Minimal residual disease monitoring by flow cytometry in children with acute lymphoblastic leukemia

The cells that have avoided the action of antitumor drugs may be retained after remission achievement during induction therapy and consolidation. A combination of these cells is given the name minimal residual disease (MRD). Multicolor flow cytometry has recently attracted considerable interest as the most promising method for measuring the content of residual tumor blasts. This technique is based on the detection of the so-called leukemia-associated immunophenotype (LAIP), i.e., a tumor-specific combination of the expression of membrane and cytoplasmic markers. Flow cytometry may be successfully used to monitor MRD in 90-95% cases of acute lymphoblastic leukemia (ALL) and in 80-85% of patients with acute myelocytic leukemia. The sensitivity of flow cytometry, which is real for routine flow techniques, is a possibility of identifying one cell among 10(4)-10(5) cells. Multicolor flow cytometry (that involves the simultaneous analysis of the expression of a few markers) is the most reasonable tool for MRD monitoring. The monoclonal antibody panels recommended by different groups of investigators for MRD monitoring in B-lineage ALL include antibodies to the pan-B-cell antigen CD19, markers of different stages of differentiation of B-lineage precursors of CD10, CD34, and CD20 and leukemia-associated markers different for each panel, such as CD22, CD38, CD58, CD45, TdT, CD13, CD33. The hyperexpression of CD10, CD34, CD19, TdT, the decreased expression of CD38, CD45, CD22, CD19, the simultaneous expression of markers of different stages of differentiation of B lymphocytes, such as CD10 and CD20, and the lymphoblast coexpression of myeloid markers of CD13, CD33, CDS15 are the most frequently described immunophenotype aberrations in B-lineage ALL. The selection of combinations of markers for MRD monitoring in children with T-ALL is based on the simultaneous expression of combinations of the antigens characteristic for early stages of differentiation of normal T lymphocytes, namely TdT and cytoplasmic CD3. Some authors consider the use of CD99 versus TdT to be most appropriate. There is recent evidence that MRD-positive patients have a higher cumulative risk for recurrences as compared with those without residual blasts. Moreover, the longer the tumor cells are retained during therapy, the worse the prognosis is. Thus, for choice of the adequate intensity of antitumor therapy, it is necessary to qualitatively and quantitatively assess MRD by multicolor flow cytometry at different stages of therapy.     

1140919448CD200-dependent suppression of human NK activity by IVIG requires CD200 receptor type 2

Problem: IVIG prepared from plasma of stored human blood can be efficacious in improving pregnancy success in a selected subgroup of patients but RCTs using an IVIG showing inferior suppression of NK activity in vitro have been negative (J Assist Reprod Genet 2006). A significant component of NK suppression by IVIG appears to be due to CD200 released into plasma from PBL during storage at 4C. CD200 receptors (CD200R) are expressed at the fetomaternal interface prior to onset of abortion; CD200R1 mediates direct effects on gamma‐delta T cell development and suppresses alpha‐beta T cell responses in vitro, whereas CD200R2 alters DC so as to facilitate development of alpha‐beta Treg cells. Which receptor(s) mediate NK cell suppression? Methods: Purified human PBL or the CD56⁺ NK cell subset of PBL were used to lyse 51Cr‐labeled K562 cells in vitro. Different IVIG preparations were tested for suppressive ability, and suppression was blocked by either anti‐huCD200 mAb or rabbit anti‐huCD200R1 or R2 antibodies. Results: CD200‐dependent IVIG NK suppressive potency differed among IVIG types (Gammagard>Gamunex>>Gamimmune). CD200‐dependent suppression was blocked by anti‐CD200R antibody able to react with the type 2 receptor. K562 cells did not express receptor, and purified CD56⁺ NK cells were suppressed effectively without the need for non‐NK cells. Conclusions: IVIG may directly express NK cell activity via CD200 binding to CD200R2.     

<Emphasis Type="Italic">In vitro</Emphasis> study of the role of lenticular epithelial calcium channels and aquaporins in the development of cataract

The role of calcium and water channels (aquaporins) in the pathogenesis of cataract was studied in vitro. Aquaporin blockade caused opacity of the lens sooner than changes in calcium ion concentration in culture medium. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 2, pp. 144–147, February, 2008     

IGF1R signalling in testicular germ cell tumour cells impacts on cell survival and acquired cisplatin resistance

Testicular germ cell tumours (TGCTs) are the most frequent malignancy and cause of death from solid tumours in the 20‐ to 40‐year age group. Although most cases show sensitivity to cis‐platinum‐based chemotherapy, this is associated with long‐term toxicities and chemo‐resistance. Roles for receptor tyrosine kinases other than KIT are largely unknown in TGCT. We therefore conducted a phosphoproteomic screen and identified the insulin growth factor receptor‐1 (IGF1R) as both highly expressed and activated in TGCT cell lines representing the nonseminomatous subtype. IGF1R was also frequently expressed in tumour samples from patients with nonseminomas. Functional analysis of cell line models showed that long‐term shRNA‐mediated IGF1R silencing leads to apoptosis and complete ablation of nonseminoma cells with active IGF1R signalling. Cell lines with high levels of IGF1R activity also showed reduced AKT signalling in response to decreased IGF1R expression as well as sensitivity to the small‐molecule IGF1R inhibitor NVP‐AEW541. These results were in contrast to those in the seminoma cell line TCAM2 that lacked IGF1R signalling via AKT and was one of the two cell lines least sensitive to the IGF1R inhibitor. The dependence on IGF1R activity in the majority of nonseminomas parallels the known role of IGF signalling in the proliferation, migration, and survival of primordial germ cells, the putative cell of origin for TGCT. Upregulation of IGF1R expression and signalling was also found to contribute to acquired cisplatin resistance in an in vitro nonseminoma model, providing a rationale for targeting IGF1R in cisplatin‐resistant disease. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

VEGF-C表达和微淋巴管密度与胃癌淋巴转移的关系及意义

目的探讨胃癌组织血管内皮生长因子C(VEGF-C)表达和微淋巴管密度(MLVD)及两者与胃癌淋巴转移的关系。方法应用免疫组织化学方法检测208例人胃癌组织、40例癌浸润前缘组织及139例人胃正常粘膜组织中VEGF-C、D2-40的表达,对D2-40阳性脉管进行MLVD计数,并结合病理资料进行统计学分析。结果胃癌组织VEGF-C的表达明显高于正常胃粘膜组织(χ2=109.199,P<0.01);胃癌组织中有淋巴结转移(χ2=14.496,P<0.01)或浸润透浆膜(χ2=11.586,P<0.01)组VEGF-C表达水平分别较无转移或浸润未及浆膜组增高。癌浸润前缘组织中MLVD(18.36±15.60个/mm2)明显高于胃癌组(9.41±9.32个/mm2,t=-3.681,P<0.01)和胃正常粘膜组织(7.70±7.69个/mm2,t=-4.180,P<0.01);胃癌淋巴结转移组MLVD(9.81±9.97个/mm2)高于无转移组(6.41±7.85个/mm2,t=2.516,P<0.01),而在浸润透浆膜组(11.20±10.55个/mm2)和未及浆膜组(8.54±9.36个/mm2)MLVD无差别(t=1.467,P=0.472)。另外,在胃癌组织中VEGF-C表达与MLVD呈正相关(F=2.910,P<0.05)。结论VEGF-C在胃癌中的高表达与胃癌浸润深度、淋巴转移密切相关。     

The EuroPrevall surveys on the prevalence of food allergies in children and adults: background and study methodology

Background:  The epidemiological surveys in children and adults of the EU-funded multidisciplinary Integrated Project EuroPrevall, launched in June 2005, were designed to estimate the currently unknown prevalence of food allergy and exposure to known or suspected risk factors for food allergy across Europe. We describe the protocol for the epidemiological surveys in children and adults. This protocol provides specific instructions on the sampling strategy, the use of questionnaires, and collection of blood samples for immunological analyses.
Methods:  The surveys were performed as multi-centre, cross-sectional studies in general populations. Case–control studies were nested within these surveys. The studies in children aged 7–10 years and adults aged 20–54 years were undertaken in eight centres representing different social and climatic regions in Europe.
Results:  After a community-based survey collecting basic information on adverse reactions to foods, all those stating they had experienced such reactions, as well as of a random sample of those stating 'no reactions' to foods, completed a detailed questionnaire on potential risks and exposures. Also a blood sample was taken to allow serological analysis to establish patterns of food and aeroallergen sensitization. We also included a questionnaire to schools on their preparedness for dealing with food allergy amongst pupils. Subjects reporting adverse reactions to foods and sensitized to the same food(s) were called in for a full clinical evaluation that included a double blind placebo controlled food challenge (DBPCFC), following a protocol which is described in detail elsewhere.
Conclusions:  The outcome of these studies will help to improve our understanding of several important aspects of food allergies in the European Community, providing for more well-informed policies and effective measures of disease prevention, diagnosis and management.     

Simultaneous MRI and PET imaging of a rat brain

Multi-modality imaging is rapidly becoming a valuable tool in the diagnosis of disease and in the development of new drugs. Functional images produced with PET fused with anatomical structure images created by MRI will allow the correlation of form with function. Our group is developing a system to acquire MRI and PET images contemporaneously. The prototype device consists of two opposed detector heads, operating in coincidence mode. Each MRI-PET detector module consists of an array of LSO detector elements coupled through a long fibre optic light guide to a single Hamamatsu flat panel position-sensitive photomultiplier tube (PSPMT). The use of light guides allows the PSPMTs to be positioned outside the bore of a 3T MRI scanner where the magnetic field is relatively small. To test the device, simultaneous MRI and PET images of the brain of a male Sprague Dawley rat injected with FDG were successfully obtained. The images revealed no noticeable artefacts in either image set. Future work includes the construction of a full ring PET scanner, improved light guides and construction of a specialized MRI coil to permit higher quality MRI imaging.     

Dendritic cell-based vaccine strategy against human immunodeficiency virus clade C: skewing the immune response toward a helper T cell type 2 profile

Given the continued spread of human immunodeficiency virus (HIV)-1 worldwide, developing efficient vaccine strategies against HIV-1 is a key task. We tested the safety and immunogenicity of a multicomponent, cell-based vaccine that consisted of antigen-expressing apoptotic bodies with or without autologous dendritic cells (DCs). The vaccine strategy involved transfection of human 293T cells with codon-optimized DNA vectors expressing env of HIV1084i, a newly transmitted pediatric HIV-1 clade C strain; SHIV89.6P tat; and SIVmac239 gag-protease. Apoptotic bodies were generated by heat shock and ultraviolet irradiation and mixed either with mouse DCs (DC-cell vaccine) or given directly (cell-only vaccine) to BALB/c mice for initial priming; boosts consisted of apoptotic bodies only. The immunogens were well tolerated with or without DCs. Compared with the cell-only vaccine, the DC-cell vaccine induced higher antibody titers against all three antigens, whereas virus-specific cytotoxic T lymphocyte responses were equally strong in both groups. Iso-type analysis of viral antigen-specific antibodies revealed a skewing toward helper T type 2 responses induced by the DC-cell vaccine but not by the cell-only vaccine. In summary, both vaccine strategies were safe and induced cellular as well as humoral antiviral immunity; the DC-based approach had the advantage of significantly stronger antibody responses.     

C-kit expression in renal oncocytomas and chromophobe renal cell carcinomas

C- kit encodes the membrane-bound tyrosine kinase KIT, whose expression has been identified in several types of human neoplasms. Recently, KIT has been reported to be a marker for chromophobe renal cell carcinoma (RCC) and renal angiomyolipoma. However, expression of this molecule has not been adequately studied in other renal tumors, particularly oncocytoma, which may morphologically resemble chromophobe RCC. In this study, we analyzed c- kit messenger RNA (mRNA) levels in 17 chromophobe RCCs and 20 renal oncocytomas obtained from complementary DNA (cDNA) microarrays. Furthermore, comprehensive immunohistochemical analysis of KIT protein using a monoclonal antibody was performed in 226 renal tumors including chromophobe RCC (n=40), oncocytoma (n=41), clear-cell RCC (n=40), renal angiomyolipoma (n=29), and papillary RCC (n=21) on tissue microarrays (TMAs) and was compared with immunostaining results from 25 chromophobe RCCs and 30 oncocytomas using standard sections. The staining intensity was semiquantitatively graded on a 3-tier scoring system. All chromophobe RCCs and oncocytomas showed significant overexpression of c- kit mRNA. The average increase of mRNA compared with normal kidney tissue was 7.4-fold for chromophobe RCCs and 7.4-fold for oncocytomas. Immunohistochemical expression of KIT was found in most chromophobe RCCs (95% in TMAs and 96% in conventional sections) and oncocytomas (88% in TMAs and 100% in conventional sections) but was infrequently observed in renal angiomyolipomas (17%), papillary RCCs (5%), and clear-cell RCCs (3%). Furthermore, the average KIT immunoreactivity in TMAs was stronger in chromophobe RCC (1.93) and oncocytoma (2.07) than in other subtypes of renal tumors tested, including angiomyolipomas (0.17), papillary RCCs (0.05), and clear-cell RCCs (0.03). In conclusion, we found a significant elevation of c- kit mRNA by cDNA expression microarrays and overexpression of KIT protein by immunohistochemistry not only in chromophobe RCCs but also in oncocytomas. In contrast, immunohistochemical expression of KIT was not detected in most other types of renal cell tumors evaluated. The differential expression of c- kit in these renal tumors may have diagnostic and therapeutic implications.