Combating non-Hodgkin lymphoma by targeting both CD20 and HLA-DR through CD20–243 CrossMab

Although rituximab has revolutionized the treatment of hematological malignancies, the acquired resistance is one of the prime obstacles for cancer treatment, and development of novel CD20-targeting antibodies with potent anti-tumor activities and specificities is urgently needed. Emerging evidence has indicated that lysosomes can be considered as an “Achilles heel” for cancer cells, and might serve as an effective way to kill resistant cancer cells. HLA-DR antibody L243 has been recently reported to elicit potent lysosome-mediated cell death in lymphoma and leukemia cells, suggesting that HLA-DR could be used as a potential target against lymphoma. In this study, we generated a bispecific immunoglobulin G-like antibody targeting both CD20 and HLA-DR (CD20–243 CrossMab) through CrossMab technology. We found that the CrossMab could induce remarkably high levels of complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and anti-proliferative activity. Notably, although HLA-DR is expressed on normal and malignant cells, the CrossMab exhibited highly anti-tumor specificity, showing efficient eradication of hematological malignancies both in vitro and in vivo. Our data indicated that combined targeting of CD20 and HLA-DR could be an effective approach against malignancies, suggesting that CD20–243 CrossMab would be a promising therapeutic agent against lymphoma.     

Combating non-Hodgkin lymphoma by targeting both CD20 and HLA-DR through CD20–243 CrossMab

Although rituximab has revolutionized the treatment of hematological malignancies, the acquired resistance is one of the prime obstacles for cancer treatment, and development of novel CD20-targeting antibodies with potent anti-tumor activities and specificities is urgently needed. Emerging evidence has indicated that lysosomes can be considered as an “Achilles heel” for cancer cells, and might serve as an effective way to kill resistant cancer cells. HLA-DR antibody L243 has been recently reported to elicit potent lysosome-mediated cell death in lymphoma and leukemia cells, suggesting that HLA-DR could be used as a potential target against lymphoma. In this study, we generated a bispecific immunoglobulin G-like antibody targeting both CD20 and HLA-DR (CD20–243 CrossMab) through CrossMab technology. We found that the CrossMab could induce remarkably high levels of complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and anti-proliferative activity. Notably, although HLA-DR is expressed on normal and malignant cells, the CrossMab exhibited highly anti-tumor specificity, showing efficient eradication of hematological malignancies both in vitro and in vivo. Our data indicated that combined targeting of CD20 and HLA-DR could be an effective approach against malignancies, suggesting that CD20–243 CrossMab would be a promising therapeutic agent against lymphoma.     

Population growth and allergen accumulation of Dermatophagoides farinae cultured at 20 and 25 °C

House dust mites are cultured to obtain mite allergen material to produce allergen extracts (vaccines) for diagnostic tests, immunotherapy, and research purposes. Research laboratories and manufacturers have their own culturing protocols to grow these mites and these may vary between manufacturers and between research laboratories. The temperature at which mites are cultured may influence the allergen composition, allergen ratio of Der 1: Der 2 and endotoxin levels in the extracts produced from these cultured mites. In order to produce standardized and uniform extracts, across the industry and in various research laboratories, the influence of culture conditions must be understood. Here we determined how temperature affects mite population growth rates, dynamics of allergen production, Der f 1: Der f 2 ratio and endotoxin levels in extracts made from Dermatophagoides farinae mites cultured at 20 and 25 °C. We found that Der f 1 and Der f 2 accumulated exponentially in the cultures with Der f 1 accumulating faster than Der f 2. When the live mite populations peaked, the ratios for Der f 1: Der f 2 were 4.1 and 4.7 for cultures reared at 20 and 25 °C, respectively. Most of the Der f 1 and Der f 2 allergen in whole cultures is not in mite bodies and is lost when the mite material is washed. Thus, if the ratio of Der f 1 and Der f 2 is an important consideration for commercial and research extracts, then the temperature at which the mites are cultured and the collection procedure are important considerations.     

Sarcoplasmic reticulum and excitation-contraction coupling at 20 and 10 °C in rainbow trout myocardium

Summary Isometric force and series membrane potential were recorded in isolated ventricular strips from rainbow trout at 20 and 10 °C. Preparations were electrically stimulated to contract at either 0.5 or 0.2 Hz. Single extrastimulations elicited a twitch force which diminished when the preceding diastole was shortened below the regular value. The stimulation following this extra stimulation evoked no potentiation of force. Apart from a marginal effect on the post extrasystolic force at 20 °C, ryanodine did not affect either of these responses or the steady-state force at 0.5 Hz. At 0.2 Hz the steady-state force was somewhat depressed by ryanodine at 20 but not at 10 °C. In contrast, extrastimulations preceded by diastoles of up to 1 h more than doubled extrasystolic force at 20 °C. This effect was removed by ryanodine. Both the potentiations and the effect of ryanodine were strongly reduced at 10 °C. Apparently, temperature acts on the release of Ca²⁺ from the sarcoplasmic reticulum, since Ca²⁺ seems to be taken up at both temperatures. Hence, at both 20 and 10 °C, the contractures evoked in a solution inhibiting sarcolemmal Ca²⁺ transfer and releasing Ca²⁺ from the sarcoplasmic reticulum were diminished by pretreatment with 15 mM caffeine. Action potential duration at 20 °C was less than half of that at 10 °C. At both temperatures it tended to be prolonged by periods of prolonged rest. No effect of ryanodine on action potential configuration was detected. The results suggest that trout myocardial sarcoplasmic reticulum, although powerful at unphysiologically low stimulation rates, does not partake in the beat-to-beat regulation of force at heart rates encountered in vivo.Abbreviations ESF extrasystolic force - SR sarcoplasmic reticulum - v _F maximal rate of force development - v _R maximal rate of relaxation - TPF time to peak force - TR _0.5 time for half relaxation - TTF duration of force development     

Photo-conversion of two epimers (20R and 20S) of pregna-5,7-diene-3β, 17α, 20-triol and their bioactivity in melanoma cells

Pregna-5,7-dienes and their hydroxylated derivatives can be formed in vivo when there is a deficiency in 7-dehydrocholesterol (7-DHC) Δ-reductase function, e.g., Smith-Lemli-Opitz syndrome (SLOS). Ultraviolet B (UVB) radiation induces photoconversion of 7-DHC to vitamin D₃, lumisterol₃ and tachysterol₃. Two epimers (20R and 20S) of pregna-5,7-diene-3β,17α,20-triol (4R and 4S, respectively) were synthesized and their UVB photo-conversion products identified as corresponding 9,10-secosteroids with vitamin D-like and tachysterol-like structures, and 5,7-dienes with inverted configuration at C-9 and C-10 (lumisterol-like). The number and character of the products and the dynamics of the process were dependent on the UVB dose. At high UVB doses, the formation of multiple oxidized derivatives of the primary products, and the formation of 5,7,9(11)-triene, were observed. The production of vitamin D-like, tachysterol-like and lumisterol-like derivatives was also observed in human skin treated with 4R and 4S, and subjected to UV irradiation, as shown by RP-HPLC. Newly synthesized compounds inhibited melanoma growth in dose dependent manner, and some of them showed equal or higher potency than 1,25(OH)₂D₃. In summary, we have characterized for the first time the products of UV induced conversion of pregna-5,7-diene-3β,17α,20-triols and documented that the newly synthesized compounds have antiproliferative properties against melanoma cells.     

Conformational distributions of denatured and unstructured proteins are similar to those of 20?×?20 blocked dipeptides

Understanding intrinsic conformational preferences of amino-acids in unfolded proteins is important for elucidating the underlying principles of their stability and re-folding on biological timescales. Here, to investigate the neighbor interaction effects on the conformational propensities of amino-acids, we carried out (1)H NMR experiments for a comprehensive set of blocked dipeptides and measured the scalar coupling constants between alpha protons and amide protons as well as their chemical shifts. Detailed inspection of these NMR properties shows that, irrespective of amino-acid side-chain properties, the distributions of the measured coupling constants and chemical shifts of the dipeptides are comparatively narrow, indicating small variances of their conformation distributions. They are further compared with those of blocked amino-acids (Ac-X-NHMe), oligopeptides (Ac-GGXGG-NH(2)), and native (lysozyme), denatured (lysozyme and outer membrane protein X from Escherichia coli), unstructured (Domain 2 of the protein 5A of Hepatitis C virus), and intrinsically disordered (hNlg3cyt: intracellular domain of human NL3) proteins. These comparative investigations suggest that the conformational preferences and local solvation environments of the blocked dipeptides are quite similar to not only those of other short oligopeptides but also those of denatured and natively unfolded proteins.     

A simple synthesis of 3β-acetoxy-20-oxomethylpregn-5-ene and 3β-acetoxy-20-hydroxymethylpregn-5-ene

3β-Acetoxy-20-oxomethylpregn-5-ene and 3β-acetoxy-20-hydroxymethylpregn-5-ene were synthesized from (22R, 23R)-sitost-5-ene-3β,22,23-triol in 66% overall yields.     

Keratin 20 - a diagnostic and prognostic marker in colorectal cancer?

Colorectal cancer cells characteristically show strong expression of keratin 20 (K20) and lack expression of keratin 7 (K7). The biological significance of reduced K20 expression, however, is unclear. 381 colorectal cancers with 148 corresponding metastases were evaluated for K20 and K7 expression by immunohistochemistry using a tissue microarray technique. K20 immunoreactivity was assessed semiquantitatively as either negative, low (<50% of cancer cells) or high (≥50% of cancer cells). Progression-free and cancer-specific survivals were determined using the Kaplan-Meier method. Expression of K20 was observed in 348 out of 372 (94%) evaluable primary tumors, with 135 (36%) cases showing low K20 and 213 (57%) cases high K20 expression, while 24 (6%) tumors completely lacked K20 immunoreactivity. Reduced K20 expression (lack of staining or low expression) was significantly associated with poor differentiation, large tumor size and mismatch repair deficiency, but did not significantly affect patients' outcome. Immunoreactivity of K20 and K7 in metastatic tissues matched well with that of corresponding primary tumors, with high concordance for lymph node (p<0.001) and distant metastases (p<0.001), respectively. In conclusion, our data illustrate the value of keratin subtyping in carcinoma of unknown primary (CUP) syndrome: K20 expression is common in colorectal cancer and the K20 high / K7 negative immunoprofile represents the predominant phenotype. Reduced K20 expression may, however, lead to false-negative assessment of metastatic deposits if only small amounts of tissue are obtained (e.g. in needle biopsies), particularly in poorly differentiated cancers. Reduced expression of K20 may be used to select tumors for microsatellite instability testing.     

Population increase and damage by three species of mites on wheat at 20°C and two humidities

Twenty bisected grains of wheat infested with five pairs of the three commonest British grain-storage mites,Acarus siro L., Glycyphagus destructor (Shrank) andTyrophagus longior (Gervais), were examined every week for 20 weeks. Mite populations, the resulting damage to germ and endosperm, and visible fungal growth were observed at 20°C and relative humidities (r.h.) of 90% and 75%.At 90% r.h.,A. siro populations reached nearly 14000 per test-tube before slowly dropping to 5000. The mites ate the germ before the endosperm, leaving an impenetrable layer of crushed endosperm cells between these regions. TheG. destructor population reached only 800 before declining to 300; these mites ate over 75% of the germ and small amounts of endosperm.Tyrophagus longior populations rose to 2200 mites before crashing at week 12 to the initial population level; these mites ate over 75% of the germ and small amounts of endosperm.At 75% r.h., bothA. siro andT. longior populations were lower than at the higher r.h., peaking at 3000 and 1000 respectively and decreasing to 500 and 600 mites respectively.Glycyphagus destructor did markedly better than at 90% r.h., reaching 1500 before falling to 400. The damage at this humidity was slower to occur but was similar to that at 90% r.h. at the end of 20 weeks.At both humidities visible fungus was always less abundant on infested grain that uninfested grain.     

The distribution and nutrient status of phytoplankton in the Southern Ocean between 20° and 70° W

Summary The distribution of phytoplankton along transects amounting to about 10,000 nautical miles in the sector of the Southern Ocean between 20° and 70°W was determined during the austral summer of 1978/79. Chlorophyll a concentration was monitored by the continuous measurement of in vivo fluorescence (IVF). Surface samples were collected for the determination of temperature, salinity, chlorophyll a concentration, carbon fixation rate and species of the phytoplankton. Phytoplankton distribution was found to be extremely patchy both locally and regionally. High phytoplankton concentrations were often associated with either hydrographic features, such as upwelling or the presence of sea-ice, or with bathymetric features, such as shelf breaks, submarine mountain ranges or islands. Enrichment experiments, in which the effects of various nutrient additions on the rate of ¹⁴C fixation by the natural phytoplankton were compared, and bioassay experiments, in which the growth of Thalassiosira pseudonana (Hustedt) Hasle and Heimdal in enriched water samples was measured, were carried out using water samples collected at various stations throughout the study area. Although these techniques were effective in demonstrating nutrient limitation elsewhere, the results suggest that availability of nitrate, phosphate, silicate, trace metals or vitamins exerts no primary control over phytoplankton abundance south of the Polar Front.     

Effects of 20-hydroxyecdysone and KK-42 on chitinase and β-N-acetylglucosaminidase during the larval-pupal transformation of Bombyx mori

The appearance of chitinolytic enzymes, chitinase and β-N-acetylglucosaminidase, in integuments of fifth-larval instars of the silkworm, Bombyx mori, was investigated by injection of 20-hydroxyecdysone into the hemolymph of the ligated larvae, and by topical application of an imidazole compound (KK-42, 1-benzyl-5[(E)-2,6-dimethyl-1,5-heptadienyl] imidazole) along the dorsal vessel of the larvae at the beginning of spinning behavior. 20-Hydroxyecdysone induced both enzyme activities. However, the induction patterns were different between two types of chitinolytic enzymes. Chitinase was rapidly induced only by high hormone levels (30 μg/insect, 7.5 μg/g live wt) and soon decreased, while β-N-acetylglucosaminidase was gradually induced even by low hormone levels (6 μg/insect, 1.5 μg/g live wt). KK-42 suppressed both the larval-pupal transformation and appearance of chitinolytic enzymes. Application of KK-42 (50 μg/insect) caused 1-day delay in β-N-acetylglucosaminidase and 2-day delay in chitinase. It was shown by immunoblotting and activity staining that the appearance of the enzyme activities was associated with that of the respective enzyme molecules. The molecular species of β-N-acetylglucosaminidase appeared was mainly the 67.5 kDa subunit. In the case of chitinase, several molecular species including active forms (88 and 65 kDa) and zymogenic form (about 215 kDa) were observed. These results suggest that β-N-acetylglucosaminidase is induced in an active form by relatively low ecdysteroid levels, whereas chitinase is induced through activation of the zymogen by higher levels of hormone.     

Comorbidity among HHT patients and their controls in a 20 years follow-up period

Background

Hereditary Haemorrhagic Telangiectasia (HHT) is an autosomal dominant genetic disorder with a wide variety of clinical manifestations due to the presence of multiple arteriovenous malformations in various tissues and organs.

Objective

To study the need for hospital admittance in a group of HHT patients and matched controls during a 20?years follow-up period commencing in 1995.

Methods

All HHT patients in the County of Funen, Denmark, were included. For each patient, three age and sex matched controls were identified at the time of enrolment. Data on all hospitalisations were extracted from the national health registers and compared with clinical records. The hospitalisations were grouped as HHT relevant or not HHT relevant based on the discharge diagnosis (International Classification of Diseases, ICD10) and with particular focus on infections, bleedings and thromboembolic events.Patients with HHT were compared with controls concerning the first time incidence of each discharge diagnosis.

Results

We included 73 HHT patients and 219 controls of which one control was lost to follow-up. HHT-patients had significantly more hospitalisations per person caused by infections in joints and bones, but not caused by infections in general. Bleeding episodes were, as expected, more frequent among the HHT-patients. The study revealed a similar incidence of abscesses and thromboembolisms, including in the central nervous system, among the HHT patients and controls.

Conclusions

Based on this study Danish HHT patients had an increased comorbidity of infections in joints and bones and of bleeding episodes. However, the incidence of thromboembolisms, cerebral abscesses and other conditions commonly considered related to HHT was comparable between the patients and the controls. The patients included in this study were closely monitored at a highly specialised HHT Centre where they received relevant diagnostic evaluation, treatment and counselling. Since this is assumed to benefit the overall health of the patients, it may explain why these patients were less prone to comorbidity than other studies have suggested.

     

Ovarian and placental expression of 20α-hydroxysteroid dehydrogenase during pregnancy in deer

The enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD) catalyzes the conversion of progesterone to its inactive form, 20α-hydroxyprogesterone. This enzyme has been shown to play a critical role in the regulation of luteal function in experimental animals. In this study, we cloned and expressed the gene encoding elk deer 20α-HSD from reproductive placental and ovarian tissues. PCR, 3'- and 5'-RACE, and northern blot analysis were performed for the cloning and characterization of deer 20α-HSD gene. We expressed recombinant deer 20α-HSD protein and used western blot analysis to determine protein expression levels in the placenta and ovary during pregnancy. The full cDNA sequence of 20α-HSD was used to clone an open reading frame encoding 323 amino acids and consisting of 1142 bp. The nucleotide sequence of deer 20α-HSD showed high homology with the sequences of the bovine (96%), goat (96%), and human (83%) 20α-HSD genes. 20α-HSD mRNA was strongly expressed in the placenta on days 30, 60, and 70 of pregnancy. A high level of the protein was also detected in the placenta but not in fetal skin tissue. The recombinant 20α-HSD protein produced in mammalian cells and bacterial systems had a molecular weight of approximately 37-kDa. The deer 20α-HSD protein signal was specifically localized in the basal part of the primary chorionic villi and chorionic stem villus of the placenta during early pregnancy. The 20α-HSD protein was also intensively localized in the larger luteal cells of the corpus luteum during pregnancy.     

Novel taxa-4(20),12-diene and 2(3→20)abeotaxane from needles of Taxus canadensis

A novel 6/8/6-membered taxane with a rare C-12(13)-double bond and rare 2(3→20)abeotaxane were isolated from the needles of Taxus canadensis. Their structures were characterized as 7β,9α,10β-triacetoxytaxa-4(20),12-diene-2α,5α,11β-triol (1) and 2α,7β,10β-triacetoxy-5α-hydroxy-2(3→20)abeotaxa-4(20),11-diene-9,13-dione (2) on the basis of 1D and 2D spectroscopic data. 1 is the first example of a natural taxane without substitution at both C-13 and C-14.     

Interferon-γ–inducible Rab20 regulates endosomal morphology and EGFR degradation in macrophages

Little is known about the molecular players that regulate changes in the endocytic pathway during immune activation. Here we investigate the role of Rab20 in the endocytic pathway during activation of macrophages. Rab20 is associated with endocytic structures, but the function of this Rab GTPase in the endocytic pathway remains poorly characterized. We find that in macrophages, Rab20 expression and endosomal association significantly increase after interferon-γ (IFN-γ) treatment. Moreover, IFN-γ and Rab20 expression induce a dramatic enlargement of endosomes. These enlarged endosomes are the result of homotypic fusion promoted by Rab20 expression. The expression of Rab20 or the dominant-negative mutant Rab20T19N does not affect transferrin or dextran 70 kDa uptake. However, knockdown of Rab20 accelerates epidermal growth factor (EGF) trafficking to LAMP-2–positive compartments and EGF receptor degradation. Thus this work defines a function for Rab20 in the endocytic pathway during immune activation of macrophages.     

Novel Taxa-4(20),12-diene and 2(3→20)Abeotaxane from Needles of Taxus canadensis

A novel 6/8/6-membered taxane with a rare C-12(13)-double bond and rare 2(3→20)abeotaxane were isolated from the needles of Taxus canadensis. Their structures were characterized as 7β,9α,10β-triacetoxytaxa-4(20),12-diene-2α,5α,11β-triol (1) and 2α,7β,10β-triacetoxy-5α-hydroxy-2(3→20)abeotaxa-4(20),11-diene-9,13-dione (2) on the basis of 1D and 2D spectroscopic data. 1 is the first example of a natural taxane without substitution at both C-13 and C-14.