RESUMEN
T cell depletion prevents graft-versus-host disease (GVHD) but also removes T cell-mediated support of hematopoietic cell engraftment. A chimeric molecule composed of IL-2 and caspase-3 (IL2-cas) has been evaluated as a therapeutic modality for GVHD and selective ex vivo depletion of host-reactive T cells. IL2-cas does not affect hematopoietic cell engraftment and significantly reduces the clinical and histological severity of GVHD. Early administration of IL2-cas reduced the lethal outcome of haploidentical transplants, and survivor mice displayed markedly elevated levels of X-linked forkhead/winged helix (FoxP3(+); 50%) and CD25(+)FoxP3(+) T cells (35%) in the lymph nodes. The chimeric molecule induces in vitro apoptosis in both CD4(+)CD25(-) and CD4(+)CD25(+) subsets of lymphocytes from alloimmunized mice, and stimulates proliferation of cells with highest levels of CD25 expression. Adoptive transfer of IL2-cas-pretreated viable splenocytes into sublethally irradiated haploidentical recipients resulted in 60% survival after a lethal challenge with lipopolysaccharide, which is associated with elevated fractions of CD25(high)FoxP3(+) T cells in the lymph nodes of survivors. These data demonstrate that ex vivo purging of host-presensitized lymphocytes is effectively achieved with IL2-cas, and that IL-2-targeted apoptotic therapy reduces GVHD severity in vivo. Both approaches promote survival in lethal models of haploidentical GVHD. The mechanism of protection includes direct killing of GVHD effectors, prevention of transition to effector/memory T cells, and induction of regulatory T cell proliferation, which becomes the dominant subset under conditions of homeostatic expansion.
Asunto(s)
Trasplante de Médula Ósea/inmunología , Enfermedad Injerto contra Huésped , Proteínas Recombinantes de Fusión/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T/trasplante , Traslado Adoptivo , Animales , Antígenos CD/biosíntesis , Antígenos CD/inmunología , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Caspasa 3/genética , Caspasa 3/inmunología , Células Cultivadas , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/prevención & control , Enfermedad Injerto contra Huésped/terapia , Interleucina-2/genética , Interleucina-2/inmunología , Depleción Linfocítica , Ratones , Ratones Endogámicos , Terapia Molecular Dirigida , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Índice de Severidad de la Enfermedad , Bazo/citología , Bazo/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Trasplante Isogénico , Irradiación Corporal TotalRESUMEN
Mobilized peripheral blood cells (MPBCs) graft and peripheral blood cells apheresis are used for bone marrow transplantation and for treatment of graft versus host disease (GvHD). We demonstrate that a short treatment of MPBCs with Fas ligand (FasL, CD95L) for 2 h using a closed automated cell processing system selectively induces apoptosis of specific donor T cells, B cells and antigen presenting cells, but, critically, not CD34+ hematopoietic stem cells and progenitors, all of which may contribute to an increased likelihood of graft survival and functionality and reduced GvHD. Treated cells secreted lower levels of interferon-gamma as compared with control, untreated, cells. Moreover, FasL treatment of immune cells increased signals, which led to their phagocytosis by activated macrophages. FasL treated immune cells also reduced the ability of activated macrophages to secrete pro-inflammatory cytokines. Most importantly, FasL ex vivo treated MPBCs prior to transplantation in NOD-SCID NSG mice prevented GvHD and improved stem cell transplantation in vivo. In conclusion, MPBCs, as well as other blood cell products, treated with FasL by automated manufacturing (AM), may be used as potential treatments for conditions where the immune system is over-responding to both self and non-self-antigens.
Asunto(s)
Enfermedad Injerto contra Huésped , Animales , Células Sanguíneas , Proteína Ligando Fas , Enfermedad Injerto contra Huésped/prevención & control , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
Pathogenic lymphocytes in the enteric wall of inflammatory bowel disease patients display various abnormalities, including reduced sensitivity to apoptosis. We evaluated a therapeutic approach to elimination of cytotoxic cells, using two IL-2 fusion proteins, a diphtheria toxin (IL2-DT) and a caspase-3 (IL2-cas) conjugate. In models of acute (dextran sodium sulfate and trinitrobenzene sulfonic acid) and chronic (dextran sodium sulfate) toxic colitis, therapeutic doses of the fusion proteins improved survival and prevented colon shortening. While both chimeric proteins eradicated CD4(+)CD25(+)Foxp3(+) T cells in mesenteric LN, IL2-DT caused severe lymphopenia. In contrast, IL2-cas was equally protective and increased fractional expression of Foxp3. Similar effects of the fusion proteins were observed in healthy mice: IL2-DT caused lymphopenia and IL2-cas increased fractional expression of FoxP3. The fusion proteins induced apoptosis in CD25(+) T cells in vitro, with lower toxicity of IL2-cas to Foxp3(+) T cells. These data infer that targeted depletion of cells expressing the IL-2 receptor has therapeutic potential in models of inflammatory colitis, despite depletion of CD25(+) Treg. The IL2-cas fusion protein is particularly relevant to inflammatory bowel disease, as direct internalization of toxic moieties overcomes multiple pathways of resistance to apoptosis of colitogenic T cells.
Asunto(s)
Caspasa 3/uso terapéutico , Toxina Diftérica/uso terapéutico , Sistemas de Liberación de Medicamentos/métodos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Receptores de Interleucina-2/metabolismo , Proteínas Recombinantes de Fusión/uso terapéutico , Linfocitos T Reguladores/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Peso Corporal/efectos de los fármacos , Caspasa 3/administración & dosificación , Caspasa 3/genética , Caspasa 3/farmacología , Proliferación Celular/efectos de los fármacos , Colon/efectos de los fármacos , Colon/patología , Sulfato de Dextran/administración & dosificación , Sulfato de Dextran/farmacología , Toxina Diftérica/administración & dosificación , Toxina Diftérica/genética , Toxina Diftérica/farmacología , Factores de Transcripción Forkhead/metabolismo , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Interleucina-2/genética , Interleucina-2/metabolismo , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/patología , Análisis de Supervivencia , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patología , Ácido Trinitrobencenosulfónico/administración & dosificación , Ácido Trinitrobencenosulfónico/farmacologíaRESUMEN
A large body of evidence on the activity of regulatory T (Treg) cells was gathered during the last decade, and a similar number of reviews and opinion papers attempted to integrate the experimental findings. The abundant literature clearly delineates an exciting area of research but also underlines some major controversies. A linear cause-result interpretation of experimental maneuvers often ignores the fact that the activity of Treg cells is orchestrated with the effector T (Teff) cells within an intricate network of physiological immune homeostasis. Every modulation of the activity of the effector (cytotoxic) immune system revolves to affect the activity of regulatory (suppressive) cells through elaborate feedback loops of negative and positive regulation. The lack of IL-2 production by innate Treg cells makes this cytokine a prime coupler of the effector and suppressive mechanisms. Here we attempt to integrate evidence that delineates the involvement of IL-2 in primary and secondary feedback loops that regulate the activity of suppressive cells within the elaborate network of physiological immune homeostasis.
Asunto(s)
Homeostasis/inmunología , Interleucina-2/inmunología , Linfocitos T Reguladores/inmunología , Animales , Humanos , Interleucina-10/inmunología , Subunidad alfa del Receptor de Interleucina-2/inmunología , Activación de Linfocitos , Ratones , Ratones Transgénicos , Modelos Inmunológicos , Receptores de Interleucina-2/inmunología , Subgrupos de Linfocitos T/inmunología , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
Graft versus host disease (GvHD) remains a limiting factor for successful hematopoietic stem cell transplantation (HSCT). T cells and antigen-presenting cells (APCs) are major components of the hematopoietic G-CSF mobilized peripheral blood cell (MPBC) graft. Here we show that a short incubation (2 h) of MPBCs with hexameric Fas ligand (FasL) selectively induces apoptosis of specific donor T cell subsets and APCs but not of CD34+ cells. FasL treatment preferentially induces apoptosis in mature T cell subsets which express high levels of Fas (CD95), such as T stem cell memory, T central memory, and T effector memory cells, as well as TH1 and TH17 cells. Anti-CD3/CD28 stimulated T cells derived from FasL-treated-MPBCs express lower levels of CD25 and secrete lower levels of IFN-γ as compared to control cells not treated with FasL. FasL treatment also induces apoptosis of transitional, naïve, memory and plasmablastoid B cells leading to a reduction in their numbers in the graft and following engraftment in transplanted mice. Most importantly, ex vivo treatment of MPBCs with FasL prior to transplant in conditioned NOD-scid IL2Rγnull (NSG) mice prevented GvHD while preserving graft versus leukemia (GvL) effects, and leading to robust stem cell engraftment.
Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Animales , Proteína Ligando Fas , Enfermedad Injerto contra Huésped/prevención & control , Ligandos , Ratones , Ratones Endogámicos NOD , Células MadreRESUMEN
Regulatory T cells (Treg) play a pivotal role in tolerance to self-antigens and tissue grafts, and suppression of autoimmune reactions. These cells modulate the intensity and quality of immune reactions through attenuation of the cytolytic activities of reactive immune cells. Treg cells operate primarily at the site of inflammation where they modulate the immune reaction through three major mechanisms: a) direct killing of cytotoxic cells through cell-to-cell contact, b) inhibition of cytokine production by cytotoxic cells, in particular interleukin-2, c) direct secretion of immunomodulatory cytokines, in particular TGF-beta and interleukin-10. In addition to differential contributions of these mechanisms under variable inflammatory conditions, mechanistic complexity and diversity evolves from the diverse tasks performed by various Treg cell subsets in different stages of the immune reaction. Here we attempt to integrate the current experimental evidence to delineate the major suppressive pathways of Treg cells.
Asunto(s)
Citocinas/metabolismo , Inflamación/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Animales , Antígenos/inmunología , Citocinas/inmunología , Humanos , Inflamación/metabolismo , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Fas-L is a TNF family member known to trigger cell death. It has recently become evident that Fas-L can transduce also non-apoptotic signals. Mesenchymal stem cells (MSCs) are multipotent cells that are derived from various adult tissues. Although MSCs from different tissues display common properties they also display tissue-specific characteristics. Previous works have demonstrated massive apoptosis following Fas-L treatment of bone marrow-derived MSCs both in vitro and following their administration in vivo. We therefore set to examine Fas-L-induced responses in adipose-derived stem cells (ASCs). Human ASCs were isolated from lipoaspirates and their reactivity to Fas-L treatment was examined. ASCs responded to Fas-L by simultaneous apoptosis and proliferation, which yielded a net doubling of cell quantities and a phenotypic shift, including reduced expression of CD105 and increased expression of CD73, in association with increased bone differentiation potential. Treatment of freshly isolated ASCs led to an increase in large colony forming unit fibroblasts, likely produced by early stem cell progenitor cells. Fas-L-induced apoptosis and proliferation signaling were found to be independent as caspase inhibition attenuated Fas-L-induced apoptosis without impacting proliferation, whereas inhibition of PI3K and MEK, but not of JNK, attenuated Fas-L-dependent proliferation, but not apoptosis. Thus, Fas-L signaling in ASCs leads to their expansion and phenotypic shift toward a more potent stem cell state. We speculate that these reactions ensure the survival of ASC progenitor cells encountering Fas-L-enriched environments during tissue damage and inflammation and may also enhance ASC survival following their administration in vivo.
Asunto(s)
Tejido Adiposo/citología , Proteína Ligando Fas/farmacología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Antígenos CD/metabolismo , Apoptosis/efectos de los fármacos , Huesos/citología , Inhibidores de Caspasas/farmacología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Fenotipo , Fosfatidilinositol 3-Quinasas/metabolismo , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismoRESUMEN
BACKGROUND: Cisplatin (CDDP) dose-limited by its side-effects is, in some instances, synergistically amplified when combined with tamoxifen (TAM). TAM has been shown to modulate apoptotic pathways of normal endometrial cells, whereas CDDP induces apoptosis in malignant endometrial cells. Their combined effect on normal or malignant endometrium is as yet unknown. This study aimed to evaluate the combined CDDP and TAM's apoptotic effect on normal endometrial tissue in the context of hormonal milieu. MATERIALS AND METHODS: Primary endometrial cell cultures were established and maintained both in the presence and absence of steroidal hormones. The cultures were treated for 24 hours with 20 microM TAM and 50 microM CDDP as single drugs and in combination. Apoptosis was determined by evaluation of pre G1 cell populations in the cell cycle analysis with flow cytometer. RESULTS AND CONCLUSION: CDDP induced apoptosis in all cultures regardless of hormonal environment, while TAM significantly enhanced CDDP-induced apoptosis in steroidal deficient media in an additive manner. These are novel findings depicting CDDP's effect on normal endometrium, singularly and combined with TAM.
Asunto(s)
Antineoplásicos Hormonales/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Cisplatino/farmacología , Endometrio/efectos de los fármacos , Moduladores de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , Ciclo Celular/efectos de los fármacos , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Sinergismo Farmacológico , Endometrio/citología , Femenino , Humanos , Esteroides/farmacologíaRESUMEN
BACKGROUND: Accumulating reports indicate that statins widely prescribed for hypercholesteromia have antineoplastic activity. We hypothesized that because statins inhibit farnesylation of Ras that is often mutated in multiple myeloma (MM), as well as the production of interleukin (IL)-6, a key cytokine in MM, they may have antiproliferative and/or proapoptotic effects in this malignancy. METHODS: U266, RPMI 8226, and ARH77 were treated with simvastatin (0-30 microM) for 5 days. The following aspects were evaluated: viability (IC50), cell cycle, cell death, cytoplasmic calcium ion levels, supernatant IL-6 levels, and tyrosine kinase activity. RESULTS: Exposure of all cell lines to simvastatin resulted in reduced viability with IC50s of 4.5 microM for ARH77, 8 microM for RPMI 8226, and 13 microM for U266. The decreased viability is attributed to cell-cycle arrest (U266, G1; RPMI 8226, G2M) and cell death. ARH77 underwent apoptosis, whereas U266 and RPMI 8226 displayed a more necrotic form of death. Cytoplasmic calcium levels decreased significantly in all treated cell lines. IL-6 secretion from U266 cells was abrogated on treatment with simvastatin, whereas total tyrosine phosphorylation was unaffected. CONCLUSIONS: Simvastatin displays significant antimyeloma activity in vitro. Further research is warranted for elucidation of the modulated molecular pathways and clinical relevance.
Asunto(s)
Anticolesterolemiantes/farmacología , Antineoplásicos/farmacología , Mieloma Múltiple/tratamiento farmacológico , Simvastatina/farmacología , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Interleucina-6/metabolismo , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Necrosis , Fosforilación , Tirosina/metabolismoRESUMEN
One of the therapeutic approaches to type 1 diabetes (T1D) focuses on enhancement of regulatory T cell (Treg) activity, either by adoptive transfer or supplementation of supporting cytokines such as interleukin-2 (IL-2). In principle, this therapeutic design would greatly benefit of concomitant reduction in pathogenic cell burden. Experimental evidence indicates that physiological recovery from lymphopenia is dominated by evolution of effector and cytotoxic cells, which abolishes the therapeutic efficacy of Treg cells. Targeted and selective depletion of effector T cells has been achieved with killer Treg using Fas ligand protein and a fusion protein composed of IL-2 and caspase-3, which showed remarkable efficacy in modulating the course of inflammatory insulitis in NOD mice. We emphasize a critical consideration in design of therapeutic approaches to T1D, immunomodulation without lymphoreduction to avoid the detrimental consequences of rebound recovery from lymphopenia.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Linfopenia/inmunología , Linfocitos T Reguladores/inmunología , Animales , Caspasa 3/inmunología , Diabetes Mellitus Tipo 1/terapia , Humanos , Inmunomodulación , Interleucina-2/inmunología , Ratones Endogámicos NODRESUMEN
Prophylactic approaches to graft versus host disease (GvHD) have employed both phenotypic reduction of T cells and selective elimination of host-primed donor T cells in vitro and in vivo. An additional approach to GvHD prophylaxis by functional depletion of apoptosis-sensitive donor T cells without host-specific sensitization ex vivo showed remarkable reduction in GHD incidence and severity. We address the role and significance of antigen-specific sensitization of donor T cells and discuss the mechanisms of functional T cell purging by apoptosis for GvHD prevention. Host-specific sensitization is dispensable because migration is antigen-independent and donor T cell sensitization is mediated by multiple and redundant mechanisms of presentation of major and minor histocompatibility complex and tissue antigens by donor and host antigen-presenting cells. Our data suggest that potential murine and human GvH effectors reside within subsets of preactivated T cells susceptible to negative regulation by apoptosis prior to encounter of and sensitization to specific antigens.
Asunto(s)
Antineoplásicos Hormonales/efectos adversos , Apoptosis/efectos de los fármacos , Endometrio/efectos de los fármacos , Tamoxifeno/efectos adversos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Células Cultivadas , Endometrio/patología , Femenino , Fase G1/efectos de los fármacos , Humanos , PosmenopausiaRESUMEN
Targeted depletion of immune cells expressing the interleukin-2 (IL-2) receptor can exacerbate inflammatory bowel disease (IBD) through elimination of regulatory T (Treg) cells, or ameliorate its course by depletion of cytotoxic cells. To answer this question we used a fusion protein composed of IL-2 and caspase-3 (IL2-cas) in an experimental model of DSS-induced toxic colitis. In a preventive setting, co-administration of DSS with a daily therapeutic dose of IL2-cas for seven days improved all disease parameters. Although CD4(+)CD25(+) T cells were depleted in the mesenteric lymph nodes, a fractional increase in CD4(+)FoxP3(+) T cells was observed in the spleen. Likewise, IL2-cas therapy improved the outcome of established disease in a chronic model of colitis. These data demonstrate that therapies that use IL-2 as a targeting moiety exert a protective effect over the colon under conditions of inflammation. The efficacy of IL-2-targeted therapy is attributed to reduced activity of reactive T cells, which ameliorates the secondary inflammatory infiltration. IL2-cas evolves as a potential therapeutic tool in IBD.
Asunto(s)
Caspasa 3/metabolismo , Colitis/prevención & control , Interleucina-2/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Animales , Peso Corporal/efectos de los fármacos , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Caspasa 3/genética , Colitis/inducido químicamente , Sulfato de Dextran , Factores de Transcripción Forkhead/metabolismo , Inyecciones Intravenosas , Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/metabolismo , Bazo/citología , Bazo/efectos de los fármacos , Bazo/metabolismo , Factores de Tiempo , Resultado del TratamientoRESUMEN
Activation-induced cell death is a general mechanism of immune homeostasis through negative regulation of clonal expansion of activated immune cells. This mechanism is involved in the maintenance of self- and transplant tolerance through polarization of the immune responses. The Fas/Fas-ligand interaction is a major common executioner of apoptosis in lymphocytes, with a dual role in regulatory T cell (Treg) function: Treg cell homeostasis and Treg cell-mediated suppression. Sensitivity to apoptosis and the patterns of Treg-cell death are of outmost importance in immune homeostasis that affects the equilibrium between cytolytic and suppressor forces in activation and termination of immune activity. Naive innate (naturally occurring) Treg cells present variable sensitivities to apoptosis, related to their turnover rates in tissue under steady state conditions. Following activation, Treg cells are less sensitive to apoptosis than cytotoxic effector subsets. Their susceptibility to apoptosis is influenced by cytokines within the inflammatory environment (primarily interleukin-2), the mode of antigenic stimulation and the proliferation rates. Here, we attempt to resolve some controversies surrounding the sensitivity of Treg cells to apoptosis under various experimental conditions, to delineate the function of cell death in regulation of immunity.
Asunto(s)
Apoptosis , Citocinas/inmunología , Homeostasis , Linfocitos T Reguladores/inmunología , Animales , Antineoplásicos/farmacología , Ciclofosfamida/farmacología , Citocinas/metabolismo , Proteína Ligando Fas/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Humanos , Inmunosupresores/farmacología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismo , Vidarabina/análogos & derivados , Vidarabina/farmacologíaRESUMEN
Chronic graft-versus-host disease (cGvHD) and systemic sclerosis (scleroderma [SSc]) share clinical characteristics, including skin and internal organ fibrosis. Fibrosis, regardless of the cause, is characterized by extracellular matrix deposition, of which collagen type I is the major constituent. The progressive accumulation of connective tissue results in destruction of normal tissue architecture and internal organ failure. In both SSc and cGvHD, the severity of skin and internal organ fibrosis correlates with the clinical course of the disease. Thus, there is an unmet need for well-tolerated antifibrotic therapy. Halofuginone is an inhibitor of collagen type I synthesis in cells derived from various tissues and species and in animal models of fibrosis in which excess collagen is the hallmark of the disease. Halofuginone decreased collagen synthesis in the tight skin mouse (Tsk) and murine cGvHD, the 2 experimental systems that show many features resembling those of human GvHD. Inhibition of collagen synthesis by halofuginone is achieved by inhibiting transforming growth factor beta-dependent Smad3 phosphorylation. Dermal application of halofuginone caused a decrease in collagen content at the treated site of a cGvHD patient, and reduction in skin scores was observed in a pilot study with SSc patients. The results of the human studies provide basis for using halofuginone treatment for dermal fibrosis. As a first step toward future treatment of internal organ involvement, an oral administration study was performed in which halofuginone was well tolerated and plasma levels surpassed the predicted therapeutic exposure.
Asunto(s)
Enfermedad Injerto contra Huésped/tratamiento farmacológico , Inhibidores de la Síntesis de la Proteína/uso terapéutico , Quinazolinas/uso terapéutico , Esclerodermia Sistémica/tratamiento farmacológico , Administración Cutánea , Animales , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Colágeno Tipo I/efectos de los fármacos , Fibrosis , Enfermedad Injerto contra Huésped/metabolismo , Enfermedad Injerto contra Huésped/patología , Humanos , Ratones , Piperidinas , Quinazolinonas , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patologíaRESUMEN
OBJECTIVES: This study was designed to examine whether cytokeratin 20 (CK20) is expressed in molar pregnancies and may therefore be used in the diagnosis of gestational trophoblastic disease (GTD). The potential of CK20 expression in predicting the evolution and the prognosis of the different subtypes of GTD was also assessed. METHODS: A total of 48 samples were studied for CK20 expression by RT-PCR methodology. Among these, 24 samples were obtained by curettage of the uterine cavity of patients diagnosed with hydatidiform mole (14 complete moles and 10 partial moles), 4 samples were obtained from choriocarcinoma cell lines (2 JAR and 2 JEG), and 20 samples were of normal trophoblast (control group) obtained from patients that underwent elective termination of pregnancy. RESULTS: Expression of CK20 was identified in all the samples of complete mole (CM), all choriocarcinoma cell lines, and 50% of the patients with partial mole (PM). None of the preparations of normal trophoblastic tissue from the control group expressed the CK20. A significant difference (P < 0.00001) was found in CK20 expression between samples of patients with GTD and control samples. Comparison between CK20 expression in CMs and PMs revealed a significantly more frequent expression of CK20 in CMs (P = 0.006). More than 50% of the patients with PMs that were positive for CK20 had an invasive evolution. CONCLUSIONS: In our opinion, CK20 may assist in distinguishing between molar and normal trophoblastic tissue and may be considered a marker of GTD. In cases in which pathological classification of different subtypes of GTD is in doubt, CK20-positive expression is suggestive for a CM whereas CK20-negative is more indicative for PM.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Enfermedad Trofoblástica Gestacional/metabolismo , Mola Hidatiforme/metabolismo , Proteínas de Filamentos Intermediarios/biosíntesis , Neoplasias Uterinas/metabolismo , Femenino , Enfermedad Trofoblástica Gestacional/diagnóstico , Humanos , Queratina-20 , Embarazo , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Neoplasias Uterinas/diagnósticoRESUMEN
OBJECTIVE: Clonal B-lymphocytes of chronic lymphocytic leukemia (B-CLL) are characterized by decreased sensitivity to programmed cell death and, therefore, they accumulate in vivo. However, these malignant cells die rapidly in vitro. In the current study we concentrated on the contribution of autologous serum (AS) and lymphocyte subsets to the survival of the malignant cells in vitro. METHODS: Mononuclear cells from the peripheral blood of 26 CLL patients and 24 controls were incubated overnight in the presence or absence of AS and heat-inactivated AS (HI-AS) or fetal calf serum (FCS). Also, isolated B cells were incubated at different concentrations in the presence of AS and/or isolated T cells. The level of apoptosis of CD19+ cells was measured by flow cytometry. RESULTS: Spontaneous apoptosis of unfractionated B-CLL cells incubated with AS, FCS or without serum was significantly lower than the rate of B-cell death in the control group, in similar culture conditions. AS had an antiapoptotic effect on unfractionated B-CLL cells when compared with FCS. The rate of apoptosis of B-CLL cells was directly associated with stage. HI of AS had a variable effect, which was related to the stage of the disease. High concentrations of B cells and the addition of autologous T cells reduced the rate of apoptosis when incubated without serum. The antiapoptotic effect of T cells was most prominent in progressive stages. CONCLUSIONS: B-CLL cells exhibit decreased spontaneous apoptosis, which is partially prevented by humoral (AS) and cellular (T cells and B-CLL cells) factors. The equilibrium between apoptotic and antiapoptotic factors changes with disease progression.
Asunto(s)
Apoptosis , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/patología , Anciano , Anciano de 80 o más Años , Animales , Antígenos CD19/metabolismo , Linfocitos B/patología , Estudios de Casos y Controles , Bovinos , Supervivencia Celular , Medios de Cultivo , Humanos , Técnicas In Vitro , Persona de Mediana Edad , Estadificación de Neoplasias , Linfocitos T/patologíaRESUMEN
Thalidomide (Thd), a potent teratogen, was shown to have therapeutic potential in cancer, primarily in multiple myeloma (MM), yet its mechanism of action has not been elucidated. It was recently suggested that its teratogenicity is derived from interference in expression of genes regulated by GC-rich promoters by blocking the binding of SP1 transcription factor to its motif. We explored the validation of the proposed model by focusing on potential molecular targets associated with MM pathogenesis. Cell lines RPMI 8226, U266, and ARH-77 were exposed for 24 h to racemic Thd and analyzed for apoptosis, membranal expression of CD29 and CD63, transcript level of hTERT, CD63, and IGFI-R (characterized by GC-rich motifs) and telomerase activity. Analysis of an hTERT core promoter reporter gene expression [enhanced green fluorescent protein (EGFP)] in transiently transfected RPMI 8226 incubated with racemic and steric (+/-)-enantiomers of Thd was performed. A consistent reduction ( approximately 10-40%) in transcript levels of all three assayed genes in all three cell lines was demonstrated in the presence of racemic Thd. Significant reduction of EGFP was demonstrated in cells transfected with hTERT reporter gene and treated with racemic and (S)-Thd. Our results show that Thd's antimyeloma activity can be ascribed to the same mechanism responsible for its teratogenic effect and that the inhibition of GC-rich promoter genes is mostly attributed to the S-racemate. Indeed, this selectivity delineates GC-rich promoter genes as a unique group eligible for specific drug targeting.
Asunto(s)
Secuencia Rica en GC/efectos de los fármacos , Mieloma Múltiple/patología , Regiones Promotoras Genéticas/efectos de los fármacos , Talidomida/farmacología , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Proteínas de Unión al ADN , Secuencia Rica en GC/genética , Humanos , Inmunosupresores/farmacología , Integrina beta1/metabolismo , Plasmacitoma , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Regiones Promotoras Genéticas/genética , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Receptores de Somatomedina/genética , Receptores de Somatomedina/metabolismo , Telomerasa/genética , Telomerasa/metabolismo , Tetraspanina 30 , Células Tumorales CultivadasRESUMEN
Phosphatidylserine's (PS) membranal distribution is associated with an expanding variety of biological processes. We studied the relevance of preliminarily exposed membranal PS levels to cellular effects of cytotoxic agents. PBL of normal controls (n = 18) and patients with doxorubicin-treated breast carcinoma (n = 27) or 5'-fluorouracil-treated colorectal cancer (n = 32) were assayed before and after drug infusion. Membranal expression levels of PS, adhesion molecules (CD18, CD11a-c, CD63) and Fas-R of leukocyte subtypes were assessed by flow cytometer. Statistical analysis was implemented. Our results demonstrate external expression of PS on all leukocyte subpopulations despite non-apoptotic light scatter characteristics. Several distinct features were observed of which the more prominent were: leukocyte subtypes each display characteristic PS levels; cancer patients' PBL display higher preliminary PS levels than normal controls in all cell groups; and existence of negative correlations between initial membranal PS levels and drug-induced changes in its expression. Our findings underscore the complex involvement of PS in PBL apoptosis and possibly drug resistance.
Asunto(s)
Antineoplásicos/farmacología , Membrana Celular/química , Leucocitos/efectos de los fármacos , Fosfatidilserinas/metabolismo , Anciano , Análisis de Varianza , Antineoplásicos/administración & dosificación , Apoptosis , Biomarcadores/sangre , Neoplasias de la Mama/sangre , Neoplasias de la Mama/complicaciones , Neoplasias de la Mama/tratamiento farmacológico , Estudios de Casos y Controles , Moléculas de Adhesión Celular/metabolismo , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/complicaciones , Neoplasias Colorrectales/tratamiento farmacológico , Femenino , Humanos , Leucocitos/ultraestructura , Leucopenia/etiología , Masculino , Persona de Mediana EdadRESUMEN
Radiation-induced fibrosis is an untoward effect of high dose therapeutic and inadvertent exposure to ionizing radiation. Transforming growth factor-beta (TGF-beta) has been proposed to be critical in tissue repair mechanisms resulting from radiation injury. Previously, we showed that interruption of TGF-beta signaling by deletion of Smad3 results in resistance to radiation-induced injury. In the current study, a small molecular weight molecule, halofuginone (100 nm), is demonstrated by reporter assays to inhibit the TGF-beta signaling pathway, by Northern blotting to elevate inhibitory Smad7 expression within 15 min, and by Western blotting to inhibit formation of phospho-Smad2 and phospho-Smad3 and to decrease cytosolic and membrane TGF-beta type II receptor (TbetaRII). Attenuation of TbetaRII levels was noted as early as 1 h and down-regulation persisted for 24 h. Halofuginone blocked TGF-beta-induced delocalization of tight junction ZO-1, a marker of epidermal mesenchymal transition, in NMuMg mammary epithelial cells and suggest halofuginone may have in vivo anti-fibrogenesis characteristics. After documenting the in vitro cellular effects, halofuginone (intraperitoneum injection of 1, 2.5, or 5 microg/mouse/day) efficacy was assessed using ionizing radiation-induced (single dose, 35 or 45 Gy) hind leg contraction in C3H/Hen mice. Halofuginone treatment alone exerted no toxicity but significantly lessened radiation-induced fibrosis. The effectiveness of radiation treatment (2 gray/day for 5 days) of squamous cell carcinoma (SCC) tumors grown in C3H/Hen was not affected by halofuginone. The results detail the molecular effects of halofuginone on the TGF-beta signal pathway and show that halofuginone may lessen radiation-induced fibrosis in humans.