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The study analyzed the socio-economic determinants of net-income in aquaculture of Kainji, Lake Basin, Nigeria. Specifically, the study examined the; fish farming systems; cost and returns, socio-economic determinants of net-farm income and challenges of fish farming in the area. The study adopted a two-stage sampling procedure to select 120 table-size fish farmers. Data were collected with questionnaires that were administered through face-to-face interview and analyzed using descriptive statistics, budgetary technique and multiple regression analysis. The results showed that 35.00 % of the fish farmers were within the age bracket of 31-40 years, 53.34 % were men, 91.67 % were married, 55.83 % had between 1-5 years of experience in fish farming and 75.83 % had tertiary educational qualification. Majority (92 %) of the fish farmers practiced the monoculture of catfish using earthen ponds system. The estimated total expenses were N 14,953,330.74 while the total revenue generated from 9 fish ponds, each stocked with an average of 3883.986 fingerlings in 2 cycles per year was N 20,188,142.00. The estimated net-farm income after tax was N 5,234,811.26 while the net profit margin and return on investment was 25.93 % and 35% respectively. Age, experience and household size were positive and significant (p<0.05) socio-economic factors that affected netfarm income while the challenges of fish farming were high cost of feed (x̅=3.24), poor pricing (x̅=3.11), poor access to capital (x̅=3.09) and persistent poaching/theft (x̅=2.67). Based on the findings of the study, it is recommended that there should be an intensive research by the fish nutrition division of National Institute for Freshwater Fisheries Research (NIFFR) on the possible alternatives of crude protein source that could be a perfect substitute to Clupeids in fish feeds. [ABSTRACT FROM AUTHOR]

The study examined economics of smoked farmed Catfish in Kainji Lake Basin, Nigeria. Random sampling technique was used to select 80 farmed-catfish processors from 20 communities. Primary data were collected through interview schedule and presented using percentages, mean, and 2-stage least square regression analysis. Results showed that the use of local oven (banda kilns) constitute the majority (at least 67%) of the method used in fish smoking. Roles such as gutting, folding, salting/brining, setting of fire and fish monitoring were mostly performed by the women, while the men and youths supply fire woods as well as fish arrangement on racks. The average gender ratio between the men, women and youths was 0.80, indicating a near gender equality in terms of value of fixed assets, revenue, employees and wage. Profitability indicators showed that smoke fish processing is a viable business with return on investment of 11.71 % for the men, 9.99 % for the women and 8.48 % for the youths respectively. The major determinants of net-income were age, experience and initial capital investment. Hence, it is recommended that the processing industry should be strengthened through subsidy on improved smoking kilns to enable processors produce high quality processed farmed catfish.

Both ethanol (EtoH) and atrazine (ATZ) have hepatic and nephro-toxic effects in rats. In the present study, the toxicity of EtoH (5 g kg−1) on the kidney and liver in the absence or in the presence of different doses of ATZ (50, 100, 300 mg kg−1) was evaluated after 21 days in rats. Results showed that the mixture effects on catalase and superoxide dismutase activities were more severe in both tissues compared to EtoH alone, especially as the dose of ATZ was increased. Hepatic malondialdehyde level (an index of lipid peroxidation) was increased from 20.32% in the EtoH +50 mg kg−1ATZ-treated rats to 34% in the EtoH +300 mg kg−1ATZ-treated rats compared to the EtoH values. Renal malondialdehyde values remain as high as 81% in the EtoH-treated rats and the different combine exposure groups. Furthermore, as the dose of ATZ in the mixture was increased, serum uric acid level increased compared to the EtoH values. When the EtoH +300 mg kg−1ATZ-animals were pretreated with curcumin (an antioxidant), the histopathological changes and peroxidative damages in both tissues were blocked. The exposure of EtoH-treated rats to ATZ enhanced renal and hepatic peroxidative damages in rats. [ABSTRACT FROM PUBLISHER]

Issues arising with social media and its influence on women entrepreneurs trigged this investigation. The research will examine the impact of social media on online small businesses owned and run by women entrepreneurs with reference to London-based female e-entrepreneurs. The work is a proposal for a PhD research and while waiting for the University for confirmation to conduct this area of investigation, I have decided to present this paper as part of my initial investigation on the issues around social media online on small businesses run by women entrepreneurs. Work used mixed research methods to investigate the impact of social media online on female entrepreneurs, their experiences and perceptions on their business activities and performance. The growth of social media as part of innovative technological development has enabled entrepreneurs to effectively carry out their business activities online, and an increasing number of female business owners are taking their businesses online and abandoning traditional, brink and mortar business model. Social media platforms such as Facebook, Twitter, Instagram, Snap chat, YouTube and LinkedIn along with other websites are incorporated into the businesses of modern-day women entrepreneurs. This research will examine, analyse and evaluate relevant theoretical framework on female entrepreneurship and social media engagement and impact on business performance. The research will develop a framework for best practice that can be used by women e-entrepreneurs across Greater London.

Abstract Background Tuberculosis is the world’s deadliest infectious disease and a leading cause of death in Nigeria. The availability of a functional healthcare system is critical for effective TB service delivery and attainment of national and global targets. This study was designed to assess readiness for TB service delivery in Oyo and Anambra states of Nigeria. Methods This was a facility-based study with a mixed-methods convergent parallel design. A multi-stage sampling technique was used to select 42 primary, secondary, and tertiary healthcare facilities in two TB high burden states. Data were collected using key informant interviews, a semi-structured instrument adapted from the WHO Service Availability and Readiness Assessment tool and facility observation using a checklist. Quantitative data were analysed using descriptive and inferential statistics while qualitative data were transcribed and analysed thematically. Data from both sources were integrated to generate conclusions. Results The domain score for basic amenities in both states was 48.8%; 47.0% in Anambra and 50.8% in Oyo state with 95% confidence interval [− 15.29, 7.56]. In Oyo, only half of the facilities (50%) had access to constant power supply compared to 72.7% in Anambra state. The overall general service readiness index for both states was 69.2% with Oyo state having a higher value (73.3%) compared to Anambra with 65.4% (p = 0.56). The domain score for availability of staff and TB guidelines was 57.1% for both states with 95% confidence interval [− 13.8, 14.4]. Indicators of this domain with very low values were staff training for the management of HIV and TB co-infection and training on MDR -TB. Almost half (47.6%) of the facilities experienced a stock out of TB drugs in the 3 months preceding the study. The overall tuberculosis-specific service readiness index for both states was 75%; this was higher in Oyo (76.5%) than Anambra state (73.6%) (p = 0.14). Qualitative data revealed areas of deficiencies for TB service delivery such as inadequate infrastructure, poor staffing, and gaps with continuing education on TB management. Conclusions The weak health system remains a challenge and there must be concerted actions and funding by the government and donors to improve the TB healthcare systems.

The purpose of this paper is to analyse the experience and challenges faced by female in multi-tasking, combing full-time academic work with being an entrepreneur; the impact on professional practice and personal development. Becoming an entrepreneur can be a challenging and daunting task, but it can also come with good experience in managing own small businesses and putting theory into practice in teaching students. The work draws on experiences of how entrepreneurs were able to combine academic work with managing their own small businesses and challenges confronted in realities. Drawing from experience, moving back and forth between the academic and business spheres, this paper considers the impact of being an entrepreneur and lecturer, and what it meant for the students, institutions and own professional development. The work uses qualitative and quantitative research paradigms to seek the perceptions of some professionals who were able to combine academic work and being an entrepreneur, challenges faced in the combined roles on their students, institution and professional development. The analysis of findings from few concludes that there are many benefits, as well as challenges, confronting professionals in multi-tasking of being a business owner and academic Lecturer, of which, most of them became very successful through hard work and determination and some failed to cope due to combination of two ultimately complementary roles. This presents further areas for research to investigate why some professionals who try to combine job roles of academic and being a business owner fail and if this may be the case for the male teachers.

The epidemiology of food borne disease is changing. New pathogens have emerged, and some have spread worldwide. The broad spectrum of food borne infections has changed dramatically over time, as well-established pathogens have been controlled or eliminated, and new ones have emerged. Food pathogens have been a cause of a large number of diseases worldwide and more so in developing countries. This has a major economic impact. The burden of food borne disease remains substantial. Most of these illnesses are not accounted for by known pathogens, so must remain to be discovered. Among the known food borne pathogens, those more recently identified predominate, suggesting that as more and more is learned about pathogens, and they come under control. In addition to the emergence or recognition of new pathogens, other trends include global pandemics of some food borne pathogens, the emergence of antimicrobial resistance, the identification of pathogens that are highly opportunistic, affecting only the most high-risk sub populations, and the increasing identification of large and dispersed outbreaks. Outbreak investigations and case-control studies of sporadic cases can identify sources of infection and guide the development of specific prevention strategies. Better understanding of how pathogens persist in animal reservoirs is also critical to successful long-term prevention. In the past, the central challenge of foodborne disease lay in preventing the contamination of human food with sewage or animal manure. In the future, prevention of foodborne disease will increasingly depend on controlling contamination of feed and water consumed by the animals themselves.

Leaving home countries to study in another country can impose a big cultural challenge to students and teachers in the classroom. This study will aim to share the experience of teaching and interacting with international students, with main focus on Romanian students studying at QA higher education in partnership with Northumbria, Ulster, Roehampton and Middlesex Universities based in London. The main purpose for this study was to compare our previous studies that focused on Nigerian students’ experiences studying in the UK intuitions. The students concerned for this current study are both on undergraduate and postgraduate programmes in Business Studies. QAHE has over 3,000 foreign students on their Business programmes comprising Romanian, Bulgarian, Italian, and others from Mexico, Columbia, China, Vietnam and few from the UK. Majority of the undergraduate students are from Romania studying undergraduate Business programme and the rest studying postgraduate Business programme are mainly from other foreign countries.

Patent Medicine Vendors (PMVs) can play a critical role in increasing access to emergency contraceptive pills (ECPs) in developing countries, but few studies have examined their knowledge and dispensing practices. Using cluster sampling, the authors selected and interviewed 97 PMVs (60.8 per cent female) in Oyo and Ogun States of Nigeria to assess their knowledge, dispensing practices, and referral for ECPs. About one-third (27.8 per cent) of respondents were not aware of ECPs, and only half knew that ECPs could prevent pregnancy. Forty per cent had ever dispensed ECPs. Reasons proffered by those who do not dispense ECPs included barriers from the State Ministry of Health, police, other regulatory agencies, and religious beliefs. Only 50.5 per cent have referral arrangements for clients. Strategies to increase access to ECPs through PMVs include training on counseling techniques and referral, effective government regulation, and community involvement. Where unsafe abortion is a major cause of maternal mortality, these strategies offer protection for many women in the future. [ABSTRACT FROM AUTHOR]

BACKGROUND: Human immunodeficiency virus (HIV) and mental illness are interlinked health problems; mental illness may pose a risk for contracting HIV and HIV-positive individuals are at higher risk of mental illness. However, in countries with high HIV prevalence, the main focus of HIV-related health programmes is usually on prevention and treatment of somatic complications of HIV, and mental illness is not given high priority. We examined HIV prevalence, uptake of HIV services, and HIV-related risk behaviour among people attending a mental health clinic in rural Malawi. METHODOLOGY: Semi-structured interviews were performed with patients capable to consent (94%), and with those accompanied by a capable caregiver who consented. HIV counselling and testing was offered to participants. FINDINGS: Among 174 participants, we collected 162 HIV test results (91%). HIV prevalence was 14.8%. Women were three times as likely to be HIV-positive compared to men. Two-thirds of participants reported having been tested for HIV prior to this study. The uptake of HIV-services among HIV-positive patients was low: 35% did not use recommended prophylactic therapy and 44% of patients not receiving antiretroviral treatment (ART) had never been assessed for ART eligibility. The reported rate of sexual activity was 61%, and 9% of sexually active participants had multiple partners. Inconsistent condom use with stable (89%) and occasional (79%) sexual partners, and absence of knowledge of the HIV status of those partners (53%, 63%) indicate high levels of sexual risk behaviour. CONCLUSIONS: HIV-prevalence among persons attending the clinic, particularly men, was lower than among the general population in a population survey. The rate of HIV testing was high, but there was low uptake of preventive measures and ART. This illustrates that HIV-positive individuals with mental illness or epilepsy constitute a vulnerable population. HIV programmes should include those with neuropsychiatric illness.

Abstract Background In dogs in the western world neoplasia constitutes the most frequently diagnosed cause of death. Although there appear to be similarities between canine and human cancers, rather little is known about the cytogenetic and molecular alterations in canine tumours. Different dog breeds are susceptible to different types of cancer, but the genetic basis of the great majority of these predispositions has yet to be discovered. In some retriever breeds there is a high incidence of soft tissue sarcomas and we have previously reported alterations of chromosomes 11 and 30 in two poorly differentiated fibrosarcomas. Here we extend our observations and present a case report on detail rearrangements on chromosome 11 as well as genetic variations in a tumour suppressor gene in normal dogs. Results BAC hybridisations on metaphases of two fibrosarcomas showed complex rearrangements on chromosome 11, and loss of parts of this chromosome. Microsatellite markers on a paired tumour and blood DNA pointed to loss of heterozygosity on chromosome 11 in the CDKN2B-CDKN2A tumour suppressor gene cluster region. PCR and sequencing revealed the homozygous loss of coding sequences for these genes, except for exon 1β of CDKN2A, which codes for the N-terminus of p14ARF. For CDKN2B exon 1, two alleles were observed in DNA from blood; one of them identical to the sequence in the dog reference genome and containing 4 copies of a 12 bp repeat found only in the canine gene amongst all species so far sequenced; the other allele was shorter due to a missing copy of the repeat. Sequencing of this exon in 141 dogs from 18 different breeds revealed a polymorphic region involving a GGC triplet repeat and a GGGGACGGCGGC repeat. Seven alleles were recorded and sixteen of the eighteen breeds showed heterozygosity. Conclusion Complex chromosome rearrangements were observed on chromosome 11 in two Labrador retriever fibrosarcomas. The chromosome alterations were reflected in the loss of sequences corresponding to two tumour suppressor genes involved in cell-cycle progression. Sequencing of CDKN2B across many different breeds revealed a widespread polymorphism within the first exon of the gene, immediately before the ankyrin coding sequences.

Abstract: Corynespora cassiicola has a unique but unexplored ability amongst fungi, in that it can hydroxylate 17α-hydroxyprogesterone at the highly hindered C-8 position of the steroid nucleus. In order to gain greater understanding of the mechanistic basis and capability of the 8β-hydroxylase we have transformed a range of structurally diverse androgens and progestogens with this organism. This has revealed that both steroid types can be hydroxylated at the 8β-position. The collective data has demonstrated the first time that 8β-hydroxylation occurs through inverted binding within a 9α-hydroxylase of the fungus. In the case of the progestogens, for this to occur, the presence of 17α-oxygen functionality (alcohol or epoxide) was essential. Remarkably monohydroxylation of 17α-hydroxyprogesterone at carbons 8β and 15β has strongly indicated that the responsible hydroxylase has 2 different binding sites for the ring-A ketone. Unusually, with one exception, all hydroxylation occurred at axial protons and in the case of the progestogens, all above the plane of the ring system. In general all maximally oxidised metabolites contained four oxygen atoms. The importance of these findings in relation to 8β-hydroxylation of these steroids is discussed. [Copyright &y& Elsevier]

South Korea has one of the highest rates of monthly alcohol consumption, high-risk drinking, and alcohol-related problems. Global viewers of Korean dramas consume messages about the cultural norms regarding alcohol consumption. There is limited data on the portrayal of alcohol in Korean dramas. The purpose of this embedded mixed methods study is to explore the nature of the portrayal of alcohol consumption in Korean dramas. Content analysis was conducted on a random selection of six drama series. The portrayal of alcohol consumption is ubiquitous, with a reference to alcohol approximately every 12 minutes of programming. The primary messages include the ritualistic importance of alcohol, the over consumption of alcohol by males and females, alcohol as a stress reliever, alcohol as a relationship facilitator, intoxication as a positive valence, unrealistic consequences of intoxication, males as reliable caretakers of intoxicated females, and nondepiction of driving while intoxicated. The results of this study further our understanding of the frequency of the portrayal of alcohol and the prevailing messages about alcohol consumption and intoxication in Korean dramas.

Over 10 years ago, the standards for cardiac monitoring were set forth by the Councils on Cardiovascular Nursing, Clinical Cardiology, and Cardiovascular Disease in the Young. The standards were endorsed by the International Society of Computerized Electrocardiology and the American Association of Critical-Care Nurses. The American Heart Association printed the standards as an American Heart Association Scientific Statement. The standards provided direction related to remote telemetry monitoring to acute care hospitals. Since the standards were published, remote monitoring of cardiac patients has increased dramatically prompting research and literature related to appropriate utilization. Appropriate and safe telemetry monitoring requires clearly written evidence-based facility policies. This article describes the process whereby a team of Veterans Hospital Administration nurses from across the country reviewed 70 remote telemetry policies representing 75 Veterans Hospital Administration hospitals for clarity, consistency, and congruency to existing levels of evidence found in the literature. This article describes the processes, successes, and challenges of compiling an evidence-based remote telemetry policy guideline.

Expression of fibroblast growth factor 2 (FGF2) is believed to be a contributing factor to the growth of a number of tumor types, including hepatocellular carcinoma (HCC). However, the potential of monoclonal antibodies that neutralize FGF2 for treatment of patients with cancer has not yet been explored in clinical trials. We therefore generated a novel monoclonal antibody (mAb), GAL-F2, specific for FGF2 and characterized its properties in vitro and in vivo. GAL-F2 binds to a different epitope than several previous anti-FGF2 mAbs tested. This novel epitope was defined using chimeric FGF1/FGF2 proteins and alanine scanning mutagenesis and was shown to comprise amino acids in both the amino and carboxy regions of FGF2. GAL-F2 blocked binding of FGF2 to each of its four cellular receptors, strongly inhibited FGF2-induced proliferation and downstream signaling in human umbilical vein endothelial cells, and inhibited proliferation and downstream signaling in two HCC cell lines. Moreover, GAL-F2, administered at 5 mg/kg i.p. twice weekly, potently inhibited growth of xenografts of the SMMC-7721, HEP-G2, and SK-HEP-1 human HCC cell lines in nude mice, and in some models, had a strong additive effect with an anti-VEGF mAb or sorafenib. Treatment with GAL-F2 also blocked angiogenesis and inhibited downstream cellular signaling in xenografts, indicating its antitumor mechanism of action. Our report supports clinical testing of a humanized form of the GAL-F2 mAb for treatment of HCC and potentially other cancers., (©2012 AACR.)

Background: In dogs in the western world neoplasia constitutes the most frequently diagnosed cause of death. Although there appear to be similarities between canine and human cancers, rather little is known about the cytogenetic and molecular alterations in canine tumours. Different dog breeds are susceptible to different types of cancer, but the genetic basis of the great majority of these predispositions has yet to be discovered. In some retriever breeds there is a high incidence of soft tissue sarcomas and we have previously reported alterations of chromosomes 11 and 30 in two poorly differentiated fibrosarcomas. Here we extend our observations and present a case report on detail rearrangements on chromosome 11 as well as genetic variations in a tumour suppressor gene in normal dogs., Results: BAC hybridisations on metaphases of two fibrosarcomas showed complex rearrangements on chromosome 11, and loss of parts of this chromosome. Microsatellite markers on a paired tumour and blood DNA pointed to loss of heterozygosity on chromosome 11 in the CDKN2B-CDKN2A tumour suppressor gene cluster region. PCR and sequencing revealed the homozygous loss of coding sequences for these genes, except for exon 1beta of CDKN2A, which codes for the N-terminus of p14ARF. For CDKN2B exon 1, two alleles were observed in DNA from blood; one of them identical to the sequence in the dog reference genome and containing 4 copies of a 12 bp repeat found only in the canine gene amongst all species so far sequenced; the other allele was shorter due to a missing copy of the repeat. Sequencing of this exon in 141 dogs from 18 different breeds revealed a polymorphic region involving a GGC triplet repeat and a GGGGACGGCGGC repeat. Seven alleles were recorded and sixteen of the eighteen breeds showed heterozygosity., Conclusion: Complex chromosome rearrangements were observed on chromosome 11 in two Labrador retriever fibrosarcomas. The chromosome alterations were reflected in the loss of sequences corresponding to two tumour suppressor genes involved in cell-cycle progression. Sequencing of CDKN2B across many different breeds revealed a widespread polymorphism within the first exon of the gene, immediately before the ankyrin coding sequences.

An explicitly polarizable force field based exclusively on quantum data is applied to calculations of relative binding affinities of ligands to proteins. Five ligands, differing by replacement of an atom or functional group, in complexes with three serine proteases-trypsin, thrombin, and urokinase-type plasminogen activator-with available experimental binding data are used as test systems. A special protocol of thermodynamic integration was developed and used to provide sufficiently low levels of systematic error along with high numerical efficiency and statistical stability. The calculated results are in excellent quantitative (rmsd = 1.0 kcal/mol) and qualitative (R(2) = 0.90) agreement with experimental data. The potential of the methodology to explain the observed differences in the ligand affinities is also demonstrated.

We have constructed a very large virtual diversity space containing more than 10(13) chemical compounds. The diversity space is built from about 400 combinatorial libraries, which have been expanded by choosing sizeable collections of suitable R-groups that can be attached to each link point of their scaffolds. These R-group collections have been created by selecting reagents that have drug-like properties from catalogs of available chemicals. As members of known combinatorial libraries, the compounds in the diversity space are in general synthetically accessible and useful as potential drug leads. Hence, the diversity space can be used as a vast source of compounds by a de novo drug design program. For example, we have used such a program to generate inhibitors of HIV integrase enzyme that exhibited activity in the micromolar range.

Purpose: This article describes the progress of our study to examine the advantages and costs of using self-managed nurse aide teams in nursing homes, steps that are being taken to implement such teams, and management strategies being used to manage the teams., Design and Methods: A quasi-experimental design is underway where certified nurse aide (CNA) teams are being established in five nursing homes (NHs) in the Dallas-Fort Worth metropolitan area, and five additional NHs are being treated as comparison NHs., Results: As of March 2004 CNA teams were established in five NHs, and baseline survey data were collected from the CNAs, nurses, residents, and family members in each of these NHs as well as from those in the five comparison homes., Implications: Qualitative analyses show positive effects of CNA teams. Quantitative analyses will not be complete until follow-up survey data are collected 12 months after team implementation. Steps for implementing teams include surveying management to be sure that they want nurse teams; orienting and training the managers, nurses, and nurse aides; and facilitating the teams. Management of the teams includes routine feedback from management to the teams and vice versa while using a give-and-take approach.

AF2 is a high affinity murine Ab possessing potent neutralizing activity against human IFN-gamma. In carrying out the modifications to humanize this Ab, we discovered that an initial version displayed affinity for IFN-gamma that was slightly less than that of AF2, but exhibited IFN-gamma-neutralizing activity that was severely diminished. Characterization via site-directed mutagenesis revealed that the majority of this loss in IFN-gamma-neutralizing activity was due to altering the V(H) framework residue at position 11. V(H) position 11 is distal to the binding surface of the Ab; however, it, along with residues 110 and 112, have been identified as forming the socket of a molecular ball-and-socket joint between the V and C domains of the Ig Fab, which influences the elbow angle between these domains. To determine whether disrupting the structure of this joint was the basis for reduced IFN-gamma-neutralizing capacity, we altered residue 148 of C(H1), which with residue 149 comprises the corresponding ball portion of the joint. Changing this single C(H1) domain residue diminished the ability of the Ab to neutralize IFN-gamma to a level similar to that observed with the V(H) alteration. Thus, an intact ball-and-socket joint between the V and C domains in AF2 is required for potent neutralization of IFN-gamma. These results suggest the importance of the elbow angle between Ig V and C domains in Ab activity, and support the hypothesis that this joint can be an important functional element of Ab structure.

Background: The participation of L-selectin in leukocyte recruitment during inflammation has suggested the use of L-selectin inhibitors as potential anti-inflammatory therapeutics. Blocking monoclonal antibodies could serve as such therapeutic agents, particularly if humanized to reduce their immunogenicity and improve their serum half-life., Objectives: For this purpose, two mouse monoclonal antibodies, DREG-55 and DREG-200, that block human L-selectin were humanized and characterized., Study Design: The resulting humanized antibodies, HuDREG-55 and HuDREG-200, constructed with human IgG4 constant regions, were evaluated for their specificity, affinity and ability to block L-selectin-dependent adhesion in in vitro assays. Their pharmacokinetic behavior in rhesus monkeys was also studied., Results: HuDREG-55 and HuDREG-200 were found to retain the specificity and affinity, within 2-fold, of the parent murine antibodies. HuDREG-55 and HuDREG-200 block L-selectin-dependent adhesion of human lymphocytes to high endothelial venules in frozen sections of lymph nodes. In addition, HuDREG-55 and HuDREG-200 are inhibitory in a novel L-selectin-dependent adhesion assay. This assay utilizes flow cytometry to measure binding of polymerized liposomes containing an analog of sialyl Lewis X, sialyl Lewis X glycoliposomes, to peripheral blood neutrophils and lymphocytes. Studying the pharmacokinetics of HuDREG-55 and HuDREG-200 in rhesus monkeys showed terminal elimination half-lives at 12.0 and 20.3 days, respectively., Conclusion: The shorter terminal elimination half-life of HuDREG-55 in rhesus monkeys may be due to the ability of HuDREG-55 but not HuDREG-200 to bind rhesus monkey L-selectin.

The objective of this work is to compare the three-dimensional structures of "humanized" and mouse-human chimeric forms of a murine monoclonal antibody elicited against human gamma-interferon. It is also to provide structural explanations for the small differences in the affinities and biological interactions of the two molecules for this antigen. Antigen-binding fragments (Fabs) were produced by papain hydrolysis of the antibodies and crystallized with polyethylene glycol (PEG) 8,000 by nearly identical microseeding procedures. Their structures were solved by X-ray analyses at 2.9 A resolution, using molecular replacement methods and crystallographic refinement. Comparison of these structures revealed marked similarities in the light (L) chains and near identities of the constant (C) domains of the heavy (H) chains. However, the variable (V) domains of the heavy chains exhibited substantial differences in the conformations of all three complementarity-determining regions (CDRs), and in their first framework segments (FR1). In FR1 of the humanized VH, the substitution of serine for proline in position 7 allowed the N-terminal segment (designated strand 4-1) to be closely juxtaposed to an adjacent strand (4-2) and form hydrogen bonds typical of an antiparallel beta-pleated sheet. The tightening of the humanized structure was relayed in such a way as to decrease the space available for the last portion of HFR1 and the first part of HCDR1. This compression led to the formation of an alpha-helix involving residues 25-32. With fewer steric constraints, the corresponding segment in the chimeric Fab lengthened by at least 1 A to a random coil which terminated in a single turn of 310 helix. In the humanized Fab, HCDR1, which is sandwiched between HCDR2 and HCDR3, significantly influenced the structures of both regions. HCDR2 was forced into a bent and twisted orientation different from that in the chimeric Fab, both at the crown of the loop (around proline H52a) and at its base. As in HCDR1, the last few residues of HCDR2 in the humanized Fab were compressed into a space-saving alpha-helix, contrasting with a more extended 310 helix in the chimeric form. HCDR3 in the humanized Fab was also adjusted in shape and topography. The observed similarities in the functional binding activities of the two molecules can be rationalized by limited induced fit adjustments in their structures on antigen binding. While not perfect replicas, the two structures are testimonials to the progress in making high affinity monoclonal antibodies safe for human use., (Copyright 1999 John Wiley & Sons, Ltd.)

The ability to produce monoclonal antibodies (Mabs) in plants offers the opportunity for the development of an inexpensive method of mucosal immunoprotection against sexually transmitted diseases. To investigate the suitability of plant-expressed Mabs for vaginal preventive applications, we compared a humanized anti-herpes simplex virus 2 (HSV-2) Mab expressed in mammalian cell culture with the same antibody expressed in soybean. We found these Mabs to be similar in their stability in human semen and cervical mucus over 24 h, their ability to diffuse in human cervical mucus, and their efficacy for prevention of vaginal HSV-2 infection in the mouse.

E- and P-selectin (CD62E and CD62P) are cell adhesion molecules that mediate leukocyte-endothelial cell and leukocyte-platelet interactions and are involved in leukocyte recruitment during inflammation. We previously developed a murine mAb, EP-5C7 (or mEP-5C7), that binds and blocks both E- and P-selectin. When used in humans, murine mAbs have short circulating half-lives and generally induce potent human anti-mouse Ab responses. We therefore engineered a humanized, complementarity determining region-grafted version of mEP-5C7 incorporating human gamma4 heavy and kappa light chain constant regions (HuEP5C7.g4). HuEP5C7.g4 retains the specificity and avidity of mEP-5C7, binding to human E- and P-selectin but not to human L-selectin, and blocking E- and P-selectin-mediated adhesion. Surprisingly, when administered to rhesus monkeys, HuEP5C7.g4 was eliminated from the circulation very rapidly, even faster than the original murine Ab. To isolate the cause of the short serum half-life of HuEP5C7.g4, several Ab variants were constructed. A chimeric IgG4 Ab was made by replacing the humanized V regions with murine V regions. A humanized IgG2 Ab, HuEP5C7.g2, was also made by replacing the human gamma4 with a gamma2 constant region. Results from pharmacokinetic studies in rhesus monkeys demonstrated that the chimeric IgG4 is also rapidly eliminated rapidly from serum, similar to the humanized IgG4 Ab, while the humanized IgG2 Ab displays a long circulation half-life, typical of human Abs.

The ability of guanosine-3',5'-cyclic monophosphate (cGMP) to induce increases in the intracellular free calcium ion concentration ([Ca2+]i) was studied at the single cell level in fura-2-loaded rat hepatocytes. Both 8-bromo-cGMP (Br-cGMP) and dibutyryl cGMP (db-cGMP) produced oscillatory [Ca2+]i increases in hepatocytes. In addition, Br-cGMP increased the frequency of agonist-induced spiking or converted [Ca2+]i oscillations into sustained nonoscillatory [Ca2+]i responses. Addition of the nitric oxide donor sodium nitroprusside also produced oscillatory [Ca2+]i increases similar to those generated by cGMP analogues. In the absence of extracellular Ca2+, cGMP-induced [Ca2+]i responses were significantly reduced and mainly appeared as single transient [Ca2+]i increases. The effects of cGMP analogues do not appear to be mediated by a secondary increase in cAMP or activation of cAMP-dependent protein kinase (PKA), since [Ca2+]i responses to cGMP analogues were inhibited by the G-kinase inhibitor 8-bromoguanosine-3',5'-cyclic monophosphorothioate (Rp-Br-cGMP[S]). Both Br-cGMP and db-cGMP also increased [Ca2+]i in the presence of the PKA inhibitor 8-bromoadenosine-3',5'-cyclic monophosphorothioate (Rp-Br-cAMP[S]) and when the cGMP-inhibitable cAMP phosphodiesterase activity was inhibited by pretreatment with siguazodan. Br-cGMP stimulated the Mn2+-induced quench of compartmentalized fura-2 in intact hepatocytes, indicating a site of action at the level of the Ca2+ stores. This locus was further supported by the finding that pretreatment of hepatocytes with Br-cGMP potentiated submaximal inositol 1,4,5-trisphosphate (InsP3)-induced Mn2+ quench in subsequently permeabilized hepatocytes. db-cGMP also decreased PKA-mediated back phosphorylation of the hepatic type-1 InsP3 receptor, indicating that G-kinase phosphorylates the InsP3 receptor at sites targeted by PKA. These data indicate that phosphorylation of the hepatic InsP3 receptor by G-kinase increases the sensitivity to InsP3 for [Ca2+]i release and is associated with the production of [Ca2+]i oscillations in single rat hepatocytes.

ABL 364 is a murine monoclonal IgG3 antibody directed against the Lewis Y carbohydrate antigen (Le(y)) expressed on the surface of many epithelial cell tumors. The antibody mediates cytotoxicity via activation of human complement or human effector cells, and has been evaluated in several clinical trials including two Phase I/II trials in relapsed small cell lung cancer and metastatic breast cancer. To improve the effector functions of the antibody, increase its half-life in circulation, and avoid the human antimouse antibody response, two chimeric and several humanized antibodies were constructed for evaluation. The chimeric IgG1 is more potent than the murine IgG3 in tumor cell lysis via activation of human peripheral mononuclear cells (10-fold), but somewhat less effective in complement-dependent lysis (2-3 fold). The chimeric IgG3 is slightly less potent than the IgG1. A humanized IgG1 was constructed by combining the complementarity-determining regions of the ABL 364 antibody with human framework and constant regions. Several additional variants were subsequently constructed to improve the binding affinity and increase expression of the antibody. Two of the variants, designated I and K, differ by a single amino acid at position 75 of the heavy chain. Both variants have affinity within 2-fold of the chimeric IgG1 antibody and retain the cytolytic activities toward tumor cell lines. However, it was possible to express variant K at a significantly higher level (5- 10-fold) than variant I. Pharmacokinetics of the humanized ABL 364 antibody variant K was compared with that of the parent murine antibody in rhesus monkeys. It was shown that the terminal half-life of the humanized antibody in rhesus monkeys is 14-20 days, with a mean of 16.3 days, while that of the parent murine antibody is only 1.9 days.

Polymorphonuclear leukocytes (i.e. neutrophils) significantly mediate damage in myocardial ischemia followed by reperfusion. In the present study, the cardioprotective effects of a humanized form of a monoclonal antibody directed against L-selectin designated monoclonal antibody (mAb) HuDREG-200 were examined in a feline model of 90-min myocardial ischemia followed by 270 min of reperfusion. In preliminary studies, flow cytometric analysis indicated that HuDREG-200 binds to feline neutrophils. In vitro administration of mAb HuDREG-200 significantly inhibited (P < .01) adherence of unstimulated neutrophils to ischemic-reperfused coronary endothelium in a concentration-dependent manner. Humanized DREG-200 (2 mg/kg) administered 10 min before reperfusion significantly attenuated myocardial necrosis compared to an isotype-matched humanized control mAb (HuABL364) which does not bind to L-selectin (14 +/- 3 vs. 29 +/- 3% necrosis/area-at-risk, P < .01), representing a 52% reduction in myocardial necrosis. This myocardial preservation also was related to reduced creatine kinase release and improved recovery of cardiac contractility (i.e. left ventricular dP/dtmax). Moreover, endothelial function, as assessed by relaxation to acetylcholine, also was significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with mAb HuDREG-200 compared to mAb HuABL364 (68 +/- 6 vs. 18 +/- 5, P < .01). Thus, a humanized anti-L-selectin mAb appears to be an effective means of preserving the ischemic myocardium from reperfusion injury and of preserving myocardial contractile function, at least during the early reperfusion period.

We have produced a single plasmid encoding both the heavy chain Fd domain (VH + CH1) of the anti-interleukin-2 receptor (IL2R) monoclonal antibody anti-Tac, and the anti-Tac light chain fused to PE40, a truncated derivative of Pseudomonas exotoxin. The active immunotoxin anti-Tac(Fab)-PE40 could be recovered from E. coli from either periplasm or renatured inclusion bodies. The double-chain immunotoxin was very cytotoxic toward IL2R-bearing cell lines, human activated T cells and fresh adult-T-cell-leukemia cells. The cytotoxicity was similar to that of anti-Tac(Fv)-PE40, the single-chain recombinant toxin containing only the variable domains of anti-Tac. IL2R-binding affinity was also equivalent to that of anti-Tac(Fv)-PE40, which is one-third that of anti-Tac. The serum half-life in mice was significantly prolonged as compared with anti-Tac(Fv)-PE40, with a beta phase of 430 vs. 57 minutes, but the LD50s were equivalent when the immunotoxins were administered in 3 daily doses. Anti-Tac(Fab)-PE40 was very cytotoxic in vitro toward transfected ATAC-4 carcinoma cells which express IL2Rs. In mice bearing ATAC-4 tumors, anti-Tac(Fab)-PE40 showed significant anti-tumor activity, inducing complete remissions in 80 and 100% of treated animals at approximately 7 and 14% respectively of the LD50. Anti-Tac(Fab)-PE40 was much more effective in vitro and in vivo than chemical conjugates between anti-Tac and truncated PE molecules. The recombinant Fab toxin should be studied further as potential treatment for IL2R-related malignancies, particularly if smaller recombinant immunotoxins have insufficient half-life in humans.

Long-term survival rates of patients with acute myelogenous leukemia treated with intensive chemotherapy are 15-20%, despite efforts to develop new treatment strategies. Murine M195 (131I-M195), an anti-CD33, immunoglobulin (Ig) G2a monoclonal antibody has reactivity restricted to early myeloid cells and myeloid leukemic blasts but not hematopoietic progenitors. Previous trials in patients with relapsed or refractory myeloid leukemia showed that 131I-M195 rapidly targeted to the bone marrow and internalized into target cells. This article describes a therapeutic dose escalation study in which 24 patients received from 50 mCi/m2 to 210 mCi/m2 of 131I-M195 in divided doses. Cytoreduction of peripheral cell counts and bone marrow blasts occurred without nonhematopoietic toxicity. Doses of 131I-M195 greater than 135 mCi/m2 were associated with marrow cytoreduction sufficient to necessitate bone marrow transplant. However, 37% of the patients developed human anti-mouse antibody, preventing retreatment. To decrease immunogenicity and improve effector function, chimeric IgG1 and IgG3, and complementarity-determining region-grafted, humanized IgG1 and IgG3 versions of mouse M195 were developed by genetic engineering techniques. The new versions maintained specificity and biologic function, and they were superior to the mouse M195 in their ability to perform antibody-dependent cellular cytotoxicity against leukemia cells. Humanized M195, but not chimeric M195, showed a 4-8.6 times higher avidity than its mouse counterpart. Because effector function of IgG depends to a large extent on Fc clustering, a homodimeric HuG1 also was developed. Homodimeric HuG1 showed an ability to cause additional dramatic improvements in effector functions, as well as an ability to internalize and retain radioisotope in target leukemia cells. Monomeric and dimeric forms of humanized M195 may be advantageous in the therapy of acute myelogenous leukemia.

Nasal and tonsillar samples were collected from 14 free-ranging clinically healthy Rocky Mountain bighorn sheep (Ovis canadensis canadensis) and 10 domestic sheep (Ovis aries). We identified 194 bacterial isolates, including 101 from bighorn and 93 from domestic sheep. Of these isolates, 115 were gram-positive and 79 were gram-negative. Staphylococcus species were the most numerous gram-positive organisms and had a higher incidence in samples from domestic than from bighorn sheep. In contrast Streptococcus species were present in higher numbers in samples from bighorn sheep. Pasteurella haemolytica, the most common gram-negative bacterium, was isolated from five of five tonsillar but from none of ten nasal samples of domestic sheep, and from seven of eight tonsillar and three of ten nasal samples of bighorn sheep. Most bacteria isolated were considered opportunistic pathogens. However, of the bacteria isolated, P. haemolytica, P. multocida, and Actinomyces pyogenes are most frequently associated with respiratory disease.

M195 is a murine monoclonal antibody that binds to the CD33 antigen and is being tested for the treatment of myeloid leukemia. Surprisingly, a complementarity determining region (CDR)-grafted, humanized M195 antibody displayed a several-fold higher binding affinity for the CD33 antigen than the original murine antibody. Here we show that the increase in binding affinity resulted from eliminating an N-linked glycosylation site at residue 73 in the heavy chain variable region in the course of humanization. Re-introducing the glycosylation site in the humanized antibody reduces its binding affinity to that of the murine antibody, while removing the glycosylation site from the murine M195 variable domain increases its affinity. The removal of variable region carbohydrates may provide a method for increasing the affinity of certain monoclonal antibodies with diagnostic and therapeutic potential.

Human-mouse chimeric immunoglobulins G1 and G3 (IgG1 and IgG3) (ChG1, ChG3) and "complementarity-determining region"-grafted, humanized IgG1 and IgG3 (HuG1, HuG3) constructs of the mouse monoclonal antibody (mAb) M195 were characterized. M195 is a murine immunoglobulin G2a (IgG2a), anti-CD33 mAb, specifically reactive with acute myelogenous leukemia cells, that is active as an antileukemia agent in humans. The new mAb constructs maintained specificity and biological function, including rapid internalization after binding to the cell surface, which has been important for delivery of therapeutic isotopes in patients. Although previously reported complementarity-determining region-grafted mAbs had reduced avidities, the HuG1 and HuG3 M195 showed up to an 8.6- and 4-fold higher binding avidity, respectively, than the original murine mAb. All constructs were effective at mediating rabbit complement-mediated cytotoxicity against HL60 targets. Fibroblasts transfected with CD33 genes and expressing high levels of CD33 antigen were also lysed in the presence of human complement, but HL60 cells or fibroblasts with lower CD33 levels were not killed. Thus, the inability of M195 and constructs to kill HL60 targets with human complement is due to the much lower antigen density on HL60 cells compared to CD33+ fibroblasts. Unlike the murine M195, the chimeric and humanized M195 demonstrated antibody-dependent cell-mediated cytotoxicity using human peripheral blood mononuclear cells as effectors. Because the chimeric and humanized M195 have improved avidities as compared to the original M195 and have, in addition, the potential to avoid human anti-mouse antibody responses and to recruit human effector functions, these new constructs may be useful therapeutically, either alone or conjugated to toxins or isotopes, in the treatment of acute myelogenous leukemia.

Mik-beta 1 is a mAb that binds to the beta subunit of the IL-2R. We have constructed a recombinant single chain immunotoxin Mik-beta 1(Fv)-PE40 by genetically fusing the H and L V domains of Mik-beta 1 to each other via a peptide linker, and then to PE40, a derivative of Pseudomonas exotoxin. Mik-beta 1(Fv)-PE40 was selectively cytotoxic for cells expressing high levels of IL-2R beta (p75) subunit. Mik-beta 1(Fv)-PE40 was cytotoxic to the NK cell line YT-S, which expresses p75 but not p55 subunits, with an IC50 of 6 ng/ml. The ATL line HUT-102 was less sensitive, with an IC50 of 200 ng/ml. However, the IC50 could be lowered to 11 ng/ml when Mik-beta 1(Fv)-PE40 was allowed to bind to HUT-102 cells at 4 degrees C for 4 h before overnight incubation at 37 degrees C. An excess of Mik-beta 1 but not of anti-Tac, the anti-p55 mAb, prevented the cytotoxicity of Mik-beta 1(Fv)-PE40. We constructed a more active version of Mik-beta 1(Fv)-PE40, designated Mik-beta 1(Fv)-PE40KDEL, by converting the carboxyl-terminus of the toxin from -REDLK to -KDEL. Mik-beta 1(Fv)-PE40KDEL showed an IC50 of 2 ng/ml toward YT-S cells and 35 ng/ml toward HUT-102 cells. Binding studies using radioiodinated Mik-beta 1 showed that Mik-beta 1(Fv)-PE40 bound to the p75 receptor subunit with 11% of the affinity of the native Mik-beta 1 antibody. Mik-beta 1(Fv)-PE40 may be a useful reagent to study cells that express IL-2R, and it deserves further study as a possible treatment for cancers in which the malignant cells express high numbers of p75 subunit.

Humanized IgG1 M195 (HuG1-M195), a complementarity determining region-grafted recombinant monoclonal antibody, is reactive with CD33, an antigen expressed on myelogenous leukemia cells. M195 is in use in trials for the therapy of acute myelogenous leukemia. Since biological activity of IgG may depend, in part, on multimeric Fab and Fc clustering, homodimeric forms of HuG1-M195 were constructed by introducing a mutation in the gamma 1 chain CH3 region gene to change a serine to a cysteine, allowing interchain disulfide bond formation at the COOH terminal of the IgG. Despite similar avidity, the homodimeric IgG showed a dramatic improvement in the ability to internalize and retain radioisotope in target leukemia cells. Moreover, homodimers were 100-fold more potent at complement-mediated leukemia cell killing and antibody-dependent cellular cytotoxicity using human effectors. Therefore, genetically engineered multimeric constructs of IgG may have advantages relative to those forms that are found naturally.

L and H chain cDNAs of M195, a murine mAb that binds to the CD33 Ag on normal and leukemic myeloid cells, were cloned. The cDNAs were used in the construction of mouse/human IgG1 and IgG3 chimeric antibodies. In addition, humanized antibodies were constructed which combined the complementarity-determining regions of the M195 antibody with human framework and constant regions. The human framework was chosen to maximize homology with the M195 V domain sequence. Moreover, a computer model of M195 was used to identify several framework amino acids that are likely to interact with the complementarity-determining regions, and these residues were also retained in the humanized antibodies. Unexpectedly, the humanized IgG1 and IgG3 M195 antibodies, which have reshaped V regions, have higher apparent binding affinity for the CD33 Ag than the chimeric or mouse antibodies.

We describe a method for expressing an antibody Fab fragment on the surface of M13 filamentous bacteriophage. The L chain gene, preceded by an Escherichia coli signal sequence, is linked to the 5' end of the gene for the M13 major coat protein. A partial H chain gene, preceded by an E. coli signal sequence and truncated at the end of the CH1 region, is inserted on a plasmid adjacent to the fused L chain gene, so both genes are under the transcriptional control of an inducible promoter. The plasmid also contains the M13 origin of replication. When the promoter is induced, functional antibody Fab fragment appears on the surface of the E. coli inner membrane, as shown by specific binding to an Ag-affinity matrix. When the E. coli are further infected with a helper phage, allowing replication and packaging of the plasmid into phage particles, the resulting phage specifically bind to an Ag-coated plate, indicating they have antibody Fab fragment on their surface. The antibody-expressing phage can also be specifically bound to and eluted from an Ag-affinity column. These observations support the possibility of a new way of generating antibodies, in which amplified Ig cDNA from an appropriate B cell population is cloned into a suitable M13 vector, and phage containing the genes for desired antibodies are selected directly by binding to an Ag-affinity matrix.

Protein engineering has made it possible to combine the binding sites of murine antibodies with human antibody regions. Antibodies constructed in this way have important advantages for therapy.