Human papillomavirus 16/18 infections in lung cancer patients in Mexico.

BACKGROUND/AIMS: Human papillomavirus (HPV) is an epitheliotropic, double-stranded DNA virus, and its high-risk genotypes are associated with human cancer. HPV genome has been detected in lung carcinomas in certain places around the world, including Mexico; however, the prevalence of this is unclear. In this study, we examine the frequency of high-risk HPV 16/18 in lung cancer tissues from a Mexican population. METHODS: 39 lung cancer specimens were analyzed by polymerase chain reaction (PCR) using HPV GP5+/GP6+ primers and then were genotyped using specific primers to HPV 16/18. Additionally, in situ hybridization (ISH) was performed using BIO-labeled oligonucleotide probes. RESULTS: Our results identified 15 positive cases (38.46%) for HPV 16 and 1 positive case (2.56%) for HPV 18 by PCR. ISH showed the presence of HPV DNA in 13 of 16 (81%) samples, in agreement with the PCR results. CONCLUSIONS: In this study, we detected HPV 16/18 gene sequences in lung cancer samples obtained from Mexican patients by PCR and ISH. We found the highest prevalence of HPV 16 infection in lung adenocarcinomas, suggesting that HPV infection may be associated with lung cancer. However, further studies are needed to elucidate the role of HPV in lung carcinogenesis.

Intracellular antigens released from Balb/c mice kidneys under chemically induced apoptosis and/or necrosis.

The present investigation assesses the possible role of apoptosis and necrosis in intracellular antigen exposure of kidneys from Balb/c mice. Renal tissues were cultured and treated with chemicals to induce apoptosis and /or necrosis. The expression of intracellular antigens Sm, RNP, Ro and La were monitored with antibodies against these antigens. Main results confirm that renal intracellular antigens are released and exposed onto the surface of apoptotic and necrotic cells, therefore these antigens become an easy target of autoantibodies. This mechanism may be important in the lupus nephritis pathogenesis.

Apoptosis and necrosis increase antigenicity of proteins recognized by antinuclear antibodies.

OBJECTIVE: Present study addresses the issue whether apoptosis and necrosis increases the antigenicity of proteins recognized by antinuclear antibodies. MATERIAL AND METHODS: HEp-2 cells were cultured in standard conditions; apoptosis was induced by camptothecin and necrosis by mercuric chloride. Protein antigenicity of cell extracts was tested onto nitrocellulose membranes and probed with positive or negative sera for antinuclear antibodies by a luminescent-dot-ELISA system. RESULTS: Apoptotic changes in HEp-2 cells appeared by 24 hours of camptothecin exposure, meanwhile the necrotic features become visible earlier. Luminescence was significantly superior in ANA positive sera than in ANA negative controls. Antinuclear antibody sera recognized better the antigens from the apoptotic and necrotic cells than controls without chemical treatments. CONCLUSIONS: Apoptosis and necrosis increase the ANA binding by better availability of intracellular antigens, or by disclosing cryptic epitopes.

Transplacental passage of maternal pemphigus foliaceus autoantibodies induces neonatal pemphigus.

The association of maternal pemphigus foliaceus (PF) with neonatal PF is rare and may be secondary to transplacental passage of PF autoantibodies. We describe a 25-year-old patient with PF who was delivered of two consecutive babies, one with classic skin lesions of PF and another that was normal. The neonate with PF was born when the mother had widespread skin disease; the normal newborn was born when the mother was in partial remission. The titers of PF autoantibodies were higher in the mother's serum and the cord serum of the baby with PF than in the mother during partial remission and the unaffected baby. The mother and affected baby had autoantibodies to desmoglein 1. Furthermore, cord blood from the baby with PF induced skin disease when injected into mice. In this case, maternal PF was associated with neonatal PF when the titers of maternal anti-desmoglein 1 autoantibodies were elevated. The cutaneous disease in neonatal PF is due to anti-desmoglein 1 autoantibodies.

Fas ligand and Bax gene transcription contributes to Ro60 ribonucleoprotein redistribution in UV-A irradiated human keratinocytes.

OBJECTIVES: Ro ribonucleoproteins are of particular interest because they are serologic markers of photosensitive variants of lupus such as the subacute cutaneous lupus erythematosus (SCLE), in which the polycyclic skin lesions are triggered by exposure to the sun. We study the role of apoptosis in the expression of Ro antigen. METHODS: We used UV-A irradiated keratinocytes. RESULTS: We demonstrate in cultured human UVA-irradiated keratinocytes that the enhanced expression of Ro60 ribonucleoprotein is caused by antigenic redistribution consecutive to Fas-L and Bax gene activation.

Ro60 ribonucleoprotein inhibits transcription by T3 RNA polymerase in vitro.

BACKGROUND: Ro60 ribonucleoprotein is a conserved molecule belonging to the family of Ro ribonucleoproteins and targeted by autoantibodies produced in patients with systemic lupus erythematosus or Sjogren's syndrome. Ro60 plays a role in postranscription events, as well as in the nucleocytoplasmic shuttling of RNA polymerase III transcripts. OBJECTIVE: To evaluate the in vitro effects of Ro60 on T3 RNA polymerase transcription. METHODS: Ro60 ribonucleoprotein was affinity-purified from human spleen extracts using 4B-Sepharose linked to anti-Ro60 monoclonal antibodies. Purified Ro60 was incorporated into the T3 RNA polymerase transcription reaction system using pTRI-beta-Actin-human DNA as a template. RESULTS: Ro60 inhibited initiation of the transcription process in a dose-dependent manner; neither elongation nor termination was affected by Ro. CONCLUSION: In vitro, Ro60 appears to inhibit the transcription of a T3 RNA polymerase-dependent template.

Anti-NuMA antibodies: an uncommon specificity in scleroderma sera.

BACKGROUND: Serum antibodies in scleroderma patients are generally directed against the nucleolus and centromeres. A small proportion of patients have serum antibodies to the centrioles and mitotic apparatus. OBJECTIVE: To determine the prevalence of serum autoantibodies against the mitotic apparatus in scleroderma patients. MATERIAL AND METHODS: Sera from 113 patients with various forms of scleroderma were tested for antinuclear antibodies by indirect immunofluorescence on HEp-2 cells. The specificity of the antibodies was determined by Western blot. RESULTS: Only two scleroderma sera recognized the mitotic apparatus. Western blot results showed that in both cases the target was an about 235 kDa protein corresponding to the NuMA determinant. Affinity-purified anti-NuMa antibodies were used to perform immunolocalization in synchronized HEp-2 cells using scanning laser confocal microscopy. The anti-NuMA autoantibodies recognized the mitotic asters but neither the centrioles nor the microtubules. CONCLUSION: Our data suggest that anti-NuMA autoantibodies may be devoid of clinical significance in scleroderma. However, they remain useful as probes in cell biology studies.

UV-A irradiation induces transcription of IL-6 and TNF alpha genes in human keratinocytes and dermal fibroblasts.

OBJECTIVE: To determine whether UV-A irradiation induces synthesis of inflammatory cytokines in the skin. METHODS: Human keratinocytes and dermal fibroblasts were cultured and exposed to various doses of UV-A radiation. The cellular distribution of IL-6 and TNF alpha was determined by indirect immunofluorescence and by flow cytometry with monoclonal anti-IL-6 and anti-TNF alpha antibodies. Cytokine production was measured in the supernatants using an ELISA. IL-6 and TNF alpha transcription induced by UV-A was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification. RESULTS: IL-6 and TNF alpha were detected in small amounts in nonUV-A-irradiated cell. UV-A exposure was followed by significant increases in IL-6 and TNF alpha expression and by small increases in IL-6 and TNF alpha levels in culture supernatants. RT-PCR demonstrated a UV-A-mediated increase in the transcription of IL-6 and TNF alpha genes. CONCLUSION: Synthesis of IL-6 and TNF alpha can be induced by UV-A irradiation. This effect of UV-A may contribute to the inflammatory skin changes seen during lupus flare-ups after sun exposure.

Renal expression of IL-6 and TNFalpha genes in lupus nephritis.

These studies were carried out to examine the presence of the inflammatory cytokines IL-6 and TNFalpha in kidneys of patients with lupus nephritis as an indicator of their possible role in its pathogenesis. A total of 19 kidney biopsies from patients with type III or IV lupus nephritis were processed by direct immunofluorescence using monoclonal anti-IL-6 and TNFalpha antibodies. Local expression of these genes was demonstrated both by in situ hybridization and by reverse transcriptase-PCR amplification of total RNA isolated from kidney tissue. Fifty-two percent of the biopsies exhibited IL-6 and TNFalpha deposited along the glomeruli and tubules; in situ expression of these cytokines was demonstrated in 6 biopsies with type IV, and 1 with type III nephritis. Inflammatory cytokines are actively synthesized in the kidneys of patients with lupus nephritis and therefore, may play a role in its pathogenesis.

The expression and localization of Scl-70/DNA topoisomerase I vary throughout the cell cycle.

PURPOSE AND METHODS: We investigated the expression and localization of topoisomerase I by Western blot and indirect fluorescent antibody assay, respectively, using anti-Scl-70/topo I from patients with diffuse scleroderma. The contribution of topoisomerase I to DNA replication was assessed using cells treated with the topoisomerase I inhibitor camptothecin. RESULTS: Scl-topo I was detected at all cell cycle phases as a single immunoreactive band of 100 kDa. Extracts from cells in the S phase contained the largest amount of immunoreactive Scl-70/topo I. Variations in the subcellular distribution of Scl-70/topo I were seen throughout the cell cycle, with a speckled nucleoplasmic distribution during G1 contrasting with concentration within the nucleolus during S. Camptothecin exposure blocked topoisomerase I expression and caused a significant decrease in DNA production. CONCLUSION: These data suggest (1) that topomerase I is active mainly during the S phase and contributes to DNA replication, and (2) that topoisomerase I may be involved in ribosomal gene transcription.

Tyrosine kinase participates in phosphorylation of the Ro60 ribonucleoprotein.

Studies of Ro ribonucleoprotein are important in rheumatology, since anti-Ro antibodies are probably involved in the pathogenesis of congenital heart block and subacute cutaneous lupus erythematosus. In addition, the phosphorylation-dephosphorylation cycle modulates binding of ribonucleoproteins to RNA, a process that might affect the antigenicity and function of the Ro protein. The present study was designed to determine whether Ro can be phosphorylated by tyrosine kinase. To answer this question, synchronized HEp-2 cells were phosphorylated in vivo with exogenous 32P, and Ro ribonucleoprotein previously subjected to metabolic radiolabeling was immunoprecipitated by monoclonal anti-Ro antibodies and examined by SDS-PAGE and autoradiography. The main results were as follows: first, Ro ribonucleoprotein was phosphorylated in vivo; second, Ro was found to have phosphorylable tyrosine residues; third, tyrosine kinase participated in the phosphorylation of Ro; and fourth, phosphorylation did not change the recognition pattern of Ro by anti-Ro antibodies. In conclusion, Ro60 is phosphorylated by tyrosine kinase.

Cross-reactivity of anti-nDNA antibodies with nuclear envelope proteins. Isolation of a cDNA encoding the 70 kDa annular protein recognized by autoantibodies from patients with systemic lupus erythematosus.

OBJECTIVES: To determine whether annular rDNA is complexed with the nuclear envelope proteins. METHODS: From a batch of lupus sera with anti-nDNA, we selected a lupus serum containing annular anti-nDNA autoantibodies resistant to DNase digestion and used it to isolate several cDNA clones from a lambda gt11 HeLa cell library. RESULTS: The cloned fusion protein immunoadsorbed the annular anti-nDNA autoantibodies, and the immunoaffinity autoantibodies eluted from the recombinant filters produced an annular pattern around the nucleus in fluorescent assays on HEp-2 cells; by Western blot, they also recognized a 70 kDa protein from HEp-2 cell extracts. Annular-lambda gt11 lysogens generated in E. coli Y1089 produced a fusion protein that recognized annular anti-nDNA autoantibody-containing lupus sera by Western blot. The recombinant filters and annular fusion protein were also recognized by a prototype anti-lamin serum. To determine whether the annular recombinant protein bound DNA, an interaction assay was performed in vitro using DNA minicircles and DNA from HEp-2 cells; this assay resulted in a slowing of the electrophoretic mobility of the DNA. CONCLUSIONS: 1) The annular DNA in eukaryotic cells is complexed with nuclear envelope proteins. 2) Annular anti-nDNA autoantibodies from lupus patients cross-react with perinuclear proteins. 3) Perinuclear proteins recognized by anti-nDNA are lamins. 4) An interaction between DNA and the 70 kDa protein is inducible in vitro. Whether this interaction affects cell function is still unknown.

Fate of human IgG from lupus erythematosus antinuclear antibody positive sera passively transfered to mice.

An animal model to study the fate of antinuclear antibodies (ANA) in vivo is reported. Newborn Balb/c mice were passively transferred by injection with sera from patients with autoimmune disease. The following antinuclear antibodies were found in the liver, spleen, kidneys and skin shortly after the injection: anti-dsDNA, nRNP, Ro/SSA and La/SSB. Clearance of all these antibodies occurred within 48 hours. The cytoskeleton, Fc immunoglobulin domain and Fc immunoglobulin-cell surface interaction may play a major role in the entry of antinuclear antibodies into cells, which may occur via endocytosis. Our animal model may be useful for studying the kinetics of binding of antinuclear antibodies to nuclear components and the effects of such binding in vivo.

Compartimiento nucleolar de la DNA-Topoisomerasa I en células HEp-2.Estudio con los sueros de pacientes con esclerodermia difusa / Nucleolar DNA Topoisomerase I: Compartiment with HEp-2 Serum study in patients with Diffuse Scleroderma

Estudiamos la prevalencia de anticuerpos contra la DNA-topoisomerasa y su restricción a isotipos, en 20 anticuerpos anti-nucleolares positivos.Los isotipos fueron determinados en pruebas de inmunofluorescencia indirecta con células HEp-2 y anticuerpos monoclonales anti-IgG1, IgG2, IgG3 e IgG4:La especialidad anti-DNA-topoisomerasa I se determinó por pruebas de doble difusión en agar y por Western blot,utilizando como antígeno, extracto de timo de ternera y un suero prototipo anti-Scl-70. Resultados: Los patrones de fluorescencia tuvieron una localización nucleolar en todos los casos y el 75 por ciento presentó conjuntamente patrón nucleosomal. En pruebas de doble difusión, de los 20 sueros, siete (35 por ciento)reconocieron los extractos de timo de ternera y de ellos, 4(20 por ciento) presentaron especificidad anti-DNA-Topoisomerasa I. Por Western blot, 15 sueros (75 por ciento) mostraton bandas reactivas a un antígeno de 70 kD (DNA-Topo I) y fueron de subclase IgG1.Estos resultados nos permiten concluir que los sueros de Esclerodermia con anticuerpos anti-nucleolares presentan , en gran proporción , especificidad anti- DNA- topoisomerasa I; no obstante, las pruebas de precipitación en agar, no son lo suficientemente sensibles para detectar su prevalencia real; para este propósito, se requiere de pruebas sensitivas como el Western blot

Compartimiento nucleolar de la DNA-Topoisomerasa I en células HEp-2.Estudio con los sueros de pacientes con esclerodermia difusa / Nucleolar DNA Topoisomerase I: Compartiment with HEp-2 Serum study in patients with Diffuse Scleroderma

Estudiamos la prevalencia de anticuerpos contra la DNA-topoisomerasa y su restricción a isotipos, en 20 anticuerpos anti-nucleolares positivos.Los isotipos fueron determinados en pruebas de inmunofluorescencia indirecta con células HEp-2 y anticuerpos monoclonales anti-IgG1, IgG2, IgG3 e IgG4:La especialidad anti-DNA-topoisomerasa I se determinó por pruebas de doble difusión en agar y por Western blot,utilizando como antígeno, extracto de timo de ternera y un suero prototipo anti-Scl-70. Resultados: Los patrones de fluorescencia tuvieron una localización nucleolar en todos los casos y el 75 por ciento presentó conjuntamente patrón nucleosomal. En pruebas de doble difusión, de los 20 sueros, siete (35 por ciento)reconocieron los extractos de timo de ternera y de ellos, 4(20 por ciento) presentaron especificidad anti-DNA-Topoisomerasa I. Por Western blot, 15 sueros (75 por ciento) mostraton bandas reactivas a un antígeno de 70 kD (DNA-Topo I) y fueron de subclase IgG1.Estos resultados nos permiten concluir que los sueros de Esclerodermia con anticuerpos anti-nucleolares presentan , en gran proporción , especificidad anti- DNA- topoisomerasa I; no obstante, las pruebas de precipitación en agar, no son lo suficientemente sensibles para detectar su prevalencia real; para este propósito, se requiere de pruebas sensitivas como el Western blot

Transplacental transfer of human antinuclear antibodies in mice by injection of lupus IgG in pregnant animals.

Pregnant female Balb/c mice were injected with IgG fractions from patients with systemic lupus erythematosus, in order to study the in vivo passage of antinuclear antibodies (ANA) across the placenta. After injection of monospecific sera directed against nDNA, Sm, nRNP, Ro(SSA) and La(SSB), ANA were found in fetal circulation and trapped in the liver, spleen kidney and skin of fetus. Also, ANA were demonstrated in placental tissue and cord. The placental IgG-Fc receptors apparently played a major role in ANA entry into the fetus. Our study demonstrates that human ANA can be passively transferred into experimental animals to study their kinetics during pregnancy.

Perfil de anticuerpos antinucleares en la esclerosis sistemica progresiva Estudio multicentrico. / Antinuclear antibodies profile in progressive systemic sclerosis. A multicenter study

Se realizo un estudio multicentrico en 41 pacientes con esclerosis sistemica progresiva (ESP) para definir la frecuencia e inmunoespecificidad de los anticuerpos antinucleares (AAN). Cuando se empleo rinon de raton y celulas humanas HEp-2 como sustratos, la prueba de inmunoflourescencia indirecta resulto positiva en el 27 y 47% de los casos respectivamente.Los anticuerpos tuvieron especificidad contra acidos nucleicos en el 20% cuando se utilizo como fuente de antigeno DNA sonicado, que contiene fragmentos de DNA nativo y desnaturalizado. Sin embargo, los anticuerpos a DNA nativo fueron negativos al usar el metodo de Chritidia luciliae. La reactividad a proteinas nucleares no histonas se encontro en un tercio de los casos con especificidad a RNPn y no hubo anticuerpos anti-Sm, SS-B ni otros.Los anticuerpos anti-uracilo se encontraron en el 15% de los enfermos y no se hallaron anticuerpos anti-centromero. El estudio de los AAN en la ESP debe realizarse en sustrato homologo.El perfil de autoanticuerpos tiene una especificidad limitada, predominando su reactividad a acidos nucleicos, poli-uracilo y proteinas acidicas no histonas que incluyen al RNPn y Scl-70