Quantitative and qualitative characterization of plasma DNA identifies primary and recurrent colorectal cancer.

Because plasma DNA may be a useful tool for cancer detection, we screened primary tumors and related multiple plasma samples at the time of surgery and during the follow-up period for plasma DNA level as well as for K-Ras mutations and p16INK4a promoter hypermethylation in colorectal cancer patients. At the time of surgery, DNA levels were higher in tumor patients than in healthy donors, and K-Ras and p16INK4a alterations were detected in 7 and 11 cancers respectively, and in all related plasma samples. During the follow-up, plasma DNA levels decrease progressively but rapidly increased when a relapse occurred, whereas K-Ras and p16INK4a alterations were detected only in relapsed patients. Therefore, combined quantitative and qualitative analyses of plasma DNA confirm the presence of colorectal cancer, define disease-free status and indicate the presence of relapse.

The chaperone-like sodium phenylbutyrate improves factor IX intracellular trafficking and activity impaired by the frequent p.R294Q mutation.

Essentials Missense mutations often impair protein folding, and thus intracellular trafficking and secretion. Cellular models of severe type I hemophilia B were challenged with chaperone-like compounds. Sodium phenylbutyrate improved intracellular trafficking and secretion of the frequent p.R294Q. The increased coagulant activity levels (∼3%) of p.R294Q would ameliorate the bleeding phenotype. SUMMARY: Background Missense mutations often impair protein folding and intracellular processing, which can be improved by small compounds with chaperone-like activity. However, little has been done in coagulopathies, where even modest increases of functional levels could have therapeutic implications. Objectives To rescue the expression of factor IX (FIX) variants affected by missense mutations associated with type I hemophilia B (HB) through chaperone-like compounds. Methods Expression studies of recombinant (r)FIX variants and evaluation of secreted levels (ELISA), intracellular trafficking (immunofluorescence) and activity (coagulant assays) before and after treatment of cells with chaperone-like compounds. Results As a model we chose the most frequent HB mutation (p.R294Q, ~100 patients), compared with other recurrent mutations associated with severe/moderate type I HB. Immunofluorescence studies revealed retention of rFIX variants in the endoplasmic reticulum and negligible localization in the Golgi, thus indicating impaired intracellular trafficking. Consistently, and in agreement with coagulation phenotypes in patients, all missense mutations resulted in impaired secretion (< 1% wild-type rFIX). Sodium phenylbutyrate (NaPBA) quantitatively improved trafficking to the Golgi and dose dependently promoted secretion (from 0.3 ± 0.1% to 1.5 ± 0.3%) only of the rFIX-294Q variant. Noticeably, this variant displayed a specific coagulant activity that was higher (~2.0 fold) than that of wild-type rFIX in all treatment conditions. Importantly, coagulant activity was concurrently increased to levels (3.0 ± 0.9%) that, if achieved in patients, would ameliorate the bleeding phenotype. Conclusions Altogether, our data detail molecular mechanisms underlying type I HB and candidate NaPBA as affordable 'personalized' therapeutics for patients affected by the highly frequent p.R294Q mutation, and with reduced access to substitutive therapy.

Abnormal bactericidal, metabolic, and lysosomal functions of Chediak-Higashi Syndrome leukocytes.

Phagocytic, antimicrobial, and metabolic functions were studied in leukocytes obtained from three patients with the Chediak-Higashi syndrome (CHS) and compared to normals, individuals, heterozygous for Chediak-Higashi syndrome, and two subjects with chronic granulomatous disease of childhood (CGD). Chediak-Higashi syndrome leukocytes showed normal ingestion of a variety of bacteria, Candida albicans, and polystyrene latex particles. Intracellular destruction was significantly impaired for Staphylococcus aureus, Group D streptococci, and a rough strain of Type II pneumococci over a 2 hr incubation. Killing of Serattia marcescens was consistently delayed at 1 hr whereas that of Escherichia coli and C. albicans appeared normal, unless the incubations were shortened to 20 min. Examination of the rates of killing indicated that the greatest defect occurred in the first 20 min of contact between Chediak-Higashi syndrome cells and bacteria. Separation of Chediak-Higashi syndrome granulocytes from monocytes revealed that the former were most defective in bactericidal activity. After phagocytosis, Chediak-Higashi syndrome granulocytes displayed a normal burst in oxygen consumption and oxidation of glucose-1-(14)C and glucose-6-(14)C and formate-(14)C. Oxidation of glucose-1-(14)C by non-phagocytizing Chediak-Higashi syndrome granulocytes and monocytes averaged 2-3 times normal, whereas glucose-6-(14)C and formate-(14)C oxidation were not significantly increased by resting cells. Iodination of intracellular protein by Chediak-Higashi syndrome leukocytes was significantly increased above normal in both the resting and phagocytizing state. Electron microscopic histochemistry revealed that almost all peroxidase activity was localized to the giant granules in Chediak-Higashi granulocytes, and after bacterial ingestion there was a failure of delivery of peroxidase to many phagosomes. Upon longer incubation more phagosomes acquired peroxidase activity, presumably through a fusion process, although many giant granules remained intact. The contrasting patterns and kinetics of the killing defects and the differing metabolic properties of Chediak-Higashi syndrome and chronic granulomatous disease leukocytes emphasize the pleiomorphic nature of inherited disorders of leukocyte function.

Adult chronic lead intoxication. A clinical review.

A stained-glass artisan with depression and a retired junkyard worker with congestive cardiomyopathy had increased mobilizable body burdens of lead by calcium ethylenediaminetetaacetic acid testing. Although both patients improved with several months of intramuscular chelation therapy, the efficacy of such therapy in chronic lead poisoning is controversial. Recognition of unusual manifestations of chronic lead poisoning may at least interrupt further exposure, even if specific therapy is not undertaken.

Clinical prediction of Pneumocystis pneumonia.

Pneumocystis carinii pneumonia is often difficult to diagnose in an ambulatory care setting. Previous reports have identified elements of the clinical history, physical examination, and clinical testing that are useful predictors of P carinii pneumonia. We analyzed published data on these predictors and measured them against methodologic standards for clinical prediction rules. Variables with high negative or positive predictive value for P carinii pneumonia, low error rates, or compelling biologic credibility were then selected to develop an untested clinical prediction rule for P carinii pneumonia. We suggest that dyspnea, oral lesions, chest roentgenographic examination, and pulse oximetry may be used to select patients requiring sputum testing and/or bronchoscopy for the diagnosis of P carinii pneumonia. The role of pulse oximetry in the diagnosis of P carinii pneumonia merits further study.

An engineered U1 small nuclear RNA rescues splicing defective coagulation F7 gene expression in mice.

BACKGROUND: The ability of the spliceosomal small nuclear RNA U1 (U1snRNA) to rescue pre-mRNA splicing impaired by mutations makes it an attractive therapeutic molecule. Coagulation factor deficiencies due to splicing mutations are relatively frequent and could therefore benefit from this strategy. However, the effects of U1snRNAs in vivo remain unknown. OBJECTIVES: To assess the rescue of the F7 c.859+5G>A splicing mutation (FVII+5A), causing severe human factor VII (hFVII) deficiency, by the modified U1snRNA+5a (U1+5a) in a murine model. METHODS: Mice expressing the human F7 c.859+5G>A mutant were generated following liver-directed expression by plasmid or recombinant adeno-associated viral (AAV) vector administration. The rescue of the splice-site defective pre-mRNA by U1+5a was monitored in liver and plasma through hFVII-specific assays. RESULTS: Injection of plasmids encoding the U1+5a rescued plasma hFVII levels, which increased from undetectable to ~8.5% of those obtained with the wild-type hFVII plasmid control. To assess long-term effects, mice were injected with low and high doses of two AAV vectors encoding the FVII+5A splice site mutant as template to be corrected by U1+5a. This strategy resulted in hFVII plasma levels of 3.9 ± 0.8 or 23.3 ± 5.1 ng mL⁻¹ in a dose-dependent manner, corresponding in patients to circulating FVII levels of ~1-4.5% of normal. Moreover, in both experimental models, we also detected correctly spliced hFVII transcripts and hFVII-positive cells in liver cells. CONCLUSIONS: Here we provide the first in vivo proof of-principle of the rescue of the expression of a splicing-defective F7 mutant by U1snRNAs, thus highlighting their therapeutic potential in coagulation disorders.

Should adult tetanus immunization be given as a single vaccination at age 65? A cost-effectiveness analysis.

OBJECTIVE: To compare three vaccination strategies for the prevention of adult tetanus. Each strategy includes childhood primary immunization and wound prophylaxis, and one of the following: 1) the currently recommended booster every ten years; 2) a single booster at 65 years of age; or 3) no intervention after age 6 except for wound prophylaxis. METHODS: Cost-effectiveness analysis was used to compare the three different strategies. A Markov model, cycled annually from age 5 through age 85, was applied to each strategy to predict the incidence and costs of tetanus for the U.S. adult population. RESULTS: The three strategies have very similar effects on life expectancy but different costs. Expressed incremental to no intervention after childhood primary immunization, the decennial booster strategy is least cost-effective, with a discounted incremental cost-effectiveness ratio of $143,138 per year of life saved compared with $4,527 for the single-booster strategy. Sensitivity analysis demonstrates that the decennial strategy is more effective but more costly over a wide range of model assumptions. CONCLUSIONS: The current policy of recommending tetanus booster vaccinations every ten years is effective but much more costly than a more easily implemented policy that also provides considerable protection against tetanus. The authors recommend forsaking decennial boosters in favor of a policy of including a single booster at age 65 along with other recommended health maintenance maneuvers reserved for that age.

Ulcerative colitis and steroid-responsive, diffuse interstitial lung disease. A trial of N = 1.

We describe a patient with ulcerative colitis and extracolonic manifestations in whom diffuse interstitial pulmonary disease developed that was responsive to glucocorticoid therapy one year after total proctocolectomy. The patient presented in December 1983 with a subacute course marked by cough and progressive exertional dyspnea, abnormal chest examination results, and a chest roentgenogram that revealed diffuse interstitial and alveolar infiltrates. A transbronchial biopsy specimen revealed a polymorphic interstitial infiltrate, mild interstitial fibrosis without apparent intraluminal fibrosis, and no vasculitis, granulomas, or significant eosinophilic infiltration. Within one week of the initiation of daily high-dose steroid therapy, the patient's symptoms dramatically improved; chest roentgenogram and forced vital capacity (60%) improved at a slower rate. All three measures deteriorated when alternate-day prednisone therapy was started but once again improved until the patient was totally asymptomatic, chest roentgenograms were normal, and forced vital capacity was 80% of the predicted value 2 1/2 years later.

Tailored media can enhance the success of smoking cessation clinics.

Smokers recruited through the medical outpatient clinics of two similar Veterans Hospitals over two successive years participated in a smoking cessation study which randomized them between a group assigned to behavior modification clinics and a group receiving a packet of smoking cessation material in the mail. Following the second year's clinics at the site of one of the two hospitals, an intensive media campaign, based on the content of the behavior modification program, was targeted at the study population over commercial television and radio. The six-month abstinence rate for clinic participants measured by self-report, serum thiocyanate and exhaled air carbon monoxide was 36.8% in the group assigned to clinic followed by media, 20.2% in the group assigned to clinic alone, and 10.6% in the group receiving materials in the mail. The difference in cessation rates between the clinic participants who were and those who were not exposed to the media following their clinics was significant at the 0.05 significance level (chi 2 = 3.9, 1 d.f.). Logistic analysis confirmed the benefit of the media campaign.

Reducing bone lead content by chelation treatment in chronic lead poisoning: an in vivo X-ray fluorescence and bone biopsy study.

A stained-glass artist with longstanding exposure to lead presented with neuropsychiatric symptoms. He was evaluated before and after chelation treatment by the CaNa2 EDTA lead mobilization test, iliac crest bone lead measurement, and in vivo tibial X-ray fluorescence (XRF). The three methods showed a progressive fall in body lead stores during chelation therapy in association with improvement in symptoms and a fall in blood lead and zinc protoporphyrin levels. In vivo tibial XRF is a safe, rapid, and noninvasive technique for detecting excessive body lead burdens. XRF measurement of bone lead content is a practical method for monitoring the efficacy of therapy as well as for establishing the diagnosis.

Comparison of sulfasalazine and placebo in the treatment of reactive arthritis (Reiter's syndrome). A Department of Veterans Affairs Cooperative Study.

OBJECTIVE: To determine whether sulfasalazine (SSZ) at a dosage of 2,000 mg/day is effective in the treatment of reactive arthritis (ReA) that has been unresponsive to nonsteroidal antiinflammatory drug (NSAID) therapy. METHODS: One hundred thirty-four patients with ReA who had failed to respond to NSAIDs were recruited from 19 clinics, randomized (double-blind) to receive either SSZ or placebo, and followed up for 36 weeks. The definition of treatment response was based on joint pain/tenderness and swelling scores and physician and patient global assessments. RESULTS: Longitudinal analysis revealed improvement in the patients taking SSZ compared with those taking placebo, which appeared at 4 weeks and continued through the trial (P = 0.02). At the end of treatment, response rates were 62.3% for SSZ treatment compared with 47.7% for placebo treatment. The Westergren erythrocyte sedimentation rate declined more with SSZ treatment than with placebo (P < 0.0001). Adverse reactions were fewer than expected and were mainly due to nonspecific gastrointestinal complaints. CONCLUSION: SSZ at a dosage of 2,000 mg/day is well tolerated and effective in patients with chronically active ReA.