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.

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.

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.

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.

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.

Synthesis and properties of 5-fluorouracil oligonucleotides.

Oligonucleotides of 5-fluoro-2'-deoxyuridine (FUdR) 1 and 5-fluorouridine (FUR) 2 have been prepared by the standard phosphoramidite method. Homo oligomers (24 mer) of 1, 2 and hetero oligomers of those will be described.

[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.

Role of antibody and T cells in the long-term inhibition of IgE synthesis.

We have shown that the long-term inhibition of IgE synthesis associated with perinatal inoculation of syngeneic IgE is accompanied by the synthesis of autoantibodies to IgE. Synthesis of IgE can also be inhibited by passive transfer of syngeneic anti-IgE antibodies. In the present investigation we made use of adoptive transfer experiments to assess the relative roles of antibodies and T cells in the inhibitory process. It was found that spleen cells from IgE-suppressed mice (synthesizing anti-IgE antibodies) could adoptively transfer the state of inhibition to syngeneic adult mice. The inhibition occurred only under conditions in which the recipient mice synthesized anti-IgE antibodies. Separated B cells, CD4+ T cells, CD8+ T cells, or a mixture of B and CD8+ T cells were ineffective. However, strong inhibition of IgE synthesis (as indicated by serum levels and numbers of IgE-secreting cells in the spleen) was observed after transfer of a mixture of B cells and CD4+ (helper) T cells. The results indicate that in this experimental model anti-IgE antibodies are the suppressive agent and that T cells do not play a role other than that of providing help to B cells for anti-IgE synthesis.

Effects of syngeneic anti-IgE antibodies on the development of IgE memory and on the secondary IgE response.

The prolonged inhibition of IgE synthesis in mice caused by perinatal inoculation of IgE is attributable, at least in part, to the formation of anti-IgE antibodies. The induction of unresponsiveness with respect to IgE synthesis requires that IgE be administered during a brief interval (2 to approximately 10) days after birth, that corresponds with the time period during which anti-IgE antibodies are induced. Passive administration of syngeneic anti-IgE also inhibits IgE synthesis. We have now investigated the effect of anti-IgE on the induction of memory for IgE production and on secondary IgE responses. Syngeneic anti-IgE antibodies were found to inhibit secondary IgE responses directly during immunization or after adoptive transfer of primed cells. Anti-IgE did not, however, prevent the induction of memory cells for IgE synthesis or cause the loss of memory for IgE synthesis. Inhibition of a secondary IgE response was found to require the presence of anti-IgE and was lost when anti-IgE antibodies were cleared from the mouse. After the transfer of primed cells and secondary challenge anti-IgE was inhibitory only when given during the first 3 to 5 days, after which the primed cells became resistant to inhibition. The failure of anti-IgE to prevent the induction of IgE memory cells is discussed in terms of class switches that occur during the transition from IgM to IgE production.

IgE-secreting cells in the thymus: correlation with induction of tolerance to IgE.

We have shown previously that normal mice become tolerant to endogenous IgE when they are approximately 2 weeks old and that this corresponds closely with the initial appearance of IgE in serum. Tolerance evidently is restricted to T cells, since B cells responsive to IgE are present in neonatal and adult mice. The present report shows that IgE-secreting cells can be detected in the thymus between days 7 and 11 after birth and that the onset of tolerance to IgE occurs at the age of 11 days. Similar results were obtained in A/J and (BALB/c x A/J)F1 mice. This suggests that tolerance is induced in the thymus, probably by cells bearing peptide fragments of IgE. The order of appearance of IgE-secreting cells is thymus, spleen, and mesenteric lymph nodes.

Mechanisms of induction of tolerance to syngeneic IgE.

We have previously shown that T cell tolerance to syngeneic IgE in mice is delayed after birth for 2-3 weeks and is induced concurrently with the appearance of IgE in serum. Although inoculation of IgE in saline on the day of birth caused T cell tolerance, an apparent inconsistency was our inability to tolerize mice after day 2, despite evidence indicating that endogenously-produced IgE is effective at the age of 2-3 weeks. Data presented here indicate that repeated i.p. inoculations, during this period, of smaller doses of IgE in saline induce tolerance in many individual mice, thus mimicking to a degree the naturally occurring events. Another earlier finding was that normal adult B cells, but not T cells, can tolerize T cells to IgE when administered on the day of birth. We have now observed that B cells bearing surface IgE are much more tolerogenic than the remaining B 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.

Induction of tolerance to syngeneic IgE in neonatal mice.

We have previously shown that adult A/J mice are tolerant to syngeneic IgE at the level of T cells, but not B cells. T cells of mice are responsive until the age of 2 to 3 wk, which correlates with the time of appearance of serum IgE. Tolerance can be induced earlier by neonatal administration of IgE in saline. We report here that purified nonimmune adult B cells, but not T cells, can transfer the state of tolerance to neonatal mice. As few as 2 x 10(6) B cells are effective. If IgE-bearing or IgE-secreting cells prove to be responsible, the amount of cell-bound IgE that can induce tolerance must be very small. The results also indicate that suppressor T cells do not have a major role in maintenance of self-tolerance to IgE.

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.

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.

Three-dimensional structure of Fab R19.9, a monoclonal murine antibody specific for the p-azobenzenearsonate group.

The crystal structure of Fab R19.9, derived from an anti-p-azobenzenearsonate monoclonal antibody, has been determined and refined to 2.8-A resolution by x-ray crystallographic techniques. Monoclonal antibody R19.9 (IgG2b kappa) shares some idiotopes with a major idiotype (CRIA) associated with A/J anti-p-azobenzenearsonate antibodies. The amino acid sequences of the variable (V) parts of the heavy (VH) and light (VL) polypeptide chains of monoclonal antibody R19.9 were determined through nucleotide sequencing of their mRNAs. The VL region is very similar to that of CRIA-positive anti-p-azobenzenearsonate antibodies as is VH, except for its third complementarity-determining region, which is three amino acids longer; it makes a loop, unique to R19.9, that protrudes into the solvent. A large number of tyrosine residues in the complementarity-determining region of VH and VL, with their side chains pointing towards the solvent, may have an important function in antigen binding.