Role of the htrA gene in Klebsiella pneumoniae virulence

We recently described the use of mini-Tn5 to generate complement-sensitive mutants derived from a complement-resistant Klebsiella pneumoniae clinical isolate deficient in the lipopolysaccharide O side chain. One mutant with a reduced capacity to survive in nonimmune human sera carried the transposon inserted in the htrA gene. We cloned and sequenced the gene and predicted from the deduced amino acid sequence that the putative HtrA homolog contains structural features similar to those of previously described HtrA proteins. To investigate the biological functions and the role of the htrA gene in the virulence of K. pneumoniae, we constructed an isogenic mutant by insertion-duplication mutagenesis. Characterization of the mutant showed that it had greater sensitivity to temperature (50 degrees C) and oxidative stress (H(2)O(2)) than the parent strain. Furthermore, the htrA mutant produced less capsule, bound more molecules of complement component C3, and was more sensitive to complement and whole-blood killing than was the parent strain. Finally, disruption of the htrA gene in a virulent K. pneumoniae strain caused a reduction of its virulence in a mice model. Our results indicate that the htrA gene plays an important role in the virulence of K. pneumoniae.     

消化道癌症患者血浆纤维连接蛋白的变化及其与微血管的关系

分析27例消化道癌症患者(其中胃癌18例,食道癌与结肠癌9例)和6例胃溃疡病人的血浆纤维连接蛋白(Fn)的变化。结果发现三者间无明显差异(P>0.05),但胃癌病人中有转移病灶者(n=7)及伴胃炎者(n=10)与相应对照组比,血浆Fn有非常明显降低(p<0.01)。胃癌组织中Fn免疫荧光主要集中在胃癌病人胃壁组织多形核白细胞浸润的炎症区及微血管周围,癌变区Fn免疫荧光很弱,甚至消失。对上述变化的意义进行了讨论。     

卵巢宫内膜样腺癌的病理组织学及组织化学分析

本文通过对２４肺卵巢宫内膜样腺癌的巨检，镜及组织化学染色等结果分析，提出按该瘤所含组织成分，将其分为单纯型及混合型两大类。单纯型中分化程度不同又再分成Ⅰ，Ⅱ，ⅢⅣ级。混合型中根据所含成分又再分为腺棘癌，腺鳞癌，透明细胞腺癌型，含粘液腺癌型及含浆液腺癌型。讨论了该瘤的临床表现，发病率，组织来源，病理特征，诊断步骤及各类型与预后之间的关系。     

Metabolism and physiological behaviour of Leuconostoc mesenteroides subsp. mesenteroides growing on raffinose

The α-galactosidase activities of 11 strains of Leuconostoc mesenteroides subsp. mesenteroides grown on glucose, lactose or raffinose were determined. The inductive effects of lactose and raffinose differed depending on the strain. In four of the strains, α-galactosidase activity of cells grown on lactose was higher than that of cells grown on raffinose, while the reverse was true for one other strain. The study of two strains exhibiting a different behaviour was extended to all raffinose-component sugars: melibiose, sucrose, glucose, galactose and fructose. The apparent fermentation yields with raffinose-component sugars complied with heterofermentation theory, except for sucrose, where there was a 40% deficit in fermentation products. Raffinose hydrolysis enzymes (α-galactosidase and β-fructosidase) underwent a catabolic repression by glucose. Growth on raffinose was considerably stimulated if the inoculum was first grown on melibiose or raffinose (induction of α-galactosidase).     

Catecholamines (CA) and adenosine triphosphate (ATP) are separately stored in bovine adrenal medulla, both in ionic linkage to granule sites, and not as a non-diffusible CA-ATP-protein complex

On superfusion of chromaffin granules from bovine adrenals with isotonic sodium and potassium salts, catecholamines and ATP were released in parallel and both in accordance with ion exchange kinetics. An artificial model was prepared by mixing a cationic (IRC-50) and an anionic (IR-4B) ion exchanger with COO- and NH+3 groups, respectively, as binding sites. This mixed ion exchanger showed in its storage and release of CA+ and ATP- striking similarities to the chromaffin granules. Within the pH range given for the interior of the granules--5.5-6--the artificial model even stored and released CA+ and ATP- within the same molar ratio as observed for the granules. We hypothesize that the chromaffin granule matrix in its storage and release functions operates as an amphoteric ion exchanger with COO- and NH+3 groups as the binding sites.     

CD4+ T cells determine the ability of spleen cells from F1 hybrid mice to induce neonatal tolerance to alloantigens and autoimmunity in parental mice

Spleen cells from F₁ hybrid mice injected into newborn parental mice induce a state of cytolytic unresponsiveness to the corresponding alloantigens. However, these mice develop a transient autoimmune syndrome characterized by the production of multiple autoantibodies and glomerulonephritis. Previous reports indicated that the depletion of F₁ donor T cells, shortly prior the injection into parental mice, does not interfere with any of these events. Here, we have explored whether the continuous absence of T cells in F₁ mice influences the ability of their spleen cells to induce neonatal tolerance to alloantigens and the associated autoimmune manifestations. Our results revealed that spleen cells from athymic (BALB/c × C57BL/6) F₁ hybrid (CB6F₁) nulnu mice or from euthymic CB6F₁ mice depleted from birth of CD4⁺ T cells, but not of CD8⁺ T cells, are unable to induce neonatal tolerance to alloantigens and autoimmune manifestations. By contrast, the partial reconstitution of T cells in CB6F¹ nulnu mice, after the neonatal graft of a syngeneic thymus, restored the capacity of spleen cells from these mice to induce tolerance and autoimmunity when injected into newborn BALB/c mice. These results demonstrate that the functional defect of spleen cells from athymic CB6F₁ nulnu mice to induce neonatal tolerance to alloantigens is directly related to the long-term absence of mature CD4⁺ T cells. Interestingly, a new increase in the titers of anti-DNA Ab was observed when spleen cells from athymic CB6F₁ nulnu mice were injected into adult BALB/c mice that had been tolerized at birth with normal CB6F₁ spleen cells. This finding indicates that B cells from CB6F₁ nulnu mice recover their capacity to interact with alloreactive Th2 cells when they are placed into mice having functional CD4⁺ T cells. These data indicate that the continuous absence of CD4⁺ T cells causes a reversible functional defect in F₁ spleen cells that determines their inability to induce neonatal tolerance and autoimmunity.     

Dynamics of the Murine Humoral Immune Response to Neisseria meningitidis Group B Capsular Polysaccharide

Immunization with Neisseria meningitidis group B capsular polysaccharide (CpsB) elicited responses in adult mice that showed the typical dynamic characteristics of the response to a thymus-independent antigen, in contrast to the thymus-dependent behavior of antibody responses to CpsC. The former had a short latent period and showed a rapid increase in serum antibodies that peaked at day 5, and immunoglobulin M (IgM) was the major isotype even though IgG (mainly IgG2a and IgG2b) was also detectable. This response was of short duration, and the specific antibodies were rapidly cleared from the circulation. The secondary responses were similar in magnitude, kinetics, IgM predominance, and IgG distribution. Nevertheless, a threefold IgG increase, a correlation between IgM and IgG levels, and dose-dependent secondary responses were observed. Hyperimmunization considerably reinforced these responses: 10-fold for IgM and 300-fold for IgG. This favored isotype switch was accompanied by a progressive change in the subclass distribution to IgG3 (62%) and IgG1 (28%), along with the possible generation of B-cell memory. The results indicate that CpsB is being strictly thymus independent and suggest that unresponsiveness to purified CpsB is due to tolerance.

The capsular polysaccharide (Cps) of Neisseria meningitidis group B (CpsB), the major cause of meningococcal disease in developed countries (38), is a linear homopolymer of α(2→8)-linked sialic acid on host sialogangliosides and sialoproteins (12, 16) causes immunological tolerance to sequential CpsB epitopes, with the anti-CpsB antibodies being mainly, if not solely, directed against conformational determinants preferably expressed by chains of eight or more residues (10). The conformational antigenic nature and metastable spatial structure of CpsB (10, 19), in combination with its neuraminidase sensitivity, tendency to internal lactonization, and intramolecular self-cleavage under mild acidic conditions (22, 29), were proposed to explain its poor immunogenicity (35). According to this hypothesis, the interaction of CpsB with B cells is transitory and therefore unable to elicit an antibody response (34). Alternatively, the high expression of longer sialic acid polymers (>12 residues), having the same α(2→8) linkage in polysialylated glycoproteins of vertebrate fetal tissues as well as limited areas of the adult neural system (21, 42), has been proposed to induce tolerance also to the conformational epitopes of CpsB (11). A feasible mechanism for inducing and maintaining tolerance, however, is not known. In any event, the poor immunogenicity of CpsB is associated with the α(2→8) linkage. Purified CpsC, a homopolymer of α(2→9)-linked sialic acid, has been shown to be immunogenic in mice (48).Bacterial Cps complexed to protein carriers induces long-lasting immunoglobulin G (IgG) antibody responses in young children and mice, which is indicative of the Cps conversion to a T-cell-dependent (TD) antigen (18). In contrast, CpsB conjugated to tetanus toxoid (3, 8, 20) or complexed with meningococcal outer membrane proteins (OMPs) (23, 24) is able to induce only low levels of CpsB-specific IgM. In these responses, however, CpsB-specific IgG was detectable (3, 8, 23). Since in simple terms protection from these infectious agents is due to the presence of circulating specific antibodies (13) and bearing in mind that an artificial IgG immune response may initiate an autoimmune process (11), we studied the evolution over time of the serum antibodies and changes in isotype distribution obtained by immunization with the native form of CpsB—namely, live N. meningitidis—in order to further explore the underlying mechanisms in the generation of the immune responses to this peculiar autoantigen which has both epitopes disseminated in the host and epitopes of ontogenetic and topologically restricted expression, a situation reproduced in the mouse model.     

血清、腹水中AFP、CEA及CA125水平对良、恶性腹水的诊断价值

目的 :探讨检测血清、腹水AFP、CEA及CA1 2 5对良、恶性腹水的诊断价值。方法 :放射免疫分析 86例患者血清和腹水AFP、CEA及CA1 2 5的含量。结果 :恶性腹水组血清、腹水AFP、CEA及CA1 2 5水平较之良性腹水组差异非常显著 (p <0 0 1 )。在鉴别结核、肝硬化腹水和恶性腹水时 ,以CA1 2 5≥ 5 0 0U/ml、AFP≥ 30 0ng/ml为临界值[1 ] ,可提高诊断的特异性和准确性。结论 :血清和腹水中AFP、CEA及CA1 2 5水平具有鉴别良、恶性腹水的重要意义。     

SCTA在微小脑动脉瘤诊断中的应用

目的 探讨螺旋CT血管造影（SCTA）在直径小于10mm的脑动脉瘤诊断中的临床应用和价值。方法 选择12例直径小于10mm的脑动脉瘤患，进行SCTA，DSA或MRA检查，SCTA成像技术为表面遮盖法（SSD）和最大密度投影法（MIP）。结果 12例微小脑动脉瘤患术前均准确定性，定位，全部经神经外科手术证实。结论 SCTA是一种无创伤性血管成像方法，在诊断脑动脉瘤方面，具有分辨率高，快速，准确，经济等优点，可为临床诊断与治疗提供更多的信息。     

Cytokine flow cytometry differentiates the clinical status of multiple sclerosis (MS) patients

In this study we have examined intracellular cytokines in peripheral blood mononuclear cells (PBMC) of MS patients by flow cytometry (cytokine flow cytometry). MS progressive patients showed an increased number of cells producing interferon-gamma (IFN-gamma) after activation with phorbol 12-myristate 13-acetate and ionomycin, compared with patients with clinically inactive forms (P < 0001) and with healthy controls (P = 0001). These cells belonged to the CD4+ and CD8+ subsets in similar proportions. Clinically inactive patients showed a lower level of cells producing IL-2 than controls (P = 0.03) and active MS patients (P = 0.03). Most IL-2-producing cells were CD4+ lymphocytes, although a small part of the IL-2 was also produced by CD8+ cells. The percentage of cells producing simultaneously IL-2 and IFN-gamma was increased in active MS and they were mainly CD4+ lymphocytes. No differences in the production of IL-4 were observed between groups. However, we found an increased IL-10 production in clinically active MS patients (P = 0.03). Treatment with IFN-beta of active MS patients showed lower levels of cytokines when compared with untreated MS patients. This methodological approach could help in the follow up and therapeutic monitoring of MS patients.