Salivary IgA subclasses and bacteria-reactive IgA in patients with aggressive periodontitis.

The local salivary immunoglobulin A (IgA) response in patients with aggressive periodontitis to oral microorganisms and its role for the pathogenesis has not been determined. This study investigated the hypothesis that aggressive periodontitis patients have impaired oral secretory immunity. Our test group was made-up of 19 aggressive periodontitis patients and 19 age- and gender-matched periodontally healthy controls. Total IgA, IgA subclass 1, IgA subclass 2 and IgA reactive to Actinobacillus actinomycetemcomitans Y4, Treponema denticola ATCC 35404 and Candida albicans DSM 3454 were determined by enzyme-linked immunosorbent assay in whole unstimulated and stimulated saliva. A statistically significantly lower concentration and secretion rate of total salivary IgA (P < 0.01) and IgA1 (P < 0.001) was found in the aggressive periodontitis group in resting and stimulated saliva. A decrease of IgA2 (P < 0.05) was seen in resting saliva. Although only minor differences were detected in the concentration and secretion of bacteria-reactive IgA in both groups, the proportion of bacteria-reactive IgA from the total IgA was significantly higher (P < 0.01) in the aggressive periodontitis group in all three microorganisms tested. Our results indicate an inhibition of total secretory IgA. In particular an IgA subclass 1-specific decrease in aggressive periodontitis was noted, while the bacteria-reactive humoral immune system in saliva was activated. The role of the decrease of IgA1 immunoglobulins in aggressive periodontitis with respect to susceptibility for periodontal diseases has to be elucidated.

Structural and functional stability of the mature transferrin receptor from human placenta.

The transferrin receptor (TfR) is a N- and O-glycosylated transmembrane protein mediating the cellular iron uptake by binding and internalization of diferric transferrin. In this study, rate constants and dissociation constants of 125I-ferri-transferrin binding to the human TfR were examined dependent on receptor glycan composition, pH, bivalent cations, and temperature. To do so, purified human placental TfR was noncovalently immobilized to polystyrene surfaces and subjected to alterations in various parameters. We found that transferrin binding was clearly dependent on a receptor pretreatment with buffers of various pH in that most of the TfR molecules irreversibly lost transferrin binding activity below pH 6.5. However, the dissociation constant of the remaining active binding sites was not affected. Similarly, we were able to define the thermal stability of the receptor as a function of transferrin binding ability. Binding of transferrin was completely lost provided that the receptor was pretreated at temperatures of at least 65 degrees C. Treatment with EDTA also caused an irreversible loss of transferrin binding activity, indicating that the functionally active conformation of the mature TfR depends on bivalent cations. In order to examine the role of the receptor glycans, we enzymatically removed the sialic acid residues, the hybrid and oligomannosidic N-glycans, or all types of N-glycans. In contrast to the parameters described above, all desialylated and N-deglycosylated TfR variants had exactly the same transferrin binding properties as the native TfR. To assess changes in the secondary structure of the receptor, circular dichroic spectra were recorded from TfR at pH 5.0, from heat pretreated receptor and from deglycosylated TfR. Since the receptor did not exhibit detectable changes in the CD spectrum of the deglycosylated receptor, it can be concluded that the N-linked carbohydrates of the mature, fully processed TfR are not essential for transferrin binding and conformational stability.

Identification of novel proteins associated with the development of chemoresistance in malignant melanoma using two-dimensional electrophoresis.

A model system for studying chemoresistance in human melanoma cells (MeWo) has been established utilizing the four commonly used cytotoxic drugs vindesine, cisplatin, fotemustine and etoposide to yield stable drug-resistant sublines. We analyzed phenotypical differences between MeWo cells and their chemoresistant counterparts using two-dimensional electrophoresis. Proteins that were overexpressed in chemoresistant cell lines were purified and identified using matrix assisted laser desorption/ionization-time of flight - mass spectrometry (MALDI-TOF-MS) and microsequencing. Here we show that four proteins, namely the translationally controlled tumor protein, the human elongation factor 1-delta, tetratricopeptide repeat protein and the isoform 14-3-3-gamma of the 14-3-3-family are overexpressed in chemoresistant melanoma cell lines. The significance of these findings is now being verified using transfection experiments with the aim of developing more effective chemotherapy protocols.

Total IgA and Porphyromonas gingivalis-reactive IgA in the saliva of patients with generalised early-onset periodontitis.

Generalised early-onset periodontitis (GEOP) is characterized by acute inflammatory bursts, resulting in rapid destruction of the periodontal apparatus in young adults. An impaired host defense seems to play an important role as etiological factor of periodontitis, especially in the development of GEOP. As the gram-negative Porphyromonas gingivalis has been identified as one of the causative anaerobic bacteria, the humoral immune response to this micro-organism is of particular interest in patients with GEOP. To evaluate the local immune status, we measured total and P. gingivalis-reactive salivary IgA in GEOP patients and in age- and gender-matched periodontally normal controls. We found a significantly lower concentration and secretion rate of total salivary IgA in the GEOP group. Although no differences were detected in the concentration or secretion of P. gingivalis-reactive IgA between groups, the specific fraction of P. gingivalis-reactive IgA of the total IgA was significantly higher in the GEOP group. These findings indicate an inhibition of total secretory IgA in GEOP, while the P. gingivalis-reactive humoral immune system in saliva is, however, activated. P. gingivalis seems to selectively activate IgA lymphocyte clones and induces a switch in the fraction of specific IgA.

Increased expression of epidermal fatty acid binding protein, cofilin, and 14-3-3-sigma (stratifin) detected by two-dimensional gel electrophoresis, mass spectrometry and microsequencing of drug-resistant human adenocarcinoma of the pancreas.

In order to study possible mechanisms leading to chemoresistance in pancreatic adenocarcinoma we examined the global protein expression of pancreatic cancer cells in vitro. We used a cell culture model derived from the adenocarcinoma of the pancreas (EPP85-181P). A classical multidrug-resistant subline, EPP85-181RDB, selected in presence of daunorubicin, and an atypical multidrug-resistant cell variant, EPP85-181RNOV, selected in presence of mitoxantrone, were analyzed using two-dimensional electrophoresis. After staining and image analysis, spots of interest were isolated using preparative two-dimensional electrophoresis and subjected to mass spectrometry and microsequencing. Three proteins, E-FABP, cofilin, and 14-3-3-sigma (stratifin), were overexpressed in chemoresistant cell lines. Cofilin was present in both multidrug in chemoresistant cell lines. Cofilin was present in both multidrug-resistant cell lines. E-FABP and 14-3-3-sigma (stratifin) was found to be overexpressed only in the mitoxantrone-selected atypical multidrug-resistant cell line. The possible significance of these findings is discussed.

Search for novel proteins involved in the development of chemoresistance in colorectal cancer and fibrosarcoma cells in vitro using two-dimensional electrophoresis, mass spectrometry and microsequencing.

In search of novel mechanisms that may lead to the development of chemoresistance of malignant tumors of the large bowel we used two-dimensional electrophoresis to identify proteins that were overexpressed in colorectal and fibrosarcoma cell lines that were resistant towards mitoxantrone. This cytostatic drug is known to lead to atypical multidrug resistance, i.e., the classical mechanism of multidrug resistance (MDR) accompanied by the overexpression of P-glycoprotein (P-gp) is ineffective. Using mass spectrometry and microsequencing we found adenine phosphoribosyl transferase and breast cancer specific gene 1 (BCSG1) overexpressed in the resistant colorectal tumor cell line. In the chemoresistant fibrosarcoma cell line we found two proteins that were overexpressed. One was identified as Rho-guanine dinucleotide phosphate (Rho-GDP) dissociation inhibitor and the other had sequence homologies with yeast protein yer-7. The putative role of these proteins is discussed.

HFE gene mutation and transferrin saturation in very low birthweight infants.

AIM: To determine if there is an association between high transferrin saturation and the C282Y HFE gene mutation in very low birthweight (VLBW) infants. METHODS: One hundred and forty three VLBW infants receiving recombinant erythropoietin and 3 to 9 mg/kg/day of enteral iron were studied. Genomic DNA was extracted from filter paper cards. The C282Y mutation was determined by restriction fragment length polymorphism analysis. RESULTS: Six infants were heterozygous for the mutation; none was homozygous. Ten infants had a transferrin saturation above 80% at least once. No infant was positive for both transferrin saturation above 80% and the mutation. CONCLUSIONS: The data strongly suggest that there is no association between high transferrin saturation and the HFE gene mutation in VLBW infants during the first weeks of life.

Fetal fibronectin as a marker for an imminent (preterm) delivery. A new technique using the glycoprotein lectin immunosorbent assay.

We have developed a new 'glycoprotein lectin immunosorbent assay' (GLIA) which permits the obstetrician to identify accurately pregnant women at risk for preterm delivery. This GLIA uses two lectins for the quantitative detection of glycosylation variants of fibronectins, namely, Maackia amurensis lectin (MAA) for the detection of fetal fibronectin (fFN), and Sambucus nigra lectin (Elderberry bark lectin; SNA). Fibronectin was quantitated in cervicovaginal secretions, amniotic fluid, and plasma of pregnant women. Detection of fFN in cervicovaginal secretions was considered to indicate a high risk of imminent delivery. The results were as follows: (1) The GLIA could differentiate between pregnant women after the onset of labour and/or with rupture of membranes and women without any signs of an imminent delivery (sensitivity 94%, specificity 96%, p < 0.001). (2) Differentiation was possible between asymptomatic pregnant women delivering within 10 days of sampling or after more than 10 days (sensitivity 93%, specificity 99%; p < 0.001). (3) If fFN was present in the cervicovaginal secretions, delivery occurred within 10 days of sampling irrespective of preterm delivery or delivery at term (p < 0.001). Thus, this GLIA is a useful assay for identifying those asymptomatic pregnant women who will deliver within 10 days of sampling.

Increased expression of annexin I and thioredoxin detected by two-dimensional gel electrophoresis of drug resistant human stomach cancer cells.

The therapy of advanced cancer using chemotherapy alone or in combination with radiation or hyperthermia yields an overall response rate of about 20-50%. This success is often marred by the development of resistance to cytostatic drugs. Our aim was to study the global analysis of protein expression in the development of chemoresistance in vitro. We therefore used a cell culture model derived from the gastric carcinoma cell line EPG 85-257P. A classical multidrug-resistant subline EPG85-257RDB selected to daunorubicin and an atypical multidrug-resistant cell variant EPG85-257RNOV selected to mitoxantrone, were analysed using two-dimensional electrophoresis in immobilized pH-gradients (pH 4.0-8.0) in the first dimension and linear polyacrylamide gels (12%) in the second dimension. After staining with coomassie brilliant blue, image analysis was performed using the PDQuest system. Spots of interest were isolated using preparative two-dimensional electrophoresis and subjected to microsequencing. A total of 241 spots from the EPG85-257RDB-standard and 289 spots from the EPG85-257RNOV-standard could be matched to the EPG85-257P-standard. Microsequencing after enzymatic hydrolysis in gel, mass spectrometric data and sequencing of the peptides after their fractionation using microbore HPLC identified that two proteins annexin I and thioredoxin were overexpressed in chemoresistant cell lines. Annexin I was present in both the classical and the atypical multidrug-resistant cells. Thioredoxin was found to be overexpressed only in the atypical multidrug-resistant cell line.

A case of Crohn's disease with increased CD8 T-cell activation and remission during therapy with intravenous immunoglobulins.

BACKGROUND: Crohn's disease (CD) represents a chronic inflammatory bowel disease with abnormal CD8 T-cell function in a subgroup of patients. METHODS: A 55-year-old woman presented with CD on the basis of clinical, endoscopic, radiologic, histologic, and sonographic examination. Since the disease could not be controlled with conventional anti-inflammatory therapy, a detailed analysis of cellular and humoral immune functions was performed and showed a dysbalanced T-cell activation pattern with an inverse CD4/CD8 ratio due to an increased number of CD8 T cells. Additionally, high IgM and low IgG2 antibody levels were detected. Treatment with intravenous immunoglobulin (IVIG) was started as immunomodulatory therapy. During this therapy the condition improved markedly. CD8 T-cell levels returned to normal, and IgM decreased as well. CONCLUSION: This case shows that selected cases of CD may be associated with abnormal functions and that such patients may benefit from IVIG treatment.

K-ras mutations in stools and tissue samples from patients with malignant and nonmalignant pancreatic diseases.

Mutant-enriched PCR and reverse dot blot hybridization in microplates were applied for examining K-ras status in stools and tissue samples from patients with pancreatic tumors and chronic pancreatitis. In tissue samples, K-ras mutations were found in 32 of 35 cases of ductal adenocarcinoma, in 5 of 7 periampullary cancers, in 1 cystadenocarcinoma, and in 3 of 5 patients with chronic pancreatitis. In stools, mutated K-ras was seen in 10 of 25 cases of ductal adenocarcinoma, in 1 case of cystadenocarcinoma, and in 2 of 6 cases of chronic pancreatitis. These data indicate that the K-ras status of stool samples may help identify pancreatic carcinoma and persons at risk for cancer development; however, it does not allow discrimination of malignant from nonmalignant diseases.

Epithelial uptake and transport of cell-free human immunodeficiency virus type 1 and gp120-coated microparticles.

Cell-free human immunodeficiency virus type 1 (HIV-1) can be taken up and released by a monolayer of primary human gingival cells and remain infectious for CD4+ cells. Virus-sized latex particles covalently coated with purified native HIV-1 envelope glycoprotein gp120 are also transported through the primary epithelial cells. This process is significantly stimulated by increasing the intracellular cyclic AMP (cAMP) concentration. Inhibition experiments with mannan and alpha-methyl-mannopyranoside indicated that mannosyl groups are involved in the interaction between gp120 and gingival cells. An increase of cellular oligomannosyl receptors by incubation with the mannosidase inhibitor deoxymannojirimycin augmented transcellular transport of the gp120-coated particles. The results suggest that infectious HIV can penetrate gingival epithelia by a cAMP-dependent transport mechanism involving interaction of the lectin-like domain of gp120 and mannosyl residues on glycoproteins on the mucosal surface. Penetration of HIV could be inhibited by soluble glycoconjugates present in oral mucins.

A human-SCID mouse model for allergic immune response bacterial superantigen enhances skin inflammation and suppresses IgE production.

Chronic skin colonization with Staphylococcus aureus is a well-known feature in atopic dermatitis. The aim of this study was to develop a human-SCID mouse model to analyze the possible role of bacterial superantigens in human allergic immune responses under in vivo conditions. SCID mice were reconstituted with peripheral blood mononuclear cells (between 2 and 9 x 10(7) cells per mouse) from atopic dermatitis patients sensitized to house dust mite allergen (Der p). Total and Der p specific antibody production required the following conditions: (i) injection of Der p; (ii) presence of CD14+ antigen-presenting cells; and (iii) IL-4 as shown by the inhibitory effect of human soluble IL-4 receptor on immunoglobulin E production. This model was used to study the immunomodulatory effects of the superantigen staphylococcal enterotoxin B in comparison with Der p. In intraperitoneally reconstituted human-SCID mice, topical treatment was ineffective in inducing skin inflammation. Therefore, additionally to intraperitoneal transfer, peripheral blood mononuclear cells from atopic donors were also injected intradermally. Such reconstituted SCID mice were then exposed via the skin to either Der p, staphylococcal enterotoxin B, or a combination of both. Maximal effects on epidermal inflammation and dermal T cell infiltration were obtained with staphylococcal enterotoxin B and Der p. Staphylococcal enterotoxin B alone was less effective and Der p only stimulated dermal T cell infiltration. These findings support the hypothesis that bacterial superantigens can act as trigger factors in allergic skin inflammation.

Verrucosis of hands and feet in a patient with combined immune deficiency.

In immunocompromised patients, warts occur frequently and can be extensive. We describe a 24-year-old patient with severe therapy-resistant warts. In addition to human papillomavirus infection, he had chronic sinusitis, candidiasis, and atopic dermatitis. Anergy to delayed-type hypersensitivity skin test reaction, significant CD4 lymphopenia, and diminished in vitro T-cell proliferative response and interferon-gamma production indicated a deficiency of cellular immunity. Extremely low concentrations of serum IgM and IgG2 and a severe deficiency of in vitro IgM production pointed also to a humoral immunodeficiency syndrome. This case represents a combination of cellular and humoral immunodeficiencies that has not been previously described in association with warts.

In an epidemiological sample the apolipoprotein E4 allele is associated to dementia and loss of memory function only in the very old.

The apolipoprotein E4 allele has been reported to be associated with late onset Alzheimer's disease. Here we report the relation of several neuropsychological test parameters and the diagnosis of dementia to the apolipoprotein E polymorphism in an epidemiological sample of 477 subjects aged 70-103 years. The apolipoprotein E4 allele was found to be associated with reduced performance in several sensitive neuropsychological memory tests and with diagnosis of dementia only in the oldest subjects (> 84 years). The association with dementia in this population based sample was much weaker than previously described and became only significant in a logistic regression analysis when age was included in the model.

The release of cytokines, adhesion molecules, and extracellular matrix parameters during and after reperfusion in human liver transplantation.

Poor initial graft function may increase postoperative morbidity including the risk of early allograft rejection. Various mediators, including immunostimulatory cytokines, may be released during reperfusion in relation to the extent of preservation and reperfusion injury. For this purpose, 81 patients with 85 liver transplants were monitored for cytokines, adhesion molecules, extracellular matrix (ECM) parameters, and neopterin at predefined time-points during and after transplantation. To estimate the origin of cytokine release, blood was obtained central and hepatic venously for the first 48 hr after reperfusion and subsequently from a peripheral vein. One-year patient survival was 88.9%; no relation to initial graft function was observed. Poor initial graft function failed to increase the risk for subsequent infectious complications but was associated with an increased risk of early allograft rejection. The incidence of steroid-resistant rejection was significantly increased in patients with poor initial graft function (35.7% versus 12.7% in patients with good and moderate initial graft function; P < or = 0.05). Various cytokines, adhesion molecules, and ECM parameters including sTNF-RII, sIL-2R, IL-8, IL-10, sVCAM-1, E-selectin, hyaluronic acid, sialic acid, and laminin correlated significantly with the extent of preservation and reperfusion injury. Although none of these parameters was more appropriate in determining the extent of preservation and reperfusion injury than currently established parameters (AST, ALT, and color and amount of bile production), the combined increase in these parameters may not only promote tissue repair but may also perpetuate liver allograft injury and thereby cause significant morbidity. Besides cytokines and adhesion molecules, the ECM may play a pivotal role in determining repair or ongoing tissue injury. Ongoing changes at the microvasculature and basement membrane may result in an increase of local and circulating cytokines and adhesion molecules, which increase the risk of subsequent early allograft rejection. Furthermore, the increase in sTNF-RII, E-selectin, and laminin during reperfusion was predictive of subsequent development of acute allograft rejection. These observations may be of value for further strategies to decrease reperfusion injury and prevent early allograft rejection.

The relevance of murine animal models to study the development of allergic bronchial asthma.

Bronchial asthma (BA) develops on the basis of a genetic predisposition and involves a characteristic sequence of changes in immune functions. In the immunopathogenesis, several phases can be distinguished: the initial stage is defined as the development of allergic sensitization. This step is dependent on: (i) T cell activation; (ii) IL-4 production; (ii) IgE synthesis; and (iv) mediator release by effector cells. The second phase of allergic inflammation as a consequence of the T cell dependent sensitization is characterized by IL-5 production and eosinophil activation and recruitment. Airway mucosa remodelling is the consequence of chronic inflammatory processes and represents the final stage of BA. In this article animal models will be discussed with regard to their relevance for these different phases in development of chronic allergic BA.