Prognostic value of immunohistochemical surfactant protein A expression in regenerative/hyperplastic alveolar epithelial cells in idiopathic interstitial pneumonias.

BACKGROUND: It is difficult to predict survival in patients with idiopathic pulmonary fibrosis. Recently, several proteins, such as surfactant protein (SP) and KL-6, have been reported to be useful biologic markers for prediction of prognosis for interstitial pneumonias. It is not clear whether there is any relationship between expression of these proteins in regenerative/hyperplastic alveolar epithelial cells and prognosis of idiopathic interstitial pneumonias (IIPs). OBJECTIVES: This study aimed to elucidate the clinical significance of the expression of such lung secretory proteins as SP-A and KL-6 in lung tissues of patients with IIPs. METHODS: We retrospectively investigated the immunohistochemical expression of SP-A, KL-6, cytokeratin (CK), and epithelial membrane antigen (EMA) in alveolar epithelial cells in lung tissues obtained from surgical lung biopsy in 43 patients with IIPs, and analyzed the correlation between expression of these markers and the prognosis of each IIP patient. CK and EMA were used as general markers for epithelial cells. RESULTS: In patients with usual interstitial pneumonia (UIP), the ratio of SP-A positive epithelial cells to all alveolar epithelial cells (SP-A positive ratio) in the collapsed and mural fibrosis areas varied, ranging from cases where almost all alveolar epithelial cells expressed SP-A to cases where only a few did. On the other hand, in many patients with nonspecific interstitial pneumonia (NSIP), many of the alveolar epithelial cells in the diseased areas expressed SP-A. The SP-A positive ratio was significantly lower in patients who died from progression of UIP than in patients with UIP who remained stable or deteriorated but did not die. In NSIP patients, a similar tendency was noted between the SP-A positive ratio and prognosis. CONCLUSIONS: The results suggest that the paucity of immunohistochemical SP-A expression in alveolar epithelial cells in diseased areas (i.e. regenerative/hyperplastic alveolar epithelial cells) may predict a worse prognosis for patients with IIPs, especially patients with UIP. A prospective study is needed to confirm these results.

[Evaluation of the incidence of pneumothorax and background of patients with pneumothorax during noninvasive positive pressure ventilation].

Pneumothorax (PTX) can be a serious complication of noninvasive positive pressure ventilation (NPPV). However, the exact incidence or background of PTX during a following NPPV therapy remains unclear. In our hospital, we examined patients with PTX related to NPPV therapy and compared them with non-PTX patients over a period of 7 years. Until 2004, 5 (5 patients) of 72 episodes (63 patients) of NPPV in acute chronic respiratory failure (RF) were accompanied by PTX (incidence, 6.9 percent). The 5 patients consisted of 4 men and 1 woman (mean age, 78.4 years and range, 74-84 years). The underlying diseases were interstitial lung diseases (ILDs) in 3 patients, chronic obstructive pulmonary disease in 1, and pneumonia in 1. The stage of RF in the 5 patients was acute in 4 and chronic in 1, and the duration between the initiation of NPPV and occurrence of PTX was within 10 days in 3 patients, 11-20 days in 1, and more than 20 days in 1 (mean duration, 15.6 days). The mean inspiratory positive airway pressure/expiratory positive airway pressure (IPAP/EPAP) of the 5 patients (10.6/4.1 cm H2O) was similar to that of 58 non-PTX patients (9.9/4.0 cm H2O). With regard to underlying diseases, the ratio of ILDs was higher in PTX patients (60%) than in non-PTX patients (8.6%) (p < 0.02). Patients at a high risk of PTX should be treated carefully with NPPV. Further, the therapy should be appropriately managed, especially in the early stage of NPPV initiation.

[Clinicopathologic study of 50 autopsy cases of idiopathic pulmonary fibrosis and non-diffuse usual interstitial pneumonia].

We investigated 50 autopsy cases of idiopathic pulmonary fibrosis (IPF) and non-diffuse usual interstitial pneumonia (UIP), and subgrouped them into three subtypes based on morphologic differences in the fibrosis (honeycomb): typical thick-walled honeycomb type (16 cases), atypical thin-walled honeycomb type (27 cases) and atelectatic indurated type (6 cases), with one undetermined case. In the thin-walled type, the percentage of males (93%), the percentage of smokers (89%), and the percentage of lung cancer cases (52%) were significantly higher than in the other two subtypes (p < 0.02, p < 0.001 and p < 0.05, respectively). However, in the thick-walled and indurated types there were significantly higher percentages of DAD (38% and 67%, respectively) than in the thin-walled type (15%) (P < 0.05). Accordingly, each subtype was thought to be closely related to its IPF clinical features, and showed differences in the development of acute exacerbation and lung cancer. This study proposes the existence of a UIP subset and suggests that this subgrouping can help in the management of the disease.

Sequence analysis of two ScFv genes of MAbs against Streptococcus mutans glucosyltransferase.

Streptococcus mutans glucosyltransferases (GTFs) are considered to be the principal etiological agents of dental caries. Water-insoluble glucans (WIG) synthesized by those GTFs mediate sucrose-enhanced colonization for the bacterium on tooth surfaces and form dental plaque. GTFs have two functional domains, that is, an N-terminal catalytic sucrose-binding domain involved in sucrose hydrolysis and a C-terminal glucan-binding domain involved in the binding of the synthesized glucan polymer. Two hybridomas, each producing a monoclonal antibody (MAb) that inhibits the WIG synthesis by WIG synthesized GTF (GTF-I), were constructed. Those MAbs, P126 and P136, were shown to be able to recognize the different epitope domains in GTF; P126 recognized the N-terminal region, whereas P136 recognized the C-terminal region. We previously constructed two single chain fragments of immunoglobulin variable regions (ScFvs), which are capable of inhibiting GTF activity, from mice hybridomas producing P126 and P136. In the present study, we analyzed the nucleotide sequences of molecularly cloned ScFv genes (named ScFv/P126 and ScFv/P136), compared them in three complementarity- determining regions (CDRs), and also located their gene loci originate. Our results showed no particular relationship between the two ScFvs, and suggested the use of a certain type of VH or VL gene segment as well as possible evidence of the ability of these two MAbs to recognize different epitopes of GTF proteins.

Expression of the gtfI gene from Streptococcus sobrinus in Streptococcus anginosus using integration-mediated transformation system.

We have constructed a Streptococcus anginosus transformant expressing the gtfI gene from Streptococcus sobrinus, using a previously developed integration-mediated transformation system to introduce foreign genes onto the oral streptococcal chromosome, and attempted to evaluate the gene expression. In this system, one cloning plasmid and three pACYC184 derivatives, anchor, heterodimer, and integration plasmids were used for the construction of a series of integrants via homologous recombination. A portion of S. sobrinus gtfI gene devoid of approximately 1 kb of the 5'-region derived from pMD39 was cloned into the integration plasmid and introduced onto the S. anginosus chromosome. Next, the polymerase chain reaction product corresponding to 2.0 kb of the 5'-region of the gtfI gene from S. sobrinus chromosome was further cloned into the cloning plasmid, and the intact gtfI gene was reconstructed following integration. The final S. anginosus integrant successfully secreted the enzymatically active gtfI gene products and extracellular enzyme was characterized. This enzyme produced water-insoluble glucans and glucan-forming activity was stimulated by the addition of dextranT10. When this integrant was grown in Todd-Hewitt broth supplemented with sucrose, the integrant adhered to the glass surface in vitro and this integrant exhibited the different colony morphology on Mitis-Salivarius agar plates compared to S. sobrinus and S. anginosus. These observations strongly suggest that the construction of S. anginosus integrant expressing S. sobrinus gtfI gene using this transformation system may be an effective means of analysis of cariogenic biofilm formation.

Feasibility of eradication of mutans streptococci from oral cavities.

OBJECTIVES: Dental caries prevention programs using chlorhexidine (CHX) have been proposed, but CHX's effect in reducing levels of mutans streptococci (S. mutans and S. sobrinus) appears to last for only a few months. The aim of this study was to attempt to eradicate mutans streptococci from the oral cavity using intensive professional mechanical tooth cleaning (PMTC) and topical application of CHX in custom-made trays. METHODS: Seven adult dentate subjects participated in this study (mean age 53.7+/-5.6, age range 46 to 62, mean DMFT, 9.1+/-4.2). For each subject, PMTC was carried out eight times within ten days. After each PMTC, 1% CHX was applied twice to the tooth surface using custom-made trays. In addition, as home treatment, subjects were required to carry out tooth brushing three times a day, and apply 0.2% CHX in custom trays after brushing in the morning and evening. In addition, subjects rinsed with 0.2% CHX solution after lunch. Salivary levels of mutans streptococci were evaluated using Dentocult-SM at baseline and on days 9, 20, 70, 120. RESULTS: Mutans streptococci were eradicated by day 120 from 4 of the 7 seven subjects participating in this study. Those 3 subjects still harboring mutans streptococci exhibited deep periodontal pocketing. CONCLUSIONS: Eradication of mutans streptococci from the oral cavity is feasible using a combination of CHX application in custom-made trays and intensive PMTC.

Usefulness of endobronchial ultrasonography for transbronchial lung biopsies of peripheral lung lesions.

BACKGROUND: Peripheral lung lesions are increasing in numbers. Endoscopic diagnosis is essential for the prevention of unnecessary operations. Conventional diagnostic procedures have limitations in availability and results. OBJECTIVES: Endobronchial ultrasonography (EBUS) was investigated as a means to guide transbronchial lung biopsy, to reduce the discomfort during the procedure and to improve diagnostic accuracy. METHODS: In 50 cases, we performed transbronchial lung biopsy combined with EBUS and fluoroscopic guidance. The results were compared to 42 controls assessed by fluoroscopy only. RESULTS: In 38 cases (76%), EBUS could describe the peripheral lesion (33 from inside, including 9 cases with difficulties in fluoroscopic observation, and 5 from an adjacent bronchus, indicating the correct location of the lesion). If successfully placed inside, a change in the patient's position was not required, which helped to reduce patient discomfort. Lung cancer was diagnosed in 24 patients and benign disease in 25 patients; in 1 case diagnosis remained unknown. When the EBUS probe could be introduced inside the lesion, the sensitivity for cancer diagnosis and specificity for cancer exclusion were 100%, respectively (15/15, 18/18). Compared to the controls in whom the biopsy site was determined by fluoroscopy only, the sensitivity tended to be superior by EBUS, although it did not reach statistical significance (p = 0.06). However, specificity and accuracy were statistically significant (both p = 0.02). CONCLUSIONS: When the lesion can be correctly described by EBUS from inside the lesion, EBUS is useful to guide transbronchial lung biopsy, can contribute to a reduction in patient discomfort and improves the accuracy of diagnosis. Additional navigation tools to increase correct positioning of the EBUS probe are desirable.

Production of functional ScFv inhibiting Streptococcus mutans glucosyltransferase activity from a hybridoma P126.

Streptococcus mutans has been considered the principal etiologic agent of dental caries in humans. The glucosyltransferase-I (GTF-I), which synthesized adhesive water-insoluble glucans from sucrose, has been demonstrated to be an important cariogenic property. Water-insoluble glucans (WIG) synthesized by S. mutans GTF-I can mediate sucrose-enhanced colonization on tooth surfaces and form dental plaque. It has been suggested that inhibition of WIG synthesis decreases bacterial colonization and cariogenicity. Indeed, the use of GTF enzymes as a vaccine antigen resulted in protection from experimental dental caries in rodents. However, it is preferable to eliminate unwanted immune response during active immunization of humans. To prevent this incidence, we attempted to produce the single-chain variable fragment (ScFv) antibody against GTF-I to develop passive immunization for dental caries. Hybridomas producing monoclonal antibody (MAb) that inhibited WIG synthesis by GTF-I have been constructed. Using mRNA from an IgG1 hybridoma P126, cDNAs encoding the variable fragments of the L and H chains of IgG1 from the hybridoma P126 were cloned by RT-PCR-based techniques and then transformed into an Escherichia coli expression system. The purified ScFv antibody recognized the recombinant (r) GTF-I proteins and was capable of inhibiting the WIG synthesis of rGTF-I.

Cellular events involved in butyric acid-induced T cell apoptosis.

We have previously demonstrated that butyric acid induces cytotoxicity and apoptosis of murine thymocytes, splenic T cells, and human Jurkat T cells. Therefore, to determine the apoptotic signaling pathway induced by butyric acid, we investigated the contribution of reactive oxygen species (ROS), mitochondria, ceramide, and mitogen-activated protein kinases in butyric acid-induced human Jurkat cell apoptosis. After exposure of cells to butyric acid, a pronounced accumulation of ROS was seen. Pretreatment of cells with the antioxidant N-acetyl-cysteine or 3-aminobenzamide attenuated butyric acid-induced apoptosis through a reduction of ROS generation. Cytochrome c, apoptosis-inducing factor, and second mitochondria-derived activator of caspases protein release from mitochondria into the cytosol were detected shortly after butyric acid treatment. Exposure of cells to butyric acid resulted in an increase in cellular ceramide in a time-dependent fashion. In addition, butyric acid-induced apoptosis was inhibited by DL-threo-dihidrosphingosine, a potent inhibitor of sphingosine kinase. Using anti-extracellular signal-regulated kinase (ERK), anti-c-Jun N-terminal kinase (JNK), and anti-p38 phosphospecific Abs, we showed a decrease in ERK, but not in JNK and p38 phosphorylation after treatment of cells with butyric acid. Pretreatment of cells with the JNK inhibitor SP600125 attenuated the effect of butyric acid on apoptosis, whereas no effect was seen with the p38 inhibitor SB202190 or the ERK inhibitor PD98059. Taken together, our results indicate that butyric acid-induced T cell apoptosis is mediated by ceramide production, ROS synthesis in mitochondria, and JNK activation in the mitogen-activated protein kinase cascade. Finally, these results were further substantiated by the expression profile of butyric acid-treated Jurkat cells obtained by means of cDNA array.

Production of a single-chain variable fraction capable of inhibiting the Streptococcus mutans glucosyltransferase in Bacillus brevis: construction of a chimeric shuttle plasmid secreting its gene product.

Periodontitis and dental caries are common oral diseases, in these days, and the passive immunization is one of the most effective approaches for prevention. For this purpose, we have constructed mouse and human monoclonal antibodies to inhibit the Porphyromonas gingivalis-associated hemagglutination and coaggregation. In addition, an artificial antibody, single-chain variable fraction, or scFv, which also inhibited the hemagglutination, was constructed. Specifically for dental caries, mouse and human monoclonal antibodies that inhibited the glucosyltransferase (GTF) activity, responsible for biofilm formation, were also constructed. The advantage of scFv over the native antibody is that the former molecule does not induce possible side-effects due to Fc, such as autoimmune disease, because it consists only of variable regions originating from both heavy and light chains. To increase the abilities of the antibody preparations, we attempted to construct an additional scFv using Bacillus brevis, a secretion-proficient gram-positive bacterium, as a host cell. An scFv protein possessing the same biological activity as that of the parental antibody was successfully secreted from a B. brevis transformant following the construction of a chimeric shuttle plasmid, which was accomplished by employing a new heterodimer system.

Anti-cariogenic properties of a water-soluble extract from cacao.

The addition of a water-soluble extract from cacao-extracted powder (CEPWS) to a cariogenic model food, a white chocolate-like diet that contains 35% sucrose, significantly reduced caries scores in SPF rats infected with Streptococcus sobrinus 6715, compared to control rats fed a white chocolate-like diet. CEPWS markedly inhibited water-insoluble glucan (WIG) synthesis through crude glucosyltransferases (GTFs) from Streptococcus sobrinus B13N in vitro. GTF-inhibitor(s) in CEPWS was prepared through three-step fractionation, and was termed CEPWS-BT, which is a high molecular weight (>10 kDa) heat-stable matrix of sugar, protein, and polyphenol. When the inhibitory effect of CEPWS-BT on glucan synthesis was examined using the purified GTF-I, GTF-T, and GTF-U enzymes from S. sobrinus B13N, significant reduction in GTF-I and GTF-T activity as a result of adding CEPWS-BT at low concentrations was observed. These results suggest that the addition of CEPWS to cariogenic food could be useful in controlling dental caries.

Cloning and nucleotide sequence analysis of the Streptococcus sobrinus gtfU gene that produces a highly branched water-soluble glucan.

Streptococcus sobrinus has four gtf genes, gtfI, gtfS, gtfT, and gtfU, on the chromosome. These genes correspond respectively to the enzymes GTF-I, GTF-S1, GTF-S2, and GTF-S3. An Escherichia coli MD66 clone that contained the S. sobrinus gtfU gene was characterized. Immunological properties showed that the protein produced by the E. coli MD66 clone was similar to S. sobrinus GTF-S1. Biological properties and a linkage analysis of the glucans by 13C NMR spectrometry revealed that the protein produced by the E. coli MD66 clone was GTF-S1.

Human gingival fibroblasts rescue butyric acid-induced T-cell apoptosis.

We previously demonstrated that butyric acid, an extracellular metabolite from periodontopathic bacteria, induces cytotoxicity and apoptosis in murine thymocytes, splenic T cells, and human Jurkat T cells. In this study, we used a cell-to-cell interaction system to examine the contribution of gingival fibroblasts to the regulation of T-cell death induced by butyric acid. Butyric acid slightly suppressed fibroblast viability in a concentration-dependent fashion. However, DNA fragmentation assays indicated that butyric acid did not induce apoptosis for up to 21 h in human gingival fibroblasts (Gin 1, F41-G, and H. pulp cells). The culture supernatants were assayed for interleukin 1alpha (IL-1alpha), IL-1beta, IL-6, IL-8, IL-11, tumor necrosis factor alpha, and transforming growth factor beta, but only the IL-6, IL-8, and IL-11 levels were significantly increased by addition of butyric acid. Butyric acid- or Fas-induced Jurkat-cell apoptosis was attenuated when Jurkat cells were cocultured with either F41-G or Gin 1 cells that had been preincubated for 6 h with butyric acid. IL-8 slightly stimulated butyric acid- or Fas-induced Jurkat-cell apoptosis in a dose-dependent manner, although a low dose of IL-8 had a mildly inhibitory effect on apoptosis. In contrast, IL-6 and IL-11 significantly suppressed butyric acid- or Fas-induced apoptosis in a dose-dependent fashion. Furthermore, the addition of monoclonal antibodies against human IL-6 and IL-11 to cocultures of gingival fibroblasts and Jurkat cells partially eliminated T-cell recovery. These results suggest that the proinflammatory cytokines such as IL-6 and IL-11, produced in fibroblasts stimulated with butyric acid, are involved in the attenuation of T-cell apoptosis by gingival fibroblasts.