TGF-ß and stem cell factor regulate cell proliferation in the proximal stem cell niche.

BACKGROUND: Stem cells are located in specific regulatory environments termed niches, which modulate the survival and proliferation of the cells through a variety of both mitogenic and inhibitory cytokines. In the murine prostate, stem cells are located in the proximal region of prostatic ducts. We examined the regulation of murine prostate cells in the stem cell niche by transforming growth factor beta (TGF-ß) and stem cell factor (SCF). METHODS: Prostate cells from the proximal and distal regions of prostatic ducts were cultured in the presence and absence of TGF-ß and SCF, both on collagen-coated wells and in collagen gels. Cell growth on collagen was assessed by determining cell number. Cell growth in collagen gels was quantified by determining the number, size and complexity of prostatic ducts. The basal and luminal phenotype of the cells was determined by immunohistochemistry. RESULTS: Endogenous TGF-ß inhibited proliferation and promoted differentiation of proximal cells towards a luminal phenotype. It also inhibited duct-forming capacity and promoted differentiation of prostatic ducts towards a luminal phenotype. Addition of SCF enhanced proximal cell proliferation on collagen-coated wells and duct formation in collagen gels. Proliferation was further increased by ablation of endogenous TGF-ß. CONCLUSION: Proliferation and the basal/luminal cell composition of cells isolated from the proximal region of prostatic ducts, the stem cell niche, is regulated in part by opposing effects of SCF and endogenous TGF-ß.

Perinatal social work during the Covid-19 pandemic: Reflecting on concepts of time and liminality.

This article reflects upon the experiences of two perinatal, hospital social workers during the unprecedented time of the Covid-19 in Ireland, as discussed with their academic colleague. This encounter revealed the complexity of service delivery that emerged, when managing the needs of vulnerable clients whilst being mindful of personal safety. One of the social workers was pregnant so was conscious of possible risks to her unborn child, as well as her young family at home. The second social worker, her line manager, discusses the dilemmas associated with the management of risk when allocating staff to contexts where they would be in direct contact with Covid-19. At the core of the analysis of these situations is the notion of liminal space and the realisation that time appears to have a new meaning; what we once knew as normal no longer exists, but we have yet to reach the 'new normal'.

A prospective audit of adjunctive zonisamide in an everyday clinical setting.

This audit examined outcomes for 203 patients prescribed zonisamide (ZNS) for various uncontrolled seizure types at a specialist outpatient service. Forty-two (20.7%) patients achieved 6 months of seizure freedom, and an additional 37 (18.2%) had a 50% seizure reduction for 6 months on a stable ZNS dose. Seizure freedom was more likely in patients with primary generalized (24/61, 39%) than in those with partial-onset (18/141, 12.7%) seizures (P<0.001). Eight patients (5 seizure free) were maintained on ZNS monotherapy. More patients became seizure free with ZNS as monotherapy or first add-on, compared with those in whom ZNS was the second, third, or fourth adjunctive drug (P=0.001). Seizure freedom was less likely in patients treated with hepatic enzyme-inducing agents (13/113, 11.5%) than in those receiving noninducing AEDs (24/82, 29.3%) (P=0.002). ZNS was discontinued in 72 (35.5%) patients largely because of side effects (n=58, 28.6%). Commonest complaints leading to withdrawal were sedation (n=14), nausea and vomiting (n=13), neuropsychiatric symptoms (n=12), rash (n=6), and weight loss (n=6). Around 80% of patients who became seizure free on ZNS or had the drug withdrawn did so on a dose 200mg. ZNS is an effective broad-spectrum AED that can also produce a range of dose-dependent and idiosyncratic side effects.

KLF4 as a rheostat of osteolysis and osteogenesis in prostate tumors in the bone.

We previously showed that KLF4, a gene highly expressed in murine prostate stem cells, blocks the progression of indolent intraepithelial prostatic lesions into aggressive and rapidly growing tumors. Here, we show that the anti-tumorigenic effect of KLF4 extends to PC3 human prostate cancer cells growing in the bone. We compared KLF4 null cells with cells transduced with a DOX-inducible KLF4 expression system, and find KLF4 function inhibits PC3 growth in monolayer and soft agar cultures. Furthermore, KLF4 null cells proliferate rapidly, forming large, invasive, and osteolytic tumors when injected into mouse femurs, whereas KLF4 re-expression immediately after their intra-femoral inoculation blocks tumor development and preserves a normal bone architecture. KLF4 re-expression in established KLF4 null bone tumors inhibits their osteolytic effects, preventing bone fractures and inducing an osteogenic response with new bone formation. In addition to these profound biological changes, KLF4 also induces a transcriptional shift from an osteolytic program in KLF4 null cells to an osteogenic program. Importantly, bioinformatic analysis shows that genes regulated by KLF4 overlap significantly with those expressed in metastatic prostate cancer patients and in three individual cohorts with bone metastases, strengthening the clinical relevance of the findings in our xenograft model.

Molecular and functional characterization of goldfish (Carassius auratus L.) transforming growth factor beta.

Transforming growth factor beta (TGF-beta) is a pleiotropic cytokine with important roles in the regulation of cell proliferation, differentiation, survival, migration, activation and de-activation. It is one of the first cytokines released during an immune response and plays a strong immunomodulatory role in the activation and subsequent de-activation of macrophages and other immune cells. TGF-beta is a highly conserved molecule, and members of the TGF superfamily can be found in organisms as evolutionarily distant as arthropods. In this manuscript, we described the identification of a goldfish TGF-beta molecule, which was highly expressed in the skin, kidney and spleen of the goldfish and its expression was up-regulated in macrophages treated with LPS or recombinant goldfish TNF-alpha. Goldfish TGF-beta shared a high amino acid identity with, and was phylogenetically related to, TGF-beta1 of other teleost fish, birds, amphibians and mammals. Recombinant goldfish TGF-beta (rTGF-beta) induced the proliferation of a goldfish fibroblast cell line (CCL71) in a dose-dependent manner. In addition, rTGF-beta down-regulated the nitric oxide response of TNF-alpha-activated macrophages. This is the first report of teleost TGF-beta function in an ectothermic vertebrate.

KLF4, A Gene Regulating Prostate Stem Cell Homeostasis, Is a Barrier to Malignant Progression and Predictor of Good Prognosis in Prostate Cancer.

There is a considerable need to identify those individuals with prostate cancer who have indolent disease. We propose that genes that control adult stem cell homeostasis in organs with slow turnover, such as the prostate, control cancer fate. One such gene, KLF4, overexpressed in murine prostate stem cells, regulates their homeostasis, blocks malignant transformation, and controls the self-renewal of tumor-initiating cells. KLF4 loss induces the molecular features of aggressive cancer and converts PIN lesions to invasive sarcomatoid carcinomas; its re-expression in vivo reverses this process. Bioinformatic analysis links these changes to human cancer. KLF4 and its downstream targets make up a gene signature that identifies indolent tumors and predicts recurrence-free survival. This approach may improve prognosis and identify therapeutic targets for advanced cancer.

An evaluation of the immunohistochemistry benefits of boric acid antigen retrieval on rat decalcified joint tissues.

This paper describes the evaluation and optimisation of boric acid antigen retrieval (AR) in rat joint tissue immunohistochemistry (IHC), with reference to two sample IHC targets, CD31 (PECAM-1) and Proliferating Cell Nuclear Antigen (PCNA). Sections of buffered formalin-fixed arthritic tibial/talus joints, decalcified with EDTA, EDTA/formalin or Surgipath(R) Decalcifier I(R), were subjected to one of a number of pre-treatments (none, 0.1% trypsin, 0.2 M acetic acid pH 7.0 or 0.2 M boric acid pH 7.0) and then immunostained for CD31 or PCNA. Of the pre-treatment AR regimens, boric acid gave the most consistent and specific immunostaining of both antigens in joints from the three different decalcification protocols. Satisfactory CD31 and PCNA staining was also achieved in EDTA decalcified joints with no pre-treatment. Likewise, PCNA could be demonstrated in Surgipath(R) decalcified tissue without pre-treatment, albeit at slightly lower staining intensity than achieved following boric acid. The remaining decalcification/pre-treatment conditions were unsatisfactory for IHC of the two antigens investigated because of lack of staining, non-specific staining or consistent loss of sections from the slides. Boric acid pre-treatment provides a valuable alternative low temperature AR method where conventional heat-mediated AR methods are normally required but cannot be used due to tissue type.

The specific monocarboxylate transporter-1 (MCT-1) inhibitor, AR-C117977, induces donor-specific suppression, reducing acute and chronic allograft rejection in the rat.

BACKGROUND: In a search for immunosuppressive drugs having novel mechanisms, monocarboxylate transporter (MCT-1) inhibitors were identified that markedly inhibited immune responses. Here, we report the effects of AR-C117977, a potent MCT-1 inhibitor, on alloimmune responses in the rat. METHODS: In vitro activity was determined in a rat mixed lymphocyte response (MLR). In vivo activity was tested in a graft versus host response (GVHR) and in both high (DA to PVG) and low (PVG to DA) responder cardiac allograft models. To assess induction of donor-specific suppression recipients of allogeneic hearts surviving longer than 100 days received a second transplant either of the same donor strain or a third-party donor strain. Effects on chronic graft rejection were assessed histologically by evaluating vasculopathy in long-term surviving grafts and in an obliterative bronchiolitis (OB) model. RESULTS: AR-C117977 inhibited the rat MLR and was more potent than cyclosporin A (CsA). In the rat GVHR model, AR-C117977 gave a dose-related inhibition. In the high responder cardiac allograft model, graft survival in excess of 100 days was achieved with AR-C117977 compared with 20 days with CsA and all the long-term survivors exhibited donor-specific suppression on retransplantation. In the low responder model, both AR-C117977 and CsA induced survival in excess of 100 days. Histology of the long-term surviving grafts suggested reduced vasculopathy associated with chronic rejection. Furthermore, AR-C117977 inhibited the occlusion of transplanted trachea in a OB model. CONCLUSION: This report describes a MCT-1 specific inhibitor having immunosuppressive activity on alloimmune responses and inducing donor-specific suppression.

Sodium valproate versus lamotrigine: a randomised comparison of efficacy, tolerability and effects on circulating androgenic hormones in newly diagnosed epilepsy.

We have performed a randomised, prospective study to compare the efficacy and tolerability of sodium valproate (VPA) and lamotrigine (LTG) monotherapy, and their effects on circulating androgenic hormones, in newly diagnosed epilepsy. A total of 225 patients (116 male; median age 35 years, range 13-80 years) were followed-up at 6-weekly intervals until they reached an end-point (12 months' seizure freedom; withdrawal due to intolerable side-effects; lack of efficacy despite adequate dosing). Twelve month seizure-free rates were identical (47%) in the VPA (n=111) and LTG (n=114) treatment arms. More patients taking VPA withdrew from the study due to adverse events (26 VPA versus 15 LTG; p=0.046). Eight patients, all taking VPA, dropped out during the first 6 months due to weight gain. There were no changes in mean serum concentrations of testosterone, sex-hormone binding globulin and androstenedione or in the free androgen index after 6 or 12 months' treatment with either drug in 112 patients who fulfilled the criteria for hormone analysis. No difference in efficacy was found between VPA and LTG in our patients with newly diagnosed epilepsy. LTG appeared to be better tolerated. Neither drug appeared to alter the circulating levels of androgenic hormones.

Shape discrimination in age-related macular degeneration.

PURPOSE: Recent studies suggest that a global shape-discrimination task is sensitive to neural undersampling and/or irregular sampling, but is not affected by normal aging. In this study, the ability of patients with age-related macular degeneration (AMD) to perform the shape-discrimination task was examined. METHODS: Twenty patients with AMD (age range, 66-81 years) were selected on the basis of Snellen visual acuity of 20/50 or better in at least one eye and prior clinical documentation. A control group consisted of 10 older subjects (age range, 61-93 years) with normal findings in a fundus examination. Radial frequency (RF) patterns were used as stimuli. A spatial paradigm and a temporal two-alternative, forced-choice (2AFC) staircase paradigm were used. In each trial, two RF patterns (one deformed and one undeformed) were presented, and patients were asked to identify the deformed pattern. The peak spatial frequency of RF patterns was 5 cyc/deg; the radial modulation frequency was 8 cyc/360 degrees; mean radii were 0.5 degrees, 1 degrees, 2.0 degrees, or 2.5 degrees; and stimulus contrast was 80%. Thresholds for detecting the deformation were estimated by a maximum-likelihood fitting procedure. RESULTS: Thirty-five of 40 eyes with AMD had 20/50 or better acuity. Among them, 29 eyes had early AMD (drusen, hyperpigmentation, hypopigmentation), 5 had extrafoveal geographic atrophy, and 1 had exudative AMD. With the spatial 2AFC, 91% (32/35) of eyes with AMD showed significant elevation of the threshold for detecting radial deformation of RF patterns when compared with normal control eyes. With the temporal 2AFC, 97% (31/32) of eyes with AMD showed significant threshold elevations, and the degree of the deficit in the shape discrimination did not correlate significantly with visual acuity loss (r = 0.3, P = 0.094). Comparison of the severity of AMD with shape-discrimination performance revealed that the average detection threshold of the eyes with extrafoveal geographic atrophy was significantly higher than that of the eyes with drusen only (P < 0.01), even though average acuity showed no significant difference. CONCLUSIONS: Patients with AMD had significant deficits in performing the global shape-discrimination task. The dissociation of shape discrimination with visual acuity suggests that the shape-discrimination task may provide distinguishable information about the integrity of the photoreceptor mosaic in AMD.

The quality of drinking water from private water supplies in Aberdeenshire, UK.

The quality of private water supplies within Aberdeenshire sampled between 1992 and 1998 was analysed with respect to the presence of total coliforms (TC), faecal coliforms (FC), and nitrate. Of the approximately 1750 samples analysed, which included multiple samples from larger supply categories, the individual failure rate was 41%, 30% and 15% for TC, FC and nitrate, respectively. A combined failure rate for these samples was 48%. Failure rates on microbiological grounds displayed a seasonal trend being greater during the latter half of the year. Although this observation is likely to be due to a combination of local and regional scale factors, part of the variability in failure rate was explained by a significant positive relationship with rainfall amount. Concentrations of nitrate tended to display an opposite trend with a greater number of failures occurring during the spring period and no relationship with rainfall was immediately apparent. A relatively small number of samples (< 50) failed simultaneously for both coliforms and nitrate suggesting that the mechanism responsible for the contamination differed. A similar failure rate for samples collected directly from the source (i.e. well) compared with those taken from the potable tap (usually kitchen cold water tap) suggests that it is the groundwater source itself that contributes much of the microbiological and nitrate contamination rather than a storage or supply line contamination mechanism. A more frequent and random sampling of category one F supplies suggested a greater overall failure rate, which has its own implications for deciding an appropriate sampling frequency.

Using web technology to support population-based diabetes care.

BACKGROUND: Managed clinical networks have been used to coordinate chronic disease management across geographical regions in the United Kingdom. Our objective was to review how clinical networks and multidisciplinary team-working can be supported by Web-based information technology while clinical requirements continually change. METHODS: A Web-based population information system was developed and implemented in November 2000. The system incorporates local guidelines and shared clinical information based upon a national dataset for multispecialty use. Automated data linkages were developed to link to the master index database, biochemistry, eye screening, and general practice systems and hospital diabetes clinics. Web-based data collection forms were developed where computer systems did not exist. The experience over the first 10 years (to October 2010) was reviewed. RESULTS: The number of people with diabetes in Tayside increased from 9694 (2.5% prevalence) in 2001 to 18,355 (4.6%) in 2010. The user base remained stable (~400 users), showing a high level of clinical utility was maintained. Automated processes support a single point of data entry with 10,350 clinical messages containing 40,463 data items sent to external systems during year 10. The system supported quality improvement of diabetes care; for example, foot risk recording increased from 36% in 2007 to 73.3% in 2010. CONCLUSIONS: Shared-care datasets can improve communication between health care service providers. Web-based technology can support clinical networks in providing comprehensive, seamless care across a geographical region for people with diabetes. While health care requirements evolve, technology can adapt, remain usable, and contribute significantly to quality improvement and working practice.

Genetic variants in microsomal epoxide hydrolase influence carbamazepine dosing.

OBJECTIVES: The dose of carbamazepine required to achieve optimal seizure control varies widely from patient to patient. We investigated polymorphic variants in various genes involved in the pharmacokinetics and pharmacodynamics of carbamazepine in an effort to identify predictors of maintenance dose. METHODS: : A total of 70 patients with epilepsy (49% were males; median age, 34 years; range, 14-72 years) who had benefited (>50% reduction in seizure frequency for at least 12 months) from treatment with carbamazepine monotherapy were included in the analysis. Known variants in drug-metabolizing enzyme genes, including those encoding cytochrome P450s, uridine 5'-diphosphate-glycosyltransferase, and microsomal epoxide hydrolase, together with a sodium channel polymorphism in SCN2A, were screened using polymerase chain reaction-restriction fragment length polymorphism or direct sequencing. Associations between demographic and genetic variables and carbamazepine dose were identified by univariate and multivariate regression analyses. RESULTS: All genotype frequencies were consistent with Hardy-Weinberg equilibrium (P > 0.05). No single demographic or genetic variable was of sufficient strength to independently influence carbamazepine dosing requirements. However, a multivariate model, incorporating patient age and specific genotypes (c.337T>C, c.416A>G) of the EPHX1 gene encoding microsomal epoxide hydrolase, revealed a significant association with the maintenance dose of carbamazepine (r(2) = 0.362, P= 0.002). CONCLUSIONS: This proof-of-principle study suggests that genetic variants in EPHX1 can be used to predict maintenance doses of carbamazepine. A large-scale prospective investigation of genetic influences on drug dosing strategies in epilepsy, with specific focus on whole gene variability for those proteins involved in the pharmacokinetics and pharmacodynamics of antiepileptic agents, is warranted.

Prion removal by nanofiltration under different experimental conditions.

Manufacturing processes used in the production of biopharmaceutical or biological products should be evaluated for their ability to remove potential contaminants, including TSE agents. In the present study, we have evaluated scrapie prion protein (PrP Sc) removal in the presence of different starting materials, using virus removal filters of different pore sizes. Following 75 nm filtration, PrP Sc was detected in the filtrate by Western blot (WB) analysis when a "super-sonicated" microsomal fraction derived from hamster adapted scrapie strain 263K (263K MF) was used as the spike material. In contrast, no PrP Sc was detected when an untreated 263K MF was used. By using spike materials prepared in a manner designed to optimize the particle size distribution within the preparation, only 15 nm filtration was shown to remove PrP Sc to below the limits of detection of the WB assays used under all the experimental conditions. However, infectious PrP Sc was recovered following 15 nm filtration under one experimental condition. The results obtained suggest that the nature of the spike preparation is an important factor in evaluating the ability of filters to remove prions, and that procedures designed to minimize the particle size distribution of the prion spike, such as the "super-sonication" or detergent treatments described herein, should be used for the preparation of the spike materials.

Reducing minors' access to tobacco: eight years' experience in Hawaii.

OBJECTIVE: This paper reports the findings of annual Synar inspections to assess compliance with federal and state legislation to limit minors' access to tobacco products in Hawaii. We also report on factors associated with selling tobacco to minors for the most recent year of inspections (2003). METHODS: Annual, random, unannounced inspections were conducted by minors (ages 14-17 years) over an 8-year period (1996-2003). Stores were randomly selected from a list of stores that sell tobacco products in Hawaii. RESULTS: There was a significant decrease in the percent of successful purchases made over the period from 1996 (44.5%) to 2003 (6.2%). Based on multivariate analyses, only two variables were associated with whether a successful purchase was made in 2003: whether the minor's identification or age was requested. CONCLUSION: Findings suggest that surveillance, education, and enforcement efforts in Hawaii have been successful at making substantial reductions in noncompliance rates. Even with the current low rate of sales to minors, failing to request the minor's identification or age was associated with making a successful purchase, while characteristics of the minor and retail environment were not.

Effects of structural variation in xyloglucan polymers on interactions with bacterial cellulose.

A cellulose/xyloglucan framework is considered to form the basis for the mechanical properties of primary plant cell walls and hence to have a major influence on the biomechanical properties of growing, fleshy plant tissues. In this study, structural variants of xyloglucan have been investigated as components of composites with bacterial cellulose as a simplified model for the cellulose/xyloglucan framework of primary plant cell walls. Evidence for molecular binding to cellulose with perturbation of cellulose crystallinity was found for all xyloglucan types. High molecular mass samples gave homogeneous centimeter-scale composites with extensive cross-linking of cellulose with xyloglucan. Lower molecular mass xyloglucans gave heterogeneous composites having a range of microscopic structures with little, if any, cross-linking. Xyloglucans with reduced levels of galactose substitution had evidence of self-association, competitive with cellulose binding. At comparable molecular mass, fucose substitution resulted in a modest promotion of microscopic features characteristic of primary cell walls. Taken together, the data are evidence that galactose substitution of the xyloglucan core structure is a major determinant of cellulose composite formation and properties, with additional fucose substitution acting as a secondary modulator. These conclusions are consistent with reported structural and mechanical properties of Arabidopsis mutants lacking specific fucose and/or galactose residues.

Comparison of select innate immune mechanisms of fish and mammals.

The study of innate immunity has become increasingly popular since the discovery of homologs of many of the innate immune system components and pathways in lower organisms including invertebrates. As fish occupy a key position in the evolution of the innate and adaptive immune responses, there has been a great deal of interest regarding similarities and differences between their defense mechanisms and those of higher vertebrates. This review focuses on describing select mechanisms of the innate immune responses of fish and the implications for evolution of immunity in higher vertebrates.

Lack of association between the C3435T polymorphism in the human multidrug resistance (MDR1) gene and response to antiepileptic drug treatment.

PURPOSE: P-glycoprotein (P-gp) has been implicated in the causation of refractory epilepsy. The expression and efflux efficiency of P-gp is influenced by a polymorphism (C3435T) in the encoding gene (MDR1). Recent evidence suggests that the homozygous C-variant, which is associated with higher expression and increased activity of P-gp, is more common in patients with pharmacoresistant epilepsy. We have investigated the prevalence of this polymorphism in a series of patients attending a specialist epilepsy clinic. METHODS: DNA samples were obtained from 400 patients, irrespective of seizure type or drug treatment. Genotype of the C3435T polymorphism was determined by traditional polymerase chain reaction (PCR) followed by restriction digest. Classification of response to treatment was determined in a blinded fashion by an independent physician. Results were expressed as genotype and allele frequencies per response group and compared by logistic regression analysis. RESULTS: In total, 170 patients were classified as responders, with > or =12 months seizure freedom on current treatment. The remaining 230 patients were classified as nonresponders. Comparison of responders and nonresponders revealed no significant difference in allele frequency (C vs. T; odds ratio, 1.03; 95% CI, 0.78-1.37; p = 0.83) or genotype frequency (CC vs TT; odds ratio, 1.07; 95% CI, 0.60-1.91; p = 0.81). Subanalyses of individual seizure types were similarly unremarkable. CONCLUSIONS: This study failed to corroborate a previously reported association between the C3435T polymorphism in the human MDR1 gene and pharmacoresistant epilepsy. Whether the C3435T polymorphism can act as a marker for the natural history of treated epilepsy remains to be determined.

Pregabalin drug interaction studies: lack of effect on the pharmacokinetics of carbamazepine, phenytoin, lamotrigine, and valproate in patients with partial epilepsy.

PURPOSE: Pregabalin (PGB) is an alpha2-delta ligand with demonstrated efficacy in epilepsy, neuropathic pain, and anxiety disorders. PGB is highly efficacious as adjunctive therapy in patients with refractory partial seizures. METHODS: Given its efficacy as adjunctive therapy, the potential for interaction of PGB with other antiepileptic drugs (AEDs) was assessed in patients with partial epilepsy in open-label, multiple-dose studies. Patients received PGB, 600 mg/day (200 mg q8h) for 7 days, in combination with their individualized maintenance monotherapy with valproate (VPA), phenytoin (PHT), lamotrigine (LTG), or carbamazepine (CBZ). RESULTS: Trough steady-state concentrations of CBZ (and its epoxide metabolite), PHT, LTG, and VPA were unaffected by concomitant PGB administration. Likewise, PGB steady-state pharmacokinetic parameter values were similar among patients receiving CBZ, PHT, LTG, or VPA and, in general, were similar to those observed historically in healthy subjects receiving PGB alone. The PGB-AED combinations were generally well tolerated. PGB may be added to VPA, LTG, PHT, or CBZ therapy without concern for pharmacokinetic drug-drug interactions.

Recombinant transferrin induces nitric oxide response in goldfish and murine macrophages.

We have previously demonstrated that the serum protein transferrin plays a key role in the activation of primary goldfish macrophages. The ability of this protein to activate goldfish macrophages was also shown to be dependent on enzymatic cleavage of the native (i.e. full-length) protein into immunostimulatory fragments. In this study, we report that immunostimulatory fragments of recombinant goldfish transferrin (rGTf), induced a potent nitric oxide (NO) response in goldfish as well as in murine macrophages. Specifically, recombinant goldfish transferrin N- and C-lobe fragments expressed in Escherichia coli induced the NO response in goldfish and murine macrophages. As little as 75-150 ng of the recombinant proteins (N- or C-lobe) had biological activity, even in the presence of 10 microg/ml of the LPS inhibitor polymixin B sulphate (PMB). These findings indicate that transferrin is a key conserved component required for induction of macrophage antimicrobial responses of fish and provide evidence that a similar induction pathway exists in mammals, which has not been reported previously.