Confirmation of genital herpes simplex viral infection by an immunoperoxidase technique

Over the 12-month period from April 1984 to April 1985, 512,000 gynecologic (Papanicolaou) smears were examined in the Provincial Screening Program in British Columbia. During this time, 307 patients were found to have smears that contained cells consistent with, or suggestive of, a herpes simplex viral (HSV) infection. The Papanicolaou-stained smears from these 307 cases were subsequently restained, without prior destaining, using an immunoperoxidase technique specific for type 2 HSV (HSV-2) and cross reactive with HSV-1. Of the 205 smears containing cells considered to be consistent with a herpes infection, 187 were positive using the immunoperoxidase technique. Of the 102 smears showing reactive cell changes though unlikely to be causes by an HSV infection, only 5 were positive using the immunoperoxidase technique. The results show that the immunoperoxidase technique is a rapid and reliable method of confirming a suspected diagnosis of herpetic infection and that it is particularly useful in those patients in whom the Papanicolaou smear findings are equivocal.     

Cytodiagnosis of herpes simplex keratitis by means of an immunoperoxidase technique. A case report

Typical herpes simplex keratitis that developed in a 5-year-old boy was initially diagnosed cytologically in Papanicolaou-stained samples. Subsequently, an immunoperoxidase staining technique was used to identify the specific type of herpes simplex virus (HSV) in the destained cellular samples. The positive staining helped to establish the diagnosis of a type 1 HSV infection, permitting early treatment with acyclovir and subsequent complete recovery from the ocular herpetic infection. Emphasis is placed on the value of the immunoperoxidase technique for the rapid and specific diagnosis of cases of suspected HSV infection.     

Immuohistochemical demonstration of alpha-atrial natriretic plypeptide-containing neurons in the rat brain

Using the immunoperoxidase technique in conjunction with specific antisera to alpha-atrial natriuretic polypeptide (alpha-ANP), it was shown that immunoreactive cell bodies and varicose fibers are widely distributed throughout the rat brain. The highest concentrations of alpha-ANP-containing neuronal cell bodies and fibers were found in the hypothalamus and septum. This result confirms the radioimmunological determination of alpha-ANP immunoreactivity in the rat brain.     

Immunoperoxidase technique for identification of Mycoplasma gallisepticum and M. synoviae.

The direct immunoperoxidase technique was applied to the identification of Mycoplasma gallisepticum and M. synoviae by staining colonies on the agar plate. The results of this technique applied to 50 isolates of M. gallisepticum and M. synoviae correlated with those of the agar gel precipitation test to the same isolates. The immunoperoxidase technique was proved to be a specific and reliable method for the identification of M. gallisepticum and M. synoviae.     

Study of neutralizing monoclonal antibodies to bovine herpes virus Type-1 (Cooper strain) by immunoperoxidase and immunoelectron microscopy

Abstract Three neutralizing monoclonal antibodies (mAbs) that are specific against bovine herpes virus Type-1 (BHV-1) were studied as to their viral specificity by immunoperoxidase and immunoelectron microscopy. Microscopic examination of GBK BHV-1 infected cells revealed peroxidase activity represented by red-brown granular deposits in the nucleus and cytoplasm. No immunoperoxidase activity was observed in negative controls. For the ultrastructural observations, two approaches were used. Firstly we tested a pre-embedding technique using GBK infected cells, mAbs and gold conjugated-protein A. Gold particles were observed linked to the viral envelopes and to the host cell membrane. Alternatively, a second technique employed BHV-1 purified by potassium tartrate gradients, mAbs and gold conjugated-protein A. After performing the immune reaction, the samples were adsorbed to formvar-coated grids, stained with phosphotungstic acid and observed in a transmission electron microscope. Gold particles were mainly attached to the virion envelope.     

The effects of different fixatives for immunofluorescent and immunoperoxidase localization of Factor VIII-related antigen in canine carotid artery and vascular prostheses

Summary The evaluation of vascular grafts seeded with autologous endothelial cells requires a reliable method of endothelial cell identification. Previous attempts to identify positively tissue Factor VIII-related antigen, found in relatively large amounts in vascular endothelial cells, proved to be inconsistent when immunoperoxidase and immunofluorescent staining techniques were tried. Because the Factor VIII antigen is very labile, this study was performed to determine an optimal fixation technique for demonstrating this antigen in frozen sections of endothelial tissue. Unfixed, acetone-fixed, and formalin-fixed sections of canine carotid artery as well as vascular grafts fixed in 1-ethyl-3-(3-diaminopropyl)-carbodiimide were examined by an indirect immunofluorescence technique. Also, the immunoperoxidase method of Factor VIII identification was applied to unfixed, acetone-fixed, and carbodiimide-fixed endothelial cell seeded vascular grafts. Acetone was the preferred fixative, resulting in excellent antigen preservation with minimal background staining. The immunoperoxidase technique of Factor VIII-related antigen identification was found to be the method of choice because of its sensitivity.     

Entamoeba histolytica and Entamoeba dispar: prevalence infection in a rural Mexican community

The frequency of Entamoeba histolytica and Entamoeba dispar infection was analyzed in a rural community in the state of Morelos, Mexico, through PCR technique by using specie specific primer. The E. histolytica specie was detected in 33 of 290 analyzed stool samples (11.4%), E. dispar specie was observed in 21 samples (7.2%) and both species of Entamoeba were detected in seven samples (2.4%). So a higher E. histolytica than E. dispar frequency infection was detected (13.8 versus 9.6%). Even though in our design we did not considered the follow-up of included individuals, the absence of invasive amebiasis cases in the studied population during our stay in town was unexpected.     

Recognition of 29 kDa surface-associated adhesive molecule of Entamoeba histolytica by monoclonal antibodies

Abstract Monoclonal antibodies have been developed and used as specific probe to locate and identify a 29-kDa molecule of axenic Entamoeba histolytica trophozoites. Monoclonal antibody produced by clone C8 (MoAb C8) strongly agglutinated the amoebic trophozoites. THe immunofluorescence of live E. histolytica trophozoites and surface fluorescence of acetone-fixed trophozoites by MoAb C8 indicated existence of a 29-kDa molecule on surface-associated plasma membrane of E. histolytica . The monoclonal antibody belonged to IgG₁ isotype. The prior treatment of E. histolytica trophozoites with MoAb C8 resulted in significant ( P < 0.01) reduction in adherence of amoebic trophozoites to cultured Chinese Hamster Ovary cells and significant ( P < 0.01) reduction in cytotoxicity to cultured Baby Hamster Kidney cells. Pretreatment of amoebic trophozoites with MoAb C8 prior to cultivation in TPS-1 medium resulted in significant ( P < 0.01) reduction in growth of the parasite. Thus, the data suggested that the surface-exposed 29-kDa molecule may be one of the receptors involved in E. histolytica host cell interactions and may possibly modulate amoebic disease processes.     

Recognition of 29 kDa surface-associated adhesive molecule of Entamoeba histolytica by monoclonal antibodies

Monoclonal antibodies have been developed and used as specific probe to locate and identify a 29-kDa molecule of axenic Entamoeba histolytica trophozoites. Monoclonal antibody produced by clone C8 (MoAb C8) strongly agglutinated the amoebic trophozoites. The immunofluorescence of live E. histolytica trophozoites and surface fluorescence of acetone-fixed trophozoites by MoAb C8 indicated existence of a 29-kDa molecule on surface-associated plasma membrane of E. histolytica. The monoclonal antibody belonged to IgG1 isotype. The prior treatment of E. histolytica trophozoites with MoAb C8 resulted in significant (P less than 0.01) reduction in adherence of amoebic trophozoites to cultured Chinese Hamster Ovary cells and significant (P less than 0.01) reduction in cytotoxicity to cultured Baby Hamster Kidney cells. Pretreatment of amoebic trophozoites with MoAb C8 prior to cultivation in TPS-1 medium resulted in significant (P less than 0.01) reduction in growth of the parasite. Thus, the data suggested that the surface-exposed 29-kDa molecule may be one of the receptors involved in E. histolytica host cell interactions and may possibly modulate amoebic disease processes.     

间接免疫过氧化物酶技术鉴定猪和牛的肥大细胞

用小鼠抗人肥大细胞类胰蛋白酶单克隆抗体ＡＡ１,ＡＡ３及ＡＡ５的间接免疫过氧化物酶技术对经Ｃａｒｎｏｙ液或中性缓冲福尔马林固定的猪和犊牛空肠,舌及胸腺的石蜡切片进行了免疫染色。对猪和牛的肥大细胞特异性免疫染色与常规的组织化学染色的结果进行了比较。     

Interaction between pathogenic amebas and fibronectin: substrate degradation and changes in cytoskeleton organization

Invasion of human tissues by the parasitic protozoan Entamoeba histolytica is a multistep process involving, as a first step, the recognition of surface molecules on target tissues by the amebas or trophozoites. This initial contact is followed by the release of proteolytic and other activities that lyse target cells and degrade the extracellular matrix. In other parasitic diseases, as well as in certain cancers, the interaction of invasive organisms or cells with fibronectin (FN) through specific receptors has been shown to be the initial step in target cell recognition. Interaction with FN triggers the release of proteolytic activities necessary for the effector cell migration and invasion. Here, we describe the specific interaction of Entamoeba histolytica trophozoites with FN, and identify a 37-kD membrane peptide as the putative receptor for FN. The interaction between the parasite and FN leads to a response reaction that includes the secretion of proteases that degrade the bound FN and the rearrangement of amebic actin into "adhesion plates" at sites of contact with FN-coated surfaces. The kinetics of the interaction was determined by measuring the binding of soluble 125I-FN to the trophozoites and visualization of the bound protein using specific antibodies. Degradation of FN was measured by gel electrophoresis and the release of radioactivity into the incubation medium. Focal degradation of FN was visualized as black spots under the trophozoites at contact sites with fluorescent FN. We conclude that the interaction of E. histolytica with FN occurs through a specific surface receptor. The interaction promotes amebic cytoskeleton changes and release of proteases from the parasite. The binding and degradation of extracellular matrix components may facilitate the migration and penetration of amebas into tissues, causing the lesions seen in human hosts.     

The ontogeny of mullerian inhibiting substance in granulosa cells of the bovine ovarian follicle

Mullerian Inhibiting Substance (MIS) previously detected in the Sertoli cells of the calf testis has been localized in the granulosa cells of the ovarian Graafian follicle by using an immunoperoxidase technique and a monoclonal antibody (IG8) to MIS that almost completely blocks its biological activity. The immunoperoxidase technique (avidin-biotin complex method) demonstrated specific localization of MIS in the cytoplasm of the ovarian granulosa cells in the bovine Graafian follicles over a wide age span, i.e. one day, one week, three months, two-and-a-half years and five years. The presence of MIS in the ovary implies a function that is as yet unknown.     

Identification of gastrin-producing cells in cell cultures and smears: an avidin-biotin-peroxidase complex (ABC) technique

A method is described that uses the avidin-biotin complex (ABC) immunoperoxidase technique to provide a rapid, sensitive, and specific means to quantitate isolated G cells in cultures and suspensions.     

Molecular differentiation of Entamoeba histolytica and Entamoeba dispar from Tunisian food handlers with amoeba infection initially diagnosed by microscopy

The purpose of the study was to obtain more reliable epidemiological data concerning Entamoeba (E.) histolytica infection in Tunisian food handlers using established molecular tools able to differentiate E. histolytica from E. dispar. From 2002 to 2005, 4,266 fresh stools specimens received in the setting of the National program of food handlers' control were analysed by optical microscopy. Twelve (2.8 per thousand) were positive for the presence of four nuclei cysts identified as E. histolytica/E. dispar. Extraction of DNA from the 12 samples, followed by specific amplifications of E. histolytica and E. dispar SSU rDNA, showed that 11 samples (92%) were positive for E. dispar and negative for E. histolytica. Sequencing analysis of 8 PCR products permitted to verify the results obtained with conventional PCR. The remaining sample was negative by PCR amplifying E. histolytica DNA or E. dispar DNA specifically, although it did not show any inhibition. It probably contains protozoan cysts genetically distinct from these two species but morphological similar. Estimation of relative proportions between E. histolytica and E. dispar in cyst carriers showed that all explored individuals harboured the non pathogenic E. dispar strains. This result highlights the need of use in this population of complementary tests that allow specific diagnosis and obviate unnecessary chemotherapy.     

Immunohistochemical localization of 6-keto-PGF1-alpha in canine coronary vasculature

The localization of the prostacyclin metabolite, 6-keto-PGF1-alpha, in canine coronary vasculature was accomplished using immunohistochemical techniques (avidin-biotin method of immunoperoxidase staining). Six-keto-PGF1-alpha was localized to the intimal endothelial cell layer of epicardial and intramyocardial arteries and veins. No specific staining was seen in the the media or adventitia of canine coronary vasculature, or in capillaries, or myocardial fibers. To our knowledge these studies represent the first immunohistochemical demonstration of the endothelial cell localization of the prostacyclin metabolite, 6-keto-PGF1-alpha. The described technique allows the cellular localization of prostaglandin metabolites in histologic sections.     

High-resolution electron microscopical study of cyst walls of Entamoeba spp

Knowledge of the fine structural organization, molecular composition and permeability properties of the cell surface of intestinal protozoan cysts is important to understand the biologic basis of their resistance. Recent studies on the biology of the cyst walls of Entamoeba histolytica and Entamoeba invadens have considerably advanced knowledge on the cellular processes involved in the transport and surface deposition of the main cyst wall components. Using transmission electron microscopy, cytochemistry, scanning electron microscopy and freeze-fracture techniques, we have obtained new information. In mature cysts the permeability of Entamoeba cysts is limited to small molecules not by the cyst wall, but by the plasma membrane, as demonstrated with the use of ruthenium red as an electron-dense tracer. Cell walls of E. histolytica cysts are made up of five to seven layers of unordered fibrils 7-8 nm thick. Alcian blue stains a regular mesh of fibrils approximately 4 nm thick, running perpendicularly to the cyst wall. In addition, abundant ionogenic groups are seen in cyst walls treated with cationized ferritin. In the mature cysts of E. histolytica and E. invadens small cytoplasmic vesicles with granular material were in close contact with the plasma membrane, suggesting a process of fusion and deposition of granular material to the cell wall. The plasma membrane of mature cysts is devoid of intramembrane particles when analyzed with the freeze-fracture technique. When viewed with scanning electron microscopy the surface of E. histolytica cysts clearly differs from that of Entamoeba coli and E. invadens.     

EB病毒转化Hu－TLC－SCID小鼠脾细胞的实验研究

利用ＥＢ病毒转化可产生较高水平人ＩｇＧ和特异性抗２型登革病毒人抗体的Ｈｕ－ＴＬＣ－ＳＣＩＤ小鼠脾细胞,通过免疫组化、免疫荧光和ＰＣＲ法检测转化细胞的人Ｂ细胞表面标志、ＥＢ病毒抗原和ＥＢ病毒基因。结果表明,被转化的Ｈｕ－ＴＬＣ－ＳＣＩＤ小鼠脾细胞能继续产生抗２型登革病毒的特异性人抗体,并具有人Ｂ细胞的、ＳｍＩｇＧ标志及ＥＢ病毒潜伏膜蛋白－１（ＬＭＰ－１）基因,可表达ＬＭＰ－１和ＥＢ病毒核抗原（ＥＢＮＡ）。     

Antibody-induced capping of measles virus antigens on plasma membrane studied by electron microscopy.

Antibodies specific for measles virus could redistribute ("cap") virus antigens on infected HeLa cells as shown by transmission and scanning electron microscopy. Using an indirect immunoperoxidase technique, infected cells showed diffuse, circumferential distribution of virus antigens over the cell surface when mixed with antibody at 4 C. At 37 C, virus-coated microvilli concentrated on one pole of the cell, leaving the remainder of the plasma membrane devoid of both viral antigens and microvillus projections. Whereas extreme polar displacement of virus-antibody complexes frequently occurred, endocytosis was rarely seen. The findings indicate that antiviral antibodies can move and cluster virus on plasma membranes and suggest that virus-antibody complexes are stripped and shed from the cell surface.     

Survival strategies of Entamoeba histolytica: Modulation of cell-mediated immune responses

Tissue invasion and disease associated with the protozoan Entamoeba histolytica has long been connected with suppression of host cellular immunity. Dampening of the host's defences may facilitate survival of amoebae in extraintestinal sites and development of the characteristic amoebic abscesses. In recent years, several studies have begun to clarify, at the cellular level, the specific effects E. histolytica has on immune cell accessory and effector cell functions. Here, Darren Campbell and Kris Chadee discuss the parasite's multiple modulatory effects on macrophages and T cells and how this manipulation of immune defences may enable the parasite to remain viable in the host. They suggest the putative amoebic molecules involved and potential modulation by the cytokines: interleukins IL-4 and IL-10 and transforming growth factor-beta.