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1.  Olive Oil and Vitamin D Synergistically Prevent Bone Loss in Mice 
PLoS ONE  2014;9(12):e115817.
As the Mediterranean diet (and particularly olive oil) has been associated with bone health, we investigated the impact of extra virgin oil as a source of polyphenols on bone metabolism. In that purpose sham-operated (SH) or ovariectomized (OVX) mice were subjected to refined or virgin olive oil. Two supplementary OVX groups were given either refined or virgin olive oil fortified with vitamin D3, to assess the possible synergistic effects with another liposoluble nutrient. After 30 days of exposure, bone mineral density and gene expression were evaluated. Consistent with previous data, ovariectomy was associated with increased bone turnover and led to impaired bone mass and micro-architecture. The expression of oxidative stress markers were enhanced as well. Virgin olive oil fortified with vitamin D3 prevented such changes in terms of both bone remodeling and bone mineral density. The expression of inflammation and oxidative stress mRNA was also lower in this group. Overall, our data suggest a protective impact of virgin olive oil as a source of polyphenols in addition to vitamin D3 on bone metabolism through improvement of oxidative stress and inflammation.
PMCID: PMC4281074  PMID: 25551374
2.  Impact of MIF Gene Promoter Polymorphism on F508del Cystic Fibrosis Patients 
PLoS ONE  2014;9(12):e114274.
Macrophage migration Inhibitory Factor (MIF) is a pro-inflammatory cytokine sustaining the acute response to gram–negative bacteria and a regulatory role for MIF in Cystic Fibrosis has been suggested by the presence of a functional, polymorphic, four-nucleotide repeat in this gene's promoter at position −794, with the 5-repeat allele displaying lower promoter activity. We aimed at assessing the association of this polymorphism with disease severity in a group of Cystic Fibrosis patients homozygous for F508del CFTR gene mutation. Genotype frequencies were determined in 189 Cystic Fibrosis and 134 control subjects; key clinical features of patients were recorded and compared among homozygous 5-allele patients and the other MIF genotypes. Patients homozygous for the 5-repeat allele of MIF promoter displayed a slower rate of lung function decline (p = 0.027) at multivariate survival analysis. Multiple regression analysis on age-normalized respiratory volume showed no association of the homozygous 5-repeat genotype with lung function under stable conditions and no correlation with P.aeruginosa chronic colonization. Therefore, only the Homozygous 5-repeat genotype at MIF −794 is associated with milder disease in F508del Cystic Fibrosis patients.
PMCID: PMC4264759  PMID: 25503271
3.  MyD88 in DNA Repair and Cancer Cell Resistance to Genotoxic Drugs 
MyD88 is an adaptor molecule in Toll-like receptor and interleukin 1 receptor signaling implicated in tumorigenesis through proinflammatory mechanisms. We have recently reported that MyD88 also directly promotes optimal activation of the Ras/Erk pathway. Here we investigate MyD88 implication in the maintenance of the transformation of Ras-dependent tumors.
RNA interference was used to inhibit MyD88 expression in the colon cancer cell lines HCT116 and LS513. Apoptosis, DNA damage, p53 function, ERCC1 levels, and Ras and inflammatory signaling pathways were analyzed. Using in vitro assays and xenotransplantation in nude mice (five per group), HCT116 tumor growth was assessed following MyD88 knockdown in presence or absence of chemotherapy.
MyD88 exerts antiapoptotic functions in colon cancer cells via the Ras/Erk, but not the NF-κB, pathway. MyD88 inhibition leads to defective ERCC1-dependent DNA repair and to accumulation of DNA damage, resulting in cancer cell death via p53. Furthermore, we show that knocking down MyD88 sensitizes cancer cells to genotoxic agents such as platinum salts in vitro and in vivo. Indeed, HCT116 tumor growth following treatment with a combination of suboptimal MyD88 inhibition and suboptimal doses of cisplatin (fold tumor increase = 5.4±1.6) was statistically significantly reduced in comparison to treatment with doxycycline alone (12.4±3.1) or with cisplatin alone (12.5±2.6) (P = .005 for both, one-sided Student t test).
Collectively, these results indicate a novel and original link between inflammation, DNA repair, and cancer, and provide further rationale for MyD88 as a potential therapeutic target in Ras-dependent cancers, in the context of concomitant genotoxic chemotherapy.
PMCID: PMC3699436  PMID: 23766530
4.  dsRNA induces apoptosis through an atypical death complex associating TLR3 to caspase-8 
Cell Death and Differentiation  2012;19(9):1482-1494.
Toll-like receptor 3 (TLR3) is a pattern-recognition receptor known to initiate an innate immune response when stimulated by double-stranded RNA (dsRNA). Components of TLR3 signaling, including TIR domain-containing adapter inducing IFN-α (TRIF), have been demonstrated to contribute to dsRNA-induced cell death through caspase-8 and receptor interacting protein (RIP)1 in various human cancer cells. We provide here a detailed analysis of the caspase-8 activating machinery triggered in response to Poly(I:C) dsRNA. Engagement of TLR3 by dsRNA in both type I and type II lung cancer cells induces the formation of an atypical caspase-8-containing complex that is devoid of classical death receptors of the TNFR superfamily, but instead is physically associated to TLR3. The recruitment of caspase-8 to TLR3 requires RIP1, and is negatively modulated by cellular inhibitor of apoptosis protein (cIAP)2–TNF receptor-associated factor (TRAF)2–TNFR-associated death domain (TRADD) ubiquitin ligase complex, which regulates RIP1 ubiquitination. Intriguingly, unlike Fas- or TRAILR-dependent death signaling, caspase-8 recruitment and activation within the TLR3 death-signaling complex appears not to be stringently dependent on Fas-associated with death domain (FADD). Our findings uncover a novel aspect of the molecular mechanisms involved during apoptosis induced by the innate immune receptor TLR3 in cancer cells.
PMCID: PMC3422471  PMID: 22421964
Toll-like receptor 3; apoptosis; caspase-8; RIP1; ubiquitination; cancer cells
5.  Dysregulated Proinflammatory and Fibrogenic Phenotype of Fibroblasts in Cystic Fibrosis 
PLoS ONE  2013;8(5):e64341.
Morbi-mortality in cystic fibrosis (CF) is mainly related to chronic lung infection and inflammation, uncontrolled tissue rearrangements and fibrosis, and yet the underlying mechanisms remain largely unknown. We evaluated inflammatory and fibrosis responses to bleomycin in F508del homozygous and wild-type mice, and phenotype of fibroblasts explanted from mouse lungs and skin. The effect of vardenafil, a cGMP-specific phosphodiesterase type 5 inhibitor, was tested in vivo and in culture. Responses of proinflammatory and fibrotic markers to bleomycin were enhanced in lungs and skin of CF mice and were prevented by treatment with vardenafil. Purified lung and skin fibroblasts from CF mice proliferated and differentiated into myofibroblasts more prominently and displayed higher sensitivity to growth factors than those recovered from wild-type littermates. Under inflammatory stimulation, mRNA and protein expression of proinflammatory mediators were higher in CF than in wild-type fibroblasts, in which CFTR expression reached similar levels to those observed in other non-epithelial cells, such as macrophages. Increased proinflammatory responses in CF fibroblasts were reduced by half with submicromolar concentrations of vardenafil. Proinflammatory and fibrogenic functions of fibroblasts are upregulated in CF and are reduced by vardenafil. This study provides compelling new support for targeting cGMP signaling pathway in CF pharmacotherapy.
PMCID: PMC3667188  PMID: 23734196
6.  The objective assessment of cough frequency: accuracy of the LR102 device 
The measurement of cough frequency is problematic and most often based on subjective assessment. The aim of the study was to assess the accuracy of the automatic identification of cough episodes by LR102, a cough frequency meter based on electromyography and audio sensors.
Ten adult patients complaining of cough were recruited in primary care and hospital settings. Participants were asked to wear LR102 for 4 consecutive hours during which they were also filmed.
Measures of cough frequency by LR102 and manual counting were closely correlated (r = 0.87 for number of cough episodes per hour; r = 0.89 for number of single coughs per hour) but LR102 overestimated cough frequency. Bland-Altman plots indicate that differences between the two measurements were not influenced by cough frequency.
LR102 offers a useful estimate of cough frequency in adults in their own environment, while significantly reducing the time required for analysis.
PMCID: PMC3268712  PMID: 22132691
cough; validation study; cough meter
7.  Prognostic value of the expression of C-Chemokine Receptor 6 and 7 and their ligands in non-metastatic breast cancer 
BMC Cancer  2011;11:213.
Chemokines and chemokine receptors are major actors of leukocytes trafficking and some have been shown to play an important role in cancer metastasis. Chemokines CCL19, CCL20 and CCL21 and their receptors CCR6 and CCR7, were assessed as potential biomarkers of metastatic dissemination in primary breast cancer.
Biomarker expression levels were evaluated using immunohistochemistry on paraffin-embedded tissue sections of breast cancer (n = 207).
CCR6 was expressed by tumor cells in 35% of cases. CCR7 was expressed by spindle shaped stromal cells in 43% of cases but not by tumor cells in this series. CCL19 was the only chemokine found expressed in a significant number of breast cancers and was expressed by both tumor cells and dendritic cells (DC). CCR6, CCL19 and CCR7 expression correlated with histologic features of aggressive disease. CCR6 expression was associated with shorter relapse-free survival (RFS) in univariate and but not in multivariate analysis (p = 0.0316 and 0.055 respectively), and was not associated with shorter overall survival (OS). Expression of CCR7 was not significantly associated with shorter RFS or OS. The presence of CCL19-expressing DC was associated with shorter RFS in univariate and multivariate analysis (p = 0.042 and 0.020 respectively) but not with shorter OS.
These results suggest a contribution of CCR6 expression on tumor cells and CCL19-expressing DC in breast cancer dissemination. In our series, unlike what was previously published, CCR7 was exclusively expressed on stromal cells and was not associated with survival.
PMCID: PMC3130701  PMID: 21624121
early breast cancer; chemokine; chemokine receptor; prognosis; metastasis
8.  Omalizumab for exacerbations of allergic bronchopulmonary aspergillosis in patients with cystic fibrosis 
BMJ Case Reports  2009;2009:bcr07.2008.0379.
Anti-IgE therapy was proposed to two teenagers with cystic fibrosis (CF) with allergic bronchopulmonary aspergillosis (ABPA) exacerbation, reluctant to a further course of oral steroids. Both patients experienced ABPA exacerbations within the past 3 years, requiring oral steroid bursts. Clinical, laboratory and radiographic features were consistent with ABPA exacerbations (representing at the time of evaluation the fourth and third episodes for patient 1 and 2, respectively). Total serum IgE was very high, >1000 kU/litre in both cases. Treatment consisting of subcutaneous injections of 375 mg anti-IgE (omalizumab) twice monthly was successful in rapidly improving respiratory symptoms and lung function. Based on clinical and functional improvement, interval between injections was progressively increased and treatment could be withdrawn after 11 injections, without recurrence at 20 weeks of follow-up after withdrawal.
PMCID: PMC3029439  PMID: 21686833
9.  Comparison of culture and qPCR for the detection of Pseudomonas aeruginosa in not chronically infected cystic fibrosis patients 
BMC Microbiology  2010;10:245.
Pseudomonas aeruginosa is the major respiratory pathogen causing severe lung infections among CF patients, leading to high morbidity and mortality. Once infection is established, early antibiotic treatment is able to postpone the transition to chronic lung infection. In order to optimize the early detection, we compared the sensitivity of microbiological culture and quantitative PCR (qPCR) for the detection of P. aeruginosa in respiratory samples of not chronically infected CF patients.
In this national study, we followed CF patients during periods between 1 to 15 months. For a total of 852 samples, 729 (86%) remained P. aeruginosa negative by both culture and qPCR, whereas 89 samples (10%) were positive by both culture and qPCR.
Twenty-six samples were negative by culture but positive by qPCR, and 10 samples were positive by culture but remained negative by qPCR. Five of the 26 patients with a culture negative, qPCR positive sample became later P. aeruginosa positive both by culture and qPCR.
Based on the results of this study, it can be concluded that qPCR may have a predictive value for impending P. aeruginosa infection for only a limited number of patients.
PMCID: PMC2949703  PMID: 20868481
10.  Dual function of MyD88 in RAS signaling and inflammation, leading to mouse and human cell transformation 
The Journal of Clinical Investigation  2010;120(10):3663-3667.
Accumulating evidence points to inflammation as a promoter of carcinogenesis. MyD88 is an adaptor molecule in TLR and IL-1R signaling that was recently implicated in tumorigenesis through proinflammatory mechanisms. Here we have shown that MyD88 is also required in a cell-autonomous fashion for RAS-mediated carcinogenesis in mice in vivo and for MAPK activation and transformation in vitro. Mechanistically, MyD88 bound to the key MAPK, Erk, and prevented its inactivation by its phosphatase, MKP3, thereby amplifying the activation of the canonical RAS pathway. The relevance of this mechanism to human neoplasia was suggested by the finding that MyD88 was overexpressed and interacted with activated Erk in primary human cancer tissues. Collectively, these results show that in addition to its role in inflammation, MyD88 plays what we believe to be a crucial direct role in RAS signaling, cell-cycle control, and cell transformation.
PMCID: PMC2947228  PMID: 20941850
11.  Elevated MMP‐12 protein levels in induced sputum from patients with COPD 
Thorax  2005;61(3):196-201.
Several matrix metalloproteinases (MMPs) are involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). In mice, MMP‐12 plays a crucial role in the development of cigarette smoke induced emphysema. A study was undertaken to investigate the role of MMP‐12 in the development of COPD in human smokers.
Induced sputum samples were collected from patients with stable COPD (n = 28), healthy smokers (n = 14), never smokers (n = 20), and former smokers (n = 14). MMP‐12 protein levels in induced sputum were determined by ELISA and compared between the four study groups. MMP‐12 enzymatic activity in induced sputum was evaluated by casein zymography and by cleaving of a fluorescence quenched substrate.
Median (IQR) MMP‐12 levels were significantly higher in COPD patients than in healthy smokers, never smokers, and former smokers (17.5 (7.1–42.1) v 6.7 (3.9–10.4) v 4.2 (2.4–11.3) v 6.1 (4.5–7.6) ng/ml, p = 0.0002). MMP‐12 enzymatic activity was significantly higher in patients with COPD than in controls (4.11 (1.4–8.0) v 0.14 (0.1–0.2) μg/μl, p = 0.0002).
MMP‐12 is markedly increased in induced sputum from patients with stable COPD compared with controls, suggesting a role for MMP‐12 in the development of COPD in smokers.
PMCID: PMC2080750  PMID: 16308335
chronic obstructive pulmonary disease; matrix metalloproteinases; induced sputum; MMP‐12; smoking
12.  Reference Ranges for Spirometry Across All Ages 
Rationale: The Third National Health and Nutrition Examination Survey (NHANES III) reference is currently recommended for interpreting spirometry results, but it is limited by the lack of subjects younger than 8 years and does not continuously model spirometry across all ages.
Objectives: By collating pediatric data from other large-population surveys, we have investigated ways of developing reference ranges that more accurately describe the relationship between spirometric lung function and height and age within the pediatric age range, and allow a seamless transition to adulthood.
Methods: Data were obtained from four surveys and included 3,598 subjects aged 4–80 years. The original analyses were sex specific and limited to non-Hispanic white subjects. An extension of the LMS (lambda, mu, sigma) method, widely used to construct growth reference charts, was applied.
Measurements and Main Results: The extended models have four important advantages over the original NHANES III analysis as follows: (1) they extend the reference data down to 4 years of age, (2) they incorporate the relationship between height and age in a way that is biologically plausible, (3) they provide smoothly changing curves to describe the transition between childhood and adulthood, and (4) they highlight the fact that the range of normal values is highly dependent on age.
Conclusions: The modeling technique provides an elegant solution to a complex and longstanding problem. Furthermore, it provides a biologically plausible and statistically robust means of developing continuous reference ranges from early childhood to old age. These dynamic models provide a platform from which future studies can be developed to continue to improve the accuracy of reference data for pulmonary function tests.
PMCID: PMC2643211  PMID: 18006882
spirometry; pulmonary function; reference values
13.  Azithromycin reduces spontaneous and induced inflammation in ΔF508 cystic fibrosis mice 
Respiratory Research  2006;7(1):134.
Inflammation plays a critical role in lung disease development and progression in cystic fibrosis. Azithromycin is used for the treatment of cystic fibrosis lung disease, although its mechanisms of action are poorly understood. We tested the hypothesis that azithromycin modulates lung inflammation in cystic fibrosis mice.
We monitored cellular and molecular inflammatory markers in lungs of cystic fibrosis mutant mice homozygous for the ΔF508 mutation and their littermate controls, either in baseline conditions or after induction of acute inflammation by intratracheal instillation of lipopolysaccharide from Pseudomonas aeruginosa, which would be independent of interactions of bacteria with epithelial cells. The effect of azithromycin pretreatment (10 mg/kg/day) given by oral administration for 4 weeks was evaluated.
In naive cystic fibrosis mice, a spontaneous lung inflammation was observed, characterized by macrophage and neutrophil infiltration, and increased intra-luminal content of the pro-inflammatory cytokine macrophage inflammatory protein-2. After induced inflammation, cystic fibrosis mice combined exaggerated cellular infiltration and lower anti-inflammatory interleukin-10 production. In cystic fibrosis mice, azithromycin attenuated cellular infiltration in both baseline and induced inflammatory condition, and inhibited cytokine (tumor necrosis factor-α and macrophage inflammatory protein-2) release in lipopolysaccharide-induced inflammation.
Our findings further support the concept that inflammatory responses are upregulated in cystic fibrosis. Azithromycin reduces some lung inflammation outcome measures in cystic fibrosis mice. We postulate that some of the benefits of azithromycin treatment in cystic fibrosis patients are due to modulation of lung inflammation.
PMCID: PMC1637104  PMID: 17064416
14.  Disruption of the langerin/CD207 Gene Abolishes Birbeck Granules without a Marked Loss of Langerhans Cell Function 
Molecular and Cellular Biology  2005;25(1):88-99.
Langerin is a C-type lectin expressed by a subset of dendritic leukocytes, the Langerhans cells (LC). Langerin is a cell surface receptor that induces the formation of an LC-specific organelle, the Birbeck granule (BG). We generated a langerin−/− mouse on a C57BL/6 background which did not display any macroscopic aberrant development. In the absence of langerin, LC were detected in normal numbers in the epidermis but the cells lacked BG. LC of langerin−/− mice did not present other phenotypic alterations compared to wild-type littermates. Functionally, the langerin−/− LC were able to capture antigen, to migrate towards skin draining lymph nodes, and to undergo phenotypic maturation. In addition, langerin−/− mice were not impaired in their capacity to process native OVA protein for I-Ab-restricted presentation to CD4+ T lymphocytes or for H-2Kb-restricted cross-presentation to CD8+ T lymphocytes. langerin−/− mice inoculated with mannosylated or skin-tropic microorganisms did not display an altered pathogen susceptibility. Finally, chemical mutagenesis resulted in a similar rate of skin tumor development in langerin−/− and wild-type mice. Overall, our data indicate that langerin and BG are dispensable for a number of LC functions. The langerin−/− C57BL/6 mouse should be a valuable model for further functional exploration of langerin and the role of BG.
PMCID: PMC538791  PMID: 15601833
16.  Macrophage Inflammatory Protein 3α Is Expressed at Inflamed Epithelial Surfaces and Is the Most Potent Chemokine Known in Attracting Langerhans Cell Precursors 
Dendritic cells (DCs) form a network comprising different populations that initiate and differentially regulate immune responses. Langerhans cells (LCs) represent a unique population of DCs colonizing epithelium, and we present here observations suggesting that macrophage inflammatory protein (MIP)-3α plays a central role in LC precursor recruitment into the epithelium during inflammation. (a) Among DC populations, MIP-3α was the most potent chemokine inducing the selective migration of in vitro–generated CD34+ hematopoietic progenitor cell–derived LC precursors and skin LCs in accordance with the restricted MIP-3α receptor (CC chemokine receptor 6) expression to these cells. (b) MIP-3α was mainly produced by epithelial cells, and the migration of LC precursors induced by the supernatant of activated skin keratinocytes was completely blocked with an antibody against MIP-3α. (c) In vivo, MIP-3α was selectively produced at sites of inflammation as illustrated in tonsils and lesional psoriatic skin where MIP-3α upregulation appeared associated with an increase in LC turnover. (d) Finally, the secretion of MIP-3α was strongly upregulated by cells of epithelial origin after inflammatory stimuli (interleukin 1β plus tumor necrosis factor α) or T cell signals. Results of this study suggest a major role of MIP-3α in epithelial colonization by LCs under inflammatory conditions and immune disorders, and might open new ways to control epithelial immunity.
PMCID: PMC2193271  PMID: 10974036
dendritic cell; chemokine; migration; regulation; in vivo expression
17.  In Breast Carcinoma Tissue, Immature Dendritic Cells Reside within the Tumor, Whereas Mature Dendritic Cells Are Located in Peritumoral Areas 
The Journal of Experimental Medicine  1999;190(10):1417-1426.
We have analyzed the presence of immature and mature dendritic cells (DCs) within adenocarcinoma of the breast using immunohistochemistry. Immature DCs were defined by expression of CD1a-, Langerin-, and intracellular major histocompatibility complex class II–rich vesicles. Mature DCs were defined by expression of CD83 and DC-Lamp. Breast carcinoma cells were defined by morphology and/or cytokeratin expression. We demonstrate two levels of heterogeneity of DCs infiltrating breast carcinoma tissue: (a) immature CD1a+ DCs, mostly of the Langerhans cell type (Langerin+), were retained within the tumor bed in 32/32 samples and (b) mature DCs, CD83+DC-Lamp+, present in 20/32 samples, are confined to peritumoral areas. The high numbers of immature DCs found in the tumor may be best explained by high levels of macrophage inflammatory protein 3α expression by virtually all tumor cells. Confirming the immature/mature DC compartmentalization pattern, in vitro–generated immature DCs adhere to the tumor cells, whereas mature DCs adhere selectively to peritumoral areas. In some cases, T cells are clustering around the mature DCs in peritumoral areas, thus resembling the DC–T cell clusters of secondary lymphoid organs, which are characteristic of ongoing immune reactions.
PMCID: PMC2195690  PMID: 10562317
breast cancer; dendritic cells; tumor immunity; MHC class II; chemokines
18.  Selective Recruitment of Immature and Mature Dendritic Cells by Distinct Chemokines Expressed in Different Anatomic Sites  
DCs (dendritic cells) function as sentinels of the immune system. They traffic from the blood to the tissues where, while immature, they capture antigens. They then leave the tissues and move to the draining lymphoid organs where, converted into mature DC, they prime naive T cells. This suggestive link between DC traffic pattern and functions led us to investigate the chemokine responsiveness of DCs during their development and maturation. DCs were differentiated either from CD34+ hematopoietic progenitor cells (HPCs) cultured with granulocyte/macrophage colony–stimulating factor (GM-CSF) plus tumor necrosis factor (TNF)-α or from monocytes cultured with GM-CSF plus interleukin 4. Immature DCs derived from CD34+ HPCs migrate most vigorously in response to macrophage inflammatory protein (MIP)-3α, but also to MIP-1α and RANTES (regulated on activation, normal T cell expressed and secreted). Upon maturation, induced by either TNF-α, lipopolysaccharide, or CD40L, DCs lose their response to these three chemokines when they acquire a sustained responsiveness to a single other chemokine, MIP-3β. CC chemokine receptor (CCR)6 and CCR7 are the only known receptors for MIP-3α and MIP-3β, respectively. The observation that CCR6 mRNA expression decreases progressively as DCs mature, whereas CCR7 mRNA expression is sharply upregulated, provides a likely explanation for the changes in chemokine responsiveness. Similarly, MIP-3β responsiveness and CCR7 expression are induced upon maturation of monocyte- derived DCs. Furthermore, the chemotactic response to MIP-3β is also acquired by CD11c+ DCs isolated from blood after spontaneous maturation. Finally, detection by in situ hybridization of MIP-3α mRNA only within inflamed epithelial crypts of tonsils, and of MIP-3β mRNA specifically in T cell–rich areas, suggests a role for MIP-3α/CCR6 in recruitment of immature DCs at site of injury and for MIP-3β/CCR7 in accumulation of antigen-loaded mature DCs in T cell–rich areas.
PMCID: PMC2212459  PMID: 9670049
dendritic cells; chemokines; migration; maturation; regulation; in vivo expression
19.  The Normal Counterpart of IgD Myeloma Cells in Germinal Center Displays Extensively Mutated IgVH Gene, Cμ–Cδ Switch, and λ Light Chain Expression  
The Journal of Experimental Medicine  1998;187(8):1169-1178.
Human myeloma are incurable hematologic cancers of immunoglobulin-secreting plasma cells in bone marrow. Although malignant plasma cells can be almost eradicated from the patient's bone marrow by chemotherapy, drug-resistant myeloma precursor cells persist in an apparently cryptic compartment. Controversy exists as to whether myeloma precursor cells are hematopoietic stem cells, pre–B cells, germinal center (GC) B cells, circulating memory cells, or plasma blasts. This situation reflects what has been a general problem in cancer research for years: how to compare a tumor with its normal counterpart. Although several studies have demonstrated somatically mutated immunoglobulin variable region genes in multiple myeloma, it is unclear if myeloma cells are derived from GCs or post-GC memory B cells. Immunoglobulin (Ig)D-secreting myeloma have two unique immunoglobulin features, including a biased λ light chain expression and a Cμ–Cδ isotype switch. Using surface markers, we have previously isolated a population of surface IgM−IgD+CD38+ GC B cells that carry the most impressive somatic mutation in their IgV genes. Here we show that this population of GC B cells displays the two molecular features of IgD-secreting myeloma cells: a biased λ light chain expression and a Cμ–Cδ isotype switch. The demonstration of these peculiar GC B cells to differentiate into IgD-secreting plasma cells but not memory B cells both in vivo and in vitro suggests that IgD-secreting plasma and myeloma cells are derived from GCs.
PMCID: PMC2212232  PMID: 9547329
20.  The Interleukin-17 Gene of Herpesvirus Saimiri 
Journal of Virology  1998;72(7):5797-5801.
In comparison to wild-type herpesvirus saimiri, viral interleukin-17 gene knockout mutants have unaltered behavior regarding viral replication, T-cell transformation in vitro, and pathogenicity in cottontop tamarins. Thus, this gene is not required for T-cell lymphoma induction but may contribute to apathogenic viral persistence in the natural host, the squirrel monkey.
PMCID: PMC110381  PMID: 9621039
21.  Polymerase Chain Reaction Selects a Novel Disintegrin Proteinase from CD40-Activated Germinal Center Dendritic Cells  
To identify genes expressed by a specific subset of dendritic cells found in vivo a polymerase chain reaction–based cDNA subtraction technique was applied to the recently described germinal center dendritic cells. A novel member of the disintegrin metalloproteinase family was cloned which comprises a not typical zinc-chelating catalytic site most similar to a bacterial metalloproteinase. Dendritic cell precursors or immature dendritic cells express no or low levels of the message. It is induced to high levels upon spontaneous or CD40-dependent maturation and in a mixed lymphocyte reaction. In situ hybridization showed distinct expression of this gene in the germinal center. This, together with the findings that certain disintegrin metalloproteinases regulate the activity of tumor necrosis factor α and that metalloproteinases have also been implicated in FasL processing, suggest that this novel molecule may play an important role in dendritic cell function and their interactions with germinal center T cells.
PMCID: PMC2199019  PMID: 9271581
22.  Germinal Center Founder Cells Display Propensity for Apoptosis before Onset of Somatic Mutation 
B lymphocytes undergo affinity maturation of their antigen receptors within germinal centers. These anatomical structures develop in secondary lymphoid organs from the clonal expansion of a few antigen-specific founder B cells, whose isolation and characterization are reported here. Human germinal center founder cells express the naive B cell markers surface IgM and IgD as well as the germinal center B cell markers CD10 and CD38. They express low levels of Bcl-2, high levels of Fas, and undergo rapid apoptosis in culture. The smaller nonproliferating sIgM+IgD+CD38+ B cells displayed a lower level of somatic mutation in their immunoglobulin variable region genes compared with the large proliferating ones. Unmutated sIgM+IgD+CD38+ tonsillar B cells may thus represent germinal center founder cells in which the program for apoptotic cell death is triggered before the onset of somatic mutation, allowing the selection of the germline antibody repertoire at an early stage.
PMCID: PMC2196025  PMID: 9053456
23.  Follicular Dendritic Cells Specifically Express the Long CR2/CD21 Isoform 
This paper describes an antibody (mAb 7D6) that specifically recognizes human follicular dendritic cells (FDCs). By expression cloning, a cDNA clone encoding for the long human CR2/ CD21 isoform (CD21L) that contains an additional exon (10a) was isolated. We demonstrated that FDCs selectively express CD21L, while B cells selectively express the short CR2/CD21 lacking exon 10a (CD21S). By screening mouse Ltk− cells transfected with the CD21L cDNA, we further showed that the other two anti–human FDC mAbs DRC-1 and KiM4 also recognize CD21L. Thus, CD21L represents the first characterized human FDC-specific molecule, which may confer unique functions of FDCs in germinal center development.
PMCID: PMC2196095  PMID: 8996252
24.  Allergic bronchial asthma due to Dermatophagoides pteronyssinus hypersensitivity can be efficiently treated by inoculation of allergen-antibody complexes. 
Journal of Clinical Investigation  1990;85(4):1024-1035.
Antigen-antibody complexes were made from allergens of the common house dust mite, Dermatophagoides pteronyssinus (Dpt) and an excess of purified autologous specific antibodies. These complexes have been used to treat Dpt-hypersensitive patients who suffered from chronic bronchial asthma. Clinical symptoms and medication intake were followed by filling in diary cards. Peak expiratory flow, measured four times a day, was also followed. Intradermal skin tests and bronchial challenge tests were performed with allergen together with an evaluation of nonspecific bronchial reactivity. Specific IgE and IgG antibodies were assayed after separation from the bulk of serum immunoglobulins by immunoadsorption. The study was carried out over two years according to a double-blind protocol. Intradermal inoculation of antigen-antibody complexes resulted in a marked reduction of both clinical and medication scores. No systemic side-effects were observed and only mild wheal and flare reactions were noted at the injection site. The treatment showed a drastic reduction of specific skin and bronchial reactivities with only marginal effects on nonspecific bronchial reactivity. Concentrations of specific IgE antibodies decreased significantly during the first weeks of treatment and remained at these lower values throughout the study. Specific IgG antibodies actually decreased in the majority of treated patients. The total amount of allergen used in this study was less than 1% of the amount currently used for conventional hyposensitization with the same allergen. These findings show that antigen-antibody complex inoculation is an efficient and safe means of treating allergic bronchial asthma and that the mechanism of action is likely to differ from conventional hyposensitization.
PMCID: PMC296531  PMID: 2318962

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