Towards gene therapy of postoperative adhesions: fiber and transcriptional modifications enhance adenovirus targeting towards human adhesion cells.

Postoperative abdominal/pelvic peritoneal adhesions are a major source of morbidity (bowel obstruction, infertility, ectopic gestation as well as chronic pelvic pain) in women. In this study, we screened various transduction and transcription modifications of adenovirus (Ad) to identify those that support maximal Ad-mediated gene delivery to human adhesion fibroblasts, which in turn would enhance the efficacy of this novel treatment/preventative strategy for postoperative adhesions. We transduced primary cultures of human peritoneal adhesion fibroblasts with fiber-modified Ad vectors Ad5-RGD-luc, Ad5-Sigma-luc, Ad5/3-luc and Ad5-CAV2-luc as well as transcriptional targeting viruses Ad5-survivin-luc, Ad5-heparanase-luc, Ad5-mesothelin (MSLN)-CRAd-luc and Ad5-secretory leukoprotease inhibitor (SLPI)-luc, and compared their activity to wild-type Ad5-luc. At 48 h, luciferase activity was measured and normalized to the total protein content in the cells. Among the fiber-modified Ad vectors, Ad5-Sigma-luc and among the transcriptional targeting modified Ad vectors, Ad5-MSLN-CRAd-luc showed significantly increased expression levels of luciferase activity at 5, 10 and 50 plaque forming units/cell in adhesion fibroblast cells compared with wild-type Ad5-luc (p < 0.05). Specific modifications of Ad improve their gene delivery efficiency towards human peritoneal adhesion fibroblasts. Developing a safe localized method to prevent/treat postoperative adhesion formation would have a major impact on women health.

Uterine fibroids are characterized by an impaired antioxidant cellular system: potential role of hypoxia in the pathophysiology of uterine fibroids.

PURPOSE: Fibroids are the most common smooth muscle overgrowth in women. This study determined the expression and the effect of hypoxia on two potent antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) on human fibroid cells. METHODS: Immortalized human leiomyoma (fibroid) and myometrial cells were subjected to hypoxia (2 % O2, 24 h). Total RNA and cell homogenate were obtained from control and treated cells; CAT and SOD mRNA and activity levels were determined by real-time RT-PCR and ELISA, respectively. RESULTS: Fibroid cells have significantly lower antioxidant enzymes, SOD and CAT mRNA and activity levels than normal myometrial cells (p < 0.05). Hypoxia treatment significantly increased SOD activity in myometrial cells while significantly decreasing CAT activity in fibroid cells (p < 0.05). There was no significant difference in CAT mRNA levels or activity in response to hypoxia in myometrial cells. Also, there was no significant difference in SOD mRNA levels in response to hypoxia in myometrial cells. CONCLUSION: This is the first report to show that uterine fibroids are characterized by an impaired antioxidant cellular enzymatic system. More importantly, our results indicate a role for hypoxia in the modulation of the balance of those enzymes in fibroid and myometrial cells. Collectively, these results shed light on the pathophysiology of fibroids thereby providing potential targets for novel fibroid treatment.

A simple and rapid purification of kallikrein from rat submandibular gland.

Rat submandibular kallikrein was isolated in an 87% yield by a very quick and simple procedure involving hydrophobic interaction chromatography. Furthermore, that purification method was superior to both aprotinin-affinity chromatography and immunoaffinity chromatography for the purification of rat submandibular kallikrein. The kallikrein purified by hydrophobic interaction chromatography consisted of a number of isoenzymes. The major component of Mr 38,000 seen on SDS-gel electrophoresis was found to be the glycosylated kallikrein, whereas the minor component of Mr 26,000 represented the non-glycosylated enzyme.

Mycosis fungoides exhibits a Th1-type cell-mediated cytokine profile whereas Sezary syndrome expresses a Th2-type profile.

We determined T-cell cytokine profiles in the epidermis, dermis, and blood of cutaneous T-cell lymphoma to differentiate whether unique cytokine profiles were associated with mycosis fungoides (MF) versus Sezary syndrome. Punch biopsy specimens from plaque stage MF (n = 7) were compared to Sezary skin (n = 3) after undergoing rapid heat-saline separation of epidermis from dermis. Normal adult skin (n = 11), neonatal foreskin (n = 4), untreated psoriatic plaques (n = 6), and normal donor peripheral blood leukocytes (n = 3) were studied as controls. Total RNA was extracted from all skin specimens, as well as peripheral blood leukocytes from MF (n = 3) and Sezary patients (n = 7), and was converted to cDNA by reverse transcriptase. Polymerase chain reaction amplification of cDNAs using interleukin 2 (IL-2), IL-4, IL-5, IL-10, and interferon gamma-specific primers was used to differentiate Th1-type responses (IL-2+ and interferon gamma +) from Th2-type responses (IL-4+, IL-5+, and IL-10+). beta-actin specific primers were included as a positive control for mRNA integrity. All MF specimens contained mRNAs for IL-2 and interferon gamma limited to epidermis but not IL-4, IL-5, or IL-10. In contrast, Sezary skin and blood showed a cytokine mRNA pattern dominated by IL-4, IL-5, and IL-10. MF blood showed a pattern similar to normal peripheral blood T cells with mixed detection of all T-helper cell cytokine mRNAs. All psoriasis samples contained mRNAs for IL-2 and interferon gamma in both epidermis and dermis with no IL-4 or IL-10 in either compartment. These findings demonstrate that the cutaneous lesions of MF are characterized by an epidermal Th1-type cytokine profile, whereas both the blood and skin of patients with Sezary syndrome is characterized by a Th2-type profile. This work suggests that differences in cytokine production may be related to the pathophysiology and clinical presentation in cutaneous T-cell lymphoma.

T-cell receptor gene rearrangement in canine mycosis fungoides: further support for a canine model of cutaneous T-cell lymphoma.

Canine cutaneous T-cell lymphoma (CTCL) is a morphologic and immunophenotypic simulant of human mycosis fungoides (MF) characterized by an infiltrate of atypical, hyperconvoluted, epidermotropic T cells. To further support our hypothesis that canine MF is a useful model for the study of human CTCL, we have used Southern blotting to search for clonal T-cell proliferations in canine MF. Cellular DNA was extracted from normal dog buffy coat cells (n = 8), lesional canine MF skin (n = 8), canine MF buffy coat cells (n = 7), normal dog skin (n = 3), and normal human buffy coat cells (n = 5), digested with a panel of restriction enzymes and Southern blotted onto nylon membranes. All cases of canine MF were also immunophenotyped with anti-canine monoclonal antibodies to CD4, CD8, CD18, CD45RA, canine class II, T-cell activation antigens, and pan-B-cell antigens. Normal dogs gave reproducible digestion patterns in blood and skin, which differed from the human germline patterns when probed with a human T-cell receptor (TCR), beta chain constant region (C beta) cDNA. Common germline bands between the species included the 3.5-kb Eco RI, 3.4-kb Bam HI, 5.4-kb Sac I. These results confirmed that the TCR-beta gene is evolutionarily conserved between dog and man. Immunostaining revealed that 3/7 cases were CD4+ canine CTCL and 4/7 were CD8+ canine CTCL. Rearranged bands, deletion of germline bands, as well as minor alterations in electrophoretic mobility were observed in lesional DNA from seven of eight cases of canine MF, with at least two restriction digests in each case. Dog rearrangements were best detected with Bgl II, Eco RI, Eco RV, and Sac I, whereas deletions were detected with Bgl II, Sac I, Eco RV, and Bam HI. These studies demonstrate the presence of clonal TCR rearrangement in canine MF, further supporting the similarity of this tumor to human MF and its role as an animal model of CTCL.

Adrenal kallikrein.

Kallikrein was identified in the adrenal glands of the rat. The enzyme was present in active and inactive forms (n = 9), since preincubation with trypsin increased kininogenase activity from 54.8 +/- 11.8 to 230 +/- 23 pg bradykinin per milligram protein per minute. Adrenal kininogenase activity was inhibited by 91% by phenylmethylsulfonyl fluoride (2 mM), 81% by D-Phe-Phe-Arg-chloromethyl ketone (1 microM), 88% by aprotinin (1,000 KIU), and only 16% by soybean trypsin inhibitor (50 microM). Preincubation with antibodies against rat urinary kallikrein resulted in over 90% inhibition of kininogenase activity. Immunoreactive glandular kallikrein was 30.7 +/- 4.8 ng/mg protein (n = 11). The apparent molecular weight of the adrenal kininogenase on gel filtration chromatography was 33,000 +/- 500 D. Both the adrenal enzyme and the purified submandibular gland kallikrein used as a control had the same mobility on alkaline polyacrylamide gel electrophoresis. To determine whether messenger RNA (mRNA) for glandular kallikrein is present in adrenal gland RNA, we used the polymerase chain reaction employing oligonucleotide primers and glandular kallikrein 32P complementary DNA (cDNA) as a probe, which should give a cDNA fragment of 370 bp. Southern blots of the amplified products revealed a fragment of the predicted size. In conclusion, glandular kallikrein has been identified in the adrenal glands. The presence of mRNA for glandular kallikrein suggests that kallikrein is synthesized locally in this tissue. This provides an anatomic basis for possible participation of a local kallikrein-kinin pathway in the regulation of adrenal function.

Submandibular enzymatic vasoconstrictor messenger RNA in rat kidney.

Recently, we reported the isolation and identification of a potent vasoconstrictor enzyme from the rat submandibular gland, a member of the rat kallikrein gene family, which we named submandibular enzymatic vasoconstrictor (SEV). We studied whether messenger RNA (mRNA) for SEV is present in the kidney and isolated glomeruli, using the polymerase chain reaction assay with primers specific to the entire rat kallikrein family that would amplify a 430-bp fragment from their mRNA. As a probe we used a phosphorus-32-labeled oligonucleotide specific for SEV mRNA. A fragment of the predicted size was obtained on Southern blot for amplified renal RNA; however, no signal was obtained with glomerular RNA. To further confirm the presence of SEV mRNA in the kidney, polymerase chain reaction was repeated using primers specific to SEV mRNA that would amplify a 372-bp fragment from SEV mRNA alone. Again, a fragment of the predicted size was obtained on Southern blot after amplification of renal RNA but not RNA from the glomeruli. Southern blot of polymerase chain reaction-amplified RNA with primers that amplified the entire kallikrein gene family, using kallikrein complementary DNA that recognizes all members of the kallikrein gene family as a probe, revealed a 430-bp fragment for both renal and glomerular RNA, indicating that glomeruli contain mRNA for a member or members of the kallikrein family other than SEV. When the Southern blots were hybridized with a 32P-labeled oligonucleotide probe specific for glandular kallikrein, a fragment of the predicted size was obtained from amplified renal RNA but not glomerular RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuronal beta-amyloid precursor protein gene expression: regulation by aurintricarboxylic acid.

beta-Amyloid precursor protein (beta-APP) and its derivative, amyloid beta-protein (beta-A4), may cause death of differentiated neurons and aurintricarboxylic acid (ATA), a metabolic inhibitor, improves neuronal survival. Therefore, we studied the effect of ATA on neuronal beta-APP gene expression. ATA decreased beta-APP mRNA levels by increasing its degradation, without changing the rate of transcription. ATA decreased both steady state and interleukin-1 (IL1)-induced increase in beta-APP mRNA levels. These effects of ATA were associated with rounding of cells suggestive of decreased cell adhesion or neurite retraction that was completely reversible when ATA was removed. However, beta-APP mRNA levels continued to remain suppressed in neurons that were actively regrowing neurites following discontinuation of ATA. In studies carried out upto 24 h, ATA did not damage cells as determined by Trypan blue exclusion, lactate dehydrogenase (LDH)-release and transmission electron microscopy. The findings suggest that constitutive or steady state levels of beta-APP mRNA may not be essential for the survival and growth of neurons and that ATA suppresses beta-APP expression without causing cell damage. These observations may be a basis for studying whether ATA or a related compound could beneficially regulate beta-APP levels in vivo.

Molecular characterization of fibroblasts isolated from human peritoneum and adhesions.

OBJECTIVE: To determine the response of adhesion and peritoneal fibroblasts to hypoxia. DESIGN: Prospective experimental study. SETTING: University medical center. PATIENT(S): Primary cultures of fibroblasts established from the peritoneal and adhesion tissues of the same patients (n = 2) to minimize genetic variations. INTERVENTION(S): Hypoxia treatment of the primary cultured fibroblast. MAIN OUTCOME MEASURE(S): Analyze the expression of extracellular matrix (ECM) components, metalloproteinases and their tissue inhibitors, growth factors, and cytokines in adhesion and peritoneal fibroblasts under normal and hypoxic conditions by reverse transcriptase/polymerase chain reaction analysis. RESULT(S): Compared to peritoneal fibroblasts, adhesion fibroblasts had a significant increase in the basal mRNA levels for collagen I, fibronectin, MMP-1, TIMP-1, TGF-beta 1, TGF-beta 2, and IL-10. Hypoxia resulted in a further increase in collagen 1, fibronectin, TIMP-1, TGF-beta 1, TGF-beta 2, IL-10, and IFN-gamma mRNA levels in both peritoneal and adhesion fibroblasts. The increase was more profound in adhesion fibroblasts. CONCLUSION(S): Hypoxia induces molecular changes in both peritoneal and adhesion fibroblasts, creating a milieu that favors adhesion development. The effect of hypoxia was more profound on adhesion fibroblasts.

Analysis of p53 gene mutations in keloids using polymerase chain reaction-based single-strand conformational polymorphism and DNA sequencing.

BACKGROUND: Keloids are the result of a dysregulated wound healing process. They are characterized by the formation of excess scar tissue that proliferates beyond the boundaries of the original wound. Somatic mutations of p53 have been implicated as causal events in up to 50% of all human malignancies. In addition, p53 has been shown to play an important role in controlling cell proliferation and apoptosis. We hypothesize that mutations in p53 can lead to a hyperproliferative state that can result in keloid formation. OBJECTIVE: To detect p53 DNA mutations in tissues and cultured fibroblasts from skin lesions of 7 patients with keloids. DESIGN: The polymerase chain reaction followed by single-strand conformational polymorphism analysis and direct DNA sequencing were used to detect p53 gene mutations. SETTING: The Department of Dermatology, Henry Ford Hospital, Detroit, Mich. PATIENTS: Seven patients with keloids seen for routine surgical excision of their lesions. Normal DNA specimens were obtained from buccal smears and healthy skin samples from these patients. RESULTS: Mutations in the p53 were identified in all patients by polymerase chain reaction followed by single-strand conformational polymorphism analysis and subsequently confirmed by DNA sequencing. A mutation in exon 5 resulting in amino acid substitution was found in 1 of the patients in keloid tissue and cultured keloid fibroblasts (codon 156, CGC-->CCC, arginine-->proline). Frameshift mutations in exons 5 and 6 caused by the insertion or deletion of a nucleotide at different positions were found in 6 patients with keloids in both keloid tissues and cultured fibroblasts. Mutations in exon 4 resulting in amino acid substitution were found in all patients in both keloid tissues and cultured fibroblasts (all in codon 72, CGC-->CCC, arginine-->proline). No p53 mutations were detected in buccal smears or cultured fibroblasts from healthy skin samples of any of the patients. CONCLUSIONS: Focal mutations in p53 may increase cell proliferation and decrease cell death in the dysregulated growth patterns that have been clinically documented. An understanding of the pattern of all growth dysregulation related to keloids may lead to new therapeutic strategies.

Prospective, single-blind, randomized, controlled study to assess the efficacy of the 585-nm flashlamp-pumped pulsed-dye laser and silicone gel sheeting in hypertrophic scar treatment.

OBJECTIVE: To determine the efficacy of the 585-nm flashlamp-pumped pulsed-dye laser and silicone gel sheeting in the treatment of hypertrophic scars in lighter- and darker-skinned patients. DESIGN: Prospective, single-blind, randomized, internally controlled, comparison investigation. SETTING: Large academic dermatology department. PATIENTS: Twenty patients with hypertrophic scars (19 completed the laser treatments and 18 completed the silicone gel sheeting treatments). MAIN OUTCOME MEASURES: Clinical measurements included hypertrophic scar blood flow, elasticity, and volume. Patients' subjective complaints of pruritus, pain, and burning were also monitored. Histological assessment of fibrosis, number of telangiectasias, and number of mast cells was performed. Statistically significant improvements in clinical measurements and patients' subjective complaints determined treatment success. RESULTS: Mean scar duration was 32 months (range, 4 months to 20 years). There was an overall reduction in blood flow, volume, and pruritus over time (P = .001, .02, and .005, respectively). However, no differences were detected among treatment and control groups. There was no reduction in pain or burning (0-40 weeks), elasticity (8-40 weeks), or fibrosis (0-40 weeks, n = 5 biopsies) in the treated or control sections of the scars. Unlike in a previous study, the number of mast cells in the scars was similar to the number of mast cells in healthy skin. CONCLUSION: Clinical results demonstrate that the improvements in scar sections treated with silicone gel sheeting and pulsed-dye laser were no different than in control sections.

Effect of hypoxia on stimulatory effect of TGF-beta 1 on MMP-2 and MMP-9 activities in mouse fibroblasts.

OBJECTIVE: Because chronic low-grade hypoxia has been implicated in the pathogenesis of fibrosis and postoperative adhesion formation, we hypothesized that hypoxia may modulate the effect of transforming growth factor-beta 1 (TGF-beta 1) on matrix metalloproteinase (MMP)-2 and MMP-9 at both transcriptional and translational levels. METHODS: Mouse fibroblasts were placed in a hypoxic environment with or without 1 ng/mL TGF-beta 1 for varying periods of time. Zymography was performed on cell supernatants collected after each treatment. Gelatinolytic bands corresponding to MMP-2 and MMP-9 were quantiated by densitometry. Multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) was also performed for MMP-2 and MMP-9 on total RNA extracted from cells after each treatment. Analysis of PCR-amplified products was performed by 2% agarose gel followed by ethidium bromide staining of DNA bands. Scanning densometry was used to determine the ratio of intensity of each band relative to beta-actin. RESULTS: Hypoxia resulted in a 64% decrease in MMP-9 activity and 80% decrease in MMP-9 mRNA level but did not affect MMP-2 mRNA level or activity. TGF-beta 1 treatment resulted in 180% and 50% increases in MMP-2 and MMP-9 activities, respectively. Increases of 37.5% and 40% in MMP-2 and MMP-9 mRNA levels, respectively, were seen. However, under hypoxic conditions, TGF-beta1 resulted in a 160% increase and 45% decrease in MMP-2 and MMP-9 activities and a 37.5% increase and 71% decrease in MMP-2 and MMP-9 mRNA levels, respectively. CONCLUSION: Hypoxia suppresses the stimulatory effect of TGF-beta1 on the activity of MMP-9 but not MMP-2. This may suggest an important role for MMP-9 under hypoxic conditions in the pathogenesis of tissue fibrosis and postoperative adhesion formation.

Effects of prostaglandin E(2) on proliferation and apoptosis of epithelial ovarian cancer cells.

OBJECTIVE: There is strong evidence indicating that prostaglandins (PG) and their synthesizing enzyme cyclooxygenase-2 (COX-2) play an important role in tumorigenesis. The purposes of the present study were to determine the pattern of expression of COX-2 and the effect of PG treatment on proliferation and apoptosis in epithelial ovarian cancer cells. METHODS: Two epithelial ovarian cancer cell lines, MDAH-2774 and SKOV3, were grown in flasks to confluence. Cells were then treated with exogenous dimethyl prostaglandin E(2) (dmPGE(2)) at increasing concentrations of 0-10 microg/mL. Total RNA was extracted from cells at different treatment doses and subjected to reverse transcriptase-polymerase chain reaction for the semiquantitative analysis of COX-2, Bcl-2, and bax expression. Flow cytometry was performed to assess effect of treatment on the cell cycle. The TUNEL assay was used to assess apoptosis. RESULTS: We found that COX-2 was constitutively expressed in the MDAH-2774 and SKOV3 epithelial ovarian cancer cells as determined by detection of a 304-bp amplified fragment using specific primers for the COX-2 gene. Treatment of both cell lines with dmPGE(2) resulted in dose-dependently higher expression of COX-2, Bcl-2, and bax mRNA compared with untreated cells. These changes were associated with an increase in the proliferative fraction and with a simultaneous reduction in apoptosis. CONCLUSIONS: Prostaglandin E(2) stimulated proliferation and reduced apoptosis in epithelial ovarian cancer cells. These effects were associated with overexpression of COX-2 and an increase in the ratio of Bcl-2:bax mRNA.

Munchausen's syndrome. A case of factitious hemoptysis.

Factitious hemoptysis is the bleeding type of Munchausen's syndrome, rarely reported in the literature (only seventeen cases). After a careful and detailed literature review, the authors report the case of a 22-year-old working-woman, with a history of asthma, Mediterranean anaemia and recurrent hemoptysis, who was admitted several times to the cardiovascular and Respiratory Sciences Department in the Carlo Forlanini Hospital in 1994 for an asthmatic attack and wheeziness at rest. During the admissions the patient underwent laboratory tests (such as the examination of sputum specimens, urinalysis, tuberculin test, cold agglutinins and pneumotropic virus tests) and diagnostic studies (fiberoptic bronchoscopy with bronchoalveolar lavage, computerized tomography and radiography of the chest, bronchial arteriography, bronchography, perfusion and ventilation lung scan), because she continually presented with hemoptysis, in order to spot and discover the nature of the bleeding. Since such examinations failed (a few of them-namely fiberoptic bronchoscopies--were even performed when she was coughing up blood) and psychiatric consultations revealed the presence of psychologically traumatic events in the patient's history which could explain the psychopathic traits of her personality (in fact she was aggressive and unstable in interpersonal relations), a diagnosis of factitious hemoptysis in Munchausen's syndrome was made.

Pulmonary hamartoma. A rare case report.

Pulmonary hamartoma is a rare lung neoformation, usually symptomless and by chance discovered, of a probable dysontogenetic origin with prevailing cartilaginous tissue and adult, onset age. The Authors report a rare case of a 25-year-old student, symptomless and fortuitously found by means of a radiograph of the chest. Many interesting features characterize the case report: histological nature of the pulmonary hamartoma, mainly vascular, so much as to feign an angiosarcoma at the macroscopical examination, and with small peripheral calcifications as shown by lung CT scan; the measures (about 7 cm) plentifully above the parameters usually reported in the literature (from 2 cm to 4 cm); the young onset age (about 10 years old). We may consider a case exceptionally reported in the literature. Besides, on the base of a few studies and of our experience, the results of the pulmonary hamartoma growth rate and doubling time are reported.

[Hepato-splenic and peritoneal tuberculosis. A difficult diagnosis]. / La tubercolosi epato-splenica e peritoneale. Una diagnosi non agevole.

Peritoneal tuberculosis is a rare extra-pulmonary location of Mycobacterium tuberculosis infection arising in the gastrointestinal tract mostly as a complication of the pulmonary location or seldom as a primary involvement. The authors report the case of a 18-year old girl admitted in 1996 to the Infectious Diseases Department of the Umberto I Hospital, "La Sapienza" State University of Rome for the persistence of fever and dry cough, despite a protracted antibiotic treatment performed in previous hospital admissions for a suspicious diagnosis of a "broncho-pneumonia". As the fever didn't decrease and a pain at the right ilium arose, an anti-tuberculous chemotherapeutic treatment was performed (isoniazid and rifampicin), that improved the state of the patient. The pain was resolved by means of a celioscopic operation, showing the evidence of various white nodes on the peritoneal, hepatic and lienal surfaces; all these pathognomonic signs and the anti-tuberculous chemotherapy confirmed the diagnosis of "hepatic, lienal and peritoneal tuberculosis". The patient was subsequently admitted to our institute, where an anti-tuberculous treatment (isoniazid, rifampicin and pyrazinamide) was performed, which caused a further resolution of the clinical and radiological picture.

Regulation of inducible nitric oxide synthase in post-operative adhesions.

BACKGROUND: The deficiency of the inducible nitric oxide synthase (iNOS) substrate, L-arginine (L-Arg), the co-factor tetrahydrobiopterin (H4B) or molecular oxygen may lead to lower NO levels, which enhances the development of adhesion phenotype. METHODS: We utilized high-performance liquid chromatography (HPLC) and immunoprecipitation with nitrotyrosine antibody to determine the levels of H4B, citrulline and protein nitration in fibroblasts established from normal peritoneal and adhesion tissues. RESULTS: The level of H4B was dramatically attenuated in adhesion fibroblasts. The immunoprecipitation with nitrotyrosine antibody revealed higher protein nitration in adhesion compared with normal fibroblasts. There were higher accumulations of citrulline in adhesion fibroblasts as compared with normal fibroblasts. In addition, peritoneal fibroblasts treated with 2% oxygen for 24 h and implanted back into the peritoneal cavity of the rats exhibited marked increase in severity of adhesion as well as extensive distribution involving many sites and organs. CONCLUSIONS: Control of the catalytic activity of iNOS in adhesion fibroblasts may be because of subsaturating amounts of L-Arg and H4B which allow iNOS to generate a combination of reactive oxygen species in addition to NO, thereby influencing NO bioavailability and function.

Quantitation of interferon gamma mRNA levels in psoralen/UVA-treated HUT-78 cells by competitive PCR.

We have developed a method to accurately quantitate IFN gamma mRNA in HUT-78 cells before and after PUVA treatment by the competitive RT/PCR technique, which could be utilized to accurately quantitate any mRNA species of interest. Total RNA was isolated from HUT-78 cells before and after PUVA treatment. A synthetic IFN gamma mRNA was made to contain a 54 bp deletion in the middle of IFN gamma cDNA gene and used as an internal standard. 0.5 microgram of target RNA was co-reverse transcribed and co-amplified with increasing concentrations of synthetic IFN gamma RNA using the same primers. The products of the synthetic RNA were separated from that of the target RNA by gel electrophoresis. This allowed determination of the amount of target IFN gamma mRNA to be quantitated by extrapolating against a standard curve. PUVA treatment of HUT-78 cells resulted in an increase in IFN gamma mRNA level from 32 to 80 pg/microgram of total RNA, suggesting that PUVA induces transcription of T-helper 1 cytokines as part of its mechanism of action.