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Optical imaging comprises a broad and diverse range of technology modalities, which aim to assess the morphology, dynamics, and pathophysiology state of organs, tissues, cells, organelles, and molecules, in their natural state and real time. The development of pioneer instruments for optical imaging is referenced to the seventeenth- to eighteenth-century era (circa 1632–1723), when it is presumed that Antonie van Leeuwenhoek invented and used a prototype of the light microscope (Wollman AJM, Nudd R, Hedlund EG, Leake MC, Open Biol 5:150019, http://dx.doi.org/10.1098/rsob.150019, 2016). The groundbreaking discovery and its application was reported by Leeuwenhoek and Robert Hooke (Hooke R, Micrographia: or some physiological descriptions of minute bodies made by magnifying glasses, with observations and inquiries thereupon. Courier Corporation, New York, 1665; Baker H, Leeuwenhoek M, Philos Trans 41:503–519, doi: 10.1098/rstl.1739.0085, 1739), who used a combination of stage, light source, and lenses similar to those currently used (Wollman AJM, Nudd R, Hedlund EG, Leake MC, Open Biol 5:150019, http://dx.doi.org/10.1098/rsob.150019, 2016). Fast-forward to the twentieth to twenty-first century, innovative advances underscore the extraordinary progression into imaging technologies, such as 3-D electron microscopy, confocal fluorescent imaging, mass spectrometry, bioluminescence, and optoacoustics, to name a few (Weissleder R, Nahrendorf M. Proc Natl Acad Sci 112(47):14424–14428, 2015). Collectively, the new imaging modalities enable researchers to reveal complex structures and dynamic interactive processes happening deep inside cellular compartments, which can provide invaluable basic and clinical science information. The present chapter outlines over three centuries of optical imaging technology, as it relates to the rationale that led to the development of innovative methods, which have transformed the means to observe, analyze, study, and diagnose the nature of cellular structures and functions. The paradigm shift inherent to the progressive advances of optical imaging and their impact on bench to bedside applications are accordingly discussed.

After the demonstration that surrogate JCkappa polypeptides could covalently bind mu heavy chain and upon the characterization of the Vkappa-like component of the kappa-like pre-B cell receptor, it became evident that germline transcription is not sterile. The present review discusses the concept of the alternative usage of kappa-like pre-B cell receptors and classical pre-B cell receptors utilizing the lambda-like surrogate light chain composed of lambda5 and VpreB. We propose that both kappa-like and lambda-like pre-B cell receptors work in concert in a fail-safe mechanism to promote light chain rearrangement, heavy chain allelic exclusion and B-lymphocyte maturation.

We previously showed that the human AKNA gene encodes an AT-hook transcription factor that regulates the expression of costimulatory cell surface molecules on lymphocytes. However, AKNA cDNA probes hybridize with multiple transcripts, suggesting either the existence of other homologous genes or a complex regulation operating on a single gene. Here we report evidence for the latter, as we find that AKNA is encoded by a single gene that spans a 61-kb locus of 24 exons on the fragile FRA9E region of human chromosome 9q32. This gene gives rise to at least nine distinct transcripts, most of which are expressed in a tissue-specific manner in lymphoid organs. Many of the AKNA transcripts originate from alternative splicing; others appear to derive from differential polyadenylation and promoter usage. The alternative AKNA transcripts are predicted to encode overlapping protein isoforms, some of which (p70 and p100) are readily detectable using a polyclonal anti-AKNA antisera that we generated. We also find that AKNA PEST-dependent cleavage into p50 polypeptides is targeted to mature B cells and appears to be required for CD40 upregulation. The unusual capacity of the AKNA gene to generate multiple transcripts and proteins may reflect its functional diversity, and it may also provide a fail-safe mechanism that preserves AKNA expression.

By confining germline transcription as a byproduct of the mechanisms inherent to genetic rearrangements, the translation of respective mRNAs and their biological relevance might have been overlooked. Here we report the identification, cloning, and biochemical characterization of a human Vkappa-like protein that is encoded by a germline transcript. This surrogate protein assembles with the immunoglobulin mu heavy chain at the surface of B cell progenitors and precursors to form a kappa-like antigen receptor. These findings support the notion that germline transcription is not futile and stress the flexibility in eukaryotic gene usage and expression. In addition, the present study confirms the co-existence of surrogate lambda and kappa receptors that are proposed to work in concert to promote B lymphocyte maturation.

Mice deficient in the enzyme adenosine deaminase (ADA) have small lymphoid organs that contain reduced numbers of peripheral lymphocytes, and they are immunodeficient. We investigated B cell deficiency in ADA-deficient mice and found that B cell development in the bone marrow was normal. However, spleens were markedly smaller, their architecture was dramatically altered, and splenic B lymphocytes showed defects in proliferation and activation. ADA-deficient B cells exhibited a higher propensity to undergo B cell receptor-mediated apoptosis than their wild-type counterparts, suggesting that ADA plays a role in the survival of cells during Ag-dependent responses. In keeping with this finding, IgM production by extrafollicular plasmablast cells was higher in ADA-deficient than in wild-type mice, thus indicating that activated B cells accumulate extrafollicularly as a result of a poor or nonexistent germinal center formation. This hypothesis was subsequently confirmed by the profound loss of germinal center architecture. A comparison of levels of the ADA substrates, adenosine and 2′-deoxyadenosine, as well resulting dATP levels and S-adenosylhomocysteine hydrolase inhibition in bone marrow and spleen suggested that dATP accumulation in ADA-deficient spleens may be responsible for impaired B cell development. The altered splenic environment and signaling abnormalities may concurrently contribute to a block in B cell Ag-dependent maturation in ADA-deficient mouse spleens.

LILIANA GUZMAN-ROJAS, JENNIFER C. SIMS-MOURTADA, ROBERTO RANGEL &HECTOR MARTINEZ-VALDEZ Department of Immunology, The University of TexasMD Anderson Cancer Center, Houston, Texas, USASUMMARYWithin germinal centres, B lymphocytes are destined to die by apoptosis via Fas signalling, unlessthey are positively rescued by antigen and by signals initiated by CD40–CD154 interactions. Thus,while the germinal centre microenvironment can become a virtual graveyard for most B lympho-cytes that fail to bind antigen with high affinity, it concomitantly provides the necessary stimuli forthe survival of cells that successfully accomplish affinity maturation. Such dichotomy in thephysiology of germinal centre reaction that results in survival of the functional B-cell repertoire andthe elimination of abnormal cells, dictates the fate towards B-cell homeostasis or disease.Consequently, the death and survival-signalling arms within germinal centres predominantlyreside on the timely and controlled expression of Fas and its ligand (FasL), and CD40 andCD154, respectively. In keeping with this notion, lymphoproliferation or deficient immunity aredocumented landmarks of inactivation of either the Fas/FasL or CD40/CD154 signalling pathways.The present review considers two different scenarios in the control of B-cell survival and deathwithin germinalcentres. Thefirst is anidealistic scenario, inwhicha discriminatory andco-ordinatesignalling initiated by the CD40/CD154 and Fas/FasL pairs, respectively, leads the rescue of thefunctional B-cell repertoire and the elimination of the abnormal phenotype. The second is a gloomyscenario in which both the lack and the hyperexpression of either receptor/ligand pairs, are seen asequally deleterious.INTRODUCTIONJust like a good education in a prestigious university campus,the acquisition of B-cell memory from nai¨ve cells takes placewithin a highly specialized microenvironment, namely thegerminal centres (GC) of the secondary lymphoid organs.The screening process for recruitment of GC candidates isinitiated by the induction of immunoglobulin (Ig)M

Population size is governed through cells reacting to a variety of intrinsic and extrinsic cues. Tumors, while liberated from many of the homeostatic constraints placed on physiologic counterparts, can nonetheless remain subject to both social and environmental control. Burkitt lymphoma cells faithful to the biopsy phenotype were used to model the reliance of the colony, if any, on an inbuilt population sensor. Below a normally suicidal threshold number of cells, low picomolar quantities of exogenous CD40 ligand (CD40L/CD154) were found to sustain the clone without the discernible shift in phenotype that accompanies high CD40L encounter. Although CD154 was undetectable in populous cultures, message was induced as numbers became limiting. Correspondingly, attempts to neutralize endogenous CD40L activity failed to perturb cells at optimal densities but resulted in their marked decline as the critical threshold was approached. These data reveal an auto-inducible survival mechanism seemingly regulated through the monitoring of population size, a process somewhat akin to that of “quorum sensing” among gram-negative bacteria in which diffusible molecules provide a means of communication to coordinate gene expression with population density. This process could be activated as cells discern depletions in their community or when deprived of signals otherwise furnished within an appropriate environmental niche.

We identified, cloned, and biochemically characterized the full-length cDNAs encoding the heavy and light chains of a human monoclonal antibody (mAb) from the Epstein-Barr virus (EBV)-cell line P22. The cell line P22, which originated from a patient with bullous pemphigoid (an autoimmune disease causing skin blistering) expressed immunoglobulin-G (IgG) with a lambda light chain. Although the variable heavy (IGHV) chain gene family could not be assigned by IGHV repertoire analysis, the determination of its nucleotide sequence demonstrated that the heavy chain of P22 belonged to the IGHV4 family. The limited IGHV4 gene usage by memory IgG, IGA and IgE-expressing cells supports the notion of the autoreactivity-associated IGHV4 genes and stresses the strong selection pressure within germinal centres towards IGHV4 family. Alignment of P22 IGHV4 cDNA sequence to germline sequences from gene databases, revealed a remarkable divergence, suggesting that the heavy chain of the P22 mAb encodes a distinct IGHV4 gene. The variable light chain (IGLV) encodes a IGLV4 gene and is 98% similar to a previously reported IGLV gene. Furthermore, fluorescent staining with the recombinant mAb showed the same reactivity to that of the native antibody. The data reported herein, (a) reveal an autoantibody encoding a distinct IGHV4 gene, (b) confirm the notion that autoantibodies preferentially use IGHV4 genes, and (c) hypothesize that somatic hypermutation within GC may be a mechanism by which autoreactive B lymphocytes escape negative selection.

Human tonsillar B cells were separated into naive IgD + CD38 − CD23 − (Bm1) and IgD + CD38 − CD23 + (Bm2), germinal center IgD − CD38 + CD23 − centroblasts (Bm3) and IgD − CD38 + CD77 − centrocytes (Bm4), and memory IgD − CD38 − (Bm5) subsets. Previous IgV H sequence analysis concluded that the triggering of somatic mutations occurs during the transition from Bm2 subset into the Bm3 subset. To determine the initiation of isotype switching, sterile transcript expression was analyzed by amplification, cloning, and sequencing. A selective sterile Iγ, Iα, and Ie expression was observed at centrocyte (Bm4) stage, suggesting that isotype switch is triggered within germinal centers, after somatic mutation is initiated within centroblasts (Bm3). Finally, the high level of 5′Sγ–Sμ3′ DNA switching circles observed in germinal center B cells indicates that within human tonsils, germinal center is a major location for isotype switching.

During T cell-dependent antibody responses, B cells within germinal centers (GC) alter the affinity of their antigen receptor by introducing somatic mutations into variable region of immunoglobulin (IgV) genes. During this process, GC B cells are destined to die unless positively selected by antigens and CD40-ligand. To understand survival/death control of germinal center B cell, the expression of four apoptosis-inducing genes, Fas, c-myc, Bax, and P53, together with the survival gene bcl-2, has been analyzed herein among purified tonsillar naive, GC, and memory B cells. IgD+CD38- naive B cells were separated into CD23- (mature B cell [Bm]1) subset and CD23+ (Bm2), IgD-CD38+ GC B cells were separated into subsets of CD77+ centroblasts (Bm3) and CD77- centrocytes (Bm4), whereas IgD-CD38- cells represented the Bm5 memory B cell subset. Sequence analysis of IgV region genes indicated that somatic hypermutation was triggered in the Bm3 centroblast subset. Here we show that bcl-2 is only detectable with naive (Bm1 and 2) and memory B cell (Bm5) subsets, whereas all four apoptosis-inducing genes were most significantly expressed within GC B cells. Fas was equally expressed in Bm3 centroblasts and Bm4 centrocytes, whereas Bax was most significantly expressed in Bm4 centrocytes. c-myc, a positive regulator of cell cycle, was most significantly expressed in proliferating Bm3 centroblasts, whereas P53, a negative regulator of cell cycle, was most signficantly expressed in nonproliferating Bm4 centrocytes. The present results indicate that the survival/death of GC B cells are regulated by the up- and downregulation of multiple genes, among which the expression of c-myc and P53 in the absence of bcl-2 may prime the proliferating Bm3 centroblasts and nonproliferating Bm4 centrocytes to apoptosis.

B-chronic lymphocytic leukemias (B-CLL) represent expanded clones of resting B lymphocytes that mostly express surface IgM (sIgM). The present study shows that B-CLL cells, freshly isolated from two patients, were sIgM+, sIgG-, and sIgA- but expressed IgG and IgA transcripts. cDNA cloning and sequencing showed that the VDJ segments associated to gamma and alpha heavy chain transcripts are identical to those from mu transcripts, thus showing that B lymphocytes giving rise to CLL cells have undergone isotype switching in vivo. Stimulation of these B-CLL cells through surface CD40 in the presence of interleukin- 10 induced them to secrete IgG and IgA, proving that they can also differentiate into Ig-secreting cells. Finally, CD40-stimulated B-CLL cells were induced to switch towards IgE in response to interleukin-4, as shown by the presence of specific VDJ-C epsilon transcripts and the secretion of IgE. Therefore, B-CLL cells tested herein can undergo isotype switching in vivo and can be induced to undergo further isotype switching and differentiation in vitro.

Upon activation, B lymphocytes can change the isotype of the antibody they express by immunoglobulin (Ig) isotype switch recombination. In previous studies on the regulation of human IgG expression, we demonstrated that interleukin 10 (IL-10) could stimulate IgG1 and IgG3 secretion by human CD40-activated naive (sIgD+) tonsillar B cells. To assess whether IL-10 actually promotes the DNA recombination underlying switching to these isotypes, we examined the effect of IL-10 on the generation of reciprocal products that form DNA circles as by-products of switch recombination. The content of reciprocal products characteristic of mu-gamma recombination was elevated after culture of CD40-activated tonsillar sIgD+ B cells with either IL-4 or IL-10, although high levels of IgG secretion were observed only with IL-10. Unlike IL-4, IL-10 did not induce reciprocal products of mu-epsilon and gamma-epsilon switch recombination. These results demonstrate that IL-10 promotes both switching to gamma and IgG secretion.

Gene expression can be regulated by chromatin modifiers, transcription factors and proteins that modulate DNA architecture. Among the latter, AT-hook transcription factors have emerged as multifaceted regulators that can activate or repress broad A/T-rich gene networks. Thus, alterations of AT-hook genes could affect the transcription of multiple genes causing global cell dysfunction. Here we report that targeted deletions of mouse AKNA, a hypothetical AT-hook-like transcription factor, sensitize mice to pathogen-induced inflammation and cause sudden neonatal death. Compared with wild-type littermates, AKNA KO mice appeared weak, failed to thrive and most died by postnatal day 10. Systemic inflammation, predominantly in the lungs, was accompanied by enhanced leukocyte infiltration and alveolar destruction. Cytologic, immunohistochemical and molecular analyses revealed CD11b(+)Gr1(+) neutrophils as major tissue infiltrators, neutrophilic granule protein, cathelin-related antimicrobial peptide and S100A8/9 as neutrophil-specific chemoattracting factors, interleukin-1β and interferon-γ as proinflammatory mediators, and matrix metalloprotease 9 as a plausible proteolytic trigger of alveolar damage. AKNA KO bone marrow transplants in wild-type recipients reproduced the severe pathogen-induced reactions and confirmed the involvement of neutrophils in acute inflammation. Moreover, promoter/reporter experiments showed that AKNA could act as a gene repressor. Our results support the concept of coordinated pathway-specific gene regulation functions modulating the intensity of inflammatory responses, reveal neutrophils as prominent mediators of acute inflammation and suggest mechanisms underlying the triggering of acute and potentially fatal immune reactions.

We have investigated the role of two signal transduction pathways on the regulation of the gamma and delta T cell antigen receptor (TcR) gene expression, in the acute lymphocytic leukemic cell line DND41. Protein kinase C (PKC) activation, and intracellular free Ca2+ mobilization, initiated by phorbol esters and calcium ionophores, respectively, not only acted independently but, more interestingly, their effects were antagonistic, suggesting a role for these signals during T cell differentiation. The Ca2+ ionophore, ionomycin, increased the levels of intracellular free Ca2+ and induced the expression of the gamma and delta chains of the T cell antigen receptor in a concentration-dependent manner. The phorbol ester 12-myristate 13-acetate down-regulated the basal gamma TcR expression with marginal effect on delta TcR mRNA, but diminished the induction of both gamma and delta TcR, initiated by the Ca2+ ionophore. These antagonistic effects of the two arms of the phospholipase C-mediated signal transduction pathways, i.e. PKC activation and increased intracellular free Ca2+, were specific to the regulation of the gamma and delta TcR, since the same signals exerted a synergistic effect on the mRNA levels of interleukin 2 receptor. These data confirm our hypothesis that the antagonistic regulation on the gamma and delta TcR gene expression by phorbol esters and calcium ionophores occurs in the same cell, and stresses the biological significance of PKC activation and intracellular free calcium mobilization during intrathymic differentiation and selection.

Purpose: Heat shock protein 90 (HSP90) is a chaperone for several client proteins involved in transcriptional regulation, signal transduction, and cell cycle control. HSP90 is abundantly expressed by a variety of tumor types and has been recently targeted for cancer therapy. The objective of this study was to determine the role of HSP90 in promoting growth and survival of Hodgkin's lymphoma and to determine the molecular consequences of inhibiting HSP90 function by the small-molecule 17-allylamino-17-demethoxy-geldanamycin (17-AAG) in Hodgkin's lymphoma. Experimental Design: HSP90 expression in Hodgkin's lymphoma cell lines was determined by Western blot and in primary lymph node sections from patients with Hodgkin's lymphoma by immunohistochemistry. Cell viability was determined by the 3-(4,5-dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Apoptosis and cell cycle fractions were determined by flow cytometry. Expression of intracellular proteins was determined by Western blot. Results: HSP90 is overexpressed in primary and cultured Hodgkin's lymphoma cells. Inhibition of HSP90 function by 17-AAG showed a time- and dose-dependent growth inhibition of Hodgkin's lymphoma cell lines. 17-AAG induced cell cycle arrest and apoptosis, which were associated with a decrease in cyclin-dependent kinase (CDK) 4, CDK 6, and polo-like kinase 1 (PLK1), and induced apoptosis by caspase-dependent and caspase-independent mechanisms. Furthermore, 17-AAG depleted cellular contents of Akt, decreased extracellular signal–regulated kinase (ERK) phosphorylation, and reduced cellular FLICE-like inhibitory protein levels (FLIP), and thus enhanced the cytotoxic effect of doxorubicin and agonistic anti–tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) death receptor antibodies. Conclusion: Inhibition of HSP90 function induces cell death and enhances the activity of chemotherapy and anti–tumor necrosis factor–related apoptosis-inducing ligand death receptor antibodies, suggesting that targeting HSP90 function might be of therapeutic value in Hodgkin's lymphoma.

T-cell homing within germinal centres (GCs) is required for humoral B-cell responses. However, the mechanisms implicated in the recruitment of T cells into the GC are not completely understood. Here we show, by immunohistology, and Northern and Western blots, that in vivo human GC B lymphocytes can express CxC and CC chemokines. Moreover, B-cell subset-specific experiments reveal that interleukin (IL)-8 and regulated on activation, normal, T-cell expressed, and secreted (RANTES) are predominantly expressed by GC centroblast and centrocytes, suggesting that chemokine expression is essential at stages in which B-lymphocytes engage in active antigen-dependent interactions with T lymphocytes. In keeping with this hypothesis, we show that the T cells recruited into the GC correlatively express the receptors for IL-8 and RANTES. We propose that chemokine expression is a key B-cell function that facilitates T-lymphocyte recruitment into the GCs and supports cognate B-cell : T-cell encounters. Moreover, our data are consistent with the impaired homing of T cells to secondary lymphoid organs in mice that are either deficient in CC and CxC chemokines or their receptors.

Proteins containing AT hooks bind A/T-rich DNA through a nine-amino-acid motif and are thought to co-regulate transcription by modifying the architecture of DNA, thereby enhancing the accessibility of promoters to transcription factors. Here we describe AKNA, a human AT-hook protein that directly binds the A/T-rich regulatory elements of the promoters of CD40 and CD40 ligand (CD40L) and coordinately regulates their expression. Consistent with its function, AKNA is a nuclear protein that contains multiple PEST protein-cleavage motifs, which are common in regulatory proteins with high turnover rates. AKNA is mainly expressed by B and T lymphocytes, natural killer cells and dendritic cells. During B-lymphocyte differentiation, AKNA is mainly expressed by germinal centre B lymphocytes, a stage in which receptor and ligand interactions are crucial for B-lymphocyte maturation. Our findings show that an AT-hook molecule can coordinately regulate the expression of a key receptor and its ligand, and point towards a molecular mechanism that explains homotypic cell interactions.

We report the identification of a human cDNA encoding a 25 kDa protein of relevant evolutionary and lymphoid interest (PRELI). PRELI was cloned by screening a B lymphocyte-specific cDNA library with a probe generated by mRNA differential display. PRELI amino acid sequence is 85% similar to the avian px19 protein, expressed within the blood islands and in the liver during avian embryo development. PRELI and px19 contain tandem repeats (A/TAEKAK) of the late embryogenesis abundant (LEA) motif, characteristic of a group of survival molecules and originally thought to be present only in plant proteins. Interestingly, PRELI expression is high in the fetal liver, a major site for B cell lymphopoiesis, while the mRNA levels in other fetal tissues such as the brain, lung, and kidney are comparatively low. At the adult stage, PRELI expression is drastically reduced in the liver but exhibits high mRNA levels in the spleen, brain, lung and kidney tissues, suggesting that PRELI expression may be important for the development of vital and immunocompetent organs. Moreover, PRELI is also highly expressed in the adult lymph nodes and peripheral blood leukocytes, further stressing that at the adult stage, PRELI expression may be important during secondary immune responses. Consistent with this hypothesis, the expression of PRELI is predominant within germinal centers (GC), a stage in which B lymphocytes are under a stressful selection pressure. Taken together these data: (i) strongly support the notion that the conserved LEA motif represents a phylogenetic link between plants and animals, (ii) reveal a novel molecule whose expression may play a role in the maturation of distinct human tissues, and (iii) suggest that PRELI expression may be important for GC B lymphocytes.

Using an approach similar to that used to study primary B-lymphocyte development within bone marrow and primary T-lymphocyte development within thymus, the peripheral B-cell maturation pathway within secondary lymphoid tissue (human tonsils) was analysed on the expression of discrete surface antigens. sIgD and CD38 permit the identification of four subpopulations of tonsillar B lymphocytes, including sIgD+ CD38-, sIgD+, CD38+, sIgD-CD38+ and sIgD-CD38- B cells. Further phenotypic, functional and Ig gene analysis (IgV gene sequences, expression of sterile transcripts and DNA switch circles) allowed us to conclude the following: (1) sIgM+ IgD+ CD38- B cells are naive B cells (Bm1 + 2), which carry unmutated antigen-receptors; (2) sIgM+ IgD+ CD38+ B cells are germinal center founder cells (Bm2'), which become prone to undergo apoptosis before the onset of somatic mutation; (3) sIgM-IgD+ CD38+ are germinal center B cells (Bm 3 delta), that have accumulated the highest number of somatic mutations ever reported in normal B cells; these cells may have undergone C mu-deletion by homologous recombination through sigma mu-sigma delta sequences: (4) sIgD-CD38+ CD77+ B cells are centroblasts (Bm3), in which somatic mutation machinery is activated; (5) sIgD-CD38+ CD77- B cells are centrocytes (Bm4), in which the isotype switching machinery is activated; (6) sIgD-CD38- cells (Bm5) represent somatically mutated resting memory B cells. In conclusion, human peripheral B-cell subpopulations corresponding to the differentiation stages before, during and after the triggering of apoptosis program, somatic mutation and isotype switch have been identified and isolated using a combination of surface markers.

Background LEA (late embryogenesis abundant) proteins encode conserved N-terminal mitochondrial signal domains and C-terminal (A/TAEKAK) motif repeats, long-presumed to confer cell resistance to stress and death cues. This prompted the hypothesis that LEA proteins are central to mitochondria mechanisms that connect bioenergetics with cell responses to stress and death signaling. In support of this hypothesis, recent studies have demonstrated that mammalian LEA protein PRELI can act as a biochemical hub, which upholds mitochondria energy metabolism, while concomitantly promoting B cell resistance to stress and induced death. Hence, it is important to define in vivo the physiological relevance of PRELI expression. Methods and findings Given the ubiquitous PRELI expression during mouse development, embryo lethality could be anticipated. Thus, conditional gene targeting was engineered by insertion of flanking loxP (flox)/Cre recognition sites on PRELI chromosome 13 (Chr 13) locus to abort its expression in a tissue-specific manner. After obtaining mouse lines with homozygous PRELI floxed alleles (PRELI(f/f)), the animals were crossed with CD19-driven Cre-recombinase transgenic mice to investigate whether PRELI inactivation could affect B-lymphocyte physiology and survival. Mice with homozygous B cell-specific PRELI deletion (CD19-Cre/Chr13 PRELI(-/-)) bred normally and did not show any signs of morbidity. Histopathology and flow cytometry analyses revealed that cell lineage identity, morphology, and viability were indistinguishable between wild type CD19-Cre/Chr13 PRELI(+/+) and CD19-Cre/Chr13 PRELI(-/-) deficient mice. Furthermore, B cell PRELI gene expression seemed unaffected by Chr13 PRELI gene targeting. However, identification of additional PRELI loci in mouse Chr1 and Chr5 provided an explanation for the paradox between LEA-dependent cytoprotection and the seemingly futile consequences of Chr 13 PRELI gene inactivation. Importantly, PRELI expression from spare gene loci appeared ample to surmount Chr 13 PRELI gene deficiency. Conclusions These findings suggest that PRELI is a vital LEA B cell protein with failsafe genetics.

We have previously shown that human B lymphocytes cultured in the CD40 system, composed of an anti-CD40 mAb presented by a CD32-transfected fibroblastic cell line, proliferate but do not secrete antibodies. However, the addition of particles of Staphylococcus aureus Cowan (SAC) induces B cell differentiation even in the absence of exogenous cytokines (CD40/SAC system). Additionally, B lymphocytes cultured in the CD40 system in the presence of human IL-10, produce IgM, IgG, and IgA, and Ig levels are further increased by SAC. Here, we have studied the capacity of peripheral blood lymphocytes from patients with IgA deficiency (IgA-D) to secrete Igs, particularly IgA after CD40 triggering. Peripheral blood mononuclear cells (PBMNC) from IgA-D patients cultured in the CD40/SAC system produced IgM and IgG, but not IgA. The addition of IL-10 to the cultures, enhanced the production of IgM and IgG and most strikingly induced the production of high amounts of IgA. The addition of IL-10 to PBMNC from IgA-D patients activated through CD40 alone resulted in the production of IgA. Thus, SAC and anti-CD40 mAb stimulate B cells to differentiate into cells secreting IgG and IgM whereas IL-10 plays a central role in inducing B cells from IgA-D patients to differentiate into IgA secreting cells.

OBJECTIVE: To assess the spontaneous production of proinflammatory cytokines and immunoglobulins in rheumatoid arthritis (RA) synovitis and modulation by interleukin-4 (IL-4). METHODS: We developed an ex vivo model of RA synovitis using pieces of RA synovium, and have studied the regulation of the production of IL-1 beta, IL-6, tumor necrosis factor alpha (TNF alpha), IgM, and IgG. RESULTS: Spontaneous production of proinflammatory cytokines in vitro was active, with prolonged cytokine gene transcription and translation. IL-6 was produced at higher levels than either IL-1 beta or TNF alpha, and explants produced more IgG than IgM. In contrast, IL-4 and interferon-gamma were undetectable. When pieces of synovium were incubated in the presence of IL-4, reduction of spontaneous proinflammatory cytokine and Ig production was observed. CONCLUSION: These results extend the observations of the antiinflammatory properties of IL-4 to an ex vivo situation, and provide the rationale for the clinical use of IL-4 in RA.

Intrathymic development and selection of the T lymphocyte repertoire is restricted by the interactions of the T cell antigen receptor and CD4 or CD8 co-receptors with self major histocompatibility complex molecules. Positive or negative selection depends on a tight regulatory control of CD4 and CD8 expression. Determining the intracellular signals that differentially regulate the expression of CD4 and CD8 is important to understand the mechanisms that are implicated in selection of single positive CD4+CD8- or CD4-CD8+. The present study shows that stimulation of human thymocytes by phorbol esters or cAMP result in a differential regulation of CD4 and CD8 expression, both at the mRNA and cell surface glycoprotein level. The differential regulation of CD4 and CD8 gene expression suggests that the selective activation of protein kinase C (PKC) and cAMP-dependent protein kinases (PKA) may be required for the selection of single positive CD4+CD8- and CD4-CD8+ cells during Intrathymic differentiation

Somatic hypermutation in immunoglobulin variable region genes occurs within germinal centers. Here, we describe a subset of germinal center dark zone centroblasts that express only sIgD and have accumulated up to 80 mutations per heavy chain variable region (IgVH delta gene). Over half of the hypermutated IgVH delta sequences were found to be clonally related. This level of mutation is not observed in either IgVH gamma transcripts from the same sample or IgVH delta transcripts from peripheral blood, suggesting that these cells neither undergo isotype switch nor mature into circulating memory B cells. Optimal growth of these cells in vitro depends on CD40 ligand, T cell cytokines, and a fibroblast stroma, a combination possibly mimicking the dark zone microenvironment. Our hypothesis is that these cells may be sequestered within germinal centers, where their somatic mutation machinery is triggered. The isolation of these hypermutated B cells may represent a critical step for studying both the biology and biochemistry of somatic hypermutation.

Incubation of human thymocytes in the presence of phorbol esters caused a reversible decrease in the mRNAs encoding terminal deoxynucleotidyltransferase (TdT; EC 2.7.7.31) and adenosine deaminase (ADA; EC 3.5.4.4) and an increase in the mRNA encoding purine nucleoside phosphorylase (PNP; EC 2.4.2.1). The effect of phorbol esters on TdT and ADA mRNA levels can be attributed to an apparent decrease in the stability of the mRNAs. The changes in ADA, TdT, and PNP mRNAs closely simulate changes in the activities of these enzymes that occur during T-cell differentiation in vivo, suggesting a role for protein kinase C activation in the regulation of the expression of these genes during intrathymic T-cell differentiation. A role for these purine degradation enzymes in the regulation of intracellular pools of the deoxynucleotide substrates of TdT is discussed.

The effect of adenosine and the time response on adenine nucleotide and Pi levels in rat blood was investigated. an increase in adenine nucleotide with a concommitant decrease in Pi concentration 30, 60, and 90 minutes after the nucleoside administration were observed. Though the 100 mg/Kg dose showed the highest effect on nucleotide concentration, the maximal response on Pi content was achieved with the 50 mg/Kg dose. The results are discussed at the light of previous data obtained in hepatocytes, and using as indicators the energy charge and the phosphorylation potential.

23 COORDINATE REGULATION OF ADENOSINE DEAMINASE, PURINE NUCLEOSIDE PHOSPHORYLASE AND TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE mRNA LEVELS BY PHORBOL ESTERS IN HUMAN THYMOCYTES

A nucleotide analysis of Bloom's syndrome lymphocytes and fibroblasts has revealed a partial depletion in adenine nucleotide pools. ATP/ADP ratio in these cells was at least three-fold lower than in primary cultures of normal human cells. Fractionation of each adenine nucleotide by high performance liquid chromatography showed that differences did not reflect an excess in ADP but rather low levels of ATP in Bloom's syndrome cultures. Moreover, such low energy state has been correlated with their low growth. Recent data from our laboratory indicate that Bloom's syndrome cells may reflect an impaired salvage pathway of purine biosynthesis since these cells appear to be defective or even lack adenine phosphoribosyl transferase (APRT) activity. Our work mainly focuses on the kinetics of purine salvage pathway in Bloom's syndrome cells, as well as on drug resistance (cytotoxic purine analogs), in order to characterize the extent of the defect.