can cause both acute and chronic human leukemias. Bcr/Abl-encoded proteins exhibit elevated kinase activity compared to c-Abl, but the mechanisms of transformation are largely unknown. Some of the biological effects of Bcr/Abl overlap with those of hematopoietic cytokines, particularly interleukin 3 (IL-3). Such effects include mitogenesis, enhanced survival, and enhanced basophilic differentiation. Therefore, it has been suggested that p210Bcr/Abl and the IL-3 receptor may activate some common signal transduction pathways. An important pathway for IL-3 signaling involves activation of the Janus family kinases (JAKs) and subsequent tyrosyl phosphorylation of STAT proteins (signal transducers and activators of transcription). This pathway directly links growth factor receptors to gene transcription. We analyzed JAK activation, STAT protein phosphorylation, and the formation of specific DNA-binding complexes containing STAT proteins, in a series of leukemia cell lines transformed by Bcr/Abl or other oncogenes. We also examined these events in cell lines transformed by a temperature sensitive (ts) mutant of Bcr/Abl, where the kinase activity of Abl could be regulated. STAT1 and STAT5 were found to be constitutively phosphorylated in 32D, Ba/F3, and TF-1 cells transformed by Bcr/Abl, but not in the untransformed parental cell lines in the absence of IL-3. Phosphorylation of STAT1 and STAT5 was also observed in the human leukemia cell lines K562 and BV173, which express the Bcr/Abl oncogene, but not in several Bcr/Abl-negative leukemia cell lines. Phosphorylation of STAT1 and STAT5 was directly due to the tyrosine kinase activity of Bcr/Abl since it could be activated or deactivated by temperature shifting of cells expressing the Bcr/Abl ts mutant. DNA-STAT complexes were detected in all Bcr/Abl-transformed cell lines and they were supershifted by antibodies against STAT1 and STAT5. DNA-STAT complexes in 32Dp210Bcr/Abl cells were similar, but not identical, to those formed after IL-3 stimulation. It is interesting to note that JAK kinases (JAK1, JAK2, JAK3, and Tyk2) were not consistently activated in Bcr/Abl-positive cells. These data suggest that STATs can be activated directly by Bcr/Abl, possibly bypassing JAK family kinase activation. Overall, our results suggest a novel mechanism that could contribute to some of the major biological effects of Bcr/Abl transformation.

We searched for immediate early cytokine responsive genes and isolated a novel gene, CIS (Cytokine Inducible SH2 containing protein) that is induced in hematopoietic cells by a subset of cytokines including interleukin-2 (IL-2), IL-3, and erythropoietin (EPO). The mutant IL-2 receptor that fails to activate STAT5 could not induce CIS, suggesting that STAT5 is involved in the cytokine-inducible expression of CIS. We cloned the 5'-flanking region of the CIS gene and found that about 200 bases upstream of the transcription-initiation site contain four potential STAT5 binding sites (MGF boxes). Luciferase reporter assays showed that these MGF boxes were essential for EPO-dependent promoter activity. Expression of STAT5 and the EPO receptor in HEK293 cells conferred EPO-dependent activation of the CIS promoter. These data indicate that CIS is a target of the JAK-STAT5 pathway of cytokine receptors. CIS contains an SH2 domain and binds to tyrosine-phosphorylated EPO and IL-3 receptors. In HEK293 cells expressing STAT5 and the EPO receptor, EPO-dependent tyrosine phosphorylation of STAT5, as well as EPO-dependent CIS-promoter activation, was suppressed when CIS was coexpressed. Moreover, the induction of oncostatin M, another STAT5 target, as well as the tyrosine-phosphorylation of STAT5, were partially suppressed by CIS expression in Ba/F3 cells. Thus, CIS is a feedback modulator of STAT5; its expression is induced by STAT5 and it negatively modulates STAT5 activation.

Integrin activation, the rapid conversion of integrin adhesion receptors from low to high affinity, occurs in response to intracellular signals that act on the short cytoplasmic tails of integrin beta subunits. Talin binding to integrin beta tails provides one key activation signal, but additional factors are likely to cooperate with talin to regulate integrin activation. The integrin beta tail-binding proteins kindlin-2 and kindlin-3 were recently identified as integrin co-activators. Here we report an analysis of kindlin-1 and kindlin-2 interactions with beta1 and beta3 integrin tails and describe the effect of kindlin expression on integrin activation. We demonstrate a direct interaction of kindlin-1 and -2 with recombinant integrin beta tails in pulldown binding assays. Our mutational analysis shows that the second conserved NXXY motif (Tyr(795)), a preceding threonine-containing region (Thr(788) and Thr(789)) of the integrin beta1A tail, and a conserved tryptophan in the F3 subdomain of the kindlin FERM domain (kindlin-1 Trp(612) and kindlin-2 Trp(615)) are required for direct kindlin-integrin interactions. Similar interactions were observed for integrin beta3 tails. Using fluorescence-activated cell sorting we further show that transient expression of kindlin-1 or -2 in Chinese hamster ovary cells inhibits the activation of endogenous alpha5beta1 or stably expressed alphaIIbbeta3 integrins. This inhibition is not dependent on direct kindlin-integrin interactions because mutant kindlins exhibiting impaired integrin binding activity effectively inhibit integrin activation. Consistent with previous reports, we find that when co-expressed with the talin head, kindlin-1 or -2 can activate alphaIIbbeta3. This effect is dependent on an intact integrin-binding site in kindlin. Notably however, even when co-expressed with activating levels of talin head, neither kindlin-1 or -2 can cooperate with talin to activate beta1 integrins; instead they strongly inhibit talin-mediated activation. We suggest that kindlins are adaptor proteins that regulate integrin activation, that kindlin expression levels determine their effects, and that kindlins may exert integrin-specific effects.

The ability of an environmental exposure to induce an epigenetic transgenerational adult onset disease phenotype is discussed in the current mini-review in the context of defining this phenomenon and the associated reproductive toxicology. A gestating female (F0 generation) exposure to an environmental compound results in the F1 generation embryo and F2 generation germ-line being directly exposed, such that the F3 generation is the first not directly exposed to the environmental compound. In contrast, postnatal or adult exposure (F0 generation) results in the F1 generation germ-line being exposed, such that F2 generation is the first to not be directly exposed to the environmental compound. The unequivocal transgenerational transmission of an adult onset disease phenotype through the germ-line requires assessment of the F3 generation for embryonic exposure, and F2 generation for postnatal exposure. This is in contrast to a number of F1 and F2 generation studies referred to as transgenerational. The reproductive toxicology associated with this transgenerational phenotype generally involves the reprogramming of the germ-line epigenome. The biological phenomenon involved in this reproductive toxicology deals with embryonic gonadal development and germ-line differentiation, or postnatally the gametogenesis process and germ cell development. The ability of an environmental compound (e.g. endocrine disruptor) to promote this reprogramming of the germ-line appears to be the causal factor in the epigenetic transgenerational phenotype.

Metabolomic profiling and the integration of whole-genome genetic association data has proven to be a powerful tool to comprehensively explore gene regulatory networks and to investigate the effects of genetic variation at the molecular level. Serum metabolite concentrations allow a direct readout of biological processes, and association of specific metabolomic signatures with complex diseases such as Alzheimer's disease and cardiovascular and metabolic disorders has been shown. There are well-known correlations between sex and the incidence, prevalence, age of onset, symptoms, and severity of a disease, as well as the reaction to drugs. However, most of the studies published so far did not consider the role of sexual dimorphism and did not analyse their data stratified by gender. This study investigated sex-specific differences of serum metabolite concentrations and their underlying genetic determination. For discovery and replication we used more than 3,300 independent individuals from KORA F3 and F4 with metabolite measurements of 131 metabolites, including amino acids, phosphatidylcholines, sphingomyelins, acylcarnitines, and C6-sugars. A linear regression approach revealed significant concentration differences between males and females for 102 out of 131 metabolites (p-values<3.8×10(-4); Bonferroni-corrected threshold). Sex-specific genome-wide association studies (GWAS) showed genome-wide significant differences in beta-estimates for SNPs in the CPS1 locus (carbamoyl-phosphate synthase 1, significance level: p<3.8×10(-10); Bonferroni-corrected threshold) for glycine. We showed that the metabolite profiles of males and females are significantly different and, furthermore, that specific genetic variants in metabolism-related genes depict sexual dimorphism. Our study provides new important insights into sex-specific differences of cell regulatory processes and underscores that studies should consider sex-specific effects in design and interpretation.

OBJECTIVE

Development of new therapeutic drug delivery systems is an area of significant research interest. The ability to directly target a therapeutic agent to a tumor site would minimize systemic drug exposure, thus providing the potential for increasing the therapeutic index.

METHODS

Photodynamic therapy (PDT) involves the uptake of a sensitizer by the cancer cells followed by photoirradiation to activate the sensitizer. PDT using Photofrin has certain disadvantages that include prolonged cutaneous photosensitization. Delivery of nanoparticles encapsulated with photodynamic agent specifically to a tumor site could potentially overcome the drawbacks of systemic therapy. In this study, we have developed a multifunctional polymeric nanoparticle consisting of a surface-localized tumor vasculature targeting F3 peptide and encapsulated PDT and imaging agents.

RESULTS

The nanoparticles specifically bound to the surface of MDA-435 cells in vitro and were internalized conferring photosensitivity to the cells. Significant magnetic resonance imaging contrast enhancement was achieved in i.c. rat 9L gliomas following i.v. nanoparticle administration. Serial magnetic resonance imaging was used for determination of pharmacokinetics and distribution of nanoparticles within the tumor. Treatment of glioma-bearing rats with targeted nanoparticles followed by PDT showed a significant improvement in survival rate when compared with animals who received PDT after administration of nontargeted nanoparticles or systemic Photofrin.

CONCLUSIONS

This study reveals the versatility and efficacy of the multifunctional nanoparticle for the targeted detection and treatment of cancer.

Sleep and waking differ significantly in terms of behavior, metabolism, and neuronal activity. Recent evidence indicates that sleep and waking also differ with respect to the expression of certain genes. To systematically investigate such changes, we used mRNA differential display and cDNA microarrays to screen approximately 10000 transcripts expressed in the cerebral cortex of rats after 8 h of sleep, spontaneous waking, or sleep deprivation. We found that 44 genes had higher mRNA levels after waking and/or sleep deprivation relative to sleep, while 10 were upregulated after sleep. Known genes that were upregulated in waking and sleep deprivation can be grouped into the following categories: immediate early genes/transcription factors (Arc, CHOP, IER5, NGFI-A, NGFI-B, N-Ras, Stat3), genes related to energy metabolism (glucose type I transporter Glut1, Vgf), growth factors/adhesion molecules (BDNF, TrkB, F3 adhesion molecule), chaperones/heat shock proteins (BiP, ERP72, GRP75, HSP60, HSP70), vesicle- and synapse-related genes (chromogranin C, synaptotagmin IV), neurotransmitter/hormone receptors (adrenergic receptor alpha(1A) and beta(2), GABA(A) receptor beta(3), glutamate NMDA receptor 2A, glutamate AMPA receptor GluR2 and GluR3, nicotinic acetylcholine receptor beta(2), thyroid hormone receptor TRbeta), neurotransmitter transporters (glutamate/aspartate transporter GLAST, Na(+)/Cl(-) transporter NTT4/Rxt1), enzymes (aryl sulfotransferase, c-jun N-terminal kinase 1, serum/glucocorticoid-induced serine/threonine kinase), and a miscellaneous group (calmodulin, cyclin D2, LMO-4, metallothionein 3). Several other genes that were upregulated in waking and all the genes upregulated in sleep, with the exception of the one coding for membrane protein E25, did not match any known sequence. Thus, significant changes in gene expression occur across behavioral states, which are likely to affect basic cellular functions such as RNA and protein synthesis, neural plasticity, neurotransmission, and metabolism.

BMS-354825 (dasatinib) and AMN107 (nilotinib) are potent alternate Abl inhibitors with activity against many imatinib mesylate-resistant BCR-ABL kinase domain (KD) mutants, except T315I. We used N-ethyl-N-nitrosourea (ENU)-exposed Ba/<em>F3</em>-p210(BCR-ABL) cells to compare incidence and types of KD mutants emerging in the presence of imatinib mesylate, dasatinib, and nilotinib, alone and in dual combinations. Although ENU is expected to induce mutations in multiple proteins, resistant clones were almost exclusively BCR-ABL KD mutant at relevant concentrations of nilotinib and dasatinib, consistent with a central role of KD mutations for resistance to these drugs. Twenty different mutations were identified with imatinib mesylate, 10 with nilotinib (including only 1 novel mutation, E292V) and 9 with dasatinib. At intermediate drug levels the spectrum narrowed to <em>F3</em>17V and T315I for dasatinib and Y253H, E255V, and T315I for nilotinib. Thus, cross-resistance is limited to T315I, which is also the only mutant isolated at drug concentrations equivalent to maximal achievable plasma trough levels. With drug combinations maximal suppression of resistant clone outgrowth was achieved at lower concentrations compared with single agents, suggesting that such combinations may be equipotent to higher-dose single agents. However, sequencing uniformly revealed T315I, consistent with the need for a T315I inhibitor, to completely block resistance.

The PTPN11 gene encodes SHP-2 (Src homology 2 domain-containing protein tyrosine Phosphatase), a nonreceptor tyrosine protein tyrosine phosphatase (PTPase) that relays signals from activated growth factor receptors to p21Ras (Ras) and other signaling molecules. Mutations in PTPN11 cause Noonan syndrome (NS), a developmental disorder characterized by cardiac and skeletal defects. NS is also associated with a spectrum of hematologic disorders, including juvenile myelomonocytic leukemia (JMML). To test the hypothesis that PTPN11 mutations might contribute to myeloid leukemogenesis, we screened the entire coding region for mutations in 51 JMML specimens and in selected exons from 60 patients with other myeloid malignancies. Missense mutations in PTPN11 were detected in 16 of 49 JMML specimens from patients without NS, but they were less common in other myeloid malignancies. RAS, NF1, and PTPN11 mutations are largely mutually exclusive in JMML, which suggests that mutant SHP-2 proteins deregulate myeloid growth through Ras. However, although Ba/F3 cells engineered to express leukemia-associated SHP-2 proteins cells showed enhanced growth factor-independent survival, biochemical analysis failed to demonstrate hyperactivation of the Ras effectors extracellular-regulated kinase (ERK) or Akt. We conclude that SHP-2 is an important cellular PTPase that is mutated in myeloid malignancies. Further investigation is required to clarify how these mutant proteins interact with Ras and other effectors to deregulate myeloid growth.

The aim of this study was to assess the feasibility of using non-invasive MR elastography for determining the stage of liver fibrosis. Twenty-five consecutive patients who had liver biopsy for suspicion of chronic liver disease were included in the study. The stage of fibrosis on the biopsies was assessed according to the METAVIR scoring system from F0, no fibrosis, to F4, cirrhosis. MR elastography was performed by transmitting low-frequency (65 Hz) mechanical waves into the liver with a transducer placed at the back of the patients. The MR pulse sequence was a motion-sensitized spin-echo sequence, phase-locked to the mechanical excitation. The phase maps were processed to obtain shear elasticity and shear viscosity maps. The mean hepatic shear elasticity increased with increasing stage of fibrosis. The mean elasticity was 2.24 +/- 0.23 kPa in the 11 patients without substantial fibrosis (F0-F1 grades), 2.56 +/- 0.24 kPa in the four patients with substantial fibrosis (F2-<em>F3</em>) and 4.68 +/- 1.61 kPa in the 10 patients with cirrhosis (F4). The differences between groups were statistically significant (p <or= 0.05). The mean shear viscosity was significantly higher in the patients with cirrhosis (5.19 +/- 1.85 Pa x s) than in the patients without cirrhosis (2.39 +/- 0.86 Pa x s in F0-F1 and 2.27 +/- 0.38 Pa x s in F2-<em>F3</em> patients). It is concluded that non-invasive MR elastography is a feasible method to assess the stage of liver fibrosis.

The BCR/ABL oncogene causes chronic myelogenous leukemia (CML) in humans and a CML-like disease, as well as lymphoid leukemia, in mice. p210 BCR/ABL is an activated tyrosine kinase that phosphorylates itself and several cellular signaling proteins. The autophosphorylation site tyrosine 177 binds the adaptor Grb2 and helps determine the lineage and severity of BCR/ABL disease: Tyr177 mutation (BCR/ABL-Y177F) dramatically impairs myeloid leukemogenesis, while diminishing lymphoid leukemogenesis. The critical signal(s) from Tyr177 has remained unclear. We report that Tyr177 recruits the scaffolding adaptor Gab2 via a Grb2/Gab2 complex. Compared to BCR/ABL-expressing Ba/F3 cells, BCR/ABL-Y177F cells exhibit markedly reduced Gab2 tyrosine phosphorylation and association of phosphatidylinositol-3 kinase (PI3K) and Shp2 with Gab2 and BCR/ABL, and decreased PI3K/Akt and Ras/Erk activation, cell proliferation, and spontaneous migration. Remarkably, bone marrow myeloid progenitors from Gab2 (-/-) mice are resistant to transformation by BCR/ABL, whereas lymphoid transformation is diminished as a consequence of markedly increased apoptosis. BCR/ABL-evoked PI3K/Akt and Ras/Erk activation also are impaired in Gab2 (-/-) primary myeloid and lymphoid cells. Our results identify Gab2 and its associated proteins as key determinants of the lineage and severity of BCR/ABL transformation.

Fusion of the TEL gene on 12p13 to the JAK2 tyrosine kinase gene on 9p24 has been found in human leukemia. TEL-mediated oligomerization of JAK2 results in constitutive activation of the tyrosine kinase (JH1) domain and confers cytokine-independent proliferation on interleukin-3-dependent Ba/F3 cells. Forced expression of the JAK inhibitor gene SOCS1/JAB/SSI-1 induced apoptosis of TEL-JAK2-transformed Ba/F3 cells. This suppression of TEL-JAK2 activity was dependent on SOCS box-mediated proteasomal degradation of TEL-JAK2 rather than on kinase inhibition. Degradation of JAK2 depended on its phosphorylation and its high affinity binding with SOCS1 through the kinase inhibitory region and the SH2 domain. It has been demonstrated that von Hippel-Lindau disease (VHL) tumor-suppressor gene product possesses the SOCS box that forms a complex with Elongin B and C and Cullin-2, and it functions as a ubiquitin ligase. The SOCS box of SOCS1/JAB has also been shown to interact with Elongins; however, ubiquitin ligase activity has not been demonstrated. We found that the SOCS box interacted with Cullin-2 and promoted ubiquitination of TEL-JAK2. Furthermore, overexpression of dominant negative Cullin-2 suppressed SOCS1-dependent TEL-JAK2 degradation. Our study demonstrates the substrate-specific E3 ubiquitin-ligase-like activity of SOCS1 for activated JAK2 and may provide a novel strategy for the suppression of oncogenic tyrosine kinases.

The BCR/ABL oncogene causes chronic myelogenous leukemia, a myeloproliferative disorder characterized by clonal expansion of hematopoietic progenitor cells and myeloid cells. It is shown here that transformation of the hematopoietic cell lines Ba/F3, 32Dcl3, and MO7e with BCR/ABL results in an increase in reactive oxygen species (ROS) compared with quiescent, untransformed cells. The increase in ROS was directly due to BCR/ABL because it was blocked by the ABL-specific tyrosine kinase inhibitor STI571. Oxidative stress through ROS is believed to have many biochemical effects, including the potential ability to inhibit protein-tyrosine phosphatases (PTPases). To understand the significance of increased production of ROS, a model system was established in which hydrogen peroxide (H(2)O(2)) was added to untransformed cells to mimic the increase in ROS induced constitutively by BCR/ABL. H(2)O(2) substantially reduced total cellular PTPase activity to a degree approximately equivalent to that of pervanadate, a well known PTPase inhibitor. Further, stimulation of untransformed cells with H(2)O(2) or pervanadate increased tyrosine phosphorylation of each of the most prominent known substrates of BCR/ABL, including c-ABL, c-CBL, SHC, and SHP-2. Treatment of the BCR/ABL-expressing cell line MO7/p210 with the reducing agents pyrrolidine dithiocarbamate or N-acetylcysteine reduced the accumulation of ROS and also decreased tyrosine phosphorylation of cellular proteins. Further, treatment of MO7e cells with H(2)O(2) or pervanadate increased the tyrosine kinase activity of c-ABL. Drugs that alter ROS metabolism or reactivate PTPases may antagonize BCR/ABL transformation.

This study was conducted under a working hypothesis that the repeating of selective breeding of normal rats with a slight impairment of glucose tolerance would produce spontaneous diabetes rats. This is the second report, 18 rats were selected by an oral glucose tolerance test from 211 normal Wistar rats obtained from an experimental animal farm. By the breeding 162 F1 rats were obtained. By the selective breeding in this way, 204 F2, 174 F3 and 215 F4 rats were obtained. The mean glucose tolerance curve became more diabetic with the increasing number of generations. The percentage of cases of a positive test for urine sugar during the glucose tolerance test increases with the number of generation. The results indicate the possibility of production of spontaneous diabetes from normal rats by the repetition of the selective breeding.

BACKGROUND

Liver biopsy is considered as the gold standard for assessing non-alcoholic fatty liver disease (NAFLD) histologic lesions. The aim of this study was to determine the diagnostic utility of non-invasive markers of fibrosis, validated in chronic viral hepatitis and alcoholic liver disease (FibroTest, FT), in patients with NAFLD.

METHODS

170 patients with suspected NAFLD were prospectively included in a reference center (Group 1), 97 in a multicenter study (Group 2) and 954 blood donors as controls. Fibrosis was assessed on a 5 stage histological scale validated by Kleiner et al from F0 = none, F1 = perisinusoidal or periportal, F2 = perisinusoidal and portal/periportal, F3 = bridging and F4 = cirrhosis. Histology and the biochemical measurements were blinded to any other characteristics. The area under the ROC curves (AUROC), sensitivity (Se), specificity (Sp), positive and negative predictive values (PPV, NPV) were assessed.

RESULTS

In both groups FT has elevated and not different AUROCs for the diagnosis of advanced fibrosis (F2F3F4): 0.86 (95%CI 0.77-0.91) versus 0.75 (95%CI 0.61-0.83; P = 0.10), and for F3F4: 0.92 (95%CI 0.83-0.96) versus 0.81 (95%CI 0.64-0.91; P = 0.12) in Group 1 and Group 2 respectively. When the 2 groups were pooled together a FT cutoff of 0.30 had a 90% NPV for advanced fibrosis (Se 77%); a FT cutoff of 0.70 had a 73% PPV for advanced fibrosis (Sp 98%).

CONCLUSIONS

In patients with NAFLD, FibroTest, a simple and non-invasive quantitative estimate of liver fibrosis reliably predicts advanced fibrosis.

Embryonic exposure to the endocrine disruptor vinclozolin during gonadal sex determination promotes an epigenetic reprogramming of the male germ-line that is associated with transgenerational adult onset disease states. Further analysis of this transgenerational phenotype on the brain demonstrated reproducible changes in the brain transcriptome three generations (F3) removed from the exposure. The transgenerational alterations in the male and female brain transcriptomes were distinct. In the males, the expression of 92 genes in the hippocampus and 276 genes in the amygdala were transgenerationally altered. In the females, the expression of 1,301 genes in the hippocampus and 172 genes in the amygdala were transgenerationally altered. Analysis of specific gene sets demonstrated that several brain signaling pathways were influenced including those involved in axon guidance and long-term potentiation. An investigation of behavior demonstrated that the vinclozolin F3 generation males had a decrease in anxiety-like behavior, while the females had an increase in anxiety-like behavior. These observations demonstrate that an embryonic exposure to an environmental compound appears to promote a reprogramming of brain development that correlates with transgenerational sex-specific alterations in the brain transcriptomes and behavior. Observations are discussed in regards to environmental and transgenerational influences on the etiology of brain disease.

The role and interdependence of CD8+ and CD4+ alpha beta-T cells in the acute response after respiratory infection with the murine parainfluenza type 1 virus, Sendai virus, has been analyzed for H-2b mice. Enrichment of CD8+ virus-specific CTL effectors in the lungs of immunologically intact C57BL/6 animals coincided with the clearance of the virus from this site by day 10 after infection. Removal of the CD4+ T cells by in vivo mAb treatment did not affect appreciably either the recruitment of CD8+ T cells to the infected lung, or their development into virus-specific cytotoxic effectors. In contrast, depletion of the CD8+ subset delayed virus clearance, although most mice survived the infection. Transgenic H-2b F3 mice homozygous (-/-) for a beta 2 microglobulin (beta 2-m) gene disruption, which lack both class I MHC glycoproteins and mature CD8+ alpha beta-T cells, showed a comparable, delayed clearance of Sendai virus from the lung. Virus-specific, class II MHC-restricted CTL were demonstrated in both freshly isolated bronchoalveolar lavage populations and cultured lymph node and spleen tissue from the beta 2-m (-/-) transgenics. Treatment of the beta 2-m (-/-) mice with the mAb to CD4 led to delayed virus clearance and death, which was also the case for normal mice that were depleted simultaneously of the CD4+ and CD8+ subsets. These results indicate that, although classical class I MHC-restricted CD8+ cytotoxic T cells normally play a dominant role in the recovery of mice acutely infected with Sendai virus, alternative mechanisms involving CD4+ T cells exist and can compensate, in time, for the loss of CD8+ T cell function.

Treatment of human diseases such as cancer generally involves the sequential use of diagnostic tools and therapeutic modalities. Multifunctional platforms combining therapeutic and diagnostic imaging functions in a single vehicle promise to change this paradigm. in particular, nanoparticle-based multifunctional platforms offer the potential to improve the pharmacokinetics of drug formulations, while providing attachment sites for diagnostic imaging and disease targeting features. We have applied these principles to the delivery of small interfering RNA (siRNA) therapeutics, where systemic delivery is hampered by rapid excretion and nontargeted tissue distribution. Using a PEGlyated quantum dot (QD) core as a scaffold, siRNA and tumor-homing peptides (F3) were conjugated to functional groups on the particle's surface. We found that the homing peptide was required for targeted internalization by tumor cells, and that siRNA cargo could be coattached without affecting the function of the peptide. Using an EGFP model system, the role of conjugation chemistry was investigated, with siRNA attached to the particle by disulfide cross-linkers showing greater silencing efficiency than when attached by a nonreducible thioether linkage. Since each particle contains a limited number of attachment sites, we further explored the tradeoff between number of F3 peptides and the number of siRNA per particle, leading to an optimized formulation. Delivery of these F3/siRNA-QDs to EGFP-transfected HeLa cells and release from their endosomal entrapment led to significant knockdown of EGFP signal. By designing the siRNA sequence against a therapeutic target (e.g., oncogene) instead of EGFP, this technology may be ultimately adapted to simultaneously treat and image metastatic cancer.

To investigate the hepatitis B virus (HBV) genotype-related differences in the progression of liver disease, 585 patients with chronic HBV infection including 258 with histologically verified chronic liver disease (CLD) and 74 with hepatocellular carcinoma (HCC) were examined. The mean ages of both patients with advanced fibrosis (F3 or F4) and with HCC were significantly older in genotype B than in genotype C patients (P =.018, P =.024, respectively). Both the hepatitis B e antigen (HBeAg) negativity rate at biopsy and the cumulative HBe seroconversion rate in patients with CLD were significantly higher in genotype B patients than genotype C patients (P <.01, P =.022, respectively). Multivariate analysis revealed that genotype B, presence of precore mutation, high ALT levels, and severe histologic activity were independent factors for HBe seroconversion. Among all the biopsy-proven CLD patients, the ratio of patients with advanced fibrosis in genotype B was significantly lower than that in genotype C (4/30 vs. 74/224, respectively; P =.034). This difference was more remarkable in younger patients (< or =45 years; 1/25 vs. 47/180, respectively; P =.020), and there was no difference in older patients (>45 years). The distribution of each genotype between CLD and HCC was very similar (B and C: 11.2% and 87.0% vs. 10.8% and 89.2%, respectively). In conclusion, our results suggest that, although the patients with genotype B experience earlier HBe seroconversion, slower progression of liver fibrosis, and slower development of HCC, the life-long risk of progression to advanced fibrosis and development of HCC may not differ among genotypes B- and C-related chronic liver disease.

IgM is the first antibody to be produced in a humoral immune response and plays an important role in the primary stages of immunity. Here we describe a mouse Fc receptor, designated Fc alpha/microR, and its human homolog, that bind both IgM and IgA with intermediate or high affinity. Fc alpha/microR is constitutively expressed on the majority of B lymphocytes and macrophages. Cross-linking Fc alpha/microR expressed on a pro-B cell line Ba/F3 transfectant with soluble IgM or IgM-coated microparticles induced internalization of the receptor. Fc alpha/microR also mediated primary B lymphocyte endocytosis of IgM-coated Staphylococcus aureus. Thus, Fc alpha/microR is involved in the primary stages of the immune response to microbes.

Members of the fibroblast growth factor receptor family of kinases (FGFR1-4) are dysregulated in multiple cancers. Ponatinib (AP24534) is an oral multitargeted tyrosine kinase inhibitor being explored in a pivotal phase II trial in patients with chronic myelogenous leukemia due to its potent activity against BCR-ABL. Ponatinib has also been shown to inhibit the in vitro kinase activity of all four FGFRs, prompting us to examine its potential as an FGFR inhibitor. In Ba/F3 cells engineered to express activated FGFR1-4, ponatinib potently inhibited FGFR-mediated signaling and viability with IC(50) values <40 nmol/L, with substantial selectivity over parental Ba/F3 cells. In a panel of 14 cell lines representing multiple tumor types (endometrial, bladder, gastric, breast, lung, and colon) and containing FGFRs dysregulated by a variety of mechanisms, ponatinib inhibited FGFR-mediated signaling with IC(50) values <40 nmol/L and inhibited cell growth with GI(50) (concentration needed to reduce the growth of treated cells to half that of untreated cells) values of 7 to 181 nmol/L. Daily oral dosing of ponatinib (10-30 mg/kg) to mice reduced tumor growth and inhibited signaling in all three tumor models examined. Importantly, the potency of ponatinib in these models is similar to that previously observed in BCR-ABL-driven models and plasma levels of ponatinib that exceed the IC(50) values for FGFR1-4 inhibition can be sustained in patients. These results show that ponatinib is a potent pan-FGFR inhibitor and provide strong rationale for its evaluation in patients with FGFR-driven cancers.

Bim, the Bcl-2 interacting mediator of cell death, is a member of the BH3-only family of pro-apoptotic proteins. Recent studies have demonstrated that the apoptotic activity of Bim can be regulated through a post-translational mechanism whereby ERK phosphorylation serves as a signal for Bim ubiquitination and proteasomal degradation. In this report, we investigated the signaling pathways leading to Bim phosphorylation in Ba/F3 cells, an interleukin-3 (IL-3)-dependent B-cell line. IL-3 stimulation induced phosphorylation of Bim(EL), one of the predominant isoforms of Bim expressed in cells, at multiple sites, as evidenced by the formation of at least three to four bands by Western blotting that were sensitive to phosphatase digestion. The appearance of multiple, phosphorylated species of Bim(EL) correlated with Akt, and not ERK, activation. The PI3K inhibitor, LY294002, blocked IL-3-stimulated Akt activity and partially blocked Bim(EL) phosphorylation. In vitro kinase assays showed that recombinant Akt could directly phosphorylate a GST-Bim(EL) fusion protein and identified the Akt phosphorylation site in the Bim(EL) domain as Ser(87). Further, we demonstrated that cytokine stimulation promotes Bim(EL) binding to 14-3-3 proteins. Finally, we show that mutation of Ser(87) dramatically increases the apoptotic potency of Bim(EL). We propose that Ser(87) of Bim(EL) is an important regulatory site that is targeted by Akt to attenuate the pro-apoptotic function of Bim(EL), thereby promoting cell survival.

Evidence from animal studies and human famines suggests that starvation may affect the health of the progeny of famished individuals. However, it is not clear whether starvation affects only immediate offspring or has lasting effects; it is also unclear how such epigenetic information is inherited. Small RNA-induced gene silencing can persist over several generations via transgenerationally inherited small RNA molecules in C. elegans, but all known transgenerational silencing responses are directed against foreign DNA introduced into the organism. We found that starvation-induced developmental arrest, a natural and drastic environmental change, leads to the generation of small RNAs that are inherited through at least three consecutive generations. These small, endogenous, transgenerationally transmitted RNAs target genes with roles in nutrition. We defined genes that are essential for this multigenerational effect. Moreover, we show that the F3 offspring of starved animals show an increased lifespan, corroborating the notion of a transgenerational memory of past conditions.