Arterio-venous malformations of uterus - diagnostic and management dilemmas.

Keeping in mind the life-threatening consequences of curettage in cases of undiagnosed uterine arterio-venous malformation (AVM), its possibility should be considered in patients presenting with abnormal heavy uterine bleeding and negative Human Chorionic Gonadotropin (ß-hCG) values. We collected a series of cases in which the patients presented with abnormal heavy uterine bleeding, some not responding to conservative treatment. In the presence of declining or low serum ß-hCG levels and ultrasound Doppler showing increased vascularity, patients were investigated to detect the possible presence of uterine AVM. In those patients in whom angiographic confirmation of uterine AVM was made, embolisation was done and the outcome was followed. In those patients in whom hysterectomy was done the histopathogy specimen was studied for the possible cause of increased vascularity. Arterio-venous shunting seen on ultrasound does not always imply a uterine AVM and some cases can present diagnostic and management dilemmas.

A critical review of atypical cerebellum-type Creutzfeldt-Jakob disease: its relationship to "new variant" CJD and bovine spongiform encephalopathy.

Shortly after the appearance of bovine spongiform encephalopathy (BSE), Creutzfeldt-Jakob disease (CJD) was identified in young patients with nonclassical presentation such as difficulty in balancing and ataxia. The classical CJD in older patients starts with dementia. To distinguish between the two types, CJD in young persons has been termed "new variant" (nvCJD). The distinguishing features of classical CJD include initial presentation with dementia, confluent spongiform changes are very unusual in the cerebellum, and PrP plaques are rarely observed. For nvCJD, initially, difficulty with balancing and ataxia occurs, confluent spongiform changes are seen in the cerebellum, and a large number of PrP plaques are seen. The icelandic observation of sheep scrapie revealed a predominantly ataxic form of scrapie, termed Type II, rather than the itchy form termed Type I. Both types have been known to exist in Europe. Since the clinical signs of Type II scrapie in sheep with trembling and ataxia are similar to those seen in BSE and nvCJD, this suggests that Type II is the cause of BSE and nvCJD. Over 8 years, from 1989 to 1996, I examined the clinical histories of 33 CJD cases aged between the ages of 18 and 84. Six under the age of 40 and 15 over the age of 40 had leading clinical features such as difficulty in balancing and ataxia similar to those seen in the young cases classified as "nvCJD." Brains were examined from the six of 15 cases over the age of 40, which revealed similar pathology to that seen in young patients classified as "nvCJD." These findings suggest that all age groups are susceptible to the strain of the agent derived from BSE cattle.

Lingering doubts about spongiform encephalopathy and Creutzfeldt-Jakob disease.

Bovine spongiform encephalopathy (BSE) is an infectious disease and has been transmitted orally to many other animals, including humans. There is clear evidence of maternal transmission, although disagreement on the source of the BSE agent remains. The current theories link the origin of BSE to common scrapie in sheep. Twenty different strains of the scrapie agent have been isolated from sheep. A search of the literature indicates two distinct clinical syndromes in sheep, both of which have been called scrapie. I have designated these Type I (the common type), which exhibits itchiness and lose their wool, and Type II, which exhibits trembling and ataxia. Sheep inoculated with BSE develop Type II scrapie and they exhibit trembling. When cattle or mink are injected with the Type I strain, only a few will develop a clinical disease. By contrast, no clinical disease has so far been shown in cattle or mink by feeding them with Type I-infected sheep brains. However, either by injecting or feeding with the BSE strain, 100% of calves and mink develop the clinical disease. Evidence suggests that Type II is the cause of BSE. Identical clinical signs of Type II trembling are found in kuru and many of the recent cases of Creutzfeldt-Jakob disease. The BSE agent has caused spongiform encephalopathies (SEs) in domestic cats, tigers, and in some species of ruminants in zoos. The nature of the BSE agent remains unchanged when passaged through a range of species, irrespective of their genetic make up, demonstrating that variations in the host PrP gene are not a major factor in the susceptibility to the BSE agent. Since more than 85 zoo animals of many species have been diagnosed with SEs, from these studies it seems reasonable to conclude that the BSE agent can infect almost all mammalian species, including humans. For eradication of BSE and to reduce the risk of infection to humans, the development of a vaccine against BSE is suggested. Such a possibility should be fully explored.

Evidence that single-stranded DNA wrapped around the tubulofilamentous particles termed "nemaviruses" is the genome of the scrapie agent.

Homogenized brain tissue from scrapie-infected hamsters and uninfected hamsters was subjected to subcellular fractionation to isolate unique tubulofilamentous particles termed "nemaviruses". Nucleic acid was purified from the concentrate by the phenol/chloroform extraction procedure and by alkaline gel electrophoresis; a single band of ssDNA corresponding to about 1.2 kb was visualized. The gel-purified ssDNA was mixed before inoculation with (a) MgCl2, (b) lipofectin, (c) ssDNA-binding protein and (d) normal brain homogenate. Hamsters in group b, c and d injected with a mixture of ssDNA developed the clinical disease. Brain pathology revealed generalized vacuolation, while animals injected with ssDNA mixed with MgCl2 and controls remained healthy. These results suggest that ssDNA is the genome of the scrapie agent.

Efficacy of herpes vaccine and acyclovir (ACV) in a rabbit model following intraocular inoculation of herpes simplex virus.

It has been shown that injection of herpes simplex virus (HSV) type I into the vitreous body of the eye in 18-day-old albino rabbits consistently induced encephalitis. In the untreated group the lesions followed a defined anatomical pathway in the central nervous system and produced a chronic progressive disease with 95% survival. Detailed observations in the spread of HSV along the optic pathway determined the extent of damage at any given day. Some of the old rabbits developed typical herpetic lesions on nose and lips. HSV was demonstrated from these lesions by electron microscopy and also by tissue culture isolation. The combined efficacy of heat-killed herpes vaccine prepared from the same isolate and acyclovir (ACV) in this animal model was studied by starting treatment four days before or four days after the challenge. Ten animals immunised before the challenge were protected. However, immunisation after the challenge not only did not confer protection, but surprisingly, appeared to enhance the primary disease. All 10 rabbits immunised after the challenge developed weakness of the hind legs and progressed very rapidly to paralysis. ACV treatment alone did not completely abrogate the HSV infection, there appears to be reactivation of HSV which produced fresh small lesions. However, a combination of immunisation and treatment with ACV after the challenge of the 10 rabbits in the group prevented the development of weakness of the hind legs or paralysis. Detailed observations on the spread of HSV along the optic pathway revealed that pathological lesions and damage were limited in the ACV and combined treatment with ACV and vaccine group.

Progression of cytomegalovirus in the rabbit following the intraocular injection of the virus.

There is growing recognition that cytomegalovirus (CMV) is one of the opportunistic infections in patients afflicted with AIDS. The purpose of the present study was to establish a chronic CMV infection of the central nervous system (CNS) in rabbits after intraocular inoculation, to evaluate the therapeutic value of the model for investigation of the effect of antiviral drugs. It was possible to establish a chronic human cytomegalovirus (HCMV) encephalitis after 14-day old rabbits were injected into the vitreous body of the eye with the HCMV. One control and two experimental rabbits were killed at 16 days and 1, 3, 6 and 9 months post-inoculation. HCMV was demonstrated from the retina, optic nerve and brain by electron microscopy. HCMV infection did not cause apparent clinical symptoms or signs in the injected animals. At the morphological level the virus-induced lesion revealed the following features: that the HCMV travels slowly along the optic nerve and crosses at the optic chiasma showing a few thickly myelinated and many demyelinated axons with astroglial scar tissue. There was no evidence of inflammatory response in the lesions as no lymphocytes, plasma cells or phagocytic cells with dead myelin or lipid contents were observed. Long term observations of HCMV inoculated rabbits showed that the intraocular lesions followed a defined anatomical pathway in the optic nerve, chiasma and brain, leading to progressive chronic disease. The rabbit model would be very suitable for the evaluation of the therapeutic value of the effect of antiviral drugs.

Evidence that homologous ssDNA is present in scrapie, Creutzfeldt-Jakob disease, and bovine spongiform encephalopathy.

Homogenized brain tissue from scrapie-infected hamsters and uninfected hamsters was subjected to sub-cellular fractionation to isolate nemavirus. Nucleic acid was extracted from these fractions, which also contained mitochondria. Agarose-gel electrophoresis revealed a band corresponding to the size of circular hamster mtDNA in both infected and uninfected samples, but slower migrating bands were observed only in samples from scrapie-infected brain. A single band of ssDNA corresponding to about 1.2 kb was purified by alkaline gel electrophoresis from the nucleic acid content of the enriched preparations of nemavirus. The ssDNA was synthesized into double-stranded DNA, cloned and sequenced. An unusual palindromic six base TACGTA repeat sequence was observed suggesting that 1.2 kb molecules consist of multiple copies of (TACGTA)n spaced along the length of the ssDNA with a preceding sequence TATATA. The comparison of the nucleotide sequence of the inserted DNA to the GenBank nucleotide database revealed no significant homology to other sequences. A probe prepared from the Nar 50 clone was hybridized against DNA prepared from scrapie, CJD, BSE and normal brains under various salt and temperature conditions. The probe reacted with a band of about 1.2 kb in scrapie, CJD and BSE but not with control normal DNA specimens, thereby confirming the presence of ssDNA in these SEs. The results suggest an intimate association between the presence of nemavirus particles and scrapie, CJD and BSE.

Molecular cloning of single-stranded DNA purified from scrapie-infected hamster brain.

Homogenized normal and scrapie-infected hamster brains were subjected to subcellular fractionation. A single band of ssDNA corresponding to about 1.2 kb was purified by alkaline gel electrophoresis from the nucleic acid content of enriched preparations of mitochondria/tubulofilamentous particles. The ssDNA was synthesized into double-stranded DNA using Taq polymerase with four dNTP for extension. The cDNA synthesized was inserted in M13mp10, cloned and sequenced. An unusual palindromic six-base TACGTA repeat sequence was obtained and confirmed by an independent automated pathway and by cutting with a specific restriction enzyme. Comparison of the nucleotide sequence of the inserted DNA with the GenBank nucleotide database revealed no significant homology with those sequences. A probe prepared from the Nar 50 clone hybridized with the scrapie DNA band of about 1.2 kb noted above; however, no hybridization was observed with normal DNA, thus confirming the presence of ssDNA in scrapie. The presence of palindromic sequences in the scrapie genome could explain why many previous searches have revealed no evidence for a scrapie-specific nucleic acid.

Evidence that scrapie-associated tubulofilamentous particles contain a single-stranded DNA.

Unique virus-like tubulofilamentous particles, termed nemavirus, have been consistently observed in spongiform encephalopathic brains by electron microscopy in thin sections. Sodium dodecyl sulphate treatment of unfixed infected brain tissue on grids revealed a twisted fibril core of tubulofilamentous particles. The unmasked fibrils were identified as scrapie-associated fibrils by immunogold labelling. Both tubulofilamentous particles and scrapie-associated fibrils are not artefacts of protease treatment of protein formed in vitro during purification. Treatment with protease and nucleases revealed that each tubule consists of three layers: (i) an outer coat of protease-sensitive material; (ii) an intermediate layer that is digested by DNase and mung bean nuclease, and (iii) inner protease-resistant protein scrapie-associated fibril.

Scrapie-associated tubulofilamentous particles in human Creutzfeldt-Jakob disease.

Scrapie-associated fibrils (SAF) were demonstrated by a simple negative staining method for electron microscopy from fresh and frozen brains with naturally occurring human Creutzfeldt-Jakob disease (CJD). The findings confirm that SAF occur as an internal part of a larger three-layer particle. The two outer coats of SAF can be disrupted by detergent alone or can be digested in two stages by a combination of proteolytic enzymes and subsequent treatment with DNase and mung bean nuclease. Examination of thin sections from the cerebral cortex of brains from patients with CJD revealed the presence of 26-30-nm diameter tubulofilamentous particles, identical to those previously described in natural scrapie of sheep and bovine spongiform encephalopathy and also in experimentally induced scrapie in mice and hamsters and CJD-infected mice and chimpanzees. Thus, it would appear that the particles are not contaminants passaged in experimental animals.

Relationship of protease-resistant protein, scrapie-associated fibrils and tubulofilamentous particles to the agent of spongiform encephalopathies.

Tubulofilamentous particles and scrapie-associated fibrils (SAF) are ultrastructural markers, while protease-resistant protein (PrP) is a molecular biological marker for all spongiform encephalopathies. Review of all published work has suggested that PrP molecules aggregate to form a three-dimensional SAF. Further reports have suggested that a single-stranded DNA wraps round SAF and acquires an outer protein coat to form tubulofilamentous particles. As incubation period increases in the infected animals, larger amounts of PrP molecules are committed to form SAF, interfering with the normal supply of PrP to cell membranes which become disrupted and eventually fragment, resulting in vacuoles typical of those found in spongiform encephalopathies.

Scrapie-associated tubulofilamentous particles in scrapie hamsters.

Examination of thin sections from the cerebral cortex of scrapie-infected hamster brains revealed characteristic circular 26-30 nm diameter tubulofilamentous particles, identical to those previously described in both experimentally induced scrapie in mice, hamsters and natural scrapie of sheep, bovine spongiform encephalopathy and human Creutzfeldt-Jakob disease and mice and chimpanzees infected with Creutzfeldt-Jakob disease. Longitudinal forms of tubulofilamentous particles were also observed in dendrites and myelinated axons. Both transverse and longitudinally cut particles were readily distinguished from microtubules and synaptic vesicles, thus there appears to be no relationship between tubulofilamentous particles, and microtubules or synaptic vesicles.

Increased multimeric mitochondrial DNA in the brain of scrapie-infected hamsters.

We observed a marked increase in multimeric mitochondrial DNA (mtDNA) in brains of scrapie-infected hamsters compared with those of uninfected hamsters. Homogenized brain tissue was subjected to subcellular fractionation to isolate scrapie-associated fibrils and tubulofilamentous structures. Nucleic acids were extracted from the scrapie-associated fibril/tubulofilament fraction which also contained mitochondria. Agarose gel electrophoresis revealed a band corresponding to the size of circular hamster mtDNA in both infected and uninfected samples, but slower migrating bands were observed only in samples from scrapie-infected brain. We showed by molecular cloning, nucleotide sequencing, and Southern blotting that the slower migrating bands are mtDNA. These findings confirm the recent demonstration by differential hybridization that multimeric mtDNA occurs in hamster scrapie brain. Elevated levels of multimeric circular mtDNA have been reported previously in various tumors and cultured cell lines.

Evidence of ssDNA in tubulofilamentous particles: their relationship to scrapie-associated fibrils.

Abnormal tubulofilamentous particles were identified by electron microscopy using a simple touch negative staining technique from brains of mice infected with four strains of the scrapie agent. Treatment by three proteolytic enzymes and subsequent treatment with DNase and mung bean nuclease of grids prepared from the infected animals confirmed previous observations that the tubulofilamentous particles observed in scrapie-effected brains are complex structures. The core of the tubulofilamentous particle scrapie-associated fibrils was revealed by treatment with SDS. Treatment with proteolytic enzymes and subsequent treatment with DNase or mung bean nuclease or S1 nuclease also revealed typical and transitional stages of scrapie-associated fibrils. However, treatment with RNase A had no effect. The data suggest that nucleic acid is a single-stranded DNA protected by a protein coat.

Detection of single-stranded DNA in scrapie-infected brain by electron microscopy.

The nucleic acid content of enriched preparations of mitochondria/tubulofilamentous particles from normal and scrapie-infected hamster brains were examined by electron microscopy. After spreading on collodion-coated grids circular molecules of approximately 15.7 kb corresponding in size to mitochondrial DNA (mtDNA) were observed both in normal and scrapie-infected brains. In nucleic acid preparations from scrapie-infected brains multimeric mtDNA and single-stranded DNA strands of about 0.49 X 10(6) daltons were also visualized. These findings demonstrate the presence of a single-stranded DNA in scrapie-infected brains and are consistent with previous data based on enzyme digestion of nucleic acids isolated from scrapie-infected brains.

Competitive enzyme-linked immunosorbent assay for Treponema pallidum antibodies.

A competitive enzyme-linked Treponema pallidum immunosorbent assay (CETPIA) was compared with the standard serological tests for syphilis. Of 3081 serum samples submitted, 2883 gave negative results in the CETPIA and the routine screening tests. Positive results were obtained in the CETPIA and in one or more of the specific treponemal tests with 115 samples. Discrepancies in the results of the CETPIA and standard serological tests were found with 83 serum samples, most of these were attributed to biological false positive reactions in the Venereal Disease Research Laboratory (VDRL) test. CETPIA may have a role in the serological diagnosis of syphilis.

Evidence that DNA is present in abnormal tubulofilamentous structures found in scrapie.

Abnormal tubulofilamentous structures have been identified in electron micrographs of thin sections and negatively stained impression grids prepared from brains of animals with scrapie and other spongiform encephalopathies, and we showed that such tubules contain a core of filamentous structures resembling scrapie-associated fibrils (SAF). We treated impression grids from brains of scrapie-infected hamsters with several substances that bind to or cleave proteins and nucleic acids to see if they had any effect on the abnormal tubulofilamentous structures. Treatment with three proteolytic enzymes reduced the caliber of the tubules from about 50 nm to 30 nm; subsequent treatment of the 30-nm tubules with DNase I left many typical SAF as well as transitional forms in which twisted SAF emerged from tubules. DNase treatment of the original thicker tubules had no effect, and no SAF were seen on grids. Treatment of the 30-nm tubules with any of three other nucleases (micrococcal, mung bean, and BAL-31) also produced SAF. However, treatment with RNase A had no effect either on the original 50-nm tubules or on the 30-nm tubules produced by proteolysis. Detergent treatment of any of the preparations produced SAF. Treatment with ethidium bromide resulted in staining of the tubules that was inhibited by magnesium ions. The data suggest that the abnormal tubulofilamentous particles found in spongiform encephalopathies may consist of an outer cylinder of protein, an inner cylinder of DNA, and an innermost core of SAF.

A chronological study of experimental scrapie in mice.

The development of scrapie-associated particles and lesions in four regions of the brain was studied in mice over a period of 30 weeks. Characteristic tubulovesicular particles, identical to those previously described, were first found about half way through the incubation period in mice inoculated by four different routes. The particles are found in brains with scrapie and other spongiform encephalopathies; they have never been seen in other conditions, and potentially represent the infectious agent.