Indirect Indirect Immunofluorescence Research Articles

Abstract 2679: Immuno-oncology panel optimization for imaging mass cytometry and digital image analysis of tumor tissues

Abstract Introduction: Imaging mass cytometry (IMC) enables highly multiplexed assessment of more than 30 proteins at a time for the quantification of cell phenotypes within the tumor microenvironment of formaldehyde fixed-paraffin embedded (FFPE) tumor tissue. This methodology provides a deep understanding of the immune-contexture while preserving their spatial information in regards to malignant cells. We report a detailed workflow for optimization of a 27-plex immune-oncology (IO) panel, immune-phenotyping, and spatial analysis of tumor tissue. Methods: IO-panel includes 27 antibodies targeting lymphoid (CD3e, CD4, CD8a, CD19, CD45RO, LAG3, ICOS, Granzyme-B), myeloid (CD11b, CD14, CD33, CD68, CSF1R, IDO-1), immune-regulatory (HLA-DR, OX-40, VISTA, TIM-3, CD73, B7-H3), epithelial (Cytokeratin), proliferative (Ki-67), and constitutive (GAPDH, NaKATPase, Vimentin, aSMA, Histone H3) markers that were selected by singlet automated chromogenic immunohistochemistry (IHC) staining. BSA-free formulation from the same clones of antibodies were isotope-metal conjugated and manual indirect immunofluorescence (IF) staining was used to confirm the stability of metal-tagged antibodies. The optimization by singlet-IF and multiplexed-IMC staining was performed with normal and malignant tissues (4mm core TMA with tonsil, placenta, prostate, breast carcinoma, ovarian carcinoma, and endometrioid carcinoma tissues). All images from IHC (HALO software), indirect IF (InForm software), and IMC (MCD viewer software) slides were evaluated by two pathologists. Cell densities and spatial analysis of a breast carcinoma core was performed using HALO software. Results: All antibodies included in the panel were individually optimized by IHC under the same protocol conditions. A significant correlation of optimal antibody concentrations where observed across the different staining techniques (IHC vs IF, r=0.51; IHC vs IMC, r= 0.54; IF vs IMC, r=0.992; all p values <0.01). The breast carcinoma core (total area=0.308mm2) was segmented into epithelial (area=0.216 mm2) and stroma (area=0.092mm2) compartments by digital image analysis. Individual cell segmentation was obtained with based on iridium nuclei signal (total cells=2420). Automated immune-phenotyping showed 20 different cell phenotypes with a predominance of malignant cells expressing B7-H3 (120.1 cells/mm2) and T-lymphocytes expressing LAG3 (3.2 cells/mm2). The average distance between proliferative cytotoxic T-lymphocytes to malignant cells (MCs) was higher when MCs were proliferating (22.94 μm, and 55.4 μm, respectively). Conclusions: IMC is a powerful platform for highly multiplexed detection of protein and cell phenotypes quantification in FFPE samples. Our developed IMC panel and digital image analysis of tissues allow the quantification of immune cells and spatial analysis of the tumor tissue microenvironment. Citation Format: Pedro Rocha, Luisa Solis, Edwin Parra, Ou Shi, Barbara Mino, Vidya C. Sinha, Amanda Rinkenbaugh, Auriole Tamegnon, Mak Duncan, Wei Lu, Mei Jiang, Lakshimi Kakarala, Jared K. Burks, Helen Piwnica-Worms, Cara Haymacker, Michael Teztlaff, Ignacio Wistuba, Alejandro Francisco-Cruz. Immuno-oncology panel optimization for imaging mass cytometry and digital image analysis of tumor tissues [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2679.

Regulatory T-cell deficiency leads to pathogenic bullous pemphigoid antigen 230 autoantibody and autoimmune bullous disease

Autoimmune bullous diseases/dermatoses (AIBDs) are severe autoantibody-mediated skin diseases. The pathogenic relevance of autoreactive CD4+ T cells for the induction of autoantibody production remains to be fully evaluated. Scurfy mice lack functional regulatory T (Treg) cells, experience spontaneous activation of autoreactive CD4+ T cells, and display severe erosive skin lesions suggestive of AIBDs. We sought to determine whether AIBDs develop in Treg cell-deficient scurfy mice. Histology, indirect immunofluorescence (IF) microscopy, direct IF, and ELISA were used to prove the presence of AIBDs in scurfy mice. Monoclonal autoantibodies from sera of scurfy mice were screened by using indirect IF on murine skin, and immunoprecipitation and mass spectrometry were used for target antigen identification, followed by confirmation in modified human embryonic kidney cells and murine keratinocytes. Pathogenicity was determined by injecting the autoantibody into neonatal mice and transferring scurfy CD4+ T cells into nu/nu mice. Autoantibodies against different known autoantigens of AIBDs spontaneously develop in scurfy mice. Histology reveals subepidermal blisters, and direct IF of skin of scurfy mice shows a predominant linear staining pattern. The mAb 20B12 shows a linear staining pattern in indirect IF, recognizes the murine hemidesmosomal protein bullous pemphigoid antigen 230 (BP230) as the target antigen, and cross-reacts with human BP230. Purified mAb 20B12 induces subepidermal blisters in neonatal mice. Transfer of scurfy CD4+ T cells is sufficient to induce antibodies with reactivity to AIBD autoantigens and subepidermal blisters in the skin of recipient T cell-deficient nu/nu mice. We show that the absence of Treg cells leads toAIBDs by pathogenic autoantibodies targeting BP230.

Comparison between direct and indirect immunofluorescence method for determination of somatic cell count

A sensitive indirect immunofluorescence (IF) assay for bovine neutrophil and somatic cell counting was developed. The obtained indirect IF was compared with direct IF. The direct IF method uses a single-fluorophore-conjugated antibody directed against the target of interest. The indirect IF method uses two antibodies. The primary antibody is unconjugated. The secondary antibody is directed against the primary and has fluorescent marker. Calibration curves for somatic cells (SC) in buffer model solutions were obtained by both methods, using fluorescence spectrophotometry. The measured linear range of somatic cells by the direct method was from 2 × 104 to 3 × 106 cells and by the indirect method was from 3 × 104 to 3 × 106 cells. The signal obtained by the indirect method was higher than the direct method at low concentrations of SC (from 30,000 to 100 000 cells/mL). That signal amplification probably is due to more than one fluorescent secondary antibody coupling to bound primary antibody. The same effect is observed by fluorescence microscopy. Consequently, the indirect IF method is more sensitive than the direct IF method. Conjugated primary antibodies are more expensive than their unconjugated counterparts, and secondary antibodies are relatively inexpensive. Therefore, using the same conjugated secondary antibody to detect different primary antibodies is cost-effective. Furthermore, a two-color staining microscopic procedure was proposed for simultaneous estimation of total SC count and neutrophil cell count.

358 Treg dysfunction leads to spontaneous production of autoantibodies to bullous pemphigoid antigens in mice

Bullous pemphigoid (BP) is the most common autoimmune blistering disease caused by autoantibodies to hemidesmosomal proteins; type XVII collagen (COL17 or BP180) and BP230. Regulatory T cells (Tregs) are crucial for peripheral immune tolerance and maintaining cutaneous immune homeostasis. However, it remains largely unknown whether failure of peripheral immune tolerance has an influence on the production of autoantibodies to the epidermal components. To address this, we investigated scurfy mice, which lack functional Tregs due to the mutation in Foxp3, a master transcriptional factor of Tregs. Utilizing direct and indirect immunofluorescence (IF) studies, we found that scurfy mice developed autoantibodies to the dermal-epidermal junction of the skin. The production of IgG autoantibodies, which had been class-switched from IgM, started within 12 days after birth. The autoantibodies reacted to the epidermal side of artificial blisters induced by 1M NaCl by indirect IF. From these findings, we focused on BP antigens. Immunoblotting using recombinant proteins of murine BP230 and COL17 demonstrated the presence of autoantibodies to those proteins in most scurfy sera. However, autoantibodies in scurfy sera have no reactivity to NC14A domain of murine COL17, corresponding to NC16A domain of human COL17, a domain responsible for subepidermal blister formation. This result may explain the fact that blister formation is not observed in scurfy mice. Furthermore, by using a CD4+ T cell-transferred model we revealed that CD4+ T cells mediate the production of those autoantibodies in scurfy mice. These results suggested that Tregs contribute to maintain immune tolerance to COL17 and BP230 at a steady state in mice.

Immune-complex deposits in anti-neutrophil cytoplasmic antibody associated crescentic glomerulonephritis; a report of two cases

Anti-neutrophil cytoplasmic antibody (ANCA)-associated crescentic glomerulonephritis is an important cause for rapidly progressing glomerulonephritis. It is generally classified under pauci-immune glomerulonephritis. However, 12%-18% of ANCA-associated crescentic glomerulonephritis show immune-complex deposits causing a diagnostic dilemma. We report 2 cases of ANCA-mediated glomerulonephritis with associated immune-complex deposits. First case is a 19-year-old female patient presented with fever and bilateral lower limb purpura since one day. Immunologic work-up was normal except positivity for cytoplasmic or c-ANCA by indirect immunofluorescence (IF). Kidney biopsy showed presence of segmental cellular crescent with fibrinoid necrosis. IF showed strong fine granular positivity for IgG, IgA, C3, C1q, kappa and lambda along the glomerular capillary walls. Second case is a 20-year-old male presented with low grade fever for last one month and vomiting for last two days. Immunologic work-up was unremarkable except positivity for cytoplasmic or c-ANCA by indirect IF. Kidney biopsy showed 14 glomeruli of which 8 glomeruli showed cellular crescents. IF for IgG, IgA, C3, kappa and lambda was done, which showed strong fine granular positivity along the glomerular capillary walls. Both the cases were treated with intravenous methylprednisolone and oral prednisone on a weaning regimen, and monthly therapy of intravenous cyclophosphamide. The maintenance phase consisted of mycophenolate mofetil and oral prednisone administrated in alternate-day low-dose regimen. Both the patients are on regular follow-up and are doing well.These immunecomplexes act synergistically with ANCA to cause more severe damage to the kidneys with a poorer outcome. Thus a prompt diagnosis and management of these patients is crucial.

Clinical and Immunologic Characterization in 26 Indian Pemphigus Patients

Pemphigus shows geographically variable characteristics. To study the clinical and immunologic characteristics of Indian pemphigus patients before and after treatment. Twenty-six Indian pemphigus patients were analyzed with regard to age, gender, clinical features, treatments and response, the results of histopathology, direct and indirect immunofluorescence (IF), enzyme-linked immunosorbent assay (ELISA), and immunoblot analyses. There were 22 pemphigus vulgaris (PV) and 4 pemphigus foliaceus (PF) patients. Direct and indirect IF was positive in 95.8% and 56% of patients, respectively. Indices of ELISA were lower in our study. Immunoblot assays detected the 130 kDa desmoglein-3 in 10 PV patients and the 160 kDa desmoglein-1 in 1 PV patient; 190 kDa periplakin was unexpectedly detected in 8 patients. Indian pemphigus patients showed several unique characteristics, including younger population, predominance of PV, low ELISA indices, lower sensitivity of indirect IF and immunoblotting, and the presence of the 190 kDa periplakin in nearly one-third of patients.

Usefulness of BP230 and BP180-NC16a Enzyme-Linked Immunosorbent Assays in the Initial Diagnosis of Bullous Pemphigoid

To investigate the diagnostic value of commercially available BP230 and BP180-NC16a enzyme-linked immunosorbent assays (ELISAs) in routine practice in patients with bullous pemphigoid (BP). Single-center retrospective study. French academic dermatology department. The study population comprised 138 patients, who were admitted from January 1998 through December 2008. Sera samples were analyzed by ELISA; clinical and immunopathological data were recorded from the patients' medical charts. BP230 and BP180-NC16a ELISA scores were evaluated with respect to clinical characteristics (number of blisters, mucosal involvement, localized or generalized disease, and outcome) and routine indirect immunofluorescence (IF). Of the 138 study patients, 81 (59%) had a positive BP230 ELISA result and 119 (86%) had a positive BP180 ELISA result. There was no relationship between a positive ELISA BP230 result and the disease extent at diagnosis or the presence of mucosal involvement. Serum anti-basement membrane zone autoantibodies (indirect IF) were more frequently detected when the BP230 ELISA result was positive (P < .001). The median anti-basement membrane autoantibody titer as detected by indirect IF was higher in patients with a positive BP230 result (P < .001). The BP180 ELISA result was associated with disease extent at diagnosis as estimated by both the percentage of patients with extensive BP (P = .01) and the mean number of blisters (P = .03) but was not associated with mucosal involvement. The currently available BP230 ELISA is a reliable although less-sensitive test than BP180 ELISA in BP, and its diagnostic added value compared with BP180 ELISA alone is approximately 5%. Our results support the predominant contribution of the BP230-specific autoantibodies to anti-basement membrane zone antibody titer as detected by indirect IF.

Homing of transplanted bone marrow cells in livers of Schistosoma mansoni‐infected mice

The efficiency of differentiation of bone marrow cells (BMCs) into hepatocytes in vivo and its importance in physiopathological processes is still debated. Murine schistosomiasis was used as a liver injury model and unfractionated male mice BMCs were transplanted through intrahepatic injection into non-irradiated Schistosoma mansoni-infected female mice on their 16th week post-infection. Two weeks after bone marrow transplantation, mice were sacrificed on a weekly basis until 10 weeks. Tracing of male donor-derived cells in female recipient mice livers was carried out by the detection of Y chromosome expression by fluorescent in situ hybridization (FISH) and also of chromodomain Y-linked (CDYL) protein by indirect immunofluorescence (IF). Their transformation into hepatocytes was studied by double labelling indirect IF using antibodies directed against CDYL and mouse albumin. Histopathological and electron microscopic examinations revealed the presence of small hepatocyte-like cells in the periportal tracts and in between the hepatocytes facing the sinusoids. Donor-derived cells showing Y chromosome by FISH and expressing CDYL protein by IF were recovered in the infected transplanted livers. The initial number of these cells increased with increased post-transplantation time. Cells were mainly localized in the periphery of schistosoma granuloma. Few donor-derived cells appeared within the hepatic parenchymal tissue and showed positivity for albumin secretion by double labelling with IF. We suggest that transplanted bone marrow stem cells can repopulate the Schistosoma-infected liver of immunocompetent mice. Their differentiation is a complex event controlled by many factors and needs to be further characterized extensively. The extent and type of liver injury and the number of transplanted cells are important variables in the process of stem cell engraftment and differentiation into functioning hepatic cells that still need to be defined.

Oncogenic function for the Dlg1 mammalian homolog of the Drosophila discs-large tumor suppressor

The fact that several different human virus oncoproteins, including adenovirus type 9 E4-ORF1, evolved to target the Dlg1 mammalian homolog of the membrane-associated Drosophila discs-large tumor suppressor has implicated this cellular factor in human cancer. Despite a general belief that such interactions function solely to inactivate this suspected human tumor suppressor protein, we demonstrate here that E4-ORF1 specifically requires endogenous Dlg1 to provoke oncogenic activation of phosphatidylinositol 3-kinase (PI3K) in cells. Based on our results, we propose a model wherein E4-ORF1 binding to Dlg1 triggers the resulting complex to translocate to the plasma membrane and, at this site, to promote Ras-mediated PI3K activation. These findings establish the first known function for Dlg1 in virus-mediated cellular transformation and also surprisingly expose a previously unrecognized oncogenic activity encoded by this suspected cellular tumor suppressor gene.

Comparative study for the detection of peritubular capillary C4d deposition in human renal allografts using different methodologies

Detection of peritubular capillary (PTC) C4d deposition in tissue sections of renal allograft biopsies became an important aid in the diagnosis of antibody-mediated rejection. Pathologists in many major transplant centers now routinely stain renal allograft biopsies for C4d. Currently, there are 3 commercially available antibodies. Two of these antibodies are monoclonal and are usually used with either a 3- or a 2-step indirect immunofluorescence (IF) methodology on frozen sections. A polyclonal antibody is used on formalin-fixed, paraffin-embedded tissue section with an immunoperoxidase detection system. The goal of our study was to compare these antibodies and methodologies in our renal allograft biopsy material. Twenty renal allograft biopsies with diffuse or focal PTC C4d staining, using immunofluorescence methods on frozen sections, were selected for this study. These biopsies were tested with the 3 commercially available anti-C4d antibodies (Biogenesis, Brentwood, Calif, cat no. 222-8004; Quidel Corporation, Santa Clara, Calif, cat no. A213; and ALPCO Diagnostic, Windham, NH, cat no. 004-BI-RC4D). Both monoclonal antibodies (Biogenesis and Quidel) were tested with a 3- and a 2-step indirect IF method on frozen sections. The polyclonal antibody (ALPCO) was applied to formalin-fixed paraffin sections using immunoperoxidase methodology. In selected cases, the polyclonal antibody was tested on frozen sections with a 3-step indirect IF method. To exclude possible false-negative staining with the IF method, we selected 10 additional biopsies that showed PTC margination of inflammatory cells, but were C4d-negative or only focally positive, and tested them with the ALPCO antibody on paraffin sections. We have found that all methodologies and antibodies tested provided adequate results with only minor differences between them. Perhaps the most sensitive method is the 3-step indirect IF on frozen sections using one of the monoclonal antibodies. We prefer the 2-step indirect IF method with the Quidel monoclonal antibody because of its simplicity, quick turnaround time, and relatively low cost. The advantages and disadvantages of the individual methodologies are discussed.

Autoantibodies to a 140‐kd polypeptide, CADM‐140, in Japanese patients with clinically amyopathic dermatomyositis

To identify novel autoantibodies specific for dermatomyositis (DM), especially those specific for clinically amyopathic DM (C-ADM). Autoantibodies were analyzed by immunoprecipitation in 298 serum samples from patients with various connective tissue diseases (CTDs) or idiopathic pulmonary fibrosis (IPF). Antigen specificity of the sera was further examined by immunoblotting and indirect immunofluorescence (IF). The disease specificity and clinical features associated with the antibody of interest were determined. Eight sera recognized a polypeptide of approximately 140 kd (CADM-140 autoantigen) by immunoprecipitation and immunoblotting. Immunoreactivity was detected in the cytoplasm, and indirect IF revealed a granular or reticular pattern. Anti-CADM-140 antibodies were detected in 8 of 42 patients with DM, but not in patients with other CTDs or IPF. Interestingly, all 8 patients with anti-CADM-140 antibodies had C-ADM. Among 42 patients with DM, those with anti-CADM-140 autoantibodies had significantly more rapidly progressive interstitial lung disease (ILD) when compared with patients without anti-CADM-140 autoantibodies (50% versus 6%; P = 0.008). These results indicate that the presence of anti-CADM-140 autoantibodies may be a novel marker for C-ADM. Further attention should be directed to the detection of rapidly progressive ILD in those patients with anti-CADM-140 autoantibodies.

Spectrum of autoimmune bullous diseases in Kuwait

Autoimmune bullous diseases (ABDs) are a rare but significant group of dermatoses that pose great challenges to the treating dermatologist. Most epidemiological studies have focused on a single ABD. Few surveys have been carried out to describe the whole spectrum of ABDs in a region, and no such studies are available from the Arabian Peninsula. To determine the clinico-epidemiological features of various ABDs in Kuwait, and to compare the results with those reported elsewhere. A total of 128 cases of ABDs were studied over a span of 11.5 years. The diagnosis in all cases was confirmed by histopathology, and direct and indirect immunofluorescence (IMF). The diagnosis of various subepidermal ABDs was further confirmed by indirect IMF on salt-split skin (SSS) and that of pemphigus by desmoglein 1 and 3 enzyme-linked immunosorbent assay (ELISA). Eighty seven per cent of patients were of Arab ethnicity. Pemphigus was observed to be the commonest ABD (47%), followed by pemphigoid (22%), pemphigoid gestationis (PG) (19%), linear IgA bullous disease (LABD) (7%), lichen planus pemphigoides (LPP) (3%), and epidermolysis bullosa acquisita (EBA) (2.3%). The minimum estimated incidence in the local population was 4.6, 2.14, 1.83, 0.69, 0.30, and 0.23 cases per million per year, respectively. Pemphigus patients were observed to have a younger age of onset (36.50 +/- 11.36 years) than reported elsewhere. BP, although the second commonest ABD, was less prevalent than in Europe and Singapore, and BP patients were observed to have a striking female predominance (85%). The prevalence of PG was much higher than that reported elsewhere. LABD was the fourth commonest ABD, and 89% of patients were children. The study suggests that similar surveys from different regions would expand our understanding of ABD.

Antibody response to the patient's own Haemophilus influenzae isolate can support the aetiology in lower respiratory tract infections.

In order to understand the clinical importance of Haemophilus influenzae isolated from sputum samples, an indirect immunofluorescence (IF) assay was developed, using the patient's own isolate as the antigen. The method was tested on samples from six patients with lower respiratory tract infection (LRTI) and H. influenzae isolated from blood (n=2), sputum (n=3) or both (n=1), and on two healthy adults with H. influenzae isolated from the nasopharynx. Between acute and convalescent sera, a four-fold IgG antibody increase was achieved in five of six LRTI patients, including the three blood culture-positive patients. One LRTI patient and the two asymptomatic carriers showed stable antibody levels against their own isolate. Although small, the study indicates that indirect IF can be a promising tool for determining whether a H. influenzae strain represents the probable cause of infection or just a strain colonising the airways. More extensive studies should be performed in order to establish the usefulness of the assay.

Paraneoplastic pemphigus in association with Castleman's disease.

Paraneoplastic pemphigus (PNP) is an autoimmune mucocutaneous disease associated with lymphoproliferative neoplasms, and frequently with a very rare tumour, Castleman's disease. To analyse the clinical history, immunopathological and histopathological findings in 28 patients with a confirmed diagnosis of PNP and Castleman's disease. Sera from all patients were assayed by indirect immunofluorescence (IF) and immunoprecipitation (IP) for plakin autoantibodies, immunoblotting for detection of plectin autoantibodies, and enzyme-linked immunosorbent assay for detection of desmoglein (Dsg)1 and Dsg3 autoantibodies. Severe oral mucositis was observed in all patients, and lichenoid cutaneous lesions were seen in 19 of 28. Twenty cases of Castleman's disease were of the hyaline vascular type, four were of plasmacytoid type and four were of mixed type. Striking findings included pulmonary destruction leading to bronchiolitis obliterans in 26 patients and fatal outcome due to respiratory failure in 22 patients with pulmonary involvement. Histological findings included lichenoid and interface dermatitis with variable intraepithelial acantholysis. Direct IF showed deposition of IgG and C3 in the mouth and skin in 24 of 28 patients. However, indirect IF detected serum IgG autoantibodies in all patients. IP revealed IgG autoantibodies against desmoplakin I, envoplakin and periplakin in all cases, and against desmoplakin II and the 170-kDa antigen in 19 patients. Dsg3 and Dsg1 autoantibodies were present in 22 and 11 patients, respectively, and plectin autoantibodies in 23 patients. PNP in association with Castleman's disease presents with severe oral mucositis and cutaneous lichenoid lesions. Serum autoantibodies against plakin proteins are the most diagnostic markers. Pulmonary injury with respiratory failure is the cause of death in most cases.

Mixed immunobullous disease of childhood: a good response to antimicrobials.

Immunobullous diseases are uncommon in childhood. In contrast to adults, the most commonly seen is IgA-mediated chronic bullous disease of childhood (CBDC), while IgG-mediated bullous pemphigoid (BP), cicatricial pemphigoid (CP) and epidermolysis bullosa acquisita (EBA) are rare. We have demonstrated both IgG and IgA autoantibodies to basement membrane zone target antigens in eight children with 'mixed immunobullous disease of childhood'. To elucidate whether a dual antibody response makes these patients distinct regarding their presentation, immunopathology, course and prognosis. We compared the eight children showing the double antibody response with 62 children with CBDC, BP, CP and EBA in whom only one antibody isotype was demonstrated. Clinical information at presentation, clinical course and response to treatment were recorded, and immunoblotting and direct and indirect immunofluorescence (IF) were performed. Six of the eight patients presented with clinical features of CBDC. In two others, it was uncertain whether they had CBDC or BP. Seven of the eight demonstrated a dual antibody response on indirect IF and three on direct IF. Immunoblotting revealed a variety of epidermal and dermal target antigens (BP230, BP180, 97-kDa protein and laminin 5). Five of the eight responded well to dapsone, two to sulphonamides, and one to systemic erythromycin alone. The clinical course was not protracted. Five are in remission 1-4 years following treatment, and three still have active disease suppressed by treatment after 6 months-2 years. Although we do not know why these children have 'mixed immunobullous disease' (the dual antibody response), our results indicate that the presence of IgA is associated with a good response to treatment with antimicrobials (dapsone, sulphonamides, erythromycin), and the clinical course is no more protracted than that found in children with a single antibody response.

Herpes Simplex Keratitis in South India: Clinico-Virological Correlation

Purpose: A retrospective cross-section study to analyze the prevalence of herpes simplex virus–induced keratitis (HSK) among 3,000 patients attending a corneal clinic in South India between 1995 and 1997, and to evaluate laboratory techniques for detecting HSK. Methods: The clinico-virological correlation was studied using herpes simplex virus (HSV) isolation on the Vero cell line, HSV-specific antigen detection by indirect immunofluorescence (IF) microscopy, and serum anti-HSV IgG quantitation, IgM estimation, and tear secretory IgA (sIgA) detection by ELISA. Observations: HSK had a prevalence of 7.8% (234 patients) in this study. A virological correlation could be obtained in 44.4% of the cases that had epithelial manifestations and in 14.8% of the cases that had only stromal disease. In 161 cases where both culture and IF microscopy were used, IF detected 27 cases (26.8%) more than cell culture. The difference in sensitivity between cell culture and IF was found to be statistically significant (McNemar's test, P < .05). An elevation in IgG titer was seen in 17 (30.4%) cases. IgM was detected in only 2 cases of the 62 (3.2%) analyzed. Of the 138 cases analyzed, sIgA was positive in 28 (20.3%) cases. A proved diagnosis could be made in 58% of cases when the specimen was collected during the first week after disease onset, and in only 5% when the time interval increased to 4 weeks. Conclusions: HSV antigen detection by indirect IF is a rapid and sensitive diagnostic tool for HSK. Tear secretory IgA (sIgA) is a specific marker for acute herpetic keratitis, and the detection of HSV-specific tear sIgA is a valuable adjunct to virus isolation and antigen detection in the laboratory diagnosis of HSK. For a successful diagnosis, the specimen should be collected as soon as possible after HSK onset.

Improved method for serological testing in celiac disease: IgA anti-endomysium antibody test: a comparison between monkey oesophagus and human umbilical cord as substrate in indirect immunofluorescence test

The IgA anti-endomysium antibody (IgA-EmA) test has been studied using the indirect immunofluorescence (IF) method with human umbilical cord (HUC) as substrate; 247 sera from patients investigated for celiac disease were tested. The results were in accordance with those obtained using monkey oesophagus (MOE) as substrate. IgA-EmA testing using HUC is shown to have similar reproducibility, sensitivity and specificity as MOE. By avoiding both fixation in chloroform and the blocking step, the processing time for this improved method is decreased by almost 1 h, and the results are stable for reading by fluorescence microscopy for at least 24 h. The indirect IF method using HUC as substrate is easy, reliable and inexpensive. We conclude that the IgA-EmA test using HUC as a substrate can be routinely used for celiac disease screening and follow up.

Detection and typing of herpes simplex viruses by using recombinant immunoglobulin fragments produced in bacteria.

Thirty-seven bacterial clones producing human recombinant monoclonal antibody Fab fragments (rFabs) reactive to herpes simplex virus (HSV) antigens were selected from a human combinatorial antibody library constructed in a phage-display vector by a panning procedure against an HSV lysate. Thirty-four of the HSV-specific rFabs were able to specifically recognize HSV-infected cells in indirect immunofluorescence (IF) assays; of these, 25 recognized cells infected by either HSV type 1 (HSV-1) or HSV-2, while 9 recognized only HSV-1-infected cells. One HSV type-common rFab (rFab H37) and one HSV-1-specific rFab (rFab H85) were further evaluated as reagents for viral detection and typing by IF staining in 134 HSV-positive (72 HSV-1 and 62 HSV-2) viral cultures from clinical specimens. The results obtained with these two rFabs were fully consistent with those obtained with a commercial preparation of fluorescein-labeled anti-HSV type-specific murine monoclonal antibodies. The detection sensitivity with the type-common rFab in indirect IF assays was higher overall than that provided by the type-specific murine monoclonal antibodies. Preparations of rFabs suitable for IF staining can be easily and inexpensively obtained in a clinical microbiology laboratory from Escherichia coli cultures. Similar HSV-specific rFabs, therefore, could be advantageous for in vitro diagnostic purposes.

The presence of anti-basement membrane zone antibodies in the sera of patients with non-bullous lupus erythematosus

We performed indirect immunofluorescence (IF) studies using 1 mol/l sodium chloride split skin to determine whether or not a positive IF is specific to patients with bullous lupus erythematosus (LE). We examined the sera from 21 patients with systemic LE (SLE), three of which were obtained from two SLE patients and one subacute cutaneous LE (SCLE) patient with bullous eruptions. As a comparison, we also studied the sera from patients with discoid LE (DLE, n = 7), SCLE (n = 1), systemic sclerosis (SSc, n = 20), bullous pemphigoid (n = 2) and normal individuals (n = 10). Sera from 16 SLE, four DLE and two SSc revealed a linear deposition of IgG isotype antibody at the epidermal side and/or the dermal side on indirect IF of split skin. The sera from three patients with bullous eruption and from 12 patients of SLE, SCLE, DLE without bullous eruption or SSc were further analysed by immunoblotting using five defined antigens, i.e. dermal extract, epidermal extract, three fusion proteins of 230 kDa bullous pemphigoid antigen (BPAG). 180 kDa BPAG, and human epidermolysis bullosa acquisita (EBA) antigen. Two SLE sera as well as one of the SCLE and the DLE serum reacted with 230 kDa BPAG in epidermal extract, and one of the SCLE and the DLE serum also reacted with the fusion protein of 180 kDa BPAG. No serum reacted with the dermal extract or the fusion protein of 230 kDa BPAG or EBA antigen. There was no consistent correlation between split-skin IF results and immunoblotting results. These results may suggest that even non-bullous LE patients often have autoantibodies to the basement membrane zone antigens, most of which are less pathogenic. Although we rarely examine the sera from non-bullous LE patients, we should keep this phenomenon in mind to avoid overestimating the results of split-skin test and immunoblotting.

Analysis of antigens recognized by autoantibodies in herpes gestationis. Usefulness of immunoblotting using a fusion protein representing an extracellular domain of the 180 kD bullous pemphigoid antigen

Herpes gestationis (HG) is a rare pregnancy-associated disease. The aim of this study was to compare various immunohistochemical and immunobiochemical techniques with respect to their diagnostic sensitivity for HG. We studied 43 HG sera; only half of these reacted with the basement membrane zone (BMZ) with both indirect immunofluorescence (IF) and complement IF of normal human skin. 81% of the sera reacted with the epidermal side of 1 M NaCl-split skin. In general, titers of anti-BMZ antibodies in HG sera were lower than those in bullous pemphigoid (BP) sera. Immunoblot analysis of human epidermal extracts showed that 51% of HG sera recognized the 180 kD BP antigen (BP180) and 26% recognized the 230 kD BP antigen (BP230). We also studied the reactivity of HG sera with fusion proteins representing either the NC16a domain of human BP180 or the C-terminal region of mouse BP230. Whereas 79% of HG sera reacted with the BP180 fusion protein, only 5% recognized the BP230 fusion protein. Our results suggest that indirect IF of 1 M NaCl-split skin and immunoblotting of a fusion protein representing the BP180 NC16a domain are more sensitive techniques for the diagnosis of HG than conventional and complement IF or immunoblotting of crude epidermal extracts.