The effects of freezing, boiling and degreasing on the microstructure of bone.

The histology of bone has been a useful tool in research. It is commonly used to estimate the age of an individual at death, to assess if the bone is of human or non-human origin and in trauma analysis. Factors that affect the histology of bone include age, sex, population affinity and burning to name but a few. Other factors expected to affect bone histology are freezing, boiling and degreasing but very little information is available for freezing and the effect thereof, and it is unknown if boiling and degreasing affects bone histology. The aim of this study was to assess the effects of freezing, freezing and boiling, and freezing, boiling and degreasing on the histological structure of compact bone. Five cadaver tibiae were frozen at -20°C for 21 days followed by segments being boiled in water for three days and degreased in trichloroethylene at 82°C for three days. Anterior midshaft sections were prepared as ground sections and for Scanning Electron Microscopy (SEM). Quantitatively, there were no significant differences between freezing, boiling and degreasing; however, qualitative differences were observed using SEM. After being frozen the bone displayed cracks and after boiling the bones displayed erosion pits on the surface. It is suggested that further research, using different durations and temperatures for boiling and freezing be undertaken on bone samples representing different ages and various skeletal elements.

Complete genome sequences of two feline leukemia virus subgroup B isolates with novel recombination sites.

It is generally accepted that all primary isolates of feline leukemia virus (FeLV) contain a subgroup A virus (FeLV-A) that is essential for transmission. In contrast, FeLV-B is thought to arise de novo in the infected animal through RNA recombination events with endogenous FeLV transcripts, presumably through copackaging of RNA from endogenous FeLV and exogenous FeLV-A. Here, we report the complete genome sequences of two novel strains of FeLV-B (FeLV-2518 and FeLV-4314) that were isolated in the absence of FeLV-A. The env genes of these isolates have been characterized previously, and the 3' recombination sites have been identified. We describe herein the 5' recombination breakpoints of each virus. These breakpoints were found to be within the signal peptide of the env gene and the reverse transcriptase-coding region, respectively. This is the first report of a recombination site within the pol gene of an FeLV-B genome and the first genetic characterization of multiple independently arising FeLV-B isolates that have been identified without a functional FeLV-A ancestral virus.

Are endogenous feline leukemia viruses really endogenous?

Full length endogenous feline leukemia virus (FeLV) proviruses exist within the genomes of many breeds of domestic cat raising the possibility that they may also exist in a transmissible exogenous form. Such viruses would share receptor usage with the recombinant FeLV-B subgroup, a viral subgroup that arises in vivo by recombination between exogenous subgroup A virus (FeLV-A) and endogenous FeLV. Accordingly, all isolates of FeLV-B made to date have contained a "helper" FeLV-A, consistent with their recombinatorial origin. In order to assess whether endogenous viruses are transmitted between cats, we examined primary isolates of FeLV for which the viral subgroup had been determined for the presence of a subgroup B virus that lacked an FeLV-A. Here we describe the identification of two primary field isolates of FeLV (2518 and 4314) that appeared to contain subgroup B virus only by classical interference assays, raising the possibility of between-host transmission of endogenous FeLV. Sequencing of the env gene and U3 region of the 3' long terminal repeat (LTR) confirmed that both viral genomes contained endogenous viral env genes. However the viral 3' LTRs appeared exogenous in origin with a putative 3' recombination breakpoint residing at the 3' end of the env gene. Further, the FeLV-2518 virions also co-packaged a truncated FeLV-A genome containing a defective env gene, termed FeLV-2518(A) whilst no helper subgroup A viral genome was detected in virions of FeLV-4314. The acquisition of an exogenous LTR by the endogenous FeLV in 4314 may have allowed a recombinant FeLV variant to outgrow an exogenous FeLV-A virus that was presumably present during first infection. Given time, a similar evolution may also occur within the 2518 isolate. The data suggest that endogenous FeLVs may be mobilised by acquisition of exogenous LTRs yielding novel viruses that type biologically as FeLV-B.

Isolation and partial characterization of Brazilian samples of feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) causes a slow progressive degeneration of the immune system which eventually leads to a disease comparable to acquired immune deficiency syndrome (AIDS) in humans. FIV has extensive sequence variation, a typical feature of lentiviruses. Sequence analysis showed that diversity was not evenly distributed throughout the genome, but was greatest in the envelope gene, env. The virus enters host cells via a sequential interaction, initiated by the envelope glycoprotein (env) binding the primary receptor molecule CD134 and followed by a subsequent interaction with chemokine co-receptor CXCR4. The purpose of this study was to isolate and characterize isolates of FIV from an open shelter in São Paulo, Brazil. The separated PBMC from 11 positive cats were co-cultured with MYA-1 cells. Full-length viral env glycoprotein genes were amplified and determined. Chimeric feline × human CD134 receptors were used to investigate the receptor utilization of 17 clones from Brazilian isolates of FIV. Analyses of the sequence present of molecular clones showed that all clones grouped within subtype B. In contrast to the virulent primary isolate FIV-GL8, expression of the first cysteine-rich domain (CRD1) of feline CD134 in the context of human CD134 was sufficient for optimal receptor function for all Brazilian FIV isolates tested.

Phylogenetic characterisation of naturally occurring feline immunodeficiency virus in the United Kingdom.

Feline immunodeficiency virus (FIV) is a significant pathogen of domestic and non-domestic felids worldwide. In domestic cats, FIV is classified into five distinct subtypes (A-E) with subtypes A and B distributed most widely. However, little is known about the degree of intrasubtype viral diversity and this may prove critical in determining whether monovalent vaccines are likely to protect against FIV strains within a single subtype. Here, we characterise novel env sequences from 47 FIV strains recovered from infected cats in the United Kingdom and its environs. Phylogenetic analyses revealed that all bar one sequence belonged to subtype A, the predominant subtype in Western Europe. A single sequence was identified as a likely subtype A/C recombinant, intriguing given that subtype C does not appear to exist in either the UK or North Western Europe and suggestive of a recombination event predating its introduction into the UK. Subtype A strains from the UK were not significantly differentiated from representative subtype A isolates found elsewhere suggesting multiple introductions of FIV into the country. Divergence among isolates was comparable to that observed for subtype A isolates worldwide, indicating that FIV in the UK covers the full spectrum of subtype A diversity seen globally. This study demonstrates that while subtype A is predominant in the UK, novel introductions may result in the emergence of novel subtypes or intersubtype recombinants, potentially circumventing vaccine strategies. However, the dominance of subtype A suggests that the development of a regional or subtype-specific protective vaccine for the UK could be achievable.

Lymphocyte migration through the blood-brain barrier (BBB) in feline immunodeficiency virus infection is significantly influenced by the pre-existence of virus and tumour necrosis factor (TNF)-alpha within the central nervous system (CNS): studies using an in vitro feline BBB model.

AIMS: In human immunodeficiency virus infection, macrophage-tropic and lymphotropic viruses exist in the host. Central nervous system (CNS) infection is an early and ongoing event, important to understand when developing strategies to treat infection. Some knowledge exists on macrophage-tropic virus interactions with the blood-brain barrier (BBB), and the aim of this study was to investigate lymphotropic lentivirus interactions with the BBB. METHODS: Interactions of the lymphotropic feline immunodeficiency virus (FIV) with an in vitro model of the feline BBB were evaluated in scenarios to mimic in vivo infections. RESULTS: Cell-free FIV crossed the BBB in very low quantities, and in the presence of tumour necrosis factor (TNF)-alpha, BBB integrity was unaffected. However, cell-associated FIV readily crossed the BBB, but BBB integrity was not significantly altered. Transmigration of uninfected and infected lymphocytes increased in response to TNF-alpha, accompanied by a moderate disruption of barrier integrity and an upregulation of vascular cell adhesion molecule-1 rather than intercellular adhesion molecule-1. Significant enhancement of migration and disruption of BBB tight junctions occurred when infected cells and TNF-alpha were added to the brain side of the BBB and this enhancement was not mediated through additional TNF-alpha production. CONCLUSIONS: Small quantities of virus in the brain together with TNF-alpha have the potential to stimulate greater cell and viral entry into the CNS and this is likely to involve important factors other than further TNF-alpha production. Lymphotropic lentivirus entry to the CNS is governed by many factors similar to macrophage-tropic strains.

Ability of antibodies to two new caliciviral vaccine strains to neutralise feline calicivirus isolates from the UK.

This study examined a panel of 110 UK field isolates of feline calicivirus (FCV) for susceptibility to cross-neutralisation by a panel of eight antisera raised in cats infected with FCV strains F9, 255, FCVG1 and FCV431. The pairs of antisera raised against F9 or 255, neutralised 20 and 21 per cent or 37 and 56 per cent of field strains of virus respectively. In contrast, the pairs of antisera raised against the newer vaccine strains FCVG1 or FCV431 neutralised 29 and 70 per cent or 67 and 87 per cent of field strains respectively. Antisera raised against the two newer strains, namely FCVG1 and FCV431, neutralised a greater proportion of field strains of calicivirus than antisera raised against the older FCV vaccine strains F9 and 255.

Clomiphene citrate modulates the expression of endometrial carbohydrates (especially N-acetyl-d-glucosamine and sialic acid) in pseudopregnant rats.

This study investigated the presence of carbohydrates N-acetyl-d-glucosamine and sialic acid oligosaccharides, in the uterine epithelium of pseudopregnant rats treated with ovarian hormones and Clomiphene citrate (CC) a commonly used fertility drug associated with low pregnancy rates. Ovariectomized sexually mature rats were given 0.25mg CC prior to the implantation-priming hormone sequence of 5mg progesterone for 3 days and a single dose of 0.5microg estradiol-17beta (E(2)) on day 3 (PP(PE)) and sacrificed 24h after the last hormone treatment. Uterine tissue was incubated with the lectin Triticum vulgare (wheat germ agglutinin, WGA), associated with avidin and subsequently labelled with biotinylated-ferritin for electron microscopy, a combined alcian blue/PAS technique for light microscopy and RT-PCR was run for progesterone-associated endometrial protein (PAEP) gene, a pregnancy related endometrial gene that is associated with the protein thought to express carbohydrates in the uterus and suppress immune function. The results indicate that CC administration decreases the expression of these carbohydrates both at a cellular level and genetic level when compared to the PP(PE) group. However, the lowest expression of N-acetyl-d-glucosamine and sialic acid was seen in the placebo group. The ovarian hormones were therefore shown to be important for the synthesis of these carbohydrates that are important in the implantation period and the maintenance of pregnancy. The results suggest that the effect of CC on the expression of N-acetyl-d-glucosamine and sialic acid is a significant reason why there are low pregnancy rates with CC use.

Vaccine protection against feline immunodeficiency virus: setting the challenge.

Since feline immunodeficiency virus (FIV) was first isolated, international research efforts have been directed towards developing a protective vaccine, not least because it may provide a model for a candidate human immunodeficiency virus (HIV) vaccine. This article reviews the challenges facing vaccine development, the current state of knowledge and future prospects for FIV vaccination.

Changes in oviductal morphology of the skink, Lampropholis guichenoti, associated with egg production.

We describe changes in the morphology of the oviductal epithelium of an oviparous skink, Lampropholis guichenoti, during the course of egg production and oviposition: to characterize the luminal epithelial changes; to provide a baseline for understanding uterine changes in viviparous species; and to establish whether the plasma membrane transformation of uterine epithelial cells is indeed a feature restricted to viviparous species. Oviducts from vitellogenic, gravid, and postgravid females were observed using scanning electron microscopy. Cellular characteristics of the oviductal epithelium previously used to determine the plasma membrane transformation were assessed morphologically. Three anatomically different areas were defined within the oviduct, but no plasma membrane transformation was observed in the oviparous skink, suggesting that this is a phenomenon particular to viviparity.

Diagnosis and management of B cell chronic lymphocytic leukaemia in a cat.

A four-year-old, female neutered domestic shorthair cat had a history of chronic intermittent vomiting and lymphocytosis. B cell chronic lymphocytic leukaemia was diagnosed by flow cytometry, which revealed abnormally large numbers of mature B lymphocytes in the peripheral blood. The cat was treated conservatively with antiemetic drugs and remained stable without chemotherapy for over a year.

Dynamics of viral and proviral loads of feline immunodeficiency virus within the feline central nervous system during the acute phase following intravenous infection.

Animal models of human immunodeficiency virus 1, such as feline immunodeficiency virus (FIV), provide the opportunities to dissect the mechanisms of early interactions of the virus with the central nervous system (CNS). The aims of the present study were to evaluate viral loads within CNS, cerebrospinal fluid (CSF), ocular fluid, and the plasma of cats in the first 23 weeks after intravenous inoculation with FIV(GL8). Proviral loads were also determined within peripheral blood mononuclear cells (PBMCs) and brain tissue. In this acute phase of infection, virus entered the brain in the majority of animals. Virus distribution was initially in a random fashion, with more diffuse brain involvement as infection progressed. Virus in the CSF was predictive of brain parenchymal infection. While the peak of virus production in blood coincided with proliferation within brain, more sustained production appeared to continue in brain tissue. In contrast, proviral loads in the brain decreased to undetectable levels in the presence of a strengthening PBMC load. A final observation in this study was that there was no direct correlation between viral loads in regions of brain or ocular tissue and the presence of histopathology.

Human uterodomes (pinopods) do not display pinocytotic function.

BACKGROUND: The term 'pinopod' or 'pinopode' has been used indiscriminately since the 1970s to describe most apical structures on uterine epithelial cells and as such suggests a cross species structural functionality. This study looks at the apical cellular protrusions in rats and humans and compares their pinocytotic ability. METHODS: We have utilized standard tracer techniques in an attempt to determine the functionality of the uterine surface protrusions in the human based on results reported in rats. RESULTS: Pinopods in rat tissue demonstrated tracer uptake, but no tracer uptake in the apical protrusions of human uterine epithelium was evident. CONCLUSIONS: These findings indicate that the uterine surface protrusions observed in the human are not pinocytotic and therefore probably perform a function different from similar structures observed in rats and mice. This highlights the need to alter nomenclature from pinopods to uterodomes.

Immunolocalization of occludin and claudin-1 to tight junctions in intact CNS vessels of mammalian retina.

The distributions of occludin and claudin-1, two tight junction-associated integral membrane proteins were investigated by immunohistochemical analysis of whole-mount preparations of the blood vessels in the myelinated streak of the rabbit retina. Light microscopy revealed that occludin and claudin-1 immunoreactivities were abundant along the interface of adjacent endothelial cells of all blood vessels. Electron microscopy revealed that both proteins were distributed in a regular pattern (at regular intervals of approximately 80 nm) along the length of tight junctions, probably in the regions of tight junction strands. No other structures or cell types expressed either of these two proteins in the myelinated streak. Whereas occludin immunoreactivity was concentrated only at the tight junction interface, claudin-1 immunoreactivity also extended into the cytoplasm of the endothelial cells, suggesting a different structural role for claudin-1 than for occludin at tight junctions. Retinal pigment epithelial cells expressed occludin around their entire circumference, consistent with the function of these cells as a barrier separating the retina from the leaky vessels of the choroid. Also consistent with the association of occludin expression with vessels that exhibit functional tight junctions, this protein was expressed at only a low level in, and showed an irregular distribution along, the vessels of the choroid, a vascular bed that lacks blood-barrier properties. Further, the distribution of occludin was examined during formation and remodelling of the rat retinal vasculature. Occludin expression was evident at the leading edge of vessel formation and was found on all vessels in both the inner and outer vascular plexus. Numerous vascular segments at the early stage of vascular formation and regression lost occludin expression. The biological significance of this transient loss of occludin expression in terms of barrier function remains to be elucidated.

The effect of acute oxidative stress on the ultrastructure of the perfused rat liver.

Ageing and liver disease are associated with ultrastructural changes in the hepatic sinusoid. Because of the possibility that reactive oxygen species could mediate these processes, we examined the effect of acute oxidative stress on the ultrastructure of the intact liver. Rat livers were perfused ex vivo, in situ with hydrogen peroxide via the portal vein. The livers were then fixed and the ultrastructure of the liver tissue examined with transmission and scanning electron microscopy. The effects of hydrogen peroxide were largely confined to the perisinusoidal areas. The sinusoidal endothelial cells became swollen and more porous, with large gaps replacing sieve plates. The space of Disse showed an increase in volume and the density of hepatocyte projections decreased. Kupffer cell activation was noted. Little or no ultrastructural change was observed within the hepatocytes. Oxidative stress delivered via the portal vein dramatically alters the ultrastructure of the perisinusoidal regions of the liver. This process may contribute to the pathogenesis of disease and age-related changes in the liver.

Expression of glucosamine trisaccharides on the rat uterine surface is altered by clomiphene citrate. II. Combination with ovarian hormones.

We used a single administration of clomiphene citrate (CC), a synthetic oestrogen that is prescribed for infertility treatment, in combination with either a single administration of oestradiol 17beta (E2) or progesterone (P4) to assess the combined effects of these hormones on the uterine surface. The aim of these experiments was to investigate how CC in combination with these hormones affected both expression of oligosaccharides on the uterine surface and membrane architecture further elucidating CC's agonistic/antagonistic properties. Ovariectomized sexually mature rats were given combinations of E2 and CC (E2 + CC) or P4 and CC (P4 + CC) or P4 and E2 (P4 + E2) and were killed 24 h later. Uterine tissue was labelled with the lectin Phytolacca americana conjugated with avidin and subsequently labelled with biotinylated ferritin and prepared for transmission electron microscopy. Results of the administration of these hormone combinations indicate that CC, when administered in conjunction with E2, had the ability to downregulate expression of oligosaccharides on the membrane surface caused by E2. When administered with P4, CC had the ability to upregulate the effects of P4. Thus, when combined with E2, CC has an antagonistic effect but when combined with P4, CC has an agonistic effect.

Vaccination with inactivated virus but not viral DNA reduces virus load following challenge with a heterologous and virulent isolate of feline immunodeficiency virus.

It has been shown that cats can be protected against infection with the prototypic Petaluma strain of feline immunodeficiency virus (FIV(PET)) using vaccines based on either inactivated virus particles or replication-defective proviral DNA. However, the utility of such vaccines in the field is uncertain, given the absence of consistent protection against antigenically distinct strains and the concern that the Petaluma strain may be an unrepresentative, attenuated isolate. Since reduction of viral pathogenicity and dissemination may be useful outcomes of vaccination, even in the absence of complete protection, we tested whether either of these vaccine strategies ameliorates the early course of infection following challenge with heterologous and more virulent isolates. We now report that an inactivated virus vaccine, which generates high levels of virus neutralizing antibodies, confers reduced virus loads following challenge with two heterologous isolates, FIV(AM6) and FIV(GL8). This vaccine also prevented the marked early decline in CD4/CD8 ratio seen in FIV(GL8)-infected cats. In contrast, DNA vaccines based on either FIV(PET) or FIV(GL8), which induce cell-mediated responses but no detectable antiviral antibodies, protected a fraction of cats against infection with FIV(PET) but had no measurable effect on virus load when the infecting virus was FIV(GL8). These results indicate that the more virulent FIV(GL8) is intrinsically more resistant to vaccinal immunity than the FIV(PET) strain and that a broad spectrum of responses which includes virus neutralizing antibodies is a desirable goal for lentivirus vaccine development.

The plasma membrane transformation facilitates pregnancy in both reptiles and mammals.

Mechanisms of placentation are very diverse in mammals and range from types in which the uterine epithelium is breached by the implanting blastocyst to those where the epithelium remains intact. Despite these differences in mechanisms, the initial response of the plasma membrane of uterine epithelial cells is remarkably similar across mammalian species which has led to the term 'plasma membrane transformation' to encapsulate the concept of a common beginning to implantation. Membrane phenomena similar to those of mammals have now been observed in some viviparous lizards at the ultrastructural level during early pregnancy, and we propose extending the concept of 'plasma membrane transformation' to lizards with live birth.