Incidence of groin hernia repairs in women and parity: a population-based cohort study among women born in Sweden between 1956 and 1983.

INTRODUCTION: The aim of this study was to evaluate the association between parity and the incidence rate of groin hernia repair in women. METHOD: This study was based on two Swedish national registers, the Medical Birth Register (MBR), and the Swedish Hernia Register (SHR). The cohort constituted of women born between 1956 and 1983. Data on vaginal and cesarean deliveries were retrieved from the MBR. The birth and hernia registers were cross matched to identify hernia repairs carried out after deliveries. RESULTS: A total of 1,535,379 women were born between 1956 and 1983. Among these, 1,417,237 (92.3%) were registered for at least one birth. The incidence rate for Inguinal Hernia Repair (IHR) and Femoral Hernia Repair (FHR) was 10.7 per 100,000 person-year and 2.6 per 100,000 person-year, respectively. Compared with women registered for one delivery, the incidence rate ratio for IHR was 1.31 (95% Confidence Interval: 1.23-1.40) among women registered for two deliveries, 1.70 (1.58-1.82) among women registered for ≥ 3 deliveries. Additionally, the incidence rate ratios were higher 1.30 (1.14-1.49) and 1.70 (1.49-1.95) for FHR among women with two and ≥ 3 registered deliveries, respectively. CONCLUSION: In the present cohort, higher parity was associated with a higher incidence of inguinal as well as FHRs.

Survey of surgical training and experience of associate clinicians compared with medical officers to understand task-shifting in a low-income country.

Background: A workforce crisis exists in global surgery. One solution is task-shifting, the delegation of surgical tasks to non-physician clinicians or associate clinicians (ACs). Although several studies have shown that ACs have similar postoperative outcomes compared with physicians, little is known about their surgical training. This study aimed to characterize the surgical training and experience of ACs compared with medical officers (MOs) in Tanzania. Methods: All surgical care providers in Pwani Region, Tanzania, were surveyed. Participants reported demographic data, years of training, and procedures assisted and performed during training. They answered open-ended questions about training and post-training surgical experience. The median number of training cases for commonly performed procedures was compared by cadre using Wilcoxon rank sum and Student's t tests. The researchers performed modified content analysis of participants' answers to open-ended questions on training needs and experiences. Results: A total of 21 ACs and 12 MOs participated. ACs reported higher exposure than MOs to similar procedures before their first independent operation (median 40 versus 17 cases respectively; P = 0·031). There was no difference between ACs and MOs in total training surgical volume across common procedures (median 150 versus 171 cases; P = 0·995). Both groups reflected similarly upon their training. Each cadre relied on the other for support and teaching, but noted insufficient specialist supervision during training and independent practice. Conclusions: ACs report similar training and operative experience compared with their physician colleagues in Tanzania.

Antecedentes: La falta de cirujanos en determinadas áreas geográficas es flagrante. Una posible solución es el intercambio de tareas, es decir, la delegación de tareas quirúrgicas en personal sanitario no médico o en clínicos asociados (associate clinicians, AC). Si bien varios estudios han demostrado que los AC obtienen resultados postoperatorios similares a los de los médicos, hay poco información acerca de su entrenamiento quirúrgico. Este estudio tuvo como objetivo caracterizar la capacitación quirúrgica y la experiencia de los AC en comparación con los médicos titulados (medical officer, MO) en Tanzania. Métodos: En este estudio, se encuestaron todos los proveedores de atención quirúrgica de la Región de Pwani, Tanzania. Los participantes proporcionaron datos demográficos, años de entrenamiento y número y tipo de procedimientos realizados y a los que se había asistido durante el periodo de capacitación. Además, respondieron a preguntas abiertas sobre el entrenamiento y su experiencia quirúrgica posterior al entrenamiento. Se comparó la mediana del número de procedimientos más realizados por cada grupo mediante la suma de rangos de Wilcoxon y la prueba de la t de Student. Los investigadores realizaron un análisis del contenido de las respuestas a las preguntas abiertas sobre las necesidades y la experiencia durante la etapa de entrenamiento. Resultados: En el estudio participaron 21 ACs y 12 MOs. Los CA estuvieron expuestos a un mayor número procedimientos del mismo tipo antes de efectuar su primera operación de forma independiente en comparación con los OM (40 versus 17 casos, P = 0,031). No hubo diferencias en el volumen operatorio total de los procedimientos comunes entre los AC y los MO (150 versus 171 casos, P = 0,995). Las opiniones de los dos grupos sobre el entrenamiento fueron similares. Los dos grupos se dieron soporte entre ellos, pero quedó patente que la supervisión por parte de un especialista durante el entrenamiento y la práctica independiente era insuficiente. Conclusiones: En Tanzania, los asociados clínicos tienen entrenamientos y experiencias quirúrgicas similares a las de sus colegas médicos.

Cost-effectiveness of groin hernia repair from a randomized clinical trial comparing commercial versus low-cost mesh in a low-income country.

BACKGROUND: Over 200 million people worldwide live with groin hernia and 20 million are operated on each year. In resource-scarce settings, the superior surgical technique using a synthetic mesh is not affordable. A low-cost alternative is needed. The objective of this study was to calculate and compare costs and cost-effectiveness of inguinal hernia mesh repair using a low-cost versus a commercial mesh in a rural setting in Uganda. METHODS: This is a cost-effectiveness analysis of a double-blinded RCT comparing outcomes from groin hernia mesh repair using a low-cost mesh and a commercially available mesh. Cost-effectiveness was expressed in US dollars (with euros in parentheses, exchange rate 30 December 2016) per disability-adjusted life-year (DALY) averted and quality-adjusted life-year (QALY) gained. RESULTS: The cost difference resulting from the choice of mesh was $124·7 (€118·1). In the low-cost mesh group, the cost per DALY averted and QALY gained were $16·8 (€15·9) and $7·6 (€7·2) respectively. The corresponding costs were $58·2 (€55·1) and $33·3 (€31·5) in the commercial mesh group. A sensitivity analysis was undertaken including cost variations and different health outcome scenarios. The maximum costs per DALY averted and QALY gained were $148·4 (€140·5) and $84·7 (€80·2) respectively. CONCLUSION: Repair using both meshes was highly cost-effective in the study setting. A potential cost reduction of over $120 (nearly €120) per operation with use of the low-cost mesh is important if the mesh technique is to be made available to the many millions of patients in countries with limited resources. TRIAL REGISTRATION NUMBER: ISRCTN20596933 (http://www.controlled-trials.com).

Reproducibility of (18)F-FDG PET uptake measurements in head and neck squamous cell carcinoma on both PET/CT and PET/MR.

OBJECTIVE: To investigate reproducibility of fluorine-18 fludeoxyglucose ((18)F-FDG) uptake on (18)F-FDG positron emission tomography (PET)/CT and (18)F-FDG PET/MR scans in patients with head and neck squamous cell carcinoma (HNSCC). METHODS: 30 patients with HNSCC were included in this prospective study. The patients were scanned twice before radiotherapy treatment with both PET/CT and PET/MR. Patients were scanned on the same scanners, 3 days apart and according to the same protocol. Metabolic tumour activity was measured by the maximum and peak standardized uptake value (SUVmax and SUVpeak, respectively), and total lesion glycolysis from the metabolic tumour volume defined from ≥50% SUVmax. Bland-Altman analysis with limits of agreement, coefficient of variation (CV) from the two modalities were performed in order to test the reproducibility. Furthermore, CVs from SUVmax and SUVpeak were compared. The area under the curve from cumulative SUV-volume histograms were measured and tested for reproducibility of the distribution of (18)F-FDG uptake. RESULTS: 24 patients had two pre-treatment PET/CT scans and 21 patients had two pre-treatment PET/MR scans available for further analyses. Mean difference for SUVmax, peak and mean was approximately 4% for PET/CT and 3% for PET/MR, with 95% limits of agreement less than ±20%. CV was small (5-7%) for both modalities. There was no significant difference in CVs between PET/CT and PET/MR (p = 0.31). SUVmax was not more reproducible than SUVpeak (p = 0.09). CONCLUSION: (18)F-FDG uptake in PET/CT and PET/MR is highly reproducible and we found no difference in reproducibility between PET/CT and PET/MR. ADVANCES IN KNOWLEDGE: This is the first report to test reproducibility of PET/CT and PET/MR.

Whole-body PET/MRI: the effect of bone attenuation during MR-based attenuation correction in oncology imaging.

PURPOSE: In combined PET/MRI standard PET attenuation correction (AC) is based on tissue segmentation following dedicated MR sequencing and, typically, bone tissue is not represented. We evaluate PET quantification in whole-body (WB)-PET/MRI following MR-AC without considering bone attenuation and then investigate different strategies to account for bone tissue in clinical PET/MR imaging. To this purpose, bone tissue representation was extracted from separate CT images, and different bone representations were simulated from hypothetically derived MR-based bone classifications. METHODS: Twenty oncology patients referred for a PET/CT were injected with either [18F]-FDG or [18F]-NaF and imaged on PET/CT (Biograph TruePoint/mCT, Siemens) and PET/MRI (mMR, Siemens) following a standard single-injection, dual-imaging clinical WB-protocol. Routine MR-AC was based on in-/opposed-phase MR imaging (orgMR-AC). PET(/MRI) images were reconstructed (AW-OSEM, 3 iterations, 21 subsets, 4mm Gaussian) following routine MR-AC and MR-AC based on four modified attenuation maps. These modified attenuation maps were created for each patient by non-linear co-registration of the CT images to the orgMR-AC images, and adding CT bone mask values representing cortical bone: 1200HU (cortCT), spongiosa bone: 350HU (spongCT), average CT value (meanCT) and original CT values (orgCT). Relative difference images of the PET following AC using the modified attenuation maps were compared. SUVmean was calculated in anatomical reference regions and for PET-positive lesions. RESULTS: The relative differences in SUVmean across patients following orgMR-AC and orgCT in soft tissue lesions and in bone lesions were similar (range: 0.0% to -22.5%), with an average underestimation of SUVmean of 7.2% and 10.0%, respectively when using orgMR-AC. In bone lesions, spongCT values were closest to orgCT (median bias of 1.3%, range: -9.0% to 13.5%) while the overestimation of SUVmean with respect to orgCT was highest for cortCT (40.8%, range: 1.5% to 110.8%). For soft tissue lesions the bias was highest using cortCT (13.4%, range: -2.3% to 17.3%) and lowest for spongCT (-2.2%, range: 0.0% to -13.7%). CONCLUSIONS: In PET/MR imaging using standard MR-AC PET uptake values in soft lesions and bone lesions are underestimated by about 10%. In individual patients this bias can be as high as 22%, which is significant during clinical follow-up exams. If bone segmentation is available, then assigning a fixed attenuation value of spongious bone to all bone structures appears reasonable and results in only a minor bias of 5%, or less in uptake values of soft tissue and bone lesions.

Prevalence of treated and untreated groin hernia in eastern Uganda.

BACKGROUND: Hernia repair is the most commonly performed general surgical procedure worldwide. The prevalence is poorly described in many areas, and access to surgery may not be met in low- and middle-income countries. The objectives of this study were to investigate the prevalence of groin hernia and the surgical repair rate in a defined sub-Saharan region of Africa. METHODS: A two-part study on hernia prevalence was carried out in eastern Uganda. The first was a population-based prevalence study with 900 randomly selected men in a Health and Demographic Surveillance Site. The second was a prospective facility-based study of all surgical procedures performed in the two hospitals providing surgical care in the region. RESULTS: The overall prevalence of groin hernia (current hernia or scar after groin hernia surgery) in men was 9.4 per cent. Less than one-third of men with a hernia had been operated on. More than half had no pain symptoms. The youngest age group had an overall prevalence of 2.4 per cent, which increased to 7.9 per cent in the age range 35-54 years, and to 37 per cent among those aged 55 years and above. The groin hernia surgery rate at the hospitals investigated was 17 per 100,000 population per year, which corresponds to a surgical correction rate of less than 1 per cent per year. Based on hospital records, a considerable number of patients having surgery for groin hernia were women (20 of 84 patients, 24 per cent). CONCLUSION: Groin hernia is a common condition in men in this east Ugandan cohort and the annual surgical correction rate is low. Investment is needed to increase surgical capacity in this healthcare system.

Canine awake head-out plethysmography (HOP): characterization of external resistive loading and spontaneous laryngeal paralysis.

We applied a novel head-out plethysmographic (HOP) method to study awake canine responses to external resistive loading and natural laryngeal paralysis. Measurements of inspiratory and expiratory specific airway resistance (sRaw(insp), sRaw(exp)) were obtained before and after uni- and bidirectional loading (R(add) = 5 cmH(2)O/L/s) in large-breed dogs (n = 9). Mean sRaw(insp) after inspiratory, and sRaw(exp) after expiratory loading were 31.4 and 33.3 cmH(2)Os, respectively. Bidirectional loads induced a significantly greater rise in both sRaw(insp) and sRaw(exp) (55.1 and 61.3 cmH(2)Os) compared to unidirectional loading (P < 0.001). Yet, type of loading did not affect flow-volume indices. The mean R(aw) of dogs was 4.81 cmH(2)O/L/s. Expiratory loading resulted in a significant 8.8% increase in functional-residual-capacity (FRC), compared to FRC(baseline) (76.7 ml/kg). Dogs (n = 5) with laryngeal paralysis demonstrated a significant increase in sR(aw) and R(aw) compared to controls without changes in FRC. In conclusion, HOP precisely characterized sR(aw) in response to external resistive loading. Hence, we could accurately quantify airway obstruction in awake dogs with laryngeal paralysis.

Adult blood lead epidemiology and surveillance--United States, 1998-2001.

PROBLEM/CONDITION: Elevated blood lead levels (BLLs) in adults can damage the cardiovascular, central nervous, reproductive, hematologic, and renal systems. The majority of cases are workplace-related. U.S. Department of Health and Human Services recommends that BLLs among all adults be reduced to < 25 microg/dL. The highest BLL acceptable by standards of the U.S. Occupational Safety and Health Administration is 40 microg/dL. The mean BLL of adults in the United States is < 3 microg/dL. REPORTING PERIOD: This report covers cases of adults (aged > or = 16 years) with BLLs > or = 25 microg/dL, as reported by 25 states during 1998-2001. DESCRIPTION OF SYSTEM: Since 1987, CDC has sponsored the state-based Adult Blood Lead Epidemiology and Surveillance (ABLES) program to track cases of elevated BLLs and provide intervention consultation and other assistance. Overall ABLES program data were last published in 1999 for the years 1994-1997. This report provides an update with data from 25 states reporting for > or = 2 years during 1998-2001. During that period, the ABLES program funded surveillance in 21 states - Alabama, Arizona, Connecticut, Iowa, Maryland, Massachusetts, Michigan, Minnesota, New Jersey, New York, North Carolina, Ohio, Oklahoma, Oregon, Pennsylvania, Rhode Island, South Carolina, Texas, Washington, Wisconsin, and Wyoming. Four additional states - California, Nebraska, New Hampshire, and Utah contributed data without CDC funding. RESULTS: During 1998-2001, the overall program's annual mean state prevalence rate for adults with BLLs > or = 25 microg/dL was 13.4/100,000 employed adults. This compares with 15.2/100,000 for 1994-1997. Yearly rates were 13.8 (1998), 12.9 (1999), 14.3 (2000), and 12.5 (2001). For adults with BLLs > or = 40 microg/dL, the overall program's annual mean state prevalence rare during 1998-2001 was 2.9/ 100,000 employed adults. This compares with 3.9/100,000 for 1994-1997. Yearly rates were 3.3 (1998), 2.5 (1999), 2.9 (2000), and 2.8 (2001). INTERPRETATION: Although certain limitations exist, the overall ABLES data indicate a declining trend in elevated BLLs among employed adults. PUBLIC HEALTH ACTIONS: ABLES-funded states increased from 21 to 35 in 2002, and more detailed reporting requirements were put into effect. These, and other improvements, will enable the ABLES program to work more effectively toward its 2010 target of eliminating all cases of BLLs > or = 25 microg/dL in adults caused by workplace exposures.

Respiratory distress syndrome: evaluation of genetic susceptibility and protection by transmission disequilibrium test.

Based on epidemiological data and genetic association studies, neonatal respiratory distress syndrome (RDS) is a complex disease with a multigenic background. The genes coding for surfactant proteins (SP) A and B have been assigned as the most likely genes in the etiology of RDS. The major factor predisposing to RDS is prematurity, and thus the phenotype of a very premature newborn infant that does not develop the disease can be regarded as hypernormal. Altogether 107 father-mother-offspring trios were divided into two sets according to the proband's phenotype, to evaluate familial segregation of candidate gene polymorphisms by the transmission disequilibrium test. A set of 76 trios were analyzed for transmission disequilibrium from parents to affected offspring. Another set of 31 trios were studied for allele transmission from parents to hypernormal offspring born very prematurely before the gestational age of 32 weeks. SP-A1-A2 haplotype 6A(2)-1A(0) showed significant excess transmission to affected infants and SP-A1 allele 6A(2) decreased transmission to the hypernormals. The present family study provides strong support for a direct or indirect role of the SP-A alleles as genetic predisposers to RDS in premature infants. The inclusion of parent-hypernormal offspring trios in transmission disequilibrium test is a useful approach to test for genetic protection against a disease.

Surfactant protein-A gene locus associated with recurrent otitis media.

Surfactant protein-A, which plays a role in innate host defense in the lung, is also expressed in the Eustachian tube. We report that the frequency of specific surfactant protein-A haplotypes and genotypes differs between children with recurrent otitis media compared with a control population.

Ammonium alters N-glycan structures of recombinant TNFR-IgG: degradative versus biosynthetic mechanisms.

The effect of ammonium on the glycosylation pattern of the recombinant immunoadhesin tumor necrosis factor-IgG (TNFR-IgG) produced by Chinese hamster ovary cells is elucidated in this study. TNFR-IgG is a chimeric IgG fusion protein bearing one N-linked glycosylation site in the Fc region and three complex-type N-glycans in the TNF-receptor portion of each monomer. The ammonium concentration of batch suspension cultures was adjusted with glutamine and/or NH(4)Cl. The amount of galactose (Gal) and N-acetylneuraminic acid (NANA) residues on TNFR-IgG correlated in a dose-dependent manner with the ammonium concentration under which the N-linked oligosaccharides were synthesized. As ammonium increased from 1 to 15 mM, a concomitant decrease of up to 40% was observed in terminal galactosylation and sialylation of the molecule. Cell culture supernatants contained measurable beta-galactosidase and sialidase activity, which increased throughout the culture. The beta-galactosidase, but not the sialidase, level was proportional to the ammonium concentration. No loss of N-glycans was observed in incubation studies using beta-galactosidase and sialidase containing cell culture supernatants, suggesting that the ammonium effect was biosynthetic and not degradative. Several biosynthetic mechanisms were investigated. Ammonium (a weak base) is known to affect the pH of acidic intracellular compartments (e.g., trans-Golgi) as well as intracellular nucleotide sugar pools (increases UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine). Ammonium might also affect the expression rates of beta1, 4-galactosyltransferase (beta1,4-GT) and alpha2,3-sialyltransferase (alpha2,3-ST). To separate these mechanisms, experiments were designed using chloroquine (changes intracellular pH) and glucosamine (increases UDP-GNAc pool [sum of UDP-GlcNAc and UDP-GalNAc]). The ammonium effect on TNFR-IgG oligosaccharide structures could be mimicked only by chloroquine, another weak base. No differences in N-glycosylation were found in the product synthesized in the presence of glucosamine. No differences in beta1, 4-galactosyltransferase (beta1,4-GT) and alpha2,3-sialyltransferase (alpha2,3-ST) messenger RNA (mRNA) and enzyme levels were observed in cells cultivated in the presence or absence of 13 mM NH(4)Cl. pH titration of endogenous CHO alpha2,3-ST and beta-1,4-GT revealed a sharp optimum at pH 6.5, the reported trans-Golgi pH. Thus, at pH 7.0 to 7.2, a likely trans-Golgi pH range in the presence of 10 to 15 mM ammonium, activities for both enzymes are reduced to 50% to 60%. Consequently, ammonium seems to alter the carbohydrate biosynthesis of TNFR-IgG by a pH-mediated effect on glycosyltransferase activity.

A potential role for activated HER-2 in prostate cancer.

The epidermal growth factor (also known as HER or ErbB) family of receptor tyrosine kinases are important mediators of cell growth, differentiation, and survival. At present there are 10 ligands that bind directly to epidermal growth factor, HER-3, or HER-4. Although none of these ligands bind directly to HER-2, it is recruited to these receptor complexes and also becomes activated. A monoclonal antibody directed against HER-2, 2C4, inhibits the association of HER-2 with other HER family members. Ligand-activated HER-2 may also play a role in cancers, particularly those that do not overexpress HER-2 at high levels. For example, when prostate cancers progress from an androgen-dependent to an androgen-independent phenotype, epidermal growth factor pathways are frequently activated. 2C4 will inhibit the growth of both androgen-dependent and androgen-independent prostate tumors grown as xenografts in athymic mice.

Engineering Chinese hamster ovary cells to maximize sialic acid content of recombinant glycoproteins.

We have engineered two Chinese hamster ovary cell lines secreting different recombinant glycoproteins to express high levels of human beta1,4-galactosyltransferase (GT, E.C. 2.4.1.38) and/or alpha2, 3-sialyltransferase (ST, E.C. 2.4.99.6). N-linked oligosaccharide structures synthesized by cells overexpressing the glycosyltransferases showed greater homogeneity compared with control cell lines. When GT was overexpressed, oligosaccharides terminating with GlcNAc were significantly reduced compared with controls, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. As expected, GT overexpression resulted in reduction of oligosaccharides terminating with GlcNAc, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. The more highly sialylated glycoproteins had a significantly longer mean residence time in a rabbit model of pharmacokinetics. These experiments demonstrate the feasibility of genetically engineering cell lines to produce therapeutics with desired glycosylation patterns.

Nonclinical studies addressing the mechanism of action of trastuzumab (Herceptin).

HER2 is a ligand-less member of the human epidermal growth factor receptor or ErbB family of tyrosine kinases. In normal biological systems, HER2 functions as a co-receptor for a multitude of epidermal growth factor-like ligands that bind and activate other HER family members. HER2 overexpression is observed in a number of human adenocarcinomas and results in constitutive HER2 activation. Specific targeting of these tumors can be accomplished with antibodies directed against the extracellular domain of the HER2 protein. One of these antibodies, 4D5, has been fully humanized and is termed trastuzumab (Herceptin; Genentech, San Francisco, CA). Treatment of HER2-overexpressing breast cancer cell lines with trastuzumab results in induction of p27KIP1 and the Rb-related protein, p130, which in turn significantly reduces the number of cells undergoing S-phase. A number of other phenotypic changes are observed in vitro as a consequence of trastuzumab binding to HER2-overexpressing cells. These phenotypic changes include downmodulation of the HER2 receptor, inhibition of tumor cell growth, reversed cytokine resistance, restored E-cadherin expression levels, and reduced vascular endothelial growth factor production. Interaction of trastuzumab with the human immune system via its human immunoglobulin G1 Fc domain may potentiate its antitumor activities. In vitro studies demonstrate that trastuzumab is very effective in mediating antibody-dependent cell-mediated cytotoxicity against HER2-overexpressing tumor targets. Trastuzumab treatment of mouse xenograft models results in marked suppression of tumor growth. When given in combination with standard cytotoxic chemotherapeutic agents, trastuzumab treatment generally results in statistically superior antitumor efficacy compared with either agent given alone. Taken together, these studies suggest that the mechanism of action of trastuzumab includes antagonizing the constitutive growth-signaling properties of the HER2 system, enlisting immune cells to attack and kill the tumor target, and augmenting chemotherapy-induced cytotoxicity.

Binding interaction of the heregulinbeta egf domain with ErbB3 and ErbB4 receptors assessed by alanine scanning mutagenesis.

Individual residues of the heregulinbeta (HRG) egf domain were mutated to alanine and displayed monovalently on phagemid particles as gene III fusion proteins. Wild type HRGbeta egf domain displayed on phage was properly folded as evidenced by its ability to bind ErbB3 and ErbB4 receptor-IgG fusion proteins with affinities close to those measured for bacterially produced HRGbeta egf domain. Binding to ErbB3 and ErbB4 receptors was affected by mutation of residues throughout the egf domain; including the NH2 terminus (His2 and Leu3), the two beta-turns (Val15-Gly18 and Gly42-Gln46), and some discontinuous residues (including Leu3, Val4, Phe13, Val23, and Leu33) that form a patch on the major beta-sheet and the COOH-terminal region (Tyr48 and Met50-Phe53). Binding affinity was least changed by mutations throughout the Omega-loop and the second strand of the major beta-sheet. More mutants had greater affinity loss for ErbB3 compared with ErbB4 implying that it has more stringent binding requirements. Many residues important for HRG binding to its receptors correspond to critical residues for epidermal growth factor (EGF) and transforming growth factor alpha binding to the EGF receptor. Specificity may be determined in part by bulky groups that prevent binding to the unwanted receptor. All of the mutants tested were able to induce phosphorylation and mitogen-activated protein kinase activation through ErbB4 receptors and were able to modulate a transphosphorylation signal from ErbB3 to ErbB2 in MCF7 cells. An understanding of binding similarities and differences among the EGF family of ligands may facilitate the development of egf-like analogs with broad or narrow specificity.

Selection of heregulin variants having higher affinity for the ErbB3 receptor by monovalent phage display.

Heregulins (HRGs) are epidermal growth factor (egf) domain containing polypeptide growth factors that bind and activate several members of the ErbB receptor family. Although HRG can bind to ErbB3 and ErbB4 homodimers, the highest affinity and most intracellularly active receptor complexes are hetero-oligomers containing ErbB2. The HRGbeta egf domain was displayed on the surface of M13 phage to facilitate mutagenic analysis and optimize for binding to a homodimeric ErbB3-immunoglobulin (IgG) fusion. Nine libraries were constructed in which virtually the entire sequence was randomized in stretches of four to six amino acids. These were selected separately for binding to immobilized ErbB3-IgG. Analysis of the resulting sequences revealed some areas that diverged radically from the wild-type, whereas others showed strong conservation. The degree of wild-type conservation correlated strongly with the functional importance of the residues as determined by alanine scanning mutagenesis (Jones, J. T., Ballinger, M. D., Pisacane, P. I., Lofgren, J. A., Fitzpatrick, V. D., Fairbrother, W. J., Wells, J. A., and Sliwkowski, M. X. (1998) J. Biol. Chem. 273, 11667-11674). Some variants from several libraries showed significant improvements in binding affinity to the ErbB3-IgG. These optimized segments were combined in various ways in the same molecule to generate variants (containing up to 16 mutations) that had >50-fold higher affinity than wild-type HRGbeta. The optimized variants stimulated ErbB2 phophorylation on MCF7 cells at levels similar to wild-type. This indicates wild-type affinity is optimized for potency and that factors other than affinity for ErbB3 are limiting. These variants showed enhanced affinity toward the ErbB4 homodimer, suggesting these receptors use very similar binding determinants despite them having 65% sequence identity.

Chinese hamster ovary cells with constitutively expressed sialidase antisense RNA produce recombinant DNase in batch culture with increased sialic acid.

Under some cell culture conditions, recombinant glycoprotein therapeutics expressed in Chinese hamster ovary (CHO) cells lose sialic acid during the course of the culture (Sliwkowski et al., 1992; Munzert et al., 1996). A soluble sialidase of CHO cell origin degrades the expressed recombinant protein and has been shown to be released into the culture fluid as the viability of the cells decreases. To reduce the levels of the sialidase and to prevent desialylation of recombinant protein, a CHO cell line has been developed that constitutively expresses sialidase antisense RNA. Several antisense expression vectors were prepared using different regions of the sialidase gene. Co-transfection of the antisense constructs with a vector conferring puromycin resistance gave rise to over 40 puromycin resistant clones that were screened for sialidase activity. A 5' 474 bp coding segment of the sialidase cDNA, in the inverted orientation in an SV 40-based expression vector, gave maximal reduction of the sialidase activity to about 40% wild-type values. To test if this level of sialidase would lead to increased sialic acid content of an expressed recombinant protein, the 474 antisense clone was employed as a host for expression of human DNase as a model glycoprotein. The sialic acid content of the DNase produced in the antisense cultures was compared with material made in the wild-type parental cell line. About 20-37% increase in sialic acid content, or 0.6-1.1 mole of additional sialic acid out of a total of 3.0 mole on the product, was found on the DNase made in the antisense cell lines.

Heregulins and the ErbB-2/3/4 receptors in gliomas.

The activation of autocrine loops involving proto-oncogene related receptor tyrosine kinases has led to the analysis of a large number of growth factor systems in human glioma specimens and cell lines. The ErbB-2 system, also called HER-2 or neu, is analogous to the epidermal growth factor receptor system (EGF-R, ErbB-1). Neuregulins consist of a large family of proteins arising from alternative mRNA splicing of a single gene located at 8p22-p11. Activation of ErbB-2 by neuregulins occurs in heterodimeric complexes with ErbB-3 and ErbB-4. A panel of human glioma cell lines, which had previously been analyzed for ErbB-2 expression, was examined for ErbB-3 and ErbB-4 expression. Coordinate expression of ErbB-2, -3 or -4 was not observed in these cell lines. Despite the presence of a complete system capable of signaling in about half the cell lines, no constitutive activation of ErbB-2, -3 or -4 was observed, and autophosphorylation of ErbB-2 in response to heregulin was observed only in one cell line from the panel, NCE-G84. Moreover, the addition of recombinant heregulin or antibodies capable of disrupting ErbB-2/ErbB-3 complexes had no effect on cell proliferation. We conclude that the role of neuregulins and its receptors in the control of glioma cell proliferation may be limited or may be context dependent on in situ conditions which are lost in vitro. Alternatively, neuregulins may be involved in cell differentiation or survival in the central nervous system. Data supporting these conclusions are described in more detail herein.

Induced expression of the new cytokine, activin A, in human monocytes: inhibition by glucocorticoids and retinoic acid.

The capacity of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), glucocorticoids or all-trans-retinoic acid to modulate production of activin A by human monocytes was studied. It was shown that GM-CSF stimulated monocytes to accumulate activin A RNA after as few as 4 hr of incubation, reaching a peak of stimulation at approximately 16 hr of incubation. The activin A transcripts accumulated in the monocytes after stimulation with only 5 U/ml of GM-CSF and reached a maximum plateau level of expression between 25 and 50 U/ml of GM-CSF. Biologically active activin A molecules were detected in the conditioned media by a bioassay, performed both in the absence and presence of a neutralizing antiserum for activin A. Accumulation of bioactive activin A in conditioned medium of monocyte cultures was detected after 24 hr of incubation with GM-CSF and high levels of activin A were maintained for 72 hr. The production of the dimeric beta A beta A in these monocytes was further confirmed by sandwich enzyme-linked immunosorbent assay (ELISA) specific for activin A. In contrast to the stimulatory effect of GM-CSF, hydrocortisone, dexamethasone or all-trans-retinoic acid at 1 x 10(-7) to 1 x 10(-5) M inhibited the constitutive expression of activin A and greatly suppressed the GM-CSF-stimulated production. Thus, the expression of activin A is modulated in monocytes by different agents. These observations may imply new roles for activin A at sites of inflammation where monocytes accumulate.