Gillian E. Wu

Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities.

PU.1 is a member of the ets family of transcription factors and is expressed exclusively in cells of the hematopoietic lineage. Mice homozygous for a disruption in the PU.1 DNA binding domain are born alive but die of severe septicemia within 48 h. The analysis of these neonates revealed a lack of mature macrophages, neutrophils, B cells and T cells, although erythrocytes and megakaryocytes were present. The absence of lymphoid commitment and development in null mice was not absolute, since mice maintained on antibiotics began to develop normal appearing T cells 3–5 days after birth. In contrast, mature B cells remained undetectable in these older mice. Within the myeloid lineage, despite a lack of macrophages in the older antibiotic‐treated animals, a few cells with the characteristics of neutrophils began to appear by day 3. While the PU.1 protein appears not to be essential for myeloid and lymphoid lineage commitment, it is absolutely required for the normal differentiation of B cells and macrophages.

Targeted expression of a human pituitary tumor–derived isoform of FGF receptor-4 recapitulates pituitary tumorigenesis

3 3 0 3 jci.org Volume 125 Number 8 August 2015 Retraction Targeted expression of a human pituitary tumor–derived isoform of FGF receptor-4 recapitulates pituitary tumorigenesis Shereen Ezzat, Lei Zheng, Xian-Feng Zhu, Gillian E. Wu, and Sylvia L. Asa Original citation: J Clin Invest. 2002;109(1):69–78. doi:10.1172/JCI14036. Citation for this retraction: J Clin Invest. 2015;125(8):3303. doi:10.1172/JCI83399. An investigation by the University Health Network recently found that portions of the RT-PCR gels shown in Figure 1, B (PGK-1 panel) and C (FGFR1 panel), are duplicated in this publication and in a subsequent publication (1). The samples were labeled differently in the panels, and the marker was shifted in Figure 1B. The corresponding author has indicated that other data from the initial screen of these samples support the conclusions made in the paper; however, the original data for the RT-PCR gels shown in Figure 1 are no longer available. The JCI’s policies prohibit data manipulation and data duplication. Therefore, the JCI is retracting this article. No issues have been raised in regard to any of the other data in this manuscript. Gillian E. Wu has agreed with the journal’s decision to retract the paper. Sylvia Asa, Shereen Ezzat, and Lei Zheng dissent from the retraction. Coauthor Xian-Feng Zhu could not be reached. 1. Ezzat S, Yu S, Asa SL. Ikaros isoforms in human pituitary tumors: distinct localization, histone acetylation, and activation of the 5′ fibroblast growth factor receptor-4 promoter. Am J Pathol. 2003;163(3):1177–1184.

Caspase-3 regulates cell cycle in B cells: a consequence of substrate specificity.

Caspases are important for apoptosis but are also involved in mammalian cell survival and cell division. Here we report that caspase-3 is a negative regulator of B cell cycling. Mice deficient in caspase-3 (Casp3−/− mice) have increased numbers of splenic B cells that show normal apoptosis but enhanced proliferation in vivo and hyperproliferation after mitogenic stimulation in vitro. Cdkn1a encodes p21 (also called Waf1 or Cip1), a cyclin-dependent kinase (CDK) inhibitor. Although expression of p21 was increased, CDK activities and proliferating cell nuclear antigen (PCNA) were increased in Casp3−/− B cells. Using Casp3−/−Cdkn1a−/− mice, we show that the hyperproliferation of Casp3−/− B cells is abolished when Cdkn1a is also deleted. Our genetic and biochemical data demonstrate that caspase-3 is essential in the regulation of B cell homeostasis.

Enumeration and characterization of DJH structures in mouse fetal liver.

The primary immunoglobulin (Ig) repertoire in the mouse develops during fetal life in the liver. The first Ig gene rearrangement‐‐the joining of a DH to a JH gene segment‐‐contributes largely to the diversity found in CDR3, as well as potentially encoding the D mu protein which is believed to function in the development of a B cell. In this report, the number of DJH joins in two mouse strains, C57BL/6 and BALB/c, were enumerated from days 12 to 16 of fetal development. It was found that the number of DJH structures increased from less than 300 per liver on day 12 to greater than 700,000 (C57BL/6) and 300,000 (BALB/c) on day 16. Each JH gene segment was used approximately equally on each day examined. When the DJH structures were examined by cloning and sequencing it was found that the DJH reading frame (RF) usage (with respect to JH) was not random‐‐RF1 was used 70% of the time. Moreover, a single D gene segment, DFL16.1, was used in greater than 50% of all joins reinforcing the notion that the fetal repertoire is restricted in its antigen binding potential.

Secretion of a λ2 immunoglobulin chain is prevented by a single amino acid substitution in its variable region

Abstract We have studied two derivatives of the IgA ( λ 2 ) secreting myeloma cell line MOPC315: MOPC315.26, which produces and secretes a λ 2 light chain, and MOPC315.37, which produces but does not secrete the λ 2 chain. It has been reported that the only alteration in the MOPC315-37 λ 2 chain is located in the variable region (Mosmann and Williamson, (1980) Cell 20, 283–292). In order to determine the nature of this alteration, we cloned the fragment of the chromosome containing the rearranged λ 2 gene from both the nonsecreting variant MOPC315-37 and the normal λ 2 -secreting parent MOPC315-26 and determined their nucleotide sequence. We found that the nucleotide sequences coding for the leader peptide and for the constant region of the λ 2 chain were identical in the secretor and nonsecretor. The sequences of the variable region differed at a single base pair corresponding to the first nucleotide in the codon for amino acid number 15. MOPC315-26 has a G in this position creating the codon GGT which codes for glycine, and MOPC315-37 has a C in this position creating the codon CGT which codes for arginine. Thus, we have demonstrated that a single amino acid substitution of a neutral amino acid, glycine, for a positively charged amino acid, arginine, results in the failure of a protein to be secreted.

Role of Positive Selection Pressure on the Evolution of H5N1 Hemagglutinin

The surface glycoprotein hemagglutinin (HA) helps the influenza A virus to evade the host immune system by antigenic variation and is a major driving force for viral evolution. In this study, the selection pressure on HA of H5N1 influenza A virus was analyzed using bioinformatics algorithms. Most of the identified positive selection (PS) sites were found to be within or adjacent to epitope sites. Some of the identified PS sites are consistent with previous experimental studies, providing further support to the biological significance of our findings. The highest frequency of PS sites was observed in recent strains isolated during 2005–2007. Phylogenetic analysis was also conducted on HA sequences from various hosts. Viral drift is almost similar in both avian and human species with a progressive trend over the years. Our study reports new mutations in functional regions of HA that might provide markers for vaccine design or can be used to predict isolates of pandemic potential.

Somatic mutations in the mitochondria of rheumatoid arthritis synoviocytes

Somatic mutations have a role in the pathogenesis of a number of diseases, particularly cancers. Here we present data supporting a role of mitochondrial somatic mutations in an autoimmune disease, rheumatoid arthritis (RA). RA is a complex, multifactorial disease with a number of predisposition traits, including major histocompatibility complex (MHC) type and early bacterial infection in the joint. Somatic mutations in mitochondrial peptides displayed by MHCs may be recognized as non-self, furthering the destructive immune infiltration of the RA joint. Because many bacterial proteins have mitochondrial homologues, the immune system may be primed against these altered peptides if they mimic bacterial homologues. In addition, somatic mutations may be influencing cellular function, aiding in the acquirement of transformed properties of RA synoviocytes. To test the hypothesis that mutations in mitochondrial DNA (mtDNA) are associated with RA, we focused on the MT-ND1 gene for mitochondrially encoded NADH dehydrogenase 1 (subunit one of complex I – NADH dehydrogenase) of synoviocyte mitochondria from RA patients, using tissue from osteoarthritis (OA) patients for controls. We identified the mutational burden and amino acid changes in potential epitope regions in the two patient groups. RA synoviocyte mtDNA had about twice the number of mutations as the OA group. Furthermore, some of these changes had resulted in potential non-self MHC peptide epitopes. These results provide evidence for a new role for somatic mutations in mtDNA in RA and predict a role in other diseases.

Development of B lymphocytes from lymphoid committed and uncommitted progenitors

Rapid experimental advances in B-cell biology are yielding an increasingly clear image of B-cell progenitors as they develop from multipotential stem cells to immunoglobulin (Ig)-secreting plasmacytes. Intermediate cells have been identified, growth and difTerentiation factors have been purified, stromal elements have been cloned, regulatory mechanisms of gene expression have been described. Not surprisingly, a wide consensus has been reached regarding many key elements of the developmental process. Despite this progress, other critical issues remain either obscure or controversial. These include fundamental questions such as the origins of lymphopoiesis, the genetic basis for lymphoid commitment, and the role of Ig in lymphoid progression, as well as detailed issues such as the role of particular growth factors or adhesion molecules. In this review, we will summarize the findings we have accumulated using in vitro and in vivo approaches to B-ceil development, and note the current issues which arise from this work. Particular emphasis is placed on the emergence of B-cell progenitors during fetal development.

A linkage map of the mouse immunoglobulin lambda light chain locus

The mouse immunoglobulin lambda light chain locus has been linked using field inversion gel electrophoresis. The lambda light chain locus classically contains two V and four J-C gene segments in inbred mouse strains, and was physically mapped in the BALB/c cell line Wehi-3 which contains unrearranged lambda light chain gene segments. The locus is relatively small and spans 300 kb, as defined by a variety of single and double digests using methylation-sensitive restriction enzymes. The order of the lambda gene segments is V2-J2C2J4C4-V1-J3C3J1C1, as was originally proposed. No evidence for nonmethylated CpG rich areas (HTF islands) within the region was found. Fine mapping using the λ1, λ3 rearranged cell line J558 mapped the gap between the V and J-C gene segments in the lambda 1 gene cluster (VI-J3C3JIC1) to approximately 70 kb. The similar distance (60–100 kb) found in the lambda 2 gene cluster (V2-J2C2J4C4) is further evidence that duplication of an ancestral locus occurred.

VH gene replacement occurs in the spleen and bone marrow of non‐autoimmune quasi‐monoclonal mice

Genes encoding the heavy chain portion of immunoglobulin molecules arise from the combi natorial association of V, D and J gene segments, which occurs during discrete stages of B lineage development in the bone marrow. Recently, VH replacement, a form of receptor editing, has been described, in which the variable region of an existing VDJH rearrangement is replaced by another VH gene segment in a recombination event believed to involve an embedded heptamer within the coding region of the VH . Studies of transgenic mice with “knocked‐in” VDJH genes encoding anti‐DNA specificity have demonstrated that receptor editing of the heavy chain is one mechanism by which autoreactive B cell receptors can be modified. Another mouse, the “quasi‐monoclonal”, which encodes a “knocked‐in” VDJH for the hapten NP also contains B lineage cells that undergo VH replacement. This suggests that VH replacement may play a role in the normal diversification of the antibody repertoire. Using a ligation‐mediated PCR assay, we have identified VQM double‐stranded DNA breaks indica tive of VH replacement intermediates from bone marrow and splenic B lineage cells of quasi‐monoclonal mice in the absence of immunization. VQM to J558 recombination deletion products consistent with VH replacement were also detected in both the bone marrow and spleen of non‐immunized quasi‐monoclonal mice. Moreover, RAG‐1 transcripts were detected in the spleen. These data suggest that VH replacement can be part of the mechanism(s) used by B lineage cells to generate diversity throughout B lineage development, including later stages occurring in secondary lymphoid tissues.