Effects of difructose anhydride III on serum immunoglobulin G concentration and health status of newborn Holstein calves during the preweaning period.

This experiment was performed to investigate the effects of increases in passively acquired immunoglobulin G (IgG) by difructose anhydride (DFA) III supplementation on subsequent serum IgG concentration and health status in calves during the preweaning period. Thirty newborn female Holstein calves were paired by birth order, and 2 calves in each pair were fed 2 L of the same batch of colostrum within 2 h and at 10 h after birth, and followed by 2 L of the same batch of pooled colostrum at 20 h after birth. One calf from each pair was assigned to the control (n = 15) or treatment (n = 15) group. All calves in the treatment group received 18 g of DFA III at each feeding from birth to 7 d of age, whereas calves in the control group did not receive DFA III. Blood samples were collected before feeding at 0, 10, 20, and 36 h, and 4 and 7 d of age, and sampling was repeated at 7-d intervals thereafter until 49 d of age for serum IgG analysis. Calves were monitored daily for diarrhea and respiratory diseases. Serum IgG concentrations peaked at 36 h of age in both groups. Apparent efficiency of IgG absorption and peak serum IgG concentration were higher in the treatment group than in the control group. Using multiple regression analysis, we showed that peak serum IgG concentration in the newborn calves was positively correlated with colostral IgG concentration and DFA III supplementation. Moreover, peak serum IgG concentration (36 h of age) positively influenced subsequent serum IgG concentration until 35 d of age for all calves in both groups. The treatment group had higher serum IgG concentration from 20 h to 21 d of age than the control group. However, we detected no differences between the groups in number of calves with diarrhea or respiratory disease.

Breastfeeding: a key modulator of gut microbiota characteristics in late infancy.

The objective of this study was to investigate the impact of the most commonly cited factors that may have influenced infants' gut microbiota profiles at one year of age: mode of delivery, breastfeeding duration and antibiotic exposure. Barcoded V3/V4 amplicons of bacterial 16S-rRNA gene were prepared from the stool samples of 52 healthy 1-year-old Australian children and sequenced using the Illumina MiSeq platform. Following the quality checks, the data were processed using the Quantitative Insights Into Microbial Ecology pipeline and analysed using the Calypso package for microbiome data analysis. The stool microbiota profiles of children still breastfed were significantly different from that of children weaned earlier (P<0.05), independent of the age of solid food introduction. Among children still breastfed, Veillonella spp. abundance was higher. Children no longer breastfed possessed a more 'mature' microbiota, with notable increases of Firmicutes. The microbiota profiles of the children could not be differentiated by delivery mode or antibiotic exposure. Further analysis based on children's feeding patterns found children who were breastfed alongside solid food had significantly different microbiota profiles compared to that of children who were receiving both breastmilk and formula milk alongside solid food. This study provided evidence that breastfeeding continues to influence gut microbial community even at late infancy when these children are also consuming table foods. At this age, any impacts from mode of delivery or antibiotic exposure did not appear to be discernible imprints on the microbial community profiles of these healthy children.

Clinical significance of increased plasma concentration of macrophage colony-stimulating factor in patients with angina pectoris.

OBJECTIVES: To determine the effect of macrophage colony-stimulating factor (MCSF) on atherogenesis in patients with coronary artery disease (CAD), we assessed the relation between the plasma concentration of MCSF and the incidence of acute coronary events in patients with CAD. BACKGROUND: Cytokines such as MCSF play a central role in inflammatory and proliferative responses in patients with acute coronary syndromes. However, the effect of MCSF on the clinical course in patients with CAD is still not known. METHODS: We measured the plasma MCSF concentration in 142 patients with documented CAD (62 +/- 9 years) and followed up for a mean period of 14 +/- 6 months. The study included 97 patients with stable angina (SA), 45 patients with unstable angina (UA) and 22 age-matched control subjects. The predictors of coronary events were analyzed by using a Cox proportional hazards model. RESULTS: The mean plasma MCSF concentration in patients with UA was significantly higher than that in patients with SA and in control subjects (981 +/- 277 vs. 693 +/- 223 vs. 680 +/- 158 pg/ml, p < 0.001). The mean plasma MCSF concentration in the 20 patients with coronary events was significantly higher than that in patients without coronary events (1,192 +/- 232 vs. 690 +/- 213 pg/ml, p < 0.001). The predictors of unfavorable outcome were an increased MCSF concentration, the presence of CAD and a low ejection fraction. CONCLUSIONS: These findings suggest that an increased circulating MCSF concentration reflects atherosclerotic progression in patients with CAD and predicts future cardiac events.

Monoclonal thyroglobulin autoantibodies: variable region analysis and epitope recognition.

A panel of human monoclonal thyroglobulin (Tg) autoantibodies (TgAAb) has been used to analyze autoantigenic determinants on human Tg and to investigate the relationship between variable (V) region gene sequences and epitope specificity. Two monoclonal TgAAb bound to the same (or closely related) epitope on Tg, and these were defined as type I TgAAb. Three other monoclonals bound to a different site and were defined as type II TgAAb. Inhibition studies with mixtures of type I and type II monoclonal TgAAb (Fab)2 preparations indicated that a mixture of the (Fab)2s almost completely inhibited (> 75%) labeled Tg binding to intact TgAAb in the sera of apparently healthy blood donors and patients with autoimmune thyroid disease (AITD). Type I TgAAb predominated in apparently healthy blood donors' sera, whereas type II TgAAb predominated in AITD sera. Analysis of V region gene sequences of the TgAAb indicated that a range of light chain and heavy chain genes from different gene families was used. Furthermore, the same germline genes that are used by TgAAb are also well represented in the genes coding for other self- and nonself-reactive antibodies. No homology in terms of light chain and heavy chain gene families, germline gene usage, or complementarity determining region sequences was observed in TgAAb directed to the same or closely related epitopes. Our studies show that TgAAb are directed to two major conformational epitopes on the Tg molecule and that the proportion of TgAAb directed to these epitopes in apparently healthy blood donors and that in patients with AITD appear to be different. TgAAb derived from different germline genes and with different complementarity determining region sequences can display similar epitope specificity, and this indicates that AAb directed to the same or a closely related epitope show considerable heterogeneity at the molecular level.

Human monoclonal thyroglobulin autoantibodies of high affinity. I. Production, characterisation and interaction with murine monoclonal thyroglobulin antibodies.

Four hybridomas secreting human thyroglobulin (Tg) autoantibodies of different IgG subclasses and light chain types (IgG1 lambda, IgG1 kappa, IgG2 lambda and IgG2 kappa) were obtained by direct fusion of Hashimoto thyroid lymphocytes with the mouse myeloma X63-Ag.653. The autoantibodies were specific for human Tg and the functional affinities were high (only 2.6-3.9 log10 pM Tg required to give 50% inhibition of binding in ELISA). Using thyroid lymphocytes, 4 lines secreting Tg autoantibodies were obtained from 11 fusions compared with 1 line from 32 fusions of Epstein Barr virus infected blood lymphocytes, which emphasises the importance of using lymphocytes derived from a tissue known to be enriched in thyroid autoantibody secreting precursor B cells. These 4 human Tg autoantibodies, as well as an IgG2 lambda Tg antibody previously derived from Hashimoto blood B cells and an IgG4 kappa monoclonal Tg antibody present in a Hashimoto serum, were used in attempts to probe the interaction between human Tg autoantibodies and the Tg molecule (2 polypeptides of 330 KD). The binding to 125-I Tg by 3/7 murine monoclonal antibodies was inhibited (36-78%) by an IgG2 lambda and an IgG4 kappa human monoclonal Tg autoantibody, indicating an overlap between the epitopes recognised by these 3 murine monoclonal Tg antibodies and 2 monoclonal human Tg autoantibodies. None of the human Tg autoantibodies (or the murine monoclonal Tg antibodies) bound to Tg denatured by reduction and alkylation. Although the number of observations is limited, our study demonstrates that high affinity human monoclonal Tg autoantibodies, like polyclonal serum Tg autoantibodies, recognise non-linear B cell epitopes on conformationally intact human Tg.

Human monoclonal thyroglobulin autoantibodies of high affinity. II. Interaction between thyroglobulin and thyroglobulin autoantibodies of different IgG subclasses.

The interaction of human thyroglobulin (Tg) autoantibodies of different IgG subclasses with Tg was investigated using four high affinity human monoclonal thyroglobulin (Tg) autoantibodies, secreted by human-mouse hybridomas, of subclasses IgG1 (kappa and lambda) and IgG2 (kappa and lambda) and an IgG4 kappa serum monoclonal Tg antibody. With exception of a low level of interference in binding between one IgG1 lambda Tg antibody and one IgG2 kappa Tg antibody (27% decrease), binding by human monoclonal Tg antibodies of one IgG subclass was unaffected by pre-incubation of 125-I Tg (or Tg on an ELISA plate) with a human monoclonal Tg antibody of a different IgG subclass. Furthermore, preincubation of Tg-coated ELISA plates with an IgG1 human monoclonal Tg antibody had little effect on binding to Tg by IgG2, IgG3 and IgG4 Tg antibodies present in the sera of 6 Hashimoto patients. Comparable observations were made using an IgG2 monoclonal Tg antibody and serum Tg antibodies of subclasses IgG1, IgG3 and IgG4. Binding of an IgG1 kappa Tg antibody was inhibited (> 80%) by pre-incubation of Tg with an IgG1 lambda Tg antibody derived by fusion of lymphocytes from the same Hashimoto patient. In contrast, pre-incubation of Tg with an IgG2 kappa Tg antibody had little effect on subsequent binding by an IgG2 lambda Tg antibody derived from lymphocytes of a different Hashimoto patient.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between thyroid autoantibody spectrotype and IgG subclass.

The relationship between spectrotype and IgG subclass of autoantibodies to thyroglobulin (Tg) and thyroid peroxidase (TPO) has been investigated using sera from Hashimoto and Graves' patients. Isoelectric focussing (IEF) was carried out in gels over the range pI 3.5-9.5 followed by transblotting to nitrocellulose and probing the filters using 125I-labelled TPO and Tg. As has been shown previously for Tg antibodies, TPO antibody focussed over different pI values in different patients but the spectrotypes for individuals were constant over 2-5 years. Further, the pI values for TPO and Tg autoantibodies appeared to be related to IgG subclass, for example IgG1 Tg antibodies tended to focus nearer the cathode (pI 8.0-9.5) than IgG4 antibodies (pI 6.5-8.5) while antibodies of subclasses IgG1 + 2 + 4 produced spectrotypes covering a broad pI range (5.7-9.5). Consequently, it seems likely that the characteristic spectrotypes described by others for Tg autoantibodies, and those we now report for TPO antibodies, reflect the IgG subclass "fingerprints" which we suggest may be a measure of the ability of an individual to respond to different epitopes on these two thyroid antigens.

Production and characterisation of a human monoclonal thyroid peroxidase autoantibody.

A human-mouse hybridoma has been produced by fusion of Hashimoto thyroid lymphocytes with the mouse myeloma line X63-Ag8.653. The cloned hybridoma secreted 2.5 micrograms per 10(6) cells per day of an IgG kappa thyroid peroxidase (TPO) autoantibody (2G4) with high affinity (2.5 x 10(9) molar-1) and specificity for human TPO. 2G4 did not react with lactoperoxidase, horseradish peroxidase or human myeloperoxidase or with porcine TPO or with human thyroglobulin. Plastic tubes coated with 2G4 bound about 50% of 125I-labelled human TPO added and the binding was inhibited by IgGs prepared from 18/18 TPO autoantibody-positive sera. This indicated that all 18 sera contained autoantibodies which recognised the same (or closely related) epitope as 2G4. Plastic tubes coated with IgGs from different TPO autoantibody-positive patient sera also bound 125I-labelled TPO but inhibition by 2G4 in this system was not complete. This suggested that the sera contained at least 2 types of TPO autoantibodies, with only one type of autoantibody reactive with the same epitope as 2G4.

A human-mouse hybridoma which secretes monoclonal thyroglobulin autoantibody with properties similar to those of the donor patient's serum autoantibody.

Human monoclonal antibodies produced by Epstein Barr (EB) virus transformation and/or cell fusion are frequently IgM antibodies which tend to cross react with a range of antigens and often bear little relationship to the highly specific IgG antibodies associated with human autoimmune disease. By fusing EB virus transformed B lymphocytes from a Hashimoto patient with a mouse myeloma line and selecting for synthesis of IgG class thyroglobulin (Tg) antibody, we have developed a hybridoma (VB/5) secreting Tg antibody of IgG2 subclass and lambda light chain type which has the characteristics of a monoclonal antibody on isoelectric focussing. The antibody has a high affinity for human Tg and recognises Tg from other primates but not non-primate Tg. However, it does not react with human thyroid peroxidase or a panel of other autoantigens. In terms of affinity constant, functional affinity and affinity heterogeneity, the antibody closely resembles the IgG2 lambda Tg antibody present in the serum of the Hashimoto patient whose B lymphocytes were used to develop the hybridoma. In addition to providing a useful reference standard for Tg antibody IgG subclass assays, VB/5 antibody and the hybridoma line provide a valuable starting point for detailed studies of Tg autoantibodies and the genes coding for the variable regions of their heavy and light chains.

Surrogate thyroglobulin receptors and T cell proliferation in Hashimoto's thyroiditis.

Immunoglobulin molecules on the surface of a B lymphocyte are the endogenous "receptors" to which specific antigens bind. Studies in mice have shown that a monoclonal antibody, conjugated with palmitate to provide a lipid tail, can be inserted into the cell membrane to provide a "surrogate" antigen receptor. We have investigated whether a palmitate conjugate of a human monoclonal antibody specific for thyroglobulin (TG) could function as a surrogate TG receptor on blood mononuclear cells separated into fractions enriched for T cells or depleted of T cells (non-T cells). Using flow cytometry, we detected surrogate TG receptors on non-T (but not on T) cells from 11 of 11 individuals studied (5 Hashimoto patients and 6 control donors). In contrast, endogenous TG receptors could only be detected on non-T cells from 1 of 3 Hashimoto patients and from 0 of 4 control donors. Because of the efficient binding of TG by surrogate receptors on non-T cells, we assessed the ability of such cells to present TG to T cells. Proliferation in response to TG was observed in T cells from only 1 of 5 Hashimoto patients. This low frequency of response was no different from that previously detected using cultures of T cells and autologous dendritic cells. Therefore, the successful generation of surrogate receptors on non-T cells is not associated with more efficient TG presentation of T cells. Furthermore, the significance of the present study is that the T cells, not the antigen-presenting cells, are likely to be the limiting element in the T cell proliferative response to TG and other thyroid autoantigens.

Studies on thyrotropin releasing hormone in cerebellar ataxia mutant mouse brain.

The effects of cathecholamine on the regional TRH distribution in the brain was studied in rolling mouse Nagoya (RMN) and non-affected C3H mice. TRH was extracted from the hypothalamus, brain stem, cerebellum, and cerebrum one hour after i.p. injection of the precursor or inhibitors of cathecholamine. TRH was distributed throughout the brain of both affected and non-affected mice; however, in RMN, TRH levels were lower in the hypothalamus and higher in other areas. 1-Dopa caused a decrease of TRH in the brain stem but no change in other regions in the RMN brain, whereas it caused an increase in TRH levels in all areas of the C3H brain. Fusaric acid increased TRH in the hypothalamus of RMN and decreased it in the cerebellum; alpha-MPT also caused a decrease in the TRH level in the cerebellum. Reserpine increased the TRH level in the hypothalamus and decreased it in the cerebrum. From these results, it appears that cerebellar ataxia in RMN does not result from a decrease in the TRH, which is actually increased in the cerebellum. Catecholamine had different effects on TRH levels in RMN and the controls; this might be due to the excess accumulation of noradrenaline in the RMN brain.

Circadian variation of ischemic threshold in patients with chronic stable angina.

Circadian variation of ischemic threshold in chronic stable exertional angina was determined in 51 patients with documented coronary artery disease from the Holter monitor results. The peak favored time zones of ischemic attacks were 8 a.m. and 9 a.m. There was no difference in frequency of ischemic attacks, magnitude of ST-segment depression, or duration of ST-segment depression between the two time zones for ischemic attacks, 6-9 a.m. and 0-3 p.m., but the ischemic threshold was lower in the morning than in the afternoon. These observations suggest that the pathogenesis of ischemic attacks differs from one time zone to the other and is considered helpful in planning therapeutic strategies for myocardial ischemia.

Attachment of Vibrio parahaemolyticus strains to estuarine algae.

Attachment of Vibrio parahaemolyticus strains to estuarine microalgae was examined in artificial seawater by viable counts of the organism and direct counts of the bacterial cells after immunoperoxidase staining. Thermostable direct hemolysin (TDH)-producing and TDH-non-producing strains of V. parahaemolyticus were found to attach to five estuarine strains of Navicula (diatom alga) in similar levels. The level of the bacterial attachment depended on salinity and temperature of the water, in which the maximum attachment was observed in 15% artificial seawater at 25 degrees C, a typical condition of Hashizu estuary in Japan during summer months. The attachment was inhibited by pectinase digestion of the algal cells. These evidences confirmed the participation of the microalgae to the ecological cycle of V. parahaemolyticus at the estuary.

[Cold-induced reversible brain ischemia in mixed connective tissue disease, a case report].

A 19-year-old woman with long-standing mixed connective tissue disease was admitted for dizziness. We examined cerebral blood flow quantitation using 99mTc-hexamethylpropyleneamine oxime (HMPAO) and single photon emission computed tomography (SPECT) at rest and after cold pressor test. Mean cerebral blood flow reduced remarkably when she complained dizziness and showed peripheral Raynaud's phenomenon after cold exposure. We concluded cold-induced reversible brain ischemia was the reason of dizziness. Our finding suggests brain Raynaud's phenomenon. Further studies are necessary to clarify this phenomenon.

Role of perceptive threshold in myocardial infarction patients without previous angina.

The purpose of this study is to clarify the mechanism of sudden onset myocardial infarction (MI) without previous angina and the relationship of MI without previous angina to the mechanism of onset of postinfarction asymptomatic myocardial ischemia. The mean initial time of ischemic pain in the upper arm under the tourniquet test was significantly prolonged in the MI patients without previous angina, compared with that for the MI patients with previous angina and normal control subjects, although there are some overlapping cases (74 +/- 37 sec versus 52 +/- 20 sec (p less than 0.01), and versus 56 +/- 15 sec (p less than 0.05), respectively). The tolerance time for ischemic pain also was similarly prolonged. There was no significant difference between the groups of MI patients (with and without previous angina) with respect to age, frequency of complications of diabetes mellitus, severity of coronary artery lesions or site of MI. The incidence of post-infarction myocardial ischemia was 50% for the previous angina group and 39.5% for the group without previous angina, but the frequency of asymptomatic ischemia was significantly higher in patients without previous angina, at 66.7%, than in those with previous angina, 32.3% (p less than 0.05). These results suggest that there is a close relationship between the mechanism of MI with sudden onset and that of asymptomatic myocardial ischemia during the pre- and post-infarction periods in patients with low sensitivity to pain.

[Cytomegalovirus infection associated with immunosuppressive therapy in collagen vascular diseases].

OBJECTIVE: We investigated the frequency of cytomegalovirus (CMV) infection during immunosuppressive therapy in collagen vascular disease by using CMV antigenemia assay. METHODS: CMV antigenemia in fifteen patients with collagen vascular disease were analyzed before and one month after immunosuppressive therapy with more than 30 mg/day of prednisolone. RESULTS: CMV antigenemia were detected in 9 patients (60%), however no CMV antigenemia detected in all patients before treatment. In 7 of 9 patients CMV infection occurred such as fever, leucocytopenia, thrombocytopenia, liver injury, interstitial pneumonia. In 2 patients of polymyositis/dermatomyositis, creatine phosphokinase (CPK) levels which had decreased once just after treatment started, elevated again although initial dose of prednisolone continued. After ganciclovir administration because of the positive results of CMV antigenemia, elevated CPK levels were normalized immediately. CONCLUSION: Our results showed that CMV infection occurred with high frequency during immunosuppressive therapy, and might mimic the exacerbation of collagen vascular disease. It is important to differentiate CMV infection from increased activity of collagen vascular disease during the treatment.

Suppression of temperature-sensitivity of a dnaA46 mutant by excessive DNA supercoiling.

We report here that the high-temperature sensitivity of a dnaA46 mutant was suppressed by addition of high concentrations of NaCl into the culture medium. This suppression was also observed with other high-temperature-sensitive dnaA mutants, except dnaA167 and dnaA508 mutants, which have mutations in the N-terminal region of DnaA protein. Since high concentrations of NaCl in the medium increased negative DNA supercoiling in a dnaA46 mutant, we hypothesized that the increase in DNA supercoiling is involved in the suppression of the temperature-sensitivity of the dnaA46 mutant by high concentrations of NaCl. This hypothesis was supported by in vitro and in vivo results as follows. A low DNA replication activity of purified DnaA46 protein at high temperatures was increased in line with an increase in DNA supercoiling of template DNA. The dnaA46 mutant showed higher sensitivity to nalidixic acid, a DNA-relaxing drug, than did the wild-type cells under the conditions of high temperatures and high concentrations of NaCl.

Significant characteristics of variant angina patients with associated syncope.

To determine whether syncope predisposes to sudden cardiac death, variant angina patients with syncope during cardiac attacks were compared with those without syncope. There were 240 consecutive patients (193 males and 47 females) diagnosed with variant angina pectoris. Thirty patients had a history of syncope during cardiac attacks while the remaining 210 had none. The incidence of cardiac events in the former group was 10.0% (3 of 30) and 10.5% in the latter. There were 3 cases of sudden cardiac death, all in the latter group. Significant clinical variables in the syncope patients included inferior ST-segment elevation and serious arrhythmias. We conclude that there is no relationship between syncope during variant angina and sudden cardiac death.

[Silent myocardial ischemia in myocardial infarction patients: its prognostic significance].

To evaluate the prognostic and clinical significance of silent myocardial ischemia (SMI), we examined cardiac events in 160 patients with old myocardial infarction who underwent ambulatory Holter monitoring, treadmill exercise testing and coronary angiography. Using the Cox's proportional hazard regression model and the survival curves with the Kaplan-Meier method, we identified the predictors of cardiac events. The incidence of cardiac events for all the patients during the 44-month follow-up period was 18%. The significant predictors of unfavorable outcomes were severe coronary lesions and SMI. The incidence of SMI was 38%. The cardiac event rate in patients with SMI was higher than in those without SMI (32 vs 9%, p < 0.05). The most frequent cardiac event in patients with SMI was reinfarction, and the significant predictors of cardiac events for these SMI patients were lower ejection fraction and maximum ST depression on Holter monitoring. In conclusion, SMI proved to be a significant predictor of unfavorable outcome in patients with old myocardial infarction. It was, therefore, suggested that revascularization (PTCA/CABG) should be used as early as possible in patients with SMI whether anginal symptoms are present or not.