Intrathecal drug delivery in the era of nanomedicine.

Administration of substances directly into the cerebrospinal fluid (CSF) that surrounds the brain and spinal cord is one approach that can circumvent the blood-brain barrier to enable drug delivery to the central nervous system (CNS). However, molecules that have been administered by intrathecal injection, which includes intraventricular, intracisternal, or lumbar locations, encounter new barriers within the subarachnoid space. These barriers include relatively high rates of turnover as CSF clears and potentially inadequate delivery to tissue or cellular targets. Nanomedicine could offer a solution. In contrast to the fate of freely administered drugs, nanomedicine systems can navigate the subarachnoid space to sustain delivery of therapeutic molecules, genes, and imaging agents within the CNS. Some evidence suggests that certain nanomedicine agents can reach the parenchyma following intrathecal administration. Here, we will address the preclinical and clinical use of intrathecal nanomedicine, including nanoparticles, microparticles, dendrimers, micelles, liposomes, polyplexes, and other colloidalal materials that function to alter the distribution of molecules in tissue. Our review forms a foundational understanding of drug delivery to the CSF that can be built upon to better engineer nanomedicine for intrathecal treatment of disease.

Cathinone increases body temperature, enhances locomotor activity, and induces striatal c-fos expression in the Siberian hamster.

Cathinone is a ß-keto alkaloid that is the major active constituent of khat, the leaf of the Catha edulis plant that is chewed recreationally in East Africa and the Middle East. Related compounds, such as methcathinone and mephedrone have been increasing in popularity as recreational drugs, resulting in the recent proposal to classify khat as a Class C drug in the UK. There is still limited knowledge of the pharmacological effects of cathinone. This study examined the acute effects of cathinone on core body temperature, locomotor and other behaviors, and neuronal activity in Siberian hamsters. Adult male hamsters, previously implanted with radio telemetry devices, were treated with cathinone (2 or 5mg/kg i.p.), the behavioral profile scored and core body temperature and locomotor activity recorded by radio telemetry. At the end of the study, hamsters received vehicle or cathinone (5mg/kg) and neuronal activation in the brain was determined using immunohistochemical evaluation of c-fos expression. Cathinone dose-dependently induced significant (p<0.0001) increases in both temperature and locomotor activity lasting 60-90min. Cathinone (2mg/kg) increased rearing (p<0.02), and 5mg/kg increased both rearing (p<0.001) and lateral head twitches (p<0.02). Both cathinone doses decreased the time spent at rest (p<0.001). The number of c-fos immunopositive cells were significantly increased in the striatum (p<0.0001) and suprachiasmatic nucleus (p<0.05) following cathinone, indicating increased neuronal activity. There was no effect of cathinone on food intake or body weight. It is concluded that systemic administration of cathinone induces significant behavioral changes and CNS activation in the hamster.

Increased responses to the actions of fibroblast growth factor 21 on energy balance and body weight in a seasonal model of adiposity.

The present study aimed to investigate the actions of fibroblast growth factor 21 (FGF21) on energy balance in a natural model of relative fatness, the Siberian hamster. Hamsters were studied under long days (LD) to promote weight gain, or short days to induce weight loss, and treated with rhFGF21 (3 mg/kg/day) via s.c. minipumps for 14 days. On days 7-9, detailed assessments of ingestive behaviour, metabolic gas exchange and locomotor activity were made. FGF21 caused substantial (P < 0.0001) weight loss in the fat LD state but not in the lean SD state: at the end of the study, FGF21-treated hamsters in LD lost 18% of body weight compared to vehicle controls, which is comparable to the natural body weight loss observed in SD. Epididymal fat pads, a correlate of total carcass fat content, were reduced by 19% in FGF21 treated hamsters in LD, whereas no difference was found in SD. Body weight loss in LD was associated with a reduction in food intake (P < 0.001) and a decreased respiratory exchange ratio (P < 0.001), indicating increased fat oxidation. Treatment with FGF21 maintained the normal nocturnal increase in oxygen consumption and carbon dioxide production into the early light phase in hamsters in LD, indicating increased energy expenditure, although locomotor activity was unaffected. These data suggest a greater efficacy of FGF21 in hamsters in LD compared to those in SD, which is consistent with both the peripheral and possibly central actions of FGF21 with respect to promoting a lean phenotype. The observed differences in FGF21 sensitivity may relate to day length-induced changes in adipose tissue mass.

Short-days induce weight loss in Siberian hamsters despite overexpression of the agouti-related peptide gene.

Many vertebrates express profound annual cycles of body fattening, although it is not clear whether these represent differential activity of the central pathways known to mediate homeostatic control of food intake and energy expenditure, or whether the recent discovery of a major role for pars tuberalis-ependymal signalling points towards novel mechanisms. We examined this in the Siberian hamster (Phodopus sungorus) by using gene transfection to up-regulate a major orexigenic peptide, agouti-related peptide (AgRP), and then determined whether this increased anabolic drive could prevent the short-day induced winter catabolic state. Infusions of a recombinant adeno-associated virus encoding an AgRP construct into the hypothalamus of hamsters in the long-day obese phase of their seasonal cycle produced a 20% gain in body weight over 6 weeks compared to hamsters receiving a control reporter construct, reflecting a significant increase in food intake and a significant decrease in energy expenditure. However, all hamsters showed a significant, prolonged decrease in body weight when exposed to short photoperiods, despite the hamsters expressing the AgRP construct maintaining a higher food intake and lower energy expenditure relative to the control hamsters. Visualisation of the green fluorescent protein reporter and analysis of AgRP-immunoreactivity confirmed widespread expression of the construct in the hypothalamus, which was maintained for the 21-week duration of the study. In conclusion, the over-expression of AgRP in the hypothalamus produced a profoundly obese state but did not block the seasonal catabolic response, suggesting a separation of rheostatic mechanisms in seasonality from those maintaining homeostasis of energy metabolism.

ECG interference suppressed using a harmonic generator.

Non-linear loading of the utilities supply introduced significant electromagnetic field (EMF) interference severe enough to disrupt electrocardiograph (ECG) monitoring and recordings in the new Emergency Department (ED) at the Royal Melbourne Hospital (RMH). This interference was evident even though standard Mu-metal shielding had been installed over the main hospital power feed which runs underneath the department. Investigations revealed that the source of the interference was due to 3rd harmonic currents flowing in the mains cable. This interference was suppressed by introducing third harmonic current into the main power cable in anti-phase to the interfering signal.

Rationale for and study design of the sulodexide trials in Type 2 diabetic, hypertensive patients with microalbuminuria or overt nephropathy.

BACKGROUND: Patients with Type 2 diabetes and albuminuria are at high risk to progress to end-stage renal disease (ESRD). Although angiotensin receptor blockers confer renoprotection, many diabetic patients still develop overt nephropathy and reach ESRD. Glycosaminoglycans belong to the same family as heparin and heparinoids. Pilot studies with sulodexide, a glycosaminoglycan, have shown that sulodexide can reduce urinary albumin excretion rates in diabetic patients. No hard renal end-point data are available. METHODS: Two multicentre, double-masked, randomized placebo controlled trials were designed to study the renoprotective potential of sulodexide. The Sulodexide Microalbuminuria Trial examined the efficacy of sulodexide given over 26 weeks in 1000 patients with Type 2 diabetes, hypertension and microalbuminuria. The Sulodexide Overt Nephropathy Trial examined the efficacy of sulodexide in 2240 patients with Type 2 diabetes, hypertension and proteinuria > or = 900 mg/24 h. RESULTS: The primary outcome of The Sulodexide Microalbuminuria Trial was (i) conversion to normoalbuminuria and at least a 25% decrease in the urinary albumin creatinine ratio (UACR), or (ii) at least a 50% reduction in UACR. The primary outcome of The Sulodexide Overt Nephropathy Trial was time to a composite end point of doubling of serum creatinine or ESRD. CONCLUSIONS: The sulodexide nephropathy programme will document whether therapy with sulodexide confers renal protection in Type 2 diabetes and nephropathy.

Effects of neuropathic and non-neuropathic isomers of tricresyl phosphate and their microsomal activation on the production of axon-like processes by differentiating mouse N2a neuroblastoma cells.

The aim of this work was to investigate the sublethal neuropathic effects of tricresyl phosphate (TCP: mixed isomers), triorthocresyl phosphate (TO:CP) and triparacresyl phosphate (TP:CP) on differentiating mouse N2a neuroblastoma cells. This was achieved by a combination of measurements of cell viability, axon outgrowth and the levels of cytoskeletal proteins detectable on western blots of extracts from cells induced to differentiate in the presence and absence of the compounds. In a time-course experiment TCP inhibited the outgrowth of axon-like processes following exposure times of 24 h or longer. Dose-response experiments indicated that TCP and TO:CP exhibited similar sustained levels of toxicity following both 24 and 48 h exposure, with no significant difference between their respective IC(50) values. By contrast, TP:CP demonstrated a transient effect on the outgrowth of axon-like processes, which was detectable after 24 but not 48 h of exposure. Isomer-specific patterns of toxicity were also evident at earlier time-points, with only the ortho isomer showing significant levels of inhibition of axon outgrowth following 4-8 h exposure. Probing of western blots with antibodies against cytoskeletal proteins indicated that the inhibition of axon outgrowth by these compounds was associated with a sustained reduction in the levels of phosphorylated neurofilament heavy chain. The inhibitory effect on axon outgrowth of TO:CP but not TP:CP was enhanced in the presence of a microsomal activation system. Since TO:CP is the most neuropathic of the isomers of TCP in vivo, differentiating N2a cells provide a useful cellular system for mechanistic studies of the neurodegenerative effects of this organophosphate.

The effects of carbaryl and trichlorphon on differentiating mouse N2a neuroblastoma cells.

The ability of the carbamate pesticide carbaryl (CB) and the organophosphate pesticide trichlorphon (TCL) to inhibit the outgrowth of axon-like processes was studied using mouse N2a neuroblastoma cells induced to differentiate by serum withdrawal. At concentrations of 1 and 2 microg/ml (4.97 and 9.94 microM), CB did not cause cell death but inhibited the outgrowth of axon-like processes from N2a cells. This effect was noted as early as 24 h after exposure of the cells to CB. A similar effect was observed with TCL at concentrations of 1 and 2 microg/ml (3.89 and 7.78 microM). Western blot analysis of cell extracts treated with the pesticides showed decreased cross reactivities with the monoclonal antibody RMd09 compared to control extracts. The results indicate that CB and TCL are both able to inhibit axon development and that this effect is associated with reduced levels of the neurofilament high molecular weight protein subunit (NFH).

Induction of bone morphogenetic protein-2 by interleukin-1 in human fibroblasts.

Rheumatoid arthritis and periodontitis are chronic inflammatory diseases associated with tissue destruction that is mediated in part by elevated levels of cytokines (e.g., interleukin-1 and tumor necrosis factor). Differential screening of a human synovial fibroblast cDNA library for interleukin-1 induced genes revealed a clone identical to the gene encoding human bone morphogenetic protein-2. Northern blot analysis of human synovial fibroblast mRNA confirmed up-regulation of bone morphogenetic protein-2 in the presence of interleukin-1. Utilizing a specific antibody, levels of bone morphogenetic protein-2 protein in conditioned medium from synovial fibroblasts were also up-regulated in the presence of interleukin-1. This is the first report of the production of bone morphogenetic protein-2 by synovial fibroblasts, and the first report of its up-regulation in response to interleukin-1. However, interleukin-1 did not induce bone morphogenetic protein-2 mRNA in human gingival fibroblasts.

Tricresyl phosphate inhibits the formation of axon-like processes and disrupts neurofilaments in cultured mouse N2a and rat PC12 cells.

Tricresyl phosphate (1 microg/ml) inhibited the outgrowth of axon-like processes in mouse N2a neuroblastoma and rat PC12 pheochromocytoma cell lines induced to differentiate by serum withdrawal and nerve growth factor addition, respectively. By contrast, it had no effect on the outgrowth of processes by rat C6 glioma cells induced to differentiate with sodium butyrate. The effect on axon outgrowth in the two neuronal cell lines correlated with altered distribution of neurofilament proteins, as determined by indirect immunofluorescence with monoclonal antibody RMd09. Western blots of neuronal cell extracts probed with the same antibody revealed decreased cross-reactivity after exposure to tricresyl phosphate. The results suggest that tricresyl phosphate has a selective effect on neuronal cell differentiation, which involves impaired axon outgrowth, reduced levels of the neurofilament heavy chain and disruption of the neurofilament network.

Hepatocellular carcinoma: clinical, aetiological and pathological features in British patients.

We have examined the clinical, prognostic, aetiological and pathological features of 41 consecutive British patients with hepatocellular carcinoma (HCC). Presenting symptoms were often vague, and patients were generally in poor condition at diagnosis, 56% being of WHO grade 2 performance score or higher. Ascites (46%) and jaundice (35%) were often present at diagnosis, and the median duration of survival from diagnosis was only 6 weeks. Only initial performance grade (p less than 0.0001) and serum bilirubin concentration (p = 0.02) significantly affected prognosis. Serum alphafoetoprotein (AFP) was positive by counter-immune electrophoresis in only 34%, but increased to 68% when radioimmunoassay was used. Cirrhosis was present in 33 patients (80%) and was most often cryptogenic (12) or alcoholic (7) in origin. Serum HBsAg was detected in 5 patients (12%) and, of the remainder tested, 21% had serological evidence of past HBV exposure. None of 8 serum HBsAg-negative tumour specimens had detectable HBV-DNA integration into the tumour cell genome. Liver-cell dysplasia was noted in 56% of patients with liver biopsies predating the diagnosis of HCC, and in 78% of biopsies taken at the time of HCC diagnosis. The commonest histological pattern was trabecular (71%). Other forms were rare; the fibrolamellar pattern was only seen in 2 patients.

Hepatocellular carcinoma and the hepatitis B virus: a study of British patients.

We have examined the relationship between hepatitis B virus infection and hepatocellular carcinoma in a series of 50 British patients with histologically-proven hepatocellular carcinoma. Serum HBsAg was detected in 10 patients (20 per cent), and of the remainder, 21 per cent of those tested had serological evidence of past hepatitis B virus exposure. None of nine tumour specimens from serum HBsAg negative patients had detectable HBV-DNA integration into the tumour cell genome. The presence or absence of serum HBsAg was of no prognostic significance in British patients with hepatocellular carcinoma. Liver cell dysplasia, a possible pre-malignant lesion, was noted in 53 per cent of patients who had had liver biopsies before the diagnosis of hepatocellular carcinoma, and in 79 per cent of biopsies taken at the time of diagnosis of hepatocellular carcinoma. The presence of liver cell dysplasia was not more common in serum HBsAg positive patients.

Hepatitis B virus and primary liver cell carcinoma. The application of molecular biology.

Worldwide, primary liver cell carcinoma (PLC) is one of the most common tumours. Epidemiological evidence has implicated hepatitis B virus (HBV) in its aetiology and the mechanisms whereby HBV could operate at the genomic level have been investigated using the techniques of molecular biology. The resemblance of certain features of HBV to the retroviruses has also suggested mechanisms whereby malignant transformation may take place, but as yet there is no clear evidence for HBV being directly oncogenic. This has suggested to some that it is the persistent inflammatory reaction caused by HBV infection that is instrumental in causing PLC. We believe, however, that HBV can act independently of this mechanism and that the failure so far to show this at the molecular level may be due to technical reasons.

Cloning and analysis of integrated hepatitis B virus DNA of the adr subtype derived from a human primary liver cell carcinoma.

A 10 kb genomic DNA fragment derived from a human primary liver cell carcinoma (PLC) and containing integrated hepatitis B virus (HBV) DNA was cloned and analysed. Physical mapping showed the viral DNA to comprise a linear sequence of at least 2.8 kb (87%) of the HBV genome and to be of the adr subtype. Integration appeared to have occurred in the region of the viral genome spanning the cohesive ends. The cellular flanking DNA sequences to one side of the integrated viral DNA contained repeats of the Alu family. The finding of no apparent rearrangements of the integrated HBV DNA sequences in this clone is in contrast to the situation in the huSP and PLC/PRF/5 PLC cell lines in which the integrated viral DNA sequences are greatly rearranged and suggests that such rearrangements may be atypical of solid PLCs.

Integration of HBV-DNA may not be a prerequisite for the maintenance of the state of malignant transformation. An analysis of 110 liver biopsies.

Hundred and ten liver biopsy specimens from various parts of the world were examined for episomal and integrated HBV-DNA sequences. In 54 patients with HBsAg chronic liver disease episomal HBV-DNA was found in 83% of HBeAg-positive patients, compared to only 22% of patients with anti-HBe. Furthermore episomal HBV-DNA in the latter predominated among the Asians. Integrated HBV-DNA was found only in 5.5% of HBeAg-positive patients but in 16.5% of patients with anti-HBe. In 28 HBsAg-positive patients with hepatoma, episomal HBV-DNA was found in 50% of HBeAg-positive patients but in only 11% of anti-HBe patients. Conversely integrated sequences were less common (25%) in HBe-Ag-positive patients than in anti-HBe patients (50%), giving an overall incidence of integration in this group of 45%. No episomal, and only one case of integrated sequences of HBV-DNA, could be detected among 10 patients with HBsAg-negative hepatoma. In addition neither episomal nor integrated HBV-DNA could be detected in 18 patients with non-HBV-related liver disease. Our data suggests that stable integration of HBV-DNA into the host's genome is not necessarily a prerequisite for the maintenance of the state of malignant transformation but may be necessary for its initiation. Alternatively, the detection of integrated HBV-DNA may represent a 'snap shot' of a random integration event amplified by clonal expansion promoted by other factors.

Hepatitis B virus DNA in saliva, urine, and seminal fluid of carriers of hepatitis B e antigen.

Concentrated samples of saliva, urine, and seminal fluid from 23 men with chronic liver disease who were positive for hepatitis B e antigen were examined for the presence of hepatitis B virus deoxyribonucleic acid (HBV-DNA) by molecular hybridisation. HBV-DNA was detected in saliva from 15 of 17 men (88%), urine from 12 of 22 men (55%), and seminal fluid from 13 of 21 men (62%). The presence of hepatitis B virus in such secretions has important epidemiological implications for heterosexual and homosexual contact.

Natural history of chronic hepatitis B virus infection in Taiwan: studies of hepatitis B virus DNA in serum.

Hepatitis B virus DNA (HBV DNA) in serum was measured by a Spot hybridization technique in a consecutive series of 79 cases with chronic HBV infection from Taiwan. HBV DNA was found in 96.3% (52/54) of HBeAg-positive, 66% (2/3) with neither HBeAg or anti-HBe and in 63.6% (14/22) of anti-HBe positive patients. The levels of HBV DNA in the HBe-Ag-positive patients were significantly higher than in the anti-HBe positive patients (median, 944 vs. 58 pg per ml, p less than 0.001). The mean ages increased from 28.7 years for the cases with high levels of HBV DNA, to 34.7 years for those with low levels (p less than 0.01) and to 41.0 years in those without HBV DNA in serum (p less than 0.05 when compared with those with low level of HBV DNA). Ninety per cent of patients (27/30) with high levels of HBV DNA showed only minor hepatic inflammatory activity, as did 91% (10/11) of those without HBV DNA. In contrast, histologic signs of chronic active hepatitis or chronic lobular hepatitis were demonstrated in 76% of cases (29/38) with low levels of HBV DNA. These data are consistent with the hypothesis that liver damage occurs during the period of clearance of hepatocytes supporting HBV replication, and are inconsistent with the view that HBV may be directly cytopathic. Thus, the natural history of chronic HBV infection may be divided into three phases.(ABSTRACT TRUNCATED AT 250 WORDS)