The soluble IL2-receptor (sIL2R) serum level, which indicates T-cell activation, analogously increased after each trAb application. Comparing the sIL2R level on day 1 after trAb application, the maximum sIL2R level was found after the third trAb application, indicating an ongoing and increasing cellular immune activation during trAb therapy. Figure 1 Serum levels (mean, +/-

SEM) of TNF-α (A), soluble IL-2R (B), and IL-6 (C) immediately before the first, second and third trAb application, and corresponding I-BET-762 cell line serum levels on day one and two dafter trAb therapy. Serum levels were measured by ELISA (Biosource, Fleurs, Belgium). * p < 0.05. HAMA was measured after trAb therapy in 7 of 9 patients. In all these patients, HAMA was significantly increased (above the threshold of 40 ng/ml), representing an immunological reaction (Table 4). Table 4 Restimulation and response Patient Increase of IFN-γ secreting T-lymphocytes HAMA

(ng/ml) Chemotherapy after trAb therapy Survival after trAb therapy (months) A + 801 – 1 B + 230 + 21 C – 30512 + 31 D – n.d. – 4 E + 7870 + 7 F – 50730 + 12 G + 2540 + 15 H AMN-107 concentration – 400 + 8 I + n.d. – 7 Increase of IFN-γ secreting T-lymphocytes compared to baseline values before therapy. HAMA = human anti-mouse antibody reaction (values measured 4 weeks after trAb therapy). Immunological anti-tumor reactivity All patients were restimulated 4 weeks after i.p.-application of trAb. Patients revealed a base value of 0.4% (mean) CD4+/CD8+ IFN-γ secreting T-lymphocytes in

PBMC before trAb-treatment. Five of nine patients showed an increase of IFN-γ secreting T-lymphocytes, reflecting 4-Aminobutyrate aminotransferase autologous anti-tumor reactivity (Figure 2). In these 5 patients, the number of tumor reactive T-lymphocytes increased from baseline value of 0.4% to 2.9% (mean) after trAb therapy and restimulation. All control experiments with unstimulated PBMC or PBMC incubated with allogeneic tumor cells showed no increase compared to the corresponding baseline values. In patient B, the IFN-γ secretion assay was performed twice after intradermal restimulation (Figure 3). Here, IFN-γ secreting T-lymphocytes increased from 0.4% before therapy to 2.8% after restimulation, followed by a value of 2.8% on day 110 after stimulation, indicating long-term immunity. This patient also had a substantial decrease of tumor markers (CA 125 decreased from 57.8 U/ml to 29.7 U/ml). Figure 2 Individual percentage values presenting the relative proportion of IFN-γ secreting T-lymphocytes in 10 × 10 6 PBMC after stimulation with 5 × 10 5 autologous tumor cells before and 3–4 weeks after trAb therapy using the Miltenyi IFN-γ secretion assay. Figure 3 Analysis of tumor reactive IFN-γ secreting CD4+/CD8+ T lymphocytes before trAb therapy and on day 39 and 110 after boost stimulation in patient B using the Miltenyi IFN-γ secretion assay.

The level of aldehydes did not differ (P > 0.05) between urine samples of T-CD and HC. Compared to faecal samples of HC, some alcohols (e.g., 1-octen-3-ol, ethanol and 1-propanol) were present at higher level in T-CD. Median values of alkane and alkene did not significantly (P > 0.05) differ between T-CD and HC. Overall, faecal samples of T-CD showed the lowest levels of aromatic organic compounds. The median value of total short chain fatty acids (SCFA) was significantly

(P < 0.05) higher in faecal samples HC compared to T-CD. Major differences were found for isocaproic, butyric and propanoic acids (P < 0.038, 0.021, and 0.012, respectively). On the contrary, acetic acid was higher in T-CD compared to HC samples. The Angiogenesis inhibitor differences of the metabolomes between faecal or urine samples of T-CD and HC was highlighted through CAP analysis which considered only significantly different compounds (Figure 7A and 7B). Variables appearing with negative values represent bins whose values decreased in T-CD compared to HC samples. On the check details contrary, variables represented with bars pointing to the right indicate bins whose values were the highest in T-CD samples. Table 3 Median values and ranges of the concentration (ppm) of volatile organic compounds (VOC) of faecal and urine samples from treated celiac disease (T-CD) children and non-celiac children (HC) as determined by gas-chromatography mass spectrometry/solid-phase

microextraction (GC-MS/SPME) analysis Chemical class Treated celiac disease (T-CD)children Non-celiac children (HC)   Faeces Urines Faeces Urines   Median Range Median Range Median Range Median Range Esters 20.31b 0 – 846.97 0.47c 0 – 40.00 47.73a 1.83 – 496.83 0.99c 0 – 8.05 Sulfur compounds 214.83b 0 – 890.86 1.46c 0 – 25.44 387.07a 0 – 499.88 3.49c 0 – 63.67 Ketones 90.88b 0 – 2402.50 54.01c 0 – 295.03 112.83a 0 – 416.20 64.49c 0 – 458.78 Hydrocarbons

16.69b 0 – 1327.15 4.25c 0 crotamiton – 67.07 119.13a 0.22 – 635.25 3.14c 0.15 – 62.56 Aldehydes 17.59c 0 – 512.28 64.31a 0.34 – 166.31 37.46b 2.08 – 365.25 73.37a 0.50 – 199.56 Alcohols 230.14a 0 – 2311.29 2.25c 0 – 17.5 122.56b 0 – 934.22 2.14c 0 – 34.96 Alkane 6.73a 0 – 653.61 0.3b 0.05 – 1.57 9.37a 0 – 432.74 0.43b 0 – 1.47 Alkene 0a 0 – 32.51 0a 0 0a 0 – 31.99 0a 0 Aromatic organic compounds 178.24b 0 – 143.67 2.10c 0.04 – 28.16 480.20a 233.74 – 993.94 2.78c 0 – 16.30 Heptane 23.01a 0 – 837.50 0c 0 – 1.37 26.37a 0 – 65.75 0.34b 0 – 2.37 Short chain fatty acids (SCFA) 21.64a 0 – 1438.28 3b 0.08 – 31.14 27.85a 0 – 1037.50 3.82b 1.44 – 24.87 Data are the means of three independent experiments (n = 3) for each children. a-cMeans within a row with different superscript letters are significantly different (P < 0.05).

pastoris extracellular β-D-galactosidase production for a thermostable enzyme from Alicyclobacillus acidocaldarius IWP-2 mw [23]. There are several examples of cold active β-D-galactosidases isolated from Pseudoalteromonas

strains [5, 10, 11] and Arthrobacter strains [7–9, 12, 13] with molecular mass above 110 kDa of monomer and forming an active enzyme of over 300 kDa. Most of them belong to the family 42 β-D-galactosidases. However, the β-D-galactosidase belonging to family 2 obtained from the Antarctic Arthrobacter isolate appears to be one of the most cold-active enzymes characterized to date [8]. All of the known cold-adapted β-D-galactosidases, except two of them isolated from Planococcus sp. strains [4, 14] and from

Arthrobacter sp. 32c (this study), form very large oligomers and therefore are of minor interest in industrial application probably because of many problems in effective overexpression. The β-D-galactosidases isolated from psychrophilic Planococcus sp. strains have low molecular weight of about 75 kDa of monomer and about 155 kDa of native protein. The β-D-galactosidase isolated from Planococcus sp. L4 is particularly thermolabile, loosing its activity within only 10 min at 45°C [14] and therefore larger scale production of this enzyme by recombinant yeast strains PKC inhibitor cultivated at 30°C might be economically not feasible. Only the β-D-galactosidase from Planococcus sp. isolate SOS orange [4] displays interesting activity and might be considered in biotechnological production on a larger scale. In comparison with known β-D-galactosidases, the Arthrobacter Baf-A1 chemical structure sp. 32c β-D-galactosidase is a protein with a relatively low molecular weight.

Molecular sieving revealed that the active enzyme is a trimmer with a molecular weight of approximately 195 ± 5 kDa. Relatively low molecular weight of the protein did not interfere with extracellular production of the protein by P. pastoris. Therefore the constructed recombinant strains of P. pastoris may serve to produce the protein extracellularly with high efficiency and in a cheap way. The calculated production cost of 1 mg of purified β-D-galactosidase was estimated at 0.03 €. The same Pichia pastoris expression systems had been unsuccessfully used for extracellular expression of previously reported β-D-galactosidase from Pseudoalteromonas sp. 22b [10, 11]. This enzyme is much bigger than Arthrobacter sp. 32c β-D-galactosidase and forms a tetramer of approximately 490 kDa. It is worth noting that we have tried to secrete this enzyme with three different secretion signals (α-factor from Saccharomyces cerevisiae, glucoamylase STA2 from Saccharomyces diastaticus or phosphatase PHO5 from S. cerevisiae) with no success. It seems that the molecular mass of the desired recombinant protein is limited to extracellular production by P. pastoris host, whereas the used secretion signal is without any influence.