Lessons learnt from the construction and implementation of a prospective ventral hernia database.

PURPOSE: The New England VA Hernia Registry was created in 2011 to prospectively collect relevant details of ventral hernia repairs, with the intention to assess and improve long term outcomes. The goal of this study is to assess registry compliance. METHODS: All ventral hernia operations performed in five VA hospitals between 2011-2022 were obtained. We assessed compliance at the hospital and surgeon level. RESULTS: 3,516 cases were performed. Overall compliance with registry entry was 37.5%, ranging from 10.8% to 67.2% across hospitals. At the hospital level, there was a negative correlation between average yearly hernia volume per surgeon and registry compliance (r2 = 0.53). Surgeon compliance varied within hospitals and over time. CONCLUSION: Registry compliance was low and highly variable. Lack of interest, incentives, oversight, and surgeon turnover are possible factors for noncompliance. Building a registry with these factors in mind, providing timely feedback, and conducting frequent audits may improve compliance.

Maspin: synthesis by human cornea and regulation of in vitro stromal cell adhesion to extracellular matrix.

PURPOSE: Maspin, a tumor-suppressor protein that regulates cell migration, invasion, and adhesion, is synthesized by many normal epithelial cells, but downregulated in invasive epithelial tumor cells. The purpose of this study was to determine whether cells in the normal human cornea express maspin and whether maspin affects corneal stromal cell adhesion to extracellular matrix molecules. METHODS: Maspin expression was analyzed by immunodot blot, Western blot, and RT-PCR analyses in cells obtained directly from human corneas in situ. Maspin protein and mRNA were also studied in primary and passaged cultures of corneal stromal cells using Western blot analysis, RT-PCR, and immunofluorescence microscopy. Maspin cDNA was cloned and sequenced from human corneal epithelial cells and expressed in a yeast system. The recombinant maspin was used to study attachment of cultured human corneal stromal cells to extracellular matrices. RESULTS: Maspin mRNA and micromolar amounts of the protein were found in all three layers of the human cornea in situ, including the stroma. Maspin was also detected in primary and first-passage corneal stromal cells, but its expression was downregulated in subsequent passages. Late-passage stromal cells, which did not produce maspin, responded to exogenous recombinant maspin as measured by increased cell adhesion not only to fibronectin, similar to mammary gland tumor epithelial cells, but also to type I collagen, type IV collagen, and laminin. CONCLUSIONS: The corneal stromal cell is the first nonepithelial cell type shown to synthesize maspin. Loss of maspin expression in late-passage corneal stromal cells in culture and their biological response to exogenous maspin suggests a role for maspin on the stromal cells in the cornea. Maspin may function within the cornea to regulate cell adhesion to extracellular matrix molecules and perhaps to regulate the migration of activated fibroblasts during corneal stromal wound healing.

Regulation of nitric oxide synthase 2 in rabbit corneal cells.

PURPOSE: The purpose of these studies was to investigate the role of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), and transforming growth factor-beta (TGF-beta) in the regulation of inducible nitric oxide synthase (NOS2) activity in rabbit corneal cells. METHODS: Rabbit corneal epithelial, stromal, and endothelial cells were grown in culture and treated with cytokines and growth factors, alone or in combination. NOS activity was measured at times up to 72 hours after treatment by assaying the culture medium for nitrite using the Griess reaction. Cell lysates were analyzed by Western blot analysis for NOS2 protein. RNA was isolated and amplified with NOS1-, NOS2-, and NOS3-specific primers by RT-PCR. RESULTS: NOS2 expression was induced by combined cytokine treatment from nondetectable levels to abundant levels in low passage (<4) stromal cells and to low levels in corneal endothelial cells but not in corneal epithelial cells. In the absence of IFN-gamma, little or no nitrite accumulation was induced by TNF-alpha, IL-1beta, or lipopolysaccharide (LPS) treatment. The inductive effects of IFN-gamma were antagonized in a dose-dependent manner by the myxoma virus rabbit IFN-gamma receptor homolog, M-T7. rRaIFN-gamma, in combination with IL-1beta and TNF-alpha, induced the appearance of NOS2 mRNA within 24 hours but detectable nitrite did not accumulate in large amounts (>10 microM) until after 24 hours postinduction. NOS2 was identified as a 130 kDa protein on Western blot analysis using monoclonal antibody against murine NOS2. TGF-beta(1) and beta(2) inhibited the accumulation of cytokine-induced nitrite in a dose-dependent manner while not significantly reducing the steady state level of NOS2 mRNA. The activity of the induced NOS was inhibited by 1400W, a NOS2-selective inhibitor, but not 7-nitroindazole, a NOS1-selective inhibitor. CONCLUSIONS: In cultured corneal stromal cells, NOS2 expression was upregulated by IFN-gamma in combination with IL-1beta and TNF-alpha but not by any of these cytokines alone, while TGF-beta downregulated the activity. Cultures of corneal epithelial cells could not be induced to express NOS2, yet cultures of endothelial cells produced low amounts of NO in response to cytokines. The NOS1 and NOS3 isoforms were not detected in any of these corneal cells.

Interferon coordinately inhibits the disruption of PML-positive ND10 and immediate-early gene expression by herpes simplex virus.

Interferons (IFNs) are important components of the innate immune response, limiting herpes simplex virus (HSV) infection. In recombinant HSV-infected cells, IFN inhibited expression of beta-galactosidase from the immediate-early gene, ICP4, promoter. The extent of inhibition was dependent on IFN dose, IFN type, cell type, and multiplicity of infection (moi). IFN inhibited gene transcription, leading to a complete block in ICP4 promoter-driven gene expression in 90% of cells. The same IFN treatments resulted in an increase in the size and number of nuclear domain 10 (ND10) structures that stained positive by immunofluorescence for the promyelocytic leukemia (PML) protein. In cultures infected at low moi with a recombinant HSV producing ICP4 as a fusion protein with green fluorescence protein, the appearance of green fluorescence in the nucleus coincided with loss of PML-positive ND10 in the same nucleus, even in the rare ICP4-expressing IFN-treated cells. IFN-dependent inhibition was nearly complete when the immediate-early promoter was in the viral genome but was minimal when the promoter was stably integrated into the cellular genome. These data reveal that IFN can completely block viral gene expression in infected cells and that enhancement of the ND10 structure, which is the site of initiation of HSV replication, correlates with the block in viral gene expression.

Cellular adaptation to down-regulated iron transport into lymphoid leukaemic cells: effects on the expression of the gene for ribonucleotide reductase.

Ribonucleotide reductase is an iron-containing enzyme that is essential for DNA synthesis. Whereas previous studies have used various iron chelators to examine the relationship between cellular iron metabolism and ribonucleotide reductase activity in cells, they have not elucidated the relationship between iron transport into cells and the expression of the gene for ribonucleotide reductase. To investigate this, we examined ribonucleotide reductase mRNA, protein and enzyme activity in a novel line of CCRF-CEM cells (DFe-T cells) that display an approx. 60% decrease in their uptake of iron compared with the parental wild-type cell line. We found that DFe-T cells displayed an approx. 40% decrease in ribonucleotide reductase specific enzyme activity relative to wild-type cells without a change in their proliferation. Kinetic analysis of CDP reductase activity revealed an approx. 60% decrease in V(max) in DFe-T cells without a change in K(m). Despite the decrease in enzyme activity, the mRNA and protein for the R1 and R2 subunits of ribonucleotide reductase in DFe-T cells were similar to those of wild-type cells. ESR spectroscopy studies revealed that DFe-T cells had a 22% decrease in the tyrosyl free radical of the R2 subunit, suggesting that a larger amount of R2 protein was present as functionally inactive apo-R2 in these cells. Our studies indicate that ribonucleotide reductase activity in CCRF-CEM cells can be down-regulated by more than 50% in response to down-regulated iron transport without an adverse effect on cell proliferation. Furthermore, our studies suggest a regulatory link between ribonucleotide reductase activity and iron transport into these cells.

Sepsis increases skeletal muscle sodium, potassium-adenosinetriphosphatase activity without affecting messenger RNA or protein levels.

BACKGROUND: Previous studies suggest that sodium, potassium-adenosinetriphosphatase (Na+, K(+)-ATPase) activity in skeletal muscle is increased during sepsis, but the molecular mechanisms are not well understood. We tested the hypothesis that increased muscle Na+,K(+)-ATPase activity during sepsis is associated with increased expression of messenger RNA for the Na+,K(+)-ATPase alpha-1, alpha-2, and beta-1 subunits and increased amounts of the enzyme protein. STUDY DESIGN: Extensor digitorum longus muscles were harvested from rats 16 hours after induction of sepsis by cecal ligation and puncture or sham operation. The Na+,K(+)-ATPase activity was determined spectrophotometrically. Messenger RNA levels for the alpha-1, alpha-2, and beta-1 subunits of Na+,K(+)-ATPase were determined by Northern blot analysis. Enzyme protein levels were measured by Western blot analysis and tritium-ouabain binding assay. RESULTS: Muscle Na+,K(+)-ATPase activity was 46 percent higher in rats that had sepsis than in rats that underwent sham operation (p < .05). No significant differences between septic and control groups were noted in messenger RNA levels for the Na+,K(+)-ATPase subunits. Western blot analysis and tritium-ouabain binding revealed no difference in enzyme protein expression between septic and control animals. CONCLUSIONS: Results suggest that sepsis increases skeletal muscle Na+,K(+)-ATPase activity without affecting enzyme messenger RNA or protein levels. The results are consistent with an increased catalytic constant for individual Na+,K(+)-ATPase protein units, an atypical mechanism for this enzyme.

Endothelial barrier function and Na+/K(+)-ATPase pump density in herpetic stromal disease.

PURPOSE: Corneal edema is a significant component of the various forms of herpes simplex virus type 1 (HSV-1)-induced stromal disease. Maintenance of corneal thickness, a reflection of corneal hydration, depends on a physical barrier formed by endothelial cell-cell junctions and by the activity of Na+/K(+)-ATPase pumps that regulate ion flux and thus influence water movement through this cell layer. These functions were measured in corneas with increased corneal thickness caused by HSV-1-induced stromal disease to determine their contribution to the pathogenesis of the edema. METHODS: Stromal disease with corneal edema was induced in rabbits by intrastromal injection of the RE strain of HSV-1. At various times after infection, during the development of and recovery from stromal disease, endothelial barrier function and Na+/K(+)-ATPase pump sites were measured in excised rabbit corneas. RESULTS: The endothelial permeability coefficient, Ktrans, for 14C-dextran, 3H-inulin, and 14C-mannitol, were not altered significantly during periods of maximal corneal edema and stromal disease. Endothelial Na+/K(+)-ATPase pump density, as measured by ouabain binding, showed a statistically significant (P < 0.05) decrease in HSV-1-infected corneas during peak edema compared to mock antigen-injected or uninjected control corneas. Pump density returned to baseline values by 24 days after infection, concurrent with the resolution of corneal edema. CONCLUSIONS: These results indicate that corneal endothelial barrier function was not altered in this form of HSV-1-induced stromal edema; however, pump density was reduced significantly.

Corneal ablations produced by the neodymium doped yttrium-lithium-fluoride picosecond laser.

This study examines corneal ablations produced by the neodymium doped yttrium-lithium-fluoride (Nd:YLF) picosecond laser. The laser delivers a 1-KHz, 40-ps pulsed, 1,053-nm wavelength beam (with energy measured in microjoules) to a 15-microns diameter spot size. The ablation mechanism is by plasma formation, which generates acoustic shock waves. Using enucleated rabbit (n = 25) and human donor eyes (n = 29), corneas were examined after tissue ablation at energies ranging from 40 to 300 microJ per pulse with various programmed ablation depths and patterns. The histologic data were collected using light microscopy and transmission electron microscopy. The tissue effects and Nd:YLF laser functions studied were ablation thresholds, cutting ability, programmed ablation depth accuracy, and acute endothelial effects. Our study showed histologic ablation thresholds for the following human corneal layers: epithelium = 7.15 +/- 0.05 x 10(11) W/cm2 (34.1 +/- 8.1 J/cm2 per pulse, 50 microJ per pulse); Bowman's layer = 1.33 +/- 0.29 x 10(12) W/cm2 (58.5 +/- 3.3 J/cm2 per pulse, 100-110 microJ per pulse); stroma and endothelium = 7.10 x 10(11) W/cm2 (28.4 J/cm2 per pulse, 50 microJ per pulse). Depth of corneal ablation was found to be directly related to energy and independent of programmed ablation depth. This study shows the endothelial loss in rabbit corneas by energy beams (50 microJ per pulse) focused 100 microns from this layer.

The mechanism of ablation of corneal tissue by the neodymium doped yttrium-lithium-fluoride picosecond laser.

This study examines the structural changes in cornea resulting from plasma formation and propagated acoustic shock waves produced by the neodymium doped yttrium-lithium-fluoride (Nd:YLF) picosecond laser. Human donor eyes and enucleated rabbit eyes were subjected to various ablation patterns at energies ranging from 40 to 300 microJ per pulse. Two distinctly different patterns were produced depending on the location of initial plasma formation. Plasmas initiated at the corneal surface produced smooth, straight-edged ablations of corneal tissue that consisted of collagen fibril fragmentation, fibril organizational disruption, and possible thermal effect observed along the lateral borders and wound apex. The extent of lateral damage was directly related to the energy applied. The range of acute collagen disorganization observed at the ablation edge in rabbit corneas at various pulsed energies was as follows: 50 microJ = 1.0-12 microns, 150 microJ = 3.8-12.5 microns, 250 microJ = 6.2-23.7 microns, and 300 microJ = 7.5-45.0 microns. Plasma formation initiated within the stroma at or above threshold energies (50-150 microJ per pulse) produced an inter- or intralamellar separation effect with little evidence of ablation or collagen fibril fragmentation. Intrastromal plasmas generated from higher energies (200-300 microJ per pulse) produced tissue ablation, along with ablation or disruption of tissue anterior to the intrastromal target area.

Catheter-induced urinary bladder rupture presenting with pneumoperitoneum.

Neonatal bladder injury is rare and usually associated with umbilical artery catherization. Patients may present with apparent renal failure, abdominal distension, and respiratory distress. Treatment involves operative closure of the perforation and bladder drainage. A case of Foley catheter-induced bladder rupture in a premature infant, not previously reported in the literature, is detailed.

Traumatic delivery: a risk factor for bleeding during ECMO?

Traumatic delivery is associated with orthopedic, nervous, and soft tissue injuries. Anticoagulation for extracorporeal membrane oxygenation (ECMO) support may predispose to hemorrhage from these birth injuries. A case of delayed hemoperitoneum secondary to hepatic laceration after delivery and ECMO, not previously reported, is detailed herein.

Regulation of herpes simplex virus thymidine kinase in cells treated with a synergistic antiviral combination of alpha interferon and acyclovir.

Alpha interferon (IFN-alpha) and acyclovir (ACV) are synergistic in their anti-herpes simplex virus activities. IFN-alpha treatment reduced the herpes simplex virus thymidine kinase (TK) activity present in cells 6 h postinfection, while steady-state levels of TK mRNA remained at or above the amount in infected, untreated cells. The inhibition of TK production by IFN-alpha treatment appeared to be transient and translational, not transcriptional.

Ouabain binding kinetics of the rat alpha two and alpha three isoforms of the sodium-potassium adenosine triphosphate.

The Na,K-ATPase has three alpha isoforms which differ in cardiac glycoside sensitivity and tissue distribution. The rodent alpha 1 isoform is relatively resistant to cardiac glycosides, while the alpha 2 and alpha 3 isoforms are quite sensitive. Because both the alpha 2 and alpha 3 isoforms are generally expressed in the same tissue, it has been difficult to differentiate and accurately determine the kinetics of ouabain binding to these isoforms. To more fully understand the interactions of the alpha 2 and alpha 3 isoforms with cardiac glycosides, the association and dissociation rates of ouabain binding were measured in transfected cell lines. cDNA's coding for the rat alpha 2 and alpha 3 isoforms were transfected into NIH 3T3 cells and characterized by Na,K-ATPase activity and [3H]ouabain binding. By individually expressing the alpha 2 and alpha 3 isoforms in ouabain-insensitive NIH 3T3 cells, the ouabain-binding characteristics of each isoform could be accurately determined. The association rate constants of the alpha 2 and alpha 3 isoforms were similar while the dissociation rate constant was 33 times slower for the alpha 3 isoform than the alpha 2 isoform. Calculation of the dissociation constant (Kd) from these rate constants yielded values of 115 and 1.6 nM for rat alpha 2 and alpha 3 isoforms, respectively. Scatchard analysis of the rat alpha 2 isoform produced a similar value for Kd of 37 +/- 9 nM. Inhibition of Na,K-ATPase activity indicates the rodent alpha 1 isoform has an IC50 1000-fold higher than the alpha 2 or alpha 3 isoform at 4.8 x 10(-5) M. The results are consistent with the hypothesis that the order of ouabain affinity between the rat alpha isoforms of the Na,K-ATPase is alpha 3 > alpha 2 >> alpha 1.

Amino acid residues of the Na,K-ATPase involved in ouabain sensitivity do not bind the sugar moiety of cardiac glycosides.

The identification of amino acid substitutions in the alpha subunit of the Na,K-ATPase that alter cardiac glycoside sensitivity provides a unique opportunity to examine the role these residues play in binding to the structural domains of these drugs. Substitution of a residue(s) involved in binding to the sugar moiety of cardiac glycosides would be expected to yield similar affinities for ouabain and its aglycone, ouabagenin. Sheep Na,K-ATPase alpha 1 subunit amino acid substitutions previously shown to influence ouabain sensitivity were tested for activity in the presence of ouabain and ouabagenin. These substitutions included both transmembrane and extracellular regions: C104F, D121E, N122D, Q111K, N122K, and Q111R, A112S. Na,K-ATPase activity versus drug concentration curves yielded I50 values over a 1000-fold range for wild type HeLa and HeLa transfectants. Interestingly, the I50 ratio for ouabagenin to ouabain in all mutants tested indicated a 19-24-fold lower affinity of the Na,K-ATPase for ouabagenin. This constant ratio among all mutations tested implies that the first transmembrane region and the first extracellular loop (H1-H2) do not participate in the binding of the sugar moiety of cardiac glycosides.

Inhibition of ribonucleotide reductase by gallium in murine leukemic L1210 cells.

Our previous studies of the mechanism of cell growth inhibition by gallium have suggested that the block in cellular iron uptake induced by transferrin-gallium results in an inhibition of the iron-dependent M2 subunit of ribonucleotide reductase. However, it is not known whether the inhibitory effect of gallium on ribonucleotide reductase is solely the result of limiting iron availability for enzyme activity or whether a direct effect of intracellular gallium on the enzyme is also involved. In the present study, utilizing a cell-free assay, we show that gallium nitrate directly inhibits CDP and ADP reductase activity. Inhibition of DNA synthesis by gallium nitrate thus appears to be due to a combination of a block in iron availability to ribonucleotide reductase and a direct inhibition of the enzyme by gallium.

Patterns of antigen expression in hepatoblastoma and hepatocellular carcinoma in childhood.

Two hepatocellular carcinomas and six hepatoblastomas were examined for the presence of 13 antigens using immunoperoxidase, avidin-biotin, staining techniques. Primary antibodies were directed against alpha-fetoprotein (AFP), alpha-1-antitrypsin (AAT), lysozyme (LYS), carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG), glial fibrillary acidic protein (GFAP), neuron specific enolase (NSE), epithelial membrane antigen (EMA), hepatitis B surface antigen (HbSA), lactoferrin (LF), desmin (DES), vimentin (VIM), and keratin (KER). Except for HbSA, the antigen staining pattern was unable to differentiate between hepatoblastoma and hepatocellular carcinoma. Both neoplasms where positive for AFP, AAT, CEA, EMA, and KER; however, neither stained for GFAP, NSE, LYS, LF, HCG, or DES. Vimentin was weakly positive in those hepatoblastomas where mesenchymal tissue was present in the tumor. Only the tissue adjacent to hepatocellular carcinomas stained positively for HbSA and correlated with the elevated serum levels of HbSA.

Inhibition of leukemic HL60 cell growth by transferrin-gallium: effects on ribonucleotide reductase and demonstration of drug synergy with hydroxyurea.

Cellular requirements for iron during DNA synthesis are related to the increased activity of the iron-containing M2 subunit of ribonucleotide reductase, the enzyme responsible for the reduction of ribonucleotides to deoxyribonucleotides. We have previously shown that transferrin-gallium (Tf-Ga) inhibits cellular iron incorporation. In the present study, Tf-Ga-induced inhibition of HL60 cell growth and upregulation of Tf receptor density was reversed with hemin. Cells exposed to 2 mumol/L Tf-Ga for six hours or longer displayed a diminution in the electron spin resonance (ESR) spectroscopy signal of the tyrosyl radical of the M2 subunit of ribonucleotide reductase. The effect of Tf-Ga on the ESR signal was reversed by hemin. Tf-Ga decreased the incorporation of 14C-adenosine into DNA and decreased intracellular deoxyribonucleotide pools, with the maximum diminution seen in deoxyadenosine triphosphate (dATP) and deoxycytidine triphosphate (dCTP) pools. Exposure of cells to combinations of Tf-Ga and hydroxyurea (a known inhibitor of ribonucleotide reductase) resulted in a marked inhibition of cell growth that was consistent with drug synergy. Our studies suggest that Tf-Ga inhibits DNA synthesis through action on the M2 subunit of ribonucleotide reductase and that combinations of Ga and hydroxyurea should be further evaluated in in vivo tumor models.

Adenosine deaminase in herpes simplex virus induced corneal stromal disease.

The development of herpes simplex virus (HSV)-induced disciform stromal disease produced by the intrastromal injection of the RE strain of HSV-1 is characterized by concurrent increases in adenosine deaminase (ADA) activity and corneal thickness. ADA activity increased from 8.6 units/cornea in normal corneas to about 14.1 units and 31.1 units in mock-infected controls and HSV-infected corneas respectively by 15 days postinjection. The molecular weight species of ADA in the corneas of HSV-infected rabbits appeared altered relative to that present in corneas which were not infected. A single topical dose with as low as 0.1% 2'-deoxycoformycin (dCF), an ADA inhibitor possessing immunosuppressive activity, was capable of totally inhibiting the corneal ADA activity. Titration of the free dCF in the cornea following a single topical dose of 0.25% dCF indicated that enough dCF remained in the cornea 24 hrs after instillation to totally inhibit all corneal ADA plus 66 units of additional enzyme. These data suggest that ADA may be a suitable target for immunosuppressive therapy during HSV-induced disciform stromal disease.