Clinical and Genetic Spectrum of Inborn Errors of Immunity in a Tertiary Care Center in Southern India

Objectives To study the incidence, clinical manifestations, and genetic spectrum of primary immunodefciency diseases (PID)/inborn errors of immunity (IEI) in a tertiary care hospital in Southern India. Methods A retrospective analysis of all patients with a clinical suspicion of PID/IEI seen at a tertiary care hospital was performed. All patients had at least one or more warning signs of PID. Serum immunoglobulin levels and other targeted investigations were performed as warranted by the clinical presentation. All families with suspected PID were counseled and ofered genetic testing. Results A total of 225 children were evaluated for PID during the study period of 6 y. Fifty-six of them did not meet the European Society of Immunodefciencies (ESID) criteria (working defnition of clinical diagnosis) and were excluded. An IEI was found in 30/49 (61.2%) patients. The most frequent reason for referral was recurrent/unusual or serious infections (28%), or cytopenia (16%). Group IV diseases of immune dysregulation was the most common category (19%), followed by group III predominant antibody defciencies in 23/163 (14%), as per the International Union of Immunological Societies (IUIS) classifcation. Conclusions This study highlights the heterogeneity of the present cohort, the underuse of genetic tests, and eforts to provide optimal care for children with possible IEI in this center.

Anti-tumorimmune effects of WT1 polypeptide vaccine on SCID mice with xenografted human monocytic leukemia / 西安交通大学学报(医学版)

Objective: To study the anti-tumor immune effects of WT1 peptide vaccine in SCID mice with xenografted human monocytic leukemia. Methods: Twenty-four hours after intraperitoneal injection of human peripheral blood lymphocytes, the xenograft human monocytic leukemia model in SCID mice was established by subcutaneous inoculation of THP1 cells. The mice were randomly divided into three groups with eight mice in each. Blank control group was vaccinated with incomplete Freund's adjuvant (IAF). Helper T cell epitope group was vaccinated with helper T cell epitopes and IAF. WT1 group was vaccinated with WT1 peptide, helper T cell epitopes and IAF. When the tumor volume grew to 100 mm3, intraperitoneal injection of vaccine components started. The SCID mice were killed 14 days after vaccination. LDH release method was adopted to detect the specific CTL killing activity of spleen cells. Histological characteristics of tumor tissue were observed under microscope after HE staining. Flow cytometry was used to test the levels of peripheral blood CD3+/CD4+T cells, CD3+/CD8+T cells and CD4+CD25+ Treg cells. ELISA method was applied to detect the levels of serum immunoglobulin, IL-2, γ-interferon, TGF-β and IL-10. Results: The xenograft human monocytic leukemia model was successfully established in SCID mice and tumor developed in all the SCID mice. In WT1 group, the activity of mouse spleen cells on THP1 cells was significantly higher than that in helper T cell epitope group and control group (P<0.05). The mean weight and volume of tumor were significantly lower in WT1 group than in helper T cell epitope group and control group (P<0.05). A large amount of tumor cell degeneration and necrosis was observed under the microscope in WT1 group mice and few tumor cells survived. Peripheral blood levels of CD3+/CD4+T cells, CD3+/CD8+T cells, IgG, IFN-γ and IL-2 were all higher in WT1 group than in helper T cell epitope group and control group (P<0.05). However, peripheral blood levels of CD4+/CD25+Treg cells, TGF-β and IL-10 were all lower in WT1 group than in helper T cell epitope group and control group (P<0.05). Conclusion: WT1 polypeptide vaccine can effectively produce anti-tumor immunity and kill leukemia cells in SCID mice with exnografted human monocytic leukemia.

Efficient derivation of extended pluripotent stem cells from NOD-scid Il2rg mice

Recently we have established a new culture condition enabling the derivation of extended pluripotent stem (EPS) cells, which, compared to conventional pluripotent stem cells, possess superior developmental potential and germline competence. However, it remains unclear whether this condition permits derivation of EPS cells from mouse strains that are refractory or non-permissive to pluripotent cell establishment. Here, we show that EPS cells can be robustly generated from non-permissive NOD-scid Il2rg mice through de novo derivation from blastocysts. Furthermore, these cells can also be efficiently generated by chemical reprogramming from embryonic NOD-scid Il2rg fibroblasts. NOD-scid Il2rg EPS cells can be expanded for more than 20 passages with genomic stability and can be genetically modified through gene targeting. Notably, these cells contribute to both embryonic and extraembryonic lineages in vivo. More importantly, they can produce chimeras and integrate into the E13.5 genital ridge. Our study demonstrates the feasibility of generating EPS cells from refractory mouse strains, which could potentially be a general strategy for deriving mouse pluripotent cells. The generation of NOD-scid Il2rg EPS cell lines permits sophisticated genetic modification in NOD-scid Il2rg mice, which may greatly advance the optimization of humanized mouse models for biomedical applications.

Mouse models for hepatitis B virus research / 한국실험동물학회지

Hepatitis B virus (HBV) infection remains a major global health problem; indeed, there are 250 million carriers worldwide. The host range of HBV is narrow; therefore, few primates are susceptible to HBV infection. However, ethical constraints, high cost, and large size limit the use of primates as suitable animal models. Thus, in vivo testing of therapies that target HBV has been hampered by the lack of an appropriate in vivo research model. To address this, mouse model systems of HBV are being developed and several are used for studying HBV in vivo. In this review, we summarize the currently available mouse models, including HBV transgenic mice, hydrodynamic injection-mediated HBV replicon delivery systems, adeno-associated virus-mediated HBV replicon delivery systems, and human liver chimeric mouse models. These developed (or being developed) mouse model systems are promising and should be useful tools for studying HBV.

Establishment of Metrigel-VEGF-SW982 complexes and application of the complexes in synovial sarcoma model in mice / 西安交通大学学报(医学版)

Objective To establish Metrigel-VEGF-SW982 complexes and use the complexes to produce animal models of synovial sarcoma so as to provide new ideas for establishing other models of soft tissue tumors. Methods After the SW982 cells were cultured and collected,they were resuspended with Metrigel,and VEGF was added.The suspension was seeded into transwell to establish the scaffold complexes of Metrigel-VEGF-SW982.The complexes were cultured overnight.Cryosections were made and HE staining was carried out to observe the cell scaffold complexes.We randomly divided 10 female SCID mice(4 week old)into scaffold group and control group. The mice in the scaffold group were transplanted with cell-scaffold complexes,and the control group with cell suspension.After 8 weeks,the success rate of modeling was compared between two groups.The mice were sacrificed and the tumors were obtained.HE staining was carried out to observe the histopathological features of tumors in both scaffold group and control group.Results The SW982 cells were cultured with Matrigel in a 3D way,which could simulate the growth condition of cells in vivo.Establishing synovial sarcoma animal model with cell scaffold complex could increase the success rate.The tumors in scaffold group had a larger volume,higher density of tumor cells and greater vascularization(P＜0.05).Conclusion Establishing synovial sarcoma animal model with Metrigel-VEGF-SW982 complex can greatly improve the success rate of modeling,which can provide basis for the study of synovial sarcoma.

The Establishment of SCID Mice Model on Endometriosis / 昆明医科大学学报

Objective To set up a favorable animal model for the drug treatment research of endometriosis by establishing the animal model of endometriosis in SCID and nude mice so as to compare the influences on implantation of human endometrial tissue derived from the eutopic and ectopic sources. Methods Eutopic and ectopic endometrium were transplanted to the lower abdominal parts subcutaneously of 30 sexually matured BALB/c-nu/nu nude mice and SCID mice respectively. The ectopic lesion sizes were under the regular observation before they were removed 6 weeks after the operation for pathological examinations. Results Nude mice and SCID mice were able to be used to establish a successful animal models of endometriosis. The success rate of SCID mice was higher than that of nude mice. The success rate of the eutopic endometrium group was significantly higher than that of the ectopic endometrium group. Nude and SCID mice endometriosis implantation models were successfully established. The modeling success rate of SCID mice is higher than that of the nude mice.The success rate of transplantation was higher in the ectopic endometrium than in the eutopic endometrium.Conclusion The SCID mice endometriosis endometriosis model provides a favorable animal model of endometriosis.

TRIAGEM NEONATAL DE IMUNODEFICIÊNCIAS GRAVES COMBINADAS POR MEIO DE TRECS E KRECS: SEGUNDO ESTUDO PILOTO NO BRASIL / NEWBORN SCREENING FOR SEVERE COMBINED IMMUNODEFICIENCIES USING TRECS AND KRECS: SECOND PILOT STUDY IN BRAZIL

RESUMO Objetivo: Validar a quantificação de T-cell receptor excision circles (TRECs) e kappa-deleting recombination circles (KRECs) por reação em cadeia de polimerase (polymerase chain reaction, PCR) em tempo real (qRT-PCR), para triagem neonatal de imunodeficiências primárias que cursam com defeitos nas células T e/ou B no Brasil. Métodos: Amostras de sangue de recém-nascidos (RN) e controles foram coletadas em papel-filtro. O DNA foi extraído e os TRECs e KRECs foram quantificados por reação duplex de qRT-PCR. O valor de corte foi determinado pela análise de Receiver Operating Characteristics Curve, utilizando-se o programa Statistical Package for the Social Sciences (SSPS) (IBM®, Armonk, NY, EUA). Resultados: 6.881 amostras de RN foram analisadas quanto à concentração de TRECs e KRECs. Os valores de TRECs variaram entre 1 e 1.006 TRECs/µL, com média e mediana de 160 e 139 TRECs/µL, respectivamente. Três amostras de pacientes diagnosticados com imunodeficiência grave combinada (severe combined immunodeficiency, SCID) apresentaram valores de TRECs abaixo de 4/µL e um paciente com Síndrome de DiGeorge apresentou TRECs indetectáveis. Os valores de KRECs encontraram-se entre 10 e 1.097 KRECs/µL, com média e mediana de 130 e 108 KRECs/µL, e quatro pacientes com diagnóstico de agamaglobulinemia tiveram resultados abaixo de 4 KRECs/µL. Os valores de corte encontrados foram 15 TRECs/µL e 14 KRECs/µL, e foram estabelecidos de acordo com a análise da Receiver Operating Characteristics Curve, com sensibilidade de 100% para detecção de SCID e agamaglobulinemia, respectivamente. Conclusões: A quantificação de TRECs e KRECs foi capaz de diagnosticar crianças com linfopenias T e/ou B em nosso estudo, validando a técnica e dando o primeiro passo para a implementação da triagem neonatal em grande escala no Brasil.

ABSTRACT Objective: To validate the quantification of T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs) by real-time polymerase chain reaction (qRT-PCR) for newborn screening of primary immunodeficiencies with defects in T and/or B cells in Brazil. Methods: Blood samples from newborns and controls were collected on filter paper. DNA was extracted and TRECs, and KRECs were quantified by a duplex real-time PCR. The cutoff values were determined by receiver operating characteristic curve analysis using SPSS software (IBM®, Armonk, NY, USA). Results: Around 6,881 samples from newborns were collected and TRECs and KRECs were quantified. The TRECs values ranged between 1 and 1,006 TRECs/µL, with mean and median of 160 and 139 TRECs/µL, respectively. Three samples from patients with severe combined immunodeficiency (SCID) showed TRECs below 4/µL and a patient with DiGeorge syndrome showed undetectable TRECs. KRECs values ranged from 10 to 1,097 KRECs/µL, with mean and median of 130 and 108 KRECs/µL. Four patients with agammaglobulinemia had results below 4 KRECs/µL. The cutoff values were 15 TRECs/µL and 14 KRECs/µL and were established according to the receiver operating characteristic curve analysis, with 100% sensitivity for SCID and agammaglobulinemia detection, respectively. Conclusions: Quantification of TRECs and KRECs was able to diagnose children with T- and/or B-cell lymphopenia in our study, which validated the technique in Brazil and enabled us to implement the newborn screening program for SCID and agammaglobulinemia.

The identifiability for depressive symptoms of different methods in suicide prevention research / 中国神经精神疾病杂志

Objective To compare the identifiability for depressive symptoms using different instruments while interviewing with different respondents in suicide prevention research in China. Methods One hundred and fifty-one suicide death cases (suicide group) and one hundred and twenty suicide attempt cases (attempt group) were recruited. For each identified cases, one family member proxy respondent, and another associate proxy respondent (friend or neighbor) and suicide attempter (only for attempt group) were interviewed separately by qualified psychiatrists. The Di-agnostic Screening Instrument for Depression (DSID) and the Structured Clinical Interview for DSM-Ⅳ Axis Ⅰ Disorders (SCID-Ⅰ) were administered to each respondent to identify the depressive symptoms based on diagnostic criteria for major depressive episode in DSM-Ⅳ. Data collected from family members and associate respondents were merged as proxy data. The concordances of the DSID and SCID-Ⅰfor identifying depressive symptoms, meeting for criteria of Major Depressive Episode (MDE) and Mild and Major Depressive Episode (MMDE), were calculated based on different respondents' data. The prevalence of depressive symptoms, MDE and MMDE, were compared among merged proxy data, family member respondent's data, and associate respondent's data in suicide group and attempt group, and between self-respondent's data and merged proxy data in suicide attempt group. Results In suicide group, based on merged proxy data, the prevalence of MDE was 41.1%(62 cases) for DSID and 41.7%(63 cases) for SCID-Ⅰ, and the Kappa coeffi-cient was 0.77. Based on suicide attempters' self-raported data, the prevalence of MDE was 23.7% (27 cases) and 22.0% (24 cases) for DSID and SCID-Ⅰ respectively, with a Kappa of 0.74. Based on merged proxy report in attempt group, 16 (13.3%) and 15 (12.5%) cases were met for criteria of MDE (Kappa=0.89), using the 2 instruments. In both of the suicide and attempt groups, the merged proxy data got higher prevalence of depressive symptoms, MDE and MMDE than that only based on family respondent's data or associate's respondent's data using both of the 2 instruments (all P<0.05). Compared with merged proxy data, attempters' self-reported data got higher prevalence of MMD and MMDE using both of the 2 instruments (all P<0.05). Conclusions Based on same respondent's data, SCID-Ⅰ performs as well as DSID in identifying depressive symptoms. Collecting data from 2 respondents would get higher prevalence of MDE or MMDE than only from one family member or one associate. In attempt group, the prevalence of MDE or MMDE based on merged proxy data were lower than that based on attempters' self-reported data.

Neonatal screening for severe combined immunodeficiency in Brazil / Triagem neonatal para imunodeficiência combinada grave no Brasil

Abstract Objective To apply, in Brazil, the T-cell receptor excision circles (TRECs) quantification technique using real-time polymerase chain reaction in newborn screening for severe combined immunodeficiency and assess the feasibility of implementing it on a large scale in Brazil. Methods 8715 newborn blood samples were collected on filter paper and, after DNA elution, TRECs were quantified by real-time polymerase chain reaction. The cutoff value to determine whether a sample was abnormal was determined by ROC curve analysis, using SSPS. Results The concentration of TRECs in 8,682 samples ranged from 2 to 2,181 TRECs/µL of blood, with mean and median of 324 and 259 TRECs/µL, respectively. Forty-nine (0.56%) samples were below the cutoff (30 TRECs/µL) and were reanalyzed. Four (0.05%) samples had abnormal results (between 16 and 29 TRECs/µL). Samples from patients previously identified as having severe combined immunodeficiency or DiGeorge syndrome were used to validate the assay and all of them showed TRECs below the cutoff. Preterm infants had lower levels of TRECs than full-term neonates. The ROC curve showed a cutoff of 26 TRECs/µL, with 100% sensitivity for detecting severe combined immunodeficiency. Using this value, retest and referral rates were 0.43% (37 samples) and 0.03% (3 samples), respectively. Conclusion The technique is reliable and can be applied on a large scale after the training of technical teams throughout Brazil.

Resumo Objetivo Aplicar no Brasil a técnica de quantificação de T-cell Receptor Excision Circles (TRECs) por PCR em tempo real para triagem neonatal de imunodeficiência combinada grave (SCID) e avaliar se é possível fazê-la em grande escala em nosso país. Métodos Foram coletadas em papel filtro 8.715 amostras de sangue de recém-nascidos e, após eluição do DNA, os TRECs foram quantificados por PCR em tempo real. O valor de corte para determinar se uma amostra é anormal foi determinado pela análise de curva ROC com o programa SSPS. Resultados A concentração de TRECs em 8.682 amostras analisadas variou entre 2 e 2.181 TRECs/µL de sangue, com média e mediana de 324 e 259 TRECs/µL, respectivamente. Das amostras, 49 (0,56%) ficaram abaixo do valor de corte (30 TRECs/µL) e foram requantificadas. Quatro (0,05%) mantiveram resultados anormais (entre 16 e 29 TRECs/µL). Amostras de pacientes com diagnóstico clínico prévio de SCID e síndrome de DiGeorge foram usadas para validar o ensaio e todas apresentaram concentração de TRECs abaixo do valor de corte. Recém-nascidos prematuros apresentaram menores níveis de TRECs comparados com os nascidos a termo. Com o uso da curva ROC em nossos dados, chegamos ao valor de corte de 26 TRECs/µL, com sensibilidade de 100% para detecção de SCID. Com o uso desse valor, as taxas de repetição e encaminhamento ficaram em 0,43% (37 amostras) e 0,03% (3 amostras), respectivamente. Conclusão A técnica é factível e pode ser implantada em grande escala, após treinamento técnico das equipes envolvidas.

Sensibilidad y especificidad entre la Entrevista Diagnóstica Internacional Compuesta versión 3. 0 (World Mental Health, CIDI) con la Evaluación Clínica Estandarizada versión I (SCID-I) en la Encuesta de Salud Mental de la ciudad de Medellín, 2012 / Sensitivity and specificity between the Composite International Diagnostic Interview Version 3. 0 (World Mental Health, CIDI) and the Standardised Clinical Evaluation version I (SCID-I) in a mental health survey of the city of Medellin, 2012

Introducción: El abordaje de los problemas de salud mental de la población colombiana hace necesario disponer de instrumentos diagnósticos válidos, fáciles de aplicar y comparables (local e internacionalmente). Objetivo: Comparar la sensibilidad y la especificidad diagnóstica entre el CIDI 3.0 y el SCID-! para el trastorno depresivo mayor, el trastorno afectivo bipolar I y II y el trastorno por dependencia de sustancias. Metodología: Estudio transversal que comparó en 100 sujetos las prevalencias de vida de tres trastornos mentales por medio del CIDI 3.0 y el SCID-I. La investigación fue aprobada por el Comité de Ética Institucional. Se midieron la sensibilidad, la especificidad, el valor predictivo positivo y el valor predictivo negativo (con sus respectivos intervalos de confianza del 95%) de las dos entrevistas diagnósticas. Para el análisis de la información se utilizó el software SPSS® versión 21.0. Resultados: La mediana de edad fue 43,5 [intervalo intercuartílico, 30] anos. La sensibilidad (Se) y la especificidad (Es) más altas se observaron en el diagnóstico de trastorno por dependencia de drogas -Se, 80% (IC95%, 34,94%-100%); Es, 98,46% (IC95%, 94,7%-100%)-. Conclusiones: El SCID-I y el CIDI 3.0 mostraron diferentes niveles de sensibilidad y especificidad para los tres trastornos estudiados así: altas para el trastorno por dependencia de sustancias, moderadas para el trastorno afectivo bipolar I y II y bajas para el trastorno depresivo mayor.

Introduction: In order to address the mental health problems of the Colombian population it is necessary to have diagnostic tools (local and international) that are valid, easy to apply, and comparable. Objective: To compare the sensitivity and specificity between the CIDI 3.0 and the SCID-I for major depressive disorder, bipolar I and II disorder, and substance dependence disorder. Methodology: Cross-sectional study comparing the life prevalence of three mental disorders in 100 subjects using the CIDI 3.0 and the SCID-I. The study was approved by the Institutional Ethics Committee. The two diagnostic interviews were performed that measured by sensitivity, specificity, positive predictive value and negative predictive value with confidence intervals of 95%. The SPSS version 21.0 software was used for data analysis. Results: The median age was 43.5 years, with an interquartile interval of 30 years. The highest sensitivity (Se) and specificity (Sp) was observed for drug dependence diagnosis - with 80%, (95%CI, 34.94-100), and 98.46 (95%CI, 94.7-100), respectively. Conclusions: SCID-I and CIDI 3.0 showed different levels of sensitivity and specificity for the three disorders studied with: high for substance dependence disorder, moderate for bipolar disorder I and II, and low for major depressive disorder.

Observation of the estrous cycle in female NOD/SCID mice / 中国实验动物学报

Objective To observe the changes in estrous cycle and vaginal smears in ovarectomized NOD/SCID mice.Methods To continuously observe the estrous cycle time by vaginal smears of NOD/SCID mice in consecutive nine days, twice daily.After ovariectomy, the changes of estrous cycle were observed by vaginal smears for 7 days.Results The estrous cycle in NOD/SCID mice was 4-6 days.Regular estrous mice accounted for 80%.There was no significant correlation between vaginal opening and estrous cycle status.After ovariectomy, the vaginal smears showed characteristics of metestrus or diestrus.Conclusions Vaginal smear cytology can be used to determine the estrous cycle and characteris-tics of NOD/SCID female mice.The ovariectomized operation of NOD/SCID female mice is effective.

Cancer stem cells: a new approach to tumor development / Células-tronco de câncer: um nova abordagem do desenvolvimento tumoral

Many theories have been proposed to explain the origins of cancer. Currently, evidences show that not every tumor cell is capable of initiating a tumor. Only a small part of the cancer cells, called cancer stem cells (CSCs), can generate a tumor identical to the original one, when removed from human tumors and transplanted into immunosuppressed mice. The name given to these cells comes from the resemblance to normal stem cells, except for the fact that their ability to divide is infinite. These cells are also affected by their microenvironment. Many of the signaling pathways, such as Wnt, Notch and Hedgehog, are altered in this tumoral subpopulation, which also contributes to abnormal proliferation. Researchers have found several markers for CSCs; however, much remains to be studied, or perhaps a universal marker does not even exist, since they vary among tumor types and even from patient to patient. It was also found that cancer stem cells are resistant to radiotherapy and chemotherapy. This may explain the re-emergence of the disease, since they are not completely eliminated and minimal amounts of CSCs can repopulate a tumor. Once the diagnosis in the early stages greatly increases the chances of curing cancer, identifying CSCs in tumors is a goal for the development of more effective treatments. The objective of this article is to discuss the origin of cancer according to the theory of stem cell cancer, as well as its markers and therapies used for treatment.

Diversas teorias buscam explicar a origem do câncer. Atualmente, há evidências de que nem todas as células tumorais têm poder de iniciar um tumor. Apenas uma pequena parte das células cancerígenas, chamadas de células-tronco de câncer (do inglês cancer stem cells - CSC), é capaz de iniciar um tumor idêntico ao original quando retirada de tumores humanos e enxertada em camundongos imunossuprimidos. Essas células foram assim denominadas por suas semelhanças com células-tronco normais, exceto pelo fato de que sua capacidade de dividir-se é infinita. Essas células também recebem influência de seu microambiente. Várias vias de sinalização, como WNT, NOTCH e Hedgehog, estão alteradas nessa subpopulação tumoral, contribuindo também para a desregulação de sua proliferação. Pesquisadores descobriram vários marcadores para as CSC, porém ainda há muito a ser pesquisado, ou talvez nem exista um marcador universal, já que eles variam entre cada tipo de tumor e até de paciente para paciente. Foi constatado também que as CSC são resistentes à radioterapia e à quimioterapia, podendo explicar o reaparecimento da doença, visto que, além de não eliminá-la completamente, quantidades mínimas das CSC podem repovoar um tumor. Como o diagnóstico em estágios iniciais aumenta muito as chances de cura do câncer, a identificação das CSC em meio a um tumor é alvo para o desenvolvimento de tratamentos mais eficazes. O objetivo deste artigo é discutir a origem do câncer segundo a teoria das CSC, bem como seus marcadores e as terapias utilizadas em seu tratamento.

Establishment of mouse model of orbital diffuse large B cell lymphoma / 中华实验眼科杂志

Background Recently, the morbidity of orbital lymphoma increased gradually, and we have made deeper research in pathology,therapy and pathogenesis of the disease.There were few reports of mice model of orbital lymphoma up to now for its lower morbidity and culture difficulty.Objective This study was to establish a mouse model of orbital diffuse large B cell lymphoma (DLBCL) by injection of systemic DLBCL cell line pfeiffer.Methods Ten SPF BALB/c mice and 5 nod-SCID mice were radiated firstly using 137Cs,with the absorption dose 3.5 Gy in the BALB/c mice and 2.6 Gy in the nod-SCID mice,and then pfeiffer cells were intraobitally injected in 4eyes of BALB/c mice (orbital injection group) and suncutaneously injectied in 4 eyes of BALB/c mice and 4 eyes of nod-SCID mice (subcutaneous injection group) at the concentration of 1.5×l08/ml.The developing status of tumors were examined once per day and the growth curve was drawn.The tumors and nearby lymph nodes were obtained 54 days after injection for the preparation of 4 μm thickness of serial sections.Hemotoxylin-eosin staining was used to examine the histopathology of the tumors, and immunochemistry was employed to detect the expressions of CD20,CD79α and CD45RO proteins.The tumors were typed based on the expressions of CD10, BCL-6 and mum-1 in the specimens,and the expressions of Ki-67 and survivin were assyed to assess the prognosis of the tumor.All the results were compared with 3 diagnosed human orbital DLBCL sections.The use and care of the mice complied with Chinese Administration Rule of Laboratory Animal.Results The tumor formation rates were 100％ in both the orbital injection group and subcutaneous injection group, and the tumors grew much faster in nod-SCID mice than BALB/c mice.Infiltration of tumor cells in lymph nodes were found in the subcutaneous injection group rather than the orbital injection group.The pathological features were accordant among the orbital injection group, subcutaneous injection group and human orbital DLBCL sections.The number of ＜50％ and ≥50％ CD20+ specimens was 3 and 5,CD79αwas 2 and 6,CD45RO was 8 and 0 in the BALB/c mice;while that in the nod-SCID mice was 1 and 3 in CD20,0 and 4 in CD79α,4 and 0 in CD45RO;the number of human orbital DLBCL specimens was 1 and 2 in CD20,1 and 2 in CD79α,2 and 1 in CD45RO,without significant differences among them (all at P=1.00).No significant differences were seen in Ki-67+ number and survivin+ number among the BALB/c mice, nod-SCID mice and human orbital DLBCL specimens (all at P=1.00).The detection of CD10,BCL-6 and mum-1 expressions indicated that the tumors of BALB/c mice,nod-SCID mice and human orbital DLBCL specimens all were the non-germinal center B cell-like types.Conclusions The orbital and subcutaneous DLBCL mouse models are successfully established by injection of pfeiffer cell line.There are the same findings and features in biological behavior, pathology and immunohistochemistry in orbital,subcutaneous models with human orbitl DLBCL.Nod-SCID mice appear to be more suitable for the growth of lymphoma cells.

Establishment of NOD/SCID-mouse model with multiple myeloma using myeloma cells from patients / 西安交通大学学报(医学版)

Objective To explore the feasibility of establishment of NOD/SCID-mouse model with multiple myeloma by using plasma cells from myeloma patients.Methods The femurs and tibias were removed from the New Zealand white rabbits;the muscles,periosteum and cartilage tissues were cleared.Then each bone was cut into two pieces gently along its middle.The NOD/SCID mice weighing 25 - 30 g (4 - 6 weeks)were anesthetized by intraperitoneal injection;rabbit bone was inserted into the right side of the mouse back and engraftment of the bones was allowed to take place after 4 weeks.The 5000 000 purified plasma cells which expressed CD38 +/CD45 - were immunofluorescence labeled and then injected slowly into the implanted rabbit bone through the distal end.The mice were observed weekly;the plasma cells growth in mice was screened by the living-imaging system and the tumor from the mice was determined by biopsy.Results The implanted rabbit bone survived after 4 weeks.The tumor in mice was observed 2 weeks after the purified myeloma cells were injected into the rabbit bone,and it reached 100 mm3 after 8 weeks.Results of the living-imaging system showed that the myeloma cells had uptake in the rabbit bone after 2 weeks of injection and this phenomenon was more pronounced after 8 weeks of injection (2.4×10 4 vs .1.5× 10 5 ,P < 0.05 ).The tumor infiltrated with numerous plasma cells and osteoclasts increased upon the biopsy. Conclusion Rabbit bone marrow implanted into NOD/SCID mice can effectively support local injection of plasma cells of multiple myeloma patients,and the NOD/SCID-mouse model of myeloma has been established.This model can be used to study in vivo experiments related to myeloma and clinical therapeutic approaches for this disease.

The establishment of human esophageal cancer model in Hu-PBL-SCID mice / 肿瘤研究与临床

Objective To establish a mouse model for human esophageal cancer.Methods Human PBL were isolated directly from whole blood by density gradient centrifugation.Fifteen SCID mice were randomly divided into two groups.Group A was control group,and in group B there were 12 mice intraperitoneally injected with 2×107 human PBL and subcutaneously injected with 5×106 ECA109 cells.The rate of tumor transplantation,tumor growth,metastasis and histological features were observed.After 3,4,5,6 weeks of engraftment,the level of IgG in mouse serum and the spleen weight were detected.Results The successful rate of tumor transplantation was 100 ％.Metastasis was not found.After 3,4,5,6 weeks of engraftment,the spleen weight in group B were (55.44±4.45) mg,(88.62±2.24) mg,[(125.98±2.19) mg] (P ＜ 0.05) and (213.71±2.96) mg,which had statistical significance compared with the control group (41.87±2.97) mg.The level of IgG was significantly higher than that in control group (P ＜ 0.01).Conclusion The experimental results demonstrate that human esophageal cancer models have been established in Hu-PBL-SCID mice.

Establishment of the CD19-K562 cell line over-expressing stably CD19 gene and its subcutaneous xenograft model in NOD-SCID mouse / 白血病·淋巴瘤

Objective To construct a MigR1-CD19 recombinant vector which contains CD19 gene, and to establish a CD19-K562 cell line over-expressing stably CD19 gene and its subcutaneous xenograft model in NOD-SCID mouse.Methods The CD19 gene was inserted into the retroviral vector (MigR1) through recombinant DNA technology after transfection into Plat-A packaging cells, and viral supernatant was collected to transduce K562 cell line repeatedly to obtain stable transduction CD19-K562 cell line.Flow cytometry was used to determine the transduction efficiency and RT-PCR was used to confirmed CD19 gene expression.Cell proliferation and apoptosis were detected by cell count and Annexin V/PI, respectively.Then the subcutaneous xenograft subtype of CD19-K562-a cell line was constructed through subcutaneous inoculation and was cultured in vitro and in vivo.Then its subcutaneous xenograft model in NOD-SCID mouse was established.The characteristics of CD19-K562-a cells were detected by RT-PCR, Wright staining and immunohistochemistry.Results MigR1-CD19 recombinant vector was successfully constructed, and the CD19 positive efficiency of K562 cell line was (99.80±0.17) ％ through retrovirus centrifugation transduction.The transduction and passage had no effects on proliferation and apoptosis of CD19-K562 cells.The CD19-K562-a cell line was constructed after CD19-K562 cells were injected subcutaneously and were passaged in vitro and in vivo.The CD19 positive efficiency of the xenograft subtype CD19-K562-a cell line was (99.78± 0.04) ％.CD19-K562-a and CD19-K562 cells were in an undifferentiated state.NOD-SCID subcutaneous xenografts were established through subcutaneous inoculation of CD19-K562-a cells.CD19 in the CD19-K562-a subcutaneous xenografts was positive, while it was negative in its counterparts K562 cells.Conclusion The CD19-K562 cell line over-expressing CD19 gene and its subcutaneous xenograft model in NOD-SCID mouse are successfully established.

Establishment and verification of a KCL22/NOD-SCID mouse transplantation tumor model of chronic myeloid leukemia / 中国实验动物学报

Objective To investigate the potential of chronic myeloid leukemia ( CML) cell line KCL22 in indu-cing leukemia in NOD-SCID mice for setting up a basis for constructing a CML mouse transplantation tumor model. Methods 2 ×107 KCL22 cells in logarithmic growth phase were injected via the tail vein into experimental NOD-SCID mice whereas PBS was injected to the mice of control group.General condition of the mice of both groups was observed.Wright staining was used to observe the changes of blood and bone marrow smears.PCR was conducted to detect the transcription level of BCR-ABL, and histology with HE staining was used to evaluate the tumor cell invasion in the liver and spleen. Results Four weeks after the injection of KCL22 cells, the mice in experimental group showed physical signs of decreased reactivity, depression, swollen hindlimb muscles and petechia on the hindlimb femur.Peripheral white blood cells ( WBC) began to increase after 5 weeks, with a significantly increased quantity compared with the control group (P90 days) (P<0.05).Conclusions A NOD-SCID mouse model of CML transplantation tumor is successfully established with leukemia KCL22 cells.

Imunodeficiência combinada grave: uma revisão da literatura / Severe combined immunodeficiency: review of the literature

A imunodeficiência combinada grave (SCID) é uma condição clínica caracterizada por marcante comprometimento da resposta imune envolvendo linfócitos T e/ou B e/ou células NK, que conduza aumento da susceptibilidade a infecções e alta taxa de mortalidade em crianças acometidas. Dificuldades na interpretação dos sintomas clínicos e na identificação de mutações genéticas, devido à ampla variedade fenotípica e genotípica da doença, representam obstáculos para o diagnóstico. Por outro lado, o tratamento é realizado de forma independente da identificação de mutação genética. O objetivo do presente trabalho foi revisar aspectos fisiopatológicos, métodos diagnósticos e tratamentos utilizados em pacientes com SCID. A revisão foi realizada com base em levantamento bibliográfico de banco de dados indexados disponíveis na Internet incluindo LILACS, MEDLINE, PubMed, SciELO Brasil, periódicos CAPES e Cochrane, e foi conduzida com os seguintes critérios de inclusão: artigos científicos publicados nos idiomas português e inglês, dentro do período de 1963 a 2014 e que possuíam as palavras-chave “Imunodeficiência Combinada Grave”, “SCID”, “Leucopenia”, “Diagnóstico”, “Tratamento” e “Transplante de medula óssea”. O levantamento bibliográfico revelou dificuldades no diagnóstico clínico, laboratorial e genético-molecular, e ressaltou a importância do diagnóstico precoce conduzindo ao tratamento adequado. O diagnóstico precoce da SCID tem papel crucial na melhora da qualidade de vida e na sobrevida dos pacientes, além de favorecer intervenções terapêuticas que previnem o surgimento de infecções e complicações clínicas subsequentes...

Severe combined immunodeficiency (SCID) is a clinical condition characterized by marked impairment of immune responses involving T and/or B lymphocytes and/or NK cells, leading to increased susceptibility to infections and a high mortality rate among affected infants. Difficulties in the interpretation of clinical symptoms and in the detection of genetic mutations make diagnosis a challenge because of the phenotypic and genotypic heterogeneity associated with the disease. Treatment is performed regardless of the detection of a genetic mutation. The objective of the present study was to review pathophysiological aspects, diagnostic methods, and therapies used in patients with SCID. The review included papers available in online databases, including LILACS, MEDLINE, PubMed, SciELO Brazil, Periódicos CAPES, and Cochrane. Papers were searched considering the following inclusion criteria: research articles published in Portuguese or English, between years 1963 and 2014, containing the keywords "Severe Combined Immunodeficiency," "SCID," "Leukopenia," "Diagnosis," "Treatment," and "Bone Marrow Transplantation." The review revealed difficulties in clinical, biochemical, and molecular genetic diagnosis, and emphasized the importance of early diagnosis leading to appropriate treatment. Early diagnosis of SCID is crucial to improve the quality of life and survival of patients, and it allows the use of therapeutic interventions that prevent the onset of infections and subsequent clinical complications...

Induced transformation of rat oval cells line WB-F344 and tumor formation in NOD/SCID mice / 临床与实验病理学杂志

Purpose To study the malignant transformation after treating rat oval cell line ( WB-F344 ) with chemical carcinogen N-methyl-N′-nitro-N-nitrosoguanidine ( MNNG) . Methods WB-F344 cells were cultured with MNNG for severe times. The biological characteristics of induced cells were detected through the following methods:to check proliferation activity by flow cytometry analysis, to examine malignant transformation degree of induced cells by soft agar assay and tumor formation in NOD/SCID mice, and to investi-gate the transcriptional and protein levels of hepatocellular carcinoma marker GGT, GST-P by real time-PCR. Results Oval cells in-duced by MNNG showed changes in biological characteristics and malignant molecular markers. Conclusion Hepatic oval cells model is successfully established, which can be confirmed by tumor formation in NOD/SCID mice.

Experimental study of the appropriate time to block adrenal androgens in endocrine therapy of prostate cancer / 中华泌尿外科杂志

Objective To explore the appropriate time to block adrenal androgens in endocrine therapy of prostate cancer.Methods An human androgen-dependent prostate carcinoma xenograft model in SCID mice was established with LNCaP cells.Firstly,the serum PSA and tumor volume of 2 groups (castrated and not castrated) were measured on the 1,4,7,10,14,17,21,28,35,42,49 and 56 day to determine the recurrent time of prostate cancer after castration.Secondly,3 different groups of castration and adrenalectomy at the same time,adrenalectomy in recurrence after castration and sham adrenalectomy in recurrent after castration,were set to measure the serum PSA and tumor volume on the 1,4,7,10,14,17,21,28,35,42,49 and 56 day.The tumor tissues of 5 groups were harvested to measure testosterone concentration,and tumor progress in these groups was compared.Results The recurrence time was 14 days in castrated group,21 days in group with castration and adrenalectomy at the same time and 35 days in group with adrenalectomy in recurrence after castration.The testosterone concentration in tumor tissues was (2.69± 0.21) pmol/g in the group with castration and adrenalectomy at the same time,and (2.16±0.13) pmol/g in the group with adrenalectomy in recurrence after castration,and the difference was significant (P＜0.05).Conclusion Compared with the therapy of castration and adrenalectomy at the same time,the therapy of adrenalectomy in recurrence after castration may have slower progress course in prostate cancer.