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.