Combination drug treatment prolongs survival of experimentally infected mice with silver-haired bat rabies virus.

Rabies is a lethal disease in humans and animals, killing approximately 60,000 people every year. Currently, there is no treatment available, except post-exposure prophylaxis (PEP) that can be administered whenever exposure to a rabid animal took place. Here we describe the beneficial effects of a combination treatment initiated at day 4 post infection, containing anti-viral drugs and immune modulators in infected mice. Combination therapy resulted in significant increase in survival time (P < 0.05) and significantly lowers viral RNA in the brain and spinal cord (P < 0.05). Furthermore, treatment influenced markers of pyroptosis and apoptosis and early inflammatory response as measured by the levels of TNF-α. Morphological lesions were absent in rabies virus infected mice with few signs of inflammation. However, these were not significant between the different groups.

Ancient hepatitis B viruses from the Bronze Age to the Medieval period.

Hepatitis B virus (HBV) is a major cause of human hepatitis. There is considerable uncertainty about the timescale of its evolution and its association with humans. Here we present 12 full or partial ancient HBV genomes that are between approximately 0.8 and 4.5 thousand years old. The ancient sequences group either within or in a sister relationship with extant human or other ape HBV clades. Generally, the genome properties follow those of modern HBV. The root of the HBV tree is projected to between 8.6 and 20.9 thousand years ago, and we estimate a substitution rate of 8.04 × 10-6-1.51 × 10-5 nucleotide substitutions per site per year. In several cases, the geographical locations of the ancient genotypes do not match present-day distributions. Genotypes that today are typical of Africa and Asia, and a subgenotype from India, are shown to have an early Eurasian presence. The geographical and temporal patterns that we observe in ancient and modern HBV genotypes are compatible with well-documented human migrations during the Bronze and Iron Ages1,2. We provide evidence for the creation of HBV genotype A via recombination, and for a long-term association of modern HBV genotypes with humans, including the discovery of a human genotype that is now extinct. These data expose a complexity of HBV evolution that is not evident when considering modern sequences alone.

Evaluation of synthetic infection-enhancing lipopeptides as adjuvants for a live-attenuated canine distemper virus vaccine administered intra-nasally to ferrets.

BACKGROUND: Inactivated paramyxovirus vaccines have been associated with hypersensitivity responses upon challenge infection. For measles and canine distemper virus (CDV) safe and effective live-attenuated virus vaccines are available, but for human respiratory syncytial virus and human metapneumovirus development of such vaccines has proven difficult. We recently identified three synthetic bacterial lipopeptides that enhance paramyxovirus infections in vitro, and hypothesized these could be used as adjuvants to promote immune responses induced by live-attenuated paramyxovirus vaccines. METHODS: Here, we tested this hypothesis using a CDV vaccination and challenge model in ferrets. Three groups of six animals were intra-nasally vaccinated with recombinant (r) CDV(5804P)L(CCEGFPC) in the presence or absence of the infection-enhancing lipopeptides Pam3CSK4 or PHCSK4. The recombinant CDV vaccine virus had previously been described to be over-attenuated in ferrets. A group of six animals was mock-vaccinated as control. Six weeks after vaccination all animals were challenged with a lethal dose of rCDV strain Snyder-Hill expressing the red fluorescent protein dTomato. RESULTS: Unexpectedly, intra-nasal vaccination of ferrets with rCDV(5804P)L(CCEGFPC) in the absence of lipopeptides resulted in good immune responses and protection against lethal challenge infection. However, in animals vaccinated with lipopeptide-adjuvanted virus significantly higher vaccine virus loads were detected in nasopharyngeal lavages and peripheral blood mononuclear cells. In addition, these animals developed significantly higher CDV neutralizing antibody titers compared to animals vaccinated with non-adjuvanted vaccine. CONCLUSIONS: This study demonstrates that the synthetic cationic lipopeptides Pam3CSK4 and PHCSK4 not only enhance paramyxovirus infection in vitro, but also in vivo. Given the observed enhancement of immunogenicity their potential as adjuvants for other live-attenuated paramyxovirus vaccines should be considered.

West Nile Virus: is a vaccine needed?

West Nile virus (WNV) is a neurotropic Flavivirus that was associated with sporadic outbreaks of meningoencephalitis in Africa and the Middle East until 1999, when a more virulent strain emerged in the US that caused thousands of infections among humans and horses, with reported fatality rates between 10 and 50%. Although the epidemiology of WNV is changing into a more endemic pattern in the US, and the incidence of neuroinvasive disease is decreasing, the long-term effects of resolved WNV infections in humans, characterized as persistent movement disorders and various functional disabilities, are a significant cause of morbidity. In addition, the horse industry is also negatively impacted by WNV infections, resulting in significant economic losses. Together with the fact that WNV is a potential bioterrorism agent, these factors suggest that there is a need for the development of a safe and effective vaccine against WNV. The increased understanding of WNV pathogenesis and correlates of protection enables the rational design of such a vaccine. Several experimental vaccines have been tested in preclinical models and some have undergone clinical trials. The challenges related to the development of cheaper, safer and more effective vaccines for use in both humans and horses are likely to be overcome by new technological developments in the field of vaccinology.

Animal models for the preclinical evaluation of candidate influenza vaccines.

At present, new influenza A (H1N1)2009 viruses of swine origin are responsible for the first influenza pandemic of the 21st Century. In addition, highly pathogenic avian influenza A/H5N1 viruses continue to cause outbreaks in poultry and, after zoonotic transmission, cause an ever-increasing number of human cases, of which 59% have a fatal clinical outcome. It is also feared that these viruses adapt to replication in humans and become transmissible from human to human. The development of effective vaccines against epidemic and (potentially) pandemic viruses is therefore considered a priority. In this review, we discuss animal models that are used for the preclinical evaluation of novel candidate influenza vaccines. In most cases, a tier of multiple animal models is used before the evaluation of vaccine candidates in clinical trials is considered. Commonly, vaccines are tested for safety and efficacy in mice, ferrets and/or macaques. The use of each of these species has its advantages and limitations, which are addressed here.

Aerosol measles vaccination in macaques: preclinical studies of immune responses and safety.

The comparative efficacy and safety of measles vaccination via the aerosol route versus subcutaneous injection has not been fully resolved. We vaccinated cynomolgus monkeys (Macaca fascicularis) with the live-attenuated Edmonston-Zagreb measles virus (MV) vaccine and compared different routes of administration in the immunocompetent and the immunocompromised host. Immunogenicity and protective efficacy of aerosol vaccination using devices similar to those previously used in humans were comparable to those in animals vaccinated by injection. No evidence for a safety hazard associated with the route of vaccination was detected. The results of this study support further clinical evaluation of aerosol vaccination for measles.

Antineoplastic effect and toxicity of 1,25-dihydroxy-16-ene-23-yne-vitamin D3 in athymic mice with Y-79 human retinoblastoma tumors.

OBJECTIVES: To evaluate the in vivo efficacy and toxicity of the 1,25-dihydroxy-16-ene-23-yne-vitamin D3 (16,23-D3) analogue in athymic nude mice injected with Y-79 human retinoblastoma cells and to compare the efficacy and toxicity of this compound with those of 1,25-dihydroxycholecalciferol (D3, calcitriol). METHODS: Thirty athymic nude mice (4-6 weeks old) were injected subcutaneously with 1 x 10(7) Y-79 human retinoblastoma cells suspended in a 1:1 mixture of Iscove culture medium supplemented with 20% fetal bovine serum and basement membrane matrix suspension. Five days after tumor injection, the mice were randomized to 3 groups of 10 mice each. The first group served as a control group and received intraperitoneal injections of 0.25 mL of mineral oil (vehicle) 5 times a week. The second group received intraperitoneal injections of 0.05 microg of calcitriol in 0.25 mL of mineral oil intraperitoneally 5 times a week. The third group received intraperitoneal injections of 0.5 microg of 16,23-D3 in 0.25 mL of mineral oil 5 times a week. Injections were continued for 5 weeks, during which tumor size and mouse weight were individually measured. Toxicity was assessed by clinical measures such as lethargy, weight loss, and death. The mice were then killed and the size, volume, and weight of each tumor were determined. Also, in representative animals in each group, kidneys were evaluated for calcification and serum calcium concentration was measured. RESULTS: All experimental and control animals developed tumors subcutaneously. The 16,23-D3-treated mice had significantly smaller average tumor size (1.55 cm3) than the control mice (3.45 cm3) (P = .02), less gain in average body weight from the beginning of treatment (2.4 g vs 5.5 g) (P= .06), and a 40% mortality. The calcitriol-treated mice did not have significantly smaller average tumor size (1.26 cm3) than the 16,23-D3-treated mice (P = .35), had significant body weight loss compared with the control animals (calcitriol-treated mice lost 4.03 g) (P =.001), and had a mortality of 90% by the completion of the experiment. Histologically, there was no difference in the degree of tumor necrosis and calcification between control and experimental mice. Serum calcium concentrations were equivalent between the control (2.15 mmol/L [8.6 mg/dL]) and experimental groups (calcitriol, 1.88 mmol/L [7.5 mg/dL] [P = .97]; 16,23-D3, 2.15 mmol/L [8.6 mg/dL] [P = .42]). Mild bilateral renal tubular calcification occurred in 3 of 4 mice in the calcitriol-treated group and in 2 of 4 mice in the 16,23-D3-treated group. CONCLUSIONS: The growth of subcutaneous Y-79 human retinoblastoma cells in athymic nude mice is significantly reduced by treatment with intraperitoneal injections of 16,23-D3. The antineoplastic effect of calcitriol is not statistically significantly different but is associated with significantly more toxicity. 1,25-Dihydroxy-16-ene-23-yne-vitamin D3 may be a useful chemotherapeutic adjunct in the treatment of retinoblastoma.

Radiation therapy and ferromagnetic hyperthermia in the treatment of murine transgenic retinoblastoma.

BACKGROUND: Combined modality therapy for childhood retinoblastoma holds the potential of decreasing treatment-related morbidity while maintaining excellent tumor control rates. OBJECTIVE: To evaluate the efficacy of external beam radiation therapy (EBRT), ferromagnetic hyperthermia (FMH), and the combination of both modalities in the control of ocular tumors in a transgenic murine model of retinoblastoma. METHODS: One hundred sixty-six mouse eyes from 4-week-old animals transgenically positive for simian virus 40 large T antigen were treated with a total dose of 10, 15, 20, 30, 40, 45, or 50 Gy of EBRT in 5-Gy fractions twice daily, with 48 degrees C or 54 degrees C FMH for 20 minutes, or with combined EBRT at 10 or 30 Gy and 48 degrees C or 54 degrees C FMH for 20 minutes. Serial histologic sections, obtained 8 weeks after treatment, were examined for the presence of tumor. RESULTS: The tumor control dose for 50% of eyes (TCD50) treated with EBRT occurred at 27.6 Gy. Ferromagnetic hyperthermia at 48 degrees C cured 30% (6/20) of eyes, while 54 degrees C FMH resulted in a 100% (20/20) cure rate. Combined treatment with 48 degrees C FMH and EBRT exhibited a TCD50 at 3.3 Gy. The thermal enhancement ratio was 8.4. Ferromagnetic hyperthermia at 54 degrees C exhibited tumor cure in all animals, but 25% of eyes were lost owing to secondary treatment complications. CONCLUSIONS: This represents the first documentation of tumor control via EBRT, ocular FMH, and a combination of these treatment modalities in this murine transgenic retinoblastoma model. The extent of treatment synergy in this model suggests that combined treatment application may allow a reduction in total ocular and periocular radiation dose while maintaining excellent local tumor control.

Vitamin D inhibits angiogenesis in transgenic murine retinoblastoma.

PURPOSE: Vitamin D compounds have been shown to inhibit tumor growth in a transgenic retinoblastoma murine model. The mechanism of action has not been defined clearly, although an antiangiogenic action has been proposed. METHODS: Transgenic retinoblastoma mice received high (0.05 microgram) and low (0.025 microgram) doses of vitamin D3 by intraperitoneal injection 5 times per week for 5 weeks. Control animals were injected with mineral oil vehicle alone. At 5 months of age, the animals were killed and eyes were enucleated and processed for light microscopy. Paraffin-embedded sections were stained with an immunoperoxidase stain (GS-1) specific for mammalian vascular endothelium. Sections were graded by a single masked reviewer, and intraobserver reliability was assessed. Mean vessel counts were made for each group. RESULTS: The high-dose group had the lowest mean vessel count (8.5), followed by the low-dose group (10.1). The control group had the highest mean vessel count (14.1). Vitamin D-treated animals (high- and low-dose groups combined) had significantly fewer vessels P = 0.001) than untreated controls. CONCLUSIONS: These results support the hypothesis that inhibition of angiogenesis is a mechanism of action for vitamin D in the transgenic retinoblastoma mouse model.

The antineoplastic effect of vitamin D in transgenic mice with retinoblastoma.

Vitamin D has been shown to inhibit growth of human retinoblastoma in tissue culture and nude mouse heterografts. We have described a heritable transgenic mouse model of retinoblastoma. The in vivo efficacy of 1,25-dihydroxycholecalciferol (vitamin D3) was examined by administering this agent to transgenic mice with retinoblastoma. Forty-six 8-10-week-old transgene-bearing mice were injected intraperitoneally for 5 wk. Experimental animals received 0.05 microgram (15 animals) or 0.025 microgram (15 animals) of vitamin D. Sixteen control animals received only a mineral oil vehicle. Eyes were enucleated at 5 mo and were examined histologically by two investigators in a masked fashion. All control animals demonstrated bilateral involvement of retinoblastoma. Four eyes in the low-dose group and six eyes in the high-dose group had no evidence of retinoblastoma. Eyes treated with vitamin D3 showed less extensive involvement of the retina by retinoblastoma. Vitamin D-treated animals demonstrated tumors confined to the retina, whereas control animals demonstrated larger tumors, more often invading the vitreous, anterior chamber, and choroid. Thus, Vitamin D inhibited the growth and local extension in a dose-dependent fashion.

Relative survival rates after alternative therapies for uveal melanoma.

Survival in a group of 556 patients with uveal melanoma treated by proton beam irradiation with a median follow-up of 5.3 years was compared with that of 238 patients enucleated during the same 10-year period as irradiated patients (July 1975 to December 1984) with a median follow-up of 8.8 years, and 257 patients enucleated during the preceding 10 years (January 1965 to June 1975) with a median follow-up of 17.0 years. Adjustments were made for known prognostic factors including age, tumor location, tumor height, and clinical estimate of tumor diameter (for enucleated patients this was estimated in a regression equation relating histologic to clinical measurement). The overall rate ratio for all cause mortality was 1.2 (95% confidence interval, 0.9-1.6) for the concurrent enucleation series versus proton beam, and 1.6 (95% confidence interval, 1.2-2.1) for the earlier enucleation series versus proton beam. Relative rates of metastatic death, cancer death, and all cause mortality comparing alternative treatments were found to vary with time after treatment. Interval-specific rate ratios were evaluated using proportional hazards models fitted to separate time intervals after treatment. For all three outcomes, rate ratios were over two and statistically significant for the first 2 years after treatment and closer to one and nonsignificant after year 6 comparing the two enucleation groups with proton beam. Results suggest that treatment choice has little overall influence on survival in patients with uveal melanoma.

Magnetic resonance imaging and spectroscopy of intraocular tumors.

Proton magnetic resonance imaging (1H MRI) has emerged as a clinically useful tool for the diagnosis of intraocular tumors. During the last four years 1H MRI characteristics, including spin-lattice relaxation times (T1) and spin-spin relaxation times (T2), have been established for several types of tumors. The introduction of surface coils to the imaging process has significantly improved the quality of intraocular MR images, leading some clinicians to suggest that 1H MR images are preferable to CT scans. Another MRI technique, in which sodium-23 (23Na) is imaged rather than protons, is now under development as tool for intraocular diagnosis. The potential of 23Na MRI depends upon the high concentration and "visibility" of sodium in the vitreous body, and upon the apparent differences in sodium behavior in normal cells vs. tumor cells. The metabolism of normal ocular tissues and intraocular tumors may be probed noninvasively with phosphorus-31 MR spectroscopy (31P MRS). Much progress has been made during the last few years in understanding the appearance of 31P MR spectra of many types of healthy and diseased cells and tissues. Clinical application of this technique to the diagnosis and monitoring of intraocular tumors following conservative treatment will be dependent upon the development of spectroscopy techniques that collect information from the volume of interest (tumor) only.

A perfusion system developed for 31P NMR study of melanoma cells at tissue-like density.

A perfusion culture system has been developed for 31P NMR study of human uveal melanoma metabolism by adapting the Vitafiber I cartridge system (Amicon). 31P NMR spectra collected weekly during periods of up to 10 weeks demonstrated increasing levels of phosphorus metabolites as the anchorage-dependent cells grew to tissue-like density.

Preliminary results on phosphorus-31 nuclear magnetic resonance evaluation of human uveal melanoma in enucleated eyes.

Clinical evaluation of uveal melanomas by nuclear magnetic resonance (NMR) techniques depends on ascertaining how these tumors characteristically appear in NMR images and spectra. The authors have determined NMR characteristics of suspected uveal melanomas by phosphorus-31 (31P) NMR spectroscopy of freshly enucleated human eyes. Nuclear magnetic resonance examination was performed at 8.45 Tesla within 90 minutes after enucleation. Enucleated eyes were maintained at 4 degrees C in tissue culture medium during the 30 minutes required for transport. Nuclear magnetic resonance spectra were obtained within 10 minutes, a clinically acceptable time, using a two-turn 31P surface coil. Spectral parameters included 10-kHz spectral width, 1024 data points, and 0.5-second recycle delay. Phosphorus-31 NMR spectroscopy allowed differentiation of choroidal melanomas from normal ocular structures. Differentiating features include significant peaks in tumor spectra due to the phosphodiesters glycerol 3-phosphoryl ethanolamine (GPE) and glycerol 3-phosphorylcholine (GPC), and the phosphomonoesters phosphorylethanolamine (PE) and phosphorylcholine (PC). These preliminary data are encouraging and suggest that clinical trials at the lower magnetic field strengths available in NMR imaging systems seem feasible and warrant investigation.

Proton beam irradiation and hyperthermia. Effects on experimental choroidal melanoma.

Ultrasonically induced hyperthermia (4.75 MHz) and proton irradiation (160 meV) were evaluated alone and combined to treat experimental choroidal melanoma in 58 rabbit eyes. Threshold tumoricidal doses were established for each modality. Therapy was performed combining subthreshold doses of heat and radiation. Focused ultrasonic energy via an external beam was found to deliver well-localized heat to an intraocular tumor. Ectopic temperature elevations due to soft-tissue-bone interfaces were alleviated by modifying beam alignment. The results indicate that hyperthermia (43 degrees C for one hour) potentiated the tumoricidal effects of radiation, while sparing normal ocular structures. Therefore, we believe that experimental hyperthermia is suitable as an adjuvant treatment modality. This shows that ultrasound hyperthermia has the potential to increase the efficacy of proton irradiation by lowering radiation doses and thus decreasing posttreatment ocular morbidity in human intraocular malignancies.

Characterization of human uveal melanoma cells by phosphorus 31 nuclear magnetic resonance spectroscopy.

We performed experiments to determine the potential usefulness of nuclear magnetic resonance spectra in the diagnosis and follow-up of ocular melanoma. High-resolution phosphorus 31 nuclear magnetic resonance spectra at 109.3 MHz were obtained for human uveal melanoma, Greene hamster melanoma, and normal human diploid fibroblast cells. Phosphate metabolites were identified and their concentrations were shown to vary among the different cell lines. Uveal melanoma cells contain unusually high concentrations of the phospholipid metabolite phosphorylcholine and the phosphodiesters glycerol 3-phosphoryl choline and glycerol 3-phosphoryl ethanolamine. Baseline data are thus provided for studies of the effect of various treatment modalities on uveal melanoma. These initial results suggest that the data provided by high-resolution phosphorus 31 nuclear magnetic resonance spectra can provide useful diagnostic and follow-up data with respect to ocular melanoma.

Cocaine's use in ophthalmology: our 100-year heritage.

One hundred years ago the fledgling ophthalmologist Carl Koller demonstrated that the alkaloid cocaine was a local anesthetic suitable for rendering the eye temporarily insensible to pain. It was an event of historical significance, as his discovery opened the door not only to a new era in ophthalmic surgery, but to surgery in general. In this paper we review the ancient uses of the coca leaf, the historical events leading up to and following Koller's discovery, and the present day uses of cocaine in ophthalmology and medicine.

Hyperthermic treatment of intraocular tumors.

A 5.8-gigahertz (GHz) ophthalmic microwave applicator was used to treat choroidal melanoma (Green strain) in rabbits. High-frequency electromagnetic radiation provides a favorable dose distribution to induce local hyperthermia in the treatment of intraocular tumors. Heating of the neoplasm, while sparing normal ocular structures, is best accomplished by a transscleral approach. A hyperthermia plaque is placed on the sclera at the base of the intraocular tumor. Contact (resistive) heating and electromagnetic radiation (radiofrequency and microwave) are best suited to a plaque technique. The advantages of electromagnetic heat induction, as compared with contact heating, are twofold: the depth of hyperthermic penetration can be modulated by frequency selection, and the tissues with low water content (sclera) remain relatively unaffected by microwaves. The 5.8-GHz ophthalmic microwave applicator satisfies the requirements for local hyperthermic treatment of intraocular tumors.

Perfluoroether liquid as a long-term vitreous substitute. An experimental study.

A commercial perfluoroether liquid, Fomblin-H Fluorinated Fluid, which is transparent and viscous, was investigated as a possible long-term vitreous substitute. The material belongs to a family of perfluorocarbon derivatives that are generally inert and nontoxic. A space for the vitreous substitute was created in rabbit eyes by a vitreous compression technique using perfluoropropane gas. Fomblin caused gliosis of the retina at 1 month after intravitreous injection. Preretinal membrane formation and retinal disorganization and detachment occurred in two of four eyes enucleated at 3 months, and in all five eyes enucleated at 4 or 6 months. Vacuoles presumably filled with Fomblin were localized histopathologically within the retina. In control eyes injected with medical-grade silicone oil, abnormalities were limited to moderate edema of the nerve fiber and ganglion cell layer. Fomblin was not as well-tolerated by the rabbit retina as was medical-grade silicone oil. Fomblin is not a suitable vitreous substitute.