Rickettsial Infectious Antibody Complexes: Detection by Antiglobulin Plaque Reduction Technique

Rickettsia rickettsii and R. australis formed infectious antibody complexes when reacted with homologous convalescent serum. The addition of antiglobulin to these complexes showed neutralization of plaques proportional to the concentration of rickettsial immune serum.     

Hen Fluorescein-Labeled Gonococcal Lipopolysaccharide Antibody in the Delayed Fluorescent Antibody Technique for the Confirmation of Neisseria gonorrhoeae

A fluorescent antibody reagent (termed anti-LPS conjugate) was prepared from sera obtained from hens immunized with gonococcal R-type lipopolysaccharide. The reagent was absorbed with Formalin-treated cells of Neisseria meningitidis. The anti-LPS conjugate gave uniform brilliant staining of Neisseria gonorrhoeae with little background fluorescence, thus making interpretation and reading of fluorescence simple. The conjugate did not significantly stain cultures of N. meningitidis, Neisseria lactamica, nonpathogenic Neisseria species, or other gram-negative bacteria. Several preparations of the conjugate provided the same specificity and reproducibility of staining. The anti-LPS conjugate was compared with Difco Laboratories fluorescent antibody conjugate for staining of N. gonorrhoeae. Both conjugates stained cells of the light and dark variants of gonococcal colony types 1 and 2, as well as cells of colony types 3 and 4. When used for the confirmation of N. gonorrhoeae, the anti-LPS and Difco conjugates stained 426 of 431 (98.8%) and 210 of 213 (98.6%) of the gonococcal cultures, respectively. Absorption of the anti-LPS conjugate with R-type lipopolysaccharide removed the staining of gonococci. However, absorption of Difco conjugate with R-type lipopolysaccharide did not remove the staining of gonococci, suggesting that the majority of fluorescein-labeled antibody present in the Difco conjugate is directed to gonococcal cell surface components other than lipopolysaccharide. The results of this study indicate that fluorescein-labeled gonococcal lipopolysaccharide antibody should be a reliable fluorescent antibody reagent for the confirmation of N. gonorrhoeae.     

直标法流式细胞术测定人红细胞表面CD35水平的方法学研究

为研究直标法流式细胞术测定红细胞表面CD35免疫分子水平的方法,抽取健康献血员静脉血,采用直标法流式细胞术测定红细胞CD35水平,研究测试后数据分析方法的可靠性和测试前抗凝剂、标本放置时间和抗体用量等因素对测定结果的影响.结果显示:M1界值选择对CD35阳性红细胞(CD35+-E)百分率计算结果有很大影响,CD35几何平均荧光强度(CD35-GMFI)随FL2通道电压升高而增加,而CD35相对几何平均荧光强度(CD35-rGMFI)不受电压影响;EDTA-2K、肝素锂、枸橼酸钠三种抗凝剂以及标本放置72h内CD35+-E百分率和CD35-rGMFI结果均无明显差异(P＞0.05);取5×105个人红细胞反应时,最适合的鼠抗人CD35单克隆抗体量为7μL.可见,CD35-rGMFI是描述红细胞表面CD35水平的最理想指标,抗凝剂、标本放置时间对测定结果无明显影响,当红细胞数量为5×105时,最佳抗体用量为7μL.     

Comparison of Midturbinate Flocked-Swab Specimens with Nasopharyngeal Aspirates for Detection of Respiratory Viruses in Children by the Direct Fluorescent Antibody Technique

Paired nasopharyngeal aspirate (NPA) and midturbinate flocked-swab specimens from 153 children with respiratory symptoms were examined by the direct fluorescent antibody (DFA) technique. Seventy-four infants (49%) had a viral infection documented by DFA. The flocked-swab specimens had 93% sensitivity and 96.7% agreement with the NPA specimens, with a kappa coefficient of 93.4% (95% confidence interval, 0.877, 0.991).The direct fluorescent antibody (DFA) technique revolutionized the rapid detection of respiratory viruses. Since its inception in 1968, it has been one of the mainstays in clinical virology laboratories throughout the world (4). The ability of DFA to detect respiratory viruses depends on many things, but it all begins with good specimen collection. The nasopharyngeal aspirate (NPA) has been considered the best specimen to detect respiratory viruses in infants (4). However, it is difficult to collect because it requires special equipment, such as a catheter, trap, and vacuum source, and specialized training. A traditional nasopharyngeal swab is the next best specimen, especially in older children or adults, because it utilizes common supplies; however, the collection end of the swab, comprised of wound Dacron fibers, has limited absorbent capacity to trap virus-infected exfoliated epithelial cells. A nylon nasopharyngeal flocked swab with enhanced absorptive properties introduced in 2006 compared favorably to the NPA for the detection of respiratory viruses by DFA (2). Recently, a midturbinate flocked swab developed by Smieja, et al. (7), and marketed by Copan, Inc., has offered a more intuitive approach for the collection of nasopharyngeal specimens (1). It has compared favorably to the NPA and the flocked nasopharyngeal swab in the diagnosis of respiratory viruses by culture, antigen detection, and PCR, none of which require intact exfoliated epithelial cells for visualization; there is no published experience of midturbinate flocked swabs with DFA in children (1, 5, 6). The midturbinate flocked swab differs from the nasopharyngeal swab. It has a sampling depth indication gauge and also has a larger absorptive capacity than the smaller nasopharyngeal swab.The present study was designed to compare the efficacy of the midturbinate flocked swab with the NPA in the detection of respiratory viruses by DFA.The study was conducted from 5 January 2010 through 11 March 2010. All children 2 years of age or less admitted to the infant''s floor of the hospital with respiratory symptoms were enrolled. The study was reviewed by the Children and Youth Institutional Review Board, who waived the need for a formal review because the study was deemed an evaluation comparing a new specimen collection device to the standard nasopharyngeal aspirate; parents were allowed to opt out of the use of the new specimen device. A nasopharyngeal aspirate specimen was collected through one nostril. A second specimen was collected through the other nostril with a midturbinate FLOQ swab (Copan Diagnostics, Inc., Murrieta, CA) designed for children 2 years of age or less; the swab was inserted up to the collar on the shaft. Both specimens were placed in 3 ml of Copan UTM transport medium, transported to the virus laboratory, and processed within 6 h. The suspension was centrifuged, and the cellular pellet washed. The cells were then spotted to glass slides. The cells were stained for DFA using a D3 Ultra respiratory screening identification kit (Diagnostic Hybrids, Inc. [DHI], Athens, OH). The kit screened for respiratory syncytial virus (RSV), influenza viruses (IFV) A and B, parainfluenza viruses (PFV) 1, 2, and 3, and adenovirus (AdV). An additional stain for human metapneumovirus (hMPV) (DHI) was included. The DFA readers were not blinded to the specimen source. The degree of DFA agreement between specimens collected by NPA and midturbinate flocked swabs was calculated with Cohen''s kappa coefficient of agreement.One hundred fifty-three infants entered the study. Paired specimens were collected from every infant. Respiratory viruses were identified in 74 (48.6%). Respiratory syncytial virus was most frequent, found in 47 patients (30%), with hMPV in 25 (16.3%), PFV in 1 (0.7%), AdV in 1 (0.7%), and IFV in none (0.0%). The 2009 H1N1 influenza A virus had last been identified in the laboratory in November 2009, more than 1 month before the start of the study. DFA of NPA specimens identified all the viruses. DFA of the flocked-swab specimens failed to detect 4 RSV and 1 hMPV isolate that had been detected in the NPA specimens. The negative DFA test results on flocked-swab specimens agreed with the negative DFA test results on NPA specimens. Overall, the positive and negative DFA test results on flocked-swab specimens had 96.7% agreement with the DFA test results on NPA specimens, with a Kappa coefficient of 93.4% (95% confidence interval [CI], 0.877, 0.991; P < 0.00001). The sensitivity of the flocked swab was 93.2% (95% CI, 0.849, 0.978).The midturbinate flocked swab proved to be comparable to the NPA for the detection of common respiratory viruses, such as RSV and hMPV, in a DFA test in the present study. The absence of IFV and the low numbers of AdV and PFV isolates in specimens prevented an assessment of the swab''s utility in detecting these viruses; however, earlier studies with nasopharyngeal flocked swabs suggested that the midturbinate swab would give similar results (3). In an earlier study, the sensitivity of the NPA in detecting either IFV or RSV was greater than the sensitivity of flocked nasopharyngeal swabs, although the difference was not statistically significant; the differences may be attributed to the greater number of respiratory epithelial cells available for examination in NPA specimens (2). The advantage of the midturbinate collection over nasopharyngeal collection resides in the relative ease of collection and the resultant patient cooperation, especially among the very young; however, the observations made in the present study may not extend beyond the pediatric population.     

Initial vascularisation in the pig placenta: I. Demonstration of nonglandular areas by histology and corrosion casts

The vascular interrelationship of the well-established porcine placenta has previously been described from vascular casts and histology, but not its developmental stages. This study was peformed using the same methods on 17 sows of well-known stages of gestation ranging from 9 1/2 to 43 days post coitum (p.c.). At the precontact stage, days 9 1/2 to 12 1/2 p.c., the subepithelial capillaries formed a wide open meshwork of variable diameter, 3–14 μm, without any difference between meso- and antimesometrial side. At the early contact and adhesion stages (days 13 to 18 p.c.), the first increase in vasculature was seen at the mesometrial side close to the embryonic disc of the very long blastocyst at day 15 p.c., 2 days after the first contact between trophoblast and maternal epithelium was seen. At day 18 p.c., the areas with dense capillaries increased markedly at the mesometrial side with the same parallel organization as seen at day 15 p.c., whereas the antimesometrial side still had a relative loose appearance comparable to the previous stages. At the early placental stages (days 20 1/2 to 23 p.c.), the capillary bed formed smooth folds, which in some areas at day 20 1/2 days developed into smaller folds or prerugae. Here the capillaries changed to convoluted forms with slightly bulbous dilations measuring about 30–35 μm in diameter. This developmental progress became more elaborate at day 23: capillaries of the low ridges of prerugae formed irregular dilations up to 50 μm in some areas. At this stage the parallel arrangement of the capillary meshwork characteristic of the previous stage was not longer discernable. By days 32–43 p.c., an increase in microscopic folding was present, and the maternal arterioles could be traced to the top of the ridges, creating the characteristic vascular architecture needed for an efficient exchange of oxygen, carbon dioxide, and nutrients of the basically developed porcine placenta. © 1994 Wiley-Liss, Inc.     

Fowl Antibody: I. Some Physical and Immunochemical Properties

Fowl antibodies to rabbit γ globulin, bovine serum albumin and to ε toxin were studied by means of their reactions with rabbit antisera to fowl globulin. Specific precipitates redissolved either in antigen excess or in solutions of lower salt concentration than those in which they had been formed were used to measure the sedimentation and diffusion coefficients and the electrophoretic mobilities of the soluble complexes of fowl antibody with antigen.

Fowl antiserum to any one antigen was found to contain two types of homologous antibodies, derived from different constituents of serum, having the same electrophoretic mobility but giving two distinct bands of precipitation with rabbit anti-fowl-globulin serum. The values obtained for the molecular weights of soluble complexes indicate that one type of antibody has a molecular weight of about 600,000 and the other 180,000, and also suggest that the latter type combined with only one molecule of antigen in antigen excess. Both types of antibody were precipitated by antigen in 0.9 and 8 per cent NaCl; the larger precipitates formed at the higher salt concentration contained more of the low molecular weight antibody, together with another component that was neither antigen nor antibody.

     

Atherogenesis: the role of inflammation and infection

Atherosclerosis is no longer considered a disorder of lipid accumulation, but a disease process characterized by the dynamic interaction between endothelial dysfunction, subendothelial inflammation and the 'wound healing response' of the vascular smooth muscle cells. Prospective epidemiological studies have unequivocally demonstrated increased vascular risk in individuals with elevated levels of (i) cytokines such as interleukin-6 and tumour necrosis factor-alpha, (ii) cell adhesion molecules such as intercellular adhesion molecule-1 and P-selectin, and (iii) acute-phase proteins such as C-reactive protein, fibrinogen and serum amyloid A. Furthermore, evidence from clinical trials have demonstrated that risk reduction achieved with anti-inflammatory agents such as statins is significantly greater in patients with evidence of inflammation. A number of risk factors for atherogenesis, including infectious agents, have been shown to exert their influence via inflammatory mechanisms. However, despite compelling experimental evidence, clinical studies looking at the role of infection in atherogenesis have lacked consistency. The clinical product of this dynamic process is variable and unpredictable between individuals, even those with apparently similar risk profiles.     

Specific Inhibition of Antibody Production: I. Protein-Overloading Paralysis

A state of protein-overloading paralysis has been induced to bovine γ globulin (BGG) in adult CBA mice. Paralysis was demonstrated by a lack of immune elimination of ¹³¹I labelled BGG after prior challenge with Freund's adjuvant containing BGG. The degree of paralysis was found to diminish when the time between the paralysing injection and challenge injection was increased. This loss of the paralysed state could be prevented by injections of antigen whilst the mice were still paralysed.

The superficial nature of the differences between paralysis (unresponsiveness induced in adults) and tolerance (unresponsiveness induced in neonatals) to BGG in CBA mice is discussed. The possibility that suppression of antibody production in previously immunized animals given a massive dose of antigen, is a distinct phenomenon from the induction of paralysis/tolerance in animals not previously immunized, is briefly discussed.

     

Effect of Oxidation-Modified Fibrinogen on the Formation and Lysis of Fibrin Clot in the Plasma

Turbidimetry studies showed that after addition of thrombin to fresh donor plasma light scatter in the sample increases and slowly attains a plateau. The process of fibrin formation was less intensive in the presence of oxidized fibrinogen. The formation of fibrin clot in lyophilized plasma was characterized by a biphasic kinetic of light scatter, oxidized fibrinogen inhibited both phases of the process. In the presence of streptokinase, oxidized fibrinogen did not modify the kinetics of fibrin clot lysis. Addition of oxidized fibrinogen to plasma reduced optical density of fibrin clot the more intensely the higher was the degree of oxidative modification of fibrinogen.     

Antibody production in mice: I. The analysis of immunological memory

The development of immunological memory was analysed by the use of `in vivo culture technique'. The lymphoid cells from primed mice were transferred into heavily irradiated recipients and the size of memory cell population in primed donor mice was estimated quantitatively by the magnitude of secondary responsiveness.

The production of memory cells was detected about 1 week after the primary administration of antigen, and increased gradually up to approximately 6 weeks. The development of the memory cells carrying information for the synthesis of γG₁-antibodies (γG₁-memory cells) was initiated at the early stage of priming and followed by that of the γG₂-memory cells. Although the ratio of population of γG₂- to γG₁-memory cells changed with lapse of time in primed mice, the original ratio of each population in donor, at any stage after priming, was apparently maintained when cultured in recipients for at least 4 weeks. This indicates that the conversion from γG₁- to γG₂-memory cells does not occur.

From these results, it was suggested that the γG₁- and γG₂-memory cells develop independently in primed mice under the continuous stimulation of primarily administered antigen.

     

Humoral response in Treponema pallidum-infected guinea pigs: I. Antibody specificity.

Young male inbred strain 2 guinea pigs were infected intradermally with 8 X 10(7) Treponema pallidum extracted from a rabbit orchitis, and 5 months later reinfected with 10(7) T. pallidum. Ninety percent of the animals developed symptomatic lesions after initial infection but none on challenge. Immunoblotting of sera obtained at intervals after infection or reinfection showed antibodies against T. pallidum antigen (TP), nonpathogenic treponemes--T. phagedenis biotype Reiter (TR), T. refringens strain Noguchi (TN), and T. vincentii (TV)--as well as normal rabbit serum (NRS) and normal rabbit testes extract (NRT). Antibodies reacting with TP were detected as early as 17 days (five polypeptides) and steadily rose (at 3 months 17 polypeptides were seen). Cross-reacting antibodies to TR, TN, TV, or rabbit proteins decreased within 3 to 5 months. After reinfection, the antibodies to NRS increased more sharply than the anti-treponemal antibodies. Adsorption with TR and NRS of sera obtained after infection or reinfection produced a reduction of antibodies to TP by 75-87%.     

Fluorescent Protein Tracer Studies in Allergic Reactions: I. The Fate of Fluorescent Antigen in Active and Passive Arthus Reaction in the Guinea-Pig Skin

The fate of intradermally injected bovine serum albumin, labelled with lissamine rhodamine was studied in actively and passively sensitized guinea pigs, and compared with the fate of the same protein in control animals. In the latter, the antigen is taken up by macrophages, apart from a discharge of antigen to the regional lymph nodes.

In the sensitized animals, in which severe Arthus reactions occurred, an accumulation of antigen in vessel walls and lumina of capillaries and venules was observed. Polynuclear leucocytes take antigen after a few hours. This is a specific reaction absent with simultaneously injected protein to which the animal is not sensitized.