Delayed-type Hypersensitivity Test Research Articles

Antibody to ovalbumin and delayed-type hypersensitivity to Candida albicans and mycobacteria in lactating Holstein cows using Quil A or Freund's complete adjuvant

Antibody-mediated immune response (AMIR) to ovalbumin (OVA) or hen-egg white lysozyme (HEWL) and cell-mediated immune response (CMIR) such as delayed-type hypersensitivity (DTH) to mycobacteria have been proposed as quantitative traits for selective breeding to improve animal health. However, DTH to mycobacteria may confound diagnosis of tuberculosis in cattle. Candida albicans, a yeast also known to induce DTH, was tested as an alternative for DTH induction and testing since it is not a target of regulatory diagnostic tests. Other objectives were to determine if both AMIR and CMIR in cattle receiving Quil A as adjuvant were equivalent to corresponding responses induced by Freund's complete adjuvant (FCA). Forty lactating Holstein cows were randomly assigned to two treatment groups, which received ovalbumin (OVA) and C. albicans adjuvanted with FCA and Freund's incomplete adjuvant (FIA) on days 0 and 14, respectively, or Quil A on days 0 and 14. The FCA was used as adjuvant and as a source of Mycobacterium tuberculosis-induced DTH. Testing for DTH was performed on day 21 with killed C. albicans whole cell (CaWC), a purified extract from C. albicans (candin) and M. phlei purified protein (phlein). Both primary and secondary antibody responses to OVA were statistically significant and similar in both FCA and Quil A treatment groups. No significant differences were detected in immunoglobulin G (IgG) isotypic-mediated responses to OVA or candin between groups. C. albicans adjuvanted with Quil A induced DTH reaction similar to those induced by C. albicans and mycobacteria in FCA.

Optimal dose and schedule of an HER‐2/neu (E75) peptide vaccine to prevent breast cancer recurrence

E75, a HER-2/neu-derived peptide, was administered as a preventive vaccine with granulocyte-macrophage-colony-stimulating factor (GM-CSF) in disease-free lymph node-positive (NP) and lymph node-negative (NN) breast cancer (BCa) patients. The optimal biologic dose (OBD) was determined based on toxicity and immunologic response. Patients were vaccinated over 6 months (3, 4, or 6 times) with different doses of E75 plus GM-CSF. Toxicities were graded per National Cancer Institute Common Terminology Criteria. GM-CSF was reduced for significant toxicity. Immunologic response was measured by delayed type hypersensitivity test (DTH), and E75-specific CD8+ T-cells were quantified with human leukocyte antigen-A2:immunoglobulin G dimer and flow cytometry. Ninety-nine patients (48 NP and 51 NN) were vaccinated in 7 dose groups. The OBD was 1000 microg E75 plus 250 microg GM-CSF monthly x 6. The optimal dose group (ODG, n = 29) experienced similar toxicities to the suboptimal dose group (SDG, n = 70), which was comprised of the remaining 6 groups. The ODG demonstrated a trend toward an increase in the average postvaccine dimer (0.87 +/- 0.10% vs 0.67 +/- 0.05%; P = .07), a significantly larger DTH response (21.5 +/- 2.5 mm vs 11.3 +/- 1.3 mm; P = .0002), and a trend toward decreased recurrences (3.4% vs 12.9%; P = .27). Compared with the SDG, the ODG had larger tumors (percentage > or =T2: 55% vs 23%; P = .004), more positive lymph nodes (percentage NP: 76% vs 37%; P = .001), and higher grade tumors (percentage grade 3: 52% vs 30%; P = .07), but a shorter median follow-up time (20 months vs 32 months; P < .001). Compared with suboptimally dosed patients, the optimally dosed E75 vaccine in disease-free BCa patients had similar toxicity but enhanced HER-2/neu-specific immunity that may lead to decreased recurrences with additional follow-up.

Dendritic cell therapy in combination with interferon‐α for the treatment of metastatic renal cell carcinoma

To evaluate the safety and efficacy of dendritic cell (DC) therapy in combination with interferon-alpha (IFN-alpha) in patients with advanced renal cell carcinoma. Seven patients, with progressive disease following IFN-alpha and interleukin (IL)-2 treatment, were treated with monocyte-derived DC (Mo-DC) and IFN-alpha between February 2004 and September 2006. They received Mo-DC once a week for 5 weeks and then every 2 weeks either intradermally or intratumorally. IFN-alpha (5-6 million U) was subcutaneously administered three times a week. Tumor size was evaluated by computed tomography scans before and after the 5th and 10th DC vaccination. A delayed-type hypersensitivity test was performed after the 4th and 5th DC administration for immunological monitoring. Five patients had stable disease while the remaining two patients had progressive disease following 4 months of vaccination. In six patients the time to progression was prolonged in comparison with the previous cytokine treatment. Six patients showed delayed-type hypersensitivity after the 4th or 5th immunization. Three patients developed high fever following DC immunization. Treatment was associated with transient flu-like symptoms. Our data indicate that DC therapy combined with IFN-alpha is safe and has the potential for prolonging the time to progression in patients with advanced renal cell carcinoma.

A delayed-type hypersensitivity skin-test system using the insulinoma cell line RINm5F to monitor beta cell-specific cellular autoimmune reactivity in the spontaneously diabetic BB/O rat

Islet-specific autoimmune reactivity (humoral and cell-mediated) is the basis for the insulitis process of type I diabetes mellitus. In this report a delayed-type hypersensitivity (DTH) skin test was used to monitor the presence of an islet-specific cell-mediated autoimmune component in BB/O rats. The BB/O rat is a strain characterized by the spontaneous development of type I diabetes. Intact RINm5F cells as well as a RINm5F cell membrane preparation were used as DTH skin test antigens. Rats of different ages and disease stages were tested in the ear with the insulinoma cell line and its cell membrane preparation. As control antigens, the fibroblast cell line 3Y1 and a cell membrane preparation made thereof were used. The DTH reaction system showed a positive cell-mediated reactivity in BB/O rats for membrane-bound RINm5F cell antigens, and not for the control fibroblast 3Y1 cell membrane determinants. The true DTH character of the skin test was established by the time-course of the reaction (maximum at 24 h), the histopathology (infiltration by dendritic cells, lymphocytes and macrophages), and the possibility to transfer the reaction with spleen cells and lymph node cells. The DTH test towards RINm5F cells showed the highest prevalence of positivity (100%) in BB/O rats around the onset of diabetes (3 weeks before to 3 weeks after the onset of glucosuria). The prevalence of DTH positivity was 56% in the period of more than 3 weeks before the onset of glucosuria. In BB/O rats with a duration of glucosuria of more than 3 weeks, the prevalence of positivity was around 60-70%.

Effects of Inhalation Exposure to a Binary Mixture of Benzene and Toluene on Vitamin A Status and Humoral and Cell-Mediated Immunity in Wild and Captive American Kestrels

Benzene and toluene are representative volatile organic compounds (VOC) released during production, storage, and transportation associated with the oil and gas industry and are chemicals of concern, as they are released in greater and possibly more biologically significant concentrations than other compounds. Most studies of air pollution in high oil and gas activity areas have neglected to consider risks to birds, including top-level predators. Birds can be used as highly sensitive monitors of air quality and since the avian respiratory tract is physiologically different from a rodent respiratory tract, effects of gases cannot be safely extrapolated from rodent studies. Wild and captive male American kestrels were exposed for approximately 1 h daily for 28 d to high (rodent lowest-observed-adverse-effect level [LOAEL] of 10 ppm and 80 ppm, respectively) or environmentally relevant (0.1 ppm and 0.8 ppm, respectively) levels of benzene and toluene. Altered immune responses characteristic of those seen in mammalian exposures were evident in kestrels. A decreased cell-mediated immunity, measured by delayed-type hypersensitivity testing, was evident in all exposed birds. There was no effect on humoral immunity. Plasma retinol levels as measured by high-performance liquid chromatography (HPLC) analysis were decreased in wild and captive kestrels exposed to the rodent LOAEL for combined benzene and toluene. This study indicates that American kestrels are sensitive to combined benzene and toluene. The study also illustrates the need for reference concentrations for airborne pollutants to be calculated, including sensitive endpoints specific to birds. Based on these findings, future studies need to include immune endpoints to determine the possible increased susceptibility of birds to inhaled toxicants.

Broad-spectrum sunscreens provide better protection from solar ultraviolet–simulated radiation and natural sunlight–induced immunosuppression in human beings

It is well established that ultraviolet (UV) radiation induces immunomodulatory effects that may be involved in skin cancer. Recent studies have shown that UVA (320-400 nm) and UVB (290-320 nm) radiation are immunosuppressive. As a result, sunscreens, which mainly absorb UVB, may be less effective in preventing UV radiation-induced immunosuppression than broad-spectrum products. We sought to study the effects of UVA exposure on human delayed-type hypersensitivity (DTH) response and compare the efficacy of sunscreens having different levels of sun-protection factor (SPF) and UVA protection against both solar-simulated radiation and outdoor real-life sunlight exposure conditions. DTH was assessed using a kit which includes 7 recall antigens that most of the participants encountered during childhood immunization. Evaluation of DTH test response was made 48 hours after test application before and after UV exposure with or without sunscreens. In unprotected participants, the response to DTH tests was significantly reduced irrespective of UV types of exposure (full-spectrum UVA, long UVA, solar-simulated radiation). A UVB sunscreen failed to protect from solar-simulated radiation-induced immunosuppression. In contrast, a broad-spectrum sunscreen with the same SPF but providing a high protection in the UVA range significantly reduced local UV-induced immunosuppression and prevented the distant effects. In the outdoor study, as compared with DTH responses obtained before sun exposure, no alteration of immune response was detected when the skin was protected by a broad-spectrum sunscreen having a high protection level in the UVA (SPF 25, UVA protection factor 14). Conversely a broad-spectrum sunscreen with lower protection against UVA (SPF 25, UVA protection factor 6) failed to prevent UV-impaired response. These results have been obtained after repeated exposure. Additional experiments obtained under acute exposure are in progress. These findings clearly demonstrated the role of UVA in the induction of photoimmunosuppression together with the need for sunscreen products providing efficient photoprotection throughout the entire UV spectrum.

Experimental infections with Paracoccidioides brasiliensis obtained from armadillos: comparison to clinical isolates

Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM) that is one of the most prevalent systemic human mycoses in Latin America. Armadillos show a high incidence of PCM infection and could, therefore, be a natural reservoir for this fungus. In this study were compared the virulence profiles of isolates obtained from nine-banded armadillos (Dasypus novemcinctus) (PbT1 and PbT4) and isolates from PCM patients (Pb265 and Bt83). Pathogenicity was evaluated by fungal load and analysis of colony morphology. Immunity against the fungus was tested by delayed type hypersensitivity test (DTH) and antibody quantification by ELISA. The higher virulence of PbT1 and PbT4 was suggested by higher fungal load in spleen and lungs. Armadillo isolates and Bt83 presented a cotton-like surface contrasting with the cerebriform appearance of Pb265. All isolates induced cellular and humoral immune responses in infected BALB/c mice. DTH reactions were similarly induced by the four isolates, however, a great variability was observed in specific antibody levels, being the highest ones induced by Bt83 and PbT4. The present work confirms that armadillos harbor P. brasiliensis, whose multiplication and induced immunity in experimentally infected mice are heterogeneous, resembling the behavior of isolates from human PCM. This study reinforces the possibility that armadillos play an important role in the biological cycle of this pathogen.

Introducing Percutaneous and Patch Testing to Foods in a Clinical Setting for Patients with Eosinophilic Esophagitis: A Case Series

Eosinophilic esophagitis (EE) includes both IgE and cell mediated pathophysiology. This study investigated the feasibility of using a combination of immediate and delayed-type hypersensitivity testing to foods for the initial evaluation of patients with biopsy-proven EE.

Passive Transfer of Maternal Mycoplasma hyopneumoniae -Specific Cellular Immunity to Piglets

Immunity in the neonatal animal is primarily maternally derived, either by lymphocytes that pass into the newborn across the placenta or following colostrum ingestion. However, the effect of this passively transferred cellular maternal immunity on the newborn's immune repertoire is not clearly understood. Various studies have shown that colostral lymphocytes are activated and possess functional abilities; however, no studies have shown the transfer of colostral antigen-specific T-cell-specific responses in a newborn. In this study we examined the transfer of vaccine-induced Mycoplasma hyopneumoniae cellular immunity from immune dams to newborn piglets. Newborn piglets from vaccinated and nonvaccinated dams were assessed in two ways for cellular immune responses specific to M. hyopneumoniae: (i) delayed-type hypersensitivity (DTH) testing and (ii) in vitro lymphocyte proliferation, assayed on piglet blood lymphocytes and sow colostral lymphocytes. DTH responses to M. hyopneumoniae were detected only for offspring of vaccinated sows, whereas DTH responses to the nonspecific mitogen phytohemagglutinin were seen for all piglets. M. hyopneumoniae-specific proliferation was seen for colostral lymphocytes from vaccinated sows and for blood lymphocytes from neonatal piglets of vaccinated dams but not for blood lymphocytes from piglets of nonvaccinated sows. Functional antigen-specific T cells were transferred to offspring from vaccinated sows and participated in the neonatal immune response upon stimulation. These data have implications for defining disease intervention strategies.

Phase II Trial of B7-1 (CD-86) Transduced, Cultured Autologous Tumor Cell Vaccine Plus Subcutaneous Interleukin-2 for Treatment of Stage IV Renal Cell Carcinoma

We report a single center phase II trial of sequential vaccination followed with vaccine plus interleukin-2 (IL-2). Vaccination consisted of autologous cells cultured from primary tumor or resected metastasis, transduced to express B7.1 surface molecule and then irradiated. The vaccine would hypothetically costimulate tumor-reactive T cells before IL-2 exposure. Treatment plan was 3 subcutaneous vaccine injections at 4-week intervals and subcutaneous IL-2 treatment for 6 weeks starting at week 7. Sixty-six patients enrolled, of whom 39 received at least 1 vaccine; most observed toxicity was attributable to IL-2 not vaccine; best responses were 3% pathologic complete response, 5% partial response, 64% stable disease, and 28% disease progression. Median survival was 21.8 months (95% confidence interval 17.8 to 29.6). Significant postvaccination increases in IFN-gamma responses to autologous tumor were observed in 2/26 cases. Eighty-one percent of posttreatment subdermal delayed-type hypersensitivity tests (using nontransduced, irradiated autologous tumor cells) had biopsies demonstrating injection site lymphocytic infiltration. Post hoc comparison of the median survival of subjects whose biopsies had lymphocytic infiltration appears longer than in the 19% noninfiltrated (28.4 vs. 17.8 mo, P=0.045, two-sided log-rank test). The single arm design precludes conclusive comparison of objective response rates (not different here) or median survival (longer here) versus those of historical series using similar IL-2 schedules alone. Better outcomes could be logically associated to vaccine response (detectable lymphocytic infiltrates) or to random events that a single arm study design cannot address. This vaccine approach may merit further clinical development.

Vaccination of Acute Myeloid Leukemia Patients with Dendritic Cells Electroporated with mRNA Encoding the Wilms' Tumor Protein WT1: A Phase I/II Trial.

To date, Wilms' tumor protein (WT1) is acknowledged as a valuable target for active specific immunotherapy in several solid and hematological malignancies, such as leukemia. Preclinical data from our laboratory and that of Hans Stauss have already shown that WT1 RNA-electroporated dendritic cells (DC) stimulate WT1-specific T cells in vitro (Van Driessche A et al. Leukemia 2005; 19:1863–1871). Therefore, we started a phase I/II dose-escalation trial in which patients with acute myeloid leukemia (AML) in remission received intradermal injections with WT1 RNA-loaded DC. Feasibility, safety and immunogenicity of the vaccine were investigated. Seven patients received four biweekly DC vaccines. A delayed-type hypersensitivity (DTH) test was performed 2 weeks following the last vaccination. Patients underwent an apheresis and monocytes were isolated using CD14-labeled magnetic beads by CliniMACS. DC were generated in 6-day cultures in clinical-grade medium supplemented with serum, GM-CSF and IL-4 and maturated with PGE2 and TNF-a. Keyhole limpet hemocyanin (KLH) was added during maturation as a CD4+ helper antigen. Mature DC were harvested, electroporated with WT1 mRNA and used as vaccines. Patients were monitored for minimal residual disease (MRD) by analyzing WT1 RNA expression in peripheral blood by qRT-PCR. When the patient was HLA-A2+, tetramer staining was performed to detect WT1-specific CD8+ T cells. Before and after the vaccination cycle, peripheral blood was collected for immunomonitoring purposes. There was successful DC generation and vaccine production in all patients selected. No serious adverse events or toxicity was seen and all vaccinations were well tolerated. A decrease in WT1 RNA expression was observed during the course of the vaccination in 3/5 patients who had an increased WT1 mRNA level in peripheral blood at the start of DC vaccination. A vaccine-specific immune response was demonstrated in 7/7 patients by an in vivo DTH reaction both to KLH as well as to WT1. By tetramer analysis, detectable levels of WT1-specific CD8+ T cells could be demonstrated during the course of the vaccination both in the peripheral blood as well as in the expanded DTH-infiltrating T cells from the skin biopsies. Preliminary data from immunomonitoring in pre- and post-vaccination T cell samples from 3 patients show a mixed T helper (Th)1/Th2 response towards the KLH and the WT1 protein following vaccination. We conclude that vaccination of AML remission patients with WT1 RNA-loaded DC is feasible and safe. Furthermore, the vaccine elicits anti-vaccine T-cell responses in vivo and a decrease in WT1 RNA expression levels was observed during MRD monitoring in some vaccinated patients.

Increased Langerhans cell accumulation after mycobacterial stimuli

Aims:To evaluate the role of Langerhans cells (LCs) in the local activation of leprosy lesions. LCs, acting as tolerance inducers and immune stimuli, are dendritic cells recently implicated in cutaneous homeostasis. The role of LCs in the defence against mycobacterial infection remains poorly understood.Methods and results:The number and distribution of CD1a+ skin cells and HLA-DR and intercellular adhesion molecule (ICAM)-1 expression were analysed in leprosy skin lesions and in delayed-type hypersensitivity (DTH) tests. The results showed a high number of LCs in tuberculin and lepromin tests, in tuberculoid lesions and in the epidermis and dermis during type I and II reactions. In multibacillary lesions, however, the number of LCs was consistently low in comparison with other groups. Increased numbers of LCs were accompanied by marked HLA-DR and ICAM-1 expression, suggesting a strong relationship between these immunological events.Conclusions:CD1a+ cells are implicated in the local immunological events taking place after mycobacterial stimuli and may account for the local activation of all types of reactional episodes in leprosy.

Phase I/II clinical trial of sequential subcutaneous and intravenous delivery of dendritic cell vaccination for refractory multiple myeloma using patient‐specific tumour idiotype protein or idiotype (VDJ)‐derived class I‐restricted peptides

Fifteen multiple myeloma (MM) patients who had failed maintenance therapy after tandem autologous stem cell transplantation underwent anti-idiotype (Id) vaccination with dendritic cells (DCs). CD14(+)-derived DCs were loaded with the autologous Id as whole protein (=6) or Id-derived class I-restricted peptides (=9) and keyhole limpet hemocyanin (KLH). Vaccination consisted of three subcutaneous (sc) and two intravenous injections of increasing DC doses at 2 weeks interval. DC therapy was well tolerated. Most patients developed both humoral and T-cell responses to KLH, suggesting immunocompetence. Eight of 15 patients developed an Id-specific T-cell proliferative response, 8/15 increased interferon-gamma-secreting T cells and 4/15 showed an Id-positive delayed-type hypersensitivity test. Anti-Id cytotoxic T-lymphocyte precursors increased after DC vaccination in 2/2 evaluable patients. A more robust T-cell response was observed after sc DC injections and increased Id-specific T-cell proliferation was found up to 1 year after vaccination. VDJ-derived peptides were as effective as the whole protein in stimulating T-cell responses. Clinically, 7/15 patients have stable disease after a median follow-up of 26 months, one patient achieved durable partial remission after 40 months, and seven patients progressed. In conclusion, sc injections of cryopreserved Id-pulsed DCs were safe and, in contrast with intravenous administrations, induced anti-MM T-cell responses.

Antiinflammatory effects of a combined herbal preparation (RAH13) of Phellodendron amurense and Coptis chinensis in animal models of inflammation

In an attempt to develop an antiinflammatory herbal remedy that is as potent as current synthetic medicines, the cortex of Phellodendron amurense Rupr (Rutaceae) and the rhizomes of Coptis chinensis Franch (Ranunculaceae) were combined in a 2:1 ratio. This ratio was chosen based on in vitro experiments and traditional Asian medicine prescriptions. The combined ethanol extract, named RAH13, was evaluated for antiinflammatory properties using animal models of acute inflammation such as the croton oil-induced ear edema test and an acetic acid-induced capillary permeability test. Models of chronic inflammation were also tested using the cotton pellet test and a delayed-type hypersensitivity (DTH) test. Oral administration of RAH13 at a dose of 200 mg/kg showed in vivo antiinflammatory activity as potent as the effects associated with 100 mg/mL of celecoxib or 1 mg/kg of dexamethasone. These effects were seen in both acute and chronic inflammation models, suggesting that RAH13 may be effective in controlling some inflammation-related diseases.

A clinical trial of autologous formalin-fixed tumor vaccine (AFTV) for patients with glioblastoma multiforme

2085 Background: This clinical pilot study is to investigate the safety, feasibility, plus clinical response of autologous formalin- fixed tumor vaccine (ATFV) in primary glioblastoma multiforme (GBM) patients. Methods: Eleven (8 recurrent and 3 initially treated) primary GBM patients were evaluated. AFTV was prepared from formalin-fixed tumor tissue or paraffin-embedded tissue and premixed with original adjuvant materials. A delayed-type hypersensitivity test (DTH) was performed before and after each course of ATFV treatment that comprised 3 vaccinations at a 5-site intradermal inoculation. In addition, immunohistochemical analysis of MIB-1, p53, and MHC class-I complex was performed on the tumor tissue to analyze the difference in the response to the treatment. Results: The treatment was well tolerated with only local induration and low-grade fever. Among the 11 patients, the best responses were 1 complete remission, 2 partial response, 1 no change, and 7 progressive disease. In this series, the median survival period was 7 months from the initiation of the AFTV treatment, and 3 of the 4 responders survived for more than 20 months after AFTV inoculation. DTH reactions, immunohistological analysis of p53 and MHC class-I complex, and patient status may be useful to predict the efficacy of this therapy. Conclusion: This study demonstrated that AFTV treatment is safe, feasible, and potentially beneficial. Further clinical investigation is highly desirable in order to improve the outcome of GBM patients. No significant financial relationships to disclose.

Patient-specific vaccines derived from proliferating autologous tumor cell lines and dendritic cells: Results of a phase II trial in metastatic melanoma

8555 Background: Patient-specific vaccines utilizing proliferating tumor cells, or tumor stem cells, may be the ideal products for active specific immunotherapy. Methods: Eligible patients had recurrent or metastatic melanoma from which a cell line was established, expanded to 200 million cells, incubated with interferon-gamma, irradiated and cryopreserved. Autologous dendritic cells (DC) were derived from peripheral blood mononuclear cells cultured in IL-4 and GM-CSF. DC were incubated with the irradiated tumor cells, then cryopreserved in 20- million-cell aliquots, which were thawed, washed and suspended in 500 micrograms of GM-CSF for injection. Treatment consisted of s.c. injections weekly × 3, then monthly × 5 in a 2-stage phase II trial with two stratifications. Patients were characterized as having objectively measurable disease (OMD) or non-measurable disease (NMD). Plans were to enroll 30 to 80 patients: 15 to 40 with OMD, 15 to 40 with MD. Objectives were to determine safety, frequency of conversion of delayed type hypersensitivity (DTH) reactions to irradiated autologous tumor cells, objective response rate (ORR) using RECIST criteria, progression-free survival (PFS), overall survival (OS), and comparison to a historical control group. Results: Between January 2001 and April 2006, 55 patients were enrolled; 53 were eligible and evaluable. The 30 men and 23 women had a median age of 50 years; 15 had OMD and 38 NMD. Patients received an average of 7.4 vaccinations out of a possible 8. Treatment was well- tolerated. 25% had a positive tumor DTH test: 1 at baseline, 7 after 3 injections, 5 after 8 injections. ORR was 0/15. Follow up for the 39 surviving patients ranges from 7 to 67 months with a median of 30 months. Median PFS is 7.1 months with 24 patients remaining progression-free. Only 14 patients have died; median OS has not been reached. 5-year projected survival is 70%; 20 patients are alive 2.5 to 5.5 years from start of vaccine. OS is better than observed for 48 comparable patients that we treated previously in a trial with irradiated tumor cells without DC (p=0.016). Conclusion: This patient-specific vaccine approach is feasible, safe, associated with encouraging survival, and warrants further investigation. No significant financial relationships to disclose.

Clinical trial of autologous formalin‐fixed tumor vaccine for glioblastoma multiforme patients

A pilot study was performed to investigate the safety and feasibility of autologous formalin-fixed tumor vaccines (AFTV) and the clinical responses to these vaccines by glioblastoma multiforme (GBM) patients. Twelve primary GBM patients were recruited. Eight had recurrent disease while four had been treated for primary disease but retained a visible tumor mass. AFTV were prepared from formalin-fixed and/or paraffin-embedded tumor tissue obtained upon surgery and premixed with original adjuvant materials. The patients were given three five-site intradermal inoculations at weekly intervals. A delayed-type hypersensitivity test was performed before and after each vaccination. In addition, the tumor tissues were subjected to immunohistochemical analysis to determine whether MIB-1, p53, and major histocompatibility complex (MHC) class-I complex expression could predict the response to the treatment. The treatment was well tolerated, with only local erythema, induration, and low-grade fever being reported. Of the 12 patients, one showed a complete response, one showed a partial response, two showed minor responses, one had stable disease, and seven exhibited progressive disease. The median survival period was 10.7 months from the initiation of the AFTV treatment but three of the five responders survived for 20 months or more after AFTV inoculation. Low p53 and high MHC class-I expression by the tumor may help predict the efficacy of this therapy. Thus, the AFTV is safe and feasible, and could significantly improve the outcome of GBM. Further clinical investigations to confirm this are highly desirable.

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.

Mouse Strain and Injection Site are Crucial for Detecting Linked Suppression in Transplant Recipients by Trans-Vivo DTH Assay

Chemokine-driven accumulation of lymphocytes, mononuclear and polymorphonuclear proinflammatory cells in antigenic tissue sites is a key feature of several types of T-cell-dependent autoimmunity and transplant rejection pathology. It is now clear that the immune system expends considerable energy to control this process, exemplified by the sequential layers of regulatory cell input, both innate and adaptive, designed to prevent a classical Type IV or 'delayed-type' hypersensitivity (DTH) reaction from occurring in the visual field of the eye. Yet, despite an abundance of in vitro assays currently available to the human T-cell immunologist, none of them adequately models the human DTH response and its various control features. The theme of this article is that it is relatively easy to model the effector side of the human DTH response with xenogeneic adoptive transfer models. However, we show that in order to detect inhibition of a recall DTH in response to colocalized donor antigen (linked suppression)--a characteristic feature of peripheral tolerance to an organ transplant--both the challenge site and the immunocompetence of the mouse adoptive host are critical factors limiting the sensitivity of the trans-vivo DTH test.

Phase I study of tumor Ag-loaded IL-12 secreting semi-mature DC for the treatment of pediatric cancer

Cancer vaccines employing DC in their capacity as APC have been tolerated well and have shown some efficacy in clinical studies. IL-12, a cytokine critical for type 1 T-helper (Th1) lymphocyte and cytotoxic T-lymphocyte (CTL) differentiation, when released from a DC-based cancer vaccine, may support the generation of a cellular T-cell response. We applied tumor cell lysate plus keyhole limpet hemocyanin (KLH)-loaded and 48-h lipopolysaccharide (LPS) plus IFN-gamma-stimulated fully mature DC, which do not release IL-12, subcutaneously to eight patients, and maximally 6-h stimulated semi-mature (sm) DC, which are potent producers of IL-12, subcutaneously (n=6) or intranodally (n=8) as a cancer vaccine to patients suffering from advanced solid pediatric malignancies. No serious adverse events were observed following application of IL-12-releasing smDC. Following immunization the majority of patients responded positively to KLH in a delayed-type hypersensitivity (DTH) test. In addition, three of six intranodally treated patients responded to the tumor Ag in the DTH test. We conclude that treatment with a DC-based cancer vaccine enabled to release the immune regulatory cytokine IL-12 is safe and feasible and has the potential to induce a cellular immune response in pediatric cancer patients.