A universal and reliable assay for molecular sex identification of three-spined sticklebacks (Gasterosteus aculeatus).

In heterogametic species, biological differences between the two sexes are ubiquitous, and hence, errors in sex identification can be a significant source of noise and bias in studies where sex-related sources of variation are of interest or need to be controlled for. We developed and validated a universal multimarker assay for reliable sex identification of three-spined sticklebacks (Gasterosteus aculeatus). The assay makes use of genotype scores from three sex-linked loci and utilizes Bayesian probabilistic inference to identify sex of the genotyped individuals. The results, validated with 286 phenotypically sexed individuals from six populations of sticklebacks representing all major genetic lineages (cf. Pacific, Atlantic and Japan Sea), indicate that in contrast to commonly used single-marker-based sex identification assays, the developed multimarker assay should be 100% accurate. As the markers in the assay can be scored from agarose gels, it provides a quick and cost-efficient tool for universal sex identification of three-spined sticklebacks. The general principle of combining information from multiple markers to improve the reliability of sex identification is transferable and can be utilized to develop and validate similar assays for other species.

Transumbilical laparoscopic surgery using GelPort through an umbilical zigzag skin incision.

INTRODUCTION: We report herein a new method of transumbilical laparoscopic surgery using a GelPort through an umbilical zigzag skin incision. The method involves collaborating with plastic surgeons to ensure the procedure was minimally invasive. MATERIALS AND SURGICAL TECHNIQUE: After marking a zigzag skin incision in the umbilical region, the skin was incised along this line. Then, a GelPort double-ring wound retractor was inserted through the incision, which enlarged the diameter of the fascial opening to 6 cm. The Gelport was latched on the wound retractor ring, following the inflation of the pneumoperitoneum by CO (2). One or more additional ports were inserted as necessary. All operations were performed in the standard fashion. The specimen was easily extracted from the abdomen through the umbilical incision, and anastomosis was performed. Using the above method, we performed the following procedures: one total gastrectomy, one distal gastrectomy, three gastric local resections, five right hemicolectomies, two high anterior resections, three cholecystectomies, and seven transabdominal preperitoneal hernioplasties. All cases were accomplished without any complications using this method. The wounds of the umbilical region were almost "scarless" in all cases. DISCUSSION: We developed an umbilical zigzag skin incision technique to perform abdominal laparoscopic operations using a GelPort, with a minimal number of skin incisions. We consider that our method reduces the technical difficulties associated with laparoscopic surgery and maintains cosmesis.

Antitumor efficacy of oncolytic herpes simplex virus adsorbed onto antigen-specific lymphocytes.

Although several studies have reported that locally administering oncolytic viruses effectively targets malignancies, the efficacy of systemically administered oncolytic viruses is restricted. Recently, however, it was reported that systemic administration of oncolytic vesicular stomatitis virus adsorbed onto antigen-specific lymphocytes was effective against malignancies. We hypothesized that intravenously administering such virus might have significant potential in treatment of the malignant tumors. We adsorbed oncolytic herpes simplex virus-1 mutant R3616 onto lymphocytes harvested from mice with acquired antitumor immunity. We administered adsorbed R3616 to peritoneally disseminated tumors and analyzed the efficacy of this treatment. Mice administered adsorbed R3616 survived significantly longer than mice administered R3616 adsorbed onto non-specific lymphocytes, or mice administered either virus or tumor antigen-specific lymphocytes alone. In this context, herpes oncolytic virus is a promising treatment not only for primary lesions, but also for multiple metastasizing lesions. This treatment strategy may become one of the most effective methods for systemic virus delivery.

Impact of novel oncolytic virus HF10 on cellular components of the tumor microenviroment in patients with recurrent breast cancer.

Oncolytic viruses are a promising method of cancer therapy, even for advanced malignancies. HF10, a spontaneously mutated herpes simplex type 1, is a potent oncolytic agent. The interaction of oncolytic herpes viruses with the tumor microenvironment has not been well characterized. We injected HF10 into tumors of patients with recurrent breast carcinoma, and sought to determine its effects on the tumor microenvironment. Six patients with recurrent breast cancer were recruited to the study. Tumors were divided into two groups: saline-injected (control) and HF10-injected (treatment). We investigated several parameters including neovascularization (CD31) and tumor lymphocyte infiltration (CD8, CD4), determined by immunohistochemistry, and apoptosis, determined by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Median apoptotic cell count was lower in the treatment group (P=0.016). Angiogenesis was significantly higher in treatment group (P=0.032). Count of CD8-positive lymphocytes infiltrating the tumors was higher in the treatment group (P=0.008). We were unable to determine CD4-positive lymphocyte infiltration. An effective oncolytic viral agent must replicate efficiently in tumor cells, leading to higher viral counts, in order to aid viral penetration. HF10 seems to meet this criterion; furthermore, it induces potent antitumor immunity. The increase in angiogenesis may be due to either viral replication or the inflammatory response.

A phase I dose-escalation clinical trial of intraoperative direct intratumoral injection of HF10 oncolytic virus in non-resectable patients with advanced pancreatic cancer.

In 2005, we initiated a clinical trial that examined the efficacy of the oncolytic virus HF10 to treat pancreatic cancer. Pancreatic cancer continues to have a high mortality rate, despite multimodal treatments for patients, and new therapeutic methods are greatly needed. The current mainstream methods for cancer treatment include biological therapeutics such as trastuzumab (Herceptin) for breast cancer or erlotinib (Tarceva) for non-small cell lung cancer. Oncolytic virus therapy is a new and promising treatment strategy for cancer. Oncolytic viruses are novel biological therapeutics for advanced cancer that appear to have a wide spectrum of anticancer activity with minimal human toxicity. To examine the efficacy of oncolytic virus therapy for pancreatic cancer, we initiated pilot studies by injecting six patients with non-resectable pancreatic cancer with three doses of HF10. All patients were monitored for 30 days for local and systemic adverse effects and were not administered any other therapeutics during this period. There were no adverse side-effects, and we observed some therapeutic potential based on tumor marker levels, survival, pathological findings and diagnostic radiography. The tumors were classified as stable disease in three patients, partial response in one patient and progressive disease in two patients.

High susceptibility to severe infectious complications at reinduction chemotherapy in patients who relapse after stem cell transplantation.

Patients who relapse after stem cell transplantation (SCT) usually appear to be liable to severe infectious complications at reinduction chemotherapy compared to patients at the first induction therapy, though this is not statistically substantiated. The aim of this study was to analyze episodes of infectious complications during reinduction chemotherapy among patients who relapsed after SCT compared with those at the first induction chemotherapy. Between February 1988 and March 2004, 145 children received SCT, and 17 (12 with hematologic malignancies and 5 with solid tumors) were enrolled as eligible subjects for this study. Positive blood cultures (sepsis) were present in six patients exclusively at the reinduction therapy but none at the first induction (P = .009). Three of the six patients progressed to septic shock. Moreover, all patients positive for blood cultures were those with hematologic malignancy (P = .007), and every patient with septic shock had received allogenic transplantation. Our results showed that reinduction chemotherapy needs attention for severe infectious complications, particularly among patients with hematologic malignancies receiving allogenic transplantations. Possible immaturity of the reconstructed systemic immune system and/or insufficient recovery of mucosal protective functions in the patients after SCT are discussed in view of their high susceptibility to severe infectious complications.

Suitability of a US3-inactivated HSV mutant (L1BR1) as an oncolytic virus for pancreatic cancer therapy.

Recently, the use of oncolytic viruses against cancer has attracted considerable attention. We studied the potential of the US3 locus-deficient herpes simplex virus (HSV), L1BR1, for oncolytic virus therapy. Its high specificity and potency indicate that L1BR1 is a promising candidate as a new oncolytic virus against pancreatic cancer. Moreover, the virus exhibited the unique characteristic of increasing apoptosis when used in combination with anticancer drugs. We assessed the feasibility of using the US3 locus-deficient HSV named L1BR1 as a new replication-competent oncolytic virus for the treatment of pancreatic cancer. The US3 locus of HSV has been shown to be a key gene in producing a multifunctional protein kinase that inhibits apoptosis induced by viral infections, chemicals and ultraviolet (UV) light. L1BR1 has been reported to be more than 10 000-fold less virulent than the parental virus in mice. In this study, we examined the tumor specificity and oncolytic effect of this attenuated replication-competent virus, L1BR1, in pancreatic cancers derived from SW1990, Capan2 and Bxpc-3cells compared with the parent virus and other well-known oncolytic herpes viruses (R3616 and hrR3). We also studied the efficacy of L1BR1 for the induction of apoptosis as an attribute of this virus in combination with the anticancer drugs 5FU and cisplatin. The combined treatment of the pancreatic cancer cells with L1BR1 and these anticancer drugs enhanced apoptosis significantly. More importantly, L1BR1 showed the lowest replication capacity in normal human hepatocytes, but the highest tumor-reducing effect in vivo among the oncolytic herpes viruses tested. In addition, L1BR1 significantly increased the induction of apoptosis of cancer cells when treated in combination with anticancer drugs although the parental virus inhibited the induction of apoptosis. These results suggest that L1BR1 is promising as a new anticancer oncolytic virus.

Oncolytic virus therapy--foreword.

We are very pleased and proud to be able to publish this special issue of Current Cancer Drug Targets devoted to oncolytic virus therapy covering basic and clinical research on adenovirus, vaccinia virus, herpes virus, and Newcastle disease virus. In these papers, we welcome the world's top authorities in the field who have generously contributed their latest review articles for exclusive publication in this special issue. Moreover, this issue also includes a range of opinion from government drug organizations. Here we simply wish to bring together the newest knowledge and experience in the field of cutting-edge oncolytic virus therapy for researchers and every kind of cancer therapist. The Foreword presents a historical perspective on the development of oncolytic virus together with the encouraging results of recent clinical trials (e.g., H101 has been tested in clinical trial of nearly 250 patients and approved for human use by the Chinese FDA, while PV701 has been tried in over 110 patients, as described in our special issue).

Activity of a sperm-borne oocyte-activating factor in spermatozoa and spermatogenic cells from cynomolgus monkeys and its localization after oocyte activation.

It is widely accepted that mature mammalian oocytes are induced to resume meiosis by a sperm-borne oocyte-activating factor(s) (sperm factor, SF) immediately after normal fertilization or intracytoplasmic sperm injection. The SF is most likely a soluble factor that is localized within the cytoplasm of mature spermatozoa, but the exact stage at which it appears during spermatogenesis and its localization after oocyte activation is not fully understood, except in the mouse. First, we injected mature spermatozoa and spermatogenic cells from cynomolgus monkeys into mouse oocytes to assess their oocyte-activating capacity. More than 90% of mouse oocytes were activated after injection of monkey spermatozoa. Round spermatids and primary spermatocytes (late pachytene to diplotene) also activated oocytes (93% and 79%, respectively). Injection of monkey spermatozoa and spermatids induces intracellular Ca(2+) oscillations in a pattern similar to that seen following normal fertilization. Most spermatocytes did not produce typical intracellular Ca(2+) oscillations. Second, we transferred pronuclei or cytoplasts from mouse oocytes that had been activated by monkey spermatozoa or spermatids into intact mature mouse oocytes by electrofusion in order to examine the localization of the SF after pronuclear formation. Some of the SF was localized within the pronuclei, but some stayed in the ooplasm. This study demonstrated that spermatogenic cells of cynomolgus monkeys acquire oocyte-activating capacity at much earlier stages than those of mice, and that the monkey SF has a pronucleus-directing nature, although to a lesser extent than the mouse SF.

Prolonged bone marrow failure with monosomy 7 after engraftment failure following bone marrow transplantation.

A patient with acute myelogenous leukemia developed prolonged bone marrow failure along with the monosomy 7 chromosome abnormality. The patient had undergone bone marrow transplantation with CD34+ selection following induction failure. However, she then suffered engraftment failure and long-term pancytopenia. Her white blood cell count gradually increased with supportive therapy including granulocyte colony-stimulating factor (G-CSF), and chromosomal analysis of bone marrow cells revealed an abnormal karyotype. Thirty months after the bone marrow transplantation we observed monosomy 7 together with the existing chromosomal abnormality in the patient's bone marrow cells. It has been reported that some patients with idiopathic and posthepatitis aplastic anemia develop clonal disorders such as myelodysplastic syndrome/acute myelogenous leukemia with monosomy 7. The findings in our case suggest that the appearance of monosomy 7 in patients with aplastic anemia may be caused by prolonged low-level hematopoiesis, with or without G-CSF stimulation.

Temporal changes in allele frequency, genetic variation and inbreeding depression in small populations of the guppy, Poecilia reticulata.

We established three closed lines of N = 10 for the guppy Poecilia reticulata, to evaluate the relationships among temporal changes in allele frequency, genetic variation and inbreeding depression for a fitness-related trait in small populations. Genetic variation at the allozyme loci, expressed by the proportion of polymorphic loci, number of alleles per locus and heterozygosity, decreased somewhat in two closed lines but it increased in one closed line over six generations. Effective population size (Ne) at each generation was estimated from the standardized variance in the allele frequencies. The average Ne was 24.4, 10.3 and 10.0 in the three closed lines. The inbreeding coefficient calculated from the Ne increased to 0.186, 0.321 and 0.414, respectively. As an index of the amount of inbreeding depression, changes in salinity tolerance were examined, because this trait is strongly sensitive to inbreeding depression and decreases linearly with an increase in inbreeding coefficient. The mean value of the salinity tolerance significantly decreased to 82.5%, 71.7% and 67.6% in the three closed lines during the six generations, suggesting inbreeding depression for salinity tolerance. Although a significant correlation was not observed between the amount of inbreeding depression and the genetic variation, the amount of inbreeding depression correlated with the inbreeding coefficient calculated from Ne. The regression line indicated an 8.4% decrease in the mean per 10% increase in the inbreeding coefficient and was similar to that obtained directly from full-sib matings. These results indicate that the temporal changes in the allele frequencies can provide an estimation of the amount of inbreeding depression during successive generations in small populations.

Anti-alpha-fodrin autoantibody is an early diagnostic marker for childhood primary Sjögren's syndrome.

OBJECTIVE: alpha-fodrin is a recently identified autoantigen associated with adult primary Sjögren's syndrome (SS). We tested whether anti-alpha-fodrin antibody could also be used as a diagnostic marker for childhood SS. METHODS: We performed immunoblot analysis of sera from 7 patients with childhood primary SS using glutathione-S-transferase alpha-fodrin fusion protein as an antigen. RESULTS: Anti-alpha-fodrin antibody was detected in sera from all 7 patients with childhood primary SS, 2 of 4 with secondary SS, and one of 7 with systemic lupus erythematosus, but in no other healthy controls. CONCLUSION: The anti-alpha-fodrin autoantibody was detected before anti-SSA or SSB antibody became positive; thus anti-alpha-fodrin antibody could be a useful marker for the early diagnosis of SS.

The gamete fusion process is defective in eggs of Cd9-deficient mice.

The cell-surface molecule Cd9, a member of the transmembrane-4 superfamily, interacts with the integrin family and other membrane proteins. and is postulated to participate in cell migration and adhesion. Expression of Cd9 enhances membrane fusion between muscle cells and promotes viral infection in some cells. Fertilization also involves membrane fusion, between gametes. In mammals, the sperm binds to microvilli on the egg surface, and sperm-egg membrane fusion first occurs around the equatorial region of the sperm head12. The fused membrane is then disrupted, and the sperm nucleus as well as the cytoplasm is incorporated into the egg. Cd9 is expressed on the plasma membrane of the mouse egg, and an anti-Cd9 monoclonal antibody inhibits sperm-egg surface interactions. We generated Cd9 mice and found that homozygous mutant females were infertile. Sperm-egg binding was normal, but sperm-egg fusion was almost entirely inhibited in eggs from Cd9 females. Intracellular Ca2 oscillations, which signal fertilization, were absent in almost all mutant eggs; in rare cases, a response occurred after a long time period. In normal animals, Cd9 molecules were expressed on the egg microvilli and became densely concentrated at the sperm attachment site. Thus, our results show that Cd9 is important in the gamete fusion process at fertilization.

Neurological complications after stem cell transplantation in childhood.

We analyzed the incidence of neurological complications in 77 patients receiving stem cell transplantation (SCT), and 12 patients (15.8%) had the following symptoms: convulsions, intracranial hemorrhage, and leukoencephalopathy. Although statistically not significant, neurological complications were seen more frequently in patients after allogeneic transplantation, and in those with acute graft-versus-host disease (GVHD) exceeding grade II. The most significant risk factor for neurological complications was identified as unrelated donor allogenic transplantation (P = 0.016). Complications were categorized into three groups, based on time of onset and symptoms: (1) convulsions during the conditioning period, (2) intracranial hemorrhage during the period of granulocyte recovery, and (3) leukoencephalopathy at around 2 months after SCT. We propose awareness of the risks of neurological complications in each period after SCT so that immediate and effective treatment of patients can be instigated.

[Adverse effects of anti-thymocyte globulin/anti-lymphocyte globulin therapy].

This single-centre study evaluated the adverse effects of anti-thymocyte globulin (ATG) and anti-lymphocyte globulin (ALG) as used for the treatment of aplastic anemia and/or for conditioning regimens prior to stem cell transplantation. ATG/ALG was given to 29 patients a total of 37 times. The incidence of adverse effects was 62.1% (23/37), and fever was the most frequent adverse effect. Therapy was discontinued in only 4 patients (10.8%) due to severe adverse effects. Adverse effects occurred more frequently with ATG (rabbit-derived) than with ALG (horse-derived). Seven patients underwent 2 or 3 cycles of ATG/ALG therapy, for a combined total of 8 times; 6 of those patients (75% (6/8)) experienced adverse effects. Shorter intervals between repeated cycles of therapy appeared to heighten the risk of adverse reactions.

Spatiotemporal dynamics of the [Ca2+]i rise induced by microinjection of sperm extract into mouse eggs: preferential induction of a Ca2+ wave from the cortex mediated by the inositol 1,4,5-trisphosphate receptor.

Hamster sperm extract (SE) possessing Ca2+ oscillation-inducing activity was microinjected into the peripheral or central region of mouse eggs, and the first increase in intracellular Ca2+ concentration ([Ca2+]i), together with the spread of fluorescence-labeled SE in the ooplasm, was investigated by imaging with confocal microscopy. Injection into the periphery always induced a Ca2+ wave that started from the injection site after a delay of 5 to 30 s depending on the concentration of SE. The diluted SE caused a wave of two-step [Ca2+]i rises, which was always observed at fertilization. Injection into the center could induce a radial Ca2+ wave with relatively high dose of SE, but lower dose of SE caused a [Ca2+]i rise after a longer delay which was initiated synchronously over the ooplasm or was preceded in a peripheral area. Injection of diluted SE remarkably prolonged the delay time and reduced the rate of [Ca2+]i rise. The critical concentration of SE needed to induce [Ca2+]i rise was significantly lower in the periphery. These results indicate that the sensitivity to SE is higher in the cortex. SE-induced [Ca2+]i rises were blocked by an antibody against the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R). The cortex was substantially more sensitive to injected InsP3 induction of Ca2+ release than the center. It is suggested that the cortex of mouse eggs may involve a functionally specialized organization of InsP3Rs and Ca2+ pools in which a cytosolic sperm factor(s) could act upon sperm-egg fusion to cause Ca2+ release, leading to the Ca2+ wave at fertilization.

Leukoencephalopathy in childhood acute lymphoblastic leukemia with t(1;19).

To clarify the incidence of leukoencephalopathy in patients with t(1;19) and their clinical characteristics, we studied 239 acute lymphoblastic leukemia (ALL) cases. The 1;19 translocation was found in 20 (8.5%) of the 239 children with ALL. Leukoencephalopathy occurred in 2 (10%) patients with t(1;19) during the early first remission and in one case with t(1;19) at the time of central nervous system (CNS) relapse. Leukoencephalopathy was not found during the early first remission in patients lacking t(1;19), but did develop in 4 patients lacking t(1;19) at the time of CNS relapse. There were no differences in age, sex, leukocyte count, platelet count or serum lactate dehydrogenase level between t(1;19) patients with and without leukoencephalopathy. Our results suggest the incidence of leukoencephalopathy in patients with t(1;19) during the early first remission to be 10%, but we can not predict which patients will develop leukoencephalopathy.

Long survivors with Ki-1 lymphoma having t(2;5) (p23;q35). Does the presence or absence of t(2;5) influence the prognosis of patients with Ki-1 lymphoma?

We experienced three patients with CD30+ diffuse large cell lymphoma having chromosomal abnormalities. The first patient was an 8-year-old girl with bilateral cervical lymphadenopathy. A biopsy of a cervical lymph node revealed diffuse large cell lymphoma (stage III), positive for CD30 and a chromosomal abnormality, t(2;5). She attained a remission and is now in complete remission 108 months after diagnosis, despite frequent relapses. The second patient was a 13-year-old boy with right axillar and supraclavicular lymph-node adenopathy. A biopsy of a cervical lymph node revealed diffuse large cell lymphoma (stage III), positive for CD30 and a chromosomal abnormality, t(2;5). He attained remission and was in continuous first remission 112 months after diagnosis. The third patient was an 11-year-old boy with fever and bilateral cervical lymph node revealed diffuse large cell lymphoma (stage III), positive for CD30 and chromosomal abnormality without t(2;5). He showed a very aggressive clinical course. Only the patients with Ki-1 lymphoma having t(2;5) survived over 100 months from the diagnosis, despite the advanced stage of the disease. These findings and a review of the literature showed that the presence or absence of t(2;5) may influence the outcome of Ki-1 lymphoma.

Immunolocalization of Na+/K+-ATPase in branchial epithelium of chum salmon fry during seawater and freshwater acclimation

Immunolocalization of the -subunit of Na+/K+-ATPase was examined in the gill epithelium of chum salmon (Oncorhynchus keta) fry during acclimation to brackish water (25 salinity) and reintroduction to fresh water. In freshwater fish, strong immunoreactivity was associated with the large spherical cells located on the free surface of the primary lamellae, especially in those found at the base of the secondary lamellae, and with the large spherical cells located on the secondary lamellae.The large spherical cells located near the central venous sinus at the base of the secondary lamellae and in the interlamellar regions, however, showed little or no immunoreactivity. When freshwater fish were acclimated to brackish water, immunoreactivity developed in the large spherical cells near the central venous sinus concomitant with an increase in the hypo-osmoregulatory ability of the fish. In contrast, reintroduction from brackish water to fresh water caused the disappearance of the immunoreactivity in the large spherical cells near the central venous sinus and a reduction in hypo-osmoregulatory ability. During acclimation to brackish water and reintroduction to fresh water, the hypo-osmoregulatory ability of the fish did not correlate with the total number of large spherical cells located on the primary lamellae but was closely correlated with the number of large spherical cells showing strong immunoreactivity for Na+/K+-ATPase. We conjecture that these immunopositive large spherical cells are mature differentiated chloride cells, whereas the immunonegative large spherical cells are young developing chloride cells. The development of immunoreactivity for Na+/K+-ATPase in young chloride cells may be one of the most important factors in the development of hypo-osmoregulatory ability by chum salmon fry.

Relationships of salinity tolerance to immunolocalization of Na+,K(+)-ATPase in the gill epithelium during seawater and freshwater adaptation of the guppy, Poecilia reticulata.

The relationships of salinity tolerance to immunolocalization of Na+,K(+)-ATPase in the gill epithelium were examined during seawater and freshwater adaptation of the guppy. In fresh water, immunoreactivity for Na+,K(+)-ATPase appeared in two types of chloride cells, which are located on the primary lamellae of the gills. Immunoreactivity was strong in the chloride cells located at the base of the secondary lamellae and weak in the chloride cells located at the interlamellar region. During seawater adaptation, the strongly-immunoreactive chloride-cells increased in number and size while the weakly-immunoreactive chloride-cells decreased in number with an increase in salinity tolerance. In the fish of the seawater-adapted strain, on the other hand, most of the chloride cells were located at the base of the secondary lamellae and showed strong immunoreactivity. During freshwater adaptation, the strongly-immunoreactive chloride-cells decreased in number and size while the weakly-immunoreactive chloride-cells increased in number with a decrease in salinity tolerance. A positive correlation was observed between the salinity tolerance and the occupying area of the strongly-immunoreactive chloride-cells while a negative correlation was observed between the salinity tolerance and the occupying area of the weakly-immunoreactive chloride-cells during the seawater and freshwater adaptation. These results directly suggested that not only the occupying area of chloride cells but also the expression of Na+,K(+)-ATPase protein in the cells is important with respect to the osmoregulatory function in the gills and hypoosmoregulatory ability at the individual level.