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A Cossarizza

Publications and source records attributed to A Cossarizza.

At least 19 recordsLinked to original sources
PubMed1998

Use of flow cytometry as a tool to study mitochondrial membrane potential in isolated, living hepatocytes.

The present paper describes the possibility of determination of mitochondrial membrane potential (Deltapsi) in isolated hepatocytes making use of a Deltapsi-sensitive dye, i.e., the lipophilic cationic probe 5,5',6,6'-tetrachloro-1,1',3, 3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) and of cytofluorimetry. The validity of the method was proved by treating hepatocytes with FCCP (decrease of Deltapsi) and subsequent addition of 6-ketocholestanol (increase of Deltapsi). The results indicate that the proposed method may be used in laboratory practice.

Animals↗
PubMed1997

Self gamma2a(b) protein is presented in vivo by gamma2a(b) B cells but not by dendritic cells.

We have previously shown that IgG2a(b) Ig does not induce tolerance in MHC class II restricted CD4 T cells in a TCR transgenic model and that anti-IgG2a(b) transgenic T cells specific for peptide 435-451 are indeed present in the periphery where they interact with gamma2a(b)-positive B cells. We also observed that because T cell tolerance depends on the presentation of self peptides, it was probable that IgG2a(b) was not easily processed and presented in vivo. In this study, we have investigated the presentation of naturally processed gamma2a(b) (435-451) determinants to specific T cells. Dendritic cells, macrophages, and B cells were purified from the spleens of Igh-1b mice. These cells were then functionally tested for the presence of specific peptide-MHC complexes. The results showed that, in vivo, gamma2a(b)-producing B cells, but not dendritic cells, are the only APCs able to present this self peptide. This indicates that recognition of the IgG2a(b)-self peptide is exclusively mediated by T-B cell interaction.

Animals↗
PubMed1997

JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis.

The sensitivity and specificity of three fluorescent probes used for cytofluorimetric analysis of mitochondrial membrane potential (delta psi) were studied in the U937 human cell line. First, the role of plasmamembrane in influencing the binding of the probes to mitochondria has been investigated. The depolarization of plasmamembrane with high doses of extracellular KCl had no immediate effects on the loading of JC-1, DiOC6(3) and rhodamine 123 (R123). However, after a few hours of culture in the presence of KCl, significant changes were observed only in cells stained with DiOC6(3). Second, a comparative study was performed concerning the effects of agents capable of collapsing deltapsi. While adding FCCP to cell cultures resulted in consistent changes in the fluorescence emission of both JC-1 and DiOC6(3) - but not of R123 - only cells stained with JC-1 responded to valinomycin. On the whole, our data indicate that JC-1 is a reliable probe for analyzing delta psi changes with flow cytometry, while the others show a lower sensitivity (R123), or a non-coherent behaviour, due to a high sensitivity to changes in plasmamembrane potential [DiOC6(3)]. These data cast some doubts on those studies that, using fluorescent probes that have a low sensitivity to delta psi, hypothesized that the fall in delta psi is one of the early events, if not one of the main causes, of apoptosis.

Apoptosis↗
PubMed1997

Resistance to apoptosis in Fanconi's anaemia. An ex vivo study in peripheral blood mononuclear cells.

Fanconi's anaemia (FA) is a rare autosomal recessive disease characterised by progressive pancytopoenia, a diverse assortment of congenital malformations, an increased sensitivity to reactive oxygen species and a predisposition to the development of malignancies. In the present study, we assessed the propensity to undergo apoptosis of peripheral blood mononuclear cells (PBMC) from Italian FA patients. Cells were challenged by 2-deoxy-D-ribose (dRib) or TNF-alpha plus cycloheximide as agents that induce apoptosis by interfering with cell redox status and mitochondrial membrane potential (MMP), and PBMC from FA patients resulted to be less prone to die than those from healthy subjects. The decreased susceptibility of FA cells to undergo apoptosis was also evident when another parameter highly correlated with the apoptotic process, i.e. MMP, was measured. Moreover, when N-acetylcysteine was added to dRib-treated PBMC, a strong protection was evident either in PBMC from control subjects or from FA patients. These data indicate that an alteration of unknown nature of the mechanisms favouring apoptosis is present in freshly collected cells from FA patients, and that such alteration could contribute to the pathogenesis of the disease, and particularly to the increased susceptibility to cancer.

Adolescent↗
PubMed1997

Cytometric analysis of immunosenescence.

We have been studying the immune system of healthy centenarians for many years, and they provide the best example of successful aging. They are people who have escaped major age-related diseases and reached the extreme limit of human life in good clinical condition. In most cases, histories of centenarians reveal them to be free of cancer, dementia, diabetes, cardiovascular diseases, and cataracts. Moreover, in order to reach such an advanced age, they should be equipped with well preserved and efficient immuno- and defense mechanisms, and optimal combinations of an appropriate lifestyle and genetic background. Using this approach, several paradoxes emerged as far as the immune system of centenarians is concerned, regarding: i) humoral immunity (increase in plasma immunoglobulins and nonorgan-specific autoantibodies, decrease in B cell number and lack of organ-specific autoantibodies); ii) cellular immunity (well preserved number of "virgin" T cells, a relatively intact T cell repertoire despite a thymus involuting since puberty, increased number of cells with markers of NK activity); iii) decreased peripheral blood lymphocyte tendency to programmed cell death, associated with a well preserved mitochondria functionality and intracellular bcl-2 levels. An age-related increase in the levels of adhesion molecule present on lymphocyte plasma-membrane, accompanied by a complex reshaping of the cytokine network, must be added to this scenario. All our data fit the hypothesis that a complex, unpredicted remodeling of the immune system occurs with age. In the present review it is underlined how flow cytometry has been used to study most of the above mentioned aspects of immunosenescence, and to establish new age-related reference values.

Adult↗
PubMed1997

Different pathways of apoptosis revealed by 2-chloro-adenosine and deoxy-D-ribose in mammalian astroglial cells.

Both the adenosine analogue 2-chloro-adenosine (2-CA) and the reducing sugar deoxy-D-ribose (dRib) induce apoptosis of astroglial cells in rat brain primary cultures (Abbracchio et al.: Biochem Biophys Res Commun 213:908-915, 1995). The present study was undertaken to elucidate by both morphological and cytofluorimetric analyses the intracellular mechanism(s) involved in induction of apoptosis by these two agents. The poly(ADP-ribose)polymerase (PARP) inhibitor 3-aminobenzamide did not prevent either 2-CA- or dRib-induced cell death, suggesting that activation of PARP is not critically important for induction of apoptosis in astrocytes. The radical scavenger N-acetyl-cysteine (NAC) strongly inhibited dRib- but not 2-CA-induced cell death, suggesting a differential role for radical formation in apoptosis by these two agents. A time-dependent increase of cells with depolarized mitochondria was observed in dRib-, and to a lesser extent, in 2-CA-treated cultures. NAC also prevented dRib- but not 2-CA-induced mitochondrial changes. We conclude that, in mammalian astrocytes, apoptosis can proceed through diverse and multiple pathways, depending upon the apoptotic stimulus. For dRib, apoptosis likely proceeds through generation of radicals and mitochondrial involvement. An adenosine extracellular receptor linked to an as yet unidentified signaling pathway may instead mediate 2-CA-induced cell death, which may have intriguing implications for both nervous system development and brain response to trauma and ischemia.

2-Chloroadenosine↗
PubMed1997

Relevance of clinical and laboratory findings in the diagnosis of cytomegalovirus encephalitis in patients with AIDS.

A retrospective evaluation was conducted in patients with AIDS and an autopsy diagnosis of cytomegalovirus (CMV) encephalitis to determine the relevance of clinical and laboratory findings in establishing a diagnosis. On autopsy of 100 patients, CMV encephalitis was diagnosed in 13 patients; eight had periventricular CMV encephalitis, four micronodular CMV encephalitis, and one both conditions. Seven patients had had a CMV infection previously (6 cases of retinitis, 1 case of colitis), and at the onset of encephalitis all of them were receiving a maintenance dose of ganciclovir. Examination of the CSF showed specific changes in patients with periventricular encephalitis. CT revealed no characteristic findings, while MRI showed an increased signal intensity on T2 weighted images. CMV DNA amplification by nested PCR was performed in nine patients with CMV encephalitis; PCR was positive in eight patients whose CSF was collected during CMV encephalitis, and negative in one patient whose CSF was collected six months before death. In conclusion, some clinical findings suggest a presumptive diagnosis, especially of periventricular encephalitis, and nested PCR appears to be a reliable and rapid technique for making an antemortem diagnosis.

AIDS-Related Opportunistic Infections↗
PubMed1997

T lymphocyte proliferative capability to defined stimuli and costimulatory CD28 pathway is not impaired in healthy centenarians.

It is generally assumed that T cell proliferation is impaired in aged individuals. We report data on the proliferative capability of peripheral blood mononuclear cells (PBMC) and T lymphocytes from 40 healthy people of different ages, (19-107 years), including 14 centenarians, to defined mitogenic stimuli. We observed no age-related proliferative impairment both in PBMC and in purified T cells stimulated by anti-CD3 mAb or phorbol myristate acetate (PMA). Furthermore, T cells stimulated by anti-CD3 mAb or PMA and costimulated by CD28 mAb did not proliferate differently among young, middle aged subjects and centenarians. Thus, short term T cell proliferation is not affected even at extreme age when well defined stimuli are used on cells deriving from carefully selected healthy subjects.

Adult↗
PubMed1997

Mitochondria alterations and dramatic tendency to undergo apoptosis in peripheral blood lymphocytes during acute HIV syndrome.

OBJECTIVE: To study alterations of mitochondrial membrane potential (delta psi) and the propensity to undergo apoptosis in peripheral blood lymphocytes (PBL) from subjects with acute HIV syndrome; and to evaluate possible modulations of these phenomena by antioxidants that can be used in therapy, such as N-acetyl-cysteine (NAC), nicotinamide (NAM), or L-acetyl-carnitine (LAC). METHODS: Mitochondrial function and the tendency of PBL to undergo spontaneous apoptosis were studied on freshly collected PBL from patients with symptomatic, acute HIV-1 primary infection, which were cultured for different durations in the presence of absence of NAC. NAM or LAC. By a cytofluorimetric method allowing analysis of delta psi in intact cells, we studied the function of these organelles under the different conditions. PBL apoptosis was evaluated by the classic cytofluorimetric method of propidium iodide staining, capable of revealing the typical DNA hypodiploid peak. RESULTS: Significant delta psi alterations and tendency to undergo apoptosis were present in PBL from the subjects we studied. Indeed, when cultured even for a few hours in the absence of any stimulus, a consistent number of cells died. However, the presence of even different levels of NAC, NAM or LAC was able to rescue most of them from apoptosis. Both a fall in delta psi and apoptosis were evident in PBL collected in the earliest phases of the syndrome (before seroconversion), and changed significantly after a few days. A significant correlation was found between spontaneous apoptosis and tumour necrosis factor (TNF)-alpha or p24 plasma levels, as well as between apoptosis and the percentages of circulating CD4+ or CD8+ T cells. CONCLUSIONS: PBL from patients with acute HIV syndrome are characterized by both significant mitochondrial alterations and a dramatic tendency to undergo apoptosis. The use of NAC, NAM or LAC seems to rescue cells through a protective effect on mitochondria, a well-known target for the action of TNF-alpha and for reactive oxygen species, the production of which is strongly induced by this cytokine. Thus, our data could provide the rationale for the use of such agents in addition to antiviral drugs in primary infection.

Acetylcarnitine↗
PubMed1996

Mitochondria are selective targets for the protective effects of heat shock against oxidative injury.

Heat shock (HS) proteins (HSPs) induce protection against a number of stresses distinct from HS, including reactive oxygen species. In the human premonocytic line U937, we investigated in whole cells the effects of preexposure to HS and exposure to hydrogen peroxide (H2O2) on mitochondrial membrane potential, mass, and ultrastructure. HS prevented H2O2-induced alterations in mitochondrial membrane potential and cristae formation while increasing expression of HSPs and the protein product of bcl-2. Protection correlated best with the expression of the 70-kDa HSP, hsp70. We propose that mitochondria represent a selective target for HS-mediated protection against oxidative injury.

Apoptosis↗
PubMed1996

Earthworm leukocytes that are not phagocytic and cross-react with several human epitopes can kill human tumor cell lines.

Earthworm coelomocytes (leukocytes) effect cytotoxicity at significantly high levels against the NK-sensitive, human tumor cell line, K562, and the NK-resistant targets (U937, BSM, CEM). By cytofluorimetric analyses using mouse anti-human monoclonal antibodies and by morphological evaluations, two types of coelomocytes were identified: (1) small (8-11 micron) electron-dense cells (SC): CD11a+, CD45RA+, CD45RO+, CDw49b+, CD54+, beta 2-m+ and Thy-1+; (2) large (12-15 micron) electron-lucent cells (LC) that are negative for these markers. Both cell types were negative for other CD and MHC class I and class II markers. SC were active during recognition, rapidly binding to targets; LC were phagocytic. Release of 51Cr revealed rapid, significant, and equal levels of killing at 4 degrees, 20 degrees, and 37 degrees C. We propose that primitive NK-like activity appeared early in evolution.

Animals↗
PubMed1996

CD45 isoforms expression on CD4+ and CD8+ T cells throughout life, from newborns to centenarians: implications for T cell memory.

CD4+ and CD8+ peripheral blood T lymphocytes show mutually exclusive expression of CD45RA or CD45R0, two isoforms of the common leukocyte antigen that seem to recognize so-called virgin/unprimed and memory/activated T cells. The expression of these isoforms has been studied by three colour cytofluorimetric analysis on CD4+ or CD8+ peripheral blood CD3+ cells from 22 healthy centenarians, analyzed in a context of 202 healthy donors 0-110 years old. An age-related unbalance of virgin and memory cells was found between CD4+ and CD8+ subsets. As expected, at birgh 95-99% of the CD3+ lymphocytes expressed the CD45RA isoform. A rapid increase of CD45R0+ cells was observed in the first 2-3 decades of life, this phenomenon being much more pronounced on CD4+ cells. Subsequently, the increase of the 'memory' compartment was much less rapid, so that in centenarians a consistent reservoire of CD45RA+ among CD4+ cells was still present (about 20%). In these exceptional individuals the percentage of CD45RA+ cells among CD8+ T lymphocytes was even higher (about 50%), and only slightly lower than that of young donors (about 55-60%). Thus, the main changes occurred at a different rate in CD4+ (about 20%). In these exceptional individuals the percentage of CD45RA+ cells among CD8+ T lymphocytes was even higher (about 50%), and only slightly lower than that of young donors (about 55-60%). Thus, the main changes occurred at a different rate in CD4+ and in CD8+ T cells, at an age of between 0 and 30 years, when the thymus is still functionally active. Interestingly, no difference in the usage of CD45 isoforms was observed within T cells bearing four different V beta-T cell receptor (TCR). The significance of this age-related unbalance is unknown. However, the presence of a great number of CD45RA+ T lymphocytes within the CD4+ and the CD8+ T cell subsets even in the peripheral blood of centenarians poses the problem of their origin (thymus? extrathymic sites?), of their functional role and of their lifespan. Moreover, the data on centenarians suggest that they may represent a very selected population where a slowing of immunosenescence occurs.

Adolescent↗
PubMed1996

Functional heterogeneity of an isolated mitochondrial population revealed by cytofluorometric analysis at the single organelle level.

Isolated rat liver mitochondria were incubated under various metabolic conditions to determine their membrane potential (MMP) as measured continuously by a tetraphenylphosphonium (TPP+)-selective electrode. By flow cytometry, a parallel analysis of fluorescence emissions observing single mitochondria stained with the lipophilic cation 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) revealed linear correlation between the median orange fluorescence (FL2) due to J-aggregate formations and MMP values measured by TPP+. No correlation was detected with the green fluorescence (FL1) emission. A significantly higher correlation appeared between the FL2/FL1 ratio and MMP values. Within the same mitochondrial population, cytofluorimetric analysis revealed the presence of various classes of organelles with different MMP, whose distribution was dependent on metabolic condition. The highest functional heterogeneity was found in deenergized mitochondria, while the highest homogeneity was observed during the first phase of the phosphorylative process. Thus, these data suggest that the cytofluorimetric use of JC-1 provides direct experimental evidence for the hypothesis of functional mitochondrial heterogeneity, at least with respect to their membrane potential.

Adenosine Diphosphate↗
PubMed1996

Control of apoptosis by the cellular ATP level.

Apoptosis is a physiological form of cell death. Its causes and execution mechanisms are not clearly understood. Oxidative stress, nitric oxide and its congeners, Ca2+, proteases, nucleases, and mitochondria are considered mediators of apoptosis. At present their importance and exact role are elusive but it is clear that mitochondria are both the target and the source of oxidative stress, nitric oxide, and Ca2+. The mitochondrial membrane potential (delta psi), which is the driving force for mitochondrial ATP synthesis, declines during apoptosis, and maintenance of delta psi prevents apoptosis. Since apoptosis is highly regulated and involves the activity of hydrolytic enzymes, chromatin condensation and vesicle formation apoptosis is likely to have a high energy demand. We propose that the cellular ATP level is an important determinant for cell death. This hypothesis is supported by circumstantial evidence, is consistent with the available data, has a corrolary in aging, and is amenable to direct experimental testing particularly with flow cytometry as a promising tool.

Adenosine Triphosphate↗
PubMed1996

A shift to Th0 cytokine production by CD4+ cells in human longevity: studies on two healthy centenarians.

Centenarians, particularly healthy centenarians, constitute the example of successful aging and the study of their immune status can help to define the endpoint of the changes occurring throughout life. We characterized T cell clones (TCC) of two healthy centenarians, studying their phenotypes and production of representative Th1 and Th2 cytokines (IFN-gamma and IL-4) and compared them with TCC obtained by three young normal subjects; in all 180TCC were analyzed. In young donors, 35TCC were CD4+, 56CD8+ and 2 were alpha beta +CD4-CD8- (double negative). In centenarians, we obtained 46CD4+TCC, 38CD8+, 2CD4+CD8+ (double positive) and 1 gamma delta + double negative. Of the young subjects' TCC, 71% produced IFN-gamma but no IL-4 (Th1 pattern) and this prevalence decreased to 39% in TCC from the centenarians. The number of clones showing the opposite Th2 pattern was similar in young and aged donors (3 out of 93TCC and 2 out of 87TCC, respectively). The intermediate profile of TCC producing both IL-4 and IFN-gamma (Th0) was found in 25.8% of clones from young people, but it almost doubled to 58.6% in centenarians. The analysis shows that the Th profiles of CD8+TCC is nearly superimposable in the two groups, whereas a major shift from a Th1 to a Th0 pattern is presented by CD4+TCC. The balance provided by a majority of CD4+TCC showing a Th0 pattern may ensure both humoral and cell-mediated defences. In CD8+TCC, however, a Th1 pattern still is present, possibly for efficient generation of cytotoxic responses. These findings should be extended by studying other centenarians and elderly subjects.

Aged↗
PubMed1996

Stress proteins in inflammation.

Inflammation provides those searching in the field with a number of "models" allowing them to study, in vivo, in humans and in animals, the regulation and the functions of HSP, which are being considered as a new and promising marker for the severity and the prognosis of inflammatory diseases. HSP are differentially regulated according to the type of inflammation, whether acute or chronic, whether self-limiting (inflammatory cell elimination by apoptosis) or self-perpetuating (inflammatory cell death by necrosis). We propose that mitochondria are a key organelle in determining the outcome of inflammation, because they are both the cellular "switchboard" for apoptosis and a selective target for the protective effects of HSP against the cytotoxic effects of TNF alpha and ROS. On the other hand, HSP exert multiple protective effects in inflammation, including self/non-self discrimination, enhancement of immune responses, immune protection, thermotolerance and protection against the cytotoxicity of inflammatory mediators. The latter protective effects against the deleterious effects of the mediators of inflammation, including ROS and cytokines, open new avenues for the development of original anti-inflammatory therapies, such as non-toxic inducers of a complete HS response. It may well be that the "beneficial effects of fever" already described by Hippocrates actually relate to increased HSP expression during fever, and to their protective effects....

Animals↗
PubMed1996

Prolactin increases the susceptibility of primary leukemia cells to NK and LAK effectors.

Our previous studies have shown that prolactin (PRL), a pituitary and lymphocyte hormone and a ligand of the cytokine/hemopoietin receptors (R) superfamily, acts synergistically with interleukin (IL)-2 on the development of lymphokine activated killer (LAK) cells and enhances the effects of GM-CSF and IL-3 on myeloid progenitors' proliferation and differentiation. More recently, we have demonstrated that GM-CSF and IL-3 increase the sensitivity of acute myeloid leukemic (AML) cells to LAK activity. Together, these findings have prompted us to study the role of PRL on the target arm of the LAK response. We show here that CD33+ blasts from AML patients express membrane PRL-R and that the PRL/PRL-R interaction is followed by increased susceptibility to natural killer (NK) (p < 0.02) and LAK (p < 0.001) cells. As predicted from the dimerization model of PRL-R and in agreement with previous reports, the response of AML blasts to PRL was bell-shaped with a trend peak at 25 ng/ml. Although enhanced lysis occurred at the target recognition level, it was not accompanied by changes in the MHC class I, cellular adhesion molecules, or myeloid differentiation antigens. Cell cycle recruitment and lysis increased concurrently in three cases studied, suggesting a modulatory action of PRL on the expression of putative cycle-related NK/LAK-target structures. Together, these data strengthen the role of PRL in the LAK response.

Adjuvants, Immunologic↗