Evolutionary emergence and maintenance of horizontally transmitted mutualism that do not rely on the supply of standing variation in symbiont quality.

Mutualism based on reciprocal exchange of costly services must avoid exploitation by 'free-rides'. Accordingly, hosts discriminate against free-riding symbionts in many mutualistic relationships. However, as the selective advantage of discriminators comes from the presence of variability in symbiont quality that they eliminate, discrimination and thus mutualism have been considered to be maintained with exogenous supply of free-riders. In this study, we tried to resolve the 'paradoxical' co-evolution of discrimination by hosts and cooperation by symbionts, by comparing two different types of discrimination: 'one-shot' discrimination, where a host does not reacquire new symbionts after evicting free-riders, and 'resampling' discrimination, where a host does from the environment. Our study shows that this apparently minor difference in discrimination types leads to qualitatively different evolutionary outcomes. First, although it has been usually considered that the benefit of discriminators is derived from the variability of symbiont quality, the benefit of a certain type of discriminators (e.g. one-shot discrimination) is proportional to the frequency of free-riders, which is in stark contrast to the case of resampling discrimination. As a result, one-shot discriminators can invade the free-rider/nondiscriminator population, even if standing variation for symbiont quality is absent. Second, our one-shot discriminators can also be maintained without exogenous supply of free-riders and hence is free from the paradox of discrimination. Therefore, our result indicates that the paradox is not a common feature of evolution of discrimination but is a problem of specific types of discrimination.

Stability of cervical esophagogastrostomy via hand-sewn anastomosis after esophagectomy for esophageal cancer.

The aim of the present study is to evaluate the outcome of hand-sewn esophagogastric anastomosis during radical esophagectomy for esophageal cancer. The outcomes of 467 consecutive esophageal cancer patients who underwent cervical esophagogastric anastomosis using interrupted and double-layered sutures after radical esophagectomy via right thoracotomy or thoracoscopic surgery were retrospectively reviewed. Anastomotic leakage, including conduit necrosis, occurred in 11 of 467 patients (2.4%); 7 of 11 (63.6%) cases experienced only minor leakage, whereas the other four (36.4%) patients had major leakage that required surgical or radiologic intervention, including two patients of conduit necrosis. Anastomotic leakages were more frequently observed after retrosternal reconstruction compared with the posterior mediastinal route (P < 0.0001). The median time to healing of leakage was 40 days (range: 14-97 days). Two patients (2/467, 0.4%) died in the hospital due to sepsis caused by the leakage and conduit necrosis. Twelve patients (2.6%) developed anastomotic stenosis, which was improved by dilatation in all patients. Hand-sewn cervical esophagogastric anastomosis is a stable and highly safe method of radical esophagectomy for esophageal cancer.

Cultivation of rice for animal feed with circulated irrigation of treated municipal wastewater for enhanced nitrogen removal: comparison of cultivation systems feeding irrigation water upward and downward.

To achieve enhanced nitrogen removal, we modified a cultivation system with circulated irrigation of treated municipal wastewater by using rice for animal feed instead of human consumption. The performance of this modified system was evaluated through a bench-scale experiment by comparing the direction of circulated irrigation (i.e. passing through paddy soil upward and downward). The modified system achieved more than three times higher nitrogen removal (3.2 g) than the system in which rice for human consumption was cultivated. The removal efficiency was higher than 99.5%, regardless of the direction of circulated irrigation. Nitrogen in the treated municipal wastewater was adsorbed by the rice plant in this cultivation system as effectively as chemical fertilizer used in normal paddy fields. Circulated irrigation increased the nitrogen released to the atmosphere, probably due to enhanced denitrification. Neither the circulation of irrigation water nor its direction affected the growth of the rice plant and the yield and quality of harvested rice. The yield of rice harvested in this system did not reach the target value in normal paddy fields. To increase this yield, a larger amount of treated wastewater should be applied to the system, considering the significant amount of nitrogen released to the atmosphere.

Characteristic genes in luminal subtype breast tumors with CD44+CD24-/low gene expression signature.

OBJECTIVE: Breast cancer cells with CD44+CD24-/low gene expression signature have been suggested to have stem cell-like tumor-initiating properties. The purpose of this study is to clarify the gene expression profiling of cells with CD44+CD24-/low gene expression signature in the luminal subtype. METHODS: Laser capture microdissection was used to select the isolation of cancer cells in 35 frozen tissues of breast cancer, and RNA extracted from these cells was examined by real-time RT-PCR to quantify CD44 and CD24 expressions. Human stem cell RT(2) Profiler PCR Array was used for gene expression analysis in the groups of CD44+CD24-/low and CD44+CD24+ gene expression signature. RESULTS: Thirty-five tumors were divided into 3 groups. Group A was composed of the CD44+CD24-/low type, in which the ratio of CD44/CD24 was >10.0. Group B was composed of the CD44+CD24+ type, in which the ratio was >0.1 and ≤10.0. In group C, composed of the CD44-/lowCD24+ type, the ratio was <0.1. The number of tumors in groups A, B, and C were 5, 28, and 2, respectively. Regarding the correlation of CD44/CD24 status with tumor characteristics, the tumors of group A were significantly associated with axillary lymph node metastasis compared with those of group B (p = 0.033). There were no significant differences in tumor size, nuclear grade, or HER2 status between the two groups. According to signaling pathways, the number of expression genes for the Notch pathway in group A was significantly greater than in group B (p = 0.028). Overexpressed genes for ALDH1 (p = 0.021) and SOX2 (p = 0.018) were noted in group A compared to group B. CONCLUSION: This study suggests that the Notch pathway may be an important signaling pathway in luminal subtype with CD44+CD24-/low gene expression signature. In addition, either ALDH1 or SOX2 may be a candidate marker for cancer stem cells in luminal subtype breast cancer.

Identification of lactose ureide, a urea derivative of lactose, in milk and milk products.

With the widespread consumption of milk, the complete characterization of the constituents of milk and milk products is important in terms of functionality and safety. In this study, a novel nonreducing carbohydrate was separated from powdered skim milk and was identified using electron spray ionization-mass spectrometry (m/z 385.1[M + H(+)]), ¹H, ¹³C, ¹H¹H-correlation spectroscopy, and heteronuclear single quantum-nuclear magnetic resonance spectra. The carbohydrate was identified as a lactose derivative of urea, N-carbamoyl-o-ß-D-galactopyranosyl-(1-4)-D-glucopyranosylamine (lactose ureide, LU). For the HPLC analysis of LU in milk and milk products, benzoylated LU, hepta-o-benzoyl lactose ureide (melting point 137-139°C; m/z 1,113 [M + H⁺]; wavelength of maximum absorption, λ(max), 229 nm; molar extinction coefficient, ε, 8.1037 × 107), was used as a standard. The crude nonreducing carbohydrate fraction from raw milk, thermally processed milk, and milk products such as powdered milks were directly benzoylated and subjected to HPLC analysis using an octadecylsilyl column to determine the quantity of LU. The content of LU in 10% solutions of powdered skim milk and powdered infant formula (5.0±1.1 and 4.9±1.5 mg/L, respectively) were almost 3-fold higher than that of UHT milk (1.6±0.5 mg/L) and higher than that of low-temperature, long-time-processed (pasteurized at 65°C for 30 min) milk (1.2±0.3 mg/L) and the fresh raw milk sample (0.3±0.1 mg/L). A time-course of the LU content in raw milk during heating at 110°C revealed that LU increased with time. From these results, it is likely that LU is formed by the Maillard-type reaction between the lactose and urea in milk and milk products. Because the concentration of LU in milk increased with the degree of processing heat treatment, it could serve as an indicator of the thermal deterioration of milk. Although it is known that the human intestine is unable to digest LU, the gastrointestinal bacteria in human subjects are able to digest and utilize urea nitrogen in formation of essential amino acids that are available to the host human. These findings suggest that LU in milk might have a functional role in human health.

Minimally invasive surgical enucleation for esophageal leiomyoma: report of seven cases.

Benign esophageal tumor is a rare entity, with leiomyoma being the most common lesion. We present our experience with enucleation of esophageal leiomyomas using a minimally invasive approach. Between March 1998 and June 2008, seven patients with esophageal leiomyoma underwent right thoracosopic enucleation (n=4) or laparoscopic transhiatal enucleation (n=3). A Dor (n=2) or Toupet fundoplication (n=1) were added for laparoscopic procedure. The mean tumor size was 3.9 cm (range, 1.5-5.5 cm). Tumor locations were upper (n=2), middle (n=1), and lower (n=4) thirds of the esophagus. No major morbidities including postoperative leakage or mortalities occurred. At a mean follow-up period of 60.1 months (range, 14-260 months), no evidence of recurrences were observed. Thoracoscopic and laparoscopic transhiatal enucleation for esophageal leiomyomas is a safe and feasible procedure. The optimal approaches should be tailored based on the location and size of the tumor.

Laparoscopic Heller myotomy with Dor fundoplication for achalasia: long-term outcomes and effect on chest pain.

The aim of the present study was to evaluate the long-term outcomes of laparoscopic Heller myotomy with Dor fundoplication (LHD) and its effect on chest pain. Between June 1995 and August 2009, a total of 35 patients with achalasia underwent an LHD. The symptom scores were calculated by combining the frequency and the severity. Pre- and postoperative evaluations included symptom score, radiology, manometry, and 24-hour pH manometry. Median total symptom score was significantly lower than the preoperative score (19 vs 4, P < 0.001) at a median follow-up of 94 months. Among the 35 patients, 18 (51%) had chest pain. The frequency of chest pain was similar for the pre- and postoperative scores, but the severity tended to be less. Median esophageal diameter (5.4 cm vs 3.5 cm, P < 0.001) and lower esophageal sphincter pressure (41 mmHg vs 8.9 mmHg, P < 0.001) were significantly reduced after surgery. Median age, duration of symptoms, esophageal diameter, and lower esophageal sphincter pressure were similar between patients with and without chest pain prior to surgery. No significant differences were observed between the two groups in terms of amplitude, duration, and frequency of contractions from the findings of postoperative 24-hour esophageal manometry. Chest pain resolved in three patients (17%) and improved in seven patients (39%) after surgery. LHD can durably relieve achalasic symptoms of both dysphagia and regurgitation, and it can be considered the surgical procedure of choice. However, achalasic chest pain does not always seem to be related with patient characteristics and manometric findings.

Knowledge of Cervical Cancer and Human Papillomavirus among Japanese Women.

BACKGROUND: The combination of human papillomavirus (HPV) vaccination and cervical cancer tests are globally recommended. Although knowledge regarding cervical cancer and HPV and experience of HPV vaccination are reportedly closely associated, the associations between knowledge and frequency of cervical cancer testing are unclear. METHODS: We conducted a questionnaire survey regarding the knowledge of cervical cancer and HPV and experience of HPV vaccination and frequency of cervical cancer testing including cervical cytology and HPV testing. RESULTS: Among 99 women who visited Tsuruha Festa, most of the 77 non-medical workers who received information on cervical cancer and HPV through the Internet were not more likely to have knowledge about cervical cancer and HPV than were 12 medical workers who had gotten information in vocational school or university curriculum. The rates of HPV vaccination, cervical cytology, and HPV testing were 4.0%, 14.1%, and 4.0%, respectively, among participants and did not differ significantly according to participant job and age. Knowledge about cervical cancer and HPV was associated with experience of HPV vaccination and frequency of cervical cytology and was not associated with frequency of HPV testing. CONCLUSIONS: We observed insufficient knowledge about cervical cancer and HPV among non-medical workers as well as low HPV vaccination, cervical cytology, and HPV testing rates, and knowledge about cervical cancer and HPV to which frequency of cervical cancer testing were partially related. Therefore, the government should take measures to enhance public awareness about cervical cancer and HPV through social media such as the Internet.
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Orthotopic, but reversed implantation of the liver allograft in situs inversus totalis-a simple new approach to a difficult problem.

Situs inversus totalis is a rare congenital anomaly in which the heart and abdominal organs are oriented in a mirror image of normal. It provides a unique challenge as there is no established technique for liver transplantation in these patients. Employing two major alterations from our standard technique, a liver was transplanted in the left subphrenic space of a patient with situs inversus totalis. First, the liver was flipped 180 degrees from right to left (facing backward). Second, a reversed cavaplasty (anterior, not posterior, donor suprahepatic caval incision) was performed. Otherwise, it was standard, with end-to-end anastomoses of the portal vein, hepatic artery and bile duct. Three years after the entirely uneventful transplant, the recipient continues to enjoy the benefits of a normally functioning liver. The described technique prevented torsion, kinking and tension on the anastomosed structures by allowing the liver to sit naturally in an anatomical position in the left hepatic fossa. As it required no special measurements or maneuvers, the technique was easy to execute and required no donor liver size restrictions. This novel technique, with a reversed cavaplasty and a 180 degrees right-to-left flip of the liver into a left-sided hepatic fossa, may be ideal for situs inversus totalis.

Ten-year experience of totally laparoscopic liver resection in a single institution.

BACKGROUND: Recent developments in liver surgery include the introduction of laparoscopic liver resection. The aim of the present study was to review a single institution's 10-year experience of totally laparoscopic liver resection (TLLR). METHODS: Between May 1997 and April 2008, 82 patients underwent TLLR for hepatocellular carcinoma (HCC) (37 patients), liver metastases (39) and benign liver lesions (six). Operations included 69 laparoscopic wedge resections, 11 laparoscopic left lateral sectionectomies and two thoracoscopic wedge resections. Nine patients underwent simultaneous laparoscopic resection of colorectal primary cancer and synchronous liver metastases. RESULTS: Median operating time was 177 (range 70-430) min and blood loss 64 (range 1-917) ml. Median tumour size and surgical margin were 25 (range 15-85) and 6 (range 0-40) mm respectively. One procedure was converted to a laparoscopically assisted hepatectomy. Three patients developed complications. Median postoperative stay was 9 (range 3-37) days. The overall 5-year survival rate after surgery for HCC and colorectal metastases was 53 and 64 per cent respectively. CONCLUSION: TLLR can be performed safely for a variety of primary and secondary liver tumours, and seems to offer at least short-term benefits in selected patients.

Induced expression of the glucocorticoid receptor in the rat intermediate pituitary lobe.

Synthesis and release of pro-opiomelanocortin-derived peptides are under differential regulation in the anterior and intermediate lobes of the pituitary. Glucocorticoids inhibit synthesis of pro-opiomelanocortin-related peptides in the anterior lobe but not in the intermediate lobe. These two lobes are also characterized by differences in neural innervation and blood flow, both of which may represent routes of access for regulatory factors (the intermediate lobe is avascular). Immunoreactive glucocorticoid receptor, which can be demonstrated in many tissues, is absent from the intermediate lobe. Immunocytochemistry was used to demonstrate the presence of immunoreactive glucocorticoid receptor in the intermediate lobe after pituitary stalk transection, neurointermediate lobe grafts to kidney capsule, or monolayer culture of neurointermediate pituitary cells. This appearance of the glucocorticoid receptor is presumably a consequence of removal of intermediate pituitary cells from neural influences that may be responsible for inhibiting their expression under normal conditions in vivo.

The localization of proteins encoded by CRYM, KIAA1199, UBA52, COL9A3, and COL9A1, genes highly expressed in the cochlea.

Genes that are highly expressed in the inner ear, as revealed by cDNA microarray analysis, may have a crucial functional role there. Those that are expressed specifically in auditory tissues are likely to be good candidates to screen for genetic alterations in patients with deafness, and several genes have been successfully identified as responsible for hereditary hearing loss. To understand the detailed mechanisms of the hearing loss caused by the mutations in these genes, the present study examined the immunocytochemical localization of the proteins encoded by Crym, KIAA1199 homolog, Uba52, Col9a3, and Col9a1 in the cochlea of rats and mice. Confocal microscopic immunocytochemistry was performed on cryostat sections. Ultrastructurally, postembedding immunogold cytochemistry was applied using Lowicryl sections. Crym protein was predominantly distributed in the fibrocytes in the spiral ligament, as well as the stria vascularis in rats. KIAA1199 protein homolog was localized in various supporting cells, including inner phalangeal, border, inner and outer pillar, and Deiters' cells. Uba52 protein was restrictedly localized within the surface of the marginal cells of the stria vascularis. Collagen type IX was found within the tectorial membrane as well as fibrocytes in the spiral ligament. The present results showed cell-specific localization of the encoded proteins of these highly expressed genes, indicating that the coordinated actions of various molecules distributed in different parts of the cochlea are essential for maintenance of auditory processing in the cochlea.

Skewed X chromosome inactivation failed to explain the normal phenotype of a carrier female with MECP2 mutation resulting in Rett syndrome.

Mutations in the X-linked MECP2 gene cause Rett syndrome, a neurodevelopmental disorder that exclusively affects girls. Females with the MECP2 mutations exhibit a broad spectrum of clinical presentations ranging from classical Rett syndrome to asymptomatic carriers, which can be explained by differences in X chromosome inactivation (XCI). Here, we report a family with a girl with Rett syndrome in whom a novel missense mutation in the MECP2 gene was transmitted through the maternal germ line. The carrier mother was asymptomatic and presented non-random XCI in the peripheral blood cells, which resulted in the X chromosome harboring the mutant allele that was predominantly active. Thus, the presence of non-random XCI in the peripheral blood cells did not provide an explanation for the normal phenotype of the carrier mother. This result suggests that mechanisms other than XCI may contribute to the phenotypic heterogeneity associated with MECP2 mutations.

Placental corticotropin-releasing hormone may be a stimulator of maternal pituitary adrenocorticotropic hormone secretion in humans.

To clarify the physiological role of placental corticotropin-releasing hormone (CRH), we measured plasma CRH, ACTH, and cortisol throughout pregnancy. Cerebrospinal fluid (CSF) CRH levels and ACTH responsiveness to synthetic CRH were also quantified in pregnant and nonpregnant women. Maternal plasma CRH levels, which increased progressively during pregnancy, correlated well with both ACTH and cortisol in early labor, delivery, and postpartum samples, and also with cortisol levels in samples before labor. CSF CRH levels in term pregnant women did not differ from those of nonpregnant women. CRH infusion that attained similar plasma CRH levels to those found in late pregnancy elicited significant ACTH release in vivo and regular CRH test provoked normal ACTH response during early pregnancy but no response during late pregnancy. We concluded that: (a) maternal pituitary-adrenal axis correlates well with plasma CRH levels, which are high enough to provoke ACTH release from maternal pituitary; (b) hypothalamic CRH secretion in term pregnant women is not exaggerated; and (c) maternal pituitary is responsive to synthetic CRH in early but not late pregnancy, suggesting that maternal pituitary-adrenal axis is already activated by high circulating CRH. Placental CRH may be an important stimulator of the maternal pituitary-adrenal axis during pregnancy.

Inhibition of osteolytic bone metastasis of breast cancer by combined treatment with the bisphosphonate ibandronate and tissue inhibitor of the matrix metalloproteinase-2.

Multiple steps are involved in the metastasis of cancer cells from primary sites to distant organs. These steps should be considered in the design of pharmacologic approaches to prevent or inhibit the metastatic process. In the present study, we have compared the effects of inhibiting several steps involved in the bone metastatic process individually with inhibition of both together. The steps we chose were matrix metalloproteinase (MMP) secretion, likely involved in tumor cell invasion, and osteoclastic bone resorption, the final step in the process. We used an experimental model in which inoculation of human estrogen-independent breast cancer MDA-231 cells into the left cardiac ventricle of female nude mice causes osteolytic lesions in bone. To inhibit cancer invasiveness, the tissue inhibitor of the MMP-2 (TIMP-2), which is a natural inhibitor of MMPs, was overexpressed in MDA-231 cells. To inhibit bone resorption, a potent bisphosphonate, ibandronate (4 microg/mouse) was daily administered subcutaneously. Nude mice received either; (a) nontransfected MDA-231 cells; (b) nontransfected MDA231 cells and ibandronate; (c) TIMP-2-transfected MDA-231 cells; or (d) TIMP-2-transfected MDA-231 cells and ibandronate. In mice from group a, radiographs revealed multiple osteolytic lesions. However, in mice from group b or group c, osteolytic lesions were markedly decreased. Of particular note, in animals from group d receiving both ibandronate and TIMP-2-transfected MDA-231 cells, there were no radiologically detectable osteolytic lesions. Survival rate was increased in mice of groups c and d. There was no difference in local enlargement in the mammary fat pad between nontransfected and TIMP-2-transfected MDA-231 cells. These results suggest that inhibition of both MMPs and osteoclastic bone resorption are more efficacious treatment for prevention of osteolytic lesions than either alone, and suggest that when therapies are designed based on the uniqueness of the bone microenvironment and combined with several common steps in the metastatic process, osteolytic bone metastases can be more efficiently and selectively inhibited.

Different roles of nitric oxide synthase-1 and -2 between herpetic and postherpetic allodynia in mice.

We investigated using the mice role of nitric oxide synthase (NOS) in the spinal dorsal horn in herpetic and postherpetic pain, especially allodynia, which was induced by transdermal inoculation of the hind paw with herpes simplex virus type-1 (HSV-1). The virus inoculation induced NOS2 expression in the lumbar dorsal horn of mice with herpetic allodynia, but not postherpetic allodynia. There were no substantial alternations in the expression level of NOS1 at the herpetic and postherpetic stages. Herpetic allodynia was significantly inhibited by i.p. administration of the selective NOS2 inhibitor S-methylisothiourea, but not the selective NOS1 inhibitor 7-nitroindazole. NOS2 expression was observed around HSV-1 antigen-immunoreactive cells. On the other hand, postherpetic allodynia was significantly inhibited by i.p. administration of 7-nitroindazole, but not S-methylisothiourea. The activity of reduced nicotinamide adenine dinucleotide phosphate diaphorase, an index of NOS1 activity, significantly increased in the laminae I and II of the lumbar dorsal horn of mice with postherpetic allodynia, but not mice without postherpetic allodynia. The expression level of NOS1 mRNA in the dorsal root ganglia was similar between mice with and without postherpetic allodynia. The results suggest that herpetic and postherpetic allodynia is mediated by nitric oxide in the dorsal horn and that NOS2 and NOS1 are responsible for herpetic and postherpetic allodynia, respectively. It may be worth testing the effects of NOS2 and NOS1 inhibitors on herpetic pain and postherpetic neuralgia in human subjects, respectively.

T cell receptor-beta mRNA splicing: regulation of unusual splicing intermediates.

The expression of functional T cell receptor-beta (TCR-beta) transcripts requires the activation of programmed DNA rearrangement events. It is not clear whether other mechanisms dictate TCR-beta mRNA levels during thymic ontogeny. We examined the potential role of RNA splicing as a regulatory mechanism. As a model system, we used an immature T cell clone, SL12.4, that transcribes a fully rearranged TCR-beta gene but essentially lacks mature 1.3-kb TCR-beta transcripts in the cytoplasm. Abundant TCR-beta splicing intermediates accumulate in the nucleus of this cell clone. These splicing intermediates result from inefficient or inhibited excision of four of the five TCR-beta introns; the only intron that is efficiently spliced is the most 5' intron, IVSL. The focal point for the regulation appears to be IVS1C beta 1 and IVS2C beta 1, since unusual splicing intermediates that have cleaved the 5' splice site but not the 3' splice site of these two introns accumulate in vivo. The block in 3' splice site cleavage is of interest since sequence analysis reveals that these two introns possess canonical splice sites. A repressional mechanism involving a labile repressor protein may be responsible for the inhibition of RNA splicing since treatment of SL12.4 cells with the protein synthesis inhibitor cycloheximide reversibly induces a rapid and dramatic accumulation of fully spliced TCR-beta transcripts in the cytoplasm, concomitant with a decline in TCR-beta pre-mRNAs in the nucleus. This inducible system may be useful for future studies analyzing the underlying molecular mechanisms that regulate RNA splicing.

Water quality estimation in consideration of pollutant runoff and internal production in Lake Biwa, Japan.

Lake Biwa is the largest lake in Japan, and water quality in the lake is heterogeneous. Therefore, it is important for water quality management that spatial distribution of water quality in the lake should be clearly understood. The objectives of this study are to show a methodology and to develop a simulation system to calculate COD distribution in Lake Biwa taking internal COD production into consideration. This study also aims to examine transition of COD in the lake using the simulation system. In the simulation system, runoff loads of COD from the Lake Biwa basin are calculated by Macro Model for each tributary. The external COD concentration in 233 inshore meshes of the Lake Biwa water surface was calculated using the runoff loads. The internal COD was calculated using relationships among limiting nutrients, chlorophyll-a and COD. Then, the spatial distribution of water quality in Lake Biwa was calculated both for the external and internal COD by spline technique. Simulations using the system were implemented for 1986-1998, and a clear difference in characteristics between a drought year and a flood year was shown. In the result, it was shown that the simulation system developed here was available to calculate COD distribution in Lake Biwa, and that it had the possibility to explain the recent phenomenon of COD increase in the lake.

Tumor suppressive efficacy through augmentation of tumor-infiltrating immune cells by intratumoral injection of chemokine-expressing adenoviral vector.

Our goal in the present study was to evaluate antitumor effects and frequency of tumor-infiltrating immune cells upon intratumoral injection of RGD fiber-mutant adenoviral vector (AdRGD) encoding the chemokines CCL17, CCL19, CCL20, CCL21, CCL22, CCL27, XCL1, and CX3CL1. Among eight kinds of chemokine-expressing AdRGDs, AdRGD-CCL19 injection most efficiently induced infiltration of T cells into established B16BL6 tumor parenchyma, whereas most of these T cells were perforin-negative in immunohistochemical analysis. Additionally, the growth of AdRGD-CCL19-injected tumors decreased only slightly as well as that of other tumors treated with each chemokine-expressing AdRGD, which indicated that accumulation of naive T cells in tumor tissue does not effectively damage the tumor cells. Tumor-bearing mice, in which B16BL6-specific T cells were elicited by dendritic cell-based immunization, demonstrated that intratumoral injection of AdRGD-CCL17, -CCL22, or -CCL27 could considerably suppress tumor growth and attract activated T cells. On the other hand, AdRGD-CCL19-injection in the immunized mice showed slight increase of tumor-infiltrating T cells compared to treatment using control vector. Collectively, although AdRGD-mediated chemokine gene transduction into established tumors would be very useful for augmentation of tumor-infiltrating immune cells, a combinational treatment that can systemically induce tumor-specific effector T cells is necessary for satisfactory antitumor efficacy.