Contour prognostic model for predicting survival after resection of colorectal liver metastases: development and multicentre validation study using largest diameter and number of metastases with RAS mutation status.

BACKGROUND: Most current models for predicting survival after resection of colorectal liver metastasis include largest diameter and number of colorectal liver metastases as dichotomous variables, resulting in underestimation of the extent of risk variation and substantial loss of statistical power. The aim of this study was to develop and validate a new prognostic model for patients undergoing liver resection including largest diameter and number of colorectal liver metastases as continuous variables. METHODS: A prognostic model was developed using data from patients who underwent liver resection for colorectal liver metastases at MD Anderson Cancer Center and had RAS mutational data. A Cox proportional hazards model analysis was used to develop a model based on largest colorectal liver metastasis diameter and number of metastases as continuous variables. The model results were shown using contour plots, and validated externally in an international multi-institutional cohort. RESULTS: A total of 810 patients met the inclusion criteria. Largest colorectal liver metastasis diameter (hazard ratio (HR) 1.11, 95 per cent confidence interval 1.06 to 1.16; P < 0.001), number of colorectal liver metastases (HR 1.06, 1.03 to 1.09; P < 0.001), and RAS mutation status (HR 1.76, 1.42 to 2.18; P < 0.001) were significantly associated with overall survival, together with age, primary lymph node metastasis, and prehepatectomy chemotherapy. The model performed well in the external validation cohort, with predicted overall survival values almost lying within 10 per cent of observed values. Wild-type RAS was associated with better overall survival than RAS mutation even when liver resection was performed for larger and/or multiple colorectal liver metastases. CONCLUSION: The contour prognostic model, based on diameter and number of lesions considered as continuous variables along with RAS mutation, predicts overall survival after resection of colorectal liver metastasis.

Laser-based angle-resolved photoemission spectroscopy with micrometer spatial resolution and detection of three-dimensional spin vector.

We have developed a state-of-the-art apparatus for laser-based spin- and angle-resolved photoemission spectroscopy with micrometer spatial resolution (µ-SARPES). This equipment is realized by the combination of a high-resolution photoelectron spectrometer, a 6 eV laser with high photon flux that is focused down to a few micrometers, a high-precision sample stage control system, and a double very-low-energy-electron-diffraction spin detector. The setup achieves an energy resolution of 1.5 (5.5) meV without (with) the spin detection mode, compatible with a spatial resolution better than 10 µm. This enables us to probe both spatially-resolved electronic structures and vector information of spin polarization in three dimensions. The performance of µ-SARPES apparatus is demonstrated by presenting ARPES and SARPES results from topological insulators and Au photolithography patterns on a Si (001) substrate.

Nerve growth factor derived from bronchial epithelium after chronic mite antigen exposure contributes to airway hyperresponsiveness by inducing hyperinnervation, and is inhibited by in vivo siRNA.

BACKGROUND: Bronchial asthma is a chronic allergic airway inflammatory disease. Neurotrophins, including nerve growth factor (NGF), play an important role in the pathogenesis of asthma. However, the effects of NGF derived from epithelium on airway hyperresponsiveness (AHR) after antigen sensitization/exposure remain uncertain. OBJECTIVE: In this study, we examined the role of NGF on AHR after chronic antigen exposure and the effect of inhibiting NGF by in vivo siRNA on AHR exacerbation. METHODS: We generated chronic mouse models of bronchial asthma using house-dust mite antigen (Dermatophagoides pteronyssinus; Dp). NGF concentrations in bronchoalveolar lavage fluid (BALF), lung histopathology, hyperresponsiveness, and related neuronal peptides and cytokines in supernatants of lung homogenates were determined. RESULTS: NGF in BALF was increased in a dose- and time-dependent manner, and was expressed primarily in bronchial epithelium. Nerve fibres and substance P-positive fibres were detected in subepithelium of Dp-sensitized and challenged mice over 4 weeks of mite antigen exposure. AHR was positively correlated with NGF concentration and nerve fibre innervation. AHR, modulation of innervation, and increased substance P were inhibited by in vivo administration of siRNA that targeted NGF, although the inhibition of NGF did not affect allergic inflammation and subepithelial fibrosis. CONCLUSION AND CLINICAL RELEVANCE: These findings suggest that NGF derived from bronchial and alveolar epithelium plays an important role in AHR after chronic exposure to mite antigen. NGF inhibition could potentially manage bronchial asthma, including AHR.

High-power laser experiment forming a supercritical collisionless shock in a magnetized uniform plasma at rest.

We present an experimental method to generate quasiperpendicular supercritical magnetized collisionless shocks. In our experiment, ambient nitrogen (N) plasma is at rest and well magnetized, and it has uniform mass density. The plasma is pushed by laser-driven ablation aluminum (Al) plasma. Streaked optical pyrometry and spatially resolved laser collective Thomson scattering clarify structures of plasma density and temperatures, which are compared with one-dimensional particle-in-cell simulations. It is indicated that just after the laser irradiation, the Al plasma is magnetized by a self-generated Biermann battery field, and the plasma slaps the incident N plasma. The compressed external field in the N plasma reflects N ions, leading to counterstreaming magnetized N flows. Namely, we identify the edge of the reflected N ions. Such interacting plasmas form a magnetized collisionless shock.

Blockade of Th1 chemokine receptors ameliorates pulmonary granulomatosis in mice.

Sarcoidosis is a granulomatous disease of unknown aetiology. We identified immunological targets for the treatment of pulmonary granulomatosis using a murine model generated with Propionibacterium acnes. Sensitisation and challenge using heat-killed P. acnes and dendritic cells (DCs) were performed to produce pulmonary granulomatosis in C57BL/6 mice. Immunological analyses using ELISA as well as cDNA microarray analysis were used to search for cytokines or chemokines associated with the formation of granulomas in the lungs. Co-administration of P. acnes and DCs reproducibly induced the formation of pulmonary granulomas, which resembled sarcoid granulomas. The cDNA microarray assay demonstrated that the gene expression of CXCL9 and CXCL10, ligands for CXCR3, and of CCL4, a ligand for CCR5, was strongly upregulated during granulomatosis. ELISA confirmed that levels of CXCL9 and CXCL10 as well as T-helper (Th)1 cytokines and chemokines including tumour necrosis factor-α and interferon-γ were elevated in bronchoalveolar lavage fluid (BALF). The blockade of Th1 chemokine receptors using TAK-779, a dual blocker for CXCR3 and CCR5, led to reduced numbers of CXCR3+CD4+ and CCR5+CD4+ T-cells in BALF. Furthermore, administration of TAK-779 ameliorated the granulomatosis. The targeted inhibition of Th1 chemokines might be useful for inhibiting Th1-biased granulomatous diseases, including sarcoidosis.

IκB kinase ß inhibitor IMD-0354 suppresses airway remodelling in a Dermatophagoides pteronyssinus-sensitized mouse model of chronic asthma.

BACKGROUND: Nuclear factor (NF)-κB is a transcription factor that regulates cytokine and chemokine production in various inflammatory diseases, including bronchial asthma. IκB kinase (IKK) ß is important for NF-κB activation in inflammatory conditions, and is possibly related to airway remodelling. Thus, inhibition of the IKKß-NF-κB pathway may be an ideal strategy for the management of airway remodelling. OBJECTIVE: We examined the effects of a newly synthesized IKKß inhibitor, IMD-0354, in a chronic allergen exposure model of bronchial asthma in mice. METHODS: A chronic mouse model was generated by challenge with house dust mite antigen (Dermatophagoides pteronyssinus). IMD-0354 was administrated intraperitoneally in therapeutic groups. Lung histopathology, hyperresponsiveness and the concentrations of mediators and molecules in supernatants of lung homogenates were determined. RESULTS: NF-κB activation was inhibited by prolonged periods of IMD-0354 administration. IMD-0354 reduced the numbers of bronchial eosinophils. IMD-0354 also inhibited the pathological features of airway remodelling, including goblet cell hyperplasia, subepithelial fibrosis, collagen deposition and smooth muscle hypertrophy. Inhibition of these structural changes by IMD-0354 was the result of the suppressing the production and activation of remodelling-related mediators, such as TGF-ß, via inhibition of IKKß. IMD-0354 inhibited IL-13 and IL-1ß production, and it restored the production of IFN-γ. It also ameliorated airway hyperresponsiveness. CONCLUSION: IKKß plays crucial roles in airway inflammation and remodelling in a chronic mouse model of asthma. A specific IKKß inhibitor, IMD-0354, may be therapeutically beneficial for treating airway inflammation and remodelling in chronic asthma.

Do biologics-naïve patients with rheumatoid arthritis respond better to tocilizumab than patients for whom anti-TNF agents have failed? A retrospective study.

OBJECTIVES: To determine responses to tocilizumab between patients with rheumatoid arthritis (RA) who switched to anti-TNF agents and those who are biologics-naïve. METHODS: This retrospective study investigated 107 patients with RA who were treated with tocilizumab. At baseline, 61 of them had already been treated with anti-TNF agents (switched group; 46 for inefficacy and 15 for adverse events), and 46 were biologics-naïve (naïve group). Treatment responses to tocilizumab at week 12 and 24 were compared between the switched and naïve groups using the disease activity score 28 (DAS28). RESULTS: Forty-two (91.3%) and 50 (82.0%) patients in the naïve and switched groups, respectively, completed 24 weeks of tocilizumab treatment. The DAS28-ESR and DAS28-CRP values (means±SD) at weeks 12 and 24 compared to baseline decreased significantly for the naïve and switched groups. The DAS28-ESR and DAS28-CRP values at weeks 12 and 24 were significantly decreased in the naïve group, compared to the switched group. Disease activity was improved in the naïve patients compared to the switched patients. CONCLUSIONS: Tocilizumab was safe, tolerable, and clinically effective for patients with inadequate responses to anti-TNF therapy and for those who were biologics-naïve, and it was more effective among the latter.

Duplication, deletion, and polymorphism in the sex-determining region of the mouse Y chromosome.

The ZFY gene in the sex-determining region of the human Y chromosome encodes a "zinc-finger" protein that may be the testis-determining factor, TDF. Although the Y chromosomes of most placental mammals carry a single homolog of ZFY, the mouse Y chromosome has two homologs, both in the sex-determining (Sxr) region. Zfy-1 alone may suffice to determine maleness; Zfy-2 is dispensable, as it was deleted in an Sxr variant that retains sex-determining function but has lost other genes. Both loci mapped near the centromere of the mouse Y chromosome. The Y chromosomes of the subspecies Mus musculus musculus and M. m. domesticus were distinguishable by a Zfy-1 restriction fragment polymorphism, which can be used to study their differing interactions with autosomal sex-determining genes.

Mouse globin system: a functional and evolutionary analysis.

Structural and functional analysis of the mouse alpha-globin and beta-globin genes reveals that the globin genes are encoded in discontinous bits of coding information and that each gene locus is much more complex than was originally supposed. Each seems to consist of an array of several authentic genes as well as several apparently inactive pseudogenes. Comparison of the sequences of some of these genes to one another indicates that chromosomal DNA is a dynamic structure. Flanking and intervening sequences change in two ways: quickly, by duplication and extensive insertions and deletions, and slowly, by point mutation. Active coding sequences are usually limited to the slower mode of evolution. In addition to identifying fast and slow modes of evolution, it has also been possible to test the function of several signals that surround these genes and to identify those that appear to play a role in gene expression.

Multicentre study of perioperative versus adjuvant chemotherapy for resectable colorectal liver metastases.

Background: It is not known whether perioperative chemotherapy, compared with adjuvant chemotherapy alone, improves disease-free survival (DFS) in patients with upfront resectable colorectal liver metastases (CLM). The aim of this study was to estimate the impact of neoadjuvant 5-fluorouracil, leucovorin and oxaliplatin (FOLFOX) on DFS in patients with upfront resectable CLM. Methods: Consecutive patients who presented with up to five resectable CLM at two Japanese and two French centres in 2008-2015 were included in the study. Both French institutions favoured perioperative FOLFOX, whereas the two Japanese groups systematically preferred upfront surgery plus adjuvant chemotherapy. Inverse probability of treatment weighting (IPTW) and Cox regression multivariable models were used to adjust for confounding. The primary outcome was DFS. Results: Some 300 patients were included: 151 received perioperative chemotherapy and 149 had upfront surgery plus adjuvant chemotherapy. The weighted 3-year DFS rate was 33·5 per cent after perioperative chemotherapy compared with 27·1 per cent after upfront surgery plus adjuvant chemotherapy (hazard ratio (HR) 0·85, 95 per cent c.i. 0·62 to 1·16; P = 0·318). For the subgroup of 165 patients who received adjuvant FOLFOX successfully (for at least 3 months), the adjusted effect of neoadjuvant chemotherapy was not significant (HR 1·19, 0·74 to 1·90; P = 0·476). No significant effect of neoadjuvant chemotherapy was observed in multivariable regression analysis. Conclusion: Compared with adjuvant chemotherapy, perioperative FOLFOX does not improve DFS in patients with resectable CLM, provided adjuvant chemotherapy is given successfully.

Antecedentes: Se desconoce si la quimioterapia perioperatoria en comparación con la quimioterapia adyuvante sola mejora la supervivencia libre de enfermedad (disease­free survival, DFS) en pacientes con metástasis hepáticas de origen colorrectal (colorectal liver metastases, CLM) resecables de inicio. El objetivo de este estudio fue estimar el impacto de la neoadyuvancia con 5­fluorouracilo, leucovorina y oxaliplatino (FOLFOX) sobre la DFS en pacientes con CLM resecables desde el principio. Métodos: Se incluyeron pacientes consecutivos que presentaban hasta cinco CLM resecables en dos centros japoneses y dos centros franceses entre 2008 a 2015. Ambas instituciones francesas favorecían FOLFOX perioperatorio, mientras que los dos grupos japoneses utilizaban sistemáticamente la cirugía de entrada y quimioterapia adyuvante. Se utilizaron la probabilidad inversa del tratamiento ponderado (Inverse Probability of Treatment Weighting, IPTW) y el modelo multivariable de regresión de Cox para ajustar por factores de confusión. El resultado primario fue la DFS. Resultados: Se incluyeron 300 pacientes (grupo de quimioterapia perioperatoria n = 151 y grupo de cirugía de entrada más quimioterapia adyuvante n = 149). La DFS a los 3 años ponderada fue del 33% después de quimioterapia perioperatoria versus 27% tras cirugía de entrada (cociente de riesgos instantáneos, hazard ratio HR: 0,85; i.c. del 95% (0,62­1,16); P = 0,32). Cuando se consideró el subgrupo de pacientes que (n = 165) de manera efectiva (al menos 3 meses) recibieron FOLFOX adyuvante, el efecto ajustado de la quimioterapia neoadyuvante no fue significativo (HR: 1,19 (0,74­1,90); P = 0,48). No se observó un efecto significativo de la quimioterapia neoadyuvante en el análisis de regresión multivariable. Conclusión: En comparación con la quimioterapia adyuvante, el FOLFOX perioperatorio no mejora la DFS en CLM resecables siempre y cuando la quimioterapia adyuvante se administre de forma efectiva.

Functional CD40 ligand is expressed by T cells in rheumatoid arthritis.

CD40 ligand (CD40-L), a member of the tumor necrosis family of transmembrane glycoproteins, is rapidly and transiently expressed on the surface of recently activated CD4+ T cells. Interactions between CD40-L and CD40 induce B cell immunoglobulin production as well as monocyte activation and dendritic cell differentiation. Since these features characterize rheumatoid arthritis (RA), the expression and function of CD40-L in RA was examined. Freshly isolated RA peripheral blood (PB) and synovial fluid (SF) T cells expressed CD40-L mRNA as well as low level cell surface CD40-L. An additional subset of CD4+ RA SF T cells upregulated cell surface CD40-L expression within 15 min of in vitro activation even in the presence of cycloheximide, but soluble CD40-L was not found in SF. CD40-L expressed by RA T cells was functional, since RA PB and SF T cells but not normal PB T cells stimulated CD40-L-dependent B cell immunoglobulin production and dendritic cell IL-12 expression in the absence of prolonged in vitro T cell activation. In view of the diverse proinflammatory effects of CD40-L, this molecule is likely to play a central role in the perpetuation of rheumatoid synovitis. Of importance, blockade of CD40-L may prove highly effective as a disease modifying therapy for RA.

Activation of human B cells by phosphorothioate oligodeoxynucleotides.

To investigate the potential of DNA to elicit immune responses in man, we examined the capacity of a variety of oligodeoxynucleotides (ODNs) to stimulate highly purified T cell-depleted human peripheral blood B cells. Among 47 ODNs of various sequences tested, 12 phosphorothioate oligodeoxynucleotides (sODNs) induced marked B cell proliferation and Ig production. IL-2 augmented both proliferation and production of IgM, IgG, and IgA, as well as IgM anti-DNA antibodies, but was not necessary for B cell stimulation. Similarly, T cells enhanced stimulation, but were not necessary for B cell activation. After stimulation with the active sODNs, more than 95% of B cells expressed CD25 and CD86. In addition, B cells stimulated with sODNs expressed all six of the major immunoglobulin VH gene families. These results indicate that the human B cell response to sODN is polyclonal. Active sODN coupled to Sepharose beads stimulated B cells as effectively as the free sODN, suggesting that stimulation resulted from engagement of surface receptors. These data indicate that sODNs can directly induce polyclonal activation of human B cells in a T cell-independent manner by engaging as yet unknown B cell surface receptors.

Macrophage colony-stimulating factor gene transduction into human lung cancer cells differentially regulates metastasis formations in various organ microenvironments of natural killer cell-depleted SCID mice.

We investigated whether local production of macrophage colony-stimulating factor (M-CSF), responsible for migration and activation of monocytes/macrophages at a tumor growth site, affected the metastatic pattern of lung cancer. For this, highly metastatic human squamous (RERF-LC-AI) or small (H69/VP) cell lung carcinoma cells were transduced with the human M-CSF gene inserted into pRc/CMV-MCSF to establish M-CSF-producing clones (MCSF-AI-9-18, MCSF-AI-9-24, and MCSF-VP-5). M-CSF gene transduction had no effect on the expression of surface antigen or on in vitro proliferation. After s.c. injection into SCID mice, the growth rates of M-CSF-producing cells were slower than those of parent or mock-transduced cells. In the metastatic model in SCID mice depleted of natural killer cells, RERF-LC-AI cells formed metastases mainly in the liver and kidneys, whereas H69/VP cells metastasized mainly to the liver and systemic lymph nodes. The numbers of metastatic colonies of MCSF-AI-9-18 and MCSF-AI-9-24 cells in the liver but not the kidneys were significantly reduced. The development of lymph node metastases of MCSF-VP-5 cells was also less than that of parent or mock-transduced cells. Treatment of SCID mice with anti-human M-CSF antibody resulted in a significant increase in liver metastases of their M-CSF gene transfectants. No significant differences were observed in the distributions in mice or in the in vitro invasive potentials of MCSF-AI-9-18 cells and Neo-AI-3 cells. These findings indicate that the antimetastatic effect of M-CSF may be specific to particular organs, suggesting the influence of heterogeneity of organ microenvironments on the metastasis of lung cancer.

Down-regulation by interleukin 4 of activation of human alveolar macrophages to the tumoricidal state.

The effect of recombinant human interleukin 4 (IL-4) on the expression of antitumor activity of human alveolar macrophages (AM) obtained by bronchoalveolar lavage from healthy donors was examined. AM were incubated for 16 h in medium with various macrophage activators [lipopolysaccharide, des-methyl muramyldipeptide, Nocardia rubra cell wall skeleton, and heptanoyl-gamma-D-Glu-(L)-meso-alpha,epsilon-A2pm(L)-D-Al aOH] in the presence or absence of IL-4, and then their tumoricidal activity was assayed by measuring 125I-UdR release from human melanoma (A375) cells. The spontaneous tumoricidal activity of AM was slightly suppressed by IL-4 in 3 of 7 donors. Addition of IL-4 to cultures of AM with the activators resulted in dose-dependent suppression of AM-mediated cytotoxicity against A375 cells. IL-4 also inhibited AM-mediated cytotoxicity against A375-R cells, which are resistant to interleukin 1 (IL-1) and tumor necrosis factor alpha, HT-29 colon cancer cells, and KB cells. IL-4 inhibited the early induction phase of AM activation. Pretreatment of AM with IL-4 also suppressed their expression of antitumor activity in response to lipopolysaccharide. IL-4 inhibited the production of monokines (IL-1 and tumor necrosis factor alpha) by AM at the protein and mRNA levels. These findings suggest that IL-4 may be important in vivo in the down-regulation of antitumor expression of AM in the lung by inhibiting the production of monokines and other killing mechanisms.

Induction of systemic and therapeutic antitumor immunity using intratumoral injection of dendritic cells genetically modified to express interleukin 12.

Bone marrow-derived dendritic cells (BM-DCs) retrovirally transduced with genes encoding murine interleukin (IL)-12 stably expressed bioactive IL-12 protein at high levels. Intratumoral injection with IL-12 gene-modified BM-DCs resulted in regression of day 7 established weakly immunogenic tumors (MCA205, B16, and D122). This antitumor effect was substantially better than that of IL-12-transduced syngeneic fibroblasts or nontransduced BM-DCs. Furthermore, intratumoral injection with IL-12-transduced dendritic cells (DCs) induced specific TH1-type responses to the tumor in regional lymph nodes and spleen at levels greater than those of IL-12-transduced fibroblasts or nontransduced BM-DCs. Trafficking studies confirmed that intratumorally injected IL-12-transduced DCs, but not fibroblasts, could migrate to the draining lymph node to the same extent as nontransduced BM-DCs. This strategy designed to deliver genetically modified DCs to tumor sites is associated with systemic and therapeutic antitumor immunity and is an alternative approach to those that use delivery of DCs loaded with tumor antigen. These results support the clinical application of IL-12 gene-modified DCs in patients with cancer.

Natural killer cell-dependent suppression of systemic spread of human lung adenocarcinoma cells by monocyte chemoattractant protein-1 gene transfection in severe combined immunodeficient mice.

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine with various biological activities, including augmentation of cytotoxic activity of monocytes and natural killer (NK) cells. The present study was undertaken to determine whether transfection of the MCP-1 gene into lung cancer cells affected their tumorigenicity and metastatic potential by the NK cell-mediated mechanism. The human MCP-1 gene inserted into an expression vector (BCMGSNeo) was transfected into human lung adenocarcinoma (PC-14) cells. There was no difference in in vitro proliferation between MCP-1 gene-transfected PC-14 cells and the parent cells or mock-transfected cells. The tumorigenicity and in vivo tumor growth of MCP-1 gene-transfected PC-14 cells were similar to those of the parent cells or mock-transfected cells when tumor cells were injected into the s.c. space of NK cell-intact severe combined immunodeficient (SCID) mice. Although parent cells and mock-transfected cells inoculated i.v. formed lung metastatic colonies and pleural effusion, MCP-1 gene transfectants reduced the systemic spread in NK cell-intact SCID mice. Interestingly, these modulations in a systemic spread by MCP-1 gene transfection were not observed in NK cell-depleted SCID mice. Decreased survival of MCP-1 gene transfectants in the lung was observed in NK cell-intact SCID mice but not in NK cell-depleted SCID mice. Recombinant MCP-1 or the supernatant of MCP-1 gene transfectants enhanced the cytotoxicity of human CD56+ NK cells and spleen cells of SCID mice against PC-14 cells. These findings suggest that locally produced MCP-1 suppresses tumor progression by a NK cell-mediated mechanism, depending on organ microenvironment.

Magnetic field effects of photocarrier generation in bulk heterojunctions at low temperature.

We report an experimental investigation of the magnetic field effect (MFE) in polymer bulk heterojunction devices at temperatures below 10 K using photocarrier extraction by linearly increasing voltages. The examined devices were composed of an active layer of poly(3-hexylthiophene) and [6,6]-phenyl-C61-butyric acid methyl ester. In the experiments, the delay time (td) dependence of the MFE was investigated in detail. For td < 80 µs, a positive MFE was observed in the field region B < 0.1 T and a negative MFE was observed for B > 0.2 T. For td > 8 ms, only a positive MFE proportional to B2 was observed. For the photocurrent pulse detected immediately after light irradiation, the MFE was negligibly small. In a high magnetic field of 15 T, a significant MFE exceeding 80% was observed at 1.8 K for td = 800 ms. We discuss the results based on a model of triplet-singlet (or singlet-triplet) conversion in the magnetic field and estimate the exchange integral for the charge-transfer exciton in this photovoltaic cell.

Anti-tumor immunity elicited by direct intratumoral administration of a recombinant adenovirus expressing either IL-28A/IFN-λ2 or IL-29/IFN-λ1.

Interleukin (IL)-28A/interferon (IFN)-λ2 and IL-29/IFN-λ1 have been demonstrated to elicit direct and indirect anti-tumor actions. In this study, we constructed an adenovirus vector expressing either IL-28A/IFN-λ2 (AdIL-28A) or IL-29/IFN-λ1 (AdIL-29) to evaluate the therapeutic properties of intratumoral injection of recombinant adenovirus to apply for the clinical implementation of cancer gene therapy. Despite the lack of an anti-proliferative effect on MCA205 and B16-F10 cells, a retarded growth of established subcutaneous tumors was observed following multiple injections of either AdIL-28A or AdIL-29 when compared with AdNull. In vivo cell depletion experiments displayed that both NK cells and CD8(+) T cells have a major role in AdIL-28A-mediated tumor growth suppression. A significant increase in the number of infiltrating CD8(+) T cells into the tumors treated with either AdIL-28A or AdIL-29 was observed. Moreover, specific anti-tumor cytotoxic T lymphocyte reactivity was detected in spleen cells from animals treated with either AdIL-28A or AdIL-29. In IFN-γ-deficient mice, anti-tumor activities of AdIL-28A were completely impaired, indicating that IFN-γ is critically involved in the tumor growth inhibition triggered by AdIL-28A. IL-12 provided a synergistic anti-tumor effect when combined with AdIL-28A. These results indicate that AdIL-28A and AdIL-29 could be successfully utilized as an alternative cancer immunogene therapy.

Na,K-ATPase isoform expression in sheep red blood cell precursors.

Isoform expression of mammalian red cell Na,K-ATPase was analyzed using messenger RNA isolated from red cell precursor-enriched bone marrow of anemic sheep. Expression of the catalytic alpha subunit was analyzed using rat isoform-specific cDNA probes and expression of the beta 1 subunit, using a sheep beta 1-specific cDNA probe. RNA isolated from sheep kidney and brain were analyzed concurrently. In the red cell, as in the kidney, messenger RNA encoding only one isoform (alpha 1) of the catalytic subunit is detected; neither of the other isoforms (alpha 2 or alpha 3) could be detected. This holds true for bone marrow of sheep of either the low potassium or high potassium phenotype. Relative to the expression of alpha 1, beta subunit-specific message (beta 1) was extremely low in the red cell compared to either kidney (less than 5%) or brain (less than 3%). Using a rat cDNA probe specific for a beta 1-like subunit, beta 2, message was detected in brain but not in either kidney or bone marrow.

Substrate specificity of lysophospholipase D which produces bioactive lysophosphatidic acids in rat plasma.

Previously we reported that lysophospholipase D in rat plasma hydrolyzes endogenous unsaturated lysophosphatidylcholines (LPCs) preferentially to saturated LPCs to lysophosphatidic acids with growth factor-like and hormone-like activities. In this study, we examined the possibility that association of LPCs with different proteins in rat plasma has an effect on the preference of lysophospholipase D for unsaturated LPCs. Large portions of various LPCs were found to be recovered in the lipoprotein-poor bottom fraction. Furthermore, the percentages of LPCs associated with albumin isolated from rat plasma were shown not to be consistent with their percentage conversions to lysophosphatidic acids by lysophospholipase D on incubation of rat plasma at 37 degrees C. These results indicate that distinct distributions of LPCs in the plasma protein fractions are not critical factors for the substrate specificity of lysophospholipase D. Experiments with Nagase analbuminemic rats suggested that albumin-LPC complexes are not necessarily required for the hydrolysis by lysophospholipase D; lipoprotein-associate LPCs appeared to be good substrates for the phospholipase. We found that both saturated and unsaturated LPCs are present mainly as 1-acyl isomers in rat plasma. This result indicates that the preference of lysophospholipase D for unsaturated LPCs is not attributable to a difference in position of the acyl group attached to the glycerol backbone of LPC. In addition, lysophospholipase D was also found to attack choline phospholipids with a long chain group and a short chain alkyl group, although their percentage hydrolyses were low. Taken altogether, these results suggest that lysophospholipase D shows higher affinities for free forms of unsaturated acyl type LPCs equilibrated with albumin-bound and lipoprotein-associated forms, than for free forms of saturated acyl type LPCs and analogs of platelet-activating factor.