Detection of leptospiral antigen (L. interrogans serovar copenhageni serogroup Icterohaemorrhagiae) by immunoelectron microscopy in the liver and kidney of experimentally infected guinea-pigs.

Guinea-pigs were experimentally infected with L. interrogans serovar copenhageni serogroup Icterohaemorrhagiae and their liver and kidney were studied by immunoelectron microscopy using the post embedding indirect immunogold labelling technique. Primary antibody was a purified rabbit anti-serum produced against the same leptospiral strain used in the inoculum. Gold-labelled leptospiral antigen (LAg) was found close to cell membranes of hepatocytes, kidney tubular cells and endothelial cells of the interstitial capillaries of the kidney. Afterwards it was internalized by hepatic and tubular cells, and eventually found in lysosomes. Phagolysosomes of Kupffer cells were also found to contain remnants of degraded leptospires and gold-labelled LAg. Gold-labelled intact leptospires were detected at the enlarged intercellular spaces between hepatocytes at the areas of hepatic cell plate disarray, showing the potential for leptospiral migration during the septicaemic phase of the disease potentially contributing to the pathogenesis of the lesions. The affinity of leptospiral antigenic material for cell membranes suggests an initial interaction with cell surface proteins followed by its internalization and cell damage. The nature of antigenic material detected, however, remains undefined; it may be a toxin, an enzyme or any other factor/s involved in leptospiral virulence.     

Immunohistochemical and in situ hybridization studies of the liver and kidney in human leptospirosis

An in situ hybridization (ISH) assay for the detection of leptospiral DNA in tissues was described and its diagnostic and pathogenetic usefulness in combination with immunohistochemistry (IHC) was evaluated in formalin-fixed, paraffin-embedded liver and kidney samples from human fatal cases of leptospirosis. IHC assays with anti-E-cadherin antibodies assessed the liver-plate disarray frequently observed in leptospirosis. Immunohistochemistry detected leptospiral antigen (LAg) in macrophages, both in human liver and kidney. In guinea pigs, in addition to these findings, staining on cell membranes of hepatocytes and, occasionally, in apical membrane of kidney tubular cells was demonstrated. Positive ISH signal was observed chiefly in the nuclei of human hepatocytes and in the cytoplasm and nuclei of liver cells of experimentally infected guinea pigs. Loss of E-cadherin membrane expression is associated with liver-plate disarray. These findings were discussed in the contention that, in leptospirosis, cell membrane damage might be important for the pathogenesis of the disease. Finally, it was suggested that both IHC and/or ISH might be used for both diagnostic and research purposes.     

问号钩端螺旋体侵入单核-巨噬细胞方式及其吞噬泡形成差异性研究

目的 探讨问号钩端螺旋体(简称钩体)侵入人或鼠单核-巨噬细胞方式及其吞噬泡形成差异性.方法 采用透射电镜观察问号钩体黄疸出血群赖型赖株侵入小鼠单核-巨噬样细胞J774A.1和佛波酯(PMA)激活的人单核细胞THP-1后吞噬泡形成情况.采用免疫荧光联合激光共聚焦显微镜及荧光分光光度仪等方法,观察细胞内吞抑制剂单丹磺酰尸胺(MDC)、氧化酚砷(PAO)阻断及内吞相关网格蛋白抗体封闭前后,J774A.1细胞和PMA激活的THP-1细胞内问号钩体赖株数量的变化.结果 J774A.1细胞内问号钩体存在于吞噬泡内,THP-1细胞内问号钩体无吞噬泡膜包绕.MDC和PAO能以剂量依赖方式抑制J774A.1和THP-1细胞内吞问号钩体,其中10 μmol/L以上MDC和1 μmol/L以上PAO阻断的J774A.1和THP-1细胞内问号钩体数量明显少于未阻断细胞(P＜0.05).网格蛋白抗体封闭后,J774A.1和THP-1细胞内问号钩体数量也明显减少(P＜0.05).结论 问号钩体以网格蛋白依赖性内吞途径侵入人或鼠单核-巨噬细胞.人或鼠单核-巨噬细胞内问号钩体吞噬泡形成有明显差异,这可能是人或鼠感染问号钩体后发病情况不同的原因之一.     

Detection of leptospiral antigen in the human liver and kidney using an immunoperoxidase staining procedure

Detection of leptospiral antigen using an immunoperoxidase staining (IP) procedure was carried out on fifteen samples of human liver, (nine from autopsies and six from biopsies) and nine samples of human kidneys (eight autopsies and one biopsy). The IP staining procedure to detect leptospiral antigen (L. interrogans serovar icterohaemorrhagiae) in human liver and kidney proved to be a reproducible method useful on paraffin embedded tissues after formalin, Bouin's or Helly's fluid fixation. Furthermore, the IP procedure on paraffin-embedded tissue appears to have potential as an aid to the diagnosis. IP stained leptospiral antigen was detected in portal spaces of the liver, engulfed by cells of the mononuclear phagocyte system, in the interstitium of the kidney, and lining vessel walls of both liver and kidney. The results suggest that in acute human leptospirosis (L. interrogans serovar icterohaemorrhagiae) the main factors in the pathogenesis of the lesions are related to the presence of organisms and/or their virulence, including products released by lysis.     

In vitro association of leptospires with host cells.

Interactions of Leptospira interrogans with cultured endothelial and kidney epithelial cells were assayed by examining (i) cytoadherence of intrinsically radiolabeled leptospires to eucaryotic cell monolayers and (ii) penetration of leptospires through cell monolayers grown on polycarbonate filters in chemotaxis chambers. L. interrogans serovars attached to cultured cells in a dose- and time-dependent manner. Adherence was diminished following pretreatment of organisms with proteases, rabbit immune serum, or heat. When observed by scanning electron microscopy, most leptospires attached by both ends, rather than just one tip like Treponema pallidum. In penetration assays, 9.7% of added L. interrogans migrated through the monolayer-filter barrier, while only 0.3% of L. biflexa penetrated in the same time interval. Transmission electron microscopy revealed that organisms entered the host cell cytoplasm. These in vitro results indicate that leptospires have an invasive capacity that may be related to pathogenicity in vivo and suggest that further investigation of interactions with host cells may enhance knowledge of leptospiral virulence.     

Effects of products of autolysis of tissue and urine on the viability, morphology, and antigenicity of Leptospira interrogans serovar pomona.

The effect of the products of autolysis on Leptospira interrogans serovar pomona was investigated in bovine kidney tissues and urine inoculated with this organism. No viable leptospires were found at 24 h or subsequently after inoculation of kidney tissues or urine. Leptospires and their soluble antigens were rapidly destroyed in tissues stored at 20 degrees C and could not be detected by the fluorescent-antibody test and the enzyme-linked immunosorbent assay. On the other hand, leptospires were detectable by dark-field microscopy and fluorescent-antibody test 38 days after addition to urine. The rate of destruction proceeded less rapidly in tissues stored at 4 degrees C. Under the conditions of this study, soluble leptospiral antigen levels decreased less rapidly than did the number of detectable leptospires. Similarly, breakdown of leptospires, demonstrated by dark-field microscopy and fluorescent-antibody test, progressed during the first 5 days after inoculation of urine, when levels of soluble leptospiral antigen remained constant. Although the number of intact leptospires was markedly reduced after freezing of tissues at -20 degrees C, soluble leptospiral antigen levels remained unaltered. Neither intact leptospires nor their soluble antigens were detected after preservation of tissues in Formalin. However, formolization of urine delayed the destruction of leptospires and increased the levels of soluble leptospiral antigen during at least the first 5 days after inoculation. These results indicate that assays such as the enzyme-linked immunosorbent assay should be useful for the detection of soluble leptospiral antigens in urine and in autolyzing tissues such as those taken from a bovine fetus aborted as a result of leptospiral infection.     

Direct detection of leptospiral material in human postmortem samples

Leptospiral culture, direct immunofluorescence, and the polymerase chain reaction (PCR) were used to detect leptospiral material in postmortem specimens collected from eight patients who died of leptospirosis. Diagnosis of leptospiral infection was based on clinical summary (premortem) and confirmed by serological analysis and/or culture of leptospires. Leptospiral culture was the least sensitive technique, yielding two isolates (3%) from 65 samples. Both isolates were from the aqueous humour and cerebrospinal fluid of the same patient. Direct immunofluorescence was of intermediate sensitivity for detection of leptospires, confirming the presence of leptospires in 11% (2 of 18) of tissue samples from three patients. PCR analysis was the most sensitive technique for detection of leptospiral material in tissue samples, being positive in 20% (11 of 56) of samples from eight patients. Both samples (cerebellum and liver) positive by immunofluorescence were also positive by PCR. The sensitivity of the PCR assay was 1-10 leptospires ml(-1) sample, and the assay was specific for Leptospira pathogenic species. Multi-system involvement was indicated based on successful amplification of leptospiral DNA from more than one tissue sample, which corroborated with the clinical and pathologic findings. The results suggest that in acute and/or fatal leptospirosis, the pathogenesis of the pathologic features are related to the presence of the organisms in the tissues. In conclusion, PCR combined with serology appears to be a useful tool for diagnosis of leptospirosis and may be invaluable in epidemiological studies.     

Detection of leptospiral DNA by nucleic acid hybridisation with 32P- and biotin-labelled probes

Dot hybridisation with 32P- and biotin-labelled probes prepared from leptospiral DNA was performed to develop a sensitive and specific diagnostic method for early infection with leptospires. The smallest amounts of leptospiral DNA that could be detected with 32P- and biotin-labelled probes was 1.5 pg and 5 pg, respectively, corresponding to about 750 and 2500 leptospires. Dot hybridisation with a 32P-labelled probe detected leptospiral DNA in sera from all of 14 experimentally infected golden hamsters. The smallest amount of leptospiral DNA detected in these experiments corresponded to about 2500 leptospires. In the test conditions described in this study, the sensitivity of dot hybridisation with a biotin-labelled probe was lower. Little cross-hybridisation was observed with unrelated DNAs which indicates that dot hybridisation could be a useful diagnostic method.     

Chemiluminescence and phagocytic responses of rat polymorphonuclear neutrophils to leptospires

The interaction of leptospires with polymorphonuclear neutrophils (PMN) was examined by the luminol-dependent chemiluminescence (CL) test. Whole blood CL changed in relation to the stage of leptospiral infection both in susceptible (SUS) and resistant (RES) rats. The intensity of CL grew with an increasing number of leptospires in the blood. CL responses were observed in isolated PMN upon exposure to living leptospires. In contrast, the same bacteria, having been inactivated by formalin, did not stimulate PMN. A variation was found in the CL response by different living strains of Leptospira. The CL intensity was arranged as follows: L. illini greater than L. biflexa greater than L. interrogans avirulent strains greater than L. interrogans virulent strains. The CL response was markedly enhanced by an opsonization of leptospires. Specific opsonization was shown to increase the rate of phagocytosis of leptospires with relation to the CL response.     

Pathogenesis of renal lesions in haemoglobinaemic and non-haemoglobinaemic leptospirosis

Hamsters were infected with Leptospira interrogans serovar ballum or Leptospira interrogans serovar pomona and the kidney lesions were compared by light and electron microscopy. Ballum and pomona both caused severe clinical signs and death within 6 days in some animals, although only ballum was associated with red blood cell destruction and haemoglobinaemic nephrosis. With ballum infections it is difficult to distinguish degenerate changes resulting from leptospiral "toxins" from those resulting from hypoxia and haemoglobinaemic nephrosis because large numbers of organisms and haemoglobinaemia coincide shortly before death. Although large numbers of leptospires were seen within the renal interstitium and blood vessels in animals dying shortly after infection, organisms were seen only in the proximal convoluted tubules of those surviving until 14 days. It is thought that leptospires are carried by the bloodstream and migrate at random throughout all body tissues. When antibody develops, only those in the renal tubules remain. The random migration results in some leptospires entering tubules at all levels of the nephron but there are good grounds for believing that the normal changes in composition of the glomerular filtrate as it passes through the nephron are increasingly deleterious to leptospiral survival. This probably explains why leptospires are found predominantly in the proximal convoluted tubules of animals after the development of specific immunity.     

Morphological changes in red blood cells of calves caused by Leptospira interrogans serovar pomona

Haemoglobinaemia is seen in certain hosts infected by certain serovars of Leptospira interrogans, but is absent from other serovar-host associations. Comparisons were made between calves infected with serovar pomona and those receiving a crude "toxin" prepared from the same organism. Red blood cells from "toxin"-injected calves showed discocyte-echinocyte transformation and contained portions of cytoplasm segregated within vacuoles. These animals showed increased sequestration of RBCs within the spleen but no overt haemoglobinaemia. Red blood cells from infected and haemoglobinaemic animals were spherical and pitted. They also showed vacuoles and tracts under the cell membrane in fully haemoglobinized RBCs and dark granular inclusions within the cytoplasm of those which were only partially haemoglobinized. Intracellular leptospires were not seen within the RBCs. Red blood cell sequestration and erythrophagocytosis were very pronounced within the spleen, liver and bone marrow. The changes in the RBCs are not easily explained by the previously proposed theory that RBC destruction is due to a phospholipase-like toxin acting directly upon the RBC membrane. A more appropriate hypothesis is that the RBC lesions are due to the adverse effects of leptospiral "toxin(s)" on the metabolism of the RBC causing the formation of defective portions of cytoplasm. These are then either degraded and expelled, leaving empty vacuoles, or are degraded and left within the cytoplasm as dark granular inclusions.     

Leptospirosis: aspects of innate immunity, immunopathogenesis and immune evasion from the complement system

Leptospirosis is a neglected infectious disease caused by spirochetes from the genus Leptospira. It constitutes a major public health problem in developing countries, with outcomes ranging from subclinical infections to fatal pulmonary haemorrhage and Weil's syndrome. To successfully establish an infection, leptospires bind to extracellular matrix compounds and host cells. The interaction of leptospires with pathogen recognition receptors is a fundamental issue in leptospiral immunity as well as in immunophatology. Pathogenic but not saprophytic leptospires are able to evade the host complement system, circulate in the blood and spread into tissues. The target organs in human leptospirosis include the kidneys and the lungs. The association of an autoimmune process with these pathologies has been explored and diverse mechanisms that permit leptospires to survive in the kidneys of reservoir animals have been proposed. However, despite the intense research aimed at the development of a leptospirosis vaccine supported by the genome sequencing of Leptospira strains, there have been relatively few studies focused on leptospiral immunity. The knowledge of evasion strategies employed by pathogenic leptospires to subvert the immune system is of extreme importance as they may represent targets for the development of new treatments and prophylactic approaches in leptospirosis.     

Adhesion of leptospires to mouse fibroblasts (L929) and its enhancement by specific antibody

The adhesion of leptospires (Leptospira interrogans serovar. copenhageni L45) to mouse L-cells was studied by microscopic observations. Within 3 h of infection of monolayers many leptospires adhered to 95-100% of the cells, and intracellular leptospires were demonstrated by electron microscopy. No specific site of attachment on the cells or the leptospires was observed. Avirulent or dead leptospires adhered poorly but attachment of the saprophytic leptospire L. biflexa serovar. patoc occurred on cell and glass surfaces. After adhesion, microvilli on the cell surfaces disappeared within 6 h of infection and cell damage was observed after 12 h. The adhesion was greatly enhanced by the presence of specific antiserum at a subagglutinating concentration. No direct penetration by leptospires of the host cells was observed with transmission and scanning electron microscopy. It appears that (1) adhesion of leptospires to L-cells precedes cell damage, and (2) leptospires may enter cells either through damaged membranes, or by a phagocytosis-like mechanism.     

Clinico-morphological characteristics of leptospirosis

Observations in 5 patients dying with leptospirosis (at 6-30 days after the onset) caused by leptospira of the Hebdomadis serogroup are described. Bright jaundice developed due to severe serous liver centrilobular edema with pronounced dissociation of the liver cell bands, cholestasis, and cloudy swelling of hepatocytes. Albuminuria, oliguria, and uraemia were caused by acute tubular and interstitial nephritis. Hemorrhagic diathesis with small bleedings in the skin, mucous and serous membranes was associated with the impairment of permeability of microcirculatory vessels. Focal necroses of skeletal muscles, myocardiodystrophy, and focal myocarditis, serous leptomeningitis were also seen. Few leptospirae could be detected extracellularly (often attached to the outer host cell membranes) by Warthin-Starry's method of silver impregnation of paraffin sections and by indirect immunofluorescence, much more rarely could they be demonstrated by Levaditi's method of silver impregnation of pieces of the viscera. Most leptospirae were present in the liver in the case of death on the 6th day when no antibodies were yet present in the blood. Since the 9th day when the antibodies did appear, leptospirae gradually disappeared from the liver and were found in the kidneys. It is suggested that some leptospiral cytotoxic products play a role in the pathogenesis of leptospirosis.     

Identification and characterization of the protein antigens of Leptospira interrogans serovar hardjo.

We radiolabeled Leptospira proteins with [35S]methionine. Solubilized extracts of radiolabeled L. interrogans serovar hardjo strain hardjoprajitno were analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. We compared the protein profile obtained in this manner to the protein profiles of various [35S]methionine-labeled Leptospira spp. The profiles of the pathogenic L. interrogans strains were very similar but not identical and exhibited no obvious relationship to those of the two nonpathogenic species. We used solubilized, radiolabeled hardjoprajitno extracts and a sensitive radioimmunoprecipitation procedure to identify protein antigens recognized by immunoglobulin G antibodies present in various rabbit anti-hardjo sera. Homologous hyperimmune rabbit serum efficiently precipitated a large subset of proteins, the majority of which were between 30,000 and 66,500 daltons. Radioimmunoprecipitations with sera prepared against each of four recent hardjo isolates cultured from infected cattle produced similar results. Immunoprecipitations done with various radiolabeled Leptospira extracts and anti-hardjoprajitno serum demonstrated that the pathogenic leptospires possessed a number of cross-reactive major and minor protein antigens. By cell fractionation procedures, we found that most of the major protein antigens were present in the outer envelope. These proteins were exposed on the leptospiral cell surface because intact radiolabeled leptospires bound antibodies directed against them.     

Histopathology of the human liver in yellow fever with special emphasis on the diagnostic role of the Councilman body

Liver specimens from 10 cases of yellow fever (YF) were studied by light and four by electron microscopy to review morphological aspects of the disease relevant to its diagnosis, with special emphasis on acidophilic bodies (AB) and on the possible presence of the virus within infected cells. The AB were compared with those found in 22 out of 69 liver specimens with other pathological processes. In YF the typical alteration was an acidophilic hepatocellular necrosis with a preferential midzonal distribution. Ceroid pigment was abundant, its amount was proportional to the degree of liver cell damage and it was found in altered hepatocytes and Kupffer cells in the most damaged areas. The inflammatory infiltrate was scanty, not only in portal tracts but also within the lobules. Electron microscopically, no viral particles were found in liver cells or AB. The latter appeared as round or elliptical cytoplasmic masses, surrounded by a conspicuous cellular membrane and densely packed with organelles, fat vacuoles and residual bodies. They differed from AB in other liver diseases by the presence of fat vacuoles and ceroid pigment. Some peculiarities of AB in other liver diseases such as presence of bile pigment and iron, would depend upon the presence of these pigments in the hepatocytes which originated them.     

Reaction of monoclonal antibodies with species specific determinants in Leptospira interrogans outer envelope

A set of 24 monoclonal antibodies (MABs) was produced against an outer envelope preparation from Leptospira interrogans serovar copenhageni. The MABs reacted in enzyme immunoassay with species-specific determinants of an antigen in the leptospiral outer envelope (OE) of pathogenic but not of saprophytic species of Leptospira. The MABs did not agglutinate whole leptospires, nor could they opsonise homologous leptospires for phagocytosis by mouse macrophages or protect new-born guinea-pigs against lethal infection. The MABs reacted by Western blotting with a 35 x 10(3)-mol-wt band in OE separated on SDS-polyacrylamide gels, and also reacted with other bands to a lesser extent. The determinants to which the MABs were directed were localised in the leptospiral OE by immunogold labelling techniques.     

Leptospirosis vaccines: past, present, and future

It is well known that Leptospira vaccine prevents the disease. However specificity for serovars limits the efficacy of killed whole cell vaccines. Leptospiral antigens that induce cross-protective immunity to the various serovars are sought as new vaccine candidates. In this paper, we have summarized both past and current findings about leptospiral antigens that are conserved among pathogenic leptospires and that induce protective immunity in animal models. The full-length genome sequences of two Leptospira strains have been published and reverse vaccinology has been used to identify leptospiral vaccine candidates. Although humoral immunity is thought to be dominant in protection from leptospiral infection, a role for cell-mediated immunity is now being explored.     

Uptake and killing of Leptospira interrogans and Borrelia burgdorferi, spirochetes pathogenic to humans, by reticuloendothelial cells in perfused rat liver

In situ-perfused rat livers were infused with a single dose of 1.5 x 10(7) radiolabeled cells of Leptospira interrogans serovar icterohaemorrhagiae, the agent of leptospirosis, or with Borrelia burgdorferi IRS, the agent of Lyme disease. Significant (P<0.0001) differences in the liver uptake of L. interrogans and of B. burgdorferi were observed, the uptakes being 37.4%+/-2.3% for L. interrogans and 60.5%+/-3.1% for B. burgdorferi. Leptospires, in contrast to borreliae, were recovered from the livers when liver samples were cultured in growth medium. Leptospires but not borreliae were recovered in bile within 30 min of infusion. The association of leptospires and borreliae with reticuloendothelial cells of the liver was demonstrated by immunohistochemistry. Leptospires and borreliae were found to be associated with vimentin-positive cells and not with desmin-positive cells. Few leptospires but no borreliae were also seen associated with vimentin- and desmin-negative cells, suggesting the presence of leptospires outside the sinusoidal spaces, in the liver parenchyma.     

GLP-1-IR细胞在鼠、猪和人胎儿肠道的分布

选用高特异性单克隆抗 GL P- 1- IR(7- 36 )酰胺抗体 ,用免疫组织化学方法估价了 GL P- 1- IR细胞在鼠、猪和人胎儿肠道中的分布。结果发现 :在各段肠管都可见到 GL P- 1- IR细胞 ;GL P- 1- IR细胞散在于肠腺的柱状上皮细胞之间 ,多位于肠腺的基底部 ,呈开放型内分泌细胞形态 ,即 L 细胞 (EG细胞 ) ;不同种属间 GL P-1- IR细胞密度不同 ,但分布规律相同 ,即自小肠和大肠的近端向远端逐渐增大。结果表明 ,分泌 GL P- 1的 L 细胞 (GL P- 1- IR细胞 )主要分布在肠道远端 ,这提示 ,远端肠道中大量存在的 L 细胞可以接受在近端胃肠道未被吸收的营养物质的刺激 ,使 L 细胞释放 GL P- 1入血