Healthy twin birth after autologous islet transplantation in a pancreatectomized patient due to a benign tumor.

OBJECTIVE: Autologous islet transplantation has been reported to show favorable outcomes on glucose metabolism. The objective of this study was to describe successful delivery of twins in an islet recipient who had undergone distal pancreatectomy. PATIENT: A 35-year-old woman who underwent distal pancreatectomy owing to a solid pseudopapillary neoplasm received an autologous islet transplantation (140,000 islet equivalents). After 2.5 years, she unexpectedly became pregnant. Cesarean section was performed at 35 weeks delivering male twins without complications. Plasma glucose and insulin levels, insulinogenic index, and hemoglobin A1c were measured from the preoperative to the postpartum state as the main outcome. RESULTS: The patient showed impaired glucose tolerance before pancreatectomy, but improved to a normal glucose tolerance after transplantation, maintaining euglycemia until pregnancy. Because her fasting glucose levels were within the normal range during pregnancy, fasting insulin represented insulin resistance. Her fasting insulin levels abruptly increased in the third trimester of pregnancy, but returned after delivery. Insulinogenic index increased over 1 year after transplantation, but gradually decreased thereafter. During pregnancy, it increased again, but could not compensate for the insulin resistance. Therefore, gestational diabetes mellitus developed: glucose homeostasis recovered to normal after delivery. CONCLUSIONS: The current report suggested a successful pregnancy after autologous islet transplantation that did not itself permanently deteriorate graft function.

[Lung cancer with a cystic lesion formed by the check-valve mechanism].

A 55-year-old man was admitted to our hospital with a growing cystic lesion in the left middle field of the lung. After we diagnosed it as non-small cell lung cancer, we performed left upper lobectomy. A series of chest X-ray revealed that the cyst was formed by the check-valve mechanism due to the lung cancer, retrospectively. We should keep in mind the existence of lung cancer and other malignant tumors adjacent to cystic lesions.

[Surgical therapy for impalpable small lung lesion; confirmation method by means of combining computed tomography (CT)-guided marking with soft X-ray radiography].

OBJECTIVE: We examined the usefulness of soft X-ray radiography of the specimen which was obtained by the lung wedge biopsy. PATIENTS AND METHODS: From September 2002 to September 2005, we entered the 10 cases (5 men and 5 women) which were consisted of 15 lesions. We performed lung wedge biopsy after computed tomography (CT)-guided lung marking, and then confirmed the lesion in the specimen by means of soft X-ray radiography. RESULTS: We could confirm impalpable small lung lesions in all cases. CONCLUSION: The confirmation method of impalpable small lung lesion that combined CT-guided lung marking with soft X-ray radiography was very useful.

[Acute empyema with intraoperative intractable air leaks in a child; report of a case].

We report a case of acute empyema with intraoperative intractable air leaks in a child. A 4-year-old girl was admitted with parapneumonic empyema by Staphylococcus aureus. Conventional conservative therapies such as antibiotics, chest tube drainage were failed. Then we performed dissection and debridement with video-assisted thoracoscopic surgery in fibrinopurulent phase of acute empyema. Intraoperative findings showed that the parietal pleura was very weak by Staphylococcus aureus pneumonia. Air leaks occurred,but pleural defects could not be closed by sutures and ligations. We could seal intractable air leaks to use fibrin glue soaked bioabsorbable polyglycolic acid felt sheet. Lung expansion promptly recovered and the patient was discharged on the 34th postoperative day without complications.

Characteristics of hepatocytes derived from early ES cells and treatment of surgically induced liver failure rats by transplantation.

BACKGROUND: Although liver transplantation has become a standard therapy for diseases such as fulminant hepatitis and cirrhosis, the lack of donor organs remains a major problem. One solution is the development of transplantable hepatocytes. The metabolic characteristics as well as function and adaptation of hepatocytes (R-EES-hep cell) derived from rat early embryonic stem cells were examined after transplantation into rats with surgically induced liver failure. METHODS: Rat hepatocyte cell lines were established from early embryonic stem cells cultured in the presence of embryotrophic factors by colony cloning methods. The cell lines were established from two cell embryos taken from spontaneous dwarf rats using the novel method of Ishiwata et al. Morphologic differentiation as well as albumin and bilirubin production were observed by immunostaining. R-EES-hep cells were transplanted into the spleens of 90% hepatectomized, surgically induced liver failure rats to analyze survival rates. RESULTS: When cultured in type I collagen gel the cells formed cordlike structures resembling the liver. Both albumin and bilirubin production were observed when transplanted; the spleen was converted into a liver-like structure with prolonged survival of the 90% hepatectomized rats for up to 3 months up to the time of killing. CONCLUSIONS: R-EES-hep cells showed many of the distinctive metabolic characteristics of the liver. These cells may be efficient for further research and application for hepatic cell transplantation to treat liver insufficiency patients and as biologic artificial organs.

[Non-small cell lung cancer simultaneously metastatic to the brain treated by lobectomy and stereotactic irradiation].

We performed left upper lobectomy in 64-year-old man with non-small cell lung cancer (NSCLC) simultaneously metastatic to the brain. He was treated by stereotactic irradiation (STI) 2 months later after lobectomy. He has been doing well now without recurrence for 5 years after the operation. We think that STI and radical lobectomy are good alternatives for patients with NSCLC metastatic to the brain.

Construction and expression of a recombinant adeno-associated virus that harbors a human beta-globin-encoding cDNA.

Towards a goal of using recombinant adeno-associated viruses (AAV) for the gene therapy of hemoglobinopathies we had previously constructed plasmid pAV h beta G psi 1, which contained a human beta-globin-encoding cDNA (HBB) downstream from the P40 promoter of AAV2 DNA [Ohi et al., Gene 89 (1990) 279-282]. Transfection of the plasmid into human 293 cells (embryonal kidney cell line) resulted in the expression of HBB at the mRNA level as well as rescue and replication of the recombinant AAV genome (Ohi et al., ibid.). The present study demonstrates that the replicated recombinant DNA was packaged into an intact virion by transcomplementation with pAV2 or the defective helpers, pAV delta Bam or pAVXB. The recombinant virus could be isolated by equilibrium CsCl density gradient, the density of which was about 1.4 g/cm3. The defective helpers are used to produce wild-type AAV-free recombinant AAV. The recombinant AAV were infectious and expressed chimeric mRNAs containing the HBB sequence in virus-infected 293, KB (oral epidermoid carcinoma cell line) and K562 (human erythroleukemia cell line) cells. The importance of the infectivity and expression of the recombinant AAV in hematopoietic cells is discussed in the context of gene therapy of hemoglobinopathies.

Construction and replication of an adeno-associated virus expression vector that contains human beta-globin cDNA.

With the goal of using adeno-associated viruses (AAV) as the gene-transfer vector for gene therapy of hemoglobinopathies, human beta-globin cDNA was ligated downstream from the P40 promoter of the AAV type-2 (AAV2) genome. To circumvent difficulties of cloning DNA containing palindromic sequences, two of which exist in the termini of AAV genome, a step-wise approach handling one palindrome at a time was devised to construct the chimeric expression vector. Electroporation of the construct into human 293 cells (embryonal kidney cell line) resulted in expression of the cloned human beta-globin cDNA, as evidenced by the synthesis of transcripts hybridizable to human beta-globin cDNA probe. Addition of the 3'-end region of AAV DNA that contains both the transcription termination signal and origin of DNA replication for AAV to the construct permitted the recombinant AAV genome to be rescued and replicate in the cell.

Immunochemical localization of metallothionein in rat liver and kidney.

An indirect immunoperoxidase staining technique was employed to localize the heavy metal binding protein, metallothionein, in rat liver and kidney. Immunostaining for metallothionein was observed in all hepatocytes and most renal tubular cells. This protein was not detectable, however, in cell types such as endothelial or connective tissue cells, indicating that metallothionein synthesis or accumulation is tissue- and cell-type specific. Hepatocytes from cadmium-exposed animals showed increased staining for metallothionein. The presence of metallothionein was also seen extracellularly within the liver sinusoids and renal tubules in both normal and cadmium-exposed animals, suggesting transport and excretion, respectively, of this protein.

Recombinant human granulocyte colony-stimulating factor can mobilize sufficient amounts of peripheral blood stem cells in healthy volunteers for allogeneic transplantation.

Peripheral blood stem cells (PBSC) are a good source for bone marrow reconstitution after intensive chemotherapy. The ability to transplant PBSC between allogeneic subjects would be a key step forward in the application of this procedure. For this purpose, we examined the mobilization and recovery of PBSC in healthy volunteers who were given recombinant human granulocyte colony-stimulating factor (G-CSF). Three informed healthy volunteers were injected with G-CSF subcutaneously at a dose of 2.5 micrograms/kg for 6 successive days and at 5.0 micrograms/kg for the following 4 days. The concentration of PBSC was assessed daily by CFU-GM and BFU-E assays, both of which peaked on the sixth to seventh day of G-CSF administration. Comparison between colony assays and hematological parameters revealed that flow cytometry analysis of CD34+ cells by mononuclear cell gating is a rapid and convenient method to quantify mobilized stem cells. The maximum numbers of CFU-GM were 432, 665, and 1386 colonies/ml blood. It is calculated that sufficient amounts of stem cells for transplantation (at least 1.0 x 10(5) CFU-GM/kg) could be obtained by leukapheresis of 5 to 201 blood when the peak was attained. This trial confirmed the feasibility of allogeneic transplantation using PBSC from healthy volunteers who have received G-CSF.

The role of renal dopamine in the reduction of high blood pressure by beta 1-selective beta-blocker with intrinsic sympathomimetic activity in spontaneously hypertensive rats.

The present experiments were undertaken to clarify the difference of renal dopamine production from beta 1-selective beta-blocker with and without intrinsic sympathomimetic activity (ISA). Either beta-blocker with ISA, celiprolol (100 or 300 mg/kg/day; CEL-100 or CEL-300) or beta-blocker without ISA, atenolol (50 mg/kg/day; ATE-50) was administered to the SHR from 19 to 26 weeks. Degrees of lowering blood pressure in CEL-300 SHR and in ATE-50 SHR were similar, but decrease in heart rate was significantly less in CEL-300 SHR than in ATE-50 SHR. Urine output, which was significantly less in control SHR than in control WKY, was significantly greater in CEL-100 SHR and CEL-300 SHR, but not in ATE-50 SHR. Urinary excretions of noradrenaline (u-NA) and dopamine (u-DA) were significantly higher in control SHR than in control WKY and a comparable u-DA/u-NA ratio was found in these two groups. U-DA and the ratio of u-DA/u-NA were significantly elevated in CEL-100 SHR and CEL-300 SHR, but not in ATE-50 SHR. There was a significant positive correlation between u-DA/u-NA ratio and urine output and a significant negative correlation between the ratio of u-DA/u-NA and change of blood pressure in control SHR, CEL-100 SHR and CEL-300 SHR. These results suggest that an enhancement of renal dopamine production by ISA (beta 2 stimulation) of beta 1-selective beta-blocker may contribute, at least in part, to the antihypertensive effect of this drug.

Co-administration of IL3 with G-CSF increases the CFU-S mobilization into peripheral blood.

In an attempt to establish an efficient method of collecting peripheral blood stem cells and to utilize them for allogeneic peripheral blood stem cell transplantation, the effect of a combined administration of recombinant murine interleukin-3 (IL3) and recombinant human granulocyte colony-stimulating factor (G-CSF) to mobilize bone marrow stem cells into the circulation was examined in C57BL/6 mice. Some appreciable numbers (796 +/- 112/ml) of CFU-GM were recovered 6 days after G-CSF administration (500 micrograms/kg per day), while by IL3 administration (100,000 units/kg per day), the CFU-GM count was much lower (61 +/- 9/ml) with a small peak at day 4. By a combined administration of IL3 (100,000 units/kg per day) and G-CSF (500 micrograms/kg per day), the CFU-GM count at the peak of day 5 was significantly augmented (1178 +/- 277/ml) as compared to that of G-CSF or IL3 alone (P < 0.05). The CFU-S counts at day 5 (168 +/- 12/ml) and at day 6 (172 +/- 27) were also significantly higher than those of IL3 alone (day 5, 30 +/- 15/ml; day 6, 20 +/- 10/ml) or G-CSF alone (day 5, 114 +/- 14/ml; day 6, 112 +/- 19/ml). Thus the combined administration of IL3 and G-CSF appears to be promising for high yield collection of peripheral blood stem cells.

Long-term survival and differentiation of human peripheral blood CD34+ cells in SCID mice.

Human peripheral blood progenitor cells (PBPC) are currently used as a source of hematopoietic reconstitution by autologous transplantation after myeloabrative chemotherapy for malignancies. PBPC would also be useful for allogeneic transplantation since the collection of PBPC is much safer than that of bone marrow stem cells (BM). For allogeneic transplantation, it is imperative to confirm that PBPC contains self-renewable stem cells that can sustain a long lasting hematopoiesis. In the present study, we examined the reconstitution of human hematopoiesis in severe combined immunodeficiency (SCID) mice by transplanting peripheral blood CD34+ cells in which the neo gene was transduced as a marker. In 2 of 4 mice receiving PB-CD34+ cell transplantation, the neo gene appeared as early as 4 weeks and lasted as long as 24 weeks in all DNA preparations of bone marrow, peripheral blood and spleen cells from the SCID mice, while in 2 of 4 mice receiving BM-CD34+ cell transplantation, although the neo gene also lasted as late as 24 weeks, it did not appear as early as in the mice receiving PB-CD34+ cell transplantation. A similar observation was noted in clinical trials, i.e. the white blood cell and platelet recovered earlier by transplantation of PBPC than of BM. In mice who had the neo gene, we were also able to demonstrate by FACS the presence of human lineage specific antigen in the cells as late as 24 weeks after transplantation with PB-CD34+ cells, and the presence of human IgG in the sera 10 weeks after transplantation. These findings indicate that PB-CD34+ cells contain long-term repopulating stem cells which undergo differentiation in SCID mice.

Intra-abdominal desmoplastic small round cell tumor (IDSRT).

Intra-abdominal desmoplastic small round cell tumor (IDSRT) is a rare neoplasm that develops in the abdominal cavity in young people. We experienced a 27-year-old man who visited with ascites of unknown cause. Compression of the colon was found by barium enema examination. On colonoscopic examination, diffuse white elevated lesions, about 5 mm in diameter, surrounded by rubedo, so-called aphthoid lesions, were also observed. IDSRT was diagnosed by biopsy at laparotomy, and chemotherapy with cyclophosphamide, etoposide, doxorubicin, and cisplatin was performed. The tumors shrank temporarily (partial response), but subsequently grew in size again. The patient died during the second course of chemotherapy after relapse. We present one case report, together with a review of the literature.

Effects of actinomycin D on brain RNA synthesis and discrimination learning in the goldfish (Carassius auratus).

Intracranial injection of actinomycin D 2 microgram inhibited about 70% of the brain RNA synthesis from 3 hr to 4 days after injection in the goldfish. Under these conditions, fish were given 4-day-training of visual discrimination between a card with vertical stripes and one with horizontal stripes. Fish injected intracranially with actinomycin D showed deficits in between-day retention (long-term memory) but not interruption of within-day acquisition (short-term memory). It is suggested that brain RNA synthesis is necessary only for the formation of long-term memory but not short-term memory.

Synthesis of human globin polypeptides mediated by recombinant adeno-associated virus vectors.

Adeno-associated virus, serotype 2 (AAV2)-based chimeric plasmids that harbored a near-full-length human alpha- or beta-globin cDNA were constructed. The cDNAs were spliced into an AAV plasmid, pAAV delta K, downstream from the viral P40 promoter, substituting the capsid gene region. The correctness of the insertion with regard to the transcription polarity was ascertained by both restriction enzyme analysis and DNA sequencing. One of the constructs, pAAVcHBBLCR, contained the erythroid-specific enhancer elements, the locus control region, HS1 and HS2, to ensure an efficient and tissue-specific gene expression. Use of a defective complementing helper, pAVXB (Dixit, M.; et al. Gene 1991, 104, 253-257.) and adenovirus 2 made it possible to prepare recombinant AAVs (rAAVs). Infection of human 293 cells (embryonal kidney cell line) with the resultant rAAV (AAVcHBB) and cotransfection of mouse erythroleukemia (MEL) cells with the beta-globin construct (pAAVcHBBLCR) and an alpha-globin construct (pAAVcHAB) triggered efficient synthesis of human globin polypeptides in the cells, as analyzed by biochemical and immunohistochemical means. The LCR made the construct respond to an inducer, N,N-hexamethylenebisacetamide, the amount of expressed human beta-globin reaching a similar level as the endogenous mouse beta-globin in MEL cells. Electrotransfection of mouse bone marrow hematopoietic stem/progenitor cells with the constructs dramatically increased the number of benzidine-positive cells in liquid suspension culture, indicating expression and synthesis of a human hemoglobin in these cells. Thus, the rAAV constructs may be useful for gene therapy of hemoglobinopathies.

Cloned murine fetuses produced by nuclear transfer using metaphase-arrested embryonic stem cells.

We examined whether metaphase nuclei could be used as nuclear donors in nuclear transfer in mice. The reconstructed embryos were developed to fetuses in both the metaphase-nuclear transfer and the G1-nuclear transfer. We also performed enucleation of oocytes following nuclear injection (injection-enucleation method) using microinjection method with a piezo-driven micromanipulator in order to produce the cloned murine fetuses. We found that this method could shorten time for manipulation in comparison with the conventional method performing nuclear injection following enucleation of oocytes (enucleation-injection method). We produced successfully cloned fetuses by the injection-enucleation method. Furthermore, there was no difference of developmental efficiency in reconstructed embryos from between B6D2F1 and ddY strain as oocyte donor.

Cloned mice derived from somatic cell nuclei.

In 1997, a cloned sheep "Dolly" was produced by nuclear transfer of somatic cell. The first birth of cloned mice derived from some somatic cells were succeeded in 1998. At present, it is shown that somatic cells, cumulus cells, fibroblasts and Sertoli cells can be used to the study of cloned animal as nuclear donor. In this study investigation was designed to compare with efficiency on the production of cloned embryos by using the microinjection and the electrofusion methods for nuclear transfer. Oocyte enucleation was performed with a micromanipulator. The oocyte was held by holding pipette, and was enucleated using a beveled pipette. Microinjection method: Cell's nucleus injection was carried out by piezo-micromanipulator. Cytochalasin B treated cumulus cell was aspirated into a injection pipette, and was broken its plasma membrane using the injection pipette. Then, the cumulus cell was injected into the enucleated ooplasm directly. Electrofusion method: The cell was aspirated into a beveled pipette, and then an aspirated cell was inserted into perivitelline space. Then, the pair of enucleated oocyte and cell was fused using electrical cell fusion apparatus. The reconstituted embryos were activated after nuclear transfer using St2+. Reconstituted embryos had been produced by the microinjection showed the embryonic development to over 8-cell stages. But, the rate of fragmentation of reconstituted embryos by the microinjection showed a little high rate in comparison with the electrofusion. When some reconstituted embryos by the microinjection were transplanted to pseudopregnant females' oviduct, 9 fetuses were observed at 14 days post coitum.

Mouse fetuses by nuclear transfer from embryonic stem cells.

At present, two methods for cloning mammals by nuclear transfer are employed. The first is based on cell fusion and has been applied to domestic animals, such as sheep, cows, and goats. While, nuclear microinjection has been used in mice only. Cloning by nuclear transfer has been reported mainly with cells from primary culture and freshly isolated cells. Here, using ES cell line TT2, we tried to produce clone mouse embryos by the two methods. With ES cell line TT2 (10-13 passaged), 16% of reconstructed oocytes microinjected with the nuclei developed in vitro to the morula/blastocycst stage, and 50% of these embryos developed to fetuses until 14 dpc when transferred to pseudopregnant females. At 20 dpc implanted sites were degenerated and absorbed. Also, in vitro development of embryos reconstructed by electrofusion shown similar results. But, when transferred to recipients, subsequent development of embryos showed lower rates, as compared with embryos microinjected and from recipients live-born pups could not be obtained.

Time course of gene expression after the injection of adenoviral vectors containing CTLA4IG gene.

INTRODUCTION: In vivo gene transfection using a recombinant adenoviral vector leads to diminished gene expression in a time-dependent manner that disappears within 4 weeks. CTLA4Ig blocks CD28-mediated costimulatory signal, and inhibits immune responses. We investigated the duration of transgene expression after administration of adenoviral vector containing CTLA4Ig gene (AdCTLA4Ig). METHODS: We injected 1 x 10(9) plaque forming units (pfu) of AdCTLA4Ig into rats (n = 7) via the tail vein. Thereafter, the blood samples were collected for assay of serum CTLA4Ig levels using enzyme-linked immunosorbent assay. RESULTS: The CTLA4Ig level reached the maximum (range, 65-86 microg/mL; average, 75 microg/mL) on days 3 to 5 after injection. Detectable levels of CTLA4Ig were observed up to 49 days. When we injected AdCTLA4Ig in combination with FTY720 administration, the maximum levels were higher and the detectable levels persisted longer. CONCLUSIONS: Because directly injected adenoviral transgene expression had been reported to disappear between 21 to 30 days, we conclude that AdCTLA4Ig inhibits the immune response and prolongs the transgene (CTLA4Ig gene) expression. Some additional immunosuppressants, like FTY720, may be useful to enhance AdCTLA4Ig effects.