CD83+CCR7− Dendritic Cells Accumulate in the Subepithelial Dome and Internalize Translocated Escherichia coli HB101 in the Peyer’s Patches of Ileal Crohn’s Disease

Recurrent Crohn’s disease originates with small erosions in the follicle-associated epithelium overlying the Peyer’s patches. Animal studies have illustrated mucosal immune regulation by dendritic cells located in the subepithelial dome. The aim of this study was to characterize the dendritic cells at this specific site in patients with Crohn’s disease. Ileal tissues were obtained after surgery performed on Crohn’s patients; ileal samples from noninflammatory bowel disease and ulcerative colitis served as standard and inflammatory controls, respectively. Flow cytometry of isolated intestinal mononuclear cells showed a larger subset of dendritic cells in Crohn’s samples compared with controls. This finding was corroborated by confocal microscopy, showing enhanced infiltrates of cells positive for the dendritic cell markers, DC-SIGN⁺ and CD83⁺, in the subepithelial dome. Moreover, the CD83⁺ cells in Crohn’s tissues showed reduced expression of the lymph node migratory receptor, CCR7, possibly contributing to the high numbers of dendritic cells. After exposure to nonpathogenic Escherichia coli in Ussing chambers, dendritic cells in the subepithelial dome of Crohn’s disease demonstrated increased co-localization with translocated bacteria. Immunohistochemical results revealed that DC-SIGN⁺ cells in Crohn’s tissues were found to express toll-like receptor 4 and produce tumor necrosis factor-α. In conclusion, nonmigrating dendritic cells that accumulate in the subepithelial dome and internalize nonpathogenic bacteria may be important for the onset and perpetuation of mucosal inflammation in Crohn’s disease.     

Age-related changes in responsiveness of various rat tissue lymphocytes to mitogens

The effect of age on the mitogen responses of rat lymphoid tissues was investigated. Evaluation was based on using the in vitro proliferative response of lymphocytes from various tissues of Fischer-344 rats (2, 7, 13, 19 and 25 months) using concanavalin A (Con A), phytohemagglutinin-P (PHA-P), pokeweed mitogen (PWM) and Mycoplasma neurolyticum. The stimulation index (S. I.) for cervical, mesenteric, thymic and splenic lymphocytes treated with Con A and PHA-P was greatest for 2-month-old rats and lowest for 19-month-old rats; however, no age-related change was observed with either PWM or M. neurolyticum. The levels of mitogenic responses for lymph nodes in the two different anatomical sites paralleled one another, with the cervical lymphocytes showing a greater response. The splenic lymphocytes responded less than either lymph node lymphocyte population. When PHA-P treatment of splenic lymphocytes followed the removal of the plastic adherent population, the S. I. of the resulting non-adherent population was comparable to the S.I. of other tissue lymphocytes; however, an age-related decrease was still observed. The PHA-P proliferative response of either the 7- or 19-month non-adherent population was suppressed by the 7-month adherent population and not by the 19-month adherent population, i.e., adherent population interaction with non-adherent population decreases with age.     

Characterization of altered calcium signalling in T lymphocytes from patients with rheumatoid arthritis (RA)

Abnormal function of peripheral blood T lymphocytes is characteristic of RA; diminished proliferation and secretion of cytokines following in vitro mitogen stimulation are observed. We have investigated the calcium flux initiating T cell activation in rheumatoid peripheral blood mononuclear cells (PBMC) to determine whether abnormalities in signalling are also present. We have found that both phytohaemagglutinin (PHA-P)- and anti-CD3-stimulated calcium fluxes were much reduced in the patients’ PBMC compared with controls, with a mean six-fold difference (P < 0·01) in rate of Ca²⁺ flux with PHA-P stimulation. When purified T cells were examined with PHA and CD3 stimulation, a reduction in the peak and plateau [Ca²⁺]_i was observed in RA T cells, but the rate of rise of [Ca²⁺]_i was only reduced in those cells stimulated with PHA. These results suggest that alterations in the initiating signal may underlie the functional T cell abnormalities associated with RA, and that there may be an additional extrinsic influence from non-T cells in the PBMC population.     

The role of interleukin-13 in chronic inflammatory intestinal disorders

Interleukin (IL)-13 is a cytokine playing a pivotal role in T helper (Th)2 immune response supposed to be implicated in some intestinal disorders. IL-13 is produced by Th2 cells, natural killer T cell, innate lymphoid cells and innate immune cells, which contribute to trigger and maintain a chronic idiopathic intestinal inflammation. In murine models IL-13 exerts pleiotropic functions, playing either pathogenic or protective roles according to the different experimental conditions. As regards celiac disease, IL-13 is considered to be involved mostly in the refractory phase rather than at uncomplicated stage. Discrepancies have been observed in the role of IL-13 upon the inflammation and fibrosis in ulcerative colitis (UC) and in Crohn's disease, respectively. Failure of the anti-IL-13 monoclonal antibodies tralokinumab and anrukinzumab in UC patients in clinical trials support the absence of a role for IL-13 in UC.This review deals with IL-13 in several experimental colitis models -such as oxazolone-, trinitrobenzene sulfonic acid- or dextran sodium sulphate-induced colitis- and chronic intestinal inflammatory disorders -including celiac disease, UC and Crohn's disease-, and it also highlights the attempts to modulate IL-13 as therapeutic tool.     

Human lymphocyte subpopulations. Human thymus-lymphoid tissue (HTL) antigen-positive lymphocytes forming rosettes with sheep erythrocytes and HTL antigen-negative lymphocytes interacting with antigen-antibody-complement complexes

Human lymphocytes from various lymphoid tissues were studied for the relationship between the existence of HTL (human thymus-lymphoid tissue) antigen, and binding of sheep erythrocytes (E) or sheep erythrocyte–antibody-complement complexes (EA(IgM)C43). E adhered to the majority of thymus lymphocytes and formed rosettes. These lymphocytes were shown to be HTL antigen positive by immunofluorescence performed simultaneously. In the peripheral lymphoid tissues, 10–30% of lymphocytes formed E rosettes and almost all E rosette-forming lymphocytes were HTL antigen positive. Conversely HTL antigen-negative cells did not form E rosettes.

In contrast, the cells binding EA(IgM)C43 were always HTL antigen negative.

There were very few HTL antigen-positive or rosette-forming lymphocytes either with E or EA(IgM)C43 in bone marrow.

From these data we conclude that E-rosette-forming and HTL antigen-positive lymphocytes are of thymus origin and EA(IgM)C43-rosette-forming cells are not thymus-dependent cells.

     

T lymphocyte subsets in human intestinal mucosa: the distribution and relationship to MHC-derived antigens

T lymphocytes in the normal human intestinal tract have been analysed in tissue sections by a double-marker immunofluorescence technique, combining antiserum to T lymphocyte antigen (HuTLA) with a monoclonal antibody detecting T cells of suppressor-cytotoxic phenotype (OKT8). The distribution of HLA-A -B, -C and Ia-like antigens in intestinal mucosa was also examined by a similar method. In small and large intestine 67 to 90% (mean 70%) of intraepithelial T lymphocytes were of suppressor-cytotoxic phenotype (OKT8+). In contrast, only 27 to 56% (mean 39%) of lamina propria T cells were OKT8+. Intestinal epithelial cells demonstrated strong membrane staining for HLA-A, -B, -C antigens. Ia-like antigens were detected on the epithelial cells of small intestinal villi, but not on colonic epithelial cells. Lamina propria macrophages expressed both HLA-A, -B, -C and Ia-like antigens, the latter having strong membrane and cytoplasmic fluorescence. The distribution of T cells with suppressor-cytotoxic or inducer phenotype in the intestinal epithelium and lamina propria may be related to the differential expression of Ia-like and HLA-A, -B, -C antigens in intestinal mucosa.     

Regulation of IgE receptor expression on human peripheral blood lymphocytes by lymphocytosis promoting factor (LPF), lectins and dexamethasone.

Using a monoclonal anti-human Fc epsilon R antibody (H107), we found that lymphocytosis promoting factor (LPF), phytohaemagglutinin (PHA-P) and Concanavalin A (Con A) could induce Fc epsilon R, detected by immunofluorescence study, on normal human peripheral blood lymphocytes without IgE. The number of Fc epsilon R bearing lymphocytes was increased by stimulation with 3, 10 and 10 micrograms/ml of LPF, PHA-P and Con A, respectively, from 6.0 +/- 3.0/1000 cells to 26.0 +/- 7.9, 54.0 +/- 6.7 and 24.8 +/- 7.1/1000 cells, respectively. Although the induction of Fc epsilon R occurred neither in the separated T-enriched fraction (TEF) nor the T-depleted fraction (TDF), it recovered when the two fractions were mixed. The cell free supernatants from TEF stimulated with LPF or PHA-P could increase Fc epsilon R(+) cells in TDF, whereas those from TDF failed to increase them in TEF. The results suggest that the induction of Fc epsilon R occurs mainly on B lymphocytes by the soluble factor(s) formed by T cells stimulated with LPF or PHA-P. The induction of Fc epsilon R by stimulants was completely inhibited by 10(-6) M dexamethasone. It was demonstrated that the effects of dexamethasone on lymphocytes were dual: one was on B cells to inhibit responsive increases of Fc epsilon R, and the other was on T cells to suppress the formation of the soluble factor(s) which induced Fc epsilon R on B cells.     

Acute cytomegalovirus infection and the host immune response. I. Development and maintenance of cytomegalovirus (CMV) induced in vitro lymphocyte reactivity and its relationship to the production of CMV antibodies

Cytomegalovirus-induced lymphocyte reactivity (CMV-LR) of peripheral blood lymphocytes and antibodies against CMV-early (EA) and late (LA) antigens were studied in eighteen patients with documented acute CMV infections and seventy-six healthy persons. The development of a positive CMV-LR test lagged far behind the appearance of virus-specific antibodies. Positive CMV-LR tests were shown in all fourteen patients who could be tested twice or more. The median value of twenty-two tests in the acute phase (<50 days) was 269 d/min and the median of thirty tests in the post-illness phase (>50 days) was 1301 d/min (P<0·02). Once positive CMV-LR remained so during the follow-up period, up to 479 days after the onset of illness symptoms. In the meantime the LA and EA antibody response remained positive. Only two patients studied once showed negative CMV-LR responses while their serum contained high CMV-EA antibody titres. In CMV-LA seropositive healthy individuals who also possessed CMV-EA antibodies (LA+EA+) CMV-LR was higher (P<0·01) than in the LA seropositives who lacked EA antibodies (LA+EA−). The young LA+EA+ individuals, however, showed better (P<0·02) CMV−LR test results than the aged ones while their CMV antibody levels—especially the EA antibodies (P<0·02)—were lower. This phenomenon of increased CMV antibody production in relation to depressed CMV-LR is possibly caused by age-associated impairment of T lymphocyte function. The combined CMV-LR and EA antibody tests may provide useful means to study the specific immunological host/virus relationship in different clinical situations.     

Lymphocyte culture in solid medium: I. Phytohaemagglutinin stimulation of human blood lymphocytes in chicken plasma clot cultures

Normal human blood lymphocytes were cultured in chicken plasma clots with a series of PHA-P dilutions. In this situation direct cell-to-cell contact was prevented, and thus a morphological estimate of the actual number of PHA responsive cells was possible in the absence of any cell-to-cell co-operation which might be mediated via cell contact. The distorting effects of transformed cell proliferation on quantification, and of death of the original small lymphocyte population, were similarly obviated. Between 9 and 20 per cent of those lymphocytes separated by methylcellulose and iron were PHA responsive at the optimal dose of PHA-P.     

Bacteroides ovatus as the Predominant Commensal Intestinal Microbe Causing a Systemic Antibody Response in Inflammatory Bowel Disease

To clarify what bacterial species of commensal intestinal microbes are recognized as the antigens that induce a serum antibody response in patients with inflammatory bowel disease (IBD), 72 subjects consisting of 12 Crohn’s disease patients, 30 ulcerative colitis patients, and 30 healthy volunteers were examined for their titers of serum antibody to these intestinal bacteria. In IBD patients, as a result, significant elevations of both the immunoglobulin G (IgG) and IgA titers to Bacteroides ovatus were found. Immunoblotting showed that a definite 19.5-kDa band of B. ovatus was bound to the serum antibody raised in IBD patients. It was thus concluded that B. ovatus causes serum antibody responses in IBD patients, and a 19.5-kDa molecule of this bacterium appears to be the responsible antigen, although the role of this event in pathogenesis remains unclear.     

Lectin-binding patterns of small lymphocytes in bone marrow,thymus and spleen: demonstration of lymphocyte subsets by quantitative radioautography

Cells from mouse bone marrow, thymus and spleen were exposed to ¹²⁵I-labeled concanavalin A (Con A), Lens culinaris lectin (LCL), soybean agglutinin (SBA), Helix pomatia agglutinin (HPA), phytohemagglutinin-P (PHA-P), peanut agglutinin (PNA), or wheat germ agglutinin (WGA) in a range of concentrations and examined radioautographically. Small lymphocytes in the three organs differed in the minimal concentration of each lectin which gave detectable surface labeling, while at optimal lectin concentrations, their labeling intensity profiles differed markedly. Inhibition by sugars demonstrated the labeling specificity. Major populations of bone marrow small lymphocytes bound WGA strongly, while Con A, SBA, HPA, PHA-P and LCL were bound only weakly, and PNA binding was lacking. Most thymus cells bound Con A, SBA, HPA, PHA-P and PNA strongly, WGA and LCL weakly. Subsets of bone marrow and thymus small lymphocytes differed from the major populations in their lectin-binding intensities. Spleen small lymphocytes were heterogeneous in the binding of each lectin. However, a major population bound LCL exceptionally strongly, while few cells bound PNA. Using a panel of lectins under standardized conditions, these studies show distinctive lectin-binding patterns for small lymphocytes in the bone marrow, thymus and spleen, respectively. Major and minor cell populations are distinguishable in each organ, providing an approach to discriminating lymphocyte lineages, subtypes and differentiation stages.     

CC chemokine receptor 6 expression by B lymphocytes is essential for the development of isolated lymphoid follicles

Isolated lymphoid follicles (ILFs) are organized lymphoid structures that facilitate the efficient interaction of antigen, antigen-presenting cells, and lymphocytes to generate controlled adaptive immune responses within the intestine. Because CC chemokine receptor 6 (CCR6) deficiency affects the generation of mucosal immune responses, we evaluated a potential role for CCR6 in the development of ILFs. We observed that CCR6 and its ligand CCL20 are highly expressed within ILFs and that B lymphocytes are the largest CCR6-expressing population within ILFs. ILF development was profoundly arrested in the absence of CCR6. Concordant with a block in ILF development at a stage corresponding to the influx of B lymphocytes, we observed that CCR6-deficient mice had a diminished population of intestinal B lymphocytes. Bone marrow reconstitution studies demonstrated that ILF development is dependent on CCR6-sufficient B lymphocytes, and adoptive transfers demonstrated that CCR6^−/− B lymphocytes were inefficient at localizing to intestinal lymphoid structures. Paralleling these findings, we observed that CCR6-deficient mice had a reduced proportion of Peyer’s patch B lymphocytes and an associated reduction in the number and size of Peyer’s patch follicular domes. These observations define an essential role for CCR6 expression by B lymphocytes in localizing to intestinal lymphoid structures and in ILF development.     

Lymphocyte subpopulations in peripheral blood of healthy persons. Characterization by surface markers and lack of selection during purification

Lymphocytes were isolated at 99% purity from peripheral blood of healthy persons by defibrination, gelatine sedimentation, treatment with carbonyl iron powder and centrifugation on Ficoll–Isopaque. Subpopulations were identified by three surface markers: cells forming rosettes with sheep red blood cells (SRBC) (E-binding lymphocytes) as a measure of T lymphocytes; lymphocytes with surface immunoglobulin identified by indirect immunofluorescence (B lymphocytes); lymphocytes with receptors for C3 observed by the rosette method using SRBC treated with rabbit antiserum and human complement (EAC-binding lymphocytes).

The yield of lymphocytes after purification varied from 15 to 65%. No selection of lymphocytes was observed either by counting immunoglobulin-bearing and EAC-binding lymphocytes in whole blood and in purified cells from the same sample, or by statistical analysis of lymphocytes in subpopulations as a function of the yields from twenty-six experiments. In the absence of selection during purification the total numbers of T and B lymphocytes could be calculated from the percentages and the total numbers of lymphocytes. Our normal values are close to those reported using other non-selective methods of purification.

When lymphocytes were simultaneously stained for immunoglobulin and rosetted with EAC, cells bearing either or both markers were found. In total, 27–35% cells were identified by these markers. Since about 70% of the cells were E-binding, practically all lymphocytes could be identified. A small overlap between E-binding and immunoglobulin-bearing/EAC-binding lymphocytes may occur.

Either the IgM or the IgG-containing fractions obtained after fractionation of rabbit anti-SRBC serum on Sephadex G-200 could be used for sensitization of SRBC with complement. Formation of rosettes was not prevented by pretreating the lymphocytes with aggregated IgG, while rosettes formed with EA prepared by high concentrations of IgG antibody (Fc-binding lymphocytes) were abolished. It is concluded that rosettes formed with IgG-EAC (or whole serum EAC) using diluted antiserum identify complement-reactive lymphocytes and are not caused by synergism with Fc receptors. When SRBC were sensitized with varying dilutions of whole antiserum or its IgG fraction identical plateaus for the percentages of EAC-binding lymphocytes were found. Subagglutinating concentrations of the IgM fraction was insufficient to reach the plateau and also consistently resulted in lower values for EAC-binding lymphocytes.

     

Distinctive Pattern of Cytokine Production and Adhesion Molecule Expression in Peripheral Blood Memory CD4+ T Cells from Patients with Active Crohn’s Disease

An expansion of both circulating and intestinal lamina propria CD4⁺CD45RO⁺ T cells has been described in patients with Crohn’s disease. We studied both the cytokine profile and the expression of adhesion molecules on this T-cell subset. Peripheral blood CD4⁺CD45RO⁺ T cells from patients with Crohn’s disease (n=45) were assessed by flow cytometry and RT-PCR methods. The cytokine profile was also measured in intestinal lamina propria from seven patients. They were classified according to the CDAI and the results were compared with those of patients with ulcerative colitis (n=21) and noninflammatory intestinal conditions (n=15), and healthy controls (n=39). The mean percentage of circulating CD4⁺CD45RO⁺ T cells producing intracellular TNF was higher in active than in inactive Crohn’s disease patients (p < 0.001), active (p = 0.49) and inactive ulcerative colitis (p = 0.019), and healthy controls (p =0. 017). TNF expression correlated with CDAI (p < 0.001). An increased expression of intracellular IL-2, IL-6, and IL-10 in active Crohn’s disease patients was also found. CD62L was downregulated in active Crohn’s disease patients while no differences were observed in CD49d and CD11a expression. Lamina propria CD4⁺CD45RO⁺ T cells from active Crohn’s disease lesions showed an increased intracellular staining of TNF, IFN-γ, and IL-10. Both peripheral and intestinal mucosa CD4⁺CD45RO⁺ T cells from active Crohn’s disease patients show an increased production of TNF. In addition, the circulating CD4⁺CD45RO⁺ T-cell subset expresses a pattern of adhesion molecules that promotes homing to extranodal lymphoid tissues. This T-cell subset may play a relevant role in the immunopathogenesis of Crohn’s disease.Dr. García de Tena and Dr. Manzano are joint first authors     

How Exact Are the Diagnosis and Classification of Malignant Lymphomas from Aspiration Biopsy Smears?: A Comparison Between Histologic and Cytologic Diagnoses of 20 Hodgkin's and 54 Non-Hodgkin's Lymphomas according to Rappaport (1966) and Kiel (1974) Nomenclatures

Introduction: Cytological findings from needle aspirations on 74 malignant lymphomas were compared with histological diagnoses. The two most important questions were: Cytomorphological criteria of the different types of lymphomas and the possibilities of classifying these criteria according to Rappaport's and Kiel nomenclature.Material and Methods: In 35 of the 74 patients aspiration biopsy was the first investigation of enlarged lymph nodes. In 39 patients aspiration biopsy was performed as the follow-up, and the primary diagnosis was known. Twenty cases were Hodgkin's disease. Fiftyfour cases of Non-Hodgkin's lymphoma were classified according to Rappaport and Kiel nomenclature. Biopsies and aspiration smears were examined separately by two investigators. The wet-fixed cytological smears were routinely stained according to Papanicolaou. Giemsa stain was done if necessary and was useful for the recognition of plasmocytoid features.Results: Hodgkin's disease was cytologically verified in 12 cases and suggested in five cases. In two cases no diagnosis was possible because of scanty material. One case of lymphadenitis was misinterpreted as Hodgkin's disease. There was good correlation of cytological and histological diagnoses on 50 Non-Hodgkin's lymphomas, according to the classifications mentioned above. There were only two cases in which cytological and histological diagnosis differed. Of the remaining four Non-Hodgkin's lymphomas, one case was cytologically false negative and the other was diagnosed falsely positive on cytology. In two other cases a tumor diagnosis was given, but the differential diagnosis between histiocytic lymphoma and large cell carcinoma was incorrect. In five cases out of 54 Non-Hodgkin's lymphoma, a cytologic or histologic diagnosis could not be established because of scanty or poorly preserved material.Discussion: Accurately processed smears are one of the most important means of establishing an exact cytological diagnosis of lymphoma. Hodgkin's cells and Reed-Sternberg giant cells are requiered for diagnosis of Hodgkin's disease. The mixed cellular pattern of Hodgkin's disease may differ only slightly from those cell pictures seen in reactive lymphadenopathy caused by viral infection, infectious mononucleosis, toxoplasmosis, hypersensitivity to Hydantoin medication, etc. In our laboratory, Non-Hodgkin's lymphomas are first classified according to the Kiel nomenclature. This nomenclature provides the clearest morphological criteria for the different lymphoid tumor cells. Absolute cell size, variation in size, and nuclear hyperchromasia are the most important features in recognition and differentiation of tumor cells in malignant lymphomas.     

Selective binding of peripheral blood lymphocytes to the walls of cerebral vessels in frozen sections of human brain

In order to identify the factor that control the binding of blood leucocytes to cerebral blood vessels we have modified and applied the frozen section assay of Stamper and Woodruff to the study of human brain. Cryostat sections of brain tissue obtained at post mortem were overlaid with blood lymphocytes and experimental conditions were defined which permitted optimum binding of the cells to transected blood vessel walls. The maximal binding of lymphocytes to cerebral vessels occurred when 6 × 10⁶ lymphocytes were overlaid onto brain sections for 30 min at 7°C with gentle agitation. Only a small proportion (0.01%) of the added lymphocytes bound to exposed cerebral vessels. However, lymphocytes were far more adherent than monocytes and polymorphonuclear cells (7-fold and 11-fold respectively: p < 0.001) and activation of lymphocytes with IL-2 enhanced their binding to blood vessel walls (mean 130% increase; p < 0.03). Further analysis revealed that CD4-positive T lymphocytes were the predominant cell population binding to the blood vessels. Antibody blocking studies showed that lymphocyte binding to cerebral blood vessels was inhibited by pretreating the lymphocytes with anti-CD11a, anti-CD18 or anti-CD49d (p ≤ 0.02) and immunohistochemical studies revealed the presence of the counter-receptors ICAM-1 (CD54) and VCAM-1 (CD106) for these adhesion molecules in addition to the presence of E-selectin (CD62E) and P-selectin (CD62P) on the cerebral blood vessels. The establishment of a technique in situ which measures selective binding of CD4-positive peripheral lymphocytes to sections of cerebral blood vessels will assist in the molecular characterization of factors that control the interaction of leucocytes with the blood-brain barrier in health and disease.     

The determination of numbers of T and B lymphocytes in the blood of children and adults by the direct immunofluorescence technique

Lymphocytes in the peripheral blood of sixty-five children ranging in age from newborns to 14-year-olds and twenty-one adults were studied by the direct immunofluorescence technique for B- and T-membrane determinants, with a GaHu–Fab fluorescein isothiocyanate (FITC) conjugate as a B-cell marker and a tetramethyl rhodamine isothocyanate (TRITC) labelled horse anti-human T-cell conjugate (ATC) as a T-cell marker. The ATC was prepared from a commercial horse anti-human thymocyte IgG fraction and made specific for human T lymphocytes by means of extensive absorption with all kinds of human blood cells. Lymphocytes were also tested for E-rosette formation with sheep red blood cells (SRBC). In adults, an average of 79% of peripheral blood lymphocytes reacted with the ATC, 14% with the anti-Fab conjugate, 2% with both conjugates and 5% with neither of the conjugates. An average of 76% of peripheral blood lymphocytes (PBL) formed E rosettes. Relative numbers of fluorescent B and T lymphocytes in blood from children showed no significant differences as compared to adults. The percentage of E rosettes in cord blood was lower than in any of the other age groups studied, but only in comparison with the 3–12-month-old age group was the difference significant (P<0·05). In the 3–6-month-old age group, the percentage of fluorescent T lymphocytes was highest and the percentage of fluorescent B lymphocytes lowest, but a significant difference was observed only for the B-lymphocyte percentage compared with the first month of age (P<0·02). Up to 2 years of age, absolute values for total blood lymphocytes, fluorescent T and B lymphocytes and E rosettes were significantly higher (P<0·001) than in the older age groups and adults. After the second year of life, those values were the same as in adults.     

The effect of long-term lymphatic drainage on the lympho-myeloid system in the guinea-pig

Methods are described for the long-term drainage (9 or 10 days) of the mesenteric lymph duct in the guinea-pig and for the analysis of bimodal nuclear distributions by electronic sizing of lymphocytes in lymph.

The present studies show that on the basis of nuclear volume, lymphocytes in mesenteric lymph consist of two populations (large and small) which are log-normally distributed. The relative proportions of the large and small lymphocytes and means and standard deviations of these component populations were estimated daily during the course of cannulation. The daily output of small lymphocytes was greatly reduced during the course of drainage to a base level of 110× 10⁶ on day 6 which was maintained thereafter. The daily output of large lymphocytes was relatively constant at about 50× 10⁶ throughout drainage. This finding is consistent with the view that many small lymphocytes are recirculating.

The output of cells in mesenteric duct lymph on the 1st day of cannulation (379× 10⁶) is similar to that recorded by other workers in thoracic duct lymph for the same period. Comparison of the present findings with the results of other studies shows that the level of `indirect entry' lymphocytes and lymphocyte blood concentration remain remarkably constant while blood volume may be doubled. It is suggested that `direct entry' of lymphocytes from lymphoid tissue to blood increases with age (body weight and, therefore, blood volume) of the animal.

During lymphocyte depletion the concentration of bone marrow lymphocytes was reduced from 466,500 to 173,500/mm³. There was no significant reduction in myeloid or erythroid cells. This finding suggests that either bone marrow lymphocytes are not involved in the production of erythroid and myeloid cells or that haemopoietic precursors are drawn from marrow lymphocytes produced in situ rather than from those which are haematogenous in origin.

Histological study of the lymphoid tissues of animals following long-term drainage showed a gross depletion of small lymphocytes in the cortex of nodes (particularly the mesenteric), white pulp of spleen and internodular areas of Peyers patches. The occurrence of large pyroninophilic cells with vesicular nuclei and prominent pyroninophilic nucleoli in depleted areas was noted. Mitotic figures were frequently observed in these cells. The thymus was not obviously affected by drainage.