Long-term outcomes among localized prostate cancer survivors: prospective predictors for return-to-work three years after cancer rehabilitation.

PURPOSE: This study aimed at (1) investigating the work status of men treated by radical prostatectomy due to diagnosis of localized prostate cancer (LPCa) three years after having attended a cancer rehabilitation program and (2) identifying prospective risk factors for not working at this time point. METHODS: In a longitudinal, questionnaire-based multicenter study, 519 working-age LPCa survivors reported on their work status 12 and 36 months following rehabilitation. Chi-square tests/t tests and multivariable logistic regression analysis were used to identify prospective factors associated with not working at 36 months follow-up. RESULTS: Nearly three quarter of LPCa survivors (N = 377, 73%) worked 3 years after post-acute rehabilitation. Most participants (N = 365, 71%) showed continuous return-to-work (RTW) patterns as they worked both 1 and 3 years following rehabilitation. Multivariable regression analysis revealed older age, low or middle socio-economic status as well as resigned and unambitious work behavior and fatigue at the time of attending the rehabilitation program to be prospective factors for not working at 36 months follow-up. Low socio-economic status [Odds ratio (OR) 4.81, 95% confidence interval (CI) 2.07-11.16] and unambitious work behavior [OR 4.48, 95% CI 2.16-9.31] were the strongest predictors. CONCLUSION: Long-term work retention is a realistic goal among LPCa survivors. The results contribute to the identification of at-risk LPCa survivors early in the RTW process. Special attention should be paid to social inequality. Further, interventions related to the management of fatigue and work-related coping styles could improve long-term RTW, as these were relevant, but potentially modifiable factors impeding work retention.

A randomized phase II trial of efficacy and safety of the immunotherapy ALECSAT as an adjunct to radiotherapy and temozolomide for newly diagnosed glioblastoma.

BACKGROUND: There is an urgent need for effective treatments against glioblastoma (GBM). In this trial, we investigated the efficacy and safety of an adoptive cell-based immunotherapy. METHODS: Patients with newly diagnosed GBM were recruited at 4 study sites in Sweden. The patients were randomized 1:2 to receive either radiotherapy (RT), 60 Gy/30 fractions, with concomitant and adjuvant temozolomide (TMZ) only, or RT and TMZ with the addition of Autologous Lymphoid Effector Cells Specific Against Tumor (ALECSAT) in an open-label phase II trial. The primary endpoint was investigator-assessed progression-free survival (PFS). The secondary endpoints were survival and safety of ALECSAT. RESULTS: Sixty-two patients were randomized to either standard of care (SOC) with RT and TMZ alone (n = 22) or SOC with ALECSAT (n = 40). Median age was 57 years (range 38-69), 95% of the patients were in good performance status (WHO 0-1). There was no significant difference between the study arms (SOC vs ALECSAT + SOC) in PFS (7.9 vs 7.8 months; hazard ratio [HR] 1.28; 95% confidence interval [CI] 0.70-2.36; P = .42) or in median overall survival (OS) (18.3 vs 19.2 months; HR 1.16, 95% CI 0.58-2.31; P = .67). The treatment groups were balanced in terms of serious adverse events (52.4% vs 52.5%), but adverse events ≥grade 3 were more common in the experimental arm (81.0% vs 92.5%). CONCLUSION: Addition of ALECSAT immunotherapy to standard treatment with radiochemotherapy was well tolerated but did not improve PFS or OS for patients with newly diagnosed GBM.

Does a Train-the-Trainer Approach for Enhancing Healthcare Professionals' Skills in Patient Education during Inpatient Medical Rehabilitation Improve Patient Outcomes?

This study is aimed at identifying the impact of a team-based train-the-trainer program (TTT-P) to enhance healthcare professional (HCP) skills in patient education during medical rehabilitation. Focusing on patient-reported outcomes, a prospective, sequential two-cohort study was conducted in the fields of psychosomatic and oncological rehabilitation. Two hundred fifteen patients were evaluated before (Cohort 1) and 196 post implementation of TTT-P (Cohort 2). Patients of both cohorts completed validated questionnaires on self-management (heiQ®), general self-efficacy (GSE scale), and quality of life (WHOQOL-Bref) at the beginning, at the end, and at the 6-month follow-up to analyze short- and intermediate-term effects. Analyses were conducted separately for the psychosomatic and oncological setting. Results showed that TTT-P had no impact on patient outcomes in both rehabilitation settings. Patients did report positive outcomes as a result of the whole inpatient rehabilitation programs, though effects at follow-up were mostly small to medium size. Concerning self-management competencies, cancer patients gained less benefit during rehabilitation than psychosomatic patients. In conclusion, TTT-P did not result in measurable improvements at the patient level, likely because of the limited nature of the intervention. However, these populations of rehabilitants took benefit from participating in a multimodal rehabilitation program, of which patient education is one part.

Identifying Expectations of Delayed Return to Work in Patients with Prostate Cancer at the Beginning of a Cancer Rehabilitation Program.

Purpose To investigate factors associated with expectations of delayed return to work (RTW) in patients with prostate cancer recently admitted to a cancer rehabilitation program. Methods In this multicenter study, data about expected time until RTW and potential correlates (personal, medical, psychosocial and work-related factors) were obtained from 822 employed cancer rehabilitation participants at the beginning of the program. Participants expecting early RTW (≤ 3 months) and delayed RTW (> 3 months) were compared. Hierarchical multivariate logistic regression was applied to study which factors are associated with expecting delayed RTW. Results In total, 171 cancer rehabilitation participants (21%) expected delayed RTW. Group comparison showed education, type of occupation, income, number of comorbid conditions, tumor stage according to the staging system of the Union for International Cancer Control (UICC), anxiety and depression, quality-of-life functioning scales, urinary and treatment-related symptoms, duration of sick leave, subjective work ability, perceived ability to return to the former job, intention to apply for a disability pension, effort-reward-imbalance and occupational stress to be associated in bivariate analysis with participants' expectations. Multivariate analysis revealed UICC tumor stage III (compared to stages I/II, OR 2.36), lower subjective work ability (OR 0.82), perceived inability to return to the former job (OR 1.88) and intention to apply for a disability pension (OR 1.94) to increase the likelihood of expecting delayed RTW. Conclusions Negative or non-beneficial RTW expectations, which are related to self-perception and behavioral intention, seem to be key factors for expecting delayed RTW. Interventions to early identify and adjust such expectations might empower cancer rehabilitation participants to develop appropriate expectations for work recovery.

Return to work in prostate cancer survivors - findings from a prospective study on occupational reintegration following a cancer rehabilitation program.

BACKGROUND: This prospective multicentre-study aimed to analyze return to work (RTW) among prostate cancer survivors 12 months after having attended a cancer rehabilitation program and to identify risk factors for no and late RTW. METHODS: Seven hundred eleven employed prostate cancer survivors treated with radical prostatectomy completed validated self-rating questionnaires at the beginning, the end, and 12 months post rehabilitation. Disease-related data was obtained from physicians and medical records. Work status and time until RTW were assessed at 12-months follow-up. Data were analyzed by univariate analyses (t-tests, chi-square-tests) and multivariate logistic regression models (OR with 95% CI). RESULTS: The RTW rate at 12-months follow-up was 87% and the median time until RTW was 56 days. Univariate analyses revealed significant group differences in baseline personal characteristics and health status, psychosocial well-being and work-related factors between survivors who had vs. had not returned to work. Patients' perceptions of not being able to work (OR 3.671) and feeling incapable to return to the former job (OR 3.162) were the strongest predictors for not having returned to work at 12-months follow-up. Being diagnosed with UICC tumor stage III (OR 2.946) and patients' perceptions of not being able to work (OR 4.502) were the strongest predictors for late RTW (≥ 8 weeks). CONCLUSIONS: A high proportion of prostate cancer survivors return to work after a cancer rehabilitation program. However, results indicate the necessity to early identify survivors with low RTW motivation and unfavorable work-related perceptions who may benefit from intensified occupational support during cancer rehabilitation.

Outcomes across the return-to-work process in PC survivors attending a rehabilitation measure-results from a prospective study.

PURPOSE: Approximately 60% of patients are able to work following a cancer diagnosis. The return-to-work (RTW) process after disability can be conceptualized as a multi-phase construct. This study investigated RTW outcomes throughout the RTW process among survivors of prostate cancer (PC) attending a cancer rehabilitation measure. METHODS: The study was based on a sample of 837 employed PC survivors enrolled in a longitudinal multicenter study. Data was collected at the beginning of the rehabilitation measure, at the end and at 12-month follow-up by means of self-report questionnaires. We compared outcomes with regard to age (<60 and ≥60 years) and socio-economic status (SES; lower, middle, higher) using t tests or univariate ANOVA for metrical and chi-square test or Fisher's exact test for categorical variables. RESULTS: In the off-work phase, most survivors reported positive expectations regarding future work, including responsiveness of their work environment. Nevertheless, one fourth intended to apply for a disability pension. At 12-month follow-up, the RTW rate was 87% and 62% when applying more conservative criteria of RTW. Among survivors who had returned to work, most reported stability of the work situation. Survivors with lower SES showed least favorable outcomes throughout the RTW process, while older age was less consistently of negative impact. CONCLUSIONS: Survivors reported many favorable RTW outcomes, but low SES might be a barrier at various stages of the RTW process. Thus, special attention must be paid to the role of social inequalities during rehabilitation and work reintegration to help survivors managing the RTW process.

Psychosocial and physical outcomes of in- and outpatient rehabilitation in prostate cancer patients treated with radical prostatectomy.

PURPOSE: A radical prostatectomy might lead to an impaired quality of life. Aim of the study was to analyse the impact of inpatient and outpatient rehabilitation on psychosocial and physical outcomes in patients after surgery. METHODS: Six hundred nineteen inpatients and 95 outpatients, treated for localized prostate cancer by prostatectomy, completed the Hospital Anxiety and Depression Scale (HADS) and two quality-of-life questionnaires (EORTC QLQ-C30 and EORTC QLQ-PR25) at the beginning and end of rehabilitation as well as 12 months after rehabilitation. Data were analysed by using t-tests, chi(2) - tests and analyses of variance with repeated measures. RESULTS: Compared to a population sample, patients reported a significantly worse quality of life (EORTC QLQ-C30) and more anxiety (HADS) at the beginning of rehabilitation. Physical, role and social functioning increased significantly over time for in- and outpatients. Patients still reported lower emotional functioning (EORTC QLQ-C30), anxiety (HADS) and prostate cancer-specific physical symptoms (EORTC QLQ-PR25) 1 year after rehabilitation, although symptom levels decreased significantly over time. The setting did not have an independent significant effect in the multivariate model. CONCLUSIONS: In- and outpatients reported an increased quality of life 1 year after rehabilitation with respect to their physical constitution and their reintegration into social life. Nonetheless, both groups still struggled with problems due to surgery. The results indicated that both settings seem to be supportive in the recovery process but that patients seem to require additional support with aftercare for treating surgery-related problems as well as emotional discomfort.

Global 3D imaging of Yersinia ruckeri bacterin uptake in rainbow trout fry.

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM) in rainbow trout, and the first commercially available fish vaccine was an immersion vaccine against ERM consisting of Y. ruckeri bacterin. The ERM immersion vaccine has been successfully used in aquaculture farming of salmonids for more than 35 years. The gills and the gastrointestinal (GI) tract are believed to be the portals of antigen uptake during waterborne vaccination against ERM; however, the actual sites of bacterin uptake are only partly understood. In order to obtain insight into bacterin uptake during waterborne vaccination, optical projection tomography (OPT) together with immunohistochemistry (IHC) was applied to visualize bacterin uptake and processing in whole rainbow trout fry. Visualization by OPT revealed that the bacterin was initially taken up via gill lamellae from within 30 seconds post vaccination. Later, bacterin uptake was detected on other mucosal surfaces such as skin and olfactory bulb from 5 to 30 minutes post vaccination. The GI tract was found to be filled with a complex of bacterin and mucus at 3 hours post vaccination and the bacterin remained in the GI tract for at least 24 hours. Large amounts of bacterin were present in the blood, and an accumulation of bacterin was found in filtering lymphoid organs such as spleen and trunk kidney where the bacterin accumulates 24 hours post vaccination as demonstrated by OPT and IHC. These results suggest that bacterin is taken up via the gill epithelium in the earliest phases of the bath exposure and from the GI tract in the later phase. The bacterin then enters the blood circulatory system, after which it is filtered by spleen and trunk kidney, before finally accumulating in lymphoid organs where adaptive immunity against ERM is likely to develop.

Adverse and long-term protective effects following oil-adjuvanted vaccination against Aeromonas salmonicida in rainbow trout.

Prophylactic measures against Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis, have been an active field of research for decades, with studies mainly focused on Atlantic salmon (Salmo salar). In the present study we have examined the protective and adverse effects of mineral oil-adjuvanted injection vaccines on rainbow trout (Oncorhynchus mykiss). A commercial vaccine and an experimental auto vaccine, as well as their respective adjuvant formulations alone were used to evaluate their individual effects, both prior to and during an experimental waterborne infection challenge. Macro- and microscopic examination revealed signs of vaccine-induced adverse effects from 10 weeks to 14 months post vaccination. Both vaccines induced statistically significant protection during the experimental challenge (P=0.018 for both vaccines), as well as significantly elevated levels of specific circulating antibodies prior to and during the experimental challenge when compared to an unvaccinated control group. During the early, critical time points of the infection, both vaccines appeared to protect against pathological changes to the liver and spleen, which provides a probable explanation for the reduced mortality seen in the vaccinated groups. A significant correlation was found between the level of A. salmonicida-specific antibodies measured prior to challenge and the endpoint survival of each group after the experimental infection, and furthermore, the levels of these antibodies remained elevated for at least 14 months post vaccination.

Oral and anal vaccination confers full protection against enteric redmouth disease (ERM) in rainbow trout.

The effect of oral vaccines against bacterial fish diseases has been a topic for debate for decades. Recently both M-like cells and dendritic cells have been discovered in the intestine of rainbow trout. It is therefore likely that antigens reaching the intestine can be taken up and thereby induce immunity in orally vaccinated fish. The objective of this project was to investigate whether oral and anal vaccination of rainbow trout induces protection against an experimental waterborne infection with the pathogenic enterobacteria Yersinia ruckeri O1 biotype 1 the causative agent of enteric redmouth disease (ERM). Rainbow trout were orally vaccinated with AquaVac ERM Oral (MERCK Animal Health) or an experimental vaccine bacterin of Y. ruckeri O1. Both vaccines were tested with and without a booster vaccination four months post the primary vaccination. Furthermore, two groups of positive controls were included, one group receiving the experimental oral vaccine in a 50 times higher dose, and the other group receiving a single dose administered anally in order to bypass the stomach. Each group was bath challenged with 6.3 × 10(8) CFU/ml Y. ruckeri, six months post the primary vaccination. The challenge induced significant mortality in all the infected groups except for the groups vaccinated anally with a single dose or orally with the high dose of bacterin. Both of these groups had 100% survival. These results show that a low dose of Y. ruckeri bacterin induces full protection when the bacterin is administered anally. Oral vaccination also induces full protection, however, at a dose 50 times higher than if the fish were to be vaccinated anally. This indicates that much of the orally fed antigen is digested in the stomach before it reaches the second segment of the intestine where it can be taken up as immunogenic antigens and presented to lymphocytes.

3D visualization of the initial Yersinia ruckeri infection route in rainbow trout (Oncorhynchus mykiss) by optical projection tomography.

Despite the fact that enteric redmouth disease (ERM) in farmed rainbow trout is one of the most devastating disease problems, little is known about the initial route of infection and pathogenicity of the aetiological agent, Yersinia ruckeri. In order to determine the initially infected organs, optical projection tomography (OPT), a novel three-dimensional (3D) bio-imaging technique, was applied. OPT not only enables the visualization of Y. ruckeri on mucosal surfaces but also the 3D spatial distribution in whole organs, without sectioning. Rainbow trout were infected by bath challenge exposure to 1 × 10(8) CFU/ml of Y. ruckeri O1 for 1 hour. Three fish were sampled for OPT and immunohistochemistry (IHC) 1, 10 and 30 minutes, 1, 3, 6, 12 and 24 hours, as well as 2, 3, 7 and 21 days after the start of the infection period. Y. ruckeri was re-isolated from the blood of infected fish as early as 1 minute post infection. Both OPT and IHC analysis confirmed that the secondary gill lamellae were the only tissues infected at this early time point, indicating that Y. ruckeri initially infects gill epithelial cells. The experimentally induced infection caused septicemia, and Y. ruckeri was found in all examined organs 7 days post infection including the brain, which correlated with the peak in mortality. To the best of our knowledge this is the first description of Y. ruckeri infection in the brain, which is likely to cause encephalitis. This in part could explain the lethality of ERM in rainbow trout. Using OPT scanning it was possible to visualize the initial route of entry, as well as secondary infection routes along with the proliferation and spread of Y. ruckeri, ultimately causing significant mortality in the exposed rainbow trout. These results demonstrate that OPT is a state-of-the-art technique capable of visualizing pathogenesis at high resolution.

Long-lasting protection induced by bath vaccination against Aeromonas salmonicida subsp. salmonicida in rainbow trout.

For decades Aeromonas salmonicida subsp. salmonicida (from here referred to as A. salmonicida) has been recognized as the causative agent of typical furunculosis. This disease has had a major impact on aquaculture worldwide, making it a target for international research, particularly within the field of immunoprohylaxis. Initial studies attempted vaccination via oral route and immersion. However, these vaccination methods proved insufficient when compared to intraperitoneally (i.p.) injected vaccines. The focus of vaccine research regarding A. salmonicida shifted towards the i.p.-injected vaccines during the 1980's and -90's, resulting in oil-adjuvanted vaccines providing high levels of protection over longer periods of time. The majority of this research has been conducted using salmon, while rainbow trout, which is also a commercially important species, has played a much less central role. In this study, we have examined the effect of a bath vaccination using an experimental A. salmonicida bacterin. Rainbow trout were vaccinated by a 5 min bath in a formalin-inactivated bacterin. Half of these fish was booster vaccinated using 50% of the initial vaccine dose 10 weeks post primary immunization. Along with an un-vaccinated control group, the fish were challenged by waterborne infection 24 weeks post primary immunization. Both vaccinated groups showed a significantly increased survival (>93% survival) compared to a 70% survival in the un-vaccinated control group (P = 0.005 and P = 0.019 for single and dual immunizations, respectively). When comparing the survival of the single and dual immunization groups, there was no significant difference (P = 0.531). ELISA showed no significant induction of specific circulating antibodies in either vaccinated group. These results are interesting with regard to the protective mechanisms, seen in the light of previous results obtained using bath as well as i.p. vaccination against furunculosis in salmonid fishes.

Potential role of specific antibodies as important vaccine induced protective mechanism against Aeromonas salmonicida in rainbow trout.

Furunculosis caused by infection with Aeromonas salmonicida subsp. salmonicida has been a known threat to aquaculture for more than a century. Efficient prophylactic approaches against this disease are essential for continued growth of salmonid aquaculture. Since the introduction of successful oil-adjuvanted vaccines in the early 1990's, a number of studies have been published on the protective as well as adverse effects of these vaccines. Most studies focus on vaccination of salmon (Salmo salar). However, rainbow trout (Oncorhynchus mykiss) are also very susceptible to infection and are vaccinated accordingly. In this study we have examined the protection against infection with a Danish strain of A. salmonicida in both vaccinated and non-vaccinated rainbow trout. A commercial and an experimental auto-vaccine were tested. The protective effects of the vaccines were evaluated through an A. salmonicida challenge 18 weeks post vaccination. Both vaccines resulted in a significantly increased survival in the vaccinated fish during a 28 day challenge period relative to non-vaccinated fish (P = 0.01 and P = 0.001 for the commercial and experimental vaccine, respectively). Throughout the entire experiment, the presence of specific antibodies in plasma was monitored using ELISA. A significant increase in specific antibody levels was seen in fish vaccinated with both vaccines during the 18 weeks between vaccination and challenge. Within 3 days post challenge, a significant decrease in specific antibodies occurred in vaccinated fish. A positive correlation was found between mean levels of specific antibodies pre challenge and overall survival. This correlation, along with the observed depletion of antibodies during the initial phase of infection, suggests that specific antibodies play an essential role in vaccine mediated protection against A. salmonicida in rainbow trout.

Differential immune response of rainbow trout (Oncorhynchus mykiss) at early developmental stages (larvae and fry) against the bacterial pathogen Yersinia ruckeri.

Innate immune factors play a crucial role in survival of young fish especially during early stages of life when adaptive immunity is not fully developed. In the present study, we investigated the immune response of rainbow trout (Oncorhynchus mykiss) larvae and fry at an early stage of development. We exposed 17 and 87° days post hatch larvae and fry (152 and 1118 degree days post hatch; avg. wt. 70 and 770 mg, respectively) to the bacterial pathogen, Yersinia ruckeri for 4h by bath challenge. Samples were taken at 4, 24, 72 and 96 h post exposure for qPCR and immunohistochemical analyses to elucidate the immune response mounted by these young fish. Larvae showed no mortality although infected larvae at 48 h post exposure showed hyperaemia in the mouth region and inflammation on the dorsal side of the body. Gene expression studies showed an up-regulation of iNOS and IL-22 in infected larvae 24h post exposure but most of the investigated genes did not show any difference between infected and uninfected larvae. Immunohistochemical studies demonstrated a high expression of IgT molecules in gills and CD8 positive cells in thymus of both infected and uninfected larvae. Infection of rainbow trout fry with Y. ruckeri, in contrast, induced a cumulative mortality of 74%. A high expression of cytokines (IL-1ß, TNF-α, IL-22, IL-8 and IL-10), acute phase proteins (SAA, hepcidin, transferrin and precerebellin), complement factors (C3, C5 and factor B), antimicrobial peptide (cathelicidin-2) and iNOS was found in infected fry when compared to the uninfected control. IgT molecules and mannose binding lectins in gills of both infected and uninfected fry were detected by immunohistochemistry. The study indicated that early life stages (yolk-sac larvae), merely up-regulate a few genes and suggests a limited capacity of larvae to mount an immune response by gene regulation at the transcriptional level. Based on the observed clearance of bacteria and lack of mortality it could be speculated that larvae may be covered by protective shield of different immune factors providing protection against broad range of pathogens. However, the increased susceptibility of older fry suggests that Y. ruckeri may utilize some of the immune elements to enter the naive fish. The up-regulation of iNOS and IL-22 in the infected larvae implicates an important role of these molecules in immune response at early developmental stages. A dense covering of surfaces of gill filaments by IgT antibody in the young fish suggest a role of this antibody as innate immune factor at early developmental stages.

Association between plasma antibody response and protection in rainbow trout Oncorhynchus mykiss immersion vaccinated against Yersinia ruckeri.

A key hallmark of the vertebrate adaptive immune system is the generation of antigen-specific antibodies from B cells. Fish are the most primitive gnathostomes (jawed vertebrates) possessing an adaptive immune system. Vaccination of rainbow trout against enteric redmouth disease (ERM) by immersion in Yersinia ruckeri bacterin confers a high degree of protection to the fish. The immune mechanisms responsible for protection may comprise both cellular and humoral elements but the role of specific immunoglobulins in this system has been questioned and not previously described. The present study demonstrates significant increase in plasma antibody titers following immersion vaccination and significantly reduced mortality during Y. ruckeri challenge.Rainbow trout were immersion-vaccinated, using either a commercial ERM vaccine (AquaVac™ ERM vet) or an experimental Y. ruckeri bacterin. Half of the trout vaccinated with AquaVac™ ERM vet received an oral booster (AquaVac™ ERM Oral vet). Sub-groups of the fish from each group were subsequently exposed to 1 x 109 CFU Y. ruckeri/ml either eight or twenty-six weeks post vaccination (wpv). All vaccinated groups showed 0% mortality when challenged, which was highly significant compared to the non-vaccinated controls (40 and 28% mortality eight and twenty-six weeks post vaccination (wpv), respectively) (P<0.0001). Plasma samples from all groups of vaccinated fish were taken 0, 4, 8, 12, 16 and 26 wpv. and Y. ruckeri specific IgM antibody levels were measured with ELISA. A significant increase in titers was recorded in vaccinated fish, which also showed a reduced bacteremia during challenge. In vitro plasma studies showed a significantly increased bactericidal effect of fresh plasma from vaccinated fish indicating that plasma proteins may play a role in protection of vaccinated rainbow trout.

Association between Yersinia ruckeri infection, cytokine expression and survival in rainbow trout (Oncorhynchus mykiss).

The immune response against bacterial pathogens has been widely studied in teleosts and it is evident that survival chances differ significantly within a host population. Identification of indicators for susceptibility and responsiveness will improve our understanding of this host-pathogen interaction. The present work shows that the transcripts of cytokine genes in blood cells sampled three days post-infection was significantly higher in fish which obtained a high bacteriemia and died at later time points when compared to both non-infected control fish and infected fish that survived the infection. Rainbow trout were infected by bath challenge in a bacterial suspension (LD(60) dose, 1.8 × 10(9) CFU/ml Yersiniaruckeri for 1 h) and subsequently transferred to individual aquaria for 30 days of observation. Blood samples were analyzed for presence of Y. ruckeri both by culture and quantitative RT real-time PCR (qRT-PCR) and transcript levels of 28 genes encoding molecules which are important in the immune response. The transcript levels of a number of central cytokines, chemokines and cytokine receptors (IL-1ß, IL-6, IL-8, IL-10, TNF-α, IL-receptor II) were significantly increased in infected fish that died later. In addition, a significantly higher amount of Y. ruckeri was found in the blood of the fish that died when compared to survivors. The study indicates that highly susceptible trout obtain an early heavy septicemia infection, which elicits a high up-regulation of the transcript of pro-inflammatory cytokines. Thus, less susceptible fish are protected by other factors and contract merely a weak non-lethal infection eliciting no or a weak cytokine response.

PAMP induced expression of immune relevant genes in head kidney leukocytes of rainbow trout (Oncorhynchus mykiss).

Host immune responses elicited by invading pathogens depend on recognition of the pathogen by specific receptors present on phagocytic cells. However, the reactions to viral, bacterial, parasitic and fungal pathogens vary according to the pathogen-associated molecular patterns (PAMPs) on the surface of the invader. Phagocytic cells are known to initiate a respiratory burst following an exposure to the pathogen, but the underlying and associated specific elements are poorly elucidated in fish. The present study describes the differential response of head kidney leukocytes from rainbow trout (Oncorhynchus mykiss) to different PAMPs mimicking viral (poly I:C), bacterial (flagellin and LPS) and fungal infections (zymosan and ß-glucan). Transcript of cytokines related to inflammation (IL-1ß, IL-6, IL-10 and TNF-α) was highly up-regulated following LPS exposure whereas flagellin or poly I:C induced merely moderate reactions. In contrast, IFN-γ expression was significantly higher in the poly I:C stimulated group compared to the LPS group. When head kidney cells were exposed to zymosan or ß-glucan, genes encoding IL-1ß, TNF-α, IL-6 and IL-10 became up-regulated. Their level of up-regulation was comparable to LPS but the kinetics differed. In particular, TNF-α induction was considerably slower when stimulated with zymosan or ß-glucan. The gene encoding the COX-2 enzyme, a central element during initiation of inflammatory reactions, was significantly higher in stimulated cells although a depressing effect of high concentrations of LPS and zymosan became evident after 4h exposure. This study suggests that rainbow trout leukocytes respond differently to viral, bacterial and fungal PAMPs, which may reflect activation of specific signaling cascades eventually leading to activation of different immune effector molecules.

Antigen-sampling cells in the salmonid intestinal epithelium.

Antigen uptake has been shown to occur in the teleost intestine, but so far, limited information is available on the distribution and nature of cells involved in the process, and M cells, known for their antigen-sampling abilities in mammals, have not been identified. Here, different intestinal segments from salmonid fish were exposed to gold-BSA to identify antigen-sampling cells. Sections from exposed intestine were examined by light and electron microscopy. Uptake of gold-BSA was restricted to very few dendritic-like cells and to a limited number of epithelial cells located in the mucosal folds in the second segment of the mid-intestine. Gold-positive epithelial cells displayed diverging and electron-dense microvilli with channels intruding into the cytoplasm. A lectin binding experiment demonstrated the presence of cells with mammalian M-cell characteristics in the identical regions. As the identified epithelial cells shared some morphological similarities with immature mammalian M cells, this phenotype may represent evolutionary early antigen-sampling enterocytes.

Response of rainbow trout (Oncorhynchus mykiss) in skin and fin tissue during infection with a variant of Gyrodactylus salaris (Monogenea: Gyrodactylidae).

Response mechanisms of rainbow trout Oncorhynchus mykiss (Walbaum), experimentally infected with a Danish strain of Gyrodactylus salaris Malmberg, 1957 were investigated using molecular tools (qPCR) and immunohistochemistry. Expression of ten immune-relevant genes and reactivity with five different antibodies in the epidermis of skin and fin tissue were analysed in susceptible but responding rainbow trout. Rainbow trout were susceptible with regard to the parasite strain which initially colonised fins but relocated to the body region as infection progressed. The ten investigated genes encoding the cytokines IL-1beta, TNF-alpha, IFN-gamma, IL-10 and markers for adaptive immune activity, such as CD-4, CD-8, TCR-alpha, IgM, IgT and MHC II, were not found significantly regulated during the course of infection although IFN-gamma showed a slight up-regulation. Immunohistochemical analyses showed positive reactivity with antibodies against CD3, B-lymphocytes, neutrophilic granulocytes and collectin but not with mAb against IgM. No staining differences between infected and non-infected skin and fin tissue were detected.

Innate immune response in rainbow trout (Oncorhynchus mykiss) against primary and secondary infections with Yersinia ruckeri O1.

Response mechanisms in teleosts against bacterial pathogens have been widely studied following injection procedures applying preparations of killed bacteria. In contrast, investigations on immune reactions in fish which have survived a primary infection and subsequently have been challenged are few or lacking. However, knowledge on these factors during infection and re-infection could provide the basis for development of improved vaccines. The innate immune response in rainbow trout (Oncorhynchus mykiss) against Yersinia ruckeri O1 has been studied following a primary intra-peritoneal injection with 5 x 10(5) CFU Y. ruckeri, and after bacterial clearance a secondary infection 35 days later. The number of pathogens in the liver was measured with a Y. ruckeri specific 16S ribosomal RNA quantitative real-time RT-PCR (q-PCR) during the course of infection. The bacterial counts peaked on day 3 during the primary infection and were significantly lower during the re-infection. Re-challenged fish showed a highly increased survival when compared to the naïve fish receiving a primary infection indicating development of adaptive immunity in the fish against this bacterial pathogen. We investigated the gene expression of innate immune factors in the liver during infections in order to elucidate molecules involved in survival of hosts before adaptive immunity was mounted. Transcription of mRNA was measured in liver samples taken 8 h, 1, 3, 7, 14 and 28 d post-infection using q-PCR. The investigation focused on genes encoding toll-like receptor 5 (TLR5), the pro-inflammatory cytokines IL-1beta, IL-6 and TNF-alpha, the acute phase proteins (APPs) serum amyloid protein a (SAA), trout C polysaccharide binding protein, a CRP/SAP like pentraxin, precerebellin, transferrin, hepcidin and finally the complement factors C3, C5 and factor B. Infection elicited significantly increased gene expression of all the cytokines (IL-6 > 1000-fold), some acute phase proteins (SAA > 3000-fold) and down-regulation of complement factors (C3, C5 and factor B). SAA expression was significantly earlier activated during the re-infection when compared to the primary infection. The pattern of gene activation suggested that the innate response was based on pathogen binding to toll-like receptors, production of cytokines and subsequent release of APPs. In general, both the innate immune response and the amount of Y. ruckeri measured in the liver during the re-infection was much lower compared to the first infection, probably reflecting development of adaptive immunity.