An immunoperoxidase method for the identification of B lymphocytes by light microscopy.

Peroxidase-conjugated anti-human immunoglobulin was used to determine the percentage of B lymphocytes in the mononuclear cell fraction of peripheral blood of 100 healthy persons (blood donors). Peroxidase activity was revealed by incubation with the usual mixture of 3'3 diaminobenzidine and hydrogen peroxide. Cells which were peroxidase-positive after incubation with benzidine solution alone were considered to be monocytes. The number of T lymphocytes was estimated by the formation of E rosettes. 19.6% mononuclear cells could be shown to be B cells, 4.1% were monocytes and 57.0% T cells. The use of peroxidase-conjugated anti-human immunoglobulin gives rise to the same percentage of B cells as the use of fluorescein-conjugated anti-human immunoglobulin. The advantage of the method described here is that B lymphocytes can be counted by conventional light microscopy.     

An alternative method for preparation of tissue sections from the rat mammary gland

In toxicopathological studies of the rat mammary glands, the guidelines of the Registry of Industrial Toxicology and Animal Data (RITA) recommend transverse sections of the inguinal mammary gland. However, occasionally limited amounts of mammary gland tissue are found in transverse sections. We compared transverse sectioning with an alternative method comprising horizontal sections of the rat mammary glands. Normal cycling female Sprague Dawley rats were sacrificed in proestrous, estrous, metestrous and diestrous, and samples from all mammary glands were collected. Transverse sections were prepared from the left-sided glands, and horizontal sections were cut from the right-sided glands. Sections were stained with HE, and epithelial and myoepithelial cells were visualized by immunohistochemical staining of cytokeratin 18 and alpha smooth muscle actin, respectively. Area of the mammary fat pad, mammary epithelium and connective tissue were measured in randomly sampled vision fields from each section. Horizontal sections contained a significantly larger area of mammary fat pad as well as glandular and connective tissue. No differences in tissue densities of epithelial or myoepithelial cells or connective tissue were observed between transverse sections and horizontal sections. Interestingly, densities of epithelium and connective tissue varied between cranial and caudal glands as well as the phases of the estrous cycle. In conclusion, horizontal histological sections of the rat mammary gland provided significantly larger samples of mammary gland tissue with no difference in tissue composition compared to transverse sections, which are recommended in the RITA guidelines.     

An improved method for purification of lymphocytes

A method that combines glass-bead column filtration, Ficoll-Hypaque gradient separation, discontinuous sucrose gradient, and drastic reduction of cell transfers is described. The procedure gives a high yield of pure human lymphocytes from small amounts of blood, good preservation of B cell/T cell ratio, and sufficient material for subsequent biochemical studies.     

An alternative method for cultivation of Lawsonia intracellularis

An alternative method for the cultivation of Lawsonia intracellularis, an obligate intracellular bacterium and the causative agent of proliferative enteropathy, was developed using an Original Space Bag inflated with a mixture of gas containing 10% hydrogen, 10% carbon dioxide, and 80% nitrogen. The flexibility of this protocol allows the testing of various environmental conditions for static cultivation of this bacterium and the development of diagnostic techniques.     

A new isolation method for rat intraepithelial lymphocytes.

Intraepithelial lymphocytes (IELs) play critical roles in gut immunity. In mice, gammadelta T cells are a large component of the IEL population. In the rat, gammadelta IELs are reportedly much less common, but technical issues suggest that previous analyses should be interpreted cautiously. The study of IELs in rats has been impeded by isolation procedures that are lengthy and complex, leading to small cell yields. For this reason, it is possible that rat IELs analyzed in previous studies have not been representative of the entire IEL compartment. We report a new method for the isolation of rat IELs that is based on the selective removal of intestinal epithelial cells under conditions that leave the basement membrane undisturbed. The method is rapid and requires neither enzymatic digestion, nor surgical removal of Peyer's patches, nor vigorous mechanical manipulation of the intestine. The yield of rat IELs using this method is 5- to 10-fold greater than that reported for other methods. Morphological and phenotypic analyses demonstrated that the purified cell population is comprised of IELs and is not contaminated with lamina propria or Peyer's patch lymphocytes. Phenotypic analysis revealed five major subsets of IELs based on differential cell surface expression of CD4, CD8, and alphabeta T cell receptor (TcR). Among the alphabetaTcR- cells was a population of gammadelta T cells present at levels not previously detected. The isolation of IEL sub-populations using this methodology should facilitate studies of the function of these cells in gut immunity.     

A simple panning method for the selection of cell surface antigen transfectants

Mouse Ltk- (H-2k) cells that became reactive with anti-H-2d monoclonal antibodies (McAb) after transfection with DNA from H-2d-positive cells were isolated by an indirect panning method. The cells were transfected with DNA from a plasmid containing the selective thymidine kinase (tk) gene in addition to total cell DNA, so that a first selection could be carried out in hypoxanthine/aminopterin/thymidine (HAT) medium. The HAT-selected tk+ transfectants were incubated with anti-H-2d McAb, washed and transferred to dishes coated with purified anti-immunoglobulin (anti-Ig). This 2-step method of panning has the advantage that only the anti-Ig reagent requires purification. Transfected cells clearly reactive with either anti-Ld/Rd or anti-Kd McAb were isolated after only 1 or 2 cycles of panning.     

Postnatal undernutrition: An alternative method

Rat pups were undernourished from birth by placing them for 12 hr/day with a normal lactating mother and 12 hr/day with a nipple-ligated mother each day for 25 days. The method resulted in a marked delay in the body growth of the undernourished pups, especially during the first 2 weeks of life. Observations of the behavior of the mothers towards the underfed pups were made at different times of the day and compared to the behavior of the mothers suckling well-fed pups. The results show that (1) nipple-ligated mothers are able to provide adequate maternal care for undernourished pups, and (2) both ligated and nonligated mothers caring for underfed pups spend more time with those pups than mothers caring for well-fed pups.     

An alternative method for significance testing of waveform difference potentials

Guthrie and Buchwald (1991) proposed an ad hoc procedure for assessing the statistical significance of waveform difference potentials that may arise in a variety of psychophysiology research contexts. In our paper, an alternative method is presented and demonstrated that has fewer underlying assumptions than does the Guthrie-Buchwald test and may, therefore, produce better results in some situations. In particular, the test proposed here (a) is distribution free, (b) requires no assumption of an underlying correlation structure (e.g., first-order autoregressive), (c) requires no estimate of the population autocorrelation coefficient, (d) is exact, (e) produces p values for any number of subjects and time points, and (f) is highly intuitive as well as theoretically justifiable. This procedure may be used to carry out multiple comparisons with exact specification or experimentwise error, however, this test is based on permutation principles and may require large amounts of computer time for its implementation.     

Mycoplasma neurolyticum: a potent mitogen for rat B lymphocytes.

A mitogen prepared from Mycoplasma neurolyticum has been demonstrated to induce extensive transformation of in vitro cultured rat B lymphocytes. The data summarized in this report show that rat thymus cells as well as hydrocortisone-resistant thymocytes were not activated by this mitogenic agent. On the other hand, spleen cells obtained from thymectomized, lethally irradiated and bone marrow-reconstituted rats were extensively activated by M. neurolyticum. Furthermore, M. neurolyticum was shown to induce the development of antibody-producing cells, as attested by the appearance of direct plaque-forming cells against sheep red blood cells and trinitrophenylated sheep red blood cells in spleen cell cultures exposed to this mitogen. It was also demonstrated that stimulation of rat lymphocytes by this mitogen was inhibited by anti-rat immunoglobulin antibodies. In view of these data, it was suggested that M. neurolyticum, which activates mouse B lymphocytes, is a potent mitogen for rat B lymphocytes as well. This mitogen is a significantly more powerful mitogen for rat B lymphocytes then any other known mitogens. The availability of such mitogenic material in the rat system will enable studies on control mechanisms of action and differentiation of rat B lymphocytes.     

Activation of rat B lymphocytes by Actinobacillus actinomycetemcomitans.

We examined the lymphoproliferative responses of cervical lymphocytes and splenocytes of homozygous (rnu/rnu) congenitally athymic nude and normal heterozygous (rnu/+) Rowett rats to whole cells of Actinobacillus actinomycetemcomitans, a suspected periodontal disease pathogen. Previously sensitized cells from immunized only, infected only, or immunized and infected, normal rats demonstrated proliferation in response to formalinized A. actinomycetemcomitans, but cells from nude rats did not proliferate. The maximum antigenic response was observed at day 5 of culture. A. actinomycetemcomitans caused cervical lymphocytes and splenocytes from untreated naive normal and nude rats to undergo increased DNA synthesis at day 2 of culture. Highly enriched nonsensitized spleen T cells prepared on a nylon wool column did not respond to A. actinomycetemcomitans, whereas enriched nonsensitized B cells proliferated. Differences in response were probably not attributable to contributions from macrophages in the T- or B-cell populations, since macrophage percentages were approximately the same in both preparations. T-cell reconstitution of nude rats with neonatal thymus cells from rnu/+rats resulted in partial recovery of T-cell function but had no effect on the mitogenic response to A. actinomycetemcomitans. It is suggested that the antigenic responses to A. actinomycetemcomitans are dependent on T cells and that A. actinomycetemcomitans cells have mitogenic activity for B cells. The potential importance of these findings in periodontal disease is discussed.     

Physiological significance of thymic B lymphocytes: An appraisal

Recent observations suggest that minor cell populations located in the thymus play critical roles in T-cell development and T-cell repertoire selection (Möller, 1988). Although known for a long time (Miller and Osoba, 1967), little attention has been paid to intrathymic B lymphocytes (TBL). This subpopulation is found throughout life, and represents 0.1–1% of all “thymocytes” and up to 30–60% of the intrathymic cells left after negative selection with anti-Thy-1 antibodies (Isaacson et al., 1987; Miyaba-Inaba et al., 1983; Andreu et al., submitted). In the past, thymic B lymphocytes (TBL) have been considered as “recirculating” B cells, and their thymus localization as being devoid of particular significance. However, their presence at very early ontogenic stages, as well as the appearance of subclasses of antibody-secreting cells in newborn thymuses before any other lymphoid tissues (table I), and other findings discussed below suggest their specific localization and physiological role inside the thymus.     

A new method for detachment of Dynabeads from positively selected B lymphocytes.

This paper describes a method for the detachment of immunomagnetic beads from positively selected human B lymphocytes. After rosetting of B cells using anti-CD19 coated magnetic beads (Dynabeads M-450 Pan B, Dynal), the Dynabeads were rapidly detached (efficiency 80%) from the cells using goat anti-mouse-Fab antiserum (DETACHaBEAD, Dynal) at ambient temperature. Isolated B cells did not show significant differences in the expression of a number of B cell antigens when compared to B cells stained in fresh whole blood. In contrast, positively selected B cells that had detached from the beads following overnight incubation, demonstrated a significantly reduced expression of certain of the antigens examined (CD19, CD20 and CD23). It was further demonstrated that neither anti-CD19 nor anti-Fab resided on the surface of the cells after detachment. The cells were still in G0 phase (greater than 90%) at the end of the isolation procedure. Moreover, anti-IgM antibodies stimulated the vast majority of the cells to leave the G0 phase, and to progress through S phase in the presence of growth factors. The cells could also be stimulated to differentiate, further confirming the normal functional capacity of the isolated cells. The method described in this paper can also be used for the detachment of other positively selected cells, such as CD4+ T cells, CD8+ T cells and CD34+ stem cells.     

An improved and rapid method for the isolation of rat lymph node or spleen T lymphocytes for T cell proliferation assays.

To detect and compare the capacity of antigen presenting cells to present antigen in a T cell proliferation assay, it is necessary to obtain a pure population of antigen-primed T cells that gives low background proliferative responses. Therefore in this paper we present a newly developed isolation method of antigen-primed T lymphocytes from rat spleen or lymph nodes. This method uses a nylon wool column to deplete most of the adherent cells and B cells, followed by an indirect elimination method with magnetic beads to remove contaminating Ia-positive cells. We compared this method with two commonly used isolation methods, namely a 1.5 h adherence step, followed by a nylon wool column and a Sephadex G-10 column and a 1.5 h adherence step followed by a passage through two consecutive columns of Sephadex G-10. The best T cell enrichment (98% OX-19/52-positive cells) was achieved with the newly developed method, in which the contamination of Ia-positive cells, predominantly B cells and dendritic cells (DC), was diminished to less than 2%. The background response of this population was low and differed significantly with the common methods. Antigen-specific T cell responses induced by splenic DC, expressed as stimulation index, gave very specific responses and showed a steep rise with increasing DC concentrations compared to the common methods. Therefore we conclude that we developed an improved, rapid and reproducible method for the isolation of rat spleen or lymph node T lymphocytes suitable for T cell proliferation assays.     

An alternative immunohistochemical method for detecting Leishmania amastigotes in paraffin-embedded canine tissues

Canine visceral leishmaniasis (CVL) is a zoonosis and a chronic systemic disease of the dog caused by a protozoan by the species Leishmania infantum in the Old World and Leishmania chagasi in the New World. Several methods are currently employed for the diagnosis of CVL including microscopic detection of the parasite in bone marrow and lymph node aspirates, demonstration of specific antibodies anti-Leishmania in sera from infected animals, and isolation of the parasite by in vitro culture or by inoculation of laboratory animals. However, a definitive diagnosis is based on the actual detection of the parasite, which is conventionally achieved by examining Giemsa-stained smears or histopathological sections stained with hematoxylin and eosin. These methods have a low sensitivity, and therefore, they are often inconclusive. This is particularly true in canine organs that have a low level of parasitism such as kidneys, lungs, central nervous system, and testis, or, in some cases, the skin. The technique for immunohistochemical detection of leishmanial amastigotes in canine tissues has been reported previously and has proved to be undoubtedly efficient for the diagnosis. In this paper, we describe a straightforward and inexpensive immunohistochemical approach for Leishmania detection in formalin-fixed paraffin-embedded canine tissues. Amastigote forms of Leishmania were easily observed within macrophages in several organs from naturally infected dogs using the streptavidin-biotin immunohistochemical method with canine hyperimmune serum as the primary antibody. In addition, the secondary antibody used was not specific to canine immunoglobulin, characterizing a cross-immune reaction. Our results indicate that this technique could be a useful tool for epidemiological, clinical, and histopathological studies.     

The DDT method: An alternative to the use of radiolabelled microspheres for the determination of regional blood flows in the rat

Intravenous administration of the 14C-radiolabelled insecticide DDT--1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane--can be used for the estimation of regional blood flows in small rodents. A mathematical model is presented which enables the conversion of relative into absolute blood flows.     

Further evidence for coelomic-associated B lymphocytes

Previous observations indicate that "CD5 B lymphocytes" are preferentially clustered in gut-related mesenchymal areas, such as peritoneum, thymus and tonsils. We have now found that pleuropericardial spaces contain an homogeneous population of large-sized, noncycling, nonsecretory B cells, expressing very high levels of surface IgM, little or no IgD, Mac-1 and low levels of B220. This phenotype and the over-representation of some antibody clonotypes suggest that the pleuropericardial cavity contains a pure "CD5 B cell" population. In all mouse strains analyzed, however, many of these cells are CD5-. These findings, together with the common origin of peritoneum and pleural layers in the primitive coelomic cavity, suggest that such B cells differentiate locally from intraembryonic precursors; we propose to designate them as "coelomic", to distinguish them from "stromal", bone marrow-derived B cells.     

An alternative method for calculating changes in arterial calf blood flow at rest

Summary In 20 patients we studied the changes in calf arterial blood flow (AF) following spinal and epidural blocks, using venous occlusion strain gauge plethysmography (SGP). AF was calculated both in the conventional way by drawing a tangent to the initial upslope of the curve, and by a new method which measures the time to the point when 50% of venous capacity is reached (tVC₅₀). The statistical differences within and between the spinal and epidural groups for AF and tVC₅₀ were determined. In measuring the post-block changes in AF as compared with the control values, a statistically significant correlation (r_s=–0.85, p<0.01) was observed between the two methods. We conclude that the new variable, tVC₅₀, seems to be potentially useful in calculating changes in arterial blood flow at rest, and could be used, for example, in connection with surgical and pharmacological interventions, especially when the initial upslope of the SGP curve is equivocal.