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Reduced chemotactic migration of polymorphonuclear neutrophil (PMN) in sepsis patients leads to decreased bacterial clearance and accelerates the progression of sepsis disease. Quantification of PMN chemotaxis in sepsis patients can help characterize the immune health of sepsis patients. Microfluidic microarrays have been widely used for cell chemotaxis analysis because of the advantages of low reagent consumption, near-physiological environment, and visualization of the migration process. Currently, the study of PMN chemotaxis using microfluidic chips is mainly limited by the cumbersome cell separation operation and low throughput of microfluidic chips. In this paper, we first designed an inertial cell sorting chip to achieve label-free separation of the two major cell types by using the basic principle that leukocytes (mainly granulocytes, lymphocytes and monocytes) and erythrocytes move to different positions of the spiral microchannel when they move in the spiral microchannel under different strength of inertial force and Dean's resistance. Subsequently, in this paper, we designed a multi-channel cell migration chip and constructed a microfluidic PMN inertial label-free sorting and chemotaxis analysis platform. The inertial cell sorting chip separates leukocyte populations and then injects them into the multi-channel cell migration chip, which can complete the chemotaxis test of PMN to chemotactic peptide (fMLP) within 15 min. The remaining cells, such as monocytes with slow motility and lymphocytes that require pre-activation with proliferative culture, do not undergo significant chemotactic migration. The test results of sepsis patients ( n=6) and healthy volunteers ( n=3) recruited in this study showed that the chemotaxis index (CI) and migration velocity ( v) of PMN from sepsis patients were significantly weaker than those from healthy volunteers. In conclusion, the microfluidic PMN inertial label-free sorting and chemotaxis analysis platform constructed in this paper can be used as a new tool for cell label-free sorting and migration studies.
Subject(s)
Humans , Chemotaxis , Neutrophils/metabolism , Microfluidics , Cell Movement , Sepsis/metabolismABSTRACT
This study evaluated the expression of CD14, toll-like receptor (TLR) 2 and TLR4 on the surface of milk neutrophils in bovine mammary glands infected with Corynebacterium bovis. Here, we used 23 culture-negative control quarters with no abnormal secretion on the strip cup test and milk somatic cell count lower than 1x105 cells/mL, and 14 C. bovis infected quarters. The identification of neutrophils, as well as, the percentage of neutrophils that expressed CD14, TLR2 and TLR4 were analyzed by flow cytometry using monoclonal antibodies. The present study encountered no significant difference in the percentages of milk neutrophils that expressed TLR2 and TLR4 or in the expression of TLR4 by milk neutrophils. Conversely, a lower median fluorescence intensity of TLR2 in milk neutrophils was observed in C. bovis-infected quarters. The percentage of neutrophils that expressed CD14 and the median fluorescence intensity of CD14 in milk neutrophils was also lower in C. bovis-infected quarters...
O presente estudo objetivou avaliar alterações na expressão de CD14, e dos receptores do tipo toll (TLR) 2 e 4 na superfície de neutrófilos lácteos provenientes de glândulas mamárias infectadas por Corynebacterium bovis. O presente estudo utilizou 23 quartos negativos no exame bacteriológico, sem alterações na prova de fundo escuro e com contagem automática de células somáticas menor que 1 x105 células/mL, e 14 quartos mamários infectados por C. bovis A identificação de neutrófilos, assim como a porcentagem de neutrófilos lácteos que expressaram CD14, TLR2 e 4 foram avaliadas por citometria de fluxo utilizando anticorpos monoclonais. A porcentagem de neutrófilos que expressaram TLR2 e TLR4 nos quartos mamários infectados por C. bovis não diferiu dos quartos mamários sadios, assim como na expressão de TLR4. No entanto, a intensidade de fluorescência do TLR2 na superfície dos neutrófilos foi menor nos quartos mamários infectados por C. bovis. A porcentagem de neutrófilos que expressaram CD14 e a intensidade de fluorescência da molécula de CD14 foi menor na superfície dos neutrófilos lácteos dos quartos infectados por C. bovis...
Subject(s)
Animals , Cattle , /analysis , Cattle/immunology , Corynebacterium/isolation & purification , /analysis , /analysis , Mammary Glands, Animal/physiopathology , Mastitis, Bovine/microbiologyABSTRACT
Objective To detect systematic oxidative stress in preeclampsia.Methods (1)Morphological features of placenta hypoxia were observed by histological method ; (2) Level of granulocyte intracellular reactive oxygen species was monitored by dyeing full blood with 2' ,7'-dichlorodihydrofluorescein diacetate (H2DCFDA) ; (3) Level of H_2O_2 in sera was detected by special kits.Results Compared to normal pregnancy,placentas from preeclampsia showed distinct features of hypoxic stress injury,such as more syncytial knots formation,fibrosis emerged,vein in-jury and loss its normal configuration; Fluorescence values of ROS probe in neutrophils from different women were 45.61±12.20(n =49),51.02 ± 13.60(n =56,P <0.01)and 85.10 ± 16.30(n =47,P <0.01); Concentra-tions of H_2O_2were (24.57±5.17)μmol/L(n =49),(26.61±3.25)μmol/L(n =56,P 0.01) and (39.84±9.67)μmol/L(n=47,P<0.01) respectively.Conclusion With the help of histological method,flow cytometry and special kits,systematic oxidative stress can be detected through checking placentic tissues,netrophils and sera of preeclampsia.
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Objective To investigate the effect of ambroxol administered before surgery on contents of PMN,TNF-?,IL-6 and IL-8 in bronchoalveolar lavage fluid after closed circuit anesthesia in infants.Methods 40 infants who practiced selective orthopedics or abdominal operations were randomly divided into ambroxol group (group A) and control group (group B). Two days before operation,infants in group A and group B were administed ambroxol 0.3 mg/kg or 0.9 normal saline 10 ml through intravenous injection three times per day,respectively .The last single dose was administered before anesthesia induction.The bronchoalveolar lavage fluid of intants were collected immediately after tracheal intubation(T1) and before tracheal extubation(T2),respectively.Interleukin-6 and interleukin-8 in the bronchoalveolar lavage fluid were determined by enhancement solid-phase enzyme immunoassay action(EASIA),and TNF-? in the bronchoalveolar lavage fluid by euzymelinked immunosorbent assay (EIA).The numbers of polymorphonuclear neutrophils in the bronchoalveolar lavage fluid were examined. The contents of PMN,TNF-?,IL-6 and IL-8 in two groups were compared .The time of analepsia and complications of respiratory tract were observed after operation.Results Compared with group A, the contents of PMN,TNF-?,IL-6 and IL-8 in BALF collected at T2 were significant higher in group B (P
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Objective The author seeked the suitable tidal volume in order to decrease mechanical ventilation-induced lung injury during closed circuit anesthesia in infants.Methodes 50 infants who practiced selective orthopedics operation randomly divided into traditional mode(12~15ml/kg,groupT,n=25)and low tidal volumes(7~8ml/kg,groupL,n=25).The bronchoalveolar lavage fluid of intants were collected immediately after tracheal intubation(T1)and before tracheal extubation,respectively.Interleukin-6 and interleukin-8 in the bronchoalveolar lavage fluid were determined by enhancement solid-phase enzyme immunoassay action(EASIA),TNF-? in the bronchoalveolar lavage fluid were determined by euzymelinked immunosorbent assay(EIA).The numbers of polymorphonuclear neutrophils in the bronchoalveolar lavage fluid of intants were examined.The infants' SpO2,arterial blood pressure and PetCO2 were monitored continuously.Arterial blood gas was analysised.The time of analepsia and complications of lung were observed after operation.Results PaCO2、PetCO2 in groupL were higher than that in groupT,pH in groupL lower than that in groupT at 30 min,60 min,120 min after mechanical ventilation and before extubation,respectively.The contents of PMN,TNF-?,IL-6 and IL-8 in BALF collected at T2 more than that at T1 in groupT.The contents of PMN,TNF-?,IL-6 and IL-8 in BALF in groupL less than that in groupT at T2(P
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Apoptosis of neutrophils controls the duration and the intensity of an inflammatory response and therefore the extent of neutrophil- mediated tissue damage, disturbance of neutrophil apoptosis has been associated with many diseases, underlying mechanism is not elucidated. C5a is a complement fragment that has multifunctional properties, which induces neutrophil chemoattraction, an oxidative burst, enhancement of phagocytosis, release of granule enzymes, and suppress neutrophil apoptosis. Several studies have reported calpain is involved in both neutrophil functions and apoptosis and it might play a more specific role in the regulation of neutrophil apoptosis. Diffenrent isoform of calpains is activted by diffenrent stimuli through different transduction pathway. It was reported previously that calpain is required for neutrophil migration and chemotaxis induced by C5a. In addition, autophagy is a ubiquitous physiological process that occurs in all eukaryotic cells and is considered to be a survival mechanism. Atg5 promotes autophagy and is indispensable to autophagosome formation. Upon calpain activation, Atg5 is cleaved and the resulting 24 ku Atg5 mediates apoptosis while losting the property of autophagy. Therefore, Atg5 represents a molecular switch between autophagy and apoptosis. The interaction among the C5a, calpain and Atg5 was introduced and new direction for further research was provided.
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Objective To investigate the effects of hypotonic sodium chloride hetastarh 40 injection(HSCH)on the expression of PMN Fas following hemorrhagic shock,and to explore the effective role and mechanisms of HSCH on hemorrhagic shock.Methods Modified Wiggers canine hemorrhagic shock model was used.Expression of Fas in PMN was measured by flow cytometry.Results The levels of PMN Fas expression during hemorrhagic shock were significantly lower than those in control group(P
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Objective To investigate the endotoxemia initiated systemic and pulmonary inflammation and acute lung injury in endotoxin tolerant rats MethodsEndotoxi n tolerance (ET) models of SD rats were induced by four daily intraperitoneal in jections of 0 6 mg?kg -1 ?d -1 Escherichia coli LPS (serotype 055:B5).Normal control (NC) rats received intraperitoneal injections of the sa me volume saline On the fifth day,rats were injected with high dose of LPS (6 mg/kg) to induce endotoxemia and lung inflammation Blood,left bronchoalveolar lavage fluid (BALF) and right lung tissue were collected before and 2,6,24,48 ,72 hours after the high dose injection of LPS (six rats for each time point) Cytological examination of blood and BALF and histopathological examination wer e performed Bromine methylphenol green was adopted for measurement of serum alb umin BALF albumin was measured by en z yme-linked immunosorbent assay (ELISA) and adjusted by the ratio to serum album in to evaluate the permeability of pulmonary microvascular Results The symptomes such as less activity,accelerated respiratory rate and weight loss in NC rats was not found in ET rats after the high dose injectio n of LPS BALF albumin as well as the ratio of BALF albumin to serum albumin in c reasedt 2 hours after injection of 6 mg/kg LPS and reached their zenith at 6 hou rs in NC rats,while no increase in ET rats In NC rats the blood white cell dif f erentiating shifted from lymphocyte to PMN,and PMN percentage of BALF also incr eased from (0 443?0 345)% to (8 000?2 896)% with its peak at 24 hours a fter the injection (P
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Objective To study the killing effect of polymorphonuclear neutrophils(PMNs) on Trichomonas vaginalis. Methods The vaginal secretion from a patient with vaginitis was incubated in the liver infusion liquid medium to get T. vaginalis. One ml serum was collected from the patient and heated for 30 min at 56 ℃ to inactivate complement in serum, and was absorbed three times with the parasites at 0 ℃ to make the serum free of antibodies. PMNs were separated from the patient’s blood and purified with density gradient centrifugation and polymer accelerating sedimentation. NBT and safranin O were used to stain the sample. The interaction between PMNs and the parasites was observed under microscope. 300 trichomonads and 3?104 PMNs were incubated for 10, 20, 30, 40, 50, 60 minutes under the conditions of aerobic or anaerobic, with superoxide dismutase (SOD) and catalase (CAT) or without SOD and CAT, and with complement or without complement. They were then inoculated in solid medium for another five days under the anaerobic condition, and surviving organisms were enumerated. Results PMNs were observed to surround and kill a single trichomonad. In the petri-dish containing PMNs, the surviving rate of the parasites in anaerobic condition was 85%, only 3% in aerobic condition (P
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Se investigo el efecto del consumo de basuca sobre la capacidad fagocitica y microbicida de los polimorfonucleares neutrofilos (PMN) en individuos consumidores y ex-consumidores habituales de esta sustancia y se determino, ademas, la capacidad opsonica de sus sueros. Los resultados mostraron una respuesta normal en todas estas actividades, en comparacion con celulas y sueros de individuos controles sanos, no consumidores de drogas de ningun tipo. Los promedios de los porcentajes de bacterias asociadas a los PMN de los consumidores y de sus controles, utilizando sueros de consumidores como fuente de opsoninas, fueron: 48:0 y 44:7 respectivamente; empleando sueros de ex-consumidores con celulas autologas y controles, los datos fueron: 50.3 y 48.6. Al realizar el mismo ensayo, pero en presencia de suero normal, los valores fueron: 52.0 con los PMN de consumidores, 52.9 con los ex-consumidores, 49.7 y 53.8 con los de los controles respectivos. Los porcentajes de destruccion de las bacterias asociadas a los PMN utilizando suero normal fueron 47.2, 50.5, 44.5, y 51.6 en consumidores, ex-consumidores y controles, respectivamente. Aunque el consumo habitual de basuca no afecto estas actividades en los PMN de sangre periferica, seria importante determinar los efectos secundarios de esta sustancia sobre los neutrofilos y los macrofagos presentes en las regiones broncoalveolares ya que tales celulas fagociticas, por su localizacion anatomica, reciben la maxima exposicion a esta droga durante el consumo
The effect of free base cocaine consumption on the phagocytic and microbicidal capabilities of polymorphonuclear neutrophils (PMN) was studied in habitual users and in ex-users of this substance. The opsonic capability of their sera was also determined. Results showed a normal response in all these activities when compared with cells and sera of healthy non-users. Mean percentages of bacteria associated with PMNs of users and controls, using sera of with PMNs of users, 52.9 with those of ex-users, and 49.7 and 53.8 with cells of the respective controls. Percentages of destruction of bacteria associated with PMNs in normal serum were as follows: 47.2 for users; 50.5 for ex-users, and 44.5 and 51.6 for their respective controls. Although the habitual consumption of free-base cocaine did not affect the phagocytic and microbicidal capabilities of peripheral blood PMNs, it is important to determine the effects of this substance on neutrophils and macrophages of the bronchoalveolar region, since these are the cells that receive maximum exposure to the drug during consumption.