Alkylation of nucleobases by 2-chloro-N,N-diethylethanamine hydrochloride (CDEAH) sensitizes PARP1-deficient tumors.

Targeting BRCA1- and BRCA2-deficient tumors through synthetic lethality using poly(ADP-ribose) polymerase inhibitors (PARPi) has emerged as a successful strategy for cancer therapy. PARPi monotherapy has shown excellent efficacy and safety profiles in clinical practice but is limited by the need for tumor genome mutations in BRCA or other homologous recombination genes as well as the rapid emergence of resistance. In this study, we identified 2-chloro-N,N-diethylethanamine hydrochloride (CDEAH) as a small molecule that selectively kills PARP1- and xeroderma pigmentosum A-deficient cells. CDEAH is a monofunctional alkylating agent that preferentially alkylates guanine nucleobases, forming DNA adducts that can be removed from DNA by either a PARP1-dependent base excision repair or nucleotide excision repair. Treatment of PARP1-deficient cells leads to the formation of strand breaks, an accumulation of cells in S phase and activation of the DNA damage response. Furthermore, CDEAH selectively inhibits PARP1-deficient xenograft tumor growth compared to isogenic PARP1-proficient tumors. Collectively, we report the discovery of an alkylating agent inducing DNA damage that requires PARP1 activity for repair and acts synergistically with PARPi.

MSH2-MSH3 promotes DNA end resection during homologous recombination and blocks polymerase theta-mediated end-joining through interaction with SMARCAD1 and EXO1.

DNA double-strand break (DSB) repair via homologous recombination is initiated by end resection. The extent of DNA end resection determines the choice of the DSB repair pathway. Nucleases for end resection have been extensively studied. However, it is still unclear how the potential DNA structures generated by the initial short resection by MRE11-RAD50-NBS1 are recognized and recruit proteins, such as EXO1, to DSB sites to facilitate long-range resection. We found that the MSH2-MSH3 mismatch repair complex is recruited to DSB sites through interaction with the chromatin remodeling protein SMARCAD1. MSH2-MSH3 facilitates the recruitment of EXO1 for long-range resection and enhances its enzymatic activity. MSH2-MSH3 also inhibits access of POLθ, which promotes polymerase theta-mediated end-joining (TMEJ). Collectively, we present a direct role of MSH2-MSH3 in the initial stages of DSB repair by promoting end resection and influencing the DSB repair pathway by favoring homologous recombination over TMEJ.

Regulation of BRCA1 stability through the tandem UBX domains of isoleucyl-tRNA synthetase 1.

Aminoacyl-tRNA synthetases (ARSs) have evolved to acquire various additional domains. These domains allow ARSs to communicate with other cellular proteins in order to promote non-translational functions. Vertebrate cytoplasmic isoleucyl-tRNA synthetases (IARS1s) have an uncharacterized unique domain, UNE-I. Here, we present the crystal structure of the chicken IARS1 UNE-I complexed with glutamyl-tRNA synthetase 1 (EARS1). UNE-I consists of tandem ubiquitin regulatory X (UBX) domains that interact with a distinct hairpin loop on EARS1 and protect its neighboring proteins in the multi-synthetase complex from degradation. Phosphomimetic mutation of the two serine residues in the hairpin loop releases IARS1 from the complex. IARS1 interacts with BRCA1 in the nucleus, regulates its stability by inhibiting ubiquitylation via the UBX domains, and controls DNA repair function.

Effects of early postoperative shower after cardiac surgery.

BACKGROUND: Little is known regarding the effects of early showers after cardiac surgery. We evaluated the influence of early showers on postoperative wound complications following cardiac surgery. METHODS: This was a prospective observational study of 100 cardiac surgery patients (mean age, 63.0±13.5 years) who underwent early postoperative showers from September 2020 to March 2021 at our institution. Postoperative showers were initiated after the drain was removed. Postoperative wound complications were examined and patient satisfaction was evaluated using questionnaires. RESULTS: Surgery was performed through sternotomy in 48 patients (48.0%) and through minimally invasive approaches (right or left mini-thoracotomy) in 52 patients (52.0%). The mean time from surgery to shower was 6.0±1.4 days. No wound dehiscence, superficial wound infection, or deep wound infection was observed. Questionnaires showed that more than 50% of patients thought they were not allowed to shower until more than 2 weeks after the operation. Patient satisfaction score was 7.4±2.3 out of 10 for early showers after cardiac surgery. CONCLUSIONS: Our study suggests that postoperative early showers after cardiac surgery are not associated with an increased risk of wound complications. Patient satisfaction was also high. Early postoperative showering can be considered after cardiac surgery.

Precision targeting tumor cells using cancer-specific InDel mutations with CRISPR-Cas9.

An ideal cancer therapeutic strategy involves the selective killing of cancer cells without affecting the surrounding normal cells. However, researchers have failed to develop such methods for achieving selective cancer cell death because of shared features between cancerous and normal cells. In this study, we have developed a therapeutic strategy called the cancer-specific insertions-deletions (InDels) attacker (CINDELA) to selectively induce cancer cell death using the CRISPR-Cas system. CINDELA utilizes a previously unexplored idea of introducing CRISPR-mediated DNA double-strand breaks (DSBs) in a cancer-specific fashion to facilitate specific cell death. In particular, CINDELA targets multiple InDels with CRISPR-Cas9 to produce many DNA DSBs that result in cancer-specific cell death. As a proof of concept, we demonstrate here that CINDELA selectively kills human cancer cell lines, xenograft human tumors in mice, patient-derived glioblastoma, and lung patient-driven xenograft tumors without affecting healthy human cells or altering mouse growth.

TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution.

R-loops are three-stranded, RNA-DNA hybrid, nucleic acid structures produced due to inappropriate processing of newly transcribed RNA or transcription-replication collision (TRC). Although R-loops are important for many cellular processes, their accumulation causes genomic instability and malignant diseases, so these structures are tightly regulated. It was recently reported that R-loop accumulation is resolved by methyltransferase-like 3 (METTL3)-mediated m6A RNA methylation under physiological conditions. However, it remains unclear how R-loops in the genome are recognized and induce resolution signals. Here, we demonstrate that tonicity-responsive enhancer binding protein (TonEBP) recognizes R-loops generated by DNA damaging agents such as ultraviolet (UV) or camptothecin (CPT). Single-molecule imaging and biochemical assays reveal that TonEBP preferentially binds a R-loop via both 3D collision and 1D diffusion along DNA in vitro. In addition, we find that TonEBP recruits METTL3 to R-loops through the Rel homology domain (RHD) for m6A RNA methylation. We also show that TonEBP recruits RNaseH1 to R-loops through a METTL3 interaction. Consistent with this, TonEBP or METTL3 depletion increases R-loops and reduces cell survival in the presence of UV or CPT. Collectively, our results reveal an R-loop resolution pathway by TonEBP and m6A RNA methylation by METTL3 and provide new insights into R-loop resolution processes.

SDE2 integrates into the TIMELESS-TIPIN complex to protect stalled replication forks.

Protecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances its stability, thereby aiding TIM localization to replication forks and the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.

Effects of Intraoperative Low-Dose Ketamine on Persistent Postsurgical Pain after Breast Cancer Surgery: A Prospective, Randomized, Controlled, Double-Blind Study.

BACKGROUND: Compared to acute postsurgical pain, studies regarding the role of ketamine in persistent postsurgical pain (PPSP) are limited. OBJECTIVES: The aim of this clinical trial was to test if intraoperative low-dose ketamine without postoperative infusion would reduce PPSP development after breast cancer surgery. STUDY DESIGN: We used a randomized, double-blinded, placebo study design. SETTING: This study was conducted at Pusan National University Hospital, Republic of Korea, between December 2013 and August 2016. METHODS: A total of 184 patients scheduled for breast cancer surgery were randomly assigned to either the control or ketamine group. Before skin incision, a bolus (0.5 mg/kg of ketamine or placebo), followed by a continuous infusion (0.12 mg/kg/h of ketamine or placebo), was administered until the end of the surgery. The patients were interviewed via telephone 1, 3, and 6 months after surgery. The first question was whether the patient had surgery-related pain. If answered affirmatively, questions from the Numeric Rating Scale for pain at rest (NRSr) and for coughing (NRSd) were also asked. Our primary outcome was the incidence of PPSP at 3 months after surgery. RESULTS: For PPSP analysis, 168 patients were included. The number of patients who experienced pain was significantly lower in the ketamine group at 3 months (86.9% in the control group vs 69.0% in the ketamine group, P = .005) postoperatively. However, the NRSr and NRSd did not differ between the groups throughout the follow-up. LIMITATIONS: There were no postoperative low-dose ketamine infusion groups to compare due to hospital regulations. Dosage of ketamine was too low to reduce the severity of PPSP. And by using propofol and remifentanil for anesthesia, different results can be deduced with volatile anesthetics. Data from written questionnaires would have been more specific than telephone interviews for long-term assessment. CONCLUSIONS: Though intraoperative low-dose ketamine without postoperative infusion significantly reduced the incidence of PPSP up to 3 months after breast cancer surgery, it failed to reduce clinically significant PPSP and improve patients' quality of life. KEY WORDS: Analgesia, breast cancer, chronic pain, ketamine, mastectomy, morphine, pain, postoperative, propofol.

Functional cross talk between the Fanconi anemia and ATRX/DAXX histone chaperone pathways promotes replication fork recovery.

Fanconi anemia (FA) is a chromosome instability syndrome characterized by increased cancer predisposition. Specifically, the FA pathway functions to protect genome stability during DNA replication. The central FA pathway protein, FANCD2, locates to stalled replication forks and recruits homologous recombination (HR) factors such as CtBP interacting protein (CtIP) to promote replication fork restart while suppressing new origin firing. Here, we identify alpha-thalassemia retardation syndrome X-linked (ATRX) as a novel physical and functional interaction partner of FANCD2. ATRX is a chromatin remodeler that forms a complex with Death domain-associated protein 6 (DAXX) to deposit the histone variant H3.3 into specific genomic regions. Intriguingly, ATRX was recently implicated in replication fork recovery; however, the underlying mechanism(s) remained incompletely understood. Our findings demonstrate that ATRX forms a constitutive protein complex with FANCD2 and protects FANCD2 from proteasomal degradation. ATRX and FANCD2 localize to stalled replication forks where they cooperate to recruit CtIP and promote MRE11 exonuclease-dependent fork restart while suppressing the firing of new replication origins. Remarkably, replication restart requires the concerted histone H3 chaperone activities of ATRX/DAXX and FANCD2, demonstrating that coordinated histone H3 variant deposition is a crucial event during the reinitiation of replicative DNA synthesis. Lastly, ATRX also cooperates with FANCD2 to promote the HR-dependent repair of directly induced DNA double-stranded breaks. We propose that ATRX is a novel functional partner of FANCD2 to promote histone deposition-dependent HR mechanisms in S-phase.

ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress.

Maintaining stability of replication forks is important for genomic integrity. However, it is not clear how replisome proteins contribute to fork stability under replication stress. Here, we report that ATAD5, a PCNA unloader, plays multiple functions at stalled forks including promoting its restart. ATAD5 depletion increases genomic instability upon hydroxyurea treatment in cultured cells and mice. ATAD5 recruits RAD51 to stalled forks in an ATR kinase-dependent manner by hydroxyurea-enhanced protein-protein interactions and timely removes PCNA from stalled forks for RAD51 recruitment. Consistent with the role of RAD51 in fork regression, ATAD5 depletion inhibits slowdown of fork progression and native 5-bromo-2'-deoxyuridine signal induced by hydroxyurea. Single-molecule FRET showed that PCNA itself acts as a mechanical barrier to fork regression. Consequently, DNA breaks required for fork restart are reduced by ATAD5 depletion. Collectively, our results suggest an important role of ATAD5 in maintaining genome integrity during replication stress.

Hypomorphic Mutations in TONSL Cause SPONASTRIME Dysplasia.

SPONASTRIME dysplasia is a rare, recessive skeletal dysplasia characterized by short stature, facial dysmorphism, and aberrant radiographic findings of the spine and long bone metaphysis. No causative genetic alterations for SPONASTRIME dysplasia have yet been determined. Using whole-exome sequencing (WES), we identified bi-allelic TONSL mutations in 10 of 13 individuals with SPONASTRIME dysplasia. TONSL is a multi-domain scaffold protein that interacts with DNA replication and repair factors and which plays critical roles in resistance to replication stress and the maintenance of genome integrity. We show here that cellular defects in dermal fibroblasts from affected individuals are complemented by the expression of wild-type TONSL. In addition, in vitro cell-based assays and in silico analyses of TONSL structure support the pathogenicity of those TONSL variants. Intriguingly, a knock-in (KI) Tonsl mouse model leads to embryonic lethality, implying the physiological importance of TONSL. Overall, these findings indicate that genetic variants resulting in reduced function of TONSL cause SPONASTRIME dysplasia and highlight the importance of TONSL in embryonic development and postnatal growth.

FANCI and FANCD2 have common as well as independent functions during the cellular replication stress response.

Fanconi anemia (FA) is an inherited cancer predisposition syndrome characterized by cellular hypersensitivity to DNA interstrand crosslinks (ICLs). To repair these lesions, the FA proteins act in a linear hierarchy: following ICL detection on chromatin, the FA core complex monoubiquitinates and recruits the central FANCI and FANCD2 proteins that subsequently coordinate ICL removal and repair of the ensuing DNA double-stranded break by homology-dependent repair (HDR). FANCD2 also functions during the replication stress response by mediating the restart of temporarily stalled replication forks thereby suppressing the firing of new replication origins. To address if FANCI is also involved in these FANCD2-dependent mechanisms, we generated isogenic FANCI-, FANCD2- and FANCI:FANCD2 double-null cells. We show that FANCI and FANCD2 are partially independent regarding their protein stability, nuclear localization and chromatin recruitment and contribute independently to cellular proliferation. Simultaneously, FANCD2-but not FANCI-plays a major role in HDR-mediated replication restart and in suppressing new origin firing. Consistent with this observation, deficiencies in HDR-mediated DNA DSB repair can be overcome by stabilizing RAD51 filament formation in cells lacking functional FANCD2. We propose that FANCI and FANCD2 have partially non-overlapping and possibly even opposing roles during the replication stress response.

Airway management in a patient with severe tracheal stenosis: bilateral superficial cervical plexus block with dexmedetomidine sedation.

A 54-year-old woman with a history of severe tracheal stenosis caused by papillary thyroid cancer with tracheal invasion was admitted for an elective surgery. A bilateral superficial cervical plexus block with 0.5 % ropivacaine 14 ml (7 ml per side) under dexmedetomidine sedation was performed, followed by tracheal dissection and endotracheal tube (ETT) insertion. The patient continued spontaneous respiration without any hypoxic event, and the bispectral index was maintained at a range of 50-80. After ETT insertion, a total thyroidectomy and tracheal resection with end-to-end anastomosis were performed under general anesthesia. The patient was transferred to the surgical intensive care unit after extubation, and she recovered without any complications.

A novel mouse PKCδ splice variant, PKCδIX, inhibits etoposide-induced apoptosis.

Protein kinase C (PKC) δ plays an important role in cellular proliferation and apoptosis. The catalytic fragment of PKCδ generated by caspase-dependent cleavage is essential for the initiation of etoposide-induced apoptosis. In this study, we identified a novel mouse PKCδ isoform named PKCδIX (Genebank Accession No. HQ840432). PKCδIX is generated by alternative splicing and is ubiquitously expressed, as seen in its full-length PKCδ. PKCδIX lacks the C1 domain, the caspase 3 cleavage site, and the ATP binding site but preserves an almost intact c-terminal catalytic domain and a nuclear localization signal (NLS). The structural characteristics of PKCδIX provided a possibility that this PKCδ isozyme functions as a novel dominant-negative form for PKCδ due to its lack of the ATP-binding domain that is required for the kinase activity of PKCδ. Indeed, overexpression of PKCδIX significantly inhibited etoposide-induced apoptosis in NIH3T3 cells. In addition, an in vitro kinase assay showed that recombinant PKCδIX protein could competitively inhibit the kinase activity of PKCδ. We conclude that PKCδIX can function as a natural dominant-negative inhibitor of PKCδin vivo.

Stimulation of the molecule 4-1BB enhances host defense against Listeria monocytogenes infection in mice by inducing rapid infiltration and activation of neutrophils and monocytes.

The tumor necrosis factor receptor family molecule 4-1BB (CD137) has diverse roles in adaptive and innate immune responses. However, little is known of its role in bacterial infections. Previously, we showed that 4-1BB-deficient mice have enhanced susceptibility to Listeria monocytogenes infection, and mice pretreated with agonistic anti-4-1BB antibody (3E1) were much more resistant to L. monocytogenes infection than mice treated with control antibody. In this study, we report that stimulating 4-1BB by administering 3E1 in the early phase of L. monocytogenes infection is critical for promoting the survival of mice by inducing rapid infiltration of neutrophils and monocytes into L. monocytogenes-infected livers. The levels of tumor necrosis factor alpha, interleukin 6, and monocyte chemoattractant protein 1 in the livers of 3E1-treated mice increased as early as 30 min postinfection and peaked by 1 to 2 h, while those in mice treated with control antibody started to increase only at 16 h postinfection. Monocytes and neutrophils from the 3E1-treated mice had higher levels of activation markers, phagocytic activity, and reactive oxygen species than those from control mice. In vitro stimulation of 4-1BB induced the production of the inflammatory cytokines/chemokines of neutrophils, but not those of monocytes. These results suggest that 4-1BB stimulation of neutrophils in the early phase of L. monocytogenes infection causes rapid production of inflammatory cytokines/chemokines and that the subsequent infiltration of neutrophils and monocytes is crucial for eliminating the infecting L. monocytogenes.

Pre-operative predictive factors for gallbladder cholesterol polyps using conventional diagnostic imaging.

AIM: To determine the clinical data that might be useful for differentiating benign from malignant gallbladder (GB) polyps by comparing radiological methods, including abdominal ultrasonography (US) and computed tomography (CT) scanning, with postoperative pathology findings. METHODS: Fifty-nine patients underwent laparoscopic cholecystectomy for a GB polyp of around 10 mm. They were divided into two groups, one with cholesterol polyps and the other with non-cholesterol polyps. Clinical features such as gender, age, symptoms, size and number of polyps, the presence of a GB stone, the radiologically measured maximum diameter of the polyp by US and CT scanning, and the measurements of diameter from postoperative pathology were recorded for comparative analysis. RESULTS: Fifteen of the 41 cases with cholesterol polyps (36.6%) were detected with US but not CT scanning, whereas all 18 non-cholesterol polyps were observed using both methods. In the cholesterol polyp group, the maximum measured diameter of the polyp was smaller by CT scan than by US. Consequently, the discrepancy between those two scanning measurements was greater than for the non-cholesterol polyp group. CONCLUSION: The clinical signs indicative of a cholesterol polyp include: (1) a polyp observed by US but not observable by CT scanning, (2) a smaller diameter on the CT scan compared to US, and (3) a discrepancy in its maximum diameter between US and CT measurements. In addition, US and the CT scan had low accuracy in predicting the polyp diameter compared to that determined by postoperative pathology.

Stimulation of osteoclastogenesis by enhanced levels of MIP-1alpha in BALB/c mice in vitro.

OBJECTIVES: We compared osteoclast (OC) formation in bone marrow-derived macrophages (BMM) from C57BL/6 (B/6) and BALB/c (B/c) mice. After stimulation of receptor activator of nuclear factor-kappaB ligand (RANKL), enhanced OC formation and higher level of macrophage inflammatory protein-1alpha (MIP-1alpha) were observed in the BMM from B/c mice. In this study, we determined whether MIP-1alpha is responsible for stimulated OC formation in the BMM. MATERIALS AND METHODS: OC formation was evaluated in BMM. Expression of MIP-1alpha during OC formation was analyzed at the mRNA and protein levels. Apoptosis of mature OCs was evaluated by observing the degradation of DNA. Activation of nuclear factor-kappaB (NF-kappaB) was measured by electrophoretic mobility shift assay. RESULTS: After stimulation by RANKL expression of MIP-1alpha at the mRNA and protein levels was much higher in BMM from B/c mice than in BMM from B/6 mice. Transcripts of the MIP-1alpha receptors, CCR1 and CCR5, were present at similar levels in unstimulated BMM of the two strains. Blockade of MIP-1alpha inhibited OC formation, and exogenously added MIP-1alpha stimulated it in RANKL-stimulated BMM. MIP-1alpha affected not only the early precursors but also mature OCs. It prevented apoptosis of mature OCs by activating NF-kappaB, and the effect of RANKL on survival was dependent on its ability to induce MIP-1alpha. CONCLUSIONS: MIP-1alpha, induced by RANKL during OC differentiation, increases OC formation by acting on OC progenitor cells, and prolongs survival of mature OC via signaling through NF-kappaB. The enhanced OC formation in BMM from B/c mice could be due to, at least in part, to their higher levels of MIP-1alpha.

Enhanced osteoclastogenesis in 4-1BB-deficient mice caused by reduced interleukin-10.

UNLABELLED: Enhanced osteoclastogenesis was observed in bone marrow-derived macrophage cells from 4-1BB-deficient mice than in those from wildtype mice. 4-1BB and 4-1BB ligand interaction may play a role at a certain stage of osteoclast formation through increased level of IL-10, a negative regulator of osteoclastogenesis. INTRODUCTION: 4-1BB is an inducible T-cell costimulatory molecule and a member of the TNF receptor family. The expression pattern of 4-1BB and 4-1BB ligand (4-1BBL) has suggested that 4-1BB plays a role not only in various responses related to innate immunity but also in bone metabolism. MATERIALS AND METHODS: Osteoclast formation was evaluated in bone marrow-derived macrophage cells (BMMs) from wildtype and 4-1BB-deficient (4-1BB-/-) mice. Expression of interleukin-10 (IL-10) during osteoclast formation was analyzed at the mRNA and protein levels. RESULTS: Expression of IL-10 was higher in RANKL-stimulated wildtype BMMs than 4-1BB-/- BMMs. When 4-1BBL was stimulated with 4-1BB-Fc fusion protein, the expression of IL-10 in BMMs increased. Neutralization of IL-10 was not as effective in preventing inhibition by IL-10 of osteoclast differentiation in 4-1BB-/- BMMs as in wildtype BMMs. When IL-10 was added to the culture medium, osteoclast formation was inhibited more efficiently in the 4-1BB-/- BMMs than in the wildtype BMMs. CONCLUSIONS: Interaction of 4-1BB and 4-1BBL stimulates IL-10 production through 4-1BBL signaling. 4-1BBL plays a role at a certain stage of osteoclast formation, and IL-10 may mediate this effect. The elevated level of osteoclastogenesis in 4-1BB-/- BMMs may thus be caused, in part, by a lower level of IL-10.

A signal through 4-1BB ligand inhibits receptor for activation of nuclear factor-kappaB ligand (RANKL)-induced osteoclastogenesis by increasing interferon (IFN)-beta production.

We tested whether any intracellular signals are transmitted through 4-1BB/CD137 ligand (4-1BBL), using a 4-1BB-Fc fusion protein and 4-1BB-deficient mice. Immobilized 4-1BB-Fc fusion protein strongly inhibited osteoclastogenesis induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB ligand (RANKL) derived from bone marrow macrophages (BMM). Incubation of BMM with M-CSF increased 4-1BBL mRNA and surface expression of 4-1BBL protein. Cross-linking 4-1BBL with immobilized 4-1BB-Fc also dramatically reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNC) derived from the BMM from 4-1BB-deficient mice, suggesting that the inhibitory effect of immobilized 4-1BB on osteoclastogenesis is due to a signal through 4-1BBL. Reverse signaling by 4-1BB-Fc increased the level of interferon (IFN)-beta in BMM and neutralization of IFN-beta reversed the inhibitory effect of immobilized 4-1BB-Fc. Inhibition of osteoclastogenesis by immobilized 4-1BB-Fc is, therefore, at least in part, due to elevation of the level of the negative regulator, IFN-beta in BMM.

4-1BB (CD137) signals depend upon CD28 signals in alloimmune responses.

Our previous study has demonstrated that there is a significant delay of Balb/c cardiac allograft rejection in the C57BL/6 4-1BB-deficient knockout recipient. In this study, we examined the effect of combined blockade of the 4-1BB and CD28 costimulatory pathways on cardiac allograft rejection in the C57BL/6-->Balb/c model. A long-term cardiac allograft survival was induced in CD28/4-1BB- deficient mice (>100 days survival in 3 of 4 mice), which was comparable with CD28-deficient mice (>100 days survival in 2 of 5 mice; P<0.2026). There was no long-term cardiac allograft survival in either wild-type (WT) or 4-1BB-deficient mice, even though 4-1BB-deficient recipients showed a significant delay of cardiac allograft rejection than WT mice. An in vitro mixed leukocyte reaction (MLR) assay showed that 4-1BB-deficient and WT mouse T cells had a similar responsiveness to allostimulation, whereas CD28- and CD28/4-1BB-deficient mouse T cells had a defective responsiveness to allostimulation. Furthermore, 4-1BB-deficient mice showed a similar CTL but an elevated Ab response against alloantigens as compared to WT mice, and the alloimmune responses of 4-1BB-deficient mice were abrogated in the CD28-deficient background. Overall, these results indicate that the CD28 costimulatory pathway plays a major role in the alloimmune response and that 4-1BB signals are dependent upon CD28 signals.