Partitioning of TSE infectivity during ethanol fractionation of human plasma.

The practice of validating processes for their capacity to inactivate a range of non-enveloped and enveloped viruses also provides confidence that plasma products will be safe from emerging viral pathogens with known aetiology. Of greater concern are diseases of unknown or poorly defined aetiology such as the group of neurological diseases collectively called the transmissible spongiform encephalopathies (TSEs), or prion diseases, for which the best known human disease is Creutzfeldt-Jakob Disease (CJD) and its variant form (vCJD). The goal of the current study was to investigate the potential for manufacturing steps used in the production of albumin and immunoglobulin products by Kistler-Nitschmann fractionation, and the utility of nanofiltration of immunoglobulin to remove TSE agents. Two different scrapie model systems were used. In the first system infectious material used for spiking was scrapie sheep brain homogenate with infectivity titres being measured in hamsters. In the second system purified scrapie agent was used (PrP fibrils) with Western blot analysis measuring reduction in the proteinase K resistant form being used as a measure of removal. The data demonstrated substantial removal of the infectious agent by the manufacturing process in both model systems although some differences were observed in partitioning of the two different infectious materials. The hamster infectivity studies were shown to be approximately 1000 fold more sensitive than the Western Blot assay. The data from both studies provide added confidence that these plasma products are safe with respect to their potential to transmit TSE.

Inactivation of viral and prion pathogens by gamma-irradiation under conditions that maintain the integrity of human albumin.

BACKGROUND AND OBJECTIVES: The administration of therapeutic plasma protein concentrates has been associated with the real risk of transmitting viral diseases and the theoretical risks of prion transmission. Our objective was to determine if gamma-irradiation can inactivate viral or prion infectivity without damaging a protein biotherapeutically. MATERIALS AND METHODS: Human albumin 25% solution, spiked with four model viruses (including porcine parvovirus) or with brain homogenate from scrapie-infected hamsters, was gamma-irradiated at constant low-dose rates and assayed for viral and prion infectivity or for albumin integrity. RESULTS: At a radiation dose of 50 kGy, viruses were inactivated by >/= 3.2 to >/= 6.4 log10 and scrapie by an estimated 1.5 log10, whereas albumin was only moderately aggregated and fragmented. CONCLUSIONS: gamma-Irradiation can preferentially inactivate viral and prion pathogens without excessive damage to albumin structure.

The viral anti-inflammatory chemokine-binding protein M-T7 reduces intimal hyperplasia after vascular injury.

Chemokines and IFN-gamma function as central regulators of inflammatory responses to vascular injury. Both classes of cytokines are upregulated during restenosis, a response to vascular injury that leads to recurrent atherosclerotic plaque growth, but the relative impact of each class of cytokines remains undetermined. M-T7 is a secreted myxoma viral immunomodulatory glycoprotein that functions both as a species-specific inhibitor of rabbit IFN-gamma and as a chemokine-binding protein, interacting with a wide range of C, C-C, and C-X-C chemokines in a species-nonspecific fashion. We wished to (a) assess the efficacy of purified M-T7 protein in inhibiting intimal hyperplasia after angioplasty injury and (b) exploit unique species-specific functions of M-T7 in order to judge the relative importance of each cytokine class on plaque growth. Anesthetized New Zealand white rabbits and Sprague-Dawley rats received either M-T7 or control at the time of arterial angioplasty injury. Histological analysis at 28 days demonstrated significant reductions in intimal hyperplasia with M-T7 treatment in both models, with an associated early inhibition of inflammatory cell invasion. Purified M-T7 protein inhibits intimal hyperplasia after angioplasty injury in a species-nonspecific fashion, thus implicating the chemokine-binding activity as more critical for prevention of plaque growth after vascular injury.

The distribution of infectivity in blood components and plasma derivatives in experimental models of transmissible spongiform encephalopathy.

BACKGROUND: The administration of blood components from donors who subsequently develop Creutzfeldt-Jakob disease has raised the issue of blood as a possible vehicle for iatrogenic disease. STUDY DESIGN AND METHODS: We examined infectivity in blood components and Cohn plasma fractions in normal human blood that had been "spiked" with trypsinized cells from a scrapie-infected hamster brain, and in blood of clinically ill mice that had been inoculated with a mouse-adapted strain of human transmissible spongiform encephalopathy. Infectivity was assayed by intracerebral inoculation of the blood specimens into healthy animals. RESULTS: Most of the infectivity in spiked human blood was associated with cellular blood components; the smaller amount present in plasma, when fractionated, was found mainly in cryoprecipitate (the source of factor VIII) and fraction I+II+III (the source of fibrinogen and immunoglobulin); almost none was recovered in fraction IV (the source of vitamin-K-dependent proteins) and fraction V (the source of albumin). Mice infected with the human strain of spongiform encephalopathy had very low levels of endogenous infectivity in buffy coat, plasma, cryoprecipitate, and fraction I+II+III, and no detectable infectivity in fractions IV or V. CONCLUSION: Convergent results from exogenous spiking and endogenous infectivity experiments, in which decreasing levels of infectivity occurred in cellular blood components, plasma, and plasma fractions, suggest a potential but minimal risk of acquiring Creutzfeldt-Jakob disease from the administration of human plasma protein concentrates.

Classifying cervical cells using a recurrent neural network by building basins of attraction.

This paper describes the result of classifying cervical cells employing a novel way of using a Hopfield-style neural network for classification. This method could be used as part of an automated cervical screening system. Rather than storing the exemplars (training elements) as stable points, a connection matrix is determined, using perceptron-type learning, such that the exemplars are placed in basins of attraction. The exemplars from different classes of cells are placed in different basins of attraction, with usually more than one basin per training class. A presented element is then classified by the basin of attraction it falls into. Input to the classification consists of one case of feature vectors derived from the cervical cell images; in another case, input consists of the images themselves. Good results were obtained using this technique.

Plasma pharmacokinetics and tissue distribution of paclitaxel in CD2F1 mice.

We defined the pharmacokinetics of paclitaxel after i.v., i.p., p.o., and s.c. administration of 22.5 mg/kg to CD2F1 mice. Additional mice were studied after i.v. bolus dosing at 11.25 mg/kg or 3-h continuous i.v. infusions delivered at 43.24 micrograms kg-1 min-1. Plasma was sampled between 5 min and 40 h after dosing. Brains, hearts, lungs, livers, kidneys, skeletal muscles, and, where applicable, testicles were sampled after i.v. dosing at 22.5 mg/kg. Liquid-liquid extraction followed by isocratic high-performance liquid chromatography (HPLC) with UV detection was used to determine paclitaxel concentrations in plasma and tissues. After i.v. administration to male mice, paclitaxel clearance (CLtb) was 3.25 ml min-1 kg-1 and the terminal half-life (t1/2) was 69 min. After i.v. administration to female mice, paclitaxel CLtb was 4.54 ml min-1 kg-1 and the terminal t1/2 was 43 min. The bioavailability of paclitaxel was approximately 10%, 0, and 0 after i.p., p.o., and s.c. administration, respectively. Paclitaxel bioavailability after i.p. administration was the same when the drug was delivered in a small volume to mimic the delivery method used to evaluate in vivo antitumor efficacy or when it was delivered in a large volume to simulate clinical protocols using i.p. regional therapy. Paclitaxel was not detected in the plasma of mice after i.p. delivery of the drug as a suspension in Klucel: Tween 80. Pharmacokinetic parameters were similar after i.v. delivery of paclitaxel at 22.5 and 11.25 mg/kg; however, the CLtb calculated in these studies was much lower than that associated with 3-h continuous i.v. infusions. After i.v. administration, paclitaxel was distributed extensively to all tissues but the brain and testicle. These data are useful in interpreting preclinical efficacy studies of paclitaxel and predicting human pharmacokinetics through scaling techniques.

Local versus general anesthesia for lumbar percutaneous discectomy.

Ninety-two adult patients scheduled for automated percutaneous discectomy (PERC) were assigned to receive either local anesthesia supplemented with monitored i.v. analgesia (MIVA) or general endotracheal anesthesia (GA-LITE). Patients were examined 1 week post-PERC for the presence of new paresthesias, and they completed a questionnaire 6-18 weeks after PERC about changes in their pain. Sixty-four percent of MIVA patients and 83% of GA-LITE patients had diminished pain following PERC. Results did not show any difference between the two groups for new paresthesias after PERC. There were no differences in postoperative pain medication requirements, but the GA-LITE group reported more postoperative nausea, vomiting, and sore throat. GA-LITE patients averaged 1.06 +/- 0.3 h in the recovery room compared with 0.70 +/- 0.3 h for MIVA patients. Although the use of general anesthesia for PERC has been contraindicated because of fear of damaging the nerve root in the sleeping patient, we conclude that general anesthesia does not increase nerve injuries attributable to instrumentation. However, general anesthesia did cause a higher incidence of minor complications such as nausea, vomiting, and sore throat in the immediate postoperative period than did MIVA.

ZDV delays but does not prevent the transmission of MAIDS by LP-BM5 MuLV-infected macrophage-monocytes.

LP-BM5 MuLV infection of monocyte-macrophages (MM cells) and the ability of MM cells infected with this murine oncornavirus complex to transmit murine acquired immune deficiency syndrome (MAIDS) were assessed. Adherent cells expressing Mac-1 antigen (Mac-1+) were isolated from the peritoneum of infected C57BL/6 mice at weekly intervals postinoculation. A small percentage of MM cells was infected by 7 days after inoculation with LP-BM5 MuLV and virus production could be detected in MM cells throughout the course of disease. MAIDS could be induced in naive C57BL/6 mice by i.p. injection of 0.5-3 x 10(5) MM cells derived from infected mice as early as 8 weeks postinfection. When 3 x 10(5) cells (300 infectious centers) were injected, the progression of disease was similar to that seen after inoculation with a known virus pool (log10 5.78 XC pfu/ml). Treatment with zidovudine (ZDV) at 1 mg/ml in drinking water delayed disease progression if started 24 h prior to inoculation of MM cells and given continuously.

Effect of 3'azidothymidine administered in drinking water or by continuous infusion on the development of MAIDS.

LP-BM5 MuLV infection of C57BL/6 mice induces a well characterized, lymphoproliferative, immunodeficiency disease (MAIDS), which is useful for evaluation of potential antiviral agents, because of the reproducibility of virological and clinical endpoints. This MAIDS retrovirus model was used to evaluate 3'azido-2,3'dideoxythymidine (AZT), using different doses, methods of administration and timing for initiation and continuation of therapy. AZT therapy 1 mg/ml in the drinking water given 30 days prior to virus challenge, and continued for 16 weeks, prevented LP-BM5 MuLV dissemination and disease in 13 of 15 treated mice. Efficacy was dose dependent for AZT concentrations of 1, 0.5, and 0.1 mg/ml in drinking water. One mg/ml AZT was most effective in preventing infection if therapy was begun within days prior to virus challenge or within the first four hours after virus inoculation. If treatment was initiated later, disease was delayed. Continuous infusion of AZT, 25 micrograms/h, was effective since virus was not detected in spleens of any mice during the 21 days of AZT treatment. However, after treatment was stopped treated mice became virus positive and disease progressed. Likewise, AZT administration at 1 mg/ml in the drinking water for only 21 days post virus inoculation (p.i.), was not sufficient to prevent virus dissemination or disease.

Transmission in NFS/N mice of the heritable spongiform encephalopathy associated with the gray tremor mutation.

It has been shown that the autosomal recessive mutation, gray tremor (gt) was associated in the homozygous state (gt/gt) with a rapidly fatal spongiform encephalopathy. Heterozygotes (+/gt) developed mild asymptomatic spongiform brain lesions as did recipient inbred mice inoculated with gt/gt brain homogenates, some of whom also showed behavioral abnormalities [Sidman, R. L., Kinney, H. C. & Sweet, H. O. (1985) Proc. Natl. Acad. Sci. USA 82, 253-257]. In these studies, inbred NFS/N mice inoculated intracerebrally at birth or as adults with gt/gt or first passage gt brain homogenates developed a progressive disease characterized by tremor, ataxia, and spasticity. The symptoms were milder and more slowly progressive than in the gt/gt homozygote, in the paralytic syndrome that followed neonatal inoculation of NFS/N mice with a wild murine leukemia virus (Cas-Br-M MuLV), or in the rapidly progressive ataxia and terminal bradykinesia that followed scrapie inoculation of NFS/N mice. The noninflammatory spongiform encephalopathy in affected NFS/N mice resembled that observed in gt/gt homozygotes, +/gt heterozygotes, and asymptomatic recipient inbred mice inoculated with gt/gt brain homogenates. Neither infectious MuLV nor MuLV proteins were detected in gt/gt brain homogenates or in affected recipient mouse brains. Scrapie-associated fibrils, readily identifiable in subcellular fractions of brains from scrapie-inoculated NFS/N mice, were not detected in similar brain fractions from NFS/N mice inoculated with gt brain homogenates. These results confirm and extend the suggestion that gt spongiform encephalopathy has both heritable and transmissible properties. Moreover, the transmissible agent of gt disease differs from both Cas-Br-M MuLV and scrapie in its disease-inducing properties in NFS/N mice. The capacity of NFS/N mice to express transmitted gt encephalopathy as clinical disease, to rapidly express Cas-Br-M MuLV spongiform encephalomyelopathy, and to develop mouse-adapted scrapie after a very short incubation time suggest a distinct sensitivity of NFS/N mice to transmissible spongiform encephalopathy.

Spinal cord injury in an inner city hospital.

During the first six years of existence of the rehabilitation unit in a black inner city major municipal hospital, 53 patients with spinal cord injury were admitted. A retrospective study of these patients sought answers to questions concerning etiology, patient characteristics, services provided and method of delivery, and advantages and disadvantages of rehabilitation in a community hospital. Findings revealed differences in causation of spinal cord injury between women and men and between patients with paraplegia and quadriplegia. tthe male paraplegic patients were the youngest; their life style led to spinal cord injury. Social factors such as inadequate housing, lack of transportation, and insufficient financial resources were deterrents to rehabilitation. An approach that emphasized consideration of all the psychosocial factors was developed. Evaluation and treatment were extended into the community. Lack of peer groups and multiphasic programs were the major disadvantages.

Flouride. Ten-year prospective study of deciduous and permanent dentition.

A ten-year longitudinal prospective study compared the effect of fluoride on dentition of 1,500 children from infancy through age 10 years. In Kalamazoo, Mich, and Oneida, NY, parallel groups were given a fluoride-vitamin supplement from infancy and from age 4 and compared with fluoridated water and control groups. Incidence of new caries activity in both deciduous and permanent teeth was measured by mean number of new decayed and filled teeth, as well as percentage of children with no caries throughout the periods studied. Prevalence of caries was also studied in six-year molars. The results indicate a consistent (and, for selected groups, a statistically significant) diminution in caries activity for both deciduous and permanent teeth for groups ranked from greatest retardation of caries to least: infant fluoride group, water fluoride group, age 4 fluoride group, and controls.