3.20: Apoptosis - Biology

3.20: Apoptosis - Biology

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Apoptosis is a process of programmed cell death that occurs in multicellular organisms. There are two ways in which cells die: (1) They are killed by injurious agents or (2) they are induced to commit suicide.

Death by injury

Cells that are damaged by injury, such as by mechanical damage or exposure to toxic chemicals undergo a characteristic series of changes. They (and their organelles like mitochondria) swell (because the ability of the plasma membrane to control the passage of ions and water is disrupted). The cell contents leak out, leading to inflammation of surrounding tissues.

Death by Suicide

Cells that are induced to commit suicide:

  • shrink
  • develop bubble-like blebs on their surface
  • have the chromatin (DNA and protein) in their nucleus degraded
  • have their mitochondria break down with the release of cytochrome c
  • break into small, membrane-wrapped, fragments
  • release (at least in mammalian cells) ATP and UTP
  • These nucleotides bind to receptors on wandering phagocytic cells like macrophages and dendritic cells and attract them to the dying cells (a "find-me" signal")
  • The phospholipid phosphatidylserine, which is normally hidden in the inner layer of the plasma membrane, is exposed on the surface
  • This "eat me" signal is bound by other receptors on the phagocytes which then engulf the cell fragments
  • The phagocytic cells secrete cytokines that inhibit inflammation (e.g., IL-10 and TGF-β)

The pattern of events in death by suicide is so orderly that the process is often called programmed cell death or PCD. The cellular machinery of programmed cell death turns out to be as intrinsic to the cell as, say, mitosis. Programmed cell death is also called apoptosis. (There is no consensus yet on how to pronounce it; some say APE oh TOE sis; some say uh POP tuh sis.)

Why should a cell commit suicide?

There are two different reasons.

1. Programmed cell death is as needed for proper development as mitosis is.


  • The resorption of the tadpole tail at the time of its metamorphosis into a frog occurs by apoptosis.
  • The formation of the fingers and toes of the fetus requires the removal, by apoptosis, of the tissue between them.
  • The sloughing off of the inner lining of the uterus (the endometrium) at the start of menstruation occurs by apoptosis.
  • The formation of the proper connections (synapses) between neurons in the brain requires that surplus cells be eliminated by apoptosis.
  • The elimination of T cells that might otherwise mount an autoimmune attack on the body occurs by apoptosis.
  • During the pupal stage of insects that undergo complete metamorphosis, most of the cells of the larva die by apoptosis thus providing the nutrients for the development of the structures of the adult.

2. Programmed cell death is needed to destroy cells that represent a threat to the integrity of the organism.


Cells infected with viruses
One of the methods by which cytotoxic T lymphocytes (CTLs) kill virus-infected cells is by inducing apoptosis and some viruses mount countermeasures to thwart it.
Cells of the immune system
As cell-mediated immune responses wane, the effector cells must be removed to prevent them from attacking body constituents. CTLs induce apoptosis in each other and even in themselves. Defects in the apoptotic machinery is associated with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis.
Cells with DNA damage
Damage to its genome can cause a cell
  • to disrupt proper embryonic development leading to birth defects
  • to become cancerous.
Cells respond to DNA damage by increasing their production of p53. p53 is a potent inducer of apoptosis. Is it any wonder that mutations in the p53 gene, producing a defective protein, are so often found in cancer cells (that represent a lethal threat to the organism if permitted to live)?
Cancer cells
Radiation and chemicals used in cancer therapy induce apoptosis in some types of cancer cells.

What makes a cell decide to commit suicide?

The balance between the withdrawal of positive signals; that is, signals needed for continued survival, and the receipt of negative signals.

Withdrawal of positive signals

The continued survival of most cells requires that they receive continuous stimulation from other cells and, for many, continued adhesion to the surface on which they are growing. Some examples of positive signals: growth factors for neurons and Interleukin-2 (IL-2), an essential factor for the mitosis of lymphocytes

Receipt of negative signals

  • increased levels of oxidants within the cell
  • damage to DNA by these oxidants or other agents like ultraviolet light, X-rays and chemotherapeutic drugs
  • accumulation of proteins that failed to fold properly into their proper tertiary structure
  • molecules that bind to specific receptors on the cell surface and signal the cell to begin the apoptosis program. These death activators include:
    • Tumor necrosis factor-alpha (TNF-α) that binds to the TNF receptor
    • Lymphotoxin (also known as TNF-β) that also binds to the TNF receptor
    • Fas ligand (FasL), a molecule that binds to a cell-surface receptor named Fas (also called CD95)

The Mechanisms of Apoptosis

There are 3 different mechanisms by which a cell commits suicide by apoptosis.

  1. Generated by signals arising within the cell
  2. Triggered by death activators binding to receptors at the cell surface:
    • TNF-α
    • Lymphotoxin
    • Fas ligand (FasL)
  3. Triggered by dangerous reactive oxygen species

Apoptosis triggered by internal signals

  • In a healthy cell, the outer membranes of its mitochondria display the protein Bcl-2 on their surface. Bcl-2 inhibits apoptosis.
  • Internal damage to the cell
    • causes a related protein, Bax, to migrate to the surface of the mitochondrion where it inhibits the protective effect of Bcl-2 and inserts itself into the outer mitochondrial membrane punching holes in it and causing
    • cytochrome c to leak out.
  • The released cytochrome c binds to the protein Apaf-1 ("apoptotic protease activating factor-1").
  • Using the energy provided by ATP, these complexes aggregate to form apoptosomes. The apoptosomes bind to and activate caspase-9. Caspase-9 is one of a family of over a dozen caspases. They are all proteases. They get their name because they cleave proteins — mostly each other — at aspartic acid (Asp) residues.
  • Caspase-9 cleaves and, in so doing, activates other caspases (caspase-3 and -7).
  • The activation of these "executioner" caspases creates an expanding cascade of proteolytic activity (rather like that in blood clotting and complement activation) which leads to
    • digestion of structural proteins in the cytoplasm,
    • degradation of chromosomal DNA
    • phagocytosis of the cell

Apoptosis triggered by external signals

  • Fas and the TNF receptor are integral membrane proteins with their receptor domains exposed at the surface of the cell
  • Binding of the complementary death activator (FasL and TNF respectively) transmits a signal to the cytoplasm that leads to the activation of caspase 8
  • Caspase 8 (like caspase 9) initiates a cascade of caspase activation leading to phagocytosis of the cell.
  • Example: When cytotoxic T cells recognize (bind to) their target,
    • They produce more FasL at their surface.
    • This binds with the Fas on the surface of the target cell leading to its death by apoptosis.

    The early steps in apoptosis are reversible — at least in C. elegans. In some cases, final destruction of the cell is guaranteed only with its engulfment by a phagocyte.

Apoptosis-Inducing Factor (AIF)

Neurons, and perhaps other cells, have another way to self-destruct that — unlike the two paths described above — does not use caspases. Apoptosis-inducing factor (AIF) is a protein that is normally located in the intermembrane space of mitochondria. When the cell receives a signal telling it that it is time to die, AIF is released from the mitochondria (like the release of cytochrome c in the first pathway). It migrates into the nucleus and binds to DNA, which triggers the destruction of the DNA and cell death.

Apoptosis and Cancer

Some viruses associated with cancers use tricks to prevent apoptosis of the cells they have transformed.

  • Several human papilloma viruses (HPV) have been implicated in causing cervical cancer. One of them produces a protein (E6) that binds and inactivates the apoptosis promoter p53.
  • Epstein-Barr Virus (EBV), the cause of mononucleosis and associated with some lymphomas
    • produces a protein similar to Bcl-2
    • produces another protein that causes the cell to increase its own production of Bcl-2. Both these actions make the cell more resistant to apoptosis (thus enabling a cancer cell to continue to proliferate).

Even cancer cells produced without the participation of viruses may have tricks to avoid apoptosis.

  • Some B-cell leukemias and lymphomas express high levels of Bcl-2, thus blocking apoptotic signals they may receive. The high levels result from a translocation of the BCL-2 gene into an enhancer region for antibody production.
  • Melanoma (the most dangerous type of skin cancer) cells avoid apoptosis by inhibiting the expression of the gene encoding Apaf-1.
  • Some cancer cells, especially lung and colon cancer cells, secrete elevated levels of a soluble "decoy" molecule that binds to FasL, plugging it up so it cannot bind Fas. Thus, cytotoxic T cells (CTL) cannot kill the cancer cells by the mechanism shown above.
  • Other cancer cells express high levels of FasL, and can kill any cytotoxic T cells (CTL) that try to kill them because CTL also express Fas (but are protected from their own FasL).

Apoptosis in the Immune System

The immune response to a foreign invader involves the proliferation of lymphocytes — T and/or B cells. When their job is done, they must be removed leaving only a small population of memory cells. This is done by apoptosis. Very rarely humans are encountered with genetic defects in apoptosis. The most common one is a mutation in the gene for Fas, but mutations in the gene for FasL or even one of the caspases are occasionally seen. In all cases, the genetic problem produces autoimmune lymphoproliferative syndrome or ALPS.


  • an accumulation of lymphocytes in the lymph nodes and spleen greatly enlarging them.
  • the appearance of clones that are autoreactive; that is, attack "self" components producing such autoimmune disorders as
    • hemolytic anemia
    • thrombocytopenia
  • the appearance of lymphoma — a cancerous clone of lymphocytes.

In most patients with ALPS, the mutation is present in the germline; that is, every cell in their body carries it. In a few cases, however, the mutation is somatic; that is, has occurred in a precursor cell in the bone marrow. These later patients are genetic mosaics — with some lymphocytes that undergo apoptosis normally and others that do not. The latter tend to out-compete the former and grow to become the major population in the lymph nodes and blood.

Apoptosis and Organ Transplants

For many years it has been known that certain parts of the body such as the anterior chamber of the eye and the testes are "immunologically privileged sites". Antigens within these sites fail to elicit an immune response. It turns out that cells in these sites differ from the other cells of the body in that they express high levels of FasL at all times. Thus antigen-reactive T cells, which express Fas, would be killed when they enter these sites. (This is the reverse of the mechanism described above.)

This finding raises the possibility of a new way of preventing graft rejection. If at least some of the cells on a transplanted kidney, liver, heart, etc. could be made to express high levels of FasL, that might protect the graft from attack by the T cells of the host's cell-mediated immune system. If so, then the present need for treatment with immunosuppressive drugs for the rest of the transplant recipient's life would be reduced or eliminated. So far, the results in animal experiments have been mixed. Allografts engineered to express FasL have shown increased survival for kidneys, but not for hearts or islets of Langerhans.

Apoptosis in Plants

Plants, too, can turn on a system of programmed cell death; for example, in an attempt to halt the spread of virus infection. The mechanism differs from that in animals although it, too, involves a protease that — like caspases — cleaves other proteins at Asp (and Asn) residues. Activation of this enzyme destroys the central vacuole, which is followed by disintegration of the rest of the cell.

Heat shock protein

Heat shock proteins (HSP) are a family of proteins that are produced by cells in response to exposure to stressful conditions. They were first described in relation to heat shock, [1] but are now known to also be expressed during other stresses including exposure to cold, [2] UV light [3] and during wound healing or tissue remodeling. [4] Many members of this group perform chaperone functions by stabilizing new proteins to ensure correct folding or by helping to refold proteins that were damaged by the cell stress. [5] This increase in expression is transcriptionally regulated. The dramatic upregulation of the heat shock proteins is a key part of the heat shock response and is induced primarily by heat shock factor (HSF). [6] HSPs are found in virtually all living organisms, from bacteria to humans.

Heat-shock proteins are named according to their molecular weight. For example, Hsp60, Hsp70 and Hsp90 (the most widely studied HSPs) refer to families of heat shock proteins on the order of 60, 70 and 90 kilodaltons in size, respectively. [7] The small 8-kilodalton protein ubiquitin, which marks proteins for degradation, also has features of a heat shock protein. [8] A conserved protein binding domain of approximately 80 amino-acid alpha crystallins are known as small heat shock proteins (sHSP). [9]

Monitoring the biology stability of human umbilical cord-derived mesenchymal stem cells during long-term culture in serum-free medium

Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have an immunosuppressive effect. The biological stability of MSCs in serum-free medium during long-term culture in vitro has not been elucidated clearly. The morphology, immunophenotype and multi-lineage potential were analyzed at passages 3, 5, 10, 15, 20, and 25 (P3, P5, P10, P15, P20, and P25, respectively). The cell cycle distribution, apoptosis, and karyotype of human umbilical cord-derived (hUC)-MSCs were analyzed at P3, P5, P10, P15, P20, and P25. From P3 to P25, the three defining biological properties of hUC-MSCs [adherence to plastic, specific surface antigen expression, multipotent differentiation potential] met the standards proposed by the International Society for Cellular Therapy for definition of MSCs. The cell cycle distribution analysis at the P25 showed that the percentage of cells at G0/G1 was increased, compared with the cells at P3 (P < 0.05). Cells at P25 displayed an increase in the apoptosis rate (to 183 %), compared to those at P3 (P < 0.01). Within subculture generations 3-20 (P3-P20), the differences between the cell apoptotic rates were not statistically significant (P > 0.05). There were no detectable chromosome eliminations, displacements, or chromosomal imbalances, as assessed by the karyotyping guidelines of the International System for Human Cytogenetic Nomenclature (ISCN, 2009). Long-term culture affects the biological stability of MSCs in serum-free MesenCult-XF medium. MSCs can be expanded up to the 25th passage without chromosomal changes by G-band. The best biological activity period and stability appeared between the third to 20th generations.



TGF-β1 (sc-146, lot # F262, 200 μg/ml), TGF-β2 (sc-90, lot # B202, 200 μg/ml) and TGF-β3 (sc-82, lot # A222, 200 μg/ml) polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). CDC47/MCM7 antibody was obtained from Medicorp (Montréal, QC, Canada). Phospho-Akt (Ser 473), Akt, XIAP, Cleaved caspase-3, and Phospho-Smad2 (Ser 465 / 467) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). The Keratin 8/18 antibody used to determine cell culture purity was donated by Dr Monique Cadrin (Univ. of Québec at Trois-Rivières, QC, Canada). Anti-Smad2/3 antibody was purchased from Calbiochem (San Diego, CA, USA). Vectastain ABC Kit for rabbit IgG was purchased from Vector Laboratories Inc. (Burlingame, CA, USA). In Situ Cell Death detection kit (TUNEL), POD and DAB substrate was obtained from Roche (Laval, QC, Canada). TGF-β1 recombinant protein was purchased from Biosource (Cat # PHG9104, lot # 16865-01S, 5 μg, diluted at 50 μg/ml, QC, Canada).


Sprague-Dawley female rats, 200–225 g, were obtained from Charles River Laboratories Canada. Animals were maintained on standard chow and water, which were available ad libitum, in animal facilities illuminated between 6:00 h and 20:00 h. All procedures were performed in accordance with guidelines of the Canadian Council on Animal Care for the handling and training of laboratory animals and the Good Health and Animal Care Committee of the Université du Québec à Trois-Rivières. Male and female mice were mated overnight and confirmation of pregnancy was determined by vaginal smears and/or the presence of a vaginal plug (day 1). Rats were killed on day 2, 4, 5, 6, 8, 10, 12, 14, 16, 18 and 20 of pregnancy at 10:00 h in the morning and at 18:00 h for days 5.5 and 6.5. Six to 8 different rats were used for each time of pregnancy. Uteri were collected and fixed for immunohistochemical staining (IHC) and apoptotic cell death detection by [TdT]-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) or endometrial protein extracts collected for Western blot analysis.

Rat pretreatments and decidual endometrial stromal cell culture

A total of 10 rats were ovariectomized and then allowed to recover from surgery for a minimum of 10 days. They were pre-treated with physiological doses of estradiol (1,3,5(10)-Estratriene-3,17β-diol, Sigma-aldrich) and progesterone (Laboratoire Mat, PQ) to induce decidualization as described previously [26]: 1) 0.2 ug estradiol injection per day for three days (in the morning, day -2,-1 and 0) 2) On the third day (day 0 of pseudopregnancy), another injection in the afternoon of estradiol (0.2 μg) and progesterone (1 mg) was performed 3) No treatment for 2 days (day 1 and 2 of pseudopregnancy) 4) Injections of estradiol (0.1 μg) and progesterone (4 mg) for three days (day 3, 4 and 5 of pseudopregnancy) 5) Another injection of estradiol (0.1 μg) in the afternoon on day 7 (day 4 of pseudopregnancy) 6) Rats were killed on day 8 (day 5 of pseudopregnancy). All endometrial stromal cells collected for cultures were recovered from rats treated with the protocol described above.

Uteri were removed and horns taken and immerged in HBSS solution containing HEPES (20 mM), penicillin (100 units/ml), streptomycin (100 μg/ml) and fungizone (1,25 μl/ml) (Invitrogen, ON, Canada). Further manipulations were performed in a sterile environment. The uterine horns were transferred into a sterile petri containing HBSS, slit longitudinally and immersed in trypsin type I solution (0.3%) (Roche Diagnostics, QC, Canada) in HBSS and agitated for 60 minutes at room temperature. Uterine horns were then vortexed at maximum for 5 sec and supernatant containing epithelial cells was discarded. Uterine horns were washed three times with 2.5 ml of HBSS and immersed in a HBSS solution containing trypsin type I (0.03%), DNAse I (0.016%) and collagenase type II (0.064%) for 15 minutes at 37°C in a water bath. Uterine horns were then vortexed at maximum for 5 sec. The supernatant containing stromal cells was transferred into a sterile falcon tube containing 150 μl of FBS D.C (Dextran-Charcoal extracted). Uterine horns were washed two times with 2.5 ml of HBSS and the supernatant was mixed with stromal cells. Uterine horns were discarded and stromal cells were centrifuged at 1000 g for 5 minutes. Cells were washed two times with HBSS and centrifuged. The supernatant was discarded and cells diluted with DMEM-F12 (Ph 7.1) (Invitrogen, ON, Canada) containing 2.438 g/L NaHCO3, 10% FBS D.C. and gentamycine 50 μg/ml. Cells were incubated at 37°C in an atmosphere of 5% CO2. Cells were plated in 6-well plates (Corning plates) at a density of 50% (4 × 10 5 cells per well). The medium was changed two hours after the first incubation in order to eliminate epithelial cell contamination from stromal cell cultures. The purity of stromal cells was more than 97%: cell culture contamination with epithelial cells was evaluated by cellular morphology and immunofluorescence using a Keratin 8/18 antibody. Three to 5 days after plating (more than 90% of confluency reached), cells were treated for 24 hours in the presence or absence of increasing doses of TGF-β recombinant protein. Total proteins from treated cell cultures were extracted using TRIZOL (Invitrogen, ON, Canada). For Western blot analyses, 15 μg of total protein was used for each analysis.

Immunohistochemical staining

The uterus was fixed in 4% paraformaldehyde solution and embedded in paraffin. Tissue sections 7 μm thick were mounted on polylysine-coated slides, deparaffinized, rehydrated, and then heated in 10 mM citrate buffer (pH 6) containing triton X-100 (Sigma-Aldrich) 0.1% (v/v). After two washes with PBS, slides were then incubated with 0.3 % hydrogen peroxide in methanol for 30 min to quench endogenous peroxidase activity. After washing with PBS, tissues were incubated with blocking serum (Vectastain ABC Kit) at room temperature for 1 h. Then, a primary antibody diluted in blocking serum (TGF-β1, β2 or β3 1:50 dilution or CDC47/MCM7 1:100 dilution) was added to the slides and incubated at 4°C overnight in a humidified chamber. After washing 5 min. in PBS, tissue sections were incubated for 30 min. with 3 μg/ml biotinylated antibody (anti-rabbit or anti-mouse). Subsequently, slides were washed with PBS and incubated with avidin-biotin complex reagent containing horseradish peroxidase for 30 min. Slides were washed with PBS for 5 min and color development was achieved using DAB substrate. The tissue sections were counterstained with haematoxylin. Negative controls were performed using the same protocol but substituting the primary antibody with normal rabbit IgG (Vector Laboratories Inc., Burlingame, CA, USA).


Tissues were prepared as described in the immunohistochemical section. Cleaved caspase-3 antibody was diluted 1:100 in blocking serum and slides were incubated at 4°C overnight. After washing twice for 5 min. in PBS, tissue sections were incubated for 30 min. at room temperature with 2 mg/ml Alexa Fluor 488 donkey anti-rabbit (1:50). Subsequently, slides were washed with PBS and mounted. Negative controls were performed using the same protocol but substituting the primary antibody with normal rabbit IgG. Sections were examined using an OlympusBX60 microscope equipped with a Coolsnap-pro CF digital camera (Carsen Group, ON, Canada).

TdT-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL)

Tissue sections were deparaffinized, rehydrated and rinsed with PBS. They were incubated with proteinase K (20 μg/ml) for 30 min. at room temperature. Slides were washed twice with PBS, the endogenous peroxidase was quenched with 0.3 % hydrogen peroxide in methanol for 30 min. The slides were rinsed and incubated with 10 mM citrate solution for two min on ice. Then, tissue sections were rinsed with PBS and incubated with TdT labelling reaction (In Situ Cell Death Detection, POD) for 30 min at 37°C in humidified environment. Slides were washed three times in PBS and tissue sections were blocked with 3% BSA for 20 min. at room temperature. Converter-POD solution was added to the slides and incubated for 30 min. at 37°C in humidified environment. Slides were washed for 5 min. in PBS, colour development was achieved using DAB substrate and counterstained with haematoxylin. Negative controls were performed using the same protocol without TdT enzyme.

Protein extraction and Western analysis

Protein homogenates from pregnant endometrium were isolated according to a protocol previously described [27]. Briefly, uteri from Day 2 to Day 20 pregnant rats were rapidly excisedand placed in ice-cold saline until dissected. Uteri were carefully laid on a glass plate and placed on the stage of a dissecting microscope. In early pregnancy (Day 2 to 5.5), total endometrium was scraped using a microscope glass and collected. Uteri from Day 6 to 10 the placenta and decidua were at an early stage of differentiation and could not be reliably separated. For this reason, DB dissectedfrom animals between these days of pregnancy contain some chorioallantoic cells, but antimesometrial decidua, choriovitelline tissues, fetus, and myometrium were removed. Even though we carefully dissected DB from these tissues, it is a possibility that a contamination with some antimesometrial decidual cells that regress to form the deciduas caspularis (DC) would occur. This is an important fact that we need to take into consideration. In uteri collected from Day 12 to 20 pregnant rats, DB were isolated by gently separating the placenta and myometrial regions with 23-gauge needles. Additionally, the DB began to regress on Day 14 and became too thin to reliablydissect after Day 17. The protocol for DB isolation was described previously by Ogle and George [28].

Endometrial cells from pregnant animals were homogenized using a pipette in RIPA lysis buffer (PBS 1× pH 7.4 1% Nonidet P-40 0.5% Sodium deoxycholate 0.1% SDS Protease Inhibitor Cocktail Tablets (Roche Diagnostics Canada, PQ)). Homogenates were centrifuged (12,000 × g for 20 min at 4°C) to remove insoluble material. The supernatant was recovered and stored at -20°C pending analysis. Protein content was determined with the Bio-Rad DC Protein Assay. Protein extracts (50 μg) were heated at 94°C for 3 min, resolved by 10% SDS-PAGE and electrotransferred to nitrocellulose membranes using a semidry transfer (Bio-Rad, Mississauga, ON). The membranes were then blocked 2 h at room temperature with PBS containing 5 % milk powder, then incubated with anti TGF-β 1-2-3 1:1000 P-Smad2 (Ser 465 / 467) 1:1000 and Smad 2/3 1:1000 and subsequently with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (1:3000 room temperature for 45 min). All membranes were stripped with Restore western blot stripping buffer (Pierce, # 21059, lot # FH71541), reprobed with an antibody specific to β-actin which was used as an internal standard. Peroxidase activity was visualized with the Super signal ® West Femto maximum sensitivity substrate (Pierce, Arlington Heights, IL, USA), according to the manufacturer's instructions. Signal was visualized using the Biochemi Imaging System (UVP, CA, USA). Densitometrical analyses were performed (protein of interest and β-actin) using the GelDoc 2000 and the Quantity One software (Bio-Rad, Mississauga, ON, Canada). Results are expressed as a ratio (protein of interest/β-actin) to correct for loading for each endometrial sample.

Hoechst and trypan blue exclusion staining

Following TGF-β treatment, both floating and attached cells were resuspended in PBS containing Hoechst 33258 for 24 hours at 4°C or resuspended in trypan blue solution (0,4%) for 5 minutes. Hoechst nuclear staining was viewed and photographed using a Olympus BX60 fluorescence microscope and a Coolsnap-Pro CF digital Camera (Carsen Group, ON, Canada). Cells with typical apoptotic nuclear morphology (nuclear shrinkage, condensation and fragmentation) were identified and counted using randomly selected fields on numbered photographic slides, of which the "counter" was not aware of the treatment, so as to avoid experimental bias. A minimum of 200 cells per treatment group were counted in each experiment. For trypan blue exclusion test, blue cells were counted under a regular microscope and were counted as non-living cells.

Statistical analysis

Western analyses of pregnant animals were repeated six to eight times (6 to 8 different endometrial extract per day of pregnancy from 6 to 8 different rats). Endometrial extracts from each rat were assessed individually. Western analyses of cultured decidual cells were repeated 5 times for each TGF-β dose (for each culture experiment, decidual cells were recovered from a pool of ten different ovariectomized/treated rats). Results subjected to statistical analyses were expressed as mean ± SEM. Data were subjected to one-way ANOVA (PRISM software version 4.0 GraphPad, San Diego, CA). Differences between experimental groups were determined by the Tukey's test.

Cell death during development

There are many ways to measure apoptosis and other forms of programmed cell death in development. Once nonmammalian embryos have passed the midblastula transition, or much earlier in mammalian embryos, apoptosis is similar to that seen in adult organisms, and is used to sculpt the animal, fuse bilateral tissues, and establish the structure of the nervous system and the immune system. Embryos present unique problems in that, in naturally occurring cell deaths, few cells are involved and they are frequently in very restricted regions. Thus, identification of apoptotic or other dying cells is more effectively achieved by microscopy-based techniques than by electrophoretic or cell-sorting techniques. Since embryos have many mitotic cells and are frequently more difficult to fix than adult tissues, it is best to confirm interpretations by the use of two or more independent techniques. Although natural embryonic deaths are frequently programmed and require protein synthesis, activation of a cell death pathway is often post-translational and assays for transcriptional or translational changes-as opposed to changes in aggregation of death-related molecules or proteolytic activation of enzymes-is likely to be uninformative. Also, embryos can frequently exploit partially redundant pathways, such that the phenotype of a knockout or upregulated death-related gene is often rather modest, even though the adult may develop response or regulation problems. For these reasons, the study of cell death in embryos is fascinating but researchers should be cautious in their analyses.

Subject Category List

Describe the manuscript you are submitting byselecting the most appropriate descriptor number:


1.1 Airway Receptors: Nerves and Smooth Muscle

1.2 Airway Receptors: Cytokines, Chemokines

1.3 Airway Remodeling: Asthma Mediators

1.4 Airway Remodeling: Smooth Muscle, Fibroblasts, Extracellular Matrix

1.5 Airway Remodeling: Functional Consequences

1.6 Airway Remodeling: Asthma Genes and Gene Products

1.7 Airway Responsiveness: Immunologic Mechanisms

1.8 Airway Responsiveness: Physiology

1.12 Clinical Asthma Management Programs

1.13 Education/Self-Management/Asthma Guidelines

1.14 Epidemiology: Adult Asthma: Outcomes

1.15 Epidemiology: Adult Asthma: Risk Factors (Etiology)

1.16 Epidemiology (Pediatric): Outcomes & Management

1.17 Epidemiology (Pediatric): Risk Factors

1.19 Immunology/Inflammation: Animal Models

1.20 Immunology/Inflammation: Human Studies

1.21 Infectious Mechanisms

1.22 Non-Invasive Assessment of the Airways: Functional

1.23 Non-Invasive Assessment of the Airways: Immunological

1.24 Methods on Non-Invasive Assessment of the Airways: Exhaled Breath and Condensates

1.25 Occupational and Environmental Airways Disease


2.2 Assessing and Improving Clinician Behavior

2.3 Health Education/Disease Prevention/Patient Education

2.4 Health Outcomes Assessment/Cost Effectiveness

2.5 Health Policy, Financing, and Organization

2.6 Information Technology/Informatics/Telemedicine

2.7 Professional Education/Training/Certification

2.8 Psychosocial and Behavioral Factors in Lung Disease

2.9 Racial, Ethnic, or Social Disparities in Lung Disease and Treatment

2.10 Research Design/Program Evaluation/Statistical Models

2.11 Social Determinants of Lung Health and Disease

2.12 End of Life: Critical Care

2.13 End of Life/Palliative Care


3.1 Animal Models of Pulmonary Fibrosis

3.2 Bioinformatics/Biological Computing

3.3 Developmental Lung Biology

3.6 Functional Genomics/Proteomics

3.7 Gene Regulation: Developmental Control/Mouse Models

3.8 Gene Regulation: General and Tissue Specific

3.9 Growth Factors and Receptors

3.10 Ion Transport and Membrane Channels

3.11 Pulmonary Fibrosis/Fibroblast Biology

3.12 Signal Transduction: Intracellular Pathways

3.13 Signal Transduction: Membrane Receptors

3.15 Smooth Muscle: Vascular

3.16 Transcription Factors

3.17 Cell Fate, Inflammatory Cells: Apoptosis

3.18 Cell Fate, Inflammatory Cells: Clearance of Apoptotic Cells

3.19 Cell Fate, Inflammatory Cells: Survival

3.20 Cell Fate, Inflammatory Cells: Proliferation

3.21 Cell Fate, Non-Inflammatory Cell Types: Cell Plasticity

3.22 Cell Fate, Non-Inflammatory Cell Types: Injury/Repair

3.23 Cell Fate, Non-Inflammatory Cell Types: Senescence

3.24 Cell Fate, Non-Inflammatory Cell Types: Stem Cells/Tissue Regeneration

3.25 Cell Fate, Non-Inflammatory Cell Types: Survival/Apoptosis/Cell Cycle

3.26 Cell Fate, Vascular Cell: Apoptosis

3.27 Cell Fate, Vascular Cell: Survival

3.28 Cell Fate, Vascular Cell: Proliferation

3.29 Cell-Cell Interactions

3.30 Cell-Matrix Interactions

3.32 Airway Gene Expression

3.36 Alveolar Gene Expression

3.37 Surfactant Gene Expression


4.1 ALI/ARDS: Biological Mechanisms

4.2 ALI/ARDS: Diagnosis & Clinical Issues

4.3 Cardiopulmonary Interactions/CV Performance

4.4 Clinical Trials in Critical Care Medicine

4.5 Diagnostic Techniques & Monitoring

2.12 End of Life: Critical Care

4.7 Mechanical Ventilation: Applications

4.8 Mechanical Ventilation: Physiology & Pathophysiology

4.9 Molecular Biology of Critical Care

4.10 Non-Pulmonary Critical Care

4.11 Pediatric Critical Care

4.12 Sepsis/Multiple Organ Failure

4.13 Ventilation: Non-Invasive/Long-Term/Weaning


5.1 Chemokines: Immune Effects

5.2 Eicosanoids, Immune Effects


6.1 Air Pollution: Epidemiology

6.2 Air Pollution: Mechanisms

6.3 Diet, Obesity and Lung Disease

1.14 Epidemiology: Adult Asthma: Outcomes

1.15 Epidemiology: Adult Asthma: Risk Factors (Etiology)

1.17 Epidemiology (Pediatric): Risk Factors

6.5 Functional Genomics and Proteomics of Environmental and Occupational Lung Disease

6.6 Gene-Environment Interaction

6.7 Genetic Epidemiology of Environmental and Occupational Lung Disease

6.8 Global Burden of Occupational and Environmental Lung Diseases

6.9 Health Effects of Nanomaterials

6.10 Hypersensitivity Pneumonitis

6.11 Inhalational Disasters Science and Health

6.12 Lung Cancer Epidemiology: Occupational and Environmental Factors

6.13 Lung Health and the Working Life

6.14 Medical Monitoring and Screening for Occupational and Environmental Respiratory Diseases

1.25 Occupational and Environmental Airways Disease

6.15 Occupational and Environmental Pleural and Lung Disease

6.17 Smoking Health Effects

6.18 Smoking: Prevention/Education/Cessation

6.19 Upper Airway: Occupational and Environmental Factors


7.1 Aerosols: Physiology and Biology

7.3 Animal Models of Airway Fibrosis

7.4 Complement, Defensins, and Other Humoral Mediators of Innate Immunity

3/20/21 Patient Summit – Top FAQs

Should myeloma patients get vaccinated against COVID-19 and if so, when?
Yes, myeloma patients should get any of the three COVID-19 vaccines available (that is, the Pfizer, Moderna, or the J&J vaccine). Myeloma specialists do not have any specific guidance on the exact timing (that is, before treatment, during treatment, after treatment, during maintenance, etc.) with the exception of patients who have had an autologous stem cell transplant (ASCT) or chimeric antigen receptor (CAR) T-cell therapy. For these patients, guidance on vaccination is similar to what is advised for the influenza vaccine which is to wait until 3 months after your ASCT or CAR T-cell therapy to get vaccinated. For all other myeloma patients, myeloma specialists are recommending that if you are offered a chance to get vaccinated, you should take the offer and get it when you can.

As always, myeloma patients should still practice social distancing, masking, and handwashing especially until the number of COVID-19 cases go down.

When should a myeloma patient get an autologous stem cell transplant ?
There have been a large number of trials that have investigated the benefit of autologous stem cell transplant (ASCT) as part of the initial treatment regimen (early) compared with ASCT after a patient has relapsed (late). One of the more recent studies to be published showed that patients who got an early transplant tended to have a longer time without relapse than did those who got a late transplant however, survival time (that is, how long patients lived) was similar between groups. The results of this trial had many patients and myeloma experts asking: if survival is the same, why should patients undergo ASCT early? Why not observe how long a patient responds to initial therapy and then if the patient relapses, do an ASCT at that time?

The decision to proceed with an ASCT early or late is based on the recommendation of your physician and your understanding of the role of ASCT. Patients should know that ASCT is a choice and not a necessity! To help answer the question of when a myeloma patient should get an ASCT, it is helpful to weigh the advantages and disadvantages of each:

  • Advantages
    • The patient is the youngest and healthiest they&rsquoll ever be
    • The myeloma will be at its most sensitive
    • The patient will experience the quickest recovery (or return to &ldquonew normal&rdquo)
    • About 10% to 15% of patients may not need it
    • About 20% of patients will relapse within 2 years
    • There is a 1% risk of serious, life-threatening complications
    • Recovery takes about 3 months
    • No proven impact on survival
    • Advantages
      • The patient will be able to conserve their quality of life and have minimal disruption to their current lifestyle
      • The patient can hedge their bet against future relapse (that is, better treatments may be available later on)
      • About 60% to 70% of patients will relapse from their initial treatment and will need ASCT within 2 to 3 years
      • Recovery will be harder than after an early transplant

      Ultimately, patients and their doctors should consider specific factors relating to their disease burden, comorbidities (like diseases that affect the heart, lung, liver, or kidneys), and other personal factors when making the decision on when to undergo ASCT. For example, if a patient has a high disease burden and high-risk features at diagnosis, then an ASCT is warranted in order to treat the myeloma as aggressively as possible early on in the disease course. Alternatively, if a patient has standard-risk disease and has had a major response to initial therapy, waiting for an ASCT until relapse may be the best option.

      Are myeloma patients with high-risk disease treated differently than patients with standard-risk disease?
      Risk assessment in myeloma patients is typically based on a set of clinical factors such as results from routine blood tests that include beta-2 microglobulin, albumin , and lactate dehydrogenase. In addition to these blood tests, routine cytogenetic analysis of myeloma cell chromosomes by fluorescence in situ hybridization (FISH) provides information on whether a patient has chromosomal abnormalities (such as chromosomal deletions or translocations). Patients with high levels of the routine blood markers and the presence of chromosomal abnormalities are considered high-risk and this group of patients, overall, is associated with the poorest outcomes. Unfortunately, treatment advancements made in myeloma have not helped to improve outcomes for patients considered high risk. High-risk patients typically do not have a long-lasting response to initial therapy (that is, they relapse quickly following treatment).

      There is no standard treatment approach for high-risk patients and most myeloma experts will pursue an aggressive treatment plan typically consisting of a 4-drug induction regimen followed by autologous stem cell transplantation (ASCT) and combination maintenance therapy (typically Revlimid plus one other agent). Alternately, high-risk patients are encouraged to enroll in a clinical trial. One trial that is enrolling high-risk patients is the MMRC MyDRUG trial. In this trial, patients considered functionally high-risk&mdashthat is, they have relapsed less than 18 months since initial therapy (and no ASCT) or less than three years on maintenance therapy with Revlimid following ASCT &ndash undergo genome sequencing, a test that analyzes a patient&rsquos myeloma genetic structure and can reveal the presence of cancer-related mutations. Based on the results, patients are matched to a specific treatment that targets their unique myeloma mutation and assigned to a branch of the MyDRUG trial that is studying that treatment. In addition to receiving the specific, mutation-matched treatment, all patients are given the standard-of-care regimen of Ninlaro + Pomalyst + dexamethasone . High-risk patients with no mutations detected receive a four-drug combination that includes Ninlaro-Pomalyst- dexamethasone and Darzalex, Xpovio, or Blenrep&mdasha treatment that is otherwise unavailable. To learn more about joining the MyDRUG trial, contact a Patient Navigator at the MMRF Patient Support Center at 888-841-6673.

      MMRF Patient Summit 3/20/21

      This web conference provides multiple myeloma patients and their families and caregivers the most up-to-date information on myeloma management from doctors and other experts in the field. View Patient Summit now.


      Reactive oxygen species (ROS) induction is an effective mechanism to kill cancer cells for many chemotherapeutics, while resettled redox homeostasis induced by the anticancer drugs will promote cancer chemoresistance. Natural ent-kaurane diterpenoids have been found to bind glutathione (GSH) and sulfhydryl group in antioxidant enzymes covalently, which leads to the destruction of intracellular redox homeostasis. Therefore, redox resetting destruction by ent-kaurane diterpenoids may emerge as a viable strategy for cancer therapy. In this study, we isolated 30 ent-kaurane diterpenoids including 20 new samples from Chinese liverworts Jungermannia tetragona Lindenb and studied their specific targets and possible application in cancer drug resistance through redox resetting destruction. 11β-hydroxy-ent-16-kaurene-15-one (23) possessed strong inhibitory activity against several cancer cell lines. Moreover, compound 23 induced both apoptosis and ferroptosis through increasing cellular ROS levels in HepG2 cells. ROS accumulation induced by compound 23 was caused by inhibition of antioxidant systems through targeting peroxiredoxin I/II (Prdx I/II) and depletion of GSH. Furthermore, compound 23 sensitized cisplatin (CDDP)-resistant A549/CDDP cancer cells in vitro and in vivo by inducing apoptosis and ferroptosis. Thus, the ent-kaurane derivative showed potential application for sensitizing CDDP resistance by redox resetting destruction through dual inhibition of Prdx I/II and GSH in cancer chemotherapy.


      Vision begins with the image focused on the retina at the back of the eye. One way of examining the first stage of processing of that image is to measure ability to detect elementary changes in light and dark across the image because the image probably also varies with time, we also measure the effects of different rates of change, to approximate changes that might occur in real-world viewing. We are dealing here with the effects of sex on these elementary visual sensations.

      At least one sex-effect is well-known. Color vision depends on three types of cone photoreceptors in the retina: some are more sensitive to the longer wavelengths of light (L-cones), some to the middle wavelengths (M-cones), and some to the shorter wavelengths (S-cones). The genes coding for two of these cone photoreceptors (L- and M-cones) are carried on the X-chromosome and any malformation of either gene in a female is necessarily expressed in the phenotype of a male offspring who inherits that gene. However, it seems to be tacitly assumed that there are no other major sex effects on visual capacities. (But see companion paper [1], for sex effects on color appearance.) Of course, major clinically oriented surveys that deal mostly with acuity and optical issues test large samples from a population and examine many variables, including sex. For example: this was done in the Hispanic Health and Nutrition Examination Survey of Hispanic and non-Hispanic populations – in fact this study found no significant sex-related acuity differences in children and adolescents [2]. However, other studies of visual acuity have reported significant sex differences in adults [3, 4].

      Despite mandates of major granting agencies to include, where possible, equal numbers of males and females in studies, little attention is paid to sex differences. For example, we examined all papers published in 2010 in a major journal, Vision Research. We tabulated all studies that used psychophysical or physiological measures to test humans’ basic capacities, such as spatial and temporal resolution, stereopsis, motion detection, and so on. We identified 410 such studies, of which only 96 (23.4%) broke down their samples into males and females. Also, at least in this sample of publications, only 4 studies (less than 1%) used sex as a comparison variable.

      The lack of attention to sex differences in vision is surprising given the considerable body of work comparing male and female visually based perceptual and cognitive abilities: for example, females are said to make many more fine distinctions among colored objects [5, 6]. But if the initial information is processed differently according to sex, at least some of the “higher” differences might be due to differences in the results of this processing. Despite this possibility, little attention has been lavished on sex differences in basic visual sensations that are determined at “early” stages of the eye and brain.

      Sex differences have been examined in other sensory systems: all showed clear sex differences. In the auditory system, there were differences in electrical responses of the brain (auditory evoked potentials) also ears of females were more likely to produce spontaneous emissions of sounds from the ear (otoacoustic emissions), and in those females, hearing sensitivity was better than in males. All these differences could be related to the masculinizing effects of androgens [7–9]. Similarly, for the olfactory system, it seems clear that, in most cases, females had better sensitivity, and discriminated and categorized odors better than males [10, 11]. The general conclusion is that for audition and olfaction, as well as taste and somato-sensory sensitivity, females have greater sensitivity than males [12].

      The substrate for these sex differences may be linked directly to gonadal steroid hormones: in rhesus monkeys, large numbers of androgen receptors are found on neurons throughout the cerebral cortex, including visual cortex. This androgen binding “may have considerable impact on cortical functioning in primates at postnatal as well as prenatal ages” [13]. There are similar findings for rats, in whom males have more androgen receptors than females, and these are especially plentiful in primary visual cortex [14]. A recent review has reiterated these findings and concluded that in both humans and rats the largest concentration of androgen receptors in the forebrain is in the cerebral cortex and not the hypothalamic and limbic areas associated with reproduction [15]: these findings might be general across mammals. All the above authors strongly emphasize that the distribution of androgen binding receptors may have considerable impact on cortical development and maturation of visual functioning.

      Furthermore, in rats, androgens, but not estrogen, directly modify development of the visual cortex. Androgens reduce the early post-natal cell-death (apoptosis) of the visual cortex as a consequence males have 20% more neurons in the visual cortex [16, 17]. This organizational effect is androgen-specific: early exposure of female rats to androgens (implanted capsules of dihydrotestosterone) led to these effects early exposure to estrogen (implanted capsules of estradiol) did not inhibit post-natal cell-death [16].

      Factors other than gonadal receptors may also be involved [18]. Theoretically, females might have a double “dose” of sex-related genes. To compensate for this, one of each pair of X-chromosomes is silenced [19]. Many humans have multiple L and M genes – we are polymorphic for these genes [20, 21]. As a consequence, different retinal areas might express different alleles, affecting the responses of these areas and the brain sites associated with different retinal areas. Moreover, the X-chromosome may have a loading of “male-benefit” genes: thus, any recessive alleles must, of necessity, be expressed in a male [18]. Furthermore, some of the sex effects we report here could be either organizational or activational and could depend on estrogen rather than testosterone they could even be due to other sex-related genes [22].

      In short, it seems highly unlikely that vision will differ from other sensory modalities that show sex differences in function. It seems parsimonious to assume that the plethora of androgen receptors and androgen effects in primary visual cortex exert a measurable and important impact on visual sensory capacities.

      We study differences between males and females in basic visual functions. The work we report here is not directly hypothesis-driven – it is exploratory. Our weak hypothesis is based on the fact that males have higher levels of androgens and that there are large numbers of testosterone receptors in visual cortex: therefore we anticipate sex differences. We cannot formulate a strict hypothesis because we cannot manipulate testosterone levels, nor have we measured them. This problem is endemic to most studies of sex differences in adult populations and is exacerbated by the possibility that some of the differences might not even be androgen driven.

      We have developed a Battery of Visual Tests designed to study different visual sensory capacities, each with state-of-art precision. Tests include: (i) ability to resolve/detect patterns that vary in space (from coarse to fine detail ) and time (from very fast to very slow rates of change) -- these measures determine the spatio-temporal contrast-sensitivity function (ii) detecting a small offset between two lines (vernier acuity) (iii) motion detection (iv) binocular depth perception (stereopsis) and (v) color vision -- especially color appearance (see [1]). One of the reasons for choosing these specific tests is that each emphasizes a different level and locus in the central nervous system. Specifically, the contrast-sensitivity function probably depends on the responses of neurons in primary visual cortex (discussed below) motion detection probably depends on responses of neurons in area MT of the temporal lobe and some aspects of color appearance depend on an intact infero-temporal cortex (e.g., [23, 24]).

      In this paper we report on sex differences in spatio-temporal contrast-sensitivity. Spatial contrast-sensitivity (S-CSF) describes the visual system’s ability to perceive changes in brightness across space, as in reading a letter on an eye chart, or recognizing a face. The details of our procedures are given below.

      The protocol for each measure in our Battery of Visual Tests is rigidly controlled. A consequence is that the thresholds we record from individuals now are precisely comparable to those recorded from individuals tested years ago. To minimize run-time errors, either the apparatus as a whole or the run-time stimulus sequence and data collection are computer-controlled. When these thresholds are measured, a participant adopts an internal criterion for reporting when a stimulus is visible unfortunately these criteria vary across time within and among participants. Where possible, therefore, we use criterion-free psychophysical procedures: the stimulus is presented in one of two alternative positions or time intervals after each presentation participants must choose where the stimulus occurred, regardless of confidence in their choice stimulus strength is reduced from trial to trial until correct responding reaches chance levels. This “forced-choice” procedure is criterion free. In practice, we use a modified Bayesian approach to vary the stimuli in order to find threshold trials are continued until the estimates of threshold reach an asymptote at a specified (99%) statistical confidence level (QUEST algorithm [25]). Also, we attempt to test each participant on the entire battery of tests to examine possible interactions among different capacities – those capacities whose thresholds are correlated presumably share a neuronal substrate.

      We have amassed large databases from participants who participated in most or all of the Battery’s tests. We now have sufficient data to examine possible sex effects. Even if effect magnitudes are small or subtle, they cannot be ignored because they point to our hypothesized developmental and maturational effects of gonadal hormones on very basic sensory functions.

      The following describes how, in the laboratory, we measure the S-CSF: we use grating patterns (alternating light and dark bars) the gratings vary from broad, coarsely spaced bars to fine, closely spaced bars. On each trial the grating pattern is either horizontal or vertical and the participant must choose between these alternative orientations. The spacing of the bars is measured as the number of light–dark cycles within one degree of visual space (cy/deg). The change from light to dark bars follows a sinusoidal profile in which intensity is varied symmetrically above and below a mean gray level the difference between maximal and minimal intensities of the pattern is reduced until the pattern is no longer detectably different from a uniform gray – that is, the grating’s contrast is reduced until the pattern is at threshold.

      The gratings had sinusoidal profiles because any image can be synthesized from (is equivalent to) a specific set of sinusoids of different frequencies, amplitudes, and phases (Fourier’s theorem). Limiting measures of spatial resolution only to the finest detail that can be perceived (acuity) is akin to testing auditory capacities using only the top end of the piano keyboard. High spatial frequencies are indeed important for discriminating among images that are similar to each other. However, sensitivity to the lower spatial frequencies greatly influences our ability to recognize and categorize parts of the image that refer to the diverse objects on the visual field [26–28].

      Similarly, temporal contrast-sensitivity (T-CSF) refers to the visual system’s ability to perceive changes in brightness over time, as when the stimulus flickers, or the retinal image slips across the retina due to object and/or viewer motion. However, S- and T-CSFs are strictly non-separable functions: the precise function that is obtained depends on the values of both sets of parameters – these define an entire three-dimensional sensitivity surface. Kelly, an early advocate of this non-separability [29, 30], used gratings of each spatial frequency which moved horizontally at a fixed velocity. This was impractical in our situation so we chose to measure the entire spatio-temporal (ST-CSF) surface using a series of spatial gratings, each of whose contrasts was modulated at each of a fixed series of temporal rates. The time-profile of the modulation was sinusoidal. The units for these temporal rates are variation-cycles/s (Hz).


      Apoptotic cell death is commonly induced in IV-infected cells and can be seen as a cellular response to limit spread of infection. Apoptotic signaling events are manipulated or triggered by defined viral gene products such as NS1, PB1-F2, or M2, respectively, which coevolved together with these cellular antiviral defense strategies. IVs thereby acquired the ability to make use of early and late events of the apoptotic machinery to increase their propagation efficiency and at the same time, to limit the early antiviral immune response of the cell. Manipulation of death pathways occurs in a viral strain- and host cell-specific manner, and the underlying molecular virus–host interactions are still poorly defined. Furthermore, apoptosis induced by ligand binding to DRs, expressed on immune and parenchymal respiratory cells, is a key mechanism of the cell-mediated immune response toward IVs however, it may cause substantial lung tissue injury.

      In conclusion, apoptotic signaling exerts pleiotropic functions during the antiviral immune response induced by IAV infection. In light of the current data, apoptosis induction in the infected host has to be seen primarily as an antiviral response to fight the invading pathogen. However, during long-term evolution of the virus, those variants may have been selected, which could take advantage of the existing activities or could even further manipulate these activities in the cell or organism for enhanced replication and spread. Thus, what we observe today as a result of this evolutionary process is the delicate balance between antiviral defense of the host and its suppression or even exploitation by the virus. This occurs on both a cell-intrinsic and systemic level. It will be a challenge in future analyses to understand in detail how the different molecular mechanisms described here contribute to the complex overall process by which the host and the virus balance the extent of apoptosis and immune activation in the respiratory tract to clear a pathogenic IAV without causing an overwhelming immunopathologic response. Exploring these pathways will define future targets for therapeutic interventions to limit IAV replication, to increase efficiency of the antiviral immune response, and to attenuate collateral lung damage during severe IAV infection.