Abrogation of transforming growth factor-alpha/epidermal growth factor receptor autocrine signaling by an RXR-selective retinoid (LGD1069, Targretin) in head and neck cancer cell lines.

Clinical studies have demonstrated that retinoids, including retinol (Vitamin A) and its synthetic derivatives, can eradicate leukoplakia and suppress the formation of squamous cell carcinoma of the head and neck (SCCHN). Nonselective retinoids have been shown to abrogate transcriptional activation of transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFR), which characterize SCCHN. LGD1069 (Targretin) is a potent RXR-selective retinoic acid agonist with a reduced toxicity profile compared with other nonselective retinoids. We examined the effect of LGD1069 (10 microm) on cellular proliferation and expression of putative intermediate biomarker genes including TGF-alpha, EGFR, and RAR-beta in seven SCCHN cell lines. A quantitative reverse transcription-PCR assay using a novel "primer dropping" method was used to determine expression levels of EGFR, TGF-alpha, and RAR-beta before and after treatment with LGD1069 (10 microM). SCCHN proliferation was reduced by a mean of 50% at 4 days in seven SCCHN cell lines after LGD1069 treatment (P < or = 0.05). EGFR expression levels were decreased by a mean of 58.4% (P = 0.007), TGF-alpha levels were decreased by a mean of 28.8% (P = 0.01), and RAR-beta levels were increased by a mean of 60% (P = 0.03). TGF-alpha stimulation of EGFR is associated with constitutive signal transducer and activator of transcription 3 (Stat3) activation in SCCHN. Abrogation of constitutive Stat3 activation was seen with LGD1069 treatment. These results suggest that an RXR-selective retinoic acid decreases SCCHN proliferation in part by interfering with TGF-alpha/EGFR autocrine signaling.

Unexpected similarity of pulsed-field gel electrophoresis patterns of unrelated clinical isolates of Legionella pneumophila, serogroup 1.

Phenotypic and genotypic methods identify subtypes of Legionella pneumophila, serogroup 1, and match patient and environmental isolates from suspected sources. The strength of this association is limited by the lack of information regarding the frequency and distribution of isolates belonging to various subtypes. In this study, 62 clinical isolates of L. pneumophila, serogroup 1, were subtyped by using pulsed-field gel electrophoresis (PFGE), to determine the distribution and degree of diversity of PFGE patterns among monoclonal antibody (MAb) subtypes. Unexpectedly, 8 of 21 MAb Philadelphia 1 isolates had a common PFGE pattern, and, among 12 MAb OLDA isolates, only 2 PFGE patterns were seen. Our hypothesis was that PFGE patterns were distributed randomly; however, statistical analysis showed that the distribution of subtypes was not random (Fisher's exact test 0.13; P>.05). In light of these results, researchers who do epidemiological investigations should use caution when interpreting the significance of matching PFGE patterns of L. pneumophila, serogroup 1.

Human leukocyte antigen class I allelic and haplotype loss in squamous cell carcinoma of the head and neck: clinical and immunogenetic consequences.

The expression of human leukocyte antigen (HLA) class I molecules on the cell surface is necessary for the presentation of peptide antigens to cytotoxic CD8+ T lymphocytes of the immune system. Down-regulation of HLA class I gene expression has been implicated in tumorigenesis, including squamous cell carcinoma of the head and neck (SCCHN). Loss of MHC class I antigens may be one mechanism by which tumor cells escape immune detection. We performed prospective immunostaining of 26 primary SCCHN tumors and samples of normal mucosa harvested several centimeters away from the primary tumor, using a large panel of antibodies directed against allele-specific as well as monomorphic determinants of HLA class I molecules. Loss of expression of HLA class I proteins in the tumor was found in 50% (13 of 26) of primary tumors and was highly correlated with HLA loss in the corresponding normal mucosa (P < 0.0001). Further analysis demonstrated that the loss of HLA class I expression in the tumor was significantly associated with regional lymph node metastases (nodal stage; P = 0.0388), and that the number of HLA class I alleles lost in the normal mucosa was associated with subsequent development of a new primary aerodigestive tract cancer (P = 0.042). A patient with two metachronous cancers available for analysis had no evidence of HLA loss in the first tumor, demonstrated allelic loss in the second cancer, and subsequently died of disease. These results suggest that the loss of expression of HLA class I alleles may have prognostic implications.

Epidermal growth factor receptor--mediated stat3 signaling blocks apoptosis in head and neck cancer.

OBJECTIVES: Upregulation of epidermal growth factor receptor (EGFR) is critical for the loss of growth control in a variety of human cancers including squamous cell cancers of the head and neck (SCCHN). In these tumor cells in culture, EGFR stimulation initiates signaling via persistent activation of STAT proteins, particularly Stat3. The present study was conducted to study the association between EGFR stimulation and constitutive activation of Stat3 in SCCHN in vivo and to investigate the proliferative and apoptotic consequences of Stat3 downmodulation in SCCHN cells in vitro. METHODS: SCCHN tumor xenografts were analyzed using electrophoretic mobility shift assay. A dominant-negative mutant Stat3 expression construct or a Stat3 antisense plasmid was transfected into SCCHN cells using lipofectamine. Cell growth and apoptosis were determined by vital dye exclusion and flow cytometry, respectively. RESULTS: In vivo liposome-mediated gene therapy with an EGFR antisense plasmid efficiently inhibited Stat3 activation in a head and neck xenograft model. Downmodulation of Stat3 using a dominant-negative or antisense approach inhibited tumor cell growth and stimulated apoptosis. CONCLUSIONS: These findings provide evidence that constitutively activated Stat3 is linked to EGFR signaling in SCCHN in vivo, which contributes to the loss of growth control by an anti-apoptotic mechanism.

Constitutive activation of Stat3 signaling abrogates apoptosis in squamous cell carcinogenesis in vivo.

Field cancerization predisposes the upper aerodigestive tract mucosa to the formation of multiple primary tumors, when exposed to environmental carcinogens. Up-regulation of epidermal growth factor receptor occurs early in squamous cell carcinogenesis and is critical for the loss of growth control in a variety of human cancers, including head and neck squamous cell carcinomas. In these tumor cells in culture, epidermal growth factor receptor stimulation initiates signaling via persistent activation of selective STAT proteins. To determine the timing of Stat3 activation in head and neck carcinogenesis, we studied the expression and constitutive activation of Stat3 in tumors and normal mucosa from patients with head and neck cancer compared with mucosa from controls without cancer. Stat3 was up-regulated and constitutively activated in both primary human head and neck tumors as well as in normal mucosa from these cancer patients compared with control normal mucosa from patients without cancer. In vivo liposome-mediated gene therapy with a Stat3 antisense plasmid efficiently inhibited Stat3 activation, increased tumor cell apoptosis, and decreased Bcl-x(L) expression in a head and neck xenograft model. These findings provide evidence that constitutively activated Stat3 is an early event in head and neck carcinogenesis that contributes to the loss of growth control by an anti-apoptotic mechanism.

TGF-alpha antisense gene therapy inhibits head and neck squamous cell carcinoma growth in vivo.

Unlike normal mucosal squamous epithelial cells, head and neck squamous cell carcinomas (HNSCCs) overexpress TGF-alpha mRNA and protein which is required to sustain the proliferation of HNSCC cells in vitro. To determine whether TGF-alpha expression contributes to tumor growth in vivo, cationic liposome-mediated gene transfer was used to deliver an antisense expression construct targeting the human TGF-alpha gene into human head and neck tumor cells, grown as subcutaneous xenografts in nude mice. The TGF-alpha antisense gene was immediately detected in the cytoplasm of the tumor cells, translocated to the nucleus by 12 h and remained localized to the nucleus for up to 3 days. Direct inoculation of the TGF-alpha antisense (but not the corresponding sense) construct into established HNSCC tumors resulted in inhibition of tumor growth. Sustained antitumor effects were observed for up to 1 year after the treatments were discontinued. Down-modulation of TGF-alpha was accompanied by increased apoptosis in vivo. These experiments indicate that interference with the TGF-alpha/EGFR autocrine signaling pathway may be an effective therapeutic strategy for cancers which overexpress this ligand/receptor pair.

Bcl-2 but not Bax expression is associated with apoptosis in normal and transformed squamous epithelium.

Aberrant regulation of apoptosis may contribute to tumorigenesis. Relative levels of apoptosis regulatory proteins, such as Bcl-2 and Bax as well as interactions of these proteins with other gene products, may contribute to the rate of apoptosis in neoplasia. We examined Bcl-2 expression in 104 squamous cell carcinomas of the head and neck, as well as histologically normal mucosa several centimeters away from the tumor, and in control normal mucosa from patients without cancer. Immunohistochemistry and immunoblotting demonstrated Bcl-2 expression in 30% (31 of 104) of squamous cell carcinoma, with an increase in Bcl-2 protein levels compared with control normal mucosa from noncancer patients. Bcl-2-positive tumors demonstrated a 5-fold decrease in the number of apoptotic cells compared with Bcl-2-negative tumors. Bcl-2 protein expression was associated with poorly differentiated tumor grade but was not correlated with Bax expression or patient survival. These findings demonstrate that Bcl-2 contributes to apoptosis in normal and transformed squamous epithelium.

Growth and differentiation of rat hepatocytes: changes in transcription factors HNF-3, HNF-4, STAT-3, and STAT-5.

The liver enriched transcription factors HNF-3 and HNF-4 are known to play major roles in development and differentiation of hepatocytes. STAT-3 and STAT-5 are signaling peptides activated by a variety of cytokines and growth factors including HGF and EGF. Their role in hepatocyte growth and differentiation is yet to be determined. We examined protein expression and DNA binding activities of these transcription factors in a hepatocyte culture system in which the hepatocytes first de-differentiate and proliferate. Overlaying proliferating hepatocytes with EHS-matrix led to an increase in HNF-4 protein and DNA-binding activity. STAT-5 DNA binding activity was only slightly effected by EHS-matrix. HNF-3 and STAT-3 DNA-binding activities were reduced in the presence of EHS-matrix. This is consistent with the role of HNF-3 as the major initiating transcription factor involved in embryonic liver development and suggests, that STAT-3 might also play a role in growth and differentiation of hepatocytes.

Requirement of Stat3 but not Stat1 activation for epidermal growth factor receptor- mediated cell growth In vitro.

Stimulation of epidermal growth factor receptor (EGFR) by ligand(s) leads to activation of signaling molecules including Stat1 and Stat3, two members of the signal transducers and activators of transcription (STAT) protein family. Activation of Stat1 and Stat3 was constitutive in transformed squamous epithelial cells, which produce elevated levels of TGF-alpha, and was enhanced by the addition of exogenous TGF-alpha. Targeting of Stat3 using antisense oligonucleotides directed against the translation initiation site, resulted in significant growth inhibition. In addition, cells stably transfected with dominant negative mutant Stat3 constructs failed to proliferate in vitro. In contrast, targeting of Stat1 using either antisense or dominant-negative strategies had no effect on cell growth. Thus, TGF-alpha/EGFR-mediated autocrine growth of transformed epithelial cells is dependent on activation of Stat3 but not Stat1.

Normalization of EGFR mRNA levels following restoration of wild-type p53 in a head and neck squamous cell carcinoma cell line.

Head and neck squamous cell carcinoma (HNSCC) is frequently characterized by mutation of the p53 tumor suppressor gene and increased expression of the epidermal growth factor receptor (EGFR). To determine the potential link between p53 and EGFR expression, we examined the effect of overexpressing wild-type p53 on EGFR mRNA levels in HNSCC. In these studies, a temperature-sensitive (ts) p53 mutant was transfected into an HNSCC line which contained a deletion of one allele of the p53 gene and a mutation of the remaining allele. Following selection, six clones were isolated, characterized by Southern blot analysis, and stable expression of mutant p53 protein was confirmed by immunoblotting of clones grown at the mutant temperature. Total RNA was isolated from transfectants grown at both wild-type or mutant temperatures followed by Northern blot analysis to determine levels of EGFR mRNA expression at the two p53 conformations. Clones grown at the wild-type temperature (32.5 degreesC) demonstrated a 69% 13% decrease in EGFR mRNA compared with the same cells grown at the mutant temperature (39.5 degreesC; p=0.006) indicating that restoration of wild-type p53 reduces EGFR mRNA levels in this HNSCC cell line. These findings suggest that abnormalities in p53 may contribute to activation of EGFR gene transcription.

Inhibition of human squamous cell carcinoma growth in vivo by epidermal growth factor receptor antisense RNA transcribed from the U6 promoter.

BACKGROUND: Squamous cell carcinomas of the head and neck (SCCHN), unlike normal mucosal squamous epithelial cells, overexpress epidermal growth factor receptor (EGFR) messenger RNA and protein. EGFR protein is required to sustain the proliferation of SCCHN cells in vitro. To determine whether EGFR expression contributes to tumor growth, we investigated the effect of suppressing EGFR expression in tumor xenografts through in situ expression of antisense oligonucleotides. METHODS: Intratumoral cationic liposome-mediated gene transfer was used to deliver plasmids capable of expressing sense or antisense EGFR sequences into human head and neck tumors, which were grown as subcutaneous xenografts in nude mice. The oligonucleotides were expressed under the control of the U6 RNA promoter. RESULTS: Direct inoculation of the EGFR antisense (but not the corresponding sense) plasmid construct into established SCCHN xenografts resulted in inhibition of tumor growth, suppression of EGFR protein expression, and an increased rate of apoptosis (programmed cell death). Sustained antitumor effects were observed for up to 2 weeks after the treatments were discontinued. CONCLUSION: These results suggest that interference with EGFR expression, using an antisense-based gene therapy approach, may be an effective means of treating EGFR-overexpressing tumors, including SCCHN.

Levels of TGF-alpha and EGFR protein in head and neck squamous cell carcinoma and patient survival.

BACKGROUND: The most accurate predictor of disease recurrence in patients treated for head and neck squamous cell carcinoma is, at present, the extent of regional lymph node metastasis. Since elevated levels of epidermal growth factor receptor (EGFR) and of its ligand, transforming growth factor-alpha (TGF-alpha), have been detected in primary tumors of patients with head and neck squamous cell carcinoma, we determined whether tumor levels of these proteins were of prognostic importance. METHODS: Monoclonal antibodies specific for EGFR and TGF-alpha were used for immunohistochemical detection of each protein in tissue sections of primary tumors from 91 patients who were treated by surgical resection. Levels of immunoreactive EGFR and TGF-alpha were quantified by use of a computerized image analysis system and were normalized to appropriate standards. The logrank test and proportional hazards regression analysis were used to calculate the probability that EGFR and TGF-alpha levels were associated with disease-free survival (i.e., no recurrence of cancer) and cause-specific survival (i.e., patients do not die of their disease). All P values were two-sided. RESULTS: When tumor levels of EGFR or TGF-alpha were analyzed as continuous variables, disease-free survival and cause-specific survival were reduced among patients with higher levels of EGFR (both P = .0001) or TGF-alpha (both P = .0001). In a multivariate analysis, tumor site, tumor level of EGFR, and tumor level of TGF-alpha were statistically significant predictors of disease-free survival; in a similar analysis, regional lymph node stage and tumor levels of EGFR and of TGF-alpha were significant predictors of cause-specific survival. CONCLUSION: Quantitation of EGFR and TGF-alpha protein levels in primary head and neck squamous cell carcinomas may be useful in identifying subgroups of patients at high risk of tumor recurrence and in guiding therapy.

Staphylococcus aureus nasal colonization and association with infections in liver transplant recipients.

BACKGROUND: Staphylococcus aureus has emerged as a leading cause of bacterial infections after liver transplantation. However, the role of nasal colonization in the development of S aureus infections has never been explored in liver transplant recipients. The objectives of this study were to determine whether nasal carriage of S aureus was a risk factor for S aureus infections in liver transplant recipients. METHODS: Over a 2-year period, 30 consecutive liver transplant recipients were studied. Beginning when the recipients were transplant candidates, nasal cultures were performed at each admission and monthly thereafter until discharge or death. RESULTS: Overall, 67% (20/30) of the patients were nasal carriers, 70% of the carriers had methicillin-resistant S aureus (MRSA), 15% had methicillin-sensitive S aureus, and 15% had both MRSA and methicillin-sensitive S aureus. Infections were significantly associated with the carrier state; 100% (9/9) of the infected patients were carriers as compared with 50% (11/21) of the noninfected patients (P=0.01). All infections were a result of MRSA, and 56% (5/9) of the infections were bacteremia. Median time to the onset of S aureus infections was 16 days after transplant. Pulse field gel electrophoresis (with digestion of S aureus with SmaI restriction enzyme) in seven infected patients demonstrated that the isolates from the anterior nares matched the invasive isolates in all cases. A total of 43% (3/7) of these infected patients shared the same restriction pattern. CONCLUSION: MRSA colonization of the anterior nares was a significant predictor of MRSA infections in liver transplant recipients. Infections occurred only in those colonized with MRSA and were a result of the endogenously colonizing S aureus strains in all cases.

Evaluation of a new rapid antigen detection kit for group A beta-hemolytic streptococci.

OBJECTIVE: To evaluate the use of a new rapid antigen-detection kit for group A beta-hemolytic streptococcus and compare results with previously published studies. METHODS: Throat swabs were obtained prospectively from patients, aged one to 18 years, presenting to the emergency department, acute concerns clinic, and walk-in clinic of an urban tertiary care children's hospital. Throat swabs were first inoculated on a 5% sheep blood agar plate and then used for the streptococcus A optical immunoassay (OIA) kit. Results of both the throat culture and the rapid antigen-detection test were then compared. RESULTS: Two-hundred thirty-three patients were enrolled. Seventy-three patients had a positive culture and 63 patients had a positive OIA. Fifteen patients had a false negative result for the OIA kit and five patients had a false positive result. Test sensitivity was 79.5%, specificity was 96.9%, positive predictive value was 92.1%, and negative predictive value was 91.2% CONCLUSION: Although previous studies have demonstrated OIA kit sensitivities as high as 98.9% and authors have, as a result, recommended that the performance of a backup throat culture for a negative OIA test is unnecessary, our results do not support this. A sensitivity of 79.5% is not sufficiently high to justify omission of a standard throat culture. Accordingly, all OIA tests that are negative should be confirmed by the performance of a throat culture.

Multiply antibiotic-resistant gram-negative bacilli in a long-term-care facility: a case-control study of patient risk factors and prior antibiotic use.

OBJECTIVE: To determine the relation between prior exposure to specific antimicrobials and acquisition of gram-negative bacilli resistant to multiple beta-lactam and aminoglycoside antibiotics among long-term-care patients. DESIGN: Case-control study. Cases were patients from whom multiply resistant Enterobacteriaceae or Pseudomonas aeruginosa were isolated; controls were patients from whom nonresistant bacteria of the same species were isolated. Prospectively defined risk factors included underlying illness, activity level, presence of decubitus ulcers, presence of indwelling devices, and prior exposure to specific antimicrobial agents. Resistant and control isolates of P aeruginosa were compared using pulsed-field gel electrophoresis (PFGE) of genomic DNA after digestion with XbaI. SETTING: 390-bed long-term Veterans' Affairs facility. RESULTS: We identified 35 patients with multiply resistant Enterobacteriaceae and 24 patients with multiply resistant P aeruginosa. Of the resistant Enterobacteriaceae, 87% of isolates were resistant to piperacillin, 55% to ceftazidime, and 90% to gentamicin. Acquisition of multiply resistant Enterobacteriaceae was associated with presence of decubitus ulcers (odds ratio [OR], 12.2; 95% confidence interval [CI95], 3.3-44.2; P = .0002) and prior receipt of ampicillin (OR, 13.7; CI95, 2.2-84; P = .005). Of resistant isolates of P aeruginosa, 88% were resistant to piperacillin, 25% to ceftazidime, 42% to imipenem, and 67% to ciprofloxacin. Isolation of a multiply resistant P aeruginosa was associated with total days of antimicrobial exposure (OR, 1.07; CI95, 1.01-1.12; P = .011) and not with prior receipt of any individual agent. Eleven multiply resistant isolates shared a common PFGE pattern. CONCLUSIONS: In our long-term-care facility, acquisition of multiply resistant Enterobacteriaceae was associated with the presence of decubitus ulcers and prior exposure to ampicillin. Acquisition of resistant P aeruginosa was associated with total antibiotic exposure. Molecular typing of P aeruginosa isolates implicated patient-to-patient transmission of a limited number of resistant strains.

In vitro activities of two novel oxazolidinones (U100592 and U100766), a new fluoroquinolone (trovafloxacin), and dalfopristin-quinupristin against Staphylococcus aureus and Staphylococcus epidermidis.

Two oxazolidinones (U100592 and U100766), trovafloxacin, and a streptogramin combination (dalfopristin-quinupristin) were highly active in vitro against Staphylococcus aureus and Staphylococcus epidermidis, including methicillin-resistant strains. Trovafloxacin was more active than ciprofloxacin. Time-kill synergy studies demonstrated indifference for the oxazolidinones combined with vancomycin and rifampin against methicillin-resistant staphylococci. Spontaneous resistance was observed with all agents.

Vancomycin-gentamicin synergism revisited: effect of gentamicin susceptibility of methicillin-resistant Staphylococcus aureus.

Vancomycin monotherapy of deep-seated staphylococcal infection may be associated with poor bacteriological response. We evaluated 24 unique patient isolates of methicillin-resistant Staphylococcus aureus (MRSA) for vancomycin-gentamicin synergism by determining time-kill curves for vancomycin at 10 micrograms/ml and gentamicin at 1 microgram/ml. Nine MRSA strains showed high-level gentamicin resistance (HLGR) (MIC, > 500 micrograms/ml), and 15 did not. Vancomycin-gentamicin demonstrated synergism against none of the HLGR strains. For the non-HLGR strains, gentamicin agar dilution MICs ranged from 0.5 to > 128 micrograms/ml. Vancomycin-gentamicin demonstrated synergism against six of these strains and indifference against nine of them. There was no relationship between the agar dilution MIC of gentamicin and the occurrence of synergism against non-HLGR strains. We conclude that a gentamicin MIC of > 500 micrograms/ml predicts a lack of vancomycin-gentamicin synergism for strains of MRSA. For non-HLGR strains, synergism is not predictable from the gentamicin MIC.