A platform for phenotypic discovery of therapeutic antibodies and targets applied on Chronic Lymphocytic Leukemia.

Development of antibody drugs against novel targets and pathways offers great opportunities to improve current cancer treatment. We here describe a phenotypic discovery platform enabling efficient identification of therapeutic antibody-target combinations. The platform utilizes primary patient cells throughout the discovery process and includes methods for differential phage display cell panning, high-throughput cell-based specificity screening, phenotypic in vitro screening, target deconvolution, and confirmatory in vivo screening. In this study the platform was applied on cancer cells from patients with Chronic Lymphocytic Leukemia resulting in discovery of antibodies with improved cytotoxicity in vitro compared to the standard of care, the CD20-specific monoclonal antibody rituximab. Isolated antibodies were found to target six different receptors on Chronic Lymphocytic Leukemia cells; CD21, CD23, CD32, CD72, CD200, and HLA-DR of which CD32, CD200, and HLA-DR appeared as the most potent targets for antibody-based cytotoxicity treatment. Enhanced antibody efficacy was confirmed in vivo using a patient-derived xenograft model.

In-depth neuropharmacokinetic analysis of antipsychotics based on a novel approach to estimate unbound target-site concentration in CNS regions: link to spatial receptor occupancy.

The current study provides a novel in-depth assessment of the extent of antipsychotic drugs transport across the blood-brain barrier (BBB) into various brain regions, as well as across the blood-spinal cord barrier (BSCB) and the blood-cerebrospinal fluid barrier (BCSFB). This is combined with an estimation of cellular barrier transport and a systematic evaluation of nonspecific brain tissue binding. The study is based on the new Combinatory Mapping Approach (CMA), here further developed for the assessment of unbound drug neuropharmacokinetics in regions of interest (ROI), referred as CMA-ROI. We show that differences exist between regions in both BBB transport and in brain tissue binding. The most dramatic spatial differences in BBB transport were found for the P-glycoprotein substrates risperidone (5.4-fold) and paliperidone (4-fold). A higher level of transporter-mediated protection was observed in the cerebellum compared with other brain regions with a more pronounced efflux for quetiapine, risperidone and paliperidone. The highest BBB penetration was documented in the frontal cortex, striatum and hippocampus (haloperidol, olanzapine), indicating potential influx mechanisms. BSCB transport was in general characterized by more efficient efflux compared with the brain regions. Regional tissue binding was significantly different for haloperidol, clozapine, risperidone and quetiapine (maximally 1.9-fold). Spatial differences in local unbound concentrations were found to significantly influence cortical 5-HT2A receptor occupancy for risperidone and olanzapine. In conclusion, the observed regional differences in BBB penetration may potentially be important factors contributing to variations in therapeutic effect and side effect profiles among antipsychotic drugs.

The impact of the site of blood sampling on pharmacokinetic parameters following sublingual dosing to dogs.

INTRODUCTION: Drugs are most commonly administered orally, but some potential drug candidates are not suited for oral administration due to poor absorption, high first pass metabolism or gastrointestinal side effects. The interest for transmucosal dosing for systemic drug delivery is increasing, e.g. buccal, sublingual and nasal routes. The evaluation of the systemic plasma concentration and the derivation of the pharmacokinetic parameters of candidate compounds in preclinical studies are essential for drug development. The effect of site of blood sampling on the measured drug concentration, in both animals and humans, is to some extent known but it is not always taken into consideration in the design of pharmacological and toxicological studies. METHODS: Blood samples were collected both from leg and jugular veins from beagle dogs following a single sublingual dosing of Compound A in order to determine the impact of different sites of blood sampling on plasma pharmacokinetics. Plasma was prepared by centrifugation and plasma concentrations of Compound A were determined by protein precipitation and liquid chromatography followed by mass spectrometric detection. The pharmacokinetic parameters were calculated by non-compartment methods. RESULTS: Sampling from the jugular vein resulted in higher and more variable exposure during the absorption phase compared to sampling from a leg vein. The plasma exposure in the jugular vein, in terms of C(max), was 4-fold compared to that in the leg vein and an approximately 2-fold bioavailability was observed. DISCUSSION: The aim of this investigation was to determine the impact of the different sites of blood sampling on assessing systemic plasma exposure and pharmacokinetic parameters for Compound A following sublingual dosing to dogs. The results demonstrate the significant impact that the site of blood sampling has on PK parameters, and raise concerns of using the jugular vein as a site of sampling after sublingual and other transmucosal routes of dosing in the head region.

Evaluation of a community-based drowning prevention programme in northern Islamic Republic of Iran.

This study evaluated the feasibility of a drowning intervention package in northern Islamic Republic of Iran. A quasi-experimental design used pre- and post-observations among residents and tourists in water-recreation beach areas of intervention and control regions by the Caspian Sea and in residents near the Caspian Sea coastline. The fatal drowning rate in the studied resident population in the provinces fell from 4.24 per 100 000 residents at baseline to 3.04 per 100,000 residents at endline. The risk of death from drowning in the intervention areas in the water-recreation area was greater during the pre-intervention (OR = 1.15, 95% CI: 0.66-2.01) than the implementation period (OR = 0.24, 95% CI: 0.15-0.37). The risk of drowning can be reduced by implementing increased supervision and raising community awareness.

Evaluation of a community-based drowning prevention programme in northern Islamic Republic of Iran

This study evaluated the feasibility of a drowning intervention package in northern Islamic Republic of Iran. A quasi-experimental design used pre- and post-observations among residents and tourists in water-recreation beach areas of intervention and control regions by the Caspian Sea and in residents near the Caspian Sea coastline. The fatal drowning rate in the studied resident population in the provinces fell from 4.24 per 100 000 residents at baseline to 3.04 per 100 000 residents at endline. The risk of death from drowning in the intervention areas in the water-recreation area was greater during the pre-intervention [OR = 1.15, 95% CI: 0.66-2.01] than the implementation period [OR = 0.24, 95% CI: 0.15-0.37]. The risk of drowning can be reduced by implementing increased supervision and raising community awareness

The origin of violent behaviour among child labourers in India.

We explored the causes and circumstances of violent behaviour among a group of child labourers in the Indian unorganized sectors. From 14 categories of occupations, a total of 1,400 child labourers were interviewed in both urban and rural areas. The average family size of these mostly illiterate child labourers is seven, and average family income is 3,200 INR per month. In the short term child labourers become violent, aggressive, and criminal, following a pyramid of violent behaviour, including socio-economic pressure, cultural deviance, and psychological pressure. When considering family history it seems that the problem is part of a vicious cycle of violence, which persists through generations and evolves with financial crisis, early marriage, and violence in the family and workplace. Our study demonstrates that the most vulnerable groups of child labourers belong to the following workplaces: dhabas, food stalls, rail/bus stations, rail-floor cleaning, and rag picking. Giving high priority to capacity building within the community, including support for locally-generated solutions, is warranted.

In vitro and in vivo effects of potassium and magnesium on storage up to 7 days of apheresis platelet concentrates in platelet additive solution.

BACKGROUND AND OBJECTIVE: Prolonged storage of platelets up to 7 days provides improved availability, logistical management and decreased wastage. Beside methods of bacterial detection, addition of magnesium and potassium to the platelet storage solution (SSP+) may further improve the quality of platelets with extended storage. MATERIALS AND METHODS: Apheresis platelets from 10 donors were divided and stored in two different platelet additive solutions (PAS) (Intersol and SSP+) for a paired comparison. A variety of in vitro platelet function and metabolic assays were performed both on day 1 and after 7 days of storage. For in vivo study, platelets were labelled with either (111)Indium or (51)Chromium after 7 days of storage and were injected into the corresponding donor. Serial blood samples were drawn for recovery and survival measurements. RESULTS: In vitro parameters for SSP+ showed significantly reduced glycolysis (lower glucose consumption and decreased production of lactate), a higher hypotonic shock response (HSR) and the extent of shape change reactivity and a lower degree of platelet activation by means of RANTES (regulated on activation, normal, T cell-expressed, and secreted), CD62p and CD63 expression. Platelet recovery on day 7 was higher for Intersol as compared to SSP+, 65 +/- 11 vs. 53 +/- 13% (P = 0.023), and survival showed no difference 4.2 +/- 1.9 vs. 3.6 +/- 1.4 days. CONCLUSION: In vitro characteristics of platelets stored in PAS with addition of potassium and magnesium indicated higher quality, but this could not be verified by the in vivo parameters by means of recovery and survival.

Thomsen-Friedenreich oncofetal antigen in Schistosoma mansoni : localization and immunogenicity in experimental mouse infection.

Our preliminary observation, that sera from schistosomiasis patients react with carcinomas, raised the possibility of antigenic cross-reactivity. We here extend this observation to show that mice experimentally infected with Schistosoma mansoni react with human urothelial and transitional bladder carcinomas and also with a gastric carcinoma cell line, AGS. To identify cross-reacting epitopes, we looked for the expression of carcinoma markers in schistosome worms and eggs using monoclonal antibodies against tumour antigens MUC1, Tn and TF (also known as the oncofetal Thomsen-Friedenreich antigen or T antigen). Immunohistochemical staining showed that the TF-epitope is present in adult intravascular S. mansoni worms and eggs deposited in tissues of infected animals. The localization of TF-immuno-reactive material in schistosomes was seen at the parasite surface between male and female worms and around trapped eggs in the liver. This localization is consistent with the antigen being secreted. Mice experimentally infected with S. mansoni, developed circulating antibodies against the TF-epitope (identified as Gal(beta1-3) GalNAc-O-R) as seen in ELISA using TF-expressing asialoglycophorin (AGP) as antigen. The observed anti-TF response in S. mansoni-infected mice reflects the complexity of host-parasite interactions in this infection.

Polybrominated diphenyl ethers (PBDEs) and their methoxylated derivatives in pike from Swedish waters with emphasis on temporal trends, 1967-2000.

Temporal trends of five tetra- to hexabromodiphenyl ethers [BDE47, BDE99, BDE100, BDE153 and BDE154) and two methoxy-tetraBDEs [6-methoxy-2,2',4,4'- tetraBDE (6-MeO-BDE47) and 2'-methoxy-2,3',4,5'- tetraBDE (2'-MeO-BDE68)] in pike from Lake Bolmen for the years 1967-2000, are presented. All BDE congeners show increasing trends up to the mid-1980s (Sigma5PBDE from 60 to 1600 pg/g wet weight in 1989, i.e. a more than 25-fold increase), and then decrease or level off. The decreasing trends of PBDEs after the 1980s were considerably slower in the present study than was found in a study of an environmental matrix from the Baltic Proper covering the same time period. This difference suggests local sources near Lake Bolmen. The MeO-BDEs show initially decreasing concentrations, which for 6-MeO-BDE47 continues until the early 1990s. The concentrations of 6-MeO-BDE47 in herring from five locations along the Swedish coast increased from south to north in the Baltic Sea. No correlation between the concentrations of the BDE congeners and the MeO-BDEs was observed, indicating sources other than PBDEs for these compounds. The presence of MeO-BDEs in fish from lakes with different characteristics suggests a natural production not favoured by eutrophication, or dependent on sampling season and geographical location.

Methods for atom probe analysis of microgradients in functionally graded cemented carbides.

Methods to prepare needle-shaped specimens for atom probe field ion microscopy from near surface regions have been developed. The material used was a cemented carbide with a composition gradient towards the surface, but the method is equally applicable for other materials. The preparation technique involves dimple grinding, electropolishing and focused ion beam (FIB) milling. The use of FIB milling allows for specimen preparation of materials which due to the preferential etching of different phases are difficult to electropolish. The technique also allows for preparation of specimens at well defined depth from the sample surface, selection of phase to be analysed, and to sharpen and re-use already analysed specimens. Atom probe analyses of the near surface zone region in a gradient sintered WC-Ti(C,N)-TaC-Co cemented carbide are presented.

Visualization of in vivo olfactory uptake and transfer using fluorescein dextran.

Nasal administration of a 3 kDa fluorescein dextran (FD3) solution to rats resulted in transcellular absorption across the olfactory epithelium and transfer to the olfactory bulb within 15 min. After entering the lamina propria, FD3 was transferred in the connective tissue surrounding the olfactory nerve bundles to the olfactory bulb of the brain. More FD3 was absorbed across the olfactory epithelium than across the respiratory epithelium and to the nasal associated lymphoid tissue. Further, the amount of FD3 crossing the olfactory epithelium was region-dependent, with higher amounts absorbed in the turbinates than in the nasal septum. Plastic embedding and sectioning followed by fluorescence microscopy, enabled simultaneous visualization of FD3 in the mucosa and olfactory bulb, as well as the opportunity to store the tissue blocks for a prolonged period of time.

Epilepsy and injury mortality in Sweden--the importance of changes in coding practice.

We analysed national mortality trends of injuries and poisoning in patients with epilepsy to establish the importance of changes in coding practice. Patients where epilepsy was a syndrome, either as an underlying or contributing cause-of-death at any time of life, throughout Sweden during 1975-1995 were included. All conditions mentioned on each death certificate were examined, and epilepsy and injury diagnoses were separated according to underlying, contributing or multiple cause-of-death. Annual mortality rates, case-fatality rates, and linear regression coefficients were computed. The most common underlying cause-of-death (UCD) was diseases of the circulatory system (34.4%) followed by epilepsy (31.7%). Injury and poisoning were coded as UCDs for 5.8% of the patients. Injury and poisoning as a contributory cause-of-death increased significantly, but not as an underlying cause during the study period. The proportion of each diagnostic group as an underlying cause decreased by 35% for epilepsy and 18% for injury from 1981 and onwards. These changes should be interpreted with caution since changes of coding practice are more likely to be the cause of these apparent changes. The most frequent specific external causes of injury deaths in epileptic patients were falls and drowning. Intracranial fatal injuries in connection with fall accidents predominated, especially among elderly persons. Deaths related to accidental poisoning were caused by the toxic effect of non-medicinal substances. Cause-of-death statistics from different countries must be interpreted with caution when making international comparisons. Both underlying and contributing cause-of-death statistics should be considered in analyses of mortality trends.

A prospective study of fatal occupational accidents -- relationship to sleeping difficulties and occupational factors.

Very little is known about the association between sleep and (fatal) occupational accidents. This study investigated this relationship using register data of self-rated sleep difficulties, together with occupational and demographic characteristics. The variables were related to subsequent occupational fatal accidents. A national sample of 47,860 individuals was selected at regular intervals over a period of 20 years, and interviewed over the phone on issues related to work and health. The responses were linked to the cause of death register (suicides excluded) and the data set was subjected to a (multivariate) Cox regression survival analysis. One hundred and sixty six fatal occupational accidents occurred, and the significant predictors were: male vs. female: relative risk (RR)=2.30 with a 95% confidence interval (CI) of 1.56-3.38; difficulties in sleeping (past 2 weeks): RR=1.89 with CI=1.22-2.94; and non-day work: RR=1.63 with CI=1.09-2.45. No significant effect was seen for age, socio-economic group, hectic work, overtime (>50 h per week), or physically strenuous work. It was concluded that self-reported disturbed sleep is a predictor of accidental death at work, in addition to non-day work and male gender.

Work-related acute injuries from mandatory fitness training in the Swedish Police Force.

Acute injuries in the Swedish Police Force from on-duty fitness training were selected retrospectively from the Information System of Occupational Injuries (ISA) at the National Board of Occupational Safety and Health and, if having caused a sick-leave exceeding 2 weeks, to the Labour Market Insurance (AMF Insurance). The latter included injuries from 1995 only. During the seven-year period 1992 to 1998, 920 injuries (80 % in males) from fitness training involving police officers were reported to the ISA-register. The total incidence was 1.6 for policemen per 10 000 hours of exposure and 2.2 for policewomen, which is 1.4 times higher than in men. Around 50 % of the injuries occurred in team and contact sports, with a slightly higher percentage for males, 54 % versus 49 % in females. The percentage of injuries from self-defense training was twice as high as in women than in men, 29 % versus 15 %. In 1995, 42 of the 72 injuries in males and 6 of the 21 injuries in females caused more than 14 days of sick-leave and were announced to the Occupational No Fault Liability Insurance. The major part, 32 of 48 injuries, came from team or contact sports (mainly floorball and soccer). Six policemen incurred injuries that were classified with a degree of disability ranging from 2 to 5 %. The total cost for medical treatment and production loss for the 48 injuries was Euro 248 448 and 99 336, respectively. Team and contact sports accounted for 89 % of the costs and 77 % of the production loss through sick-leave.

Epitopes on CA 125 from cervical mucus and ascites fluid and characterization of six new antibodies. Third report from the ISOBM TD-1 workshop.

CA 125 is found in body fluids in a variety of molecular weight forms. The largest species are found in normal abdominal fluid and cervical mucus. The present study therefore incorporated CA 125 derived from these sources as well as ascites fluid to investigate if the source of CA 125 influenced epitope characterization. Ascites-derived CA 125 varied in size from about 190 to about 2,700 kD. Cervical mucus-derived CA 125 treated with ultrasound changed its apparent size from more than 20,000 to 700 kD. Epitope mapping of antibodies was not grossly influenced by the size or source of CA 125 used as target. However, low-molecular-weight CA 125, i.e. ascites fractions CA 17/E, CA 17/F and CA 10/7, did show differences in certain assay combinations and cross-inhibition patterns which probably can be explained by steric effects due to the smaller size compared with the most abundant forms of CA 125 present in serum and other body fluids. The specificity of six new monoclonal antibodies to CA 125 was tested by cross-inhibition and immunometric assay combinations and compared to reference antibodies. One antibody, X306, belonged to the OC125-like antibodies. Four antibodies, X52, X75, X325 and VK8, were M11-like. The sixth antibody, 7C12, reacted with an epitope which was difficult to define. This antibody was inhibited by M11-like antibodies and OV197. However, used as an inhibitor, 7C12 inhibited only itself. We grouped it as an OV197-like antibody, but clearly different from OV197. The topography of epitopes was studied by analyzing all antibody pairs in immunoradiometric assays. These results confirmed the grouping of antibodies described above and are in accordance with previous findings that the highest signal is obtained using an OC125-like antibody or OV197 on the solid phase and an M11-like antibody as tracer. The composition of the sample in terms of high- and low-molecular-weight species of CA 125 was measured, with different responses depending on the antibody pair used. This might be one reason for discrepancies between assay results for CA 125 using different assays.

Tanning habits and sunburn in a Swedish population age 13-50 years.

As part of a skin cancer control programme, we studied the occurrence of self-reported outdoor tanning, sunbed use and ultraviolet (UV)-induced erythema in an urban area. A cross-sectional questionnaire study of 6000 adolescents aged 13-19 years, and 4000 adults aged 20-50 years was applied. Non-response was analysed for outdoor tanning and sunbed use. Results, in general, did not differ between responders and non-responders. Females aged 17-29 years tanned outdoors most frequently. Sunbed use and related erythema was twice as common in young females. In males, outdoor tanning was not age-related. In the past 12 months, 55% reported sunburn and/or burn from sunbed use, one-third were burned in Sweden, one-quarter on sunny resorts abroad. Sunburn occurs frequently. Compliance with recommendations for sunbed use is poor, especially among adolescents and young adults. To reduce the occurrence of erythema, the influence of risk settings upon behaviours is a critical issue for exploration.

Autologous chondrocytes used for articular cartilage repair: an update.

Articular cartilage in adults has a poor ability to self-repair after a substantial injury; however, it is not known whether there is a cartilage resurfacing technique superior to the existing techniques. It is not satisfactory that at the beginning of the new millennium, there still is a lack of randomized studies comparing different cartilage repair techniques and there still is little knowledge of the natural course of a cartilaginous lesion. To date, various articular cartilage resurfacing techniques have the potential to improve the repair of cartilage defects and reduce the patient's disability. One such cartilage repair technique is autologous chondrocyte transplantation combined with a periosteal graft. Since the first patient was operated on in 1987, much interest in cartilage repair and cell engineering has emerged. The experience with autologous chondrocyte transplantation during the past 13 years with in vitro chondrocyte expansion, cartilage harvest, and postoperative biopsy technique is discussed, and the latest followup of 213 consecutive patients in different subgroups with 2 to 10 years followup is presented. The technique gives stable long-term results with a high percentage of good to excellent results (84%-90%) in patients with different types of single femoral condyle lesions, whereas patients with other types of lesions have a lower degree of success (mean, 74%).

Work-related injuries from mandatory fitness training among Swedish firemen.

A considerable amount of employees incur injuries in professions with mandatory on-duty fitness training. The training is necessary for maintaining a good health status, physical condition and strength in professions requiring such qualities. Injuries in Swedish firemen from on-duty fitness training between 1992 and 1998 were selected retrospectively from the Information System of Occupational Injuries (ISA) at the National Board of Occupational Safety and Health and, if having caused a sick-leave exceeding 2 weeks, to the Labour Market Insurance (AMF Insurance). The latter comprised injuries from 1995 only. During the seven-year period 1,468 injuries from fitness training occurred in male firemen. With an estimated 147 h per man and year the injury incidence was 2.6 per 10,000 h of exposure. The mean number of days of absence from work was 24.1 days (SD 39.8). In 1995, the mean cost per injury for medical treatment was close to 7,000 Euro and for production loss almost 4,500 Euro. By far the major part of the injuries, 75 %, occurred during team and contact sports (primarily floorball and soccer) and this sport category also accounted for 83% of the medical costs and 78% of the costs for production loss. It is suggested that a restriction of team and contact sports as mandatory on-duty fitness training for firemen should be tested and evaluated concerning the injury incidence.

Correlative fine specificity of several Thomsen-Friedenreich disaccharide-binding proteins with an effect on tumor cell proliferation.

Epithelial cancer cells show increased cell surface expression of mucin antigens with aberrant O-glycosylation, notably type I core (Galbeta1-3GalNAcalpha), termed Thomsen-Friedenreich disaccharide (TFD), a chemically well-defined carbohydrate antigen with a proven link to malignancy. Several TFD-binding proteins influence the proliferation of cells to which they bind. We studied the fine specificity of TFD-binding proteins and its relationship with epithelial tumor cell proliferation. Competitive binding assays against asialoglycophorin showed that Agaricus bisporus lectin (ABL) and human anti-TFD monoclonal antibody (mAb) TF1 were inhibited only by TFD and its alpha-derivatives. Peanut agglutinin (PNA), mAb TF2, and mAb TF5 were also inhibited by other carbohydrates such as lacto-N-biose (Galbeta1-3GlcNAc), lactose, and (Mealpha or beta) Gal, indicating lower recognition of the axial C-4 hydroxyl group position of GalNAc from TFD, and the major relevance of the terminal Gal on interaction of these three TFD-binding proteins. In the direct glycolipid-binding assay, ABL bound mostly to alpha-anomeric TFD-bearing glycolipids, whereas PNA interacted mainly with beta-linked TFD. Of the three anti-TFD mAbs analyzed, all bound N5b (terminal beta-TFD), but only TF2 interacted with N6 (terminal alpha-TFD). These findings indicate that TFD-binding proteins that stimulate the proliferation of epithelial tumor cell lines recognize mainly a terminal beta-Gal region of beta-linked TFD, whereas ABL, which inhibits the proliferation of these tumor cells, binds mainly to subterminal GalNAc of alpha-anomeric TFD.

Levels of dopamine in blood and brain following nasal administration to rats.

The aim of this study was to investigate the levels of [(3)H]dopamine in blood, the cerebrospinal fluid (CSF) and brain tissue samples in rats and to find out whether the drug is transferred along the olfactory pathway to the central nervous system following nasal administration. [(3)H]Dopamine (50 microCi) was given to male Sprague-Dawley rats either intravenously or nasally to the right nostril. For the absorption study, blood samples were withdrawn from the carotid artery. The CSF samples were taken by cisternal puncture and then brain tissue samples were excised. The presence of unchanged dopamine in the samples was ascertained using thin layer chromatography (TLC). The radioactivity in the samples was measured using liquid scintillation. The greatest amount of the total radioactivity absorbed from the nasal mucosa into the systemic circulation was observed at the first sampling point 15 min after administration. The bioavailability of the total radioactivity was 68+/-30%. The uptake of [(3)H]dopamine in the brain was significantly higher 30 min after nasal administration than after intravenous administration (P<0.01). TLC data showed that approximately 59%, 14% and 68% of the radioactivity in the olfactory bulb, CSF and olfactory mucosa, respectively, coeluted with dopamine. In conclusion, these results show that unchanged dopamine is transferred into the olfactory bulb via the olfactory pathway in rats.