HIV-1 gp140 epitope recognition is influenced by immunoglobulin DH gene segment sequence.

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.

Age-independent myocardial infarct quantification by signal intensity percent infarct mapping in swine.

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.

Percent infarct mapping for delayed contrast enhancement magnetic resonance imaging to quantify myocardial viability by Gd(DTPA).

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 SI(remote+2SD), SI(remote+6SD), SI full width half maximum (SI(FWHM)), 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, IV(SIremote+2SD) and IV(SIremote+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. SI(FWHM), 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 SI(FWHM) 8.3 mL [3.79, 19.0]. SI-PIM(FWHM), however, underestimates TTC (-5.74 mL [-11.89; -2.52] (P < 0.01)). Thus, SI-PIM outperforms SI(FWHM) 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-SI(FWHM), 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.

Differentiation of acute and four-week old myocardial infarct with Gd(ABE-DTTA)-enhanced CMR.

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.

Virtual in vivo biopsy map of early prostate neoplasm in TRAMP mice by MRI.

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 (R1(norm)). The R1(norm) values and the histological grades in the corresponding areas were correlated. RESULTS: 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. CONCLUSION: This method has the potential for early noninvasive detection of developing prostate cancer.

Epigenetic reprogramming: a possible etiological factor in bladder pain syndrome/interstitial cystitis?

PURPOSE: The etiology of bladder pain syndrome/interstitial cystitis is poorly understood. The possibility that epigenetic reprogramming may have a role is discussed. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

SIRT1 is significantly elevated in mouse and human prostate cancer.

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 progression in the transgenic adenocarcinoma of mouse prostate mouse is related to energy balance, body mass, and body composition, but not food intake.

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 degrees C or 22 degrees C and pair fed the same diet for 21 weeks (95% of ad libitum intake at 27 degrees C). In the second experiment, TRAMP mice were housed at 27 degrees C or 22 degrees C and fed the same diet ad libitum for 21 weeks. Despite a similar calorie intake, pair-fed mice at 27 degrees 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 degrees 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 degrees C consumed approximately 30% more calories than ad libitum-fed mice at 27 degrees 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.

In the Absence of Central pre-B Cell Receptor Selection, Peripheral Selection Attempts to Optimize the Antibody Repertoire by Enriching for CDR-H3 Y101.

Sequential developmental checkpoints are used to "optimize" the B cell antigen receptor repertoire by minimizing production of autoreactive or useless immunoglobulins and enriching for potentially protective antibodies. The first and apparently most impactful checkpoint requires µHC to form a functional pre-B cell receptor (preBCR) by associating with surrogate light chain, which is composed of VpreB and λ5. Absence of any of the preBCR components causes a block in B cell development that is characterized by severe immature B cell lymphopenia. Previously, we showed that preBCR controls the amino acid content of the third complementary determining region of the H chain (CDR-H3) by using a VpreB amino acid motif (RDR) to select for tyrosine at CDR-H3 position 101 (Y101). In antibodies bound to antigen, Y101 is commonly in direct contact with the antigen, thus preBCR selection impacts the antigen binding characteristics of the repertoire. In this work, we sought to determine the forces that shape the peripheral B cell repertoire when it is denied preBCR selection. Using bromodeoxyuridine incorporation and evaluation of apoptosis, we found that in the absence of preBCR there is increased turnover of B cells due to increased apoptosis. CDR-H3 sequencing revealed that this is accompanied by adjustments to DH identity, DH reading frame, JH, and CDR-H3 amino acid content. These adjustments in the periphery led to wild-type levels of CDR-H3 Y101 content among transitional (T1), mature recirculating, and marginal zone B cells. However, peripheral selection proved incomplete, with failure to restore Y101 levels in follicular B cells and increased production of dsDNA-binding IgM antibodies.

Myocardial tissue characterization by combining late gadolinium enhancement imaging and percent edema mapping: a novel T2 map-based MRI method in canine myocardial infarction.

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.

beta1A integrin expression is required for type 1 insulin-like growth factor receptor mitogenic and transforming activities and localization to focal contacts.

The cells' ability to proliferate in response to growth factor stimulation is significantly altered during cancer progression. To investigate the mechanisms underlying these alterations in prostate cancer, the role and expression of beta1A integrin and type 1 insulin-like growth factor receptor (IGF-IR), known to contribute to cell proliferation and transformation, were analyzed. Using small interfering RNA oligonucleotides to down-regulate beta1A, we show that beta1A expression is required for IGF-IR-mediated prostate cancer cell proliferation and anchorage-independent growth. In vivo, using age-matched transgenic adenocarcinoma of mouse prostate (TRAMP) mice at different stages of prostate cancer [prostatic intraepithelial neoplasia, PIN; well-differentiated adenocarcinoma, WD; and poorly differentiated adenocarcinoma, PD], the expression of beta1A and of IGF-IR was studied. beta1A and IGF-IR expression levels were concurrently up-regulated in high PIN and WD, whereas their expression did not correlate in late-stage PD. In contrast to the up-regulated expression of beta1A, the levels of beta1C, a beta1 cytoplasmic variant that inhibits cell proliferation, were down-regulated in all stages of prostate cancer. A similar expression pattern was observed for a beta1C downstream effector, Grb2-associated binder-1 (Gab1) which is known to inhibit IGF-IR phosphorylation. To analyze in vitro the mechanistic implications of beta1A, beta1C, and Gab1 deregulation in prostate cancer, we investigated whether expression of either beta1 variant in beta1-null cells affected IGF-IR localization. We found that IGF-IR and beta1A were colocalized in highly specialized integrin signaling compartments, designated focal contacts. However, in the presence of beta1C, IGF-IR remained diffuse on the cell surface and did not localize to focal contacts. The findings that beta1 integrins and IGF-IR are concurrently deregulated and that expression of beta1 integrins is necessary to achieve appropriate IGF-IR intracellular distribution point to the important role that the cross-talk between these receptors may have during prostate cancer progression and will be helpful in formulating new therapeutic strategies.

Alterations in B cell development, CDR-H3 repertoire and dsDNA-binding antibody production among C57BL/6 ΔD-iD mice congenic for the lupus susceptibility loci sle1, sle2 or sle3.

Systemic lupus erythematosus (SLE) is an autoimmune disease that reflects a failure to block the production of self-reactive antibodies, especially those that bind double-stranded DNA (dsDNA). Backcrossing the lupus-prone NZM2410 genome onto C57BL/6 led to the identification of three genomic intervals, termed sle1, sle2 and sle3, which are associated with lupus susceptibility. We previously generated a C57BL/6 strain congenic for an immunoglobulin DH locus (ΔD-iD) that enriches for arginine at dsDNA-binding positions. We individually introduced the ΔD-iD allele into the three sle strains to test whether one or more of these susceptibility loci could affect the developmental fate of B cells bearing arginine-enriched CDR-H3s, the CDR-H3 repertoire created by the DH and the prevalence of dsDNA-binding antibodies. We found that the combination of the ΔD-iD allele and the sle1 locus led to a decrease in mature, recirculating B cell numbers and an increase in marginal zone cell numbers while maintaining a highly charged CDR-H3 repertoire. ΔD-iD and sle2 had no effect on peripheral B cell numbers, but the CDR-H3 repertoire was partially normalized. ΔD-iD and sle3 led to an increase in marginal zone B cell numbers, with some normalization of hydrophobicity. Mice with ΔD-iD combined with either sle1 or sle3 had increased production of dsDNA-binding IgM and IgG by 12 months of age. These findings indicate that the peripheral CDR-H3 repertoire can be categorically manipulated by the effects of nonimmunoglobulin genes.

Violation of an evolutionarily conserved immunoglobulin diversity gene sequence preference promotes production of dsDNA-specific IgG antibodies.

Variability in the developing antibody repertoire is focused on the third complementarity determining region of the H chain (CDR-H3), which lies at the center of the antigen binding site where it often plays a decisive role in antigen binding. The power of VDJ recombination and N nucleotide addition has led to the common conception that the sequence of CDR-H3 is unrestricted in its variability and random in its composition. Under this view, the immune response is solely controlled by somatic positive and negative clonal selection mechanisms that act on individual B cells to promote production of protective antibodies and prevent the production of self-reactive antibodies. This concept of a repertoire of random antigen binding sites is inconsistent with the observation that diversity (DH) gene segment sequence content by reading frame (RF) is evolutionarily conserved, creating biases in the prevalence and distribution of individual amino acids in CDR-H3. For example, arginine, which is often found in the CDR-H3 of dsDNA binding autoantibodies, is under-represented in the commonly used DH RFs rearranged by deletion, but is a frequent component of rarely used inverted RF1 (iRF1), which is rearranged by inversion. To determine the effect of altering this germline bias in DH gene segment sequence on autoantibody production, we generated mice that by genetic manipulation are forced to utilize an iRF1 sequence encoding two arginines. Over a one year period we collected serial serum samples from these unimmunized, specific pathogen-free mice and found that more than one-fifth of them contained elevated levels of dsDNA-binding IgG, but not IgM; whereas mice with a wild type DH sequence did not. Thus, germline bias against the use of arginine enriched DH sequence helps to reduce the likelihood of producing self-reactive antibodies.

HIV protease inhibitor ritonavir induces lipoatrophy in male mice.

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.

Infarct density distribution by MRI in the porcine model of acute and chronic myocardial infarction as a potential method transferable to the clinic.

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.

The link between antibodies to OxLDL and natural protection against pneumococci depends on D(H) gene conservation.

Selection and physiological production of protective natural antibodies (NAbs) have been associated with exposure to endogenous antigens. The extent to which this association depends on germline NAb sequence is uncertain. Here we show that alterations in germline D(H) sequence can sever the association between the production of self-reactive NAbs and NAbs that afford protection against a pathogen. In unmanipulated hosts, the availability of the evolutionarily conserved DFL16.1 gene segment sequence profoundly affected the serum levels of NAbs against bacterial phosphorylcholine but not oxidized low-density lipoprotein. Mice with partially altered DFL16.1 sequence could use N nucleotides to recreate the amino acid sequence associated with the classical protective T15 idiotype­positive NAbs, whereas those without DFL16.1 could not. DFL16.1 gene-deficient mice proved more susceptible to challenge with live Streptococcus pneumoniae. Our findings indicate that although production of self-reactive NAbs can be independent of germline D(H) sequence, their capacity to provide protection against pathogens cannot. The potential relevance of these findings for the rational design of vaccines is discussed.

Acute infarct selective MRI contrast agent.

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.

Determination of infarct size in ex vivo swine hearts by multidetector computed tomography using gadolinium as contrast medium.

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.

Quantification of myocardial viability distribution with Gd(DTPA) bolus-enhanced, signal intensity-based percent infarct mapping.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.