Research

Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells.

Stem Cells Transl Med. 2015 Aug 18;

Authors: Wang D, Quan Y, Yan Q, Morales JE, Wetsel RA

Abstract
: Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of β2-microglobulin on both gene alleles in hESCs. Because β2-microglobulin serves as the HLA-I light chain, disruption of the β2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8(+) T-cell-mediated destruction. Although interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression, promoting CD8(+) T cell-mediated killing of control hESCs and their derivatives, CD8(+) T-cell-mediated cytotoxicity was barely observed with β2-microglobulin-null hESCs and their derivatives treated with IFN-γ. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, β2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that β2-microglobulin-null hESCs significantly reduce immunogenicity to CD8(+) T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future.
SIGNIFICANCE: This study reports the generation of a novel β2-microglobulin (B2M)(-/-) human embryonic stem cell (hESC) line. Differentiated mature cells from this line do not express cell surface human leukocyte antigen molecules even after interferon-γ stimulation and are resistant to alloreactive CD8(+) T cells. Moreover, this B2M(-/-) hESC line contains no off-target integration or cleavage events, is devoid of stable B2M mRNA, exhibits a normal karyotype, and retains its self-renewal capacity, genomic stability, and pluripotency. Although B2M(-/-) hESC-derived cells are more susceptible to natural killer (NK) cells, murine transplantation studies have indicated that they are, overall, much less immunogenic than normal hESCs. Thus, these data show for the first time that, in vivo, the advantages provided by B2M(-/-) hESC-derived cells in avoiding CD8(+) T-cell killing appear significantly greater than any disadvantage caused by increased susceptibility to NK cells.

PMID: 26285657 [PubMed – as supplied by publisher]

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Facilitated Subcutaneous Immunoglobulin (fSCIg) Therapy – Practical Considerations.

Clin Exp Immunol. 2015 Aug 19;

Authors: Ponsford M, Carne E, Kingdon C, Joyce C, Price C, Williams C, El-Shanawany T, Williams P, Jolles S

Abstract
There is an increasing range of therapeutic options for primary antibody deficient patients who require replacement immunoglobulin. These include intravenous immunoglobulin (IVIg), subcutaneous immunoglobulin (SCIg), rapid push SCIg and most recently recombinant human hyaluronidase facilitated SCIg (fSCIg). Advantages of fSCIg include fewer needle punctures, longer infusion intervals, and an improved adverse effect profile relative to IVIg. Limited real life experience exists concerning the practical aspects of switching or starting patients on fSCIg. We describe the first 14 patients who have been treated with fSCIg at the Immunodeficiency Centre for Wales (ICW), representing over 6 patient-years of experience. The regime was well tolerated with high levels of satisfaction and no increase in training requirement – including for a treatment-naive patient. Two patients discontinued fSCIg due to pain and swelling at the infusion site, and one paused therapy following post-infusion migraines. Ultrasound imaging of paired conventional and facilitated SCIg demonstrated clear differences in subcutaneous space distribution associated with a ten-fold increase in rate and volume delivery with fSCIg. Patient profiles for those choosing fSCIg fell into two main categories: those experiencing clinical problems with their current treatment and those seeking greater convenience and flexibility. When introducing fSCIg, consideration of the type and programming of infusion pump, needle gauge and length, infusion site, up-dosing schedule, home training and patient information are important as these may differ from conventional SCIg. This paper provides guidance on practical aspects of the administration, training and outcomes to help inform decision making for this new treatment modality. This article is protected by copyright. All rights reserved.

PMID: 26288095 [PubMed – as supplied by publisher]

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Primary central nervous system lymphoma in Miyazaki, southwestern Japan, a human T-lymphotropic virus Type-1 (HTLV-1)-endemic area: clinicopathological review of 31 cases.

J Clin Exp Hematop. 2014;54(3):179-85

Authors: Maekawa K, Moriguchi-Goto S, Kamiunten A, Kubuki Y, Shimoda K, Takeshima H, Asada Y, Marutsuka K

Abstract
Primary central nervous system lymphoma (PCNSL) is a rare and aggressive brain tumor. The aim of this study was to clarify the prevalence of T-cell-type PCNSL (T-PCNSL) in a human T-lymphotropic virus type-1 (HTLV-1)-endemic area of Southwestern Japan. We retrospectively investigated 31 PCNSL cases diagnosed between 1996 and 2013 at the University of Miyazaki Hospital. These cases accounted for 4.4% of all nodal or extranodal malignant lymphomas. Histologically, most of these cases were diagnosed as diffuse large B-cell lymphoma, while only two cases were considered to be low-grade and high-grade B-cell lymphoma (not otherwise specified). No T-PCNSL was found in this series. In addition, Epstein-Barr virus-encoded RNAs were not detected by in situ hybridization in any of the cases. Overall, no T-PCNSL cases were found in 18 years in a region with a high frequency of HTLV-1 seropositivity, namely, Southwestern Japan. This suggests that PCNSL and lymphomas of other anatomical sites are biologically distinct.

PMID: 25501108 [PubMed – indexed for MEDLINE]

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Characteristics relating to the interiorization of acquired immunodeficiency syndrome in Brazil: a cross-sectional study.

Infect Dis Poverty. 2015;4(1):31

Authors: Vieira GD, Dos Reis AR, Augusto FO, Martins KR, Kern PR, de Souza TF, Basano SA, Camargo LM, de Sousa CM

Abstract
BACKGROUND: In recent years there has been changes in the social and geographic profile of acquired immunodeficiency syndrome (AIDS). The aim of this study was to evaluate the internalization of AIDS in the state of Rondônia, Brazil.
FINDINGS: In Rondônia, 1473 AIDS cases were reported, with an average annual incidence of 15.8/100,000 persons (42.7 % women). The most common mode of viral transmission was sexual (96.5 %), and the majority of the individuals had not completed their primary education (64.8 %). There was heterogeneity in relation to case distribution, involving almost all of the municipalities in the state. The average annual mortality rate was 2.5/100,000 persons.
CONCLUSION: Rondônia has a higher incidence of AIDS than the national average and the northern region. Efforts to provide access to treatment and follow-up of these individuals should be implemented, prioritizing areas where the incidence is higher and decentralizing the treatment of patients with AIDS in the state.

PMID: 26284512 [PubMed – as supplied by publisher]

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Primary immunodeficiency update: Part II. Syndromes associated with mucocutaneous candidiasis and noninfectious cutaneous manifestations.

J Am Acad Dermatol. 2015 Sep;73(3):367-81

Authors: Pichard DC, Freeman AF, Cowen EW

Abstract
Several primary immunodeficiencies (PIDs) have recently been described that confer an elevated risk of fungal infections and noninfectious cutaneous manifestations. In addition, immunologic advances have provided new insights into our understanding of the pathophysiology of fungal infections in established PIDs. We reviewed PIDs that present with an eczematous dermatitis in part I. In part II of this continuing medical education article we discuss updates on PIDs associated with fungal infections, their biologic basis in PIDs, and noninfectious cutaneous manifestations.

PMID: 26282795 [PubMed – in process]

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The Antipancreatic Cancer Activity of OSI-027, a Potent and Selective Inhibitor of mTORC1 and mTORC2.

DNA Cell Biol. 2015 Aug 18;

Authors: Chen B, Xu M, Zhang H, Xu MZ, Wang XJ, Tang QH, Tang JY

Abstract
In the present study, we investigated the potential activity of OSI-027, a potent and selective mammalian target of rapamycin (mTOR) complex 1/2 (mTORC1/2) dual inhibitor, against pancreatic cancer cells both in vitro and in vivo. We demonstrated that OSI-027 inhibited survival and growth of both primary and transformed (PANC-1 and MIA PaCa-2 lines) human pancreatic cancer cells. Meanwhile, OSI-027 induced caspase-dependent apoptotic death of the pancreatic cancer cells. On the other hand, caspase inhibitors alleviated cytotoxicity by OSI-027. At the molecular level, OSI-027 treatment blocked mTORC1 and mTORC2 activation simultaneously, without affecting ERK-mitogen-activated protein kinase activation. Importantly, OSI-027 activated cytoprotective autophagy in the above cancer cells. Whereas pharmacological blockage of autophagy or siRNA knockdown of Beclin-1 significantly enhanced the OSI-027-induced activity against pancreatic cancer cells. Specifically, a relatively low dose of OSI-027 sensitized gemcitabine-induced pancreatic cancer cell death in vitro. Further, administration of OSI-027 or together with gemcitabine dramatically inhibited PANC-1 xenograft growth in severe combined immunodeficiency mice, leading to significant mice survival improvement. In summary, the preclinical results of this study suggest that targeting mTORC1/2 synchronously by OSI-027 could be further investigated as a valuable treatment for pancreatic cancer.

PMID: 26284306 [PubMed – as supplied by publisher]

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Spontaneous bacterial and fungal infections in genetically engineered mice: Is Escherichia coli an emerging pathogen in laboratory mouse?

Berl Munch Tierarztl Wochenschr. 2015 Jul-Aug;128(7-8):278-84

Authors: Benga L, Benten WP, Engelhardt E, Gougoula C, Sager M

Abstract
The impact of particular microbes on genetically engineered mice depends on the genotype and the environment. Infections resulting in clinical disease have an obvious impact on animal welfare and experimentation. In this study, we investigated the bacterial and fungal aetiology of spontaneous clinical disease of infectious origin among the genetically engineered mice from our institution in relation to their genotype. A total of 63 mice belonging to 33 different mice strains, from severe immunodeficient to wild-type, were found to display infections as the primary cause leading to their euthanasia. The necropsies revealed abscesses localized subcutaneously as well as in the kidney, preputial glands, seminal vesicles, in the uterus, umbilicus or in the lung. In addition, pneumonia, endometritis and septicaemia cases were recorded. Escherichia coli was involved in 21 of 44 (47.72%) of the lesions of bacterial origin, whereas [Pasteurella] pneumotropica was isolated from 19 of 44 (43.18%) cases. The infections with the two agents mentioned above included three cases of mixed infection with both pathogens. Staphylococcus aureus was considered responsible for five of 44 (11.36%) cases whereas Enterobacter cloacae was found to cause lesions in two of 44 (4.54%) mice. Overall, 16 of the 44 (36.36%) cases of bacterial aetiology affected genetically engineered mice without any explicit immunodeficiency or wild-type strains. The remaining 19 cases of interstitial pneumonia were caused by Pneumocystis murina. In conclusion, the susceptibility of genetically modified mice to opportunistic infections has to be regarded with precaution, regardless of the type of genetic modification performed. Beside the classical opportunists, such as [Pasteurella] pneumotropica and Staphylococcus aureus, Escherichia coli should as well be closely monitored to evaluate whether it represents an emerging pathogen in the laboratory mouse.

PMID: 26281439 [PubMed – in process]

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Primary immunodeficiency update: Part I. Syndromes associated with eczematous dermatitis.

J Am Acad Dermatol. 2015 Sep;73(3):355-64

Authors: Pichard DC, Freeman AF, Cowen EW

Abstract
In the past decade, the availability of powerful molecular techniques has accelerated the pace of discovery of several new primary immunodeficiencies (PIDs) and revealed the biologic basis of other established PIDs. These genetic advances, in turn, have facilitated more precise phenotyping of associated skin and systemic manifestations and provide a unique opportunity to better understand the complex human immunologic response. These continuing medical education articles will provide an update of recent advances in PIDs that may be encountered by dermatologists through their association with eczematous dermatitis, infectious, and non-infectious cutaneous manifestations. Part I will discuss new primary immunodeficiencies that have an eczematous dermatitis. Part II will focus on primary immunodeficiencies that greatly increase susceptibility to fungal infection and the noninfectious presentations of PIDs.

PMID: 26282794 [PubMed – in process]

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Genetic errors of the human caspase recruitment domain-B-cell lymphoma 10-mucosa-associated lymphoid tissue lymphoma-translocation gene 1 (CBM) complex: Molecular, immunologic, and clinical heterogeneity.

J Allergy Clin Immunol. 2015 Aug 12;

Authors: Pérez de Diego R, Sánchez-Ramón S, López-Collazo E, Martínez-Barricarte R, Cubillos-Zapata C, Ferreira Cerdán A, Casanova JL, Puel A

Abstract
Three members of the caspase recruitment domain (CARD) family of adaptors (CARD9, CARD10, and CARD11) are known to form heterotrimers with B-cell lymphoma 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma-translocation gene 1 (MALT1). These 3 CARD-BCL10-MALT1 (CBM) complexes activate nuclear factor κB in both the innate and adaptive arms of immunity. Human inherited defects of the 3 components of the CBM complex, including the 2 adaptors CARD9 and CARD11 and the 2 core components BCL10 and MALT1, have recently been reported. Biallelic loss-of-function mutant alleles underlie several different immunologic and clinical phenotypes, which can be assigned to 2 distinct categories. Isolated invasive fungal infections of unclear cellular basis are associated with CARD9 deficiency, whereas a broad range of clinical manifestations, including those characteristic of T- and B-lymphocyte defects, are associated with CARD11, MALT1, and BCL10 deficiencies. Interestingly, human subjects with these mutations have some features in common with the corresponding knockout mice, but other features are different between human subjects and mice. Moreover, germline and somatic gain-of-function mutations of MALT1, BCL10, and CARD11 have also been found in patients with other lymphoproliferative disorders. This broad range of germline and somatic CBM lesions, including loss-of-function and gain-of-function mutations, highlights the contribution of each of the components of the CBM complex to human immunity.

PMID: 26277595 [PubMed – as supplied by publisher]

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IRF4 Is a Critical Gene in Retinoic Acid-Mediated Plasma Cell Formation and Is Deregulated in Common Variable Immunodeficiency-Derived B Cells.

J Immunol. 2015 Aug 14;

Authors: Indrevær RL, Moskaug JØ, Paur I, Bøhn SK, Jørgensen SF, Blomhoff R, Aukrust P, Fevang B, Blomhoff HK

Abstract
In the present study, we aimed at identifying the mechanisms whereby the vitamin A metabolite all-trans retinoic acid (RA) promotes the formation of plasma cells upon stimulation of B cells via the innate immunity receptors TLR9 and RP105. Most often, differentiation of B cells involves the sequential events of class switch recombination and somatic hypermutations characteristic of germinal center reactions, followed by plasma cell formation. By studying the regulatory networks known to drive these reactions, we revealed that RA enhances the expression of the plasma cell-generating transcription factors IFN regulatory factor (IRF)4 and Blimp1, and paradoxically also activation-induced deaminase (AID) involved in somatic hypermutations/class switch recombination, in primary human B cells. IRF4 was identified as a particularly important protein involved in the RA-mediated production of IgG in TLR9/RP105-stimulated B cells. Based on kinetic studies, we present a model suggesting that the initial induction of IRF4 by RA favors AID expression. According to this model, the higher level of IRF4 that eventually arises results in sustained elevated levels of Blimp1. Regarded as a master regulator of plasma cell development, Blimp1 will in turn suppress AID expression and drive the formation of IgG-secreting plasma cells. Notably, we demonstrated IRF4 to be deregulated in B cells from common variable immunodeficiency patients, contributing to the observed aberrant expression of AID in these patients. Taken together, the present study both provides new insight into the mechanisms whereby RA induces differentiation of B cells and identifies IRF4 as a key to understand the defective functions of B cells in common variable immunodeficiency patients.

PMID: 26276871 [PubMed – as supplied by publisher]

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