Survey of genetic authenticity of commercially produced inbred rats

Strain specific typing antisera (SSTA) were used to genetically monitor six inbred strains of rats from major US commercial producers. SSTA were used in both the hemagglutination test and the microcytotoxicity assay because results from a preliminary study showed that use of both tests gave more reliable results than either test used individually. Of 25 colonies tested (representing all six strains), one colony of LEW rats appeared to be contaminated genetically.     

Trikatu,an herbal compound as immunomodulatory and anti-inflammatory agent in the treatment of rheumatoid arthritis – An experimental study

In the present study, trikatu, an herbal compound was evaluated for its immunomodulatory and anti-inflammatory properties with reference to cell mediated immune responses (delayed type hypersensitivity reaction), humoral immune response (haemagglutination titer and plaque forming assay), macrophage phagocytic index, circulating immune complex and inflammatory mediators in rats. For comparison purposes, indomethacin was used as a reference drug for anti-inflammatory studies. The results obtained in our study showed a significant decrease in cell mediated immune responses, humoral immune responses (haemagglutination titre and plaque forming assay) and macrophage phagocytic index in trikatu treated rats (1000 mg/kg/b.wt.) compared to control animals implying its immunosuppressive property. In addition, significant anti-inflammatory effects were observed in trikatu treated adjuvant induced arthritic rats by a reduction in the levels of circulating immune complexes and inflammatory mediators (TNF-alpha and Interleukin-1beta). Thus, in conclusion, our data suggest that trikatu could be considered as a potential anti-inflammatory agent for treating autoimmune inflammatory disorders like rheumatoid arthritis with immunosuppressive property.     

Immunological Methods for the Detection of a Coryneform Isolated from Ratoon Stunted Sugarcane

A coryneform bacterium, isolated from ratoon stunted sugarcane, has been obtained in pure culture. Its identification by immunological means was investigated. Optimum conditions for the light microscopic observation of bacterial agglutination by specific antiserum, immunofluorescence and the microcapillary haemagglutination of IgG-sensitized erythrocytes by the bacterium were established. All three assay procedures are simple and rapid. By comparison, more samples can be handled at any one time bythe microcapillary haemagglutination method. Based on bacterial agglutination, the antiserum is specific for the coryneform isolated from ratoon-stunted sugarcane.     

Rapid assay for antibodies to Varicella-Zoster virus (VZV) using a VZV-HEM preparation

A freeze-dried, formalized-erythrocytes-bound VZV antigen for indirect haemagglutination, VZV-HEM, was prepared. It was used to test serologically 46 children, all of them patients of Prague paediatric clinics, with a known history of chickenpox. For comparison, the same sera were tested by the indirect haemagglutination reaction with freshly prepared VZV antigen and by ELISA. All three serological tests gave congruent results in 97.8% of cases. The advantages of the VZV-HEM assay are results obtainable within 2 hours of serum dilution and simplicity. The assay is especially suitable for emergency testing the immunity of persons at risk.     

近交系HFJ大鼠的培育及其部分表型观察

目的培育高繁殖力近交系HFJ大鼠并观察其部分表型特征。方法以一对Wistar大鼠为基代,通过全同胞近亲交配方式,采用选优法培育高繁殖力近交系HFJ大鼠。采用生化标记法、皮肤移植实验进行遗传质量检测;观察比较其部分表型特征。结果HFJ种群符合近交系标准;其窝产仔数、离乳率、胎次间隔显著高于Wistar大鼠;HFJ大鼠12周龄后生长缓慢,体重低于同龄Wistar大鼠;HFJ大鼠部分血液常规指标及血液生化指标与Wistar大鼠有显著差异。结论HFJ大鼠繁殖力强,是具有独特生物学特性的近交系大鼠。     

Evaluation of three serological tests for the detection of human plague in northeast Brazil.

The passive haemagglutination (PHA) test, enzyme-linked immunosorbent assay (ELISA) and the dot enzyme-immunosorbent assay (DOT-ELISA) were used to detect the levels of IgG antibodies against the Fraction 1 (F1) antigen of Yersinia pestis in sera of plague-infected patients from Northeast Brazil. Twenty three selected PHA-positive sera of subjects with bacteriological confirmation of plague were also positive in the DOT-ELISA but only 19 were detected by the conventional ELISA technique. Another group of 186 serum samples from subjects diagnosed as plague-infected by clinical and epidemiological parameters, but PHA-negative, were screened with DOT-ELISA and 11 gave positive results. The specificity of the assays on the serological detection of plague was confirmed in inhibition tests using purified F1 antigen. These results suggest that DOT-ELISA can be an useful, simple and more sensitive alternative for the serodiagnosis of plague in Northeast Brazil.     

Use of simple sequence length polymorphisms for genetic characterization of rat inbred strains

Genetic monitoring is an essential component of colony management and for the rat has been accomplished primarily by using immunological and biochemical markers. Here, we report that simple sequence length polymorphisms (SSLPs) are a faster and more economical way of monitoring inbred strains of rats. We characterized 61 inbred strains of rats, using primer pairs for 37 SSLPs. Each of these loci appeared to be highly polymorphic, with the number of alleles per locus ranging between 3 and 14 and, as a result, all the 61 inbred strains tested in this study could be provided with a unique strain profile. These strain profiles are also used for estimating the degree of similarity between strains. This information may provide the rationale in selecting strains for genetic crosses or for other specific purposes.     

微卫星DNA标记在近交系大鼠HFJ和MIJ遗传监测中的应用

目的 用24对引物对近交系HFJ和MIJ大鼠的微卫星位点进行多态性分析,并选用近交系Lewis和F344大鼠作为对照,进行比较分析.方法 用传统的酚-氯仿法分别提取4个近交系大鼠MIJ、HFJ、Lewis和F344 的基因组DNA,选取大鼠24个微卫星位点,通过PCR扩增,扩增产物经过非变性聚丙烯酰胺凝胶电泳和银染,根据电泳结果,比较分析4种品系近交系大鼠之间微卫星多态性.结果 4种品系及品系内不同个体的近交系大鼠在24个微卫星位点上的扩增产物均出现一个条带,MIJ和HFJ大鼠在品系间和品系内均表现为单态性,同Lewis 和F344的扩增结果比较,14个位点显示多态性,有10个位点显示单态性.结论 两个近交系大鼠品系MIJ和HFJ符合近交系要求,筛选出的14个多态性微卫星位点可用于有关近交系大鼠的遗传背景监测.     

The effects of experimental conditions on the expression of in vitro-mediated tumor cytotoxicity mediated by murine macrophages

Summary The level of target destruction by tumoricidal mouse peritoneal macrophages was studied under various culture conditions. The degree of macrophage homogeneity in the monolayer was affected by the presence of fetal calf serum in the plating medium and the time allowed for cell adherence. Macrophage tumoricidal activity was not dependent on the ratio of macrophages to target cells, but on the densities of the two cell types in the culture vessel. The development of an in vitro microcytotoxicity assay for the evaluation of tumor cell lysis by activated macrophages revealed that serum concentration and the type of target cell used in the assay may affect the degree of tumoricidal response.     

Kinetics of synthesis and immunologically active fraction of anti-tumor Immune RNA

The kinetics of synthesis of anti-tumor Immune RNA (I-RNA) in immunized rodents was determined and an immunologically active fraction of I-RNA isolated. As measured in a microcytotoxicity assay for cell mediated immunity, cytotoxic immune reactivity of syngeneic I-RNA was maximal when extracted from the spleens of Fischer rats 21–28 days following their inoculation with 10⁶ syngeneic MC3-R tumor cells. Maximum immunoreactivity of xenogeneic I-RNA extracted from the lymphoid organs of guinea pigs immunized with MC-1 mouse tumor cells was reached 14 days after immunization. Both syngeneic and xenogeneic anti-tumor I-RNA were fractionated in preparative sucrose density gradients. The highest cytotoxic immune reactivity was consistantly obtained from I-RNA fractions with sedimentation values of 12–16S. The immunologically active I-RNA comprised only 5–7% of the total RNA extracted from the lymphoid tissues of immunized animals.     

Comparative methods of detecting HbsAg in chronic glomerulonephritis patients in Nigeria

Abstract A comparative evaluation of the enzyme-linked immunosorbent assay (Elisa), passive haemagglutination (PHA) and counterimmunoelectrophoresis (CIE) methods were carried out. Approx. 6% of control samples were positive with the Elisa assay, while the values for Behring Institute (BI) passive haemagglutination, Wellcome Research Laboratory (WRL) passive haemagglutination and CIE were 2.7%, 2.2% and 3.3%, respectively. The above data contrast with values of 21.3%, 10.6, 10.3 and 12.3 obtained in sera from chronic glomerulonephritis patients.
Our observation suggests that HbsAg may be associated with chronic glomerulonephritis.     

Characterization of a genetically segregating determinant of chicken fetal antigen by a new hapten inhibition of microcytotoxicity (HIM) assay

The immunodominant structure of a chicken fetal antigen (CFA) determinant was investigated using a new hapten inhibition of microcytotoxicity (HIM) assay. This HIM assay employing avian erythrocytes was shown to be a highly sensitive, economical technique and was verified in a separate experiment using bovine serum albumin (BSA) coupled to Japanese quail peripheral red blood cells (QPRBCs). Inhibition of microcytotoxicity was measured following preincubation of 1 µl of specific antiserum with 1 µl of antigen solution. A concentration of 21.1nm BSA was found to produce effective (50%) inhibition of microcytotoxicity of BSA-coated QPRBCs. The percentage cytotoxicity was determined by estimating the proportion of intact RBCs to free nuclei using an inverted microscope. Staining of the reaction mixtures was not required for scoring. Application of this technique for the characterization of immunodominant structures was demonstrated by the analysis of a CFA determinant known to exhibit both developmental expression and genetic segregation. R-anti-CFA-10 was effectively inhibited only by d-galactose (35.5mm) and the galactose-containing sugars lactose (28.2mm), raffinose (29.9mm), and stachyose (39.8mm). Implications of the carbohydrate nature of CFA are discussed.This work was supported by Grant PCM-8109810 from the NSF and NY(C) 157424 from the USDA.     

Distribution of alloantigens on human Fc receptor-bearing lymphocytes: the presence of B cell alloantigens on sIg-positive but not sIg-negative lymphocytes.

Human peripheral blood lymphocyte subpopulations were analyzed for the presence of B cell alloantigens with a microcytotoxicity assay. B cell alloantigens were found exclusively on sIg-positive lymphocytes and were not present on sIg-negative, Fc receptor-bearing lymphocytes or sIg-negative, Fc receptor-negative T lymphocytes.     

An attempt to identify the intestinal receptor for the K88 adhesin by means of a haemagglutination inhibition test using glycoproteins and fractions from sow colostrum.

The K88 antigen of Escherichia coli specifically adheres to the piglet intestinal cell; a solution of this antigen agglutinates guinea-pig red cells at 4 degrees C. The latter reaction was used as a model of the former, using inhibition of haemagglutination as an index of specific combination with the K88 adhesin. Inhibition was found with mucous glycoproteins and chemical modification of their heterosaccharide residues by mild acid hydrolysis, periodate oxidation or the Smith degradation procedure suggested that the terminal beta-D-galactosyl structure in a heterosaccharide sidechain of a glycoprotein might combine specifically with the K88 adhesin and inhibit haemagglutination. One serum glycoprotein (fetuin), after exposure of its subterminal beta-D-galactosyl residue, also inhibited haemagglutination, but high inhibitory activity was exhibited by some submaxillary glycoproteins in which this structure was absent or not prominent. It was concluded that in some cases inhibition of haemagglutination by glycoprotein was non-specific. No inhibition was found using glycosaminoglycans, glycogen or any simple sugar or glycoside. Sow colostrum was inhibitory but this was associated mainly with its gamma-globulin fraction. Some inhibitory activity was traced to a colostral glycopeptide fraction of low molecular weight but the smaller colostral oligosaccharides were not inhibitory; the composition of these components in sow colostrum is reported.     

Respiratory responses to leukotrienes and biogenic amines in normal and hyperreactive rats

The effects of leukotriene D4, serotonin, and methacholine were studied on respiratory smooth muscle in vitro and respiratory responses in vivo in three strains of rats. These were an inbred strain of hyperresponsive rats, Sprague Dawley rats, and Fischer rats. Trachea from inbred rats responded in vitro to serotonin and methacholine but not to leukotrienes or histamine. Parenchyma from inbred rats responded to serotonin, methacholine, and leukotrienes. In vivo respiratory responses in inbred rats were observed after aerosol administration of histamine and serotonin, methacholine, and leukotriene D4. When these in vitro and in vivo experiments were repeated in Sprague Dawley and Fischer rats, a clear correlation was observed between the responses of strains of rats to aerosolized antigen and responses to spasmogenic mediators. It is concluded that inbred rats have a nonspecific bronchial hyperreactivity that contributes to their sensitivity to aerosolized antigen and that they may be a useful model for human asthmatic conditions.     

Autoimmunity to glial antigens in vitro.

Lymph nodes cells and spleen cells in a 50:50 mixture from Fischer 344 rats were cultured on syngeneic glial cells and fibroblasts. In the glia cultures, but not in the fibroblast cultures, the lymphocytes were stimulated to a vivid blast transformation and mitotic activity with a peak after 7 days, after which they reverted to small- and medium-sized lymphocytes. The stimulated lymphoid cells were not cytotoxic to glial cells when tested in a microcytotoxicity assay. A fraction of the lymphoblasts and their progeny (approximately 4%) took a positive intracytoplasmic stain for γ-globulin immunoglobulin G after direct immunofluorescence. Efforts to demonstrate immunoglobulin produced and released into the culture medium by the stimulated cells were negative. The findings may indicate that among the lymphoid cells responding to the glial antigens under these conditions, there are suppressor cells that abrogate the killer cell effect.     

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37°C for 60 minutes. After this time, the samples can then be easily scored for ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.Open in a separate windowClick here to view.^{(20M, flv)}     

Natural cytotoxicity

Summary The microcytotoxicity assay was used to examine lymphocyte subpopulations derived from healthy donors or persons suffering from non-malignant diseases for natural cytotoxicity.Part of the natural cytotoxicity may be caused by cells devoid of conventional surface markers. However, most of the natural cytotoxicity was found to be associated with cells bearing Fc receptors, irrespective of T and non-T identity.The Fc receptors appeared to be directly involved in the mechanism of natural cytotoxicity, since blocking of these receptors with Fc fragments or immune complexes significantly reduced this reactivity.Sponsored by the Danish Cancer Society     

Genetic map of 16 polymorphic markers forming three linkage groups assigned to rat Chromosome 4

Sixteen polymorphic markers, including markers for eight new loci, forming three linkage groups, were assigned to rat Chromosome (Chr) 4 by linkage analysis of the progeny of an F₂ intercross of Fischer (F344/N) and Lewis (LEW/N) inbred rats. One gene, Igk, was mapped by restriction fragment length polymorphism (RFLP) analysis. One marker for Tcrb was identified by the polymorphic insertion of a repetitive LINE element. The remaining 14 markers contained polymorphic simple sequence repeats (SSRs). Ten were identified in genes (Tgfa, Npy, Prss1, Prss2, Aldr1, Iapp, Prp, Eno2, Cacnlla1, and Il6), one was identified in a sequence related to a gene (Egr4l1), and three were identified in anonymous DNA segments. The SSR markers were highly polymorphic in 16 inbred rat strains. These markers expand the genetic map of the rat and should be useful in future genetic studies of inbred rats.     

Comparison of enzyme-linked immunosorbent assay, electron microscopy, and reversed passive haemagglutination for detection of human rotavirus in stool specimens

An enzyme-linked immunosorbent assay (ELISA) using microplates as solid phase, rabbit antiserum against human rotavirus Wa strain as catching antibody, and the same reagent labeled with beta-D-galactosidase as conjugate, has been developed for detection of human rotavirus antigen(s) in stool specimens from patients with acute gastroenteritis. The limit of detection of purified human rotavirus by ELISA was 15.6 ng/ml (1.56 ng/well) of viral protein. The sensitivities of ELISA, electron microscopy, and the reversed passive haemagglutination method (ROTA-CELL) were compared. ELISA was more sensitive than electron microscopy and the reversed passive haemagglutination method. The ELISA blocking assay was useful for detection of an antibody response to human rotavirus in paired sera from children in two institutions during outbreaks of rotavirus gastroenteritis.