RESUMEN
The actin cytoskeleton association is required for caspase 8-independent Fas/CD95 receptor internalization, a critical step for an optimal death-inducing signaling complex formation along the endocytic pathway, leading to efficient activation of the caspase cascade and, ultimately, cell death. However, the way in which this initiation phase of Fas receptor signaling is regulated is still unknown. We report herein that, in B cells, upon Fas engagement, the tyrosine phosphatase SHP-1-regulated Vav dephosphorylation, by downmodulating the Fas-ezrin-actin linkage is a fine-tune switch-off mechanism that the cell uses as a way to terminate the receptor internalization, controlling therefore the time and extent of the DISC formation and cell death.
Asunto(s)
Apoptosis , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Receptor fas/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Linfocitos B/enzimología , Linfocitos B/metabolismo , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Ratones , Fosforilación , Transducción de SeñalRESUMEN
Whole blood flow cytometry was performed among donors with various clinical forms of coccidioidomycosis using T27K, a coccidioidal antigen preparation protective in mice but not previously studied in humans. The median percent of CD3+ lymphocytes (CD3+) producing intracellular interferon-gamma (IFN-gamma) among healthy immune donors was 0.43%, significantly above that for non-immune donors (0.01%) and greater than that for subjects with other forms of coccidioidomycosis, including chronic pulmonary (0.11%), disseminated (0.09%) and concomitant human immunodeficiency virus (HIV) infection (0.07%) (P < or =0.002 for all). No increase in intracellular interleukin (IL)-10 production or apoptosis was noted in samples incubated with T27K. Among 14 HIV-infected patients with concomitant coccidioidomycosis, seven of eight patients whose peripheral blood CD4 concentration was > 200 cells microl(-1) had > 0.06% of CD3+ produce intracellular IFN-gamma, compared to none of six whose peripheral blood CD4+ lymphocyte concentration was < or =200 cells microl(-1) (P = 0.005). These data indicate that there is a specific human cellular immune response to T27K as a coccidioidal antigen and that this response can be categorized based on the clinical status of the coccidioidally infected patient.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/inmunología , Antígenos Fúngicos/inmunología , Coccidioides/inmunología , Citometría de Flujo/métodos , Inmunidad Celular , Infecciones Oportunistas Relacionadas con el SIDA/microbiología , Adulto , Anciano , Anciano de 80 o más Años , Complejo CD3/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Coccidioidomicosis/inmunología , Coccidioidomicosis/microbiología , Femenino , Humanos , Interferón gamma/biosíntesis , Masculino , Persona de Mediana EdadRESUMEN
The human factor H protein family comprises six plasma glycoproteins. Earlier we described a membranal factor H-related (mFHR) molecule that is expressed by human B lymphoblastoid cell lines and exerts cofactor activity. In our present study we screened human blood cells for the presence of mFHR proteins and further characterized these molecules. By cytofluorimetry it is shown that the factor H-specific rabbit antiserum reacts strongly with B cells and neutrophil granulocytes, but not with T cells and monocytes. On B lymphocytes mFHR is shown to be down-regulated upon activation of the cells via sIg. In experiments studying which short consensus repeat (SCR) domains are part of the cell membrane proteins we found that antibodies raised against SCRs 1-4, 19-20 and FHR-3 bound to neutrophils but not to B cells. While mFHRs derived both from B cells and granulocytes are shown to bind heparin, their size and structure are different as revealed by Western blotting. A further characteristic of the granulocyte-derived mFHR is its sensitivity to the PI-specific PLCgamma enzyme. These data demonstrate the existence of new members of the FHR protein family, as two distinct, membranal forms are identified. Based on the differences, the B cell derived molecule is termed mFHR-1 and the neutrophil derived protein mFHR-2.
Asunto(s)
Linfocitos B/metabolismo , Factor H de Complemento/biosíntesis , Neutrófilos/metabolismo , Linfocitos B/citología , Membrana Celular/metabolismo , Células Cultivadas , Regulación hacia Abajo , Heparina/metabolismo , Humanos , Isoenzimas/metabolismo , Activación de Linfocitos , Neutrófilos/citología , Fosfatidilinositol Diacilglicerol-Liasa , Fosfolipasa C gamma , Fosfolipasas de Tipo C/metabolismoRESUMEN
C1q and the outer envelope protein of HIV, gp120, have several structural and functional similarities. Therefore, it is plausible to assume that proteins that are able to interact with C1q may also interact with isolated gp120 as well as the whole HIV-1 virus. Based on this hypothesis, we studied the potential ability of the recombinant form of the 33-kDa protein, which binds to the globular "heads" of C1q (gC1q-R/p33), to inhibit the growth of different HIV-1 strains in cell cultures. gC1q-R/p33 was found to effectively and dose-dependently inhibit the production of one T-lymphotropic (X4) and one macrophage-tropic (R5) strain in human T cell lines (MT-4 and H9) and human monocyte-derived macrophage cultures, respectively. At a concentration range of 5-25 microg/ml, gC1q-R caused a marked and prolonged suppression of virus production. The extent of inhibition was enhanced when gC1q-R was first incubated with and then removed from the target cell cultures before virus infection, compared to that when the cells were infected with gC1q-R-HIV mixtures. The extent of inhibition was comparable to that of the Leu3a anti-CD4 antibody. Addition of gC1q-R to the cell cultures on day 1 or 2 after infection induced markedly less inhibition of HIV-1 growth than pretreatment of the cells just before or together with the infective HIV strains. In ELISA experiments, gC1q-R did not bind to a solid-phase recombinant gp120 while strong and dose-dependent binding of gC1q-R to solid-phase CD4 was observed. Our present findings indicate that gC1q-R is an effective inhibitor of HIV-1 infection, which prevents viral entry by blocking the interaction between CD4 and gp120. Since gC1q-R is a human protein, it is most probably not antigenic in humans. It would seem logical, therefore, to consider gC1q-R or its fragments involved in the CD4 binding as potential therapeutic agents.
Asunto(s)
Complemento C1q/metabolismo , VIH-1/efectos de los fármacos , VIH-1/fisiología , Receptores de Hialuranos , Glicoproteínas de Membrana , Receptores de Complemento/administración & dosificación , Receptores de Complemento/metabolismo , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Sitios de Unión , Antígenos CD4/metabolismo , Proteínas Portadoras , Línea Celular , Complemento C1q/química , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/patogenicidad , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/virología , Proteínas Mitocondriales , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Solubilidad , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/virología , Replicación Viral/efectos de los fármacosRESUMEN
The adjuvant effect of gamma-inulin, a strong activator of the alternative complement pathway, is well-known, but its exact mechanism is not revealed yet. Here, we show that macrophages, isolated from the peritoneal cavity of gamma-inulin-injected mice and used as antigen-presenting cells, enhance the proliferation of antigen-specific T-cells up to 2.5-fold when compared with macrophages of non-treated animals. This effect is abrogated by the presence of anti-C3 F(ab')2 fragments and by prior decomplementation of the donor animals with CVF. It is demonstrated that treatment of mice with the adjuvant results in deposition of C3-fragments onto the surface of peritoneal macrophages, as does in vitro incubation of the cells with gamma-inulin in the presence of fresh autologous serum. Prior incubation of macrophages with gamma-inulin plus serum in vitro enhances subsequent C3 production. Because it has been shown earlier that CR1/2 expressed on activated T-cells and interacting with covalently bound C3-fragments plays an important role in the augmentation of the adaptive response, our present results reveal a mechanism that contributes to the adjuvant effect of gamma-inulin and point to a further link between innate and adaptive immunity.
Asunto(s)
Presentación de Antígeno/inmunología , Complemento C3/inmunología , Inulina/inmunología , Macrófagos Peritoneales/inmunología , Animales , Activación de Linfocitos , Ratones , Linfocitos T/inmunologíaRESUMEN
Histamine is produced from histidine by histidine decarboxylase (HDC) in many cells including normal and malignant lymphocytes. We examined the expression of HDC and the effect of histamine receptor antagonists on the proliferation of a human T cell line, Jurkat and on antigen-driven proliferation of lymphocytes from ovalbumin-immunized mice. Our results demonstrate that HDC is inducible in Jurkat cells by anti-CD3. The H1 receptor antagonist triprolidine dose dependently inhibits proliferation of both Jurkat cells and ovalbumin-stimulated murine lymphocytes, while the H2 antagonist ranitidine was ineffective. Alpha-fluoro-methyl-histidine blocking HDC activity did not inhibit the T cell proliferation, suggesting an existing pool of histamine in T cells.
Asunto(s)
Antagonistas de los Receptores Histamínicos H1/farmacología , Células Jurkat/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Ovalbúmina/inmunología , Linfocitos T/inmunología , Triprolidina/farmacología , Animales , Histamina/metabolismo , Histidina Descarboxilasa/metabolismo , Humanos , Células Jurkat/enzimología , Células Jurkat/patología , RatonesRESUMEN
A 45 kDa protein was isolated from a soluble vaccine prepared from formaldehyde-killed spherules of Coccidioides immitis. From the N-terminal amino acid sequence, the protein yielded a 17-amino-acid peptide that was homologous to sequences of other fungal aspartyl proteinases. The coccidioidal cDNA encoding the proteinase was amplified using oligonucleotide primers designed from the 45 kDa N-terminal amino acid sequence and a fungal aspartyl proteinase consensus amino acid sequence. The PCR product was cloned and sequenced, and the remaining 5' upstream and 3' downstream cDNA was amplified, cloned, and sequenced. The cDNA encoding the coccidioidal aspartyl proteinase open reading frame was cloned and the fusion protein containing a C-terminal His-tag expressed in E. coli. The recombinant aspartyl proteinase was purified by immobilized metal affinity chromatography. This recombinant protein will be used for further studies to evaluate its antigenicity, including protective immunogenicity.
Asunto(s)
Ácido Aspártico Endopeptidasas/genética , Coccidioides/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Clonación Molecular , Coccidioides/genética , ADN de Hongos , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Vacunas Fúngicas/genética , Vacunas Fúngicas/inmunología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Homología de Secuencia de AminoácidoRESUMEN
The activity of the chitin synthase inhibitor lufenuron was evaluated in vitro using the spherule-endospore (SE) phase of Coccidioides immitis. The lufenuron was also used to treat mice infected with C. immitis by the respiratory route. In vitro, lufenuron had no effect upon fungal cell growth. Two formulations of lufenuron were evaluated in vivo. Neither the oral nor the injectable lufenuron extended the survival of mice infected with C. immitis when compared with placebo-treated mice.
Asunto(s)
Benzamidas/farmacología , Quitina Sintasa/antagonistas & inhibidores , Coccidioides/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Animales , Benzamidas/uso terapéutico , Coccidioidomicosis/tratamiento farmacológico , Coccidioidomicosis/mortalidad , Femenino , Ratones , Pruebas de Sensibilidad MicrobianaRESUMEN
Murine cells of the B lymphoblastoid line A20 and concanavalin A-elicited peritoneal macrophages are shown to activate and fix C3 fragments covalently when incubated in fresh, autologous serum under conditions allowing the initiation of the alternative complement pathway. For the detection of cell-bound C3, cytofluorimetry was performed using FITC-labeled F(ab')2 fragments of anti-mouse C3. Cell-bound C3 fragments are not internalized or shed by the cells under culture conditions for at least two hours. When the antigen-presenting capacity of serum-treated cells was tested using various antigens and experimental systems, augmentation of the proliferation of antigen-specific T cells was found. This enhancing effect was particularly pronounced at suboptimal antigen doses. The elevation of T cell proliferation induced by C3-opsonized antigen-presenting cells (APC) could be abrogated by F(ab')2 fragments of goat anti-mouse C3, suggesting the involvement of C3 receptors expressed by T cells in the process. Using the 7G6 mAb recognizing murine CR1/CR2, the presence of these complement receptors on activated T cells is demonstrated by cytofluorimetry and immunoprecipitation, as well. These results point to the role of C3 bound to acceptor sites on APC in the facilitation of antigen presentation, providing a further link between innate and adaptive immunity.
Asunto(s)
Células Presentadoras de Antígenos/metabolismo , Complemento C3/metabolismo , Epítopos de Linfocito T/inmunología , Activación de Linfocitos/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Presentación de Antígeno , Células Presentadoras de Antígenos/inmunología , Complemento C3/inmunología , Complemento C3/fisiología , Vía Alternativa del Complemento , Hibridomas/inmunología , Hibridomas/metabolismo , Sueros Inmunes/farmacología , Inmunidad Innata , Inmunosupresores/farmacología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos BALB C , Receptores de Complemento 3b/biosíntesis , Receptores de Complemento 3d/biosíntesisRESUMEN
OBJECTIVE: To study the mechanism of the complement-mediated antibody-dependent enhancement (C'-ADE) of HIV infection which may play a significant role in the progression of HIV-disease. METHODS: In vitro complement activating and complement-mediated HIV-infection enhancing abilities of three human anti-gp41 monoclonal antibodies (MAb) were tested. C'-ADE was estimated using HIV-1IIIB and CR2 (CD21)-carrying MT-4 target cells. Normal human serum (NHS), purified C1q, C1q-deficient (C1qD) and C2-deficient (C2D) human sera were applied as complement sources. RESULTS: All MAb mediated increased C1q binding to solid-phase gp41. All MAb had a marked dose-dependent and strictly complement-mediated HIV-infection enhancing effect. Mixtures of the MAb with purified C1q also significantly increased HIV-1 infection. C1qD serum had a markedly lower enhancing effect than NHS, which could be raised to normal level by addition of purified C1q. Pretreatment of the target cells with anti-CR2 antibodies only partially inhibited the enhancing effect of the MAb plus normal human serum. CONCLUSION: These novel findings indicate that besides the well-known facilitation of entry of HIV-1 by the interaction between virus-bound C3 fragments and CR2 present on the target cells, fixation of C1q to intact virions also results in an enhanced productive HIV-1 infection in the MT-4 cell cultures.
Asunto(s)
Complemento C1q/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Anticuerpos Monoclonales/inmunología , Complemento C1q/farmacología , Progresión de la Enfermedad , Infecciones por VIH/fisiopatología , Humanos , Células Tumorales CultivadasRESUMEN
Mast cells and basophils are known to be triggered by allergens via cross-linking with their high-affinity IgE-binding receptors, Fc epsilon RI. The anaphylatoxic activity of the complement-derived peptides C3a and C5a has been known for a long time; however, it has also been reported that serosal- and mucosal-type mast cells respond differently to peptidergic stimuli. The mechanism of mast cell activation by cross-linking of Fc epsilon RI has been the subject of intensive studies in the past few years, while the action mode of the anaphylatoxic complement peptides has been revealed only recently. We report about a novel function of C3a: its inhibitory activity on IgE-mediated triggering of the mucosal RBL-2H3 cells. Surprisingly, the other anaphylatoxic peptide C5a, which has been shown to be significantly more effective in several biological assays, did not influence antigen-induced triggering of the RBL-2H3 cell line at all.
Asunto(s)
Complemento C3a/metabolismo , Complemento C5a/metabolismo , Mastocitos/inmunología , Animales , Antígenos , Membrana Celular/inmunología , Membrana Celular/metabolismo , Humanos , Mastocitos/metabolismo , Receptores de IgE/metabolismoRESUMEN
As a result of the up to date treatment the life quality of diabetic patients is improved, their life span is prolonged. The authors recite the guidelines of vocational guidance and disability evaluation of diabetics. It is generally accepted that patients treated by diet alone may choose any profession, their lifestyle need not differ from that of healthy people. Well educated diabetic patients on oral antidiabetics or insulin, who have near normoglycemic blood sugar levels, can follow the lifestyle of a healthy individual; yet they should avoid occupations where their own or others lives might be put in danger. The authors recite effective departmental orders about the questions that influence the lives of diabetic patients-for example drivers licence, sports facilities for diabetics- and are the most frequently encountered by experts. Problems of rehabilitation that arise during the management of diabetics are also discussed.
Asunto(s)
Diabetes Mellitus/rehabilitación , Adulto , Conducción de Automóvil , Consejo , Femenino , Guías como Asunto , Humanos , Hungría , Esperanza de Vida , Estilo de Vida , Masculino , Persona de Mediana Edad , OcupacionesRESUMEN
Intracerebral inoculation of susceptible strains of mice with Theiler's murine encephalomyelitis virus (TMEV) results in a chronic, immunologically mediated demyelinating disease that shares many features with human multiple sclerosis. CD4+ T lymphocytes play a critical role in the pathogenesis of virus-induced demyelinating disease. We have identified a region within amino acid residues 24 to 37 of the VP3 capsid protein of TMEV (VP3(24-37)) that is recognized by T lymphocytes from the demyelination-susceptible SJL/J strain of mice. The T-cell response to VP3(24-37) represents a predominant Th-cell response against the virus from either TMEV-immunized or TMEV-infected SJL/J mice, and viral epitopes VP1(233-250), VP2(74-86), and VP3(24-37) account for most of the Th-cell response to TMEV.
Asunto(s)
Cápside/química , Enfermedades Desmielinizantes/virología , Epítopos/análisis , Linfocitos T/inmunología , Theilovirus/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cápside/análisis , Cápside/inmunología , Proteínas de la Cápside , Cartilla de ADN , Enfermedades Desmielinizantes/patología , Susceptibilidad a Enfermedades , Humanos , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Esclerosis Múltiple/patología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Poliomielitis/patología , Poliomielitis/virología , Reacción en Cadena de la Polimerasa , Theilovirus/inmunología , Theilovirus/patogenicidadRESUMEN
Complement regulatory proteins present on the surface of various mammalian cells play an important role in controlling homologous lysis, by interacting with C3 (and usually C4). These proteins have a similar structural motif ("short consensus repeat") (Reid, K.B.M., Bentley, R.D., Campbell, R.D., Chung, L.P., Sim, R.B., Kristensen, T. and Tack, B.F., Immunol. Today 1986. 7:230), and the genes encoding them are members of the family of regulators of complement activation. Here we describe a hitherto unknown member of this family, a molecule expressed by B lymphoblastoid cells. This protein is recognized by polyclonal antibodies to factor H and by MAH4, a monoclonal antibody reacting with the N-terminal portion of factor H. The cell surface protein is built up of two disulfide-linked chains of approximately 68 and 75 kDa. Biosynthetic labeling studies confirmed that it is synthesized by B cells only, but not by the investigated lines of other origin. When tested for its functional activity, this molecule was shown to act as cofactor for factor I-mediated cleavage of fluid-phase C3b to C3bi. The protein appears to be encoded by a 3.5-kb mRNA, hybridizing with a cDNA probe coding for the N-terminal portion of factor H. Due to its cross-reactivity with anti-H antibodies, cofactor activity for factor I and hybridization with factor H cDNA, despite its two-chain composition, it is considered a factor H-like protein.
Asunto(s)
Linfocitos B/química , Factor H de Complemento/análisis , Northern Blotting , Línea Celular , Factor H de Complemento/biosíntesis , Factor H de Complemento/inmunología , Humanos , Pruebas de Precipitina , ARN Mensajero/análisisRESUMEN
The most apical 2 mm of the root canals of periapically diseased roots were examined for microorganisms by scanning electron microscopy (SEM). Bacteria in this area were observed in 10 out of 12 (83.3%) cases. The two remaining cases exhibited bacteria more coronally, with tissue remnants between the bacterial front and the apical foramen. Rod-shaped bacteria dominated, but filaments, spirochetes and cocci were also seen. Cocci and rods sometimes formed micro-colonies. Occasionally, cocci were seen attached to filaments forming "corn-cob"-like structures. Deposits resembling bacterial plaque were also found inside the root canal. SEM is useful for studying microbial topography of the apical root canal.
Asunto(s)
Cavidad Pulpar/microbiología , Enfermedades Periapicales/microbiología , Raíz del Diente/microbiología , Bacterias/aislamiento & purificación , Infecciones Bacterianas/microbiología , Diente Premolar , Recuento de Colonia Microbiana , Cavidad Pulpar/ultraestructura , Humanos , Microscopía Electrónica de Rastreo , Diente Molar , Raíz del Diente/ultraestructuraRESUMEN
An apical dentine sampling technique was applied in order to monitor the bacteriology of the pulp canal and radicular dentine before and during treatment of teeth with chronic apical periodontitis. Twenty-three teeth with a radiographic diagnosis of apical periodontitis were studied. They were subjected to a standardized two-appointment treatment regimen of extensive apical reaming in the absence of antimicrobial agents and 1-week dressing with calcium hydroxide. Bacteriological samples were taken from the root canal at the start, and apical dentine samples at the end, of each sitting. Provision was made to allow growth of anaerobic bacteria. All root canals but one showed growth at the start of treatment. Dentine samples were positive in 14 of the 23 teeth at the end of the first appointment. Eight of the 23 canals had detectable growth from the canal at the start of the second appointment, but in sufficient numbers for quantification in only one root canal. The subsequent dentine samples were otherwise negative at the second appointment. There was a tendency for teeth causing symptoms to harbour more bacteria than symptomless teeth.
Asunto(s)
Bacterias Anaerobias/efectos de los fármacos , Hidróxido de Calcio/uso terapéutico , Dentina/microbiología , Periodontitis Periapical/terapia , Tratamiento del Conducto Radicular , Adulto , Hidróxido de Calcio/farmacología , Recuento de Colonia Microbiana , Cavidad Pulpar/microbiología , Humanos , Periodontitis Periapical/microbiología , Proyectos Piloto , Tratamiento del Conducto Radicular/instrumentaciónRESUMEN
Although not universally accepted, retrospective histological, roentgenological and microbiological studies have indicated that cross-infection can occur between infected pulps and deep periodontal pockets. This review provides examples of similarities in the microfloras of these adjacent oral sites, supporting the idea that infection spreads from one site to the other. The organisms most often involved are probably bacteroides, fusobacteria, eubacteria, spirochetes, wolinellas, selenomonas, campylobacter, and peptostreptococci. Important qualities of cross-infecting organisms may be the ability to survive in highly reduced environments and motility. Precautions should be taken to prevent in vivo seeding of such micro-organisms, particularly in compromised teeth and hosts.
Asunto(s)
Necrosis de la Pulpa Dental/microbiología , Bolsa Periodontal/microbiología , Periodontitis/microbiología , Infecciones Bacterianas/transmisión , Cavidad Pulpar/microbiología , Bacterias Anaerobias Gramnegativas , Humanos , Pulpitis/microbiologíaRESUMEN
In this study computerized image analysis procedures were applied to endodontic radiographs. Kontron IBAS 2000 is a commercially available image analysis system with processing routines applicable to radiograph digitizing and transformations. The system was evaluated for: its ability to harmonize blackening and contrast in endodontic radiographs; its ability to compensate for angulation distortion of sequential exposures of individual teeth; its potential for application of digital subtraction methods; and its use in automated gray-level analyses of diseased and healthy bone areas in endodontic radiographs. The Kontron IBAS 2000 system proved suitable for all applications. However, the specificity of the subtraction procedure was limited by some inherent problems in the harmonization of blackening and in the subtraction process itself. On the other hand, automated gray level measurements proved to be a robust method for unbiased and quantitative assessment of healing of apical periodontitis.