There is great potential in successful delivery of genetic material to somatic cells of animals and humans, not only to treat genetic deficiencies, but also to provide more effective therapies and vaccines. Concerted effort has been mounted over the past decade to validate the concept
of gene-based therapy. Delivery of genes in vitro via multiple methods has demonstrated the biologic usefulness of the basic concept. However, application of similar gene delivery strategies to whole animal systems has been more difficult. Much of the complexity associated with gene delivery
involves encounters with host biological barriers, including innate and acquired host responses to exogenous DNA, as well as specifically encoded proteins. Delivery of genetic material to somatic cells in vivo is a multi-factorial event involving variables in formulation, route, and dose,
and target species or strain. In vivo expression of these variables can result in conflicting findings with regard to gene expression and toxicity. These findings may compromise the predictive reliability of interstrain and, particularly, interspecies studies. This review will address representative
in vivo somatic cell transfection modalities, variability in species and strain responses following delivery of nucleic acids, and some of the potential mechanisms involved.
Background and Purpose: Use of high-energy near-infrared lasers is becoming more prevalent in today's industries, such as technology, medicine, and military operations. Despite wide-range use of these lasers, threshold, median effective dose (ED50), and the mechanism
of laser-tissue interaction are not well defined at the 1,318-nm wavelength for human corneal exposures. The goals of the study reported here were to establish the ED50 for single-pulse, 1,318-nm laser exposures on the Dutch Belted rabbit cornea and to characterize microscopic changes.
Results of this study were then compared with those of previous corneal studies.Methods: A neodymium:yttrium aluminum garnet (Nd:YAG) laser was used to deliver single 1,318-nm wavelength pulses to the corneas of 10 female Dutch Belted rabbits (Oryctolagus cuniculus). Single
pulses of 0.5-ms duration and radiant energy ranging from 116 to 2,250 J/cm2 irradiated the exposure sites. Sites were clinically evaluated for presence of a lesion at one hour and 24-h after exposure. Results of the 24-h evaluation were used to determine the (ED50).
Corneas were subsequently collected at the 24-h endpoint for microscopic evaluation.Results: The ED50 for 1,318-nm exposures to the rabbit cornea was determined to be 382 J/cm2, as measured at the 1/e2 (0.865 times that of the peak power per unit
area). At each exposure site, there was a small (<1 mm in diameter), white, circular, well demarcated corneal lesion characterized histologically by a band of stromal coagulative necrosis and endothelial necrosis, with sparing of the anterior epithelium. In addition, there appeared some
potential for damage to Descemet's membrane at the highest energy level tested.Conclusions: Findings indicate that the rabbit cornea is subject to injury at the 1,318-nm wavelength with the established ED50.
A simple and sensitive duplex polymerase chain reaction (PCR) assay was developed for use in detection of Helicobacter species and H. hepaticus in laboratory mice. Bacteria were extracted and concentrated from fecal pellets and intestinal segments by use of buoyant density
centrifugation. To improve quality assurance, an internal control (mimic) for detection of false-negative reactions was included. In addition, cartridges (Capillette) pre-filled with PCR reagents, were used to minimize the hands-on time required, thus reducing the risk of contamination with
previously amplified material. Laboratory mice from Swedish animal houses sent to the National Veterinary Institute for health monitoring were found to have high prevalence of H. hepaticus.
Purpose: Current animal models of diabetic microangiopathy, including diabetic retinopathy (DR), have substantial limitations. To determine whether the diabetic pig would provide an improved large animal model of DR, a 20-week porcine model of diabetic dyslipidemia that manifests
appreciable macrovasculopathy was evaluated for development of retinal microvascular changes associated with diabetes mellitus (hereafter referred to as diabetes) in humans.Methods: The effect of diabetes alone or in combination with high dietary fat intake on retinal capillary
morphology was assessed. Cell density in the retinal capillaries was evaluated by use of elastase digestion isolation of the retinal vascular bed, and retinal capillary basement membrane thickness was determined by use of electron microscopic analyses.Results: Diabetic pigs fed
a normal diet had significantly (P < 0.001) thicker retinal capillary basement membranes than did nondiabetic animals. High fat intake did not contribute to basement membrane thickening. Significant effects of diabetes or diet on retinal capillary cell densities were not observed,
nor did diabetes induce formation of microaneurysms typical of advanced diabetic retinopathy.Conclusions: The rapid development of retinal capillary basement membrane thickening in diabetic pigs makes this animal model useful for early intervention and mechanistic studies for this
diabetes-associated microvascular change.
Purpose: In an effort to develop a non-lethal model of ricin toxicosis, we studied the biochemical effects of the administration of ricin in mice.Methods: Mice received an intraperitoneal injection of ricin toxin, after which a panel of 21 biochemical parameters was
determined. Effect of ricin administration on blood glucose concentration was studied in greater detail. To determine whether results of biochemical analyses correlated with therapeutic manipulations known to protect against ricin toxicosis, the effect of immunization on blood glucose values
after ricin administration was studied.Results: Of the biochemical parameters studied, only blood glucose and amylase values were affected by ricin administration. Because blood glucose concentration can be easily and instantaneously measured on 25 to 50 μl of blood, using handheld
instruments, this parameter was further evaluated. A dose- and time-related decrease in blood glucose concentration was documented. Mice immunized with ricin had smaller changes in blood glucose concentration than did non-immunized animals after ricin administration.Conclusion:
Blood glucose concentration may be used as a surrogate for lethal challenge as a measure of ricin toxicosis after its systemic administration.
Detection of infectious viruses in clinical samples typically relies on daily examination of inoculated cell cultures for appearance of virus-induced cytopathic effect (CPE), with subsequent immunologic or genetic analysis to identify the specific virus producing the CPE. Performing
virus isolation on samples suspected of containing Cercopithecine herpesvirus 1 (monkey B virus [BV]) is dangerous due to the extreme neuropathogenicity of this virus in humans, and minimally requires biosafety level 3 (BSL-3) facilities. To provide a safer method of detecting infectious
BV in clinical samples, the eucaryotic green fluorescent protein (GFP) coding sequence was flanked with BV sequences containing transcriptional control elements. This construct was placed into a stealth vector and transfected into Vero cells, then stable transformed cell lines were selected.
These cells express GFP only when infected with BV or other related primate herpesviruses. Expression of GFP allows detection of infectious BV in cultures sooner and more reliably than does standard microscopic observation for CPE. The ability to detect BV by GFP expression eliminates the
need for further testing to identify the virus as an α-herpesvirus following development of CPE, thus allowing cell cultures to be sealed at inoculation. Although not entirely specific for BV, this cell line will make detection of infectious BV in samples collected from macaques safer
to perform.
Confirmation of transgene-coded protein expression on the surface of mouse white blood cells has traditionally involved use of spleen and lymph nodes as a cell source. However, this eliminates the mice from further analysis and increases the number of animals needed for testing. A minimally
invasive method of cell collection would allow these mice to remain in the colony, thereby conserving valuable resources. The goal of the study reported here was to detect transgene expression on mouse white blood cells collected from peripheral blood. Mice over-expressing the T-cell receptor
(TCR) transgene product Vβ8.2 on the surface of T lymphocytes were compared with wild-type control mice. Mononuclear cells were counted, using a hemacytometer. Peripheral blood cells were stained with anti-Vβ8.2 fluorescent conjugate and were evaluated, using
fluorescence-activated (FA) microscopy as well as flow cytometry (fluorescence-activated cell sorting [FACS]). Mean ± SD number of mononuclear cells was 5.34 ± 1.72 × 105 cells/100 ml from TCR Tg mice and 4.99 ± 1.99 × 105 cells/100 μl
from control mice. Expression of transgene Vβ8.2 was confirmed by use of FA microscopy and FACS. Mean population of lymphocytes expressing Vβ8.2 was 43.0 ± 12.9% from TCR Tg blood samples, and 9.4 ± 1.0% from control blood samples. Spleen and lymph
node specimens also were compared. Results of the unpaired t test and the Mann-Whitney test indicated significant difference in the amount of expressed Vβ8.2 between transgenic and wild-type mice. It is concluded that this microassay can serve as a quick, minimally invasive,
non-lethal alternative for detecting TCR transgene-encoded antigens on the surface of mouse lymphocytes, and is potentially applicable to a number of other transgene-coded cell surface proteins.
A novel Respirovirus was isolated from nasopharyngeal swab specimens from clinically normal laboratory guinea pigs, and was characterized and named caviid parainfluenza virus 3 (CavPIV-3). The CavPIV-3 is enveloped, is 100 to 300 nm in diameter, and has a characteristic 15-nm-diameter
chevron-shaped virus ribonucleocapsid protein. Sequence analysis of the fusion glycoprotein of CavPIV-3 revealed it to be 94% identical to human and guinea pig parainfluenza 3 (PIV-3) viruses and 80% identical to bovine PIV-3. To determine whether CavPIV-3 causes clinical disease in laboratory
guinea pigs and to compare the serologic response of guinea pigs to CavPIV-3 and to other paramyxoviruses, an infection study was performed, in which groups of guinea pigs were inoculated with CavPIV-3, Sendai virus, simian virus 5 (SV-5), murine pneumonia virus (PVM), or bovine PIV-3 virus.
During the course of the study, guinea pigs were maintained in an infectious disease suite, housed in Micro-Isolator™ cages, and were only manipulated under a laminar flow hood. Clinical signs of disease were not observed in any of the paramyxovirus-inoculated guinea pigs
during the eight-week course of the study, and histologic signs of disease were not evident at necropsy eight weeks after inoculation. Guinea pigs inoculated with CavPIV-3, Sendai virus, PVM, and bovine PIV-3 developed robust homologous or heterologous serologic responses. In contrast, guinea
pigs inoculated with SV-5 developed modest or equivocal serologic responses, as assessed by use of an enzyme-linked immunosorbent assay. Further, use of the SV-5 enzyme-linked immunosorbent assay resulted in the highest degree of non-specific reactivity among all of the paramyxovirus assays.
In summary, CavPIV-3 is a novel guinea pig Respirovirus that subclinically infects laboratory guinea pigs, resulting in a robust serologic response, but no observed clinical or histologic disease. The CavPIV-3 fusion glycoprotein gene sequence is available from GenBank as accession
No. AF394241, and the CavPIV-3 virus is available from the American Type Culture Collection as accession No. DR-1547.
The Japanese macaque or snow monkey (Macaca fuscata) is an autochthonous monkey in Japan. It has long been assumed that the monkey population was not infected with Cercopithecine herpesvirus 1 (monkey B virus [BV]) since cases of human BV infection have never been reported
in Japan. Although serologic testing of captive snow monkeys in Japan revealed antibodies to BV, it was thought that native Japanese macaques had either been infected with herpes simplex virus from humans or with BV from other imported macaque species. To clarify this issue, we performed polymerase
chain reaction (PCR) analysis to amplify BV sequences from trigeminal ganglia of 30 Japanese macaque monkeys that were seropositive for BV. Sequences from two BV genes, UL27 (360 bp) and UL19 (1.0 Kbp), from 3 of 30 monkeys were amplified. Results of restriction fragment length polymorphism
analysis and DNA sequencing of the fragments provided evidence that native Japanese macaques are infected with BV. Phylogenetic analysis indicated that these monkeys harbor their own genotype of BV that is different from other known BV genotypes, and provided additional evidence supporting
the co-evolution of BV and macaques.
On physical examination, a 5 × 10-cm abdominal mass was found in an eight-year-old female rhesus macaque. Radiography revealed an opaque mass in the cranial portion of the abdomen, displacing the stomach craniad. Percutaneous biopsy obtained hair with little tissue, confirming
a diagnosis of trichobezoar. Initially, the hairball was medically managed by oral administration of lubricants. Medical management proved unsuccessful, the macaque began to lose weight, and two gastric trichobezoars were subsequently removed surgically. Normal appetite and activity were regained
within one week. Gastric trichobezoars may lead to severe clinical illness, and should be considered in the differential diagnosis for anorexia and/or weight loss in any nonhuman primate. Trichobezoars may also be detected and treated prior to development of illness.
During an annual physical examination, a middle-aged adult female olive baboon (Papio anubis) in the time-mated breeding colony at the Biologic Resources Laboratory at the University of Illinois at Chicago was found to have a high serum calcium value (> 12 mg/dl). To determine
the cause of the hypercalcemia, additional diagnostic tests, including thoracic and abdominal radiographs and a parathyroid panel (parathyroid hormone (PTH), ionized calcium, and parathyroid hormone-related protein (PTH-rp) assays), were performed. The radiographs did not reveal lesions suggestive
of neoplasia. A parathyroid panel was obtained twice. Both times the PTH (23.4 and 46.4 pmol/L, normal = 2.91 to 4.57 pmol/L) and ionized calcium (1.68 and 2.10 mmol/L, normal = 1.31 to 1.37 mmol/L) were increased above values for adult females with normal calcium concentration. A tentative
diagnosis of primary hyperparathyroidism was made. After a γ-radiation scan and magnetic resonance imaging of the neck were done, exploratory surgery was performed to identify and remove the affected gland. After gland removal, the baboon's serum calcium, PTH (1.6 pmol/L), and ionized
calcium (1.59 mmol/L) values decreased. Results of histologic examination confirmed the diagnosis of benign solitary parathyroid adenoma.
Epididymal cribriform hyperplasia (ECH) is a variant of normal epididymal histologic features in men, and has also been reported in rats, mice, dogs, cats, and bulls. The epididymal change has been associated with aging, testicular atrophy, cryptorchidism, and germ cell tumors. Epididymal
cribriform hyperplasia was observed in p53 homozygous knockout mice on a mixed 129/Sv-FVB/N background, but not in wild-type or heterozygous mice. The aim of the study reported here was to determine the prevalence and characterize the morphologic, immunohistochemical, and ultrastructural features
of ECH in these mice. Epididymal cribriform hyperplasia was present in 88% (72/82) of male mice ranging in age from seven to 65 weeks. The lesion was characterized microscopically by epithelial cells with atypical hyperchromatic nuclei, vacuolization, intratubular lumina formation, infrequent
apoptosis, and rare mitotic figures. In contrast to germ cells, the cells of ECH did not express α-fetoprotein, carcinoembryonic antigen, or S-100. Ultrastructurally, the cells were pleomorphic with stereocilia at their apical borders and within intratubular lumina, and were supported
by a basement membrane. Although 14% (10/72) of mice had concomitant testicular neoplasia, ECH did not appear to be a preneoplastic change. Investigators using these mice for modeling human disease should be aware of the background prevalence of this lesion.