Chromothriptic cure of WHIM syndrome.

Chromothripsis is a catastrophic cellular event recently described in cancer in which chromosomes undergo massive deletion and rearrangement. Here, we report a case in which chromothripsis spontaneously cured a patient with WHIM syndrome, an autosomal dominant combined immunodeficiency disease caused by gain-of-function mutation of the chemokine receptor CXCR4. In this patient, deletion of the disease allele, CXCR4(R334X), as well as 163 other genes from one copy of chromosome 2 occurred in a hematopoietic stem cell (HSC) that repopulated the myeloid but not the lymphoid lineage. In competitive mouse bone marrow (BM) transplantation experiments, Cxcr4 haploinsufficiency was sufficient to confer a strong long-term engraftment advantage of donor BM over BM from either wild-type or WHIM syndrome model mice, suggesting a potential mechanism for the patient's cure. Our findings suggest that partial inactivation of CXCR4 may have general utility as a strategy to promote HSC engraftment in transplantation.

CRISPR/Cas9-mediated Cxcr4 disease allele inactivation for gene therapy in a mouse model of WHIM syndrome.

WHIM syndrome is an autosomal dominant immunodeficiency disorder caused by gain-of-function mutations in chemokine receptor CXCR4 that promote severe panleukopenia because of retention of mature leukocytes in the bone marrow (BM). We previously reported that Cxcr4-haploinsufficient (Cxcr4+/o) hematopoietic stem cells (HSCs) have a strong selective advantage for durable hematopoietic reconstitution over wild-type (Cxcr4+/+) and WHIM (Cxcr4+/w) HSCs and that a patient with WHIM was spontaneously cured by chromothriptic deletion of the disease allele in an HSC, suggesting that WHIM allele inactivation through gene editing may be a safe genetic cure strategy for the disease. We have developed a 2-step preclinical protocol of autologous hematopoietic stem and progenitor cell (HSPC) transplantation to achieve this goal. First, 1 copy of Cxcr4 in HSPCs was inactivated in vitro by CRISPR/Cas9 editing with a single guide RNA (sgRNA) that does not discriminate between Cxcr4+/w and Cxcr4+/+ alleles. Then, through in vivo natural selection, WHIM allele-inactivated cells were enriched over wild-type allele-inactivated cells. The WHIM allele-inactivated HSCs retained long-term pluripotency and selective hematopoietic reconstitution advantages. To our knowledge, this is the first example of gene therapy for an autosomal dominant gain-of-function disease using a disease allele inactivation strategy in place of the less efficient disease allele repair approach.

Severe CD8+ T Lymphopenia in WHIM Syndrome Caused by Selective Sequestration in Primary Immune Organs.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is an ultra-rare combined primary immunodeficiency disease caused by heterozygous gain-of-function mutations in the chemokine receptor CXCR4. WHIM patients typically present with recurrent acute infections associated with myelokathexis (severe neutropenia due to bone marrow retention of mature neutrophils). Severe lymphopenia is also common, but the only associated chronic opportunistic pathogen is human papillomavirus and mechanisms are not clearly defined. In this study, we show that WHIM mutations cause more severe CD8 than CD4 lymphopenia in WHIM patients and WHIM model mice. Mechanistic studies in mice revealed selective and WHIM allele dose-dependent accumulation of mature CD8 single-positive cells in thymus in a cell-intrinsic manner due to prolonged intrathymic residence, associated with increased CD8 single-positive thymocyte chemotactic responses in vitro toward the CXCR4 ligand CXCL12. In addition, mature WHIM CD8+ T cells preferentially home to and are retained in the bone marrow in mice in a cell-intrinsic manner. Administration of the specific CXCR4 antagonist AMD3100 (plerixafor) in mice rapidly and transiently corrected T cell lymphopenia and the CD4/CD8 ratio. After lymphocytic choriomeningitis virus infection, we found no difference in memory CD8+ T cell differentiation or viral load between wild-type and WHIM model mice. Thus, lymphopenia in WHIM syndrome may involve severe CXCR4-dependent CD8+ T cell deficiency resulting in part from sequestration in the primary lymphoid organs, thymus, and bone marrow.

Periodontal disease in patients with WHIM syndrome.

AIM: WHIM (warts, hypogammaglobulinaemia, infections and myelokathexis) syndrome is a rare combined primary immunodeficiency disease caused by gain-of-function (GOF) mutations in the chemokine receptor CXCR4 and includes severe neutropenia as a common feature. Neutropenia is a known risk factor for periodontitis; however, a detailed periodontal evaluation of a WHIM syndrome cohort is lacking. This study aimed to establish the evidence base for the periodontal status of patients with WHIM syndrome. MATERIALS AND METHODS: Twenty-two adult WHIM syndrome patients and 22 age- and gender-matched healthy volunteers (HVs) were evaluated through a comprehensive medical and periodontal examination. A mouse model of WHIM syndrome was assessed for susceptibility to naturally progressing or inducible periodontitis. RESULTS: Fourteen patients with WHIM syndrome (63.6%) and one HV (4.5%) were diagnosed with Stage III/IV periodontitis. No WHIM patient presented with the early onset, dramatic clinical phenotypes typically associated with genetic forms of neutropenia. Age, but not the specific CXCR4 mutation or absolute neutrophil count, was associated with periodontitis severity in the WHIM cohort. Mice with a Cxcr4 GOF mutation did not exhibit increased alveolar bone loss in spontaneous or ligature-induced periodontitis. CONCLUSIONS: Overall, WHIM syndrome patients presented with an increased severity of periodontitis despite past and ongoing neutrophil mobilization treatments. GOF mutations in CXCR4 may be a risk factor for periodontitis in humans.

Alterations in the spatiotemporal expression of the chemokine receptor CXCR4 in endothelial cells cause failure of hierarchical vascular branching.

The C-X-C chemokine receptor CXCR4 and its ligand CXCL12 play an important role in organ-specific vascular branching morphogenesis. CXCR4 is preferentially expressed by arterial endothelial cells, and local secretion of CXCL12 determines the organotypic pattern of CXCR4+ arterial branching. Previous loss-of-function studies clearly demonstrated that CXCL12-CXCR4 signaling is necessary for proper arterial branching in the developing organs such as the skin and heart. To further understand the role of CXCL12-CXCR4 signaling in organ-specific vascular development, we generated a mouse model carrying the Cre recombinase-inducible Cxcr4 transgene. Endothelial cell-specific Cxcr4 gain-of-function embryos exhibited defective vascular remodeling and formation of a hierarchical vascular branching network in the developing skin and heart. Ectopic expression of CXCR4 in venous endothelial cells, but not in lymphatic endothelial cells, caused blood-filled, enlarged lymphatic vascular phenotypes, accompanied by edema. These data suggest that CXCR4 expression is tightly regulated in endothelial cells for appropriate vascular development in an organ-specific manner.

Leukocyte chemotactic receptor Fpr1 protects against aging-related posterior subcapsular cataract formation.

Cataracts are a common consequence of aging; however, pathogenesis remains poorly understood. Here, we observed that after 3 months of age mice lacking the G protein-coupled leukocyte chemotactic receptor Fpr1 (N-formyl peptide receptor 1) began to develop bilateral posterior subcapsular cataracts that progressed to lens rupture and severe degeneration, without evidence of either systemic or local ocular infection or inflammation. Consistent with this, Fpr1 was detected in both mouse and human lens in primary lens epithelial cells (LECs), the only cell type present in the lens; however, expression was confined to subcapsular LECs located along the anterior hemispheric surface. To maximize translucency, LECs at the equator proliferate and migrate posteriorly, then differentiate into lens fiber cells by nonclassical apoptotic signaling, which results in loss of nuclei and other organelles, including mitochondria which are a rich source of endogenous N-formyl peptides. In this regard, denucleation and posterior migration of LECs were abnormal in lenses from Fpr1-/- mice, and direct stimulation of LECs with the prototypic N-formyl peptide agonist fMLF promoted apoptosis. Thus, Fpr1 is repurposed beyond its immunoregulatory role in leukocytes to protect against cataract formation and lens degeneration during aging.

A Critical Role of Formyl Peptide Receptors in Host Defense against Escherichia coli.

Formyl peptide receptors (FPRs, mouse Fprs) belong to the G protein-coupled receptor superfamily and mediate phagocyte migration in response to bacteria- and host-derived chemoattractants; however, knowledge about their in vivo roles in bacterial pathogenesis is limited. In this study, we investigated the role of Fpr1 and Fpr2 in host defense against Escherichia coli infection. In vitro, we found that supernatants from E. coli cultures induced chemotaxis of wild-type (WT) mouse bone marrow-derived neutrophils and that the activity was significantly reduced in cells genetically deficient in either Fpr1 or Fpr2 and was almost absent in cells lacking both receptors. Consistent with this, E. coli supernatants induced chemotaxis and MAPK phosphorylation in HEK293 cells expressing either recombinant Fpr1 or Fpr2 but not untransfected parental cells. WT bone marrow -derived neutrophils could actively phagocytose and kill E. coli, whereas both activities were diminished in cells lacking Fpr1 or Fpr2; again, an additive effect was observed in cells lacking both receptors. In vivo, Fpr1 and Fpr2 deficiency resulted in reduced recruitment of neutrophils in the liver and peritoneal cavity of mice infected with inactivated E. coli Moreover, Fpr1-/- and Fpr2-/- mice had significantly increased mortality compared with WT mice after i.p. challenge with a virulent E. coli clinical isolate. These results indicate a critical role of Fprs in host defense against E. coli infection.

Formyl Peptide Receptor-1 Blockade Prevents Receptor Regulation by Mitochondrial Danger-Associated Molecular Patterns and Preserves Neutrophil Function After Trauma.

OBJECTIVES: Trauma predisposes to systemic sterile inflammation (systemic inflammatory response syndrome) as well as infection, but the mechanisms linking injury to infection are poorly understood. Mitochondrial debris contains formyl peptides. These bind formyl peptide receptor-1, trafficking neutrophils to wounds, initiating systemic inflammatory response syndrome, and wound healing. Bacterial formyl peptides, however, also attract neutrophils via formyl peptide receptor-1. Thus, mitochondrial formyl peptides might suppress neutrophils antimicrobial function. Also, formyl peptide receptor-1 blockade used to mitigate systemic inflammatory response syndrome might predispose to sepsis. We examined how mitochondrial formyl peptides impact neutrophils functions contributing to antimicrobial responses and how formyl peptide receptor-1 antagonists affect those functions. DESIGN: Prospective study of human and murine neutrophils and clinical cohort analysis. SETTING: University research laboratory and level 1 trauma center. PATIENTS: Trauma patients, volunteer controls. ANIMAL SUBJECTS: C57Bl/6, formyl peptide receptor-1, and formyl peptide receptor-2 knockout mice. INTERVENTIONS: Human and murine neutrophils functions were activated with autologous mitochondrial debris, mitochondrial formyl peptides, or bacterial formyl peptides followed by chemokines or leukotrienes. The experiments were repeated using formyl peptide receptor-1 antagonist cyclosporin H, "designer" human formyl peptide receptor-1 antagonists (POL7178 and POL7200), or anti-formyl peptide receptor-1 antibodies. Mouse injury/lung infection model was used to evaluate effect of formyl peptide receptor-1 inhibition. MEASUREMENTS AND MAIN RESULTS: Human neutrophils cytosolic calcium, chemotaxis, reactive oxygen species production, and phagocytosis were studied before and after exposure to mitochondrial debris, mitochondrial formyl peptides, and bacterial formyl peptides. Mitochondrial formyl peptide and bacterial formyl peptides had similar effects on neutrophils. Responses to chemokines and leukotrienes were suppressed by prior exposure to formyl peptides. POL7200 and POL7178 were specific antagonists of human formyl peptide receptor-1 and more effective than cyclosporin H or anti-formyl peptide receptor-1 antibodies. Formyl peptides inhibited mouse neutrophils responses to chemokines only if formyl peptide receptor-1 was present. Formyl peptide receptor-1 blockade did not inhibit neutrophils bacterial phagocytosis or reactive oxygen species production. Cyclosporin H increased bacterial clearance in lungs after injury. CONCLUSIONS: Formyl peptides both activate and desensitize neutrophils. Formyl peptide receptor-1 blockade prevents desensitization, potentially both diminishing systemic inflammatory response syndrome and protecting the host against secondary infection after tissue trauma or primary infection.

WHIM Syndrome: from Pathogenesis Towards Personalized Medicine and Cure.

WHIM syndrome is a rare combined primary immunodeficiency disease named by acronym for the diagnostic tetrad of warts, hypogammaglobulinemia, infections, and myelokathexis. Myelokathexis is a unique form of non-cyclic severe congenital neutropenia caused by accumulation of mature and degenerating neutrophils in the bone marrow; monocytopenia and lymphopenia, especially B lymphopenia, also commonly occur. WHIM syndrome is usually caused by autosomal dominant mutations in the G protein-coupled chemokine receptor CXCR4 that impair desensitization, resulting in enhanced and prolonged G protein- and ß-arrestin-dependent responses. Accordingly, CXCR4 antagonists have shown promise as mechanism-based treatments in phase 1 clinical trials. This review is based on analysis of all 105 published cases of WHIM syndrome and covers current concepts, recent advances, unresolved enigmas and controversies, and promising future research directions.

Mechanisms of Sustained Neutrophilia in Patient WHIM-09, Cured of WHIM Syndrome by Chromothripsis.

WHIM-09 is the first patient described with WHIM syndrome, an autosomal dominant form of neutropenia related to bone marrow retention of neutrophils. Originally diagnosed incorrectly with autoimmune neutropenia, the patient underwent splenectomy at age 9, but the absolute neutrophil count (ANC) did not rise. Subsequently, she was spontaneously cured by chromothripsis (chromosome shattering), which deleted the disease allele CXCR4 R334X , and 163 other genes, on chromosome 2 in a single hematopoietic stem cell (HSC). Chromothriptic CXCR4 +/o HSCs replaced CXCR4 +/R334X WHIM HSCs, and the ANC rose to a new sustained and benign baseline ~ 2-3-fold above normal that had remained unexplained. Here, we show that splenectomized Cxcr4 +/o mice had sustained and benign neutrophilia, phenocopying neutrophilia in WHIM-09. In addition, WHIM-09's granulocyte-macrophage precursor cells possessed increased granulocyte colony-forming activity ex vivo. Thus, WHIM-09's neutrophilia may be multifactorial, involving neutrophil-extrinsic factors (splenectomy), as well as CXCR4 haploinsufficiency-dependent neutrophil-intrinsic factors (increased myeloid precursor cell differentiation). The strong bone marrow retention signal for neutrophils conferred by the WHIM mutation may have prevented neutrophilia after splenectomy until the mutation was deleted by chromothripsis.

WHIM Syndrome Caused by Waldenström's Macroglobulinemia-Associated Mutation CXCR4 (L329fs).

WHIM syndrome is an autosomal dominant immunodeficiency disease caused by mutations affecting the carboxy-terminus of CXCR4. To characterize novel genetic causes of the syndrome, we recruited a pediatric patient with possible WHIM syndrome, performed CXCR4 gene sequencing and compared his clinical phenotype and CXCR4 tail amino acid sequences with other patients with WHIM syndrome carrying CXCR4 (R334X) mutations. We identified and biochemically characterized a heterozygous 5 base pair deletion (nucleotides 986-990) located in the portion of the open reading frame (ORF) of CXCR4 that encodes the carboxy-terminal domain of the receptor. This CXCR4 (L329fs) mutation causes a frame-shift at codon 329 resulting in replacement of the final 24 predicted amino acids of the receptor with 12 missense amino acids. Like previously reported WHIM mutations, this frame-shift mutation CXCR4 (L329fs) decreased receptor downregulation in response to the CXCR4 agonist CXCL12 in patient PBMCs as well as in transfected K562 and HEK 293 cells, but increased calcium flux responses in K562 cells to CXCL12 stimulation. Thus, CXCR4 (L329fs) appears to be a de novo autosomal dominant frame-shift gain-of-function mutation that like other carboxy-terminus mutations causes WHIM syndrome. The same CXCR4 (L329fs) frame-shift variant has been reported to occur in tumor cells from a patient with Waldenström's Macroglobulemia (WM), but is caused by a distinct genetic mechanism: insertion of a single nucleotide in the L329 codon, providing additional evidence that the carboxy-terminus of CXCR4 is a genetic hotspot for mutation.

CXCR4 antagonist AMD3100 redistributes leukocytes from primary immune organs to secondary immune organs, lung, and blood in mice.

AMD3100 (plerixafor), is a specific CXCR4 antagonist approved by the FDA for mobilizing hematopoietic stem cells from bone marrow to blood for transplantation in cancer. AMD3100 also mobilizes most mature leukocyte subsets to blood; however, their source and trafficking potential have not been fully delineated. Here, we show that a single injection of AMD3100 10 mg/kg into C57Bl/6 mice rapidly mobilizes (peak ∼ 2.5 h) the same leukocyte subsets to blood as in humans. Using this model, we found that AMD3100 mobilization of neutrophils, lymphocytes, and monocytes to blood is not reduced by splenectomy or by blockade of lymphocyte egress from lymph node with FTY720, but is coupled to (i) reduced content of each of these cell types in the bone marrow; (ii) reduced T-cell numbers in thymuses; (iii) increased lymphocytes in lymph nodes; and (iv) increased neutrophil and monocyte content in the lung. Direct intrathymic labeling showed that AMD3100 selectively mobilizes naïve thymic CD4(+) and CD8(+) T cells to blood. Finally, AMD3100-induced neutrophil mobilization to blood did not reduce neutrophil trafficking to thioglycollate-inflamed peritoneum. Thus, AMD3100 redistributes lymphocytes, monocytes, and neutrophils from primary immune organs to secondary immune organs, peripheral tissues, and blood, without compromising neutrophil trafficking to inflamed sites.

Chemokine receptor Ccr1 drives neutrophil-mediated kidney immunopathology and mortality in invasive candidiasis.

Invasive candidiasis is the 4(th) leading cause of nosocomial bloodstream infection in the US with mortality that exceeds 40% despite administration of antifungal therapy; neutropenia is a major risk factor for poor outcome after invasive candidiasis. In a fatal mouse model of invasive candidiasis that mimics human bloodstream-derived invasive candidiasis, the most highly infected organ is the kidney and neutrophils are the major cellular mediators of host defense; however, factors regulating neutrophil recruitment have not been previously defined. Here we show that mice lacking chemokine receptor Ccr1, which is widely expressed on leukocytes, had selectively impaired accumulation of neutrophils in the kidney limited to the late phase of the time course of the model; surprisingly, this was associated with improved renal function and survival without affecting tissue fungal burden. Consistent with this, neutrophils from wild-type mice in blood and kidney switched from Ccr1(lo) to Ccr1(high) at late time-points post-infection, when Ccr1 ligands were produced at high levels in the kidney and were chemotactic for kidney neutrophils ex vivo. Further, when a 1∶1 mixture of Ccr1(+/+) and Ccr1(-/-) donor neutrophils was adoptively transferred intravenously into Candida-infected Ccr1(+/+) recipient mice, neutrophil trafficking into the kidney was significantly skewed toward Ccr1(+/+) cells. Thus, neutrophil Ccr1 amplifies late renal immunopathology and increases mortality in invasive candidiasis by mediating excessive recruitment of neutrophils from the blood to the target organ.

Regulation of motor function and behavior by atypical chemokine receptor 1.

Atypical Chemokine Receptor 1 (ACKR1), previously known as Duffy Antigen Receptor for Chemokines, stands out among chemokine receptors for high selective expression on cerebellar Purkinje neurons. Although ACKR1 ligands activate Purkinje cells in vitro, evidence for ACKR1 regulation of brain function in vivo is lacking. Here we demonstrate that Ackr1 (-/-) mice have markedly impaired balance and ataxia on a rotating rod and increased tremor when injected with harmaline, which induces whole-body tremor by activating Purkinje cells. Ackr1 (-/-) mice also exhibited impaired exploratory behavior, increased anxiety-like behavior and frequent episodes of marked hypoactivity under low-stress conditions. Surprisingly, Ackr1 (+/-) had similar behavioral abnormalities, indicating pronounced haploinsufficiency. The behavioral phenotype of Ackr1 (-/-) mice was the opposite of mouse models of cerebellar degeneration, and the defects persisted when Ackr1 was deficient only on non-hematopoietic cells. Together, the results suggest that normal motor function and behavior may partly depend on negative regulation of Purkinje cell activity by Ackr1.

[Prospective randomized controlled study on advanced primary hepatic cancer treated by ganfule prescription].

Primary hepatic cancer is one of common malignant tumors. When being diagnosed, most patients were in middle and advanced stage and missed opportunities for surgical treatment. Therefore, chemotherapy and Chinese medicines become the main therapies for advanced primary hepatic cancer. This study was designed to observe the efficacy of Ganfule prescription combined with chemotherapy in treating advanced primary hepatic cancer. In the study, 58 cases of advanced primary hepatic cancer were randomly divided into the treatment group (30 cases) and the control group (28 cases). The treatment group was administered with Ganfule prescription combining with chemotherapy, while the control group was given chemotherapy alone. The tumors progress, quality of life, serum AFP level were evaluated in every three treatment cycles; and the survival rate was followed up for one year. According to the results of this study, after the treatment, there was no statistical significance in the comparison between the two groups in terms of response rate (RR) and disease control rate (DCR) (30.0% vs 25.0%, P = 0.670; 66.7% vs 60.7%, P = 0.637). The improvement rate of KPS score in the treatment group was significantly higher than that of the control group (43.33% vs 21.43%, P < 0.05). The reduction of serum AFP level in the treatment group was more significant than that of the control group (P < 0.05). During the one-year follow-up visit, the survival rate of the treatment group was 26.67%, and the control group was 25.00%, which indicated no statistical significance. This study drew the following conclusion that the oral administration of Ganfule prescription could improve the quality of life of patients of primary hepatic cancer, decrease the serum AFP level and maintain the disease control rate and the one-year survival rate.

A chemokine receptor CXCR2 macromolecular complex regulates neutrophil functions in inflammatory diseases.

Inflammation plays an important role in a wide range of human diseases such as ischemia-reperfusion injury, arteriosclerosis, cystic fibrosis, inflammatory bowel disease, etc. Neutrophilic accumulation in the inflamed tissues is an essential component of normal host defense against infection, but uncontrolled neutrophilic infiltration can cause progressive damage to the tissue epithelium. The CXC chemokine receptor CXCR2 and its specific ligands have been reported to play critical roles in the pathophysiology of various inflammatory diseases. However, it is unclear how CXCR2 is coupled specifically to its downstream signaling molecules and modulates cellular functions of neutrophils. Here we show that the PDZ scaffold protein NHERF1 couples CXCR2 to its downstream effector phospholipase C (PLC)-ß2, forming a macromolecular complex, through a PDZ-based interaction. We assembled a macromolecular complex of CXCR2·NHERF1·PLC-ß2 in vitro, and we also detected such a complex in neutrophils by co-immunoprecipitation. We further observed that the CXCR2-containing macromolecular complex is critical for the CXCR2-mediated intracellular calcium mobilization and the resultant migration and infiltration of neutrophils, as disrupting the complex with a cell permeant CXCR2-specific peptide (containing the PDZ motif) inhibited intracellular calcium mobilization, chemotaxis, and transepithelial migration of neutrophils. Taken together, our data demonstrate a critical role of the PDZ-dependent CXCR2 macromolecular signaling complex in regulating neutrophil functions and suggest that targeting the CXCR2 multiprotein complex may represent a novel therapeutic strategy for certain inflammatory diseases.

The leukocyte chemotactic receptor FPR1 is functionally expressed on human lens epithelial cells.

BACKGROUND: Lens degeneration in Fpr1(-/-) mice prompted us to search for functional FPR1 expression directly on lens epithelial cells. RESULTS: FPR1 is functionally expressed on human lens epithelial cells but has atypical properties compared with hematopoietic cell FPR1. CONCLUSION: Lens epithelial cell FPR1 may be involved in development and maintenance of the lens. SIGNIFICANCE: This is the first link between non-hematopoietic expression of FPR1 and an ophthalmologic phenotype. Formyl peptide receptor 1 (FPR1) is a G protein-coupled chemoattractant receptor expressed mainly on leukocytes. Surprisingly, aging Fpr1(-/-) mice develop spontaneous lens degeneration without inflammation or infection (J.-L. Gao et al., manuscript in preparation). Therefore, we hypothesized that FPR1 is functionally expressed directly on lens epithelial cells, the only cell type in the lens. Consistent with this, the human fetal lens epithelial cell line FHL 124 expressed FPR1 mRNA and was strongly FPR1 protein-positive by Western blot and FACS. Competition binding using FPR1 ligands N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys (Nle = Norleucine), formylmethionylleucylphenylalanine, and peptide W revealed the same profile for FHL 124 cells, neutrophils, and FPR1-transfected HEK 293 cells. Saturation binding with fluorescein-labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys revealed ~2500 specific binding sites on FHL-124 cells (K(D) ~ 0.5 nm) versus ~40,000 sites on neutrophils (K(D) = 3.2 nm). Moreover, formylmethionylleucylphenylalanine induced pertussis toxin-sensitive Ca(2+) flux in FHL 124 cells, consistent with classic G(i)-mediated FPR1 signaling. FHL 124 cell FPR1 was atypical in that it resisted agonist-induced internalization. Expression of FPR1 was additionally supported by detection of the intact full-length open reading frame in sequenced cDNA from FHL 124 cells. Thus, FHL-124 cells express functional FPR1, which is consistent with a direct functional role for FPR1 in the lens, as suggested by the phenotype of Fpr1 knock-out mice.

[Study on inhibitory effects of Taxus chinensis var. mairei aqueous extract on the proliferation of tumor cells].

OBJECTIVE: To study the inhibitory effects of Taxus chinensis var. mairei Aqueous Extract (TAE) on SGC-7901 and MCF-7 cells, and to explore its mechanisms. METHODS: The inhibitory effects of TAT and Paclitaxel on the proliferation of SGC-7901 and MCF-7 cells were tested by MTT method. Their effects on the morphology of SGC-7901 and MCF-7 cells were observed by microscope. Its effects on the cell apoptosis were detected by flow cytometry. RESULTS: The TAE had inhibitory effects on the proliferation of tumor cells, and its mechanisms were correlated to inducing the apoptosis of tumor cells. CONCLUSION: TAE had inhibitory effects on the proliferation of tumor cells.

Clinical and Hematologic Effects of Endotoxin in Warts, Hypogammaglobulinemia, Infections, and Myelokathexis Syndrome Model Mice.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome immunodeficiency is caused by autosomal dominant gain-of-function CXCR4 mutations that promote severe panleukopenia caused by bone marrow retention of mature leukocytes. Consequently, WHIM patients develop recurrent bacterial infections; however, sepsis is uncommon. To study this clinical dichotomy, we challenged WHIM model mice with LPS. The LD50 was similar in WHIM and wild-type (WT) mice, and LPS induced acute lymphopenia in WT mice that was Cxcr4 independent. In contrast, in WHIM mice, LPS did not affect circulating T cell levels, but the B cell levels anomalously increased because of selective, cell-intrinsic, and Cxcr4 WHIM allele-dependent emergence of Cxcr4high late pre-B cells, a pattern that was phenocopied by Escherichia coli infection. In both WT and WHIM mice, the CXCR4 antagonist AMD3100 rapidly increased circulating lymphocyte levels that then rapidly contracted after subsequent LPS treatment. Thus, LPS-induced lymphopenia is CXCR4 independent, and a WHIM mutation does not increase clinical LPS sensitivity. Anomalous WT Cxcr4-independent, but Cxcr4 WHIM-dependent, promobilizing effects of LPS on late pre-B cell mobilization reveal a distinct signaling pathway for the variant receptor.

Chemokine receptor CCR5 promotes leukocyte trafficking to the brain and survival in West Nile virus infection.

The molecular immunopathogenesis of West Nile virus (WNV) infection is poorly understood. Here, we characterize a mouse model for WNV using a subcutaneous route of infection and delineate leukocyte subsets and immunoregulatory factors present in the brains of infected mice. Central nervous system (CNS) expression of the chemokine receptor CCR5 and its ligand CCL5 was prominently up-regulated by WNV, and this was associated with CNS infiltration of CD4+ and CD8+ T cells, NK1.1+ cells and macrophages expressing the receptor. The significance of CCR5 in pathogenesis was established by mortality studies in which infection of CCR5-/- mice was rapidly and uniformly fatal. In the brain, WNV-infected CCR5-/- mice had increased viral burden but markedly reduced NK1.1+ cells, macrophages, and CD4+ and CD8+ T cells compared with WNV-infected CCR5+/+ mice. Adoptive transfer of splenocytes from WNV-infected CCR5+/+ mice into infected CCR5-/- mice increased leukocyte accumulation in the CNS compared with transfer of splenocytes from infected CCR5-/- mice into infected CCR5-/- mice, and increased survival to 60%, the same as in infected CCR5+/+ control mice. We conclude that CCR5 is a critical antiviral and survival determinant in WNV infection of mice that acts by regulating trafficking of leukocytes to the infected brain.