Impact of interleukin-21 in the pathogenesis of primary Sjogren's syndrome: increased serum levels of interleukin-21 and its expression in the labial salivary glands

Kang, Kwi Young,Kim, Hyun-Ok,Kwok, Seung-Ki,Ju, Ji Hyeon,Park, Kyung-Su,Sun, Dong-Il,Jhun, Joo Yeon,Oh, Hye Jwa,Park, Sung-Hwan,Kim, Ho-Youn BioMed Central 2011 ARTHRITIS RESEARCH AND THERAPY Vol.13 No.5

<P><B>Introduction</B></P><P>Interleukin (IL)-21 is a cytokine that controls the functional activity of effector T helper cells and the differentiation of Th17 cells, and promotes B-cell differentiation. To test whether IL-21 participates in the pathogenesis of primary Sjögren's syndrome (SS), serum IL-21 level was measured and IL-21 expression in the labial salivary glands (LSG) was examined.</P><P><B>Methods</B></P><P>Serum IL-21 levels in 40 primary SS, 40 rheumatoid arthritis (RA), and 38 systemic lupus erythematosus (SLE) patients and 20 healthy controls were measured. Serum IL-21 levels of SS patients were assessed for correlations with laboratory data, including anti-nuclear antibody, anti-Ro/La antibodies, globulin, immunoglobulin (Ig) class, and IgG subclass. LSGs from 16 primary SS and 4 controls with sicca symptoms were evaluated for IL-21 and IL-21 receptor (IL-21R) expression by immunohistochemistry. Confocal microscopy was performed to further characterize the IL-21 positive cells.</P><P><B>Results</B></P><P>Primary SS patients had significantly higher serum IL-21 levels than controls, and these increments correlated positively with levels of IgG, IgG1. Serum IgG1 levels correlated with anti-Ro antibody titers. Immunohistochemical analyses showed that lymphocytic foci and the periductal area of the LSGs from SS patients expressed high levels of IL-21 and lower levels of IL-21R, whereas the control LSGs showed minimal expression of both antigens. The more the lymphocyte infiltrated, IL-21expression in LSGs showed a tendency to increase. Confocal microscopic analyses revealed that IL-21 expressing infiltrating lymphocytes in the LSGs of SS patients also expressed CXCR5.</P><P><B>Conclusions</B></P><P>Primary SS is associated with high serum IL-21 levels that correlate positively with serum IgG, especially IgG1, levels. The expression of IL-21 is increased as more lymphocytes infiltrated in LSGs. These observations suggest that IL-21 may play an important role in primary SS pathogenesis.</P>

A Novel Function of Interleukin-10 Promoting Self-Renewal of Hematopoietic Stem Cells

Kang, Young-Ju,Yang, Seung-Jip,Park, Gyeongsin,Cho, Bin,Min, Chang-Ki,Kim, Tae-Yoon,Lee, Joon-Sung,Oh, Il-Hoan Wiley (John WileySons) 2007 Stem Cells Vol.25 No.7

<P>Self-renewal of hematopoietic stem cells (HSCs) is key to their reconstituting ability, but the factors regulating the process remain poorly understood. Here, we show that Interleukin-10 (IL-10), a pleiotropic immune modulating cytokine, can also play a role in regulating HSC self-renewal. First, a quantitative decrease of primitive hematopoietic cell populations, but not more matured cells, was observed in the bone marrows of IL-10 disrupted mice as determined by long-term in vitro cultures or in vivo competitive repopulation assays. In contrast, normal HSCs from 5-fluorouracil treated marrows cultured on the IL-10 secreting stroma displayed an enhanced repopulating activity compared with cells grown on control stroma, with ninefold higher numbers of donor-derived HSCs in the reconstituted recipient marrows. Moreover, limiting dilution transplantation assay demonstrated that exogenous addition of IL-10 in the stroma-free cultures of purified Lin- Sca-1+ c-kit+ cells caused three- to fourfold higher frequencies of HSCs in the 5-day short-term culture without indirect inhibitory effect of IL-10 on tumor necrosis factor-alpha or interferon-gamma secretion. Interestingly, primitive hematopoietic cells, including Lin- Sca-1+ c-kit+ or side population cells, expressed the surface receptor for IL-10, and microenvironmental production of IL-10 was sharply increased in the osteoblasts lining the trabecular regions of the radiation-stressed marrow but not in the steady-state marrows. These results show that IL-10 may be a ligand that can stimulate self-renewal of HSCs to promote their regeneration in addition to being a ligand for immune regulation. Disclosure of potential conflicts of interest is found at the end of this article.</P>

RSV 와 인플루엔자 바이러스 A 형 감염에 의해 천명을 보이는 소아와 천식 소아에서 비인두 흡인액의 Interleukin-5 와 Interleukin-γ 치의 비교

오재원,이하백,김창렬,염명걸,문수지,박일규,강정옥 대한 소아알레르기 및 호흡기학회 1999 소아알레르기 및 호흡기학회지 Vol.9 No.2

목 적 : 호흡기 바이러스 감염은 소아에서 천식을 악화시키는 것으로 잘 알려져 있다. 그러나 영유아기의 천식이나 천명발생에 있어서 호홉기 바이러스의 역할에 대해서는 명확하게 밝혀져 있지 않다. 소아기의 천명이 천식과 달리 하나의 독립된 질환인지, 아니면 같은 질환으로 달리 표현되는 것인지 논란이 되어왔다. 이에 본 저자들은 호흡기 바이러스 감염과 천명과의 상관관계와 기전을 이해하기 위하여 RSV 감염이나 influenza A 바이러스 감염에 의해 천명이 있는 소아와 바이러스가 증명되지 않고 천명이 있는 천식소아에서의 비인두 흡인액의 IL-5와 IFN-γ치를 측정하여 비교하였다. 방 법 : 호흡기 바이러스 감염군 38명(RSV감염군 21명, influenga A virus 감염군 17명), 바이러스가 증명되지 않은 천식환아군 12명, 정상 대조군 16명을 대상으로 double sandwich ELISA를 이용하여 비인두 흡인액에서 IL-5와 IFN-γ치를 측정하여 비교하였다. 결 과 : RSV 감영군에서 비인두 흡인액의 IL-5 평균치가 influenza A 바이러스군의 평균치보다 의미있게 높았으며, 천식군보다도 높은 양상을 보이나 의미있는 차이는 없었다 반면, influenza A 바이러스 감염군의 비인두 흡인액의 IFN-γ치가 RSV군에 비해 의미있게 높았으나 천식환아군이나 정상군에 비해 의미있게 높지는 않았다. 비인두 흡입액에서 IL-5와 IFN-γ치간의 상관관계는 없었다. 결 론 : RSV 감염군은 influenza A 바이러스 감염군에 비해 Th2 반응이 우세한 것으로 추정되며, 반면에 influenza A virus 감염군은 Th1 반응을 보이는 것을 알 수 있다. Background : Infection with respiratory virus has been shown to exacerbate asthma. However, the role of a respiratory virus in the pathogenesis of chronic asthma and/or wheezing in young children has not been clearly defined. And it also has been debated whether virus-induced wheezing in young children is an entity different from allergic asthma, or just a different expression of the same disease. In this study, we attempted to evaluate the importance of eosinophilic inflammation, comparing IL-5 and IFN-γ levels in nasopharyngeal secretions in wheezing children with or without viral infection and the controls. Methods : We compared IL-5 and IFN-γ levels in nasopharyngeal secretions from 38 non-asthmatic wheezing children with viral infections (RSV in 21 children, influenza A virus in 17 children), 12 asthmatic children without viral infections and 16 children as the controls. Results : The present study reported that RSV infection in children induced more releasing of IL-5 in nasopharyngeal secretions than the influenza A virus infected ones and the controls. On the other hand, the releasing of IFN-γ levels in nasopharyngeal secretions from children with influenza A virus infection was significantly higher than those of the children with RSV infection or asthmatic children. Conclusion : RSV infection in children may play a role in the immune response toward a Th2 phenotype as increasing IL-5 secretion in nasopharyngeal secretion. Increased IFN-γ production in response to the influenza A virus infection may be related to the effective Th1 responses.

위암에서 Helicobacter pylori cagA, vacA, iceA 유전자와 숙주 Interleukin-1β및 Interleukin-1 수용체 길항제 유전자 다형성

이성훈 ( Seong Hun Lee ),김태오 ( Tae Oh Kim ),이동현 ( Dong Hyun Lee ),박원일 ( Won Il Park ),김광하 ( Gwang Ha Kim ),허정 ( Jeong Heo ),강대환 ( Dae Hwan Kang ),송근암 ( Geun Am Song ),조몽 ( Mong Cho ) 대한내과학회 2006 대한내과학회지 Vol.71 No.1

Background: Both Helicobacter pylori (H. pylori) cagA, vacA, iceA genotype and host IL-1B/IL-1RN polymorphisms play a role in determining the clinical consequences of H. pylori infection. This study aimed to investigate whether there might be any combinations of H. pylori cagA, vacA, iceA genotype and host IL-1B/IL-1RN polymorphisms that are particularly associated with the occurrence of gastric carcinoma in Korean patients. Methods: This study population was comprised of 239 patients with H. pylori infection: 122 with gastric carcinoma and 117 with gastritis only. DNA was isolated from gastric biopsy sample and H. pylori cagA, vacA and iceA genotype were determined by PCR. IL-1B-511 polymorphisms were genotyped by PCR-RFLP and IL-1RN polymorphisms were analyzed with variable number of tandom repeat after PCR. Results: H. pylori cagA, vacA, and iceA genotype were not associated with an increased risk for gastric carcinoma. IL-1B-511*T carriers and IL-1RN*2 carriers did not show increased risk for gastric carcinoma. On combination of bacterial/host genotypes, cagA+/IL-1B-511*T carriers and cagA+/IL-1RN*2 carriers, vacA s1/IL-1B-511*T carriers, vacA s1/IL-1RN*2 carriers, vacA m1/IL-1B-511*T carriers, vacA m1/IL-1RN*2 carriers, iceA1/IL-1B-511*T carriers, iceA1/IL-1RN*2 carriers showed no increased risk of gastric carcinoma. Conclusions: Combined H. pylori cagA, vacA, iceA genotype and host IL-1B/IL-1RN polymorphisms shows no increased risk of gastric carcinoma. Therefore, it seems other endogenous or exogenous factors may play more important role in the development of gastric carcinoma in Korean.(Korean J Med 71:24-37, 2006)

Effect of high-mobility group box 1 on keratinocytes and fibroblasts

( Chan-yang Lee ),( In-hye Kang ),( Seung-min Oh ),( Jin-woo Lee ),( Young Il Kim ),( Ki-heon Jeong ) 대한피부과학회 2020 대한피부과학회 학술발표대회집 Vol.72 No.1

Background: High-mobility group protein B1 (HMGB1) is a physiological activator of immune responses. In patients with chronic inflammatory skin disorders including lupus, atopic dermatitis, and psoriasis, HMGB1 is increased in the cutaneous lesions. Objectives: This study was designed to investigate the effect of HMGB1 on keratinocytes and fibroblasts. Methods: In this study, keratinocytes and fibroblasts were exposed to narrow band ultraviolet B (NBUVB). Thereafter, release of HMGB1 were measured. Then keratinocytes and fibroblasts were treated with recombinant HMGB1 (rHMGB1) and production of inflammatory factors such as interleukin (IL)-1β, IL-6, IL-8, IL-18, chemokine (C-X-C motif) ligand (CXCL)1, CXCL2 and tumor necrosis factor (TNF)-α were examined. Results: 24h after NBUVB irradiation, the level of HMGB1 mRNA was increased in the fibroblasts but it was decreased in the keratinocytes. Extracellular level of HMGB1 was significantly increased in both keratinocytes and fibroblasts, 72h after NBUVB irradiation. After rHMGB1 treatment, proinflammatory molecule such as CXCL-1, IL-6, IL-8 and IL-18 were significantly increased in the keratinocytes and CXCL-1, IL-6 and IL-8 were significantly increased in the fibroblasts. Conclusion: HMGB1 can be released from keratinocytes and fibroblasts by external stress such as NBUVB irradiation and leads to activation of inflammatory signaling pathways in the keratinocytes and fibroblasts.

RSV나 인플루엔자 A형 바이러스에 감염된 비천식 소아에게 비인두 흡인액의 Interleukin - 11 , Interferon - γ치와 eosinophil cationic protein 치의 비교

오재원(Jae Won Oh),이하백(Ha Baik Lee),박일규(Il Kyu Park),강정옥(Jung Oak Kang) 대한천식알레르기학회 2000 천식 및 알레르기 Vol.20 No.1

N/A Background: Infection with respiratory virus has been shown to exacerbate asthma in humans. However, the role of a respiratory virus in the pathogenesis of chronic asthma and/or wheezing in young children has not been clearly defined. The objective of this study was to determine whether respiratory virus infections such as RSV, and influenza A virus are related to the productions of IL-11, IFN-γ, and ECP levels in nasopharyngeal secretions. Method: We compared IL-11, IFN-γ, and ECP levels in nasopharyngeal secretions from 38 non-asthmatic wheezing children with viral infections (RSV in 21 children, influenza A virus in 17 children), and 16 non-asthmatic healthy children who were included as the controls. IL-11, and IFN-y levels were analysed by ELISA. ECP concentrations were measured by monoclonal antibody-based fluorometric assay. Result. RSV infection in children induced a greater release of IL-11 in nasopharyngeal secretions than in influenza A virus infection, and in the controls. The release of IFN-γ levels in nasopharyngeal secretions from children with influenza A virus infection was significantly higher than in nasopharyngeal secretions from children with RSV. ECP levels of subjects with viral infection were significantly higher than in control children. Conclusion. This study demonstrated that RSV is a potent inducer of IL-11 elaboration in nasal epithelium and that IL-11 is an important mediator in the pathogenesis of RSV infection. Increased IFN-γ production in response to the influenza A virus infection may be related to effective Th1 responses.

조혈모세포이식 후 골성장인자의 변화 및 골대사에 미치는 영향 : Impact on Bone Mineral Metabolism

백기현,오은숙,오기원,이원영,김혜수,권순용,한제호,강무일,차봉연,이광우,손호영,강성구,김춘추 대한내분비학회 2002 Endocrinology and metabolism Vol.17 No.5

연구배경: 각종 장기이식의 시행이 많아지고 이식 후 생존율이 증가함에 따라 이식 후 합병증에 대한 관심 또한 높아지고 있다. 조혈모세포이식 후에도 다양한 내분비적 합병증이 발생할 수 있으며 골격에 대한 합병증도 문제점으로 대두되고 있다. 조혈모세포 이식 후 발생하는 골소실에는 이식 후 초기의 골형성 저하와 골흡수 증가가 중요한 역할을 담당하리라고 추측되는데 이러한 골재형성불일치(biochemical uncoupling)에 골 성장인자들이 미치는 영향에 대해서는 알려진 바가 없다. 본 연구에서는 조혈모세포이식 전, 후로 말초 혈액에서 IGF-I, FGF-2, M-CSF같은 성장인자의 변화를 알아보고, 이들 성장인자의 변화가 조혈모세포이식 후의 골형과 골흡수에 미치는 영향 및 이식 후 발생되는 골량 소실과의 연관성을 확인해 보고자 하였다. 방법: 여러 가지 혈액질환으로 인해 동종 골수이식을 시행 받은 환자들을 전향적으로 관찰하였으며 이식 전 및 이식 후 1주, 2주, 3주, 4주 및 3개월, 6개월 1년에 말초 혈액에서 골교체표지자를 측정하였다. 이식 전 및 이식1년 후에 요추골 및 대퇴골 골밀도를 측정할 수 있었던 36명의 환자들을 대상으로 냉동 보관되어 있던 혈청을 이용하여 IGF-I, FGF-2 및 M-CSF를 시기별로 측정하였으며 이들 성장인자와 골교체표지자의 변화 및 골밀도 변화 사이의 상관관계를 확인하였다. 결과: 골흡수 표지자인 혈청 ICTP는 이식 전에 비해 이식 후 4주까지 점차 의의 있게 증가하다가 이후 6개월까지 더욱 증가한 후 감소하였다. 골형성 표지자인 osteocalcin은 이식 후 3주까지는 점차 감소하다가 이후 증가하여 이식 후 3개월 및 6개월에 기저치보다 통계적으로 유의하게 증가한 후 감소하였다. 혈청 IGF-I과 FGF-2는 각각 이식 후 3주 및 1주까지 의미있게 감소하다가 이후 증가하였으며 혈청 M-CSF는 이식 후 1주째에 기저치에 비해 의미 있게 증가하였다가 이후 기저치로 회복되었다. 이식 1년 후 평균 요추부 골밀도는 5.2% 감소하였고 평균 근위대퇴골 골밀도는 11.6% 감소하였다. 이식 전 및 이식 후 3주에 측정한 IGF-I과 같은 시기에 측정한 오스테오칼신 사이에 유의한 상관관계가 관찰되었으며 이식 후 3주째의 M-CSF와 골흡수표지자인 M-CSF 사이에서 의미 있는 양의 상관관계를 관찰할 수 있었다. 이식 후 3주 및 3개월에 IGF-I이 낮은 환자일수록 이식 1년 후 근위대퇴골에서의 골소실이 많은 것으로 분석되었다. 결론: 조혈모세포이식 후 발생하는 골소실에는 기존에 알려진 기저질환의 영향, 성호르몬의 감소, 면역억제의 투여, 골수기질세포와 조골세포의 손상 및 이식초기 사이토카인의 변화이외에도 골성장인자가 관련이 있음을 확인하였고, 이는 이식 후 발생되는 골량소실에 중요한 역할을 할 것이라고 사료된다. Background: A loss of bone mass is usually detected after a bone marrow transplantation (BMT), especially during the early post-transplant period. We recently reported that enhanced bone resorption following a BMT was related to both the steroid dose and the increase in IL-6. We also suggested damage to the marrow stromal microenvironment, by myoablation, partly explains the impaired bone formation following a BMT. It is well known that some growth factor play important role in bone growth and osteogenesis. However, the pathogenetic role of bone growth factors in post-BMT bone loss is unknown and data on the changes in the growth factors, in accordance with bone turnover markers and bone mineral density (BMD) changes are scarce. We investigated changes in bone growth factors such as IGF-I (Insulin-like growth factor-I), fibroblast growth factor-2 (FGF-2) and Macrophage colony stimulating factor (M-CSF), during the post-BMT period, and assessed whether the growth factor changes influenced the bone turnover and post-BMT bone loss. The present study is the first prospective study to describe the changes in bone growth factors following a BMT. Methods: We prospectively investigated 110 patients undergoing a BMT, and analyzed 36 patients (32.4±1.3 years, 17 men and 19 women) whose BMDs were measured before, and 1 year after, the BMT. The serum biochemical markers of bone turnover were measured before, 1, 2, 3 and 4 weeks, 3 and 6 months, and 1 year, after the BMT. The serum, FGF-2, IGF-I and M-CSF levels were measured before and 1 and 3 weeks, and 3 months after the BMT. The correlation between the changes of growth factors and various bone parameters was analyzed. Results: The mean bone losses in the lumbar spine and total proximal femur, calculated as the percentage change from the baseline to the level at 1 year, were 5.2(p<0.05) and 11.6%(p<0.01), respectively. the serum type I carboxyterminal telopeptide(ICTP), a bone resorption marker, increased progressively until 6 months after the BMT, but thereafter decreased, to the base value after 1 year. Serum osteocalcin, a bone formation marker, decreased progressively, until 3 weeks after the BMT but then increased transiently, and finally returned to the base level at 1 year. The serum IGF-I and FGF-2 also decreased progressively until 3 weeks 1 week after the BMT, respectively, then increased to the base values at 3 months. The serum M-CSF increased briskly at 1 week post-BMT, then decreased to the base level. There were positive correlations between the percentage changes from the baseline proximal femur BMD and the IGF-I levels 3 weeks and 3 months (r=0.52, p<0.01, r=0.41, p<0.05) post BMT. A significant correlation was found between the IGF-I and osteocalcin levels pre-BMT, and 3 weeks after the BMT. Another positive correlation was found between the M-CSF and the ICTP levels at 3 weeks post BMT (r=0.54, p<0.05). Conclusion: In conclusion, there were significant changes in the serum IGF-I, FGF-2 and M-CSF levels in the immediate post-BMT period, which were related to a decrease in bone formation and loss in the proximal femoral BMD during the year following the BMT (J Kor Soc Endocrinol 17:664∼674, 2002).

Anti-inflammatory effect of polyphenol-rich extract from the red alga Callophyllis japonica in lipopolysaccharide-induced RAW 264.7 macrophages

Ryu, BoMi,Choi, Il-Whan,Qian, Zhong-Ji,Heo, Soo-Jin,Kang, Do-Hyung,Oh, Chulhong,Jeon, You-Jin,Jang, Chul Ho,Park, Won Sun,Kang, Kyong-Hwa,Je, Jae-Young,Kim, Se-Kwon,Kim, Young-Mog,Ko, Seok-Chun,Kim, G The Korean Society of Phycology 2014 ALGAE Vol.29 No.4

Despite the extensive literature on marine algae over the past few decades, a paucity of published research and studies exists on red algae. The purpose of this study was to evaluate the potential therapeutic properties of the ethanol extract of the red alga Callophyllis japonica against lipopolysaccharide (LPS)-stimulated macrophage inflammation. The C. japonica extract (CJE) significantly inhibited the nitric oxide (NO) production and the induced dose-dependent reduction of the protein and mRNA levels of inducible nitric oxide synthase and cyclooxygenase-2. Additionally, the CJE reduced the mRNA levels of inflammatory cytokines, including tumor necrosis factor-${\alpha}$, interleukin (IL)-$1{\beta}$, and IL-6. We investigated the mechanism by which the CJE inhibits NO by examining the level of mitogen-activated protein kinases (MAPKs) activation, which is an inflammation-induced signaling pathway in macrophages. The CJE significantly suppressed the LPS-induced phosphorylation of c-Jun N-terminal kinase, extracellular signal-regulated kinase and p38 MAPK. Taken together, the results of this study demonstrate that the CJE inhibits LPS-induced inflammation by blocking the MAPK pathway in macrophages.

골수이식 후 사이토카인과 골교체 생화학적표지자의 변화 및 상관관계

민우성,강무일,한제호,강성구,오기원,이원영,김혜수,문성대,손현식,신완식,김춘추,윤건호,차봉연,이광우,손호영 대한내분비학회 2000 Endocrinology and metabolism Vol.15 No.1

Background : Loss of bone mass is usually detected after BMT. The causes of bone loss are related with gonadal dysfunction and immunosuppressants. Cytokines, especially IL-6, play an important role in the pathogenesis of postmenopausal osteoporosis. However, the pathogenetic role of cytokines in post-BMT bone loss is unknown and data on the changes of cytokines in accordance with bone turnover markers are scarce. The aim of this study is to assess the relationship of bone turnover markers and cytokines of peripheral blood and bone marrow before and after allogeneic BMT. Methods : This prospective study included two analyses. The first was a study of 46 BMT recipients, examining the relationship between bone turnover markers and cytokines of serum which were measured before and 1, 2, 3, 4 week and 3 months after BMT. The second was a study of 14 BMT patients, measuring bone marrow plasma cytokines such as IL-6 and TNF-? at post-BMT 3 week and bone turnover marker at the same time to assess the relationship beween two parameters. Results : Serum ICTP, bone resorption marker, increased progressively until 4 weeks (peak) after BMT and then decreased thereafter. Serum osteocalcin, bone formation marker, decreased progressively until 3 weeks after BMT and then increased thereafter. There was positive correlation between serum ICTP and bone marrow IL-6 levels at the post-BMT 3 week with a statistical significance, but the correlation between bone turnover markers and bone marrow TNF-? or peripheral blood cytokines was not found. Conclusion : Our data suggest that the progressive increase of bone resorption after BMT is related with the increase of bone marrow IL-6, which is a potent stimulator of bone resorption in vivo(J Kor Soc Endocrinol 15:85-96, 2000).

抗精神病藥物에 依한 無顆粒細胞症 治驗 1例

吳東財,申榮宇,張煥一,趙京杉,朴康圭 大韓神經精神醫學會 1985 신경정신의학 Vol.24 No.1

The authors experienced a female schizophrenic patient who revealed severely decreased leucocytes (350/㎣) with the manifestation of high fever, chillness and sore throat after the oral administration of neuroleptics. Agranulocytosis induced with the use of neuroleptic drugs has been well known complication in psychiatric practice although it is quite rare. In this case, following the combined use of neuroleptics (thioridazine halopondol and chlorproonazine), severe decrement of leucocytes (mainly lymphocytes with few neutrophils) was noticed on peripheral blood and marked decrement of erythroid series, especially of myeloid series, with decrement of total cellular elements were confirmed through the biopsy of bone marrow. She did not responded at all to the massive administration of antibiotics, but dramatic improvement was noticed both clinically and in the follow-up biopsy findings of bone marrow after two times trial of leukapheresis.