A retrospective analysis of early and late outcome of biodegradable stent placement in the management of refractory anastomotic colorectal strictures.

BACKGROUND: Benign colorectal strictures are treated conventionally by endoscopic dilation. Experience using SEMS for benign colonic strictures is limited, and outcomes to date have been disappointing. Refractory colorectal strictures remain challenging to be treated with surgery. Polydioxanone-based stent are biodegradable (BD) stent CE approved for esophageal strictures. This study was designed to investigate retrospectively the safety and the efficacy of these stents for the management of strictures refractory to multiple sessions of dilation. METHODS: Patients with postsurgical benign strictures located within 20 cm from anal verge, refractory to mechanical or pneumatic dilation (at least 3 sessions) were included in this analysis. Clinical success was defined as the absence of occlusive symptoms and the ability to pass through the stricture with a regular size colonoscope. All patients were predilated before stent placement. Stents were released under fluoroscopic control. All patients were under stool softeners for 3 months. Follow-up was scheduled with endoscopic and fluoroscopic controls within 90 days from stent deployment and afterwards by telephone interview and/or ambulatory consultation. RESULTS: Eleven patients (7 males, mean age 62.3 ± 8.5 years) were included. Technical success was achieved in all the patients. Stent migration was observed in four patients within the first 2 weeks after stent placement. Stent migration was followed by recurrence of stricture and obstructive symptoms in all the cases. Among the seven patients who completed the process of stent biodegradation, five of them had complete resolution of the stricture and relief of symptoms. Two of 11 patients required surgical treatment during the follow-up period (mean 19.8 (range 42-15) months). The overall success rate of the BD stent was 45 %. CONCLUSIONS: This retrospective analysis of a limited number of patients demonstrated that nondedicated esophageal BD stents are associated with high risk of migration and clinical success in less than 50 % of patients. Dedicated stents with large diameter and antimigration findings could potentially improve the outcome of patients with refractory benign colorectal strictures.

Randomised clinical trial: low-volume bowel preparation for colonoscopy - a comparison between two different PEG-based formulations.

BACKGROUND: Low-volume bowel preparations with polyethylene glycol (PEG) have been shown to provide an equivalent cleansing with improved tolerability as compared with standard PEG bowel preparation for colonoscopy. A new iso-osmotic sulphate-free formulation of PEG-Citrate-Simethicone (PEG-CS) in combination with bisacodyl has been recently developed. AIM: To compare the quality of bowel cleansing with PEG-CS with bisacodyl vs. PEG-Ascorbate (PEG-ASC) in adult out-patients undergoing colonoscopy. METHODS: Randomised, observer-blind, parallel group study in adult out-patients undergoing colonoscopy in five Italian centres. Both preparations were taken the evening before the procedure. Subjects were instructed to take 2-4 tablets of 5 mg bisacodyl at 16:00 hours and 2 L of PEG-CS at 20:00 hours or 2 L of PEG-ASC plus 1 L of additional water the day before colonoscopy. Bowel cleansing was evaluated according to the Boston Bowel Preparation Scale (≥6 scores were considered as 'clinical success'), and mucosal visibility according to a 3-point scale. Tolerability, acceptability and compliance were also evaluated. RESULTS: Four hundred and eight patients were randomly allocated to PEG-CS and bisacodyl (n = 204, male patient 48%, mean age 59.1 years) or PEG-ASC (n = 204, male patient 51%, age 59.4 years). In the planned per-protocol analysis, the rate of successful preparation was 79.1% following PEG-CS with bisacodyl, and 70% following PEG-ASC (P < 0.05). Mucosal visibility was evaluated as optimal in 56.1% in the PEG-CS and bisacodyl and 46.3% in the PEG-ASC group (P < 0.05). There were no serious adverse events (AE) in each of the two experimental groups. Two subjects in the PEG-ASC group discontinued the study because of AE. CONCLUSIONS: Polyethylene glycol-Citrate-Simethicone in combination with bisacodyl was more effective for bowel cleansing than PEG-ASC for out-patient colonoscopy. Tolerability, safety, acceptability and compliance of the two low-volume bowel preparations were similar.

Safety of cold polypectomy for <10mm polyps at colonoscopy: a prospective multicenter study.

BACKGROUND: Cold polypectomy techniques (without electrocautery) by means of biopsy forceps or snare are widely adopted for the removal of subcentimetric polyps. However, few data are available on the safety of this approach. The aim of this study was to assess the safety of cold polypectomy for subcentimetric polyps, as well as the rate of advanced neoplasia in these lesions. PATIENTS AND METHODS: In a prospective multicenter trial, consecutive patients with at least one < 10-mm polyp at colonoscopy were prospectively included. All of the < 10-mm polyps detected within the study period were removed by cold polypectomy. The rates of immediate or delayed bleeding and other complications were assessed at 7 and 30 days after cold polypectomy by telephone calls. The rate of advanced histology was also assessed. Predictive variables of postpolypectomy bleeding or advanced neoplasia were identified by multivariate analysis. RESULTS: A total of 1015 < 10-mm polyps in 823 patients (15.5 % on antiplatelet agents) were removed. Of these, 822 (81 %) were ≤ 5 mm and 193 (19 %) were 6 - 9 mm. Immediate postpolypectomy bleeding occurred in 18 patients, corresponding to a per-patient and per-polyp bleeding rate of 2.2 % (95 % confidence interval [CI] 1.2 % - 3.2 %) and 1.8 % (95 %CI 1 % - 2.6 %), respectively. Therapy with antiplatelet agents (odds ratio [OR] 4; 95 %CI 1.5 - 10.6) and larger polyp size (OR 2; 95 %CI 1.1 - 6.9) were independent predictors of bleeding. Bleeding was successfully treated by endoscopic hemostasis in all cases and required no further medical intervention. Advanced neoplasia prevalence in polyps ≤ 5 mm was as high as 8.7 %. CONCLUSIONS: The results from this study showed the high safety of a cold polypectomy approach for subcentimetric polyps. This was due to the low rate of postpolypectomy bleeding and to the high efficacy of endoscopic hemostasis in its treatment. The high rate of advanced neoplasia in polyps ≤ 5 mm should prompt some caution on the management of these lesions following detection at computed tomography colonography or colon capsule endoscopy.

Balanced Propofol Sedation in Patients Undergoing EUS-FNA: A Pilot Study to Assess Feasibility and Safety.

Introduction and aims. Balanced propofol sedation (BPS) administered by gastroenterologists has gained popularity in endoscopic procedures. Few studies exist about the safety of this approach during endosonography with fine needle aspiration (EUS-FNA). We assessed the safety of BPS in EUS-FNA. Materials and methods. 112 consecutive patients, referred to our unit to perform EUS-FNA, from February 2008 to December 2009, were sedated with BPS. A second gastroenterologist administered the drugs and monitorized the patient. Results. All the 112 patients (62 males, mean age 58.35) completed the examination. The mean dose of midazolam and propofol was, respectively, of 2.1 mg (range 1-4 mg) and 350 mg (range 180-400). All patients received oxygen with a mean flux of 4 liter/minute (range 2-6 liters/minute). The mean recovery time after procedure was 25 minutes (range 18-45 minutes). No major complications related to sedation were registered during all procedures. The oxygen saturation of all patients never reduced to less than 85%. Blood systolic pressure during and after the procedure never reduced to less than 100 mmHg. Conclusions. In our experience BPS administered by non-anaesthesiologists provided safe and successful sedation in patients undergoing EUS-FNA.

Anti-CD19 immunotoxin enhances the activity of chemotherapy in severe combined immunodeficient mice with human pre-B acute lymphoblastic leukemia.

The anti-CD19 immunotoxin (IT) (HD37-dgRTA) is effective in killing B-lineage leukemia cells and in curing severe combined immunodeficient mice with acute lymphoblastic leukemia. The present study aimed to identify effective combinations of HD37-dgRTA and chemotherapeutic agents. The in-vitro cytotoxicity assays demonstrate that the combination of HD37-dgRTA and either daunorubicin or vincristine is effective. The in-vivo experiments using HD37-dgRTA with vincristine prolonged the survival of mice compared to the chemotherapeutic agent or IT (90.7 vs. 147.1 days). Also, 80% of the mice treated with IT plus vincristine were long-term survivors.

A comparison of an anti-CD25 immunotoxin, Ontak and anti-CD25 microbeads for their ability to deplete alloreactive T cells in vitro.

Ex vivo depletion of alloreactive CD25(+) T cells from a stem cell transplant (SCT) can reduce the incidence of graft-versus-host disease (GVHD) while preserving antimicrobial and perhaps antileukemia activity. However, the most effective methods for allodepleting T cells prior to transplant have not been determined. In this study, we have compared three agents that deplete CD25(+) activated, alloreactive T cells. These included Ontak (Denileukin Diftitox), an IL-2 fusion toxin, anti-CD25 microbeads (MACS), an anti-CD25 immunotoxin (IT) and a combination of the IT and MACS. Peripheral blood mononuclear cells (PBMCs) activated in a primary mixed lymphocyte reaction (MLR) were allodepleted using optimal amounts of each agent, and the cells were then analyzed by flow cytometry. The treated cells were examined both for remaining alloreactivity and for the preservation of third party reactivity by testing them in a secondary MLR. Our data demonstrate that both the anti-CD25 IT and the anti-CD25 MACS were equally effective in depleting CD4(+)CD25(+) cells and in sparing T cells that were reactive with third party cells. The anti-CD25 IT was, however, superior in depleting alloreactive CD8(+)CD25(+) cells. In contrast, Ontak did not eliminate alloreactive cells and the Ontak-treated cells retained significant reactivity against the original stimulator cells.

Add-back of allodepleted donor T cells to improve immune reconstitution after haplo-identical stem cell transplantation.

Poor immune reconstitution after haplo-identical stem cell transplantation results in high mortality from viral infections and relapse. One approach to overcome this problem is to deplete alloreactive cells selectively by deleting T cells activated by recipient stimulators, using an immunotoxin directed against the activation marker CD25. However, the degree of depletion of alloreactive cells is variable following stimulation with recipient PBMC, and this can result in GvHD. We have shown that using recipient EBV-transformed LCL as stimulators to activate donor alloreactive T cells results in more consistent depletion of in vitro alloreactivity while preserving T-cell responses to viral and potential myeloid tumor Ag. Based on these data, we have embarked on a phase I clinical dose escalation study of add-back of allo-LCL-depleted donor T cells in the haplo-identical setting, to determine if the allodepletion we achieve to allow infusion of sufficient T cells to restore useful antiviral/anti-leukemic responses without causing GvHD. Fifteen patients have so far been treated. The incidence of significant acute or chronic GvHD has been low (2/15), as has mortality from infection (1/15). Preliminary data show accelerated immune reconstitution in dose level 2 patients. Infused allodepleted donor T cells appear able to expand significantly in the face of viral reactivations, and doses as low as 3 x 10(5)/kg may be sufficient to confer useful antiviral immunity in this setting. At a median follow-up of 19.5 months, nine of 15 patients are alive and disease-free. Five patients have relapsed, all of whom have died.

An anti-CD45RO immunotoxin kills HIV-latently infected cells from individuals on HAART with little effect on CD8 memory.

CD4+ CD45RO+ T cells are the major latent viral reservoir in HIV-infected individuals and hence a major obstacle in curing the disease. An anti-CD45RO immunotoxin (IT) can decrease the number of both productively and latently infected CD4+ T cells obtained from HIV-infected individuals with detectable viremia. In this study, we determined whether this IT could also kill latently infected replication-competent CD4+ T cells obtained from infected individuals without detectable plasma viremia. Our results demonstrate that ex vivo treatment with the anti-CD45RO IT significantly reduced the frequency of these cells. In contrast, the IT had only a modest effect on the cytomegalovirus-specific memory responses of CD8+ T cells. These results suggest that purging latent cells from infected individuals on highly active antiretroviral therapy with the anti-CD45RO IT might reduce the HIV latent reservoir without seriously compromising CD8+ T cell memory responses.

Clinical-scale selective depletion of alloreactive T cells using an anti-CD25 immunotoxin.

Allogeneic hematopoietic stem cell transplantation is the treatment of choice for many hematological malignancies. Its efficacy is limited by graft-versus-host disease (GVHD), the leading cause of post-transplant morbidity and mortality. GVHD is mediated by a subpopulation of T cells in the stem cell graft. Ex vivo T cell depletion of all T cells of the graft can prevent development of GVHD but can lead to a delay in immune reconstitution and an increase of potentially lethal opportunistic infections and leukemic relapses. Hypothetically, an approach that enables a selective depletion of the alloreactive donor T cells that cause GVHD while preserving third party (anti-leukemic and anti-microbial) reactivity would be optimal for recipients of HSCT. Our preliminary data demonstrated that an anti-CD25 immunotoxin, which reacts with a cell surface activation antigen, can selectively deplete alloreactive donor T cells activated by non-leukemic recipient white blood cells while preserving the beneficial third-party reactivity in vitro. In this report we describe a method for clinical-scale ex vivo selective depletion of alloreactive donor T cells using the anti-CD25 immunotoxin, RFT5-SMPT-dgRTA. Two logs of alloreactive T cells could be selectively depleted while preserving third party reactivity. This method was reproducible in 10 pre-clinical experiments with 8 HLA-mismatched healthy volunteer pairs and 2 HLA-matched sibling donor/patient pairs.

Clinical evaluation of ricin A-chain immunotoxins in patients with Hodgkin's lymphoma.

BACKGROUND: Immunotoxins (ITs) consist of cell binding ligands coupled to toxins or their subunits. Hodgkin's lymphoma (HL) is an excellent target for ITs since lymphocyte activation markers such as CD25 and CD30 are expressed in large numbers. The ITs RFT5.dgA (anti CD25) and Ki-4.dgA (anti CD30) were constructed by linking the monoclonal antibodies RFT5 and Ki-4 to deglycosylated ricin A-chain (dgA). Both ITs showed potent specific activity against HL cells in vitro and in vivo in animal models, and were subsequently evaluated in phase I/II clinical trials in humans. PATIENTS AND METHODS: In two separate trials, the ITs were administered i.v. four times every other day over 4 h. The objectives of the phase I trials included the determination of the maximum tolerated dose (MTD), dose-limiting toxicities (DLTs), pharmacokinetics, antitumor activity and immune response against the IT. RESULTS: Twenty-seven patients with refractory HL were included in the phase I/II study of RFT5.dgA and 17 patients were included in the phase I study of Ki-4.dgA. The MTD of RFT5.dgA was 15 mg/m(2), whereas that of Ki-4.dgA was 5 mg/m(2). DLTs were related to vascular leak syndrome, consisting of edema, tachycardia, dyspnea, weakness and myalgia. Measurement of serum levels of RFT5.dgA demonstrated a C(max) of 0.2-9.7 micro g/ml with a half-life (t()) varying from 4 to 10.5 h. Peak serum concentration of Ki-4.dgA ranged from 0.23 to 1.7 micro g/ml. In both trials approximately 60% of patients developed human anti-mouse and/or anti-dgA antibodies. Seventeen of 18 patients treated at the MTD of RFT5.dgA were evaluable for clinical response. Responses included two partial remissions (PR), one minor response (MR) and five stable diseases (SD). Fifteen of 17 patients treated with Ki-4.dgA were evaluable for clinical response. Responses included one PR, one MR and two SD. CONCLUSIONS: RFT5.dgA and Ki-4.dgA showed moderate efficacy in heavily pretreated refractory patients with HL. Ki-4.dgA was less well tolerated than RFT5.dgA. This might be due, at least in part, to the formation of Ki-4.dgA/sCD30 complexes.

Treatment of SCID/human B cell precursor ALL with anti-CD19 and anti-CD22 immunotoxins.

The anti-CD19 (HD37-dgRTA) and anti-CD22 (RFB4-dgRTA) immunotoxins (ITs) are murine IgG(1) monoclonal antibodies (Mabs) conjugated to a deglycosylated ricin A chain (dgRTA). They are effective in killing B-lineage non-Hodgkin's lymphoma (NHL) cells in vitro, in vivo and in adult patients with B-lineage NHL. The potential of these agents for the treatment of childhood B-precursor acute lymphoblastic leukemia (ALL) is unknown. The anti-CD19 and anti-CD22 ITs should have anti-tumor activity against childhood B-lineage ALL since both target antigens are expressed on the surface of these cells. We have previously shown that, in vitro these two ITs selectively kill leukemia cells obtained from children with leukemia. To evaluate the efficacy of our ITs in an in vivo model we injected the human pre-B ALL cell line, NALM-6-UM1, into severe combined immunodeficient (SCID) mice. We tested the ability of two ITs to prolong survival or cure mice of both early and advanced tumors. In early disease, treatment with HD37-dgRTA, RFB4-dgRTA, or Combotox (an equimolar concentration of the two ITs) significantly improved their survival. In advanced disease, treatment with RFB4-dgRTA or Combotox significantly improved survival. Overall there were 10 long-term survivors who were cured, as determined by survival beyond 150 days with no evidence of disease as determined by polymerase chain reaction (PCR) analysis.

Definitive separation of graft-versus-leukemia- and graft-versus-host-specific CD4+ T cells by virtue of their receptor beta loci sequences.

Although graft-versus-host (GVH) disease (GVHD) is usually associated with graft versus leukemia (GVL), GVL can occur in the absence of clinical GVHD. There is evidence to suggest that GVL and GVH are mediated by different clones of T cells. The objective of this study was to identify the two types of T cells based on their receptor sequences. To this end we used irradiated nonleukemic cells from recipients as stimulator cells in a primary mixed leukocyte reaction (MLR). The activated CD4(+) donor T cells that expressed CD25 were purified by cell sorting. To prepare GVL-specific T cells, alloreactive T cells in the primary MLR were first depleted with an anti-CD25 immunotoxin. The remaining T cells had negligible alloreactivity in a secondary MLR. The allodepleted cells were then stimulated by using purified leukemia cells from the same individual as stimulator cells, and the CD25(+)-activated cells were purified by cell sorting. The GVL- and GVH-specific T cells were analyzed for their T cell receptor (TCR) clonality by using anchored RT-PCR of all the TCRbeta locus complementarity-determining region 3 (CDR3) sequences. By comparing TCRbeta CDR3 sequences from transformed bacterial colonies, we were able to demonstrate that T cells mediating GVH were different from those mediating GVL in each of the eight HLA-mismatched and one HLA-matched donor/recipient pairs. By using the appropriate TCRbeta CDR3-specific primers and probes, the GVH- and GVL-specific clones were monitored in a recipient undergoing an allogeneic stem cell transplant from her HLA-matched related donor.

Optimized clinical-scale culture conditions for ex vivo selective depletion of host-reactive donor lymphocytes: a strategy for GvHD prophylaxis in allogeneic PBSC transplantation.

BACKGROUND: Ex vivo selective depletion (SD) is a strategy to prevent GvHD, in which host-reactive donor lymphocytes are selectively eliminated from a PBSC allograft while conserving useful donor immune function. Prior to testing this strategy in patients, our goal was to develop a clinical-scale SD process, which involves co-culture of donor lymphocytes and irradiated recipient cells, followed by the addition of an immunotoxin (IT) directed against the alpha-chain of the IL-2 receptor (CD25), expressed on activated donor T cells. METHODS: Stimulator cells were generated from immunomagnetically selected and expanded recipient T lymphocytes. Donor PBMCs from G-CSF-mobilized peripheral blood were co-cultured for 72 h with irradiated stimulator cells. Alloreactive T cells were targeted for elimination by the addition of the anti-CD25 IT, RFT5-SMPT-dgA, and the IT enhancer, NH(4)Cl. RESULTS: Stimulator-cell selection/expansion yielded > 2 x 10(10) highly enriched CD3(+) cells (98.9 +/- 2.2%). After SD, cell recovery was 68.5 +/- 23.3% and viability was 84.6 +/- 6.4%. This permitted a potential T-cell dose >/= 1 x 10(8) CD3(+) cells kg(-1) to transplant recipients. Although SD donor lymphocytes retained little proliferative capacity against the original stimulator cells (2.6 +/- 0.6%), responses were conserved against third party cells (107.6 +/- 18.6%), the bacterial superantigen staphylococcus enterotoxin B (108.2 +/- 4.2%), and CMV Ag (72.1 +/- 3.8%). DISCUSSION: We have demonstrated that ex vivo SD is feasible in clinical-scale culture conditions. The ability of this strategy to prevent GvHD is the subject of an ongoing clinical trial, in which the SD lymphocyte product is transplanted in conjunction with a T cell-depleted PBSC allograft.

Monitoring expression of HER-2 on circulating epithelial cells in patients with advanced breast cancer.

Nineteen breast cancer patients with measurable metastatic disease who were starting an initial or new line of therapy were evaluated for circulating epithelial cells (CECs) a minimum of 4 times over the course of treatment. In 7 of the 10 CEC+ patients, HER-2 expression was detected on the CECs. CECs expressing HER-2 varied among patients and in serial samples from the same patient including a shift from HER-2- to HER-2+ CECs. These results demonstrate that it is possible to quantify receptors essential for rationally designed therapy using CECs and that reliance on the immunophenotype of the primary tumor can be misleading.

Chemical construction of immunotoxins.

Immunotoxins are chimeric proteins consisting of an antibody linked to a toxin. The antibodies most frequently used for the preparation of immunotoxins are murine monoclonal antibodies belonging to IgG isotype. The most used toxins for the chemical construction of immunotoxins are Ricin toxin A chain in its deglycosylated form and recombinant Pseudomonas endotoxin with the cell-binding domain deleted. The linkage of the antibody to the toxin can be accomplished by chemical methods using reagents that crosslink antibody to toxin. The usual crosslinkers attach disulfide groups into the antibody molecule to form a disulfide bond between the antibody and the toxin. Disulfide bonds are susceptible to reduction in the cytoplasm of the targeted cells thereby releasing the toxin so that it can exert its cytotoxic activity only into the cells (e.g., tumor cells) binding the antibody moiety. This article describes various methods to obtain antibodies and toxins and several procedures for their crosslinking as well as "in vitro" and "in vivo" testing of the immunotoxins efficacy.

Effects of specific anti-B and/or anti-plasma cell immunotherapy on antibody production in baboons: depletion of CD20- and CD22-positive B cells does not result in significantly decreased production of anti-alphaGal antibody.

Anti-Galalpha1-3Gal antibodies (antialphaGal Ab) are a major barrier to clinical xenotransplantation as they are believed to initiate both hyperacute and acute humoral rejection. Extracorporeal immunoadsorption (EIA) with alphaGal oligosaccharide columns temporarily depletes antialphaGal Ab, but their return is ultimately associated with graft destruction. We therefore assessed the ability of two immunotoxins (IT) and two monoclonal antibodies (mAb) to deplete B and/or plasma cells both in vitro and in vivo in baboons, and to observe the rate of return of antialphaGal Ab following EIA. The effects of the mouse anti-human IT anti-CD22-ricin A (proportional to CD22-IT, directed against a B cell determinant) and anti-CD38-ricin A (proportional to CD38-IT, B and plasma cell determinant) and the mouse anti-human anti-CD38 mAb (proportional to CD38 mAb) and mouse/human chimeric anti-human anti-CD20 mAb (proportional to CD20 mAb, Rituximab, B cell determinant) on B and plasma cell depletion and antialphaGal Ab production were assessed both in vitro and in vivo in baboons (n = 9) that had previously undergone splenectomy. For comparison, two baboons received nonmyeloablative whole body irradiation (WBI) (300 cGy), and one received myeloablative WBI (900 cGy). Depletion of B cells was monitored by flow cytometry of blood, bone marrow (BM) and lymph nodes (LN), staining with anti-CD20 and/or anti-CD22 mAbs, and by histology of LN. EIA was carried out after the therapy and antialphaGal Ab levels were measured daily. In vitro proportional to CD22-IT inhibited protein synthesis in the human Daudi B cell line more effectively than proportional to CD38-IT. Upon differentiation of B cells into plasma cells, however, less inhibition of protein synthesis after proportional to CD22-IT treatment was observed. Depleting CD20-positive cells in vitro from a baboon spleen cell population already depleted of granulocytes, monocytes, and T cells led to a relative enrichment of CD20-negative cells, that is plasma cells, and consequently resulted in a significant increase in antialphaGal Ab production by the remaining cells, whereas depleting CD38-positive cells resulted in a significant decrease in antialphaGal Ab production. In vivo, WBI (300 or 900 cGy) resulted in 100% B cell depletion in blood and BM, > 80% depletion in LN, with substantial recovery of B cells after 21 days and only transient reduction in antialphaGal Ab after EIA. Proportional to CD22-IT depleted B cells by > 97% in blood and BM, and by 60% in LN, but a rebound of B cells was observed after 14 and 62 days in LN and blood, respectively. At 7 days, serum antialphaGal IgG and IgM Ab levels were reduced by a maximum of 40-45% followed by a rebound to levels up to 12-fold that of baseline antialphaGal Ab by day 83 in one baboon. The results obtained with proportional to CD38-IT were inconclusive. This may have been, in part, due to inadequate conjugation of the toxin. Cell coating was 100% with proportional to CD38 mAb, but no changes in antialphaGal Ab production were observed. Proportional to CD20 mAb resulted in 100% depletion of B cells in blood and BM, and 80% in LN, with recovery of B cells starting at day 42. Adding 150cGy WBI at this time led to 100% depletion of B cells in the BM and LN. Although B cell depletion in blood and BM persisted for > 3 months, the reduction of serum antialphaGal IgG or IgM Ab levels was not sustained beyond 2 days. Proportional to CD20 mAb + WBI totally and efficiently depleted CD20- and CD22-positive B cells in blood, BM, and LN for > 3 months in vivo, but there was no sustained clinically significant reduction in serum antialphaGal Ab. The majority of antibody secretors are CD38-positive cells, but targeting these cells in vitro or in vivo with proportional to CD38-IT was not very effective. These observations suggest that CD20-and CD22-positive B cells are not the major source of antialphaGal Ab production. Future efforts will be directed towards suppression of plasma cell function.

The toxicity of deglycosylated ricin A chain-containing immunotoxins in patients with non-Hodgkin's lymphoma is exacerbated by prior radiotherapy: a retrospective analysis of patients in five clinical trials.

A retrospective analysis of 102 patients with relapsed, non-Hodgkin's lymphoma treated with two different ricin A chain-containing immunotoxins (ITs) in five Phase I clinical trials indicates that the dose-limiting toxicity, vascular leak syndrome, was more frequent and more severe in patients who had undergone prior radiotherapy (RT). Excluding patients with prior RT from the calculations of the maximum tolerated dose indicates that the maximum tolerated doses of these ITs had not been reached in any trial and are clearly higher than reported previously. Excluding patients with prior RT from future clinical trials may increase the dose of ITs that can be given in the absence of severe vascular leak syndrome.

A role for intracellular pH in membrane IgM-mediated cell death of human B lymphomas.

We show that anti-IgM-induced cell death in a human B lymphoma cell line, B104, is associated with early intracellular acidification and cell shrinkage. In contrast, another human B cell lymphoma line, Daudi, less susceptible to B cell antigen receptor-mediated cell death, responded to anti-IgM with an early increase in intracellular pH (pH(i)). The anti-IgM-induced changes of pH(i) were associated with different levels of activation of the Na(+)/H(+) exchanger isoform 1 (NHE1) as judged by its phosphorylation status. Prevention of anti-IgM-induced cell death in B104 cells by the calcineurin phosphatase inhibitor, cyclosporin A, abrogated both intracellular acidification and cell shrinkage and was associated with an increase in the phosphorylation level of NHE1 within the first 60 min of stimulation. This indicates a key role for calcineurin in regulating pH(i) and cell viability. The potential role of pH(i) in cell viability was confirmed in Daudi cells treated with an Na(+)/H(+) exchanger inhibitor 5-(N,N-hexamethylene)amiloride. These observations indicate that the outcome of the anti-IgM treatment depends on NHE1-controlled pH(i). We suggest that inactivation of the NHE1 in anti-IgM-stimulated cells results in intracellular acidification and subsequently triggers or amplifies cell death.

Homodimers but not monomers of Rituxan (chimeric anti-CD20) induce apoptosis in human B-lymphoma cells and synergize with a chemotherapeutic agent and an immunotoxin.

In 1997, a chimeric anti-CD20 monoclonal antibody (mAb) (Rituxan) was approved for the treatment of low-grade/follicular B-cell lymphoma. Rituxan has a long half-life and low immunogenicity, and it mediates effector function. Rituxan induces apoptosis in some tumor cell lines in vitro. Previous studies with mAbs that react with neoplastic B cells have demonstrated that homodimers of immunoglobulin G ([IgG](2)) often inhibit cell growth more effectively than their monomeric (IgG)(1) counterparts. In this study, the ability of IgG or F(ab')(2) homodimers vs monomers of Rituxan were compared for their ability to inhibit the growth of several different B-lymphoma cell lines in vitro. It was found that homodimers of Rituxan had superior antigrowth activity in vitro and that F(ab')(2) homodimers were the most active. Homodimers, but not monomers, of Rituxan induced both apoptosis and necrosis of several B-cell lymphoma lines in vitro; the inhibition of cell growth was not dependent upon the presence of Fc receptors or upon 10-fold or greater differences in the density of CD20 on the target cells. Rituxan homodimers, compared with monomers, also rendered drug-resistant CD20(+) B-lymphoma cells more sensitive to chemotherapeutic agents and synergized with an anti-CD22 immunotoxin in vitro.