Comparison of preoperative chemotherapy using docetaxel, cisplatin and fluorouracil with cisplatin and fluorouracil in patients with advanced carcinoma of the thoracic esophagus.

We retrospectively compared preoperative docetaxel, cisplatin, and fluorouracil (DCF) with cisplatin and fluorouracil (CF) in patients with esophageal cancer. The study included patients with advanced thoracic esophageal carcinoma (excluding T4 tumors) receiving preoperative chemotherapy. In the DCF group, five patients received two courses of treatment every 4 weeks, and 33 patients received three courses every 3 weeks. In the CF group, 38 patients received two courses of treatment every 4 weeks. Patients underwent curative surgery 4-5 weeks after completing chemotherapy. Patient demographic characteristics did not differ between the two study groups. The incidence of a grade 3 or 4 hematologic toxicity was significantly higher in the DCF group (33 patients) than in the CF group (five patients; P < 0.001). Curative resection was accomplished in 79% of patients in the DCF group and 66% in the CF group (P = 0.305). There were no in-hospital deaths. The incidence of perioperative complications did not differ between the groups. A grade 2 or 3 histological response was attained in a significantly higher proportion of patients in the DCF group (63%) than in the CF group (5%; P < 0.001). Progression-free survival and overall survival were significantly higher in the DCF group (P = 0.013, hazard ratio 0.473; P = 0.001, hazard ratio 0.344). In conclusion, a grade 3 or 4 hematologic toxicity was common in the DCF group but was managed by supportive therapy. Histological response rate, progression-free survival, and overall survival were significantly higher in the DCF group compared with the CF group.

Array-based genomic resequencing of human leukemia.

To identify oncogenes in leukemias, we performed large-scale resequencing of the leukemia genome using DNA sequence arrays that determine approximately 9 Mbp of sequence corresponding to the exons or exon-intron boundaries of 5648 protein-coding genes. Hybridization of genomic DNA from CD34-positive blasts of acute myeloid leukemia (n=19) or myeloproliferative disorder (n=1) with the arrays identified 9148 nonsynonymous nucleotide changes. Subsequent analysis showed that most of these changes were also present in the genomic DNA of the paired controls, with 11 somatic changes identified only in the leukemic blasts. One of these latter changes results in a Met-to-Ile substitution at amino-acid position 511 of Janus kinase 3 (JAK3), and the JAK3(M511I) protein exhibited transforming potential both in vitro and in vivo. Further screening for JAK3 mutations showed novel and known transforming changes in a total of 9 out of 286 cases of leukemia. Our experiments also showed a somatic change responsible for an Arg-to-His substitution at amino-acid position 882 of DNA methyltransferase 3A, which resulted in a loss of DNA methylation activity of >50%. Our data have thus shown a unique profile of gene mutations in human leukemia.

A new operative technique for the resection of gastric tube cancer by means of lifting the anterior chest wall and videoscope-assisted surgery.

The prolonged survival of patients receiving surgery for esophageal cancer has led to an increased incidence of adenocarcinoma arising in the gastric tube used for reconstruction (gastric tube cancer). In patients with advanced gastric tube cancer, resection of the gastric tube should be considered, but currently available procedures are very invasive. In patients undergoing curative surgery for gastric tube cancer that has developed after reconstruction through the retrosternal route, the gastric tube is usually resected through a median sternotomy, followed by reconstruction with the colon. However, postoperative complications often occur and treatment outcomes remain poor. We developed a new surgical technique for gastric tube resection without performing a sternotomy in patients with gastric tube cancer who had previously undergone reconstruction through the retrosternal route. Our technique was used to treat two patients. Two Kirschner wires were passed subcutaneously through the anterior chest; the chest was lifted to extend the retrosternal space and secure an adequate surgical field. The stomach was separated from the surrounding tissue under videoscopic guidance. Total resection of the gastric tube was done. The retrosternal space was used to lift the jejunum. Roux-en-Y reconstruction was performed. Neither patient had suture line failure or surgical site infection. Their recovery was uneventful. Our surgical technique has several potential advantages including (i) reduced surgical stress; (ii) the ability to use the retrosternal space for reconstruction after gastric tube resection; and (iii) a reduced risk of serious infections such as osteomyelitis in patients with suture line failure. Our findings require confirmation by additional studies.

Minimum leakage rate (0.5%) of stapled esophagojejunostomy with sacrifice of a small part of the jejunum after total gastrectomy in 390 consecutive patients.

BACKGROUND: The development of new surgical instruments and devices has facilitated the performance of esophagojejunostomy after total gastrectomy. However, total prevention of dehiscence of anastomoses remains difficult. We introduced a new procedure for esophagojejunostomy using a circular stapler, requiring sacrifice of only a small part of the jejunum. METHODS: The study group comprised 390 consecutive patients who underwent reconstruction by Roux-en-Y esophagojejunostomy, performed with a circular stapler, sacrificing a small part of the jejunum after total gastrectomy. We assessed anastomotic leakage and anastomotic stenosis after surgery. RESULTS: Only 2 patients (0.5%) had leakage and 4 (1.0%) had anastomotic stenosis after reconstruction. All the patients were cured by conservative therapy. CONCLUSIONS: Esophagojejunostomy performed with a circular stapler after total gastrectomy, with sacrifice of only a small part of the jejunum, is a useful and easy procedure, with a leakage rate of 0.5%.

Role of Flk-1 in mouse hematopoietic stem cells.

It was reported that human hematopoietic stem cells in bone marrow were restricted to the CD34(+)KDR(+) cell fraction. We found that expression levels of Flk-1, a mouse homologue of KDR, were low or undetectable in mouse Lin(-)c-Kit(+)Sca-1(+)CD34(low/-) cells as well as Hoechst33342(-) cells (side population), which have long-term reconstitution capacity. Furthermore, neither Flk-1(+)CD34(low/-) cells nor Flk-1(+)CD34(+) cells had long-term reconstitution capacity in mouse. Taken together with other observations using Flk-1-deficient mice, these results indicate that Flk-1 is essential for the development of hematopoietic stem cells in embryo but not for the function of hematopoietic stem cells in adult mouse bone marrow.

Isolation of a novel interleukin-1-inducible nuclear protein bearing ankyrin-repeat motifs.

We isolated a novel gene termed interleukin (IL)-1-inducible nuclear ankyrin-repeat protein (INAP), of which expression was specifically induced by IL-1 in OP9 stromal cells. The INAP has ankyrin-repeat motifs and shares weak amino acid sequence homology with Bcl-3 and other IkappaB family members. The human genomic INAP gene found in the NCBI data base is located at chromosome 3q3.11. Northern blot analyses revealed that INAP was not expressed in any examined tissues without stimulation, but INAP expression was rapidly and transiently induced by IL-1 although not by tumor necrosis factor alpha nor by phorbol 12-myristate 13-acetate in OP9 cells. Immunoblots with anti-INAP-specific antibody demonstrated that INAP was rapidly and specifically produced by IL-1 stimulation and was predominantly localized in the nucleus. Immunofluorescence stainings showed that the INAP newly synthesized by IL-1 stimulation was promptly translocated into the nucleus, and FLAG-tagged INAP forcibly expressed in NIH/3T3 cells was also specifically localized in the nucleus. The possible interaction of INAP with RelA/p65, NF-kappaB1/p50, NF-kappaB2/p52, C/EBPbeta, and retinoid X receptor was examined, but we could detect none of these interactions in the nuclear extracts of IL-1-stimulated cells. Unlike Bcl-3 and other IkappaB family members, INAP may play a unique role in IL-1-induced specific gene expression and/or signal transduction in the nucleus.

Induction of light chain replacement in human plasma cells by caffeine is independent from both the upregulation of RAG protein expression and germ line transcription.

When some human plasma cell lines are cultured with concanavalin A, the original light chain is replaced with another light chain which results from secondary VJ recombination (light chain shifting). We examined various intracellular factors involved in the induction of light chain shifting. Light chain shifting can be induced upon treatment with agents with phosphatase inhibitory activity such as caffeine and okadaic acid. Although the plasma cells used express both RAG-1 and RAG-2, the expression level of these proteins was not affected by caffeine or okadaic acid. Transcription of the germ line locus, which correlates to the locus activation for rearrangement, is also not influenced by phosphatase inhibition. However, the amount of signal broken-ended DNA intermediates generated during V(D)J rearrangement was shown to increase upon caffeine or okadaic acid treatment. The inhibitory activity of caffeine on phosphatase was the same as okadaic acid. However, caffeine exhibited much higher activity for VJ coding joint formation than okadaic acid. Therefore, although phosphatase inhibition might act, in part, on a mechanism by which V(D)J recombinase activity is regulated within the human plasma cells, other factor(s) are probably also involved in the process.

Concanavalin A stimulation enhanced secondary VlambdaJlambda rearrangement in some human plasma B cells without up-regulation of recombination-activating gene expression and Vlambda germline transcription.

Light chain shifting is a phenomenon that occurs in certain human antibody-producing plasma B cells which, when stimulated with concanavalin A (Con A), shift production of the original light chain to new light chains. Here we investigated the effect of Con A stimulation on these light chain shift-inducible cells. Analysis of transcripts and VJ-coding joints for new light chains revealed that a leaky amount of secondary VlambdaJlambda rearrangement occurs spontaneously, without Con A stimulation, and that Con A stimulation markedly increases VJ-coding joints and transcripts for new light chains. It was also shown that new light chain producers, which have carried out secondary rearrangement, do not further rearrange their light chain genes, even when stimulated with Con A. Recombination-activating gene (RAG) products and Vlambda germline transcription were constitutively expressed in these cell lines and their expression levels were not affected by Con A stimulation. These results suggest that Con A stimulation enhanced secondary VlambdaJlambda rearrangement, but this was not a result of the up-regulation of RAG expression and Vlambda germline transcription, which are believed to be sufficient for the process of VlambdaJlambda rearrangement.

Serum starvation induced secondary V lambda J lambda rearrangement in a human plasma B cell line.

HB4C5 is a human antibody producing plasma B cell line that expresses the recombination activating gene-1 (RAG-1) and RAG-2 constitutively, but undergoes few secondary immunoglobulin gene rearrangements when cultured in fetal bovine serum-containing medium. Here, we found that depletion of serum from the culture media induces secondary VlambdaJlambda rearrangement in this cell line. To investigate the induction mechanism of secondary VlambdaJlambda rearrangement, we assessed the expression levels of RAG-1 and RAG-2 products, Vlambda germline transcription level and the amount of Vlambda signal broken ends (SBE) in HB4C5 cells cultured in serum-supplemented or serum-free medium. Western-blot analysis showed that the expression level for the RAG-1 and RAG-2 proteins was not affected by the serum depletion. Vlambda germline transcript was found to be constitutively expressed in HB4C5 cell line and this transcription level was not affected by the lack of serum. On the other hand, the amount of Vlambda SBE was shown to be increased in HB4C5 cells cultured in serum-free medium, suggesting that this increased formation of Vlambda SBE at least partly contributed to the enhanced occurrence of secondary VlambdaJlambda rearrangement in HB4C5 cells cultured in the serum-free condition. These results indicate that expression of RAG proteins and Vlambda germline transcription is not enough to undergo secondary VlambdaJlambda rearrangement in this cell line.

Recombinant light chain of human monoclonal antibody HB4C5 as a potentially useful lung cancer-targeting vehicle.

Recombinant lambda light chain of lung cancer-reacting human monoclonal antibody HB4C5 was expressed in Escherichia coli. Expression in bacteria ensured the generation of homogeneous light chain species devoid of activity-hampering N-linked glycosylation usually found in the light chain CDR-1 of HB4C5. Molecular engineering was also employed to eliminate the C-terminal two amino acid residues, i.e., Cys and Ser, to prevent the formation of lambda light chain dimers which are less reactive than the monomeric form. The lambda light chain was overexpressed in E. coli as inclusion bodies, which were solubilized, refolded, and treated with Aeromonas proteolytica aminopeptidase to remove the N-terminal Met with subsequent natural cyclization of the penultimate Gln residue to pyroglutamate, the same N-terminal end as that of naturally occurring lambda light chain in HB4C5. Monomeric recombinant lambda light chains, both before and after removal of the N-terminal Met residue, were 40 times more immunoreactive than the parent HB4C5. The immunostaining of lung cancer tissue sections with the recombinant lambda light chain indicated cancer-specific reactions to all specimens of adenocarcinoma, squamous cell carcinoma and large cell carcinoma histologies, but did not react with small cell carcinoma. Tumor radioimmunoimaging experiments in LC6 (lung squamous cell carcinoma line)--xenografted nude mice by the i.p. injection of 125I-labeled recombinant lambda light chain and 125I-labeled human lambda light chain control gave tumor-specific and recombinant lambda light chain-dependent images on day 5 postinjection, and images were also detectable on day 3. Biodistribution studies with 125I-labeled recombinant lambda light chain demonstrated that the lambda light chain could penetrate better into the tumor sites, both at the necrotic and solid parts of the xenograft, as compared to our previous results with 125I-labeled HB4C5 which could localize to the necrotic part only. These results suggest that the recombinant lambda light chain is potentially useful as a lung cancer-targeting vehicle, for such as radioimmunoimaging and radioimmunotherapy, with least possible adverse immunogenic effects.

Light chain shifting: identification of a human plasma cell line actively undergoing light chain replacement.

We identified an antibody-secreting human B-cell line (HTD8), which actively replaces the production of the original lambda light chain with a new lambda chain (light chain shifting) at a high rate. Loss of the original rearranged lambda light chain occurs by significantly reducing the amount of transcript expressed. Expression of the new lambda chain, which replaces the original lambda chain, occurs by rearranging new VJ segments on a previously excluded allele. V lambda gene usage of these new rearrangements are biased toward Vlambda4, Vlambda6, and Vlambda10 families, which are known to be the least frequently used. In striking contrast to the plasma cell phenotype, recombination activating genes, RAG-1 and RAG-2, were expressed in the HTD8 cells and were shown to be necessary, but insufficient for inducing expression of the new lambda chain. These results suggest that human plasma cells have the potential to actively undergo light chain replacement.

A case of abdominal aortic aneurysm associated with systemic lupus erythematosus.

A case of abdominal aortic aneurysm associated with systemic lupus erythematosus (SLE) is reported. A 45-year-old woman with a 18-year history of SLE was admitted with severe lumbago radiating to the bilateral inguinal region. CT and DSA showed a dumbbell shaped true aneurysm of the abdominal aorta. An aorto-biiliac Y shaped graft replacements was performed. SLE is rarely associated with aneurysm of the great arteries. We could find only 4 reports of abdominal aneurysm associated with SLE. Common features were the young age of the patient, the long term of the systemic disease, and administration of corticosteroid therapy for a relatively long period of time. We speculate that atherosclerosis, hypertension, and corticosteroid may all work in concert, possibly together with aortic wall involvement or vasculitic damage, to produce the rare abdominal aneurysm in SLE.

Class-switching of the IgM type anti-adenocarcinoma human antibody HB4C5 into an IgG1 type resulted in the loss of the antigen binding ability.

The heavy and light chain genes (mu and lambda) of the human anti-adenocarcinoma monoclonal antibody HB4C5 (MAb-C5) were cloned from the human-human hybridoma cell line HB4C5 and co-expressed in Chinese hamster ovary (CHO) and COS-7 cells. ELISA assay and the RI imaging of the cancer tissue xenografted into nude mice showed that the recombinant MAb-C5 retained the original antigen binding activity. We then replaced the IgM constant region of the MAb-C5 heavy chain gene with the human IgG1 constant region gene and co-expressed it with the light chain gene. This recombination was confirmed by a complete DNA sequencing and Western-blot analysis. Despite the fact that the DNA sequence, the expressed protein size, and the assembly of heavy and light chains were indicated to be normal, the IgG1 type MAb-C5 could not bind to the original antigen. This result suggest that this antibody alters its antigen binding property upon class-switching.

Metabotropic glutamate receptors and visual cortical synaptic plasticity.

Two forms of use-dependent synaptic plasticity, called long-term potentiation (LTP) and long-term depression (LTD), can be elicited in the visual cortex following different paradigms of electrophysiological stimulation. These neurobiological phenomena often are considered as necessary components of models for the alteration in function of the nervous system that must occur at some level for the establishment and (or) maintenance of memory engrams, for learning processes, or for the consolidation of active neural connections and regression of inactive contacts in the developing brain. It has been postulated that for LTP and LTD to be produced in the hippocampus, activation of a particular subtype of excitatory amino acid receptor, the metabotropic receptor, is a critical requirement. Only recently has it become possible to test this hypothesis directly, as a new compound, (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG), has been introduced and the suggestion made that it selectively antagonizes the metabotropic receptor. This substance has been tested in the present study on responses recorded from slices of rat visual cortex and has been found both to block the activation of the metabotropic receptor and to interfere selectively with the form of synaptic plasticity called LTD. It thus appears from the experiments reported in this paper as though the metabotropic receptor subtype that is blocked by MCPG is required for the expression of LTD but not for the expression of LTP, in the visual cortex of adult rats.

Induction of LTD but not LTP through metabotropic glutamate receptors in visual cortex.

Long-term potentiation (LTP) and long-term depression (LTD), often used as essential components in synaptic models for learning, memory and forgetting, can be produced in cortical tissue by repetitive activation of neural pathways under different stimulus conditions. The involvement of metabotropic glutamate receptors (mGluRs) has been postulated to be necessary for the establishment of either or both forms of synaptic plasticity in hippocampus. The recent introduction of a specific antagonist for mGluRs, (+/-)-alpha-methyl-4-carboxyphenylglycine, prompted the investigation of the respective involvement of this receptor population in the induction of LTP and LTD in visual cortex of the rat in vitro. The results suggest the critical involvement of mGluRs in producing LTD but not LTP.

Effects of an inhibitor for calcium/calmodulin-dependent protein phosphatase, calcineurin, on induction of long-term potentiation in rat visual cortex.

A role of Ca2+/calmodulin-dependent protein phosphatase (calcineurin) in induction of long-term potentiation (LTP) was investigated using its selective inhibitor, FK506, in visual cortical slices of young rats. Field potentials or excitatory postsynaptic potentials (EPSPs) to test stimulation of white matter were recorded extra- or intracellularly from layer 2/3, and tetanic stimulation (tetanus) was applied to the white matter at 5 Hz. During the application of FK506 (1 microM), short tetanus (6 s) which had rarely induced LTP in the normal medium, became effective in inducing LTP. Tetanus for 1 min in the presence of FK506 induced LTP with higher probability than in the normal medium. To test possible involvement of presynaptic mechanisms, paired pulses at 50 ms intervals were given to the white matter. The facilitation ratio of the second to first EPSPs was not significantly changed by FK506 and after the induction of LTP, suggesting that the action of FK506 may not be presynaptic. To confirm this, FK506 was injected directly into neurons through recording electrodes. In cases in which stable EPSPs were recorded, the probability of LTP induction became higher than that obtained with normal electrodes. These results suggest that calcineurin plays a role in processes antagonizing the induction of LTP in visual cortex.

FK409, a novel vasodilator isolated from the acid-treated fermentation broth of Streptomyces griseosporeus. I. Taxonomy, fermentation, isolation, and physico-chemical and biological characteristics.

FK409, a novel vasodilator with anti-platelet aggregation activity, has been isolated from the acid-treated fermentation broth of Streptomyces griseosporeus No. 16917, which was cultured on a medium containing NaNO3 for 4 days. FK409 was purified from the culture-filtrate by extraction with ethyl acetate after adjusting the pH to 3.0 with HC1, followed by silica gel chromatography. The molecular formula of this compound was determined to be C8H13N3O4. In vitro, FK409 showed a potent relaxation activity on noradrenaline induced contraction of rat aorta. In addition to the vasodilating activity, this compound also showed potent anti-aggregation activities towards rabbit platelets. In vivo, intravenously administered FK409 resulted in marked blood pressure lowering in rats.