Combined flow cytometry and confocal laser scanning microscopy for evaluation of BR96 antibody cancer cell targeting and internalization.

BACKGROUND: Monoclonal antibodies (mAb) are important tools in the management of tumor disease, and the discovery of antibodies with both specific cancer cell targeting and capacity to enter the cells by internalization are critical to improve the therapeutic efficacy. METHOD: Antibody cancer cell targeting and internalization properties of fluoroscein-conjugated mAb made against Lewis Y (BR96) were evaluated quantitatively and qualitatively by means of flow cytometry (FCM) and confocal laser scanning microscopy (CLSM), respectively, on cells from a rat tumor cell line (BN7005-H1D2). RESULTS: The study demonstrated a specific binding of BR96 to LewisY (LeY) located in the cell membrane and as BR96/LeY immunocomplexes (BR96/LeY) internalized into the cytoplasm. BR96/LeY was internalized into about 15% of the cells, usually distributed throughout the cytoplasm, but also located close to the nuclei. Cytotoxic effects by BR96 were indicated, and CLSM visualized subpopulations containing cells with bound or internalized BR96/LeY that possessed morphologically pyknotic nuclei and disrupted DNA. CONCLUSION: The spatial-temporal pattern by BR96 cell targeting and internalization processes of BR96/LeY into the cancer cells expressing LeY was demonstrated by FCM and CLSM. Used together, the FCM and CLSM techniques provide a valuable tool for preclinical analyses of antibody targeting and their capacities as carriers of cytotoxic conjugates for the use in cancer therapy.     

Specific mediator inhibition by the NO donors SNP and NCX 2057 in the peripheral lung: implications for allergen-induced bronchoconstriction

Background

The aim of this study was to examine potential therapeutic effect of the two NO donors NCX 2057 (3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid) 4-(nitrooxy)butyl ester) and SNP (sodium nitroprusside) on the early allergic airway response in the peripheral lung.

Methods

The experiments were performed in guinea pig lung parenchyma (GPLP) derived from ovalbumin (OVA) sensitized guinea pigs. The effects of NCX 2057 and SNP were evaluated by contractile responses and mediator release during OVA challenge. The generation of nitrite and nitrate was assessed by chemiluminescence. Statistical analysis was evaluated by ANOVA.

Results

Cumulatively increasing concentrations of OVA (1–10,000 ng/ml) induced concentration-dependent contractions of the GPLP that were reduced by NCX 2057 (100 μM, p < 0.001) and SNP (100 μM, p < 0.05). Antigen-induced eicosanoid release was decreased by NCX 2057 (100 μM, p < 0.001) but not by SNP (100 μM), whereas the release of histamine was reduced by SNP (100 μM, p < 0.001) but not by NCX 2057 (100 μM). In addition, NCX 2057 (0.1–100 μM), but not SNP (0.1–100 μM), relaxed leukotriene D₄ (10 nM) precontracted GPLP (p < 0.01). The guanylyl cyclase inhibitor ODQ had no effect on the NCX 2057 mediated relaxation. SNP released significantly less nitrite than NCX 2057.

Conclusion

Although both SNP and NCX 2057 reduced the release of pro-inflammatory mediators, their profiles were distinctly different. Furthermore, NCX 2057 also induced smooth muscle dilation in the GPLP. The findings point to specific anti-inflammatory effects of different NO donors in the peripheral lung tissue.     

The occurrence of refractoriness and mast cell mediator release following mannitol-induced bronchoconstriction

For several hours after exercise-induced bronchoconstriction, there is diminished responsiveness to repeated challenge. The mechanism causing this refractoriness is unclear. Inhalation of dry powder mannitol is a new bronchial provocation test that has been suggested as a surrogate for an exercise challenge. Refractoriness to repeated mannitol challenge has however not been established. Our objective was to investigate if repeated challenge with mannitol is associated with refractoriness and diminished release of mast cell mediators of bronchoconstriction. Sixteen subjects with asthma underwent repeated inhalation of mannitol 90 min apart. Lung function was assessed by forced expiratory volume in 1 s (FEV?). The urinary excretion (ng/mmol creatinine) of the mediators 9α,11β-prostaglandin (PG) F? and leukotriene (LT) E? were measured. The group mean fall in FEV? after the second challenge was 48.5 ± 5.8% of the first (P < 0.001). The protection afforded by the initial challenge, however, varied considerably between subjects (range 88-0%). Furthermore, the urinary excretion of the two mediators was increased after both challenges. The average excretion of mediators after the challenges was significantly higher for the six most refractory subjects. This was observed both for LTE? (95.6 ± 5.2 vs. 58.0 ± 2.4 for the 6 least refractory) (P < 0.001) and for 9α,11β-PGF? (137.6 ± 6.7 vs. 50.1 ± 1.1 for the 6 least refractory) (P = 0.002). As occurs with exercise-induced bronchoconstriction, repeated inhalation of mannitol induced refractoriness. We propose that refractoriness is due to tachyphylaxis at the level of the airway smooth muscle responsiveness to mediators of bronchoconstriction rather than due to fatigue of their release from mast cells.     

Increased prevalence of symptoms of rhinitis but not of asthma between 1990 and 2008 in Swedish adults: comparisons of the ECRHS and GA²LEN surveys

Background

The increase in asthma prevalence until 1990 has been well described. Thereafter, time trends are poorly known, due to the low number of high quality studies. The preferred method for studying time trends in prevalence is repeated surveys of similar populations. This study aimed to compare the prevalence of asthma symptoms and their major determinants, rhinitis and smoking, in Swedish young adults in 1990 and 2008.

Methods

In 1990 the European Community Respiratory Health Survey (ECRHS) studied respiratory symptoms, asthma, rhinitis and smoking in a population-based sample (86% participation) in Sweden. In 2008 the same symptom questions were included in the Global Allergy and Asthma European Network (GA²LEN) survey (60% participation). Smoking questions were however differently worded. The regions (Gothenburg, Uppsala, Umeå) and age interval (20–44 years) surveyed both in 1990 (n = 8,982) and 2008 (n = 9,156) were analysed.

Results

The prevalence of any wheeze last 12 months decreased from 20% to 16% (p<0.001), and the prevalence of “asthma-related symptoms” was unchanged at 7%. However, either having asthma attacks or using asthma medications increased from 6% to 8% (p<0.001), and their major risk factor, rhinitis, increased from 22% to 31%. Past and present smoking decreased.

Conclusion

From 1990 to 2008 the prevalence of obstructive airway symptoms common in asthma did not increase in Swedish young adults. This supports the few available international findings suggesting the previous upward trend in asthma has recently reached a plateau. The fact that wheeze did not increase despite the significant increment in rhinitis, may at least in part be due to the decrease in smoking.     

Extracellular carbonic anhydrase mediates hemorrhagic retinal and cerebral vascular permeability through prekallikrein activation

Excessive retinal vascular permeability contributes to the pathogenesis of proliferative diabetic retinopathy and diabetic macular edema, leading causes of vision loss in working-age adults. Using mass spectroscopy-based proteomics, we detected 117 proteins in human vitreous and elevated levels of extracellular carbonic anhydrase-I (CA-I) in vitreous from individuals with diabetic retinopathy, suggesting that retinal hemorrhage and erythrocyte lysis contribute to the diabetic vitreous proteome. Intravitreous injection of CA-I in rats increased retinal vessel leakage and caused intraretinal edema. CA-I-induced alkalinization of vitreous increased kallikrein activity and its generation of factor XIIa, revealing a new pathway for contact system activation. CA-I-induced retinal edema was decreased by complement 1 inhibitor, neutralizing antibody to prekallikrein and bradykinin receptor antagonism. Subdural infusion of CA-I in rats induced cerebral vascular permeability, suggesting that extracellular CA-I could have broad relevance to neurovascular edema. Inhibition of extracellular CA-I and kallikrein-mediated innate inflammation could provide new therapeutic opportunities for the treatment of hemorrhage-induced retinal and cerebral edema.     

Laser-Supported Dual Energy X-Ray Absorptiometry (DXL) Compared to Conventional Absorptiometry (DXA) and to FRAX as Tools for Fracture Risk Assessments

Dual X-ray and Laser (DXL) adds a measure of the external thickness of the heel, measured by laser, to a conventional measurement of bone mineral density (BMD) of the calcaneus, using Dual energy X-ray Absorptiometry (DXA). The addition of heel thickness aims at a better separation of fatty tissue from bone than the standard method of DXA, which may mistake fatty tissue for bone and vice versa. The primary aim of this study was to evaluate whether DXL of the calcaneus can be used to assess the 10-year risk of fractures. Secondary aims were to compare the predictive ability of DXL with the two most established methods, Dual energy X-ray Absorptiometry (DXA) of the hip and spine and the WHO fracture risk assessment tool, FRAX. In 1999 a cohort of 388 elderly Swedish women (mean age 73.2 years) was examined with all three methods. Prospective fracture data was collected in 2010 from health care registers. One SD decrease in BMD of the heel resulted in an age-adjusted Hazard Ratio (HR) of 1.47 for a hip fracture (95% CI 1.09–1.98). Harrell’s C is the Cox regression counterpart of the Area Under Curve (AUC) of the Receiver Operating Characteristic (ROC) as a measure of predictive accuracy. Harrell’s C for BMD of the calcaneus was 0.65 for prediction of hip fractures. These results were not significantly different from those for BMD of the femoral neck or for FRAX. The HR for a hip fracture, for one SD decrease in BMD at the femoral neck, was 1.72 (95% CI 1.21–2.44. Harrell’s C was 0.67 for BMD at the femoral neck and 0.59 for FRAX. We conclude that DXL of the calcaneus could be a useful tool for fracture risk assessments.     

Minor Cytological Abnormalities and up to 7-Year Risk for Subsequent High-Grade Lesions by HPV Type

Objective

Diagnoses of atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions (LSIL) are common, but the corresponding risk of disease varies by human papillomavirus (HPV) status, complicating management strategies. Our aim was to estimate the longer-term risk of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) among women with ASCUS/LSIL by age, HPV status, and genotype(s).

Methods

A total of 314 women with ASCUS/ LSIL were followed for a median of 3.8 years. Baseline HPV status was determined by reflex testing and women with histologically confirmed CIN2+ were identified through linkage to the Swedish National Quality Register for Cervical Cancer Prevention. Cumulative incidence and hazard ratios were estimated to explore differences between index data and associations with CIN2+.

Results

In total, 89 women (28.3%) developed CIN2+. High-risk (HR) HPV-positive women developed significantly more CIN2+ than HR-HPV-negative women (cumulative incidence 3.5 years after the index test: 42.2%, 95% CI: 32.5–53.5 for HPV16/18; 36.2%, 95% CI: 28.3–45.4 for other HR-HPV types; and 2.0%, 95% CI: 0.5–7.8 for HR-HPV-negative women; p<0.0001).

Conclusion

HPV status was of greatest importance in determining the risk of CIN2+. The risk was low among HPV-negative women during the first years of follow-up, suggesting these women could be followed less intensively. HPV16/18-positive women may need intensified follow-up as they showed the highest risk of CIN2+.     

Effects of psychophysiological stress on trapezius muscles blood flow and electromyography during static load

Mental stress was induced by the Stroop colour word task (CW task) and the effects on the micro-circulation and electromyography (EMG) in the upper portion of the trapezius muscle were studied during a series of fatiguing, standardized static contractions. A lowered blood flow of the skin recorded continuously by laser-Doppler flowmetry (LDF) was used as a stress indicator in addition to an elevated heart rate. Muscle blood flow was recorded continuously by LDF using a single optical fibre placed inside the muscle, and related to surface EMG. A group of 20 healthy women of different ages was examined. Recordings were made during a 50-min period in the following sequence: a 10-min series of alternating 1-min periods of rest and stepwise increased contraction induced by keeping the arms straight and elevated at 30, 60, 90 and 135° with a 1-kg load carried in each hand; a 10-min recovery period without load; a repeated contraction series with simultaneous performance of the CW task; a second 10-min recovery period, and a second contraction series without CW task. Signal processing was done on line by computer. The LDF and root mean square (rms)-EMG values were calculated, as well as the EMG mean power frequency (MPF) for fatigue. The CW-task added to the contraction series caused an increase in the heart rate accompanied by a decrease in the blood flow to the skin and a 30% increase in the blood flow in the exercising muscle. Both returned to normal during the subsequent recovery period and showed normal levels during the final contraction series without CW. The rms-EMG showed a 20% increase that persisted during the final contraction series performed without CW. There was no influence on MPF. This CW has previously been shown to evoke an increased secretion of adrenaline from the adrenal medullae to the blood. The increased blood flow in the exercising muscle would therefore appear to have been caused by -adrenoceptor vasodilatation, and the fall in the blood flow in the skin by -adrenoceptor vasoconstriction. The findings may have implications for work situations characterized by repetitive static loads to the shoulder muscles and psychological stress.