Understanding host-microbiota interactions in the commercial piglet around weaning.

Weaning is a critical period in the life of pigs with repercussions on their health and welfare and on the economy of the swine industry. This study aimed to assess the effect of the commercial early weaning on gut microbiota, intestinal gene expression and serum metabolomic response via an integrated-omic approach combining 16S rRNA gene sequencing, the OpenArray gene expression technology and 1H-NMR spectroscopy. Fourteen piglets from different litters were sampled for blood, jejunum tissue and caecal content two days before (- 2d), and three days after (+ 3d) weaning. A clearly differential ordination of caecal microbiota was observed. Higher abundances of Roseburia, Ruminococcus, Coprococcus, Dorea and Lachnospira genera in weaned piglets compared to prior to weaning showed the quick microbial changes of the piglets' gut microbiota. Downregulation of OCLN, CLDN4, MUC2, MUC13, SLC15A1 and SLC13A1 genes, also evidenced the negative impact of weaning on gut barrier and digestive functions. Metabolomic approach pinpointed significant decreases in choline, LDL, triglycerides, fatty acids, alanine and isoleucine and increases in 3-hydroxybutyrate after weaning. Moreover, the correlation between microbiota and metabolome datasets revealed the existence of metabolic clusters interrelated to different bacterial clusters. Our results demonstrate the impact of weaning stress on the piglet and give insights regarding the associations between gut microbiota and the animal gene activity and metabolic response.

Early socialization and environmental enrichment of lactating piglets affects the caecal microbiota and metabolomic response after weaning.

The aim of this study was to determine the possible impact of early socialization and an enriched neonatal environment to improve adaptation of piglets to weaning. We hypothesized that changes in the microbiota colonization process and in their metabolic response and intestinal functionality could help the animals face weaning stress. A total of 48 sows and their litters were allotted into a control (CTR) or an enriched treatment (ENR), in which piglets from two adjacent pens were combined and enriched with toys. The pattern of caecal microbial colonization, the jejunal gene expression, the serum metabolome and the intestinal physiology of the piglets were assessed before (-2 d) and after weaning (+ 3d). A differential ordination of caecal microbiota was observed after weaning. Serum metabolome suggested a reduced energetic metabolism in ENR animals, as evidenced by shifts in triglycerides and fatty acids, VLDL/LDL and creatine regions. The TLR2 gene showed to be downregulated in the jejunum of ENR pigs after weaning. The integration of gene expression, metabolome and microbiota datasets confirmed that differences between barren and enriched neonatal environments were evident only after weaning. Our results suggest that improvements in adaptation to weaning could be mediated by a better response to the post-weaning stress.

Short communication: Preweaning socialization and environmental enrichment affect short-term performance after regrouping in commercially reared pigs.

On-farm practices like premature weaning and frequent regrouping induce stress to pigs. Early socialization or environmental enrichment in piglets reduce weaning stress, as suggested in previous studies. Little research with both effects and in commercial settings was found. The aim was to investigate the effects of preweaning socialization and environmental enrichment on life-long performance in 661 Danbred pigs. Two treatments were distinguished during the suckling period: control (CON, 24 litters) and enriched (ENR, 24 litters). Control piglets were raised in barren farrowing pens; ENR piglets were provided with six enrichment objects from birth, and two neighboring litters were socialized from Day (D) 14. Pigs were regrouped on D25 (weaning) and D71 (fattening), while keeping the same treatment. Individual body weight was recorded on D1, 14, 23, 27, 31, 38, 69, 79, and after slaughter (carcass weight, CW). Pigs were slaughtered in six batches. Estimated slaughter weight (ESW) was calculated by CW × 1.25. Body weight, CW, and average daily gain (ADG) were analyzed by linear mixed models. Slaughter age was analyzed by Wilcox Rank-Sum test. Body weight and ESW were adjusted to non-linear models to obtain the predicted growth curves of CON and ENR, from birth to the targeted market weight (TMW, 105 kg). Average daily gain during the suckling, nursery, and fattening periods, and from birth to slaughter, did not differ between treatments. However, ADG of ENR when moving pigs from farrowing to nursery (4-day period) and from nursery to fattening (10-day period), revealed a better performance than CON (+20.6 g/day, P = 0.02; +53 g/day, P = 0.03, respectively). Enriched pigs tended to be slaughtered 2.8 days earlier than CON (P = 0.08). On the other hand, the predicted growth curves showed a non-significant 2-day window of reaching TMW between treatments (P = 0.23). Results suggested that enriching the neonatal environment improved the short-term performance after regrouping, and may benefit the life-long performance by reducing time to reach TMW.

Insulin-sparing and fungible effects of E4orf1 combined with an adipocyte-targeting sequence in mouse models of type 1 and type 2 diabetes.

Obesity impairs glycemic control and causes insulin resistance and type 2 diabetes. Adenovirus 36 (Ad36) infection can increase the uptake of excess glucose from blood into adipocytes by increasing GLUT4 translocation through the Ras-Akt signaling pathway, which bypasses PI3K-Akt-mediated insulin receptor signaling. E4orf1, a viral gene expressed early during Ad36 infection, is responsible for this insulin-sparing effect and may be an alternative target for improving insulin resistance. To deliver the gene to adipocytes only, we connected the adipocyte-targeting sequence (ATS) to the 5' end of E4orf1 (ATS-E4orf1). In vitro transfection of ATS-E4orf1 into preadipocytes activated factors for GLUT4 translocation and adipogenesis to the same extent as did Hemagglutinin (HA)-E4orf1 transfection as positive reference. Moreover, the Transwell migration assay also showed that ATS-E4orf1 secreted by liver cells activated Akt in preadipocytes. We used a hydrodynamic gene delivery technique to deliver ATS-E4orf1 into high-fat diet-fed and streptozotocin-injected mice (disease models of type 2 and type 1 diabetes, respectively). ATS-E4orf1 improved the ability to eliminate excess glucose from the blood and ameliorated liver function in both disease models. These findings suggest that ATS-E4orf1 has insulin-sparing and fungible effects in type 2 and 1 diabetes independent of the presence of insulin.

Reduced ADP-ribosylation by PARP1 natural polymorphism V762A and by PARP1 inhibitors enhance Hepatitis B virus replication.

HepG2 and Huh7 cell lines are frequently used as models to study viral hepatitis and hepatocellular carcinoma. However, they exhibit significantly different capacities in their ability to support hepatitis B virus (HBV) replication. To investigate the basis for this, transcription factor-binding motifs at the HBV core promoter (HBVCP) were tested in luciferase reporter constructs to identify the possible role of host factors. Among the transcription factors screened: PARP1, SP1, HNF4α, HNF3, hB1F and HNF1, deletion of the PARP1 binding motif abrogated transcriptional activity at the HBVCP in HepG2 but not Huh7 cells. Sequencing of the PARP1 gene revealed that HepG2 cells carried an Ala762 allele which has low ADP-ribosylation activity, which was shown to have increased PARP1 binding affinity to its cognate motif thus resulting in higher transcriptional activity. PARP1 inhibitors that are being developed as broad cancer therapeutics also target PARP1 ADP-ribosylation enzymatic function. Four PARP1 inhibitors: PJ-34, ABT888, AZD2281 and AG014699 were tested for their effect on HBV replication. All four small molecules effectively enhanced HBV replication in vitro, confirming the role of PARP1 in HBV replication and that alteration of ADP-ribosylation by mutation or drugs can affect HBV replication. Our data demonstrate that natural polymorphisms in the host affecting proteins such as PARP1 can have a significant effect on HBV replication. Hence, patients who are infected with HBV and are on clinical trials involving PARP1 inhibitors may be at risk from unintended side-effects such as exacerbation of HBV replication.

Enhancement of immune responses to neem leaf extract (Azadirachta indica) correlates with antineoplastic activity in BALB/c-mice.

An aqueous plant extract from Azadirachta indica and its chromatographic fraction F1 (neem extract) exerted immunomodulating and antimetastatic activities in BALB/ c-mice. Regular subcutaneous administration of neem extract yielded significantly increased spleen weight and significant enhancement of peritoneal macrophage activity in the chemiluminescence assay, and activation marker CD-44 expression. The thymus weight and thymocyte counts did not show significant differences in the control and neem extract-treated groups, however, determination of peripheral blood cells revealed significant up-regulations of leukocyte subsets, the lymphocytes and monocytes. Flow cytometric analaysis of lymphocyte supopulations documented increased counts of CD-4 and CD-8 cells and an inreased activation marker expression on lymphocytes (CD-25) and monocytes (MAC-3) in neem-treated mice compared to the control animals. To evaluate the antimetastatic activity of neem extract, sarcoma L-1 cells and lymphosarcoma RAW cells were intravenously inoculated into BALB/c-mice. In these model systems the number of experimental lung and liver metastases decreased relevantly, however, biometrically non-significantly in neem extract-treated animals, as compared to the control mice which received injections of saline solutions. Neem extract can be regarded as an immunomodulating and antimetastatic substance which holds promise for further experimental and clinical investigation.

Intratumoral application of standardized mistletoe extracts down regulates tumor weight via decreased cell proliferation, increased apoptosis and necrosis in a murine model.

The cytotoxic in vitro activity of standardized mistletoe extracts (ME) was examined by established assays towards the human ductal breast carcinoma cell line BT474. A dose-dependent (optimum 25 mg/mL medium) and significantly (p < 0.05) enhanced cytotoxic activity towards the BT474 cells was demonstrated. In vivo experiments on the antitumor activity of ME-A and ME-M were performed in a BALB/c-mouse / BT474 ductal breast carcinoma model. ME-A and ME-M were intratumorally administered according to an application schedule which was found to be optimal concerning dosage and time of administration. Standardized intratumoral application of ME-A and ME-M induced a significantly (p < 0.05) decreased tumor weight in experimental mice. Histological investigations were performed comprising analysis of mitosis and proliferation rates (Ki67 expression), as well as necrosis and apoptosis induction (ssDNA detection). As compared to tumors of control mice with intratumoral phosphate-buffered saline (PBS) injections, tumors of the ME-A and ME-M treated groups showed a decreased cell proliferation rate, as well as an increased cell necrosis and apoptosis rate. Standardized mistletoe extracts, interfering with defined tumor cell functions, e.g., proliferation, necrosis and apoptosis, may have an impact on local cancer treatment.

Enhancement of circulating dendritic cell activity by immunomodulators (OK432 and KP-40).

The effects of biological response modifiers (BRMs) on the functions of two types of dendritic cells (DCs) were examined in relation to anti-tumor therapy. The two BRMs studied in our assay system were OK432 (a streptococcal preparation) and KP-40 (Propionibacterium avidum: a heat-inactivated bacterial vaccine). Recently, techniques for isolating human DCs from peripheral blood have been established, and DCs can be divided into two subsets: CD11c+ DCs (myeloid DC population) and CD11c- DCs (lymphoid DC population). Both OK432 and KP-40 were found to up-regulate the activity of myeloid-lineage DCs: the expression of major histocompatibility complex (MHC) class II molecules and adhesion/costimulatory molecules increased, and the production of IL-12 also increased. Therefore, DCs pulsed with OK432 and KP-40 could be applied to vaccination as a new adjuvant for specific immunotherapies.

Standardized mistletoe extract augments immune response and down-regulates local and metastatic tumor growth in murine models.

The immunomodulatory and antimetastatic activity of standardized aqueous mistletoe extract (sME) was evaluated in BALB/c-mice. Regular subcutaneous (s.c.) applications (three times per week for 14 consecutive days; 2, 20, 100 and 500 micrograms per injection and mouse) up-regulated thymocyte and peripheral blood leukocyte counts in tumor-bearing mice. Tumor weight and tumor volume were significantly down-regulated after application of sME doses greater than 20 micrograms per injection. To check the influence of sME treatment on growth of experimental metastases, RAW 117 H 10 lymphosarcoma cells and L-1 sarcoma cells were intravenously inoculated into BALB/c-mice to establish liver and lung colonization, respectively. sME was regularly administered starting 24 hours after tumor cell challenge. Organ colonization was investigated on day 14 after tumor cell inoculation and demonstrated statistically significant (p < 0.05) reductions of experimental liver and lung metastases for sME-treated mice.

Application-dependent immunomodulating and antimetastatic efficacy of thymic peptides in BALB/c-mice.

The immunomodulating and antimetastatic activity of clinically approved, low molecular weight, standardized thymic peptide (TP) preparations was evaluated in BALB/c-mice. Daily applications (subcutaneously, s.c.; intraperitoneally, i.p.; intramusculary, i.m.) of two commercially available TP preparations (7 consecutive days, 10, 50 and 100 micrograms per mouse and injection) up-regulated the thymus weight and thymocyte counts as well as peripheral blood leukocyte and lymphocyte counts in liver metastases-bearing mice. The immunomodulating activity of TP application was most pronounced and statistically significant for thymus weight and counts of thymocytes, leukocytes and lymphocytes after s.c. administration of both TP preparations and concentrations. I.p. and i.m. TP-injections were less effective at reaching statistical significance, however, for defined dosages and parameters, only. To evaluate the influence of TP on experimental liver metastases, RAW 117 lymphosarcoma cells were intravenously inoculated into BALB/c-mice. TP (10, 50, 100 micrograms/mouse) were s.c., i.p. and i.m. administered daily for 7 consecutive days starting 24 hours after tumor cell challenge. Liver colonization was investigated on day 14 after tumor cell inoculation and demonstrated a statistically significant (p < 0.05) reduction of experimental liver metastases for s.c. (both preparations and concentrations) as well as i.p. and i.m. (dose-dependent) TP-treated mice.

Application of standardized mistletoe extracts augment immune response and down regulates metastatic organ colonization in murine models.

The immunomodulatory and antimetastatic activity of standardized aqueous mistletoe extracts from plants grown on fir trees (ME-A) and pine trees (ME-P) were evaluated in BALB/c-mice. Regular subcutaneous (s.c.) and intraperitoneal (i.p.) applications (three times per week for 14 consecutive days; 5 and 50 microg per injection and mouse) upregulated thymus weight and peripheral blood leukocyte counts in tumor bearing mice. To check the influence of ME-A and ME-P treatment on growth of experimental metastases, RAW 117 H 10 lymphosarcoma cells and L-1 sarcoma cells were intravenously inoculated into BALB/c-mice to establish liver and lung colonization. ME-A and ME-P were regularly administered starting 24 h after tumor cell challenge. Organ colonization was investigated on day 14 after tumor cell inoculation and demonstrated statistically significant (P<0.05) reductions of experimental liver and lung metastases for ME-A and ME-P treated mice.

Evaluation of absorbed radiation dose to working staff during cardiac catheterization procedures.

BACKGROUND: Cardiac catheterization has been used frequently for the evaluation and treatment of patients with heart diseases. The working staff, particularly cardiologists who perform these procedures, have the highest potential risk of receiving high radiation doses due to close contact with patients. The purpose of this study was to measure and evaluate the accumulated radiation dosage of the cardiologists while they were performing clinical procedures in the cardiac catheterization laboratory. The working environment of the catheterization laboratory was also monitored for radiation. METHODS: Thermoluminescent dosimeters (TLDs) with very high sensitivity were employed for dose evaluations. They were taped to various parts of the body of the cardiologists during catheterization procedures. For environmental monitoring, TLDs were also distributed at several sites of the catheterization rooms for a period of 2 to 4 weeks. RESULTS: The study showed that the left wrist of the cardiologists received the highest radiation dose (338 microsieverts [microSv]/procedure) and the left lens received the second highest dose (149 microSv/procedure) during the procedures. The dose to the knees was unexpectedly high (92 microSv/procedure), partly due to radiation leakage from the lead curtain shielding under the patient couch. On average, the effective radiation dose per year was 37 mSv/y for a cardiologist who performs 10 catheterization procedures per week. Compare this to the occupational exposure limit of 50 mSv/y. The estimated accumulated equivalent dose to the lens was 152 mSv/y, which exceeded the regulatory limit for occupational exposure. CONCLUSIONS: Using proper lead shielding and increasing the distance from the radiation source are good strategies for reducing the radiation dose in medical staff. The work area outside the catheterization room was considered safe because the radiation level was essentially equivalent to the background radiation level.

Microwave oscillators incorporating high performance distributed Bragg reflector microwave resonators.

We previously demonstrated a new resonator device structure that achieves Q-factors well above those currently realisable. The new structure consists of a microwave cavity, where the enclosure walls consist of distributed Bragg reflectors (DBRs) in three dimensions, made of low-loss sapphire. Theoretical analysis has demonstrated that the resonator's performance is critically dependent upon accurate alignment of the DBR components, thereby maintaining the desired symmetry of the resonant structure. Breaking of the symmetry causes mixing of the high performance Bragg reflected mode with low-Q hybrid cavity modes. The fabrication tolerances required to achieve the expected resonator performance are met with a precise but simple alignment tool. A pair of these resonators have been built at 9.0 GHz, and have demonstrated unloaded Qs in excess of 700,000 at room temperature. These resonators are incorporated into simple two-port feedback oscillator circuits. Phase noise measurements were performed on the two free-running oscillators.

Adhesive properties of P-like fimbriae in Klebsiella-species.

Clinical isolates of three encapsulated Klebsiella strains with type 1 (mannose-sensitive, MS+MR-), type 3 (mannose-resistant, MS-MR+), type 1.3 (MS+MR+) fimbriae and facultatively coexpressing P-like fimbria were investigated for their ability to adhere to uroepithelial cells (UECs) and tracheal epithelial cells (TECs). Irrespective of the type of epithelial cells, adhesion of the MS+MR+ (type 1.3) fimbriated Klebsiella strain was significantly stronger than adhesion of strains carrying only type 1 (MS+MR-) or type 3 (MS-MR+) fimbriae. The coexpression of P-like fimbriae increased the adhesive properties of Klebsiella strains to UECs but not to TECs. Adhesion of P-like fimbriated Klebsiella strains to UECs was significantly inhibited in the presence of the P+ fimbriae-specific Gal alpha-4-Gal beta (galabiose). Such adhesion was unrelated to the coexpression of type 1, type 3 or type 1.3 fimbriae. However, adhesion to TECs was only moderately inhibited.

Modulating activity of mistletoe lectins 1 and 2 on the lymphatic system in BALB/c-mice.

The galactoside-specific lectin (mistletoe lectin-1, VAA-1) and the N-acetylgalactosamine-specific lectin (mistletoe lectin-2, VAA-2) were purified from aqueous mistletoe extract and checked for their immunoactive potency. Regular subcutaneous administration of the optimal immunomodulating VAA-1 /VAA-2 dosage (1 ng lectin/kg body weight) could be shown to modulate thymocyte proliferation, maturation, emigration and activation in BALB/c-mice. Thus, the increase in thymocyte counts was statistically significant after VAA-1 treatment. However, analogue VAA-2 applications significantly decreased thymocyte proliferation. Determinations of lymphatic subsets revealed considerable upregulation (after VAA-1 treatment) and significant downregulation (after VAA-2 treatment) of immature L 3 T4(+)/Lyt-2(+) thymic cells. Counts of mature cells expressing helper/inducer (L3T4(+)) or suppressor/cytotoxic (Lyt-2(+)) phenotypes did not present remarkable differences after VAA-1/VAA-2 administration. Counts of BALB/c-mouse peripheral blood cells revealed evident (statistically non-significant) increases of lymphocytes (PBL) and monocytes (PBM) after VAA-1 treatment, however, cell counts after VAA-2 administration were comparable to non-treated control animals. The determination of activated PBL expressing interleukin IL-2 receptors proved that VAA-1 induced a potent immunostimulation, since these cells were significantly enhanced after VAA-1 administration but not after VAA-2 treatment.

Respiratory burst of human polymorphonuclear leukocytes in response to the galactoside-specific mistletoe lectin.

The phagocytic activity of human polymorphonuclear leukocytes (PMNLs) towards Staphylococcus aureus Cowan 1 was evaluated in chemiluminescence assays. As to check its activating ability, galactoside-specific mistletoe lectin (ML-1) was coincubated with PMNLs which were then challenged with S. aureus. Statistically significant (p < 0.001) chemiluminescent response (correlating with phagocytic activity) could be demonstrated at optimal experimental condition, viz: 1 x 10(6) PMNLs incubated with 0.005 ng ML-1 for 30 and 60 minutes before S. aureus challenge. Other experimental schedules (different timing and PMNL/ML-1 concentrations) did not present with statistically relevant changes in chemiluminescent response. These studies suggest that optimal ML-1 concentrations enhance the phagocytic activity of PMNLs which might be of benefit in thus treated patients as to prevent (or lower the rate of) infections under antineoplastic therapy.

Immunomodulating ability of galactoside-specific lectin standardized and depleted mistletoe extract.

Commercially available mistletoe extract standardized for the galactoside-specific lectin (ML-1; Eurixor) and a chromatographically ML-1-depleted preparation (same charge no. and composition of remaining components) were tested for their immunomodulating potency. In BALB/c-mice, regular subcutaneous administration of the optimal immunomodulating dosage (1 ng ML-1/kg body weight) could be shown to induce no influence on spleen weight, a non-significant increase of thymus weight, a significant increase of thymocyte, peritoneal macrophage, peripheral blood leukocyte, lymphocyte and monocyte and monocyte counts, and a significant decrease of peripheral blood granulocyte counts. Administration of analogue volumes (concentrations) of ML-1-depleted extract, however, did not induce any immunopotentiation. Accordingly, it may be assumed, that the galactoside-specific lectin (ML-1) represents the main immunomodulating component in commercially available mistletoe extracts.

The role of ABO blood groups in infections induced by Staphylococcus saprophyticus and Pseudomonas aeruginosa.

Recently, considerable evidence has been accumulated showing that carbohydrate-containing blood group substances represent prime candidates for the specific interaction with microbial surface lectins in infectious diseases. Accordingly, clinical studies have proved that urinary tract infections by Staphylococcus saprophyticus and outer ear canal infections by Pseudomonas aeruginosa can be positively correlated with the patients blood group. Apparently, the blood group antigens (terminal carbohydrates) represent receptors recognized by S. saprophyticus and P. aeruginosa surface lectins.

[In vitro activity of Mercurius cyanatus complex against relevant pathogenic bacterial isolates]. / In-vitro-Aktivität von Mercurius cyanatus-Komplex gegenüber relevanten pathogenen Bakterienisolaten.

The antimicrobial activity of mercurius cyanatus complex (Oligoplex) and its components Mercurius cyanatus D5, Echinacea angustifolia D1, Ailanthus glandulosa D3, Ammonium bromatum D3, Baptisia tinctoria D3, Euspongia officinalis D2, alcohol 5% (dilution: D1 = 1: 10, D2 = 1 : 100 etc.) was tested in vitro by serial dilution tests against 105 clinical isolates (grampositive/negative, aerobes and anaerobes with relevance for pharyngitis). The bactericidal activity was compared with that of vancomycin when appropriate. One component of the composition (Mercurius cyanatus) exerted a considerable bactericidal activity against S. pyogenes, S. agalactiae, S. pneumoniae, S. aureus, E. faecalis in serial dilutions of the clinical relevant concentration D5. However, growth of H. influenzae, Bacteriodes sp. and Actinobacillus actinomycetemcomitans was not inhibited by Mercurius cyanatus and any other component of the composition. The composition, however, exerted a bactericidal range similar to that of Mercurius cyanatus, but less efficient. Analysis of the bactericidal effect of Mercurius cyanatus and vancomycin revealed comparability for S. pyogenes, S. agalactiae, S. pneumoniae, S. aureus and E. faecalis for vancomycin concentrations of 0.063-2 mg/l, which are clinically relevant.

Immunostimulation by propionibacteria--effects on immune status and antineoplastic treatment.

Experimental studies were performed to investigate further the effects of immunotherapy with Propionibacterium avidum KP-40 on thymocyte proliferation, maturation and emigration in BALB/c-mice. Thymus weight and thymocyte counts, especially cells presenting the immature or cytotoxic/suppressor phenotype were significantly increased. Due to enhanced emigration, peripheral blood lymphocyte and monocyte counts as well as expression of activation markers were significantly upregulated. The antimetastatic effect of Propionibacterium avidum KP-40 was demonstrated in BALB/c-mice, where RAW 117-H10 lymphosarcoma liver colonization was significantly reduced after immunostimulation. Clinical investigations proved that surgical treatment of colorectal carcinoma induced an evident decrease of peripheral blood lymphocytes as compared with preoperative counts. However, single preoperative Propionibacterium avidum KP-40 administration induced a considerable increase of peripheral white blood cell counts, especially lymphocytes. Clinical effects of preoperative immunostimulation by Propionibacterium granulosum KP-45 were investigated in a prospective randomized trial in colorectal carcinoma patients. Positive effects on survival time, local tumor recurrence and distant metastasis could be demonstrated in stages I and II, whereas no advantage of immunotherapy was found in advanced stages III and IV. A recent prospective randomized clinical trial was initiated on the quality of life of colorectal carcinoma patients. Three months after surgical treatment negative effects could not be determined after immunotherapy. Quality of life even proved to be better in patients with abdominoperineal resection as compared to non Propionibacterium avidum KP-40 treated control patients.