IgM-bearing Cells Research Articles

Effect of lignin supplementation of a diet contaminated with Fusarium mycotoxins on blood and intestinal lymphocyte subpopulations in chickens

The objective of the study was to investigate the effects of lignin supplementation of a diet contaminated with the Fusarium mycotoxins deoxynivalenol (DON) and zearalenone (ZEA) on peripheral blood leukocytes and duodenal immunocompetent cells in broiler chickens. From day 1 after hatching, all chickens were fed an identical control diet for two weeks. Then chickens of Group 1 continued to be fed the control diet, whereas Group 2 was fed the same diet supplemented with lignin at 0.5% level. Simultaneously, Group 3 started to receive a diet contaminated with DON (2.95 mg kg-1) and ZEA (1.59 mg kg-1), while Group 4 received an identical contaminated diet supplemented with 0.5% lignin for further two weeks. Samples of blood and duodenal tissue were collected from 6 birds of each group at 4 weeks of age. Neither counts of white blood cells nor phagocytic function in the peripheral blood were significantly affected in the mycotoxin- and/or lignin-treated birds. As compared to the control, increased numbers of IgM-bearing cells were found in the peripheral blood in Group 3 fed the contaminated diet (P < 0.05) and in Group 4 given the contaminated diet supplemented with lignin (P < 0.01). While the contaminated diet led to reduced numbers of duodenal CD4+ cells, in Group 2 treated only with lignin the number of duodenal CD4+ cells was increased. Lignin enrichment of the contaminated diet did not eliminate the mycotoxin-induced reduction in the number of duodenal CD4+ cells. The results suggest that dietary supplementation of lignin as an indigestible compound to poultry feed may increase the density of some intestinal immunocompetent cells without exerting effects on that in the peripheral blood. However, when added to a diet contaminated with Fusarium mycotoxins, lignin did not prevent the mycotoxin-induced changes in the numbers of blood and intestinal immunocompetent cells.

Microhabitat preference of Anisakis simplex in three salmonid species: Immunological implications

Three salmonid fish species, Oncorhynchus mykiss, Salmo salar and Salmo trutta, were infected experimentally with the parasitic nematode Anisakis simplex (A. simplex) and the difference between in vivo behaviour of the nematode in the three fish species was investigated. Infection success rate differed between species. S. salar (Baltic salmon) showed the highest number of successfully established nematodes, whereas S. trutta (brown trout) and O. mykiss (rainbow trout) had a higher natural resistance. Microhabitat selection of nematodes differed according to fish species. In brown trout, A. simplex larvae were attached to the digestive tract (stomach, pyloric caeca, intestine), while the majority of larvae found in rainbow trout were located between the pyloric caeca. In Baltic salmon, nematodes were dispersed in and on spleen, head kidney, liver, swim bladder and musculature. Encapsulation and inflammatory cellular reactions differed accordingly. Histopathological and immunohistochemical studies using monoclonal antibodies raised against salmonid IgM, CD8 and MHCII were performed to detect the presence of immune cells around the infecting nematodes. None of the three fish species showed positive reactions for IgM-bearing cells in the inflammatory tissue connected with nematodes. CD8+ cells were detected in all three species and MHCII-bearing cells were found associated with encapsulated A. simplex in rainbow trout and brown trout, but not in Baltic salmon. Physiological, immunological and pathological implications of microhabitat differences are discussed.

Immunological status of the progeny of breeder hens kept on ochratoxin A (OTA)- and aflatoxin B1 (AFB1)-contaminated feeds

This study aimed to evaluate the immunological status of progeny of hens kept on ochratoxin A (OTA)- and aflatoxin B1 (AFB1)-contaminated feed. For this purpose, White Leghorn (WL) layer breeder hens (40-weeks-of-age) were divided into six groups (A–F). Hens in Group A were fed a commercial layer ration while those in Groups B and C were kept on a diet amended with 3 and 5 mg OTA/Kg, respectively. Group D was fed a ration containing 5 mg AFB1/Kg, while hens in Groups E and F were kept on feed amended with OTA and AFB1 each. All feedings were for 1, 2, or 3 weeks. Fertile eggs were set for hatching on a weekly basis to obtain progeny of each week separately. At 14 days-of-age, subsets of progeny were euthanized and the frequency of immunoglobulin(s)-bearing cells in their spleen and bursa of Fabricius assessed; at 16 days-of-age, other chicks in each set were utilized to determine their lymphoblastogenic responses against phytohemagglutinin (PHA-P). At 30 days-of-age, the final sub-set of chicks/group was euthanized and their peritoneal macrophages harvested for measurements of phagocytic potential and nitrite production. Relative weights of the bursa of Fabricius and of the spleen were significantly lower in the progeny of hens fed mycotoxin-contaminated diets for 14 and 21 days. The frequencies of IgA-, IgG-, and IgM-bearing cells were also significantly lower in the bursa of Fabricius and spleen of progeny chicks obtained from hens fed the OTA + AFB1 mixed diet. Feeding contaminated diets to breeder hens also resulted in significantly lower responses to PHA-P. In addition, the percentages of peritoneal macrophages displaying phagocytosis of sheep red blood cells (SRBC), the number of SRBC/macrophage, and nitrite production were each significantly lower in cells from progeny chicks from OTA- and AFB1-fed hens. The findings of the present study indicated there were severe immunosuppressive effects in progeny chicks as a result of exposure of their parent hens to OTA and AFB1 either alone or in combination. These studies provide emphasis for the need for mycotoxin regulation policy with respect to the ingredients used in poultry feed, since it is clear that feeding multi-mycotoxin-contaminated diets to breeder hens will almost certainly result in the hatching of manifestly unhealthy chicks.

Immunological status of the progeny of breeder hens kept on ochratoxin A (OTA)-contaminated feed

This study aimed to evaluate the immunological status of the progeny of breeder hens kept on ochratoxin A (OTA)-contaminated feed. For this purpose, 84 White Leghorn (WL) layer breeder hens (40-weeks-of-age) were divided into seven groups (A–G). Hens in the Group A were fed a commercial layer ration while those in Groups B–G were kept on a diet amended with 0.1, 0.5, 1.0, 3.0, 5.0, or 10.0 mg OTA/kg, respectively, for 3 weeks. Fertile eggs were set for hatching on the weekly basis to get the progeny of each week separately. Hatched chicks (n = 10 from each group) were euthanized at Day 14 of age, and their immunological organs weighed and fixed in neutral buffered formalin. An indirect immunoperoxidase method was applied to study the frequency of immunoglobulin(s)-bearing cells in the spleen and bursa of Fabricius from these progeny. From other chicks within each set, at Day 16 of age, lymphoblastogenic responses against an intradermal administration of phytohemagglutinin (PHA-P) were determined. Relative weights of the bursa of Fabricius and of the thymus were significantly lower in the progeny of hens fed OTA-contaminated diet for 14 and 21 days. The frequencies of IgA-, IgG-, and IgM-bearing cells were also significantly (P ≤ 0.05) lower in the bursa of Fabricius and spleen of the progeny chicks obtained from dams fed the OTA-mixed diet. Progeny chicks obtained from the breeder hens fed higher doses of OTA showed significantly lower responses to PHA-P than did counterpart chicks from control hens. The findings of this study suggested that there were immunosuppressive effects from OTA in the progeny obtained from breeder hens kept on OTA-contaminated diets.

Characterization of mast cell numbers and subtypes in biopsies from the gastrointestinal tract of dogs with lymphocytic-plasmacytic or eosinophilic gastroenterocolitis

It has been suggested but not proven that hypersensitivity type I reactions are involved in the pathogenesis of canine inflammatory bowel disease (IBD). The main effector cells in type I hypersensitivity reactions are mast cells (MCs). Canine MCs, as human MCs, can be subdivided into three subtypes according to their content of mast cell-specific proteases: tryptase (MC T), chymase (MC C), or tryptase and chymase bearing MCs (MC TC). In this study, numbers and subsets of mast cells were investigated in biopsies from the gastrointestinal tract of dogs with histopathologically confirmed lymphocytic-plasmacytic enteritis (LPE) ( n = 4), lymphocytic-plasmacytic colitis (LPC) ( n = 1) and eosinophilic gastroenterocolitis (EGE) ( n = 11). Paraffin sections of formalin-fixed samples from the stomach, small intestine (duodenum, jejunum, ileum) and colon were stained by using a metachromatic staining method (kresylecht-violet; KEV) and a combined enzyme histochemical and immunohistochemical technique for chymase and tryptase. Additionally, immunohistochemistry with antibodies against T cells (CD3), macrophages (myeloid/histiocyte antigen) and IgA, IgG and IgM bearing cells was conducted. Quantitative evaluation of mast cells and semiquantitative scoring of immunohistochemically stained cells were performed. Between the two histopathologically defined groups clear differences concerning mast cell numbers were detected. In most affected intestinal tissue locations of dogs with LPE/LPC a decrease in metachromatically (kresylecht-violet) stained granule-containing MCs and immunohistochemically stained MC T,C,TC was found. This reduction could be due to mast cell degranulation, a T helper cell 1 dominated reaction pattern or a “thinning out” due to increasing T cells, IgA and IgG bearing cells. Dogs with EGE displayed higher variability in mast cell numbers but most of the affected large and small intestinal locations had increased numbers of MCs. In these cases, T cells, IgA bearing cells and macrophages also increased. Increased numbers of MCs and eosinophils seen in the intestinal mucosa of dogs with EGE could indicate the presence of a type I hypersensitivity reaction (T helper cell 2 pattern) in response to dietary antigens. Changes in cell numbers occurred also in unaffected locations of dogs with LPE/LPC and EGE which showed reduced MC T,C,TC, increased KEV positive cells and partially increased leucocytes and macrophages.

Presence of immunoglobulins and antigens in serum, lung and small intestine in Ascaris suum infected and immunised pigs

The immunodetection of local Ascaris suum antigens and local and systemic antibodies were analysed in pigs reinfected with eggs or immunized with the 14, 42 and 97 kilodalton (kDa) fractions from A. suum. Twenty-one Iberian pigs were divided in 7 groups of 3 pigs. Groups 1 and 2 were uninfected and challenge control groups, respectively. Groups 3 and 4 were infected weekly with increasing doses of A. suum eggs and Group 4 was additionally treated with pyrantel pamoate. Groups 5, 6 and 7 were immunised with the 14, 42 or 97 kDa fractions from adult worms, respectively. Groups 2–7 were challenged with 10,000 infective eggs. Animals of Groups 3 and 4 showed a pulmonary granulomatous reaction with moderate number of eosinophils and leukocytes, while Groups 5–7 presented higher number of cells, especially in animals immunized with the 42 kDa fraction. These immunized groups presented abundant deposition of Ascaris body fluid (BF) and body wall (BW) antigens as well as the 14 and 42 kDa fractions in the pulmonary and intestinal tissues, while lower deposition of antigens was observed in animals of Groups 3 and 4. The immunized pigs of Groups 5 and 6 showed the highest systemic IgG titres in serum and these antibodies were negatively correlated with the number of larvae recovered in the lungs, suggesting that the IgG response may have a protective function against the ascariosis. The highest concentrations of IgA-bearing cells were observed in animals of Groups 3 and 4 compared to the immunised pigs (Groups 5–7), suggesting that local IgA production may be involved in the protection against migrating larvae. The main localisations of IgA-bearing cells were the bronchial and peribronchial areas of lungs and the lamina propia of duodenum. Low numbers of local IgG-bearing cells were observed in all animals and no IgM-bearing cells were detected in the local tissues.

Zinc induced apoptosis in bone marrow and splenic B-lineage lymphocytes of the mouse

Recently, our lab discovered that moderate concentrations of zinc could induce apoptosis in CD4 +CD8 +TCR lo murine thymocytes. To determine if B-lineage lymphocytes demonstrated similar sensitivity, bone marrow and splenic B-lymphocytes were treated with zinc at a concentration range of 100 to 200 μM for 8 hrs and analyzed by flow cytometry for apoptotic death in the B220 +IgM − and B220 +IgM + subsets of the bone marrow and the IgM + and IgG + subsets of the spleen. B220 +IgM + B-lineage cells in the bone marrow and IgM + B-cells in the spleen were found to be most susceptible exhibiting 38% and 25% levels of zinc-induced apoptosis. B220 +IgM − bone marrow B-lineage cells and IgG + B-cells in the spleen were found to be less sensitive to zinc treatment than IgM-bearing cells. Thus, it is possible that zinc might initiate apoptosis in some cells of the immune system due to fluxes of the metal in cells or the presence of intracellular pools of zinc.

Immunoanalysis of IgG and IgM molecules on the surface of BLV infected cattle lymphocytes

The BLV-infected animals were divided into lymphocytotic (9 animals: BLV+PL+) and non lymphocytotic (11 animals: BLV+PL -). BLV-free animals formed a control group (7 animals: BLV-PL-). Flow cytometry immunofluorescence (FACS) and scanning electron microscopy (SEM) were used to evaluate the expression of IgG and IgM molecules on the surface of the lymphocytes of cattle infected with the bovine leukaemia virus (BLV). The percentage of IgM-bearing cells was significantly higher in the BLV+PL+ (75% and 74%) than in BLV+PL (43% and 50%) and BLV-PL (39% and 44%) groups by FACS and SEM respectively. The percentage of IgG-bearing cells was higher in BLV+PL+ (6%) and BLV+PL (8%) compared to BLV-PL (0.7%) determined by FACS and significantly higher in BLV+PL+ (62%) in relation to BLV+PL (17%) and BLV-PL (8%) groups ascertained by SEM. A significantly higher intensity of IgM expression was observed in the BLV+PL+ group by SEM and FACS; a higher intensity of IgG expression in BLV+PL+ group was detected only by SEM. The results suggest that FACS and SEM are complementary. The finding of significant differences in the expression of IgG molecules in BLV+ groups compared with BLV - animals confirms the sensitivity of the SEM method.

Ontogeny of IgM-bearing cells and changes in the immunoglobulin M-like protein level (IgM) during larval stages in sea bass ( Dicentrarchus labrax)

The ontogenetic development of IgM positive cells was described as demonstrated by immunofluorescent staining (FITC) and flow cytometric analysis (FACS) of cells stained for IgM expression with a mouse anti-sea bass IgM monoclonal antibody. In addition, the immunoglobulin (IgM) level in larvae was measured using a sensitive ELISA and the development of the lymphoid organs was studied histologically. A small amount of IgM was detected by ELISA in the eggs, decreasing regularly until day 5 post-hatching, with no IgM being detected from day 5 to day 15. IgM was again detectable from day 15 to day 30 together with the detection of IgM positives cells by FACS analysis. During this period the thymus and the head kidney became lymphoid. From day 30 to day 95, the IgM level increased and correlated with the body weight of the larvae. These results show that the IgM detected from day 0 to day 5 derive from maternal fish while the IgM detected from day 15 are neo-synthesised IgM. Moreover, mature B cells and strong IgM synthesis are present in 50-day-old sea bass larvae when the lymphoid organs are fully developed.

Expression and quantification of IgG and IgM molecules on the surface of lymphocytes of cattle infected with bovine leukaemia virus

Immunogold-labelled antibodies were used with scanning electron microscopy ( SEM) and fluorescent-labelled antibodies were used with flow cytometry ( FACS) to evaluate the expression and quantity of IgG and IgM molecules on the surface of the lymphocytes of cattle infected with bovine leukaemia virus ( BLV). The BLV-infected animals were divided serologically and haematologically into groups with ( BLV +PL + ) and without ( BLV +PL − ) persistent lymphocytosis ( PL). The percentage of IgM-bearing cells was significantly higher in the BLV +PL + group than in the BLV −PL − and BLV +PL − groups by FACS. There was a significantly higher percentage of IgG-bearing cells in the BLV +PL + group than in the BLV −PL − and BLV +PL − groups by SEM, but no differences were found by FACS. A significantly higher intensity of IgM expression was observed in the BLV +PL + group by SEM. A higher intensity of IgG expression in some animals was detected only by SEM. An increase in the number of larger IgM cells was observed in the BLV +PL − and BLV +PL + groups by SEM. The SEM analysis was more sensitive than FACS in this experiment.

A wild-type mu s C-terminal gene is expressed in Bloom's syndrome cells.

Selective IgM deficiency is found commonly in patients with Bloom's syndrome (BS). Serum IgM concentrations were low though serum IgG and IgA concentrations were normal in both patients with BS included in the study. In a previous study the authors showed that selective IgM deficiency in BS is due to an abnormality in the maturation of surface IgM-bearing cells into IgM-secreting cells and a failure of secreted mu (mu s) mRNA synthesis. The membrane-bound mu (mu m) and mu s mRNA are produced from transcripts of a single immunoglobulin mu gene by alternative RNA processing pathways. The control of mu s mRNA synthesis depends on the addition of poly(A) to mu s C-terminal segment. The study described here demonstrated that there was no mutation or deletion in the sequence including mu s C-terminal coding sequence, the RNA splice site (GG/TAAAC) at the 5' end of mu s C-terminal segment, and the AATAAA poly(A) signal sequence, and second GT-rich element immediately down-stream of the cleavage site in both patients.

Ontogeny of IgM and IgM-bearing cells in rainbow trout

We have studied the ontogenic development of immunoglobulin M (IgM) and of IgM-bearing cells in the rainbow trout, Oncorhynchus mykiss. Lymphocytes showing cytoplasmic IgM were first observed in embryos at 12 days before hatch (14°C). At this stage, no cells positive for surface IgM were present. Lymphocytes bearing surface IgM were observed at 8 days before hatch (14°C). Unfertilized trout eggs contained detectable amounts of IgM (11.2 ± 2.6 μg/g of egg weight), indicating that transfer of IgM from mother to embryo can occur in salmonids. The levels of IgM from whole fish increase slowly after the appearance of intraembryonic cells that express surface IgM. The amount of IgM/g of tissue peaks around hatch, but this parameter shows lower values up to 2 months after hatch.

A simple method for negative and positive selection of murine and human IgM-bearing lymphocytes based on the use of antibody-coated silica microparticles

The use of octadecyl silica microparticles is proposed as an alternative method for negative and positive selection of IgM-bearing lymphocytes. SiC18 microparticles coated with affinity purified IgG anti-IgM were evaluated in their efficiency to deplete IgM-bearing cells. 6 mg of SiC18-IgG depleted an average of 97% IgM-bearing cells from a murine spleen cell suspension and of 84% IgM-bearing cells from human spleen cells. The viability of the selected cells was not affected by this procedure. We also analyzed the ability of whole serum to release IgM-bearing cells from the complex SiC18-target cell. The IgM releasing efficiency was 98%, with a resulting viability of 85%. These results indicate that SiC18 microparticles, saturated with specific antibodies, may be used as an alternative method for depletion or purification of cells bearing the corresponding antigen, from complex cell suspensions. The method is simple, efficient and inexpensive, representing an interesting alternative to the immunomagnetic selection.

Regulation of the B Cell Immune Response: Induction of Competence and Progression in a Surface IgM-Bearing Subset

We have targeted the specific surface IgM-bearing subset of human B lymphocytes within a mixed population and investigated cell cycle activation in these cells stimulated with phorbolester and antibody directed against surface immunoglobulin. Stimulation with phorbol 12.13-dibutyrate (PDB) in combination with anti-IgM led to induction of the state of competence followed by progression to proliferation of the surface IgM-bearing cells. In contrast, sequential stimulation with PDB and anti-IgM in either order did not induce either competence or progression. Brief exposure to both stimuli for 30 min did not induce significant proliferation, but did induce the state of competence such that the cells progressed to DNA synthesis after incubation with PDB alone. Furthermore, a competence-related gene, c-fos, was induced in cells that did not become fully competent. Thus, induction of competence to proliferate required simultaneous delivery of two signals whereas actual progression to proliferation by competent cells was accomplished with only one signal.

DIVERSITY IN DNA REARRANGEMENTS AND IN RNA EXPRESSIONS OF IMMUNOGLOBULIN GENE ON COMMON VARIABLE IMMUNODEFICIENCY

Six heterogeneous common variable immunodeficiency (CVID) patients were analysed for germ-line DNA, DNA rearrangements, and RNA expressions of immunoglobulin (Ig) gene by Southern or northern blotting using appropriate probes. We detected no polymorphism in neutrophil DNA hybridized to a C mu and a C gamma probe. In three patients, both serum Ig and Ig-bearing cells were scarcely detected, and by northern hybridization methods, neither mu mRNA, gamma mRNA, alpha mRNA nor kappa mRNA was detected. However, one Epstein-Barr virus-transformed B lymphoblastoid cell line (LCL) of these three patients was different from the germ line in the region of JH, C gamma, and C kappa, and expressed mu mRNA at a higher level. The B cell defects of these three patients lay on the B cell maturation stage similar to X-linked agammaglobulinaemia (XLA). In two others among the six CVID patients, serum IgM and IgM-bearing cells were detected to a certain degree, and by northern hybridization, mu mRNA was detected at a lower level, but neither mu mRNA, alpha mRNA, nor kappa mRNA was detected. One LCL of these two patients could express mu mRNA at the normal level. In the last patient, the serum IgM was normal, serum IgG and IgA were somewhat low, Ig-bearing cells were normal, mu mRNA and kappa mRNA were detected at the normal level, and gamma mRNA and alpha mRNA were detected at a lower level. The defect of this patient affected the class switch stage. These results showed that primary B cell defects in CVID occurred at several B cell differentiation stages which could be classified by expression of the Ig gene, and at the degree of clonal diversity in the B cell repertoire. Furthermore, this study provides support for the idea that the CVID defect is related to a more generalized cellular function, such as regulating the proliferation and/or clonal expansion of cells of the B lymphoid lineage.

A New Murine Model of Autoimmune Orchitis Induced by Immunization With Viable Syngeneic Testicular Germ Cells Alone.: II. Immunohistochemical Findings of Fully-Developed Inflammatory Lesion

Previous studies demonstrated that experimental autoimmune orchitis (EAO) was produced in C3H/He mice with very high incidence by two or three subcutaneous injections of viable syngeneic testicular germ cells, without the use of any adjuvants or immunopotentiators and that the disease induced was characterized by a complete lack of epididymitis despite a definite orchitis with hypospermatogenesis. In this report, immunohistochemical characterization of immune cells in the fully-developed orchitic lesion was carried out using monoclonal antibodies and immunoperoxidase staining. Thy-1.2+ cells, Mac-1+ cells, B220+ cells and cytoplasmic Ig-bearing cells in the lesion were estimated to be approximately 30, 15, 20 and 30% of all inflammatory cells, respectively. Major phenotype of T cells in the lesion was CD4+ (approximately 85%) with the remainder (approximately 15%) being CD8+. The percentages of cytoplasmic IgG-, IgA- and IgM-bearing cells were estimated as approximately 35, 60 and 5% of all cytoplasmic Ig-bearing cells, respectively. Deposits of immunoglobulins and third component of complement were identified on the basement membrane of the seminiferous tubules, interstitium between the tubules, vessel endothelium and degenerated germ cells in the lesion. Circulating antibodies directed against the acrosomal portion of germ cells were detected in IgG and IgM classes but not in IgA class. Inflammatory cells (including macrophages, B cells and, probably, activated T cells) in the lesion were Ia+, but Leydig cells, Sertoli cells and germ cells did not stain for Ia at all.

Production and distribution of immunoglobulin-bearing cells in the intestine of young pigs infected with Ascaris suum

Pigs of 10 days-1 month old were orally infected with eggs of Ascaris suum at different rates and inoculation schedules. Histological sections from various parts of the small intestines were prepared to observe the production and localization of immunoglobulin-bearing cells. Fluorescent antibody and immunoperoxidase staining methods were used to determine the number of IgM-, IgA- and IgG-producing plasma cells in the intestinal lamina propria. Significant increases in immunoglobulin-bearing cells were observed in those pigs which received single inoculations of A. suum eggs. Pigs infected every 2,4,8 and 10 days with 10,000-20,000 embryonated eggs showed numerical increases in IgM-bearing cells. Increases in IgA-bearing cells were noted in pigs which received the higher number of eggs every 8-10 days. Higher concentrations of IgA- and IgM-bearing cells were observed in the jejunal mucosa of infected pigs as compared to those in the duodenum and ileum.

Anatomic distribution and localization of immunocompetent cells in normal mouse endolymphatic sac.

The distribution and anatomic localization of immunocompetent cells in normal mouse endolymphatic sac (ES) were examined by immunohistochemical methods. Antibodies against T cells (anti-Thy-1, -Lyt-1, -Lyt-2), macrophage (anti-Mac-1) and immunoglobulins (anti-IgM, -IgG, -IgA) were employed in an indirect technique utilizing strept-avidin-biotin complex. In the normal mouse ES, each of these cells could be detected by a difference in its frequency and distribution within and around the ES. Thy-1 positive (Thy-1+) cells are the most predominant in this tissue and can be seen throughout the ES. Lyt-1+ cells were found within the ES epithelium and in the perisaccular region, whereas Lyt-2+ cells were rarely present. Mac-1+ cells were present primarily in the lumen of the distal portion of the ES. IgM-bearing (IgM+) cells were seen in the subepithelial region, IgG+ cells were occasionally detected in the lumen and only a few IgA+ cells were present in the perisaccular region. This study revealed that the ES has the necessary immunological components for antigen processing and the generation of local immune responses within the inner ear.

In vitro differentiation of B lymphocytes from primitive hemopoietic precursors present in long-term bone marrow cultures.

B lymphocytes are not produced in the Dexter long-term bone marrow cultures, but a primitive B cell precursor is present. The findings presented in this study demonstrate that this precursor can be induced to produce B lymphocytes by transferring the cultures to the Whitlock conditions for the long-term growth of B cells in vitro. Two weeks after the transfer of cultures maintained at 33 degrees C in medium supplemented with horse serum and steroids to low concentrations of fetal calf serum at 37 degrees C, marked effects can be observed. The pattern of cell growth changes from one in which the hemopoietic cells are clustered in tight foci containing several hundred cells to smaller ones in which the cells are not as densely packed. Fat cells in the adherent layer disappear and the supporting stroma becomes more uniform in appearance. This change in the culture format is accompanied by a decrease in the number of nonadherent cells and a shift from myelopoiesis to lymphopoiesis. The numbers of granulocyte-macrophage progenitors decline weekly after the change in culture conditions and are not detected by the third week. B cell colony-forming units appear by 3 wk. Cells that express the 14.8 cell surface antigen are induced by 1 wk after the change in culture conditions, followed by the appearance of surface IgM-bearing cells 2 wk later. This shift to lymphopoiesis can be confirmed morphologically. Granulocytes and macrophages disappear from the cultures by 4 wk, at which time almost all of the cells have a characteristic lymphocyte morphology. Upon switching these cultures back to the original Dexter conditions, only low levels of transient myelopoiesis can be reinitiated.

Changes in conjunctival lymphocyte populations induced by oral immunization with Chlamydia trachomatis.

The populations of lymphocyte subsets in the conjunctiva were assessed in monkeys enterically immunized with different preparations of Chlamydia trachomatis prior to ocular challenge. Subsets were identified in immunohistochemical studies employing cross-reactive anti-human monoclonal and polyclonal reagents. Ocular challenge of orally immunized animals resulted in more equal numbers of T-helper and T suppressor/cytotoxic cells, compared to the higher proportion of TS seen in naive monkeys. TH:TS/CTL ratios of 1.0-2.0 and 0.1-0.5 respectively were observed. Orally immunized monkeys also showed proportionately more IgA- and IgG-bearing cells and less IgM-bearing cells. B cells of each class were commonly seen immediately under the epithelial basement membrane. Although oral immunization induced a number of changes in the proportions and distribution of lymphocyte subsets which subsequently appeared in the conjunctiva, these changes were not correlated with disease outcome, except for the appearance of increased proportions of follicular IgA-bearing cells in partially protected monkeys.