In a model of Batten disease, palmitoyl protein thioesterase-1 deficiency is associated with brown adipose tissue and thermoregulation abnormalities

Infantile neuronal ceroid lipofuscinosis (INCL) is a fatal neurodegenerative disorder caused by a deficiency of palmitoyl-protein thioesterase-1 (PPT1). We have previously shown that children with INCL have increased risk of hypothermia during anesthesia and that PPT1-deficiency in mice is associated with disruption of adaptive energy metabolism, downregulation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), and mitochondrial dysfunction. Here we hypothesized that Ppt1-knockout mice, a well-studied model of INCL that shows many of the neurologic manifestations of the disease, would recapitulate the thermoregulation impairment observed in children with INCL. We also hypothesized that when exposed to cold, Ppt1-knockout mice would be unable to maintain body temperature as in mice thermogenesis requires upregulation of Pgc-1α and uncoupling protein 1 (Ucp-1) in brown adipose tissue. We found that the Ppt1-KO mice had lower basal body temperature as they aged and developed hypothermia during cold exposure. Surprisingly, this inability to maintain body temperature during cold exposure in Ppt1-KO mice was associated with an adequate upregulation of Pgc-1α and Ucp-1 but with lower levels of sympathetic neurotransmitters in brown adipose tissue. In addition, during baseline conditions, brown adipose tissue of Ppt1-KO mice had less vacuolization (lipid droplets) compared to wild-type animals. After cold stress, wild-type animals had significant decreases whereas Ppt1-KO had insignificant changes in lipid droplets compared with baseline measurements, thus suggesting that Ppt1-KO had less lipolysis in response to cold stress. These results uncover a previously unknown phenotype associated with PPT1 deficiency, that of altered thermoregulation, which is associated with impaired lipolysis and neurotransmitter release to brown adipose tissue during cold exposure. These findings suggest that INCL should be added to the list of neurodegenerative diseases that are linked to alterations in peripheral metabolic processes. In addition, extrapolating these findings clinically, impaired thermoregulation and hypothermia are potential risks in patients with INCL

The role of neural cell adhesion molecule in the tumorigenicity of multiple myeloma in SCID mice.

Neural Cell Adhesion Molecule (NCAM) is a glycoprotein involved in cell adhesion. NCAM is present on multiple myeloma (MM) as determined by immunohistochemistry. Molecular characterization of MM associated NCAM determined that MM expresses the 140 kd form of the molecule and the sequence of a 525bp RT-PCR product is identical to lymphoid associated forms of the molecule. The 8226 Dox40 cell line is 50% positive for NCAM. This cell line was sorted and subsequently cloned into an NCAM negative and NCAM positive population. To determine what role NCAM plays in the pathogenesis of MM SCID mice were injected with these populations. Of the mice that received the NCAM negative cells 19/23 (87%) developed tumors while only 2/23 (9%) of the mice that received NCAM positive tumors developed tumors. Increasing the number of tumor cells injected did not change these results. Injection of two other MM lines, ARH-77 (NCAM negative) and Karpas (NCAM positive) yielded the same results. The differences in tumorigenicity are not due to differences in growth kinetics of the two clones, ability to invade using an in vitro invasion assay or differences in genotype when examined karyotypically or genotypically. SCID mouse NK cells and macrophages are not cytotoxic for the human tumor cells and SCID macrophages are equally cytostatic for the human tumor cells. Neither cell line is able to stimulate the release of H₂O₂ or TNF from the macrophages. To determine what role NCAM plays in the differences in tumorigenicity the NCAM negative cells were transfected with NCAM using the LXSN retroviral vector. Injection of SCID mice with the transfected cells did not alter their tumorigenicity in SCID mice. These results suggest the NCAM may be a useful marker to isolate Multiple Myeloma cells but NCAM plays no role in the tumorigenicity of Multiple Myeloma

Mouse current vocalization threshold measured with a neurospecific nociception assay: the effect of sex, morphine, and isoflurane.

Sine-wave electrical stimulation at frequencies 2000, 250, and 5 Hz to respectively evaluate Aβ, Aδ, and C sensory neurons has recently been added to the armamentarium used to evaluate sensory neurons. We developed an automated nociception assay using sine-wave stimulation methodology to determine current vocalization threshold in response to 2000, 250, and 5 Hz and examine the effects of sex, analgesics, and anesthetics in mice. At baseline, males had significantly higher mean current vocalization thresholds compared with female mice at 2000, 250, and 5 Hz (p ≤ 0.019). By 1 h after intrathecal injections of morphine there were significant increases in current vocalization threshold percent changes from baseline that varied with doses (p = 0.0001) and frequency used (p < 0.0001). Specifically, with increasing doses of morphine, there were significantly greater increases in current vocalization threshold percent changes from baseline in response to 5 Hz compared with 250 and 2000 Hz stimulation in a significantly ordered pattern: 5 Hz > 250 Hz (p < 0.0001) and 250 Hz > 2000 Hz (p = 0.0002). Forty-five minutes after exposure, there were no effects of isoflurane on current vocalization thresholds at any frequency. Therefore, our findings suggest that this automated nociception assay using sine-wave stimulation in mice, can be valuable for measurements of the effects of sex, opioids, and anesthetics on the response to electrical stimuli that preferentially stimulate Aβ, Aδ, and C-sensory fibers in vivo. This investigation suggests the validation of this assay and supports its use to examine mechanisms of nociception in mice

The use of a cationic liposome formulation (DOTAP) mixed with a recombinant tumor-associated antigen to induce immune responses and protective immunity in mice

The cationic liposome DOTAP is a well-known transfection reagent. It has been manufactured and approved for clinical use, is readily available, and can be easily used as an adjuvant. These characteristics prompted us to investigate the effectiveness of DOTAP as an adjuvant to induce immune responses and protective immunity in mice using baculovirus-derived carcinoembryonic antigen (bV-CEA) as a model antigen. Two routes of administration and a dose-response study of bV-CEA were used in BALB/c mice to define the magnitude of the immune response as well as the most effective route of immunization. The results demonstrate differences in antibody titers, immunoglobulin (Ig)G isotype, and T-cell responses between the intravenous (i.v.) or subcutaneous (s.c.) route of immunization. The titer of the anti-CEA antibodies induced by the s.c. immunization was greater than the response by i.v. immunization. The s.c. route enhanced the IgG2a/2b isotype, whereas i.v. immunization elicited primarily IgG1. T-cell proliferation responses and cytokine production paralleled the humoral response (i.e., production was higher in the s.c. immunized animals). No differences in immunological responses were seen using either 25 or 10 mu g of bV-CEA three times. An amount of 25 mu g of bV-CEA/DOTAP given by s.c. immunization was sufficient in protecting mice from the transplant of syngeneic tumor cells transduced with the human CEA gene. We conclude that the cationic liposome DOTAP may be a useful immunoadjuvant for active anti-tumor immunotherapy in future clinical trials. This study will help to define the most effective way to use such an adjuvant

The three isoforms of nitric oxide synthase distinctively affect mouse nocifensive behavior

Nitric oxide synthases (NOSs) have been shown to modulate thermal hyperalgesia and mechanical hypersensitivity in inflammatory and neuropathic pain. However, little is known about the effect of NOSs on baseline function of sensory nerve fibers. Using genetic deficiency and pharmacologic inhibition of NOSs, we examined the impact of the three isoforms NOS1, NOS2, and NOS3 on baseline nocifensive behavior by measuring current vocalization threshold in response to electrical stimulation at 5, 250, 2000 Hz that preferentially stimulate C, Aδ, and Aβ fibers. In response to 5, 250 and 2000 Hz, NOS1-deficient animals had significantly higher current vocalization thresholds compared with wild-type. Genetic deficiency of NOS2 was associated with higher current vocalization thresholds in response to 5 Hz (C-fiber) stimulation. In contrast, NOS3-deficient animals had an overall weak trend toward lower current vocalization thresholds at 5 Hz and significantly lower current vocalization threshold compared with wild-type animals at 250 and 2000 Hz. Therefore, NOSs distinctively affect baseline mouse current vocalization threshold and appear to play a role on nocifensive response to electrical stimulation of sensory nerve fibers

Sickle cell disease in mice is associated with sensitization of sensory nerve fibers

The pain phenotype in sickle cell disease (SCD) patients is highly variable. A small percentage of SCD patients experience many vaso-occlusive crises/year, 5% of patients account for over 30% of pain episodes, while 39% report few episodes of severe pain. Clearly, a better understanding of the pathobiology of SCD is needed to improve its therapy. Humanized sickle cell mice recapitulate several phenotypes of SCD patients and provide a model for the study of SCD pain. Researchers have shown that one strain of humanized SCD mice, the BERK strain, has abnormal pain phenotype. However, the nociception phenotype of another humanized SCD mouse strain, the Townes strain, has not been described. In a large cross-sectional study of BERK and Townes SCD mice, we examined thermosensory response and sensory nerve fiber function using sine-wave electrical stimulation at 2000, 250, and 5 Hz to stimulate preferentially Aβ, Aδ, and C sensory nerve fibers, respectively. We found that BERK and Townes mice, compared to respective controls, had decreases in 2000, 250, and 5 Hz current vocalization thresholds in patterns that suggest sensitization of a broad spectrum of sensory nerve fibers. In addition, the pattern of sensitization of sensory fibers varied according to strain, sex, age, and mouse genotype. In a similarly variable pattern, Townes and BERKs also had significantly altered sensitivity to noxious thermal stimuli in agreement with what has been shown by others. In summary, the analysis of somatosensory function using sine-wave electrical stimulation in humanized sickle cell mice suggests that in SCD, both myelinated and unmyelinated, fibers are sensitized. The pattern of sensory fiber sensitization is distinct from that observed in pain models of neuropathic and inflammatory pain. These findings raise the possibility that sensitization of a broad spectrum of sensory fibers might contribute to the altered and variable nociception phenotype in SCD

The relationship between the nature of the psychosocial environment and the child's social adjustment

Thesis (M.S.)--Boston UniversityPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at [email protected]. Thank you.2031-01-0

c-fos expression, ATP concentration and NAD⁺/NADH ratio in mouse brown adipose tissue.

<p>A–D. Representative images of brain c-fos immunostaining showing c-fos positive cells in ventromedial hypothalamic nucleus of wild-type (A and B) and <i>Ppt1</i>-KO (C and D) at baseline (A and C) and after cold exposure (B and D), N = 5−6 per group. E. Number of c-fos positive cells in ventromedial hypothalamic nucleus of <i>Ppt1</i>-KO and wild-type mice indicating that both wild-type and <i>Ppt1</i>-KO mice similarly upregulated <i>c-fos.</i> F. Baseline ATP concentration in brown adipose tissue of wild-type and <i>Ppt1</i>-KO mice are similar (N = 6 per group, p = NS). G. Baseline NAD⁺/NADH ratio in brown adipose tissue of wild-type and <i>Ppt1</i>-KO mice are similar (N = 6 per group, p = NS).</p

Body temperature during basal conditions and cold and heat exposures in <i>Ppt1</i>-KO and wild-type mice.

<p>In all panels, horizontal bars and respective p values show comparisons among groups indicated. A. Longitudinal measurements (least square mean ±SE) of basal temperature of male and female <i>Ppt1</i>-KO and wild-type mice at 8–9 and 18–26 weeks (N = 10 in each group). B. Temperature changes from baseline measured every 30 min during a 6-h (all time points combined) acute cold exposure at young (8–9 weeks) and old (18–26 weeks) ages. Bars represent least square means ±SE. C. Temperature changes (least square means ±SE) from baseline measured (every 30 min) during a 6-hour (all time points combined) acute heat exposure at young (8–9 weeks) and old (18–26 weeks) ages. In acute cold and heat exposure experiments, N = 5 in each group.</p