Peripheral neuropathies in Waldenström's macroglobulinaemia.

OBJECTIVE: We sought to determine the prevalence, clinical features, and laboratory characteristics of polyneuropathies in Waldenström's macroglobulinaemia (WM), a malignant bone marrow disorder with lymphocytes that produce monoclonal IgM. METHODS: We prospectively studied 119 patients with WM and 58 controls. Medical history was taken, and neurological examinations, electrodiagnostic tests, and serum studies were performed by different examiners who were blinded to results except the diagnosis of WM. RESULTS: Polyneuropathy symptoms, including discomfort and sensory loss in the legs, occurred more frequently (p<0.001) in patients with WM (47%) than in controls (9%). Patients with WM had 35% lower quantitative vibration scores, and more frequent pin loss (3.4 times) and gait disorders (5.5 times) than controls (all p<0.001). Patients with IgM binding to sulphatide (5% of WM) had sensory axon loss; those with IgM binding to myelin associated glycoprotein (MAG) (4% of WM) had sensorimotor axon loss and demyelination. Patients with WM with IgM binding to sulphatide (p<0.005) or MAG (p<0.001) had more severe sensory axon loss than other patients with WM. Demyelination occurred in 4% of patients with WM with no IgM binding to MAG. Age related reductions in vibration sense and sural SNAP amplitudes were similar ( approximately 30%) in WM and controls. CONCLUSIONS: Peripheral nerve symptoms and signs occur more frequently in patients with WM than controls, involve sensory modalities, and are often associated with gait disorders. IgM binding to MAG or sulphatide is associated with a further increase in the frequency and severity of peripheral nerve involvement. Age related changes, similar to those in controls, add to the degree of reduced nerve function in patients with WM.

Hepatic failure as the presenting manifestation of malignant lymphoma.

Fulminant hepatic failure carries a high mortality regardless of etiology. Liver transplantation may be lifesaving. Hepatic invasion by malignant lymphoma is a rare cause of liver failure, but one that is potentially responsive to treatment. Lymphoma (non-Hodgkin's or Hodgkin's) should be included in the differential diagnosis of fulminant hepatic failure so that liver transplantation is avoided and appropriate therapy can be instituted. The findings and clinical course of 4 patients with liver failure due to hepatic lymphoma, who were referred to our institution for liver transplant evaluation, are presented and discussed. Medical records, imaging studies, and histological material were examined. Review of the literature revealed less than 40 cases of lymphoma presenting as fulminant hepatic failure. The diagnosis of malignant lymphoma may be difficult. The presenting symptoms and signs are indistinguishable from other causes of fulminant hepatic failure. Early liver biopsy with adequate tissue and immunologic studies is mandatory for diagnosis. This condition may be reversible and may respond to chemotherapy if the diagnosis is made prior to multiorgan system failure. The presence of malignant lymphoma is considered a contraindication to liver transplantation, although firm data are lacking.

NMR solution structure and backbone dynamics of the CC chemokine eotaxin-3.

Eotaxin-3 is one of three related chemokines that specifically activate chemokine receptor CCR3. We report the 3D structure and backbone dynamics of eotaxin-3 determined by NMR spectroscopy. Eotaxin-3 is monomeric under the conditions in this study and consists of an unstructured N-terminus before the first two conserved cysteine residues, an irregularly structured N-loop following the second conserved cysteine, a single turn of 3(10)-helix, a three-stranded antiparallel beta-sheet, an alpha-helix, and an unstructured C-terminal tail. As in other chemokines, the alpha-helix packs against one face of the beta-sheet. The average backbone and heavy atom rmsd values of the 20 structures (residues 9-65) are 0.44 and 1.01 A, respectively. A comparison between the structures of eotaxin-3 and related chemokines suggests that the electrostatic potential in the vicinity of a surface groove and the structure of the beta2-beta3 turn may be important for maintaining receptor specificity. The backbone dynamics of eotaxin-3 were determined from 15N NMR relaxation data using the extended model free dynamics formalism. Large amplitude motions on the picosecond to nanosecond time scale were observed in both termini and in some residues in the N-loop, the beta1-beta2 turn, and the beta3 strand; the location of these residues suggests a possible role for dynamics in receptor binding and activation. In contrast to eotaxin, eotaxin-3 exhibits no substantial mobility on the microsecond to millisecond time scale.

Growth-inhibitory effects of CD40 ligand (CD154) and its endogenous expression in human breast cancer.

CD40 binding produces multifaceted growth signals in normal and malignant B cells, whereas its physiological role is less well characterized in epithelial cancers. We examined the growth outcome of CD40 ligation in human breast cancer cells, using CD40+ (T47D and BT-20) and CD40-negative (MCF-7, ZR-75-1) cell lines as defined by flow cytometric analysis, immunohistochemistry, and reverse transcription-PCR. Treatment with the soluble recombinant CD40 ligand (CD40L) molecules gp39 or CD40L-trimer significantly reduced [3H]thymidine uptake in BT-20 and T47D cells by up to 40%, but did not affect the growth of CD40-negative MCF-7 or ZR-75-1 cells. Similarly, significant growth inhibition was observed after co-incubation with CD40L-transfected murine L cells (55.0 +/- 8.9%, P < 0.001) that express membrane CD40L constitutively, or with paraformaldehyde-fixed, CD3+ CD40L+ PBLs from three different HLA-mismatched donors (39.7 +/- 3.7%, P < 0.01). Untransfected L cells and non-CD40L-expressing lymphocytes did not produce significant growth inhibition. The in vivo antitumorigenic effects of CD40L were examined using a s.c. severe combined immunodeficient-hu xenograft model. Pretreatment with two different soluble recombinant CD40L constructs (CD40L and gp39) produced similar xenograft growth-inhibitory effects [67 +/- 24% (n = 4), and 65 +/- 14% (n = 8) inhibition, respectively], which were reversed by co-treatment with the CD40L-neutralizing antibody LL48. In vitro analysis indicated that CD40L-induced growth inhibition was accompanied by apoptotic events including cell shrinkage, rounding, and detachment from the adherent T47D culture monolayer. Thirty-one and 27% of gp39-treated T47D and BT-20 cells underwent apoptosis, respectively, as compared with 56 and 65% from the same cell lines after treatment with the Fas agonistic antibody CH-11. An up-regulation of the proapoptotic protein Bax in T47D and BT-20 cells was observed, which indicated that this Bcl-2 family member may contribute to this growth-inhibitory effect. To explore the clinical relevance of CD40L-CD40 interaction, retrospective immunohistochemical analysis was carried to characterize in situ CD40- and CD40L-expression in breast cancer patient biopsies. All of the infiltrating ductal (5 of 5 cases tested) and lobular (4 of 4 cases) breast carcinomas, carcinomas in situ (6 of 6 cases), and mucinous carcinoma tested (1 case) expressed CD40. Varying proportions of tumor cells also expressed CD40L in the majority of infiltrating ductal (3 of 5 cases) and lobular (3 of 4 cases) carcinomas, and carcinomas in situ (4 of 6 cases), as determined by immunohistochemistry and validated by RT-PCR detection of the CD40L message in only CD40L positive-staining cases. Tumor infiltrating mononuclear cells from infiltrating carcinomas and carcinomas in situ expressed CD40 (10 of 10 cases), but less commonly CD40L (1 case of infiltrating lobular carcinoma, 2 cases of carcinoma in situ). Our findings indicate that the CD40 signaling pathway is active in human breast carcinoma cells. However, tumor-infiltrating lymphocytes from primary tumor tissues may be limited in their capacity to directly modulate tumor growth through the CD40L-CD40 loop.

Identification of receptor binding and activation determinants in the N-terminal and N-loop regions of the CC chemokine eotaxin.

Eotaxin is a CC chemokine that specifically activates the receptor CCR3 causing accumulation of eosinophils in allergic diseases and parasitic infections. Twelve amino acid residues in the N-terminal (residues 1-8) and N-loop (residues 11-20) regions of eotaxin have been individually mutated to alanine, and the ability of the mutants to bind and activate CCR3 has been determined in cell-based assays. The alanine mutants at positions Thr(7), Asn(12), Leu(13), and Leu(20) show near wild type binding affinity and activity. The mutants T8A, N15A, and K17A have near wild type binding affinity for CCR3 but reduced receptor activation. A third class of mutants, S4A, V5A, R16A, and I18A, display significantly perturbed binding affinity for CCR3 while retaining the ability to activate or partially activate the receptor. Finally, the mutant Phe(11) has little detectable activity and 20-fold reduced binding affinity relative to wild type eotaxin, the most dramatic effect observed in both assays but less dramatic than the effect of mutating the corresponding residue in some other chemokines. Taken together, the results indicate that residues contributing to receptor binding affinity and those required for triggering receptor activation are distributed throughout the N-terminal and N-loop regions. This conclusion is in contrast to the separation of binding and activation functions between N-loop and N-terminal regions, respectively, that has been observed previously for some other chemokines.

Monoclonal antibodies in the prehybridoma era: a brief historical perspective and personal reminiscence.

Emil von Behring, an immunologist, received the first Nobel Prize in Physiology or Medicine in 1901 for his studies on serum therapy of diphtheria. Seventeen Nobel Prizes have been awarded to scientists for their work in immunology and related disciplines. E. Metchnikoff and P. Ehrlich were pioneers who became associated with cellular and humoral theories of immunity, respectively. Almroth Wright described opsonins and was a vigorous advocate of vaccine therapy for bacterial diseases. He was an influential scientist and mentor who served as the model for Bernard Shaw's play, The Doctor's Dilemma. Immunochemistry developed through the work of K. Landsteiner, M. Heidelberger, E. Kabat, and many others. At mid-20th century, cell-selection theories of antibody formation championed by N. Jerne and F.M. Burnet shifted the field from a chemical to a biological orientation. Myeloma immunoglobulins, Bence Jones proteins, and monoclonal macroglobulins from patients and mice played a central role in elucidation of normal immunoglobulin structure, genetics, synthesis, and metabolism. By the late 1960s, antibody activity in some human myeloma and Waldenström macroglobulin paraproteins had been documented. Subsequently, other human paraproteins were shown to have antigen-binding properties, principally to auto- or bacterial antigens. The development of hybridoma technology by G. Köhler and C. Milstein revolutionized immunology after 1975. These investigators demonstrated that antibody-producing cells of virtually any desired specificity could be fused with a myeloma cell line, the result being unlimited amounts of homogeneous (monoclonal) antibodies carrying that specificity. Monoclonal antibodies have been shown to have efficacy in cancer therapy, particularly in patients with lymphoma and breast cancer. It is likely that this approach, alone and in combination with other modalities, will prove useful for patients with additional types of malignancies.

CD40 ligand-induced apoptosis is Fas-independent in human multiple myeloma cells.

We and others previously demonstrated that human multiple myeloma (MM) cells express CD40 and have an active CD40-growth regulatory pathway. This study characterizes the growth outcome of soluble (gp39) or membrane-bound recombinant human CD40-ligand (rCD40L) and its relationship with Fas-dependent apoptosis. Contrary to the moderate growth-stimulatory effect of the CD40-MAb G28.5, gp39 inhibited 3H-thymidine uptake of the plasma dyscrasia lines ARH-77, U266, and HS-Sultan in a dose-dependent fashion by up to 82%. By comparison, RPMI 8226 cells were resistant to CD40L-growth modulation, which may be attributable to a single base substitution (TCA-->TTA, serine-->leucine) at the 3rd cysteine-rich extramembrane region of CD40. Gp39 similarly reduced myeloma clonogenic colony (MCC) formation in patient primary bone marrow cultures by 50% (40-76%; n=6). Studies using transfectant L cells that constitutively expressed CD40L showed that membrane-bound CD40L inhibited the growth of ARH-77, U266, and HS-Sultan cells (66%, 63%, and 32%, respectively), whereas untransfected L cells did not. Growth inhibition by gp39 or CD40L+ L cells was neutralized by coincubation with the CD40L antibodies 5c8 or LL48. CD40L-treatment increased apoptotic activity of MM cells, as defined by oligonucleosomal DNA fragmentation and an increased binding to annexin V (16-28%). All three untreated CD40-responsive MM lines expressed the Fas/Apo-1/CD95 antigen (65-92% CD95+). However, only ARH-77 cells responded to the growth inhibitory effect of the CD95-agonistic antibody CH-11. CD95 expression was not affected significantly by gp39 treatment, and growth inhibition by CH-11 was additive to gp39 (from 42% to 64% decrease in 3H-thmidine uptake). Conversely, the CD95 antagonist antibody ZB4 reversed the Fas-dependent growth inhibitory process but did not significantly alter gp39-mediated growth outcome. Gp39 treatment lowered the expression of TNFR-associated factors TRAF4 and TRAF6 by 38% and 32%, respectively, whereas detectable levels of TRAF1,2,3, and 5 levels remained unchanged. Our observations indicate that the CD40L-binding inhibits human MM cell growth and increases its apoptotic activity. This growth inhibitory effect corresponds to lower levels of cytoplasmic TRAF signaling elements, and appears independent of the Fas-signaling pathway. CD40 receptor mutation may lead to unresponsiveness to CD40 growth modulation in multiple myeloma cells.

Serologic prevalence of antibody to human herpesvirus type 8 in patients with various monoclonal gammopathies.

Both viral and serologic studies have consistently shown an association of human herpesvirus type 8 (HHV-8) with Kaposi's sarcoma, primary effusion lymphoma, and Castleman's disease. The presence of HHV-8 DNA in patients with myeloma has been reported by some investigators but not substantiated by others. In addition, variable results have been obtained with serologic studies for HHV-8 in patients with myeloma and certain other monoclonal gammopathies (MG). We tested 238 coded serum or plasma samples from 96 patients with various MG for antibodies to lytic and latent HHV-8 antigens by indirect immunofluorescence. Thirty-four of 96 (35%) patients were positive for the lytic antibody, but none were positive for the latent antibody. Patients with kappa or lambda light chain myeloma were often positive for the lytic antibody when compared to patients with IgG or IgA myeloma (8 of 11 [73%] vs. 12 of 38 [32%], P = 0.033). The patients with light chain myeloma also were more likely to be positive when compared to patients with Waldenström's macroglobulinemia (WM) (4 of 15 [27%], P = 0.045) or AL amyloidosis (4 of 13 [31%], P = 0.047). Four of 9 (44%) patients with monoclonal gammopathy of undetermined significance (MGUS) were positive. However, 4 other patients who progressed from MGUS to myeloma were negative. Subgroup analysis of MG may help clarify the role of HHV-8 in these disorders.

Delphi-panel analysis of appropriateness of high-dose therapy and bone marrow autotransplants in newly diagnosed multiple myeloma.

There is controversy whether high-dose therapy and a bone marrow autotransplant or conventional chemotherapy is a better treatment for newly diagnosed multiple myeloma. Data from 1 comparative study and 1 randomized trial provide insufficient subject-level data to advise specific people whether to have an autotransplant. We analyzed appropriate use of high-dose therapy and bone marrow autotransplants in people with newly diagnosed, multiple myeloma using a modified Delphi-panel group judgment process. The panel consisted of 9 myeloma experts from diverse geographic sites and practice settings who reviewed Boolean MEDLINE searches of multiple myeloma and chemotherapy or autotransplants. The panel rated a metric of 64 clinical setting developed by permuting age, performance score, disease-stage and disease-related prognostic variables and response to initial therapy. Each panelist rated appropriateness of high-dose therapy and an autotransplant versus conventional-dose chemotherapy on a 9-point ordinal scale (1, most inappropriate, 9, most appropriate). An appropriateness index was developed based on median rating and amount of disagreement. Relationship of appropriateness indices to the permuted clinical variables was considered by analysis of variance and recursive partitioning. Autotransplants were rated appropriate in persons <55 years old with stage 3 disease and a complete or partial response or stable disease after initial chemotherapy, inappropriate in persons with stage 1 or 2 disease, a performance score <70% and a complete or partial response or stable disease after initial chemotherapy and uncertain in all other settings.

Alpha-heavy chain disease, Mediterranean lymphoma, and immunoproliferative small intestinal disease: a review of clinicopathological features, pathogenesis, and differential diagnosis.

There are a number of clinical syndromes associated with chronic diarrhea, malabsorption, and lymphoplasmacytic proliferation of the small intestine. In Middle-Eastern and Mediterranean countries immunoproliferative small intestinal disease is endemic, whereas in other parts of the world (including Northwestern Europe and North America) celiac sprue, and other sprue-like syndromes refractory to dietary gluten withdrawal, predominate. All of these syndromes appear to involve chronic stimulation of intestinal mucosa-associated lymphoid tissue and are associated with a heightened risk of malignant transformation. The clinicopathological features of these diseases, and distinction of the Middle Eastern syndromes from those more common in the Western hemisphere, have been reviewed.

Active monomeric and dimeric forms of Pseudomonas putida glyoxalase I: evidence for 3D domain swapping.

3D domain swapping of proteins involves the interconversion of a monomer containing a single domain-domain interface and a 2-fold symmetrical dimer containing two equivalent intermolecular interfaces. Human glyoxalase I has the structure of a domain-swapped dimer [Cameron, A. D., Olin, B., Ridderström, M., Mannervik, B., and Jones, T. A. (1997) EMBO J. 16, 3386-3395] but Pseudomonas putida glyoxalase I has been reported to be monomeric [Rhee, H.-I., Murata, K., and Kimura, A. (1986) Biochem. Biophys. Res. Commun. 141, 993-999]. We show here that recombinant P. putida glyoxalase I is an active dimer (kcat approximately 500 +/- 100 s-1; KM approximately 0.4 +/- 0.2 mM) with two zinc ions per dimer. The zinc is required for structure and function. However, treatment of the dimer with glutathione yields an active monomer (kcat approximately 115 +/- 40 s-1; KM approximately 1.4 +/- 0.4 mM) containing a single zinc ion. The monomer is metastable and slowly reverts to the active dimer in the absence of glutathione. Thus, glyoxalase I appears to be a novel example of a single protein able to exist in two alternative domain-swapped forms. It is unique among domain-swapped proteins in that the active site and an essential metal binding site are apparently disassembled and reassembled by the process of domain swapping. Furthermore, it is the only example to date in which 3D domain swapping can be regulated by a small organic ligand.

Decreases in levels of serum fibronectin predict the severity of vascular leak syndrome in patients treated with ricin A chain-containing immunotoxins.

The major dose-limiting adverse effect of ricin A chain-containing immunotoxin (IT) therapy is vascular leak syndrome (VLS). Since plasma fibronectin (Fn) plays a role in maintaining microcirculatory integrity and since the gradient between plasma and tissue Fn can be altered in various pathological situations, we determined whether the administration of IT-ricin A chain to patients resulted in changes in the levels of serum Fn and, if so, whether these changes correlated with the severity of VLS. We also measured the serum levels of tumor necrosis factor alpha (TNFalpha), a proinflammatory cytokine which has been implicated in tissue damage and in interleukin 2-mediated VLS. Our results indicate that the most severe manifestations of VLS were associated with the highest pretreatment levels of Fn, the largest decreases in Fn immediately after starting IT therapy, increases in the levels of serum TNFalpha, higher concentrations of circulating IT, and the lowest numbers of circulating tumor cells. These parameters should, therefore, be useful for predicting which patients will have severe VLS.

A phase I study of bolus versus continuous infusion of the anti-CD19 immunotoxin, IgG-HD37-dgA, in patients with B-cell lymphoma.

IgG-HD37-SMPT-dgA is a deglycosylated ricin A chain (dgA)-containing immunotoxin (IT) prepared by conjugating the monoclonal murine (MoAb) anti-CD19 antibody, HD37, to dgA using the heterobifunctional hindered disulfide linker, N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio) toluene (SMPT). In this report, we have used two regimens for the administration of IgG-HD37-SMPT-dgA to patients with non-Hodgkin's lymphoma (NHL) in two concomitant phase I trials. One trial examined four intermittent bolus infusions administered at 48-hour intervals. The other studied a continuous infusion (CI) administered over the same 8-day period. In the intermittent bolus regimen, the maximum tolerated dose (MTD) was 16 mg/m2/8 d and the dose-limiting toxicity (DLT) consisted of vascular leak syndrome (VLS), aphasia, and evidence of rhabdomyolysis encountered at 24 mg/m2/8 d. Using the CI regimen, the MTD was defined by VLS at 19.2 mg/m2/8 d. At the MTD of both regimens, a novel toxicity, consisting of acrocyanosis with reversible superficial distal digital skin necrosis in the absence of overt evidence of systemic vasculitis, occurred in 3 patients. Of 23 evaluable patients on the bolus schedule, there was 1 persisting complete response (CR; > 40 months) and 1 partial response (PR). Of 9 evaluable patients on the continuous infusion regimen, there was 1 PR. Pharmacokinetic parameters for the bolus regimen at the MTD showed a mean maximum serum concentration (Cmax) of 1,209 +/- 430 ng/mL, with a median T1/2 beta for all courses of 18.2 hours (range, 10.0 to 80.0 hours), a volume of distribution (Vd) of 10.9 L (range, 3.1 to 34.5 L), and a clearance (CL) of 0.45 L/h (range, 0.13 to 2.3 L/h). For the CI regimen at MTD, the mean Cmax was 963 +/- 473 ng/mL, with a median T1/2 beta for all courses of 22.8 hours (range, 24.1 to 30.6 hours), a Vd of 9.4 L (range, 4.4 to 19.5 L), and a CL of 0.32 L/h (range, 0.12 to 0.55 L/h). Twenty-five percent of the patients on the bolus infusion regimen and 30% on the CI regimen made antibody against mouse Ig (HAMA) and/or ricin A chain antibody (HARA). We conclude that this IT can be administered safely and that both regimens achieve comparable peak serum concentrations at the MTD; these concentrations are similar to those achieved previously using other regimens with IgG-dgA ITs at their respective MTDs. Thus, toxicity is related to the serum level of the IT and does not differ with different targeting MoAbs.

CD40 and the effect of anti-CD40-binding on human multiple myeloma clonogenicity.

CD40 is a 48 kDa glycosylated phospoprotein that is a member of the tumor necrosis factor receptor (TNF-R) superfamily. CD40 was originally identified in B lymphocytes, and is found on monocytes, dendritic cells, some carcinoma cell lines, and the thymic epithelium. CD40 is expressed on normal pre-B through mature B stages of differentiation. For normal B cells, the cross-linking of CD40 induces cell cycle progression, long-term proliferation in vitro, IgE secretion, increased adhesion molecule (LFA-1) expression, and low level IL-6 secretion. The natural ligand of CD40 (CD40L, gp39, or T-BAM, for T-B cell activating molecule) was recently identified as an inducible molecule expressed transitionally on activated T cells. Although originally believed to be absent in normal and malignant plasma cells, CD40 has been demonstrated on the majority of myeloma cell lines and myeloma cells from plasma cell dyscrasia (PCD) patient specimens tested. CD40 activation modulated myeloma cell proliferation and clonogenicity in vitro, suggesting that the CD40 pathway is active in myeloma cell growth. For the IL-6 dependent cell line ANBL-6, CD40 activation was associated with autocrine IL-6 production. However, the IL-6 pathway does not appear to play a predominant role in CD40 activation of non-IL-6-dependent MM cell lines and patient primary bone marrow cultures. The possible pathophysiologic role of the CD40 receptor in human multiple myeloma is discussed.

Chemosensitization of human hepatocellular carcinoma cells with cyclosporin A in post-liver transplant patient plasma.

We previously showed that combined neoadjuvant doxorubicin (DOX) treatment and orthotopic liver transplantation produced a 3-year tumor-free survival rate of 54% in stage II-IVa nonresectable hepatocellular carcinomas (HCCs). These patients received posttransplant immunosuppressive doses of cyclosporin A (CsA). CsA has been shown to modify the function of a membrane P-glycoprotein (Pgp) whose overexpression is associated with a multidrug-resistant (MDR1) phenotype. This study utilized HCC cell lines to characterize the in vitro chemomodulatory properties of CsA as found in posttransplant patient plasma to consider the hypothesis that CsA may prolong posttransplant survival by enhancing the therapeutic efficacy of DOX against multidrug-resistant hepatoma cells. We characterized Pgp expression in the HCC lines Hep3B, Hep G2, and SK-HEP-1 by immunohistochemistry and the reverse transcription-polymerase chain reaction. The combined cytotoxicity of DOX + CsA was examined by [3H]thymidine uptake and flow cytometric drug-retention assays. Pgp expression was assessed further after prolonged (10-day) treatment with CsA. Hep3B and Hep G2 cells expressed low to moderate levels of Pgp. The effective DOX dose required for inhibiting MDR1(+) Hep3B and Hep G2 cell proliferation by 50% (DOX IC50) was 44.5 ng/ml and 43.5 microgram/ml, as compared with 10.7 ng/ml for Pgp-negative SK-HEP-1 cells. Optimal concentrations of CsA (0.8 micrometer) lowered DOX IC50 for Hep3B cells and Hep G2 cells by 6-fold and 4-fold, respectively. Similarly, plasma from patients containing immunosuppressive levels of CsA lowered DOX IC50 of the MDR1(+) Hep G2 cells by up to 4-fold. Prolonged exposure to CsA did not affect its chemosensitizing capacity or Pgp expression of HCC cells. PSC-833, a nonimmunosuppressive analogue of CsA, was equally effective in reducing the DOX IC50 of MDR1(+) HCC cells. CsA and PSC-833 increased drug retention by approximately 75%, but did not significantly affect hepatoma cell viability or Pgp expression. Pharmacological concentrations of cyclosporin analogues, including one nonimmunosuppressive form, enhance DOX cytotoxicity of MDR1(+) HCC cells by modulating drug retention. CsA as found in posttransplant patient plasma enhanced DOX cytotoxicity to human MDR1(+) hepatoma cells in vitro, albeit at less than optimal chemosensitizing concentrations. Prolonged exposure to CsA did not affect its chemosensitizing properties or block Pgp expression of HCC cells. These findings support our hypothesis that in vivo immunosuppressive levels of CsA may enhance DOX chemotherapeutic efficacy on MDR1(+) HCC cells.

Anti-CD40 antibody binding modulates human multiple myeloma clonogenicity in vitro.

Ligand binding of the B-cell lineage antigen CD40 enhances growth and interleukin-6 (IL-6) secretion in human B cells (the CD40/IL-6 loop). IL-6 has an autocrine and paracrine role in human multiple myeloma (MM) cell growth. With the use of the CD40 monoclonal antibody (MoAb) G28-5, we examined CD40 expression and the effect of CD40 binding on MM clonogenic colony (MCC) formation to characterize the IL-6/CD40 loop activity in MM. CD40 was expressed on plasmacytoid cells in 21 of 28 plasma cell dyscrasia (PCD) bone marrow (BM) biopsies tested (10 of 14 MM, 2 of 2 Waldenstrom's macroglobulinemia [WM], 2 of 2 plasma cell leukemia [PCL], 6 of 8 monoclonal gammopathy of undetermined significance [MGUS], and 1 of 2 primary amyloidosis [AL]). G28-5 binding increased MCCs by 35% to 150% in 11 of 17 CD40+ PCD BM cultures, but did not affect MCC formation in CD40- specimens or normal BM colony forming units (CFU-GEMM, CFU-GM, BFU-E). Responsive cultures originated from BM of patients with MM (2 of 5 cases tested), WM (2 of 2), PCL (2 of 2), and MGUS (5 of 6). CD40-responsiveness was not significantly inhibited by the presence of an anti-IL-6 MoAb (2 of 2 MGUS cultures tested), and did not correlate with the capacity to respond to IL-6 stimulation (n = 17, P > .05) or a detectable level of endogenous IL-6 (n = 15, P > .05). Additional studies were performed with PCD cell lines to characterize the interrelationship of CD40 activation and IL-6 production. Fifty percent to greater than 95% of cells from the RPMI 8226 and ARH77 lines expressed CD40, whereas 6% of U266 cells were CD40+. For RPMI 8226, ARH-77, and U266 cells, the increased MCC formation after anti-CD40 stimulation was not affected by the presence of an anti-IL-6 neutralizing MoAb and was not accompanied by detectable IL-6 secretion. There was no apparent increase in IL-6 mRNA transcription following G28-5 treatment of U266 or RPMI 8226 cells. Our observations indicate that CD40 is expressed in a subset of human myeloma cells present in various PCDs. Cell-line studies suggest that the CD40+ myeloma cell may regulate MM clonogenic colony formation without activating the IL-6 pathway.