Faculty Bibliography

PURPOSE/OBJECTIVE:To test whether the cystatin-like functional domain in tear specific lipocalin (TSL) is functionally active in tears during the normal diurnal cycle and during external ocular infections. METHODS:Capillary tube collected reflex (RTF), open (OTF) and closed (CTF) eye tear samples were recovered from six normals and semi-quantitatively western blot assayed for cystatin C and TSL. CTF samples were immunoprecipitated with antibodies raised against TSL, cystatin C and other antiproteases and screened for the co-precipitation of proteases by casein and gelatin zymography. OTF samples recovered from individuals with viral, fungal and bacterial keratitis were similarly screened for TSL-bound proteases. Human tissue was subjected to immunohistochemical study. RESULTS:Western blot analysis reveals a progressive increase in cystatin C in going from RTF to OTF to CTF samples (approximately 3, 7 and 30 ng microl(-1), respectively). In contrast, the concentration of TSL remains constant (approximately 1500 ng microl(-1)). Immunocytochemistry data show staining of the apical surface of the human conjunctiva and some intra-cellular staining for cystatin C, but not for cystatin A. Zymography confirms earlier data that CTF contains exceptionally high levels of proteases bound to a wide range of specific inhibitors. However, only trace amounts of proteases are complexed with cystatin C and no protease can be detected bound to TSL in either the pathological or CTF samples. CONCLUSION/CONCLUSIONS:Although TSL contains a functional cystatin-like domain, it is not physiologically active during the normal diurnal cycle or during external ocular infections. Reactive proteases in CTF are most likely controlled by the presence of excess levels of more reactive cystatins, especially cystatin C, which accumulates during prolonged eye closure. Immunohistochemical data suggest that the apical conjunctiva may be a contributing source for the accumulating cystatin C.

Gamma interferon (IFN-gamma) is critical in the immune response against Mycobacterium tuberculosis. In an ongoing trial of aerosol IFN-gamma in conjunction with standard drug therapy, we have observed activation of IFN signaling in bronchoalveolar lavage (BAL) cells from tuberculosis (TB) patients. We hypothesized that aerosol IFN-gamma treatment of pulmonary TB would increase expression of genes important for the control of TB. We investigated the expression of downstream genes by measuring inducible nitric oxide synthase (iNOS) and the chemokine IFN-inducible 10-kDa protein (IP-10) by real-time quantitative reverse transcription-PCR. In vitro, M. tuberculosis induced IP-10, and IFN-gamma stimulated this further, with no effect on iNOS expression. We studied 21 patients with pulmonary TB and 7 healthy subjects. Similar to the in vitro model, IP-10 mRNA was increased in BAL cells from TB patients and was augmented after treatment with aerosolized IFN-gamma. TB was also associated with elevated iNOS mRNA, but aerosolized IFN-gamma did not further enhance expression. Genomic analysis identified 1,300 of 4,058 genes expressed in BAL cells from six TB patients before and after 1 month of therapy, including aerosolized IFN-gamma. However, only 15 genes were differentially regulated by IFN-gamma. We conclude that iNOS and IP-10 mRNA expression is increased in TB but that aerosol IFN-gamma treatment increases expression of few genes in the human lung

Gene expression in skeletal muscle is regulated by a family of myogenic basic helix-loop-helix (bHLH) proteins. The binding of these bHLH proteins, notably MyoD and myogenin, to E-boxes in their own regulatory regions is blocked by protein kinase C (PKC)-mediated phosphorylation of a single threonine residue in their basic region. Because electrical stimulation increases PKC activity in skeletal muscle, these data have led to an attractive model suggesting that electrical activity suppresses gene expression by stimulating phosphorylation of this critical threonine residue in myogenic bHLH proteins. We show that electrical activity stimulates phosphorylation of myogenin at threonine 87 (T87) in vivo and that calmodulin-dependent kinase II (CaMKII), as well as PKC, catalyzes this reaction in vitro. We find that phosphorylation of myogenin at T87 is dispensable for skeletal muscle development. We show, however, that the decrease in myogenin (myg) expression following innervation is delayed and that the increase in expression following denervation is accelerated in mutant mice lacking phosphorylation of myogenin at T87. These data indicate that two distinct innervation-dependent mechanisms restrain myogenin activity: an inactivation mechanism mediated by phosphorylation of myogenin at T87, and a second, novel regulatory mechanism that regulates myg gene activity independently of T87 phosphorylation

The diagnosis of septic implant failure can be difficult to make, yet is imperative for optimal patient outcomes in revision total hip arthroplasty. In most cases, a thorough history and physical examination combined with preoperative laboratory tests and an intraoperative frozen section are sufficient to differentiate septic from aseptic failure. If preoperative laboratory test values are elevated, preoperative aspiration of the hip can be used in selected patients to confirm or exclude the diagnosis of infection. Nuclear medicine studies comprise a second-line investigation to evaluate patients with a painful total hip arthroplasty in whom revision surgery otherwise is not indicated. Intraoperative tissue appearance in combination with intraoperative Gram stains are unreliable for detecting periprosthetic sepsis, and neither is adequate when considered alone for ruling out infection at the time of revision total hip arthroplasty. It is imperative that the surgeon doing revision total hip arthroplasty thoroughly understands the relative utility of preoperative and intraoperative tests used to diagnose periprosthetic sepsis

BACKGROUND: In individuals heterozygous for ABCA1 transporter mutations, defective reverse cholesterol transport (RCT) causes low HDL-cholesterol and premature coronary heart disease (CHD). However, the extent to which impaired RCT underlies premature CHD in others with low HDL-cholesterol is not known. The primary acceptors of cell cholesterol are a minor subclass of lipid-poor pre-beta-HDLs. These are generated during remodeling of alpha-HDLs, which account for almost all HDL-cholesterol. We studied the strength of the association of CHD with pre-beta-HDL concentrations in Japanese men. METHODS: Blood was collected from 42 men with clinical CHD and 44 healthy controls 40-70 years of age. Pre-beta-HDL was assayed by crossed immunoelectrophoresis. RESULTS: Cases had lower HDL-cholesterol (-23%), total apolipoprotein A-I (-26%), and pre-beta-HDL (-55%; all P <0.001) concentrations; lower pre-beta-HDL:alpha-HDL ratios (-45%; P <0.001); and higher plasma triglycerides (20%; P <0.03) than the controls. On stepwise logistic regression, CHD was associated most strongly with pre-beta-HDL concentrations. On ROC analysis, pre-beta-HDL concentration discriminated between cases and controls better than any other lipoprotein measurement. When plasma was incubated for 16 h at 37 degrees C, mean (SD) pre-beta-HDL increased by 47 (36)% in controls, but was unchanged in cases (group difference, P <0.001). CONCLUSIONS: Our results suggest that inefficient RCT, secondary to a low pre-beta-HDL concentration and production rate in plasma, contributes to premature CHD in Japanese men with low HDL-cholesterol

Strict spatial and temporal regulation of proliferation and differentiation is essential for proper germline development and often involves soma/germline interactions. In C. elegans, a particularly striking outcome of defective regulation of the proliferation/differentiation pattern is the Pro phenotype in which an ectopic mass of proliferating germ cells occupies the proximal adult germ line, a region normally occupied by gametes. We describe a reduction-of-function mutation in the gene pro-1 that causes a highly penetrant Pro phenotype. The pro-1 mutant Pro phenotype stems from defects in the time and position of the first meiotic entry during early germline development. pro-1(RNAi) produces a loss of somatic gonad structures and concomitant reduction in germline proliferation and gametogenesis. pro-1 encodes a member of a highly conserved subfamily of WD-repeat proteins. pro-1(+) is required in the sheath/spermatheca lineage of the somatic gonad in its role in the proper establishment of the proliferation/differentiation pattern in the germline. Our results provide a handle for further analysis of this soma-to-germline interaction

OBJECTIVE: To characterize the architecture of the zona pellucida in living human eggs and embryos, noninvasively with the PolScope, a digital polarizing light microscope. DESIGN: The PolScope was used to examine zonae pellucida of living human eggs and embryos. SETTING: Academic IVF clinic. PATIENT(S): Patients undergoing fresh, nondonor infertility treatment who underwent egg aspiration, fertilization by intracytoplasmic sperm injection or traditional IVF, and cleavage-stage embryo transfer (day 3). INTERVENTION(S): The PolScope imaged the zona of eggs before intracytoplasmic sperm injection and in cleavage-stage embryos before transfer. MAIN OUTCOME MEASURE(S): Thickness and retardance of three zona layers were measured from eight quadrants. Mean and variance in thickness and retardance were calculated for individual eggs and embryos, between eggs and embryos of a cohort, and across the sample patient population. RESULT(S): Cleavage-stage (day 3) embryos have thinner zonae (15.2 +/- 2.9 microm) than both immature (20.4 +/- 2.4 microm) and mature (19.5 +/- 2.2 microm) eggs. The zona of embryos is thinner, primarily owing to thinning of the outer layer. The thicker the zona layer, the greater its retardance. Considerable variation exists in the thickness and retardance of zona layers around individual eggs and embryos and between members of a cohort. The zona of eggs and embryos from different patients differ in thickness, retardance, and variation. CONCLUSION(S): Thickness and organization of zonae pellucida of human eggs and embryos varies considerably and can be quantitatively imaged with the PolScope

The fms oncogene encodes the macrophage colony-stimulating factor receptor (CSF1R), a transmembrane tyrosine kinase receptor, which is abnormally expressed in breast cancer. Transfection of wild-type CSF1R into HC11 mammary epithelial cells (HC11-CSF1R) renders the transfectants capable of in vitro local invasion and in vivo tumorigenesis. Transfection with CSF1R mutated to express phe at the tyr-721 autophosphorylation site (HC11-CSF1R-721) creates a phenotype that lacks metastastic competence but maintains local invasiveness. Conversely, HC11 cells transfected with CSF1R mutated at tyr-807 (HC11-CSF1R-807) retain their metastatic competence, but are not locally invasive. Our aims were to determine which genes were differentially expressed with transfection of HC11 with wild-type CSF1R, and to determine the effect of mutation at the autophosphorylation sites on gene expression, using 4.6 K cDNA microarrays. Complementary DNA from HC11, HC11-CSF1R-721 and HC11-CSF1R-807 were each hybridized together with HC11-CSF1R on individual arrays. A principal component spectral method combined with prenormalization procedures was used for sample clustering. Differentially expressed genes were identified by the analysis of variance. Confirmation by Northern blotting was performed for MAP kinase phosphatase-1, WDNM1 (extracellular proteinase inhibitor), Trop 2 (tumor-associated calcium signal transducer-2), procollagen type IV alpha, secretory leukoprotease inhibitor, prenylated snare protein Ykt6, ceruloplasmin and chaperonin 10. Many of these genes have not previously been associated with tumor invasion and metastasis. We have successfully identified genes that can be linked to the invasive phenotypes or to tumorigenesis. These genes provide a basis for further studies of metastatic progression and local invasiveness, and can be evaluated as therapeutic targets