Germinal Center Activity Research Articles

Mechanisms of B cell autoimmunity in SLE

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that is known to be associated with polyclonal B-cell hyperreactivity. The underlying causes of the diffuse B-cell over-reactivity are unclear, but potential candidates include (a) intrinsic hyper-reactivity leading to polyclonal B-cell activation with disturbed activation thresholds and ineffective negative selection; (b) lack of immunoregulatory functions; (c) secondary effects of an overactive inflammatory environment, such as overactive germinal center and ectopic follicular activity; and/or (d) disturbed cytokine production by non-B immune cells. These mechanisms are not mutually exclusive and may operate to varying extents and at varying times in SLE. Phenotypic and molecular studies as well as the results of recent clinical trials have begun to provide new insights to address these possibilities. Of importance, new information has made it possible to distinguish between the contribution played by abnormalities in central checkpoints that could lead to a pre-immune repertoire enriched in autoreactive B cells, on the one hand, and the possibility that autoimmunity arises in the periphery from somatic hypermutation and abnormal selection during T cell-dependent B-cell responses on the other. There is an intriguing possibility that apoptotic material bound to the surface of follicular dendritic cells positively selects autoreactive B cells that arise from non-autoreactive B-cell precursors as a result of somatic hypermutation and thereby promotes the peripheral emergence of autoimmunity.

Epstein-Barr virus colonization of tonsillar and peripheral blood B-cell subsets in primary infection and persistence

AbstractEpstein-Barr virus (EBV) persists in the immune host by preferentially colonizing the isotype-switched (IgD−CD27+) memory B-cell pool. In one scenario, this is achieved through virus infection of naive (IgD+CD27−) B cells and their differentiation into memory via germinal center (GC) transit; in another, EBV avoids GC transit and infects memory B cells directly. We report 2 findings consistent with this latter view. First, we examined circulating non–isotype-switched (IgD+CD27+) memory cells, a population that much evidence suggests is GC-independent in origin. Whereas isotype-switched memory had the highest viral loads by quantitative polymerase chain reaction, EBV was detectable in the nonswitched memory pool both in infectious mononucleosis (IM) patients undergoing primary infection and in most long-term virus carriers. Second, we examined colonization by EBV of B-cell subsets sorted from a unique collection of IM tonsillar cell suspensions. Here viral loads were concentrated in B cells with the CD38 marker of GC origin but lacking other GC markers CD10 and CD77. These findings, supported by histologic evidence, suggest that EBV infection in IM tonsils involves extrafollicular B cells expressing CD38 as an activation antigen and not as a marker of ectopic GC activity.

IgG4-Related Disorder of the Retroperitoneum Resembling Castleman’s Disease Plasma Cell Type: A Report of 2 Cases

This study reports 2 cases of IgG4-related disorder of the retroperitoneum resembling plasma cell type of Castleman's disease. A single lesion was located in the renal hilum and ureter, respectively, in these 2 cases. Histologically, both lesions were characterized by reactive follicular hyperplasia with active germinal centers and a sheet of polyclonal mature plasma cells in the interfollicular area. The prominent sclerosis and/or fibrosis, which were characteristic histological findings of IgG4-related disorders, were absent. However, immunohistochemical study demonstrated numerous IgG4+ plasma cells accounting for more than 50% of IgG+ cells. The phlebitis that occurs with early lesions of obliterative phlebitis is one of the characteristic histological findings of IgG4-related disorders and was noted in one case. Serum IgG4 concentration was increased in one case. The serum interleukin-6 level was within the normal range in one case that was examined. From a therapeutic perspective, it is important to discriminate IgG4-related disorder from plasma cell type of Castleman's disease.

TRAF2 and TRAF3 independently mediate Ig class switching driven by CD40

The isotype switch defect in CD40(-/-) mice is corrected by wild-type (WT) CD40 transgene, but not by a mutant CD40 transgene that does not bind tumor necrosis factor receptor-associated factors (TRAF) 2 and 3. To define the individual roles of TRAF2 and TRAF3 in CD40 activation of B cells, we introduced mutant CD40 transgenes that selectively lack the ability to bind TRAF2 (DeltaTR2), TRAF3 (DeltaTR3) or both (DeltaTR2,3) into B cells of CD40(-/-) mice. Serum IgG1 and IgE levels, IgG1 antibody response to sub-optimal doses of the T cell-dependent antigen keyhole limpet hemocyanin, germinal center formation, CD40-mediated proliferation, isotype switching and activation of the non-canonical NF-kappaB pathway were partially diminished in DeltaTR2 and DeltaTR3 mice and virtually absent in DeltaTR2,3 mice. These results suggest that TRAF2 and TRAF3 can each independently mediate class switch recombination (CSR) driven by CD40, but both are required for optimal CD40-driven isotype switching.

Expression of CD320 in human B cells in addition to follicular dendritic cells

CD320 has been recently discovered and reported as a follicular dendritic cell (FDC) protein. Although CD320 is known to enhance proliferation of germinal center (GC) B cells, little other information is available. In this study, we investigated its cellular distribution in the GC. Confocal microscopy of human tonsil sections revealed co-localization of CD320 with CD19 and CD38 but not with CD3 indicating that GC B cells expressed CD320 in addition to FDC. In purified GC B cells, CD320 expression was inhibited in the nucleus, membrane and cytoplasm. Reverse transcriptase-polymerase chain reaction confirmed CD320 mRNA expression in B cells. These finding indicate that CD320 is expressed in B cells in addition to FDC, and that its GC activity may be more complicated than previously thought.

Rectal lymphoid hyperplasia resembling polypoid-type malt-lymphoma: A case report

Introduction. Lymphoid hyperplasia of the rectum is a rare benign lesion, histologically defined by large lymphoid follicles, with active germinal centers, surrounded by thin mantle and marginal zone. We report an unusual case of rectal polyp, characterized by benign lymphoid hyperplasia associated to prominent marginal zone, resembling polypoid-type of MALT lymphoma.

Epstein-Barr virus persistence in the absence of conventional memory B cells: IgM+IgD+CD27+ B cells harbor the virus in X-linked lymphoproliferative disease patients

AbstractEpstein-Barr virus (EBV) persists in healthy virus carriers within the immunoglobulin (Ig)D−CD27+ (class-switched) memory B-cell compartment that normally arises through antigen stimulation and germinal center transit. Patients with X-linked lymphoproliferative disease (XLP) lack such class-switched memory B cells but are highly susceptible to EBV infection, often developing fatal symptoms resembling those seen in EBV-associated hemophagocytic syndrome (EBV-AHS), a disease caused by aberrant virus entry into the NK- or T-cell system. Here we show that XLP patients who survive primary EBV exposure carry relatively high virus loads in the B-cell, but not the NK- or T-cell, compartment. Interestingly, in the absence of conventional class-switched memory B cells, the circulating EBV load was concentrated within a small population of IgM+IgD+CD27+ (nonswitched) memory cells rather than within the numerically dominant naive (IgM+IgD+CD27−) or transitional (CD10+CD27−) subsets. In 2 prospectively studied patients, the circulating EBV load was stable and markers of virus polymorphism detected the same resident strain over time. These results provide the first definitive evidence that EBV can establish persistence in the B-cell system in the absence of fully functional germinal center activity and of a class-switched memory B-cell compartment.

Role of TRAF2 and TRAF3 in CD40 mediated immunoglobulin class switching

The isotype switch defect in CD40−/− mice is corrected by wild type (WT) CD40 transgene, but not by a mutant CD40 transgene that does not bind TRAF2 and TRAF3. To define the individual roles of TRAF2 and TRAF3 in CD40 activation of B cells, we introduced mutant CD40 transgenes that selectively lack the ability to bind TRAF2 (ΔTR2), TRAF3 (ΔTR3) or both (ΔTR2,3) into B cells of CD40−/− mice. Serum IgG1 and IgE levels, germinal center formation, CD40‐mediated proliferation, isotype switching, and activation of the non‐canonical NFκB pathway and JNK were partially diminished in ΔTR2 and ΔTR3 mice, and virtually absent in ΔTR2,3 mice. These results suggest that TRAF3 plays a positive role in CD40 driven CSR and that both TRAF2 and TRAF3 are required for optimal CD40 driven isotype switching.

Localized lymphoid hyperplasia of the rectum representing progressive transformation of the germinal center

Histologically, benign lymphoid hyperplasia (BLH) of the rectum is usually characterized by large lymphoid follicles with active germinal centers, and a narrow surrounding mantle zone and marginal zone. We present two cases of BLH of the rectum demonstrating progressive transformation of the germinal center (PTGC). The patients were 50- and 71-year-old Japanese women. Colonoscopy demonstrated small sessile polyps in both cases. The resected specimen contained numerous lymphoid follicles with active germinal centers and a portion of the lymphoid follicles exhibited PTGC. The area showing PTGC contained a few large lymphoid cells resembling lymphocytic and histiocytic Reed-Sternberg cells of nodular lymphocyte-predominant Hodgkin lymphoma. These PTGC contained small- to-medium clusters of epithelioid cells in both cases. In situ hybridization studies demonstrated scattered Epstein-Barr virus (EBV)-encoded small RNA-positive medium and large lymphoid cells and crypt epithelium in both lesions. EBV may be involved in the etiology of a subset of BLH of the rectum. However, reactivity of lymphoid cells for EBV has been reported in lymphoid tissues in a high percentage of "normal" individuals. The etiology of BLH of the rectum remains unclear.

High Level EBV Persistence in the IgM+ IgD+ CD27+ B Cell Subset in Patients with X-Linked Lymphoproliferative Disease Lacking Isotype-Switched Memory B Cells.

Epstein-Barr virus (EBV) infects >90% of population world wide and, in healthy virus carriers, establishes life long persistence in the immunoglobulin (Ig)Dneg, CD27+ (“class-switched”) memory B cell compartment normally produced by antigen stimulation and transit through germinal centres. Patients with the X-linked lymphoproliferative disease (XLP) cannot make such class-switched memory B cells due to an inherited mutation in the slam-associated protein (SAP) gene involved in the maturation of antibody responses. Interestingly, XLP patients are highly susceptible to severe primary EBV infection and develop a fulminant infectious mononucleosis (IM) which is often fatal and where the symptoms progress to resemble those of a different (non-familial) disease, EBV-associated haemophagocytosis syndrome (EBV-AHS), caused by virus entry into the NK or T cell system. Some XLP patients survive their primary infection, but the nature of EBV carriage in these individuals (lacking conventional memory B cells) remains unresolved. To investigate this further we obtained blood samples from 8 such XLP patients. EBV load in total peripheral blood mononuclear cells (PBMC), determined by quantitative PCR, occupied a broad range but on average was 2 to 3-fold fold higher than that of healthy controls. The virus was concentrated within the B cell (CD19+) compartment, as in healthy carriers, and not within T or NK cells, as typically seen in EBV-AHS. We then confirmed that these XLP patients indeed lacked conventional class-switched memory B cells but did carry a small population of IgM+, IgD+, CD27+ (“non-switched”) memory cells; however their circulating B cell pool was dominated by naïve (IgM+, IgD+, CD27neg) cells and by expanded numbers of immature “transitional” (CD10+ CD27neg) cells. In each of 4 cases studied by cell sorting, EBV was concentrated in this small subset of “non-switched” memory B cells. To see if the high virus load detected in these patients indicated true virus persistence as opposed to recent or recurrent infection, serial samples obtained over a 3 year period from two XLP patients were assayed. Virus load was stable and, in one case with the highest load, screening with markers of virus polymorphism detected the same resident strain over time. Our results in XLP patients make it clear that EBV can persist in the absence of a conventional class-switched memory B cell compartment. Instead, the virus is sequestered in a small population of “non-class-switched memory” cells with Ig gene mutations. The origin of such cells, which are also detectable in the blood of normal donors, is uncertain; however their existence in XLP patients suggests that such cells arise independently of germinal centre activity and hence that EBV may be able to colonise its host without exploiting germinal centre transit.

Developmental anatomy of the tonsil and its implications for intracapsular tonsillectomy

To describe the developmental anatomy of the human tonsil from the embryonic period through adulthood and to use this information to define the applicability of intracapsular tonsillectomy in the management of disease. Descriptive-anatomic. (1) Normal embryos and fetuses from two archival collections, (2) children with adenotonsillar hypertrophy and (3) adult autopsy material. The second branchial pouch is visible in the 4th post-conceptional week and demonstrates canalization and branching in the 8th week. Lymphoid infiltration of the lamina propria occurs in the seventh month of intrauterine life. Primary follicles form late in gestation, but germinal center stimulation does not occur until shortly after birth. During the first year of life, there is rapid proliferation of lymphoid elements and formation of active germinal centers. During the phase of maximum tonsillar hyperplasia, the lymphoid elements proliferate rapidly, increasing tonsillar bulk. In senescence, there is involution of the lymphoid elements and proliferation of fibrous tissue in the capsule and trabeculae. The overall bulk of the tonsil is much decreased. There is an intimate relationship between the epithelial and lymphoid components of the tonsil which continues through life. To adequately control upper airway obstruction of tonsillar origin and recurrent tonsillar infection in childhood, total or near total intracapsular tonsillectomy likely will prove the minimum acceptable surgical intervention. Changes in tonsillar anatomy after the first decade may alter operative choice in older patients.

B lymphocyte physiology: the beginning and the end.

Whereas lymphatic tissues were implicated as the chief sites for antibody production almost 100 years ago, three findings cemented the role of the plasma cell as the actual producer, namely the histological and tissue culture studies of Fagraeus, the beautiful immunofluorescent approach of Coons and his group, and the micromanipulation approach to the study of antibody formation by single cells introduced by Lederberg and myself. Proof that antibody-forming cells derived from B, rather than T, cells had to await the studies of Miller and Mitchell, with some minor technical contributions from myself. The physiology of the germinal centre also has a long history, and recently much interest has surrounded the relative roles of germinal centres as sites of somatic immunoglobulin V gene hypermutation and selection of high affinity B cells, versus the roles of extra-follicular proliferative foci as sources of the primary antibody response. Tolerance and self-antigens within the primary B lymphocyte repertoire is secured by clonal deletion within the bone marrow of those cells which are operationally the most threatening to the body, and a second level of functional impairment of B cell activity and life-span, capable of being induced centrally or peripherally, termed clonal anergy. As these concepts became progressively more refined through transgenic models of immunological tolerance, we turned our attention more towards tolerance within the secondary B lymphocyte repertoire. This is generated primarily inside germinal centres, where it appears that two quite separate mechanisms act as a bulwark against the possible creation of hypermutated anti-self cells. The first is that germinal centre activity and memory cell generation are dependent on antigen-specific, germinal centre-seeking CD4+ T cells, and if a putative anti-self mutant B cell gets no help because of T cell tolerance, it will not expand further. The second is a very specific mechanism confined to the germinal centre whereby antigen-specific B cells are especially sensitive to antigen-induced apoptosis if soluble, deaggregated antigen is presented to them before they reach the 'rescue' signal of follicular dendritic-cell-bound antigen. While some of the death in germinal centres is clearly apoptotic in nature, a further phenomenon observed electron microscopically relates to the formation of type B dark cells. It is not yet clear whether the DNA in this type of dying cell is cleaved. Transgenic expression of bcl-2 in germinal centre B lymphocytes confers incomplete protection from apoptosis caused by soluble antigen. The suggestion that at least some of this apoptosis is mediated via Fas-Fas ligand interactions is prompted by the observation that the apoptotic phenomenon is markedly reduced in lpr mice.

Delayed memory B cell recovery in peripheral blood and lymphoid tissue in systemic lupus erythematosus after B cell depletion therapy

Recent data suggest that the reconstituting peripheral B cell compartment after B cell depletion therapy may be functionally immature, with a preponderance of transitional B cells and a paucity of memory B cells. This study was undertaken to determine the magnitude, duration, and cause of these defects in rituximab-treated systemic lupus erythematosus (SLE) patients. Fifteen patients with SLE previously treated with rituximab as part of a phase I/II dose-escalation study were evaluated during a long-term followup (mean followup period 41 months). B cells from peripheral blood and tonsils were assessed using multicolor flow cytometry, and their developmental pathway was classified based on the expression of defined surface markers. Reconstitution of peripheral blood CD27+ memory B cells was delayed for several years after B cell depletion therapy in a subset of patients with prolonged clinical responses and autoantibody normalization. This delay correlated with the degree of expansion of B cells of a transitional phenotype during the B cell reconstitution phase (P = 0.005) and the absence of baseline autoantibodies directed against extractable nuclear antigens (RNP, Sm, Ro antigen, La antigen). Despite the paucity of peripheral blood memory cells and the prolonged expansion of functionally immature transitional B cells, tonsil biopsy tissues revealed active germinal center (GC) reactions, but with decreased Fc receptor homolog 4-positive memory B cells. These results suggest heterogeneity in the B cell depletion and reconstitution process that impacts clinical and immunologic outcomes in SLE. The presence of GC reactions, but with altered memory B cell subpopulations in tonsils, suggests that peripheral blood memory cell reconstitution lags behind a slow secondary lymphoid tissue recovery, with important implications for immunologic competence and tolerance.

Localized lymphoid hyperplasia of the rectum resembling polypoid mucosa-associated lymphoid tissue lymphoma: A report of three cases

Histologically, benign lymphoid hyperplasia of the rectum is usually characterized by large lymphoid follicles with active germinal centers and by a narrow surrounding mantle zone and marginal zone (MZ). We report here three cases of benign lymphoid hyperplasia of the rectum associated with prominent marginal zone hyperplasia, which caused serious difficulty in the differential diagnosis from the polypoid type of mucosa-associated lymphoid tissue (MALT) lymphoma. Colonoscopy demonstrated small sessile polyps in all three cases. Histologically, the lesions were characterized by a hyperplastic germinal center and expanded MZs. The expanded MZs contained numerous monocytoid B-cells (MBC) and scattered large transformed B-cells. Initially, combined colonoscopic and histological findings strongly supported a diagnosis of polypoid MALT-type lymphoma of the rectum. However, there were neither colonized lymphoid follicles nor lymphoepithelial lesions in any of the three lesions. MBCs and large transformed B-lymphocytes were CD43- and bcl-2-. Moreover, immunohistochemical and genotypic studies proved the polytypic nature of the B-lymphocytes in all three lesions. The present cases indicated that benign lymphoid hyperplasia of the rectum should be included in the differential diagnosis for polypoid MALT-type lymphoma of the rectum.

Severe gastritis in the Peruvian Andes

To compare the Helicobacter pylori-associated pathology in gastric biopsies taken from patients living at sea level with those taken from patients living at high altitude. We included 38 patients from a hospital in the Andean city of La Oroya, Peru, located at 3700 m in altitude, and 40 control patients taken from Comas Clinic located in the city of Lima at sea level. Fibrepanendoscopy and multiple biopsies were performed in all the patients followed by histopathological examination. In the antrum, patients from the Andean town had a higher prevalence of glandular lymphoid adherence lesions, active germinal centres, moderate to severe chronic atrophic gastritis, intestinal metaplasia and moderate to severe total deep gland loss, than did patients from the coastal town. Furthermore, the severity of the histological lesions seen in the gastric body and cardia was significantly greater in the high-altitude patients than in those from sea level. This study suggests that the severity of H. pylori-associated gastric lesions seen on histopathological examination is greater in patients living at high altitude, the cause of which is most probably multifactorial but nonetheless principally altitude related.

Follicular lymphoid hyperplasia of the oral cavity representing progressive transformation of germinal center

Follicular lymphoid hyperplasia (FLH) of the oral cavity is a rare and poorly understood lymphoproliferative disorder. We present a case of FLH of the oral cavity presenting with progressive transformation of germinal center (PTGC). The patient was a 49-year-old Japanese woman presenting with a hard mass in the right cheek. The resected specimen contained numerous lymphoid follicles with active germinal centers and a portion of the lymphoid follicles exhibited PTGC. The PTGCs contained a few large lymphoid cells resembling lymphocytic and histiocytic Reed-Sternberg cells of nodular lymphocyte-predominant Hodgkin lymphoma. The PTGC was surrounded by groups of epithelioid cells. In situ hybridization studies demonstrated strong expression of Epstein-Barr virus (EBV)-encoded small RNA in scattered large lymphoid cells in the PTGC. Although the etiology of FLH of the oral cavity remains unclear, the present case suggests that a subset of FLH of the oral cavity appears to be an EBV-associated lymphoproliferative disorder.

HCV RECURRENCE AFTER LIVER TRANSPLANTATION IS ASSOCIATED WITH THE DEVELOPMENT OF INTRAHEPATIC LYMPHOID FOLLICLES DESPITE IMMUNESUPPRESSION

P796 Aims: Chronic inflammation in the liver can lead to the development of lymphoid tissue in the portal tracts. Some of these lymphoid infiltrates are highly organized, containing all characteristic components of secondary lymphoid organs, with B-lymphocytes localized in follicles and with T-cells more diffusely distributed in surrounding areas. One could speculate that these ectopic lymphoid structures fulfill similar functions as secondary lymphoid organs in the initiation and perpetuation of local immune responses. In the current study we investigated the formation of portal tract-associated lymphoid tissue (PALT) after liver transplantation, particularly in patients with hepatitis C virus (HCV) recurrence. In this setting, we can investigate a possible effect of immunosuppressive regimens on the neogenesis of lymphoid tissue induced by HCV infection of the liver graft. Results: First, the explant livers of chronic HCV patients after orthotopic liver transplantation were analysed by immunohistochemistry for the presence of PALT structures. In 9 out of 10 patients, B-cell follicles and T-cell areas were observed. Approximately one-third of all portal tracts which were examined contained B-cell follicles. Of these follicles, 4% contained a network of follicular dendritic cells (FDC) indicating of presence of an active germinal center. Most of the paracortical T cell areas contained blood vessels resembling high endothelial venules that are found in lymph nodes. These endothelial cells stained positive for the Peripheral Node Addressin (PNAD), a ligand for the adhesion receptor L-selectin. All of the microvascular endothelium in the PALT, but not the sinusoidal endothelium, stained positive for CD34 indicating neovascularization in the PALT. Next, post-transplantation biopsies of the HCV patients were analyzed. All these patients suffered from re-infection of the graft by HCV. More then a third of these patients developed PALT which contained B-cell follicles. Follicles were not found earlier as 18 months after transplantation. In this small number of post-transplantation follicles, however, no FDC-network was observed. This suggest that B-cell organization in response to HCV recurrence is relatively slow and renders inactive follicles. None of the control (HCV-negative) transplantation patients developed PALT, even at later time points after transplantation. Conclusions: HCV infection can result in the formation of ectopic lymphoid compartments in the liver graft. Immunosuppressive drugs do not prevent the formation of these PALT, although the number of B-cell follicles found in post-transplantation biopsies was significantly lower compared to liver biopsies of chronic HCV patients with end-stage liver disease. However, the role of PALT in chronic inflammatory liver diseases, like viral hepatitis, and the significance for HCV-recurrence remains unknown.

How do we avoid developing allergy: Modifications of the T H2 response from a B-cell perspective

The synthesis of IgE by B cells occurs at a low rate compared with that of other antibodies, even in allergic subjects. One rate-limiting step is the class switch, by which B lymphocytes switch to produce immunoglobulin ε heavy chains rather than μ or γ heavy chains. We propose an additional rate-limiting step: survival of the B lymphocyte after the switch to IgE. The hypothesis we present here is that the survival of the IgE-switched B cell is compromised, particularly in an active germinal center. Antigenic stimulation in the absence of a danger signal fails to induce a mature germinal center, which allows IgE-switched B cells to escape and mature into plasma cells. Antigenic stimulation in the presence of a danger signal (or under nonhygienic conditions) induces germinal centers, which eliminate IgE-switched B cells. Thus the essence of an allergen is antigenic stimulation in the absence of conditions that generate mature germinal centers. Because germinal centers are important for the generation of B-cell memory, the IgE immune response is characteristically poor in memory (but might be long lasting because of the generation of long-lived plasma cells). In addition to this direct route to IgE, typical for atopic sensitization, another type of T H2 response exists. On chronic allergen exposure with the concomitant induction of germinal centers, IgG4-switched B memory cells are induced that are slow to differentiate into plasma cells. These IgG4-switched B memory cells might occasionally undergo a secondary switch to IgE.

Immunohistochemical expression patterns of germinal center and activation B-cell markers correlate with prognosis in diffuse large B-cell lymphoma.

Recent studies with cDNA microarrays showed that diffuse large B-cell lymphoma (DLBCL) cases with gene expression profiles similar to germinal center (GC) B cells had much better prognosis than DLBCL cases with gene expression profiles resembling activated B cells. The goal of the current study is to evaluate if using a panel of GC B-cell (CD10 and Bcl-6) and activation (MUM1/IRF4 and CD138) markers by immunohistochemistry defines prognosis in patients with de novo DLBCL. Immunohistochemical stains for the above markers were performed on paraffin-embedded tissues from 42 de novo DLBCL patients. Median follow-up in all patients was 41 months (range, 1-103 months) and in surviving patients was 65 months (range, 14-103 months). These cases could be classified into three expression patterns: GC B-cell pattern (pattern A) expressing CD10 and/or Bcl-6 but not activation markers; activated GC B-cell pattern (pattern B) expressing at least one of GC B-cell markers and one of activation markers; and activated non-GC B-cell pattern (pattern C) expressing MUM1/IRF4 and/or CD138 but not GC B-cell markers. Patients with pattern A had much better overall survival than those with the other two patterns (Kaplan-Meier survival analysis, P < 0.008, log rank test). Using multivariate Cox proportional hazards regression analysis, the international prognostic index scores and the expression pattern of these markers were independent prognostic indicators. Our results suggest that expression patterns of this panel of GC B-cell and activation markers by immunohistochemistry correlate with the prognosis of patients with DLBCL. Immunohistochemical analysis on paraffin-embedded tissues is more readily available than gene expression profiling by cDNA microarray and may provide similar prognostic information.

Up-regulation of Muc4 and its complex formation with ErbB2 in murine gallbladder carcinomaȁa potential mechanism for gallbladder carcinogenesis and/or tumor growth

mphoid cells increased in the medulla during 24 to 48 h. The number of cax~cer cells in the marginal sinus reached the maximmu at 6 h, then declined until 48 h, but significant proliferation was observed front 4 days atier inoculation. The marginal sinus was then filled with cancer cells and expanded. At this period, the ED3 macrophages accumulated and intermingled with cancer ceils, Some cancer ceils resulted in apoptosis, being incorporated by the ED3 macrophages. In LN depleted of macrophages with liposomes containing DMDP, cancer cells massivdy invade the LN parenchyma TNF-e~ and IL-I[3 in the LN significanfly increased at ] and 3 h, respectively [1-2 increased significantly during 6 to 24 h. These cytokines returned to the basal level thereaher. 1FN-y or IL-12 did not increase at earlier periods, but significantly decreased from 24 to 96 h Localization of IL113 positive cells was consistent with that of ED2 macrophage in the medulla, but not of ED3 macrophage. TNF-cx positive cells were found in the germinal centen Conclusions: Immune responses in thks study include germinal center activation, followed by lymphoid cell pmlileration in the medulla and sinus histiocytosis, which mimic histological maniIestations of regional LN in cancer patients with improved survival Thus, LN did have capability to eliminate carmer cells temporarily. Depiction of immunocompetent cells due to deterioranon of cytokine induction may be responsible for later loss of the capabihty to prevent metastasis Macmphages contribute as a first line defense against cancer invasion. There is a different role in preventing metastasis between marginal atrd medullary sinus macrophages.