Investigation of in-line filter replacement intervals for infusion.

BACKGROUND: In-line filters in peripheral and central venous catheters are used to remove bacterial cells mechanically. A recent study indicated an extension of the use of infusion sets to 7 days. There is no evidence regarding replacement intervals for in-line filters. AIM: To test in-line filters that were used continuously for 7 days in order to investigate their ability to remove bacteria and assess the flow rate. METHODS: Three different in-line filters were attached to an ELNEOPA-NF No. 2 premixed infusion bag of intravenous hyperalimentation, into which Staphylococcus epidermidis ATCC12228 or Escherichia coli ATCC25922 was inoculated. These experiments were compared with a control infusion. The infusion was dropped at a flow rate of 40 mL/h and replaced at 24-h intervals for 7 days. Samples were collected 24 h after drop initiation. FINDINGS: S. epidermidis was not detected in droplets between Days 1 and 6, but In-line filters 1 and 2 showed droplets containing 6-10 colony-forming units/mL on Day 7. E. coli was not detected in any of the filters after 7 days of continuous use. Flow rates <40 mL/h were observed on Day 7 for In-line filter 3 in studies of S. epidermidis, and on Days 4 and 3 for In-line filters 2 and 3, respectively, in studies of E. coli. CONCLUSION: This study revealed differences in bacterial removal and flow rates under high inoculation between the three in-line filters tested. It is suggested that in-line filters can be used continuously for a maximum of 6 days, and reductions in flow rate after 48 h of continuous use should be noted carefully.

Impact of hospital environmental cleaning with a potassium peroxymonosulphate-based environmental disinfectant and antimicrobial stewardship on the reduction of hospital-onset Clostridioides difficile infections.

BACKGROUND: A 1% potassium peroxymonosulphate-based environmental disinfectant (PPED) produces sodium hypochlorite when combined with sodium chloride, which functions as a disinfectant. However, little is known about the impact of hospital cleaning with PPED on hospital-onset Clostridioides difficile infection (HO-CDI). AIM: To reduce HO-CDI, we promoted antimicrobial stewardship and hospital ward cleaning with PPED: this study was conducted to evaluate their impact. METHODS: We began a promotion of post-prescription review with feedback for broad-spectrum antimicrobials and hospital ward cleaning with PPED. We reviewed the ratio of HO-CDI, PPED consumption, and days of therapy (DOT) of broad-spectrum antimicrobials between July 2014 and March 2018, dividing this time into the pre-promotion (July 2014 to June 2015) and post-promotion periods (July 2015 to March 2018). FINDINGS: Using interrupted time series analysis, an immediate significant change in HO-CDI was observed after intervention (P=0.03), although a downward trend was not observed over this period (P=0.19). Trends in PPED consumption significantly changed over this period (P=0.02). DOT of carbapenems decreased immediately after the intervention began (P<0.01). A Poisson regression analysis showed that PPED consumption and DOT of carbapenems were independent factors affecting HO-CDI (P=0.039 and 0.016, respectively). CONCLUSION: We revealed that DOT of carbapenems and use of PPED were associated with the HO-CDI ratio and that both interventions reduced the rate of HO-CDI. This is the first report on the impact of hospital ward cleaning with PPED on the reduction of HO-CDI.

Bendamustine-120 plus rituximab therapy for relapsed or refractory follicular lymphoma: a multicenter phase II study.

The optimal dose, schedule, and other aspects of bendamustine plus rituximab treatment remain unclear for patients with relapsed or refractory follicular lymphoma (FL). Herein, we analyzed the efficacy of bendamustine combined with rituximab (RB-120) treatment for Japanese patients with relapsed or refractory FL. This phase II clinical trial included patients with relapsed or refractory FL who received 375 mg/m2 rituximab on day 1 and 120 mg/m2 bendamustine on days 2 and 3 every 28 days for up to 6 cycles. The primary endpoint was the overall response rate (ORR), and the secondary endpoints included the complete response (CR) rate, progression-free survival (PFS), overall survival (OS), and safety. Thirty-seven patients were enrolled in the trial (median age 62 years, range 42-75 years). All patients were previously treated with rituximab-containing chemotherapy, and 83.8% were previously treated with the R-CHOP regimen. A median of 5 cycles (range 1-6) and 48.6% of patients completed 6 cycles. The ORR was 91.9% (95% confidence interval [CI] 78.1-98.3%), with a CR rate of 86.5% (95% CI 71.2-95.5%). The 3-year PFS and OS were 70.9% (95% CI 52.3-83.3%) and 88.9% (95% CI 73.1-95.7%), respectively, with the median 39.5 months follow-up duration. The most-frequently observed grade 3/4 adverse events were hematologic: lymphopenia (95%) and neutropenia (70%). No treatment-related deaths were observed. RB-120 showed a good efficacy with equivalent toxicities, compared with the bendamustine 120 mg/m2 monotherapy. However, the problem of high drop-out incidences cannot be ignored.

Intestinal amoebiasis in a patient with acute graft-versus-host disease after allogeneic bone marrow transplantation successfully treated by metronidazole.

Amoebiasis has rarely been reported in patients undergoing hematopoietic stem cell transplantation, although it is a world-wide infection and extremely common. We present a case of intestinal amoebiasis unexpectedly revealed by colonoscopy after allogeneic bone marrow transplantation from a human leukocyte antigen-mismatched unrelated donor for acute myeloid leukemia arising from chronic myelomonocytic leukemia and successfully treated by metronidazole.

Myeloablative hematopoietic stem cell transplantation for myelodysplastic syndrome in patients younger than 55 years: impact of comorbidity and disease burden on the long-term outcome.

We retrospectively investigated 31 myelodysplastic syndrome (MDS) patients receiving myeloablative hematopoietic stem cell transplantation (HCT) and focused on prognostic factors affecting the long-term outcome. Patients were classified according to the French-American-British classification and the HCT-comorbidity index was determined. Cytosine arabinoside or thiotepa combined with cyclophosphamide and total body irradiation was used as myeloablative conditioning in eight and 23 patients respectively. After a follow-up period of 0.8-14.2 years from transplantation (median: 6.4 years), 23 patients were alive in complete remission, and the 5-year overall survival (OS) and disease-free survival (DFS) rates were 79% and 72% respectively. The cumulative nonrelapse mortality (NRM) rate was 22% at 5 years. According to multivariate analysis, > or =20% blasts in the bone marrow and an HCT-comorbidity score > or = 3 were significantly associated with poor OS and DFS. Patients with a high HCT-comorbidity score and male patients receiving transplantation from female donors were significantly more likely to have a higher NRM according to the univariate, but not the multivariate analysis. These data suggest that comorbidity and the tumor burden at the time of transplantation may be useful variables for predicting the outcome in MDS patients receiving myeloablative HCT.

Complete resolution of severe chronic active Epstein-Barr virus infection by cultured, activated donor T lymphocyte infusion after nonmyeloablative stem cells allografting.

A patient with chronic active Epstein-Barr virus (EBV) infection was treated by allogeneic SCT from an HLA-identical sibling donor, using a nonmyeloablative regimen. Even on day 70, mixed chimerism remained together with a quite high viral load. On days 76 and 90, donor lymphocytes were infused after short-term culture with OKT3 plus recombinant IL-2. At 8 days after the last dose, all hematopoietic cells were shown to be donor-type dominant; thereafter, the viral load started to decrease and finally disappeared. Anti-mHA-specific CTLs were generated in vitro, which were shown to be effective in eradicate viral-infected recipient T lymphocytes.

Increased frequency of CD3/8/56-positive umbilical cord blood T lymphocytes after allo-priming in vitro.

Umbilical cord blood (UCB) or adult peripheral blood mononuclear cells (PBMC) were repeatedly stimulated by HLA-mismatched allogeneic Epstein-Barr virus (EBV)-transformed cell lines, and the bulk responders or single-cloned cells were immunophenotypically analyzed by flow cytometry. One month after the allo-stimulation, not in PBMC but in UCB, the proportion of CD3/8/56 triple positive T lymphocytes significantly increased. Furthermore, UCB clones exhibited those unique CD3/8/56 markers at an extremely higher frequency than PB clones. They showed as much strong killing activity against allo-stimulators as conventional PB CD56-negative, CD8 T-cell clones, whereas they did not kill the other target, Raji cells. UCB CD3/8/56 T cell clones produced a smaller amount of interferon-gamma compared with PB CD4 or PB CD8 T cell clones. We concluded that CD8 T cells coexpressed with CD56 marker expanded after allo-priming in vitro and would become one of the graft-versus-host (GVH) effectors after UCB transplantation.

Efficient lentiviral transduction of human cord blood CD34(+) cells followed by their expansion and differentiation into dendritic cells.

OBJECTIVE: To support immune reconstitution after cord blood transplantation, immunotherapy using gene-modified dendritic cells (DCs), the most potent antigen-presenting cells, can be a powerful strategy for preventing infection and recurrence. To investigate the applicability of lentiviral vector-transduced DCs compared to retroviral vectors, we transduced umbilical cord blood (CB) CD34(+) cells, then expanded and differentiated them into DCs. MATERIALS AND METHODS: We transduced CB CD34(+) cells by vesicular stomatitis virus G-protein pseudotyped self-inactivating lentiviral vector or retroviral vectors carrying the enhanced green fluorescent protein gene. The cells were expanded in the stroma-dependent culture system and transferred to the culture condition for developing DCs. The efficiency of transduction and expression of the transgene in severe combined immunodeficiency (SCID) mice-repopulating cells (SRCs) and DCs were compared between lentiviral vector and retroviral vectors. Induced DCs were cocultured with allogeneic or autologous T cells to test the ability to present antigens. RESULTS: CB CD34(+) cells transduced by lentiviral vector and expanded ex vivo sustained stable transgene expression and multipotentiality by assessing SRCs assay and clonogenic assay of bone marrow cells from the transplanted mice. DCs derived from these cells expressed green fluorescent protein and surface markers CD1a, CD80, and HLA-DR and showed potent allo-stimulatory activity as well as nontransduced DCs did. On the other hand, we did not detect transgene expression in SRCs and DCs transduced by retroviral vectors. CONCLUSION: Gene-modified DCs derived from ex vivo expanded CB CD34(+) cells transduced by lentiviral vector will be useful in future immunotherapy protocols.

Extensive and long-term ex vivo production of dendritic cells from CD34 positive umbilical cord blood or bone marrow cells by novel culture system using mouse stroma.

We previously developed a system using murine strome (HESS-5), which could expand umbilical cord blood (UCB) stem and progenitor cells, especially CD34+/38- cells, in the presence of human recombinant cytokines. In this study, the ability of expanded UCB- or bone marrow (BM)-CD34+ cells to differentiate into dendritic cells (DCs) was examined. DCs could be induced either from short or long term cultured CD34+ cells after switching the cytokines from Flk-2/Flt-3 ligand, stem cell factor (SCF), thrombopoietin (TPO) to granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) (immature type) plus tumor necrosis factor (TNF)-alpha with stimulation by CD40L transfectant (mature type). Each immature or mature UCB-DCs showed a dextran uptake or a potent allo-T lymphocytes proliferative ability, respectively. Furthermore, those DCs from BM significantly stimulated auto-T lymphocytes in an antigen (varicella zoster virus) specific manner. In conclusion, a novel culture system using HESS-5 is useful to support a rapid and sustained generation of primitive myeloid cells which can develop into functional DCs.

Successful in vitro generation of Epstein-Barr virus-specific cytotoxic T lymphocytes from severe chronic active EBV patients.

Severe chronic active Epstein-Barr virus (EBV) infection (SCAEBV) is a rare but life-threatening disorder. Poor cytotoxic activity against the virus is widely believed to contribute to the development of this disease. We wished to determine whether it is possible to generate autologous EBV-specific cytotoxic T cells (CTLs) in vitro that can be infused back into the patient to treat his/her viremia. To do this, we first had to establish autologous EBV-transformed B cells (EBCL) as antigen-presenting cells, which is known to be difficult to do with B cells from SCAEBV patients. In one patient, the standard method of incubating B cells with EBV-containing B95-8 supernatant was sufficient. In a second patient, however, the B cells apoptosed too rapidly in culture to permit transformation. However, apoptosis could be blocked by the presence of CD40 ligand-transfectant cells, and EBV transformation was successful when performed with this transfectant. Indicating a native immune response to EBV, peripheral blood lymphocytes from both patients proliferated in response to autologous EBCL. Furthermore, patient T cells had higher frequencies of IFN-gamma-producing CD8 cells after stimulation with autologous EBCL than sero-positive healthy controls. EBV-specific CTLs could be generated from both patients after repeated stimulation with autologous EBCL. These CTL lines were predominantly composed of CD4+ cells, and autologous EBCL killing was largely inhibited by an antibody against HLA-DR. These findings support the possibility of adoptive immune therapy to treat SCAEBV patients.

Injection of CD4(+) and CD8(+) cells with donor or host accessory cells induces acute graft-vs-host disease in human skin in immunodeficient mice.

OBJECTIVE: We examined cell subsets with respect to cutaneous graft-vs-host disease by cell sorting selection of subsets of human mononuclear cells and injecting the subsets subcutaneously in a mouse model. MATERIALS AND METHODS: Cell suspensions containing cultured human epidermal cells and dermal fibroblasts from a single donor mixed with lymphoid cell subsets positively selected using the FACSVantage cell sorting instrument and/or MACS cell isolation kits from unrelated individuals were injected into immunodeficient mice. This model is known to generate human skin with histologic findings similar to human graft-vs-host disease. RESULTS: Donor T-cell subsets CD4(+) and CD8(+) plus either host or donor CD14(+) cells were necessary to cause acute cutaneous graft-vs-host disease. Although graft-vs-host disease can result from recognition of class I antigens expressed on human cutaneous cells by donor peripheral blood mononuclear cells, additional recognition of class II antigens expressed on host mononuclear cells resulted in more severe histologic manifestations. Dendritic cells that differentiated from donor and host monocytes also showed competent accessory cell function in this system. CONCLUSIONS: Based on this model, human cutaneous graft-vs-host disease was caused by donor CD4(+) cells and CD8(+) cells activated through recognition of host antigens, including class I and class II antigens presented by either donor or host CD14(+) cells or dendritic cells.

The efficient generation of CD83 positive immunocompetent dendritic cells from CD14 positive acute myelomonocytic or monocytic leukemia cells in vitro.

The ability of leukemic cells to differentiate to mature dendritic cells (DCs) was investigated in six acute myelomonocytic or monocytic leukemia cases. It was found that CD14 positive cells were more efficiently changed to CD83 positive mature typed DCs with granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-alpha) compared with CD14 negative cells. Such leukemia derived DCs expressed a sufficient level of costimulatory molecules (CD80 and CD86), and were shown to be monoclonal based on an the X-inactivation analysis. They also stimulated not only allo- but auto-T lymphocytes, which thereafter became cytotoxic T lymphocytes (CTLs).

Enhancement of human cord blood CD34+ cell-derived NK cell cytotoxicity by dendritic cells.

NK cells and dendritic cells (DCs) are both important in the innate host defense. However, the role of DCs in NK cell-mediated cytotoxicity is unclear. In this study, we designed two culture systems in which human cord blood CD34(+) cells from the same donor were induced to generate NK cells and DCs, respectively. Coculture of the NK cells with DCs resulted in significant enhancement of NK cell cytotoxicity and IFN-gamma production. However, NK cell cytotoxicity and IFN-gamma production were not increased when NK cells and DCs were grown together separated by a transwell membrane. Functional studies demonstrated that 1) concanamycin A, a selective inhibitor of perforin/granzyme B-based cytolysis, blocked DC-stimulated NK cytotoxicity against K562 cells; and 2) neutralizing mAb against Fas ligand (FasL) significantly reduced DC-stimulated NK cytotoxicity against Fas-positive Jurkat cells. In addition, a marked increase of FasL mRNA and FasL protein expression was observed in DC-stimulated NK cells. The addition of neutralizing mAb against IL-18 and IL-12 significantly suppressed DC-stimulated NK cell cytotoxicity. Neutralizing IFN-gamma Ab almost completely inhibited NK cell cytotoxicity against Jurkat cells. These observations suggest that DCs enhance NK cell cytotoxicity by up-regulating both perforin/granzyme B- and FasL/Fas-based pathways. Direct interaction between DCs and NK cells is necessary for DC-mediated enhancement of NK cell cytotoxicity. Furthermore, DC-derived IL-18 and IL-12 were involved in the up-regulation of NK cell cytotoxicity, and endogenous IFN-gamma production plays an important role in Fas-mediated cytotoxicity.

Recognition of small molecules by a ribonucleopeptide.

In this research, we have design an ATP binding domain that consists of an RNA subunit and a peptide subunit by means of structure-based design approach and in vitro selection method. The RNA subunit is designed to consist of two functional domains; an ATP binding domain with 20 randomized nucleotides and an adjacent stem region that serves as a binding site for the RNA-binding peptide. RNA-peptide receptors to ATP were isolated from a pool of different RNAs by selection with affinity column and enzymatic amplification. Effects of the RNA-binding peptide for the specific ATP binding to the selected RNA-peptide receptors are discussed.

Seasonal changes in the hypothalamo-pituitary-testes axis of the Japanese wood mouse (Apodemus speciosus).

Seasonal changes in the hypothalamo-pituitary-testes axis of the Japanese wood mice (Apodemus speciosus) were studied. The testes, epididymis, pituitary and hypothalamus were compared between mice in the breeding season (July) and non-breeding season (October) using morphological techniques, and the plasma testosterone level was evaluated by enzyme immunoassay. Significant differences in these tissues were observed between the breeding season and the non-breeding season. Specifically, differences in the non-breeding season included 1) a decline in testicular and epididymal weights, arrest of spermatogenesis and decrease of serum testosterone concentration; 2) a decrease in the number of luteinizing hormone (LH)-, follicle stimulating hormone (FSH)-, prolactin (PRL)-, and growth hormone (GH)-immunoreactive cells, and decrease in the size of FSH, PRL, and GH-immunoreactive cells; and 3) an increase in the size of gonadotropin-releasing hormone (GnRH)-immunoreactive neurons. Our findings indicate that the male adult Japanese wood mouse exhibits unique seasonal changes in the hypothalamo-pituitary-testes axis which are not found in laboratory mice.

Homo-oligomer formation by basigin, an immunoglobulin superfamily member, via its N-terminal immunoglobulin domain.

Basigin (Bsg) is a highly glycosylated transmembrane protein with two immunoglobulin (Ig)-like domains. A number of studies, including gene targeting, have demonstrated that Bsg plays pivotal roles in spermatogenesis, implantation, neural network formation and tumor progression. In the present study, to understand the mechanism of action of Bsg, we determined its expression status on the plasma membrane. Cotransfection of Bsg expression vectors with two different tags clarified that Bsg forms homo-oligomers in a cis-dependent manner on the plasma membrane. If the disulfide bond of the more N-terminally located Ig-like domain was destroyed by mutations, Bsg could not form oligomers. In contrast, the mutations of the C-terminal Ig-like domain or N-glycosylation sites did not affect the association. The association of mouse and human Bsgs, which exhibit high homology in the transmembrane and intracellular domains but low homology in the extracellular domain, was very weak as compared with that within the same species, suggesting the importance of the extracellular domain in the association. If the extracellular domain of the human Ret protein was replaced with the N-terminal Ig-like domain of Bsg, the resulting chimera protein was associated with intact wild-type Bsg, but not if the C-terminal Ig-like domain, instead of the N-terminal one, of Bsg was used. No oligomer formation took place between the intact wild-type Ret and Bsg proteins. In conclusion, these data indicate that the N-terminal Ig-like domain is necessary and sufficient for oligomer formation by Bsg on the plasma membrane.

Immunological analysis in xenogeneic fetal porcine liver transplantation into a beagle.

BACKGROUND/AIMS: While allogeneic organ transplantation has been performed safely, a major barrier in xenogeneic transplantation is how to inhibit hyperacute rejection. METHODOLOGY: We challenged xenogeneic fetal liver transplantation from pig to dog. The graft was investigated by immunohistochemical analysis on recipient's IgG, IgM and C3. RESULTS: In 1 of 4 cases, the graft escaped hyperacute rejection for about 4 hours after transplantation, however, the recipient died next day due to hemorrhage from the torn capsule of the liver due to the arterial blood pressure of the recipient. Histologically, the parenchyma showed good countenance and no congestion nor hemorrhage was shown in the vessels. On immunohistochemical analysis, canine IgG, IgM and C3 were deposited on the sinusoidal epithelium of the fetal liver more moderately than that of adult control. Fetal porcine liver showed less expression of major histocompatability complex class I antigen than that of the adult one. CONCLUSIONS: We consider that the hyperacute rejection occurred more slowly in xenogeneic fetal liver transplantation than in the adult one due to not only less expression of major histocompatability complex class I, but also lower expression of the epitope recognized by a natural antibody of the recipient.

The serum soluble HLA-DR antigens as a predictive marker of the response to interferon-alpha treatment in patients with chronic hepatitis C.

The serum concentrations of soluble HLA-DR antigens (sDR) were monitored in 40 patients with chronic hepatitis C (CHC) who received interferon treatment. The expression of HLA-class II antigens in liver tissues was also studied by immunohistochemistry. The sDR levels in patients with chronic hepatitis C were significantly higher than those in healthy subjects (416+/-236 [mean +/- S.D.] ng/ml vs. 286+/-163 ng/ml) (P<0.05). There was no correlation between the sDR levels and serum alanine aminotransferase levels, suggesting that sDR do not reflect the extent of liver necrosis. Although there was no difference in pretreatment sDR levels between interferon complete responders and non-responders, sDR significantly declined in complete responders, while they did not in non-responders. The hepatic expression of HLA-DR antigens was observed in dendritic cells, lymphocytes and Kupffer cells in portal area, while in Kupffer cells and endothelial cells in central acinus. These expression significantly decreased in complete responders. From these results, sDR, reflecting the hepatic expression of HLA-DR antigens, could be a predictive marker of response to inteferon treatment.

Inhibition of Epstein-Barr virus (EBV)-specific CD8+ cytotoxic T lymphocyte (CTL) activity by soluble HLA class I in vitro.

In the present study, the effects of soluble HLA (sHLA) class I molecules against EBV-specific CTL were examined. Two different sources of sHLA class I, either bioengineered spliced form of HLA-B7 (sB7) or natural production from EBV-transformed B cells (natural sHLA), were added during the induction of CTL or incubated with MHC-restricted CD8+ CTL, which were selected by immunobeads just before testing for their cytotoxic activity. Both sB7 and natural sHLA class I blocked the generation of CD8+ CTL and also inhibited the cytotoxic activity of established CTL in a dose-dependent manner. In both ways, natural sHLA class I was effective in 10-fold lower concentrations compared with sB7. The inhibitory effect did not require a sharing of the HLA allotypes between sHLA and the CTL. CTL, after being treated with sHLA, underwent apoptosis, which was considered here as the main mechanism.

Delayed hyperacute xenograft rejection in porcine to canine fetal liver transplantation.

Fetal tissues are generally considered to express weaker antigenic cell-surface molecules than adult tissues. We have reported that transplantation of porcine fetal liver tissue (fragments) is useful for acute and chronic hepatic failure in rats. We further investigated, in the present study, whether transplantation of a porcine fetal liver has the advantage of delayed hyperacute xenograft rejection (HAR) in comparison with that of an adult liver. Porcine fetal liver heterotopically transplanted into dogs was compared. Haematoxylin-eosin (HE) and immunohistochemical studies using IgM, C3, IgG antibodies were performed in serial biopsies of the liver grafts. Lectin binding to target antigen epitopes on pig and dog tissues was studied by flow cytometry. Carbohydrate expression on the liver was also studied by immunohistochemistry. The macroscopic and HE section findings indicate that HAR started 15 min postgraft in fetal and adult liver grafts. Thereafter, vascular changes and parenchymal damage progressed more rapidly in the adult grafts. The final HAR time in adult liver transplantation was determined to be 60 min, while it was determined to be 180 min in fetal liver transplantation. IgM, C3 and IgG were deposited more strongly in the adult grafts than in the fetal grafts up until 60 min after xenografting. Phaseolus vulgaris erythroagglutinin lectin competitively blocked dog sera binding to porcine PBLs. The fetal liver expressed oligosaccharide at a significantly lower level than the adult liver. We conclude that porcine fetal liver xenografts had a significantly delayed HAR.