Kou Sakabe

Nucleus pulposus allograft retards intervertebral disc degeneration

Autogenous implantation of nucleus pulposus or nucleus pulposus cells that were activated by coculture retards intervertebral disc degeneration, but harvesting such grafts causes disc degeneration at the donor site. This study examined whether nucleus pulposus allografts similarly retard disc degeneration and whether such allografting induces immunologic rejection. Japanese White rabbits served as donors and recipients for allografts. Lumbar disc degeneration was induced by aspirating the nucleus pulposus. Two weeks later, intact nucleus pulposus or nucleus pulposus cells were injected and compared with a sham procedure and normal control. The recipients’ discs were examined histologically and immunologically at intervals for 16 weeks. Discs receiving an intact nucleus pulposus showed the least degeneration, followed by discs receiving nucleus pulposus cells, both of which were better than no treatment. These findings correlated directly with the intensity of immunochemical staining for Type II collagen. Allogeneic grafts did not induce any appreciable host-versus-graft response. Injection of nucleus pulposus and nucleus pulposus cells retards intervertebral disc degeneration. However, injection of intact nucleus pulposus is more effective than injection of nucleus pulposus cells alone. The intercellular matrix plays an important, but poorly understood, role in preserving intervertebral discs.

Localization of the ATP-sensitive potassium channel subunit (Kir6.1/uKATP-1) in rat brain

The Kir6.1/uK(ATP)-1, subunit of ATP-sensitive K(+) channels (K(ATP)), was localized in adult rat brain by in situ hybridization and immunohistochemistry. The mRNA of this subunit was ubiquitously expressed in various neurons and nuclei of the adult rat brain. Interestingly, Kir6.1/uK(ATP)-1 mRNA was also expressed in glial cells. Kir6.1/uK(ATP)-1 protein staining gave a dispersed array of fine dots throughout all neurons and glial cells examined. Under electron microscope, the immunoreactive products were specifically restricted to the mitochondria. The present study indicates that this K(ATP) subunit is localized in the mitochondria and may play a fundamental role in vital brain function.

Symptom profile of multiple chemical sensitivity in actual life.

Objective: This study was conducted to confirm the definition of multiple chemical sensitivity (MCS) in actual life: that multiple symptoms are provoked in multiple organs by exposure to, and ameliorated by avoidance of, multiple chemicals at low levels. We used the Ecological Momentary Assessment to monitor everyday symptoms and the active sampling and passive sampling methods to measure environmental chemical exposure. Methods: Eighteen patients with MCS, diagnosed according to the 1999 consensus criteria, and 12 healthy controls participated in this study. Fourteen patients and 12 controls underwent 1-week measurement of physical and psychologic symptoms and of the levels of exposure to various chemicals. Linear mixed models were used to test the hypotheses regarding the symptom profile of MCS patients. Results: Some causative chemicals were detected in 11 of 14 MCS patients. Two other patients did not report any hypersensitivity episodes, whereas passive sampling showed far less exposure to chemicals than control subjects. Another subject reported episodic symptoms but was excluded from the following analyses because no possible chemical was detected. Eleven of the 17 physical symptoms and all four mood subscales examined were significantly aggravated in the interview based on “patient-initiated symptom prompts.” On the other hand, there were no differences in physical symptoms or mood subscales between MCS patients and control subjects in the interview based on “random prompts.” Conclusions: MCS patients do not have either somatic or psychologic symptoms under chemical-free conditions, and symptoms may be provoked only when exposed to chemicals. AS = active sampling; AS–PS method = active sampling and passive sampling methods; CAS = the concentration of exposure estimated by the AS method; CFS = chronic fatigue syndrome; CPS = the concentration of exposure estimated by the PS method; CS = chemical sensitivity; DAMS = Depression and Anxiety Mood Scale; DNPH = 2,4-dinitrophenyl-hydrazine; ED = electronic diary; EESI = Environmental Exposure and Sensitivity Inventory; EMA = Ecological Momentary Assessment; FM = fibromyalgia; M.I.N.I. = Mini International Neuropsychiatric Interview; MCS = multiple chemical sensitivity; PS = passive sampling; RSD = relative standard deviation; RSDAS = RSD of repeatability test in the AS method; RSDPS = RSD of repeatability test in the PS method; VOCs = volatile organic compounds.

Effect of Reinsertion of Activated Nucleus Pulposus on Disc Degeneration: An Experimental Study on Various Types of Collagen in Degenerative Discs

We examined the emergence and sequential changes in type I, II, and VI collagen production in an experimental rabbit model of disc degeneration. Type I collagen was minimally present initially and did not change over 24 weeks. Type I collagen seemed to have no effect on the degenerative process in this model. Staining for type II collagen was positive circumferentially in chondrocytelike cells and was mild in the early phase of disc degeneration, when the chondrocytelike cells began to appear in the inner layers of the annulus fibrosus. The stain became stronger during the middle phase when the chondrocytelike cells arranged themselves in cluster. Compared with type II collagen, the staining for type VI collagen was relatively strong early in the degenerative process. These findings led us to speculate that these chondrocytelike cells play an active role in the degenerative process. The reinsertion of nucleus pulposus cells cocultured with annulus fibrosus delayed disc degeneration and the emergence of chondrocytelike cells. Considering that the emergence of chondrocytelike cells which produce type II and type VI collagen is delayed in discs with the injection of cocultured nucleus pulposus cells by annulus fibrosus cells, we conclude that chondrocytelike cells that produce type VI collagen also seems to accelerate degeneration. Type VI collagen is produced at an earlier phase than type II collagen and may be both active agent and a marker for disc degeneration.

Effects of sex steroids on the proliferation of thymic epithelial cells in a culture model: A role of protein kinase C

Using a rat thymic epithelial cell line (TEC; IT‐45R1), the present study attempted to elucidate the mechanism of action of sex steroid hormones (SH) on the proliferation of TEC The findings were as follows: (a) the proliferation of TEC in response to SH was mediated through protein kinase C activity introduced as a result of interaction between SH and plasma‐borne inhibitors; (b) the strong inhibitory effect of SH on TEC proliferation might be mediated through the SH receptor pathway because the proliferative response was triggered by progesterone (P) and androgen (A), whereas the inhibitory response was triggered by P, A and oestrogen. These results clearly suggest that the control of TEC proliferation is a ‘shut‐off’ mechanism triggered by high plasma levels of SH. This further refers to the speculation that the development of the normal thymus may be due to a lack of this ‘shut‐off’ mechanism so that development occurs at the adequate plasma SH levels that are often observed before puberty. However, this development is inhibited at the high plasma SH levels after puberty and/or during pregnancy.

The MerE protein encoded by transposon Tn21 is a broad mercury transporter in Escherichia coli

In order to clarify the physiological role of the merE gene of transposon Tn21, a pE4 plasmid that contained the merR gene of plasmid pMR26 from Pseudomonas strain K‐62, and the merE gene of Tn21 from the Shigella flexneri plasmid NR1 (R100) was constructed. Bacteria with plasmid pE4 (merR‐o/p‐merE) were more hypersensitive to CH3Hg(I) and Hg(II), and took up significantly more CH3Hg(I) and Hg(II), than the isogenic strain. The MerE protein encoded by pE4 was localized in the membrane cell fraction, but not in the soluble fraction. Based on these experimental results, we suggest for the first time that the merE gene is a broad mercury transporter mediating the transport of both CH3Hg(I) and Hg(II) across the bacterial membrane.

Overexpression of GRP78 protects glial cells from endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress induces apoptotic cell death by causing the accumulation of structurally abnormal proteins. The 78-kDa glucose-regulated protein (GRP78) is an ER chaperone that regulates protein folding in the ER and has been suggested to contribute to cell survival. Using the rat C6 glioma cell line and flow cytometry, we assessed GRP78 expression following tunicamycin- and glutamate-induced ER stress. The results showed that GRP78 expression is upregulated following ER stress and has protective effects on injured glial cells. Annexin V and propidium iodide labeling revealed cells transiently expressing GRP78 prior to injury were protected against high-concentrations of tunicamycin and glutamate within 72 h. Our findings support the hypothesis that GRP78 inhibits cell death associated with ER stress.

Metallothionein is a crucial protective factor against Helicobacter pylori-induced gastric erosive lesions in a mouse model

Infection with the gastric pathogen Helicobacter pylori (H. pylori) causes chronic gastritis, peptic ulcer, and gastric adenocarcinoma. These diseases are associated with production of reactive oxygen species (ROS) from infiltrated macrophages and neutrophiles in inflammatory sites. Metallothionein (MT) is a low-molecular-weight, cysteine-rich protein that can act not only as a metal-binding protein, but also as a ROS scavenger. In the present study, we examined the role of MT in the protection against H. pylori-induced gastric injury using MT-null mice. Female MT-null and wild-type mice were challenged with H. pylori SS1 strain, and then histological changes were evaluated with the updated Sydney grading system at 17 and 21 wk after challenge. Although the colonization efficiency of H. pylori was essentially the same for MT-null and wild-type mice, the scores of activity of inflammatory cells were significantly higher in MT-null mice than in wild-type mice at 17 wk after challenge. Histopathological examination revealed erosive lesions accompanied by infiltration of inflammatory cells in the infected MT-null mice but not in wild-type mice. Furthermore, activation of NF-kappaB and expression of NF-kappaB-mediated chemokines such as macrophage inflammatory protein-1alpha and monocytes chemoattractant protein-1 in gastric cells were markedly higher in MT-null mice than in wild-type mice. These results suggest that MT in the gastric mucosa might play an important role in the protection against H. pylori-induced gastric ulceration.

Inhibitory effect of natural and environmental estrogens on thymic hormone production in thymus epithelial cell culture

The present study was carried out to assess the direct effect of natural estrogen and environmental estrogens on thymus epithelial cell (TEC) production/secretion of the thymic hormone thymosin-alpha 1 by using the technique of quantitative high-performance liquid chromatography. The presence of estrogen receptors in the TECs was also investigated. Murine TECs were cultured in the experimental DMEM medium containing various concentrations of natural or environmental estrogens, which was followed by determining the production of thymosin-alpha 1. The production of thymosin-alpha 1 by TECs was significantly inhibited by increasing concentrations of 17beta-estradiol (natural estrogen) over 3 x 10(-11) M, genistein (phytoestrogen) over 3 x 10(-9) M, coumestrol (phytoestrogen) over 3 x 10(-9) M, alpha-zearalanol (livestock anabolic) over 3 x 10(-7) and bisphenol-A (plastic) over 3 x 10(-6) M. Small amounts of estrogen receptor were present in the TECs. The above results clearly indicate that natural and environmental estrogens directly modulate TECs to produce thymic hormone probably through an estrogen receptor mechanism. Furthermore, our finding may be useful for evaluating biological effects of chemicals with estrogenic activity.

Evaluation of subjective symptoms of Japanese patients with multiple chemical sensitivity using QEESI

ObjectivesThe Quick Environment Exposure Sensitivity Inventory (QEESI©) has been used as a questionnaire to evaluate subjective symptoms of patients with multiple chemical sensitivity (MCS), also known as idiopathic environmental intolerance, in Japan. However, no cutoff value for Japanese subjects has yet been established. We designed this study to establish a cutoff value for Japanese subjects using QEESI© for screening of MCS patients.MethodsA questionnaire using the QEESI© was administered to 103 MCS patients and 309 healthy control subjects matched for age and sex. QEESI© scores of the two groups were compared using logistic regression analysis, receiver operating characteristic analysis, and the Mann–Whitney test.ResultsCutoff values for Japanese subjects were determined for the Chemical Intolerance subscale (40), Symptom Severity subscale (20), and Life Impact subscale (10). The subjects whose scores exceeded the cutoff values in any two subscales accounted for 88.4% of the patients but only 14.5% of the controls.ConclusionsOur results suggest that subjects meeting two out of three subscale criteria can be screened as “patients suffering from a low level of environmental chemicals such as MCS” in Japan.