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A clonal population of B cells expressing a V(H) 1-69-encoded idiotype accumulates in hepatitis C virus (HCV) associated mixed cryoglobulinemia (MC). These cells are phenotypically heterogeneous, resembling either typical marginal zone (MZ) B cells (IgM(+) IgD(+) CD27(+) CD21(+) ) or the exhausted CD21(low) B cells that accumulate in HIV infection or in common variable immunodeficiency. We show that both the MZ-like and the CD21(low) V(H) 1-69(+) B cells of MC patients are functionally exhausted, since they fail to respond to TLR and BCR ligands. The proliferative defect of V(H) 1-69(+) B cells can be overcome by co-stimulation of TLR9 and BCR in the presence of interleukin(IL)-2 and IL-10. The MZ-like V(H) 1-69(+) B cells do not express the inhibitory receptors distinctive of CD21(low) B cells, but display constitutive activation of extracellular signal regulated kinase (ERK) and attenuated BCR/ERK signaling. These cells also express abundant transcripts of Stra13 (DEC1, Bhlhb2, Sharp2, Clast5), a basic helix-loop-helix transcription factor that acts as a powerful negative regulator of B-cell proliferation and homeostasis. Our findings suggest that MZ B cells activated by HCV undergo functional exhaustion associated with BCR signaling defects and overexpression of a key antiproliferative gene, and may subsequently become terminally spent CD21(low) B cells. Premature exhaustion may serve to prevent the outgrowth of chronically stimulated MZ B cells., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A subset of patients with common variable immunodeficiency (CVID), group 1a of the Freiburg classification, is characterized by increased B cells expressing low levels of CD21 (CD21(low) ), lymphoproliferation and autoimmunity. The CD21(low) B cells have been shown to be profoundly anergic, and defects of BCR-mediated calcium signaling and of T cells have been described in CVID 1a. We found that also the classical naïve B cells from CVID 1a patients, but not from CVID non-1a patients, proliferated poorly. The B cells of CVID 1a patients had a reduced capacity to divide reminiscent of the proliferative arrest associated with replicative senescence. Thus, we investigated whether lymphocyte dysfunction in CVID 1a was related to telomere-dependent replicative senescence, and found that both the B and the T cells from CVID 1a patients had significantly shorter telomeres compared with B and T cells from CVID non-1a patients. Telomere lengths in B and T cells were significantly correlated, indicating that the rate of telomere attrition in lymphocytes is an individual characteristic of CVID patients. Our findings suggest that telomere-dependent replicative senescence contributes to the immune dysfunction of CVID 1a patients, and may provide an important clue for a better understanding of the pathogenesis of CVID., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Peptide nucleic acid (PNA) probes hybridize to denatured telomeric sequences in cells permeabilized in hot formamide. In reported protocols, the hybridization was conducted in solutions with high formamide concentrations to avoid the DNA renaturation that can hamper binding of the oligo-PNA probe to specific sequences. We postulated that telomeric DNA, confined in the nuclear microvolume, is not able to properly renature after hot formamide denaturation. Therefore, to improve hybridization conditions between the probe and the target sequences, it might be possible to add probe to sample after the complete removal of formamide., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We evaluated the relationship between cytokine profile and the expression of the lymphocyte activation gene-3 (LAG-3) in both T cell clones and polyclonal T cell lines; LAG-3 is a CD4-like protein whose expression is reportedly restricted to Th1/0 cells and dependent upon IFN-gamma. We found that, while LAG-3 was expressed only by CD4+ T cell clones producing IFN-gamma, most CD8+ clones producing IL-4 but not IFN-gamma (i.e., with a T cytotoxic-2-like profile) were LAG-3+. The intensity of LAG-3 expression by CD8+ clones correlated with the amount of released IFN-gamma, suggesting that this cytokine is not required for expression but rather for the up-regulation of LAG-3. Flow cytometric analyses of polyclonal T cell lines confirmed that LAG-3 could be expressed by both CD4+ and CD8+ cells that did not contain cytoplasmic IFN-gamma. In these cell lines, large proportions of CD4+ and CD8+ cells coexpressed LAG-3 and CD30, a putative marker of Th2-like cells. Overall, our data do not support the earlier suggestion that LAG-3 and CD30 are selective markers of T cells with type-1 and type-2 cytokine profiles, respectively.

Destruction of immune cells in peripheral lymphoid tissues plays presumably a pivotal role in acquired immune deficiency syndrome pathogenesis. We found that cell suspensions obtained from lymph nodes of eight human immunodeficiency virus (HIV)-infected individuals contained variable proportions (2.1% to 18.3%, median 11.2%) of dead lymphocytes permeable to supravital dyes, represented by CD4+, CD8+, and B cells. The frequency of dead cells correlated directly (R = 0.847) with the amount of HIV provirus in the cell populations, and HIV provirus was enriched in the dead cell fractions. Similar proportions of dead cells were observed in cell suspensions from lymphadenopathic lymph nodes of HIV- donors, but not from small resting HIV- lymph nodes. Electron microscopic and flow cytometric analyses revealed that most dead cells from HIV+ lymph nodes lacked internucleosomal DNA fragmentation but displayed combined features of apoptosis and necrosis, eg, chromatin condensation and mitochondrial swelling. Cells with similar morphology were readily identified in lymph node tissue sections, and marked mitochondrial swelling could be occasionally observed in cells with otherwise normal morphology. Our findings have two major implications. One is that the in vivo cell death in HIV-infected lymph nodes occurs predominantly through a novel pathway, related to but distinct from classical apoptosis and characterised by early and severe mitochondrial damage. The second implication is that HIV-related lymphadenopathy is accompanied in vivo by massive destruction of uninfected lymph node cells. Comparable levels of cell death were observed in other inflammatory lymphadenopathies not related to HIV; however, the uniquely endless and generalized nature of HIV lymphadenopathy might render this "inflammatory" cell destruction a powerful pathogenetic mechanism, accounting for the progressive disruption and depletion of lymphoid tissues seen in HIV infection.

Perturbations of the repertoire of variable-beta (Vbeta) regions of the T cell receptor have been observed in patients infected by HIV and have been attributed to stimulation by viral antigens or superantigens. We further sought for traces of HIV-induced perturbations by comparing Vbeta repertoire in peripheral blood and in lymphoid tissues of six infected patients. Vbeta expression was studied with a panel of 17 anti-Vbeta antibodies covering about 50% of the entire repertoire. We observed major divergences between lymph nodes and peripheral blood in the expression of several Vbeta segments, and these differences were significantly more frequent in CD8+ than in CD4+ T cells (P = 0.0097). Vbeta2 was perturbed in CD8 cells from all but one patient. One HIV-negative subject with localized reactive lymphadenopathy of unknown etiology had four perturbed Vbeta segments, including Vbeta2, in CD8+ cells, while another uninfected subject with an unreactive lymph node architecture had no perturbations. Our findings suggest that stimulation by HIV or by other antigens determines divergences in the Vbeta repertoire between lymphoid tissues and peripheral blood predominantly in CD8+ T cells.

The measurement of apoptosis in peripheral blood might represent a useful tool in acquired immunodeficiency syndrome (AIDS) and cancer research. Among the many assays that are currently used to identify apoptotic leukocytes, flow cytometric methods are the most valuable in terms of rapidity, simplicity, and level of analytical detail. Some flow cytometric assays may also offer the additional advantage of detecting the earliest phases of apoptosis, which is paramount importance for measuring apoptotic cells in vivo before they are destroyed by phagocytes.

Lymphocytes from HIV-1-infected subjects undergo massive apoptosis when cultured in vitro, and this phenomenon might reflect pathogenetic mechanisms leading to immune dysfunction in vivo. However, (1) lymphocyte death is not restricted to CD4+ cells but seems to involve predominantly CD8+ cells, and (2) the same phenomenon occurs in other viral infections. Furthermore, it is not known whether a relationship exists between the HIV-1 burden and this type of cell death. In this work we sought to determine whether the HIV-1 provirus load correlates with the propensity to apoptosis of CD4+ and CD8+ cells. We studied 10 HIV-1-infected patients with CD4+ cell counts above 500/mm3 and free of concomitant infections. We correlated the frequency of HIV-1-infected CD4+ cells with the extent of culture-induced apoptosis as well as with the phenotype of the apoptotic lymphocytes. We found that the magnitude of apoptosis correlated with the frequency of HIV-1-infected CD4+ cells (p = 0.0007), and that increasing viral load and apoptosis were associated with a shift to the selective death of CD8+ cells. Our data support the view that, in addition to CD4+ cell killing, another immunopathogenic effect of HIV might be that of priming CD8+ cells to apoptosis. In vivo, this could eventually lead to the exhaustion of the cytotoxic T cell compartment.

We have developed a quantitative and sensitive flow cytometric method for the detection of human apoptotic lymphocytes that, unlike previously described assays, allows their identification in mixed populations of peripheral blood leukocytes as well as their immunophenotyping. Apoptotic lymphocytes are identified on the basis of peculiar light scatter changes, reflecting their smaller size and their modified nucleus/cytoplasm organization, and of the decreased expression of surface CD45 molecules. Based on these criteria, apoptotic lymphocytes generated by exposure to ionizing radiation can be easily distinguished from viable cells and from necrotic lymphocytes generated by treatment with antibody and complement. Using this assay, we reappraised the phenomenon of the in vitro apoptosis of lymphocytes from patients with human immunodeficiency virus (HIV) infection. Lymphocytes from HIV patients, unlike those from normal HIV-negative subjects, undergo apoptosis upon simple in vitro culture. We found that the percentages of lymphocytes undergoing apoptosis were significantly higher in patients with low CD4 cell counts (< 400/microL) than in patients at earlier stages (> 400 CD4 cells/microL). However, phenotypic analysis disclosed that apoptotic lymphocytes generated in these cultures were mostly CD8+ T cells and CD19+ B cells. Thus, in contrast to what has been previously suggested, the phenomenon of in vitro lymphocyte apoptosis might not be pathogenetically related to the depletion of CD4+ T cells in acquired immunodeficiency syndrome. Nevertheless, it might represent an useful marker of disease progression. Our assay allows the analysis of unfractionated peripheral blood leukocytes and thus the identification of apoptotic lymphocytes circulating in vivo. Apoptotic lymphocytes could indeed be detected in the circulation of a patient with cancer shortly after high-dose cytotoxic chemotherapy. By contrast, no apoptotic lymphocytes could be detected in vivo in patients with early or advanced HIV infection.

The effect of the organic sulphated polyanions, pentosan sulphate (SP54), dextran sulphate 500 (DS500) and suramin, have been tested on golden Syrian hamsters infected with the 263K strain of scrapie by the intraperitoneal (i.p.) or the intracerebral route. SP54 had the greatest effect in prolonging the incubation period of the disease when administered within 2 h of the i.p. inoculum. The same amount of SP54 given 24 h after scrapie inoculation had a potent effect in some animals and no effect in others. This result suggests that SP54 inhibits the uptake of the scrapie agent into the nerve endings and/or carrier cells at the site of the inoculum, i.e. the peritoneum, and that this event occurs in about 24 h. DS500 had a similar although less potent effect (22.4 days delay during the incubation period) than SP54 (54.4 days) when administered within 2 h of scrapie injection by the i.p. route, and suramin had only a minimal effect (10 days). This study suggests that treatment of scrapie and related spongiform encephalopathies of animals and man is possible only before the agent has reached the clinical target areas of the brain.