Direct Immunofluorescence Staining Research Articles

Evaluation of the Binax NOW, BD Directigen, and BD Directigen EZ assays for detection of respiratory syncytial virus.

The Binax NOW assay (Binax, Inc., Portland, Maine) and the BD Directigen EZ assay (Becton Dickinson and Company, Sparks, Md.), two new rapid immunoassays for detection of respiratory syncytial virus (RSV), as well as the BD Directigen RSV assay (DRSV) (Becton Dickinson and Company) and direct immunofluorescence staining (DFA) were compared with culture for detection of RSV in fresh specimens from both children and adults during the 2002-2003 respiratory virus season. The majority (95%) of specimens were nasal or nasopharyngeal washes or aspirates. A total of 47 (26%) were culture positive for RSV. The overall sensitivities of DFA (n = 149), NOW (n = 118), EZ (n = 88), and DRSV (n = 180) compared with culture (n = 180) were 93, 89, 59, and 77%, respectively. The specificities of DFA, NOW, EZ, and DRSV were 97, 100, 98, and 96%, respectively. However, when results were separated into those from children and those from adults, DFA was the only rapid test adequate for detection of RSV (sensitivity of 100% compared to 0, 0, and 25% for NOW, EZ, and DRSV, respectively) in adults. For children the sensitivities of DFA, NOW, EZ, and DRSV were 93, 94, 72, and 81%. The NOW assay was the most sensitive and specific and the easiest to perform of the kit tests for detecting RSV in children. None of these three rapid kit tests was sensitive for detecting RSV in specimens from adults. DFA remains the rapid method of choice for detecting RSV in the adult population.

Quantitative histology by multicolor slide-based cytometry.

In lymphatic organs, the quantitative analysis of the spatial distribution of leukocytes by tissue cytometry would give relevant information about alterations during diseases (leukemia, HIV, AIDS) and their therapeutic regimen, as well as in experimental settings. We have developed a semiautomated analysis method for laser scanning cytometry (LSC) termed "multiple thresholding," which is suitable for archived or fresh biopsy material of human lymph nodes and tonsils. Sections are stained with PI for nuclear DNA and up to four antigens using direct or indirect immunofluorescence staining. Measurement is triggered on DNA-fluorescence (argon laser, Ar) or on specific cell labeling. Due to the heterogeneity of cell density, measurements are performed repeatedly at different threshold levels (low threshold: regions of low cellular density, germinal center; high threshold: dense regions, mantle zone). Data are acquired by single- (Ar) or dual-laser excitation (Ar-HeNe) in order to analyze single- (FITC) up to four-color (FITC/PE/PECy5/APC) stained specimen. Percentage and cellular density of cell-subsets is quantified in different microanatomical regions of the specimen. These data were highly correlated with manual scoring of identical specimens (r(2) = 0.96, P < 0.0001). With LSC, semiautomated operator-independent immunophenotyping in tissue sections of lymphatic organs with up to three antibodies simultaneously is possible. We expect this tissue cytometric approach to yield new insight into processes during diseases and help to quantify the success of therapeutic interventions.

601. HIV-1 TAT Protein Transduction Domain-Mediated Enhancement of HSV-TK/Ganciclovir Suicide Gene Therapy

Top of pageAbstract INTRODUCTION Suicide gene therapy using herpes simplex thymidine kinase gene combined to prodrug ganciclovir (GCV) is one of the most prominent approaches in cancer gene therapy. However, efficacy of the therapy is often insufficient due to the poor rate of gene transfer and due to the limited bystander effect. Recent studies have shown that a protein transduction domain from HIV-1 transactivator protein (TAT-PTD) can translocate through plasmamembrane and deliver proteins into cell. Aim of this study was to make intercellularly spreading HSV-TK protein with the aid of cell penetrating peptide TAT and thus enhance the cytotoxic effect of HSV-TK/GCV gene therapy.METHODS We constructed a novel triple fusion protein containing TAT protein transduction domain (TAT-PTD), HSV-TK suicide gene and GFP marker gene (TAT-TK-GFP) to study effect of TAT-PTD on HSV-TK/GCV-suicide gene therapy. Sensitivity to ganciclovir was tested in different cell lines by comparing cells expressing TAT-TK-GFP to cells expressing TK-GFP. Biodistribution of TAT-TK-GFP from transfected cell to surrounding cells was analyzed by direct fluorescence microscopy or by immunofluorescence staining.RESULTS We found that TAT-peptide enhances cytotoxic effect of HSV-TK/GCV-suicide gene therapy in rapidly dividing cells e.g. in BTC4 rat glioma cell line or in SKOV3.ip1 ovarian cancer cell line. However, enhancement was abolished after serum deprevation. Intercellular spreading of TAT-TK-GFP-protein was not visualized by direct fluorescence microscopy or immunofluorescence staining. Control fusion protein, VP22-GFP, contrast to TAT-fusion protein showed spreading from transfected cell to adjacent cells after methanol fixation.CONCLUSION This study shows that the triple fusion gene TAT-TK-GFP is useful tool for investigating the utility of TAT fusion proteins in suicide gene therapy. Our data with TAT-TK-GFP demonstrate that HSV-TK/GCV cytotoxicity can be enhanced in rapidly growing cancer cells, despite the fact that intercellular spreading cannot be visualized.

Long-term preservation of direct immunofluorescence staining in slides stored at room termperature

To determine whether reproducible results from direct immunofluorescent tissue staining could be obtained after storing the slides at room temperature for a period of greater than 3 years, and up to 5 years, we examined the original slides of 20 cases noted to be positive for direct immunofluorescence. Diagnoses include pemphigus, pemphigoid, lupus, dermatitis herpetiformis, lichen planus and vasculitis. These specimens, initially evaluated during the period January 1997 to October 1998, were prepared with a standard immunofluorescence staining technique, and then a permanent aqueous mounting medium was added. All specimens were stored at room temperature in vertical slide holding trays. The Phase I evaluation was performed in April 1999 and published in 2000. The focus in both Phase I and Phase II of the study was to observe the presence and relative intensity of the immunofluorescence staining, as well as the ability to make a final diagnosis. The readings of Phase I and Phase II were then compared to the original reports. Our results suggest that direct immunofluorescent studies, using a crystalline permanent aqueous mounting medium, can be stored over periods as long as 4-5 years at room temperature without significant degradation of staining. To determine whether reproducible results from direct immunofluorescent tissue staining could be obtained after storing the slides at room temperature for a period of greater than 3 years, and up to 5 years, we examined the original slides of 20 cases noted to be positive for direct immunofluorescence. Diagnoses include pemphigus, pemphigoid, lupus, dermatitis herpetiformis, lichen planus and vasculitis. These specimens, initially evaluated during the period January 1997 to October 1998, were prepared with a standard immunofluorescence staining technique, and then a permanent aqueous mounting medium was added. All specimens were stored at room temperature in vertical slide holding trays. The Phase I evaluation was performed in April 1999 and published in 2000. The focus in both Phase I and Phase II of the study was to observe the presence and relative intensity of the immunofluorescence staining, as well as the ability to make a final diagnosis. The readings of Phase I and Phase II were then compared to the original reports. Our results suggest that direct immunofluorescent studies, using a crystalline permanent aqueous mounting medium, can be stored over periods as long as 4-5 years at room temperature without significant degradation of staining.

GeneScan reverse transcription-PCR assay for detection of six common respiratory viruses in young children hospitalized with acute respiratory illness.

A reverse transcription-PCR (RT-PCR) assay based on automated fluorescent capillary electrophoresis and GeneScan software analysis was developed to detect six common respiratory viruses in clinical specimens from young children. Assays for human respiratory syncytial virus (HRSV); human parainfluenza viruses 1, 2, and 3 (HPIV1, -2, and -3, respectively); and influenza A and B viruses were incorporated into a single standard assay format. The optimized assay panel was used to test 470 respiratory specimens obtained from 462 children hospitalized with acute respiratory illness that had been previously tested by viral culture (405 specimens) or direct immunofluorescence staining (DIF) (65 specimens). Of 93 specimens positive for respiratory viruses by culture or DIF, 86 (92%) were positive by RT-PCR, including 66 HRSV, 2 HPIV2, 5 HPIV3, 3 influenza A virus, and 10 influenza B virus specimens. An additional 119 respiratory viruses were identified by RT-PCR in 116 patients for whom results were negative by viral isolation or DIF. We conclude that the GeneScan RT-PCR panel can markedly improve detection of acute respiratory virus infections in young children.

Discoid lupus erythematosus presenting as madarosis

Purpose Discoid lupus erythematosus (DLE) is an autoimmune disorder that usually affects the sun–exposed skin. Periocular involvement occurs uncommonly and may progress from eyelid erythema to scarring and madarosis. Method Observational case report. Result A case of DLE that presented with madarosis alone in the absence of preceding skin erythema and scarring. Conclusion Our case demonstrates that DLE may present with madarosis alone in the absence of a history of preceding erythema and scarring. Discoid lupus erythematosus should therefore be considered as a differential diagnosis in chronic blepharitis that persists despite usual medical management and eyelid hygiene. Biopsy should be considered in the presence of clinical features such as erythematous scale on the face and alopecia and sent for direct immunofluorescence staining.

The reliability of long-term storage of direct immunofluorescent staining slides at room temperature.

Long-term preservation of immunofluorescence is important for re-examinations. We investigated whether the storage of direct immunofluorescent (DIF)-positive slides at room temperature was reliable in daily practice. One hundred and twenty-five DIF-positive slides from the skin of 52 patients were evaluated. Sections were examined for the presence of immunoglobulin A (IgA), IgG, IgM, C3, and fibrinogen using fluorescein isothiocyanate-conjugated antisera and mounted with a ready-to-use permanent mounting medium containing an antifading reagent and sodium azide (DAKO, Glostrup, Denmark, S3023). The slides were stored at room temperature for 16-24 months. Changes in diagnostic pattern, fluorescence intensity, and the form and location of accumulation of immunoreactants and technical deformation were investigated. Over the entire observation period, 49.6% of the slides faded away; the median length of survival was 16 months. Before 12 months, the survival rate of slides was 92.0%, whereas after 20 months it was 28.0%. In the early faded slides, which faded away before 16 months following the first examination, C3 and IgA were the most frequently observed immunoreactants. The technical deformations did not prevent the diagnosis. The preservation of fluorescence in DIF-positive slides using mounting media with an antifading reagent is possible for 2 years at room temperature. However, in daily practice, storage for longer than 11 months prevents a reliable diagnosis.

A novel method to analyze viral antigen-specific cytolytic activity in the chicken utilizing flow cytometry

In order to overcome some of the main drawbacks that have emerged in the conventional assays for cytotoxic T-lymphocytes (CTLs) in the chicken, a novel approach to analyze viral antigen-specific cytolytic activity utilizing flow cytometry was developed. In this method, the target cells were distinguished from the effector cells by pre-labelling them with a fluorescent dye PKH67. Cell death was detected with propidium iodide which labels the DNA of damaged cells. Flow cytometric assay also enables phenotyping of the effector cells by direct or indirect immunofluorescence staining of lymphocyte surface molecules. The results showed that specific cytotoxic T cells were observed in the blood of chickens primed with fixed avian reticuloendotheliosis virus strain T transformed MHC-compatible B cells. Phenotypic analysis of the effector cells from blood demonstrated CTL activity both in CD8 + and CD4 + T cell populations and the majority CTLs were TCR2 + cells.

Catheter-based adenovirus-mediated local intravascular gene delivery of a soluble TGF-beta type II receptor using an infiltrator in porcine coronary arteries: efficacy and complications.

Enhanced extracellular matrix (ECM) accumulation is an important finding in human restenotic arterial neointima after angioplasty. Transforming growth factor beta1(TGF-beta1) is known to regulate the synthesis and turnover of a variety of ECM components, and may play an important role in restenosis. Recombinant adenoviral vector expressing an ectodomain of the TGF-beta type II receptor fused to the human immunoglobulin Fc portion (AdTbeta-ExR) inhibits the action of TGF-beta probably either by adsorbing TGF-beta or by acting as a dominant negative receptor. We carried out a catheter-based local adenovirus mediated gene delivery using an Infiltrator in porcine coronary arteries to know the pattern of gene expression, efficacy and procedural complications. Twenty four coronary arteries in 13 pigs were used for intravascular gene delivery by intramural injection with either AdTbeta-ExR or adenovirus expressing beta-galactosidase (AdCALacZ). Direct immunofluorescent staining and reverse transcription polymerase chain reaction (RT-PCR) were used for detection of type II TGF-beta receptor and its mRNA respectively. X-Gal histochemistry was performed to identify beta-galactosidase. Both soluble TGF-beta receptor and beta-galactosidase were expressed locally in the media and adventita at injected arterial segments without any significant dissemination to remote area. Intravascular gene transfection performed with various titer of each adenoviral vector showed that AdTbeta-ExR of 5 x 10(8) pfu and AdCALacZ of 2.5 x 10(8) pfu were the minimum titer for the expression of each transgene. Infiltration of CD3 positive T cells was detected by immunohistochemical staining in the area of each transgene expression, and tends to decrease over time after gene delivery. Pathological study of 24 treated arteries showed complications such as disruption of external elastic lamina with hemorrhage (n = 4), minimal disruption of internal elastic lamina and endothelial layer, and medial thickening. In conclusion, catheter-based local intravascular gene delivery of adenoviral vector is feasible and effective in a selected artery, but must be undertaken with caution due to possible lethal complications. Local delivery of soluble TGF-beta type II receptor in this way may provide an effective intravascular gene therapy to inhibit TGF-beta signal pathway without any significant systemic side effect.

CD28 knockout mice as a useful clue to examine the pathogenesis of chronic graft-versus-host reaction

CD28 knockout mice as a useful clue to examine the pathogenesis of chronic graft-versus-host reaction.BackgroundInjection of BALB/c or DBA/2 spleen cells into F1 C57BL/6 (B6) hybrids induces a graft-versus-host reaction (GVHR) of a chronic stimulatory type that results in clinical and pathologic manifestations that resemble the human systemic lupus erythematosus (SLE). The aim of the present study was to examine the role of a major T-cell costimulatory signal receptor, CD28, in the production of autoantibody and the development of an immune complex glomerulonephritis, which are common in SLE pathology.MethodsFor this purpose, CD28-deficient (CD28KO) mice were used for the source of donor lymphocytes. Chronic GVHR was induced by an injection of BALB/c or BALB. CD28KO donor cells into normal BCF1 mice. Serum titers of anti-dsDNA antibodies were assessed by enzyme-linked immunosorbent assay (ELISA) and major histocompatibility complex (MHC) class II antigen expression on B cells were tested by flow cytometry. In addition, depositions of immunoglobulin (Ig) were examined by direct immunofluorescence staining on frozen kidney sections.ResultsWhen (BALB/c × B6)F1 mice were injected with parental BALB/c lymphocytes, serum anti-dsDNA titer was significantly increased in association with nonspecific B-cell activation and IgG deposition in the glomerular basement membrane. In sharp contrast, none of these signs were observed in F1 mice, which were injected with CD28KO spleen cells.ConclusionThe CD28-mediated T-cell costimulatory pathway plays a pivotal role in the development of polyclonal B-cell activation, autoantibody production, and an immune complex glomerulonephritis. We propose that CD28KO mice are useful clues in examining the pathogenesis of experimental lupus nephritis. CD28 knockout mice as a useful clue to examine the pathogenesis of chronic graft-versus-host reaction. Injection of BALB/c or DBA/2 spleen cells into F1 C57BL/6 (B6) hybrids induces a graft-versus-host reaction (GVHR) of a chronic stimulatory type that results in clinical and pathologic manifestations that resemble the human systemic lupus erythematosus (SLE). The aim of the present study was to examine the role of a major T-cell costimulatory signal receptor, CD28, in the production of autoantibody and the development of an immune complex glomerulonephritis, which are common in SLE pathology. For this purpose, CD28-deficient (CD28KO) mice were used for the source of donor lymphocytes. Chronic GVHR was induced by an injection of BALB/c or BALB. CD28KO donor cells into normal BCF1 mice. Serum titers of anti-dsDNA antibodies were assessed by enzyme-linked immunosorbent assay (ELISA) and major histocompatibility complex (MHC) class II antigen expression on B cells were tested by flow cytometry. In addition, depositions of immunoglobulin (Ig) were examined by direct immunofluorescence staining on frozen kidney sections. When (BALB/c × B6)F1 mice were injected with parental BALB/c lymphocytes, serum anti-dsDNA titer was significantly increased in association with nonspecific B-cell activation and IgG deposition in the glomerular basement membrane. In sharp contrast, none of these signs were observed in F1 mice, which were injected with CD28KO spleen cells. The CD28-mediated T-cell costimulatory pathway plays a pivotal role in the development of polyclonal B-cell activation, autoantibody production, and an immune complex glomerulonephritis. We propose that CD28KO mice are useful clues in examining the pathogenesis of experimental lupus nephritis.

CD28 knockout mice as a useful clue to examine the pathogenesis of chronic graft-versus-host reaction

Injection of BALB/c or DBA/2 spleen cells into F1 C57BL/6 (B6) hybrids induces a graft-versus-host reaction (GVHR) of a chronic stimulatory type that results in clinical and pathologic manifestations that resemble the human systemic lupus erythematosus (SLE). The aim of the present study was to examine the role of a major T-cell costimulatory signal receptor, CD28, in the production of autoantibody and the development of an immune complex glomerulonephritis, which are common in SLE pathology. For this purpose, CD28-deficient (CD28KO) mice were used for the source of donor lymphocytes. Chronic GVHR was induced by an injection of BALB/c or BALB. CD28KO donor cells into normal BCF1 mice. Serum titers of anti-dsDNA antibodies were assessed by enzyme-linked immunosorbent assay (ELISA) and major histocompatibility complex (MHC) class II antigen expression on B cells were tested by flow cytometry. In addition, depositions of immunoglobulin (Ig) were examined by direct immunofluorescence staining on frozen kidney sections. When (BALB/c x B6)F1 mice were injected with parental BALB/c lymphocytes, serum anti-dsDNA titer was significantly increased in association with nonspecific B-cell activation and IgG deposition in the glomerular basement membrane. In sharp contrast, none of these signs were observed in F1 mice, which were injected with CD28KO spleen cells. The CD28-mediated T-cell costimulatory pathway plays a pivotal role in the development of polyclonal B-cell activation, autoantibody production, and an immune complex glomerulonephritis. We propose that CD28KO mice are useful clues in examining the pathogenesis of experimental lupus nephritis.

Long-term preservation of direct immunofluorescence staining in slides stored at room temperature.

To determine whether reproducible results from direct immunofluorescent tissue staining could be obtained after storing the slides at room temperature. We examined the original slides of 22 cases noted to be positive for direct immunofluorescence. Diagnoses include pemphigus, pemphigoid, lupus, dermatitis herpetiformis, lichen planus, and vasculitis. These specimens, initially evaluated during the period January 1997 to September 1998, were prepared with a standard immunofluorescence staining technique, and then a permanent aqueous mounting medium was added. All specimens were stored at room temperature in vertical slide holding trays. We focused on the presence and relative intensity of the immunofluorescence staining, as well as the final diagnosis. We then compared our readings to that of the original reports. Twenty of the 22 cases studied (91%) were read as having the same diagnosis as the initial immunopathology report. Seventeen of the 22 cases (77%) were found to have the identical or slightly less fluorescence intensity. The original reports in 3 of the cases did not comment on the original intensity of fluorescence. Thus, a comparison of fluorescence preservation could not be made. In 2 of the cases, the quality of tissue preservation was poor, and though fluorescent staining was noted, we were unable to render a diagnosis. Our results suggest that direct immunofluorescent studies, using a permanent aqueous mounting medium, can be stored over long periods of time at room temperature without significant degradation of staining.

Chronic oral mucosal ulceration in a 54-year-old female

Pemphigus vulgaris most often begins in the mouth but is often overlooked in the differential diagnosis of chronic, multiple oral ulcerations and erosions. Accurate diagnosis requires perilesional biopsy including intact epithelium, submitted for hematoxylin and eosin as well as direct immunofluorescence staining. Early and aggressive treatment with moderate to high dose prednisone in combination with steroid sparing drugs such as azathioprine and mycophenolate allow complete remission in most patients.

Interaction between turkey monocytes and avian Chlamydia psittaci in the presence of Mycoplasma sp.: the importance of nitric oxide

The interaction between Chlamydia psittaci and turkey monocytes was studied in vitro. Purified monocytes were inoculated with C. psittaci, in the presence or absence of Mycoplasma hyorhinis. Whereas turkey monocytes produced high amounts of nitric oxide (NO) following the inoculation with M. hyorhinis, inoculation with C. psittaci did not induce NO production in these phagocytes. The monocytes strongly supported chlamydial growth, as demonstrated by the presence of inclusion forming units, the positive direct immunofluorescence staining and transmission electron microscopy. In contrast, upon co-inoculation of the monocytes with C. psittaci and M. hyorhinis, a reduced replication rate of C. psittaci was observed. N G-monomethyl- L-Arginine, a competitive inhibitor for the enzyme NO-synthase, inhibited the NO production and reversed the antichlamydial activity of the M. hyorhinis co-inoculated turkey monocytes. These results imply two considerations. First, as chlamydiae are obligate intracellular bacteria, special care should be taken to guard chlamydial cultures from mycoplasmal contamination, in order to prevent false results when investigating the response of immunomodulating cells to chlamydial infection. Secondly, as a mycoplasmal co-infection in vitro has the capacity of inducing antichlamydial activity in turkey monocytes, through the action of NO, it could be suggested that a similar interaction might take place in vivo. Moreover, it was shown that avian M. gallisepticum strains were also able to induce NO in turkey monocytes. Considering the high prevalence of both C. psittaci and Mycoplasma sp. in turkeys, this interaction, through the pivotal role of NO, might influence the outcome of respiratory diseases in turkeys.

Expression of estrogen receptors in desquamative gingivitis.

Most cases of chronic desquamative gingivitis (CDG) are shown by direct immunofluorescence (DIF) to be immune mediated diseases. Some patients present with similar clinical and microscopic findings as CDG but DIF staining is negative. It has been suggested that those cases of CDG may be hormone (estrogen) mediated and may be treated with estrogens with favorable results. Gingival tissue from 24 cases of CDG and one case of ordinary gingivitis were studied for estrogen receptor (ER) expression using immunohistochemical techniques. Twenty-four of the 25 cases were female. Using standard DIF analysis, 11 of the CDG cases were diagnosed as benign mucous membrane pemphigoid, 10 as lichen planus or lichenoid mucositis (LP), and one as pemphigus. The remaining 3 cases were not diagnostic for a specific disorder (idiopathic). Five of the females had a history of estrogen substitute therapy. Twenty-two of 23 female CDG cases were positive for ER, although the degree of staining varied. A 32-year-old female with ordinary gingivitis, whose gingivitis varied with her menstrual cycle, did not stain for ER. A 50-year-old male and a 76-year-old female, both with gingival LP, also had negative staining for ER. There appears to be no correlation between diagnosed diseases (immunological versus idiopathic) and expression of ER in CDG gingiva. ER expression in the gingiva is probably not related to the presence or absence of estrogen supplementation. The results of this study do not support the use of estrogen in the treatment of idiopathic CDG.

Effect of dexamethasone on lymphocyte subpopulations in premature infants with bronchopulmonary dysplasia.

This study was designed to determine the effect of dexamethasone treatment on peripheral blood lymphocyte counts and subpopulations in premature infants with bronchopulmonary dysplasia (BPD). Peripheral blood lymphocyte subpopulations in 12 premature infants with BPD were analyzed before treatment with a 6-week course of dexamethasone (day 0), on days 3 and 10 of treatment, and 2 weeks after discontinuing dexamethasone therapy (day 56). Lymphocyte immunophenotypes were determined using direct two-color immunofluorescent staining followed by flow cytometry. The percentage of lymphocytes was significantly lower on days 3 (17.55 +/- 2.55) and 10 (20 +/- 11.8) of dexamethasone therapy compared with before (30.36 +/- 6.41) or after treatment. The percentage of T cells was significantly lower on days 3 and 10 of dexamethasone therapy (mean +/- SEM; 58.09 +/- 1.93 and 60.09 +/- 2.47, respectively) compared with before (67.09 +/- 4.24) or after treatment. The absolute number of T cells was significantly lower on day 10 of therapy. The percentage of CD4+ cells was significantly lower on days 3 (38.91 +/- 2.49) and 10 (40.45 +/- 2.24) of therapy, and this decrease persisted after dexamethasone was stopped (36.73 +/- 3.41). The absolute number of CD4 cells was significantly lower on day 10 (1328 +/- 216) of therapy and reached a nadir on day 56 (1143 +/- 106). Similarly, the CD4/CD8 ratio was also significantly lower on days 3 and 10 of treatment (1.56 +/- 0.18 and 1.64 +/- 0.14, respectively) and reached a nadir on day 56 (1.04 +/- 0.13). Dexamethasone significantly reduced the percentage and absolute number of lymphocytes, T cells, and CD4 cells, as well as the CD4/CD8 ratio. A reduction in CD4 cells and in the CD4/CD8 ratio persisted 2 weeks after dexamethasone therapy was stopped. In contrast, the absolute number of B cells increased transiently, and CD8 cells were unaffected by dexamethasone. This alteration in lymphocyte subpopulations may help account for the clinically beneficial anti-inflammatory effect of dexamethasone in the treatment of BPD complicated by respiratory failure. The dexamethasone-induced decrease in CD4 cells may also increase the susceptibility of these infants to infection.

High levels of HIV-1 replication show a clear correlation with downmodulation of Bcl-2 protein in peripheral blood lymphocytes of HIV-1-seropositive subjects.

Peripheral blood lymphocytes (PBLs) from 51 HIV-1-seropositive subjects with different levels of HIV-1 replication and 20 healthy blood donors were examined for the expression of the antiapoptotic Bcl-2 protein. All the plasma samples from HIV-1 patients were characterized for the presence of HIV-1 p24 and HIV, RNA viral load. Bcl-2 protein expression in fresh peripheral blood lymphocytes was studied by different tests, including Western blot and indirect immunofluorescence techniques. Direct immunofluorescence staining, revealed by flow cytometry, was applied to quantify the number of specific anti-Bcl-2 antibody epitope binding sites, thus extrapolating the relative number of Bcl-2 into the cells. The results indicate that the expression of Bcl-2 protein is significantly lower in peripheral blood lymphocytes of HIV-1-seropositive patients showing high levels of viral replication, detected by means of HIV-1 p24 and RNA viral load, with respect to HIV-1 patients with low levels of virus replication and healthy blood donors. The clear-cut inverse correlation between viral replication and Bcl-2 expression reinforces the view that HIV-1-mediated apoptosis probably represents a key mechanism in AIDS pathogenesis.

The significance of the 'dust-like particles' pattern of immunofluorescence. A study of 66 cases.

Subacute cutaneous lupus erythematosus (SCLE) is a marker of a unique subset of lupus erythematosus patients. A 'dust-like particles' direct immunofluorescence (DIF) staining pattern, which consists of fine granular particles of immunoglobulin(s) scattered through the epidermis and the cellular infiltrates of the dermis, was reported to be specific for SCLE. In this study, we assessed the real specificity of this staining pattern, which had not yet been evaluated. We systematically searched for the dust-like particles staining pattern among the 4374 skin biopsy specimens submitted for direct cutaneous immunofluorescence during a 7-year period (1989-96). The corresponding patient records were reviewed. Dust-like particles were observed in 66 samples originating from 60 patients. Only 53% of the patients had SCLE. The remaining patients had systemic lupus erythematosus with visceral involvement (17%), discoid lupus erythematosus (3%), mixed connective tissue disease (2%). Sjögren syndrome (2%) and other diseases. Eighty-five per cent of the patients had connective tissue disease. Seventy-seven per cent of the patients were positive for antinuclear antibodies, but only 36% were positive for anti-Ro (SSA) antibodies. This study shows that the dust-like particles staining pattern is not specific for SCLE, but is highly suggestive of connective tissue disease. The nature of the antigen responsible for the immunoglobulin deposition and the prognostic significance of this DIF pattern remain to be established.

Levels of expression of CD19 and CD20 in chronic B cell leukaemias.

AIMS: To investigate whether the antigen levels of the B cell lineage markers CD19 and CD20 can distinguish between normal and neoplastic B cells or characterise distinct expression patterns among...

Early detection of epidermal dust-like particles in experimentally UV-induced lesions in patients with photosensitivity and lupus erythematosus.

Dust-like particles, producing a specific fine-speckled, epidermo-subepidermal direct immunofluorescence staining pattern, have been associated mainly with subacute cutaneous lupus erythematosus (LE). Under experimental conditions the appearance of immunoglobulins along the basement membrane in ultraviolet (UV) light-induced lesions has been reported as a late phenomenon. In this study, photoprovocations with UVA and UVB light were carried out in 16 photosensitive patients with discoid (n = 13), subacute cutaneous (n = 2) or systemic LE (n = 1) and serial biopsies from UV-induced lesions were processed for direct immunofluorescence. A specific, fine-speckled epidermal staining was detected within 7 to 14 days after UV provocation in 7/16 of the patients; in the majority of those patients associated with anti-SSA antibodies adn discoid LE without systemic manifestations of their disease.