The functional genetic variation in the PTPN22 gene has a negligible effect on the susceptibility to develop inflammatory bowel disease.

The aim of this study was to assess the possible association between the protein tyrosine phosphatase non-receptor 22 (PTPN22) gene 1858C-->T (rs2476601, encoding R620W) polymorphism and inflammatory bowel disease (IBD). Our study population consisted of 1113 IBD [544 ulcerative colitis (UC) and 569 Crohn's disease (CD)] patients and 812 healthy subjects. All the individuals were of Spanish white origin. Genotyping of the PTPN22 gene 1858C-->T polymorphism was performed by real time polymerase chain reaction technology, using TaqMan 5'-allelic discrimination assay. The frequency of the PTPN22 1858T allele in healthy subjects was 6.2% compared with 6.7% in the UC patients and 5.1% in Crohn's patients. No statistically significant differences were observed when the PTPN22 1858C-->T allele and genotype distribution among CD patients, UC patients and healthy controls were compared. These results indicate that the PTPN22 1858C-->T polymorphism does not appear to play a major role in IBD predisposition in our population.

CTLA4/CT60 polymorphism is not relevant in susceptibility to autoimmune inflammatory intestinal disorders.

The aim of this work was to investigate the possible influence of the recently described CT60 A/G dimorphism of the CTLA4 (cytotoxic T-lymphocyte antigen 4) gene in the susceptibility to two different autoimmune inflammatory intestinal disorders, inflammatory bowel disease (IBD) and celiac disease. We analyzed a case-control cohort composed of 528 Spanish patients with IBD (284 with Crohn disease and 244 with ulcerative colitis) and 454 unrelated healthy individuals, and additionally a group of 90 celiac disease families. CT60 genotyping was performed with a TaqMan 5' allelic discrimination assay. After comparing patients with IBD with the control population, we found no significant deviation in the distribution of the alleles or genotypes of CTLA4/CT60 dimorphism. In addition, by means of familial and case-control analysis, no evidence for a statistically significant association was observed between CTLA4/CT60 and celiac disease susceptibility. Therefore, our results suggest that the CTLA4/CT60 polymorphism does not play a major role in inflammatory intestinal disorders.

Isolation, maturational level, and functional capacity of human colon lamina propria plasma cells.

BACKGROUND AND AIMS: Large numbers of plasma cells (PC) localise in the intestinal lamina propria (LP) where they play a critical role in the defence against pathogens. This study analyses the level of maturation reached by normal human colon LPPC in comparison with that of bone marrow (BM) PC. METHODS: A technique was designed to purify LPPC by combining collagenase digestion of the mucosal layer and immunomagnetic selection of CD54(+) LP cells. It provided highly purified PC, as demonstrated by morphology, CD38(h) phenotype, and cytoplasmic IgA staining criteria. This procedure allowed comparison of in vitro functional capacities and a broad phenotypic analysis of BMPC and LPPC. RESULTS: LPPC and BMPC exhibited identical expression of differentiation markers (CD19(-/+), CD20(-), HLA-DR(low/-), VS38c(high)), survival molecules (CD95 (low/-), Bcl-2(+)), and B cell transcription factor profile, as well as similar in vitro Ig secreting kinetics (14 days) and lack of susceptibility to apoptosis by CD95 ligation. In contrast, they markedly differed in adhesion molecule expression, as LPPC showed higher levels of CD44 and CD21 and were alpha 4 beta 7(+) whereas BMPC lacked this integrin and expressed higher levels of CD49d and CD31. CONCLUSION: These data indicate that PC at effector sites of the humoral response (BM and LP) show similar high differentiation, survival, and functional features but display a distinctive pattern of adhesion molecules, probably related to their respective homing locations.

[Development, validity and reliability of a general addiction scale. A preliminary study]. / Desarrollo, validez y seguridad de una escala de adicción general. Un estudio preliminar.

INTRODUCTION: The authors develop a General Scale to measure the intensity of the addiction to substances (not alcohol, not opiates) and addictive behaviors. METHODS: The General Scale is a self-scale compound by eleven ítems that was delivered to fifty and five students of the courses 5 masculine and 6 masculine of the Medicine of the University of Alcalá (Madrid, Spain), and was them requested that applied said scale from different supposed addictive: tobacco; tea, coffee or cola drinks; chocolate; others sweet; alcohol; sex; use of the personal computer and/or Internet and/or videoplay; and to practice sports. Of that manner, each subject provided a total of 440 complimented scales.Finally, it was requested the subjects that indicated in a scale apart, their degree of addiction from the different exposed concepts. Those data served of external criterion. RESULTS AND DISCUSSION: The Scale is monodimensional, and shows a high construct validity (account 63% of the total variance obtained by a Factorial Analysis), a high alpha reliability (alpha: 0.94) and a good internal consistency (split-half method with the Spearman-Brown correction; R: 0.92). All items share with the general addiction concept that represents the total score of the Scale, an common variance proportion equal or over the 52%. CONCLUSION: The Scale seems be a valid and reliable instrument to compare groups of the calls new addictions of a measurable manner.

Thyroid-infiltrating B lymphocytes in Graves' disease are related to marginal zone and memory B cell compartments.

B lymphocytes that infiltrate the thyroid (Thy-B cells) in Graves' patients appear to be implicated in the pathophysiology of this disorder. The goal of the present study was to examine the nature of these Thy-B cells. To this end, Thy-B lymphocytes were isolated from surgical thyroidal samples, and their phenotype was determined by using mouse monoclonal antibodies (mAb) directed against a wide variety of surface markers, followed by flow cytometry multicolor analysis. The results show that most Thy-B cells (approximately 60%) exhibited IgM(+) IgD(low to -) surface immunoglobulin (Ig) profile, whereas the minor cell fraction (approximately 30%) consisted of switched IgG(+) memory B lymphocytes. Thy-B cells expressed low levels of CD5, CD23, and CD62L, which distinguished them from the resting B-cell pool, the major B-cell subset in the blood. In addition, they lacked CD38, CD10, and CD71, characteristic molecules for the germinal center B lymphocytes. In addition, Thy-B lymphocytes showed peculiar patterns both of adhesion molecules (CD62L(-), CD44(intermediate)), and of activation molecules (CD69(+), CD80(+), and, in part, CD95(+)). Taken together, these results suggest that the Thy-B lymphocyte subset consists of a combination of IgM(+) B cells resembling marginal zone B lymphocytes, and isotype-switched memory B cells.

Chronic lymphocytic leukemia B cells inhibit spontaneous Ig production by autologous bone marrow cells: role of CD95-CD95L interaction.

A variable degree of humoral immunodeficiency is a common feature in patients with B-cell chronic lymphocytic leukemia (B-CLL). The aim of this study was to explore the possibility that B-CLL cells play a direct role in this phenomenon. To this end, patients' bone marrow (BM) immunoglobulin (Ig)-secreting cells were cocultured with autologous purified B-CLL cells. The results show that tumoral cells inhibited the spontaneous IgG secretion by BM plasma cells, and this effect increased after PMA-induction of B-CLL cells. This inhibitory process was proportional to the number of B-CLL cells added and depended on cellular contact. Adhesion molecules did not appear to be involved in the cellular interaction, because the inclusion of blocking antibody to a variety of these proteins did not reverse the inhibitory phenomenon. However, the addition of monoclonal antibody that blocked the function of either CD95 or CD95L clearly reversed B-CLL cell inhibition on autologous BM plasma cells. These latter cells were shown to express CD95, and B-CLL cells contained detectable quantities of CD95L at the level of messenger RNA and protein. Annexin V-binding experiments revealed increased apoptosis of BM Ig-secreting cells when cocultured with autologous B-CLL cells. Finally, this inhibitory phenomenon might be operative in vivo because (a) there was a good correlation between the intensity of the inhibitory effect in vitro and the serum IgG level exhibited by every patient and (b) B-CLL cells also inhibited in vivo antigen-induced IgG-tetanus toxoid-secreting cells obtained from normal immunized subjects. Collectively, these data suggest that B-CLL cells inhibit autologous CD95-bearing Ig-secreting cells by the interaction with CD95L present on B-CLL cells and, hence, contribute to the state of humoral immunodeficiency that occurs in these patients.

Immunohistochemical detection of ribosomal transcription factor UBF and AgNOR staining identify apoptotic events in neoplastic cells of Hodgkin's disease and in other lymphoid cells.

Ribosomal RNA synthesis is a key molecular process for understanding the mechanisms that drive cell proliferation. In this process, the upstream binding factor (UBF) is involved in regulating rDNA transcription at the nucleolus, together with RNA polymerase I. Recently, UBF was demonstrated to be a substrate for selective cleavage by specific proteases during apoptosis. Here we studied the expression of UBF in several cases of Hodgkin's disease (HD) by immunostaining and found it to be absent or clearly diminished in a high proportion of Reed-Sternberg cells and Hodgkin cells compared to small reactive lymphocytes. This result contrasted with labeling of those cells by the AgNOR technique, a marker of cell proliferation dependent on increased amounts of several proteins related to ribosome assembly. Disappearance of UBF and preservation of other NOR proteins is consistent with the pattern of selective proteolysis by caspases described in early stages of apoptosis. This correlates well with our results observed on induction of apoptosis in Jurkat cells treated with anti-FAS/APO-1 serum and with those in aged germinal center B-cells, in which UBF was no longer seen although the staining signal of other NOR proteins was maintained. These results support the concept that the rate of apoptosis is higher in neoplastic cells of HD than in the benign reactive lymphocyte population. Differential proteolysis of NOR proteins, as revealed by double staining of UBF and AgNOR, may prove valuable for identification of early stages of apoptosis in cytological and histopathological samples.

Regulation of immunoglobulin secretion by plasma cells infiltrating nasal polyps.

OBJECTIVE/HYPOTHESIS: To learn more about the role of plasma cells infiltrating nasal polyps in the pathogenesis of nasal polyposis, we examined their function by analyzing immunoglobulin (Ig) production and the factors implicated in the secretion. STUDY DESIGN: A series of 19 consecutive nasal polyp tissue samples and, as a control, peripheral blood samples from the same patients, were studied by histopathological and immunological examination. METHODS: Hematoxylin-eosin and immunohistochemical staining was carried out to identify plasma cells infiltrating nasal polyps. Nasal polyp mononuclear cells (NPMNCs) were purified from nasal polyp tissue samples, and Ig-secreting cells were identified in cytospin preparations stained with fluorescein isothiocyanate-conjugated antibodies against IgA, IgG, IgM, and IgE. Purified NPMNCs were cultured in basal conditions and after the addition of several stimuli. Ig secreted into the culture supernatants was evaluated by an enzyme-linked immunosorbent assay. RESULTS: Plasma cells accounted for an important fraction of the inflammatory infiltrate. The main Ig isotype synthesized by these cells was IgA, whereas little IgE was detected. In vitro cultures demonstrated that the plasma cells actively secreted Ig for a short period. When cytokine dependence was analyzed, interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-alpha) were shown to be partially responsible for the Ig production. Dependence on CD95-mediated apoptosis was not observed. CONCLUSIONS: Nasal polyp-infiltrating plasma cells are mainly IgA-secreting cells, the latter property being related to the mucosal immune system. The IgA production is partly dependent on IL-10 and TNF-alpha. The absence of IgE-secreting cells in most of the samples suggests that a type I hypersensitivity reaction is not essential for the development of nasal polyp.

[Reconstitution of peripheral blood lymphocytes in patients treated with bone marrow transplantation: comparison between allogeneic and autologous transplantation]. / Reconstitución de los linfocitos de sangre periférica en pacientes tratados con trasplante de médula ósea: comparación entre el trasplante alogénico y el autólogo.

BACKGROUND: This study compares the immune reconstitution of total T cells, CD4 and CD8 cell subsets, activated T cells, NK cells and B cells in 66 patients who underwent allogeneic or autologous bone marrow transplantation (BMT). PATIENTS, MATERIAL AND METHODS: The reconstitution of peripheral lymphocytes subsets was studied using two-color flow cytometry. The study group consisted of 39 patients who received allogeneic BMT compared with 27 patients who received autologous BMT. Peripheral blood was examined at different time intervals. As a measure of immune function, the response to the mitogen phytohemaglutinin (PHA) was determined. RESULTS: The pattern of recovery of CD3+, CD4+ and CD8+ T cells, as well as the PHA response, was similar for each type of transplant. CD3+CD5- cells were significantly higher following autologous BMT than after allogeneic BMT and during more time. An overexpression of DR on T cells following autologous or allogeneic BMT demonstrates an increasing degree of T-lymphocyte activation. This activated T-cell subset was more stable in patients transplanted with allogeneic BM than in patients treated with autologous BM. The levels of total B cells and CD19+CD5+ B-cells were increased during 2 to 12 months following autologous MBT, remaining normal afterwards; in contrast, the levels of CD19+ lymphocytes and CD19+CD5+B-cells remained higher than normal ranges until 36 months in patients transplanted with allogeneic BM. The percentage of NK cells was significantly increased following both autologous and allogeneic BMT. The highest percentage of NK cells were detected about 2 and 6 months post-transplant in patients treated with autologous or allogeneic BM, respectively. CONCLUSIONS: Allogeneic BMT appears to induce a slight delay recovery of B and NK cells in comparison to autologous BMT. In contrast, T-cells recovery was similar for each type of transplant, although a higher percentage of CD3+CD5- T cells and a faster recovery of activated CD3+DR+ cells to normal levels were observed in patients transplanted with autologous BM.

T lymphocytes that infiltrate nasal polyps have a specialized phenotype and produce a mixed TH1/TH2 pattern of cytokines.

BACKGROUND: Nasal polyps (NPs) are inflammatory reactions in the nasal mucosa the etiology and pathogenesis of which remains unknown. OBJECTIVE: The purpose of this study was to study in detail the phenotype and function of T lymphocytes infiltrating NPs by analyzing the expression of surface markers and cytokine secretion. METHODS: NP tissue samples and peripheral blood were obtained from 18 patients. Mononuclear cells were purified from these samples, and their phenotype was investigated by triple-color immunofluorescence and flow cytometric analysis. Cytokine production was determined in cultures by using an ELISA technique. RESULTS: NP lymphoid cells mainly consisted of T lymphocytes. These T lymphocytes showed a CD45RO+CD45RA- phenotype and expressed pan-T cell molecules; the CD8+ subset was predominant. NP T cells showed a lower density of CD28, CD3, and TCR-alphabeta compared with T cells from peripheral blood. NP T lymphocytes expressed the activation markers DR and CD69 and exhibited the adhesion molecule profile CD54+, CD62L-, and CD103+ CD49dlow. Virtually all NP T cells bore CD95 (FAS), but they did not undergo apoptosis, either spontaneously or induced by CD95 cross-linking with the mAb CH11. The pattern of cytokines secreted by NP T lymphocytes was characterized by the spontaneous and simultaneous production of IFN-gamma and IL-5. Neither IL-2 nor IL-4 were detectable in nonstimulated cultures. CONCLUSION: This study defines the T lymphocytes that infiltrate NPs as memory T cells in an activated status, with homing properties related to the mucosal immune system. They are resistant to anti-CD95-mediated apoptosis and produced a mixed TH1 /TH2 cytokine pattern as defined by the simultaneous production of IFN-gamma and IL-5.

Normal and clonal B lineage cells can be distinguished by their differential expression of B cell antigens and adhesion molecules in peripheral blood from multiple myeloma (MM) patients--diagnostic and clinical implications.

Human MM is a haematologic disorder characterized by the accumulation of malignant plasma cells (PC), primarily in the bone marrow (BM). Although these cells characteristically home to the BM, in recent years several groups have detected the presence of related malignant B cells in the peripheral blood (PB) which could be implicated in the progression and spread of the disease. However, the proportion and origin of these clonotypic circulating B cells is still controversial. In this study, using a triple-staining flow cytometric procedure and a whole blood lysis method, PB B lineage cells could be divided into two populations according to their distinct repertoires of cell adhesion molecules and B cell antigens in untreated MM patients. The results show that: (i) the percentage and the absolute number of PB CD19+ B cells were decreased in MM patients compared with controls; (ii) the quantity and percentage of B cell antigens (CD20, CD22, CD24, DR, CD138) and adhesion molecules (beta1- and beta2-integrins, CD44, CD54, CD56, CD61 and CD62L) expressed by these PB CD19+ cells of MM patients and healthy subjects were similar and all of them were virtually polyclonal cells; (iii) a very minor circulating CD19-CD38++CD45-/dim subset was also detected which expressed CD138 (B-B4) (high intensity), monoclonal cytoplasmic immunoglobulin (cIg), and was negative for pan-B antigens (CD19, CD20, CD24, DR), surface immunoglobulin (sIg) and several adhesion molecules such as CD62L, CD18 and CD11a; this CD19-CD38++CD45-/dim CD138++ subset was not found in normal blood and exhibited a phenotypic profile which was closely related to that of malignant BM plasma cells, with the exception of the CD56 antigen. Polymerase chain reaction (PCR) analysis of IgH clonotypic rearrangements confirmed these results. We postulate that, in MM patients, circulating B lineage cells may be divided into two different categories: polyclonal CD19+ B cells and a very minor proportion of clonal CD138++ PC that escape from the BM.

Regulatory role of CD95 ligation on human B cells induced in vivo capable of spontaneous and high-rate Ig secretion.

CD95 ligation elicits apoptotic signals in many cell systems. This study analyzes the effect of anti-CD95 mAb on human cells capable of spontaneous and high-rate Ig secretion. Such cells have been induced in vivo and represent a highly mature B cell stage. Addition of the anti-CD95 monoclonal antibody (mAb) CH11 to tonsil B cells inhibited 50-60% of their spontaneous Ig secretion. The effect was exerted early in the culture and could be reversed by a pre-treatment with a neutralizing mAb. N-acetyl-D-sphingosine (C2-ceramide), although not a close analog, also reduced Ig secretion to a similar extent. The inclusion of a tetrapeptide inhibitor for certain interleukin-1beta-converting enzyme proteases prevented the inhibitory effect of CH11 mAb on tonsil B cells. B cells capable of spontaneous Ab secretion obtained from blood of recently-immunized volunteers were also inhibited by CH11 mAb and C2-ceramide. In contrast, bone marrow (BM) B cells capable of spontaneous Ig secretion were unaffected by these agents. This CD95 ligation-mediated inhibition of tonsil and blood Ig-secreting B cells could not be reversed by cytokines with demonstrated activity on these B cells. Human mature B cells induced in vivo are identifiable as CD38hi cells. Flow cytometric analysis revealed that a fraction of tonsil CD38hi cells expressed low levels of CD95. Moreover, about 20% of these cells exhibited basal apoptosis, as defined by annexin V binding. This phenomenon was markedly increased by CD95 ligation. On the other hand, BM CD38hi cells showed neither CD95 expression nor CD95-induced annexin V binding. These data suggest that CD95 ligation might play a role in the control of human humoral responses by inducing apoptosis in susceptible mature B cells.

Deficient expression of adhesion molecules by human CD5- B lymphocytes both after bone marrow transplantation and during normal ontogeny.

Despite the relatively early reconstitution of blood B-lymphocyte counts observed in patients treated with bone marrow transplantation (BMT), these patients undergo a prolonged phase of humoral immunodeficiency. Adhesion molecules perform relevant functions in many cell types. The present study examines the expression of several adhesion molecules on human B lymphocytes newly formed after BMT. Blood B cells from 38 patients were studied by flow cytometry and three-color analysis. Blood CD5- B lymphocytes obtained at an early stage after BMT (2 to 4 months) showed a markedly low expression of the adhesion molecules CD54, CD44, CD11a, and CD62L. However, these cells exhibited a normal expression of other molecules including CD29, CD19, CD20, and DR. This deficiency was progressively corrected, reaching normal levels in the late post-BMT period (12 to 15 months). In contrast, CD54, CD44, CD11a, and CD62L expression on the patients' CD5+ B lymphocytes was found to be consistently normal. Deficient adhesion molecule expression on CD5- B cells in the early post-BMT period was similarly observed in patients treated with either an allo-BMT (n = 24) or an auto-BMT (n = 14). Because the post-BMT period mimics normal ontogeny, adhesion molecule expression was also investigated in cord-blood B lymphocytes. Cord-blood CD5- B lymphocytes, in contrast to CD5+, also expressed CD54, CD44, CD11a, and CD62L at levels much lower than those found in normal adults. Present data suggest that progressive expression of CD54, CD44, CD11a, and CD62L seems to be a part of the maturational program of CD5- B lymphocytes during both post-BMT and normal development periods. This observation may help to explain the humoral immunodeficiency observed in both conditions.

[Immunologic reconstitution of peripheral blood lymphocytes in patients treated by bone marrow transplantation]. / Reconstitución inmunológica de los linfocitos de sangre periférica en pacientes tratados con trasplante de médula ósea.

BACKGROUND: Lymphocyte subset reconstitution was studied in 65 patients undergoing allogeneic and autologus bone marrow transplantation (BMT). PATIENTS AND METHODS: The expression of molecules on the membrane of lymphocyte subsets was assessed by two-colour flow cytometry and a direct immunofluorescence assay. The functional capacity of the patient's T lymphocytes following transplantation was identified by stimulation whit peripheral blood lymphocytes; B cells from BMT recipients were tested for their ability to respond, in vitro, to pokeweed (PWD) mitogen. RESULTS: 1) The proportion of CD8+ T lymphocytes was higher than the CD4+ T lymphocytes until 1 1/2 year after-BMT, with high percentage of immature T cells (CD3+, CD8+, HLA-DR+, CD25-) in the first nine months post-transplant. Moreover, a large proportion of T lymphocytes lacked CD5 expression in the first year following BMT. 2) T-cell proliferative response to PHA was low with subsequent recovery until normality. 3) Low numbers of B cells in the first two months with a significant increase since then until 1 1/2 year after-BMT; the phenotype of these B cells was mainly CD19+, CD5+. 4) High in vitro spontaneous immunoglobulin production by peripheral blood B lymphocytes and an impaired response to PWM was observed. 5) Increased percentage of cells with natural killer (CD56) cell phenotype was seen during the 2nd and 3rd months after the graft infusion. After 1 1/2 year postgrafting, this percentage returned to normal level. CONCLUSIONS: Taken together, these data indicate the existence of numerous abnormalities in several subsets of peripheral blood lymphocytes after BMT and suggest a slow kinetics of immune recovery after human marrow transplantation being complete between 18 and 24 months following BMT.

B cells capable of spontaneous IgG secretion in cerebrospinal fluid from patients with multiple sclerosis: dependency on local IL-6 production.

Cerebrospinal fluid (CSF) from multiple sclerosis (MS) patients contains B cells capable of spontaneous IgG secretion in vitro. This study analyses the function and regulation of these cells. CSF cells obtained from nine MS patients actively produced IgG during 2-3 days in culture, and the activity decreased when CSF cells were cultured in serum-free medium. CSF cells from four controls did not secrete detectable IgG in vitro. Further experiments revealed that IL-6 played a role on MS CSF IgG-secreting cells, as can be deduced from the following findings: (i) the addition of exogenous IL-6, but not of other cytokines, to serum-free cultures restored missing CSF cell IgG secretion (ii) the inclusion of anti-IL-6, but not of control, blocking MoAb reduced IgG secretion by CSF cells in fetal calf serum (FCS)-containing cultures; and (iii) CSF cells were capable of active IL-6 production in the presence of FCS. These results suggest that endogenous IL-6 production by MS CSF cells seems to be responsible for inducing CSF IgG-secreting B cells to reach terminal differentiation.

Human tonsil, blood and bone marrow in vivo-induced B cells capable of spontaneous and high-rate immunoglobulin secretion in vitro: differences in the requirements for factors and for adherent and bone marrow stromal cells, as well as distinctive adhesion molecule expression.

Human B cells capable of spontaneous IgG secretion are commonly found in circulation and in lymphoid tissues such as tonsil and bone marrow (BM). The present study compares the mechanisms that regulate tonsil, blood and BM B cells capable of spontaneous IgG secretion. The BM cell subset produced IgG during a markedly longer period of time (14 days) than did tonsil and blood cell subsets (2-3 days). Blood and BM, but not tonsil, B cell IgG secretion depended on the presence of adherent cells, as demonstrated by adherent cell depletion and re-addition experiments. Stromal BM cells supported linear IgG secretion by non-adherent BM cells for 2 weeks, but were unable to prolong the short-term IgG secretion by tonsil and blood cells. Different factors induced IgG secretion in each of the three B cell populations as optimal IgG secretion by tonsil, blood or BM cell subsets required either tumor necrosis factor-alpha, interleukin-6 or fibronectin + interleukin-6, respectively. Finally, these populations also showed differences in the expression of adhesion molecules; the tonsilar cell subset was PNA+/- CD44+ CD49d+ CD49e- Leu-8+/-, the blood cell subset was PNA- CD44+/- CD49d+ CD49e- Leu-8+ and the BM cell subset was PNA- CD44+/- CD49d+ CD49e- Leu-8-. These results suggest that the mechanisms controlling the final differentiation and the expression of adhesion molecules in these B lymphocytes exhibit territorial specificity.

Circulating stem cell collection in lymphoma and myeloma after mobilization with cyclophosphamide and granulocyte colony-stimulating factor for autologous transplantation.

We report the results of 72 leukapheresis procedures performed for autologous peripheral blood stem cell collection in 18 patients with lymphoma and myeloma, after combined mobilization with cyclophosphamide and granulocyte colony-stimulating factor (G-CSF). The numbers of mononuclear cells (MNCs), CD34+ cells and granulocyte-macrophage colony-forming units (CFU-GM) either in the peripheral circulation (preleukapheresis sample) or in the product obtained from leukapheresis (leukapheresis sample) were evaluated. A highly superior proportion of CD34+ cells (14-fold) and CFU-GM (5-fold) resulted from the mobilization therapy. CFU-GM and CD34+ cells were highly enriched with respect to all MNCs (relative recoveries: 2.13, range 0.3-41, and 1.08, range 0.2-8.5, respectively) due to an additional mobilization effect by the leukapheresis procedure. Also, a relatively strong linear correlation between the three different parameters was found in the leukapheresis product (CD34+:CFU-GM, r = 0.81; MNCs:CD34, r = 0.69; MNCs:CFU-GM, r = 0.75; CFU-GM:CD34+, and MNCs, r = 0.85). Our data suggest that the number of MNCs and CD34+ cells obtained after combined mobilization with cyclophosphamide and G-CSF can be used as predictor of the number of granulomonocytic progenitors.

[Allogenic bone marrow transplantation in Wiskott-Aldrich syndrome]. / Trasplante de medula ósea alogénico en el síndrome de Wiskott-Aldrich.

Two males of 3 and 3 1/2 years of age with the Wiskott-Aldrich syndrome who underwent bone marrow transplantation from an HLA compatible brother following conditioning treatment with busulphan and cyclophosphamide are described. In both patients the taking of the graft was proven by study of blood subgroups and correction of the immunodeficiency, normalization of platelet number and function and disappearance of cutaneous eczema were seen. At 3 and 1 year respectively of the transplantation the patients showed no evidence of graft versus host disease and no severe infections or hemorrhagic episodes have seen.

Essential role of tumor necrosis factor-alpha in the differentiation of human tonsil in vivo induced B cells capable of spontaneous and high-rate immunoglobulin secretion.

Human tonsils contain B cells capable of spontaneous and high-rate immunoglobulin (Ig) secretion in vitro. These cells are in vivo induced mature B cells, and, as such, they provide and adequate model for studying tonsil B cell differentiation. The present report analyzes the effect of a variety of factors on purified tonsil B cells capable of spontaneous IgG secretion in fetal calf serum (FCS)-containing and serum-free supplemented cultures. Tumor necrosis factor-(TNF) alpha was found to be important for these B cells to reach the high-rate IgG-secreting stage, as is indicated by the following findings: (a) none of the factors used modified tonsil B cell IgG secretion in FCS-containing cultures; (b) TNF-alpha (5-20 ng/ml), but not other cytokines or factors including interleukin (IL)-6, was capable of restoring missing IgG production in serum-free supplemented cultures of tonsil B cells; and (c) IgG secretion in FCS-containing cultures was inhibited by the addition of blocking anti-TNF-alpha antibodies, but not anti-IL-6 antibodies, and this inhibition could be specifically reversed by exogenous TNF-alpha. TNF-alpha was actively produced by tonsil B cells (range 120-750 pg/ml) in the presence, but not in the absence, of FCS. The TNF-alpha inductive effect occurred during the first 12 h of culture and did not require DNA synthesis. These results indicate that the early and endogenous generation of TNF-alpha seems to be essential for tonsil in vivo induced B cells to differentiate into the high-rate Ig-secreting stage.

Cytokine network regulating terminal maturation of human bone marrow B cells capable of spontaneous and high rate Ig secretion in vitro.

Human bone marrow (BM) B cells capable of spontaneous and high rate Ig secretion for 14 days in vitro have been described previously. We have shown recently that Ig secretion by these BM cells depends on stromal adherent BM cell-derived factors identified as IL-6 and fibronectin. Our report shows that the endogenous generation of IL-1 beta and TNF-alpha in serum-containing cultures of BM mononuclear cells (BMMC) is also involved in the control of Ig-secreting cells, because their blockade with specific antibodies markedly reduced Ig production. Further experiments revealed that IL-1 beta and TNF-alpha acted by regulating IL-6 production, as can be deduced from the following findings: 1) the inhibition of Ig secretion caused by either anti-IL-1 beta or anti-TNF-alpha antibodies could be reversed by exogenous IL-6; 2) the addition of either of these antibodies inhibited endogenous IL-6 production in BMMC cultures; 3) IL-1 beta plus TNF-alpha, but neither one alone, restored complete IL-6 and Ig production by BMMC in serum-free cultures. Moreover, adherent, but not nonadherent, BM cells were responsible for endogenous IL-1 beta and TNF-alpha secretion. Finally, IL-1 beta plus TNF-alpha induced the production of IL-6, but not of Ig, by adherent BM cells. Neither IL-6 nor Ig production was induced by adding this cytokine combination to nonadherent BM cell cultures, despite the fact that this fraction contained all the Ig-secreting cells. However, the addition of IL-6 restored Ig secretion in this cell fraction. These results suggest that IL-1 beta and TNF-alpha produced by adherent BM cells synergistically induce early IL-6 generation, which, in turn, drives BM B cell producers into the high rate Ig-secreting state.