Rotational Energy as Mass in H_{3}

We have made precise measurements of the cyclotron frequency ratios H_{3}^{+}/HD^{+} and H_{3}^{+}/^{3}He^{+} and observe that different H_{3}^{+} ions result in different cyclotron frequency ratios. We interpret these differences as due to the molecular rotational energy of H_{3}^{+} changing its inertial mass. We also confirm that certain high J, K rotational levels of H_{3}^{+} have mean lifetimes exceeding several weeks. From measurements with the lightest H_{3}^{+} ion we obtain lower limits on the atomic masses of deuterium and helium-3 with respect to the proton.

Prolonged regulatable expression of EPO from an HSV vector using the LAP2 promoter element.

We previously reported regulated expression of erythropoietin (EPO) over 4 weeks in the peripheral nerve in vivo, using a herpes simplex virus (HSV)-based vector containing a Tet-on regulatable gene expression cassette. To create a vector that would be appropriate for the treatment of chronic neuropathy, we constructed a HSV vector with expression of EPO under the control of the Tet-on system in which the HSV latency-associated promoter 2 element was used to drive the expression of the Tet-on transactivator. EPO expression from the vector was tightly controlled by administration of doxycycline (DOX) in vitro. One month after inoculation of the vector to transduce dorsal root ganglion (DRG) in vivo, administration of DOX-containing chow-induced expression of EPO. Mice with streptozotocin-induced diabetes, inoculated with the vector, were protected against the development of neuropathy by continuous administration of DOX-containing chow over the course of 3 months. Identical results were achieved when DOX was administered every other week over 3 months of diabetes, but administration of DOX, 1 week out of 3, provided only partial protection against the development of neuropathy. Taken together, these results suggest such a vector is well suited for clinical trial for the treatment of chronic or subacutely developing neuropathy.

Transgene-mediated expression of tumor necrosis factor soluble receptor attenuates morphine tolerance in rats.

Opiate/narcotic analgesics are the most effective treatments for chronic severe pain, but their clinical utility is often hampered by the development of analgesic tolerance. Recent evidence suggests chronic morphine may activate glial cells to release proinflammatory cytokines. In this study, we used herpes simplex virus (HSV) vector-based gene transfer to dorsal root ganglion to produce a local release of p55 tumor necrosis factor (TNF) soluble receptor in the spinal cord in rats with morphine tolerance. Subcutaneous inoculation of HSV vectors expressing p55 TNF soluble receptor into the plantar surface of the hindpaws enhanced the antinociceptive effect of acute morphine in rats. Subcutaneous inoculation of those vectors into hindpaws also delayed the development of chronic morphine tolerance in rats. TNF soluble receptor expressed by HSV vector reduced gene transcription of spinal TNFα and interleukin-1ß (IL-1ß) induced by repeated morphine. Furthermore, we found that TNF soluble receptor mediated by HSV reversed the upregulation of protein level of TNFα and IL-1ß and phosphorylation of p38 mitogen-activated protein kinase induced by repeated morphine. These results support the concept that proinflammatory cytokines may have an important role in the pathogenesis induced by morphine. This study provides a novel approach to treating morphine tolerance.

Vector-mediated expression of erythropoietin improves functional outcome after cervical spinal cord contusion injury.

We evaluated the therapeutic effect of erythropoietin (EPO) delivered by direct injection of a nonreplicating herpes simplex virus (HSV)-based vector coding for EPO (vEPO) in a model of cervical hemicord contusion at C7. At 1 h after spinal cord injury (SCI), either vEPO or control vector carrying a reporter gene (vC) was injected into the cord above and below the lesion. Animals injected with vEPO showed a statistically significant improvement in the ipsilateral forelimb function, as measured by open-field evaluation of motor performance, forelimb reaching in the cylinder test and misplacement in grid walk. This correlated with preservation of gray matter in the area of the lesion. There was also mild but significant improvement of hindlimb motor function measured by Basso-Beattie-Bresnahan score and computerized gait analysis in vEPO compared with control vector-injected animals. Microtubule-associated protein tau, phosphorylated and nonphosphorylated neurofilament protein and the synaptic proteins synaptophysin and PSD-95 were all significantly increased in the spinal cord of vEPO-treated animals compared with control vector-injected animals. These data suggest that gene transfer of EPO after cervical SCI by minimizing the injury size and enhancing tissue sparing preserves large-caliber axons and promotes synaptogenesis.

Engineering herpes simplex virus vectors for gene transfer to neurons.

HSV has a unique relationship with neurons which the virus naturally utilizes for efficient gene transfer and long-term gene expression. Progress in developing defective viral mutants with reduced cytotoxicity, and increasing insight into the state of the viral genome during latency and the functional elements of the latency promoter system, suggest that HSV vectors may be developed in which these natural features are exploited for effective transgene expression in brain.

CD8(+) T cells can block herpes simplex virus type 1 (HSV-1) reactivation from latency in sensory neurons.

Recurrent herpes simplex virus type 1 (HSV-1) disease usually results from reactivation of latent virus in sensory neurons and transmission to peripheral sites. Therefore, defining the mechanisms that maintain HSV-1 in a latent state in sensory neurons may provide new approaches to reducing susceptibility to recurrent herpetic disease. After primary HSV-1 corneal infection, CD8(+) T cells infiltrate the trigeminal ganglia (TGs) of mice, and are retained in latently infected ganglia. Here we demonstrate that CD8(+) T cells that are present in the TGs at the time of excision can maintain HSV-1 in a latent state in sensory neurons in ex vivo TG cultures. Latently infected neurons expressed viral genome and some expressed HSV-1 immediate early and early proteins, but did not produce HSV-1 late proteins or infectious virions. Addition of anti-CD8alpha monoclonal antibody 5 d after culture initiation induced HSV-1 reactivation, as demonstrated by production of viral late proteins and infectious virions. Thus, CD8(+) T cells can prevent HSV-1 reactivation without destroying the infected neurons. We propose that when the intrinsic capacity of neurons to inhibit HSV-1 reactivation from latency is compromised, production of HSV-1 immediate early and early proteins might activate CD8(+) T cells aborting virion production.

A human trial of HSV-mediated gene transfer for the treatment of chronic pain.

Gene transfer to the dorsal root ganglion using replication defective herpes simplex virus (HSV)-based vectors reduces pain-related behaviors in rodent models having inflammatory pain, neuropathic pain and pain caused by cancer in bone. HSV vectors engineered to produce inhibitory neurotransmitters, including the delta opioid agonist peptide enkephalin, the mu opioid agonist peptide endomorphin-2 and glutamic acid decarboxylase (GAD), to effect the release of gamma amino butyric acid (GABA) act to inhibit nociceptive neurotransmission at the first synapse between primary nociceptive and second-order neuron in the dorsal horn of the spinal cord. HSV vectors engineered to release anti-inflammatory peptides, including interleukin (IL)-4, IL-10 and the p55 soluble tumor necrosis factor alpha (TNFalpha) receptor reduce neuroimmune activation in the spinal dorsal horn. The path leading from preclinical animal studies to the ongoing phase 1 human trial of the enkephalin-producing vector in patients with pain from cancer, and plans for an efficacy trial with an opioid-producing vector in inflammatory pain and an efficacy trial with a GAD-producing vector in diabetic neuropathic pain are outlined.

Axonal transport of proteins. A new view using in vivo covalent labeling.

The injection of [2,3-3H]N-succinimidyl propionate ([3H]N-SP) into the rat sciatic nerve was used to covalently label both intra- and extra-axonal proteins. While extra-axonal proteins (e.g., myelin proteins) remained in the injection site, the intra-axonal proteins were transported in both the anterograde and retrograde directions. The mobile labeled proteins appeared to move by normal axonal transport processes because: (a) autoradiographic studies showed that they were localized exclusively within the axon at considerable distances from the injection site, (b) specific and identifiable proteins (by SDS gel electrophoresis) moved at expected rates in the anterograde direction, and (c) an entirely different profile of proteins moved in the anterograde vs. retrograde direction. This novel experimental approach to axonal transport, which is independent of de novo protein synthesis, provided a unique view of slow anterograde transport, and particularly of retrograde transport of endogenous proteins. A large quantity of a 68,000 mol wt proteins, moving at approximately 3-6 mm/day, dominated the retograde transport profile. [3H]N-SP, therefore, represents a new and unique "vital stain" which may find many applications in cell biology.

Retrograde axonal transport of endogenous proteins in sciatic nerve demonstrated by covalent labeling in vivo.

Extracellularly applied N-succinimidyl [2,3-3H]propionate was used in vivo to covalently label intra-axonal proteins in the rat sciatic nerve. This technique permitted a unique view of axonal transport of proteins independent of biosynthesis. The proteins detected in slow anterograde transport (1 to 2 millimeters per day) correspond to cytoskeletal proteins described in previous papers. The slowly retrogradely transported component (3 to 6 millimeters per day) was composed primarily of a single protein with a molecular weight of 68,000.

Intermolecular interactions in collagen self-assembly as revealed by Fourier transform infrared spectroscopy.

When a solution of collagen molecules, at neutral pH and moderate ionic strength, is warmed from 4 degrees to 30 degrees C, a spontaneous self-assembly process takes place in which native-type collagen fibers are produced. Events occurring during thermally induced fibrillogenesis process can be monitored, in aqueous media and in real time, by Fourier transform infrared spectroscopic techniques. Tentative assignments of observed spectral bands are given.

Metabolic mapping of functional activity in the hypothalamo-neurohypophysial system of the rat.

Physiological stimulation of the hypothalamo-neurohypophysial system by salt loading of rats resulted in a dramatically increased glucose utilization in the posterior pituitary but not in the paraventricular or supraoptic nuclei. The good correlation between glucose utilization and neural activity in the posterior pituitary (that is, nerve terminals) contrasted with the lack of correlation in the paraventricular and supraoptic nuclei (that is, the sites of the cell bodies of the same neurons). This difference in the metabolic response to functional activity between the two regions of these neurons can be explained by the differences in surface-to-volume ratios of these regions.

Innovative materials processing strategies: a biomimetic approach.

Many organisms construct structural ceramic (biomineral) composites from seemingly mundane materials; cell-mediated processes control both the nucleation and growth of mineral and the development of composite microarchitecture. Living systems fabricate biocomposites by: (i) confining biomineralization within specific subunit compartments; (ii) producing a specific mineral with defined crystal size and orientation; and (iii) packaging many incremental units together in a moving front process to form fully densified, macroscopic structures. By adapting biological principles, materials scientists are attempting to produce novel materials. To date, neither the elegance of the biomineral assembly mechanisms nor the intricate composite microarchitectures have been duplicated by nonbiological processing. However, substantial progress has been made in the understanding of how biomineralization occurs, and the first steps are now being taken to exploit the basic principles involved.

HSV-mediated transfer of interleukin-10 reduces inflammatory pain through modulation of membrane tumor necrosis factor alpha in spinal cord microglia.

To dissect the molecular basis of the neuroimmune response associated with the genesis of inflammatory (nociceptive) pain, we constructed a herpes simplex virus-based gene transfer vector to express the antiinflammatory cytokine interleukin-10 (IL-10), and used it to examine the effect of IL-10 expression in activated microglial cells in vitro, and in inflammatory pain in vivo. IL-10 reduced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and decreased the expression of full-length membrane spanning tumor necrosis factor-alpha (mTNFalpha) following lipopolysaccharide stimulation of microglia in vitro. IL-10 also reduced intracellular cleavage of mTNFalpha and release of the soluble cleavage product sTNFalpha. Similar effects on TNFalpha expression were observed when the cells were pretreated with a p38 MAPK inhibitor. In animals, injection of a dilute solution of formalin in the skin resulted in an increase in mTNFalpha in spinal dorsal horn, without detectable sTNFalpha. Local release of IL-10 achieved by gene transfer reduced the number of spontaneous flinches in the early and delayed phases of the formalin test of inflammatory pain. The effect of IL-10 on nocisponsive behavior correlated with a block in phosphorylation of p38 and reduced expression of 26 kDa mTNFalpha in spinal microglia. The results emphasize the key role played by membrane TNFalpha in the spinal neuroimmune response in pain caused by peripheral inflammation.

In vivo metabolism of complement. I. Metabolism of the third component (C'3) in acquired hemolytic anemia.

The in vivo metabolism of purified third component of complement labeled with (125)-iodine (C'3-(125)I) was studied in normal subjects and in patients with acquired hemolytic anemias. 27 such studies were performed; in addition, three studies were performed using C'3i, the biologically inactive reaction product of C'3. In normal subjects the mean fractional catabolic rate of C'3 was 2.12%/hr and the normal range (defined throughout as the mean +/- 2 SD) was from 1.56 to 2.68. The mean percentage of C'3 that was intravascular was 66.6% and the normal range was from 51 to 83. The C'3 synthesis rate averaged 1.16 mg/kg per hr with a normal range of from 0.90 to 1.42. The mean serum concentration of C'3 was 1.43 mg/ml with a normal range of from 1.00 to 1.87. The fractional catabolic rate and synthesis rate of C'3 were at the upper limit of normal or were increased above normal in patients who had warm antibody autoimmune hemolytic anemia with complement on their erythrocytes and in patients with paroxysmal nocturnal hemoglobinuria studied during periods of active hemolysis. An increased C'3 synthesis rate was also found in one patient who was hematologically normal but had an active peptic ulcer and elevated serum concentration of C'3.A normal fractional catabolic rate and C'3 synthesis rate were found in patients with autoimmune hemolytic anemia associated with alpha-methyldopa administration, atypical cold antibody autoimmune hemolytic anemia, and in paroxysmal nocturnal hemoglobinuria during an asymptomatic interval. The three studies with C'3i-(125)I revealed a very rapid removal of the labeled protein from the plasma with less than 10% remaining after 2 hr and with a corresponding increase in urinary excretion rate of the label. The fractional catabolic rate of C'3i averaged 37%/hr. The findings are consistent with the previously elucidated in vitro reaction mechanism of C'3 and strengthen the concept that serum complement participates in immune reactions in vivo.

Autoimmune thyroiditis and exposure to iodine 131 in the Ukrainian cohort study of thyroid cancer and other thyroid diseases after the Chornobyl accident: results from the first screening cycle (1998-2000).

CONTEXT: Due to the Chornobyl accident, millions were exposed to radioactive isotopes of iodine and some received appreciable iodine 131 (131I) doses. A subsequent increase in thyroid cancer has been largely attributed to this exposure, but evidence concerning autoimmune thyroiditis (AIT) remains inconclusive. OBJECTIVE: The objective of the study was to quantify risk of AIT after 131I exposure. DESIGN/SETTING/PARTICIPANTS: Baseline data were collected from the first screening cycle (1998-2000) of a large cohort of radiation-exposed individuals (n = 12,240), residents of contaminated, iodine-deficient territories of Ukraine. Study individuals were under the age of 18 yr on April 26, 1986, and had thyroid radioactivity measurements made shortly after the accident. OUTCOMES: AIT was defined a priori based on various combinations of elevated antibodies to thyroid peroxidase (ATPO), TSH, and clinical findings; elevated ATPO were considered to be an indicator of thyroid autoimmunity. RESULTS: No significant association was found between 131I thyroid dose estimates and AIT, but prevalence of elevated ATPO demonstrated a modest, significant association with 131I that was well described by several concave models. This relationship was apparent in individuals with moderately elevated ATPO and euthyroid, thyroid disease-free individuals. CONCLUSIONS: Twelve to 14 yr after the Chornobyl accident, no radiation-related increase in prevalence of AIT was found in a large cohort study, the first in which 131I thyroid doses were estimated using individual radioactivity measurements. However, a dose-response relationship with ATPO prevalence raises the possibility that clinically important changes may occur over time. Thus, further follow-up and analysis of prospective data in this cohort are necessary.

Cancer overview.

Cancer is a very common group of diseases. There are 700,000 cases of the disease reported each year, and about 350,000 persons die of these diseases. There is extensive literature and documentation relative to alcohol as a probable factor in the development of head, neck, and esophageal cancers among others. However, there is little in the literature relative to the interrelationship of alcohol and cancer in the area of cancer treatment. Dignity of the dying patient and his family is maintained, and home care is a vital component. While alcohol use is not generally written about, it is used as desired by the patient. A "Brompton's-type cocktail," a mixture of morphine, cocaine, and alcohol, forms a basis of pain control in English and many United States' hospices. One purpose in today's conference is to look at another lifestyle factor, alcohol, to determine its role alone or in conjunction with other factors in the causation of cancer. Are there high risk groups which need attention in alcohol cancer prevention programs?

Alterations in retrograde axonal transport in streptozocin-induced diabetic rats.

Retrograde axonal transport in the sciatic nerve of rats with streptozocin-induced diabetes was studied by the [3H]N-succinimidyl propionate [( 3H]NSP) method. The accumulation of retrogradely transported labeled proteins in the dorsal root ganglia and the ventral horn of spinal cord 1 day after [3H]NSP injection was not statistically significantly different from controls in rats diabetic for 1 or 14 days at the time of [3H]NSP injection. However, accumulation of labeled proteins in the dorsal root ganglia 7 days after [3H]NSP injection was reduced by 35% and transport to the ventral horn of spinal cord 7 days after [3H]NSP injection was reduced by 70% at the same time points. Partial control of the diabetes with insulin resulted in a partial reversal of these deficits. The early occurrence of defects in retrograde transport suggests that such defects may play a role in the pathogenesis of the neuropathy.

Activation of TLR-4 to produce tumour necrosis factor-α in neuropathic pain caused by paclitaxel.

BACKGROUND: Neuropathic pain is a common complication of treatment with the anti-neoplastic drug paclitaxel. Animal studies suggest neuroinflammation and transient receptor potential channels TRPA1 and TRPV4 are involved in the pathogenesis of pain in this condition. However, how neuroinflammation and TRPA1 and TRPV4 are linked to cause pain in paclitaxel-treated animals is not known. METHODS: Paclitaxel-induced pain was modelled by IP injection of paclitaxel (16 mg/kg) once a week for 5 weeks. The role of toll-like receptor 4 (TLR-4) in tumour necrosis factor-α (TNF-α) production and the effect of TNF-α on the expression of TRPA1 and TRPV4 were evaluated in vitro and in vivo. TNF-α signalling in dorsal root ganglion (DRG) was blocked by expressing soluble TNF receptor I (TNFsR) from a herpes simplex virus (HSV)-based vector (vTNFsR). RESULTS: Paclitaxel treatment increased the expression and release of TNF-α in satellite glial cells and increased the expression of TRPA1 and TRPV4 in DRG neurons in animals. In vitro, paclitaxel enhanced the expression and release of TNF-α in enriched primary satellite glial cells, an effect that was blocked by an inhibitor of TLR-4. Direct application of TNF-α to primary DRG neurons in culture up-regulated the expression of TRPA1 and TRPV4. In vivo, vector-mediated TNFsR release from DRG neurons reduced paclitaxel-induced up-regulation of TRPA1 and TRPV4 expression and prevented paclitaxel-induced pain. CONCLUSION: These results suggest that paclitaxel activation of TLR-4 to cause release of TNF-α from satellite glial cells increases the expression of TRPA1 and TRPV4 in DRG neurons to cause neuropathic pain.

In vivo expression of beta-galactosidase in hippocampal neurons by HSV-mediated gene transfer.

Stereotactic inoculation of a herpes simplex virus (HSV) gene transfer vector into the hippocampus and caudate of rat brain resulted in limited and transient viral replication and the establishment of latency. Virus attenuation was achieved by insertional inactivation of a viral gene, Us3. Insertion of a lacZ reporter gene, under the control of the HSV glycoprotein C (gC) late gene promoter, allowed viral replication to be monitored in vivo. Unlike unattenuated virus, the Us3::pgC-lacZ recombinant caused little apparent damage to normal hippocampal morphology. Transient lacZ expression was detected in a considerable population of neurons of the dentate gyrus following hippocampal injection, whereas few positively staining neurons were present within the caudate after injection at that site. Latency-associated transcripts, the hallmark of latent infection, were detected in the brain 10 months after injection. This recombinant virus may be useful as a gene transfer vector for long-term expression of foreign genes in the central nervous system.