MicroRNA-106b-25 cluster expression is associated with early disease recurrence and targets caspase-7 and focal adhesion in human prostate cancer.

The miR-106b-25 microRNA (miRNA) cluster is a candidate oncogene in human prostate cancer. Here, we report that miRNAs encoded by miR-106b-25 are upregulated in both primary tumors and distant metastasis. Moreover, increased tumor miR-106b expression was associated with disease recurrence and the combination of high miR-106b and low CASP7 (caspase-7) expressions in primary tumors was an independent predictor of early disease recurrence (adjusted hazard ratio=4.1; 95% confidence interval: 1.6-12.3). To identify yet unknown oncogenic functions of miR-106b, we overexpressed it in LNCaP human prostate cancer cells to examine miR-106b-induced global expression changes among protein-coding genes. The approach revealed that CASP7 is a direct target of miR-106b, which was confirmed by western blot analysis and a 3'-untranslated region reporter assay. Moreover, selected phenotypes induced by miR-106b knockdown in DU145 human prostate cancer cells did not develop when both miR-106b and CASP7 expression were inhibited. Further analyses showed that CASP7 is downregulated in primary prostate tumors and metastatic lesions across multiple data sets and is by itself associated with disease recurrence and disease-specific survival. Using bioinformatics, we also observed that miR-106b-25 may specifically influence focal adhesion-related pathways. This observation was experimentally examined using miR-106b-25-transduced 22Rv1 human prostate cancer cells. After infection with a miR-106b-25 lentiviral expression construct, 22Rv1 cells showed increased adhesion to basement membrane- and bone matrix-related filaments and enhanced soft agar growth. In summary, miR-106b-25 was found to be associated with prostate cancer progression and disease outcome and may do so by altering apoptosis- and focal adhesion-related pathways.

Reproductive outcome of fresh or frozen-thawed embryo transfer is similar in high-risk patients for ovarian hyperstimulation syndrome using GnRH agonist for final oocyte maturation and intensive luteal support.

BACKGROUND: Triggering ovulation by GnRH agonist (GnRHa) in GnRH antagonist IVF protocols coupled with adequate luteal phase support has recently been suggested as a means to prevent ovarian hyperstimulation syndrome (OHSS). Our objective was to examine the outcome of fresh embryo transfer (f-ET) after triggering ovulation by GnRHa and providing intensive luteal phase supplementation, compared with that of the next first frozen-thawed embryo transfer (ft-ET) after cycles with the same protocol and cryopreservation of all the embryos. METHODS: We performed a cohort study at a university-based IVF clinic. The study population was patients at high risk for OHSS. A daily dose of 50 mg i.m. progesterone in oil and 6 mg of oral 17-ß-estradiol initiated on oocyte retrieval day in the f-ET group (n= 70). In the ft-ET group (n= 40) the embryos were cryopreserved and transferred in the next cycle. RESULTS: The live birth rate per f-ET was 27.1 versus 20% in the ft-ET groups [P = 0.4; rate ratio = 1.36 (0.65-2.81)]. The implantation, pregnancy and spontaneous abortion rates were comparable in both groups. None of the patients developed OHSS. CONCLUSIONS: In this observational cohort study, we showed that triggering ovulation with GnRHa and intensive luteal phase support is a promising new modality to prevent OHSS without the cost of cycle cancellation, ET deferral and reduced clinical pregnancy rates. Confirmation of these findings by RCTs is now required.

Reproductive outcome of men with azoospermia due to cryptorchidism using assisted techniques.

The aetiology of cryptorchidism is still undiscernible in the majority of cases. It has long been argued that cryptorchidism reflects a primary testicular maldevelopment, where the contralateral scrotal testis also suffers from aspermatogenesis and low spermatogonia count. The aim of the study was to determine the reproductive outcome of ex-cryptorchid men with azoospermia post-orchidopexy after testicular sperm extraction (TESE) and intracytoplasmatic sperm injection (ICSI). In a retrospective analysis, we compared the sperm retrieval, fertilization, pregnancy and live birth rates after ICSI of consecutive ex-cryptorchid azoospermic patients (n = 15) undergoing TESE between Jan 2000 and Dec 2007 vs. non-cryptorchid azoospermic men (n = 142). Sperm retrieval rate of ex-cryptorchid men by TESE (66%) was comparable with non-cryptorchid men (47%) (p = 0.15) despite significantly higher FSH levels (30.7 +/- 25.4 vs. 17.9 +/- 14.8 respectively) (p = 0.018) and a more prevalent histopathology diagnosis of aspermatogenesis (75% vs. 40%, p = 0.046). Fertilization (43.3%), pregnancy (30%) and live birth (20%) rates after TESE-IVF-ICSI in the ex-cryptorchid group were not different from the non-cryptorchid group (48.7, 43 and 29%, p = 0.26, p = 0.21, p = 0.29 respectively). We conclude that the reproductive outcome of ex-cryptorchid men with azoospermia post-orchidopexy employing TESE-IVF-ICSI is comparable with non-cryptorchid azoospermic men.

[Assisted reproduction technologies and the risk of fetal, chromosomal and genetic malformations].

BACKGROUND: Assisted reproduction techniques allowed thousands of otherwise infertile couples to attain pregnancy. As this technology moves into the mainstream of infertility treatment, it has become more critical to reassess its safety. OBJECTIVE: To review the birth outcome of patients undergoing conventional in-vitro fertilization and intracyto- plasmic sperm injection regarding fetal malformations, chromosomal and genetic abnormalities. METHODS: Selective review of the literature. RESULTS: Most of the published data is from observational studies and is not randomized or blinded. Unfortunately, most articles are inherently biased. Chromosomal and genetic abnormalities are increased probably only as a direct corollary to the underlying parental risk and not due to the technology itself. There is a slight increase in the congenital malformations rate, but inspection of these malformations reveal no clustering of any specific abnormality. CONCLUSIONS: Children born after assisted reproduction technologies have an increased risk of a major congenital malformation and chromosomal abnormalities compared with those born after natural conception. The risk is mainly due to paternal and maternal risk factors, which are more prevalent in couples who use assisted reproduction techniques for reproduction. Infertility-linked risk is highly probable for the observed findings. A technique-related risk, however, cannot be ruled out. Intracytoplasmic sperm injection appears to be a safe alternative for couples who otherwise would be unable to achieve pregnancy. The inherent risks associated with these genetically "at risk" couples mandate thorough evaluation and counseling before undertaking ICSI.

Spontaneous ovarian hyperstimulation syndrome and hyperreactio luteinalis are entities in continuum.

Hyperreactio luteinalis (HL) and spontaneous ovarian hyperstimulation syndrome (OHSS) are both rare conditions during pregnancy. The clinical presentation of HL and OHSS are comparable and both should be differentiated from ovarian carcinoma. We present a case of a 32-year-old woman who was initially seen with markedly enlarged multicystic ovaries and ascites in the 13th week of a spontaneously conceived pregnancy. Ultrasonographic follow-up and magnetic resonance imaging of the ovaries were employed in order to avoid exploratory laparotomy and rule out ovarian carcinoma. The patient received supportive therapy and delivered a healthy child at term. The increasing use of ultrasonography may lead to more frequent findings of multicystic ovaries in spontaneously conceived pregnancies. Making the distinction between HL and spontaneous OHSS in these cases may be difficult though clinically irrelevant as the approach to treatment is similar in both.

Regulation of cyclooxygenase activity and progesterone production in the rat corpus luteum by inducible nitric oxide synthase.

This study explores interactions between the nitric oxide synthase (NOS) and the cyclooxygenase (COX) pathways in the regulation of progesterone production in early corpus luteum cells of rats. Nitric oxide (NO), prostaglandin E (PGE) and progesterone production was analysed in luteal cells of the rat corpus luteum exposed to inhibitors of non-specific NOS, inhibitors of inducible NOS (iNOS) and inhibitors of COX. Equine chorionic gonadotrophin (eCG)/hCG-primed rat corpus luteum cells produced NO, PGE and progesterone in a linear manner during 66 h of culture. Exposure of the cells to the non-specific NOS inhibitor, N(omega)-nitro-L-arginine (0.15 mmol l(-1)) for 48 h reduced NO, PGE and progesterone production to 21, 32 and 60% of that of the controls, respectively (P < 0.05 to P < 0.01). Another non-specific NOS inhibitor, N(omega)-methyl-L-arginine, produced similar inhibitions. Exposure of the cultured cells to S-ethylisothiourea (1 mmol l(-1)), a selective inhibitor of iNOS, suppressed the production of NO by 63%, PGE by 69% and progesterone by 48%. These findings indicate that production of PGE is regulated partly by iNOS, and that progesterone is probably regulated indirectly by the secondary changes in PGE. The addition of arachidonic acid to N(omega)-methyl-L-arginine-treated cells resulted in a significant increase in PGE and progesterone production (273 and 186%, respectively) without stimulating NO production. In contrast to the regulation exerted by the NO system on COX activity, the COX system does not modulate NO production in this model. This notion stems from the observation that the COX inhibitors acetylsalicylic acid (5 mmol l(-1)) and indomethacin (5 micromol l(-1)) suppressed PGE by 86 and 89%, respectively, and progesterone by 34 and 57%, respectively, but failed to inhibit NO production. The results from the present study indicate that iNOS-mediated NO production is involved in stimulating PGE synthesis in rat luteal cells, which may upregulate progesterone production.

A modification of the quininium resin test for assessing gastric acidity.

BACKGROUND: Gastric acid is an important defence against enteric infection. Studies investigating the relationship between hypochlorhydria and enteric infections or gastric malignancy have been limited by difficulties in the non-invasive measurement of gastric acidity. AIM: To develop a blood test for hypochlorhydria based on the quininium resin test. METHODS: Quininium resin dissociates to liberate free quinine at pH

Engagement of OX40 enhances antigen-specific CD4(+) T cell mobilization/memory development and humoral immunity: comparison of alphaOX-40 with alphaCTLA-4.

Increasing the long-term survival of memory T cells after immunization is key to a successful vaccine. In the past, the generation of large numbers of memory T cells in vivo has been difficult because Ag-stimulated T cells are susceptible to activation-induced cell death. Previously, we reported that OX40 engagement resulted in a 60-fold increase in the number of Ag-specific CD4(+) memory T cells that persisted 60 days postimmunization. In this report, we used the D011.10 adoptive transfer model to examine the kinetics of Ag-specific T cell entry into the peripheral blood, the optimal route of administration of Ag and alphaOX40, and the Ag-specific Ab response after immunization with soluble OVA and alphaOX40. Finally, we compared the adjuvant properties of alphaOX40 to those of alphaCTLA-4. Engagement of OX-40 in vivo was most effective when the Ag was administered s.c. Time course studies revealed that it was crucial for alphaOX40 to be delivered within 24-48 h after Ag exposure. Examination of anti-OVA Ab titers revealed a 10-fold increase in mice that received alphaOX40 compared with mice that received OVA alone. Both alphaOX40 and alphaCTLA-4 increased the percentage of OVA-specific CD4(+) T cells early after immunization (day 4), but alphaOX40-treated mice had much higher percentages of OVA-specific memory CD4(+) T cells from days 11 to 29. These studies demonstrate that OX40 engagement early after immunization with soluble Ag enhances long-term T cell and humoral immunity in a manner distinct from that provided by blocking CTLA-4.

Monopaternal superfecundation of quintuplets after transfer of two embryos in an in vitro fertilization cycle.

OBJECTIVE: To present the first genetically proven identity of quintuplets in an IVF treatment cycle after transferring only two embryos. DESIGN: Case report. SETTING: IVF unit and obstetrics department of university-affiliated general hospital. PATIENT(S): Twenty-five-year-old patient undergoing IVF treatment for unexplained infertility. INTERVENTION(S): In vitro fertilization with intracytoplasmic sperm injection performed on 50% of oocytes, resulting in successful production of nine early-cleavage embryos. Transfer of two embryos on day 3 and freezing of the remaining embryos. MAIN OUTCOME MEASURE(S): Development of five separate embryonic sacs. Fetal reduction of three embryos at 12 weeks of gestation. RESULT(S): Successful completion of the twin pregnancy and full genetic analysis of the three embryos and the twins that were born at term. CONCLUSION(S): Despite transferring only two embryos, superfecundation occurred, resulting in five embryos. Genetic analysis can be used to determine paternity and identity of all the embryos.

Elucidating the autoimmune and antitumor effector mechanisms of a treatment based on cytotoxic T lymphocyte antigen-4 blockade in combination with a B16 melanoma vaccine: comparison of prophylaxis and therapy.

We have previously shown that small B16 melanomas can be successfully treated using a combination of anti-cytotoxic T lymphocyte antigen (CTLA)-4 monoclonal antibody with a granulocyte/macrophage colony-stimulating factor (GM-CSF) producing irradiated tumor cell vaccine. Regression of tumors results in long-lasting immunity and is frequently accompanied by autoimmune depigmentation. Here we examine the cellular and molecular mechanisms of this combined treatment. Histological examination of depigmented lesions revealed infiltration of polymorphonuclear cells and deposition of antibody. The combination therapy also induced tumor rejection and skin depigmentation in B cell-deficient and in CD4(+) T cell-depleted mice. Both effects of the treatment absolutely required CD8(+) T cells. Analysis of the response in successfully treated mice revealed elevated levels of CD8(+) T cells specific for a nonameric peptide consisting of residues 180-188 of the melanocyte differentiation antigen tyrosinase-related protein (TRP)2. There was no evidence of reactivity to the melanocyte antigens gp100, tyrosinase, Mart1/MelanA, or TRP1. Fas-FasL interactions and perforin played a role in mounting the effector response, whereas the tumor necrosis factor pathway was not required. The cellular requirements for tumor rejection in this therapeutic setting were strikingly different from those in a prophylactic setting. In particular, if mice received a prophylactic vaccine consisting of anti-CTLA-4 and B16-GM-CSF before tumor challenge, full protection was obtained even in the absence of CD8(+) T cells. Our data demonstrate that therapeutic autoreactive CD8(+) T cell responses can effectively be generated in tumor-bearing mice and stresses the value of studying tumor immunity in a therapeutic rather than a prophylactic setting.

Successful pregnancy following replacement of embryos previously refrozen at blastocyst stage: case report.

We present the first reported clinical pregnancy following transfer of embryos that had been subjected to two freeze-thaw cycles: the first at day 3 after insemination, and the second after culturing to the blastocyst stage. A 25-year-old woman undergoing IVF treatment for male factor infertility opted for intracytoplasmic sperm injection (ICSI). ICSI treatment resulted in the successful production of 19 early cleavage embryos, all of which were frozen. After thawing, the embryos were cultured to the blastocyst stage. Thereafter, the blastocysts were refrozen and again thawed prior to embryo transfer. Embryos surviving a day 3 freeze-thaw cycle developed to the blastocyst stage and survived the second freeze-thaw cycle. Successful clinical pregnancy resulted following two sequential freeze-thaw cycles. This finding shows that it is possible to refreeze supernumerary blastocysts for subsequent transfer.

Costimulatory wars: the tumor menace.

Advances in our understanding of T cell costimulatory molecules have provided a vast array of novel approaches to tumor immunotherapy. In the past year, combinatorial immunotherapy based on earlier studies of CTLA-4 blockade, the identification of novel B7-family members, the modulation of CD40 to reverse tolerance to tumor-associated antigens and the use of OX40 to enhance antitumor responses of CD4+ T cells have all contributed to the development of more-powerful immunomodulatory cancer therapies.

Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade.

We have previously shown that antibodies to CTLA-4, an inhibitory receptor on T cells, can be effective at inducing regression of transplantable murine tumors. In this study, we demonstrate that an effective immune response against primary prostate tumors in transgenic (TRAMP) mice can be elicited using a strategy that combines CTLA-4 blockade and an irradiated tumor cell vaccine. Treatment of TRAMP mice at 14 weeks of age resulted in a significant reduction in tumor incidence (15% versus control, 75%), as assessed 2 months after treatment. Histopathological analysis revealed that treated mice had a lower tumor grade with significant accumulation of inflammatory cells in interductal spaces when treated with anti-CTLA-4 and a granulocyte-macrophage colony-stimulating factor-expressing vaccine. Vaccination of nontransgenic mice with this regimen resulted in marked prostatitis accompanied by destruction of epithelium, indicating that the immune response was, at least in part, directed against normal prostate antigens. These findings demonstrate that this combinatorial treatment can elicit a potent antiprostate response and suggest potential of this approach for treatment of prostate cancer.

Elimination of residual metastatic prostate cancer after surgery and adjunctive cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade immunotherapy.

Cancer relapse after surgery is a common occurrence, most frequently resulting from the outgrowth of minimal residual disease in the form of metastases. We examined the effectiveness of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade as an adjunctive immunotherapy to reduce metastatic relapse after primary prostate tumor resection. For these studies, we developed a murine model in which overt metastatic outgrowth of TRAMP-C2 (C2) prostate cancer ensues after complete primary tumor resection. Metastatic relapse in this model occurs reliably and principally within the draining lymph nodes in close proximity to the primary tumor, arising from established metastases present at the time of surgery. Using this model, we demonstrate that adjunctive CTLA-4 blockade administered immediately after primary tumor resection reduces metastatic relapse from 97.4 to 44%. Consistent with this, lymph nodes obtained 2 weeks after treatment reveal marked destruction or complete elimination of C2 metastases in 60% of mice receiving adjunctive anti-CTLA-4 whereas 100% of control antibody-treated mice demonstrate progressive C2 lymph node replacement. Our study demonstrates the potential of adjunctive CTLA-4 blockade immunotherapy to reduce cancer relapse emanating from minimal residual metastatic disease and may have broader implications for improving the capability of immunotherapy by combining such forms of therapy with other cytoreductive measures including surgery.

Three-dimensional MRI atlas of the human cerebellum in proportional stereotaxic space.

We have prepared an atlas of the human cerebellum using high-resolution magnetic resonance-derived images warped into the proportional stereotaxic space of Talairach and Tournoux. Software that permits simultaneous visualization of the three cardinal planes facilitated the identification of the cerebellar fissures and lobules. A revised version of the Larsell nomenclature facilitated a simple description of the cerebellum. This atlas derived from a single individual was instrumental in addressing longstanding debates about the gross morphologic organization of the cerebellum. It may serve as the template for more precise identification of cerebellar topography in functional imaging studies in normals, for investigating clinical-pathologic correlations in patients, and for the development of future probabilistic maps of the human cerebellum.

Combination immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF)-producing vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation.

We examined the effectiveness of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade, alone or in combination with a granulocyte/macrophage colony-stimulating factor (GM-CSF)-expressing tumor cell vaccine, on rejection of the highly tumorigenic, poorly immunogenic murine melanoma B16-BL6. Recently established tumors could be eradicated in 80% (68/85) of the cases using combination treatment, whereas each treatment by itself showed little or no effect. Tumor rejection was dependent on CD8(+) and NK1.1(+) cells but occurred irrespective of the presence of CD4(+) T cells. Mice surviving a primary challenge rejected a secondary challenge with B16-BL6 or the parental B16-F0 line. The same treatment regimen was found to be therapeutically effective against outgrowth of preestablished B16-F10 lung metastases, inducing long-term survival. Of all mice surviving B16-BL6 or B16-F10 tumors after combination treatment, 56% (38/68) developed depigmentation, starting at the site of vaccination or challenge and in most cases progressing to distant locations. Depigmentation was found to occur in CD4-depleted mice, strongly suggesting that the effect was mediated by CTLs. This study shows that CTLA-4 blockade provides a powerful tool to enhance T cell activation and memory against a poorly immunogenic spontaneous murine tumor and that this may involve recruitment of autoreactive T cells.

A flexible protocol for artificial preparation of the endometrium without prior gonadotropin-releasing hormone agonist suppression in women with functioning ovaries undergoing frozen-thawed embryo transfer cycles.

OBJECTIVE: To present our experience with a flexible and convenient protocol for artificial endometrial preparation without prior GnRH agonist suppression in patients with functioning ovaries undergoing frozen ET. DESIGN: Case series. SETTING: An IVF unit in a university hospital. PATIENT(S): All patients who underwent IVF with embryo cryopreservation from December 1997 to June 1998 and requested transfer of their frozen-thawed embryos. INTERVENTION(S): Controlled endometrial preparation for ET entailed the use of a fixed dose of 6 mg/d of micronized E2 started on day 1 of the cycle, followed by concomitant administration of micronized P placed in the vagina. MAIN OUTCOME MEASURE(S): Hormonal and endometrial profiles throughout the cycle, pregnancy rate per ET, implantation rate, and pregnancy outcome. RESULT(S): Of 185 treatment cycles in 140 patients, 8 cycles (4.3%) were canceled. In another 2 cycles, no embryos were suitable for transfer. For the remaining 175 ET cycles, the calculated pregnancy rate and implantation rate were 21.7% and 9%, respectively. The proliferative phase could be extended up to 20 days but was a mean (+/-SD) of 15+/-1.9 days. CONCLUSION(S): For patients with functioning ovaries, controlled endometrial preparation for the transfer of frozen-thawed embryos can be done successfully by using oral E2 from day 1 of the cycle followed by P preparation. Prior suppression with GnRH agonist is not necessary.

Transfer of frozen-thawed embryos in artificially prepared cycles with and without prior gonadotrophin-releasing hormone agonist suppression: a prospective randomized study.

Transfer of frozen-thawed embryos is usually carried out in a natural cycle or in a programmed cycle in which the endometrium is exogenously stimulated following down-regulation of the hypophysis. To analyse the possibility that the programmed cycle for embryo transfer can still be hormonally manipulated without the use of gonadotrophin-releasing hormone agonist (GnRHa) we have conducted a prospective randomized study that compared the outcome of frozen-thawed embryo transfer cycles using micronized 17beta-oestradiol and micronized progesterone preparations with and without the concomitant use of GnRHa. One hundred and six patients were randomly divided into two groups. In group A (53 patients) 4 mg/day of micronized 17beta-oestradiol was initiated following down-regulation of hypophysis. In group B (53 patients) oestrogen stimulation started on day 1 of the cycle without prior pituitary down-regulation using a dose of 6 mg/day for 7 days. In both groups, micronized progesterone in a dose of 900 mg/day was administered vaginally after at least 12 days of oestrogen stimulation. Embryo transfer embryo transfer took place 48-72 h thereafter according to the cryopreserved embryonic stage. Overall, none of the patients had any follicular development and only one cycle in group B had to be cancelled because of premature progesterone secretion. The two groups did not differ in age (31+/-5.6 and 31+/-5.0 years), number of embryos transferred per patient (3.4+/-1.2 and 3.3+/-1.0), and day of progesterone initiation (15+/-2.2 and 15+/-1.9 for groups A and B respectively). The endometrial thickness on the day of progesterone initiation was comparable in both groups (11 +/-1.6 and 10+/-1.6 mm for groups A and B respectively). Similarly, the pregnancy rate per embryo transfer and implantation rate in group A (26.4% and 9.5%) were comparable to those of group B (21.1% and 9%). These results indicate that programmed cycles can be successfully applied by administering a high dose of micronized 17beta-oestradiol starting on day 1 of the cycle. Compared to GnRHa programmed cycles, this approach is simpler, more convenient for both the patient and medical staff, and results in a similar success rate at a lower cost.

A role for CTLA-4-mediated inhibitory signals in peripheral T cell tolerance?

Occupancy of the antigen receptor is not sufficient for activation of naïve T cells--additional co-stimulatory signals are required that can be provided only by 'professional' antigen-presenting cells. This two-signal model for T cell activation has been thought to provide a mechanism for the induction and maintenance of peripheral tolerance. Work over the past six years has demonstrated that the relevant co-stimulatory receptor on T cells is the molecule CD28. Recent data shows that the CD28 homologue CTLA-4 plays a role in negative regulation of T cell responses. Here we suggest that CTLA-4 may also serve as an attenuator of T cell-activating signals, raising the threshold of stimulation required to obtain full activation. The inhibitory signals mediated by CTLA-4 may provide an additional mechanism for the maintenance of peripheral tolerance.

CTLA-4 blockade synergizes with tumor-derived granulocyte-macrophage colony-stimulating factor for treatment of an experimental mammary carcinoma.

Generation of a T cell-mediated antitumor response depends on T cell receptor engagement by major histocompatibility complex/antigen as well as CD28 ligation by B7. CTLA-4 is a second B7 receptor expressed by T cells upon activation that, unlike CD28, appears to deliver an inhibitory signal to T cells. Recently, we and others demonstrated that administration of an anti-CTLA-4 antibody was sufficient to promote regression of several murine tumors. However, certain tumors, such as the SM1 mammary carcinoma, remain refractory to this type of immunotherapy. In the present study, we report that the combination of both CTLA-4 blockade and a vaccine consisting of granulocyte-macrophage colony-stimulating factor-expressing SM1 cells resulted in regression of parental SM1 tumors, despite the ineffectiveness of either treatment alone. This synergistic therapy resulted in long-lasting immunity to SM1 and depended on both CD4(+) and CD8(+) T cells. Interestingly, synergy was not observed between CTLA-4 and a B7-expressing SM1 vaccine. Given that granulocyte-macrophage colony-stimulating factor promotes differentiation and activation of dendritic cells as well as enhances cross-priming of T cells to tumor-derived antigens and that SM1 is major histocompatibility complex class II-negative, our findings suggest that CTLA-4 blockade acts at the level of a host-derived antigen-presenting cell. In addition, these results also support the idea that the most effective and synergistic vaccine strategy targets treatments that enhance T cell priming at the level of host-derived antigen-presenting cells.