Identification of a Potent Sodium Hydrogen Exchanger Isoform 1 (NHE1) Inhibitor with a Suitable Profile for Chronic Dosing and Demonstrated Cardioprotective Effects in a Preclinical Model of Myocardial Infarction in the Rat

Sodium-hydrogen exchanger isoform 1 (NHE1) is a ubiquitously expressed transmembrane ion channel responsible for intracellular pH regulation. During myocardial ischemia, low pH activates NHE1 and causes increased intracellular calcium levels and aberrant cellular processes, leading to myocardial stunning, arrhythmias, and ultimately cell damage and death. The role of NHE1 in cardiac injury has prompted interest in the development of NHE1 inhibitors for the treatment of heart failure. This report outlines our efforts to identify a compound suitable for once daily, oral administration with low drug-drug interaction potential starting from NHE1 inhibitor sabiporide. Substitution of a piperidine for the piperazine of sabiporide followed by replacement of the pyrrole moiety and subsequent optimization to improve potency and eliminate off-target activities resulted in the identification of N-[4-(1-acetyl-piperidin-4-yl)-3-trifluoromethyl-benzoyl]-guanidine (60). Pharmacological evaluation of 60 revealed a remarkable ability to prevent ischemic damage in an ex vivo model of ischemia reperfusion injury in isolated rat hearts.     

Natural Killer Cells Promote Tissue Injury and Systemic Inflammatory Responses During Fatal Ehrlichia-Induced Toxic Shock-Like Syndrome

Human monocytotropic ehrlichiosis is caused by Ehrlichia chaffeensis, a Gram-negative bacterium lacking lipopolysaccharide. We have shown that fatal murine ehrlichiosis is associated with CD8⁺T cell-mediated tissue damage, tumor necrosis factor-α, and interleukin (IL)-10 overproduction, and CD4⁺Th1 hyporesponsiveness. In this study, we examined the relative contributions of natural killer (NK) and NKT cells in Ehrlichia-induced toxic shock. Lethal ehrlichial infection in wild-type mice induced a decline in NKT cell numbers, and late expansion and migration of activated NK cells to the liver, a main infection site that coincided with development of hepatic injury. The spatial and temporal changes in NK and NKT cells in lethally infected mice correlated with higher NK cell cytotoxic activity, higher expression of cytotoxic molecules such as granzyme B, higher production of interferon-γ and tumor necrosis factor-α, increased hepatic infiltration with CD8αCD11c⁺ dendritic cells and CD8⁺T cells, decreased splenic CD4⁺T cells, increased serum concentrations of IL-12p40, IL-18, RANTES, and monocyte chemotactic protein-1, and elevated production of IL-18 by liver mononuclear cells compared with nonlethally infected mice. Depletion of NK cells prevented development of severe liver injury, decreased serum levels of interferon-γ, tumor necrosis factor-α, and IL-10, and enhanced bacterial elimination. These data indicate that NK cells promote immunopathology and defective anti-ehrlichial immunity, possibly via decreasing the protective immune response mediated by interferon-γ producing CD4⁺Th1 and NKT cells.Human monocytotropic ehrlichiosis (HME) is an emerging tick-borne and a life threatening illness caused by Ehrlichia chaffeensis, an obligatory intracellular bacterium.^1,2 HME can manifest as either mild disease with nonspecific flu-like symptoms or severe and fatal disease that presents as toxic shock-like syndrome, multi-organ failure, or adult respiratory distress syndrome.^2,3,4,5,6 Severe HME in immunocompetent patients is thought to be due to immune-mediated pathology, which is attributed to severe inflammation in the absence of large quantities of ehrlichiae in the tissues.⁴ Doxycycline treatment is most effective if administered early in the course of illness.^1,7 The lack of complete understanding of the pathogenic mechanisms of Ehrlichia-induced toxic shock-like syndrome is a major limitation in successful management of these patients. Ehrlichia muris and Ixodes ovatus Ehrlichia (IOE), which are closely related to each other and to E. chaffeensis, stimulate protective or pathogenic immune responses, respectively, in C57BL/6 mice.^8,9 Intradermal (i.d.) and intraperitoneal (i.p.) infection with a high dose of IOE induces mild and fatal disease, respectively.⁹ Using these models, we and others have shown that antigen-specific interferon (IFN)-γ producing CD4⁺Th1 cells and IgG2a antibodies play important roles in protective immunity against Ehrlichia.^4,10,11 In contrast, fatal murine ehrlichiosis, which recapitulates the pathological and laboratory manifestations of fatal HME is associated with a suppressed or weak CD4⁺Th1 immune response, marked lymphopenia, high levels of serum tumor necrosis factor (TNF)-α and interleukin (IL)-10, and severe liver injury, characteristics consistent with toxic shock syndrome.^4,5,12 β₂m^−/− mice, which are deficient in CD8⁺T cells and natural killer T (NKT) cells, are resistant to fatal IOE-induced toxic shock when compared with wild-type and TAP^−/− mice that are deficient in only CD8⁺T cells, suggesting that CD8⁺T cells mediate immunopathology and fatal ehrlichiosis.¹² Interestingly, absence of NKT cells in CD1d^−/− mice did not protect mice from fatal ehrlichiosis even though their absence prevented the development of liver injury and systemic inflammation.¹³ LPS-lacking Ehrlichia can directly stimulate IFN-γ production by NKT cells in a CD1 days-dependent, but toll-like receptor 4-independent, manner.¹⁴ Further studies showed that NKT cells are critical for clearance of the bacterial burden in vivo, which may account for death of CD1d^−/− mice following lethal ehrlichial infection.¹³NK cells are abundant in the liver, able to produce high levels of pro- and anti-inflammatory cytokines, and play an important role in innate immunity to microbial pathogens.¹⁵ NK cells contribute to the capacity of CD8⁺T cells to produce IFN-γ and to lyse Listeria monocytogenes- and Mycobacterium tuberculosis-infected monocytes.^16,17 Cross talk between NK cells and dendritic cells (DC) is essential for maturation of DC and activation of NK cells. IL-12, IL-15, and IL-18 produced by activated macrophages or mature DC activate cytotoxic NK cells and stimulate their IFN-γ production, which in turn promotes the expansion of CD4⁺Th1 cells.^{17,18,19,20,21,22,23,24} While NK cells play a protective role in some viral infections, NK cells cause hepatocyte apoptosis in models of viral hepatitis.^25,26The current study was undertaken to specifically delineate the contributions of NK cells and NKT cells to the immunopathogenesis of Ehrlichia-induced toxic shock-like syndrome using animal models of nonlethal and lethal ehrlichiosis caused by i.d. and i.p inoculation of IOE, respectively. Our data provide a new finding suggesting that NK cells contribute to severe tissue injury and suppressed protective immunity during infection with these LPS-lacking, obligately intracellular bacteria.     

C-arm flat detector computed tomography: the technique and its applications in interventional neuro-radiology

Introduction  

Flat detector computed tomography (FDCT) is an imaging tool that generates three-dimensional (3-D) volumes from data obtained during C-arm rotation using CT-like reconstruction algorithms. The technique is relatively new and, at current levels of performance, lags behind conventional CT in terms of image quality. However, the advantage of its availability in the interventional room has prompted neuro-radiologists to identify clinical settings where its role is uniquely beneficial.     

Radiation therapy induces circulating serum Hsp72 in patients with prostate cancer

Background and purpose

Hsp72 found in the extracellular milieu has been shown to play an important role in immune regulation. The impact of common cancer therapies on extracellular release of Hsp72 however, has been to date undefined.

Materials and methods

Serum from 13 patients undergoing radiation therapy (XRT) for prostate cancer with or without hormonal therapy (ADT) was measured for levels of circulating serum Hsp72 and pro-inflammatory cytokines (IL-6 and TNF-α) using the classical sandwich ELISA technique and the relative expression of CD8⁺ T lymphocytes and natural killer (NK) cells was measured using flow cytometry. Mouse orthotopic xenograft of human prostate cancer tumors (DU-145 and PC-3) were used to validate and further characterize the response noted in the clinical setting. The biological significance of tumor released Hsp72 was studied in human dendritic cells (DC) in vitro.

Results

Circulating serum Hsp72 levels increased an average of 3.5-fold (median per patient 4.8-fold) with XRT but not with ADT (p = 0.0002). Increases in IL-6 (3.3-fold), TNF-α (1.8-fold), CD8⁺ CTL (2.1-fold) and NK cells (3.2-fold) also occurred. Using PC-3 and DU-145 human prostate cancer xenograft models in mice, we confirmed that XRT induces Hsp72 release primarily from implanted tumors. In vitro studies using supernatant recovered from irradiated human prostate cancer cells point to exosomes containing Hsp72 as a possible stimulator of pro-inflammatory cytokine production and costimulatory molecules expression in human DC.

Conclusions

The current study confirms for the first time in an actual clinical setting elevation of circulating serum Hsp72 with XRT. The accompanying studies in mice and in vitro identify the released exosomes containing Hsp72 as playing a pivotal role in stimulating pro-inflammatory immune responses. These findings, if validated, may lead to new treatment paradigms for common human malignancies.     

Pooled genetic analysis in ultrasound measured non-alcoholic fatty liver disease in Indian subjects:A pilot study

AIM: To investigate genetic susceptibility in Indian subjects with non-alcoholic fatty liver disease(NAFLD) by performing a pooled genetic study.METHODS: Study subjects(n = 306) were recruited and categorized into NAFLD and control groups based on ultrasound findings of fatty infiltration. Of the 306 individuals, 156 individuals had fatty infiltration and thus comprised the NAFLD group. One hundred and fifty(n = 150) individuals were normal, without fatty infiltration of the liver, comprising the control group. Blood samples, demographic and anthropometric data from the individuals were collected after obtaining informed consent. Anthropometric data, blood glucose, lipids and liver function tests were estimated using standard methods. Genome wide association stud-ies done to date on NAFLD were identified, 19 single nucleotide polymorphisms(SNPs) were selected from these studies that were reported to be significantly associated with NAFLD and genotyping was performed on the Sequenom platform. Student's t test for continuous variables and χ2 test was applied to variant carriers from both groups. Required corrections were applied as multiple testing was done.RESULTS The mean age of the control group was 39.78 ± 10.83 and the NAFLD group was 36.63 ± 8.20 years. The waist circumference of males and females in the control and NAFLD groups were 80.13 ± 10.35; 81.77 ± 13.65 and 94.09 ± 10.53; 92.53 ± 8.27 respectively. The mean triglyceride and alanine transaminase(ALT) levels in the control and NAFLD groups were 135.18 ± 7.77; 25.39 ± 14.73 and 184.40 ± 84.31; 110.20 ± 67.05 respectively. When χ2 test was applied to the number of individuals carrying the variant risk alleles between the control and NAFLD group, a significant association was seen between rs738409 of the patatin-like phospholipase domain containing 3(PNPLA3) gene(P = 0.001), rs2073080 of the PARVB gene(P = 0.02), rs2143571 of SAMM50 gene(P = 0.05) and rs6487679 of the pregnancy zone protein(PZP) gene(P = 0.01) with the disease. Variant single nucleotide polymorphisms(SNPs) in NCAN and PNPLA3 gene were associated with higher levels of ALT, whereas variant SNPs in APOC3, PNPLA3, EFCAB4 B and COL13A1 were associated with high triglyceride levels. Apart from the above associations, rs2073080, rs343062 and rs6591182 were significantly associated with high BMI; rs2854117 and rs738409 with high triglyceride levels; and rs2073080, rs2143571, rs2228603, rs6487679 and rs738409 with high ALT levels.CONCLUSION: Pooled genetic analysis revealed an association of SNPs in PNPLA3, PARVB, SAMM50 and PZP genes with NAFLD. SNPs in NCAN and PNPLA3gene were associated with higher levels of ALT,whereas variant SNPs in APOC3, PNPLA3, EFCAB4 B and COL13A1 were associated with high triglyceride levels.     

Escherichia coli O157 in the rectoanal mucosal region of cattle

The rectoanal junction mucosal region is the site of colonization of Escherichia coli O157 in cattle. Our objective was to determine the genetic relatedness of E. coli O157 in the mucosa of the rectoanal junction to isolates from colon contents and feces. Colon contents and rectums were collected from cattle at harvest. Rectums were opened and feces were sampled with a cotton swab. The mucosa of the rectum was cleansed free of visible feces with water and saline. The region, 2 to 5 cm proximal to the rectoanal junction, was swabbed with a foam-tipped applicator and then incisions were made in this region and the submucosa was swabbed with an applicator. Isolation and identification of E. coli O157 was performed in accordance with well-documented methods. Prevalence of E. coli O157 in the colon contents, feces, rectal mucosa, and rectal submucosa was 21%, 29%, 54%, and 34%, respectively. Pulsed-field gel electrophoresis was used to compare clonal similarity among isolates from different sampling regions. Sixty-seven cattle had E. coli O157 isolated from the rectal mucosa swab and feces of which 82% were clonally similar (dice similarity >95%) within animal. Escherichia coli O157 isolates from feces and colon contents were similar in 76% of cattle, but E. coli O157 isolates from the rectoanal mucosal swab and colon contents were only similar in 61.4% of cattle. Our results suggest that E. coli O157 in the feces may be from two sources, colonized in the rectoanal mucosa or transient in the gastrointestinal tract.     

Transcriptional Dependencies in Diffuse Intrinsic Pontine Glioma

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Paraffin-embedded tissue as a source of RNA for gene expression analysis in oral malignancy

OBJECTIVE: To assess the feasibility of using archival oral mucosal tissue to examine gene expression at the ribonucleic acid (RNA) level.
MATERIALS AND METHODS: We describe the isolation of RNA from 8 nm sections of formalin-fixed paraffin-embedded oral mucosal tissue. RNA was reverse transcribed and three candidate genes amplified by polymerase chain reaction (PCR). The ribosomal protein S14 gene is a housekeeping gene which has been used as an internal standard in several quantitative PCR protocols. The thymidine kinase (TK) gene is expressed at low levels in most tissues and, with a well-documented genomic organisation, is a useful tool for discrimination between genomic DNA and cDNA. The RIa gene is reported to be overexpressed in many cancer cell lines, in malignant tissue and in vitro transformed cellS. RESULTS: The S14 gene, the TK gene and the RIα gene of the cAMP-dependent protein kinase (PKA) were amplified successfully from formalin-fixed paraffin-embedded tissue sections. The TK primer pair is a useful additional tool in the unambiguous identification of RNA-derived species.
CONCLUSION: RNA suitable for reverse transcribed (RT)-PCR was extracted from archival oral mucosal tissue. This should permit rapid sequence analysis of transcribed tumor suppressor genes and oncogenes in this material. Furthermore, the RT-PCR approach described may allow quantification of gene expression in oral mucosal archival material processed in a standard fashion.