Immunoglobulin gene analysis in polyneuropathy associated with IgM monoclonal gammopathy

Antineural antibody activity is the implicated pathogenic mechanism in polyneuropathy associated with monoclonal gammopathy. Recognition of antigen depends on immunoglobulin variable regions, encoded by V genes. We studied V(H)DJ(H) and V(L)J(L) gene use in monoclonal B cells by clonal analysis in 20 patients with polyneuropathy and IgM monoclonal gammopathy. V genes associated with bacterial responses appear over-represented and V(H)3-23 was preferentially used, without association with specific D, J(H) or V(L)J(L). V genes revealed somatic mutation and intraclonal variation was found in 9 of 20 patients. Polyneuropathy associated with monoclonal gammopathy may be caused by an immune response to bacterial antigens, which recruit somatically mutated autoreactive B cells.     

Neurologic and hematologic response to fludarabine treatment in IgM MGUS polyneuropathy

We studied the efficacy of fludarabine in 16 patients with immunoglobulin M monoclonal gammopathy of unknown significance polyneuropathy in a prospective uncontrolled trial. The modified Rankin scale improved in 5/16 patients, all of whom had a demyelinating polyneuropathy. The motor conduction velocity improved by more than 10% in two or more nerves for four of five of these patients. Hematologic response in bone marrow occurred in three of five of these patients, whereas two of five already had small polyclonal B cell populations. There were no serious side effects.     

Antigens shared by malignant plasma cells and normal B cells may be involved in graft versus myeloma

Cytotoxic T cells play an important role in graft-versus-host-disease (GvHD) and graft-versus-leukaemia/myeloma, which may occur in patients treated with an allogeneic stem cell transplantation (ASCT). Here, we describe the selection of a myeloma reactive CD4+ cytotoxic T cell-line (CTL) and two CD4+ clones from this CTL. The CTL was generated from the blood from a patient with multiple myeloma (MM) with graft versus myeloma/GvHD, following an ASCT. The CTL was stimulated using irradiated peripheral blood mononuclear cells and EBV transformed B cells from the myeloma patient (EBVp), both of which were obtained prior to ASCT. Both the CTL and the two T cell clones specifically lysed EBVp and secreted IFN-gamma after coculture with EBVp and autologous myeloma tumour cells in a class II restricted fashion. These results show that myeloma tumour cells and autologous B cells present a common polymorphic peptide that functions as a target for graft derived cytotoxic T cells. Identification of these proteins will give insight into the relationship between graft versus myeloma (GvM) and GvHD and may provide immunotherapeutical targets in the treatment of MM.     

Gene-expression profiling of CD34+ cells from various hematopoietic stem-cell sources reveals functional differences in stem-cell activity

The replacement of bone marrow (BM) as a conventional source of stem cell (SC) by umbilical cord blood (UCB) and granulocyte-colony stimulating factor-mobilized peripheral blood SC (PBSC) has brought about clinical advantages. However, several studies have demonstrated that UCB CD34(+) cells and PBSC significantly differ from BM CD34(+) cells qualitatively and quantitatively. Here, we quantified the number of SC in purified BM, UCB CD34(+) cells, and CD34(+) PBSC using in vitro and in vivo assays for human hematopoietic SC (HSC) activity. A cobblestone area-forming cell (CAFC) assay showed that UCB CD34(+) cells contained the highest frequency of CAFC(wk6) (3.6- to tenfold higher than BM CD34(+) cells and PBSC, respectively), and the engraftment capacity in vivo by nonobese diabetic/severe combined immunodeficiency repopulation assay was also significantly greater than BM CD34(+), with a higher proportion of CD45(+) cells detected in the recipients at a lower cell dose. To understand the molecular characteristics underlying these functional differences, we performed several DNA microarray experiments using Affymetrix gene chips, containing 12,600 genes. Comparative analysis of gene-expression profiles showed differential expression of 51 genes between BM and UCB CD34(+) SC and 64 genes between BM CD34(+) cells and PBSC. These genes are involved in proliferation, differentiation, apoptosis, and engraftment capacity of SC. Thus, the molecular expression profiles reported here confirmed functional differences observed among the SC sources. Moreover, this report provides new insights to describe the molecular phenotype of CD34(+) HSC and leads to a better understanding of the discrepancy among the SC sources.     

Effects of Ethanol on Cultured Fetal Serotonergic Neurons

In utero ethanol exposure impairs the development of several neurotransmitter systems, including the serotonergic system. However, at present the mechanism by which in utero ethanol exposure damages the developing brain is unknown. This research examined the possibility that ethanol directly impairs the development of serotonergic neurons. This hypothesis was assessed by examining the content of serotonin (5-HT), 5-HT uptake, and 5-HT immunopositive neurons in cultures of fetal rhombencephalic neurons that were exposed to ethanol for 4 days in vitro. In addition, the effects of in vitro ethanol exposure on protein and DNA content of cultured rhombencephalic neurons were determined.
These studies demonstrated that a 4-day exposure of cultured rhombencephatic neurons to 50 to 300 mg ethanol/dl did not affect 5-HT content, 5-HT uptake, or the proportion of 5-HT immunopositive neurons. In addition, this ethanol exposure had no significant effect on protein or DNA content. Additional studies, using a 4-day exposure to 450 mg ethanol/dl also did not detect significant differences in 5-HT uptake or in protein or DNA content.
The marked differences in the findings of the present in vitro and previous in vivo studies may be due to the fact that the ethanol exposure in vivo was longer than that in vitro, and included the period of early development of serotonergic neurons and their progenitors. Alternatively, the differences may be due to ethanol-associated alterations in maternal or fetal factors (e.g., hormones, amino acids, and growth factors) that are necessary for the normal development of the serotonergic system in vivo. Normal concentrations of such factors in the serum-containing media may have protected the cultured neurons from the damaging effects of ethanol.     

Analysis of multidrug-resistance (MDR-1) glycoprotein and CD56 expression to separate monoclonal gammopathy from multiple myeloma

Summary Monoclonal gammopathy of undetermined significance (MGUS) is different from multiple myeloma (MM) by a low proliferation and by its indolent clinical course. In this study, two biological parameters were investigated which mark the transition from MGUS to MM, i.e. expression of the P-170 glycoprotein associated with the multidrug resistance phenotype (MDR-1) and expression of the natural killer cell antigen, CD56. Strong MDR-1 expression was found in plasma cells of 32/38 untreated MGUS as compared with 33/105 untreated MM stage I–III (84% v 32%, P <0.001) and in 0/10 normal plasma cell samples. CD56 expression in high density was present in 43/57 analysed untreated MM but in none of 23 MGUS (78% v 0% P <0.0001). Plasma cells did characteristically show a low Ki-67 proliferation index in 14/15 MGUS patients (mean 0.05%, range 0–0.2%) and a higher index in 25 analysed MM patients (mean 2.31%, range 1–7%, P <0.03).
These data indicate that MDR-1 expression together with absence of CD56 expression and a low proliferation index can be used to separate MGUS from MM.     

Outcome of allogeneic transplantation in newly diagnosed and relapsed/refractory multiple myeloma: long‐term follow‐up in a single institution

Allogeneic stem cell transplantation (allo‐SCT) has the potential to induce long‐term remission in multiple myeloma (MM), but the role of allo‐SCT in MM is controversial due to the high rate of treatment‐related mortality (TRM). However, although proteasome inhibitors and immunomodulatory drugs have improved the outcome of patients with MM, high‐risk patients still have a very poor prognosis. This indicates the need for new treatment strategies and identification of patients who might benefit from allo‐SCT. We therefore analyzed the outcome of one hundred and forty‐seven patients with MM who received an allo‐SCT at our institution (58 in first line, 89 in relapsed/refractory setting) after a median follow‐up of 88.8 months. For the first‐line setting, median progression‐free survival (PFS) and overall survival (OS) were remarkably good, with a CR rate of 48.3%, median PFS of 30.2 months, and 10‐yr OS of 51%. We found no difference in outcome for patients with high‐risk metaphase cytogenetics or FISH del(13q14), but efficacy in current standard high‐risk patients could not be determined. The outcome in the relapsed/refractory setting was poor, especially in the subgroup of patients relapsing within 18 months after auto‐SCT. Therefore, if applied at all in these patients, improvement of allo‐SCT is needed, focusing on reduction of TRM and more effective immunotherapy.