Squirrel monkeys (Saimiri sciureus) infected with the agent of bovine spongiform encephalopathy develop tau pathology.

Squirrel monkeys (Saimiri sciureus) were infected experimentally with the agent of classical bovine spongiform encephalopathy (BSE). Two to four years later, six of the monkeys developed alterations in interactive behaviour and cognition and other neurological signs typical of transmissible spongiform encephalopathy (TSE). At necropsy examination, the brains from all of the monkeys showed pathological changes similar to those described in variant Creutzfeldt-Jakob disease (vCJD) of man, except that the squirrel monkey brains contained no PrP-amyloid plaques typical of that disease. Constant neuropathological features included spongiform degeneration, gliosis, deposition of abnormal prion protein (PrP(TSE)) and many deposits of abnormally phosphorylated tau protein (p-Tau) in several areas of the cerebrum and cerebellum. Western blots showed large amounts of proteinase K-resistant prion protein in the central nervous system. The striking absence of PrP plaques (prominent in brains of cynomolgus macaques [Macaca fascicularis] with experimentally-induced BSE and vCJD and in human patients with vCJD) reinforces the conclusion that the host plays a major role in determining the neuropathology of TSEs. Results of this study suggest that p-Tau, found in the brains of all BSE-infected monkeys, might play a role in the pathogenesis of TSEs. Whether p-Tau contributes to development of disease or appears as a secondary change late in the course of illness remains to be determined.

High activity Rhenium-186 HEDP with autologous peripheral blood stem cell rescue: a phase I study in progressive hormone refractory prostate cancer metastatic to bone.

We tested the feasibility and toxicity of high activities Rhenium-186 hydroxyethylidene diphosphonate, with peripheral blood stem cell rescue in patients with progressive hormone refractory prostate cancer metastatic to bone. Twenty-five patients received between 2500 and 5000 MBq of Rhenium-186 hydroxyethylidene diphosphonate followed 14 days later by the return of peripheral blood peripheral blood stem cells. Activity limiting toxicity was defined as grade III haematological toxicity, lasting at least 7 days, or grade IV haematological toxicity of any duration or any serious unexpected toxicity. Activity limiting toxicity occurred in two of six who received activities of 5000 MBq and maximum tolerated activity was defined at this activity level. Prostate specific antigen reductions of 50% or more lasting at least 4 weeks were seen in five of the 25 patients (20%) all of whom received more than 3500 MBq of Rhenium-186 hydroxyethylidene diphosphonate. The actuarial survival at 1 year is 54%. Administered activities of 5000 MBq of Rhenium-186 hydroxyethylidene diphosphonate are feasible using autologous peripheral blood peripheral blood stem cell rescue in patients with progressive hormone refractory prostate cancer metastatic to bone. The main toxicity is thrombocytopaenia, which is short lasting. A statistically significant activity/prostate specific antigen response was seen. We have now commenced a Phase II trial to further evaluate response rates.

Assessment of interlaboratory and intralaboratory sperm morphology readings with the use of a Hamilton Thorne Research integrated visual optical system semen analyzer.

OBJECTIVE: To evaluate the level of variance produced in a multicenter study with the use of a computer-assisted sperm morphology analyzer. DESIGN: A multicenter, prospective, blinded study. SETTING: Assisted reproduction research laboratories. PATIENT(S): Semen samples produced for assisted reproductive procedures. INTERVENTION(S): Hamilton Thorne Research (Beverly, MA) integrated visual optical system semen analyzers were used at five different centers to evaluate the same set of 30 slides that were prepared and numerically coded at Tygerberg Hospital in Tygerberg, South Africa. MAIN OUTCOME MEASURE(S): The percentage of normal sperm. RESULT(S): Interlaboratory coefficients of variation (CVs) ranged between 16.31% and 23.09%. One of the participating laboratories produced an approximately 14% (-6.5-7.7) limits of agreement analysis, with a CV of 11.36%, for its duplicate readings. The use of a 10% normal sperm morphology cutoff point to determine discordance levels produced rates ranging between 10% and 23.3% for the interlaboratory and intralaboratory readings. This level of discordance equates with < or = 7 of the corresponding readings from two laboratories falling into a different normal sperm morphology group (< or = 10% or >10%). CONCLUSION(S): The magnitudes of variation produced by the readings performed in our study reached the same level as for the manual evaluation of sperm morphology. A < 10% CV can be obtained if the correct quality control measures are implemented.

Genetic stability of Sabin 1 strain of poliovirus: implications for quality control of oral poliovirus vaccine.

The Sabin vaccine strains of poliovirus, like all RNA viruses, exist as a quasispecies of genomic sequences whose composition can be altered during virus propagation. Since changes in vaccine virus during manufacture can enhance the neurovirulent potential of the vaccine, each monovalent lot of oral poliovirus vaccine (OPV) undergoes several tests to ensure consistency of manufacture, including the monkey neurovirulence test (MNVT). Recently, we proposed a new molecular approach for direct quantification of vaccine variants with neurovirulent potential as an alternative way to monitor consistency of OPV production. Analysis of the Sabin 1 genome allowed us to identify a limited number of specific loci that exhibit significant change during viral propagation in vitro and in vivo. Here we explore the possible roles of these changes and show that 7427-U-->C and 7441-G-->A alterations in the 3'-UTR of the Sabin 1 virus do not increase monkey neurovirulence. These, as well as our previous results, suggest that only mutations in the 5'-UTR play a significant role in the limited increase in Sabin 1 monkey neurovirulence observed after extended propagation of the virus beyond the passage level used in vaccine production. Our studies with high-passage batches of the Sabin 1 strain confirmed the stability of this strain, which retains acceptable levels of monkey neurovirulence even after serial passages at elevated temperature. Compared to the MNVT, molecular analysis of the genetic composition of Sabin 1 poliovirus provides a more sensitive analytical approach to monitor consistency of vaccine production.

Search for viral nucleic acid sequences in brain tissues of patients with schizophrenia using nested polymerase chain reaction.

BACKGROUND: We used polymerase chain reaction to search for nucleic acid sequences of several viruses in DNA and RNA extracted from brain tissues of schizophrenic and control subjects. METHODS: We extracted DNA and RNA templates from frozen brain specimens of 31 patients with schizophrenia and 23 nonschizophrenic control patients with other diseases. The extracts were subjected to polymerase chain reaction with oligonucleotide primers for 12 different viruses (cytomegalovirus, Epstein-Barr virus, herpes simplex virus type 1, human herpesvirus type 6, varicellazoster virus, measles virus, mumps virus, rubella virus, the picornavirus group, influenza A virus, human T-cell lymphotropic virus type I, and St Louis encephalitis virus), several of which have been suspected of involvement in schizophrenia. Nested primers were used to increase the sensitivity of the method. RESULTS: No amplified nucleic acid sequences encoded by the selected viral genomes were detected in extracts of any brain specimens from either schizophrenic or control patients. CONCLUSIONS: These data agree with previous studies that failed to find sequences of a number of viruses in the cerebrospinal fluid or selected areas of the brains of schizophrenic patients. Additional efforts should be undertaken to identify other known and unknown pathogens in schizophrenia, sampling more areas of the brain from subjects with a variety of clinical types of schizophrenia.

Evaluation of shell vial cell culture technique for the detection of bovine coronavirus.

The effect of blind passage and centrifugation on the isolation of bovine coronavirus in human rectal tumor cells cultured in shell vials was investigated. A total of 68 fecal samples known to be positive for bovine coronavirus by transmission electron microscopic (TEM) examination were used. The samples were centrifuged onto human rectal tumor cell monolayers and incubated in the presence of trypsin. The growth of bovine coronavirus in infected cells was demonstrated by fluorescent antibody staining, and the extracellular virus was detected and confirmed by hemagglutination and hemagglutination-inhibition tests, respectively. Of the 68 TEM-positive samples, 51 (75%), 58 (85%), and 61 (90%) grew in shell vial cell cultures at first, second, and third passages, respectively. Of the 51 cultures positive on first passage, 19 were examined by TEM; 18 of these were positive for bovine coronavirus. The shell vial technique was also compared with direct detection of bovine coronavirus by staining cryostat sections of infected tissues in a direct fluorescent antibody assay. The results of direct fluorescent antibody assay were available for 54 of the 68 samples, of which 53 (98%) and 43 (80%) were positive by shell vial technique and direct fluorescent antibody assay, respectively. For identification of bovine coronavirus, shell vials using human rectal tumor cells in the presence of trypsin is more sensitive than direct fluorescent antibody assay but is relatively less sensitive than transmission electron microscopy.

Relative changes in blood flow with functional electrical stimulation during exercise of the paralyzed lower limbs.

Eight spinal cord injured (SCI) patients performed three sets of exercise with two conditions, 60% and 80% of VO2peak, with an arm crank ergometer. Functional neuromuscular stimulation was used to induce static leg contractions in two of the above sets of exercise. The three exercise sets were performed with no functional neuromuscular stimulation (NOS); with functional neuromuscular stimulation at 40 milliamperes; and with functional neuromuscular stimulation at 80 milliamperes (HIS). The lower limb blood flow was estimated by a photoelectric plethysmograph. Results showed that the lower limb blood flow was consistently reduced across both functional neuromuscular stimulation levels (17.4% from NOS to LOS; 13.8% from LOS to HIS; and 28.8% from NOS to HIS), and work loads (15.3% from rest to 60% VO2peak; 38.0% from 60% VO2peak to 80% VO2peak; and 47.5% from rest to 80% VO2peak). Rate-pressure product was decreased by 8.3% between NOS and HIS at 60% VO2peak (15.7 +/- 3.4 to 14.4 +/- 3.8), by 6.8% between NOS and HIS at 80% VO2peak (18.9 +/- 53 to 17.6 +/- 4.8), and by 12.4% between LOS and HIS at 80% VO2peak (20.1 +/- 6.7 to 17.6 +/- 4.8). These data indicate that in SCI (a) functional neuromuscular stimulation-induced contractions of the lower limb muscles can increase blood flow and thus reduce venous blood pooling in the paralyzed muscles, and (b) such improvements are associated with a reduced rate pressure product.(ABSTRACT TRUNCATED AT 250 WORDS)

Solubilization and characterization of diacylglycerol acyltransferase from microspore-derived cultures of oilseed rape.

Particulate fractions prepared from microspore-derived (MD) embryos of oilseed rape (Brassica napus L. cv. Reston) and an embryogenic MD cell suspension culture of oilseed rape (B. napus L. cv. Jet Neuf) were used as a source of diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) for enzyme characterization and development of a solubilization procedure. DGAT activity in the 1500-100,000 g fraction from MD embryos was stimulated 4-5-fold by 3 to 4 mg of BSA/ml of reaction mixture. DGAT activity from MD embryos was stimulated 2-3-fold by fluoride salts and 1.4-fold by NaCl, whereas iodide salts caused substantial inhibition of enzyme activity. The effect of the various 1:1 electrolytes on enzyme activity appeared to be related more to their differential effects on solution structure rather than ionic strength. DGAT was solubilized from membranes of MD embryos and the cell suspension culture by about 80 and 50% respectively, using 2 M NaCl in 1% (w/v) octanoyl-N-methyl-glucamide (MEGA-8) (pH 8.0 buffer) at a detergent to protein ratio of 2:1. The specific activity of solubilized DGAT was about 2-fold greater than that of the particulate enzyme. The mechanism of solubilization appeared to be related to the lowering of the critical micellar concentration of MEGA-8 in the presence of NaCl. DGAT, solubilized from MD embryos, eluted with an M(r) of about 2 x 10(6) during gel-filtration chromatography on a Superose 6 column equilibrated in buffer containing 0.1% (w/v) MEGA-8. The solubilized enzyme exhibited optimal activity at pH 7. At concentrations above 2 microM acyl-CoA, the specificity of solubilized DGAT for oleoyl-CoA and palmitoyl-CoA was considerably greater than for stearoyl-CoA.

Cryopreservation of transgenic mouse lines.

A transgenic animal represents an enormous investment in time and money. Animals can be destroyed through disease, fire, malfuncnons in the control of the environment, negligence, sabotage, or accidental disposal. Researchers can protect valuable transgenic lines from accrdental destruction by "banking" them in liquid nitrogen. Cryopreservation can also reduce animal costs by decreasing the number of live animals investigators must maintain. Often, when one is trying to produce a transgenic animal, some lines will be derived that may not initially appear interesting. These animals can be stored in liquid nitrogen for future recovery and study. The maintenance of just one line of mice, say 25 mice at 15 cents/d, can cost over $1000 (US) in a single year.

Cryopreservation of transgenic rat lines.

The following protocol is for freezing rat blastocysts by direct plunging into liquid nitrogen. Although this protocol is designed for freezing the embryos in polypropylene cryotubes, it could readily be adapted to 0.5-mL straws. For a general discussion of cryopreservation, the reader is referred to Chapter 25.

Cryopreservation of transgenic sheep lines.

Although a method exists for freezing sheep embryos directly into liquid nitrogen without a programmable freezing machine, a more traditional slow cooling method is described here. Either dimethyl sulfoxide (DMSO) or ethylene glycol can be used as a cryoprotectant in this slow cooling method. Pregnancy rates for frozen embryos are similar to those of fresh embryos. For a general discussion of cryopreservation, the reader is referred to Chapter 25.

Cryopreservation of transgenic mice.

Advances in cryopreservation enable one to freeze embryos without the use of a programmable freezing machine or complex protocols. These methods achieve high rates of survival when mouse embryos are frozen. Understanding the factors that influence the survival of cryopreserved embryos can aid troubleshooting and in adapting freezing strategies from mice to other species. Frozen stocks of transgenics can be maintained indefinitely in liquid nitrogen. Cryopreservation of these valuable animals not only protects them from environmental catastrophes, but also is an economical method of storing lines for future detailed analysis.

Detection of flaviviruses by reverse-transcriptase polymerase chain reaction.

RNA sequences of five flaviviruses were detected by a modified polymerase chain reaction (PCR) that incorporated a reverse transcriptase and RNase inhibitor. Oligonucleotide primer pairs were synthesized to amplify sequences from St. Louis encephalitis (SLE), Japanese encephalitis (JBE), yellow fever (YF), dengue 2 (DEN-2), and dengue 4 (DEN-4) viruses. The amplified products were visualized as bands of appropriate size on ethidium bromide-stained agarose gels. The identity of these products was confirmed by restriction endonuclease cleavage to generate fragments of predicted lengths. The reverse-transcriptase PCR (RT-PCR) successfully amplified flavivirus sequences from cell cultures, frozen brain tissue, and formalin-fixed, paraffin-embedded brain tissue. The reactions were highly specific, and the method compared favorably to two conventional assays of viral infectivity. RT-PCR followed by PCR with nesting primers (N-PCR) was 1,000-fold more sensitive in detecting virus than classical infectivity titration by intracerebral inoculation of suckling mice and nearly 1,000-fold more sensitive than amplification of virus in cell culture followed by inoculation of mice.

Clinicopathologic and pathologic findings of herpesvirus-induced urinary tract infection in conventionally reared cats.

Clinicopathologic manifestations of induced infection of the feline lower urinary tract with bovid herpesvirus-4 (BHV-4, strain FCAHV) were characterized in 6 conventionally reared adult cats (2 sexually intact males, 2 castrated males, and 2 females). Two additional control cats were exposed with noninfected cell culture control inoculum. Clinical and radiographic signs of lower urinary tract disease were not observed in exposed or control cats. Microscopic hematuria was detected in urine samples collected by cystocentesis from 4 to 6 exposed cats and 1 of 2 control cats. Results of culture of urine for bacteria, mycoplasmas, ureaplasmas, and viruses were consistently negative. Low titer of serum BHV-4 (strain FCAHV)-neutralizing antibodies was detected in 4 of 6 exposed cats, but not in controls. Gross abnormalities of the urinary tract were not observed in any cat. Light microscopic examination of serial sections of the lower urinary tract revealed mild focal lymphoid cystitis in 2 of 6 exposed cats, one of which also had increased amounts of connective tissue and proliferation of blood vessels in the urinary bladder lamina propria. Ninety days after initial exposure, BHV-4 (strain FCAHV) was reisolated from explanted urinary bladder tissues of 5 of 6 exposed cats. Virus was not isolated from tissues of control cats. It was concluded that BHV-4 (strain FCAHV) establishes persistent urinary tract infection in conventionally reared adult male and female cats. However, persistent BHV-4 infection in cats may remain clinically inapparent.

Clinicopathologic analysis of herpesvirus-induced urinary tract infection in specific-pathogen-free cats given methylprednisolone.

The clinicopathologic manifestations of bovid herpesvirus-4 (BHV-4; FCAHV strain)-induced infection of the lower portion of the urinary tract were characterized in 12 adult neutered male and 6 female specific-pathogen-free cats, and were compared with those in 12 neutered male control cats. Six neutered male and 6 female cats were given immunosuppressive doses of methylprednisolone acetate prior to inoculation of their urinary bladders with BHV-4. Six neutered male control cats were given immunosuppressive doses of methylprednisolone acetate prior to inoculation of their urinary bladders with uninfected tissue culture control inoculum. Six additional neutered male control cats were exposed only to uninfected tissue culture control inoculum. All cats were observed for 90 days following inoculation. Dysuria and gross hematuria were observed in only 1 BHV-4-exposed cat. Radiographic abnormalities of the lower portion of the urinary tract were not observed. Microscopic hematuria, crystalluria, and lipiduria were identified with similar frequency in BHV-4-exposed and control cats. Results of urine culturing for bacteria, mycoplasma, ureaplasma, and viruses were negative. Viruses were not isolated from blood leukocytes collected from exposed or control cats. Three to 6 weeks after inoculation, high concentrations of BHV-4 serum antibodies were detected in all exposed cats by an indirect fluorescent antibody test. Light microscopic examination of the urinary tract revealed multifocal lymphoid cystitis in 2 BHV-4-exposed cats. Except for suppurative bronchitis in 1 BHV-4-exposed cat given glucocorticoids, morphologic differences in urinary and extraurinary tissues were not observed. In urinary bladder tissue collected 90 days after inoculation, BHV-4 was reisolated from urinary bladder explants of all but 1 exposed cat. Virus was also isolated from a kidney explant of 1 exposed male cat, and spleen cell co-cultures of 1 exposed female cat given glucocorticoids.(ABSTRACT TRUNCATED AT 250 WORDS)

Scrapie-induced diabetes mellitus in hamsters.

Scrapie-infected hamsters had slightly elevated non-fasting plasma glucose levels, markedly abnormal glucose tolerance tests, and impaired release of insulin in response to a glucose load. Plasma cortisol levels were essentially the same in infected and uninfected animals. Histological examination of the pancreas revealed no morphological changes in infected animals with no alteration in distribution of cells secreting insulin, glucagon and somatostatin. In contrast, brains of scrapie-infected animals had the diffuse vacuolation typical of spongiform encephalopathy. These experiments suggest that scrapie-induced diabetes mellitus in hamsters may result from damage to the central nervous system.

Antibodies to HTLV-I in populations of the southwestern Pacific.

Sera collected from 1,102 individuals in 14 populations of the southwestern Pacific between 1956 and 1979 were tested by ELISA for antibodies to human T-cell leukemia virus type I (HTLV-I). Selected sera were also tested by particle agglutination and immunoblotting. Six of the populations had prevalences of antibodies greater than 4%, two populations had prevalences greater than 15%. Six populations had antibody prevalences of 2% or less. Three populations from the coast and northern islands of New Guinea had high prevalences of antibodies, while three New Guinea highland groups had virtually none. One population from the Solomon Islands had a high prevalence, while two others had very low prevalences. Two populations from small remote islands in Vanuatu both had high prevalences. Pacific sera did not neutralize a standard strain of virus readily neutralized by Japanese, European, and American sera. We conclude that infections with HTLV-I, some acquired more than 20 years ago, are widespread throughout the southwestern Pacific, even in several very isolated populations, although others have been spared. Some strains of HTLV-I in populations of the Pacific may have substantially different envelope proteins from prototype strains of America, Europe, and Japan.

Caffeine promotes in vitro fertilization of mouse ova within 15 minutes.

Epididymal sperm were collected from C57Bl6/J X DBA2/J (B6D2) males and allowed to capacitate for 2 hr. When cumulus-free oocytes were exposed to sperm for 15 min in either the presence (6.0 mM) or absence of caffeine, fertilization did not occur. However, when cumulus cells were left intact, 23% of oocytes were fertilized in caffeine-free medium and 62% in caffeine-containing medium. When cumulus-free oocytes were incubated with sperm for 30 min, none was fertilized in the absence of caffeine, but 33% were fertilized when 6.0 mM caffeine was present (P less than .02). These effects of caffeine were on the sperm, as sperm exposed to caffeine and then coincubated with oocytes for 15 min in essentially caffeine-free media fertilized a similar percent of oocytes (93%) as when sperm and oocytes were exposed to caffeine during the fertilization period (86%). When sperm were capacitated in caffeine-containing medium, the percentage of ova fertilized was similar to capacitation without caffeine. We conclude that both cumulus cells and caffeine speed up the fertilization process with mouse gametes and that the effect of caffeine is on the sperm, but not due to more rapid capacitation.