Effects of milnacipran, a 5-HT and noradrenaline reuptake inhibitor, on C-fibre-evoked field potentials in spinal long-term potentiation and neuropathic pain.

BACKGROUND AND PURPOSE: The analgesic action of 5-HT and noradrenaline reuptake inhibitors (SNRIs) on nociceptive synaptic transmission in the spinal cord is poorly understood. We investigated the effects of milnacipran, an SNRI, on C-fibre-evoked field potentials (FPs) in spinal long-term potentiation (LTP), a proposed synaptic mechanism of hypersensitivity, and on the FPs in a neuropathic pain model. EXPERIMENTAL APPROACH: C-fibre-evoked FPs by electrical stimulation of the sciatic nerve fibres were recorded in the spinal dorsal horn of anaesthetized adult rats, and LTP was induced by high-frequency stimulation of the sciatic nerve fibres. A rat model of neuropathic pain was produced by L5 spinal nerve ligation and transection. KEY RESULTS: Milnacipran produced prolonged inhibition of C-fibre-evoked FPs when applied spinally after the establishment of LTP of C-fibre-evoked FPs in naïve animals. In the neuropathic pain model, spinal administration of milnacipran clearly reduced the basal C-fibre-evoked FPs. These inhibitory effects of milnacipran were blocked by spinal administration of methysergide, a 5-HT½ receptor antagonist, and yohimbine or idazoxan, α2-adrenoceptor antagonists. However, spinal administration of milnacipran in naïve animals did not affect the basal C-fibre-evoked FPs and the induction of spinal LTP. CONCLUSION AND IMPLICATIONS: Milnacipran inhibited C-fibre-mediated nociceptive synaptic transmission in the spinal dorsal horn after the establishment of spinal LTP and in the neuropathic pain model, by activating both spinal 5-hydroxytryptaminergic and noradrenergic systems. The condition-dependent inhibition of the C-fibre-mediated transmission by milnacipran could provide novel evidence regarding the analgesic mechanisms of SNRIs in chronic pain.

In vivo delivery of interferon-α gene enhances tumor immunity and suppresses immunotolerance in reconstituted lymphopenic hosts.

T cells recognize tumor-associated antigens under the condition of lymphopenia-induced homeostatic proliferation (HP); however, HP-driven antitumor responses gradually decay in association with tumor growth. Type I interferon (IFN) has important roles in regulating the innate and adaptive immune system. In this study we examined whether a tumor-specific immune response induced by IFN-α could enhance and sustain HP-induced antitumor immunity. An intratumoral IFN-α gene transfer resulted in marked tumor suppression when administered in the early period of syngeneic hematopoietic stem cell transplantation (synHSCT), and was evident even in distant tumors that were not transduced with the IFN-α vector. The intratumoral delivery of the IFN-α gene promoted the maturation of CD11c(+) cells in the tumors and effectively augmented the antigen-presentation capacity of the cells. An analysis of the cytokine profile showed that the CD11c(+) cells in the treated tumors secreted a large amount of immune-stimulatory cytokines including interleukin (IL)-6. The CD11c(+) cells rescued effector T-cell proliferation from regulatory T-cell-mediated suppression, and IL-6 may have a dominant role in this phenomenon. The intratumoral IFN-α gene transfer creates an environment strongly supporting the enhancement of antitumor immunity in reconstituted lymphopenic recipients through the induction of tumor-specific immunity and suppression of immunotolerance.

Oncolytic virus therapy for pancreatic cancer using the adenovirus library displaying random peptides on the fiber knob.

A conditionally replicative adenovirus is a novel anticancer agent designed to replicate selectively in tumor cells. However, a leak of the virus into systemic circulation from the tumors often causes ectopic infection of various organs. Therefore, suppression of naive viral tropism and addition of tumor-targeting potential are necessary to secure patient safety and increase the therapeutic effect of an oncolytic adenovirus in the clinical setting. We have recently developed a direct selection method of targeted vector from a random peptide library displayed on an adenoviral fiber knob to overcome the limitation that many cell type-specific ligands for targeted adenovirus vectors are not known. Here we examined whether the addition of a tumor-targeting ligand to a replication-competent adenovirus ablated for naive tropism enhances its therapeutic index. First, a peptide-display adenovirus library was screened on a pancreatic cancer cell line (AsPC-1), and particular peptide sequences were selected. The replication-competent adenovirus displaying the selected ligand (AdDeltaCAR-SYE) showed higher oncolytic potency in several other pancreatic cancer cell lines as well as AsPC-1 compared with the untargeted adenovirus (AdDeltaCAR). An intratumoral injection of AdDeltaCAR-SYE significantly suppressed the growth of AsPC-1 subcutaneous tumors, and an analysis of adenovirus titer in the tumors revealed an effective replication of the virus in the tumors. Ectopic liver gene transduction following the intratumoral injection of AdDeltaCAR-SYE was not increased compared with the AdDeltaCAR. The results showed that a tumor-targeting strategy using an adenovirus library is promising for optimizing the safety and efficacy of oncolytic adenovirus therapy.

Direct selection of targeted adenovirus vectors by random peptide display on the fiber knob.

Targeting of gene transfer at the level of cell entry is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success because proper targeting ligand-receptor systems on the cells of interest are generally unknown. Systematic approaches to generate adenovirus vectors targeting any given cell type need to be developed to achieve this goal. Here, we constructed an adenovirus library that was generated by a Cre-lox-mediated in vitro recombination between an adenoviral fiber-modified plasmid library and genomic DNA to display random peptides on a fiber knob. As proof of concept, we screened the adenovirus display library on a glioma cell line and observed selection of several particular peptide sequences. The targeted vector carrying the most frequently isolated peptide significantly enhanced gene transduction in the glioma cell line but not in many other cell lines. Because the insertion of a pre-selected peptide into a fiber knob often fails to generate an adenovirus vector, the selection of targeting peptides is highly useful in the context of the adenoviral capsid. This vector-screening system can facilitate the development of a targeted adenovirus vector for a variety of applications in medicine.

Adenovirus-mediated interferon alpha gene transfer induces regional direct cytotoxicity and possible systemic immunity against pancreatic cancer.

We previously demonstrated a characteristically high sensitivity of pancreatic cancer cells to interferon alpha (IFN-alpha) gene transfer, which induced a more prominent growth suppression and cell death in pancreatic cancer cells than in other types of cancers and normal cells. The IFN-alpha protein can exhibit both direct cytotoxicity and indirect immunological antitumour activity. Here, we dissected and examined the two mechanisms, taking advantage of the fact that IFN-alpha did not show any cross-species activity in its in vivo effect. When a human IFN-alpha adenovirus was injected into subcutaneous xenografts of human pancreatic cancer cells in nude mice, tumour growth was significantly suppressed due to cell death in an adenoviral dose-dependent manner. The IFN-alpha protein concentration was markedly increased in the injected subcutaneous tumour, but leakage of the potent cytokine into the systemic blood circulation was minimal. When a mouse IFN-alpha adenovirus was injected into the same subcutaneous tumour system, all mice showed significant tumour inhibition, an effect that was dependent on the indirect antitumour activities of IFN-alpha, notably a stimulation of natural killer cells. Moreover, in this case, tumour regression was observed not only for the injected subcutaneous tumours but also for the untreated tumours at distant sites. This study suggested that a local IFN-alpha gene therapy is a promising therapeutic strategy for pancreatic cancer, due to its dual mechanisms of antitumour activities and lack of significant toxicity.

Adenovirus-mediated gene transfer of interferon alpha improves dimethylnitrosamine-induced liver cirrhosis in rat model.

Several lines of evidence suggest that interferon (IFN)-alpha is effective in suppression of liver cirrhosis (LC) as well as hepatitis C virus (HCV) infection, which is a major cause of LC in Japan. However, IFN-alpha often causes systemic toxicity such as flu-like symptoms, which precludes the IFN-alpha dose escalation required for clinical efficacy. Since IFN-alpha is rapidly degraded in the blood circulation, only a small amount of subcutaneously injected IFN-alpha protein can reach the target organ, the liver. It is expected that on-site IFN-alpha production in the liver overcomes the limitation of the conventional parenteral IFN-alpha administration. An adenovirus vector expressing the rat IFN-alpha gene (AxCA-rIFN) was injected intravenously into rats with dimethylnitrosamine-induced LC. While the subcutaneous IFN-alpha protein injection led to a transient elevation of the cytokine both in the liver and serum, the vector-mediated IFN-alpha gene transduction induced a significant amount of IFN-alpha detected in the liver but not in the serum. The injection of AxCA-rIFN prevented the progression of the rat LC, and improved the survival rate of the treated rats. Although no significant toxicity was noted in the animals, we showed that IFN-alpha gene expression in the liver can be efficiently downregulated by the Cre/loxP-mediated shut-off system, in case the IFN-alpha overdose becomes a problem. The study suggested for the first time the advantage and feasibility of IFN-alpha gene therapy for LC.

Suppression of colorectal cancer growth using an adenovirus vector expressing an antisense K-ras RNA.

In human colorectal cancer, K-ras point mutations occur in approximately 40-50% of the cases, a frequency second only to pancreatic cancer (80-90%). Unlike pancreatic and lung cancers, however, the tumor-suppressive effect of antisense K-ras RNA expression has not been examined for colorectal cancers. A recombinant adenovirus vector expressing an antisense or sense K-ras gene fragment (AxCA-AS-K-ras or AxCA-S-K-ras) was first transduced into seven human colorectal cancer cell lines. Stable expression of antisense or sense K-ras RNA was detected by RNA blot analysis. Western blot analysis confirmed a reduction of up to 25% of K-ras-specific p21 protein in the antisense K-ras-transduced HCT-15 cells. In contrast to our previous findings on pancreatic cancer, the status of K-ras point mutations was not correlated with the growth-suppressive effect of the antisense K-ras vector: both the K-ras-mutation-positive and -negative colorectal cancer cell lines were suppressed for their growth in vitro. There was no growth-inhibitory effect on normal cells such as hepatocytes. Next, to test the efficacy in vivo, HCT-15 cells were inoculated subcutaneously into the left flank of SCID mice, and AxCA-AS-K-ras was injected intratumorally three times after the tumor mass was established. The infection of AxCA-AS-K-ras, but not the control AxCA-S-K-ras, significantly suppressed the growth of the HCT-15 subcutaneous tumor. This study shows that the adenovirus-mediated in vivo gene transfer of the antisense K-ras construct may be a useful therapeutic strategy for colorectal cancer.

Identification of genes showing differential expression in antisense K-ras-transduced pancreatic cancer cells with suppressed tumorigenicity.

K-ras point mutation occurs in >80% of pancreatic cancer. We reported previously that the transduction of an antisense K-ras RNA expression vector suppressed the growth of pancreatic cancer cells with K-ras point mutations in vitro and in vivo. The RNA differential display method (DD) was used to compare the mRNA expression profile of the pancreatic cancer cell line AsPC-1 and that of the antisense K-ras-transduced, growth-retarded AsPC-1 cells. cDNA fragments were isolated from 20 bands on the DD gel, and their differential expression between the two cell lines was confirmed. A sequence analysis revealed that all of the 11 clones up-regulated in the antisense-transduced cells were mitochondrial genes. The other nine cDNA clones that were down-regulated in the antisense-transduced AsPC-1 cells included an oncogene PTI-1 (prostate tumor inducing gene-1), matrix metalloproteinase (MMP)-7, the beta3 chain of laminin-5, lysosome-associated membrane protein-2, the H chain of apoferritin, ribosomal protein S6, proteasome subunit XAPC7, and two cDNA fragments with no homology to the GenBank database. In addition to the AsPC-1 cells, reverse transcription-PCR analysis on surgical specimens of pancreatic cancer revealed that the PTI-1 and MMP-7 genes were overexpressed in three and four cases, respectively, of five cases examined. This method offers a unique opportunity to identify a set of genes that may be modulated by K-ras activation, at least in a subset of the pancreatic cancer. The information on such genes may facilitate our understanding of the spectrum of the functional genetic changes in pancreatic cancer.

[Peptide hormone].

Clinical studies have confirmed the efficacy of peptide hormone as a routine tumor marker in the diagnosis, monitoring, and assessment of the prognosis of patients with endocrine tumors. However, in a large number of cancer patients, including those with ectopic hormone-producing tumors, the peptide hormone has limited clinical use as a routine tumor marker because of its poor specificity for tumor tissue and its low concentration in the patient sera. A new analysis product, ProGRP, is a specific and reliable serum tumor marker for small cell lung carcinoma (SCLC). It is useful not only in the evaluation of prognosis but also in the detection of SCLC at an early stage. On the other hand, recently, the RET proto-oncogene has been identified as a gene responsible for multiple endocrine neoplasia (MEN) syndromes: MEN 2A and MEN 2B. In the future, serum peptide hormone markers and molecular genetic markers may be useful in the diagnosis of endocrine tumors.

Immunoradiometric assay for prolactin in serum and tissue--comparison with radioimmunoassay.

Prolactin (PRL) concentrations in sera and tumors of patients with various pituitary tumors were measured by both immunoradiometric assay (IRMA) and radioimmunoassay (RIA). PRL concentrations in sera and tumor tissues measured by IRMA were well correlated with those measured by RIA. PRL concentrations in sera reflected those of tumors removed. This IRMA is a simple and useful method for PRL determination in serum and tissue.

[Estrogen receptors in human gastric, hepatocellular, and gallbladder carcinomas and normal liver tissues].

Steroid binding assay using the dextran coated charcoal (DCC) method was applied to human tissues including tumors of the digestive organs, and the results were compared with those of enzymeimmunoassay (EIA) and immunocytochemical assay (ICA) with monoclonal antibody against human estrogen receptor of MCF-7 breast cancer cells. Using the DCC method, estrogen receptor activity was detected in 6 of 26 cases (23.1%) with gastric carcinoma, 3 of 16 hepatocellular carcinoma cases (18.8%), 1 of 3 gallbladder carcinoma cases (33.3%), and both of the 2 cases (100%) with normal liver tissue. However, using EIA, no ER activity was detected in any case. Moreover, ER positive cells were not found by immunohistochemical staining in the gastric carcinoma cases or in normal liver tissue, both of which showed ER activity by the DCC method. These results suggest that the estrogen receptor like material exists in cytosol of the human digestive tumors and normal liver tissue, but that the specificity of the antibodies against estrogen receptor molecules in these tumors may be different from that of the breast tumors.

Characterization of "big big prolactin" in serum and tumor extract in patients with PRL-secreting tumor.

Molecular size heterogeneity of immunoreactive prolactin (PRL) in sera of pregnant women and patients with PRL-secreting pituitary tumor as well as pituitary tumor extract was investigated. "Big big PRL" in the vicinity of the void volume was identified by PRL radioimmunoassay. The ratio of "big big PRL" to "little PRL" was almost constant in normal pregnant women, while the proportion varied from case to case in patients with pituitary tumor. The ratio of "big big PRL" to "little PRL" was significantly (P less than 0.01) larger in these patients (43.3 +/- 9.8%, mean +/- SE) than in pregnant women (13.0 +/- 0.77%). Neither radiation therapy nor thyrotropin releasing hormone (TRH) stimulation significantly affected the ratio of "big big PRL" to "little PRL", while it decreased from 20.5% to 9.5% one month after tumor resection. The elution patterns of the normal human pituitary extract and the tumor extract obtained from patients with PRL-secreting pituitary tumor revealed peaks in identical positions, although the ratio of "big big PRL" to "little PRL" in the tumor extract (38.2 +/- 3.6%) was significantly (P less than 0.01) larger than that in normal human pituitary extract (5.8 +/- 2.8%). These results indicate that "big big PRL" is present in sera of pregnant women and patients with PRL-secreting pituitary tumor, normal pituitary extract and tumor extract. It is also suggested that the increased circulating "big big PRL" observed in patients with PRL-secreting tumor may originate in tumor tissue.

[Enzyme immunoassay and radioimmunoassay using monoclonal antibody for the determination of estrogen receptors in human breast cancer].

Enzymeimmunoassay (EIA) with monoclonal antibody against human estrogen receptor (ER) from MCF-7 breast cancer cells and radioimmunoassay (RIA) with monoclonal antibody against ER D-5 antigen from human myometrium were applied to human breast tumor, and the results were compared to those of steroid binding assay using the dextran coated charcoal (DCC) method. The rates of coincidence of positivity and negativity of ER between the DCC method and EIA or RIA in 30 human breast tumors were 96.7% (29/30) and 86.7% (26/30), respectively. A highly significant positive correlation was observed between ER values obtained by the DCC method and those by EIA (r = 0.87, P less than 0.001). On the other hand, the correlation between ER values obtained by the DCC method and those by RIA was less significant (r = 0.41, P less than 0.05). The discrepancy between these ER values may be due to the difference of the monoclonal antibody used in each new assay. The above results indicate that EIA and RIA with monoclonal antibody for human ER are useful for clinical use, although the reason for the partial discrepancy of the data between the DCC method and RIA remains to be elucidated.

The presence of macromolecular prolactin in serum of pregnant women and patients with pituitary adenoma.

The heterogeneity of immunoreactive Prolactin (IR-PRL) in serum of pregnant women and patients with pituitary adenoma were studied by Sephadex G-100 gel chromatography. The major IR-PRL peaks in serum of pregnant women were eluted at the position corresponding to that of 125I-PRL and the small amount of IR-PRL peaks were eluted near the void volume (peak 1) and between the void volume and 125I-PRL (peak 2). On the other hand, the proportion of peak 1 and peak 2 to total immunoreactivity was significantly increased in serum of patients with pituitary adenoma compared to those in serum of pregnant women. The rechromatographic studies under conditions of protein denaturation and sulfide cleavage on Sepharose CL-6B column revealed that the peak 1 of the pregnant women and patients with pituitary adenoma were eluted at the position between 125I-AFP and ovalbumine and the molecular weight was estimated to be about 54,000. This macromolecular PRL was also bound specifically to Sepharose coupled with anti-PRL, indicating that this macromolecule contained a sequence of PRL in its structure.