Influence of comorbidities on transplant outcomes in patients aged 50 years or more after myeloablative conditioning incorporating fludarabine, BU and ATG.

Non-myeloablative (MA) and reduced intensity allo-SCT regimens are offered to older patients and/or those with comorbidities because the morbidity and mortality attributable to fully MA conditioning is thought to be unacceptably high. A total of 207 patients aged 50-66 years were treated between 1999 and 2008 with SCT after MA conditioning with fludarabine 50 mg/m(2) daily × 5 and i.v. BU 3.2 mg/kg daily × 4.90 (43%) had additional TBI 200 cGy × 2. GVHD prophylaxis was CsA, MTX and thymoglobulin (4.5 mg/kg total dose). As defined by the hematopoietic cell transplantation co-morbidity index (HCT-CI) scoring system 117 (57%) pts scored 0 and 90 (43%) 1. At 5 years OS was 39 vs 54% (P=0.008), disease-free survival 38 vs 49% (P=0.03), TRM 39 vs 19% (P=0.003) and relapse 36 vs 39% (P=ns) in those with scores of 0 and 1, respectively. Multivariate analysis confirmed the influence of HCT-CI scores on TRM (subhazard ratios=2.29; 95% confidence interval=1.29-4.08; P=0.005). We conclude that comorbidities as assessed by the HCT-CI do influence TRM with this regimen but that age alone should not be an indication to prefer a less intense protocol.

A feasibility, pharmacokinetic and frequency-escalation trial of intraperitoneal chemotherapy in high risk gastrointestinal tract cancer.

AIMS: To assess the feasibility, pharmacokinetics and maximum tolerable frequency (MTF) of intraperitoneal (IP) 5-fluorouracil and leucovorin (FU/LV) added, as a regional boost, to intravenous chemotherapy after resection of gastrointestinal cancer. METHODS: Fifty-three patients were recruited following gastrointestinal cancer resection (43 colon; 10 stomach/small bowel) with serosal involvement. Peritoneal ports were implanted and IP fluid distribution evaluated ultrasonically. Twelve patients were studied for pharmacokinetics; 44 (41 evaluable) for MTF. Treatment was weekly intravenous bolus FU/LV for 6 months; to this was added IP FU/LV (400/20 mg/m(2) in 1500 ml 4% icodextrin) with increasing frequency from 4 weekly to 1 weekly in four successive cohorts. RESULTS: Peritoneal fluid distribution was excellent. Intraperitoneal FU exposure (AUC) after IP treatment was >1000-fold plasma AUC after IP treatment (regional pharmacokinetic advantage), and >100-fold plasma AUC after intravenous treatment (regional therapeutic advantage). IP therapy was well tolerated if given every 4, 3 or 2 weeks, but not weekly: 11/13, 7/8, 10/13 and 0/7 patients respectively completed treatment without IP modification in these cohorts. Problems with peritoneal access occurred in 20% of patients. CONCLUSION: Adding fortnightly IP FU/LV to a standard intravenous regimen is safe, tolerable and provides high peritoneal FU exposure. More reliable peritoneal access is needed to improve the feasibility of this otherwise promising therapeutic approach.

Prednisolone and azathioprine in membranous nephropathy: a 10-year follow-up study.

BACKGROUND: Idiopathic membranous nephropathy (IMN) is the most common cause of nephrotic syndrome in adults. Although its clinical course is usually benign, some patients develop chronic renal failure. Combination of corticosteroids with cytotoxic drugs and cyclosporin have been used in the treatment of the disease. Conflicting results are reported with the use ofprednisolone and azathioprine. In this study, the effect of treatment with prednisolone and azathioprine and the parameters related to a poor outcome over a follow-up period of 10 years is estimated. METHODS: 50 patients were included in this study; 33 were treated with prednisolone (initially 60 mg/day) and azathioprine (initially 2 mg/kg body weight/day) in gradually reduced doses for 26 +/- 9 months, whereas 17 patients received no immunosuppressive drugs. The clinical course was estimated using the end-points of doubling of baseline serum creatinine and/or end-stage renal failure (ESRF). The contribution of clinical and histological parameters in the clinical outcome was examined by univariate and multivariate analyses. RESULTS: Doubling of baseline serum creatinine was observed in 20 of 50 patients (40%), 14 from treated and 6 from the untreated group (42% vs. 35%, p=NS). ESRF developed in 10 of 50 patients (20%), 7 from treated and 3 from the untreated group (21% vs. 18%, p=NS). Most patients from both groups who reached the end-points had impaired renal function at presentation and persistent nephrotic syndrome during the follow-up period. Both parameters were identified as independent risk factors related to an unfavorable clinical outcome. No difference in the remission rate of nephrotic syndrome was observed between treated and untreated patients (51% vs. 58%, p=NS). CONCLUSION: Treatment with prednisolone and azathioprine seems to be of no long-term benefit in ameliorating the clinical course of nephrotic patients with membranous nephropathy. Thus, other therapeutic regimens including cyclophosphamide, chlorambucil or cyclosporin should be used in nephrotic IMN patients with poor prognostic features.

Unrelated donor BMT recipients given pretransplant low-dose antithymocyte globulin have outcomes equivalent to matched sibling BMT: a matched pair analysis.

Fifty-seven patients receiving unrelated donor (UD) BMT were matched for disease and stage with 57 recipients of genotypically matched related donor (MRD) BMT. All UD recipients were matched serologically for A and B and by high resolution for DR and DQ antigens. All patients received CsA and 'short course' methotrexate with folinic acid. Unrelated donor BMT patients also received thymoglobulin 4.5 mg/kg (6 mg/kg if <30 kg) in divided doses over 3 days pretransplant. For UD and RD BMT, respectively, incidence of acute GVHD grade II-IV was 19 +/- 6% vs 36 +/- 8%, grade III-IV 10 +/- 6% vs 18 +/- 7%, chronic GVHD 44 +/- 8% vs 51 +/- 8%, non-relapse mortality 15 +/- 5% vs 8 +/- 4% at 100 days, 28 +/- 8% vs 36 +/- 7% at 3 years. At 3 years, relapse was 45 +/- 7% vs 42 +/- 7%, and disease-free survival 39 +/- 7% vs 37 +/- 7%. None of these differences are significant. Three-year overall survival was identical at 42 +/- 7%. For 29 patients with low/intermediate risk leukemia, disease-free survival was 68 +/- 10% after UD BMT vs 59 +/- 9% for RD BMT recipients (P = NS). Corresponding figures for high risk patients were 14 +/- 7% and 21 +/- 8%, respectively. We conclude that UD BMT recipients matched as above and given pretransplant ATG have similar outcomes to recipients of MRD BMT using conventional drug prophylaxis. Unrelated donor BMT should be considered in any circumstance where MRD BMT is routine.

Successful treatment of post-transplant lymphoproliferative disorder in autologous blood stem cell transplant recipients.

We report three cases of post-transplant lymphoproliferative disorder (PTLD) in the context of autologous stem cell transplantation (ASCT) for multiple myeloma (MM) and non-Hodgkin's lymphoma. The first two cases received ASCT for MM, one with a CD34-selected autograft and the other with an unmanipulated autograft. Both these cases of PTLD achieved a complete response following treatment with IVIG, gancyclovir, solumedrol and interferon (IFN). The third case received ASCT with an unmanipulated autograft for relapsed angioimmunoblastic lymphoma. He also achieved a complete response but only after rituximab was added to IVIG, gancyclovir, solumedrol and IFN. None of these patients experienced a relapse of their PTLD with follow-up ranging from 1.5 to 5 years. These cases highlight the importance of considering PTLD in the differential diagnosis of lymphadenopathy and fever post ASCT. They also demonstrate the possibility of durable complete remission of post-ASCT PTLD following antiviral and immune modulating therapy.

Early human renal allograft fibrosis: cellular mediators.

BACKGROUND: The development of early renal interstitial fibrosis (IF) in renal allografts is likely to depend on multiple factors. We studied retrospectively renal biopsies from cadaveric human renal allografts, transplanted from 1996 to 1998, with the intention of detecting early fibrotic changes and determining the underlying cellular mediators. We studied 23 transplant patients whose 46 renal biopsies were analysed, including a donor biopsy taken routinely at implantation from each patient and 23 follow-up biopsies, taken as clinically indicated over a period of 3 months following transplantation. METHODS: Histological evaluation of induction and progression of fibrosis relied on point count analysis of conventional (Masson's trichrome/MT) and immunohistochemical staining for collagen III and IV, and alpha-smooth muscle actin (alpha-SMA) as a marker of myofibroblast differentiation. Mast cells (MC) were counted in sections stained with an anti-human mast cells tryptase monoclonal antibody. Activated macrophages as well as total, helper and cytotoxic T-lymphocytes were identified on frozen sections by direct immunofluorescence using mouse anti-CD71, CD3, CD4 and CD8 antibodies respectively. Eosinophils (E) were counted in hematoxylin and eosin (HE)-stained sections. Changes in interstitial fibrosis (IF) scores were evaluated and correlated with myofibroblasts, MC, E and lympho-monocytic cells. RESULTS: We noted a significant increase in IF over a 3 months period following transplantation. There was also a significant increase in alpha-SMA+ cells, MC and E counts from implantation to follow-up biopsies. Similarly, there was a significant increase in interstitial infiltration by T-lymphocytes (modal category = 2 versus 0, p = 0.012) but not by macrophages. MC at implantation and follow-up were found to be predictive of IF (immunostainable collagen III) at follow-up (R(2) = 0.510, p = 0.023 and p = 0.030). Further, the predictive value for total T-lymphocyte infiltration at follow-up was also significant (R(2) = 0.617, p = 0.036). A strong correlation was found between alpha-SMA+ cells and MC counts at implantation (r = 0.7259, p < 0.001) and in follow-up biopsies (r = 0.5183, p < 0.01). However, there was no correlation between E counts and either alpha-SMA+ cells or MC either at implantation or in follow-up biopsies. Based on changes in interstitial immunostainable alpha-SMA, our patients were divided arbitrarily into 2 groups; group 1 (n = 12) with >100% increase in alpha-SMA and group 2 (n = 11) with <100% increase. Group 1 patients differed significantly from group 2 regarding the degree of MC infiltration at follow-up (t = 0.4519, p < 0.05) with the mean increase in MC count from implantation to follow-up biopsies being +5.1 cells/high power field (HPF) in group 1 and +0.8 cell/HPF in group 2 (p = 0.0237). MC counts in group 1 were associated with a higher modal category (greater than one) of cytotoxic: helper T-lymphocytes ratio compared to group 2 (2:1 versus 1:1 respectively). CONCLUSION: Multiple cells may contribute to early interstitial fibrosis in a subgroup of human renal allograft recipients with MC playing a major role.

A randomized, controlled pilot study of the safety and efficacy of 4% icodextrin solution in the reduction of adhesions following laparoscopic gynaecological surgery.

BACKGROUND: Adhesion-related readmissions are frequent sequelae to gynaecological surgery. Attempts to prevent adhesions by separating healing peritoneal surfaces include site-specific barriers and hydroflotation by instilled solutions. Rapid absorption limits the effectiveness of solutions such as Ringer's lactated saline (RLS). This pilot study assessed the safety, tolerability and preliminary effectiveness of a non-viscous, iso-osmolar solution of 4% icodextrin, an alpha-1,4 glucose polymer with prolonged intraperitoneal residence, in reducing adhesions after laparoscopic gynaecological surgery. METHODS: Women aged > or = 18 years, requiring laparoscopic adnexal surgery (n = 62), were entered into a randomized, open-label, assessor-blinded, multicentre study to compare 4% icodextrin with RLS. Treatments were coded in blocks of four with equal randomization to each group, and pre-allocated to consecutively numbered patients. At least 100 ml per 30 min was used for intra-operative lavage, with 1 l instilled post-operatively. Per protocol analysis included all eligible patients (n = 53); reformation analysis required one or more baseline adhesion (n = 42). Incidence, extent and severity of post-operative adhesions were assessed at second-look laparoscopy after 6-12 weeks. Procedures were video-taped for third party, blinded assessment. RESULTS: Safety and tolerability (laboratory variables, adverse events, clinical follow-up) were good with no difference between treatments. A shift analysis of incidence-ranked adhesions (n = 53) showed apparent improvements in more patients with icodextrin than RLS (37 versus 15%; not significant). Adhesion score reduction (n = 42) was more frequent in icodextrin- than RLS-treated patients: incidence (52 versus 32%), extent (52 versus 47%), and severity (65 versus 37%). Despite greater baseline adhesions, median reformation was less after icodextrin (24%) than RLS (60%). The pilot study group sizes were not powered for statistical significance. CONCLUSIONS: In this preliminary study, 4% icodextrin lavage plus instillation was well tolerated and reduced adhesion formation and reformation following laparoscopic gynaecological surgery. A Phase III pivotal study is currently in progress.

Early progressive interstitial fibrosis in human renal allografts.

BACKGROUND: Early fibrosis has been described in renal allografts and implicated in the progression of chronic allograft nephropathy (CAN). The precise factors implicated in the initiation and progression of early allograft fibrosis remain uncertain. PATIENTS AND METHODS: We studied retrospectively 23 cadaveric renal allograft recipients over a 3-year period, who had paired renal biopsies (Bx) (at implantation and as clinically indicated) within 3 months of transplantation (Tx). Eight of them have progressed over an average period of 3.16 +/- 0.83 years to CAN. Histological evaluation of interstitial fibrosis (IF) relied on point count analysis of Masson's trichrome (MT) staining as well as immunostainable collagens III (iCol III) and IV (iCol IV). The severity of the IF scores was correlated with the clinical, biochemical and histological parameters. The nature and severity of the interstitial inflammatory infiltrate were also evaluated by immunofluorescence. In addition, patients were subdivided into those whose fibrosis progressed (> 50% increase in IF/iCol IlI; Group 1) and non-progressors (< 50% increase in fibrosis score; Group 2) in an attempt to determine discriminatory features. RESULTS: In the whole group, there was a significant increase in the IF score, as estimated by MT staining and iCol III, from implantation to follow-up Bx (p = 0.0027 and p = 0.0088, respectively). The changes in iCol IV were not significant. Further, the increase in interstitial inflammatory infiltrate of total T lymphocytes, and not of macrophages, from implantation (modal category = 2) to follow-up (modal category = 0) was significant (p = 0.0121). The predictive value of such increase was significant (R2 = 0.617, p = 0.03). The donor's age (R2 = 0.892, p = < 0.0001), death from cerebrovascular accident (CVA) (R2 = 0.822, p = 0.047), as well as recipient's body weight (R2 = 0.892, p = 0.001), male gender (R2 = 0.687, p = 0.041) and elevated mean arterial pressure (MAP) (R2 = 0.892, p = < 0.0001) were all significant risk factors for early IF. Delayed graft function (DGF) proved to be a significant predictor of early IF (R2 = 0.822, p = 0.003) and became more significant in the presence of superimposed acute rejection (AR) (p = 0.0001). Proteinuria > 1 g/day (R2 = 0.882, p = 0.004) and hypertriglyceridemia > 2.25 mmol/l (R2 = 0.808, p = < 0.0001) were also associated with early IF. Of the implantation histological parameters, iCol III proved to be a highly significant predictor of early IF (R2 = 0.892, p = < 0.0001). Interestingly, the predictive value of iCol III for graft survival in terms of CAN was significant (Cox p = 0.088). Group 1 progressor patients (n = 10) were all males (p = 0.038) and received their kidneys from donors who died from CVAs in 90% of cases (p = 0.011). They had, compared to non-progressors, a lower cyclosporin A level (p = 0.047), a higher incidence of AR episodes (80% versus 54%), a higher serum creatinine at 10 days post-Tx (p = 0.005), a higher proteinuria (2.07 +/- 3.89 g/l vs 0.96 +/- 0.97 g/l, p = 0.041) and a higher serum triglyceride (2.48 +/- 1.37 mmol/l vs 1.69 +/- 0.81 mmol/l, p = 0.039) level. 8% of Group 1 patients had DGF compared to 30% in Group 2 (p = 0.023). Of note, the modal category of cytotoxic: helper T lymphocytes ratio was greater than 1 in Group 1 (2:1) patients and not in Group 2 (1:1). CONCLUSION: Implantation histology, and in particular iCol III, is a predictor of early IF in a subgroup of patients with DGF and AR. Additional risk factors include hypertension, proteinuria and hypertriglyceridemia especially in patients receiving kidneys from older donors who died of CVAs.

Targeted disruption of semaphorin 3C leads to persistent truncus arteriosus and aortic arch interruption.

Semaphorin 3C is a secreted member of the semaphorin gene family. To investigate its function in vivo, we have disrupted the semaphorin 3C locus in mice by targeted mutagenesis. semaphorin 3C mutant mice die within hours after birth from congenital cardiovascular defects consisting of interruption of the aortic arch and improper septation of the cardiac outflow tract. This phenotype is similar to that reported following ablation of the cardiac neural crest in chick embryos and resembles congenital heart defects seen in humans. Semaphorin 3C is expressed in the cardiac outflow tract as neural crest cells migrate into it. Their entry is disrupted in semaphorin 3C mutant mice. These data suggest that semaphorin 3C promotes crest cell migration into the proximal cardiac outflow tract.

PlexinA2 and semaphorin signaling during cardiac neural crest development.

Classic studies using avian model systems have demonstrated that cardiac neural crest cells are required for proper development of the cardiovascular system. Environmental influences that perturb neural crest development cause congenital heart defects in laboratory animals and in man. However, little progress has been made in determining molecular programs specifically regulating cardiac neural crest migration and function. Only recently have complex transgenic tools become available that confirm the presence of cardiac neural crest cells in the mammalian heart. These studies have relied upon the use of transgenic mouse lines and fate-mapping studies using Cre recombinase and neural crest-specific promoters. In this study, we use these techniques to demonstrate that PlexinA2 is expressed by migrating and postmigratory cardiac neural crest cells in the mouse. Plexins function as co-receptors for semaphorin signaling molecules and mediate axon pathfinding in the central nervous system. We demonstrate that PlexinA2-expressing cardiac neural crest cells are patterned abnormally in several mutant mouse lines with congenital heart disease including those lacking the secreted signaling molecule Semaphorin 3C. These data suggest a parallel between the function of semaphorin signaling in the central nervous system and in the patterning of cardiac neural crest in the periphery.

Lipopolysaccharide-stimulated or granulocyte-macrophage colony-stimulating factor-stimulated monocytes rapidly express biologically active IL-15 on their cell surface independent of new protein synthesis.

Although IL-15 shares many of the biological activities of IL-2, IL-2 expression is primarily under transcriptional regulation, while the mechanisms involved in the regulation of IL-15 are complex and not completely understood. In the current study, we found that CD14(+) monocytes constitutively exhibit both IL-15 mRNA and protein. IL-15 protein was found stored intracellularly and stimulation of CD14(+) monocytes with either LPS or GM-CSF resulted in mobilization of IL-15 stores to the plasma membrane. This rapidly induced surface expression was the result of a translocation of preformed stores, confirming that posttranslational regulatory stages limit IL-15, because it was not accompanied by an increase in IL-15 mRNA and occurred independent of de novo protein synthesis. After fixation, activated monocytes, but not resting monocytes, were found to support T cell proliferation, and this effect was abrogated by the addition of an IL-15-neutralizing Ab. The presence of preformed IL-15 stores and the ability of stimulated monocytes to mobilize these stores to their surface in an active form is a novel mechanism of regulation for IL-15.

The CD3- 16+ 56+ NK cell count independently predicts autologous blood stem cell mobilization.

Better predictive factors for autologous blood stem cell mobilization (BSCM) are needed. The purpose of this study was to determine if an independent association exists between lymphocyte or NK cell counts and BSCM. Data were analyzed on 141 consecutive patients aged 19-69 years (median 45) who received combined chemotherapy plus G-CSF for BSCM, and who had measurements of immune cells prior to BSCM. Of the 141 patients, 41% had breast cancer, 14% Hodgkin's disease, 34% non-Hodgkin's lymphoma, and 11% other diagnoses. BSCM involved dose-intensive cyclophosphamide, etoposide, cisplatin (DICEP) plus G-CSF 300 microg (<70 kg) or 480 microg (>70 kg) for 45% of patients, while the remaining 55% received other chemotherapy plus similar doses of G-CSF. Only a single apheresis was performed for 94% of patients. The following factors were analyzed for predictors of BSCM: age, gender, prior chemotherapy, prior radiotherapy, diagnosis, disease status, marrow involvement, mobilization regimen, Hb, WBC, platelet count, B cell, T cell, and NK cell counts. The peripheral blood CD34+ counts on the first day of apheresis (PBCD34) were 6-1783 x 10(6)/l (median 150). The PBCD34 count correlated strongly with the number of CD34+ cells collected/l blood apheresed and with the number of CD34+ cells collected/kg. By multivariate analysis using continuous variables, relapsed status (P = 0.0003), not using DICEP mobilization (P = 0.0001), female gender (P = 0.0057), low platelet count (P = 0.051), and low CD3- 16+ 56+ count (P = 0.0158) were associated with low PBCD34 counts. Using categorical variables, the only factors that independently predicted a PBCD34 count <150 x 10(6)/l were: >1 prior chemotherapy regimen (odds ratio = 5.12, P = 0.0003), not using DICEP mobilization (odds ratio = 4.94, P = 0.0001), and CD3- 16+ 56+ count <125 x 10(6)/l (odds ratio= 2.58, P = 0.0157). In conclusion, the CD3- 16+ 56+ count may be a useful additional predictor of BSCM and warrants further study.

Fluid dynamics in man of an intraperitoneal drug delivery solution: 4% icodextrin.

Interest in targeting drugs into the peritoneal cavity for intra-abdominal cancers or infections is undergoing a revival as recent clinical trials have demonstrated, not only a regional advantage in concentration of the active agent, but also improved long-term outcomes. Solutions currently used for intraperitoneal (IP) drug delivery have short residence times, however, which can limit the exposure of all areas of the peritoneum to the active agent. Icodextrin 4% solution was compared with saline and a glucose-based peritoneal dialysis solution in a clinical study of IP residence time. The study was carried out during the fortnightly rest phase in 9 patients undergoing 5-fluorouracil (5-Fu) IP treatment for colorectal cancer. The volume remaining in the peritoneal cavity was measured at 0, 12, 24, 48, 72, and 96 hr after an instillation of 2 liters of each fluid. Saline (n = 3 dwells) and glucose (n = 3 dwells) peritoneal dialysis solutions were almost fully absorbed by 24 hr, and the patients experienced discomfort when using these solutions. In contrast, icodextrin 4% solution (n = 188 dwells) maintained its instilled volume for up to 48 hr, and half the instilled volume remained after 72 and 96 hr. This result would allow extensive and prolonged coverage of the peritoneal surface. Icodextrin 4% solution may be an effective vehicle to deliver therapeutic agents into the peritoneal cavity.

Factors involved in clearance of genital warts.

Consecutive patients with first presentation of external anogenital warts had the location, duration at presentation, number of warts and wart area, age, smoking and sun bed use, and presence of coexistent sexually transmitted infections (STIs) recorded. The number of treatment episodes, and number of weeks, to clear the warts were documented. Number of warts and wart area at presentation were associated with time and number of treatments to clear. Those with 1-3 warts required significantly fewer treatment episodes and less time to clear than those with 11-41 warts, as did those with warts area 2-19 mm(2) compared with wart area 100-1038 mm(2). Using survival analysis, the number of warts was significant for the number of treatments and weeks to clear. The hazard ratio for clearance with double the number of warts after 4 treatments was 0.53 (95% confidence interval [CI] 0.37-0.76), and at 4 weeks was 0.70 (0.45-0.86). The clearance rates in non-smokers compared with smokers were higher, but not significantly different. Wart burden at presentation is an indicator of time to clearance. The number of warts is the best predictor - fewer warts results in earlier clearance.

Double high-dose therapy for Hodgkin's disease with dose-intensive cyclophosphamide, etoposide, and cisplatin (DICEP) prior to high-dose melphalan and autologous stem cell transplantation.

We previously reported a 50% (95% CI = 33-76%) 5 year event-free survival (EFS) rate for 23 patients with Hodgkin's disease (HD) who received salvage therapy with single agent high-dose melphalan (HDM) and autologous stem cell transplantation (ASCT). Predictors of poor outcome included bulky disease and initial remission <1 year. Since 1995, similar poor prognosis patients have been treated with double high-dose therapy consisting of dose-intensive cyclophosphamide 5.25 g/m2, etoposide 1.05 g/m2, cisplatin 105 mg/m2 (DICEP) for tumor cytoreduction and stem cell mobilization followed by HDM/ASCT. The purpose of the present study is to determine if the use of DICEP is associated with improved event-free (EFS) and overall survival (OAS) for patients treated with HDM/ASCT. From February 1981 to June 1999, 46 consecutive patients received HDM/ASCT for relapsed (n = 35) or refractory (n = 11) HD. DICEP re-induction and blood stem cell mobilization was used for 21 patients. Factors considered for univariate and multivariate analyses included age at transplant, number of failed chemotherapy regimens, prior radiotherapy, length of initial remission, relapsed or refractory disease status, extranodal relapse, B symptoms at relapse, bulk, post-ASCT radiotherapy, and DICEP re-induction therapy. Cox proportional hazards models were constructed for both event and death. DICEP and HDM were well tolerated with no early treatment-related mortality or toxicity requiring life-sustaining measures. For all 46 patients, the projected 5 year EFS was 52% (95% CI = 38-72%) and OAS was 57% (95% CI = 40-82). Factors independently associated with relapse in multivariate analysis included bulk >5 cm (RR = 6.38, P = 0.002), prior radiotherapy (RR = 3.59, P = 0.027), and not using DICEP (RR = 5.29, P = 0.005). Factors independently associated with death included bulk >5 cm (RR = 5.13, P = 0.009), > or =3 prior chemotherapy regimens (RR = 4.72, P = 0.019), and not using DICEP (RR = 7.49, P = 0.015). This study demonstrates that DICEP re-induction prior to HDM/ASCT is feasible. The preliminary data are sufficiently encouraging to warrant a multicenter phase II or a phase III trial evaluating DICEP followed by HDM/ASCT as salvage therapy for HD.

Development of a novel glucose polymer solution (icodextrin) for adhesion prevention: pre-clinical studies.

Intra-abdominal adhesion formation causes significant post-operative morbidity. Controlled studies using animal models have been carried out to assess the tolerability and preventive efficacy of icodextrin solution (a biodegradable, biocompatible, glucose polymer). Reduction of adhesion formation was first evaluated in a rabbit double uterine horn model, applying 10-75 ml of 7.5 and 20%, or 50 ml of 2.5-20% icodextrin solution post-operatively. Significant increases in adhesion free sites (P < 0.005) were observed with volumes > or =25 ml, and at concentrations > or =4%. Efficacy of 50 ml 4 and 20% icodextrin was then evaluated both during and after surgery, demonstrating significant reductions in adhesion formation (P < 0. 002). In one study, intra- plus post-operative use of 4% icodextrin produced the greatest reduction of non-surgical site adhesions; in others, the post-operative effect was predominant. Post-surgical administration of 50 ml 4% icodextrin in a rabbit sidewall model also resulted in more adhesion-free animals, and a significant reduction (P < 0.001) in areas of adhesion formation and reformation. In a rat infection potentiation model, 4% icodextrin produced no difference in mortality, abscess formation or overall abscess score. These data suggest that 4% icodextrin offers a well-tolerated and effective means of reducing post-surgical adhesion formation.

Activin receptor-like kinase 2 can mediate atrioventricular cushion transformation.

Epithelial-mesenchymal transformation in the atrioventricular (AV) cushion of the tubular heart is a critical step in the formation of the valves and membranous septa. Transforming growth factor beta (TGFbeta) ligands are a primary signal of this transformation. To investigate the expression and function of specific Type I TGFbeta receptors during AV cushion transformation, we cloned and characterized the chicken homologues of two mammalian activin receptor-like kinases (ALK), ALK2 and ALK5, and generated specific, polyclonal antibodies against the extracellular binding domains of each. Both the chicken ALK2 (ChALK2) and the chicken ALK5 (ChALK5) cDNAs encode proteins that bind TGFbeta1 in the presence of the Type II TGFbeta receptor. However, as expected, only ChALK5 stimulated the TGFbeta-responsive PAI-1 promoter. These data establish that ChALK2 and ChALK5 are the chicken homologues of the mammalian receptors ALK2 and ALK5. Both ChALK2 and ChALK5 are expressed by AV endocardial cells. AV cushion explants harvested from stage 13-18 embryos were incubated with antisera to ChALK2 or ChALK5. Anti-ChALK2 antisera inhibited mesenchyme formation by 34-50% while neutralizing anti-ChALK5 antisera were without effect. These data demonstrate that ChALK2 can mediate transformation in the AV cushion.

Migration of cardiac neural crest cells in Splotch embryos.

Pax3 encodes a transcription factor expressed during mid-gestation in the region of the dorsal neural tube that gives rise to migrating neural crest populations. In the absence of Pax3, both humans and mice develop with neural crest defects. Homozygous Splotch embryos that lack Pax3 die by embryonic day 13.5 with cardiac defects that resemble those induced by neural crest ablation in chick models. This has led to the hypothesis that Pax3 is required for cardiac neural crest migration. However, cardiac derivatives of Pax3-expressing precursor cells have not been previously defined, and Pax3-expressing cells within the heart have not been well demonstrated. Hence, the precise role of Pax3 during cardiac development remains unclear. Here, we use a Cre-lox method to fate map Pax3-expressing neural crest precursors to the cardiac outflow tract. We show that although Pax3 itself is extinguished prior to neural crest populating the heart, derivatives of these precursors contribute to the aorticopulmonary septum. We further show that neural crest cells are found in the outflow tract of Splotch embryos, albeit in reduced numbers. This indicates that contrary to prior reports, Pax3 is not required for cardiac neural crest migration. Using a neural tube explant culture assay, we demonstrate that neural crest cells from Splotch embryos show normal rates of proliferation but altered migratory characteristics. These studies suggest that Pax3 is required for fine tuning the migratory behavior of the cardiac neural crest cells while it is not essential for neural crest migration.