Prospective clinical study of endoscopic ultrasound-guided choledochoduodenostomy with direct metallic stent placement using a forward-viewing echoendoscope.

A prospective clinical study was conducted to evaluate the safety, feasibility, and efficacy of endoscopic ultrasound (EUS)-guided choledochoduodenostomy (CDS) with direct metallic stent placement using a prototype forward-viewing echoendoscope. The indication for EUS - CDS in this study was lower biliary obstruction only, and not failed endoscopic biliary drainage, because the aim was to evaluate EUS - CDS for first-line biliary drainage therapy. The technical and functional success rates were 94 % (17 /18) and 94 % (16 /17), respectively. Early complications (focal peritonitis) were encountered in two patients (11 %). No patients developed late complications. EUS - CDS with direct metallic stent placement using a forward-viewing echoendoscope was generally feasible and effective for malignant distal biliary tract obstruction. The forward-viewing echoendoscope was useful, especially for deploying the metallic stent.

Immunological responsiveness of neonatal A/J mice to isotypic determinants of syngeneic IgE.

We have previously shown that adult A/J mice produce high titers of anti-IgE with isotypic or idiotypic specificities in response to challenge with a conjugate of KLH with syngeneic monoclonal IgE. Thus, B cells that can synthesize anti-IgE are present in the mice. Adult mice are unresponsive to unconjugated IgE in CFA, suggesting that tolerance exists at the level of T cells. The present study shows that neonatal mice produce anti-IgE antibodies in response to unconjugated IgE in CFA, but that this capacity is lost after the age of 2-3 wk. The loss of responsiveness corresponds closely with the appearance of detectable IgE in serum, suggesting that the IgE may induce tolerance. The affinities of anti-IgE antibodies produced by neonatal mice fall in the range of values obtained with KLH-IgE in adult mice. Tolerance to unconjugated IgE in CFA can be induced in neonatal mice by administration of IgE in saline. In addition, the tolerant state can be induced by adoptive transfer of spleen cells from adult mice. The time-dependent acquisition of tolerance provides a useful model for studying mechanisms of tolerance and autoimmunity.

Inhibition of an IgE response by secondary B cells of a different isotype.

We found that the synthesis of IgE anti-Ars antibodies is strongly inhibited by the presence of secondary non-IgE-producing cells that are specific for the Ars hapten. Such B cells can be induced by inoculation of a protein-Ars conjugate in CFA. The effect is seen after inoculation of OVA-Ars in CFA followed by KLH-Ars in alum, or, more convincingly, after adoptive transfer of B cells induced by antigen in CFA. Dose-response data indicated that inhibition can be effected by B cells containing noninhibitory numbers of contaminating T cells. Possible synergistic effects of carrier-specific regulatory T cells were ruled out by using a different protein carrier for immunization of donor and recipient mice. The effect was shown to be specific for the hapten used for immunization of donor mice.

Structure of idiotopes associated with antiphenylarsonate antibodies expressing an intrastrain crossreactive idiotype.

We have explored the structural basis of idiotopes associated with the major idiotype (CRIA) of A/J anti-p-azobenzenearsonate antibodies, with emphasis on the regions of contact with anti-idiotypic antibody. The analysis was facilitated by a recent description of the three-demensional structure of the Fab portion of a CRIA-related antibody molecule. Direct binding measurements failed to reveal idiotopes associated exclusively with the L chain. However, the L chain participated in the formation of approximately 80% of the idiotopes recognized by polyclonal anti-Id. This indicates that multiple complementarity-determining regions (CDRs) participate in the formation of idiotopes. The affinity of anti-Id for CDRs on L chains must be appreciable but insufficient to permit direct binding (i.e., less than approximately 10(4) M-1). Approximately 20-35% of polyclonal anti-Id reacted with high affinity with H chains recombined with non-CRIA-related L chains. This interaction was found to involve the D region as well as one or both CDRs in the VH segment, again indicating the contribution of multiple CDRs. It is suggested that a typical idiotope may be similar in size to that of protein epitopes whose three-dimensional structures are known; such epitopes comprise a substantial fraction of the surface area occupied by the CDRs of an antibody. The expression of an idiotope recognized by the mAb AD8, which interacts with the VH segment, was found to be unaffected by major changes in the neighboring D and VL regions. This observation is relevant to efforts to predict three-dimensional structure from the amino acid sequence of CRIA+ molecules.

Primary structure of IgE monoclonal antibodies expressing an intrastrain crossreactive idiotype.

We have obtained amino acid sequences (by mRNA and amino acid sequencing) for two IgE kappa mAb that have specificity for the Ars hapten group and are related to the major idiotypic family, CRIA (crossreactive idiotype A), in the A strain of mouse. One mAb, SE20.2, fully expresses CRIA; the other, SE1.3, possesses some but not all of the characteristic idiotopes. Both IgE proteins contain VH and V kappa segments that are closely related to those associated with CRIA. The D segment of SE20.2 is also typical of CRIA+ mAb, but that of SE1.3 is one amino acid residue longer. Chain recombination experiments indicated that the L chain of SE1.3 is fully capable of supporting CRIA expression. Its deficiency with respect to idiotopes of CRIA was attributed to the extra amino acid in the D region and/or substitutions in the VH segment. A major objective was to ascertain the frequency of somatic mutations in IgE. For the VH segment (amino acids 1-98) of SE20.2, there are only three nucleotide differences and one uncertainty with respect to the nucleotide sequence of the germline gene associated with CRIA. A somewhat higher frequency of substitutions is present in the VH segment of SE1.3. The VK amino acid sequences of the IgE proteins are nearly identical to those of a prototype of the CRIA family, mAb R16.7. The results are discussed with reference to the mechanism of the IgM to IgE switch.

Endoscopic retrograde cholangiography versus peroral cholangioscopy to evaluate intraepithelial tumor spread in biliary cancer.

BACKGROUND AND STUDY AIMS: Localized-type bile duct carcinoma (LBDC) is often accompanied by extensive intraepithelial tumor spread (ITS) of 2 cm or more, which makes radical resection more difficult. This retrospective case review compares the diagnostic accuracy of endoscopic retrograde cholangiography (ERC) and peroral cholangioscopy (POCS) to detect ITS beyond the visible LBDC. PATIENTS AND METHODS: Forty-four consecutive patients with LBDC diagnosed between April 2004 and October 2008 who underwent radical resection with histopathological analysis were included in this study. Extensive ITS was found histopathologically in one-third of the cases (32 %). The outcome parameters were the presence or absence of extensive ITS and the extent of extensive ITS proximal and distal to the main tumor. RESULTS: In six cases it was not possible to pass the cholangioscope through the tumor sites. ERC correctly identified the presence of extensive ITS in 11/14 cases and did not yield any false-positive results. The three cases in which ERC was negative were all correctly identified by POCS plus biopsy since the cholangioscope could be passed in all three cases. The extent of extensive ITS was correctly diagnosed by ERC alone, ERC with POCS, and ERC with POCS plus mapping biopsy in 22 %, 77 %, and 100 % of cases, respectively. CONCLUSIONS: The presence of extensive ITS was correctly detected in 80 % of cases by ERC alone. POCS with mapping biopsy provided perfect diagnostic accuracy not only of the presence or absence but also of the extent of extensive ITS. However, POCS has the limitation that the cholangioscope cannot be passed through the tumor sites in approximately 15 % of cases.

Production of syngeneic autoreactive monoclonal antibodies specific for isotypic determinants of IgE.

Methods are described for the production of syngeneic mouse anti-IgE monoclonal antibodies (mAb). Hybridomas were prepared by using spleen cells from mice immunized with a conjugate of keyhole limpet hemocyanin with monoclonal IgE. Serum titers varied from approximately 40 to 1000 micrograms/ml. The anti-IgE mAb were isolated by affinity chromatography on columns containing immobilized monoclonal IgE. The mAb are specific for isotypic determinants of IgE and do not react with other immunoglobulin isotypes. One of the mAb, which has a high affinity for IgE (Ka = 4.7 X 10(8) M-1), should be useful for studies of regulation of IgE. The applicability of the mAb to quantitative assays for IgE was demonstrated.

Efficient production of syngeneic anti-IgE monoclonal antibodies with high affinity and diverse specificity.

Syngeneic monoclonal anti-IgE antibodies are of value in studies of the suppression of IgE synthesis. Procedures are described here for the production of high titers of murine anti-IgE antibodies by initiating immunization in the perinatal period, before mice develop tolerance to their autologous IgE. This in turn facilitates the production of monoclonal anti-IgE antibodies. Properties of some of these mAbs are reported, including affinity, fine specificity and ability to bind to IgE on B lymphoma cells or mast cells.

Proteolytic digestion of mouse IgE.

Conditions are described for the preparation of F(ab')2 and Fab fragments of mouse IgE. Papain, pepsin or trypsin each produced F(ab')2 fragments with Mr approximately equal to 130,000 which yielded Fab fragments on further digestion. The release of Fab fragments from F(ab')2 resulted from further cleavage of the H chain. Pepsin, and especially trypsin appear more suitable for the preparation of F(ab')2 because of the difficulty of separating a 93 kDa by-product from the F(ab')2 produced by papain. The best yields of purified Fab were obtained with papain. Rates of digestion were in the order, pepsin approximately equal to trypsin much greater than papain.

Quantitation of IgE antibodies by radioimmunoassay in the presence of high concentrations of non-IgE antibodies of the same specificity.

Radioimmunoassays for mouse IgE antibodies, based on adherence to an antigen-coated surface, are precise and sensitive, but errors can be introduced by the presence of relatively high concentrations of non-IgE antibodies of the same specificity. Such errors are caused by competition for the limited number of antigenic determinants on the antigen-coated surface. In this paper we explore further the quantitative aspects of the 'competition effect'. An easily applied method is described, based on preferential precipitation of non-IgE antibodies by ammonium sulfate, that permits analysis of IgE antibodies in the presence of large amounts of non-IgE antibodies (that are principally IgG). For IgE anti-Ar, the maximum permissible ratio is extended from approximately 1500:1 to at least 40,000:1. We have also determined the effect of IgG antibodies or whole mouse serum on PCA reactions of mouse IgE antibodies, carried out in rats.

In vitro investigation of factors influencing IgE synthesis.

Cytokines play an important role in the induction or inhibition of synthesis of the particular isotypes of immunoglobulin after antigenic stimulation. Studies of these effects in vivo require substantial amounts of reagents that are difficult and time-consuming to prepare. The present paper describes methods for immunization and in vitro culture that permit the investigation of effects of cytokines and anti-cytokines on a secondary, antigen-specific IgE response, using much lower quantities of materials than are required in vivo. The results are compared with those reported from in vivo studies.

Quantitative measurements of an intrastrain cross-reactive idiotype in IgE antibodies.

This paper describes the development of methods for quantitation of an intrastrain cross-reactive idiotype (CRIA) in IgE antibodies directed to the p-azophenylarsonate (Ar) group and their application to normally immunized and idiotypically suppressed mice. The data were validated by the use of monoclonal and serum anti-Ar antibodies known to possess or lack CRIA. The idiotype was absent in the IgE as well as the total anti-Ar population of idiotypically suppressed mice. A good correlation was noted between the CRIA content of total and IgE anti-Ar with a trend toward a somewhat lower content in the IgE population. Irradiated mice that received immune cells prior to further immunization expressed relatively low concentrations of the idiotype (per weight unit of anti-Ar). The method will be useful in studies of the regulation of the switch to IgE synthesis.

Prolongation of the responsiveness of newborn mice to syngeneic IgE by inhibition of IgE synthesis.

Between the ages of 2 to approximately 11 days mice respond to a challenge with syngeneic IgE by producing anti-IgE antibodies; by the age of 2 weeks they are unresponsive. Even adult mice, however, produce high titers of anti-IgE antibodies when immunized with a conjugate of syngeneic IgE and a foreign antigen such as keyhole limpet hemocyanin (KLH), indicating that adult tolerance to unconjugated IgE resides in the T-cell compartment. The loss of responsiveness in 2-week-old mice follows closely after the first appearance of IgE-secreting cells and detectable serum IgE. This suggests that the delayed onset of tolerance is attributable to the delay in synthesis of IgE. Data presented here provide support for this hypothesis. A further delay in the initial synthesis of IgE, induced by neonatal administration of anti-IgM antibodies, caused a corresponding extension of the period after birth during which mice remain responsive to unconjugated IgE.

[Comparative studies on the evaluation of vibratory perception thresholds using three different instruments, vibratron II, TM-31A and SMV-5--reliability, correlation with age and interrelationship].

Vibratory perception thresholds of 40 (13 males and 27 females) subjects without sensory disturbances were evaluated using three different instruments, Vibratron II, TM-31A and SMV-5, successively three times every third or fourth day to compare their reliability, correlation with age and interrelationship. The intraclass correlation coefficients of the threshold, obtained at the palmar distal phalanx of the index finger of the predominant hand by using Vibratron II and SMV-5 were 0.77 and 0.88, respectively, and those at the radial styloid process on the same side by using TM-31A and SMV-5 were 0.62 and 0.84, respectively. There was a similarity of the intraclass correlation coefficients in the measurement at both sites between the subjects of ages < or = 40 and those of ages > 40, when different instruments were used. However, the thresholds were lower among the subjects of ages < or = 40 than among those of ages > 40. A significantly positive correlation was found between the threshold and the age at both sites when using different instruments. A significantly positive correlation was also found between the thresholds obtained by Vibratron II and SMV-5 at the palmar distal phalanx and between those obtained by TM-31A and SMV-5 at the radial styloid process. The above data indicate that each instrument is applicable not only for diagnosing and evaluating the vibratory sensation disturbances, but also for the follow-up study and evaluation of the efficacy of the specific treatment for the patients with sensory disturbances. They are likewise applicable for the screening and diagnosis of peripheral neuropathy in the occupational and environmental medicines. However, the reliability was lowest in the evaluation by TM-31A, and the correlation coefficient was smallest in the relationship between the age and the threshold obtained by TM-31A.

Induction of high titers of anti-IgE by immunization of inbred mice with syngeneic IgE.

We have generated high titers (up to 1 mg/ml) of antibodies to isotypic determinants of IgE by immunization of A/J mice with syngeneic monoclonal IgE conjugated to keyhole limpet hemocyanin. As much as 3 mg of anti-idiotypic antibodies per ml was induced at the same time. In contrast to conventional rheumatoid factors, the anti-isotypic antibodies are of moderately high affinity (10(7)-10(8) M-1). Assays of the anti-IgE antisera indicated the presence of IgE, both free and in the form of immune complexes; the latter values are minimum estimates owing to masking of isotypic determinants. Regulatory effects of these high titers of anti-IgE can now be investigated. Such studies will be facilitated by the availability of monoclonal, syngeneic anti-IgE antibodies.

Inhibition of IgE synthesis by anti-IgE: role in long-term inhibition of IgE synthesis by neonatally administered soluble IgE.

Inoculation of syngeneic IgE into 2- to 12-day-old mice results in prolonged synthesis of anti-IgE antibodies without further challenge. These anti-IgE antibodies may be largely responsible for the long-term inhibition of synthesis of IgE that is known to result from a perinatal challenge with IgE. This conclusion is supported by the effect of passive inoculation of syngeneic polyclonal anti-IgE antibodies into young mice, which similarly results in selective inhibition of IgE synthesis. Further evidence is the close relationship between the age dependency of IgE-induced inhibition of subsequent IgE synthesis and the ability of IgE to induce anti-IgE antibodies. IgE synthesis was monitored at the level of secretion by B cells as well as serum IgE levels and IgE antibody responses.