Biological activities of a recombinant adenovirus p53 (SCH 58500) administered by hepatic arterial infusion in a Phase 1 colorectal cancer trial.

The major focus of intrahepatic arterial (IHA) administration of adenoviruses (Ad) has been on safety. Currently, there is little published data on the biological responses to Ad when administered via this route. As part of a Phase I study, we evaluated biological responses to a replication-defective adenovirus encoding the p53 transgene (SCH 58500) when administered by hepatic arterial infusion to patients with primarily colorectal cancer metastatic to the liver. In analyzing biological responses to the Ad vector, we found that both total and neutralizing Ad antibodies increased weeks after SCH 58500 infusion. The fold increase in antibody titers was not dependent on SCH 58500 dosage. The proinflammatory cytokine interleukin-6 (IL-6) transiently peaked within 6 h of dosing. The cytokine sTNF-R2 showed elevation by 24 h post-treatment, and fold increases were directly related to SCH 58500 doses. Cytokines TNF-alpha, IL-1beta, and sTNF-R1 showed no increased levels over 24 h. Predose antibody levels did not appear to predict transduction, nor did serum Ad neutralizing factor (SNF). Delivery of SCH 58500 to tumor tissue occurred, though we found distribution more predominantly in liver tissues, as opposed to tumors. RT-PCR showed significantly higher expression levels (P<0.0001, ANOVA) for adenovirus type 2 and 5 receptor (CAR) in liver tissues, suggesting a correlation with transduction. Evidence of tumor-specific apoptotic activity was provided by laser scanning cytometry, which determined a coincidence of elevated nuclear p53 protein expression with apoptosis in patient tissue. IHA administration of a replication defective adenovirus is a feasible mode of delivery, allowing for exogenous transfer of the p53 gene into target tissues, with evidence of functional p53. Limited and transient inflammatory responses to the drug occurred, but pre-existing immunity to Ad did not preclude SCH 58500 delivery.

Adenovirus-mediated wild-type p53 gene transfer in patients receiving chemotherapy for advanced non-small-cell lung cancer: results of a multicenter phase II study.

PURPOSE: To study the additional benefit from adenoviral p53 gene therapy in patients undergoing first-line chemotherapy for advanced non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Twenty-five patients with nonresectable NSCLC were enrolled in an open-label, multicenter phase II study of three cycles of regimen A, carboplatin (area under the curve, 6; day 1) plus paclitaxel (175 mg/m(2), day 1), or regimen B, cisplatin (100 mg/m(2), day 1) plus vinorelbine (25 mg/m(2), days 1, 8, 15, and 22) in combination with intratumoral injection of 7.5 x 10(12) particles of SCH 58500 (rAd/p53, day 1). Responses of individual tumor lesions were assessed after each cycle, and gene transfer was examined in posttreatment tumor biopsies using reverse transcriptase polymerase chain reaction. RESULTS: There was no difference between the response rate of lesions treated with p53 gene therapy in addition to chemotherapy (52% objective responses) and lesions treated with chemotherapy alone (48% objective responses). Subgroup analysis according to the chemotherapy regimens revealed evidence for increased mean local tumor regressions in response to additional p53 gene therapy in patients receiving regimen B, but not in patients receiving regimen A. There was no survival difference between the two chemotherapy regimens, and the median survival of the cohort was 10.5 months (1-year survival, 44%). Transgene expression was confirmed in tumor samples from 68% of patients, and toxicities attributable to gene therapy were mild to moderate. CONCLUSION: Intratumoral adenoviral p53 gene therapy appears to provide no additional benefit in patients receiving an effective first-line chemotherapy for advanced NSCLC.

Promoting responsiveness between mothers with depressive symptoms and their infants.

PURPOSE: To test the efficacy of an interactive coaching intervention to promote responsiveness between mothers experiencing postpartum depressive symptoms (PPDS) and their infants. DESIGN: An experimental design with 117 postpartum women in the Northeastern United States. METHODS: Participants were randomly assigned either to the treatment or control group. Both groups had home visits at 4-8 weeks, 10-14 weeks, and 14-18 weeks postpartum and mother-infant interaction was videotaped and coded for responsiveness. The treatment group also received a coached behavioral intervention designed to promote maternal-infant responsiveness. Measures included the Edinburgh Postnatal Depression Scale, the Beck Depression Inventory-II, and the Dyadic Mutuality Code. FINDINGS: The hypothesis, that the treatment group would show significantly higher maternal-infant responsiveness after the intervention, was supported. No effect of the intervention on depression scores was found. A significant increase in responsiveness and a significant decrease in depression scores occurred over time for both treatment and control groups. No interaction between group and time was detected. CONCLUSIONS: The study showed that a coaching strategy had a positive effect on maternal-infant interaction in this sample. Future research is needed to test coaching interventions in conjunction with other strategies targeted to promote maternal-infant responsiveness and to reduce PPDS.

An international study exploring levels of postpartum depressive symptomatology.

OBJECTIVE: Differences in postpartum depressive symptomatology (PPDS) among an international sample of 892 women from nine countries representing five continents were explored. METHOD: Edinburgh Postnatal Depression Scale (EPDS) and Beck Depression Inventory (BDI) were used to assess PPDS among a convenience sample that completed the two questionnaires twice, yielding a total of four sets of scores per subject. Women sampled were primiparae with no obstetrical complications, and had a healthy baby. Depression history and therapy were ruled out as exclusion criteria. RESULTS: Mean scores for EPDS and BDI varied across sites at both time points (P value<.001). European and Australian women had the lowest levels of PPDS, USA women fell at the midpoint, and women from Asia and South America had the highest depressive symptom scores. The moderate concordance between the EPDS and BDI suggested that the measures have complementary uses for screening and assessment. CONCLUSION: Utility of EPDS and BDI for yielding profiles of postpartum women's depressive symptomatology was demonstrated. Further research to validate depressive symptom measures with diverse international populations is indicated.

Biological activity and safety of adenoviral vector-expressed wild-type p53 after intratumoral injection in melanoma and breast cancer patients with p53-overexpressing tumors.

p53 mutations are common genetic alterations in human cancer. Gene transfer of a wild-type (wt) p53 gene reverses the loss of normal p53 function in vitro and in vivo. A phase I dose escalation study of single intratumoral (i.t.) injection of a replication-defective adenoviral expression vector containing wt p53 was carried out in patients with metastatic melanoma or breast cancer with increased p53 protein immunoreactivity in pretreatment tumor biopsies. The biological activity of the injected wt p53 was assayed by reverse transcriptase-polymerase chain reaction in tumor tissue. A total of six (five melanoma and one breast adenocarcinoma) patients were treated at dose levels dependent upon tumor size/dose escalation sequence. Five of six patients became positive for the transfer of wt p53 into tumor tissue 2 days after injection of the vector. Of the four patients assayed, all developed anti-adenoviral antibodies. Adverse reactions associated with i.t. injection were mild, with no obvious correlation between the incidence, severity, or relationship of the events and drug dose. p53 gene therapy by i.t. injection of a replication-defective adenoviral expression vector is safe, feasible, and biologically effective (with respect to transduction frequency) in patients with either metastatic melanoma or breast cancer.

Mother's perceptions of postpartum stress and satisfaction.

OBJECTIVE: To examine mothers' postpartum perceptions of stress and satisfaction. DESIGN: Methodologic triangulation with quantitative and qualitative data in a nonexperimental design. PARTICIPANTS AND SETTING: A convenience sample of 95 women was obtained during normally scheduled postpartum appointments at a health maintenance organization. MAIN OUTCOME MEASURES: The self-administered questionnaire included the Mothers' Information Tool (MIT), What Being the Parent of a Baby Is Like (WPL-R), and the Brief Symptom Inventory (BSI). Open-ended MIT items revealed mothers' perceptions of stress and satisfaction. The WPL-R provided maternal satisfaction scores, and the BSI yielded Global Stress Index scores. RESULTS: Content analysis identified the following categories: Roles, Tasks, Resources, and Relationships. Subcategories identified as areas of stress were Work/School, Sleep/Rest, Adjustment/Own Needs, Health/Body Image, Organization of Life, Child Care, Day Care, Housework, Future Challenges, Finances, Housing, Time, Partner, and Family. Subcategories identified as areas of satisfaction were Participating in Relationships, Sharing the Future, Being Proud to Be a Mother, Enjoying a Healthy Baby, and Caring for a Child. Levels of stress and satisfaction of mothers who scored high and low on quantitative measures were compared. CONCLUSION: The outcomes contribute to the knowledge concerning postpartum women's perceptions of the mothering experience and suggest approaches to nursing assessment and intervention to prevent postpartum adjustment difficulties.

Negotiating couplehood: the process of resolving the December dilemma among interfaith couples.

Christmas forces interfaith couples to address questions concerning holiday observances. The purpose of this investigation was to explore the experience of the "December dilemma," that is, the experience of Christmas and Hanukah among couples in which one partner is Jewish. A qualitative design based on the continuous comparison method of Grounded Theory analysis was used. Participants were solicited through interfaith couples' programs, referral, and snowballing. Unstructured interactive interviews of 22 couples were audiotaped, transcribed, and analyzed. The categories generated were: Ghosts of Christmas and Hanukah Past, Coming Together, and Holiday Observances as a Couple. The basic problem facing these couples was how to bridge religious backgrounds with differing holiday traditions in a way that integrated respect for each partner's needs, heritage, and identity. The basic social process of negotiating "couplehood," that is, moving from individuality to partnership emerged when mutual agreement could be reached to solve problems about how to celebrate the December holidays. The data indicated that exploration of the ways these couples managed the dilemmas created by the December holidays provided a window to how they negotiated other challenges in their relationships.

A phase I study of adenovirus-mediated wild-type p53 gene transfer in patients with advanced non-small cell lung cancer.

Mutations of the tumor suppressor gene p53 are the most common genetic alterations observed in human cancer. Loss of wild-type p53 function impairs cell cycle arrest as well as repair mechanisms involved in response to DNA damage. Further, apoptotic pathways as induced by radio- or chemotherapy are also abrogated. Gene transfer of wild-type p53 was shown to reverse these deficiencies and to induce apoptosis in vitro and in preclinical in vivo tumor models. A phase I dose escalation study of a single intratumoral injection of a replication-defective adenoviral expression vector encoding wild-type p53 was carried out in patients with incurable non-small cell lung cancer. All patients enrolled had p53 protein overexpression as a marker of mutant p53 status in pretreatment tumor biopsies. Treatment was performed either by bronchoscopic intratumoral injection or by CT-guided percutaneous intratumoral injection of the vector solution. Fifteen patients were enrolled in two centers, and were treated at four different dose levels ranging from 10(7) to 10(10) PFU (7.5 x 10(9) to 7.5 x 10(12) particles). No clinically significant toxicity was observed. Successful transfer of wild-type p53 was achieved only with higher vector doses. Vector-specific wild-type p53 RNA sequences could be demonstrated in posttreatment biopsies of six patients. Transient local disease control by a single intratumoral injection of the vector solution was observed in four of those six successfully transduced patients. There was no evidence of clinical responses at untreated tumor sites. Wild-type p53 gene therapy by intratumoral injection of a replication-defective adenoviral expression vector is safe, feasible, and biologically effective in patients with advanced non-small cell lung cancer.

Nuclear domains in skeletal myotubes: the localization of transferrin receptor mRNA is independent of its half-life and restricted by binding to ribosomes.

The retention of mRNAs near the nuclei that synthesize them may be an important feature of the organization of multinucleated skeletal myotubes. Here, we assess the possible role of two factors in this localization. First, we examine the role of mRNA half-life, by studying the distribution of the mRNA for the transferrin receptor (TfR), whose half-life can be manipulated in culture by changing the availability of iron. In situ hybridization of myotubes of the mouse muscle cell line C2 shows that TfR mRNA is concentrated in the core of the myotubes. Its distribution around the nuclei is often asymmetric and its concentration changes abruptly. Stable transcripts display the same asymmetric localization as unstable ones, suggesting that half-life does not determine subcellular localization of TfR mRNA. Differential effects of the protein synthesis inhibitors puromycin and cycloheximide suggest that the mRNA is retained in position by its association with ribosomes. We then examine the distribution of the rough endoplasmic reticulum (RER) and find it to be broader than the distribution of TfR mRNA. In contrast to TfR mRNA, the mRNA for a secreted immunoglobulin kappa light chain has a more uniform distribution. Taken together, the results suggest that TfR mRNA may associate with RER subdomains by specific targeting.

Identification of symptoms of postpartum depression: linking research to practice.

This paper presents a research approach for identification of symptoms of postpartum depression that could be adapted for use in practice settings. Women (n = 95) who had recently given birth completed a self-administered questionnaire at a health center at the time of their regularly scheduled postpartum visit. The questionnaire was composed of The Mothers' Information Tool to obtain demographic data, personal history, and information about the woman's birth and postpartum experience and the Depression Adjective Check Lists (DACL) and Brief Symptom Inventory (BSI) to assess symptoms suggestive of postpartum depression. Follow-up assessment of women whose scores were elevated on the DACL or BSI provided confirmation of symptoms and opportunity for additional evaluation and treatment of postpartum depression when indicated. The authors conclude that paper-and-pencil instruments could be used effectively in clinical practice to identify depressive symptoms among postpartum women.

Evidence that the pathway of transferrin receptor mRNA degradation involves an endonucleolytic cleavage within the 3' UTR and does not involve poly(A) tail shortening.

The stability of transferrin receptor (TfR) mRNA is regulated by iron availability. When a human plasma-cytoma cell line (ARH-77) is treated with an iron source (hemin), the TfR mRNA is destabilized and a shorter TfR RNA appears. A similar phenomenon is also observed in mouse fibroblasts expressing a previously characterized iron-regulated human TfR mRNA (TRS-1). In contrast, mouse cells expressing a constitutively unstable human TfR mRNA (TRS-4) display the shorter RNA irrespective of iron treatment. These shorter RNAs found in both the hemin-treated ARH-77 cells and in the mouse fibroblasts are shown to be the result of a truncation within the 3' untranslated regions of the mRNAs. The truncated RNA is generated by an endonuclease, as most clearly evidenced by the detection of the matching 3' endonuclease product. The cleavage site of the human TfR mRNA in the mouse fibroblasts has been mapped to single nucleotide resolution to a single-stranded region near one of the iron-responsive elements contained in the 3' UTR. Site-directed mutagenesis demonstrates that the sequence surrounding the mapped endonuclease cleavage site is required for both iron-regulated mRNA turnover and generation of the truncated degradation intermediate. The TfR mRNA does not undergo poly(A) tail shortening prior to rapid degradation since the length of the poly(A) tail does not decrease during iron-induced destabilization. Moreover, the 3' endonuclease cleavage product is apparently polyadenylated to the same extent as the full-length mRNA.

The secondary structure of the regulatory region of the transferrin receptor mRNA deduced by enzymatic cleavage.

The secondary structure of the portion of the transferrin receptor mRNA responsible for the regulation of the transcript's half-life has been deduced by ribonuclease H cleavage directed by antisense oligodeoxyribonucleotides as well as with other ribonucleases sensitive to RNA secondary structure. The data indicate that both a synthetic 252-nucleotide RNA and the comparable portion of a 2.7-kb cellular mRNA contain three stem-loops referred to as iron-responsive elements (IREs). This secondary structure appears to be relatively static, with little interconversion with another possible structure having a similar calculated free energy but involving longer-range base pairing. Deletion of a selected cytosine residue from each of the IRE loops has been shown to yield an unregulated, unstable mRNA. This altered RNA has a secondary structure similar, if not identical, to that of the RNA that is competent in regulation.

Translation and the stability of mRNAs encoding the transferrin receptor and c-fos.

Turnover of the full-length human transferrin receptor (TfR) mRNA is regulated by iron, and this regulation is mediated by the transcript's 3' untranslated region. Alterations in the sequence of the TfR mRNA regulatory region have been identified that render the mRNA unregulated by iron and intrinsically unstable. When cells expressing this unstable mRNA are treated with inhibitors of protein synthesis (cycloheximide or puromycin), the steady-state level of the encoded human TfR mRNA is increased due to a stabilization of the transcript. A similar set of observations has been made using a chimeric mRNA in which the rapid turnover determinant of the TfR mRNA is replaced by the (A+U)-rich region from the 3' untranslated region of c-fos mRNA. To distinguish between a labile protein participant in the degradation of these mRNAs and a requirement for their translation per se, we introduced a ferritin iron-responsive element into the 5' untranslated region of each of these mRNAs. The presence of the 5' iron-responsive element allowed us to use iron availability to alter the translation of the mRNAs in question without global effects on cellular protein synthesis. Although specific translation of these mRNAs could be inhibited by iron chelation to a degree comparable to that seen with cycloheximide (approximately 95% inhibition), no effects on mRNA turnover were observed. These data support a model in which a trans-acting labile protein is necessary for the turnover of these mRNAs rather than there being a requirement for the translation of the mRNAs themselves.

Targeting, dosimetry, and radioimmunotherapy of B-cell lymphomas with iodine-131-labeled LL2 monoclonal antibody.

Sixteen patients with non-Hodgkin's lymphoma were infused with 6.2 to 58.2 mCi (0.2 to 3.9 mg) doses of radioactive iodine (131I)-labeled LL2 immunoglobulin G (IgG) or F(ab')2, in order to study antibody distribution, pharmacokinetics, dosimetry, toxicity, tumor targeting, and therapy. LL2 is a murine IgG2a monoclonal antibody (MAb) reactive with B cells and non-Hodgkin's B-cell lymphoma. In a series of five assessable therapy patients, doses as small as 30 mCi 131I-LL2 IgG or F(ab')2 resulted in tumor responses (two partial remissions, two mixed and minor responses, and one no response), while one patient receiving diagnostic doses as low as 6.2 mCi showed a partial remission for 1 year and a complete remission after a second low radiation dose. No acute toxicities were noted, and only myelotoxicity accompanied therapeutic doses, with grade IV marrow toxicity seen in three of seven patients receiving total doses of about 50 mCi. Dosimetry calculations showed spleen and tumor dose rules of about 4.6 cGy/mCi, which was three to four times the dose to other organs. Despite the administration of relatively low doses of LL2 (0.2 to 3.9 mg), 82% of 60 known extrasplenic lymphoma sites were imaged. Serum clearance showed an average distribution half-life (T1/2) of 2.1 hours and an elimination T1/2 of 32.0 hours. The average total-body clearance T1/2 was 43 to 45 hours. LL2's antigenic target does not appear to be shed in high amounts into the circulation. Three of eight patients having at least two injections showed a human antimouse antibody response. These patients may have been presensitized to animal protein. An interesting observation in this study was the marked drop in circulating B lymphocytes after the administration of radioiodinated LL2 or anticarcinoembryonic antigen MAbs, suggesting that this is a nonspecific radiation effect and not necessarily related to the binding of MAb to normal B cells.

A new Acanthamoeba myosin heavy chain. Cloning of the gene and immunological identification of the polypeptide.

An Acanthamoeba myosin heavy chain has been identified whose tail domain amino acid sequence distinguishes it from Acanthamoeba myosins IB, IC, and II. The gene for this novel myosin heavy chain spans approximately 6.8 kilobases, is split by 17 introns, and encodes a 177-kDa polypeptide. While the amino-terminal approximately 90 kDa of this polypeptide is highly similar to the globular head sequences of myosins I and II, its approximately 87-kDa tail domain shows essentially no similarity to the tail sequences of either type of myosin. The only exception to this is the carboxyl-terminal approximately 50-amino acid region of the polypeptide, which is homologous to the carboxyl termini of the myosins I. Interestingly, this approximately 50-residue segment has been shown to exist in a diverse family of cytoskeleton-associated proteins that include nonreceptor tyrosine kinases, phospholipase C gamma, and fodrin (Rodaway, A. R. F., Sternberg, M. J. E., and Bentley, D. L. (1989) Nature 342, 624). Sequence analysis indicates that the tail domain of this new myosin is incapable of forming a myosin II-like coiled-coil structure, implying that the protein is single-headed and nonfilamentous. For this reason we have tentatively classified it as a high molecular weight form of myosin I (HMWMI). To determine if HMWMI exists in cells, antiserum was raised against a bacterially expressed fusion peptide made using a cDNA clone encoding most of the unique HMWMI tail domain. This antiserum does not recognize Acanthamoeba myosins IB, IC, or II but does recognize a single polypeptide in whole cell extracts with the mobility predicted for the HMWMI heavy chain. This protein is precipitated from crude extracts using F-actin and released from the pellet by ATP, supporting its classification as a member of the myosin family of proteins.

Tumor, red marrow, and organ dosimetry for 131I-labeled anti-carcinoembryonic antigen monoclonal antibody.

Tumor-, red marrow-, and organ-absorbed doses were calculated for patients receiving 131I-labeled monoclonal antibodies against carcinoembryonic antigen for either diagnosis or therapy. Ten patients with confirmed liver tumors who received doses ranging from 10.79 to 200 mCi were evaluated. Urine and blood samples were taken in order to determine total body and red marrow activity, respectively. Anterior and posterior gamma camera images were obtained at multiple times postinjection in order to quantitate activity uptake using the conjugate view counting method for the following organs and regions: lungs, liver, spleen, kidneys, and the liver tumors. In addition, sacral regions of interest were drawn to generate red marrow-absorbed dose estimates for comparison to those obtained by blood sampling. Tumor volumes were obtained from volumetric analysis of the patient's computed tomographic study and tumor S values were obtained by assuming uniform distribution of the 131I-labeled monoclonal antibody in spherical tumor regions considering all emitted electrons, beta-particles, and photons. The following mean absorbed doses in rads/mCi injected were obtained: lungs, 2.3 +/- 1.6 (SD); liver, 1.4 +/- 0.7; spleen, 2.6 +/- 1.4; kidneys, 3.1 +/- 1.5; total body, 0.7 +/- 0.5; red marrow from blood sampling, 2.9 +/- 1.9; red marrow from sacral scintigraphy, 1.7 +/- 1.2; and liver tumors, 69.3 +/- 92.5. Tumor volumes ranged from 1 to 216 g and the percentage of uptake/g of monoclonal antibody into these tumors ranged from 0.0006 to 1.040. There was a statistically significant difference between the two techniques for estimation of red marrow dose (P less than 0.01). This methodology, permits calculation of tumor, red marrow, and organ dosimetry using planar gamma camera imaging.

Clinical studies of cancer radioimmunodetection with carcinoembryonic antigen monoclonal antibody fragments labeled with 123I or 99mTc.

Seventy-three patients with diverse cancers containing carcinoembryonic antigen received 123I-labeled anti-carcinoembryonic antigen monoclonal antibody F(ab')2 fragment [38 patients], 99mTc-labeled anti-carcinoembryonic antigen monoclonal antibody Fab' fragment [23 patients], or both reagents at different times [6 patients] for evaluation of antibody targeting and imaging [radioimmunodetection (RAID)], using planar and single-photon emission computed tomography. The results indicated that antibody fragments are preferred for early tumor imaging (within 24 h). Rapid targeting and clearance from blood and normal organs of the antibody fragments (blood median t1/2 elimination of 26.5 and 13.2 h for the F(ab')2 and Fab' fragments respectively) permitted the use of short-lived radionuclides, such as 123I (13.3 h) and 99mTc (6 h), and confirmed that selective antibody accretion in tumors occurred very soon after administration, such as between 2 and 5 h. Scan interpretations at 24 h for the 123I-labeled F(ab')2 and at 2-5 h for the 99mTc-labeled Fab' revealed overall sensitivities, on a tumor site basis, of 95.9 and 94.9%, respectively. On a site basis, the overall accuracies were 94.2 and 93.8% for the 123I and 99mTc immunoconjugates, respectively. In the 6 patients studied with both radioimmunoconjugates, a high concordance in detection was found. Both imaging agents also revealed a high number of putatively new tumor sites not disclosed by other radiological methods at the time of the RAID studies, of which 40.0 and 20.5% were subsequently confirmed as tumor for the 123I and 99mTc agents, respectively, within an 11-month follow-up period. This represented 24 proven occult tumor sites in 19 patients given the 123I-immunoconjugate and 16 proven occult tumor sites in 9 patients receiving the 99mTc agent. The new lesions were found up to 17 and 7 months earlier for 123I-RAID and 99mTc-RAID, respectively, than with other detection methods. The smallest tumors identified were below 0.5 cm, especially with the 99mTc immunoconjugate and single-photon emission computed tomography imaging. The findings of this study confirm previous evidence that RAID is a safe and a potentially useful new method of cancer detection. Despite the excellent results with the 123I-F(ab')2 antibody fragment, its poor availability and high cost limit its clinical use. Therefore, the 99mTc agent, which is made by an instant, 1-step, 1-vial, direct labeling method, appears to be the method of choice for rapid and accurate detection of cancer by RAID.