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The article presents a discussion about the loss of the BH3-only protein Bid. The BH3-only members of the Bcl-2 protein family are considered as important initiators of developmentally programmed cell death and stress-induced apoptosis, by acting as molecular sensors for intracellular stress signals. The BH3-only protein Bid is capable of activating through proteolytic cleavage by caspases or other proteases.

A letter to the editor is presented in response to the article "Bcl-2 transgene expression fails to prevent fatal hepatocyte apoptosis induced by endogenous TNFα in mice lacking RelA" that was published online in February 17, 2006.

Objective: To estimate cost savings after implementation of customized electronic duplicate order alerts., Design: Alerts were implemented for microbiology tests at the largest public hospital in Victoria, Australia. These alerts were designed to pop up at the point of test ordering to inform the clinician that the test had previously been ordered and to suggest appropriate reordering time frames and indications., Results: In a 6-month audit of urine culture (our most commonly ordered test) after alert implementation, 2,904 duplicate requesters proceeded with the request and 2,549 tests were cancelled, for a 47% reduction in test ordering. For fecal polymerase chain reaction (PCR), our second most common test, there was a 54% reduction in test ordering. For our most commonly ordered expensive test, hepatitis C PCR, there was a 42% reduction in test ordering: 25 tests were cancelled.Cancelled tests resulted in estimated savings of AU$52,382 (US$33,960) for urine culture, AU$34,914 (US$22,442) for fecal PCR, AU$4,506 (US$2,896) for hepatitis C PCR. For cancelled hepatitis B PCR and Epstein-Barr virus (EBV) and cytomegalovirus (CMV) serology, the cost savings was AU$8,472 (US$5445). The estimated financial cost saving in direct hospital costs for these 6 assays was AU$100,274 (US$67,925) over the 6-month period. Environmental waste cost saving by weight was estimated to be 280 kg. Greenhouse gas footprint, measured in carbon dioxide equivalent emissions for cancelled EBV and CMV serology tests, resulted in a saving of at least 17,711 g, equivalent to driving 115 km in a standard car., Conclusion: Customized alerts issued at the time of test ordering can have enormous impacts on reducing cost, waste, and unnecessary testing.

Non-optimal vaginal microbiota, as observed in bacterial vaginosis (BV), is typically characterized by a depletion of beneficial lactobacilli and an abundance of numerous anaerobes. These non-optimal conditions are associated with subclinical cervicovaginal inflammation and an increased risk of HIV infection compared to women colonized with optimal vaginal microbiota dominated by lactobacilli. Lactic acid (LA) is a major organic acid metabolite produced by vaginal lactobacilli that elicits anti-inflammatory effects from cervicovaginal epithelial cells and is dramatically depleted during BV. However, it is unclear if LA retains its anti-inflammatory activity in the presence of vaginal microbiota metabolites comprising short chain fatty acids (SCFAs) and succinic acid, which are also produced by an optimal vaginal microbiota. Furthermore, the immunomodulatory effect of SCFAs and succinic acid on cervicovaginal epithelial cells at higher concentrations present during BV is unknown. Here we report that in the presence of physiologically relevant concentrations of SCFAs and succinic acid at pH 3.9 (as found in women with lactobacillus-dominated microbiota) LA induced an anti-inflammatory state in cervicovaginal epithelial cells and inhibited inflammation elicited by the toll-like receptor (TLR) agonists polyinosinic:polycytidylic acid and Pam3CSK4. When cervicovaginal epithelial cells were treated with a vaginal microbiota metabolite mixture representative of BV, containing a lower concentration of LA but higher concentrations of SCFA/succinic acid at pH 7, no anti-inflammatory was observed. Rather, the vaginal microbiota metabolite mixture representative of BV dysregulated the immune response of cervicovaginal epithelial cells during prolonged and sustained treatments. This was evidenced by increased basal and TLR-induced production of pro-inflammatory cytokines including tumor necrosis factor-α, but decreased basal production of chemokines including RANTES and IP-10. Further characterization of individual components of the BV vaginal microbiota mixture suggested that acetic acid is an important vaginal microbiota metabolite capable of eliciting diverse immunomodulatory effects on a range of cervicovaginal epithelial cell targets. These findings indicate that elevated levels of SCFAs are a potential source of cervicovaginal inflammation in women experiencing BV, and support the unique anti-inflammatory properties of LA on cervicovaginal epithelial cells as well as a role for LA or LA-producing lactobacilli to reverse genital inflammation associated with increased HIV risk., (Copyright © 2020 Delgado-Diaz, Tyssen, Hayward, Gugasyan, Hearps and Tachedjian.)

Polymorphisms in NFKB1 that diminish its expression have been linked to human inflammatory diseases and increased risk for epithelial cancers. The underlying mechanisms are unknown, and the link is perplexing given that NF-κB signaling reportedly typically exerts pro-tumorigenic activity. Here we have shown that NF-κB1 deficiency, even loss of a single allele, resulted in spontaneous invasive gastric cancer (GC) in mice that mirrored the histopathological progression of human intestinal-type gastric adenocarcinoma. Bone marrow chimeras revealed that NF-κB1 exerted tumor suppressive functions in both epithelial and hematopoietic cells. RNA-seq analysis showed that NF-κB1 deficiency resulted in aberrant JAK-STAT signaling, which dysregulated expression of effectors of inflammation, antigen presentation, and immune checkpoints. Concomitant loss of STAT1 prevented these immune abnormalities and GC development. These findings provide mechanistic insight into how polymorphisms that attenuate NFKB1 expression predispose humans to epithelial cancers, highlighting the pro-tumorigenic activity of STAT1 and identifying targetable vulnerabilities in GC., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The properties of CD4(+) regulatory T cell (Treg) subsets are dictated by distinct patterns of gene expression determined by FOXP3 and different combinations of various transcription factors. Here we show the NF-κB transcription factor RelA is constitutively active in naïve and effector Tregs. The conditional inactivation of Rela in murine FOXP3(+) cells induces a rapid onset, multi-focal autoimmune disease that depends on RelA being expressed in conventional T cells. In addition to promoting Treg lineage stability, RelA determines the size of the effector Treg population, a function influenced by the presence or absence of RelA in conventional T cells. These findings showing that RelA controls Treg stability and promotes the competitive fitness of effector Tregs highlight the importance of RelA activity in peripheral Treg induced tolerance., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Resistance to chemotherapy is a major problem in cancer treatment, and it is frequently associated with failure of tumor cells to undergo apoptosis. Birinapant, a clinical SMAC mimetic, had been designed to mimic the interaction between inhibitor of apoptosis proteins (IAPs) and SMAC/Diablo, thereby relieving IAP-mediated caspase inhibition and promoting apoptosis of cancer cells. We show that acute myeloid leukemia (AML) cells are sensitive to birinapant-induced death and that the clinical caspase inhibitor emricasan/IDN-6556 augments, rather than prevents, killing by birinapant. Deletion of caspase-8 sensitized AML to birinapant, whereas combined loss of caspase-8 and the necroptosis effector MLKL (mixed lineage kinase domain-like) prevented birinapant/IDN-6556-induced death, showing that inhibition of caspase-8 sensitizes AML cells to birinapant-induced necroptosis. However, loss of MLKL alone did not prevent a caspase-dependent birinapant/IDN-6556-induced death, implying that AML will be less likely to acquire resistance to this drug combination. A therapeutic breakthrough in AML has eluded researchers for decades. Demonstrated antileukemic efficacy and safety of the birinapant/emricasan combination in vivo suggest that induction of necroptosis warrants clinical investigation as a therapeutic opportunity in AML., (Copyright © 2016, American Association for the Advancement of Science.)

We examined the role of NFκB1 in the homeostasis and function of peripheral follicular (Fo) B cells. Aging mice lacking NFκB1 (Nfκb1(-/-)) develop lymphoproliferative and multiorgan autoimmune disease attributed in large part to the deregulated activity of Nfκb1(-/-)Fo B cells that produce excessive levels of the proinflammatory cytokine interleukin 6 (IL-6). Despite enhanced germinal center (GC) B cell differentiation, the formation of GC structures was severely disrupted in the Nfκb1(-/-)mice. Bone marrow chimeric mice revealed that the Fo B cell-intrinsic loss of NFκB1 led to the spontaneous generation of GC B cells. This was primarily the result of an increase in IL-6 levels, which promotes the differentiation of Fo helper CD4(+)T cells and acts in an autocrine manner to reduce antigen receptor and toll-like receptor activation thresholds in a population of proliferating IgM(+)Nfκb1(-/-)Fo B cells. We demonstrate that p50-NFκB1 represses Il-6 transcription in Fo B cells, with the loss of NFκB1 also resulting in the uncontrolled RELA-driven transcription of Il-6.Collectively, our findings identify a previously unrecognized role for NFκB1 in preventing multiorgan autoimmunity through its negative regulation of Il-6 gene expression in Fo B cells., (© 2016 de Valle et al.)

Lactic acid and short chain fatty acids (SCFAs) produced by vaginal microbiota have reported antimicrobial and immune modulatory activities indicating their potential as biomarkers of disease and/or disease susceptibility. In asymptomatic women of reproductive-age the vaginal microbiota is comprised of lactic acid-producing bacteria that are primarily responsible for the production of lactic acid present at ~110 mM and acidifying the vaginal milieu to pH ~3.5. In contrast, bacterial vaginosis (BV), a dysbiosis of the vaginal microbiota, is characterized by decreased lactic acid-producing microbiota and increased diverse anaerobic bacteria accompanied by an elevated pH>4.5. BV is also characterized by a dramatic loss of lactic acid and greater concentrations of mixed SCFAs including acetate, propionate, butyrate, and succinate. Notably women with lactic acid-producing microbiota have more favorable reproductive and sexual health outcomes compared to women with BV. Regarding the latter, BV is associated with increased susceptibility to sexually transmitted infections (STIs) including HIV. In vitro studies demonstrate that lactic acid produced by vaginal microbiota has microbicidal and virucidal activities that may protect against STIs and endogenous opportunistic bacteria as well as immune modulatory properties that require further characterization with regard to their effects on the vaginal mucosa. In contrast, BV-associated SCFAs have far less antimicrobial activity with the potential to contribute to a pro-inflammatory vaginal environment. Here we review the composition of lactic acid and SCFAs in respective states of eubiosis (non-BV) or dysbiosis (BV), their effects on susceptibility to bacterial/viral STIs and whether they have inherent microbicidal/virucidal and immune modulatory properties. We also explore their potential as biomarkers for the presence and/or increased susceptibility to STIs.

Although the diverse functions served by the nuclear factor-κB (NF-κB) pathway in virtually all cell types are typically employed to deal with stress responses, NF-κB transcription factors also play key roles in the development of hemopoietic cells. This review focuses on how NF-κB transcription factors control various aspects of thymic T-cell and myeloid cell differentiation that include its roles in hemopoietic precursors, conventional αβ T cells, CD4(+) regulatory T cells, natural killer T cells, γδ T cells, macrophages, and dendritic cells., (© 2012 John Wiley & Sons A/S.)

The role of specific members of the NF-κB family of transcription factors in CD8 T-cell selection and development is largely unknown. Here, we show that mice lacking NF-κB1 develop a unique population of conventional CD8 single-positive (SP) thymocytes with memory T cell-like properties that populate peripheral immune organs. Development of this memory-like population is not due to PLZF(+) thymocytes and instead coincides with changes in CD8 T-cell selection. These include a reduction in the efficiency of negative selection and a dependence on MHC class Ia or Ib expressed by haematopoietic cells. These findings indicate that NF-κB1 regulates multiple events in the thymus that collectively inhibit the excess development of CD8(+) thymocytes with memory cell characteristics.

The nuclear factor (NF)-κB signalling pathway is known to be critical for natural killer T (NKT) cell differentiation; however, the role of individual NF-κB transcription factors and the precise developmental stages that they control remain unclear. We have investigated the influence of the classical NF-κB transcription factors NF-κB1, c-Rel and RelA on NKT cell development and function, using gene-deleted mice. Individually, none of these factors were essential for the requirement of NF-κB signalling in early NKT cell development before NK1.1 expression, in contrast to earlier reports in which the classical NF-κB pathway was globally disrupted. Instead, we found that each factor played a non-redundant role in later stages of NKT cell maturation and function. Although NF-κB1 deficiency resulted in a moderate reduction in mature NK1.1+ NKT cells, this was found to be more subtle than previously reported. RelA deficiency had a more profound effect on the NK1.1+ stage of NKT cell development, whereas c-Rel-deficient mice had normal NKT cell numbers. All three factors (NF-κB1, RelA and c-Rel) were necessary for normal NKT cell cytokine production. Notably, IL-17, which is produced by a specific subset of NKT cells (NKT-17 cells), defined as NK1.1(-)CD4(-), was not impaired by a lack of these individual NF-κB transcription factors, nor was this subset depleted, suggesting that NKT-17 cells are regulated independently of the NF-κB pathway. Thus, individual NF-κB family members have a largely redundant role in early NKT cell development, but each of them has an important and distinct role in NKT cell maturation and/or function.

During thymopoiesis, a unique program of gene expression promotes the development of CD4 regulatory T (T reg) cells. Although Foxp3 maintains a pattern of gene expression necessary for T reg cell function, other transcription factors are emerging as important determinants of T reg cell development. We show that the NF-kappaB transcription factor c-Rel is highly expressed in thymic T reg cells and that in c-rel(-/-) mice, thymic T reg cell numbers are markedly reduced as a result of a T cell-intrinsic defect that is manifest during thymocyte development. Although c-Rel is not essential for TGF-beta conversion of peripheral CD4(+)CD25(-) T cells into CD4(+)Foxp3(+) cells, it is required for optimal homeostatic expansion of peripheral T reg cells. Despite a lower number of peripheral T reg cells in c-rel(-/-) mice, the residual peripheral c-rel(-/-) T reg cells express normal levels of Foxp3, display a pattern of cell surface markers and gene expression similar to those of wild-type T reg cells, and effectively suppress effector T cell function in culture and in vivo. Collectively, our results indicate that c-Rel is important for both the thymic development and peripheral homeostatic proliferation of T reg cells.

Apoptotic death of hepatocytes, a contributor to many chronic and acute liver diseases, can be a consequence of overactivation of the immune system and is often mediated by TNFalpha. Injection with lipopolysaccharide (LPS) plus the transcriptional inhibitor D(+)-galactosamine (GalN) or mitogenic T cell activation causes fatal hepatocyte apoptosis in mice, which is mediated by TNFalpha, but the effector mechanisms remain unclear. Our analysis of gene-targeted mice showed that caspase-8 is essential for hepatocyte killing in both settings. Loss of Bid, the proapoptotic BH3-only protein activated by caspase-8 and essential for Fas ligand-induced hepatocyte killing, resulted only in a minor reduction of liver damage. However, combined loss of Bid and another BH3-only protein, Bim, activated by c-Jun N-terminal kinase (JNK), protected mice from LPS+GalN-induced hepatitis. These observations identify caspase-8 and the BH3-only proteins Bid and Bim as potential therapeutic targets for treatment of inflammatory liver diseases.

The nuclear factor-kappaB (NF-kappaB) pathway is crucial for the survival of B cells stimulated through Toll-like receptors (TLRs). Here, we show that the heightened death of TLR4-activated nfkb1(-/-) B cells is the result of a failure of the Tpl(2)/MEK/ERK pathway to phosphorylate the proapo-ptotic BH3-only protein Bim and target it for degradation. ERK inactivation of Bim after TLR4 stimulation is accompanied by an increase in A1/Bim and Bcl-x(L)/Bim complexes that we propose represents a c-Rel-dependent mechanism for neutralizing Bim. Together these findings establish that optimal survival of TLR4-activated B cells depends on the NF-kappaB pathway neutralizing Bim through a combination of Bcl-2 prosurvival protein induction and Tpl2/ERK-dependent Bim phosphorylation and degradation.

Monocytic leukemia zinc finger protein (MOZ), a transcriptional coactivator and member of the MYST family of histone acetyltransferases, is the target of recurrent translocations in acute myeloid leukemia. Since genes associated with translocations in leukemia are typically important regulators of blood formation, we investigated if Moz has a role in normal hematopoiesis. We generated mice carrying a mutation in the Moz gene. Homozygous Moz mutant mice died at birth. Moz mutant fetal liver hematopoietic cells were incapable of contributing to the hematopoietic system of recipients after transplantation. We observed profound defects in the stem cell compartment of Moz-deficient mice. Progenitors of all lineages were reduced in number. However, blood cell lineage commitment was unaffected. Together, these results show that Moz is essential for a fundamental property of hematopoietic stem cells, the ability to reconstitute the hematopoietic system of a recipient after transplantation and that Moz is specifically required in the stem cell compartment.

Engaging mammalian Toll-like receptors (TLRs) activate both the NF-kappaB and mitogen-activated protein kinase signaling pathways. Here we establish that mitogen-activated protein 3 kinase Tpl2, levels of which are markedly reduced in nfkb1(-/-) cells, is required for extracellular signal-regulated kinase (ERK) activation in bone marrow-derived macrophages and B cells stimulated with diverse TLR ligands. Despite rescuing TLR-dependent ERK activation in nfkb1(-/-) bone marrow-derived macrophages by using an estrogen receptor-regulated version of the mitogen-activated protein 3 kinase, c-Raf (Raf:ER), CpG or LPS induction of IL-10 was only partially restored in nfkb1(-/-) cells expressing Raf:ER, a finding consistent with NF-kappaB1 regulating IL-10 by a combination of ERK-independent and -dependent mechanisms. Collectively, our findings indicate that the Tpl2/MEK/ERK signaling module is a master regulator of ERK-dependent gene expression downstream of TLRs in different hemopoietic cells.

Reticuloendotheliosis viral oncogene homolog/nuclear factor of kappa light polypeptide gene enhancer in B cells 1 (Rel/NF-kappaB) activation is a ubiquitous outcome of engaging Toll-like receptors (TLRs), yet the cell-type-specific functions of this pathway in response to particular microbial signals remain poorly defined. Here we show that NF-kappaB1 and C-Rel, Rel/NF-kappaB proteins induced in conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) by cytosine-phosphate-guanosine (CpG) DNA, a TLR-9 ligand, serve markedly different functions in these DC subsets. With the exception of impaired interleukin-12 (IL-12) production, cultured Nfkb1(-/-)C-Rel(-/-) cDCs responded relatively normally to CpG DNA. In contrast, CpG-treated Nfkb1(-/-)C-Rel(-/-) pDCs, which were still able to produce type I interferon and regulated on activation normal T-cell expressed and secreted (RANTES), but not IL-6 or IL-12, failed to acquire an activated dendritic phenotype and underwent apoptosis. Although the TLR-9-mediated death of Nfkb1(-/-)C-Rel(-/-) pDCs, which coincided with a failure to up-regulate the prosurvival proteins B-cell lymphoma apoptosis regulator xL (Bcl-x(L)) and A1, was blocked by Bcl-2 transgene expression, this inhibition of apoptosis still failed to rescue the differentiation defects. This indicated that these NF-kappaB transcription factors independently regulate TLR-9-mediated pDC morphogenesis and survival. Collectively, these findings establish that NF-kappaB1 and c-Rel, while largely dispensable for TLR-9-induced cDC activation, are critical for regulating differentiation and survival programs during pDC activation.

Determining the roles of Rel/NF-kappaB transcription factors in mouse skin development with loss-of-function mutants has been limited by redundancy among these proteins and by embryonic lethality associated with the absence of RelA. Using mice lacking RelA and c-rel, which survive throughout embryogenesis on a tumor necrosis factor alpha (TNF-alpha)-deficient background (rela(-/-) c-rel(-/-) tnfalpha(-/-)), we show that c-rel and RelA are required for normal epidermal development. Although mutant fetuses fail to form tylotrich hair and have a thinner epidermis, mutant keratinocyte progenitors undergo terminal differentiation to form an outer cornified layer. Mutant basal keratinocytes are abnormally small, exhibit a delay in G(1) progression, and fail to form keratinocyte colonies in culture. In contrast to the reduced proliferation of mutant keratinocytes during embryogenesis, skin grafting experiments revealed that the mutant epidermis develops a TNF-alpha-dependent hyperproliferative condition. Collectively, our findings indicate that RelA and c-rel control the development of the epidermis and associated appendages during embryogenesis and regulate epidermal homeostasis in a postnatal environment through the suppression of innate immune-mediated inflammation.

Downstream of kinase (Dok)-related protein (DokR, also known as p56(dok)/FRIP/Dok-R) is implicated in cytokine and immunoreceptor signaling in myeloid and T cells. Tyrosine phosphorylation induces DokR to bind the signal relay molecules, RasGTPase-activating protein (RasGAP) and Nck. Here, we have examined the function of DokR during hematopoietic development and the requirement for RasGAP and Nck binding sites in its biological function. Retroviral-mediated expression of DokR in bone marrow cells dramatically inhibited their capacity to form colonies in vitro in response to the cytokines macrophage colony-stimulating factor and stem cell factor, whereas responses to interleukin-3 and granulocyte macrophage colony-stimulating factor were only weakly affected. When introduced into lethally irradiated mice, hematopoietic cells expressing DokR showed a drastically reduced capacity to repopulate lymphoid tissues. Most notably, DokR dramatically reduced repopulation of the thymus, in part by reducing the number of T cell precursors seeding in the thymus, but equally, through inhibiting the transition of CD4(-)CD8(-) to CD4(+)CD8(+) T cells. Consequently, the number of mature peripheral T cells was markedly reduced. In contrast, a minimal effect on B cell and myeloid lineage development was observed. Importantly, functional RasGAP and Nck binding sites were found to be essential for the biological effects of DokR in vitro and in vivo.

Transcription factors NF-kappaB1 and c-Rel, individually dispensable during embryogenesis, serve similar, yet distinct, roles in the function of mature hemopoietic cells. Redundancy among Rel/NF-kappaB family members prompted an examination of the combined roles of c-Rel and NF-kappaB1 by using mice that lack both proteins. Embryonic development and the maturation of hemopoietic progenitors were unaffected in nfkb1(-/-)c-rel(-/-) mice. Peripheral T cell populations developed normally, but follicular, marginal zone, and CD5(+) peritoneal B cell populations all were reduced. In culture, a failure of mitogen-stimulated nfkb1(-/-)c-rel(-/-) B cells to proliferate was caused by a cell cycle defect in early G(1) that prevented growth. In vivo, defects in humoral immunity and splenic architecture seen in nfkb1(-/-) and c-rel(-/-) mice were exacerbated in the double mutant mice. These findings demonstrate that in the B lineage overlapping roles for NF-kappaB1 and c-Rel appear to be restricted to regulating the activation and function of mature cells.

Interleukin 12 (IL-12) is a 70-kD proinflammatory cytokine produced by antigen presenting cells that is essential for the induction of T helper type 1 development. It comprises 35-kD (p35) and 40-kD (p40) polypeptides encoded by separate genes that are induced by a range of stimuli that include lipopolysaccharide (LPS), DNA, and CD40 ligand. To date, the regulation of IL-12 expression at the transcriptional level has mainly been examined in macrophages and restricted almost exclusively to the p40 gene. Here we show that in CD8(+) dendritic cells, major producers of IL-12 p70, the Rel/nuclear factor (NF)-kappaB signaling pathway is necessary for the induction of IL-12 in response to microbial stimuli. In contrast to macrophages which require c-Rel for p40 transcription, in CD8(+) dendritic cells, the induced expression of p35 rather than p40 by inactivated Staphylococcus aureus, DNA, or LPS is c-Rel dependent and regulated directly by c-Rel complexes binding to the p35 promoter. This data establishes the IL-12 p35 gene as a new target of c-Rel and shows that the regulation of IL-12 p70 expression at the transcriptional level by Rel/NF-kappaB is controlled through both the p35 and p40 genes in a cell type-specific fashion.

Rel and RelA, individually dispensable for lymphopoiesis, serve unique functions in activated B and T cells. Here their combined roles in lymphocyte development were examined in chimeric mice repopulated with c-rel(-/-) rela(-/-) fetal liver hemopoietic stem cells. Mice engrafted with double-mutant cells lacked mature IgM(lo)IgD(hi) B cells, and numbers of peripheral CD4(+) and CD8(+) T cells were markedly reduced. The absence of mature B cells was associated with impaired survival that coincided with reduced expression of bcl-2 and A1. bcl-2 transgene expression not only prevented apoptosis and increased peripheral B-cell numbers, but also induced further maturation to an IgM(lo)IgD(hi) phenotype. In contrast, the survival of double-mutant T cells was normal and the bcl-2 transgene could not rectify the peripheral T-cell deficit. These findings indicate that Rel and RelA serve essential, albeit redundant, functions during the later antigen-independent stages of B- and T-cell maturation, with these transcription factors promoting the survival of peripheral B cells in part by upregulating Bcl-2.

The protein hen egg white lysozyme (HEL) contains two segments, in tandem, from which two families of peptides are selected by the class II molecule I-Ak, during processing. These encompass peptides primarily from residues 31-47 and 48-63. Mutant HEL proteins were created with changes in residues 52 and 55, resulting in a lack of binding and selection of the 48-63 peptides to I-Ak molecules. Such mutant HEL proteins donated the same amount of 31-47 peptide as did the unmodified protein. Other mutant HEL molecules containing proline residues at residue 46, 47, or 48 resulted in extensions of the selected 31-47 or 48-62 families to their overlapping regions (in the carboxyl or amino termini, respectively). However, the amount of each family of peptide selected was not changed. We conclude that the presence or absence of the major peptide from HEL does not influence the selection of other epitopes, and that these two families are selected independently of each other.

The Rel/nuclear factor (NF)-kappaB family of transcription factors have been implicated in the regulation of a wide variety of genes, in particular those encoding proteins crucial to the function of the immune system. Through the use of mutant mice that lack one or more of these proteins, we have begun to examine the individual and combined roles of Rel, RelA and NF-kappaB1 in B-cell development and function. Here we outline and discuss how these transcription factors operate as differentiation stage-specific regulators of B-cell development, survival, division and immunoglobulin expression, emphasizing those Rel/NF-kappaB-regulated genes that mediate these functions.

We report here the identification and quantitation of a minor epitope from hen egg white lysozyme (HEL) isolated from the class II MHC molecule I-Ak of APCs. We isolated and concentrated the peptides from the I-Ak extracts by a peptide-specific mAba, followed by their examination by electrospray mass spectrometry. This initial step improved the isolation, recovery, and quantitation and allowed us to identify 13 different minor peptides using the Ab specific for the HEL tryptic fragment 34-45. The HEL peptides varied on both the amino and carboxy termini. The shortest peptide was a 13-mer (residues 33-45), and the longest peptide was a 19-mer (residues 31-49). The two most abundant were 31-47 (1.3 pmol) and 31-46 (1 pmol), while the least abundant were 31-45 (40 fmol) and 32-45 (4 fmol). Only 0.3% of the total class II molecules were occupied by this family of HEL peptides. The amount of the 31-47 peptide, the predominant member of this series, was 22 times lower than that of 48-62, the major epitope of HEL. The 31-47 peptide bound about 20-fold weaker to I-Ak compared with the dominant 48-62 peptide. Thus, the lower abundance of the minor epitope correlated with its weaker binding strength.