Influence of the Ity/Lsh/Bcg gene on the development of suppressor cell precursors in the early phase of the infection of mice with Mycobacterium lepraemurium.

In vitro inducible suppressor cell precursors were detected in the spleen of BALB/c but not in DBA/2 mice infected intraperitoneally with 10(8) Mycobacterium lepraemurium bacilli, thus suggesting that their development is genetically controlled. Two pairs of mouse strains congenic at the Ity/Lsh/Bcg locus (BALB/c-C.D2 and B10.A-B10.A.Bcgr) were used to investigate whether this phenomenon is influenced by this gene known to control the relative susceptibility of mice to Myco. lepraemurium infection. This seems likely, as the detection of culture-induced suppressor activity was delayed for 5-6 weeks in C.D2 and B10.A.Bcgr mice infected intravenously with 10(4) Myco. lepraemurium bacilli. However, despite the retardation in the detection of suppressor cell precursors, the level of in vitro induced suppressor activity at onset in spleen cell suspensions of mice carrying the resistant allele was higher than in cell cultures derived from susceptible mice. As the resistant allele has a different effect when found on BALB/c or DBA/2 background, other genetic factors are apparently involved in the development of suppressor cell precursors. We finally observed that, in spleen cell cultures from intravenously infected Ity/Lsh/Bcg congenic mice on the BALB/c background, adherent and non-adherent cells were required in the inductive phase of suppressor cell development, whereas in vitro induced suppressor activity was found exclusively in the adherent cell fraction. Given these properties, we thus conclude that suppressor cell precursors detected in the spleen of these intravenously infected mice are similar to those previously observed in C3H mice infected intraperitoneally with a thousand times more bacilli.     

Influence of macrophage resistance gene Lsh/Ity/Bcg (candidate Nramp) on Toxoplasma gondii infection in mice.

Functional studies have shown that the murine macrophage resistance gene Lsh/Ity/Bcg (candidate Nramp) regulates macrophage priming/activation for antimicrobial activity via the tumour necrosis factor-alpha (TNF-alpha)-dependent production of reactive nitrogen intermediates. Since Toxoplasma gondii also parasitizes macrophages, is a stimulator of endogenous TNF-alpha release, and is sensitive to nitric oxide-mediated killing in activated macrophages, studies were carried out using chromosome 1 congenic mouse strains to determine whether Lsh influences T. gondii infection. Two interesting observations were made: (i) contrary to expectation, mice carrying the Lsh-resistant allele died earlier over the acute phase of infection than Lsh-susceptible mice; and (ii) Lsh-resistant mice which survived this acute phase of infection showed lower brain cyst numbers than the Lsh-susceptible mice. Whilst the latter occurred independently of route of inoculation (oral, intraperitoneal, or subcutaneous), the former was influenced both by the route of inoculation and the genetic background on which the Lsh-resistant allele had been isolated. Hence, following oral administration of 20 brain cysts of the RRA strain of T. gondii, mice carrying the Lsh-resistant allele on a B10 genetic background showed a significantly enhanced rate of mortality over the acute (first 8-12 days) phase of infection than B10 Lsh-susceptible mice. Although this acute phase of infection in B10 background mice was accompanied by an increase in serum TNF-alpha levels in both Lsh-resistant and -susceptible mouse strains, early mortality preceded the TNF-alpha peak, and administration of neutralizing rabbit anti-TNF-alpha did not significantly enhance survival. Hence, inflammatory mediators other than TNF-alpha appear to be responsible for the increased rate of acute mortality observed in resistant mice. Infection intraperitoneally led to delayed mortality in B10 mice, with the mean time to 50% mortality now being significantly longer in Lsh-resistant than in Lsh-susceptible mice. On a BALB genetic background, it was the i.p. route of infection which led to acute mortality and more rapid death in the Lsh-resistant strain. When a less virulent inoculum was used and mortality delayed, Lsh-susceptible mice died more rapidly, and i.p. administration of rabbit anti-TNF-alpha led to 100% mortality between days 8 and 10 of infection in both susceptible and resistant mouse strains, consistent with a crucial protective role for TNF-alpha during this phase of infection.(ABSTRACT TRUNCATED AT 400 WORDS)     

Disseminated Salmonella paratyphi infection in a rheumatoid arthritis patient treated with infliximab

Anti-tumour necrosis factor (TNF)-α treatments are immunosuppressant and represent an important risk factor for developing infections. We report a case of Salmonella paratyphi bacteraemia associated with soft tissue infection in a patient who used infliximab and had recently travelled to India. This case report provides supporting evidence, essentially based only on case reports, that patients receiving anti-TNF-α treatments have an increased susceptibility to Salmonella infections, which may develop at unusual and disseminated sites. We emphasize the importance of appropriate counselling of patients undergoing anti-TNF treatment and travelling to areas in which Salmonellae are endemic, as well as the importance of advice to these patients concerning food hygiene.     

A recombination event in the closely linked plasminogen and apolipoprotein(a) gene loci

Genetic studies as well as in situ hybridisation data have strongly demonstrated that the genes coding for apoprotein(a) and plasminogen are linked and localised to chromosome 6 at band 6q26-27. We describe in this report the presence of a recombination event in a region of approximately 50 kb of DNA separating the two genes. The recombination was found in an Italian family, in which a mutation affecting both plasminogen plasma level and activity of plasminogen activity has been detected. Polymerase chain reaction and sequencing analysis showed the presence of a mutation different from those previouly reported in two Japanese families.     

Dry gangrene of the extremities in calves associated with Salmonella dublin infection; a possible immune-mediated reaction

Dry gangrene of the extremities in calves is a circulatory error that may occur after infection with Salmonella dublin. This report describes an examination of three affected, 12 in-contact and five control calves, a main objective being to investigate the possible role of cold agglutination in pathogenesis. The lesions included dry gangrene of the hind legs, ears and tail. A cold agglutination test gave positive results in all animals examined except the controls. The three affected calves had high titres of S. dublin antibodies, as also did four of the in-contact animals. The results suggested a relationship between cold agglutination and the occurrence of the disease.     

Study of some of the mechanisms involved in the prevention against Salmonella enteritidis serovar Typhimurium infection by lactic acid bacteria

The possible mechanism exerted by different lactic acid bacteria (LAB) in the protection against Salmonella enteritidis serovar Typhimurium (S. typhimurium) infection was determined. LAB was administered to BALB/c mice, and the animals were subsequently challenged with S. typhimurium. The inhibition of the translocation of S. typhimurium in the liver was correlated with a decrease in cellular apoptosis determined in slices from the small intestine of mice. The microbiocidal activity of peritoneal macrophages was increased by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus, but not for the probiotic strain L. casei CRL 431. The levels of IFNγ and Bcl-2 positive cells in the small intestine of mice fed with the LAB were also determined by immunofluorescence. Using in vivo studies, we demonstrated that the biological and immune mechanisms induced by the LAB studied were different for each bacterium and were mediated by anti-S. typhimurium S-IgA microbiocidal activity and/or cellular apoptosis inhibition of infected immune cells.     

Resistance to subcutaneous infection with Mycobacterium lepraemurium is controlled by more than one gene.

The resistance of C57BL (high) and BALB/c (low) mice, their F1 hybrids, and the offspring derived from backcrosses of the F1 to both parental strains was assessed at 20 weeks after subcutaneous infection with 10(7) Mycobacterium lepraemurium organisms. The numbers of bacilli recovered from the infected foot and draining lymph node indicated that resistance to subcutaneous infection is controlled by more than one non-H-2-linked gene of intermediate dominance. In general, female mice were more resistant than males.     

A gene for pachyonychia congenita is closely linked to the keratin gene cluster on 17q12-q21.

Pachyonychia congenita (PC) is a group of hereditary syndromes which have in common a hypertrophic dystrophy of the distal nail, and are associated with a variety of additional features, notably various dyskeratoses of skin and mucous membranes. The pathology is unknown but the array of clinical features suggests the possibility of a keratin abnormality. In the present report we describe linkage analyses in a large PC pedigree of the Jackson-Lawler type, a subtype which is characterised by multiple epidermal cysts, hair abnormalities, and natal teeth. The disease locus in this family was found to be tightly linked to markers mapping within, or very close to, the keratin type I cluster at 17q12-q21; maximum lod scores for linkage of the disease to a KRT10 polymorphism and to D17S800, a marker known to be very tightly linked to KRT10, were respectively +4.51 and +7.73, both at theta = 0.00. Although always likely, our findings provide strong evidence of a keratin gene anomaly underlying an inherited disorder affecting epidermis, nail, hair, and mucosa. These findings permit testing to see if pachyonychia congenita shows any locus heterogeneity and suggest specific candidate keratin genes for mutation searching studies. In addition, they suggest a role for keratins in the phenomenon of natal dentition.     

The ERAP gene is associated with HCV chronic infection in a Chinese Han population

Endoplasmic reticulum aminopeptidases (ERAPs), ERAP1 and ERAP2, are critical components in the antigen-presentation system and are specialized to produce optimal-sized peptides for HLA I binding. ERAP gene polymorphisms have been correlated with HLA-associated diseases. To investigate the association between ERAP gene polymorphisms and HCV chronic infection, a TaqMan assay was used to genotype 4 SNPs (rs27044, rs30187, rs26618 and rs26653) in ERAP1 and 2 SNPs (rs2248374 and rs2549782) in ERAP2 genes in 376 Chinese Han HCV chronic infections and 324 healthy Chinese Han controls. The allelic distribution of rs26618 in the ERAP1 gene and rs2248374 in ERAP2 gene were both significantly different in case and control groups. The C-allele of rs26618 had an increased HCV chronicity risk compared with the T-allele (P = .025, OR = 1.318, 95%CI: 1.035–1.677), and the same effect was found in A-allele of rs2248374 compared with G-allele (P = 0.046, OR = 1.244, 95%CI: 1.004–1.540). There were notable differences in the genotype distribution in analysis using the dominant genetic model in rs26618 (CC + CT vs. TT; P = 0.007, OR = 1.473, 95%CI: 1.091–1.989) and recessive genetic model in rs2248374 (AA vs. AG + GG; P = 0.003, OR = 1.548, 95%CI: 1.026–2.335). In addition, rs26618 and rs2248374-genotype combination played noteable effects on the clinical parameters. These results indicated that the ERAP gene may play a critical role in HCV chronicity in this Chinese Han population.     

甲型副伤寒杆菌鞭毛蛋白H1a基因原核表达系统构建及其表达产物的免疫原性和保护作用

目的 构建甲型副伤寒杆菌（Salmonella paratyphi A）H1a基因原核表达系统，确定表达产物rH1a免疫原性和保护作用，检测甲型副伤寒杆菌临床菌株H1a基因携带和表达情况。方法 采用高保真PCR从甲型副伤寒杆菌临床株JH01中扩增H1a基因，T-A克隆后测序，构建H1a基因原核表达系统pET32a-H1a-E．coli B121DE3。采用SDS-PAGE和BioRad凝胶图象分析系统检查rH1a表达情况，Ni-NTA亲和层析法收集rH1a。采用Western blot鉴定其免疫反应性和免疫原性。建立PCR和ELISA检测98株甲型副伤寒杆菌临床菌株H1a基因携带和表达频率。观察rH1a对甲型副伤寒杆菌50001株感染小鼠的免疫保护作用。结果 与报道的相关序列比较，所克隆的H1a基因核苷酸和氨基酸序列相似性均为99．59％。rH1a表达量为细菌总蛋白的60％左右。甲型副伤寒杆菌全菌抗血清能识别rH1a并与之结合。rH1a免疫家兔可产生抗体。100％（98／98）甲型副伤寒杆菌临床菌株均含有H1a基因并表达H1a。500μg rH1a灌喂或皮下注射免疫小鼠受甲型副伤寒杆菌50001株攻击后的存活率均为50．0％，若加入5μg rLTB，存活率分别上升至75．0％和66．7％。结论 本研究成功地从甲型副伤寒杆菌临床菌株中构建了H1a基因高效原核表达系统。rH1a有良好的免疫原性和一定的免疫保护作用。甲型副伤寒杆菌临床菌株广泛存在H1a基因并高频率表达。     

甲型副伤寒杆菌nmpC基因原核表达及表达产物免疫保护作用

目的 构建甲型副伤寒杆菌外膜蛋白基因nmpC的原核表达系统,确定其重组表达产物rNmpC免疫原性和保护作用,了解甲型副伤寒杆菌临床菌株nmpC基因携带及表达率.方法 采用PCR和T-A克隆法从甲型副伤寒杆菌临床株JH01中获得nmpC基因克隆并构建其原核表达系统.采用SDS-PAGE和Bio-Rad凝胶图像分析系统检测rNmpC表达情况及其产量,采用免疫扩散法、Western blot和微量肥达试验鉴定其抗原性和免疫应答性.采用PCR和ELISA分别检测98株甲型副伤寒杆菌临床菌株nmpC基因携带及表达率.采用小鼠感染模型了解rNmpC对甲型副伤寒杆菌致死性感染的免疫保护作用.结果 与报道的相关序列比较,所克隆的nmpC基因核苷酸和氨基酸序列相似性均为100%.rNmpC表达量约为细菌总蛋白的30%.rNmpC免疫家兔可产生抗体并能与甲型副伤寒杆菌全菌抗血清产生阳性Western杂交信号.所有甲型副伤寒杆菌菌株均携带nmpC基因并表达NmpC蛋白,但伤寒杆菌、乙型及丙型副伤寒杆菌未检出nmpC基因.100μg和200μgrNmpC对感染小鼠的免疫保护率分别为41.7%(5/12)和66.7%(8/12).rNmpC免疫小鼠或保护试验存活小鼠血清仅对甲型副伤寒杆菌H抗原产生1∶5～1∶40的凝集效价.结论 NmpC是甲型副伤寒杆菌独有的序列保守、分布广泛且自然表达的外膜蛋白抗原,该外膜蛋白具有良好的免疫原性和一定的免疫保护作用,可作为多价甲型副伤寒杆菌基因工程疫苗候选抗原.     

The expression of the virulence-associated effector protein gene avrA is dependent on a Salmonella enterica-specific regulatory function

Although the avrA gene is prevalent among 80% of the Salmonella enterica serovars, only a small number of them usually express the respective virulence-associated effector protein AvrA. However, under culture conditions below pH 6.0 many of the AvrA non-producer strains (e.g. S. Agona, S. Bovismorbificans, S. Virchow) begin to produce AvrA, while some others remain silent. Four phenotypical classes of S. enterica were identified under defined standard culture conditions: class 1 comprises strains with a constitutive synthesis of AvrA; class 2 comprises strains with an acid induction of AvrA; class 3 comprises strains with silent avrA genes; and the fourth class comprises strains which do not contain the avrA gene (class 0 strains). The expression of avrA was found to be controlled by a Salmonella-specific mechanism because cloned avrA genes from classes 1, 2, and 3 strains remain silent in Escherichia coli strains, while easily expressed in S. enterica strains. The expression of AvrA in classes 1, 2, and 3 strains does not coincide with the nucleotide sequences of the respective promoter or structural regions of the avrA genes, but depends directly on this Salmonella-specific regulatory system which appears to be differently modulated in the distinct Salmonella serovars.     

A rapid method to identify Salmonella enterica serovar Gallinarum biovar Pullorum using a specific target gene ipaJ

Salmonella enterica serovar Gallinarum biovar Pullorum (S. Pullorum) is the pathogen of pullorum disease, which leads to severe economic losses in many developing countries. Traditional methods to identify S. enterica have relied on biochemical reactions and serotyping, which are time-consuming with accurate identification if properly carried out. In this study, we developed a rapid polymerase chain reaction (PCR) method targeting the specific gene ipaJ to detect S. Pullorum. Among the 650 S. Pullorum strains isolated from 1962 to 2016 all over China, 644 strains were identified to harbour ipaJ gene in the plasmid pSPI12, accounting for a detection rate of 99.08%. Six strains were ipaJ negative because pSPI12 was not found in these strains according to whole genome sequencing results. There was no cross-reaction with other Salmonella serotypes, including Salmonella enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum), which show a close genetic relationship with S. Pullorum. This shows that the PCR method could distinguish S. Gallinarum from S. Pullorum in one-step PCR without complicated biochemical identification. The limit of detection of this PCR method was as low as 90?fg/μl or 10² CFU, which shows a high sensitivity. Moreover, this method was applied to identify Salmonella isolated from the chicken farm and the results were consistent with what we obtained from biochemical reactions and serotyping. Together, all the results demonstrated that this one-step PCR method is simple and feasible to efficiently identify S. Pullorum.     

Exogenous tumor necrosis factor alpha and interleukin-1 alpha increase resistance to Salmonella typhimurium: efficacy is influenced by the Ity and Lps loci.

Interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor alpha (TNF-alpha) administered prior to infection with Salmonella typhimurium increases survival in mice that are Ityr, not in susceptible Lpsd or Itys mice. Combined IL-1 alpha and TNF-alpha pretreatment results in greater survival than that seen with either cytokine alone in Ityr mice. Treatment after infection with TNF-alpha and/or IL-1 alpha increases the mean time to death but not the survival fraction of Lpsd mice and was ineffective in either Ityr or Itys mice.     

利用减毒沙门菌递送的柯萨奇病毒A16型VP1基因重组质粒的构建和鉴定

目的:构建能够稳定携带柯萨奇病毒A16型(CoxA16)VP1基因的重组减毒伤寒沙门菌并鉴定目的抗原的表达,研发利用减毒沙门菌递送的口服疫苗.方法:利用DNA重组技术,构建表达CoxA16 VP1蛋白的真核表达质粒,体外转染Vero细胞后检测VP1蛋白表达情况及抗原活性,并将该质粒通过电转化构建重组减毒伤寒沙门菌.结果:构建了表达CoxA16 VP1蛋白的重组质粒,转染vero细胞后检测到CoxA16 VP1蛋白的表达并具有抗原活性;获得能稳定携带该质粒的重组减毒伤寒沙门菌.结论:成功构建稳定携带柯萨奇病毒A16型(CoxA16)VP1基因的重组减毒伤寒沙门菌,为口服CoxA16疫苗的研究打下了基础.     

Rheumatoid arthritis is caused by a Proteus urinary tract infection

Genetic, molecular and biological studies indicate that rheumatoid arthritis (RA), a severe arthritic disorder affecting approximately 1% of the population in developed countries, is caused by an upper urinary tract infection by the microbe, Proteus mirabilis. Elevated levels of specific antibodies against Proteus bacteria have been reported from 16 different countries. The pathogenetic mechanism involves six stages triggered by cross‐reactive autoantibodies evoked by Proteus infection. The causative amino acid sequences of Proteus namely, ESRRAL and IRRET, contain arginine doublets which can be acted upon by peptidyl arginine deiminase thereby explaining the early appearance of anti‐citrullinated protein antibodies in patients with RA. Consequently, RA patients should be treated early with anti‐Proteus antibiotics as well as biological agents to avoid irreversible joint damages.     

Isolation and characterization of the Treponema denticola prtA gene coding for chymotrypsinlike protease activity and detection of a closely linked gene encoding PZ-PLGPA-hydrolyzing activity.

The chymotrypsinlike protease gene (prtA) from Treponema denticola ATCC 35405 was isolated from a lambda gt11 clone bank as one of several clones expressing protease activity. The DNA from one positive clone capable of hydrolyzing type IV collagen was subcloned into plasmid vector pUC119 for further analysis. Deletion analysis of subclone pXQ27.2 revealed the approximate location of the prtA gene on the DNA insert. DEAE-Sephadex chromatography of crude cell extracts of the subclone revealed two distinct T. denticola enzymes, one hydrolyzing SAAPNA (succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide [chymotrypsin substrate]) and the other hydrolyzing PZ-PLGPA (phenylazobenzyl-oxycarbonyl-L-leucylglycyl-L-prolyl-D -arginine [collagenase substrate]). Each activity was purified to near homogeneity and exhibited by sodium dodecyl sulfate-polyacrylamide gel electrophoresis estimated molecular sizes of 67 and 36 kDa, respectively. Western blot (immunoblot) analysis demonstrated that only the 67-kDa SAAPNA-hydrolyzing enzyme reacted with antibody against the T. denticola chymotrypsinlike protease. The purified SAAPNA-hydrolyzing enzyme degraded type IV collagen, laminin, and fibronectin, but not type I collagen. These results indicate that the prtA gene coding for the chymotrypsinlike protease from T. denticola has been isolated. Another distinct gene encoding an enzyme hydrolyzing PZ-PLGPA appears to be adjacent to the prtA gene.     

一个单纯家族性嗜铬细胞瘤家系的VHL基因突变筛查

目的检测一个单纯家族性嗜铬细胞瘤家系的VHL基因突变情况。方法对一个单纯家族性嗜铬细胞瘤家系进行VHL基因突变检测，抽取该家系5例患者及15名血缘亲属外周血基因组DNA，对VHL基因3个外显子进行PCR，产物进行DNA测序。结果该家系5例患者均检测出VHL基因第2外显子上第587位核苷酸A—C突变，该突变导致第125位编码氨基酸由组氨酸（H）转变为脯氨酸（P）。15名家系成员中筛查出7名成员为该突变基因携带者，B超检查发现1例为双侧肾上腺肿瘤，1例为右肾囊肿。该突变为首次报道。结论该嗜铬细胞瘤家系中检测到可能的致病突变，VHL基因检测可早期发现致病基因携带者，建议对单纯家族性嗜铬细胞瘤患者常规进行VHL基因突变筛查。     

Enhanced serum methylated p16 DNAs is associated with the progression of gastric cancer

Objective: The present study is to evaluate the effect of methylated p16 on the progression in patients with gastric cancer (GC), and develop a useful biomarker for predicting patient’s prognosis. Design and methods: Methylation status of p16 in GC, their corresponding para-cancerous histological normal tissues (PCHNTs), preoperative peritoneal washes (PPWs) and serum were assessed using real-time methylation specific-PCR (MSP). Results: The frequency of p16 methylation was significantly higher in GC tissues (85.9%; 79/92) than that in paired PCHNTs (12.0%; 11/92) (P<0.0001). p16 methylation correlated closely with lymph node metastasis, peritoneal metastasis, TNM stage, et al (all P<0.05). Both frequency of p16 methylation in PPWs and serum were 79.7% (63/92). The Aζ value of the receiver-operator characteristic curve for methylated p16 was 0.899 for serum and PPWs, compared to that in GC tissues. The patients with elevated methylated p16 levels in tumor tissues had poorer disease-free survival (DFS) rates than those without (P=0.042). There is a narrow significant difference in median survival time of more than 30 months between patients with and without preoperatively detectable methylated p16 in serum (P=0.057). Methylated p16 in PPWs revealed no significant association with survival (P=0.129). Cox regression analysis showed that serum methylated p16 DNAs was an independent risk factor for GC patients, with a remarkable decrease in DFS 30 months after surgical resection of the gastric tumor. Conclusions: Serum methylated p16 DNAs might serve as a potential biomarker for the progression and a prognostic factor in gastric cancer patients.