Immunoglobulin-secreting Cells Research Articles

Medroxyprogesterone acetate enhances in vivo and in vitro antibody production.

In the present study we examine the effects of medroxyprogesterone acetate (MPA) on the specific antibody secretion to T-dependent antigens. Our results show that the in vivo administration of MPA to mice, 7 or 90 days before immunization with sheep red blood cells (SRBC), significantly enhanced both, primary and secondary antibody responses, without affecting delayed-type hypersensitivity (DTH). These effects could be counteracted by the anti-progestin onapristone or ZK 98299 (ZK) suggesting that MPA interacted with progesterone (PRG) receptors to increase B-cell response. To better understand the mechanisms involved in MPA activity we carried out cultures of splenocytes, bone marrow cells or lymph node cells from immunized mice in the presence of MPA, and evaluated the amount of antibody release to supernatants. We found that low doses of MPA (10(-9) M and 10(-10) M) significantly enhanced the in vitro production of specific immunoglobulin G (IgG) antibodies, an effect that appears to involve the interaction of the progestin with PRG receptors, as judged by the inhibition of MPA effects with ZK (10(-8) M) or RU486 (10(-9) M). These receptors were detected by flow cytometry analysis in a proportion of T lymphocytes. Because MPA did not increase the number of immunoglobulin-secreting cells, our findings suggest that MPA enhanced the capacity of individual cells to produce specific immunoglobulin.

Regulation of CCR6 chemokine receptor expression and responsiveness to macrophage inflammatory protein-3α/CCL20 in human B cells

The regulation of CCR6 (chemokine receptor 6) expression during B-cell ontogeny and antigen-driven B-cell differentiation was analyzed. None of the CD34+Lin− hematopoietic stem cell progenitors or the CD34+CD19+ (pro-B) or the CD19+CD10+ (pre-B/immature B cells) B-cell progenitors expressed CCR6. CCR6 is acquired when CD10 is lost and B-cell progeny matures, entering into the surface immunoglobulin D+ (sIgD+) mature B-cell pool. CCR6 is expressed by all bone marrow–, umbilical cord blood–, and peripheral blood–derived naive and/or memory B cells but is absent from germinal center (GC) B cells of secondary lymphoid organs. CCR6 is down-regulated after B-cell antigen receptor triggering and remains absent during differentiation into immunoglobulin-secreting plasma cells, whereas it is reacquired at the stage of post-GC memory B cells. Thus, within the B-cell compartment, CCR6 expression is restricted to functionally mature cells capable of responding to antigen challenge. In transmigration chemotactic assays, macrophage inflammatory protein (MIP)-3α/CC chemokine ligand 20 (CCL20) induced vigorous migration of B cells with differential chemotactic preference toward sIgD− memory B cells. These data suggest that restricted patterns of CCR6 expression and MIP-3α/CCL20 responsiveness are integral parts of the process of B-lineage maturation and antigen-driven B-cell differentiation.

Regulation of CCR6 chemokine receptor expression and responsiveness to macrophage inflammatory protein-3α/CCL20 in human B cells

AbstractThe regulation of CCR6 (chemokine receptor 6) expression during B-cell ontogeny and antigen-driven B-cell differentiation was analyzed. None of the CD34+Lin− hematopoietic stem cell progenitors or the CD34+CD19+ (pro-B) or the CD19+CD10+ (pre-B/immature B cells) B-cell progenitors expressed CCR6. CCR6 is acquired when CD10 is lost and B-cell progeny matures, entering into the surface immunoglobulin D+ (sIgD+) mature B-cell pool. CCR6 is expressed by all bone marrow–, umbilical cord blood–, and peripheral blood–derived naive and/or memory B cells but is absent from germinal center (GC) B cells of secondary lymphoid organs. CCR6 is down-regulated after B-cell antigen receptor triggering and remains absent during differentiation into immunoglobulin-secreting plasma cells, whereas it is reacquired at the stage of post-GC memory B cells. Thus, within the B-cell compartment, CCR6 expression is restricted to functionally mature cells capable of responding to antigen challenge. In transmigration chemotactic assays, macrophage inflammatory protein (MIP)-3α/CC chemokine ligand 20 (CCL20) induced vigorous migration of B cells with differential chemotactic preference toward sIgD− memory B cells. These data suggest that restricted patterns of CCR6 expression and MIP-3α/CCL20 responsiveness are integral parts of the process of B-lineage maturation and antigen-driven B-cell differentiation.

Airway inflammation in a murine model of chronic asthma: evidence for a local humoral immune response.

Asthma is an acute-on-chronic inflammatory disease of the airways characterized by recruitment of eosinophils into the epithelial layer, chronic inflammation in the lamina propria, as well as variable accumulation of mast cells in the airway wall. The role of local production of allergen-specific immunoglobulins in triggering mast cell-mediated asthmatic inflammation is unknown. We used a chronic inhalational exposure model of asthma in ovalbumin-sensitized BALB/c mice to examine the phenotype of immunoglobulin-secreting cells and mast cells in the airway wall. In parallel, we assayed ovalbumin-specific IgG and total IgE in the plasma of these animals. In sensitized mice exposed to aerosolized ovalbumin for 6 weeks, aggregates of chronic inflammatory cells consisted of a majority of plasmacytoid cells, including numerous IgG-synthesizing cells, which were significantly increased in sensitized animals compared to controls. IgA-synthesizing cells were also present, but were not increased in the sensitized exposed mice. Immunoglobulins in the cytoplasm of the plasma cells were demonstrated to be antigen-specific. No IgM-or IgE-synthesizing cells were observed, although levels of total IgE in the plasma were significantly increased. There was no recruitment of mast cells of either the mucosal or the connective tissue phenotype into the lamina propria or the epithelium. In this experimental model of chronic asthma, the pattern of inflammation in the airway wall is consistent with development of a local IgG-mediated humoral immune response. However, there is no evidence of local production of IgE or recruitment of mast cells.

Effects of primary- and secondary-treated bleached kraft mill effluents on the immune system and physiological parameters of roach

The present study was designed to examine, whether, effluents from a modern pulp and paper mill using elemental chlorine-free/total chlorine-free (ECF/TCF) bleaching, exert effects on the immune system of fish and, in addition, to relate these findings to physiological parameters known to be affected by bleached kraft-mill effluents (BKME). Roach ( Rutilus rutilus) were exposed in laboratory conditions to primary- or secondary-treated effluent from a pulp and paper mill. In order to study their capability to respond to foreign antigens they were immunised with bovine γ-globulin (BGG) prior to exposure. The number of anti-BGG antibody-secreting cells (ASC) and the number of immunoglobulin-secreting cells (ISC) in the spleen and blood as well as the level of anti-BGG specific antibodies and concentration of plasma immunoglobulin (IgM) were studied. Phagocytosis and migration of granulocytes of the head kidney were also determined. In addition to the immunological parameters, the activity of hepatic biotransformation enzymes, the carbohydrate metabolism and osmoregulation were examined. Exposure of roach for 21 days to BKME affected several immunological parameters. Both effluents, primary- and secondary-treated, impaired the immunoreactivity of the fish. Sex-related differences in the immune responses were evident in many parameters e.g. in the number of blood ISC and splenic ASC. Sex also had effects on cortisol levels and in the induction of 7-ethoxyresorufin O-deethylase (EROD). These results demonstrate that both primary- and secondary-treated effluent from a pulp and paper mill using ECF/TCF bleaching have effects on fish immune functions. Further, these findings suggest that steroids may contribute to immunomodulation in fish.

Switch from cyclosporine A to tacrolimus in renal transplant recipients: impact on Th1, Th2, and monokine responses

We showed previously that pretransplant CD4 helper defects and low in-vitro IL-10 responses predict a low risk of acute kidney graft rejection. To compare the effect of tacrolimus (Tacr) and cyclosporine A (CsA) on the humoral immune response we assessed T helper function, B cell/monocyte responses and in-vitro cytokine responses (TNF-α, GM-CSF, IL-1β, IL-2, IL-4, IL-6, IL-10) in 20 renal transplant recipients before and 3 months after they were switched from CsA to Tacr because of hyperlipoproteinemia, hirsutism, or gum hyperplasia. T helper function was assessed using a PWM-driven allogeneic coculture system of patient T cells together with control B cells. B cell/monocyte responses were determined using a PWM-stimulated allogeneic coculture system, SAC I-stimulated B-cell cultures and LPS-stimulated monocyte cultures. Immunoglobulin-secreting cell (ISC) responses were assessed in a reverse hemolytic plaque assay, and ELISA were used to determine cytokine secretion. Treatment with Tacr resulted in a decreased expression of costimulatory ligands and adhesion molecules (T cells: CD40L, p < 0.05; CD28 and CD54, p ⩽ 0.01; B cells: CD25, p = 0.05; CD40, p < 0.001; monocytes: CD40, p < 0.05), which coincided with decreased PHA-stimulated T cell IL-2 responses (398 ± 153 versus 43 ± 15 pg/ml, p < 0.05), impaired CD4 helper activity (117% ± 22% versus 73% ± 19%, p < 0.05) and increased CD4 suppressor activity (−120% ± 28% versus −18% ± 27%, p = 0.02). We observed enhanced CD4 IL-10 responses ( p < 0.01) and LPS-stimulated monocyte responses (TNF-α, IL-1β, and IL-6, p < 0.005; IL-10, p < 0.05), indicating an increased humoral immune responsiveness under treatment with tacrolimus. Our data show that switching of immunosuppressive therapy from CsA to tacrolimus results in suppression of costimulatory ligands, adhesion molecules, Th1 responses and CD4 helper activity. However, enhanced humoral immune responses, Th2 and monokine responses, might have a negative impact on long-term graft function.

Pollution and the immune response: atopic diseases--are we too dirty or too clean?

The effect of pollutants on the immune system has filled tomes and is almost always controversial whether the topic is disinfectants in drinking water or particulates and lung cancer. Even whether outdoor and indoor air quality is improving or declining has been a source of considerable investigation, consultation and often litigation. Few areas, however, yield so much uncertainty and debate as the role of airborne pollutants in atopy. Atopy is defined as an immunoglobulin E- (IgE) mediated allergic response to environmental antigens and can be manifested as rhinitis, asthma or atopic dermatitis. While there is some debate over whether there has been a real increase in atopy over the last decades, it is indisputable that there has been a dramatic change over the last 200 years. While isolated cases of anaphylaxis had been noted before, allergies were unheard of until the early 19th century. Only 54 years after the first description of hay fever in England in 1819, it was considered an epidemic and a clear association with urbanization had been reported.1 What aspect of urbanization is involved has been much debated. There has been much discussion amongst both experimental immunologists and epidemiologists over whether pollutants are responsible for increased allergic sensitization and/or severity.2,3 In the last few years new studies have supported an alternative explanation, namely the decline in infectious diseases in industrialized countries.4,5 Does this ‘hygiene theory’ toll the death knell for the ‘pollution theory’? In this review I will argue that there is a compelling case to be made for both theories and that even this is not the entire picture.

Epstein-Barr virus-dependent lymphoproliferative disease: critical role of IL-6

Epstein-Barr virus (EBV)-induced lymphoproliferative disease (lpd) is a B cell neoplasm that affects patients who are immunosuppressed in the context of organ transplantation or HIV infection. A model for the aggressive form of this entity was generated by xenotransplantation of SCID mice with human peripheral blood leukocytes from individuals with prior contact with EBV. This model, where large B cell lymphoma occurs, was used to test the hypothesis that IL-6 has a major role in EBV-induced B cell tumorigenesis. IL-6 is known to differentiate B cells into immunoglobulin-secreting plasma cells and induce EBV replication, and xenochimeric animals have detectable serum levels of human IL-6. Human IL-6 inhibition with a neutralizing monoclonal antibody decreased tumor incidence from 62 % to 27 %. In addition, anti-IL-6 treatment significantly improved xenotransplanted animal survival, with median survival at > 245 days when compared to that of controls at 132 days. In conclusion, IL-6 plays a critical role in the pathogenesis of EBV-induced human lpd, and IL-6 inhibition may represent a new and promising preventive or therapeutic approach against this malignancy.

Spontaneous monoclonal immunoglobulin-secreting peripheral blood mononuclear cells as a marker of disease severity in multiple myeloma.

Peripheral blood from patients with multiple myeloma (MM) contains a small number of plasma cells related to the bone marrow tumour cells by their cytoplasmic immunoglobulin (Ig), their cell membrane antigen expression and/or their gene rearrangements, but hitherto the monoclonal Ig (M-Ig) production by circulating cells has not been reported. Using a two-colour ELISPOT assay, Ig-secreting cells (Ig-SCs) were detected in the blood of 28 MM and five Waldenstrom's macroglobulinaemia (WM) patients. The number of cells that spontaneously produced an Ig isotype similar to that of the M-Ig in serum was greater than that of the other Ig-SCs. MM patients presented an excess of circulating heavy-chain (alpha or gamma) Ig-SCs (0.38% of the PBMC) with kappa or lambda light chains (0.48%) compared with the number of cells secreting the other heavy- (0.02%) and light-chain isotypes (0.03%). WM patients also presented high numbers of cells secreting the mu-heavy-chain isotype (0.66%). The Ig synthesized in vitro was characterized as monoclonal, and the M-Ig secretory capacity of the peripheral blood cells was similar to that observed for Ig-SCs from polyclonal activated B cells in vivo. The number of these monoclonal cells was significantly increased in patients in an advanced stage of MM (I/II vs. III, P < 0.001) and correlated with the serum beta-2 microglobulin concentration (r = 0. 69; P < 0.0003). The number of M-Ig-SCs in MM patients could be a useful marker for evaluating the progression of multiple myeloma.

Influence of various antibiotics on B cell immunoglobulin secretion and lymphocyte growth

Influence of various antibiotics on B cell immunoglobulin secretion and lymphocyte growth

Probiotics and gastrointestinal health

Evidence for positive health benefits of Lactobacilli applies to only a few strains used for commercial applications. It is generally agreed that a probiotic must be capable of colonizing the intestinal tract to influence human health; this requirement disqualifies many of the strains currently used in fermented dairy products. Lactobacillus GG, a variant of L. casei sps rhamnosus, has been studied extensively in adults and children. When consumed as a dairy product or as a lyophilized powder, LGG colonizes the gastrointestinal tract for 1–3 days in most individuals and up to 7 days in about 30% of subjects. Traveler’s diarrhea, antibiotic-associated diarrhea, and relapsing Clostridium difficile colitis are improved with LGG. In infantile diarrhea, the severity and duration of the attack is reduced. LGG-fermented milk lessens the intestinal permeability defects caused by exposure to cows milk or rotavirus infection. LGG has proven beneficial effects on intestinal immunity. It increases the numbers of IgA and other immunoglobulin-secreting cells in the intestinal mucosa. LGG stimulates local release of interferon. It facilitates antigen transport to underlying lymphoid cells, which serves to increase antigen uptake in Peyer’s patches. LGG also acts as an immunoadjuvant for oral vaccines. In an animal model of colon cancer, LGG reduced the incidence of chemically induced tumors in the large bowel of rodents. Extensive safety testing has shown no pathogenic potential in humans or animals. Probiotic cultures of Lactobacilli have the potential to bring substantial health benefits to the consumer. The purported benefits for any probiotic must pass the highest standards of scientific scrutiny before the claims can be accepted.

Modulation of immune parameters of roach, Rutilus rutilus, exposed to untreated ECF and TCF bleached pulp effluents

The present study was designed to assess the effects of elemental chlorine free (ECF) and totally chlorine free (TCF) pulp and paper mill effluents on the immune defence of the roach ( Rutilus rutilus). Fish were exposed for 5 weeks to concentrations of 0, 0.6, 2, 6 and 20% of untreated effluents from ECF or TCF bleaching processes. In order to study the capability to respond to foreign antigens the fish were immunised with bovine γ-globulin (BGG) 3 weeks before sampling. The numbers of anti-BGG antibody-secreting cells and immunoglobulin-secreting cells in the spleen and blood, as well as the levels of the anti-BGG antibodies and concentrations of immunoglobulin in plasma and the proliferation of lymphocytes after mitogenic activation were determined. In addition, phagocytosis-related functions, respiratory burst activity and migration of leukocytes, were measured. Formation of specific anti-BGG antibody-secreting cells in the blood and immunoglobulin-secreting cells in the spleen and blood were suppressed in the fish exposed to a high concentration (20%) of ECF effluent. Similarly, the level of anti-BGG antibody and the stimulation indices of Concanavalin A-stimulated proliferation responses in vitro were lower in these fish. A decreased migration of granulocytes was observed in the fish exposed to 0.6–2% of ECF and TCF effluents. These results demonstrate that untreated ECF and TCF effluents significantly affected the immune parameters of the roach. Taken as a whole, fish exposed to TCF effluent showed less immunomodulation than those exposed to EFC effluent.

Cow’s milk challenge through human milk evokes immune responses in infants with cow’s milk allergy

Objectives: In order to measure the immune response evoked in breast-fed infants with cow’s milk allergy (CMA) by cow’s milk challenge through human milk, mothers were given increasing doses of cow’s milk after they had been on a cow’s milk elimination diet. Another objective was to study the secretion of β-lactoglobulin (BLG) into human milk before and during milk challenge in relation to the appearance of symptoms in infants. Study design: Seventeen asymptomatic mothers who had infants with challenge-proven CMA and 10 asymptomatic mothers who had healthy infants were recruited. Infants ranged in age from 1.8 to 9.4 months. A solid-phase enzyme-linked immunoassay (ELISPOT) was used to assess the total number of immunoglobulin-secreting and specific antibody-secreting cells. Flow cytometry was used to enumerate different lymphocyte subpopulations among peripheral blood lymphocytes primed during provocation by cow’s milk antigens. BLG levels were assessed in human milk before the challenge and 1, 2, 3, and 4 hours after the commencement of the challenge. Results: All but one of the infants with CMA showed symptoms of CMA during cow’s milk challenge through human milk. There was a significant rise in the total number of immunoglobulin-secreting cells in the IgA and IgG classes associated with a positive cow’s milk challenge response, but the proportions of peripheral blood B cells bearing CD19, CD23, CD19 and 23, CD5, or CD19 and CD5 were comparable. BLG levels were comparable in both study groups. Conclusions: Most of the infants with CMA reacted to cow’s milk challenge through human milk. Hypersensitivity reactions to food antigens through human milk may be more common than previously thought. (J Pediatr 1999;135:506-12)

Reversal of Blimp-1-mediated apoptosis by A1, a member of the Bcl-2 family.

Blimp-1 (B lymphocyte-induced maturation protein 1) is strongly expressed during the late stages of B cell differentiation to immunoglobulin-secreting plasma cells. Overexpression of Blimp-1 in B lymphoma cells has been reported to induce either growth arrest and cell death or Ig secretion and terminal differentiation, depending on the developmental stage of the recipient lymphomas. By using a retroviral expression system we show that Blimp-1-transduced immature WEHI 231 murine B lymphoma cells produce J chain, increased levels of the secretory form of micro heavy chain mRNA and secrete IgM for a short period of time. Concomitantly, they exhibit altered ratios of c-myc/mad4 mRNA levels, a reduction in the expression of the anti-apoptotic bcl-2 family member A1 and a distinct growth disadvantage, followed by cell death. Reintroduction of A1 by retroviral transduction greatly extends the life span of Blimp-1-expressing WEHI 231 cells which continue to secrete IgM. These data suggest that levels of A1 may determine the checkpoint between death and survival of Blimp-1-expressing B cells at different stages of differentiation.

Development of the natural response of immunoglobulin secreting cells in the pig as a function of organ, age and housing

We analysed the development of the natural immunoglobulin-secreting cell (Ig-SC) response in systemic- and mucosal-lymphoid tissues of specified pathogen free pigs between 1 and 40 weeks of age. As antigen exposure may influence the development of the Ig-SC repertoire we also compared the frequencies of Ig-SC in various lymphoid tissues of 40 weeks old specified pathogen free pigs and conventional pigs. A procedure to isolate lamina propria cells from porcine intestine was adapted for this study. The frequencies of IgM-, IgG-, and IgA-secreting (spot forming) cells were determined with a reversed enzyme linked immunospot assay, which was also adapted for detection of Ig-SC in pigs. The Ig-SC frequencies were calculated as percentage of the mononuclear leukocytes isolated from the various organs. The observations till 40 weeks of age were as follows: Splenic IgM-SC predominated at all ages and reached a plateau of 0.1–0.2% of the mononuclear leukocytes already at 4 weeks of age. The IgM-SC of mesenteric lymph node (MLN) predominated up till 12 weeks of age and reached an optimum of 0.15% reached at 4 weeks of age. The frequencies of IgG-SC of spleen and MLN had dips around 4 weeks of age and increased thereafter till 40 weeks of age (spleen 0.025%, MLN 0.05% at 40 weeks of age). The frequencies of IgA-SC were low in the spleen (⩽0.003%) and moderate in the MLN (0.01–0.02%) at all ages tested. In peripheral lymph node (PLN) and bone marrow (BM), the frequencies of IgM-SC (0.03–0.05%) were much lower than in the spleen. The IgG-SC frequencies of BM and MLN also had dips around 4 weeks of age and increased thereafter. The IgG-SC frequency of BM reached a plateau at 12 weeks of age (0.15%) and for PLN the highest frequency was observed at 40 weeks of age (0.05%). The frequencies of IgA-SC were low in BM and PLN (<0.003%). High frequencies of IgA-SC were observed in mucosa associated tissue like Peyer’s patches (PP) and intestinal lamina propria (till 20% of the mononuclear leukocytes in intestinal lamina propria of 12–40 weeks of age). IgM and IgA are both important isotypes in mucosal lymphoid organs in the pig. The shift from IgM to IgA as predominant, mucosal isotype was first observed in duodenum and jejunum (12 weeks) and later in ileum (40 weeks). The influence of ageing on the frequency of Ig-SC in PP was only observed in jejunal PP, whereas in ileal PP the frequencies of Ig-SC did not vary over time. We combined our data about the frequencies of IgM-, IgG-, and IgA-SC in various organs with data obtained by others about the distribution of lymphocytes over porcine lymphoid organs at about 12 weeks of age. Based on these calculations we concluded that the small intestine, with more than 80% of all Ig-SC, is far most the major site of Ig production in the pig. We also concluded that the small intestine is the major site of IgA and IgM production cells in the pig. Although IgA becomes predominant along the intestine, the results demonstrated that in the pig IgM is more a mucosal isotype compared with other species. With 40% of all IgG-SC the porcine BM appeared to be the major site of IgG production. Unexpected results were obtained for IgG-SC in the systemic lymphoid organs. In these organs the frequencies of IgG-SC dropped firstly from 1 to 4 weeks of age and steadily increased thereafter till 40 weeks of age. This observation is discussed in relation to the possibility that systemic IgG-SC at one week of age were passively acquired from maternal colostrum. The influence of housing/antigenic load at 40 weeks of age was mainly expressed by an increase (2–8×) of the frequency of IgG-SC in spleen, PLN, BM, and intestinal lamina propria, whereas the typical mucosal IgA-SC frequencies in the lamina propria were hardly affected.

CAESAREAN SECTION DELIVERY IS ACCOMPANIED WITH DEFECTIVE CAPACITY TO GENERATE IGA IN NEONATES

63 AIM Microbial antigens preferentially expand T helper 1-type cells and thereby provide optimal conditions to redirect the immunologic memory away from the T helper 2-type phenotype of atopy. Caesarean section delivery interferes with the development of normal intestinal microflora which contribute to the development of mucosal immunity in the gut. This study aimed to evaluate the effect of caesarean delivery on IgA generation. METHODS Blood samples were obtained during 12 vaginal deliveries (VD) and 16 elective caesarean sections (ECS), and 2 and 6 months later. The number of immunoglobulin secreting cells (ICS)/ 106 cord and peripheral blood mononuclear cells were determined by the enzyme-linked immunospot method, the immunophenotypes by a flow cytometer and the cytokine concentrations in plasma/serum samples by ELISA. RESULTS The number of ISC/106 IgA-secreting cells expressed in median (interquartile ranges) in cord blood samples of VD vs. ECS were 19.5 (5.0-25.7) vs. 4.9 (0.0-8.4) respectively, p=0.03. The percentage of NK cells in ECS samples tended to be lower than that in VD; 12.2 (14.6-9.7) in ECS and 18.3 (25.1-11.3) in VD. The concentration of IL6 of ECS samples was lower than that of VD. These differences at birth were no longer significant when the infants were 2 and 6 months old. CONCLUSIONS These results indicate that ECS predisposes neonates to low capacity to generate IgA concomitantly with low circulating levels of IL6 and NK cells. Caesarean section delivery can hinder the first-line nonspecific immunity to enteral antigens of microbial or dietary origin at a critical period of life when the immune memory is yet to be established. This may explain the observation that the incidence of atopic diseases increases concurrently with the frequency of elective caesarean section deliveries in the developed countries.

Characterization of Human Ovarian Carcinomas in a SCID Mouse Model

This study characterizes a murine model which is promising for the study of the growth and natural history of ovarian cancer and for testing of new therapies for its treatment. Intact portions of 20 different human ovarian cancer surgical specimens were implanted in over 60 severe combined immunodeficient (SCID) mice using techniques previously developed in our laboratory. Growth of xenografts was evaluated by gross examination and histopathologic analysis. Confirmation of the human origin of the tumor outgrowth was obtained usingin situhybridization analysis. By histological evaluation, all of the patients' tumors showed evidence of invasive growth in at least 1 of the mice implanted with portions of each surgical specimen and these tumors remained morphologically similar to the parent tumors for a long period of time. Furthermore, 65% (13/20) of the xenografts grew rapidly enough (i.e., reached a diameter of 1–2 cm within 2–6 months) to allow passage to subsequent SCID mice. Among the passaged xenografts, 3 eventually developed metastases in a distribution pattern similar to that of naturally occurring ovarian cancer and 2 developed ascites without evidence of further metastatic spread. Upon evaluation of sera from tumor-bearing mice, human antibodies presumably derived from immunoglobulin-secreting cells present in the original tumor specimen were identified. In support of this, human B cells and plasma cells could be seen within the tumor xenograft for more than 6 months following implantation. In summary, transplantation of surgical specimens from ovarian cancer patients into SCID mice results in an attractive model for the study of the natural history of ovarian cancer and may also be useful for analysis or new experimental therapeutic approaches for the treatment of this disease.

Preferential enhancement of B cell IgA secretion by intestinal epithelial cell-derived cytokines and interleukin-2

Intestinal epithelial cells (IEC) are known to secrete a number of important cytokines. Recently, we determined that IEC-derived IL-6 and TGF-β could enhance IgA secretion and suppress IgM secretion by isolated mucosal B cells. However, since the IEC-derived cytokines must function in the context of locally produced T cell cytokines, the effect of IEC-and T cell-derived cytokines on mucosal B cell immunoglobulin secretion was determined. Using 4 day culture supernatants (IEC-SN) from the rat IEC-6 intestinal epithelial cell line and lipopolysaccharide (LPS) stimulated Peyer's patch or mesenteric lymph node B cells, the IEC-SN was found to act with IL-2 to greatly enhance IgA secretion but limit or suppress IgM secretion as compared to cultures of LPS stimulated B cells alone. However, neither IL-4, IL-5, nor IFN-γ affected IgA secretion with the IEC-SN. Deletion of the IEC-SN with specific anti-cytokine antibodies suggested that IEC-derived TGF-β and IL-6 were both responsible for the enhancing effect along with IL-2 on IgA secretion, whereas IEC-derived TGF-β alone limited or suppressed IgM secretion. These results suggest that cytokines derived from local IEC and T cells may create an environment which may contribute to the preferential enhancement of IgA secretion seen in mucosal tissues.

Immunoglobulin-secreting Cells in the Surface Secretion on the Pharyngeal Tonsils

As B-lymphocytes on the pharyngeal tonsils constitute a considerable part of the leukocytes in the surface secretion, and their biological role is obscure, we explored their possible function with respect to immunoglobulin production. Twenty children scheduled for routine adenoidectomy participated. Surface secretion from 10 children was analysed for presence of plasma cells and cells from the secretions of the other 10 children were tested in enzyme-linked immunosorbent spot assays (ELISPOT-assays) for their capacity to secrete and produce IgA, IgM and IgG. Plasma cells and cells that secreted IgA, IgM and IgG respectively were present in the secretions of all tested children. In eight of ten children the IgG immunocytes, Ig-producing blasts and plasma cells, outnumbered the IgA immunocytes. The number of immunoglobulin secreting cells (ISCs) was reduced by half or more in cell suspensions exposed to the reversible protein synthesis inhibitor cycloheximide. It is concluded that immunocytes that produce and secrete immunoglobulin are present in the surface secretion on the pharyngeal tonsils. The production represents an addition to the immunoglobulins transported to the secretion by the poly-Ig receptor and by passive diffusion. The results shed new light on the pathogenesis of mucosal infections in the upper airways.

Cellular Immune Response of Adenoidal and Tonsillar Lymphocytes to the P6 Outer Membrane Protein of Non-typeable Haemophilus Influenzae and its Relation to Otitis Media

Cellular immune responses to the P6 outer membrane protein of non-typeable Haemophilus influenzae (NTHi) were determined in vitro by measuring immunoglobulin (Ig) secreting cells and lymphocyte proliferation in adenoidal and tonsillar lymphocytes from 19 children. Preliminary tests showed that P6 did not stimulate naïve cells such as cord blood lymphocytes, but did stimulate sensitized cells in adenoids and tonsils. Cellular proliferation was significantly higher in adenoidal lymphocytes than in tonsillar lymphocytes (median: quadratile of stimulation index=3.7:2.3-5.5 vs. 1.2:1.0-2.1, p<0.02). A comparison between children with or without otitis media revealed that proliferative responses to P6 of adenoidal lymphocytes from children with otitis media were significantly decreased (2.0:1.8-3.6 vs. 3.7:2.3-5.5, p<0.04). P6-specific antibody secreting cells were identified in a total of 14 adenoids and the number of cells secreting IgA was decreased in the otitis media group compared to controls (median: quadratile/106 cells=435:359-499 vs. 755:593-1870, p<0.05). Cultivation with P6 stimulated IgA secretion in children without otitis media, while no response was seen in children with otitis media (median: quadratile/106 cells=1323:915-2410 vs. 2240:1900-2830, p<0.02). These preliminary data demonstrate that lymphocytes from adenoids and tonsils recognize P6 as a specific antigen and that the adenoid is the more reactive of the two organs. Impaired P6-specific cellular immune responses of adenoids in children with otitis media may explain the recurrent nature of otitis media due to NTHi in the otitis prone population.