Development of immunochromatographic colloidal gold test strip for rapid detection of Haemophilus influenzae in clinical specimens.

AIMS: A simple and fast test strip for simultaneous detection of Haemophilus influenzae, based on the theory of colloidal gold immunochromatography was developed. METHODS AND RESULTS: In this study, monodispersional colloidal gold and gold-labelled polyclonal antibody (AbP6Line) specific to the linear antigenic determinant OMP6 of H. influenzae were prepared, and identified by UV-visible spectroscopy (200-700 nm) and transmission electron microscopy. The test strip was assembled on a plastic backing containing a sample pad, a conjugate pad, an absorbent pad and a nitrocellulose membrane, on which test and control lines were stuck. The strip showed us specific recognization of H. influenzae and did not identify other common respiratory pathogens such as Streptococcus pneumoniae, Mycoplasma pneumoniae, Moraxella catarrhalis and Legionella pneumophila. It was shown that the detection limit of the test strip was as low as 1 × 106 CFU per ml and the whole process can be completed within 10 min. The strips could be stored at 4°C for at least 6 months without losing sensitivity or specificity. CONCLUSIONS: The test strip was specific, simple and convenient for rapid detection of H. influenzae presenting good stability and reproducibility. SIGNIFICANCE AND IMPACT OF THE STUDY: The test strip provides a simple, convenient and specific tool for the on-site surveillance and diagnosis of H. influenzae infection in clinical samples.

Dielectric analysis of the APG/n-butanol/cyclohexane/water nonionic microemulsions.

The nonionic APG/n-butanol/cyclohexane/water microemulsions with different microstructure, which is induced by the variation of water contents, are investigated by the dielectric spectroscopy. An appropriate dielectric theory, Hanai theory and the corresponding analytical method are applied to obtain the internal properties of the constituent phases of microemulsions, such as the relative permittivity and conductivity of continuous and dispersed phases and the volume fraction of dispersed phase. Using these parameters, the distribution of n-butanol in constituent phases, which is of important in the study field of the microstructure of microemulsion, is obtained quantitatively. It is found that the n-butanol molecules not only distribute in the interfacial APG layer but also in the continuous and dispersed phases. In addition, the percolation threshold is interpreted by using the dynamic percolation model. The structural and dynamic information are obtained, for instance, the critical volume fraction of water when percolation occurs and the characteristic time for the rearrangement of clusters. These parameters are intimately related to the properties of microemulsions, especially the characteristics of the interfacial layer.

Dielectric properties of E. coli cell as simulated by the three-shell spheroidal model.

Dielectric properties of E. coli cell have been re-studied by means of the three-shell spheroidal model, where the three shells correspond to the outer membrane, the periplasmic space and the inner membrane, respectively. With the model, a curve-fitting procedure has been developed to analyze the dielectric spectra. Although E. coli cell has been studied before, its special morphological structure was taken into account more comprehensively than any previous model in the present work. Dielectric properties of various cell components have been estimated from the observed dielectric spectra, especially the permittivity of the outer membrane, which was evaluated quantitatively for the first time. The values of epsilon(om) were 12 for kappa(om) of 0 to 10(-4) S/m and 34 for kappa(om) of 10(-3) S/m. The specific capacitance of the inner membrane was 0.6-0.70 microF/cm(2). The relative permittivity and the conductivity of the cytoplasm were about 100 and 0.22 S/m, respectively, and the conductivity of the periplasmic space was 2.2-3.2 S/m.

Dielectric analysis of nanofiltration membrane in electrolyte solutions: influences of electrolyte concentration and species on membrane permeation.

Dielectric properties of a nanofiltration membrane immersed in dilute aqueous electrolyte solutions were measured, and frequency dependence of capacitance and conductance of the systems was analyzed, based on the interfacial polarization theory, giving values of permittivity and conductivity of the membrane and the solutions. Permittivity, epsilon m, of the membrane slightly decreased whereas conductivity, km, of the membrane increased with increasing electrolyte concentration, as a result of entrance of ions into the membrane. The ratio of membrane/solution conductivity, km/kw, also depended on the electrolyte concentration, showing that distribution of ions in the membrane and in solutions follow Donnan equilibrium, due to the presence of negative fixed charges in the membrane. New expressions were derived from Donnan equilibrium principle to explain this phenomenon, and negative fixed charge concentration ce of the membrane was obtained; thus the Donnan potential, DeltaPhi Don, of the membrane in solutions at various concentrations could be calculated. The new expressions could be expected to be usable to analyze ion permeation property through membrane.

Peroxynitrite leads to arteriolar smooth muscle cell membrane hyperpolarization and low vasoreactivity in severe shock.

This paper aimed to study the mechanism of vascular hyporeactivity during severe hemorrhagic shock. Rats were divided into control and shock group. Membrane potential of arteriolar strips was measured with intracellular recording method and membrane potential changes in arteriolar smooth muscle cells (ASMC) were recorded with membrane potential sensitive fluorescent dye (DiBAC4) and confocal microscopy. Hyperpolarization of ASMC membrane appeared at the late stage of shock, which correlated to low vasoreactivity. Glybenclamide, an inhibitor of K(ATP) channel reversed the hyperpolarizing effect. S-nitroso-N-acetylpenicillamine (SNAP), a donor of NO, in a higher concentration (400 mol/l) caused membrane hyperpolarization in control and shock group, which was completely reversed by application of Tiron, a scavenger of O2-. The hyperpolarizing effect of SNAP was decreased by ODQ, glybenclamide and (or) charybdotoxin. It is concluded that hyperpolarization of ASMC leads to vascular hyporeactivity. Peroxynitrite (OONO-) involves in the development of hyperpolarization in severe shock. The production of cGMP and activation of K(ATP) and K(Ca) channel contribute to the hyperpolarizing effect of OONO-*.

Dysregulation of the humoral immune response in old mice.

The increase in autoantibodies with age of both experimental animals and humans has been thought to reflect a shift in the antibody repertoire from foreign to self antigens. In mice, before immunization, the age-associated increase in antibodies reactive with a prototypic autoantigen, bromelain-treated autologous erythrocytes (BrMRBC), reflected a 3-fold increase in serum IgM and the number of IgM-secreting spleen cells in old compared with young mice. However, the percentage of the IgM-secreting spleen cell repertoire reactive with BrMRBC in old mice was actually approximately 50% that in young mice. In contrast, after immunization with sheep erythrocytes (SRBC), old mice showed a 5-fold increase in the percentage of IgM-secreting cells reactive with BrMRBC while young mice showed no significant increase. The converse is true for the percentage of IgM-secreting spleen cells in old mice specific for SBRC, which is 10% the number generated by young mice. The increased autoantibody response of old mice is not, however, linked to their poor response to the nominal antigen. Thus, immunization with phosphorylcholine (PC) conjugated keyhole limpet hemocyanin, an antigen that induces a comparable anti-PC response in old and young mice, also induced more autoantibody forming cells in old than young mice. The increased autoantibody response of old mice after immunization can be accounted for by both an increased number of Ig-secreting spleen cells as well as an increased percentage of the expressed repertoire of IgM-secreting spleen cells that react with autoantigens.(ABSTRACT TRUNCATED AT 250 WORDS)

[Changes in adhesion features of leukocytes in rats with severe burns].

Twenty-four rats were divided into 3 groups: normal controls (8), simple burned (8), burned plus Polygonum cuspidatum (p.c.)-treated (8). Three hours after burn, 3ml of whole blood was drawn to isolate polymorphonuclear neutrophilic leucocytes (PMNs). Confluent monolayer of rat microvessel endothelial cells was grown in a parallel plate of flow chamber. PMNs were driven through the flow chamber under different wall shear stresses. Leukocyte-endothelia adhesion was observed with an Olympus inverted microscope and recorded with a colour microscopic TV set. The adhesive characteristic curve of leukocyte was significantly different between the normal and burned groups. The number of PMNs adhesive to endothelium increased obviously 3 hours after burn. Under 0.196 Pa of wall shear stress, the adhesion rate of PMNs was 81.2 + 4.1% in the burned group and 58.8 + 4.6% in the normal group. The PNMs adhesion rate was decreased to near normal in p.c.-treated group. The average survival time in the burned group was 4.1 + 1.2 hours, much lower than that in the p.c. treated group (33.2 + 15.8 hours). However, in the simple burned group, one rat survived for 24 hours and its PMNs adhesive curve was similar to that of the normal or p.c.-treated groups. The findings indicate that PMNs adhesion features increase apparently after severe burn, which is closely related to the survival rate.

Computer assisted analysis of leukocyte rheological behavior in microvasculature.

A new approach called Time-Space Scheme has been recommended for measuring the velocity of moving blood cells in the microvasculature. In each sampling time, the data are put into a row, then rows are integrated in sequential times as sequential rows. The motion of various objects in the field of microcirculation can be analyzed from the time-space image. In the time-space image RBC's flow appears as a diagonal dark stripe and WBC's flow as a bright stripe. The slope at different points on those stripes reflects the local velocity of RBCs and WBCs. Using the time-space image analyzing method, the velocities of erythrocyte and leukocyte in microcirculation are measured simultaneously by a self-designed interactive image processing system. According to characteristic of leukocyte flowing movement, leukocytes in microvasculature are divided into the three kinds: central axial-flowing leukocyte, rolling leukocyte and adhesive leukocyte. On this basis, we introduce the leukocyte-endothelium contact time as a quantitative index for leukocyte-endothelium interaction. During hemorrhagic shock, the phenomenon of leukocyte-endothelium adhesion has been observed by this system, which suggests that the method is effective and useful for the research of microcirculation.

Enhancement of the immune response in mice by Astragalus membranaceus extracts.

Astragalus membranaceus extracts injected into normal mice or mice immunodepressed by cyclophosphamide or radiation treatment or by aging are able to enhance the antibody response to a T-dependent antigen. Optimal conditions for maximum effect have been established in normal C3H/HeJ and (C57B1/10 x DBA/2)F1 mice. Enhancement of the antibody response was associated with increase of Th cell activity in normal and immunodepressed mice. The biological activity of endotoxin-free extracts is dependent on the carbohydrate content.

[Effect of Polygonum cuspidatum No 4 on the enhancement of cardiac function during burn shock].

A burn shock model was produced in rats by scalding the lumbus and lower extremities with 80 degrees C water for 30 seconds. The variable of cardiac functions were measured with a Cardiac-II-R cardio-output computer. It was shown that heart functions decreased as early as 10 min following scalding. Cardiac output (CO), cardiac index (CI), and stroke volume index (SVI) were reduced by 40% of normal, and stroke work (SW) by 47%. With the decrease of CO, total peripheral resistance (TPR) increased. Two hour later TPR was increased 2.6-fold more than pre-burn level, which made BP maintain nearly normal. TPR started to reduce at 3 hr post-burn, resulting in decrease of BP. The decreased heart function returned to near normal by administration of crystal of Polygonum cuspidatum No 4 (p.c), e.g, CO and CI were increased to 91.0% of pre-burn value, SW to 100.1%, and SVI to 102.0%. The increased TPR was reduced to near normal. Half of p.c.-treated animals survived within 24 hr, but all of simple burn animals and n.s.-treated animals died within 24 hr. Under the condition of no fluid infusion, administration of p.c. could restore the decreased heart function, which showed that besides loss of plasma, direct myocardial depression was one of the reason for the decrease of cardiac output during burn shock. The survival rate of animals with burn shock was enhanced by injection of p.c., which indicated that increasing heart function was a very important measure for the treatment of burn shock.

Effect of naloxone on microcirculatory behavior during irreversible hemorrhagic shock.

Thirty-six male Sprague-Dawley rats were divided into three groups (n = 12): simple hemorrhagic shock group, saline-treated group, and naloxone-treated group. Synchronous videotape recording with two cameras was used to observe the relationship between the change of blood pressure and that of microcirculation of cremaster muscle. It has been suggested that the temporary plugging of capillaries by WBC may be the explanation for no reflow in microvessels after transfusion and infusion during the irreversible stage of shock. Administration of naloxone (2 mg/kg iv) followed by reinfusion of the shed blood is associated with an increase in blood pressure and pulse pressure. Coincidentally, a pulsatile movement of the blood, impacted on the stationary blood cells in capillaries, eventually dislodges them and leads to reperfusion of capillaries. Thereafter systemic blood pressure and carotid artery red blood cell velocity were increased and remained stable. The 24-hr survival rate was significantly increased in the naloxone-treated group (10/12), as compared with that in the saline-treated group (1/12). The mechanism of naloxone treatment is discussed. The suggestion is advanced that the effects on cardiac and microcirculatory performance may influence each other, and that both contribute to the pathogenesis of irreversible hemorrhagic shock.

Microvascular adjustments during irreversible hemorrhagic shock in rat skeletal muscle.

A study was made of microvessel response to hemorrhagic hypotension and the subsequent restoration of blood volume. The experiments were conducted on anesthetized rats in which the cremaster muscle was exteriorized for intravital microscopy. Variables measured during hypotension (40 mm Hg for 60 min) and after blood restoration (120 min observation) included systemic blood pressure, heart rate, arteriolar and venular diameter, sensitivity to epinephrine, velocity and volumetric flow rate. These findings were correlated with 24-hr survival statistics. The response to hemorrhagic hypotension is a reflection of two separate adjustments, cardiac output and peripheral vascular behavior. In survivors, the microvascular sequelae following blood replacement was one of continuous improvement of muscle perfusion, whereas in nonsurvivors the picture was one of progressive deterioration. The consistent hallmark of irreversibility, arteriolar hyposensitivity, was associated with a continuous falling off in mean arterial pressure despite restoration of normal blood volume.