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Biomedical subjects

John W Harmon

Publications and source records attributed to John W Harmon.

12 recordsLinked to original sources
PubMed2006

Selective decrease in the DNA base excision repair pathway in squamous cell cancer of the esophagus.

OBJECTIVES: Oxidative damage can lead to a highly mutagenic 8-oxoguanine lesion, which mispairs with adenosine residues, leading to G:C-->T:A transversions. In mammalian cells 8-oxoguanine glycosylase initiates the DNA base excision repair pathway to repair the 8-oxoguanine lesion. To date, there is no information regarding oxidative DNA damage and repair pathways in esophageal cancer. Therefore we designed the current study to demonstrate the DNA damage and repair pathways in esophageal cancer by expression of 8-oxoguanine glycosylase in reflux-induced and mutagen (methyl-n-amyl nitrosamine)-induced DNA damage and apoptosis in esophageal tumors. METHODS: Gastroduodenal reflux was surgically created in male Sprague Dawley rats (n = 120). Half of the animals received methyl-n-amyl nitrosamine. Animals not undergoing operations served as control animals (n = 10). The experiment concluded 30 weeks postoperatively. Immunohistochemistry for 8-oxoguanine and 8-oxoguanine glycosylase was assessed by 2 independent observers. Protein expression was assessed by using the Western blot method. RESULTS: There was significantly more DNA damage in both adenocarcinoma (n = 15) and squamous cell carcinoma (n = 19), as exemplified by positive 8-oxoguanine expression compared with that seen in control animals (P < .05). 8-Oxoguanine glycosylase was several folds upregulated in adenocarcinoma (P < .05), but there was significantly decreased expression in squamous cell carcinoma (P < .01). The apoptosis was assessed as caspase-dependent and caspase-independent pathways, and both were active and correlated well with 8-oxoguanine expression. CONCLUSION: These results demonstrate the selective decrease in the DNA base excision repair pathway in combined reflux and methyl-n-amyl nitrosamine-induced squamous cell cancer of the esophagus.

Adenocarcinoma↗
PubMed2006

Epidermal growth factor receptor and hedgehog signaling pathways are active in esophageal cancer cells from rat reflux model.

BACKGROUND: Advancements in experimental therapeutics for esophageal cancers have been hampered by the lack of a reliable preclinical model that recapitulates the biology of human cancer, including in vivo growth in an animal model. METHODS: Bilious reflux was induced by esophago-jejunostomy in Sprague-Dawley rats. Nine of 12 (75%) Sprague-Dawley rats developed squamous or adenosquamous cancers, and three cell lines were created by in vitro propagation of freshly resected tumors, JA and JB lines from one cancer, and the AMY cell line from another. We subsequently tested the ability of these cell lines to propagate long-term in vitro and form xenografts in vivo, both hallmarks of transformed cells. In addition, we determined the effects of small molecule inhibitors of two important oncogenic pathways-the epidermal growth factor receptor (EGFR) and Hedgehog (Hh) signaling pathways, in vitro, as a "proof of principle" of using these unique cell lines for developing targeted therapies for esophageal cancer. Mechanism-based growth inhibition was assessed by down-regulation of activated downstream targets of EGFR in the case of Iressa, and by Hh luciferase reporter activity with cyclopamine. RESULTS: JA, JB, and AMY cell lines were able to grow continuously in vitro and consistently form xenografts in vivo in athymic mice, both subcutaneously, as well as in the "orthotopic" location at the gastroesophageal serosal junction (n = 2 mice per line, six of six engrafted). By histology, the tumors grow in vivo as well-differentiated keratinizing squamous cell carcinomas. JB cells had the highest expression of EGFR protein and also the most profound response to Iressa (gefitinib), an EGFR inhibitor (IC50 < 1 microm). Growth inhibition by Iressa was mirrored functionally by down-regulation of activated targets of the EGFR pathway, phospho-ERK1/2 and phospho-MEK levels. AMY cells expressed approximately 900-fold elevation of the Hh ligand, Indian Hh (Ihh), compared with normal esophageal epithelium, whereas expression of another Hh ligand, Sonic Hh (Shh), was not detected. On treatment with the specific Hh small molecule inhibitor cyclopamine, AMY cells demonstrated growth inhibition, which was accompanied by significant down-regulation of endogenous Hh luciferase reporter activity at 24 h and increased apoptosis in treated cells. CONCLUSIONS: We have established a model of esophageal carcinogenesis, capable of long-term in vitro and in vivo passage, and demonstrated therapeutic potential of targeting the EGFR and Hh pathways in the cell lines created from the rodent cancers. These unique cell lines should provide a platform for rapid preclinical validation of novel therapeutics for esophageal cancers.

Animals↗
PubMed2005

Wound healing enhancement: electroporation to address a classic problem of military medicine.

The major goal of wound healing biology is to determine how a wound can be induced to repair damaged tissue faster and more efficiently. Enhancement of dermal and epidermal regeneration is an extremely important goal for the treatment of many different types of wounds. Exogenous application of growth factors to the wound site has been shown to have potential to improve wound healing. Frequent applications of large amounts of growth factor have been required. This is because proteases in the wound quickly destroy peptide growth factor. Gene therapy has the potential to produce growth factors deep within the wound, where they can be effective as well as able to constantly replenish growth factor that is destroyed by peptidases. We have shown that application of plasmid DNA expression vectors directly into the wound is an inefficient modality. Electroporation, the application of an electrical field across cells to permeabilize the cell membrane has led us to explore the possibility of utilizing the technique to enhance transfection efficiency. We have identified electroporation parameters that improve the efficiency of DNA transfection in cutaneous wounds, and we have shown that electroporation itself does not impair wound healing. We are now on the threshold of exploring whether electroporation-assisted transfection with DNA plasmid expression vectors for growth factors will be an effective modality for enhancing cutaneous wound healing.

Animals↗
PubMed2005

The role of magnetic resonance cholangiography in the management of patients with gallstone pancreatitis.

OBJECTIVE: To examine the utility of magnetic resonance cholangiography (MRC) in the preoperative evaluation of patients with gallstone pancreatitis. SUMMARY BACKGROUND DATA: Gallstone pancreatitis is often associated with the presence of common bile duct (CBD) stones that may require endoscopic removal prior to planned laparoscopic cholecystectomy. No reliable clinical criteria exist, however, that can accurately predict CBD stones and the need for preoperative endoscopic retrograde cholangiopancreatography (ERCP). METHODS: Sixty-four patients were identified with gallstone pancreatitis based on clinical presentation and imaging studies over a three-and-a-half-year period. All patients underwent MRC, and the images were evaluated for gallstones, CBD stones, cholecystitis, and pancreatitis RESULTS: Seventeen of the 64 patients (27%) with gallstone pancreatitis were found to have CBD stones confirmed by ERCP. MRC correctly predicted CBD stones in 16 of the 17 patients (sensitivity = 94%). In 1 additional patient, MRC demonstrated CBD stones not seen at ERCP, consistent with probable passage. By comparison, the sensitivities of other criteria for predicting CBD stones were (1) elevated bilirubin >or=2.0 mg/dL = 65%; (2) dilated duct on ultrasound = 55%; and (3) CBD stones on ultrasound = 27%. MRC was able to visualize gallbladder stones in 57 of 62 patients (94%) and correctly predicted acute cholecystitis in 6 of 8 patients. MRC also detected peripancreatic edema and inflammatory changes consistent with acute pancreatitis in 45 of 64 patients (70%). CONCLUSIONS: These results demonstrate that MRC can accurately identify CBD stones preoperatively in patients with gallstone pancreatitis and provide valuable information with respect to other biliary pathology, including cholelithiasis, acute cholecystitis, and pancreatitis. MRC is an effective noninvasive screening tool for CBD stones, appropriately selecting candidates for preoperative ERCP and sparing others the need for an endoscopic procedure with its associated complications.

Acute Disease↗
PubMed2003

Duodenal reflux produces hyperproliferative epithelial esophagitis--a possible precursor to esophageal adenocarcinoma in the rat.

Esophageal reflux of duodenal contents converts a rat nitrosamine esophageal cancer model from squamous cell carcinoma to adenocarcinoma. Further, there was a tendency for male rats to have a higher incidence of cancer than female rats. However, chemical castration with the gonadotropin-releasing hormone analog leuprolide did not protect male or female animals from developing cancer. We have identified an early (6-week) hyperproliferative epithelial cell reaction to duodenal reflux. We carried out experiments to assess the specificity of duodenal reflux in producing the hyperproliferative epithelial precursor lesion. Animals underwent specific surgical procedures to produce esophageal reflux of pure duodenal contents, mixed gastroduodenal, or bland intestinal contents. A hyperproliferative mucosal esophagitis developed in the group with duodenal reflux but not in the other groups. Mucosal thickness in the duodenal reflux group reached seven times that of normal mucosa at 6 weeks. These results suggest that esophageal reflux of duodenal contents plays an important role in the pathogenicity of proliferative esophagitis and the potential development of esophageal adenocarcinoma.

Adenocarcinoma↗
PubMed2002

A nuclear targeting peptide, M9, improves transfection efficiency in fibroblasts.

BACKGROUND: Nonviral transfection of eukaryotic cells remains inefficient. Liposomes can transport DNA plasmid into the cytoplasm, but the nuclear membrane remains a barrier to efficient plasmid DNA transfection. But normal cells have mechanisms to transport nucleic acids across the nuclear membrane. Cells routinely utilize a transporter to carry mRNA from nucleus to cytoplasm. MATERIALS AND METHODS: We used a modified mRNA transporter, the M9 component of heterogeneous nuclear ribonucleoprotein-A1, complexed to a DNA carrier to facilitate DNA transfer into the nucleus. We examined the effect of M9 on transfection in 3T3 fibroblasts. Our hypothesis was that the M9 shuttle would increase transfection efficiency by delivering plasmid to the nucleus, after cytoplasmic entry was facilitated by Lipofectamine. Transfection was assessed using plasmids expressing beta-galactosidase and green fluorescent protein (GFP). Intracellular location of rhodamine-labeled plasmid was determined by fluoroscopic microscopy. RESULTS: In the fluorescent microscopy experiments, we found that rhodamine-labeled DNA plasmid was sequestered in the cytoplasm in the Lipofectamine-treated cells, but gained access to the nucleus with the addition of M9. At concentrations where neither M9 nor Lipofectamine individually increased plasmid mediated transfection, as evidenced by beta-galactosidase activity; their combination increased transfection dramatically by approximately 20-fold, from 2 +/- 1 to 32 +/- 5. CONCLUSIONS: As expected, based on their presumed actions, Lipofectamine and the M9 shuttle synergistically promote efficient cellular transfection. Efficient cellular transfection will be required in clinical applications of gene therapy.

3T3 Cells↗
PubMed2002

Increased laparoscopic experience does not lead to improved results with acute cholecystitis.

BACKGROUND: As laparoscopic experience increases, ever more challenging cases are attempted. Enlarged surgeon experience, along with better technology, has been lauded as improving outcomes. The purpose of this study is to see if this applies to the management of acute cholecystitis. METHODS: We reviewed our experience over a 7 and a half year period. Information was obtained from a prospectively maintained computer database containing patient presentation, demographics, workup, laboratory values, and outcomes. Diagnosis of gangrene was based on pathologic examination of the specimen. RESULTS: There were 305 patients admitted to our institution with acute cholecystitis. Group I (n = 111) was admitted during the first half of the study and group II (n = 194) during the second half. Demographics were similar in the two groups. While slightly more patients were attempted laparoscopically in group II (90% versus 82%), conversion rates were virtually identical (27.1% versus 27.5%). There was a trend toward improved results with group II versus group I in mortality (3% versus 4%) and morbidity (14% versus 21%; P = not significant). Deaths were divided between sepsis and cardiac events. Gangrenous cholecystitis was less frequent in group II patients (29% versus 40%; P = 0.06). Analysis of gangrene versus non-gangrene patients within each group showed that conversion rates remained twice as high (40% versus 20%; P < 0.05) in those with gangrene. Interestingly, gangrene had no effect on morbidity or mortality. CONCLUSIONS: Morbidity and mortality for acute cholecystitis remain relatively high. These seem to be determined by the degree of acute and chronic illness present at the time of diagnosis. As conversion rates remain unchanged, increased surgeon experience and further advances in laparoscopic technology are unlikely to dramatically affect results. Efforts to improve outcomes for this common disease should therefore focus on better and earlier identification of patients for operation.

Acute Disease↗
PubMed2002

Diverticulitis presenting as a strangulated inguinal hernia.

BACKGROUND/AIM: Inguinal hernia is a common diagnosis for patients presenting with a painful groin mass; other potentially dangerous diagnoses may mimic a groin hernia. We present 3 unusual cases of diverticulitis with perforation. The resulting abscess presented clinically as an atypical strangulated inguinal hernia. METHODS/RESULTS: From July 1, 1999, to June 30, 2000, 344 patients were admitted to the Johns Hopkins Bayview Medical Center with the diagnosis of diverticulitis. Of these patients, 44 (12.8%) required surgical intervention. We report here 3 cases in that academic year of diverticulitis complicated by perforation and abscess formation that presented as atypical strangulated inguinal hernias. In addition to being rare and difficult to diagnose, such cases illustrate that a tender inguinal mass may not represent a hernia. CONCLUSIONS: The diagnosis of strangulated inguinal hernias remains primarily one of clinical suspicion. Radiologic and laboratory studies should augment, not replace, one's clinical diagnosis. In addition, the differential diagnosis of inguinal masses is discussed.

Abdominal Abscess↗
PubMedDate not supplied

Novel nuclear shuttle peptide to increase transfection efficiency in esophageal mucosal cells.

The major barrier to successful transfection appears to be passage of the DNA plasmid from the cytoplasm into the cell nucleus. The M9 nuclear localization peptide, a fragment of the naturally occurring heterogeneous nuclear ribonucleoprotein A1, which serves to shuttle messenger RNA across the nuclear membrane, has been proposed as a tool for enhancing transfection efficiency. We tested three different reporter plasmids to assess the ability of M9 to improve transfection efficiency in esophageal mucosal cells. The effect of M9 on the intracellular movement of plasmid was also assessed using fluorescent microscopy to trace rhodamine-labeled plasmid. The M9 nuclear shuttle peptide consistently increased the transfection efficiency. When transfection was carried out with specific plasmids, beta-galactosidase enzyme activity, keratinocyte growth factor-1 growth factor levels, and the number of transfected cells expressing growth factor peptides were progressively increased with increasing M9 to plasmid ratios. Fluorescent microscopy demonstrated that the M9 shuttle allowed rhodamine-tagged plasmid to gain access to the nucleus, while it was located exclusively in the cytoplasm without the peptide. The M9 shuttle peptide increases transfection efficiency in esophageal mucosal cells, and therefore may have a useful role in gene therapy applications involving the esophagus.

Analysis of Variance↗
PubMedDate not supplied

Electroporation enhances transfection efficiency in murine cutaneous wounds.

Transfection of wounds with DNA-encoding growth factors has the potential to improve healing, but current means of nonviral gene delivery are inefficient. Repeated high doses of DNA, necessary to achieve reliable gene expression, are detrimental to healing. We assessed the ability of in vivo electroporation to enhance gene expression. Full-thickness cutaneous excisional wounds were created on the dorsum of female mice. A luciferase- encoding plasmid driven by a CMV promoter was injected at the wound border. Following plasmid administration, electroporative pulses were applied to injection sites. Pulse parameters were varied over a range of voltage, duration, and number. Animals were euthanized at intervals after transfection and the luciferase activity measured. Application of electric pulses consistently increased luciferase expression. The electroporative effect was most marked at a plasmid dose of 50 micro g, where an approximate tenfold increase was seen. Six 100- micro s-duration pulses of 1750 V/cm were found to be the most effective in increasing luciferase activity. High numbers of pulses tended to be less effective than smaller numbers. This optimal electroporation regimen had no detrimental effect on wound healing. We conclude that electroporation increases the efficiency of transgene expression and may have a role in gene therapy to enhance wound healing.

Animals↗
PubMedDate not supplied

Delivery of plasmid DNA expression vector for keratinocyte growth factor-1 using electroporation to improve cutaneous wound healing in a septic rat model.

We have previously shown that wound healing was improved in a diabetic mouse model of impaired wound healing following transfection with keratinocyte growth factor-1 (KGF-1) cDNA. We now extend these findings to the characterization of the effects of DNA plasmid vectors delivered to rats using electroporation (EP) in vivo in a sepsis-based model of impaired wound healing. To assess plasmid transfection and wound healing, gWIZ luciferase and PCDNA3.1/KGF-1 expression vectors were used, respectively. Cutaneous wounds were produced using an 8 mm-punch biopsy in Sprague-Dawley rats in which healing was impaired by cecal ligation-induced sepsis. We used National Institutes of Health image analysis software and histologic assessment to analyze wound closure and found that EP increased expression of gWIZ luciferase vector up to 53-fold compared with transfection without EP (p < 0.001). EP-assisted plasmid transfection was found to be localized to skin. Septic rats had a 4.7 times larger average wound area on day 9 compared with control (p < 0.001). Rats that underwent PCDNA3.1/KGF-1 transfection with EP had 60% smaller wounds on day 12 compared with vector without EP (p < 0.009). Quality of healing with KGF-1 vector plus EP scored 3.0 +/- 0.3 and was significantly better than that of 1.8 +/- 0.3 for treatment with vector alone (p < 0.05). We conclude that both the rate and quality of healing were improved with DNA plasmid expression vector for growth factor delivered with EP to septic rats.

Analysis of Variance↗