Specific and non-overlapping functions of testosterone and 11-ketotestosterone in the regulation of professional phagocyte responses in the teleost fish gilthead seabream.

Sex hormones, both estrogens and androgens, have a strong impact on immunity in mammals. In fish, the role of androgens in immunity has received little attention and contradictory conclusions have been obtained. However, it is well known that sex steroids are involved in fish growth, osmoregulation and gonad remodelation. In this study, we examine the in vitro effects of testosterone and 11-ketotestosterone, the two main fish androgens, on the professional phagocytes of the teleost fish gilthead seabream (Sparus aurata L.). Although both testosterone and 11-ketotestosterone failed to modulate the respiratory burst of seabream phagocytes, testosterone but not 11-ketotestosterone was able to increase the phagocytic ability of non-activated phagocytes. Curiously, 11-ketotestosterone was more powerful than testosterone at inducing the expression of its own receptor, namely androgen receptor b (ARb), in acidophilic granulocytes (AGs), but none of them affected the basal ARb expression levels in macrophages (MØ). Furthermore, although physiological concentrations of testosterone exerted a pro-inflammatory effect on both AGs and MØs, 11-ketotestosterone showed an anti-inflammatory effect in AGs and a strong pro-inflammatory effect in MØs. Interestingly, both androgens modulated the expression of toll-like receptors in these two immune cell types, suggesting that androgens might regulate the sensitivity of phagocytes to pathogens and damage signals. Testosterone and 11-ketotestosterone have a competitive effect, at least, on the modulation of the expression of some genes. Therefore, our results show for the first time a non-overlapping role for testosterone and 11-ketotestosterone in the regulation of professional phagocyte functions in fish.

In situ forming microparticle implants for delivery of sex steroids in fish: Modulation of the immune response of gilthead seabream by testosterone.

Current knowledge on the sensitivity of marine fish to androgenic environmental chemicals is limited, despite the growing interest in the effects of endocrine disrupting chemicals. To study in vivo the effects of testosterone (T) on the fish immune response, we used a microencapsulation implant technique, the in situ forming microparticle system, containing 1 mg T/kg body weight (T-ISM), in adult specimens of gilthead seabream (Sparus aurata L.), a species of great economic interest. We demonstrated that implants themselves (without T) have no significant effect on most of the parameters measured. In T-ISM implanted fish, T serum levels reached supraphysiological concentrations accompanied by a slight increase in 11-ketotestosterone and 17ß-estradiol levels 21 days post-implantation (dpi). Liver and head-kidney samples were processed 7 and 21 dpi to assess T-ISM effect on (i) the mRNA expression of genes involved in the metabolism of steroid hormones and in the immune response, and (ii) phagocyte activities. The expression profile of cytokines, chemokines and immune receptors was altered in T-ISM implanted animals that showed an early pro-inflammatory tendency, and then, a mixed pro-/anti-inflammatory activation during longer exposure. Furthermore, the enhancement of phagocytic activity and the production of reactive oxygen species by leukocytes 21 dpi in T-ISM implanted specimens suggest fine modulation of the innate immune response by T. Taken together, these data demonstrate for the first time the feasibility of using ISM implants in an aquatic species, and provide new data on the role played by T on the immune response in fish.

Identification of T-cell stimulating antigens from Giardia lamblia by using Giardia-specific T-cell hybridomas.

T-cell immune response plays an important role in controlling Giardia lamblia infections. Little is known about the G. lamblia-specific antigens that stimulate a cell-mediated immune response. The aim of the present study was to identify T-cell stimulating G. lamblia antigens. For this purpose, we generated a group of Giardia-specific T-cell hybridomas (2F9, 4D5, 6D10, 8B9, 9B10, 10F7 and 10G5). Hybridomas were screened for reactivity with G. lamblia protein extract by the CTLL bioassay. These T-cell hybridomas did not exhibit any significant activation either in the absence of G. lamblia protein extract or in the presence of irrelevant antigen (hen white egg lysozyme). To further characterize the T-cell hybridomas generated, we selected three hybridomas (10G5, 4D5 and 9B10). Giardia lamblia proteins of 90-110, 65-77 and 40-64 kDa showed T-cell stimulating activity for the hybridomas 10G5, 4D5 and 9B10, respectively, in a concentration-dependent manner. Protein extract obtained from different G. lamblia strains (GS/M-83-H7, WB C6 and a clinical isolate (YJJ)) stimulated all T-cell hybridomas, indicating that T-cell-stimulating antigens are expressed among different G. lamblia strains. In conclusion, we identified T-cell stimulating G. lamblia antigens by using Giardia-specific T-cell hybridomas. To our knowledge, these hybridomas are the first-described T-cell hybridomas specific for G. lamblia.

A role for matrix metalloproteinases in granulocyte infiltration and testicular remodelation in a seasonal breeding teleost.

The testicular cystic structure and the abrupt morphological changes that the fish testis undergoes during the reproductive cycle (RC) make it an interesting model for studying the regulation of spermatogenesis, in particular the role of matrix metalloproteinases (Mmps). The gilthead seabream is a seasonal breeding teleost whose testis undergoes drastic remodeling events, especially during the post-spawning stage when a massive infiltration of a immune cell type, the acidophilic granulocytes, occurred. Bearing this in mind, we studied the gilthead seabream testis gelatinolytic activities involved in migration and tissue remodeling and its regulation by endocrine, immune and tissue stimuli. Thus, we demonstrated that the germinal epithelium of the testis showed gelatinolytic activity during spermatogenesis and post-spawning but not during resting, when only scarce interstitial cells were stained. Moreover, the precursor and mature forms of two gelatinases, Mmp2- and Mmp9-like, were active in the gonad, whose activities were up-regulated by 17beta-estradiol (E(2)) but not by lipopolysaccharide (LPS)/bacterial DNA (VaDNA) in testicular cell suspensions. E(2) and LPS/VaDNA also up-regulated a variety of cytokines and chemokines. We also cloned mmp9, mmp13, tissue inhibitors of Mmps (timp)-2a and timp2b genes and found that all of them were expressed in the gonad in a RC stage-dependent manner. Interestingly, mmps and timps were highly expressed by the testicular acidophilic granulocytes. Moreover, in these cells, the gelatinolytic activity seemed to correspond to the precursor and mature forms of putative Mmp2 and Mmp9 gelatinases, while the main gelatinolytic activity seemed to correspond to the mature form of Mmp2 in head-kidney acidophilic granulocytes. Finally, although none of the stimuli used were able to induce the gelatinolytic activity of Mmp9-like in head-kidney acidophilic granulocytes, the expression of mmp9, timp2a and timp2b were all up-regulated by LPS/VaDNA in these cells, while only mmp9 and timp2a expression increased upon stimulation with gelatin.

Laparoscopic jejunostomy with an 18-mm trocar.

We describe our technique to perform laparoscopic jejunostomies with an 18-mm trocar. This procedure facilitates the exteriorization of the proximal bowel and construction of the jejunostomy. We describe our laparoscopic technique in nine patients with severe neurologic conditions (two in the postoperative period of a cerebral aneurysm in a coma, three patients with severe head injury, and four patients with cerebrovascular strokes). The operative time ranged from 20 to 75 min (average, 44.38 min). Nutrition was initiated 24 h after the placement of the jejunostomy. Tolerance of the enteral nutrition was excellent in all cases. One major complication occurred, minor leakage around the feeding tube 3 weeks after the jejunostomy was constructed. The jejunostomy was removed without further consequences. Laparoscopy is an effective technique for the creation of feeding jejunostomies. We believe that this minimally invasive approach is an alternative for patients requiring long-term postpyloric enteral feeding.