Development and Validation of a Brain Phantom for Therapeutic Cooling Devices.

Tissue cooling has been proven as a viable therapy for multiple conditions and injuries and has been applied to the brain to treat epilepsy and concussions, leading to improved long-term outcomes. To facilitate the study of temperature reduction as a function of various cooling methods, a thermal brain phantom was developed and analyzed. The phantom is composed of a potassium-neutralized, superabsorbent copolymer hydrogel. The phantom was tested in a series of cooling trials using a cooling block and 37 deg water representing nondirectional blood flow ranging up to 6 gph, a physiologically representative range based on the prototype volume. Results were compared against a validated finite difference (FD) model. Two sets of parameters were used in the FD model: one set to represent the phantom itself and a second set to represent brain parenchyma. The model was then used to calculate steady-state cooling at a depth of 5 mm for all flow rates, for both the phantom and a model of the brain. This effort was undertaken to (1) validate the FD model against the phantom results and (2) evaluate how similar the thermal response of the phantom is to that of a perfused brain. The FD phantom model showed good agreement with the empirical phantom results. Furthermore, the empirical phantom agreed with the predicted brain response within 3.5% at physiological flow, suggesting a biofidelic thermal response. The phantom will be used as a platform for future studies of thermally mediated therapies applied to the cerebral cortex.

An unexpected role for a Wnt-inhibitor: Dickkopf-1 triggers a novel cancer survival mechanism through modulation of aldehyde-dehydrogenase-1 activity.

It is widely accepted that canonical Wnt (cWnt) signaling is required for the differentiation of osteoprogenitors into osteoblasts. Furthermore, tumor-derived secretion of the cWnt-antagonist Dickkopf-1 (Dkk-1) is known to cause bone destruction, inhibition of repair and metastasis in many bone malignancies, but its role in osteosarcoma (OS) is still under debate. In this study, we examined the role of Dkk-1in OS by engineering its overexpression in the osteochondral sarcoma line MOS-J. Consistent with the known role of Dkk-1 in osteoblast differentiation, Dkk-1 inhibited osteogenesis by the MOSJ cells themselves and also in surrounding tissue when implanted in vivo. Surprisingly, Dkk-1 also had unexpected effects on MOSJ cells in that it increased proliferation and resistance to metabolic stress in vitro and caused the formation of larger and more destructive tumors than controls upon orthotopic implantation. These effects were attributed in part to upregulation of the stress response enzyme and cancer stem cell marker aldehyde-dehydrogenase-1 (ALDH1). Direct inhibition of ALDH1 reduced viability under stressful culture conditions, whereas pharmacological inhibition of cWnt or overexpression of ALDH1 had a protective effect. Furthermore, we observed that ALDH1 was transcriptionally activated in a c-Jun-dependent manner through a pathway consisting of RhoA, MAP-kinase-kinase-4 and Jun N-terminal Kinase (JNK), indicating that noncanonical planar cell polarity-like Wnt signaling was the mechanism responsible. Together, our results therefore demonstrate that Dkk-1 enhances resistance of OS cells to stress by tipping the balance of Wnt signaling in favor of the non-canonical Jun-mediated Wnt pathways. In turn, this results in transcriptional activation of ALDH1 through Jun-responsive promoter elements. This is the first report linking Dkk-1 to tumor stress resistance, further supporting the targeting of Dkk-1 not only to prevent and treat osteolytic bone lesions but also to reduce numbers of stress-resistant tumor cells.

Compact inline optical electron polarimeter.

A compact optical electron polarimeter using a helium target is described. It offers a maximum fluorescence detection efficiency of ~20 Hz/nA, which is an order of magnitude higher than that of earlier designs. With an argon target, this device is expected to have a polarimetric figure-of-merit of 270 Hz/nA. By relying on a magnetic field to guide a longitudinally spin-polarized electron beam, the present instrument employs fewer electrodes. It also uses a commercially available integrated photon counting module. These features allow it to occupy a smaller volume and make it easier to operate.

Efficacy of caspofungin as salvage therapy for invasive aspergillosis compared to standard therapy in a historical cohort.

In a non-comparative study, caspofungin was effective salvage therapy for approximately half of the patients refractory to or intolerant of standard antifungal agents for invasive aspergillosis. To establish a frame of reference for these results, we compared the response to caspofungin with responses to other antifungal agents in a historical cohort of similar patients. The efficacy could be evaluated in 83 patients who received caspofungin 50 mg daily after a 70-mg loading dose. The historical control group, identified through a retrospective review of medical records, included 214 evaluable patients possibly refractory to or intolerant of ≥1 week of standard antifungal therapy. All patients had documented invasive aspergillosis. Favorable response was defined as a complete or partial response to therapy. Underlying diseases, baseline neutropenia, corticosteroid use, and sites of infection were similar in both studies. Most patients had received amphotericin B formulations and/or itraconazole, and were refractory to standard therapy. Favorable response rates were 45% with caspofungin and 16% with standard therapy. The unadjusted odds ratio for a favorable response (caspofungin/standard therapy) was 4.1 (95% confidence interval: 2.2, 7.5). After adjusting for potential imbalances in the frequency of disseminated infection, neutropenia, steroid use, and bone marrow transplantation between groups, the odds ratio remained at 4.1 (2.1, 7.9). Although only tentative conclusions about relative efficacy can be drawn from retrospective comparisons, caspofungin appeared to be at least as efficacious as an amphotericin B formulation and/or itraconazole for the treatment of invasive aspergillosis in patients refractory to or intolerant of their initial antifungal therapy.

ASP1 (BACE2) cleaves the amyloid precursor protein at the beta-secretase site.

Sequential proteolytic processing of the Amyloid Precursor Protein (APP) by beta- and gamma-secretases generates the 4-kDa amyloid (A beta) peptide, a key component of the amyloid plaques seen in Alzheimer's disease (AD). We and others have recently reported the identification and characterisation of an aspartic proteinase, Asp2 (BACE), as beta-secretase. Here we describe the characterization of a second highly related aspartic proteinase, Asp1 as a second beta-secretase candidate. Asp1 is expressed in brain as detected at the mRNA level and at the protein level. Transient expression of Asp1 in APP-expressing cells results in an increase in the level of beta-secretase-derived soluble APP and the corresponding carboxy-terminal fragment. Paradoxically there is a decrease in the level of soluble A beta secreted from the cells. Asp1 colocalizes with APP in the Golgi/endoplasmic reticulum compartments of cultured cells. Asp1, when expressed as an Fc fusion protein (Asp1-Fc), has the N-terminal sequence ALEP..., indicating that it has lost the prodomain. Asp1-Fc exhibits beta-secretase activity by cleaving both wild-type and Swedish variant (KM/NL) APP peptides at the beta-secretase site.

Identification of a novel aspartic protease (Asp 2) as beta-secretase.

The Alzheimer's disease beta-amyloid peptide (Abeta) is produced by excision from the type 1 integral membrane glycoprotein amyloid precursor protein (APP) by the sequential actions of beta- and then gamma-secretases. Here we report that Asp 2, a novel transmembrane aspartic protease, has the key activities expected of beta-secretase. Transient expression of Asp 2 in cells expressing APP causes an increase in the secretion of the N-terminal fragment of APP and an increase in the cell-associated C-terminal beta-secretase APP fragment. Mutation of either of the putative catalytic aspartyl residues in Asp 2 abrogates the production of the fragments characteristic of cleavage at the beta-secretase site. The enzyme is present in normal and Alzheimer's disease (AD) brain and is also found in cell lines known to produce Abeta. Asp 2 localizes to the Golgi/endoplasmic reticulum in transfected cells and shows clear colocalization with APP in cells stably expressing the 751-amino-acid isoform of APP.

Restoration of cardiac contraction by angiotensin II during raised [K+]O in the rabbit.

Catecholamines restore cardiac contraction depressed by hyperkalaemia (raised [K+]O) and acidosis, yet in exercise hyperkalaemia and acidosis are tolerated during beta adrenergic blockade. To test whether the negative effects of raised [K+]O are offset by a non-adrenergic hormone, angiotensin II (AII) was given to rabbit papillary muscle (All 75 nM, n = 9) and rabbit isolated working hearts (All 5 nM, n = 8) perfused with 8 and 10 mM K+ Tyrode at 37 degrees C. A similar protocol was also performed in a further nine isolated hearts treated with propranolol (1 microM) and prazosin (1 microM). All caused a significant (P < 0.01) increases in contraction and aortic flow in normal Tyrode and maintained aortic flow during high [K+]O. In the papillary muscle and isolated heart treated with adrenergic blockers, high [K+]O reduced the stimulatory effects of All, but contraction and aortic flow was still significantly greater (P < 0.01) than in high [K+]O alone. These results show that All can ameliorate the depressive effects of high [K+]O on the heart. The local release of All in the heart during activation of the sympathetic nervous system and the rise in circulating All during exercise could therefore play a role in protecting the heart from hyperkalaemia.

GG167 (4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid) is a potent inhibitor of influenza virus in ferrets.

GG167 (4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid) is a novel viral neuraminidase (sialidase) inhibitor which, following intranasal administration in ferrets, is at least 100 to 1,000 times more effective than ribavirin and amantadine against influenza A and B viruses. It retains its activity even when treatments are delayed until 24 h postinfection and has no effect on the serum antibody response to infection.

Effect of verapamil on restoration of cardiac performance in raised [K+]o by adrenergic stimulation in the rabbit.

Modulation of the L-type calcium channel by catecholamines improves action potential parameters in single ventricular myocytes depolarized by high [K+]o Tyrode. Whether this modulation is important in offsetting the negative effects of hyperkalaemia in the whole heart is not known. We tested the effects of the calcium channel antagonist, verapamil, on restoration of cardiac performance by adrenergic stimulation in high [K+]o in anaesthetized rabbits and isolated perfused working rabbit hearts. Raised [K+]o decreased SBP, LVP and LVdP/dtmax in vivo ([K+]a 8.6 +/- 0.2 mM; n = 10) and aortic flow (AF) in the isolated heart (8 mM [K+]o Tyrode; n = 25). However, the negative effects of raised [K+]a were offset by isoprenaline (Iso, 1 microgram kg-1 min-1 i.v.) in vivo and by noradrenaline (NA, 80 nM) in the isolated heart. Verapamil (0.15 mg kg-1 i.v.; 15 nM isolated heart) markedly potentiated the negative inotropic effects of raised [K+]o in both preparations. Verapamil attenuated the effect of isoprenaline in vivo but in the isolated heart, the protective effect of NA in 8 mM [K+] Tyrode (AF 97 +/- 10 mL min-1 in 8 mM [K+]o compared with AF 141 +/- 8.5 mL min-1 in 8 mM [K+]o + NA) was offset by the drug (90 +/- 8 mL min-1 in 8 mM [K+]o + NA + V). Furthermore, verapamil abolished aortic flow in 8 mM [K+]o alone. These findings suggest that the heart may be critically dependent on modulation of intracellular calcium in order to tolerate concentrations of K+ similar to those seen during a short burst of intensive exercise ([K+]a 8.6 mM).

Inhibition of influenza virus replication in mice by GG167 (4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid) is consistent with extracellular activity of viral neuraminidase (sialidase).

We demonstrate the potent antiviral activity of a novel viral neuraminidase (sialidase) inhibitor, 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (GG167), administered by the intranasal route in comparison with those of amantadine and ribavirin in experimental respiratory tract infections induced with influenza A and B viruses. In an extended study in which mice were infected (day 0) with influenza A/Singapore/1/57 virus, with treatments given prophylactically plus twice daily over days 0 to 3 and with mice observed to day 10, we show that intranasally administered GG167 at 0.4 and 0.01 mg/kg of body weight per dose reduced mortality, lung consolidation, and virus titers in the lung, with no virus growing back following the cessation of treatment. In other studies with influenza B/Victoria/102/85 virus in which infected mice were culled after the cessation of treatment, the calculated intranasal dose required to reduce virus titers in the lungs of treated animals to 10% of that seen in untreated controls (EDAUC10 [where AUC is area under the virus titer days curve]) was 0.085 mg/kg per dose. GG167 was inactive against influenza viruses A and B when given by the intraperitoneal or oral route (EDAUC10, > 100 mg/kg per dose). GG167 was metabolically stable, with an elimination half-life of 10 min following intravenous administration. While readily bioavailable by systemic routes, it was poorly bioavailable by the oral route. Its potent efficacy by the intranasal route but lack of efficacy by other routes, relative to those of amantadine and ribavirin, was explicable in terms of its in vitro activity, bioavailability, and pharmacokinetic properties and with the extracellular activity of viral sialidase.

Baclofen-induced dyskinesia.

Baclofen (Lioresal) is a derivative of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). It is used to treat spasticity particularly for the relief of flexor spasms, pain, clonus, and muscular rigidity. There have been many rare neurologic side effects reported with its use. These side effects, in particular, hallucinations and seizures, have been observed predominantly following precipitous withdrawal of the drug. We present a case demonstrating a muscular dyskinetic side effect when baclofen treatment was first initiated. The mechanism by which baclofen affects spasticity and how the resulting side effect of dyskinesia developed in our patient is not known. They are, however, most probably related to dopamine receptor hypersensitivity and the resulting imbalance of the dopaminergic/cholinergic systems. Clinicians should be aware of this additional adverse effect of muscular dyskinesia, with the use of baclofen, and its reversibility when baclofen is discontinued.

4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid is a highly effective inhibitor both of the sialidase (neuraminidase) and of growth of a wide range of influenza A and B viruses in vitro.

The sialidase (neuraminidase) inhibitor 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (4-guanidino-Neu5Ac2en) has been examined for the ability to inhibit the growth of a wide range of influenza A and B viruses in vitro in comparison with amantadine, rimantadine, and ribavirin. 4-Guanidino-Neu5Ac2en inhibited plaque formation by laboratory-passaged strains of influenza A and B viruses, with 50% inhibitory concentrations ranging from 0.005 to 0.014 microM. A wider range of values (0.02 to 16 microM) was obtained with more recent clinical isolates, but in all cases 4-guanidino-Neu5Ac2en inhibited influenza A and B virus replication at lower concentrations than amantadine, rimantadine, or ribavirin. Inhibition by 4-guanidino-Neu5Ac2en was not obviously affected by the passage history of the viruses or by resistance to amantadine or rimantadine. 4-Guanidino-Neu5Ac2en was a very potent inhibitor of the sialidases of all the influenza viruses examined, with 50% inhibitory concentrations ranging from 0.00064 to 0.0079 microM. No cytotoxicity was observed with 4-guanidino-Neu5Ac2en at up to 10 mM. 4-Guanidino-Neu5Ac2en therefore represents a new potent and selective inhibitor of influenza A and B virus sialidase activity and replication in vitro.

Pharmacokinetics of antibiotics in natural and experimental superficial compartments in animals and humans.

Transcapillary exchange of antibiotics and other small molecules is diffusion driven and occurs in the capillary beds of the tissues. Small polar molecules are ionized at physiological pH and diffusion is pore-mediated. More lipophilic substances can also leave capillaries by the transcellular route. Splanchnic tissues have fenestrated capillary walls while somatic tissues have mainly micropores in their capillary walls. Under normal physiological conditions the ratio of capillary surface area to volume of fluid present (SA/V) is very large (> 100) and the rate of exchange of substances between capillaries, interstitial fluid and tissue fluid is extremely rapid. The structure of the interstitial space or ground substance, linking the capillary to the tissue cells, is designed to regulate the exchange of water, albumen and other solutes between the plasma and tissues. Interstitial space fluid (tissue fluid) is not simply an ultrafiltrate of plasma and has a specific chemical imbalance with plasma. Some antibiotics bind to serum albumen and it is claimed that this impairs their ability to penetrate into tissue fluids. However, the reduced concentration of albumen in the tissue fluid, relative to the plasma, is the main reason why percentage tissue penetration data based on total levels present are misleading and perpetuate the misconception that highly bound antibiotics (> 80%) have a reduced penetration potential. The majority of infections are localized in extracellular fluid. Several models have been developed to sample serially the extracellular compartment. They have yielded diverse concentration/time profiles even for the same antibiotic at similar sites (skin and subcutaneous tissues). It has been shown that the various profiles are a direct consequence of compartment SA/V, which can range from > 100 to < 10. In the preclinical situation, the blister model has proven popular and reproducible but it should be remembered that the delayed profile seen is an artefact of blister geometry (SA/V < 10). On the evidence available it is likely that bacterial infections, in the presence of acute inflammation, will enhance the rate of entry of agents, while the reverse is true in areas of chronic inflammation where pathological barriers are already in place.

Epididymal sarcoidosis.

Sarcoidosis is a multisystem disorder that rarely involves the genitourinary tract. To date only 28 cases of histologically proved sarcoidosis involving the epididymis have been described in the literature. Although uncommon, sarcoidosis should be considered in any differential diagnosis of testicular lesions. We present 2 cases of epididymal sarcoidosis. A 27-year-old asymptomatic black man had multiple nontender scrotal nodules on routine physical examination. A 34-year-old black man was initially diagnosed with sarcoidosis by transbronchial biopsy. He received 10 months of prednisone therapy before noticing a mass in the right testicle. Surgical exploration of both patients demonstrated noncaseating granulomatous inflammation consistent with sarcoidosis.

Fluorocarbocyclic nucleosides: synthesis and antiviral activity of 2'- and 6'-fluorocarbocyclic 2'-deoxy guanosines.

A series of four isomeric 2'- and 6'-fluorocarbocyclic guanosine analogues have been prepared and evaluated as potential anti-herpes agents. The racemic 2' beta-fluoro isomer 2-amino-1,9-dihydro- 9-[(1 alpha, 2 alpha, 3 beta, 4 alpha)-2-fluoro-3-hydroxy-4- (hydroxymethyl)cyclopentyl]-6H-purin-6-one (11a, C-AFG) and its 2' alpha-fluoro epimer 11b plus the chiral 6' beta-fluoro isomer 2-amino-1,9-dihydro-9-[[1S-(1 alpha, 2 alpha, 3 alpha, 4 beta)]- 2-fluoro-4-hydroxy-3-(hydroxymethyl)cyclopentyl]-6H-purine-6-one (11c) and its 6' alpha-fluoro epimer 11d were prepared from their respective fluoro amino diol hydrochlorides (6a,d). For comparison, the furanosyl compound 9-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)guanine (17, AFG) was prepared by coupling 2-amino-6-chloropurine with 2-deoxy-2-fluoro-3,5-di-O-benzoyl-alpha- D-arabinofuranosyl bromide followed by base hydrolysis. The 6' alpha-fluoro derivative 11d exhibited comparable activity to that of acyclovir (ACV) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in vitro but was greater than 30-fold more active than ACV against HSV-1 and HSV-2 in vivo in the mouse systemic model. The 2' beta-fluoro derivative (11a, C-AFG) was extremely potent in vitro against HSV-1 and HSV-2 (ID50 0.006 and 0.05 micrograms/mL) and in vivo it was greater than 2 orders of magnitude more potent than ACV against HSV-1 and 70-fold more potent against HSV-2. The 2' alpha-fluoro 11b and 6' beta-fluoro 11c isomers were much less active.

Uptake, distribution and immunotoxicological effects of mercury in mice.

The uptake and distribution of mercury in various organs and tissues of mice were examined after administration with mercuric chloride (HgCl2) or mercuric sulphide (HgS). The results show that mice treated with HgCl2 were found to have significantly higher levels of mercury in various organs and tissues as compared to HgS-treated animals. Except for the kidneys, no significant differences were found in mercury levels between the HgS-treated and control mice. This appears to be due to the higher solubility of HgCl2, allowing for its greater absorption into the body. Irrespective of the mercurial administered, the kidneys contained the highest concentration of mercury, followed by the liver and brain. Mercury was also found to confer protection against Trypanosoma evansi, possibly due to its toxicity. When treated with HgS, enhanced antibody production and increased levels of circulating leucocytes was seen. HgCl2 and HgS-treated mice showed no signs of anorexia, no significant changes being found in growth and food intake.

Fatal accidents and blood ethanol levels in adolescents and adults. The Wayne County experience, 1978-1988.

The files of 874 fatal traumatic accident victims, aged 12-25 years, examined at the Wayne County Medical Examiner's Office during the period 1978-1988 were reviewed. Postmortem blood alcohol results of individuals who died after less than 15 min of hospitalization were utilized to approximate alcohol levels at the time of the fatal injury. Relationships between types of accidents, sex, age, race, and time of accident were examined. White victims were far more likely to have been drinking than blacks, and the data indicated that underaged drinkers were involved in fatal accidents at lower levels of blood alcohol than their counterparts of legal drinking age. Consistent racial differences in average alcohol levels were not observed, however. Unlike female and black victims, who much less frequently tested positive for alcohol when underage, white male victims 16-21 years of age were just as likely to have been drinking as those aged 21-25. The results of the study show that postmortem blood alcohol level can be used to identify differences in alcohol consumption among groups of accident victims in a major metropolitan area.

Ribonucleotide reductase encoded by herpes simplex virus is a determinant of the pathogenicity of the virus in mice and a valid antiviral target.

The role of the herpes simplex virus (HSV)-encoded ribonucleotide reductase (RR) in the pathogenicity of the virus has been examined by use of mutants with lesions in either the large or small subunit of the enzyme. The virulence of the mutants in mice was reduced by about 10(6)-fold when compared with that of the parental virus (HSV type 1 strain 17), while the virulence of a revertant of one of the mutants was restored to within about 100-fold of that of the parent virus. These experiments demonstrate that activity of the HSV RR is essential for virus pathogenicity in mice and suggests that the enzyme is a valid target for specific antiviral compounds.

A morphological study of the effect of treatment with the antibiotic ceftazidime on experimental staphylococcal endocarditis and aortitis.

The morphological effects of antibiotic therapy with either single or repeated (8 hourly) injections of ceftazidime were examined in rabbits with experimentally induced staphylococcal endocarditis and aortitis. At 3 h after initiating treatment, many of the bacteria, irrespective of the location of the colony, showed evidence of abnormal ultrastructural changes of the cytoplasm and/or cell wall. By 8 h many degenerate lysed bacteria were present. By 24 h, in rabbits which received a single injection, bacterial colonies contained many normal and dividing bacteria. In comparison, bacterial colonies at 24 h in rabbits receiving repeated injections consisted of large masses of lysed bacteria with only a few viable appearing thick-walled 'persistent' cells. At 48 and 72 h, no viable appearing bacteria were observed although they could be isolated by culture methods. Treatment was associated with an increased inflammatory cell response at the surface of the vegetation and within the vasculature. In the later stages there was evidence of healing with endothelialization of the lesions. It would appear, therefore, that ceftazidime penetrates efficiently into the vegetations with only a short transitory phase at sub-bactericidal concentrations. The few 'persistent' bacteria appear to be protected from the host defences by the surrounding thrombus which prevents their eradication.