Secukinumab-associated localized granuloma annulare (SAGA): a case report and review of the literature.

Granuloma annulare (GA) is a benign, usually self-limited inflammatory skin dermatosis characterized clinically by pink-red to brown dermal papules or annular plaques. The main histologic feature is the presence of palisading or interstitial granulomas composed of necrobiotic collagen, elastic fibers, and mucin surrounded by a lymphohistiocytic infiltrate. Granuloma annulare is commonly associated with trauma, infections, diabetes mellitus, dyslipidemia, malignancy, thyroid disease, and a variety of medications. Two cases of GA have been reported in association with the use of secukinumab, a monoclonal antibody directed against interleukin 17A (IL17A), for the treatment of moderate-to-severe plaque psoriasis. We report the third case of secukinumab-associated GA in a 52-year-old woman with a history of diabetes mellitus type II, dyslipidemia, and non-alcoholic steatohepatitis. After four months of therapy with secukinumab, she presented with pink papules coalescing to plaques involving the antecubital fossae. Histology demonstrated a lymphohistiocytic palisading granuloma with central necrobiotic collagen and mucin, consistent with GA. Physicians should be aware of the possibility of GA developing in patients receiving secukinumab, especially in those with predisposing factors for GA. A better understanding of secukinumab-associated GA may lead to discoveries in GA pathogenesis and reveal broader immunomodulatory effects of secukinumab.

Interlaboratory comparison of cytomegalovirus viral load assays.

To assess interlaboratory variability in qualitative and quantitative cytomegalovirus (CMV) viral load (VL) testing, we distributed a panel of samples to 33 laboratories in the USA, Canada and Europe who performed testing using commercial reagents (n = 17) or laboratory-developed assays (n = 18). The panel included two negatives, seven samples constructed from purified CMV nucleocapsids in plasma (2.0-6.0 log(10) copies/mL) and three clinical plasma samples. Interlaboratory variation was observed in both actual (range, 2.0-4.0 log(10) copies/mL) and self-reported lower limits of detection (range, 1.0-4.0 log(10) copies/mL). Variation observed in reported results for individual samples ranged from 2.0 log(10) (minimum) to 4.3 log(10) (maximum)(.) Variation was greatest at low VLs. Assuming +/- 0.5 log(10) relative to the expected result represents an acceptable result, 57.6% of results fell within this range. Use of commercially available reagents and procedures was associated with less variability compared with laboratory-developed assays. Interlaboratory variability on replicate samples was significantly greater than intralaboratory variability (p < 0.0001). The significant interlaboratory variability in CMV VL observed may be impacting patient care and limiting interinstitutional comparisons. The creation of an international reference standard for CMV VL assay calibration would be an important step in quality improvement of this laboratory tool.

Interlaboratory comparison of epstein-barr virus viral load assays.

To assess interlaboratory variability in qualitative and quantitative Epstein-Barr virus (EBV) viral load (VL) testing, we distributed a panel of samples to 28 laboratories in the USA, Canada and Europe who performed testing using commercially available reagents (n = 12) or laboratory-developed assays (n = 18). The panel included two negatives, seven constructed samples using Namalwa and Molt-3 cell lines diluted in plasma (1.30-5.30 log(10) copies/mL) and three clinical plasma samples. Significant interlaboratory variation was observed for both actual (range 1.30-4.30 log(10) copies/mL) and self-reported (range, 1.70-3.30 log(10) copies/mL) lower limits of detection. The variation observed in reported results on individual samples ranged from 2.28 log(10) (minimum) to 4.14 log(10) (maximum). Variation was independent of dynamic range and use of commercial versus laboratory-developed assays. Overall, only 47.0% of all results fell within acceptable standards of variation: defined as the expected result +/- 0.50 log(10). Interlaboratory variability on replicate samples was significantly greater than intralaboratory variability (p < 0.0001). Kinetics of change in VL appears more relevant than absolute values and clinicians should understand the uncertainty associated with absolute VL values at their institutions. The creation of an international reference standard for EBV VL assay calibration would be an initial important step in quality improvement of this laboratory tool.

Chronic hyperplastic and neoplastic cutaneous lesions (Marjolin's ulcer) in hot-brand sites in adult beef cattle.

Linear exophytic cutaneous lesions (brand keratomas) are a chronic sequel to hot-iron branding in a small proportion of beef cattle in the western United States. Rarely, brand keratomas progress to form large ulcerated masses. Samples of chronically thickened skin were collected from 8 adult cattle with hot-brand lesions and from 2 cattle with ulcerated masses at brand sites. Cutaneous thickening was attributable to abrupt transition from normal haired skin to regular epidermal hyperplasia with marked orthokeratotic hyperkeratosis, acanthosis, hypopigmentation, and loss of adnexae. Epithelial atypia was absent. Normal dermal collagen was replaced by mature granulation tissue containing islands of dense hyalinized collagen. Two cows, aged 5 and 13 years, developed large, slow-growing squamous cell carcinomas at brand sites. Malignancy in branded skin is a rare complication of hot-iron branding in cattle and may arise because of malignant transformation of brand keratomas.

Molecular-based strategies for assessment of CMV infection and disease in immunosuppressed transplant recipients.

OBJECTIVES: Molecular assays are now considered to be the "gold standard" for assessment of human cytomegalovirus (CMV) infection and disease in those at risk from severe associated clinical manifestations. There is, however little consistency in the methods used in different centres. This study was undertaken to compare different qualitative molecular-based approaches for assessment of CMV activation from latency in samples from immunosuppressed transplant recipients. METHODS: Nucleic acid amplification techniques based on the polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA) were undertaken for the assessment of CMV replication and associated disease in immunosuppressed transplant recipients. Samples from 32 transplant recipients were tested during this study using three molecular-based strategies: (1) detection of CMV DNA in whole blood extracts (positive after a single round of PCR considered "high-level" positive, N = 55); (2) detection of cell-free CMV DNA in plasma (two methods, N = 55 for each); and (3) detection of late pp67 CMV mRNA after NASBA (N = 51). Results using a commercial pp65 antigenemia assay were available for comparison from 40 samples. RESULTS: Seven samples were positive for CMV by all methods and 36 were negative by all methods undertaken. The other 12 samples gave discordant results using different molecular methods. The correlation between whole blood "high-level" PCR, NASBA for pp67 mRNA and antigenemia results was generally good. Results presented show that plasma PCR results do not always correlate with methods utilizing whole blood as the substrate and that inhibitors in these samples could be problematic. Whole blood PCR gave more positive results than the other assays but use of a nested assay on whole blood or plasma led to detection of CMV in individuals who had no other indicators of virus replication and who did not develop associated disease (low specificity). Although the number of confirmed CMV disease episodes was low in this study, the problems of low positive predictive value for sensitive, qualitative PCR assays was clearly demonstrated. CONCLUSION: Assays based on qualitative detection of viral nucleic acid may provide information useful for management of CMV but caution is necessary when making comparisons between results using different molecular strategies. It remains to be proven in large, comparative clinical studies in which the approach and method give the best balance between sensitivity, specificity and clinical relevance for different patient groups.

Single amino acid substitutions on the surface of Escherichia coli maltose-binding protein can have a profound impact on the solubility of fusion proteins.

Proteins are commonly fused to Escherichia coli maltose-binding protein (MBP) to enhance their yield and facilitate their purification. In addition, the stability and solubility of a passenger protein can often be improved by fusing it to MBP. In a previous comparison with two other highly soluble fusion partners, MBP was decidedly superior at promoting the solubility of a range of aggregation-prone proteins. To explain this observation, we proposed that MBP could function as a general molecular chaperone in the context of a fusion protein by binding to aggregation-prone folding intermediates of passenger proteins and preventing their self-association. The ligand-binding cleft in MBP was considered a likely site for peptide binding because of its hydrophobic nature. We tested this hypothesis by systematically replacing hydrophobic amino acid side chains in and around the cleft with glutamic acid. None of these mutations affected the yield or solubility of MBP in its unfused state. Each MBP was then tested for its ability to promote solubility when fused to three passenger proteins: green fluorescent protein, p16, and E6. Mutations within the maltose-binding cleft (W62E, A63E, Y155E, W230E, and W340E) had little or no effect on the solubility of the fusion proteins. In contrast, three mutations near one end of the cleft (W232E, Y242E, and I317E) dramatically reduced the solubility of the same fusion proteins. The mutations with the most profound effect on solubility were shown to reduce the global stability of MBP.

Curing coupled-bunch instabilities with uneven fills.

A new, unified theoretical description of coupled-bunch instabilities in unevenly filled storage rings is presented. Uneven-fill longitudinal dynamics are explained in terms of two physical phenomena: fill-induced tune-spread damping and modulation coupling of strong even-fill eigenmodes. The latter is also present in the transverse plane. The analysis yields simple criteria for optimizing fill shapes to reduce the growth rates of the most unstable modes. Experimental results from the ALS and PEP-II are shown to be in good agreement with the theory.

Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency.

Because of its stringent sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV) is a useful reagent for cleaving genetically engineered fusion proteins. However, a serious drawback of TEV protease is that it readily cleaves itself at a specific site to generate a truncated enzyme with greatly diminished activity. The rate of autoinactivation is proportional to the concentration of TEV protease, implying a bimolecular reaction mechanism. Yet, a catalytically active protease was unable to convert a catalytically inactive protease into the truncated form. Adding increasing concentrations of the catalytically inactive protease to a fixed amount of the wild-type enzyme accelerated its rate of autoinactivation. Taken together, these results suggest that autoinactivation of TEV protease may be an intramolecular reaction that is facilitated by an allosteric interaction between protease molecules. In an effort to create a more stable protease, we made amino acid substitutions in the P2 and P1' positions of the internal cleavage site and assessed their impact on the enzyme's stability and catalytic activity. One of the P1' mutants, S219V, was not only far more stable than the wild-type protease (approximately 100-fold), but also a more efficient catalyst.

Development and application of a PCR-based method including an internal control for diagnosis of congenital cytomegalovirus infection.

Cytomegalovirus (CMV) is the most common cause of congenital infection in the developed world. We have designed and evaluated an assay that includes an internal control for amplification and detection of CMV DNA in amniotic fluid and neonatal urine samples. We present data on the use of this assay in the diagnosis of congenital CMV infection. A total of 145 amniotic and fetal fluid samples were examined by this assay; 83 were from healthy pregnant women and 62 were from women who were being investigated because of concerns over the pregnancy (diagnostic group). CMV DNA was detected in three amniotic fluid samples from the diagnostic group but was not detected in any samples taken from healthy pregnant women. Thirty-nine urine samples were obtained from 19 neonates with suspected congenital infection; CMV DNA was detected in urine from 6 of these patients. The assay provides useful information about CMV infection in the fetus and the neonate; when used in conjunction with other diagnostic tools it will enable mothers and obstetricians to make informed decisions about the management of pregnancies complicated by CMV infection.

The measurement of stress-related immune dysfunction in psychoneuroimmunology.

In recent years there has been a dramatic increase in research dedicated to the psycho-behavioural modulation of immune function, i.e. the field of Psychoneuroimmunology (PNI). This has led, necessarily, to the use of several in vitro and in vivo techniques in attempts to delineate the relationship between these two phenomena. However, since the field's inception, considerable uncertainty has existed over the significance of the immune outcomes detected and this has been compounded by the equivocal nature of some of the published data. A great deal of this uncertainty could, however, be overcome if a clearer understanding was achieved on the advantages and limitations conferred by the manifold immune assays described in the literature. This would, in turn, encourage their more appropriate use within PNI. Hence, in this review we describe the rationale behind, and offer an evaluation of, some of the more frequently used in vitro and in vivo immunological and virological techniques. We hope that a clear understanding of the rationale behind such assays and their inherent advantages and limitations will inform the discussion of the significance of stress-related immune impairment.

Detection of Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus DNA in CSF from persons infected with HIV who had neurological disease.

OBJECTIVES: To determine the frequency and clinical relevance of Epstein-Barr virus (EBV) and Kaposi's sarcoma associated herpesvirus (KSHV) DNA detection in the CSF from patients infected with HIV. METHODS: Cerebrospinal fluid was obtained prospectively from 115 consecutive patients infected with HIV undergoing diagnostic lumbar puncture for investigation of neurological disease. Amplification of DNA was performed using a nested polymerase chain reaction (PCR) for detection of EBV internal repeat and KSHV minor capsid sequences. RESULTS: EBV DNA was detected in the CSF supernatant of 18 patients. This included all patients with primary CNS lymphoma (seven patients) or a combination of systemic and CNS lymphoma (two patients). By contrast EBV DNA was not detected in the CSF supernatant of any patient with systemic, but not CNS, lymphoma (10 patients). EBV DNA was also detected in the supernatant of nine further patients without a diagnosis of lymphoma at the time of lumbar puncture, two of whom subsequently developed CNS lymphoma. No EBV DNA was detected in CSF supernatant from the remaining 87 samples (two of these patients subsequently developed lymphoma). KSHV DNA was detected in the CSF of two patients, one had systemic (but not CNS) lymphoma and the other did not have lymphoma. CONCLUSION: A diagnosis of CNS lymphoma is strongly associated with the presence of EBV DNA in CSF. In the absence of clinical and radiological features of CNS lymphoma, the presence of detectable CSF EBV DNA may predict subsequent tumour development. KSHV DNA is rarely detected in CSF in this patient group and shows no correlation with lymphoma or other neurological disease.

Detection of human TNF-alpha mRNA by NASBA.

A method to amplify and detect TNF-alpha mRNA from primed Mono Mac 6 cells is described. A silica-based extraction system was utilised for preparation of cell extracts and specific oligonucleotide primers were designed for amplification of TNF-alpha mRNA by the NASBA process. Amplification products were detected using either a liquid hybridisation assay, with analysis by polyacrylamide gel electrophoresis, or a plate hybridisation system. The method has many potential applications for the study of inflammatory cytokines and cellular mRNAs in cell culture and clinical samples.

Development and application of a new method for amplification and detection of human rhinovirus RNA.

A method based on nucleic acid sequence based amplification (NASBA) was developed for detection of rhinovirus RNA. Appropriate collection and storage conditions for maintenance of rhinovirus RNA integrity in clinical samples was determined. Two silica-based extraction methods were evaluated for preparation of RNA from virus isolates and clinical samples. Primers and probes were selected from the non-translated region at the 5' end and from VP4 of sequenced rhinoviruses. Amplified products were detected by 'in-solution' hybridization, with analysis by polyacrylamide gel electrophoresis (enzyme linked gel assay or ELGA), and by a microtitre-based plate hybridization assay. Using propagated picornavirus isolates in vitro the rhinovirus NASBA, with detection of amplified sequences by ELGA or plate hybridization, was confirmed as sensitive and specific for detection of rhinovirus RNA. The method was applied successfully to analysis of rhinovirus sequences in clinical samples from individuals with respiratory-tract symptoms. Rhinovirus NASBA will be useful for studies of the molecular epidemiology of respiratory infections and monitoring of response to anti-rhinovirus therapy.

Detection of varicella-zoster virus DNA by nested PCR in CSF from HIV-infected patients: A prospective evaluation.

The aim of this prospective study was to determine the frequency and clinical significance of detection of varicella-zoster virus (VZV) DNA in cerebrospinal fluid (CSF) from 120 HIV-infected individuals. Six of 8 CSF samples from patients with recent (up to 8 months previously) or concurrent cutaneous zoster contained detectable VZV DNA using the polymerase chain reaction. No detectable CSF VZV DNA was present in two patients who had an encephalopathy complicating cutaneous zoster or in 112 other patients without a history of recent of concurrent zoster. In conclusion, VZV DNA may be detected in CSF of patients with neurological disease and concurrent or recent zoster. However, the absence of detectable VZV DNA in CSF does not preclude the possibility of VZV associated neurological complications.

Consequences of live poliovirus vaccine administration in chronic fatigue syndrome.

The effect of live oral polio virus vaccination on chronic fatigue syndrome (CFS) patients was examined in a double-blind study. CFS patients were allocated randomly to placebo (N = 7) or vaccine (N = 7) conditions. All controls subjects received the vaccine (9). Vaccine administration was not associated with clinical exacerbation of CFS. However, objective responses to the vaccine revealed differences between patients and controls: increased poliovirus isolation, earlier peak proliferative responses, lower T-cell subsets on certain days post vaccination and a trend for reduced gamma-interferon in the CFS-vaccine group. Polio vaccination was not found to be clinically contraindicated in CFS patients, however, there was evidence of altered immune reactivity and virus clearance.

Polymeric carrier of proline analogue with antifibrotic effect in pulmonary vascular remodeling.

The proline analogue cis-4-hydroxy-L-proline (cHyp) inhibits collagen accumulation but diffuses out of tissues. To prolong the antifibrotic effect, we used a copolymer of cHyp attached to a backbone of poly(ethylene glycol) (PEG) and lysine. The copolymer was encapsulated in liposomes conjugated with PEG or in liposomes coated with the polysaccharide amylopectin to improve uptake by lungs after intravenous infusion. Amylopectin-liposomes had approximately 3-fold greater uptake in cultured endothelial cells compared with PEG-liposomes and greater lung retention 1 wk after infusion (5.2 +/- 0.8% versus 2.7 +/- 0.2%, p < 0.05). Sustained antifibrotic activity, assayed by growth inhibition of smooth muscle cells and fibroblasts over 4 d, was greater for amylopectin-liposomes/copolymer than PEG-liposomes/copolymer. Inhibition of collagen accumulation in pulmonary arteries of hypoxic (10% O2) rats was used to assess antifibrotic activity. Amylopectin-liposomes/copolymer attenuated increased right ventricular pressure by approximately 50% and completely prevented excess vascular collagen 1 wk after a single intravenous injection. The copolymer in liposomes was > 1,000-fold more effective by weight than unencapsulated monomeric cHyp. Thus, the copolymer, a potent, long-acting antifibrotic agent, totally prevented collagen accumulation for 1 wk in pulmonary arteries undergoing vascular remodeling when delivered in amylopectin-liposomes.

Polymer of proline analogue with sustained antifibrotic activity in lung fibrosis.

Inhibitors of collagen such as cis-4-hydroxy-L-proline (cHyp) may ameliorate bleomycin (bleo)-induced pulmonary fibrosis. An alternating polymer of poly(ethylene glycol) (PEG)-lysine (PEG-Lys) with cHyp attached as a pendant side chain was prepared for intratracheal delivery with bioinactive trans-Hyp (tHyp) polymer as control. To test whether the cHyp polymer has prolonged lung retention and sustained antifibrotic activity, we first instilled 3H- and 14C-labeled cHyp polymer in normal rats. Lung retention was 86 +/- 9% at 6 h and 29 +/- 3% at 7 d (n = 5). Next, rats were instilled intratracheally with either saline (sal) or 1.2 U bleo, and the following treatment groups were studied: Bleo/sal; Bleo/cHyp polymer; Bleo/tHyp polymer; and Bleo/PEG-Lys + cHyp. The dose of the test agents was 150 mg/kg polymer containing 8.5 mg/kg cHyp or tHyp instilled intratracheally at 7 and 14 d after bleo. At 21 d, hydroxyproline content (mg/lung) was: Control, 1.8 +/- 0.1; Bleo/sal 4.0 +/- 0.1*; Bleo/cHyp polymer, 2.8 +/- 0.3*+; Bleo/tHyp polymer, 4.4 +/- 0.2*; and Bleo/PEG-Lys + cHyp, 4.0 +/- 0.1* (*p < 0.05 versus Control; +p < 0.05 versus Bleo/sal; n = 5/group). The cHyp polymer also reduced lung total protein content, but the decrease was not significant. The dose required to produce 50% inhibition of lung collagen was approximately 700-fold less than monomeric cHyp. Thus, the cHyp polymer is a potent, long-acting antifibrotic agent which may be useful in treating lung fibrosis.

Acute lumbosacral polyradiculopathy due to cytomegalovirus in advanced HIV disease: CSF findings in 17 patients.

OBJECTIVES: To describe the abnormalities in CSF from HIV infected patients with acute lumbosacral polyradiculopathy (ALP) caused by cytomegalovirus (CMV) infection. METHODS: Retrospective case notes and laboratory records were reviewed for 17 consecutive patients with CMV associated ALP admitted to specialist HIV/AIDS units at UCL Hospitals and Chelsea and Westminster Hospital. RESULTS: Infection with CMV was confirmed by detection of CMV DNA by polymerase chain reaction amplification in 15 patients (all of whom were negative by culture), by culture in one patient, and by objective clinical response to anti-CMV treatment in one patient. Only nine patients had a CSF pleocytosis 28-1142 (median 150) cells/mm3; in seven there was a polymorphonuclear (PMN) leucocyte preponderance. Protein concentrations in CSF were moderately or considerably raised in 13 patients; CSF: plasma glucose ratios were < or = 50% in five patients. Two patients had no pleocytosis, normal CSF: plasma glucose, and normal or near normal protein values. CONCLUSIONS: Abnormalities in CSF in CMV associated ALP are varied: only 50% of patients have a "typical" PMN preponderant pleocytosis. The diagnosis of this condition should not rely on demonstration of a PMN preponderant pleocytosis, but on identification of CMV DNA in CSF and the exclusion of other opportunistic infections and lymphoma in order that specific anti-CMV treatment may be instituted.

Characterization of the region encoding the CO-induced hydrogenase of Rhodospirillum rubrum.

In the photosynthetic bacterium Rhodospirillum rubrum, the presence of carbon monoxide (CO) induces expression of several proteins. These include carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. Together these enzymes catalyze the following conversion: CO + H2O --> CO2 + H2. This system enables R. rubrum to grow in the dark on CO as the sole energy source. Expression of this system has been shown previously to be regulated at the transcriptional level by CO. We have now identified the remainder of the CO-regulated genes encoded in a contiguous region of the R. rubrum genome. These genes, cooMKLXU, apparently encode proteins related to the function of the CO-induced hydrogenase. As seen before with the gene for the large subunit of the CO-induced hydrogenase (cooH), most of the proteins predicted by these additional genes show significant sequence similarity to subunits of Escherichia coli hydrogenase 3. In addition, all of the newly identified coo gene products show similarity to subunits of NADH-quinone oxidoreductase (energy-conserving NADH dehydrogenase I) from various eukaryotic and prokaryotic organisms. We have found that dicyclohexylcarbodiimide, an inhibitor of mitochondrial NADH dehydrogenase I (also called complex I), inhibits the CO-induced hydrogenase as well. We also show that expression of the cooMKLXUH operon is regulated by CO and the transcriptional activator CooA in a manner similar to that of the cooFSCTJ operon that encodes the subunits of CODH and related proteins.

Detection of polyomaviral DNA in clinical samples from immunocompromised patients: correlation with clinical disease.

Clinical samples from immunocompromised patients were screened for polyomaviral sequences by nested polymerase chain reaction (PCR) to evaluate the association of these viral infections with progressive multifocal leukoencephalopathy (PML). JC virus (JCV) DNA was detected in 19 of 23 CSF samples and all four brain samples from patients with PML. Neither BK virus (BKV) nor simian virus 40 (SV40) DNA were detected in these samples. No evidence was found to support the hypothesis that polyomaviral DNA is present in the central nervous system of immunosuppressed patients without PML (CSF n = 67, brain n = 19). JCV DNA was not detected in any peripheral blood sample included in this study. JCV DNA was detected in urine from three of eight patients with PML, but was also amplified from three of 29 urine samples from patients without PML, JCV, and not SV40 or BKV, was associated with PML in this study.