Interleukin (IL) 31 induces in cynomolgus monkeys a rapid and intense itch response that can be inhibited by an IL-31 neutralizing antibody.

BACKGROUND: Overexpression or administration of interleukin 31 (IL-31) has been shown to induce a profound itch response in mice and dogs. The chronic pruritus observed in mouse IL-31 transgenic mice results in the development of skin lesions and alopecia through excoriation from excessive scratching, a condition similar to that observed in patients with atopic dermatitis (AD). OBJECTIVE: To test whether IL-31 induces pruritus in non-human primates and, if so, whether treatment with an anti-IL-31 neutralizing monoclonal antibody (mAb) can block the response. METHODS: A series of studies was conducted in cynomolgus monkeys to evaluate the itch response to recombinant cynomolgus IL-31 (cIL-31) administration. Three routes of cIL-31 administration (intravenous, intradermal, and subcutaneous) were evaluated. Subcutaneous treatment with a humanized anti-human IL-31 mAb cross-reactive to cIL-31 was subsequently tested for its ability to block the response to intradermal cIL-31 administration. RESULTS: Each route of cIL-31 delivery elicited a scratching response immediately after cIL-31 administration and lasted at least 3 h. Treatment with the IL-31 mAb inhibited the cIL-31-mediated scratching response in a dose-dependent manner. CONCLUSION: These results demonstrate that an IL-31 mAb can inhibit IL-31-mediated pruritus in vivo, and could be an effective therapy for pruritic skin conditions like AD where IL-31 upregulation may play a role.

The receptor tyrosine kinase MerTK regulates dendritic cell production of BAFF.

The MerTK receptor tyrosine kinase is an important negative regulator of dendritic cell function and is required to prevent B cell autoimmunity in vivo. It is not currently known however, if any causal relationship exists between these two aspects of MerTK function. We sought to determine if dendritic cells (DC) from mice lacking MerTK (mertk(- / - ) mice) have characteristics that may aid in the development of B cell autoimmunity. Specifically, we found that mertk(- / - ) mice contain an elevated number of splenic DC, and this population contains an elevated proportion of cells secreting the critical B cell pro-survival factor, B cell activating factor (BAFF). Elevated numbers of BAFF-secreting cells were also detected among mertk(- / - ) bone marrow-derived dendritic cell (BMDC) populations. This was observed in both resting BMDC, and BMDC stimulated with lipopolysaccharide (LPS) or treated with exogenous apoptotic cells. We also found that DC in general have a pro-survival effect on resting B cells in co-culture. However, despite containing more BAFF-secreting cells, mertk(- / - ) BMDC were not superior to C57BL/6 or baff-deficient BMDC at promoting B cell survival. Furthermore, using decoy receptors, we show that DC may promote B cell survival and autoimmunity through a BAFF-and a proliferation-inducing ligand-independent mechanism.

TACI-Ig prevents the development of airway hyperresponsiveness in a murine model of asthma.

BACKGROUND: Increased levels of serum IgE are associated with greater asthma prevalence and disease severity. IgE depletion using an anti-IgE monoclonal antibody has met with success in the treatment of moderate-to-severe and severe persistent allergic asthma. OBJECTIVE: To test whether B cell-targeted therapy is a more effective treatment for airway hyperresponsiveness (AHR) in a murine model compared with IgE-depletion. METHODS: We delivered soluble mTACI-Ig, a receptor for the B cell survival factors BLyS (B Lymphocyte Stimulator) and APRIL (A PRoliferation-Inducing Ligand), or anti-IgE to allergen-sensitized mice before airway challenge with allergen. RESULTS: mTACI-Ig treatment reduced circulating mature B cell levels in the blood, while anti-IgE treatment had no effect on B cell counts. Both mTACI-Ig and anti-IgE decreased the levels of total and allergen-specific IgE in the serum. Histopathologic analysis of lungs showed a reduction in disease severity scores for both treatment groups, but results were more pronounced in mTACI-Ig-treated mice. Neutrophil and eosinophil numbers in the bronchoalveolar lavage (BAL) were significantly reduced following mTACI-Ig treatment, but not after anti-IgE delivery. BLyS and APRIL blockade also resulted in a significant decrease in IL-4 and eotaxin mRNA and IL-4 and KC protein levels in total lung homogenates and BAL fluid, respectively. Finally, mTACI-Ig treatment was more effective than anti-IgE treatment in reducing AHR to inhaled antigen. CONCLUSIONS: Our data demonstrate that delivery of mTACI-Ig is a more effective treatment than anti-IgE mAb in a murine model of AHR.

Atacicept (TACI-Ig) inhibits growth of TACI(high) primary myeloma cells in SCID-hu mice and in coculture with osteoclasts.

APRIL (a proliferation-inducing Ligand) and BLyS/BAFF (B-lymphocyte stimulator/B-cell-activating factor of the TNF (tumor necrosis factor) family have been shown to be the survival factors for certain myeloma cells in vitro. BAFF binds to the TNF-related receptors such as B-cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) and BAFFR, whereas APRIL binds to TACI and BCMA and to heparan sulfate proteoglycans (HSPG) such as syndecan-1. TACI gene expression in myeloma reportedly can distinguish tumors with a signature of microenvironment dependence (TACI(high)) versus a plasmablastic signature (TACI(low)). We tested the effect of atacicept (formerly TACI-Ig, which blocks APRIL and BAFF) and BAFFR-Ig (which blocks BAFF only) on primary myeloma growth in the SCID-hu model and in coculture with osteoclasts. With only few exceptions, atacicept and to a lesser extent BAFFR-Ig, inhibited growth of TACI(high) but not TACI(low) myeloma samples in vivo and ex vivo, and the response rate was inversely correlated with TACI expression. Most TACI(high) myeloma cells were molecularly classified as being low risk with our recently described 70-gene model. APRIL and BAFF were highly expressed by osteoclasts and were upregulated in myeloma cells after coculture with osteoclasts. Our findings suggest that APRIL plays an essential role in the survival of TACI(high) bone marrow-dependent myeloma cells and TACI gene expression may be a useful predictive marker for patients who could benefit from atacicept treatment.

TACI-Ig neutralizes molecules critical for B cell development and autoimmune disease. impaired B cell maturation in mice lacking BLyS.

BLyS and APRIL have similar but distinct biological roles, mediated through two known TNF receptor family members, TACI and BCMA. We show that mice treated with TACI-Ig and TACI-Ig transgenic mice have fewer transitional T2 and mature B cells and reduced levels of circulating immunoglobulin. TACI-Ig treatment inhibits both the production of collagen-specific Abs and the progression of disease in a mouse model of rheumatoid arthritis. In BLyS-deficient mice, B cell development is blocked at the transitional T1 stage such that virtually no mature B cells are present, while B-1 cell numbers are relatively normal. These findings further elucidate the roles of BLyS and APRIL in modulating B cell development and suggest that BLyS is required for the development of most but not all mature B cell populations found in the periphery.

A soluble class II cytokine receptor, IL-22RA2, is a naturally occurring IL-22 antagonist.

IL-22 is an IL-10 homologue that binds to and signals through the class II cytokine receptor heterodimer IL-22RA1/CRF2-4. IL-22 is produced by T cells and induces the production of acute-phase reactants in vitro and in vivo, suggesting its involvement in inflammation. Here we report the identification of a class II cytokine receptor designated IL-22RA2 (IL-22 receptor-alpha 2) that appears to be a naturally expressed soluble receptor. IL-22RA2 shares amino acid sequence homology with IL-22RA1 (also known as IL-22R, zcytor11, and CRF2-9) and is physically adjacent to IL-20Ralpha and IFN-gammaR1 on chromosome 6q23.3-24.2. We demonstrate that IL-22RA2 binds specifically to IL-22 and neutralizes IL-22-induced proliferation of BaF3 cells expressing IL-22 receptor subunits. IL-22RA2 mRNA is highly expressed in placenta and spleen by Northern blotting. PCR analysis using RNA from various tissues and cell lines showed that IL-22RA2 was expressed in a range of tissues, including those in the digestive, female reproductive, and immune systems. In situ hybridization revealed the dominant cell types expressing IL-22RA2 were mononuclear cells and epithelium. Because IL-22 induces the expression of acute phase reactants, IL-22RA2 may play an important role as an IL-22 antagonist in the regulation of inflammatory responses.

Annexin V binds to positively selected B cells.

Recombinant annexin V (rAnV) has been used in flow cytometry to identify cells undergoing apoptosis, based on its ability to bind to phosphatidylserine, a negatively charged lipid normally restricted to the cytoplasmic face of the plasma membrane but externalized early during apoptosis. When we stained murine bone marrow (BM) cells with fluorescently labeled rAnV, we found that a surprisingly large fraction of BM B cells bearing selectable transgenic Ag receptors bind significant amounts of rAnV, but that these cells are not apoptotic. Here, we show that binding of rAnV to developing B cells in normal mice correlates with B cell receptor-dependent selection events at several stages of development within both B-1 and B-2 cell subsets. In fact, nearly all B-1 B cells and splenic marginal zone B cells bind rAnV, suggesting that the externalization of phosphatidylserine occurs once mature B cells are selected through BCR-mediated signaling. However, this plasma membrane alteration is apparently not shared by all lymphocytes, because we did not find a parallel population of rAnV-binding viable T cells in vivo in normal or TCR transgenic mice. We also show that BM stromal cell lines can influence the extent of rAnV binding by viable BM B cells during coculture in vitro. We suggest that rAnV detects a potentially important membrane alteration that occurs as B cells develop in the BM and are readied for export to the peripheral lymphoid organs and again among mature B cells recruited to the marginal zone or the B-1 compartment.

Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function.

Cytokines are important in the regulation of haematopoiesis and immune responses, and can influence lymphocyte development. Here we have identified a class I cytokine receptor that is selectively expressed in lymphoid tissues and is capable of signal transduction. The full-length receptor was expressed in BaF3 cells, which created a functional assay for ligand detection and cloning. Conditioned media from activated human CD3+ T cells supported proliferation of the assay cell line. We constructed a complementary DNA expression library from activated human CD3+ T cells, and identified a cytokine with a four-helix-bundle structure using functional cloning. This cytokine is most closely related to IL2 and IL15, and has been designated IL21 with the receptor designated IL21 R. In vitro assays suggest that IL21 has a role in the proliferation and maturation of natural killer (NK) cell populations from bone marrow, in the proliferation of mature B-cell populations co-stimulated with anti-CD40, and in the proliferation of T cells co-stimulated with anti-CD3.

TACI and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease.

B cells are important in the development of autoimmune disorders by mechanisms involving dysregulated polyclonal B-cell activation, production of pathogenic antibodies, and co-stimulation of autoreactive T cells. zTNF4 (BLyS, BAFF, TALL-1, THANK) is a member of the tumour necrosis factor (TNF) ligand family that is a potent co-activator of B cells in vitro and in vivo. Here we identify two receptors for zTNF4 and demonstrate a relationship between zTNF4 and autoimmune disease. Transgenic animals overexpressing zTNF4 in lymphoid cells develop symptoms characteristic of systemic lupus erythaematosus (SLE) and expand a rare population of splenic B-Ia lymphocytes. In addition, circulating zTNF4 is more abundant in NZBWF1 and MRL-lpr/lpr mice during the onset and progression of SLE. We have identified two TNF receptor family members, TACI and BCMA, that bind zTNF4. Treatment of NZBWF1 mice with soluble TACI-Ig fusion protein inhibits the development of proteinuria and prolongs survival of the animals. These findings demonstrate the involvement of zTNF4 and its receptors in the development of SLE and identify TACI-Ig as a promising treatment of autoimmune disease in humans.

Annexin V binds to viable B cells and colocalizes with a marker of lipid rafts upon B cell receptor activation.

Recombinant annexin V (rAnV) has been used to identify apoptotic cells based on its ability to bind phosphatidylserine (PS), a lipid normally restricted to the cytoplasmic face of the plasma membrane, but externalized early during apoptosis. However, this association of rAnV binding and apoptosis is not an obligatory one. We demonstrate that rAnV binds to a large fraction of murine B cells bearing selectable Ag receptors despite the fact that these cells are not apoptotic. Phosphatidylserine, which is uniformly distributed on resting B cells, is mobilized to co-cap with IgM on anti-IgM-treated B cells and to colocalize with GM1, a marker of lipid rafts. Cross-linking PS before anti-IgM treatment sequesters this lipid and alters signaling through IgM. Thus, PS exposed on the majority of B cells in vivo does not reflect early apoptosis, but, instead, plays a role in receptor-mediated signaling events.

Anergic CD8+ T cells can persist and function in vivo.

Using a mouse model system, we demonstrate that anergic CD8+ T cells can persist and retain some functional capabilities in vivo, even after the induction of tolerance. In TCR Vbeta5 transgenic mice, mature CD8+Vbeta5+ T cells transit through a CD8lowVbeta5low deletional intermediate during tolerance induction. CD8low cells are characterized by an activated phenotype, are functionally compromised in vitro, and are slated for deletion in vivo. We now demonstrate that CD8low cells derive from a proliferative compartment, but do not divide in vivo. CD8low cells persist in vivo with a t1/2 of 3-5 days, in contrast to their in vitro t1/2 of 0.5-1 day. During this unexpectedly long in vivo life span, CD8low cells are capable of producing IFN-gamma in vivo despite their inability to proliferate or to kill target cells in vitro. CD8low cells also accumulate at sites of inflammation, where they produce IFN-gamma. Therefore, rather than withdrawing from the pool of functional CD8+ T cells, anergic CD8low cells retain a potential regulatory role despite losing their capacity to proliferate. The ability of anergic cells to persist and function in vivo adds another level of complexity to the process of tolerance induction in the lymphoid periphery.

V(D)J recombinase activity in a subset of germinal center B lymphocytes.

Reexpression of the V(D)J recombinase-activating genes RAG1 and RAG2 in germinal center B cells creates the potential for immunoglobulin gene rearrangement and the generation of new antigen receptor specificities. Intermediate products of V(D)J recombination are abundant in a subset of germinal center B cells, demonstrating that the kappa immunoglobulin light-chain locus becomes a substrate for renewed V(D)J recombinase activity. This recombinationally active cell compartment contains many heavy-chain VDJ rearrangements that encode low-affinity or nonfunctional antibody. In germinal centers, secondary V(D)J recombination may be induced by diminished binding to antigen ligands, thereby limiting abrupt changes in receptor specificity to B cells that are usually eliminated from the germinal center reaction. This restriction preserves efficient antigen-driven selection in germinal centers while allowing for saltations in the somatic evolution of B cells.

A functionally compromised intermediate in extrathymic CD8+ T cell deletion.

We have established a model system for analyzing the induction of self-tolerance among mature peripheral T cells in V beta 5 TCR Tg mice. Both CD4+V beta 5+ and CD8+ V beta 5+ cells undergo a superantigen-driven chronic deletion in the periphery of I-E mice. Prior to their disappearance, CD4+ transgene-expressing cells are activated and then rendered anergic to further stimulation through their TCRs. This scenario differs strikingly in the CD8+ cellular compartment, which is characterized by a distinct population of CD8loV beta 5lo cells localized to the blood and spleen. CD8lo cells are small, express the surface phenotype of memory cells, and rapidly incorporate BrdU in vivo. The kinetics of their appearance and disappearance in adult thymectomized mice, the rapid chasing of BrdU from labeled cells, and their in vivo cortisone sensitivity all suggest CD8lo cells are slated for deletion. Furthermore, their functional incompetence can be documented in vitro in the absence of internucleosomal DNA fragmentation. Thus, we have identified an intermediate population of T cells targeted for peripheral deletion that, although functionally compromised, has not yet undergone programmed cell death.

Thymic selection events mediated by the pre-TCR do not depend upon a limiting ligand.

Thymocyte differentiation requires the production of a functional TCR, the culmination of a carefully orchestrated series of events in which TCR beta chain gene rearrangement precedes that of TCR alpha genes. The product of a successful rearrangement of the TCR beta locus associates with an invariant protein in immature thymocytes to form the 'pre-TCR' complex, which is required for allelic exclusion at the TCR beta locus, the expression of CD4 and CD8 co-receptors, and the clonal expansion of immature thymocytes. The pivotal role for the beta chain protein during early thymocyte development led us to investigate the relative differentiation efficiency within the same thymus of cells which do and cells which do not possess productive TCR gene rearrangements. Using mixed radiation bone marrow chimeras to establish an in vivo competition between TCR beta transgenic (Tg) and non-Tg bone marrow cells, we show that the prior productive rearrangement of a TCR beta chain gene only subtly enhances the efficiency of intrathymic differentiation. Further, we have compared the relative differentiation efficiency of TCR alpha beta and TCR beta Tg cells within the mixed chimera system by altering the proportion of TCR Tg bone marrow cells in the reconstituting inoculum. As expected, Tg cells carrying both alpha and beta chains of a selectable TCR are developmentally hindered compared with their non-Tg counterparts by the lack of ample numbers of intrathymic positively selecting ligands or niches. In contrast, parallel experiments using TCR beta Tg bone marrow cells demonstrate that the early selection events mediated by the pre-TCR do not similarly depend upon a ligand present in limiting quantities.

V beta 5+ T cell receptors skew toward OVA+H-2Kb recognition.

T cells recognize a complex of peptide Ag bound within the groove of MHC-encoded molecules. Although many studies have attempted to correlate TCR gene expression with specificity for particular Ag/MHC combinations, it is still not clear exactly how the TCR physically interacts with its cognate ligand. We have analyzed transgenic mice that carry a rearranged gene encoding a V beta 5.2+ TCR beta-chain derived from the CD8+ CTL clone B3, which is specific for chicken OVA+H-2Kb. Surprisingly, we have found that peripheral lymphocytes isolated from naïve V beta 5.2 transgenic mice can generate a strong primary anti-OVA CTL response when stimulated in vitro with OVA+H-2b, whereas generation of even a weak anti-OVA response from nontransgenic littermates requires in vivo priming. This response is Ag specific, because the transgenic mice are unable to respond with or without priming to vesicular stomatitis virus, which contains a dominant epitope presented in the context of H-2Kb. The precursor frequency of OVA-specific CTL in unprimed V beta 5.2 transgenic mice is approximately 30-fold higher than that in nontransgenic littermate controls. Reverse transcription-PCR analyses demonstrate that OVA-specific CTL lines derived from unprimed V beta 5.2 transgenic mice express a variety of TCR V alpha elements, indicating that the transgenic anti-OVA response is not solely due to the reconstitution of the original B3 TCR. In fact, our data suggest that even a nontransgenic V beta 5+ TCR is intrinsically OVA specific. First, five separate OVA-specific oligoclonal CTL lines derived from individual nontransgenic mice demonstrate dramatic skewing toward expression of V beta 5.1+ or V beta 5.2+ TCR over the course of several in vitro stimulations. Second, sorting for V beta 5+CD8+ nontransgenic cells enriches for OVA-specific CTL. However, peptide antagonism experiments using mutant forms of the Kb-restricted OVA peptide reveal distinct differences between the recognition patterns of two individual OVA-specific CTL lines derived from unprimed V beta 5.2 transgenic mice. These experiments support the notion that a discrete portion of the responding TCR can heavily influence but not necessarily be solely sufficient for the recognition of a peptide Ag presented in the cleft of an MHC-encoded molecule.

Wocko, a neurological mutant generated in a transgenic mouse pedigree.

Naturally occurring mutations involving the nervous system have provided virtually all of our current understanding of the genetic regulation of neural development (Caviness and Rakic, 1978). The difficulty of isolating the corresponding genes, however, has precluded a molecular analysis of these mutants. Insertional mutagenesis, induced by microinjection of DNA into fertilized ova to produce transgenic animals, provides a molecular tag that marks the site of the mutational event. In this article, we describe a transgenic neurological mutation, designated wocko (Wo), which disrupts the development of the inner ear. These mutant mice display a dominant behavioral phenotype that consists of circling, hyperactivity, and head tossing, reminiscent of the shaker/waltzer class of mutants, and they display a recessive homozygous sublethal phenotype. Anatomical analyses showed that both structural and neural components of the vestibular system were disrupted, while analyses of mutant fetuses showed that these morphological abnormalities were due to aberrant development. Although low levels of transgene expression were detected using a sensitive PCR assay, several nonmutant pedigrees that contain the same construct also expressed the transgene in the inner ear, suggesting that low levels of transgene expression alone were not responsible for the wocko phenotype. Because the integrated transgene provides a marker to clone the wocko mutation, the analysis of this mutant will give unique insight into the molecular genetics of inner ear development and into a broad class of neurological mutations that affect the inner ear.

Effectiveness of the dietitian-technician team on a burn unit.

A study was conducted to determine whether the clinical registered dietitian (R.D.) on the burn and trauma unit of an 863-bed medical center was able to perform more efficiently when a part-time dietetic technician (D.T.) was employed and whether the R.D.-D.T. team had an influence on the nutritional status of burn patients. The authors audited a random sample of medical records of burn patients from the year prior to employment of the D.T. (year 1, N = 44) and the year following her employment (year 2, N = 41) to determine the quantity and frequency of nutrition information charted. Results indicated that the percentage of records charted by the R.D. in year 2 increased significantly over the percentage in year 1, as did recommendations for nutrition support. Mean percentage of nutrition recommendations that the R.D. documented for total patient days also increased significantly. Data were insufficient to determine the influence of the R.D.-D.T. team on the nutritional status of patients. With part-time technician assistance, the dietitian had more time to screen and monitor patient records; to plan, implement, and evaluate nutrition care; and to make recommendations for aggressive nutrition support.

Methods of nutritional support for hospitalized patients.

Major components of intravenous nutritional solutions include crystalline amino acids as the protein source, and monohydrous dextrose and lipid emulsions as the energy sources. Intravenous nutrition is indicated in patients without gastrointestinal tract function. Patients with a functioning gastrointestinal tract may be supported enterally by tube feeding. A number of enteral feeding formulas are available, including modular components that may be combined to satisfy specific needs.