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Positive practice overcorrection (PPOC) has long played a significant role in the behavioral treatment of serious self-stimulatory behavior. Three experiments comparing the effectiveness of 30-second, 2-minute, and 8-minute PPOC on reduction of stereotypic hand behavior of adults with severe to profound developmental disabilities were conducted to resolve inconsistencies in previously reported findings concerning the role of PPOC duration in response suppression. Experiment 1, which used an alternating treatments--multiple baseline design, suggested that the different durations were equally effective in reducing the stereotypic behaviors to near-zero levels. Experiment 2, which used a reversal design, supported the findings of Experiment 1. Experiment 3, which used a reversal design to test the shortest and longest durations, generally confirmed the results of the first two experiments. This study therefore failed to support the oft-claimed superiority of long-duration PPOC. The possible factors underlying these findings and their implications for future research and practice are discussed.

We have investigated the homotypic associations of major histocompatibilty, class II and class I molecules using immunoprecipitation from detergent solubilised cell extracts. A 120-kDa structure corresponding to an HLA-DR dimer of dimers was immunoprecipitated by the HLA-DR specific mAb L243 from both biotinylated cell-surface and metabolically labeled B cells and transfectant fibroblasts. The thermostability of this structure in SDS was examined. It was detected at 4 degrees C, 22 degrees C, and 37 degrees C, but not at 50 degrees C or 100 degrees C. Experiments performed with L243 Fab fragments and with purified HLA-DR molecules, indicated the presence of HLA-DR dimers of dimers and single heterodimers on B cells. HLA-DQ was also found to form SDS-stable dimers of dimers and single heterodimers on the cell surface of B cells, demonstrating that HLA class II isotypes, other than HLA-DR, also form homotypic associations. Similar experiments performed with HLA class I specific mAb, W632, revealed the existence of a 90 kDa and a 135-kDa structure corresponding to a MHC class I multimers. Under the same conditions, non-MHC molecules such as CD14 were found not to self-associate. These findings indicate that major histocompatibility molecules have the intrinsic ability to form homotypic associations at the cell surface of antigen presenting cells.

Integrins are transmembrane molecules involved in numerous cell matrix, cell-cell adhesion phenomena and also utilised as viral receptors. These interactions with integrins are mediated by brief oligopeptide recognition sequences. The Arg-Gly-Asp sequence (RGD), is recognized by many integrins, including integrin alphavbeta3 (CD51/61). Coxsackievirus A9 (CAV-9), a human pathogen that has an Arg-Gly-Asp sequence in the VP1 capsid protein, has been known to be one of the many viruses that utilise integrin alphavbeta3 as a receptor. In order to determine important binding sites of CAV-9 on integrin alphavbeta3, we performed binding studies of CAV-9 on CHO-alphavbeta3, CHO-alphavbeta1 and CHO-alphavbeta1-3-1 mutant cell line, in the presence of function blocking mAb specific for integrin alphavbeta3 and natural ligand vitronectin. Our experiments show that the CYDMKTTC sequence (187-193 residue) of integrin beta3, which has been shown to be involved in ligand specificity, is an important binding site for CAV-9. We also report that an RGD-less Coxsackievirus A9 mutant can bind efficiently on the ligand binding site of integrin alphavbeta3. Thus documenting the capability of this RNA virus to interact with integrin alphavbeta3, without the presence of an Arg-Gly-Asp sequence.

Objective: To examine the factors associated with use of the emergency department (ED) as the only source of health care among adolescents., Design: Analyses of the 1997 Commonwealth Fund Survey of the Health of Adolescent Girls, a nationally representative sample of 6748 in-school male and female adolescents in 5th through 12th grade. The X2 statistics and logistic regression analyses were computed with the use of SUDAAN., Results: Overall, 4.6% of the adolescents in the survey, or 1.5 million adolescents in the United States, reported that the ED was their usual source of health care. In multivariate models, factors associated with the use of the ED included male sex, African American ethnicity, fewer financial resources, and living in a rural area. Adolescents with higher levels of risky behaviors, a history of physical or sexual abuse, and higher depression scores were all more likely to use the ED as their usual source of care. Adolescents who reported using the ED as their usual care source were also less likely to have had regular well visits and were more likely to report having missed needed care than those with other sources of primary care., Conclusions: Adolescents who use the ED as their usual source of care are often from vulnerable populations. Many have special mental or physical needs that are unlikely to be met with ED visits only, and they are likely to have missed care they needed. Creating linkages between EDs and other services could help at-risk adolescents identify and use more appropriate sources of primary care.

Cylindrospermopsis raciborskii is a toxic-bloom-forming cyanobacterium that is commonly found in tropical to subtropical climatic regions worldwide, but it is also recognized as a common component of cyanobacterial communities in temperate climates. Genetic profiles of C. raciborskii were examined in 19 cultured isolates originating from geographically diverse regions of Australia and represented by two distinct morphotypes. A 609-bp region of rpoC1, a DNA-dependent RNA polymerase gene, was amplified by PCR from these isolates with cyanobacterium-specific primers. Sequence analysis revealed that all isolates belonged to the same species, including morphotypes with straight or coiled trichomes. Additional rpoC1 gene sequences obtained for a range of cyanobacteria highlighted clustering of C. raciborskii with other heterocyst-producing cyanobacteria (orders Nostocales and Stigonematales). In contrast, randomly amplified polymorphic DNA and short tandemly repeated repetitive sequence profiles revealed a greater level of genetic heterogeneity among C. raciborskii isolates than did rpoC1 gene analysis, and unique band profiles were also found among each of the cyanobacterial genera examined. A PCR test targeting a region of the rpoC1 gene unique to C. raciborskii was developed for the specific identification of C. raciborskii from both purified genomic DNA and environmental samples. The PCR was evaluated with a number of cyanobacterial isolates, but a PCR-positive result was only achieved with C. raciborskii. This method provides an accurate alternative to traditional morphological identification of C. raciborskii.

The intracellular and cell-surface heterotypic associations of HLA-DR in the presence and absence of the invariant chain were investigated. Simultaneous confocal microscopy imaging of the Golgi apparatus and HLA-DR molecules revealed that cells transfected only with HLA-DR and not the invariant chain or HLA-DM, accumulate class II molecules mostly in the Golgi apparatus, proximal to the cell nucleus. In contrast, in cells transfected with both HLA-DR and the invariant chain, or HLA-DR, the invariant chain and HLA-DM, the class II molecules are more evenly distributed in intracellular compartments. Confocal microscopy and flow cytometry revealed that in the absence of the invariant chain, a greater number of HLA-DR molecules are transported to the cell surface. Biochemical experiments and nonequilibrium pH gradient electrophoresis revealed that HLA-DR associates with surface invariant chain in the presence of HLA-DM. In cells that lack HLA-DM, no cell-surface association of HLA-DR and Ii was observed. Taken together, these results reveal two separate and distinct functions for surface and intracellular invariant chain subsets. The intracellular invariant chain "arrests" the class II molecules in the endocytic pathway. In contrast, cell-surface invariant chain associates with class II molecules at the cell surface, possibly playing a role in recycling empty class II molecules or as an accessory molecule.

It is becoming apparent that many viruses employ more than one cell surface molecule for their attachment and cell entry. In this study, we have tested the role of integrin alpha(v)beta3 and MHC class I molecules in the coxsackievirus A9 (CAV-9) infectious cycle. Binding experiments utilizing CHO cells transfected and expressing human integrin alpha(v)beta3, revealed that CAV-9 particles were able to bind to cells, but did not initiate a productive cell infection. Antibodies specific for integrin alpha(v)beta3 molecules significantly reduced CAV-9 infection in susceptible cell lines. Moreover, MAbs specific for beta2-microglobulin (beta2-m) and MHC class I molecules completely inhibited CAV-9 infection. To assess the effect of these antibodies on virus binding, we analysed CAV-9 binding by flow cytometry in the presence of alpha2-m- or integrin alpha(v)beta3-specific antibodies. The results showed a reduction in CAV-9 binding in the presence of integrin alpha(v)beta3-specific antibodies while there was no reduction in the presence of beta2-m-specific MAb. Taken together, these data suggest that integrin alphavbeta3 is required for CAV-9 attachment but is not sufficient for cell entry, while beta2-m, although not directly involved in CAV-9 binding, plays a post-attachment role in the CAV-9 infectious process, possibly being involved in virus entry.

Purpose: To assess the influence of demographic variables and health risk status on adolescents' preferences and actual receipt of services regarding provider gender, sharing a physician with parents, and private examinations., Methods: Data from students participating in the Commonwealth Fund 1997 Survey of the Health of Adolescent Girls were analyzed. The weighted sample included 6748 students from grades 5-12. The influence of demographic variables and health risk status on preferences regarding physician gender, sharing a physician with parents, and parental presence during examinations and on actual physician gender, sharing a physician with parents, and receipt of confidential care was assessed for the 5067 students who indicated that they had a health check-up or physical examination within the past 2 years. Associations were examined using SAS to determine preliminary estimates of significance and correlation coefficients, and SUDAAN to generate proportions and Cochran Mantel-Haenszel Chi-squared values. A multiple logistic regression procedure in SUDAAN was used to assess interaction among demographic variables., Results: Gender, race/ethnicity, grade level, and risk status were associated with preferences regarding provider gender and sharing a physician with parents. 50% of girls preferred a female provider; 48% had no preference. 23% of boys preferred a male provider; 65% had no preference. Most adolescents had no preference regarding whether they shared a physician with parents. Gender, race/ethnicity, grade level, and risk status were associated with preference regarding parental presence during examinations. Most younger girls preferred to have a parent present; most younger boys had no preference. Most older girls and boys preferred private examinations. For actual care situation, most adolescents were cared for by male health providers and did not share a physician with parents. 57% of girls and 66% of boys spoke privately with their health provider. Girls who had a female physician were more likely to have private time than were girls receiving care from a male physician. Gender, grade level, and risk status were associated with having private time with a physician., Conclusions: Gender was a significant variable in adolescents' preferences regarding health care. Preferences were also influenced by race/ethnicity, grade level, and risk status. A substantial proportion of adolescents, including those involved in health risk activities, report not having private time with their health provider.

Purpose: This study examined the factors associated with access to care among adolescents, including gender, insurance coverage, and having a regular source of health care., Methods: Analyses were done on the 1997 Commonwealth Fund Survey of the Health of Adolescent Girls, a nationally representative sample of in-school adolescents in 5th through 12th grade. Access to health care, missing needed care, and whether the adolescent had private time with their provider were assessed. Cochran-Mantel-Haenszel chi-square statistics were computed using SUDAAN., Results: Nearly a third of the 6748 adolescents surveyed had missed needed care. The most common reason for missing care was not wanting a parent to know (35%). Girls were more likely than boys to miss care (29% vs. 24%). Most adolescents reported using a source of primary health care (92%); girls were more likely than boys to use a physician's office rather than another site (65% vs. 60%). Eleven percent of adolescents reported having no health insurance. Uninsured adolescents were more likely to have missed needed care (46% vs. 25%) [corrected]., Conclusions: Certain groups of adolescents have less access to health care. Girls have more emotional barriers, such as not wanting parents to know about care, and embarrassment. Adolescents without health insurance are at high risk for missing care because of financial strain. States, insurers, and advocates can influence policies around confidentiality and insurance coverage to address these issues.

The serratus anterior fascia was used as a free-tissue transfer in four patients for the reconstruction of dorsal hand defects. All patients had multiple open metacarpal fractures with extensor tendon injuries. The fascia was used to "sandwich" the extensor tendons in a bed of areolar gliding tissue to avoid adhesions. The mean follow-up was 2 years. There were no complications and all flaps survived completely. All flaps were grafted with meshed split-thickness skin at the time of transfer with a 100 percent take in all cases. A good functional result was noted in all patients. This free-tissue transfer is recommended for complex injuries to the dorsum of the hand associated with soft-tissue defects.

Single-particle tracking (SPT) was used to determine the mobility characteristics of MHC (major histocompatibility complex) class I molecules at the surface of HeLa cells at 22 degrees C and on different time scales. MHC class I was labeled using the Fab fragment of a monoclonal antibody (W6/32), covalently bound to either R-phycoerythrin or fluorescent microspheres, and the particles were tracked using high-sensitivity fluorescence imaging. Analysis of the data for a fixed time interval suggests a reasonable fit to a random diffusion model. The best fit values of the diffusion coefficient D decreased markedly, however, with increasing time interval, demonstrating the existence of anomalous diffusion. Further analysis of the data shows that the diffusion is anomalous over the complete time range investigated, 4-300 s. Fitting the results obtained with the R-phycoerythrin probe to D = D0talpha-1, where Do is a constant and t is the time, gave D0 = (6.7 +/- 4.5) x 10(-11) cm2 s-1 and alpha = 0.49 +/- 0.16. Experiments with fluorescent microspheres were less reproducible and gave slower anomalous diffusion. The R-phycoerythrin probe is considered more reliable for fluorescent SPT because it is small (11 x 8 nm) and monovalent. The type of motion exhibited by the class I molecules will greatly affect their ability to migrate in the plane of the membrane. Anomalous diffusion, in particular, greatly reduces the distance a class I molecule can travel on the time scale of minutes. The present data are discussed in relation to the possible role of diffusion and clustering in T-cell activation.

Objective: To assess the quality of life and functional status of patients who have undergone hemimandibulectomy based on the type of reconstructive procedure performed., Study Design: Survey, retrospective., Methods: Twenty-one patients who had undergone hemimandibulectomy and had similar defects were divided into two groups based on the reconstructive technique utilized. Eleven patients were placed in the soft tissue reconstruction group. Ten patients were placed in the mandible reconstruction group. All patients were assessed for: function, utilizing the Performance Status Scale, and quality of life, using a general cancer questionnaire (FACT-G) and a series of questions specific for head and neck cancer patients., Results: Mandible reconstruction produced a perceived better physical appearance (P = .02), better eating ability (P = .04), and a better overall quality of life (P = .002). The mandible reconstruction cohort consistently outscored the soft tissue cohort on all questionnaires., Conclusion: Restoration of mandibular continuity after hemimandibulectomy leads to improved function and a superior quality of life in appropriately selected patients.

Objective: To determine the safety and efficacy of an immunogenic gene therapy using a drug designed to produce expression of a foreign class I major histocompatibility complex protein in patients with head and neck cancer., Design: Phase 1 prospective clinical trial., Setting: Academic medical setting., Patients: Nine patients with advanced head and neck squamous cell carcinoma who had failed conventional therapy and did not express HLA-B7, a class I major histocompatibility complex protein., Intervention: Patients were treated with Allovectin-7 (Vical Inc, San Diego, Calif) by direct intratumoral injection. Allovectin-7 contains a plasmid complementary DNA complexed with a cationic lipid, which results in expression of HLA-B7., Main Outcome Measures: Patients were assessed for any toxic effects and for any change in tumor volume. Biopsy specimens obtained before and after therapy were evaluated by immunohistochemistry to detect HLA-B7 expression and with the terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay to detect any induction of apoptosis., Results: There were no toxic effects of the gene therapy. In 4 of these 9 patients there was a partial response to treatment, evidenced by a gradual reduction in tumor size. One patient has remained alive for more than 17 months since commencing treatment, with no clinical evidence of disease but with persistent histological evidence of cancer. Analysis of the biopsy specimens from 2 of the patients who responded to therapy demonstrated HLA-B7 expression. The TUNEL assay demonstrated extensive apoptosis in both of these patients, suggesting that this may be the mechanism of tumor reduction., Conclusions: These data demonstrate the potential efficacy and lack of toxicity of this form of alloantigen gene therapy. A multi-institutional study has been initiated to expand on these findings.

A method for analyzing flecainide in plasma was developed and assessed. Based on the use of C18 extraction columns eight or fewer times, the rapid and simple extraction procedure provided consistent, high-efficiency flecainide extraction (>85%). Using reverse-phase, high-performance liquid chromatography with fluorometric detection, flecainide acetate was detectable to approximately 15 ng/ml. Retention times of the internal standard and flecainide were 8.9 to 9.2 and 9.8 to 10.4 minutes, respectively, and short sample preparation and run times enabled results to be delivered within 2 to 3 hours of receiving samples. The assay was linear for the standard range 20 ng/ml to 2 microg/ml (r2 with three standards, >0.999) and delivered a high level of accuracy. Quality control concentrations obtained from eight assays consistently fell within 5% of nominal values (100 ng/ml and 1 microg/ml). Based on four assays, the assay was also reproducible with calculated-between and within-assay coefficients of variation of less than 1% and 3%, respectively. The authors found that the performance of this assay was excellent and that the solid-phase extraction technique was simple, rapid, and cost effective.

The administration of drugs by subcutaneous infusion is routinely practiced in palliative medicine for the management of patients who are no longer able to take oral medication. It is not uncommon for two or more drugs to be combined in subcutaneous infusion solutions. The combination of an opioid and a short-acting benzodiazepine is frequently required. Unfortunately, the stability of benzodiazepines and newer opioids, such as fentanyl, has not been determined. This study examined the stability of solutions containing either fentanyl alone or fentanyl and midazolam in combination. Eight different solutions were assessed for up to 7 days following preparation. The solutions were prepared in polypropylene syringes using 0.9% saline as a diluent. Duplicate syringes were stored at approximately 5 degrees C, 22 degrees C, and 38 degrees C. High performance liquid chromatography was the analytical technique used to measure fentanyl and midazolam. Initial concentrations ranges were 13.2-38.9 micrograms/mL for fentanyl and 282-959 micrograms/mL for midazolam. It was found that fentanyl (+/- midazolam) was very stable (> 95%) when stored at temperatures ranging from 5 degrees C to 38 degrees C for at least 1 week. Midazolam (+ fentanyl) was not as stable as fentanyl under the same storage conditions and underwent time-dependent decomposition of up to 12.1% (observed at 7 days when stored at 38 degrees C). When stored at 22 degrees C and 38 degrees C, more than 90% of initial midazolam concentrations were retained for 4 days following preparation and for 7 days when stored at 5 degrees C. The clinical implications of these results are that, on the basis of physicochemical stability, subcutaneous infusion solutions containing fentanyl and midazolam may be prepared at intervals of 4 days (or 7 days if stored under refrigerated conditions).

The technique of single-particle fluorescence imaging was used to investigate the oligomeric state of MHC class II molecules on the surface of living cells. Cells transfected with human leukocyte antigen (HLA)-DR A and B genes were labeled at saturation with a univalent probe consisting of Fab coupled to R-phycoerythrin. Analysis of the intensities of fluorescent spots on the cell surface revealed the presence of single and double particles consistent with the simultaneous presence of HLA-DR heterodimers and dimers of dimers. The proportion of double particles was lower at 37 degrees C than at 22 degrees C, suggesting that the heterodimers and dimers of dimers exist in a temperature-dependent equilibrium. These results are discussed in the context of a possible role for HLA-DR dimers of dimers in T cell receptor-MHC interactions. The technique is validated by demonstrating that fluorescence imaging can distinguish between dimers and tetramers of human erythrocyte spectrin deposited from solution onto a solid substrate. The methodology will have broad applicability to investigation of the oligomeric state of immunological and other membrane-bound receptors in living cells.

Spasmodic dysphonia is a focal laryngeal dystonia characterized by inappropriate contractions of the intrinsic laryngeal musculature. The prevalence of associated neurological findings has led to detailed investigation of the central nervous system. Previous research revealed latency abnormalities in patients' auditory brainstem responses. The present study further investigated central auditory findings in patients with spasmodic dysphonia, including brainstem and cortical function. Fourteen normal-hearing patients with spasmodic dysphonia were tested using the auditory brainstem response (ABR) and SCAN-A test of central auditory processing. The ABR estimated brainstem transmission time and evaluated auditory pathway integrity at a high stimulus rate. SCAN-A assessed the auditory cerebral cortex. Implications of these findings are discussed. We found no ABR abnormalities in subjects with spasmodic dysphonia. Positive SCAN-A findings were negligible. The ABR findings contradict previous reports.

The "bystander effect," produced by ganciclovir-mediated killing of cells transduced with a herpes simplex virus thymidine kinase (HSVtk) gene, defines the cooperative killing of non-HSVtk-transduced cells. In vitro, a major contributor to this phenomenon is metabolic cooperation involving transfer of cytotoxic small molecules between cells via gap junctions. In this study, the bystander effect was assessed in vivo using cells of oral squamous cell carcinoma origin. Mixtures of HSVtk+ and HSVtk- tumor cells were implanted subcutaneously in the left flank of nude mice, and naive HSVtk- cells were implanted subcutaneously in the right flank. When tumors attained a size of 0.5 to 1 cm, the animals were treated with ganciclovir on a daily basis. The tumors comprised of mixed cells in the left flank resolved, consistent with a predicted bystander effect. The naive tumors in the right flank either resolved or became cytostatic showing little further growth compared to controls. Similar results were obtained when naive tumors were grown in both flanks and the tumor in the left flank received intratumoral injection of HSVtk retroviral producer cells or PA317 (HSVtk+) packaging cells, but not parental NIH 3T3 cells. Concomitant treatment with dexamethasone impaired the antitumor effect on the contralateral side. When these experiments were performed in SCID-Beige mice, there was a reduced antitumor effect on the ipsilateral flank and no antitumor response in the contralateral flank. Together with histology of regressing tumors, which showed an infiltration of lymphoid cells, these results are suggestive of an immune-related antitumor response that could account for the distant bystander effect.

Purpose: To correlate magnetic resonance (MR) imaging findings of pelvic fluid, peritoneal enhancement, and peritoneal nodules with the presence of benign or malignant pelvic disease., Materials and Methods: Pelvic MR imaging findings in 87 women (mean age, 46 years) who subsequently underwent surgery were retrospectively reviewed. Phased-array multicoil, axial spin-echo T1-weighted MR images and axial fast spin-echo T2-weighted images were obtained. The largest pelvic fluid pocket was measured on the T2-weighted images in three orthogonal dimensions. Peritoneal enhancement and nodules were evaluated on gadolinium-enhanced MR images in 54 women., Results: Pelvic fluid was shown in 21 of 25 patients with malignant neoplasms and 48 of 62 patients with benign disorders. Larger fluid pockets were associated with malignancy rather than with benign disorders, but there was substantial overlap. Use of peritoneal enhancement to diagnose peritoneal spread of tumor had a sensitivity, specificity, accuracy, and positive predictive value of 92% (11 of 12), 86% (36 of 42), 87% (47 of 54), and 65% (11 of 17), respectively. Use of peritoneal nodules for peritoneal spread had a higher specificity (90% [38 of 42]) and positive predictive value (69% [nine of 13]), although the sensitivity was lower (75% [nine of 12])., Conclusion: Large peritoneal fluid pockets are moderately predictive of malignancy or peritoneal spread of tumor. Peritoneal enhancement and enhancing peritoneal nodules are more sensitive and more specific.

The mobility of cell surface MHC molecules and their ability to form dynamic associations may be related to the physiological status of the cell and to the potential to bind effector T lymphocytes. To investigate these properties, we have prepared HLA DR specific monoclonal antibodies coupled in a 1:1 mole ratio to the fluorescent phycobiliprotein, R-phycoerythrin (PE). We show that these small particles can be sequentially imaged using a cooled slow-scan charge coupled device camera and hence can be used for single particle tracking experiments. We have applied this technique to investigate the movements of HLA DR molecules on fibroblasts transfected with human DR alpha and DR beta genes. PE-IgG was bound to the transfected fibroblasts and particle tracks were obtained by sequential imaging over a period of typically 30 minutes. Analysis of particle tracks revealed the presence of directed motion and domain-limited diffusion in addition to random diffusion. The contributions of these three types of motion showed cell to cell variability. Velocities of directed motion were of the order of 2 nm second-1 whilst domain diameters were in the range 200-800 nm. Diffusion coefficients for random diffusion were in the range 1 x 10(-13)-5 x 10(-12) cm2 second-1. The higher mobilities were observed for the lower intensity fluorescent spots, which possibly correspond to images of single particles. Much lower mobility was observed with a cell where the spot intensities were approximately double that of the lower intensity spots. These spots could be images of double particles implying the association of at least two HLA DR alpha beta dimers. These data are relevant to the study of MHC class II cell surface redistribution and antigen presentation in specific immunity.

Objectives: To determine whether the bystander effect demonstrated in vitro for ganciclovir-mediated killing of a herpes simplex virus thymidine kinase (HSV-tk) gene-infected human squamous cell carcinoma is operative in vivo in a nude mouse model., Design: Prospective study in a murine model., Intervention: Human head and neck squamous cell carcinoma tumors were grown as xenografts on the flanks of 20 nude mice. The tumors in the left flank were then infected with the HSV-tk gene. Then, after 48 hours, the animals were treated with intraperitoneal ganciclovir twice daily. Assessment of the tumors on both flanks was performed over a 31-day period., Main Outcome Measures: Resolution of tumors infected with HSV-tk gene in animals treated with ganciclovir; resolution of tumors uninfected with HSV-tk gene on the contralateral flank in animals treated with ganciclovir., Results: Following HSV-tk gene therapy in nude mice, complete resolution of HSV-tk-gene-infected human head and neck squamous cell carcinoma tumors was observed following ganciclovir treatment. Uninfected tumors were also noted to regress, but not completely resolve, in response to intraperitoneal ganciclovir (distant bystander effect)., Conclusions: This study confirms that the local and distant bystander effects exist in this murine model, enhancing the possibility of its role for treatment of human squamous cell carcinoma of the head and neck.

Objective: To compare computed tomography (CT) with open neck exploration in determining prevertebral invasion by squamous cell carcinoma of the oropharynx or hypopharynx., Design: Retrospective analysis using the findings at open neck exploration and results of histopathologic studies as the criterion standards., Setting: Tertiary care referral center., Patients: Twenty-nine of 40 patients with advanced squamous cell carcinoma of the oropharynx or hypopharynx treated between January 1, 1986, and December 31, 1994, were selected for analysis based on CT findings of posterolateral extension of the primary tumor placing the prevertebral muscle (PVM) at risk. All study patients had no previous therapy and underwent neck exploration to determine resectability., Results: Overall accuracy of CT in predicting PVM status was 55.2%. The sensitivity of preoperative CT for PVM invasion was 50%; the specificity was 61%. Using an estimate of 21% for the prevalence of PVM invasion, the predictive value of a positive CT scan was 0.254 and the predictive value of a negative CT scan was 0.821. Open neck exploration correctly predicted PVM status in all cases., Conclusions: Open neck exploration is superior to CT to evaluate possible PVM invasion by squamous cell carcinoma of the oropharynx or hypopharynx. The predictive value of a negative CT scan for PVM invasion is high, so it may be useful in treatment planning. Patients with advanced squamous cell carcinoma of the oropharynx or hypopharynx at risk for PVM invasion who are otherwise surgical candidates should be considered for open neck exploration to determine resectability most accurately.

Objective: To determine the efficacy of toluidine blue in assessing margin status after removal of squamous cell carcinomas of the upper aerodigestive tract., Design: A prospective study of 50 consecutive patients undergoing surgical resection of squamous cell carcinomas of the upper aerodigestive tract was performed during February 1 to December 1, 1993. After tumor resection, toluidine blue was applied directly to the remaining unresected mucosa. The staining characteristics of the mucosa were then compared with those of frozen-section biopsy specimens of the margins and with the permanent histologic findings of the resected tumor specimen., Results: In three cases, toluidine blue identified a positive margin, which was confirmed on frozen and permanent section. In six cases, false-positive staining was noted, which was most frequently related to traumatic handling of the mucosa during the resection. In no case was a positive margin found on histologic staining that failed to stain with toluidine blue. During routine staining of surrounding unresected mucosa, three cases of a second primary tumor that was not seen on routine evaluation before tumor removal were identified with toluidine blue. In one case, a second T1 oral cavity lesion was found, while in the other cases, separate pharyngeal lesions were identified., Conclusion: Based on these findings, it appears that toluidine blue improved the ability to assess margin status at the time of resection, and we advocate its use after resection of tumors to the upper aerodigestive tract.

Purpose: Sinonasal undifferentiated carcinoma (SNUC) is a rare and aggressive malignancy of the paranasal sinuses and nasal cavity. Of the few reported series, most indicate a dismal prognosis. In this report, the clinical presentation, histopathologic criteria used for diagnosis, mode of treatment, and outcome are evaluated in seven patients with SNUC., Materials and Methods: Seven patients with SNUC treated at the University of Cincinnati between 1983 and 1993 were analyzed retrospectively., Results: Most of the patients presented with extensive local disease, and two patients also had cervical metastases. All except one were treated using a multimodality approach. Four of the seven patients died of disease (DOD), with a mean survival of only 11.5 months following treatment. Inability to eradicate local disease was responsible for treatment failure in all cases. Three patients have achieved short-term control of disease following combined therapy, but one is at high risk for recurrence., Conclusion: SNUC was associated with an overall poor prognosis in our series despite aggressive treatment. Control of local disease was the central therapeutic consideration. Intensive multimodality therapy is recommended for all patients with SNUC.

The objective of this study was to determine whether elevated levels of N-ras correlated with clinicopathological data. Complete clinical data were available on 133 of 481 patients surgically treated for squamous cell carcinoma of the head and neck (SCCHN) who had immunohistochemical data for N-ras. Advanced stages of disease were strongly related to the staining for N-ras in tumour cells (P = 0.0031). The stage of disease was inversely related to duration of survival (P = 0.0017). Initial statistical evaluation revealed an apparent correlation between survival and N-ras staining. However, duration was found to be independent of the level of N-ras. The illusory relationship initially was a result of the confounding effect of the stage of disease.

This study investigated the expression of the N-ras oncogene in routinely processed tissue sections from 133 patients with squamous cell carcinoma of the head and neck (SCCHN) by immunohistochemistry using anti-N-ras monoclonal antibody. N-ras expression was present in 67 of 133 (49.6%) cases. There was a highly significant correlation between N-ras expression and clinical stage of disease (P=0.003). This study confirmed that overexpression of the N-ras oncogene is common in SCCHN and that it may be an important event in the late stage of disease.

A physical map has been constructed of the human genome containing 15,086 sequence-tagged sites (STSs), with an average spacing of 199 kilobases. The project involved assembly of a radiation hybrid map of the human genome containing 6193 loci and incorporated a genetic linkage map of the human genome containing 5264 loci. This information was combined with the results of STS-content screening of 10,850 loci against a yeast artificial chromosome library to produce an integrated map, anchored by the radiation hybrid and genetic maps. The map provides radiation hybrid coverage of 99 percent and physical coverage of 94 percent of the human genome. The map also represents an early step in an international project to generate a transcript map of the human genome, with more than 3235 expressed sequences localized. The STSs in the map provide a scaffold for initiating large-scale sequencing of the human genome.

Aims-To determine whether mutant p53 alleles harboured by malignant tumours of the oral cavity were also present in previous premalignant lesions at the same site.Methods-Paraffin embedded tumour specimens along with their premalignant counterparts were analysed for p53 alterations using immunohistochemistry, microdissection, polymerase chain reaction amplification, and DNA sequencing.Results-Malignant lesions from five of eight patients showed overexpression of p53 protein by immunohistochemistry. Upon DNA sequencing, two of these five specimens had p53 mutations. Of the five patients whose cancers showed p53 overexpression by immunohistochemistry, three had previous premalignant lesions that also had immunohistochemically detectable p53 protein. However, DNA sequencing showed that none of these three had mutations in the p53 gene. The remaining five premalignant lesions had no immunohistochemically detectable p53 protein.Conclusions-Some premalignant lesions have increased p53 protein which can be detected by staining with antibody to p53. This staining is not caused by mutations in p53 that are found in subsequent tumours at the same site.

In order to investigate the relationship between stimulus equivalence class formation and age differences, two groups of elderly (ages 62-81) and young (ages 19-22) adult men and women mastered a series of simple and conditional discriminations. Subsequently, they were administered a test for the emergence of equivalence relations. There was a significant age difference in the emergence of equivalence relations, with the elderly subjects less likely to demonstrate equivalence relations. Measures of response latencies evidenced a general slowing of behavior for the elderly group across tasks. There were no gender or Age by Gender interactions. Possible explanations for the age differences are discussed.

Silicone has been used in a variety of forms as a replacement for soft tissue for more than three decades in the United States. The safety of this practice has come under scrutiny recently as our knowledge of the local, systemic, and immune effects of silicone has expanded. Contrary to what was once thought, silicone is not biologically inert. Silicone elastomer, however, has not been implicated in the development of systemic immune disease or carcinogenesis. The evidence to date supports the continued use of silicone elastomer implants in the larynx. It is clear, however, that continued investigation into the basic biologic properties and mechanisms of the host-silicone implant interaction is warranted.

I prospectively evaluated the results of 30 consecutive patients with bilateral carpal tunnel release using two techniques. The first release was performed with a standard incision while the opposite hand underwent release by a double incision open technique. Postoperatively, subjective complaints of pain, grip strength, pinch strength, and pillar tenderness were evaluated at 1, 2, 4, 6, and 10 weeks. All patients expressed complete relief of preoperative numbness in both hands. The improvement in pinch and grip strength and lack of pillar tenderness in the hands that underwent the double incision open technique closely matched the reported results of endoscopic carpal tunnel release. There were no complications with either technique.

The rapid whole blood test, developed for the detection of circulating antibodies to human immunodeficiency virus type 1 (HIV-1), is based on agglutination of autologous red blood cells using an anti-human glycophorin antibody conjugated to the HIV-1 immunodominant epitope of gp41 (579-613). A simplified procedure for preparing antibody-peptide conjugates for use in the autologous red cell agglutination test is described. F(ab')2 fragments of the anti-glycophorin antibody were prepared by pepsin digestion and reduced to F(ab') fragments with the use of tri-n-butylphosphine (TBP). This permitted the simultaneous reduction of the F(ab') fragments and coupling of a bromoacetyl derivative of the synthetic immunodominant peptide gp41 (579-613) [Cys-Acm 598, Lys-BrAc 604] containing epsilon-bromoacetyl-lysine at residue 604 to the resultant F(ab') fragment. Conjugation to the F(ab') fragment resulted in a stable thio-ether linkage between the peptide Lys-604 and the inter heavy chain cysteines of the F(ab'). The resultant F(ab')-peptide conjugate was comparable to the previously described disulfide coupled conjugate when used in the autologous red cell agglutination test. This simplified conjugation chemistry may also be useful for the development of reagents for FACS analysis as well as targetted vaccines.

The T-lymphotropic lentivirus, feline immunodeficiency virus (FIV) is now recognised as a major viral pathogen affecting domestic cat populations worldwide. A rapid, autologous red cell agglutination test for antibodies to FIV has been developed. A synthetic peptide analog corresponding to the immunodominant epitope within the FIV transmembrane glycoprotein gp40 residues (680-715) KVEAMEKFLYTAFAMQELGC (Acm)NQNQFFK(BrAc)KIPLELWTR was conjugated to an anti-feline erythrocyte antibody using a thio-ether linkage. Within 3 min of adding this reagent to 20 microliters of whole blood, circulating antibody to the peptide epitope caused agglutination of the red blood cells. The performance of this simple test is comparable with the two commercially available enzyme immunoassay (EIA) kits and an EIA based on this peptide. A variant of the gp40 (680-715) peptide corresponding to the FIV, PPR strain gp40 (678-716) sequence was also synthesised and no difference in reactivity was observed in an EIA on 211 seropositive samples, indicating that the peptide-based test may be applicable to other known strains of the virus.

Pleomorphic adenoma (PA), the most common neoplasm of the parotid gland in adults, undergoes malignant transformation in only two to six percent of cases. In order to determine whether or not the p53 gene is involved in the malignant transformation of PA of the parotid gland, we have analyzed archival specimens from four patients with carcinoma ex-pleomorphic adenoma (CEPA) by immunohistochemistry and sequencing of the amplified DNA. Tissue specimens from each patient contained both the CEPA and the precursor PA. Immunostaining revealed that while all CEPA were p53 positive, only one of the four precursor PA was positive for p53. In this case, DNA was isolated from both the PA and CEPA and exons five through eight were amplified using the polymerase chain reaction. The same p53 mutation (single base substitution) was found in both the PA and the CEPA at codon 248. Adjacent normal tissue contained the wild-type p53 sequence. To our knowledge, this is the first report of the same p53 point mutation in both a PA and the associated CEPA of the parotid. These results strongly suggest a role for p53 gene mutation in the malignant transformation of some PA of the parotid gland.

This Head and Neck Intergroup analysis was undertaken to evaluate further previously reported observations linking blood transfusions, which were given to patients with head and neck cancer, to a worse prognosis. This study population represents those patients registered to the Head and Neck Intergroup Trial 0034 for previously untreated resectable squamous cell carcinoma. Additional transfusion data were obtained by one of us (D.E.S.) on 217 patients and added to the Head and Neck Intergroup data set, providing an opportunity for assessing the impact of survival by other variables. The study group was separated using 13 variables. Analysis demonstrated that transfusion did not significantly decrease the locoregional control (P = .60). Multivariate analysis indicated that T stage (P = .015), N stage (P = .004), treatment received (P = .004), and Karnofsky Performance Scale (P = .031) were the only factors that did significantly influence survival. This multivariate analysis controlling for these variables demonstrated no significant effect on survival for those patients receiving transfusion during surgery (P = .55) or after surgery (P = .39). This study of 217 patients, controlled for other variables, does not demonstrate any significant negative relation between blood transfusions and either locoregional control or survival.

The kinetics of the autologous red cell agglutination test for detecting circulating antibodies to HIV-1 were studied. Two monoclonal anti-red blood cell antibodies (1C3/86 and 10F7MN) were used to construct Fab-peptide conjugates for the test. Both antibodies recognize glycophorin alpha on the surface of erythrocytes by immunoprecipitation or immunoblotting techniques. The number of binding sites, association and dissociation constants of 1C3/86 Fab and 10F7MN Fab' fragments were determined (n = 4.80 X 10(5) sites/erythrocyte, Ka = 0.43 X 10(7) M-1, Kd = 23 X 10(-8) M for 1C3/86, n = 4.66 X 10(5) sites/erythrocyte, Ka = 1.05 X 10(7) M-1, Kd = 9.5 X 10(-8) M for 10F7MN. The binding studies were performed under the same conditions as the autologous red blood cell agglutination test. When 0.9 microgram of anti-glycophorin Fab was added to 10 microliters of blood 0.25 microgram of 1C3/86 Fab was bound whereas 0.29 microgram for 10F7MN Fab' was bound. Antibody binding reached a plateau after 2 min and once bound did not exchange with unbound Fab over the time scale of the test. The binding of the anti-peptide antibody (cross-linking antibody) was also complete within 2 min. Addition of approximately 0.1 microgram of anti-peptide antibody gave half a maximal agglutination score. This is equivalent to 10 micrograms/ml circulating antibody. Under the agglutination test conditions, Fab-peptide conjugate was bound to 14% of available glycophorin molecules. Half maximal agglutination occurred when approximately 1.1% of the bound Fab-peptide conjugates were cross-linked. A maximum agglutination score of four occurred in the presence of 1 microgram of anti-peptide antibody equivalent to 100 micrograms/ml circulating antibody whereas an agglutination score of 1+ was elicited by only 0.32 microgram anti-peptide antibody and involved the cross-linking of approximately 160 glycophorin molecules per red cell.

The human histocompatibility leucocyte antigen (HLA) class II-associated invariant chain comprises at least four polypeptides. One of these is expressed as a membrane-bound subunit and has been designated as CD74. We investigated the expression of CD74 on B- and T-cell types utilizing monoclonal antibodies (mAb) specific for the c-terminus of CD74. All B cells tested expressed CD74 regardless of their HLA phenotypes. High and low CD74 expressors were provisionally assigned based on quantitative variations found in some haplotypes. A mutant cell with selective deletions of class II loci DR and DQ but with retention of DP and additional class II heterodimers synthesize normal amounts of CD74. In contrast, however, a mutant with a total class II deletion had diminished expression of CD74, suggesting that at least one of the class II isotypes is essential for full constitutive expression of CD74. Major histocompatibility complex (MHC) class II negative T-cell lines and CD3+ T cell isolated from peripheral blood did not express CD74. However, allostimulated T cells which express de novo induced class II determinants also did not express CD74. Taken together, these results show that CD74 is expressed on B cells and unexpectedly is not synthesized on de novo-induced class II positive T-cell clones.

A technique of nerve block in the distal forearm is described for use in carpal tunnel release operations. In 70 operations, the extremities of 52 patients have been successfully anesthetized in this manner without complication. This simple anesthetic technique is recommended for use by the hand surgeon.

Objective: To evaluate the safety and efficacy of E5 (Xomen-E5), a murine monoclonal immunoglobulin M antibody, in reducing mortality in patients with serious Gram-negative infections. Phase II, single-site study., Design: Randomized, double-blind, placebo-controlled study., Setting: Surgical, neurosurgical and medical ICUs, comprising approximately 30 beds in a multidisciplinary university hospital., Patients: Patients with clinical evidence of serious infection admitted to the ICU for monitoring of vital signs and for intensive care nursing. Of the 39 patients enrolled in the study, 23 had documented Gram-negative infection., Methods: Patients suspected of having life-threatening Gram-negative infections received one of two doses of E5 or placebo. Safety and efficacy were assessed by survival on days 3, 7, and 21, appearance of adverse reactions, development of antimurine antibodies, and effects on BP, urine output, WBC count, and temperature., Measurements and Main Results: Mortality rate from Gram-negative infection 3 days after the last drug (or placebo) infusion was two (22%) of nine deaths in the placebo group compared with 0 of nine for E5 2.5 mg/kg and 0 of five for E5 7.5 mg/kg. At 21 days after therapy, three patients treated with E5 had died. Only one of these three deaths resulted from infection. Eight of 15 E5 patients tested had immunoglobulin G antimurine antibodies by 3 wks after therapy began, but all were asymptomatic., Conclusions: E5 was well tolerated and may have the potential to reduce early morbidity and the mortality rate in seriously ill patients with Gram-negative infections. Results from larger phase III studies are needed to confirm these findings.

Water intake, urine output, and fluid exchange (water intake less urine output) were measured in rats at hourly intervals for 7 hours and at 24 hours following acute administration of angiotensin II (AII, 200 micrograms/kg SC). AII induced the expected abrupt increase in water intake and a more gradual increase in urine output. The change in fluid exchange (fluid exchange of the AII-treated group less fluid exchange of controls) became positive within the first hour after treatment with AII, decreased linearly with time, and reached 0 at approximately 10 to 12 hours after treatment with AII. When AII was administered intracerebroventricularly (50 ng), similar results were observed. In this case, the change in fluid exchange (delta F) reached 0 in about 6 hours. Imposition of a water load (1% of body weight, IP) on the group receiving AII SC failed to affect the time required for delta F to reach 0 if the water load was disregarded. However, inclusion of the load as a part of intake extended the time the rats remained in positive fluid balance beyond that of the nonloaded, AII-treated control group. In the case of the larger water load (3% of body weight, IP), delta F returned to that of controls in about 4 to 5 hours if the water load was disregarded. However, inclusion of the load as part of intake extended the period of hyperhydration well beyond that of both the nonloaded, AII-treated group and the AII-treated group given the 1% load.(ABSTRACT TRUNCATED AT 250 WORDS)

The single dose pharmacokinetics of 4 commercially available 100 mg oral aspirin formulations were studied in 6 healthy men and 6 healthy women. Two of the formulations were rapid release ('Cardiprin' 100, 'Platelin') and the other 2 were enteric coated formulations ('Astrix' 100, 'Cartia'). There were marked differences in the plasma concentration-time profiles between the rapid release and the enteric coated formulations. There were no significant differences (p greater than 0.05) in the mean time to achieve maximum aspirin concentrations between 'Cardiprin' 100 (0.48 h) and 'Platelin' (0.35 h), but this was significantly prolonged (p less than 0.001) for 'Astrix' 100 (3.73 h) and even more prolonged for 'Cartia' (6.84 h). Similar between-formulation differences were seen in the areas under the plasma concentration-time curves, for which the rank order was 'Cardiprin' 100 (1.60 mg/L.h) = 'Platelin' (1.54 mg/L.h) greater than 'Astrix' 100 (0.73 mg/L.h) greater than 'Cartia' (0.56 mg/L.h). For 'Cardiprin' 100, 'Platelin' and 'Astrix' 100 plasma aspirin concentrations were below 5 micrograms/L by 7 h after ingestion, whereas for 'Cartia' aspirin was detectable for up to 16 h, giving the appearance of sustained release. The enteric coated formulations produced the greatest variability in the plasma aspirin concentration vs time profiles. The urinary recovery of salicylate was greater than 80% of the administered dose for all 4 formulations. The clinical significance of the marked pharmacokinetic differences observed with these 4 low-dose aspirin formulations is not known.

A rapid whole blood test has been developed for circulating antibodies to human immunodeficiency virus type 1 (HIV-1), based on agglutination of autologous red blood cells. Evaluation of the test revealed that 100% of seropositive HIV-1 patients (both asymptomatic and AIDS cases) were detected (n = 94) with a specificity of 99.5% in healthy blood donors (n = 596). The assay uses an Fab fragment of a monoclonal antibody specifically directed against glycophorin (a transmembrane glycoprotein present on the surface of human red blood cells). This anti-red blood cell Fab is conjugated via the inter-heavy chain cysteines to a synthetic peptide corresponding to the immunodominant epitope of the HIV-1 viral coat protein gp41 (579-613). Addition of this reagent to 10 microliters of whole blood results in the Fab-peptide conjugate coating the red blood cells with peptide. In the presence of circulating antibodies to the HIV-1 peptide, red cell agglutination occurs within 2 min. The sensitivity and specificity of this reagent indicate that it is appropriate for use as a rapid diagnostic test for HIV-1 seropositivity.

Norepinephrine (NE)-stimulated 3H-inositol phosphate (3H-InsP) formation in primary glial cell cultures is thought to be due to alpha 1-adrenergic receptor activation. Surprisingly, the alpha 1-selective agonists phenylephrine and methoxamine showed only 12-21% of the intrinsic activity of NE in activating this response. Although the alpha 2-selective agonist UK 14,304 was itself inactive, inclusion of UK 14,304 increased the response to the alpha 1-selective agonists by about threefold. This increase was concentration-dependent and occurred at all time points examined. 6-Fluoro-NE and alpha-methyl-NE mimicked the effect of NE in glial cultures, although with lower potencies. However, several partial agonists were ineffective in activating this response, in both the presence and absence of UK 14,304. Synergistic interactions were not observed for alpha 1-mediated responses in slices of rat cerebral cortex, either for formation of 3H-InsPs or potentiation of isoproterenol- or adenosine-stimulated cyclic AMP accumulation. Both UK 14,304 and phenylephrine inhibited NE-stimulated 3H-InsP formation in concentrations similar to those necessary to activate this response directly. These results suggest that NE activates 3H-InsP formation in primary glial cultures by synergistic actions on both alpha 1- and alpha 2-adrenergic receptors. The agonists UK 14,304 and phenylephrine also can act to inhibit the response to NE competitively.