Melanoma cells can be eliminated by sialylated CD43 × CD3 bispecific T cell engager formats in vitro and in vivo.

Targeted cancer therapy with monoclonal antibodies has proven successful for different cancer types but is limited by the availability of suitable antibody targets. CD43s, a unique sialylated form of CD43 expressed by hematologic malignancies, is a recently identified target and antibodies interacting with CD43s may have therapeutic potential against acute myeloid leukemia (AML) and myelodysplastic syndrome. CD43s is recognized by the human antibody AT1413, that was derived from a high-risk AML patient who successfully cleared leukemia after allogeneic stem cell transplantation. Here we observed that AT1413 binds also to certain non-hematopoietic tumor cells, particularly melanoma and breast cancer. AT1413 immune precipitated CD43s from melanoma cells confirming that it recognizes the same target on melanoma as on AML. AT1413 induced antibody-dependent cellular cytotoxicity against short-term cultured patient-derived melanoma samples. However, AT1413 was unable to affect the growth of melanoma cells in vivo. To increase the efficacy of AT1413 as a therapeutic antibody, we generated two different formats of bispecific T-cell engaging antibodies (TCEs): one binding bivalently (bTCE) and the other monovalently (knob-in-hole; KiH) to both CD43s and CD3ε. In vitro, these TCEs redirected T-cell cytotoxicity against melanoma cells with differences in potencies. To investigate their effects in vivo, we grafted mice that harbor a human immune system with the melanoma cell line A375. Treatment with both AT1413 bTCE and AT1413 KiH significantly reduced tumor outgrowth in these mice. These data indicate a broad therapeutic potential of AT1413 that includes AML and CD43s-expressing solid tumors that originate from CD43-negative tissues.

Broadly Reactive Anti-Respiratory Syncytial Virus G Antibodies from Exposed Individuals Effectively Inhibit Infection of Primary Airway Epithelial Cells.

Respiratory syncytial virus (RSV) causes severe respiratory disease in young children. Antibodies specific for the RSV prefusion F protein have guided RSV vaccine research, and in human serum, these antibodies contribute to >90% of the neutralization response; however, detailed insight into the composition of the human B cell repertoire against RSV is still largely unknown. In order to study the B cell repertoire of three healthy donors for specificity against RSV, CD27+ memory B cells were isolated and immortalized using BCL6 and Bcl-xL. Of the circulating memory B cells, 0.35% recognized RSV-A2-infected cells, of which 59% were IgA-expressing cells and 41% were IgG-expressing cells. When we generated monoclonal B cells selected for high binding to RSV-infected cells, 44.5% of IgG-expressing B cells and 56% of IgA-expressing B cells reacted to the F protein, while, unexpectedly, 41.5% of IgG-expressing B cells and 44% of IgA expressing B cells reacted to the G protein. Analysis of the G-specific antibodies revealed that 4 different domains on the G protein were recognized. These epitopes predicted cross-reactivity between RSV strain A (RSV-A) and RSV-B and matched the potency of antibodies to neutralize RSV in HEp-2 cells and in primary epithelial cell cultures. G-specific antibodies were also able to induce antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis of RSV-A2-infected cells. However, these processes did not seem to depend on a specific epitope. In conclusion, healthy adults harbor a diverse repertoire of RSV glycoprotein-specific antibodies with a broad range of effector functions that likely play an important role in antiviral immunity.IMPORTANCE Human RSV remains the most common cause of severe lower respiratory tract disease in premature babies, young infants, the elderly, and immunocompromised patients and plays an important role in asthma exacerbations. In developing countries, RSV lower respiratory tract disease has a high mortality. Without an effective vaccine, only passive immunization with palivizumab is approved for prophylactic treatment. However, highly potent RSV-specific monoclonal antibodies could potentially serve as a therapeutic treatment and contribute to disease control and mortality reduction. In addition, these antibodies could guide further vaccine development. In this study, we isolated and characterized several novel antibodies directed at the RSV G protein. This information can add to our understanding and treatment of RSV disease.

The modified FACS calcein AM retention assay: A high throughput flow cytometer based method to measure cytotoxicity.

Current methods to determine cellular cytotoxicity in vitro are hampered by background signals that are caused by auto-fluorescent target and effector cells and by non-specific cell death. We combined and adjusted existing cell viability assays to develop a method that allows for highly reproducible, accurate, single cell analysis by high throughput FACS, in which non-specific cell death is corrected for. In this assay the number of living, calcein AM labeled cells that are green fluorescent are quantified by adding a fixed number of unlabeled calibration beads to the analysis. Using this modified FACS calcein AM retention method, we found EC50 values to be highly reproducible and considerably lower compared to EC50 values obtained by conventional assays, displaying the high sensitivity of this assay.

Possible involvement of phosphatidylinositol 3-kinase/Akt pathway in insulin-like growth factor-I-induced alkaline phosphatase activity in osteoblasts.

In the present study, we investigated whether Akt is involved in insulin-like growth factor-I (IGF-I)-stimulated activity of alkaline phosphatase, a marker of mature osteoblast phenotype, in osteoblast-like MC3T3-E1 cells. IGF-I induced the phosphorylation of Akt in these cells. Akt inhibitor significantly suppressed the IGF-I-stimulated alkaline phosphatase activity. The phosphorylation of Akt induced by IGF-I was reduced by the Akt inhibitor. LY294002 and wortmannin, inhibitors of phosphatidylinositol 3-kinase, significantly suppressed the IGF-I-induced alkaline phosphatase activity. The phosphorylation of Akt induced by IGF-I was markedly reduced by LY294002 and wortmannin. These results strongly suggest that phosphatidylinositol 3-kinase/Akt plays a role in the IGF-I-stimulated alkaline phosphatase activity in osteoblasts.

Determinación mediante inmunohistoquímica de infección por virus de la fiebre amarilla y virus de la hepatitis B / Yellow fever and hepatitis B determination using immunohistochemical markers

La inmunohistoquímica (IHQ) constituye una herramienta para definir la etiología en el síndrome icterohemorrágico. Objetivo: Identificar mediante IHQ la presencia en hígado del antígeno de superficie de la Hepatitis B y del virus de la fiebre amarilla. Materiales y métodos: se incluyeron muestras de tejido hepático procedente de 55 fallecidos por sindrome icterohemorrágico, remitidos entre enero del 2000 y diciembre del 2001 al Instituto Nacional de Salud, (Lima, Perú). Resultados: La prueba se realizó unicamente en 46 muestras, 33 por ciento (15/46) fueron diagnosticados por histopatología como fiebre amarilla, de ellos, 9 casos fueron positivos por IHQ para fiebre amarilla, siendo 2 de ellos también positivos para HBsAg. Histológicamente se presentaron 6 casos como hepatitis con necrosis submasiva o masiva, que por IHQ fueron sólo dos casos positivos para fiebre amarilla. En 39 por ciento (18/46) no se llegó al diagnóstico etiológico de la Hepatitis, y finalmente hubo dos casos con siagnóstico histopatológico de ádenocarcinoma hepático y sólo se obtuvo positividad por IHQ para HBsAg en uno. Conclusiones: La IHQ es una alternativa para la confirmación diagnóstica de hepatitis B y fiebre amarilla

Cellular localization of group IIA phospholipase A2 in rats.

It has been known that group II phospholipase A2 (PLA2) mRNA and protein are present in the homogenates of the spleen, lung, liver, and kidney in normal rats, but the cellular origin of this enzyme has not been yet identified. At present, five subtypes of group II PLA2 have been identified in mammals. Antibodies or mRNA probes previously used for detecting group II PLA2 need to be evaluated to identify the subtypes of group II PLA2. In this study we tried to identify group IIA PLA2-producing cells in normal rat tissues by in situ hybridization (ISH) using an almost full-length RNA probe for rat group IIA enzyme. Group IIA PLA2 mRNA was detected in megakaryocytes in the spleen and Paneth cells in the intestine by ISH. These cells were also immunopositive for an antibody raised against group IIA PLA(2) isolated from rat platelets. Group IIA PLA2 mRNA-positive cells were not detected in lung, liver, kidney, and pancreas. Under normal conditions, group IIA PLA2-producing cells are splenic megakaryocytes and intestinal Paneth cells in rats.

Curing properties of furfuryl alcohol condensate with carbonaceous fine particles under ultrasonication.

Ultrasonic treatment (sonication) was carried out through the curing process of furan resin by using an ultrasonic homogenizer at the frequency of 20 kHz and the various intensities (0-90 W). Various carbonaceous fine particles were added to furan resin to investigate the change of polymerization degree. The curing rate of furan resin was accelerated by sonication, which increased the polymerization degree with an increase in ultrasound intensity. The increase of curing rate was also observed by small additions of carbonaceous fine particles. In this case, the curing rate was increased with an increase in the specific surface area on additives.

Immunohistochemical demonstration of L-serine distribution in the rat brain.

L-Serine has been suggested by in vitro studies to be an important neurotrophic factor which supports survival and neurite outgrowth of neurons. It is also a precursor of D-serine, a putative neurotransmitter. In the present study, we raised antibodies against L-serine in a rabbit and examined immunohistochemical distribution of the amino acid in the rat brain. In the hippocampus and the cerebellar cortex, where neurotrophic effects of L-serine have been indicated, L-serine immunoreactivity was found primarily in astrocytes. In the brain stem, where neuronal distribution of D-serine was reported, positive staining for L-serine was located primarily in neurons. Regional differences of cellular distribution of L-serine were indicated.

Immunohistochemical evidences for localization and production of D-serine in some neurons in the rat brain.

D-Amino acids are thought not to occur in mammalian tissues. However, previous studies reported D-serine was present only in astrocytes in the rat brain. In the present study, it was indicated by a highly sensitive immunocytochemical method with a D-serine specific antibody that D-serine was contained not only in astrocytes but also in some neurons, such as pyramidal neurons in the cerebral cortex, and neurons in the nucleus of the trapezoid body. Some amacrine cells also showed strong immunoreactivity for D-serine in the eyes which were injected with colchicine into the corpus vitreum.

Effect of soy milk and bifidobacterium-fermented soy milk on plasma and liver lipids in ovariectomized Syrian hamsters.

The effects of soy milk and fermented soy milk on lipid metabolism were studied in ovariectomized Syrian hamsters. Five mo-old Syrian hamsters were randomly assigned to four treatment groups: ovariectomized (OVX)+control diet (OVX-C); OVX+soy milk diet (OVX-SM); OVX+fermented soy milk diet (OVX-FSM); and sham-operated+control diet (Sham-C). The hamsters were fed on these diets for 4 wk. The atherogenic index value of the OVX-FSM group was lower than that of the OVX-C group. The plasma triglyceride level of the OVX-FSM group was significantly lower than that of the OVX-C group. The liver total cholesterol contents in the OVX-SM and OVX-FSM groups were significantly lower than that in the OVX-C group. Thus, these results demonstrate that bifidobacterium-fermented soy milk had a hypolipidemic effect in ovariectomized hamsters.

High viral eradication with a daily 12-week natural interferon-beta treatment regimen in chronic hepatitis C patients with low viral load. IFN-beta Research Group.

Virological sustained response (SR) is achieved in 31-49% of patients with chronic hepatitis C with combination therapy using interferon (IFN)-alpha and ribavirin for 24-48 weeks. However, as a period of 24-48 weeks is a burden for patients, we investigated the effect of daily intravenous administration of natural IFN-beta for 12 weeks in this study. In all, 112 patients were enrolled and received a daily administration of 6 MU of natural IFN-beta intravenously for 12 weeks. Serum HCV-RNA before treatment was assessed by the competitive reverse-transcription polymerase chain reaction assay. The patients were divided into two groups according to pretreatment viral load: the low viral load group (N = 25, <6.3 x 10(5) copies/ml), and the high viral load group (N = 87, > or =6.3 x 10(5) copies/ml) who were additionally administered IFN-beta thrice weekly for subsequent 14 weeks at the patients' request. Virological SR was obtained in 37% (41/112) of all the patients; 88% of those with a low viral load, and 22% of patients with a high viral load. Virological SR was attained in 21% of patients with HCV subtype 1, and in 67% of those with subtype 2a. In patients with HCV subtype 1b, virological SR was obtained in patients with the mutant-type (> or =4 amino acid mutations in the NSSA2209-48) having a low viral load (4/4), but not in those having a high viral load (0/3). The results suggest that a daily intravenous administration of natural IFN-beta for 12 weeks showed high therapeutic efficacy in patients with a low viral load despite the shorter treatment period and that the therapeutic efficacy depends on viral load rather than on the number of NS5A2209-48 amino acid mutations.

Cytotoxic effects of NSL-1406, a new thienopyrimidine derivative, on leukocytes and osteoclasts.

We synthesized a series of thienopyrimidine derivatives and examined their cytotoxic effects on several cell lines. One of the derivatives, NSL-1406, was shown to exert potent cytotoxic effects on leukemia cell line including P388 cells and J774 cells. It was also inhibitory on mouse osteoclasts and suppressed the in vitro bone resorption by osteoclasts at nanomolar concentrations.

N-[2,2-dimethyl-3-(N-(4-cyanobenzoyl)amino)nonanoyl]-L-phenylalanine ethyl ester as a stable ester-type inhibitor of chymotrypsin-like serine proteases: structural requirements for potent inhibition of alpha-chymotrypsin.

We introduce a new potent inhibitor, N-[2, 2-dimethyl-3-(N-(4-cyanobenzoyl)amino)nonanoyl]-L-phenylalanine ethyl ester (3), which preferentially inhibits serine proteases belonging to a chymotrypsin superfamily. This inhibitor, despite consisting of a stable ethyl ester structure, showed strong inhibitory activities toward bovine alpha-chymotrypsin, human cathepsin G, and porcine elastase by acting as an acylating agent. The calculated inactivation rate constant (kinact) and enzyme-inhibitor dissociation constant (Ki) against alpha-chymotrypsin were 0.0028 s-1 and 0.0045 microM, respectively (kinact/Ki = 630 000 M-1 s-1). These kinetic parameters indicate that this inhibitor is one of the most powerful alpha-chymotrypsin inactivators ever reported. On the basis of structure-activity relationship (SAR) and structure-stability relationship studies of analogues of 3, which were modified in three parts of the molecule, i.e., the 4-cyanophenyl group, beta-substituent at the beta-amino acid residue, and ester structure, we suggest that the potent inhibitory activity of 3 is due to the following structural features: (1) the ethyl ester which enforces specific acyl-enzyme formation, (2) the n-hexyl group at the beta-position and 4-cyanophenyl group which stabilize the acyl-enzyme, and (3) the phenylalanine residue which functions for the specific recognition of S1 site in the enzyme. In particular, the action of 3 as a potent inhibitor, but poor substrate, can be ascribed largely to the very slow deacylation rate depending on the structure factors cited in feature 2. The results of inhibition by 3 and its analogues against different serine proteases such as chymase, cathepsin G, and elastase suggest that these compounds recognize common parts in the active sites among these chymotrypsin-like serine proteases, and 3 is one of the most suitable structures to recognize those common parts. Our results provide an intriguing basis for further developments in the design of a stable ester-based selective serine protease inhibitor.

Effects of various 5-HT3 receptor antagonists, granisetron, ondansetron, ramosetron and azasetron on serotonin (5-HT) release from the ferret isolated ileum.

The object of this study was to evaluate the involvement of 5-HT3 receptors in the regulation of 5-HT release from the small intestine using ferrets, an animal model of emesis. 2-Methyl-5-HT, a 5-HT3 receptor agonist, produced a concentration-dependent increase of 5-HT from the ferret ileum. This increase in 5-HT release was significantly inhibited by granisetron (10(-7) and 10(-6) M) or azasetron (10(-7) and 10(-6) M) in a concentration-dependent manner. Ondansetron (10(-7) M) and ramosetron (10(-6) M) also significantly inhibited the 2-methyl-5-HT-induced increase in 5-HT release. When the concentration of ondansetron was increased from 10(-7) M to 10(-6) M, inhibition of 5-HT release was reduced. Ramosetron, for which 5-HT3 receptor binding of the rat brain is remarkably stronger than for any other 5-HT3 receptor antagonists, inhibited the 5-HT release at only the highest concentration of 10(-6) M. Based on these observations that the mode of action on the 2-methyl-5-HT induced 5-HT release is different among 5-HT3 receptor antagonists, it is suggested that there is a possibility that the neuronal 5-HT3 receptors and the 5-HT3 receptors on the EC cells may represent two distinct subtypes.

Effects of soy milk and bifidobacterium fermented soy milk on lipid metabolism in aged ovariectomized rats.

The effects of soy milk and fermented soy milk on lipid metabolism were studied in aged ovariectomized rats. Twenty 8-mo-old Wistar rats were randomly assigned to four treatment groups: sham-operated + control diet (sham-C); ovariectomized (OVX) + control diet (OVX-C); OVX + soy milk diet (OVX-SM); and OVX + fermented soy milk diet (OVX-FSM). The rats were fed on these diets for 6 weeks. Ovariectomy induced an increase in the plasma cholesterol level by 40%. The plasma total cholesterol level of the OVX-FSM rats was decreased by 20% compared to that of the OVX-C rats. The plasma total cholesterol level of the OVX-SM group was not significantly different from that of the OVX-C and sham-C rats. The plasma triglyceride level of the OVX-FSM rats was lower than that of the sham-C rats. The liver cholesterol content in OVX-SM and OVX-FSM rats was lower than that of the OVX-C rats. The liver triglyceride contents of the sham-C, OVX-SM, and OVX-FSM groups were lower than that of the OVX-C group. Fecal steroid excretion did not differ among the groups. Ovariectomy decreased the uterus weight. The OVX-SM and OVX-FSM groups had the same uterus weights as those of the OVX-C group. Thus, the diet including fermented soy milk prevented the cholesterol elevation induced in rats by ovarian hormone deficiency.

GPIIb/IIIa integrin antagonists with the new conformational restriction unit, trisubstituted beta-amino acid derivatives, and a substituted benzamidine structure.

Ethyl N-[3-(2-fluoro-4-(thiazolidin-3-yl(imino)methyl)benzoyl)amino-2, 2-dimethylpentanoyl]piperidine-4-acetate 40 (NSL-96184) is a highly potent and orally active fibrinogen receptor antagonist, which is characterized by the presence of the trisubstituted beta-amino acid residue, 3-ethyl-2,2-dimethyl-beta-alanine. This compound was developed on the basis of the SAR study of N-[3-(N-4-amidinobenzoyl)amino-2, 2-dimethyl-3-phenylpropionyl]piperidine-4-acetic acid 1(NSL-95301) with the derivatization focused on the central trisubstituted beta-amino acid unit as well as the basic amidinobenzoyl unit, and the esterification of the carboxyl group for prodrug composition. Compound 1, which was reported in our previous study, was discovered by the application of combinatorial chemistry. The molecular modeling study suggests that the trisubstituted beta-amino acid unit is responsible for fixing the molecule to its active conformation. Compound 40 showed an excellent profile in the in vitro and in vivo studies for its human platelet aggregation inhibitory activity and oral availability in guinea pigs. This oral availability largely depends on the modification of the amidino group with a cyclic secondary amine, i.e., thiazolidine in 40. In in vivo studies, the onset of the antiplatelet action of 40 is very fast after oral administration, whereas its duration of action is relatively short. These results suggest that 40 has an excellent therapeutic potential, especially for antithrombotic treatment in the acute phase. 3-Substituted-2,2-dimethyl-beta-amino acid residues would serve as new and useful linear templates to restrict the conformational flexibility of peptidomimetics.

Discovery and structure--activity relationship studies of a novel and specific peptide motif, Pro-X-X-X-Asp-X, as a platelet fibrinogen receptor antagonist.

A novel hexapeptide, H-Pro-Ser-Nva-Gly-Asp-Trp-OH 6, a specific antagonist of platelet fibrinogen receptor (GpIIb/IIIa), was discovered in a structure-activity relationship (SAR) study where the role of the N-terminal Pro moiety of an RGD-containing peptide, H-Pro-Ser-Arg-Gly-Asp-Trp-OH 1, which is a potent but not specific antagonist toward GpIIb/IIIa integrin, was investigated. This novel peptide 6 exhibits very high activity as a human platelet aggregation inhibitor (IC50 0.59 microM, human PRP/collagen) as well as marked specificity for GpIIb/IIIa. A series of substitutions at the third position (Nva residue) in this hexapeptide, focused on the conformational rigidity, led to compounds which are superior to the original novel peptide 6 with regard to anti-platelet activity. The peptides, H-Pro-Ser-Hyp-Gly-Asp-Trp-OH 17 and H-Pro-Ser-delta Pro-Gly-Asp-Trp-OH 18 with the 5-membered ring structure, which restricted the conformation of the peptide backbone at the third position, inhibited the aggregation of human platelets at submicromolar concentrations (IC50 0.39 and 0.30 microM, respectively). Further structure-activity relationship studies at each position of the peptide sequence suggest a novel motif sequence, Pro-X1-X2-X3-Asp-X4, for specific GpIIb/IIIa integrin recognition, in which the N-terminal free Pro residue and the Asp residue at the fifth position are essential to the activity. This motif sequence is summarized as follows: (1) a small amino acid such as Ser, Ala or Gly is preferable at X1 position; (2) X2 may be any amino acid, preferably a bulky amino acid such as Tle or a cyclic amino acid such as Pro; (3) X3 must be a small amino acid such as Gly; and (4) X4 is preferably an amino acid with an aromatic side chain.

Effects of soya milk and Bifidobacterium-fermented soya milk on plasma and liver lipids, and faecal steroids in hamsters fed on a cholesterol-free or cholesterol-enriched diet.

The effects of freeze-dried soya milk (SM) and Bifidobacterium-fermented soya milk (FSM) on plasma and liver lipids, and faecal steroid excretion were estimated in hamsters fed on a cholesterol-free or cholesterol-enriched diet. Hamsters fed on the cholesterol-free diet containing 300 g FSM/kg had lower levels of plasma VLDL + LDL cholesterol than the animals fed on the control diet. SM in the diet produced a similar pattern without significant differences. In the cholesterol-enriched diet group, SM and FSM decreased the levels of plasma total cholesterol and VLDL + LDL-cholesterol. SM and FSM decreased the plasma triacylglycerol level in both the cholesterol-free and -enriched diet groups. The liver total cholesterol contents in the SM and FSM groups were lower than that in the control group, for hamsters fed on the cholesterol-free diet. The liver triacylglycerol content was not modified by SM or FSM in hamsters fed on either the cholesterol-free or -enriched diet. SM and FSM increased the total bile acid excretion and the proportion of cholesterol entering the cholic acid biosynthesis pathway in both the cholesterol-free and -enriched diet groups. SM and FSM did not affect neutral steroid excretion in the cholesterol-free or -enriched diet group. There was an inverse relationship between VLDL + LDL-cholesterol and faecal bile acid excretion in hamsters fed on the cholesterol-free (r -0.670, P < 0.01) and cholesterol-enriched (r -0.761, P < 0.001) diets respectively. These results indicated that SM had an anti-atherogenic effect, and that this effect was not diminished by prior fermentation.

Root canal system of the mandibular incisor.

To better assess the efficiency of the mechanical preparation of root canals, 1085 transparent specimens of extracted mandibular incisors were examined for canal configuration, thickness and curvature of the root canals, condition of any accessory canals, and location of the apical foramen. Greater than 85% of the root canals possessed a single canal (Type I). Of specimens in which furcation was observed, only 3% possessed two separate canals (Type III and IV). Fewer than 30% of the specimens showed accessory canals that were mechanically impossible to clean. The majority of the lateral branches were small, greater than 80% of the specimens were smaller than a #15 reamer, and none of the branches were larger than a #30 reamer. Although apical foramina located distal to the apex were observed in about 50% of the specimens, 83.6% of all apical foramina were within 0.5 mm of the apex, and 99.5% were within 1.0 mm. Data on the thickness of the root and main canal in the apical portion and curvature of the root canal suggest that for adequate apical preparation, a #40 reamer must be able to reach the apical constriction.