Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells.

Inadequate presentation of tumor antigens by host professional antigen-presenting cells (APCs), including dendritic cells (DCs), is one potential mechanism for the escape of tumors from the host immune system. Here, we show that human cancer cell lines release a soluble factor or factors that dramatically affect DC maturation from precursors without affecting the function of relatively mature DCs. One factor responsible for these effects was identified as vascular endothelial growth factor (VEGF). Thus, VEGF may play a broader role in the pathogenesis of cancer than was previously thought, and therapeutic blockade of VEGF action may improve prospects for immunotherapy as well as inhibit tumor neovasculature.

Redox phospholipidomics of enzymatically generated oxygenated phospholipids as specific signals of programmed cell death.

High fidelity and effective adaptive changes of the cell and tissue metabolism to changing environments require strict coordination of numerous biological processes. Multicellular organisms developed sophisticated signaling systems of monitoring and responding to these different contexts. Among these systems, oxygenated lipids play a significant role realized via a variety of re-programming mechanisms. Some of them are enacted as a part of pro-survival pathways that eliminate harmful or unnecessary molecules or organelles by a variety of degradation/hydrolytic reactions or specialized autophageal processes. When these "partial" intracellular measures are insufficient, the programs of cells death are triggered with the aim to remove irreparably damaged members of the multicellular community. These regulated cell death mechanisms are believed to heavily rely on signaling by a highly diversified group of molecules, oxygenated phospholipids (PLox). Out of thousands of detectable individual PLox species, redox phospholipidomics deciphered several specific molecules that seem to be diagnostic of specialized death programs. Oxygenated cardiolipins (CLs) and phosphatidylethanolamines (PEs) have been identified as predictive biomarkers of apoptosis and ferroptosis, respectively. This has led to decoding of the enzymatic mechanisms of their formation involving mitochondrial oxidation of CLs by cytochrome c and endoplasmic reticulum-associated oxidation of PE by lipoxygenases. Understanding of the specific biochemical radical-mediated mechanisms of these oxidative reactions opens new avenues for the design and search of highly specific regulators of cell death programs. This review emphasizes the usefulness of such selective lipid peroxidation mechanisms in contrast to the concept of random poorly controlled free radical reactions as instruments of non-specific damage of cells and their membranes. Detailed analysis of two specific examples of phospholipid oxidative signaling in apoptosis and ferroptosis along with their molecular mechanisms and roles in reprogramming has been presented.

Vascular endothelial growth factor effects on nuclear factor-kappaB activation in hematopoietic progenitor cells.

Vascular endothelial growth factor (VEGF) inhibits of the activation of transcription factor nuclear factor-kappaB (NF-kappaB) in hematopoietic progenitor cells (HPCs), and this is associated with alterations in the development of multiple lineages of hematopoietic cells and defective immune induction in tumor-bearing animals. Antibodies to VEGF have been shown to abrogate this effect. The mechanism by which VEGF antagonizes the induction of NF-kappaB was investigated in this study. Using supershift electrophoretic mobility shift analysis, we found that although tumor necrosis factor alpha (TNF-alpha) induced the nuclear translocation and DNA binding of p65-containing complexes, VEGF alone induced nuclear translocation and DNA binding of the complexes containing RelB. These results were confirmed by immunofluorescence confocal microscopy. VEGF effectively blocked TNF-alpha-induced NF-kappaB activation in HPCs from RelB-/- mice, however, similar to the effect observed in HPCs obtained from RelB+/- and RelB+/+ mice. This suggests that RelB is not required for VEGF to inhibit NF-kappaB activation. However, although TNF-alpha induced rapid activation of IkappaB kinase (IKK) as expected, this activity was substantially reduced in the presence of VEGF. This decreased IKK activation correlated with the inhibition of IkappaB alpha phosphorylation and degradation of IkappaB alpha and IkappaB epsilon in HPCs. VEGF alone, however, did not have any effect on phosphorylation of IkappaB alpha or degradation of IkappaB alpha and other inhibitory molecules IkappaB beta, IkappaB epsilon, or Bcl-3. SU5416, a potent inhibitor of the VEGF receptor I (VEGFR1) and VEGFR2 receptor tyrosine kinases, did not abolish the inhibitory effect of VEGF, indicating that the VEGF effect is mediated by a mechanism unrelated to VEGFR1 or VEGFR2 tyrosine kinase activity. Thus, VEGF appears to inhibit TNF-alpha-induced NF-kappaB activation by VEGFR kinase-independent inhibition of IKK. Therapeutic strategies aimed at overcoming VEGF-mediated defects in immune induction in tumor-bearing hosts will need to target this kinase-independent pathway.

Decreased antigen presentation by dendritic cells in patients with breast cancer.

We evaluated T-cell responses to mitogens and to defined antigens in breast cancer patients. Significant defects in responses to tetanus toxoid and influenza virus were observed in patients with advanced-stage breast cancer. To define whether these defects were associated with a defect in antigen presentation [dendritic cells (DCs)] or effector function (T cells), these cells were studied separately. Purified DCs from 32 patients with breast cancer demonstrated a significantly decreased ability to stimulate control allogeneic T cells, but stimulation of patient T cells with either control allogeneic DCs or immobilized anti-CD3 antibody resulted in normal T-cell responses, even in patients with stage IV tumors. These data suggest that reduced DC function could be one of the major causes of the observed defect in cellular immunity in patients with advanced breast cancer. We then tested whether stem cells from these patients could give rise to functional DCs after in vitro growth with granulocyte/macrophage colony-stimulating factor and interleukin 4. Normal levels of control allogeneic and tetanus toxoid-dependent T-cell proliferation were observed when DCs obtained from precursors were used as stimulators. Those cells also induced substantially higher levels of influenza virus-specific CTL responses than mature DCs from the peripheral blood of these patients, although responses did not quite reach control values. Thus, defective T-cell function in patients with advanced breast cancer can be overcome by stimulation with DCs generated from precursors, suggesting that these cells may better serve as autologous antigen carriers for cancer immunotherapy than mature peripheral blood DCs.

Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function.

Inadequate function of dendritic cells (DCs) in tumor-bearing hosts is one mechanism of tumor escape from immune system control and may compromise the efficacy of cancer immunotherapy. Vascular endothelial growth factor (VEGF), produced by most tumors, not only plays an important role in tumor angiogenesis but also can inhibit the maturation of DCs from hematopoietic progenitors. Here, we investigate a novel combination of antiangiogenic and immunotherapy based on this dual role of VEGF. Two s.c. mouse tumor models were used: D459 cells, expressing mutant human p53; and MethA sarcoma with point mutations in the endogenous murine p53 gene. Therapy with anti-mouse VEGF antibody (10 microg i.p. twice a week over 4 weeks) was initiated when tumors became palpable. Treatment of established tumors with anti-VEGF antibody alone did not affect the rate of tumor growth. However, anti-VEGF antibody significantly improved the number and function of lymph node and spleen DCs in these tumor-bearing animals. To investigate the possible effects of this antibody on the immunotherapy of established tumors, tumor-bearing mice were immunized with DCs pulsed with the corresponding mutation-specific p53 peptides, together with injections of anti-VEGF antibody. Therapy with peptide-pulsed DCs alone resulted in considerable slowing of tumor growth but only during the period of treatment, and tumor growth resumed after the end of the therapy. Combined treatment with peptide-pulsed DCs and anti-VEGF antibody resulted in a prolonged and much more pronounced antitumor effect. This effect was associated with the induction of significant anti-p53 CTL responses only in this group of mice. These data suggest that inhibition of VEGF may be a valuable adjuvant in the immunotherapy of cancer.

Clinical significance of defective dendritic cell differentiation in cancer.

Defective dendritic cell (DC) function has been described previously in cancer patients and tumor-bearing mice. It can be an important factor in the escape of tumors from immune system control. However, the mechanism and clinical significance of this phenomenon remain unclear. Here, 93 patients with breast, head and neck, and lung cancer were investigated. The function of peripheral blood and tumor draining lymph node DCs was equally impaired in cancer patients, consistent with a systemic rather than a local effect of tumor on DCs. The number of DCs was dramatically reduced in the peripheral blood of cancer patients. This decrease was associated with the accumulation of cells lacking markers of mature hematopoietic cells. The presence of these immature cells was closely associated with the stage and duration of the disease. Surgical removal of tumor resulted in partial reversal of the observed effects. The presence of immature cells in the peripheral blood of cancer patients was closely associated with an increased plasma level of vascular endothelial growth factor but not interleukin 6, granulocyte macrophage colony-stimulating factor, macrophage colony-stimulating factor, interleukin 10, or transforming growth factor-beta and was decreased in lung cancer patients receiving therapy with antivascular endothelial growth factor antibodies. These data indicate that defective DC function in cancer patients is the result of decreased numbers of competent DCs and the accumulation of immature cells. This effect may have significant clinical implications.

Dendritic cells transduced with full-length wild-type p53 generate antitumor cytotoxic T lymphocytes from peripheral blood of cancer patients.

Accumulation of wild-type or mutant p53 protein occurs in approximately 50% of human malignancies. This overexpression may generate antigenic epitopes recognized by CTLs. Because normal cells have undetectable levels of p53, these CTLs are likely to be tumor specific. Here, for the first time, we test the hypothesis that full-length wild-type p53 protein can be used for generation of an immune response against tumor cells with p53 overexpression. T cells obtained from nine HLA-A2-positive cancer patients and three HLA-A2-positive healthy individuals were stimulated twice with dendritic cells (DCs) transduced with an adenovirus wild-type p53 (Ad-p53) construct. Significant cytotoxicity was detected against HLA-A2-positive tumor cells with accumulation of mutant or wild-type p53 but not against HLA-A2-positive tumor cells with normal (undetectable) levels of p53 or against HLA-A2-negative tumor cells. This response was specific and mediated by CD8+ CTLs. These CTLs recognized HLA-A2-positive tumor cells expressing normal levels of p53 protein after their transduction with Ad-p53 but not with control adenovirus. Stimulation of T cells with Ad-p53-transduced DCs resulted in generation of CTLs specific for p53-derived peptide. These data demonstrate that DCs transduced with the wild-type p53 gene were able to induce a specific antitumor immune response. This offers a new promising approach to immunotherapy of cancer.

The synthetic peptide from HIV increases functional activity of granulocytes in healthy subjects.

The influence of HIV lysate and eight synthetic peptides which are fragments of HIV proteins on the functional activity of polymorphonuclear neutrophils (PMN) was tested in 12 healthy subjects. PMN activity in nitroblue tetrazolium reduction (NBT test) and PMN chemiluminescence (CL) was studied. Only one peptide was found to result in a significant increase in NBT test on the whole blood. This was the oligopeptide (G-97) from the CD4-binding site of HIV-1 gp120. The increase of CL response of PMN in the presence of G-97 was revealed after only 15 min preincubation. The same effect in the presence of sera from healthy or infected patients at the persistent generalized lymphadenopathy stage was achieved by increasing the time of preincubation to 30 min. G-97 did not influence the proliferative activity of lymphocytes.

Clinical significance of HIV DNA in polymorphonuclear neutrophils from patients with HIV infection.

HIV DNA was detected by the polymerase chain reaction technique in polymorphonuclear neutrophils (PMNs) in 11 of 37 (29.7%) HIV-infected patients. A detectable level of HIV DNA in PMNs was more common in symptomatic than asymptomatic HIV infected patients (46.7% and 18.2%, respectively; p < 0.05). HIV DNA in PMNs was detected most frequently in patients with recurrent bacterial pneumonia or Pneumocystis carininii pneumonia. An association between HIV DNA in PMNs and a low CD4/8 ratio as well as high levels of immunoglobulins in the sera was noted. Detectable HIV DNA was found more frequently in patients with neutropenia than in those with a normal level of neutrophils in peripheral blood (44.4% and 28.0%, respectively; p < 0.05). These data suggest that infection of PMNs by HIV may be associated with PMN impairment during HIV infection.

Chemoattraction of femoral CD34+ progenitor cells by tumor-derived vascular endothelial cell growth factor.

Patients and animals with GM-CSF-producing tumors have an increased number of mobilized CD34+ progenitor cells within their peripheral blood and tumor tissue. These CD34+ cells are inhibitory to the activity of intratumoral T-cells. The present study used the murine Lewis lung carcinoma (LLC) model to assess mechanisms that could lead to the accumulation of CD34+ cells within the tumor tissue. In vitro analyses showed that LLC tumor explants released chemoattractants for normal femoral CD34+ cells. The LLC tumor cells contributed to the production of this activity since CD34+ cell chemoattractants were also released by cultured LLC cells. Antibody neutralization studies showed that most, although not all, of the chemotactic activity that was produced by LLC cells could be attributed to VEGF. In vivo studies with fluorescent-tagged CD34+ cells showed their accumulation within the tumor tissue, but not within the lungs, spleen or bone marrow, suggesting a selective accumulation within the tumor. Whether or not VEGF could chemoattract CD34+ cells in vivo was measured with a VEGF-containing Matrigel plug assay. Infusion of fluorescent-tagged CD34+ cells into mice after the plugs became vascularized revealed the accumulation of fluorescent-tagged cells within the plugs. However, these CD34+ cells failed to accumulate within the VEGF-containing Matrigel plugs when they were infused together with neutralizing anti-VEGF antibody. Through a combination of in vitro and in vivo analyses, the LLC cells were shown to be capable of chemoattracting CD34+ cells, with most of the tumor-derived chemotactic activity being due to tumor release of VEGF.

Peripheral blood neutrophils from HIV-1-infected individuals are armed with factors that cause inhibition of their migration in response to specific antigens.

In this study, synthetic peptides that copy conserved regions of the HIV-1 envelope proteins gp120 and gp41 were tested for their impact on the chemotaxis of leukocytes and neutrophils from HIV-1-infected individuals, while neutrophils from HIV-1-infected patients were tested for their effect on the chemotaxis of neutrophils from healthy donors. The synthetic peptides (corresponding to the 251-272-amino acid sequence of gp120 and the 584-618-amino acid sequence of gp41) were capable of specifically inhibiting the formyl peptide-induced chemotaxis of cells from HIV-1-infected patients, and such inhibition was observed both with a total leukocyte population and with pure neutrophils. The migration of neutrophils from healthy donors was specifically inhibited in the presence of either of the synthetic peptides of HIV-1 envelope proteins after their incubation with neutrophil supernatants obtained from HIV-1-infected individuals. As shown by ELISA tests, the neutrophil supernatants from HIV-1-infected individuals contain antibodies to a recombinant env-1 protein that might be one of the reasons for the specific arming of neutrophils from HIV-1-infected persons.

Human squamous cell carcinomas of the head and neck chemoattract immune suppressive CD34(+) progenitor cells.

CD34(+) progenitor cells have previously been shown to be mobilized in patients with squamous cell carcinoma of the head and neck (HNSCC). The present study showed that these CD34(+) cells inhibit the capacity of intratumoral lymphoid cells to become activated in response to stimulation through the TCR/CD3 complex. The mechanisms that could lead to the accumulation of CD34(+) cells within the tumor tissue were assessed. This was accomplished through in vitro studies that determined if HNSCC produce soluble factors that chemoattract CD34(+) cells. The migration of cord blood CD34(+) cells, which were used as a readily available source of progenitor cells, was stimulated by products derived from HNSCC explants and primary HNSCC cultures. This stimulated migration was due to chemotaxis because it was dependent on an increasing gradient of HNSCC-derived products. CD34(+) cells that were isolated from the peripheral blood of HNSCC patients were similarly chemoattracted to the HNSCC-derived products. The majority of the chemotactic activity produced by HNSCC could be attributed to vascular endothelial cell growth factor (VEGF). These studies indicate that HNSCC can chemoattract immune inhibitory CD34(+) progenitor cells through their production of VEGF.

[Clinical manifestations and the problems of classification of HIV infection]. / KLinicheskie proiavleniia i voprosy klassifikatsii VICh-infektsii.

The clinical manifestations and some immunological parameters (CD4 lymphocytes, CD4/CD8 ratio, IgM, IgA, IgG levels, skin test) were examined in 226 adult patients (148 males and 78 females) infected with HIV. These included 58 (26%) asymptomatic patients with seropositive test, 109 (48%) with the only clinical manifestation generalized lymphadenopathy; 54 (24%) with AIDS-related infections, 5 (2%) with AIDS. A subsequent follow-up of 3 months to 3 years demonstrated that AIDS developed in 7 patients, 9 died. The period of infection with HIV and death ranged from 1.5 to 9 years. The signs of cell immunodeficiency were found in 70% of the examinees. Recommendations are given on the classification of HIV infection.

[Lymphocyte and neutrophil cytochemistry in the dynamics of different forms of diphtheria in adults]. / Tsitokhimiia limfotsitov i neitrofilov v dinamike razlichnykh form difterii u vzroslykh.

The cytochemical study of lymphocytes and neutrophils isolated from fractionated blood of diphtheria patients and carriers has revealed that a decrease in the activity of lymphocyte succinic dehydrogenase and myeloperoxidase can be observed at all periods of the disease and in all its forms. A decrease in the activity of lymphocyte nonspecific esterase has been noted only in patients with toxic and subtoxic diphtheria and a decrease in the activity of neutrophil alkaline phosphatase, in carriers. The analysis of correlations between the parameters of five enzymes under study (lymphocyte succinic dehydrogenase, lymphocyte acid phosphatase, lymphocyte non-specific esterase, myeloperoxidase, neutrophil alkaline phosphatase) and enzymatic rosette parameters has been made. The analysis has revealed an essential increase in the number of correlations in comparison with donors, changes in the qualitative nature of these correlations and sometimes the reversion of the correlations. Carriers have shown the greatest number of correlations. By the end of the terms of observations no restoration of normal correlations has been observed.

[Effect of reaferon on the functional activity of peripheral blood lymphocytes and neutrophils of volunteers]. / Vliianie reaferona na funktsional'nuiu aktivnost' limfotsitov i neitrofilov perifericheskoi krovi dobrovol'tsev.

The administration of Reaferon, the analog of human alpha 2-interferon obtained by means of gene engineering technigues, to 25 volunteers revealed the ability of small doses of this preparation to produce a stimulating effect on the capacity of lymphocytes and neutrophils for rosette formation. When introduced in large doses, the preparation sharply suppressed this capacity. A significant decrease in the T- and B-lymphocyte count, as well as in the neutrophil count, was noted. These changes persisted for 7 days after the course of Reaferon injections had been over. The intravenous injection of the preparation produced more pronounced effect than its intramuscular injection in the same doses. No essential changes in the count of the precursor cells of both T- and B-lymphocytes were registered.

[Blood lymphocyte and neutrophil function in patients with viral non-A, non-B hepatitis and a fecal-oral mechanism of the transmission of infection]. / Funktsional'noe sostoianie limfotsitov i neitrofilov krovi u bol'nykh virusnym gepatitom ni A ni B s fekal'no-oral'nym mekhanizmom peredachi infektsii.

A decrease in the capacity of lymphocytes for rosette formation, accompanied by a rise in the leukocyte count, has been found in patients with the severe form of hepatitis non A, non B. Neutrophils in these patients have shown a tendency towards an increased rosette formation. All altered characteristics normalized by the period of convalescence. As noted in this investigation, a considerable increase in the leukocyte count, along with an increase in the number of active rosette-forming neutrophils and a decrease in the capacity of leukocytes for rosette formation, is an unfavorable sign predicting the possibility of acute hepatic encephalopathy.

[The cellular immune response in different forms of diphtheria in adults]. / Kletochnyi immunnyi otvet pri razlichnykh formakh difterii u vzroslykh.

In 109 adult diphtheria patients with different forms of the disease (toxic, subtoxic, localized) and carriers, the latter including 6 persons having had the localized form of diphtheria, 12 different subpopulations of T and B lymphocytes and neutrophils were studied by the methods of rosette formation with sheep, mouse and bovine red blood cells. The study showed that, starting from week 2 of the disease, an increase in the content of the subpopulations under study was registered in all forms of diphtheria; the disease, as well as in patients with the localized form of the disease accompanied by complications and in long-term carriers. In all forms of diphtheria a decrease in the content of neutrophil rosette-forming cells was shown. The marker tests for prognostication of the complicated course of the disease, viz. the elevated content of M-rosette-forming cell and T gamma-rosette-forming cells during the first week of then disease, were established.

[The effect of the blood serum factors of HIV-infected persons on the functional activity of the neutrophils in healthy donors]. / Vliianie faktorov syvorotki krovi VICh-infitsirovannykh lits na funktsional'nuiu aktivnost' neitrofilov zdorovykh donorov.

The influence of 64 blood serum samples from HIV-infected patients at different stages of the infection on the functional activity of polymorphonuclear neutrophils (PMN) of 9 healthy donors was studied. In this study an increase in the production of oxygen radicals by PMN of the donors was revealed (in the chemiluminescence test), positive correlations between this increase and stages of HIV infection were followed. The presence of HIV-positive serum in the incubation medium led to the inhibition of phagocytosis and produced almost no changes in the capacity of PMN for its completion. A decrease in phagocytosis correlated with the level of complement and did not depend on other serum factors; on the contrary, an increase in chemiluminescence was not linked with the levels of complement, but had correlative relationships with the levels of anti-HIV antibodies and circulating immune complexes.

[The presence of HIV DNA in the neutrophils of persons infected with the human immunodeficiency virus and the clinical parallels of this phenomenon]. / Prisutstvie DNK VICh v neitrofilakh lits, infitsirovannykh virusom immunodefitsita cheloveka, i klinicheskie paralleli étogo fenomena.

Relationships between some clinical data and the detection of human immunodeficiency virus (HIV) DNA in polymorphonuclear neutrophils (PMN) of 37 HIV-infected persons were studied. HIV DNA was determined with the use of polymerase chain reaction. The level of HIV DNA in PMN, sufficient for determination, was mostly detected in persons with clinical manifestations of HIV infection and less frequently in asymptomatic patients (46.7% and 18.2% respectively, p < 0.05). The significant relationship between the presence of HIV DNA in NP and a decrease in the CD4/CD8 ratio, as well as an increase in the level of immunoglobulins in the blood serum, has been established. The presence of HIV DNA in PMN was positively related to the decrease of the functional activity of these cells and the level of PMN in peripheral blood. The infection of PMN with HIV may be one of important mechanisms of their damage in HIV infection.

[The comparative characteristics of the indices of lymphocyte and neutrophil functional activity in patients with HIV infection and chronic viral hepatitis B]. / Sravnitel'naia kharakteristika nekotorykh pokazatelei funktsional'noi aktivnosti limfotsitov i neitrofilov u bol'nykh s infektsiei VICh i khronicheskim virusnym gepatitom B.

In 34 patients with human immunodeficiency virus (HIV) infection at the asymptomatic stage and 29 patients with chronic viral hepatitis B at the period of exacerbation (of these 14 patients had chronic persistent hepatitis and 15 patients had chronic active hepatitis) the complex study of the functional activity of lymphocytes and neutrophils was carried out by cytochemical methods with the simultaneous determination of the content of immunoregulating lymphocyte subpopulations. In patients with chronic active hepatitis a decrease in the percentage and the absolute number of helper T-lymphocytes and the ratio of CD4/8 in comparison with those in patients with HIV infection were revealed. At the same time patients with HIV infection exhibited more pronounced decrease in the activity of all lymphocytic enzymes under study (neutrophil esterase, acidic phosphatase and succinate dehydrogenase in lymphocytes), as well as in the activity of myeloperoxidase and the content of cation proteins and glycogen in neutrophils in comparison with patients having chronic active hepatitis.