Role of adherence in the pathogenesis of Haemophilus influenzae type b infection in infant rats.

We evaluated the role of pili in the pathogenesis of disease due to Haemophilus influenzae type b (HiTb), using the infant rat model. Piliated and nonpiliated HiTb strains were isolated from the nasopharynx and cerebrospinal fluid, respectively, of three children. Infant rats inoculated intranasally with nonadherent HiTb developed bacteremia and meningitis more frequently (P = 0.005) than animals inoculated with companion adherent HiTb strains. When analyzed separately, only one HiTb pair (884/880) demonstrated significant differences in the incidence of bacteremia and meningitis between the adherent and nonadherent strains. Blood or cerebrospinal isolates recovered from infant rats inoculated with piliated adherent HiTb strains were not piliated and were not adherent in vitro. Adherent and nonadherent HiTb colonized the nasopharynx of infant rats equally. The piliated strains of HiTb were not adherent in vivo or in vitro to rat nasal or buccal epithelial cells, respectively. Piliated strains of HiTb have no apparent advantage over nonpiliated HiTb strains for colonization or invasion of infant rats. Furthermore, the loss of piliation is noted for cerebrospinal fluid, blood, and nasal isolates of HiTb cultured from infant rats inoculated with an adherent piliated HiTb strain. Thus, the loss or suppression of pili may be an important prerequisite for the invasion of the host by HiTb strains that are highly piliated.     

Aberrant T-cell function in vitro and impaired T-cell dependent antibody response in vivo in vitamin A-deficient rats.

We have previously reported that vitamin A deficiency resulted in a reduced IgA antibody response to cholera toxin (CT) after per-oral immunization. In the present investigation we have studied the in vivo and in vitro immune response in vitamin A-deficient rats to two parenterally applied antigens, beta-lactoglobulin (beta-LG) and picrylsulphonic acid (TNP)-Ficoll. The serum IgG and IgM antibody responses to the T-cell dependent antigen beta-LG were significantly lower in the vitamin A-deficient rats than in the pair-fed control rats. No such differences were seen with the IgG and IgM responses to the T-cell independent antigen TNP-Ficoll. However, the biliary IgA and the serum IgE antibodies against both antigens were decreased in the vitamin A-deficient rats. In vitro lymphocyte stimulation with concanavalin A (Con A) or beta-LG gave higher T-cell proliferation rates in the vitamin A-deficient than in the control rats. Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) levels in supernatants from Con A-stimulated mesenteric lymph node cells were also higher in the vitamin A-deficient rats, while IL-6 levels were decreased, which is consistent with an up-regulated Th1 activity. Proliferation studies on purified accessory cells and T cells from the deficient and the control rats, mixed in different combinations, showed that the T cells, but not the accessory cells, were disturbed in the vitamin A-deficient rats. Despite the increased T-cell activity in vitro the vitamin A-deficient rats had a lower delayed-type hypersensitivity (DTH) reaction than the pair-fed control rats. In conclusion, the increased IL-2 and IFN-gamma levels may reflect an up-regulation of Th1 cell function, while the decreased IgA, IgE and IL-6 levels indicate a suppression of Th2 cells. The disturbed T-lymphocyte function is manifested in vivo as a decreased DTH reaction and suppressed antibody production, the latter possibly due to a lack of B-cell switching and proliferation factors in vitamin A-deficient rats.     

Recombinant dissection of myosin heavy chain of Toxocara canis shows strong clustering of antigenic regions

Myosins from nematode parasites elicit strong humoral and cellular immune responses and have been investigated as vaccine candidates. In this study we cloned and sequenced a cDNA coding for myosin heavy chain from Toxocara canis, a nematode parasite of canids which may also infect humans and cause various unspecific symptoms. To determine the major antigenic regions the myosin heavy chain was systematically dissected into ten overlapping recombinant fusion polypeptides which were purified by metal chelate chromatography. Single fragments were then tested for their IgG reactivity in sera from toxocarosis patients and healthy probands. Two regions, one region at the mid to carboxy-terminal end of the head domain and one region in the rod domain, were identified as major antigens, which in combination were positive with 86% of the sera. The other domains were less reactive. This shows that the patients' IgG reactivity was not directed evenly against all parts of the molecule, but was rather clustered in few regions.     

Impaired mucosal antibody response to cholera toxin in vitamin A-deficient rats immunized with oral cholera vaccine.

To investigate the importance of vitamin A in the ability to respond to oral antigen administration, rats were fed a vitamin A-free diet. The animals were immunized perorally three times with a mixture of cholera toxin (CT) and a commercial cholera vaccine. The total immunoglobulin A (IgA) concentration as well as the specific IgA anti-CT antibody levels in serum and bile was significantly lower in the vitamin A-deficient animals than in the paired fed controls (animals that were fed a normal commercial diet in an amount equal to the amount the deficient animals consumed), while the levels of total and specific anti-CT IgG were not affected to the same extent by the vitamin A deficiency. The number of IgA anti-CT antibody-producing cells in the mesenteric lymph nodes after immunization was also significantly lower in the vitamin A-deficient rats than in the control rats. Supplementation of the diet with retinyl palmitate restored the ability to mount an IgA antibody response to the antigen, since the level of specific IgA anti-CT antibodies in relation to the total IgA concentration was as high in the vitamin A-supplemented group as in the paired fed control group. Restricted diet intake by itself did not affect the ability to respond adequately to the antigen since there was no difference in IgA anti-CT antibody level between paired fed rats and those being fed ad libitum. Assessment of transforming growth factor beta in cell cultures revealed no difference between vitamin A-deficient and paired fed animals. In summary, vitamin A deficiency resulted in a decreased number of IgA-producing cells, decreased IgA production, and a reduced ability to respond with IgA antibodies to the oral cholera vaccine.     

Frequency and properties of naturally occurring adherent piliated strains of Haemophilus influenzae type b.

We found that 41 of 75 (55%) children with Haemophilus influenzae type b disease (70 cases of meningitis, 2 of cellulitis, 2 of septic arthritis, and 1 of epiglottitis) and 2 of 120 (1.7%) children with upper respiratory infection were colonized with H. influenzae type b in the nasopharynx (NP). Of these 43 NP strains from children with systemic H. influenzae type b disease, 7 (16%) adhered to human buccal epithelial cells. The strains isolated from the systemic site of all children, including children from whose NP adherent bacteria were isolated, did not adhere to buccal epithelial cells in vitro. Each adherent NP strain had biotype (I), serotype (b), and antibiotic susceptibility (sensitive) similar to that of the corresponding nonadherent systemic isolate. With one exception, all NP-systemic pairs had similar major outer membrane proteins. Six of the seven NP strains had a protein band in the whole cell lysate preparation with a molecular weight between 22,000 and 23,000, which could not be seen in the nonadherent cerebrospinal fluid strains. Electron micrographs of all adherent strains showed that more than 95% of the organisms examined were highly piliated, whereas the nonadherent strains were not piliated. All piliated strains agglutinated human erythrocytes. Adherence to buccal epithelial cells and agglutination of erythrocytes could not be blocked by mannose or alpha-methyl-D-mannoside. We speculate that piliation is not important for NP colonization by H. influenzae type b and that the loss of pili may be required for host invasion.     

Functional expression of chemokine receptor 2 by normal human eosinophils

BACKGROUND: Within the granulocytes, the CC chemokines preferentially activate basophils and eosinophils on binding to chemokine receptors (CCRs). In vivo administration of neutralizing anti-monocyte chemoattractant protein 1 (MCP-1) antibodies can block accumulation of eosinophils in the lungs of antigen-challenged animals. OBJECTIVE: We studied a panel of chemokines for chemotactic activity in normal human eosinophils from healthy donors with a special focus on MCP-1, identified the respective receptor required for the biological response of eosinophils, and investigated mediators used for signal transduction. METHODS: Cells were enriched by magnetic cell sorting. Receptor expression in eosinophils was shown by RT-PCR and fluorescence-activated cell sorting. The biological response was tested in chemotaxis and calcium mobilization assays. RESULTS: Eosinophils have detectable mRNA for CCR2, and the receptor protein is expressed on cell surfaces. MCP-1 induces chemotaxis and calcium mobilization in eosinophils. The chemotactic activity of MCP-1 revealed a double-peaked dose-response curve; one of the peaks is abolished by addition of a blocking antibody to CCR2, but it is insensitive to blocking of CCR1 or CCR3. Specific enzyme inhibitors ruled out signaling characteristics of CCR2 in eosinophils. CONCLUSION: Normal human eosinophils express functional CCR2 on cell surfaces.     

Central nervous system inflammatory response after cerebral infarction as detected by magnetic resonance imaging

Brain inflammation contributes to the tissue injury caused by ischemic stroke. Macrophages as the most abundant inflammatory cell population in stroke lesions can be visualized using ultrasmall superparamagnetic iron oxide (USPIO) as a cell-specific contrast agent for magnetic resonance imaging (MRI). The aim of our present study was to delineate the inflammatory response during experimental cerebral infarction by means of USPIO-enhanced MRI and to correlate the spatial distribution of USPIO-induced MR signal alterations with cellular infiltration and iron deposition. To this end USPIOs were administered to Wistar rats 5 days after photothrombotic cerebral infarction. MR imaging at 7 T performed 24 h later displayed a rim-like signal loss around the infarction in the USPIO treated animals. On histological brain sections obtained from the same animals after MRI the distribution of iron and ED1+ phagocytes was in full spatial agreement with the signal loss seen on T2*-weighted images. Our study validates USPIO-enhanced MRI as an important tool for the noninvasive visualization of brain inflammation in stroke and other CNS pathologies.     

Humoral immune response against a 70-kilodalton heat shock protein of Entamoeba histolytica in a group of patients with invasive amoebiasis.

In order to investigate the humoral immune response against Entamoeba histolytica a lambda ZapII cDNA library was constructed from trophozoites of the pathogenic E. histolytica strain SFL-3. The library was screened with serum IgG from a patient with invasive amoebiasis. Forty-nine immunopositive lambda clones were isolated and partial sequences from the inserts were obtained. By comparison of the sequences with the merged database MIPSX from the Martinsried Institute for Protein Sequences we were able to identify homologous proteins for 36 of the clones. Twenty-six of the clones encoded intracellular proteins, among these, the major part (16 clones) were highly homologous to the eukaryotic 70-kDa heat shock proteins (Hsps). The open reading frame of one complete clone encodes a protein of 656 amino acid residues of 71.5 kDa which has 69.8% sequence identity with the human Hsp70 protein. In a larger screening experiment only 3 out of 12 patients detected with their IgG the phage which expressed the 70-kDa heat shock protein(s).     

Influence of surface hapten density of sheep red blood cells on the hemolytic or blocking activity of IgG antibodies.

The hapten density on the surface of sheep red cells, coated with benzylpenicilloic acid (BPO) was determined. This was achieved by comparison of the inhibitory capacity of the test cells with that of a standard cell preparation of known antigen density. IgG-induced hemolysis was much more affected by the recution of haptenic sites/cell than IgM-induced hemolysis. It was concluded that more than 18,000 protein islets on the sheep red cell surface bearing at least four haptenic sites were necessary for IgG-induced lysis. Target cells with 190,000 BPO groups/cell could be protected by anti-BPO-IgG against the lytic effect of simultaneously added IgM. Using target cells with 240,000 haptenic groups/cell or more a synergistic hemolytic action of IgG-and IgM-anti-BPO antibodies could be observed.