Generation of functional and mature dendritic cells from cord blood and bone marrow CD34+ cells by two-step culture combined with calcium ionophore treatment

The object of this study is to explore a culture method to generate a large number of functional and mature dendritic cells (DC) from human CD34+ hematopoietic progenitor cells. In the present study, we used a two-step method combined with calcium ionophore to induce DC from cord blood (CB) or normal human bone marrow (BM) CD34+ progenitor cells. The two-step method consists of 10 days of first step culture for the expansion and proliferation of CD34+ hematopoietic progenitor cells in the presence of SCF, IL-3, IL-6, G-CSF, and 7--11 days of second step culture for the induction of DC in the presence of GM-CSF, IL-4 and TNF-alpha. By the two-step culture, total nucleated cells were increased 208+/-66 (+/-SD, n=13), or 94+/-29 (n=5)-fold in the culture of CB or BM cells, respectively, compared with the number of CD34+ cells at the time of starting culture. Out of the total nucleated cells, 23 +/-10.4% of cells in CB cell culture and 25 +/-5% of cells in the BM cell culture acquired DC characteristic phenotypes, which were marked expressions of CD1a, HLA-DR, co-stimulatory molecules such as CD80, CD40, and adhesion molecule such as CD58. In allogeneic mixed leukocyte reaction (MLR), two-step cultured cells showed potent allo-stimulatory capacity. With this two-step culture, the absolute number of CD1a+ cells that co-expressed HLA-DR, CD80, CD40 and CD58 was enhanced approximately 3 times in CB cell culture and 1.9 times in BM cell culture, compared with the commonly used one-step culture method for the generation of DC from CD34+ cells using SCF, GM-CSF and TNF-alpha. However, on these DC generated in the two-step culture, the expressions of co-stimulatory molecule CD86 and mature DC marker CD83 were not sufficient. By the treatment of two-step cultured cells with calcium ionophore agent (A23187), the expression of co-stimulatory molecules such as CD86 and CD80 (especially CD86) was up-regulated. Besides, the expression of mature DC marker CD83 was remarkably induced by treatment with A23187 for a short duration (24 h). Consistent with the up-regulation of surface molecules CD86, CD80 and CD83, the two-step cultured cells treated with A23187 also showed a stronger allo-stimulatory capacity compared with the cells without A23187 treatment. In conclusion, the present study demonstrated that the two-step culture method effectively improved the yield of CD1a+ DC generated from CD34+ cells, and the phenotypes and functions of these CD1a+ DC could be enhanced efficiently by treatment with a calcium ionophore agent.     

The distinction between Burkitt lymphoma and diffuse large B-Cell lymphoma with c-myc rearrangement.

To compare immunophenotypic and molecular features between Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) with c-myc rearrangements (c-mycR DLBCL), we analyzed 18 cases of B-cell non-Hodgkin's lymphoma with c-mycR that were confirmed by chromosomal and/or Southern blotting analyses. The cases were histologically classified into 10 BLs and five DLBCLs. The remaining three cases could not be classified because of suboptimal quality of the surgical materials. BLs were from five adults and five children, whereas all DLBCLs were from adults. BLs were positive for CD20 (10/10 cases examined), CD10 (9/10), Bcl-2 (1/9), and Bcl-6 (10/10), whereas they were negative for CD3 (0/10) and EBV (0/8), by Epstein-Barr virus (EBV) EBER-1 RNA in situ hybridization. c-MycR DLBCLs were positive for CD20 (5/5), CD10 (2/5), Bcl-2 (3/4), and Bcl-6 (4/4), whereas none of them were positive for CD3 and EBV. A mean of MIB-1 index (MIB-1+ cells/neoplastic cells, %) of BLs (98.1%) was higher than that of c-mycR DLBCLs (66.3%; P <.0001). Somatic mutation of immunoglobulin heavy-chain gene variable region (VH gene) in BLs (four cases) ranged from 0.7 to 4.9% with an average value of 2.3%, whereas those in DLBCLs (three cases) from 8.2 to 32.0% with an average value of 17.0%. It is, therefore, concluded that a growth fraction of nearly 100%, as well as a monotonous proliferation of medium-sized cells and c-myc(R), should be of value in the diagnosis of BL, which is probably different from c-myc(R) DLBCL. In addition, CD10+, Bcl-2-, and low frequency of mutation of the VH gene could be helpful for the histologic distinction of BL from (c-mycR) DLBCL.     

Chromophobe renal cell carcinoma

Chromophobe renal cell carcinoma (RCC) is a recently established subtype of RCC, which has rarely been reported in Japan. In this communication, the authors report two Japanese cases of chromophobe RCC together with the immunohistochemical findings. The tumors were composed of sheets and cribriform glands formed by tumor cells with cloudy and reticular cytoplasm. Ultrastructurally, the cytoplasm was filled with numerous microvesicles. The tumor cells were positive for cytokeratin, epithelial membrane antigen, and Tamm-Horsfall protein. Occasionally, LeuM1-positive cells were also noted. Vimentin was negative, unlike the usual RCC. Reactivity for peanut agglutinin was more frequent than that to Lotus tetragonolobus agglutinin. The results of this study suggest that the tumor cellq possessed phenotypes similar to the distal nephron rather than to the proximal tubular cells.     

Establishment and Characterization of Cell Lines from Human Adenovirus Type 12-induced Murine Tumors Producing Endogenous Virus Particles

Two cell lines designated IC KMS and D KMS were established from human adenovirus type 12 induced tumors of C3Hf/OK mouse. The cell lines retained the characteristics of the original tumor i.e., production of numerous C type and intracisternal A-type particles, integration of Adl2 El region DNA and amplification of the myc gene family. Chromosomal analysis revealed chromosome aberrations in both IC KMS and D KMS cells. The modal chromosome number of IC KMS cells was 54 and that of D-KMS cells was 48. Metacentric chromosomes and mini-chromosomes were found. Trisomy of chromosome 3, 7 and 12 was seen frequently in D KMS cells. Although DNA aneuploidy was revealed by flow cytometry, the DNA indices of these cells showed no relation to the copy number of integrated Adl2 DNA. These cells have been propagated by serial culture during the past 17 months. Production of endogenous virus particles is a unique characteristic of IC KMS and D KMS cells. These cell lines would be useful materials for examining the contribution of Adl2 carcinogenesis to activation of endogenous virus particles, and also the correlation between Adl2 carcinogenesis and cancer related genes. Acta Pathol Jpn 42: 242-248, 1992.     

ERBIN associates with p0071, an armadillo protein,at cell-cell junctions of epithelial cells

BACKGROUND: ERBIN, an ErbB2 receptor-interacting protein, belongs to a recently described family of proteins termed the LAP [leucine-rich repeats and PSD-95/dLg-A/ZO-1 (PDZ) domains] family which has essential roles in establishment of cell polarity. RESULTS: To identify new ERBIN-binding proteins, we screened a yeast two-hybrid library, using the carboxyl-terminal fragment of ERBIN containing PDZ domain as the bait, and we isolated p0071 (also called plakophilin-4) as an ERBIN-interacting protein. p0071 is a member of the p120 catenin family, which are defined as proteins with 10 armadillo repeats, and localizes along the cell-cell border. The ERBIN PDZ domain binds the COOH-terminus of p0071 containing the PDZ domain-binding sequence. Endogenous ERBIN was co-immunoprecipitated with p0071. In fully polarized Madin-Darby canine kidney (MDCK) cells, ERBIN co-localized largely with beta-catenin and partly with desmoplakin along the lateral plasma membrane domain. At these cell-cell contact regions, ERBIN co-localizes with p0071. Over-expression of the dominant active forms of Cdc42, Rac1 or RhoA, Rho family small GTPases, resulted in a marked accumulation of ERBIN at the cell-cell contacts of MDCK and HeLa cells. CONCLUSION: These results show that ERBIN interacts in vivo with p0071 and that it may be involved in the organization of adherens junctions and the desmosomes of epithelia. In addition, we demonstrated that the subcellular localization of ERBIN might be regulated by Rho family small GTPases.     

De novo interstitial deletion of 1p (pter----p34.1::p32.3----qter).

We report a case of a 9 month old girl with a de novo interstitial deletion of 1p, karyotype 46,XX, del(1)(pter----p34.1::p32.3----qter). She had dysmorphic features including upward slanting palpebral fissures, a bulbous nose, a long philtrum, low set and malformed ears, a short neck, hypoplastic nails on both index fingers, widened interdigital spaces between the toes, dilated lateral ventricles, right hydronephrosis, a dilated right ureter, mental and motor developmental delay, and generalised hypotonia.     

Phosphorylation of Ser-3 of cofilin regulates its essential function on actin

Background: Cofilin is a low-molecular weight actin-modulating protein, and is structurally and functionally conserved in eucaryotes from yeast to mammals. The functions of cofilin appear to be regulated by phosphorylation and dephosphorylation. Results: A proteolytic study of phosphorylated porcine cofilin and expression of a mutated cofilin in cultured cells revealed that Ser-3 is the unique phosphorylation site. Phosphorylated cofilin was found not to bind to either F-or G-actin while unphosphorylated cofilin binds to both. S3D-cofilin, in which Ser-3 was replaced with Asp, did not bind in vitro to actin while S3A-cofilin did. The transient overexpression of wild-type or S3A-cofilin in cultured cells caused disruption of pre-existing actin structures and induced cytoplasmic actin bundles. Heat shock-induced nuclear or NaCl buffer-induced cytoplasmic actin/cofilin rods contained the expressed cofilin. In contrast, the overexpression of S3D-cofilin did not alter the actin structures. Induced actin rods did not contain S3D-cofilin. S3D-porcine cofilin did not complement the lethality associated with Δcof1 mutations in Saccharomyces cerevisiae while wild-type and S3A-cofilin did. Furthermore, we found that S2A/S4D- and S2D/S4D-yeast cofilin mutants were not viable. Conclusion: We conclude that the function of cofilin is negatively regulated in vivo by phosphorylation of Ser-3 and that cells require the functions of unphosphorylated cofilin for viability.     

Detection of Helicobacter pylori associated antigen and heat shock protein 60 on follicular dendritic cells in the germinal centres of low grade B cell lymphoma of gastric mucosa associated lymphoid tissue (MALT).

AIMS: To investigate the localisation of Helicobacter pylori antigens and the expression of human heat shock proteins (HSP) in stomachs affected by MALT lymphoma. METHODS: Surgically resected stomachs from 24 patients with MALT lymphoma were immunostained with anti-H pylori rabbit antibodies (ORP-1 and ORP-2) and anti-human HSP60 mouse monoclonal antibodies (mAb) (LK-1 and LK-2). RESULTS: Follicular dendritic cells of germinal centres in the stomachs affected by MALT lymphoma were immunostained with anti-H pylori polyclonal antibodies and with anti-human HSP60 mAb, as were the epithelial cells. None of the lymph node samples reacted. CONCLUSIONS: Human HSP60, which cross reacts with anti-H pylori polyclonal antibodies, is often expressed on follicular dendritic cells in gastric MALT lymphoma tissues and may be aetiologically relevant to lymphomagenesis of MALT lymphoma.     

Abnormal expression of the human CD44 gene in early colorectal malignancy with special reference to variant exon 9 (9v).

AIMS: To examine the expression of CD44 variant exon 9 in early colorectal malignancies. METHODS: Formalin fixed, paraffin wax embedded tissue sections from 30 cases of tubular adenoma and 35 cases of adenoma with focal carcinoma of the colon were examined immunohistochemically using a monoclonal antibody (MAb 11.24) directed against CD44-9v. RESULTS: In the normal colorectal mucosa immunoreactivity was confined to the basal part of the crypts and was expressed in less than 10% of crypt cells. CD44-9v was expressed in the superficial part of tubular adenoma with mild atypia in 67% of the cases and in 19% of the tumour cells. The immunoreactivity was observed along the basement membrane in mild atypia, as in the non-neoplastic crypts. In the course of progression to severe atypia the spatial polarity of immunoreactivity was lost, and the extent of CD44-9v expression increased in intensity and in the percentage of positive cases and positive cells. In the carcinomatous lesions of adenoma with focal carcinoma, 94% of the cases and 44% of the tumour cells were positive for CD44-9v protein. CONCLUSION: CD44-9v may be overexpressed at the early stage of colorectal tumorigenesis and this increase continues throughout the course of the disease.