The MAGUK family protein CARD11 is essential for lymphocyte activation

Members of the MAGUK family proteins cluster receptors and intracellular signaling molecules at the neuronal synapse. We report that genetic inactivation of the MAGUK family protein CARD11/Carma1/Bimp3 results in a complete block in T and B cell immunity. CARD11 is essential for antigen receptor- and PKC-mediated proliferation and cytokine production in T and B cells due to a selective defect in JNK and NFkappaB activation. Moreover, B cell proliferation and JNK activation were impaired upon stimulation of TLR4 with lipopolysaccharide, indicating that CARD11 is involved in both the innate and adaptive immune systems. Our results show that the same family of molecules are critical regulators of neuronal synapses and immune receptor signaling.     

Activation of dendritic cells through the interleukin 1 receptor 1 is critical for the induction of autoimmune myocarditis

Dilated cardiomyopathy, resulting from myocarditis, is the most common cause of heart failure in young patients. We here show that interleukin (IL)-1 receptor type 1-deficient (IL-1R1(-/-)) mice are protected from development of autoimmune myocarditis after immunization with alpha-myosin-peptide(614-629). CD4(+) T cells from immunized IL-1R1(-/-) mice proliferated poorly and failed to transfer disease after injection into naive severe combined immunodeficiency (SCID) mice. In vitro stimulation experiments suggested that the function of IL-1R1(-/-)CD4(+) T cells was not intrinsically defect, but their activation by dendritic cells was impaired in IL-1R1(-/-) mice. Accordingly, production of tumor necrosis factor (TNF)-alpha, IL-1, IL-6, and IL-12p70 was reduced in dendritic cells lacking the IL-1 receptor type 1. In fact, injection of immature, antigen-loaded IL-1R1(+/+) but not IL-1R1(-/-) dendritic cells into IL-1R1(-/-) mice fully restored disease susceptibility by rendering IL-1R1(-/-) CD4(+) T cells pathogenic. Thus, IL-1R1 triggering is required for efficient activation of dendritic cells, which is in turn a prerequisite for induction of autoreactive CD4(+) T cells and autoimmunity.     

Lower HER‐2/chromosome enumeration probe 17 ratio in cytologic HER‐2 fluorescence in situ hybridization for breast cancers

BACKGROUND

Fluorescence in situ hybridization (FISH) is the gold standard for assessing HER‐2 status for breast cancers, and paraffin‐embedded tissue sections are used routinely for HER‐2 FISH. Cytologic samples also are used, but to the authors' knowledge, little is known regarding the reliability of these samples. The objective of this study was to elucidate the usefulness of cytologic specimens for HER‐2 FISH testing.

METHODS

Histologic and cytologic specimens from 32 patients with invasive ductal carcinoma of the breast were subjected to comparative analysis of HER‐2 status by FISH. FISH was performed by using a PathVysion HER‐2 DNA Probe Kit according the manufacturer's instructions. The signal ratios of chromosome enumeration probe 17 (CEP17) and HER‐2 were estimated and compared. In 15 cytologic specimens, the distance between signals (HER‐2 and CEP17) and the nearest nuclear membrane were measured by using 3‐dimensional image analysis and confocal microscopy.

RESULTS

Cytologic and histologic FISH results were compared. Signal ratios of HER‐2/CEP17 were lower in cytologic specimens from 26 of 32 patients compared with the histologic material. Three‐dimensional image analysis demonstrated that the distance between the CEP17 signal and the nuclear membrane was shorter than the distance between the HER‐2 gene and the nuclear membrane.

CONCLUSIONS

The current results demonstrated that CEP17 could be lost more easily through histologic sectioning compared with the cytology results, because CEP17 is closer to the nuclear membrane. FISH analysis in cytologic specimens produced more accurate HER‐2/CEP17 ratios, because the whole nucleus was subjected to FISH testing, compared with matched histologic specimens. Cancer (Cancer Cytopathol) 2008. © 2008 American Cancer Society.     

Intrathecal high-dose histamine induces spinally-mediated nociceptive behavioral responses through a polyamine site of NMDA receptors

Previous research has demonstrated that a high dose of histamine (1600 pmol) injected i.t. in mice can evoke nociceptive behaviors consisting of biting/licking along with occasional scratching. The present study was undertaken to examine the involvement of spinal N-methyl-d-aspartate (NMDA) and histamine H(1) and H(2) receptors in the nociceptive behaviors evoked by high-dose histamine. Co-administration of the histamine H(1) receptor antagonists, d-chlorpheniramine and pyrilamine, or the histamine H(2) receptor antagonists, ranitidine and zolantidine, failed to suppress the histamine-evoked nociceptive behaviors. Moreover, following histamine administration, nociceptive behaviors in histamine H(1) receptor-knockout and histamine H(2) receptor-knockout mice were indistinguishable from those in wild-type mice, suggesting that histamine-induced nociceptive behaviors are not mediated through histamine H(1) and H(2) receptors in the spinal cord. The histamine-induced nociceptive behaviors were inhibited by co-administration of the competitive NMDA receptor antagonists, d-(-)-2-amino-5-phosphonovaleric acid (D-APV) and 3-((+)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPPA), and the ion channel blocker, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine maleate (MK-801). Co-administration of ifenprodil, an antagonist for both the polyamine site and the NR2B subunit of NMDA receptors, also inhibited the histamine-induced nociceptive behaviors. (R-[R, S])-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidinepropanol hydrochloride (Ro25-6981), an antagonist of the NMDA receptor subtype containing the NR2B subunit, did not inhibit histamine-induced nociceptive behaviors, whereas these behaviors were attenuated by pretreatment with an antisense oligodeoxynucleotide against the mRNA for the NR1 subunit of the NMDA receptor. Moreover, agmatine and arcaine, antagonists for a polyamine site on the NMDA receptor, inhibited nociceptive behaviors induced by histamine. These results suggest that a polyamine site on spinal NMDA receptors is involved in eliciting the nociceptive behavioral episode following intrathecal injection of histamine.     

Bone marrow stromal cells prepared using AB serum and bFGF for hematopoietic stem cells expansion

BACKGROUND: An ex vivo culture system was previously established for stem cell expansion using human marrow stromal cells and serum-free medium. However, the stromal cells were prepared using long-term culture medium containing horse serum and FCS, which may transmit infectious diseases of xenogeneic origin. In this study, therefore, a method was established to prepare stromal cells using an AB serum-based medium. In the case that serum from a transplant recipient or PBPC donor is available, additional infectious diseases would not be transmitted. STUDY DESIGN AND METHODS: Cord blood CD34+ cells were cultured with thrombopoietin, stem cell factor, and flt3/flk2 ligand on a monolayer of human marrow primary stromal cells prepared using long-term culture medium or AB serum-based medium. After 2 weeks, clonogenic progenitor activity and SCID mouse-reconstituting cell activity were assayed. mRNA expression of cytokines and Notch ligand by stromal cells was also examined. RESULTS: There were no remarkable differences in expansion-supporting activity and mRNA expression between stromal cells established by the two methods. CONCLUSION: An ex vivo expansion system completely based on AB serum has been established.     

Attempt at off-label balloon valvuloplasty post-dilation for intuity sutureless valve

Alternatives to traditional aortic valve replacement now form part of the valve surgeon's armamentarium. Sutureless valves offer decreased bypass and crossclamp times, excellent maneuverability, and promising outcomes. We present a case of a sutureless aortic valve replacement for a late failed David procedure, complicated by postoperative development of severe paravalvular regurgitation. We attempted off-label balloon post-dilation to improve expansion of the valve, however paravalvular regurgitation persisted. The patient underwent subsequent aortic valve replacement using a mechanical valve and experienced no further paravalvular leak.     

Synergistic activity of Card11 mutant and Bcl6 in the development of diffuse large B‐cell lymphoma in a mouse model

Diffuse large B‐cell lymphoma (DLBCL) is the most common subtype of malignant lymphoma; it derives from germinal center B cells. Although DLBCL harbors many genetic alterations, synergistic roles between such alterations in the development of lymphoma are largely undefined. We previously established a mouse model of lymphoma by transplanting gene‐transduced germinal center B cells into mice. Here, we chose one of the frequently mutated genes in DLBCL, Card11 mutant, to explore its possible synergy with other genes, using our lymphoma model. Given that BCL6 and BCL2 expression and/or function are often deregulated in human lymphoma, we examined the possible synergy between Card11, Bcl6, and Bcl2. Germinal center B cells were induced in vitro, transduced with Card11 mutant, Bcl6, and Bcl2, and transplanted. Mice rapidly developed lymphomas, with exogenously transduced Bcl2 being dispensable. Although some mice developed lymphoma in the absence of transduced Bcl6, the absence was compensated by elevated expression of endogenous Bcl6. Additionally, the synergy between Card11 mutant and Bcl6 in the development of lymphoma was confirmed by the fact that the combination of Card11 mutant and Bcl6 caused lymphoma or death significantly earlier and with higher penetrance than Card11 mutant or Bcl6 alone. Lymphoma cells expressed interferon regulatory factor 4 and PR domain 1, indicating their differentiation toward plasmablasts, which characterize activated B cell‐like DLBCL that represents a clinically aggressive subtype in humans. Thus, our mouse model provides a versatile tool for studying the synergistic roles of altered genes underlying lymphoma development.     

New mouse model of acute adult T‐cell leukemia generated by transplantation of AKT,BCLxL, and HBZ‐transduced T cells

Adult T‐cell leukemia/lymphoma (ATL) develops in human T‐cell leukemia virus type 1 (HTLV‐1) carriers. Although the HTLV‐1‐encoded HBZ gene is critically involved, HBZ alone is insufficient and additional, cooperative “hits” are required for the development of ATL. Candidate cooperative hits are being defined, but methods to rapidly explore their roles in ATL development in collaboration with HBZ are lacking. Here, we present a new mouse model of acute type ATL that can be generated rapidly by transplanting in vitro‐induced T cells that have been retrovirally transduced with HBZ and two cooperative genes, BCLxL and AKT, into mice. Co‐transduction of HBZ and BCLxL/AKT allowed these T cells to grow in vitro in the absence of cytokines (Flt3‐ligand and interleukin‐7), which did not occur with any two‐gene combination. Although transplanted T cells were a mixture of cells transduced with different combinations of the genes, tumors that developed in mice were composed of HBZ/BCLxL/AKT triply transduced T cells, showing the synergistic effect of the three genes. The genetic/epigenetic landscape of ATL has only recently been elucidated, and the roles of additional “hits” in ATL pathogenesis remain to be explored. Our model provides a versatile tool to examine the roles of these hits, in collaboration with HBZ, in the development of acute ATL.     

Health Services for Behavioral Problems in Pediatric Primary Care

The aim of this research was to explore primary care pediatricians’ experiences in delivering behavioral health services in their own practices within the Nebraska context. An online survey was sent to the 154 primary care pediatricians who are members of the Nebraska chapter of the American Academy of Pediatrics. Questions explored their management of behavioral problems, attitudes, and perceived barriers to providing behavioral health services in their practices. Seventy pediatricians completed the survey (47%). The majority of pediatricians reported seeing substantial numbers of children with behavioral problems. Eighty-five percent believed that most emotional and behavioral complaints could be managed by the pediatrician. Eighty-eight percent believed that the parents would prefer to receive services for their children’s behavioral problems in the primary care office. Most felt that their training in mental health issues was inadequate. Pediatricians in this survey feel that pediatric behavioral problems are best managed in the primary care office and perceive that parents also prefer this setting. Improving training in behavioral health in pediatrics is necessary to meet the delivery of much needed behavioral health care to children and families.