Direct Immunofluorescence Technique Research Articles

Fibrin/fibrinogen and fibrinolytic activity of the palmar fascia in Dupuytren's contracture

Considering the proved interaction of fibrin with fibroblasts and the seemingly decisive role of structural and functional changes (“modulation”) of these cells in the evolution of Dupuytren's contracture, research has been carried out in order to investigate the fibrinolytic capacity and the possible presence of fibrin/fibrinogen in the palmar fascia of subjects operated upon for Dupuytren's Disease. Fibrin/fibrinogen were detected by a direct immunofluorescence technique and fibrinolytic activity was assessed by a fibrin plate method. A remarkable decrease of fibrinolytic activity and the presence of fibrin/fibrinogen were observed in small nodules in the early stage of disease, whereas large nodules showed a high amount of plasminogen activator enzymes. Small nodules seem to form and increase by progressive adhesion of fibroblasts to the polymerizing fibrin, while high fibrinolytic activity of large nodules probably results from “modulation” of many fibroblasts into contractile myofibroblasts and could therefore be considered as a biochemical sign of the evolutionary phase of Dupuytren's contracture.

Acute synovitis induced by preformed immune complexes.

The morphology of acute immune complex (IC) elicited synovitis in rabbit knee joints was studied, as well as IC-induced leukocyte activation in vivo and in vitro. Acute synovitis was induced by intra-articular injection of in vitro preformed, complement activating bovine serum albumin (BSA)-anti-BSA IC. Within 30 min, migration of polymorphonuclear granulocytes (PMNGs) was observed. Scanning electron microscopy showed that adhering, apparently activated leukocytes were attached to the synovial lining, often forming clusters. Phagocytosis of IC was evident, as immunoglobulins were detected within the leukocyte cytoplasm by the direct immunofluorescence technique. At the peak accumulation of PMNGs, focal erosions of the synovial lining were observed. Later, monocytes and macrophages appeared and degenerated PMNGs were found, sometimes within the cytoplasm of large macrophages. Chemiluminescence experiments showed a maximum in vitro activation of leukocytes by complement activating IC formed near optimal precipitation proportions and in slight antigen excess, whereas IC in large antigen excess gave a smaller and later response.

かいよう性大腸炎に合併した増殖性化のう性口内炎の１症例

A case of pyostomatitis vegetans associated with ulcerative colitis of a 22-year old woman has been reported, and clinical appearances, etiology and immunological mechanisms of development were discussed. Ulcerative colitis, as well as Crolm's disease and proctitis, is one of the inflammatory bowel diseases which have many local and systemic complications. Oral manifestations of ulcerative colitis arc as follows; 1) recurrent aphthous ulceration, 2) pyostomatitis vegetans, 3) ulcers analogous to pyoderma gangrenosum of the skin and 4) hemorrhagic ulcers of the oral mucosa and skin.The present case showed clinical appearances of above pyostomatitis vegetans, indicating numerous small papillary projections that contained miliary abscesses occurring in an area of intense erythema on the gingiva. The mucosa had been thrown into folds along gingivobuccal sulucus. Bacteriologic studies revealed no specific findings except normal oral microorganisms. Little subjective discomfort was associated with this condition. The histopathologic appearances were also compatible with pyostomatitis vegetans in which acanthosis and hyperkeratosis could be seen with elongation of the rete pegs. Small pustules existed in and under the epithelium of papillary projections. A dense lymphocytic and plasms cellular infiltration was found in the lamina propria, and cellular infiltration was reconfirmed by direct immunofluorescence technique using antihuman IgA antibodies, etc.The oral manifestation became worse and regressed according to the conditions of recurrence and covalescence of ulcerative colitis. The lesion responded well to topical application of a gluco-corticoid steroid hormone.The present case indicates that, in findings this kind of lesion on the oral mucosa, a possibility of gastrointestinal disturbance due to inflammatory bowel disease should be considered.

Amyloid P component in normal human placentae

The presence of tissue amyloid P component was determined by using a direct immunofluorescence technique on frozen sections of normal human placentae and umbilical cords from gestations of various durations. Amyloid P component was first detected at the 16th week of gestation and appeared to increase progressively in amount so as to be present in abundance in the term placenta. Placental amyloid P component was present in the perifetal capillary zone where basement membrane-like material and reticulin fibres are also found. Amyloid P component may be related to the maturation of the placenta.

IgE deposition in normal skin of patients with rheumatoid arthritis in relation to clinical and laboratory findings.

Biopsy specimens from apparently normal skin of 28 patients with classical or definite rheumatoid arthritis (RA) were examined for the presence of IgE deposition by a direct immunofluorescence technique. IgE deposition was found in 12 patients (43%) and in none of the 10 controls. This deposition was mainly localised on mast cells, and in three patients perivascular IgE staining was also noted. The skin from nine of the 12 patients also showed deposition of IgM and complement C3 or C4 factor, or both. All 12 patients with skin IgE deposition had raised levels of IgM rheumatoid factor (RF) in the serum. Nine of these also had IgE RF. IgE-containing circulating immune complexes (IgE CIC), raised serum IgE levels, and extra-articular (EA) manifestations were present in respectively 10, nine, and eight skin IgE positive patients. It is suggested that IgE and IgE CIC may be involved in the pathogenesis of RA and its EA manifestations.

Natural Killer Cells Against Human Corneal Endothelium

Human corneal endothelial cell monolayer preparations were incubated with fresh nonadherent peripheral blood lymphocytes. Vital and supravital staining and direct immunofluorescent techniques were used to analyze lymphocyte binding, endothelial cell viability, and the phenotypes of the lymphocyte populations binding to endothelial cells. Spontaneous lymphocyte-endothelial cell conjugate formation appeared maximal after six hours of incubation (86 lymphocytes per 1,000 endothelial cells). Cytotoxicity assays showed decreased endothelial viability after exposure to the lymphocytes when compared to controls incubated in media alone. The binding-lymphocyte population was a large granular nonadherent lymphocyte lacking surface immunoglobulin and stained positively for Leu-11 antigen, a surface marker expressed by natural killer cells. These data demonstrate that nonsensitized peripheral blood lymphocytes recognize and kill corneal endothelial cells in vitro. Thus, the lymphocyte subpopulations responsible for the destruction of corneal endothelial cells have functional and phenotype characteristics similar to natural killer cells and may be important participants in corneal graft injury.

Expression of a second Epstein-Barr virus-determined nuclear antigen in mouse cells after gene transfer with a cloned fragment of the viral genome.

Large Epstein-Barr virus (EBV) DNA restriction fragments corresponding to regions transcribed in transformed, proliferating cells were cloned in a cosmid derivative of the dominant-acting selection vector pSV2-gpt. Recombinant vectors carrying the EcoRI A fragment of EBV DNA were modified in the region corresponding to the deletion of the virion DNA in the non-transforming viral substrain P3HR-1, to create a series of recombinants lacking parts of this region. The recombinant vectors were introduced into 3T3 mouse fibroblasts under selective conditions, and resistant clones shown to contain EBV DNA sequences were analyzed for the expression of EBV-related antigens detectable by direct, indirect, and anticomplement immunofluorescence techniques. Cells that contained the BamHI K fragment expressed the EBV-determined nuclear antigen (EBNA) as expected. Cells transfected with recombinant vectors containing the BamHI W, Y, and H fragment part of the EcoRI A fragment also express a nuclear antigen detectable with certain anti-EBNA-positive human sera in anticomplement immunofluorescence tests. The BamHI WYH-induced EBNA polypeptide is similar in size to the EBNA2 polypeptide in Raji cells, as shown by gel electrophoresis and immunoblotting. The antigen is not detected in cells transfected with EcoRI A-derived vectors in which the BamHI H fragment has been deleted or in cells transformed with vectors carrying the BamHI H fragment alone. Direct and indirect immunofluorescence did not reveal the presence of antigens associated with productive infection in any of the EBV DNA-transfected fibroblast clones.

Posttraumatic autoimmune reaction in peripheral nerve: Effect of a single injury

This study was conducted to show that local autoimmune reactions could be observed in rat sciatic nerve after a single injury. Furthermore, we attempted to correlate the intensity of the immunological reaction with the severity of nerve damage, with the type of surgical treatment and with the degree of functional recovery. Through the use of direct immunofluorescence techniques, we found that the severity of the initial damage was associated with the intensity of the local immunological response assessed 2.5 months after surgery. There was an association between type of surgical treatment and intensity of the autoimmune reaction. A correlation between autoimmune reaction and degree of long-term functional impairment was not immediately clear. The probable factors that underlie these results are discussed.

Specific binding of cholera toxin to rat erythrocytes revealed by analysis with a fluorescence-activated cell sorter.

The direct binding of cholera toxin to the receptor on the native cell surface was analyzed with a fluorescence-activated cell sorter (FACS) by the direct membrane immunofluorescence technique using FITC-conjugated cholera toxin B subunit as a ligand and erythrocytes, but the binding was significantly affected by a change in pH, showing optimum pH of 7.2. The optimum conditions for analysis of the cholera toxin-binding with a FACS were reaction of the target cells with 0.2 M phosphate-buffer (pH 7.2) containing 0.025% of BSA and 0.175 M of NaCl at 4 degrees C for 40 min. The binding of cholera toxin B subunit to rat erythrocytes was linear in the range of 1.2 ng to 80 ng, which corresponded to 2,469 to 163,500 molecules of toxin per cell, and the latter was almost the saturated level of binding. although erythrocytes from different strains of rats possessed equal binding ability for the cholera toxin, no binding was observed with erythrocytes from mouse, guinea pig, cow, pig, man, or rabbit, indicating that the cholera-toxin binding occurs specifically on rat erythrocytes. This is in accord with our previous analytical deta on the absence of GM1 in erythrocytes of these animals except rat, of which erythrocytes contain GM1. Also, the structural specificity of the receptor for cholera toxin was assessed by a binding inhibition experiment using glycolipid-containing liposomes as inhibitors and GM1 was found to be the most potent inhibitor, showing complete inhibition of toxin (40 ng) binding to 5 x 10(6) erythrocytes at 505.6 pmol of GM1.

Comparison of the Scott Selecticult-HSV kit with conventional culture and direct immunoperoxidase staining for detection of herpes simplex virus in cultures of clinical specimens

In a comparative study, clinical specimens were cultured for herpes simplex virus (HSV). The presence of virus was noted by the appearance of characteristic cytopathic effect, as determined by standard direct immunofluorescence techniques, by using a direct immunoperoxidase stain for viral antigen, or by using the Selecticult-HSV (SC-HSV) stain for viral antigen. There was 100% correlation between the SC-HSV stain and immunofluorescence staining in recognizing HSV-infected cells (81 of 81 positive specimens). In comparison with observation of cytopathic effect, the SC-HSV system and conventional culture detected 93 and 78% of positive cultures at 48 h postinoculation and 76 and 32%, respectively, at 24 h. By 5 days postinoculation, SC-HSV detected 100% of the positive specimens. As compared with the direct immunoperoxidase stain, SC-HSV stain was slightly more sensitive and gave less background stain. HSV serotypes 1 and 2 were both detected by the SC-HSV stain. The Scott SC-HSV kit appears to be an effective system for the diagnosis of HSV infections.

Colloid body formation in bullous pemphigoid

Thirty-eight biopsy specimens from 18 cases of bullous pemphigoid (BP) were observed using direct immunofluorescence (IF) techniques with fluorescein isothiocyanate (FITC)-labelled antisera against human serum factors. In addition to deposits of immunoglobulins and serum components at the basement-membrane zone (BMZ), 15 specimens from eight cases displayed homogeneous and globular fluorescent bodies in the uppermost dermis and/or the blisters when FITC-labelled antisera to human IgM and other serum factors were used. Using immunoperoxidase staining, haematoxylin/eosin (HE) and periodic acid-Schiff (PAS) staining, these immunoglobulin and/or complement-positive cell-sized bodies were shown to be slightly eosinophilic and PAS positive. Electron microscopy revealed entangled networks of microfilaments approximately 7-8 nm in diameter. These homogeneous, fibrillar bodies were histologically, immunohistologically and ultrastructurally indistinguishable from the colloid bodies found in lesional skins of lichen planus, lupus erythematosus, dermatomyositis and several other dermatoses. In BP, degenerated keratinocytes adjacent to the blister roof, may, after undergoing a filamentous change, drop off into the dermis and subsequently form homogeneous, fibrillar bodies in the uppermost dermis when reepithelization is completed.

Direct and indirect immunofluorescent findings in dermatomyositis.

Three skin biopsies (proximal nailfold, extensor forearm and buttock) and serum samples were studied by direct and indirect immunofluorescence, immunodiffusion and radioimmunoassay techniques in 6 patients with dermatomyositis. A variety of serologic and immunopathologic abnormalities was observed in the patients. Three of 6 patients had antinuclear antibodies (ANA) of a speckled pattern mixed with a peculiar dot pattern. Epidermal intercellular deposits of immunoglobulins were seen in the nailfold biopsies of 2 patients; one of them also had IgG deposits in the cytoplasm of epidermal cells. Forearm and buttock biopsies were negative.

Reliability of cytodiagnosis in oral pemphigus vulgaris. A study of 30 cases.

ABSTRACTThe aim of this study was to verify the reliability of the Tzanck test, performed by both traditional cytomorphology and the direct immunofluorescence technique, in diagnosis of oral pemphigus vulgaris.Cytologic smears were obtained from oral erosions of thirty patients with well ascertained pemphigus vulgaris. Control smears were taken from thirty patients affected by various other diseases of oral mucosa and from thirty healthy subjects. Cytologic diagnosis of oral pemphigus was based upon significant findings of acantholytic cells by cytomorphology, and of epithelial cells with pericellular deposition of IgG (which persisted after cytocentrifugation) by direct immunofluorescence.The cytomorphological test gave positive results in twenty‐eight patients with pemphigus and in one patient with herpetic stomatitis, and negative results in all other cases.The immunocytologic test gave positive results in all patients with pemphigus, and negative results in all controls.These findings indicate that the cytomorphological test may be a useful method for screening oral pemphigus, while the immunocytologic test may provide a reliable method for definitive diagnosis.

Immunopathological correlation between mesangial C3d-deposition and C3d-fixing circulating immune complexes in lupus nephritis

By a direct immunofluorescent technique, glomerular C3d deposition was examined in a total of 50 renal biopsy specimens from patients with lupus nephritis. C3d deposition was then compared with disease activity, glomerular IgG and C3c deposition, and the levels of circulating immune complexes (CIC) measured by a solid-phase anti-C3d assay. There was a good correlation between disease activity and the positivity of glomerular C3d deposits ( P < 0.001), as well as C3c deposits ( P < 0.001). Even in clinically inactive patients, a relatively high percentage (59%) of C3d deposits were positive compared with C3c deposits (17%). Mesangial C3d deposition correlated with clinical disease activity more significantly ( P < 0.005) than capillary wall C3d deposition ( P < 0.025). C3d deposits were detected in all of the 30 cases with positive C3c deposits, and moreover, in 15 of the 20 (75%) cases with negative C3c deposits. Glomerular IgG deposits were almost always associated with C3d deposits, both in mesangial areas and along capillary walls, with statistical significance ( P < 0.005, P < 0.001, respectively). The serum levels of C3d-fixing immune complexes (IC) were significantly correlated with the positivity and intensity of mesangial C3d deposits. This study demonstrates glomerular deposition of C3d in patients with lupus nephritis and reveals a significant correlation between mesangial C3d deposition and disease activity.

Papular Polymorphous Light Eruption

Biopsy specimens of papules taken from eight patients with polymorphous light eruption ( PMLE ) were examined by a direct immunofluorescence technique. Extensive intervascular and focal perivascular deposits of fibrin were detected in each case. Slight vascular deposition of C3 and IgM were observed in five and two patients, respectively. The lupus band test was negative in all cases. The findings suggest that venular injury with activation of the clotting system is involved in the development of lesions in PMLE .

Ferritin in normal human peripheral blood T-lymphocyte subpopulations

Six peripheral blood lymphoid fractions (total lymphocytes, non-T, T, Tar (autologous rosetteforming T cells/precursor), Tμ (helper), and Tγ (suppressor) lymphocytes) isolated through rosetting procedures were examined for the presence of ferritin by a direct immunofluorescence technique. Although ferritin was present in all lymphoid fractions studied, a significantly higher proportion of ferritin-containing cells were detected in the T-cell fraction than in the non-T-cell fraction, (mean ± SD = 7.9 ± 1.6% and 5.0 ± 1.2%, respectively). Tμ- and Tγ-cell fractions showed a twofold increase in the number of ferritin-positive cells (14.1 ± 1.4% and 15.4 ± 2.6%, respectively), as compared with Tar (7.0 ± 0.9%)- and total lymphocyte (6.9 ± 1.3%)-cell fractions. These results indicate that ferritin is preferentially distributed in Tμ and Tγ lymphocytes and may constitute the basis for explaining some of the roles exercised by these cells in the control of other biological systems.

Restoration of antigenicity of tissue antigens, cell-bound immunoglobulins and immune deposits in paraffin-embedded tissue. The influence of fixation and proteolytic enzymatic digestion.

The possibility of preservation and restoration of antigenicity of some antigens in paraffin-embedded tissue was evaluated by direct immunofluorescent technique on deparaffinized sections. Fixation with 96% ethanol-1% acetic acid, 10% neutral buffered formalin and p-formaldehyde was useful for the preservation of tissue antigens and immune deposits, whose antigenicity could be easily restored by trypsin digestion. Neutral buffered formalin was also a satisfactory fixative in immunofluorescent staining on lymphocyte/plasma cell-bound immunoglobulins. Fixation with alcohol-Bouin's fluid showed contrast results; feasible for staining of cell-bound immunoglobulins, but poor for that of glomerular immune deposits. After papain digestion, BSA and lysozyme, antigens of immune complexes, were easily detected in experimental chronic serum sickness glomerulonephritis. Pepsin was more efficient than trypsin in restoring the antigenicity of renal tissue antigens such as fibronectin and polyantigenic basement membrane, but the brush border antigen of the proximal renal tubules was frail to the pepsin digestion. In general, the enzymatic digestion time necessary for the restoration of antigenicity was in parallel with fixation time. Results obtained have shown that deparaffinized sections could be used as satisfactory substrate for immunohistochemistry when proper fixation and efficient proteolytic enzymatic pretreatments were performed.

Demonstration of the presence of IgA in the human chorioamniotic membrane

The presence of IgA was detected by direct immunofluorescence techniques in the chorion and decidua of eight fragments of chorioamniotic membranes obtained near the rupture edge from eight normal patients. It is suggested that, similar to what occurs in other organic systems, a first barrier against infection of the amniotic chamber exists at this site.

Deposition of the Membrane Attack Complex of Complement in Bullous Pemphigoid

Bullous pemphigoid is associated with deposition of IgG and C3 at the dermal-epidermal junction. In order to see whether complement activation in bullous pemphigoid resulted in deposition of membrane attack complex (MAC) at the basement membrane zone, skin biopsies from patients with bullous pemphigoid were examined using a direct immunofluorescence technique. By employing a monoclonal antibody to a neoantigen of C9, the MAC was demonstrated in linear pattern at the basement membrane zone. These deposits were seen in both involved and uninvolved skin but the amount of MAC was greater in involved skin as judged by intensity of staining. Stippled deposits of MAC were also present in or around epidermal basal cells. The MAC could be generated in vitro by reaction of normal plasma with antibasement membrane antibody bound to sections of monkey esophagus. The IgG antibody activated complement and this complement activation proceeded all the way to the terminal step.

Deposits of IgG and C 3 in the spinal cord and motor cortex of ALS patients

Deposits of IgG and complement were demonstrated by direct immunofluorescent techniques with sections of motor cortex and spinal cord from amyotrophic lateral sclerosis (ALS) patients. Six of 16 ALS patients showed deposits within the spinal cord while 5 of 13 showed similar deposits within the motor cortex. The specificity of this staining was shown by blocking experiments and the use of conjugated F(ab′) 2. Similar deposits were found in the CNS in disease states associated with possible immune or infectious etiologies and were not found in the CNS of normal controls.