In situ hybridization for the detection of hepatitis C virus RNA in human liver tissue.

In situ hybridization (ISH) enables visualization of specific nucleic acid in morphologically preserved cells and tissue sections. Detection of the HCV genomes in clinical specimens is useful for differential diagnosis, particularly between recurrent HCV infection and acute cellular rejection in transplant specimens. We optimized an ISH protocol that demonstrated sensitivity and specificity for detecting genomic and replicative form of HCV RNA in tissue biopsies. Digoxigenin (Dig)-labelled sense and anti-sense riboprobes were synthesized using a plasmid containing a fragment of the highly conserved HCV noncoding region as a template. The efficiency of the Dig-labelled riboprobes in detecting genomic and replicative-intermediate HCV RNA was analysed in 30 liver biopsies from patients infected or uninfected with HCV in a blinded study. A Huh7 cell line that stably replicates genome-length HCV RNA was developed to be used as a positive control. Negative control riboprobes were used in parallel to evaluate and control for background staining. The anti-sense probe detected HCV RNA in 20/21 specimens from HCV-infected liver tissues obtained from patients and in 0/9 samples from patients with non-HCV-related liver diseases, resulting in a sensitivity and specificity of 95% and 100%, respectively. HCV genomic RNA was variably distributed in tissue sections and was located primarily in the perinuclear regions in hepatocytes. Detection of HCV RNA by our optimized ISH protocol appears to be a sensitive and specific method when processing clinical specimens. It may also be revealing when exploring the pathophysiology of HCV infection by verifying the presence of viral genetic material within heptocytes and other cellular elements of diseased liver tissue. This methodology might also evaluate the response to antiviral therapies by demonstrating the absence or alteration of genetic material in clinical specimens from successfully treated patients.

Effect of rehabilitation on the long-term efficacy of botulinum toxin-A for spastic cerebral palsy.

OBJECTIVE: To investigate the long-term effects of botulinum toxin-A (BTX-A) nerve block on relaxation of spasticity in cerebral palsy. PATIENTS AND METHODS: From June 2015 to December 2018, 52 children, aged 20-56 months, with spastic cerebral palsy were treated with BTX-A. The dose of BTX-A was selected based on the weight of the child and the modified Ashworth scale (MAS). The injection dose ranged from 45 IU to 150 IU (average 68.0±31.6 IIU). The muscle tone and motor functions of all children were evaluated before the block. The spasticity was measured using the MAS, and the motor function was measured using the Physician Rating Scale (PRS) and the gross motor function measure (GMFM). After two years, all children were re-evaluated. RESULTS: No significant difference was observed between the trial and control groups in terms of age, weight, MAS, PRS, and GMFM measurements before the block (p>0.05). The PRS and GMFM improved significantly in both groups after two years (p<0.05). The PRS and GMFM in the trial group increased more significantly than those in the control group (p<0.05). CONCLUSIONS: The BTX-A block showed a long-term positive effect. Rehabilitation training after the block could help children to improve their motor functions.

Isolation and characteristics of autoreactive T cells specific to aggrecan G1 domain from rheumatoid arthritis patients.

Our previous work showed that the cartilage proteoglycan aggrecan could induce an erosive polyarthritis and spondylitis in BALB/c mice and the G1 globular domain of the aggrecan (G1) contained the arthritogenic region. To elucidate whether autoreactive T cells to G1 are expressed in rheumatoid arthritis patients, we analyzed the frequency of human G1-specific T cells in the peripheral blood of five rheumatoid arthritis patients and tried to establish G1-reactive T cell lines from these rheumatoid arthritis patients. The results showed that the G1-specific T cells in PBL were detectable at the range of 4.97 +/- 0.5 x 10(-6) in peripheral blood lymphocytes. We have also generated 15 G1-specific T lymphocyte lines from these patients with a standard split-well method. All these cells expressed fine specificity to human recombinant G1, but not to unrelated antigen. All the 15 lines expressed a pan-T cell marker and 13 of them selectively used the alphabeta T cell receptor. Two of them used gammadelta T cell receptor. The 13 of these T cell lines was CD4 positive. One line expressed CD8. One line expressed both CD4 and CD8. Moreover, 14 out of 15 lines expressed the Th-1 cytokine profile, characterized by interferon-gamma positivity and IL-4 negativity. No Th-2 type cell line was generated. These data provide strong evidence in favor of the presence of autoreactive T cells in the rheumatoid arthritis patients. What is the mechanism(s) that these autoreactive T cells attack self-target and whether these G1-specific, Th-1 type T cell lines can induce arthritis in immune deficiency mice are currently under investigation.

Comparison of hemodynamics and recovery of sevoflurane and isoflurane anesthesia in Chinese adult patients.

BACKGROUND: The new inhalational anesthetic sevoflurane would be expected to provide a rapid emergence from anesthesia due to its low blood/gas partition coefficient. In this study, we compared the hemodynamic effects, speed and quality of emergence, in ASA class I-II Chinese adult surgical patients receiving either sevoflurane or isoflurane anesthesia. METHODS: Eighty adult Chinese patients, ASA class I-II, scheduled for elective gynecological or general surgical procedures, were randomized to receive sevoflurane (n = 40) or isoflurane (n = 40) anesthesia. Ventilation is controlled via endotracheal intubation with anesthesia facilitated by either agent at anesthetic concentration of 1-1.5 MAC under the fresh gas flow 2 L/min. Heart rate, arterial blood pressure, temperature, SpO2 and end-tidal CO2 were continuously monitored. Any adverse effect such as airway irritation, nausea or vomiting was recorded during induction and emergence from anesthesia. The emergence time was assessed by various questionales for orientation during recovery. In the post-anesthetic recovery period, pain was monitored and managed by objective pain discomfort scale for analgesic supplements. Complaints of nausea and vomiting were recorded and followed up by a research nurse who visited the patient within 24 h following surgery. RESULTS: The extent of exposure to anesthetic (MAC x hours) was similar in both groups. Sevoflurane and isoflurane caused similar alterations in systolic and diastolic arterial pressure during maintenance. After surgical incision, the heart rate accelerated more in patients receiving isoflurane (p < 0.05). During emergence, time of response to command was significantly shorter in patients receiving sevoflurane than patients receiving isoflurane (5.6 +/- 0.4 min versus 15.2 +/- 3.0 min, p < 0.001). Side effects such as nausea and vomiting were comparable in both groups. CONCLUSIONS: Compared with isoflurane, sevoflurane anesthesia had the clinical advantages of maintaining stable hemodynamics and rapid recovery in Chinese adult patients.

The relief effect of botulinum toxin-A for spastic iliopsoas of cerebral palsy on children.

OBJECTIVE: This work intended to observe the effect of injecting botolinum toxin type A (BTX-A) for relieving spastic iliopsoas of cerebral palsy on children, and to investigate the improvement of this method for the motor function in these children. PATIENTS AND METHODS: From July 2006 to August 2012, 37 children with spastic iliopsoas cerebral palsy were received rehabilitation therapy. The age ranged from 3 to 15 years. The control group were treated by conventional physical therapy (PY). The experimental group were treated not only by the conventional physical therapy, but also BTX-A injection. The dose of BTX-A injection was according to the weight of the child and the Modified Ashworth Scale (MAS). The dose of the injection ranged from 15 IU to 45 IU with the average dose 31.2±13.9 IU. RESULTS: There was no significant difference between two the groups on ages, weight and MAS, GMFM (Gross Motor Function Measure) and extension angle of hip joints before treatment. In both groups, the Modified Ashworth Scale decreased, GMFM and extension angle of hip joints increased after eight weeks. In the control group, the GMFM improved significantly. In the experimental group, MAS, GMFM and extension angle of hip joints changed significantly after therapy. There was significant difference between two groups in MAS, GMFM and extension angle of hip joints after two months. CONCLUSIONS: The BTX-A injection can relieve iliopsoas spasticity of cerebral palsy on children efficiently. This therapy can help children to correct abnormal gait and to improve their motor function.

[Modulation of T-cell-mediated immune responses by trichosanthin via antigen processing and presentation].

It has been shown in our previous study that the lymphoproliferation to allo- or soluble antigens could be inhibited when T cells were cocultured with antigen presenting cells (APCs) pulsed with Trichosanthin (Tk), a plant protein purified from a Chinese medicinal herb Trichosanthes kirilowii Maximovich. In this paper, data are presented dealing with the mechanism by which the Tk functioned as a down modulator. APCs of human peripheral blood were first treated with one of inhibitors of antigen processing (chloroquine, leupeptin or cycloheximide), followed by pulsed with Tk, and then added into a T cell culture with stimulators PMA and A 23187. The suppression of lymphoproliferation was observed to be obviously diminished or totally disappeared. In contrast, when CyA was used to replace the Tk for pulse-treatment of APCs that had received the same pretreatment with the inhibitors, no significant change was detected for the suppression, suggesting that the Tk might use a different pathway to induce hyporeactivity and that the pathway was concerned in APC's function of antigen presentation. This view obtained support from our immuno-histochemical examination of the Tk-pulsed APCs. By preparing colloidal gold-labelled Tk (Tk-G particle), we were able to show that the Tk-G, when incubated with APCs and T cells at 37 degrees C for two hours and washed, was visualized under the electronic microscope to be bound to APCs', instead of T cells', membrane surface and internalized cells' into endosomes. And then the Tk-G particles were further identified within lysosomes. In this way, the Tk molecules were subjected to be processed as an external antigen and might be presented to T cells to activate certain T subsets that in turn mediated the immunosuppression. This course was capable of, as expected, being interrupted by antigen processing inhibitor, especially by chloroquine.

Cis-trans complementation of DQA1-DQB1 genes are modulated by DQ alleles: an immunogenetics analysis of DQ association with the down-regulatory function of CD8 cells in trichosanthin-induced immunosuppression.

The initiation of a CD8 cell-mediated pathway (M+) was adopted as a phenotypic trait to analyse genetic predisposition in trichosanthin (Tk)-induced immuno-suppression. Tk is a natural protein antigen with 247 amino acid residues. Based on DNA typing for DR, DQ, DP and TAP genes, data in this paper indicate that only DQ genes were primarily involved and that the alleles DQA1*0501 and DQB1*0201 were strongly associated with the M+ phenotype in cis (on DR3 haplotype) or trans (on DR5/DR7 heterozygotes) complementation. This is consistent with our observation that only the DQ-positive cells were capable of expanding after being co-cultured with Tk for 96 h. Two points of interest were noted. (1) The susceptible haplotype DRB1*0301-DQA1*0501-DQB1*0201 showed an association with the M+ phenotype only if combined with DRB1*04-, DRB1*08-, or DRB1*09-related haplotypes. When co-presented with DRB1*11-, DRB1*15-, DRB1*07-related haplotypes, however, no cis complementation could be detected. A detailed analysis of the association patterns indicated that the DQB1 locus of the non-susceptible haplotypes was the main factor for up- or down-modulation. (2) For M+ phenotype-related trans complementation in Tk-induced suppression, it was found that not only DQA1*0501-DQB1*0201 (DR5/7) alleles, but also associated DQA1*0301-DQB1*0201 (DR4/7, 9/7) alleles, were involved. The allele DQB1*0201 was not associated with the DQA1 alleles in DRB1*01-, DRB1*15-, DRB1*13-, DRB1*07-related haplotypes. The results obtained indicate that there are some additional genetic factors involved in the functional expression of cis and trans complementation of DQA1 and DQB1 genes, among which the DQ alleles play a critical role as self-regulators.

[Isolation of a gene related to trichosanthin-induced apoptosis (GRETA)].

Disturbance of the apoptosis-related signaling pathway is regarded as one of the critical factors for tumorigenesis. Isolation of the genes involved in the process of apoptosis would be thereby helpful to explore the mechanism of tumor transformation and to develop novel therapeutic approaches. Here we report a gene fragment GRETA, the gene related to trichosanthin-induced apoptosis, isolated from a leukemia cell line U937 undergoing apoptosis induced by a plant protein Trichosanhin (TCS). A 293bp segment of GRETA was revealed to be 78.3% homologous to Bruton's tyrosine kinase at nucleic acid level. And Northern blot analysis showed that three messengers of RNA with the size of about 0.8-kb, 2.0-kb and 7.0-kb, respectively, were detected in TCS-untreated U937 cells when probed with GRETA, but there were only 0.8-kb and 2.0-kb transcripts appeared in apoptotic U937 cells. In addition, the abundance of each transcript changed apparently. The 0.8-kb transcript, for example, was the main band in Northern analysis in apoptotic U937 cells while was only detected marginally in TCS-untreated cells. These data suggested a possible relationship between the alternative splicing patterns of GRETA and the apoptosis.