Human papillomavirus typing with GP5+/6+ polymerase chain reaction reverse line blotting and with commercial type-specific PCR kits.

BACKGROUND: Infection with human papillomavirus (HPV) is a necessary step in the progression to cervical cancer. Many methods for HPV testing are currently available, most developed to detect pools of HPV types. OBJECTIVES: To evaluate the HPV typing by molecular methods and to compare commercial kits with an established laboratory method. STUDY DESIGN: Eighty-four cervical samples found to be positive for HPV DNA by GP5+/6+-polymerase chain reaction-enzyme immunoassay-reverse line blotting (PCR-EIA-RLB) were re-tested with two commercial methods, INNO-LiPA and Amplisense HPV typing, able to identify the HPV type predicted by PCR-EIA-RLB in 76 and 67 samples, respectively. RESULTS: The INNO-LiPA assay revealed HPV DNA in 75/76 samples (98.7%; 95% CI, 0.93-0.99) that would contain HPV types identifiable by this assay. The Amplisense HPV assay revealed HPV DNA in 58/67 samples (86.6%; 95% CI, 0.76-0.93) containing HPV types detectable by this assay. For samples with a single infection, the unweighted kappa for concordance of HPV typing was 0.87 (95% CI, 0.78-0.97) for PCR-EIA-RLB versus INNO-LiPA, 0.94 (95% CI, 0.87-0.99) for INNO-LiPA versus Amplisense HPV, and 0.82 (95% CI, 0.70-0.94) for PCR-EIA-RLB versus Amplisense HPV typing. PCR-EIA-RLB revealed 12 multiple infections, INNO-LiPA revealed 14, and Amplisense HPV revealed 5. The agreement among tests for samples with multiple infections was lower, giving kappa values of 0.44 (95% CI, 0.18-0.70) for PCR-EIA-RLB versus INNO-LiPA, 0.52 (95% CI, 0.19-0.85) for PCR-EIA-RLB versus Amplisense HPV and 0.43 (95% CI, 0.12-0.74) for INNO-LiPA versus Amplisense HPV. CONCLUSIONS: In HPV-positive samples, the agreement among tests for HPV typing was high for single infections but markedly lower for infections with multiple HPV types.     

Drug resistance mutations and newly recognized treatment-related substitutions in the HIV-1 protease gene: prevalence and associations with drug exposure and real or virtual phenotypic resistance to protease inhibitors in two clinical cohorts of antiretroviral experienced patients

This study aimed at identifying HIV-1 protease amino acid changes associated with protease inhibitor (PI) exposure and susceptibility. New amino acid substitutions were correlated with the number of experienced PIs, reaching statistical significance only for those at positions 3, 44, and 74. The correspondence multivariate model demonstrated that > or =3 experienced PIs and substitutions or mutations at positions 3, 46, 54, 73, 74, and 84 were correlated with PI cross-resistance, including resistance for lopinavir and amprenavir in this cohort of patients who were naive for these drugs.     

Protease-activated receptors: contribution to physiology and disease

Proteases acting at the surface of cells generate and destroy receptor agonists and activate and inactivate receptors, thereby making a vitally important contribution to signal transduction. Certain serine proteases that derive from the circulation (e.g., coagulation factors), inflammatory cells (e.g., mast cell and neutrophil proteases), and from multiple other sources (e.g., epithelial cells, neurons, bacteria, fungi) can cleave protease-activated receptors (PARs), a family of four G protein-coupled receptors. Cleavage within the extracellular amino terminus exposes a tethered ligand domain, which binds to and activates the receptors to initiate multiple signaling cascades. Despite this irreversible mechanism of activation, signaling by PARs is efficiently terminated by receptor desensitization (receptor phosphorylation and uncoupling from G proteins) and downregulation (receptor degradation by cell-surface and lysosomal proteases). Protease signaling in tissues depends on the generation and release of proteases, availability of cofactors, presence of protease inhibitors, and activation and inactivation of PARs. Many proteases that activate PARs are produced during tissue damage, and PARs make important contributions to tissue responses to injury, including hemostasis, repair, cell survival, inflammation, and pain. Drugs that mimic or interfere with these processes are attractive therapies: selective agonists of PARs may facilitate healing, repair, and protection, whereas protease inhibitors and PAR antagonists can impede exacerbated inflammation and pain. Major future challenges will be to understand the role of proteases and PARs in physiological control mechanisms and human diseases and to develop selective agonists and antagonists that can be used to probe function and treat disease.     

Age-associated alterations in CXCL1 chemokine expression by murine B cells

Background  

The CXCL1 chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC), have been shown to play a role in a number of pathophysiological disease states including endotoxin-induced inflammation and bacterial meningitis. While the expression of these chemokines has been identified in a variety of cell types in the mouse, little is known about their expression with murine B-lymphocytes.     

Two cases of misinterpretation of molecular results in incontinentia pigmenti,and a PCR-based method to discriminate NEMO/IKKgamma dene deletion

Familial incontinentia pigmenti (IP) is a rare X-linked dominant disorder that affects ectodermal tissues. Over 90% of IP carrier females have a recurrent genomic deletion of exons 4-10 of the NEMO (IKBKG-IKKgamma) gene, which encodes a regulatory component of the IkB kinase complex, required to activate the NF-kB pathway. In IP, mutations in NEMOlead to the complete loss of NF-kB activation creating a susceptibility to cellular apoptosis in response to TNF-alpha. This condition is lethal for males during embryogenesis while females, who are mosaic as a result of X-inactivation, can survive. Recently, a second nonfunctional copy of the gene, DeltaNEMO, was identified, opposite in direction to NEMO in a 35.5-kb duplicated sequence tract. PCR-based detection of the NEMO deletion is diagnostic for IP disease. However, we present instances in which ex 4-10 DeltaNEMO pseudogene deletion occurs in unaffected parents of two females with clinically characteristic IP. These were missed by the currently standard PCR-based method, but can be easily discriminated by a new PCR-based test reported here that permits unambiguous molecular diagnosis and proper familial genetic counseling for IP.     

Ultrastructural changes in the dorsal skin of Wistar-derived hypotrichotic WBN/ILA-Ht rats exposed to subchronic ultraviolet B (UVB)-irradiation

Ultrastructural changes in the dorsal skin were examined in Wistar-derived hypotrichotic WBN/ILA-Ht rats exposed to subchronic UVB-irradiation (10 kJ/m2 per rat per day for up to 3 months). Epidermal hyperplasia developed at I month of UVB-irradiation and progressed thereafter, resulting in epidermal thickening and formation of epidermal ingrowths projecting into the dermis. In some portions of the epidermal ingrowths at 2 and 3 months, keratinocytes were somewhat pleomorphic. In addition, some of the keratinocytes showing cytoplasmic projections migrated into the dermis. The basement membrane and hemidesmosomes at the epidermal-dermal junction became to disappear along with the development of edema spreading from the upper dermis to the epidermis. However, Langerhans cells were still detected in the hyperplastic epidermis even at 3 months. In the dermis, in addition to edema, fibroblast proliferation and mast cell infiltration progressed with time, and degranulation of mast cells was obvious at 2 and 3 months. Only a few basophils as well as eosinophils were also found. In the upper dermis, especially beneath the epidermis, decrease in diameter and disintegration of collagen fibrils were observed. Ultrastructural characteristics of the dorsal skin responses to subchronic UVB-irradiation were clarified in the present study.     

Congenital posterior cervical spine malformation due to biallelic c.240‐4T>G RIPPLY2 variant: A discrete entity

The clinical and radiological spectrum of spondylocostal dysostosis syndromes encompasses distinctive costo‐vertebral anomalies. RIPPLY2 biallelic pathogenic variants were described in two distinct cervical spine malformation syndromes: Klippel–Feil syndrome and posterior cervical spine malformation. RIPPLY2 is involved in the determination of rostro‐caudal polarity and somite patterning during development. To date, only four cases have been reported. The current report aims at further delineating the posterior malformation in three new patients. Three patients from two unrelated families underwent clinical and radiological examination through X‐ray, 3D computed tomography and brain magnetic resonance imaging. After informed consent was obtained, family‐based whole exome sequencing (WES) was performed. Complex vertebral segmentation defects in the cervico‐thoracic spine were observed in all patients. WES led to the identification of the homozygous splicing variant c.240‐4T>G in all subjects. This variant is predicted to result in aberrant splicing of Exon 4. The current report highlights a subtype of cervical spine malformation with major atlo‐axoidal malformation compromising spinal cord integrity. This distinctive mutation‐specific pattern of malformation differs from Klippel–Feil syndrome and broadens the current classification, defining a sub‐type of RIPPLY2‐related skeletal disorder. Of note, the phenotype of one patient overlaps with oculo‐auriculo‐vertebral spectrum disorder.     

Sequence-based typing of Legionella pneumophila serogroup 1 offers the potential for true portability in legionellosis outbreak investigation

Seven gene loci of Legionella pneumophila serogroup 1 were analyzed as potential epidemiological typing markers to aid in the investigation of legionella outbreaks. The genes chosen included four likely to be selectively neutral (acn, groES, groEL, and recA) and three likely to be under selective pressure (flaA, mompS, and proA). Oligonucleotide primers were designed to amplify 279- to 763-bp fragments from each gene. Initial sequence analysis of the seven loci from 10 well-characterized isolates of L. pneumophila serogroup 1 gave excellent reproducibility (R) and epidemiological concordance (E) values (R = 1.00; E = 1.00). The three loci showing greatest discrimination and nucleotide variation, flaA, mompS, and proA, were chosen for further study. Indices of discrimination (D) were calculated using a panel of 79 unrelated isolates. Single loci gave D values ranging from 0.767 to 0.857, and a combination of all three loci resulted in a D value of 0.924. When all three loci were combined with monoclonal antibody subgrouping, the D value was 0.971. Sequence-based typing of L. pneumophila serogroup 1 using only three loci is epidemiologically concordant and highly discriminatory and has the potential to become the new "gold standard" for the epidemiological typing of L. pneumophila.