Endotoxin contamination in wound dressings made of natural biomaterials.

Contamination by endotoxin of nine kinds of wound dressings made of natural biomaterials (calcium alginate, collagen, chitin, and poly-L-leucine) was examined with the use of water extracts. By applying the Limulus amoebocyte lysate (LAL) test, high concentrations of endotoxin were detected in extracts from three kinds of products made of calcium alginate. These extracts evoked fever in rabbits and induced the release of a proinflammatory (pyrogenic) cytokine, interleukin-6 (IL-6), from human monocytic cells (MM6-CA8). The effects disappeared when the extracts were treated with endotoxin-removing gel column chromatography or with an endotoxin antagonist, B464, confirming that the contaminating pyrogen was endotoxin. A noteworthy finding was that one of the endotoxin-containing extracts showed very weak IL-6-inducibility in human monocytic cells in contrast to its high pyrogenicity to rabbits. The discrepancy could be explained based on differences between humans and rabbits in sensitivity to the endotoxin, because the extract showed higher proinflammatory-cytokine (TNF-alpha)-inducibility in rabbit whole-blood cells (WBCs) than human WBCs. The results suggest that the LAL test is a useful method of detecting endotoxin contamination in wound dressings and the MM6-CA8 assay is a good supplement to the LAL test for evaluating pyrogenicity in humans accurately.

Dose distributions in a human head phantom for neutron capture therapy using moderated neutrons from the 2.5 meV proton-7Li reaction or from fission of 235U.

The feasibility of neutron capture therapy (NCT) using an accelerator-based neutron source of the 7Li(p,n) reaction produced by 2.5 MeV protons was investigated by comparing the neutron beam tailored by both the Hiroshima University radiological research accelerator (HIRRAC) and the heavy water neutron irradiation facility in the Kyoto University reactor (KUR-HWNIF) from the viewpoint of the contamination dose ratios of the fast neutrons and the gamma rays. These contamination ratios to the boron dose were estimated in a water phantom of 20 cm diameter and 20 cm length to simulate a human head, with experiments by the same techniques for NCT in KUR-HWNIF and/or the simulation calculations by the Monte Carlo N-particle transport code system version 4B (MCNP-4B). It was found that the 7Li(p,n) neutrons produced by 2.5 MeV protons combined with 20, 25 or 30 cm thick D20 moderators of 20 cm diameter could make irradiation fields for NCT with depth-dose characteristics similar to those from the epithermal neutron beam at the KUR-HWNIF.

Combinatorial expression patterns of LIM-homeodomain and other regulatory genes parcellate developing thalamus.

The anatomical and functional organization of dorsal thalamus (dTh) and ventral thalamus (vTh), two major regions of the diencephalon, is characterized by their parcellation into distinct cell groups, or nuclei, that can be histologically defined in postnatal animals. However, because of the complexity of dTh and vTh and difficulties in histologically defining nuclei at early developmental stages, our understanding of the mechanisms that control the parcellation of dTh and vTh and the differentiation of nuclei is limited. We have defined a set of regulatory genes, which include five LIM-homeodomain transcription factors (Isl1, Lhx1, Lhx2, Lhx5, and Lhx9) and three other genes (Gbx2, Ngn2, and Pax6), that are differentially expressed in dTh and vTh of early postnatal mice in distinct but overlapping patterns that mark nuclei or subsets of nuclei. These genes exhibit differential expression patterns in dTh and vTh as early as embryonic day 10.5, when neurogenesis begins; the expression of most of them is detected as progenitor cells exit the cell cycle. Soon thereafter, their expression patterns are very similar to those that we observe postnatally, indicating that unique combinations of these genes mark specific cell groups from the time they are generated to their later differentiation into nuclei. Our findings suggest that these genes act in a combinatorial manner to control the specification of nuclei-specific properties of thalamic cells and the differentiation of nuclei within dTh and vTh. These genes may also influence the pathfinding and targeting of thalamocortical axons through both cell-autonomous and non-autonomous mechanisms.

Chemical and biological evaluation of endotoxin contamination on natural rubber latex products.

Relationship between pyrogenicity and bacterial endotoxin contamination on latex products was demonstrated by chemical analysis and biological assays. In commercially available latex products' surveillance, water extracts prepared from one surgical glove and two silicone elastomer-coated Foley catheters sterilized by gamma-irradiation were obviously pyrogenic in rabbits. The induced fever was monophasic at low dose of the pyrogenic extracts and biphasic at high dose. These extracts exhibited limulus amebocyte lysate gelation activity, and induced inflammatory cytokine (interleukin-1, interleukin-6, and tumor necrosis factor-alpha) production from MM6-CA8 human monocytoid cells. These biological properties, including pyrogenicity, completely disappeared by treating the pyrogenic extracts with endotoxin-adsorbent affinity column. Limulus amebocyte lysate activity and cytokine production from MM6-CA8 cells induced by the extracts were significantly decreased by endotoxin inhibitors, an active fragment peptide of an 18-kDa cationic antimicrobial protein and a synthetic lipid A B464 analogue. Furthermore, very small amounts of 2-keto-3-deoxyoctonate and 3-hydroxy fatty acid, which are common constituents of bacterial endotoxins, were detected by gas chromatography-mass spectrometry analysis of the pyrogenic extracts. These findings clearly showed that the pyrogenicity found in these latex products originated from endotoxins contaminating the products.

yam8(+), a Schizosaccharomyces pombe gene, is a potential homologue of the Saccharomyces cerevisiae MID1 gene encoding a stretch-activated Ca(2+)-permeable channel.

The Saccharomyces cerevisiae MID1 gene encodes a stretch-activated Ca(2+)-permeable channel. In a protein database, we found a Schizosaccharomyces pombe gene whose predicted protein shows 26% identical and 62% similar to the Mid1 channel in amino acid sequence. cDNA derived from this gene, designated yam8(+), was isolated by reverse transcription-polymerase chain reaction (RT-PCR). Further analysis showed that the Yam8 protein consists of 486 amino acids and has 6 hydrophobic segments. The yam8(+) cDNA, placed under the S. cerevisiae TDH3 promoter, partially complemented the mating pheromone-induced death (mid) phenotype of the S. cerevisiae mid1 mutant. The expression of the yam8(+) cDNA in the mid1 mutant cells partially remediated the mid phenotype and resulted in a slight increase in Ca(2+) uptake activity. These findings suggest that Yam8 is a potential homologue of Mid1.

Efficacy of yeast phytase in improving phosphorus bioavailability in a corn-soybean meal-based diet for growing pigs.

Crossbred barrows (n = 66; 6 wk old) were used in a 6-wk experiment to evaluate the efficacy of phytase from yeast or Aspergillus niger on performance, tibial characteristics, and serum inorganic P concentration. We also investigated the stability of these phytases in acidic solutions with pepsin, which simulated gastric conditions. Pigs were fed a P-adequate diet containing .34% nonphytate-P or a low-P diet containing .20% nonphytate-P. The low-P diet was supplemented with 0, 1,000, 2,000, or 4,000 phytase units (PU; the activity at optimal pH, i.e., pH 4.2 for yeast phytase and pH 5.5 for phytase from Aspergillus niger)/kg of yeast phytase, or 1,000 PU/kg phytase from Aspergillus niger. The graded level of yeast phytase linearly increased ADG (P = .047), tibial weight (P = .091), tibial density (P < .001), and P concentration in tibial cortex (P = .018). Aspergillus niger phytase also increased ADG (P = .022), serum inorganic P concentration (P < .001), tibial density (P = .007), and tibial P concentration (P = .025). The pigs given 1,000 PU/kg Aspergillus niger phytase showed greater ADG (P = .091), tibial density (P= .001), and tibial P concentration (P = .062) than those given 1,000 PU/kg yeast phytase. No measurements differed (P > .31) between the pigs given 1,000 PU/kg Aspergillus niger phytase and those given 4,000 PU/kg yeast phytase. These results suggested that yeast phytase improves bioavailability of P in the diet for growing pigs but the efficacy of yeast phytase is less than that of Aspergillus niger phytase. During incubation in acidic solutions with pepsin, yeast phytase (P < .001) lost more of its activity than Aspergillus niger phytase. This lesser stability of yeast phytase may be responsible for the poorer efficacy of yeast phytase than that of Aspergillus niger. In summary, supplementation of swine diets with yeast phytase is beneficial, but its efficacy is less than that of Aspergillus niger phytase.

Graded and areal expression patterns of regulatory genes and cadherins in embryonic neocortex independent of thalamocortical input.

The differentiation of areas of the mammalian neocortex has been hypothesized to be controlled by intrinsic genetic programs and extrinsic influences such as those mediated by thalamocortical afferents (TCAs). To address the interplay between these intrinsic and extrinsic mechanisms in the process of arealization, we have analyzed the requirement of TCAs in establishing or maintaining graded or areal patterns of gene expression in the developing mouse neocortex. We describe the differential expression of Lhx2, SCIP, and Emx1, representatives of three different classes of transcription factors, and the type II classical cadherins Cad6, Cad8, and Cad11, which are expressed in graded or areal patterns, as well as layer-specific patterns, in the cortical plate. The differential expression of Lhx2, SCIP, Emx1, and Cad8 in the cortical plate is not evident until after TCAs reach the cortex, whereas Cad6 and Cad11 show subtle graded patterns of expression before the arrival of TCAs, which later become stronger. We find that these genes exhibit normal-appearing graded or areal expression patterns in Mash-1 mutant mice that fail to develop a TCA projection. These findings show that TCAs are not required for the establishment or maintenance of the graded and areal expression patterns of these genes and strongly suggest that their regulation is intrinsic to the developing neocortex.

Reduced crystallization inhibition by urine from men with nephrolithiasis.

BACKGROUND: Human urine is known to inhibit growth, aggregation, nucleation, and cell adhesion of calcium oxalate monohydrate (COM) crystals, the main solid phase of human kidney stones. This study tests the hypothesis that low levels of inhibition are present in men with calcium oxalate stones and could therefore promote stone production. METHODS: In 17 stone-forming men and 17 normal men that were matched in age to within five years, we studied the inhibition by dialyzed urine proteins of COM growth, aggregation, and binding to cultured BSC-1 renal cells, as well as whole urine upper limits of metastability (ULM) for COM and calcium phosphate (CaP) in relationship to the corresponding supersaturation (SS). RESULTS: Compared with normals, patient urine showed reduced COM growth inhibition and reduced ULM in relationship to SS. When individual defects were considered, 15 of the 17 patients were abnormal in one or more inhibition measurements. ULM and growth inhibition defects frequently coexisted. CONCLUSIONS: Reduced COM growth and CaP and CaOx ULM values in relationship to SS are a characteristic of male stone formers. Both defects could promote stones by facilitating crystal nucleation and growth. Abnormal inhibition may be a very important cause of human nephrolithiasis.

Dural arteriovenous malformation manifesting as dementia due to ischemia in bilateral thalami. A case report.

BACKGROUND: Although dementia is one of the curable manifestations of a dural arteriovenous malformation (AVM), the pathophysiology remains unclear. CASE DESCRIPTION: We describe an elderly patient who had an AVM in the tentorium and manifested signs of dementia from ischemia, predominantly in the bilateral thalami. Intravascular embolization of the dural AVM resulted in amelioration of signs of dementia, and this improvement was consistent with that of neuroradiological and hemodynamic conditions in the thalami. CONCLUSION: The coincidental improvement of CBF in the thalami and signs of dementia after embolization of the AV shunt supports the concept of primary participation of the thalami in the pathophysiology of dementia of vascular origin in our patient.

Defects in thalamocortical axon pathfinding correlate with altered cell domains in Mash-1-deficient mice.

We have analyzed the pathfinding of thalamocortical axons (TCAs) from dorsal thalamus to neocortex in relation to specific cell domains in the forebrain of wild-type and Mash-1-deficient mice. In wild-type mice, we identified four cell domains that constitute the proximal part of the TCA pathway. These domains are distinguished by patterns of gene expression and by the presence of neurons retrogradely labeled from dorsal thalamus. Since the cells that form these domains are generated in forebrain proliferative zones that express high levels of Mash-1, we studied Mash-1 mutant mice to assess the potential roles of these domains in TCA pathfinding. In null mutants, each of the domains is altered: the two Pax-6 domains, one in ventral thalamus and one in hypothalamus, are expanded in size; a complementary RPTP(delta) domain in ventral thalamus is correspondingly reduced and the normally graded expression of RPTP(delta) in that domain is no longer apparent. In ventral telencephalon, a domain characterized in the wild type by Netrin-1 and Nkx-2.1 expression and by retrogradely labeled neurons is absent in the mutant. Defects in TCA pathfinding are localized to the borders of each of these altered domains. Many TCAs fail to enter the expanded, ventral thalamic Pax-6 domain that constitutes the most proximal part of the TCA pathway, and form a dense whorl at the border between dorsal and ventral thalamus. A proportion of TCAs do extend further distally into ventral thalamus, but many of these stall at an aberrant, abrupt border of high RPTP(delta) expression. A small proportion of TCAs extend around the RPTP(delta) domain and reach the ventral thalamic-hypothalamic border, but few of these axons turn at that border to extend into the ventral telencephalon. These findings demonstrate that Mash-1 is required for the normal development of cell domains that in turn are required for normal TCA pathfinding. In addition, these findings support the hypothesis that ventral telencephalic neurons and their axons guide TCAs through ventral thalamus and into ventral telencephalon.

Epidemiology of canine calcium oxalate uroliths. Identifying risk factors.

Calcium oxalate uroliths are most commonly encountered in Miniature Schnauzers, Lhaso Apsos, Yorkshire Terriers, Bichons Frises, Shih Tzus, and Miniature Poodles. They are more common in males than females, and more common in older than young dogs. Dogs that form abnormal nephrocalcin are also predisposed to calcium oxalate uroliths. Dietary risk factors for calcium oxalate uroliths include excessive calcium supplementation or excessive calcium restriction, excessive oxalic acid, high protein, high sodium, restricted phosphorus, restricted potassium, and restricted moisture (dry formulations). Dogs with hyperadrenocorticism or hypercalcemia are predisposed to calcium oxalate urolith formation.

Ondine's curse and Hirschsprung's disease: neurocristopathic syndrome.

We report a female newborn with Ondine's curse and Hirschsprung's disease--neurocristopathic syndrome. The female infant required endotracheal intubation and mechanical ventilation due to apnea which developed soon after birth. She had abdominal distension with bilious vomiting. A barium enema revealed a caliber change at the rectum and rectal biopsies showed no ganglion cells. Colostomy was performed at the age of 17 days. Hypoxemia with hypercapnia was noted during her sleep, and tracheostomy was performed at the age of 55 days. In addition, deafness and pupillary autonomic dysfunction were observed. The definitive surgery for Hirschsprung's disease was performed at the age of 4 months. She is now 2 years old with normal growth but needs ventilator support at home. In this case, we detected no mutation in the RET gene and EDNRB gene.

[Combination chemotherapy (CVP) in a patient with refractory idiopathic thrombocytopenic purpura (ITP)].

A 54-year-old woman, who had been given a diagnosis of idiopathic thrombocytopenic purpura (ITP) refractory to steroid therapy, was admitted to our hospital because of severe bleeding tendency. Splenectomy, high dose vitamin-C and interferon-alpha were not effective, although high-dose gamma-globulin had some effect. Since high-dose glucocorticoid was effective temporarily, we decided to perform chemotherapy. Seven courses of CVP chemotherapies (CVP; CPM 500 mg, VCR 2 mg, PSL 50 mg) prevented severe bleeding and did not have serious toxicity. Chemotherapy can be indicated for refractory ITP reactive to immunosuppressive therapy such as high-dose glucocorticoid.

Long-term follow-up of a girl with the neonatal form of Bartter's syndrome.

We followed up a girl with the neonatal form of Bartter's syndrome for sixteen years and determined the sensitivity to angiotensin II before and during the indomethacin treatment. A 4-month-old girl was admitted to our hospital, because of severe hypokalemia and growth retardation. Initially we treated her with spironolactone and potassium supplements. This treatment increased plasma potassium levels and her growth. At the age of one year she was diagnosed as having Bartter's syndrome. Since then she has been treated with indomethacin at an initial dose of 3 mg/kg/day combined with spironolactone and potassium. After the start of the indomethacin treatment, her growth increased dramatically, and her final height was normal adult height. Her puberty developed normally and menarche occurred at the age of 12 years. Levels of serum sodium, chloride, plasma aldosterone and urinary prostaglandin E2 were also normalized. Levels of angiotensin I and II were improved but not within the normal range, but plasma potassium levels slightly decreased after plasma aldosterone levels were normalized and did not change during the treatment period. Plasma renin activity remained high until about the age of 8 years, after which it decreased to almost within the normal range. At 5 months after the start of indomethacin (3 mg/kg/day), her vascular sensitivity to angiotensin II had been improved, and after 2 years and 5 months, her vascular sensitivity was further improved. At this time renin activity had decreased after angiotensin II infusion, but plasma aldosterone did not change. At the age of 16 years (dose of indomethacin: 0.5 mg/kg/day), plasma aldosterone increased after angiotensin II infusion. These data suggest that indomethacin and spironolactone are effective treatments for the neonatal form of Bartter's syndrome, especially during childhood.

[A case of predominant right thalamic infarction with severe verbal memory disturbance].

We encountered a 45-year-old right-handed man who had suffered a predominant right thalamic infarction and complained of memory loss. Performance on the Miyake Test (recall of ten pairs of related and unrelated words), the Rey Osterrieth Complex Figures, the Benton Test of Visual Retention and the Wechsler Memory Scale-R disclosed a severe verbal memory disturbance associated with a little, if any, visual memory disturbance. An MRI study revealed bilateral lesions limited to the thalamus involving most of the right anterior nucleus (AN), mediodorsal nucleus (MD), ventrolateral nucleus (VL), and centromedial nucleus (CM), as well as a small part of the left MD, and CM. HM-PAO-SPECT scans showed areas of decreased cerebral blood flow not only in the right thalamus but in the medial and basilar region of the right temporal lobe. It is noteworthy that our patient had a predominant right thalamic lesion and exhibited a severe verbal memory disturbance rather than visual memory disturbance. This suggests that the right hemisphere is dominant for verbal memory function in this patient.

Protein tyrosine kinase Syk is associated with and activated by the IL-2 receptor: possible link with the c-myc induction pathway.

The IL-2 receptor (IL-2R) consists of three subunits, the IL-2R alpha, IL-2R beta, and IL-2R gamma chains. The IL-2-induced proliferative signals emanate from the cytoplasmic domains of IL-2R beta and IL-2R gamma, but the nature and function of the signaling molecules that transmit these signals are not fully understood. Here, we report that Syk protein tyrosine kinase (PTK) is physically associated with IL-2R in peripheral blood lymphocytes. cDNA expression studies further revealed that this association is critical for the IL-2-induced activation of Syk PTK, which occurs primarily via the serine-rich region of the IL-2R beta chain, which is essential for proliferative signal transmission. Furthermore, we provide evidence that in the hematopoietic cell line, BAF-B03, the activation of Syk PTK results in the induction of the c-myc gene, an event critical for the cell proliferation. Thus, Syk PTK may be a critical integral member of the signaling molecules engaged by the IL-2R.

Functional activation of Jak1 and Jak3 by selective association with IL-2 receptor subunits.

The interleukin-2 receptor (IL-2R) consists of three subunits: the IL-2R alpha, IL-2R beta, and IL-2R gamma chains, the last of which is also used in the receptors for IL-4, IL-7, and IL-9. Stimulation with IL-2 induces the tyrosine phosphorylation and activation of the Janus kinases Jak1 and Jak3. Jak1 and Jak3 were found to be selectively associated with the "serine-rich" region of IL-2R beta and the carboxyl-terminal region of IL-2R gamma, respectively. Both regions were necessary for IL-2 signaling. Furthermore, Jak3-negative fibroblasts expressing reconstituted IL-2R became responsive to IL-2 after the additional expression of Jak3 complementary DNA. Thus, activation of Jak1 and Jak3 may be a key event in IL-2 signaling.

On the mechanisms of 3-tert-butyl-4-hydroxyanisole- and its metabolites-induced cytotoxicities in isolated rat hepatocytes.

The cytotoxic effects of 3-tert-butyl-4-hydroxyanisole (BHA) and its metabolites, 3-tert-butylhydroquinone (tBHQ) and 3-tert-butyl-4,5-dihydroxyanisole (BHA-OH), were investigated in freshly isolated rat hepatocytes. These compounds caused a time-dependent cell death accompanied by loss of intracellular ATP, glutathione (GSH) and protein thiols at concentration of 0.5 mM. Supplementation of the hepatocyte suspension with 5 mM N-acetylcysteine, a precursor of intracellular GSH, significantly delayed the onset of cytotoxicity induced by BHA-OH and tBHQ; the loss of intracellular ATP, GSH and protein thiols was also prevented. Although N-acetylcysteine did not affect BHA disappearance in the cell suspension, disappearance of tBHQ and formation of tBHQ-GSH conjugate were stimulated by N-acetylcysteine. In addition, N-acetylcysteine prevented BHA-OH disappearance and 3-tert-butyl-5-methoxy-1,2-benzoquinone (BHA-Q) formation. In isolated hepatic mitochondria, BHA, tBHQ and BHA-OH impaired respiration related to oxidative phosphorylation; tert-butylquinone (tBQ) and BHA-Q, quinones derived from tBHQ and BHA-OH, resulted in the significant inhibition of mitochondrial respiration. These results indicate that BHA-OH is the most cytotoxic followed by tBHQ and BHA and that protein thiols and mitochondrial respiratory system are important targets for BHA and its intermediates.

Immunohistochemical demonstration of calcium-dependent cysteine proteinase (calpain) in collagen-induced arthritis in mice.

The mechanisms whereby destructive changes in joints develop in rheumatoid arthritis (RA) are not fully understood, but cartilage degradation is considered one of the most important factors. Calpain, a calcium-dependent cysteine proteinase that degrades a variety of substrate proteins in various tissues, may be among the proteolytic enzymes that mediate cartilage degradation associated with RA and other inflammatory arthritides. The object of this study is to ascertain immunohistochemically whether calpain is present in the inflamed joints of mice with collagen-induced arthritis (CIA), as an experimental animal model of inflammatory arthritis such as RA. Murine hindpaws with CIA were decalcified, frozen-sectioned, and stained by immunoperoxidase method (PAP) using a polyclonal antibody against calpain II. Calpain was present in the articular tissues affected by CIA: positive staining was observed in 1) proliferating synovia, and 2) surface, matrix, and chondrocyte lacunae of articular cartilage. It was postulated that calpain functions as one of the enzymes responsible for the cartilage degradation associated with CIA.