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We previously described the processing of antibodies to CD74 (the major histocompatibility complex class II-associated invariant chain, Ii), by B-cell lymphoma cell lines. These cells expressed relatively low levels of Ii on the surface, but the molecules were rapidly internalized and replaced by new molecules, so that approximately 8 x 10(6) antibody molecules per cell were taken up per day. We herein report the results of similar studies with other cell types, namely a melanoma, a colon carcinoma, a T-cell lymphoma and B-lymphoblastoid cell lines. The melanoma and the carcinoma were treated with interferon-gamma to induce high levels of the antigen. The T-cell lymphoma, HUT 78, was selected specifically because it was previously reported to lack cell surface Ii, while expressing the molecule intracellularly. However, HUT 78 displayed Ii on the cell surface, as did the other cell lines tested, and catabolism of the antibody was very fast on all of the cell lines. The capacity of four of the cell lines for cumulative antibody uptake was evaluated, using 'residualizing' radiolabels, which are trapped within the cell after catabolism of the antibody to which they were conjugated. A high level of uptake was observed in all cases, although there was significant variation between the cell lines. With melanoma SK-MEL-37, the total LL1 uptake in 24 hr was nearly 10(7) molecules per cell and the average turnover time for Ii on the cell surface was 4 min; with carcinoma HT-29, the total LL1 uptake in 24 hr was approximately 10(6) molecules per cell, and the average turnover time for Ii on the cell surface was 27 min. Based on the cell content of mature class II antigens (alphabeta), these data suggest that a large fraction, or all, of immature class II molecules (alphabetaIi) reach the cell surface before entering the peptide-loading compartment, independent of the particular cell type.

The catabolism of 99Tcm-antibody conjugates after internalization by B-cell lymphomas was investigated, using antibody LL1, an antibody to the MHC class II invariant chain which is internalized and catabolized very rapidly. Intact IgG antibodies were labelled with 99Tcm after mild reduction. The 99Tcm label was strongly retained within cells, similar to 'residualizing' labels such as 111In-diethylenetriamine pentaacetate (111In-DTPA), but different from a conventional iodine label. Unlike 111In-DTPA, 99Tcm was not retained in a low molecular weight form, but instead was found to be bound to a large number of different cellular proteins, and was retained in the cytoplasm rather than in lysosomes. Therefore, this form of 99Tcm represents a new paradigm of intracellular retention of a radiolabel.

NZW x BXSB F1 mice develop a systemic autoimmune syndrome with various lupus-like manifestations. Male animals develop a degenerative coronary disease with myocardial infarction, resulting in death before 6 mo of age. The presence in these mice of anti-phospholipid Abs reacting with beta2-glycoprotein I may contribute to the pathogenesis of the cardiovascular lesions. beta2-glycoprotein I, a plasma protein implicated in various aspects of the coagulation pathway, is also the target of autoantibodies in humans with the anti-phospholipid syndrome. We obtained several mAbs from NZW x BXSB F1 mice that were selected for binding to cardiolipin. Two mAbs are specific for beta2-glycoprotein I and display a species-dependent pattern with preferential reactivity to mouse beta2-glycoprotein I. The other mAbs display charge-mediated interactions with anionic phospholipids in the absence of beta2-glycoprotein I. The analysis of the V region sequences of the mAbs suggests that cationic residues in the H chain complementarity-determining region 3 are important for their phospholipid reactivity. The structural features of the V(H)-D-J(H) junctions of these mAbs further support the view that an increased frequency of unusual V(D)J rearrangements directly contributes to the development of murine autoimmunity.

Evaluation of laparoscopic myotomy with or without an anti-reflux (Dor) procedure in patients with achalasia.Retrospective.Data were collected from patients who underwent a laparoscopic myotomy for achalasia, following repeated pneumatic dilations. In the period 1993-1998, seven patients were treated in the Dijkzigt location of the Erasmus University Hospital (Rotterdam, the Netherlands). In the period 1995-1999 seven patients were treated in the Free University Hospital (Amsterdam, the Netherlands) by means of a laparoscopic myotomy followed by Dor fundoplication. All of the patients received a check-up from their specialist according to a protocol and in 2000 they were requested to complete a questionnaire concerning how satisfied they were with the operation.In both groups the age ranged from 20 to 60 years (mean ages were 39 and 36 respectively) and the number of men and women was almost equal. There was no conversion. Average operation time was 1.5 hours for the group without fundoplication and 2.5 hours for the group with fundoplication. No perioperative or postoperative complications occurred. During the follow-up period (mean: 3.5 years; range: 1-7) one recurrence was encountered. In the group without fundoplication, reflux oesophagitis was observed in five of the patients. No reflux was encountered in the group with the added fundoplication. All of the patients preferred laparoscopic myotomy to pneumatic dilations.Laparoscopic myotomy was an effective and safe treatment for achalasia following repeated pneumatic dilations. The patients themselves were also satisfied. Without fundoplication, reflux oesophagitis was more prevalent.

We demonstrated previously that human B-cell lymphomas were effectively and specifically killed in vitro by an antibody to CD74 (LL1) linked to (111)In or other Auger electron emitters. This study was intended to more accurately compare the potency and specificity of 3Auger electron emitters, (111)In, 67Ga, and 125I, and to evaluate beta-particle emitters, 131I and 90Y. The unique property of LL1 is its high level of intracellular uptake.Raji B-lymphoma cells were incubated with serial dilutions of the radiolabeled Abs for 2 d and then monitored for cell growth by 2 assays: a cell counting assay and a clonogenic assay. The uptake of radioactivity per cell was monitored at various time points, and the radiation dose was calculated using published S values for radioactivity located in the cytoplasm. Both specific and nonspecific toxicity were evaluated.The beta-particle emitters had considerably higher levels of nonspecific toxicity than the Auger electron emitters, but both 131I and 90Y, and particularly 131I, still had high levels of specificity. Both of these results were consistent with dosimetry calculations. Relative to the delivered disintegrations per cell, 131I and 67Ga were the most potent of the radionuclides tested, with 125I and (111)In being significantly weaker and 90Y being intermediate. The high potency of 67Ga, together with its low nonspecific toxicity, caused this radionuclide to have the highest specificity index.When delivered by Ab LL1, both Auger electron and beta-particle emitters can produce specific and effective toxicity. The choice of the optimal radionuclide for therapy may depend on the ease and efficiency of labeling, the specific activity obtained, the nature of the tumor being targeted, and other factors, but the high specificity indices of the Auger electron emitters may be an advantage.

We previously described the processing of antibodies to CD74 (the major histocompatibility complex class II-associated invariant chain, Ii), by B-cell lymphoma cell lines. These cells expressed relatively low levels of Ii on the surface, but the molecules were rapidly internalized and replaced by new molecules, so that approximately 8 x 10(6) antibody molecules per cell were taken up per day. We herein report the results of similar studies with other cell types, namely a melanoma, a colon carcinoma, a T-cell lymphoma and B-lymphoblastoid cell lines. The melanoma and the carcinoma were treated with interferon-gamma to induce high levels of the antigen. The T-cell lymphoma, HUT 78, was selected specifically because it was previously reported to lack cell surface Ii, while expressing the molecule intracellularly. However, HUT 78 displayed Ii on the cell surface, as did the other cell lines tested, and catabolism of the antibody was very fast on all of the cell lines. The capacity of four of the cell lines for cumulative antibody uptake was evaluated, using 'residualizing' radiolabels, which are trapped within the cell after catabolism of the antibody to which they were conjugated. A high level of uptake was observed in all cases, although there was significant variation between the cell lines. With melanoma SK-MEL-37, the total LL1 uptake in 24 hr was nearly 10(7) molecules per cell and the average turnover time for Ii on the cell surface was 4 min; with carcinoma HT-29, the total LL1 uptake in 24 hr was approximately 10(6) molecules per cell, and the average turnover time for Ii on the cell surface was 27 min. Based on the cell content of mature class II antigens (alphabeta), these data suggest that a large fraction, or all, of immature class II molecules (alphabetaIi) reach the cell surface before entering the peptide-loading compartment, independent of the particular cell type.

To increase tumor-to-nontumor localization ratios of injected radiolabeled antibodies (Abs), several interrelated methods were used.The model systems used were two human carcinoma xenografts grown in nude mice, targeted by antibodies RS11 (antiepithelial glycoprotein-2) or MN-14 (anticarcinoembryonic antigen). The Abs were conjugated with biotin and 111In-benzyl diethylenetriamine pentaacetic acid, and, at various times after injection, were cleared by intraperitoneal injection of galactosylated streptavidin, which delivers the complexes to hepatocytes. The radiolabel used was selected because it is retained within tumors after catabolism of the Ab by the tumor cell but is quite rapidly excreted from hepatocytes into bile.With blood clearance induced at 24 h, and dissection 5 h later, high tumor-to-nontumor ratios were attained. Depending on the model used, tumor-to-blood ratios were 16:1 to 31:1, and tumor-to-nontumor ratios for the kidney, lungs and bone were also high and greatly increased by the clearance regimen. Despite clearance into the liver, tumor-to-liver ratios remained1, due to fairly rapid biliary excretion of the label. The absolute antibody uptake by the tumors was also high, because 24 h was allowed for the Ab to penetrate and bind to cells within the subcutaneous tumors.The method described produced high tumor-to-nontumor ratios at 1 d after injection and may be advantageous for tumor imaging with antibodies. Radiation dosimetry calculations indicate that there is only a slight advantage with this approach for radioimmunotherapy.

We investigated the in vitro cytotoxic potential of Auger electron-emitting radionuclides delivered to the cytoplasm or, more specifically, to lysosomes, via antibodies. The antibody (Ab) used was LL1, which is specific for CD74, an epitope of the major histocompatibility complex (MHC) class II antigen invariant chain, Ii, present on the cell surface. It is taken up in large amounts, approximately 10(7) Ab molecules per cell per day, and delivered to lysosomes. The radioisotopes tested included (111)In, 99mTc and 125I. With sufficient specific activity, approximately 10 mCi/mg Ab, all of these isotopes were potent cytotoxic agents. 125I was active only if a "residualizing" form was used, meaning a form that is trapped within cells after catabolism of the Ab to which it was conjugated (conventional oxidative iodination produces a non-residualizing label). The conjugates of (111)In and 99mTc used are known to be residualizing. One hundred percent cell kill in vitro was obtained with (111)In and 125I, under conditions in which a non-reactive control Ab, conjugated in the same way, produced no significant toxicity. 99mTc was also potent and specific, but appeared somewhat less active than the other isotopes under the conditions evaluated. Although few Abs are accreted by cells at the same rate as LL1, it may be possible to use other Abs to deliver similar amounts of radioactivity, if Abs with higher specific activity can be produced. Such conjugated radioisotopes may be useful for attacking tumor cells in vivo, particularly for single cells or micrometastases.

In two patients, men aged 35 and 69 years admitted postoperatively to the intensive care unit, fever of unknown origin developed. One had been admitted because aspiration was suspected. He had been treated immediately with amoxicillin and clavulanic acid. The other had undergone oesophageal excision and gastric reconstruction because of oesophageal carcinoma and had been subjected to antibiotic decontamination (amphotericin B, norfloxacine en fungizone). No cause for the fever was detected, but it quickly subsided after discontinuation of the amoxicillin-clavulanic acid and the norfloxacine, respectively. When encountering fever of unknown origin in intensive care patients it is always important to think of drug fever.

Previous experiments demonstrated that some human B-cell lymphoma cell lines were unusual in that antibodies bound to the cell surface dissociated at high levels. This did not occur with non-B-cell hematologic tumors or with carcinomas. In this study, additional B-cell lymphoma and lymphoblastoid (Epstein-Barr virus-transformed) cell lines were tested.The antibodies selected for most experiments, MA103 and anti-CD45, react with relatively high avidity to the cell surface. Antibodies to CD19, CD20, and CD22 also were tested on certain cell lines. The antibodies were labeled with 125I. After binding to the surface of viable cells, unbound antibody was washed away, and the fate of the bound antibody was investigated for 2-3 days.Of the eight B-cell lymphomas tested, three had high levels of dissociation, two had low levels of dissociation, and three had intermediate levels of dissociation. The six lymphoblastoid cell lines had only slightly elevated levels of dissociation, relative to non-B cell lines. Sublines of Raji and Ramos cells were identified that varied greatly in the level of antibody dissociation. The level of dissociation from lymphomas was correlated with the tendency of the cell lines to cluster, with single cells displaying less dissociation than clustered cells. However, some exceptions to this correlation were noted. Cell lines such as Ramos, which showed little dissociation of anti-CD20, displayed relatively rapid catabolism of this antibody.The level of antibody dissociation as well as the rate of antibody catabolism will affect the results of radioimmunotherapy strongly because these factors affect the time interval for which the cells are in contact with the radioisotope. Different B-cell lines display markedly different levels of dissociation. There is some evidence suggesting that antibody dissociation is high with fresh human tumor cells, but further investigation of this point is required.

We have attempted to improve the therapeutic index of radioimmunotherapy by manipulating the blood clearance rate and the catabolism of the radiolabel. The general strategy is to allow the antibody (Ab) to circulate in the blood for 2-3 days, then to clear it rapidly by a method that delivers the Ab to hepatocytes. In addition, the radiolabel selected has two key properties: it is a residualizing label (which is lysosomally trapped after catabolism), so it is retained well by tumor cells, but is excreted rapidly by hepatocytes into bile.In initial experiments, three residualizing radiolabels were tested for their rate of excretion after specific delivery in vivo to either hepatocytes, via galactosylated Ab, or Kupffer cells, via immune complexes. A label showing rapid biliary excretion only after delivery to hepatocytes, (111)In-benzyl-diethylenetriamine tetraacetic acid, was then used for radioimmunodetection in a protocol of delayed rapid blood clearance in which clearance was by hepatocytes. This was achieved by using galactosylated Ab, combined with temporary inhibition of the asialo-glycoprotein receptor on hepatocytes. Ab RS11 and the lung adenocarcinoma Calu-3 xenograft in nude mice were used. Control experiments were performed with a conventional 125I label and with 125I-dilactitol-tyramine.Indium-benzyl-diethylenetriamine tetraacetic acid was identified as a label that was excreted more rapidly from hepatocytes than from Kupffer cells, by biliary excretion. Using this radiolabel with delayed rapid blood clearance, very high tumor/blood ratios were obtained, 166:1 at day 3, but tumor/normal tissue ratios for other tissues were not as high. There appeared to be some uptake of the radiolabel by all normal tissues tested, including the lungs and muscle. Dosimetry calculations suggested that the therapeutic index was no better than with a simple Ab injection.Antibody catabolism can be directed towards either hepatocytes or Kupffer cells, and this difference can strongly affect the excretion rate of radiolabels, since only hepatocytes can excrete degradation products into bile. Processing will also depend on the particular radiolabel. These factors are particularly important for protocols involving delayed rapid blood clearance, since liver uptake is so rapid. The methods described should stimulate other approaches of manipulating Ab blood clearance and radiolabel catabolism to achieve improved therapeutic results.

CD22 antibodies (Abs) bound to B-cell lymphomas are known to be internalized and catabolized rapidly. Therefore, it would be expected that use of CD22 as a target for radioimmunotherapy should be enhanced by the use of "residualizing" radiolabels, which are trapped within the cell after catabolism of the Ab to which they had been conjugated. Our study was intended to evaluate this hypothesis using Ab LL2. In initial experiments, we found that LL2 binding was strongly temperature dependent, with approximately 15-fold greater binding at 37 degrees C than at 0 degrees C. A series of experiments suggested that this difference is due to a conformational change in the antigen at low temperature, so that the LL2 epitope is partially blocked. In vitro, residualizing labels-including 125I-dilactitol tyramine and 111In-DTPA-were retained by cells much longer than a conventional iodine label. In vivo, residualizing labels also showed a marked advantage in terms of uptake by Ramos B-cell lymphoma xenografts in nude mice. However, the absolute Ab uptake by xenografts was quite low, in comparison with results obtained with many carcinoma xenografts, which appears to be due in part to vascular properties of the B-cell lymphoma xenografts.

A new cell line derived from a human adenocarcinoma of the colon, GS-7, was propagated as a s.c. tumor in nude mice. This tumor histologically is a mucinous adenocarcinoma (also designated mucoid or colloid) with characteristic large mucin pools that are not lined by an epithelial layer but may contain scattered, randomly distributed cancer cells. Ten to 20% of human colorectal adenocarcinomas are of this histological type, but rapidly growing xenografts with this histology have been rarely used experimentally. This tumor, therefore, constitutes a useful model for similar human tumors. The mucin pools contain large amounts of carcinoembryonic antigen and tumor-associated glycoprotein 72, and the cells express epithelial glycoprotein 2 on their surface. The ability of antibodies injected i.v. to penetrate this tumor was investigated, using both biotinylated and radioiodinated antibodies (Abs). The results demonstrate that Abs can effectively penetrate the mucin pools, and that large amounts of Ab can localize there. This tumor type may have advantages as a target for certain forms of experimental immunotherapy.

In an attempt to recognize general patterns in the processing of Abs (antibodies) bound to the surface of tumor cells, eight monoclonal antibodies were tested on 10 hematological malignancies of various histological types. The results were compared with previous findings obtained with carcinomas, melanomas, and gliomas, using some of the same antibodies. The data demonstrated that some B-cell lymphomas appear to be unusual in that Abs were unable to bind to them irreversibly; except for those Abs that were rapidly internalized, none of the Abs tested was able to bind irreversibly to the B-cell lymphomas Raji or RL. In contrast, most Abs bound irreversibly to the tumors of other histological types, including other hematological tumors. Irreversible Ab binding to B-cell lymphomas was achieved by cross-linking the Abs on the cell surface. Such differences between cell lines may be due to differences in the supramolecular structure of the surface membrane, which affect the frequency or stability of bivalent Ab binding. The Ab binding interaction could not be described in terms of "functional affinity." These results may lead to improvements in the use of Abs for tumor immunotherapy and for other purposes.

NZW x BXSB F1 mice develop a systemic autoimmune syndrome with various lupus-like manifestations. Male animals develop a degenerative coronary disease with myocardial infarction, resulting in death before 6 mo of age. The presence in these mice of anti-phospholipid Abs reacting with beta2-glycoprotein I may contribute to the pathogenesis of the cardiovascular lesions. beta2-glycoprotein I, a plasma protein implicated in various aspects of the coagulation pathway, is also the target of autoantibodies in humans with the anti-phospholipid syndrome. We obtained several mAbs from NZW x BXSB F1 mice that were selected for binding to cardiolipin. Two mAbs are specific for beta2-glycoprotein I and display a species-dependent pattern with preferential reactivity to mouse beta2-glycoprotein I. The other mAbs display charge-mediated interactions with anionic phospholipids in the absence of beta2-glycoprotein I. The analysis of the V region sequences of the mAbs suggests that cationic residues in the H chain complementarity-determining region 3 are important for their phospholipid reactivity. The structural features of the V(H)-D-J(H) junctions of these mAbs further support the view that an increased frequency of unusual V(D)J rearrangements directly contributes to the development of murine autoimmunity.

Processing radiolabeled degradation products is the key factor affecting retention of antibodies within the cell. In this study, we have analyzed the processing of antibodies labeled in nine different ways.Antibodies were labeled with three different radioisotopes and seven different forms of 125I. Eight of the radiolabels (except 188Re) were conjugated to the same antibody, MA103, and tested on the renal carcinoma cell line SK-RC-18 and/or the ovarian carcinoma cell line SK-OV-6. Rhenium conjugation utilized the antibody RS7, the target cell line ME180 and three of the other radiolabels were also tested with this antibody-target cell combination for comparison.Iodine conjugated to antibodies by conventional methods was rapidly released from the cell after antibody catabolism. In contrast, iodinated moieties, such as dilactitol-tyramine and inulin-tyramine were retained within cells four to five times longer.The use of radiolabels that are trapped within cells after antibody catabolism can potentially increase the dose of radiation delivered to the tumor, from the same amount of radioactivity deposited by a factor of four or five. The prolonged retention of 111In relative to 125I is not due to deiodination of iodine conjugates, but rather to intracellular retention of catabolic products containing 111In, perhaps within lysosomes.

Previous experiments indicated that most antibodies binding to cell surface antigens are internalized gradually and degraded within lysosomes, with a half-life of degradation of approximately 1 day, for most antibodies. The research discussed in this article extended our studies to eight additional antibodies reacting with six different antigens, including three antigens anchored in the membrane by glycosyl-phosphatidylinositol. The authors also tested antibodies labeled with 111indium, as well as 125iodine, to determine whether different radiolabels would be processed differently.Antibodies were radiolabeled with 125I or with 111In bound to benzyl-DTPA. After binding to the surface of tumor cells in vitro, excess antibody was washed away, and the fate of the radiolabel was investigated over periods of 3-7 days. Radiolabel released into the supernatant or retained by the cells was analyzed to determine whether it was still on intact antibody.In 13 of the 15 antibodies that were tested, a similar pattern of irreversible binding and gradual catabolism was observed. Iodine conjugated to antibodies was released rapidly from the cell after antibody catabolism. In contrast, the 111In was retained within the cell much longer than 125I, with the rate of degradation and release into the medium being at least fivefold slower. More than 50% of the bound 111In was still present on the cells after 7 days. Biochemical analysis of the retained 111In extracted cells after 4-6 days demonstrated that it was no longer associated with antibodies and was in a low molecular weight form, probably still associated with the chelator benzyl-DTPA.Different radiolabels are processed by tumor cells differently, after catabolism of the antibody to which they originally were conjugated. The data suggest that the prolonged retention of 111In, relative to that of 125I, is due not to deiodination of iodine conjugates, but rather to intracellular retention of catabolic products containing 111In, perhaps within lysosomes. The use of radioisotopes that are retained within cells after antibody internalization and degradation may improve both radioimmunodetection and radioimmunotherapy of cancer.

Sera from 20 species of mammals were tested for their ability to lyse erythrocytes from 18 species of mammals and birds by the alternative complement pathway. Erythrocytes were not lysed by homologous complement, with one minor exception, but all erythrocytes tested were lysed by at least one complement source, and all sera tested except that of the horse lysed at least one type of erythrocyte. Control experiments indicated that lysis was via the alternative complement pathway and that antibodies were not involved. Complement from the various species could be ranked from most active to least active, and erythrocytes could be ranked from most susceptible to least susceptible. There was an inverse correlation between complement activity and erythrocyte susceptibility. The ranking of the orders of placental mammals, from strongest to weakest complement, was carnivore > artiodactyl (ruminants and swine) > primate = armadillo > rodent > rabbit > horse. Opossum serum had activity that placed it in the centre of this range. Ferret complement, the most potent tested, lysed all erythrocytes tested except for homologous erythrocytes, with APCH50 titres as high as 4000. Although the overall reactivity pattern was clear, there were several striking exceptions. For example, the only complement source which lysed ferret erythrocytes was sera of the mouse. The amount of sialic acid present on erythrocytes of 14 mammals was determined, and was, in general, directly correlated with resistance to alternative complement pathway lysis, although there were prominent exceptions to this correlation, involving erythrocytes of the horse, burro and human. All 20 types of complement were also tested for their ability to lyse antibody-coated human tumour cells, under conditions in which both the classical and alternative complement pathways were functional. The data obtained suggest that alternative pathway activation is, in some cases, a major factor determining the effectiveness of a particular complement source in the lysis of xenogeneic tumour cells.

BUB/BnJ mice were previously identified as having exceptionally potent complement activity, relative to common mouse strains, in the lysis of antibody-coated human tumor cells. We describe herein our investigation into the molecular and genetic basis for this difference between mouse strains, and also our results with wild mice and mouse strains recently derived from the wild, to determine whether low complement levels are characteristic of wild mice. BUB complement was compared with complement from BALB/c and C57BL/6 mice. BUB mice had higher levels of most individual classical pathway components, except for C1, than the other two strains, but the difference was generally only 2-3-fold, so insufficient to fully explain the difference observed with tumor target cells. CH50 titers on antibody-coated sheep erythrocytes also demonstrated only a 2-4-fold difference. However, CH50 titers on antibody-coated human erythrocyte target cells demonstrated a difference similar in magnitude to that seen with human tumor targets. These results suggest that the difference between mouse strains depends partly on the use of human, rather than sheep, target cells. In an assay for alternative complement pathway activity using neuraminidase-treated human erythrocytes as targets, complements of BALB/c and BUB mice were similar in activity, suggesting that the difference between mouse strains is manifested in the early steps of complement activation. Analysis of F1 and backcross mice suggested that the difference in complement level between BUB and BALB/c or C57BL/6 mice is controlled by semi-dominant genes, and cannot be attributed to a single gene. Wild mice and mice recently derived from the wild generally had low complement levels, similar to most laboratory mice. However, three strains of aboriginal mice, including Mus hortulanus (spicilegus) and Mus spretus, had complement levels higher than that of BUB mice, and as high as sera from the rabbit or rat, which are the most potent known complement sources for the lysis of human tumor cells. In comparison with BUB mouse sera, M. hortulanus sera had at least four-fold higher levels of C3, C6, C8 and C9, and some or all of these differences may explain its higher total complement activity. In the lysis of antibody-coated human erythrocytes, M. hortulanus serum was more potent than any other complement source tested, including sera of the guinea pig, rat, rabbit or human. These strains may be useful in investigating the role of complement in various pathological processes, and in investigating the genetic regulation of the complement system.

The fate of monoclonal antibodies binding to the surface of human tumor cells in vitro was investigated. Seven antibodies, labeled with 125I, were tested on four cell lines, which included a melanoma and carcinomas of the ovary, kidney, and lung. The antibodies were selected only by the criterion that they not be rapidly internalized via coated pits, so that they would be representative of most antibodies reacting with cell surface antigens. After allowing binding during a 2-h incubation, unbound antibody was removed, and the release of intact or degraded antibody in the supernatant was monitored. The data demonstrate that most bound antibody was gradually degraded and released from the cell over a 2-3-day period, probably via internalization, while only a small fraction, less than 20% for most antibodies, appeared to dissociate intact. One exceptional antibody, MW207, dissociated largely intact. The release of intact antibody was virtually complete within 4 h, and radioactivity released after this time was predominantly in degraded form. These results demonstrate that antibody binding to the surface of viable cells must in general be considered irreversible, and hence the concept of affinity is not applicable. Since an Fab fragment of one of the antibodies dissociated rapidly, such irreversible binding appears to require bivalent attachment. Another conclusion of this study is that most antibodies binding to the cell surface are gradually internalized, which we suggest is due to the normal turnover of cell surface constituents via non-clathrin-dependent endocytosis. Several experimental approaches indicated that a large fraction of antibody retained by the cells, for at least 2 days after binding, was present at the cell surface.

To determine the incidence of oesophageal carcinoma in patients with achalasia and to establish the efficacy of endoscopic surveillance, 195 consecutive patients with achalasia (90 men and 105 women, mean age 52 years), who were treated by pneumatic dilatation in our institution between 1973 and 1988 were prospectively studied. None of the patients had undergone cardiomyotomy. Follow up totalled 874 person years after pneumatic dilatation. In this period three patients developed an oesophageal squamous cell carcinoma. The mean age at diagnosis of the oesophageal carcinoma was 68 years (37, 77, and 89 years). The mean period between the onset of dysphagia and the diagnosis of the tumour was 17 years (19, 28, and 5 years); the mean interval between the diagnosis of achalasia and carcinoma was 5.7 years (5, 8, and 4 years). The incidence of oesophageal squamous cell carcinoma in this series (3.4/1000 patients per year) is significantly higher than the statistically expected incidence (0.104/1000 patients per year) using age and sex specific incidence data from the population of the Netherlands (Poisson statistics: p less than 0.001). The risk of developing oesophageal squamous cell carcinoma in patients with achalasia is therefore increased 33 fold. Periodic endoscopy showed the potential for detecting early stage oesophageal carcinoma in two cases but a larger study with a longer follow up is required to determine the efficacy of endoscopic screening in improving the prognosis for patients with achalasia who develop oesophageal squamous cell carcinoma.

Galactose conjugation of antibodies causes them to be recognized by the hepatic asialoglycoprotein receptor and therefore cleared very rapidly from the blood. In these investigations, some effector functions of galactose-conjugated antibodies were assayed, and several applications to experimental tumors in vivo were demonstrated. Galactose conjugation did not interfere with two antibody functions in addition to antigen binding, namely complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity. This conjugation procedure was originally developed for its potential use in localized immunotherapy, such as i.p. Injection of galactose-antibody conjugates i.p. demonstrated, more conclusively than other methods that have been used, that the presence of ascites causes prolonged retention of antibody in the peritoneal cavity and that this effect is correlated with the volume of ascites present. In mice bearing i.p. tumor xenografts, i.p. injection of galactose-antibody conjugates resulted in high tumor/nontumor ratios at 28 h after antibody injection, with values of 40:1, 43:1, 77:1, and 11:1 for the blood, kidney, lung, and spleen, respectively, although the ratio was only 4:1 for the liver. Control experiments demonstrated that i.p. injection of unconjugated antibody or a galactose-conjugated nonreactive antibody produced much lower tumor/nontumor ratios. In investigations of possible systemic application of galactose-antibody conjugates, we found that injection of large amounts of an inhibitor that binds competitively to the hepatic receptor, asialo-bovine submaxillary mucin, can block clearance of galactose-conjugated antibodies for 2-3 days. In this way, high blood levels of antibody can be maintained for 2-3 days, thus allowing penetration and binding to solid tumors, followed by very rapid blood clearance. With this approach, using a human carcinoma growing s.c. in nude mice, high tumor/nontumor ratios were obtained 4 days after injection, with mean values of 43:1, 18:1, 17:1, and 15:1 for the blood, kidney, lung, and spleen, respectively, although the ratio for the liver was only 1.7:1. The blood level at this time was 0.04 +/- 0.02% (SD) of the injected dose/g, while the tumor level was 1.69 +/- 1.29% of the injected dose/g. In conclusion, galactose-conjugated antibodies appear to have diverse applications in regional or systemic immunotherapy.

The simultaneous occurrence of hypercorticolism and increased plasma catecholemia due to pheochromocytoma does not fit into the concept of the known combinations of multiple endocrine adenomatosis. In this paper we communicate the very rare picture of a adrenocorticotropic (ACTH)-producing pheochromocytoma, giving rise to both clinical entities simultaneously. It is hypothesized that the severity of the clinical picture of this patient was caused by hypercorticolism, which affects the metabolism of catecholamines resulting in a relatively increased production of dopamine.

Hereditary Antithrombin III (AT III) deficiency was found in a large Hindustani family, living partly in the Netherlands, partly in Suriname. Of 201 members investigated 35 were found to affected: AT III activity (chromogenic substrate) and AT III antigen (immuno-electrophoresis according to Laurell) were about 45 %. Analysis of this fanily clearly demonstrated the autosomal dominant inheritance of the condition. Six non-investigated members (1 living, 5 non-living) were diagnosed as being affected on the basis of affected offspring.Seventeen affected members had no signs of thrombo-embolic(TE) processes (age group 0-10 years old, n=2; 11-20, n=5; 21-30, n=4; 31-40, n=4; 41-50, n=2). Thirteen showed clinical or proven signs of TE processes (first time in age group 0-10 years old, n=0; 11-20, n=l; 21-30, n=G; 31-40, n=4; 41-50,n=l; 51-60, n=0; 61-70, n=l). No clinical information is yet available on the remaining affected members. Deep venous thrombosis (DVT) occurred in 9 patients (age group 21-30, n=5; 31-40, n=3; 61-70, n=l). Triggering factors were none 4, surgery 1, oral contraceptives and preg- nancy4. Pulmonary embolism occurred in 6 patients (2 clinical, 4 proven) and was fatal in 4; ages were 19, 21, 26, 37, 48 and 68 years old. Pregnancy was uncomplicated in 3 women (total of 4 pregnancies), one of these women was treated prophylactically with anticoagulants during pregnancy (1 pregnancy). Two women (9 pregnancies) had a thrombotic episode (1st and 3rd pregnancy respectively) and 1 woman died suddenly 7 days after her 7th childbirth. DVT occurred in 2 of 4 women who used oral contraceptive pills.In some symptomless patients (age 22, 26, 32, 33, 40 years old) impedance plethysmography (n=5), 125I-fibrinogen leg scanning (n=3), 125I-fibrinogen T½(n=3) and 51C-platelet survival (n=l) were normal

After a mean follow-up period of 6.1 years, a series of 862 patients who had undergone surgery for suspected hyperparathyroidism were evaluated to determine the reasons treatment failed. Incorrect diagnosis was found to be the cause of failure in 27 cases (3%). In 89 patients who required reoperation to achieve euparathyroidism, the three major reasons for failure were inexperience with the surgical procedure (n = 37), abnormal localization (n = 36), and multiglandular disease (n = 39). In patients with monoglandular involvement, the disease never recurred. However, of the 41 patients with more than one enlarged parathyroid gland, 20 had persistent disease and 21 others had recurrent disease with a normocalcemic interval of 1.2 to 17 years. In general, recurrent hyperparathyroidism occurs more frequently than is usually realized and thus patients with multiglandular involvement require very long follow-up periods. Persistent monoglandular disease is largely avoidable.

The effect of some gelatin plasma substitutes (Haemaccel, plasmagel and Plasmion (Geloplasma), which are widely used in Europe) on collagen-, ADP- and adrenaline-induced platelet aggregation in human PRP in vitro was studied under controlled conditions (pH, electrolyte composition). Haemaccel inhibited these aggregations, both in citrated as well as in heparinised PRP, whereas they were enhanced by both Plasmagel and Plasmion as compared to the appropriate control. Increasing teh concentration of the inducer overcame the inhibition by Haemaccel. Haemaccel inhibited, while Plasmion enhanced 14C-serotonin release induced by collagen, ADP or adrenaline. Also in the presence of indomethacin (90 muM) Haemaccel inhibited aggregation induced by high concentrations of collagen and the primary aggregation induced by ADP and adrenaline, while Plasmion enhanced these aggregations induced by ADP and adrenaline, while Plasmion enhanced these aggregations. The inhibition by Haemaccel was not caused by binding of Ca2+ to haemaccel.

Early postlaparotomy needle jejunostomy feeding with an elemental diet resulted in a typical clinical entity in six of 160 patients (4 per cent). In all, an acute condition of the abdomen developed with a grossly distended intestine, filled with fluid and gas, and an empty stomach as confirmed roentgenographically or at a second laparotomy. This complication is presumably caused by carbohydrate hyperosmolarity of the elemental diet and carbon dioxide production by fermentation. The complication was seen predominantly in patients with a Roux-en-Y reconstructed pancreatectomy. In two patients, a negative second laparotomy was performed. In two others, a pancreaticojejunostomy suture line blowout followed, resulting in death. One patient died after aspiration of the gastric contents.

Five patients were treated with dopamine in doses over 30 micrograms/kg . min in severe circulatory shock and developed fixed dilated pupils. This phenomenon was most likely attributed to the use of dopamine. Further neurological examination gave no support to cerebral damage. Therefore, dilated pupils, unreactive to light, bear no relation to the neurological status during treatment with high doses of dopamine.

Monoclonal antibody localization to human carcinoma xenografts in nude mice was investigated by injecting biotinylated antibody. The antibody used in most experiments, MA103, reacts with a widely distributed human antigen present at a high density on the cell surface and has a relatively high effective affinity, 5.8 x 10(9) M-1. Various doses of antibody were injected, and tumors were collected at various times after injection. Frozen sections were stained by the immunoperoxidase method to detect bound antibody, and adjacent sections were stained with the same antibody in vitro to demonstrate total antigen distribution. In complementary experiments, unconjugated MA103 was injected in high doses to determine whether subsequent in vitro staining of frozen sections by biotinylated MA103 would be inhibited. The results demonstrate that: (a) approximately 0.5 mg antibody was sufficient to saturate antigenic sites on viable tumors cells; (b) saturation was achieved on viable tumor cells throughout the tumor 3 days after injection; (c) in necrotic areas, some antigen was accessible to antibody in vivo, but a considerable fraction of the antigen was inaccessible; (d) at 1 day after injection, stained cells were confined to areas near the blood vessels in the stroma; (e) for i.p. tumors, there was no difference between i.p. and i.v. injection of antibody and no indication of solid tumor penetration directly from the peritoneal cavity; and (f) nonspecific localization of a nonreactive biotinylated monoclonal antibody was weakly detectable and when present was seen as diffuse staining in the connective tissue only. A more limited range of experiments was performed with a second biotinylated antibody, MH99, which reacts with an epithelial cell surface antigen also recognized by many other antibodies, including 17-1A, AUA1, and KS1/4. Results were generally similar, except that a higher dose, 1.5 mg, was required to obtain homogeneous dark staining of tumor cells, and there appeared to be a considerable amount of antigen in viable areas of the tumor that was not accessible in vivo, possibly because it was intracellular. This approach appears to demonstrate tumor localization of antibody more impressively than other methods that have been used. This is partly because of the excellent resolution attained, since the reactive antibody produces a sharp membrane-staining pattern that is totally absent using a nonreactive control antibody. In addition, use of a target antigen that is predominantly on the cell surface, as opposed to cytoplasmic or secreted, is probably important. These data will be of value in attempting to use antibodies for immunotherapy.