Therapeutic Stimulation of Glycolytic ATP Production for Treating ROS-Mediated Cellular Senescence.

Cellular senescence is conditioned through two interrelated processes, i.e., a reduction in adenosine triphosphate (ATP) and the enhancement of reactive oxygen species (ROS) production levels in mitochondria. ATP shortages primarily influence the energy-intensive synthesis of large biomolecules, such as deoxyribonucleic acid (DNA). In addition, as compared to small biomolecules, large biomolecules are more prone to ROS-mediated damaging effects. Based on the available evidence, we suggest that the stimulation of anaerobic glycolytic ROS-independent ATP production could restrain cellular senescence. Consistent with this notion, non-drug related intermittent hypoxia (IH)-based therapy could be effectively applied in sports medicine, as well as for supporting the physical activity of elderly patients and prophylactics of various age-related disorders. Moreover, drug therapy aiming to achieve the partial blockade of respiratory chain and downstream compensatory glycolysis enhancement could prove to be useful for treating cardiovascular, neurological and hormonal diseases. We maintain that non-drug/drug-related therapeutic interventions applied in combination over the entire lifespan could significantly rejuvenate and prolong a high quality of life for individuals.

Clinically feasible and prospective immunotherapeutic interventions in multidirectional comprehensive treatment of cancer.

INTRODUCTION: The immune system is able to exert both tumor-destructive and tumor-protective functions. Immunotherapeutic technologies aim to enhance immune-based anti-tumor activity and (or) weaken tumor-protective immunity. AREAS COVERED: Cancer vaccination, antibody (Ab)-mediated cytotoxicity, Ab-based checkpoint molecule inhibition, Ab-based immunostimulation, cytokine therapy, oncoviral therapy, drug-mediated immunostimulation, exovesicular therapy, anti-inflammatory therapy, neurohormonal immunorehabilitation, metabolic therapy, as well as adoptive cell immunotherapy, could be coherently used to synergize and amplify each other in achieving robust anti-cancer responses in cancer patients. Tumor-specific immunotherapy applied at early stages is capable of eliminating remaining tumor cells after surgery, thus preventing the development of minimal residual disease. Patients with advanced disease stages could benefit from combined immunotherapy, which would be aimed at providing tumor cell/mass dormancy. Traditional therapeutic anti-cancer interventions (chemoradiotherapy, hyperthermia, anti-hormonal therapy) could significantly enhance tumor sensitivity to anti-cancer immunotherapy. It is important that lower-dose (metronomic) chemotherapy regimens, which are well-tolerated by normal cells, could advance immune-mediated control over tumor growth. EXPERT OPINION: We envisage that combined immunotherapy regimens in the context of traditional treatment could become the mainstream modality for treating cancers in all phases of the tumorigenesis. The effectiveness of the anti-cancer treatment could be monitored by the following blood parameters: C-reactive protein, lactate dehydrogenase, and neutrophil-to-lymphocyte ratio.

Inhibitory effect of IQ-1S, a selective c-Jun N-terminal kinase (JNK) inhibitor, on phenotypical and cytokine-producing characteristics in human macrophages and T-cells.

c-Jun N-terminal kinase (JNK) is a critical mitogen activated protein kinase (MAPK) implicated in inflammatory processes, with IQ-1S (11H-indeno[1,2-b]quinoxalin-11-one oxime sodium salt) being a high-affinity JNK inhibitor with pronounced anti-inflammatory properties. Here, we studied direct effects of IQ-1S on phenotypical and cytokine-producing characteristics of activated human monocytes/macrophages and T cells in vitro. Purified monocyte/macrophage cells were activated by bacterial lipopolysaccharide (LPS, 1 µg/ml) for 24 h, while T cells were activated by particles conjugated with antibodies (Abs) against human CD2, CD3, and CD28 for 48 h. Treatment with IQ-1S (0.5-25 µÐœ) in the presence of LPS reduced percentages of CD197 (CCR7)-positive cells in macrophage cultures, without affecting CD16+ (FcγRIII, low-affinity Fc-receptor), CD119+ (interferon-γ receptor 1), and CD124+ (IL-4 receptor α-subunit) cells. In addition, IQ-1S reduced production of tumour necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-6, and IL-10 in macrophage cultures. In activated T cell cultures, IQ-1S decreased CD25+ cell numbers in both CD4-positive and CD4-negative T cell compartments. Central memory СD45RA-/СD197+ and effector memory СD45RA-/СD197- T cells were more sensitive to IQ-1S-mediated suppression, as compared to naïve СD45RA+/СD197+ and terminally-differentiated effector СD45RA+/СD197- T cells. IQ-1S also suppressed T-cell cytokine production (IL-2, interferon-É£, IL-4, and IL-10). Collectively, the results suggest that both human macrophage and T cells could be immediate cell targets for IQ-1S-based anti-inflammatory immunotherapy. IQ-1S-mediated suppressive effects were unlikely to be associated with macrophage/T helper polariation.

Total threshold cytotoxicity of therapeutic antibodies for selective destruction of pathogenic memory T cells: implications for immunotherapy of autoimmune and allergenic disorders.

Introduction: Pathogenic memory CD4+ T cells are the mainspring of autoimmune and allergic disorders, suggesting that effective pathogenetic immunotherapy should be primarily directed onto their direct inactivation without affecting normal cells. Areas covered: A novel immunotherapeutic concept is proposed that applies suboptimal doses of several cytotoxic antibodies (Abs) against membrane antigens (Ags) (such as CD4, СD45RO, СD69, CD103, CD27, CD38, DR, etc.) with a view to achieve a threshold density of immune complexes on pathogenic memory CD4+ T cells for their selective elimination. During disease exacerbations, a complex Ab formulation could be applied to combine Abs against CD4, СD45RO, and СD69 to selectively destroy both activated memory CD4+ T cells located in lymphoid tissues and resident memory CD4+ T cells present in local inflammatory sites in situ. In contrast, normal T cells are spared from destruction as being recognized only by some Abs leading to Ab-Ag complexes below cytolytic threshold levels. Inactivation of pathogenic CD4+ T cells will also withdraw T helper support to pathogenic memory B cells and memory CD8+ T cells, thus effectively diminishing their activity. Expert opinion: The described approach benefits from universality and potential availability of a vast library of monoclonal Abs with relevant specificity.

Attaining threshold antibody cytotoxicity for selective tumor cell destruction: an opinion article.

We propose a novel immunotherapeutic paradigm that justifies application of several antibodies to various membrane-associated antigens to achieve a critical threshold density of immune complexes on the surface of cancer cells sufficient for triggering downstream cytolytic pathways. Indeed, some cancer-associated antigens (such as cancer/testis antigens) were found to be expressed on many cancer (but not normal) cells, with their baseline membrane expression levels being originally quite low for some of them, or even further down-regulated due to immune-driven cell selection. To achieve the mandatory threshold density of membrane-associated immune complexes on malignant cells, the concept stipulates combined application of antibodies specific for a cancer-associated antigen along with antibodies against an antigen expressed not only on tumor, but also on normal cells. In the proposed scenario it is of vital importance that the latter antibodies should be applied in suboptimal dosage to exclude the destruction of normal cells devoid of a cancer-associated antigen. Malignant cells often co-express antigens not present concurrently on normal cells at high levels. In such cases, suboptimal dosages of antibodies specific for those antigens could also be applied to achieve cumulative effect leading to selective destruction of tumour cells. Hence, the described immunotherapeutic technology could be used metaphorically speaking as a kind of 'immunological knife', which is capable of highly selective destruction of cancer cells without destroying normal cells.

Erythropoietin Directly Affects Human Macrophage Functionality.

OBJECTIVE: We studied direct effects of Erythropoietin (Epo) on functional properties of human monocytes/macrophages (Mc/Mphs) in vitro. METHODS: Cells expressing CD14 marker were isolated from human peripheral blood mononuclear cells (PBMCs) by positive magnetic separation. Mc/Mphs were cultured without or with bacterial lipopolysaccharide (LPS) in the absence or presence of Epo for 24 h. RESULTS: We showed that Epo treatment hoticeably reduces the percentages of CD14+ cells, CD124 (alpha subunit of IL-4 receptor)+ cells and CD197 (CCR7)+ cells in non-activated Mph cultures without affecting the levels of CD16 (low-affinity Fc-receptor)+ and CD119 (interferon-γ (IFN-γ) receptor)+ cells. Epo also markedly reduced percentages of CD197+ cells in LPS-activated Mc/Mphs, without significantly affecting the expression of all other molecular markers studied. In addition, Epo caused moderate up-regulation of interleukin-1ß (IL-1ß) and IL-6 production in resting Mc/Mph cultures, as compared to the down-regulation of IL-1ß and IL-6 production in LPS-activated cells. No Epomediated effects on tumor necrosis factor-α (TNF-α) and IL-10 production were observed. CONCLUSION: Our data suggests that Epo effects on Mph functionality are largely dependent on the baseline activation status of these cells, and that Epo exerts no distinct direct effects on the particular Mph polarization pathway.

A Possible Role for Idiotype/Anti-idiotype B-T Cell Interactions in Maintaining Immune Memory.

Variable regions of both B-cell receptors (BCRs) and T-cell receptors (TCRs) are completely formed in the postnatal period, and, consequently, no innate immune tolerance against these structures exists in adulthood. Indeed, antibodies (Abs) specific to TCRs have been found in both animals and humans. These facts clearly indicate the existence of B cells able to directly interact with T cells through binding of BCRs to TCRs without implicating major histocompatibility complex molecules. A novel paradigm is proposed in that the immune memory is based on idiotype/anti-idiotype interactions occurring between BCRs and TCRs following clearance of the antigen that elicited immune responses. It is envisaged that direct contact between memory T and B cells could provide co-stimulatory signals needed to sustain viability, growth, and differentiation of the interacting immune cells. In contrast, plasma cells originating from memory B-cells could produce anti-TCR Abs that inhibit direct BCR-to-TCR interactions, thereby downregulating the B- to T-cell contact-based immune memory via a negative feedback mechanism.

Immune responses to polyclonal T-cell vaccination in patients with progressive multiple sclerosis.

The overall objective of disease management in autoimmune diseases is to suppress chronic inflammation and prevent organ damage. Therapies often revolve around five drug classes: non-steroidal anti-inflammatory drugs (NSAIDS), anti-malarials, steroids, immunosuppressants, and bio-therapies. However, none of these is a 'cure' and each displays a potential for adverse events. In particular, while all of them suppress harmful autoimmune responses, they also impact on useful protective immune responses. T-Cell receptor (TCR) immunogenicity provides a rationale for T-cell vaccinations to induce anti-idiotypic immune responses with the purpose of down-regulating functionality of idiotype-bearing self-reactive T-cells. To explore this, in this study, 39 patients with progressive (chronic) multiple sclerosis (MS) were multiply immunized with autological polyclonal T-cell vaccines (TCVs). None of the TCV-treated patients experienced any significant side-effects during the entire follow-up period (2 years). T-Cell vaccination had no significant effects on T-cell sub-population contents in the blood of MS patients after 2 years of immunotherapy initiation. However, a substantial reduction in the frequency of CD4+ and CD8+ memory T-cells able to produce interferon (IFN)-γ following activation were noted in the blood of TCV-treated patients. Moreover, significant and sustained reduction in plasma IFNγ levels and concomitant increases in interleukin (IL)-4 levels were documented in these samples. The TCV-treated subjects, however, exhibited no significant changes in plasma IL-17 and IL-18. More importantly was a significant decline in proliferative T-cell responses to myelin antigens in the TCV-treated patients, indicating attenuation of myelin-specific T-cell activity. Collectively, the results suggest that polyclonal T-cell vaccination is safe to use, able to induce measurable, long-lasting, anti-inflammatory immune effects in patients with advanced MS.

Xenogeneic cell-based vaccine therapy for stage III melanoma: safety, immune-mediated responses and survival benefits.

New therapies for melanoma have yielded promising results, but their application is limited because of serious side-effects and only moderate impact on patient survival. Vaccine therapies may offer some hope by targeting tumor-specific responses, considering the immunogenic nature of melanomas. To investigate the safety profile and efficiency of a xenogeneic cell-based vaccine therapy in stage III melanoma patients and evaluate the survival rate in treated patients. Twenty-seven stage III melanoma patients were immunized with a lyophilized xenogeneic polyantigenic vaccine (XPV) prepared from murine melanoma B16 and carcinoma LLC cells. Neither grade III/IV toxicities, nor clinically significant changes in blood and biochemical parameters were noted after an induction course of 10 XPV subcutaneous immunizations. No laboratory or clinical signs of systemic autoimmunity were documented. Following 10 vaccinations, a relative increase in the numbers of circulating memory CD4+CD45RO+ T cells (but not CD8+ CD45RO+ T cells) was observed. Peripheral blood mononuclear cells obtained from XPV-treated patients demonstrated increased proliferative responses to human BRO melanoma-associated antigens and marked increases in serum levels of IFN-γ and IL-8. Serum levels of TNF-α, IL-4 and IL-6 were not affected. The overall five-year survival rate in the treated patients was significantly higher than that in 27 control patients with matched clinical and prognostic characteristics (55% vs 18%). XPV-based immunotherapy could be maximally effective when started as early as possible before or after surgical excision of the primary tumor and local metastases, i.e. when tumor-mediated suppressive effects on immunity are minimal.

A novel dual NO-donating oxime and c-Jun N-terminal kinase inhibitor protects against cerebral ischemia-reperfusion injury in mice.

The c-Jun N-terminal kinase (JNK) has been shown to be an important regulator of neuronal cell death. Previously, we synthesized the sodium salt of 11H-indeno[1,2-b]quinoxalin-11-one (IQ-1S) and demonstrated that it was a high-affinity inhibitor of the JNK family. In the present work, we found that IQ-1S could release nitric oxide (NO) during its enzymatic metabolism by liver microsomes. Moreover, serum nitrite/nitrate concentration in mice increased after intraperitoneal injection of IQ-1S. Because of these dual actions as JNK inhibitor and NO-donor, the therapeutic potential of IQ-1S was evaluated in an animal stroke model. We subjected wild-type C57BL6 mice to focal ischemia (30min) with subsequent reperfusion (48h). Mice were treated with IQ-1S (25mg/kg) suspended in 10% solutol or with vehicle alone 30min before and 24h after middle cerebral artery (MCA) occlusion (MCAO). Using laser-Doppler flowmetry, we monitored cerebral blood flow (CBF) above the MCA during 30min of MCAO provoked by a filament and during the first 30min of subsequent reperfusion. In mice treated with IQ-1S, ischemic and reperfusion values of CBF were not different from vehicle-treated mice. However, IQ-1S treated mice demonstrated markedly reduced neurological deficit and infarct volumes as compared with vehicle-treated mice after 48h of reperfusion. Our results indicate that the novel JNK inhibitor releases NO during its oxidoreductive bioconversion and improves stroke outcome in a mouse model of cerebral reperfusion. We conclude that IQ-1S is a promising dual functional agent for the treatment of cerebral ischemia and reperfusion injury.

Direct effects of interleukin-7 on the function of human T cells in vitro.

СD3+ T lymphocytes were isolated by positive magnetic separation from the peripheral blood of healthy donors. In the absence of any additional activating stimuli, interleukin-7 (IL-7) was shown to augment the levels of T cells expressing CD25 activation marker both in СD4-positive and in CD4-negative effector memory (CD45RA-CD197-) T cell subsets, as well as in terminally differentiated (CD45RA+CD197-) Т cells, without significantly affecting the activation status of naive (CD45RA+CD197+) and central memory (CD45RA-CD197+) T cells. In addition, IL-7 noticeably enhanced the production of IL-2, interferon-γ (IFN-γ), and IL-10, but not IL-4, in T cells. The direct effects of IL-7 on T cell activation induced in vitro by MACSiBead™ particles coated with CD2, CD3, and CD28 antibodies (Abs) were also investigated. Upon cell activation, IL-7 significantly augmented the levels of CD25+ T cells in naive (CD45RA+CD197+), central memory (CD45RA-CD197+), and effector memory (CD45RA-CD197-) T-cell compartments. In addition, IL-7 facilitated activation of СD4- (but not CD4+) terminally differentiated effector (CD45RA+CD197-) Т cells. Finally, IL-7 was found to upregulate the production of IL-2, IFN-γ, IL-4, and IL-10 by activated T cells. In conclusion, we speculate that IL-7 is capable of enhancing functional T cell activity without causing significant functional inbalance between various T cell subsets.

Xenovaccinotherapy for colorectal cancer.

The objectives of this phase I-II trial were to assess the toxicity, immunological and clinical responses induced in 37 patients with stage IV colorectal cancer by the subcutaneous administration of a xenogenic polyantigenic vaccine (XPV) prepared from disrupted murine melanoma (B16) and carcinoma (LLC) cells. An inducing course of vaccinotherapy consisted of 10 immunizations (5 at weekly and 5 at fortnight intervals). Twenty-four hours later each of first 5 vaccinations the patient was subcutaneously given a low dose of the recombinant interleukin-2 (IL-2). A consolidating course of vaccinotherapy consisted of monthly vaccinations. No grade III or IV toxicities, but also laboratory and clinical signs of developing systemic autoimmune disorders were noted in any XPV-treated patient. A significant increase in cell-mediated immunoreactivity to both LLC and B16 antigens (Ags) occurred in the patients after inducing vaccinations, as determined by delayed-type hypersensitivity (DTH) skin reactions, as well as by blood lymphocyte proliferation responses. Vaccinations also led to increased cell-mediated reactivity to murine non-tumor, spleen cell (SC)-associated Ags. This reactivity, however, was not as significant as that to tumor-associated antigens (TAAs). XPV was also found to capable of generating IgG antibody-mediated responses. With immunotherapy concentrations of both interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) detectably elevated in patients' sera, suggesting intensification of T helper 1-/T helper 2-mediated responses in the XPV-treated patients. The average survival of the XPV-treated patients was noticeably superior than was that of the clinically comparable control patients (17 vs 7 months). Collectively the results suggest that xenogenic TAAs are safe to use, able to induce measurable cellular and humoral immune responses, and may be clinically effective in certain colorectal cancer patients.

Xenovaccinotherapy for melanoma.

The objectives of this phase I-II trial were to assess the toxicity, immunological and clinical responses induced in stage III/IV melanoma patients by the subcutaneous administration of xenogenic polyantigenic vaccine (XPV) prepared from disrupted murine melanoma (B16) and carcinoma (LLC) cells. An inducing course of vaccinotherapy consisted of ten immunizations (five at weekly and five at fortnight intervals). Twenty-four hours following each of the first five vaccinations, the patient was subcutaneously given a low dose of the recombinant interleukin-2 (IL-2). A consolidating course of the vaccinotherapy consisted of monthly vaccinations. Grade 3 or 4 toxicities, as well as laboratory and clinical signs of developing autoimmune disorders, were recorded in none of the 40 XPV-treated evaluable patients. A significant increase in delayed-type hypersensitivity (DTH) skin reaction to vaccinal B16, but not to LLC antigens (Ags), occurred in patients after inducing vaccinations. At the same time, those patients demonstrated a marked augmentation of blood lymphocyte proliferation responses not only to B16 but also to LLC Ags. Vaccinations also led to increased cell-mediated reactivity to murine non-tumor, spleen cell (SC)-associated Ags, which, however, was not as significant as that to tumor-associated antigens (TAAs). Of great importance was the fact that XPV administration resulted in increased blood lymphocyte proliferative reactivity of patients to human melanoma-associated Ags, while not affecting their reactivity to the control alloantigens. With immunotherapy, concentrations of both interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) were elevated in patients' sera, suggesting an intensification of the T helper1/ T helper 2-mediated responses in the XPV-treated patients. The average survival of the 32 stage IV melanoma XPV-treated evaluable patients was noticeably higher than that of the 32 clinically comparable control patients (13 vs. 5 months). The overall 3 year-survival rate in the XPV-treated group and the control group was 25% (8 patients) and 3% (1 patient), respectively. In general, the results suggest that xenogenic tumor cells may provide a novel feasible approach to constructing clinically effective vaccines.

Cell transplantation therapy in re-animating severely head-injured patients.

The results of controlled, retrospective clinical investigation of applying cell transplantation (CT) therapy in 38 severely head-injured patients are presented. The patients initially were in state of coma (Glasgow coma scale score 3--7), owing to their traumatic brain injuries. Cells prepared from fetal nervous and hematopoietic tissues were grafted subarachnoidally via lumbar puncture. The control group consisted of 38 patients and was clinically comparable with the trial one. From the results obtained it appears that CT treatment promoted both wakening consciousness of the patients and their following neurological rehabilitation. A death-rate in the trial and control group was 5% (two cases) and 45% (17 cases), respectively. According to a Glasgow scale, favorable (good+satisfactory) outcomes of a disease were noted in 33 (87%) cell-grafted and only in 15 (39%) control patients. Statistical analysis revealed that CT treatment generally improved the outcomes by 2.5-fold. No serious complications of CT therapy were noted. The results point out a possible rationality of applying CT therapy in severely head-injured patients as early as within acute period of a disease.

Erythroid cells in immunoregulation: characterization of a novel suppressor factor.

Nucleated erythroid cells (EC) have been previously reported to possess a potent natural suppressor (NS) activity for B-cell responses. In this study, we demonstrate that murine EC are able to reduce not only lipopolysaccharide (LPS)-driven B-cell proliferation, but also proliferative and cytotoxic T-cell responses generated in a primary allogeneic mixed lymphocyte culture (MLC); and that a soluble low molecular weight factor may be involved in such EC-derived immunoregulation. In addition, the erythroid cell-derived suppressor factor (ESF) was found to be capable of effectively reducing the allergen-driven proliferation of peripheral blood mononuclear cells (PBMC) isolated from allergic patients. From the data presented herein, it appears that ESF is heat-stable (80 degrees C for 20 min) and has molecular weight (MW) lower or close to 0.5 kDa. ESF activity is resistant to both enzyme (trypsin plus chymotrypsin) proteolysis and action of the enzymes such as lipase and phospholipase C. On the other hand, ESF is effectively inactivated by neuraminidase treatment, suggesting the presence in its structure of sialic residue(s). The neuraminidase-sensitive, ESF-like activity is readily detected in the medium conditioned with normal mouse bone marrow (BM) cells. On fractionation of low MW erythroid products on a reversed-phase C16 column in a linear acetonitrile gradient (5-95%), ESF activity is detected in the first peak alone with the shortest time of its retention by the column. The results suggest that (1) by producing ESF, EC may regulate both B- and T-cell-mediated immune processes and (2) based on its physicochemical and biological characteristics, ESF can be distinguished from each of earlier characterised suppressor mediators of bone marrow origin.

Transplantation treatment of spinal cord injury patients.

The minimally manipulated cells from fetal nervous and hemopoietic tissues (gestational age 16-22 weeks) were subarachnoidally implanted into 15 patients (18-52 years old) with severe consequences of traumatic spinal cord injury (SCI) at cervical or thoracic spine level. The times after SCI were from 1 month to 6 years. Each patient underwent from one to four cell transplantations (CT) with various time intervals. In 11 of 15 cases, CT was combined with an operative partial disruption of a connective tissue cyst and with implantation into a spinal cord lesion of a spinal cord fragment together with olfactory ensheathing cells. Before CT the patients showed complete motor and sensory function disorder consistent with a grade A of SCI according to Frankel classification. With CT treatment, six patients improved their neurological status from A to C grade of SCI, exhibiting incomplete restoration of both motor and sensory function. The status of other five CT-treated patients became consistent with SCI grade B and was characterized by appearance of contracting activity in some muscles and incomplete restoration of sensitivity. The remaining four patients did not exhibit any clinical improvements. No serious complications of CT were noted. The results suggest a clinical relevance of the CT-based approach to treating severe consequences of SCI.