Alterations of microRNAs throughout the malignant evolution of cutaneous squamous cell carcinoma: the role of miR-497 in epithelial to mesenchymal transition of keratinocytes.

Skin carcinogenesis is known to be a multi-step process with several stages along its malignant evolution. We hypothesized that transformation of normal epidermis to cutaneous squamous cell carcinoma (cSCC) is causally linked to alterations in microRNAs (miRNA) expression. For this end we decided to evaluate their alterations in the pathologic states ending in cSCC. Total RNA was extracted from formalin fixed paraffin embedded biopsies of five stages along the malignant evolution of keratinocytes towards cSCC: Normal epidermis, solar elastosis, actinic keratosis KIN1-2, advanced actinic keratosis KIN3 and well-differentiated cSCC. Next-generation small RNA sequencing was performed. We found that 18 miRNAs are overexpressed and 28 miRNAs are underexpressed in cSCC compared to normal epidermis. miR-424, miR-320, miR-222 and miR-15a showed the highest fold change among the overexpressed miRNAs. And miR-100, miR-101 and miR-497 showed the highest fold change among the underexpressed miRNAs. Heat map of hierarchical clustering analysis of significantly changed miRNAs and principle component analysis disclosed that the most prominent change in miRNAs expression occurred in the switch from 'early' stages; normal epidermis, solar elastosis and early actinic keratosis to the 'late' stages of epidermal carcinogenesis; late actinic keratosis and cSCC. We found several miRNAs with 'stage specific' alterations while others display a clear 'gradual', either progressive increase or decrease in expression along the malignant evolution of keratinocytes. The observed alterations focused in miRNAs involved in the regulation of AKT/mTOR or in those involved in epithelial to mesenchymal transition. We chose to concentrate on the evaluation of the molecular role of miR-497. We found that it induces reversion of epithelial to mesenchymal transition. We proved that SERPINE-1 is its biochemical target. The present study allows us to further study the pathways that are regulated by miRNAs along the malignant evolution of keratinocytes towards cSCC.

Low ALT blood levels predict long-term all-cause mortality among adults. A historical prospective cohort study.

BACKGROUND: Increased blood levels of alanine amino transferase (ALT, also known as SGPT; serum glutamic pyruvic transaminase) serve as a marker of liver injury by various mechanisms. Less is known about the clinical implications associated with low-normal ALT levels. Previous studies showed low ALT levels to be associated with poor long-term outcomes among elderlies, serving as a biomarker for increased incidence of frailty and subsequent risk of mortality. However, it has not been determined yet whether low-normal ALT values might be predictive of frailty and mortality in younger, middle-aged adults. METHODS: We conducted a historical prospective cohort analysis. RESULTS: A total of 23,506 adults with ALT levels within the normal range, at the mean age of 48 ± 11 years, participating in an annual screening program for preventive medicine, were followed-up for a median period of 8.5 years during which 638 died. Low-normal ALT values (serum ALT activity <17IU/L) were found to be predictive for increased risk of all-cause mortality (HR=1.6; 95% CI 1.34-1.92; p<0.001). Statistically significant correlation was demonstrated even after applying a multifactorial model correction for age, gender, eGFR, low albumin, arterial hypertension, diabetes mellitus and ischemic heart disease. CONCLUSIONS: We suggest that low-normal ALT values may serve as an independent predictive marker for increased long-term mortality in middle-aged adults.

Small-interfering RNA targeted at antiapoptotic mRNA increases keratinocyte sensitivity to apoptosis.

BACKGROUND: Gene silencing RNA interference technology concentrates on downregulation of gene expression using specific double-stranded RNA molecules (small-interfering RNA, siRNA), which induce the degradation of complementary mRNA. SiRNA therapeutics is currently being tested in several clinical trials. Skin disorders are an attractive target for siRNA-based technology because effective agents can be delivered by topical application. In several inflammatory skin disorders, the barrier function of the skin is markedly impaired and this may increase the delivery efficiency. Psoriasis is a very common skin disorder. The characteristics of this disorder are abnormal keratinocyte proliferation and inflammation. Keratinocytes from psoriatic epidermis express much higher levels of the antiapoptotic protein, Bcl-xL, compared with normal keratinocytes. Insulin-like growth factor 1 receptor (IGF-1R) plays a major role in cell growth, differentiation and apoptosis in many cell types, including keratinocytes and IGF-1R activation plays an important role in the pathogenesis of psoriasis. Keratinocytes from patients with psoriasis are more susceptible to IGF-1-stimulated proliferation compared with normal keratinocytes. IGF-1R is expressed by proliferating basal and suprabasal keratinocytes and is more abundant in psoriatic lesions. OBJECTIVES: To prove the validity of IGF-1R and Bcl-xL as useful targets for siRNA-based therapeutics and to deliver siRNA selectively and efficiently to primary human keratinocytes; this is a primary essential step in the development of siRNA. METHODS: Primary normal human keratinocytes were transfected with various siRNA molecules, and transfection efficiency was monitored by fluorescent labelling. Cell growth and apoptosis induction were evaluated in the transfected cells. RESULTS: We were able to deliver efficiently siRNA targeting Bcl-xL or IGF-1R to primary human keratinocyte cultures. We also showed that siRNAs targeting Bcl-xL and IGF-1R induce growth inhibition, apoptosis and increased sensitivity to ultraviolet B in keratinocytes. CONCLUSIONS: The present findings demonstrate that Bcl-xL and IGF-1R are valid, important targets for siRNA-based technology directed at the suppression of keratinocyte hyperproliferation.

Lipoid pneumonia: a preventable form of drug-induced lung injury.

We report a case where recurrent "pneumonia" was eventually diagnosed as lipoid pneumonia in an elderly patient with cerebrovascular disease. The discontinuation of paraffin oil laxative led to clinical improvement. Lipoid pneumonia, a foreign body-type reaction to the presence of lipid within lung parenchyma, is probably underdiagnosed and underreported, and paraffin oil laxative is the main causative agent. Paraffin oil is marketed as a food additive, and no information about its hazards is provided to clinicians or patients. We suggest that a change in paraffin oil licensing may decrease the incidence of lipoid pneumonia.

Thrombophilic factors are not the leading cause of thrombosis in Behçet's disease.

BACKGROUND: Venous and arterial thromboses occur in patients with Behçet's disease and are associated with significant morbidity and mortality. Studies on a possible association between the occurrence of thrombosis and thrombophilia in patients with this disease have been controversial. OBJECTIVE: To determine the prevalence of the most common thrombophilias and dyslipidaemia in patients with Behçet's disease with and without thrombosis. METHODS: Blood samples from 107 patients with Behçet's disease who had or did not have thrombosis were analysed for factor V Leiden, prothrombin G20210A polymorphism, methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, factor VIII level, homocysteine and C reactive protein concentrations, dyslipidaemia, and plasma glucosylceramide. RESULTS: There was no difference between patients with and without thrombosis in the prevalence of prothrombin G20210A polymorphism, factor V Leiden, homozygous MTHFR C677T, or plasma concentrations of homocysteine, C reactive protein, or glucosylceramide. In contrast, patients with thrombosis were found to have significantly higher mean levels of factor VIII, total cholesterol, triglycerides, VLDL cholesterol, and apolipoproteins B-100, C-II, and C-III than those without thrombosis. Multistepwise logistic regression analysis showed that triglyceride concentration was the best marker associated with thrombosis (p = 0.008), with an estimated odds ratio of 1.58 (95% confidence interval, 1.09 to 2.30) for a difference of 40 mg/dl. CONCLUSIONS: Thrombophilia does not seem to play a major role in the tendency to thrombosis in Behçet's disease. However, dyslipidaemia, predominantly hypertriglyceridaemia, might be a risk factor.

Altered subcellular localization of p53 in estrogen-dependent and estrogen-independent breast cancer cells.

LCC2, an estradiol-independent tamoxifen (Tax)-resistant subline of MCF-7 human breast cancer cell line, is resistant relatively towards Tax and methotrexate (Mtx). The purpose of the present study is to evaluate the role of p53 in determining this resistance. While MCF-7 is sensitive to and undergoes apoptosis, as determined by propidium iodide stain, by Tax and Mtx, LCC2 is resistant to apoptosis induction by these agents. Both cell lines undergo apoptosis and are sensitive equally to doxorubicin (Adr). p53 cDNA of both sublines was evaluated by polymerase chain reaction (PCR) amplification and sequencing and was found to be of wild-type. p53 mRNA, as well as protein, are elevated markedly in LCC2 as compared to MCF-7 cells. p53 expression was increased by estradiol and Adr, not changed by Mtx, and decreased by Tax and estradiol-deprivation in both sublines. p53 modulation by the various agents, in both sublines, was evaluated by cytochemical staining and subcellular fractionation. This analysis showed that p53 is localized mainly in the nuclear fraction in MCF-7 cells, and in the cytoplasmatic fraction in LCC2 cells. Doxorubicin induced apoptosis in MCF-7 cells along with increase in its nuclear fraction. In contrast, LCC2 underwent apoptosis by Adr despite its cytoplasmatic sequestration. These experiments demonstrate that p53 is sequestered to cytoplasm in the estrogen-independent, Tax-resistant LCC2 cells. However, the differences in apoptotic rate between MCF-7 and LCC2 cells do not seem to be dependent on p53. The LCC2 cell line may serve as a useful model for the study of the mechanism of cytoplasmatic sequestration of wild type (wt) p53, its physiologic consequences, and its relation to estrogen-independence or Tax resistance of breast cancer cells.

The changing epidemiology of leptospirosis in Israel.

We reviewed all serologically confirmed cases of leptospirosis from 1985 to 1999 in Israel, where the disease is endemic. There were 59 cases, with an average annual incidence of 0.05/100,000. The dominant serogroup, Leptospira icterohemorrhagica, occurred in 29% of patients; in an earlier study (1970-1979), it accounted for only 2%. Serogroups that occurred mainly in rural areas accounted previously for 79% but had declined to 32%.

Pulmonary-renal syndrome in systemic sclerosis.

BACKGROUND AND OBJECTIVE: Renal failure, pulmonary hypertension, and interstitial lung disease are major causes of morbidity and mortality in systemic sclerosis (SSc). However, the concomitant occurrence of pulmonary hemorrhage associated with acute renal failure in SSc has been rarely described. The present study is the first analysis of pulmonary-renal syndrome in SSc. PATIENT AND METHODS: We present a 44-year-old woman with SSc who died of a fulminant course of acute renal failure associated with diffuse alveolar hemorrhage. We termed this uncommon and fatal complication of SSc scleroderma-pulmonary-renal syndrome (SPRS). A search of the English-written literature yielded reports of 10 additional similar cases. These patients, together with our present case, form the basis of the present analysis. RESULTS: The average age of the patients with SPRS was 46 years. The majority of the patients (80%) were women, and most had diffuse SSc. SPRS occurred an average of 6.4 years after disease onset and was associated with prior fibrosing alveolitis and/or D-penicillamine treatment. Interestingly, normotensive renal failure seems to characterize the scleroderma patients, because 9 of 11 (82%) had normal blood pressure. SPRS bears a poor prognosis: all of the 11 patients (100%) died within 12 months of admission. However, only 60% of the 5 patients for whom we have treatment data received corticosteroids. CONCLUSIONS: Pulmonary-renal syndrome is a rare but fatal complication of SSc. Because the treatment data are scarce and the prognosis is poor, aggressive treatment with pulse corticosteroids, cyclophosphamide, and possibly plasmapheresis is suggested.

Differential sensitivity of MCF-7 and LCC2 cells, to multiple growth inhibitory agents: possible relation to high bcl-2/bax ratio?

Comparison of LCC2, the E(2)-independent, tamoxifen-resistant subline of the MCF-7 human breast cancer cell line with its parent line, disclosed that it is more resistant to growth inhibition and apoptosis induction by a variety of agents acting by diverse mechanisms. Thus, LCC2 cells can serve as a useful in-vitro model for the study of the molecular mechanisms of this resistance. It was found that bcl-2 protein and mRNA were elevated and that bax protein and mRNA were reduced in LCC2 compared with MCF-7 cells. Incubation of both lines in the presence of bcl-2 antisense caused growth inhibition and reduced bcl-2 protein levels only in MCF-7 cells, suggesting the involvement of bcl-2 in the regulation of normal growth of breast cancer cells. Increased bcl-2 expression in breast cancer cells may correlate with their resistance to growth inhibitory agents. Bcl-2 is a useful target for enhancing the effects of growth inhibitory agents.

Aortic aneurysm and dissection are not associated with an increased risk for giant cell arteritis/ polymyalgia rheumatica.

It has recently been claimed that giant cell arteritis (GCA) is associated with a markedly increased risk of aortic aneurysm formation or rupture. In the present study, the opposite approach was taken, by looking for the incidence of GCA and polymyalgia rheumatica (PMR) in patients with aortic aneurysm, aortic dissection, or both (AA/D). The records of 315 consecutive patients admitted with the diagnosis of AA/D were reviewed. In addition, follow up information was obtained in 82 patients by examination in the outpatient clinic. After careful examination and assessment of clinical and laboratory data, it was found that none of the 82 patients who survived hospitalisation and were available for examination had GCA or PMR. Moreover, review of the retrospective data available from hospital records of the total consecutive 315 patients with AA/D failed to find any patient with a diagnosis of GCA/PMR. In conclusion, the present study did not find an increased prevalence of GCA/PMR among a cohort of Israeli patients with AA/D. Therefore, it is suggested that a thorough investigation aiming to diagnose GCA/PMR is not cost effective in most of the elderly patients presenting with AA/D.

Aspergillus niger endocarditis in an immunocompetent patient: an unusual course.

Aspergillus is an opportunistic nosocomial fungus generally associated with a high mortality rate. A niger has been rarely associated with infection, and most cases have occurred in patients who have recently undergone heart surgery or in immunocompromised patients. We present a case of an immunocompetent patient with A niger endocarditis which illustrates the difficulties in diagnosis and the possible insidious course of fungal endocarditis.

Systemic vasculitis presenting as a tumorlike lesion. Four case reports and an analysis of 79 reported cases.

Systemic vasculitis might present as a tumorlike lesion that initially could misdirect the correct diagnosis and the appropriate medical treatment. The aim of the present study is to summarize all reported cases of tumorlike presentation in systemic vasculitides, in order to have comprehensive data on the characteristics of this unusual phenomenon. We report 4 cases of systemic vasculitis presenting as a tumorlike lesion. In addition, we performed a MEDLINE search of all English-language papers published from 1966 to 1999, looking for vasculitis presenting as tumorlike lesion. Details were included concerning vasculitis classification, specific characteristics, location of the "tumor," the presence or absence of systemic involvement, and whether surgery was performed before diagnosis. Seventy-nine cases of vasculitis presenting as a tumorlike lesion were found in the literature, in addition to the 4 new cases described. The average age of the reported cases was 50.5 +/- 15.8 years, and 51% were female. In 82% of the cases the "tumor" was associated with constitutional symptoms and elevated erythrocyte sedimentation rate (ESR). The most common vasculitis categories with tumorlike presentation were Wegener granulomatosis (WG; 28 cases) and giant cell arteritis (GCA; 17 cases). In almost half the patients, surgery was performed before diagnosis. All patients with GCA presented with either a breast or an ovarian tumor. The most common location of a tumorlike lesion was the breast (22%), followed by central nervous system lesions (16%). Other frequent locations were the ovary (10%), caused exclusively by GCA and polyarteritis nodosa (PAN), and the male genitourinary system, almost all caused by PAN. Including vasculitis in the differential diagnosis of a tumorlike lesion might lead to an earlier diagnosis and consequently to prompt and appropriate treatment, avoiding needless operations. Constitutional symptoms and elevated ESR should alert clinicians to the possible diagnosis of vasculitis rather than a tumor. The association of GCA with ovarian pseudotumor is distinct and has not been emphasized before. We therefore suggest that GCA should be included in the list of differential diagnosis of an ovarian or breast tumor in an elderly woman, particularly when systemic symptoms and parameters of inflammation are present.

Efficacy and safety of intravenous amiodarone in recent-onset atrial fibrillation: experience in patients admitted to a general internal medicine department.

We examined the efficacy and safety of intravenous amiodarone in 20 unselected patients with recent-onset atrial fibrillation who were admitted to a general internal medicine department during a 6-month period. The treatment protocol included a loading dose of 1200 mg intravenous amiodarone in 24 hours, after which amiodarone treatment was continued orally. Eleven of the 20 patients (55%) converted to sinus rhythm within 48 hours of intravenous amiodarone treatment and were discharged in sinus rhythm, while 9/20 (45%) patients failed to convert during hospitalisation. Six patients (30%) failed to convert to sinus rhythm even after one further month of oral treatment. There was one death and a high frequency (25%) of thrombophlebitis during hospitalisation. The in-hospital non-convertors had a significantly lower ejection fraction and initial low ventricular response rate than the convertors. In conclusion, the acute conversion rate by intravenous amiodarone was at best modest. It is suggested that intravenous amiodarone is probably more effective in patients with rapid recent-onset atrial fibrillation and good left ventricular function.

Polyarteritis nodosa: a report from Israel.

BACKGROUND: The clinical manifestations and outcome of all adult patients with polyarteritis nodosa (PAN), allocated during a 15-year period in the largest medical center in Israel, were examined. METHODS: A retrospective analysis of patients with PAN who fulfilled the American College of Rheumatology (ACR) 1990 Classification Criteria and were either biopsy- or angiography-proven. RESULTS: Nine patients were included in the report. The clinical and laboratory manifestations were similar to those in previous studies. All patients were treated with combinations of cyclophosphamide and corticosteroids. There were two (22%) deaths, 2 and 5 months after initiation of treatment in patients who probably had microscopic polyangiitis (MPA) rather than classical PAN. Considering the patients with a complete follow-up, 71% had a complete and long-term remission. Moreover, by exclusion of the two patients with probable MPA who died, all of the five patients with classical PAN were alive and well as of this writing. Two patients (22%) had a long history, since childhood, of familial Mediterranean fever (FMF). CONCLUSIONS: The clinical presentation and course of PAN in Israeli patients is comparable with reports elsewhere. However, a distinction should be made between PAN and MPA. The present report emphasizes the good long-term prognosis of patients with typical PAN who are treated adequately. In addition, a possible association of PAN with FMF in Israeli patients is suggested.

Rheumatic manifestations preceding adult acute leukemia: characteristics and implication in course and prognosis.

The manifestations and outcome of adult patients with acute leukemia (AL) were examined to study the characteristics of the rheumatological prodrome of AL and to find whether it is a marker of a distinct clinical and laboratory course and whether it has any prognostic implication. During a 10-year period, 8/139 (5.8%) of AL patients presented with rheumatic manifestations. The average duration of the arthritis syndrome preceding the diagnosis of AL was 3.25 months. The most common pattern of presentation was a reactive arthritis-like syndrome involving the large joints asymmetrically and associated with low back pain. Distinctive features suggesting a paraneoplastic arthritis were severe pain disproportionate to physical findings, a poor response to conventional antirheumatic treatment, and early significant osteopenia or lytic bone lesions. The epidemiological, clinical and laboratory characteristics of patients with or without rheumatic manifestations were comparable, except for fever on presentation, the presence of transient metabolic derangement following chemotherapy, and the initial average hemoglobin, hematocrit and serum uric acid values. Moreover, the initial outcome of the two groups was similar, as the early mortality rates were comparable (42.8% vs. 45% for patients with or without arthritis, respectively). In conclusion, rheumatic syndrome presentation of adult AL is uncommon, and apparently has no deleterious effects on initial prognosis. A timely diagnosis requires an increased awareness to distinctive features.