Influence of ultraviolet-B irradiation on engraftment, graft-versus-host disease and graft-versus-leukemia effect in a rat model for allogeneic bone marrow transplantation

Ultraviolet-B (UVB) irradiation is known to inhibit lymphocyte activity and consequently to reduce the incidence of graft-versus-host disease (GVHD) in experimental models for allogeneic bone marrow transplantation (BMT). GVHD is frequently associated with morbidity and mortality, but also with the beneficial graft-versus-leukemia (GVL) effect, demonstrated by a reduction in the incidence of leukemia relapse. In this study, we investigated whether UVB treatment of allogeneic T cells could prevent GVHD while sparing the beneficial GVL effect following allogeneic BMT in the Brown Norway myelocytic leukemia (BNML) rat model analogous to human acute myelocytic leukemia (AML). The dose of UVB required to abolish lethal GVHD in the rat allogeneic BMT model (WAG/Rij donors into BN recipients) was 4000 J/m2. However, this UVB dose simultaneously abrogated all GVL activity mediated by the T cells in the graft, while the radio-protective capacity of rat BM cells was strongly reduced. The number of allogeneic BM cells required to protect lethally irradiated BN rats was increased 50 to 100-fold. It is concluded that UVB acts as a non-selective form of T cell inactivation, and that UVB pretreatment of an allogeneic marrow graft is unlikely to be useful clinically as a preventive measure for GVHD, since other means of reduction of the number of functional T cells are less damaging to bone marrow stem cells.

Relationship between the magnitude of intraocular pressure during an episode of acute elevation and retinal damage four weeks later in rats

Purpose: To determine relationship between the magnitude of intraocular pressure (IOP) during a fixed-duration episode of acute elevation and the loss of retinal function and structure 4 weeks later in rats.

Methods: Unilateral elevation of IOP (105 minutes) was achieved manometrically in adult Brown Norway rats (9 groups; n = 4 to 8 each, 10–100 mm Hg and sham control). Full-field ERGs were recorded simultaneously from treated and control eyes 4 weeks after IOP elevation. Scotopic ERG stimuli were white flashes (26.04 to 2.72 log cd.s.m^-2). Photopic ERGs were recorded (1.22 to 2.72 log cd.s.m22) after 15 min of light adaptation (150 cd/m2). Relative amplitude (treated/control, %) of ERG components versus IOP was described with a cummulative normal function. Retinal ganglion cell (RGC) layer density was determined post mortem by histology.

Results: All ERG components failed to recover completely normal amplitudes by 4 weeks after the insult if IOP was 70 mmHg or greater during the episode. There was no ERG recovery at all if IOP was 100 mmHg. Outer retinal (photoreceptor) function demonstrated the least sensitivity to prior acute IOP elevation. ERG components reflecting inner retinal function were correlated with post mortem RGC layer density.

Conclusions: Retinal function recovers after IOP normalization, such that it requires a level of acute IOP elevation approximately 10 mmHg higher to cause a pattern of permanent dysfunction similar to that observed during the acute event. There is a ‘threshold’ for permanent retinal functional loss in the rat at an IOP between 60 and 70 mmHg if sustained for 105 minutes or more.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) as a therapeutic target to prevent retinal vasopermeability during diabetes

Lipoprotein-associated phospholipase A2 (Lp-PLA2) hydrolyses oxidized low-density lipoproteins into proinflammatory products, which can have detrimental effects on vascular function. As a specific inhibitor of Lp-PLA2, darapladib has been shown to be protective against atherogenesis and vascular leakage in diabetic and hypercholesterolemic animal models. This study has investigated whether Lp-PLA2 and its major enzymatic product, lysophosphatidylcholine (LPC), are involved in blood-retinal barrier (BRB) damage during diabetic retinopathy. We assessed BRB protection in diabetic rats through use of species-specific analogs of darapladib. Systemic Lp-PLA2 inhibition using SB-435495 at 10 mg/kg (i.p.) effectively suppressed BRB breakdown in streptozotocin-diabetic Brown Norway rats. This inhibitory effect was comparable to intravitreal VEGF neutralization, and the protection against BRB dysfunction was additive when both targets were inhibited simultaneously. Mechanistic studies in primary brain and retinal microvascular endothelial cells, as well as occluded rat pial microvessels, showed that luminal but not abluminal LPC potently induced permeability, and that this required signaling by the VEGF receptor 2 (VEGFR2). Taken together, this study demonstrates that Lp-PLA2 inhibition can effectively prevent diabetes-mediated BRB dysfunction and that LPC impacts on the retinal vascular endothelium to induce vasopermeability via VEGFR2. Thus, Lp-PLA2 may be a useful therapeutic target for patients with diabetic macular edema (DME), perhaps in combination with currently administered anti-VEGF agents.

A new model of posterior capsule opacification in rodents

PURPOSE. To describe a new model of posterior capsule opacification (PCO) in rodents METHODS. An extracapsular lens extraction (ECLE), by continuous curvilinear capsulorrhexis and hydrodissection, was performed in 42 consecutive Brown Norway rats. Animals were killed at 0, 6, and 24 hours and 3, 7, and 14 days after surgery. Eyes were enucleated and processed for light microscopy and immunohistochemistry. RESULTS. In 34 (81%) of the animals the operated eye appeared well healed before death, with a clear cornea and a well-formed anterior chamber. In eight (19%) there was no view of anterior segment structures because of hyphema, fibrin, or corneal opacification. PCO was clinically evident 3 days after ECLE and was present in all animals at 2 weeks. Immediately after ECLE, lens epithelial cells (LECs) were present in the inner surface of the anterior capsule and lens bow. Twenty-four hours after surgery, LECs started to migrate toward the center of the posterior capsule. At 3 days, multilayered LECs, some spindle shaped, were present throughout the lens capsule. Capsular wrinkling was apparent. Lens fibers and Soemmering's ring were observed in all animals 14 days after surgery, indicating some degree of cellular differentiation. Activated macrophages were present in greater numbers at 3 and 14 days after surgery (P <0.05), when proliferation and migration of LECs appeared to be greatest, and lens fiber differentiation was evident, respectively. CONCLUSIONS. In rodents PCO occurs after ECLE and is associated with low-grade inflammation, mostly of mononuclear macrophages. Although no intraocular lens implantation was performed, this model appears to be valuable for studying the sequence of events that leads to PCO after cataract surgery and the extracellular matrix cues that promote lens fiber differentiation.

Amolopkinins W1 and W2 – novel bradykinin-related peptides (BRPs) from the skin of the Chinese torrent frog, Amolops wuyiensis: antagonists of bradykinin-induced smooth muscle contraction of the rat ileum.

Bradykinin-related peptides (BRPs) represent one of the most widespread and closely studied families of amphibian defensive skin secretion peptides. Apart from canonical bradykinin (RPPGFSPFR) that was first reported in skin extracts of the European brown frog, Rana temporaria, many additional site-substituted, N- and/or C-terminally extended peptides have been isolated from skin extracts and secretions from representative species of the families Ranidae, Hylidae, Bombinatoridae and Leiopelmatidae. The most diverse range of BRPs has been found in ranid frog skin secretions and this probably reflects the diversity and number of species studied and their associated life histories within this taxon. Amolops (torrent or cascade frogs) is a genus within the Ranidae that has been poorly studied. Here we report the presence of two novel BRPs in the skin secretions of the Chinese Wuyi Mountain torrent frog (Amolops wuyiensis). Amolopkinins W1 and W2 are dodecapeptides differing in only one amino acid residue at position 2 (Val/Ala) that are essentially (Leu1, Thr6)-bradykinins extended at the N-terminus by either RVAL (W1) or RAAL (W2). Amolopkinins W1 and W2 are structurally similar to amolopkinin L1 from Amolops loloensis and the major BRP (Leu1, Thr6, Trp8)-bradykinin from the skin of the Japanese frog, Rana sakuraii. A. wuyiensis amolopkinins were separately encoded as single copies within discrete precursors of 61 amino acid residues as deduced from cloned skin cDNA. Synthetic replicates of both peptides were found to potently antagonize the contractile effects of canonical bradykinin on isolated rat ileum smooth muscle preparations. Amolopkinins thus appear to represent a novel sub-family of ranid frog skin secretion BRPs.

Ranachensinin: A Novel Aliphatic Tachykinin from the Skin Secretion of the Chinese Brown Frog, Rana chensinensis

Amphibian skin secretions contain a plethora of pharmacologically-active substances and represent established sources of bioactive peptides, including tachykinins. Tachykinins are one of the most widely-studied peptide families in animals and are found in neuroendocrine tissues from the lowest vertebrates to mammals. They are characterized by the presence of a highly-conserved C-terminal pentapeptide amide sequence motif (-FXGLM-amide) that also constitutes the bioactive core of the peptide. Amidation of the C-terminal methioninyl residue appears to be mandatory in the expression of biological activity. Here, we describe the isolation, characterization and molecular cloning of a novel tachykinin named ranachensinin, from the skin secretion of the Chinese brown frog, Rana chensinensis. This peptide, DDTSDRSN QFIGLM-amide, contains the classical C-terminal pentapeptide amide motif in its primary structure and an Ile (I) residue in the variable X position. A synthetic replicate of ranachensinin, synthesized by solid-phase Fmoc chemistry, was found to contract the smooth muscle of rat urinary bladder with an EC50 of 20.46 nM. However, in contrast, it was found to be of low potency in contraction of rat ileum smooth muscle with an EC50 of 2.98 µM. These data illustrate that amphibian skin secretions continue to provide novel bioactive peptides with selective effects on functional targets in mammalian tissues.