慢性肝病、原发性肝癌病人血清、肝组织纳米细菌的检测

目的 研究慢性肝病和原发性肝癌病人血清、组织中纳米细菌（NB）感染，为其发生机制提供新的认识。方法 55例慢性肝病（25例慢性乙肝和30例肝炎后肝硬化）、43例肝癌病人和336例健康人血清采用ELISA、免疫组化和钙染色；53例慢性肝病（28例慢性乙肝和25例肝硬化）、43例肝癌和15例对照肝组织行免疫组化染色，部分阳性组织透射电镜观察。结果 （1）慢性肝病、肝癌病人和健康人血清ELISA检测阳性率分别为20．0％、9．3％和8．0％，慢性肝病NB感染率高于健康人（P〈0．05）。免疫组化染色感染率分别为14．5％、4．7％和5．7％（P〈0．05）。钙染色分别为7．3％、4．7％和6．5％（P〉0．05）。（2）慢性肝病、癌及癌旁、对照组免疫组化阳性率分别为：20．8％、16．3％、14．0％和0％（P〉0．05）。部分肝癌和癌旁组织透射电镜观察发现纳米细菌样微生物结构（3／5）。结论 部分慢性肝病、肝癌病人血清、组织中存在纳米细菌感染，慢性肝病血清中纳米细菌感染率高于健康人。     

An experimental painful peripheral neuropathy due to nerve constriction. I. Axonal pathology in the sciatic nerve.

A constriction injury to the sciatic nerve of the rat produces a painful peripheral neuropathy that is similar to the conditions seen in man. The pathology of the sciatic nerve in these animals was examined at 10 days postinjury, when the abnormal pain sensations are near maximal severity. The nerves were examined with (1) complete series of silver-stained longitudinal sections of pieces of the nerve (3 cm or more) that contained the constriction injury in the center, (2) toluidine blue-stained semithin sections taken at least 1 cm proximal and 1 cm distal to the constriction, and (3) EM sections taken adjacent to those stained with toluidine blue. One centimeter or more proximal to the constriction, both myelinated and unmyelinated axons were all normal. Nearer to the constriction, extensive degeneration of myelinated axons became increasingly common, as did signs of endoneurial edema. Distal to the constriction, the nerve was uniformly edematous and full of myelinic degeneration. There was a profound loss of large myelinated axons and a distinctly less severe loss of small myelinated and unmyelinated axons. These observations show that at 10 days postinjury the constriction produces a partial and differential deafferentation of the sciatic nerve's territory. The absence of degeneration in the nerve 1 cm proximal to the constriction indicates the survival of the primary afferent neurons whose axons are interrupted.     

Interaction of nanobacteria with cultured mammalian cells

Nanobacteria were recently isolated from human blood and commercial fetal bovine serum (FBS) and were located in the -2 subgroup of proteobacteria based upon their 16S rRNA gene sequence. They can be cultured even in the absence of mammalian cells, and have extraordinary properties, like very slow growth rate and an impermeable cell wall, making their detection difficult by standard microbiological techniques. Since they are present in FBS, and thus in cell cultures, it is essential to clarify their effects on cultured mammalian cells. In this study, we show that four out of six nanobacterial isolates from different sera exerted a cytotoxic effect on 3T6 fibroblasts verified by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] viability assay, lactate dehygrogenase (LDH) release and by direct microscopy. The cytotoxic effect of nanobacteria was attenuated after they had been subcultured several times. The cytotoxic effect was similar with all tested murine and human fibroblastoid cell lines. Differential interference contrast and electron microscopy, and FITC staining with specific monoclonal antibodies indicated selective, possibly receptor-mediated adherence, followed by internalization and cytotoxicity in the 3T6 fibroblasts used as a model in these interaction studies. Thus, nanobacteria have a special way of invading mammalian cells: they trigger cells that are not normally phagocytic to engulf them. These organisms seem to be an important cause for cell vacuolization, poor thriving and unexpected cell lysis, problems frequently encountered in mammalian cell culture.     

Mutational analysis of the SOX9 gene in campomelic dysplasia and autosomal sex reversal: lack of genotype/phenotype correlations

It has previously been shown that, in the heterozygous state, mutations in the SOX9 gene cause campomelic dysplasia (CD) and the often associated autosomal XY sex reversal. In 12 CD patients, 10 novel mutations and one recurrent mutation were characterized in one SOX9 allele each, and in one case, no mutation was found. Four missense mutations are all located within the high mobility group (HMG) domain. They either reduce or abolish the DNA-binding ability of the mutant SOX9 proteins. Among the five nonsense and three frameshift mutations identified, two leave the C-terminal transactivation (TA) domain encompassing residues 402-509 of SOX9 partly or almost completely intact. When tested in cell transfection experiments, the recurrent nonsense mutation Y440X, found in two patients who survived for four and more than 9 years, respectively, exhibits some residual transactivation ability. In contrast, a frameshift mutation extending the protein by 70 residues at codon 507, found in a patient who died shortly after birth, showed no transactivation. This is apparently due to instability of the mutant SOX9 protein as demonstrated by Western blotting. Amino acid substitutions and nonsense mutations are found in patients with and without XY sex reversal, indicating that sex reversal in CD is subject to variable penetrance. Finally, none of 18 female patients with XY gonadal dysgenesis (Swyer syndrome) showed an altered SOX9 banding pattern in SSCP assays, providing evidence that SOX9 mutations do not usually result in XY sex reversal without skeletal malformations.      

Increased neuropeptide Y (NPY)-like immunoreactivity in rat sensory neurons following peripheral axotomy.

The effects of peripheral axotomy (sciatic nerve transection) on the presence and distribution of neuropeptide Y (NPY) in rat dorsal root ganglion (DRG) and spinal grey matter were examined using immunocytochemistry. In normal rats and on the sham-operated side of experimental rats, NPY-like immunoreactivity (NPYir) was observed in all laminae of the lumbar spinal cord, with an especially dense concentration of immunostained axons and axonal varicosities in laminae I-II of the dorsal horn. There was no detectable NPYir in L4-L5 DRG cells from normal rats or from the sham-operated side of experimental rats. At 14 days after axotomy, there was a large ipsilateral increase in the density of NPYir axons and varicosities in the lumbar spinal cord on the side of the nerve injury; this was especially apparent in laminae III-V. In the same rats, NPYir was observed in many small, medium, and large neurons in the L4-L5 DRGs on the side of the severed nerve.     

Human mtDNA sublimons resemble rearranged mitochondrial genoms found in pathological states

Sublimons, originally identified in plant mitochondria, are defined as rearranged mtDNA molecules present at very low levels. We have analysed the primary structures of sublimons found in human cells and tissues and estimated their abundance. Each tissue of a given individual contains a wide range of different sublimons and the most abundant species differ between tissues in a substantially systematic manner. Sublimons are undetectable in rho(0) cells, indicating that they are bona fide derivatives of mtDNA. They are most prominent in post-mitotic tissue subject to oxidative stress. Rearrangement break-points, often defined by short direct repeats, are scattered, but hotspot regions are clearly identifiable, notably near the end of the D-loop. The region between the replication origins is therefore frequently eliminated. One other hotspot region is located adjacent to a known site of protein binding, suggesting that recombination may be facilitated by protein-protein interactions. For a given primary rearrangement, both deleted and partially duplicated species can be detected. Although each sublimon is typically present at a low level, at most a few copies per cell, sublimon abundance in a given tissue can vary over three orders of magnitude between healthy individuals. Collectively, therefore, they can represent a non-negligible fraction of total mtDNA. Their structures are very similar to those of the rearranged molecules found in pathological states, such as adPEO and MNGIE; therefore, we propose that, as in plants, human mtDNA sublimons represent a pool of variant molecules that can become amplified under pathological conditions, thus contributing to cellular dysfunction.     

Nanobacteria: an infectious cause for kidney stone formation

BACKGROUND: Nanobacteria are cytotoxic, sterile-filterable, gram-negative, atypical bacteria detected in bovine and human blood. Nanobacteria produce carbonate apatite on their cell walls. Data on Randall's plaques suggest that apatite may initiate kidney stone formation. We assessed nanobacteria in 72 consecutively collected kidney stones from Finnish patients. METHODS: Nanobacteria and kidney stone units were compared using scanning electron microscopy (SEM). Demineralized kidney stones were screened for nanobacteria using a double-staining method and a specific culture method. Isolated nanobacteria were analyzed for mineral formation in vitro with Ca and 85Sr incorporation tests. RESULTS: SEM highlighted the resemblance in size and morphology of nanobacteria and the smallest apatite units in the kidney stones. Nanobacterial antigens could be detected after the demineralization of the stones in 1 N HCl. Nanobacteria were surprisingly resistant to this treatment, and cultures could be established from 93.1% of the stones. Only struvite stones had common bacteria, in addition to the nanobacteria. When the results of all of the assays were combined, 70 of the 72 stones (that is, 97.2%) were nanobacteria positive. Although apatite stones indicated highest nanobacteria antigen signals, the overall nanobacteria positivity did not depend on the stone type. The isolated nanobacteria produced apatite stones in vitro, measured by Ca and 85Sr incorporation. CONCLUSIONS: We propose that kidney stone formation is a nanobacterial disease analogous to Helicobacter pylori infection and peptic ulcer disease. Both diseases are initiated by bacterial infection and subsequently endogenous and dietary factors influence their progression.