Rabbit Anti-human Serum Research Articles

High levels of serum mannose-binding lectin are associated with the severity of clinical signs of leptospirosis

The clinical heterogeneity observed in leptospirosis may be associated with host factors or bacteria virulence. Human serum mannose-binding lectin (MBL) recognizes many pathogens, and low levels of this lectin are associated with susceptibility to infection. MBL is also implicated in the modulation of the inflammatory process. We determined the levels of serum MBL during leptospirosis infection. A double-antibody sandwich ELISA was used to detect the immunoreactive serum MBL. The ELISA plates were coated with monoclonal antibody to MBL and bound MBL or recombinant human MBL were detected by rabbit anti-human MBL serum. HRPO-conjugated goat anti-rabbit antibody was used for detection of the reaction. Two groups of patients seen at referral hospitals in Recife, PE, Brazil, were divided according to the year of infection, 2001 (N = 61) or 2002 (N = 57) and compared in terms of disease severity and levels of serum MBL. A group of healthy volunteers (N = 97) matched by age, gender, and ethnic background was used as control. Patients infected in 2001 had more severe outcomes than those infected in 2002, including jaundice, hemorrhage, respiratory alteration, and renal complication (P = 0.0009; chi-square test). The frequency of patients producing serum MBL >1000 ng/mL was higher in the 2001 group than in the 2002 and control groups (P < 0.01), suggesting an association of MBL level with disease severity. The involvement of MBL and genetic variation of the MBL2 gene should be further evaluated to establish the role of this lectin in the pathogenesis of leptospirosis.

Group A Streptococcal Cysteine Protease Degrades C3 (C3b) and Contributes to Evasion of Innate Immunity

A relative lack of neutrophils around Streptococcus pyogenes is observed in streptococcal toxic shock syndrome (STSS). Because the bacteria spread rapidly into various organs in STSS, we speculated that S. pyogenes is equipped with molecules to evade the host innate immune system. Complement C3b opsonizes the pathogen to facilitate phagocytosis, and a complex of C3b converts C5 into anaphylatoxin. Because we found that C3 (C3b) is degraded in sera from patients with STSS, we investigated the mechanism of C3 (C3b) degradation by S. pyogenes. We incubated human C3b or serum with recombinant SpeB (rSpeB), a wild-type S. pyogenes strain isolated from an STSS patient or its isogenic DeltaspeB mutant and examined the supernatant by Western blotting with anti-human C3b. Western blot and Biacore analyses revealed that rSpeB and wild-type S. pyogenes rapidly degrade C3b. Additionally, C3 (C3b) was not detected in sera collected from infected areas of STSS patients. Furthermore, the survival rate in human blood and in mice was lower for the DeltaspeB mutant than the wild-type strain. Histopathological observations demonstrated that neutrophils were recruited to and phagocytosed the DeltaspeB mutant, whereas with the wild-type strain, few neutrophils migrated to the site of infection, and the bacteria spread along the fascia. We observed the degradation of C3 (C3b) in sera from STSS patients and the degradation of C3 (C3b) by rSpeB. This suggests that SpeB contributes to the escape of S. pyogenes from phagocytosis at the site of initial infection, allowing it to invade host tissues during severe infections.

Complement-mediated killing of Vibrio species by the humoral fluids of amphioxus Branchiostoma belcheri: Implications for a dual role of O-antigens in the resistance to bactericidal activity

The functional properties of complement in invertebrate deuterostomes are rather ill-defined. Here we showed that the humoral fluids from amphioxus Branchiostoma belcheri were capable of causing lysis of some Vibrio species including Vibrio alginolyticus HW284, Vibrio parahaemolyticus HW458 and Vibrio harvey SF-1, the first such data in the invertebrate deuterostomes. The fluid bacteriolytic activity was abolished by pre-incubation with heat-inactivated rabbit anti-human C3 serum, heating at 45 °C for 30 min, and repeated thawing and freezing. Additionally, the bacteriolytic activity was Mg 2+-dependent and Ca 2+-independent, and selective activation of the alternative pathway by zymosan A induced a loss of bacteriolytic activity. This strongly suggests that activation of the alternative complement pathway is responsible for the fluid bacteriolytic activity. It was also shown that some Vibrio species like Vibrio cincinnatiensis HW287 appeared resistant to the complement-mediated lysis. The LPS profiling revealed that the fluid-resistant V. cincinnatiensis HW287 had an LPS profile with a ladder of both high-molecular-weight (HMW) and low-molecular-weight (LMW) O-antigen bands, whereas the fluid-sensitive V. alginolyticus HW284 had few HMW O-antigen bands, suggesting a positive correlation between O-antigen size and humoral fluid resistance. Moreover, complement consumption assays demonstrated that both V. alginolyticus HW284 and V. cincinnatiensis HW287 consumed complement, with the former consuming significantly higher complement than the latter. Overall, it is suggested that HMW O-antigens may protect the fluid-resistant Vibrio species by a dual act of avoiding initiating complement activation as well as sterically hindering complement from gaining access to and damaging the cell membrane.

Purification and Localization of Nitric Oxide Synthases from Hybrid Tilapia (Nile Tilapia × Mozambique Tilapia)

The aims of this study were to purify and localize the nitric oxide synthases (NOSs) from hybrid tilapia (Nile tilapia Oreochromis niloticus x Mozambique tilapia O. mossambicus). The purification procedures involved affinity chromatography with a 2', 5'-ADP-agarose 4B column and ion exchange with a diethylaminoethanol Bio-Gel A column. The results from gel filtration assays showed that the molecular weights of neuronal NOS (nNOS) and inducible NOS (iNOS) were 178 and 120 kDa, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed that there were three bands with molecular weights of 89, 47, and 29 kDa from the purified nNOS. However, only one band, with a molecular weight of 120 kDa, appeared on the gel from the purified iNOS. Hybrid tilapia nNOS was a dimer structure, while iNOS appeared to be a monomer structure. Moreover, our results revealed that the activities of nNOS and iNOS were significantly higher after the addition of Ca+2 or Mg+2 ions individually. However, when L-arginine and NADPH were present, the addition of 1 mM of either ion did not further increase the activity. The chemical L-N(G)-methyl-L-arginine could inhibit the activities of the purified NOSs with or without L-arginine. Western blot analyses showed only an 89-kDa immunoreactive band from the extracts of cerebrum; however, we did not find the specific bands in other tissues, such as gill, intestine, liver, spleen, and anterior kidney. We found another 120-kDa immunoreactive protein band with the rabbit antirat iNOS serum against iNOS from the extracts of anterior kidney and spleen. The results of immunohistochemistry with the rabbit antihuman nNOS serum indicated that the nNOS existed in the cerebellum, olfactory bulb, diencephalons, and nerve cell bodies and neuronal fibers of the spinal cord. Interestingly, only macrophages from anterior kidney and spleen showed positive reactions with the rabbit antirat iNOS serum. In the same way, the endothelial NOS (eNOS) located in the heart and epithelial cells of the blood vessels reacted positively with the rabbit antibovine eNOS serum.

Observational Study of Hepatic Cytochrome P-450 Protein Expression and Activity in Summer Flounder (Paralichtys dentatus) after Combination Ormetoprim-Sulfadimethoxine Treatment

The metabolism of aquaculture antibiotics on the piscine, hepatic cytochrome P-450 (CYP) system has not yet been defined. Fifty summer flounder, maintained at 20°C, were fed ormetoprim-sulfadimethoxine (Romet-30®) at 1% body weight daily and were randomly sampled before treatment and on days 1, 6, 10 and 21 after treatment. Western blotting of hepatic microsomes included goat antirat CYP1A1 and rabbit antihuman CYP3A4 serum. Catalytic activities comprised: 3-cyano-7-ethoxycoumarin (CEC), 7-benzyloxy-4-trifluoromethylcoumarin (BFC), resorufin benzyl ether (BzRes). Treatment induced CYP1A1 and CYP3A4 expression. Dealkylation of CEC (CYP1A2) was increased after treatment. Romet-30 inhibited CYP3A4 activity measured by BFC, but induced BzRes CYP3A4. The usefulness of mammalian antibodies for piscine P-450 Western blotting was demonstrated. The hepatic P-450 1A2 and 3A4 metabolism was quantifiable by kits developed for mammalian microsomes.

Site-specific 99mTc-labeling of antibody using dihydrazinophthalazine (DHZ) conjugation to Fc region of heavy chain.

The development of an antibody labeling method with 99mTc is important for cancer imaging. Most bifunctional chelate methods for 99mTc labeling of antibody incorporate a 99mTc chelator through a linkage to lysine residue. In the present study, a novel site-specific 99mTc labeling method at carbohydrate side chain in the Fc region of 2 antibodies (T101 and rabbit anti-human serum albumin antibody (RPAb)) using dihydrazinophthalazine (DHZ) which has 2 hydrazino groups was developed. The antibodies were oxidized with sodium periodate to produce aldehyde on the Fc region. Then, one hydrazine group of DHZ was conjugated with an aldehyde group of antibody through the formation of a hydrazone. The other hydrazine group was used for labeling with 99mTc. The number of conjugated DHZ was 1.7 per antibody. 99mTc labeling efficiency was 46-85% for T101 and 67-87% for RPAb. Indirect labeling with DHZ conjugated antibodies showed higher stability than direct labeling with reduced antibodies. High immunoreactivities were conserved for both indirectly and directly labeled antibodies. A biodistribution study found high blood activity related to directly labeled T101 at early time point as well as low liver activity due to indirectly labeled T101 at later time point. However, these findings do not affect practical use. No significantly different biodistribution was observed in the other organs. The research concluded that DHZ can be used as a site-specific bifunctional chelating agent for labeling antibody with 99mTc. Moreover, 99mTc labeled antibody via DHZ was found to have excellent chemical and biological properties for nuclear medicine imaging.

KClO 4 inhibits thyroidal activity in the larval lamprey endostyle in vitro

An in vitro experimental system was devised to assess the direct effects of the goitrogen, potassium perchlorate (KClO 4), on radioiodide uptake and organification by the larval lamprey endostyle. Organification refers to the incorporation of iodide into lamprey thyroglobulin (Tg). Histological and biochemical evidence indicated that endostyles were viable at the termination of a 4 h in vitro incubation. A single iodoprotein, designated as lamprey Tg, was identified in the endostylar homogenates by polyacrylamide gel electrophoresis and Western blotting. Lamprey Tg was immunoreactive with rabbit anti-human Tg serum and had an electrophoretic mobility similar to that of reduced porcine Tg. When KClO 4 was added to the incubation medium, both iodide uptake and organification by the endostyle were significantly reduced relative to controls as determined by gamma counting, and gel-autoradiography and densitometry, respectively. Western blotting showed that KClO 4 significantly lowered the total amount of lamprey Tg in the endostyle. Based on the results of this in vitro investigation, we conclude that KClO 4 acts directly on the larval lamprey endostyle to inhibit thyroidal activity. These data support a previous supposition from in vivo experimentation that KClO 4 acts directly on the endostyle to suppress the synthesis of thyroxine and triiodothyronine, resulting in a decrease in the serum levels of these two hormones.

Radioimmunoassays of Follicle Stimulating Hormone and Luteinizing Hormone in the Dog

Heterologous radioimmunoassays for canine FSH and LH were developed and used to quantitate these hormones during proestrus, estrus and diestrus of the bitch. The assay for FSH consists of a rabbit anti-human FSH serum, radioiodinated rat FSH and dog FSH-LER-l685-3A as standard. Inhibition curves produced by the standard, crude extracts of canine pituitary glands and various canine plasma were parallel. Specificity of FSH assay was confirmed by testing for binding inhibition with canine LH (LER-l685-l). Binding was hardly affected by canine LH. The intra- and interassay coefficients of variation was 3.1% and 10.8%. Sensitivity of the FSH assay was 0.20 ng/tube which was adequate to quantify the concentration of FSH in 100 μl or less of canine plasma. The LH assay consists of a rabbit anti-ovine LH serum, radioiodinated rat LH and dog LH-LER-l685-l as standard. Crude extracts of canine pituitary glands and various canine plasma p roduced inhibition curves which were parallel to the standard curve. Binding inhibition was no affected by canine FSH(LER-l685-3A). The intra- and interassay coefficients of variation was 5.8% and 14.3%. Sensitivity of the LH assay was 10.7 pg/tube which allowed estimation of LH concentrations in 100 μl or less of canine plasma. During the reproductive cycle, levels of FSH were lowest during proestrus (51.1 ± 7.4 ng/ml) and increased to 305.5 ± 39.6 ng/ml on a day after LH peak. Levels of FSH declined by Day 4 (63.5 ± 10.4 ng/ml) then increased to 117.4 ± 11.9 ng/ml on Day 18 after LH peak. Levels of LH increased only during preovulatory LH surge, during proestrus and diestrus, were less than 0.5 ng/ml.

Placental protein 14 production by human Fallopian tube epithelial cells in vitro.

We studied the in-vitro secretory function of non-polarized and polarized cultured Fallopian tube epithelial cells by measurement of the placental protein 14 (PP14) secretion in primary cultures and subcultures from Fallopian tubes obtained from eight premenopausal women in different phases of the ovarian cycle. Primary cultures were established in minimal essential medium in Earle's salts supplemented with fetal bovine serum and the cells were subcultured for six passages, in the polarized cell cultures, the cells being seeded on an extracellular matrix system. Cell freezing was carried out using 10% dimethyl sulphoxide. PP14 secretion into the culture media was measured by a radioimmunoassay using 125I-PP14 as label and rabbit anti-human PP14 serum. There was a large amount of PP14 secretion into the culture media in primary cultures, the secretion decreasing considerably after subculture 1. PP14 secretion after subculture 2 was not different from the control values. Polarized and non-polarized cells secreted similar amounts of PP14 and frozen-thawed cells did not appear to secrete PP14. Epithelial cells from Fallopian tubes obtained at different phases of the ovarian cycle did not appear to show any difference in PP14 secretion rates. Our data suggest that the in-vitro secretion of PP14 by human Fallopian tube epithelial cells is adversely affected by cell ageing and freezing.

Stimulation of Intracellular Ca2+ Levels in Human Neutrophils by Soluble Immune Complexes

Soluble immune complexes bind to unprimed neutrophils and generate intracellular Ca2+ transients but fail to activate the NADPH oxidase. Following priming of the neutrophils with either tumor necrosis factor alpha or granulocyte-macrophage colony-stimulating factor, stimulation of the cells with the soluble immune complexes leads to an enhanced Ca2+ signal and significant secretion of reactive oxidants. The enhanced Ca2+ signal observed in primed neutrophils results from the influx of Ca2+ from the external environment and is partly sensitive to tyrosine kinase inhibitors. This is in contrast to the Ca2+ signal observed in unprimed neutrophils, which arises from the mobilization of intracellular stores. When the surface expression of FcgammaRIIIb on primed neutrophils was decreased either through incubation with Pronase or phosphoinositide-specific phospholipase C, the extra enhanced Ca2+ mobilization seen in primed cells was significantly lowered, while the initial rise in intracellular Ca2+ was unaffected. Depletion of FcgammaRIIIb had no significant effect on the Ca2+ transients in unprimed neutrophils. Cross-linking FcgammaRII, but not FcgammaRIIIb, induced increases in intracellular Ca2+ in unprimed neutrophils, while cross-linking either of these receptors increased Ca2+ levels in primed neutrophils. The FcgammaRII-dependent intracellular Ca2+ rise in primed cells was unaffected by incubation in Ca2+-free medium, whereas the FcgammaRIIIb-dependent transient was significantly decreased when Ca2+ influx was prevented in Ca2+-free medium supplemented with EGTA. Cross-linking either FcgammaRII or FcgammaRIIIb in primed or unprimed cells failed to stimulate substantial levels of inositol 1,4,5-trisphosphate production. These results indicate that following stimulation of primed neutrophils with soluble immune complexes the enhanced Ca2+ mobilization observed is the result of a functional activation of the glycosylphosphatidylinositol-linked FcgammaRIIIb.

Modification of antibody isoelectric point affects biodistribution of 111-indium-labeled antibody

We evaluated the influence of changes in charge on the biodistribution of 111In-labeled purified rabbit antihuman serum albumin (R-HSA) IgG conjugated to diethylenetriaminepentaacetic dianhydride (DTPA). Optimization of isoelectric point (pI) may influence the biodistribution profile, especially retention in vital organs, which ultimately affects radioimmunoimaging. Experiments were designed to modify the pI of R-HSA by conjugating various molar ratios of DTPA (DTPA:R-HSA ratios 5:1 to 100:1). The pI of the conjugates was determined by isoelectricfocusing (IEF). 111In-labeled DTPA:R-HSA with known pI range was injected intraperitoneally into BALB/c mice to evaluate biodistribution. There was a proportional relationship between the molar ratio of DTPA to R-HSA IgG and the number of DTPA substituted. Molar ratios of 5, 10, 20, 50 and 100 gave, on average, 2.0, 3.6, 5.1, 9.5 and 16.0 DTPA per R-HSA IgG, respectively. An anodal shift in the pI of 111In-labeled R-HSA IgG was noted with increased number of DTPA conjugation. Biodistribution studies at both 4 and 24 h showed sequential increase in the liver activity with increasing number of DTPA per antibody, whereas colon and small intestine showed a decrease in the activity at 4 h. The organ-specific increase (e.g., liver) or decrease (e.g., colon and small intestine) in the activity may depend on a critical balance of charge of a particular organ and its interaction with the amount of negative charge carried by the antibody conjugate. The results suggest that pI optimized 111In-labeled antibody could be used to increase or decrease colon and hepatic retention for more efficient radioimmunoimaging of colon tumors and their hepatic metastasis.

Monoclonal antibody against an urinary protein secreted from the renal tubules--immunohistochemical analysis of renal cell carcinoma

Monoclonal antibodies (mABs) against urinary proteins originated from renal tubules were obtained in order to find a possible tumor marker for renal cell carcinoma. Urine samples were collected from healthy adult males. After removal of Thamm-Horsfall protein (THP) by sodium chloride precipitation, supernatant urine was ultrafiltered, thus, THP-free urinary proteins were obtained. Then, proteins of plasma origin were removed by circulating the urinary proteins through an affinity column which contained immobilized rabbit anti-human whole serum immunoglobulins. BALB/c mice were immunized with the THP- and plasma protein-free urinary proteins. The spleen cells were fused with mouse myeloma cells. Hybridomas were screened by indirect immunofluorescence of fresh frozen kidney tissue sections using culture supernatants. Five hybridomas producing mAbs which reacted with renal tubular segments were established. One mAb reacted strongly with distal renal tubules. Using this mAb, formalin fixed and paraffin embedded sections of 39 renal cell carcinomas were stained by immunoperoxidase method. Twenty-six of the 39 cancer tissues (66.7%) were stained positive. According to the histological classification, positive staining rate of clear cell subtype, and mixed subtype was 75% (6/8), 80% (8/10) and 66.7% (6/9), respectively. According to the tumor grade, positive staining rate of G1, G2, G3 was 66.7% (6/9), 71.4% (15/21) and 55.5% (5/9), respectively. These findings indicate that most of renal cell carcinomas were originated from common precursor cells for both proximal and distal tubules, because normal proximal tubular cells were not stained by the mAb.

Association between breast cancer patient survival and levels of creatine kinase BB in tumor cytosols

Fatty acid-binding proteins (FABPs) are abundant low-molecular-weight cytosolic proteins in tissues involved in fatty acid (FA) metabolism. Because epidermis is also an active lipogenic tissue, we examined cytosols from murine and porcine epidermis and cultured human keratinocytes and fibroblasts for FABPs. High-affinity FA-binding activity was present in both epidermis and differentiated keratinocytes, whereas no high-affinity FA-binding activity was found in cultured human fibroblasts or undifferentiated keratinocytes. By column chromatography, a single binding peak was identified in the high (90–100 kDa)-molecular-weight range and no binding activity was evident in the low (14–15 kDa)-molecular-weight range, where conventional FABPs elute. Moreover, rabbit anti-rat heart FABP, anti-rat intestine FABP, and anti-rat liver FABP antisera did not identify proteins in the 14–15-kDa range in murine epidermal cytosol by Western immunoblots, whereas the anti-rat-heart antibody recognized a protein of approximately 32 kDa. Isoelectric focusing of differentiated keratinocyte cytosol demonstrated a single FA-binding peak having a pI of ≈ 4.0. Analysis of this binding peak by SDS-PAGE revealed peptides of approximately 66 and 38 kDa. These findings suggest the possibility that the FA-binding protein in keratinocyte cytosol normally exists as a heterodimer. Western immunoblots of both differentiated keratinocyte cytosol and keratinocyte-conditional media stained with a rabbit anti-human serum albumin antibody identified a protein of approximately 67 kDa, but the electrofocused fraction did not react with this antibody. Thus, epidermis and differentiated keratinocytes possess high-affinity cytosolic FA-binding activity that cannot be ascribed either to conventional low-molecular-weight FABPs or to albumin.

Prostate specific antigen gene expression in ovarian cancer post-liver transplantation

Fatty acid-binding proteins (FABPs) are abundant low-molecular-weight cytosolic proteins in tissues involved in fatty acid (FA) metabolism. Because epidermis is also an active lipogenic tissue, we examined cytosols from murine and porcine epidermis and cultured human keratinocytes and fibroblasts for FABPs. High-affinity FA-binding activity was present in both epidermis and differentiated keratinocytes, whereas no high-affinity FA-binding activity was found in cultured human fibroblasts or undifferentiated keratinocytes. By column chromatography, a single binding peak was identified in the high (90–100 kDa)-molecular-weight range and no binding activity was evident in the low (14–15 kDa)-molecular-weight range, where conventional FABPs elute. Moreover, rabbit anti-rat heart FABP, anti-rat intestine FABP, and anti-rat liver FABP antisera did not identify proteins in the 14–15-kDa range in murine epidermal cytosol by Western immunoblots, whereas the anti-rat-heart antibody recognized a protein of approximately 32 kDa. Isoelectric focusing of differentiated keratinocyte cytosol demonstrated a single FA-binding peak having a pI of ≈ 4.0. Analysis of this binding peak by SDS-PAGE revealed peptides of approximately 66 and 38 kDa. These findings suggest the possibility that the FA-binding protein in keratinocyte cytosol normally exists as a heterodimer. Western immunoblots of both differentiated keratinocyte cytosol and keratinocyte-conditional media stained with a rabbit anti-human serum albumin antibody identified a protein of approximately 67 kDa, but the electrofocused fraction did not react with this antibody. Thus, epidermis and differentiated keratinocytes possess high-affinity cytosolic FA-binding activity that cannot be ascribed either to conventional low-molecular-weight FABPs or to albumin.

Immunohistochemical Analysis of Spontaneous Pancreatic Islet Amyloid Deposits in Nonhuman Primates

Pancreatic islet amyloidosis has been previously reported in aged diabetic and nondiabetic humans, nonhuman primates, cats, raccoons (Procyon lotor), and degus (Ocrodon degus, a South American rodent).h In nonhuman primates, islet amyloidosis, often accompanied by diabetes mellitus, has been reported in Celebes crested macaques (Macaca nigYU)~ rhesus monkeys (Macaca ~nulatta), cynomolgus monkeys (Macaca fascicularis), a Formosan rock macaque (Macaca cyclopis), and a drill baboon (Mandrillus leucophams). 1.2.7.8 Human and feline islet amyloid deposits are derived from the polymerization of islet amyloid polypeptide (IAPP; amylin), whereas in the degu, they are derived from insulin.69 IAPP is an islet beta-cell product secreted with insulin by humans and many other ~pecies.~ The definitive biological functions of IAPP and the significance of IAPP in the pathogenesis of human type 2 (non-insulin dependent) diabetes mellitus and feline diabetes mellitus are unre~olved.~.~-~ The purpose of this study was to examine the immunoreactivity of pancreatic islet amyloid in captive nonhuman primates. A review of necropsy records from several sources identified four cynomolgus monkeys, three Celebes crested macaques, and one orangutan (Pongo pygmaeus) with islet amyloid deposits. All of the nonhuman primates were adults ranging from 7 to 26 years of age. One of the Celebes crested macaques had diabetes mellitus, but the metabolic status of the other animals was not known. Formalin-fixed, paraffin-embedded tissues were sectioned at 5 lm and stained with either hematoxylin and eosin (HE) or Congo red with or without KMnO, pretreatment. Congo red-stained sections were examined by both bright field and polarized light microscopy. Using a modified avidin-biotin-peroxidase complex (ABC) method,> pancreatic tissues were evaluated for immunoreactivity with guinea pig anti-porcine insulin serum (Dako Corp., Carpinteria, CA) diluted 1 : 800 in phosphate-buffered saline (PBS), pH 7.4; 1 : 400 rabbit anti-human glucagon serum (Dako); 1 : 100 rabbit anti-human somatostatin serum (Dako); 1 : 400 rabbit anti-human pancreatic polypeptide serum (Dako); 1 : 800 rabbit anti-human calcitonin gene-related peptide (CGRP) serum (Peninsula Laboratories, Belmont, CA); and 1 : 200 rabbit anti-human amylin serum (Peninsula). Positive controls included internal controls in the tissue sections containing islet amyloid and application of the primary antisera to tissue sections from normal bonnet macaque (Macaca radiata) pancreas. Negative controls included replacing the primary antisera with the appropriate nonimmune sera and applying the primary antisera to tissue sections from a canine kidney containing glomerular amyloid. Five-micrometer tissue sections were collected on positively charged glass slides (Fisher Scientific, Pittsburgh, PA), deparaffinized, rehydrated, and incubated in 1.5% normal goat serum in PBS for 20 minutes at room temperature in a humidified chamber. Sections were incubated with primary antibody for 1 hour under the same conditions, followed by a 30-minute incubation in 3% hydrogen peroxide in methanol to block endogenous peroxidase activity and 30-minute incubations of biotinylated goat anti-rabbit or anti-guinea pig IgG (Vector Laboratories, Burlingame, CA), and ABC (Vector). The chromagen was 0.0 16% diaminobenzidine tetrahydrochloride (Sigma Chemical Co., St. Louis, MO) with NiCI, (Digene Diagnostics, Silver Spring, MD) in 0.024% hydrogen peroxide and PBS. Sections were counterstained with nuclear fast red (Digene). All the animals in this study had either multifocal or diffuse pancreatic islet amyloidosis. The extent and distribution of amyloid ranged from mild multifocal deposition surrounding islet capillaries with little or no loss of islet cells to marked diffuse deposition in enlarged islets with severely reduced

Cathepsin E in follicle associated epithelium of intestine and tonsils: localization to M cells and possible role in antigen processing

A specific rabbit anti-human serum was used selectively to localize the aspartic proteinase cathepsin E to follicle associated epithelium (FAE) of human and rat intestine, including jejunum, ileum, appendix, colon and rectum, as well as of human palatine, pharyngeal and lingual tonsils. Coexpression of class II histocompatibility antigen HLA-DR antigen has been observed in some of the cathepsin E-positive epithelial cells. In addition, cathepsin E has been detected in a few mononuclear cells of intestinal lymphoid structures and tonsils resembling interdigitating reticulum cells of lymph nodes. Another aspartic proteinase, cathepsin D, has been found to be poorly represented in FAE and intensely expressed by macrophages. Electron immunocytochemistry localized cathepsin E to endosomal vesicles and endoplasmic reticulum of M cells in rat and human ileum as well as of M-like cells in human palatine tonsil. The results suggest a possible role of endosomal cathepsin E in the processing of macromolecules and microorganisms transported by M cells and related epithelial cells to mucosal associated lymphoid tissue (MALT).

Insulin-like growth factor immunoreactivity increases in muscle after acute eccentric contractions

The purpose of the study was to note whether insulin-like growth factor (IGF) immunoreactivity increased after eccentric contractions. IGF immunoreactivity in the rat tibialis anterior muscle was measured on 5 successive days (4-5 rats/group, n = 28) after an acute bout of 192 eccentric contractions elicited by electrical stimulation. The muscle tissue sections were immunocytochemically processed with rabbit anti-human IGF-I serum. Immunoreactivity was analyzed with videomicroscopy and computer-aided image processing. Four days after eccentric contractions, IGF immunoreactivity was significantly higher than control [0.081 +/- 0.073 (SD) absorbance at 480 nm vs. 0.026 +/- 0.018; P < 0.05]. The increases in IGF-I immunoreactivity were mostly within the muscle fibers. These results suggest that an acute bout of eccentric exercise increases IGF-I immunoreactivity in rat type II muscle 4 days postexercise.

High-Affinity Fatty Acid-Binding Activity in Epidermis and Cultured Keratinocytes Is Attributable to High-Molecular-Weight and Not Low-Molecular-Weight Fatty Acid-Binding Proteins

Fatty acid-binding proteins (FABPs) are abundant low-molecular-weight cytosolic proteins in tissues involved in fatty acid (FA) metabolism. Because epidermis is also an active lipogenic tissue, we examined cytosols from murine and porcine epidermis and cultured human keratinocytes and fibroblasts for FABPs. High-affinity FA-binding activity was present in both epidermis and differentiated keratinocytes, whereas no high-affinity FA-binding activity was found in cultured human fibroblasts or undifferentiated keratinocytes. By column chromatography, a single binding peak was identified in the high (90-100 kDa)-molecular-weight range and no binding activity was evident in the low (14-15 kDa)-molecular-weight range, where conventional FABPs elute. Moreover, rabbit anti-rat heart FABP, anti-rat intestine FABP, and anti-rat liver FABP antisera did not identify proteins in the 14-15-kDa range in murine epidermal cytosol by Western immunoblots, whereas the anti-rat-heart antibody recognized a protein of approximately 32 kDa. Isoelectric focusing of differentiated keratinocyte cytosol demonstrated a single FA-binding peak having a pI of approximately 4.0. Analysis of this binding peak by SDS-PAGE revealed peptides of approximately 66 and 38 kDa. These findings suggest the possibility that the FA-binding protein in keratinocyte cytosol normally exists as a heterodimer. Western immunoblots of both differentiated keratinocyte cytosol and keratinocyte-conditional media stained with a rabbit anti-human serum albumin antibody identified a protein of approximately 67 kDa, but the electrofocused fraction did not react with this antibody. Thus, epidermis and differentiated keratinocytes possess high-affinity cytosolic FA-binding activity that cannot be ascribed either to conventional low-molecular-weight FABPs or to albumin.

Red cell alterations associated with virucidal methylene blue phototreatment

Several red cell storage properties were evaluated following phototreatment with methylene blue (MB) under conditions that inactivated > or = 6 log10 of added vesicular stomatitis virus. Red cell 2,3 DPG levels were similar to untreated controls throughout conventional 42-day storage at 4 degrees C. Plasma hemoglobin levels were elevated approximately twofold in MB-phototreated samples, and morphology scores were 5 percent lower after 42-day storage. ATP levels declined 30 percent in phototreated samples and in a control sample containing MB and not exposed to light. Lipid peroxidation was not observed in treated or control cells, nor were differences observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ghost membranes derived from phototreated and control samples. Phototreated cells exhibited enhanced ion permeability; sodium and potassium levels approached equilibrium with the suspending medium within 4 to 7 days after treatment. Direct agglutination tests using rabbit anti-human IgG or rabbit anti-human serum albumin on MB-phototreated cells indicated that serum proteins had absorbed to the surface of treated red cells. Plasma depletion by washing red cells prior to phototreatment did not prevent protein binding upon subsequent addition of untreated autologous or group AB plasma. To a much smaller extent, phototreatment of plasma resulted in IgG association with untreated red cells. The addition of glutathione to red cell suspensions prevented IgG binding to phototreated red cells but did not prevent enhanced ion permeability. Taken together, these data suggest that the red cell surface is altered by virucidal MB phototreatment of vesicular stomatitis virus.

Affinity Purification of Human τ Proteins and the Construction of a Sensitive Sandwich Enzyme‐Linked Immunosorbent Assay for Human τ Detection

Immunoaffinity chromatography with a monoclonal antibody produced against bovine tau protein was used to purify tau proteins from human brain. Fifty grams of brain tissue yielded approximately 2 mg of pure tau proteins. The affinity-purified human tau was used to produce a high-titered rabbit anti-human tau serum. The monoclonal anti-tau antibody and the polyclonal rabbit anti-tau serum were then used to construct a sandwich enzyme-linked immunosorbent assay for detection of human tau proteins, with a sensitivity of 1 ng/ml.