Rationale Several studies provide indirect evidence that already the prenatal environment plays an important and decisive role for the development of allergy later in life. Since the underlying mechanisms still remain unclear, we established a murine model of prenatal allergen-sensitization and/or allergen-exposure to study the impact of maternal allergy on the development of an allergic immune response in early life. Methods An allergic TH-2 response was induced in pregnant mice by sensitization and aerosol allergen exposure. Results Both, IgG1 and IgG2a, but not IgE antibodies cross the placental barrier. Free allergen also crosses the placental area and was detected in serum and amniotic fluids of neonatal F1 mice. These F1 mice demonstrated a suppressed TH-1 response as reflected by lowered frequencies and reduced levels of IFN- production. Development of an IgE response against the same allergen was completely prevented early in life. This effect was mediated by diaplacental transfer of allergen-specific IgG1 antibodies. In contrast, allergic sensitization against a different allergen early in life was accelerated in these mice. This effect was mediated by maternal CD4 and OVA-specific TH-2 cells induced by allergic sensitization during pregnancy. Conclusions These data indicate a critical role for maternal adaptive immune response in shaping pre- and postnatal development of an allergic immune response.

Apolipoprotein A-IV (apo A-IV) is a 46-kDa plasma protein (1) with different isoforms (2). It is secreted with chylomicrons into the circulation (3). Within the plasma compartment, apo A-IV dissociates from chylomicrons (4) and associates with HDL, triglyceride-rich lipoproteins, and the lipoprotein-free fraction (5). Apo A-IV is influenced by dietary intervention, and a low-fat, low-cholesterol diet (NCEP Step II diet) and is able to decrease apo A-IV secretion rates significantly (6). Apo A-IV is catabolized rapidly with a fractional catabolic rate of ∼2.4 pools/day (7). Apo A-IV is higher in patients with renal …

Many studies have shown carbohydrate-deficient transferrin (CDT) to be a sensitive and specific marker of chronic alcohol abuse. We present the case of a 23-year-old, healthy professional soccer player who caused a car accident due to alcohol consumption. Several CDT test results were elevated above the laboratory reference range and were considered to be caused by alcohol intake at a level commensurate with misuse and thus license reapplication was refused. In addition, assuming chronic alcohol abuse, the young man suffered from increasing social isolation. He was finally referred to our out-patient clinic for further evaluation on the assumption of a liver disease. Since chronic alcohol consumption was denied, and there was no evidence of liver disease, a qualitative characterization of the transferrin isoforms was performed. Isoelectric focusing of serum transferrin revealed a pattern atypical for chronic alcohol intake but detected a genetically determined transferrin (Tf)-D-variant. The changed amino acid sequence caused an overlapping of transferrin isoforms with different degrees of sialylation, thus revealing false-positive serum CDT values. Determination of this Tf-D-variant heterozygosity resulted in his social rehabilitation and license reinstatement. Thus, where the evidence for alcohol dependency is either uncertain or uncorroborated, qualitative isoelectric focusing of transferrin is a useful method for analyzing unexplained CDT elevations, thus increasing the value of CDT as a marker for chronic alcoholic abuse.

A boy with an unspecific symptomatology consisting of mental retardation, strabismus, hypotonia and mild ataxia was diagnosed with a congenital disorder of glycosylation (CDG). Neither cerebellar atrophy nor dysmorphic features were present. The serum transferrin band pattern obtained by isoelectric focusing(IEF) showed a strongly elevated disialotransferrin band together with only slightly elevated asialotransferrin, thus a type I pattern. This is a new CDG classified CDG-x since CDG-la, -b, -c, -d and -e were excluded. Quantitative differences to the type 1 pattern of a CDG-la patient with a moderate to severe course were confirmed by densitometric evaluation of the gels and by SDS gel electrophoresis. Liver biopsy showed lysosomal inclusions suggesting a pre-Golgi defect. This patient's case supports the approach to include isoelectric focusing of serum transferrin in the diagnostic work-up of patients with unexplained symptoms.

We report the clinical findings and the diagnostic work-up of a 17-month-old girl with CDG-x. Predominant clinical signs were, besides psychomotor retardation and truncal hypotonia, stereotyped dystonic hand movements and ophthalmological abnormalities such as optic atrophy, nystagmus and strabismus. Other symptoms that are often found in patients with CDG were not present, such as seizures, microcephaly, cerebellar hypoplasia, dysmorphic features, hepatointestinal disease, coagulopathy or multiorgan involvement. Isoelectric focusing (IEF) of the patient's serum showed a marked elevation of disialotransferrin, thus confirming an IEF type 1 pattern. A generalized glycosylation defect was confirmed also by IEF of a further glycoprotein (α 1 -antitrypsin), an increased carbohydrate deficient transferrin (CDT) serum concentration and an increased CDT/transferrin ratio. All known types of CDG-I, secondary glycosylation abnormalities and variants of amino acid sequence were excluded.

CDG (congenital disorders of glycosylation) ist eine genetisch bedingte Multisystemerkrankung bedingt durch unterschiedliche Enzym- bzw. Transportdefekte an verschiedenen Stellen der Glykoproteinsynthese. Der am häufigsten diagnostizierte Typ ist das CDG-Ia, bei dem neurologisch eine zerebelläre Ataxie dominiert. Bisher gibt es nur geringes Wissen über die Manifestation des CDG-Ia nach der Pubertät. Wir stellen ein erwachsenes Geschwisterpaar vor, bei dem erst im Erwachsenenalter die Diagnose gestellt wurde und bei dem es sich unseres Wissens nach um die ältesten Patienten in Deutschland handelt. Die klinischen Symptome sind zwar typisch, der Krankheitsverlauf jedoch milder als bisher beschrieben. Labordiagnostisch lag der CDT (carbohydrate deficient transferrin)-Wert oberhalb des Referenzbereichs, war jedoch deutlich niedriger als bei anderen CDG-Patienten. Die isoelektrische Fokussierung des Transferrins zeigte einen typischen Befund für ein CDG, die des α1-Antitrypsins geringgradige Veränderungen des Bandenmusters. Dies kann Ausdruck der milderen Verlaufsform sein oder auf eine Stabilisierung der Erkrankung nach der Pubertät hindeuten. Bei der differenzialdiagnostischen Abklärung einer hereditären zerebellären Ataxie sollte im Erwachsenenalter auch das CDG-Syndrom berücksichtigt werden.

8.45–9.00 Opening and welcome 9:00–11:10 Immunology of the male reproductive system, male infertility and contraception 11:10–11:40 Coffee break 11:40–13:00 Aspects of Allergy before and during pregnancy 13:00–14:00 Lunch 14:00–15:15 Autoimmunity and immunological disorders during pregnancy 15:15–15:45 Coffee break 15:45–17:30 Immuno-endocrine interactions 17:30–19:30 Poster session with German beer and brezels

Epidemiological studies indicate the importance of pre‐ and postnatal factors in shaping the T‐cell effector response in early childhood. To study the underlying mechanisms we developed a murine model of prenatal sensitization. BALB/c mice were sensitized to Ovalbumin (OVA) before mating followed by OVA‐aerosol‐exposure during pregnancy (BALB/cOVA). The following key results have been recently obtained: (i) free allergen crosses the placental barrier in pregnant mice; (ii) T‐cells from (BALB/cOVA × BALB/c) F1 demonstrated a lowered frequency and reduced level of IFN‐γ production; (iii) F1 mice were challenged with the heterlogous allergen BLG. The antibody response and type I hyperreactivity reaction were accelerated and augmented in these (BALB/cOVA × BALB/c)F1 [=wild‐type phenotype]. This phenotype depends on the maternal adaptive immune system, since (SCIDOVA × BALB/c)F1 showed a normal immune response. The wild‐type phenotype could be reconstituted in (SCIDOVA × BALB/c)F1 by prenatal transfer of CD4 + OVA T‐cells. These data suggest a critical role for maternal T/B cells in shaping prepostnatal maturation of specific immunity to allergens.

A 1.5-year-old boy with macrocephaly due to a Dandy-Walker malformation presented with progressive hydrocephalus, extensive muscular hypotonia, transient cholestatic syndrome, extensive coagulation abnormalities and elevated creatine kinase indicating myopathy. Diagnostic work-up indicated a congenital disorder of glycosylation (CDG, formerly carbohydrate deficient glycoprotein syndrome). The serum transferrin pattern obtained by automated isoelectric focusing (IEF) showed an hitherto unreported pattern with strongly elevated tri-, di-, mono- and asialotransferrin bands, increasing in this order together with markedly decreased tetrasialotransferrin. Investigation of two additional glycoproteins, alpha(1)-antitrypsin and alpha(1)-antichymotrypsin, confirmed a generalised defect of glycosylation. All known glycosylation defects could be ruled out by enzymatic analyses in either leukocytes or fibroblasts or by the results obtained by IEF. SDS-electrophoresis demonstrated a marked difference in the molecular weight of transferrin, suggesting the lack of parts or of all oligosaccharide chains. The defect could be delineated to a deficiency of beta-1,4-galactosyltransferase (E.C.2.4.1.38) due to a homozygous insertion (1031 - 1032 insC). Details of the biochemical and molecular findings will be described elsewhere.

Congenital disorders of glycosylation (CDG) are a group of hereditary multisystem diseases due to different defects of enzymes or transport molecules involved in the synthesis of glycoproteins. CDG-la is the most common subtype, with cerebellar ataxia as the main neurological symptom. Currently there is little information about CDG-la manifestation in adulthood. Here we present two sisters in whom the diagnosis of CDG-la was made in the fourth decade of life and who to our knowledge are the oldest known patients with the disorder in Germany. The clinical course of the disease was typical, although less severe than previously described. The carbohydrate-deficient transferrin (CDT) level was increased but lower than in other CDG patients. Isoelectric focusing of transferrin revealed changes typical of CDG, whereas those of alpha 1-antitrypsin were only moderately pathologic. This might be due to the milder manifestation of the disease in our patients or it could be indicative of a stabilization of the disease after puberty. The CDG should be included in the differential diagnostic workup of hereditary cerebellar ataxia in adults.

BACKGROUND:The introduction of a new set of reagents for the determination of carbohydrate-deficient transferrin (CDT) as a marker of chronic alcohol abuse requires an independent evaluation of the analytic specificity of the test. This information is needed for correct interpretation and classification of test results. METHODS:Isoelectric focusing on the PhastSystem(TM) followed by immunofixation, silver staining, and densitometry was used to validate the initial transferrin isoform fractionation step on anion-exchange microcolumns involved in the ChronAlcoI.D. assay. RESULTS:The in vitro transferrin iron load was complete and stable. The CDT and non-CDT transferrin fractionation on anion-exchange microcolumns was reliable and reproducible (CV < or = 10%). Except for quantitatively unimportant traces of trisialo-Fe(2)-transferrin (<5% of total CDT), only asialo-, mono-, and disialo-Fe(2)-transferrin were detected in the microcolumn eluates (n = 170). There was a loss of proportionally similar amounts of asialo-Fe(2)-transferrin (during column rinsing) and disialo-Fe(2)-transferrin (on the anion exchanger). Thus, the peak height ratios for disialo- and asialo-Fe(2)-transferrin did not change from >1 (serum) to <1 (eluates) as described for the CDTect assays. The transferrin patterns in the ChronAlcoI.D. eluates were representative of those in serum. Transferrin D variants with isoelectric points close to that of trisialo-Fe(2)-transferrin C1 did not cause overdetermination of CDT by the ChronAlcoI.D. test. CONCLUSIONS:The initial CDT and non-CDT fractionation step involved in determination of CDT by the ChronAlcoI.D. assay is efficient for eliminating non-CDT transferrins from serum before quantification of CDT in the final turbidimetric immunoassay. We recommend IEF for validation of other (commercial) CDT analysis methods and of odd CDT results.

We report the observation of abnormal protein glycosylation in a patient with achondroplasia in whom known causes of impaired glycosylation could not be found. Automated isoelectric focusing (IEF) was performed in our laboratory for the investigation of glycosylation disturbances of transferrin (1)(2). All procedures were carried out in accordance with the Helsinki Declaration of 1975 as revised in 1996. We used surplus serum samples as controls, and surprisingly, in one of those, IEF revealed a moderate increase in disialotransferrin and a slight increase in asialotransferrin (Fig. 1⇓ , lane 1) compared with control serum (Fig. 1⇓ , lane 2). A similar IEF pattern has been described, e.g., in alcohol abuse (3) or in carbohydrate-deficient glycoprotein (CDG) syndrome (4). The patient’s isotransferrin pattern had been stable in different blood samples for more than 3 years. The hypoglycosylation of transferrin was confirmed by an increased carbohydrate-deficient transferrin (CDT) concentration of 44 units/L (upper reference limit, 20 units/L) determined by the CDTect assay (Pharmacia & Upjohn). Figure 1. Fe2-isotransferrin ( Fe 2 -Tf ) band patterns in serum and cerebrospinal fluid ( CSF ) obtained by automated IEF. CSF serves as reference for the localization of isotransferrin bands. Lane 1 , patient’s serum; lane 2 , control serum. Clinically, the patient was a 41-year-old man in good general health. His past history included well-controlled arterial …

Apolipoproteins (apo) C-I, C-II, and C-III play crucial roles in intravascular lipid metabolism. Whereas apo C-II is an obligate cofactor for lipoprotein lipase, apo C-III was shown to inhibit its action. Apo C-I can be a potent cofactor of human lecithin:cholesterol acyltransferase. Structural mutants and deficiencies of apo C-II lead to hypertriglyceridemia. A similar phenotype is associated with apo C-III mutants and is inducible by overexpression of human apo C-III in transgenic animals. No structural variant has so far been reported for apo C-I. The present paper describes a rapid semi-automated procedure for isoelectric focusing analysis of these C-apolipoproteins from whole plasma or serum and their visualization by immunofixation and silver staining. The procedure allows detection of charged variants of C-apolipoproteins. As applied to 295 patients with coronary heart disease and 85 controls, it also serves to detect deficiency syndromes of these apolipoproteins. The procedure provides reliable, easy and quick analysis of C-apolipoproteins applicable as a routine or screening procedure not restricted to specialized laboratories.

The aim of the study was to establish reliable cut-off values, indicating chronic alcohol abuse, of the relative (and absolute) serum carbohydrate-deficient transfemn (CDT) concentrations determined by the ChronAIco I.D.™-assay.Serum samples from 88 women and 48 men, with the daily alcohol consumption, acute or chronic (liver) diseases and medication ascertained by means of a questionnaire, were analyzed.The cut-off values of the CDT/transferrin ratios and the CDT serum concentrations (95 th percentiles) for women consuming < 20 g ethanol/day and men drinking < 50 g ethanol/day were 2.5% and 102 mg/1.Pathological CDT results were confirmed by isoelectric focusing.Taking into account the intra-individual variance of serum CDT and the analytical imprecision, the use of a borderline of 2.5-2.7% as decision criterion indicating chronic alcohol abuse is suggested.

Isoelectric focusing (IEF) and immunofixation of murine serum amyloid P component (SAP), purified and in serum, showed a distinct and strain‐dependent isoform pattern with up to seven bands (pI 5.1–5.7). Neuraminidase treatment caused a shift of the isoforms to more basic pI values, but did not affect their number. When the acute‐phase response was analysed in three mouse strains, CBA/J and C3H/HeN initially showed seven SAP isoforms in serum and C57BL/6 J three or four. The responses in all three strains peaked at day 2 and were normalized within 14 days. On days 2 and 4, CBA/J and C3H/HeN mice showed one more acidic isoform and an increase in the concentration of the most basic isoform. C57BL/6 J mice exhibited two to three new isoforms during the acute‐phase response. This appears to be the first demonstration of the physiological existence of SAP isoforms. In contrast, demonstration of isoforms of human SAP required the presence of urea and higher SAP concentrations. IEF and immunofixation of SAP monomers showed five to eight isoforms, ranging from pI 4.7–5.7. IEF of SAP in human serum resulted in a less distinct pattern and more acidic isoforms. As with murine SAP, neuraminidase treatment caused a shift of the isoforms, but no reduction in isoform number. Two‐dimensional gel electrophoresis confirmed the existence of multiple isoforms of human SAP monomers.