Plasma ctDNA RAS mutation analysis for the diagnosis and treatment monitoring of metastatic colorectal cancer patients.

BACKGROUND: RAS assessment is mandatory for therapy decision in metastatic colorectal cancer (mCRC) patients. This determination is based on tumor tissue, however, genotyping of circulating tumor (ct)DNA offers clear advantages as a minimally invasive method that represents tumor heterogeneity. Our study aims to evaluate the use of ctDNA as an alternative for determining baseline RAS status and subsequent monitoring of RAS mutations during therapy as a component of routine clinical practice. PATIENTS AND METHODS: RAS mutational status in plasma was evaluated in mCRC patients by OncoBEAM™ RAS CRC assay. Concordance of results in plasma and tissue was retrospectively evaluated. RAS mutations were also prospectively monitored in longitudinal plasma samples from selected patients. RESULTS: Analysis of RAS in tissue and plasma samples from 115 mCRC patients showed a 93% overall agreement. Plasma/tissue RAS discrepancies were mainly explained by spatial and temporal tumor heterogeneity. Analysis of clinico-pathological features showed that the site of metastasis (i.e. peritoneal, lung), the histology of the tumor (i.e. mucinous) and administration of treatment previous to blood collection negatively impacted the detection of RAS in ctDNA. In patients with baseline mutant RAS tumors treated with chemotherapy/antiangiogenic, longitudinal analysis of RAS ctDNA mirrored response to treatment, being an early predictor of response. In patients RAS wt, longitudinal monitoring of RAS ctDNA revealed that OncoBEAM was useful to detect emergence of RAS mutations during anti-EGFR treatment. CONCLUSION: The high overall agreement in RAS mutational assessment between plasma and tissue supports blood-based testing with OncoBEAM™ as a viable alternative for genotyping RAS of mCRC patients in routine clinical practice. Our study describes practical clinico-pathological specifications to optimize RAS ctDNA determination. Moreover, OncoBEAM™ is useful to monitor RAS in patients undergoing systemic therapy to detect resistance and evaluate the efficacy of particular treatments.

Crushing injuries of the foot and ankle, with complex open fractures: result of a prospective study with a 3 year follow-up.

The objective of this study was to determine the compared results of both the reconstruction surgery and the amputation in severe crushing of the foot, which led to open fractures. The type of study. Prospective. Background. Two major trauma hospitals (Floreasca Clinical Emergency Hospital and "Bagdasar Arseni" Clinical Emergency Hospital) from the university center in Bucharest. Patients. 21 patients, who sustained crushing of the foot with resulting Gustilo type III open fractures, were involved. The exclusion criteria were represented by open fractures that had very gross destructions of the neurovascular bundle, for which the amputation was the only solution, with no modality to reconstruct whatsoever. Treatment. An immediate amputation (at 24, 48 hours after a thorough debridement, proper patient resuscitation, and detailed imaging investigation - the technique of delayed emergency) and reconstruction surgery were performed. Methods of evaluation. Three variables were used: the Sickness Impact Profile (SIP) score, the Visual Analogue Scale (VAS) for the residual pain and the number of rehospitalizations for secondary surgical procedures. Results. When comparing the two lots of patients, first in which the amputation patients were included and second in which the reconstruction patients were included, it was noticed that there was a less favorable prognostic in the second lot for a three-year follow up period. Conclusions. The patients with a mangled foot, in which reconstruction surgery of the bone and soft tissue envelope was performed, had a worse prognostic than those who had an amputation as a first intention. Abbreviations: SIP = Sickness Impact Profile, VAS = Visual Analogue Scale, MVA = Motor Vehicle Accident, STSG = Split Thickness Skin Graft.

Oral benfotiamine plus alpha-lipoic acid normalises complication-causing pathways in type 1 diabetes.

AIMS/HYPOTHESIS: We determined whether fixed doses of benfotiamine in combination with slow-release alpha-lipoic acid normalise markers of reactive oxygen species-induced pathways of complications in humans. METHODS: Male participants with and without type 1 diabetes were studied in the General Clinical Research Centre of the Albert Einstein College of Medicine. Glycaemic status was assessed by measuring baseline values of three different indicators of hyperglycaemia. Intracellular AGE formation, hexosamine pathway activity and prostacyclin synthase activity were measured initially, and after 2 and 4 weeks of treatment. RESULTS: In the nine participants with type 1 diabetes, treatment had no effect on any of the three indicators used to assess hyperglycaemia. However, treatment with benfotiamine plus alpha-lipoic acid completely normalised increased AGE formation, reduced increased monocyte hexosamine-modified proteins by 40% and normalised the 70% decrease in prostacyclin synthase activity from 1,709 +/- 586 pg/ml 6-keto-prostaglandin F(1alpha) to 4,696 +/- 533 pg/ml. CONCLUSIONS/INTERPRETATION: These results show that the previously demonstrated beneficial effects of these agents on complication-causing pathways in rodent models of diabetic complications also occur in humans with type 1 diabetes.

Hyperglycaemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells.

AIMS/HYPOTHESIS: Hyperglycaemia is a primary cause of vascular complications in diabetes. A hallmark of these vascular complications is endothelial cell dysfunction, which is partly due to the reduced production of nitric oxide. The aim of this study was to investigate the regulation of endothelial nitric oxide synthase (eNOS) activity by acute and chronic elevated glucose. METHODS: Human aortic endothelial cells were cultured in 5.5 mmol/l (NG) or 25 mmol/l glucose (HG) for 4 h, 1 day, 3 days or 7 days. Mouse aortic endothelial cells were freshly isolated from C57BL/6J control and diabetic db/db mice. The expression and activity of eNOS were measured using quantitative PCR and nitrite measurements respectively. The binding of activator protein-1 (AP-1) to DNA in nuclear extracts was determined using electrophoretic mobility-shift assays. RESULTS: Acute exposure (4 h) of human aortic endothelial cells to 25 mmol/l glucose moderately increased eNOS activity and eNOS mRNA and protein expression. In contrast, chronic exposure to elevated glucose (25 mmol/l for 7 days) reduced total nitrite levels (46% reduction), levels of eNOS mRNA (46% reduction) and eNOS protein (65% reduction). In addition, AP-1 DNA binding activity was increased in chronic HG-cultured human aortic endothelial cells, and this effect was reduced by the specific inhibition of reactive oxygen species production through the mitochondrial electron transport chain. Mutation of AP-1 sites in the human eNOS promoter reversed the effects of HG. Compared with C57BL/6J control mice, eNOS mRNA levels in diabetic db/db mouse aortic endothelial cells were reduced by 60%. This decrease was reversed by the overexpression of manganese superoxide dismutase using an adenoviral construct. CONCLUSIONS/INTERPRETATION: In diabetes, the expression and activity of eNOS is regulated through glucose-mediated mitochondrial production of reactive oxygen species and activation of the oxidative stress transcription factor AP-1.

Sepragel sinus (hylan B) as a postsurgical dressing for endoscopic sinus surgery.

OBJECTIVE: To assess the safety and efficacy of Sepragel sinus, a hylan B gel (cross-linked hyaluronic acid molecule), when used as a postoperative dressing after endoscopic sinus surgery as a facilitator of healing and a preventative for scarring and stenosis. STUDY DESIGN: Ten patients undergoing bilateral endoscopic ethmoidectomy in an outpatient specialty hospital operating room underwent complete filling of a randomly selected right or left ethmoidectomy cavity with Sepragel sinus. Outcome measures were synechiae, middle meatal stenosis, mucosal status, mucosal regeneration, transparency of Sepragel sinus, and subjective pain and congestion. RESULTS: Sepragel sinus significantly improved all outcome measures by week 2 and remained statistically significant for reduction of synechiae and stenosis. CONCLUSION: Sepragel sinus is useful as a space-occupying gel stent to separate sinus mucosal surfaces. The data strongly support the superiority of Sepragel sinus over no treatment in the control of postethmoidectomy synechiae and middle meatal stenosis, as well as early improvement in mucosal healing and postoperative pain. SIGNIFICANCE: Because of its biocompatibility, lack of inflammatory response, transparency, and ability to fill any complex volume, Sepragel sinus offers distinct advantages over currently used stenting materials.

Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site.

Endothelial nitric oxide synthase (eNOS) is activated by phosphorylation of serine 1177 by the protein kinase Akt/PKB. Since hyperglycemia-induced mitochondrial superoxide overproduction increases O-linked N-acetylglucosamine modification and decreases O-linked phosphorylation of the transcription factor Sp1, the effect of hyperglycemia and the hexosamine pathway on eNOS was evaluated. In bovine aortic endothelial cells, hyperglycemia inhibited eNOS activity 67%, and treatment with glucosamine had a similar effect. Hyperglycemia-associated inhibition of eNOS was accompanied by a twofold increase in O-linked N-acetylglucosamine modification of eNOS and a reciprocal decrease in O-linked serine phosphorylation at residue 1177. Both the inhibition of eNOS and the changes in its post-translational modifications were reversed by antisense inhibition of glutamine:fructose-6-phosphate amidotransferase, the rate-limiting enzyme of the hexosamine pathway, or by blocking mitochondrial superoxide overproduction with uncoupling protein-1 (UCP-1) or manganese superoxide dismutase (MnSOD). Immunoblot analysis of cells expressing myc-tagged wild-type human eNOS confirmed the reciprocal increase in O-linked N-acetylglucosamine and decrease in O-linked serine 1177 phosphorylation in response to hyperglycemia. In contrast, when myc-tagged human eNOS carried a mutation at the Akt phosphorylation site (Ser1177), O-linked N-acetylglucosamine modification was unchanged by hyperglycemia and phospho-eNOS was undetectable. Similar changes in eNOS activity and covalent modification were found in aortae from diabetic animals. Chronic impairment of eNOS activity by this mechanism may partly explain the accelerated atherosclerosis of diabetes.

Hyperglycemia potentiates collagen-induced platelet activation through mitochondrial superoxide overproduction.

Alteration of platelet function contributes to microthrombus formation and may play an important role in the pathogenesis of diabetic micro- and macroangiopathies. However, the molecular mechanism for platelet dysfunction observed in patients with diabetes has not been fully elucidated. In this study, the direct effects of hyperglycemia on platelet function in vitro were investigated. Hyperglycemia increased reactive oxygen species generation in human platelets, and this effect was additive with that of collagen. Thenoyltrifluoroacetone (TTFA), an inhibitor of mitochondrial electron transport chain complex II, and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation, completely prevented the effects of hyperglycemia, suggesting that reactive oxygen species arise from the mitochondrial electron transport chain. Hyperglycemia potentiated both platelet aggregation and the subsequent release of platelet-derived growth factor AB induced by a nonaggregating subthreshold concentration of collagen, which were also completely inhibited by TTFA or CCCP. Furthermore, hyperglycemia was found to inhibit protein tyrosine phosphatase (PTP) activity and increase phosphorylation of the tyrosine kinase Syk in platelets exposed to collagen. Hyperglycemia-induced PTP inhibition and Syk phosphorylation were found to be completely prevented by TTFA, CCCP, or Mn(III)tetrakis (4-benzoic acid) porphyrin, a stable cell-permeable superoxide dismutase mimetic. These results suggest that hyperglycemia-induced mitochondrial superoxide generation may play an important role in platelet dysfunction observed in patients with diabetes.

Leptin induces mitochondrial superoxide production and monocyte chemoattractant protein-1 expression in aortic endothelial cells by increasing fatty acid oxidation via protein kinase A.

Leptin, a circulating hormone secreted mainly from adipose tissues, is involved in the control of body weight. The plasma concentrations are correlated with body mass index, and are reported to be high in patients with insulin resistance, which is one of the major risk factors for cardiovascular disease. However, the direct effect of leptin on vascular wall cells is not fully understood. In this study, we investigated the effects of leptin on reactive oxygen species (ROS) generation and expression of monocyte chemoattractant protein-1 (MCP-1) in bovine aortic endothelial cells (BAEC). We found that leptin increases ROS generation in BAEC in a dose-dependent manner and that its effects are additive with those of glucose. Rotenone, thenoyltrifluoroacetone (TTFA), carbonyl cyanide m-chlorophenylhydrazone (CCCP), Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), uncoupling protein-1 (UCP1) HVJ-liposomes, or manganese superoxide dismutase (MnSOD) HVJ-liposomes completely prevented the effect of leptin, suggesting that ROS arise from mitochondrial electron transport. Leptin increased fatty acid oxidation by stimulating the activity of carnitine palmitoyltransferase-1 (CPT-1) and inhibiting that of acetyl-CoA carboxylase (ACC), pace-setting enzymes for fatty acid oxidation and synthesis, respectively. Leptin-induced ROS generation, CPT-1 activation, ACC inhibition, and MCP-1 overproduction were found to be completely prevented by either genistein, a tyrosine kinase inhibitor, H-89, a protein kinase A (PKA) inhibitor, or tetradecylglycidate, a CPT-1 inhibitor. Leptin activated PKA, and the effects of leptin were inhibited by the cAMP antagonist Rp-cAMPS. These results suggest that leptin induces ROS generation by increasing fatty acid oxidation via PKA activation, which may play an important role in the progression of atherosclerosis in insulin-resistant obese diabetic patients.

Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation.

The hexosamine pathway has been implicated in the pathogenesis of diabetic complications. We determined first that hyperglycemia induced a decrease in glyceraldehyde-3-phosphate dehydrogenase activity in bovine aortic endothelial cells via increased production of mitochondrial superoxide and a concomitant 2.4-fold increase in hexosamine pathway activity. Both decreased glyceraldehyde-3-phosphate dehydrogenase activity and increased hexosamine pathway activity were prevented completely by an inhibitor of electron transport complex II (thenoyltrifluoroacetone), an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenylhydrazone), a superoxide dismutase mimetic [manganese (III) tetrakis(4-benzoic acid) porphyrin], overexpression of either uncoupling protein 1 or manganese superoxide dismutase, and azaserine, an inhibitor of the rate-limiting enzyme in the hexosamine pathway (glutamine:fructose-6-phosphate amidotransferase). Immunoprecipitation of Sp1 followed by Western blotting with antibodies to O-linked GlcNAc, phosphoserine, and phosphothreonine showed that hyperglycemia increased GlcNAc by 1.7-fold, decreased phosphoserine by 80%, and decreased phosphothreonine by 70%. The same inhibitors prevented all these changes. Hyperglycemia increased expression from a transforming growth factor-beta(1) promoter luciferase reporter by 2-fold and increased expression from a (-740 to +44) plasminogen activator inhibitor-1 promoter luciferase reporter gene by nearly 3-fold. Inhibition of mitochondrial superoxide production or the glucosamine pathway prevented all these changes. Hyperglycemia increased expression from an 85-bp truncated plasminogen activator inhibitor-1 (PAI-1) promoter luciferase reporter containing two Sp1 sites in a similar fashion (3.8-fold). In contrast, hyperglycemia had no effect when the two Sp1 sites were mutated. Thus, hyperglycemia-induced mitochondrial superoxide overproduction increases hexosamine synthesis and O-glycosylation of Sp1, which activates expression of genes that contribute to the pathogenesis of diabetic complications.

Changes in diabetic retinal matrix protein mRNA levels in a common transgenic mouse strain.

Recently, all the structural features of non-proliferative diabetic retinopathy have been demonstrated in mice fed 30% galactose for 21-26 months. To determine whether changes in retinal matrix protein mRNA levels occur early in the course of murine diabetes we used a competitive RT-PCR method to quantitate retinal mRNA levels in an inbred mouse strain (FVB) commonly used for transgenic studies. Retinal mRNA was prepared from STZ-diabetic and non-diabetic FVB mice at 4, 8, 12 and 16 weeks and cDNA encoding basement membrane components was quantitated using MIMIC constructs that compete for the same primer pairs. alpha1 (IV) collagen, the beta1 and gamma1 chains of laminin, fibronectin, and vitronectin mRNAs were quantitated. For alpha1 (IV) collagen, statistically significant diabetes-induced increases were apparent by 8 weeks (3.11 +/- 0.20 vs. 1.29 +/- 0.19 x 10(6) molecules/mg total RNA, p < 0.005). Similarly, diabetes-induced increases were observed by 8 weeks for the beta1 chain of laminin (4.54 +/- 0.22 vs. 1.85 +/- 0.43 x 10(5) molecules/mg total RNA, p < 0.005), the gamma1 chain of laminin (7. 33 +/- 0.29 vs. 4.84 +/- 0.76 x 10(4)/microg total RNA, p < 0.05), and for fibronectin (2.22 +/- 0.21 vs. 1.35 +/- 0.15 x 10(6) molecules/mg total RNA, p < 0.05). The magnitude of change was greatest for alpha1 (IV) collagen (2.4-fold) and beta1 laminin (2. 5-fold) at 8 weeks, and least for fibronectin (1.6-fold). A smaller diabetes-induced increase in vitro nectin mRNA was also observed, but it failed to reach statistical significance at 12 and 16 weeks. These data provide the basis for assessing the effects of genetic manipulation on diabetic retinopathy in transgenic mouse models.

Ultrastructure of submucosal glands in human anterior middle nasal turbinates.

The abundant glands situated in the lamina propria of the human anterior middle nasal turbinate were complex tubules that consist of serous, seromucous, and mucous cells, either singly or in combination. Serous granules were homogeneously dense, but could have a small lighter core. Seromucous granules had a dense rim and a large compartment of appreciably lighter density. Gradation between serous and seromucous granules made precise identification of these secretory cell types difficult. Mucous cells were of conventional morphology. The secretory tubules, which possessed a complement of myoepithelial cells, gradually transformed into ducts or the changeover was relatively sudden. The ductular portions of the tubules consisted either of tall prismatic cells or of shorter columnar cells, both of which lacked secretory granules, but had many mitochondria in their supranuclear cytoplasm. In many cases the ducts, for most of their length, consisted of secretory cells. These glands clearly participate in the elaboration of the glycoconjugate coat that serves to protect the nasal mucosa and keeps it from drying out.

The missing link: a single unifying mechanism for diabetic complications.

A causal relationship between chronic hyperglycemia and diabetic microvascular disease, long inferred from various animal and clinical studies, has now been definitely established by data from the Diabetes Control and Complications Trial (DCCT), a multicenter, randomized, prospective, controlled clinical study. A relationship between chronic hyperglycemia and diabetic macrovascular disease in patients with non-insulin-dependent diabetes mellitus (NIDDM) is also supported by the Kumamoto study. How does hyperglycemia induce the functional and morphologic changes that define diabetic complications? Vascular endothelial cells are a major target of hyperglycemic damage, but the mechanisms underlying this damage remain incompletely understood. Three seemingly independent biochemical pathways are involved in the pathogenesis: glucose-induced activation of protein kinase C (PKC) isoforms: increased formation of glucose-derived advanced glycation end products; and increased glucose flux through the aldose reductase pathway. The relevance of each of these three pathways is supported by animal studies in which pathway-specific inhibitors prevent various hyperglycemia-induced abnormalities. Hyperglycemia increases reactive oxygen species (ROS) production inside cultured bovine aortic endothelial cells. In this paper, we show that ROS may activate aldose reductase, induce diacylglycerol, activate PKC, induce advanced glycation end product formation, and activate the pleiotropic transcription factor nuclear factor-kappa B (NF-kappaB). These data demonstrate that a single unifying mechanism of induction, increased production of ROS, serves as a causal link between elevated glucose and each of the three major pathways responsible for diabetic damage.

Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage.

Diabetic hyperglycaemia causes a variety of pathological changes in small vessels, arteries and peripheral nerves. Vascular endothelial cells are an important target of hyperglycaemic damage, but the mechanisms underlying this damage are not fully understood. Three seemingly independent biochemical pathways are involved in the pathogenesis: glucose-induced activation of protein kinase C isoforms; increased formation of glucose-derived advanced glycation end-products; and increased glucose flux through the aldose reductase pathway. The relevance of each of these pathways is supported by animal studies in which pathway-specific inhibitors prevent various hyperglycaemia-induced abnormalities. Hyperglycaemia increases the production of reactive oxygen species inside cultured bovine aortic endothelial cells. Here we show that this increase in reactive oxygen species is prevented by an inhibitor of electron transport chain complex II, by an uncoupler of oxidative phosphorylation, by uncoupling protein-1 and by manganese superoxide dismutase. Normalizing levels of mitochondrial reactive oxygen species with each of these agents prevents glucose-induced activation of protein kinase C, formation of advanced glycation end-products, sorbitol accumulation and NFkappaB activation.

Transfection of human pancreatic islets with an anti-apoptotic gene (bcl-2) protects beta-cells from cytokine-induced destruction.

Apoptosis has been identified as a mechanism of pancreatic islet beta-cell death in autoimmune diabetes. Proinflammatory cytokines are candidate mediators of beta-cell death in autoimmune diabetes, and these cytokines can induce beta-cell death by apoptosis. In the present study, we examined whether transfection of human islet beta-cells with an anti-apoptotic gene, bcl-2, can prevent cytokine-induced beta-cell destruction. Human islet beta-cells were transfected by a replication-defective herpes simplex virus (HSV) amplicon vector that expressed the bcl-2 gene (HSVbcl-2) and, as a control, the same HSV vector that expressed a beta-galactosidase reporter gene (HSVlac). Two-color immunohistochemical staining revealed that 95+/-3% of beta-cells transfected with HSVbcl-2 expressed Bcl-2 protein compared with 14+/-3% of beta-cells transfected with HSVlac and 19+/-4% of nontransfected beta-cells. The bcl-2-transfected beta-cells were fully protected from impaired insulin secretion and destruction resulting from incubation for 5 days with the cytokine combination of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma. In addition, the bcl-2-transfected islet cells were significantly protected from cytokine-induced lipid peroxidation and DNA fragmentation. These results demonstrate that cytokine-induced beta-cell dysfunction and death involve mechanisms subject to regulation by an anti-apoptotic protein, Bcl-2. Therefore, bcl-2 gene therapy has the potential to protect human beta-cells in pancreatic islets, or islet grafts, from immune-mediated damage in type 1 diabetes.

Cyclooxygenase-2 expression is up-regulated in squamous cell carcinoma of the head and neck.

The purpose of this study was to determine whether cyclooxygenase-2 (COX-2) was overexpressed in squamous cell carcinoma of the head and neck (HNSCC). Quantitative reverse transcription-PCR, immunoblotting, and immunohistochemistry were used to assess the expression of COX-2 in head and neck tissue. Mean levels of COX-2 mRNA were increased by nearly 150-fold in HNSCC (n = 24) compared with normal oral mucosa from healthy volunteers (n = 17). Additionally, there was about a 50-fold increase in amounts of COX-2 mRNA in normal-appearing epithelium adjacent to HNSCC (n = 10) compared with normal oral mucosa from healthy volunteers. Immunoblotting demonstrated that COX-2 protein was present in six of six cases of HNSCC but was undetectable in normal oral mucosa from healthy subjects. Immunohistochemical analysis showed that COX-2 was expressed in both HNSCC and adjacent normal-appearing epithelium. Taken together, these results suggest that COX-2 may be a target for the prevention or treatment of HNSCC.

In vivo interleukin-2 gene therapy of established tumors with herpes simplex amplicon vectors.

In vivo cytokine gene transfer may greatly simplify autologous tumor vaccine production. Herpes simplex viral amplicon vectors (HSV) are efficient gene-transfer vehicles and may overcome many limitations of prior gene-transfer methods. The interleukin-2 (IL-2) and beta-galactosidase genes (lac) were inserted into an HSV amplicon vector and tested in a subcutaneous squamous cell carcinoma of lung origin to determine the efficiency of in vivo gene transfer and the utility of such a direct gene transfer approach in cancer therapy. Gene transfer and expression were assessed by histochemical staining and enzyme-linked immunosorbent assay (ELISA). Growth of injected tumors as well as non-injected tumors remote from the site of injection was assessed. Assessment of lymphocytic infiltrates into tumors was performed by immunohistochemistry. Survival was recorded. Direct in vivo injection of established tumors with a HSVi12 resulted in efficient gene transfer and production of IL-2 in the injected tumor but not at tumors remote from the sites of injection. There was a significant suppression of growth of the tumors injected with HSVi12 (P<0.01) when compared with tumors injected with HSV without i12. Of note, growth of tumors remote from sites of HSVi12 injection was also retarded and treatment was associated with a significant (P<0.05) improvement in survival. Direct intratumoral administration of HSV amplicon vectors can result in efficient transfer of cytokine genes and have antitumor efficacy. HSV vectors are therefore potentially useful agents in such in vivo gene-therapy strategies and simplify cytokine antitumor gene-therapy strategies.

Inhibition of cyclooxygenase-2 expression. An approach to preventing head and neck cancer.

Cyclooxygenase (COX) catalyzes the formation of prostaglandins (PG) from arachidonic acid. A large body of evidence has accumulated to suggest that COX-2, the inducible form of COX, is important in carcinogenesis. In this study, we determined whether (1) COX-2 was overexpressed in squamous cell carcinoma of the head and neck (HNSCC) and whether (2) retinoids, a class of chemopreventive agents, blocked epidermal growth factor (EGF)-mediated activation of COX-2 expression. Levels of COX-2 mRNA were determined in 15 cases of HNSCC and 10 cases of normal oral mucosa. Nearly a 100-fold increase in amounts of COX-2 mRNA was detected in HNSCC. By immunoblot analysis, COX-2 protein was detected in 6 of 6 cases of HNSCC but was undetectable in normal mucosa. Because retinoids protect against oral cavity cancer, we investigated whether retinoids could suppress EGF-mediated induction of COX-2 in cultured oral squamous carcinoma cells. Treatment with EGF led to increased levels of COX-2 mRNA, COX-2 protein, and synthesis of PG. These effects were suppressed by a variety of retinoids. Based on the results of this study, it will be important to establish whether newly developed selective COX-2 inhibitors are useful in preventing or treating HNSCC. Moreover, the anticancer properties of retinoids may be due, in part, to inhibition of COX-2 expression. Combining a retinoid with a selective COX-2 inhibitor may be more effective than either agent alone in preventing cancer of the upper aerodigestive tract.

Changes in nasal epithelium in patients with severe chronic sinusitis: a clinicopathologic and electron microscopic study.

OBJECTIVE: Defective ciliary ultrastructure and impaired mucociliary clearance play an important role in the development of respiratory disease and sinusitis. Changes in the ciliary ultrastructure of the sinonasal epithelium have been documented in patients with primary ciliary dyskinesia. However, secondary ciliary dyskinesias and epithelial cytopathologic changes have been underappreciated as a consequence of respiratory dysfunction and chronic sinusitis. STUDY DESIGN: Thirty-two patients with severe chronic sinusitis were evaluated for ciliary and epithelial abnormalities. MATERIALS AND METHODS: Fourteen patients (44%) were children who underwent full allergy, sweat, and immunologic workups. Eighteen patients (56%) were adults who had severe refractory sinusitis and had failed previous sinus surgery. All patients underwent nasal epithelium biopsies of the middle turbinate and evaluation by light and transmission electron microscopy. RESULTS: Ciliated cells were found in 23 patients (72%) with 9 patients (28%) having no cilia. Foci of normal ciliated epithelium were found in only 19% of the patients, often in epithelial invaginations. Variable numbers (usually a minor population) of cilia in 20 cases (87%) exhibited ultrastructural defects including compound cilia and microtubule and dynein arm defects. All of the patients showed variable loss of differentiated epithelial cells ranging from denuded epithelium to basal cell hyperplasia often associated with squamous metaplasia, secondary to chronic sinonasal disease. The lamina propria was often edematous with dilated capillaries, plasma cells, lymphocytes, and hyperplastic seromucous glands. CONCLUSIONS: This study demonstrates that ciliary dyskinesias are primarily the result rather than the cause of chronic sinusitis. Patients with chronic sinusitis of uncertain origin exhibit a prominent loss of differentiated epithelial cells, as well as ciliary defects, most of which are likely to be secondary to the chronic disease process. These changes slow down mucociliary clearance and lead to a vicious cycle leading to chronicity.

Green tea regulates cell cycle progression in oral leukoplakia.

BACKGROUND: A complete in vitro multi-stage carcinogenesis model for oral cancer was developed to examine chemopreventive strategies. In the present study, the effects of EGCG [(-)-epigallocatechin-3-gallate], the major constituent of green tea, is being examined to understand mechanisms of action. METHODS: Effects of EGCG on the cell populations were examined with growth assays, cell cycle analysis, and western blots for retinoblastoma protein (pRB). RESULTS: In each cell type, EGCG inhibited growth, with a decrease in efficacy as cells progressed from normal to cancer. A G1 block was induced with an increase in the underphosphorylated form of pRB; EGCG-induced inhibition was not permanent, cells recovered, and no resistance developed. CONCLUSIONS: Our multistage carcinogenesis model for chemoprevention was effective in defining the chemopreventive value of EGCG. The observation that cancerous oral epithelium was less responsive than normal or dysplastic tissues has implication in the use of this agent, and the mechanisms responsible for this result remain to be defined.

Transduction of non-dividing adult human pancreatic beta cells by an integrating lentiviral vector.

Pancreatic islet cells are terminally differentiated endocrine cells and are refractory to stable infection by retroviral vectors, which require the breakdown of the nuclear membrane during cell division in order to insert the transgene into the host cell genome. Thus, attempts to render beta-cell allografts less immunogenic have had to rely on stable transfection of surrogate cells. Similarly, this problem has precluded the development of conditionally immortalized human beta cells for clinical allotransplantation. In this report, we demonstrate that adult human islet beta cells can be transduced by a new three-plasmid integrating lentiviral vector with an efficiency of 62 +/- 1.8 % at a multiplicity of infection (MOI) of 2.5 in vitro. This work makes genetic engineering of adult human pancreatic beta cells possible for the first time, allowing strategies to render beta-cell allografts non-immunogenic to be optimized and to creating conditionally immortalized human beta cells for clinical transplantation.