Pedro O. de Campos-Lima

Efficient human hematopoietic cell transduction using RD114- and GALV-pseudotyped retroviral vectors produced in suspension and serum-free media.

Retroviral vectors derived from the Moloney murine leukemia virus have been used in successful and promising gene therapy clinical trials. However, platforms for their large-scale production must be further developed. As a proof of principle, we reported the generation of a packaging cell line that produces amphotropic retroviral vectors in suspension and serum-free medium (SFM). In the present study, we have constructed and characterized two retroviral packaging cell lines designed for gene transfer in hematopoietic cells. These cell lines grow in suspension and SFM, and produce high-titer RD114- and gibbon ape leukemia virus (GALV)-pseudotyped vectors for a 3-month culture period. Viral particles released are as robust during repeated freeze-thaw cycles and on thermal inactivation at 37 degrees C as their counterparts produced in cells cultured adherently with serum. We also show that RD114- and GALV-pseudotyped vectors produced in suspension and SFM efficiently transduce human lymphocytes and hematopoietic stem cells. As these retroviral packaging cell lines distinctively maintain high vector titers while growing in suspension and SFM, we conclude that these cell lines are uniquely suitable for large-scale clinical-grade vector production for late-phase clinical trials involving gene transfer into hematopoietic cells.

Immunosuppressive Effect of Isopropanol: Down-Regulation of Cytokine Production Results from the Alteration of Discrete Transcriptional Pathways in Activated Lymphocytes

Isopropanol (IPA) is widely used in household applications and constitutes a leading cause of acute alcohol intoxication second only to ethanol. Although the effects of ethanol on the immune system have been extensively studied, far fewer data are available on IPA. Given the structural similarity between the two molecules, we hypothesized that IPA could as well have immunomodulatory properties. We report here that acute IPA exposure is detrimental to human T lymphocyte and NK cell activity in vitro in concentrations as low as 0.08–0.16% (13–26 mM). IPA treatment did not affect receptor-mediated early signaling but had a reproducible and dose-dependent effect on the nuclear translocation of NFAT and AP-1. Furthermore, we show in a model of acute IPA intoxication that animals became immunosuppressed as judged by their reduced ability to release IL-2 and IFN-γ in the serum in response to staphylococcal enterotoxin B. This effect was also associated to the down-regulation of TNF-α production and was sufficiently strong to rescue susceptible animals from enterotoxin-induced toxic shock. Our results suggest that IPA is potentially immunosuppressive to the adaptive and innate immune system and have broad significance given the exposure of the general population to this ubiquitous chemical.

The Dysregulation of the Monocyte/Macrophage Effector Function Induced by Isopropanol Is Mediated by the Defective Activation of Distinct Members of the AP-1 Family of Transcription Factors

Isopropanol is the second most common cause of short-chain alcohol acute intoxication. Nonethanolic short-chain alcohols mediate their immunomodulatory effect by interfering with nuclear factor of activated T cells (NFAT) activation with or without additional activator protein-1 (AP-1) involvement. In the present study, we examined the immunomodulation induced by isopropanol in conditions that are not reliant on NFAT: the inflammatory cytokine response of lipopolysaccharide (LPS)-stimulated monocytes. Our hypothesis was that isopropanol acute exposure would have an attenuated effect or no consequence in this setting. To our surprise, the impairment of AP-1 activation was sufficient to mediate a severe and dose-dependent phenotype in human monocytes in vitro at alcohol concentrations as low as 0.16% (or 26 mM). There were three outcomes: interleukin (IL)-1β/IL-8 were unaltered; IL-6 was upregulated; and tumor necrosis factor alpha (TNF-α)/CCL2 were downregulated. The effector function of human monocyte-derived macrophages was also compromised. Our results showed that Toll-like receptor 4 early signaling was preserved, as isopropanol did not change the kinase activity of the IL-1 receptor-associated kinase 1 in LPS-stimulated cells. The nuclear factor-κB signaling cascade and the p38/c-Jun N-terminal kinase modules of the mitogen-activated protein kinase pathway were alcohol insensitive. Conversely, the activation of extracellular signal-regulated protein kinase and, ultimately, of c-Fos and JunB were impaired. The alcohol-induced cytokine dysregulation was confirmed in a mouse model of isopropanol intoxication in which the production of TNF-α in response to LPS challenge was virtually abolished. The magnitude of this alcohol effect was sufficiently high to rescue animals from LPS-induced toxic shock. Our data contribute to the dismal body of information on the immunotoxicology of isopropanol, one of the most ubiquitous chemicals to which the general population is significantly exposed.

The idiotype (Id) cascade in mice elicited the production of anti-R24 Id and anti-anti-Id monoclonal antibodies with antitumor and protective activity against human melanoma

Gangliosides have been considered as potential targets for immunotherapy because they are overexpressed on the surface of melanoma cells. However, immunization with purified gangliosides results in a very poor immune response, usually mediated by IgM antibodies. To overcome this limitation, we immunized mice with R24, a monoclonal antibody (mAb) that recognizes the most tumor‐restricted ganglioside (GD3); our goal was to obtain anti‐idiotype (Id) antibodies bearing the internal image of GD3. Animals produced anti‐Id and anti‐anti‐Id antibodies. Both anti‐Id and anti‐anti‐Id antibodies were able to inhibit mAb R24 binding to GD3. In addition, the anti‐anti‐Id antibodies were shown to recognize GD3 directly. Anti‐Id and anti‐anti‐Id mAb were then selected from two fusion experiments for evaluation. The most interesting finding emerged from the characterization of the anti‐anti‐Id mAb 5.G8. It was shown to recognize two different GD3‐expressing human melanoma cell lines in vitro and to mediate tumor cell cytotoxicity by complement activation and antibody‐dependent cellular cytotoxicity. The biological activity of the anti‐anti‐Id mAb was also tested in a mouse tumor model, in which it was shown to be a powerful growth inhibitor of melanoma cells. Thus, activity of the anti‐anti‐Id mAb 5.G8 matched that of the prototypic anti‐GD3 mAb R24 both in vitro and in vivo. Altogether, our results indicate that the idiotype approach might produce high affinity, specific and very efficient antitumor immune responses. (Cancer Sci 2011; 102: 64–70)

Nilotinib and imatinib inhibit cytarabine cellular uptake: implications for combination therapy.

The tyrosine kinase inhibitor (TKI) imatinib has been used for a decade to treat chronic myeloid leukemia (CML). A very efficient response is obtained with patients in chronic phase, but its efficacy in late phase patients is often transient. The combination of imatinib or of the new TKI nilotinib with cytarabine is a new treatment approach proposed for CML. We have investigated the effect of imatinib and nilotinib on cytarabine uptake, and have found that both molecules inhibit cytarabine transport. These results should impact on the design of clinical trials that investigate the combination of TKIs and nucleoside analogs.

Methanol Induces a Discrete Transcriptional Dysregulation that Leads to Cytokine Overproduction in Activated Lymphocytes

Methanol is an important cause of acute alcohol intoxication; it is ubiquitously present at home and in the workplace. Although the existing literature provides a reasonable insight into the immunological impact of ethanol and to a much lesser extent of isopropanol, much less data are available on methanol. We hypothesized on structural grounds that methanol would share the immunosuppressive properties of the two other short-chain alcohols. We report here that methanol increases the proliferative capacity of human T lymphocytes and synergizes with the activating stimuli to augment cytokine production. The cytokine upregulation was observed in vitro at methanol concentrations as low as 0.08% (25mM) as measured by interleukin-2, interferon-γ, and tumor necrosis factor-α release in T cells. Methanol did not affect the antigen receptor-mediated early signaling but promoted a selective and differential activation of the nuclear factor of activated T cells family of transcription factors. These results were further substantiated in a mouse model of acute methanol intoxication in which there was an augmented release of proinflammatory cytokines in the serum in response to the staphylococcal enterotoxin B. Our results suggest that methanol has a discrete immunological footprint of broad significance given the exposure of the general population to this multipurpose solvent.

Short-term immunological effects of non-ethanolic short-chain alcohols.

Short-chain alcohols are embedded into several aspects of modern life. The societal costs emanating from the long history of use and abuse of the prototypical example of these molecules, ethanol, have stimulated considerable interest in its general toxicology. A much more modest picture exists for other short-chain alcohols, notably as regards their immunotoxicity. A large segment of the general population is potentially exposed to two of these alcohols, methanol and isopropanol. Their ubiquitous nature and their eventual use as ethanol surrogates are predictably associated to accidental or deliberate poisoning. This review addresses the immunological consequences of acute exposure to methanol and isopropanol. It first examines the general mechanisms of short-chain alcohol-induced biological dysregulation and then provides a tentative model to explain the molecular events that underlie the immunological dysfunction produced by methanol and isopropanol. The time-related context of serum alcohol concentrations in acute poisoning, as well as the clinical implications of their short-term immunotoxicity, is also discussed.

The size of the unbranched aliphatic chain determines the immunomodulatory potency of short and long chain n-alkanols.

Background: Aliphatic n-alkanols have multiple biological effects not yet completely characterized. Results: n-Alkanols are immunomodulators that act downstream of the cell membrane by dysregulating the activation of the NFAT and NF-κB transcription factors. Conclusion: There is a correlation between the immunomodulatory capacity of the homologous series of n-alkanols and the size of the aliphatic chain/hydrophobicity. Significance: This work contributes to the understanding of the biological activity of ubiquitous n-alkanols. Aliphatic n-alkanols are a family of ubiquitous substances that display general anesthetic properties in accordance to their degree of hydrophobicity. In addition, the immunomodulatory activity of one of its members, ethanol, has long been recognized. We reasoned that because unbranched aliphatic n-alkanols are structurally very similar they might have an immunological impact that mirrors their anesthetic potency. We report the impact of the homologous C1–C12 alcohol series on the ability of activated primary human lymphocytes to produce IFN-γ. Methanol enhanced IFN-γ production whereas C2–C10 alcohols reduced the release of this cytokine. The activity of the n-alkanol series was observed within a wide concentration window ranging from millimolar levels for short chain alcohols to micromolar amounts for C7–C10 alcohols. There was a clear correlation between immunomodulatory activity and hydrophobicity of the compounds, but a cutoff effect was evident at C11. n-Alkanols were shown to act downstream of the cell membrane because T cell receptor early signaling was preserved. The activation of the nuclear factor of activated T cells (NFAT) was down-regulated progressively in accordance to the size of the n-alkanol aliphatic chains with a clear downward trend that was interrupted at C11. The nuclear factor-κB (NF-κB) signaling was also compromised, but the cutoff appeared earlier at C10. The pattern of immunomodulation and transcriptional dysregulation induced by the n-alkanol series suggested the existence of interaction pockets of defined dimensions within intracellular targets that compromise the activation of NFAT and NF-κB transcription factors and ultimately modulate the effector function of the T lymphocyte.

Characterization of an alternative packaging system derived from the cat RD114 retrovirus for gene delivery.

Retroviral vectors derived from the Moloney murine leukemia virus (MLV) are widely used in gene therapy. Pseudotyping of these vectors with the cat RD114 retrovirus envelope increases their potential for delivering genes into human hematopoietic cells. In the present study, we have further investigated the potential of the RD114 retrovirus in gene therapy. We describe and characterize an alternative retroviral packaging system derived from the RD114 retrovirus.