Local Humoral Immune Response Research Articles

Specific antibody levels in the aqueous humor and serum of two distinct populations of patients with ocular toxoplasmosis

The population of Erechim, Southern Brazil, is characterized by a high incidence of ocular toxoplasmosis, which is presumed to be of acquired origin. We wished to compare the local specific humoral immune response of individuals from this region with that of Swiss patients suffering from the same disease. Paired samples of aqueous humor and serum were withdrawn from 27 Brazilian and 50 Swiss patients presenting consecutively with active ocular toxoplasmosis. The total and specific levels of IgG in each of these were determined. The populations did not differ with respect either to age or sex. The serum levels of total IgG in Brazilian (10.8 g/l) and Swiss patients (11.1 g/l) were similar ( p = 0.499 ), but the aqueous humor ones were higher in the former group (95 vs. 20 mg/l; p = 0.0001 ). The systemic and local levels of specific IgG were likewise higher in Brazilian patients [206 i.u. vs. 72 i.u. ( p = 0.001 ) and 14 i.u. vs. 4 i.u. ( p = 0.005 ), respectively] and the number of individuals without detectable levels of local specific IgG was correspondingly lower (11% vs. 54%; p = 0.0005 ). The Goldmann–Witmer coefficient (an index of local specific antibody production) did not differ between Brazilian and Swiss patients (2.1 vs. 0.08, respectively; p = 0.107 ). Our findings are indicative of a more pronounced uveovascular barrier breakdown in Brazilians than in Swiss patients with active ocular toxoplasmosis. That the systemic and local specific immune response is weaker in Swiss than in Brazilian patients has not been hitherto documented. This finding may reflect differences in the immunological handling of the infection.

Humorale Mukosa-Immunantwort bei der Rhinitis allergica

Mucosa-immunologic aspects are gaining an increasing awareness in the pathophysiology of type I allergies. Humoral mucosal immune responses are dominated by secretory IgA, but there is evidence for a relevant role of IgG in nasal mucosa-associated lymphoid tissue. was to measure allergen-specific immunoglobulins (IgA and IgG) in nasal secretions as an expression of a humoral mucosal immune response in allergic rhinitis. For tissue eosinophilia we studied nasal Eosinophilic Cationic Protein (ECP) and for mast cell activation nasal tryptase. Nasal secretions of 40 patients suffering from allergic rhinitis were analyzed for allergen-specific IgA, IgG, and IgE, and for ECP and tryptase. Patients were highly sensitized against the major allergens of house dust mites, timothy, and birch pollen. 43 non-atopic individuals served as controls. In order to study possible effects of the actual pollen season on the studied parameter we secondly compared patients allergic to seasonal allergens co- (n = 28) and extra-seasonally (n = 41). In order to determine a possible influence of allergen-specific IgA in eosinophilic degranulation we additionally studied 5 patients after nasal allergen challenge. In allergic rhinitis we found significantly increased levels of allergen-specific immunoglobulins of all studied subclasses and allergens in nasal secretions. Comparison of nasal ECP and tryptase showed significantly increased concentrations in allergic individuals as well. Co-seasonally we found elevated allergen-specific IgE, ECP, and tryptase but lower concentrations of allergen-specific IgA and IgG. There was no association between late phase eosinophilia and IgA concentrations after local allergen challenge. The occurrence of allergen-specific immunoglobulins in nasal secretions is interpreted as a local humoral mucosal immune response. The physiologic role of local allergen-specific immunoglobulins is not clear to date. Involvement in degranulation of eosinophils or mast cells, like suggested before, seems unlikely.

Phenotype of hepatic infiltrates and hepatic lymph nodes of lambs primarily and challenge infected with Fasciola hepatica, with and without triclabendazole treatment

The phenotype of the hepatic inflammatory infiltrate and hepatic lymph nodes (HLN) was analysed in lambs primarily and challenge infected with Fasciola hepatica. Group 1 was primarily and challenge infected with two doses of 200 metacercariae (mc) each and was non-treated. Trickle infection was administered to five groups: group 2 was challenge infected and non-treated; group 3 was primarily infected and non-treated; group 4 was primarily infected and treated with triclabendazole (TCBZ) at 12 weeks postinfection (wpi); group 5 was treated at 4 wpi and challenge infected and group 6 was treated at 12 wpi and challenge infected. An uninfected group was used as the control. The distribution of T cell subpopulations (CD3, CD4 and CD8), and B cells (CD79alpha, IgM, IgG) was analysed. The hepatic inflammatory infiltrate was represented mainly by CD3 and CD4 T cells, and B cells (CD79alpha, IgG). These infiltrates were more severe (P < 0.05) in primarily (group 3) or challenge (groups 2, 5 and 6) trickle infected lambs than in the group single challenge infected (group 1). Cellular changes in HLN consisted in an increase of CD4 over CD8 T cells and an increase of B cells and IgG+ plasma cells, and they were more severe in primarily and challenge trickle infected groups than in the group infected with two larger doses of mc, although significant differences were not found with respect to all challenge trickle infected groups. The strong local cellular and humoral immune responses did not protect against subsequent infections, neither in non-treated lambs (group 2) nor in lambs treated with TCBZ at 4 wpi (group 5) or 12 wpi (group 6).

Aqueous humor and serum immunoblotting for immunoglobulin types G, A, M, and E in cases of human ocular toxoplasmosis.

The purpose of this study was to compare the local and systemic Toxoplasma-specific humoral immune responses in individuals with ocular toxoplasmosis (OT). To this end, paired aqueous humor and serum samples from 46 individuals with active OT and from 30 individuals without inflammatory eye disease (controls) were analyzed by immunoblotting for anti-Toxoplasma immunoglobulin G (IgG), IgA, IgM, and IgE directed against 20- to 120-kDa antigens. The presence in the aqueous humor of a unique band, or of at least three bands that were at least three times more intense in aqueous humor than in serum, was taken as evidence of local antibody production. IgG bands were detected in 98% of the aqueous humor samples, while IgA bands were detected in 76%, IgM bands were detected in 8%, and IgE bands were not detected in any. Evidence of local production of specific antibodies was found in 32 cases (70%) (IgG in 23 [50%]; IgA in 16 [35%]). In 10 instances (22%), routine laboratory tests were not indicative of OT. In 14 cases (30%), no local antibody production was detected by immunoblotting; 3 of these cases yielded evidence of local antibody production according to the Goldmann-Witmer coefficient. Local antibody production was revealed for 7 of the 30 controls (23%). Hence, the sensitivity of immunoblotting for IgG and IgA is 70%, and the specificity is 77%. We conclude that immunoblotting for local specific IgG and IgA supports the clinical diagnosis of OT in 70% of cases. In 22% of these, the diagnosis is not confirmed by other laboratory tests. Hence, immunoblotting increases the sensitivity of routine laboratory tests and should be considered for samples that register negative by such tests.

High-risk papillomavirus infection is associated with altered antibody responses in genital tract: non-specific responses in HPV infection.

In order to gain more information about local humoral immune responses to HPV infection, we quantified IgG, IgM, secretory-IgA (S-IgA), and total-IgA by ELISA, and lysozyme and lactoferrin by TR-IFMA, in cervical and cervicovaginal secretions of 40 healthy women and 28 high-risk HPV infected patients (11 were HPV16+). IgG, total-IgA, and S-IgA concentrations in cervicovaginal secretions (p < 0.0001) and high IgG and total-IgA concentrations (p < 0.001 and p < 0.01, respectively) in endocervical secretions were significantly higher in HPV+ patients than in the healthy group. Since the S-IgA/total-IgA ratio was significantly lower in cervicovaginal (7.5%) and endocervical secretions (36.5%) in HPV+ women compared to the control group (p < 0.003 and p < 0.001, respectively), HPV could be responsible for an increase in local production of non-secretory IgA (monomeric and dimeric forms). IgG and total-IgA concentrations in cervicovaginal and endocervical secretions fell in the same general percentage range in both HPV16+ and HPV+ groups (80% and 15%, respectively). However, the S-IgA/total-IgA ratio was much lower in HPV16+ than in HPV+ women, in both cervicovaginal secretions (3.4%) (p < 0.003) and in endocervical secretions (23.3%) (p < 0.001). Innate immunity proteins and local S-IgA response could not stop the spread of HPV infection in spite of high lysozyme and lactoferrin concentrations. HPV16+ disturbed the local humoral immune system, which could partly explain its low clearance.

High-Risk Papillomavirus Infection Is Associated with Altered Antibody Responses in Genital Tract: Non-specific Responses in HPV Infection

In order to gain more information about local humoral immune responses to HPV infection, we quantified IgG, IgM, secretory-IgA (S-IgA), and total-IgA by ELISA, and lysozyme and lactoferrin by TR-IFMA, in cervical and cervicovaginal secretions of 40 healthy women and 28 high-risk HPV infected patients (11 were HPV16+). IgG, total-IgA, and S-IgA concentrations in cervicovaginal secretions (p <0.0001) and high IgG and total-IgA concentrations (p <0.001 and p < 0.01, respectively) in endocervical secretions were significantly higher in HPV+ patients than in the healthy group. Since the S-IgA/total-IgA ratio was significantly lower in cervicovaginal (7.5%) and endocervical secretions (36.5%) in HPV+ women compared to the control group (p < 0.003 and p < 0.001, respectively), HPV could be responsible for an increase in local production of non-secretory IgA (monomeric and dimeric forms). IgG and total-IgA concentrations in cervicovaginal and endocervical secretions fell in the same general percentage range in both HPV161 and HPV+ groups (80% and 15%, respectively). However, the S-IgA/total-IgA ratio was much lower in HPV16+ than in HPV+ women, in both cervicovaginal secretions (3.4%) (p < 0.003) and in endocervical secretions (23.3%) (p < 0.001). Innate immunity proteins and local S-IgA response could not stop the spread of HPV infection in spite of high lysozyme and lactoferrin concentrations. HPV16+ disturbed the local humoral immune system, which could partly explain its low clearance.

Mucosal immunization with purified flagellin from Salmonella induces systemic and mucosal immune responses in C3H/HeJ mice

This study investigated the immune response elicited in C3H/HeJ mice after oral, parenteral and nasal immunization with purified flagellin from Salmonella enterica serovar Enteritidis alone or conjugated to starch microparticles as adjuvant or together with the uptake-enhancer recombinant cholera toxin B-subunit (rCTB). Systemic (IgM–IgG, IgA, IgG2a, IgG2b, IgG1) and local (s-IgA) humoral immune responses in the mice were analyzed using enzyme-linked immunosorbent assays (ELISA). Primed splenocytes were also stimulated in vitro with flagellin and the supernatants analyzed for cytokine production. Finally, immunized mice were challenged orally with live Salmonella. A high flagellin-specific IgM–IgG response was seen in all groups, especially in mice immunized nasally with flagellin plus rCTB or subcutaneously, but a strong systemic antibody response was also induced when free antigen was given orally. Intranasal or subcutaneous immunization of mice with flagellin plus rCTB or oral immunization with flagellin plus microparticles resulted in a significantly greater mucosal response (higher s-IgA titers in feces) than seen in the control group ( P<0.05). The mucosal IgA responses were significantly correlated with the serum IgA titers. The subclass profile in serum revealed a mixed Th1/Th2-type response, with a predominance of Th1-type, as indicated by the subclass ratio (IgG1/IgG2a+IgG2b). The splenocytes stimulated in vitro produced interferon (IFN)-γ, at levels, which increased with time. The group immunized with flagellin plus rCTB subcutaneously had a relatively higher IFN-γ response than the other groups. Interleukin (IL)-2 was also produced, especially in mice immunized nasally or subcutaneously with flagellin conjugated to microparticles. However, neither IL-4 nor IL-5 was produced in any of the groups. After oral challenge with live serovar Enteritidis, the groups immunized orally or nasally with free flagellin had significantly lower degree of infection than the control group ( P<0.05).

Local and systemic humoral immune response during acute and chronic Acanthamoeba keratitis in rabbits

Purpose. To study local and systemic antibody responses during the course of acute and chronic Acanthamoeba keratitis in a rabbit model. The role of secretory IgA (sIgA) in innate and adaptive immunity is investigated.Methods. Acanthamoeba keratitis was induced by intrastromal injection of live trophozoites in rabbits with and without prior oral immunization with aqueous Acanthamoeba antigen. The severity score of the ensuing keratitis was followed. Anti-Acanthamoeba antibodies in tears and sera were determined before infection and on a weekly basis postinfection for 6 weeks. The role of mucosal IgA as a link between the innate and adaptive immunity to Acanthamoeba was evaluated.Results. Acanthamoeba keratitis was significantly milder in animals infected after oral immunization than in naïve animals. Mucosal sIgA bound Acanthamoeba in a concentration-dependent, mannose-mediated manner. It significantly inhibited Acanthamoeba binding to rabbit corneal epithelium in vitro and in vivo. Anti-Acanthamoeba IgA significantly influenced amoebic lysis by neutrophils.Conclusions. Mucosal IgA protects the external ocular surface by virtue of anti-adhesin properties displayed by the mannosylated residues on the heavy chains of IgA molecule (innate immunity) as well as specific antigen-binding sites (adaptive immunity). Immune IgA also augmented neutrophil-mediated amoebic lysis (adaptive immunity).

Induction of Partial Protection in Mice after Oral Administration of Lactococcus lactis Producing Brucella abortus L7/L12 Antigen

The Brucella abortus ribosomal protein L7/L12 is an immunodominant antigen and an interesting candidate for the development of oral live vaccines against brucellosis. Here, a recombinant Lactococcus lactis strain producing L7/L12 under the control of nisin inducible promoter was orally administered to BALB/c mice. Significant levels of anti-L7/L12 specific IgA detected in feces revealed an induced local humoral immune response. However, serum analysis did not reveal any anti-L7/L12 antibodies suggesting the absence of a systemic response. Nevertheless, the vaccinated mice showed a partial protective immunity against B. abortus virulent strain (S2308) challenged by intraperitoneal inoculation.

Herpes simplex virus infection of the human eye induces a compartmentalized virus-specific B cell response.

Intraocular infection with herpes simplex virus (HSV) can cause uveitis, a potentially sight-threatening disease. The disease is characterized by an ocular infiltration of inflammatory cells such as macrophages and B and T cells. The characteristics of the local humoral and cellular immune responses elicited on intraocular HSV infection are poorly understood. The local HSV-specific antibody production, which are used routinely for confirmation of a clinical diagnosis of herpetic uveitis, has never been analyzed in detail. This study analyzed the humoral immune response against HSV type 1 (HSV-1) in paired samples of intraocular fluid and serum of patients with intraocular herpesvirus infection. In addition, the B cell epitope distribution on a single HSV-1 type-specific antigen, glycoprotein G, was compared for these paired samples. The results presented here indicate that the inflamed eyes of patients with HSV-induced uveitis display a compartmentalized B cell response directed toward the triggering virus.

Evidence for a bias toward intracellular antigens in the local humoral anti-tumor immune response of a colorectal cancer patient revealed by phage display.

Many patients with colorectal carcinoma (CRC) mount a cellular as well as a humoral immune response to the tumor. To investigate the nature and specificity of the humoral immune response in a CRC patient, lymphocytes infiltrating the primary colorectal tumor and lymph nodes draining the tumor were used as antibody variable (V)-gene pools for the construction of phage antibody repertoires. These libraries were first validated by selection on the antigen tetanus toxoid and shown to contain antibodies that were probably derived from both naive and memory B cells. The repertoires were then screened for the presence of antibodies directed to CRC by selection on the cell line CaCo2. For comparison, the same selections were performed with a phage antibody repertoire made from B cells of healthy donors. Striking differences were observed in the panel of specificities selected from these different repertoires: although a large panel of antibodies reactive with patient-derived primary tumors was obtained from the immune repertoires, none of these discriminated between normal colonic epithelium and colon cancer and none were reactive with cell-surface antigens. However, selections using the non-immune library did result in numerous antibodies that recognized cell surface markers on CaCo2. These data suggest a bias in the local humoral immune response in this CRC patient, directed primarily toward intracellular epithelial-cell specific target antigens.

Periodontal diseases: to protect or not to protect is the question?

For decades, investigations have identified local and systemic humoral immune responses to microorganisms comprising the supra- and subgingival biofilms in the oral cavity. Inflammation and tissue destruction in the periodontium are accompanied by alterations in the quantity, quality, and specificity of antibody. The conundrum in this scenario is the existence of a substantial plasma cell infiltrate at sites of periodontal lesions and a seemingly robust antibody response in the oral cavity and the serum, apparently coincident with progressing disease. Consequently, much effort has been expended to elucidate the critical characteristics of protective humoral responses and to develop strategies for enhancing these unique features. We and others have conducted studies attempting to distinguish disease susceptibility associated with: i) variations in response levels-significantly increased to some species with disease, minimal response to others; ii) functional comparisons of antibody-subclass differences, genetic regulation, and maturation of responses; iii) microbial and antigenic specificity of the antibody-focus on specific pathogens and identification of selected antigens as targets for immunoprotection; and, iv) kinetics of responses during disease and therapeutic interventions-linking immune changes with infection and as a measure of treatment success. This report summarizes varied research designs and results, to provide a profile of antibody in health, gingivitis, and periodontitis. These profiles may be used to provide a framework focusing on the humoral response to commensal microorganisms and likely pathogens, as they emerge in the biofilm-etiologic for or in response to disease processes. Models for antibody as a diagnostic adjunct and for predicting protective antibody responses are suggested. These concepts are likely relevant for considering vaccine approaches to periodontitis.

Adventitial infiltrates associated with advanced atherosclerotic plaques: structural organization suggests generation of local humoral immune responses.

Advanced atherosclerotic lesions often contain adventitial lymphoid infiltrates, which occasionally contain nodular aggregates resembling lymphoid follicles. The structural organization suggests that local maturation of B cells may take place at these sites, as described for the mucosa-associated lymphoid tissue (MALT). This concept was evaluated by studying the micro-anatomy and cellular composition of adventitial infiltrates associated with advanced atherosclerosis of the aorta. Sections of 22 atherosclerotic aortas were stained immunohistochemically for cellular markers characteristic for lymphoid follicles, such as HECA-452-positive endothelial cells, CD20-positive B cells, CD21-positive follicular dendritic cells, and CD68-positive macrophages. Ki-67 was used as a proliferation marker. The TUNEL technique was used to study the presence of apoptotic cells. Specimens containing MALT served as comparison and positive controls. Seven of the 22 atherosclerotic aortas contained adventitial infiltrates resembling lymphoid follicles. The organized nodular centres were composed of CD45RA+ B cells, follicular dendritic cells (CD21+), a few T lymphocytes (CD3+) and 'tingible body' macrophages (CD68+). A large number of cells were Ki-67-positive; apoptotic bodies were numerous and phagocytosed by macrophages. The parafollicular area contained CD45RO-positive T cells and HECA-452-positive vessels. Vessels elsewhere were always HECA-452-negative. Specimens with MALT showed similar features. This study reveals a close resemblance between adventitial lymphoid infiltrates in advanced atherosclerotic aortic disease and MALT, suggesting local generation of a humoral immune response, likely to be initiated by antigens released during a process of long-standing tissue injury and inflammation as part of advanced atherosclerosis.

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.

Evaluation of live, cold-adapted influenza A and B virus vaccines in elderly and high-risk subjects

We have evaluated the use of live cold-adapted influenza A and B virus vaccines in the elderly. Cold-adapted influenza A and B virus vaccines are safe and modestly immunogenic in individuals over 65 years of age. However, our studies and those of other groups have shown that immune response to cold-adapted vaccines in this age group are modest. Administration of combined cold-adapted influenza A and inactivated influenza vaccine has resulted in slightly higher frequencies of local and systemic humoral immune responses than inactivated vaccine alone in some, but not all, studies. In a double-blind field trial conducted in nursing homes over a 3 year period, combined cold-adapted influenza A (H3N2) and trivalent inactivated influenza vaccine resulted in a 60% decrease (95% CI, 18–82%) in the rate of laboratory documented influenza A compared with inactivated vaccine alone. Further studies of multivalent cold-adapted influenza vaccines used in combination with inactivated vaccine should be performed.

T cell- and perforin-dependent depletion of B cells in vivo by staphylococcal enterotoxin A.

Bacterial superantigens bind to major histocompatibility complex (MHC) class II and subsequently activate both CD4+ and CD8+ T lymphocytes expressing certain T-cell receptor (TCR)-Vbeta chains. In response to superantigen exposure these subsets proliferate, produce large amounts of proinflammatory cytokines and in addition CD8+ cytotoxic T lymphocytes (CTL) are induced. Previous studies in vitro have shown that these CTL effectively lyse MHC class II-expressing cells presenting the proper superantigen. However, it is unknown whether superantigens induce a similar response towards MHC class II+ antigen-presenting cells in vivo. In this study we demonstrate that administration of repeated injections of the superantigen staphylococcal enterotoxin A (SEA) to TCR-Vbeta3 transgenic mice results in a loss of MHC class II-expressing cells in the spleen. Analysis of different MHC class II+ subsets revealed a selective depletion of CD19+ B cells, while F4/80+ macrophages increased in number. Depletion of T cells with anti-CD4 or anti-CD8 monoclonal antibody indicated that CD8+ T cells were crucial for SEA-induced cytotoxicity in vivo. Repeated injections of SEA to perforin-deficient mice resulted in significantly less B-cell depletion compared with control mice. This suggests that superantigen-activated CD8+ T cells lyse MHC class II+ antigen-presenting cells in a perforin-dependent manner in vivo. It is suggested that this represents a novel bacterial immune escape mechanism, which may particularly impair local humoral immune responses.

Immunohistochemical study of the local immune response to Fasciola hepatica in primarily and secondarily infected goats

The distribution of CD2, CD4, CD8, γ/δ T-lymphocytes, B-cells and IgG lambda-light chain (λ-IgG) containing cells were analysed in the inflammatory infiltrate associated to hepatic lesions and gallbladder (HL), and in hepatic lymph nodes (HLN) of goats primarily and secondarily infected with Fasciola hepatica. In the HL, CD2 and CD8 T-cells were more numerous ( p=0.01) in secondarily rather than in primarily infected goats, whereas CD4 T-lymphocytes were less numerous than CD8 and showed no significant change in both groups. The ratio CD4/CD8 was 0.66 and 0.39 for primarily and secondarily infected goats, respectively. In contrast, in the HLN, CD4 were more numerous than CD8 T-cells, the ratio CD4/CD8 was 2.0 in control, 1.5 and 1.3 in primary and secondary infections, respectively. γ/δ T-lymphocytes were scarce in the HL and moderate in the HLN of both primarily and secondarily infected animals. B-cells (IgM +, λ-IgG + or CD79 +) varied from scarce or moderate in the HL to abundant in the HLN, where CD79 +-cells were mainly located in lymphoid follicles and IgM and IgG in plasma-cells of the medullary cords, suggesting an intense local humoral immune response. However, this response did not prevent the hepatic damage in secondarily infected goats, in which hepatic lesions were more severe than in primarily infected ones.

Systemic and local immune responses againstHelicobacter pylori urease in patients with chronic gastritis: distinct IgA and IgG productive sites

Background—Helicobacter pylori urease is a major target for immune responses among various bacterial components in H pyloriinfected patients.Aims—To analyse the relation between systemic and local humoral immune responses toH pylori...

Immunohistochemical characterization of hepatic lesions associated with Elaeophora elaphi parasitism in red deer (Cervus elaphus)

This study was based on the immunohistochemical characterization of hepatic lesions caused by Elaeophora elaphi in 15 red deer. In eight animals filariae were found in branches of the portal vein. "Web-like" endophlebitis (15 cases), lymph nodule-like thrombi associated with filarial debris (12 cases) and fibrinous thrombi (three cases) were also observed in the portal vein. Multifocal granulomatous hepatitis (six cases) and lymph nodule-like tissue (three cases) were found in the liver parenchyma. The distribution of CD3 antigen, IgG-lambda light chains, and S-100 protein in lymphoid tissue in the portal vein thrombi and hepatic parenchyma was similar to that found in the cortex of the lymph nodes. In these lesions, lymphoid follicles with large germinal centres (mainly composed of IgG+ B lymphocytes, with occasional S-100+ follicular dendritic cells) predominated over interfollicular lymphoid tissue (mainly composed of CD3+ T lymphocytes and S-100+ interdigitating cells). This suggested that the local humoral immune response was more important than the cellular response in this parasitic disease. The presence of interdigitating and follicular dendritic cells confirmed the high degree of organization of the abnormal lymphoid tissue and suggested that antigen presentation to T and B lymphocytes and the subsequent proliferation of the latter was particularly efficient in such tissue. The latter may therefore be superior to diffuse granulomatous infiltrate in terms of the production of a local immune response.

Analysis of VH gene rearrangements from synovial B cells of patients with rheumatoid arthritis reveals infiltration of the synovial membrane by memory B cells.

The VH gene (Variable gene segments of the heavy chain locus) repertoire can be investigated by DNA analysis of rearranged immunoglobulin VH genes, which also allows for an indirect estimation of antibody selection by analysis of somatic mutations. Using a polymerase chain reaction (PCR) it is also possible to analyse these genes in small numbers of cells or even single cells. This approach was chosen to investigate germinal centre like lymphocyte follicles in the synovial membranes of two patients with rheumatoid arthritis (RA) in order to analyse the local humoral immune response in RA. Individual B-cell aggregates of synovial membrane of two patients with RA were isolated by micromanipulation from microscopic slides. VH-DH-JH (variable, diversity, and joining segments of the heavy chain locus) rearrangements in all possible VH-JH combinations were amplified from these B cell foci, cloned and subjected to sequence analysis. Sequence analysis revealed that most of the rearranged VH genes were somatically mutated with at least 1% (range 1.3-14.9%) somatic mutations and therefore were derived from antigen-selected memory B cells. Intraclonal diversity in one-third of the clones indicated the generation of memory B cells in the synovial membrane and characterized the synovial membrane as lymphatic tissue where secondary immune responses to an as yet unknown antigen take place.