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Complementarity Determining Region 3 of the immunoglobulin (Ig) H chain (CDR-H3) lies at the center of the antigen-binding site where it often plays a decisive role in antigen recognition and binding. Amino acids encoded by the diversity (DH) gene segment are the main component of CDR-H3. Each DH has the potential to rearrange into one of six DH reading frames (RFs), each of which exhibits a characteristic amino acid hydrophobicity signature that has been conserved among jawed vertebrates by natural selection. A preference for use of RF1 promotes the incorporation of tyrosine into CDR-H3 while suppressing the inclusion of hydrophobic or charged amino acids. To test the hypothesis that these evolutionary constraints on DH sequence influence epitope recognition, we used mice with a single DH that has been altered to preferentially use RF2 or inverted RF1. B cells in these mice produce a CDR-H3 repertoire that is enriched for valine or arginine in place of tyrosine. We serially immunized this panel of mice with gp140 from HIV-1 JR-FL isolate and then used enzyme-linked immunosorbent assay (ELISA) or peptide microarray to assess antibody binding to key or overlapping HIV-1 envelope epitopes. By ELISA, serum reactivity to key epitopes varied by DH sequence. By microarray, sera with Ig CDR-H3s enriched for arginine bound to linear peptides with a greater range of hydrophobicity but had a lower intensity of binding than sera containing Ig CDR-H3s enriched for tyrosine or valine. We conclude that patterns of epitope recognition and binding can be heavily influenced by DH germ line sequence. This may help explain why antibodies in HIV-infected patients must undergo extensive somatic mutation in order to bind to specific viral epitopes and achieve neutralization.

Purpose To test whether signal intensity percent infarct mapping (SI-PIM) accurately determines the size of myocardial infarct (MI) regardless of infarct age. Materials and Methods Forty-five swine with reperfused MI underwent 1.5T late gadolinium enhancement (LGE) magnetic resonance imaging (MRI) after bolus injection of 0.2 mmol/kg Gd(DTPA) on days 2–62 following MI. Animals were classified into acute, healing, and healed groups by pathology. Infarct volume (IV) and infarct fraction (IF) were determined by two readers, using binary techniques (including 2–5 standard deviations [SD] above the remote, and full-width at half-maximum) and the SI-PIM method. Triphenyl-tetrazolium-chloride staining (TTC) was performed as reference. Bias (percent under/overestimation of IV relative to TTC) of each quantification method was calculated. Bland–Altman analysis was done to test the accuracy of the quantification methods, while intraclass correlation coefficient (ICC) analysis was done to assess intra- and interobserver agreement. Results Bias of the MRI quantification methods do not depend on the age of the MI. Full-width at half-maximum (FWHM) and SI-PIM gave the best estimate of MI volume determined by the reference TTC (P-values for the FWHM and SI-PIM methods were 0.183, 0.26, 0.95, and 0.073, 0.091, 0.73 in Group 1, Group 2, and Group 3, respectively), while using any of the binary thresholds of 2–4 SDs above the remote myocardium showed significant overestimation. The 5 SD method, however, provided similar IV compared to TTC and was shown to be independent of the size and age of MI. ICC analysis showed excellent inter- and intraobserver agreement between the readers. Conclusion Our results indicate that the SI-PIM method can accurately determine MI volume regardless of the pathological stage of MI. Once tested, it may prove to be useful for the clinic. J. Magn. Reson. Imaging 2016;43:911–920

Background Assessing the extent of ischemic and reperfusion-associated myocardial injuries remains challenging with current magnetic resonance imaging (MRI) techniques. Our aim was to develop a tissue characterization mapping (TCM) technique by combining late gadolinium enhancement (LGE) with our novel percent edema mapping (PEM) approach to enable the classification of tissue represented by MRI voxels as healthy, myocardial edema (ME), necrosis, myocardial hemorrhage (MH), or scar. Methods Six dogs underwent closed-chest myocardial infarct (MI) generation. Serial MRI scans were performed post-MI on days 3, 4, 6, 14, and 56, including T2 mapping and LGE. Dogs were sacrificed on day 4 (n = 4, acute MI) or day 56 (n = 2, chronic MI). TCMs were generated based on a voxel classification algorithm taking into account signal intensity from LGE and T2-based estimation of ME. TCM-based MI and MH were validated with post mortem triphenyl tetrazolium chloride (TTC) staining. Pearson’s correlation and Bland-Altman analyses were performed. Results The MI, ME, and MH measured by TCM were 13.4% [25th–75th percentile 1.6–28.8], 28.1% [2.1–37.5] and 4.3% [1.0–11.3], respectively. TCM measured higher MH and MI compared to TTC (p = 0.0033 and p = 0.0007, respectively). MH size was linearly correlated with MI size by both MRI (r = 0.9528, p < 0.0001) and TTC (r = 0.9625, p < 0.0001). MH quantification demonstrated good agreement between TCM and TTC (r = 0.8766, p < 0.0001, 2.4% overestimation by TCM). A similar correlation was observed for MI size (r = 0.9429, p < 0.0001, 6.1% overestimation by TCM). Conclusions Preliminary results suggest that the TCM method is feasible for the in vivo localization and quantification of various MI-related tissue components.

A substantial, common shortcoming of the currently used semiautomated techniques for the quantification of myocardial infarct with delayed enhancement magnetic resonance imaging is the assumption that the whole myocardial slab that corresponds to the hyperenhanced tomographic area is 100% nonviable. This assumption is, however, incorrect. To resolve this conflict, we have recently proposed the signal intensity percent-infarct mapping method and validated it in an ex vivo, canine experiment. The purpose of the current study has been the validation of the signal intensity percent-infarct mapping method in vivo, using a porcine model of reperfused myocardial infarct.In swines (n=6), reperfused myocardial infarct was generated occluding for 90 min by an angioplasty balloon either the left anterior descending or the left circumflex coronary artery. To obtain DE images, Gd(DTPA) enhanced inversion-recovery fast gradient-echo acquisitions were carried out on day 28 after myocardial infarction. Scanning started 15 min after intravenous injection of 0.2 mmol/kg Gd(DTPA). At the end of the MRI session, the animal was sacrificed and 2,3,5-triphenyltetrazolium chloride staining was used to validate the existence and to determine the accurate size of the myocardial infarct. Tissue samples were taken and stained with hematoxylin-eosin and Masson's trichrome for histological assessment of the infarct and the periinfarct zone. The signal intensity percent-infarct mapping data were compared with corresponding data from the delayed enhancement images analyzed with SI(remote+2S.D.) thresholding, and with corresponding triphenyltetrazolium-chloride staining data using Friedman's repeated measure analysis of variance on ranks.The infarct volume determined by the triphenyltetrazolium chloride, SI(remote+2S.D.) and signal intensity percent-infarct mapping methods was 3.04 ml [2.74, 3.45], 13.62 ml [9.06, 18.45] and 4.27 ml [3.45, 6.33], respectively. Median infarct volume determined by SI(remote+2S.D.) significantly differed from that determined by triphenyltetrazolium chloride (P.05). The Bland-Altman overall bias was 12.49% of the volume of the left ventricle. Median infarct volume determined by signal intensity percent-infarct mapping, however, did not differ significantly (NS) from that obtained by triphenyltetrazolium chloride. Signal intensity percent-infarct mapping yielded only a 1.99% Bland-Altman overall bias of the left ventricular volume.This in vivo study in the porcine reperfused myocardial infarct model demonstrates that signal intensity percent-infarct mapping is a highly accurate method for the determination of the extent of myocardial infarct. MRI images for signal intensity percent-infarct mapping are obtained with the pulse sequence of conventional delayed enhancement imaging and are acquired within clinically acceptable scanning time. This makes signal intensity percent-infarct mapping a practical method for clinical implementation.

To determine the infarct affinity of a low molecular weight contrast agent, Gd(ABE-DTTA), during the subacute phase of myocardial infarct (MI). Dogs (n = 7) were examined, using a closed-chest, reperfused MI model. MI was generated by occluding for 180 min the Left Anterior Descending (LAD) coronary artery with an angioplasty balloon. DE-MRI images with Gd(ABE-DTTA) were obtained on days 4, 14, and 28 after MI. Control DE-MRI by Gd(DTPA) was carried out on day 27. T2-TSE images were acquired on day 3, 13 and 27. Triphenyltetrazolium chloride (TTC) histomorphometry validated postmortem the existence of infarct. Gd(ABE-DTTA) highlighted the infarct on day 4, but not at all on day 14 or on day 28, following MI. On day 4, the mean ± SD signal intensity (SI) of infarcted myocardium in the presence of Gd(ABE-DTTA) significantly differed from that of healthy myocardium (45 ± 6.0 vs. 10 ± 5.0, P < 0.05), but it did not on day 14 (11 ± 9.4 vs. 10 ± 5.7, P = NS), nor on day 28 (7 ± 1.5 vs. 7 ± 2.4, P = NS). The mean ± SD signal intensity enhancement (SIE) induced by Gd(ABE-DTTA) was 386 ± 165% on day 4, significantly different from mean SIE on day 14 (9 ± 20%), and from mean SIE on day 28 (12 ± 18%), following MI (P < 0.05). The last two mean values did not differ significantly (P = NS) from each other. As control, Gd(DTPA) was used and it did highlight the infarct on day 27, inducing a mean SIE value of 312 ± 40%. The mean SIE on day 3, 13, or 27 did not vary significantly (P = NS) on the T2-TSE images (114 ± 41%, 123 ± 41%, and 150 ± 79%, respectively). Post mortem, the existence of infarcts was confirmed by TTC staining. The infarct affinity of Gd(ABE-DTTA) vanishes in the subacute phase of scar healing, allowing its use for infarct age differentiation early on, immediately following the acute phase.

Purpose To demonstrate the advantages of signal intensity percent-infarct-mapping (SI-PIM) using the standard delayed enhancement (DE) acquisition in assessing viability following myocardial infarction (MI). SI-PIM quantifies MI density with a voxel-by-voxel resolution in clinically used DE images. Materials and Methods In canines (n= 6), 96 hours after reperfused MI and administration of 0.2 mmol/kg Gd(DTPA), ex vivo DE images were acquired and SI-PIMs calculated. SI-PIM data were compared with data from DE images analyzed with several thresholding levels using SIremote+2SD, SIremote+6SD, SI full width half maximum (SIFWHM), and with triphenyl-tetrazolium-chloride (TTC) staining. SI-PIM was also compared to R1 percent infarct mapping (R1-PIM). Results Left ventricular infarct volumes (IV) in DE images, IVSIremote+2SD and IVSIremote+6SD, overestimated (P < 0.05) TTC by medians of 13.21 mL [10.2; 15.2] and 6.2 mL [3.79; 8.23], respectively. SIFWHM, SI-PIM, and R1-PIM, however, only nonsignificantly underestimated TTC, by medians of −0.10 mL [−0.12, −0.06], −0.86 mL [−1.04; 1.54], and −1.30 mL [−4.99; −0.29], respectively. The infarct-involved voxel volume (IIVV) of SI-PIM, 32.4 mL [21.2, 46.3] is higher (P < 0.01) than IIVVs of SIFWHM 8.3 mL [3.79, 19.0]. SI-PIMFWHM, however, underestimates TTC (−5.74 mL [−11.89; −2.52] (P < 0.01)). Thus, SI-PIM outperforms SIFWHM because larger IIVVs are obtained, and thus PIs both in the rim and the core of the infarcted tissue are characterized, in contradistinction from DE-SIFWHM, which shows mainly the infarct core. Conclusion We have shown here, ex vivo, that SI-PIM has the same advantages as R1-PIM, but it is based on the scanning sequences of DE imaging, and thus it is obtainable within the same short scanning time as DE. This makes it a practical method for clinical studies. J. Magn. Reson. Imaging 2010;32:859–868. © 2010 Wiley-Liss, Inc.

BACKGROUND The noninvasive, early detection of Prostate Intraepithelial Neoplasia (PIN), a precancerous neoplasia of the prostate, would be highly desirable. In our experiments, we used TRAMP mice to model PIN in the range of grade 1 through grade 4. METHODS Contrast enhanced pixel-by-pixel R1 mapping of the prostate was used to detect areas with the different prostate neoplasia grades. After anesthesia, Gd(ABE-DTTA) was injected I.V. A series of MRI images with varying TI were then acquired to create R1 maps in a 2 mm transversal tomographic slice that included the prostate. After euthanasia and the excision of the prostate, a 2 mm slice, corresponding to the tomographic slice, was selected and prepared for histological analysis. The microscopic sections of this slice were scanned and analyzed along with the R1 maps. The R1 values were normalized to that measured in muscle tissue in each individual mouse to account for possible variations among the mice in contrast agent uptake (R1norm). The R1norm values and the histological grades in the corresponding areas were correlated. RESULTS A significant difference was found between the R1norm values measured in areas with grade 1–2 versus those observed in areas with grades 3–4. Also, a significant correlation was found between the area size of the ROIs differentiated by MRI, and those determined by histology. CONCLUSION This method has the potential for early noninvasive detection of developing prostate cancer. Prostate 69:449–458, 2009. © 2008 Wiley-Liss, Inc.

The phenomenological tissue kinetics of Gd(ABE-DTTA) was investigated in myocardial infarction (MI). Reperfused infarction was generated by balloon catheter in closed-chest canines (N = 11). Forty-eight hours thereafter, inversion-recovery (IR)-prepared fast gradient-echo control images were acquired with varying inversion times (TIs). Precontrast R1 maps were calculated from the TI dependence of signal intensity (SI) using nonlinear curve fitting. Then 0.05 mmol/kg Gd(ABE-DTTA) was administered I.V. In 11 dogs postcontrast R1 maps were generated at 24 hr and 48 hr postcontrast. In five dogs measurements were also repeated at 108 hr and 12 days. In one dog early measurement was carried out at 4 hr. ΔR1 values for blood and viable and infarcted myocardium were calculated at each time point by subtracting the precontrast R1 from the postcontrast R1. Gd(ABE-DTTA) showed significant, progressive accumulation into infarcts during the first 2 days (kin = 0.39 hr–1) and a delayed clearance (kout = 0.005 hr–1). Among the time points sampled, the maximum infarct ΔR1 was detected at 48 hr (1.72 s–1). Contrast agent (CA) in infarcted tissue was detectable for 12 days. Clearance from blood and viable myocardium occurred in parallel and was completed by 108 hr. Gd(ABE-DTTA) displays slow, tissue-persistent kinetics and partly intravascular, partly extravascular characteristics. It demonstrates high affinity for infarcted myocardium and induces highlighting of infarcts between 4 hr and 12 days following administration. Magn Reson Med 58:55–64, 2007. © 2007 Wiley-Liss, Inc.

To test whether signal intensity percent infarct mapping (SI-PIM) accurately determines the size of myocardial infarct (MI) regardless of infarct age.Forty-five swine with reperfused MI underwent 1.5T late gadolinium enhancement (LGE) magnetic resonance imaging (MRI) after bolus injection of 0.2 mmol/kg Gd(DTPA) on days 2-62 following MI. Animals were classified into acute, healing, and healed groups by pathology. Infarct volume (IV) and infarct fraction (IF) were determined by two readers, using binary techniques (including 2-5 standard deviations [SD] above the remote, and full-width at half-maximum) and the SI-PIM method. Triphenyl-tetrazolium-chloride staining (TTC) was performed as reference. Bias (percent under/overestimation of IV relative to TTC) of each quantification method was calculated. Bland-Altman analysis was done to test the accuracy of the quantification methods, while intraclass correlation coefficient (ICC) analysis was done to assess intra- and interobserver agreement.Bias of the MRI quantification methods do not depend on the age of the MI. Full-width at half-maximum (FWHM) and SI-PIM gave the best estimate of MI volume determined by the reference TTC (P-values for the FWHM and SI-PIM methods were 0.183, 0.26, 0.95, and 0.073, 0.091, 0.73 in Group 1, Group 2, and Group 3, respectively), while using any of the binary thresholds of 2-4 SDs above the remote myocardium showed significant overestimation. The 5 SD method, however, provided similar IV compared to TTC and was shown to be independent of the size and age of MI. ICC analysis showed excellent inter- and intraobserver agreement between the readers.Our results indicate that the SI-PIM method can accurately determine MI volume regardless of the pathological stage of MI. Once tested, it may prove to be useful for the clinic.

Studies in vitro suggest that osteopontin (OPN), an extracellular matrix protein secreted by macrophages infiltrating prostate tumors, and by tumor cells, may have a role in the transition from clinically insignificant tumors to metastatic prostate cancer (PC). Latent PC occurs at equal rates in Western and Asian men, but the incidence of advanced PC is many-fold higher in Western men. Our earlier studies in TRAnsgenic Mouse Prostate adenocarcinoma (TRAMP) mice showed that genistein, an isoflavone found in soybeans, lowered the incidence of advanced PC. This suggested that lower intake of dietary soy may be one possible cause for higher incidence of advanced PC in Western men. The objective of the present study was to test the hypothesis that genistein may exert its preventive effect by inhibiting OPN expression. From 5 to 28 wk of age, 80, 68, and 30 TRAMP mice were fed AIN-76A diet containing 0, 250, or 500 mg genistein/kg body weight, respectively. Organ weights were measured. The steady-state level of OPN mRNA was evaluated by RT-PCR in a longitudinal study in 74 TRAMP and 32 nontransgenic litter mates (NTM). Administration of 250 and 500 mg genistein/kg AIN-76A improved survival (P = 0.008 and P = 0.005, respectively) and reduced mean weight of prostates with poorly differentiated cancer (PD) (P < 0.001), as well as the mean weight of periaortic lymph nodes (LN), although the latter was not significant. OPN was upregulated 10-fold in PD compared with prostates with a lower pathological score from TRAMP or NTM of any age (P = 0.003). OPN mRNA levels in the dorsolateral prostate and metastasis to LN were significantly correlated (r = 0.643; P = 0.00006). Genistein had a dose-dependent, significant inhibitory effect on OPN transcript levels in prostates displaying advanced prostate cancer (PD; score 6; P = 0.05). Studies are consistent with the possibility that dietary genistein may delay the progression from benign to malignant tumors by inhibiting OPN expression.

Mycoplasma arthritidis causes acute polyarthritis in rats and chronic proliferative arthritis in mice. M. arthritidis -induced arthritis serves as a model for arthritis caused by infectious agents and as a model for examining the role of the superantigen MAM ( M. arthritidis T-cell mitogen) in the development of autoimmunity. M. arthritidis strain 158-1 is a spontaneous mutant of strain 158 that has a drastic reduction in virulence. We show that the mutant is missing a major antigen of 47 kDa (P47) and has acquired a protein of 67 kDa (P67). P47 and P67 partitioned into the detergent phase by extraction with Triton X-114. Coomassie blue staining of sodium dodecyl sulfate-polyacrylamide gels show that P67 is produced in abundance. Analysis of gel-purified P67 by mass spectrometry led to its identification as a lipoprotein (the open reading frame [ORF] 619 gene product) predicted from the genome sequence of M. arthritidis . PCR analysis of genomic DNA from 158 and 158-1 indicates that P47 and P67 are encoded by the same ORF 619 gene and differ only in the number of repeats in a tandem repeat region. By two-dimensional polyacrylamide gel analysis, no protein differences were detectable between 158 and 158-1 other than P47 and P67. Collectively, the data suggest that the tandem repeat region of P47 and P67 influences disease outcome.

Background Apoptosis is disrupted in prostate tumor cells, conferring a survival advantage. p53 is a nuclear protein believed to regulate cancer progression, in part by inducing apoptosis. To test this possibility in future studies, the objective of the present study was to generate a transgenic mouse model expressing mutant p53 in the prostate (PR). Methods Transgene incorporation was tested using Southern analysis. Expression of mutant p53 protein was examined using immunofluorescence microscopy. Apoptosis in the PR was evaluated using the Tunnel method. Results A construct, consisting of the rat probasin promoter and a mutant human p53 fragment, was prepared and used to generate transgenic mice. rPB-mutant p53 transgene incorporation, as well as nuclear accumulation of mutant human p53 protein, was demonstrated. Prostatic intraepithelial neoplasia (PIN) III and IV were found in PR of 52-week old transgenic mice, whereas no pathological changes were found in the other organs examined. PR ability to undergo apoptosis following castration was reduced in rPB-mutant p53 mice as compared to non transgenic littermates. Conclusions Transgenic rPB-mutant p53 mice accumulate mutant p53 protein in PR, resulting in neoplastic lesions and reduced apoptotic potential in the PR. Breeding rPB-mutant p53 mice with mice expressing an oncogene in their PR will be useful in examining interactions of multiple genes that result in progression of slow growing prostate tumors expressing oncogenes alone to metastatic cancer. © 2004 Wiley-Liss, Inc.

The lysogenic bacteriophage MAV1 has been shown to be a virulence factor for the development of arthritis in rats infected with Mycoplasma arthritidis. In the present study, arthritis was evaluated by histopathologic examination to demonstrate that MAV1 is a virulence factor not only in the rat but also in the mouse. Specifically, the MAV1 lysogen 158L3-1 was more virulent than the nonlysogen strain 158 in DBA/2NCr, C3H/HeNCr, C3H/HeJ, and C3Smn.CB17-Prkdc(scid)/J mice, as well as in LEW rats.

To study the feasibility of a myocardial infarct (MI) quantification method [signal intensity-based percent infarct mapping (SI-PIM)] that is able to evaluate not only the size, but also the density distribution of the MI. In 14 male swine, MI was generated by 90 min of closed-chest balloon occlusion followed by reperfusion. Seven (n = 7) or 56 (n = 7) days after reperfusion, Gd-DTPA-bolus and continuous-infusion enhanced late gadolinium enhancement (LGE) MRI, and R1-mapping were carried out and post mortem triphenyl-tetrazolium-chloride (TTC) staining was performed. MI was quantified using binary [2 or 5 standard deviation (SD)], SI-PIM and R1-PIM methods. Infarct fraction (IF), and infarct-involved voxel fraction (IIVF) were determined by each MRI method. Bias of each method was compared to the TTC technique. The accuracy of MI quantification did not depend on the method of contrast administration or the age of the MI. IFs obtained by either of the two PIM methods were statistically not different from the IFs derived from the TTC measurements at either MI age. IFs obtained from the binary 2SD method overestimated IF obtained from TTC. IIVF among the three different PIM methods did not vary, but with the binary methods the IIVF gradually decreased with increasing the threshold limit. The advantage of SI-PIM over the conventional binary method is the ability to represent not only IF but also the density distribution of the MI. Since the SI-PIM methods are based on a single LGE acquisition, the bolus-data-based SI-PIM method can effortlessly be incorporated into the clinical image post-processing procedure.

Gram-positive bacteria are recognized pathogens in urinary tract infections. Lipoteichoic acids, major components of the cell wall of gram-positive bacteria, are important virulence attributes, but their mechanism of action is not well understood. We have postulated that infection-induced altered function of progenitors of urothelial cells (UT) residing in the basal layer is likely to have long-lasting effects on the architecture and function of the urothelium. Our earlier in vitro studies in UT of basal type, grown under growth restricting conditions, have shown that (1) treatment with lipoteichoic acid from Streptococcus faecalis (LT-2) stimulates a subpopulation of progenitors of urothelial cells to proliferate, and (2) resulting large colonies differentiated at an increased rate under conditions simulating those in the basal layer of the urothelium. The hypothesis underlying the present studies was that nitric oxide (NO) mediated LT-2 action on these functions of UT. Immunocytochemical studies using an antibody against inducible nitric oxide synthase (iNOS) confirmed expression of iNOS in LT-2-treated UT. Our hypothesis was tested by treating UT grown under growth restricting conditions (0.005% bovine pituitary extract) with LT-2 (25 μg/ml), in the presence or absence of inhibitors of NOS (1 mM NG-nitro-L-arginine methyl ester [L-NAME]; 1 μM dexamethasone [DEXA]) or 25 μM hemoglobin, a potent inactivator of NO. Treatment with LT-2 in the presence of these agents prevented the following effects of LT-2 alone: (1) the stimulatory effect on proliferation of single cells, as well as within the resulting large colonies; (2) the subsequent differentiation of large colonies resulting from this proliferative activity, as indicated by distribution of β1 subunit-containing integrins to cell-cell contacts; (3) the inhibitory effect on the subsequent ability of LT-2-treated UT to attach to extracellular matrix proteins. These studies suggest that induction of NOS by LT-2, initially aimed at restricting the replication of infectious agents, may have potential cost of damage to the host bladder by interfering with urothelial differentiation. © 1996 Wiley-Liss, Inc.

Gram-positive bacteria are recognized pathogens in urinary tract infections. Cellular mechanisms triggered by lipoteichoic acids (LTs), cell well components of gram-positive bacteria, have not been completely defined. We have postulated that infection-induced altered function of progenitors of urothelial cells residing in the basal layer is likely to have long lasting effects on the architecture and function of the urothelium. Our recent studies in vitro showed that treatment of poorly differentiated urothelial cells of basal type with LT from Streptococcus faecalis (LT-2) stimulated rapid proliferation of a subpopulation of progenitors of urothelial cells, supporting this possibility (Elgavish et al., 1996, J. Cell. Physiol., 169:42-51). The hypothesis underlying the present studies was that, following LT-triggered increase in proliferation of progenitors, the rate of differentiation of the resulting progeny was also stimulated. We proposed that this mechanism may allow rapid removal of cells from the injured area and replacement by cells that have not been exposed to infection. To simulate in vitro conditions in the basal layer that inhibit terminal differentiation, cells grew on fibronectin or collagen-coated substrate, in medium containing low Ca2+ (0.2 mM) and low levels of growth factors (0.005% bovine pituitary extract [BPE]). During the last 3 days in culture, cells grew in the same low Ca2+ (0.2 mM) medium, but without BPE, with or without LT-2. In a positive control group, cells grew during their last 3 days in culture in medium without BPE and LT-2 but in which levels levels of Ca2+ were higher (2 mM), a condition known to stimulate differentiation in other cell types. Several lines of evidence supported the possibility that long-term treatment with LT-2 stimulated progression of large colonies (i.e., the progeny resulting from LT-triggered proliferation) to a more differentiated state: (1) the rate of their differentiation, determined by criterion of intense cytokeratin 8 expression, was increased; (2) steady-state level of beta mRNA and expression of beta 1 subunit of integrins at the protein level were inhibited; (3) in contrast to large colonies in control cultures, the entire population of LT-2-treated large colonies contained beta 1 integrins distributed at cell-cell contacts. Raising extracellular Ca2+ concentration to 2 mM induced similar effects, suggesting that LT-2 may act by stimulating an increase in intracellular levels of Ca2+. However, further studies will be needed to elucidate the molecular mechanisms underlying the stimulatory effect of LT-2 on proliferation of progenitors of urothelial cells in the basal layer of the urothelium and subsequent differentiation of their progeny. We propose that these processes may have a causative role in the pathological changes that occur in the aftermath of chronic or recurrent suburothelial infection in the urinary bladder.

Urinary tract infection with gram-positive bacteria is common. Avenues for ingress of bacteria into the bladder include luminal and suburothelial infection. Terminally differentiated superficial urothelial cells lining the lumen of the bladder are often shed in response to infection. In contrast, infection-induced altered function of progenitors of urothelial cells residing in the basal layer of the urothelium is likely to have long lasting effects on the structure and function of the urothelium. The main objective of the present studies was to investigate in vitro the possibility that exposure to lipoteichoic acid, a cell wall component of the gram-positive Streptococcus faecalis (LT-2), stimulates basal urothelial cells to proliferate. To simulate conditions that restrict proliferation and inhibit terminal differentiation of urothelial cells in the basal layer, secondary cultures of urothelial cells (UT) were grown on collagen or fibronectin-coated substrate in medium containing low levels of Ca2+ (0.2 mM) and growth factors (0.005% bovine pituitary extract [BPE]). Under these conditions, UT cultures displayed a highly reproducible colony size distribution, possibly due to the fact that colonies were progeny of basal cells with various proliferative potentials, retained in vitro. In cultures grown under growth-restricting conditions, the majority of progenitors appeared to be quiescent, just like stem cells in the basal layer of the urothelium. Thus, the population of large colonies (more than six cells/colony), was small when a steady state of growth was achieved, 3–7 days after seeding. Growth factors (0.005–0.5% BPE) caused a dose-dependent increase in this population of large colonies. Moreover, treatment of UT grown under growth-restricting conditions (0.005% BPE) with LT-2 increased steady-state levels of the population of large colonies to levels obtained in cultures growing under optimal conditions with respect to growth factors. These results indicated that the subpopulation of progenitors, quiescent under normal conditions, could be stimulated to proliferate. Two lines of evidence were consistent with the possibility that treatment with LT-2 stimulated proliferation of the subpopulation of progenitors and that large colonies were the progeny of this subpopulation of single cells: (1) treatment with LT-2 increased the percentage of single cells that incorporated bromodeoxyuridine (i.e., proliferated) in a time-dependent manner; (2) An increase in the percentage of large colonies was found following LT-2-triggered proliferation of single cells. We propose that, under normal conditions, cells produced in response to LT-2-triggered proliferation of stem cells are removed from the system due to an increased rate of differentiation followed by apoptosis. Recurrent infection and inflammation may not allow these processes to proceed effectively, resulting in chronic injury to the bladder. Moreover, under conditions in which stem cells accumulate mutations that incapacitate their progeny to undergo apoptosis, LT-triggered proliferation could be a contributing factor to tumorigenesis. © 1996 Wiley-Liss, Inc.

OBJECTIVE To demonstrate the feasibility of using multidetector computed tomography with gadolinium contrast (Gd-MDCT) for the quantification of myocardial infarct (MI). MATERIALS AND METHODS MI was induced in male swine (n = 6). One week later, the animals received 0.2-mmol/kg gadopentetate dimeglumine and were sacrificed. On the excised hearts, Gd-MDCT with several tube voltages (80, 120, and 140 kV), late gadolinium enhancement MRI (LGE-MRI), and triphenyl-tetrazolium-chloride staining were then conducted. We used a 2-SD threshold for the CT images and several threshold limits (2, 3, 4, 5, 6 SD, and full width at half-maximum [FWHM]) for the LGE-MRI images to delineate the infarct area. Total infarct volume and infarct fraction of each heart were calculated. RESULTS MI size measured by MDCT at 140 kV showed good correlation with the reference triphenyl-tetrazolium-chloride value. Applying an 80-kV tube voltage, however, significantly underestimated MI size. In our study, the LGE-MRI method, using the 6-SD threshold, provided the most accurate determination of MI size. LGE-MRI, using the 2- and 3-SD threshold limits, significantly overestimated infarct size. CONCLUSIONS The Gd-MDCT technique has been found suitable for the evaluation of MI in an ex vivo experimental setting. Gd-MDCT has the ability to detect MI even at low kV settings, but accuracy is limited by a high image noise because of reduced photon flux.

Background Standard extracellular cardiovascular magnetic resonance (CMR) contrast agents (CA) do not provide differentiation between acute and older myocardial infarcts (MI). The purpose of this study was to develop a method for differentiation between acute and older myocardial infarct using myocardial late-enhancement (LE) CMR by a new, low molecular weight contrast agent. Dogs (n = 6) were studied in a closed-chest, reperfused, double myocardial infarct model. Myocardial infarcts were generated by occluding the Left Anterior Descending (LAD) coronary artery with an angioplasty balloon for 180 min, and four weeks later occluding the Left Circumflex (LCx) coronary artery for 180 min. LE images were obtained on day 3 and day 4 after second myocardial infarct, using Gd(DTPA) (standard extracellular contrast agent) and Gd(ABE-DTTA) (new, low molecular weight contrast agent), respectively. Triphenyltetrazolium chloride (TTC) histomorphometry validated existence and location of infarcts. Hematoxylin-eosin and Masson's trichrome staining provided histologic evaluation of infarcts. Results Gd(ABE-DTTA) or Gd(DTPA) highlighted the acute infarct, whereas the four-week old infarct was visualized by Gd(DTPA), but not by Gd(ABE-DTTA). With Gd(ABE-DTTA), the mean ± SD signal intensity enhancement (SIE) was 366 ± 166% and 24 ± 59% in the acute infarct and the four-week old infarct, respectively (P < 0.05). The latter did not differ significantly from signal intensity in healthy myocardium (P = NS). Gd(DTPA) produced signal intensity enhancements which were similar in acute (431 ± 124%) and four-week old infarcts (400 ± 124%, P = NS), and not statistically different from the Gd(ABE-DTTA)-induced SIE in acute infarct. The existence and localization of both infarcts were confirmed by triphenyltetrazolium chloride (TTC). Histologic evaluation demonstrated coagulation necrosis, inflammation, and multiple foci of calcification in the four day old infarct, while the late subacute infarct showed granulation tissue and early collagen deposition. Conclusions Late enhancement CMR with separate administrations of standard extracellular contrast agent, Gd(DTPA), and the new low molecular weight contrast agent, Gd(ABE-DTTA), differentiates between acute and late subacute infarct in a reperfused, double infarct, canine model.

Sulfate transport studies were carried out in secondary cultures of epithelial cells isolated from the human ureter. Results demonstrate the presence of carrier-mediated SO4(2-) transport as supported by three lines of evidence: 1) saturation kinetics, 2) substrate specificity, and 3) inhibition by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The DIDS-insensitive component of SO4(2-) transport was markedly lower than the DIDS-sensitive component and was not affected by changes in extracellular pH (pHo) or Cl- concentration. The mechanism of this DIDS-insensitive component is not clear. The DIDS-sensitive component of SO4(2-) uptake was a saturable function of the extracellular sulfate concentration ([SO4(2-)]o). Increasing the extracellular chloride concentration ([Cl-]o) inhibited DIDS-sensitive SO4(2-) uptake competitively. Taken together with the fact that increasing [Cl-]o stimulated SO4(2-) efflux, these results suggest that SO4(2-) uptake in uroepithelial cells occurs via SO4(2-)-Cl- anion exchange. Cis-inhibition studies with a variety of anions indicate that this anion-exchange system may be shared by S2O3(2-) and MoO4(2-) but not by NO3- and H2PO4-. SO4(2-) uptake was stimulated at decreasing pHo with a pK approximately 7.4. Decreasing pHo from 7 to 6 lowered the apparent Michaelis constant significantly but had no significant effect on kcat, suggesting that protons may increase the affinity of the SO4(2-) transporter for SO4(2-). SO4(2-) efflux was inhibited at low pHo and was stimulated by increasing [Cl-]o. This study is the first to demonstrate an ion transport process in epithelial cell cultures isolated from the human ureter. In contrast to epithelial cells from the upper urinary tract, no Na(+)-dependent SO4(2-) transport could be demonstrated in these lower urinary tract epithelial cells. In conclusion, the major mechanism for SO4(2-) transport in ureteral epithelial cells is a carrier-mediated, DIDS-sensitive, pHo-sensitive SO4(2-)/Cl- anion-exchange mechanism. These studies suggest that varying [SO4(2-)]o and [Cl-]o or pHo in the ureteral lumen will affect SO4(2-) influx and efflux and may influence the size of the intracellular pool of SO4(2-) available for macromolecular sulfation in these cells.

Expression of CFTR from a retroviral vector in CFPAC, a pancreatic adenocarcinoma cell line derived from a patient with Cystic Fibrosis, causes a decrease in the average intracellular pH (pHi) in these transduced clones (PLJ-CFTR), as compared to CFPAC or CFPAC transduced with control virus (PLJ clones). Whereas the average pHi calculated based on results obtained in two PLJ-CFTR clones, PLJ-CFTR-20 (n = 2) and PLJ-CFTR-6 (n = 5), was 7.46 +/- 0.07, the average pHi calculated from results obtained in CFPAC (n = 13), PLJ-6 (n = 11) and PLJ-10 (n = 3) was pH 7.83 +/- 0.11. This finding suggests that CFTR may be involved, directly or indirectly, in the regulation of pHi in the pancreas.

The noninvasive, early detection of Prostate Intraepithelial Neoplasia (PIN), a precancerous neoplasia of the prostate, would be highly desirable. In our experiments, we used TRAMP mice to model PIN in the range of grade 1 through grade 4.Contrast enhanced pixel-by-pixel R1 mapping of the prostate was used to detect areas with the different prostate neoplasia grades. After anesthesia, Gd(ABE-DTTA) was injected I.V. A series of MRI images with varying TI were then acquired to create R1 maps in a 2 mm transversal tomographic slice that included the prostate. After euthanasia and the excision of the prostate, a 2 mm slice, corresponding to the tomographic slice, was selected and prepared for histological analysis. The microscopic sections of this slice were scanned and analyzed along with the R1 maps. The R1 values were normalized to that measured in muscle tissue in each individual mouse to account for possible variations among the mice in contrast agent uptake (R1(norm)). The R1(norm) values and the histological grades in the corresponding areas were correlated.A significant difference was found between the R1(norm) values measured in areas with grade 1-2 versus those observed in areas with grades 3-4. Also, a significant correlation was found between the area size of the ROIs differentiated by MRI, and those determined by histology.This method has the potential for early noninvasive detection of developing prostate cancer.

Purpose To compare a novel method, percent-infarct-mapping (PIM), with conventional delayed enhancement (DE) of contrast for accurate myocardial viability assessment. Contrary to signal intensity (SI), the longitudinal relaxation-rate enhancement (ΔR1) is an intrinsic parameter linearly proportional to the concentration of contrast agent (CA). Determining ΔR1 voxel-by-voxel, after administering an infarct-avid CA, allows determination of per-voxel percentage of infarcted tissue. The feasibility of generating PIM is demonstrated in canine reperfused infarction using an infarct-avid, persistent-CA (PCA), (Gd)(ABE-DTTA). PIM is compared to the DE method using Gd(DTPA), and both to triphenyltetrazolium chloride (TTC) staining histochemistry. Materials and Methods In six dogs, 48 hours following closed-chest, 180-minute coronary occlusion, DE imaging was carried out. PCA was administered immediately thereafter. Pixel-by-pixel R1 maps of the entire left ventricle (LV) were generated 48 hours after PCA using inversion-recovery with multiple inversion times. R1, ΔR1, and percent-infarct values were calculated voxel-by-voxel. Results Significant correlations (P < 0.01, R ≥ 0.8) were obtained for percent-infarct-per-slice (PIS) determined by DE or PIM vs. those from corresponding TTC-stained slices. Compared to TTC, DE overestimated PIS by 32 ± 18%. PIM underestimated PIS by 2.5 ± 4.9%. Infarction fraction overestimation was 29 ± 6% of LV by DE, but only 1.0 ± 2.3% by PIM. Errors with PIM were significantly smaller than those with DE. Infarct area determined by signal intensity was similarly overestimated whether using Gd(DTPA) or Gd(ABE-DTTA). Conclusion The ΔR1-based PIM method is highly reproducible and more accurate than DE for quantifying myocardial viability in vivo. J. Magn. Reson. Imaging 2008;28:1386–1392. © 2008 Wiley-Liss, Inc.

The etiology of bladder pain syndrome/interstitial cystitis is poorly understood. The possibility that epigenetic reprogramming may have a role is discussed.A literature search was performed with the Entrez-PubMed(R) database using the key words urinary bladder, epigenetics, epigenetic mechanisms, interstitial cystitis, diagnosis, etiology, urothelial cells, mast cells, nerve fibers, nerves, nerve growth factor, recurrent injury, stem cells, inflammatory mediators and demethylases.The uroepithelium is intimately associated with the nervous system. Sensory input at the apical surface of umbrella cells regulates bladder function via a transmural signaling pathway. When umbrella cells are shed in response to noxious stimuli, stem cells in the basal layer become exposed. The polycomb group genes are key in the maintenance of adult stem cells. The polycomb group genes mediate gene silencing and repress transdifferentiation by methylating lysine 27 of histone H3 (H3K27me3). Jmjd3, an enzyme demethylating H3K27me3, antagonizes polycomb group genes mediated silencing. Inflammatory stimuli are strong inducers of Jmjd3 and may reverse gene silencing in stem cells, modifying the differentiation pattern. Epigenetic processes involving H3K27 methylation are multistable processes. Transient signaling, eg by lipopolysaccharide, triggers epigenetic reprogramming and establishes one of the alternative regulatory states. Once established such states can be maintained and propagated even in the absence of the initial signal.We postulate that similar epigenetic reprogramming mechanisms in the bladder may provide an explanation for uroepithelial, mast cells and nerve cell abnormalities in bladder pain syndrome/interstitial cystitis, as well as propagation of this altered state in the absence of the signal that may have triggered it. It also provides a new experimental paradigm for exploring the etiology of bladder pain syndrome/interstitial cystitis. Data supporting this hypothesis would provide a rationale for new diagnostic as well as treatment options for bladder pain syndrome/interstitial cystitis.

Evidence suggests that the histone deacetylase, SIRT1, is a mediator of life span extension by calorie restriction; however, SIRT1 may paradoxically increase the risk of cancer. To better understand the relationship among SIRT1, energy balance, and cancer, two experiments were done. First, a transgenic mouse model of prostate cancer (transgenic adenocarcinoma of mouse prostate; TRAMP) was used to determine the role of energy balance on SIRT1 expression and the effect of cancer stage on SIRT1 and hypermethylated in cancer-1 (HIC-1). Second, immunohistochemistry was done on human prostate tumors to determine if SIRT1 was differentially expressed in tumor cells versus uninvolved cells. Results show that SIRT1 is not increased in the dorsolateral prostate (DLP) of calorie-restricted mice during carcinogenesis. In contrast, when examined in the DLP as a function of pathologic score, SIRT1 was significantly elevated in mice with poorly differentiated adenocarcinomas compared with those with less-advanced disease. HIC-1, which has been shown to regulate SIRT1 levels, was markedly reduced in the same tumors, suggesting that a reduction in HIC-1 may be in part responsible for the increased expression of SIRT1 in prostatic adenocarcinomas. Furthermore, immunostaining of human prostate tumors showed that cancer cells had greater SIRT1 expression than uninvolved cells. In conclusion, DLP SIRT1 expression from calorie-restricted mice was not altered during carcinogenesis. However, SIRT1 expression was increased in mice with poorly differentiated adenocarcinomas and in human prostate cancer cells. Because SIRT1 may function as a tumor promoter, these results suggest that SIRT1 should be considered as a potential therapeutic target for prostate cancer. [Cancer Res 2007;67(14):6612–8]

A convenient method is introduced for myocardial perfusion research by combining multislice short-axis cine cardiovascular magnetic resonance (MSA-CMR) and colored microspheres (CM). In canine control-ischemia-reperfusion (n = 11), Cardiac output (CO) was measured using MSA-CMR (COMSA), Phase Contrast CMR (PC-CMR, COPC) and CM (from reference blood samples, COmicro) in 3 experimental periods per animal. COmicro significantly and systematically overestimated COMSA (median deviation: 291 mL/min, p0.01), while there was excellent agreement between PC-CMR and MSA-CMR without significant over-or underestimation (median deviation: -14 ml/min, p = NS). In quantitative myocardial perfusion assay by CM, CMR should be used instead of reference blood sampling. This should improve the accuracy of absolute regional perfusion measurement.

Calorie restriction can inhibit or delay carcinogenesis, reportedly due to a reduction in calorie intake rather than by concurrent changes in body mass and/or composition. Our objective was to test the hypothesis that body mass and/or composition have an important effect, independent of energy intake, on the benefits or hazards associated with calorie restriction or overeating, respectively. In the first experiment, transgenic mice that spontaneously develop prostate cancer [transgenic adenocarcinoma of mouse prostate (TRAMP)] were housed at 27°C or 22°C and pair fed the same diet for 21 weeks (95% of ad libitum intake at 27°C). In the second experiment, TRAMP mice were housed at 27°C or 22°C and fed the same diet ad libitum for 21 weeks. Despite a similar calorie intake, pair-fed mice at 27°C (PF27) were heavier (28.3 ± 3.3 versus 17.6 ± 1.6 g at 21 weeks; P < 0.001; mean ± SD) and had greater fat (6.4 ± 2.1 versus 1.9 ± 0.3 g; P < 0.001) and lean mass (P < 0.001) than pair-fed mice at 22°C. Furthermore, PF27 mice had greater levels of serum leptin (P < 0.001), lower levels of adiponectin (P < 0.05), and a greater frequency of prostatic adenocarcinoma (P < 0.05). In contrast, ad libitum–fed mice housed at 22°C consumed ∼30% more calories than ad libitum–fed mice at 27°C, but there was no difference between groups in body composition or cancer progression. These results imply that the ability of calorie restriction to inhibit or delay cancer incidence and progression is mediated in part by changes in energy balance, body mass, and/or body composition rather than calorie intake per se, suggesting that excess calorie retention, rather than consumption, confers cancer risk. [Cancer Res 2007;67(1):417–24]

Viability detection is crucial for the management of myocardial infarction (MI). Signal intensity (SI)-based MRI methods may overestimate infarct size in vivo. In contrast to SI, the longitudinal relaxation-rate enhancement (ΔR1) is an intrinsic parameter that is linearly proportional to the concentration of contrast agent (CA). Determining ΔR1 in the presence of an infarct-avid persistent CA (PCA) allows determination of the per-voxel percentage of infarcted tissue. Introduced here is a ΔR1-based CE-MRI method, termed percent infarct mapping (PIM), for quantifying myocardial viability following delayed PCA accumulation. In a canine MI model (N = 6), PIMs were generated using a persistent CA (PCA) and validated using triphenyltetrazolium-chloride (TTC) histochemistry. Voxel-by-voxel R1 maps of the entire left ventricle (LV) were generated 24 and 48 hr after PCA administration using inversion recovery (IR) with multiple inversion times (TIs). PI values were calculated voxel by voxel. Significant correlations (P < 0.01, R = 0.97) were obtained for PI per slice (PIS) determined using PIM vs. corresponding TTC-based values. Median deviations of PIS with PIM from that with TTC were only 1.01% and –0.53%, at 24 hr and 48 hr. Median deviations from the true infarction fraction (IF) were 1.23% and 0.49% of LV at 24 hr and 48 hr, respectively. No significant difference was found between PIM24 hr and PIM48 hr. ΔR1-based PIM is an accurate and reproducible method for quantifying myocardial viability distribution, and thus enhances the clinical utility of CE-MRI. Magn Reson Med, 2006. © 2006 Wiley-Liss, Inc.

Purpose To demonstrate the usefulness of normalized ΔR1 (ΔR1n) mapping in myocardial tissue following the administration of the contrast agent (CA) Gd(ABE-DTTA). Materials and Methods Ischemia-reperfusion experiments were carried out in 11 dogs. The method exploited the relatively long tissue lifetime of Gd(ABE-DTTA), and thus no fast R1 measurement technique was needed. Myocardial perfusion was determined with colored microspheres (MP). Results With varying extent of ischemia, impaired wall motion (WM) and lower ΔR1n values were detected in the ischemic sectors, as opposed to the nonischemic sectors where normal WM and higher ΔR1n were observed. Based on the ΔR1n, data from the myocardial perfusion assay and the ΔR1n maps were compared in the ischemic sectors. A correlation analysis of these two parameters demonstrated a significant correlation (R = 0.694, P < 0.005), validating the ΔR1n-mapping method for the quantitation of ischemia. Similarly, pairwise correlations were found for the MP, ΔR1n, and wall thickening (WT) values in the same areas. Based on the correlation between ΔR1n and MP, ΔR1n maps calculated with a pixel-by-pixel resolution can be converted to similarly high-resolution myocardial perfusion maps. Conclusion These results suggest that the extent of the severity of ischemia can be quantitatively represented by ΔR1n maps obtained in the presence of our CA. J. Magn. Reson. Imaging 2006. © 2006 Wiley-Liss, Inc.

Lysogenization of Mycoplasma arthritidis with the MAV1 bacteriophage increases the virulence of the mycoplasma in rats. The MAV1 vir gene is one of only two constitutively transcribed phage genes in the lysogen. We show here that Vir is a lipoprotein and is located on the outer surface of the cell membrane. To investigate whether Vir is a virulence factor, the vir gene was cloned into the transposon vector Tn 4001T and inserted in the genome of the nonlysogen strain 158. The virulence of the resulting transformants was no different from that of the parent strain. Interestingly, all vir -containing transformants were resistant to infection by MAV1. Vir had no effect on MAV1 adsorption. We conclude that Vir is not a virulence factor but functions to exclude superinfecting phage, possibly by blocking the injection of phage DNA into the bacterial cytoplasm.

We investigated the effects of the HIV protease inhibitor ritonavir on body composition, serum lipids, and gene expression in C57BL/6 mice. Dual-energy X-ray absorptiometry measurements in ritonavir-treated male mice revealed whole-body lipoatrophy. In female mice fat reduction was restricted to the gonadal depot. A histopathological analysis showed no visible abnormalities in liver or adipose tissue from ritonavir-treated mice, although adipocytes were significantly smaller in diameter. Serum triglyceride levels were increased in ritonavir-treated male mice. Ritonavir was coadministered with the peroxisome proliferator-activated receptor alpha (PPARalpha) agonist gemfibrozil and the PPARgamma agonist rosiglitazone for 8 weeks. Neither drug alleviated the hypertriglyceridemia or lipoatrophy in ritonavir-treated male mice. Rather, gemfibrozil exacerbated the lipoatrophy. Ritonavir reduced basal expression of two PPARalpha target genes in liver, as well as the PPARgamma target gene phosphoenolpyruvate carboxykinase (PEPCK) in adipose tissues. Ritonavir partially inhibited induction of PPAR target genes by gemfibrozil and rosiglitazone. Gemfibrozil induced expression of fatty acid oxidation genes in liver, and this induction was less substantial when ritonavir was coadministered. Similarly, rosiglitazone induced expression of uncoupling protein-1, uncoupling protein-2, and PEPCK in adipose tissues, and this effect was partially inhibited by ritonavir. Thus, the effects of ritonavir on serum triglycerides and body composition may be due, at least in part, to an inhibition of PPAR function.

Interstitial cystitis (IC) is a chronic inflammatory disease of the bladder wall. The etiology is obscure.18,19,35,44,47,56 Several theories for the etiology of IC have been proposed over the years, including infection,6,56 immunological etiology,1 leaky urothelium resulting from a deficiency of its surface coat of glycosaminoglycans,41,42 activated mast cells,6,49 and neurogenic etiology.6,17 However, none of these theories have been proven beyond doubt.6 The rapid onset of IC, its appearance in mid-life, and the absence of strong family association have suggested to us8–11 and many others6 that IC could be an acquired disease, possibly caused by injury. Recurrent exposure to bacterial infection, which is common in the bladder, has been proposed as a putative mechanism of injury.47,56 Earlier studies were based on the assumption that the sole avenue for the interaction of noxious agents with the bladder is exposure of terminally differentiated “umbrella” cells to agents in the lumen. However, mere exposure of the superficial layer to injury, such as bacterial infection, may not result in chronic changes in the urothelium. These terminally differentiated cells are often shed in response to infectious agents,2,38 and unexposed progenitors of urothelial cells located in the basal layer (stem cells) divide and re-epithelize the injured area.

We hypothesized that liver fatty acid oxidation (FAO) is compromised in the leptin-deficient obese (Lep(ob)/Lep(ob)) mouse model, and that this would be further challenged when these mice were fed a high-fat diet. Obese mice had a 3.8-fold increased body fat content and a 9-fold increased liver fat content as compared to control mice when both groups were fed a low-fat diet. The expression of liver FAO enzymes, carnitine palmitoyltransferase-1a, long-chain acyl-CoA dehydrogenase, medium-chain acyl-CoA dehydrogenase, and short-chain acyl-CoA dehydrogenase, was not affected in obese mice as compared to controls on either a low-fat or a high-fat diet. The expression of very-long-chain acyl-CoA dehydrogenase was elevated in obese mice on the control diet, as compared to control mice. For all measures evaluated, increasing the level of fat in the diet had a smaller effect than leptin deficiency. In summary, despite obese mice having an excess of fat available for mitochondrial beta-oxidation in liver, overall energy balance appeared to dictate that the net liver FAO remained at control levels.

We investigated the potential of genistein, the primary isoflavone of soy, to protect against breast and prostate cancers in animal models. For mammary cancer studies, Sprague-Dawley rats were fed AIN-76A diet plus minus 250 mg genistein/kg diet. Dimethylbenz[a]anthracene was administered by gavage at d 50 postpartum to induce mammary tumors. Mammary cancer chemoprevention was demonstrated after prepubertal and combined prepubertal and adult genistein treatments but not after prenatal- or adult-only treatments, demonstrating that the timing of exposure to genistein is important for mammary cancer chemoprevention. The cellular mechanism of action was found to be mammary gland and cell differentiation, as shown by whole-mount analysis and beta-casein expression. An imprinting effect was shown for epidermal growth factor receptor expression in mammary terminal end buds. For prostate cancer studies, we used two models. The first was a chemically (N-methylnitrosourea) induced prostate cancer rat model. Genistein in the diet inhibited the development of invasive adenocarcinomas in a dose-dependent manner. The second model was a transgenic mouse model that resulted in spontaneously developing adenocarcinoma tumor of the prostate. Genistein in the diet reduced the incidence of poorly differentiated prostatic adenocarcinomas in a dose-dependent manner and down-regulated androgen receptor, estrogen receptor-alpha, progesterone receptor, epidermal growth factor receptor, insulin-like growth factor-I, and extracellular signal-regulated kinase-1 but not estrogen receptor-beta and transforming growth factor-alpha mRNA expressions. We conclude that dietary genistein protects against mammary and prostate cancers by regulating specific sex steroid receptors and growth factor signaling pathways.

BACKGROUND Osteopontin (OPN) is a secreted extracellular matrix (ECM) protein found in bone, as well as associated with epithelial cells. The main objective of these studies was to test in vitro the hypothesis that interaction with OPN stimulates proliferation of a quiescent subpopulation of prostate epithelial cells with high proliferative potential. METHODS To simulate conditions that restrict proliferation and inhibit terminal differentiation of basal cells in vivo, control cultures grew on substrate coated with collagen (CO) or fibronectin (FN), in medium containing low levels of growth factors. RESULTS Under growth-restricting conditions, most prostate epithelial cells with high proliferative potential, seeded in control secondary cultures, were quiescent within the time frame of the studies, as indicated by the small number of large colonies in these cultures. Growing prostate epithelial cells (PR) under the same growth-restricting conditions, but on substrate coated with OPN instead of CO or FN, stimulated proliferation of a subpopulation of single cells with high proliferative ability as indicated by: 1) dose-dependent increase in the percentage of single cells incorporating bromodeoxyuridine, i.e., proliferating PR; and 2) subsequent dose-dependent increase in the percentage of large colonies. The OPN effect was not merely due to preferential attachment to OPN, because PR attachment to OPN, CO, or FN was identical. PR attachment to OPN was inhibited in the presence of GRGDTP or an antibody against the integrin subunit αv, but not in the presence of an RGES peptide or a nonspecific IgG. CONCLUSIONS Integrin-mediated OPN/PR interaction stimulates proliferation of a quiescent subpopulation of prostate epithelial cells with high proliferative potential, possibly stem cells. Prostate 35:83–94, 1998. © 1998 Wiley-Liss, Inc.

Secondary cultures of basal urothelial cells isolated from patients with stress incontinence (7 patients), neurogenic bladder (2 patients), interstitial cystitis (IC) (27 patients), bladder rupture (1 patient) and bacterial cystitis (3 patients) grew under growth restricting conditions. All groups displayed reproducible colony size distribution, reflecting the proliferative potential distribution in the population of progenitor cells seeded. The percentage of large colonies (6 cells/colony), progeny of basal cells with high proliferative potential, was low in cultures from control patients with stress incontinence, neurogenic bladder or bladder rupture. Exposure of cultures from control patients with stress incontinence to lipoteichoic acid from Streptococcus faecalis, in vitro, increased the percentage of large colonies to levels statistically indistinguishable from those in untreated IC cultures. This supported the possibility that exposure of progenitors of urothelial cells to infection in vivo may cause the persistent increase in the percentage of large colonies in 80% of the IC patients tested. Given these findings, it was not surprising that the percentage of large colonies was also high in cultures from patients with acute bacterial cystitis. In conclusion, the present findings support the theoretical model for the etiology of IC we proposed based on our studies in normal urothelial cells (Elgavish et al., Journal of Cellular Physiology 169: 42-51, 52-65, 66-77, 1996): (1) The proliferative ability of a subpopulation of progenitors of urothelial cells is increased in IC; and (2) This change may be the result of recurrent exposure of progenitors of urothelial cells to injury due, possibly but not exclusively, to infection and chronic inflammation. We propose to use this change as a diagnostic tool for IC.

In vitro tissue culture techniques were employed to study the effects of bacterial endotoxins on the growth of normal epithelial cells from the human ureter (NHU). Primary cultures of NHU cells were initiated from explant outgrowth cultures of human ureteral tissue and cultured on collagen gel in F-12* medium containing 1% fetal calf serum (FCS). Optimal clonal growth of secondary cultures of NHU cells seeded at relatively low seeding cell densities, directly on plastic dishes, was achieved in F-12* medium containing bovine pituitary extract (0.5% BPE) and 0.05% BSA. Results indicated that insulin in the F-12* medium could be replaced by three orders of magnitude less IGF-1. Further clonal growth experiments demonstrated that PGE1 is growth stimulatory and can replace BPE as a growth factor requirement. This finding was in agreement with the fact that BPE growth requirement could be replaced by cholera toxin or dibutyryl cAMP. These results suggested that both BPE and cholera toxin operated by activation of a cAMP-dependent mitogenic pathway. Seven gram-negative bacterial lipopolysaccharides (LPS) and three gram-positive bacterial lipotechoic acids (LT) were tested for their effects on NHU clonal growth. Three out of the five LPS derived from Escherichia coli (strains 055:B5, 0128:B12, and 0127:B8), LPS from Klebsiella pneumoniae, and LPS from Pseudomonas aeruginosa all showed significant growth inhibitory effects at minimally effective doses ranging from 5 to 25 micrograms/ml. LPS derived from E. coli strain (0111:B4) had no growth effects at the highest concentration tested (100 micrograms/ml). In contrast, LT derived from Streptococcus pyogenes, S. faecalis, Staphylococcus aureas, and Bacillus subtilis all markedly enhanced clonal growth at concentrations ranging from 1 microgram/ml less than [LT] less than 50 micrograms/ml. LT from Strep. pyogenes was inhibitory to clonal growth at 100 micrograms/ml. The growth inhibitory effects of LPS were shown to be sensitive to the presence of hydrocortisone in the growth medium, indicating that LPS effects on growth are mediated via the arachidonic acid cascade. We speculate that these results indicate a link between the susceptibility of uroepithelial tissue to the pathogenic microflora seen in urinary tract diseases and the differential sensitivity of proliferation-competent uroepithelial cells to growth inhibition by LPS produced by gram-negative bacteria. However, further studies with uropathogenic serotypes will be necessary to corroborate this possibility. The growth-stimulating activity of LTs produced by gram-positive bacteria may be due to their ability to bind to cell-associated fibronectin and to activate the fibronectin receptor as part of ligand receptor-induced mitogenic transmembrane signalling pathway.

We previously reported the presence of a carrier-mediated sulfate transport system in human lung fibroblasts (IMR-90) (A. Elgavish, J. B. Smith, D. J. Pillion, and E. Meezan. J. Cell. Physiol. 125: 243-250, 1985). Kinetic studies carried out in the lung fibroblasts show that Cl- inhibits SO4(2-) uptake in a competitive manner. Taken together with the fact that high extracellular Cl- stimulates SO4(2-) efflux, these results suggest that SO4(2-) uptake into lung fibroblasts occurs via a SO4(2-)-Cl- exchange mechanism. Extracellular HCO3- inhibits sulfate influx in a competitive manner (pH 7.5) but has no marked effect on sulfate efflux. SO4(2-) and HCO3- may therefore have the ability to bind to a common extracellular anion binding site, but they do not appear to exchange for one another. Lowering extracellular pH has a stimulatory effect on the initial rate of sulfate uptake. The pK of the extracellular pH effect is around pH 7.0, indicating that small changes in the extracellular pH around the ambient levels encountered under physiological conditions will markedly affect sulfate influx into the cell. Kinetic studies suggest that lowering extracellular pH increases the initial rate of sulfate influx by increasing the affinity of the carrier for sulfate twofold. Lowering intracellular pH inhibits the initial rate of sulfate influx into the cell. The pK of this intracellular pH effect is also around pH 7.0, indicating that physiological levels of intracellular protons are necessary for the normal activity of the anion exchanger.(ABSTRACT TRUNCATED AT 250 WORDS)

Peridinium cinctum f. westii (Lemm.) Lef., a dominant species in the phytoplankton of Lake Kinneret was studied in batch cultures. External orthophosphate concentrations were depleted by successive transfers to increasingly lower initial orthophosphate concentrations (P0) of 200, 50, 5, 0.6 and 0.2 μM (culture cycles of 45–64 days). Lower cell yields were obtained as P0 decreased, but growth rates were unaffected. The decrease in an intra-cellular P pool unextractable by cold 6% trichloroacetic acid (TCA) was shown to be responsible for the effect on cell yields. A 31P nuclear magnetic resonance (NMR) study revealed that the major constituents of this internal phosphorus pool in phosphate-rich cells were the polyphosphates, organized in rigid aggregates of low mobility. Electron-dense bodies which were observed in phosphate-enriched P. cinctum cells further supported the existence of an aggregate structure of the polyphosphates. It is concluded that P. cinctum stores excess orthophosphate as polyphosphates which accumulate in a rigid morphological structure.

/sup 31/P nuclear magnetic resonance (NMR) spectroscopy of intact Cosmarium sp. cells is presented as a suitable tool for the differentiation of intracellular accumulation pools of polyphosphates. The cold trichloroacetic acid (TCA) insoluble fraction is shown to contain most of the total cellular phosphate in the phosphate rich Cosmarium cells. Moreover, evidence from a /sup 31/P-NMR study and electron microscopic observations of cold TCA treated Cosmarium cells indicate that this fraction consists mostly of polyphosphates which seem to retain the native morphological structure observed in the untreated cells. The determination of orthophosphate in the hot water extract of Cosmarium cells did not measure the polyphosphate pools. Determination of total phosphorus content in the hot water extract rendered a value three times higher than the frequently used orthophosphate determination procedure. However, as revealed by the /sup 31/P-NMR spectra and the chemical analyses of the extract and of the treated cells, even total phosphorus in the extract measured only 30% of the total cellular phosphorus. /sup 31/P-NMR enabled the unequivocal chemical identification of the major phosphate compounds in the hot water extract (Surplus P) as orthophosphate and polyphosphates of about 10 phosphate units chainlength. More than 70% of the accumulation pool ofmore » polyphosphates was still in the cells after extraction. However, the electron microscopy study revealed that the native granular structure of polyphosphates had been destroyed by the hot water extraction procedure.« less