Immunoglobulin (Ig)G purified from human sera mirrors intravenous Ig human leucocyte antigen (HLA) reactivity and recognizes one's own HLA types, but may be masked by Fab complementarity-determining region peptide in the native sera.

Intravenous immunoglobulin (IVIg) reacted with a wide array of human leucocyte antigen (HLA) alleles, in contrast to normal sera, due possibly to the purification of IgG from the pooled plasma. The reactivity of IgG purified from normal sera was compared with that of native sera to determine whether any serum factors mask the HLA reactivity of anti-HLA IgG and whether IgG purified from sera can recognize the HLA types of the corresponding donors. The purified IgG, unlike native sera, mirrored IVIg reactivity to a wide array of HLA-I/-II alleles, indicating that anti-HLA IgG may be masked in normal sera - either by peptides derived from soluble HLA or by those from antibodies. A < 3 kDa peptide from the complementarity-determining region (CDR) of the Fab region of IgG (but not the HLA peptides) masked HLA recognition by the purified IgG. Most importantly, some of the anti-HLA IgG purified from normal sera - and serum IgG from a few donors - indeed recognized the HLA types of the corresponding donors, confirming the presence of auto-HLA antibodies. Comparison of HLA types with the profile of HLA antibodies showed auto-HLA IgG to the donors' HLA antigens in this order of frequency: DPA (80%), DQA (71%), DRB345 (67%), DQB (57%), Cw (50%), DBP (43%), DRB1 (21%), A (14%) and B (7%). The auto-HLA antibodies, when unmasked in vivo, may perform immunoregulatory functions similar to those of therapeutic preparations of IVIg.

Suppression of blastogenesis and proliferation of activated CD4(+) T cells: intravenous immunoglobulin (IVIg) versus novel anti-human leucocyte antigen (HLA)-E monoclonal antibodies mimicking HLA-I reactivity of IVIg.

Activated CD4(+) T cells undergo blastogenesis and proliferation and they express several surface receptors, including ß2-microglobulin-free human leucocyte antigen (HLA) heavy chains (open conformers). Intravenous immunoglobulin (IVIg) suppresses activated T cells, but the mechanism is unclear. IVIg reacts with HLA-Ia/Ib antigens but its reactivity is lost when the anti-HLA-E Ab is adsorbed out. Anti-HLA-E antibodies may bind to the peptides shared by HLA-E and the HLA-I alleles. These shared peptides are cryptic in intact HLA, but exposed in open conformers. The hypothesis that anti-HLA-E monoclonal antibodies (mAbs) that mimic HLA-I reactivity of IVIg may suppress activated T cells by binding to the shared peptides of the open conformers on the T cell surface was tested by examining the relative binding affinity of those mAbs for open conformers coated on regular beads and for intact HLA coated on iBeads, and by comparing the effects on the suppression of phytohaemagglutinin (PHA)-activated T cells of three entities: IVIg, anti-HLA-E mAbs that mimic IVIg [Terasaki Foundation Laboratory (TFL)-006 and (TFL)-007]; and anti-HLA-E antibodies that do not mimic IVIg (TFL-033 and TFL-037). Suppression of blastogenesis and proliferation of those T cells by both IVIg and the anti-HLA-E mAbs was dose-dependent, the dose required with mAbs 50-150-fold lower than with IVIg. TFL-006 and TFL-007 significantly suppressed blastogenesis and proliferation of activated CD4(+) T cells, but neither the non-IVIg-mimicking mAbs nor control antibodies did so. The suppression may be mediated by Fab-binding of TFL-006/TFL-007 to the exposed shared peptides. The mAb binding to the open conformer may signal T cell deactivation because the open conformers have an elongated cytoplasmic tail with phosphorylation sites (tryosine(320)/serine(335)).

Effect of an isoflavones-containing red clover preparation and alkaline supplementation on bone metabolism in ovariectomized rats.

The aim of this study was to test the combined effect of a quality-controlled red clover extract (RCE) standardized to contain 40% isoflavones by weight (genistein, daidzein, biochanin A, and formononetin present as hydrolyzed aglycones) together with a modified alkaline supplementation on bone metabolic and biomechanical parameters in an experimental model of surgically-induced menopause. Sprague-Dawley female rats were maintained under controlled standard conditions of light and fed with conventional food of standard calcium content and no alfalfa or soybean components. Rats were randomized into four groups: Group A represented normal rats (sham operated) while three other groups were ovariectomized (OVX) and fed for three months as follows: standard food (group B), 6 mg/kg/day food mixed with RCE (Group C), or given 6 mg/kg/day of RCE plus a modified alkaline supplementation (BP) through a nasogastric tube at a dose of 16 mg (group D). The animals were killed 90 days after surgery. As compared to group B, RCE or RCE + BP treatments brought about significantly higher level of estradiol and mitigated the weight loss of the uterus and improved maximum load of the femoral neck. Osteocalcin level showed an over 65% increase in group B but both RCE and RCE + BP treatments prevented such abnormality with a significantly better result in RCE + BP group which virtually normalized such parameter as well as urinary excretion of DPD. Group C and D reduced the over 20% loss of bone mineral density and bone mineral content/body weight ratio observed in untreated post-ovariectomy group. Untreated ovariectomy caused about 48% decrease of cancellous bone mass in the femoral neck while this abnormality was prevented at similar extent by both RCE and RCE + BP treatments. Ovariectomy determined an over 80% increase of bone alkaline phosphatase (BALP) level but both RCE and RCE + BP treatments significantly mitigated such variable. The BALP decrease yielded by the combined RCE + BP treatment was statistically lower than RCE alone. Taken together these data show that red clover preparation in dosages amenable to clinical practice do improve OVX-induced osteoporosis while a mild metabolic alkalosis might further synergize some therapeutic aspects.

Preventive strategy for Candida gut translocation during ischemia-reperfusion injury supervening on protein-calorie malnutrition.

OBJECTIVE: The aim of the present study was to test the hypothesis that protein-calorie malnutrition aggravates the gut translocation of Candida albicans triggered by mesenteric ischemia-reperfusion (IR) injury in an experimental model while testing a natural product containing the antifungal anethole/polygodial mixture (Kolorex). METHODS: MFI strain white mice (n = 90) were randomly allocated to a 4-week dietary regimen: (1) standard pellet diet containing 25% casein; (2) low-protein (2.5%) casein diet; (3) as group 2 plus oral supplementation with 20 microL of a 5% solution of Kolorex during the last 4 days. Twenty rats from each of these groups (termed 1a, 2a and 3a) were orally inoculated with Candida suspension 6 h prior to mesenteric IR injury. Animals of each group but without Candida inoculation (termed 1b, 2b and 3b) served as control. A colon permeability study was carried out as well. Rats were killed prior to the IR injury and 3 h afterwards. Control rats were killed at the same time. RESULTS: Over 60% of the mesenteric lymph nodes and 30% of kidney samples were positive for C. albicans in the low-protein-fed rats after IR injury. Kolorex significantly decreased that rate of positivity and also significantly reduced the concentration of C. albicans per gram of each positive tissue sample examined. Protein-calorie malnourished animals showed a statistically significant increase in colon permeability and this phenomenon further increased after IR injury. The groups of rats treated with Kolorex compound showed a partial, although significant, improvement of this parameter. CONCLUSIONS: These results suggest that Kolorex might exert a competitive effect against with C. albicans colonization. The present study represents the first experimental in vivo investigation of the anethole/polygodial-containing compound under the specific conditions of calorie-protein malnutrition and the results have potential clinical interest.

Metal-induced oxidative damage in cultured hepatocytes and hepatic lysosomal fraction: beneficial effect of a curcumin/absinthium compound.

OBJECTIVE: Metals undergo redox cycling and there is increasing evidence of free radical generation and oxidative injury in the pathogenesis of liver injury and fibrosis in metal storage diseases. The aim of the present study was to test a natural hepatoprotective compound in metal-induced liver injury. METHODS: Hepatocytes were isolated from Wistar rats by collagenase perfusion method and cultured as such and also with alpha-linolenic acid (LNA)-bovine serum albumin (BSA). Hepatocytes were then cultured with a graded dilution of PN-M001 (100 microg/mL and 200 microg/mL), which is a curcuma/absinthium-containing compound, or sylibin (100 microg/mL) dissolved in dimethyl sulfoxide for 10 min before the addition of metallic salts (iron, copper and vanadium). Lysosomal fractions were prepared for lysosome fragility tests in which beta-galactosidase activity and lactate dehydrogenase (LDH) leakage were measured, as well as oxidative damage tests in the presence of hydrophilic and lipophilic free radical generators. Quenching activity by 1,1-diphenyl-2-picrylhydrazyl (DPPH) was also assessed. RESULTS: Malonildialdehyde accumulation in the medium showed a direct time-course increase with incubation time. Both PN-M001 and sylibin showed a significant protective effect against all challenge metal ions, as expressed by the half inhibition concentration (IC(50)) against lipid peroxidation. However, on a molar ratio, sylibin seemed to be more effective than PN-M001 in Fe-induced peroxidative damage (P < 0.05). Both test compounds, irrespective of the concentration, significantly reduced the LDH and beta-galactosidase concentration in the lysosomal fractions. As compared with untreated lysosomal fractions challenged with the two peroxide radicals generators, either PN-M001 or sylibin exerted significant protection However, PN-M001 was significantly better than sylibin in suppressing acid phosphatase enzyme activity. Both compounds showed comparable and significant DPPH radical-scavenging activity. CONCLUSION: These data support the potential clinical application of curcumin-containing compounds.

Single-blind follow-up study on the effectiveness of a symbiotic preparation in irritable bowel syndrome.

OBJECTIVE: Experimental and clinical studies have shown that a novel symbiotic (known as SCM-III) exerts a beneficial effect on gut translocation and local and systemic inflammatory and microbial metabolic parameters. The present investigation was a preliminary trial on the effectiveness of SCM-III for irritable bowel syndrome (IBS). METHODS: Sixty-eight consecutive adult patients with IBS who were free from lactose malabsorption, abdominal surgery, overt psychiatric disorders and ongoing psychotropic drug therapy or ethanol abuse were studied prospectively and divided into 2 groups that were comparable for age, gender, body size, education and pattern of presenting symptoms. The 2 groups were blindly given for 12 weeks either SCM-III 10 mL t.i.d or the same dosage of heat-inactivated symbiotic. RESULTS: Treatment with SCM-III was 'effective' or 'very effective' in more than 80% of the patients (P < 0.01 vs baseline values and control). Less than 5% reported 'not effective' as the final evaluation compared with over 40% of patients in the control group. After 6 weeks of treatment, a significant improvement of pain and bloating was reported in the treatment group compared with control and baseline values. There was also a benefit for bowel habits, mostly for patients with constipation or alternating bowel habits. No overt clinical or biochemical adverse side-effects were recorded. CONCLUSION: Compared with baseline values and the control group, SCM-III resulted in a significant increase in lactobacilla, eubacteria and bifidobacteria, which suggests that some selected IBS patients could benefit substantially from symbiotics, but the treatment may need to be given on a cyclic schedule because of the temporary modification of the fecal flora.

Scanning electron microscopic study of the muscle fiber ends at the myotendinous junction in the posterior cricoarytenoid and cricothyroid muscles in rats.

The fine structural organization of muscle fiber ends at the myotendinous junction in the posterior cricoarytenoid (PCA) and cricothyroid (CT) muscles in adult rats was studied by scanning electron microscopy, after removal of tendon connective tissues using the HCI-hydrolysis method. The muscle fiber ends in the PCA muscle had a relatively simple conical appearance and contained a great number of longitudinal slits on the surface. In the CT muscle, the muscle fiber ends were classified into two types. One type had a conical appearance similar to the PCA muscle with many slits on the surface, while the other type was characterized by a complex arrangement of cylindrical cytoplasmic processes and deep clefts, as seen in common skeletal muscles. These findings suggest that the PCA muscle is evolutionarily primitive, and that the CT muscles represent a transitional form between primitive and evolved muscles.

Scanning electron microscopic study of the neuromuscular junctions of the cricothyroid and thyroarytenoid muscles in rats.

Neuromuscular junctions were observed in the cricothyroid (CT) and thyroarytenoid (TA) muscles of adult rats by scanning electron microscopy after removing the intramuscular connective tissue components using the HCI hydrolysis method. Morphologically, the junctions were classified into three types in the CT muscle and two types in the TA muscle, based on the structural characteristics of the subneural apparatuses, including junctional folds. In the CT muscle, type 1 junctions (32%) consisted of more than 15 cup-like depressions with slit-like junctional folds. Type 2 junctions (20%) were characterized by approximately 10 cup-like depressions with a small number of pit- or slit-like junctional folds. Type 3 junctions (48%) had irregular labyrinthine gutters with slit-like junctional folds. In the TA muscle, type 1 (82%) and 2 (18%) junctions had similar structures to type 1 and 2 junctions in the CT muscle, respectively. Histochemical studies using myosin adenosine triphosphatase staining showed that both CT and TA muscles predominantly consisted of type II muscle fibers (78% and 82%, respectively), and that the diameter of type II fibers was larger than that of type I fibers. These findings suggest that the type 2 junction belongs to type I muscle fibers, while both type 1 and type 3 junctions belong to type II fibers, and that the type 3 junction is a structural variation of the type 1 junction. The significance of the structural differences of the subneural apparatuses in the intrinsic laryngeal muscles is discussed briefly.

Postnatal morphodifferentiation of the subneural apparatuses of the posterior cricoarytenoid muscle in rats: a scanning electron microscopy study.

The subneural apparatus, i. e., the post-synaptic component of the neuromuscular junction, in the posterior cricoarytenoid muscle of the rat was studied by scanning electron microscopy, with special attention given to its postnatal differentiation along with the functional development of the muscle. Primitive synaptic troughs observed in the first postnatal week consisted of single cup-like depressions 5-6 microm in diameter. On the 7th day, low sarcoplasmic ridges appeared in the trough. In the second postnatal week, muscle fibers could be classified into two groups: large (10-15 microm in diameter) and small (less than 10 microm in diameter). In the large muscle fibers, many low ridges became circular and protruded to transform the single trough into numerous cup-like depressions (2-5 microm in diameter). In contrast, the subneural apparatus in the small muscle fibers consisted of a small number of cup-like depressions. The two types of subneural apparatus differentiated into adult forms by the 28th postnatal day, although they remained smaller in size than those of adults. In the large muscle fibers, the number of pit-like or elongated invaginations increased and gradually transformed into slit-like junctional folds by the 28th postnatal day, while the small muscle fibers still possessed a few pit-like or elongated junctional folds at this point in time. The two types of morphodifferentiation of the subneural apparatus are thought to reflect the two types of muscle fibers in the rat posterior cricoarytenoid muscle.

Effect of the FAA1 gene disruption of sake yeast on the accumulation of ethyl caproate in sake mash.

Fatty acid activation gene (FAA1) in sake yeast Kyokai no. 701 (K701) was disrupted to investigate the accumulation of ethyl caproate in sake mash. Ethyl caproate, recognized as an important apple-like flavor in sake, is generated by fatty acid synthesis in yeast cells. The disruptant for the FAA1 gene (K701deltafaa1) exhibited a reduced growth rate in a medium containing cerulenin and myristic acid or oleic acid compared with that of the parental strain (K701). In a sake brewing test in which the rice used was polished to 60% of its original size, the fermentation ability of K701deltafaa1 was inferior to that of K701 but the production of ethyl caproate by K701deltafaa1 was 1.6-fold higher than that by K701. These results suggest that the FAA1 gene in sake yeast plays an important role in sake brewing and the accumulation of ethyl caproate.

Postnatal changes in the types of muscle fibre in the canine inferior pharyngeal constrictor.

Deglutition is considered to be immature in infants and to mature postnatally. We evaluated postnatal changes in muscle fibre type composition in the canine inferior pharyngeal constrictor muscle, which consists of the thyropharyngeal (TP) and cricopharyngeal (CP) muscles, using ATPase staining with respect to the maturation of deglutition. In the TP muscle type IIA and type IIB fibres, the main components in the adult, were already predominant at 1 week postnatally. The percentage of primitive type IIC fibre showed a rapid reduction and reached the adult level within 6 weeks. In the CP muscle, the majority of fibres were type IIC at 2 weeks. At 2 months, more than 20% of the fibres were still type IIC and the proportion of type I fibres as a main component in the adult was smaller than that of the adult. None of the puppies younger than 9 weeks old had a fibre type composition similar to that of the adult. In the extensor digitorum longus and flexor digitorum superficialis, the compositions of muscle fibre types became similar to that of the adult at 6 and 9 weeks of age, respectively. Thus, the TP muscle matured more rapidly than the limb muscles, while the CP muscle matured more slowly. We speculated that the TP and CP muscles have specific individual differentiation patterns associated with their functional roles before and after birth, compared with the limb muscles.

Improvement of isoamyl acetate productivity in sake yeast by isolating mutants resistant to econazole.

Sake yeast strains were improved so as to produce larger amounts of isoamyl acetate than the parental strain by isolating econazole-resistant mutants. Econazole, an imidazole antimycotic, directly interacts with unsaturated fatty acids in the yeast cell membrane, where it also inhibits the synthesis of ergosterol and decreases the ratio of unsaturated to saturated fatty acids. In contrast, alcohol acetyltransferase (AATase), which catalyzes the synthesis of isoamyl acetate, is inhibited by unsaturated fatty acids. Fifty econazole-resistant mutants were isolated from a sake yeast, Kyokai no. 701, several of which produced approximately 1.4 to 2.4 times more isoamyl acetate and an almost equal amount of isoamyl alcohol compared with the parental strain. The AATase activities of the mutants in koji extract were 1.2 to 1.4 times higher, and the unsaturated to saturated fatty acid ratios were lower, than in the parental strain.

Effect of NAD+-dependent isocitrate dehydrogenase gene (IDH1, IDH2) disruption of sake yeast on organic acid composition in sake mash.

The ratio of organic acids in sake mash is a very important factor affecting the taste of alcoholic beverages. To alter the organic acid composition in sake and investigate the mechanism of producing organic acids in sake mash, we examined the effect of NAD+-dependent isocitrate dehydrogenase (IDH) activity deficiency in sake yeast by disrupting the IDH1 or IDH2 gene. Two haploid strains (MATa or MATa genotype) isolated from sake yeast Kyokai no. 701 (K701) were disrupted using the aureobasidin A resistant gene (AUR1-C) as a selection marker. These disruptants were defective in the activity of IDH and failed to grow on medium containing glycerol as a sole carbon source. Sake meter, alcohol concentration, and glucose consumption in sake brewed with the disruptants were reduced in comparison with those of the parental strains. The production of citrate (including isocitrate), malate, and acetate by the disruptants was increased, but succinate production was reduced to approximately half in comparison with the parental strains. These results indicate that approximately half the amount of succinate in sake mash is produced via the oxidative pathway of the TCA cycle in sake yeast. While the diploid strain constructed by mating haploid disruptants for the IDH gene exhibited stronger fermentation ability than the haploid disruptants, almost similar profiles of components in sake were obtained for both strains.

A novel muscle spindle containing muscle fibres devoid of sensory innervation in the posterior cricoarytenoid muscle of the normal adult guinea pig.

A novel muscle spindle composed of five thin intrafusal muscle fibres and four thick muscle fibres was encountered in the posterior cricoarytenoid muscle of the normal adult guinea pig. Four intrafusal muscle fibres of the five were innervated by the annulospiral type of sensory ending, but lacked a distinct aggregation of equatorial nuclei such as bags and chains. On the other hand, one intrafusal fibre and four thick fibres between the layers of the spindle capsule absolutely lacked sensory innervation and were almost similar in the fine structural features to neighbouring extrafusal fibres. These fibres are considered to fail to receive the sensory innervation in an early stage of development, and to retain an undifferentiated feature of intrafusal fibre in the intracapsular space into adulthood or to follow the course of extrafusal differentiation between the layers of the spindle capsule. It is suggested therefore that this muscle spindle may be primitive.

Histochemical study of the canine inferior pharyngeal constrictor muscle: implications for its function.

The inferior pharyngeal constrictor muscle (IPC), which consists of the thyropharyngeal (TP) and cricopharyngeal (CP) muscles. plays an important role during deglutition. The histochemical properties of the canine IPC muscle were investigated. The motor endplates of the TP muscle clustered at the midlength of the muscle, while those of the CP muscle were scattered diffusely. The glycogen depletion technique suggested that most of the CP muscle fibers terminated into the belly of the muscle and fiber lengths varied. With ATPase stain, type II fibers were shown to be predominant in the TP muscle, while type I fibers were predominant in the CP muscle. The diameter of the TP muscle fibers was significantly larger than that of the CP muscle. Although the histochemical characteristics of these two muscles were markedly different, they gradually changed, resulting in their coordinated physiological movements.

Motor innervation of the guinea pig interarytenoid muscle: reinnervation process following unilateral denervation.

The authors investigated the process of denervation and reinnervation of the interarytenoid (IA) muscle in the guinea pig using transmission electron microscopy and glycogen depletion technique after unilateral transection of the recurrent laryngeal nerve (RLN) and superior laryngeal nerve to clarify the innervation pattern of the unpaired IA muscle. Anastomosis between the bilateral arytenoid branches was confirmed in the belly of the IA muscle. Five weeks after transection, all of the IA muscle fibers appeared to have been reinnervated by the contralateral RLN. As the arytenoid branch of the RLN runs together with that of the contralateral RLN in a single intramuscular nerve funiculus, it is possible that collateral sprouting branches grow and extend into the adjacent denervated Schwann's sheaths. The authors conclude that the unpaired IA muscle, as a whole, receives specific motor nerve supply from the bilateral RLNs, although each muscle fiber is innervated unilaterally.

Extravesical tumor implantation caused by perforation during transurethral resection of a bladder tumor: a case report.

We report a case of invasive bladder cancer in which cancer dissemination occurred through a perforation of the vesical wall during transurethral resection of the tumor. A radical cystectomy was performed 1 month later and several clusters of viable cancer cells were histologically identified in a fibrous foreign body granuloma in the paravesicular adipose tissue of the lymphadenectomy specimen. The patient received adjuvant chemotherapy, but developed right inguinal lymph node metastasis 21 months after cystectomy.

[Diagnostic value of magnetic resonance imaging in postoperative maxillary cyst].

Magnetic resonance imaging (MRI) is superior to conventional X-ray tomography and CT scanning in detecting postoperative maxillary cysts (POMCs). We analyzed the MRI features of 51 cases (72 sides) and compared them with the operative findings of 42 cases (52 sides) of POMC. The total number of cysts diagnosed was 121. Multiple cysts were found in 37 sides (51%) and bilateral cysts were found in 21 cases (45%). According to classification of POMC based on location in the maxilla the central cysts were found in only 38% of all cysts (peripheral ones in 62%). In the 42 patients operated on, 69 of 83 cysts which had been detected by MRI were confirmed, whereas there were two additional cysts which could not be diagnosed preoperatively. Sixty four of 71 cysts were opened to the nasal cavity under endonasal endoscopic control. The other seven cysts (five sides) were operated on via a buccogingival incision. Detailed and accurate diagnosis by MRI and development of endoscopic instruments enabled endonasal surgery in most (91%) sides of the POMC. In five cysts of the superior type which are small isolated and distant from the lateral wall of the nasal cavity, endoscopic endonasal surgery was not indicated. In conclusion, MRI for POMC was extremely helpful in selecting a surgical approach.

[Relationship of Onodi cell to optic neuritis--radiological anatomy on coronal CT scanning].

The relationship of paranasal sinusitis to optic neuritis remains controversial. One of the major sources of this controversy is that there are some reports of rhinogenic optic neuritis (RON) in patients with mild paranasal sinusitis or with almost normal paranasal sinuses. The Onodi cell is a posterior ethmoid cell which pneumatized far laterally and to some degree superiorly to the sphenoid sinus and is intimately associated with the optic nerve. Coronal CT scanning is requisite to detect the Onodi cell; when it is present, an image of the sphenoid sinus just as if it were divided into top and bottom is characteristic. In our material from 200 patients (direct coronal CT scans 10 mm in width), the Onodi cell was observed in 7%. A case of RON whose pathogenesis was considered to be a direct spread of inflammation from the localized infection of the Onodi cell is reported. Ethmoiditis localized to the Onodi cell seems to play an important role in the pathogenesis of RON. Continued careful documentation of the localized posterior paranasal sinus lesion around the optic canal by detailed diagnostic imaging and endoscopic sinus surgery is necessary to resolve the disease entity of RON.