ALBERT

Description: The effect of human spleen(L-rich) and heart(H-rich) ferritins on the proliferation and differentiation of human B lymphocytes was studied in comparison with that of holo- and apo-transferrins. Ferritins rich in H and L chain, as well as the transferrins, did not inhibit the proliferative response of resting and activated B cells stimulated with polyclonal B-cell mitogen, Staphylococcus aureus Cowan strain I. In contrast, the ferritins, but not the transferrins, clearly suppressed the antibody production by B blasts in T-cell-independent as well as T- cell-dependent system. Kinetic study showed that inhibitory action of ferritins on immunoglobulin (Ig) production was caused at an early stage of B-cell differentiation. The cytoplasmic Ig-containing cells decreased in proportion to the reduction of Ig secretion. The evidence that ferritin inhibited Ig synthesis of Epstein-Barr virus-transformed human B-lymphoblastoid cell line also supported the idea that the effect of ferritin was directed toward the antibody-producing B lymphocytes. The molecular analysis showed that the inhibitory effect of ferritin was regulated at the transcriptional level of the Ig generation signal. Our results suggest that H- and L-rich ferritins exert their inhibitory action on the differentiation of B cells maturing into Ig-producing cells.

Description: The effect of human spleen(L-rich) and heart(H-rich) ferritins on the proliferation and differentiation of human B lymphocytes was studied in comparison with that of holo- and apo-transferrins. Ferritins rich in H and L chain, as well as the transferrins, did not inhibit the proliferative response of resting and activated B cells stimulated with polyclonal B-cell mitogen, Staphylococcus aureus Cowan strain I. In contrast, the ferritins, but not the transferrins, clearly suppressed the antibody production by B blasts in T-cell-independent as well as T- cell-dependent system. Kinetic study showed that inhibitory action of ferritins on immunoglobulin (Ig) production was caused at an early stage of B-cell differentiation. The cytoplasmic Ig-containing cells decreased in proportion to the reduction of Ig secretion. The evidence that ferritin inhibited Ig synthesis of Epstein-Barr virus-transformed human B-lymphoblastoid cell line also supported the idea that the effect of ferritin was directed toward the antibody-producing B lymphocytes. The molecular analysis showed that the inhibitory effect of ferritin was regulated at the transcriptional level of the Ig generation signal. Our results suggest that H- and L-rich ferritins exert their inhibitory action on the differentiation of B cells maturing into Ig-producing cells.

Description: The biologic effects of retinoids such as all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid on proliferation and differentiation of hematopoietic cells are mediated by binding and activating two distinct families of transcription factors: the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). The RARs require heterodimerization with RXRs; in addition, RXRs can form homodimers, which can bind to DNA response elements that are either distinct or the same as those bound by the RAR/RXR heterodimers. Therefore, the two retinoid pathways provide sequences that are specific for effective DNA binding and activation of target genes. We have developed several series of novel synthetic retinoids that selectively interact with RXR/RXR homodimers and RAR/RXR heterodimers. We show here that SR11236 and SR11246, which are RXR-selective analogs, had little ability to inhibit clonal growth and induce differentiation of leukemic cells (HL- 60 cells and fresh acute myeloid leukemia cells). However, SR11249, SR11256, and LGD1069, which activated both RXR/RXR homodimers and RAR/RXR heterodimers, could inhibit clonal growth and induce differentiation of HL-60 cells as well as leukemic cells from patients, including those with acute promyelocytic leukemia (APL). This is similar to results observed with RAR/RXR-specific ligands. Interestingly, the combination of ATRA and either SR11249, SR11256, or LGD1069 showed synergistic effects in inducing differentiation of HL-60 cells. A retinoid (SR11238) with strong anti-AP-1 activity that did not activate the RARs and RXRs for gene transcription from the response element TREpal was inactive in our assay systems, suggesting that the antiproliferative effects of retinoids on leukemic cells is not mediated by inhibiting the AP-1 pathway. We conclude that the RAR/RXR pathway is more important than RXR/RXR pathway for differentiation and proliferation of acute myeloid leukemic cells, and certain retinoids or combination of retinoids with both RAR and RXR specificities may synergistically enhance the differentiation activity of ATRA, which may be relevant in several clinical situations.

Description: Thrombopoietin and its receptor (MPL) are important regulators of megakaryopoiesis. We have identified an activating mutation of MPL using a combination of a retrovirus-mediated gene transfer and polymerase chain reaction-driven random mutagenesis. This point mutation causes a single amino acid substitution from Ser498 to Asn498 in the transmembrane region and abrogates factor-dependency of all interleukin-3-dependent cell lines tested. Murine interleukin-3- dependent Ba/F3 cells expressing the mutated but not the normal form of MPL were tumorigenic when transduced into syngeneic mice. Analysis of intracellular signaling pathways indicated that the mutant MPL protein constitutively activated two distinct signaling pathways, SHC-Raf-MAPK and JAK2-STAT3/STAT5.

Description: Abstract 4542 Background: Norovirus-gastroenteritis (NV-GE) is considered as a highly transmittable disease that can lead to fatal outcomes in vulnerable populations. Therefore, prompt detection of norovirus in stool specimens is important for patients who undergo hematopoietic stem cell transplantation (HSCT). The commercially available immunochromatography kit (Denka Seiken, Tokyo, Japan) is a diagnostic tool that can easily and rapidly detect norovirus antigens with high specificity and relatively less sensitivity compared with reverse transcription polymerase chain reaction (RT-PCR). Although previous studies used RT-PCR to detect norovirus in stool, this method is not necessarily a standardized technique for the clinical use, and only limited information is available about clinical significance of norovirus infection among the HSCT recipients. Here, we report an analysis of patients with NV-GE in our HSCT unit using the immunochromatography method. Patients and Methods: We prospectively examined stool specimens to detect norovirus antigens in patients who developed diarrhea in our HSCT unit between December 2008 and June 2011. We also retrospectively examined stool specimens which had been collected for Clostridium difficile (CD) toxin test and frozen at –40°C between January 2007 and November 2008. During the study period, a total of 468 patients underwent HSCT (autologous 83, allogeneic 385) at our center and we tested stool specimens from 355 patients who developed diarrhea. Results: Norovirus was detected by the immunochromatography method in 11 patients among the HSCT recipients, and in 1 patient who died before HSCT because of disease progression. Among the 12 patients with NV-GE, 3 were retrospectively diagnosed using the frozen specimens. The CD toxin test was also positive in 1 of the 12 patients with NV-GE. The median age of the 12 patients was 56 years (range, 29–66). The median duration of symptoms was 30 days (2–134). Among the 11 patients who developed NV-GE after HSCT, primary disease included lymphoma (6 patients), acute leukemia (4 patients), and multiple myeloma (1 patient), and 9 of them were not in remission at HSCT. One patient developed NV-GE after autologous HSCT, and 10 patients after allogeneic HSCT. Among the 10 allo-HSCT recipients, 6 received grafts from unrelated bone marrow donor and 4 received grafts from related donor. Five patients received myeloablative conditioning and 5 received reduced-intensity conditioning before allo-HSCT. The median time from HSCT to the onset of symptoms was 36 days (4–93). The median time from HSCT to diagnosis of NV-GE was 37 days (11–101). At diagnosis of NV-GE, all allo-HSCT recipients were given immunosuppressive agents, and 2 of them received corticosteroids for intestinal graft-versus host disease (GVHD). The volume of diarrhea was more than 500 ml per day in 4 patients. Among 4 patients who underwent endoscopy of the lower gastrointestinal tract, intestinal GVHD was diagnosed in 2 patients by histopathology findings, whereas the other 2 patients had no evidence of intestinal GVHD, which resulted in no need for an intensification of immunosuppression. Of the 12 patients with NV-GE, 6 were alive with a median follow-up of 826 days (17–1168) after the diagnosis of NV-GE. No patients died of NV-GE, and 6 died of other causes (disease progression, 4; GVHD, 1; multiple organ failure, 1). There was no outbreak of NV-GE as we promptly implemented isolation of infected patients and enhanced hygiene strategy within an hour of collection of stool specimens in patients with diarrhea. Conclusions: In this study, we detected 11 patients who developed NV-GE after HSCT using the immunochromatography method. Our results suggested that this method is helpful in the differential diagnosis of patients with diarrhea after HSCT and enable us to take an appropriate and prompt preventive measure. In the future study, validation with RT-PCR and immunochromatography method is warranted among the immune-compromised populations. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Oral tacrolimus (Tac) was first developed as a twice-daily formulation (Tac BID) and has been widely used in solid organ and allogeneic hematopoietic stem cell transplantation (allo-SCT). Lifelong immunosuppression is required to preserve graft function after solid organ transplantation, and medical nonadherence of transplant recipients has been identified as a major cause of allograft failure. It has been well documented that frequent drug dosing affects patients' adherence. In response to this potential nonadherence problem, a once-daily modified release formulation of oral tacrolimus (Tac QD) has been developed. Results of randomized prospective phase III studies have indicated that Tac QD is well tolerated with similar efficacy and safety profiles as those of Tac BID in recipients after organ transplantation. Although Tac BID has been widely used in allo-SCT recipients, there are no available data on the use of Tac QD in this population. In this study, we compared retrospectively the efficacy of Tac QD versus Tac BID administration. The objective of this study was to investigate whether Tac QD is as effective as Tac BID in the setting of allo-SCT from unrelated donors. Methods: The study cohort included 77 consecutive patients who received allogeneic bone marrow transplantation from unrelated donors for treatment of hematologic malignancies between 2000 and 2014: AML (n=35), ALL (n=15), CML (n=8), MDS (n=12), NHL (n=5), and MPN (n=2). Patients received Tac iv from day -1 with short-term methotrexate (MTX) on days 1, 3, 6, and 11; Tac iv was converted to either Tac QD (n=37) or Tac BID (n=40) when the patients were engrafted and could tolerate oral medication. Doses were modified to maintain a whole-blood trough concentration of 8-12 ng/ml. Tac BID was administrated until Oct 2008, and Tac QD was used from Nov 2008. Kaplan-Meier estimates were used for overall survival (OS), and cumulative incidence estimates were used for acute GVHD, chronic GVHD, and non-relapse mortality (NRM). Results: Median age was 53 years (range: 23-66) for Tac QD cohorts and 39 years (range: 17-56) for Tac BID cohorts. Tac QD patients were older (P

Description: In six patients with Epstein-Barr virus (EBV) induced infectious mononucleosis (IM) and in two patients with IM-like syndrome, peripheral blood lymphocytes in the acute and convalescent phase were tested for human la-like antigens as well as other cell surface markers. The major population of T lymphocytes in the acute phase showed la-like antigens, as detected by indirect immunofluorescence with heteroantisera, and the number of la-like antigen-positive T lymphocytes decreased with convalescence. The crossabsorption study indicated that the amount of la-like antigen on the surface of IM T cells was less than that of Raji cells.

Description: The biologic effects of retinoids such as all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid on proliferation and differentiation of hematopoietic cells are mediated by binding and activating two distinct families of transcription factors: the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). The RARs require heterodimerization with RXRs; in addition, RXRs can form homodimers, which can bind to DNA response elements that are either distinct or the same as those bound by the RAR/RXR heterodimers. Therefore, the two retinoid pathways provide sequences that are specific for effective DNA binding and activation of target genes. We have developed several series of novel synthetic retinoids that selectively interact with RXR/RXR homodimers and RAR/RXR heterodimers. We show here that SR11236 and SR11246, which are RXR-selective analogs, had little ability to inhibit clonal growth and induce differentiation of leukemic cells (HL- 60 cells and fresh acute myeloid leukemia cells). However, SR11249, SR11256, and LGD1069, which activated both RXR/RXR homodimers and RAR/RXR heterodimers, could inhibit clonal growth and induce differentiation of HL-60 cells as well as leukemic cells from patients, including those with acute promyelocytic leukemia (APL). This is similar to results observed with RAR/RXR-specific ligands. Interestingly, the combination of ATRA and either SR11249, SR11256, or LGD1069 showed synergistic effects in inducing differentiation of HL-60 cells. A retinoid (SR11238) with strong anti-AP-1 activity that did not activate the RARs and RXRs for gene transcription from the response element TREpal was inactive in our assay systems, suggesting that the antiproliferative effects of retinoids on leukemic cells is not mediated by inhibiting the AP-1 pathway. We conclude that the RAR/RXR pathway is more important than RXR/RXR pathway for differentiation and proliferation of acute myeloid leukemic cells, and certain retinoids or combination of retinoids with both RAR and RXR specificities may synergistically enhance the differentiation activity of ATRA, which may be relevant in several clinical situations.

Description: Lymphocytes in umbilical cord blood and neonatal peripheral blood have been shown to have less ability in an immune reaction. In our present experimental approach to address this issue, we made use of the cord blood of full-term birth infants to investigate the expression of the interleukin- 2 receptor gamma (IL-2Rgamma) chain that is shared with receptors for IL-4, IL-7, IL-9, and IL-15 as well as IL-2. The gamma chain expression in cord blood lymphocytes was about one-third that in the lymphocytes of adults, whereas no significant difference between cord blood and adult monocytes was observed. A reduced expression of the gamma chain was observed in all of the CD4+ T cells, CD8+ T cells, gamma-delta T cells, B cells, CD16+ natural killer (NK) cells, and CD56(bright) NK cells of the cord blood lymphocytes. The reduced gamma chain expression reached two-thirds of that in adults after 3 days of culture in vitro and in infants 3 days after birth, thus implying that the increase in the gamma chain may significantly contribute to the prevention of neonatal infection.

Description: Retinoic acid modulates proliferation and differentiation of a wide variety of normal and leukemic cells through two distinct families of transcriptional factors: the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). A stereoisomer of retinoic acid, 9-cis- retinoic acid, is a high-affinity ligand for RXR and binds efficiently to RAR. In contrast, all-trans-retinoic acid interacts 40-fold less efficiently with RXR as compared with RAR. To clarify the biologic role of retinoic acid compounds (all-trans,- 9-cis-, and 13-cis-retinoic acid) in hematopoietic cells, we studied their effects on clonal growth, differentiation, and expression of RAR-alpha and RXR-alpha genes in HL-60 cells. At very low concentrations (10(-15) to 10(-12) mmol/L), each retinoid enhanced clonal growth of HL-60 cells. These concentrations of the retinoids had no capacity to induce differentiation of leukemic cells as measured by ability either to reduce nitroblue tetrazolium and to express CD11b antigens, suggesting that retinoids at very low concentrations may stimulate proliferation of leukemic cells rather than induce their differentiation. These findings may help explain why patients with acute promyelocytic leukemia may relapse while receiving retinoic acids. With continuous therapy, retinoids are metabolized rapidly with increased urinary excretion, lowering their plasma levels to a range that may stimulate proliferation without inducing differentiation of leukemic cells. In contrast, we found that at higher concentrations (〉 or = 10(-11) mmol/L) each retinoid inhibited clonal growth, reduced c-myc RNA levels, and induced differentiation of HL-60 cells. 9-cis-retinoic acid was a slightly more potent inducer of differentiation than all-trans- retinoic acid; the mechanism for this increased potency and its clinical potential requires additional studies. Steady-state levels of RAR-alpha mRNA in HL-60 cells were not affected by either 9-cis- and all-trans-retinoic acid. In contrast, 9-cis-retinoic acid, but not all- trans-retinoic acid, reduced RXR-alpha mRNA accumulation in a dose- dependent manner.