Assessment of the acid phosphatase CP01850 from Corynebacterium pseudotuberculosis in DNA and subunit vaccine formulations against caseous lymphadenitis / Avaliação da fosfatase ácida recombinante CP01850 de Corynebacterium pseudotuberculosis em formulações vacinais de subunidade e de DNA contra a linfadenite caseosa

The target cp1002_RS01850 from Corynebacterium pseudotuberculosis was used to construct a DNA and recombinant subunit vaccine against caseous lymphadenitis. Recombinant protein rCP01850 was expressed in Escherichia coli using pAE vector, and DNA vaccine was engineered with pTARGET vector. BALB/c mice were divided in five groups containing eight animals each, inoculated with: pTARGET/cp01850 as DNA vaccine (G1); rCP01850 plus Al (OH)3 as recombinant subunit vaccine (G2); pTARGET/cp01850 and a boost with rCP01850 plus Al (OH)3 (G3); pTARGET (G4); or Al (OH)3 (G5). Mice were inoculated and blood samples were collected on days 0, 21, and 42 for the analysis of total IgG, IgG1 and IgG2a by ELISA. In each group, five animals were challenged with Mic-6 C. pseudotuberculosis strain, and three were used for cytokine quantification by qPCR. Although no group has been protected by vaccines against lethal challenge, G2 showed an increase in the survival rate after challenge. Significantly higher levels of IL-4, IL-12, IFN-γ, total IgG, IgG1 and IgG2a were also detected for G2, evidencing a mixed Th1/Th2 immunological profile. In conclusion, despite no protection level provided by different vaccinal strategies using cp1002_RS01850 from C. pseudotuberculosis, G2 developed a Th1/Th2 immune response with an increase in survival rate.(AU)

O alvo cp1002_RS01850 de Corynebacterium pseudotuberculosis foi utilizado para construir uma vacina recombinante de subunidade e de DNA contra a linfadenite caseosa. A proteína recombinante rCP01850 foi expressa em Escherichia coli usando o vetor pAE, e a vacina de DNA foi construída com o vetor pTARGET. Camundongos BALB/c foram divididos em grupos de oito animais, inoculados com: pTARGET/cp01850 como vacina de DNA (G1); rCP01850 e Al (OH)3 como vacina recombinante de subunidade (G2); pTARGET/cp01850 e um boost com rCP01850 e Al (OH)3 (G3); pTARGET (G4); ou Al (OH)3 (G5). Os animais foram inoculados e amostras de sangue foram coletadas nos dias 0, 21, e 42 do experimento para a análise de IgG total, IgG1 e IgG2a por ELISA. De cada grupo, cinco animais foram desafiados com a cepa Mic-6 de C. pseudotuberculosis, e três foram usados para a quantificação de citocinas por qPCR. Apesar de nenhum grupo ter sido protegido pelas vacinas testadas contra o desafio letal, G2 apresentou taxa de sobrevida e níveis de IL-4, IL-12, IFN-γ, IgG total, IgG1 e IgG2a significativamente mais altos, evidenciando um perfil imunológico misto Th1/Th2. Conclui-se que apesar das diferentes estratégias vacinais utilizando cp1002_RS01850 de C. pseudotuberculosis não terem sido capazes de gerar proteção, G2 desenvolveu uma resposta Th1/Th2 e elevou a taxa de sobrevida.(AU)

Desarrollo de una prueba de ELISA para detectar anticuerpos en carneros vacunados o infectados con Corynebacterium pseudotuberculosis / Development of an ELISA test to detect antibodies in vaccinated sheep or infected Corynebacterium pseudotuberculosis

El objetivo de este trabajo fue evaluar un ELISA indirecto desarrollado para medir la respuesta inmune humoral en carneros vacunados contra la linfoadenitis caseosa (LC) y/o desafiados con una cepa de Corynebacterium pseudotuberculosis homóloga. Se distribuyeron corderos de 4 meses clínicamente sanos en 4 grupos: grupo 1, corderos vacunados (G1, n = 5); grupo 2, corderos vacunados e inoculados (G2, n = 8); grupo 3, corderos inoculados (G3, n = 2); y grupo 4, control (G4, n = 2). Los animales del G1 y del G2 recibieron dos dosis de una bacterina experimental; los del G2 y del G3 fueron desafiados con una cepa de C. pseudotuberculosis cuatro semanas posvacunación. Se estudiaron por ELISA los títulos serológicos durante 7 meses y se efectuaron las necropsias en los grupos G2, G3 y G4. Se tomaron muestras de pulmón y linfonódulos para efectuar estudios bacteriológicos e histopatológicos. La cepa inoculada en los animales del G2 y del G3 reprodujo las lesiones macroscópicas y microscópicas típicas de la LC; ésta fue aislada del sitio de inoculación, de linfonódulos o de pulmón en 7/8 animales del G2 y en 2/2 animales del G3. La prueba de ELISA, con una sensibilidad del 98% y una especificidad del 100%, detectó diferencias significativas entre los serorreactores de los diferentes grupos experimentales y permitió establecer una relación con el tipo de tratamiento aplicado. Se concluye que el ELISA desarrollado puede ser una herramienta útil para identificar animales infectados y con clínica positiva a la LC.

The aim of this study was to evaluate an indirect specific ELISA developed for the detection of humoral immune response in vaccinated sheep and/or challenged with a Corynebacterium pseudotuberculosis strain. Healthy 4 month-old lambs were distributed into 4 groups: Group 1 immunized (G1, n = 5), Group 2 vaccinated/inoculated (G2, n = 8), Group 3 inoculated (G3, n = 2) and Group 4 control (G4, n = 2). Groups G1 and G2 received two doses of an experimental bacterin. Four weeks postvaccination, G2 and G3 groups were challenged with a C. pseudotuberculosis strain. Serological titers were studied by ELISA for 7 months and pathological studies were performed in groups G2, G3 and G4 by taking lung and lymph node samples for bacteriology and histopathology. The inoculated strain in G2 and G3 animals reproduced the macroscopic and microscopic lesions typical of caseous lymphadenitis (CL) and was isolated from the inoculation site, lymph nodes and/or lung in 7/8 animals from G2, and 2/2 animals of G3. The developed ELISA test had sensitivity and specificity of 98% and 100% respectively, detected significant differences between serological reactors of different experimental groups and allowed to establish a relationship with the type of treatment. We conclude that the developed ELISA may be a useful tool to identify infected animals with positive clinical CL.

Interleukin-12 and interferon gamma levels in infants with post BCG lymphadenitis

Bacille Calmette Guerin [BCG] is a live bacterial vaccine used in many countries to prevent tuberculosis [TB]. However, because the vaccine consists of live attenuated bacteria, there is still a risk that inoculation with this Mycobacterium bovis strain in immunodeficent infant will cause localized [BCGitis] or disseminated infection, referred to as 'BCG-osis'[1,2] This was prospective hospital based study aiming to determine the serum levels of interleukin-12 and interferon-y among infants with post BCG lymphadenitis. The study included 20 cases [mean +/- SD of age was 4.2 +/- 1.7 months] with newly diagnosed post BCG lymphadenitis group in addition to 20 apparently healthy infants age and sex matched [mean +/- SD of age was 3.9 +/- 1.9 months] as a control group. The study was conducted during the period from March 2008 to November 2010. Both patients and controls were recruited from Paediatric Outpatients Clinics and Paediatric Emergency Department in Assiut University Children Hospital, Egypt. Written informed consents were obtained from the parents of both patients and controls. All cases were subjected to a thorough history, full clinical examinations and investigations which include routine blood tests, immunological studies and serum levels of Interleukin -12 and interferon- gamma. In the post BCG lymphadenitis group, 85% of cases came from rural areas, 45% have positive consanguinity, while 20% of the same group have positive family history of post BCG lymphadenitis. Serum IL-12 and IFN-gamma levels were significantly deceased in cases of post BCG lymphadenitis compared with control group [P< 0.01]. Serum IL-12 and IFN-gamma levels were significantly and positively correlated with age in studied cases. In addition, IL-12 was positively and significantly correlated with IFN-gamma. Serum IL-12 and IFN-gamma levels should be assessed in infants with post BCG lymphadenitis to detect IL-12/ IFN-gamma axis abnormalities. BCG vaccination should be delayed in every newborn and infant with a family history of post BCG lymphadenitis until immunodeficiency diseases and IL-12/IFN-gamma axis abnormalities have been ruled out

Soft tissue abscess and lymphadenitis due to Mycobacterium avium Complex as an expression of immune reconstitution inflammatory syndrome after a second scheme of highly active antiretroviral therapy

Immune reconstitution inflammatory syndrome (IRIS) is an atypical and unexpected reaction related to highly active antiretroviral therapy (HAART) in human immunodeficiency virus (HIV) infected patients. IRIS includes an atypical response to an opportunistic pathogen (generally Mycobacterium tuberculosis, Mycobacterium avium complex, cytomegalovirus and herpes varicella-zoster), in patients responding to HAART with a reduction of plasma viral load and evidence of immune restoration based on increase of CD4+ T-cell count. We reported a case of a patient with AIDS which, after a first failure of HAART, developed a subcutaneous abscess and supraclavicular lymphadenitis as an expression of IRIS due to Mycobacterium avium complex after starting a second scheme of HAART.

El síndrome inflamatorio de reconstitución inmune (SIRI) es una reacción atípica e inesperada relacionada con el tratamiento antirretroviral de gran actividad (TARGA) en pacientes infectados por el virus de la inmunodeficiencia humana (VIH). El SIRI representa una respuesta inflamatoria frente a un patógeno oportunista (generalmente Mycobacterium tuberculosis, Complejo Mycobacterium avium, citomegalovirus y herpes varicela-zóster) en pacientes que responden a la TARGA con una marcada reducción de la carga viral en plasma y evidencia de una recuperación inmunológica expresada por el incremento de los niveles de linfocitos T CD4+. Presentamos el caso de un paciente con síndrome de inmunodeficiencia adquirida que desarrolló un absceso subcutáneo en muslo derecho y una adenitis supraclavicular izquierda como manifestación de SIRI por Complejo Mycobacterium avium luego del inicio de un segundo esquema de TARGA.

Apoptosis y proliferación celular en infecciones por el virus Herpes humano 6 (HHV-6). Mecanismos de regulación a través de interacciones de p53, bcl-2, ras y p21WAF / Apoptosis and celular proliferation in human herpes virus 6 (HHV-6) infection. Mechanism as of regulation by interactions of p53, bcl2, ras and p21WAF

Se analizaron inmunohistológicamente líneas de cultivo celular infectadas con el HHV-6: HSB2-células T inmaduras y HDLM2-células de Hodgkin, así como ganglios linfáticos de pacientes con enfermedad de Hodgkin y linfadenitis de Kikuchi-Fujimoto (LKF) en relación a la expresión de los productos oncógenos/antioncógenos p53, bcl-2, ras y p21WAF. Se comprobó la proliferación celular inmunohistológicamente mediante anticuerpos contra PCNa (antígeno nuclear de proliferación celular) y la apoptosis se investigó en cortes finos de tejido ganglionar, analizando el ADN fragmentado con marcación final in situ. La LKF mostró alta incidencia de focos de células muertas (linfadenitis histiocítica necrozante), mientras que en la enfermedad de Hodgkin se observó proliferación celular. Con las técnicas utilizadas no se logró mostrar diferencias significativas en la expresión de ADN viral no de antígenos en las líneas celulares, ni en las biopsias de enfermedad de Hodgkin y de LKF. Las células HDLM2 con mejor viabilidad posterior a la infección con HHV-6 y un grado de apoptosis inferior, mostraron una expresión de p53 y de PCNA mucho menor que las células HSB2. Las biopsias de LKF no expresaron p53; ras se observó en menores células que en la enfermedad de Hodgkin y la positividad de PCNA fue tres veces mayor en enfermedad de Hodgkin en comparación con LKf. Sin embargo el bcl-2 se observó con mayor frecuencia en LKF que en enfermedad de Hodgkin. Los resultados no son de fácil interpretación; los datos sugieren la implicación de otros factores exógenos (por ejemplo, citoquinas y factores de crecimiento) en el mecanismo regulatorio de la proliferación celular y de la apoptosis, ambas inducidas probablemente por virus

Subacute necrotizing lymphadenitis: a collective clinicopathological and immunohistochemical study

Subacute necrotizing lymphadenitis (SNL) is a well documented and unique clinicopathologic entity, although its etiology and pathogenesis have not been clearly established. Microscopically, cortical and paracortical necrotizing lesions with karyorrhexis, abundant nuclear debris and infiltration of large mononuclear cells are characteristic. This study analyzed the common clinical and pathological features of 118 patients with SNL and the nature of the mononuclear cells. Patients were generally young women and revealed cervical lymphadenopathy with tenderness, fever, leukopenia and elevation of the erythrocyte sedimentation rate. Features of the adjacent uninvolved area in the lymph node included a starry sky pattern, follicle centers, sinus histiocytosis or aggregation of foamy histicoytes. There was an inverse relationship between the extent of necrosis and of histocytic infiltration but not between the extent of necrosis and the duration from the onset of symptoms to the diagnosis. Immunohistochemically the infiltrated mononuclear cells of the affected foci were T lymphocytes and histiocytes. The clinical, histological and immunohistochemical features suggest that SNL represents a hypersensitivity reaction to certain infectious agent without forming granuloma.