[Correlation of vasogenic white matter lesions and retinal vascular network parameters].

Objective: To investigate the correlation of vasogenic white matter lesions with retinal vascular network parameters using fully automatic retinal image analysis of fundus photographs. Methods: A total of 106 patients with cerebral small vessel disease who were hospitalized in Department of Neurology, the First Affiliated Hospital of Sun Yat-sen University during March and October 2015, and were able to undertake cerebral MRI and fundus photography in a sitting position were included. They were divided into two groups (mild or moderate-severe) according to the Fazekas scores of periventricular white matter lesions and deep white matter lesions shown by MRI. The clinical data and retinal vascular network parameters were compared between mild and moderate-severe groups. Results: According to the severity of periventricular white matter lesions, Logistic regression analysis showed that after adjusting baseline information, decreased asymmetry index of artery (OR=1.71, 95%CI 1.02-2.88, P<0.05)was associated with periventricular white matter lesions. As for deep white matter lesions, Logistic regression analysis showed that after adjusting baseline information, decreased central retinal artery equivalent(OR=5.19, 95%CI 1.06-25.44, P<0.05), decreased asymmetry index of artery (OR=2.96, 95%CI 1.42-6.17, P<0.05), decreased asymmetry index of venule (OR=2.99, 95%CI 1.48-6.02, P<0.05) and increased central retinal vein equivalent (OR=0.14, 95%CI 0.03-0.67, P<0.05) were associated with deep white matter lesions. Conclusions: White matter lesions of different places could be contributed to different pathological process. Therefore, the early diagnosis and observation of them are applicable to different retinal vascular network parameters.

Identification of a tobacco protein interacting with tomato mosaic virus coat protein and facilitating long-distance movement of virus.

The protein-protein interaction of virus and host is essential for virus infection and host defense. The coat protein (CP) of tomato mosaic virus (ToMV) has been proved to be involved in cell-to-cell and long-distance movements of viruses that are presumably related with the protein-protein interactions. However, the host proteins that interact with the ToMV coat protein (ToCP) are largely unknown. In this study, we isolated a cDNA from a tobacco library through yeast two-hybrid system, which encodes a protein designated the ToMV CP-interacting protein-L (IP-L) that interacted with ToCP in vitro and in vivo. Sequencing analysis revealed that the putative coding region of IP-L gene was identical to that of an 'elicitor responsive protein' gene from N. tabacum (Genbank: #AB040409). A homology was also found between the cDNA sequence of IP-L and two senescence-related cDNAs (SENU1: Z75523 and AY479987) isolated from tomato and pepper. Northern blotting analysis showed that the mRNA level of IP-L was elevated after infection of ToMV. Then, we investigated the in vivo function of IP-L using virus-induced gene silencing (VIGS) and virus challenging assay. Semi-quantitative RT-PCR and Northern blotting results showed that the endogenous mRNA of IP-L in N. benthamiana plant was silenced at 10 days post inoculation with the in vitro transcripts of PVX-IP-L that were produced from the potato virus X (PVX)-based gene silencing plasmid pPC2S.IP-L. The IP-L silent plant developed a delayed systemic symptom at 7 days post challenging with ToMV, indicating that a high expression of IP-L was necessary for the interaction with ToCP to assist the viral transportation. Together, our data suggested that IP-L is a novel plant factor that interacts with the coat protein of ToMV and facilitates the long-distance movement of virus, which may provide a valuable clue for us to further investigate the mechanisms of plant virus infection and to control plant virus diseases.

Delivery of glucocorticoid conjugate in rat gastrointestinal tract and its treatment for ulcerative colitis.

AIM: To evaluate colonic delivery and therapeutic effect of the newly synthesized dexamethasone (DX)-dextran (500 000) conjugate (DXD50) in the rat. METHODS: The amount of dexamethasone was measured in the contents from different parts of rat gastrointestinal tract and in plasma after ig conjugate. Therapeutic effect of conjugate and DX was tested in trinitrobenzenesulfonic acid-induced colitis in rat. Repair of colitis was assessed by measuring colonic ulceration area, colon weight, and colonic myeloperoxidase (MPO) activity. Systemic immunosuppression of DX was evaluated with weight of thymus and spleen and lymphocyte count in peripheral blood from rat with ulcerative colitis. RESULTS: Dexamethasone released from conjugate was mainly distributed in contents of cecum and colon. When DXD50 and DX 0.25 micromol . kg-1 . d-1 were used ig to treat ulcerative colitis in rat, the ulcerative area of colon was reduced by 55.6 % and 33.3 %, respectively whereas colon weight was reduced by 17.9 % and 2.6 %, respectively. The conjugate had no effect on lymphocyte count in peripheral blood, spleen weight, and thymus weight of rat which could be reduced markedly by the same dose of DX (P < 0.05 vs control). CONCLUSION: DXD50, which could specifically deliver DX to large intestine, is a promising agent in the treatment of human inflammatory bowel disease.

[Function of the leaky UGA codon in ssRNA plant virus genomes].

The opal mutation (UGA) at nucleotide 2670-2672 in the replicase gene of the attenuated tomato mosaic virus K (ToMV-K) mainly contributes to the virus attenuation based on a series of studies on the viral attenuation mechanism. From analysis and comparison between the replicase gene mutation point of ToMV-K and the related regions of all plant viruses containing the leaky UGA, we have found that some characters, including the CGG motif, alpha-helix structure and some specific amino acids, are, presumably, able to helpfully confer the readthrough mechanism. Finally, some other ssRNA plant viruses like PVX, PVY, CMV have been analyzed. We found that their genomic modifications and viral attenuations could be explored according to the mutation mode of the ToMV-K replicase.

[Complete nucleotide sequences and genome structure of two Chinese tobacco mosaic virus isolates deduced from full-length infectious cDNA clones].

The complete nucleotides of two Chinese tobacco mosaic virus (TMV) isolates, TMV-Cv (vulgare strain) and TMV-N14 (an attenuated virus originated from a tomato strain), were determined from their respective full-length infectious cDNA clones and compared with published TMV sequences. The genome structure of TMV-Cv contained 6395 nucleotides, in which four functional open reading frames (ORF), coding for replicase (126 kD/183 kD), movement protein (MP, 30 kD) and coat protein (CP, 17.6 kD) respectively, could be recognized. TMV-N14 contained 6384 nucleotides in its genome. In contrast to TMV-Cv, five functional ORFs encoding the replicase 98.5 kD/126 kD/183 kD, MP(27 kD) and CP(17.6 kD), respectively, were detected in the TMV-N14 genome. TMV-Cv is 99% homologous to a Korean TMV isolate belonging to the vulgare strain at the nucleotide level. TMV-N14 is 99% homologous to a highly virulent Japanese isolate TMV-L (tomato strain) at the nucleotide level. In TMV-N14, one opal nulation (UGA) occurred in the replicase gene and one ochre nutation (UAA) in the MP gene. The former mutation created a potential, additional ORF within the replicase gene, the latter reduced the size of the MP to 27 kD. In addition, there were also 13 amino acid substitutions in the replicase gene of TMV-N14 when compared to that of TMV-L. Collectively, these changes may have significant implications in the attenuation of the virulence of TMV-N14.

[Cloning and infectivity analysis of the cDNAs of tobacco mosaic virus (tomato strain) and its attenuated virus(N14) genomes].

An oligonucletide primer with SP6 transcription promoter and the other primer with a KpnI site at 5' terminuses were used to prepare the cDNAs of tobacco mosaic virus(TMV, Chinese isolate) and its attenuated virus(N14, nitrite-treated mutant, tomato strain) genomic RNAs by RT-PCR. The cDNAs were cut into two fragments by BamHI and first cloned separately then recombined into pUC18 by the restriction endonucleases of BamHI, SmaI, HincII, PstI, SphI. The sequences of 5' and 3' terminal nucleotides (< 50 bp) were consistent with the reported references. The infectious clones(pTMV-Cv/pN14) have been obtained and corroborated by in vitro transcription with SP6 RNA polymerase and tobacco infection. Necrotic local lesions were observed in Nicotiana samsun NN induced by the transcripts from both pTMV and pN14 within 3-5 days after inoculation, systemic symptoms showed in single-lesion infected Nicotiana tobacum cv, coincided with symptoms of tobaccoes infected by TMV and N14.

The role of CD4+ lymphocytes in the susceptibility of mice to stress-induced reactivation of experimental colitis.

Idiopathic inflammatory bowel disease is a chronic relapsing condition. The role of stress in causing relapses of inflammatory bowel disease remains controversial. We now show that colitis induced in mice by dinitrobenzenesulfonic acid (DNBS) resolves by 6 weeks, but can subsequently be reactivated by stress plus a sub-threshold dose of DNBS, but not by DNBS alone. Stress reduced colonic mucin and increased colon permeability. Susceptibility to reactivation by stress required CD4+ lymphocytes and could be adoptively transferred. We conclude that stress reactivates experimental colitis by facilitating entry of luminal contents that activate previously sensitized CD4 cells in the colon.

IL-11 inhibits Clostridium difficile toxin A enterotoxicity in rat ileum.

Interleukin-11 (IL-11) is a stromal cell-derived cytokine with several biological activities against hematopoietic cells. Recent results indicated that IL-11 reduced mucosal damage in animal models of colitis. This study aimed to explore the action of recombinant human IL-11 (rhIL-11) on the intestinal effects of Clostridium difficile toxin A, an inflammatory enterotoxin, and cholera toxin, a noninflammatory enterotoxin in rat ileum. We administered rhIL-11 subcutaneously to rats before injection of toxin A into ileal loops and measured fluid secretion, epithelial permeability to mannitol, histopathological damage, and release of rat mast cell protease II (RMCP II) from intestinal mast cells and of tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein-2 (MIP-2) from lamina propria macrophages. rhIL-11 (50-1,000 micrograms/kg) inhibited toxin A but not cholera toxin-mediated secretion and permeability in a dose-dependent fashion and reduced toxin A-induced epithelial cell damage. Rats treated with rhIL-11 also showed reduced RMCP II, TNF-alpha, and MIP-2 release in response to toxin A. Exposure of rat peripheral monocytes in vitro to rhIL-11 (1 microgram/ml) inhibited lipopolysaccharide and toxin A-mediated increases in TNF-alpha mRNA and protein levels. We conclude that rhIL-11 blocks the intestinal effects of C. difficile toxin A, possibly by inhibiting release of inflammatory mediators from mucosal mast cells and intestinal macrophages.

Cellular pathology of experimental colitis induced by trinitrobenzenesulphonic acid (TNBS): protective effects of recombinant human interleukin-11.

The objective of this study was to elucidate colonic mucosal ultrastructural effects of trinitrobenzene-sulphonic acid (TNBS) with and without co-administration of recombinant human interleukin-11 (rhIL-11). Using a standard colitis model (ir alcoholic TNBS), rats were sacrificed at 3~14 days after TNBS. Co-administration of rhIL-11 (100, 300 or 1000 mug/kg sc) was given for protection, and controls received saline or alcohol ir, or rhIL-11 sc alone. Transmission electron microscopy revealed that early TNBS-induced cytopathology was primarily in absorptive cells, changes which occurred prior to goblet cell damage. Progressive cellular changes included vacuolization and increased multivesicular bodies in cell apices, disconfiguration of microvilli, enlarged Golgi apparatuses, enlargement of basal inter-cellular spaces, and eventual desquamation of epithelium and apical bursting.Organelle damage preceded surface changes and resembled ultrastructural changes reported for human ulcerative colitis. The principal effect of rhIL-11 was apparent massive release of mucus from goblet cells, filling the colonic crypts, and suggesting a mode of its protection.

Tungstic acid reduction of cold-resistant stress-induced ulceration in rats.

Sprague-Dawley rats were restrained at 4 degrees C for 2 h (stress). Tungstic acid in a single dose of 0.01, 0.1, 1, 10, 100 or 300 mg/kg (dissolved in distilled water) was administered intragastrically to animals 30 min prior to stress. Stress induced significant gastric mucosal damage, whereas tungstic acid pretreatment dose-dependently reduced lesion formation. Doses of tungstic acid of 1 mg/kg and higher significantly (P < 0.05-0.001) decreased ulcers. The mucosal mast cell counts in rats pretreated with tungstic acid were significantly higher than those of control rats. In motility experiments using oral administration of amberlite pellets, pretreatment with tungstic acid dose-dependently reduced the gastric emptying rate during a 1 h period of stress. Gastric mucosal xanthine oxidase and superoxide dismutase (SOD) activities, after pretreatment with a single dose of tungstic acid, were not altered in stressed animals. It is suggested that tungstic acid effectively antagonizes stress-induced gastric ulcers, possibly by decreasing motility and mass cell degranulation. Xanthine oxidase and SOD activities and mucous content were not changed in the gastric mucosa by the present method of tungstic acid administration.

Protection by recombinant human interleukin-11 against experimental TNB-induced colitis in rats.

The potential effect of recombinant human interleukin-11 (rhIL-11) on trinitrobenzene sulfonic acid (TNB)-induced colitis was investigated in rats. Intrarectal TNB (40 mg in 0.25 ml 40% ethanol) produced significant ulcerative colitis. The lesions were most severe at three days after TNB instillation, and then declined, but lesions were still observed after two weeks. TNB administration also significantly enhanced the colonic mucosal myeloperoxidase (MPO) levels, which paralleled the severity of colitis. The rhIL-11 at subcutaneous doses of 300 or 1000 micrograms/kg daily for seven days, or 1000 micrograms/kg for three days when given after TNB significantly decreased lesion formation in TNB-induced colitis. These treatments also significantly reduced colonic mucosal MPO levels. TNB enhanced colonic mucosal levels of PGE2, LTB4, and TxB2, but these arachidonic acid derivatives were not affected by the present rhIL-11 treatments. TNB administration for three days caused a body weight loss that returned to normal after 14 days. The rhIL-11 significantly reduced colonic lesion severity and reduced colonic fecal blood loss. Given alone, rhIL-11 did not influence body weight. It can be concluded that rhIL-11 was protective against TNB-induced colitis and reactions of colonic MPO, but that these responses were not mediated through modulation of eicosanoid metabolism.

Effects of chronic nitric oxide synthase inhibition in cold-restraint and ethanol-induced gastric mucosal damage in rats.

Gastric actions of Nw-nitro-1-arginine methyl ester (L-NAME) were investigated in rats, as this agent is a reliable nitric oxide synthase inhibitor L-NAME solutions were placed in subcutaneous osmotic minipumps which continuously released L-NAME at 0.1, 1.0, 10, or 40 mg/kg/day. L-NAME dose and time-dependently enhanced stress-induced gastric ulceration but did not affect mucosal mast cell population. Ulcerogenic actions of L-NAME were reversed by L-arginine but not by D-arginine. Ten L-NAME treatment also enhanced the ethanol-induced gastric mucosal damage, depressed gastric mucosal blood flow but did not alter gastric mucus, secretory volume, or acid output. It is concluded that in the present models, chronic nitric oxide synthase inhibition enhanced ulcerogenesis by decreasing mucosal resistance due to reduced mucosal blood perfusion. This implicates nitric oxide as a mucosal defense factor which acts in part by maintaining mucosal blood flow.

Influence of repeated prazosin administration on cardiovascular responses in rats and rabbits.

Prazosin was injected i.v. at a dose of 50 micrograms/kg every 2 h for 8 h in conscious rats. Its hypotensive action significantly declined. A similar effect was also observed in rabbits pretreated with prazosin (40 micrograms/kg, i.v.) every 1 h for 4 h. In prazosin-treated rabbits, the total peripheral resistance became less responsive to phentolamine stimulation. Repeated prazosin administration abolished its ability to block receptors in a model of anococcygue muscle contraction after noradrenaline (NA) stimulation. The alpha-adrenoceptors in anococcygue muscle exhibited lower pD2 to NA and lower pA2 to prazosin in prazosin-treated rats. The results demonstrate that repeated prazosin administration reduces the effectiveness of alpha-adrenoceptors blockers.

Mucosal nitric oxide is not responsible for the hemodynamic changes induced by nicotine in rat stomachs.

It has been shown that chronic nicotine treatment decreases gastric mucosal blood flow (GMBF). The mechanism for this action is still not defined. In this study, nicotine treatment (5, 25 or 50 µg/ml drinking water) for 10 days dose dependently reduced the GMBF and volume of hemoglobin but increased ethanol-induced gastric damage. These effects were potentiated by N(ω)-nitro-l-arginine methyl ester (l-NAME), a nitric oxide (NO) synthase inhibitor. l-arginine but not the d-analog restored the actions of l-NAME, indicating that the selective action of l-NAME. However, the potentiating actions of l-NAME were significantly attenuated in the nicotine-pretreated rats. When the basal mucosal NO synthase (both iNOS and cNOS) activity and its second messenger cyclic GMP levels were measured, no difference was found between the nicotine and the non-nicotine groups. Furthermore, high dose of l-arginine could not reverse the action of nicotine. These findings suggest that the adverse action of chronic nicotine treatment on GMBF and lesion formation is probably mediated through a NO independent mechanism.

Effects of chronic nitric oxide synthase inhibition on TNB-induced colitis in rats.

Nitric oxide (NO) synthesis is increased in ulcerative colitis, but the role of NO in colitis is poorly understood. The present study employed Nw-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, in rats to evaluate the effect of NO on 2,4,6-trinitrobenzenesulphonic acid (TNB)-induced colitis. L-NAME solutions were placed in subcutaneous, osmotic mini-pumps which continuously released L-NAME at 0.042, 0.208, 0.417, or 1.667 mg kg-1 h-1. L-NAME dose-dependently enhanced lesions in TNB-induced colitis. The two higher doses of L-NAME significantly increased colonic mucosal damage, although there was slight, nonsignificant reduced lesion formation with the lowest dose of L-NAME. 0.042 mg kg-1 h-1. A single dose of L-NAME at 100 mg kg-1 subcutaneously injected daily in TNB-treated rats also increased lesions, and these ulcerogenic actions of L-NAME were reversed by L-arginine but not by D-arginine (both at 500 mg kg-1, s.c.). Only the highest dose of L-NAME (mini-pump) significantly depressed myeloperoxidase (MPO) activity. Faecal occult bleeding showed a close relationship with severity of colitis. These findings suggest that there may exist a balance between NO protective and aggressive effects. In TNB-induced colitis, antagonism of endogenous NO generation was intensified, whereas slight inhibition of NO synthesis reduced lesions. Variations in responses, related to timing or dose changes in L-NAME, may reflect the differences in inducible vs constitutive NO synthase isoforms.

Expression of proliferating cell nuclear antigen in cutaneous malignant lymphomas and lymphoid infiltrates.

Paraffin-embedded tissue of skin biopsy specimens taken retrospectively from 24 patients with cutaneous malignant lymphomas (CML) and 8 patients with cutaneous lymphoid infiltrates (CLI) and other dermatoses were studied retrospectively with PC10 immunostaining. The results show a statistical significant difference among PC10 indices for cutaneous genuine histiocytic lymphoma (CGHL), cutaneous germinal center cell-derived lymphomas (CGCCL), cutaneous peripheral T-cell lymphomas (CPTL), non-mycosis fungoides (MF) and Sezary's syndrome (SS), and MF when compared with those for CLI. There is a linear relationship between PC10 index and square root of PC10 density, both of which seem to have a parallel relationship to the severity of malignancy in CML. The nuclear volume of the positive tumor cell or lymphocyte with PC10 immunostaining may be also useful in differentiating CML from CLI.

Expression of human hepatitis B virus surface antigen gene in transgenic tobacco.

Expression of Human hepatitis B virus surface antigen (HBsAg) gene in plant was reported for the first time. The recombinant plasmid pRoKII-HBsAg was constructed by inserting HBsAg gene into the downstream of CaMV 35S promoter of binary vector pRoKII and then introduced into Agrobacterium tumefaciens LBA4404. The kanamycin-resistant plants were obtained by Agrobacterium-mediated transformation system. It was shown that HBsAg gene was expressed in transgenic tobacco plants and their progenies by ELISA. The spherical particles of psi 22 nm is the leaf extract of transgenic tobacco were observed by immunosorbent electron microscopy.

DNA flow cytometry from paraffin-embedded tissue in diagnosis of cutaneous malignant lymphomas and pseudolymphomas.

DNA content and cell cycle distributions in paraffin-embedded blocks of 111 skin biopsy specimens of 70 patients with cutaneous malignant lymphomas (CML) and 41 patients with cutaneous pseudolymphomas (CPL) including chronic actinic dermatitis (CAD) were estimated by DNA flow cytometry. A statistical significant difference between DNA indices (DIs) or proliferative indices (PIs) for CML including mycosis fungoides (MF) II, MF III, Sezary's syndrome (SS), cutaneous peripheral T-cell lymphomas other than MF and SS, cutaneous germinal center cell-derived lymphomas and cutaneous genuine histiocytic lymphoma from CPL. DIs were also helpful in differentiating MF I from CPL. There was a linear relationship between DIs and PIs, both of which had a parallel relationship to the degrees of malignancy and mortality of varieties of CML. The finding of aneuploidy is likely to be useful in differentiating CML from CPL. It is worth noting that DIs, PIs and proportions of aneuploidy in CAD were all higher than those of higher malignancy of CML. These data could not be considered as markers of malignancy.

An analysis of 24 patients with IgA deposition at the BMZ.

A study of 24 patients with IgA deposition at the BMZ of the skin showed that five conditions could be recognized: 1) linear IgA bullous dermatosis in adults (LAD, 7 cases); 2) linear IgA and IgG bullous dermatosis in adults (LAGD, 10 cases); 3) chronic bullous disease of childhood (CBDC, 3 cases); 4) dermatitis herpetiformis (DH, 1 case), and 5) systemic lupus erythematosus (SLE, 3 cases). Histopathologically, 5 of 7 patients with LAD were similar to the DH group, but 7 of 10 patients with LAGD were similar to the BP group. Half the patients with LAD and LAGD had oral lesions, and most of them had excellent responses to dapsone and Tripterygium Wilfordii, but the patients with CBDC did not respond to these treatments. In the patients with LAD and LAGD, the positivity rates of IgA anti-BMZ antibodies examined by indirect immunofluorescence (IIF) on intact skin and NaCl split skin were 41% and 64%, respectively. The heterogeneity of the histopathologic pictures of LAD and LAGD, the incidence of DH, and the value of using NaCl split skin for IIF are discussed.

Nitric oxide inhibition intensifies cold-restraint induced gastric ulcers in rats.

Treatment 20 min beforehand with an inhibitor of nitric oxide (NO) synthesis NW-nitro-1-arginine methyl ester (L-NAME) (12.5, 25, 50 or 100 mg/kg, s.c.), dose-dependently intensified gastric glandular mucosal ulceration produced by cold-restraint stress. Hexamethonium (20 mg/kg) or atropine (1 mg/kg) pretreatment s.c. 20 min before stress strongly antagonised stress-evoked ulceration, as well as the ulcer-potentiating effects of L-NAME when either cholinoceptor antagonist was given concurrently with the NO inhibitor. Stress-induced mast cell degranulation was not worsened by L-NAME pretreatment. The findings suggest that NO could confer partial protection against stress-induced gastric ulcer formation; its activity is triggered off by the ulcerogenic mechanism of stress.