The second cell membrane: the basal lamina and the tissue cell theory of metazoa.

We suggest that the basal lamina is essentially a second plasma or cell membrane appearing at the next higher level of biological organization; that together with associated cell monolayers it creates a tissue level membrane which is used to form multicellular cells and that collections of these provide the essential structure of metazoa. Thus when the histological structure of multicellular organisms is viewed in a topologically simplified form such organisms appear to be sets of multicellular cells (m-cells) formed by a unit tissue membrane built around the basal lamina. Not only are m-cells in this way structurally isomorphous (homeomorphic) to unit or classical biological cells (u-cells) but the two cellular levels are also functionally isomorphous. This suggests a “General Principle of Hierarchical Isomorphism or Iteration”, i.e. that multicellular evolution recapitulates unicellular evolution. This principle of structural and functional isomorphic mappability of unicellular onto multicellular organisms then governs the organization of matter all the way from molecules to man. Just as cytoplasm precipitates the bimolecular plasma membrane to form u-cells for the purpose of achieving reaction sequestration, in turn, these u-cells precipitate a common basal lamina to form m-cells, the histologist's acini, to produce sequestered “tissue plasms”. Thus, the “generalized acinus” with its basal laminar complex seem to constitute a second level (multicellular) cell and cell membrane, respectively.Four operators, ultimately under genetic control, can generate both u and m-cells from planar configurations of their respective unit membranes therewith providing the essential structure of all cells, tissues, organs and organisms. These are the ply, permeability vector, topological and stratificational operators. They are collected into a set of “organ formulae”. Both the plasma membrane and the basal lamina act as covering membranes and, again, as membranes for subcells so that a complete multicellular organism is a tetrahierarchical cell in which the molecule is the element of the first two cellular domains and the cell is the element of the last two. The analysis identifies a new transport organ group which together with the classical endocrine and exocrine groups comprises nearly the whole of the soft tissue organs. In a major reduction, all these organs are continuously (topologically) transformable into each and into hollow spheres, cells or acini thus greatly simplifying the histology of metazoa. Given this emphasis on cellularization it would seem that life, i.e. the autonomous chemoservo, results from the cooperation of cellularization and replication operations on the catalyzation process. Through cellularization, the lipid bilayer and basal laminar membranes provide the essential catalytic reaction sequestration demanded by chemical reaction theory while through complementary base pairing the DNA double helix provides the essential memory which stores the patterns of the variations of the sequestered reactions.     

REVIEW: Is the “flagellate” pattern of spermatogenesis plesiomorphic in Metazoa?

Summary

The phenomenon of “flagellate spermatogenesis” typically known among marine invertebrates with “primitive” sperm and external or external-internal fertilization is discussed. It is suggested that “flagella bearing” in early germinative cells might be explained by plesiomorphic similarity between these cells and flagellate somatic epithelial cells. The early germ cells of more apomorphic multicellular animals using internal fertilization with “modified” and “aberrant” sperm typically have no flagella and this organelle, as the sperm tail, first appears in spermatids. It is speculated that the “flagellate” pattern typifies the basal level and that the transition between “flagellate” and “specialized” spermatogenesis constitutes a significant step in evolution.     

Programmed Cell Death–Ligand 1 Overexpression in Thyroid Cancer

Objective: Programmed cell death–ligand 1 (PD-L1) expression on tumor tissue has been associated with favorable response to anti–programmed cell death–receptor 1/PD-L1 therapy in many human cancers. Studies have reported that PD-L1 is also expressed in thyroid cancer. The objective of this paper is to introduce the potential predictive and therapeutic values of PD-L1 in thyroid cancer.Methods: A literature search was conducted in the PubMed database using the terms “PD-L1,” “B7-H1,” and “thyroid cancer.” PD-L1 positivity was determined by immunohistochemical assay.Results: The frequency of PD-L1 positivity in different studies ranged from 6.1 to 82.5% in papillary thyroid cancer (PTC) patients and 22.2 to 81.2% in anaplastic thyroid cancer (ATC) patients. PD-L1 positivity rate was higher in ATC than in PTC within the same studies, and its expression intensity was significantly higher in tumor tissue than in the corresponding nontumor thyroid tissues. Moreover, PD-L1 expression was positively associated with the aggressiveness and recurrence of thyroid cancers and negatively associated with the differentiation status and outcomes. PD-L1 checkpoint pathway blockade may emerge as a promising therapeutic target in the treatment of thyroid cancers.Conclusion: PD-L1 is a potential biomarker to predict the recurrence and prognosis of thyroid cancers. It is also a novel immunotherapy target for optimizing the management landscape of radioiodine-refractory and ATCs.Abbreviations: ATC = anaplastic thyroid cancer; DTC = differentiated thyroid cancer; IHC = immunohistochemical; OS = overall survival; PD-1 = programmed cell death–receptor 1; PD-L1 = programmed cell death–ligand 1; PD-L2 = programmed cell death–ligand 2; PTC = papillary thyroid cancer; TNM = tumor-node-metastasis; Treg = regulatory T cell     

The costs and benefits of multicellular group formation in algae*

The first step in the evolution of complex multicellular organisms involves single cells forming a cooperative group. Consequently, to understand multicellularity, we need to understand the costs and benefits associated with multicellular group formation. We found that in the facultatively multicellular algae Chlorella sorokiniana: (1) the presence of the flagellate Ochromonas danica or the crustacean Daphnia magna leads to the formation of multicellular groups; (2) the formation of multicellular groups reduces predation by O. danica, but not by the larger predator D. magna; (3) under conditions of relatively low light intensity, where competition for light is greater, multicellular groups grow slower than single cells; (4) in the absence of live predators, the proportion of cells in multicellular groups decreases at a rate that does not vary with light intensity. These results can explain why, in cases such as this algae species, multicellular group formation is facultative, in response to the presence of predators.     

Can tumorigenesis result from in vivo “transfection” of a normal cell with DNA escaped from disintegrating nontransformed cells?

In this paper I present a hypothetical step on the route to turmorigenesis which may be common to many of the tumorogenic events taking place in vivo. According to this hypothesis portions of DNA from normal nontransformed dying cells may escape degradation, “transfect” other cells and under appropriate conditions also induce transformation. When various high risk factors for carcinogenesis are examined this hypothesis may easily fit into each of them. In addition, recent reports of experimental “transfection” indirectly support this hypothesis. It could now be argued that aging, ionizing radiation, immunosuppressive drugs, genetic errors, defects in DNA repair, immunodeficiency states, chronic infections and chronic inflammatory diseases may all increase the availability of free DNA or the readiness of cells to accommodate “transfection” DNA, or both. Thus the risk of cancer associated with these situations may be explained by increased chance for “transfection” with DNA.It is suggested that the minimal requirements for cell transformation consist of a certain sequence of DNA which does not have to be integrated into the nucleus at once, it may instead accumulate piece by piece so that the first “transfection” of a cell may occur long before transformation takes place. If the “transfecting” DNA sequence does not fulfil the minimal requirements for maintaining a state of repetitive uncontrolled mitosis, then this cell may wait silently or remain a benign tumor until “boosted” with an ultimate complementary DNA sequence to develop into a fully fledged cancer.     

Will the clinical development of 4th-generation “double mutant active” ALK TKIs (TPX-0131 and NVL-655) change the future treatment paradigm of ALK+ NSCLC?

Our current treatment paradigm of advanced anaplastic lymphoma kinase fusion (ALK+) non-small cell lung cancer (NSCLC) classifies the six currently approved ALK tyrosine kinase inhibitors (TKIs) into three generations. The 2nd-generation (2G) and 3rd-generation (3G) ALK TKIs are all “single mutant active” with varying potencies across a wide spectrum of acquired single ALK resistance mutations. There is a vigorous debate among clinicians which is the best upfront ALK TKI is for the first-line (1L) treatment of ALK+ NSCLC and the subsequent sequencing strategies whether it should be based on the presence of specific on-target ALK resistance mutations or not. Regardless, sequential use of “single mutant active” ALK TKIs will eventually lead to double ALK resistance mutations in cis. This has led to the creation of fourth generation (4G) “double mutant active” ALK TKIs such as TPX-0131 and NVL-655. We discuss the critical properties 4G ALK TKIs must possess to be clinically successful. We proposed conceptual first-line, second-line, and molecularly-based third-line registrational randomized clinical trials designed for these 4G ALK TKIs. How these 4G ALK TKIs would be used in the future will depend on which line of treatment the clinical trial design(s) is adopted provided the trial is positive. If approved, 4G ALK TKIs may usher in a new treatment paradigm for advanced ALK+ NSCLC that is based on classifying ALK TKIs based on the intrinsic functional capabilities (“singe mutant active” versus “double mutant active”) rather than the loosely-defined “generational” (first-, second-,third-,fourth-) classification and avoid the current clinical approaches of seemingly random sequential use of 2G and 3G ALK TKIs.     

Capacity of different cell types to prime in vivo for secondary in vitro cytotoxic T-cell responses against non-major-histocompatibility antigens

The capacity of several types of cell preparations to induce in vivo a state of memory for a secondary in vitro cytotoxic response against non-major-histocompatibility antigen was markedly reduced (on a per cell basis) by uv-irradiation. This indicated that memory induction requires metabolically active stimulator cells. An “adherent cell preparation” (AC) that was enriched for dendritic cells was among the most effective memory-inducing cell populations; but concanavalin A-activated nylon-wool-nonadherent spleen cells (Con A-NWT) or concanavalin A-activated unfractionated spleen cells (Con A-spl) were on the average equally effective. Normal unfractionated spleen cells (spl) or nonactivated nylon-wool-nonadherent cells (NWT) were markedly less effective on a per cell basis. This pattern of stimulatory activity was in line with the relative stimulatory activity of these cell types in primary cytotoxic responses in the presence of interleukin 2 (IL-2) and also in line with the relative capacity to induce IL-2-dependent proliferation in H-2D-incompatible T-cell populations (cf. W. Dröge et al., J. Immunol.132, 2749, 1984). These differences in the immunogenic potential and the requirement for metabolically active stimulator cells suggested that these cells stimulated the CTL system directly and not indirectly through antigen processing cells of the immunized host. Nevertheless, the secondary cytotoxic response after injection of low numbers of Con A-spl into H-2 heterozygous recipients, (BALB/c × BALB/b)F1, or into recipients with recombinant H-2 haplotype (A.J) was only preferentially but not exclusively restricted to the H-2 haplotype of the immunizing cell populations. Restriction was considerably more complete when AC were used for immunization.     

Cellular interactions in lymphocyte proliferation: effect of syngeneic and xenogeneic macrophages.

Secondary cell-mediated responses to ectromelia virus infection were studied using an in vitro system. Lymphoid “responder” cells from mice which had recovered from intravenous primary infection at various times prior to sacrifice, were cultured with syngeneic, virus-infected macrophages or spleen cells as “stimulator” cells at 39 °C, a temperature which prevented the virus from exerting cytopathic effects against responder cells. This restrictive temperature and medium with 2-mercaptoethanol at 10^?4M often gave viable cell yields of more than 100% of the original responder cells over 4 days of culture. Preliminary experiments showed that spleen cells from primed mice, cultured with syngeneic, infected spleen cells from normal mice gave the most powerful secondary cytotoxic cell responses as measured by ⁵¹Cr release from virusinfected H-2-compatible target cells. The cytotoxic cells were sensitive to anti-θ and complement treatment and lysed H-2-compatible, virus-infected target cells much more efficiently than infected, allogeneic target cells, thus indicating that they were T cells. Some activity against uninfected H-2-compatible target cells was also generated, but this was largely independent of the presence of virus-induced antigen, (i.e. infected stimulator cells were unnecessary) and therefore seemed to be a consequence of the cultural conditions. Cold target competition showed that this activity was the responsibility of a T cell subset separate from the virus-specific cytotoxic T cells. The peak of cytotoxic activity against virus-infected targets occurred at 4 days of culture and DNA synthesis was maximal on day 3. The concentration of cytotoxic T cells at the peak was eight-fold higher than at the peak of the splenic primary response in vivo, Memory T cells (precursors of secondary cytotoxic T cells) appeared in spleen within 12–14 days of primary infection in vivo, reached a plateau at 5–6 weeks and persisted for at least 16 months. Spleen cells appeared partly refractory to secondary stimulation in vitro at 8–10 days post-priming. This did not seem to be due to cellular migration from spleen to lymph nodes or peritoneal cavity, but its cause was not determined. Primary responses in vitro were not detectable under conditions optimal for secondary responses, thus suggesting a major quantitative, or qualitative difference between virgin and memory T cells.     

The human LT system. IV. Studies on the large MW LT complex class: association of these molecules with specific antigen binding receptor(s) in vitro.

The present studies demonstrate that a portion of lymphotoxin (LT) cell-lytic activity present in supernatants from: 1) lectin (Con A, PHA) stimulated nonimmune; or 2) antigen (soluble or cellular) stimulated immune human lymphocytes in vitro, is associated with immunoglobulin (Ig) or “Ig-like” receptor molecule(s). This concept was supported by three findings: 1) LT activity in these supernatants was partially inhibited by heterologous anti-human (IgG) Fab′₂ antisera; 2) LT activity present in soluble antigen stimulated immune human lymphocyte supernatants could specifically bind to and be eluted from Sepharose 4B columns to which the specific stimulating antigen was covalently attached; and 3) LT activity present in primary one-way mixed lymphocyte culture (MLC) supernatants could be removed by absorption on the specific stimulator cells. The amount of total LT activity found to be associated with “Ig” in these supernatants was variable, but ranged from 5 to 20% in lectin stimulated cell supernatants to 20 to 50% in antigen or MLC stimulated supernatants. Physical-chemical studies on the molecular weight class of LT molecules having reactivity with anti-Fab′₂ sera, as well as antigen binding capacity, revealed these properties reside in the large (>200,000) MW LT class, termed complex. The nature and biological significance of these “antigen specific” LT complexes, as they relate to mechanisms of cytotoxicity in vitro, will be discussed.     

Insect Seed Predators in Erythrina falcata (Fabaceae): Identification of Predatory Species and Ecological Consequences of Asynchronous Flowering

Seed predation by insects exerts negative effects on plant reproduction by limiting the supply of seeds and preventing germination. Seed predators of the family Fabaceae are usually generalists, which increases the rate of predation. One strategy to minimize seed predation, developed by plants from temperate regions, is “escape in time,” i.e., flowering before or after the peak of predation. For tropical species, few studies have investigated the strategies used by plants to minimize seed predation. Here, using Erythrina falcata, a tropical species of Fabaceae, we test three main hypotheses: (i) escape in time is a mechanism used by E. falcata to minimize seed predation, (ii) the predators of E. falcata seeds are generalists, and (iii) the biometric variables of the pods can influence seed predation. In order to test these hypotheses, we determined the flowering time of E. falcata, rate of seed predation, the predators insects, and biometric variables of the pods. The analyzed trees were grouped into three classes: “early,” “peak,” and “late” flowering. The average seed predation rates on trees in the early and late classes were 65% and 50%, respectively, and in the peak class, 80%; thus, our first hypothesis can be accepted. Three species of Lepidoptera and two of Coleoptera were found preying on E. falcata seeds. These species were observed to be generalist predators; thus, our second hypothesis can be accepted. The biometric variables of the pods cannot influence seed predation rate. The ecological consequences of asynchronous flowering on plants and insects are discussed.     

Updated Review of Nuclear Molecular Imaging of Thyroid Cancers

ObjectivesWe initiate this comprehensive review to update the advances in this field by objectively elucidating the efficacies of promising radiopharmaceuticals.MethodsWe performed a comprehensive PUBMED search using the combined terms of “thyroid cancer” and “radiopharmaceuticals” or “nuclear medicine”, yielding 3273 and 11026 articles prior to December 31, 2020, respectively.ResultsBased on the mechanism of molecular metabolism, the evaluation of differentiated thyroid cancer and dedifferentiated thyroid cancer is largely centered around radioiodine and fluorine 18 (¹⁸F)-fludeoxyglucose, respectively. Further, ¹⁸F-L-dihydroxyphenylalanine and gallium 68 DOTATATE are the preferred tracers for medullary thyroid cancer. In dedifferentiated medullary thyroid cancer and anaplastic thyroid cancer, ¹⁸F-fludeoxyglucose is superior.ConclusionsThe future lies in advances in molecular biology, novel radiopharmaceuticals and imaging devices, paving ways to the development of personalized medication for thyroid cancer patients.     

Which aging in yeast is “true”?

Two model systems, “replicative aging” and “chronological aging” (CA), which are used for gerontological research on the yeast Saccharomyces cerevisiae, are compared. In the first case, the number of daughter cells generated by an individual mother cell before cell propagation irreversibly stops is analyzed. This makes the model very similar to the well-known Hayflick model. In the case of CA, the survival of yeast cell population in the stationary phase of growth is studied. It is noted that the second model is similar to the “stationary phase aging” model, which is used in the author’s laboratory for cytogerontological studies on animal and human cells. It is assumed that the concept of cell proliferation restriction as the main cause of age-related accumulation in the cells of multicellular organisms of macromolecular defects (primarily DNA damage) leading to deterioration of tissue and organ functioning and, as a result, to an increase in the death probability allows explaining how the aging process proceeds in almost any living organisms. Apparently, in all cases, this process is initiated by the appearance of slow propagating (or not propagating at all) cells, which leads to the termination of “dilution,” with the help of new cells, of macromolecular defects accumulating at the level of whole cell population. It is concluded that data on the geropromoter or geroprotector activity of various factors obtained in tests on the yeast CA model can be used with a high reliability to understand the mechanisms of human aging and longevity.     

CXCR4 Expression and Treatment with SDF-1α or Plerixafor Modulate Proliferation and Chemosensitivity of Colon Cancer Cells

BACKGROUND: Signaling through stromal cell-derived factor-1α (SDF-1α), strongly secreted by bone marrow stromal cells and the CXC chemokine receptor 4 (CXCR4) exposed on tumor cells has pivotal roles in proliferation, metastasis, and tumor cell “dormancy.” Dormancy is associated with cytostatic drug resistance and is probably a property of tumor stem cells and minimal residual disease. Thus, hampering the SDF-1α/CXCR4 cross talk by a CXCR4 antagonist like Plerixafor (AMD3100) should overcome tumor cell dormancy bymobilization of tumor cells from “sanctuary” niches. Our aim was to elucidate the direct effects exerted by SDF-1α and Plerixafor on proliferation, chemosensitivity, and apoptosis of CXCR4-expressing tumor cells. METHODS: The ability of SDF-1α and Plerixafor to regulate intracellular signaling, proliferation, and invasion was investigated using two colon cancer cell lines (HT-29 and SW480) with either high endogenous or lentiviral expression of CXCR4 compared to their respective low CXCR4-expressing counterparts as a model system. Efficacy of Plerixafor on sensitivity of these cell lines against 5-fluorouracil, irinotecan, or oxaliplatin was determined in a cell viability assay as well as stroma-dependent cytotoxicity and apoptosis assays. RESULTS: SDF-1α increased proliferation, invasion, and ERK signaling of endogenously and lentivirally CXCR4-expressing cells. Exposure to Plerixafor reduced proliferation, invasion, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. Combination of chemotherapy with Plerixafor showed an additive effect on chemosensitivity and apoptosis in CXCR4-overexpressing cells. An SDF-1-secreting feeder layer provideda“protective niche” for CXCR4-overexpressing cells resulting in decreased chemosensitivity. CONCLUSION: CXCR4-antagonistic therapy mobilizes and additionally sensitizes tumor cells toward cytoreductive chemotherapy.     

Metabolic control analysis indicates a change of strategy in the treatment of cancer

Much of the search for the “magic cancer bullet” or “block buster” has followed the expectation of a single gene or protein as “the rate-limiting step” for tumor persistence. Examples continue to abound: EGFR, VEGFR, Akt/PI3K, HIF-1α, PHD, PDK, or FAS continue to be targeted individually. However, many such attempts to block a metabolic or signal transduction pathway by targeting, specifically, a single rate-limiting molecule have proven to be unsuccessful. Metabolic control analysis (MCA) of cancer cells has generated a generic explanation for this phenomenon: several steps share the control of energy metabolism (for glycolysis: glucose transporter, hexokinase, glycogen synthesis and ATP demand; for oxidative phosphorylation: respiratory complex I and ATP demand), i.e., there is no single “rate-limiting step”. Targeting a type of step that does not exist is unlikely to be a successful paradigm for continued research into drug targeting of cancer.MCA establishes how to determine, quantitatively, the degrees of control that the various enzymes in the intracellular network exert on vital flux (or function) and on the concentration of important metabolites, substituting for the intuitive, qualitative and most often erroneous concept of single rate-limiting step. Moreover, MCA helps to understand (i) the underlying mechanisms by which a given enzyme exerts high or low control, (ii) why the control of the pathway is shared by several pathway enzymes and transporters and (iii) what are the better sets of drug targets. Indeed, by applying MCA it should now be possible to identify the group of proteins (and genes) that should be modified to achieve a successful modulation of the intracellular networks of biotechnological or clinical relevance. The challenge is to move away from the design of drugs that specifically inhibit a single controlling step, towards unspecific drugs or towards drug mixtures, which may have multiple target sites in the most exacerbated, unique and controlling pathways in cancer cells. Successful nonspecific drugs should still be specific for the networks of cancer cells over those of normal cells and to establish such cell-type specificity within molecular non-specificity will continue to require sophisticated analyses. Clinical practice has anticipated the latter strategy of mixtures of drugs: combinations of anti-neoplastic drugs are already administered with encouraging results. Therefore, the most promising strategy for cancer treatment seems to be that of a multi-targeted, MCA-advised, therapy.     

Personalized In Vitro Cancer Modeling — Fantasy or Reality?

With greater technological advancements and understanding of pathophysiology, “personalized medicine” has become a more realistic goal. In the field of cancer, personalized medicine is the ultimate objective, as each cancer is unique and each tumor is heterogeneous. For many decades, researchers have relied upon studying the histopathology of tumors in the hope that it would provide clues to understanding the pathophysiology of cancer. Current preclinical research relies heavily upon two-dimensional culture models. However, these models have had limited success in recreating the complex interactions between cancer cells and the stroma environment in vivo. Thus, there is increasing impetus to shift to three-dimensional models, which more accurately reflect this phenomenon. With a more accurate in vitro tumor model, drug sensitivity can be tested to determine the best treatment option based on the tumor characteristics. Many methods have been developed to create tumor models or “tumoroids,” each with its advantages and limitations. One significant problem faced is the replication of angiogenesis that is characteristic of tumors in vivo. Nonetheless, if three-dimensional models could be standardized and implemented as a preclinical research tool for therapeutic testing, we would be taking a step towards making personalized cancer medicine a reality.     

Characterization of oligosaccharides of highly purified glycoprotein gC of herpes simplex virus type 1 (HSV-1)

The envelope membrane glycoprotein gC of HSV-1 was purified from Triton X-100 extracts of virus-infected BHK-21 or HEp-2 cells by a single step immuno-affinity column using monoclonal anti-gC antibody. The analysis of the purified [³H]G1cN labeled glycoprotein gC (by gel filtration on Bio-Gel P4) before and after digestion with endo-β-N-acetylglucosaminidase (endo D) indicated that gC contains Asn-linked “complex type” oligosaccharides. No “high mannose” type oligosaccharides were detected. Fractionation of radio-labeled glycopeptides of gC on a column of concanavalin A-sepharose suggested that glycopeptides have “diantennary” and “triantennary” and/or “tetra antennary” structures. Tunicamycin inhibited the incorporation of [¹⁴C]GalN or [³H]GlcN into gC in HSV-1 infected BHK-21 or HEp-2 cells. Gel filtration analysis of [³H]GlcN labeled gC following β-elimination reaction failed to indicate O-glycosidically linked oligosaccharides.     

Multicellular group formation in response to predators in the alga Chlorella vulgaris

A key step in the evolution of multicellular organisms is the formation of cooperative multicellular groups. It has been suggested that predation pressure may promote multicellular group formation in some algae and bacteria, with cells forming groups to lower their chance of being eaten. We use the green alga Chlorella vulgaris and the protist Tetrahymena thermophila to test whether predation pressure can initiate the formation of colonies. We found that: (1) either predators or just predator exoproducts promote colony formation; (2) higher predator densities cause more colonies to form; and (3) colony formation in this system is facultative, with populations returning to being unicellular when the predation pressure is removed. These results provide empirical support for the hypothesis that predation pressure promotes multicellular group formation. The speed of the reversion of populations to unicellularity suggests that this response is due to phenotypic plasticity and not evolutionary change.     

Analysis of lymphocytes reactive to histocompatibility antigens. II. Exponential alloantigen-dependent lymphocyte growth in vitro

Rat thoracic duct lymphocytes were maintained in continual blast transformation and cell division by repeated in vitro stimulation with allogeneic cells. This resulted in increases in responder cell numbers of up to 10,000-fold in 10-day periods. Growth of responder lymphocyte populations was dependent upon cell density, culture medium nutrients, and the presence of antigen in the form of allogeneic cells. A titration assay for mixed lymphocyte interactions (MLI) was used to relate absolute growth of cells in preparative cultures to [³H]thymidine incorporation in analytical MLI. Growth of lymphocyte populations derived by repeated stimulation with cells bearing a single foreign MHC haplotype was supported to lesser, variable degrees by stimulation with unrelated “third party” stimulator cells. The extent of this operational cross-reactivity was assessed by parallel line analysis of MLI titrations of responder lymphocytes enriched for specific alloreactivity.     

Evolution of the term “cellular senescence” and its impact on the current cytogerontological research

The term “cellular/cell senescence” was first introduced by Leonard Hayflick to describe the “age-related” changes in normal eukaryotic cells during aging in vitro, i.e., over the exhaustion of their mitotic potential. In the “classic” variant, it was assumed that cells “grow old” with the help of some internal mechanism, which leads to accumulation of various macromolecular defects (DNA damage in the first place). Currently, as a rule, “cellular senescence” means accumulation/appearance of particular “biomarkers of aging” in cells (they are most often transformed cells that do not demonstrate any replicative senescence) under the influence of various external factors (oxidative stress, H₂O₂, mitomycin C, ethanol, ionizing radiation, doxorubicin, etc.) that cause DNA damage. This phenomenon has been called DDR (DNA Damage Response). Among the said biomarkers, there are senescence-associated beta-galactosidase activity, expression of p53 and p21 proteins as well as of proteins involved in the regulation of inflammation, such as IL-6 or IL-8, activation of oncogenes, etc. Thus, “aging/senescence” of cells does not occur simply by itself—it takes place because of the influence of DNA-damaging agents. This approach, in my opinion, despite being very important to define a strategy to fight cancer, distracts us, yet again, from the study of the real mechanisms of aging. It should be emphasized that the “stationary phase aging” model developed in my laboratory also allows registering the occurrence of certain biomarkers of aging in cultured cells, but in this case they arise due to the restriction of their proliferation by contact inhibition, i.e., due to a rather physiological impact, which does not cause any damage to cells by itself (the situation is similar to what we observe in a whole multicellular organism).