Typical Plasma Cells Research Articles

A novel human multiple myeloma cell line with a 1q21 gain genetic abnormality and CKS1B overexpression.

Multiple myeloma (MM) is an incurable hematologic malignancy mainly due to its cytogenetic abnormalities. Therefore, it is important to establish permanent malignant MM cell lines as tools to develop more effective therapies. Pleural effusion cells of a 70-year-old patient was collected to establish the CZ2 cell line. Characterization of CZ2 was determined with nephelometry, flow cytometry, fluorescence in situ hybridization (FISH), and chromosomal microarray analysis (CMA). Western blotting analysis was adopted to determine protein expression. Cell viability was measured by the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. We established and characterized a new MM cell line, CZ2. Using nephelometry and flow cytometry, cells with typical plasma cell morphology but not classical plasma cell phenotype were found to be non-immunoglobulin-secretary cells. FISH analysis of cells revealed a unique characteristic, namely, that there was only gain of the 1q21 region (1q21+). No other common cytogenetic abnormalities in MM, such as deletion of 17p (17p-), deletion of 13q (13q-), or translocation of immunoglobulin heavy chain (IgH), were observed. In addition, the original cell line maintains its single cytogenetic abnormality. Meanwhile, we observed through western blotting that CDC28 protein kinase regulatory subunit 1B (CKS1B), an adverse prognostic gene located in the 1q21 region, was highly expressed in CZ2. Knockdown of CKS1B reduced cell viability and also increased the levels of cleaved-poly(ADP-ribose) polymerase (cleaved-PARP) and cleaved-caspase3. CZ2 provides a suitable material for cellular and molecular studies of MM with only a 1q21 abnormality. This cell line is characterized by a gain of 1q21, and the high expression of CKS1B is an important model for studies of myeloma cell growth and drug resistance during therapy.

A simplified method for calculating spectral emission of nonequilibrium air plasmas in hypersonic shock-layers

A simplified method for calculating the spectral emission of nonequilibrium air plasmas is developed. In order to obtain the nonequilibrium energy level populations, the nonequilibrium coefficients are introduced into the Saha-Boltzmann equation. These nonequilibrium coefficients are calculated by using several significant radiative processes. An approach to the determination of nonequilibrium electronic energy level populations of diatomic molecules is also presented. Based on the method, spectral emission of atoms and molecules in a typical air plasma cell is investigated. The results reveal that there is a significant difference between the nonequilibrium and equilibrium emission. We apply the method to the nonequilibrium AVCO R-156 experiment. Good agreement with the NEQAIR code and the measured data is shown, indicating that the method is reasonable and has good accuracy.

RNA-Seq Based Immunoglobulin Repertoire Analysis of Normal Plasma Cells Generated in an in Vitro B Cell Differentiation System

Antibody secreting plasma cells (PCs) play an important role in effective humoral immune responses. The low frequency of bone marrow PCs in humans makes it challenging to obtain sufficient numbers of PCs for biologic studies. Previous studies have employed in vitro model systems to generate cells that morphologically, phenotypically, and functionally resemble normal polyclonal PCs. Gene expression profiles of in vitro generated PCs (IVPCs) mirror their normal counterparts, however to date extensive immunoglobulin (Ig) repertoire analysis of IVPCs is lacking. Here, we used a modified 3-step protocol to generate IVPCs and used RNA-seq to explore the transcriptome with emphasis on the Ig repertoire of plasmablasts and PCs. Total B cells were isolated from 3 normal donors and cultured with various cytokines and the B cell activators CpG ODN and CD40L. RNA was obtained from freshly isolated B cells (Day 0; D0) as well as from Day 4 (D4) plasmablasts, and Day 10 (D10) IVPCs. Morphologically, D10 cells exhibited typical PC morphology, including an eccentric nucleus and perinuclear hof. RNA-seq was performed on total RNA from all 3 donors and time points using the Standard TRuSeq v2 library prep and with paired end sequencing on the Illumina HiSeq 4000 platform. Principle component analysis of gene expression data showed that D0, D4 and D10 cells could be clearly segregated across all 3 normal donors. Of importance, transcripts previously described as distinguishing B cells from PCs were found to be differentially expressed including overexpression of CXCR5, CD19, EBF, CD83, PAX5, IRF8 in D0 B cells and overexpression of IRF4, Blimp-1, XBP1, BCMA, SLAMF7, Syndecan-1, CD38 and CD27 in IVPCs, thus validating our in vitro model for generating PCs. Furthermore, expression of cell cycle related transcripts such as CKS1, CDK1, and CCDN2 followed the pattern of low expression in resting B cells, increased expression in plasmablasts, and decreased expression in IVPCs confirming the cells are actively cycling in a manner comparable to cells in vivo. D10 IVPCs also overexpressed transcripts known to be upregulated during the unfolded protein response. As expected from Ig secreting cells, D10 IVPCs had an over-representation of Ig transcripts. At D0, resting B cells had high levels of IgD and IgM heavy chain (HC) transcripts. At D10, IgM transcripts modestly increased with Log2 fold change (FC) = 3 and as expected, IgD levels decreased significantly (Log2 FC = -2.2). IgA and IgG isotype transcripts significantly increased at D10 (Log2 FC > 6.0) with the IgG4 subtype having the greatest Log2 FC at 8.4. Next we focused on the Ig repertoire of D0, D4, and D10 cells. By aligning to known germline Ig sequences in IMGT/V-Quest (www.imgt.org) and then assembling the paired ends of D0, D4 and D10 Ig transcripts, we were able to analyze the Ig repertoire. Since the Ig HC variable (V) region is encoded by V, diversity (D) and joining (J) segments, only fragments that could be confidently determined were considered. All but 3 IGHV transcripts (IGHV3-35, IGHV3-47 and IGHV7-8) and 2 IGHD transcripts (IGHD4-4 and IGHD5-5) were found and all IGHJ segments were represented across the differentiation spectrum. In D0 cells, the number of unique VDJ combinations ranged from 643 to 863 across all 3 normal samples and increased to a range of 2524 to 2867 in D10 IVPCs. When looking at the differential expression of each VDJ combination from D0 to D10, a pairwise t-test for relative frequency showed that there was no significant change greater than 1%, suggesting the repertoire diversity was not skewed, thus proving the conditions for stimulation were not targeting any one starting B cell. Our data also allowed us to track clonal expansions during differentiation as defined by the increasing frequency of sequences with identical nucleotide sequence in the V region and CDR3 (including D and J regions). Hence, a single sequence could be tracked from D0 to D10. Of interest, in a small sampling of the total available sequences, only those B cells with a mutated IGHV region, characteristic of a memory B cell, went on to expand in this system whereas B cells with an unmutated IGHV did not. Our analysis of the Ig repertoire of IVPCs suggests this system provides a functional model to study Ig repertoire along the B cell differentiation process and further delineate the conditions that may result in a clonal expansion, a hallmark of many hematologic malignancies including multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Autologous stem cell transplantation as a treatment modality for type 1 cryoglobulinemia in monoclonal gammopathy of renal significance.

e19500 Background: Type I cryoglobulinemia (CRYO) can be driven by disorders such as plasma cell dyscrasias. Monoclonal gammopathy of renal significance (MGRS) represents a group of kidney disorders characterized by the presence of a paraprotein; though not fitting the criteria for a hematologic malignancy. There exists no standard approach to manage these patients. Our patient is a 56-year-old man who was initially evaluated for edema. Workup revealed worsening renal function and proteinuria (Figure 1). Renal biopsy revealed abundant cryofibrinogen deposits and cryoglobulinemic glomerulonephritis with membranoproliferative pattern. Bloodwork showed type I (IgG Kappa) CRYO. Bone marrow evaluation revealed 7% clonal plasma cells. Further evaluation did not confirm a diagnosis of myeloma. Methods: Treatment was initiated with bortezomib, cyclophosphamide and dexamethasone. After 3 cycles of therapy the patient had normalization of his creatinine and marked improvement in edema. Peripheral blood stem cells were mobilized following the administration of high-dose cyclophosphamide (2gm/m2) plus filgrastim. He was subsequently conditioned for transplantation with high-dose melphalan (200mg/m2; HDM) followed by autologous stem cell rescue. He tolerated the procedure well without any unexpected toxicities; achieving prompt hematologic recovery Results: Evaluations post transplant revealed resolution of any circulating cryoglobulins and reduction in proteinuria. At 1 year post transplant, the patient has returned to his premorbid condition. Conclusions: Type 1 CRYO associated MGRS represents a complicated disorder for which there is no consensus treatment approach. As this process may be driven by clonal plasma cells, classical anti-myeloma therapy may provide a strategy for management. Given the typical low plasma cell burden in these patients, HDM has the potential to offer deep and durable remissions. Prospective studies are needed to validate this approach. [Table: see text]

Autologous Stem Cell Transplantation As a Treatment Modality for Type 1 Cryoglobulinemia in Monoclonal Gammopathy of Renal Significance

Cryoglobulins (CGs) are immunoglobulins that precipitate at temperature below 37 C and re-dissolve after rewarming. Type I cryoglobulinemia is characterized by monoclonal immunoglobulins and is often driven by a lymphoproliferative disorder or plasma cell dyscrasia. Although CGs are often associated with hematologic malignancies which independently require therapy; this phenomenon can also be associated with more benign disorders. Monoclonal gammopathy of renal significance (MGRS) represents a group of disorders characterized by the presence of a paraprotein; although not fitting the diagnostic criteria for a symptomatic hematologic malignancy such as lymphoma or myeloma, can manifest by renal impairment through a variety of mechanisms. There exists no standard approach to manage these patients. As the driving force of these processes are characterized by the proliferation of malignant plasma cells (overproducing a nephrotoxic paraprotein) high-dose melphalan followed by autologous stem cell rescue represents an ideal approach to achieve organ function recovery and long term disease controlOur patient is a 56-year-old man who was initially evaluated for fatigue, lower extremity swelling and leg ulcers in December of 2011. Workup revealed worsening renal function and proteinuria. A renal biopsy was performed showing abundant cryofibrinogen deposits and cryoglobulinemic glomerulonephritis with membranoproliferative pattern. Labs showed type I cryoglobulinemia composed of monoclonal IgG kappa proteins. He was treated by his rheumatologist with a short course of prednisone and cyclophosphamide with transient improvement in proteinuria and renal function.He was monitored without therapy until 2015 when the gradual worsening of his edema led him to be re-evaluated by a nephrologist who ultimately referred the patient for hematologic workup upon the discovery of a monoclonal protein. Bone marrow evaluation revealed 7% clonal plasma cells with FISH studies positive for gain of CKS1B, trisomy 9 and monosomy 13. Further evaluation did not confirm a diagnosis of symptomatic myeloma. At this time however, the creatinine had risen to over 2 with over 35 grams of proteinuria on a 24-hour urine specimen. Lab work confirmed the presence of a cryoglobulin.As the recurrent cryoglobulinemia was felt to be plasma cell driven and the etiology of the renal impairment; the decision was made to treat with Bortezomib, Cyclophosphamide and Dexamethasone (CyBorD). After 3 cycles of therapy the patient had normalization of his serum creatinine, marked improvement in edema, as well as significant improvement in proteinuria. Peripheral blood stem cells were mobilized following the administration of high-dose cyclophosphamide (2gm/m2) plus high dose filgrastim. He was subsequently conditioned for transplantation utilizing high-dose melphalan (HDM/200mg/m2) followed by autologous stem cell rescue. He tolerated the procedure well without any unexpected toxicities.Evaluations post transplant revealed no evidence of a circulating cryoglobulin and continued clinical improvement and reduction in proteinuria. (figure 1). Now, 5 months post transplant, the patient has no evidence of a detectable paraprotein and has returned to his premorbid condition.Type 1 Cryoglobulinemia associated renal disease represents a complicated disorder for which there is no standard of care. As this may be driven by clonal plasma cells; classical anti-myeloma therapy may provide an optimal strategy for management. Given the typical low plasma cell burden in these patients HDM has the potential to offer deep and durable remissions. Prospective studies are needed to validate this approach. [Display omitted] DisclosuresBiran:Amgen: Speakers Bureau; Takeda: Speakers Bureau; Celgene: Speakers Bureau; Novartis: Speakers Bureau. Skarbnik:Pharmacyclics: Consultancy; Gilead Sciences: Speakers Bureau; Genentech: Speakers Bureau; Seattle Genetics: Speakers Bureau; Abbvie: Consultancy. Siegel:Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Merck: Honoraria. Vesole:Amgen: Speakers Bureau; Takeda: Speakers Bureau; Novartis: Speakers Bureau; Celgene: Speakers Bureau; Janssen: Speakers Bureau. Richter:Celgene: Consultancy, Speakers Bureau; Jannsen: Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau.

Leishmaniasis: A Reminder in the Face of Forgotten Travel

Figure 2. Dermal lymphohistiocytic infiltrate with typical plasma cells (arrows). A 10-year old boy presented with a slowly growing (over 1 year) painless nodule on his left temple, pre-existing for 1 year (Figure 1). His general health was excellent; travel outside Germany was denied. The skin biopsy showed a diffuse granulomatous dermatitis mainly composed of epitheloid cells, and occasional multinucleated foreign body type giant cells. A granulomatous foreign body reaction after a ruptured epidermal cyst was diagnosed. After ineffective topical corticosteroid therapy, the first of a scheduled sequential excision was done. This specimen once more showed epitheloid granulomas now consuming the whole dermis. However, in contrast to the first biopsy, plasma cells were now spotted within the lymphohistiocytic infiltrate that prompted us to re-evaluate the diagnosis (Figure 2). Investigations for Borrelia and Mycobacteria were negative, but polymerase chain reaction revealed the presence of Leishmania donovani complex DNA. After confronting the patient and his mother with this result, the boy recalled a 2-week journey to a rural area near Barcelona, Spain, approximately 1 year before the skin lesion had occurred, thus, pointing at Leishmania infantum as the culprit infectious agent. Laboratory tests, clinical, and ultrasound investigations did not reveal any signs of systemic disease. Consequently, cutaneous leishmaniasis was diagnosed and topical 15% paromomycin/12% methylbenzethonium chloride in white petrolatum treatment under occlusion was initiated. After 2 months of treatment, the lesion completely resolved, leaving an atrophic scar.

Long-Lived Plasma Cells from Human Small Intestine Biopsies Secrete Immunoglobulins for Many Weeks In Vitro

To understand the biology of Ab-secreting cells in the human small intestine, we examined Ab production of intestinal biopsies kept in culture. We found sustained IgA and IgM secretion as well as viable IgA- or IgM-secreting cells after >4 wk of culture. The Ab-secreting cells were nonproliferating and expressing CD27 and CD138, thus having a typical plasma cell phenotype. Culturing of biopsies without tissue disruption gave the highest Ab production and plasma cell survival suggesting that the environment regulates plasma cell longevity. Cytokine profiling of the biopsy cultures demonstrated a sustained presence of IL-6 and APRIL. Blocking of the activity of endogenous APRIL and IL-6 with BCMA-Fc and anti-human IL-6 Ab demonstrated that both these factors were essential for plasma cell survival and Ab secretion in the biopsy cultures. This study demonstrates that the human small intestine harbors a population of nonproliferating plasma cells that are instructed by the microenvironment for prolonged survival and Ab secretion.

The Murine Vk*MYC Myeloma Shares Defining Genetic Lesions with Human Multiple Myeloma.

Abstract 1808Poster Board I-834We have developed the first mouse model of multiple myeloma (MM), Vk*MYC, in an immune competent mouse, with indolent plasma cell (PC) growth confined to the bone marrow, and in which the major biological and clinical features of the human disease are recapitulated, including the post germinal center origin of the tumor cells, the presence of a M-spike in the serum, anemia, diffuse bone disease and drug sensitivity.To further characterize karyotypically and phenotypically the Vk*MYC tumors, we performed gene expression profile (GEP) and aCGH analysis on CD138+ purified MM cells from Vk*MYC mice. For GEP, we analyzed 33 individual tumors on the Affymetrix 430_2 array for which publicly available datasets can be used to compare levels of gene expression. For aCGH, we used the Agilent 244K array to analyze 19 individual tumors.By GEP we first confirmed the PC origin of the Vk*MYC tumors, that clearly express the typical PC markers, including CD138, IRF4, BLIMP, XBP1, IgG or IgA, but not B cell markers like IgM, IgD, PAX5, or BCL6. Next, we analyzed the expression of the three cyclin D, that are almost universally dysregulated in human myeloma patients and cell lines, but are not expressed in normal human or murine PCs. We found that the Vk*MYC tumors express high levels of cyclin D2, and occasionally cyclin D1, but never cyclin D3 that, instead, is commonly expressed in B cells. Interestingly, in one sample lacking any D type cyclin expression we detected loss of Rb expression (and homozygous deletion of the gene, see below), as in the “TC none” group of human MM patients, highlighting once again the importance of this pathway in both human and mouse myeloma.aGCH analysis indicated that the genomic complexity of Vk*MYC tumors is low, and complex rearrangements were rarely observed. In contrast with the recurrent trisomy of odd number chromosomes present in approximately 50% of human MM patients, we found no evidences of hyperdiploidy in Vk*MYC mice, except perhaps in one tumor with trisomy 15 and 17. Instead, we detected monosomy 5 in 10/19, monosomy 14 in 4/19 and trisomy 16 in 4/19 tumors. Interestingly a large portion of the murine chr.14 is syntenic to the human chr.13, frequently lost in MM patients. After monosomy 13, the most frequent chromosomal change in human MM is loss of a sex chromosome. In the mouse, the most common numerical abnormality identified was loss of a sex chromosome: 10/11 male had loss of Y and 5/8 female had loss of X.From the analysis of aCGH data publicly available on the MMRC portal, we have generated a list of the most common bi-allelic deletions present in human myeloma patients and cell lines. Interestingly, several of these lesions are also present in Vk*MYC tumors. The Rb1 gene, frequently affected by monosomy 13 in human MM, is bi-allelically lost in 3/19 Vk*MYC tumors. In one case, we also detected a focal monoallelic deletion encompassing Rb1, in addition to the four tumors with monosomy 14. Other focal bi-allelic deletion identified are: Cdkn2a (p16 and p19ARF), FHIT and PTEN. The latter was found in a Vk*MYC tumor selected in-vivo to be bortezomib resistant. On chr.X we identified recurrent (5/19 tumors) bi-allelic deletions of UTX, the histone demethylase gene recently reported to be deleted in various cancers, including MM tumors and human myeloma cell lines. While we are in the process of performing functional studies in human cell lines to identify a role for UTX in myeloma pathogenesis, we took advantage of the relative genetic uniformity of the Vk*MYC mice to identify by GEP a signature associated with UTX lost. We separated our cohort of Vk*MYC tumors for which GEP data are available in two groups based on expression of UTX and generated by volcano plot a list of genes that distinguished the two groups. Strikingly, loss of UTX was associated with loss of expression of a subset of lymphoid genes and acquisition of a “macrophage signature”, suggesting a role for UTX in lineage commitment. These data provide a genetic basis for tumor plasticity similar to epithelial mesenchymal transition observed in some solid tumors, in which the tumor gradually acquires supporting characteristics of its microenvironment.We conclude that the Vk*MYC provide a remarkably faithful model of MM not only phenotypically but also genetically, that provides an excellent framework for functional characterization of the molecular pathogenesis of MM DisclosuresBergsagel:Genentech: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Merck: Research Funding.

Intracerebral Manifestation of an Atypical Monoclonal Plasma Cell Hyperplasia Depicted by MR Perfusion and Diffusion Tensor Imaging and MR Spectroscopy

Intracerebral Manifestation of an Atypical Monoclonal Plasma Cell Hyperplasia Depicted by MR Perfusion and Diffusion Tensor Imaging and MR SpectroscopyMarc-André Weber1, Andrea Viehoever2, Bram Stieltjes1, Saida Zoubaa3, Brigitte Wildemann2, Klaus Baudendistel4 and Hans-Ulrich Kauczor1Audio Available | Share

Extremely Peculiar Nuclear Changes in Cultured Primary Myeloma Cells, Including Bulging and Duplication/Multiplication of Nuclei and Cell Expansion without Apparent Mitotic Forms.

Using our new cytokine-free, mitogen-free in vitro culture system for primary multiple myeloma cells (see abstract by Cohen Y and Zipori D), we have observed peculiar nuclear changes occurring during the first days in culture. After 3–4 days small lymphocytic-like cells are frequently seen, accompanied by typical plasma cells together with plasma-like cells possessing a single bulge in one edge of the nucleus (Figure 1). This nuclear bulging has been seen only in cultures from primary myeloma cases therefore it might represent a pathognomonic disease related phenomenon. Later in culture, multiple cells show nuclear bulging of variable shapes and degrees. It seems that this heterogeneity represents a spectrum of the same nuclear process (Figure 2). In addition, binucleated cells with eccentric nuclei are commonly seen at this stage, whereas multinucleated cells can be seen occasionally. Another observation is an apparent burrier-like structure between those nuclei in binucleated cells. Most interestingly, despite extensive expansion of the cultured myeloma cells (whose clonality was confirmed by FACS analysis, PCR and FISH-Bioview), mitotic forms are very rare. Furthermore, by combined FISH/morphology analysis of some cultured binucleated myeloma cells from a patient with del 13, a single signal was seen in each of the two nuclei using a probe for chromosome 13. Altogether, these unusual myeloma cell forms can now explain the yet unexplained finding frequently seen by FACS analysis of nuclear hyperploidity in myeloma cells. Currently, we use time lapse cinematography to assess the possibility that cultured myeloma cells undergo nuclear replication, through budding, and examine the possibility that these changes lead to the emergence of two daughter cells. [Display omitted]

Multiple Myeloma Stem Cells and Plasma Cells Display Distinct Drug Sensitivities.

We recently demonstrated that multiple myeloma (MM) is organized in a hierarchical manner in which clonogenic MM progenitors or stem cells resembling post-germinal B cells give rise to MM plasma cells (PC). To study the potential biologic differences between MM stem cells and MM PC, we examined each cellular subset for characteristics found in normal stem cells as well as their responses to various antitumor agents. The human MM cell lines RPMI 8226 and NCI-H929 were initially studied as we previously found that they recapitulate clinical MM specimens and consist of distinct cell populations based on the expression of the PC surface antigen CD138; CD138+ cells resemble typical MM PC, whereas CD138neg cells express B cell surface antigens and have greater clonogenic capacity. Examination of these cellular subpopulations by flow cytometry demonstrated that CD138neg cells were smaller and less granular by light scatter than CD138+ PC and expressed higher levels of the intracellular enzyme aldehyde dehydrogenase that is present in normal hematopoietic progenitors with self-renewal potential. Furthermore, cells expressing the side population phenotype after staining with the DNA binding dye Hoechst 33342 were exclusively CD138neg. We also investigated the effects of different clinically applicable agents on CD138+ and CD138neg cells. CD138+ and CD138neg cells isolated from RPMI 8226 and NCI-H929 cells by fluorescence activated cell sorting were treated with dexamethasone (dex, 100nM), bortezomib (velcade, 10nM), CC5013 (revlimid, 1μM), rituximab (10μg/ml) or alemtuzumab (campath,10μg/ml) for 72 hours followed by plating in methylcellulose to assess clonogenic capacity. CD138+ PC were significantly inhibited by dex (27 ± 11% recovery compared to untreated control cells), velcade (14 ± 6%) and revlimid (44 ± 27%), whereas rituximab (92 ± 25%) and campath (97 ± 18%) had little activity. In contrast, clonogenic growth of CD138neg cells was not significantly inhibited by dex (82 ± 19%), velcade (88 ± 29%), or revlimid (91 ± 14%), but was significantly decreased by rituximab (63 ± 22%) and campath (47 ± 27%). Similarly, clonogenic MM growth of CD138neg cells from 4 clinical MM samples was not affected by dex (84 ± 9%), velcade (82 ± 24%), or revlimid (93 ± 11%), but was significantly inhibited by rituximab (19 ± 7%) or campath (15 ± 11%). Clonogenic MM precursors may be distinguished from MM PC by a variety of biological parameters typically expressed by normal stem cells. Furthermore, these cellular subsets have different susceptibilities to a variety of clinical agents, and agents with activity against MM PC may be ineffective against MM stem cells. Moreover, agents without activity agasint MM PC may have major activity against MM stem cells. The divergent sensitivities of MM stem cells and PC may explain the dramatic, but transient, responses seen with many agents. Therapeutic strategies that result in long-term remissions may require the inhibition of both MM PC to reduce clinical symptoms and MM stem cells responsible for relapse.

Induction of CD28 on the new myeloma cell line MOLP-8 with t(11;14)(q13;q32) expressing δ/λ type immunoglobulin

The novel multiple myeloma (MM) cell line MOLP-8 carrying the t(11;14) (q13;q32) was established from the peripheral blood of a 52-year-old Japanese male patient with Bence–Jones δ/λ type MM (stage IIIA with hyperammonemia). The growth of MOLP-8 cells is constitutively independent of exogenous growth factors or feeder cells. MOLP-8 cells grow mainly as free floating single cells and slightly adherent on the bottom of the plastic culture flask. Wright–Giemsa-stained MOLP-8 cells show the typical plasma cell morphology with abundant cytoplasm, heterogeneous cell size and one to three nuclei. The immunoprofile of MOLP-8 corresponds to that seen typically in primary MM cells: positive for cytoplasmic immunoglobulin (Ig) δ/λ chains, CD10, CD29, CD38, CD40, CD44, CD49b, CD49d, CD54, CD56, CD58, CD71, CD138 and PCA-1; the cells were negative for surface Igs and various other B-cell, T-cell and myelomonocyte-associated immunomarkers. CD28 became positive after co-culture of MOLP-8 cells with bone marrow adherent stromal (BST) feeder cells for a week. About 30% of MOLP-8 cells adhered strongly to the BST cells, but the cellular adhesion was clearly inhibited by addition of either anti-CD29 or anti-CD106 monoclonal antibody, suggesting a specific cellular adhesion through α4β1-integrin–VCAM-1 interaction. The novel MOLP-8 cell line together with the present myeloma cell lines will present useful model systems in the investigation of the biology of MM.

A case of squamous cell carcinoma associated with plasmacytoma

A case of squamous cell carcinoma associated with plasmacytoma

Spontaneous production of anti-IFN-alpha and anti-IL-12 autoantibodies by thymoma cells from myasthenia gravis patients suggests autoimmunization in the tumor.

Myasthenia gravis (MG) is mediated by autoantibodies to the acetylcholine receptor (AChR), expressed in muscle and rare thymic myoid cells. Most early-onset cases show thymic lymph node-type infiltrates, including pre-activated plasma cells spontaneously producing anti-AChR antibodies. Since these are not evident in the associated thymomas found in another 10% of MG patients, AChR-specific B cells must be autosensitized elsewhere. Unexpectedly, at diagnosis, >70% of MG/thymoma patients also have high-titer neutralizing autoantibodies to IFN-alpha, and >50% to IL-12; moreover, titers increase strikingly if the thymomas recur, indicating a closer tumor relationship than for anti-AChR. To investigate this, we have measured autoantibody production by cells cultured from thymomas, any available thymic remnants and blood, with or without the B cell stimulant pokeweed mitogen (PWM). To check autoantibody specificity and clonal origins, we isolated Fabs from two combinatorial libraries from producer thymus/thymoma cells. Surprisingly, thymoma cells spontaneously produced antibodies to IFN-alpha and/or IL-12 in >40% of seropositive cases, showing typical plasma cell behavior, whereas they produced anti-AChR only after PWM stimulation. We isolated 15 combinatorial Fabs to IFN-alpha (versus only one to AChR). Their strong binding in radio-immunoprecipitation and Western blots implies high affinities. The four Fabs tested neutralized anti-viral actions of IFN-alpha. The diverse V genes clearly showed ongoing antigen-driven selection. These results imply pre-activation in situ by native IFN-alpha/IL-12 expressed within a 'dangerous' tumor microenvironment. With these molecules, it should be easier to identify provoking cell type(s) that may give novel additional clues to autoimmunization against T-cell epitopes from the more complex AChR.

CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

CCL28 is a CC chemokine signaling via CCR10 and CCR3 that is selectively expressed in certain mucosal tissues such as exocrine glands, trachea, and colon. Notably, these tissues commonly secrete low-salt fluids. RT-PCR analysis demonstrated that salivary glands expressed CCL28 mRNA at the highest levels among various mouse tissues. Single cells prepared from mouse parotid glands indeed contained a major fraction of CD3(-)B220(low) cells that expressed CCR10 at high levels and CCR3 at low levels and responded to CCL28 in chemotaxis assays. Morphologically, these cells are typical plasma cells. By immunohistochemistry, acinar epithelial cells in human and mouse salivary glands were strongly positive for CCL28. Furthermore, human saliva and milk were found to contain CCL28 at high concentrations. Moreover, the C terminus of human CCL28 has a significant sequence similarity to histatin-5, a histidine-rich candidacidal peptide in human saliva. Subsequently, we demonstrated that human and mouse CCL28 had a potent antimicrobial activity against Candida albicans, Gram-negative bacteria, and Gram-positive bacteria. The C-terminal 28-aa peptide of human CCL28 also displayed a selective candidacidal activity. In contrast, CCL27, which is most similar to CCL28 and shares CCR10, showed no such potent antimicrobial activity. Like most other antimicrobial peptides, CCL28 exerted its antimicrobial activity in low-salt conditions and rapidly induced membrane permeability in target microbes. Collectively, CCL28 may play dual roles in mucosal immunity as a chemoattractant for cells expressing CCR10 and/or CCR3 such as plasma cells and also as a broad-spectrum antimicrobial protein secreted into low-salt body fluids.

Auer rod-like inclusions in multiple myeloma

We report a case of IgA multiple myeloma, in which the plasma cells showed multiple azurophilic, Auer rod-like intracytoplasmic inclusions in May-Grünwald-Giemsa-stained marrow smears. Cytochemical stainings revealed a strong alpha-N-esterase activity of these inclusions, whereas the reactions for peroxidase, Sudan black, chloroacetate esterase, and PAS were negative. Immunostaining verified IgA-kappa inside the plasma cells. The inclusions, however, were negative. Amyloid and lysozyme were also not detectable. Electron microscopy showed Auer rod-like inclusions with a smooth surface in the neighborhood of a well-developed rough endoplasmic reticulum, but with no direct relation to it. The inclusions showed a fine lamellar substructure, and the periodicity of the filamentous striations was about 10 nm, comparable with the substructure of typical Auer rods. Our findings suggest that the azurophilic inclusions in multiple myeloma are Auer rod-related structures, which likewise consist of active lysosomal enzymes. In contrast to the Auer rods in acute myeloblastic leukemia (AML), however, the inclusions in multiple myeloma consist of typical plasma cell enzymes.

Two Different Extramammary Neoplasms Metastatic to the Breast

Two Different Extramammary Neoplasms Metastatic to the Breast

Prognostic value of histopathological grading in canine extramedullary plasmacytomas.

The canine extramedullary plasmacytoma (cEMP) has recently been the subject of numerous investigations, indicating that the histomorphologic diagnosis is often difficult because of the variety of morphologic features. Therefore, the objective of this study was to establish a subclassification scheme for cEMPs and to evaluate correlations between the types and malignancy. Retrospectively, 117 cEMPs, all immunohistochemically characterized by a monoclonal immunoglobulin light-chain expression, were collected and assigned to morphologic types. These types were compared using data from a follow-up study on metastasis and tumor recurrence, then compared by proliferation rate, determined by immunohistochemical detection of the antigen Ki-67. Histopathologic typing revealed five different types of cEMPs, ranging from the mature type with typical plasma cells to the polymorphous-blastic type. Between these two forms, three additional types were established: hyaline, cleaved, and asynchronous. Most of the cEMPs were of the cleaved and asynchronous types. In all cEMPs, mononuclear and multinuclear giant cells were present to varying degrees. Although the results of cell proliferation and the follow-up study indicated less benign behavior by the polymorphous-blastic type, the proliferation rate revealed no statistically significant differences among the cEMP types. The clinical data therefore confirmed previous findings that the risk of tumor recurrence and metastasis in general can be classed as low. The established cEMP typing system is probably a very helpful diagnostic tool, although the types cannot be used for a tumor grading system.

Intestinal plasmacytosis in rabbits: a histologic and ultrastructural study.

A retrospective study revealed intestinal plasmacytosis in 53 of 102 rabbits used in various experimental studies and as controls. The breeds affected included New Zealand white (n = 46), Dutch-belted (n = 6), and Watanabe (n = 1) rabbits. Sex predisposition was not found in any breed. The mean (+/-SD) ages were 3.1 +/- 1.4 years for New Zealand white rabbits, 1.3 +/- 1.1 for Dutch-belted rabbits, and 2 years for the Watanabe rabbit. The severity increased with animal age. The incidence was higher (P < 0.05) in rabbits used in antibody production and cholesterol studies. The lesions were characterized by multifocal to diffuse infiltration of well-differentiated plasma cells in the intestinal mucosa. Electron microscopic examination revealed typical plasma cell morphology of the infiltrating cells. Small intestine and cecum were the major sites affected. In severe cases, colon, rectum, trachea, esophagus, mesenteric lymph node, and spleen were also involved.

Cell Surface Markers in Multiple Myeloma

To describe the immunophenotype of normal and myelomatous plasma cells (PCs) and the changes in immunoregulatory nonmyelomatous cells in multiple myeloma (MM). The cell surface markers (antigens) associated with this common cancer were reviewed. Immunophenotypic characterization of both normal PCs and their counterpart malignant hematopoietic cells can be achieved by using monoclonal antibodies and either flow cytometry or immunocytochemical techniques. Normal PCs are heterogeneous and express, in addition to cytoplasmic immunoglobulins, the antigens CD9, CD10, CD13, CD19, CD20, CD33, CD38, and D-related human leukocyte antigen (HLA-DR). This heterogeneity also occurs in malignant PCs. Myelomatous PCs may express, in addition to CD38 (the most typical PC marker), the antigens CD9, CD10, HLA-DR, and CD20. Other non-B-cell lineage markers such as myeloid (CD13, CD14, CD15, CD33, CD41, and glycophorin A), T-cell (CD2 and CD4), and natural killer-associated (CD56) antigens, as well as CD23, CD24, CD25, CD37, CD39, CDw40, CD45R, CD71, and certain unclustered antigens (R1-3, PCA-1, PCA-2, PC1, 62B1, 8A, 8F6, and MM4), have been noted in myelomatous PCs. The presence of these antigens in the myeloma cells may have a prognostic value--for example, the expression of CD20 and of myelomonocytic antigens (CD11b, CD13, CD14, CD15, and CD33) may be related to a poor prognosis. The adverse prognostic implication of the expression of CD10 initially described in MM has not been subsequently confirmed. Patients with MM may have mononuclear cells in their peripheral blood that express the same antigens as those expressed by the myeloma cells in their bone marrow. The presence of such cells or their therapy-associated decrease or disappearance may be related to the prognosis of patients with MM. The presence of cell surface markers on PCs and their prognostic significance in patients with MM warrant further investigation.