Osseointegration of zirconia and titanium implants in a rabbit tibiae model evaluated by microtomography, histomorphometry and fluorochrome labeling analyses.

OBJECTIVES: This study compares the osseointegration of machined-zirconia implants containing yttria (M-Y-TZP) with machined (M-Ti) and resorbable blast media (RBM-Ti) titanium implants. MATERIAL AND METHODS: M-Y-TZP, M-Ti and RBM-Ti implants were randomly placed in rabbit tibiae. Fluorochrome bone labels (tetracycline, alizarin and calcein) were administered at different time periods. After 8 weeks, osseointegration was evaluated in terms of bone-to-implant contact (BIC), new bone area (nBA), remaining cortical bone area (rBA) and temporal quantification of fluorochromes, using micro-CT and histomorphometric analyses. RESULTS: RBM-Ti implants showed higher resorption of the remaining cortical bone and bone formation (rBA = 36.9% and nBA = 38.8%) than M-Y-TZP implants (rBA = 48% and nBA = 26.5%). The BIC values showed no differences among the groups in the cortical region (mean = 52.2%) but in the medullary region, they were 0.45-fold higher in the RBM-Ti group (51.2%) than in the M-Y-TZP group (35.2%). In all groups, high incorporation of tetracycline was observed (2nd to 4th weeks), followed by alizarin (4th to 6th weeks) and calcein (6th to 8th weeks). In the cortical region, incorporation of tetracycline was similar between RBM-Ti (49.8%) and M-Y-TZP (35.9%) implants, but higher than M-Ti (28.2%) implants. Subsequently, alizarin and calcein were 1.1-fold higher in RBM-Ti implants than in the other implants. In the medullary region, no significant differences were observed for all fluorochromes. CONCLUSION: All implants favored bone formation and consequently promoted primary stability. Bone formation around the threads was faster in RBM-Ti and M-Y-TZP implants than in M-Ti implants, but limited bone remodeling with M-Y-TZP implants over time can have significant effects on secondary stability, suggesting caution for its use as an alternative substitute for titanium implants.

PARP inhibitors enhance replication stress and cause mitotic catastrophe in MYCN-dependent neuroblastoma.

High-risk and MYCN-amplified neuroblastomas are among the most aggressive pediatric tumors. Despite intense multimodality therapies, about 50% of these patients succumb to their disease, making the search for effective therapies an absolute priority. Due to the important functions of poly (ADP-ribose) polymerases, PARP inhibitors have entered the clinical settings for cancer treatment and are being exploited in a variety of preclinical studies and clinical trials. PARP inhibitors based combination schemes have also been tested in neuroblastoma preclinical models with encouraging results. However, the expression of PARP enzymes in human neuroblastoma and the biological consequences of their inhibition remained largely unexplored. Here, we show that high PARP1 and PARP2 expression is significantly associated with high-risk neuroblastoma cases and poor survival, highlighting its previously unrecognized prognostic value for human neuroblastoma. In vitro, PARP1 and 2 are abundant in MYCN amplified and MYCN-overexpressing cells. In this context, PARP inhibitors with high 'PARP trapping' potency, such as olaparib or talazoparib, yield DNA damage and cell death preceded by intense signs of replication stress. Notwithstanding the activation of a CHK1-CDC25A replication stress response, PARP-inhibited MYCN amplified and overexpressing cells fail to sustain a prolonged checkpoint and progress through mitosis in the presence of damaged DNA, eventually undergoing mitotic catastrophe. CHK1-targeted inhibition of the replication stress checkpoint exacerbated this phenotype. These data highlight a novel route for cell death induction by PARP inhibitors and support their introduction, together with CHK1 inhibitors, in therapeutic approaches for neuroblastomas with high MYC(N) activity.

Correlation between terminal restriction fragments and flow-FISH measures in samples over wide range telomere lengths.

OBJECTIVES: Terminal restriction fragment (TRF) analysis of human telomeres was used to calibrate flow-fluorescence in situ hybridization (FF) measures of telomere lengths to expand the range of measures and increase power of resolution of our previously published protocol. TRF data used as the gold standard should be obtained by electrophoresis with suitable resolution applied to appropriately isolated genomic DNA. When we considered TRF attained by correct methods, we found our method to be insufficiently accurate, thus we have reviewed our previously published FF protocol to obtain the best coefficient of determination (r(2)) between our experimental results and valid TRF lengths. MATERIALS AND METHODS: Using human telomere-specific PNA probe, Cy5-OO-(CCCTAA)3 , we measured telomere lengths of continuous cell line and of peripheral blood lymphocytes by FF. We modified hybridization, stringency, negative control handling, stoichiometric DNA staining and telomere fluorescence assessment of the protocol. RESULTS: We realized a procedure with increased power of resolution, improved TRF versus FF r(2) values that allowed simultaneous analysis of DNA and telomere duplication. Notwithstanding multiple steps in formamide sampling, recovery was satisfactory. DISCUSSION: The reviewed FF protocol appeared at least as suitable as the TRF method. Measures obtained by TRF can be affected by chromosome end variability, DNA fragmentation, incomplete digestion and unsuitable electrophoresis. In contrast, the FF technique analyses telomeric sequences confined to preserved nuclei thus overcome most previous limitations. As yet, however, the FF telomere measure cannot be performed together with immunophenotyping and/or generation study by the dye dilution method.

Improved procedure for the measurement of telomere length in whole cells by PNA probe and flow cytometry.

OBJECTIVES: 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. MATERIALS AND METHODS: After telomeric DNA denaturation in hot formamide solution and several washes to remove the ionic solvent, cells were hybridized overnight at room temperature with human telomere-specific PNA probe conjugated with Cy5 fluorochrome, Cy5-OO-(CCCTAA)(3) . After stringency washes and staining with ethidium bromide, the cells were analysed by flow cytometry and by using a confocal microscope. RESULTS: Using three continuous cell lines, different in DNA content and telomere length, and resting human peripheral blood T and B lymphocytes, we demonstrated that the oligo-PNA probe hybridized to telomeric sequences after complete removal of formamide and that in the preserved nucleus, telomeric sequence denaturation is irreversible. CONCLUSION: According to our experience, oligo-PNA binding results is efficient, specific and proportional to telomere length. These, our original findings, can form the technological basis of actual in situ hybridization on preserved whole cells.

A unified procedure for conservative (morphology) and integral (DNA and immunophenotype) cell staining for flow cytometry.

BACKGROUND: Current methods for multiparameter DNA flow cytometry suffer from several limitations. These include significant modifications of cell morphological parameters, the impossibility to counterstain cells with certain fluorochromes, and laborious tuning of the instrument that, for some procedures, must be equipped with an ultraviolet (UV) laser. To overcome these problems, we developed a novel method for the simultaneous analysis of morphological parameters, four-color immunophenotyping, and stoichiometric DNA labeling using a bench-top flow cytometer. METHODS: The method consists of a mild permeabilization/fixation treatment at room temperature, followed by labeling with fluorochrome-conjugated monoclonal antibodies (mAbs) and with the DNA dye 7-aminoactinomycin D (7-AAD) at 56 degrees C. RESULTS: Using this method, we analyzed resting peripheral blood mononucleated cells (PBMC), proliferating T cells cultured in the presence of interleukin-2 (IL-2), and lymphoblastoid B cells. Lymphocytes, monocytes, and lymphoblasts treated by this procedure retained differential light scattering (DLS) characteristics virtually identical to those of untreated cells. This allowed regions to be drawn on forward scatter (FSC) and side scatter (SSC) cytograms resolving different cell populations. DLS were preserved well enough to distinguish large lymphoblasts in the S or G2/M phases from small G0/G1 cells. Also, stainability with fluorescein-isothiocyanate (FITC), R-phycoerythrin (PE), allophycocyanin (APC)-conjugated mAbs was generally preserved. DNA labeling with 7-AAD was of quality good enough to permit accurate cell cycle analysis. CONCLUSIONS: The method described here, which we called integral hot staining (IHS), represents a very simple, reproducible, and conservative assay for multiparameter DNA analysis using a bench-top flow cytometer. Last but not least, the cytometer tuning for multiparameter acquisition is straightforward.

Peptide analogues as a strategy to induce tolerance in T cells with indirect allospecificity.

BACKGROUND: It has been demonstrated that indirect recognition of allogeneic MHC molecules might play an important role in provoking graft rejection. Although direct recognition of allogeneic molecules on antigen presenting cells of the graft may induce a state of tolerance, the continuous presentation of processed alloantigens by specialized antigen presenting cells does not allow the same phenomenon to occur. Tolerance to interleukin-2 secreting T cells can be achieved in different ways, among these is the exposure to mutants of the wild type allopeptide. We have investigated whether peptide analogues of the allopeptide can induce tolerance in T cells with indirect allospecificity. METHODS: T cell clones with indirect anti-HLA-A2-specificity generated from a HLA-A2-DRB1*1502+ patient who chronically rejected a HLA-A2-expressing kidney allograft were used for this study. Nine peptide analogues of HLA-A2 (residues: 103-120) were produced with single amino acid substitutions at the putative T cell receptor for antigen contact positions. Their effect on the proliferation of a panel of T cell clones was evaluated. RESULTS: Peptide analogues and wild type peptide had similar capacity to bind to the restriction molecule HLA-DRB1*1502. Co-presentation of the peptide analogues 111R/A, H, K and 114H/K, with the wild type peptide inhibited T cell responses, indicative of antagonism. In addition, one analogue 112G/S induced unresponsiveness in the T cells to subsequent culture with the wild type peptide. CONCLUSIONS: The data presented here suggest that using reagents such as altered peptides may represent a strategy to prevent the activation of T cells with indirect alloreactivity and allograft rejection in vivo.

Human T cells with a type-2 cytokine profile are resistant to apoptosis induced by primary activation: consequences for immunopathogenesis.

The mechanisms leading to a relative dominance of T cells producing type 2 cytokines in certain human immune disorders are still unclear. We investigated the relative susceptibility to apoptosis induced by primary in vitro activation of human type 1 (producing interferon-gamma (IFN-gamma)) or type 2 (producing IL-4) T cells. Peripheral blood lymphocytes were isolated from patients with immune disorders characterized by expansion of type 2 cells (four with AIDS and hyper-IgE/hypereosinophilia, one with Churg-Strauss syndrome, and one with idiopathic hypereosinophilic syndrome) or from individuals with normal cytokine balances. Cells were stimulated for 16 h with ionomycin and phorbol ester, and apoptosis of cytokine-producing cells was assessed by flow cytometry. T cells with a type-2 cytokine profile, i.e. producing IL-4 alone, were significantly more resistant to activation-induced apoptosis than those producing IFN-gamma alone. This was observed in AIDS patients, whose type 2 cells were mostly CD8+, as well as in the patients with Churg-Strauss and with hypereosinophilic syndrome. CD4+ and CD8+ IL-4-producing cells were equally resistant to apoptosis. Lower susceptibility to apoptosis of type-2 T cells was also observed in subjects with normal cytokine balances. Bcl-2 expression was high in type-2 cells and in viable type-1 cells, whereas it was low in apoptotic type-1 cells. Resistance to activation-induced apoptosis may explain the expansion of cells producing type-2 cytokines in certain immune disorders.

Hypervariable region 1 variants act as TCR antagonists for hepatitis C virus-specific CD4+ T cells.

In various human viral infections, the appearance of mutated epitopes displaying TCR antagonistic activity has been correlated with the severity and persistence of infection. In hepatitis C virus (HCV) infection, where the virus persistence has been associated with the rapid and substantial Ag modifications occurring during replication, TCR antagonism has been evidenced in CD8+ T cell responses. However, CD4+ T cell antagonism may be another important strategy by which HCV eludes a protective response, because sustained Th responses directed against several HCV Ags are associated with a self-limited course of infection. The data reported here represent the first evidence that variants of the hypervariable region (HVR1) of the putative Envelope 2 protein of HCV can act as powerful TCR antagonists for HVR1-specific CD4+ T cells isolated from HCV-infected individuals. Using classical antagonism assays, we observed strong inhibition of cellular proliferation and cytokine production when the agonist and the antagonist ligands were simultaneously presented by the same APCs. The presence in HVR1 of conserved residues, critical for binding to HLA-DR molecules, supports the function of HVR1 variants as TCR antagonists. In conclusion, our data evidence an antagonism phenomenon, which was achieved by naturally occurring class II-restricted T cell epitopes whose mechanism was addressed in terms of the antagonist capacity to inhibit agonist-mediated TCR down-regulation and early signal transduction.

Lymphocyte activation gene-3 (LAG-3) expression and IFN-gamma production are variably coregulated in different human T lymphocyte subpopulations.

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.

Death of bystander cells by a novel pathway involving early mitochondrial damage in human immunodeficiency virus-related lymphadenopathy.

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.

Comparison of the Vbeta repertoire in peripheral blood and in lymph nodes of HIV-infected subjects reveals skewed usage predominantly in CD8+ T cells.

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.

Measurement of apoptotic cells in peripheral blood.

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.

TCL1 oncogene activation in preleukemic T cells from a case of ataxia-telangiectasia.

The TCL1 oncogene on human chromosome 14q32.1 is involved in chromosome translocations [t(14;14)(q11;q32.1) and t(7;14)(q35;q32.1)] and inversions [inv14(q11;q32.1)] with TCR alpha/beta loci in T-cell leukemias, such as T-prolymphocytic (T-PLL). It is also involved in T-acute and -chronic leukemias arising in cases of ataxia-telangiectasia (AT), an immunodeficiency syndrome. Similar chromosomal rearrangements occur also in the clonally expanded T cells in AT patients before the appearance of the overt leukemia. We have analyzed the expression of TCL1 mRNA and protein in peripheral blood lymphocytes (PBLs) from four AT cases and from healthy controls. We found that the TCL1 gene was overexpressed in the PBLs of an AT patient with a large clonal T-cell population exhibiting the t(14;14) translocation but not in the lymphocytes of the other cases. Fluorescence in situ hybridization of the TCL1 genomic locus to lymphocyte metaphases from the AT patient with the T-cell clonal expansion showed that the breakpoint of the t(14;14) translocation lies within the TCL1 locus and is accompanied by an inverted duplication of the distal part of chromosome 14. These data indicate that TCL1 is activated in preleukemic clonal cells as a consequence of chromosome translocation involving sequences from the TCR locus at 14q11. Deregulation of TCL1 is the first event in the initiation of malignancy in these types of leukemias and represents a potential tool for clinical evaluation.

Frequency of provirus-bearing CD4+ cells in HIV type 1 infection correlates with extent of in vitro apoptosis of CD8+ but not of CD4+ cells.

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.

Ability of HIV to promote a TH1 to TH0 shift and to replicate preferentially in TH2 and TH0 cells.

Both interferon gamma (IFN-gamma) produced by T helper 1 (TH1) lymphocytes and interleukin-4 (IL-4) produced by TH2 lymphocytes were reduced in either bulk circulating mononuclear cells or mitogen-induced CD4+ T cell clones from the peripheral blood of individuals infected with human immunodeficiency virus (HIV). There was a preferential reduction in clones producing IL-4 and IL-5 in the advanced phases of infection. However, enhanced proportions of CD4+ T cell clones producing both TH1-type and TH2-type cytokines (TH0 clones) were generated from either skin-infiltrating T cells that had been activated in vivo or peripheral blood T cells stimulated by antigen in vitro when cells were isolated from HIV-infected individuals. All TH2 and most TH0 clones supported viral replication, although viral replication was not detected in any of the TH1 clones infected in vitro with HIV. These results suggest that HIV (i) does not induce a definite TH1 to TH2 switch, but can favor a shift to the TH0 phenotype in response to recall antigens, and (ii) preferentially replicates in CD4+ T cells producing TH2-type cytokines (TH2 and TH0).

Activated and "memory" phenotype of circulating T lymphocytes in intrauterine life.

We examined the expression of T cell markers in the peripheral blood of five immunologically normal human fetuses at 18-20 weeks of gestational age. The distribution of T cells expressing CD1, CD3, CD4, CD8, CD56, and the alpha/beta and gamma/delta receptors for antigen was comparable to that of newborns and normal adults, except for the absence of gamma/delta cells expressing the delta TCS-1 epitope. The V beta repertoire, as evaluated by two-color flow cytometry using mAbs to specific V beta families, was also comparable to that of adult samples. A significant fraction (8.9 to 16.4%) of fetal CD3+ T cells expressed the alpha chain of IL-2R (CD25) in the absence of HLA-DR; this suggests that antigenic stimuli trigger, during intrauterine life, an unusual pathway of T cell activation. Consistent with this, 7 to 27% of fetal T cells were found to express the CD45R0 marker of "memory" cells.

Detection and characterization of apoptotic peripheral blood lymphocytes in human immunodeficiency virus infection and cancer chemotherapy by a novel flow immunocytometric method.

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.