Serotyping of Chlamydia trachomatis by indirect fluorescent-antibody staining of inclusions in cell culture with monoclonal antibodies.

A new method for determining the serovars of Chlamydia trachomatis isolates by utilizing fluorescent-antibody staining of inclusions in cell culture is described. Monoclonal antibodies which have been successfully used previously for serotyping in the microimmunofluorescence test were employed. The cell culture method offers two advantages over the microimmunofluorescence test for many laboratories. It requires less antigen of the new isolate, about 10% cell culture infectivity versus 50% for the microimmunofluorescence test. Although fewer isolates can be typed at one time in cell culture, the technical requirements of the test are less rigorous.     

Comparison of monoclonal antibody staining and culture in diagnosing cervical chlamydial infection.

We compared a fluorescein-conjugated monoclonal antibody (FA) direct specimen test (MicroTrak; Syva Co., Palo Alto, Calif.) with culture (TC) in McCoy cells (vials, with blind passage and iodine staining of inclusions) for diagnosis of Chlamydia trachomatis infection in the cervix. Duplicate specimens were collected from 1,230 women, but for 262 of these subjects, both results were unavailable (150 FA smears were inadequate, indicating a need for clinical training in specimen collection), leaving 968 comparisons. Prevalence of chlamydiae by culture was 13% (126/968). Compared with TC results, the sensitivity of FA was 70% (88/126) and the specificity was 94% (795/842). There was a 91% agreement (883/968). The predictive value of a positive FA test was 65% (88/135), and that of a negative FA was 95% (795/833). We reexamined 38 smears for which paired results were discrepant, and the reread would have changed the result in only 5 of these. TC is less than 100% sensitive and some FA-positive, TC-negative specimens represent positive specimens not detected by TC. Unfortunately, it is not possible to identify which results in this group are truly false-positive. Clearly, the FA procedure has a performance profile which would make it a useful tool in screening high-risk populations (particularly when TC is not available) but it is less suited to screening low-risk populations, for which false-positive results are more important. The greater utility of the FA procedure in a venereal disease clinic was confirmed by testing 172 evaluable specimen pairs, of which 34 (20%) were Chlamydia isolate positive. The FA sensitivity was 76% (26/34) and specificity was 96% (133/138), giving a predictive value of 84% (26/31) for a positive test.     

Early detection of chlamydial inclusions combining the use of cycloheximide-treated McCoy cells and immunofluorescence staining.

Detection of Chlamydia trachomatis inclusions only 21 h after a specimen reaches the laboratory has been achieved by the combined use of cycloheximide-treated McCoy cells and immunofluorescence staining. Moreover, cells exposed to cycloheximide were more sensitive for detecting chlamydial inclusions than those pretreated by irradiation, since larger numbers of inclusions were found in the former cells. The application of this rapid and sensitive method allows a diagnosis of chlamydial infection to be made before antibiotic therapy is started. In this way, it should enable the treatment of nonspecific genital infections to be placed on a more rational basis.     

Detection of viral and chlamydial antigens in open-lung biopsy specimens

The recovery of viruses and Chlamydia trachomatis from cell cultures and the detection of their antigens in impression smears prepared from open-lung biopsy (OLB) specimens from immunocompromised adults were compared. Touch impression smears were prepared on three slides, each containing eight wells. OLB tissue was homogenized (Stomacher) and inoculated into MRC-5, primary monkey kidney, and McCoy cell cultures. The direct and indirect immunofluorescence (IF) tests were used to detect antigens to the following organisms: herpes simplex virus, adenovirus, parainfluenza types 1 and 3, respiratory syncytial virus, cytomegalovirus (CMV), Chlamydia trachomatis, influenza types A and B, and varicella-zoster virus. Of 105 OLB specimens, 21 viral isolates (20%) were recovered in cell culture; 20 were CMV and one was an influenza virus type A (H3N2). Both culture and IF results were positive with 12 specimens, but in nine instances a virus was isolated and IF was negative or eight times culture results did not yield the organism but IF test results were positive. Chlamydia trachomatis was never isolated or detected by IF. The authors recommend that for optimal detection of CMV from OLB specimens a new rapid centrifugation-enhanced cell culture system be used in preference, or in conjunction with a preliminary IF screen of impression smears for CMV detection.     

Identification of proliferating lymphocyte subpopulations by combined alkaline phosphatase anti-alkaline phosphatase (APAAP) staining and autoradiography

An improvement in the classification of proliferating ([³H]thymidine incorporating) lymphocyte subpopulations in mitogen- or antigen-stimulated microcultures is described. The binding of subset-specific monoclonal antibodies is detected by the alkaline phosphatase anti-alkaline phosphatase method (APAAP). There are two advantages compared to the peroxidase anti-peroxidase (PAP) method; (1) endogenous enzyme (peroxidase)_activity exhibited by some cells causes no interference, and (2) the red alkaline phosphatase staining obtained with new fuchsin provides a far superior contrast to silver grains than conventional peroxidase staining.     

Knockdown of tissue nonspecific alkaline phosphatase impairs neural stem cell proliferation and differentiation

In the adult mammalian brain the subependymal layer of the lateral ventricles houses neural stem cells giving rise to young neurons migrating towards the olfactory bulb. The molecular cues controlling essential functions within the neurogenesis pathway such as proliferation, short and long distance migration, differentiation and functional integration are poorly understood. Neural progenitors in situ express the tissue nonspecific form of alkaline phosphatase (TNAP), a cell surface-located nonspecific phosphomonoesterase capable of hydrolyzing extracellular nucleotides. To gain insight into the functional role of TNAP in cultured multipotent neural stem cells we applied a knockdown protocol using RNA interference with shRNA and retroviral infection. We show that TNAP knockdown reduces cell proliferation and differentiation into neurons or oligodendrocytes. This effect is abrogated by addition of alkaline phosphatase to the culture medium. Our results suggest that TNAP is essential for NSC proliferation and differentiation in vitro and possibly also in vivo.     

Improvement of rotavirus isolation in the cell culture by immune peroxidase staining.

Peroxidase-labeled monoclonal antibody against rotavirus group-specific antigen (inner capsid) was used for the detection of rotavirus by immunoperoxidase staining (IPS) in trypsin-free MA104 cells within 18 h post-inoculation with clinical specimens. One hundred and twenty-one fecal samples from children with acute gastroenteritis were evaluated by IPS, conventional virus isolation in cell culture and a commercially available group A-antigen ELISA (Rotazyme II, Abbott Laboratories). Fifty-eight (47.9%) stool samples were found positive by IPS. In contrast, rotavirus was isolated from only 4 (3.3%) fecal specimens by conventional cell culture (i.e. demonstration of a cytopathogenic effect). A total of 93 (76.9%) samples were positive by ELISA. IPS permits rapid detection of rotavirus infections and detects shedding of infectious virus. The method should be useful for the investigation of nosocomial spread of rotavirus infection in hospitals, contamination of environmental surfaces and desinfectants.     

Dephosphorylation of endotoxin by alkaline phosphatase in vivo.

Natural substrates for alkaline phosphatase (AP) are at present not identified despite extensive investigations. Difficulties in imagining a possible physiological function involve its extremely high pH optimum for the usual exogenous substrates and its localization as an ecto-enzyme. As endotoxin is a substance that contains phosphate groups and is usually present in the extracellular space, we studied whether AP is able to dephosphorylate this bacterial product at physiological pH levels. We tested this in intestinal cryostat sections using histochemical methods with endotoxin from Escherichia coli and Salmonella minnesota R595 as substrate. Results show that dephosphorylation of both preparations occurs at pH 7.5 by AP activity. As phosphate residues in the lipid A moiety determine the toxicity of the molecule, we examined the effect of the AP inhibitor levamisole in vivo using a septicemia model in the rat. The results show that inhibition of endogenous AP by levamisole significantly reduces survival of rats intraperitoneally injected with E. coli bacteria, whereas this drug does not influence survival of rats receiving a sublethal dose of the gram-positive bacteria Staphylococcus aureus. In view of the endotoxin-dephosphorylating properties of AP demonstrated in vitro, we propose a crucial role for this enzyme in host defense. The effects of levamisole during gram-negative bacterial infections and the localization of AP as an ecto-enzyme in most organs as well as the induction of enzyme activity during inflammatory reactions and cholestasis is in accordance with such a protective role.     

Comparison of standard tissue culture, tissue culture plus staining, and direct staining for detection of genital herpes simplex virus infection.

Genital herpes simplex virus infection in women was studied by using conventional tissue culture (TC) virus isolation compared with short-term (24-h) TC on Lab-Tek chamber slides followed by fluorescent-antibody (FA) staining. Three different staining techniques were used after TC: (i) staining with biotin-avidin (TC-BA/FA), (ii) direct FA (TC-FA), and (iii) indirect FA. The TC-BA/FA method showed complete correlation with the TC method. The TC-FA method showed no false-positive results but 31.5% false-negative results compared with the TC method. In contrast, the TC-indirect FA method showed 11.9% false-positive results and 11.7% false-negative results. The direct staining of specimens by the biotin-avidin technique (direct BA/FA) without prior tissue culture showed 37.7% false-positive results and 11.1% false-negative results. The TC-BA/FA technique thus was as sensitive as, but more rapid than, the TC method. The quality of fluorescence was far superior in TC-BA/FA staining as compared with TC-FA or TC-indirect FA procedures. The TC-BA/FA appears to be a valuable technique in laboratory diagnosis of genital herpes infections, especially in clinical situations requiring rapid detection of the virus.     

Distribution of alkaline phosphatase activity in healthy and diseased human liver tissue.

Histochemical demonstration of the alkaline phosphatase activity with an azo-dye method in human livers showed that the activity, which is normally localized to endothelial cells of portal and central veins as well as of sinusoids around the central veins and to a lesser extent of sinusoids around the portal connective tissue, is increased in various liver diseases. Appreciable canalicular activity is rare, and when it occurs it is more prominent in parenchyma involved by malignant tumours.     

Comparative cytochemical study of succinic dehydrogenase and alkaline phosphatase activity in primarily explanted and transplantable tissue culture cells

Summary The activity of succindehydrogenase and of alkaline phosphatase was studied in the transplantable strains of amniotic epithelium, NEr-2 and the heart ofMacacca cynomolgus, as well as in the first generation cells of amnion and the second generation cells of the monkey kidney. In difference from the primarily explanted amniotic cells, the activity of the enzymes does not disappear from the cells of transplantable strains and the second generation of the monkey kidney. The endogenous activity of dehydrogenase was relatively high in the primarily explanted amniotic cells and the cells of the second generation of monkey kidney tissue. A possibility of connection between the changes of the enzyme activity and the processes of cellular adaptation to the conditions of culturing is discussed.(Presented by Active Member AMN SSSR V. V. Parin) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 51, No. 2, pp. 107–110, February, 1961     

Differential staining of interspecific chromosomes in somatic cell hybrids by alkaline Giemsa stain

Staining of chromosome preparations of Chinese hamster-human hybrid cells and mouse-chimpanzee hybrids with alkaline Giemsa has yielded color differentiation of the interspecific chromosomes. Bicolor chromosomes, indicating apparent translocations also are observed for each of these hybrids. The specific color differences observed provide a rapid means of recognizing and aiding in the identification of the interspecific chromosomes and apparent translocations in these somatic cell hybrids.     

Correlation of alkaline phosphatase staining of cortisol-producing adrenocortical tumors with dexamethasone suppression and ACTH stimulation.

The histochemical activity of alkaline phosphatase (Al-Pase), the induction of which is one of the effects of ACTH on the adrenocortical cells, was examined in 10 adrenocortical tumors causing Cushing's syndrome and in 65 adrenal cortices. All of the compact cells in every gland, and almost all, about half, or a small proportion in the four tumors showed Al-Pase activity. These tumors decreased in steroidogenesis after the administration of dexamethasone. No compact cells exhibited the activity in six tumors, none of which was "dexamethasone-suppressible". Three of the seven attached glands examined were halfway between those of typical Cushing's syndrome and those of other than Cushing's syndrome from the viewpoint of compact/clear cell morphology. All of the tumors that had Al-Pase-positive clear cells increased in steroidogenesis after ACTH administration. These results suggested that Al-Pase activity of tumor cells was also ACTH effect and that a decrease in steroidogenesis of tumors after dexamethasone administration was due not to fluctuations but to suppression of intrinsic ACTH.     

Clinical significance of an increased or decreased serum alkaline phosphatase level.

An increase in the level of serum alkaline phosphatase (ALP) may be caused by a wide variety of pathologic lesions that involve multiple organs, since the enzyme is an ubiquitous one. Before one attempts to identify a significant pathologic abnormality, the clinician should consider the possibility of a physiologic or spurious cause for an increased level of ALP. A decreased ALP level also has diagnostic value.     

T lymphocyte tissue culture lines produced by cell hybridization.

This study describes the generation of permanent T cell tissue culture lines by cell fusion techniques. The AKR strain-derived T cell tumor BW 5147 was hybridized in the presence of polyethylene glycol with various T cell populations isolated from antigen-sensitized mice. Surface analysis of resulting hybrid cell lines showed expression of both Thy-1.2 and H-2 antigens which are characteristic for the lymphocyte used for fusion. In contrast, in hybrids derived from spleen cells neither expression of Ig nor of B cell-typicyl Ia determinants was found suggesting either preferential hybridization of BW 5147 cells with T lymphocytes or extinction of B cell markers in hybrid cells. These hybrid lines which may display the immunological properties of the T cell population chosen are presently investigated for their antigen reactivity.