Redox active or thiol reactive? Optimization of rapid screens to identify less evident nuisance compounds

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity are routinely encountered in assays at various stages of drug discovery. We observed that assays for the investigation of thiol-reactive and redox-active compounds have not been collected in a comprehensive review. Here, we review these assays and subject them to experimental optimization to improve their reliability. We demonstrate the usefulness of our assay cascade by assaying a library of bioactive compounds, chemical probes, and a set of approved drugs. These high-throughput assays should complement the array of wet-lab and in silico assays during the initial stages of hit discovery campaigns to pursue only hit compounds with tractable mechanisms of action.     

A Decision Rule for Predicting Bacterial Meningitis in Children with Cerebrospinal Fluid Pleocytosis When Gram Stain Is Negative or Unavailable

Objectives:  Among children with cerebrospinal fluid (CSF) pleocytosis, the task of separating aseptic from bacterial meningitis is hampered when the CSF Gram stain result is unavailable, delayed, or negative. In this study, the authors derive and validate a clinical decision rule for use in this setting.
Methods:  This was a review of peripheral blood and CSF test results from 78 children (<19 years) presenting to Children's Hospital Columbus from 1998 to 2002. For those with a CSF leukocyte count of >7/μL, a rule was created for separating bacterial from viral meningitis that was based on routine laboratory tests, but excluded Gram stain. The rule was validated in 158 subjects seen at the same site (Columbus, 2002–2004) and in 871 subjects selected from a separate site (Boston, 1993–1999).
Results:  One point each (maximum, 6 points) was assigned for leukocytes >597/μL, neutrophils >74%, glucose <38 mg/dL, and protein >97 mg/dL in CSF and for leukocytes >17,000/mL and bands to neutrophils >11% in peripheral blood. Areas under receiver-operator-characteristic curves (AROCs) for the resultant score were 0.98 for the derivation set and 0.90 and 0.97, respectively, for validation sets from Columbus and Boston. Sensitivity and specificity pairs for the Boston data set were 100 and 44%, respectively, at a score of 0 and 97 and 81% at a score of 1. Likelihood ratios (LRs) increased from 0 at a score of 0 to 40 at a score of ≥4.
Conclusions:  Among children with CSF pleocytosis, a prediction score based on common tests of CSF and peripheral blood and intended for children with unavailable, negative, or delayed CSF Gram stain results has value for diagnosing bacterial meningitis.     

甘肃省1995年急性弛缓性麻痹病例病毒学监测

甘肃省１９９５年共报告１５岁以下儿童急性弛缓性麻痹（ＡＦＰ）病例９０例，发病率为１．３２／１０万。省脊髓灰质炎（脊灰）实验室收到ＡＦＰ病例的粪便标本共７９例，占８８％；双份合格粪便标本采集率为６８％（６１／９０）；粪便标本７天内送省脊灰实验室的占６５％（５２／７９）收到粪便标本后７天内分离培养的占８９％（７０／７９）；分离培养结果及时报告率为８７％（６９／７９）。病毒分离阳性率１３．９２％（１１／７９），其中脊灰病毒（ＰＶ）Ⅱ型５株，Ⅲ型１株，非脊灰肠道病毒（ＮＰＥＶ）５株。收到ＡＦＰ病例密切接触者粪便标本３ｌ８份，病毒分离阳性率５．６６％（１８／３１８），其中ＰＶⅡ型８株，ＮＰＥＶ１０株。全部送检粪便标本ＮＰＥＶ分离率３．７８％（１５／３９７）。所分离的ＰＶ均为疫苗株，且Ⅱ型居多。     

Acute Myocarditis

ABSTRACT In a prospective study, 57 patients with a preliminary diagnosis of myocarditis were investigated. Twenty-four patients were considered to have an acute myocarditis, 14 had a suspected myocarditis, while in 19 patients myocarditis was excluded. Episodes of frequent supraventricular and/or ventricular extrasystoles during hospital stay were seen in 8/24 cases (33%) with myocarditis and in 1/19 cases (5%) without myocarditis. On follow-up 1 month later, no supraventricular extrasystoles were observed in either group. Echocardiographic signs consistent with left ventricular insufficiency were noted in 7/24 cases (29%) with myocarditis, in 1/14 cases (7%) with suspected myocarditis and in no case without myocarditis. With a “routine” serologic test battery covering influenza viruses A and B, adenovirus, Coxsackie virus group B, ECHO viruses, Chlamydia psittaci, Mycoplasma pneumoniae and hemolytic streptococci group A, a possible etiology could be documented in 9/24 cases (38%) with myocarditis and in 4/19 cases (21%) without myocarditis. Enterovirus-specific IgM was detected with solid-phase reverse immunosorbent test (SPRIST) in 12/23 (48%) cases with myocarditis and in 3/16 cases (19%) without myocarditis. In SPRIST-IgM-positive cases, IgM antibodies were detected in 15/20 (75%) of the sera taken on admission. The overall serological results indicated a recent infection in 16/24 cases (67%) with myocarditis and in 5/19 cases (26%) without myocarditis (p < 0.05).     

Characterization and phenotypic analysis of differentiating CD34+human bone marrow cells in liquid culture

Abstract: Our current understanding of human haematopoietic stem cell biology is based in part on the characterization of human CD34⁺ bone marrow cell differentiation in vitro. CD34 is highly expressed on early stem cells and haematopoietic progenitor cells with clonogenic potential and is gradually lost during differentiation and commitment. However, CD71 (transferrin receptor) is expressed at low levels on early stem cells and generally increases during haematopoietic progenitor cell proliferation. We reasoned that the combination of these surface markers would provide a useful framework for the simultaneous analysis of multiple lineage differentiation of CD34⁺ haematopoietic progenitor cells in liquid culture. In this report, we identify the phenotype of distinct subpopulations of myeloid, erythroid and lymphoid cells in liquid suspension culture using differential expression of CD34 vs. CD71 in combination with specific lineage markers. Freshly isolated human CD34⁺ bone marrow cells were introduced into suspension culture and monitored over a 6-d period using 3-colour flow cytometry. This is the first demonstration that differential expression of CD34 vs. CD71 can be used to simultaneously monitor differentiation of multiple haematopoietic cell lineages in liquid suspension culture, facilitating the study of cytokine-, drug- or chemical-induced alterations in haematopoietic progenitor cell differentiation in vitro.     

Molecular and biological analysis of echovirus 9 strain isolated from a diabetic child

The full-length infectious cDNA clone was constructed and sequenced from the strain DM of echovirus 9, which was recently isolated from a 6-week-old child at the clinical onset of type 1 diabetes. Parallel with the isolate DM, the full-length infectious cDNA clone of the prototype strain echovirus 9 Barty (Barty-INF), was constructed and sequenced. Genetic relationships of the sequenced echo 9 viruses to the other members of the human enterovirus type B species were studied by phylogenetic analyses. Comparison of capsid protein sequences showed that the isolate DM was closely related to both prototype strains: Hill and Barty-INF. The only exception was the inner capsid protein VP4 where serotype specificity was not evident and the isolate DM clustered with the strain Hill and the strain Barty-INF with echovirus 30 Bastianni. Likewise, the nonstructural protein coding region, P2P3, of isolate DM was more similar to strain Hill than to strain Barty-INF. However, like echovirus 9 Barty, the isolate DM contained the RGD-motif in the carboxy terminus of capsid protein VP1. By blocking experiments using an RGD-containing peptide and a polyclonal rabbit antiserum to the alpha(v)beta(3)-integrin, it was shown that this molecule works as a cellular receptor for isolate DM. By using primary human islets, it was shown that the isolate DM is capable of infecting insulin-producing beta-cells like the corresponding prototype strains did. However, only isolate DM was clearly cytolytic for beta-cells. The infectious clones that were made allow further investigations of the molecular features responsible for the diabetogenicity of the isolate DM.     

Prevalence of viral infection markers by polymerase chain reaction amplification and interferon-alpha measurements among patients undergoing lumbar puncture in an emergency department

Aseptic meningitis is a frequent diagnosis in emergency departments. Nevertheless, viral investigations are not carried out currently and the viral etiology in adult population has not been studied extensively. We conducted a prospective study including all consecutive patients undergoing lumbar puncture during a 15 months period in an adult emergency department. Bloody and purulent cerebrospinal fluid (CSF) were excluded. The main tests undertaken were: CSF genomic amplification by the polymerase chain reaction (PCR) for neurotropic viruses and serum and CSF interferon-alpha (IFN-alpha) measurements. Among 194 patients included, 45 had and 149 did not have aseptic meningitis. Of 45 patients with aseptic meningitis, 10 had alternative non-virological final diagnosis, and 35/45 were presumed to have neurological disorders of viral origin. Patients (27/35) completed virological analysis: 21/27 (78%) had either positive viral PCR (enterovirus: 8 patients, Varicella zoster virus (VZV): 5, Epstein-Barr virus (EBV): 2, herpes simplex virus (HSV): 1, human herpes virus 6: 1) or only raised serum or CSF IFN-alpha (4 patients). Overall, 59% of patients with a positive viral PCR had either CSF or serum raised IFN-alpha. Twentyone patients without meningitis had either positive viral PCR (enterovirus: 3 patients) or only high serum IFN-alpha level (18 patients). In the setting of aseptic meningitis diagnosed in an adult emergency department, viruses are the most common agents encountered, with enterovirus and VZV as the two main etiological agents. Current CSF viral genome amplification and IFN-alpha measurement are informative and could be useful to confirm the viral origin of various neurological disorders, although the sensitivity and specificity of IFN-alpha measurement for the diagnosis of viral infection need further confirmation.     

Coxsackievirus B3 replication and persistence in intestinal cells from mice infected orally and in the human CaCo-2 cell line

Although the transmission of coxsackievirus B3 occurs mainly via the oral route, little is known about the primary replication and persistence of this agent in the intestine. To address this question, BALB/c mice were inoculated by gavage with coxsackievirus B3, Nancy strain. The mice were killed from 1 hr to 90 days after infection. The viral markers were detected in the small intestine using RT-PCR, cell culture and detection of VP1 protein. Coxsackievirus B3 was detected positive by the three methods from hr 2 to day 45 after infection. By using monoclonal antibodies directed towards VP1, CD40 and CD26, the virus was shown to be present in the lymphocytes of the mucosa as soon as 2 hr after infection; in contrast, no virus was detected in the epithelial cells lining the intestinal lumen. Further experiments were performed to evaluate the capacity of coxsackievirus B3 to establish a persistent infection in two intestinal cell lines. In contrast to HT29 cells, the CaCo-2 cells were shown to develop a persistent infection for up to 20 passages, as demonstrated by the detection of viral RNA and VP1 protein. This study provides further evidence that, after infection by the oral route, the viral particles are concentrated in the lymphocytes of the mucosal layer. In addition, the results suggest that coxsackievirus B3 is capable of establishing a persistent infection in the small intestine that may act as a reservoir of viral particles for the delayed spread of the virus to other target organs.     

Separation of human basophils into two fractions with different density and histamine content

We designed experiments in this study to test the hypothesis suggested by recent purification data that blood basophils comprise two populations of different density, which circulate in numbers characteristic for each human subject. Basophils were separated into two density bands by single step centrifugation on a discontinuous Percoll gradient. Band 1 cells were at the interface between plasma and Percoll of density 1.070 gm/ml. Band 2 cells were at the Percoll 1.070 to 1.080 interface. When the number of band 1 basophils was expressed as a percentage of the total in bands 1 and 2, this relative amount generally remained in a narrow range for blood obtained from the same donor on 3 successive days but differed markedly in different individuals. In a series of leukapheresis experiments, we demonstrated that the percentage of band 1 basophils in postleukapheresis venous blood was strikingly similar to the preleukapheresis value. If basophils that repopulated the leukapheresis-depleted circulation came from the bone marrow, we can conclude that blood levels of basophils in bands 1 and 2 are under physiologic control and that the two types of basophils are released in amounts characteristic for each human subject. Additional evidence for two distinct blood basophil populations was provided by histamine measurements. The histamine content per basophil was consistently higher in cells from band 1 than from band 2, the mean difference between pairs of values for 30 subjects being 0.3 +/- 0.04 pg or about 27% of the band 1 basophil histamine content of 1.1 pg.