Evaluation of quantitative real-time PCR for rapid assessments of the exposure of sentinel fish to Myxobolus cerebralis

Pathogen-free rainbow trout (Oncorhynchus mykiss) aged 735 degree days were experimentally exposed to a low dose of infectious Myxobolus cerebralis (20 triactinomyxons fish⁻¹). Three time periods were chosen for sampling that included 10 days (d), 67 d, and 5 months (mo) post exposure. Five diagnostic assays were used: (1) conventional single-round polymerase chain reaction (PCR), (2) nested PCR, (3) real-time TaqMan PCR, (4) pepsin–trypsin digest, and (5) histopathology. M. cerebralis was detected among individual rainbow trout by all of the PCR diagnostic tests employed at each of the three sampling time points. This result demonstrates that any of these three diagnostic approaches are capable of detecting the parasite from infected fish tissues under the conditions tested. Real-time PCR provided good biological evidence that parasite replication increases temporally as shown by quantification values that were significantly different (P<0.0001) at 10 d as compared to 67 d and 5 mo postexposure. Although sampling at 10 d by real-time PCR may be too early to accurately predict quantities of the parasite that will be present at 5 mo, it does forecast the proportions of fish that are likely to be infected at 67 d and 5 mo postparasite exposure. Real-time PCR could potentially be used as a quantitative diagnostic PCR tool to predict parasite load and outcome of M. cerebralis infection.     

Role of Asp104 in the SHV beta-lactamase

Among the TEM-type extended-spectrum beta-lactamases (ESBLs), an amino acid change at Ambler position 104 (Glu to Lys) results in increased resistance to ceftazidime and cefotaxime when found with other substitutions (e.g., Gly238Ser and Arg164Ser). To examine the role of Asp104 in SHV beta-lactamases, site saturation mutagenesis was performed. Our goal was to investigate the properties of amino acid residues at this position that affect resistance to penicillins and oxyimino-cephalosporins. Unexpectedly, 58% of amino acid variants at position 104 in SHV expressed in Escherichia coli DH10B resulted in beta-lactamases with lowered resistance to ampicillin. In contrast, increased resistance to cefotaxime was demonstrated only for the Asp104Arg and Asp104Lys beta-lactamases. When all 19 substitutions were introduced into the SHV-2 (Gly238Ser) ESBL, the most significant increases in cefotaxime and ceftazidime resistance were noted for both the doubly substituted Asp104Lys Gly238Ser and the doubly substituted Asp104Arg Gly238Ser beta-lactamases. Correspondingly, the overall catalytic efficiency (kcat/Km) of hydrolysis for cefotaxime was increased from 0.60 +/- 0.07 microM(-1) s(-1) (mean +/- standard deviation) for Gly238Ser to 1.70 +/- 0.01 microM(-1) s(-1) for the Asp104Lys and Gly238Ser beta-lactamase (threefold increase). We also showed that (i) k3 was the rate-limiting step for the hydrolysis of cefotaxime by Asp104Lys, (ii) the Km for cefotaxime of the doubly substituted Asp104Lys Gly238Ser variant approached that of the Gly238Ser beta-lactamase as pH increased, and (iii) Lys at position 104 functions in an energetically additive manner with the Gly238Ser substitution to enhance catalysis of cephalothin. Based on this analysis, we propose that the amino acid at Ambler position 104 in SHV-1 beta-lactamase plays a major role in substrate binding and recognition of oxyimino-cephalosporins and influences the interactions of Tyr105 with penicillins.     

Cooperative induction of CXCL10 involves NADPH oxidase: Implications for HIV dementia

With the increasing prevalence of HIV‐associated neurocognititve disorders (HAND), understanding the mechanisms by which HIV‐1 induces neuro‐inflammation and subsequent neuronal damage is important. The hallmark features of HIV‐encephalitis, the pathological correlate of HIV‐associated Dementia (HAD), are gliosis, oxidative stress, chemokine dysregulation, and neuronal damage/death. Since neurons are not infected by HIV‐1, the current thinking is that these cells are damaged indirectly by pro‐inflammatory chemokines released by activated glial cells. CXCL10 is a neurotoxic chemokine that is upregulated in astroglia activated by HIV‐1 Tat, IFN‐γ, and TNF‐α. In this study we have demonstrated that HIV‐1 Tat increases CXCL10 expression in IFN‐γ and TNF‐α stimulated human astrocytes via NADPH oxidase. We have shown that the treatment of astrocytes with a mixture of Tat and cytokines leads to a respiratory burst that is abrogated by apocynin, an NADPH oxidase inhibitor. Pretreatment of Tat, IFN‐γ, and TNF‐α stimulated astrocytes with apocynin also resulted in concomitant inhibition of CXCL10 expression. Additionally, apocynin was also able to reduce Tat and cytokine‐mediated activation of the corresponding signaling molecules Erk1/2, Jnk, and Akt with a decrease in activation and nuclear translocation of NF‐κB, important regulators of CXCL10 induction. Understanding the mechanisms involved in reducing both oxidative stress and the release of pro‐inflammatory agents could lead to the development of therapeutics aimed at decreasing neuro‐inflammation in patients suffering from HAD. © 2009 Wiley‐Liss, Inc.     

Extensive Gastric Heterotopia of the Small Intestine Resulting in Massive Gastrointestinal Bleeding, Bowel Perforation, and Death: Report of a Case and Review of the Literature

Gastric heterotopia of the small intestine is a rare occurrence outside of Meckel's diverticulum and intestinal duplication. The vast majority of cases of gastric heterotopia occur as polypoid or tumorous lesions in the duodenum. These lesions have been associated with clinical symptoms including diarrhea, obstruction, dyspepsia, ulceration, and gastrointestinal bleeding. We present a case of gastric heterotopia that is unique because the lesions occurred as multiple, carpet-like, nonpolypoid areas throughout a large portion of the small intestine. A review of the literature is included. Received March 10, 1999; accepted June 28, 1999.