Low Levels Of Apoptosis Research Articles

Assessing the Role of Neutrophil Apoptosis in the Resolution of Particle-Induced Pulmonary Inflammation

Following inflammatory-cell recruitment in the lung, neutrophil apoptosis and subsequent engulfment by macrophages are regarded as important components in the resolution of pulmonary inflammation. The goal of this study was to further investigate the role of apoptosis and its influence, if any, on the pulmonary inflammatory process following exposures to the following particulate types: amorphous (AMO) or crystalline silica (Si), lipopolysaccharide (LPS), or pigment-grade titanium dioxide (TiO 2) . Rats were intratracheally instilled either with TiO 2, AMO, or Si particles at doses of 1 or 5 mg/kg or 6 µg LPS. Following exposures, bronchoalveolar lavage fluids and lung tissues were collected and evaluated at 12, 24, 48, or 168 h (i.e., 1 wk). At the 1 mg/kg dose, AMO instillation produced the highest pulmonary inflammatory response, concomitant with a rise in apoptotic cells that mirrored temporally the transient nature of the inflammatory response. At 5 mg/kg, amorphous silica and crystalline silica particles induced substantial pulmonary inflammation [~50-60% neutrophils (PMNs)] at 12 h postexposure (pe). A fundamental difference between the two inflammatory patterns, however, was the subsequent reversibility of inflammation in the AMO-exposed rats at 168 h postexposure and the sustained inflammatory effect in the Si-exposed animals measured through 168 h pe (~40% PMNs). Pulmonary apoptotic responses in AMO-exposed rats mirrored temporally and correlated with the time-course reduction of inflammatory responses, leading to resolution. In the Si-exposed rats, apoptotic levels remained elevated, concomitant with sustained inflammation measured through 168 h pe. High doses of TiO 2 particles produced transient lung inflammation, but with low levels of apoptosis. In addition, instillation of LPS produced a transient inflammatory response which mirrored the time course of apoptosis levels and was resolved by 168 h pe. cDNA microarray methods demonstrated that gene expression was altered for several apoptosis-related genes in AMO-, Si-, and LPS-exposed animals at 24 h pe. The results of these studies demonstrate that, following exposures, the resolution of lung inflammation correlated temporally with apoptotic levels of neutrophils in AMO- and LPS-exposed rats. Alternatively, instillation of crystalline silica resulted in sustained pulmonary inflammation and measurable apoptosis at 1 wk postexposure, but the apoptotic cell processes were not effective in resolving the inflammatory response. The findings suggest that the coordination between the resolution of inflammation and inflammatory cell apoptosis in the lung is dependent on the particle-type and that other factors, such as particle cytotoxicity, may also be important.

Cardiomyocyte apoptosis and ischemic preconditioning in open heart operations

Background The aim of the present study was to ascertain the percentage of left apical myocardial apoptosis in three-vessel coronary artery bypass grafting patients quantitatively and the impact of ischemic preconditioning. Methods Twenty-one patients with three-vessel disease who had elective coronary artery bypass grafting were randomized in a ratio of 2:1 to ischemic preconditioning (n = 14) or a control group (n = 7). The ischemic preconditioning protocol was established by two cycles of ascending aorta occlusion for 2 minutes followed by 3 minutes of reperfusion. Myocardial samples from the apex of the left ventricle were taken using a Tru-Cut needle before aortic cross-clamping and immediately after declamping. The percentage of apoptosis was analyzed by TUNEL methods. Data on hemodynamics and biochemical markers were collected. Results Low levels of myocardial apoptosis were found before the operation (0.01% ± 0.00%). During the early reperfusion period, the percentage of myocardial apoptotic cells significantly increased (0.15% ± 0.05%, p = 0.008). Ischemic preconditioning significantly improved cardiac index and right ventricular ejection fraction recovery after the operation ( p = 0.036 and 0.001 respectively, repeated measure) but had no effect on myocardial apoptosis before and after the operation (0.01 ± 0.00 versus 0.01 ± 0.00, p = 0.658 and 0.12% ± 0.04% versus 0.23% ± 0.14%, p = 0.302). Conclusions Cardioplegic myocardial ischemia during open heart operation was associated with induction of cardiomyocyte apoptosis in humans. Attenuation of postoperative cardiac dysfunction by ischemic preconditioning appeared to be independent of apoptosis.

Plasticity and Cytokinetic Dynamics of the Hair Follicle Mesenchyme: Implications for Hair Growth Control

The continuously remodeled hair follicle is a uniquely exploitable epithelial-mesenchymal interaction system. In contrast to the cyclical fate of the hair follicle epithelium, the dynamics of the supposedly stable hair follicle mesenchyme remains enigmatic. Here we address this issue using the C57BL/6 hair research model. During hair growth, increase in total follicular papilla size was associated with doubling of papilla cell numbers, much of which occurred before intra-follicular papilla cell proliferation, and subsequent to mitosis in the proximal connective tissue sheath. This indicates that some papilla cells originate in, and migrate from, the proliferating pool of connective tissue sheath fibroblasts. Follicular papilla cell number and total papilla size were maximal by anagen VI, but intriguingly, decreased by 25% during this period of sustained hair production. This cell loss, which continued during catagen, was not associated with intra-follicular papilla apoptosis, strongly indicating that fibroblasts migrate out of the late anagen/early catagen papilla and re-enter the proximal connective tissue sheath. Low-level apoptosis occurred only here, along with the "detachment" of cells from the regressing connective tissue sheath. Thus, the hair follicle mesenchyme exhibits significant hair cycle-associated plasticity. Modulation of these cell interchanges is likely to be important during clinically important hair follicle transformations, e.g. vellus-to-terminal and terminal-to-vellus during androgenetic alopecia.

A mechanistic role for cardiac myocyte apoptosis in heart failure.

Heart failure is a common, lethal condition whose pathogenesis is poorly understood. Recent studies have identified low levels of myocyte apoptosis (80-250 myocytes per 10(5) nuclei) in failing human hearts. It remains unclear, however, whether this cell death is a coincidental finding, a protective process, or a causal component in pathogenesis. Using transgenic mice that express a conditionally active caspase exclusively in the myocardium, we demonstrate that very low levels of myocyte apoptosis (23 myocytes per 10(5) nuclei, compared with 1.5 myocytes per 10(5) nuclei in controls) are sufficient to cause a lethal, dilated cardiomyopathy. Interestingly, these levels are four- to tenfold lower than those observed in failing human hearts. Conversely, inhibition of cardiac myocyte death in this murine model largely prevents the development of cardiac dilation and contractile dysfunction, the hallmarks of heart failure. To our knowledge, these data provide the first direct evidence that myocyte apoptosis may be a causal mechanism of heart failure, and they suggest that inhibition of this cell death process may constitute the basis for novel therapies.

A mechanistic role for cardiac myocyte apoptosis in heart failure

Heart failure is a common, lethal condition whose pathogenesis is poorly understood. Recent studies have identified low levels of myocyte apoptosis (80–250 myocytes per 105 nuclei) in failing human hearts. It remains unclear, however, whether this cell death is a coincidental finding, a protective process, or a causal component in pathogenesis. Using transgenic mice that express a conditionally active caspase exclusively in the myocardium, we demonstrate that very low levels of myocyte apoptosis (23 myocytes per 105 nuclei, compared with 1.5 myocytes per 105 nuclei in controls) are sufficient to cause a lethal, dilated cardiomyopathy. Interestingly, these levels are four- to tenfold lower than those observed in failing human hearts. Conversely, inhibition of cardiac myocyte death in this murine model largely prevents the development of cardiac dilation and contractile dysfunction, the hallmarks of heart failure. To our knowledge, these data provide the first direct evidence that myocyte apoptosis may be a causal mechanism of heart failure, and they suggest that inhibition of this cell death process may constitute the basis for novel therapies.

Decreased neutrophil apoptosis in tracheal fluids of preterm infants at risk of chronic lung disease

Objective: To investigate the hypothesis that preterm infants who are more susceptible to lung damage have decreased neutrophil apoptosis, and to explore its relation to interleukin 10 (IL10) concentration. Design:...

Prognostic use of growth characteristics of early gastric cancer and expression patterns of apoptotic, cell proliferation, and cell adhesion proteins.

Selection of suitable treatment for early gastric cancers, such as endoscopic mucosal resection or the major surgical option of resection of the cancer together with a radical lymph node dissection, may be assisted by comparing the growth characteristics of the cancer with selected molecular characteristics. The results could be used to predict those cases that have a higher risk of developing secondary metastases. A total of 1,196 Japanese patients with early gastric cancers (648 mucosal cancers and 548 submucosal) were included in the selection of two groups: a metastatic group made up 57 cancers with lymph node metastasis (9 mucosal, 48 submucosal), and a nonmetastatic group of 61 cases (6 mucosal, 55 submucosal) without lymph node metastasis. Growth characteristics of the cancers (superficially spreading, penetrating or invasive, lymph node metastasis) were compared with immunohistochemical expression of single-stranded DNA (ssDNA) protein (apoptosis indicator), bcl-2 and p53 (apoptosis-associated), Ki-67 (cell proliferation), and E-cadherin (cell adhesion) proteins. The lesions in the nonmetastatic group had higher levels of apoptosis and lower expression of bcl-2 than in the metastatic group, indicating an inhibitory role for apoptosis in malignant progression. Apoptosis was also higher in the superficial compared with the invasive lesions of both groups. The lesions in the metastatic group had higher p53 expression than that of the nonmetastatic group, whereas apoptosis in the metastatic group was lower than in the nonmetastatic group. An unproved explanation for this finding may be that, although increased, p53 was mutated and ineffective in promoting apoptotic control of metastatic progression. E-cadherin was decreased in the invasive lesions of both groups, indicating a greater ability of these cells to lose adhesion, to invade the submucosa, and to metastasize. Cell proliferation was highest in the superficial lesions of both metastatic and nonmetastatic groups. Early gastric cancers with low levels of apoptosis, increased bcl-2, and high levels of p53 expression are more likely to invade and metastasize.

Assessing the Role of Neutrophil Apoptosis in the Resolution of Particle-Induced Pulmonary Inflammation

Following inflammatory-cell recruitment in the lung, neutrophil apoptosis and subsequent engulfment by macrophages are regarded as important components in the resolution of pulmonary inflammation. The goal of this study was to further investigate the role of apoptosis and its influence, if any, on the pulmonary inflammatory process following exposures to the following particulate types: amorphous (AMO) or crystalline silica (Si), lipopolysaccharide (LPS), or pigment-grade titanium dioxide (TiO 2) . Rats were intratracheally instilled either with TiO 2, AMO, or Si particles at doses of 1 or 5 mg/kg or 6 µg LPS. Following exposures, bronchoalveolar lavage fluids and lung tissues were collected and evaluated at 12, 24, 48, or 168 h (i.e., 1 wk). At the 1 mg/kg dose, AMO instillation produced the highest pulmonary inflammatory response, concomitant with a rise in apoptotic cells that mirrored temporally the transient nature of the inflammatory response. At 5 mg/kg, amorphous silica and crystalline silica particles induced substantial pulmonary inflammation [~50-60% neutrophils (PMNs)] at 12 h postexposure (pe). A fundamental difference between the two inflammatory patterns, however, was the subsequent reversibility of inflammation in the AMO-exposed rats at 168 h postexposure and the sustained inflammatory effect in the Si-exposed animals measured through 168 h pe (~40% PMNs). Pulmonary apoptotic responses in AMO-exposed rats mirrored temporally and correlated with the time-course reduction of inflammatory responses, leading to resolution. In the Si-exposed rats, apoptotic levels remained elevated, concomitant with sustained inflammation measured through 168 h pe. High doses of TiO 2 particles produced transient lung inflammation, but with low levels of apoptosis. In addition, instillation of LPS produced a transient inflammatory response which mirrored the time course of apoptosis levels and was resolved by 168 h pe. cDNA microarray methods demonstrated that gene expression was altered for several apoptosis-related genes in AMO-, Si-, and LPS-exposed animals at 24 h pe. The results of these studies demonstrate that, following exposures, the resolution of lung inflammation correlated temporally with apoptotic levels of neutrophils in AMO- and LPS-exposed rats. Alternatively, instillation of crystalline silica resulted in sustained pulmonary inflammation and measurable apoptosis at 1 wk postexposure, but the apoptotic cell processes were not effective in resolving the inflammatory response. The findings suggest that the coordination between the resolution of inflammation and inflammatory cell apoptosis in the lung is dependent on the particle-type and that other factors, such as particle cytotoxicity, may also be important.

Mechanisms of cytotoxicity in human ovarian carcinoma cells exposed to free Mce6 or HPMA copolymer-Mce6 conjugates.

It is essential to understand cellular responses on photodynamic therapy (PDT) to design delivery systems that maximize cytotoxic effects coupled with minimal induction of side effects or protective mechanisms (or both). Here, we investigated mechanisms of toxicity in human ovarian carcinoma A2780 cells treated with structurally diverse N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (P)-mesochlorin e6 monoethylenediamine (Mce6) conjugates that possessed differential subcellular accumulation or covalent attachments of photosensitizers (or both). Apoptosis and necrosis were observed after photoactivation, with increased apoptotic responses observed in cells exposed to conjugates possessing Mce6 linkage via a lysosomally degradable tetrapeptide spacer (HPMA copolymer-Mce6 conjugates containing Mce6 bound via glycylphenylalanylleucylglycine [GFLG] linker [P-GFLG-Mce6], HPMA copolymer-Mce6 conjugates containing Mce6 bound via a GFLG spacer and containing nuclear localization sequence, PKKKRKV132K(FITC)C [NLS(fluorescein-5-isothiocyanate [FITC])] bound via a thioether linkage [P-NLS(FITC)-GFLG-Mce6]). Furthermore, the induction of necrosis was more pronounced in cells exposed to conjugates containing both a nuclear localization sequence (NLS) and Mce6 bound by a degradable linker (P-NLS(FITC)-GFLG-Mce6). Caspase-independent mechanisms of cell death were identified in cells treated with nuclear-targeted conjugates possessing Mce6 attached using a nondegradable tether (HPMA copolymer-Mce6 conjugates containing Mce6 bound via a GG spacer and containing NLS(FITC) bound via a thioether linkage [P-NLS(FITC)-GG-Mce6]), whereas low levels of apoptosis and necrosis were detected in cells exposed to photoactivated nontargeted HPMA copolymer-Mce6 conjugates containing Mce6 coupled through a nondegradable spacer (HPMA copolymer-Mce6 conjugates containing Mce6 bound via GG linker [P-GG-Mce6]). Variations in gene expression were observed in cells on PDT. Specifically, HSP70 expression was solely detected in cells treated with P-GFLG-Mce6, whereas the loss of detection of several genes were observed in cells treated with P-NLS(FITC)-GFLG-Mce6. Variations in cellular responses on PDT using different HPMA copolymer-Mce6 conjugates will prove useful in the design of optimal HPMA copolymer PDT delivery systems.

Preexisting Antiadenoviral Immunity and Regional Myocardial Gene Transfer: Modulation by Nitric Oxide

The utility of adenoviral vectors, currently used in cardiovascular gene transfer protocols, is limited by the brevity of transgene expression and by antiadenoviral immune responses. The effect of preexisting antiadenoviral immunity on intracardiac gene transfer or its modulation by nitric oxide is unknown. Adenoviral vectors, expressing the firefly luciferase gene (AdLuc) or the human nitric oxide synthase 3 (NOS3) gene (AdNOS3), were infused into the great cardiac vein of naive pigs or immunized pigs. Pigs were immunized by intravenous injection of control virus AdRR5 and the resulting neutralizing antibody titers (median, 1:178; p < 0.0001 vs. baseline) were similar to preexisting titers in 54% of randomly selected coronary artery bypass graft patients. In naive animals distribution of transgene expression in the left ventricular free wall was focal. In immunized pigs myocardial luciferase expression 3 days after AdLuc gene transfer was more than 1000-fold lower than in naive pigs, whereas no change in NOS3 transcript levels was detected after AdNOS3 gene transfer. Severe, grade III-IV mononuclear cell infiltration and myocyte apoptosis were observed in four of five AdLuc-infected, immunized animals, compared with low-level inflammation and apoptosis in five of six AdNOS3-infected pigs. Coinfusion of AdLuc and AdNOS3 in immunized pigs resulted in spatially colocalized transgene expression, reduced T cell-mediated inflammation, and myocyte apoptosis and was associated with 200-fold greater median reporter transgene expression levels in the subendocardium (1.0 x 10(3) light units [LU]/mg protein, n = 8, vs. 4.5 x 10(1) LU/mg protein in AdLuc- and AdRR5-coinfected pigs, n = 7, p = 0.02). Preexisting antiadenoviral immunity abrogates myocardial gene expression in pigs and is associated with severe inflammation and myocyte apoptosis. Intracardiac NOS3 gene transfer may reduce these barriers to adenovirus-mediated myocardial gene transfer.

Transgenic expression in mouse lung reveals distinct biological roles for the adenovirus type 5 E1A 243- and 289-amino-acid proteins.

Little is known about the biological significance of human adenovirus type 5 (Ad5) E1A in vivo. However, Ad5 E1A is well defined in vitro and can be detected frequently in the lungs of patients with pulmonary disease. Transgenic expression of the Ad5 E1A gene targeted to the mouse lung reveals distinct biological effects caused by two Ad5 E1A products. Either of two Ad5 E1A proteins was preferentially expressed in vivo in the transgenic lungs. The preferential expression of the Ad5 E1A 243-amino-acid (aa) protein at a moderate level was associated with cellular hyperplasia, nodular lesions of proliferating lymphocyte-like cells, and a low level of p53-dependent apoptosis in the lungs of transgenic mice. In contrast, the preferential expression of the Ad5 E1A 289-aa protein at a moderate level resulted in a proapoptotic injury and an acute pulmonary proinflammation in the lungs of transgenic mice, mediated by multiple apoptotic pathways, as well as an enhancement of the host immune cell response. Expression of the Ad5 E1A 243-aa protein resulted in proliferation-stimulated p53 upregulation, while expression of the Ad5 E1A 289-aa protein led to DNA damage-induced p53 activation. These data suggest that the Ad5 E1A 243- and 289-aa proteins lead to distinct biological roles in vivo.

Phenotype and regulation of persistent intracerebral T cells in murine Toxoplasma encephalitis.

Toxoplasma gondii is a parasite causing asymptomatic, persistent encephalitis. Protective CD4 and CD8 T cells are recruited to and accumulate in the brain in acute Toxoplasma encephalitis (TE), with slowly decreasing numbers in chronic TE. It is unclear how the size of the intracerebral T cell pool is regulated. Conceivably, permanent recruitment, proliferation, and apoptosis may be involved. We observed that in murine TE recruitment of T cells to the brain was terminated in chronic TE. In vivo 5-bromo-2'-deoxyuridine incorporation and in vitro T cell proliferation experiments revealed that intracerebral T cells did not proliferate, which was explained by the expression of the cell cycle inhibitors p21(Waf/cip1) and p27(Kip1) and the inhibitory activity of intracerebral F4/80(+) cells. TUNEL staining detected apoptotic T cells at low frequency corresponding to an increased expression of the anti-apoptotic molecules Bcl-2 and Bcl-x(L) and a reduced expression of the pro-apoptotic molecules Bad, Bax, and Fas ligand in CD4 and CD8 T cells. During progression from acute to chronic TE, both CD4 and CD8 T cells down-regulated CD45RB expression and expressed a differential pattern of cytokines. From these experiments it is concluded that the number of intracerebral T cells increases by recruitment of T cells during acute infection, whereas proliferation of intracerebral T cells does not play a role. In chronic TE, T cell recruitment is terminated, the phenotype of intracerebral T cells changes, and their number is gradually downsized by low level apoptosis, which, however, does not completely resolve the T cell infiltrates.

Differential regulation of spontaneous and immune complex‐induced neutrophil apoptosis by proinflammatory cytokines. Role of oxidants, Bax and caspase‐3

Neutrophil apoptosis represents a crucial step in the mechanisms governing the resolution of neutrophilic inflammation. Several soluble mediators of inflammation modulate neutrophil survival, retarding their apoptosis, whereas neutrophil activation by immune complexes (IC) results in the acceleration of apoptosis. To investigate neutrophil fate at the site of inflammation, we studied the effects of interleukin (IL)-2, IL-6, IL-8, IL-15, GM-CSF, and fMLP on spontaneous and IC-induced neutrophil apoptosis and the mechanisms regulating the survival of these cells. Spontaneous apoptosis was inhibited by GM-CSF, IL-6, and IL-15, but only GM-CSF overturned IC-induced apoptosis. No role of oxidants on the modulation of IC-dependent apoptosis was found. Indeed, fMLP or GM-CSF augmented the IC-dependent oxidative response, whereas the other compounds were ineffective. CGD neutrophils showed low levels of spontaneous apoptosis, but when exposed to IC, underwent a sharp increment of the apoptotic rate in a GM-CSF-inhibitable manner. Conversely, the expression of the proapoptotic protein Bax in 18-h aged neutrophils was down-regulated by GM-CSF, IL-6, and IL-15. Furthermore, IC induced a nearly threefold Bax up-regulation, which was completely reversed only by GM-CSF. Accordingly, the spontaneous activity of caspase-3 was inhibited by GM-CSF, IL-6, and IL-15. Furthermore, IC induced a sharp increment of enzymatic activity, and only GM-CSF inhibited the IC-dependent acceleration. Our results show that apoptosis of resting and IC-activated neutrophils is regulated differently, GM-CSF being the most potent neutrophil antiapoptotic factor. The results also unveil the existence of an oxidant-independent, Bax- and caspase-3-dependent, intracellular pathway regulating neutrophil apoptosis.

Low levels of apoptosis and high FLIP expression in early rheumatoid arthritis synovium

Low levels of apoptosis and high FLIP expression in early rheumatoid arthritis synovium

Apoptosis and Expression of Bcl-2, Bcl-XL, and Bax in Renal Cell Carcinomas

There are at present disparate published results with regard to the relevance of the Bcl-2 gene family, levels of apoptosis, and cell proliferation in the development and progression of renal cell carcinoma (RCC). The present study analyses the inter-relationship between the expression of representatives of the anti-apoptotic (Bcl-2, Bcl-XL) or pro-apoptotic (Bax) Bcl-2 proteins, incidence of apoptosis, and mitosis in a selected small group of 22 graded RCCs that had paired normal renal tissue, or non-neoplastic tissue in the renal biopsy specimen. The cases were chosen to determine the feasibility of measuring these parameters as potential surrogate markers of progression or treatment failure of the cancers. The results showed that in approximately 50% of the RCCs, where Bcl-2 and/or Bcl-XL expression was high, apoptosis was not detected, and when expression of these proteins was low or not found, increased levels of apoptosis were seen. In most of the remaining 50% of samples, high levels of Bcl-XL but not Bcl-2 were negatively correlated with low levels of apoptosis (Bcl-XL: r=−0.437, P=0.07 and Bcl-2: r=+0.560, P=0.02). For the same group of samples, high Bax expression was found in association with apoptosis (r=+0.578, P=0.02). A novel finding was an association between low expression of Bcl-2 and/or Bcl-XL in normal tissue and the level of expression of these proteins in the RCCs, an intrinsic variation that may be an individual patient factor. The results indicate that, in RCCs with increased expression of Bcl-2 and/or Bcl-XL, levels of apoptosis are minimal and these combined factors may assist in progression of the cancers and resistance to treatments.

Activation of a T cell hybridoma by an alloligand results in differential effects on IL-2 secretion and activation-induced cell death.

The molecular nature of the interaction of T cell receptors (TCR) with alloligands is not well understood. Although a role for groove-bound peptide(s) has been clearly demonstrated for major histocompatibility complex (MHC) class I alloreactivity, this has not been established for MHC class II-induced alloresponses. In the present study, we have analyzed the interaction of a nominal peptide-self MHC complex and of an alloligand with their cognate TCR (1934.4 TCR for autoantigen recognition and qCII85.33 TCR for allorecognition). Our results demonstrate that 1934.4 TCR recognition of the N-terminal epitope of myelin basic protein (Ac1-11, Ac=acetylated at position 1) complexed with the MHC class II molecule I-A(u) involves contacts with both chains of the MHC molecule. In contrast, qCII85.33 TCR recognition of an allopeptide:I-A(u) complex appears to predominantly involve the beta chain of the MHC molecule. Thus, the two TCR appear to have different footprints on the I-A(u) molecules. Unexpectedly, this differential involvement of the two chains of the I-A(u) molecule affects activation induced cell death, with allostimulation resulting in poor induction of FasL expression and relatively low levels of apoptosis. Significantly, stimulation of cognate T cells with alloantigen or autoantigen results in similar levels of IL-2 secretion. The reduced apoptosis of T cells in response to allostimulation may be one of the mechanisms that favors the expansion of a relatively large repertoire of alloreactive T cells.

Exogenous interleukin-2 administration corrects the cell cycle perturbation of lymphocytes from human immunodeficiency virus-infected individuals.

Human immunodeficiency virus (HIV)-induced immunodeficiency is characterized by progressive loss of CD4(+) T cells associated with functional abnormalities of the surviving lymphocytes. Increased susceptibility to apoptosis and loss of proper cell cycle control can be observed in lymphocytes from HIV-infected individuals and may contribute to the lymphocyte dysfunction of AIDS patients. To better understand the relation between T-cell activation, apoptosis, and cell cycle perturbation, we studied the effect of exogenous interleukin-2 (IL-2) administration on the intracellular turnover of phase-dependent proteins. Circulating T cells from HIV-infected patients display a marked discrepancy between a metabolic profile typical of G(0) and a pattern of expression of phase-dependent proteins that indicates a more-advanced position within the cell cycle. This discrepancy is enhanced by in vitro activation with ConA and ultimately results in a marked increase of apoptotic events. Conversely, treatment of lymphocytes with IL-2 alone restores the phase-specific pattern of expression of cell cycle-dependent proteins and is associated with low levels of apoptosis. Interestingly, exogenous IL-2 administration normalizes the overall intracellular protein turnover, as measured by protein synthesis, half-life of newly synthesised proteins, and total protein ubiquitination, thus providing a possible explanation for the effect of IL-2 on the intracellular kinetics of cell cycle-dependent proteins. The beneficial effect of IL-2 administration is consistent with the possibility of defective IL-2 function in vivo, which is confirmed by the observation that lymphocytes from HIV-infected patients show abnormal endogenous IL-2 paracrine/autocrine function upon in vitro mitogen stimulation. Overall these results confirm that perturbation of cell cycle control contributes to HIV-related lymphocyte dysfunction and, by showing that IL-2 administration can revert this perturbation, suggest a new mechanism of action of IL-2 therapy in HIV-infected patients.

In vivo expression of p53 and Bcl-2 and their role in programmed cell death in premalignant and malignant lung lesions

Forty-four specimens of non-malignant and malignant human lung tissue, taken from patients with non-small cell lung cancer (NSCLC), were examined for the expression of wild-type p53, mutant p53, and bcl-2 and the occurrence of programmed cell death (apoptosis). Wild-type p53 expression peaked in peritumoral and metaplastic samples, whereas mutant p53, bcl-2 and apoptosis were first detected in metaplasia and increased with progression to carcinoma. Bcl-2 positive samples had lower levels of apoptosis than bcl-2 negative samples and was independent of wild-type or mutant p53 expression. These results suggest that the over-expression of wild-type p53 may be an early cellular response to an alteration in normal cellular homeostasis. The ensuing increase in apoptosis appears to be relatively independent of mutant or wild-type p53 expression, but does not occur in cells expressing bcl-2.

Inhibition of matrix metalloproteinases blocks lethal hepatitis and apoptosis induced by tumor necrosis factor and allows safe antitumor therapy.

Acute and fulminant liver failure induced by viral hepatitis, alcohol or other hepatotoxic drugs, are associated with tumor necrosis factor (TNF) production. In a mouse model of lethal hepatitis induced by TNF, apoptosis and necrosis of hepatocytes, but also lethality, hypothermia and influx of leukocytes into the liver, are prevented by a broad-spectrum matrix metalloproteinase (MMP) inhibitor, BB-94. Mice deficient in MMP-2, MMP-3 or MMP-9 had lower levels of apoptosis and necrosis of hepatocytes, and better survival. We found induction of MMP-9 activity and fibronectin degradation. Our findings suggest that several MMPs play a critical role in acute, fulminant hepatitis by degrading the extracellular matrix and allowing massive leukocyte influx in the liver. BB-94 also prevented lethality in TNF/interferon-gamma therapy in tumor-bearing mice. A broad-spectrum MMP inhibitor may be potentially useful for the treatment of patients with acute and perhaps chronic liver failure, and in cancer therapies using inflammatory cytokines.

Multidrug-resistance phenotype and clinical responses to gemtuzumab ozogamicin

Expression of multidrug resistance (MDR) features by acute myeloid leukemia (AML) cells predicts a poor response to many treatments. The MDR phenotype often correlates with expression of P-glycoprotein (Pgp), and Pgp antagonists such as cyclosporine (CSA) have been used as chemosensitizing agents in AML. Gemtuzumab ozogamicin, an immunoconjugate of an anti-CD33 antibody linked to calicheamicin, is effective monotherapy for CD33(+) relapsed AML. However, the contribution of Pgp to gemtuzumab ozogamicin resistance is poorly defined. In this study, blast cell samples from relapsed AML patients eligible for gemtuzumab ozogamicin clinical trials were assayed for Pgp surface expression and Pgp function using a dye efflux assay. In most cases, surface expression of Pgp correlated with Pgp function, as indicated by elevated dye efflux that was inhibited by CSA. Among samples from patients who either failed to clear marrow blasts or failed to achieve remission, 72% or 52%, respectively, exhibited CSA-sensitive dye efflux compared with 29% (P =.003) or 24% (P <.001) among samples from responders. In vitro gemtuzumab ozogamicin--induced apoptosis was also evaluated using an annexin V--based assay. Low levels of drug-induced apoptosis were associated with CSA-sensitive dye efflux, whereas higher levels correlated strongly with achievement of remission and marrow blast clearance. In vitro drug-induced apoptosis could be increased by CSA in 14 (29%) of 49 samples exhibiting low apoptosis in the absence of CSA. Together, these findings indicate that Pgp plays a role in clinical resistance to gemtuzumab ozogamicin and suggest that treatment trials combining gemtuzumab ozogamicin with MDR reversal agents are warranted. (Blood. 2001;98:988-994)