Development and Characterization of Novel Chronic Eosinophilic Inflammation- Mediated Murine Model of Malignant Pancreatitis.

AIMS: Develop a novel murine models of malignant pancreatitis. BACKGROUND: Although patients with chronic pancreatitis are at a greater risk of developing pancreatic cancer, there is no definitive mouse model that currently develops chronic pancreatitis-induced pancreatic cancer. OBJECTIVE: Characterization of eosinophilic inflammation-mediated malignant pancreatitis in novel murine model. METHODS: We developed a murine model of chronic eosinophilic inflammation associated with pancreatitis that also shows characteristic features of pancreatic malignancy. The mouse received cerulein and azoxymethane via intraperitoneal administration developed pathological malignant phenotype, as well as concomitant lung inflammation. RESULTS: We discovered pathological alterations in the pancreas that were associated with chronic pancreatitis, including a buildup of eosinophilic inflammation. Eosinophil degranulation was reported nearby in the pancreas tissue sections that show acinar-to-ductal metaplasia and acinar cell atrophy, both of which are characteristic of pancreatic malignancies. Additionally, we also observed the formation of PanIN lesions after three initial doses of AOM and eight weeks of cerulein with the AOM treatment regimen. We discovered that persistent pancreatic eosinophilic inflammation linked with a pancreatic malignant phenotype contributes to pulmonary damage. The RNA seq analysis also confirmed the induction of fibro-inflammatory and oncogenic proteins in pancreas and lung tissues. Further, in the current manuscript, we now report the stepwise kinetically time-dependent cellular inflammation, genes and proteins involved in the development of pancreatitis malignancy and associated acute lung injury by analyzing the mice of 3 AOM with 3, 8, and 12 weeks of the cerulein challenged protocol regime. CONCLUSION: We first show that sustained long-term eosinophilic inflammation induces time-dependent proinflammatory, profibrotic and malignancy-associated genes that promote pancreatic malignancy and acute lung injury in mice.

Vaccine efficacy in mutant SARS-CoV-2 variants.

Many aspects of the SARS-CoV-2 virus remain poorly understood, including its rapid mutation and its effects on populations of different ages. The present literature of review is focused on the effectiveness of current available vaccines in view of immerging several SARS-CoV-2 variants. The most dangerous and infectious SARS-CoV-2 strain, B117, was recently discovered in the United Kingdom, and another new variant, 501.V2, was discovered in South Africa. In countries such as the United States, Japan, India, and Brazil, the variant B117 spread far more quickly than the original strain. The new SARS-CoV-2 mutations have made producing a universal and effective vaccine more difficult. SARS-CoV-2's S protein, which aids in receptor identification and membrane fusion, is a primary target for vaccine development using its mRNA or inactivated virus. Currently, in the interval of few days new more infectious SARS-CoV-2 mutant is detected, started from SARS-CoV-2 Alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), delta plus, gamma (P.1) and now variant lamda. The variant detected first in Peru and spread almost 27 countries including UK that accounts for 82% of new infections. These mutant variants are posing new challenge even to the fully vaccinated individuals and a challenge for the public health. Thus, a need to review current treatment vaccination guideline and strategy as early as possible. Reporting all new SARS-CoV-2 variants and their effectiveness in response to several available vaccines, we would like to draw the attention of health care provider, and all developed countries health care agencies including WHO to frame new guidelines for vaccination and immediate intervention to control the development of new SARS-CoV-2 variants from the third world countries by providing vaccines to the poor countries as early as possible.

Eosinophils and T cell surface molecule transcript levels in the blood differentiate eosinophilic esophagitis (EoE) from GERD.

We recently rereported that blood mRNA levels of T cells and IgE receptors are the novel non-invasive biomarkers for eosinophilic esophagitis (EoE) with the aim to establish the panel of T cells and IgE receptor as the novel non-invasive biomarkers for EoE. In addition to earlier proposed cell surface molecules, we now added T cell receptor CXCR6 and eosinophils expressed cell surface molecules CD101 and CD274 mRNA levels. The mRNA levels of eosinophils cell surface molecule CD101 and CD274 and T cell receptor CXCR6, Vß11, CD1d and chemokine CXCL16 levels were examined using the blood of normal, EoE and GERD patients. The analysis showed statistically significant induced mRNA levels of CD274, CD101 and reduced CXCR6 will be an additional molecule with respective 95%, 90% and 90% positive predictive value in between EoE and GERD patients. In brief, these additional data will be critical to establish a complete panel of earlier published TCRδ (95%), Jα18 (83%) and FCεRII (100%) non-invasive biomarker to monitor the EoE severity and treatment effect in EoE patients. In conclusion, we now propose both induced and reduced transcript levels of cell surface molecules of the cell surface molecules along with earlier reported molecules that will be useful for monitoring EoE status before and following treatment. Most importantly, the complete predictive non-invasive biomarker panel will also serve to differentiate EoE from GERD.

Tacrolimus (FK506) treatment protects allergen-, IL-5- and IL-13-induced mucosal eosinophilia.

Eosinophils are a common clinical feature associated with chronic allergic diseases, and elemental diets, systemic steroids, anti-IL-5 and anti-IL-13 treatment have shown some therapeutic promise. Herein, we present evidence that pre- and post-intraperitoneal administration of tacrolimus (FK506) is very effective in reducing CCR3/Siglec-F+ eosinophils in Aspergillus-challenged asthma and EoE, CD2-IL-5 induced global eosinophilia, and DOX regulated IL-13-induced asthma. We used flow cytometry and anti-major basic protein (MBP) immunostaining to examine eosinophils in the spleen, bone marrow, BALF, lung, oesophagus and intestine. Additionally, we also performed ELISA and Western blot analyses to show that tacrolimus treatment also reduces the levels of eosinophil-specific cytokines IL-4, IL-5, IL-13 and TGF-ß, eosinophil-specific chemokines Eotaxin-1 and Eotaxin-2, and progenitors of target RCAN1 mRNA and protein levels. Additionally, the current investigations also show that the TGF-ß-mediated oesophageal and lung fibrosis is also reduced in Aspergillus-challenged, CD2-IL-5 transgenic and DOX-responsive IL-13 mice. Mechanistically, we show that tacrolimus in vitro treatment inhibited bone marrow-derived eosinophil proliferation and viability by promoting eosinophil apoptosis that may be associated with downregulation of RCAN1. Taken together, we provide in vivo and in vitro evidence that tacrolimus ameliorates eosinophil levels and associated pathogenesis in allergen-, IL-5- and IL-13-induced EoE, EG and asthma pathogenesis. Considering tacrolimus side-effects and reactivity to several other drugs, we propose the topical use of tacrolimus for paediatric and low-dose oral for adult patients as a novel therapeutic strategy for the clinical trial to reduce mucosal eosinophilia first in steroid-refractory or elemental diet non-responsive adult EoE, EG and asthma patients.

IL-15 immunotherapy is a viable strategy for COVID-19.

Coronavirus disease 2019 (COVID-19) is a pulmonary inflammatory disease induced by a newly recognized coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 infection was detected for the first time in the city of Wuhan in China and spread all over the world at the beginning of 2020. Several millions of people have been infected with SARS-CoV-2, and almost 382,867 human deaths worldwide have been reported so far. Notably, there has been no specific, clinically approved vaccine or anti-viral treatment strategy for COVID-19. Herein, we review COVID-19, the viral replication, and its effect on promoting pulmonary fibro-inflammation via immune cell-mediated cytokine storms in humans. Several clinical trials are currently ongoing for anti-viral drugs, vaccines, and neutralizing antibodies against COVID-19. Viral clearance is the result of effective innate and adaptive immune responses. The pivotal role of interleukin (IL)-15 in viral clearance involves maintaining the balance of induced inflammatory cytokines and the homeostatic responses of natural killer and CD8+ T cells. This review presents supporting evidence of the impact of IL-15 immunotherapy on COVID-19.

Possible novel non-invasive biomarker for inflammation mediated pancreatic malignancy.

OBJECTIVES: Pancreatic malignancy is a major public health problem worldwide and recent reports indicated that pancreatic cancer will be second most common cause of cancer-related deaths by the end of 2021. The cause of increasing death rate is due to the nonexistence of detection tools to early diagnose, poor prognosis, resistance to chemotherapy and also lack in understanding the mechanism of PDAC pathogenesis. Circulating tumor cells (CTCs) play a major role in metastatic step of intravasation and presence of these cells are strong prognostic marker for the progression of pancreatic malignancy in chronic pancreatitis (CP). GOAL: Identifying the novel CTCs in the chronic inflammation mediated experimental model for the progression of malignancy in CP. METHODS: We have performed flow cytometer and immunofluorescence analyses in the lymphoid and lung samples was performed o detect CTCs in the chronic inflammation induced mouse model CP. RESULTS: We report that induced SOX9 positive cells were observed in the blood, lymph node and spleen samples of cerulein with azoximethane (AOM) treated mouse model of CP compared to cerulein alone. Further, we provide evidence that early metastasis through the migration and homing of mega merged SOX9+ and PDX+ ductal stem cells (CTCs) in the lungs of cerulein with AOM treated mice. These identified CTCs in experimentally induced malignant pancreatitis may serve as a novel finding to identify a non-invasive biomarker that needs to be examined in the blood of human pancreatic cancer. CONCLUSIONS: Taken together, the presented data of identified mega merged SOX9+ and PDX+ ductal stem cells (CTCs) may serve a non-invasive biomarker for the early detection of pancreatic malignancy and metastasis.

Significance of Interleukin (IL)-15 in IgE associated eosinophilic Esophagitis (EoE).

BACKGROUND AND AIM: IgE-mediated immune responses contribute to the pathogenesis of eosinophilic esophagitis (EoE). Interleukin (IL)-4 is a well-established cytokine involved in B cell activation, immunoglobulin (Ig) E production and isotype class switching. Earlier reports indicated that IL-15, B cells and IgE are induced in EoE pathogenesis. Therefore, we hypothesized that induced IL-15 and IgE may have a significant correlation in promoting EoE pathogenesis. METHODS: Accordingly, we performed ELISA, qPCR, flowcytometric and immunostaining analyses to examine IgE, B cells, eosinophils and mast cells in the esophagus of IL-15 overexpressed mice following EoE induction. RESULTS: Herein, we show that IL-15 overexpressed mice indeed have induced baseline IL-4, B cells, eosinophils, mast cells and IgE levels in the blood and esophagus. Further, we observed that IL-15 overexpressed mice show induction of IgE, and accumulation of degranulated eosinophils and mast cells in allergen-induced experimental EoE. Notably, despite induced blood IgE, esophageal eosinophilia is not induced in intestinal fatty acid binding protein IL-15 overexpressed gene (Fabpi-IL-15) mice. Fabpi-IL-15 transgenic mice showed IgE in the blood and intestine and intestinal eosinophilia, but no esophageal eosinophilia at baseline and comparable eosinophils in the esophagus of saline and allergen challenged Fabpi-IL-15 mice. Similarly, allergen challenged IL-15 gene-deficient mice show reduced IgE and esophageal eosinophilia in allergen-induced experimental EoE. CONCLUSIONS: Taken together, we for the first time provide direct evidence that tissue-specific IL-15 induced IgE mediated responses, not systemic IgE is critical in promoting EoE pathogenesis.

Interleukin-18 has an Important Role in Differentiation and Maturation of Mucosal Mast Cells.

A significant amount of correlational evidence has linked increased levels of IL-18 with allergic diseases in both human and animal models, and, as mast cells are major mediators of allergies, we hypothesized that IL-18 may have a role in mast cell biology. Rationale for our hypothesis is based on the evidence that IL-3 deficient mice are not devoid of mast cells, even though IL-3 is a major differentiation and growth factor for mast cells. Accordingly, we cultured IL-18 responsive bone marrow CD34+ cells in vitro under a variety of conditions and cytokine combinations to examine mast cell differentiation and maturation using flow cytometry, quantitative PCR,and immunostaining techniques. Additionally, in vivo mast cell transformation and maturation were also analysed using endogenous IL-18 gene-deficient or Fabpi-IL-18 overexpressed mice. Our data indicate that both IL-3 and IL-18 exposed CD34+ bone marrow precursors differentiate and mature into mast cells. Further, we observed that IL-18 differentiates mast cells independent of IL-3, as pharmacologic blockade of IL-3 does not prevent in vitro IL-18-driven mast cell differentiation. Further, we found that endogenous IL-18 deficiency restricts maturation of IL-3 generated mast cells and IL-18 derived mast cells require IL-3 for their survival. Additionally, we observed IL-18 intestinal overexpression promotes tissue mast cell proliferation and mucosal mast cell development. Taken together, we provide the evidence that IL-18 has an important contributory role in mast cell differentiation, maturation and in vivo development of mucosal mast cells. Therefore, IL-18 may represent a future pharmacologic target for treating mast cell-mediated allergic diseases.

Role of eosinophils in the initiation and progression of pancreatitis pathogenesis.

Eosinophilic pancreatitis (EP) is reported in humans; however, the etiology and role of eosinophils in EP pathogenesis are poorly understood and not well explored. Therefore, it is interesting to examine the role of eosinophils in the initiation and progression of pancreatitis pathogenesis. Accordingly, we performed anti-major basic protein immunostaining, chloroacetate esterase, and Masson's trichrome analyses to detect eosinophils, mast cells, and collagen in the tissue sections of mouse and human pancreas. Induced eosinophils accumulation and degranulation were observed in the tissue sections of human pancreatitis, compared with no eosinophils in the normal pancreatic tissue sections. Similarly, we observed induced tissue eosinophilia along with mast cells and acinar cells atrophy in cerulein-induced mouse model of chronic pancreatitis. Additionally, qPCR and ELISA analyses detected induced transcript and protein levels of proinflammatory and profibrotic cytokines, chemokines like IL 5, IL-18, eotaxin-1, eotaxin-2, TGF-ß1, collagen-1, collagen-3, fibronectin, and α-SMA in experimental pancreatitis. Mechanistically, we show that eosinophil-deficient GATA1 and endogenous IL-5-deficient mice were protected from the induction of proinflammatory and profibrotic cytokines, chemokines, tissue eosinophilia, and mast cells in a cerulein-induced murine model of pancreatitis. These human and experimental data indicate that eosinophil accumulation and degranulation may have a critical role in promoting pancreatitis pathogenesis including fibrosis. Taken together, eosinophil tissue accumulation needs appropriate attention to understand and restrict the progression of pancreatitis pathogenesis in humans. NEW & NOTEWORTHY The present study for the first time shows that eosinophils accumulate in the pancreas and promote disease pathogenesis, including fibrosis in earlier reported cerulein-induced experimental models of pancreatitis. Importantly, we show that GATA-1 and IL-5 deficiency protects mice form the induction of eosinophil active chemokines, and profibrotic cytokines, including accumulation of tissue collagen in an experimental model of pancreatitis. Additionally, we state that cerulein-induced chronic pancreatitis is independent of blood eosinophilia.

Regulatory effects of IL-15 on allergen-induced airway obstruction.

BACKGROUND: Airway obstruction is a physiologic feature of asthma, and IL-15 might have an important role in asthma pathogenesis. OBJECTIVE: We tested the hypothesis that regulation of IL-15 is critical for preservation of allergen-induced airway hyperresponsiveness (AHR), airway resistance, and compliance in response to methacholine. METHODS: Airway inflammation, AHR, resistance, and compliance were assessed in Il15 gene-deficient mice and IL-15-overexpressing mice in an allergen-induced murine model of asthma. We assessed eosinophil numbers by using anti-major basic protein immunostaining, goblet cell hyperplasia by using periodic acid-Schiff staining, and cytokine and chemokine levels by performing quantitative PCR and ELISA. RESULTS: We made a novel observation that IL-15 deficiency promotes baseline airway resistance in naive mice. Moreover, rIL-15 delivery to the lung downregulates expression of proinflammatory cytokines and improves allergen-induced AHR, airway resistance, and compliance. These observations were further validated in doxycycline-inducible CC10-IL-15 bitransgenic mice. Doxycycline-exposed, Aspergillus species extract-challenged CC10-IL-15 bitransgenic mice exhibited significantly reduced levels of proinflammatory cytokines (IL-4, IL-5, and IL-13) and decreased goblet cell hyperplasia. Airway obstruction, including AHR and airway resistance, was diminished in allergen-challenged doxycycline-exposed compared with non-doxycycline-exposed CC10-IL-15 bitransgenic mice. Mechanistically, we observed that IL-15-mediated protection of airway obstruction is associated with induced IFN-γ- and IL-10-producing regulatory CD4+CD25+ forkhead box p3 (Foxp3)+ T cells. Additionally, we found that a human IL-15 agonist (ALT-803) improved airway resistance and compliance in an experimental asthma model. CONCLUSION: We report our novel finding that IL-15 has a potent inhibitory effect on the airway obstruction that occurs in response to environmental allergens.

Pathogenic mechanisms of pancreatitis.

Pancreatitis is inflammation of pancreas and caused by a number of factors including pancreatic duct obstruction, alcoholism, and mutation in the cationic trypsinogen gene. Pancreatitis is represented as acute pancreatitis with acute inflammatory responses and; chronic pancreatitis characterized by marked stroma formation with a high number of infiltrating granulocytes (such as neutrophils, eosinophils), monocytes, macrophages and pancreatic stellate cells (PSCs). These inflammatory cells are known to play a central role in initiating and promoting inflammation including pancreatic fibrosis, i.e., a major risk factor for pancreatic cancer. A number of inflammatory cytokines are known to involve in promoting pancreatic pathogenesis that lead pancreatic fibrosis. Pancreatic fibrosis is a dynamic phenomenon that requires an intricate network of several autocrine and paracrine signaling pathways. In this review, we have provided the details of various cytokines and molecular mechanistic pathways (i.e., Transforming growth factor-ß/SMAD, mitogen-activated protein kinases, Rho kinase, Janus kinase/signal transducers and activators, and phosphatidylinositol 3 kinase) that have a critical role in the activation of PSCs to promote chronic pancreatitis and trigger the phenomenon of pancreatic fibrogenesis. In this review of literature, we discuss the involvement of several pro-inflammatory and anti-inflammatory cytokines, such as in interleukin (IL)-1, IL-1ß, IL-6, IL-8 IL-10, IL-18, IL-33 and tumor necrosis factor-α, in the pathogenesis of disease. Our review also highlights the significance of several experimental animal models that have an important role in dissecting the mechanistic pathways operating in the development of chronic pancreatitis, including pancreatic fibrosis. Additionally, we provided several intermediary molecules that are involved in major signaling pathways that might provide target molecules for future therapeutic treatment strategies for pancreatic pathogenesis.

Significance of Eosinophils in Promoting Pancreatic malignancy.

BACKGROUND: Several reports indicate that eosinophils are induced in chronic pancreatitis including patients with pancreatic malignancy. However, significance of eosinophilic pancreatitis (EP) is poorly understood and unexplored. AIM: Accumulation and degranulation of eosinophils promote pancreatic fibrosis and malignancy. METHOD: Human pancreatic tissue biopsy samples including chronic pancreatitis (n=3), malignant (n=4), non-malignant (n=3), and normal (n=3) were used for H&E, anti-MBP staining, anti-tryptase staining, anti-IgE staining and Mason's trichrome staining. RESULTS: We show induced eosinophils and degranulated eosinophils indicated by the presence of anti-MBP stained extracellular granules in the malignant pancreatic (pancreatic cancer) and non-malignant human pancreatic tissues. A comparable number of eosinophils were observed in non-malignant and malignant pancreatic tissue sections, but the sections differed in degranulated eosinophils and the presence of extracellular granules. Additionally, induced mast cells and tissue-specific IgE positive cells were also detected in the tissue sections of malignant pancreatitis patients compared to non-malignant human pancreatic patients. Tissue-specific IgE induction is critical for the degranulation of eosinophils and mast cells that may lead to increased accumulation of collagen in malignant compared to non-malignant human pancreatic tissue samples. We show a large number of anti-tryptase stained extracellular granules in the tissue sections of malignant pancreatic cancer patients. Both IgE and eosinophil major basic proteins (MBP) are reported for the activation and degranulation of mast cells in tissues. CONCLUSION: Taken together, our investigation concludes that eosinophils and mast cells accumulation and degranulation are critical in promoting pancreatitis pathogenesis that may lead to the development of pancreatic fibrosis and malignancy.

Chronic Pancreatitis Associated Acute Respiratory Failure.

Pancreatitis is a condition characterized by parenchymal inflammation of the pancreas, which is often associated with lung injury due to low level of oxygen and the condition is termed as acute pancreatitis-associated lung injury (APALI). Clinical reports indicated that ~ 20% to 50% of patients from low oxygen levels in blood with acute respiratory distress syndrome (ARDS). ARDS is a severe form of acute lung injury (ALI), a pulmonary disease with impaired airflow making patients difficult to breathe. ALI is frequently observed in patients with severe acute pancreatitis. Approximately one third of severe pancreatitis patients develop acute lung injury and acute respiratory distress syndrome that account for 60% of all deaths within the first week. The major causes of ALI and ARDS are sepsis, trauma, aspiration, multiple blood transfusion, and most importantly acute pancreatitis. The molecular mechanisms of ALI and ARDS are still not well explored, but available reports indicate the involvement of several pro-inflammatory mediators including cytokines (TNF-α, IL-1ß, IL-6) and chemokines [like interleukin-8 (IL-8) and macrophage inhibitory factor (MIF)], as well as macrophage polarization regulating the migration and pulmonary infiltration of neutrophils into the pulmonary interstitial tissue, causing injury to the pulmonary parenchyma. Acute lung injury and acute respiratory distress syndrome in acute pancreatitis remains an unsolved issue and needs more research and resources to develop effective treatments and therapies. However, recent efforts have tested several molecules in an experimental model and showed promising results as a treatment option. The current review summarized the mechanism that is operational in pancreatitis-associated acute respiratory failure and respiratory distress syndrome in patients and current treatment options.

Possible Noninvasive Biomarker of Eosinophilic Esophagitis: Clinical and Experimental Evidence.

Eosinophilic esophagitis (EoE) diagnosis and follow-up response to therapy is based on repeated endoscopies and histological examination for eosinophils/HPF. The procedure is invasive and risky in particular for the pediatric population. Presently, there is no highly sensitive and specific noninvasive blood test available to monitor the disease pathogenesis. Reports indicate the expression of PDL1 (CD274) on the eosinophils in allergic patients. Herein, we report that CD274-expressing and -nonexpressing eosinophils were detected in both examined pediatric and adult EoE patients. We show that CD274 expression on blood eosinophils and blood mRNA expression levels increase in the blood of EoE patients and decrease following treatment. These observations are consistent with the esophageal eosinophilia of before and after treatment in both examined patients. These two clinical and experimental analysis reports provide the possibility that the CD274 mRNA and CD274-expressing esinophil levels may be novel possible noninvasive biomarkers for EoE.

Elements Involved In Promoting Eosinophilic Gastrointestinal Disorders.

Eosinophilic gastrointestinal disorders (EGID) are food allergen-induced allergic gastrointestinal disorders, characterized by accumulation of highly induced eosinophils in different segments of gastrointestinal tract along with eosinophil microabssess and extracellular eosinophilic granules in the epithelial layer. EGID are both IgE- and cell-mediated group of diseases that include eosinophilic esophagitis (EoE), eosinophilic gastritis (EG), eosinophilic gastroenteritis (EGE) and eosinophilic colitis (EC). Despite the increased incidences and considerable progress made in understanding EGID pathogenesis. The mechanism is still not well understood. It has been shown that IL-4, IL-5, IL-13, IL-15, IL-18, eotaxin-1, eotaxin-2 and eotaxin-3 play a critical role in EGID pathogenesis. Currently, the only criterion for diagnosing EoE, EGE and EC are repetitive endoscopic and histopathological evaluation of biopsies along with other clinical characteristics/manifestations. Antigen elimination and corticosteroid therapies are the most effective therapies currently in practice for the treatment of EGID. The cytokines (anti-IL-5 and anti-IL-13) therapy trials were not very successful in case of EoE. Most recently, a clinical trial using anti-IL-13 reported only 60% reduced esophageal eosinophilia without achieving primary endpoint. This clinical finding is not surprising and is in accordance with our earlier report indicating that IL-13 is not critical in the initiation of EoE. Notably, EGID still has no reliable noninvasive diagnostic biomarkers. Hence, there is a great necessity to identify novel noninvasive diagnostic biomarkers that can easily diagnose EGID and provide an effective therapy. Now, the attention is required to target cell types like iNKT cells that produce eosinophil active cytokines and is found induced in the pathogenesis of both experimental and human EoE. iNKT cell neutralization is shown to protect allergen-induced EoE in experimental model. In this review, we have discussed the key elements that are critical in the disease initiation, progression, pathogenesis and important for future diagnostic and therapeutic interventions for EGID.

Role of Bruton's tyrosine kinase in myeloma cell migration and induction of bone disease.

Myeloma cells typically grow in bone, recruit osteoclast precursors and induce their differentiation and activity in areas adjacent to tumor foci. Bruton's tyrosine kinase (BTK), of the TEC family, is expressed in hematopoietic cells and is particularly involved in B-lymphocyte function and osteoclastogenesis. We demonstrated BTK expression in clinical myeloma plasma cells, interleukin (IL)-6- or stroma-dependent cell lines and osteoclasts. SDF-1 induced BTK activation in myeloma cells and BTK inhibition by small hairpin RNA or the small molecule inhibitor, LFM-A13, reduced their migration toward stromal cell-derived factor-1 (SDF-1). Pretreatment with LFM-A13 also reduced in vivo homing of myeloma cells to bone using bioluminescence imaging in the SCID-rab model. Enforced expression of BTK in myeloma cell line enhanced cell migration toward SDF-1 but had no effect on short-term growth. BTK expression was correlated with cell-surface CXCR4 expression in myeloma cells (n = 33, r = 0.81, P < 0.0001), and BTK gene and protein expression was more profound in cell-surface CXCR4-expressing myeloma cells. BTK was not upregulated by IL-6 while its inhibition had no effect on IL-6 signaling in myeloma cells. Human osteoclast precursors also expressed BTK and cell-surface CXCR4 and migrated toward SDF-1. LFM-A13 suppressed migration and differentiation of osteoclast precursors as well as bone-resorbing activity of mature osteoclasts. In primary myeloma-bearing SCID-rab mice, LFM-A13 inhibited osteoclast activity, prevented myeloma-induced bone resorption and moderately suppressed myeloma growth. These data demonstrate BTK and cell-surface CXCR4 association in myeloma cells and that BTK plays a role in myeloma cell homing to bone and myeloma-induced bone disease. Am. J. Hematol. 88:463-471, 2013. © 2013 Wiley Periodicals, Inc.

NAMPT/PBEF1 enzymatic activity is indispensable for myeloma cell growth and osteoclast activity.

Multiple myeloma (MM) cells typically grow in focal lesions, stimulating osteoclasts that destroy bone and support MM. Osteoclasts and MM cells are hypermetabolic. The coenzyme nicotinamide adenine dinucleotide (NAD(+)) is not only essential for cellular metabolism; it also affects activity of NAD-dependent enzymes, such as PARP-1 and SIRT-1. Nicotinamide phosphoribosyltransferase (NAMPT/PBEF/visfatin, encoded by PBEF1) is a rate-limiting enzyme in NAD(+) biosynthesis from nicotinamide. Coculture of primary MM cells with osteoclasts induced PBEF1 upregulation in both cell types. PBEF1 expression was higher in experimental myelomatous bones than in nonmyelomatous bone and higher in MM patients' plasma cells than in healthy donors' counterparts. APO866 is a specific PBEF1 inhibitor known to deplete cellular NAD(+). APO866 at low nanomolar concentrations inhibited growth of primary MM cells or MM cell lines cultured alone or cocultured with osteoclasts and induced apoptosis in these cells. PBEF1 activity and NAD(+) content were reduced in MM cells by APO866, resulting in lower activity of PARP-1 and SIRT-1. The inhibitory effect of APO866 on MM cell growth was abrogated by supplementation of extracellular NAD(+) or NAM. APO866 inhibited NF-κB activity in osteoclast precursors and suppressed osteoclast formation and activity. PBEF1 knockdown similarly inhibited MM cell growth and osteoclast formation. In the SCID-rab model, APO866 inhibited growth of primary MM and H929 cells and prevented bone disease. These findings indicate that MM cells and osteoclasts are highly sensitive to NAD(+) depletion and that PBEF1 inhibition represents a novel approach to target cellular metabolism and inhibit PARP-1 and bone disease in MM.

Galectin-3 in urine of cancer patients: stage and tissue specificity.

PURPOSE: Galectin-3 has been implicated in advanced stage of cancer disease. In the current study we examined the possibility of urinary galectin-3 levels to stage cancer disease and to follow up therapy. EXPERIMENTAL DESIGN: Urine was collected from all types of cancer patients at different stages including patients undergoing radio/chemotherapy. Galectin-3 level was determined by anti-galectin-3 based ELISA and agglutination assays. Immunoblotting and purification on lactosyl affinity column further confirmed the presence of galectin-3. RESULTS: Cancer samples exhibited stage dependent expression of galectin-3 approx. ranging from 1.0 to 3.3, 4.4 to 5.4, 5.4 to 24.7, 13.1 to 31.9, 13.9 to 32.9 ng/mg C (creatinine) for stage I-V, respectively, at P approximately <0.05 level. Galectin-3 levels were decreased by approx. threefolds after 5th day of therapy. CONCLUSIONS: Sample collection being simple and non-invasive, urinary galectin-3 may be used as a potential diagnostic tool for monitoring or follow up of the stage of cancer disease.