The control of apoptosis in lymphocyte selection

Summary: The stochastic nature of rearrangement and diversification of the gene segments encoding immunoglobulins (Igs) and T cell receptors (TCRs) inevitably gives rise to immature B and T lymphocytes that lack antigen receptors or express useless or dangerous (self‐antigen‐specific) ones. Signaling through antigen receptors promotes survival, proliferative expansion and further differentiation of useful cells and deletion of the useless and dangerous ones. During immune responses, pathogen‐specific B and T lymphocytes, as well as cells of the innate immune system, undergo extensive proliferation and develop effector functions, such as antibody secretion, cytotoxicity or cytokine production. To prevent tissue damage by these effector molecules, activated lymphocytes are removed when an infection has been overcome. Together with other mechanisms, including developmental arrest and induction of unresponsiveness (anergy), programmed cell death (apoptosis) of autoreactive lymphocytes safeguards immunological tolerance to self and assists in the development of an effective immune system. We have been investigating the molecular mechanisms that control programmed cell death. This review describes some of our experiments using transgenic and knockout mice, which overexpress or lack apoptosis regulators, that led to discoveries on how life and death decisions are made during development and functioning of the immune system.     

The dendritic cell-tumor cross-talk in cancer

The question as to whether the tumor grows because of or despite the host immune system is being progressively addressed with refined technology, gene targeting in mice and human translational research. The productive interplay between major actors of the antitumor immunity is actively compromised by the tumor microenvironment subverting the links between innate and cognate immunity and/or generating devastating new players. The complexity of the host-tumor equilibrium could be dissected at the reduced level of the dialogue between professional antigen presenting cells (APC), more precisely dendritic cells, and tumor cells that may profoundly dictate the outcome of the neoplasma. This review will summarize the novel mechanisms by which tumor cells regulate DC recruitment, differentiation, activation and cross-presenting functions in tumor beds and how innate players might counterbalance these interactions. Finally, we will highlight interesting strategies that harness the DC potential to fight against cancer.     

Contribution of cholinergic systems to state-dependent modulation of respiratory control

Respiration is altered during different stages of the sleep-wake cycle. We review the contribution of cholinergic systems to this alteration, with particular reference to the role of muscarinic acetylcholine receptors (MAchRs) during rapid eye movement (REM) sleep. Available evidence demonstrates that MAchRs have potent excitatory effects on medullary respiratory neurones and respiratory motoneurones, and are likely to contribute to changes in central chemosensitive drive to the respiratory control system. These effects are likely to be most prominent during REM sleep, when cholinergic brainstem neurones show peak activity levels. It is possible that MAchR dysfunction is involved in sleep-disordered breathing, such as obstructive sleep apnea.     

The therapeutic potential of immune cross-talk in leishmaniasis

Veterans of infection, Leishmania parasites have been plaguing mammals for centuries, causing a morbidity toll second only to that of malaria as the most devastating protozoan parasitic disease in the world. Cutaneous leishmaniasis (CL) is, by far, the most prevalent form of the disease, with symptoms ranging from a single self-healing lesion to chronic metastatic leishmaniasis (ML). In an increasingly immunocompromised population, complicated CL is becoming a more likely outcome, characterized by severely inflamed, destructive lesions that are often refractory to current treatment. This is perhaps because our ageing arsenal of variably effective antileishmanial drugs may be directly or indirectly immunomodulatory and may thus have variable effects in each type and stage of CL. Indeed, widely differing immune biases are created by the various species of Leishmania, and these immunological watersheds are further shifted by extrinsic disturbances in immune homeostasis. For example, we recently showed that a naturally occurring RNA virus (Leishmania RNA virus (LRV)) within some Leishmania parasites creates hyperinflammatory cross-talk, which can predispose to ML: a case of immunological misfire that may require a different approach to immunotherapy, whereby treatments are tailored to underlying immune biases. Understanding the intersecting immune pathways of leishmaniasis and its co-infections will enable us to identify new drug targets, and thereby design therapeutic strategies that work by untangling the immunological cross-wires of pathogenic cross-talk.     

Metabolic control through hepatocyte and adipose tissue cross-talk in a multicompartmental modular bioreactor

Physiological processes involve a complex network of signaling molecules that act through paracrinal or endocrinal pathways; however, traditional in vitro models cannot mimic these interactions because of the lack of a dynamic cross-talk between cells belonging to different tissues. The multicompartmental modular bioreactor is a novel cell culture system where hepatocytes and adipose tissue are shown to interact in a more physiological manner. In the multicompartmental modular bioreactor, cells and tissues can be cultured in a common medium, which flows through the system acting as the bloodstream. Primary rat hepatocytes and adipose tissue were cultured separately and together in conventional conditions and in the bioreactor. Urea synthesis, albumin secretion, glycerol, free fatty acid, and glucose concentrations were analyzed and compared. The dynamic connected culture of adipose tissue and hepatocytes led to a significant enhancement of hepatic function in terms of increase of albumin and urea production with respect to conventional cultures. Interestingly, the glycerol gradually released from adipose tissue was buffered in the dynamic connected culture, manifesting a homeostatic-like control. These data show that the dynamic culture not only improves hepatocyte function, but also allows a cross-talk between tissues, leading to enhanced metabolic regulation in vitro.     

A simple method to control aspects of fluence modulation in IMRT planning

Many inverse-planning algorithms and commercial systems generate intensity-modulated beam profiles that have considerable structure. This is the desirable outcome of the quest for high dose-space conformality. However, when these profiles are realized experimentally using the dynamic multileaf collimator (DMLC) method of delivery the monitor-unit efficiency can be quite small, with unwanted consequences. Also the interpretation of these fields leads to the generation of small field segments, again with undesirable consequences. In this note it is shown that the features of beam-space can be user-controlled to minimize these problems. There is a tradeoff between obtaining desirable features in beam-space and high conformality in dose-space.     

The apoptosis of non-small cell lung cancer induced by cisplatin through modulation of STIM1

Cis-diamminedichloroplatinum (II) (cisplatin) is one of the most active antitumor agents used in human chemotherapy of non-small cell lung cancer. Cisplatin forms crosslinked DNA adducts and its cytotoxicity has been shown to be mediated by propagation of DNA damage recognition signals to downstream pathways prompting apoptosis. The steps involved in the process include changes in Ca²⁺ signaling with dysregulated tumor cell turn-over. Stromal interaction molecules 1 (STIM1), as one of the most potent tumor suppressor genes, are identified as the endoplasmic-reticulum (ER) Ca²⁺ sensor controlling store-operated Ca²⁺ entry (SOCE) in non-excitable cells, which is main pathway to extracellular Ca²⁺ influx. Its role in STIM1 cisplatin-induced apoptosis of non-small cell lung cancer was the focus of study with focus on SOCE inhibitors 2-APB- and SKF96365-cisplatin-induced apoptosis in the non-small cell lung cancer (NSCLC) cell lines A549 and H460. In this experimental model, cisplatin-induced apoptosis and decreased concentration of intracellular Ca²⁺ was demonstrated. The expression of STIM1 was significantly higher in carcinoma tissue than in the adjacent non-neoplastic lung tissue. These findings support the conclusion that STIM1 may play an important role in the development of NSCLC which makes drugs that repress the expression of STIM1 to be a potential target for lung cancer therapy.     

Morphine-induced macrophage apoptosis: oxidative stress and strategies for modulation

Occurrence of macrophage apoptosis has been implicated for the altered immune function found in an opiate milieu. In the present study, we evaluated the role of oxidative stress in morphine-induced macrophage apoptosis. Morphine promoted the apoptosis of macrophages. This effect of morphine was associated with the production of superoxide and nitric oxide (NO). Antioxidants provided protection against morphine-induced macrophage injury. In addition, diphenyleneiodonium chloride, an inhibitor of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, attenuated the proapoptotic effect of morphine. Antitransforming growth factor-beta (anti-TGF-beta) antibody and propranolol (an inhibitor of the phospholipase D pathway) inhibited morphine-induced superoxide generation as well as apoptosis. N'-Tetraacetic acid tetra (acetoxymethyl) ester, a calcium-chelating agent, inhibited morphine-induced apoptosis, whereas thapsigargin (a calcium agonist) stimulated macrophage apoptosis under basal as well as morphine-stimulated states. These studies suggest that morphine-induced macrophage apoptosis is mediated through downstream signaling involving TGF-beta and NO production. Moreover, there is NADPH oxidation activation involving phospholipase D and Ca(2+), leading to the generation of superoxide. In in vivo studies, administration of N-acetyl cysteine and preinduction of heme oxygenase activity and epoetin alpha prevented morphine-induced peritoneal macrophage apoptosis, thus further confirming the role of oxidative stress in morphine-induced macrophage apoptosis.     

The autoinhibitory control element and calmodulin conspire to provide physiological modulation of endothelial and neuronal nitric oxide synthase activity

NO production by the endothelial and neuronal isoforms of nitric oxide synthase (cNOS) is regulated on a moment-to-moment basis by calmodulin binding, triggered by transient elevations in intracellular-free calcium levels. Nonetheless, additional modes of cNOS regulation are implicit in the discoveries of stimuli that elicit a sustained increase in cNOS activity despite undetectable or transient increases in intracellular Ca2+ in endothelial cells; such stimuli include shear-stress, oestrogen, insulin or insulin-like growth factor treatment of endothelial cells. Recently, we identified a peptide insertion within the FMN-binding domain of mammalian NOSs that is unique to calcium-dependent isoforms, and not shared with inducible NOS or ancestral flavoproteins. Evidence suggests that this insertion serves as a fundamental control element, analogous to intrinsic autoinhibitory peptides that have been demonstrated to regulate activity of other calmodulin-dependent enzymes. Thus, the peptide insertion of cNOSs appears to function as structural element that is displaced upon calmodulin binding, resulting in dysinhibition of NO synthesis. Once displaced, the peptide may also be subject to transient chemical modifications and protein-protein interactions that modulate autoinhibitory function. Herein we summarize our present knowledge and speculate on mechanisms by which calmodulin and the autoinhibitory peptide conspire to regulate cNOS activity.     

Redox control of apoptosis: an update

The redox environment of the cell is currently thought to be extremely important to control cell growth, differentiation, and apoptosis as many redox-sensitive proteins characterize these networks. A recent, widely accepted theory is that free radicals are not only dangerous species but, at low concentration, they have been designed by evolution to participate in the maintenance of cellular redox (reduction/oxidation) homeostasis. This notion derives from the evidence that cells constantly generate free radicals both as waste products of aerobic metabolism and in response to a large variety of stimuli. Free radicals, once produced, provoked cellular responses (redox regulation) against oxidative stress transducing the signals to maintain the cellular redox balance. Growing evidence suggests that in many instances the production of radical species is tightly regulated and their downstream targets are very specific, indicating that reactive oxygen species and reactive nitrogen species actively participate in several cell-signalling pathways as physiological "second messengers." In this review, we provide a general overview and novel insights into the redox-dependent pathways involved in programmed cell death.     

Allergen-dependent CD14 modulation and apoptosis in monocytes from allergic patients

BACKGROUND: CD14 is a most important monocyte surface molecule. Recently, it has been reported that there is an important relationship between CD14 and immunoglobulin E, and that regulation of CD14 expression is an effector mechanism mediating apoptosis of monocytes. OBJECTIVE: The present study was designed to determine whether specific allergens were able to modulate CD14 expression and apoptosis by monocytes from allergic patients or whether specific immunotherapy (IT) might affect these processes. METHODS: One group of adult allergic asthmatic patients had received IT for the previous 3 years. Another similar group was not treated with IT. We challenged peripheral blood monocytes from both groups of asthmatic patients in vitro with the specific allergen that produced clinical symptoms in asthmatic patients. The cells were also challenged with allergen to which the patients were not sensitive. Monocytes from normal subjects were also challenged with allergens. Expression of CD14 on the monocyte surface was analyzed by flow cytometry, and soluble CD14 (sCD14) in culture supernatant by enzyme-linked immunosorbent assay. The three groups of subjects were challenged with allergens, and apoptosis was analyzed by flow cytometry. RESULTS: When monocytes from non-IT-treated asthmatic patients were cultivated with the allergens to which the patients were sensitive, a significant up-regulation on the monocyte surface was observed compared with results from the healthy group (P < 0.003) and from the IT asthmatic group (P < 0.003). A significantly higher sCD14 level was observed in the culture supernatant of the monocytes from the IT asthmatic group were observed compared with those from the healthy group (P < 0.001) and those from the non-IT asthmatic group (P < 0.001). A significantly higher apoptosis level was observed in monocytes from the IT asthmatic group compared with those from the healthy group (P < 0.001) and those from the non-IT asthmatic group (<0.001). CONCLUSIONS: We present evidence that the expression of CD14 on the surface of monocytes and the apoptosis of the same cells can be modulated by an allergen-dependent mechanism. These processes can be affected by IT.     

The role of anakinra in the modulation of intestinal cell apoptosis and inflammatory response during ischemia/reperfusion

Background/aim Even though interleukin-1 receptor antagonist, IL-1Ra, is used in certain inflammatory diseases, its effect on ischemia-reperfusion injury is a current research topic. We aimed to investigate the protective effects of anakinra, an IL-1Ra, on the I/R induced intestinal injury.Materials and methods The rat model of intestinal ischemia-reperfusion was induced. Rats were randomized into 4 groups: (group 1) control group, (group 2) I/R group, (group 3 and 4) treatment groups (50 mg/kg and 100 mg/kg, respectively). Gene expressions of caspase-3, TNF-α, IL-1α, IL-6, and apoptotic cells in tissue samples were evaluated by PCR and TUNEL methods, respectively. Plasma levels of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were studied by the ELISA method and tissue samples were examined histopathologically as well.Results Anakinra inhibited the expression of IL-1α, IL-6, and TNF-α and decreased the SOD, CAT, and MDA caused by ischemia-reperfusion injury in both treatment groups. Caspase-3 expression and TUNEL-positive cell number in treatment groups were also less. Histopathologically, anakinra better preserved the villous structure of the small intestine at a dose of 100 mg/kg than 50 mg/kg. Conclusion Anakinra decreased the intestinal damage caused by ischemia-reperfusion and a dose of 100 mg/kg was found to be histopathologically more effective.     

Histaminergic modulation of hormonal control in the exocrine guineapig pancreas

The effects of histamine upon secretin- or cholecystokinin (CCK)-evoked exocrine pancreatic secretion were investigated in the anaesthetised guinea pig. Histamine (0.1 µmol/kg/min) induced a slight increase in pancreatic juice flow and total protein release compared to saline controls. Secretin (0.5 pmol/kg/min) and CCK-8 (0.75 pmol/kg/min) evoked marked time course increases in both the rate of pancreatic juice flow and total protein output in the anaesthetised guinea pig. Administration of either secretin or CCK-8 simultaneously with histamine elevated the exocrine pancreatic secretion compared to the smaller response obtained when administered separately. These results indicate that histamine may play an important physiological role in modulating the hormonal control of exocrine guinea pig pancreas.     

Hypothetical neural control of human bipedal walking with voluntary modulation

A hypothetical neuromusculoskeletal model is developed to simulate human normal walking and its modulated behaviors. A small set of neural periodic patterns drive spinal muscle synergies which in turn lead to specific pattern of muscle activation and supraspinal feedback systems maintain postural balance during walking. Then, the model demonstrates modulated behaviors by superimposing voluntary perturbations on the underlying walking pattern. Motions of kicking a ball and obstacle avoidance during walking are simulated as examples. The superposition of the new pulse command to a set of invariant pulses representing spino-locomotor is sufficient to achieve the coordinated behaviors. Also, forward bent walking motion is demonstrated by applying similar superposition. The composition of activations avoids a complicated computation of motor program for a specific task and presents a simple control scheme for different walking patterns.