68Ga-PSMA 11 ligand PET imaging in patients with biochemical recurrence after radical prostatectomy - diagnostic performance and impact on therapeutic decision-making.

OBJECTIVE: To evaluate the diagnostic performance of [68Ga]Ga-PSMAHBED-CC conjugate 11 positron emission tomography (PSMA-PET) in the early detection of metastases in patients with biochemical recurrence (BCR) after radical prostatectomy (RP) for clinically non-metastatic prostate cancer, to compare it to CT/MRI alone and to assess its impact on further therapeutic decisions. MATERIAL AND METHODS: We retrospectively assessed 117 consecutive hormone-naïve BCR patients who had 68Ga-PSMA 11 PET/CT (n = 46) or PET/MRI (n = 71) between May 2014 and January 2017. BCR was defined as two PSA rises above 0.2 ng/ml. Two dedicated uro-oncological imaging experts (radiology/nuclear medicine) reviewed separately all images. All results were presented in a blinded sequential fashion to a multidisciplinary tumorboard in order to assess the influence of PSMA-PET imaging on decision-making. RESULTS: The median time from RP to BCR was 36 months (IQR 16-72). Overall, 69 (59%) patients received postoperative radiotherapy. Median PSA level at the time of imaging was 1.04 ng/ml (IQR 0.58-1.87). PSMA-positive lesions were detected in 100 (85.5%) patients. Detection rates were 65% for a PSA value of 0.2 to <0.5 ng/ml, 85.7% for 0.5 to <1, 85.7% for 1 to <2 and 100% for ≥2. PSMA-positive lesions could be confirmed by either histology (16%), PSA decrease in metastasis-directed radiotherapy (45%) or additional information in diffusion-weighted imaging when PET/MRI was performed (18%) in 79% of patients. PSMA-PET detected lesions in 67 patients (57.3%) who had no suspicious correlates according to the RECIST 1.1 criteria on MRI or CT. PSMA-PET changed therapeutic decisions in 74.6% of these 67 patients (p < 0.001), with 86% of them being considered for metastases-directed therapies. CONCLUSIONS: We confirm the high performance of PSMA-PET imaging for the detection of disease recurrence sites in patients with BCR after RP, even at relatively low PSA levels. Moreover, it adds significant information to standard CT/MRI, changing treatment strategies in a significant number of patients.

[Osteosarcoma of the prostate after radiotherapy]. / Osteosarkom der Prostata nach Strahlentherapie.

Osteosarcoma of the prostate is a rare finding. These tumours usually occur years after radiotherapy for prostate cancer. We report the case of a 74-year-old man with prostate cancer who had been treated with radiotherapy and androgen deprivation therapy. The man presented with urinary retention and his prostate was transurethrally resected. The histopathological investigation showed formations of a poorly differentiated osteosarcoma in the prostate. Because of serious comorbidities we decided to withhold chemotherapy considering its potential side effects. The man died a few months after the diagnosis of osteosarcoma in the prostate with the disease in a metastatic stage. In conclusion, osteosarcoma of the prostate is a rarely reported consequence of radiotherapy in patients with prostate cancer and is characterised by poor life expectancy.

Perimodiolar electrodes in cochlear implant surgery.

Perimodiolar-positioned cochlear implant electrodes have been developed in order to bring the electrode contacts as close as possible to the spiral ganglion cells, which are the target of electrostimulation. This results in lower electrical thresholds, higher dynamic ranges and less channel interaction when compared with normal implant electrodes which are usually located peripherally within the scala tympani. In this study we evaluated 4 different types of perimodiolar electrode: the Clarion Preformed electrode, the Clarion Preformed electrode with positioner, the Nucleus Contour electrode and the Med-El Perimodiolar Combi 40 electrode. These devices require different approaches to achieve a perimodiolar electrode position. The electrodes were inserted in fresh human temporal bones. After processing these bones with the electrodes in situ by employing a sawing, grinding and polishing technique, the inner ear structures as well as the electrode positions could be evaluated in detail. All electrode types studied had a more or less perimodiolar position; however, each type produced a certain amount of trauma to cochlear structures which is discussed in relation to mechanical properties. Further human temporal bone studies with improved perimodiolar cochlear implant electrodes are necessary in order to find an optimized type of electrode.

Transperineal radiofrequency interstitial tumor ablation (RITA) of the prostate.

The purpose of this study was to investigate the safety and feasibility of radiofrequency interstitial tumor ablation (RITA) in localized prostate cancer (PCa) and to assess the predictability of the lesions obtained. In 10 patients with localized PCa (mean age 70.4 years), a total of 21 marker lesions were induced under general (n = 3), spinal (n = 4), or local anesthesia only (n = 3). Radiofrequency energy was delivered transperineally under transrectal ultrasound (TRUS) guidance. Radical prostatectomy was performed in all patients 1-7 days after RITA. The findings of intraoperative TRUS and histologic examination of the specimen were correlated. Lesions 2 x 2 x 2 cm were targeted. Postoperatively, patients were catheterized for an average of 1.8 days (range 1-3). Average lesion diameters defined by coagulative necrosis at histologic examination were 2.20 +/- 0.23 x 2.10 +/- 0.31 x 2.38 +/- 0.14 cm (average volume 5.86 +/- 1.63 cm3). Lesions were well defined and did not extend beyond the prostatic capsule. No complications (e.g., rectal wall injury) were noted. RITA-induced lesions were safe, feasible, technically simple, and resulted in lesions well predictable in size and location. On histologic examination, well-defined areas of coagulative necrosis were documented. No damage to the periprostatic tissue was noted. The procedure can be performed with spinal or local anesthesia only.

Inhibition of allergen-induced histamine release from human basophils by cyclosporine A and FK-506.

A number of structurally different allergens trigger the release of mediators from basophils by cross-linking of IgE receptors. In this study, we analyzed the effects of cyclosporine A (CSA) and FK-506 on allergen-induced histamine release in human blood basophils obtained from birch- or grass-pollen-allergic donors (n = 12). Preincubation of basophils with CSA (0.003-3 microg/ml) or FK-506 (0.003-3 microg/ml) led to inhibition of histamine release induced by purified recombinant tree pollen allergens (r Bet v 1, r Bet v 2) and timothy grass pollen allergens (r Ph1 p 1, r Ph1 p 2, r Ph1 p 5). The effects of CSA and FK-506 were dose dependent, with IC50 values ranging between 0.03 and 0.3 microg/ml for both CSA and FK-506. Cyclosporine H, an inactive CSA analog, did not show any effect on allergen-induced histamine secretion. IgE dependency of the reaction was demonstrated in passive transfer experiments using highly enriched human basophils (> 95% pure) and specific IgE from a patient allergic to Bet v 2. In summary, our data show that CSA and FK-506 inhibit recombinant-allergen-induced histamine release from peripheral blood basophils in allergic donors.

Recombinant Bet v 1, the major birch pollen allergen, induces hypersensitivity reactions equal to those induced by natural Bet v 1 in the airways of patients allergic to tree pollen.

BACKGROUND: Atopic allergens produced by recombinant DNA methods are promising tools for diagnosis and therapy of Type I allergy. To evaluate the immunologic properties of these molecules, it is necessary to compare them with natural allergens in vitro and in vivo. OBJECTIVE: The study was carried out to determine whether the potency of recombinant Bet v 1 (rBet v 1) is comparable to that of natural Bet v 1 (nBet v 1) in inducing allergic reactions in the nose and bronchi. METHODS: Thirteen patients allergic to birch pollen with bronchial asthma and/or rhinitis were investigated. Skin prick tests and nasal and bronchial challenges were performed with rBet v 1 and nBet v 1. RESULTS: In patients allergic to birch pollen, both allergens induced comparable skin reactions. In subjects with rhinitis rBet v 1 was equally potent in inducing nasal reactions (mean PD(+60)NR +/- SD, 10.48 +/- 17.42 microg vs 7.98 +/- 8.9 microg, p > 0.05). In patients with asthma, rBet v 1 was equally potent in inducing bronchial reactions (PD20 FEV1, 0.81 +/- 1.74 microg vs 0.62 +/- 1.44 microg, p > 0.05) as nBet v 1. CONCLUSION: No significant differences were observed between natural and recombinant allergen. We conclude that allergens produced by recombinant techniques can induce typical allergic reactions in important target organs of Type I allergy: the nose and bronchi.

High-level expression and purification of the major birch pollen allergen, Bet v 1.

Bet v 1, the single major allergen from birch pollen, shares IgE epitopes with all major tree pollen allergens from closely related species such as alder, hazel, hornbeam, beech, and European chestnut. Because of high sequence homologies among these allergens and the well-studied cross-reactivities on B cell epitopes, Bet v 1 is a representative model protein which can be used for in vitro studies. The cDNA coding for Bet v 1, the single major allergen from birch pollen, was cloned into the T7-based Escherichia coli expression system pMW 175/BL21(DE3) and synthesized as a nonfusion protein. In contrast to other E. coli systems (e.g., pKK233-2/JM105), this system produces high levels of readily extractable proteins corresponding to 5-10% of E. coli total protein, the percentage varying with culture conditions. The overall yield was 8-10 mg of purified recombinant protein per liter of culture medium. The recombinant allergen was purified by several steps, including ion-exchange and hydrophobic interaction chromatography. The purified recombinant allergen showed identical immunological properties with the respective natural counterpart. The use of recombinant allergens of high purity is expected to result in more accurate diagnostic procedures, but possibly also in a superior immunotherapy of Type I allergic diseases when compared with methods using crude allergen extracts containing various amounts of allergen concentrations.

Induction of IgE antibodies in mice and rhesus monkeys with recombinant birch pollen allergens: different allergenicity of Bet v 1 and Bet v 2.

BACKGROUND: Serologic measurements with recombinant birch pollen allergens, rBet v 1 and rBet v 2 (birch profilin), have shown that more than 95% of patients allergic to tree pollen mount high levels of IgE against rBet v 1, whereas only approximately 10% of the patients display rather low levels of IgE against rBet v 2. OBJECTIVE: In this study an attempt was made to determine whether the different allergenicity of the major birch pollen allergen, rBet v 1, and a minor birch pollen allergen, rBet v 2, might be related to a different immunogenicity of the proteins as evaluated in experimental animal systems (mice and rhesus monkeys). METHODS: Purified recombinant allergens were injected into mice and rhesus monkeys with aluminum hydroxide as adjuvant for elicitation of specific IgE responses. Antibody responses to the allergens were detected by immunoblotting, and time courses of immune responses were measured by ELISA. RESULTS: In both animal models more than the 10-fold dose of rBet v 2 was required to induce IgE antibodies, and even then, the amount of specific IgE antibodies elicited with rBet v 1 was substantially higher than that induced by rBet v 2. It was noted that rBet v 2 formed stable polymers through disulfide bonds. CONCLUSION: In two different animal models (mice and rhesus monkeys) the major birch pollen allergen, rBet v 1, induced substantially higher levels of IgE than rBet v 2. A reduced allergenicity of Bet v 2 caused by polymer formation would be in agreement with previous studies indicating reduced allergenicity of proteins on chemical polymerization.

Comparison of recombinant timothy grass pollen allergens with natural extract for diagnosis of grass pollen allergy in different populations.

BACKGROUND: Complementary DNAs coding for the major timothy grass pollen (Phleum pratense) allergens Phl p 1, Phl p 2, and Phl p 5 and birch profilin were isolated, expressed as recombinant nonfusion proteins in Escherichia coli, and purified. OBJECTIVE: In this study the in vitro IgE-binding capacity of recombinant Phl p 1, Phl p 2, Phl p 5, and birch profilin and their IgE recognition frequencies were investigated by using sera from different populations. METHODS: One hundred eighty-three sera from patients allergic to grass pollen were obtained from different populations in Europe, Japan, and Canada. The sera were selected according to clinical criteria, skin testing, and RAST (CAP system; Pharmacia, Uppsala, Sweden) and then tested for IgE reactivity with natural and purified recombinant timothy grass pollen allergens by ELISA and Western blot. RESULTS: Most (94.5%) of the patients allergic to grass pollen could be diagnosed with a combination of recombinant Phl p 1, Phl p 2, Phl p 5, and profilin by means of ELISA. Sera that did not react with the recombinant allergens contained low levels of timothy grass pollen-specific IgE. Although considerable variability in IgE recognition frequency of the recombinant allergens was observed in certain populations, a good correlation was found between natural timothy CAP results and the combination of recombinant allergens in all 183 tested sera (r = 0.87). CONCLUSIONS: Despite considerable variability in the IgE recognition frequency, purified recombinant timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 5) and profilin permitted successful in vitro diagnosis of grass pollen allergy in 94.5% of allergic individuals from different populations. The addition of other recombinant allergens (e.g., recombinant Phl p 4) would only slightly improve the in vitro test sensitivity.

High-level expression of tree pollen isoallergens in Escherichia coli.

cDNAs coding for the major allergen of alder (Alnus glutinosa) pollen Aln g 1, for nine isoforms of Bet v 1, the major birch (Betula verrucosa) pollen allergen, and for four isoforms of Cor a 1, the major allergen of hazel (Corylus avellana) pollen, were inserted into the plasmid pMW175 or pMW 172 and expressed in Escherichia coli as recombinant non-fusion proteins. These constructs produced between 20 and 160 mg protein/l. The recombinant tree pollen isoallergens were tested in immunoblots for their antibody binding properties. For this purpose, we used two monoclonal antibodies (BIP 1 and BIP 4) raised against natural Bet v 1, a polyclonal rabbit anti-recombinant Bet v 1a, as well as serum IgE from allergic patients. Our results show that this expression system is suitable for the production of milligram amounts of tree pollen isoallergens which can be used for the characterization of allergenic epitopes recognized by T and B cells.

Immunologic characterization of purified recombinant timothy grass pollen (Phleum pratense) allergens (Phl p 1, Phl p2, Phl p 5).

BACKGROUND: Grass pollen allergens belong to the potent elicitors of type I allergy. Approximately 40% of allergic individuals display IgE reactivity with grass pollen allergens. In previous studies we have reported the complementary DNA cloning and expression in Escherichia coli of three of the most relevant timothy grass pollen allergens: Phl p 1, Phl p 2, and Phl p 5. OBJECTIVE: To achieve high level expression of immunologically active timothy grass pollen allergens in E. coli, the cDNAs were inserted into expression plasmids. METHODS: The three recombinant grass pollen allergens were expressed at high levels in E. coli as recombinant nonfusion proteins, purified by conventional protein chemical methods and tested for their IgE-binding capacity by immunoblot and ELISA, as well as in histamine release assays. RESULTS: Milligram amounts of pure recombinant allergens were obtained from cultured E. coli. IgE binding to purified recombinant Phl p 1, Phl p 2, and Phl p 5 could be demonstrated by immunoblot and ELISA. With ELISAs the percentage of grass pollen-specific IgE directed against the individual recombinant allergens could be estimated. In addition, the purified recombinant timothy grass pollen allergens induced dose-dependent and specific histamine release from patients' blood basophils. CONCLUSION: Purified recombinant timothy grass pollen allergens represent useful tools for diagnosis and therapy of grass pollen allergy.

Common IgE-epitopes of recombinant Phl p I, the major timothy grass pollen allergen and natural group I grass pollen isoallergens.

Grass pollen allergens are potent elicitors of Type I allergy. More than 95% of grass pollen allergic patients display IgE-cross-reactivity to group I grass pollen allergens of different grass species. A cDNA coding for the major timothy grass pollen allergen, Phl p I, was isolated previously. To investigate the presence of common IgE-epitopes among naturally occurring group I grass pollen isoallergens, Phl p I was expressed in Escherichia coli and used for IgE-absorption experiments. Recombinant Phl p I was able to inhibit IgE-binding to most of group I isoallergens from seven grass species as identified by two dimensional electrophoresis. When tested in competitive ELISA experiments, recombinant Phl p I bound a high percentage of grass pollen specific IgE. The results indicate that recombinant Phl p I shares many of the IgE-epitopes with natural group I grass pollen allergens and hence may represent a useful tool for specific diagnosis and therapy of grass pollen allergy.

Molecular characterization of Api g 1, the major allergen of celery (Apium graveolens), and its immunological and structural relationships to a group of 17-kDa tree pollen allergens.

Individuals suffering from immediate hypersensitivity (type-I allergy) to a particular pollen frequently display intolerance to several foods of plant origin. In this respect, individuals sensitized to birch pollen and/or mugwort pollen frequently display type-I allergic symptoms after ingestion of celery. In this study, we expressed the major allergenic protein of celery, Api g 1, which is responsible for the birch-celery syndrome, in the form of a non-fusion protein. The open reading frame of the cDNA of Api g 1 codes for a protein of 153 amino acids with a molecular mass of 16.2 kDa and 40% identity (60% similarity) to the major allergen of birch pollen, Bet v 1. Furthermore, Api g 1 exhibited similar characteristics to (a) two proteins in parsley induced by fungal infection, (b) the major tree pollen allergens and (c) pathogenesis-related and stress-induced proteins in other plant species. The reactivity of recombinant Api g 1 with IgE antibodies present in sera from celery intolerant patients was comparable to that of the natural celery allergen. Cross-reactivity with Bet v 1 was proven by cross-inhibition experiments, which provides further support for the existence of the birch-celery syndrome and for the suggestion that allergies to some vegetable foods are epiphenomena to allergies caused by inhalation of tree pollen.

High level expression of birch pollen profilin (Bet v 2) in Escherichia coli: purification and characterization of the recombinant allergen.

Up to 20% of the population in industrialized countries suffer from type I allergic symptoms (rhinitis, conjunctivitis, and bronchial asthma). The cDNA coding for birch pollen profilin, a highly conserved cross-reactive allergen and actin-binding protein was expressed in Escherichia coli. Upon induction with IPTG up to 30 mg recombinant profilin per liter culture could be obtained. A single step purification protocol based on the high affinity of profilin to poly-(L-proline) Sepharose was used to obtain large amounts of soluble and pure recombinant birch profilin. Recombinant birch pollen profilin specifically bound IgE, elicited dose dependent histamine release from patients basophils and could be used for skin prick testing without toxic effects. The results indicate that by using purified recombinant profilin, specific diagnosis of type I allergy might be improved.

Effects of IL-4 and IL-13 on total and allergen specific IgE production by cultured PBMC from allergic patients determined with recombinant pollen allergens.

BACKGROUND: Interleukin (IL)-4 and IL-13 have been shown to be potent switch factors for IgE synthesis in human B cells. OBJECTIVE: In this study we investigated the effects of recombinant human IL-4 and IL-13 on total and allergen specific IgE synthesis by peripheral blood mononuclear cells (PBMC) from pollen allergic patients and healthy control individuals. METHODS: Peripheral blood mononuclear cells (PBMC) from allergic patients were investigated for their capacity to produce allergen specific IgE in vitro. Total protein extracts from birch pollen and timothy grass pollen as well as purified recombinant birch pollen allergens, Bet v I, birch profilin (Bet v II) and recombinant timothy grass pollen allergens, Phl p I, Phl p II, and Phl p V were used to measure specific IgE-antibody synthesis in cell culture supernatants by IgE-immunoblot and ELISA. RESULTS: PBMC obtained from allergic patients spontaneously secreted allergen specific IgE in the culture supernatants. Addition of Interleukin 4, Interleukin 13 and anti-CD40 antibody to the cultures alone or in combinations significantly induced total IgE production whereas allergen specific IgE production was not affected. CONCLUSION: Our results indicate that the peripheral blood of allergic individuals contains long lived allergen specific B cells which have already switched to IgE production and which are not sensitive to IL-4 and IL-13 treatment. These results may have implications on attempts to use cytokines or cytokine antagonists in therapy of Type I allergy.

Humoral immune responses to recombinant tree pollen allergens (Bet v I and Bet v II) in mice: construction of a live oral allergy vaccine.

Recombinant tree pollen allergens (recombinant Bet v I and recombinant birch profilin, Bet v II) were purified and used to immunize BALB/c and B6D2F1 mice with Al(OH)3 to elicit a specific IgE response. Serum from immunized mice was then used to detect immunoblotted natural tree pollen allergens. The onset of the humoral immune response was monitored using antimouse IgE, IgG1, IgG2a/b, IgG3 and IgA. In both strains, a specific and long-lasting IgE response could be elicited with both recombinant allergens. Mice immunized continuously with recombinant Bet v I + Al(OH)3 showed a significant decrease of specific IgE antibodies indicating that continuous application of allergens can reduce specific IgE responses. The possibility of inducing a different type of immune responses is indicated by the fact that mice fed with Bet v I expressed in apathogenic Salmonella strains showed a Th1 immune response to Bet v I accompanied by specific IgG2a/b without detectable IgG1 or IgE. Recombinant allergens can hence be used to decrease or even modulate specific IgE responses in vivo.

Isolation of an immunodominant IgE hapten from an epitope expression cDNA library. Dissection of the allergic effector reaction.

An epitope expression cDNA library was constructed from the randomly fragmented cDNA coding for Phl p I, the major grass pollen allergen. Using IgE from allergic patients, epitope clones were isolated and immunodominant fragments were selected. Among three epitope clones coding for a similar region of Phl p I, one clone expressed a 15-amino-acid epitope which was target for IgE antibodies from approximately 30% of grass pollen allergic patients. According to the prevalence of grass pollen allergy, 22% of all allergic patients are expected to display IgE reactivity with this epitope. Although the purified recombinant epitope specifically bound IgE, it did not release histamine from basophiles of most grass pollen allergic patients and thus represents an IgE hapten. Immunodominant IgE haptens may be useful as therapeutic agents to saturate mast cell-bound IgE prior to allergen exposure and may represent candidates for a safe immunotherapy of allergic diseases by reducing anaphylactic side effects.

Complementary DNA cloning of the major allergen Phl p I from timothy grass (Phleum pratense); recombinant Phl p I inhibits IgE binding to group I allergens from eight different grass species.

BACKGROUND: Grass pollens, such as pollen from timothy grass (Phleum pratense), represent a major cause of type I allergy. OBJECTIVE: In this report we attempted to determine how cross-reactive allergenic components of grass pollens from different species can be represented by a minimum number of recombinant allergens. METHODS: We isolated and sequenced a timothy grass pollen cDNA coding for the major allergen Phl p I. A recombinant Phl p I-beta-galactosidase fusion protein, which bound to IgE in 87% of patients with grass pollen allergy, was produced in Escherichia coli. Using recombinant Phl p V and Phl p I, we defined representative patients' sera that bound to group I but not to group V allergens, as well as sera with reactivity against group I and group V allergens. IgE immunoblot inhibition studies were done with nitrocellulose-blotted pollen extracts from eight grass species with different geographic distribution. RESULTS: Preadsorption of patients' sera with recombinant nonfusion Phl p I strongly reduced IgE binding to group I allergens from the eight grasses, showing extensive cross-reactivity between species. CONCLUSION: A single recombinant group I allergen contains many of the IgE epitopes of group I isoallergens from a number of different grass species.