Topical formulations of novel angiostatic steroids inhibit rabbit corneal neovascularization

PURPOSE: To evaluate the antiangiogenic potential of topical ophthalmic formulations of the novel angiostatic steroids AL-3789 and AL-4940, using a rabbit model of corneal neovascularization. METHODS: Neovascularization was induced in the rabbit cornea by surgical implantation of a standard ethylene vinyl acetate copolymer (Elvax-40) pellet containing 1 microg lipopolysaccharide. Coded formulations of the control vehicle or the following test agents were administered in prevention and intervention treatment protocols: 1% formulations of AL-3789, AL-4940, and cortisol acetate as a positive drug control. Three doses of AL-3789 (0.01%, 0.1%, and 1%) were also evaluated in a prevention treatment protocol. Corneal responses were monitored throughout a 2-week treatment period, and 1 week after the last treatment dose. Observations included quantitative measurement of the area of new blood vessel growth and qualitative assessment of cellular infiltrate and edema. All treatments and observations were performed in a double-masked manner. RESULTS: All tested formulations, except the vehicle and the 0.01% AL-3789 preparation, significantly inhibited corneal neovascularization and other lipopolysaccharide-induced responses in the various treatment protocols employed. AL-4940, the free alcohol form of AL-3789, was slightly less effective than cortisol acetate or AL-3789. The extent of inhibition of the angiogenic response by the 1% and 0.1% AL-3789 suspensions ranged from 76% to 100% 1 week after the last treatment. CONCLUSIONS: The antiangiogenic steroid AL-3789 may be a therapeutically useful angiostatic agent for corneal neovascularization and potentially could be effective in other ocular neovascular diseases.     

Different immunological mechanisms contribute to cartilage destruction in antigen-induced arthritis

Antigen-induced arthritis in guinea pigs was used as a model to investigate the pathogenic mechanisms responsible for cartilage destruction in chronic joint inflammation. The activation of macrophages, their effects on cartilage metabolism, and the development of autoimmunity to cartilage constituents were studied during the progression of arthritis. The results show that in arthritic animals the macrophages are systemically activated, with a peak in the early phase of inflammation. Interleukin 1, produced by the activated cells, suppresses the proteoglycan synthesis in cartilage explants and cultured chondrocytes and increases the proliferation of the cells in vitro. During the progression of arthritis humoral and cell-mediated immune responses to collagen type II and cartilage proteoglycans occur correlating with the severity of arthritis. It is concluded that different immunological mechanisms may be involved in cartilage destruction during antigen-induced arthritis. Mediator-induced metabolic reactions dominate in the early phase, whereas autoimmunity to cartilage might play an essential role in later phases of arthritis.     

Post-transfusion hyperhaemolysis in a patient with sickle cell disease: use of steroids and intravenous immunoglobulin to prevent further red cell destruction

Forty nine multiple drug resistant strains of E. coli isolated from UTI were serotyped. The pattern was found to be 057 (eight strains); 0109 (four strains); 020, 038, 068, 0106, 0148. Rough (three each). 012, 054, 0101, 0160 (two each) and 02, 032, 046, 053, 060, 065, 090, 091, 0117, 0118, untypable (one each). The resistance pattern of all E. coli were identified and matted with recepient strain in penassay broth and in human urine. In a penassay broth transfer of resistance was demonstrated in 38 strains (77.5%) while in human urine transfer was demonstrated only in 14 strains (28.57%).     

The use of pimonidazole to characterise hypoxia in the internal environment of an in vivo tissue engineering chamber

The distribution of hypoxic cells in an in vivo tissue engineering chamber was investigated up to 28 days post-implantation.

Methods

Arteriovenous loops were constructed and placed into bi-valved polycarbonate chambers containing 2×10⁶ rat fibroblasts in basement membrane gel (BM gel). Chambers were inserted subcutaneously in the groin of male rats and harvested at 3 (n=6), 7 (n=6), 14 (n=4) or 28 (n=4) days. Ninety minutes before harvest, pimonidazole (60 mg/kg) was injected intraperitoneally. Chamber tissue was removed, immersion fixed, paraffin embedded, sectioned and stained immunohistochemically using hypoxyprobe-1 Mab that detects reduced pimonidazole adducts forming in cells, where pO₂<10 mmHg.

Results

At 3 days a fibrin clot/BM gel framework filled the chamber. Seeded fibroblasts had largely died. The majority of 3 day chambers did not demonstrate tissue growth from the AV loop nor was pimonidazole binding present in these chambers. In one chamber in which tissue growth had occurred strong pimonidazole binding was evident within the new tissue. In four out of six 7 day chambers a broader proliferative zone existed extending up to 0.4 mm (approximately) from the AV loop endothelium which demonstrated intense pimonidazole binding. The two remaining 7 day chambers displayed even greater tissue growth (leading edge>0.7 mm from the AV loop endothelium), but very weak or no pimonidazole binding. At 14 and 28 days the fibrin/BM gel matrix was replaced by mature vascularised connective tissue that did not bind pimonidazole.

Conclusion

Employing a tissue engineering chamber, new tissue growth extending up to 0.4 mm from the AV loop endothelium (chambers≤7 days) demonstrated intense pimonidazole binding and, therefore, hypoxia. Tissue growth greater than 0.5 mm from the AV loop endothelium (7–28 days chambers) did not exhibit pimonidazole binding due to a significant increase in the number of new blood vessels and was, therefore, adequately oxygenated.     

The use of anabolic steroids by athletes to increase body weight and strength

Over the past 20 years the taking of anabolic steroids by healthy athletes for the purpose of increasing body weight and strength has become very widespread. The ability of these agents to cause potentially serious side effects is discussed. In a series of 20 subjects studied over 18 months, no side effects of significance were recorded, and marked increases in strength and body weight were achieved.     

The chick embryo chorioallantoic membrane as an in vivo wound healing model

The chick embryo chorioallantoic membrane (CAM) was used as an in vivo wound healing model. A full excision of a 1 mm2 CAM area was filled by a granulation tissue after 96-120 h, which eventually formed a scar in 75% of the cases. In the remaining 25%, a solution of continuity was left which, however, was smaller in size than the one observed immediately after the excision. Under the microscope, the CAM area involved in the repair process showed: i. hyperplasia of the chorionic epithelium; ii. about three times as many microvessels and fibroblasts in the mesenchyme as in the normal adjacent control regions; iii. an inflammatory infiltrate mostly consisting of macrophages; and iv. a strong positivity for fibronectin in the extracellular matrix. The validity of this experimental model appears to be confirmed by the fact that we were able to reproduce all the critical events controlling the wound healing process, such as re-epithelization, angiogenesis, formation of an inflammatory infiltrate and deposition of one of the main constituents of the extracellular matrix, such as fibronectin.     

Targeting the receptor-Gq interface to inhibit in vivo pressure overload myocardial hypertrophy

Hormones and neurotransmitters may mediate common responses through receptors that couple to the same class of heterotrimeric guanine nucleotide-binding (G) protein. For example, several receptors that couple to Gq class proteins can induce cardiomyocyte hypertrophy. Class-specific inhibition of Gq-mediated signaling was produced in the hearts of transgenic mice by targeted expression of a carboxyl-terminal peptide of the alpha subunit Galphaq. When pressure overload was surgically induced, the transgenic mice developed significantly less ventricular hypertrophy than control animals. The data demonstrate the role of myocardial Gq in the initiation of myocardial hypertrophy and indicate a possible strategy for preventing pathophysiological signaling by simultaneously blocking multiple receptors coupled to Gq.     

The in vivo use of dithiothreitol in cystinosis

Two male patients with late stage (uremic) infantile nephropathic cystinosis (INC) (Table 1) were treated by mouth with the reducing agent dithiothreitol (DTT), at doses not exceeding 25 mg-kg-1 body weight three times per day. Three sequential periods of observation were obtained in both patients: on thiol (8.5 months); off thiol (8-9 months); on thiol again (7 months or longer). Other than nausea and vomiting at the maximum dose range, no apparent toxicity was observed. One subject died in uremia in the 24th month of the study. The half-cystine concentration in peripheral blood leukocytes decreased during both treatment periods in each patient from initial pretreatment levels in excess of 8 nmol-mg-1 protein (normal less than 0.1 nmol-mg-1) to 10-20% of initial values (Table 2 and Fig. 1, A and B). Reduction in total number of blood leukocytes or in the neutrophil fraction, where cystine storage occurs selectively in cystinosis, did not occur (Table 3) as a possible explanation for these findings; nor did storage of samples, a possible artifact, influence the cystine content of cystinotic cells (Fig. 2). Multiple site rectal mucosa biopsy clearly revealed cystine storage but serial biopsies did not reflect a positive DTT response when compared with the leukocyte assay (Table 4). High intersample variation in cystine content, even between samples taken at one time, prevented measurement of a treatment response. DTT had no apparent detrimental effect on the concentration of representative proteins, including hemoglobin (Table 3), serum insulin, and serum immunoglobulin during the treatment trials. Renal function (glomerular and tubular) was severely depressed and did not improve during the period of observation in either patient (Table 2; Fig. 3, A and B). Postmortem tissues from one patient revealed 10-40-fold excess cystine accumulation in kidney cortex and liver (Table 5). However, these levels of accumulation are at the lower range of or even below published values for cystine in cystinotic kidney and liver. Whereas chemical methods are not reliable for detecting and measuring DTT in biologic fluids, preliminary evidence indicates that a silylated derivative of oxidized DTT can be detected in the urine of patients receiving DTT by mouth (Fig. 4). This finding suggests that the thiol is absorbed and excreted.     

The use of ketamine in an experimental model of generalized cerebral ischemia

Thus far, a sufficiently effective cerebroprotective substances has not been discovered. Glutamate overproduction plays a key role in ischemic brain lesion. Ketamine is assigned to the group of commonly used clinical anesthetics, being also familiar as NMDA antagonist. Sodium fluoride-induced cerebral ischemia in mice is used as a model of circulatory ischemic lesion. As shown by the experimental data, simultaneous administration of NaF + ketamine has no effect whatsoever on the survivorship of animals, as compared to that in the control group treated with NaF alone. Beforehand treatment of mice with 150 mg/kg ketamine brings about considerable prolongation of the survival term (15 per cent of the animals survive for more than 2 hours). The inference is reached that ketamine is endowed with cerebroprotective activity largely attributable to glutamate antagonism at the level of ischemia involved neurons.     

Monoclonal antibodies directed to the erbB-2 receptor inhibit in vivo tumour cell growth

Four monoclonal antibodies (MAbs) specific for the extracellular domain of the human erbB-2/HER2 protein (FRP5, FSP16, FWP51 and FSP77) have been isolated (Harwerth et al., J. Biol. Chem., 267, 15160-15167, 1992). In this paper we describe the effects of erbB-2 specific MAb administration on the tumorigenic growth of human erbB-2 transformed NIH3T3 cells implanted into athymic nude mice. Two antibodies, FWP51 and FSP77, inhibited the onset of tumour growth, while the administration of FRP5 and FSP16 did not affect tumour growth. In addition, administration of MAbs FWP51 and FSP77 led to a retardation in the growth of established tumours. Treatment was not curative in that tumours regrew within two weeks of the final treatment. The administration of a combination of MAbs FWP51 and FSP77 which react with two distinct regions on the erbB-2 molecule was more effective than treatment with either MAb alone. The two growth-inhibitory antibodies were also effective in the treatment of tumours established from SKOV3 cells, a human ovarian tumour cell line with high levels of the erbB-2 protein. The effect of the MAbs on the anchorage-independent growth of erbB-2 transformed cells and on erbB-2 receptor turnover was also measured.     

T1rho-relaxation in articular cartilage: effects of enzymatic degradation

Spin-lattice relaxation in the rotating frame (T1rho) dispersion spectroscopy and imaging were used to study normal and enzymatically degraded bovine articular cartilage. Normal specimens demonstrate significant T1rho "dispersion" (approximately 60 to approximately 130 ms) in the 100 Hz to 9 kHz frequency range. Proteoglycan-degraded specimens have 33% greater T1rho values than collagen-degraded or normal samples. T1rho-weighted images reveal structure not found in conventional T1- or T2-weighted images. Our results suggest that T1rho measurements are selectively sensitive to proteoglycan content. The potential of this method in distinguishing the early degenerative changes in cartilage associated with osteoarthritis is discussed.     

Action of pentamidine-bound nanoparticles against Leishmania on an in vivo model

The efficiency of antileishmanial agents may be enhanced by improving their bioavailability with a colloidal drug carrier. We have investigated the action of free pentamidine, compared with pentamidine bound to polymethacrylate nanoparticles, in a rodent model. BALB/c mice were infected, via the tail vein, with 4 x 10(7) L. major (MON 74) promastigotes. Twelve days after infection, seven groups of mice were treated respectively with methylglucamine antimoniate (Glucantime) 5.56 mg/kg i.p. x 5 d., pentamidine bound nanoparticles (100 microM), unloaded polymethacrylate nanoparticles, unloaded nanoparticles associated with free pentamidine (100 microM) 0.1 ml i.v. x 3 d and free pentamidine isethionate (2.28 mg/kg and 0.17 mg/kg i.v. x 3 d.). Twenty-one days post infection, the mice were sacrificed and the Leishmania load in the liver calculated from the number of amastigotes/500 liver cells and total liver weight in treated and untreated mice. Results demonstrated a 77% amastigote reduction in the group treated with targeted pentamidine relative to the control group. The ratio free pentamidine/bound-pentamidine was approx. 12.     

Observations on seminal vesicle dynamics in an in vivo rat model

PURPOSE: To gain understanding of the seminal vesicle as a muscular organ, seminal vesicle compliance and contractile properties were quantified with an in vivo, microsurgical rat model. MATERIALS AND METHODS: Microsurgical dissection was performed on anesthetized rats to enable simultaneous organ filling and monitoring of intraluminal pressures. The reliability and reproducibility of post-ganglionic hypogastric nerve-induced ipsilateral (4 rats) and bilateral (5 rats) seminal vesicle contractile responses were assessed during repeated nerve stimulation. Seminal vesicle resting compliance was assessed during a constant saline infusion (10 rats). Functional performance curves were obtained at fixed fill-volumes by measuring organ contraction after nerve stimulation (4 rats). RESULTS: A reproducible seminal vesicle contractile response was obtained with a nerve stimulation interval > 15 minutes. Bilateral seminal vesicle responses were observed with unilateral nerve stimulation. The resting organ compliance curve with saline filling exhibited a characteristic, triphasic response. Functional performance studies revealed that contractile performance improves as the fill-volume increases until the distensibility limit of the organ is reached. CONCLUSIONS: A reliable, in vivo, rat model of seminal vesicle organ compliance and contractility is described. The seminal vesicle is a highly contractile, compliant smooth muscular organ with dynamic properties analogous to that of the urinary bladder. This experimental system may allow for the investigation of pharmacologic and other physiological influences on in vivo organ activity.     

The use of collagen-based model peptides to investigate platelet-reactive sequences in collagen

Simple collagen-like peptides comprising a repeat Gly-Pro-Hyp sequence are highly platelet-reactive when presented to platelets in triple-helical and polymeric form. This activity is not mediated by the platelet collagen receptor integrin alpha 2 beta 1. This may imply the existence of an intrinsic platelet reactivity associated with the collagen triple helix as such or perhaps that the Gly-Pro-Hyp sequence in collagen serves as a specific cell-recognition site. In our view this basic alpha 2 beta 1-independent reactivity is modulated by the presence in collagen of sequences that may either enhance or diminish the interaction with platelets. Inhibition studies with short linear peptides have allowed the tentative identification of sequences in collagen such as XPGEP(Q)GPX and D(N)GE(Q)X that may promote the activation of platelets and so enhance collagen-platelet interaction. Sequences serving as integrin alpha 2 beta 1-binding sites may also promote platelet reactivity by permitting interaction with the collagen receptor. Using triple-helical peptides based on the sequence of the platelet-reactive collagen type III fragment alpha 1(III)CB4, we have been able to locate an alpha 2 beta 1-binding site in collagen type III within a 30-mer sequence representing residues 508-537 of the alpha 1(III) constituent alpha-chain. Despite their alpha 2 beta 1-independent platelet reactivity, signalling by the (Gly-Pro-Hyp)n-based peptides shows many features in common with signalling by collagen fibers, including activation of p72SYK and p125FAK the latter of which has until now been considered a specific consequence of ligand binding to alpha 2 beta 1.     

Influence of transducer frequency on Doppler microemboli signals in an in vivo model

The purpose of this study was to apply virtual reality technology to spiral computed tomographic (CT) angiogram images in a rabbit model of atherosclerosis and to correlate the images with histopathologic evaluation of the aorta. Image data were transferred to the virtual endoscope system in a graphics workstation. "Virtualized angioscopy" includes an interactive graphic user interface, which controls the viewpoint, the direction of the observation, and rendering and navigation functions. The virtual angioscopy system demonstrated irregularities of the luminal surface of the ascending aorta and a smooth luminal surface in the descending aorta. These observations were correlated with histopathologic findings. The results of this study indicate that the potential and real benefits of virtualized angioscopy far outweigh several technical limitations.     

The importance of physicochemical swelling in cartilage illustrated with a model hydrogel system

The physicochemically derived swelling stress in articular cartilage plays a crucial role in determining the pattern of stress sharing between the exudable fluid and the 'solid' components comprising its matrix. This pattern of stress sharing in turn influences the manner in which cartilage consolidates or deforms in compression via the outflow of fluid. Synthetic hydrogels exposed to a variety of cationic blocking solutions provide simplified model systems for exploring quantitatively the influence of the intrinsic swelling parameter on consolidation behaviour, thus yielding further insights into the fundamental parameters controlling the biomechanical properties of complex tissues such as articular cartilage.     

The use of clays as an aid to water purification

The conditions under which cationic species will adsorb intensely onto silicate surfaces have been investigated with a view to specifying basic requirements for successful operation of a water purification process based on silicate/cation interactions. Zeta-potential measurements have been related to effectiveness of cation precipitation. It is shown that clay additions enhance ferric iron removal, optimum pH range being pH 9 to 10. Coal measures shale addition increases the rate of settling of precipitates several-fold. Electro-kinetic measurements show that clay additions also remove uncharged particles efficiently. The precipitation/clay adsorption process is reversible with respect to pH.