[Comparison of EB virus infection between short term and long term use of mycophenolate mofetil for prophylaxis of graft versus host disease after haploidentical hematopoietic stem cell transplantation].

Objective: To investigate the role of short-term use of mycophenolate mofetil (MMF) in EB viral infection and acute graft-versus host disease (GVHD) in patients receiving haploidentical hematopoietic stem cell transplantation (haplo-HSCT) . Method: Adult patients (≥14 years) who were diagnosed with hematological malignancies received haplo-HSCT in Peking University Institute of Hematology from May 2016 to December 2017 were retrospectively reviewed. The median age was 30 (14-60) years old. A total of 498 patients including 277 males and 221 females were enrolled. Donors' median age was 38 (8-66) years old. All patients were classified into long-term use of MMF (n=199), which was defined as 500 mg every 12 hours from day 9 pre-transplant to 250 mg every 12 hours from day 30 after transplant then withdrawal on day 45 to 60 after transplant, and short-term use of MMF (n=299), which was defined as 500 mg every 12 hour from day 9 pre-transplant then withdrawal till neutrophil engraftment. Kaplan-Meier model was used to analyze the cumulative incidence of EBV infection, and the Cox proportional regression model for multivariate analysis. Result: Characteristics including sex, age, disease types, mismatched HLA loci, donor-recipient relationship, donor-recipient blood type, donor age, and donor sex were comparable between two groups (all P＞0.05). According to once, the incidence of EBV viremia, defined as EBV＞103 copies/ml at least once, in short-term group and long-term group was 19.4% (58/299) and 27.6% (55/199) respectively (P=0.046).Donor age and the duration of MMF prophylaxis (short-term group as reference) were associated with EBV viremia according to multivariate analysis [HR=1.022(95%CI 1.006-1.038),1.600(95%CI 1.059-2.418);P=0.006 and 0.026, respectively]. The incidence of grade Ⅱ-Ⅳ and Ⅲ/Ⅳ acute GVHD in long-term and short-term group was 32.2% (64/199) versus 20.7% (62/299)(P=0.005) and 10.1% (20/199) versus 8.0% (24/299) (P=0.427), respectively. Donor sex (female as reference) and duration of MMF prophylaxis (short-term group as reference) were associated with grade Ⅱ-Ⅳ acute GVHD [HR=1.908(95%CI 1.079-3.373),1.752(95%CI 1.161-2.643);P=0.026 and 0.008, respectively].There were no statistical differences in the incidence of CMV viremia, refractory CMV viremia and hemorrhagic cystitis (all P＞0.05) between the two groups. Conclusion: Short-term use of MMF can reduce EBV viremia without increasing the development of acute GVHD in haplo-HSCT patients.

Systematic administration of B vitamins attenuates neuropathic hyperalgesia and reduces spinal neuron injury following temporary spinal cord ischaemia in rats.

BACKGROUND: B vitamins have been demonstrated to be effective in treating chronic pain due to peripheral nerve injury. We investigated whether B vitamins could alleviate neuropathic pain and reduce neuron injury following temporary ischaemia in a rat model of spinal cord ischaemia-reperfusion injury (SCII). METHODS: SCII was produced by transiently blocking the unilateral lumbar arteries in adult male Sprague-Dawley rats. Behavioural and neurochemical signs of neuropathic pain and spinal neuron injury were analysed with and without B vitamin treatment. RESULTS: SCII caused behavioural thermal hyperalgesia and mechanical allodynia and neurochemical alterations, including increased expression of the vanilloid receptor 1 (VR1) and induction of c-Fos, as well as activation of the astrocytes and microglial cells in the spinal cord. Repetitive systemic administration of vitamin B complex (B1/B6/B12 at 33/33/0.5 mg/kg, i.p., daily, for 7-14 consecutive days) significantly reduced thermal hyperalgesia and the increased expression of VR1 and c-Fos, as well as activation of the astrocytes and microglial cells. SCII caused a dramatic decrease of the expression of the rate-limiting enzyme glutamic acid decarboxylase-65 (GAD65), which synthesizes γ-aminobutyric acid (GABA) in the axonal terminals, and ß-III-tubulin, and also caused loss of Nissl bodies in the spinal cord. These alterations were largely prevented and rescued by the B vitamin treatment. CONCLUSIONS: These findings support the idea that the B vitamins are capable of neuroprotection and antinociception during spinal cord injury due to temporary ischaemia. Rescuing the loss of inhibitory GABAergic tone may reduce spinal central sensitization and contribute to B vitamin-induced analgesia.

Cloning expression, monoclonal antibody preparation and serologic study of mammaglobin in breast cancer.

UNLABELLED: Mammaglobin may be a potential serum biomarker for the differential diagnosis of breast cancer. 260 serum samples were collected from 127 untreated breast cancer patients and 133 healthy volunteers to analyze the sera expression of mammaglobin and its implications for both. The expression vector of pGEX-4T-2-Mammaglobin and pBVIL1-Mammaglobin were constructed and transformed into E.coli.HB101 for expression. The mice were immunized with the purified recombinant protein to prepare monoclonal antibody and to detect by ELISA the serum of normal people and breast cancer patients. Recombinant mammaglobin antigen was effectively expressed in E.coli. Two hybridoma cell lines were obtained after the mice were immunized by pGEX-4T-2-mammaglobin. 133 cases of normal serum and 127 cases of breast cancer serum were analyzed by ELISA. The sera expression level of mammaglobin in breast cancer group (average OD value 0.645±0.223) was significantly (p KEYWORDS: mammaglobin; cloning expression; monoclonal antibody; serologic study; breast cancer.

Interleukin-6 is essential for Staphylococcal exotoxin B-induced T regulatory cell insufficiency in nasal polyps.

BACKGROUND: The pathogenesis of nasal polyps is still unclear. There is increasing evidence indicating that Staphylococcal aureus (S. aureus) is associated with the formation of nasal polyps, but the mechanism has not been well documented to date. METHODS: We stimulated cultured nasal polyps and turbinate tissues with Staphylococcal exotoxin B (SEB), detected the expression of pro-inflammatory cytokines (IL-2, IL-6, and IL-8) and T cell cytokines (IFN-gamma, IL-4, IL-5, IL-10, and IL-17) in the supernatants, and evaluated mRNA expression (T-bet, GATA-3, Foxp3, and RORgammat) and frequencies of CD4+CD25+ T regulatory cells (Tregs) in nasal tissues. We also evaluated the effects of blocking IL-6 with monoclonal antibodies to T cell profiles in cultured nasal tissues stimulated by SEB. RESULTS: Levels of IL-6, IFN-gamma and IL-4 increased significantly in SEB-stimulated nasal polyps. Meanwhile, mRNA expressions of T-bet and GATA-3 were significantly up-regulated, while Foxp3 was inhibited and the frequencies of CD4+CD25+ Tregs were decreased after SEB stimulation. After blocking IL-6, the levels of IL-10 and Foxp3 mRNA, as well as the frequencies of CD4+CD25+ Tregs, were significantly increased, while IFN-gamma and IL-4 production and the mRNA expression of T-bet and GATA-3 were significantly inhibited. CONCLUSIONS: SEB is able to modulate pro-inflammatory factors, T-helper type 1/Th2 profiles and suppress Treg activity in cultured nasal polyps, which were rescued by blocking IL-6 activity. Therefore, IL-6 is essential for SEB-induced Treg insufficiency in nasal polyps.

Dual inhibitory roles of geldanamycin on the c-Jun NH2-terminal kinase 3 signal pathway through suppressing the expression of mixed-lineage kinase 3 and attenuating the activation of apoptosis signal-regulating kinase 1 via facilitating the activation of Akt in ischemic brain injury.

It is well documented that heat-shock protein (hsp90) plays an essential role in maintaining stability and activity of its clients. Recent studies have shown that geldanamycin (GA), an inhibitor of hsp90, could decrease the protein of mixed-lineage kinase (MLK) 3 and activate Akt; our previous research documented that MLK3 and Akt and subsequent c-Jun N-terminal kinase (JNK) were involved in neuronal cell death in ischemic brain injury. Here, we investigated whether GA could decrease the protein of MLK3 and activate Akt in rat four-vessel occlusion ischemic model. Our results showed that global cerebral ischemia followed by reperfusion could enhance the association of hsp90 with MLK3, the association of hsp90 with Src, and JNK3 activation. As a result, GA decreased the protein of MLK3 and down-regulated JNK activation. On the other hand, Src kinase was activated and phosphorylated Cbl, which then recruited the p85 subunit of phosphatidylinositol 3-kinase (PI-3K), resulting in PI-3K activation, and as a consequence increased Akt activation, which inhibited ASK1 activation and down-regulated JNK3 activation. In summary, our results indicated that GA showed a dual inhibitory role on JNK3 activation and exerted strong neuroprotection in vivo and in vitro, which provides a new possible approach for stroke therapy.

Periodic mesoporous organosilica hollow spheres with tunable wall thickness.

Periodic mesoporous organosilica (PMO) hollow spheres with tunable wall thickness have been successfully synthesized by a new vesicle and a liquid crystal "dual templating" mechanism, which may be applicable for drug and DNA delivery systems, biomolecular encapsulation, as well as nanoreactors for conducting biological reactions at the molecular levels.

Successful allogeneic bone marrow transplantation (BMT) by injection of bone marrow cells via portal vein: stromal cells as BMT-facilitating cells.

We examined the importance of the coadministration of bone marrow (BM) stromal cells with BM cells via the portal vein. A significant increase in the number of day-14 colony-forming unit-spleen (CFU-S) was observed in the recipient mice injected with hemopoietic stem cells (HSCs) along with donor BM stromal cells obtained after three to four weeks of culture. Histological examination revealed that hematopoietic colonies composed of both donor hemopoietic cells and stromal cells coexist in the liver of these mice. However, when donor HSCs plus BM stromal cells were administered i.v., neither the stimulatory effects on CFU-S formation nor the hemopoietic colonies in the recipient liver were observed. These findings suggest that the interaction of HSCs with stromal cells in the liver is the first crucial step for successful engraftment of allogeneic HSCs. It is likely that donor stromal cells and HSCs trapped in the liver migrate into the recipient BM and spleen, where they form CFU-BM and CFU-S, respectively.

Exophiala jeanselmei causing late endophthalmitis after cataract surgery.

PURPOSE: To report two cases of late endophthalmitis caused by Exophiala jeanselmei after cataract surgery. METHODS: Case reports, including clinical evaluation, direct examination, and culture of the aqueous humor. RESULTS: In each case, samples from the anterior chamber had positive growth of yeasts with toruloid hyphae and pseudohyphae. Intravitreal and anterior chamber amphotericin B were used in both cases. Apparent clinical resolution was achieved, but after 3 months in one case and 6 months in the other the infection recurred more aggressively, with severe endophthalmitis leading to ocular atrophy. CONCLUSION: E. jeanselmei causes a severe intraocular infection and isolation, and identification of the agent ensures proper diagnosis and treatment. After clinical resolution of the infection, careful and long-term follow-up is recommended to promptly detect relapse and immediately reintroduce treatment.

Stimulatory effects of hepatocyte growth factor on hemopoiesis of SCF/c-kit system-deficient mice.

In this study, we report that W/W mutant mice, which have severe macrocytic anemia caused by a deficit of extracellular domain in c-kit molecules and therefore die perinatally, have hemopoietic stem cells (HSCs) and mature hematolymphoid cells in the bone marrow (BM), thymus, and spleen, although there are significant decreases in cell counts. Moreover, the mitogen-induced proliferative response, mixed lymphocyte reaction, and anti-SRBC plaque formation of spleen cells in W/W mice are similar to those in age-matched +/? littermates and normal mice, suggesting that the SCF/c-kit system is necessary for cell proliferation but not essential for HSCs to differentiate. We next examine the stimulatory effects of hepatocyte growth factor (HGF) on hemopoiesis in W/W mice. HGF has a stimulatory effect on the colony formation (CFU-C) of W/W BM cells when cultured using either a methylcellulose assay (containing cytokines) or a long-term culture (LTC) assay. A similar stimulatory effect of HGF is observed in the other W or SI locus-mutant mice (W/Wv and SI/SId mice), which show less severe anemia than W/W. The numbers of nonadherent cells and cobblestone colonies significantly increase in the LTCs using their BM cells. In addition, in vivo administration of HGF shows a transient increase in the CFU-C counts in BM cells and peripheral blood cells. RBC, WBC, and platelet counts also increased. These results suggest that the SCF/c-kit system is not essential to hemopoiesis but that a compensatory system such as the HGF/c-met system functions in the SCF/c-kit system-deficient mice.

Physical manipulation of subcutaneously implanted cardiac muscle in rat increased plasma atrial natriuretic peptide concentration.

UNLABELLED: To examine the effects of physical stretch on cardiac muscle endocrine activity, the authors transplanted whole neonatal hearts subcutaneously into the back and the ears of the correspondent mother (n = 9). Seven days later, physical manipulation was applied on the implanted heart by stretching the skin and the subcutaneous tissue encasing the implanted cardiac muscle, for a period of five to ten minutes. Such manipulation was repeated approximately every seven days postoperatively for a total of two to four times for each rat. The plasma atrial natriuretic peptide (ANP) levels were measured by radioimmunoassay prior to and immediately following manipulation. Postmanipulation plasma ANP levels were found to increase from the premanipulation levels. At two weeks postimplant, the average increase was 290% with the highest single-specimen increase being nearly twelvefold. The increases observed at two and three weeks following implantation had Signed Rank Test p values of 0.015 and 0.042 respectively. The viability of the implanted hearts was confirmed by cell culture. Light microscopic immunocytochemistry detected ANP immunoreactivity in the implanted cardiocytes. The elevated plasma ANP concentration induced by the manipulation appeared to be correlated with the functional status of the implanted cardiocytes. IN CONCLUSION: (1) Subcutaneously transplanted neonatal myocardiocytes survived for at least three to four weeks while retaining the ability to produce ANP. (2) Physical manipulation of implanted heart induced ANP release. Therefore, cardiac ANP production and release is indeed stimulated by physical stretching.

[A morphological study of retinal damages by intraocular lead].

The morphological changes in the retina were observed by light and electron microscopy at intervals after implantation of lead into the vitreous of rabbit eyes. The results revealed progressive structural disorders under both modalities of examination, indicating that lead had toxic effects on the retina.

Detection of endothelin-like immunoreactivity in epithelium and fibroblasts of the human umbilical cord.

We studied tissue sections of freshly obtained full-term and premature human umbilical cords using polyclonal antibody to endothelin and immunocytochemistry. Endothelin immunoreactivity was detected in the cytoplasm of epithelial cells and primitive fibroblasts, but not in the endothelial cells of both full-term and premature umbilical cords. Immunoelectron microscopy using indirect immunogold staining technique localized endothelin immunoreactivity to the cytoplasm of the epithelial cells and fibroblasts but not confined to any particular structures. No endothelin immunoreactivity was detected in the nucleus or on the cell membrane. Pre-absorption tests with synthetic endothelin-1, -2, and -3 independently established that the immunoreactivity represented endothelin-1 and -2, but not -3. The presence of endothelin-1 and -2-like immunoreactive materials in epithelial cells and fibroblasts of human umbilical cord suggests a role of endothelin in parturition.

Presence of a lysosomal enzyme, arylsulfatase-A, in the prelysosome-endosome compartments of human cultured fibroblasts.

Although endosomes and lysosomes are associated with different subcellular functions, we present evidence that a lysosomal enzyme, arylsulfatase-A, is present in prelysosomal vesicles which constitute part of the endosomal compartment. When human cultured fibroblasts were subfractionated with Percoll gradients, arylsulfatase-A activity was enriched in three subcellular fractions: dense lysosomes, light lysosomes, and light membranous vesicles. Pulsing the cells for 1 to 10 min with the fluid-phase endocytic marker, horseradish peroxidase, showed that endosomes enriched with the marker were distributed partly in the light lysosome fraction but mainly in the light membranous fraction. By pulsing the fibroblasts for 10 min with horseradish peroxidase conjugated to colloidal gold and then staining the light membranous and light lysosomal fractions for arylsulfatase-A activity with a specific cytochemical technique, the endocytic marker was detected under the electron microscope in the same vesicles as the lysosomal enzyme. The origin of the lysosomal enzyme in this endosomal compartment was shown not to be acquired through mannose 6-phosphate receptor-mediated endocytosis of enzymes previously secreted from the cell. Together with our recent finding that the light membranous fraction contains prelysosomes distinct from bona fide lysosomes and was highly enriched with newly synthesized arylsulfatase-A molecules, these results demonstrate that prelysosomes also constitute part of the endosomal compartment to which intracellular lysosomal enzymes are targeted.

Effect of ammonium chloride on subcellular distribution of lysosomal enzymes in human fibroblasts.

Three subcellular fractions enriched in lysosomal enzyme activities have been isolated recently from human cultured fibroblasts with Percoll gradients: the dense lysosomes (DL), light lysosomes (LL), and light membranous vesicles (LM). They were shown to have different morphological, cytochemical, biochemical, and immunological properties. We now report on the dramatic but different effects of a primary amine, NH4Cl, on these subfractions. The lysosomes, as detected with a specific ultrastructural cytochemical stain for the lysosomal enzyme, arylsulfatase A, were swollen significantly in all these fractions, increasing their volumes by 64% (DL), 53% (LL), and 95% (LM), respectively. When arylsulfatase A enzyme activity was monitored, about half of the DL content was diverted to the LL. However, when newly synthesized arylsulfatase A enzyme protein was monitored with metabolic labeling and immunoprecipitation, about 80% of the enzyme protein was depleted from both the DL and LL. In contrast, neither the enzyme activity nor the newly synthesized enzyme protein of arylsulfatase A was greatly altered in the LM fraction by the treatment. Since primary amines caused newly synthesized lysosomal enzymes to diverge from the lysosomal route to a secretory pathway, it was deduced that (i) the LM fraction corresponded to a prelysosomal compartment whose lysosomal enzyme content was not affected by the amine and was thus proximal to the point of diversion between the secretory and lysosomal pathways; (ii) the LL and DL fractions were distal to the point of diversion since both fractions were depleted of their newly synthesized lysosomal enzyme; and (iii) the sorting of newly synthesized lysosomal enzyme may be different from that of the preexisting pool of the same enzyme since the LL fraction was depleted of its newly synthesized enzyme protein while accumulating excessive enzyme activity.