The cost of childhood immunisation in general practice

AIM: To estimate the cost, to general practices in the Wellington Immunisation Network, of the audit process of recalling and immunising children according to the New Zealand Immunisation Schedule. METHOD: Practices recorded all clinical and clerical time spent on immunisation as well as the materials used throughout one audit cycle. Staff time and materials were costed directly. Practice overheads were apportioned to immunisation according to the actual time spent on each of the tasks relating to immunisation relative to the total staff hours at the practice. RESULTS: The average cost of immunising a child who attended a non-capitated practice after a single reminder or recall was $15.15. The cost to the practice after taking the practice nurse subsidy and GMS into account was $8.51. The cost of immunising children who were not immunised at the first recall increased in proportion to the number of recall reminders. The annual average cost of immunisation to practices in the study exceeded the revenue obtained from the Immunisation Benefit. CONCLUSION: Overall, given the frequency of recall reminders, there was a net cost to practices for childhood immunisation after deducting the current immunisation benefit rate of $9.78 excluding GST. Thus, the practices in this study made a "loss" in carrying out childhood immunisations.     

Mucosal immunogenicity of genetically detoxified derivatives of heat labile toxin from Escherichia coli

Using a fixed dose of antigen, the immune response to detoxified mutants of LT-WT following intranasal (i.n.), subcutaneous (s.c.) and oral (i.g.) immunisation has been studied. When given i.n., both LT-WT and mutant toxin, K63, generated significant levels of toxin-specific IgG in the serum, and the levels of IgA in nasal and lung lavages were greater than those induced by rLT-B. In comparison, i.g. immunisation of mice with a similar quantity of either LT-WT or K63 toxin induced barely detectable levels of IgG in the sera. However, if the amount of protein used for i.g. immunisation was increased tenfold, relatively good levels of toxin-specific IgG were induced in the sera by both LT-WT or K63. Low levels of toxin-specific IgA were also observed in intestinal washes from these mice. Western blotting of the sera, using the native toxin as an antigen, demonstrated the presence of both anti-A and anti-B subunit antibodies. Most significantly, toxin-neutralising antibodies were induced in the serum, with the strongest activity being induced by the LT-WT, an intermediate activity induced by mutant K63 and a lower response by rLT-B. Together, these data show that ADP-ribosyltransferase is not necessary for mucosal immunogenicity of these proteins, and that the i.n. route of immunisation is more effective than the i.g. route of immunisation for the generation of both systemic (IgG) and mucosal (IgA) immune responses.     

Development of snake antivenom antibodies in chickens and their purification from yolk

Adult white leghorn hens hyperimmunised with Brazilian snake venoms of the genus Bothrops and/or Crotalus produced antibodies capable of recognising, combining with and neutralising the toxic and lethal components of the venoms. The antibodies were first detected by an enzyme-linked immunosorbent assay two weeks after starting the immunisation schedule, reached the highest titres by the third week and remained high for at least 24 weeks. These antibodies are transferred to the egg yolk from which they were isolated as enriched IgY preparations by a combination of methods using positive and negative precipitation with sodium sulphate and/or caprylic acid. The yolk-derived IgY preparations contained antibodies which blocked the phospholipase A2-dependent haemolytic activity of both venoms and the haemorrhagic activity of Bothrops venom, and neutralised the toxic lethal activities of the venoms with good efficacy. The median effective dose (ED50) of the IgY anti-Bothrops venom was 592.5 microliters/2LD50 and, 1.0 ml neutralised 0.0675 mg of venom. The ED50 of the IgY anti-Crotalus venom was 457.5 microliters/3LD50 and 1.0 ml neutralised 0.075 mg of venom.     

Control of hypercalcaemia of parathyroid carcinoma by immunisation

BACKGROUND: Patients with parathyroid tumours can develop extreme hypercalcaemia and osteitis fibrosa cystica. Clinical features result from the action of parathyroid hormone (PTH) on bone receptors. Because this hormone is produced in microgram quantities, inhibition of its metabolic effects with potent PTH antibodies should be possible. We tested whether an immunisation with synthetic human and bovine PTH peptides could stimulate autoantibodies against PTH. METHODS: A patient with metastatic parathyroid carcinoma in the lungs and pleura developed severe bone disease and extreme hypercalcaemia that proved resistant to conventional therapy. She was immunised with 200 microg human and bovine PTH peptides and 50 microg human PTH. Booster doses were also given at 4 weeks and 11 weeks. The patient was then seen every week. FINDINGS: Antibodies against PTH were produced within 4 weeks of initial immunisation and titres increased with repeated doses of immunogens. Total serum calcium concentrations, which had ranged from 3.5 mmol/L to 4.2 mmol/L over the previous 18 months, fell to between 2.5 mmol/L and 3.0 mmol/L over 6 months of therapy. This fall was accompanied by striking clinical improvement. INTERPRETATION: We believe this is the first use of immunotherapy to control remote, non-metastatic complications of malignant disease. B-cell tolerance to human PTH was broken by immunisation with PTH peptides in adjuvant. This therapeutic approach could be used to control excess hormone production in several types of endocrine tumour and may have applications in other diseases.     

Electrophysiologic effects of civamide (zucapsaicin) on canine cardiac tissue in vivo and in vitro

Mucins are highly expressed in many different human cancers and numerous murine monoclonal antibodies (MAbs) to human mucins, particularly Mucin 1 (MUC1), have been produced. However, no such antibodies to murine mucin 1 (muc1) have been described and we now describe 6 different antibodies produced to murine muc1 and to human MUC1 cytoplasmic tail, either by immunising rats, or muc1 o/o mice with synthetic peptides or a fusion protein composed of glutathione-s-transferase (GST) linked to the tandem repeat region of muc1. The antibodies to both the extracellular tandem repeat region and to the cytoplasmic tail were found to react with mucin-containing murine tissues such as breast, stomach, colon, ovary, kidney and pancreas, and the staining patterns were similar to those found in humans. The reagents reacted specifically with muc1 peptides and tissues; however, some cross reactivity with other mucin-derived peptides was noted, particularly those containing the amino acid sequence TSS. Three different epitopes (TSS, TAVLSGTS and LSGTSSP) of the M30, M70 and MFP25 MAbs were detected. Of interest was the finding that some of the antibodies reacted with murine lymphocytes; it was not clear whether these reactions were due to mucin 1 on mouse lymphocytes (MUC1 was considered to be absent from human lymphocyte), or due to cross reaction with a sialic adhesion molecule on lymphocytes. The antibodies should prove valuable reagents when studying differentiation and expression in murine glandular tissues and the ontogeny of mucin-secreting tumours.     

Antigenic determinants of Staphylococcus aureus type 5 and type 8 capsular polysaccharide vaccines

Bacterial capsular polysaccharides (CP) are carbohydrate polymers comprised of repeating saccharide units. Several of these CP have side chains attached to their backbone structures. The side chains may include O-acetyl, phosphate, sialic acid, and other moieties. Those moieties represent the immunodominant epitopes and the most functional ones. The clinically significant Staphylococcus aureus type 5 CP (CP 5) and type 8 CP (CP 8) are comprised of a trisaccharide repeat unit with one O-acetyl group attached to each repeat unit. The immunogenicity of these CP and the functionality of antibodies to the backbone and the O-acetyl moieties were investigated. Immunization with the native CP conjugates (CP with 75% O-acetylation) elicited a high proportion of antibodies directed against the O-acetyl moiety. Nonetheless, all of the vaccinees produced antibodies to the backbone moieties as well. Conjugate vaccines made of de-O-acetylated CP elicited backbone antibodies only. Antibodies to both backbone and O-acetyl groups were found to be opsonic against S. aureus strains which varied in their O-acetyl content. Absorption studies with O-acetylated and de-O-acetylated CP showed that (i) native CP conjugates generated antibodies to both backbone and O-acetyl groups and (ii) O-acetylated isolates were opsonized by both populations of antibodies while the non-O-acetylated strains were predominantly opsonized by the backbone antibodies. These results suggest that S. aureus CP conjugate vaccines elicit multiple populations of antibodies with diverse specificities. Moreover, the antibodies of different specificities (backbone or O-acetyl) are all functional and efficient against the variations in bacterial CP that may occur among clinically significant S. aureus pathogenic isolates.     

Identification and synthesis of altered peptides modulating T cell recognition of a synthetic peptide antigen

In studies of T cell responses to synthetic peptides we have observed agonist and antagonist activities associated with contaminants identified within the parent synthesis. The synthesis of two candidate analogues implied by a peptide contaminant formed during the synthesis of La 51-58 (IMIKFNRL) has been carried out. The peptide contaminant was 17-18 Da smaller than the parent peptide consistent with a modified asparagine residue at position 6 and so we synthesised both an aspartimide and a nitrile analogue, representing cyclisation or dehydration of the asparagine residue. The candidate aspartimide and nitrile analogues both bound empty MHC class I molecules to form allo determinants recognised by monoclonal antibodies. These results demonstrate that altered synthetic peptides can bind class I MHC molecules and prompt caution in the use of synthetic peptides as a source of immunising antigen.     

Affinity analysis of idiotype-positive and idiotype-negative Ars-binding hybridoma proteins and Ars-immune sera

The possibility that idiotype dominance may be associated with increased affinity for hapten was investigated in the murine A/J anti-p-azophenylarsonate (Ars) response. Fluorescence quenching of 14 Ars-binding hybridoma proteins by Ars-tyrosine was measured and Ka calculated using computer-assisted curve fitting. There was a 200-fold range in Ka for idiotype-positive hybridoma proteins, with 2 IgM hybridoma proteins being near the median. No clear difference in Ka was apparent between idiotype-positive (Id+) and idiotype-negative (Id-) hybridoma proteins. Ka was measured by fluorescence quenching on affinity-purified anti-Ars antibodies from 6 conventional antisera; there was no difference between Id+ and Id- (idiotype suppressed) sera. The affinities of the hybridoma proteins were correlated with the ratio of binding to Ars36-BSA and Ars10-BSA by direct radioimmunoassay. With this calibration, functional affinities of Ars-immune sera could be determined from relative binding ratios without the need for prior affinity purification. This was done for 18 Ars-immune sera, and again there was no clear difference between Id+ and Id- sera. Studies from this laboratory have identified the amino acid sequence of a hybridoma protein which corresponds to the germ line DNA sequence for the cross-reactive idiotype family. The present study shows that the protein directly encoded by the germ line gene has low affinity for hapten suggesting that somatic diversification operating on the germ line sequence can produce antibodies with increased affinity for hapten within the cross-reactive idiotype family. The present study also suggests that affinity is not the driving force behind idiotype dominance of the Ars-immune response.     

Cytotoxic effects of islet cell surface antibodies

Antibodies against rat islet cells were produced by immunisation of rabbits with neonatal rat islet cells. In the presence of complement the rabbit anti-rat islet cell surface sera were strongly cytotoxic for both, neonatal rat islet cells and spleen lymphocytes as revealed by the high percentage of 51Cr release from these cells. However, after absorption with rat lymphocytes and rat liver powder the cytotoxicity of the islet cell antisera for rat lymphocytes was drastically reduced while the release of 51Cr from islet cells was only slightly influenced. These data indicate that islet cell specific antibodies were still present in the antisera after absorption. Native normal rabbit serum was also cytotoxic for neonatal rat islet cells and spleen lymphocytes. The release of 51Cr from islet cells and lymphocytes was vigorously reduced after absorption of the normal rabbit serum with lymphocytes and was paralleled by a similar decrease of insulin release from intact islets under conditions where the active insulin secretion was blocked pharmacologically. Intact islets prelabeled with 51Cr were also used as targets but this approach was less suitable for the detection of cytotoxic islet cell surface antibodies.     

Genetic organization, nucleotide sequence and regulation of expression of genes encoding phenol hydroxylase and catechol 1,2-dioxygenase in Acinetobacter calcoaceticus NCIB8250

Hapten refers to a chemical compound of small molar mass (typically less than 1000 daltons) that can bind with an antibody, but cannot initiate an immune response by itself unless it is conjugated to a protein carrier of larger molar mass. A novel method to prepare a hapten to generate anti-hapten immunity without covalent conjugation to a carrier was developed. Coating both water-soluble and -insoluble haptens onto a nitrocellulose membrane effectively presented haptens to the system and caused the generation of specific anti-hapten B lymphocytes and antibodies by immunization both in vitro and in vivo. This method has a potential to substitute for conventional hapten carrier conjugation to generate anti-hapten immunity.     

Techniques for purification of rabbit osteoclasts and analysis of their functions

Autoantibodies to the human thyrotropin receptor (TSH-R) are pathogenic in a number of autoimmune thyroid diseases including Graves' disease. We have characterised polyclonal antisera to TSH-R for antibodies which may mimic those present in autoimmune thyroid disease. For immunisations, recombinant extracellular region of human TSH-R which does not interact with its ligand TSH was used. The induced antibodies react with the full length membrane receptor in transfected mammalian cells by flow cytometry showing the presence of antibody capable of recognising the native functional receptor. The properties of the generated antibodies have been compared after two injections or following a multiple immunisation protocol with the receptor in adjuvant. High titre antisera were readily generated after the short injection protocol and further immunisations did not lead to any change in antibody titers. Analysis of the epitopes recognised using synthetic peptides confirmed previous observations that the immunodominant determinants localise to the amino and the carboxyl terminal part of the extracellular region of the receptor. Antisera from both rabbits contain TSH blocking antibody as assessed by inhibition of TSH mediated cAMP stimulation. There was an increase in TSH binding inhibitory immunoglobulin (TBII) activity with multiple injections. Furthermore, the increase in TBII activity was not related to spreading of the antibody response to new determinants on TSH-R. Our results support previous observations on the difficulties in reproducing, by adjuvant immunisation with recombinant TSH-R preparations, the fine specificity of antibodies to TSH-R present in autoimmune disorders such as Graves' disease or primary myxoedema.     

Anaphylactic IgE and IgG1 production for hapten can be enhanced by contrast media

Various side effects have been associated with the clinical use of contrast media. Immunological mechanisms have been proposed but there have been very few experimental studies with animal models. We have attempted to develop murine models to determine whether or not anaphylactic antibodies such as IgE and IgG1 against hapten (DNP) were enhanced with contrast medium (iopamidol) as an adjuvant or if the contrast medium itself produced antibodies of the IgE class. The results showed that anti-hapten IgE and IgG1 production was greatly enhanced with immunogen plus contrast medium. Anti-contrast medium antibodies of the IgE class could not be detected by PCA reactions. The enhancement of IgE and IgG1 production for hapten was associated with IL-4 release by the neutralization test used by monoclonal anti-IL-4 antibodies. This is the first observation to show that contrast media may have a strong adjuvant effect for the production of IgE and IgG1. This murine model demonstrates a possible immunological function of contrast media in vivo.     

Suppression of anti-hapten antibody response in vitro by hapten-carrier conjugates

Production of antibodies was stimulated or suppressed arbitrarily by antigen treatment in vitro of spleens cultured at various time intervals after in vivo immunization. Spleens of mice immunized to the 2,4-dinitrophenyl or (4-hydroxy-3-iodo-5-nitrophenyl)acetyl haptenic determinants produced antibodies in culture when no antigen was applied in vitro. When a conjugate of the hapten to the same carrier employed for priming was given in vitro, an initial reduction of the response was observed, the level of which was dependent on antigen dose. Subsequently, increased amounts of antibodies were measured. In contrast, in vitro exposure to the hapten conjugated to an unrelated carrier resulted in significant reduction of the response for the entire period of the test. This suppressive effect manifested with various carrier proteins (ovalbumin, bovin IgG, bovine and rabbit serum albumin and keyhole limpet hemocyanin), when when applied to cultures in doses which were potentially immunogenic.     

Specificity of antibodies produced against native or desialylated human immunodeficiency virus type 1 recombinant gp160

In a previous report we have shown that, in contrast to antibodies produced against native or fully deglycosylated human immunodeficiency virus type 1 (HIV-1) gp160 in rabbits, antibodies raised against desialylated HIV-1 gp160 also recognize gp140 from HIV-2 at high titers. Here, we characterize the fine specificity of these cross-reactive antibodies. Inhibition assays with a panel of synthetic peptides as competitors showed that cross-reactivity to gp140 was due to antibodies that were specific for the region encompassing HIV-1 gp41 immunodominant epitope, mimicked by peptide P39 (residues 583 to 609), the latter being able to totally inhibit the formation of complexes between radiolabeled HIV-2 gp140 and antibodies elicited by desialylated HIV-1 gp160. In addition, anti-desialylated gp160 antibodies retained on a P39 affinity column still bound HIV-2 gp140. Fine mapping has enabled us to localize the cross-reactive epitope within the N-terminal extremity of the gp41 immunodominant region. Interestingly, this cross-reactive antibody population did not recognize glycosylated or totally deglycosylated simian immunodeficiency virus gp140 despite an amino acid homology with HIV-1 within this region that is comparable to that of HIV-2. This cross-reactivity between HIV-1 and HIV-2 did not correlate with cross-neutralization. These results illustrate the influence of carbohydrate moieties on the specificity of the antibodies produced and clearly indicate that such procedures may be an efficient way to raise specific immune responses that are not type specific. Moreover, this cross-reactivity might explain the double-positive reactivity observed, in some human sera, against both HIV-1 and HIV-2 envelope antigens.     

Properties of a single-chain antibody containing different linker peptides

Single-chain antibodies were constructed using six different linker peptides to join the VH and VL domains of an anti-2-phenyloxazolone (Ox) antibody. Four of the linker peptides originated from the interdomain linker region of the fungal cellulase CBHI and consisted of 28, 11, six and two amino acid residues. The two other linker peptides used were the (GGGGS)3 linker with 15 amino acid residues and a modified IgG2b hinge peptide with 22 residues. Proteolytic stability and Ox binding properties of the six different scFv derivatives produced in Escherichia coli were investigated and compared with those of the corresponding Fv fragment containing no joining peptide between the V domains. The hapten binding properties of different antibody fragments were studied by ELISA and BIAcoreTM. The interdomain linker peptide improved the hapten binding properties of the antibody fragment when compared with Fv fragment, but slightly increased its susceptibility to proteases. Single-chain antibodies with short CBHI linkers of 11, six and two residues had a tendency to form multimers which led to a higher apparent affinity. The fragments with linkers longer than 11 residues remained monomeric.     

Characterization of morphine-specific monoclonal antibodies showing minimal cross-reactivity with codeine

Six murine monoclonal antibodies against morphine were produced using N-(4-aminobutyl)normorphine as a hapten. Most of the antibodies obtained distinguished the substituents at the 3 and 6 positions of morphine. This property of the antibodies led to a reduction in cross-reactivity with codeine, morphine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G) to negligible levels. However, one of the antibodies distinguished the substituent only at the 3 position of morphine, which cross-reacted with M-6-G, naloxone and naltrexone. In the competitive inhibition enzyme-linked immunosorbent assay, morphine was detected at concentrations as low as circa 100 pg/ml.     

复合轧制SM45钢板的组织性能

 连铸坯直接轧制生产特厚钢板时,由于压缩比的限制,很难生产出厚度超过100 mm的高质量钢板。采用复合轧制工艺可生产出厚度为260 mm的SM45复合钢板。对钢板进行探伤、冷弯、拉伸、冲击及硬度等试验检验其结合度和力学性能。结果表明,复合轧制生产的SM45钢板结合度良好,未发现明显的缺陷存在。钢板复合界面与基体的强度均在600 MPa以上;[Z]向试样的强度也达到600 MPa以上,断面收缩率在30%以上;冲击功在37 J以上。钢板不同位置处的基本组织都为铁素体与珠光体,但晶粒尺寸不同。复合界面处的组织为一条铁素体为主的带状组织,该组织的产生是由先共析铁素体导致的。     

Specific V gene usage in anti-phosphorylcholine IgE antibodies

Three hybridomas from phosphorylcholine(PC)-KLH immunized BALB/c mice producing IgE antibodies against the PC hapten were investigated for their fine specificity to the hapten and usage of V gene segments in H- and L-chains. All three IgE antibodies recognize the entire azophenyl-PC hapten. They are T15 Id negative and do not bind to the natural PC determinant expressed by the Streptococcus carbohydrate R36A. T15 Id positive IgE antibodies could neither be elicited by immunization in detectable amounts nor generated by the cell fusion technique. By using the Southern blot technique and nucleotide sequence analysis of PCR amplified VHDJH and VLJL rearrangements, we have demonstrated that the three IgE anti-PC hybridomas use the VH1-DSP2-JH2, the VHOX1-DSP2-JH3 or the VH36-60-D-JH2 gene segment combinations for the H chain together with the V kappa 1C-J kappa 1, V kappa 1C-J kappa 2 or V lambda 1-J lambda 1 genes for the L chains. Except for the VH36-60, the same gene segments were found in different combinations in anti-PC antibodies of other Ig classes than IgE. However, high rates of somatic mutations are expressed in both VH1 of the H chain and in V kappa 1C of the L chain. The VH36-60 is expressed in antibodies with the major Id of the azophenyl-arsonate (Ars) response and VHOX1 generally contributes to the phenyl-oxazolone specificity. This suggests that these V genes are involved in the recognition of the azophenyl moiety of the coupled PC hapten. Thus PC-KLH specific IgE antibodies utilize mutated VH1 and/or VH/VL gene segment combinations which are involved in binding of the azophenyl spacer. These IgE are therefore specific for azophenyl-phosphorylcholine, unlike antibodies normally expressed against the Streptococcus PC determinant in mice. The genetic diversity and the high mutation rates indicate that the specific B cells develop later in the immune response. Thus, they represent newly generated specificities of so-called group II anti-PC antibodies and are not isotype-switch descendants from already existing T15 Id positive IgM antibodies.     

涂覆制坯对不锈钢/低合金钢复合界面影响

 为探究涂覆铁钴镍夹层法制备不锈钢/低碳钢复合板坯对复合板界面洁净度的影响,运用Procast仿真软件,利用正交试验法,确立夹层涂覆优选工艺参数。依据仿真结果制备带涂覆夹层复合板坯,通过与固态夹层嵌入法制得复合板坯对比,分析不同组坯方式对轧制成形后微观组织和力学性能的影响。试验结果表明,两类组坯方式的复合板夹层均可阻隔碳、铬元素扩散,试样拉剪强度符合相关标准要求;涂覆法较嵌入法能明显提升阻隔铬元素扩散能力,获得更平直的结合界面,夹杂物和氧化物含量也能得到不同程度的改善,其中涂覆法氧元素质量分数(4.1%)显著低于嵌入法(11.1%),涂覆法拉剪强度(531 MPa)高于嵌入法(503 MPa)。     

Antibody catalysis of peptidyl-prolyl cis-trans isomerization in the folding of RNase T1

An antibody generated to an alpha-keto amide containing hapten 1 catalyzes the cis-trans isomerization of peptidyl-prolyl amide bonds in peptides and in the protein RNase T1. The antibody-catalyzed peptide isomerization reaction showed saturation kinetics for the cis-substrate, Suc-Ala-Ala-Pro-Phe-pNA, with a kcat/Km value of 883 s-1.M-1; the reaction was inhibited by the hapten analog 13 (Ki = 3. 0 +/- 0.4 microM). Refolding of denatured RNase T1 to its native conformation also was catalyzed by the antibody, with the antibody-catalyzed folding reaction inhibitable both by the hapten 1 and hapten analog 13. These results demonstrate that antibodies can catalyze conformational changes in protein structure, a transformation involved in many cellular processes.