Disrupted lymphocyte homeostasis in hepatitis-associated acquired aplastic anemia is associated with short telomeres.

Hepatitis-associated aplastic anemia (HAA) is a variant of acquired aplastic anemia (AA) in which immune-mediated bone marrow failure (BMF) develops following an acute episode of seronegative hepatitis. Dyskeratosis congenita (DC) is an inherited BMF syndrome characterized by the presence of short telomeres, mucocutaneous abnormalities, and cancer predisposition. While both conditions may cause BMF and hepatic impairment, therapeutic approaches are distinct, making it imperative to establish the correct diagnosis. In clinical practice, lymphocyte telomere lengths (TL) are used as a first-line screen to rule out inherited telomeropathies before initiating treatment for AA. To evaluate the reliability of TL in the HAA population, we performed a retrospective analysis of TL in 10 consecutively enrolled HAA patients compared to 19 patients with idiopathic AA (IAA). HAA patients had significantly shorter telomeres than IAA patients (P = 0.009), including four patients with TL at or below the 1st percentile for age-matched controls. HAA patients had no clinical features of DC and did not carry disease-causing mutations in known genes associated with inherited telomere disorders. Instead, short TLs were significantly correlated with severe lymphopenia and skewed lymphocyte subsets, features characteristic of HAA. Our results indicate the importance of caution in the interpretation of TL measurements in HAA, because, in this patient population, short telomeres have limited specificity.

Repair of DNA double-strand breaks is not modulated by low-dose gamma radiation in C57BL/6J mice.

In this study, we sought to determine whether low-dose ionizing radiation, previously shown to induce a systemic adaptive response in C57BL/6J mice, is capable of enhancing the rate of DNA double-strand break repair. Repair capacity was determined by measuring γ-H2AX levels in splenic and thymic lymphocytes, using flow cytometry, at different times after a challenge irradiation (2 Gy, (60)Co). Irradiation with low doses (20 and 100 mGy) was conducted in vivo, whereas the challenge dose was applied to primary cultures of splenocytes and thymocytes in vitro 24 h later. Obtained kinetics curves of formation and loss of γ-H2AX indicated that cells from low-dose irradiated mice did not express more efficient DNA double-strand break repair compared to controls. Immunoblot analysis of γ-H2AX and Phospho-Ser-1981 ATM confirmed that DNA damage signaling was not modulated by preliminary low-dose radiation. Mouse embryonic fibroblasts of C57BL genetic background failed to show clonogenic survival radioadaptive response or enhanced repair of DNA double-strand breaks as evaluated by immunofluorescence microscopy of γ-H2AX foci. Our results indicate that radiation adaptive responses at systemic levels, such as increases in the tumor latency times in aging mice, may not be mediated by modulated DNA repair, and that the genetic background may affect expression of a radioadaptive response.

Rapid cell senescence and apoptosis in lymphocytes and granulocytes and absence of GM-CSF receptor in congenital dysgranulopoietic neutropenia.

A girl with congenital dysgranulopoietic neutropenia (CDN) and her non-neutropenic mother with aphthae (A) were investigated. Apoptosis in lymphocytes and granulocytes of both patients (mother A+) were documented by high annexin and electron microscopic morphology. CD11b/CD18 of the daughter's granulocytes ranged between low to normal while that of the mother changed between very low to high levels through A(-) to A(+) periods. In both patients, CD11b/CD18 on lymphocytes were high; GM-CSF receptor was negative; CD4-/CD8- lymphocytes were high and the leukocytes which showed abnormal cell cycle were stained by senescence associated beta-galactosidase. We think that increased apoptosis and rapid cell senescence of leukocytes underlies the pathophysiology of CDN.

Polyclonal expansion of large granular lymphocytes in common variable immunodeficiency - association with neutropenia.

Common variable immunodeficiency (CVID) is the most frequent symptomatic primary immunodeficiency disease, characterized by low levels of circulating immunoglobulins and recurrent bacterial infections, particularly of the respiratory tract. T cell dysfunction is often present, and lymphoproliferative and autoimmune disorders as well as haematological cytopenias are frequently observed. In this study, we report a polyclonal expansion of large granular lymphocytes (LGL) in a substantial proportion of CVID patients, associated with splenomegaly, increased numbers of CD8(+) T cells, inverted CD4 : CD8 T cell ratios and neutropenia. CVID patients who had both increased numbers of LGL and granulocytopenia had elevated levels of soluble Fas ligand (sFasL). Our observations indicate that CVID may be added to the list of inflammatory diseases associated with increased numbers of LGL. Furthermore, our findings suggest common pathogenic mechanisms of granulocytopenia in CVID and lymphoproliferative disease of granular lymphocytes.

Granulocyte colony-stimulating factor perturbs lymphocyte mitochondrial function and inhibits cell cycle progression.

Sera from healthy subjects receiving recombinant human granulocyte colony-stimulating factor (rHuG-CSF) to mobilize CD34(+) peripheral blood progenitors (PBPC) have been recently shown to induce unresponsiveness of allogeneic lymphocytes to mitogenic challenge. In the present investigation, the effects of rHuG-CSF on the early stages of lymphocyte activation-induced apoptosis and on lymphocyte cell cycle entry were evaluated. Sera were obtained from HLA-identical donors receiving rHuG-CSF to mobilize CD34(+) PBPC for allogeneic transplantation. Normal peripheral blood mononuclear cells (PBMC) were challenged with phytohemagglutinin (PHA) in the presence of serum collected before (preG) or after rHuG-CSF administration (postG). Mitochondrial function, that is, incorporation of 3,3'-dihexyloxacarbocyanine iodide [DiOC(6)(3)] and generation of reactive oxygen species (ROS) as well as expression of c-Myc and Bcl-2 family members (Bcl-2, Bcl-X(L), Bax) were evaluated by multiparameter flow cytometry. The activation-induced fragmentation of genomic DNA was detected by highly sensitive LM-PCR assay.CD4(+)DiOC(6)(3)(low) and CD8(+)DiOC(6)(3)(low) T lymphocytes increased and reached 32% (range 27%-38%) and 20% (range 15%-23%) of circulating T cells, respectively, on day 4 of rHuG-CSF administration. Hypergeneration of ROS could be demonstrated in 65% (range 58%-82%) of CD4(+) T lymphocytes and in 0.4% (range 0.2%-0. 8%) of circulating CD8(+) T cells. rHuG-CSF determined no alteration of mitochondrial function if added to allogeneic PBMC in vitro, thus suggesting indirect effects mediated by soluble factors; on the contrary, when PBMC were challenged with PHA in the presence of postG serum, both perturbation of mitochondrial transmembrane potential (Deltapsi(m)) and hypergeneration of ROS were induced, and lymphocytes were predominantly arrested in a G(0) -like phase of the cell cycle and displayed genomic DNA fragmentation. Interestingly, the preincubation of PBMC with a blocking antibody directed against CD95 abrogated the perturbation of lymphocyte Deltapsi(m), suggesting that the CD95 signaling pathway might play a role in the induction of apoptosis after PHA stimulation in the presence of postG serum. Moreover, Bax protein was overexpressed in postG (median fluorescence intensity = 180, range 168-186) compared with preG cultures (median fluorescence intensity = 75, range 68-80; p < 0.01), while no differences in Bcl-2, Bcl-X(L), and c-Myc staining intensity were observed. Our findings demonstrate a humoral-mediated rHuG-CSF-induced dissipation of lymphocyte mitochondrial Deltapsi(m); these effects might be mediated by Bax overexpression, with imbalance between apoptosis-promoting and apoptosis-inhibiting Bcl-2 family members and with subsequent induction of mitochondrial permeability transition. Whether immune dysfunction will favorably impact on incidence and severity of acute graft vs host disease after allogeneic PBPC transplantation remains to be determined.

Lineage involvement of stem cells bearing the philadelphia chromosome in chronic myeloid leukemia in the chronic phase as shown by a combination of fluorescence-activated cell sorting and fluorescence in situ hybridization.

Chronic myeloid leukemia (CML) is thought to arise from a pluripotent hematopoietic stem cell that has undergone a reciprocal translocation between the BCR gene on chromosome 22 and the ABL proto-oncogene on chromosome 9. This rearrangement results in a shortened chromosome 22, designated the Philadelphia (Ph) chromosome. The Ph chromosome has been found in cells from all hematopoietic lineages except mature T lymphocytes. To examine this issue, we combined fluorescence-activated cell sorting (FACS) and fluorescence in situ hybridization (FISH) to study lineage involvement of mature cells and stem cells in 12 patients with CML in the chronic phase. We found Ph chromosomes in myeloid cells and most B lymphocytes (CD19(+)) but not in mature T cells (CD3(+)) or natural killer (NK) cells (CD3(-)56(+)). Moreover, evidence of BCR/ABL fusion was found in pluripotent stem cells (CD34(+)Thy-1(+)), B-progenitor cells (CD34(+)CD19(+)), T/NK progenitor cells (CD34(+)CD7(+) cells), and T progenitor cells (CD34(+)CD7(+)CD5(+)) with a frequency equal to that in all CD34(+) cells isolated by FACS from bone marrow cells. T lymphocytes showed a marked decrease in Ph+ cells between progenitor cells and mature cells. Moreover, the ratios of Ph+ to Ph- cells in mature T cells and NK cells were below background levels, whereas Ph+ B lymphocytes also decreased during their maturation. These data suggest that Ph+ lymphocytes are eliminated during differentiation. In contrast to FISH of blood and bone marrow, which gives information principally about mature cells, the technique of "sorter FISH (FACS + FISH)" provides a powerful tool to explore the cytogenetic changes in immature cell populations of stem cell diseases based on immunophenotypes. Further clarification of genetic changes in stem cells could be achieved by using sorter FISH with monoclonal antibodies.

Valores de referencia de las subpoblaciones de linfocitos T en la sangre del cordón umbilical / Reference values of T lymphocyte subpopulations in cord blood

Con el objeto de determinar el porcentaje de las subpoblaciones de linfocitos T en la sangre del cordón umbilical y establecer los valores de referencia en el área metropolitana de la ciudad de Puebla, se seleccionaron al azar 80 recién nacidos de un total de 400; éstos eran consecutivos a término, eutróficos y libres de enfermedad. Se determinó el porcentaje de subpoblaciones CD3, CD4 y CD8 de linfocitos T en la sangre del cordón umbilical por citometría de flujo. Los límites de referencia se obtuvieron a partir de las percentilas 25 y 75 en los parámetros de distribución gaussiana. Los valores de referencia para CD3 (por ciento) fueron de 45.5-91.3, para CD4 (por ciento) de 26.2-69.4, para CD8 (por ciento) de 16.2-24.1 y para NK (por ciento) de 2.0-8.5. Los resultados obtenidos representan los límites de referencia de las subpoblaciones de linfocitos T para compararse con los valores observados en los niños enfermos o en riesgo de desarrollar una enfermedad inmunológica

Development and cell phenotypes in primary follicles of foetal sheep lymph nodes.

Lymph nodes from sheep foetuses and postnatal lambs were examined to determine the participation of different leucocyte populations in primary follicle formation, with special emphasis on the emergence and subsequent development of follicular dendritic cells during late gestation and early postnatal life. A series of immune and enzyme histochemical markers was used. The first 5'-nucleotidase-positive primary follicles were found at 80 days gestational age (gestation in sheep is 150 days) in superficial cervical lymph nodes. In the last month of gestation the primary follicles possessed follicular dendritic cells, macrophages, dendritic cells, and CD5-positive lymphocytes, in addition to IgM-positive cells. Follicular dendritic cells in primary follicles were found to be ultrastructurally immature. These follicular dendritic cells were characterised by a few, course surface projections and many ribosomes attached to the endoplasmic reticulum. A final differentiation to mature follicular dendritic cells was coincident with the postnatal germinal centre reaction. Computer-assisted morphometric analysis demonstrated that the size of 5'-nucleotidase-positive primary follicles in the distal jejunal lymph node, but not in the superficial cervical lymph node, increased significantly during late gestation. It was concluded that stromal cells in primary follicles of foetal sheep lymph nodes were a continuously developing population but that ultrastructural maturity was only achieved in the germinal centres of postnatal lambs.

The impairment of natural killer function in the healthy aged is due to a postbinding deficient mechanism.

In order to study the fine mechanisms that underlie the impairment of non-MHC-restricted cytolytic activity which occurs during human aging, we examined by multiparametric flow cytometry the binding and lytic activities of human natural killer cells. The flow analysis revealed a striking increase of the CD16+8- subset, together with a significant decrease of CD8bright cells and total T cells (CD3+). Aging had no influence on the CD8dim subset. The total lytic activity expressed by PBL as well as their binding efficiency to K562 targets were moderately but not significantly increased in the elderly. In contrast, the cytotoxicity of the single target-bound natural killer cell (i.e., lytic efficiency) was deeply impaired in aged subjects, suggesting that the NK functional impairment observed in aging is located at postbinding level.

Lymphocyte subsets associated with T cell receptor beta-chain gene rearrangement in patients with rheumatoid arthritis and neutropenia.

OBJECTIVE: To determine the incidence of a clonal lymphoid disease in patients with chronic rheumatoid arthritis (RA) and neutropenia. METHODS: Lymphocytes from 23 RA patients with either current neutropenia or a history of this complication were studied. RESULTS: Eight patients had a clonal rearrangement of the T cell receptor beta-chain gene. Phenotypically, they showed a distinctive pattern characterized by an inverted CD4+:CD8+ cell ratio and an increased number and percentage of CD57+/CD8+ and CD3+/DR+ lymphocytes. None had evidence of a lymphoid malignancy. CONCLUSION: Among RA patients with neutropenia, there is a subset who have a subclinical disease resembling T gamma lymphoproliferative disease.

The role of interleukin 2 (IL-2) and serum-soluble IL-2 receptor cells in idiopathic IgA nephropathy.

Interleukin 2 (IL-2) plays a central role in the immune response and may be involved in the derangement of cellular immunoregulation of idiopathic IgA nephropathy (IgAN). The aim of this study was to investigate the serum levels and production of IL-2 from peripheral blood mononuclear cells (PBMC) and the distribution of IL-2 receptor cells and serum-soluble IL-2 receptor cells (sIL-2R) in patients with IgAN. Twenty-four patients with IgA nephropathy and 11 healthy controls (age and sex matched) were studied during an infection-free period without signs of clinical activity at the moment of the study. Serum IL-2 concentrations did not differ between patients and controls. The supernatant levels of IL-2 taken from 24-hr cultures of PBMC stimulated with phytohemagglutinin or tumor necrosis factor increased significantly in the patients but not in the controls. The percentage of IL-2R positive cells (CD25+) was increased in patients compared with controls. Moreover, IgAN patients had increased activated CD4+ lymphocytes when compared with the controls. Serum levels of sIL-2R were significantly higher in patients than in controls. There were no correlations among renal function, serum IgA levels, and urinary findings with cellular subsets or with IL-2 levels. However, sIL-2R was higher in the subgroup of patients with episodic macrohematuria and was closely related with the presence of red blood cells in the urinary sediment. We conclude that PBMC of IgA nephropathy patients have an overproduction of IL-2 after mitogenic stimulation, an increased helper T cell activity, increased IL-2R+ cells, and elevated serum levels of sIL-2R. These alterations are present in periods of apparent clinical inactivity. Finally, sIL-2R is closely related with hematuria, providing a good marker for disease activity. Our results suggest a pivotal role of IL-2 in cellular immune responses with regard to T cell activation in patients with IgAN.

Natural killer cell surface markers on cells containing parallel tubular structures.

Parallel tubular structures (PTS) and/or the associated electron-dense granules, thought to contain molecules responsible for target cell lysis, can be detected in the cytoplasm of lymphocytes with large granular lymphocyte (LGL) morphology. In the present study we compared PTS presence in freshly isolated peripheral blood lymphocytes and peripheral blood lymphocytes incubated overnight in the presence of human pooled serum, sera from patients with Hodgkin's disease and interferon-alpha. Under all conditions we found PTS in the majority of CD16+ cells (64.3-74.8%) but less than 41.8% in CD57+ cells. In the case of double-labeled lymphocytes, 41.0-61.7% CD16+/8+ but only 24.2-27.5% CD57+/8+ cells were PTS+. Thus, in all cases where lymphocytes expressed CD16 antigen there was a high percentage of PTS positivity. Although the PTS+ cells exhibited phenotypic heterogeneity there was, except for a proportion of CD57+ lymphocytes which exhibited less of the characteristic LGL features, generally LGL morphological homogeneity. CD16 lymphocytes are potentially more cytotoxic than CD57 and CD8 cells. Taking this into consideration, the presence of the PTS in the majority of CD16 cells suggests an important role for PTS in target cell lysis.

Preferential binding of Chlamydia trachomatis to subsets of human lymphocytes and induction of interleukin-6 and interferon-gamma.

The interactions between Chlamydia trachomatis and human blood mononuclear leukocytes were studied using flow cytometry, immunofluorescence, electron microscopy and cytokine assays. Under serum-free conditions, elementary bodies (EB) of C. trachomatis were found to bind to human T lymphocytes as well as to B cells and monocytes/macrophages (M phi). For all cell types the binding was saturable, rapid, temperature-independent and independent of the chlamydia-specific serological status of the donor. Similar proportions of T and B cells bound EB at similar levels. In the T cell population, proportionally less CD8+ cells bound EB. Whereas M phi phagocytosed and destroyed the bound micro-organisms for lymphocytes, the Chlamydia remained at the surface, adherent to morphologically featureless membrane areas and showed no evidence of uptake even after long periods at 37 degrees C. Host molecules modulated these basic binding patterns: a heat-stable serum factor inhibited EB binding to T cells and a heat-labile serum factor enhanced binding to B cells. Stimulation with C. trachomatis EB rapidly elicited cytokine production by lymphocytes including interleukin-6 from B cells and interferon-gamma (IFN-gamma) from T and/or nonT/nonB cells. The responses were irrespective of the serological status of the donor. The findings suggest that C. trachomatis-leucocyte interactions may differ from the interactions of other bacteria and human leucocytes. The possible relationship between leucocyte-binding, cytokine induction, and the pathognomonic development of lymphoid follicles during mucosal C. trachomatis infections is discussed.

Immunoelectron microscopy of cell populations in regional and central lymph of sheep.

The immunoreactivity and the ultrastructural localization of monoclonal anti-sheep lymphocyte antibodies conjugated with colloidal gold particles were examined in free-floating cells of sheep central lymph from the thoracic duct, postnodal lymph draining either the popliteal nodes or the mesenteric nodes, and prenodal lymph draining the pregnant uterus. The monoclonal antibodies used in this study were SBU-T1 (CD5), SBU-T4 (CD4), SBU-T8 (CD8), SBU-II (anti DR antibody), and E53 which are reported to be sheep homologues of human T1, T4, T8, HLA-DR, and pan B cell antibodies, respectively. Colloidal gold particles were evenly distributed or segmentally aggregated on the surfaces of lymphocytes and macrophages incubated with monoclonal antibodies and in vesicles in the cytoplasm of anti DR antibody labeled macrophages. Not only did CD5 labeled cells show a high percentage in each regional lymph examined, but the percentage of CD4 labeled cells was consistently higher than that of CD8 labeled cells. Moreover, the immunoreactivity of CD8 labeled cells was specific among lymph from the different regions. The sum of the percentages of CD4 and CD8 labeled cells was less than the percentage of CD5 labeled cells, indicating the presence of a minor T cell subpopulation which was CD5+, CD4-, and CD8-. A characteristic finding was a high percentage of CD8 labeled cells and many abnormal eosinophils in uterine prenodal lymph in pregnant sheep. Taken together the results showed that variously labeled immunoreactive cells are distributed somewhat differently in lymph derived from different organ sites.

Single laser three color immunofluorescence staining procedures based on energy transfer between phycoerythrin and cyanine 5.

Monoclonal antibodies specific for phycoerythrin (PE) were covalently labeled with the fluorescent dye cyanine 5 (Cy5). Excitation at 488 nm of immune complexes obtained by mixing Cy5-anti-PE with PE resulted in a 4-fold reduction of PE fluorescence measured at 565 nm and an increase of fluorescence measured at 655 nm. The observed energy transfer between PE and Cy5-anti-PE was used to develop three color immunofluorescence staining procedures for flow cytometers equipped with an Argon laser tuned at 488 nm. Mouse IgG1 monoclonal antibodies specific for cell surface antigens were cross-linked with either unlabeled or Cy5 labeled mouse IgG1 anti-PE using F(ab')2 fragments of monoclonal rat anti-mouse IgG1. PE was added to these immune complexes in sufficient amounts to saturate all PE binding sites. Cells were incubated with PE-labeled and PE/Cy5-labeled tetrameric antibody complexes together with FITC labeled antibodies and analyzed by flow cytometry. The emission from FITC, PE and PE/Cy5 could be readily separated and bright three color immunofluorescence staining of mononuclear cells from human peripheral blood and bone marrow was observed. The results of these experiments demonstrate that useful probes for single laser three color staining of cell surface antigens can be readily obtained by mixing of selected reagents. Compared to standard procedures for the covalent labeling of PE (tandem) molecules to antibodies, the non-covalent procedures described in this report provide significant advantages in terms of the amount of reagents, time and equipment required to obtain suitable reagents for three color immunofluorescence staining.

Analysis of immunocompetent cells in the bat, Pteropus giganteus: isolation and scanning electron microscopic characterization.

Immunocompetent cells of the Indian fruit bat (P. giganteus) were characterized by differential surface adhesiveness and surface topography. We observed three cell types: (1) plastic adherent with pseudopodia; (2) nylon wool adherent with small microvilli and pits; and (3) a nylon wool nonadherent with comparatively smooth surfaces. These cell types resemble, respectively, macrophages, B cells and T cells of other mammals, including mice and humans. The disposition of microvilli on the B-type cell surface changes significantly after immunization, which suggests modulation of the molecular organization of the cell membrane and its fluidity. The proportions of these three cell types are not much different from those found in mice. Follicular dendritic cells, capable of retaining antigen for long periods in mice, have also been detected in bats. Further characterization of immunocompetent cells wil help in understanding the mechanism of immune responses in bats.