Analysis of neutrophil-lymphocyte ratio as a prognostic element in the response to neoadjuvant therapy in rectal cancer / Análise da relação neutrófilo-linfócito como elemento prognóstico na resposta à terapia neo-adjuvante no câncer do reto

INTRODUCTION: The previous radio-chemotherapy approach is highly relevant in the management of rectal cancer, collaborating on organ functional preservation, being performed prior to surgery. The inflammatory response plays an important role in this treatment. OBJECTIVE: It consists in correlating the number of peripheral lymphocytes and the neutrophil/lymphocyte ratio in the peripheral blood with tumor response to neoadjuvant therapy. METHODS: Review of medical records of patients with rectal cancer in HMSJ and HSJ Oncology Services since 2009 - cases submitted to neoadjuvant treatment with radio-chemotherapy. RESULTS: Of those 96 patients with this disease who underwent neoadjuvant therapy with radio-chemotherapy, 35 patients were eligible; complete tumor response was observed in 11 cases (31%), and 9 were submitted to surgical treatment. Comparing the leukocyte parameters between patients with complete response (CR) and incomplete response (IR) the following values were observed: total number of leukocytes (mean) CR 7390.9 × IR 7220.4 (p = 0.8); total lymphocytes CR 2103 × IR 1960.9 (p = 0.4); neutrophil/lymphocyte ratio CR 3.55 × IR 3.79 (p = 0.5). The mean radiotherapy dose was 49.1 Gy, with CR 47.3 × IR 50.0 (p = 0.06). CONCLUSION: It was not possible to demonstrate in this study a significant relationship between complete tumor response to neoadjuvant therapy with respect to blood leukocyte parameters analyzed. (AU)

INTRODUÇÃO: A abordagem radio-quimioterápica previa apresenta grande relevância no manuseio do câncer de reto, colaborando na preservação funcional do órgão, sendo realizada previamente à cirurgia. A resposta inflamatória tem papel importante neste tratamento. OBJETIVO: Consiste em correlacionar o número de linfócitos periféricos e a relação neutrófilos/linfócitos no sangue periférico com a resposta tumoral à terapia neo-adjuvante. MÉTODOS: Revisão de prontuários dos pacientes portadores de câncer retal dos serviços de Oncologia do HMSJ e HSJ, desde 2009, casos submetidos ao tratamento neo-adjuvante com radio-quimioterapia. RESULTADOS: Do total de 96 pacientes portadores desta enfermidade, submetidos à terapia neo-adjuvante com radio-quimioterapia foram elegíveis 35 pacientes, tendo sido observada resposta completa tumoral em 11 casos (31%), e nove foram submetidos ao tratamento cirúrgico. Na comparação dos parâmetros leucocitários entre os pacientes com resposta completa (RC) e resposta incompleta (RI) foram observados os seguintes valores: número total de leucócitos (média) RC 7390,9 × RI 7220,4 (p = 0,8); linfócitos totais RC 2103 × RI 1960,9 (p = 0,4); relação neutrófilo/linfócito RC 3,55 × RI 3,79 (p = 0,5). A dose radioterápica média foi de 49.1 Gy, sendo RC 47,3 × RI 50,0 (p = 0,06). CONCLUSÃO: Não foi possível demonstrar no presente estudo relação significativa entre resposta completa tumoral à terapia neo-adjuvante nos parâmetros analisados do perfil leucocitário. (AU)

Evaluation of Toll-like, chemokine, and integrin receptors on monocytes and neutrophils from peripheral blood of septic patients and their correlation with clinical outcomes

Recognition of pathogens is performed by specific receptors in cells of the innate immune system, which may undergo modulation during the continuum of clinical manifestations of sepsis. Monocytes and neutrophils play a key role in host defense by sensing and destroying microorganisms. This study aimed to evaluate the expression of CD14 receptors on monocytes; CD66b and CXCR2 receptors on neutrophils; and TLR2, TLR4, TLR5, TLR9, and CD11b receptors on both cell types of septic patients. Seventy-seven septic patients (SP) and 40 healthy volunteers (HV) were included in the study, and blood samples were collected on day zero (D0) and after 7 days of therapy (D7). Evaluation of the cellular receptors was carried out by flow cytometry. Expression of CD14 on monocytes and of CD11b and CXCR2 on neutrophils from SP was lower than that from HV. Conversely, expression of TLR5 on monocytes and neutrophils was higher in SP compared with HV. Expression of TLR2 on the surface of neutrophils and that of TLR5 on monocytes and neutrophils of SP was lower at D7 than at D0. In addition, SP who survived showed reduced expression of TLR2 and TLR4 on the surface of neutrophils at D7 compared to D0. Expression of CXCR2 for surviving patients was higher at follow-up compared to baseline. We conclude that expression of recognition and cell signaling receptors is differentially regulated between SP and HV depending on the receptor being evaluated.

Characterization of nCD64 expression in neutrophils and levels of s-TREM-1 and HMGB-1 in patients with suspected infection admitted in an emergency department / Caracterización de la expresión de nCD64 en neutrófilos y de los niveles de s-TREM-1 y HMGB-1 en pacientes con sospecha de infección admitidos en el departamento de emergencias

Introduction: The nCD64 receptor, the soluble triggering receptor expressed in myeloid cells (s-TREM-1), and the high mobility group-box 1 protein (HMGB-1) have been proposed as significant mediators in sepsis. Objective: To evaluate the prognostic value of these markers in patients with suspected infection recently admitted in an emergency department (ED). Materials and methods: All patients who presented to the ED with suspected infection were eligible for enrollment in this study. Baseline clinical data, Sequential Organ Failure Assessment score (SOFA) score, APACHE II score, HMGB-1 levels, s-TREM-1 levels, and nCD64 levels were analyzed. The HMGB-1 and sTREM-1 serum concentrations were determined using commercially available ELISA kits, and CD64 on the surface of neutrophils was measured by flow cytometry. Results:. A total of 579 patients with suspected infection as their admission diagnosis were enrolled in this study. The median patient age was 50 years (IQR = 35-68). Morbidity during the 28-day followup period was 11.1% (n=64). The most frequent diagnosis at the time of admission was communityacquired pneumonia (CAP) in 23% (n=133) patients, followed by soft tissue infection in 16.6% (n=96), and urinary tract infection in 15% (n=87). After multivariable analysis, no significant association was identified between any biomarker and 28-day mortality. Conclusion: In the context of a tertiary care hospital emergency department in a Latin-American city, the nCD64 receptor, s-TREM-1, and HMGB-1 biomarkers do not demonstrate prognostic utility in the management of patients with infection. The search continues for more reliable prognostic markers in the early stages of infection.

Introducción. El receptor CD64, receptor soluble ´desencadenador´ expresado en células mieloides (sTREM-1) y la proteína del grupo Box-1 de alta movilidad (HMGB-1), se han propuesto como mediadores en la sepsis. Objetivo. Evaluar el valor pronóstico de estos marcadores en pacientes con sospecha de infección, recientemente admitidos en un departamento de emergencias. Materiales y métodos. Se incluyeron en el estudio pacientes que consultaron al hospital con sospecha de infección. Se analizó la base de datos clínica, el puntaje SOFA, el puntaje APACHE II, los niveles de HMGB-1, los niveles de sTREM-1 y los niveles de nCD64. Se determinaron las concentraciones en suero de HMGB-1 y sTREM-1, usando kits de ELISA disponibles comercialmente, y la de CD64 se midió por citometría de flujo. Resultados. Se analizaron 579 pacientes con sospecha de infección al ingreso. La edad media fue de 50 años (rango intercuartílico=35-68), y 11,1 % (n=64) murieron durante el seguimiento de 28 días. El diagnóstico más frecuente en el momento del ingreso fue neumonía adquirida en la comunidad, en 23 % (n=133) de los pacientes, seguida de infección de tejidos blandos, en 16,6 % (n=96), e infección urinaria, en 15 % (n=87). Después de un análisis multivariado, no hubo asociación significativa entre ningún biomarcador y la mortalidad a los 28 días. Conclusión. Los resultados sugieren que en el contexto de un departamento de emergencias de tercer nivel de una ciudad latinoamericana típica, los tres marcadores evaluados no ofrecieron ninguna ventaja en el pronóstico de infección. La búsqueda de marcadores pronósticos más confiables en estadios tempranos de la infección aún continúa abierta.

Delayed Human Neutrophil Apoptosis by Trichomonas vaginalis Lysate

Neutrophils play an important role in the human immune system for protection against such microorganisms as a protozoan parasite, Trichomonas vaginalis; however, the precise role of neutrophils in the pathogenesis of trichomoniasis is still unknown. Moreover, it is thought that trichomonal lysates and excretory-secretory products (ESP), as well as live T. vaginalis, could possibly interact with neutrophils in local tissues, including areas of inflammation induced by T. vaginalis in humans. The aim of this study was to investigate the influence of T. vaginalis lysate on the fate of neutrophils. We found that T. vaginalis lysate inhibits apoptosis of human neutrophils as revealed by Giemsa stain. Less altered mitochondrial membrane potential (MMP) and surface CD16 receptor expression also supported the idea that neutrophil apoptosis is delayed after T. vaginalis lysate stimulation. In contrast, ESP stimulated-neutrophils were similar in apoptotic features of untreated neutrophils. Maintained caspase-3 and myeloid cell leukemia-1 (Mcl-1) in neutrophils co-cultured with trichomonad lysate suggest that an intrinsic mitochondrial pathway of apoptosis was involved in T. vaginalis lysate-induced delayed neutrophil apoptosis; this phenomenon may contribute to local inflammation in trichomoniasis.

Fibroplasia after polypropylene mesh implantation for abdominal wall hernia repair in rats / Fibroplasia após implante de tela de polipropileno para correção de hérnia da parede abdominal em ratos

PURPOSE: This study assessed the collagen deposition and correlated it with local inflammatory responses to evaluate the length of time required for fibroplasia when polypropylene meshes are used to repair incisional abdominal wall hernias in rats. METHODS: Thirty-six male Wistar rats underwent longitudinal resection of a peritoneal and musculoaponeurotic tissue segment (3x2 cm) of the abdominal wall followed by defect reconstruction with polypropylene mesh bridging over aponeurosis. The animals were divided into 6 groups according to the time points for the analysis of fibroplasia: 1, 2, 3, 7, 21 and 30 days post-implantation. Animals were sacrificed at each time point, and the site where the polypropylene mesh was implanted was evaluated histologically to assess inflammatory response and percentage of collagen using computer-assisted videomorphometry. RESULTS: Total collagen was found at the mesh site on the 3rd day post-implantation, and increased progressively on all subsequent days up to the 21st day, when it reached its highest percentage (p<0.001). Type III collagen increased progressively from the 3rd to the 21st days, when it reached its highest percentage (p<0.001); on the 30th day, it decreased significantly (p>0.001). Type I collagen was first found between the 7th and 21st days; it reached its highest percentage on the 21st day and then remained stable until the 30th day. The type I to type III collagen ratio increased significantly and progressively up to the 30th day (p<0.001). Neutrophils were found at the mesh site from the 1st to the 21st day post-implantation. Macrophages, giant cells and lymphocytes were seen on the 2nd day. Thirty days after mesh implantation, neutrophils disappeared, but the percentages of macrophages, giant cells and lymphocytes remained stable (p<0.001). CONCLUSIONS: This study showed that total collagen was first seen on the 3rd day post-implantation, with a higher percentage of type I collagen...

OBJETIVO: Avaliar o tempo de fibroplasia em tela de polipropileno na correção de hérnias incisionais da parede abdominal, em ratos, através da quantidade de colágeno, correlacionando-o com a resposta inflamatória local. MÉTODOS: Trinta e seis ratos machos da linhagem Wistar foram submetidos à ressecção longitudinal de um segmento músculo-aponeurótico e peritoneal (3x2 cm) da parede abdominal, seguida por reforço com tela de polipropileno, em forma de ponte sobre a aponeurose. Os animais foram distribuídos em seis grupos, de acordo com o tempo de fibroplasia a ser estudado (1, 2, 3, 7, 21 e 30 dias de pós-operatório). Após os prazos estabelecidos para estudo da fibroplasia, os animais foram submetidos à eutanásia, e a área de fixação da tela de polipropileno foi avaliada histologicamente quanto à reação inflamatória e à percentagem de colágeno pela técnica videomorfométrica assistida por computador. RESULTADOS: Houve aparecimento de colágeno total junto à tela no 3º dia pós-implante, com aumento progressivo na sua proporção em todos os dias subseqüentes até o 21º dia, quando atingiu sua proporção máxima (p<0,001). A partir do dia 3, o colágeno III sofreu um aumento progressivo até o dia 21, quando atingiu sua proporção máxima (p<0,001), e no 30º dia apresentou uma redução significativa (p<0,001). O colágeno tipo I surgiu entre o 7º e o 21º dia, apresentou sua máxima proporção no 21º dia e manteve-se inalterado até o final do período de observação. A relação colágeno tipo I/tipo III aumentou progressivamente e inverteu-se no 30º dia de observação (p<0,001). Os neutrófilos foram identificados no 1º dia pós-implante, mantendo-se junto à tela até o 21º dia. Os macrófagos, gigantócitos e linfócitos foram identificados no 2º dia. Trinta dias após a implantação da tela, desapareceram os neutrófilos e mantiveram-se estáveis as proporções de macrófagos, gigantócitos e linfócitos (p<0,001). CONCLUSÕES: Os resultados do presente estudo evidenciaram...

Increased oxygen free radical production from isolated human pmnls and whole blood by luminol-enhanced chemiluminescence in autistic children

To investigate the estimated capacity of polymorphonuclear leucocytes [PMNLs] and whole blood to produce reactive oxygen species [ROS] in children with autism and mental retardation, and compare it with normal children. Cohort study conducted between January and April 2007. Chemiluminescence laboratory [CL], Department of Physiology, Faculty of Medicine, King Khaled University Hospital, Riyadh, Saudi Arabia. Forty autistic and eight mentally retarded children. Oxygen free radical production [O[2]-, H[2]O[2], OH-] was detected by luminol-enhanced chemiluminescence, from isolated PMNLs and whole blood, stimulated by phorbol myristate acetate [PMA] and opsinized zymazan [OPZ]. Oxygen free radical production from whole blood and PMNLs. Forty autistic [35 male and fivefemale],andeightmentally retarded children [study group] were compared with forty six normal Saudi children [control group]. The mean age was 7.4 +/- 0.5 years. The CL peak response of whole blood and PMNLs stimulated with PMA and OPZ, in autistic children was significantlyhigher [p < 0.05]compared to control children. However, the CL peak response in children with mental retardation did not show any significantdifferenceswhencomparedtothecontrolgroup. There is an increase in oxygen free radicals production from whole blood and from PMNLs in autistic children. Therefore, an increase the antioxidant consumption in autistic children is strongly recommended

Use of mannanoligosacharides as an adjuvant treatment for gastrointestinal diseases and this effects on E. coli inactivated in dogs

PURPOSE: To evaluate the effects of mannanoligosacharides on dogs showing gastroenteritis. METHODS: Sixteen dogs, 2-6 month-old, distributed into two groups: T1 - animals with gastroenteritis receiving treatment for the disease + mannanoligosacharides (2 g/animal).; and T2 - animals with gastroenteritis receiving just treatment for the disease. The animals were randomly included in the sample and all of them were submitted to blood and feces collection for coproculture. In the treatment for gastroenteritis antibiotic, antihemetic, vermifuge, vitamins and sorotheraphy were used. The parameters evaluated were the numbers of leucocytes, neutrophils, lymphocytes and the presence of enteropathogenic bacteria in feces. RESULTS: The mannanoligosacharides was effective in eliminating pathogenic E. coli in 85.71 percent of the animals, while in the no-treated group only 25 percent of the animals were negative to E. coli. CONCLUSION: The mannanoligosacharides is effective in the control of pathogenic E. coli and it can be indicated as an adjuvant treatment for gastroenteritis in dogs.

OBJETIVO: Avaliar os efeitos da adição de mananoligossacarídeos fosforilados (MOS) que apresentavam gastroenterite , pacientes de uma Clínica Veterinária. MÉTODOS: foram estudados 16 cães de 2 a 6 meses de idade, de várias raças. Os animais foram distribuídos em 2 grupos, sendo o grupo T1 composto por animais com gastroenterite, que receberam o tratamento para a doença e Mos (2,0 g/animal) e o grupo T2, animais com gastroenterite, que somente receberam o tratamento para a doença sem o MOS. Os animais foram incluídos aleatoriamente na amostra e todos eles foram submetidos à coleta de sangue e de fezes para coprocultura.O tratamento para a gastroenterite constituiu-se de antibiótico,antiemético,vermífugo,vitaminas e soroterapia. No experimento, foram avaliados os leucócitos, neutrófilos, linfócitos e a presença de bactérias enteropatogênicas nas fezes. RESULTADOS: Constatou-se a efetividade do Mos no grupo tratado, quando houve a eliminação da Escherichia coli patogênica em 85,71 por cento dos animais,enquanto que, no grupo sem o Mos, só 25 por cento, não apresentaram o microorganismo. CONCLUSÃO: O Mos é efetivo no controle da E. coli patogênica, sendo indicado como tratamento adjuvante nas gastroenterites.

Polymorphonuclear leukocyte migration across intestinal epithelium induces epithelial cytokine expression

La migración de neutrófilos polimorfonucleares es un rasgo común de la inflamación activa, que precede la formación de abscesos. La contribución relativa de células epiteliales como fuente de quimioquinas en la infiltración de leucocitos durante la inflamación intestinal no ha sido estudiada. Para evaluar esta contribución nosotros diseñamos un modelo heterólogo de migración transepitelial, “in vitro”, haciendo uso de PMN de rata y células epiteliales de origen humano. Nosotros demostramos que neutrófilos polimorfonucleares luego de su activación quimiotáctica, inducen el incremento en los niveles de ARNm de IL-1ß, IL-8 en las células epiteliales de intestino, mientras que no afecta al ARNm de ENA-78. Estos resultados sugieren que quimiocinas y citoquinas sintetizados por la célula epitelial podrían jugar un papel en el mantenimiento de la respuesta inflamatoria.

Polymorphonuclear neutrophil migration is a common feature of active inflammation that precedes the formation of abscesses. The relative contribution of epithelial cells as a source of chemokines in the recruitment of leukocytes during intestinal inflammation has not been studied. To evaluate this contribution, we have designed a heterologous “in vitro” model for transepithelial PMN migration, based on the use of rat neutrophils and human epithelial cells. We show that polymorphonuclear neutrophil, upon chemotactic activation, induces an increase in IL-1ß, IL-8 levels in intestinal epithelial cells, while not changed ENA-78 mRNA. These results suggest that chemokines and cytokines synthesized by intestinal epithelial cells could play a role in the maintenance of the inflammatory response.

Evaluación de los mecanismos oxidativos del PMN neutrófilo de saliva de pacientes periodontalmente sanos por reducción de NBT / An evaluation of the oxidative mechanisms of salivary PMN neutrophil in saliva of periodontally healthy patients by NBT reduction

Antecedentes: en general, las enfermedades periodontales se caracterizan por cambios, tanto estructurales como bioquímicos en el ámbito tisular, y en su patogenia, por contraste de infecciones bacterianas, el neutrófilo es considerado como la célula más importante de la inmunidad innata. La importancia de esta célula radica en el hecho de estar presente en cada uno de los cuatro estadios que caracterizan el avance de la lesión, así como en los hallazgos de susceptibilidad a infecciones bacterianas recurrentes, incluyendo enfermedad periodontal, en pacientes con defectos cualitativos y cuantitativos en PMN neutrófilos de sangre periférica. Una de las alteraciones funcionales del neutrófilo asociada a la pérdida de la capacidad de defensa ejercida por esta célula contra bacterias extracelulares, la constituye el hecho de ser incapaz de acivar sus mecanismos oxidativos para producir la muerte intracelular de los microorganismos ingeridos. Objetivo: evaluar la capacidad del PMN neutrófilo de saliva y sangre periférica de pacientes periodontalmente sanos para activarse y utilizar losmecanismos microbicidas dependientes de oxígeno, para reconocer si el PMN neutrófilo, al migrar a través del surco gingival, conserva intacta su funcionalidad. Métodos: el estudio fue descriptivo comparativo, in vitro. Para su desarrollo se formaron muestras de saliva y de sangre periférica de 5 individuos. Los neutrófilos aislados fueron sometidos a activación con phorbol miristato acetato (PMA) y posteriormente a las pruebas de reducción del nitroazul de tetrazolio, el cual se puede cuantificar por observación directa al microscopio de luz. Resultados: los PMN neutrófilos obtenidos de sangre y saliva reducen el NBT en un porcentaje más ato que los PMN neutrófilos de sangre periférica, en ausencia de activación. Se encontró que los PMN neutrófilos de saliva tienen un estado de activación de base debido a su constante exposición a las bacterias de la cavidad bucal, y una vez que se activan, dichas células reducen el NBT en menor cantidad que los PMN neutrófilos de sangre periférica bajo iguales condiciones. Conclusiones: los PMN neutrófilos de saliva son células funcionales capaces de activarse, ya que mantienen intactos los mecanismos oxidativos una vez llegan a la saliva a través del surco gingival

Leucocitos, neutrofilos e indice I: T en recien nacidos sanos de la altura / Leukocytes, neutrophiles and I/T index index in healthy high altitude newborns / Leukocytes, neutrophiles and I/T index index in healthy high altitude newborns

Objetivo. Establecer los valores normales de leucocitos, neutroófilos e índice I:T en recien nacidos sanos de la altura a 4100 m.s.n.m.Diseño. Corte transversal. Lugar. Hospital dela Mujer de la ciudad de La Paz, Participantes 300 recien nacidos de término seleccionados bajo criterios de inclusión. Mediciones principales. Recolección de datos y antecedentes perinatales del recien nacidos seleccionado, toma de muestra de angre de cordón despues de la ligadura, previo consentimiento informado de lospadres. Procesamiento de la muestras por 2 bioquímicos estandarizados en el procedimiento.Cálculo manual de los indices I:T. Resultados. Tratamiento de varianles contínuas se obtuvieron valores de promedio Hto 48.09 (IC95 porciento 47.58-48.61) Hb 15.31 (IC95 porciento 15.14-15.48) leucocitos 12.350 (IC 95 porciento 11.152-12948) Indice I:T /neotrófilos inmaduros/totales) 0.04 (IC95 porciento 0-0.24). conclusiones. Los valores obtenidos no muestran diferencia significativa con aquellos obtenidos sobre alturas bajas ( 1600 m.s.n.m.) o sobre la costa en muestras de cordón y en la primera hora de vida. Existen sin embargo la neccesidad de laborar nuevas curvas para niños después del primer día y después de la primera semana, lo mismo ara neonatos sometidos a condiciones de hipoxia, hipertensión o infección excluidos en este estudio.

Quantities of Receptor Molecules for Colony Stimulating Factors on Leukocytes in Measles

We analyzed the comparative amounts of granulocyte-colony stimulating factor (G-CSFr) and granulocyte macrophage CSF (GM-CSFr) receptors expressed on neutrophils and monocytes in measles patients to investigate the role of these CSFrs in the development of leukopenia including neutropenia and monocytopenia in measles. EDTA-anticoagulated peripheral blood of 19 measles patients, 10 children with other infections showing leukopenia and 16 children with normal complete blood cell counts (CBC)s were analyzed using flow cytometry and QuantiBRITE. The leukocyte (5260 +/- 2030/uL vs. 9900 + 2680/uL, p=0.000), neutrophil (2580 +/- 960/uL vs. 4250 +/- 2750/uL, p=0.024) and the lymphocyte counts of measles patients (1810 +/- 1430/uL vs. 4530 +/- 3450/uL, p= 0.006) were lower than in the normal controls. The neutrophils of measles patients expressed similar amounts of G- CSFr (1858 +/- 355) as normal children (1764 +/- 477, p= 0.564) and leukopenic patients (1773 +/- 673, p=0.713), but lower levels of GM-CSFr (535 +/- 118) than normal children (957 +/- 344, p=0.000) and leukopenic patients (832 +/- 294, p=0.002). The monocytes of measles patients expressed similar amounts of G-CSFr (916 +/- 336) and GM-CSFr (3718 +/- 906) as normal children (1013 +/- 391 and 4125 (2645, p > 0.05) but less than leukopenic patients (1454 +/- 398 and 5388 +/- 806, p > 0.05). The neutrophil and monocyte counts of measles patients did not correlate with the amount of G-CSFr or GM-CSFr expressed on neutrophils or monocytes (p > 0.05), but in the normal children, the monocyte count correlated with the levels of GM-CSFr on monocytes (r=0.951, p=0.049). In conclusion, neutropenia is one of the more important characteristics of measles patients, which could be due to the decreased GM-CSFr expression on neutrophils. However, the monocytopenia found in measles patients is not due to the decreased expression of CSFr on the monocytes.

Purification of tryptic peptides for mass spectrometry using polyvinylidene fluoride membrane.

A simple procedure for the purification of tryptic peptides, prior to mass spectrometric analysis, using polyvinylidene fluoride membrane (PVDF) is described. The sensitivity of mass spectrometric analysis is such that minor impurities in tryptic peptide digests suppress the signal obtained. However, we obtained useful signal, from a sample that did not yield any spectra earlier, by purifying the sample using PVDF membrane. For this, the tryptic peptide digest was first spotted on the membrane which was then air-dried and washed. Further, the membrane was extracted with trifluoroacetic acid (TFA) and acetonitrile and subjected to mass spectrometric analysis. This procedure enabled us to identify a cross-reactive D1 antigen on the neutrophil surface that bound antibodies that targeted 60 kD Ro autoantigen in systemic lupus erythematosus, an autoimmune disorder.

Varying expression levels of colony stimulating factor receptors in disease states and different leukocytes

Administration of G-CSF may not always respond in rise of neutrophil counts in different patient population. In order to understand a possible inter-relationship between the G-CSF and GM-CSF induced leukocyte responses and expression levels of receptors for G-CSF (G-CSFr) and GM-CSF (GM-CSFr), the levels of each receptor and CSF were measured in patients with basophilia (8), eosinophilia (14) and bacterial infection showing neutrophilia (12) in comparison with normal healthy adults (12) and children (14). G-CSFr was expressed in neutrophils in the largest amount followed by monocytes, but GM-CSFr was expressed more in monocytes than neutrophils. Lymphocytes and basophils did not express G-CSFr or GM-CSFr. The amount of GM-CSFr in neutrophils was present less in patients with infection than normal control (P = 0.031). The neutrophils expressed more G-CSFr than GM-CSFr. The quantity of G-CSFr in eosinophil showed marked interval change, higher in acute stage. The plasma concentrations of G-CSF in patients with infection were much higher than normal adults or children (117.95 +/- 181.16 pg/ml, P < 0.05). Binding assay with excess amount of CSFs could discriminate the patient who did not show any response to G-CSF or GM-CSF administration. After incubation with excess CSFs, more receptors were blocked in children than in adults (G-CSF P = 0.024, GM-CSF P = 0.006). These results indicate that the amount of CSFr in leukocyte varies in different types of leukocyte, and changes according to the patients' condition even in the same type of leukocyte, and the CSFrs of children bind to CSFs more than those of adults.

Avaliaçäo de neutrófilos na desnutriçäo moderada / Evaluation of polymorphonuclear neutrophils in moderate malnutrition

Objetivo. Avaliar a etapa de ingestao da fagocitose e do metabolismo oxidativo de neutrófilos em crianças portadoras de desnutriçao moderada. Métodos. Foi analisado o sangue periférico de vinte e duas crianças portadoras de desnutriçao moderada, sem infecçao, comparando-se os resultados aos de vinte crianças eutróficas da mesma faixa etária (2 a 5 anos de idade). A ingestao fagocitária por neutrófilos foi avaliada através da ingestao de partículas de zimosan e o metabolismo oxidativo avaliado pela reduçao do nitro blue tetrazolium entre um número fixo de 200 neutrófilos. Resultados. As médias aritméticas da ingestao por neutrófilos de partículas de zimosan, zimosan incubado com soro homólogo e zimosan incubado com soro autólogo foram 18, 41 e 46 em desnutridos e 20, 57 e 63 em eutróficos. A reduçao espontânea e estimulada de nitro blue tetrazolium foi de 6 e 11 em desnutriçao e 12 e 17 em eutróficos. Conclusao. Concluiu-se haver uma diminuiçao da etapa da ingestao e do metabolismo oxidativo de neutrófilos nos pacientes estudados portadores de desnutriçao moderada.

Anticuerpos anti-citoplasma de neutrófilos: aspectos bioquímicos y metodológicos, importancia en diferentes patologías / Antineutrophil cytoplasmic antibodies: biochemical and methodological aspects, their importance in diferents pathologies

Los anticuerpos anticitoplasma de neutrófilos, ANCA, se encuentran involucrados en la patogénesis de las diferentes formas de las vasculitis inmunes. Su descubrimiento y posterior estudio ha permitido su utilización como elemento de ayuda diagnóstica y de seguimiento en enfermedades tales como la granulomatosis de Wegener, panarteritis microscópica y glomerulonefritis cresenticas necrotizantes. El objetivo de esta revisión es tratar de comprender las diferentes formas de vasculitis y así orientarse con respecto al diagnóstico de las mismas. La técnica de inmunofluorescencia indirecta (IFI) que hasta el momento es la única normatizada, sigue siendo una herramienta fundamental para la detección de ANCA dada su elevada especificidad; pero no debe olvidarse que posee como interferentes a los anticuerpos antinucleares (ANA). Se desarrolló un absorvente a partir de un extracto nucleoproteico de timo vacuno que permite eliminar la interferencia que producen los ANA cuando están presente en una muestra donde se quiere determinar ANCA. De esta manera se logró optimizar la IFI-ANCA, lo cual quedó demostrado que los resultados obtenidos cuando se utilizó esta técnica en conjunto con ELISA antígeno específico, LIA blot antígeno específico y Dot blot gránulos Ó desarrollados. Resultados preexistentes en relación con los ANCA y colangenopatías como el lupus eritematoso sistémico (LES) pediátrico y la artritis reumatoidea (AR) mostraban resultados dispares entre diferentes autores, lo que generó la inquietud de estudiar la seroprevalencia en estas enfermedades y su importancia clínica. Se demostró relación entre ANCA e insuficiencia renal en LES pediátrico, no así en AR. Se generó la hipótesis que la presencia de ANCA sea un epifenómeno derivado de la respuesta inflamatoria. También se pudo demostrar la presenciade ANCA en pacientes con glomerulopatías sin evidencias clínicas ni anatomopatológicas de vasculitis como son las glomerulopatías: de la diabetes, IgA, membranoproliferativas, mesangial, membranosa, nefroesclerosis, esclerosis focal y segmentaria, todas ellas asociadas a microhematuria y/o proteinuria en diferentes grados. Finalmente la revisión de este tema y en particular el desarrollo y optimización de los métodos de detección ha permitido ampliar y profundizar el campo de estudio en relación con los ANCA arribando a las conclusiones aquí expresadas

Normal cerebrospinal fluid values in full-term gestation and premature neonates

Results of cerebrospinal fluid (CSF) examinations from 77 high-risk neonates were reviewed. The mean CSF white cells (WBC) count was 4.5 cell/mm3, being two standard deviations above the mean 11.7 cells/mm3 in the full-term gestation neonate group; in the premature neonate one, the mean CSF WBC count was 5.1 cells/mm3, being two standard deviations above the mean 16.7 cell/mm3. PMNs (polymorphonuclear leukocytes) were present in less than 40 per cent of those children, being the mean PMN percentage 4.2 per cent and 0.6 per cent, the mean ANC (absolute neutrophil count) was 0.3/mm3 and 0.06/mm3, in full-term gestation neonate group and premature neonate one, respectively. The mean CSF protein concentration is significantly greater in those premature neonates (101.2 mg/dl) compared with that in term neonates (77.6 mg/dl). The average glucose was just the same in both groups (67 mg/dl). All of these values were from patients who underwent nontraumatic cisternal puncture, with no red blood cells (RBC/mm3=0). Traumatic puncture, even up to 500 RBC/mm3, interfered on CSF parameters.

Bilirubin crystals in neutrophils in neonatal hyperbilirubinaemia.

Serum unconjugated bilirubin (UCB) levels > 205.2 mumol/L were present in 120 of 143 cases of neonatal hyperbilirubinaemia. Sixty-five per cent (78/120) cases showed bilirubin crystals in the neutrophils of Leishman's stained smears from EDTA blood. The proportion of crystal positive (CP) neutrophils was higher in septicaemia than in Haemolytic Disease of Newborn (HDN). Mortality rate was significantly higher (P < 0.001) in CP septicaemic cases when compared with those which were crystal negative (CN).

Lysozyme content of bovine neutrophils

The fractionation of bovine blood neutrophil and the isolation of granules was achieved through homogenization of neutrophil and differential centrifugation of whole homogenate. Lysozyme was assayed in neutrophil whole homogenate the granule fraction and the nuclear pellet. It was possible to detect small quantities of lysozyme because of high sensitivity of spectrophotometer and techniques which were used. Neutrophil granule contained twice as much lysozyme as whole homogenate suggesting that lysozyme was localized in granules. Lysozyme could be also detected in minute quantitites in pellet only if the whole homogenate was washed in 0.34 m sucrose inadequately the quantity of lysozyme detected varied between a total of 0.12 to 0.67 micro g/10[6] neutrophil. Although this amount seems small it can be considered as important in removal of sensitive bacteria as the amount found in some other animal species e.g. rat [0.82 to 0.87 micro g per 10[6] cell]