Enhanced LPS-induced peritonitis in mice deficiency of cullin 4B in macrophages.

Cullin 4B (CUL4B), a member of the cullin protein family, is a scaffold protein of the CUL4B-RING-E3 ligase complex that ubiquitinates intracellular proteins.CUL4B's targets include cell cycle-regulated proteins and DNA replication-related molecules. In this study, we generated myeloid-specific Cul4b-deficient mice (Cul4b(f/y);LysM-Cre(KI/KI)) to investigate the influence of Cul4b deficiency on innate immunity, especially on the function of macrophages. Our results show that an intraperitoneal injection of lipopolysaccharide (LPS) led to a significant decrease in body weights and increased leukocyte infiltrates with increased chemokines in the peritoneal cavity of Cul4b(f/y);LysM-Cre(KI/KI) mice. However, the proinflammatory cytokines, IL-6 and TNF-α did not increase in LPS-injected Cul4b(f/y);LysM-Cre(KI/KI) mice. Furthermore, bone marrow-derived macrophages from Cul4b(f/y);LysM-Cre(KI/KI) mice secreted higher levels of chemokines but lower levels of TNF-α and IL-6 upon LPS stimulation. Of note, increased proliferation of Cul4b-deficient macrophages was also observed. These results show that myeloid-specific Cul4b deficiency worsens LPS-induced peritonitis. In addition, Cul4b deficiency leads to enhanced DNA replication and proliferation, increased production of chemokines but a decreased production of proinflammatory cytokines of macrophages. Our data highlight a new role of cullin family, CUL4B, in the immune system.

Innate immunity drives xenobiotic-induced murine autoimmune cholangitis.

Although primary biliary cirrhosis (PBC) is considered a model autoimmune disease, it has not responded therapeutically to traditional immunosuppressive agents. In addition, PBC may recur following liver transplantation, despite the absence of major histocompatibility complex (MHC) matching, in sharp contrast to the well-known paradigm of MHC restriction. We have suggested previously that invariant natural killer T (iNK T) cells are critical to the initiation of PBC. In this study we have taken advantage of our ability to induce autoimmune cholangitis with 2-octynoic acid, a common component of cosmetics, conjugated to bovine serum albumin (2-OA-BSA), and studied the natural history of pathology in mice genetically deleted for CD4 or CD8 following immunization with 2-OA-BSA in the presence or absence of α-galactosylceramide (α-GalCer). In particular, we address whether autoimmune cholangitis can be induced in the absence of traditional CD4 and CD8 responses. We report herein that CD4 and CD8 knock-out mice immunized with 2-OA-BSA/PBS or 2-OA-BSA/α-GalCer develop anti-mitochondrial antibodies (AMAs), portal infiltrates and fibrosis. Indeed, our data suggest that the innate immunity is critical for immunopathology and that the pathology is exacerbated in the presence of α-GalCer. In conclusion, these data provide not only an explanation for the recurrence of PBC following liver transplantation in the absence of MHC compatibility, but also suggest that effective therapies for PBC must include blocking of both innate and adaptive pathways.

Identification and characterization of IgA antibodies against ß2-glycoprotein I in childhood Henoch-Schönlein purpura.

BACKGROUND: Henoch-Schönlein purpura (HSP) is a common IgA-mediated vasculitis in children. The antigenic target for IgA is to be determined. OBJECTIVE: To test whether ß2-glycoprotein I (ß2GPI) is an antigenic target for IgA in childhood HSP, and to evaluate the clinical implications and pathogenic role of such IgA autoantibodies. METHODS: The reactivity of patients' plasma samples and purified polyclonal IgA with ß2GPI, ß2GPI-derived peptides and endothelial cells was tested by enzyme-linked immunosorbent assay. The association between clinical manifestations and IgA anti-ß2GPI antibodies was also analysed. Finally, IgA-mediated cytotoxicity on endothelial cells was further evaluated. RESULTS: At the acute stage, patients with HSP had significantly higher plasma levels of IgA antibodies against ß2GPI than healthy controls [reference units (RU) 1.14 ± 0.8 vs. 0.42 ± 0.24, P < 0.001]. IgA anti-ß2GPI antibodies were associated with the presence of joint manifestations (with vs. without joint involvement, 1.15 ± 0.64 vs. 0.85 ± 0.47, P = 0.0341) and heavy proteinuria (with vs. without heavy proteinuria, 2.09 ± 2.02 vs. 1.04 ± 0.62, P = 0.0028). Polyclonal IgA from plasma samples positive for IgA anti-ß2GPI antibodies bound well not only to ß2GPI with Kd values < 10(-5) mol L(-1), but also to some ß2GPI-dereived linear peptides (P3, P5, P7, P11 and P12). Moreover, ß2GPI-reactive polyclonal IgA also bound well to endothelial cells and induced complement-dependent cell lysis. CONCLUSION: These findings reveal the clinical and pathogenic relevance of IgA anti-ß2GPI antibodies in childhood HSP and suggest that ß2GPI may be an important autoantigen for HSP.

Aggressive digital papillary adenocarcinoma: a review.

Vigorous treatment of aggressive digital papillary adenocarcinoma (ADPA), including amputation, has been recommended by most authors, but the appropriateness and effectiveness of excision as an alternative to amputation has not been systematically evaluated. To evaluate the appropriateness and effectiveness of excision as an alternative to amputation in the treatment of ADPA, we reviewed the clinical presentations, treatments and patient outcomes presented in case reports on ADPA available on Ovid MEDLINE. We also assessed the results of immunohistochemical staining for proliferation markers in one patient in order to explain the nonaggressive nature of ADPA noted in that patient. Except for the duration of lesions, there was no significant difference in clinical outcome between the excision and amputation groups. We also found that p63 may be a useful marker for distinguishing primary ADPA from metastatic adenocarcinomas. In addition, the intensity of Ki67 expression in tumour cells may be a marker of aggressive behaviour and thus be helpful in therapeutic decision-making. Wide excision with or without sentinel lymph-node biopsy is a feasible alternative to amputation. It should be considered in patients who present with a long-standing history of ADPA without evidence of underlying bone invasion or distant metastasis and with low-intensity expression of proliferation markers.

Amphotericin B up-regulates angiogenic genes in hepatocellular carcinoma cell lines.

BACKGROUND: Amphotericin B (AmB) has a discordant influence on epirubicin (4'-epidoxorubicin) cytotoxicity in hepatocellular carcinoma (HCC). This indicates that the cellular function of HCC may be significantly influenced by AmB. Whether the influence of AmB on HCC has any possibility to influence cancer growth has not been studied. This study was to try and clarify this issue. MATERIALS AND METHODS: Two HCC cell lines including one without augmentation of the epirubicin cytotoxicity by AmB (cell line A; HCC24/KMUH) and one with this effect (cell line B; HCC38/KMUH) were studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and whole human genome microarray (experimental group: 2.5 microg mL(-1) AmB). RESULTS: Differential expressions of genes induced by AmB in two cell lines had no influence on cell proliferation as determined by MTT assay. Only cell line B showed up-regulation of genes related to oxidative stress, acute phase reaction, cytokine-cytokine receptor interaction and complement and coagulation cascades. Among the chemokine genes up-regulated by AmB, five genes (CCL2, CXCL1, CXCL5, CXCL6, IL8) were angiogenic. Cell line B also showed up-regulation of one angiogenic C10orf10 gene and down-regulation of one angiostatic chemokine gene (CXCL10). Up- or down-regulation of other genes in cell line A and B did not show any evidence to promote angiogenesis. CONCLUSION: AmB has the capacity to concomitantly up-regulate angiogenic genes in HCC cells susceptible to AmB-induced oxidative stress.

Induction of IL-10 producing CD4+ T cells with regulatory activities by stimulation with IL-10 gene-modified bone marrow derived dendritic cells.

Dendritic cells (DCs) can induce both tolergenic as well as effective immune responses in the lung. Pulmonary DCs producing interleukin (IL)-10 mediated tolerance induced by respiratory exposure to antigen. IL-10 is an important immunosuppressive cytokine, which inhibits maturation and function of DC. To assess whether IL-10 producing DCs can exert the tolergenic effect through the differentiation of regulatory T cells, bone marrow derived DCs were genetically modified by IL-10 expressing adenovirus. IL-10 gene modified DCs (Ad-IL-10-DC) displayed a characteristic phenotype of immature DCs. Here we showed that in vitro repetitive stimulation of naïve DO11.10 CD4(+) T cells with Ad-IL-10-DCs resulted in a development of IL-10 producing T-cell regulatory cells. These T cells could not proliferate well but also lost their ability to produce interferon-gamma upon restimulation with irradiated splenocytes and ovalbumin peptide. Furthermore, in co-culture experiments these T cells inhibited the antigen-driven proliferation of naïve CD4+ T cells in a dose-dependent manner. Our findings demonstrated that IL-10 producing DCs had the potential to induce the differentiation of Tr1-like cells and suggested their therapeutic use.

Diffusion-Weighted Magnetic Resonance Imaging in Liver Graft Rejection.

OBJECTIVE: The purpose of this study is to evaluate the use of diffusion-weighted magnetic resonance imaging (DWMRI) in the assessment of graft rejection after liver transplantation (LT). METHODS: From June 2017 to January 2018, 32 patients were included in the study with a mean age of 52.3 years. All patients underwent LT. The DWMRI was performed using the apparent diffusion coefficient map and measuring the different b-values (b-400, b-600, b-800, and b-1000). These measurements were compared with the histopathology results. Statistical analysis included t test, analysis of variance, and area under the curve for receiver operating characteristic (ROC). RESULTS: There were 17 patients without rejection and 15 patients with liver graft rejection diagnosed by histopathology. The mean (SD) results between the nonrejection and rejection groups were as follows: b-400 = 1.568 (0.265) vs 1.519 (0.119) (P = .089), b-600 = 1.380 (0.181) vs 1.284 (0.106) (P = .039), b-800 = 1.262 (0.170) vs 1.170 (0.086) (P = .035), b-1000 = 1.109 (0.129) vs 1.098 (0.078) (P = .095); B-values × 10-3 mm2/s. Only b-600 (P = .04) and b-800 (P = .04) values have significant differences between the 2 groups. B-600 showed 90.48% sensitivity and 83.33% specificity (ROC area under the curve = 0.784; P < .001), and b-800 showed 90.38% sensitivity and 83.03% specificity (ROC area under the curve = 0.816; P < .001). The values obtained with the apparent diffusion coefficient in b-800 were clearly differentiated between the mild, moderate, and severe degrees of rejection (P < .001). CONCLUSION: Measurement of b-600 and b-800 values using DWMRI may be used for the diagnosis of graft rejection after LT.

Generation of functionally distinct B lymphocytes from common myeloid progenitors.

Current models of adult haematopoiesis propose that haematopoietic stem cells (HSCs) differentiate into common lymphoid (CLP) and common myeloid (CMP) progenitors and establish an early separation between myeloid and lymphoid lineages. Nevertheless, the developmental potential of CMP-associated B cells suggests the existence of alternate pathways for B lymphopoesis. The aim of this study was to compare the developmental and functional properties of CMP- and CLP-derived B cells. While both populations matured through pro-B cell and transitional B cell intermediates in the bone marrow and spleen, respectively, following transfer into irradiated mice, mature CMP- and CLP-derived B cells exhibit distinct functional responses. Specifically, CMP-derived B cells did not respond to mitogenic stimulation to the same degree as their CLP-derived counterparts and secrete lower levels of IgM and the inflammatory cytokines such as interleukin (IL)-6 and IL-10. Together, these data suggest the existence of multiple pathways for generating functionally distinct B cells from bone marrow precursors.

Dystrophin immunostaining of muscle from Chinese patients with various neuromuscular diseases.

The localization of dystrophin was studied using the immunohistochemical method of diagnostic muscle specimens from 68 patients, aged 9 days to 65 years, with various neuromuscular disorders. Additionally muscle specimens from 2 normal humans and 2 normal mice were used as positive controls, and those from 2 mice with x-linked muscular dystrophy as negative controls. The specimens from all 14 Duchenne muscular dystrophy (DMD) patients, including one with preclinical DMD, showed negative dystrophin staining except for two which had 0.2% to 0.8% positive fibers. The mdx mice also showed negative dystrophin staining. In Becker muscular dystrophy (BMD), muscle fibers stained in a patchy or discontinuous fashion. Two symptomatic DMD carriers exhibited a distinct mosaic pattern of dystrophin positive and negative fibers. In contrast, dystrophin was present in all 7 biopsies from patients with 4 other types of muscular dystrophy (limb-girdle, congenital, myotonic and facioscapulohumeral). Other specimens, those from normal humans and control mice, revealed homogeneous immunostaining along the surface membranes of all muscle fibers. We thus conclude that immunohistochemical dystrophin staining can aid in differentiating DMD from preclinical DMD or BMD, as well as in the detection of DMD carriers.

Spinal anesthesia in MELAS syndrome: a case with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes.

MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) is one of the classic mitochondrial encephalomyopathies with variable clinical presentation and multisystem involvement. Enhanced sensitivity to neuromuscular blockade or anesthetic agents and susceptibility to malignant hyperthermia in these patients have ever been reported, all of which complicate the management of general anesthesia. To avoid these appalling troubles in general anesthesia, we chose spinal anesthesia for a patient with MELAS syndrome receiving appendectomy. The patient obtained adequate anesthesia and good recovery without neurologic sequelae. Although there is little information about the application of regional anesthesia in MELAS patients, we demonstrate that it may be a satisfactory choice. However, it is suggested that regional anesthesia is performed only when neurological abnormalities of spinal cord or peripheral nerves are definitely ruled out.

The role of CD11c(+) hepatic dendritic cells in the induction of innate immune responses.

The role of the liver in the initiation and maintenance of tolerance is a critical immune function that involves multiple lineages of immune cells. Included within these populations are liver dendritic cells (DCs). Although there has been significant work on the phenotypic and functional roles of splenic and bone marrow dendritic cells, as well as their subsets, comparable studies in liver have often been difficult. To address this issue we have isolated, from C57BL/6 mice, relatively pure populations of DCs and compared phenotype and function to the data from spleen using flow cytometry, cell sorter assisted purification and culture, morphology by cytospin and May-Giemsa staining, cell cycle progression, antigen uptake, cytokine production and allo-activation potential. natural killer (NK)1.1(-)CD11c(+) liver DC subsets (conventional DCs, T cell receptor (TcR)beta(-)NK1.1(-)CD11c(+)B220(-) and plasmacytoid DCs, TcRbeta(-)NK1.1(-)CD11c(+)B220(+)) efficiently endocytose dextran and produce significant levels of tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-12 p40 in response to Toll-like receptor (TLR) ligands, with responses higher than splenic DCs. There is also a differential capability of hepatic DCs to respond to innate signals. Indeed, CD11c(+) hepatic DCs have a greater capacity to respond to innate stimulation but are less capable of inducing CpG activated-allogeneic T cells. These data suggest that hepatic dendritic cells function as a critical bridge between innate and adaptive immunity and are capable of inducing stronger innate responses with a lower capacity for allo-stimulation than splenic dendritic cells. These properties of liver dendritic cells contribute to their unique role in the induction of tolerance.

Circulating IgA from acute stage of childhood Henoch-Schönlein purpura can enhance endothelial interleukin (IL)-8 production through MEK/ERK signalling pathway.

Recently, sera from children with active Henoch-Schönlein purpura (HSP) have been found to enhance interleukin (IL)-8 production by human umbilical venous endothelial cells (HUVEC). To further determine the possible factor with the ability to enhance endothelial IL-8 production in sera from acute stage of HSP, 10 children with HSP at the acute stage and 10 healthy controls were enrolled. IgA antiendothelial cell antibodies (AECA) were detected by cell-based ELISA. Active sera with or without pretreatment with anti-human IgA antibody, sera of controls, and immunoglobulin A (IgA) derived from sera were used to stimulate the HUVEC. The ability of these factors to enhance endothelial IL-8 production was evaluated. Furthermore, signalling pathways were also assayed by different inhibitors, and confirmed by immunoblotting. Serum levels of IgA AECA in HPS patients at the acute stage were significantly higher than in controls (P < 0.001). The active sera could enhance endothelial IL-8 production (P = 0.004, compared with control sera), and the ability of these sera was mostly abolished when pretreated with fixed anti-human IgA antibody. The supernatant IL-8 levels of endothelial cells stimulated by IgA derived from acute stage of HSP were statistically higher than controls (P < 0.001). PD98059, an inhibitor of ERK phosphorylation, significantly reduced IgA AECA-stimulated endothelial IL-8. IgA AECA also enhanced the phosphorylation of ERK1 with a time-dependent manner. Together with these findings, it is concluded that IgA AECA derived from acute stage of HSP may bind to endothelial and enhance endothelial cells to produce IL-8 via MEK/REK signalling pathway.

Adenovirus expressing interleukin-1 receptor antagonist alleviates allergic airway inflammation in a murine model of asthma.

Interleukin-1 (IL-1) is a proinflammatory cytokine and IL-1 receptor antagonist (IL-1ra) is a natural inhibitor that binds to IL-1 receptor type I without inducing signal transduction. It is suggested that IL-1 is required for allergen-specific T helper type 2 cell activation and the development of airway hyper-responsiveness (AHR), but the immunologic effect of exogenous IL-1ra in allergic asthma remains unclear. To examine the effect of IL-1ra on airway inflammation and immunoeffector cells in allergic asthma, recombinant adenovirus expressing human IL-1ra (Ad-hIL-1ra) was delivered intranasally into ovalbumin (OVA)-immunized mice. Single intranasal administration of Ad-hIL-1ra before airway antigen challenge in OVA-immunized mice significantly decreased the severity of AHR and reduced pulmonary infiltration of eosinophils and neutrophils. Suppression of IL-5 and eotaxin with concomitant enhancement of interferon gamma in bronchoalveolar lavage fluid was also noted in OVA-immunized mice by administration of Ad-hIL-1ra. In addition, histological studies showed that Ad-hIL-1ra was able to decrease OVA-induced peribronchial inflammation. Taken together, our results indicated that administration of Ad-hIL-1ra may have therapeutic potential for the immunomodulatory treatment of allergic asthma.

A nationwide survey on epidemiological characteristics of childhood Henoch-Schönlein purpura in Taiwan.

OBJECTIVE: To evaluate the annual incidence and other epidemiological characteristics of Henoch-Schönlein purpura (HSP) among children in Taiwan. METHODS: The records of patients were derived from the research database of the Bureau of National Health Insurance, Taiwan, Republic of China, from January 1999 to December 2002. Children younger than 17 yr of age with the diagnosis of HSP were included into this study. Data for each patient including sex, age, date of onset and length of hospitalization were recorded and analysed. RESULTS: A total of 2759 cases were included with an annual incidence of 12.9 (11.8-13.4) per 100 000 children <17 yr of age. The occurrence of HSP had a peak at the age of 5 to 6 yr. In this study, 1118 (40.5%) patients had been hospitalized at some stage. There were 1454 males and 1305 females, for a male to female ratio of 1.11. Males had a higher annual incidence before the age of 10 yr (P = 0.04), and had a lower incidence than females at older ages (P = 0.02). Disease onset was more common in autumn and winter, and no apparent change in seasonal pattern was noted over 4 yr. CONCLUSIONS: Insurance claim data provide useful information on the epidemiology of HSP in Taiwan. Childhood HSP in Taiwan, with an incidence of 12.9 per 100 000 children, occurs commonly in autumn and winter; and at the age of 5 to 6 yr. The characteristics presented in this study may provide valuable data for understanding and further studies of HSP.

In vitro and in vivo functional analysis of CD5+ and CD5- B cells of autoimmune NZB x NZW F1 mice.

Polyclonal B cell activation has been thought to play the critical role in production of autoantibodies, and possible activation of autoreactive T cells in murine lupus, especially abnormal expansion of CD5+ B cells, is one of the characteristic findings in these mice. The aim of this study was to investigate further the characteristics and function of CD5+ and CD5- B cells. Both CD5+ and CD5- B cells were isolated for in vitro autoantibody production, cytokine expression and in vivo anti-DNA antibody production with reconstitution of severe combined immunodeficient (SCID) mice. The data showed: (i) both CD5+ and CD5- B cells produced a high level of anti-DNA antibody after stimulation with lipopolysaccharide (LPS) plus IL-5; (ii) both peritoneal CD5+ and CD5- B cells expressed a high level of IL-10 mRNA after stimulation with LPS, while in contrast CD5- B cells of non-autoimmune BALB/c mice did not express IL-10 mRNA after stimulation; (iii) SCID mice reconstituted with either CD5+ or CD5- B cells all produced significant levels of anti-DNA antibodies in vivo and manifested with proteinuria. These data suggest both CD5+ and CD5- B cells play important roles in polyclonal B cell activation and subsequent autoantibody production. Generalized polyclonal B cell activation, instead of expanding a certain subpopulation of B cells, contributed to the pathogenesis of autoimmunity in murine lupus.

Nonresonant chi((3))(1111)obtained by nearly degenerate four-wave mixing using chirped-pulse technology.

Picosecond chirped-pulse technology is used to generate two spectrally separate, time-synchronized pulses for chi((3)) measurements by nearly degenerate four-wave mixing. Near 1053 nm, nonresonant relative measurements of chi((3))(1111)are carried out on three model substances, nitrobenzene, alpha chloronaphthalene, and 4'-decyloxynaphthyl-1'-(4-decyloxybenzoate). Their chi((3)) values are normalized to CS(2).

Highly efficient conversion of picosecond Nd laser pulses with the use of group-velocity-mismatched frequency doubling in KDP.

We report an increase in the conversion efficiency from approximately 40% to approximately 75% for type II frequency doubling of 1.053-microm, 1.6-ps-duration pulses in KDP when a delay of approximately 1.45 +/- 0.1 ps between the extraordinary and ordinary pulses is introduced at the input to the doubling crystal. The delay compensates for the effect of group-velocity dispersion in the crystal with the result that the effective interaction length in the crystal is approximately doubled.

Over-expression of apoptosis-related proteins contributes to muscular damage in the obstructed ureter of the rat.

OBJECTIVE: To determine the role of apoptosis-related proteins (Myc, Bax, Bcl-2, and Bcl-X(L)) in muscular damage in obstructed rat ureters. MATERIALS AND METHODS: The in situ end-labelling of DNA fragments and the expression of apoptosis-related proteins were assessed, using immunohistochemistry, in 54 female Sprague-Dawley rats in which unilateral ureteric obstruction was caused by ureteric ligation. RESULTS: The severity of ureteric damage increased during the period of obstruction. Apoptotic cells and the expression of Bax were detected in the smooth muscle layer from 14 days after ligation. The percentage of apoptotic cells and the expression of Bax in the smooth muscle layer increased and reached a peak 21 days after ligation, and then declined. The expression of Myc was also detected in the smooth muscle layer 14 days after ligation but reached a peak at 28 days. The expressions of Bcl-2 and Bcl-X(L) in the smooth muscle layer were only detected 21 and 28 days after ligation. The numbers of apoptotic cells in the smooth muscle layer correlated significantly with the expressions of Myc and Bax (r = 0.7360 and 0.7432, respectively; both P < 0.005), and with the expression index of Bax/Bcl-2 and Bax/Bcl-X(L) (r = 0.8909 and 0.8592, respectively; both P < 0.001). CONCLUSIONS: Apoptosis-related proteins might be important in regulating cell apoptosis in ureteric damage during the development of obstructive uropathy.