Analysis of All 34 Exons of the SPINK5 Gene in Japanese Atopic Dermatitis Patients.

Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a large multidomain serine protease inhibitor that is expressed in epidermal keratinocytes. Nonsense mutations of the SPINK5 gene, which codes for LEKTI, cause Netherton syndrome, which is characterized by hair abnormality, ichthyosis, and atopy. A single nucleotide polymorphism (SNP) of SPINK5, p.K420E, is reported to be associated with the pathogenesis of atopic dermatitis (AD). We studied all 34 exons of the SPINK5 gene in Japanese 57 AD patients and 50 normal healthy controls. We detected nine nonsynonymous variants, including p.K420E; these variants had already been registered in the SNP database. Among them, p.R654H (n=1) was found as a heterozygous mutation in the AD patients, but not in the control. No new mutation was detected. We next compared the data of the AD patients with data from the Human Genetic Variation Database provided by Kyoto University; a significant difference was found in the frequency of the p.S368N genotype distribution. PolyPhen-2 and SIFT, two algorithms for predicting the functional effects of amino acid substitutions, showed significant scores for p.R654H. Therefore, R654H might be a risk factor for epidermal barrier dysfunction in some Japanese AD patients.

Addition Rate Constants of Phosphorus- and Carbon-Centered Radicals to Double Bond of Monomers as Studied by a Pulsed Electron Paramagnetic Resonance Method.

Addition reaction of a radical to the double bond of a monomer, which is important at early stage of photopolymerization, has been studied by time-resolved (TR-) and pulsed electron paramagnetic resonance (EPR) spectroscopic methods. Reactions of one phosphorus-centered and three carbon-centered radicals attacking to several monomers have been employed. Intermediate radicals were identified by analyzing the recorded TR-EPR spectra, and the reaction rate constants were determined by the electron spin-echo detection method proposed by Weber and Turro [J. Phys. Chem. A, 2003, 107 (18), 3326 - 3334]. The quantum chemical calculation shows that the rate constants for the addition reactions are well-explained by introducing two factors of "enthalpy effect" and "polar effect" to control the activation barrier height. It was observed that the rate constants of the phosphorus-centered radical were larger than those of carbon-centered radicals for some monomers. The difference in the rate constants was argued on the basis of frequency factor and activation barrier both of which are influenced by an atom of radical center.

Vaccination with NY-ESO-1 overlapping peptides mixed with Picibanil OK-432 and montanide ISA-51 in patients with cancers expressing the NY-ESO-1 antigen.

We conducted a clinical trial of an NY-ESO-1 cancer vaccine using 4 synthetic overlapping long peptides (OLP; peptides #1, 79-108; #2, 100-129; #3, 121-150; and #4, 142-173) that include a highly immunogenic region of the NY-ESO-1 molecule. Nine patients were immunized with 0.25 mg each of three 30-mer and a 32-mer long NY-ESO-1 OLP mixed with 0.2 KE Picibanil OK-432 and 1.25 mL Montanide ISA-51. The primary endpoints of this study were safety and NY-ESO-1 immune responses. Five to 18 injections of the NY-ESO-1 OLP vaccine were well tolerated. Vaccine-related adverse events observed were fever and injection site reaction (grade 1 and 2). Two patients showed stable disease after vaccination. An NY-ESO-1-specific humoral immune response was observed in all patients and an antibody against peptide #3 (121-150) was detected firstly and strongly after vaccination. NY-ESO-1 CD4 and CD8 T-cell responses were elicited in these patients and their epitopes were identified. Using a multifunctional cytokine assay, the number of single or double cytokine-producing cells was increased in NY-ESO-1-specific CD4 and CD8 T cells after vaccination. Multiple cytokine-producing cells were observed in PD-1 (-) and PD-1 (+) CD4 T cells. In conclusion, our study indicated that the NY-ESO-1 OLP vaccine mixed with Picibanil OK-432 and Montanide ISA-51 was well tolerated and elicited NY-ESO-1-specific humoral and CD4 and CD8 T-cell responses in immunized patients.

Photochemistry of the ozone-water complex in cryogenic neon, argon, and krypton matrixes.

The photochemistry of ozone-water complexes and the wavelength dependence of the reactions were studied by matrix isolation FTIR spectrometry in neon, argon, and krypton matrixes. Hydrogen peroxide was formed upon the irradiation of UV light below 355 nm. Quantitative analyses of the reactant and product were performed to evaluate the matrix cage effect of the photoreaction. In argon and krypton matrixes, a bimolecular O((1)D) + H2O → H2O2 reaction was found to occur to form hydrogen peroxide, where the O((1)D) atom generated by the photolysis of ozone diffused in the cryogenic solids to encounter water. In a neon matrix, hydrogen peroxide was generated through intracage photoreaction of the ozone-water complex, indicating that a neon matrix medium is most appropriate to study the photochemistry of the ozone-water complex.

Analysis of low-lying gerade Rydberg states of acetylene using two-photon resonance fluorescence excitation spectroscopy.

The np gerade Rydberg states of acetylene were analyzed using two-photon resonance fluorescence excitation spectroscopy in the 72,000-93,000 cm(-1) energy region. The npπ(1)Σ(g)(+) and npπ(1)Δ(g) Rydberg series (n = 3-5) were identified in the fluorescence excitation spectrum measured by monitoring the C(2) d(3)Π(g)-a(3)Π(u) Swan system. Some vibronic bands were assigned to the npπ(1)Δ(g)-X̃(1)Σ(g)(+) transition on the basis of rotational analysis. The 5pσ(1)Π(g) state was observed, which is the first such observation in an npσ(1)Π(g) series. Rotational analysis of the 5pσ(1)Π(g)-X̃(1)Σ(g)(+) transition showed e/f-symmetry dependent predissociation of acetylene in the 5pσ(1)Π(g) state. The 0(0)(0) band of the deuterated acetylene (C(2)D(2)) 4pπ(1)Σ(g)(+)-X̃(1)Σ(g)(+) transition exhibits an atypical structure, which was satisfactorily reproduced by a simple model of quantum interference between the discrete and quasi-continuum states. The predissociative lifetimes of the npπgerade Rydberg states were estimated from the spectral profiles. The predissociation mechanism of acetylene in the Rydberg states is discussed.

Regulatory T cells in γ irradiation-induced immune suppression.

Sublethal total body γ irradiation (TBI) of mammals causes generalized immunosuppression, in part by induction of lymphocyte apoptosis. Here, we provide evidence that a part of this immune suppression may be attributable to dysfunction of immune regulation. We investigated the effects of sublethal TBI on T cell memory responses to gain insight into the potential for loss of vaccine immunity following such exposure. We show that in mice primed to an MHC class I alloantigen, the accelerated graft rejection T memory response is specifically lost several weeks following TBI, whereas identically treated naïve mice at the same time point had completely recovered normal rejection kinetics. Depletion in vivo with anti-CD4 or anti-CD25 showed that the mechanism involved cells consistent with a regulatory T cell (T reg) phenotype. The loss of the T memory response following TBI was associated with a relative increase of CD4+CD25+ Foxp3+ expressing T regs, as compared to the CD8+ T effector cells requisite for skin graft rejection. The radiation-induced T memory suppression was shown to be antigen-specific in that a third party ipsilateral graft rejected with normal kinetics. Remarkably, following the eventual rejection of the first MHC class I disparate skin graft, the suppressive environment was maintained, with markedly prolonged survival of a second identical allograft. These findings have potential importance as regards the immunologic status of T memory responses in victims of ionizing radiation exposure and apoptosis-inducing therapies.

Hydroa vacciniforme is associated with increased numbers of Epstein-Barr virus-infected γδT cells.

Hydroa vacciniforme (HV) is a rare photosensitivity disorder of childhood associated with Epstein-Barr virus (EBV)(+) T-cell infiltration. We have summarized clinical manifestations of HV, and analyzed EBV(+) T-cell subsets as well as EBV DNA load in lymphocyte fractions, in comparison with hypersensitivity to mosquito bites (HMB), an EBV-associated T/natural killer (NK) lymphoproliferative disorder. We found that 31 of 33 (93.9%) HV lesions were composed of EBV(+) T cells and reactive EBV(-) cytotoxic T cells, without significant CD56(+) cell infiltration, whereas many CD56(+) cells were present in 8 of 9 (88.9%) HMB lesions. Of 13 (20.6%) HMB patients with or without HV, 12 (92.3%) showed increased percentages (>32%) of NK cells in the peripheral blood, whereas in the 16 patients with HV alone, 14 (87.5%) showed no increase. Of the 11 HV patients, 10 (90.9%) had increased percentages (>5%) of circulating γδT cells, with a mean value of 15.7 ± 2.9%, and the γδT-cell fractions contained larger amounts of EBV DNA than non-γδT-cell fractions. A triple-labeling method revealed that all three HV patients examined had increased percentages of EBER(+), T-cell receptor (TCR)γδ(+), and TCRαß(-) cells. Our observations indicate that HV is associated with increased numbers of EBV(+) γδT cells, whereas HMB is associated with EBV(+) NK cells.

A phase I study of vaccination with NY-ESO-1f peptide mixed with Picibanil OK-432 and Montanide ISA-51 in patients with cancers expressing the NY-ESO-1 antigen.

We conducted a phase I clinical trial of a cancer vaccine using a 20-mer NY-ESO-1f peptide (NY-ESO-1 91-110) that includes multiple epitopes recognized by antibodies, and CD4 and CD8 T cells. Ten patients were immunized with 600 µg of NY-ESO-1f peptide mixed with 0.2 KE Picibanil OK-432 and 1.25 ml Montanide ISA-51. Primary end points of the study were safety and immune response. Subcutaneous injection of the NY-ESO-1f peptide vaccine was well tolerated. Vaccine-related adverse events observed were fever (Grade 1), injection-site reaction (Grade 1 or 2) and induration (Grade 2). Vaccination with the NY-ESO-1f peptide resulted in an increase or induction of NY-ESO-1 antibody responses in nine of ten patients. The sera reacted with recombinant NY-ESO-1 whole protein as well as the NY-ESO-1f peptide. An increase in CD4 and CD8 T cell responses was observed in nine of ten patients. Vaccine-induced CD4 and CD8 T cells responded to NY-ESO-1 91-108 in all patients with various HLA types with a less frequent response to neighboring peptides. The findings indicate that the 20-mer NY-ESO-1f peptide includes multiple epitopes recognized by CD4 and CD8 T cells with distinct specificity. Of ten patients, two with lung cancer and one with esophageal cancer showed stable disease. Our study shows that the NY-ESO-1f peptide vaccine was well tolerated and elicited humoral, CD4 and CD8 T cell responses in immunized patients.

Markedly elevated serum levels of calcium-binding S100A8/A9 proteins in psoriatic arthritis are due to activated monocytes/macrophages.

BACKGROUND: Serum levels of S100A8/A9 may correlate with disease activity in psoriasis. OBJECTIVE: We sought to elucidate the association of serum levels of S100A8/A9 heterodimers with the clinical subtypes of psoriasis and the major cell source. METHODS: Serum samples were collected from patients with psoriasis vulgaris (n = 30), psoriatic arthritis (PA) (n = 16), pustular psoriasis (n = 24), and atopic dermatitis (n = 14) and from healthy control subjects (n = 21). Serum concentrations of S100A8/A9 were measured, and the expression levels were examined in psoriatic lesions. The messenger RNA levels were quantified in circulating monocytes and neutrophils. RESULTS: Serum levels of S100A8/A9 were significantly increased in all subtypes of psoriasis as compared with healthy controls and atopic dermatitis. Among the psoriatic subtypes, PA and pustular psoriasis showed remarkably high concentrations of S100A8/A9 heterodimers. The higher serum levels were associated with the presence of articular symptoms, but not significantly correlated with body surface areas of psoriatic lesions. S100A8 was expressed by both keratinocytes and infiltrating mononuclear cells, whereas S100A9 was predominantly expressed by neutrophils. The expression levels of S100A8 and S100A9 messenger RNA in monocytes were increased by approximately 2.25- and 1.91-fold in PA, respectively, whereas no significant increase was observed in psoriasis vulgaris and pustular psoriasis. LIMITATIONS: Difficulty in acquisition of clinical and laboratory samples in untreated patients, and of a sufficient number of subjects, were limitations. CONCLUSIONS: Although serum levels of S100A8/A9 were increased in all types of psoriasis examined, patients with PA had higher levels of S100A8/A9, probably because of an activated monocyte/macrophage system.

Production of proinflammatory cytokines without invocation of cytotoxic effects by an Epstein-Barr virus-infected natural killer cell line established from a patient with hypersensitivity to mosquito bites.

OBJECTIVE: Cumulative evidence supports that Epstein-Barr virus (EBV)-infected natural killer (NK) cells induce severe systemic and cutaneous inflammation in patients with hypersensitivity to mosquito bites (HMB). In order to understand the pathogenesis of HMB, we established an EBV-infected cell line and characterized the cytological profiles. MATERIALS AND METHODS: A novel EBV-infected NK-cell line, designated NKED, was established from a patient with HMB and used for the present study along with two other NK-cell lines, KAI3 and KHYG-1. RESULTS: NKED expressed the latency II-related transcripts. NKED cells were positive for CD2 and CD161 antigens, and negative for CD3, CD16, CD34, CD56, and T-cell receptor α/ß and γ/δ antigens. Although NKED cells contained several cytotoxic molecules, the cells had an extremely poor cytotoxic activity. The majority of NKED cells were negative for perforin, major histocompatibility complex class I-restricted NK-cell receptors, CD94 and KIR2D, and an activating receptor, NKG2D. NKED cells, however, secreted higher levels of tumor necrosis factor-α. Stimulation with phorbol 12-myristate 13-acetate or tumor necrosis factor-α induced expression of BZLF1 messenger RNA in the NKED and KAI3 cells, indicating the transition from the latent- to the lytic-cycle infection. CONCLUSIONS: These data suggested that NKED cells revealed a very low cytotoxic effect probably because of the low expression levels of perforin, but had the ability to release proinflammatory cytokines. NKED cells did not reflect the characteristics of HMB, as they were different from pathogenic NK cells proliferating in the HMB patient, but the difference indicated that pathogenic NK cells could change their character in the presence of interleukin-2.

Infrared spectroscopy and quantum chemical calculations of OH-(H2O)n complexes.

Infrared spectra of OH-(H2O)n (n = 1, 2) isolated in solid Ne were measured by FT-IR spectroscopy. Complexes of OH-(H2O)n were prepared by vacuum ultraviolet (VUV) photolysis of water clusters, and the OH radical stretch and HOH bending vibrations of OH-H2O and OH-(H2O)2 complexes were identified with the aid of quantum chemical calculations. Observation of the recombination reaction OH-H2O + H --> (H2O)2 under dark conditions provides undisputed evidence for our spectroscopic assignment. Quantum chemical calculations predict the cyclic structure to be the most stable for OH-(H2O)2 and OH-(H2O)3. The strength of the hydrogen bond within OH-(H2O)n depends on cluster size.

Tracing of the molecular remnants of herpes virus infections in necrotic skin tissue.

We have established an assay system to detect herpesvirus-derived transcripts in lesional crusts. Fifteen patients with herpes simplex (HS), 21 with herpes zoster (HZ), 2 with varicella, and 20 with irrelevant diseases were enrolled in the present study. Total RNA was extracted from crusts or scales, and converted to cDNA. Virus-encoded transcripts were amplified using reverse transcriptase (RT)-PCR. Housekeeping gene transcripts such as beta2-microglobulin (beta2-MG) and beta-actin (beta-actin) mRNA were also examined, and an efficient preservative condition of the crusts was determined. With extracted RNAs, beta2-MG and beta-actin mRNA were successfully amplified in all crust samples. Herpes simplex virus (HSV)-specific, lytic cycle-related transcript, UL30 mRNA was detected in all 15 HS samples, including 13 samples of HSV-1- and 2 of HSV-2-encoded UL30 mRNA, respectively. Of 23 samples, including 21 HZ and 2 varicella cases, varicella zoster virus (VZV)-specific, lytic cycle-related transcript, ORF40 mRNA was detected in 22 samples. In a control group, no UL30 and ORF40 mRNA were detected. Crust samples that had been stored without any pretreatment or preservative for 6 months at room temperature (RT) were available for the present assay. When compared with the freshly obtained materials, the amount of beta2-MG mRNA was reduced to 51% in the stored samples covered with adhesive tape, to 48% in a sample left at R.T. without any treatment, and to 1.2% in the samples stocked in saline for 5 days. Herpes virus- and host-derived transcripts contained in crusts can be detected by RT-PCR amplification. Crusts or dry epidermal necrosis with inflammatory cells may provide beneficial diagnostic information.

Karyotypic analysis of bone marrow cells in pyodermic lesions associated with myelodysplastic syndrome.

BACKGROUND: Recalcitrant pyodermic lesions and neutrophilic dermatoses are often associated with subclinical myelodysplastic syndrome (MDS). In this case series, we assessed the diagnostic importance of karyotypic analysis of bone marrow cells in 4 patients with MDS-associated pyodermic eruptions treated at our university hospital. Karyotypic analysis was performed in bone marrow cells and peripheral blood lymphocytes obtained. Serum levels of granulocyte colony-stimulating factor were measured. OBSERVATIONS: Four patients with pyodermic eruptions or neutrophilic dermatosis had chromosomal abnormalities in bone marrow cells, including del(20)(q11;q13.3) in 2 patients, trisomy 8 in 1 patient, and t(11;22)(q23;q11) in 1 patient. Three patients without morphologic findings suggestive of MDS were diagnosed as having refractory anemia. One female patient had refractory anemia with ringed sideroblasts associated with del(20). Two patients with refractory anemia had a normal karyotype in peripheral blood lymphocytes. Two patients with elevated serum levels of granulocyte colony-stimulating factor had more active or widespread cutaneous diseases. CONCLUSIONS: Karyotypic analysis of bone marrow cells, but not of peripheral blood lymphocytes, is essential in proving a diagnosis of MDS-associated pyodermic lesions. The overexpression of granulocyte colony-stimulating factor, which may compensate for impaired hematopoiesis in patients with MDS, seems to be a key cytokine leading to neutrophilic infiltration.

Induction of immune response against NY-ESO-1 by CHP-NY-ESO-1 vaccination and immune regulation in a melanoma patient.

BACKGROUND: NY-ESO-1 is a cancer/testis antigen highly immunogenic in cancer patients. Cholesterol-bearing hydrophobized pullulan (CHP) is a nanoparticle-forming antigen-delivery vehicle and CHP complexed with NY-ESO-1 protein (CHP-NY-ESO-1) efficiently activates CD4 and CD8 T cells in vitro. AIM: In this study we report on a 50-year-old male melanoma patient with multiple skin and organ metastases (T4N3M1c) who was vaccinated with CHP-NY-ESO-1 at biweekly intervals and who had an unusual disease course. We characterized in this patient humoral and cellular immune responses, immune regulatory cells, and cytokine profiles in the peripheral blood and at local tumor sites. RESULTS: Ten days after the second CHP-NY-ESO-1 vaccination (day 25), blisters appeared on the skin at the metastatic lesions associated with inflammatory changes. A skin biopsy showed the presence of many NY-ESO-1-expressing apoptotic melanoma cells as determined by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) test. However, the tumors continued to grow, and the patient died of pulmonary failure due to multiple metastases on day 48. Serum antibody responses were detected after the second CHP-NY-ESO-1 vaccination and antibody titer increased with subsequent vaccinations. Th1 dependent IgG1 was the predominant immunoglobulin subtype. Both, NY-ESO-1-specific CD4 and CD8 T cell responses were detected in PBMC by IFN-gamma secretion assays. After CHP-NY-ESO-1 vaccination a slight decrease in CD4(+)CD25(+)Foxp3(+) Tregs was observed in PBMC but significantly increased numbers of CD4(+)CD25(+)Foxp3(+) Tregs and CD68(+) immunoregulatory macrophages were detected at the local tumor sites. CD4(+)CD25(+)Foxp3(+) Tregs were also increased in the blister fluid. Cytokines in the serum suggested a polarization towards a Th1 pattern in the PBMC and those in the blister fluid suggested a Th2-type response at the tumor site. CONCLUSIONS: Our observations indicate induction of specific humoral and cellular immune responses against NY-ESO-1 after CHP-NY-ESO-1 vaccination in a melanoma patient. The concomitant appearance of regulatory T cells and of immune regulatory macrophages and cytokines at the local tumor sites in this patient may explain immune escape.

Prospective study of clinical symptoms and skin test reactions in medical students exposed to formaldehyde gas.

Previous investigators have reported the occurrence of both allergic and non-allergic systemic complications due to exposure to formaldehyde gas. However, little is known about the pathogenic link between formaldehyde-induced clinical symptoms and patch test results, or about the long-term effects of formaldehyde exposure. In the present study, a questionnaire was administered to 143 medical students, and 60 of them were tested by patch test for formaldehyde at the beginning and end of a human anatomy laboratory course. Another group of 76 students who had finished the course 2-4 years previously were administered another questionnaire, and the patch test was carried out on 58 of them. The frequencies of skin irritation, eye soreness, lacrimation, eye fatigue, rhinorrhea, throat irritation, general fatigue and mood swings increased after repeated exposure. Two (3.3%) of 60 students became positive to 1% formaldehyde at the end of the anatomy course (one male with allergic hand dermatitis due to direct contact with formaldehyde, and one female with an atopic background with unbearable physical symptoms) while the remaining 58 showed a negative reaction throughout the study period. The vast majority of students complained of various non-allergic, physical symptoms, and recovered from such symptoms without subsequent complications. No progression to multiple chemical sensitivity was found. Students with an episode of atopic dermatitis and allergic rhinitis were susceptible to formaldehyde exposure, and developed mucocutaneous symptoms, probably due to the impaired barrier function and remodeling of the skin and mucosa.

T cell immunomonitoring and tumor responses in patients immunized with a complex of cholesterol-bearing hydrophobized pullulan (CHP) and NY-ESO-1 protein.

We recently showed that vaccination with a complex of cholesterol-bearing hydrophobized pullulan and NY-ESO-1 protein (CHP-NY-ESO-1) elicited antibody responses in 9 of 9 patients vaccinated in a clinical trial. In this study, we performed T cell immunomonitoring and analyzed tumor responses in these patients. To evaluate CD4 and CD8 T cell responses, an IFN-gamma secretion assay was used. The assay showed low background and was sensitive for detecting antigen-specific T cells. An increase in the CD4 T cell response was observed in 2 of 2 initially sero-positive and 5 of 7 initially sero-negative patients after vaccination. An increase in the CD8 T cell response was also observed in 2 of 2 sero-positive and 5 of 7 sero-negative patients after vaccination. Analysis of peptides recognized by CD4 and CD8 T cells revealed two dominant NY-ESO-1 regions, 73-114 and 121-144. Tumor responses were observed in 3 esophageal cancer patients and a malignant melanoma patient. In 3 of 4 prostate cancer patients, prostate-specific antigen (PSA) values stabilized during the course of vaccination. The use of whole protein, containing multiple CD4 and CD8 epitopes, may be beneficial for cancer vaccines to prevent tumors from evading the immune response.

Infrared spectroscopy of ozone-water complex in a neon matrix.

Matrix isolation infrared spectroscopy has been applied to study an ozone-water complex of atmospheric interest. The complex was identified in the spectral region of three normal modes of ozone and water. Ab initio calculation at MP4(SDQ), QCISD, and CCSD(T) levels indicates the existence of only one stable conformer, which accords with the present experimental result. This conformer belongs to the Cs symmetry group where two molecular planes of ozone and water are perpendicular to the Cs symmetry plane. The binding energy was calculated to be 1.89 kcal/mol at the CCSD(T)/6-311++G(3df,3pd)//CCSD(T)/6-311++G(d,p) level of theory. The formation constant and atmospheric abundance of the ozone-water complex are estimated using the thermodynamic and spectroscopic data obtained.

Bovine apolipoprotein B-100 is a dominant immunogen in therapeutic cell populations cultured in fetal calf serum in mice and humans.

Recent studies have demonstrated that cell populations intended for therapeutic purposes that are cultured in heterologous animal products can acquire xenoantigens, potentially limiting their utility. In investigations of the immune response to murine embryonic stem cells, we found that a strong antibody response was generated after the second infusion. Both polyclonal and monoclonal antibody responses, derived from immunized mice, were found to be specific for bovine apolipoprotein B-100, which binds to abundant low-density lipoprotein receptors on the cell surface and is internalized. Here we show that in the majority of patients administered 3 different types of cell-based therapies using cells grown in fetal calf serum-containing media, an antibody response to bovine apolipoprotein B-100 develops after the second infusion and is the dominant specificity. The known and potential clinical effects of such antibodies are discussed.

Antibody response against NY-ESO-1 in CHP-NY-ESO-1 vaccinated patients.

NY-ESO-1 specific humoral responses are frequently observed in patients with various types of NY-ESO-1 antigen expressing tumors. In a large proportion of NY-ESO-1 antibody-positive patients of NY-ESO-1-specific CD8 T-cells can also be detected suggesting that monitoring of the NY-ESO-1 specific humoral immune response may be a relevant and more practical surrogate for estimating the overall immune response against NY-ESO-1 in clinical vaccine studies. We have immunized 9 cancer patients with full length NY-ESO-1 protein formulated with cholesterol-bearing hydrophobized pullulan (CHP-NY-ESO-1) and investigated the humoral immune responses against NY-ESO-1. Seven patients were NY-ESO-1 antibody-negative and 2 patients were positive prior to vaccination. Vaccination with CHP-NY-ESO-1 resulted in the induction or increase of NY-ESO-1 antibody responses in all 9 patients immunized. Epitope analysis revealed 5 regions in the NY-ESO-1 protein molecule that were recognized by antibodies induced after vaccination. The 5 regions were also recognized by antibodies present in nonvaccinated, NY-ESO-1 antibody-positive cancer patients. A peptide spanning amino acids 91-108 was recognized in 6 out of 9 vaccinated patients and in 8 out of 9 nonvaccinated, sero-positive patients, being the most dominant antigenic epitope in NY-ESO-1 for antibody recognition in cancer patients. In conclusion, we showed that CHP-NY-ESO-1 protein vaccination had a potent activity for inducing humoral immune responses against NY-ESO-1 antigen in cancer patients. The antigenic epitopes recognized by antibodies in the vaccinated patients were similar to those recognized in cancer patients with spontaneous humoral immunity against NY-ESO-1.