Positive Delayed-type Hypersensitivity Reaction Research Articles

A Novel Murine Model of Early Abdominal Aortic Aneurysm Reflects Immunopathology in Human Abdominal Aortic Aneurysms

The pathogenesis of abdominal aortic aneurysm (AAA) remains poorly understood but there is mounting evidence that AAA may be an immune mediated condition. Current mouse models of AAA were developed primarily to reproduce mature aneurysms and may poorly reflect the pathology found in early AAA. We propose a novel murine model of AAA incorporating sensitization to aortic antigens, which Results in more robust AAA and duplicates the proposed autoimmune mechanisms in humans. C57B/L6 mice were randomized to sensitization to fragmented elastin (fELA) or control. One week later AAA was induced in all mice with the topical elastase method. Prior to sacrifice, development of a delayed-type hypersensitivity (DTH) reaction was assessed by cutaneous challenge in the ear pinna with fELA. Blood and periaortic lymph nodes (LN) were collected for quantification of type 1 regulatory T-cells (Tr1) and T-helper 17 (Th17) cells by flow cytometry. The mice were perfused with a silicone injection compound to fill and opacify their vasculature and microcomputed tomography scans were obtained for three-dimensional reconstruction of the infrarenal aorta (Fig 1) to evaluate volume and surface area of the aneurysms. Mice sensitized to fELA had larger aneurysms: volume 5.31 mm3 versus 4.16 mm3 (P = .034) and surface area 34.55 mm2 versus 30.87 mm2 (P = .038). They had positive DTH reactions to fELA represented by a greater weight difference between challenged and unchallenged ears: 4.13 mg vs 0.21 mg (P = .0004). The degree of DTH response was found to positively correlate with both volume (Fig 2) (R = 0.79; P = .00025) and surface area of aneurysm (R = 0.71; P = .0019). Flow cytometry Results were noTable for a significantly decreased Tr1/Th17 ratio in the sensitized group both in the blood (P = .0005) and LNs (P = .031) and in periaortic LNs this Tr1/Th17 ratio negatively correlated with AAA volume (R = –0.52; P = .021) with a trend toward a similar correlation in the blood (R = –0.49; P = .056). The addition of sensitization to fELA to a surgical topical elastase model of AAA Results in the development of larger aneurysms whose size correlates both with the DTH response to the antigen and with a decreasing Tr1/Th17 ratio in blood and periaortic LNs. The decreased Tr1/Th17 ratio is of particular interest as this was noted to be highly predictive of the presence of AAA in a study of Veterans Affairs patients presenting for screening ultrasound examination (0.5287 in AAA vs 2.6570 in non-AAA; P < .00001; receiver operating characteristic curve with an area under the curve of 0.925; 58 patients).Fig 2The strength of the delayed type-hypersensitivity (DTH) reaction to cutaneous challenge with elastin fragments correlates with aneurysm volume as calculated from three-dimensional reconstructions of the infrarenal aorta on microcomputed tomography scans (MicroCT). Cutaneous challenge was performed on the pinna of the ear and reaction quantified by weight difference compared to contralateral ear which was injected with vehicle.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Therapeutic efficacy of artemisinin-loaded nanoparticles in experimental visceral leishmaniasis

Visceral leishmaniasis (VL) is a fatal vector-borne parasitic syndrome attributable to the protozoa of the Leishmania donovani complex. The available chemotherapeutic options are not ideal due to their potential toxicity, high cost and prolonged treatment schedule. In the present study, we conjectured the use of nano drug delivery systems for plant-derived secondary metabolite; artemisinin as an alternative strategy for the treatment of experimental VL. Artemisinin-loaded poly lactic co-glycolic acid (ALPLGA) nanoparticles prepared were spherical in shape with a particle size of 220.0±15.0nm, 29.2±2.0% drug loading and 69.0±3.3% encapsulation efficiency. ALPLGA nanoparticles administered at doses of 10 and 20mg/kg body weight showed superior antileishmanial efficacy compared with free artemisinin in BALB/c model of VL. There was a significant reduction in hepatosplenomegaly as well as in parasite load in the liver (85.0±5.4%) and spleen (82.0±2.4%) with ALPLGA nanoparticles treatment at 20mg/kg body weight compared to free artemisinin (70.3±0.6% in liver and 62.7±3.7% in spleen). In addition, ALPLGA nanoparticle treatment restored the defective host immune response in mice with established VL infection. The protection was associated with a Th1-biased immune response as evident from a positive delayed-type hypersensitivity reaction, escalated IgG2a levels, augmented lymphoproliferation and enhancement in proinflammatory cytokines (IFN-γ and IL-2) with significant suppression of Th2 cytokines (IL-10 and IL-4) after in vitro recall, compared to infected control and free artemisinin treatment. In conclusion, our results advocate superior efficacy of ALPLGA nanoparticles over free artemisinin, which was coupled with restoration of suppressed cell-mediated immunity in animal models of VL.

Mutated Ras-Transfected, EBV-Transformed Lymphoblastoid Cell Lines as a Model Tumor Vaccine for Boosting T-Cell Responses Against Pancreatic Cancer: A Pilot Trial

Genetically modified lymphoblastoid cell lines (LCL) have been shown to be an attractive alternative source of antigen-presenting cells for cancer vaccination in vitro. We tested their application in patients with pancreatic cancer in a phase I clinical trial. As a model tumor antigen, we selected the point-mutated (codon 12) Ki-Ras p21 oncogene (muRas) frequently (∼85%) present in pancreatic adenocarcinoma. Autologous LCLs were established in vitro by spontaneous outgrowth from peripheral blood lymphocytes of seven pancreatic carcinoma patients and were genetically modified with an episomal Epstein-Barr virus (EBV)-based expression vector to express muRas (muRas-LCL). Weekly vaccinations with subcutaneous injection of 5×10(6) muRas-LCL were done. In six of seven patients, therapeutic vaccination elicited a T-cell response with an increase in the frequency of muRas-specific precursor cytotoxic T lymphocytes in the peripheral blood and positive delayed-type hypersensitivity reactions at the injection site. Besides local reactions and flu-like symptoms, there were no signs of toxicity and no acute EBV infection, onset of EBV-associated lymphoma, or other severe complications. A clinical response (stable disease) was observed for a short time period (2-4 months) in four of seven patients (57%), mostly in earlier tumor stages. Our results indicate that LCL presenting genetically modified antigen represent a valuable and easily available tool for in vivo autologous tumor vaccination. LCL can be transfected with any known tumor antigen and therefore should be further clinically investigated.

Heat-Shock Induction of Tumor-Derived Danger Signals Mediates Rapid Monocyte Differentiation into Clinically Effective Dendritic Cells

This study characterizes, biologically and clinically, a novel type of dendritic cells (DC) produced in the short term and called tumor antigen-presenting cells (TAPCells). In particular, we identified factors present in a lysate derived from heat-shocked allogeneic melanoma cells (TRIMEL) that are associated with TAPCells' enhanced capability to induce CD8(+) T-cell responses in vitro and in vaccinated melanoma patients. First, extensive phenotypic and functional characterization of TAPCells was performed, followed by vaccination of 45 melanoma patients with four doses of TAPCells over a period of 2 months. Specific delayed-type hypersensitivity (DTH) reaction was analyzed posttreatment and correlated with overall survival rates. Furthermore, heat-shock (HS)-induced factors present in TRIMEL and their effects on DC activation were identified and studied. TRIMEL induced a committed, mature, DC-like phenotype in TAPCells and effectively activated melanoma-specific CD4(+) and CD8(+) T cells. Clinically, 64% of vaccinated patients showed positive DTH reaction against TRIMEL, and this was associated with improved overall survival. HS treatment of tumor cells increased calreticulin (CRT) plasma membrane translocation and induced the release of high-mobility group box 1 proteins (HMGB1). Both CRT and HMGB1 mobilization were associated with enhanced TAPCells' maturation and antigen (Ag) cross-presentation, respectively. DTH infiltration analysis revealed the presence of CD8(+)/CD45RO(+) T cells, thus confirming TAPCells' ability to cross-present Ags in vivo. Our results indicate that lysates derived from heat-shocked tumor cells are an optimal source of tumor-associated Ags, which are crucial for the generation of DCs with improved Ag cross-presentation capacity and clinically effective immunogenicity.

Exposure to Mycobacterium avium Decreases the Protective Effect of the DNA Vaccine pVAXhsp65 Against Mycobacterium tuberculosis-Induced Inflammation of the Pulmonary Parenchyma

This work investigated the effect of previous Mycobacterium avium exposure on the protective ability of the DNA vaccine pVAXhsp65 against inflammation in the pulmonary parenchyma. BALB/c mice were presensitized with heat-killed M.avium and then immunized with three doses of pVAXhsp65 prior to challenge with Mycobacterium tuberculosis. M.avium sensitization induced high levels of spontaneous IL-5 production that were concomitant with a positive delayed-type hypersensitivity reaction; antigen-specific IFN-γ production was also observed upon splenocyte stimulation. Prior exposure to M.avium resulted in altered cytokine and antibody production induced by immunization with pVAXhsp65; instead of a Th1 response, vaccinated mice previously exposed to M.avium developed a strong Th2 response. This switch to a Th2 response coincided with the loss of the anti-inflammatory effect of pVAXhsp65 vaccination previously observed in the pulmonary parenchyma of mice infected with M.tuberculosis. These results suggest that exposure to environmental mycobacteria can modulate immune responses induced by mycobacterial vaccines other than bacillus Calmette-Guérin.

An experimental protocol for the establishment of dogs with long-term cellular immune reactions to Leishmania antigens

Domestic dogs are considered to be the main reservoirs of zoonotic visceral leishmaniasis. In this work, we evaluated a protocol to induce Leishmania infantum/Leishmania chagasi-specific cellular and humoral immune responses in dogs, which consisted of two injections of Leishmania promastigote lysate followed by a subcutaneous inoculation of viable promastigotes. The primary objective was to establish a canine experimental model to provide positive controls for testing immune responses to Leishmania in laboratory conditions. After inoculation of viable promastigotes, specific proliferative responses of peripheral blood mononuclear cells (PBMCs) to either Leishmania lysate or recombinant proteins, the in vitro production of interferon-γ by antigen-stimulated PBMCs and a significant increase in circulating levels of anti-Leishmania antibodies were observed. The immunized dogs also displayed positive delayed-type hypersensitivity reactions to Leishmania crude antigens and to purified recombinant proteins. An important finding that supports the suitability of the dogs as positive controls is that they remained healthy for the entire observation period, i.e., more than seven years after infection. Following the Leishmania antigen lysate injections, the infection of dogs by the subcutaneous route appears to induce a sustained cellular immune response, leading to an asymptomatic infection. This provides a useful model for both the selection of immunogenic Leishmania antigens and for immunobiological studies on their possible immunoprotective activities.

Results from a Phase I Clinical Study of the Novel Ii-Key/HER-2/neu(776–790) Hybrid Peptide Vaccine in Patients with Prostate Cancer

Active immunotherapy is emerging as a potential therapeutic approach for prostate cancer. We conducted the first phase I trial of an Ii-Key/HER-2/neu(776-790) hybrid peptide vaccine (AE37) with recombinant granulocyte macrophage colony-stimulating factor as adjuvant in patients with HER-2/neu(+) prostate cancer. The primary end points of the study were to evaluate toxicity and monitor patients' immune responses to the vaccine. Thirty-two HER-2/neu(+), castrate-sensitive, and castrate-resistant prostate cancer patients were enrolled. Of these, 29 patients completed all six vaccination cycles with AE37. Immunologic responses in the total patient population were monitored by delayed-type hypersensitivity and IFN-gamma ELISPOT and intracellular staining. Regulatory T-cell (Treg) frequency and plasma HER-2/neu and transforming growth factor-beta levels were also determined. Immunologic responses were also analyzed among groups of patients with different clinical characteristics. Local/systemic toxicities were monitored throughout the study. Toxicities beyond grade 2 were not observed. Seventy-five percent of patients developed augmented immunity to the AE37 vaccine and 65% to the unmodified AE36 peptide as detected in the IFN-gamma-based ELISPOT assay. Intracellular IFN-gamma analyses revealed that AE37 elicited both CD4(+) and CD8(+) T-cell responses. Eighty percent of the patients developed a positive delayed-type hypersensitivity reaction to AE36. Additionally, significant decreases could be detected in circulating Treg frequencies, plasma HER-2/neu, and serum transforming growth factor-beta levels. Patients with less extensive disease developed better immunologic responses on vaccination. AE37 vaccine is safe and can induce HER-2/neu-specific cellular immune responses in patients with castrate-sensitive and castrate-resistant prostate cancer, thus emphasizing the potential of AE37 to target HER-2/neu for the immunotherapy of prostate cancer.

Abstract B18: Induction of Th1/Th17 and inhibition of Th3 cellular profile by DC-based immunotherapy is associated with immunological and clinical anti-tumor responses in melanoma patients

Abstract The ability of DCs to induce a primary immune response makes them ideal candidates for cancer vaccines against tumors. Recently, in a serie of phase I/II clinical studies, we showed the effectiveness of autologous DCs loaded with a conditioned allogeneic melanoma cell lysate named TRIMEL. Our results demonstrated a correlation between positive tumor-specific delayed type hypersensitivity (DTH) reaction induced by DC-vaccination, and improved long-term patient survival in late-stage melanoma patients. In fact, 60% of patients showed a positive DTH reaction showing a three-fold prolonged survival compared to non-responder patients (33 and 11 months respectively). Herein, we investigated whether cellular and cytokine profiles in patients PBL before and after vaccination reflects differential responses associated to the clinical outcome. Analysis of T cell populations in blood showed that melanoma patients (n=28) have an increased proportion of Th3 (CD4+TGF-β+) regulatory T lymphocytes (2.64 % ± 0.16) compared to normal donors (n=14) (1.72 % ± 0.22). However, we didn't observed differences in TGF-β concentrations at serum level. Moreover, we analyzed vaccine effect on cytokine and cellular responses in PBL. Interestling, although there was not differences in Th3 population proportions between responder (n=14) and non-responder patients (n=14) before vaccination, after treatment responder patients showed a three-fold reduction of CD4+TGf-β+ T cells compared to non-responders (1.65 % ± 0.19 and 5.54 % ± 0.28 respectively). Furthermore, our results show that DC-vaccination resulted in a three-fold augment of IFN-γ releasing by Th1 population and a two-fold increase of IL-17 producing Th17 lymphocyte population in responder patients, but not in non-responders. A direct correlation between IFN-γ and IL-17 production in responder patients blood was observed (p &amp;lt; 0.0001) suggesting that those cytokines may favour an anti melanoma response. Taken together our results indicates that despite undetectable differences in the cellular and cytokine profiles in melanoma patients before DC-vaccination, immunization resulted in two different pattern of response associated to the immunological and clinical outcome. Our study may contribute to a better understanding of clinical immunological responses and to an earlier prognosis of patient outcome. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B18

S86 Results from the first phase I clinical study of the novel Ii-Key/HER-2(776–790) hybrid peptide vaccine in patients with prostate cancer

Purpose: Active immunotherapy is emerging as a potential therapeutic approach for prostate cancer. We conducted the first phase I trial of an Ii-Key/HER-2/neu(776–790) hybrid peptide vaccine (AE37) with recombinant granulocyte macrophage colony-stimulating factor as adjuvant in patients with HER-2/neu + prostate cancer. The primary end points of the study were to evaluate toxicity and monitor patients' immune responses to the vaccine. Experimental Design: Thirty-two HER-2/neu + , castrate-sensitive, and castrate-resistant prostate cancer patients were enrolled. Of these, 29 patients completed all six vaccination cycles with AE37. Immunologic responses in the total patient population were monitored by delayed-type hypersensitivity and IFN-γ ELISPOT and intracellular staining. Regulatory T-cell (Treg) frequency and plasma HER-2/neu and transforming growth factor-β levels were also determined. Immunologic responses were also analyzed among groups of patients with different clinical characteristics. Local/systemic toxicities were monitored throughout the study. Results: Toxicities beyond grade 2 were not observed. Seventy-five percent of patients developed augmented immunity to the AE37 vaccine and 65% to the unmodified AE36 peptide as detected in the IFN-γ–based ELISPOT assay. Intracellular IFN-γ analyses revealed that AE37 elicited both CD4 + and CD8 + T-cell responses. Eighty percent of the patients developed a positive delayed-type hypersensitivity reaction to AE36. Additionally, significant decreases could be detected in circulating Treg frequencies, plasma HER-2/neu, and serum transforming growth factor-β levels. Patients with less extensive disease developed better immunologic responses on vaccination. Conclusion: AE37 vaccine is safe and can induce HER-2/neu–specific cellular immune responses in patients with castrate-sensitive and castrate-resistant prostate cancer, thus emphasizing the potential of AE37 to target HER-2/neu for the immunotherapy of prostate cancer. Clin Cancer Res; 16(13); 3495–506. ©2010 AACR.

Phase II Trial of Dendritic Cells Loaded with Antigens from Self-Renewing, Proliferating Autologous Tumor Cells as Patient-Specific Antitumor Vaccines in Patients with Metastatic Melanoma: Final Report

Between January 2001 and September 2007, we treated 54 metastatic melanoma patients with patient-specific tumor cell vaccines consisting of dendritic cells (DCS), derived from their peripheral blood cells that were cultured in interleukin (IL)-4 and granulocyte macrophage colony-stimulating factor (GM-CSF), which had phagocytosed irradiated autologous tumor cells from a continuously proliferating, self-renewing, autologus tumor cell (TC) culture. The loaded DCs were injected subcutaneously in 500 microg of GM-CSF weekly x three, and then monthly for 5 months, for a total of up to 8 injections. The 34 men and 20 women had a median age of 50.5 years; 32 had M1c (visceral metastases and/or elevated lactate dehydrogenase) as their most advanced disease stage. Overall, 83% had received other systemic therapies, including interferon-alpha (n = 20), biochemotherapy (n = 19), GM-CSF (n = 19), chemotherapy (n = 16), IL-2 (n = 13), and other investigational vaccines (n = 7). Patients received an average of 7.4 vaccinations. Treatment was well-tolerated, with most patients experiencing only mild local pruritus and/or erythema. A positive delayed-type hypersensitivity reaction to purified autologous tumor cells was observed at baseline in only 1 of 54 patients, compared to 12 of 54 following vaccination (p = 0.001). The projected 5-year survival rate is an impressive 54% at a median follow-up of 4.5 years (range, 2.4-7.4) for the 30 surviving patients. This survival was superior to that observed following vaccination with irradiated TC in 48 melanoma patients in a previous trial (64 versus 31 months, p = 0.016). This patient-specific vaccine warrants further investigation, based on its safety and encouraging survival rates. ( NCI-V01-1646).

Postoperative change of anti-Thomsen-Friedenreich and Tn IgG level: The follow-up study of gastrointestinal cancer patients

To study the influence of tumor removal on the serum level of IgG antibodies to tumor-associated Thomsen-Friedenreich (TF), Tn carbohydrate epitopes and xenogeneic alphaGal, and to elucidate on the change of the level during the follow-up as well as its association with the stage and morphology of the tumor and the values of blood parameters in gastrointestinal cancer. Sixty patients with gastric cancer and 34 patients with colorectal cancer in stages I-IV without distant metastases were subjected to follow-up. The level of antibodies in serum was determined by the enzyme-linked immunosorbent assay (ELISA) using synthetic polyacrylamide (PAA) glycoconjugates. Biochemical and haematological analyses were performed using automated equipment. In gastrointestinal cancer, the TF antibody level was found to have elevated significantly after the removal of G3 tumors as compared with the preoperative level (u = 278.5, P < 0.05). After surgery, the TF and Tn antibody level was elevated in the majority of gastric cancer patients (sign test, 20 vs 8, P < 0.05, and 21 vs 8, P < 0.05, respectively). In gastrointestinal cancer, the elevated postoperative level of TF, Tn and alphaGal antibodies was noted in most patients with G3 tumors (sign test, 22 vs 5, P < 0.01; 19 vs 6, P < 0.05; 24 vs 8, P < 0.01, respectively), but the elevation was not significant in patients with G1 + G2 resected tumors. The postoperative follow-up showed that the percentage of patients with G3 resected tumors of the digestive tract, who had a mean level of anti-TF IgG above the cut-off value (1.53), was significantly higher than that of patients with G1 + G2 resected tumors (c2 = 3.89, all patients; c2 = 5.34, patients without regional lymph node metastases; P < 0.05). The percentage of patients with a tumor in stage I, whose mean anti-TF IgG level remained above the cut-off value (1.26), was significantly higher than that of patients with the cancer in stages III-IV (c2 = 4.71, gastric cancer; c2 = 4.11, gastrointestinal cancer; P < 0.05). The correlation was observed to exist between the level of anti-TF IgG and the count of lymphocytes (r = 0.517, P < 0.01), as well as between the level of anti-Tn IgG and that of serum CA 19-9 (r = 0.481, P < 0.05). No positive delayed-type hypersensitivity reaction in skin test challenges with TF-PAA in any of the fifteen patients, including those with a high level of anti-TF IgG, was observed. The surgical operation raises the level of anti-carbohydrate IgG in most patients, especially in those with the G3 tumor of the gastrointestinal tract. The follow-up demonstrates that after surgery the low preoperative level of TF antibodies may be considerably increased in patients with the carcinoma in its early stage but remains low in its terminal stages. The stage- and morphology-dependent immunosuppression affects the TF-antibody response and may be one of the reasons for unresponsiveness to the immunization with TF-antigens.

Immunotherapy against experimental canine visceral leishmaniasis with the saponin enriched-Leishmune® vaccine

In order to assess the immunotherapeutic potential on canine visceral leishmaniasis of the Leishmune® vaccine, formulated with an increased adjuvant concentration (1 mg of saponin rather than 0.5 mg), 24 mongrel dogs were infected with Leishmania (L.) chagasi. The enriched-Leishmune® vaccine was injected on month 6, 7 and 8 after infection, when animals were seropositive and symptomatic. The control group were injected with a saline solution. Leishmune®-treated dogs showed significantly higher levels of anti-FML IgG antibodies (ANOVA; p < 0.0001), a higher and stable IgG2 and a decreasing IgG1 response, pointing to a TH1 T cell mediated response. The vaccine had the following effects: it led to more positive delayed type hypersensitivity reactions against Leishmania lysate in vaccinated dogs (75%) than in controls (50%), to a decreased average of CD4+ Leishmania-specific lymphocytes in saline controls (32.13%) that fell outside the 95% confidence interval of the vaccinees (41.62%, CI95% 43.93–49.80) and an increased average of the clinical scores from the saline controls (17.83) that falls outside the 95% confidence interval for the Leishmune® immunotherapy-treated dogs (15.75, CI95% 13.97–17.53). All dogs that received the vaccine were clustered, and showed lower clinical scores and normal CD4+ counts, whereas 42% of the untreated dogs showed very diminished CD4+ and higher clinical score. The increase in clinical signs of the saline treated group was correlated with an increase in anti-FML antibodies (p < 0.0001), the parasitological evidence (p = 0.038) and a decrease in Leishmania-specific CD4+ lymphocyte proportions (p = 0.035). These results confirm the immunotherapeutic potential of the enriched-Leishmune® vaccine. The vaccine reduced the clinical symptoms and evidence of parasite, modulating the outcome of the infection and the dog's potential infectiosity to phlebotomines. The enriched-Leishmune® vaccine was subjected to a safety analysis and found to be well tolerated and safe.

Immunotherapy against metastatic renal cell carcinoma with mature dendritic cells

We performed a clinical trial of immunotherapy using autologous mature dendritic cells (DC) pulsed with autologous tumor lysate, for patients with metastatic renal cell carcinoma (RCC). Patients with refractory metastatic RCC were enrolled in the study. All of them received interferon (IFN)-alpha treatment after nephrectomy and were followed over 3 months prior to this study. Autologous monocyte-derived immature DC were pulsed with lysate from autologous primary tumor as the antigen and keyhole limpet hemocyanin (KLH) as immunomodulator, and cultured in the presence of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and prostaglandin (PG)E2 to generate mature DC. Mature DC were injected intradermally near bilateral inguinal lymph nodes of the patients. A delayed-type hypersensitivity (DTH) test and enzyme-linked immunospot (ELISPOT) assay were performed to evaluate the immunological response. After 4 months from first injection, the clinical effect was evaluated by diagnostic imaging. The treatments were well tolerated without significant toxicity by the patients who were an average of 65.7 years old and had multiple metastases in the lung and other organs. One of the two patients developed a positive DTH reaction to tumor lysate and the other patient only to KLH. The patient with a positive DTH reaction to tumor lysate had stable disease in the clinical evaluation. We confirmed the safety of DC therapy in this clinical trial. The DTH test revealed that the DC therapy induced immunological response to RCC. On the other hand, it was necessary to reconsider the patient selection criteria.

Interest of FDG-PET Scanning for the Monitoring of Immunotherapies for Follicular Lymphoma.

Except in the subset of patients with localized disease, follicular lymphoma (FL), the most common type of indolent non-Hodgkin's lymphoma, should still be regarded as an incurable malignancy with a relentless relapsing course. New therapeutic approaches have been developed including anti-tumor vaccination strategies with induced humoral and cellular immune responses revealed by sensitive biological analyses but no imaging of the lymphocyte activation developed so far. Because [18F]fluorodeoxyglucose (FDG) is sequestred in metabolically active cells, we prospectively investigated the use of FDG-PET (positron emission tomography) to kinetically monitor cell activation in 7 FL patients vaccinated by repeated intradermal (ID) and intravenous injections of tumor lysate-pulsed autologous dendritic cells (DC-lysate). Patients were administered 5 vaccines and all patients developed a positive delayed-type hypersensitivity reaction at the site of DC-lysate ID injections after several vaccines administration, testifying of the induction of a biological response. Among the 4 patients with measurable disease at time of vaccination, 2 complete remissions (CR) and 2 disease progressions were observed. Three patients with initial normal computed tomographies (CT) were still in CR 12 months after the last vaccine. PETs were performed before vaccination, 14 days and 1 month after the last vaccine. PET revealed initial malignant nodes infracentimetric on concomitant CT, and was very sensitive in documenting disease progression even in patients with no significant variations of CT findings. A transient marked increase in FDG uptake within malignant nodes was observed early after vaccine administration in a patient who achieved a CR (fig 1-3-4-5), while ranges of tracer uptake variations were less important with a continuous increase of FGD uptake, size and number of involved loci in non responding patients (fig 2: follow up of intensity of FDG uptake by 6 tumoral nodes in a non responding pt). In conclusion, FDG-PET is a non invasive imaging procedure which besides being useful for the staging and the evaluation of treatment efficacy in FL, might be of crucial interest to detect cell responses induced by immunotherapies. [Display omitted]

Vaccination of colorectal cancer patients with carcinoembryonic antigen peptide-loaded dendritic cells

2544 Dendritic cells (DC) are the most potent antigen presenting cells of the immune system, capable of inducing an effective anti-tumor immune response. They can be cultured in large quantities from peripheral blood monocytes from cancer patients, loaded with tumor antigen ex vivo and subsequently reinjected into the patients. Currently DC are employed by us and others in clinical vaccination protocols and objective clinical and immunological responses have been observed. Recent investigations indicate that carcinoembryonic antigen (CEA) is an effective target for DC-based immunotherapy. However, very little data exist concerning the induction of an in vivo immune response, in colorectal cancer patients. We have initiated a clinical study with keyhole limpet hemocyanin (KLH) and CEA-peptide loaded DC in HLA-A2.1 positive patients with resectable liver metastases of recurrent colorectal cancer. Patients are vaccinated 3 times intravenously and intradermally with autologous CEA-loaded mature DC prior to surgical resection of the metastases. Immuno-monitoring consists of T cell responses in peripheral blood, delayed type hypersensitivity (DTH) reactions, and in resected tumor and lymph node material. To date, 7 patients have completed vaccination. Treatment was well tolerated with minor side effects. Vaccination resulted in an antibody response and a proliferative T cell response against KLH in all tested patients. Furthermore, CEA-peptide specific T cells were observed in T cell cultures from positive DTH reaction sites in 5 pts. These T cells were not detected in biopsy specimens of the first vaccination site, indicating a de novo induction. CEA-tetramer positive T cells were also detected in blood (1 pt) and abdominal lymph nodes (1 pt) obtained after vaccination. Together, these results demonstrate that CEA-pulsed dendritic cells are capable of inducing specific immune reactions in colorectal cancer patients. Our previous results have shown that specific T cell responses in DTH infiltrating T lymphocytes in stage IV melanoma patients were correlated with survival (de Vries et al. submitted for publication). Therefore these observations warrant further confirmation in our ongoing study. No significant financial relationships to disclose.

Papular dermatitis due to Leishmania spp. infection in dogs with parasite‐specific cellular immune responses

Papular dermatitis due to Leishmania spp. infection was diagnosed in three boxers and two Rottweilers with Leishmania-specific cellular immunity. Diagnosis was based on histological and immunohistochemical examination of papules in four dogs and on cytological examination in one dog. Serum protein electrophoresis was within reference ranges and low antibody levels to Leishmania infantum were detected. Delayed-type hypersensitivity (DTH) reaction to leishmanin was evaluated before treatment in three dogs with positive results. After meglumine antimoniate therapy for 3 to 4 weeks and allopurinol treatment for 6 to 10 months, all dogs were clinically normal, had positive DTH reactions to leishmanin and reduced antibody titres. In conclusion, we suggest that this previously unreported cutaneous presentation of canine leishmaniosis appears to be associated with specific immunocompetence and, consequently, with a favourable prognosis.

Novel adjuvant based on a proteoliposome‐derived cochleate structure containing native lipopolysaccharide as a pathogen‐associated molecular pattern

Proteoliposomes (PL) from Neisseria meningitidis B have been widely used as a core antigen for antimeningococcal vaccination. PL contain major outer membrane proteins, LPS and phospholipids, and they induce a strong Th1 immune response, but they have low stability in solution. Attending to the need for new vaccine adjuvants, we developed a highly stable cochleate structure (CS) from PL using a technology that allows easy incorporation of new antigens. We explored the ability of PLCS to activate the immune system and its possible application as an adjuvant for parenteral and mucosal routes. Our results showed that PLCS were able to upregulate the expression of MHC class II and costimulatory molecules on human dendritic cells, as well as being able to stimulate the production of soluble mediators of a Th1 response, such as IL-12 and nitric oxide. High levels of anti-PL IgG were detected in serum after i.m. or mucosal (oral and nasal) administration, but also anti-PL secretory IgA was produced in saliva following nasal delivery. The immune response polarization to a Th1 pattern was confirmed by the induction of IgG2a antibodies, positive delayed type hypersensitivity reactions, and IFN-gamma production by splenocytes from immunized mice. The adjuvant potential was explored using PLCS containing ovalbumin (Ova). PLCS-Ova was able to elicit a substantial increase in anti-Ova IgG compared with Ova alone. In addition, a significant reduction in lesion size was observed in mice immunized with Leishmania major antigens in PLCS after challenge with virulent protozoa, suggesting at least partial modulation of the Th2 environment induced by this parasite. In conclusion, our results support the use of PLCS as a potent Th1 adjuvant for parenteral and mucosal vaccines.

Immunization of colorectal cancer patients with recombinant baculovirus-derived KSA (Ep-CAM) formulated with monophosphoryl lipid A in liposomal emulsion, with and without granulocyte-macrophage colony-stimulating factor

KSA (Ep-CAM) is highly expressed by colorectal cancers. The safety and immunologic effects of a vaccine consisting of recombinant baculovirus-derived KSA formulated with monophosphoryl lipid A (MPL) in liposomes and emulsified in mineral oil were evaluated, with and without co-administration of granulocyte-macrophage colony-stimulating factor (GM-CSF). Eleven patients with metastatic colorectal cancer received three subcutaneous (s.c.) injections of the vaccine at 4-week intervals. Six patients were randomized to also receive human recombinant GM-CSF (rGM-CSF) by subcutaneous injection daily for 4 days with each vaccination. Immunizations with and without rGM-CSF were well tolerated. Seven of the 11 patients developed significant KSA-specific cellular immune responses as assessed by lymphoproliferation and interferon-γ (IFN-γ) ELISPOT assays. All nine tested patients developed positive delayed type hypersensitivity reactions. Eight of the 11 patients developed KSA-specific antibody responses. The highest levels of cellular immune responses were observed in patients who received GM-CSF. Immunization with baculovirus-derived KSA formulated with monophosphoryl lipid A in liposomal emulsion is safe and can elicit KSA-specific immune responses. Co-administration of GM-CSF with this formulation is an effective method of generating KSA-specific T-helper (Th) 1-associated cellular immune responses.

The prevalence of immediate and delayed type hypersensitivity reactions to Microsporum canis antigens in cats

Spontaneous recovery from Microsporum canis infections in cats is thought to be dependent on the development of a competent immune response. The purpose of this study was to determine the prevalence of positive delayed type hypersensitivity reactions in cats with and without dermatophytosis. Four groups of cats were intradermally skin tested with M canis extract and test sites were evaluated both subjectively and objectively at 0, 24 and 48 h after injection. Delayed intradermal testing (IDT) reactions were absent in cats not exposed to dermatophytosis ( n=20); infected–recovered cats ( n=38 culture negative lesion negative and n=43 lesion negative but culture positive) had significantly larger IDT reactions than unexposed cats and cats that were still actively infected ( n=18). Based on the results of this study, IDT with M canis extract can be used to assess the cellular immune response of cats with dermatophytosis.

Dendritic cell immunotherapy for patients with metastatic renal cell carcinoma: University of Tokyo experience.

Dendritic cells (DC) are the most potent antigen-presenting cells and induce host antitumor immunity through the T-cell response. A clinical study of immunotherapy using cultured DC loaded with tumor antigen, for patients with metastatic renal cell carcinoma (RCC) was performed. Dendritic cells were generated by culturing monocytes from peripheral blood for 7 days in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. On day 6 the DC were pulsed with lysate from autologous tumor as the antigen and with keyhole limpet hemocyanin (KLH) as immunomodulator. The patients were given four doses of lysate-pulsed DC by intradermal injection with a 2-week interval between doses. Clinical effect and immune response were, respectively, evaluated by radiological examination and delayed-type hypersensitivity (DTH) test. Three patients were enrolled and the immunotherapy was well tolerated without significant toxicity. The vaccination induced a positive DTH reaction to tumor lysate in two patients and to KLH in all patients. Clinical responses consisted of one case of no change and two cases of progression of disease. However, we did not see a significant reduction of tumor volume in any case. Dendritic cell vaccination can safely induce an immunological response against RCC. Further trials are needed to fully evaluate its efficacy.