Nagahito Saito

Helicobacter pylori : longer survival in deep ground water and sea water than in a nutrient-rich environment

Helicobacter pylori can infect the human stomach through the ingestion of water. Only a few studies on the morphological changes and viability of this bacteria in DGW (deep ground water) have been reported and none in natural seawater (SW). Morphological changes and the culturability of H. pylori after storage in DGW or SW for a week were examined to see if they could be a factor associated with the high infection rate in Japan. An H. pylori strain, ATCC 43504, stored in DGW and in SW at 4 °C for 7 days, was examined daily for any ultrastructural changes and culturability. The same H. pylori strain was also cultured in conventional Brucella broth culture liquid medium as control and was similarly observed. Bacteria kept in DGW and SW were found not only to retain their spiral form but also to show better culturability than those kept in the control nutrient‐rich medium (p<0.01). Thus, natural waters were found to be more conducive to H. pylori survival than a nutrient‐rich medium.

Morphogenetic Study on the Maturation of Osteoblastic Cell as Induced by Inorganic Polyphosphate

Since inorganic polyphosphates [poly(P)] have an activity to induce bone differenciation in vitro and in vivo, we examined an effect of poly(P) on organelle by light microscopy and electron microscopy in Murine MC3T3-E1 osteoblastic cells. The MC3T3-E1 cells were ultrastructurally observed to possess morphological characteristics of osteoblasts. Cells cultured with poly(P) were strongly stained with an anti-collagen type I antibody but not in those cultured without poly(P). Ultrastructural analysis of cells cultured with poly(P) revealed a well-developed Golgi apparatus, swollen and elongated rough endoplasmic reticulum, large mitochondria and many coated pits. Since MC3T3-E1 cells can be transformed from a resting phase to an active blastic cell phase after supplementation with poly(P), it implies that poly(P) can be an effective material for bone regeneration.

Attributable Risk of H. pylori in Peptic Ulcer Disease

Recent reports in the United States have found that fewer peptic ulcers are due to Helicobacter pylori than previously believed. The aim of this study is to determine if the declining prevalence of H. pylori infection in the general population can account for the apparent increase in the frequency of non-H. pylori ulcers. A total of 396 patients with peptic ulcer or ulcer scar were enrolled in this study. The pre-1950 population consisted of 149 patients with gastric ulcers and with 44 duodenal ulcers. The post-1950 population consisted of 96 patients with gastric ulcers and 107 with duodenal ulcers. The frequency of H. pylori-negative gastric ulcers was 5.4% in patients born before 1950 and 4.2% in patients born after 1950, and the frequency of H. pylori-negative duodenal ulcers was 0% and 1.9%, respectively. There are no statistical differences between the two populations in gastric and duodenal ulcers. H. pylori seropositivity was 74.9% in asymptomatic volunteers born before 1950 and 20.7% in those born after 1950 (P < 0.01) in the general population. The attributable risk of H. pylori infection in peptic ulcer diseases was not affected by the prevalence of H. pylori infection in the general population in Japan. This suggests that the apparent increase in frequency of non-H. pylori ulcers in the United States is not simply due to the declining prevalence of infection. Other explanations for non-H. pylori ulcers should be sought.

Antigen retrieval trial for post-embedding immunoelectron microscopy by heating with several unmasking solutions

A novel antigen retrieval procedure was carried out in the post-embedding immunogold electron microscopy method to improve the stainability of the samples. This was done by weakly fixing cultured Helicobacter pylori (ATCC43504) and embedding in Lowicryl K4M. Before staining with the anti-H. pylori antibody, the ultrathin sections were mounted on a nickel grid and heated at 121C for 15 min, 99C for 40 min, and 65C for 24 hr in distilled water, 0.1 M phosphate buffer (pH 7.4), 0.01 M EDTA (pH 7.2), 0.05 M Tris buffer (pH 10.0), 0.8 M urea (pH 7.2), 0.01 M citric acid (pH 6.0), or a commercially available target unmasking fluid (S1699; pH 6.0). Antigen retrieval in the Tris buffer solution generally showed better stainability than the classical post-embedding method without any antigen retrieval. At 65C for 24 hr, better stainability of the ultrasections was observed for each of the solutions used except for the phosphate buffer compared to the control. We suggest that the antigen retrieval method should be applied for routine use even by in postembedding immunogold electron microscopy.

Removal of Mucus for Ultrastructural Observation of the Surface of Human Gastric Epithelium Using Pronase

Background. Helicobacter pylori adhering to the human gastric epithelium causes gastric diseases such as ulcer, carcinoma and lymphoma. It is thus important to observe in detail both the surface of the epithelial cells and the H. pylori that adhered to it for the elucidation of H. pylori‐induced diseases by scanning electron microscopy (SEM). Since the thick mucus layer blocks the observation of the cell surface and the bacteria, it is generally eliminated during the processing for SEM by roughly mechanical methods, but these treatments also demolish the ultrastructure of the cells. We studied the nonmechanical method for removal of mucus layer of gastric epithelium using pronase.

Plural light chains in a single plasma cell of a monoclonal gammopathy undetermined significance case: An ultrastructural study

A 44-year-old man was found to have M-proteins of IgG consisting of κ- and λ-chains in serum without lymphadenopathy or splenomegaly. The serum concentrations of IgG, IgA and IgM were within normal limits. Bone marrow examination showed normal cellular marrow containing 6.3% of plasma cells with no abnormal features. No chromosomal abnormality was observed at all. The patient was diagnosed as having monoclonal gammopathy of undetermined significance. The bone marrow plasma cells possessed free κ- and λ-chains in Golgi apparatus, rough endoplasmic reticula and cytoplasmic matrices. Plural light chains were simultaneously produced with the same heavy chain in a plasma cell by immunoelectron microscopy. This is the first report in the world of a monoclonal gammopathy of undetermined significance producing plural light chains with the same heavy chain.

Can proton pump inhibitors be used as bactericidal drugs in Helicobacter pylori-positive patients?

It was recently reported by Nakao and Malfertheiner [1] that proton pump inhibitors (PPIs) could inhibit the growth of Helicobacter pylori. This is an interesting, important, and useful report for clinicians involved in the eradication of H. pylori. The ideal method for H. pylori eradication, including new triple [2] or quadruple [3] therapy, has not yet been determined, as was asserted in the same issue of this journal. While the primary drugs for eradication of the bacteria are antibiotics, the infection cannot be cured reliably by any single antibiotic, and newly resistant H. pylori strains often will be produced by conventional eradication therapies. PPIs have been used together with antibiotics to change the acid environment in the gastric mucosa. If the report by Nakao and Malfertheiner [1] means that one of the pharmacological functions of PPIs is to assist directly in the eradication of H. pylori in vivo, clinicians throughout the world would be encouraged. However, we have three questions related to understanding the effects of PPIs as described by these authors. They report that PPIs show a growth-inhibitory activity when there is direct contact between H. pylori and the drug employed. PPIs are protected so that they can be absorbed not in the acid gastric mucosa but in the nonacid lower tract and can circulate to the stomach to act on the parietal cells. Therefore, the mechanism whereby direct contact occurs in vivo between PPIs and the bacterial organisms in sufficient amount and duration to exhibit growth inhibition should be discussed. Second, the authors demonstrated the H. pylori by using scanning electron microscopy. Whenever we observe such organisms from the clinical specimens of nonmalignant disorders, we find flagella together with the bacterial body (Fig. 1). It is possible that flagella play many roles for motility in the mucus and also for attachment to the epithelial cells. The organisms that were not exposed to PPIs appear not to have flagella (Figure 3a in the report by Nakao and Malfertheiner [1]). What was the source of the H. pylori CPY433 used in their report? Had it previously survived in special circumstances (e.g., been isolated from a special disorder)? Finally, it is common knowledge that the spiral form of H. pylori may change into a coccoid form under a slightly unsuitable condition [4,5]. In the report being discussed here, each kind of H. pylori retained spiral structures even under the condition of being exposed to PPIs. Can the authors explain why the stress of antimicrobial activity of PPIs would allow the organisms to retain the spiral form? It seems that the growth-inhibited or bactericidal environment created by PPIs would be the truly unsuitable condition for H. pylori.