Effects of macrophage colony-stimulating factor (M-CSF) on anti-fungal activity of mononuclear phagocytes against Trichosporon asahii

Trichosporon asahii is an emerging opportunistic pathogen in immunocompromised patients. Little is known about the mechanisms of host defence against T. asahii. We investigated the fungicidal activity of human peripheral blood monocytes and murine peritoneal macrophages against T. asahii isolates, and the effects of M-CSF on the anti-fungal activity of mononuclear phagocytes. We also established a neutropenic mouse model of disseminated trichosporonosis with T. asahii. M-CSF enhanced the phagocytic fungicidal activity of mononuclear cells, and infected mice treated with human M-CSF at 10 x 106 U/kg showed a significant improvement in survival rate, with fewer fungal colony counts in the lung compared with control mice. Mice treated with human M-CSF showed higher concentrations of tumour necrosis factor-alpha (TNF-alpha) in the lung and plasma compared with control mice. The survival rate was significantly reduced in mice treated with anti-mouse TNF-alpha. Our results showed that M-CSF enhanced the fungicidal activity of mononuclear phagocytes partly by production of TNF-alpha, and suggest that the administration of M-CSF to patients with disseminated trichosporonosis may be a useful adjunct to conventional anti-microbial therapy and prophylaxis.     

Application of Bead-ELISA method to detect Helicobacter pylori VacA

Helicobacter pylori is an etiological agent of gastritis, gastric ulcer and gastric cancer. In order to clarify the significance of vacuolating cytotoxin (VacA) for the pathogenesis of Helicobacter pylori infection, we established and applied the sandwich bead enzyme-linked immunosorbent assay (Bead-ELISA) for quantitative determination of VacA in the culture mediums of H. pylori and other species of Helicobacter. The minimum concentration of VacA in culture medium detected by Bead-ELISA was 25 pg VacA/ml and its sensitivity was found to be quite high compared to vacuolation assay and Western blot analysis, e.g. the minimum concentrations of VacA in culture medium required for detection by vacuolation assay and Western blotting were 11 ng/ml and 38 ng/ml, respectively. All the H. pylori strains used were found to produce VacA in the culture medium by Bead ELISA, even though some strains were negative by Western blot and vacuolation assay. The results obtained by Bead-ELISA was consistent with those by PCR amplification of a 785 bp vacA fragments. A toxin immunologically similar to VacA produced by other strains of Helicobacter such as H. muridarum (ATCC49282), H. mustelae (F10) and H. felis (ATCC49179) could not be detected by Bead-ELISA as well as Western Blot.     

Heat shock protein 70 (hsp70) as a major target of the antibody response in patients with pulmonary cryptococcosis

Cryptococcus neoformans causes infection in individuals with defective T cell function, such as AIDS, as well as without underlying disease. It has been suggested that humoral as well as cellular immunity might play an important role in the immune response to C. neoformans infection. We have recently shown, using immunoblotting, that the 70-kD hsp family of C. neoformans was the major target molecule of the humoral response in murine pulmonary cryptococcosis. In this study we also used immunoblotting to define the antibody responses in the sera of 24 patients with pulmonary cryptococcosis: 21 proven and three suspected diagnoses. Anti-C. neoformans hsp70 antibody was detected in 16 of 24 (66.7%) patients with pulmonary cryptococcosis. Fourteen of 17 (82.3%) patients with high antigen titres (> or = 1:8) and two of seven (28.6%) patients with low titres (< or = 1:4) had detectable levels of anti-hsp70 antibody. Sera from patients positive for anti-hsp70 antibody showed high titres in the Eiken latex agglutination test for the detection of serum cryptococcal antigen. Our results indicate that the 70-kD hsp family from C. neoformans appears to be a major target molecule of the humoral response, not only in murine pulmonary cryptococcosis, but also in human patients with pulmonary cryptococcosis.     

Peptide 19-containing neurons in the medullary dorsal horn, subnuclei interpolaris and oralis, and nucleus principalis of the rat

Peptide 19 (PEP 19) is a 7.6 kDa polypeptide which can bind to calmodulin and inhibit calcium-calmodulin signaling. In this study, PEP 19-immunoreactivity (ir) was examined in the rat trigeminal sensory nuclei. Numerous PEP 19-immunoreactive (ir) neurons were detected in the medullary dorsal horn (MDH) and rostral parts of the trigeminal sensory nuclei (subnuclei interpolaris and oralis, and nucleus principalis). The mean numbers ± S.D. per section of PEP 19-ir neurons were 104.2 ± 30.4 in the MDH, 137.8 ± 39.5 in the subnucleus interpolaris, 129.2 ± 46.9 in the subnucleus oralis and 157.2 ± 34.1 in the nucleus principalis. In the MDH, small to medium-sized PEP 19-ir neurons were abundant within superficial laminae. PEP 19-ir neurons with various cell body sizes were also distributed in the rostral parts of the trigeminal sensory nuclei. A double immunofluorescence analysis also demonstrated that many PEP 19-ir neurons co-expressed parvalbumin (PV)-ir in the MDH (9.0%), subnucleus oralis (7.7%) and nucleus principalis (19.7%). In the subnucleus interpolaris, such neurons were relatively rare (1.7%). PEP 19-ir neurons were mostly devoid of calbindin D-28k. In addition, a retrograde tracing method revealed that a substantial number of PEP 19-ir neurons projected to the thalamus. PV-ir was common in thalamus-projecting PEP 19-ir neurons. These findings suggest that PEP 19-ir neurons in the MDH may have a function in modulation of nociceptive and thermo-receptive signaling. It is also likely that PEP 19-ir neurons in rostral parts of the trigeminal sensory nuclei are related to transduction of mechano-receptive information from facial regions to the thalamus.     

Clinical significance of DJ-1 as a secretory molecule: retrospective study of DJ-1 expression at mRNA and protein levels in ductal carcinoma of the breast

Tsuchiya B, Iwaya K, Kohno N, Kawate T, Akahoshi T, Matsubara O & Mukai K
(2012) Histopathology  61, 69–77 Clinical significance of DJ‐1 as a secretory molecule: retrospective study of DJ‐1 expression at mRNA and protein levels in ductal carcinoma of the breast Aims: DJ‐1 is a molecule secreted into serum by some breast cancer cells. However, little is known about the clinical significance of the DJ‐1 expression. Methods and results: Expression of DJ‐1 protein was examined by immunohistochemistry, and expression of DJ‐1 mRNA was detected using in‐situ hybridization in 273 invasive ductal carcinomas (IDCs) and 41 ductal carcinomas in situ (DCISs) of the breast, and also in breast cancer cell lines. Breast cancer cells were examined for their secretion of DJ‐1 using immunoblot analysis. By immunohistochemistry DJ‐1 protein expression was lower than adjacent non‐cancerous epithelium in 6 (14.6%) of the 41 DCISs and 146 (53%) of the 273 IDCs, even although all 314 carcinomas retained expression of DJ‐1 mRNA, which was higher than that in adjacent non‐cancerous epithelium in 220 cases (70%). Patients with IDC whose cancer cells showed low expression of DJ‐1 protein had significantly shorter disease‐free survival (P = 0.0152) and overall survival (P = 0.0196) than those whose cancer cells retained DJ‐1 expression. MDA‐MB‐231 cells, which secreted DJ‐1, showed low expression of DJ‐1 protein. Conclusions: Low expression of DJ‐1 protein with high expression of its mRNA, which may reflect a secretory expression pattern, is predictive of poor outcome in patients with IDC.     

Cyclooxygenase 2 expression in the spared nerve injury model of neuropathic pain

Cyclooxygenase-2 (COX-2) after induction peripherally, and within the CNS, plays an important role in producing inflammatory pain. However, its role in neuropathic pain models is controversial. Recently a robust and persistent model of partial nerve injury pain, the spared nerve injury (SNI) model, has been developed. The aim of the present study was to examine the regulation of COX-2 in the rat SNI model and to evaluate the effectiveness of the selective COX-2 inhibitor rofecoxib in preventing neuropathic allodynia and hyperalgesia. RNase protection assays revealed only a very small and transient increase in COX-2 mRNA in the dorsal horn of the spinal cord in the SNI model with a maximum change at 24 h. Immunohistochemical analysis showed a small increase in COX-2 protein in the deep layers of the dorsal horn 10 h following SNI surgery. Rofecoxib (100 microM) did not affect spontaneous excitatory postsynaptic currents or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propanoic acid (AMPA) and N-methyl-d-aspartate (NMDA) responses in lamina II neurons from spinal cords of animals with SNI indicating no detectable action on transmitter release or postsynaptic activity. Furthermore, rofecoxib treatment (1 and 3.2 mg/kg for 5 and 3 days respectively starting on the day of surgery) failed to modify the development of allodynia and hyperalgesia in the SNI model. However, rofecoxib significantly reduced inflammatory hypersensitivity evoked by injection of complete Freund's adjuvant into one hindpaw, indicating that the doses used were pharmacologically active. The pain hypersensitivity produced by the SNI model is not COX-2-dependent.     

Identification of chromosome arm 9p as the most frequent target of homozygous deletions in lung cancer

Genome scanning at a 1-Mb resolution was undertaken in 29 lung cancer cell lines to clarify the distribution of homozygous (i.e., both allele) deletions along lung cancer genomes, using a high-resolution single nucleotide polymorphism array. Eighteen regions, including two known tumor suppressor loci, CDKN2A at 9p21 and FHIT at 3p14, were found homozygously deleted. Frequencies of deletions at the 18 regions were evaluated by genomic polymerase chain reaction in 78 lung cancer cell lines. Seven regions, 2q24, 3p14, 5q11, 9p21, 9p23, 11q14, and 21q21, were homozygously deleted in two or more cell lines. The CDKN2A locus at 9p21 was most frequently deleted (20/78, 26%), and the deletions were detected exclusively in non-small-cell lung carcinomas (NSCLCs). The PTPRD (protein tyrosine phosphatase receptor type D) locus at 9p23 was the second-most frequently deleted (8/78, 10%), and the deletions were detected in both small-cell lung carcinomas (SCLC) and NSCLC. In addition, the 9p24 region was deleted in a NSCLC. In total, 24 (31%) cell lines carried at least one deletion on chromosome arm 9p, while deletions on the remaining chromosome arms were observed at most in four (5%) cell lines. Deletions at 9p24, 9p23, and 9p21 were not contiguous with one another, and preferential co-occurrence or mutual exclusiveness for the deletions at these three loci was not observed. Thus, it was indicated that 9p is the most frequent target of homozygous deletions in lung cancer, suggesting that the arm contains multiple lung tumor suppressor genes and/or genomic features fragile during lung carcinogenesis.     

Detecting tissue-specific alternative splicing and disease-associated aberrant splicing of the PTCH gene with exon junction microarrays

Mutations in the human ortholog of Drosophila patched (PTCH) have been identified in patients with autosomal dominant nevoid basal cell carcinoma syndrome (NBCCS), characterized by minor developmental anomalies and an increased incidence of cancers such as medulloblastoma and basal cell carcinoma. We identified many isoforms of PTCH mRNA involving exons 1-5, exon 10 and a novel exon, 12b, generated by alternative splicing (AS), most of which have not been deposited in GenBank nor discussed earlier. To monitor splicing events of the PTCH gene, we designed oligonucleotide arrays on which exon probes and exon-exon junction probes as well as a couple of intron probes for the PTCH gene were placed in duplicate. Probe intensities were normalized on the basis of the total expression of PTCH and probe sensitivity. Tissue-specific regulation of AS identified with the microarrays closely correlated with the results obtained by RT-PCR. Of note, the novel exon, exon 12b, was specifically expressed in the brain and heart, especially in the cerebellum. Additionally, using these microarrays, we were able to detect disease-associated aberrant splicings of the PTCH gene in two patients with NBCCS. In both cases, cryptic splice donor sites located either in an exon or in an intron were activated because of the partial disruption of the consensus sequence for the authentic splice donor sites due to point mutations. Taken together, oligonucleotide microarrays containing exon junction probes are demonstrated to be a powerful tool to investigate tissue-specific regulation of AS and aberrant splicing taking place in genetic disorders.     

Association study of the C3 gene with adult and childhood asthma

Bronchial asthma (BA) is a multifactorial disorder, the development of which is affected by both environmental and genetic factors. The complement system plays an important role in immunological response against invading microorganisms. It has been shown that complement-C3-deficient mice have reduced inflammation of asthmatic airways. Previously, we reported the association of four single nuclear proteins (SNPs) in the exons of the C3 gene with childhood and adult BA. The C3 gene, however, is a large gene, and functional SNPs associated with susceptibility to BA have not yet been identified. We analyzed 26 SNPs in the C3 gene and its promoter region to narrow down the regions showing association with childhood and adult BA. Childhood and adult atopic BA patients and healthy child and adult controls were recruited from urban cities in Japan and genotyped. In SNP analysis, an SNP (SNP24, rs11569562) located in intron 31 of the C3 gene was associated with adult BA [corrected P (Pcor) = 0.030]. In linkage disequilibrium (LD) block 4 spanning exons 24-41, the frequency of the CCC haplotype in adult BA was significantly higher than that in adult controls (Pcor = 0.038). Neither the SNP nor the haplotype showing association with adult BA demonstrated a significant association with serum total immunoglobulin E (IgE) level in BA patients and controls. Our results suggest that LD block 4 confers susceptibility to adult BA with mechanisms relevant to the effector phase of allergic inflammation.     

Enhanced expression of CCL20 in human Helicobacter pylori-associated gastritis

CC chemokine ligand 20 (CCL20) attracts CC chemokine receptor 6 (CCR6)-expressing cells. Using endoscopic biopsies taken from the gastric antrum of 42 subjects infected with H. pylori and 42 uninfected subjects, mucosal CCL20 mRNA and protein levels were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. CCL19 mRNA and protein levels, as well as CCL21 mRNA levels, were also measured. The CCL20 mRNA and protein levels were significantly elevated in H. pylori-positive patients and substantially decreased after successful eradication. CCL19 and CCL21 expression levels were comparable in the H. pylori-infected and the uninfected groups. The CCL20 concentrations correlated with the degree of chronic gastritis. Immunohistochemistry and the in vitro infection assay showed that CCL20 was principally produced by the gastric epithelium. CCR6-expressing cells, including CD45RO⁺ memory T lymphocytes and fascin⁺-CD1a⁺ immature dendritic cells, infiltrated close to the CCL20-expressing epithelial cells. The CCL20/CCR6 interaction may be involved in the development of H. pylori-associated gastritis.