The piglet as a model for B cell and immune system development.

The ability to identify factors responsible for disease in all species depends on the ability to separate those factors which are environmental from those that are intrinsic. This is particularly important for studies on the development of the adaptive immune response of neonates. Studies on laboratory rodents or primates have been ambiguous because neither the effect of environmental nor maternal factors on the newborn can be controlled in mammals that: (i) transmit potential maternal immunoregulatory factors in utero and (ii) are altricial and cannot be reared after birth without their mothers. Employing the newborn piglet model can address each of these concerns. However, it comes at the price of having first to characterize the immune system of swine and its development. This review focuses on the porcine B cell system, especially on the methods used for its characterization in fetal studies and neonatal piglets. Understanding these procedures is important in the interpretation of the data obtained. Studies on neonatal piglets have (a) provided valuable information on the development of the adaptive immune system, (b) lead to important advances in evolutionary biology, (c) aided our understanding of passive immunity and (d) provided opportunities to use swine to address specific issues in veterinary and biomedical research and immunotherapy. This review summarizes the history of the development of the piglet as a model for antibody repertoire development, thus providing a framework to guide future investigators.

Investigation of copper adsorption to peat using the simple metal sorption model.

The Simple Metal Sorption (SiMS) equilibrium model was used to simulate the proton/cation exchange behavior of peat with dissolved copper. The SiMS model represents proton binding and metal binding as cation exchange for heterogeneous sorbents as a function of pH, salt concentration, total metal concentration and total ligand concentration. The SiMS model uses fewer parameters than other cation exchange models for multidimensional datasets and can be executed on a standard spreadsheet. The cation exchange selectivity coefficient, K(Me,app), is represented as K(Me,app) = KMe[H+](alpha)(LT/MeT)(beta) I(psi). The model is similar to standard surface complexation approaches, with an intrinsic relationship described by mass action laws (KMe = metal equilibrium constant) and variable terms that are expressed as simple power functions of proton concentration, ligand to metal ratio (LT/MeT), and ionic strength (I). The model successfully simulated the proton exchange behavior of acid-washed, Sphagnum peats over a range of 4 to 8 pH units with ionic strength differing by three orders of magnitude (I = 0.001 to 0.1). Simulation of copper binding on five peat data sets and the dried biomass of Potamogeton lucens was also successful (0.94 < r2 < 0.99). However, there was no apparent relationship between model parameters and peat characteristics. Incorporation of the SiMS model into a framework for predicting metals removals in wetlands will require more work.

Renal medullary carcinoma.

PURPOSE: Renal medullary carcinoma is a rare and extremely aggressive neoplasm that almost always develops in young patients with sickle cell trait. To our knowledge all cases to date have been metastatic at surgical resection. Pathological examination reveals an aggressive tumor mainly involving the renal medulla with a varied morphology. The prognosis is dismal. Mean survival from the time of resection is 15 weeks (range 2 to 52). The disease course has not been altered by surgery, radiotherapy or various regimens of chemotherapeutic agents. MATERIALS AND METHODS: We add to the literature our experience treating renal medullary carcinoma in 2 cases and review the existing literature on this disease. RESULTS: Both patients whom we treated died of the disease, as have the other 35 patients described in the literature. CONCLUSIONS: A high index of suspicion may lead to earlier diagnosis and treatment, and survival of patients with renal medullary carcinoma.

Sonographic prediction of viability in testicular torsion: preliminary observations.

This study was performed to determine if sonography can assist in predicting testicular viability in the setting of testicular torsion. Sixteen patients with sonographically diagnosed and surgically proved testicular torsion were studied. The preoperative sonograms were reviewed retrospectively to determine testicular echogenicity and homogeneity, testicular size and vascularity, scrotal skin thickness and vascularity, and the presence or absence of a hydrocele. These findings were correlated with the viability of the testis at surgery. All nine patients with normal homogeneous testicular echogenicity had viable testes at surgery. All seven patients with hypoechoic or inhomogeneous testes had nonviable testes at surgery and pathologic evidence of necrosis. The other findings were less helpful in predicting viability. In the setting of testicular torsion, normal testicular echogenicity is a strong predictor of viability. Immediate surgical detorsion in these patients carries a very high likelihood of salvaging the affected testis.

Molecular mechanisms of murine Fc epsilon RII/CD23 regulation.

The low affinity receptor for IgE (Fc epsilon RII or CD23), expressed primarily on mouse B cells, is known to be upregulated by interleukin-4 (IL-4) at both the mRNA and protein levels. Fc epsilon RII expression is superinduced when the IL-4 is combined with cell activation. In order to explore the molecular regulation of Fc epsilon RII expression, mouse B cell lines were screened to develop a cell line model. The B cell lymphoma A20.1, was found to behave in a manner similar to mouse B cells in that Fc epsilon RII levels are very low on cells cultured in media alone (< 10(3)/cell), increased by culture in the presence of IL-4, and superinduced by LPS and IL-4 (> 10(5)/cell). The steady state mRNA levels for Fc epsilon RII corresponded to the level of cell surface expression. Transcription assays indicated that the Fc epsilon RII level increases could be explained entirely by increased transcription rates. The A20.1 cell line was subsequently used to analyse the Fc epsilon RII promoter. Nested deletion analysis of the 1.3 kB 5' of the mouse Fc epsilon RII transcription start site, using CAT reporter plasmids transfected into A20.1 cells, identified major elements activating the Fc epsilon RII promoter within 250 bp of the transcription start site. Constructs containing greater than 250 bp of 5' sequence showed significantly reduced CAT activity suggesting negative regulatory regions. Coincident with the restricted tissue expression of murine Fc epsilon RII, the promoter was B cell specific in that little CAT expression was seen in fibroblast, mast cells or T cell lines. Expression was seen, however, in both mouse and human B cell lines. Finally, the promoter was analysed for response to IL-4. Stimulation with IL-4 plus LPS resulted in only a modest increase in CAT activity (approximately 2-fold), in contrast to transcription assays, where increases approximated that seen at the cell surface. Thus, the IL-4 response must also require sequences distal to the regions examined.

The oligomeric nature of the murine Fc epsilon RII/CD23. Implications for function.

The low affinity receptor for IgE (Fc epsilon RII/CD23) is a type II integral membrane protein with an extracellular C-terminal region homologous to C-type animal lectins. Immediately adjacent to this lectin homology region is a sequence that is predicted to form an alpha-helical coiled-coil stalk leading to dimer or trimer formation. This provides an explanation for the known self-associative capacity for the Fc epsilon RII. In this study the self-association to a trimer or tetramer is shown with rFc epsilon RII by chemical cross-linking and affinity purification on IgE columns. The data indicate that only the oligomeric form of Fc epsilon RII has sufficient affinity/avidity to bind to an IgE adsorbent. In contrast, Fc epsilon RII that is purified using anti-Fc epsilon RII mAb adsorbents has largely lost its capacity to bind IgE, as well as its capacity to self-associate, indicating that IgE recognizes the oligomeric form of the Fc epsilon RII. This phenomenon was further examined by performing detailed binding analysis of the mouse IgE/Fc epsilon RII interaction. A biphasic binding curve with high (2-7 x 10(7) M-1) and low (2-7 x 10(6) M-1) affinity binding was seen. Fc epsilon RII mutants were prepared that lack one or more of the 21 amino acid homologous repeat domains in the stalk region of the molecule. These mutant Fc epsilon RII molecules bound IgE with only a single low affinity (5-10 x 10(6) M-1). In addition, cross-linking analysis of one of these mutants demonstrated that it does not exhibit the receptor self-association seen for the intact Fc epsilon RII. Two chimeric Fc epsilon RII molecules were prepared having the mouse Fc epsilon RII lectin homology (carboxyl-terminal) region and the stalk region of either the related human Fc epsilon RII or the corresponding domain of Ly-49. Chimeric molecules using the former (alpha-helical coiled-coil) stalk supported normal binding of IgE although the Ly-49/Fc epsilon RII chimera failed to bind IgE. Taken together, the results indicate that high (approximately 10(8) M-1) affinity IgE binding results from interaction of multiple lectin domains with (presumably) symmetrical sites on the IgE molecule. Specificity for IgE is determined by the lectin domain although the binding avidity is determined by oligomerization through the coiled coil stalk.

Fc epsilon receptors.

Advances in our understanding of the molecular structure of Fc receptors have been made at a rapid pace. Details of how Fc receptors are involved in cell triggering, e.g. allergic mediator release from mast cells, and IgE synthesis are also continuing to be elucidated, although much work is still required. Recent highlights of investigations of mast-cell and lymphocyte IgE Fc receptors will be outlined.

Laparoscopic nephrectomy: initial case report.

A tumor-bearing right kidney was completely excised from an 85-year-old woman using a laparoscopic approach. A newly devised method for intra-abdominal organ entrapment and a recently developed laparoscopic tissue morcellator made it possible to deliver the 190 gm. kidney through an 11 mm. incision.

Endopyelotomy for secondary ureteropelvic junction obstruction in children.

Percutaneous endopyelotomy has been shown to be successful in treating ureteropelvic junction obstruction in adults. Little data have been published regarding this procedure in children. We describe 4 patients 6.5 weeks to 5.5 years old who underwent percutaneous endopyelotomy to treat ureteropelvic junction obstruction following failed open dismembered pyeloplasty. Preoperative obstruction was demonstrated by a nephrostogram, diuretic renogram and/or ultrasonography. Percutaneous endopyelotomy was successful in relieving the obstruction in all 4 patients, although 2 required secondary endoscopic procedures. One patient had persistent obstruction 40 days after endopyelotomy at the ureteropelvic junction and, subsequently, required percutaneous resection of a persistent flap of obstructing tissue. In another patient a ureterovesical stricture was noted at the time of stent removal, which was treated by endoscopic incision. All patients have been followed from 1.5 to 3 years postoperatively. Followup diuretic renograms, ultrasound and/or excretory urography demonstrated a patent ureteropelvic junction in all patients and all have remained asymptomatic. Endopyelotomy appears to be safe and effective in treating secondary ureteropelvic junction obstruction in children.

The immunochemistry of solid-phase sandwich enzyme-linked immunosorbent assays.

Multivalent antigens (Ags) such as membrane proteins can be quantitated by using sandwich enzyme-linked immunosorbent assays (ELISAs), which typically show sensitivity from 0.1 to 50 ng/ml. The percentage of antigen that binds in the log-log linear region reflects the affinity of the capture antibody (CAb), and the range of linearity for assays conducted with a particular CAb is proportional to the antibody (Ab) concentration. The sandwich ELISA titration plot reflects the actual amount of Ag bound when asymmetrical configurations are used; steric hindrance that occurs with certain symmetrical configurations, especially when enzyme-Ab conjugates of greater than or equal to 10(6) daltons are used, can alter this relationship. Monoclonal CAbs bind less Ag than polyclonal CAbs. Immobilization of monoclonal CAbs by using a modified avidin-biotin system can result in greater antigen capture capacity (AgCC) than when the Abs are directly adsorbed on plastic. Adsorption of proteins on polystyrene is noncovalent and proportional to the amount added for up to 150 ng/200 microliter in a microtiter well. Adsorption can result in substantial loss of antigenic or antibody activity. Desorption is continuous at a low level and can negatively influence the results of an immunoassay. Data from microtiter sandwich ELISAs can be readily acquired and analyzed by using a computer-based analysis system (ELISANALYSIS) written for the IBM PC. This analytical system considers the immunochemical principles of sandwich ELISAs predicted theoretically and demonstrated empirically.

The immunochemistry of sandwich ELISAs--I. The binding characteristics of immunoglobulins to monoclonal and polyclonal capture antibodies adsorbed on plastic and their detection by symmetrical and asymmetrical antibody-enzyme conjugates.

Radiolabelled bovine IgG1, IgG2, SIgA and IgM and heavy-chain specific polyclonal and monoclonal antibodies to these isotypes were employed as models to investigate immunochemical aspects of sandwich enzyme immunoassays (ELISAs). The titration plots obtained by measuring enzyme activity paralleled those obtained when the binding of radiolabelled immunoglobulins to solid-phase capture antibodies was quantitated. As predicted from the Mass Law, the percentage of labelled immunoglobulin which was bound remained constant over the range in which the sandwich ELISA titration was linear on a log-log plot. Also as predicted from the Mass Law, increasing the solid-phase concn of polyclonal antibodies by affinity purification increased the linear region of the log-log ELISA plot and the corresponding region over which a constant percentage of immunoglobulin binding was observed. When used as capture antibodies adsorbed on plastic at equal concns, the best monoclonal antibodies were 1/8- less than 1/16 as effective as their polyclonal counterparts in binding iodinated bovine immunoglobulins; these differences can be directly interpreted to result from an 8 and greater than 16-fold higher functional, relative affinity of the polyclonal reagents. Steric hindrance was shown to occur when symmetrical sandwich ELISAs, i.e. capture and detection antibody are both heavy-chain specific, are used to measure monomeric but not IgM immunoglobulins. The use of an asymmetrical configuration, i.e. anti-Fab antibody-enzyme conjugates, avoids this problem. Symmetrical conjugates based on the avidin-biotin system, horseradish peroxidase or alkaline phosphatase, were less effective than their asymmetrical (anti-Fab) counterparts. Evidence that the lower activity of symmetrical conjugates was due to steric hindrance was illustrated using horseradish peroxidase-antibody conjugates of different sizes. Sandwich assays using affinity-purified, polyclonal solid-phase antibodies and an asymmetrical conjugate were judged to be immunochemically and economically optimal. Using an asymmetrical configuration, the non-linear nature of sandwich ELISA titration plots is the predictable result of changing antibody to antigen ratios in an antibody-limiting system, and not the result of steric hindrance of the detection system.

Altered recognition of surface-adsorbed compared to antigen-bound antibodies in the ELISA.

We found in preliminary studies using 125I-labelled antibodies that an antibody bound to a solid-phase antigen was recognized more efficiently than an antibody adsorbed directly to the solid phase. The present study was designed therefore to quantitate the differential recognition of an antibody adsorbed directly to the solid phase and an antibody bound to antigen on the solid phase using the amplified enzyme-linked immunosorbent assay (a-ELISA), and to compare results with the amounts of specific antibody determined by quantitative immunoprecipitation. The degree of differential recognition was quantitated for rabbit IgG and SIgA anti-ovalbumin (anti-OA) and anti-fluorescein, and was found to be dependent upon the isotype of the antibody and not its specificity. The ratio describing the differential recognition of SIgA antibodies (1.8) was much less than for IgG antibodies (greater than 30) and remained constant over the titration range analyzed while the ratios obtained for IgG varied substantially (25-60) over the same range. These ratios of differential recognition were used to estimate rabbit IgG antibody levels to OA, bovine serum albumin, ferritin and alpha-lactalbumin. The estimates obtained were consistently much less than total antibody levels measured by quantitative precipitation. The use of glutaraldehyde-aggregated OA in the ELISA, however, increased the amount of IgG anti-OA and SIgA anti-OA capable of recognizing OA adsorbed on plastic from 12 to 50 and from 30 to 80%, respectively.

Cyclosporine immunomodulation in a rabbit model of chronic hypersensitivity pneumonitis.

Rabbit models of chronic experimental hypersensitivity pneumonitis and desensitization were used to evaluate the effects of systemic cyclosporine. When administered 12 to 18 h before each inhalational challenge with aerosolized antigen and the adjuvant muramyl dipeptide, cyclosporine suppressed the development of disease as well as the anamnestic antibody response, particularly in bronchoalveolar lavage fluids. When administered at the time of sensitization only, cyclosporine suppressed the primary antibody response but not the anamnestic antibody response or the disease. Antigen- and mitogen-induced blastogenesis was inhibited by cyclosporine in vitro, but antigen-specific blastogenesis was not abrogated by cyclosporine previously administered in vivo. These results indicate that cyclosporine caused profound immunomodulation in this model, which can be at least partially explained by transient suppressive effects on T cells, particularly the helper/inducer and delayed hypersensitivity subset(s).

Characterization of the soluble immune complex (EIC) of the amplified enzyme-linked immunosorbent assay (a-ELISA) and an evaluation of this assay for quantitation by reaction stoichiometry.

The molecular composition of the soluble enzyme immune complex (EIC) of alkaline phosphatase (AP) and anti-AP which comprises the detection system of the amplified ELISA (a-ELISA) was investigated. The EIC appeared relatively homogeneous in sucrose density gradients and sedimented as a protein of 600-650 K daltons. Based on size and the results of double-label experiments, the EIC was shown to be composed of two moles of anti-AP and three moles of AP. During reaction with substrate at pH 9.6, greater than 50% of the AP is released as free enzyme and the released enzyme has the same activity as enzyme found in the EIC. The maximum yield of EIC is produced by solubilization of the antibody-AP equivalence precipitate with a 9-fold excess of the amount of AP required for precipitation at equivalence. EICs show no significant loss of activity when stored for one year at 4 degrees C, -20 degrees C or -70 degrees C. The EIC is most stable during long term storage (five years) in 50% glycerol at -20 degrees. Over the linear region of titration curve for dimeric and monomeric M315, the ratio of AP or EIC to M315 fails to show a constant stoichiometry. Using 131I-EIC and 125I-M315, it was determined that the lack of a constant stoichiometry in the linear region was due to differences in the amount of enzyme bound. Hence, stoichiometric quantitation of the primary antibody is not possible using the current a-ELISA.

Interaction of porcine immunoglobulin M with protein A of Staphylococcus aureus.

Intrigued by reports that the mitogenic effect of protein A on B lymphocytes was due to a direct interaction of cell surface immunoglobulin with protein A, the binding of 19 S, 8 S, and Fab mu fragments of 125I-labeled IgM isolated from porcine serum was investigated. Approx. 60% of purified 19 S porcine IgM interacted specifically with Protein A-Sepharose. Mild reduction and alkylation of 19 S IgM to yield monomeric IgM did not appear to alter its ability to bind to protein A. Elution of either molecular species of IgM from protein A and subsequent repassage over Protein A-Sepharose resulted in nearly quantitative rebinding of the IgM to protein A. Fab mu fragments prepared by digestion of 19 S IgM with pepsin exhibited binding characteristics similar to that observed for intact and monomeric IgM. These results suggest that: (1) there are at least two populations of porcine serum IgM, one that binds to protein A and one that does not; (2) these populations are not interconverting; (3) the ability of IgM to bind to protein A is not dependent on the 19 S pentameric structure extant in sera, but rather is an intrinsic property of some and not all four chain IgM protomers; and (4) a binding site for protein A on porcine IgM is localized in the Fab mu (including the C mu 2 domain) regions of the molecule.