c-Src regulation of fibroblast growth factor-induced proliferation in murine embryonic fibroblasts

Activated fibroblast growth factor receptor 1 (FGFR1) propagates FGF signals through multiple intracellular pathways via intermediates FRS2, PLCgamma, and Ras. Conflicting reports exist concerning the interaction between FGFR1 and Src family kinases. To address the role of c-Src in FGFR1 signaling, we compared proliferative responses of murine embryonic fibroblasts (MEF) deficient in c-Src, Yes, and Fyn to MEF expressing either endogenous levels or overexpressing c-Src. MEF with endogenous c-Src had significantly greater FGF-induced DNA synthesis and proliferation than cells lacking or overexpressing c-Src. This was related directly to c-Src expression by analysis of c-Src-deficient cells transfected with and sorted for varying levels of a c-Src expression vector. This suggests an "optimal" quantity of c-Src expression for FGF-induced proliferation. To determine if this was a general phenomenon for growth factor signaling pathways utilizing c-Src, responses to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and lysophosphatidic acid (LPA) were examined. As for FGF, responses to EGF were clearly inhibited when c-Src was absent or overexpressed. In contrast, varying levels of c-Src had little effect on responses to PDGF or LPA. The data show that mitogenic pathways activated by FGF-1 and EGF are regulated by c-Src protein levels and appear to differ significantly from those activated by PDGF and LPA.     

Enhanced pulmonary natural killer cell activity during murine encephalitozoonosis

Experimental infection with the protozoan parasite Encephalitozoon cuniculi induced an augmentation of pulmonary natural killer cell (NK) activity in C57BL/6 mice. Enhanced clearance of 51Cr-labelled YAC-1 lymphoma cells peaked on day 4 of infection and returned to normal levels by the tenth day of infection. Infected mice also demonstrated heightened resistance to pulmonary tumor formation compared to uninfected control mice following challenge with lung-homing B16F10 melanoma cells.     

Natural killer cell depletion enhances virus synthesis and virus-induced hepatitis in vivo

The role of natural killer (NK) cells in the natural resistance of mice to infections by several viruses was examined. Mice were specifically depleted of NK cells by i.v. injection of rabbit antiserum to asialo GM1, a neutral glycosphingolipid present at high concentrations on the surface of NK cells. Control mice were left untreated or were injected with normal rabbit serum. Four to 6 hr later, these mice were infected with lymphocytic choriomeningitis virus (LCMV), mouse hepatitis virus (MHV), murine cytomegalovirus (MCMV), or vaccinia virus. The mice were sacrificed 3 days post-infection and assayed for virus in liver and spleen, spleen NK cell activity, and plasma interferon (IFN). All mice treated with anti-asialo GM1 antibody had drastically reduced NK cell-mediated lysis. Correlating with NK cell depletion, these mice had significantly higher (up to 500-fold) titers of MCMV, MHV, or vaccinia virus in their livers and spleens as compared to control mice. NK cell-depleted MCMV and MHV-infected mice had higher levels of plasma IFN than controls, correlating with the higher virus titers. These NK cell-depleted, virus-infected mice had more extensive hepatitis, assayed by the number of inflammatory foci in their livers, as compared to control virus-infected mice; these foci were also larger and contained more degenerating liver cells than those in control mice. In contrast to the results obtained with MHV, MCMV, and vaccinia virus, NK cell depletion had no effect on virus titers in the early stages of acute LCMV infection or during persistent LCMV infection. Mice depleted of NK cells had similar amounts of LCMV in their spleens and similar plasma IFN levels. Because this antibody to asialo GM1 does not impair other detectable immunologic mechanisms, these data support the hypothesis that NK cells act as a natural resistance mechanism to a number of virus infections, but suggest that their relative importance may vary from virus to virus.     

Generation of a conditionally transformed murine embryonic fibroblast cell line using doxycycline-dependent IGF-1R overexpression

The insulin-like growth factor I receptor (IGF1-R) system has long been implicated in cancer and is a promising target for tumor therapy. Besides in vitro screening assays, the discovery of specific inhibitors against IGF-1R requires relevant cellular models, ideally applicable to both in vitro and in vivo studies. With this aim in mind, the authors generated an inducible cell line using the tetracycline-responsive gene expression system to mimic the effects of therapeutic inhibition of the IGF-1R both in vitro and on established tumors in vivo. Inducible overexpression of IGF-1R in murine embryonic fibroblasts was achieved and resulted in the transformation of the cells as verified by their ability to grow in soft agar and in nude mice. Continuous repression of exogenous IGF-1R expression completely prevented outgrowth of the tumors. Furthermore, induced repression of IGF-1R expression in established tumors resulted in regression of the tumors. Interestingly, however, IGF-1R-independent relapse of tumor growth was observed upon prolonged IGF-1R repression. The IGF-1R cell line generated using this approach was successfully employed to test reference small-molecule inhibitors in vitro and an IGF-1R-specific inhibitory antibody, EM164, in vivo. Besides efficacy as a read-out, phospho-AKT could be identified as a pharmacodynamic biomarker, establishing this cell line as a valuable tool for the preclinical development of IGF-1R inhibitors.     

Reduced mitotic activity at the periphery of human embryonic stem cell colonies cultured in vitro with mitotically-inactivated murine embryonic fibroblast feeder cells

This study attempted to investigate whether different levels of mitotic activity exist within different physical regions of a human embryonic stem (hES) cell colony. Incorporation of 5-bromo-2-deoxyuridine (BrdU) within newly-synthesized DNA, followed by immunocytochemical staining was used as a means of detecting mitotically-active cells within hES colonies. The results showed rather surprisingly that the highest levels of mitotic activity are primarily concentrated within the central regions of hES colonies, whereas the peripheral regions exhibited reduced levels of cellular proliferation. Two hypothetical mechanisms are therefore proposed for hES colony growth and expansion. Firstly, it is envisaged that the less mitotically-active hES cells at the periphery of the colony are continually migrating outwards, thereby providing space for newly-divided daughter cells within the more mitotically-active central region of the hES colony. Secondly, it is proposed that the newly-divided hES cells within the central region of the colony somehow migrate to the outer periphery. This could possibly explain why the periphery of hES colonies are less mitotically-active, since there would obviously be an extended time-lag before newly-divided daughter cells are ready again for the next cell division. Further investigations need to be carried out to characterize the atypical mechanisms by which hES colonies grow and expand in size.     

Natural killer cells in patients with pulmonary tuberculosis]

In the existent literature the number of works devoted to the subpopulation of natural killer cells (NK) is not significant. The purpose of the present study was to determine the NK content in the blood of pulmonary tuberculosis patients; to establish correlation of the level of their content with the content of the previously studied T-lymphocyte subpopulations; to determine the intensity of the fluorescence of NK, CD3+, CD4+, CD8+ lymphocytes. The data were obtained on the significant increase in the NK mean level in tuberculosis patients (20.37 +/- 1.74) as compared with that in healthy subjects (12.77 +/- 2.56). The NK fluorescence intensity (56.33 +/- 2.28) conditioned by the Fc-receptor expression intensity is significantly lower than the analogous index in healthy volunteers (82.4 +/- 7.69). The NK level in the blood of tuberculosis patients correlates with the content of CD3+, CD8+ lymphocytes as well as with the CD4+/CD8+ index.     

NK细胞和NKT细胞发育的转录调控

自然杀伤（natural killer,NK）细胞和自然杀伤T（natural killer T,NKT）细胞是参与机体抗病毒免疫和肿瘤免疫的两群淋巴细胞亚群,是介导先天性免疫（innate immunity）应答和调节适应性免疫（adaptive immunity）应答的重要效应细胞。近年来,随着对NK细胞和NKT细胞及其转录调控因子研究的不断深入,NK细胞和NKT细胞的发育机制逐步被阐明,这将为提高NK细胞和NKT细胞的抗病毒和肿瘤免疫疗效提供新的策略。     

Natural killer and lymphokine-activated killer cell functions in chronic myeloid leukemia

Summary The natural killer (NK) and lymphokine-activated killer (LAK) cell activities of peripheral blood lymphocytes from chronic myeloid leukemia (CML) patients in remission and from healthy donors have been studied. Regression analysis to compare both cytotoxic responses in individual donors and the frequency of LAK cell precursors was also carried out. About 42% of CML patients in remission showed low NK activity (less than the mean percentage NK activity of healthy donors — 2 SD) and were categorised as low NK responders. The stage of remission or the drugs used to bring about remission did not influence the NK status. The LAK activity of low NK as well as normal NK responder CML patients was significantly low against the NK-sensitive K562 cell line and the NK-resistant VIP (melanoma) and T-24 (bladder carcinoma) tumor targets, as assessed by linear regression analysis. Allogeneic leukemic cells were more resistant to killing, especially by patients' LAK cells. The frequency analysis of LAK cell precursors revealed a significant reduction in the LAK cell progenitor frequency in CML patients in remission.     

Role of pulmonary microvascular endothelial cell apoptosis in murine sepsis-induced lung injury in vivo

Background

Sepsis remains a common and serious condition with significant morbidity and mortality due to multiple organ dysfunction, especially acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Sepsis-induced ALI is characterized by injury and dysfunction of the pulmonary microvasculature and pulmonary microvascular endothelial cells (PMVEC), resulting in enhanced pulmonary microvascular sequestration and pulmonary infiltration of polymorphonuclear leukocytes (PMN) as well as disruption of the normal alveolo-capillary permeability barrier with leak of albumin-rich edema fluid into pulmonary interstitium and alveoli. The role of PMVEC death and specifically apoptosis in septic pulmonary microvascular dysfunction in vivo has not been established.

Methods

In a murine cecal ligation/perforation (CLP) model of sepsis, we quantified and correlated time-dependent changes in pulmonary microvascular Evans blue (EB)-labeled albumin permeability with (1) PMVEC death (propidium iodide [PI]-staining) by both fluorescent intravital videomicroscopy (IVVM) and histology, and (2) PMVEC apoptosis using histologic fluorescent microscopic assessment of a panel of 3 markers: cell surface phosphatidylserine (detected by Annexin V binding), caspase activation (detected by FLIVO labeling), and DNA fragmentation (TUNEL labeling).

Results

Compared to sham mice, CLP-sepsis resulted in pulmonary microvascular barrier dysfunction, quantified by increased EB-albumin leak, and PMVEC death (PI+ staining) as early as 2 h and more marked by 4 h after CLP. Septic PMVEC also exhibited increased presence of all 3 markers of apoptosis (Annexin V+, FLIVO+, TUNEL+) as early as 30 mins – 1 h after CLP-sepsis, which all similarly increased markedly until 4 h. The time-dependent changes in septic pulmonary microvascular albumin-permeability barrier dysfunction were highly correlated with PMVEC death (PI+; r = 0.976, p < 0.01) and PMVEC apoptosis (FLIVO+; r = 0.991, p < 0.01). Treatment with the pan-caspase inhibitor Q-VD prior to CLP reduced PMVEC death/apoptosis and attenuated septic pulmonary microvascular dysfunction, including both albumin-permeability barrier dysfunction and pulmonary microvascular PMN sequestration (p < 0.05). Septic PMVEC apoptosis and pulmonary microvascular dysfunction were also abrogated following CLP-sepsis in mice deficient in iNOS (Nos2^−/−) or NADPH oxidase (p47^phox−/− or gp91^phox−/−) and in wild-type mice treated with the NADPH oxidase inhibitor, apocynin.

Conclusions

Septic murine pulmonary microvascular dysfunction in vivo is due to PMVEC death, which is mediated through caspase-dependent apoptosis and iNOS/NADPH-oxidase dependent signaling.     

Natural killer cell activity in mouse aggregation chimeras

The activity of natural killer (NK) cells in spleen against syngeneic and allogeneic tumor cells was studied by the use of tetraparental mouse chimeras. Chimeras were produced by aggregation of early embryos of histoincompatible mouse strains of “high” and “low” NK cell activity. NK activities of spleen cells were assayed in vitro by the ⁵¹Cr-release method. Coat color distribution and isozymal analysis (glucose-phosphate isomerase) of several lymphoid organs (thymus, lymph nodes, and bone marrow) revealed a predominant share of the “high”-NK-reactive genotype in the chimeras. However, the cellular NK activity against two target cell lines differing in their susceptibility to lysis was significantly lower in chimeras than in the “high”-reactive strain. Addition of “low”-NK spleen cells or of NH₄Cl-inactivated “high”-NK spleen cells to “high”-NK spleen cells inhibited their cytolytic activity. Possible mechanisms of the suppression of the cytolytic capacity of NK cells in chimeras are discussed.     

Natural killer cell exhaustion in SARS-CoV-2 infection

At the end of 2019, an outbreak of a severe respiratory disease occurred in Wuhan China, and an increase in cases of unknown pneumonia was alerted. In January 2020, a new coronavirus named SARS-CoV-2 was identified as the cause. The virus spreads primarily through the respiratory tract, and lymphopenia and cytokine storms have been observed in severely ill patients. This suggests the existence of an immune dysregulation as an accompanying event during a serious illness caused by this virus. Natural killer (NK) cells are innate immune responders, critical for virus shedding and immunomodulation. Despite its importance in viral infections, the contribution of NK cells in the fight against SARS-CoV-2 has yet to be deciphered. Different studies in patients with COVID-19 suggest a significant reduction in the number and function of NK cells due to their exhaustion. In this review, we summarize the current understanding of how NK cells respond to SARS-CoV-2 infection.     

Natural killer cell mediated missing-self recognition can protect mice from primary chronic myeloid leukemia in vivo

Background

Natural Killer (NK) cells are thought to protect from residual leukemic cells in patients receiving stem cell transplantation. However, multiple retrospective analyses of patient data have yielded conflicting conclusions regarding a putative role of NK cells and the essential NK cell recognition events mediating a protective effect against leukemia. Further, a NK cell mediated protective effect against primary leukemia in vivo has not been shown directly.

Methodology/Principal Findings

Here we addressed whether NK cells have the potential to control chronic myeloid leukemia (CML) arising based on the transplantation of BCR-ABL1 oncogene expressing primary bone marrow precursor cells into lethally irradiated recipient mice. These analyses identified missing-self recognition as the only NK cell-mediated recognition strategy, which is able to significantly protect from the development of CML disease in vivo.

Conclusion

Our data provide a proof of principle that NK cells can control primary leukemic cells in vivo. Since the presence of NK cells reduced the abundance of leukemia propagating cancer stem cells, the data raise the possibility that NK cell recognition has the potential to cure CML, which may be difficult using small molecule BCR-ABL1 inhibitors. Finally, our findings validate approaches to treat leukemia using antibody-based blockade of self-specific inhibitory MHC class I receptors.     

Exercise stress and murine natural killer cell function

Male C3He mice were trained to run on a treadmill (final speed, slope, and duration of 30 m/min, 8 degrees, 30 min/day, 5 days/week, respectively) for 10 weeks or they remained sedentary. At the end of the training program, half of the mice were sacrificed and half were given a single bout of exercise to exhaustion (50% stepwise increases in final running speed for 2-min intervals). Splenic catecholamine concentrations, splenic natural killer cell cytolytic activity against YAC-1 tumor targets, and frequency of asialo GM1 (a murine natural killer cell surface glycolipid)-positive splenocytes were assessed. Exhaustive exercise in both trained and untrained mice reduced the in vitro killing of tumor targets by splenic natural killer cells relative to killing by splenocytes from mice which did not undergo the acute exercise bout (P less than 0.05). The frequency of asialo GM1-positive splenocytes was also reduced in the exhaustively exercised animals (P less than 0.05). Training alone, without the additional stress of exhaustive exercise, reduced the frequency of asialo GM1-positive splenocytes relative to a sedentary condition (P less than 0.05), but did not compromise natural killer cell cytolytic activity against the tumor targets. Splenic epinephrine concentrations in the exhaustively exercised animals were elevated 3- to 5-fold above the concentrations observed in trained and sedentary mice. These results suggest that a single, acute exercise bout reduces the capacity of splenic natural killer cells to kill tumor targets in vitro and that training enhances splenic natural killer cell cytolytic activity, on a per cell basis, against tumor targets.     

Natural killer cell cytotoxicity: role of calmodulin

The phenothiazine derivatives, fluphenazine and trifluoperazine which are known to bind to calmodulin and to inhibit its activity, abrogate the development of both spontaneous and interferon-enhanced cytotoxicity of mouse splenic lymphocytes enriched for NK cell activity. Phenothiazines also inhibit the rapid increase in cyclic GMP levels in interferon-treated splenic lymphocytes. Furthermore, treatment of mouse splenic lymphocytes with electrophoretically pure interferon, alpha/beta caused a marked decrease in the level of calmodulin within 1 to 4 hours. These results provide evidence that calmodulin may play a role in the development of NK cell cytotoxicity and that the effect of interferon on calmodulin may constitute part of the molecular mechanism of interferon action.     

Redox regulation of cell proliferation by pyrrolidine dithiocarbamate in murine thymoma cells transplanted in vivo.

Pyrrolidine dithiocarbamate (PDTC) is a synthetic compound largely used in cell biological studies and known to exert either antioxidant or pro-oxidant effects. Recently, its antitumoral activity has been proposed on the basis of its antioxidant and proapoptotic effects. In the present study, we evaluated the effect of increasing i.p. doses of PDTC on the growth of a strain of highly malignant thymoma cells inoculated in the peritoneum of inbred Balb/c mice. PDTC treatment increased the number of thymoma cells in a dose-dependent manner, enhancing the percentage of proliferating tumor cells. PDTC exerted regulatory effects on cell cycle distribution, decreasing the expression of cell cycle inhibitors. Alterations in the production of intracellular reactive oxygen species, levels of oxidized glutathione, and intracellular levels of the redox-active metals iron and copper were also observed. The above results represent the first evidence that PDTC may induce in vivo cell proliferation in a murine thymoma cell model. In addition, we suggest that the ability of PDTC to bind and transport metals inside the cell and its pro-oxidant property may be factors underlying its effects on thymoma cell proliferation and cell cycle distribution.     

Natural killer cell function in HIV-1 infected patients

A cross-talk between dendritic cells (DC) and resting natural killer (NK) cells leads to the activation of both cell populations, a process requiring cell-cell contact. When the number of activated NK cells overwhelms surrounding DC, they became able to kill specifically immature DC, a feedback mechanism to shut off DC-mediated immune responses. DC, at the mucosal site, can capture HIV and transfer it to CD4+ T lymphocytes present in the regional lymph node thus giving rise to a productive infection; on the other hand, NK cells represent the first line of defence against viral infection. Our preliminary results suggest that during the early phases of an HIV infection, NK cell activity is not functionally compromised, but that infected cells might escape natural immune surveillance through several mechanisms, including a reduced lysis of autologous DC.