James R. Wright

Histological study of the development of the embryo and early larva of Oreochromis niloticus (Pisces: Cichlidae)

The developmental stages of Oreochromis niloticus are similar to those described in other mouth‐breeding tilapias except that, as in zebrafish, no cavity was found in the blastula. Variation in the rate of development of the embryo and larva of O. niloticus was found within a clutch of eggs as well as between clutches. Hatching glands are described for the first time in tilapias. They are widely distributed within the ectoderm covering the head, body, tail, and surface of the yolk sac near its attachment to the embryo. Timing of larval development is similar to that in other mouthbrooding tilapias, but is slower than that found in substrate‐spawning tilapias. A pneumatic duct connects the swimbladder to the digestive tract and swimbladder inflation and initiation of feeding occurs at about the same time. The digestive tract of the larva 8 and 9 days after fertilization is similar to that found in the adult, except that there are no digestive glands. An endocrine pancreatic islet was first seen 76 h after fertilization. A prominent thymus gland is present at 100 h. Hematopoietic tissue develops in the vicinity of the pronephros during early larval development. A spleen develops later, 7 days after fertilization. J. Morphol. 247:172–195, 2001.

Oxygen Free-Radical Scavengers and Immune Destruction of Murine Islets in Allograft Rejection and Multiple Low-Dose Streptozocin-Induced Insulitis

We examined the effects of desferrioxamine (DFX), a potent inhibitor of the formation of oxygen-derived hydroxyl radicals, and nicotinamide (NIC), a poly(ADP-ribose) synthetase inhibitor and a weak free-radical scavenger, on two models of immune destruction of murine islets [i.e., allograft rejection and multiple low-dose streptozocin (STZ)-induced insulitis]. Freshly isolated or low-temperature-cultured BALB/cJ islets were transplanted beneath the kidney capsules of C57BL/6J recipients. The recipients were treated with NIC alone (500 mg · kg−1 · day−1), DFX alone (4.2 mg/day × 14 days), or NIC + DFX. Only recipients treated with NIC + DFX, receiving cultured islets, showed a mean graft survival time significantly longer than control mice receiving freshly isolated or cultured islets. Control CD-1 mice treated with multiple low doses of STZ developed insulitis and diabetes. Treatment with NIC alone, DFX alone, or NIC + DFX decreased the severity of hyperglycemia relative to the controls. Treatment with DFX alone was more effective than NIC alone or NIC + DFX. Only the group treated with DFX alone had a lower incidence of diabetes (mean plasma glucose level >200 mg/dl) than the controls after 4 wk. Histologically, islets from control mice showed severe insulitis, islet destruction, and absence of stainable insulin, whereas islets from DFX-treated mice showed only mild peri-insulitis and a relative preservation of β-cell granulation. Our study showed that NIC and DFX partially protect islets from immune destruction in allograft rejection and in low-dose STZ-induced insulitis. Apparently, hydroxyl radicals play important roles in both of these models.

Histological analysis of achilles tendons in an overuse rat model

The purpose of this study was to design an animal model that induces histological changes in Achilles tendons consistent with those cited in the literature for human Achilles tendon disease. Sprague‐Dawley rats were subjected to 10° uphill treadmill running on a custom‐designed rodent treadmill and at a speed of 17 meters per minute for 1 h, five times per week, over a 12‐week treatment period. Subsequent histological analysis revealed alterations in the rat Achilles tendon that were generally consistent with those described in the literature for diseased human tendon tissues. These features include: decreased collagen fiber organization, more intense collagen staining, and increased cell nuclei numbers. Interestingly, though, immunohistochemical cell typing suggests that the observed increased cellularity does not include a significant inflammatory component but is secondary to increased numbers of endothelial cells (i.e., vascularization) and fibroblasts. These histological features likely represent a biological repair/remodeling response resulting from overuse running.

Co-encapsulation of Sertoli enriched testicular cell fractions further prolongs fish-to-mouse islet xenograft survival.

BACKGROUND We previously demonstrated that alginate microencapsulation can prolong fish (tilapia) islet xenograft survival in diabetic animals. However, at present, microencapsulation does not provide complete immune protection to discordant islet xenografts, and long-term graft survival requires supplemental low-dose systemic immunosuppression. In the present study, fish islets were co-encapsulated with Sertoli enriched testicular cell fractions to find out whether this would further prolong fish islet graft survival in diabetic mice. METHODS Sertoli enriched testicular cell fractions were enzymatically harvested from adult Balb/c or Wistar-Furth rats. They were cultured and co-encapsulated with fragmented tilapia islets in alginate microcapsules. Encapsulated islets alone or islets co-encapsulated with Sertoli cells were then intraperitoneally transplanted into streptozotocin-diabetic Balb/c mice, and graft survival times were compared. Encapsulated and co-encapsulated islet function was also confirmed in streptozotocin-diabetic athymic nude mice. RESULTS Co-encapsulation with Sertoli enriched testicular cell fractions further prolonged mean fish islet graft survival time from 21+/-6.7 days (encapsulated islet cells alone) to >46+/-6.3 days (co-encapsulated with syngeneic murine Sertoli cells), without additional systemic immunosuppression. Testicular cells harvested from xenogeneic Wistar-Furth rats produced similar protective results (>46+/-10.9 days). CONCLUSIONS Our results support the theory that Sertoli cells produce local immunosuppressive factors. These factors supplement the immune protective feature of alginate microcapsules in our model. Testicular cell fractions may be an important naturally occurring facilitator in the development of new microencapsulation systems for islet xenotransplantation.

The Nlrp3 inflammasome promotes myocardial dysfunction in structural cardiomyopathy through interleukin-1β.

•  What is the central question of this study? Heart failure is associated with persistent sterile inflammation that worsens disease severity; however, the molecular mechanisms behind cytokine recruitment and their relevance in the diseased myocardium remain unknown. •  What is the main finding and its importance? We show that interleukin‐1β is activated downstream of the Nlrp3 inflammasome in calcineurin‐transgene‐induced structural heart disease. Genetic deletion of Nlrp3 abrogated inflammasome signalling and interleukin‐1β release, improving function. The role of Nlrp3 in non‐ischaemic cardiomyopathy and the utility of inflammasome antagonism have not yet been explored, revealing potential for translational application.

Liposomal tacrolimus lotion as a novel topical agent for treatment of immune-mediated skin disorders: experimental studies in a murine model

Summary    Background Systemic but not topical tacrolimus (TAC) is effective against psoriasis. Mechanical methods that enhance skin penetration by TAC increase its topical antipsoriatic effect. Liposomal delivery of TAC would increase its penetration of skin, allow for slow release and diminish its toxicity.

Prolongation of rat islet allograft survival by treatment with monoclonal antibodies against VLA-4 and LFA-1.

In this study, we investigated the effects of treatment with monoclonal antibodies against the VLA-4 and LFA-1 adhesion molecules on rat islet allograft rejection. TA-2 and TA-3 are function-blocking mAb against rat VLA-4 and LFA-1, respectively. Lewis rats were made diabetic (plasma glucose levels > 22.2 mmol/L) with streptozotocin. One week later, 1500 freshly isolated Wistar Furth rat islets were transplanted under the left kidney capsule of each rat. Monoclonal antibodies were administered intravenously at a dosage of 2 mg on the day of islet transplantation and then intraperitoneally every second day for 3 weeks or until graft rejection. Plasma glucose levels were monitored at least 3 times a week and blood leukocyte counts were monitored every 4 days. Rejection was defined as 2 plasma glucose levels > 11.1 mmol/L. Mean graft survival times in untreated and control mAb-treated rats were 5.3 and 6.0 days, respectively. Treatment with anti-VLA-4 or anti-LFA-1 resulted in only modest prolongation of mean graft survival time (9.3 and 7.4 days, respectively). However, treatment with the combination of anti-VLA-4 plus anti-LFA-1 resulted in long-term (i.e., 60-day) graft survival in 5 of 7 rats. Graft nephrectomy and histology confirmed islet graft survival at 60 days. A second Wistar Furth rat islet graft under the opposite renal capsule after graft nephrectomy did not show full tolerance; however, the function of the second graft was significantly prolonged without any immunosuppression. Combined blockade of VLA-4 and LFA-1 also markedly prolonged islet graft survival when islets were transplanted via the portal vein. In conclusion, both VLA-4 and LFA-1 play a role in islet allograft rejection and blockade of both prevents or greatly delays graft rejection.

Experimental Transplantation With Principal Islets of Teleost Fish (Brockmann Bodies): Long-Term Function of Tilapia Islet Tissue in Diabetic Nude Mice

Certain teleost fish have macroscopically visible islets called BBs that are anatomically discrete. BBs were harvested from Oreochromis nilotica (tilapia) with microscissors, divided, and cultured overnight at 37°C before transplantation into STZ-induced diabetic nude mice. Each mouse received BB fragments from 3-5 fish weighing in aggregate ∼1.7 kg. Non-FPGs were monitored 5 days/wk. Recipients remained normoglycemic (plasma glucose <11.1 mM) for 50 days posttransplantation. Mice bearing 50-day-old grafts had essentially normal GTTs. Left nephrectomies then were performed to remove the grafts, and plasma glucose levels in recipient mice rose to >22.2 mM. Histological examination of graft-bearing kidneys showed viable, vascularized islet tissue containing numerous well-granulated β-cells; examination of recipient native pancreases revealed small islets composed predominately of non-β-cells.

Long-term function of fish islet xenografts in mice by alginate encapsulation.

BACKGROUND Large, anatomically discrete pancreatic islets, Brockmann bodies (BBs), exist in certain teleost fish. When transplanted under the renal capsules of streptozotocin-diabetic athymic nude mice, BB grafts produce uniform normoglycemia for 50 days and mammalian-like glucose tolerance profiles; however, these very discordant islets reject in 7-8 days when transplanted into euthymic BALB/c mice. METHODS In the present study, BBs were mass harvested, minced into <1-mm tissue fragments, and encapsulated in alginate-based macrospheres (5 mm diameter) or noodles (0.5x3 cm). Nonencapsulated and encapsulated BB fragments were transplanted intraperitoneally into streptozotocin-diabetic (nonfasting blood glucose >400 mg/dl) nu/nu and BALB/c mice. Glucose levels were monitored at least 3 times a week. RESULTS Encapsulated BB grafts uniformly survived >50 days (10/10) or >100 days (3/3) in nu/nu recipients. The mean graft survival time was 27+/-13 days in BALB/c recipients (n=7). Daily intraperitoneal administration of 2.5 mg/kg 15-deoxyspergualin, in combination with encapsulation, resulted in uniform long-term BB graft function in BALB/c recipients (n=5). Similarly, long-term function was achieved in four of six BALB/c recipients with daily intraperitoneal administration of 10 mg/kg cyclosporine (two grafts failed after 39 and 45 days). Nonencapsulated BB grafts transplanted intraperitoneally into BALB/c or nu/nu recipients functioned for <7 days; immunosuppression alone did not permit graft survival in BALB/c recipients. In all cases of graft survival of >50 days, grafts were surgically removed from the peritoneal cavity, and blood sugar levels returned to a diabetic state within a few days. Historical sections of grafts, stained with hematoxylin and eosin and immunoperoxidase for insulin, showed viable, well-granulated BB tissue. CONCLUSIONS This study demonstrates that tilapia BBs are suitable for encapsulation and that encapsulated BBs can be made to function long term in diabetic mice.

Tilapia—A Source of Hypoxia-Resistant Islet Cells for Encapsulation

Encapsulation of pancreatic islets prevents graft revascularization after transplantation, resulting in graft hypoxia and attrition. Hypoxia-resistant islets would be ideal for encapsulation. Tilapia, a tropical teleost fish, have large, anatomically discrete islets that can be easily harvested without expensive, fickle islet isolation procedures and that provide mammalian-like glucose tolerance profiles when transplanted into diabetic recipients. Because tilapia can live in stagnant water, we speculated that tilapia islets might tolerate lower oxygen tensions than mammalian islets. Tilapia and rat islets (n = 30) were placed in paired 60-mm Petri dishes containing 10 mL of deoxygenated CMRL-1066 media and cultured together in sealed chambers gassed with 95% N2/5% CO2. Islet viability was determined by fluorscein diacetate/ethidium bromide staining at intervals varying from 2.5 h to 7 days; blood gas measurements were obtained on media samples at the end of selected incubation intervals. Rat islets underwent near-total necrosis and fragmentation in <24 h; occasional viable single cells could be identified until about 72 h. On the other hand, the fish islets showed no loss of viability until about 72 h when some showed mild central necrosis. Even at 7 days, all fish islets appeared roughly 50% viable. Fish islets cultured under hypoxic conditions for 72 h (media, pO2 = 27.8 mmHg) and then transplanted into streptozotocin-diabetic athymic nude mice were viable (6/6) but showed some diminished function (3/6) over a 25-day follow-up period. Our results suggest that tilapia islets will survive and function at lower oxygen tensions than mammalian islets.