Neurolysis of celiac plexus and splanchnic nerve under ultrasonographic guidance

Key words  intractable pain - celiac plexus neurolysis - ultrasonography     

A new HPLC fluorimetric method to monitor urinary delta-aminolevulinic acid (ALA-U) levels in workers exposed to lead

Summary A new sensitive HPLC method for the determination of urinary delta-aminolevulinic acid (ALA-U) was used to evaluate the relationship between blood-lead (Pb-B) and ALA-U levels in male workers exposed to lead. The differences between the ALA-U levels determined by this method (ALAU-HP) and by a colorimetric method (ALA-U-CL) are discussed. The HPLC method gave values similar to the ALA-U-CL values at high ALA-U level. However, at low blood-lead levels (58 ± 22 g/l, n = 23), the mean ALA-U-HP level corrected by urinary creatinine level was one-third of the corrected ALA-UCL level (0.83 ± 0.14 and 2.4 ± 0.5 mg/g creatinine, respectively). A significant increase of the mean corrected ALA-U-HP level was observed at 162 ± 22 g/l Pb-B (P < 0.05, n = 26), while that of ALA-UCL was observed at 245 ± 30 g/l Pb-B (P < 0.01, n = 37). The regression equation based on the logistic model fitted well to the relationship data between the Pb-B level and the percentage of the subjects with corrected ALA-U-HP above the cut-off point (1.12 mg/g creatinine) and the expected Pb-B level for 50% response was 270 g/l Pb-B, while it did not fit well to the relationship data between Pb-B level and the percentage of the subjects with corrected ALAU-CL above the cut-off point (3.5 mg/g creatinine). The maximum responses for the two sets of corrected ALA-U levels were both observed at 625 ± 25 g/l. The corrected ALA-U level by HPLC method seems to be a useful indicator for biological monitoring of exposure to lead at low levels (< 400 g/l Pb-B = health-based biological limit, WHO) as well as high ones.     

Semaphorin3A signalling is mediated via sequential Cdk5 and GSK3beta phosphorylation of CRMP2: implication of common phosphorylating mechanism underlying axon guidance and Alzheimer's disease

Collapsin response mediating protein-2 (CRMP2) has been identified as an intracellular protein mediating Semaphorin3A (Sema3A), a repulsive guidance molecule. In this study, we demonstrate that cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3beta (GSK3beta) plays a critical role in Sema3A signalling. In In vitro kinase assay, Cdk5 phosphorylated CRMP2 at Ser522, while GSK3beta did not induce any phosphorylation of CRMP2. Phosphorylation by GSK3beta was exclusively observed in Cdk5-phosphorylated CRMP2, but barely in CRMP2T509A. These results indicate that Cdk5 primarily phosphorylates CRMP2 at Ser522 and GSK3beta secondarily phosphorylates at Thr509. The dual-phosphorylated CRMP2, but not non-phosphorylated or single-phosphorylated CRMP2, is recognized with the antibody 3F4, which is highly reactive with the neurofibrillary tangles of Alzheimer's disease. 3F4 recognized the CRMP2 in the wild-type but not cdk5-/- mouse embryonic brain lysates. The phosphorylation of CRMP2 at Ser522 caused reduction of its affinity to tubulin. In dorsal root ganglion neurones, Sema3A stimulation enhanced the levels of the phosphorylated form of CRMP2 detected by 3F4. Over-expression of CRMP2 mutant substituting either Ser522 or Thr509 to Ala attenuates Sema3A-induced growth cone collapse response. These results suggest that the sequential phosphorylation of CRMP is an important process of Sema3A signalling and the same mechanism may have some relevance to the pathological aggregation of the microtubule-associated proteins.     

Immunocytochemical Evidence of Amyloid-enhancing Factor (AEF) in Polymorphonuclear Leukocytes

Amyloid enhancing factor (AEF) was extracted from spleens of mice that had received amyloidogenic stimulation. Sephacryl S 300 gel filtration of the crude AEF yielded five fractions, among which strong AEF activity was present in the first peak (Fl), and confirmed by an amyloid induction experiment. An anti AEF antiserum was obtained from a rabbit by immunization with Fl. This antibody reacted strongly with splenic polymorphonuclear leukocytes (PML) from mice given amyloidogenic stimulation, and weakly with those from normal untreated mice. Isoelectric focusing (IEF) analysis of both Fl and sera from mice given amyloidogenic stimulation was performed. A single band was observed on IEF analysis of Fl, whereas many bands were seen on IEF analysis of the sera. After the substances in the gel had been transferred to nitrocellulose membranes by capillary blotting, the membranes were made to react with the anti-AEF antiserum. The results suggested that AEF is a high molecular-weight substance derived from PML and increases in the serum at the time of, or shortly prior to, amyloid deposition in the spleen. Acta Pathol Jpn 39: 349∼355, 1989.     

Immunohistochemical localization of the secretory products of rat Clara cells

We used proteins in rat lung lavage fluid to successfully produce an antiserum against Clara cell secretory products. When used with the immunoperoxidase method, the antiserum specifically stained cells of the bronchiolar lining, which are morphologically consistent with Clara cells, as well as a few columnar cells in the bronchial and tracheal mucosa. B-5-fixed lung tissue furthermore demonstrated the immunoreactive layer over the bronchiolar epithelium. The alveolar and bronchial lining layers, on the other hand, were not immunoreactive, although a trace of granular immunoreactivity was seen in the latter. It was suggested that Clara cells produce and secrete some proteinaceous materials, which are mainly localized in the bronchiolar area after secretion and are seldom transferred into the alveolar lining layer. Our antibody cross-reacted with the Clara cells of mice, but not with those of hamsters, guinea pigs, rabbits, dogs, cats, monkeys, and man. The high degree of specificity of this antisera to Clara cells in formalin-fixed materials should make it a valuable tool for identifying Clara cell change in non-neoplastic lung pathology and in obtaining some insights into cell origin in neoplastic diseases.     

Relationship between glucose transporter and changes in the absorptive system in small intestinal absorptive cells during the weaning process

In the present study, we investigated the changes in the localization of the glucose transporter GLUT2 and the fructose transporter GLUT5 in small intestinal absorptive cells during postnatal development, especially during the weaning period, using immunohistochemistry and confocal laser scanning microscopy. In the jejunum, GLUT2 was observed within the apical and basolateral membrane domain of absorptive cells, especially in the middle part of the villi. In the suckling rat ileum, GLUT2 was found within the apical and basolateral membrane domain of absorptive cells, but after 18 or 19 days after birth, GLUT2 was found mainly within the apical membrane domain. GLUT5 was observed within the apical membrane domain of absorptive cells in the suckling rat jejunum. In the 18- or 19-day-old rat jejunum, GLUT5 was localized within the apical and basolateral membrane domain of absorptive cells in the lower part of the villi, but after weaning, GLUT5 was found within the apical and basolateral membrane domain of absorptive cells throughout the entire villi. In the suckling rat ileum, there was little GLUT5 in the absorptive cells. In the 18- or 19-day-old rat ileum, GLUT5 was localized within the apical membrane domain of absorptive cells in the lower part of the villi, but after weaning, GLUT5 was observed mainly within the apical membrane domain of absorptive cells throughout the entire villi. These results suggest that the localization of glucose transporters corresponds with a shift from neonatal-suckling to weaned absorptive cells during postnatal development.     

A differential assay of NK-cell-mediated cytotoxicity in K562 cells revealing three sequential membrane impairment steps using three-color flow-cytometry

Annexin V and propidium iodide (PI) staining is a general technique for detecting apoptosis by flow-cytometry (FCM). The release of 2',7'-bis-(2-carboxyethyl)-5- (and-6)-carboxyfluorescein (BCECF), a non-lipophilic membrane-impermeable labeling dye, from the cytoplasm of target cells is an indicator of increased membrane permeability. This study aimed to devise a three-color FCM technique involving the BCECF-release parameter in addition to conventional Annexin V and PI staining for the analysis of target K562 cells undergoing cytotoxic/apoptotic processes mediated by natural killer (NK) cells. The results demonstrated the following step-wise process of membrane impairment: (1) initiation of Annexin V staining accompanied by increasing forward scatter (FSC) before BCECF-release, indicating membrane impairment without permeabilization by necrosis; (2) BCECF-release with decreasing FSC before PI influx; and (3) PI staining with the lowest FSC state. Therefore, the early stage of cytotoxicity/apoptosis conventionally defined by the flow-cytometric criteria of Annexin V staining before PI staining could be sub-divided into two stages before and after BCECF-release. Annexin-V staining in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis was also initiated without BCECF-release. Although the underlying mechanism of the transition process from stage 1 to stage 2 is still unknown, this FCM technique should be a useful tool for differential assays of target cells regarding the sequential processes of NK-induced cytotoxicity.     

Phenytoin promotes Th2 type immune response in mice

The effects of chronic administration of phenytoin, a common anticonvulsive drug, on immune responses were studied in mice. Anti-keyhole limpet haemocyanin (KLH) IgE antibody response after KLH-immunization was enhanced in phenytoin-treated mice. Proliferative responses of spleen cells induced with KLH, concanavalin A (ConA), lipopolysaccharide and anti-CD3 antibody were reduced in phenytoin-treated mice. Accessory function of spleen adherent cells on ConA-induced T cell proliferative response was reduced in phenytoin-treated mice. KLH-induced IL-4 production of spleen cells was enhanced, while IFN-gamma production was reduced in phenytoin-treated mice. In addition, production of IL-1 alpha, but not IL-6 and IL-12 by spleen adherent cells from phenytoin-treated mice was reduced. Natural killer cell activity was reduced in phenytoin-treated mice. These results suggest that phenytoin treatment preferentially induces a Th2 type response. We also observed that plasma ACTH and corticosterone levels were increased in phenytoin-treated mice, and speculated that phenytoin might act directly and indirectly, through HPA axis activation, on the immune system to modulate Th1/Th2 balance.     

Effects of dietary iodine on chemical induction of thyroid carcinoma

Effects of dietary iodine on the induction of thyroid carcinoma using N-nitrosobis(2-hydroxypropyl)amine (BHP) were studied. Male Wistar rats were fed with an iodine-adequate diet (IAD group), an iodine-rich diet (IRD group) and an iodine-deficient diet (IDD group), respectively, until the time of sacrifice. From the 2nd experimental month, animals were injected with BHP once a week for 10 weeks. In the IAD and IRD groups, benign nodules and papillary carcinoma were found. The incidence of rats with benign nodules was 100% in both groups and animals with papillary carcinoma in the IAD and IRD groups comprised 33% and 29%, respectively. The area of the thyroid gland occupied by nodular lesions was much narrower in the IRD group than in the IAD group. In the IDD group, the thyroid showed marked enlargement due to multiple nodular proliferation of follicle cells. The incidence of rats with carcinoma was 100%, and not only papillary but also follicular carcinoma and one pulmonary metastasis were found. As the iodine content of the diet decreased, the nodular lesions increased in width and number, and the incidence of carcinoma in rats became higher. These effects of dietary iodine are probably related to the goitrogenic and/or promoting effects of TSH.     

Agarose for a bioartificial pancreas.

Islets were encapsulated into 5% concentration agarose microbeads. The effect of microencapsulation on islet allograft survivals was determined using a streptozotocin-induced diabetic (STZ) mouse and a nonobese diabetic (NOD) mouse as recipients. All five STZ BALB/c mice receiving microencapsulated islets (C57BL/6) maintained normoglycemia indefinitely. When NOD mice were used as recipients of the bioartificial pancreas, four of five grafts (islets from C3H/He) functioned for more than 80 d. Two of five NOD mice maintained normoglycemia until animals were sacrificed at 102 and 192 postoperative d. Microbeads made of commercially available agarose can effectively prolong alloislets functioning in the STZ-diabetic mouse and even in the NOD mouse (animal model of human type I diabetes) without the use of any immunosuppressive drug.