A Mini-Pig Mandibular Defect Model for Evaluation of Craniomaxillofacial Bone Regeneration.

Craniomaxillofacial bone defects represent a clinical challenge in the fields of maxillofacial surgery and (implant) dentistry. Regeneration of these bone defects requires the application of bone graft materials that facilitate new bone formation in a safe, reliable, and predictive manner. In addition to autologous bone graft, several types of (synthetic) bone substitute materials have become clinically available, and still major efforts are focused on improving such bone substitute materials by optimizing their properties. Given the regulatory necessity to evaluate the performance of new bone substitute materials for craniomaxillofacial bone regeneration in a large animal model with similarity to human bone before clinical application, we here describe a mini-pig mandibular bone defect model that allows for the creation of multiple (critical-size) bone defects within the mandibular body of a single animal. As examples of bone substitute materials, we utilize both the clinically used BioOss granules and an experimental calcium phosphate cement for filling the created defects. Regarding the latter, its advantages are the injectable application within the defect site, in which the material rapidly sets, and the tailorable degradation properties via the inclusion of hydrolytically degrading polymeric particles. For both bone substitute materials, we show the suitability of the bone defect model to assess bone regeneration via histology and micro-computed tomography. Impact statement Given the regulatory necessity to evaluate the performance of new bone substitute materials for craniomaxillofacial bone regeneration in a large animal model with similarity to the human bone before clinical application, we here describe a mini-pig mandibular bone defect model that allows for the creation of multiple (critical-size) bone defects within the mandibular body of a single animal that can be used for the evaluation of the bone regenerative capacity of new bone grafting materials as well as tissue-engineered products for alveolar bone regeneration.

S100A8/A9 is not essential for the development of inflammation and joint pathology in interleukin-1 receptor antagonist knockout mice.

BACKGROUND: Excessive osteoclast activity, which is strongly stimulated by pro-inflammatory mediators, results in bone and cartilage degeneration as central features of many arthritides. Levels of the alarmin S100A8/A9 and interleukin (IL)-1ß are both increased in arthritis patients and correlate with disease activity and progression of tissue erosion. We previously presented S100A8/A9 as a good biomarker for joint inflammation and arthritis pathology under circumstances of high IL-1 signaling in mice that lack the gene encoding IL-1 receptor antagonist (Il1rn-/- mice). Here, we investigated whether S100A8/A9 is also actively involved in the development of joint inflammation and both cartilage and bone pathology under these conditions by comparing Il1rn-/- mice with mice that have an additional deficiency for S100a9 (Il1rn-/-XS100a9-/-). METHODS: Il1rn-/-XS100a9-/- on a BALB/c background were obtained by crossing S100a9-/- mice and Il1rn-/- mice. Arthritis incidence and severity were macroscopically scored. Myeloid cell populations in the bone marrow and spleen were determined using flow cytometry. In vitro osteoclastogenesis of bone marrow cells was evaluated with TRAP staining. Microscopic joint inflammation, cartilage degeneration, and bone destruction were evaluated using histology of ankle joints of 12- and 20-week-old mice. RESULTS: Macroscopically scored arthritis severity was comparable between Il1rn-/- and Il1rn-/-XS100a9-/- mice. Inflammation, cartilage erosion, and bone erosion were clearly present in 12-week-old mice of both strains lacking Il1rn-/-, but not significantly different between Il1rn-/-XS100a9-/- and Il1rn-/-. Moreover, we observed that the numbers of neutrophils and monocytes were increased by the absence of Il1rn, which was affected by the absence of S100a9 only in the spleen but not in the bone marrow. In line with our other findings, the absence of S100a9 did not affect the osteoclastogenic potential of osteoclast precursors in the absence of Il1rn. Finally, in agreement with the findings in early arthritis development in 12-week-old mice, cartilage and bone erosion in 20-week-old mice was significantly higher in both Il1rn-/- strains, but the additional absence of S100a9 did not further affect tissue pathology. CONCLUSION: S100A8/A9 deficiency does not significantly affect inflammation and joint destruction in mice with high IL1ß signaling suggesting that S100A8/A9 is not essential for the development of arthritis under these conditions.

Sustained release of ancillary amounts of testosterone and alendronate from PLGA coated pericard membranes and implants to improve bone healing.

Testosterone and alendronate have been identified as two bone healing compounds which, when combined, synergistically stimulate bone regeneration. This study describes the development of a novel ultrasonic spray coating for sustained release of ancillary amounts of testosterone and alendronate encapsulated in PLGA 5004A as a carrier. Due to the low amounts of testosterone and alendronate used, sensitive in vitro assays were developed to determine in vitro release. The ultrasonic spray coating technology was optimized for coating titanium screws and pericardial collagen membranes, with the aim to improve osseo-integration and (guided) bone regeneration, respectively, without interfering with their primary mode of action. In vitro release analysis of collagen membranes and screws showed up to 21 days sustained release of the compounds without a burst release. Subsequent preclinical studies in rat and rabbit models indicated that testosterone and alendronate coated membranes and screws significantly improved bone regeneration in vivo. Coated membranes significantly improved the formation of new bone in a critical size calvarial defect model in rats (by 160% compared to controls). Coated screws implanted in rabbit femoral condyles significantly improved bone implant contact (69% vs 54% in controls), bone mineral density (121%) and bone volume (119%) up to 1.3 mm from the implant. Based on the results obtained, we suggest that implants or membranes enabled with local sustained delivery of ancillary amounts of testosterone and alendronate can be a promising system to stimulate local bone regeneration resulting in improved osseo-integration of implants and improved healing of bone defects and fractures.

Simlukafusp alfa (FAP-IL2v) immunocytokine is a versatile combination partner for cancer immunotherapy.

Simlukafusp alfa (FAP-IL2v, RO6874281/RG7461) is an immunocytokine comprising an antibody against fibroblast activation protein α (FAP) and an IL-2 variant with a retained affinity for IL-2Rßγ > IL-2 Rßγ and abolished binding to IL-2 Rα. Here, we investigated the immunostimulatory properties of FAP-IL2v and its combination with programmed cell death protein 1 (PD-1) checkpoint inhibition, CD40 agonism, T cell bispecific and antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. The binding and immunostimulatory properties of FAP-IL2v were investigated in vitro and compared with FAP-IL2wt. Tumor targeting was investigated in tumor-bearing mice and in a rhesus monkey. The ability of FAP-IL2v to potentiate the efficacy of different immunotherapies was investigated in different xenograft and syngeneic murine tumor models. FAP-IL2v bound IL-2 Rßγ and FAP with high affinity in vitro, inducing dose-dependent proliferation of natural killer (NK) cells and CD4+/CD8+ T cells while being significantly less potent than FAP-IL2wt in activating immunosuppressive regulatory T cells (Tregs). T cells activated by FAP-IL2v were less sensitive to Fas-mediated apoptosis than those activated by FAP-IL2wt. Imaging studies demonstrated improved tumor targeting of FAP-IL2v compared to FAP-IL2wt. Furthermore, FAP-IL2v significantly enhanced the in vitro and in vivo activity of therapeutic antibodies that mediate antibody-dependent or T cell-dependent cellular cytotoxicity (TDCC) and of programmed death-ligand 1 (PD-L1) checkpoint inhibition. The triple combination of FAP-IL2v with an anti-PD-L1 antibody and an agonistic CD40 antibody was most efficacious. These data indicate that FAP-IL2v is a potent immunocytokine that potentiates the efficacy of different T- and NK-cell-based cancer immunotherapies.

Evaluation of Collagen Membranes Coated with Testosterone and Alendronate to Improve Guided Bone Regeneration in Mandibular Bone Defects in Minipigs.

OBJECTIVES: The purpose of the present in vivo study was to evaluate whether pericard collagen membranes coated with ancillary amounts of testosterone and alendronate in a poly-lactic glycolic acid (PLGA) carrier as compared to uncoated membranes will improve early bone regeneration. MATERIAL AND METHODS: In each of 16 minipigs, four standardized mandibular intraosseous defects were made bilaterally. The defects were filled with Bio-Oss® granules and covered with a non-coated or coated membrane. Membranes were spray-coated with 4 layers of PLGA containing testosterone and alendronate resulting in 20, 50 or 125 µg/cm2 of testosterone and 20 µg/cm2 alendronate (F20, F50, F125). Non-coated membranes served as controls (F0). Animals were sacrificed at 6 and 12 weeks after treatment. Qualitative and quantitative histological evaluations of bone regeneration were performed. Differences between groups were assessed by paired Student's t-test. RESULTS: Light microscopical analysis showed new bone formation that was in close contact with the Bio-Oss® surface without an intervening non-mineralized tissue layer. Histomorphometric analysis of newly formed bone showed a significant 20% increase in area in the F125 coated membrane treated defects (40 [SD 10]%) compared to the F0 treated defects after 6 weeks (33 [SD 10]%, P = 0.013). At week 12, the total percentage of new bone was increased compared to week 6, but no increase in newly formed bone compared to F0 was observed. CONCLUSIONS: The data from this in vivo study indicate that F125 collagen membranes coated with testosterone and alendronate resulted in superior bone formation (+24%) when normalized to control sites using uncoated membranes.

IL-1ß-Mediated Activation of Adipose-Derived Mesenchymal Stromal Cells Results in PMN Reallocation and Enhanced Phagocytosis: A Possible Mechanism for the Reduction of Osteoarthritis Pathology.

Background: Injection of adipose-derived mesenchymal stromal cells (ASCs) into murine knee joints after induction of inflammatory collagenase-induced osteoarthritis (CiOA) reduces development of joint pathology. This protection is only achieved when ASCs are applied in early CiOA, which is characterized by synovitis and high S100A8/A9 and IL-1ß levels, suggesting that inflammation is a prerequisite for the protective effect of ASCs. Our objective was to gain more insight into the interplay between synovitis and ASC-mediated amelioration of CiOA pathology. Methods: CiOA was induced by intra-articular collagenase injection. Knee joint sections were stained with hematoxylin/eosin and immunolocalization of polymorphonuclear cells (PMNs) and ASCs was performed using antibodies for NIMP-R14 and CD271, respectively. Chemokine expression induced by IL-1ß or S100A8/A9 was assessed with qPCR and Luminex. ASC-PMN co-cultures were analyzed microscopically and with Luminex for inflammatory mediators. Migration of PMNs through transwell membranes toward conditioned medium of non-stimulated ASCs (ASCNS-CM) or IL-1ß-stimulated ASCs (ASCIL-1ß-CM) was examined using flow cytometry. Phagocytic capacity of PMNs was measured with labeled zymosan particles. Results: Intra-articular saline injection on day 7 of CiOA increased synovitis after 6 h, characterized by PMNs scattered throughout the joint cavity and the synovium. ASC injection resulted in comparable numbers of PMNs which clustered around ASCs in close interaction with the synovial lining. IL-1ß-stimulation of ASCs in vitro strongly increased expression of PMN-attracting chemokines CXCL5, CXCL7, and KC, whereas S100A8/A9-stimulation did not. In agreement, the number of clustered PMNs per ASC was significantly increased after 6 h of co-culturing with IL-1ß-stimulated ASCs. Also migration of PMNs toward ASCIL-1ß-CM was significantly enhanced (287%) when compared to ASCNS-CM. Interestingly, association of PMNs with ASCs significantly diminished KC protein release by ASCs (69% lower after 24 h), accompanied by reduced release of S100A8/A9 protein by the PMNs. Moreover, phagocytic capacity of PMNs was strongly enhanced after priming with ASCIL-1ß-CM. Conclusions: Local application of ASCs in inflamed CiOA knee joints results in clustering of attracted PMNs with ASCs in the synovium, which is likely mediated by IL-1ß-induced up-regulation of chemokine release by ASCs. This results in enhanced phagocytic capacity of PMNs, enabling the clearance of debris to attenuate synovitis.

S100A8/A9 increases the mobilization of pro-inflammatory Ly6Chigh monocytes to the synovium during experimental osteoarthritis.

BACKGROUND: Monocytes are dominant cells present within the inflamed synovium during osteoarthritis (OA). In mice, two functionally distinct monocyte subsets are described: pro-inflammatory Ly6Chigh and patrolling Ly6Clow monocytes. Alarmins S100A8/A9 locally released by the synovium during inflammatory OA for prolonged periods may be dominant proteins involved in stimulating recruitment of Ly6Chigh monocytes from the circulation to the joint. Our objective was to investigate the role of S100A8/A9 in the mobilization of Ly6Chigh and Ly6Clow monocytic populations to the inflamed joint in collagenase-induced OA (CiOA). METHOD: S100A8 was injected intra-articularly to investigate monocyte influx. CiOA was induced by injection of collagenase into knee joints of wild-type C57BL/6 (WT), and S100a9-/- mice. Mice were sacrificed together with age-matched saline-injected control mice (n = 6/group), and expression of monocyte markers, pro-inflammatory cytokines, and chemokines was determined in the synovium using ELISA and RT-qPCR. Cells were isolated from the bone marrow (BM), spleen, blood, and synovium and monocytes were identified using FACS. RESULTS: S100A8/A9 was highly expressed during CiOA. Intra-articular injection of S100A8 leads to elevated expression of monocyte markers and the monocyte-attracting chemokines CCL2 and CX3CL1 in the synovium. At day 7 (d7) after CiOA induction in WT mice, numbers of Ly6Chigh, but not Ly6Clow monocytes, were strongly increased (7.6-fold) in the synovium compared to saline-injected controls. This coincided with strong upregulation of CCL2, which preferentially attracts Ly6Chigh monocytes. In contrast, S100a9-/- mice showed a significant increase in Ly6Clow monocytes (twofold) within the synovium at CiOA d7, whereas the number of Ly6Chigh monocytes remained unaffected. In agreement with this finding, the Ly6Clow mobilization marker CX3CL1 was significantly higher within the synovium of S100a9-/- mice. Next, we studied the effect of S100A8/A9 on release of Ly6Chigh monocytes from the BM into the circulation. A 14% decrease in myeloid cells was found in WT BM at CiOA d7. No decrease in myeloid cells in S100a9-/- BM was found, suggesting that S100A8/A9 promotes the release of myeloid populations from the BM. CONCLUSION: Induction of OA locally leads to strongly elevated S100A8/A9 expression and an elevated influx of Ly6Chigh monocytes from the BM to the synovium.

The teleost head kidney: Integrating thyroid and immune signalling.

The head kidney, analogous to the mammalian adrenal gland, is an organ unique for teleost fish. It comprises cytokine-producing lymphoid cells from the immune system and endocrine cells secreting cortisol, catecholamines, and thyroid hormones. The intimate organization of the immune system and endocrine system in one single organ makes bidirectional signalling between these possible. In this review we explore putative interactions between the thyroid and immune system in the head kidney. We give a short overview of the thyroid system, and consider the evidence for the presence of thyroid follicles in the head kidney as a normal, healthy trait in fishes. From mammalian studies we gather data on the effects of three important pro-inflammatory cytokines (TNFα, IL-1ß, IL-6) on the thyroid system. A general picture that emerges is that pro-inflammatory cytokines inhibit the activity of the thyroid system at different targets. Extrapolating from these studies, we suggest that the interaction of the thyroid system by paracrine actions of cytokines in the head kidney is involved in fine-tuning the availability and redistribution of energy substrates during acclimation processes such as an immune response or stress response.

S100A8/A9, a potent serum and molecular imaging biomarker for synovial inflammation and joint destruction in seronegative experimental arthritis.

BACKGROUND: Seronegative joint diseases are characterized by a lack of well-defined biomarkers since autoantibodies are not elevated. Calprotectin (S100A8/A9) is a damage-associated molecular pattern (DAMP) which is released by activated phagocytes, and high levels are found in seronegative arthritides. In this study, we investigated the biomarker potential of systemic and local levels of these S100 proteins to assess joint inflammation and joint destruction in an experimental model for seronegative arthritis. METHODS: Serum levels of S100A8/A9 and various cytokines were monitored during disease development in interleukin-1 receptor antagonist (IL-1Ra)-/- mice using ELISA and multiplex bead-based immunoassay, and were correlated to macroscopic and microscopic parameters for joint inflammation, bone erosion, and cartilage damage. Local expression of S100A8 and S100A9 and matrix metalloproteinase (MMP)-mediated cartilage damage in the ankle joints were investigated by immunohistochemistry. In addition, local S100A8 and activated MMPs were monitored in vivo by optical imaging using anti-S100A8-Cy7 and AF489-Cy5.5, a specific tracer for activated MMPs. RESULTS: Serum levels of S100A8/A9 were significantly increased in IL-1Ra-/- mice and correlated with macroscopic joint swelling and histological inflammation, while serum levels of pro-inflammatory cytokines did not correlate with joint swelling. In addition, early serum S100A8/A9 levels were prognostic for disease outcome at a later stage. The increased serum S100A8/A9 levels were reflected by an increased expression of S100A8 and S100A9 within the ankle joint, as visualized by molecular imaging. Next to inflammatory processes, serum S100A8/A9 also correlated with histological parameters for bone erosion and cartilage damage. In addition, arthritic IL-1Ra-/- mice with increased synovial S100A8 and S100A9 expression showed increased cartilage damage that coincided with MMP-mediated neoepitope expression and in vivo imaging of activated MMPs. CONCLUSIONS: Expression of S100A8 and S100A9 in IL-1Ra-/- mice strongly correlates with synovial inflammation, bone erosion, and cartilage damage, underlining the potential of S100A8/A9 as a systemic and local biomarker in seronegative arthritis not only for assessing inflammation but also for assessing severity of inflammatory joint destruction.

Therapy response monitoring of the early effects of a new BRAF inhibitor on melanoma xenograft in mice: evaluation of (18) F-FDG-PET and (18) F-FLT-PET.

Inhibition of the V600E mutated BRAF kinase gene (BRAF(V600E) ) is an important and effective approach to treating melanomas. A new specific small molecule inhibitor of BRAF(V600E) , PLX3603, showed potent melanoma growth-inhibiting characteristics in preclinical studies and is currently under clinical investigation. In this study we investigated the feasibility of (18) F-FDG and (18) F-FLT-PET to monitor the early effects of the BRAF(V600E) inhibitor in mice with melanoma xenografts. SCID/beige mice with subcutaneous (s.c.) A375 melanoma xenografts, expressing BRAF(V600E) , received the BRAF(V600E) inhibitor twice daily orally (0, 25, 50 and 75 mg/kg). At 1, 3 and 7 days after start of therapy, the uptake of (18) F-FDG and (18) F-FLT in the tumor and normal tissues was determined in ex vivo tissue samples. Serial (18) F-FDG and (18) F-FLT-PET scans were acquired of animals at 1 day before and 1, 3 and 7 days after start of treatment with 75 mg/kg BRAF(V600E) inhibitor. A dose-dependent decrease in (18) F-FDG uptake in the A375 tumors was observed by ex vivo biodistribution analysis. Administration of 75 mg/kg BRAF inhibitor for 1, 3 and 7 days resulted in a significantly decreased (18) F-FDG uptake in A375 tumors (41, 35 and 51%, respectively). (18) F-FLT uptake in the A375 tumors was low at baseline and no significant changes in (18) F-FLT uptake were observed at any of the doses administered. These effects were corroborated by serial in vivo (18) F-FDG and (18) F-FLT-PET imaging. These data demonstrate that (18) F-FDG-PET can be used as an imaging biomarker to noninvasively evaluate the early effects of PLX3603.

Kinetics and thiol requirements of iodothyronine 5'-deiodination are tissue-specific in common carp (Cyprinus carpio L.).

Iodothyronine deiodinases determine the biological activity of thyroid hormones. Despite the homology of the catalytic sites of mammalian and teleostean deiodinases, in-vitro requirements for the putative thiol co-substrate dithiothreitol (DTT) vary considerably between vertebrate species. To further our insights in the interactions between the deiodinase protein and its substrates: thyroid hormone and DTT, we measured enzymatic iodothyronine 5'-deiodination, Dio1 and Dio2 mRNA expression, and Dio1 affinity probe binding in liver and kidney preparations from a freshwater teleost, the common carp (Cyprinus carpio L.). Deiodination rates, using reverse T3 (rT3, 3,3',5'-triiodothyronine) as the substrate, were analysed as a function of the iodothyronine and DTT concentrations. In kidney rT3 5'-deiodinase activity measured at rT3 concentrations up to 10 µM and in the absence of DTT does not saturate appreciably. In the presence of 1mM DTT, renal rT3 deiodination rates are 20-fold lower. In contrast, rT3 5'-deiodination in liver is potently stimulated by 1mM DTT. The marked biochemical differences between 5'-deiodination in liver and kidney are not associated with the expression of either Dio1 or Dio2 mRNA since both organs express both deiodinase types. In liver and kidney, DTT stimulates the incorporation of N-bromoacetylated affinity labels in proteins with estimated molecular masses of 57 and 55, and 31 and 28 kDa, respectively. Although primary structures are highly homologous, the biochemistry of carp deiodinases differs markedly from their mammalian counterparts.

Central and peripheral integration of interrenal and thyroid axes signals in common carp (Cyprinus carpio L.).

In teleostean fishes the hypothalamic-pituitary-thyroid axis (HPT axis) and the hypothalamic-pituitary-interrenal axis (HPI axis) regulate the release of thyroid hormones (THs) and cortisol respectively. Since many actions of both hormones are involved in the regulation of metabolic processes, communication between both signal pathways can be anticipated. In this study, we describe central and peripheral sites for direct interaction between mediators of both neuroendocrine axes in the common carp (Cyprinus carpio). Despite suggestions in the literature that CRH is thyrotropic in some fish; we were not able to establish stimulatory effects of CRH on the expression of the pituitary TSHbeta subunit gene. In preoptic area tissue incubated with 10(-7) M thyroxine (T(4)) a 2 x 9-fold increase in the expression of CRH-binding protein (CRHBP) was observed. Thus, T(4) could reduce the bioavailable hypothalamic crh via the up regulation of crhbp expression and hence down regulate the HPI axis. At the peripheral level, cortisol (10(-6) M), ACTH (10(-7) M), and alpha-MSH (10(-7) M) stimulate the release of T(4) from kidney and head kidney fragments, which contain all functional thyroid follicles in carp, by two- to fourfold. The substantiation of three pituitary thyrotropic factors, viz. TSH, ACTH, and alpha-MSH, in common carp, allows for an integration of central thyrotropic signals. Clearly, two sites for interaction between the HPT axis, the HPI axis, and alpha-MSH are present in common carp. These interactions may be key to the proper regulation of general metabolism in this fish.

Comparative thyroidology: thyroid gland location and iodothyronine dynamics in Mozambique tilapia (Oreochromis mossambicus Peters) and common carp (Cyprinus carpio L.).

In teleosts, the thyroid gland is mostly found in the subpharyngeal region. However, in several species thyroid follicles are found in, for example, heart, head kidney and kidney. Such heterotopic thyroid follicles are active, and considered to work in concert with the subpharyngeal thyroid. In Mozambique tilapia (Oreochromis mossambicus) thyroid activity is, indeed, restricted to the subpharyngeal region; in common carp (Cyprinus carpio) the functional endocrine thyroid is associated with renal tissues. The subpharyngeal follicles of carp comprise only 10% of the total thyroid tissue, and these follicles neither accumulate iodide nor synthesize or secrete thyroid hormones to a significant degree. Although the shape and size of carp subpharyngeal and renal follicles vary, the epithelial cell height of the thyrocytes and thyroxine immunoreactivity do not differ, which suggests that the activity of the carp subpharyngeal thyroid follicles is dormant. Differences in thyroid physiology between the two fish species were further assessed at the level of peripheral thyroid hormone metabolism. Carp clears plasma of thyroid hormones faster than tilapia does. Furthermore, a significant amount of conjugated thyroid hormones was observed in the plasma of tilapia, which was preceded by the occurrence of thyroid hormone conjugates in the subpharyngeal region and coincides with the appearance of conjugates in the surrounding water. Apparently, plasma thyroid hormone conjugates in tilapia originate from the thyroid gland and function in the excretion of thyroid hormones. Our data illustrate the variability in teleostean thyroidology, an important notion for those studying thyroid physiology.

Experimental hyperthyroidism and central mediators of stress axis and thyroid axis activity in common carp (Cyprinus carpio L.).

The effect of experimental hyperthyroidism, realized by T(4) injection, on central mediators of the hypothalamo-pituitary-interrenal axis (HPI-axis) in common carp (Cyprinus carpio L.) was studied. Our results show that hyperthyroidism evokes a marked 3.2-fold reduction in basal plasma cortisol levels. Corticotropin-releasing hormone-binding protein (CRH-BP) mRNA levels in the hypothalamus, measured by real-time quantitative PCR, were significantly elevated by 40%, but CRH, urotensin-I, prepro-TRH, prohormone convertase-1 (PC1), and POMC mRNA levels were unchanged. In the pituitary pars distalis, PC1, CRH receptor-1, and POMC mRNA levels were unaffected, as was ACTH content. Plasma alpha-MSH concentrations were significantly elevated by 30% in hyperthyroid fish, and this was reflected in PC1 and POMC mRNA levels in pituitary pars intermedia that were increased 1.5- and 2.4-fold respectively. The alpha-MSH content of the pars intermedia was unchanged. Hyperthyroidism has profound effects on the basal levels of a central mediator, i.e., CRH-BP, of HPI-axis function in unstressed carp in vivo, and we conclude that HPI- and hypothalamo-pituitary-thyroid-axis functions are strongly interrelated. We suggest that the changes in plasma cortisol, thyroid hormone, and alpha-MSH levels reflect their concerted actions on energy metabolism.

Increased leptin expression in common Carp (Cyprinus carpio) after food intake but not after fasting or feeding to satiation.

Leptin is a key factor in the regulation of food intake and is an important factor in the pathophysiology of obesity. However, more than a decade after the discovery of leptin in mouse, information regarding leptin in any nonmammalian species is still scant. We report the identification of duplicate leptin genes in common carp (Cyprinus carpio). The unique gene structure, the conservation of both cysteines that form leptin's single disulfide bridge, and stable clustering in phylogenetic analyses substantiate the unambiguous orthology of mammalian and carp leptins, despite low amino acid identity. The liver is a major yet not the only site of leptin expression. However, neither 6 d nor 6 wk of fasting nor subsequent refeeding affected hepatic leptin expression, although the carp predictably shifted from carbohydrate to lipid metabolism. Animals that were fed to satiation grew twice as fast as controls; however, they did not show increased leptin expression at the termination of the study. Hepatic leptin expression did, however, display an acute and transient postprandial increase that follows the postprandial plasma glucose peak. In summary, leptin mRNA expression in carp changes acutely after food intake, but involvement of leptin in the long-term regulation of food intake and energy metabolism was not evident from fasting for days or weeks or long-term feeding to satiation. These are the first data on the regulation of leptin expression in any nonmammalian species.

ACTH, alpha-MSH, and control of cortisol release: cloning, sequencing, and functional expression of the melanocortin-2 and melanocortin-5 receptor in Cyprinus carpio.

Cortisol release from fish head kidney during the acute phase of the stress response is controlled by the adrenocorticotropic hormone (ACTH) from the pituitary pars distalis (PD). Alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin, from the pars intermedia (PI), have been implicated in cortisol release during the chronic phase. The present study addresses the regulation of cortisol release by ACTH and alpha-MSH in common carp (Cyprinus carpio) and includes characterization of their receptors, namely, the melanocortin-2 and melanocortin-5 receptors (MC2R and MC5R). We could not demonstrate corticotropic activity of alpha-MSH, beta-endorphin, and combinations of these. We do show a corticotrope in the PI, but its identity is as yet uncertain. Carp restrained for 1 and 7 days showed elevated plasma cortisol and alpha-MSH levels; cortisol is still elevated but lower at day 7 than day 1 of restraint. Interrenal response capacity is unaffected, as estimated by stimulation with a maximum dose ACTH in a superfusion setup. MC2R and MC5R appear phylogenetically well conserved. MC2R is predominantly expressed in head kidney; a low abundance was found in spleen and kidney. MC5R is expressed in brain, pituitary PD, kidney, and skin. Quantitative PCR analysis of MC2R and MC5R expression in the head kidney of restrained fish reveals MC2R mRNA downregulation after 7 days restraint, in line with lower plasma cortisol levels seen. We discuss regulation of corticosteroid production from a phylogenetic perspective. We propose that increased levels of alpha-MSH exert a positive feedback on hypothalamic corticotropin-releasing hormone release to sustain a mild stress axis activity.

The involvement of thyroid hormone metabolism in gilthead sea bream (Sparus auratus) osmoregulation.

We have investigated the effect of adaptation to low salinity water on the thyroid status of the euryhaline teleost, Sparus auratus. We show that, following low salinity adaptation, the plasma T(4) concentration increases and branchial deiodination activities of T(4), T(3), and rT(3) decrease. Moreover, branchial and hepatic enzyme activities that are putatively involved in thyroid hormone metabolism respond differentially in low salinity conditions. Our results indicate the involvement of thyroid hormones in Sparus auratus osmoregulation. Moreover, the gills appear well equipped to play an important role in the modulation of plasma thyroid hormone titers.