Direct Immunofluorescence Technique Research Articles

Oral pemphigus vulgaris: an immunofluorescent study of fifty-eight cases.

Oral mucosa biopsy specimens and sera from fifty-eight patients with pemphigus vulgaris limited to the oral cavity were studied by the direct and indirect immunofluorescence technique. Direct immunofluorescence demonstrated intercellular substance (ICS) deposition of IgG, either alone or in combination with C3, IgA, and IgM, in fifty-seven out of fifty-eight (98.3 per cent) oral mucosa biopsy specimens examined. At the time of initial evaluation circulating ICS antibodies were detected in twenty-eight out of fifty-eight patients (48.3 per cent) when a compound animal epithelial tissue was used as substrate. However, when normal human oral mucosa was used as substrate, circulating ICS antibodies were observed in fifty out of fifty-eight patients (86.2 per cent). It is concluded that direct and indirect immunofluorescence is a valuable diagnostic test in the early diagnosis of oral pemphigus vulgaris. In addition, normal human oral mucosa is a more appropriate substrate than animal epithelial tissue.

Immunofluorescent study of cytologic smears in oral pemphigus: A simple diagnostic technique

This study was undertaken in order to explore the diagnostic efficacy of the direct immunofluorescent technique for demonstration of in vivo bound immunoglobulins to desquamated epithelial cells from patients with oral pemphigus. Cytologic smears were obtained with a wooden spatula from the oral lesions of thirty patients with known pemphigus vulgaris. Control smears were taken from thirty-five patients twenty suffering from various other mucosal diseases and fifteen devoid of oral lesions. The smears were stored at −30° C. and prepared within 48 hours for examination by the direct immunofluorescent technique. Deposition of IgG was demonstrated in all thirty smears from the oral pemphigus lesions. None of the control smears gave positive results. These findings suggest that the direct immunofluorescent technique using cytologic smears may provide an additional simple and reliable method in the diagnosis of oral pemphigus vulgaris.

Immunofluorescent staining of poly(ADP-ribose) in situ in HeLa cell chromosomes in the M phase.

Randomly and synchronously growing HeLa cells were tested for poly(ADP-ribose) by direct and indirect immunofluorescent antibody techniques. Fluorescence of poly(ADP-ribose) was seen only in the nuclei of intact cells when the direct immunofluorescent antibody technique was used but in both the nuclei and cytoplasm when the indirect immunofluorescent antibody technique was used; fluorescence in the cytoplasm was nonspecific. When randomly or synchronously growing HeLa cells were fixed in acetone and treated with DNase I before incubation with fluorescein-labeled antibody, intense fluorescence was observed only in the nuclei when the direct immunofluorescent staining technique was used. Addition of 3-aminobenzamide, a potent inhibitor of poly(ADP-ribose) polymerase, with the DNase I completely abolished the fluorescence in the nuclei of synchronously and randomly growing HeLa cells, except in M-phase nuclei. These results suggest that poly(ADP-ribose) can be synthesized even in the nuclei of acetone-fixed HeLa cells from "endogenous NAD+" during incubation with fluorescent antibody and also that the fluorescence of chromosomes of HeLa cells in the M phase is, in fact, due to the in situ presence of poly(ADP-ribose), not to poly(ADP-ribose) synthesized during incubation with antibody.

HBsAg glomerular deposits in glomerulonephritis: Fact or artifact?

To evaluate the role of the hepatitis B virus infection in the pathogenesis of glomerulonephritis, we screened 136 consecutive patients with various types of glomerular lesions for the presence of HBsAg either in serum or in kidney biopsy specimens by means of radioimmunoassay and direct immunofluorescence technique, respectively. In selected patients, we carried out the search for antiglobulin activity (AA) factor(s) within the renal tissue with a fluoresceinated aggregated human IgG preparation (FITC-IgGagg). Eleven HbsAg seropositive patients were found, none of whom showed HBsAg deposits within their kidney biopsy specimen. Five of the remaining 125 seronegative patients, 3 with mixed cryoglobulinemia and 2 with lupus nephritis, showed glomerular-positive staining with a rabbit fluoresceinated anti-HBs serum and deposits of IgM endowed with AA, along with IgG and complement factors. The presence of HBsAg within the glomeruli was not confirmed, however, when a fluoresceinated anti-HBs Fab' preparation was used instead of the fluoresceinated anti-HBs serum. The pretreatment of the tissue sections with unconjugated anti-IgM serum blocked the direct staining for HBsAg and FITC-IgGagg, but not that of the other immunoreactants. Conclusion. The IgM AA deposits may interfere with the direct immunofluorescence testing for HBsAg, leading to erroneous interpretation.

Continuous cell culture from chick embryos inoculated with rev strain t

Oncogenic reticuloendotheliosis virus strain T (REV-T) was inoculated into 9-day-old embryonated eggs of specific pathogen free chickens. A continuous cell line was established from the liver cells of one embryo harvested the day before hatching which had the following properties: It formed loose aggregates and propagated floating in the medium. The cell doubling time was about 24 hours. Viral antigen was demonstrated in the cytoplasm by the direct immunofluorescent antibody technique. C-type particles were observed outside the cells. Virus-induced cell surface antigen was demonstrated by the immunofluorescent antibody technique. The cell line grew in soft agar. Virus production continued in the cell line for 17 months. When the cells or the culture fluid were inoculated into 1-day-old chickens, they died from neoplastic disease. The chickens which survived after infection showed abnormal feathering ("nakanuke"). The virus released from this cell-line was inoculated into 9-day-old embryonated eggs and three cell lines were established. These cell lines did not contain avian leukosis/sarcoma viruses.

Experimentelle Candidiasis: Pathogenese und serologische Untersuchungen in einem Kaninchenmodell

Three methods of inducing candidiasis in a rabbit model were studied in an effort to better understand the host responses and pathogenesis in this infection. Serological studies of both antigen and antibody were included. Endocarditis and cystitis were induced by inserting catheters containing viableCandida albicans into the right carotid artery and bladder, respectively, and pyelonephritis was induced by intravenous inoculation of viableCandida albicans. All rabbits with cystitis had evidence of infection only in the urinary bladder. Blood cultures in only one (with endocarditis) of 31 rabbits were positive forCandida albicans. All rabbits withCandida albicans endocarditis and pyelonephritis developed agglutinating antibody and immunodiffusion bands within seven to fourteen days from induction of infection, while none of the rabbits withCandida albicans cystitis developed agglutinating antibody titer changes or immunodiffusion bands. All rabbit urine and serum samples were examined forCandida albicans antigen by a latex agglutination and gel diffusion test. No free antigen was detected in any of the specimens using the methods described. A direct immunofluorescence technique was utilized to detect antibody coated yeast cells in the urine of infected animals as evidence of invasive disease. All rabbits with endocarditis, and four of eight rabbits with pyelonephritis had positive direct immunofluorescence studies of their urine, while all rabbits with cystitis had negative urines for antibody coated yeast cells.

An Immunofluorescence Study of the Skin in Diabetes Mellitus

The clinically normal skin of the lower back of 30 patients with diabetes mellitus was examined, using the direct immunofluorescence technique. No deposit of immunoglobulins or complement (C3) could be demonstrated, while other authors have previously reported lupus-like deposits in diabetes mellitus. As other discrepant studies of skin immunofluorescence have been published, it is suggested that the standards of the various immunopathology laboratories are different. This may explain why the actual value of the lupus band test remains controversial.

Proliferative glomerulonephritis in experimental Leishmania donovani infection of the golden hamster

Extensive kidney involvement is reported in golden hamsters infected with Leishmania donovani. The lesion is characterised grossly by pale and enlarged kidneys, and histologically by thickening of the basal lamina of the glomerulus, distended convuluted tubules containing protein casts, constricted glomerular capillaries and infiltration of the interstitial tissues with mononuclear cells. Quantitative measurement of the glomerular cells labelled in vivo with tritiated thymidine showed a striking increase in the glomerular cell proliferation as the infection progressed. Using direct immunofluorescent technique, granular deposits of IgG, complement C 3 and fibrinogen were detected in the glomeruli of the infected animals. A positive protamine sulphate test, as well as histological evidence, showed that disseminated intravascular coagulation had occurred in the infection. Although the antigens have not been identified, antigen-antibody complexes are strongly implicated in the pathogenesis of the proliferative glomerulonephritis observed in this study.

Cellular sites of immunoglobulins. VII. Localization of immunoglobulins in female mammary gland.

Direct immunofluorescent techniques are utilized to identify the type of infiltrating immunoglobulin in cells of the mature female mammary gland before menopause.In the average normal premenopausal breast, there are some immunoglobulin-containing cells infiltrating the intra- and interlobular stroma. They are predominantly IgA-containing cells, some are IgM-containing cells, and only a few are IgG-containing cells. Some secretory IgA and IgM immunoglobulins are also found in the apical portion of the acinar epithelial cells, but no IgG is detected.In the lactating mammary gland, the immunoglobulins, chiefly secretory IgA and IgM, are accumulated in the acinar epithelial cells and secreted into the lumen.Our present studies prove that the antibodies in mother's milk and colostrum are selectively secreted by IgA- and IgM-containing cells in the mammary glands, but they are probably carried by the lymphatics.

Immunopathological Studies on a Case of Sweet’s Syndrome

Immunopathological studies in a case of Sweet's syndrome revealed IgM- and IgE-bearing polymorphonuclear leucocytes in involved skin when the direct immunofluorescence technique was used. Circulating polymorphonuclear neutrophils showed defective chemotaxis with normal random migration, phagocytosis, killing, and hexose monophosphate shunt activity.

Cicatricial pemphigoid: Direct and indirect immunofluorescent studies

Oral mucosa, skin tissue, and serum samples from thirty-three patients with cicatricial pemphigoid were studied by the direct and indirect immunofluorescent techniques to determine the presence of tissue-bound and circulating antibodies. A linear continuous basement membrane zone pattern was observed in 96.9 percent of the oral mucosa biopsy specimens studied. This pattern is indistinguishable from the pattern observed in bullous pemphigoid. In 97 percent of the cases clinically healthy skin biopsy specimens were negative for basement membrane zone staining. Circulating anti-basement membrane zone antibodies were present in 36.4 percent of thirty-three patients with cicatricial pemphigoid in low titer (1:10 to 1:40), when normal oral mucosa was used as substrate. The demonstration of tissue-bound and circulating basement membrane zone antibodies in cicatricial pemphigoid morphologically identical to those found in bullous pemphigoid provides further support for the concept that the two diseases may represent variants of the same entity.

Isolation of actinomyces israelii in a case of gallbladder infection and comparison of identification methods

In a case of gallbladder infection, bacteriological studies revealed the presence ofActinomyces israelii, an organism rarely encountered in this site. While direct or indirect immunofluorescence techniques would undoubtedly provide the best identification criteria in the shortest time, the antisera were not available when the study was conducted. The most rapid technique for a tentative identification of isolated colonies of the strain was based upon detection of glycosidases together with catalase activity. Although not sufficient sinceA. israelii andA. naeslundii present the same enzymatic profiles, the other species and the most closely related genera can nevertheless be excluded by this method. Gas liquid chromatography — indeed useful yet much slower — contributes to identification but is not suitable for differentiation at the species level. Accurate identification was obtained by classical, time-consuming techniques. DistinguishingA. israelii fromA. naeslundii ten days following pathogen isolation can only be of limited practical interest.

Time of appearance of immunoglobulin-containing cells in the mucosa of the neonatal intestine.

In 46 intestinal specimens from infants 2 hr to 6 months old, the numbers of immunoglobulin-containing cells were counted by the direct immunofluorescent or direct immunoperoxidase technique. A restudy was made of biopsy specimens taken for diagnostic purposes, 34 from the rectum and 3 from the transverse colon of 30 infants at the Children's Hospital, Helsinki in 1971-1977, and in addition, of autopsy material recently collected from the intestines of 9 infants. Up to the age of 12 days, no immunoglobulin-containing cells were seen. A small number (1 to 2) of IgM-containing cells was seen in the lymph nodes of the 2 earlier specimens taken at the ages of 6 and 12 days. A rectal specimen from a 12-day-old infant showed 24 IgA- and 60 IgM-containing cells/mm2. In rectal specimens of infants less than 1 month old, the mean number of IgM-containing cells (26/mm2) was higher than that of IgA-containing cells (14/mm2), but older infants had a significantly higher mean number of IgA-containing cells (P < 0.01). The mean number of IgM-containing cells was the same in children 1 to 3 months (53 cells/mm2) and 3 to 6 months of age (59 cells/mm2), whereas the mean number of IgA-containing cells increased with age up to 6 months (112 and 163 cells/mm2). The youngest infant who had IgG-containing cells was 13 days old, although positive staining of intercellular spaces in the lamina propria and of the capillary endothelium by anti-IgG serum was observed in all specimens. The mean number of IgG-containing cells was low (5 cells/mm2) in all age groups. Sparse IgE-containing cells (less than 12 /mm2) were seen in 4 of the 46 specimens. In 5 patients, 2 or 3 consecutive specimens were available for the study; in these, the increase in the numbers of IgA- and IgM-containing cells was similar to the mean increase in cell numbers for the series as a whole.

Immunological studies of plasma protease inhibitors associated with human blood platelets.

Abstract Associations between whole human platelets and plasma protease inhibitors were studied by direct immunofluorescence techniques. Tested conjugates of anti-human antisera to α 1 -antitrypsin, α 2 -macroglobulin, α 1 -antichymotrypsin, inter-α-trypsin inhibitor, Cl inhibitor and antithrombin III were used. Only α 1 -antitrypsin and Cl inhibitor were found to be associated with platelets. The biological role of platelet-associated protease inhibitors is discussed.

Long-term photochemotherapy: histopathological and immunofluorescence observations in 243 patients.

Skin biopsies of 243 patients treated with photochemotherapy (PUVA) for 1--4 years were examined histologically. Two hundred and six patients were examined retrospectively after total cumulative UV-A doses of 579 . 6 +/- 598 . 0 J/cm2 (mean +/- s.d.). An eosinophilic homogenization and a reduction of elastic fibres at the dermo--epidermal junction, and an increase of dermal macrophages were found as possible abnormalities. However, except for the increase of melanophages there was no statistically significant correlation between the incidence of these changes, the total UV-A dose applied and the skin type of the patients. Neither were such correlations found in thirty-seven patients biopsies twice after 394 . 8 +/- 267 . 6 J/cm2 and 808 . 5 +/- 458 . 9 J/cm2 (mean +/- s.d.), respectively. Studies with direct immunofluorescence techniques revealed no immunoglobulin deposits in PUVA treated skin in fifty-six patients after 469 . 2 +/- 370 . 2 J/cm2; antinuclear antibodies were observed in 4 . 6% of 129 patients after 169 J/cm2 (mean); in 11% of fifty-three patients reexamined after 381 J/cm2 (mean) and in 13 . 6% of twenty-two patients reexamined a second time after 643 J/cm2 (mean). Thirteen out of a total of 572 patients developed a peculiar mottling of skin in areas previously overdosed by PUVA. Subepidermal homogenization and reduction of elastic fibres were found in 45% of the patients, indicating that these changes indeed are a consequence of PUVA. Nuclear and cellular irregularities were found in 45% of the biopsies and 63% showed a disturbed epidermal architecture, but no carcinomas were observed. PUVA-induced mottling was reversible in 31%, partially reversible in 15%, but continued to be present in 54%.

Pityriasis lichenoides, an immune complex disease?

Nine biopsies from skin lesions of 5 patients with pityriasis lichenoides acuta and three biopsies from skin lesions of 3 patients with pityriasis lichenoides chronica were examined by means of the direct immunofluorescence technique. IgM deposits along the dermoepidermal junction were found in only two biopsies. In the majority of bioipsies, complement (C3) deposits were found along the dermo-epidermal junction and in the vessel walls. Immunoglobulin and C3 deposits were not found concomitantly in the vessel walls.

Immunofluorescent study on middle ear mucosa.

To clarify the developmental course of the immunological defense system in the middle ear, immunoglobulin-forming cells of different classes and secretory proteins, such as secretory component (SC) and lactoferrin (Lf), were investigated in the middle ear mucosa of 20 developing and 5 normal adult guinea pigs by use of direct immunofluorescence technique. Changes in the middle ear mucosa were also observed after antigenic challenges directly to the tympanic cavity of 6 developing and 39 adult guinea pigs. IgA- and IgM-forming cells began to appear in the tubal mucosa at the seventh postnatal day, while it was scarcely possible to find IgG1- and IgG2-forming cells in developing guinea pigs. Immunoglobulin-forming cells of all classes increased in the middle ear mucosa after the antigenic stimuli. The injection of antigens to the tympanic cavity of developing animals induced the most striking accumulation of immunoglobulin-forming cells in the middle ear mucosa. Results of this study showed that local synthesis of IgA, as well as other classes, is latent in the middle ear, that the middle ear of immature animals is vulnerable to antigenic stimuli, and that the middle ear of developing animals possesses potential immune responsiveness.

C3b receptor in normal human oral mucosa

The presence of a receptor for C3b in normal human oral mucosa was studied using C3b produced by treating normal human serum with cobra venom factor. Cryostat sections of normal human oral mucosa were incubated with C3b, followed by a direct immunofluorescence technique using monospecific goat-antihuman C3. The histologic localization of C3b fluorescence was determined by fixing cryostat sections with glutaraldehyde and staining with hematoxylin and eosin. The endothelial cells of the capillaries and smaller venules in the oral mucosa showed staining by anti-C3. C3b did not bind to the intercellular substance nor to the basement membrane zone of normal human oral mucosa. Normal human serum treated with EDTA was negative, thus indicating that native C3 did not bind to the receptor. The endothelial C3b receptor of the oral mucosa may have an important function in the localization of immune complexes that occur in systemic diseases, as well as in diseases restricted to the oral mucosa.

Presence of multiple isotypes on the surface of human tonsillar lymphocytes.

Formaldehyde-fixed small lymphocytes from human tonsils were investigated for the presence of the Ig heavy chain class isotypes alpha, gamma, delta and mu on their membranes by means of a direct immunofluorescence technique. About 50% of the small lymphocytes were defined as B cells with an anti-Fab antiserum. Most of the B lymphocytes carried more than one isotype on their membrane, and all combinations of two isotypes were observed. The existence of three or four isotypes on the membrane of individual lymphocytes was deduced. The sIgD, sIgM and sIgA, sIgD, sIgM combinations were the most common. The isotypes were present as separate molecules since they showed independent distribution patients undeesent as separate molecules since they showed independent distribution patterns under capping conditions.

Benign and neoplastic eosinophilic staining cells: an immunofluorescence study.

Deposition of immunoglobulins, complement and fibrinogen on eosinophilic staining cells was investigated using direct immunofluorescence techniques. Serum factor deposition was detected on benign epidermal eosinophilic cells seen in pityriasis lichenoides et varioliformis acuta, sunburn erythema and, in addition, on subepidermal hyaline bodies in lichen planus; no such deposition occurred on neoplastic eosinophilic cells in Bowen's disease and squamous cell carcinoma. The qualitative findings of immunofluorescence microscopy seem to be different in inflammatory and malignant dermatoses.