RESUMO
OBJECTIVE: Diminish interleukin-1ß (IL-1ß) signaling in a model of primary osteoarthritis by RNA interference-based transcript reduction or receptor blockade, and quantify changes incurred on transcript expression of additional mediators. METHODS: Knees of Hartley guinea pigs were collected at 120 and 180 days of age following injection with viral vectors (N = 4/treatment group/date) at 60 days. Two groups received either adeno-associated viral serotype 5 vector containing a knockdown sequence (TV), or adenoviral vector encoding for IL-1 receptor antagonist protein (Ad-IRAP); treatments were contrasted with opposite knees administered corresponding vector controls. A third group evaluated TV relative to saline-only injected knees. Chondropathy and immunohistochemistry findings were compared to untreated guinea pigs. Transcript expression levels in cartilage were calculated using the comparative CT (2(-ΔΔCT)) method and analyzed by one-way analysis of variance (ANOVA) with pairwise comparisons using Tukey 95% confidence intervals. RESULTS: Vector transduction was confirmed at both harvest dates. TV and Ad-IRAP, relative to vector controls, significantly decreased IL-1ß. Inflammatory mediators [tumor necrosis factor-α (TNF-α), IL-8, interferon-γ (IFN-γ)], and catabolic matrix metalloproteinase 13 (MMP13) were also decreased, while anabolic transforming growth factor-ß1 (TGF-ß1) was increased. IL-1ß was also decreased by TV vs saline, with a decrease in MMP13 and increase TGF-ß1; TNF-α, IL-8, and IFN-γ were transiently increased. CONCLUSIONS: This work confirmed that a reduction in IL-1ß signaling was accomplished by either method, resulting in decreased expression of three inflammatory mediators and one catabolic agent, and increased expression of an anabolic molecule. Thus, evidence is provided that IL-1ß serves a role in vivo in spontaneous osteoarthritis and that these translational tools may provide beneficial disease modification.
Assuntos
Artrite Experimental/metabolismo , Cartilagem Articular/metabolismo , Regulação da Expressão Gênica , Mediadores da Inflamação/metabolismo , Interleucina-1beta/antagonistas & inibidores , Osteoartrite do Joelho/metabolismo , RNA Interferente Pequeno/genética , Animais , Artrite Experimental/patologia , Cartilagem Articular/patologia , Condrócitos/metabolismo , Condrócitos/patologia , Cobaias , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Masculino , Osteoartrite do Joelho/patologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Oxidative stress reportedly plays a role in sepsis-induced organ dysfunction and failure in many species. In septic horses, laminae are targeted; evidence of laminar oxidative stress has been reported experimentally in the black walnut extract (BWE) model. Carbohydrate (CHO)-induced laminitis may be more similar to clinical sepsis-related laminitis than the BWE model in that animals with CHO-induced disease commonly develop laminar failure. The role of oxidative stress in the CHO model remains unknown. HYPOTHESIS/OBJECTIVES: Markers of oxidative stress will be increased in laminae from horses with BWE- and CHO-induced laminitis. ANIMALS: Banked laminar tissue from various time points from animals subjected to BWE (n = 15) and CHO (n = 20) protocols. METHODS: Laminar 4-hydroxynonenal (4-HNE) and protein carbonyl content were evaluated by slot blot analysis. Laminar 3-nitrotyrosine (3-NT) immunohistochemistry was performed. RESULTS: The number of laminar 3-NT (+) cells was increased at developmental and Obel grade 1 (OG1) time points in the BWE model (versus control [CON]; P= .013) and lower in OG1 tissues than CON in the CHO model (P = .04). No change in 4-HNE content was observed in the CHO model, and no increase in laminar protein carbonyl content was present in either model (P > .05). CONCLUSIONS AND CLINICAL IMPORTANCE: These results do not support a prominent role for oxidative stress at examined time points in CHO-overload laminitis and support transient oxidative stress in the BWE model. Tissue oxidation does not appear to be a central early pathophysiologic event in CHO-associated laminitis.
Assuntos
Doenças do Pé/veterinária , Casco e Garras/efeitos dos fármacos , Doenças dos Cavalos/metabolismo , Inflamação/veterinária , Estresse Oxidativo/fisiologia , Aldeídos/análise , Aldeídos/metabolismo , Animais , Biomarcadores , Modelos Animais de Doenças , Doenças do Pé/induzido quimicamente , Doenças do Pé/metabolismo , Doenças do Pé/fisiopatologia , Doenças dos Cavalos/induzido quimicamente , Doenças dos Cavalos/fisiopatologia , Cavalos , Inflamação/induzido quimicamente , Inflamação/fisiopatologia , Juglans/química , Peroxidação de Lipídeos , Extratos Vegetais/química , Extratos Vegetais/toxicidade , Proteínas/metabolismo , Espécies Reativas de Nitrogênio , Espécies Reativas de Oxigênio , Amido/administração & dosagem , Amido/toxicidade , Tirosina/metabolismoRESUMO
OBJECTIVE: To provide a comprehensive immunohistochemical (IHC) map of the temporal expression and tissue distribution of interleukin-1ß (IL-1ß) through progression of osteoarthritis (OA) in two strains of guinea pigs with varying propensity for spontaneous knee joint disease. METHODS: OA-prone Hartley and OA-resistant Strain 13 guinea pigs were collected at 60, 120, 180, 240, 360, and 480 days of age (N=4 animals per strain per date). IHC was performed on whole joint preparations; the distribution of IL-1ß expression on coronal sections was mapped, semi-quantitatively scored, and correlated to OA grade using Mankin criteria with guinea pig-specific modifications. OA and IHC indices were compared among times and between strains using the Kruskal-Wallis one-way analysis of variance by ranks followed by Dunn's post test. RESULTS: OA indices for both strains increased from 60 to 480 days of age; a statistically higher score (P ≤ 0.01) was found in Hartley animals at 180, 240, 360, and 480 days. At 60 days of age, IL-1ß expression was detected in cartilage, menisci, synovium, and subchondral bone in both strains. Persistent and statistically increased (P<0.05) IL-1ß expression was found in these same tissues in Hartley animals at 120 and 180 days, while Strain 13 animals demonstrated a significant reduction in positive immunostaining. Statistical differences in IHC indices between strains beyond 240 days of age were restricted to synovium (days 240 and 480) and subchondral bone (days 360 and 480). CONCLUSIONS: As expected, histologic OA proceeded in an accelerated manner in Hartley animals relative to Strain 13 animals. The OA-prone strain did not demonstrate reduced IL-1ß expression during adult maturity as occurred in the OA-resistant strain, and this persistent expression may have corresponded to early incidence of OA. Future interventional studies are warranted to explore whether dysregulation of IL-1ß expression may contribute to premature onset of spontaneous disease in the Hartley guinea pig.
Assuntos
Interleucina-1beta/metabolismo , Articulação do Joelho/metabolismo , Osteoartrite do Joelho/metabolismo , Animais , Cartilagem Articular/metabolismo , Cobaias , Imuno-Histoquímica , Articulação do Joelho/patologia , Osteoartrite do Joelho/patologia , Membrana Sinovial/metabolismoRESUMO
BACKGROUND: While there is evidence of laminar leukocyte infiltration in black walnut extract (BWE)-induced laminitis, there is no such evidence for carbohydrate overload (CHO) laminitis. OBJECTIVE: To assess presence of leukocytes and signs of epidermal stress/injury in the laminar tissue from horses with CHO-induced laminitis. ANIMALS: Twenty-four adult horses. METHODS: Immunohistochemistry for myeloid cell markers calprotectin (CP) and monocyte-specific marker (CD163) was performed on laminar sections obtained from 2 groups of horses in the CHO model: the developmental time point (DTP) group (n = 6) and the onset of lameness (LAM) group (n = 6), and a control (CON) group (n = 8). RESULTS: DTP was characterized by an increase in CP(+) leukocytes (7.8-fold increase versus CON, P < .001), and LAM time point was characterized by a more marked increase in laminar CP(+) (108.5-fold, P < .001) and mild increase in CD163(+) (1.9-fold, P = .007) cell counts. Increased CP epidermal signal (indicating epidermal stress or injury) occurred consistently at the LAM time point, although histological evidence of basement membrane (BM) detachment was minor, only being present in 3/6 horses. CONCLUSIONS AND CLINICAL RELEVANCE: Maximal laminar leukocyte infiltration and epithelial stress occurred at the onset of lameness in the CHO model showing a different temporal pattern from the BWE model, where maximal leukocyte infiltration clearly precedes epithelial stress. Leukocyte infiltration before major histological changes in the CHO model indicates that leukocyte infiltration can be a cause of and not a reaction to BM degradation and structural failure.
Assuntos
Doenças do Pé/veterinária , Doenças dos Cavalos/imunologia , Leucócitos/imunologia , Animais , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Membrana Basal/citologia , Membrana Basal/imunologia , Modelos Animais de Doenças , Doenças do Pé/imunologia , Doenças do Pé/patologia , Doenças dos Cavalos/patologia , Cavalos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica/veterinária , Coxeadura Animal/imunologia , Coxeadura Animal/patologia , Complexo Antígeno L1 Leucocitário/imunologia , Leucócitos/citologia , Distribuição Aleatória , Receptores de Superfície Celular/imunologia , Estatísticas não ParamétricasRESUMO
REASONS FOR PERFORMING STUDY: There is increasing evidence of involvement of inflammatory cells in acute laminitis. OBJECTIVE: To immunolocalise monocytes/macrophages and B and T lymphocytes in the laminar tissue of normal horses and those with black walnut extract (BWE)-induced laminitis. METHODS: Immunohistochemistry was used in archived laminar tissue samples from 20 horses divided equally into 4 groups: control animals (CON), and those administered BWE at 1.5 h (1.5H DTP group), at the onset of leucopenia (3H DTP group) and at the onset of lameness (LAM group). Antibodies against CD3, CD20 and CD163 were used to recognise lymphocytes (T and B) and monocytes/macrophages, respectively. RESULTS: Mononuclear cells were present in laminar tissue of normal horses. The majority of CD3- and CD20-positive lymphocytes were localised around the deep dermal vessels but were also evident around vessels of the primary dermal laminae. CD163-positive macrophages were primarily perivascular in deep dermis or in dermal laminae. No changes in the number of laminar B or T lymphocytes occurred at any time point post BWE administration. However, increases (P=0.0016) in laminar CD163-positive cells occurred in the secondary dermal laminae (SDL) in the 1.5H DTP and 3H DTP groups, returning to basal values in LAM group. CONCLUSIONS: Lymphocyte and macrophage populations are present in the laminar tissue of clinically normal horses and BWE administration induces an increase in CD163-positive macrophages in SDL. POTENTIAL RELEVANCE: Both the host tissue population of mononuclear cells and the influx of monocytes may play an important role in the pathophysiological changes leading to laminar injury.
Assuntos
Doenças do Pé/veterinária , Casco e Garras , Doenças dos Cavalos/patologia , Inflamação/veterinária , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Extratos Vegetais/toxicidade , Animais , Antígenos/metabolismo , Doenças do Pé/patologia , Casco e Garras/patologia , Cavalos , Inflamação/patologia , Juglans/toxicidade , Leucócitos Mononucleares/classificaçãoRESUMO
BACKGROUND: C-X-C motif ligand 1 (CXCL1) is an important chemokine of epithelial origin in rodents and humans. OBJECTIVES: To assess in vivo and in vitro the regulation of CXCL1 in equine laminitis. ANIMALS: Twenty adult horses. METHODS: Real-time quantitative polymerase chain reaction (PCR) was used to assess expression of CXCL1 in samples of laminae, liver, skin, and lung from the black walnut extract (BWE) model of laminitis, and in cultured equine epithelial cells (EpCs). Tissue was obtained from control animals (CON, n = 5), and at 1.5 hours (early time point [ETP] group, n = 5), at the onset of leukopenia (developmental time point [DTP] group, n = 5), and at the onset of lameness (LAM group, n = 5) after BWE administration. EpCs were exposed to Toll-like/Nod receptor ligands, oxidative stress agents, and reduced atmospheric oxygen (3%). In situ PCR was used to localize the laminar cell types undergoing CXCL1 mRNA expression. RESULTS: Increases in laminar CXCL1 mRNA concentrations occurred in the ETP (163-fold [P= .0001]) and DTP groups (21-fold [P= .005]). Smaller increases in CXCL1 expression occurred in other tissues and organs. In cultured EpCs, increases (P < .05) in CXCL1 mRNA concentration occurred after exposure to lipopolysaccharide (LPS [28-fold]), xanthine/xanthine oxidase (3.5-fold), and H(2)O(2) (2-fold). Hypoxia enhanced the LPS-induced increase in CXCL1 mRNA (P= .007). CXCL1 gene expression was localized to laminar EpCs, endothelial cells, and emigrating leukocytes. CONCLUSION AND CLINICAL IMPORTANCE: These findings indicate that CXCL1 plays an early and possibly initiating role in neutrophil accumulation in the BWE laminitis model, and that laminar keratinocytes are an important source of this chemokine. New therapies using chemokine receptor antagonists may be indicated.
Assuntos
Quimiocina CXCL1/imunologia , Doenças do Pé/veterinária , Doenças dos Cavalos/imunologia , Coxeadura Animal/imunologia , Animais , Hipóxia Celular/imunologia , Quimiocina CXCL1/biossíntese , Quimiocina CXCL1/genética , Doenças do Pé/genética , Doenças do Pé/imunologia , Doenças dos Cavalos/genética , Cavalos , Coxeadura Animal/genética , Estresse Oxidativo/imunologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Receptores Toll-Like/imunologiaRESUMO
BACKGROUND: Laminar inflammation is one of the earliest events in equine laminitis. Calprotectin (CP), a Damage-Associated Molecular Pattern protein, is overexpressed in inflammatory conditions of human skin. HYPOTHESIS: CP is overexpressed in the laminar epidermis of horses with black walnut extract (BWE)-induced laminitis. ANIMALS: Twenty adult horses. METHODS: Experimental study. Horses were allocated to one of 4 groups. BWE was administered to horses in 3 groups, which were sampled 1.5, 3, and 12 hours (LAM) later. CP was visualized by immunohistochemistry. Laminar leukocyte counts and intensity of laminar epithelial staining were scored for all animals and statistically analyzed. RESULTS: Laminar epidermal CP signal was significantly increased (P= .02) at the LAM time point, compared with other groups. Rare leukocytes were detected in laminae with CP staining in CON group, but there were marked increases in number of leukocytes in BWE-treated groups (P= .003). Sequential hematoxylin and eosin staining demonstrated that the majority of CP-positive leukocytes were perivascular polymorphonuclear neutrophils (PMN) at each of the developmental time points. CP-positive PMN and mononuclear cells were detected in perivascular locations and close to the epidermal basement membrane in the LAM group. CONCLUSIONS AND CLINICAL IMPORTANCE: CP expression in the laminar epidermis occurs after extravasation of leukocytes, indicating that leukocyte emigration might be an initiating factor in laminar epithelial stress and inflammation in BWE-induced laminitis. These results indicate a possible role of CP in laminitis pathophysiology and laminar failure.
Assuntos
Células Epiteliais/metabolismo , Doenças do Pé/veterinária , Doenças dos Cavalos/induzido quimicamente , Complexo Antígeno L1 Leucocitário/metabolismo , Células Mieloides/metabolismo , Animais , Doenças do Pé/induzido quimicamente , Doenças dos Cavalos/patologia , Cavalos , Juglans/química , Extratos Vegetais/toxicidade , Transporte ProteicoRESUMO
BACKGROUND: The immune response to Mycobacterium tuberculosis is complex and multifactorial, the cytokine system being a major factor in M. tuberculosis immunity. AIM: To analyze the immunohistochemical aspects of tuberculous lymph nodes in immunocompetent patients and search for associations between SOCS and cytokine expression in human tuberculous lymphadenitis. METHODS: Thirteen lymph nodes were assayed by immunohistochemistry for SOCS-1 and 3, STAT-3, RANTES, MIP-1-alpha, ICAM-1, IFN-gamma as well as CD45RO, CD20, CD34, CD68, trypsin and lysozyme. Additionally, the RT in situ PCR was performed for SOCS-1 and 3 mRNA detection. RESULTS: Decreased MIP-1 alpha expression together with reduced SOCS-3 (p=0.042), lysozyme (p=0.024) and CD45RO (p=0.05) was observed in the TB lymph nodes compared to the control lymph nodes. In conclusion, the lymphadenitis due to M. tuberculosis was associated with a downregulation of memory T cells (CD45RO), activated lysozymes and SOCS-3 compared to controls, which may play a role in the long-term bacterial replication and altered immune modulation characteristic of the disease.
Assuntos
Citocinas/biossíntese , Doenças Endêmicas , Linfonodos/metabolismo , Proteínas Supressoras da Sinalização de Citocina/biossíntese , Tuberculose dos Linfonodos/metabolismo , Adolescente , Adulto , Idoso , Antígenos CD/metabolismo , Citocinas/imunologia , Humanos , Imuno-Histoquímica , Linfonodos/imunologia , Linfonodos/patologia , Pessoa de Meia-Idade , Muramidase/metabolismo , Mycobacterium tuberculosis , RNA Mensageiro/imunologia , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/imunologia , Tripsina/metabolismo , Tuberculose dos Linfonodos/imunologia , Tuberculose dos Linfonodos/patologiaRESUMO
AIMS: Immune factors influencing the progression of cervical intraepithelial neoplasia (CIN) to cancer remain poorly defined. This study investigates the expression of RANTES, MIP1alpha, COX1, COX2, STAT3, TGFbetaRI, IL10R, TNFalphaRII and TLR4 in the cervical immune response in HIV/HPV (human papillomavirus) co-infected women. METHODS: Cervical biopsies of 36 patients were assayed by immunohistochemistry, and the Ventana Benchmark System was used for HIV-nef detection. RESULTS: Cervices from HIV-positive patients exhibited nef in cells mainly around blood vessels, and showed a decreased expression of all the immune factors tested except IL10R and STAT3, while RANTES (5.54 cells/mm(2)) was highly expressed in comparison with controls (1.41 cells/mm(2), p = 0.028). COX1 was decreased in the HIV/HPV- (0.32 cells/mm(2), p = 0.017) and HPV-infected patients (0.21 cells/mm(2), p = 0.015) compared with controls (3.28 cells/mm(2)). CONCLUSIONS: It is suggested that RANTES in HIV/HPV co-infection may influence the development of CIN leading to progression to cervical cancer.
Assuntos
Infecções por HIV/imunologia , HIV-1 , Infecções por Papillomavirus/imunologia , Displasia do Colo do Útero/imunologia , Neoplasias do Colo do Útero/imunologia , Adolescente , Adulto , Terapia Antirretroviral de Alta Atividade , Quimiocina CCL5/metabolismo , Ciclo-Oxigenase 1/metabolismo , Progressão da Doença , Feminino , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Humanos , Técnicas Imunoenzimáticas , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Infecções por Papillomavirus/complicações , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia , Displasia do Colo do Útero/patologia , Displasia do Colo do Útero/virologiaRESUMO
The purpose of this study was to look for associations between a newly described class of suppressors of cytokine signaling (SSI/SOCS) and cytokine expression in the uterine cervix from HIV/HPV coinfected women. We examined the pro-inflammatory cytokines TNF-alpha and IL-6 since their expressions are linked and responsible for many aspects of both localized and systemic inflammatory responses. Further, expression of SSI/SOCS has been implicated in the negative feedback regulation of cytokine receptor signaling. PCR-amplified HIV-1 cDNA was noted mainly in the stroma, showing a perivascular distribution, and most of the infected cells colabeled with the macrophage marker CD68. The distribution of IL-6 and TNF-alpha was in the same area to HIV-1 and much greater than normal cervices from women with no evidence of viral infection. SOCS/SSI-1 and -3 mRNA positive cells in the uterine cervix were commonly detected in these noninfected cervical tissues; however, very few cells that contained SOCS were evident in areas where HIV-1, TNF-alpha, and IL-6 expressing cells were found. This suggests that viral-related suppression of SOCS/SSI-1-3 expression may be a factor in the marked local enhancement of TNF-alpha and IL-6 production which, in turn, may help facilitate viral spread; however, further studies should be done in order to elucidate the exact mechanisms of SOCS in the cervix.
Assuntos
Colo do Útero/metabolismo , Infecções por HIV/metabolismo , Interleucina-6/metabolismo , Papillomaviridae , Infecções por Papillomavirus/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Antígeno B7-2/genética , Antígeno B7-2/metabolismo , Colo do Útero/imunologia , Colo do Útero/patologia , Citocinas/genética , Citocinas/metabolismo , DNA Viral/análise , Feminino , Infecções por HIV/complicações , Infecções por HIV/imunologia , HIV-1/isolamento & purificação , Humanos , Interleucina-6/genética , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/imunologia , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Proteínas Supressoras da Sinalização de Citocina/genética , Fator de Necrose Tumoral alfa/genéticaAssuntos
Hibridização In Situ/métodos , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Reação em Cadeia da Polimerase/métodos , Infecções Tumorais por Vírus/diagnóstico , DNA Complementar/isolamento & purificação , DNA Viral/isolamento & purificação , Humanos , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Infecções Tumorais por Vírus/virologiaRESUMO
OBJECTIVE: To determine the clinical significance in normal Pap smears of HPV detection as determined by Hybrid Capture (HC) and in situ hybridization analyses. STUDY DESIGN: We studied 135 consecutive Pap smears as well as 46 other smears from high-risk patients each initially diagnosed as within normal limits. RESULTS: The 135 "normal" Pap smears were rescreened, and 6 (4%) where found to be either ASCUS or SIL. In the remaining 129 cases, HPV DNA was detected in 0% and 9%, respectively, using in situ hybridization and HC I. Upon rescreening the high-risk patients, nine (20%) were reclassified as having SIL/ASCUS; each was in situ hybridization positive, and eight were HC positive; six (67%) of these women developed SIL on follow-up. In the 37 Pap smears in high-risk women still within normal limits after manual rescreening, HPV was detected in 2% by in situ hybridization and 46% by HC; 6% of the HC-positive women developed SIL on follow-up. CONCLUSION: In situ hybridization rarely detects HPV in Pap smears diagnosed as within normal limits after manual rescreening. In situ hybridization is very effective in detecting rare, atypical cells in Pap smears diagnosed as within normal limits and, in a high-risk population, is predictive of SIL on clinical follow-up.
Assuntos
DNA Viral/análise , Teste de Papanicolaou , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Infecções Tumorais por Vírus/diagnóstico , Esfregaço Vaginal , Adolescente , Adulto , Idoso , Feminino , Humanos , Hibridização In Situ/métodos , Pessoa de Meia-Idade , Papillomaviridae/genética , Controle de Qualidade , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Displasia do Colo do Útero/diagnóstico , Displasia do Colo do Útero/virologia , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/virologiaRESUMO
BACKGROUND: The authors noticed a significant increase in the rate of atypical squamous cells of undetermined significance (ASCUS) diagnoses in postmenopausal women receiving hormone replacement therapy with ThinPrep (monolayer) Papanicolaou (Pap) tests (8.3%) as compared with conventional Pap tests (2.3%; P = 0.025). The purpose of this study was to correlate these morphologic changes with human papillomavirus (HPV) DNA detection by in situ hybridization and clinical outcome. METHODS: Twenty-five monolayer Pap tests diagnosed as ASCUS in postmenopausal women receiving hormone replacement, as well as 2 cases of low-grade squamous intraepithelial lesion (SIL) from the same group, were destained in acid alcohol. In situ HPV hybridization was performed with a consensus probe that can detect greater than 90% of the HPV types that occur in cervical SIL. In addition, 11 of the ASCUS tests were analyzed with the periodic acid-Schiff (PAS) stain. RESULTS: All 25 ASCUS tests were negative for HPV DNA as compared with a detection rate of 15 of 17(88%) for low-grade SIL. Glycogen that was PAS positive was present in the atypical squamous cells of the postmenopausal women for whom HPV was not detected. Clinical follow-up demonstrated that only 2 of the 25 women with ASCUS had a subsequent biopsy proven SIL. CONCLUSIONS: The ThinPrep preparation can produce artifactual HPV-like changes in postmenopausal women receiving hormone replacement therapy that may be caused by glycogenization and, because of the high incidence of HPV negativity and negative clinical outcome, are best considered as benign cellular changes.
Assuntos
Carcinoma de Células Escamosas/virologia , Terapia de Reposição Hormonal , Teste de Papanicolaou , Infecções por Papillomavirus/diagnóstico , Infecções Tumorais por Vírus/diagnóstico , Neoplasias do Colo do Útero/virologia , Esfregaço Vaginal , Idoso , Artefatos , Carcinoma de Células Escamosas/patologia , DNA Viral/análise , Reações Falso-Positivas , Feminino , Humanos , Hibridização In Situ , Incidência , Pessoa de Meia-Idade , Papillomaviridae/genética , Papillomaviridae/patogenicidade , Pós-Menopausa , Neoplasias do Colo do Útero/patologiaRESUMO
The purpose of this study is to report an unusual variant of cervical squamous cell carcinoma, not associated with either human papillomavirus infection or antecedent squamous intraepithelial lesions. Five women had a diagnosis of invasive cervical cancer discovered at hysterectomy performed for prolapse (two cases), leiomyoma (one case), or a vaginal fistula (two cases). The women ranged in age from 47 to 78 years (mean 59 years). Four of the five had a history of normal Papanicolaou (Pap) smears; the other had a Pap smear diagnosis of atypical squamous cells of undetermined significance (ASCUS). All had large cervical tumors (two with parametrial involvement and one with vaginal involvement) that showed extensive keratin formation, an inverted pattern of growth, and, except for one case, minimal cytologic atypia. There was extensive hyperkeratosis and parakeratosis adjacent to each tumor; none had evidence of squamous intraepithelial lesion. Human papillomavirus testing by polymerase chain reaction in situ hybridization and reverse-transcribed polymerase chain reaction in situ was negative in each case, compared with a detection rate of 107 of 108 (99%) for squamous intraepithelial lesion-associated cervical squamous cell and adenocarcinomas. Two of the women died of extensive local recurrence; two other women were recently diagnosed. We conclude that highly differentiated keratinizing squamous cell carcinoma of the cervix is a rare entity not associated with human papillomavirus infection or squamous intraepithelial lesion and thus difficult to detect on routine cervical cancer screening.
Assuntos
Carcinoma de Células Escamosas/patologia , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/patologia , Infecções Tumorais por Vírus/patologia , Displasia do Colo do Útero/patologia , Neoplasias do Colo do Útero/patologia , Adulto , Idoso , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/virologia , DNA de Neoplasias/análise , DNA Viral/análise , Feminino , Humanos , Hibridização In Situ , Queratinas , Pessoa de Meia-Idade , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/virologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Displasia do Colo do Útero/genética , Displasia do Colo do Útero/virologiaRESUMO
OBJECTIVE: To investigate longterm responsiveness to interferon-alpha (lFN-alpha) of patients with extrahepatic manifestations of hepatitis C virus (HCV) in a nonendemic area. METHODS: We prospectively evaluated 11 patients with extrahepatic manifestations of HCV infection, including 10 with Type II cryoglobulins, treated with IFN-alpha--9 had cutaneous vasculitis, 6 arthralgias, 7 neuropathy, and 4 glomerulonephritis. Liver biopsies were performed on all patients, although 6/11 had normal liver function tests. All received 3 M units IFN-alpha tiw, with total length of treatment ranging from 3 mo to 5 yrs. Periodic assessments were made of clinical activity, biochemical variables, cryoglobulin quantitation, and HCV copy number. RESULTS: Three patients were withdrawn because of toxicity. Three were nonresponders at 6, 16, and 17 mo of therapy, based on persistence of HCV RNA in blood, cryoprecipitates, and peripheral blood mononuclear cells. One patient was a partial responder at 3 yrs, with 2 major flares of cutaneous vasculitis occurring on separate attempts to withdraw IFN-alpha. Three patients (27.2%) were complete responders based on resolution of symptoms (purpura, neuropathy) and disappearance of cryoprecipitates and HCV RNA, but only one successfully tapered IFN-alpha after 3 yrs of treatment, with sustained resolution at followup 15 mo later. CONCLUSION: IFN-alpha is safely tolerated for prolonged periods in patients with extrahepatic HCV infection, and is particularly effective for treatment of cutaneous vasculitis. Careful monitoring is needed for evolution of liver pathology to cirrhosis, or for progression of renal or neurologic disease.
Assuntos
Antivirais/uso terapêutico , Hepatite C Crônica/complicações , Hepatite C Crônica/tratamento farmacológico , Interferon-alfa/uso terapêutico , Adulto , Idoso , Artralgia/tratamento farmacológico , Artralgia/etiologia , Crioglobulinemia/tratamento farmacológico , Crioglobulinemia/etiologia , Feminino , Dosagem de Genes , Genes Virais , Glomerulonefrite/tratamento farmacológico , Glomerulonefrite/etiologia , Hepacivirus/classificação , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Hepatite C Crônica/sangue , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Doenças do Sistema Nervoso/tratamento farmacológico , Doenças do Sistema Nervoso/etiologia , Estudos Prospectivos , RNA Viral/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Resultado do Tratamento , Vasculite/tratamento farmacológico , Vasculite/etiologia , Viremia/tratamento farmacológicoRESUMO
Hypermethylation of the MLH1 promoter underlies most sporadic colorectal cancers with microsatellite instability (MSI). To investigate the role of hypermethylation in the normal colonic mucosa as a possible precursor lesion, we studied 700 bp upstream of MLH1 covering 51 CpG sites. We found partially methylated alleles in 15 of 34 (44%) patients <60 years of age and 20 of 24 (83%) patients > or =80 years of age (P = 0.0026). Fully methylated alleles were present in 18 of 33 (55%) patients with MSI+ tumors but in only 18 of 90 (20%) patients with MSI- tumors (P = 0.00019). By in situ analysis, methylation was patchy and located mainly in the cryptal regions close to the lumen. We conclude that the spread of methylation in the MLH1 promoter in the normal colonic mucosa is closely associated with age and the development of sporadic MSI+ colorectal cancers.
Assuntos
Colo/fisiologia , Neoplasias Colorretais/genética , Metilação de DNA , Mucosa Intestinal/fisiologia , Repetições de Microssatélites/genética , Proteínas de Neoplasias/genética , Lesões Pré-Cancerosas/genética , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Proteínas de Transporte , Neoplasias Colorretais Hereditárias sem Polipose/genética , Humanos , Hibridização In Situ/métodos , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Proteínas Nucleares , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas , Análise de Sequência de DNARESUMO
In situ amplification permits the histological localization of low-copy DNA and RNA targets. However, in many instances it would be useful to know the specific phenotype of the target-containing cell or to ascertain the distribution of a different nucleic acid sequence in the same tissue section. This review describes a methodology that allows co-in situ localization of two nucleic acid targets or a DNA/RNA sequence and a protein in paraffin-embedded, formalin-fixed tissue. The key variable for detection of low-copy RNA targets by RT in situ PCR is optimal protease digestion to permit cDNA target-specific incorporation of the reporter nucleotide. This is achieved via inactivation of nonspecific DNA synthesis by overnight DNase digestion. The key variable for immunohistochemical localization of proteins is to determine the effect of protease digestion on the antigen-based signal intensity. Background for DNA targets by in situ hybridization or, for targets present in 1-10 copies per cell, PCR ISH is dependent primarily on probe concentration and the stringency of the post-hybridization wash. Radioactive 3H-labeled nucleotides permit an excellent distinction with colorimetric signals for co-localization, although two distinct chromogens can in many instances allow successful localization of two different targets.
Assuntos
Reação em Cadeia da Polimerase/métodos , DNA/análise , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/genética , Humanos , Imuno-Histoquímica , Hibridização In Situ , Proteínas/análise , RNA/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The authors describe a reverse transcriptase in situ polymerase chain reaction (RT in situ PCR) method that allows the determination of B-cell clonality as defined by expression of kappa and lambda mRNA as well as the different VH families that comprise the entire heavy chain (IgH) sequence using paraffin-embedded, formalin-fixed tissue. Polyclonal expression of B-cell light and heavy chains was documented in two reactive lymph nodes and a spleen in the same histologic distribution as the B-cell marker CD20. Monoclonal expression of kappa versus lambda mRNA was documented in 11 cases of B-cell lymphoma and was corroborated in 5 cases by flow cytometry; each case showed monoclonal expression of IgH. The authors analyzed seven additional tissues where a definitive diagnosis of B-cell lymphoma could not be rendered based on histologic, immunohistologic. and clinical analysis. RT in situ PCR for IgH and kappa versus lambda expression differentiated the B-cell lymphomas (n = 2) from reactive B-cell processes (n = 3) and from two cases of B-cell-rich T-cell lymphoma. The described RT in situ PCR methodology allows routine determination of the monoclonal versus multiclonal expression patterns of B cells, which will facilitate the diagnosis of B-cell lymphomas and aid in understanding the pathogenesis of B-cell malignancies using archival, paraffin-embedded tissues.
Assuntos
Rearranjo Gênico de Cadeia Pesada de Linfócito B/genética , Rearranjo Gênico de Cadeia Leve de Linfócito B/genética , Linfoma de Células B/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Antígenos CD20/análise , Linfócitos B/patologia , Células Clonais , Primers do DNA/química , Diagnóstico Diferencial , Feminino , Humanos , Imuno-Histoquímica , Linfonodos/metabolismo , Linfonodos/patologia , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Linfoma de Zona Marginal Tipo Células B/genética , Linfoma de Zona Marginal Tipo Células B/patologia , Linfoma de Células T/genética , Linfoma de Células T/patologia , RNA Neoplásico/análise , Baço/metabolismo , Baço/patologiaRESUMO
Rotaviral infection is the most common cause of gastroenteritis in young children. Although rotavirus infection has a high morbidity and mortality rate in animals, in most cases in the United States the disease appears to be self-limited in humans. We report on 2 cases of fatal rotaviral infection in a 1 year old and a 4 year old. In each case, the illness showed a rapid systemic course dominated by cardiac and central nervous system involvement; in one case, rotaviral infection was documented by stool culture. Viral RNA was localized by reverse transcriptase in situ polymerase chain reaction to the mucosal cells of the small and large intestine and to many other tissues, including the heart and central nervous system, where it was noted in the endothelial cells of the microvasculature. Immunohistochemical analysis for the virus showed an identical histologic distribution in the intestinal epithelial cells and the systemic microvasculature. It is concluded that rotaviral infection can lead to a fatal disseminated infection in humans and the mechanism of this complication is based on a diffuse endothelialitis and concomitant tissue damage.
Assuntos
Imuno-Histoquímica/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções por Rotavirus/patologia , Rotavirus/patogenicidade , Pré-Escolar , Primers do DNA/química , Endotélio Vascular/patologia , Endotélio Vascular/virologia , Evolução Fatal , Feminino , Humanos , Lactente , Masculino , RNA Viral/análise , Rotavirus/isolamento & purificação , Infecções por Rotavirus/fisiopatologiaRESUMO
A definitive diagnosis of T-cell lymphoma may be contingent on the rearrangement profile of the T-cell receptor. This is most accurately done by molecular analysis of the beta-chain of the T-cell receptor (TCR beta) by Southern blotting hybridization that requires unfixed tissue. We describe a reverse transcriptase in situ PCR (RT in situ PCR) method that permits the target-specific direct incorporation of the reporter nucleotide into the different transcripts that comprise the TCR beta, using paraffin-embedded, formalin-fixed tissue. Each of the 25 possible V beta segment rearrangements was documented in three lymph nodes with nonspecific lymphadenitis, with clonal expansion evident in a case of metastatic melanoma. Monoclonal expression was documented in seven tissues diagnostic of a T-cell lymphoma. We analyzed five additional tissues for which a definitive diagnosis of T-cell vs B-cell lymphoma could not be rendered on the basis of histological, immunohistological, and flow cytometric analysis. RT in situ PCR for TCR beta expression with CD3 co-labeling demonstrated which of these lesions was a B-cell-rich T-cell lymphoma. We conclude that the RT in situ PCR methodology will allow the routine determination of monoclonal vs multiclonal expression patterns of the TCR beta using archival paraffin-embedded tissues.(J Histochem Cytochem 49:139-145, 2001)