Zhong-Ping He

Background: Amyotrophic Lateral Sclerosis (ALS) is a progressive, adult onset, fatal neurodegenerative disease of motor neurons. There is emerging evidence that alterations in RNA metabolism may be critical in the pathogenesis of ALS. MicroRNAs (miRNAs) are small non-coding RNAs that are key determinants of mRNA stability. Considering that miRNAs are increasingly being recognized as having a role in a variety of neurodegenerative diseases, we decided to characterize the miRNA expression profile in spinal cord (SC) tissue in sporadic ALS (sALS) and controls. Furthermore, we performed functional analysis to identify a group of dysregulated miRNAs that could be responsible for the selective suppression of low molecular weight neurofilament (NFL) mRNA observed in ALS. Results: Using TaqMan arrays we analyzed 664 miRNAs and found that a large number of miRNAs are differentially expressed in ventral lumbar SC in sALS compared to controls. We observed that the majority of dysregulated miRNAs are down-regulated in sALS SC tissues. Ingenuity Pathway Analysis (IPA) showed that dysregulated miRNAs are linked with nervous system function and cell death. We used two prediction algorithms to develop a panel of miRNAs that have recognition elements within the human NFL mRNA 3′UTR, and then we performed functional analysis for these miRNAs. Our results demonstrate that three miRNAs that are dysregulated in sALS (miR-146a*, miR-524-5p and miR-582-3p) are capable of interacting with NFL mRNA 3′UTR in a manner that is consistent with the suppressed steady state mRNA levels observed in spinal motor neurons in ALS. Conclusions: The miRNA expression profile is broadly altered in the SC in sALS. Amongst these is a group of dysregulated miRNAs directly regulate the NFL mRNA 3′UTR, suggesting a role in the selective suppression of NFL mRNA in the ALS spinal motor neuron neurofilamentous aggregate formation.

Background: Severe acute respiratory syndrome (SARS) caused a large outbreak of pneumonia in Beijing, China, in 2003. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect and quantify SARS-CoV in 934 sera and self-collected throat washes and fecal samples from 271 patients with laboratory-confirmed SARS managed at a single institution. Results: SARS-CoV detection rates in sera were highest in the first 9 days of illness, whereas detection was highest in throat washes 5–14 days after onset of symptoms. The highest SARS-CoV RT-PCR rates (70.4–86.3%) and viral loads (log₁₀ 4.5–6.1) were seen in fecal samples collected 2–4 weeks after the onset of clinical illness. Fecal samples were frequently SARS-CoV RT-PCR positive beyond 40 days, and occasional sera still had SARS-CoV detected after 3 weeks of illness. Conclusion: In the context of an extensive outbreak with major pressure on hospital resources, patient self-collected samples are an alternative to nasopharyngeal aspirates for laboratory confirmation of SARS-CoV infection.

To study the dynamics of peripheral blood B lymphocytes and natural killer (NK) cells in patients with severe acute respiratory syndrome (SARS). The absolute numbers of peripheral blood B lymphocytes and NK cells in 602 serial samples from 240 patients with SARS were counted, using flow cytometry, and compared with that of normal population. The absolute numbers of peripheral blood B lymphocytes and NK cells in SARS patients were significantly lower than that of the normal population (P < 0.001) and were much lower in SARS patients with severe or extremely severe types, as compared with that of moderate or mild type cases (P < 0.001). The amount of B lymphocytes in recovery SARS patients increased at the 2nd week after onset, and gradually becoming normal at the 5th week of the disease onset. The number of NK cells was in the low level at onset, and keep decreasing at the 2nd week. However, it was increasing with the recovery of the disease, but did not reach to normal level at the 5th week after onset. The absolute numbers of peripheral blood B lymphocytes and NK cells were associated with the severity of the disease, and detection of these two kinds of cells was useful for predicting the prognosis of SARS.

The sensitivities and specificities of an immunofluorescence assay and an enzyme immunoassay for detection of antibodies specific for severe acute respiratory syndrome coronavirus (SARS-CoV) were compared for 148 laboratory-confirmed SARS cases. The appearance and persistence of SARS-CoV-specific antibodies were assessed, with immunoglobulin G detected in 59% of samples collected within 14 days and persisting for 60 to 95 days after the onset of illness.

To investigate excretion of severe acute respiratory syndrome coronavirus RNA (SARS-CoV) in stool of SARS patients. SARS-CoV RNA was detected in stool specimens with fluorescent quantitative polymerase chain reactions (FQ-PCR) in 101 SARS patients on the 10 to 55 days after onset, 27 non-SARS patients and 400 individuals with health check-up. SARS-CoV RNA was positive in stool specimens by FQ-PCR in 58 of 101 SARS patients (57.4%), and all negative in 27 non-SARS patients and 400 healthy individuals. Positive rate of SARS-CoV RNA was 100% (8/8), 67.7% (21/31), 47.4% (27/57) and 40.0% (2/5) on the 10 - 19, 20 - 29, 30 - 39 and 40 - 55 days after onset of fever, respectively, with values of logarithm of SARS-CoV RNA load of 6.06 +/- 2.05, 4.51 +/- 1.23, 3.82 +/- 1.44 and 3.57 +/- 1.25, respectively. Positive rate and load of SARS-CoV RNA in stool of SARS patients was the highest at their acute phase, and decreased with the extension of its course.

To study on the dynamics of peripheral blood lymphocytes and their subpopulations in patients with severe acute respiratory syndrome. Using flow cytometry, the absolute numbers of peripheral blood lymphocytes and their subpopulations in 240 SARS patients (696 specimens) and 51 individuals as controls, were counted and compared. The absolute numbers of peripheral blood lymphocytes and their subpopulations (CD45, CD3, CD4, CD8) were 1298 +/- 785, 897 +/- 606, 510 +/- 372, 362 +/- 263/mm(3), respectively, significantly lower in SARS patients as compared to the normal controls (2024 +/- 423, 1391 +/- 289, 795 +/- 129, 551 +/- 183/mm(3)). Of SARS patients, severe group (1095 +/- 740, 740 +/- 562, 419 +/- 346, 304 +/- 244/mm(3)) had lower counts than that of mild group (1404 +/- 788, 991 +/- 612, 564 +/- 378, 396 +/- 267/mm(3)), and in group with deaths (587 +/- 493, 369 +/- 371, 204 +/- 191, 150 +/- 130/mm(3)) was lower than that of recovery group (1355 +/- 776, 948 +/- 603, 539 +/- 375, 382 +/- 263/mm(3)). There were significant differences (P < 0.01) for CD45, CD3, CD4, CD8, but with no significant difference (P > 0.05) for CD4/CD8 ratio between severe and mild, recovery and death groups. The lymphocytes and their subpopulations (CD45, CD3, CD4, CD8) declined in the 1st week and to the lowest level (977 +/- 579, 641 +/- 466, 360 +/- 275, 270 +/- 216/mm(3)) in the 2nd week. Then the lymphocytes and their subpopulations gradually increased during the recovery of the disease. The absolute numbers of peripheral blood lymphocytes and their subpopulations in SARS patients might be used as one of the methods for diagnosis on the severity and prognosis of the disease.

To establish a simple, sensitive, specific and less-costly method for detecting genotypes of TT virus (TTV). TTV DNA was tested by nested polymerase chain reaction (nPCR) in sera from 180 patients with different types of viral hepatitis and 96 normal individuals in Beijing. TTV genotypes were determined in 40 sera collected from TTV DNA positive patients by heteroduplex mobility assay (HMA) and through sequencing. The positive rates of TTV DNA in viral hepatitis patients and normal individuals were 22.2% (40/180) and 19.8% (19/96), respectively (chi(2) = 0.220, P = 0.639). TTV DNA positive rates of patients with hepatitis A, B, C, E and non-A to E were 20.0% (6/30), 16.7% (5/30), 23.3% (7/30), 36.7% (11/30) and 18.3% (11/60), respectively. Of 40 TTV DNA positive patients, 20 (50.0%) were TTV G1, 7 (17.5%) TTV G2, 10 (25.0%) coinfected with different genotypes of TTV, and 3 untyped by HMA. Twenty G1 and 7 G2 detected by HMA were confirmed by sequence analysis. Of 10 patients coinfected with different genotypes of TTV, 5 were G1 and G2, 2 G1 and G3, 1 G1 and G4, 1 G1 and G3, and 1 with G1, G2 and G3 coinfections. HMA was recognized as simple, sensitive, specific and less-costly, thus could be used for genotyping of TTV.

To study the predictive factors associated with clinical deterioration in SARS patients. The clinical data of 60 SARS patients were analyzed by logistic regression and Cox's proportional hazards analysis. In logistic regression models, both older age (P=0.009) and severe lymphopenia (P=0.004) were significant predictors of clinical deterioration. In Cox's proportional hazard models, severe lymphopenia was significant predictor associated with prolongation of stay in hospital. Older age and severe lymphopenia seem to be statistically significant for predicting the clinical deterioration in SARS patients.