Darstellung und polymerisation von O-vinylacetalen. Untersuchung von O-, S- und N-vinylverbindungen. VI

Formaldehyde- and acetaldehyde-O-vinylacetals were synthesized by dehydrohalogenation of the analogous β-hydroxyethyl-compounds. If initiated radically the monomers do not homopolymerize well but they form regular alternating copolymers with maleic anhydride. In case of the kationic polymerization the O? CH₂? O-groups act as chain transfer agents. On heating the poly-O-vinyl-acetals especially with LEWIS acids a hydride shift takes place, and polymers with ester-side-chains are formed.     

Induction of female sexual behavior by GTP in ovariectomized estrogen primed rats

The effect of both intrahypothalamic and systemic administration of guanosine triphosphate (GTP) on lordosis behavior was studied in ovariectomized and ovariectomized-adrenalectomized, estrogen-primed rats (estradiol benzoate, 4 μg). This estrogen dose per se induced only weak or no lordosis behavior. Injection of GTP into the medial hypothalamic area (100 μg in 2.5 μl) elicited lordosis behavior with relatively short latency in 6 out of 7 rats. Systemic administration of GTP in a dose range of 0.8 mg to 5.0 mg to ovariectomized estrogen-primed rats, stimulated intense lordosis behavior in all subjects. Weak lordosis responses were displayed within the first 12 hr after GTP injection, but at 48 hr all rats were highly estrous. Lordosis behavior remained for up to eight days, its duration being related to the dose of GTP administered. GTP (2 mg) induced lordosis behavior in ovariectomized, adrenalectomized estrogen-primed rats, thus excluding the participation of adrenal steroids in this effect. The results are interpreted in terms of the stimulation of adenyl cyclase-cAMP systems by GTP.     

Multiple Separations in Microfabricated Channels: From Biological Microenvironments to DNA

A continuous sample introduction and separation scheme is presented as an alternative to the current slab gel and microfabricated chip technologies for biological separations. This new technique involves continuous sample introduction via a conventional small bore capillary coupled to a small dimension rectangular channel. Both free zone and size based separations have been carried out in the rectangular channel. Laser induced fluorescence and electrochemical detection schemes have been employed with this technique. Some of the areas this technology has been used to investigate include monitoring dynamic events from microenvironments, monitoring analytes over long periods of time, and performing DNA separations.     

Impeded Th1 CD4 memory T cell generation in chronic-persisting liver infection with Echinococcus multilocularis

Memory T cells of the CD4 lineage coordinate immune responses against pathogens via the antigen-induced secretion of potent effector cytokines. The efficacy of these responses is thought to depend on both the overall number of pathogen-specific memory T cells and the particular array of cytokines that these cells are programmed to secrete. It is unknown to what extent cellular immunity can be induced by Echinococcus multilocularis infection. To examine the immunological memory provided by the adaptive cellular immune system in control of the chronic-persisting infection, peripheral lymphocytes of patients with alveolar echinococcosis (AE) were studied ex vivo. Stimulation of memory cells was performed with E. multilocularis vesicular fluid, purified protein derivative as recall antigen and phytohemagglutinin. Cytomegalovirus latency served as disease control. Frequencies of circulating CD4(+) T cells secreting IFN-gamma, IL-2, tumor necrosis factor-alpha, IL-4, IL-5 and IL-10 were determined by both cytokine flow cytometry and ELISPOT assays. Most strikingly, in chronic AE the frequencies of E. multilocularis antigen-specific cells committed to T(h)1-cytokine production were low (mean 0.5% of CD4(+) T cells). However, an E. multilocularis-specific response of CD4(+) T cells at frequencies of >/=0.1% was detected in the majority of AE patients (68%). Low numbers of cells committed to T(h)1 cytokine secretion were invariably seen in patients with active and inactive disease. Interestingly, the number of specific CD4(+) memory T cells was not increased in cured AE patients after complete surgical removal of the metacestode. Hyporesponsiveness during the chronic helminth infection was E. multilocularis specific. Thus, our results demonstrate that antigen-specific memory function against E. multilocularis is markedly different from that against viral or bacterial pathogens. Whether the antigen-specific cellular hyporesponsiveness with impeded T(h)1 CD4(+) memory T cell generation is a cause or a result of the progressive metacestode activity remains to be determined.     

Activities of Pseudomonas aeruginosa effectors secreted by the Type III secretion system in vitro and during infection

Pseudomonas aeruginosa utilizes a number of distinct pathways to secrete proteins that play various roles during infection. These include the type II secretion system, which is responsible for the secretion of the majority of exoproducts into the surrounding environment, including toxins and degradative enzymes. In contrast, the type III secretion system mediates the delivery of protein effectors directly into the cytoplasm of the host cell. Using tissue culture assays and a mouse acute-pneumonia model, we have determined the contribution of each of the type III effectors during infection. In strain PAK, ExoS is the major cytotoxin required for colonization and dissemination during infection. ExoT confers protection of tissue culture cells from type III-dependent lysis, while ExoY seemed to have little effect on cytotoxicity. ExoU is over 100-fold more cytotoxic than ExoS. The cytotoxicity of type II secretion was determined following deletion of the genes for the more toxic type III secretion system. The participation of these secretion systems during lifelong colonization of cystic fibrosis (CF) patients is unclear. By comparing clonal strains from the same patient isolated at the initial onset of P. aeruginosa infection and more than a decade later, after chronic colonization has been established, we show that initial strains are more cytotoxic than chronic strains that have evolved to reduce type III secretion. Constitutive expression of genes for the type III secretion system restored ExoS secretion but did not always reestablish cytotoxicity, suggesting that CF strains accumulate a number of mutations to reduce bacterial toxicity to the host.     

Analysis of the α/β T-cell receptor repertoire by competitive and quantitative family-specific PCR with exogenous standards and high resolution fluorescence based CDR3 size imaging

The characterization of the human T-cell receptor (TCR) repertoire in various physiological and pathological conditions has become an important tool in studies of the immune response. Therefore, a number of PCR based strategies for the semiquantitative analysis of the TCR repertoire have been described. Family specific amplification of TCR cDNA has been employed in a number of studies often with contradictory results. We have developed a strategy utilizing exogenous standards with homologous primer binding sites for the quantitative analysis of the α/β T-cell receptor repertoire. This system allows the detection of even minute differences in T-cell populations based on quantitative PCR (Q-PCR) and competitive PCR (C-PCR). Results presented here demonstrate that expansions of T-cell subsets as defined by the specificity of the variable gene segments can be readily monitored when exceeding 1% of the total repertoire. In addition, the proposed method reveals direct information of CDR3 size heterogeneity and can be used to estimate the T-cell repertoire complexity and monitor clonal expansions. We discuss variables such as cell number and experimental conditions influencing accuracy and reproducibility of the analyses. We have used this protocol based on non-radioactive techniques for characterization of the fine specificity of the T-cell repertoire in peripheral and organ-infiltrating T-lymphocytes. The analyses revealed information about polyclonal or clonal expansion of T-cells in vivo and in vitro following various stimuli such as superantigenic stimulation of T-cell subsets as well as antigen-driven shaping of the α/β T-cell repertoire in autoimmune and infectious diseases.     

Immunization of Aotus Monkeys with Recombinant Plasmodium falciparum Hybrid Proteins Does Not Reproducibly Result in Protection from Malaria Infection

Plasmodium falciparum antigens SERP, HRPII, MSAI, and 41-3 have shown promise as vaccine components. This study aimed at reproducing and extending previous results using three hybrid molecules. Antibody responses were reproduced in Aotus monkeys, but solid protection from a P. falciparum blood-stage challenge that showed an unintendedly enhanced pathogenicity was not observed.The increasing drug resistance of Plasmodium falciparum, the most pathogenic human malaria parasite, underlines the need for an effective malaria vaccine. Identification, testing, and optimization of candidate molecules originating from all developmental stages of the parasite are under way. Previously, a successful trial in Aotus monkeys employed the Escherichia coli-expressed hybrid proteins MS2/SERP/HRPII and SERP/MSAI/HRPII (11). Both hybrid proteins contain a region of the serine repeat protein SERP (1, 9), including two putative T-cell epitopes (13) and previously shown to induce a partial protective response in Aotus monkeys (5), and the C-terminal half of the histidine-rich protein HRPII (14), which has also been shown to induce a partially protective response (5, 8). SERP/MSAI/HRPII contains in addition a conserved N-terminal region of the merozoite surface antigen MSAI (7) that includes at least four T-cell epitopes (3, 6). Here we report on further analysis of three hybrid proteins of this type in a vaccination trial with Aotus monkeys. Two of the proteins, SERP/HRPII and SERP/MSAI/HRPII, are improved versions of the hybrid proteins mentioned above, obtained by deleting nonmalaria protein regions and changing an internal restart residue (methionine-729 of SERP) into alanine. Thus, the SERP/HRPII hybrid protein comprises residues 630 to 893 of SERP fused to the 189 C-terminal residues of HRPII, and SERP/MSAI/ HRPII comprises residues 630 to 764 of SERP fused to residues 146 to 259 of MSAI, which is fused to the 189 C-terminal residues of HRPII. SERP/41-3/HRPII contains the same components as SERP/HRPII, and additionally includes residues 77 to 188 of antigen 41-3 (10), which was previously shown to confer protection against a P. falciparum challenge (5). The internal restart residue (methionine-100) was also mutagenized into alanine and another residue, arginine-319, was changed into leucine to prevent proteolytic degradation. SERP/MSAI/HRPII was partially purified to a final purity of about 30%, as described previously (8), in order to match the quality of the proteins used in the successful previous trials (5, 11). The other two hybrid proteins were purified from bacterial lysates to over 90% purity by size exclusion chromatography (SERP/41-3/HRPII) or by sequential cation and anion exchange and then size exclusion chromatography (SERP/ HRPII) (data not shown). The final products were dialyzed against phosphate-buffered saline–3 M urea and adjusted to 100 μg of protein per ml. Efficacy was tested following an experimental protocol identical to the one used in the previous successful trial (11).Fifteen laboratory-raised Aotus azarae boliviensis karyotype VI monkeys were randomly assigned to one of four experimental groups (three groups of four and one group of three monkeys) and immunized with 1 ml of antigen or with the diluent alone (control group), both mixed with 100 μl of polyalphaolefin (4) as an adjuvant, on days 0, 21, and 42. Each vaccine dose was administered subcutaneously at two separate sites in the right and left flank and was well tolerated. The seroconversion results, as measured by enzyme-linked immunosorbent assay with SERP/HRPII as the solid-phase antigen and peroxidase-labelled rabbit anti-human immunoglobulin G (1:10,000 dilution; Pierce) as the secondary antibody, are shown in Fig. ​Fig.1.1. All experimental monkeys developed comparable antibody responses to SERP/HRPII, irrespective of the immunogen. Control monkey sera did not react significantly (not shown). A boosting effect is obvious after the second injection in all three groups (Fig. ​(Fig.1),1), as well as after the third SERP/41-3/HRPII injection (Fig. ​(Fig.1C).1C). This is similar to the seroconversion pattern observed previously (11). Prechallenge sera were also tested by immunofluorescence (IFA) for reactivity with P. falciparum schizonts. All preimmune sera and control group immune sera were negative (1:100 dilution). IFA titers from the experimental animals were all 1:1,600, except for animals A381 and A462 (titer, 1:800) and A452 and A292 (titer, 1:3,200). Thus, antibodies specific for native parasite determinants were induced. The relatively low IFA titers were comparable to those obtained in previous successful trials (5, 11). Open in a separate windowFIG. 1Development of antibody responses in Aotus monkeys during the immunization period as determined by enzyme-linked immunosorbent assay. Monkeys in different immunization groups were immunized at weeks 0, 3, and 6 (indicated by arrows) and challenged at week 8 (indicated by an asterisk). All sera were tested for reactivity with SERP/HRPII in a 1:100 dilution. The hybrid antigens used for immunization were SERP/MSAI/HRPII (A), SERP/HRPII (B), and SERP/41-3/HRPII (C). Sera of the three control monkeys remained negative in this assay (not shown). OD, optical density.At week 7 all monkeys were splenectomized, and at week 8 they received intravenously 2 × 10⁶ parasitized erythrocytes, which had been isolated from an Aotus monkey infected with an in vivo-passaged FUP-Cayenne isolate of P. falciparum (a kind gift of W. E. Collins) (Fig. ​(Fig.1).1). Monkey A293 appeared to have no spleen, although there was no prior history of splenectomy. The immunoglobulin G response of monkey A293 was nevertheless comparable to that of the other animals (Fig. ​(Fig.1B).1B). Figure ​Figure22 shows the course of parasitemia after challenge. Two of three control animals rapidly developed a parasitemia which required mefloquine therapy (20 mg/kg of body weight orally) when parasitemia reached 10% (day 8 for A371 and day 10 for A320). In A432, parasitemia developed to 8.6% (day 10) and then fluctuated until rapidly reaching 19% (day 21), at which point mefloquine was administered (Fig. ​(Fig.2A).2A). None of the three immunized groups showed a solid protective response (Fig. ​(Fig.2B2B to D). A340 (SERP/HRPII group) (Fig. ​(Fig.2C)2C) and A292 (SERP/41-3/HRPII group) (Fig. ​(Fig.2D)2D) showed low fluctuating parasitemias with a peak around 2.5% at the end of the observation period (day 25). Otherwise, parasitemias of the experimental animals did not significantly differ from those of the controls. No obvious correlation between prechallenge antibody levels and protection was evident. Open in a separate windowFIG. 2Course of infection with the FUP-Cayenne isolate of P. falciparum in control Aotus monkeys (A) and in Aotus monkeys immunized with SERP/MSAI/HRPII (B), SERP/HRPII (C), and SERP/41-3/HRPII (D). Parasitemias of ≥10% were cured with mefloquine.It seems unlikely that small conservative changes designed to improve SERP/HRPII and SERP/MSAI/HRPII expression in E. coli and to remove nonrelevant sequences adversely affected immune response development. After challenge, parasitemia developed markedly faster than in the previous trial, which had shown protection. Challenge with 2 × 10⁶ parasitized erythrocytes now resulted in high parasitemias on days 7 to 9 in 2 of the 3 controls and in 6 of the 12 experimental animals, whereas previously controls were untreated until day 14 (11). Also, one control and three experimental animals suffered recrudescence, which was not seen previously (11) or with later infections with the same parasite stock (5). It is remarkable that this apparent enhanced pathogenicity developed after a single passage in A. nancymai just before the present trial started. It is likely that this unintended pathogenicity influenced the experimental outcome. The protection of two monkeys in the SERP/HRPII and SERP/41-3/HRPII group may, however, reveal some protective effect of these vaccine candidates.For demonstration of the protective potential of antigens in the primate model the pathogenicity of the challenge strain in the respective primate (sub)species, i.e., the equilibrium of immune response and pathogenicity, seems to be crucial (2, 12). The disturbance of this equilibrium may explain the discrepancy between previous successful trials (5, 11) and the present study. Recombinant proteins shown to be protective in the Aotus model (5, 11) failed to protect Saimiri monkeys, in which the course of parasitemia is quite different from that observed in Aotus monkeys. Similarly, no protection could be demonstrated in A. nancymai against the same challenge strain as was used in the successful trials with A. azarae boliviensis and A. lemurinus griseimembra (7a). The poor standardization of these models due to the scarcity of monkeys susceptible to human malaria remains an obstacle for the evaluation of human malaria vaccine candidates.     

Evaluation of the clonidine-suppression test in the diagnosis of pheochromocytoma

Summary In this study we examined the preoperative value of the clonidine-suppression test in 15 patients with surgically proved pheochromocytomas. The result of the clonidine-suppression test was pathological (epinephrine plus norepinephrine above 500 ng/l 3 h after clonidine) in 10 of 15 patients (66%). These patients had relatively large tumors and higher basal norepinephrine plasma levels. Out of the 5 cases without a pathological clonidine test 4 had normal basal plasma catecholamine levels with the result that the clonidine test could not be properly applied and 1 case produced a false negative result. These 5 cases generally had smaller tumors and lower plasma catecholamine levels. Two of these cases had basally raised epinephrine values. The other three cases had either a paradoxical increase or a suspiciously low fall (less than 25%) in norepinephrine within the normal range. We conclude that the clonidine-suppression test is only reliable for the diagnosis of relatively large pheochromocytomas.Abbreviations MIBG metaiodobenzylguanidine - HPLC High-performance liquid chromatography     

Flush solution 2, a new concept for one-to-three-day hypothermic renal storage preservation. Functional recovery after preservation in Euro-Collins, Collins' C2, hypertonic citrate, and F.2 solution

Experiments were performed on the quality of renal functional recovery after 24, 48, or 72-hr hypothermic storage preservation of canine kidneys in Euro-Collins solution (EC), Collins' solution C2, hypertonic citrate solutions (HC, HC-D2O), or our new flush solution 2 (F.2). Clearance tests (inulin, paraaminohippuric acid, and creatinine) and resorption rates for sodium, potassium, and glucose indicated a high superiority in the early functional recovery of F.2-preserved kidneys after all preservation periods tested. The excellent function after preservation in F.2 contrasted especially with the poor or even absent function after 72-hr preservation in HC and HC-D2O or EC. Thus F.2--a hyperosmolar solution containing sucrose with a balanced Na-K relation on the basis of "heavy water" (D2O)--is especially suitable for preservation up to 72 hr if cyclosporine is used for immunosuppression in the recipient. The recipient can be supplied with an organ with immediate good functional recovery because cyclosporine banishes the higher risk for rejection of these well-functioning organs; simultaneously, the possibility for continuous functional supervision allows avoidance of nephrotoxic side effects from the immunosuppressant.     

Issues and advances in pain control in children

Increased interest in the topic of pain has resulted in pharmacologic advances that provide new possibilities for pain relief in children. It has also resulted in new nonpharmacologic therapies that are now being used more frequently with children. It is hoped that these advances will continue so that health care providers will have a larger repertoire of pain control methods effective with children, and so that the traumatic aspects of hospitalization and health care can be reduced greatly. The pain experienced by children has heretofore been on the backroads of scientific and scholarly development. As a result, we actually know very little about pediatric pain, its measurement, and its treatment. The literature and research cited previously are strikingly limited for providing a base of knowledge to guide clinical practice. We avoided a "cookbook" approach to the discussion on pain control, because there are many of those available. Instead, we presented a summary of the research that is currently available in the attempt to help nurses better recognize the limitations in what we know with certainty about this important topic. We hope that this knowledge will spur readers to examine their own beliefs and knowledge, question former assumptions about pediatric pain, and promote a more inquiring approach to assessment and management of children's pain. Pain is a multidisciplinary problem. Although health care providers from the various disciplines each approach the pain problem from different angles, each approach has its place in the overall picture of solving the problems of pediatric pain control. Nurses have been and will continue to be a vital part of clinical and scientific advancements to move pediatric pain out of the realm of mystery and into the realm of the known.