Observations of a streak texture in the hybrid-aligned smectic-C phase.

A novel structure was observed below the smectic-A-smectic-C phase transition in a very thin open cell having an air interface above and enforced planar anchoring at the substrate below. The structure appears as periodic dark and light streaks running perpendicular to the oily streaks, which are present in the smectic-A phase [D. Coursault et al., Soft Matter, 2016, 12, 678]. These new streaks, which we call "soapy streaks", form by extending from one oily streak to the next in discrete steps, eliminating optical evidence at visible wavelengths of the oily streaks. At lower temperatures the streaks can undulate and exhibit a sawtooth-like structure; such a structure is chiral in two dimensions. A possible scenario for the origin of these streaks is presented.

Immunomodulatory effects of therapeutic preparations of human albumin.

BACKGROUND AND OBJECTIVES: Albumin is the most abundant protein in plasma and is considered to be immunologically inert. However, we recently observed that therapeutic human albumin preparations, used as protein control in studies involving high doses of IVIg, modulated the MHC II-restricted activation of antigen-specific T cells. In the present work, we characterized this effect in more details. MATERIALS AND METHODS: An in vitro antigen presentation assay using mouse cells was used to evaluate the effect of therapeutic human albumin preparations on the activation of ovalbumin-specific T cells. Flow cytometry and quantitative real-time PCR were used to monitor the expression of genes involved in this process. RESULTS: Therapeutic human albumin preparations increased T cell activation in a dose-dependent manner. The effect was explained by an increase in the expression of MHC II and of two other genes (CIITA and H2-M) involved in antigen presentation by murine monocytic cells. Similarly, the expression of HLA-DR on the surface of human monocytic cells was increased following incubation with therapeutic human albumin preparations. CONCLUSION: Altogether, these results reveal a possible physiological role of albumin in immunological processes, leading to an increased ability of antigen presenting cells to trigger T cell activation. This immunomodulatory effect needs to be considered, at least in studies in which albumin is used as a presumably inert control protein.

Set-up and routine use of a database of 10,555 genotyped blood donors to facilitate the screening of compatible blood components for alloimmunized patients.

BACKGROUND AND OBJECTIVES: Large-scale genotyping of blood donors for red blood cell and platelet antigens has been predicted to replace phenotyping assays in the screening of compatible blood components for alloimmunized patients. Although several genotyping platforms have been described, novel procedures and processes are needed to perform genotyping efficiently and to maximize its benefits for blood banks. MATERIALS AND METHODS: Here we describe the processes and procedures developed to introduce large-scale genotyping in our routine operations. RESULTS: Preliminary cost-benefit analysis indicated that genotyping must target frequent blood donors (> 3 donations/year) to be efficiently used. A custom-designed computer application was developed to manage the whole project. It selects frequent donors among recent donations, prints coded labels to identify blood samples sent to the external genotyping laboratory, and stores genotyping results. It can search for donors compatible for any combination of the 22 genotyped antigens as well as consult the current inventory for the presence of the corresponding blood components. The phenotype of recovered components is confirmed by standard serology techniques prior to shipment to hospitals. CONCLUSION: Since October 2007, 10 555 blood donors have been genotyped. The database is used on a regular basis to find compatible blood components with a genotype-phenotype concordance of 99.6%.

Improved leucoreduction of red blood cell units prepared after a 24-h hold with the platelet-rich plasma method using newly developed filters.

BACKGROUND AND OBJECTIVES: A previous study indicated that the extension of whole blood (WB) storage from 8 to 24 h at 20-24 degrees C before the processing of platelet-rich plasma (PRP)-depleted red blood cell (RBC) units had a negative effect on the efficacy of leucoreduction filters. In this study, we further characterized the phenomenon and tested the leucoreduction capacity of two newly developed filters. MATERIALS AND METHODS: Whole blood was stored at 20-24 degrees C and processed at 4-h intervals between 8 and 24 h postcollection. Components were leucoreduced before storage. Efficacy of novel filters to leucoreduce 24-h-hold PRP-depleted RBC units was also evaluated. RESULTS: Using a conventional filter, the mean residual white blood cell (WBC) counts in leucoreduced PRP-depleted RBCs were comparable in units prepared within 12 h from collection but gradually increased upon extended preprocessing storage from 0.36 +/- 0.03 at 12 h to 0.46 +/- 0.21, 0.76 +/- 0.54 and 1.72 +/- 1.76 x 10(6) per unit at 16, 20 and 24 h, respectively. However, the mean residual WBC content in 24-h-hold RBCs was reduced to 0.60 +/- 0.39 x 10(6) and 0.46 +/- 0.13 x 10(6) per units using RC2D and the prototypes B-1582 rev B filters, respectively. CONCLUSION: For PRP-depleted RBC units, the extension of the WB room temperature storage from 8 to 24 h before processing is likely to require the introduction of newly developed filters having an increased leucoreduction capacity in order to meet the maximal residual WBC guideline in the RBCs.

LFA-1 as a key regulator of immune function: approaches toward the development of LFA-1-based therapeutics.

Over the past decade, Lymphocyte Function-Associated Antigen-1 (LFA-1, alphaLbeta2, CD11a/CD18) has emerged as an attractive therapeutic target for the treatment of multiple inflammatory diseases. Its established role in the trafficking and activation of leukocytes coupled with the recent elucidation of the global conformational changes that govern its function continue to drive pharmaceutical interest in this target. This sustained interest has led to the implementation of numerous drug discovery strategies leading to the development of antibodies, peptidomimetics, and small molecules that block LFA-1 function. The most successful demonstration of clinical efficacy to date has been with Raptiva, a humanized anti-LFA-1 antibody. In clinical trials of patients with moderate to severe psoriasis, improvements in several disease specific parameters including the Psoriasis Area and Severity Index (PASI) were observed. This review article will provide an overview of LFA-1 biology and structural regulation, as well as strategies that have been adopted in pursuit of effective therapies. Recent findings with different classes of small molecule antagonists will be highlighted with an emphasis on how their different mechanisms of action on the inserted domain (I domain) of CD11a have impacted our understanding of LFA-1 function and illuminated other potential avenues for therapeutic intervention.

Role of bcl-X(L) in the control of apoptosis in murine myeloma cells.

The development of an increasing number of tumors has been shown to involve the deregulation of not only cell proliferation but also normal cell death by apoptosis. Expression of the bcl-2 proto-oncogene has been shown to inhibit the apoptotic cell death of many types of cells. Recent work also has revealed the existence of several bcl-2-related genes that also can inhibit (e.g., bcl-X(L) and Mcl-1) or activate (e.g., bax, bcl-X(s), bag, and bad) apoptosis in several systems. Myelomas are antibody-secreting tumor cells derived from terminally differentiated B lymphocytes, and previous work from our laboratory showed that murine SP2/0 myeloma cells and derived B-cell hybridomas were highly sensitive to apoptosis induction by a block of gene expression (cycloheximide). Additional work revealed that a related murine myeloma cell line, P3X63Ag8.653, was resistant to apoptosis induction in similar conditions. To understand the genetic basis of this differential susceptibility, we examined the expression of apoptosis-related genes in these cell lines. Northern blot experiments showed no significant difference in the expression of myc and bax apoptosis-promoting genes in susceptible (SP2/0 and D5) and resistant (P3X63) cell lines. Also, no significant expression of the bcl-2 gene could be detected in these cell lines. However, a much higher expression level of bcl-X(L) mRNA was observed in apoptosis-resistant P3X63Ag8.653 cells. The role of bcl-X(L) was supported by the finding that expression of bcl-X(L) cDNA in transfected, apoptosis-sensitive D5 cells increased the viability of these cells greatly and reduced DNA fragmentation following apoptosis induction. Significant bcl-X(L) but not bcl-2 expression was also detected in three other murine myeloma cell lines (MOPC 315, RPC 5.4, and J558) derived from different plasmacytoma tumors. These results indicate a predominant role of bcl-X(L) in preventing apoptosis in myeloma cells and suggest that the expression of bcl-2 or bcl-X(L) genes in B-cell tumors depends on the differentiation stage of the precursor normal cell.

Tolerance to intravenous nitroglycerin in patients with congestive heart failure: role of increased intravascular volume, neurohumoral activation and lack of prevention with N-acetylcysteine.

To better understand the mechanism of nitrate tolerance in patients with congestive heart failure, 13 patients received a 24 h infusion of nitroglycerin (1.5 micrograms/kg body weight per min) with or without N-acetylcysteine (225 mg/kg per 24 h). The infusions were separated by a 24 h nitrate-free interval. By the end of the nitroglycerin infusion, mean arterial pressure had returned to baseline values and there was a significant increase in ventricular filling pressures and systemic vascular resistance compared with values after 1 h of treatment. The simultaneous infusion of N-acetylcysteine had no effect on these changes. Although a strict fluid restriction of 1.5 liters/day was maintained for 1 week before and throughout the study, after 24 h of nitroglycerin infusion there was a significant and similar degree of hemodilution whether nitroglycerin was infused alone (9.1 +/- 4.3%) or with N-acetylcysteine (8.7 +/- 4.1%). This hemodilution corresponded to an increase in intravascular volume of 745 +/- 382 ml, most of which occurred during the 1st h. Plasma renin activity increased and plasma atrial natriuretic peptide decreased during the infusion. The results of this study suggest that nitrate tolerance is multifactorial. In addition to the previously described pharmacologic tolerance to the effect of nitroglycerin on vascular smooth muscle, a capillary fluid shift from the extravascular to intravascular space appears to be involved, especially during the 1st h of the infusion. A third mechanism, reflex neurohumoral activation, also seems to contribute to the genesis of nitroglycerin tolerance.

The combining site of anti-I Ma (group 1).

Evidence is provided that the trisaccharide beta DGal(1----4)beta DGlcNAc(1----6)beta DGal is bound by the monoclonal anti-I Ma antibody beginning with a basically nonpolar cleft at the surface of the protein which comes into contact with a weakly polar region of the trisaccharide that extends from the C-5 methylene group of the reducing unit about the surfaces involving the acetamido grouping and the OH-3' of the beta DGlcNAc unit intramolecularly hydrogen bonded to the O-5'' of the nonreducing beta DGal unit and up to the C-5'' methylene group. The combining site then terminates with a polar grouping at its periphery which is disposed to react with OH-6'' and likely OH-4'' in a highly specific manner. The hydroxyl groups at positions 1, 2, 3, 4, 6', 3'' and 4'' remain in contact with the aq. phase. This conclusion was deduced from the relative potencies as inhibitors of a wide number of synthetic compounds that bear varying structural relationships to the trisaccharide. It appears that the stability of the complex is mainly related to attractive interactions between two large complementary and essentially hydrophobic surfaces relative to those when these surfaces are exposed to water.

Effect of the elapsed time after the final antigen boost on the specificity of monoclonal antibodies produced by B cell hybridomas.

We have studied the effect of the number of days following the last antigen boost on the specificity of monoclonal antibodies produced by B cell hybridomas using spleen cells of mice immunized with human red cells of the A blood group. We showed, as previously observed by others, that the highest numbers of monoclonal anti-human red blood cells were obtained in fusions done 3 and 4 days after the final boost. However differential screening of the hybridoma cultures showed that the majority of the monoclonal antibodies reacting with the A blood group antigen were obtained in fusions done only 2 days after the last antigen injection. These results show that the delay between the final boost and the fusion experiment can influence not only the total number of antibody-secreting hybridomas but also the specificity of the antibodies produced.

Increased proportion of B cell hybridomas secreting monoclonal antibodies of desired specificity in cultures containing macrophage-derived hybridoma growth factor (IL-6).

The addition of macrophage feeder cells or conditioned medium has been shown to increase the yield of murine hybridomas obtained after the fusion of myeloma cells and activated B lymphocytes. It has been shown recently that the conditioned medium contains a growth factor (HGF) active on newly formed hybridomas and that the human HGF is similar to B cell stimulatory factor 2 which can induce the synthesis of antibodies in transformed B cells. We have compared in several fusion experiments the stimulatory effects of HGF both on the yield of hybridomas and on the number of antibody-secreting hybridomas. The results obtained clearly showed that while the stimulatory effect of HGF on the yield of growth-positive wells was variable and sometimes barely detectable, the proportion of growth-positive wells containing monoclonal antibodies was consistently much higher in the HGF-containing cultures. These results suggest that the majority of the antibody-secreting newly formed hybridomas are sensitive to HGF and indicate that HGF is a very useful culture supplement for the generation of a high number of antibody-producing hybridomas even if it may not increase significantly the yield of viable hybridomas.

Efficient preparation of human monoclonal antibody-secreting heterohybridomas using peripheral B lymphocytes cultured in the CD40 system.

The use of peripheral B lymphocytes in the successful preparation of human monoclonal antibodies by hybridoma technology is highly dependent on lymphocyte activation procedures. We studied the ability of peripheral human B lymphocytes cultured in vitro and activated through their CD40 antigen (CD40 system) (Banchereau et al., 1991) to form antibody-secreting heterohybridomas after fusion with murine X63Ag8.653 myeloma cells. The frequency of antibody-secreting heterohybridomas formation was greatly increased (15 times) by culture of B cells in the CD40 system. The CD40 system offers many advantages over other procedures of B lymphocyte activation representing a significant technological advance in the preparation of human monoclonal antibodies by standard hybridoma technology.

Use of hu-IgG-SCID mice to evaluate the in vivo stability of human monoclonal IgG antibodies.

Human and in vitro modified mAbs such as humanized rodent mAbs and immunotoxins are now considered for a variety of applications in humans. The adequate in vivo stability of these Ig preparations is not easily predicted from in vitro studies and may be essential for many therapeutic applications. In this study, we report the development and characterization of an in vivo model for testing this parameter using SCID mice containing a physiological concentration of human IgG (hu-IgG-SCID). The model was tested with several IgG1 and IgG3 human mAbs reacting with the human Rh(D) red cell antigen. It is known that human IgG have a shorter half-life in SCID mice than in humans. However, our results showed that the half-life of IgG3 mAbs (1.5 +/- 0.5 days) was much shorter than the one of IgG1 mAbs (5.8 +/- 1.4 days), indicating that the relative stability of IgG1 and IgG3 human mAbs in hu-IgG-SCID mice is similar to the one previously reported in humans (21 days vs. 7 days respectively). The IgG catabolism rate in humans is known to be inversely proportional to serum IgG concentrations. Accordingly, the dilution of the mAbs in a large excess (200-fold) of human IgG was found to be an important parameter of the hu-IgG-SCID mouse model since much longer (3-4-fold) mAb half-lives were obtained in the presence of a lower dose or in the absence of co-injected human IgG. This study show the usefulness of this animal model for the evaluation of human antibody stability in an in vivo environment.

Scintigraphic "doughnut sign" on skeletal imaging due to a hemangioendothelioma of bone.

A 61-yr-old patient was referred to our hospital for investigation of pain and tenderness in his left lower limb. Bone scan revealed multiple lesions of tibia and foot, several of which appeared as doughnut-like lesions and corresponded to lytic abnormalities on radiographs. Pathologic examination revealed multiple epithelioid hemangioendothelioma of bone.

Technetium-99m glucoheptonate in brain-tumor detection: an important advance in radiotracer techniques.

We have compared [Tc-99m] sodium pertechnetate with Tc-99m glucoheptonate in 52 patients studied for various brain lesions. Flow studies as well as delayed scans were performed in all. Especially in primary and metastatic lesions of the posterior fossa, the diagnostic yield was improved by the delayed glucoheptonate (GH) scans. In contrast, no advantage of GH over pertechnetate could be detected in the study of infarcts or other ischemic lesions. Various hypotheses are discussed to explain the observed differences in behavior between the two tracers.

Lymphocytotoxic definition of combined ABH and Lewis antigens and their transfer from sera to lymphocytes.

Analysis of lymphocytotoxic reactions with peripheral blood lymphocytes from 74 donors, typed for ABO, secretor, and Lewis phenotypes, identified clusters of reactions distinguishing six antigens resulting from the interactions of Lewis, secretor, and ABO systems: Lea, Leb, ALed, BLed, ALeb, and BLeb. In these experiments, Led on O lymphocytes and Lec were not detected as expected from the experience of other authors with A antigen, B and Leb were detected only on the lymphocytes of ABH secretors, demonstrating that all the ABH antigens of lymphocytes are controlled by the secretor system as are the ABH antigens in external secretions. The ABH and Lewis antigens identified on lymphocytes could be transferred in vitro to lymphocytes, cultured for 2 to 7 days at 37 degrees C in the serum of donors of selected ABO, Lewis, and secretor phenotypes, confirming that ABH and Lewis antigens are not synthesized by lymphocytes but are acquired from circulation as are the Lewis antigens on erythrocytes. As expected, the HLA antigens of lymphocytes were not modified after culture.

Noninvasive evaluation of supraventricular tachycardias.

In this article we discuss the role of noninvasive methods in evaluation of supraventricular tachycardias. The limitation of Holter monitoring and exercise testing is discussed. A significant portion of the article is devoted to the role of esophageal recording, body surface potential mapping, and phase image analysis, areas that are often underutilized but that have potential in the diagnosis of supraventricular tachycardias.

Synthetic, conformational, and immunochemical studies of modified Lewis b and Y human blood-group determinants to serve as probes for the combining site of the lectin IV of Griffonia simplicifolia.

Syntheses of the methyl glycosides of the Lewis b [alpha-L-Fuc-(1----2)-beta-D-Gal-(1----3) [alpha-L-Fuc-(1----4)]-beta-D-GlcNAc-] and Y [alpha-L-Fuc-(1----2)-beta-D-Gal-(1----4) [alpha-L-Fuc-(1----3)]-beta-D- GlcNAc-] human blood-group determinants and both their 6a-deoxy and N-deacetylated derivatives are reported. In the case of the Lewis b structure (Leb-OMe), the 6a-O-mesyl and 6a-deoxy-6a-iodo derivatives were also prepared. The conformational preferences predicted by HSEA calculation are shown to be in good agreement with expectations based on 1H- and 13C-n.m.r. spectroscopy. The immunochemical data based on inhibition and thermodynamic studies require that the binding of Leb-OMe and Y-OMe by the lectin IV of Griffonia simplicifolia does not involve recognition of the OMe, NHAc, or 6a-OH group and, consequently, occurs at a cleft at the surface of the protein. The complex formed between the lectin and 6a-deoxy-6a-iodo-Leb-OMe provided the heavy nuclei required for the solution of the X-ray crystal structure.

Synthesis and conformational properties of methyl 6,6-di-C-methyl-beta-D-galactopyranoside. Probes for the combining sites of D-galactosyl-binding proteins.

The binding of D-galactopyranosyl groups by lectins and antibodies can involve the 5-hydroxymethyl group. In order to examine the nature of these binding reactions, it was of interest to synthesize 6,6-di-C-methyl-D-galactose which was found to exist, like D-galactose, extensively in the pyranose forms. 2,3,4,6-Tetra-O-acetyl-7-deoxy-6-C-methyl-alpha-D-galacto-heptopyranosyl bromide was prepared under standard conditions and converted into methyl 6,6-di-C methyl-beta-D-galactopyranoside (6). Evidence based on 13C-n.m.r. studies indicates that the favored conformer of 6 has O-4 and O-6 in syn-axial-like relationship. General comments are presented on the nature of the binding of oligosaccharides by proteins.

Synthesis of type 2 human blood-group antigenic determinants. The H, X, and Y haptens and variations of the H type 2 determinant as probes for the combining site of the lectin I of Ulex europaeus.

Chemical syntheses of the human blood-group antigenic determinants derived from N-acetyllactosamine are described; namely, the 6-deoxy derivative, the 4'-epimer, and the 5 H type 2 [alpha LFuc(1 to 2)beta DGal-(1 to 4)beta DGlcNAc], X [beta DGal(1 to 4)[alpha LFuc(1 to 3)[beta DGlcNAc], and Y [alpha LFuc-(1 to 2)beta DGal(1 to 4)[alpha LFuc(1 to 3)]beta DGlcNAc] determinants as glycosides of 8-carboxymethyloctanol. In order to study the binding of the H type 2 determinant with the lectin I of Ulex europaeus, structures designed to specifically alter the hydrophilic and hydrophobic portions of the H type 2 determinant were also prepared; namely, the 6-deoxy derivative, the 4'-epimer, and the 5"-nor-homolog. The use of these structures, together with the H type 1 hapten and the N-deacetylated forms of both the H type 1 and H type 2 determinants, as inhibitors of the agglutination of O red cells by the lectin allowed the conclusion that the binding of the H type 2 determinant is hydrophobic; the binding involves a wedge-like portion of the determinant that is basically hydrophobic, except for the 5-hydroxymethyl group, which is at the tip of the wedge and forms an intramolecular hydrogen-bond with O-5 for acceptance by a hydrophobic cleft at the surface of the lectin. Blocking procedures involving alkoxymethyl groups and new experiences involving glycosylation reactions are described.

Synthesis of the t [beta-D-Gal-(1 goes to 3)-alpha-D-GalNAc]-antigenic determinant in a form useful for the preparation of an effective artificial antigen and the corresponding immunoadsorbent.

The T antigenic determinant was synthesized in the form 8-methoxycarbonyloctyl 2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-alpha-D-galactopyranoside (6) (beta-D-Gal-(1 goes to 3)-alpha-D-GalNAcO(CH2)8CO2Me). This T-hapten was used to prepare a T-BSA artificial antigen (7) and an immunoadsorbent (8), which were shown to possess the expected immunological properties. Nuclear Overhauser enhancements of the signals for anti-periplanar H-2' and the syn-axial H-3' of the beta-D-galactopyranosyl group were observed on saturation of H-1'. The signal for H-3 of the 2-acetamido-2-deoxy-alpha-D-galactopyranoside residue was also enhanced.