Koji Furuya

Congenital Hydrocephalus Revealed in the Inbred Rat, LEW/Jms

We studied the development of congenital hydrocephalus found in a colony of an inbred strain of Wistar-Lewis rats (LEW/Jms) at various intervals after birth. The disorder was transmitted as a simple recessive mendelian character. Hydrocephalic neonates were recognized 2 days after birth by stretching of the skin over the head. Death usually occurred between 10 and 20 days of age. The findings suggested the possibility of a disturbance of cerebrospinal fluid circulation resulting from primary occlusion of the 3rd or lateral ventricles during embryological development. In later phases, the hydrocephalus was aggravated by obliteration of the subarachnoid space and by stenosis of the aqueduct occurring secondary to compression of these structures from increased pressure within the brain. In some animals, external hydrocephalus occurred as a result of rupture of the occipital pole and the establishment of a direct communication between the lateral ventricles and the subdural space. We looked for antibodies against viruses that have been known to produce hydrocephalus in experimental animals and obtained entirely negative results.

Establishment of a primary culture of Echinococcus multilocularis germinal cells.

This study was designed to establish an in vitro primary culture of germinal cells ofEchinococcus multilocularis, a parasite that causes alveolar echinococcosis of the liver (AEL). We also investigated the temperature-dependency of the cultured cells. The germinal cells, which originated from a human lesion, were cultured by an original fluid-suspension method at 25°C or 37°C for 4 weeks. Anchorage-dependent and- independent cells were observed by light microscopy, transmission electron microscopy, and immunocytochemistry to confirm their origin. Cell number and viability were examined by immunocytochemistry and mitochondrial exclusion test. The cultured cells were also inoculated into jirds (Meriones unguiculatus) to evaluate metacestode formation. Morphology and immunocytochemistry showed that the cultured cells were typically germinal cells. The cell number declined gradually over the 4-week culture period, but viability remained at 50% at 3 weeks. These findings were not associated with either of the two culture temperatures; moreover, host-associated cells were not noted in the cultured cells at 25°C. The implanted cells formed metacestodes in the jird peritoneal cavity, and their histology demonstrated mature and typical alveolar-type echinococcal cysts. We successfully established an in vitro primary culture of germinal cells. This should contribute to future studies, and, hence, a better outcome for patients with AEL.

CONTRIBUTION OF MASS SCREENING SYSTEM TO RESECTABILITY OF HEPATIC LESIONS INVOLVING ECHINOCOCCUS MULTILOCULARIS

The prognosis for patients with alveolar echinococcosis of the liver (AEL) is excellent when the lesion is completely resected. Early detection of the disease and subsequent resection of the lesion are thus indispensable; however, the usefulness of screening systems is now controversial. This study was designed to compare screened and non-screened patients according to stage classification and to re-evaluate the effect of screening. We studied a total of 82 patients (63 screened and 19 non-screened). The stage classification showed a significant intergroup difference (P<0.002). The largest tumors ranged from 30 to 100 mm, and there was a significant intergroup difference (P<0.0014). Ultrasonography showed even small lesions in the screened patients. The complete resection rate was 74.6% for the screened patients, and 21.1% for the nonscreened patients, showing a significant difference (P<0.0001). The rate of unresectable lesions was higher in the non-screened patients (32%) than in the screened patients (11%), showing a significant difference (P<0.04). The present screening system contributes to early detection and subsequent resection of AEL, leading to a better outcome.

Encephalitozoon-Like Organisms in Patients with Alveolar Hydatid Disease: Cell Culture, Ultrastructure, Histoimmunochemical Localization and Seroprevalence

ABSTRACT. We found Encephalitozoon‐like organisms in an in vitro culture of a human liver lesion which was due to larval Echinococcus multilocularis. The organisms developed in the same fashion as an Encephalitozoon cunculi. The spores that developed in parasitophorous vacuoles were 2.0–2.6 × 1.1–1.5 μm: each contained a single nucleus and 4–5 polar tubule coils, closely resembling E. cuniculi in its ultrastructure. Mature spores were collected from the supernatants by the use of Percoll centrifugation resulting in the banding of the spores on continuous gradients. We prepared three sorts of spores which were different in buoyant density in 0.15 M NaCl: 1.05–1.07 g/ml spores, 1.12 g/ml spores, and spores of over 1.14 g/ml. Polyclonal antibodies to a pool of each spore preparation were produced in a rabbit and applied to the detection of microsporidian antigen in situ. The histoimmunoperoxidase (HIP) procedure was used to detect the microsporidian antigen in echinococcal liver lesions from patients with alveolar hydatid disease (AHD). Ten echinococcal liver lesions from different AHD patients were examined and four were found to be positive in the HIP test. The Percoll‐separated spores were also used as an antigen to detect for antibodies in the sera from the patients with AHD by Western blotting. Antibodies were detected in 62 (52%) of the 119 AHD patients and in only 8 (5%) of the 159 normal healthy individuals.

Nucleotide sequences of DNA fragments of Encephalitozoon cuniculi amplified by polymerase chain reaction with primers regarded as specific for Echinococcus

Encephalitozoon‐like spores were separated from a human echinococcal liver lesion, which was caused by Echinococcus multilocularis. They were found to fall into the species Encephalitozoon cuniculi, which was shown to have En. cunniculi specific DNA by way of polymerase chain reaction (PCR). We also used PCR to genetically discriminate between the En. cuniculi spores and the Ec. multilocularis larvae. Two primer sets, known to be specific for Echinococcus, were examined. These primers were expected to work normally when the two quite different DNA preparations were tested as templates, i.e. only Echinococcus DNA could give a positive signal in the PCR tests. However, it was found that the two Echinococcus‐specific primer sets could amplify not only EC. multilocularis DNA, but also En. cuniculi spore DNA. We then tried to determine the order of nucleotides in the Echinococcus‐specific primers‐amplified En. cuniculi PCR products and compared the determined sequences with those of Ec. multilocularis. The results clearly indicated that sequencing made little difference between En. cuniculi and Ec. multilocularis.

Primers Designed for Amplification of Echinococcus multilocularis DNA Amplify the DNA of Encephalitozoon-Like Spores in the Polymerase Chain Reaction

ABSTRACT. Microsporidian spores were developed from cells which were grown in vitro from a human liver lesion which was due to larval Echinococcus multilocularis. The microsporidian spores developed in the same fashion as an Encephalitozoon cuniculi. The Encephalitozoon‐like spores were completely separated on Percoll gradients. The separated spores contained DNA capable of amplification by two different primer sets designed for the polymerase chain reaction (PCR) of E. multilocularis DNA. However, the cell DNA from which microsporidium developed was thoroughly insensitive to the PCR using the E. multilocularis primer sets. The results strongly suggested that Encephalitozoon should be taken into consideration, when DNA isolated from larval E. multilocularis is analyzed.

Gas-liquid chromatographic demonstration of the specificity of rabbit IgG antibody to the pesticide DDT and its metabolites

Abstract The specificity of rabbit IgG antibody to DDT was studied by primary binding interactions between the antibody and DDT and each of its metabolites (DDD, DDE and DDT-OH) which are haptens. The principal association constants of DDT-antibody for DDT, DDD, DDE and DDT-OH were computed at 1.06 × 10 8 1/mol, 1.61 × 10 8 1/mol, 1.09 × 10 7 1/mol and 0.75 × 10 7 1/mol, respectively. To confirm the antibody specificity further, the binding interactions between the antibody and DDT were also tested in the presence of DDD, DDE, DDT-OH, o , p ′-DDT and their mixture, respectively. Significant differences in the DDT-displacing ability among DDT and its structurally related haptens were observed to be related to the van der Waals contours and perhaps hydrophobicities of these haptens. The DDT-displacing power of each metabolite was dependent on the stronger of the two different association constants of the DDT-antibody for the hapten. In conclusion, it was demonstrated that rabbit IgG antibody to DDT discriminates minor differences in structure among DDT and its structurally related haptens with regard to the position of the chlorine atom on the diphenyl nuclei, p , p ′-and o , p ′-; the number of chlorine atoms added to 2-carbon, dichloro- and trichloro-; and the structures ethylene (>C=C C−C ); ethanol ( C−C OH) and ethane ( C−C H). ECD gas-liquid chromatography was applied successfully to determine the amount of unbound free hapten molecules remaining in the supernatant separated by the Farr technique after equilibrium was established between antibody and hapten. Thus, the gas Chromatographie method appears to be useful for the analysis of the specificity of hapten-antibody reactions, especially of binding reactions between antibody and hydrophobic haptens for which radioisotope-labeling is difficult.

Chromatographic analysis of the hydrophobic interactions of rabbit IgG immunoglobulins and their papain-digested fragments by bis-(p-chlorophenyl)-acetic acid coupled to aminohexyl sepharose

The present study revealed that the IgG immunoglobulins of normal or non-immune rabbit IgG and anti-bovine serum albumin, anti-ovalbumin, anti-bovine IgG and anti-p-chlorobenzoic acid antibodies could non-specifically bind to bis-(p-chlorophenyl)-acetic acid (DDA) coupled to omega-amino-hexyl Sepharose (AHS) by the use of strengthened hydrophobic interactions dependent on the concentration of NaCl. The main binding site on the adsorbent of DDA-substituted AHS (DDA-AHS) was found to be the DDA ligand. The hydrophobic potency of the DDA ligand was thought to be more effective than that of p-chlorobenzoic acid coupled to AHS. Out study also demonstrated that two different binding sites capable of interacting the DDA ligand were contained in IgG molecules. One was located in the Fc region and the other in the Fab region. The former had an ability to adhere to the DDA-AHS adsorbent in the presence of NaCl of 3 M or over, while the latter showed heterogeneous binding behavior depending upon its antibody specificity. Differences in the chromatographic distribution among the whole IgG immunoglobulins including anti-DDA antibody were found by a hydrophobic salting-out chromatography (HSOC) method on a DDA-AHS column. It was therefore assumed that when whole IgG proteins were subjected to HSOC on a DDA-AHS column, the hydrophobic binding site in the Fc region played a decisive role at high salt concentrations of 3 M or over, while the hydrophobic binding site in the Fab region played a major role at intermediate and low salt concentrations of 2 M or below. Thus, by taking advantage of this HSOC method, whole IgG or its Fab molecules possessing very strongly hydrophobic binding sites to promote high quantum yields of 8-anilinonaphthalene-1-sulfonate (ANS) fluorescence can be easily separated. We concluded that the ligand of DDA is a probe for the hydrophobic regions in IgG immunoglobulins.

Intraperitoneal Dissemination Probably Caused by Needle Biopsy of Alveolar Echinococcosis of the Liver: Experimental Study

Abstract. Alveolar echinococcosis of the liver (AEL) is a parasitosis with a potential for malignant tumor-like behavior. The disease is diagnosed by a combination of serologic tests, diagnostic images, and the histology of needle biopsy specimens. It remains unresolved whether the biopsy induces subsequent troubles. We designed this study to investigate critical problems after needle biopsy of AEL lesions using an experimental model. Five samples were prepared from the resected lesions of AEL patients: (A) 10% suspension of trypsin digests of the minced lesion; (B) 10% suspension of mesh-filtered sediment of the minced lesion; (C) 10% sediment suspension after washing the nonminced lesion; (D) supernatant after centrifuging intracystic fluid; (E) 10% sediment suspension after centrifuging intracystic fluid. A 1-ml aliquot of each sample was injected intraperitoneally into jirds (gerbils) or cotton rats, respectively. The animals were sacrificed 12 weeks later, and intraperitoneal metacestodes were observed. All samples except D developed metacestodes, and their histologies were all lesions of typical alveolar echinococcosis. These results suggest that a needle biopsy may cause intraperitoneal dissemination or tracial implantation of the parasites along the track of the needle.

Electrostatic and hydrophobic effects in chromatography of rabbit IgC immunoglobulins on aminohexyl sepharose substituted with bis-(p-chlorophenyl)-acetic acid

The binding interactions of rabbit IgG immunoglobulins with bis-(p-chlorophenyl)-acetic acid (DDA) substituted aminohexyl Sepharose (AHS) (DDA-AHS) at low ionic strength are under the influence of pH, temp, ionic strength and their antibody specificity. The IgG molecules held on a DDA-AHS column in the presence of 0.01 M phosphate buffer (pH 6.8) at 4 degrees C could be stepwise eluted by the addition of 0.01 M acetate buffers (pH 5.5, 5.0 and 4.5) followed by 3 M NaClO4, a chaotropic reagent. The adsorbability of IgG molecules by the DDA-AHS column was reinforced at temps higher than 4 degrees C and at increasing NaCl concns ranging from 0.05 to 0.2 M. The antibody specificity and, perhaps, binding affinity greatly affected this binding system. On the basis of these results, it was evident that both the DDA ligand and charged groups (omega-amino and isourea groups) took part in the binding interactions between IgG molecules and the agarose derivative at low ionic strength. Thus the binding interactions of rabbit IgG immunoglobulins with DDA-AHS at low ionic strength are assumed to be due to hydrophobic plus electrostatic interactions. It was also observed that the binding site on the surface of IgG molecules at low ionic strength was located only in the Fab region.