Hanna Wald

Involvement of the CXCL12/CXCR4 pathway in the advanced liver disease that is associated with hepatitis C virus or hepatitis B virus

Chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) infection is accompanied by inflammation and fibrosis eventually leading to cirrhosis. The chemokine CXCL12 is involved in chronic inflammatory conditions. The role of the CXCL12/CXCR4 pathway in HCV‐ and HBV‐associated liver inflammation and fibrosis was therefore studied. The levels and tissue localization of CXCL12 in liver and plasma of HCV and HBV patients were tested using immunohistochemistry and ELISA. The expression and function of CXCR4 on liver‐infiltrating lymphocytes (LIL) were tested by FACS and transwell migration assays. We found that CXCL12 is expressed by bile duct epithelial cells in normal liver tissue. Bile duct proliferation and liver fibrosis in chronic HCV and HBV infection result in the anatomical re‐distribution of CXCL12 in the liver. Moreover, CXCL12 is up‐regulated in the endothelium of neo‐blood‐vessels formed in active inflammatory foci and is significantly elevated, compared with controls, in the plasma of patients with advanced liver fibrosis. Complementing these observations were others indicating that over 50% of LIL express CXCR4 and, in response to CXCL12, migrated and adhered to fibronectin. These observations suggest an important role for the CXCL12/CXCR4 pathway in recruitment and retention of immune cells in the liver during chronic HCV and HBV infection.

Enhanced Unique Pattern of Hematopoietic Cell Mobilization Induced by the CXCR4 Antagonist 4F‐Benzoyl‐TN14003

An increase in the number of stem cells in blood following mobilization is required to enhance engraftment after high‐dose chemotherapy and improve transplantation outcome. Therefore, an approach that improves stem cell mobilization is essential. The interaction between CXCL12 and its receptor, CXCR4, is involved in the retention of stem cells in the bone marrow. Therefore, blocking CXCR4 may result in mobilization of hematopoietic progenitor and stem cells. We have found that the CXCR4 antagonist known as 4F‐benzoyl‐TN14003 (T‐140) can induce mobilization of hematopoietic stem cells and progenitors within a few hours post‐treatment in a dose‐dependent manner. Furthermore, although T‐140 can also increase the number of white blood cells (WBC) in blood, including monocytes, B cells, and T cells, it had no effect on mobilizing natural killer cells. T‐140 was found to efficiently synergize with granulocyte colony‐stimulating factor (G‐CSF) in its ability to mobilize WBC and progenitors, as well as to induce a 660‐fold increase in the number of erythroblasts in peripheral blood. Comparison between the CXCR4 antagonists T‐140 and AMD3100 showed that T‐140 with or without G‐CSF was significantly more potent in its ability to mobilize hematopoietic stem cells and progenitors into blood. These results demonstrate that different CXCR4 antagonists may have different therapeutic potentials.

IFN-γ Acts on T Cells to Induce NK Cell Mobilization and Accumulation in Target Organs

The mechanism(s) that regulates NK cell mobilization and the significance of this process to NK cell activity are unknown. After Con A-induced hepatitis, NK cells are mobilized from the spleen and bone marrow into the periphery in an IFN-γ-dependent fashion. Intraperitoneal administration of IFN-γ stimulates the mobilization of NK cells into the circulation, but not their cell death or proliferation. Increased number of circulating NK cells was coupled with their accumulation in the peritoneum, liver, and tumor-bearing lung tissue. Furthermore, increased number of NK cells in the lung reduced metastasis of Lewis lung carcinoma cells (3LL cell line) resulting in significantly extended NK-dependent survival. Mobilization of NK cells was specific and required the presence of T cells. Moreover, mobilization and migration of spleen NK cells in response to IFN-γ treatment is dependent on the chemokine receptor CXCR3. Mechanistic insights regarding the role of IFN-γ in the regulation of NK cell mobilization and their accumulation at sites of tumor metastasis may lead to the development of novel immunotherapy for cancer.

Differential regulation of ROMK expression in kidney cortex and medulla by aldosterone and potassium

This study explores the role of K+ and aldosterone in the regulation of mRNA of the ATP-sensitive, inwardly rectifying K+ channel, ROMK, in the rat kidney. K+ deficiency downregulated ROMK mRNA in cortex to 47.1 +/- 5.1% of control (P < 0.001) and in medulla to 56.1 +/- 3. 4% (P < 0.001). High-K+ diet slightly increased ROMK mRNA in medulla to 122 +/- 9% (P < 0.05 vs. control). Adrenalectomy (Adx) downregulated cortical ROMK mRNA to 30.7 +/- 6.8% (P < 0.001 vs. control), and increased it in medulla to 138 +/- 12.9% (P < 0.02 vs. control). In Adx rats, K+ deficiency decreased ROMK mRNA in cortex and medulla similar to intact rats. The alpha1- and beta1-Na-K-ATPase subunits were regulated in parallel to that of ROMK. In medulla, ROMK mRNA correlated with serum K+ concentration at R = 0.9406 (n = 6, P < 0.001) and alpha1-Na-K-ATPase mRNA at R = 0.9756 (n = 6, P < 0.001). ROMK2 also correlated with serum K+ concentration (R = 0.895; n = 6, P < 0.01). These results show that cortical ROMK expression is regulated by aldosterone and K+, whereas the medullary ROMK mRNA is regulated by serum K+.This study explores the role of K+ and aldosterone in the regulation of mRNA of the ATP-sensitive, inwardly rectifying K+ channel, ROMK, in the rat kidney. K+ deficiency downregulated ROMK mRNA in cortex to 47.1 ± 5.1% of control ( P < 0.001) and in medulla to 56.1 ± 3.4% ( P < 0.001). High-K+ diet slightly increased ROMK mRNA in medulla to 122 ± 9% ( P < 0.05 vs. control). Adrenalectomy (Adx) downregulated cortical ROMK mRNA to 30.7 ± 6.8% ( P < 0.001 vs. control), and increased it in medulla to 138 ± 12.9% ( P < 0.02 vs. control). In Adx rats, K+ deficiency decreased ROMK mRNA in cortex and medulla similar to intact rats. The α1- and β1-Na-K-ATPase subunits were regulated in parallel to that of ROMK. In medulla, ROMK mRNA correlated with serum K+ concentration at R = 0.9406 ( n = 6, P < 0.001) and α1-Na-K-ATPase mRNA at R = 0.9756 ( n = 6, P < 0.001). ROMK2 also correlated with serum K+ concentration ( R = 0.895; n = 6, P < 0.01). These results show that cortical ROMK expression is regulated by aldosterone and K+, whereas the medullary ROMK mRNA is regulated by serum K+.

CD4+CXCR4highCD69+ T cells accumulate in lung adenocarcinoma.

The chemokine receptor CXCR4 is involved in the growth and metastasis of tumor cells. However, the expression of its ligand, the chemokine CXCL12, in tumors and its role in regulating the accumulation of immune cells within the tumors is not clear. Using ELISA and immunohistochemistry we found that CXCL12 is expressed in the majority of nonsmall cell lung cancer tissue sections obtained from stage IA to IIB nonsmall cell lung cancer patients undergoing operation. Histopathologic examination of these sections indicated that high CXCL12 expression correlated with increased tumor inflammation. In addition, disease recurrence rates in a subgroup of adenocarcinoma patients showed a tendency to correlate with high CXCL12 expression in the tumor. Isolation of adenocarcinoma-infiltrating immune cells demonstrated an increase in the percentage of CD4+CD69+CXCR4+ T cells as compared with normal lung tissue. About 30% of these cells expressed the regulatory T cell markers CD25high and FoxP3. The percentage of CD8 T cells within the tumor did not change, however; the percentage of NK and NK T cells was significantly reduced. In correlation with CXCR4 expression, CD4 T cells showed increased migration in response to CXCL12 compared with CD8 T cells and NK cells. Overall, these observations suggest that CXCL12 expression may influence tumor progression by shaping the immune cell population infiltrating lung adenocarcinoma tumors.

CXCR4 antagonist 4F-benzoyl-TN14003 inhibits leukemia and multiple myeloma tumor growth

OBJECTIVE The chemokine receptor CXCR4 and its ligand CXCL12 are involved in the progression and dissemination of a diverse number of solid and hematological malignancies. Binding CXCL12 to CXCR4 activates a variety of intracellular signal transduction pathways that regulate cell chemotaxis, adhesion, survival, proliferation, and apoptosis. MATERIALS AND METHODS Here, we demonstrate that the CXCR4 antagonist, 4F-benzoyl-TN14003 (BKT140), but not AMD3100, exhibits a CXCR4-dependent preferential cytotoxicity toward malignant cells of hematopoietic origin. BKT140 significantly and preferentially stimulated multiple myeloma apoptotic cell death. BKT140 treatment induced morphological changes, phosphatidylserine externalization, decreased mitochondrial membrane potential, caspase-3 activation, sub-G1 arrest, and DNA double-stranded breaks. RESULTS In vivo, subcutaneous injections of BKT140 significantly reduced, in a dose-dependent manner, the growth of human acute myeloid leukemia and multiple myeloma xenografts. Tumors from animals treated with BKT140 were smaller in size and weights, had larger necrotic areas and high apoptotic scores. CONCLUSIONS Taken together, these results suggest a potential therapeutic use for BKT140 in multiple myeloma and leukemia patients.

IFN-γ treatment at early stages of influenza virus infection protects mice from death in a NK cell-dependent manner

Influenza pandemics are imminent and represent a major world health concern. Since vaccinations are expected to be less efficient in the coming years due to newly emerging influenza virus strains, novel antiviral therapies are urgently needed. Here, we show that influenza-infected mice, capable of clearing the virus in the early stages of infection, failed to control inflammation and death. Sequential administration of Interferon-gamma (IFN-gamma) at early stage of the infection protected infected mice from death in a NK cell-dependent manner. IFN-gamma treatment stimulated NK cell proliferation and function and increased their number in the bone marrow, blood, spleen, and infected lungs, keeping viral clearance intact. In parallel, IFN-gamma treatment significantly reduced the number of T cells and NKT cells in the lungs at the inflammatory phase following infection. Thus, rapidly clearing the virus and reducing inflammation by shaping the cellular and cytokine profiles in the early stages of infection may favorably change the fate of influenza pathogenesis.

Interaction between CXCR4 and CCL20 pathways regulates tumor growth

The chemokine receptor CXCR4 and its ligand CXCL12 is overexpressed in the majority of tumors and is critically involved in the development and metastasis of these tumors. CXCR4 is expressed in malignant tumor cells whereas its ligand SDF-1 (CXCL12) is expressed mainly by cancer associated fibroblasts (CAF). Similarly to CXCR4, the chemokine CCL20 is overexpressed in variety of tumors; however its role and regulation in tumors is not fully clear. Here, we show that the chemokine receptor CXCR4 stimulates the production of the chemokine CCL20 and that CCL20 stimulates the proliferation and adhesion to collagen of various tumor cells. Furthermore, overexpression of CCL20 in tumor cells promotes growth and adhesion in vitro and increased tumor growth and invasiveness in vivo. Moreover, neutralizing antibodies to CCL20 inhibit the in vivo growth of tumors that either overexpress CXCR4 or CCL20 or naturally express CCL20. These results reveal a role for CCL20 in CXCR4-dependent and -independent tumor growth and suggest a therapeutic potential for CCL20 and CCR6 antagonists in the treatment of CXCR4- and CCL20-dependent malignancies.

The effect of streptozotocin-induced diabetes mellitus on urinary excretion of sodium and renal Na+−K+-ATPase activity

Renal sodium handling and microsomal Na+−K+-ATPase activity in kidney cortex, medulla and papilla of rats with streptozotocin-induced diabetes mellitus (DM) was studied.During 7 days following the administration of streptozotocin GFR, urinary excretion, filtered load and tubular reabsorption of Na+ averaged (mean±SE) 1.18±0.016 ml/min, 1.74±0.14, 177.3±8.9 and 175.6±8.9 mEq/min respectively in experimental rats as compared to corresponding rates of 0.85±0.04 (P<0.001), 0.85±0.03 (P<0.001), 129.8±5.8 (P<0.001) and 129±5.8 (P<0.001) respectively in the control rats.The activity of microsomal Na−K-ATPase in the kidney cortex, medulla and papilla of the control group was (mean±SE) 44.7±1.7, 150±7.5 and 37.4±3.6 (μmoles Pi/mg prot/h) respectively. 24 h after DM induction Na−K-ATPase activity in the cortex rose to 59.3±2.4 (P<0.001) and remained high after 3 and 7 days. Medullary Na−K-ATPase activity was unchanged 24 h after streptozotocin administration but was markedly increased to 260±9 (P<0.001) after 3 days and remained high after 7 days.These findings show that stretozotocin-induced DM in rats causes a substantial increase in GFR which is associated with a net increase in filtered and reabsorbed load of Na+ and natriuresis. These alterations are accompanied by a marked increase in Na−K-ATPase activity in renal medulla and in the cortex.

The High-Affinity CXCR4 Antagonist BKT140 Is Safe and Induces a Robust Mobilization of Human CD34+ Cells in Patients with Multiple Myeloma

Purpose: CXCR4 plays an important role in the retention of stem cells within the bone marrow. BKT140 (4F-benzoyl-TN14003) is a 14-residue bio stable synthetic peptide, which binds CXCR4 with a greater affinity compared with plerixafor (4 vs. 84 nmol/L). Studies in mice demonstrated the efficient and superior mobilization and transplantation of stem cells collected with GCSF-BKT140, compared with those obtained when using stem cells obtained with each one of these mobilizing agent alone. These results have served as a platform for the present clinical phase I study. Experimental Design: Eighteen patients with multiple myeloma who were preparing for their first autologous stem cell transplantation were included. Patients received a standard multiple myeloma mobilization regimen, consisting of 3 to 4 g/m2 cyclophosphamide (day 0), followed by granulocyte colony—stimulating factor (G-CSF) at 5 μg/kg/d starting on day 5 and administered between 8 and 10 pm until the end of stem cell collection. A single injection of BKT140 (0.006, 0.03, 0.1, 0.3, and 0.9 mg/kg) was administered subcutaneously on day 10 in the early morning, followed by G-CSF 12 hours later. Results: BKT140 was well tolerated at all concentrations, and none of the patients developed grade 3 and 4 toxicity. A single administration of BKT140 at the highest dose, 0.9 mg/kg, resulted in a robust mobilization and collection of CD34+ cells (20.6 ± 6.9 × 106/kg), which were obtained through a single apheresis. All transplanted patients received ∼5.3 × 106 CD34+ cells/kg, which rapidly engrafted (n = 17). The median time to neutrophil and platelet recovery was 12 and 14 days, respectively, at the highest dose (0.9 mg/kg). Conclusions: When combined with G-CSF, BKT140 is a safe and efficient stem cell mobilizer that enabled the collection of a high number of CD34+ cells in 1 and 2 aphaeresis procedures, resulting in successful engraftment. Clin Cancer Res; 20(2); 469–79. ©2013 AACR.