Faculty Bibliography

HIV DNA vaccines are potent inducers of cell-mediated immune (CMI) response in mice but elicit poor HIV-specific IFN-gamma-producing T cells in monkeys and humans. In this study, we performed kinetic analyses on splenocytes of BALB/c mice that were immunized by a single injection with a unique DNA vaccine. Using IFN-gamma-ELISPOT and multiparametric FACS analysis, we characterized the induced CMI response. We found that the response was detectable for at least 63 wk. ELISPOT detection of IFN-gamma-producing T cells showed a profile with two waves separated by a long period of minimal response. Multiparametric FACS analysis showed two populations of CD3(+)CD8(+) T cells that were specific for all HIV Ags. These cells had similar robust proliferation abilities and contained granzyme B. However, only a few produced IFN-gamma. Both IFN-gamma-producing and non-IFN-gamma-producing HIV-specific CD8(+) T cells were detected in the early stage (week (W)1 and W2 postimmunization (PI)), in the prolonged intermediate period of minimal response (W4-W26 PI), and in the final late phase of increased response (W30-W63 PI). Our longitudinal characterization showed that both subsets of cells underwent expansion, contraction, and memory generation/maintenance phases throughout the lifespan of the animal. Altogether, these findings bring insight to the heterogeneity of the immune T cell response induced by a single immunization with this DNA and strengthen the concept that used of the IFN-gamma-ELISPOT assay alone may be insufficient to detect critical T cell responses to candidate HIV vaccines.

BACKGROUND: Fibroblast growth factor receptor 1 (FGFR1) mRNA can be alternatively spliced to generate isoforms containing (FGFR1alpha) or lacking (FGFR1beta) the first immunoglobulin-like domain. We examined which isoforms are expressed by cultured prostate cells, their affinities for FGF-2, and the effect of heparin on FGF-2 binding. METHODS: FGFR1 isoform expression was examined by RT-PCR. FGFR1alpha and FGFR1beta were expressed in CHO cells mutant in heparan sulfate synthesis, and their affinities for FGF-2, FGF-1, FGF-4, and FGF-6 were determined in the presence and absence of heparin. RESULTS: FGFR1alpha was expressed in luminal epithelial cells, whereas FGFR1beta was expressed in basal epithelial and smooth muscle cells. FGFR1beta bound FGF-2 with three-fourfold higher affinity than FGFR1alpha both in the presence and absence of heparin. Heparin increased affinity of both receptor isoforms for FGF-2 approximately four-fivefold. CONCLUSIONS: Prostate smooth muscle and basal epithelial cells are likely to be more sensitive than luminal epithelial cells to the low concentrations of FGFs present in vivo

We present a fast, versatile and adaptive-multiscale algorithm for analyzing a wide-variety of DNA microarray data. Its primary application is in normalization of array data as well as subsequent identification of 'enriched targets', e.g. differentially expressed genes in expression profiling arrays and enriched sites in ChIP-on-chip experimental data. We show how to accommodate the unique characteristics of ChIP-on-chip data, where the set of 'enriched targets' is large, asymmetric and whose proportion to the whole data varies locally. SUPPLEMENTARY INFORMATION: Supplementary figures, related preprint, free software as well as our raw DNA microarray data with PCR validations are available at http://www.math.umn.edu/~lerman/supp/bioinfo06 as well as Bioinformatics online

Little is known about the formation of the interventricular septum (IVS), a central event during cardiogenesis. Here, we describe a novel population of myocardial progenitor cells in the primitive ventricle of the mouse embryo, which is characterized by expression of lysozyme M (lysM). Using LysM-Cre mice we show that lysozyme expressing cells give rise to the IVS and to a part of the left ventricular free wall, demonstrating that these heart regions are developmentally related. LysM+ precursors are not of hematopoietic origin and develop in the absence of transcription factors that regulate lysozyme expression in macrophages. LysM-deficient mice lack an overt cardiac phenotype, perhaps due to compensation by the related lysozyme P, which we also found to be expressed in the developing heart. Direct visualization of lysM expression, using LysM-EGFP knock-in mice, showed that ventricular septation is initiated at embryonic day 9 by the movement of myocardial trabeculae from the primitive ventricle towards the bulbo-ventricular groove and revealed the dynamics of IVS formation at later stages. Our studies predict that LysM-Cre mice will be useful to inactivate genes in the developing IVS

Earlier studies suggested that TSP50 is a testis-specific gene that encodes a protein, which is homologous to serine proteases but differs in that threonine replaces serine in its catalytic triad. Most importantly, it was abnormally reactivated in many breast cancer biopsies tested. While further investigating its biochemical and cell biological natures, we found that TSP50 exhibited enzyme activity and was located in the endoplasmic reticulum and cytosol membrane. During our studies to elucidate the regulatory mechanisms related to its differential expression, we discovered a putative p53-binding site and several Sp1-binding sites in the TSP50 promoter, which led us to test if it was regulated by the p53 gene. We found that the p53 transgene negatively regulated the TSP50 promoter in diverse types of cell lines. This result was consistent with other observations: (a) p53 overexpression reduced endogenous TSP50 expression; and (b) breast cancer cell lines containing mutated p53, such as MCF7/Adr, or normal p53, such as MCF7, produced high or low levels of TSP50 transcripts, which was consistent with the fact that TSP50 promoter activity was much higher in MCF7/Adr than that in MCF7 cells. We also found that the quantity of Sp1 transcription factor was lower in MCF7/Adr than in MCF7 cells, which suggested that another mechanism (i.e., transcription factor modulation) was also involved in TSP50 differential expression.

Retinoic acid (RA) signaling is important for multiple aspects of embryonic development and tissue homeostasis. Heterodimers of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) transduce RA signaling. It is not yet clear how the diversity of receptor combinations relates to the diversity of functions for RA. The expression patterns of three zebrafish RARs and four RXRs were reported recently. Here, we identify an additional RAR, a zebrafish RARgamma paralog, and two additional RXRs, duplicates of the previously identified RXRalpha and RXRgamma. Thus, the zebrafish genome contains duplicates of each RAR and RXR gene. All zebrafish RAR and RXR paralogs have overlapping and distinct areas of expression, as might be expected for duplicate genes in the process of diverging in function. By representing what is potentially the complete set of zebrafish RARs and RXRs, this study provides a valuable reference for future functional studies of the individual zebrafish RARs and RXRs

Epithelia can adjust the permeability of their paracellular permeation route to physiological requirements, pathological conditions, and pharmacological challenges. This is reflected by a transepithelial electrical resistance (TER) ranging from a few tenth to several thousands Omega.cm(2), depending on the degree of sealing of the tight junction (TJ). The present work is part of an effort to understand the causes and mechanisms underlying these adaptations. We observed that an extract of human urine (hDLU) increases TER in a concentration- and time-dependent manner and is more effective when added from the basolateral side of cultured monolayers of Madin-Darby canine kidney cells than from the apical one. We found that its main TER-increasing component is epidermal growth factor (hEGF), as depletion of this peptide with specific antibodies, or inhibition of its receptor with PD153035, abolishes its effect. Since the permeability of the TJ depends on the expression of several species of membrane proteins, chiefly claudins, we explored whether hDLU can affect five members of the claudin family, the three known members of the ZO family, and occludin. EGF present in hDLU decreases the content of claudins-1 and -2 as well as delocalizes them from the TJ and increases the content of claudin-4. As expected from the fact that the degree of sealing of the TJ must be a physiologically regulated parameter, besides of hEGF, we also found that hDLU appears to contain also other components that decrease TER, claudin-4 and -7, and that seem to act with different kinetics than the TER-increasing ones.

Although ras is a potent mitogenic oncogene, its tumorigenicity depends on cellular context and cooperative events. Here we show that low-level expression of a constitutively active Ha-ras in mouse urothelium induces simple urothelial hyperplasia that is resistant to progression to full-fledged bladder tumors even in the absence of Ink4a/Arf. In stark contrast, doubling of the gene dosage of the activated Ha-ras triggered early-onset, rapidly growing, and 100% penetrant tumors throughout the urinary tract. Tumor initiation required superseding a rate-limiting step between simple and nodular hyperplasia, the latter of which is marked by the emergence of mesenchymal components and the coactivation of AKT and STAT pathways as well as PTEN inactivation. These results indicate that overactivation of Ha-ras is both necessary and sufficient to induce bladder tumors along a low-grade, noninvasive papillary pathway, and they shed light on the recent findings that ras activation, via point mutation, overexpression, or intensified signaling from FGF receptor 3, occurs in 70%-90% of these tumors in humans. Our results highlight the critical importance of the dosage/strength of Ha-ras activation in dictating its tumorigenicity - a mechanism of oncogene activation not fully appreciated to date. Finally, our results have clinical implications, as inhibiting ras and/or its downstream effectors, such as AKT and STAT3/5, could provide alternative means to treat low-grade, superficial papillary bladder tumors, the most common tumor in the urinary system

A web-based survey was developed to evaluate joint arthroplasty surgeon's preferences for the return to sporting activities after total hip arthroplasty. This survey listed 30 groups of activities (37 specific sports) and was sent to all members of the Hip Society and American Association of Hip and Knee Surgeons. All surgeons were asked to grade each activity as follows: allow, allow with experience, not allowed, or undecided. Results were computed using a power analysis, Z test, and chi(2) test to determine statistical significance. There were a total of 549 responses giving an overall response rate of 72%, with 93% (92/99) of the Hip Society members and 72% (522/727) of American Association of Hip and Knee Surgeons members responding to the survey. Consensus guidelines and postoperative timing for the return to specific activities are presented

FKBP52 is a member of the FK506-binding family of immunophilins and serves as a co-chaperone for steroid hormone nuclear receptors to govern appropriate hormone action in target tissues. Male mice missing Fkbp52 are infertile, and this infertility has been ascribed to compromised sensitivity of the anterior prostate, external genitalia, and other accessory sex organs to androgen. Here, we show additional defects contributing to infertility. We found that epididymal Fkbp52(-/-) sperm are sparse often with aberrant morphology, and they have reduced fertilizing capacity. This phenotype, initially observed in null males on a C57BL/6/129 background, is also maintained on a CD1 background. Expression studies show that while FKBP52 and androgen receptor are co-expressed in similar cell types in the epididymis, FKBP52 is also present in epididymal sperm flagella. Collectively, our results suggest that reduced number and abnormal morphology contribute to compromised fertilizing capacity of Fkbp52(-/-) sperm. This study is clinically relevant because unraveling the role of immunophilin signaling in male fertility will help identify new targets for male contraceptives and/or alleviate male infertility.