Autophagy-inducing peptide increases CHO cell monoclonal antibody production in batch and fed-batch cultures.

The development of generic biopharmaceuticals is increasing the pressures for enhanced bioprocess productivity and yields. Autophagy ("self-eating") is a cellular process that allows cells to mitigate stresses such as nutrient deprivation. Reputed autophagy inhibitors have also been shown to increase autophagic flux under certain conditions, and enhance recombinant protein productivity in Chinese Hamster Ovary (CHO) cultures. Since peptides are commonly added to bioprocess culture media in hydrolysates, we evaluated the impact on productivity of an autophagy-inducing peptide (AIP), derived from the cellular autophagy protein Beclin 1. This was analyzed in CHO cell batch and fed-batch serum-free cultures producing a human Immunoglobulin G1 (IgG1). Interestingly, the addition of 1-4 µM AIP enhanced productivity in a concentration-dependent manner. Cell-specific productivity increased up to 1.8-fold in batch cultures, while in fed-batch cultures a maximum twofold increase in IgG concentration was observed. An initial drop in cell viability also occurred before cultures recovered normal growth. Overall, these findings strongly support the value of investigating the effects of autophagy pathway modulation, and in particular, the use of this AIP medium additive to increase CHO cell biotherapeutic protein production and yields.

Role of phosphatidylinositol-3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) signalling pathways in porcine reproductive and respiratory syndrome virus (PRRSV) replication.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a positive sense, single-stranded RNA genome virus that has become a major infection in swine, exerting huge economic losses to the industry worldwide. Detailed knowledge concerning the molecular mechanisms by which the virus manipulates the host cell signals transduction machinery is not only critical to further our understanding of viral replication and pathogenesis, but also guides our efforts to design new and improved therapeutic strategies. The phosphatidylinositol-3-kinase (PI3K)-dependent Akt and the mammalian target of rapamycin (mTOR) (PI3K/Akt/mTOR) are major host cell signalling pathways that regulate protein synthesis, cell growth, proliferation, migration and survival. It is also established that many viruses exploit these signalling cascades for their own benefit, driving viral protein expression, replication, as well as the suppression of the host's antiviral activities. In this article, we will review the role of these signalling pathways during PRRSV replication, and discuss some of our recent findings implicating mTOR.

CpG protects human monocytic cells against HIV-Vpr-induced apoptosis by cellular inhibitor of apoptosis-2 through the calcium-activated JNK pathway in a TLR9-independent manner.

Monocytic cells survive HIV replication and consequent cytopathic effects because of their decreased sensitivity to HIV-induced apoptosis. However, the mechanism underlying this resistance to apoptosis remains poorly understood. Lymphocytic cells are exposed to microbial products because of their translocation from the gut in persons with chronic HIV infections or following coinfections. We hypothesized that activation of monocytic cells by such microbial products through interaction with corresponding TLRs may confer antiapoptotic signals. Using HIV-viral protein R (Vpr)(52-96) peptide as a model apoptosis-inducing agent, we demonstrated that unlike monocyte-derived macrophages, undifferentiated primary human monocytes and promonocytic THP-1 cells are highly susceptible to Vpr(52-96)-induced apoptosis. Interestingly, monocytes and THP-1 cells stimulated with TLR9 agonist CpG induced almost complete resistance to Vpr(52-96)-induced apoptosis, albeit through a TLR9-independent signaling pathway. Moreover, CpG selectively induced the antiapoptotic cellular inhibitor of apoptosis (c-IAP)-2 protein and inhibition of the c-IAP-2 gene by either specific small interfering RNA or synthetic second mitochondrial activator of caspases mimetic reversed CpG-induced resistance against Vpr(52-96)-mediated apoptosis. We demonstrated that c-IAP-2 is regulated by the JNK and calcium signaling pathway, in particular calmodulin-dependent protein kinase-II. Furthermore, inhibition of JNK and the calcium signaling including the calmodulin-dependent protein kinase-II by either pharmacological inhibitors or their specific small interfering RNAs reversed CpG-induced protection against Vpr(52-96)-mediated apoptosis. We also show that CpG induced JNK phosphorylation through activation of the calcium signaling pathway. Taken together, our results suggest that CpG-induced protection may be mediated by c-IAP-2 through the calcium-activated JNK pathway via what appeared to be TLR9-independent signaling pathways.

Inverse association of repressor growth factor independent-1 with CD8 T cell interleukin (IL)-7 receptor [alpha] expression and limited signal transducers and activators of transcription signaling in response to IL-7 among [gamma]-chain cytokines in HIV patients.

BACKGROUND: CD8 T lymphocytes from chronically infected HIV-positive patients degenerate into a preapoptotic state and exhibit impaired functionality. Particularly in viremic patients, this was associated with an increased proportion of interleukin-7 receptor-alpha low-expressing (IL-7Ralpha(low)) effector-like CD8 T cells. As cytokine signaling through signal transducers and activators of transcription (STAT) is essential for cellular function, we hypothesized that activation of this pathway may be impaired in these cells. OBJECTIVES: To evaluate cytokine-induced STAT activation in IL-7Ralpha(low) and IL-7Ralpha(high) CD8 T cells from chronically infected HIV-positive patients and investigate the potential molecular mechanism involved in the reduced IL-7Ralpha expression. METHODS: CD8 T cells from HIV-positive patients on and off antiretroviral therapy were assayed respectively for STAT activation, cytokine receptor, and transcription factor expression by flow cytometry and real-time PCR. RESULTS: IL-7 stimulation failed to activate STAT5 in a substantial proportion of patient CD8 T cells. This correlated with reduced IL-7Ralpha mRNA and surface protein expression. Interestingly, IL-7Ralpha(low) cells appeared to be fully capable of recruiting the STAT pathway in response to IL-2, IL-4, IL-10, and IL-15. mRNA expression suggested a potential role for growth factor independent (Gfi)-1 as an IL-7Ralpha transcriptional repressor, but not that of other transcriptional regulators studied, including Gfi-1B and GA-binding protein alpha. Programmed death-1 inhibitory receptor, though upregulated in CD8 T cells from HIV-positive patients, appeared unrelated to IL-7Ralpha expression and STAT signaling capacity.

Anti-apoptotic genes in the survival of monocytic cells during infection.

Macrophages are cells of the immune system that protect organisms against invading pathogens by fulfilling critical roles in innate and adaptive immunity and inflammation. They originate from circulating monocytes and show a high degree of heterogeneity, which reflects the specialization of function given by different anatomical locations. Differentiation of monocytes towards a macrophage phenotype is also accompanied by an increase of resistance against various apoptotic stimuli, a required characteristic that allows macrophages to accomplish their function in a stressful environment.Apoptosis, a form of programmed cell death, is a tightly regulated process, needed to maintain homeostasis by balancing proliferation with cellular demise. Caspases, a family of cysteine proteases that are highly conserved in multicellular organisms, function as central regulators of apoptosis. FLIP (FLICE-inhibitory protein), anti-apoptotic members of the Bcl2 family and inhibitors of apoptosis (IAP) are the main three groups of anti-apoptotic genes that counteract caspase activation through both the extrinsic and intrinsic apoptotic pathways.Modulation of the apoptotic machinery during viral and bacterial infections, as well as in various malignancies, is a wellestablished mechanism that promotes the survival of affected cells. The involvement of anti-apoptotic genes in the survival of monocytes/macrophages, either physiological or pathological, will be described in this review. How viral and bacterial infections that target cells of the monocytic lineage affect the expression of anti-apoptotic genes is important in understanding the pathological mechanisms that lead to manifested disease. The latest therapeutic approaches that target anti-apoptotic genes will also be discussed.

Amplification of the signal transducer and activator of transcription I signaling pathway and its association with apoptosis in monocytes from HIV-infected patients.

BACKGROUND: Monocytes/macrophages play a major role in inflammation and pathogen clearance. However, chronic immune activation observed during HIV infection may also cause cellular dysfunction and tissue pathology. Indeed, several defects have been reported in these cells during HIV infections. As cytokine responsiveness via the signal transducer and activator of transcription (STAT1) signaling pathway is critical for these functions, we hypothesized that its activation in monocytes from HIV-positive patients may be disrupted. OBJECTIVES: To evaluate cytokine-dependent STAT signaling in monocytes from HIV-positive patients and study the biological impact and molecular mechanisms responsible for the alterations in the interferon (IFN)-gamma-induced STAT1 pathway observed. METHODS: Monocytes from chronically infected HIV-positive patients on and off antiretroviral therapy were assayed respectively for STAT activation, apoptosis, and other downstream effects by flow cytometry, real-time PCR and enzyme-linked immunosorbent assay. RESULTS: Unlike IFN-alpha, interleukin-10, granulocyte macrophage colony-stimulating factor, and interleukin-4, only IFN-gamma-induced STAT1 activation was upregulated in monocytes from off-therapy patients compared with those on antiretroviral therapy and HIV-negative controls, correlating with increased total STAT1 expression. Among the IFN-gamma responsive genes (IRF-1, CXCL9, CXCL10) studied, differential effects were observed, likely reflecting the more complex regulatory control over their expression. Interestingly, spontaneous monocyte apoptosis was elevated in HIV-positive patients off-therapy compared with HIV-negative controls and correlated with STAT1 expression. IFN-gamma-induced apoptosis was also increased and persisted despite seemingly effective antiretroviral therapy. CONCLUSION: Amplification of STAT1 signaling and apoptosis may reflect the chronic nature of immune activation in HIV-positive patients and contribute to the functional impairment observed in monocytes through the course of the disease.

Disruption of the gamma c cytokine network in T cells during HIV infection.

The common gamma chain (gammac)-sharing cytokines (IL's-2, 4, 7, 9, 15, and 21) play a vital role in the survival, proliferation, differentiation and function of T lymphocytes. As such, disruption of their signaling pathways would be expected to have severe consequences on the integrity of the immune system. Indeed, it appears that the signaling network of these cytokines is both disrupted and exploited by HIV at various stages of infection. IL-2 secretion and signaling downstream of its receptor are impaired in T cells from chronically-infected HIV+ patients. Elevated plasma IL-7 levels and decreased IL-7Ralpha expression in patient T cells results in significantly decreased responsiveness to this critical cytokine. Interestingly, IL-2 and IL-15 are also able to render CD4+ T cells permissive to HIV infection through their influence on the activity of the APOBEC3G deaminase enzyme. Herein, we describe the current state of knowledge on how the gammac cytokine network is affected during HIV infection, with a focus on how this impairs CD4+ and CD8+ T cell function while also benefiting the virus itself. We also address the use of cytokines as adjuncts to highly active antiretroviral therapy to bolster immune reconstitution in infected patients.

Interleukin-2: from T cell growth and homeostasis to immune reconstitution of HIV patients.

Interleukin (IL)-2 was initially described as a major stimulant of T lymphocytes in vitro. Later, the characterization of IL-2 knockout animals showed that the ability to stimulate T cells could be replaced by other cytokines. In vivo, IL-2 plays a unique role in controlling lymphoproliferation. This is partly explained by its role in the generation and maintenance of T regulatory cells (Treg). In HIV-infected patients, the IL-2/IL-2 receptor (IL-2R) system is dysregulated. The fact that IL-2 is underproduced along with defective IL-2R signaling detected in patient lymphocytes, may explain the progressive impairment of the immune system that occurs during chronic infection with this virus. These defects are partly reversed by highly active antiretroviral therapy (HAART). However, in some patients IL-2R defects persist and the CD4 counts remain low despite good control of the viral load. These patients benefit from HAART given in conjunction with IL-2 therapy.

Potency of cell-mediated immune responses to different combined HIV-1 immunogens in a humanized murine model.

In this study, cell-mediated immune responses were evaluated in HLA-A2.1 mice that received polycistronic vector expressing HIV-1 gp120, gag and pol or single vectors expressing gp120 + gag/pol as well as recombinant structural proteins and adjuvants. Mice primed with the polycistronic DNA/CpG and boosted with the same regimen plus proteins induced a higher T-cell proliferative response to gp120. However, a very high frequency of IFN-gamma was detected in mice receiving the mixture of gp120 + gag/pol DNA constructs, recombinant proteins and CpG. We also measured specific CD8+T cells in PBMCs by intracellular cytokine and HLA-A2.1-peptide dimer staining in response to HLA-A2.1-restricted HIV-1 epitopes (gp120, gag and pol). The group that received single gp120 + gag/pol DNA constructs, recombinant proteins and CpG had a higher CD8+T cell response to the combination of peptides compared to the other groups that received the polycistronic construct. The present study reveals an optimal combination of immunogens to enhance immune responses against HIV-1.

Recent advances in the regulation of CD44 expression and its role in inflammation and autoimmune diseases.

Interaction of CD44, an adhesion molecule, with its extracellular matrix ligand, hyaluronan (HA), has been suggested to play a critical role in a number of biological manifestations, including cell migration, tumorigenesis, metastasis, and regulation of immune responses. CD44 comprises a large family of transmembrane glycoproteins that exhibit extensive molecular heterogeneity. This heterogeneity in size is generated by alternative RNA splicing of variable exons as well as by post-translational modifications. Most cell types express CD44 but do not bind HA. The biological functions of CD44, including the regulation of lymphocyte recruitment to the sites of inflammation, have been attributed to the generation of a functionally active, HA-adhesive phenotype. The molecular mechanisms underlying the regulation of CD44 expression and the generation of a functionally active HA-binding phenotype are not well understood. Recently, CD44-HA interactions have been reported to play a critical role in a number of autoimmune diseases in humans and experimental animal models. Initial studies have taken advantage of anti-CD44 antibodies which specifically block CD44-HA interactions. Administration of these antibodies in several experimental murine models of autoimmune diseases resulted in alleviation of inflammatory reactions. In addition, the generation of CD44-deficient animals has facilitated our understanding of the involvement of CD44 in inflammation and autoimmune diseases. This review will focus on the recent advances in the molecular mechanisms regulating CD44 expression, ligand binding, as well as the contribution of CD44 to the development of inflammation and autoimmune disorders.

Defective interleukin-2-dependent STAT5 signalling in CD8 T lymphocytes from HIV-positive patients: restoration by antiretroviral therapy.

BACKGROUND: CD8 T lymphocytes are critical in the control of HIV replication and disease progression. Our previous studies demonstrated that CD8 T cells from chronically infected patients with high virus load proliferated poorly in response to interleukin-2 (IL-2), a cytokine critical in CD8 T cell growth and differentiation, even though relatively high levels of IL-2 receptor (IL-2R) were expressed. This suggested that signal transduction defects in response to IL-2 might be involved. The STAT5 transcription factor is important in IL-2-dependent biological responses and it is known that there are defects in unstimulated CD3 and CD4 cells in HIV-infected patients. OBJECTIVE: To investigate whether the induction of STAT5 by IL-2 is altered in the CD8 T cells from HIV-positive individuals and the impact of highly active antiretroviral therapy (HAART) on its status. METHODS: CD8 T lymphocytes were purified from the peripheral blood mononuclear cells of HIV-positive patients before and after HAART. Ex vivo IL-2-induced STAT5 activation was evaluated by immunoblotting and electrophoretic mobility shift assays. RESULTS: CD8 T cells from a subset of untreated HIV-positive patients were unable to activate STAT5a and STAT5b proteins functionally in response to IL-2. This defect was not a result of alterations in IL-2R expression but correlated with an impaired activation of the upstream kinase Jak-3, known to mediate STAT5 activation. Overall, HAART restored Jak/STAT signalling in such patients. CONCLUSIONS: These results further uncover a potential mechanism by which CD8 T cell function is impaired in HIV-infected patients.