Dabigatran for Extended Treatment of Venous Thromboembolism.


In clinical trials, the drug was compared with warfarin and placebo.

After an initial course of treatment for venous thromboembolism (VTE), extended warfarin therapy lowers risk for recurrent VTE — but at the expense of excess risk for bleeding. Dabigatran (Pradaxa) now has been examined for this indication in two placebo-controlled, industry-sponsored trials.

One trial included nearly 2900 VTE patients at especially high risk for recurrence; after initial treatment (mean, 7 months), they were randomized to receive either dabigatran or warfarin. During extended treatment that averaged 16 months, the incidence of symptomatic or fatal VTE was 1.8% with dabigatran and 1.3% with warfarin, a result that met criteria for “noninferiority” of dabigatran. Major or clinically relevant bleeding was significantly less common with dabigatran than with warfarin (5.6% vs. 10.2%).

Another trial included about 1300 VTE patients at lower risk for recurrence than patients in the first trial; after initial treatment (mean, 10 months), they were randomized to receive either dabigatran or placebo. During extended treatment that averaged 5.5 months, the incidence of symptomatic or fatal VTE was significantly lower with dabigatran than with placebo (0.4% vs. 5.6%). Major or clinically relevant bleeding was significantly more common with dabigatran than with placebo (5.3% vs. 1.8%).

Comment: Dabigatran’s efficacy was similar to that of warfarin and superior to that of placebo; dabigatran caused less bleeding than warfarin but more bleeding than placebo. One worrisome observation was a small excess of adverse coronary events with dabigatran in the high-risk trial — an outcome that has been noted in previous dabigatran trials (JW Cardiol Feb 15 2012). Dabigatran has not yet been FDA approved for deep venous thrombosis treatment or prophylaxis.

Source: Journal Watch General Medicine

Brain-to-brain interface allows transmission of tactile and motor information between rats.

braintobrainResearchers have electronically linked the brains of pairs of rats for the first time, enabling them to communicate directly to solve simple behavioral puzzles. A further test of this work successfully linked the brains of two animals thousands of miles apart—one in Durham, N.C., and one in Natal, Brazil.

The results of these projects suggest the future potential for linking multiple brains to form what the research team is calling an “organic computer,” which could allow sharing of motor and sensory information among groups of animals. The study was published Feb. 28, 2013, in the journal Scientific Reports. “Our previous studies with brain-machine interfaces had convinced us that the rat brain was much more plastic than we had previously thought,” said Miguel Nicolelis, M.D., PhD, lead author of the publication and professor of neurobiology at Duke University School of Medicine. “In those experiments, the rat brain was able to adapt easily to accept input from devices outside the body and even learn how to process invisible infrared light generated by an artificial sensor. So, the question we asked was, ‘if the brain could assimilate signals from artificial sensors, could it also assimilate information input from sensors from a different body?'” To test this hypothesis, the researchers first trained pairs of rats to solve a simple problem: to press the correct lever when an indicator light above the lever switched on, which rewarded the rats with a sip of water. They next connected the two animals’ brains via arrays of microelectrodes inserted into the area of the cortex that processes motor information. One of the two rodents was designated as the “encoder” animal. This animal received a visual cue that showed it which lever to press in exchange for a water reward. Once this “encoder” rat pressed the right lever, a sample of its brain activity that coded its behavioral decision was translated into a pattern of electrical stimulation that was delivered directly into the brain of the second rat, known as the “decoder” animal.

The decoder rat had the same types of levers in its chamber, but it did not receive any visual cue indicating which lever it should press to obtain a reward. Therefore, to press the correct lever and receive the reward it craved, the decoder rat would have to rely on the cue transmitted from the encoder via the brain-to-brain interface. The researchers then conducted trials to determine how well the decoder animal could decipher the brain input from the encoder rat to choose the correct lever. The decoder rat ultimately achieved a maximum success rate of about 70 percent, only slightly below the possible maximum success rate of 78 percent that the researchers had theorized was achievable based on success rates of sending signals directly to the decoder rat’s brain. Importantly, the communication provided by this brain-to-brain interface was two-way. For instance, the encoder rat did not receive a full reward if the decoder rat made a wrong choice. The result of this peculiar contingency, said Nicolelis, led to the establishment of a “behavioral collaboration” between the pair of rats. “We saw that when the decoder rat committed an error, the encoder basically changed both its brain function and behavior to make it easier for its partner to get it right,” Nicolelis said. “The encoder improved the signal-to-noise ratio of its brain activity that represented the decision, so the signal became cleaner and easier to detect. And it made a quicker, cleaner decision to choose the correct lever to press. Invariably, when the encoder made those adaptations, the decoder got the right decision more often, so they both got a better reward.” In a second set of experiments, the researchers trained pairs of rats to distinguish between a narrow or wide opening using their whiskers. If the opening was narrow, they were taught to nose-poke a water port on the left side of the chamber to receive a reward; for a wide opening, they had to poke a port on the right side. The researchers then divided the rats into encoders and decoders. The decoders were trained to associate stimulation pulses with the left reward poke as the correct choice, and an absence of pulses with the right reward poke as correct. During trials in which the encoder detected the opening width and transmitted the choice to the decoder, the decoder had a success rate of about 65 percent, significantly above chance. To test the transmission limits of the brain-to-brain communication, the researchers placed an encoder rat in Brazil, at the Edmond and Lily Safra International Institute of Neuroscience of Natal (ELS-IINN), and transmitted its brain signals over the Internet to a decoder rat in Durham, N.C. They found that the two rats could still work together on the tactile discrimination task. “So, even though the animals were on different continents, with the resulting noisy transmission and signal delays, they could still communicate,” said Miguel Pais-Vieira, PhD, a postdoctoral fellow and first author of the study. “This tells us that it could be possible to create a workable, network of animal brains distributed in many different locations.” Nicolelis added, “These experiments demonstrated the ability to establish a sophisticated, direct communication linkage between rat brains, and that the decoder brain is working as a pattern-recognition device. So basically, we are creating an organic computer that solves a puzzle.” “But in this case, we are not inputting instructions, but rather only a signal that represents a decision made by the encoder, which is transmitted to the decoder’s brain which has to figure out how to solve the puzzle. So, we are creating a single central nervous system made up of two rat brains,” said Nicolelis. He pointed out that, in theory, such a system is not limited to a pair of brains, but instead could include a network of brains, or “brain-net.” Researchers at Duke and at the ELS-IINN are now working on experiments to link multiple animals cooperatively to solve more complex behavioral tasks. “We cannot predict what kinds of emergent properties would appear when animals begin interacting as part of a brain-net. In theory, you could imagine that a combination of brains could provide solutions that individual brains cannot achieve by themselves,” continued Nicolelis. Such a connection might even mean that one animal would incorporate another’s sense of “self,” he said. “In fact, our studies of the sensory cortex of the decoder rats in these experiments showed that the decoder’s brain began to represent in its tactile cortex not only its own whiskers, but the encoder rat’s whiskers, too. We detected cortical neurons that responded to both sets of whiskers, which means that the rat created a second representation of a second body on top of its own.” Basic studies of such adaptations could lead to a new field that Nicolelis calls the “neurophysiology of social interaction.” Such complex experiments will be enabled by the laboratory’s ability to record brain signals from almost 2,000 brain cells at once. The researchers hope to record the electrical activity produced simultaneously by 10-30,000 cortical neurons in the next five years. Such massive brain recordings will enable more precise control of motor neuroprostheses—such as those being developed by the Walk Again Project—to restore motor control to paralyzed people, Nicolelis said. The Walk Again Project recently received a $20 million grant from FINEP, a Brazilian research funding agency, to allow the development of the first brain-controlled whole-body exoskeleton aimed at restoring mobility in severely paralyzed patients. A first demonstration of this technology is scheduled for the opening game of the 2014 Soccer World Cup in Brazil.

Read more at: http://medicalxpress.com/news/2013-02-brain-to-brain-interface-transmission-tactile-motor.html#jCp

Novartis collaboration aims to eliminate rheumatic heart disease (RHD) in Zambia, Africa.

  • novartisRHD has been eliminated in most developed nations, but sub-Saharan Africa studies show at least 2-3% of school-age children suffer from this often fatal disease.

  • Collaboration between Novartis physicians, Zambian healthcare providers, cardiologists from Massachusetts General Hospital (MGH) and the Pan-African Cardiology Society will promote RHD prevention by treating children with streptococcal infections and silent RHD
  • The collaboration will screen 3,000 Zambian children by echocardiography and provide monthly penicillin injections to children with silent RHD to prevent recurrent strep throat and associated cardiac damage

Novartis today announced that it has launched an effort to eliminate rheumatic heart disease in Zambia in collaboration with the Lusaka University Teaching Hospital (UTH), the Ministry of Health in Zambia, the Pan-African Cardiology Society and Massachusetts General Hospital (MGH).

RHD is a complication of untreated streptococcal infections in which the valves of the heart are scarred and eventually degenerate, leading to heart failure. Eliminated by antibiotic treatment in most developed nations, in the developing world an estimated 15 million children suffer from this debilitating and often fatal disease[1].

“The toll of heart failure in young children with RHD in Zambia is immense, for the patient, their families, and the nation,” said Mark C. Fishman, Cardiologist and President of the Novartis Institutes for BioMedical Research (NIBR). “It is entirely preventable. For the past several years Novartis has been working with colleagues in Lusaka to help understand and treat asthma in young children. We are expanding the collaboration to raise awareness, educate, and provide antibiotic therapy to prevent RHD.”

To measure RHD prevalence and identify those in need of secondary prophylaxis, teams of health care professionals from Lusaka UTH, the MGH, and Novartis will use portable echocardiography machines to evaluate 3,000 children, ages 9-10, in Lusaka-area public schools. Echocardiography screening is estimated to detect more than 10 times as many cases as clinical screening[1].

Images from the echocardiography screens will be analyzed in Zambia and at the MGH using a cloud-based electronic registry developed by Dimagi Inc, a Cambridge, MA-based company that designs open-source electronic healthcare systems for low resource environments.

Children identified as having RHD will be treated with monthly penicillin injections (termed “secondary prophylaxis”) to prevent recurrent streptococcal infections and additional valve damage.
Primary prevention, the treatment of children with streptococcal infection to prevent RHD, is key to elimination of the disease. To this end, all children diagnosed with strep throat will be treated with injectable penicillin in the community-based study sites. Prevalence of RHD and adherence to secondary prophylaxis will be determined via the mobile electronic registry.

“We have assembled an experienced team from MGH who are excited to bring the mobile heart imaging technology to Zambia,” stated Michael H. Picard, MD, Director of Echocardiography at the Massachusetts General Hospital and a Past President of the American Society of Echocardiography. “We are creating a model for country-wide screening through schools that will not only raise awareness of the magnitude of this disease but also offer a simple method to identify those who will benefit from a very simple and safe treatment. The MGH Cardiology Division and its Cardiac Ultrasound Laboratory are delighted to be a partner in this initiative.”

The Pan-African Cardiology Society will assist with the development of the study protocol and ethics approval. Based on the experience of the initial Lusaka-based effort, Novartis plans to support the rollout of the RHD training and treatment effort to Provinces across Zambia, with the ultimate goal of eliminating RHD in Zambia.

“Rheumatic heart disease is the most common acquired heart ailment in Zambian children, but statistics are spotty and the disease is certainly diagnosed late when damage to the heart valves has already reached advanced stage,” said John Musuku, Principal Investigator and UTH pediatrician. “Our hope is that the collaboration with Novartis will lay the foundation to detect the disease early so preventative measures are instituted.  This is an effort to eradicate the disease across Zambia in our life time.”


Source: Novartis

Major Psychiatric Disorders Linked to Genes Involved with Brain’s Calcium Balance.

Five major psychiatric illneses — autism, ADHD, bipolar disorder, depression, and schizophrenia — seem related to calcium-signaling pathways in the brain, according to a Lancet study.

Researchers performed genome-wide analyses on some 30,000 patients with the disorders and a roughly equal number of controls. They identified four genetic variants — all related to calcium signaling — that were significantly associated with the presence of one of the five disorders.

Commentators, noting the importance of calcium signaling to nerve growth and development, conclude that the results could help identify new targets for psychotropic drugs.

Source: Lancet


Ondansetron Safe During Pregnancy.

A large Danish study in the New England Journal of Medicine shows no significant association between the antiemetic ondansetron and adverse pregnancy outcomes.

In this retrospective cohort study, ondansetron had been prescribed for nausea and vomiting in almost 2000 of some 600,000 pregnancies. Ondansetron users were no more likely than nonusers to experience spontaneous abortion or stillbirth, or to have preterm delivery, a small-for-getational-age infant, or an infant with a major birth defect.

Source: Journal Watch Women’s Health


It’s a Trap. 10 Interview Questions Designed To Trick You .


Hiring managers are tasked with the impossible job of learning a candidate inside and out after just a few interactions. That’s why they’re always coming up with new tactics to extract every last drop of information from a candidate. It’s important to keep your guard up!  You can almost be sure some of the questions asked will be “interview traps” – interview questions designed to get you to reveal some critical bit of information about yourself that you might have preferred to remain covered. They come in many forms, but all have the common goal of getting you to expose some character flaw that will bump you down a few rungs in the rankings.

Hold it together! Here are 10 of the most popular “interview traps” and tips on how to use them to your advantage.

The setup: Why is there a gap in your work history?

The trap: Does all this time off work mean you’re lazy?

It’s not necessarily a problem to have a gap on your resume. If you pursued personal projects, took care of a sick relative, volunteered for charity or otherwise used your time off in a productive manner, let them know. They don’t care that you haven’t spent any recent time in an office – only that you haven’t spent it all on the couch.

The setup: What would the person who likes you least in the world say about you?

The trap: Are you aware of your own weaknesses – and how to work around them?

A cousin to “what’s your biggest weakness?,” this question also requires framing your dominant personality traits in a positive light. Perhaps your enemy would say you’re neurotic and controlling, when in fact you just have a completionist’s eye for detail, which will ensure no project is finished until all loose ends are tied and re-tied for peace of mind.

The setup: Describe when you were part of a team that could not get along.

The trap: Do you work well with people you don’t like?

No matter whose fault it actually was, the interviewer will assume you can’t work well with others if you complain about a dysfunctional team buried in your work history. What matters to them is how you handled the situation – did you allow room for discussions and ideas you may not have agreed with? Did you learn any lessons about give-and-take from clashing with a coworker?

The setup: If you could change one thing about your last job, what would it be?

The trap: Are you holding on to any lingering issues you couldn’t resolve at your last job?

Can you vocalize your problems in a professional manner and come to a diplomatic understanding with your coworkers / bosses? This question tests whether you let problems stew and boil over, or whether you can address them rationally with the benefit of a positive work environment in mind.

The setup: Explain ________ (your industry) to your nephew / grandmother / totally oblivious client.

The trap: Sure, you know your line of work – but can you communicate your responsibilities to others?

Are you a good communicator? As a developer, can you explain how the newest product feature operates in a way that the marketing team can process, so they can in turn pitch it to customers? If you can’t explain your job duties in plain English, you probably aren’t well-versed enough in the field to effectively communicate your needs to the coworkers you will interact with on a daily basis.

The setup: Tell me about yourself.

The trap: Are you lying on your resume? Are you confident you’re qualified for this job?

Don’t meander. This also tests your communication skills – whether you know how to pitch, and whether you know when to stop talking. Succinctly list education history, skills gained from previous jobs, and perhaps a personal project or two which enhances your skill set and demonstrates motivation outside of the workplace. Then, stop talking. Rambling indicates a lack of confidence, suggesting you’re not sure whether what you’ve listed is “enough” to qualify you for the job.

The setup: Why should we hire you?

The trap: Are you a good fit for this specific role and company?

If you can’t answer this question, you probably didn’t research the company you’re trying to work for. Make sure you know the specific functions your future role will entail, and the short- and long-term goals of the organization itself. Then, frame your skills in a context which aligns with the job description and the company’s direction.

It also doesn’t hurt to research the hiring board to find out what makes them tick, so you can carry the conversation if they mention a project from their background.

The setup: What’s your ideal job?

The trap: …Is it something other than this one?

It’s okay to have career aspirations, so long as the things you want to do overlap with the things you’ll be doing here. Avoid mentioning a title – it may not carry the clout in this company’s role structure that you think it does. Instead, discuss the problems you’d like to solve, platforms you want to work with, and other active engagements that encompass both your dream work and the work in front of you.

The setup: What annoys you about coworkers / bosses?

The trap: Are you easy to work with, or are you a Negative Nancy?

It’s never a good idea to badmouth a coworker, whether peer or superior. It’s best to say you’ve been fortunate to navigate amicable work relationships. If pressed, mention an attribute that highlights dedication to the company cause, and say that you will expect and encourage that same dedication from your peers.

The setup: If you won the lottery, would you still work?

The trap: Are you motivated to succeed?

Most people know this question aims to trap candidates for whom work is merely a means to an end, rather than a passion to which they will be dedicated. But it’s also facetious to say you’d stay in your current position if you were to be blessed with such fortunes. It’s perfectly acceptable to say you’d start your own company, charity or project to further your personal development. This question really gets at whether you’re naturally inclined to work, so make sure those imaginary piles of cash would enable some form of future productivity.

Source: smarterer.com




Pentoxifylline reduces fibrin deposition and prolongs survival in neonatal hyperoxic lung injury.


Bronchopulmonary dysplasia is a leading cause of mortality and morbidity in preterm infants despite improved treatment modalities. Pentoxifylline, a phosphodiesterase inhibitor, inhibits multiple processes that lead to neonatal hyperoxic lung injury, including inflammation, coagulation, and edema. Using a preterm rat model, we investigated the effects of pentoxifylline on hyperoxia-induced lung injury and survival. Preterm rat pups were exposed to 100% oxygen and injected subcutaneously with 0.9% saline or 75 mg/kg pentoxifylline twice a day. On day 10, lung tissue was harvested for histology, fibrin deposition, and mRNA expression, and bronchoalveolar lavage fluid was collected for total protein concentration. Pentoxifylline treatment increased mean survival by 3 days (P = 0.0018) and reduced fibrin deposition by 66% (P < 0.001) in lung homogenates compared with untreated hyperoxia-exposed controls. Monocyte chemoattractant protein-1 expression in lung homogenates was decreased, but the expressions of TNF-α, IL-6, matrix metalloproteinase-12, tissue factor, and plasminogen activator inhibitor-1 were similar in both groups. Total protein concentration in bronchoalveolar lavage fluid was decreased by 33% (P = 0.029) in the pentoxifylline group. Pentoxifylline treatment attenuates alveolar fibrin deposition and prolongs survival in preterm rat pups with neonatal hyperoxic lung injury, probably by reducing capillary-alveolar protein leakage.

bronchopulmonary dysplasia (BPD) is a leading cause of mortality and morbidity in preterm newborn infants with respiratory distress syndrome despite improved treatment modalities (22). Nowadays, BPD, defined by oxygen requirement at 36 wk of gestation, affects especially newborn infants born at <30 wk of gestation with a birth weight <1,200 g (1, 13). BPD is a multifactorial disease, characterized by decreased alveolarization and abnormal vascularization and associated with oxidative stress, barotrauma, surfactant deficiency, inflammation, alveolar fibrin deposition, nutrition, and genetic background (13–15). The (im)balance between initiating factors and host characteristics probably determines whether BPD will actually occur in the preterm infant.

The inflammatory response and coagulation cascade play important roles in the pathophysiology of acute and chronic lung disease in newborn infants and animal models. Increased levels of proinflammatory cytokines (TNF-α and IL-6) have been observed in tracheal aspirates of infants developing BPD (16–18). In addition, intra-alveolar and intravascular fibrin deposition has been detected in acute lung injury (2, 10, 11). In animal models, including premature baboons and neonatal mice and rats, exposure to hyperoxia results in chronic lung disease closely resembling BPD in preterm infants (3, 5, 8, 28, 37, 39).

In a previous study (37), our group demonstrated that genes involved in inflammation, coagulation, fibrinolysis, fibrosis, extracellular matrix turnover, alveolar development, edema, cell cycle, and oxidative stress response are differentially expressed in preterm rats with BPD secondary to exposure to hyperoxia (37). Expression profiles were in line with histopathology. The importance of extravascular fibrin deposition and upregulation of the coagulation cascade and inflammatory response in this model suggests a potential role for compounds with anti-inflammatory and anticoagulant properties. The methylxantine derivative pentoxifylline (PTX) has been used in the treatment of peripheral arterial diseases because of its positive effects on the capillary blood flow by reducing blood viscosity and improving erythrocyte deformability (27, 38). Moreover, PTX, a phosphodiesterase inhibitor, has significant anti-inflammatory effects, resulting in accumulation of intracellular cAMP. PTX decreases neutrophil sequestration, prevents the pulmonary vascular permeability to protein (12, 41, 43), and inhibits the production of free oxygen radicals (33). The inhibitory effect of PTX on proinflammatory responses and diffuse intravascular coagulation was demonstrated in baboons with lipopolysaccharide-induced endotoxemia (23). PTX was also effective in a radiation-induced lung model in mice by inhibiting TNF-α mRNA and protein production in lung tissue (29). Modulatory effects of PTX on the production of IL-6 were demonstrated in fetal rat type II pneumocytes exposed to hyperoxia and nitric oxide (6).

Because PTX inhibits multiple processes that contribute to the development of BPD, including inflammation, coagulation, and edema, we investigated the effects of PTX treatment on hyperoxia-induced lung injury. We demonstrate that PTX treatment in an experimental model of neonatal hyperoxic lung injury reduces extravascular fibrin deposition and prolongs survival.



Timed-pregnant Wistar rats were kept in a 12:12-h dark-light cycle and fed a standard chow diet (Special Diet Services, Witham, Essex, UK) ad libitum. After a gestation of 21.5 days (spontaneous birth occurs 22 days after conception), pregnant rats were killed by decapitation and pups were delivered by hysterectomy through a median abdominal incision. Immediately after birth, pups were dried and stimulated. Pups from two to three litters, with a maximum of 12 pups, were pooled and exposed to 100% oxygen in a transparent 50 × 50 × 70 cm Plexiglas chamber for 10 days or until death occurred (survival experiments). Pups were fed by lactating foster rats. To avoid oxygen toxicity of the foster rats, two groups of five pups exposed to room air were included and used to generate data on room air-exposed controls. Foster rats were rotated daily between the three cages. The oxygen concentration, weight, evidence of disease, and mortality were checked twice a day. Hyperoxia-exposed pups were injected subcutaneously, starting on day 2, via a 0.5-ml syringe (U-100 Micro-Fine insulin 29G syringe, Becton Dickinson, NY) at the lower back. Pups received either 150 μl of 75 mg/kg PTX (Sigma, St. Louis, MO) in 0.9% saline or 150 μl of 0.9% saline (age-matched control) twice a day. The research protocol was approved by the Institutional Animal Care and Use Committee of the Leiden University Medical Center.

Tissue preparation.

Pups were anesthetized with an intraperitoneal injection of ketamine (50 mg/kg body wt; Ketanest-S, Parke-Davis/Pfizer, New York, NY) and xylazine (50 mg/kg bodyweight; Rompun, Bayer, Leverkusen, Germany). To avoid postmortem fibrin deposition in the lungs, heparin (100 units; Leo Pharma, Breda, the Netherlands) was injected intraperitoneally. After 5 min, pups were exsanguinated by transection of the abdominal blood vessels. The thoracic cavity was opened, and the lungs were removed, snap-frozen in liquid nitrogen, and stored at −80°C until use for real-time RT-PCR or the fibrin deposition assay. For histology studies, the trachea was cannulated (Bioflow 0.6-mm intravenous catheter, Vygon, Veenendaal, the Netherlands), and the lungs were fixed in situ via the trachea cannula with buffered formaldehyde (3.8% paraformaldehyde in PBS, pH 7.4) at 25 cmH2O pressure for 3 min. Lungs were removed, fixed additionally in formaldehyde for 24 h at 4°C, and embedded in paraffin after dehydration in a graded alcohol series and xylene.

Brochoalveolar lavages.

Pups were anesthetized with an intraperitoneal injection of ketamine and xylazine and injected intraperitoneally with heparin. A cannula (Bioflow 0.6 mm intravenous catheter) was positioned in the trachea, and the pups were exsanguinated by transection of the abdominal blood vessels. Lungs were slowly lavaged four times with 500 μl NaCl, 0.9%, 1 mM EDTA (pH 8.0). Samples were pooled and centrifuged for 10 min at 5,000 rpm. Supernatants were stored at −20°C until further use.

Lung histology.

Paraffin sections (4 μm) from the left upper lobe were cut and mounted onto SuperFrost plus-coated slides (Menzel-Gläzer). After deparaffinization, sections were stained with hematoxylin and eosin or with a monoclonal anti-human fibrin antibody (59D8, Boston Research Services, Winchester, MA) that specifically recognizes the β-chain of fibrin (9, 40). For immunohistochemistry, sections were incubated with 0.3% H2O2 in methanol to block endogenous peroxidase activity. After a graded alcohol series, sections were boiled in 0.01 M sodium citrate (pH 6.0) for 10 min. Sections were incubated overnight with 59D8, stained with EnVision-HRP (Dako, Glostrup, Denmark), using NovaRed (Vector, Burlingame, CA) as chromogenic substrate, and counterstained briefly with hematoxylin.

Fibrin detection assay.

Fibrin deposition in lungs was detected as described previously (40). Briefly, frozen lungs were homogenized with an Ultra-Turrax T8 tissue homogenizer (IKA-Werke, Staufen, Germany) for 1 min at full speed in a cold 10 mM sodium phosphate buffer (pH 7.5), containing 5 mM EDTA, 100 mM ε-aminocaproic acid, 10 U/ml aprotinin, 10 U/ml heparin, and 2 mM phenylmethanesulfonyl fluoride. The homogenate was incubated for 16 h on a top over top rotor at 4°C. After centrifugation (10,000 rpm, 4°C, 10 min), the pellet was resuspended in extraction buffer [10 mM sodium phosphate buffer (pH 7.5), 5 mM EDTA, and 100 mM ε-aminocaproic acid] and recentrifuged. Pellets were suspended in 3 M urea, extracted for 2 h at 37°C, and centrifuged at 14,000 rpm for 15 min. After the supernatant was aspirated and discarded, the pellet was dissolved at 65°C in reducing sample buffer (10 mM Tris, pH 7.5, 2% SDS, 5% glycerol, 5% β-mercaptoethanol, and 0.4 mg/ml bromophenol blue) for 90 min with vortexing every 15 min. Hereafter, samples were subjected to SDS-PAGE (7.5%; 5% stacking) and blotted onto PVDF membrane (Immobilon-P, Millipore, Bredford, MA). The 56-kDa fibrin β-chains were detected with a monoclonal anti-human fibrin antibody (59D8, Boston Research Services, Winchester, MA) that specifically recognizes β-fibrin (9, 40), using enhanced chemiluminescence (Amersham, Arlington Heights, IL) and Kodak X-OMAT Blue XB-1 films (Kodak, Rochester, NY). Exposures were quantified with a Bio-Rad GS-800 calibrated densitometer using the Quantity One, version 4.4.1 software package (Bio-Rad, Veenendaal, the Netherlands). Fibrin deposition was quantified in lungs of at least four rats per experimental group. As a reference, fibrin standards were generated from rat fibrinogen (Sigma, St. Louis, MO). After rat fibrinogen was solubilized in two-thirds strength PBS (pH 7.4), human α-thrombin (Sigma, St. Louis, MO) was added, vortexed, and incubated at 37°C for 10 min. After addition of 2× SDS sample buffer, the fibrin sample was vortexed and incubated at 65°C for 90 min; aliquots were frozen at −80°C until use.

Real-time RT-PCR.

Total RNA was isolated from lung tissue homogenates using guanidium-phenol extraction (RNAzol, Campro Scientific, Veenendaal, the Netherlands). Briefly, after tissue homogenization in RNAzol B, RNA was isolated with phenol-chloroform extraction and isopropanol precipitation. The RNA sample was dissolved in RNase-free water and quantified spectrophotometrically. The integrity of the RNA was studied by gel electrophoresis on a 1% agarose gel, containing ethidium bromide. Samples showing degradation of ribosomal RNA by visual inspection under ultraviolet light were discarded. First-strand cDNA synthesis was performed with the SuperScript Choice System (Life Technologies, Breda, the Netherlands) by mixing 2 μg of total RNA with 0.5 μg of oligo(dT)12–18 primer in a total volume of 10 μl. After the mixture was heated at 70°C for 10 min, a solution containing 50 mM Tris·HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM dNTPs, 0.5 μl RNase inhibitor, and 200 U Superscript reverse transcriptase was added, resulting in a total volume of 20 μl. This mixture was incubated at 42°C for 1 h; total volume was adjusted to 100 μl with RNase-free water and stored at −80°C until further use. For real-time quantitative PCR, 1 μl of first-strand cDNA, diluted 1:10 in RNase-free water, was used in a total volume of 25 μl, containing 12.5 μl of 2× SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and 200 ng of each primer. Primers, designed with the Primer Express software package (Applied Biosystems), are listed in Table 1. PCR reactions, consisting of 95°C for 10 min (1 cycle), 94°C for 15 s, and 60°C for 1 min (40 cycles), were performed on an ABI Prism 7700 sequence detection system (Applied Biosystems). Data were analyzed with the ABI Prism 7700 sequence detection system version 1.9 software and quantified with the comparative threshold cycle method with β-actin as a housekeeping gene reference (26).

Protein assay.

Total protein concentration was measured in bronchoalveolar lavage fluid (BALF) using the Dc protein assay (Bio-Rad), according to the manufacturer’s instructions. The detection limit was 31 μg/ml.

Statistical analysis.

Values are expressed as means ± SE. Differences between groups were analyzed with the Student’s t-test. For comparison of survival curves, Kaplan-Meier analysis followed by a log rank test was performed. P values of <0.05 were considered statistically significant.


Fibrin deposition.

Because fibrin deposition correlates well with the severity of tissue damage in hyperoxia-induced lung injury, we quantified fibrin deposition by Western blot analysis in lungs after exposure to different oxygen concentrations to determine the optimal experimental conditions for intervention. Fibrin deposition in lung homogenates of preterm rat pups exposed to 60, 80, and 100% oxygen is shown in Fig. 1A. Significant fibrin deposition was absent in lungs of pups exposed to 60 and 80% oxygen for 14 days (Fig. 1B). However, fibrin deposition increased from 18 ± 8 in room air-exposed pups to 296 ± 42 ng fibrin/mg of tissue in lungs of pups exposed to 100% oxygen for 10 days (Fig. 1B; P < 0.001), demonstrating that this is the optimal experimental condition. Therefore, intervention studies were performed in preterm rat pups exposed to 100% oxygen. Treatment of oxygen-exposed pups with PTX for 10 days resulted in a reduction of fibrin deposition from 296 ± 42 in oxygen-exposed controls to 100 ± 23 ng fibrin/mg of tissue in lungs of PTX-treated pups exposed to 100% oxygen (Fig. 1B; P < 0.001). The localization of fibrin deposits in the lungs was studied immunohistochemically on formaldehyde-fixed paraffin sections. Fibrin deposits were mainly observed in the extravascular compartment in the alveolar lumen and were associated with the alveolar wall. In control lungs, fibrin deposits were absent.


A: Western blot analysis of fibrin deposition in lung homogenates of preterm rat pups exposed to 60 and 80% oxygen for 14 days and 100% oxygen for 10 days. RA, room air control; C, oxygen-exposed pup treated with 0.9% saline; P, oxygen-exposed pup treated with pentoxifylline (PTX). Lung homogenates of pups exposed to 60 and 80% oxygen were not diluted, whereas lung homogenates of pups exposed to 100% oxygen were diluted 1:5. Fibrin standards were used to quantify fibrin deposition in the lung homogenates. B: quantification of fibrin deposition in lung homogenates on day 10. Experimental groups include room air controls (white bars), oxygen-exposed pups treated with 0.9% saline (black bars), and oxygen-exposed pups treated with PTX (gray bars). Oxygen-treated pups were exposed to 60% oxygen (n = 4), 80% oxygen (n = 4), and 100% oxygen (n = 20). Data are expressed as means ± SE. ***P < 0.001, oxygen vs. room air-exposed controls. ΔΔΔP < 0.001, PTX vs. oxygen-exposed controls. C and D: formaldehyde-fixed paraffin lung section, 400-fold magnification, of a rat pup exposed to room air for 10 days (C) and after exposure to 100% oxygen for 10 days (D). Sections were immunohistochemically stained with monoclonal anti-human fibrin antibody 59D8, which specifically detects rat β-fibrin. Arrows in D indicate fibrin deposits in the alveolar lumen of the lung.

mRNA expression in lung homogenates.

Because PTX has anti-inflammatory, anti-coagulant and anti-fibrinolytic properties, we studied mRNA expression of key enzymes involved in these processes in lung homogenates after lung injury by hyperoxia. TNF-α mRNA expression did not change after oxygen exposure (P = 0.934) or PTX treatment (P = 0.35;. IL-6 (P < 0.001), matrix metalloproteinase-12 (MMP-12; P = 0.019), and monocyte chemoattractant protein-1 (MCP-1; P < 0.001) expressions after 10 days of hyperoxia were increased compared with room air-exposed controls). IL-6 expression (P = 0.55) and MMP-12 expression (P = 0.29) did not change by PTX treatment during oxygen exposure. However, MCP-1 mRNA expression decreased 1.6-fold in pups exposed to hyperoxia and 10 days of PTX treatment. The expression of the physiological initiator of coagulation tissue factor (TF; P = 0.008) and the anti-fibrinolytic factor plasminogen activator inhibitor-1 (PAI-1; P < 0.001) increased during exposure to hyperoxia compared with room air-exposed controls. However, PTX treatment did not change TF (P = 0.20) and PAI-1 (P = 0.93) expression after exposure to 100% oxygen for 10 days .


Relative mRNA expression, determined with real-time RT-PCR, of genes related to inflammation [TNF-α, IL-6, matrix metalloproteinase-12 (MMP-12), and monocyte chemoattractant protein-1 (MCP-1); A] and coagulation and fibrinolysis [tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1); B] in 10-day-old room air-exposed pups (white bars), oxygen-exposed pups treated with 0.9% saline (black bars), and oxygen-exposed pups treated with PTX (gray bars). Data are expressed as means ± SE of 6 (room air) or 20 (both oxygen-exposed groups) rats. *P < 0.05, **P < 0.01, ***P < 0.001, oxygen vs. room air-exposed controls. ΔP < 0.05, PTX vs. oxygen-exposed controls.


Total protein concentration was measured in BALF as a marker for capillary leakage. Protein concentration on postnatal day 10 was 103 ± 7 μg/ml in pups exposed to room air, 328 ± 37 μg/ml in pups exposed to 100% oxygen treated with saline (Fig. 3; P < 0.001), and 218 ± 18 μg/ml in pups exposed to 100% oxygen treated with PTX (Fig. 3; P = 0.029). PTX treatment decreased the white blood cell count in BALF from 55 ± 16 × 105 cells/ml in oxygen-exposed pups treated with saline to 16 ± 5 × 105 cells/ml in oxygen-exposed pups treated with PTX (P = 0.05).


Total protein concentration in bronchoalveolar lavage fluid (BALF) of 10-day-old preterm room air-exposed pups injected with 0.9% saline (white bars), oxygen-exposed pups injected with 0.9% saline (black bars), and oxygen-exposed pups injected with PTX (gray bars). Data are expressed as means ± SE of 5 rats per group. ***P < 0.001, oxygen vs. room air-exposed controls. ΔP < 0.05, PTX vs. oxygen-exposed controls.


Survival of PTX-treated hyperoxia-exposed pups was prolonged compared with oxygen-exposed controls (Fig. 4; P = 0.0018). After 12 days of oxygen exposure, 73% of the controls had died vs. only 20% of the PTX pups. The mean survival of preterm oxygen-exposed rat pups treated with PTX was prolonged by 3 days. Subcutaneous injection of room air-exposed pups (n = 5) with 0.9% saline did not lead to illness or mortality the first 4 wk after birth (data not shown).


Kaplan-Meier survival curve of preterm rats treated with either 0.9% saline (▵, n = 26) or 75 mg/kg PTX twice a day (•, n = 25) exposed to 100% oxygen. Data are expressed as percentage ± SE of pups surviving at the observed time point. P < 0.01, PTX vs. saline controls.


The reported experiments show that PTX treatment reduces fibrin deposition threefold and prolongs survival by 3 days in preterm rat pups with hyperoxia-induced lung injury. Fibrin deposition is an important contributor to the pathogenesis of lung injury by oxidative stress. The findings of this study suggest that inhibition of fibrin deposition may have therapeutic potential in the treatment of BPD. This hypothesis is supported by studies in PAI-1 knockout mice, which have less hyperoxia-induced fibrin deposition, resulting in a less severe phenotype and increased resistance toward hyperoxia-induced mortality (2). In addition, blocking of the coagulation cascade attenuates acute lung injury induced by gram-negative sepsis in baboons (4). Fibrin may have proinflammatory and profibrotic properties, by facilitating cell migration and activating inflammatory cells and fibroblasts (31), and it hampers pulmonary gas exchange via inactivation of lung surfactant (7, 30).

Because the mechanism by which PTX reduces fibrin deposition and prolongs survival can be multifactorial, we studied the mRNA expression of key enzymes involved in inflammation, coagulation, and fibrinolysis. PTX reduced MCP-1 mRNA expression but not the expression of TNF-α, IL-6, and MMP-12, indicating that only the influx of monocytes and macrophages to the lung is inhibited during the inflammatory response. PTX did not reduce mRNA expression of TF and PAI-1, indicating that transcriptional activation of the coagulation cascade and inhibition of the fibrinolytic cascade are similar under these experimental conditions.

The effects of PTX treatment in hyperoxia-induced lung injury on the transcriptional regulation of inflammation and coagulation are minor. Because fibrin deposits are located in the extravascular compartment in alveolar septa and lumen, extravasation of plasma proteins, including fibrinogen, into the alveolar lumen may be important. This is supported by a decrease in protein concentration in BALF after PTX treatment, indicating that extravasation of plasma proteins is inhibited by PTX, probably resulting in a lower fibrinogen content in the alveolar fluid and, as a result, less fibrin deposition.

Conflicting results have been found on survival after PTX treatment in lung injury models. In contrast to our findings, PTX treatment did not prolong survival in adult rats after exposure to >95% oxygen (25). This discrepancy may be explained by the difference in oxygen tolerance between neonates and adults in combination with a lower PTX dosage used in the adult rat experiments. Beneficial effects of PTX treatment on survival were reported after endotoxemia, even at low dosages, in mice (42).

We only found an effect of PTX on MCP-1 mRNA expression but not on TNF-α and IL-6 expression. Many other in vitro and in vivo studies demonstrate that PTX inhibits the expression of proinflammatory cytokines (6, 19, 23, 24, 29, 32, 34, 35, 42). These studies emphasize the attenuating effect of PTX on TNF-α and IL-6, the key proinflammatory targets of PTX. Moreover, three studies implicate that inhibition of the release of TNF-α is the most important outcome predictor in endotoxemia (24, 34, 42). A possible explanation for this discrepancy might be that TNF-α does not play the same pivotal role in the inflammatory response in neonatal hyperoxia-induced lung injury as it does in endotoxemia. The finding that TNF-α was not significantly different between room air-exposed and oxygen-exposed pups strengthens this hypothesis. In contrast, the importance of MCP-1 has been demonstrated in a hyperoxia-induced BPD model. Newborn rats exposed to hyperoxia were injected with anti-MCP-1, resulting in the prevention of neutrophil influx and reduced protein oxidation (36). We assume that the inhibitory effect of PTX on the expression of MCP-1 results in a reduction of the influx of macrophages and/or neutrophils to the lung, resulting in less tissue damage and contributing to an improvement in BPD.

The clinical importance of PTX treatment was recently demonstrated by preliminary data on preterm neonates, prone to develop BPD, in whom pretreatment with 40–80 mg·kg−1·day−1 of nebulized PTX in four dosages reduced treatment requirements after the first month of life (21). Limitations of the potential clinical importance of our observations are the high oxygen concentration (100%) used for a prolonged period (10 days) to induce lung injury and the relatively high PTX concentration compared with dosages used in the clinic: 150 mg·kg−1·day−1 subcutaneously in our study vs. 40–80 mg·kg−1·day−1 by nebulization and 30 mg·kg−1·day−1 intravenously in preterm human infants (20, 21). Furthermore, few human preterm infants will be exposed to 100% oxygen for 10 days, but less extreme oxygen exposures did not produce the primary indicator of injury (fibrin deposition) in the animal model used in this study.

In summary, this study shows that PTX significantly prolongs survival and attenuates alveolar fibrin deposition of preterm rat pups with experimental neonatal hyperoxic lung injury. The effect of PTX is probably the result of decreased protein leakage from the capillaries to the alveolar lumen. PTX may have the potential to prevent and/or reduce the severity of BPD in preterm infants who need ventilatory support in the neonatal intensive care unit.

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This study was supported by grant 920-03-213 from the Netherlands Organisation for Health Research and development (S. A. J. ter Horst), a grant from the Stichting Prof. AAH Kassenaar Fonds (G. T. M. Wagenaar and F. J. Walther), and Grant HL-55534 from the National Institutes of Health (F. Y. Walther).


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Efficacy of ibuprofen and pentoxifylline in the treatment of phosgene-induced acute lung injury.

Phosgene, a highly reactive former warfare gas, is a deep lung irritant which produces adult respiratory distress syndrome (ARDS)-like symptoms following inhalation. Death caused by phosgene involves a latent, 6-24-h, fulminating non-cardiogenic pulmonary edema. The following dose-ranging study was designed to determine the efficacy of a non-steroidal anti-inflammatory drug, ibuprofen (IBU), and a methylxanthine, pentoxifylline (PTX). These drugs were tested singly and in combination to treat phosgene-induced acute lung injury in rats. Ibuprofen, in concentrations of 15-300 mg kg-1 (i.p.), was administered to rats 30 min before and 1 h after the start of whole-body exposure to phosgene (80 mg m-3 for 20 min). Pentoxifylline, 10-120 mg kg-1 (i.p.), was first administered 15 min prior to phosgene exposure and twice more at 45 and 105 min after the start of exposure. Five hours after phosgene inhalation, rats were euthanized, the lungs were removed and wet weight values were determined gravimetrically. Ibuprofen administered alone significantly decreased lung wet weight to body weight ratios compared with controls (P < or = 0.01) whereas PTX, at all doses tested alone, did not. In addition, the decrease in lung wet weight to body weight ratio observed with IBU+PTX could be attributed entirely to the dose of IBU employed. This is the first study to show that pre- and post-treatment with IBU can significantly reduce lung edema in rats exposed to phosgene.

Source: Journal of applied Toxicology

Treating Skin Infections.

A noninferiority study of tedizolid phosphate provides information on this new antibiotic, as well as insights into both study design and treatment duration for skin infections.

Given the detrimental effects of antimicrobial therapy both in promoting antibiotic resistance and in altering the microbiome, shortening the duration of treatment for common infections is potentially beneficial. In a recent manufacturer-sponsored, multinational, phase III, double-blind, double-dummy trial, researchers compared a 6-day course of tedizolid phosphate — a novel oxazolidinone — with a 10-day course of linezolid for acute bacterial skin and skin-structure infections (ABSSSIs).

A total of 667 adults with ABSSSIs were randomized to receive 200 mg of oral tedizolid once daily for 6 days or 600 mg of oral linezolid twice daily for 10 days. In intent-to-treat analysis, tedizolid was noninferior to linezolid as determined at 48- to 72-hour clinical assessment (the primary efficacy outcome; 79.5% vs. 79.4%), as well as at the end of therapy (day 11; 69.3% vs. 71.9%) and 7 to 14 days later (85.5% vs. 86.0%). The posttherapy clinical response rate was high (85%) and similar between groups in the 178 patients (26.7%) with infections caused by methicillin-resistant Staphylococcus aureus.

Comment: As noted by the authors and editorialists, this study provides information both on tedizolid phosphate and on new FDA guidelines for assessing the efficacy of antibiotic therapy for ABSSSIs. It also highlights the need for clinical trials of currently approved agents to determine what treatment duration is really necessary for such infections.

Source: Journal Watch Infectious Diseases.


Respiratory distress syndrome in patients with advanced cancer treated with pentoxifylline: a randomized study.


The inappropriate endogenous secretion of tumour necrosis factor (TNF) could play a role in the pathogenesis of acute respiratory distress syndrome (ARDS), one of the most frequent causes of death in cancer patients. Because of its capacity to inhibit TNF secretion in vitro, pentoxifylline (PTX) could be extremely useful in ARDS therapy. In this study 30 advanced cancer patients with ARDS were randomized to receive either the conventional care or conventional care plus PTX (100 mg i.v. twice a day for 7 days followed by an oral administration of 400 mg three times a day) to evaluate the efficacy of PTX in reducing TNF serum levels and in improving the symptoms of this syndrome. Serum levels of TNF were measured before and after 7 days of therapy. The percentage of patients alive at 7 days was significantly higher in the PTX-treated group than in the controls (12/15 versus 3/15; P < 0.001). The mean survival time was significantly higher in the PTX-treated group than in the controls. A clinical and/or radiological improvement was obtained in 11/15 patients treated with PTX and in only 2/15 patients in the conventional care group (P < 0.01). TNF mean levels significantly decrease in the PTX-treated group. These data confirm in vivo the capacity of PTX to inhibit TNF secretion in patients with ARDS. Moreover PTX therapy may improve the symptoms related to ARDS without particular toxic effects.