Silencing of augmenter of liver regeneration inhibited cell proliferation and triggered apoptosis in U266 human multiple myeloma cells.

Augmenter of liver regeneration (ALR) is a thermostable cytokine that was originally identified to promote the growth of hepatocytes. This study was conducted to explore the expression and function of ALR in multiple myeloma (MM), a common hematologic malignancy. Real-time PCR and western blot analysis were performed to detect the expression of ALR in U266 human MM cells and healthy peripheral blood mononuclear cells (PBMCs). U266 MM cells were exposed to 20 or 40 µg/mL of recombinant ALR and tested for cell proliferation. Small interfering RNA-mediated silencing of ALR was done to investigate the role of ALR in cell proliferation, apoptosis, and cytokine production. Compared to PBMCs, U266 MM cells exhibited significantly higher levels of ALR at both the mRNA and protein levels. The addition of recombinant ALR protein significantly promoted the proliferation of U266 cells. In contrast, knockdown of ALR led to a significant decline in the viability and proliferation of U266 cells. Annexin-V/PI staining analysis demonstrated that ALR downregulation increased apoptosis in U266 MM cells, compared to control cells (20.1±1.1 vs 9.1±0.3%, P<0.05). Moreover, ALR depletion reduced the Bcl-2 mRNA level by 40% and raised the Bax mRNA level by 2-fold. Additionally, conditioned medium from ALR-depleted U266 cells had significantly lower concentrations of interleukin-6 than control cells (P<0.05). Taken together, ALR contributed to the proliferation and survival of U266 MM cells, and targeting ALR may have therapeutic potential in the treatment of MM.

An AFLP-based approach for the identification of sex-linked markers in blunt snout bream, Megalobrama amblycephala (Cyprinidae).

Sex-specific DNA markers are useful for studying sex-determination mechanisms and establishment of monosex populations. Three widely spaced geographical populations (Liangzi, Poyang and Yuni Lakes in China) of blunt snout bream (Megalobrama amblycephala) were screened with AFLPs to search for sex-linked markers. Female and male pools (10 individuals in each pool) from each population were screened using 64 different primer combinations. A total of 4789 genomic fragments were produced, with a mean frequency of 75 bands per primer pair. Three different primer combinations produced putative sex-associated amplifications and were selected for individual screening in the three populations. However, none showed sex specificity when we converted these three markers into sequence characterized amplified region markers and evaluated all the individuals from the three populations.

Transient transgenic expression of gamma interferon promotes Legionella pneumophila clearance in immunocompetent hosts.

Gamma interferon (IFN-gamma) and T1-phenotype immune responses are important components of host defense against a variety of intracellular pathogens, including Legionella pneumophila. The benefit of intrapulmonary adenovirus-mediated IFN-gamma gene therapy was investigated in a nonlethal murine model of experimental L. pneumophila pneumonia. Intratracheal (i.t.) administration of 10(6) CFU of L. pneumophila induced the expression of T1 phenotype cytokines, such as IFN-gamma and interleukin-12 (IL-12). Natural killer cells were identified as the major cellular source of IFN-gamma. To determine if enhanced expression of IFN-gamma in the lung could promote pulmonary clearance of L. pneumophila, we i.t. administered 5 x 10(8) PFU of a recombinant adenovirus vector containing the murine IFN-gamma cDNA (AdmIFN-gamma) concomitant with L. pneumophila. We observed a 10-fold decrease in lung bacterial CFU at day 2 in the AdmIFN-gamma-treated group compared to controls (P < 0.01). Alveolar macrophages isolated from AdmIFN-gamma-treated animals displayed enhanced killing of intracellular L. pneumophila organisms ex vivo. Similar improvements in bacterial clearance were observed with i.t. recombinant IFN-gamma treatment. The transient transgenic expression of IL-12, a known inducer of IFN-gamma and promoter of T1-type immune responses, resulted in more modest improvement in bacterial clearance (sixfold reduction; P < 0.05). These results demonstrate that, even in immunocompetent hosts, exogenous administration or transient transgenic expression of IFN-gamma, and to a lesser extent IL-12, may be of potential therapeutic benefit in the treatment of patients with Legionella pneumonia.

Chemokine-dependent neutrophil recruitment in a murine model of Legionella pneumonia: potential role of neutrophils as immunoregulatory cells.

The roles of CXC chemokine-mediated host responses were examined with an A/J mouse model of Legionella pneumophila pneumonia. After intratracheal inoculation of 10(6) CFU of L. pneumophila, the bacterial numbers in the lungs increased 10-fold by day 2; this increase was accompanied by the massive accumulation of neutrophils. Reverse transcription-PCR data demonstrated the up-regulation of CXC chemokines, such as keratinocyte-derived chemokine, macrophage inflammatory protein 2 (MIP-2), and lipopolysaccharide-induced CXC chemokine (LIX). Consistent with these data, increased levels of KC, MIP-2, and LIX proteins were observed in the lungs and peaked at days 1, 2, and 2, respectively. Although the administration of anti-KC or anti-MIP-2 antibody resulted in an approximately 20% decrease in neutrophil recruitment on day 2, no increase in mortality was observed. In contrast, the blockade of CXC chemokine receptor 2 (CXCR2), a receptor for CXC chemokines, including KC and MIP-2, strikingly enhanced mortality; this effect coincided with a 67% decrease in neutrophil recruitment. Interestingly, anti-CXCR2 antibody did not affect bacterial burden by day 2, even in the presence of a lethal challenge of bacteria. Moreover, a significant decrease in interleukin-12 (IL-12) levels, in contrast to the increases in KC, MIP-2, and LIX levels, was demonstrated for CXCR2-blocked mice. These data indicated that CXCR2-mediated neutrophil accumulation may play a crucial role in host defense against L. pneumophila pneumonia in mice. The increase in lethality without a change in early bacterial clearance suggested that neutrophils may exert their protective effect not through direct killing but through more immunomodulatory actions in L. pneumophila pneumonia. We speculate that a decrease in the levels of the protective cytokine IL-12 may explain, at least in part, the high mortality in the setting of reduced neutrophil recruitment.

Early recruitment of neutrophils determines subsequent T1/T2 host responses in a murine model of Legionella pneumophila pneumonia.

The contribution of neutrophils to lethal sensitivity and cytokine balance governing T1 and T2 host responses was assessed in a murine model of Legionella pneumophila pneumonia. Neutrophil depletion by administration of granulocyte-specific mAb RB6-8C5 at 1 day before infection rendered mice approximately 100-fold more susceptible to lethal pneumonia induced by L. pneumophila. However, this treatment did not alter early bacterial clearance, despite a substantial decrease in neutrophil influx at this time point. Cytokine profiles in the lungs of control mice demonstrated strong T1 responses, characterized by an increase of IFN-gamma and IL-12. In contrast, neutrophil-depleted mice exhibited significantly lower levels of IFN-gamma and IL-12, and elevation of T2 cytokines, IL-4 and IL-10. Immunohistochemistry of bronchoalveolar lavage cells demonstrated the presence of IL-12 in neutrophils, but not alveolar macrophages. Moreover, IL-12 was detected in lavage cell lysates by ELISA, which was paralleled to neutrophil number. However, intratracheal administration of recombinant murine IL-12 did not restore resistance, whereas reconstitution of IFN-gamma drastically improved bacterial clearance and survival in neutrophil-depleted mice. Taken together, these data demonstrated that neutrophils play crucial roles in primary L. pneumophila infection, not via direct killing but more immunomodulatory effects. Our results suggest that the early recruitment of neutrophils may contribute to T1 polarization in a murine model of L. pneumophila pneumonia.

Impaired pulmonary host defense in mice lacking expression of the CXC chemokine lungkine.

Lungkine (CXCL15) is a novel CXC chemokine that is highly expressed in the adult mouse lung. To determine the biologic function of Lungkine, we generated Lungkine null mice by targeted gene disruption. These mice did not differ from wild-type mice in their hematocrits or in the relative number of cells in leukocyte populations of peripheral blood or other tissues, including lung and bone marrow. However, Lungkine null mice were more susceptible to Klebsiella pneumonia infection, with a decreased survival and increased lung bacterial burden compared with infected wild-type mice. Histologic analysis of the lung and assessment of leukocytes in the bronchioalveolar lavage revealed that neutrophil numbers were normal in the lung parenchyma, but reduced in the airspace. The production of other neutrophil chemoattractants in the Lungkine null mice did not differ from that in wild-type mice, and neutrophil migration into other tissues was normal. Taken together, these findings demonstrate that Lungkine is an important mediator of neutrophil migration from the lung parenchyma into the airspace.

Effectiveness of low-dose ASA in prevention of secondary ischemic stroke, the ASA Study Group in Taiwan.

This randomized double-blind controlled study was carried out to investigate the effect of 100 mg acetylsalicylic acid (ASA) per day on the secondary prevention of ischemic stroke. Patients who suffered a first ischemic stroke from 13 participating hospitals were enrolled. They were independent or only partially dependent in activities of daily living and all had received brain CT for diagnosis. Eligible patients were randomly allocated to the 100 mg ASA or the nicametate citrate (a vasodilator) groups, and trial medications were started within three to six weeks after the onset of stroke. The primary end point was cerebral reinfarction, and intracranial hemorrhage was classified as an adverse event. Four hundred and sixty-six patients participated in this study; and 222 cases (136 males and 86 females) were allocated to the ASA group while 244 cases (150 males and 94 females) were assigned to the nicametate group. No significant difference in baseline characteristics between the two groups was observed. Cerebral reinfarction developed 6.3% (14/222) in the ASA group and 11.9% (29/244) in the nicametate group. According to the Cox's proportional hazards model, the estimated risk ratio (ASA group vs. nicametate group) was 0.538, with a 95% confidence interval of 0.284-1.019. The result was of borderline statistical significance. The risk for cerebral reinfarction was reduced by almost 50% among those who took 100 mg ASA versus those who took nicametate.

Structure and expression of fyn genes in Xenopus laevis.

By low stringency screening of a Xenopus laevis oocyte cDNA library with a probe prepared from the yes oncogene of the avian retrovirus Y73, we have isolated clones encoding a 537 amino acid protein with 96% amino acid identity to the protein product of the human fyn proto-oncogene. In addition to the high degree of conservation at the amino acid sequence level, we also find a surprisingly high degree of sequence conservation between portions of the 5' untranslated regions of the frog and human genes. Probing of total DNA from a homozygous diploid frog with a fragment from one of the cDNA clones gave a hybridization pattern which is consistent with the presence of two fyn genes in the haploid Xenopus laevis genome. Hybridization analysis revealed that the ovary contains two different sized fyn RNAs, one 3.8 kb in length and the other 3.1 kb in length. Our finding of fyn transcripts brings to three the number of protein-tyrosine kinases of the src family whose mRNAs are found in X. laevis oocytes. This suggests that multiple protein-tyrosine kinases of the src family are required for processes in oogenesis or early development.

Essential tremor: clinical, electromyographical and pharmacological studies in 146 Chinese patients.

From February 1984 to February 1988, 258 cases of various kinds of tremor were seen in our movement disorder clinic. Among them, 146 cases (57%) were diagnosed as essential tremor, and of these, 96 (65.8%) were males and 50 (34.2%) were females, ranging in age from 14 to 89 years (mean: 36 years). The main tremor occurred in the hands (100%), and in a few cases were combined with head, leg, lip, voice and neck tremors. A familial tendency was obvious in 47 cases (32%). The surface electromyographic (EMG) study of essential tremor revealed two patterns, one of synchronous type (73%) and the other of alternating type (27%). Its frequency was between 5 and 9Hz. The burst duration was short (50-100 msec) and the amplitude was low (less than 200 mu v). All cases were divided into two groups to received propranolol therapy, 78 cases with high dosages (120-240 mg/day) and 68 with low dosages (60-80 mg/day). The higher dose was better in effect than the lower one (p less than 0.01). Propranolol also had effect on tremors of alternating type (p less than 0.01), which is different from previous reports.