Influence of health insurance, hospital factors and physician volume on receipt of immediate post-mastectomy reconstruction in women with invasive and non-invasive breast cancer.

For women with breast cancer who undergo mastectomy, immediate breast reconstruction (IR) offers a cosmetic and psychological advantage. We evaluated the association between demographic, hospital, surgeon and insurance factors and receipt of IR. We conducted a retrospective hospital-based analysis with the Perspective database. Women who underwent a mastectomy for invasive breast cancer (IBC) and ductal carcinoma in situ (DCIS) from 2000 to 2010 were included. Logistic regression analysis was used to determine factors predictive of IR. Analyses were stratified by age (<50 vs. ≥ 50) and IBC versus DCIS. Of the 108,992 women with IBC who underwent mastectomy, 30,859 (28.3 %) underwent IR, as compared to 6,501 (44.2 %) of the 14,710 women with DCIS who underwent mastectomy underwent IR. In a multivariable model for IBC, increasing age, black race, being married, rural location, and increased comorbidities were associated with decreased IR. Odds ratios (OR) of IR increased with commercial insurance (OR 3.38) and Medicare (OR 1.66) insurance (vs. self-pay), high surgeon-volume (OR 1.19), high hospital-volume (OR 2.24), and large hospital size (OR 1.20). The results were identical for DCIS, and by age category. The absolute difference between the proportion of patients who received IR with commercial insurance compared to other insurance, increased over time. Immediate in-hospital complication rates were higher for flap reconstruction compared to implant or no reconstruction (15.2, 4.0, and 6.1 %, respectively, P < .0001). IR has increased significantly over time; however, modifiable factors such as insurance status, hospital size, hospital location, and physician volume strongly predict IR. Public policy should ensure that access to reconstructive surgery is universally available.

Characterization of a transforming N-ras gene in the human hepatoma cell line Hep G2: additional evidence for the importance of c-myc and ras cooperation in hepatocarcinogenesis.

The expression of the c-myc gene has previously been shown to be elevated and deregulated in the human hepatoma cell line Hep G2 (B. E. Huber and S. S. Thorgeirsson, Cancer Res., 47: 3414-3420, 1987). We now report that the Hep G2 N-ras gene is activated to a dominant-acting, transforming gene by a missense mutation in codon 61. Hep G2 DNA produced transformed foci when transfected into NIH 3T3 cells. Subsequent to a secondary round of transfection, Southern blot analysis of tumorigenic NIH 3T3 foci demonstrated the presence of human N-ras sequences. Nucleotide sequence analysis of one Hep G2 N-ras allele demonstrated that codons 12, 13, and 59 were normal and that codon 61 had a missense mutation (CAA to CTA). This mutation results in the incorporation of leucine instead of glutamine at residue 61 of the N-ras gene product, p21. N-ras sequences were amplified by the polymerase chain reaction from both Hep G2 genomic DNA and Hep G2 complementary DNA. Analysis of the amplified sequences demonstrated that only one Hep G2 N-ras allele exhibited the codon 61 mutation and that both the mutant and normal alleles were transcribed. Northern blot analysis demonstrated equivalent steady-state levels of N-ras transcripts in Hep G2 cells and normal human liver. The steady-state levels of N-ras and ornithine decarboxylase transcripts were positively correlated suggesting a positive relationship between N-ras expression and the replication rate of Hep G2 cells. c-Ki-ras and c-Ha-ras transcripts were not detected in either Hep G2 cells or normal human liver. Immunoprecipitation experiments using the monoclonal antibody Y13-259 demonstrated the presence of p21 in Hep G2 cells. Expression of a dominant-acting, transforming N-ras gene, in conjunction with the altered regulation of the c-myc gene, documents two important genetic lesions that could be responsible for the transformed phenotype of Hep G2 cells.

In vivo antitumor activity of 5-fluorocytosine on human colorectal carcinoma cells genetically modified to express cytosine deaminase.

A human colorectal carcinoma cell line, WiDr, was genetically engineered to express the nonmammalian enzyme, cytosine deaminase (CD). Expression of CD in WiDr cells (WiDr/CD) did not alter the growth rate of these cells when grown in vitro or as solid tumor xenografts in nude mice. However, expression of CD did increase the sensitivity of these cells to the nontoxic prodrug, 5-fluorocytosine (FCyt), decreasing the 50% inhibitory concentration for FCyt from 26,000 microM in parental WiDr cells to 27 microM in WiDr/CD cells. The increase in sensitivity to FCyt in WiDr/CD cells was the result of the CD-mediated conversion of FCyt to 5-fluorouracil (FUra) and subsequent FUra anabolites. The half-life of the prodrug, FCyt, was determined to be approximately 40 min in nude mice. A single i.p. injection of 500 mg FCyt/kg body weight resulted in a transient FCyt plasma level of approximately 4000 microM while osmotic minipumps or constant tail vein infusions of FCyt achieved continual FCyt plasma levels of 5 microM and 50 microM, respectively, with no overt signs of toxicity. Significant antitumor effects were observed in nude mice bearing tumors derived from WiDr/CD cells when these animals were given 500 mg FCyt/kg i.p. for 10 consecutive days. These antitumor effects were demonstrated by decreases in tumor growth rate, tumor size, tumor weight, and thymidine incorporation into tumor DNA. This antitumor effect was significant but less profound if FCyt was administered by constant tail vein infusion. WiDr and WiDr/CD cells were very sensitive to FUra in vitro (50% inhibitory concentration approximately 5 microM). However, no significant antitumor effects were observed in nude mice bearing tumors derived from either WiDr or WiDr/CD cells when these animals were treated with various doses of FUra. Taken collectively, these data indicate that nontoxic plasma levels of FCyt can be attained which can produce profound antitumor effects on tumors engineered to express CD and that these antitumor effects are significantly better than those that can be achieved using FUra. These positive data support the continued development of a gene therapy approach to colorectal carcinoma involving the selective expression of CD in colorectal tumors with subsequent administration of FCyt.

Kilham rat triggers T-cell-dependent autoimmune diabetes in multiple strains of rat.

Kilham rat virus (KRV) infection of BB/Wor diabetes-resistant (DR) RT1(u) rats induces autoimmune diabetes without direct cytolytic infection of pancreatic beta-cells and is a new model of virus-induced IDDM. To investigate genetic susceptibility to KRV-induced diabetes, major histocompatibility complex congenic and other inbred rats were infected with the virus and studied for the appearance of diabetes and insulitis. KRV infection alone induced insulitis, selective beta-cell necrosis, and diabetes in BB/Wor DR and LEW1.WR1 (RT1 A(u) B/D(u) C(a)) but not other rats. Thus, KRV, an environmentally ubiquitous rat parvovirus, can precipitate autoimmune diabetes in rats that are not susceptible to spontaneous diabetes. If rats are injected with poly(I.C) immediately before KRV infection, diabetes frequency increases to >90% in BB/Wor DR and LEW1.WR1 rats, and PVG.RT1(u) rats are converted from KRV-resistant to KRV-susceptible status. Susceptibility to KRV-induced diabetes thus requires the presence of class I A(u) and class II B/D(u) gene products, which are shared by DR, LEW1.WR1, and PVG.RT1(u) rats. The RT1(u) haplotype is not sufficient for susceptibility, however, because while WF rats are RT1(u), they resist KRV-induced diabetes. If rats are depleted of RT6.1+ regulatory T-cells before KRV infection, the frequency of diabetes is dramatically increased in DR and LEW1.WR1, but not PVG.RT1(u) or other rats. These data confirm a regulatory role of RT6.1+ T-cells in diabetes induction, but indicate that they may not operate as such in all rat strains. KRV-induced diabetes is T-cell-mediated: DR and LEW1.WR1 rats are protected from diabetes by treatment with monoclonal antibodies directed against alpha beta T-cell receptor (TCR)+, CD5+, and CD8+ T-cells. Concanavalin A-activated spleen cells from KRV-infected DR rats adoptively transfer diabetes and insulitis into class II(u) compatible rats, suggesting that KRV infection of susceptible rats leads to the activation of diabetogenic class II(u) restricted T-cells. The ability of a common rat virus to initiate IDDM in multiple strains of rats strengthens the possibility that viruses may also initiate IDDM in human populations.

Generation of a transgenic model for retrovirus-mediated gene therapy for hepatocellular carcinoma is thwarted by the lack of transgene expression.

Transgenic mice have been generated to determine the tissue-specific expression, safety, and efficacy of a novel chimeric gene that is being investigated as a test system for virus-directed enzyme prodrug therapy (VDEPT). The chimeric gene consists of the transcriptional regulatory sequences of the albumin gene and the protein-coding sequence of the varicella-zoster virus thymidine kinase (VZV-TK) gene inserted into a retroviral vector. Eight founders were obtained from microinjection of a nearly full-length proviral fragment containing the chimeric gene. Liver extracts of the founders and 12 G1 mice were analyzed by enzymatic and Western blot analysis for the presence of VZV-TK. No VZV-TK enzymatic activity or protein was detected. Methylation analysis indicated that both the chimeric gene and retroviral sequences were methylated. Treatment of newborn mice with 5-azacytidine or backcrossing into a DBA/2 genetic background did not result in detectable VZV-TK expression or a change in transgene methylation. The poor transgene expression reported here appears to reflect an inherent, continuing problem of transgenic technology with transgenes that are essentially intact retroviral shuttle vectors. These methylation and expression problems are generally applicable to other animal models for retroviral-mediated gene therapy and should be of interest to researchers as they design and evaluate preclinical safety and efficacy studies.

Transcriptional regulatory sequences of carcinoembryonic antigen: identification and use with cytosine deaminase for tumor-specific gene therapy.

The 5' sequences from the human carcinoembryonic antigen gene (CEA) were analyzed using luciferase reporter gene assays. This analysis identified important cis-acting sequences needed for selective expression in CEA-positive cells. Over 50 CEA/luciferase reporter clones were constructed and analyzed in two CEA-positive and two CEA-negative cell lines. The CEA sequences analyzed extended from the translational start to 14.5 kb 5' of the CEA gene. A 408-bp region from the CEA 3' untranslated region was also examined for its effect on reporter gene activity. The CEA promoter was located between bases -90 and +69 of the transcriptional start site. Sequences between -41 and -18 were essential for expression from the CEA promoter. Multimerization of sequences between -89 and -40 resulted in copy number-related increases in both expression level and selectivity for CEA-positive cells. Two upstream regions of CEA, -13.6 to -10.7 kb or -6.1 to -4.0 kb, when linked to the multimerized promoter led to high-level, selective expression in CEA-positive cell lines. Several CEA/luciferase constructs demonstrated 80- to 120-fold higher expression in CEA-positive cell lines compared to expression in CEA-negative Hep3B cells. The expression from these constructs was quite strong in CEA-positive cells, being two- to four-fold higher than an SV40 enhancer/promoter construct. The most promising CEA transcriptional regulatory sequences were used to regulate the expression of cytosine deaminase (CD) in stable cell lines. The expression of CD was assessed directly by an enzymatic assay and indirectly by determining the in vitro IC50 to 5-fluorocytosine (5FC). The chimeric gene pCEA/CD-145 displayed the desired expression spectrum--high-level expression in the CEA-positive cells and low-level expression in CEA-negative cells. CD expression from this chimera correlated well with the expression of the endogenous CEA gene. Treatment of mice bearing NCI H508 pCEA/CD-145 tumor xenografts with 5FC lead to significant antitumor effects in vivo. The CEA/CD chimeric gene should be useful for tumor-specific suicide gene therapy of CEA-positive tumors.

Nucleotide sequence of the dihydrofolate reductase gene of Saccharomyces cerevisiae.

The nucleotide sequence of a DNA fragment that contained the Saccharomyces cerevisiae gene DFR coding for dihydrofolate reductase (DHFR) was determined. The DHFR was encoded by a 633-bp open reading frame, which specified an Mr24264 protein. The polypeptide was significantly related to the DHFRs of chicken liver and Escherichia coli. The yeast enzyme shared 60 amino acid (aa) residues with the avian enzyme and 51 aa residues with the bacterial enzyme. DHFR was overproduced about 40-fold in S. cerevisiae when the cloned gene was present in the vector YEp24. As isolated from the Saccharomyces library, the DFR gene was not expressed in E. coli. When the gene was present on a 1.8-kb BamHI-SalI fragment subcloned into the E. coli vector, pUC18, weak expression in E. coli was observed.

The admid system: generation of recombinant adenoviruses by Tn7-mediated transposition in E. coli.

A new system has been developed for generating recombinant adenoviruses by Tn7-mediated transposition in E. coli. Low copy number E. coli plasmids containing a full-length adenoviral genome with lacZattTn7 replacing E1 have been constructed. The adenovirus plasmid or admid, as well as high copy number progenitors, were stably maintained in E. coli strain DH10B. Several transfer vectors containing a mammalian expression cassette flanked by Tn7R and Tn7L were used as donors to transpose the mini-Tn7 into the E1 region of the adenoviral genome. Transposed recombinant admids are readily identified by their beta-galactosidase phenotype. Transfection of admid DNA into producer cells resulted in the efficient production of infectious adenovirus. This easy-to-use, efficient system generates pure, clonal stocks of recombinant adenovirus without successive rounds of plaque purification.

The role of imagination in facilitating deductive reasoning in 2-, 3- and 4-year-olds.

When 4- and 6-year-olds are cued to use their imagination, they can overcome the belief bias effect and demonstrate deductive reasoning ability on syllogisms containing contrary-to-fact material. This study tested whether 2- and 3-year-olds could also reason with incongruent syllogisms when encouraged to use their imagination. Eighty-four 2-, 3- and 4-year-olds were randomly assigned to one of four conditions: no cue, word cue, fantasy planet or imagery. Children were then presented with six syllogistic reasoning problems containing incongruent information. In the imagination conditions, 2- and 3-year-olds performed as competently as 4-year-olds. The findings are discussed in relation to other research which suggests that under certain circumstances 2- and 3-year-olds have the capacity for counterfactual thinking.

Practical approaches to improve laboratory performance and transfusion safety.

Laboratory quality can be defined as "doing the right thing right." The chance of doing the right thing right is greater if basic process control systems are implemented in laboratories. The elements of process control were reviewed, and practical suggestions for organizing procedures, training, determining competency, maintaining equipment, and implementing self-assessment and process improvement activities were offered. Although process control can improve the reliability of internal operations, the quality of patient care can be further improved by extending this level of control outward to include interaction with patients. Finally, to expand the concept of quality, quality activities must be extended toward a multidisciplinary model of coordinated care.

Virus-directed enzyme/prodrug therapy (VDEPT). Selectively engineering drug sensitivity into tumors.

A gene therapy approach has been described that generates a tumor-selective qualitative difference in the metabolic capability in tumor cells. This is the result of the selective expression of a nonmammalian enzyme in tumor cells. Selective expression is achieved by utilization of a chimeric gene composed of the TRS from a tumor-associated marker gene linked to the coding domain of a gene encoding a nonmammalian enzyme. We have described the application of this approach for the treatment of metastatic CRC. This approach involves creation of a chimeric gene composed of the CEA TRS linked to the coding domain of the CD gene. Selective expression of CD in the tumor cells will allow the selective conversion of the prodrug 5-FCyt to 5-FCyt in the tumor while sparing normal cells. Most importantly, delivery and expression of CD into a small fraction of tumor cells may be sufficient to achieve a significant antitumor effect.

The histological appearances of Nissen-type fundoplication in the ferret.

Nissen fundoplication is of proven effectiveness in the surgical control of gastro-oesophageal reflux. However, our understanding of the effects of fundoplication upon foregut physiology is incomplete and post-operative symptoms are often poorly understood. This experimental study aimed systematically to characterize the tissue response to fundoplication in an animal model, to improve understanding of the effects of anti-reflux surgery upon foregut physiology. Nissen-type fundoplication was performed in the ferret, and the tissue response at 3 months examined histologically. Sham-operated animals that underwent laparotomy but no dissection or wrap, acted as controls. In fundoplicated animals, serosal fibrosis was observed in the gut wall, with patchy replacement of muscle by fibrous tissue. The ventral and dorsal vagal nerve trunks were identified intact within the wrap. In cases where the wrap had spontaneously disrupted, fibrosis was more extensive and there was evidence of nerve damage. This is the first systematic description of the histopathological response to Nissen fundoplication. In the intact wrap, the vagal trunks appear spared, but there is fibrosis in the serosa, extending into the muscularis of the distal oesophagus and region of the cardia. These findings are discussed in relation to the effects of Nissen fundoplication upon gastric physiology and postoperative symptoms.

Nissen-type fundoplication and its effects on the emetic reflex and gastric motility in the ferret.

Recurrent vomiting with failure to thrive is a common problem in neurologically impaired children. Many undergo fundoplication to control the underlying gastro-oesophageal reflux. The results of surgery are not always satisfactory and post-operative retching may be a major problem - a symptom indicative of activation of the emetic reflex. An animal model of antireflux surgery has been developed and used to investigate the effects of such surgery upon the emetic reflex and vagal influences on gastric motility. Following surgery, animals responded to a previously subemetic dose of a centrally acting opiate receptor agonist (loperamide), suggesting that fundoplication may sensitize the emetic reflex. A gastric vago-vagal reflex (tonic inhibition of corpus tone) and responses to direct stimulation of vagal motor efferents (both cholinergic and nonadrenergic noncholinergic responses) were not significantly affected by antireflux surgery. Mechanisms by which neural damage may sensitize the emetic reflex are discussed, together with the possible clinical implications for the management of post-operative symptoms in neurologically impaired children.

Effect of oral contraceptives on leukocyte phagocytic activity and plasma levels of prostaglandin E2 and thromboxane B2 in normal menstruating women.

Mononuclear and polymorphonuclear cells were isolated from the peripheral blood of normal menstruating women. Four of the subjects were not using oral contraceptives and five were taking various formulations. The women were tested once a week for 12 consecutive weeks. Plasma levels of 6-keto-prostaglandin F2 alpha (6-KF), prostaglandin E2 (PGE2), thromboxane B2 (TxB2), estrogen, and progesterone were measured by specific radioimmunoassays. The phagocytic activity of the mononuclear and polymorphonuclear cells isolated from the peripheral blood was measured with a bacterial phagocytosis and killing assay. The phagocytic activity of both types of cells was depressed perimenstrually in both groups of women. However, examination of individuals showed that those subjects not taking oral contraceptives had a worsening of phagocytic activities with approaching menses while the oral contraceptive subjects generally had an improving of these activities at this time. We were unable to correlate the phagocytic activities with either hormone or prostaglandin levels in the plasma of these subjects. However, the subjects on oral contraceptives had significantly lower levels of PGE2 and TxB2 than those women who were not using oral contraceptives.

Regulated expression of artificial chimeric genes contained in retroviral vectors: implications for virus-directed enzyme prodrug therapy (VDEPT) and other gene therapy applications.

Replication-defective retroviral vectors were created that contained chimeric genes composed of either the albumin (ALB) or the alpha-fetoprotein (AFP) transcriptional regulatory sequences linked to the coding domain of the thymidine kinase gene from Varicella zoster virus (VZV TK). These viruses were used to infect the human hepatoblastoma cell line, HepG2. Subsequent to infection, the infected cells were single-cell cloned. The level of expression of VZV TK from the chimeric genes correlated with the level of endogenous expression of ALB or AFP in most clones, indicating that the transcription of the chimeric VZV TK gene is controlled in a similar manner to the endogenous ALB or AFP genes, and that sites of viral integration are less important to overall gene expression. Most importantly, as the expression of the endogenous ALB gene was modified, so was expression of VZV TK from the ALB/VZV TK chimeric gene. This demonstrates that retroviruses can deliver a chimeric gene containing tissue-specific transcriptional regulatory sequences that can respond to endogenous cell regulatory signals resulting in regulated gene expression.

Nissen fundoplication may induce gastric myoelectrical disturbance in children.

BACKGROUND: Recurrent vomiting with failure to thrive is a common problem in neurologically impaired children. Many undergo fundoplication to control the underlying gastroesophageal reflux, but the results of surgery are not always satisfactory, and postoperative retching may be a major problem. Retching is part of the emetic reflex and is associated with nausea, which is itself associated with disturbed gastric electrical control activity, resulting in a gastric dysrhythmia. METHODS: By recording gastric electrical control activity before and after Nissen fundoplication using the noninvasive technique of surface electrogastrography, the authors have shown that (1) Neurologically impaired children with gastroesophageal reflux more commonly have a preexisting gastric dysrhythmia (65% neurologically impaired v 20% neurologically normal children with gastroesophageal reflux, P<.05), (2) Children who retch preoperatively are three times more likely to retch postoperatively, and (3) 25% of neurologically impaired children may start to retch postoperatively for the first time. CONCLUSION: The authors propose that in neurologically impaired children, loss of central inhibitory mechanisms may result in inappropriate activation of the emetic reflex, which may be heightened by antireflux surgery.

Retching and vomiting in neurologically impaired children after fundoplication: predictive preoperative factors.

BACKGROUND/PURPOSE: In neurologically impaired children, retching and recurrent vomiting are common after Nissen fundoplication. The aim of this study was to identify whether there are preoperative factors that predict their occurrence. METHODS: Twenty neurologically impaired children (8 boys, 12 girls; age range, 3 months to 8 years) were studied prospectively by taking a detailed history of behaviors and symptoms associated with feeding before and after Nissen fundoplication for gastroesophageal reflux. RESULTS: Preoperatively, children could be classified into 2 groups. Children in group A had symptoms suggestive of only gastroesophageal reflux (effortless "vomiting" or regurgitation), whereas children in group B exhibited one or more features associated with activation of the emetic reflex (pallor, sweating, retching, forceful vomiting). Postoperatively 0 of 8 in group A retched compared with 8 of 12 in group B (P <.005, Fishers Exact test). CONCLUSIONS: Children at high risk of retching, and ultimately vomiting, after antireflux surgery may be identified clinically preoperatively. They have symptoms that are specifically caused by activation of the emetic reflex rather than to gastroesophageal reflux. In these cases, antireflux surgery could be considered inappropriate and hence be avoided.

The transcriptional control region of the human carcinoembryonic antigen gene: DNA sequence and homology studies.

Phage clones containing human carcinoembryonic antigen (CEA) 5' flanking sequences were isolated from a chromosome 19 genomic library. The clones were confirmed to contain CEA sequences flanking the CEA transcriptional start site by restriction endonuclease mapping, PCR analysis, and sequence determination. Restriction endonuclease mapping determined that the clones spanned approximately 26 kbp of contiguous sequence from 14 kbp 5' to 12 kbp 3' of the CEA transcriptional start. The DNA sequence of a 11,288 bp Hind III/Sau 3A restriction endonuclease fragment was determined. This sequence extends from 10.7 kbp 5' to 0.6 kbp 3' of the transcriptional start. This sequence was analyzed for the presence of consensus transcriptional regulatory sequences, repetitive sequences, and other features. Several transcriptional regulatory consensus sequences were identified. These consensus sequences may have significance for the transcriptional regulation of CEA.