A Point Mutation Creating a 3' Splice Site in C8A Is a Predominant Cause of C8α-γ Deficiency in African Americans.

C8α-γ deficiency was examined in four unrelated African Americans. Two individuals were compound heterozygotes for a previously reported point mutation in exon 9. mRNA from the remaining six C8A alleles contained a 10 nt insertion between nt 992 and 993 corresponding to the junction between exons 6 and 7. This suggested that C8α-γ deficiency in these individuals was caused by a splicing defect. Genomic sequencing revealed a G→A point mutation in intron 6, upstream of the exon 7 acceptor site. This mutation converts a GG to an AG, generates a consensus 3' splice site that shifts the reading frame, and creates a premature stop codon downstream. To verify that the point mutation caused a splicing defect, we tested wild-type and mutant mRNA substrates, containing 333 nt of the C8α intron 6/exon 7 boundary, in an in vitro splicing assay. This assay generated spliced RNA containing the 10 bp insertion observed in the C8α mRNA of affected patients. In addition, in mutant RNA substrates, the new 3' splice site was preferentially recognized compared with wild-type. Preferential selection of the mutant splice site likely reflects its positioning adjacent to a polypyrimidine tract that is stronger than that adjacent to the wild-type site. In summary, we have identified a G→A mutation in intron 6 of C8A as a predominant cause of C8α-γ deficiency in African Americans. This mutation creates a new and preferred 3' splice site, results in a 10 nt insertion in mRNA, shifts the reading frame, and produces a premature stop codon downstream.

Evaluation of Physician Prescribing Patterns For Antibiotics in the Treatment of Nonnecrotizing Skin and Soft Tissue Infections.

PURPOSE: Skin and soft tissue infections (SSTIs) cause about 15 million cases of infection that result in more than 869,000 annual hospitalizations in the United States. Cellulitis accounted for 63% of all patients hospitalized with SSTIs between 2009 and 2011. The primary objective of this study was to evaluate physician adherence rates to evidence-based practice guidelines. Secondary objectives included evaluating antibiotic selection preferences and duration of therapy. The goal of the project was to generate data to inform the development of a hospital-based protocol for nonnecrotizing SSTI treatment. METHODS: This study was a single-center, retrospective, electronic chart review of patients admitted to the hospital for nonnecrotizing SSTI. We reviewed charts of patients who were admitted with a diagnosis of cellulitis and abscess infection from August 2014 to August 2015. RESULTS: Vancomycin, piperacillin/tazobactam, and clindamycin were the initial empiric antibiotics used most frequently. The adherence rates to guideline-recommended empiric antibiotic therapy and duration of treatment were about 40% and 70%, respectively. The median duration of antibiotic therapy was 12 days. Male gender and presence of purulent discharge as independent variables led to poor adherence to guideline-recommended empiric antibiotic therapy (male versus female gender, 35% versus 50.8%; P = 0.045; purulent discharge [yes versus no], 23.9% versus 60.4%; P < 0.0001). CONCLUSIONS: The results showed substantial noncompliance with guideline recommendations on empiric antibiotic selection for the treatment of nonnecrotizing SSTIs. There is a substantial opportunity for clinical pharmacist intervention in ensuring the efficient utilization of hospital resources to improve guideline compliance; promote appropriate antibiotic selection; reduce unnecessary antibiotic exposure; and reduce cost of hospitalization.

Antimicrobial susceptibilities of respiratory pathogens in the surgical/trauma intensive care unit compared with the hospital-wide respiratory antibiogram in a level I trauma center.

BACKGROUND: Unit-specific antibiograms have developed to guide clinicians to appropriate antibiotic choices for subsets of patients. The additional level of a unit- and respiratory-specific antibiogram for surgical and trauma patients defines key differences in susceptibility information for treating hospital-acquired pneumonia. METHODS: This was a three-year, retrospective single-center study. A total of 729 positive respiratory specimens from 612 patients were analyzed using Quality Compass Pathfinder(®), the antibiotic-reporting software. Culture and susceptibility reports were compared for the surgical/trauma intensive care unit (S/TICU) and the general hospital (excluding the S/TICU but including the medical ICU [MICU]). Data were filtered to include those genera and organisms with a high association with hospital-acquired pneumonia: Acinetobacter, Citrobacter, Enterobacter, Escherichia coli, Haemophilus, Klebsiella, Neisseria, Pseudomonas, Staphylococcus, Stenotrophomonas, Streptococcus, and Serratia. RESULTS: For gram-negative organisms, susceptibility differences were noted for Acinetobacter and Pseudomonas between the S/TICU and the rest of the hospital. In particular, Acinetobacter was significantly more susceptible to ciprofloxacin (86% vs. 43%; p<0.001), gentamicin (86% vs. 54%; p=0.001), imipenem-cilastatin (93% vs. 55%; p<0.001), trimethoprim-sulfamethoxazole (88% vs. 54%; p=0.001), and tobramycin (50% vs. 0; p=0.012). Pseudomonas isolates from the S/TICU were significantly more susceptible to aztreonam (83% vs. 68%; p=0.037), ciprofloxacin (86% vs. 69%; p=0.019), and imipenem-cilastatin (94% vs. 79%; p=0.01). For gram-positive organisms, Staphylococcus isolates from the S/TICU were significantly more susceptible to erythromycin (81% vs. 57%; p=0.007) and trimethoprim-sulfamethoxazole (98% vs. 91%; p=0.034) than were the hospital isolates. CONCLUSIONS: For key respiratory pathogens, such as Pseudomonas, Acinetobacter, and Staphylococcus, surgical and trauma patients present greater susceptibility to several antibiotics. Although this information cannot be extrapolated to other institutions, it does provide a basis for comparable institutions to consider developing unit- and collection-site-specific antibiograms for infections that affect surgical/trauma patients commonly.

Disseminated coinfection with Actinomyces graevenitzii and Mycobacterium tuberculosis: case report and review of the literature.

We report the first case of disseminated infection with both Actinomyces graevenitzii and Mycobacterium tuberculosis and review the medical literature. Concomitant actinomycosis and tuberculosis is very rare. The potential of the facultatively aerobic, newly described A. graevenitzii for disseminated invasive infection needs to be evaluated.

Modulation of vascular smooth muscle cells proteoglycan synthesis by the extracellular matrix.

In this study, we investigated the effect of the extracellular matrix (ECM) secreted by vascular cells on proteoglycan (PG) synthesis by vascular smooth muscle cells in culture. PG synthesis of human aortic smooth muscle cells plated on plastic or the matrices derived from vascular endothelial cells, vascular smooth muscle cells, or THP-1 macrophages was characterized. Smooth muscle cell and macrophage matrices increased both secreted and cellular smooth muscle cells PG production by 2.5-fold to 3.9-fold, respectively, over plastic and endothelial cell matrix. Macrophage matrix was more potent than smooth muscle cell matrix in this regard. Selective enzymatic removal of chondroitin sulfates, collagen, and elastin from smooth muscle cell matrix enhanced the stimulation of PG synthesis, as did the removal of chondroitin sulfates from macrophage matrix. PG turnover rates were similar for smooth muscle cells plated on the three matrices. The newly synthesized PG from cultures plated on smooth muscle cell-, and macrophage-derived matrices had greater charge density, larger molecular size, and longer glycosaminoglycan chains than those from endothelial cell matrix cultures. These data show that the ECM plays a major role in modulating vascular smooth muscle cell PG metabolism in vitro.

Azaftig stimulates in vitro lipolysis by rodent and human adipocytes.

Azaftig is an urinary proteoglycan present in some cancer and AIDS patients experiencing weight loss. Administration of azaftig to mice results in weight loss that is associated with loss of fat depot. So far, very little is known about the mechanism underlying loss of fat depot in mice or weight loss in patients excreting azaftig. Augmentation of lipolysis may be one mechanism that can cause reduction of fat depot. Therefore, the present study was designed to examine the effect of azaftig on lipolysis by adipocytes derived from obese rats and humans. Results show a dose-dependent potentiation of lipolysis by azaftig in both rat and human adipocytes.

Angiotensin II stimulates synthesis of vascular smooth muscle cell proteoglycans with enhanced low density lipoprotein binding properties.

One mechanism by which Angiotensin II (AII) may promote atherogenesis is through modulation of proteoglycan (PG) metabolism by vascular smooth muscle cells (SMC). To test this hypothesis, we investigated the effect of AII on PG synthesis by human aortic SMC and the ability of the newly synthesized PG to bind low density lipoprotein (LDL). AII stimulated PG synthesis by SMC in a dose- and time-dependent manner. In the presence of 1 microM AII, medium and cellular PG increased by 73 and 97%, respectively. AII caused a 55% increase in biglycan mRNA which resulted in a 52% increase in biglycan synthesis. Losartan, an AII receptor antagonist, and broad and isoform-specific protein kinase C (PKC) inhibitors abolished the AII-induced up-regulation of PG synthesis. Moreover, direct activation of PKC with phorbol ester stimulated PG synthesis significantly. Similarly, inhibitors of tyrosine kinase also caused inhibition of PG synthesis. AII increased the size and charge density of the newly synthesized PG. In addition, AII stimulated the synthesis of PG that bound LDL with very high affinity by 2.5-fold to 3-fold over control. These results suggest that the AII-mediated alterations in vascular SMC PG metabolism may contribute to the pathophysiology of atherosclerosis.