Prior stimulation of antigen-presenting cells with Lactobacillus regulates excessive antigen-specific cytokine responses in vitro when compared with Bacteroides

The development of allergy is related to differences in the intestinal microbiota. Therefore, it is suggested that the immune responses induced by different genera of bacteria might be regulated through adaptive as well as innate immunity. In this study, we examined whether antigen-specific immune responses were affected by stimulation with the different genera of intestinal bacteria in vitro. Mesenteric lymph node (MLN) cells isolated from germ-free ovalbumin (OVA)-specific T cell receptor transgenic (OVA-Tg) mice were stimulated with OVA and intestinal bacteria. Cecal contents from conventional mice but not germ-free mice could induce OVA-specific cytokine production. Among the murine intestinal bacteria, Bacteroides acidofaciens (BA) enhanced OVA-specific IFN-γ and IL-10 production while Lactobacillus johnsonii (LA) increased OVA-specific IL-10 production only. The expression of cell surface molecules and cytokine production by antigen-presenting cells (APCs) from germ-free Balb/c mice were analyzed. BA increased the expression of MHC II and co-stimulatory molecules on APCs compared with LA. BA increased IL-6 and IL-10 production but induced less IL-12p40 than LA. To examine the effects of prior stimulation of APCs by intestinal bacteria on the induction of antigen-specific immune responses, cytokine production was determined following co-culture with OVA, CD4⁺ T cells from OVA-Tg mice, and APCs which were pre-stimulated with the bacteria or not. APCs pre-stimulated with LA did not enhance OVA-specific cytokine production while BA stimulated OVA-specific IL-10 production. These results suggest that the prior stimulation of intestinal immunocytes by Lactobacillus might regulate excessive antigen-specific cytokine responses via APCs when compared with prior stimulation by Bacteroides.     

Synthesis,spectroscopic characterization and in vitro anticancer activity of new platinum(II) complexes with some thione ligands in the presence of triethylphosphine

Seven new platinum(II) complexes (1–7) of triethylphosphine (Et₃P) and thiones (L) with general formula, cis-[Pt(Et₃P)₂(L)₂]Cl₂ were prepared and characterized by elemental analysis, FTIR and NMR (¹H, ¹³C & ³¹P) measurements. The analytical and spectroscopic data suggested the formation of the desired complexes. The complexes were tested for in vitro cytotoxicity against four cell lines: Hela (human cervical adenocarcinoma), MCF-7 (human breast carcinoma), A549 (human lung carcinoma), and HTC15 (human colon carcinoma). The anticancer activity values of compounds 1–6 are much better than cisplatin and carboplatin as indicated by their IC₅₀ values.     

The proline-rich region of 18.5 kDa myelin basic protein binds to the SH3-domain of Fyn tyrosine kinase with the aid of an upstream segment to form a dynamic complex in?vitro

The intrinsically disordered 18.5 kDa classic isoform of MBP (myelin basic protein) interacts with Fyn kinase during oligodendrocyte development and myelination. It does so primarily via a central proline-rich SH3 (Src homology 3) ligand (T92–R104, murine 18.5 kDa MBP sequence numbering) that is part of a molecular switch due to its high degree of conservation and modification by MAP (mitogen-activated protein) and other kinases, especially at residues T92 and T95. Here, we show using co-transfection experiments of an early developmental oligodendroglial cell line (N19) that an MBP segment upstream of the primary ligand is involved in MBP–Fyn–SH3 association in cellula. Using solution NMR spectroscopy in vitro, we define this segment to comprise MBP residues (T62–L68), and demonstrate further that residues (V83–P93) are the predominant SH3-target, assessed by the degree of chemical shift change upon titration. We show by chemical shift index analysis that there is no formation of local poly-proline type II structure in the proline-rich segment upon binding, and by NOE (nuclear Overhauser effect) and relaxation measurements that MBP remains dynamic even while complexed with Fyn–SH3. The association is a new example first of a non-canonical SH3-domain interaction and second of a fuzzy MBP complex.     

Standardization and Prevalence of the Booster Phenomenon: Evaluation Using a Two-Step Skin Test with 43 kDa Glycoprotein in Individuals from an Endemic Region of Paracoccidioidomycosis

Background

We estimated the occurrence rate of the booster phenomenon by using an intradermal test with 43 kDa glycoprotein in an endemic area of paracoccidioidomycosis in the central-west region of Brazil.

Methods

Individuals who had a negative result on a survey performed by using an intradermal test with 43 kDa glycoprotein in an endemic area of paracoccidioidomycosis underwent a second intradermal test after 10–15 days to determine the presence or absence of the booster phenomenon. Statistical analyses were performed using the Chi-square test, Chi-square for linear trend test, Student’s t test, and binomial test; p < 0.05 was considered significant.

Results

For the first time, we reported the occurrence of the booster phenomenon to an intradermal reaction caused by 43 kDa glycoprotein at a rate of 5.8–8.4%, depending on the test’s cutoff point. This suggests that a cutoff point should be considered for the booster phenomenon in intradermal tests with 43 kDa glycoprotein: a difference of 6–7 mm between readings according to the first and second tests, depending on the purpose of the evaluation.

Conclusion

The results indicate that the prevalence of paracoccidioidal infection in endemic areas is underestimated, as the booster phenomenon has not been considered in epidemiological surveys for this infection.

     

Treatment of non-small cell lung cancer patients with the trifunctional monoclonal antibody catumaxomab (anti-EpCAM <Emphasis Type="Bold">×</Emphasis> anti-CD3): a phase I study

Purpose Catumaxomab is a trifunctional monoclonal antibody with binding sites directed to human EpCAM and the human T cell antigen CD3 (anti-EpCAM × anti-CD3). Catumaxomab demonstrated efficacy when administered intraperitoneally in patients with EpCAM positive malignant ascites from ovarian cancer in terms of tumor cell killing and reduction of ascites generation. As EpCAM is also overexpressed in NSCLC, the present study was conducted in order to evaluate safety and tolerability of intravenous treatment with catumaxomab. Patients and methods UICC stage IB–IV NSCLC patients were eligible, if they had at least one prior therapy. Other inclusion criteria were: age 18–75 years, adequate bone marrow function and AST or gamma-GT ≤ 2.5 ULN. Escalating doses of catumaxomab were administered over 8 h as a continuous intravenous infusion. Dose escalation started at 2 μg and was increased to 7.5 μg. In addition various doses of dexamethasone as premedication were investigated. All patients were pretreated with antihistamines. Results Dose limiting toxicity (DLT) was a grade 3 and 4 elevation of ALT, AST and gamma-GT, which was observed in dose level IV (10 mg dexamethasone premedication, 5 μg catumaxomab) and V (40 mg dexamethasone followed by 7.5 μg catumaxomab). Elevated liver enzymes decreased to grade 2 within 3–7 days and were at baseline level within 14 days after infusion. The maximum tolerated dose (MTD) was defined in dose level III (40 mg of dexamethasone followed by 5 μg of catumaxomab). Conclusions Five micrograms of catumaxomab with a pre-medication of 40 mg dexamethasone and antihistamines can be recommended as first dose for i.v. therapy consisting of multiple catumaxomab infusions for patients with NSCLC.     

A functional polymorphism in the promoter/enhancer region of the<Emphasis Type="Italic"> FOXP3/Scurfin</Emphasis> gene associated with type 1 diabetes

FOXP3/Scurfin, a member of forkhead/winged-helix proteins, is involved in the regulation of T-cell activation, and essential for normal immune homeostasis. The FOXP3/Scurfin gene is located on chromosome Xp11.23, which includes one of the type 1 diabetes susceptible loci. Therefore, we investigated whether the human FOXP3/Scurfin gene might be a new candidate gene for type 1 diabetes. We first screened the human FOXP3/Scurfin gene for microsatellite and single nucleotide polymorphisms. Next, we performed an association study between the FOXP3/Scurfin gene and type 1 diabetes. Then, the evaluation of promoter/enhancer activity of the intron with (GT)(n) polymorphism was performed by dual luciferase reporter assay. We demonstrated two regions contained microsatellite polymorphisms; one was (GT)(n), located on intron zero and the other (TC)(n) on intron 5, which were under linkage-disequilibrium. The (GT)(15) allele showed a significantly higher frequency in patients with type 1 diabetes than in controls (43.1% vs 32.6%, P=0.0027). The genotype frequencies of (GT)(15)/(GT)(15) in female patients and of (GT)(15) in male patients tended to be higher than those in female ( P=0.064) and male ( P=0.061) controls, respectively. A significant difference in the enhancer activity between (GT)(15) and (GT)(16) dinucleotide repeats was detected. In conclusion, the FOXP3/Scurfin gene appears to confer a significant susceptibility to type 1 diabetes in the Japanese population.     

A novel point mutation in an acceptor splice site of intron 32 (IVS32 A–12→G) but no exon 3 mutations in the glycogen debranching enzyme gene in a homozygous patient with glycogen storage disease type IIIb

Genetic deficiency of the glycogen-debranching enzyme (debrancher) causes glycogen storage disease type III (GSD III), which is divided into two subtypes: IIIa and IIIb. In GSD IIIb, glycogen accumulates only in the liver, whereas both liver and muscles are involved in GSD IIIa. The molecular basis for the differences between the two subtypes has not been fully elucidated. Recently, mutations in exon 3 of the debrancher gene were reported to be specifically associated with GSD IIIb. However, we describe a homozygous GSD IIIb patient without mutations in exon 3. Analysis of the patient’s debrancher cDNA revealed an 11-bp insertion in the normal sequence. An A to G transition at position –12 upstream of the 3′ splice site of intron 32 (IVS 32 A^–12→G) was identified in the patient’s debrancher gene. No mutations were found in exon 3. Mutational analysis of the family showed the patient to be homozygous for this novel mutation as well as three polymorphic markers. Furthermore, the mother was heterozygous and the parents were first cousins. The acceptor splice site mutation created a new 3′ splice site and resulted in insertion of an 11-bp intron sequence between exon 32 and exon 33 in the patient’s debrancher mRNA. The predicted mutant enzyme was truncated by 112 amino acids as a result of premature termination. These findings suggested that a novel IVS 32 A^–12→G mutation caused GSD IIIb in this patient. Received: 1 August 1997 / Accepted: 22 September 1997