To investigate the efficacy and safety of granulocyte transfusion combined with granulocyte colony stimulating factor (G-CSF) in severe infection patients with severe aplastic anemia (SAA).
Fifty-six patients in severe infections with SAA who had received granulocyte transfusions combined with G-CSF from 2006 to 2012 in our department were analyzed. A retrospective analysis was undertaken to investigate the survival rates (at 30 days, 90 days and 180 days), the responses to treatment (at 7 days and 30 days, including microbiological, radiographic and clinical responses), the neutrophil count and adverse events after transfusion.
All SAA patients with severe infections were treated with granulocyte transfusions combined with G-CSF. Forty-seven patients had received antithymocyte globulin/antilymphocyte globulin and cyclosporine A as immunosuppressive therapy. The median number of granulocyte components transfused was 18 (range, 3–75). The survival at 30 days, 90 days and 180 days were 50(89%), 39(70%) and 37(66%) respectively. Among 31 patients who had invasive fungal infections, the survival at 30 days, 90 days and 180 days were 27(87%), 18(58%) and 16(52%) respectively. Among the 25 patients who had refractory severe bacterial infections, the survival at 30 days, 90 days and 180 days were 23(92%), 21(84%) and 21(84%) respectively. Survival rate was correlated with hematopoietic recovery. Responses of patients at 7 and 30 days were correlated with survival rate. Common adverse effects of granulocyte transfusion included mild to moderate fever, chills, allergy and dyspnea.
Granulocyte transfusions combined with G-CSF could be an adjunctive therapy for treating severe infections of patients with SAA.
In Asia, young-onset type 2 diabetes (YOD) is characterized by obesity and increased risk for cardiovascular disease (CVD). In a genome-wide association study (GWAS) of 99 Chinese obese subjects with familial YOD diagnosed before 40-year-old and 101 controls, the T allele of rs1408888 in intron 1 of DACH1(Dachshund homolog 1) was associated with an odds ratio (OR) of 2.49(95% confidence intervals:1.57–3.96, P = 8.4×10−5). Amongst these subjects, we found reduced expression of DACH1 in peripheral blood mononuclear cells (PBMC) from 63 cases compared to 65 controls (P = 0.02). In a random cohort of 1468 cases and 1485 controls, amongst top 19 SNPs from GWAS, rs1408888 was associated with type 2 diabetes with a global P value of 0.0176 and confirmation in a multiethnic Asian case-control cohort (7370/7802) with an OR of 1.07(1.02–1.12, Pmeta = 0.012). In 599 Chinese non-diabetic subjects, rs1408888 was linearly associated with systolic blood pressure and insulin resistance. In a case-control cohort (n = 953/953), rs1408888 was associated with an OR of 1.54(1.07–2.22, P = 0.019) for CVD in type 2 diabetes. In an autopsy series of 173 non-diabetic cases, TT genotype of rs1408888 was associated with an OR of 3.31(1.19–9.19, P = 0.0214) and 3.27(1.25–11.07, P = 0.0184) for coronary heart disease (CHD) and coronary arteriosclerosis. Bioinformatics analysis revealed that rs1408888 lies within regulatory elements of DACH1 implicated in islet development and insulin secretion. The T allele of rs1408888 of DACH1 was associated with YOD, prediabetes and CVD in Chinese.
China experienced several large measles outbreaks in the past two decades, and a series of enhanced control measures were implemented to achieve the goal of measles elimination. Molecular epidemiologic surveillance of wild-type measles viruses (MeV) provides valuable information about the viral transmission patterns. Since 1993, virologic surveillnace has confirmed that a single endemic genotype H1 viruses have been predominantly circulating in China. A component of molecular surveillance is to monitor the genetic characteristics of the hemagglutinin (H) gene of MeV, the major target for virus neutralizing antibodies.
Analysis of the sequences of the complete H gene from 56 representative wild-type MeV strains circulating in China during 1993–2009 showed that the H gene sequences were clustered into 2 groups, cluster 1 and cluster 2. Cluster1 strains were the most frequently detected cluster and had a widespread distribution in China after 2000. The predicted amino acid sequences of the H protein were relatively conserved at most of the functionally significant amino acid positions. However, most of the genotype H1 cluster1 viruses had an amino acid substitution (Ser240Asn), which removed a predicted N-linked glycosylation site. In addition, the substitution of Pro397Leu in the hemagglutinin noose epitope (HNE) was identified in 23 of 56 strains. The evolutionary rate of the H gene of the genotype H1 viruses was estimated to be approximately 0.76×10−3 substitutions per site per year, and the ratio of dN to dS (dN/dS) was <1 indicating the absence of selective pressure.
Although H genes of the genotype H1 strains were conserved and not subjected to selective pressure, several amino acid substitutions were observed in functionally important positions. Therefore the antigenic and genetic properties of H genes of wild-type MeVs should be monitored as part of routine molecular surveillance for measles in China.
Different cell/tissue derived extracellular matrix (ECM) display subtle differences that might provide important cues for proliferation and differentiation of cells in vitro or in vivo. However, the bioactivities of different ECMs in vitro were not fully understood. In this study, osteoblasts-derived and fibroblast-derived ECM-coated cell culture dishes were prepared respectively by culturing osteoblastic MC3T3-E1 cells and rat fibroblast then decellularizing the cultures. We investigated the bioactivities of the two different ECMs coated on cell culture plates using cellular, biochemical and molecular method. The proliferative activity of the bone marrow-derived mesenchymal stem cells (BMSCs) cultured on osteoblast-ECM was lower than for BMSCs grown on fibroblast-ECM. Compared with the BMSCs cultured on fibroblast-derived ECM, the cells grown on osteoblastic ECM showed enhanced alkaline phosphatase (ALP) activity, higher BMP-2 and osteopontin protein levels, increased secreted calcium content, and higher levels of runt-related transcriptional factor 2 (Runx 2) and osteocalcin (OCN) mRNA. Knockdown of BMP-2 or FGF-2 with shRNA transfection hardly effected osteoblastic differentiation or proliferation of MC3T3-E1 seeded on osteoblast-ECM or fibroblast-ECM. Therefore, the osteoblastic ECM had better osteoinductive potential and lower proliferative effect than fibroblastic ECM, and the two ECM presented enough bioactivity, knockdown of growth factors had no significant effect on differentiation and proliferation of re-seeded cells.
Extracellular matrix; Bone marrow-derived mesenchymal stem cells; Osteoblasts; siRNA; Proliferation; Differentiation
Diabetes and obesity are complex diseases associated with insulin resistance and fatty liver. The latter is characterized by dysregulation of the Akt, AMP-activated protein kinase (AMPK), and IGF-I pathways and expression of microRNAs (miRNAs). In China, multicomponent traditional Chinese medicine (TCM) has been used to treat diabetes for centuries. In this study, we used a three-herb, berberine-containing TCM to treat male Zucker diabetic fatty rats. TCM showed sustained glucose-lowering effects for 1 week after a single-dose treatment. Two-week treatment attenuated insulin resistance and fatty degeneration, with hepatocyte regeneration lasting for 1 month posttreatment. These beneficial effects persisted for 1 year after 1-month treatment. Two-week treatment with TCM was associated with activation of AMPK, Akt, and insulin-like growth factor-binding protein (IGFBP)1 pathways, with downregulation of miR29-b and expression of a gene network implicated in cell cycle, intermediary, and NADPH metabolism with normalization of CYP7a1 and IGFBP1 expression. These concerted changes in mRNA, miRNA, and proteins may explain the sustained effects of TCM in favor of cell survival, increased glucose uptake, and lipid oxidation/catabolism with improved insulin sensitivity and liver regeneration. These novel findings suggest that multicomponent TCM may be a useful tool to unravel genome regulation and expression in complex diseases.
Background. Mannose-binding lectin (MBL) is a pattern-recognition molecule, which functions as a first line of host defense. Pandemic H1N1 (pdmH1N1) influenza A virus caused massive infection in 2009 and currently circulates worldwide. Avian influenza A H9N2 (H9N2/G1) virus has infected humans and has the potential to be the next pandemic virus. Antiviral function and immunomodulatory role of MBL in pdmH1N1 and H9N2/G1 virus infection have not been investigated.
Methods. In this study, MBL wild-type (WT) and MBL knockout (KO) murine models were used to examine the role of MBL in pdmH1N1 and H9N2/G1 virus infection.
Results. Our study demonstrated that in vitro, MBL binds to pdmH1N1 and H9N2/G1 viruses, likely via the carbohydrate recognition domain of MBL. Wild-type mice developed more severe disease, as evidenced by a greater weight loss than MBL KO mice during influenza virus infection. Furthermore, MBL WT mice had enhanced production of proinflammatory cytokines and chemokines compared with MBL KO mice, suggesting that MBL could upregulate inflammatory responses that may potentially worsen pdmH1N1 and H9N2/G1 virus infections.
Conclusions. Our study provided the first in vivo evidence that MBL may be a risk factor during pdmH1N1 and H9N2/G1 infection by upregulating proinflammatory response.
Background: In heart, the extracellular matrix (ECM), produced by cardiac fibroblasts, is a potent regulator of heart,s function and growth, and provides a supportive scaffold for heart cells in vitro and in vivo. Cardiac fibroblasts are subjected to mechanical loading all the time in vivo. Therefore, the influences of mechanical loading on formation and bioactivity of cardiac fibroblasts, ECM should be investigated.
Methods: Rat cardiac fibroblasts were cultured on silicone elastic membranes and stimulated with mechanical cyclic stretch. After removing the cells, the ECMs coated on the membranes were prepared, some ECMs were treated with heparinase II (GAG-lyase), then the collagen, glycosaminoglycan (GAG) and ECM proteins were assayed. Isolated neonatal rat ventricular cells were seeded on ECM-coated membranes, the viability and lactate dehydrogenase (LDH) activity of the cells after 1-7 days of culture was assayed. In addition, the ATPase activity and related protein level, glucose consumption ratio and lactic acid production ratio of the ventricular cells were analyzed by spectrophotometric methods and Western blot.
Results: The cyclic stretch increased collagen and GAG levels of the ECMs, and elevated protein levels of collagen I and fibronectin. Compared with the ECMs produced by unstretched cardiac fibroblasts, the ECMs of mechanically stretched fibroblasts improved viability and LDH activity, elevated the Na+/K+-ATPase activity, sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity and SERCA 2a protein level, glucose consumption ratio and lactic acid production ratio of ventricular cells seeded on them. The treatment with heparinase II reduced GAG levels of these ECMs, and lowered these metabolism-related indices of ventricular cells cultured on the ECMs.
Conclusions: Mechanical stretch promotes ECM formation of cardiac fibroblasts in vitro, the ECM of mechanically stretched cardiac fibroblasts improves metabolic activity of ventricular cells cultured in vitro, and the GAG of the ECMs is involved in regulating metabolic activity of ventricular cells.
mechanical stretch; cardiac fibroblast; extracellular matrix; glycosaminoglycan; metabolism.
This study aims to investigate the expression of delta-like 1 (DLK1) gene in the bone marrow cells of patients with myelodysplastic syndromes (MDS) and to explore its molecular characteristics for the early diagnosis of MDS.
The expression of DLK1 mRNA in the bone marrow cells of cases with MDS, acute myeloid leukemia (AML), and normal control groups were measured by real-time polymerase chain reaction and were analyzed for clinical significance.
Significantly higher expression of DLK1 mRNA was observed in the bone marrow cells of MDS patients (0.7342±0.3652) compared with the normal control group (0.4801±0.1759) (P<0.05). The expression of DLK1 mRNA had a positive correlation with the proportion of bone marrow blasts (r=0.467, P<0.05). Moreover, DLK1 mRNA expression was significantly increased as MDS progressed (P<0.05). Patients with abnormal karyotypes exhibited significantly higher expression of DLK1 mRNA (0.9007±0.4334) than those with normal karyotypes (0.6411±0.2630) (P<0.05). Subsequently, patients with highly expressed DLK1 (≥0.8) presented significantly higher malignant clone burden (0.4134±0.3999) than those with lower DLK1 expression (<0.8),(0.1517±0.3109), (P<0.05).
The DLK1 gene was highly expressed in MDS patients, and was increased as MDS progressed. The expression of DLK1 mRNA was positively correlated with the proportion of the bone marrow blasts. A high expression of DLK1 gene suggested a higher malignant clone burden of MDS.
DLK1 gene; myelodysplastic syndromes; expression
Telomere shortening has been reported to be related to oxidative stress (OS) associated with the aging process and aging-associated diseases, including Alzheimer’s disease (AD). We measured the methylated and non-methylated telomere lengths in the peripheral blood mononuclear cells of 34 AD patients and 49 healthy controls by a Southern blotting analysis, using methylation-sensitive and - insensitive restriction enzyme isoschizomers, MspI and HpaII. AD patients bore normal mean telomere lengths and had an unchanged distribution of the telomere length in peripheral leukocytes. However, the subtelomeres in the shortest telomeres were relatively more methylated in AD patients of both genders, compared with age-matched controls. We observed that the pathogenesis of AD was associated with the epigenetic condition of the subtelomere, but not on the overall telomere length and distribution. The relative elevation of subtelomeric methylation of short telomeres in peripheral blood leukocytes may be a characteristic of AD. This implies that leukocytes containing short telomeres with less methylated subtelomeres tend to be removed faster from the peripheral blood in AD patients.
Alzheimer’s disease; Telomere; Subtelomere; DNA methylation
To investigate the expression of TET2 mRNA and protein in the bone marrow mononuclear cells (BMMNC) of patients with myelodysplastic syndrome (MDS) and its clinical significance.
The expression of TET2 mRNA and protein in bone marrow mononuclear cells (BMMNC) of 32 patients with MDS and 20 healthy donors was examined by qPCR and Western blot.
The expression of TET2 mRNA in BMMNC was down-regulated in MDS patients compared with the donor group [(0.41±0.28)% vs. (1.07±0.56)%] (P<0.001). Compared with lower expression group (TET2<0.4) [(6.53±6.17)%], patients with higher expression of TET2 (≥0.4) presented significantly lower proportion of bone marrow blasts [(1.21±1.56)%] (P<0.05). The expression of TET2 mRNA in BMMNC of MDS patients was inversely correlated with malignant clone burden (r=-0.398, P<0.05) and IPSS (r=-0.412, P<0.05). The expression of TET2 protein was down-regulated in MDS patients compared with that in the donor group.
The mRNA and protein expression of TET2 in BMMNC of MDS patients is decreased, which might be useful as an important parameter for the evaluation of MDS clone burden.
myelodysplastic syndrome; TET2 gene; clinical features
Diagnostic information for psychiatric research often depends on both clinical interviews and medical records. Although discrepancies between these two sources are well known, there have been few studies into the degree and origins of inconsistencies.
We compared data from structured interviews and medical records on 1,970 Han Chinese women with recurrent DSM-IV major depression (MD). Correlations were high for age at onset of MD (0.93) and number of episodes (0.70), intermediate for family history (+0.62) and duration of longest episode (+0.43) and variable but generally more modest for individual depressive symptoms (mean kappa = 0.32). Four factors were identified for twelve symptoms from medical records and the same four factors emerged from analysis of structured interviews. Factor congruencies were high but the correlation of factors between interviews and records were modest (i.e. +0.2 to +0.4).
Structured interviews and medical records are highly concordant for age of onset, and the number and length of episodes, but agree more modestly for individual symptoms and symptom factors. The modesty of these correlations probably arises from multiple factors including i) inconsistency in the definition of the worst episode, ii) inaccuracies in self-report and iii) difficulties in coding medical records where symptoms were recorded solely for clinical purposes.
In European and US studies, patients with major depressive disorder (MDD) report more stressful life events (SLEs) than controls, but this relationship has rarely been studied in Chinese populations.
Sixteen lifetime SLEs were assessed at interview in two groups of Han Chinese women: 1970 clinically ascertained with recurrent MDD and 2597 matched controls. Diagnostic and other risk factor information was assessed at personal interview. Odds ratios (ORs) were calculated by logistic regression.
60% of controls and 72% of cases reported at least one lifetime SLE. Fourteen of the sixteen SLEs occurred significantly more frequently in those with MDD (median odds ratio of 1.6). The three SLEs most strongly associated with risk for MDD (OR > 3.0) preceded the onset of MDD the majority of the time: rape (82%), physical abuse (100%) and serious neglect (99%).
Our results may apply to females only. SLEs were rated retrospectively and are subject to biases in recollection. We did not assess contextual information for each life event.
More severe SLEs are more strongly associated with MDD. These results support the involvement of psychosocial adversity in the etiology of MDD in China.
Major depressive disorder; Stressful life event; Social adversity; Symptom
While few children and young adults have cross-protective antibodies to the pandemic H1N1 2009 (pdmH1N1) virus, the illness remains mild. The biological reasons for these epidemiological observations are unclear. In this study, we demonstrate that the bulk memory cytotoxic T lymphocytes (CTLs) established by seasonal influenza viruses from healthy individuals who have not been exposed to pdmH1N1 can directly lyse pdmH1N1-infected target cells and produce gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Using influenza A virus matrix protein 1 (M158-66) epitope-specific CTLs isolated from healthy HLA-A2+ individuals, we further found that M158-66 epitope-specific CTLs efficiently killed both M158-66 peptide-pulsed and pdmH1N1-infected target cells ex vivo. These M158-66-specific CTLs showed an effector memory phenotype and expressed CXCR3 and CCR5 chemokine receptors. Of 94 influenza A virus CD8 T-cell epitopes obtained from the Immune Epitope Database (IEDB), 17 epitopes are conserved in pdmH1N1, and more than half of these conserved epitopes are derived from M1 protein. In addition, 65% (11/17) of these epitopes were 100% conserved in seasonal influenza vaccine H1N1 strains during the last 20 years. Importantly, seasonal influenza vaccination could expand the functional M158-66 epitope-specific CTLs in 20% (4/20) of HLA-A2+ individuals. Our results indicated that memory CTLs established by seasonal influenza A viruses or vaccines had cross-reactivity against pdmH1N1. These might explain, at least in part, the unexpected mild pdmH1N1 illness in the community and also might provide some valuable insights for the future design of broadly protective vaccines to prevent influenza, especially pandemic influenza.
Atrial natriuretic peptide (ANP) provides a potent defense mechanism against volume overload in mammals. Its primary receptor, natriuretic peptide receptor-A (NPR-A), is localized mostly in the kidney, but also is found in hypothalamic areas involved in body fluid volume regulation. Acute glucocorticoid administration produces potent diuresis and natriuresis, possibly by acting in the renal natriuretic peptide system. However, chronic glucocorticoid administration attenuates renal water and sodium excretion. The precise mechanism underlying this paradoxical phenomenon is unclear. We assume that chronic glucocorticoid administration may activate natriuretic peptide system in hypothalamus, and cause volume depletion by inhibiting dehydration-induced water intake. Volume depletion, in turn, compromises renal water excretion. To test this postulation, we determined the effect of dexamethasone on dehydration-induced water intake and assessed the expression of NPR-A in the hypothalamus. The rats were deprived of water for 24 hours to have dehydrated status. Prior to free access to water, the water-deprived rats were pretreated with dexamethasone or vehicle. Urinary volume and water intake were monitored. We found that dexamethasone pretreatment not only produced potent diuresis, but dramatically inhibited the dehydration-induced water intake. Western blotting analysis showed the expression of NPR-A in the hypothalamus was dramatically upregulated by dexamethasone. Consequently, cyclic guanosine monophosphate (the second messenger for the ANP) content in the hypothalamus was remarkably increased. The inhibitory effect of dexamethasone on water intake presented in a time- and dose-dependent manner, which emerged at least after 18-hour dexamethasone pretreatment. This effect was glucocorticoid receptor (GR) mediated and was abolished by GR antagonist RU486. These results indicated a possible physiologic role for glucocorticoids in the hypothalamic control of water intake and revealed that the glucocorticoids can act centrally, as well as peripherally, to assist in the normalization of extracellular fluid volume.
Establishing botanical extracts as globally-accepted polychemical medicines and a new paradigm for disease treatment, requires the development of high-level quality control metrics. Based on comprehensive chemical and biological fingerprints correlated with pharmacology, we propose a general approach called PhytomicsQC to botanical quality control.
Incorporating the state-of-the-art analytical methodologies, PhytomicsQC was employed in this study and included the use of liquid chromatography/mass spectrometry (LC/MS) for chemical characterization and chemical fingerprinting, differential cellular gene expression for bioresponse fingerprinting and animal pharmacology for in vivo validation. A statistical pattern comparison method, Phytomics Similarity Index (PSI), based on intensities and intensity ratios, was used to determine the similarity of the chemical and bioresponse fingerprints among different manufactured batches.
Eighteen batch samples of Huangqin Tang (HQT) and its pharmaceutical grade version (PHY906) were analyzed using the PhytomicsQC platform analysis. Comparative analysis of the batch samples with a clinically tested standardized batch obtained values of PSI similarity between 0.67 and 0.99.
With rigorous quality control using analytically sensitive and comprehensive chemical and biological fingerprinting, botanical formulations manufactured under standardized manufacturing protocols can produce highly consistent batches of products.
The telomeres of somatic cells become shorter with individual aging. However, no significant change in subtelomeric methylation of somatic cells with aging has yet been reported.
Telomere lengths of the peripheral blood cells of 148 normal Japanese were analyzed by Southern blotting using methylation-sensitive and -insensitive isoschizomers.
With aging, long telomeres decrease and short telomeres increase, and the contents of the telomeres with methylated subtelomere increase in long telomeres, thus leading us to postulate that telomeres with less methylated subtelomeres tend to become shortened faster.
A telomere length distribution analysis with methylation-sensitive and -insensitive isoschizomer seems to be a useful tool to assess the subtelomeric methylation status of the somatic cell population. The subtelomeric methylation of peripheral blood cells is also indicated to be an indicator for aging-associated genomic changes.
Telomere; Aging; DNA methylation; Japanese
Nephrectomized rats have widely been used to study chronic renal failure. Interestingly, renal cell carcinoma occurred in the remnant kidney after uninephrectomy (UNX). In this study, we probed insulin-like growth factor (IGF)-1 signaling pathway in UNX-induced renal cancer. Adult male Sprague-Dawley rats were randomized into two groups: UNX rats (n = 22) and sham-operated rats (n = 12). Rats were killed at 3, 7, and 10 months. After 7 months after nephrectomy, the UNX rats developed renal cell carcinoma with increased expression of proliferating cell nuclear antigen, and 68.2% (15/22) of the animals exhibited invasive carcinoma. Western blot demonstrated significant down-regulation of IGF binding protein 3 contrasting with the up-regulation of protein kinase Cζ and Akt/protein kinase B in the renal cancer tissues. These findings indicate a unique rat model of UNX-induced renal cancer associated with enhanced IGF-1 signaling pathway.
A glucosamine 6-phosphate deaminase homologue from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.4 Å resolution.
The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution and belong to space group P212121, with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å.
SMU.636; Streptococcus mutans; glucosamine 6-phosphate deaminase
A number of clinical features potentially reflect an individual's familial vulnerability to major depression (MD), including early age at onset, recurrence, impairment, episode duration, and the number and pattern of depressive symptoms. However, these results are drawn from studies that have exclusively examined individuals from a European ethnic background. We investigated which clinical features of depressive illness index familial vulnerability in Han Chinese females with MD.
We used lifetime MD and associated clinical features assessed at personal interview in 1,970 Han Chinese women with DSM-IV MD between 30–60 years of age. Odds Ratios were calculated by logistic regression.
Individuals with a high familial risk for MD are characterized by severe episodes of MD without known precipitants (such as stress life events) and are less likely to feel irritable/angry or anxious/nervous.
The association between family history of MD and the lack of a precipitating stressor, traditionally a characteristic of endogenous or biological depression, may reflect the association seen in other samples between recurrent MD and a positive family history. The symptomatic associations we have seen may reflect a familial predisposition to other dimensions of psychopathology, such as externalizing disorders or anxiety states. Depression and Anxiety 0:1–6, 2011. © 2011 Wiley-Liss, Inc.
major depression; family history; symptom; life events