Differentiation of human single-positive fetal thymocytes in vitro into IL-4- and/or IFN-{gamma}-producing CD4+ and CD8+ T cells

In this study we have investigated the capacity of human fetalthymocytes to differentiate in vitro into subsets of T cellswith polarized T_h1 or T_h2 cytokine profiles. Stimulation offreshly isolated human fetal thymocytes with anti-CD3 mAb, cross-linkedonto CD32,CD58,CD80-expressing mouse fibroblasts and subsequentculture in the presence of exogenous rIL-2 for 6 days, inducedthe production of both IL-4 and IFN-, which was mainly producedby CD4⁺ single-positive (SP) and CD8⁺ SP cells respectively.Addition of rIL-4 during priming augmented IL-4 production incultures of human fetal thymocytes, which was mainly due toan increased production of IL-4 by CD8SP cells. In contrast,addition of IL-4 to the cultures only slightly enhanced IL-4production and had little effect on frequencies of IL-4-producingCD4SP cells. Both CD4SP and CD8SP cells produced IL-5, IL-10and IL-13 at comparable levels, following priming in the presenceof rIL-4. Priming in the presence of rIL-12 strongly enhancedthe production of IFN- in both CD4SP and CD8SP cells. No correlationbetween expression of CD27, CD30 and CD60, and a particularcytokine profile of differentiated thymocytes could be demonstrated.Together, these results demonstrate the full capacity of fetalhuman thymocytes to differentiate into cytokine-producing Tcells in a priming milieu with appropriate stimulatory moleculesand exogenous cytokines. In addition, CD4SP thymocytes rapidlydifferentiate into polarized T_h2 cells following stimulationin vitro in the absence of exogenous rIL-4.     

Complex Serology and Immune Response of Mice to Variant High-Molecular-Weight O Polysaccharides Isolated from Pseudomonas aeruginosa Serogroup O2 Strains

The O antigen of the Pseudomonas aeruginosa lipopolysaccharide is the optimal target for protective antibodies, but the unusual and complex nature of their sugar substituents has made it difficult to define the range of these structures needed in an effective vaccine. Most clinical isolates of P. aeruginosa can be classified into 10 O-antigen serogroups, but slight chemical differences among O polysaccharides within a serogroup give rise to subtype epitopes. These epitopes could impact the reactivity of O-antigen-specific antibodies, as well as the susceptibility of a target strain to protective, opsonic antibodies. To define parameters of serogroup and subtype-epitope immunogenicity, antigenicity, and surface expression on P. aeruginosa cells, we prepared high-molecular-weight O-polysaccharide vaccines from strains of P. aeruginosa serogroup O2, for which eight structurally variant O antigens expressing six defined subtype epitopes (O2a to O2f) have been identified. A complex pattern of immune responses to these antigens was observed following vaccination of mice. The high-molecular-weight O polysaccharides were generally more immunogenic at low doses (1 and 10 μg) than at a high dose (50 μg) and usually elicited antibodies that opsonized the homologous strain for phagocytic killing. Some of the individual polysaccharides elicited cross-opsonic antibodies to a variable number of strains that express all of the defined serogroup O2 subtype epitopes. Combination into one vaccine of two antigens that individually elicited cross-reactive opsonic antibodies to most members of the O2 serogroup inhibited, instead of enhanced, the production of antibodies broadly reactive with most serogroup O2 subtype strains. Thus, immune responses to P. aeruginosa O antigens may be restricted to a limited range of epitopes on structurally complex O antigens, and combining multiple related antigens into a single vaccine formulation may inhibit the production of those antibodies best able to protect against most P. aeruginosa strains within a given O-antigen serogroup.It has been established through animal and human experimentation that the lipopolysaccharide (LPS) O antigen of Pseudomonas aeruginosa is a target for protective antibodies (3, 36, 38). The studies of Knirel and colleagues (17, 19) on the chemical composition and structure of the major O-side-chain polysaccharides have provided important insights into the immunochemical properties of these antigens, but our understanding of their antigenic and immunogenic properties is incomplete. This point is highlighted by the inability to date to develop effective, LPS-specific immunotherapies for human P. aeruginosa infection (7).Results obtained with animals by using immunogens and antibodies specific to the O polysaccharides have indicated that slight chemical differences among bacterial strains with otherwise closely related O-side-chain structures can produce a complex pattern of reactions between antibodies and related antigens (13). With standard serologic methods using whole-cell agglutinations, strains of P. aeruginosa can be classified as members of one serogroup (serotype); members of each serogroup share a group-specific antigen. Further subdivision into subtypes, which correlate with structural variants determined by Knirel and colleagues (17), can be accomplished with appropriate antisera (22).To develop safe and effective O-antigen-specific P. aeruginosa vaccines, we have utilized the high-molecular-mass (>100,000-Da) fraction of O polysaccharides. These antigens are safe and immunogenic in humans and animals (13, 27, 37) and elicit protective antibodies to the strains from which they are isolated. However, in recent studies of animals immunized with a heptavalent high-molecular-weight O-polysaccharide vaccine whose individual components were isolated from single strains representative of the major serogroups causing P. aeruginosa infection, opsonic antibody responses to the group-specific antigens were not commonly elicited (13). Thus, in spite of chemical and serologic relatedness among subtype strains within a P. aeruginosa serogroup, single antigens isolated from one subtype strain do not always elicit opsonic antibodies to all of the strains within the serogroup (13). Previous results showed that a particular O antigen from a given serogroup may elicit group-specific immunity, while an O antigen from another serogroup may elicit only immunity specific to the subtype epitopes expressed on that particular O antigen.To explore this situation further and gain additional insight into the serologic diversity among P. aeruginosa LPS O antigens, we prepared high-molecular-weight O-polysaccharide immunogens from five strains of P. aeruginosa serogroup O2 that, together, express all six of the identified subtype antigens (Table ​(Table1).1). These polysaccharides were used to immunize mice, and the resultant sera were assessed by enzyme-linked immunosorbent assay (ELISA) and for opsonic killing activity. The results showed a complex interaction among the strains with regard to high-molecular-weight O-polysaccharide immunogenicity, antigenicity, serogroup and subtype epitope density, and susceptibility to opsonic killing. These findings indicate that the current serogroup classifications of P. aeruginosa are probably inadequate to define the full range of LPS antigens needed to elicit comprehensive immunity to a wide range of clinical isolates.

TABLE 1

Strains used for immunogen production, their serologic classification by subtype epitope, and chemical structures of the associated O antigens Open in a separate window^aBoldface type indicates a feature of a structure that distinguishes it from a related structure of the same serogroup. Abbreviations: FucNAc, 2-acetamido-2,6-dideoxygalactose (N-acetylfucosamine); Man(NAc)₂A, 2,3-diacetamido-2,3-dideoxymannuronic acid; Man(2NAc3N)A, 2-acetamido-3-acetamidino-2,3-dideoxymannuronic acid; Gul(NAc)₂A, 2,3-diacetamido-2,3-dideoxyguluronic acid. ^bThe lower structure is also part of the O antigen of strain 170007; there is about a 2:1 ratio of the upper and lower structures.      

Enhancement of an inhibitory input to the feeding central pattern generator in Lymnaea stagnalis during conditioned taste-aversion learning

To study the neuronal mechanism of a conditioned taste-aversion (CTA) learning in the pond snail Lymnaea stagnalis, we examined the synaptic connection between the neuron 1 medial (N1M) cell and the cerebral giant cell (CGC), the former is an interneuron in central pattern generator for the feeding response and the latter is a regulatory neuron to the central pattern generator. Inhibitory postsynaptic potential (IPSP) which was evoked in the N1M cell by activation of the CGC was larger and lasted longer in the conditioned animal than that in the control animal. The electrical properties of the cell body of CGC and the responses of the CGC to the chemosensory inputs were not changed during the CTA learning. These results, together with the previous report indicating the existence of excitatory projection from the N1M cell to the feeding motoneuron, suggest that enhanced IPSP in the N1M cell may underlie the suppression of feeding responses in the Lymnaea CTA learning.     

The mechanism in the asymmetrically selective oxidation of 3.4-dihydroxyphenylalanine catalyzed by the poly-L-lysine-copper(II) complex

In the oxidation of 3.4-dihydroxyphenylalanine (DOPA) the effect of the asymmetrical structure in poly-L -lysine-copper(II) complex (PLL-Cu(II)) on the asymmetrical selectivity ratio was investigated at various reaction temperatures and various preparation conditions of the catalyst, or by addition of catechol or phenylalanine. From the comparison of the asymmetric selectivity with the structure of the catalyst, the combination of two or more asymmetric cupric complex units was considered as the cause for the asymmetrically selective catalysis, where α-helical structure may serve to hold the two or more cupric complex units in a special steric configuration. Moreover a plausible mechanism for the asymmetrically selective oxidation was discussed by the evidence from the addition effects of catechol or phenylalanine and by the asymmetrically selective interaction between PLL-Cu(II) and DOPA. Then, the mechanism seems to be confirmed by an inspection with a molecular model.     

Induced circular dichroism of biphenyl and its derivatives in mesophase

The mesophase induced circular dichroism (CD) of biphenyl and its derivatives was investigated using the Tween 80 (monooleate of the ether of sorbitan with 4 mol of polyethyleneglycol)-water system, which can form a twisted smectic mesophase. The sign of CD bands in this mesophase is explained by a modified C₂-symmetry rule, since it can be considered that alignment of the solute molecules in this mesophase can be elucidated in terms of a pseudo-C₂ symmetry, when the concentration of the solute is comparatively high. The assignments of the electronic transitions of the solute molecules show a good accordance with results of magnetic circular dichroism study.     

The molecular weight dependence on the photoconductivity of poly(N-vinylcarbazole)

Poly(N-vinylcarbazole), (PNVC), was prepared, fractionated by gel permeation chromatography, and then characterized by viscometry and vapour pressure osmometry. The fractionated PNVC species with relatively narrow molecular weight distributions were successfully used to measure both their electrical dark-conductivity and photoconductivity using a surface type cell in high vacuum (ca. 10^?7 mm Hg) at room temperature. A molecular weight dependent photoconductivity was found for the fractionated PNVCs with weight average molecular weights in the range of 1,2·10³ to 2,4·10⁵. This observation is in contradiction to Epping's results who has found a molecular weight independent photoconductivity in the molecular weight range of 3·10⁵ to 7·10⁶. Our molecular weight dependence may be well understood in terms of the interrupted overlap of the π-electrons of adjacent carbazolyl groups at the terminal parts of the polymer chains, this effect being all the more stronger the smaller the molecular weight is.     

Axillary management based on American college of surgeons oncology group Z0011 criteria makes it possible to omit intraoperative diagnosis of sentinel lymph nodes in early breast cancer patients

The ACOSOG Z0011 trial has resulted in the omission of axillary lymph node dissection (ALND) in early breast cancer patients with one or two metastatic sentinel lymph nodes (SLNs). There has been increasing interest in the necessity of intraoperative assessment of SLNs in patients treated based on the Z0011 criteria. We evaluated the utility of intraoperative assessment in these eligible patients. A total of 1396 patients were treated following the Z0011 criteria from April 2012 to December 2019. We examined the proportion and clinicopathological features of patients who underwent ALND due to three or more metastatic SLNs and the sensitivity of intraoperative assessment. Only 16 (1.1%) patients had three or more metastatic SLNs diagnosed by intraoperative assessment, and they immediately underwent ALND. Of the clinicopathological factors, high clinical tumor stage (p = 0.002) and high Ki-67 labeling index value (p = 0.056) were more likely to be associated with the presence of three or more metastatic SLNs. The major independent risk factor for three or more metastatic SLNs was high clinical tumor stage (OR 3.94 [95% CI 1.42–11.0]; p = 0.009). Intraoperative assessment had low sensitivity (70.5%) and a high false-negative rate (29.5%) in detecting SLN metastases. The main finding of our study was the small proportion of patients who required ALND due to three or more metastatic SLNs according to the Z0011 criteria. The Z0011 strategy enables intraoperative assessment of SLNs to be omitted in early breast cancer patients.     

Development of a PC-based expert system for lower urinary tract pressure-flow studies

Summary An on-line personal computer (PC)-based expert system was constructed for pressure-flow study of the lower urinary tract. An empty expert-system shell and supplementary programs were used for this system; the latter served to compensate the defects of the shell and are referred to as data programs in this report. The data programs were written in the C programming language and were used for on-line data sampling, data entry using a photoscanner, calculation of parameters, saving and loading of data, graphic display, and printing. The urodynamic pressure-flow expert system necessitates on-line data transfer, since it requires a large volume of data. However, off-line data recorded on paper was also made available for ready input by a photoscanner. Reasonable diagnostic accuracy has been achieved by our new expert system for pressure-flow studies. It should be useful in the clinical setting as well as for teaching urological students.Presented at the 19th Annual Meeting of the International Continence Society, Ljubljana, 1989, and published elsewhere as an abstract (Neurourol Urodyn 8:409–410)     

In vitro antibacterial activity of FK041, a new orally active cephalosporin.

The in vitro activity of FK041, a new orally active cephem antibiotic, against a wide variety of clinical isolates of bacteria was investigated and compared with those of cefdinir (CFDN) and cefditoren (CDTR). FK041 exhibited broad spectrum activity against reference strains of Gram-positive and Gram-negative aerobes and anaerobes. FK041 was active against clinical isolates of Gram-positive organisms except Enterococcus faecalis with MIC90s less than 1.56 microg/ml. FK041 was more active than CFDN and CDTR against Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus agalactiae and was comparable to CFDN and CDTR against Streptococcus pyogenes and Streptococcus pneumoniae. FK041 had no activity against methicillin-resistant staphylococci, like CFDN and CDTR. FK041 showed moderate activity against penicillin-resistant S. pneumoniae with an MIC range of 0.05 approximately 3.13 microg/ml, and was superior to CFDN but inferior to CDTR. Against clinical isolates of many Gram-negative organisms such as Neisseria gonorrhoeae, Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis, FK041 had good activity comparable or superior to those of CFDN and CDTR. However, it was inferior to CDTR in activity against Moraxella catarrhalis, Haemophilus influenzae, Morganella morganii, and Serratia marcescens, and was inactive against Pseudomonas aeruginosa. With FK041 a small difference between MIC and MBC against S. aureus, E. coli, K. pneumoniae, and H. influenzae was found, indicating that its action is bactericidal against these species. FK041 was stable to group 2beta-lactamase hydrolysis but was unstable to group 1beta-lactamase hydrolysis. The stability of FK041 to these enzymes was similar to those of CFDN and CDTR. FK041 showed high affinity for the main penicillin-binding proteins (PBPs) of S. aureus (PBP 3, 2, and 1) and E. coli (PBP 3, 4, lbs, 2, and 1a).