Necessity of flow cytometry assessment of circulating plasma cells and its connection with clinical characteristics of primary and secondary plasma cell leukaemia.

Plasma cell leukaemia (PCL) is a rare and very aggressive plasma cell disorder. Preventing a dismal outcome of PCL requires early diagnosis with appropriate analytical tools. Therefore, the investigation of 33 patients with primary and secondary PCL was done when the quantity of circulating plasma cells (PCs) using flow cytometry (FC) and morphology assessment was evaluated. The phenotypic profile of the PCs was also analysed to determine if there is an association with clinical outcomes and to evaluate the prognostic value of analysed markers. Our results revealed that FC is an excellent method for identifying circulating PCs as a significantly higher number was identified by FC than by morphology (26·7% vs. 13·5%, P = 0·02). None of secondary PCL cases expressed CD19 or CD20. A low level of expression with similar positivity of CD27, CD28, CD81 and CD117 was found in both PCL groups. A decrease of CD44 expression was detected only in secondary PCL. Expression of CD56 was present in more than half of PCL cases as well as cytoplasmic nestin. A decreased level of platelets, Eastern Cooperative Oncology Group score of 2-3 and lack of CD20+ PC were associated with a higher risk of death. FC could be incorporated in PCL diagnostics not only to determine the number of circulating PCs, but also to assess their phenotype profile and this information should be useful in patients' diagnosis and possible prognosis.

Exosomes Play an Important Role in the Progression of Plasma Cell Mastitis via the PI3K-Akt-mTOR Signaling Pathway.

BACKGROUND: Plasma cell mastitis (PCM) is one of the most frequently encountered inflammatory diseases of the nonlactating breast. However, its pathogenesis has remained unknown. METHODS: In this study, we observed the ultrastructure changes of PCM by a transmission electron microscope. The transcriptome expression difference of exosomes was detected by RNA-Seq; then, we confirmed the key difference genes by western blot and immunohistochemistry. Finally, we established the mouse PCM model by tissue homogenate injection to validate the role of exosomes on the progression of PCM. RESULTS: The analysis of the exosomal transcriptome expression difference between PCM and normal mammary tissues using RNA-Seq showed the differential genes and enrichment pathways involved in the course of PCM. The decreased HSP90AA1 and EEF2, excessive production of p-AKT, and p-mTOR were consistent with clinical specimens. Inhibition of exosome secretion significantly inhibited inflammatory cell infiltration, and the mammary duct had maintained a better structure in the PCM mouse model. CONCLUSION: Our results revealed the role of exosomes acting as critical signal introduction facilitators in the progression of plasma cell mastitis and identified potential key genes in the regulation of this process. These results will help to dissect the molecular mechanism of PCM and provide therapeutic targets.

Proteasome inhibition with bortezomib depletes plasma cells and specific autoantibody production in primary thymic cell cultures from early-onset myasthenia gravis patients.

Bortezomib is a potent inhibitor of proteasomes currently used to eliminate malignant plasma cells in multiple myeloma patients. It is also effective in depleting both alloreactive plasma cells in acute Ab-mediated transplant rejection and their autoreactive counterparts in animal models of lupus and myasthenia gravis (MG). In this study, we demonstrate that bortezomib at 10 nM or higher concentrations killed long-lived plasma cells in cultured thymus cells from nine early-onset MG patients and consistently halted their spontaneous production not only of autoantibodies against the acetylcholine receptor but also of total IgG. Surprisingly, lenalidomide and dexamethasone had little effect on plasma cells. After bortezomib treatment, they showed ultrastructural changes characteristic of endoplasmic reticulum stress after 8 h and were no longer detectable at 24 h. Bortezomib therefore appears promising for treating MG and possibly other Ab-mediated autoimmune or allergic disorders, especially when given in short courses at modest doses before the standard immunosuppressive drugs have taken effect.

Regulated IRE1-dependent decay participates in curtailing immunoglobulin secretion from plasma cells.

Inositol-requiring enzyme 1 (IRE1) is a kinase and ribonuclease that executes the splicing of X box binding protein 1 (XBP-1) mRNA in response to the accumulation of unfolded protein in the ER, a signal cascade termed the unfolded protein response. Recently, IRE1 has been implicated in mRNA and miRNA cleavage and degradation, a pathway termed regulated IRE1-dependent decay (RIDD). Deletion of XBP-1 in the liver and pancreas strongly enhances RIDD by upregulating IRE1 protein levels and enhancing its ribo-nuclease activity. Because XBP-1 is essential for generating plasma cells with developed secretory capacity, we sought to evaluate the contribution of RIDD to this regulation. Mice were conditionally deleted for XBP-1 and/or IRE1 in their B-cell lineage. Similarly to the liver, deletion of XBP-1 induces IRE1 expression in LPS-treated B cells. In vitro, IRE1 cleaves the mRNA of secretory µ chains, which explains the reduction in secretory µ mRNA and its synthesis in XBP-1 KO plasma cells. In accordance, the IgM response is partially restored in XBP-1/IRE1 double KO mice relative to XBP-1 KO mice. Interestingly, the IgG1 response is reduced to a similar level in XBP-1 KO, IRE1 KO, and their double knockout animals. Our data demonstrate a specific contribution by RIDD in curtailing immunoglobulin synthesis and secretion.

Light and electron microscopic studies of the Harderian gland in Bilgorajska goose (Anser anser).

The Harderian gland (HG) in birds is the dominant orbital gland, which plays an important role in immunological response. Tissue sections taken from adult females of Bilgorajska goose were stained with hematoxylin-eosin, Azan, PAS, AB pH 2.5, AF and HDI. Based on the histological structure the HG in Bilgorajska geese had compound tubular structure with multiple lobules and two types of epithelial cells lining the tubules. Epithelial cells in the central part of the lobes were dark in color and contained serous fluid, while in the deeper layers, epithelial cells were lightly coloured and contained mucous fluid. Histochemical studies showed the presence of neutral mucopolysaccharides and carboxylated acid mucopolysaccharides in the secretory cells. The small number of single plasma cells were present in HDI staining below the basement membrane of the secondary and primary ducts, near the crypts of the main duct. TEM study demonstrated that plasma cells had a large nucleus with condensed heterochromatin and were rich in rough endoplasmic reticulum. The knowledge of gland's structure, and above all an analysis of the immune system components may affect clinical practice and properly conducted immunization of birds.

High detection rate of clinically relevant genomic abnormalities in plasma cells enriched from patients with multiple myeloma.

Multiple myeloma is a heterogeneous disease, which is characterized by the occurrence of specific genomic abnormalities that are both of diagnostic and prognostic relevance. Since the detection of these abnormalities through molecular-genetic techniques is hampered by the overall low percentage of plasma cells present in primary bone marrow aspirates, we assessed the efficacy of these techniques in enriched plasma cell fractions from 61 multiple myeloma patients. Using interphase FISH, genomic abnormalities could be detected in 96% of the enriched samples as compared to 61% in the cultured whole bone marrow samples. We also found that microarray-based genomic profiling of enriched plasma samples facilitates the detection of additional, possibly clinically relevant, genomic abnormalities. We conclude that the genomic delineation of enriched plasma cells from multiple myeloma patients results in a significantly increased detection rate of clinically relevant genomic abnormalities. In order to facilitate molecular-genetic data interpretation, we recommend morphological assessment of plasma cell purity after enrichment.

In multiple myeloma, bone-marrow lymphocytes harboring the same chromosomal abnormalities as autologous plasma cells predict poor survival.

Chromosomal abnormalities in plasma cells (PCs) from multiple myeloma (MM) provide a clonal signature to identify malignant cells. BM-lymphocytes from MM aspirates, defined by stringent criteria, were screened for the same chromosomal abnormalities as autologous PCs, including translocations, deletions, and amplifications. For 200 MM patients, we evaluated BM mononuclear cells to identify lymphocytes and autologous PCs on the same slide, followed by interphase fluorescence in situ hybridization to characterize their chromosomal abnormalities. Of all patients having a given chromosomal abnormality(s) in PCs, 45% showed that same abnormality(s) in 2-37% (median = 5%) of BM-lymphocytes. Most translocations, amplifications, and deletions found in MM PCs were also detected in lymphocytes, above the healthy-donor "cut-off." In patients having chromosomally abnormal CD20(-) PCs, chromosomally abnormal lymphocytes were found among CD20+ cells confirming them as B cells. Exceptions were amplification of 1q21 or p53 deletion, which characterize PCs but were undetectable in BM-lymphocytes, suggesting that processes leading to these abnormalities may be exclusive to PCs. For a set of 75 patients whose BM-lymphocytes and PCs were analyzed by all six probe sets, 58% of those with abnormal PC also had abnormal BM-lymphocytes harboring from one to five different abnormalities. Confirming the clinical significance of chromosomally abnormal BM-lymphocytes, MM patients having abnormalities in both lymphocytes and PC had significantly worse survival than those with abnormalities only in PC (HR = 2.68). The presence of at least one chromosomal abnormality in BM-lymphocytes appears to have greater clinical significance than particular abnormalities. Chromosomally abnormal BM-lymphocytes correlate with poor outcome and by extrapolation with more aggressive disease.

Generation of human long-lived plasma cells by developmentally regulated epigenetic imprinting.

Antibody secreting cells (ASCs) circulate after vaccination and infection and migrate to the BM where a subset known as long-lived plasma cells (LLPCs) persists and secrete antibodies for a lifetime. The mechanisms by which circulating ASCs become LLPCs are not well elucidated. Here, we show that human blood ASCs have distinct morphology, transcriptomes, and epigenetics compared with BM LLPCs. Compared with blood ASCs, BM LLPCs have decreased nucleus/cytoplasm ratio but increased endoplasmic reticulum and numbers of mitochondria. LLPCs up-regulate pro-survival genes MCL1, BCL2, and BCL-XL while simultaneously down-regulating pro-apoptotic genes HRK1, CASP3, and CASP8 Consistent with reduced gene expression, the pro-apoptotic gene loci are less accessible in LLPCs. Of the pro-survival genes, only BCL2 is concordant in gene up-regulation and loci accessibility. Using a novel in vitro human BM mimetic, we show that blood ASCs undergo similar morphological and molecular changes that resemble ex vivo BM LLPCs. Overall, our study demonstrates that early-minted blood ASCs in the BM microniche must undergo morphological, transcriptional, and epigenetic changes to mature into apoptotic-resistant LLPCs.

Production of type I interferons: plasmacytoid dendritic cells and beyond.

Plasmacytoid dendritic cells (pDCs) are specialized producers of type I interferons (IFNs) that respond to most viruses. Because of their antiviral activity and regulatory functions in innate and adaptive immunity, type I IFNs are important not only for antiviral resistance but also in other types of infections and in immune pathology. Here we discuss recent data that begin to reveal the unique molecular mechanisms underlying the remarkably rapid and efficient type I IFN production by pDCs.

Visualization of numerical centrosomal abnormalities by immunofluorescent staining.

The presence of multiple centrosomes in tumor cells is associated with the formation of multipolar mitotic spindles and results in aneuploidy of both daughter cells. Centrosome amplification is a feature of all cancer cells. We have previously described centrosome amplification in abnormal B cells. Further studies of centrosome amplification in different stages of B lineage development could provide important information about multiple myeloma pathogenesis.

Two Cases of Crystal-storing Histiocytosis Diagnosed by Morphology, Immunohistochemistry, and Ultrastructural Examination.

Crystal-storing histiocytosis (CSH) is a non-neoplastic histiocytic proliferation containing crystalline material, usually associated with an underlying lymphoproliferative or plasmacytic disorder. The crystalline structures are typically derived from kappa light chain immunoglobulins. The lesions of CSH are comprised of sheets of histiocytes with abundant eosinophilic cytoplasm containing variably prominent, elongated crystals. This rare phenomenon is important to recognize, as it is known to morphologically obscure an underlying neoplasm. Histologically, the cells of CSH may closely mimic Gaucher cells, as well as the "pseudo-Gaucher" cells sometimes encountered in chronic myeloid leukemia. The distinction between the cells of CSH and that of histologic mimics may be made more definitively through the use of electron microscopy, as the crystalline inclusions seen in CSH display characteristic size, shape, and localization within the cells. Here, we report 2 rare cases of CSH diagnosed by morphology, immunohistochemistry, and ultrastructural examination. The first case presented was diagnosed concurrently with plasma cell myeloma, and the second case discussed was diagnosed in association with marginal zone lymphoma.

An integrin αEß7-dependent mechanism of IgA transcytosis requires direct plasma cell contact with intestinal epithelium.

Efficient IgA transcytosis is critical for the maintenance of a homeostatic microbiota. In the canonical model, locally-secreted dimeric (d)IgA reaches the polymeric immunoglobulin receptor (pIgR) on intestinal epithelium via simple diffusion. A role for integrin αE(CD103)ß7 during transcytosis has not been described, nor its expression by intestinal B cell lineage cells. We found that αE-deficient (αE-/-) mice have a luminal IgA deficit, despite normal antibody-secreting cells (ASC) recruitment, local IgA production and increased pIgR expression. This deficit was not due to dendritic cell (DC)-derived retinoic acid (RA) nor class-switching defects, as stool from RAG-/- mice reconstituted with αE-/- B cells was also IgA deficient. Flow cytometric, ultrastructural and transcriptional profiling showed that αEß7-expressing ASC represent an undescribed subset of terminally-differentiated intestinal plasma cells (PC) that establishes direct cell to cell contact with intestinal epithelium. We propose that IgA not only reaches pIgR through diffusion, but that αEß7+ PC dock with E-cadherin-expressing intestinal epithelium to directly relay IgA for transcytosis into the intestinal lumen.

Next generation flow cytometry for MRD detection in patients with AL amyloidosis.

The treatment of AL amyloidosis aims to eradicate the plasma cell clone and eliminate toxic free light chain production. Only in a minority of patients the plasma cell clone is completely eradicated; residual light chain production may still exist while clonal relapse may occur. We used sensitive next-generation flow cytometry (NGF) to detect minimal residual disease (MRD) in AL amyloidosis patients at complete haematologic response. MRD evaluation was feasible in 51 of 52 (98%) tested patients and at a median sensitivity of 2.3 × 10-6 MRD was undetectable in 23 (45%). An organ response occurred in 86% of MRDneg vs 77% in MRDpos; renal response in 15/17(88%) of MRDneg vs in 14/16(87.5%) of MRDpos and cardiac response in 10/10(100%) of MRDneg vs 11/15(73%) of MRDpos patients. After a median follow-up of 24 months post MRD testing, no MRDneg patient had a haematologic relapse vs 6/28(21%) MRDpos (p = .029). Pooling haematologic and organ progressions, 9 (32%) MRDpos patients had disease progression vs only 1 (4%) MRDneg patient (p = .026). In conclusion, MRD detection using NGF has profound clinical implications, so that AL patients with undetectable MRD have a very high probability of organ response and a very low probability of haematologic relapse.

Plasma cell immunoglobulin secretion: arrest is accompanied by alterations of the golgi complex.

Conditions influencing Ig secretion by plasma cells have been studied with suspensions of murine plasma cells and myeloma cells by determining the release of (3)H-Ig after a pulse of biosynthetic labeling with L- [4,5-(3)H]-leucine. Ig secretion is insensitive to a variety of hormones, mediators, cyclic nucleotide derivatives, extracellular calcium depletion, and agents acting on mierotubules or microfilaments; i.e., to a number of factors which are involved in the regulation of secretion by cells with a storage compartment. On the other hand, Ig secretion is markedly inhibited by conditions which (a) lower intracellular calcium levels (ionophore A 23187 in Ca(++)-free medium), (b) induce partial sodium/potassium equilibration (the ionophores monensin and nigericin and, in the case of myeloma cells, ouabain and incubation in K(+)-free medium) or (c) uncouple oxidative phosphorylation. The first two situations are accompanied by striking alterations of the ultrastructural appearance of the Golgi complex, different in each case. These ultrastructural observations, together with autoradiographic experiments after a short pulse with L-[4,5-(3)H]-leucine, have led to the following hypothesis: (a) under Ca(++) depletion (3)H-Ig passes to Golgi vesicles but these vesicles are incapable of fusion or migration and therefore accumulate in exaggerated numbers in the Golgi area; (b) under partial Na(+)/K(+) equilibration, (3)H-Ig passes to Golgi vesicles which have an exaggerated tendency to fuse with other Golgi elements, thereby generating large vacuoles which store increasing amounts of Ig; (c) under energy block, multiple membrane fission and fusion events are inhibited and there is therefore, little intracellular transport of (3)H-Ig or alteration of cell ultrastructure.

A spontaneous rheumatoid arthritis-like disease in MRL/l mice.

MRL/l mice spontaneously develop an arthritis very similar in many respects to human rheumatoid arthritis. A detailed morphologic and serologic analysis of this disease revealed the following: (a) a 75% incidence of synovial and periarticular inflammation, very similar to human rheumatoid arthritis, in 5-6 mo-old females, (b) close associations between presence of joint inflammation and subsynovial and/or periarticular vasculitis, and (c) a close correlation between presence of circulating IgM rheumatoid factor (RF) and demonstrable synovial and/or joint pathology, i.e., 95% of mice with significant levels of IgMRF had synovitis and/or arthritis.

Bcl-2+ tonsillar plasma cells are rescued from apoptosis by bone marrow fibroblasts.

Plasma cells represent the final stage of B lymphocyte differentiation. Most plasma cells in secondary lymphoid tissues live for a few days, whereas those in the lamina propria of mucosa and in bone marrow live for several weeks. To investigate the regulation of human plasma cell survival, plasma cells were isolated from tonsils according to high CD38 and low CD20 expression. Tonsillar plasma cells express CD9, CD19, CD24, CD37, CD40, CD74, and HLA-DR, but not CD10, HLA-DQ, CD28, CD56, and Fas/CD95. Although plasma cells express intracytoplasmic Bcl-2, they undergo swift apoptosis in vitro and do not respond to CD40 triggering. Bone marrow fibroblasts and rheumatoid synoviocytes, however, prevented plasma cells from undergoing apoptosis in a contact-dependent fashion. These data indicate that fibroblasts may form a microenvironment favorable for plasma cell survival under normal and pathological conditions.