Expression and localization of Kv1 potassium channels in rat dorsal and ventral spinal roots.

We investigated the expression and localization of Kv1 channels in dorsal spinal roots (DRs) and ventral spinal roots (VRs) in rats. Among Kv1.1-1.6 tested by RT-PCR, mRNAs of Kv1.1, 1.2, and 1.5 were moderately expressed, those of Kv1.3 and Kv1.6 were weakly expressed, and that of Kv1.4 was hardly expressed at all in both DRs and VRs, whereas all six mRNAs were detected in spinal cord. Western blotting revealed that the major immunoreactive proteins were Kv1.1 and Kv1.2 in both DRs and VRs. Quantitative analysis indicated that levels of Kv1.1 and Kv1.2 protein were significantly higher in DRs than VRs. Immunohistochemical examination showed that Kv1.1 and Kv1.2 were colocalized in juxtaparanodal regions of axons in both DRs and VRs. Finally, immunoprecipitation experiments revealed that Kv1.1 and Kv1.2 were coassembled. These findings indicate that Kv1 subtypes in DRs and VRs are somewhat different from those in spinal cord, and that the numbers of Kv1.1 and Kv1.2 channels are higher in DRs than VRs.

In vitro response of immunoregulatory cytokine expression in human monocytic cells to human parvovirus B19 capsid.

Human parvovirus B19 is a clinically important pathogen in both children and adults. In adults, it frequently causes acute and chronic arthritis, which may be related to persistent infection. The effect of the capsid of human parvovirus B19 on monocytes, which are thought to be responsible for the first line of defense against parvoviral infection, is not well understood. In this study, we investigated changes in mRNA expression levels of several immunoregulatory cytokines in monocytic cells after treatment with the B19 capsid. When human monocytic cell line THP-1 cells were treated with the B19 capsid, the expression of tumor necrosis factor alpha (TNF-alpha) mRNA was suppressed independently of transforming growth factor beta (TGF-beta) mRNA. In contrast, the level of mRNA for interleukin-1 alpha (IL-1alpha) remained unchanged, and that for interleukin-1 beta (IL-1beta) was slightly increased after the capsid treatment. Flow cytometry demonstrated that THP-1 cells treated with B19 capsid showed no differences in surface expression of CD11a, CD16 and CD33, as compared with control cells. These findings that B19 capsid antigen did not promote positive responses for production of TNF-alpha and IL-1alpha may provide insight into the mechanisms of persistent infection of human parvovirus B19 and the systemic viral spread via bloodstream.

Chlamydia pneumoniae infection of microglial cells in vitro: a model of microbial infection for neurological disease.

Chlamydia pneumoniae is the aetiological cause of a wide variety of chronic inflammatory diseases and may be associated with neurological disease. Microbiological and immunological aspects of the interaction between C. pneumoniae and the central nervous system (CNS) are not well understood because of the lack of a suitable infection model for neuronal studies. In the present study, an in vitro C. pneumoniae infection model was developed in the established microglial cell line EOC 20. Infection of the cells resulted in obvious induction of proinflammatory cytokines. The infection also selectively induced matrix metalloproteinase-9 (MMP-9) but not MMP-2. Moreover, beta interferon, which is known to modulate CNS disease, inhibited induction of MMP-9 following C. pneumoniae infection. These results support the view that C. pneumoniae infection may be associated with marked alteration of the ability of microglial cells to enhance cytokine production as well as induction of an MMP.

Depletion of resident Chlamydia pneumoniae through leukoreduction by filtration of blood for transfusion.

Current studies indicate that a significant percentage of healthy blood donors carry Chlamydia pneumoniae in their blood. Although the clinical significance of such findings is unknown, eradication of such bacteria from blood components may contribute to transfusion safety. Deletion of C. pneumoniae in Red Blood Cell (RBC) units was accomplished through leukoreduction by filtration. The presence of bacteria in RBC units before and after leukoreduction was assessed by real-time PCR using primers specific for C. pneumoniae 16S rRNA. The eluates of filters used for leukoreduction were also assessed by PCR and immunostaining with fluorescein isothiocyanate-conjugated chlamydial monoclonal antibodies specific for C. pneumoniae determination. Nineteen of 30 RBC units tested showed the presence of C. pneumoniae DNA. Leukofiltration resulted in a marked reduction of leukocytes as well as C. pneumoniae in terms of bacterial number and positive rate for the bacteria. The eluates of filters showed trapped bacteria determined by both PCR and immunostaining assays. Thus, leukoreduction with a filter is an effective method to significantly reduce resident C. pneumoniae levels in RBC components but may not be completely sufficient for total eradication of this pathogen.

Chlamydia pneumoniae infection of alveolar macrophages: a model.

Chlamydia (Chlamydophila) pneumoniae is a common respiratory pathogen, and it seems likely that alveolar macrophages may have an important role in infection with this bacterium. In the present study, we examined the usefulness of a continuous cell line of murine alveolar macrophages, designated "MH-S," as an in vitro C. pneumoniae infection model. Infection of MH-S cells with C. pneumoniae resulted in the development of typical inclusion bodies in the cells, similar to that seen in primary alveolar macrophages. However, we noted that, although the number of bacteria in the cultures increased during the infection, there was a restricted production of infective elementary bodies. The analysis of bacterial messenger RNA in the cultures showed that the message levels for the omcB gene were present only at a moderate level, but the levels of hsp60 messages increased markedly during infection. Neutralization of tumor necrosis factor (TNF)-alpha induced by inoculation with antibody significantly enhanced the infection, but omcB message levels were still inhibited. These results indicate that the growth of C. pneumoniae in alveolar macrophages may be restricted. Endogenous TNF-alpha may be one of the factors responsible for such restriction, but other factors also may be involved.

Comparative study of the presence of Chlamydia pneumoniae in cerebrospinal fluid of Patients with clinically definite and monosymptomatic multiple sclerosis.

There is considerable controversy concerning the evidence for the presence of Chlamydia pneumoniae in the cerebrospinal fluid (CSF) of both multiple sclerosis (MS) patients and patients with other neurological diseases (OND). In order to clarify this issue, the laboratories at Vanderbilt University Medical Center (VUMC) and the University of South Florida (USF) examined the reproducibility of their respective PCR assays for the detection of C. pneumoniae DNA in the CSF of a common group of MS patients and OND controls. The two laboratories used different DNA extraction and PCR techniques in order to determine the prevalence of the C. pneumoniae genome in both monosymptomatic and clinically definite MS patients as well as in OND controls. In clinically definite MS patients, the VUMC and USF detection rates were 72 and 61%, respectively, and in patients with monosymptomatic MS, the VUMC and USF detection rates were 41 and 54%, respectively. The PCR signal was positive for 7% of the OND controls at VUMC and for 16% at USF. These studies confirm our previous reports concerning the high prevalence of C. pneumoniae in the CSF of MS patients. The presence of C. pneumoniae in patients with monosymptomatic MS would also suggest that infection with the organism occurs early in the course of the disease.

Analysis of Chlamydia pneumoniae growth in cells by reverse transcription-PCR targeted to bacterial gene transcripts.

Chlamydia pneumoniae is an obligate intracellular bacterium and has a unique development cycle consisting of an elementary body (EB) and reticular body (RB). EBs survive in extracellular environments as well as infect susceptible host cells. However, EBs display no measurable metabolic activity. In contrast, RBs are metabolically active and can replicate in a host cell but are noninfectious. Therefore, analysis of C. pneumoniae growth in infected cells by conventional bacterial culture may not permit sufficient information about growth of the bacteria in cells. In this study, therefore, we examined the usefulness of the reverse transcription (RT)-PCR method for analysis of bacterial transcripts to evaluate C. pneumoniae growth in HEp-2 cells because the levels of bacterial gene transcripts are known to show the metabolic activity of bacteria. The transcripts for the C. pneumoniae hsp60 gene and 16S rRNA in the cells were easily detected just after infection, followed by a marked increase. In contrast, pyk and omcB transcripts slowly increased after a latent period. The hydrocortisone treatment of C. pneumoniae-infected cells induced an increase of all bacterial transcripts tested compared with the control group. The treatment of the infected cells with the antibiotic minocycline showed a selective inhibition of bacterial gene transcripts, even though the complete inhibition of EB production determined by the bacterial culture assay was evident. These results indicate that the determination of bacterial gene transcripts by RT-PCR might be a powerful method to analyze in detail growth of C. pneumoniae in host cells, particularly altered bacterial growth caused by agents such as antimicrobials.