Considerations for successful cancer immunotherapy in aged hosts.

Immunotherapy is now experiencing unprecedented successes in treating various cancers based on new understandings of cancer immunopathogenesis. Nonetheless, although ageing is the biggest risk factor for cancer, the majority of cancer immunotherapy preclinical studies are conducted in young hosts. This review will explore age-related changes in immunity as they relate to cancer immune surveillance, immunopathogenesis and responses to immunotherapy. Although it is recognized that declining T cell function with age poses a great challenge to developing effective age-related cancer immunotherapies, examples of successful approaches to overcome this hurdle have been developed. Further, it is now recognized that immune functions do not simply decline with age, but rather change in ways than can be detrimental. For example, with age, specific immune cell populations with detrimental functions can become predominant (such as cells producing proinflammatory cytokines), suppressive cells can become more numerous or more suppressive (such as myeloid-derived suppressor cells), drugs can affect aged immune cells distinctly and the aged microenvironment is becoming recognized as a significant barrier to address. Key developments in these and other areas will be surveyed as they relate to cancer immunotherapy in aged hosts, and areas in need of more study will be assessed with some speculations for the future. We propose the term 'age-related immune dysfunction' (ARID) as best representative of age-associated changes in immunity.

Association of breast cancer and obesity in a homogeneous population from Spain.

OBJECTIVE: To evaluate for the first time in Spain if the association between obesity and breast cancer prognosis is similar to that reported in other countries with non Mediterranean dietary patterns. METHODS: Weight and height and other variables of interest, tumor characteristics and current clinical status 3 yr after diagnosis were retrieved from medical files of breast cancer women diagnosed during 2006. A total of 159 cases with complete information were studied and categorized according to the World Health Organization criteria in normal-/under-weight, overweight, and obese. RESULTS: Among breast cancer patients, 70.4% were classified as overweight/ obese and 29.6% as normal weight. Prevalence of obesity was high (38.4%) in comparison with information reported for healthy women of the same region (27.11%) and was higher among post-menopausal patients and in women with low level of alcohol and tobacco consumption. Moreover, overweight/ obese cases (79.5%) tended to have more often human epidermal growth factor receptor 2 status negative when compared with those with normal weight (70.2%; p=0.097) and the survival curves tended to be influenced by body mass index although without statistical significance. CONCLUSIONS: Overweight/obesity in a Mediterranean country is highly prevalent among breast cancer patients. Our results support a putative influence of obesity per se and not the alimentary patterns as a prognostic factor in breast cancer patients justifying the need to perform larger prospective studies.

Paclitaxel and cisplatin in the treatment of metastatic non-small-cell lung cancer during pregnancy.

The concomitant occurrence of cancer during pregnancy is a rare event. The cancers most frequently detected during pregnancy are breast, cervical, melanoma, ovarian, leukaemia and lymphoma, however the diagnosis of lung cancer during pregnancy is particularly exceptional. In this case, we report on a pregnant woman who was diagnosed with non-small-cell lung cancer and received therapy with paclitaxel and cisplatin.

Paclitaxel and cisplatin in the treatment of metastatic non-small-cell lung cancer during pregnancy

The concomitant occurrence of cancer during pregnancy is a rare event. The cancers most frequently detected during pregnancy are breast, cervical, melanoma, ovarian, leukaemia and lymphoma, however the diagnosis of lung cancer during pregnancy is particularly exceptional. In this case, we report on a pregnant woman who was diagnosed with non-small-cell lung cancer and received therapy with paclitaxel and cisplatin (AU)

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Establishment and characterization of a new mantle cell lymphoma cell line, Mino.

Mantle cell lymphoma (MCL) is a distinct type of B-cell non-Hodgkin's lymphoma characterized by cyclin D1 overexpression and the cytogenetic abnormality, the t(11;14)(q13;q32). MCL cell lines have been difficult to establish and in vitro studies of these neoplasms are scarce. We describe the establishment and characteristics of a new MCL cell line, Mino. The cells are large, growing singly and in small clumps in vitro. By flow cytometry, the immunophenotype was compatible with MCL (i.e. CD5+CD20+CD23-FMC7+). Conventional cytogenetics showed hyperdiploidy with multiple complex karyotypic abnormalities, but no evidence of the t(11;14), proven to be present only by fluorescence in situ hybridization and polymerase chain reaction (PCR) methods. Western blots showed expression of cyclin D1 but no detectable cyclin D2 and cyclin D3; the retinoblastoma protein was predominantly phosphorylated. There was expression of tumor suppressor gene products including p53, p16(INK4a), and p21(WAF1). Sequencing of the TP53 gene revealed a mutation (codon 147(valine-->glycine)) in exon 5. Epstein Barr virus was absent. In summary, Mino is a new MCL cell line that may be useful to study the pathogenesis of MCL.

Stromal-derived factor-1 in human tumors recruits and alters the function of plasmacytoid precursor dendritic cells.

Dendritic-cell (DC) trafficking and function in tumors is poorly characterized, with studies confined to myeloid DCs (DC1s). Tumors inhibit DC1 migration and function, likely hindering specific immunity. The role of plasmacytoid DCs (DC2s) in tumor immunity is unknown. We show here that malignant human ovarian epithelial tumor cells express very high levels of stromal-derived factor-1, which induces DC2 precursor (preDC2) chemotaxis and adhesion/transmigration, upregulates preDC2 very late antigen (VLA)-5, and protects preDC2s from tumor macrophage interleukin-10-induced apoptosis, all through CXC chemokine receptor-4. The VLA-5 ligand vascular-cell adhesion molecule-1 mediated preDC2 adhesion/transmigration. Tumor preDC2s induced significant T-cell interleukin-10 unrelated to preDC2 differentiation or activation state, and this contributed to poor T-cell activation. Myeloid precursor DCs (preDC1s) were not detected. Tumors may weaken immunity by attracting preDC2s and protecting them from the harsh microenvironment, and by altering preDC1 distribution.

Reciprocal regulation of plasmacytoid dendritic cells and monocytes during viral infection.

Reciprocal regulation of opposing functions characterizes biological systems. We now show that adenovirus-infected plasmacytoid dendritic cells (PDC) inhibit monocyte to myeloid dendritic cell (MDC) differentiation and function, and that adenovirus-infected monocytes inhibit PDC type I interferon secretion. Adenovirus-infected PDC secreted IFN-alpha, beta and omega in an 86:2:1 ratio. PDC type I interferons inhibited MDC differentiation and function (reduced IL-12 secretion, IFN-gamma induction, MLR and CD40 expression, and increased CD1a(+)CD14(+) cells). Type I interferon receptor blocking antibody reversed all PDC effects, and recombinant IFN-alpha, beta or omega replicated all effects, except reduced CD40. Adenovirus-infected monocytes suppressed PDC type I interferon secretion, which was reversed with anti-IL-10 neutralizing antibodies. Exogenous IL-10 suppressed PDC type I interferon secretion without reducing PDC viability. Therefore, monocyte IL-10 regulates PDC type I interferon secretion, and PDC type I interferons inhibit MDC differentiation and function. Such reciprocal regulation of potentially opposing influences may help modulate anti-pathogen immunity.

Gemcitabine plus vinorelbine for the treatment of advanced non-small cell lung cancer.

The aim of this study was to determine the clinical activity and toxicity of a novel chemotherapy regimen of weekly gemcitabine and vinorelbine in patients with advanced non-small cell lung cancer (NSCLC). 40 chemotherapy-naïve patients with stage IIIB/IV NSCLC were included. The doses of gemcitabine and vinorelbine were 1000 and 25 mg/m(2), respectively, given on days 1, 8 and 15, every 28 days. 38 patients were evaluable for response. One patient achieved a complete response (CR) and 10 attained a partial response (PR), for an overall response rate (ORR) of 29% (95% confidence interval (CI): 15-43%). 47% of patients experienced a clinical benefit. The main toxicity consisted of grade 3 anaemia and neutropenia in 5% of patients. Non-haematological toxicity was minimal. The dose-intensities were 744 mg/m(2)/week for gemcitabine and 15 mg/m(2)/week for vinorelbine. 40% of the patients survived for longer than 1 year. The median time to progression was 4 months and the median survival 8.5 months (95% CI: 3.1-13.8 months). The weekly administration of gemcitabine and vinorelbine is very well tolerated and results in an acceptable response rate for the treatment of NSCLC.

Macrophage-derived dendritic cells have strong Th1-polarizing potential mediated by beta-chemokines rather than IL-12.

Monocyte-derived dendritic cells (MDDCs) activate naive T lymphocytes to induce adaptive immunity, effecting Th1 polarization through IL-12. However, little is known about other potential DC Th1 polarizing mechanisms, or how T cell polarization may be affected by DCs differentiating in, or exposed to, a proinflammatory environment. Macrophages (MPhis) are DC precursors abundant in inflamed tissues, lymph nodes, and tumors. Thus we studied the T cell-activating and -polarizing properties of MPhi-derived DCs (PhiDCs). Monocytes were cultured in MPhi-CSF (M-CSF) to produce MPhis, which were then differentiated into DCs following culture with GM-CSF plus IL-4. PhiDCs activated a significant allogeneic MLR and were significantly better than MDDCs in activating T cells with superantigen. Most strikingly, PhiDCs elicited up to 9-fold more IFN-gamma from naive or Ag-specific T cells compared with MDDCs (with equivalent IL-4 secretion), despite producing up to 9-fold less IL-12. Neutralization of MDDC, but not PhiDC IL-12 significantly inhibited T cell IFN-gamma induction. PhiDCs produced up to 12-fold more beta-chemokines (macrophage-inflammatory protein-1alpha, -1beta, and RANTES) than MDDCs. Ab blockade of CCR5, but not CXC chemokine receptor 4, inhibited T cell IFN-gamma induction by PhiDCs significantly greater than by MDDCs. Thus DCs differentiating from MPhis induce T cell IFN-gamma through beta-chemokines with little or no requirement for IL-12. Myeloid DCs arising from distinct precursor cells may have differing properties, including different mechanisms of Th1 polarization. These data are the first reports of IFN-gamma induction through chemokines by DCs.

Liver allotransplantation after extracorporeal hepatic support with transgenic (hCD55/hCD59) porcine livers: clinical results and lack of pig-to-human transmission of the porcine endogenous retrovirus.

BACKGROUND: Whole organ extracorporeal perfusion of a genetically modified humanized (transgenic) pig liver has been proposed as a technology that may sustain patients with severe liver failure while awaiting human liver transplantation. METHODS: We report on two cases of successful extracorporeal perfusion of a transgenic pig liver in patients awaiting transplantation for fulminant hepatic failure. The pig livers used were transgenic for human CD55 (decay-accelerating factor) and human CD59. These transgenic modifications are designed to reduce or eliminate the hyperacute rejection inherent in pig-to-primate xenotransplants. We also report on the results of serial surveillance testing for presence of the porcine endogenous retrovirus (PoERV) in these two patients. RESULTS: Extracorporeal perfusion in two patients was performed for 6.5 and 10 hr, respectively, followed by the successful transplantation of a human liver and resultant healthy patients (18 and 5 months later as of this writing). The porcine livers showed evidence of synthetic and secretory function (decreasing protime and bilirubin, bile production). Serial polymerase chain reaction analysis of these patients' peripheral blood mononuclear cells has failed to show presence of PoERV DNA sequences. CONCLUSIONS: The CD55/CD59 transgenic porcine liver appears capable of safely "bridging" a patient to liver transplantation. Human PoERV infection from these livers has yet to be demonstrated.

Maturation of dendritic cells accompanies high-efficiency gene transfer by a CD40-targeted adenoviral vector.

Important therapeutic applications of genetically modified dendritic cells (DC) have been proposed; however, current vector systems have demonstrated only limited gene delivery efficacy to this cell type. By means of bispecific Abs, we have dramatically enhanced gene transfer to monocyte derived DC (MDDC) by retargeting adenoviral (Ad) vectors to a marker expressed on DC, CD40. Adenovirus targeted to CD40 demonstrated dramatic improvements in gene transfer relative to untargeted Ad vectors. Fundamental to the novelty of this system is the capacity of the vector itself to modulate the immunological status of the MDDC. This vector induces DC maturation as demonstrated phenotypically by increased expression of CD83, MHC, and costimulatory molecules, as well as functionally by production of IL-12 and an enhanced allostimulatory capacity in a MLR. In comparing this vector to other Ad-based gene transfer systems, we have illustrated that the features of DC maturation are not a function of the Ad particle, but rather a consequence of targeting to the CD40 marker. This vector approach may thus mediate not only high-efficiency gene delivery but also serve a proactive role in DC activation that could ultimately strengthen the utility of this vector for immunotherapy strategies.

Efficient gene delivery into epstein-barr virus (EBV)-transformed human B cells mediated by replication-defective herpes simplex virus-1 (HSV-1): A gene therapy model for EBV-related B cell malignancy.

Subgroups of the B cell malignancies are known to be associated with Epstein-Barr virus (EBV) infection, especially in immunocompromised patients. These are fatal and refractory to conventional antineoplastic therapy. B cells are usually post-mitotic cells and even mitogen activated or transformed B cells have shown relative resistance against viral mediated gene transfer. To address this issue, we employed a replication-defective herpes simplex virus-1 (HSV-1) to mediate gene transfer into EBV-transformed B cells. The virus expresses the herpes simplex virus thymidine kinase (HSV-TK) and the E. coli lacZ reporter genes and is designated T0Z.1. We used the lymphoblastoid cell line SWEIG as a model for human EBV-related B cell malignancy. This cell line was established by in vitro EBV infection of primary human peripheral blood mononuclear cells. When SWEIG cells were infected with T0Z.1, X-gal staining revealed lacZ expression in more than 20% cells even at multiplicity of infection (MOI) as low as 1 and the expression persisted for at least one week. Ganciclovir (GCV) administration after T0Z.1 infection effectively decreased the number of the infected tumor cells in a dose-responsive manner. Viral toxicity was analyzed by cell proliferation assay (MTS assay) and found to be little even at 10 MOI infection. Three MOI of the virus yielded maximum antineoplastic effect and more than 50% tumor cells were killed by HSV-TK/GCV. These results suggest the potential utility of replication-defective HSV-1 for the treatment of EBV-related B cell malignancies.

Antisense to the epstein-barr virus (EBV)-encoded latent membrane protein 1 (LMP-1) suppresses LMP-1 and bcl-2 expression and promotes apoptosis in EBV-immortalized B cells.

The Epstein-Barr virus (EBV)-encoded latent membrane protein (LMP-1) is required for viral transformation and functions to protect cells from apoptotic cell death, in part, by induction of antiapoptotic genes, including Bcl-2 and A20. We have used antisense oligodeoxynucleotides targeted to LMP-1 as a strategy to suppress LMP-1 expression and thereby inhibit its functions. We have shown that levels of LMP-1 protein in EBV-positive lymphoblastoid cell lines can be reduced by in vitro treatment with unmodified oligodeoxynucleotides targeted to the first five codons of the LMP-1 open-reading frame. Furthermore, suppression of LMP-1 was associated with molecular and phenotypic effects that included downregulation of the LMP-1-inducible antiapoptotic genes, Bcl-2 and Mcl-1, inhibition of proliferation, stimulation of apoptosis, and enhancement of sensitivity to the chemotherapeutic agent, etoposide. These effects were largely sequence-specific and observed in EBV-positive, but not EBV-negative cell lines. These studies suggest that lowering expression of LMP-1 in EBV-associated malignancy might have therapeutic effects and might synergize with other antitumor agents.

Selection and characterization of Toxoplasma gondii mutants resistant to artemisinin.

Toxoplasma gondii infection, like malaria, is sensitive to inhibition by artemisinin (ART). Mechanisms of action for ART in malaria treatment have been proposed, but little is known about its effects in T. gondii infection. To better understand its inhibitory effects on T. gondii, mutants resistant to ART were selected by progressive culture in permissive levels of the drug. Five clonal isolates were established and characterized. The isolates were approximately 65-fold less sensitive to ART than is the parental RH and showed cross-resistance to the ART derivatives dihydroartemisinin and artemether. In addition to ART resistance, 1 clone (C9) formed morphologically unusual parasitophorous vacuoles and another (A2) was avirulent for mice and protected mice from challenge with the wild type. These clonal T. gondii mutant isolates will be useful for the study of not only the mechanism of action of ART but also parasite vacuole biology and virulence factors.

Stimulation of human T lymphocytes obtained from Toxoplasma gondii-seronegative persons by proteins derived from T. gondii.

Toxoplasma gondii antigens are superantigens in mice. To investigate a superantigen effect in humans, lymphocytes from T. gondii-seronegative subjects were studied for proliferation to T. gondii antigens (TA). Marked cellular proliferation, predominantly of CD4+ lymphocytes, was apparent. TA elicited expansions of Vbeta-bearing lymphocytes in all subjects, but different Vbeta-bearing lymphocytes were expanded in different subjects in both CD4+ and CD8+ subpopulations. Cord blood cells also proliferated to TA. Previously fixed antigen-presenting cells were unable to present TA. Thus, T. gondii appears to produce a molecule(s) that induces polyclonal activation of human T cells and requires antigen processing to mediate this effect. That T. gondii does not appear to behave as a superantigen in humans is important in understanding the pathogenesis of T. gondii infection in immunocompromised hosts and in the design of anti-T. gondii vaccines.

Toxoplasma gondii-infected cells are resistant to multiple inducers of apoptosis.

Infection with certain intracellular pathogens, including viruses and bacteria, may induce host cell apoptosis. On the other hand, infection with some viruses inhibits apoptosis. Complex protozoan parasites, including Toxoplasma gondii and members of Plasmodium, Leishmania, and Microsporidia, are also obligate intracellular pathogens, yet relatively little is known regarding their subversion of host cell functions. We now report that cells infected with T. gondii are resistant to multiple inducers of apoptosis, including Fas-dependent and Fas-independent CTL-mediated cytotoxicity, IL-2 deprivation, gamma irradiation, UV irradiation, and the calcium ionophore beauvericin. Inhibition of such a broad array of apoptosis inducers suggests that a mechanism common to many, or perhaps all, apoptotic pathways is involved. The inhibitory activity requires live intracellular parasite and ongoing protein synthesis. Despite T. gondii-mediated inhibition of DNA fragmentation, infected cells can still be lysed by CTL.

Phenotypic knock-out of the latent membrane protein 1 of Epstein-Barr virus by an intracellular single-chain antibody.

Epstein-Barr virus (EBV) causes lymphoproliferative diseases in immunocompromised patients and is associated with endemic Burkitt lymphoma, nasopharyngeal carcinoma and some cases of Hodgkin disease. The latent membrane protein 1 (LMP1) of EBV is a transmembrane protein that is essential for the transformation of B lymphocytes. LMP1-mediated up-regulation of Bcl-2 is thought to be an important element in this process. As an approach to explore novel treatments for EBV-associated lymphomas, we constructed a single-chain antibody (sFv) directed against LMP1 to achieve functional inhibition of this oncoprotein in EBV-transformed B lymphocytes. We demonstrated that intracellular expression of an endoplasmic reticulum (ER)-targeted form of this sFv markedly reduced LMP1 protein levels. We also observed a decrease in intracellular level of this protein which correlated with a marked reduction of Bcl-2 expression in EBV-transformed B lymphocytes. We further demonstrated that anti-LMP1 sFv-mediated reduction of Bcl-2 correlated with increased sensitivity of these cells to drug-induced cell death. Therefore, these data suggest that an anti-LMP1 sFv used in combination with conventional chemotherapy may be useful for gene therapy of EBV-associated lymphomas in immunocompromised patients.

Pyrogenic reactions in patients undergoing cardiac catheterization associated with contaminated glass medicine cups.

Pyrogenic reactions are potentially life-threatening complications caused by bacterial endotoxin. After two cardiac catheterization patients developed rigors the same day, the procedures were halted and a case control study was conducted. To identify case patients (persons with rigors < or = 3 hr after catheterization during September 25-November 9, 1995), we reviewed medical records of all cardiac catheterization patients who had a blood culture or received intravenous meperidine. Twelve case patients and 40 randomly selected control patients were identified. No specific catheter was associated with case patients, but exposure to intracoronary-nitroglycerin (NTG) was (odds ratio = 12.0; 95% confidence interval 2.2, 75.6). NTG or indocyanine green dye was poured into glass medicine cups previously washed in an enzyme cleaner and then sterilized. The cleaner, used for an entire day, had elevated levels of gram-negative bacteria (> 10(4) colony forming units/mL) and endotoxin (434 endotoxin units [EU]/mL]); the reprocessed cups had no live bacteria but had elevated endotoxin levels (median 2,250 EU). Exposure to contaminated glass medicine cups probably resulted in pyrogenic reactions and contributed to death in two critically ill patients.

HIV-gp120 induced cell death in hematopoietic progenitor CD34+ cells.

Haematologic abnormalities accompany the majority of HIV-1 infections. At present it is unclear whether this is due directly to HIV infection of hematopoietic progenitor cells, or whether this results from an indirect mechanism secondary to HIV infection. Here we provide evidence for an indirect mechanism, whereby hematopoietic progenitor cells undergo HIV gp120-induced apoptosis (programmed cell death) even in the absence of HIV infection. Freshly isolated, purified human hematopoietic progenitor CD34+ cells, derived from both umbilical cord blood and bone marrow, co-expressed the CD4 marker at low density on their surface. Although these CD34+CD4+ cells theoretically should be capable of productive infection by HIV, we found that HIV-IIIB could not establish productive infection in these cells. Nonetheless, gp120 from IIIB could bind the cells. Thus, binding of gp120 did not correlate with infectivity. Furthermore, binding of gp120 was a specific event, leading to apoptosis upon crosslinking with anti-gp120 through a fas-dependent mechanism. If apoptosis is also observed in vivo even in uninfected hematopoietic cells, this could contribute significantly to the impairment in hematopoietic cell number and function. Our data suggest a novel indirect mechanism for depletion of CD34+ and CD34+-derived cells even in the absence of productive viral infection of these cells.

Gene therapy strategies for AIDS-related malignancies.

AIDS-related malignancies (ARM) include AIDS-defining cancers such as Kaposi's sarcoma, non-Hodgkin's lymphoma and cervical carcinoma. In addition, certain other malignancies are also increased with human immunodeficiency virus (HIV) infection. New antiretroviral agents and better prophylaxis and treatment of HIV-related opportunistic infections are prolonging the lives of HIV-infected individuals. There will thus likely be a continued rise in the incidence and prevalence of ARM in the long term, even if effective antiretroviral and other AIDS-related therapies reduce their appearance in the short term. There are presently no curative regimens for the common ARM, with the possible exception of some lymphomas. Survival is shortened by most, and treatment is often toxic and poorly tolerated. Gene therapies may thus offer a useful adjunct to conventional treatment strategies for selected ARM. Although some gene therapy strategies may work well in the HIV setting, the chronic viral infection, immunodeficient status of the host, the tendency for HIV-infected individuals to have altered drug metabolism and an increased rate of adverse drug reactions will likely present special challenges. This review summarizes the state-of-the-art in the fledgling field of gene therapy for ARM, and explores areas for future research.