Trigeminal primary afferent terminals containing substance P－like immunoreactivity in the paratrigeminal nucleus

With the methods of transganglionic transport of horseradish peroxidase(HRP) combined with postembedding colloidal gold stain immunoelectron microscopy, the shape and synaptic relationship of the trigeminal primary afferent terminals containing substance

Malondialdehyde treatment reduced immunoreactivity of amandin and delayed its digestion

Objectives:The secondary products of lipid oxidation are one of the main factors inducing protein oxidation.The effects of oxidation treatment with malondialdehyde(MDA)on the immunoreactivity of amandin and its digestion were studied.Materials and Methods:The rabbit IgG binding ability of amandin was analyzed by western blotting,and the changes in amandin oxidation and immunoreactivity during digestion of amandin with different degrees of oxidation were investigated in combination with an almond allergen enzyme-linked immunosorbent assay kit.Alteration of linear epitopes of amandin by oxidation was investigated by liquid chromatography-tandemmass spectrometry(LC-MS/MS).Results:The results showed that the immunoreactivity of amandin was significantly reduced after 1 mmol/L MDA and 100 mmol/L MDA treat-ment.However,the 1 mmol/L MDA treatment was owing to cleavage of linear epitope peptide in amandin and oxidation of the active amino acid.The 100 mmol/L MDA treatment was due to aggregation of amandin and significant decrease in its solubility.Oxidation also reduced di-gestibility of amandin and significantly affected immunoreactivity during digestion.LC-MS/MS also identified four oxidation-prone methionine sites(aa 264-274,298-308,220-240,and 275-297)in gamma conglutinin 1.Conclusions:MDA treatment reduced the immunoreactivity of amandin.MDA treatment also led to protein aggregation,which slowed down the digestion of amandin and altered the immunoreactivity of amandin during digestion.

Comparison of tyrosine hydroxylase immunoreactivity in cochleas of chinchilla and guinea pig

Objective To investigate the distribution of the tyrosine hydroxylase (TH) immunoreactivity in the cochleas of chinchilla and compare it with guinea pig. Methods Ten healthy chinchillas and six healthy guinea pigs were used in this study. The surface preparation of cochleas were obtained by microdissection. The TH like immunoreactivity on the surface preparation as examined by the avidin and biotinylated horseradish peroxidase macromolecular complex (ABC) technique and investigated with light microscopy. Results TH like immunoreactivity was seen in the region from modiolus to the habenula perforata. There are two different groups of fibers in this aera, one is blood vessel dependent, the other is blood vessel independent system. No TH like immunoreactivity was present in the inner spiral bundle (ISB) and the tunnel spiral bundle (TSB) regions in chinchilla cochleas. TH like immunoreactivity was found in the region from modiolus to the habenula perforata and the ISB and TSB regions in the organ of corti (OC) in guinea pig cochleas. Conclusions The presence of TH immunoreactivity on the cochlea of chinchilla and guinea pig is different. TH like immunoreactivity on chinchilla cochlea was involved only in the fibers corresponding to the sympathetic fibers on cochlea of guinea pig. No fibers corresponding to the dopaminergic olivocochlear lateral efferent system of guinea pig were found in ISB and TSB within the OC in chinchilla.

Impact of three different processing methods on the digestibility and allergenicity of Chinese mitten crab(Eriocheir sinensis)tropomyosin

The tropomyosin(TM)fractions of crab protems may cause allergic reactions in mdividuals susceptible to allergies;however,efficient and safe methods by which to reduce such allergenicity are not currently available.Therefore,in this study,the effects of three different processing methods,i.e.,microwave,ultrasound,and high temperature-pressure(HTP)treatments,on the digestion stability of TM from Chinese mitten crab muscle and the allergenicity of TM digestion products were explored.sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that microwaving had little effect on the digestion stability of TM.In contrast,ultrasound and HTP treatments facilitated the degradation of TM.Similarly,Western blotting and inhibition ELISA indicated that the IgE-binding activity of TM was significantly reduced after treatment with ultrasound or HTP.Among the three different proces sing methods,HTP was the most effective method for improving digestibility of TM and reducing immunoreactivity.This finding provides new insights into treatments for crab allergies.

Estrogen affects neuropathic pain through upregulating N-methyl-D-aspartate acid receptor 1 expression in the dorsal root ganglion of rats

Estrogen affects the generation and transmission of neuropathic pain,but the specific regulatory mechanism is still unclear.Activation of the N-methyl-D-aspartate acid receptor 1（NMDAR1） plays an important role in the production and maintenance of hyperalgesia and allodynia.The present study was conducted to determine whether a relationship exists between estrogen and NMDAR1 in peripheral nerve pain.A chronic sciatic nerve constriction injury model of chronic neuropathic pain was established in rats.These rats were then subcutaneously injected with 17β-estradiol,the NMDAR1 antagonist D（-）-2-amino-5-phosphonopentanoic acid（AP-5）,or both once daily for 15 days.Compared with injured drug na？ve rats,rats with chronic sciatic nerve injury that were administered estradiol showed a lower paw withdrawal mechanical threshold and a shorter paw withdrawal thermal latency,indicating increased sensitivity to mechanical and thermal pain.Estrogen administration was also associated with increased expression of NMDAR1 immunoreactivity（as assessed by immunohistochemistry） and protein（as determined by western blot assay） in spinal dorsal root ganglia.This 17β-estradiol-induced increase in NMDAR1 expression was blocked by co-administration with AP-5,whereas AP-5 alone did not affect NMDAR1 expression.These results suggest that 17β-estradiol administration significantly reduced mechanical and thermal pain thresholds in rats with chronic constriction of the sciatic nerve,and that the mechanism for this increased sensitivity may be related to the upregulation of NMDAR1 expression in dorsal root ganglia.

Age-related Variation in Snake Venom: Evidence from Two Snakes (Naja atra and Deinagkistrodon acutus) in Southeastern China

In this study we explored electrophoretic profiles, enzymatic activities and immunoreactivity of neonate and adult venoms from two snakes （Naja atra and Deinagkistrodon acutus） coexisting in southeastern China. Age-related variation in electrophoretic profiles was found in both species and proteolytic and fibrinogenolytic activity was higher in neonate than adult venoms. Neonate D. acutus venom had higher 5＇ nucleotidase, PLA2, hyaluronidase and gelatinolytie activity, but lower esterolytic activity, than adult venom. Neonate and adult D. acutus venoms showed identical phosphomonoesterase, LAO and fibrinolytic activities. Neonate N. atra venom had higher phosphomonoesterase and LAO activity, but lower 5＇ nucleotidase, PLA2, hyaluronidase and Ache activities than adult venom. Neonate and adult N. atra venoms showed similar gelatinolytic activity. Further, age-dependent immunoreactivity was found in both species, and cross-reactions between homologous venoms and antiserums were closely related to venom composition. We speculate that age-related variation in venom characteristics is possibly driven by evolutionary forces associated with ontogenetic shifts in dietary habits, competition and predation pressure.

血清放射免疫胰岛素和真胰岛素测定对胰岛β细胞功能评价(英文)

Objective:To study the changes of true insulin(TI) and immunoreactive insulin (IRI) in subjects with NGT, IGT and DM, to study the difference between true insulin and immunoreactive insulin in evaluating βcell function and insulin sensitivity. Methods:The levels of serum IRI and TI were determined in 54 cases with type Ⅱ diabetes mellitus(Group DM) ,43 cases with impaired glucose tolerance (Group IGT) and 75 cases with normal glucose tolerance (Group NGT). Then every group was subdivided into obese and non - obese subgroups according to body mass index. IRI was determined by RIA. TI was determined by ELISA using monoclonal antibody with no significant cross- reaction between insulin and proinsulin. The insulin resistance index (Homa- IR), pancreatic β- cell function index (Homa- B) and insulin release index (the ratio of the increment of insulin to that of plasma glucose 30 min after a glucose load,△ I30/△G30) were analyzed preliminarily. Results: The ratio of TI/IRI in non - obese subgroup with DM was lower than those in non obese subgroups with NGT and IGT (P＜0.01). The ratios of TI/Ipd in obese subgroups with IGT and DM were lower than those in obese subgroup with NGT(P＜0.05). The pancreatic β-cell function indexes (Homa-BIRI) of IRI in non- obese subgroups with IGT and DM were lower than that in non- obese subgroup with NGT(P＜0.05). The pancreatic β - cell function index (Homa- BTI) of TI in non- obese subgroup with IGT was lower than that in non obese subgroups with NGT ( P ＜ 0.05 ). The HomaBTI in non - obese subgroup with DM was lower than that in non - obese subgroups with NGT and IGT(P ＜0.01).The Homa - BIRI in obese subgroups of NGT, IGT and DM had no significant difference. Homa - BTI in obese subgroups with IGT and DM was lower than that in obese subgroup with NGT(P＜0.01). Conclusion:The ratio of TI/IRI was a good marker in evaluating β- cell function. Pancreatic β- cell function index (Homa- BTI) of TI was better than that of IRI.

In Vitro Refolding Process of Bovine Allergen β-lactoglobulin by Multispectroscopic Method

Objective To characterize the relationship between the refolding process of recombinant bovine β-1actoglobulin and its immunoreactivity for clinical purposes. To establish a spectral method which examine the extent of recombinant allergen renaturation. Methods The refolding process of recombinant bovine β-1actoglobulin was investigated by using circular dichroism, fluorescence and synchronous fluorescence spectra. IgE-binding capacity of recombinant protein was analyzed by ELISA. In addition, bioinformatic methods were used to explain the spectral characteristics and analyze the relationship between the conformational changes and the immunoreactivity of the protein during renaturation in vitro. Results Renaturation of recombinant bovine β-1actoglobulin resulted in a more compact structure resembling the natural counterpart with stronger IgE-binding capacity. Conclusion The degree of protein renaturation Results from this study may be of help for food future. correlated with the IgE-binding capacity of the protein. allergy therapy and development of vaccination in the

EXPRESSION OF GALECTIN-3 IN GASTRIC CARCINOMA AND ITS SIGNIFICANCE

Objective: To study the expression of Galectin-3 in gastric cancerous tissues and the relationship between Galectin-3 expression and biological behave of gastric carcinoma. Methods: S-P immunohistochemistry method was used to examine Galectin-3 expression in 64 primary gastric cancerous tissues and 10 normal gastric mucosa as control. The expression of Galectin-3 was examined by HPIAS- 2000 image analysis software. Results: Positive Gaiectin-3 expression was observed in 85.9% of the gastric cancer cases. In gastric cancerous tissues, about 46% of cases showed strong nuclear immunoreactivity. The degree of enhancement of immunoreactivity was different in various histopathological subtypes in cancerous tissues. The readings of average absorbency of Galectin-3 in well, moderate and poorly differentiated adenocarcinoma tissues were 0.17130.02147, 0.18730.0313 and 0.25380.1216 respectively. A stronger expression of Galectin-3 in cancerous tissues was observed. When Galectin-3 expression in gastric cancerous tissues was compared with that in normal gastric tissues, there was a significant difference (P<0.001). Conclusion: A significantly stronger expression of Galectin-3 in gastric cancerous tissues was observed. Galectin-3 might be a useful tumor marker for gastric carcinomas with respects to tumor metastasis, proliferation, cancer progression, and tumor cell adhesion.

Human Seroreactivity to Secreted Molecules of Corynebacterium pseudotuberculosis

Corynebacterium pseudotuberculosis is an infectious agent that occurs in small ruminants causing caseous lymphadenitis, and more rarely in humans causing lymphadenitis and pneumonia. The breeding small ruminants have great economic importance in Brazil. Rural farm workers and veterinary students who acquired this disease suffered from weakening symptoms for weeks, and the identification of the etiological agent was time-consuming and complex. Due to the low prevalence of case records, there is probably no available commercial diagnostic kit for C. pseudotuberculosis infection in humans. This study aimed to describe human seroreactivity to secreted antigens from C. pseudotuberculosis. Reactivity of serum from farm workers (n = 14), individuals who work with the bacillus at laboratory (n = 8) or individuals without contact (n = 25) was tested with secreted proteins from PAT10 strain of C. pseudotuberculosis by Western blotting. Samples of all (100%) farm workers showed reactivity to 31 kDa, 71 kDa and 164 kDa proteins, while laboratory workers showed 87.5%, 62.5 % and 37.5%, and no-contact 20%, 0% and 16%, respectively. All sera recognized the 275 kDa protein. Our data suggest that C. pseudotuberculosis secreted proteins are antigenic in humans and the recognition profiles allowed the identification of individuals with and without prior contact with this bacillus. This is the first paper which describes human reactivity to C. pseudotuberculosis in serum samples of workers in Brazil.

Purified Wheat Gliadin Proteins as Immunoglobulin E Binding Factors in Wheat Mediated Allergies

Some wheat gliadin proteins are strong allergens that may cause various symptoms of food allergies and baker’s asthma. The most immunoreactive ω-5 gliadin fractions are the main allergens in wheat dependent exercise induced anaphylaxis (WDEIA). While the allergenicity of ω-5 is quite well understood, knowledge about α, β, γ and ω-1.2 gliadins is much more scanty. This study examines allergenic properties of other fractions as compared to ω-5. Gliadins were extracted from flour of winter wheat (Triticum aestivum L.) cultivar Ostka strzelecka. Purified samples representing proteins belonging to α, β, γ, ω-1.2 and ω-5 classes were isolated using preparative gel electrophoresis. Immuno-reactivity and allergenic properties of these proteins were analyzed by ELISA using sera from allergic patients with elevated sIgE (> 2KU/L), and by skin prick test (SPT). ELISA showed that ω-5 and ω-1.2 differed considerabely from α-, β- and γ-gliadins in respect of immunoreactivity. Responses of both ω-gliadins were almost twice as high as for other fractions. Significant differences were also observed among individual ω-gliadin fractions as evidenced by ANOVA. SPT showed that patient with symptoms of baker’s asthma and WDEIA had a positive results to all gliadins tested. Another patient with baker’s asthma (but not WDEIA) reacted positively only to ω-5 gliadins. In two patients with skin allergy SPT were negative with all analyzed proteins. Results show ω-1.2 gliadins to be almost as immunorective as ω-5. The α-, β- and γ-gliadins also recognize specific IgE antibodies, but their binding capacity is only about half that of ω-fractions. This kind of immunoreactivity could still be important since a cumulative effect of individual fractions may intensify disease symptoms in allergic patients.

Changes in Behavior and Amino Acid Neurotransmitters in the Brain of Rats with Seizure Induced by IL-1β or IL-6

To explore the mechanism of epilepsy induced by IL-1β and IL-6, the changes of glutamic acid (Glu) and GABA immunoreaction in the cerebral cortex and hippocampus of rats with seizure induced by IL-1β or IL-6 were studied. Rats were randomly divided into 3 groups: control group (intracerebroventricular injection (icv) of NS), IL-1β group (icv injection of IL-1β) and IL-6 group (i.c.v. injection of IL-6). 120 min after the icv injection of reagents of IL-1β or IL-6, behavioral changes were observed and Glu and GABA in the cerebral cortex and hippocampus were examined by means of immunohistochemistry. Our results showed that no seizure developed in the control group, while moderate seizure was observed in IL-1β group and IL-6 group. Compared with the controls, the immunoreaction of Glu was significantly increased, while GABA was obviously decreased in IL-1β group and IL-6 group after 120 min. Our study suggested that the IL-1β and IL-6 might promote and induce epilepsy by increasing Glu and decreasing GABA in the cerebral cortex and hippocampus.

Serum cytokine profile in patients with hepatitis B e antigen-negative chronic active hepatitis B and inactive hepatitis B virus carriers

An insufficient cellular immune response seems to be critical for the immunopathogenesis of chronic hepatitis B virus infection.We have previously demonstrated no differences of T-lymphocyte subsets in blood between inactive hepatitis B s antigen(HBsAg) carriers and patients with HBeAg-negative chronic active hepatitis B.This study investigated the peripheral blood cytokine profile in patients with HBeAg-negative chronic active hepatitis B infection(Group A,n = 21) and inactive HBsAg carriers(Group B,n = 13).Serum cytokines [interferon(IFN)-γ,tumor necrosis factor-α,interleukin(IL)-1b,IL-4,IL-12,IL-10,IL-2,IL-5,IL-8] were analyzed by using flow cytometry.Patients with chronic active disease presented with significantly decreased levels of IFN-γ and IL-10 compared to inac-tive carriers(P = 0.048 and P = 0.008,respectively).In HBeAg-negative chronic active hepatitis B patients,a significant negative correlation of IFN-γ levels with serum hepatitis B viral load was noted(P = 0.021).In conclusion,patients with HBeAg-negative chronic active hepatitis B and HBsAg inactive carriers display a different cytokine profile.Decreased Th1 response observed in patients with chronic active hepatitis B could be implicated in the persistence of virus replication and ongoing progression of liver disease.

变异胰岛素症

变异胰岛素症是在糖尿病患者中发现的分子病。对变异胰岛素的研究是近年在胰岛素结构与功能,临床医学和分子遗传学方面的新课题。本文就变异胰岛素的发现及其特点、变异胰岛素的鉴定技术以及变异胰岛素与糖尿病的关系作一综述。

Changes in beta-endorphin neuron numbers and serum hormone levels in the arcuate nucleus of ovariectomized rats undergoing treadmill exercise

BACKGROUND： The arcuate nucleus, when damaged in young rats, can lead to pathological changes in adults, such as osteoporosis. Ovariectomized rats suffer from osteoporosis at eight weeks following surgery and the number of β -endorphin immunoreactive neurons in the arcuate nucleus of the hypothalamus is significantly decreased. OBJECTIVE： To establish a rat model of osteoporosis using ovariectomy and to explore changes in the number of β-endorphin neurons and to correlate any such change with serum hormone levels in response to exercise or rest. DESIGN, TIME AND SETTING： The completely randomized block design, neural morphology study was performed at the Key Laboratory of Physiology, Guangdong Medical College, China between March 2004 and January 2005. MATERIALS： Sixteen healthy female rats were selected for ovariectomy. METHODS： Following model establishment, rats were assigned to either rest or exercise groups and each rat was housed individually. Rats in the exercise group underwent an exercise regimen using a treadmill. MAIN OUTCOME MEASURES： Eight weeks following exercise, radioimmunoassay was performed to detect serum growth hormone, estrogen and osteocalcin levels. Immunohistochemistry was used to measure changes in the number of β -endorphin neurons in the arcuate nucleus of the hypothalamus. Changes in bone metabolism were assessed using bone histomorphometry. RESULTS： In the exercise group, the β -endorphin immunoreactive neurons were high in number, darkly stained, and the nucleus was not obvious. In the rest group, the β -endorphin immunoreactive neurons were low in number and lightly stained. The number of β-endorphin immunoreactive neurons in the exercise group was higher compared with the rest group （t = 2.83, P 〈 0.05）. Estrogen levels were similar between the rest and exercise groups （P 〉 0.05）. Serum osteocalcin and growth hormone levels were significantly higher in the exercise group compared with the rest group （t = 2.78, 2.32, P 〈 0.05）. Compared with the rest group, the percentage of trabecular bone area, trabecular thickness, and trabecular number were significantly increased, but trabecular separation was significantly reduced （t=2.48, 2.57, 2.32, 3.06, P 〈 0.05） in the exercise group. In the exercise group, the trabeculae of the tibia were arranged regularly and were high in number. In the rest group, the trabeculae of the tibia were organized in a disorderly manner and were low in number, with many fat particles. CONCLUSION： Exercise promotes bone growth and delays osteoporosis by inducing an increase in the number of β-endorphin immunoreactive neurons in the arcuate nucleus of the hypothalamus and by increasing serum growth hormone and osteocalcin levels.

Dietary compliance in celiac disease

Celiac disease is an immune-mediated disorder that causes severe architectural disturbance in the small intestinal mucosa of genetically-predisposed individuals.Impaired absorption of multiple nutrients results and diarrhea and weight loss develop.Evidence has accumulated that a strict gluten-free diet can result in resolution of diarrhea,weight gain and normalization of nutrient malabsorption.In addition,histopathological changes also normalize,but this histopathological res-ponse appears to be time-dependent,sex-dependent and age-dependent.Compliance to a gluten-free diet is difficult and costly resulting in poor compliance and only a limited clinical response.This poses a risk for later long-term complications,including malignancy.A major practical clinical problem is the assessment of compliance to the gluten-free diet.Although symptoms may resolve and serological antibody markers may improve,multiple studies have documented ongoing architectural disturbance and inflammatory change,and with these continued inflammatory changes,a persistent risk for long-term complications.Recent immunological studies have suggested that peptides can be detected in both urine and fecal specimens that may be indicative of limited compliance.At the same time,multiple biopsy studies have demonstrated that complete normalization of the mucosa may occur in some patients within 6 mo of initiation of a gluten-free diet,but more often,up to 2 years or more may be required before repeated biopsies eventually show mucosal recovery and mucosal healing.

H. pylori Escape Host Immunoreaction Through Inhibiting ILK Expression by VacA

Helicobacter pylori （H. pylori） persistently colonizes the gastric mucosa despite a vigorous immune response. Vacuolating cytotoxin secreted by H. pylori has turned out to be a potent immunomodulatory toxin, but the signal transduction pathways involved has not been studied in macrophages. We observed in this study that vacA-deficient H. pylori induced significantly higher expression of integrin-linked kinase （ILK） and endothelial nitric oxygen synthase （eNOS）, and significantly more production of reactive oxygen species （ROS） in monocyte/macrophage-like U937 cells, as compared with isogenic vacA^＋ H. pylori. The expression of eNOS mRNA in U937 cells overexpressing ILK was markedly increased compared with those transfected with empty vectors. Thus, vacA-deficient H. pylori appears to upregulate ILK expression, which modulates the expression of eNOS and as a result, stimulates the production of ROS. It is VacA that prevents such a process by inhibiting ILK expression, helping H. pylori escape host immunoreaction. This mechanism explains, at least in part, persistent infection of H. pylori in the stomach.

EFFECTS OF MORPHINE AND MONOAMINE NEUROTRANSMITTERS ON PAIN MODULATION OF SUBSTANCE P IN THE SPINAL CORD

Substance P (SP)in the spinal cord is unevenly distributed and is concentrated in the dorsal horn, particularly in terminals of unmyelinated nociceptive primary afferent fibers. It indicates that SP may be an excitatory neurotransmitter released from nociceptive primary afferent nerve terminals. It was reported that SP in the spinal cord took part both in

Immunoreactive growth hormone in human peripheral T lymphocytes:encoding sequence of cDNA identical to that of the pituitary human growth hormone

Objective To detect the cDNA sequence of immunoreactive growth hormone (irGH) in human lymphocytes and to compare it with that of human growth hormone (hGH) in the pituitary. Methods The mRNA of irGH in human peripheral blood mononuclear cells (PBMC) was detected by Northern blot analysis. The cDNA of irGH was obtained by reverse transcriptase and polymerase chain reaction amplification and obtained by secreening from a cDNA library of human T lmphocytes. The sequence of irGH cDNA was determined and compared with that of the pituitary hGH. Results Very low level of irGH mRNA was detected in human PBMCs by Northern blot analysis. The cDNA sequence of the entire open reading frame of human irGH was 651 bp, which is identical to that of pituitary hGH. Conclusions The same hGH molecule can be produced by human pituitary somatotrophic cells and human peripheral lymphocytes.

A Novel Chemiluminescence Immunoassay Using Solid-Phase Antigen for Free 17β-Estradiol in Human Serum

A high-performance chemiluminescence immunoassay, with long-term durability, good precision and time- saving, was proposed for the detection of free 17β-estradiol （E2） in human serum. Ninety-six microplates were coated with bovine serum albumin conjugated E2 antigen as solid phase for the immunoassay. The E2-BSA antigen coated on the microplate and the E2 antigen in the sample competed for the binding sites on the horseradish peroxi- dase （HRP） labeled anti-E2 antibody. Chemiluminescence reaction was subsequently carried out by HRP catalyzing luminol-H2O2 substrates, and the chemiluminescence intensity was inversely proportional to the amount of analyte in human sera samples. The concentration of immunoreagents, immunoreaction time, and other relevant variable conditions upon the immunoassay were studied and optimized. The proposed method exhibited detection limit as low as 5.94× 10^-3 μg·L^-1 in a linear detection range from 0.01 to 1.00μg·L^-1 good recoveries between 105% and 108%, and high precision with intra- and inter-assay coefficients between 7.9% and 14.3%.