abstract: We should not be distracted by the debate about whether or not global warming is caused by humans; whatever the answer is, we urgently need to retard or reverse the trend.
author: Gregory A Petsko
date: 2009
institute: 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA
title: A harsh climate

I might as well come right out and say it: I don't care whether global warming is caused by manmade greenhouse gas emissions. And neither should you.

Before you start reaching for your laptops, iPhones, and BlackBerrys to fire off scathing emails, give me a moment to explain why I made this statement and what it really means. I bet that, when I'm through, you will agree with me.

This column is being written because of the confluence of two events. One is a meeting in Copenhagen of representatives of most of the world's nations, aimed at formulating a new global strategy for dealing with the climate crisis. The talks have ground to a halt as I write this because the group of developing countries, known as the G-77, has accused the United States and other industrialized states of forsaking the Kyoto Protocol, the current climate agreement that imposes greenhouse gas emissions on nearly every developed nation.

The second event is 'Climategate', the release of illegally hacked emails between climatologists. As an example of giving aid and comfort to the enemy, Climategate could hardly be improved on. In late November, a computer file including more than 1,000 emails either sent from or to members of the University of East Anglia's Climate Research Unit (CRU) was stolen and released on the Internet. The emails contain language that opponents of emission curbs have seized on as alleged examples of data manipulation and outright fraud on the part of climate researchers. For example, one email apparently sent by the head of the CRU, Professor Phil Jones, refers to using "Mike's *Nature* trick of adding in the real temps to each series for the last 20 years ... to hide the decline". The CRU is one of the leading research units on climate change, and their data had a major role in the Fourth Assessment Report of the Intergovernmental Panel on Climate Change (IPCC), released in 2007, that provided unequivocal evidence for global warming (see Figure 1).

Of course, scientists use the word 'trick' all the time as a shorthand term for a method or algorithm, but professional skeptics rarely bother themselves with the way scientists work. It seems likely that the files were stolen in an attempt to undermine the Copenhagen talks, but my assessment is that there are so many other contentious issues in that meeting that this is a relatively minor matter for most of its participants. Nevertheless, Professor Jones has stepped down as head of the CRU pending an internal investigation. In my view, he should instead have been made to write on the blackboard 1,000 times: "I will never put anything into an email or text message that could be embarrassing to me or to my organization if it were read by someone else, and if I don't believe this I should ask Tiger Woods."

One of the most sensible things I have read about the climate debate is an opinion piece by Stewart Brand in the 15 December 2009 edition of *The New York Times*. He argues that the popular depiction of the combatants as belonging to two camps, the alarmists and the skeptics, is fallacious. There are actually four sides: *denialists*, a group consisting of people with a right-wing political agenda who assert that the claim that global warming is caused by manmade emissions is a lie and is not based on sound science; *skeptics*, a group largely comprising scientists who argue that climate science, particularly large-scale modeling, is far too imperfect to form the basis of a consensus; *warners*, another group of scientists who believe that the best climate models accurately predict a looming planetary disaster and that human production of greenhouse gases is the primary cause; and *calamatists*, a collection of environmental activists whose agenda, like that of the denialists, is ideologically driven, but in the opposite direction: they have a neo-luddite view of industrialization, and believe the denialists are evil. As Brand, a self-described warner, points out, understanding from which of these camps any given argument springs is useful in distinguishing propaganda from science, and appeals to emotion from evidence-based assertions.

Yet even Brand misses what I think is the crucial point, the point I want to make in this column, which is that you can't win a war if you are fighting in the wrong field. And in the war over climate change, which should be fought in the field of science, the denialists and the calamatists have dragged us into battle on their turf.

When you're in a fight with an opponent who is not above using invective and illogic, the worst mistake you can make is letting the other side define the terms of the debate. That's exactly what has happened in the argument about climate change. For decades the denialists insisted that the earth was not getting warmer. Short-term fluctuations were meaningless, they asserted. Climate modeling was worse than useless. The doomsayers were just trying to push a liberal political agenda, and so on. But after massive amounts of data were collected and analyzed by the Intergovernmental Panel on Climate Change, it became clear, on the release of their report in 2007, that no sensible person could deny that a dramatic rise in the planet's average temperature had been occurring for at least a century (see Figure 1). Largely thanks to Al Gore, this information also reached the general public, whose reaction even the staunchest denialists could not ignore.

So they did what clever, unprincipled losers often do: they changed the issue. Of course the earth is getting warmer, they said (blithely ignoring the fact that they had said exactly the opposite the day before), but human activities have nothing to do with it. It's entirely due to natural causes, and people who assert that manmade greenhouse gases are causing the problem are employing flawed science, deliberately distorting the facts (Climategate), and are using fear to advance the same old, tired environmental activism. Because global warming is not a manmade phenomenon, there is no scientific or political reason to limit manmade greenhouse gas emissions. Sarah Palin (why am I not surprised?) is one of the leaders of this chorus, stating recently that climate change occurs naturally "like gravity", while warning that reducing greenhouse gas emissions will mean "job losses" and "economic costs". (This is the same ex-Alaska governor Sarah Palin who, before becoming a national political figure, said in July of last year, "Alaska's climate is warming. While there have been warming and cooling trends before, climatologists tell us that the current rate of warming is unprecedented within the time of human civilization. Many experts predict that Alaska, along with our northern latitude neighbors, will warm at a faster pace than any other areas, and the warming will continue for decades." I don't know whether to laugh at that kind of soulless opportunism or just cry.)

This strategy is actually working, to some extent. It's much harder to establish the cause of something than it is to prove that something is happening, and the data supporting manmade emissions as the leading driver of climate change are not nearly as persuasive, or as immune to challenge, as the data demonstrating the fact of global warming. And scientists, foolishly, have allowed that to become the center of the climate crisis debate. I say foolishly because, in so doing, they have given up the victory that they already won.

The denialists have conceded the fact of climate change. And here is my central point: once you admit that the earth is warming rapidly, it does not matter in the least whether that trend is due to manmade causes or not.

Regardless of its origin, a rapidly changing climate is a very bad thing. We have built an entire civilization on the assumption of long-term climate stability. We grow wheat in Kansas rather than in the Yukon because Kansas has an ideal climate for growing wheat and the Yukon is too cold, and we assume that will still be the case 10 years from now. We build our cities on the coast because that is convenient for shipping goods, and we assume the coastline won't suddenly move 10 miles inland. We don't have air conditioning in many homes in northern California because we assume the average temperature won't suddenly rise by several degrees, making summer unbearably hot. We assume that England won't have a yearly climate like Lapland, even though its position on the globe might lead one to expect otherwise, because the Gulf Stream will always be there off the west coast, keeping things moderate. Every one of these assumptions fails in the event of significant global warming. One reason I prefer the term 'climate crisis' to 'climate change' or 'global warming' in discussing this problem is because our dependence on stable long-term climate patterns means that any change in those patterns represents a potential catastrophe on a planet-wide scale.

It doesn't matter what the cause of that crisis is; once you accept the fact that the crisis is coming, the only thing that matters is how to prevent it or slow it down. And the only way we have of doing that at the moment is to reduce our greenhouse gas emissions. Put another way, human activity may not even be causing the earth's temperature to rise, but human activity is the only means we have of doing something about it.

A simple analogy may make this point clearer. Suppose we learned tomorrow that there was one chance in ten that a huge asteroid, recently discovered, was going to crash into the earth in 5 years, killing a billion people and raining debris in such amounts as to blot out sunlight significantly for a year. (A similar event is thought to have led to the extinction of the dinosaurs.) Would anyone in his or her right mind argue that, because we couldn't prove that human activity was responsible for the asteroid, there was no reason to hurt our economy by spending hundreds of billions of dollars firing nuclear-tipped rockets at it to destroy it or alter its course? Yet that's exactly what the denialists are trying to argue now, in the case of a climate crisis that has at least an equal probability of globally devastating consequences.

True, our climate models can't predict with certainty that the steps being considered in Copenhagen will retard, halt or reverse the current warming trend. But they represent all we can do at the moment. If global warming is being caused primarily by greenhouse gases, as many thoughtful scientists believe, then they will do a lot. If global warming is actually caused by, say, sunspots or something similar, reduction of emissions may not do so much. But everyone agrees that they will do something, and my point is that something simply has to be done.

I hope you see now why I started this essay as I did. We should not be debating whether human activity is responsible for global warming or not. Given that even the denialists and skeptics have conceded the fact of global warming, the debate should be over the most effective means of doing something about it. This means, I am afraid, not just limiting our discussion to controls on CO~2~ emissions. We need to look seriously at developing technologies for carbon sequestration, alternative fuels, and carbon-neutral technologies for transportation and energy production. Much of this will involve engineering microorganisms and plants, so genomics is going to be very important in enabling these technologies as we grapple with the crisis. I also see no escape from at least investigating ideas for geoengineering - solutions involving deliberate changing in sunlight absorption, carbon capture and temperature reduction on a continent- or planet-wide scale. My gut reaction to geoengineering is that it is a terrible idea, born as much of hubris as desperation, that should be shelved permanently because we will never have the kind of models that would guarantee beforehand that it could be done safely. But the fact is, we don't know what we don't know when it comes to such projects, and, given the severity of the climate crisis, if someone wants to propose that we should at least begin to study such solutions to determine the extent of our ignorance and the possibility that we might someday be able to employ them, I wouldn't say no.

So, the next time you find yourself in a debate with someone over the climate crisis, and they say that we shouldn't reduce CO~2~ emissions because there is no definitive proof that manmade greenhouse gases are the cause of global warming, respond by saying, "Then if an alien race were threatening to exterminate mankind, you wouldn't do anything to try to stop them because human activities weren't the cause of the alien invasion, is that right?" And they'll reply, "Of course not! But this is completely different." And you'll say, "No, it's not. Let me explain why."

Given the harsh climate that has developed around the subject of global warming, you probably won't convince them that they're wrong. But at least you'll be having the right argument.


abstract: Monosodium urate crystals stimulate monocytes and macrophages to release IL-1β through the NALP3 component of the inflammasome. The effectiveness of IL-1 inhibition in hereditary autoinflammatory syndromes with mutations in the NALP3 protein suggested that IL-1 inhibition might also be effective in relieving the inflammatory manifestations of acute gout. The effectiveness of IL-1 inhibition was first evaluated in a mouse model of monosodium urate crystal-induced inflammation. IL-1 inhibition prevented peritoneal neutrophil accumulation but TNF blockade had no effect. Based on these findings, we performed a pilot, open-labeled study (trial registration number ISRCTN10862635) in 10 patients with gout who could not tolerate or had failed standard antiinflammatory therapies. All patients received 100 mg anakinra daily for 3 days. All 10 patients with acute gout responded rapidly to anakinra. No adverse effects were observed. IL-1 blockade appears to be an effective therapy for acute gouty arthritis. The clinical findings need to be confirmed in a controlled study.
author: Alexander So; Thibaut De Smedt; Sylvie Revaz; Jürg Tschopp
date: 2007
institute: 1Service of Rhumatologie, Department of Medicine, Centre Hospitalier Universtaire Vaudois and University of Lausanne, 1011 Lausanne, Switzerland; 2Apoxis SA, Avenue de Sévelin 18-20, 1004 Lausanne, Switzerland; 3Institute of Biochemistry, University of Lausanne, chemin de Boveresses 155, 1066 Epalinges, Switzerland
references:
title: A pilot study of IL-1 inhibition by anakinra in acute gout

# Introduction

Acute gout is a common cause of arthritis, affecting approximately 1% of the adult population, and epidemiological evidence suggests that its prevalence is increasing \[1\]. Current treatments during an acute attack include nonsteroidal antiinflammatory drugs (NSAIDs), colchicine and corticosteroids. Although these agents are generally effective, they also present significant risks in patients who have pre-existing renal, cardiovascular and gastrointestinal diseases.

Gouty inflammation is due to monosodium urate (MSU) crystal-induced release of proinflammatory cytokines from leukocytes. Among the many cytokines implicated \[2,3\], IL-1 may have a special role in the inflammatory network, as MSU crystals stimulate IL-1 release by monocytes and synovial mononuclear cells \[4\]. The MSU crystals trigger IL-1 release through innate immune pathways, which include TLR-2 and TLR-4, found on the surface of monocytes and macrophages, as well as the 'inflammasome' complex that leads to IL-1β activation \[5,6\]. The inflammasome acts as an intracellular sensor of inflammatory stimuli and regulates the activation of caspase-1. On assembly of the inflammasome, which consists of a member of the nucleotide-binding oligomerization domain-leucine rich repeat protein family (such as NALP1, NALP2, NALP3, or IPAF), the adaptor protein ASC and caspase-1 \[7\], caspase-1 becomes active and cleaves pro-IL-1β to release the mature p17 form of IL-1β. Activators of the NALP3 inflammasome include ATP, the microbial cell-wall component muramyl dipeptide and bacterial RNAs \[8-10\]. MSU and calcium pyrophosphate dihydrate crystals directly activate the inflammasome via NALP3 in monocytes or macrophages to release active IL-1β and cause neutrophil influx into the peritoneal space when administered by intraperitoneal injection. These responses were abrogated in ASC^-/-^ or caspase-1^-/-^ mice \[6\].

Spontaneous activation of the NALP3 inflammasome due to mutations in the *NALP3* gene has been implicated in hereditary autoinflammatory syndromes such as Muckle–Wells syndrome and chronic infantile neurologic cutaneous articular \[11\]. Affected patients respond dramatically to IL-1 inhibition \[12,13\], suggesting that IL-1β plays a crucial role in the pathogenesis of inflammation in these conditions. Based on these findings, we questioned whether IL-1 inhibition may also have a beneficial effect in gouty inflammation.

As treatment with drugs currently used in acute gout is not always well tolerated or is contraindicated due to coexistent medical problems, we investigated the validity of IL-1 blockade as therapy in acute gout. We first analyzed the effects of IL-1 inhibition using the mouse peritoneal model of MSU-induced inflammation and then assessed the effects of anakinra in patients with acute gout who presented contraindications or were refractory to standard treatment in an open study.

# Methods

## Reagents

MSU crystals were prepared based on the method described previously \[14\]. Briefly, 1.68 g uric acid was dissolved in 500 ml of 0.01 M NaOH and heated to 70°C. NaOH was added as required to maintain the pH between 7.1 and 7.2, and the solution was filtered and incubated at room temperature with little stirring slowly and continuously 24 hours.

## Animal studies

BALB/C mice were treated intraperitoneally with PBS or 0.5 mg MSU crystals in 0.5 ml sterile PBS. Some mice were injected intraperitoneally with 200 μg anti-IL-1RI mAb (clone 35F5; BD Pharmingen, San Jose, CA, USA) or with 200 μg anti-TNF mAb (clone TN3-19.12; BD Pharmingen) or with 200 μg anakinra (Kineret; Amgen, Thousand Oaks, CA, USA) at the time of MSU injection. Mice were euthanized after 6 hours by CO~2~ exposure and the peritoneal cavities were washed with 10 ml cold PBS. The lavage fluids were analyzed for neutrophil recruitment by fluorescence-activated cell sorting using the neutrophil markers Ly-6G and CD11b (BD Pharmingen). Five mice per group were used for study.

## Human studies

The diagnosis of gout arthritis was based on clinical and laboratory features. All patients fulfilled the American College of Rheumatology criteria for acute gouty arthritis \[15\], but all had a long previous history of either recurrent gouty attacks or tophaceous gout. Patients were treated with anakinra on an open-label basis. All subjects had either failed conventional treatment with NSAIDs, colchicine or corticosteroids for at least 48 hours or had developed significant side-effects on these drugs in the past. Patients with an active untreated infection, with uncontrolled diabetes, with uncontrolled heart or respiratory failure or with chronic renal failure with a creatinine clearance \<30 ml/min were not eligible. Joint infection was excluded by prior bacterial culture in all cases that underwent joint aspiration.

All patients consented to receive anakinra. Treatment was administered daily at a dose of 100 mg subcutaneously for 3 days. On starting anakinra, the NSAID or colchicine therapy was discontinued. Patients who were already on low-dose corticosteroids continued their treatment at the same dose. Clinical efficacy was assessed by clinical examination of the swollen and tender joint count as well as the patient's evaluation of the efficacy of treatment in terms of pain reduction after 3 days of therapy in comparison with their symptoms before treatment. All patients were followed up for \>1 month. During follow-up, patients were evaluated for side-effects related to the treatment and for joint symptoms related to gout. All patients were followed by physicians responsible for this study (AS and SR). The study protocol was approved by the local institution's ethics committee and has the ISRTCN trial registration number 10862635.

# Results

## MSU-induced peritoneal inflammation and its inhibition by anakinra or anti-IL-1RI mAb

To study the inflammatory response to MSU crystals *in vivo*, we used a well-described model of neutrophil infiltration in the peritoneal cavity subsequent to MSU crystal administration. In previous experiments we determined that intraperitoneal administration of MSU crystals induced a dose-dependent neutrophil accumulation at the site of crystal deposition, with a plateau effect observed at 500 μg (data not shown), and this dose was therefore used in subsequent experiments. We went on to determine whether IL-1 blockade reduced the inflammatory response in the same model. Coadministration of MSU crystals with two different IL-1 inhibitors (anti-IL-1RI mAb or IL-1R antagonist (anakinra)) had similar and marked inhibitory effects on neutrophil recruitment (Figure 1a). This effect was not observed when anti-TNF blocking mAb was administered in the same fashion as the IL-1 inhibitors (Figure 1b). The differences were statistically significant in comparison with the positive MSU control.

The same dose of TNF inhibitor was effective in suppressing liver inflammation induced by concavalin A administration, a model of hepatitis dependent on TNF production (data not shown).

## Effects of anakinra in gout patients

The effectiveness of IL-1 inhibition in suppressing the symptoms of acute gout was assessed in 10 patients who could not tolerate or did not respond to standard antiinflammatory treatments. A summary of the medical histories is presented in Table 1. All patients responded rapidly to the drug, with the most rapid onset observed within 24 hours. In all patients, subjective symptoms of gout were greatly relieved by 48 hours after the first injection. No side-effects were observed during the study period. Clinical examination of affected joints showed complete resolution of signs of arthritis in 9/10 patients on day 3 after initiation of treatment.

Clinical summary of the 10 patients studied and their response to treatment

| Patient | Clinical presentation | Affected joints | Serum uric acid (normal range, 160–390 μmol/l) | Serum creatinine (normal range, 44–80 μmol/l) | Hypouricemic treatment | Effect of anakinra (hours) | Patient assessment of improvement in pain (%) |
|----|----|----|----|----|----|----|----|
| Case 1 (female, 72 years old) | Chronic tophaceous gout, renal stones | Fingers, toes | 637 | 79 | Uricase | 36 | 70 |
| Case 2 (male, 70 years old) | Chronic tophaceous gout | Ankle, toes | 564 | 202 | Allopurinol | 24 | 90 |
| Case 3 (male, 72 years old) | Acute gout | Knee, ankle, foot | 482 | 121 | Allopurinol | 24 | 90 |
| Case 4 (male, 51 years old) | Acute gout | Ankle, toe | 396 | 84 | Allopurinol | 24 | 100 |
| Case 5 (male, 40 years old) | Acute gout | Ankle, toe | 322 | 113 | Allopurinol | 36 | 100 |
| Case 6 (female, 72 years old) | Acute gout | Feet, toe | 572 | 72 | None | 36 | 80 |
| Case 7 (male, 76 years old) | Acute gout | Ankle, foot | 338 | 79 | None | 36 | 100 |
| Case 8 (male, 70 years old) | Acute gout | Wrist, elbow, hand | 779 | 406 | None | 48 | 50 |
| Case 9 (male, 53 years old) | Chronic tophaceous gout | Elbow, finger, foot, ankle | 660 | 84 | Allopurinol | 48 | 50 |
| Case 10 (male, 38 years old) | Acute gout | Wrist, finger | 540 | 84 | None | 24 | 60 |

## Case 1

A 72-year-old woman with a 13-year history of chronic tophaceous gout and hyperuricemia was treated with rasburicase. The patient previously had a severe cutaneous reaction to allopurinol, and uricosuric treatment with benzbromazone caused renal stones. During previous gout flares, medical treatment was unsatisfactory. She could only tolerate a low dose of diclofenac (50–100 mg/day), as higher doses caused gastrointestinal side-effects including one episode of gastrointestinal hemorrhage. Colchicine at 1 mg/day provoked intolerable diarrhea, and oral corticosteroid caused severe abdominal pain.

Uricase treatment was commenced (14 mg intravenously daily for 5 days) and resulted in rapid lowering of the patient's uric acid levels. On the fourth day of treatment, arthritis developed in her hand and foot joints. As treatment with diclofenac during previous flares took more than 1 week to relieve her symptoms, treatment with anakinra was started. Her arthritis responded rapidly and she was able to continue the course of uricase. No acute flares were observed during the following 2 months.

## Case 2

A 70-year-old man with an 8-year history of chronic tophaceous gout was assessed for hypouricemic treatment. The patient's past medical history included congestive cardiac failure, severe ischemic heart disease, hypertension and renal insufficiency (serum creatinine, 202 μmol/l; normal range, 44–80 μmol/l). Previous trials of treatment with allopurinol had to be abandoned because acute gout developed after the first dose, which did not respond to small doses of NSAIDs. Higher doses of NSAIDs were contraindicated because of renal failure. Colchicine at low doses (\<1 mg/day) provoked rapid onset of diarrhea.

The patient was again started on a low dose of allopurinol (100 mg), and after the first dose developed acute arthritis of the right foot and ankle. Anakinra was administered for 3 days with rapid and complete resolution of signs and symptoms of arthritis. The patient continued on allopurinol 100 mg daily, and at follow-up 2 months later he had no further flare-ups while continuing on the same dose of allopurinol.

## Case 3

A 72-year-old man with a past history of diabetes, hypertension, renal failure and ischemic heart disease presented with polyarticular gout. Arthritis involved the knees, the right ankle and the right tarsal joints. MSU crystals were detected in the knee joint aspirate.

Owing to renal impairment and a history of rectal bleeding on colchicine, treatment with oral prednisone at 30 mg daily was started with a tapering dose over 7 days. Despite steroids, the arthritis remained active and the patient could not walk. After steroids were stopped, the patient received a 3-day course of anakinra, with complete resolution of arthritis by the second day. There had been no recurrence of arthritis at follow-up 1 month later.

## Case 4

A 50-year-old man with a 20-year history of polyarticular gout principally involving the right ankle and big toe sought medical advice because of increasingly frequent gout attacks. He had started allopurinol but could not continue treatment because it induced flare-ups of arthritis. NSAIDs caused severe gastrointestinal pain, and colchicine at 1 mg daily was ineffective. Higher doses of colchicine provoked diarrhea. On examination, he had arthritis of the right ankle joint and MSU crystals were identified in the joint aspirate.

A trial of anakinra was initiated at the same time as starting allopurinol (300 mg/day). The patient's arthritis responded rapidly and he continued on allopurinol. He was asymptomatic 2 months later.

## Case 5

A 40-year-old man with a 12-year history of gout presented with recurrent podagra of the right big toe. The attacks occurred approximately once every 2 months, and responded normally to indomethacin 150 mg daily over 7 days. The patient was taking allopurinol 300 mg daily and his uric acid level was stable at 322 μmol/l. He could not tolerate colchicine, which induced diarrhea when given at doses above 1.5 mg daily.

The patient consulted after another attack, which did not respond to indomethacin over 2 days. Joint aspiration confirmed the presence of urate crystals in the joint fluid. The patient received anakinra and his symptoms completely resolved 36 hours after the first injection. He has had no further attacks at the 6-week follow-up.

## Case 6

A 73-year-old female presented with acute gout involving the big toes of both feet for the first time. She had a past medical history of hypertension and knee osteoarthritis. On examination, both metatarsophalangeal-1 joints were red and swollen and tender to the touch. The joint aspirate did not yield any fluid. Serum uric acid was raised at 572 μmol/l and serum creatinine was normal at 72 μmol/l. A presumptive diagnosis of acute gout was made because of the patient's clinical presentation and the hyperuricemia.

The patient's symptoms had not responded to 2 days of treatment with diclofenac (100 mg daily), so anakinra was started. Her joint pains were 50% improved by 24 hours and 80% by 36 hours after the injections. No further recurrence was reported on follow-up at 1 month. The patient was started subsequently on allopurinol.

## Case 7

A 76-year-old man developed polyarticular gout affecting his right ankle, the right tarsal joint and both big toe joints during hospitalization for acute cholecystitis and bacterial sepsis. The patient had a past medical history of cardiovascular disease treated with low-dose aspirin and nitrates, of hypertension, of obstructive airway disease and of cirrhosis. On examination, joint inflammation was observed at the aforementioned affected sites. Aspiration of the ankle joint revealed uric acid crystals.

Owing to a lack of response to NSAIDs over 48 hours, anakinra treatment was started. The patient responded after the first injection, and the symptoms and signs of gout were completely abolished 48 hours afterwards.

## Case 8

A 70-year-old man developed arthritis of his right wrist, elbow, hand and shoulder. The patient's past medical history included chronic renal failure, diabetes, and cardiovascular disease with atrial fibrillation. Gout was suspected because of hyperuricemia and he was treated with colchicine, which provoked rectal bleeding and a worsening of renal failure. The patient's serum urate was raised at 779 μmol/l and his serum creatinine was 258 μmol/l. The joint aspiration of the left elbow revealed MSU crystals.

A trial of treatment with prednisone caused a decompensation of the patient's diabetes. Anakinra was started and on review, after completing the injections, the symptoms of arthritis of the knee and elbow were greatly reduced, but the right wrist and hand remained moderately painful.

## Case 9

A 53-year-old male presented with severe tophaceous gout of 17 years' duration. In the past, treatment with allopurinol and uricosurics had been attempted but discontinued because treatment provoked acute attacks of gout that were not well controlled by NSAIDs. Colchicine caused severe diarrhea at a dose of 1 mg/day. There was a positive family history of gout. On examination, tophi were detected over the elbows, fingers, knees, ankles and feet, with signs of inflammation in the hands, knees and right foot. The patient's serum urate was increased at 660 μmol/l and his renal function was normal.

Owing to the patient's prior intolerance of NSAIDs and colchicine, a further attempt to start allopurinol was made under cover of anakinra, given at 100 mg daily over 3 days. On starting anakinra, the patient reported a 50% decrease in joint pains after 2 days and he started allopurinol 200 mg daily and acetametacin 90 mg daily without complications. On follow-up 1 month later, the patient had one minor attack of gout and the allopurinol was continued.

## Case 10

A 38-year-old male presented with painful arthritis of the left wrist. In the past, the left wrist was fractured in a road traffic accident and the patient also had a past history of gout affecting the ankles and the left knee. Aspiration of the left wrist showed MSU crystals. Colchicine (2 mg/day) over 5 days was not effective in controlling the symptoms of gout.

Anakinra was administered and there was a rapid clinical response. On follow-up at day 7 after the first injection, the left wrist was still slightly swollen and tender but the ankle and knee were asymptomatic. The patient was subsequently started on allopurinol.

# Discussion

MSU crystals induce tissue inflammation via multiple mechanisms. The release of monocyte-derived cytokines, triggered by urate crystals, can in turn modulate endothelial expression of adhesion molecules that enhance neutrophil recruitment to the site of inflammation \[16\]. Within the joint, the release of chemokines, prostanoids as well as kinins further amplifies the inflammatory response \[17\]. The recent finding that MSU crystals induce IL-1 release by activation of the NALP3 inflammasome led us to investigate whether IL-1 blockade could inhibit inflammation provoked by MSU. Our results from animal studies confirmed the effectiveness of IL-1 inhibition in preventing neutrophil trafficking to the peritoneum, whereas TNF inhibition did not have any effect. Indeed, TNF inhibition aggravated neutrophil influx. At present we do not have any simple explanation for the apparent increase in neutrophil accumulation in mice treated with the anti-TNF mAb. This observation was made in the two separate experiments performed with five animals per group. The anti-TNF agent employed inhibited concavalin A-induced hepatitis, demonstrating that the reagent is biologically active.

The latter results differed from those reported by Chapman and colleagues, who found that anti-TNF inhibited MSU-induced endothelial activation, as evidenced by scintigraphy, in a porcine monoarthritis model \[16\]. These differences may be due to the different species employed in the animal models as well as to the differences in the cytokine inhibitors tested. Apart from the inflammasome, IL-1 secretion by macrophages in contact with MSU can also be induced through a TLR-dependent mechanism \[5\]. In the current experiments, IL-1 inhibitors would block the effects of activation via both the TLR and the inflammasome pathways.

As proof of concept that IL-1 inhibition may be clinically effective, we performed an open-label study of anakinra treatment in patients with acute gout. All patients had acute excacerbations of longstanding gout and three had severe tophaceous gout. All the patients had either not responded adequately to standard treatment with NSAIDs, colchicine or steroids (*n* = 7) or had significant comorbidities that made use of these treatments hazardous (*n* = 4). Three patients could not continue hypouricemic treatment with allopurinol because of the acute flares provoked at the start of treatment. Only three daily injections were administered, and in all cases the symptoms of gout responded rapidly. All patients had responded within 48 hours, and in four patients (cases 2, 3, 4 and 10) the symptoms improved within 24 hours. Patients' subjective assessment of pain due to gout was positive, and the mean diminution of pain was 79% by day 3 after the first injection. Not all patients responded so well, particularly in two of the patients who had tophaceous gout. They had a moderate response, although still with \>50% reduction of pain. As IL-1 blockade may increase the risk of infection, we excluded any patient who had proven or clinically suspected active infection. When appropriate, joint aspiration was performed to exclude septic arthritis. No treatment-related side-effects were observed during therapy and there were no infectious complications. On follow-up, three patients who had tophaceous gout reported mild joint pains but no acute flare during the first month.

Although these findings are positive, we have to stress that this was an open-label study and confirmation of these results would require a randomized controlled trial to prove that IL-1 inhibition is effective in acute gout. If the effectiveness of IL-1 is confirmed, then a short course of IL-1 inhibition may prove to be a valid addition to the therapeutic arsenal when the physician is confronted with complicated cases of gouty arthritis.

# Conclusion

In this pilot study involving 10 patients with gouty arthritis refractory to conventional therapies, anakinra given at 100 mg daily for 3 days rapidly relieved the inflammatory symptoms of gout. These results reinforce the findings that implicate IL-1β in the pathophysiology of gout and need to be confirmed by randomized controlled trials. IL-1 inhibition may be a promising therapeutic target in crystal-induced arthritis.

# Abbreviations

IL = interleukin; mAb = monoclonal antibody; MSU = monosodium urate; NALP = Nacht, LRR and Pyrin domain containing protein; NSAID = nonsteroidal antiinflammatory drug; PBS = phosphate-buffered saline; TLR = Toll-like receptor; TNF = tumor necrosis factor.

# Competing interests

The authors declare that they have no competing interests.

# Authors' contributions

The study was conceived by AS and JT. TDS performed the animal studies. AS and SR performed the clinical study.


author: Carl ZimmerCarl Zimmer is a science writer based in Guilford, Connecticut, United States of America. E-mail: <email@example.com>
date: 2006-12
references:
title: The Genome: An Outsider's View

The Buddha once told a story about a king who ordered a group of blind men to be presented with an elephant. Each man touched a different part of the animal. The king then asked them what an elephant is like.

The blind men who touched the elephant's head replied, "An elephant, your majesty, is just like a water jar." The blind men who touched its ear said, "An elephant, your majesty, is just like a winnowing basket." The blind men who touched its tusk declared, "An elephant, your majesty, is just like a plowshare." The ones who touched the trunk replied, "An elephant, your majesty, is just like a plow pole." The blind men who touched the body replied, "An elephant, your majesty, is just like a storeroom." The blind men who touched the foot replied, "An elephant, your majesty, is just like a post." The blind men who touched the hindquarters replied, "An elephant, your majesty, is just like a mortar." The blind men who touched the tail replied, "An elephant, your majesty, is just like a pestle." And the blind men who touched the tuft at the end of the tail replied, "An elephant, your majesty, is just like a broom."

The blind men fell into a fistfight, shouting, "An elephant is like this, an elephant is not like that! An elephant is not like this, an elephant is like that!" \[1\]

I am a science writer, and my chief passion is biology. I spend time with biologists of all stripes—computational biologists, paleontologists, biochemists, ecologists, and all the rest. It is a marvelous privilege. But there are times, I must confess, when I feel like I am watching a blind fistfight.

One of the first bouts I witnessed took place in the late 1990s, when I was researching the origin of whales. Whales descend from terrestrial mammals, and made the transition to water between about 50 million and 40 million years ago. In the 1990s paleontologists began unearthing a series of spectacular fossils documenting that transition, including whales with full-blown legs. Functional morphologists joined forces to investigate the transition, studying swimming otters to understand how proto-whales might have moved through the water. I spent a lot of time with scientists such as these. They are naturalists. They have to know a lot of natural history. They have to memorize the details of many species, to understand how the physiology, behavior, morphology, and ecology of each animal add up into an integrated whole. Yet these naturalists also know that they only have a slippery grasp on all of that embodied complexity.

I put what I learned from those naturalists into my first book, *At the Water's Edge* \[2\]. As I was finishing up my manuscript, I began coming across papers in which scientists were taking a radically different approach to the question of whale origins: they were comparing the DNA of whales to that of other mammals.

At the time, molecular phylogenies were still a novelty. The computational methods for calculating them were relatively new, and scientists could only use them to compare a few gene sequences of whales and other mammals. But the conclusions from these few studies were the same: the closest living cousins of whales are hippos.

This may not seem like a big deal. It certainly didn't seem to bother the scientists who carried out the studies. They were just analyzing digital code, abstracted from the animals that carried it. The results spoke for themselves. Nevertheless, they gave paleontologists and mammalian systematists conniptions.

Here's why. Hippos are artiodactyls (also known as even-toed ungulates). Other artiodactyls include cows, camels, and pigs, and goats. Zoologists have long recognized a number of anatomical features that unite artiodactyls in their own group, distinct from other hoofed mammals such as horses and rhinos. One of the most obvious hallmarks was a bone in the ankle, the astragalus. The artiodactyl astragalus has a unique double-pulley shape that allows the artiodactyl hoof to swing back and forth in a distinctive way.

In the 1990s paleontologists found a number of spectacular early whale fossils, but they had yet to find bones from the whale ankle. The bones and teeth they did find suggested that the closest relatives of whales were an extinct group of mammals called mesonychids. Mesonychids were hoofed mammals, but they did not have a double-pulley astragalus. Therefore, paleontologists concluded, they were probably not artiodactyls. And if mesonychids were not artiodactyls, then whales could not be either. And that meant that the whale–hippo link had to be wrong.

As far as I could tell, this line of reasoning caused no distress among the scientists who found the hippo–whale link in their genetic data. Frankly, I'm not sure they knew what an astragalus was. How many computational biologists are trained in anatomy? The shape of the mesonychid astragulus was irrelevant to them, really. To them, DNA was an overwhelmingly superior source of information. It could be analyzed precisely. It was strings of code, rather than a maddening blur of phenotype.

I decided that this potential whale–hippo link was important enough to mention in my book, but did so only briefly because the results were so preliminary. Still, I left the matter open, and I'm glad I did. Subsequent studies on mammal DNA continued to support the whale–hippo link. And meanwhile paleontologists discovered more fossils of ancient walking whales. In 2001 Phil Gingerich and his colleagues from the University of Michigan and from Pakistan described a skeleton of the seal-like species *Rodhocetus*. Among its bones, they found an astragulus—the first whale astragulus ever discovered. And lo and behold, the astragalus had a double-pulley shape \[3\]. The DNA, it seems, had been right all along.

This experience made a strong impression on me. I was struck by the divide between these two kinds of biologists. Each group had a profound confidence in their own sources of information, and an abiding skepticism about the other's. As I learned more about the history of modern biology, I realized that this rift did not begin in the 1990s. It was already present in the 1950s, as molecular biologists began championing their new science over more traditional ways of understanding life.

Harvard University's biology department was a microcosm of this conflict. James Watson, fresh from discovering the structure of DNA, breezed into the department in 1956 with revolution on his mind. "It was time to sweep beyond mere description of animals and plants and move into a new biology based on chemistry and physics," as Watson's biographer, Victor McElheny, writes \[4\].

Needless to say, the Harvard naturalists were not happy. Edward O. Wilson, entomologist, ecologist, and sociobiologist, pushed back hard. "Watson, having risen to historic fame at an early age, became the Caligula of biology," he writes in his autobiography, *Naturalist*. "It was foolish, we argued, to ignore principles and methodologies distinctive to the organism, population, and ecosystem, while waiting for a still formless and unproved molecular future" \[5\]. The struggle only ended when Harvard's biologists agreed to split their department in two.

Over the past fifty years, these factions of biologists have had a complex relationship. Some scientists have continued to carry out relatively traditional natural history work, with little need to delve into molecular (or computational) biology. Others have given little attention to natural history, focusing their efforts instead on deciphering the complexities of a membrane channel, or building new algorithms for identifying open reading frames. In some cases, biologists have bridged this divide, and the result has been a fruitful collaboration. But in other cases—such as the DNA studies on whales and hippos—one group moves into the other's traditional territory, sparking new conflict.

"That can't be right" is a refrain I've often heard when computational biologists report a controversial result. Along with the whale–hippo link, I can also recall the snorts that came with the first studies on human DNA suggesting that we can trace our ancestry to Africa 150,000 years ago. The picture is certainly more complex than the first "out of Africa" scenarios suggested, but the core of that idea has withstood further scrutiny. These days I sense a growing confidence in computational biologists, perhaps brought on by successes such as these. Many are no longer content merely to present an analysis of DNA and leave it at that. They use their findings to make sweeping statements about ecology and evolution.

One example of this new ambition was a paper published earlier this year on the evolution of cats \[6\]. The scientists offered a sweeping scenario for cat evolution, complete with migrations of cats out of Asia into the New World and back, along with the emergence of the major groups of felids, ranging from ocelots to bobcats to lions. The scientists based their scenario entirely on an analysis of cat DNA. They did not consider a single fossil of a cat, nor did they have a paleontologist expert on cats as a coauthor. Cat fossil experts inform me that fossils of true cats as old as 17 million years have been discovered in North America. The geneticists put the arrival of cats in North America at only 8 million years ago. Whether or not the DNA results are correct, it is striking that the report does not even mention the existence of fossils that do not fit the pattern.

Genomes unquestionably contain a rich store of information, but sometimes that information can be deceptive. Recently, two paleontologists, Philip Donoghue of the University of Bristol and Mark Purnell of the University of Leicester, described how this deception can arise in the study of vertebrate evolution \[7\]. A number of studies have suggested that vertebrates underwent several rounds of genome duplication since they diverged from their closest invertebrate ancestors. One of these duplications may have coincided with the emergence of jawed vertebrates (gnathostomes). This link has prompted some scientists to propose that this duplication triggered a leap in complexity that set gnathostomes apart from their closest vertebrate relatives, the hagfish and lampreys. Gnathostomes not only have jaws, but also have a long list of other traits that set them apart from agnathans, including an advanced immune system, a mineralized skeleton, teeth, and fins. They are also far more diverse. Perhaps a genome duplication provided the raw material for an adaptive radiation.

As Donoghue and Purnell demonstrate, this idea only makes sense if you ignore the fossil record. Paleontologists have found a rich record of fossils documenting the gradual assembly of the gnathostome body plan. Conodonts, for example, possessed a mineralized skeleton, dentine, and enamel—but nothing else that sets gnathostomes apart from agnathans. Fins evolved later, and a mineralized braincase came later still. Nowhere along the phylogeny of vertebrates is there a sudden leap of complexity in the forerunners of true gnathostomes, nor is there an abrupt leap in levels of diversity. One is left to wonder just when the alleged effects of a genome duplication occurred.

Taking natural history seriously is good not only because it can steer a computational biologist away from these sorts of errors. It also opens up fascinating new questions to which computational biologists can profitably apply their skills. Consider the coelacanth, a rare, ugly fish found in the Indian Ocean. It belongs to the lobe fins, a group of fish from which we and all other land vertebrates evolved. Scientists knew about the fossils of coelacanths long before they saw one alive. The fossil record of their ancestors reaches back 400 million years, but it was not until 1938 that living coelacanths were discovered off the coast of South Africa.

I present the coelacanth in the spirit of the Buddha. Is it a fish, or a just bag of digital code?

Earlier this year, computational biologists published a remarkable paper about the coelacanth in *Nature* \[8\]. The paper had its origins in the startling discovery that certain regions of the human genome that do not code for proteins are highly conserved. Natural selection has kept these sequences much the same in humans and in other mammals. The scientists were puzzled as to how natural selection could preserve their homology, since these sequences do not encode any protein.

The scientists decided to see how widespread these regions are among animals. Searching gene databases, they discovered related genetic sequences in the coelacanth. Despite more than 400 million years separating our ancestors, the coelacanth and human regions were 80% identical. A close examination of the coelacanth segments revealed them to be transposons—virus-like stretches of DNA that could insert new copies of themselves into the genome.

The authors conclude that these ultraconserved regions of the human genome descend from a transposon in the ancestors of lobe fins. In the tetrapod lineages, the transposons lost their ability to spread through the genome. Some became exapted for regulatory functions, and were preserved by natural selection. The others were eroded by mutations. In the coelacanth lineage, on the other hand, the transposons remained active.

As fascinating as these results are, I was also struck by how they were framed. The authors begin their paper by referring to the coelacanth as a "living fossil." They end their paper with the conclusion that by preserving this family of transposons, the fish "acts, in a sense, as a living molecular fossil. The remaining 99.9% of its genome, as yet unsequenced, may very well hold precious traces of additional events that helped shape our own evolution."

The coelacanth is a living fossil only insofar as scientists once thought it was extinct. But that is not the dictionary definition of a living fossil: an organism "that has remained essentially unchanged from earlier geologic times" \[9\]. Paleontologists have been giving the coelacanth lineage a fresh look, and they've discovered a surprising amount of morphological change \[10\]. Some ancient relatives of today's coelacanths were slender, eel-like creatures. Others had round, sunfish-like bodies. Today's coelacanths are probably not living fossils in an ecological sense, either. Early fossils of coelacanths are often found in sedimentary rocks formed in coastal waters. Living coelacanths, by contrast, only live deep under water.

I would question whether the coelacanth is much of a molecular living fossil, either. The new research certainly shows that coelacanths preserve information in their genome that reveals some of the history of ultraconserved regions in our genome that would otherwise be lost. But that's because their transposons are still "alive," while our transposons lost their ability to replicate a long time ago and have become frozen in place. In that sense, *we* are the living fossils.

If one thinks about the coelacanth's natural history as well as its genome, a new set of questions arises. Transposons are prone to mutations, which can take away their ability to insert new copies in a genome. These coelacanth transposons have been replicating for 400 million years, and now probably take up a significant fraction of the coelacanth genome. And yet they still have not diverged very much in all that time. Is natural selection conserving them? Is natural selection acting on the transposon or the coelacanth? How do these proliferating transposons affect the physiology of the coelacanth? Do they act like pathogens, or are they providing some benefit we don't yet understand? Does their expansion play any role in the ecology of living coelacanths?

None of these questions would even arise without the invaluable work of computational biologists. But if all the blind men gather together around the coelacanth, perhaps they can better understand just what this creature is.

I would like to thank Pavel Penzer, Alberto Apostolico, and Concettina Guerra for inviting me to Recomb '06, where I presented a talk on which this essay is based. Michael Coates, Phil Donoghue, John Gatesy, Phil Gingerich, David Haussler, Christine Janis, Blair Van Valkenburgh, and other scientists have generously shared their thoughts about the biological elephant.

# References


abstract: The late Quaternary megafauna extinction was a severe global-scale event. Two factors, climate change and modern humans, have received broad support as the primary drivers, but their absolute and relative importance remains controversial. To date, focus has been on the extinction chronology of individual or small groups of species, specific geographical regions or macroscale studies at very coarse geographical and taxonomic resolution, limiting the possibility of adequately testing the proposed hypotheses. We present, to our knowledge, the first global analysis of this extinction based on comprehensive country-level data on the geographical distribution of all large mammal species (more than or equal to 10 kg) that have gone globally or continentally extinct between the beginning of the Last Interglacial at 132 000 years BP and the late Holocene 1000 years BP, testing the relative roles played by glacial–interglacial climate change and humans. We show that the severity of extinction is strongly tied to hominin palaeobiogeography, with at most a weak, Eurasia-specific link to climate change. This first species-level macroscale analysis at relatively high geographical resolution provides strong support for modern humans as the primary driver of the worldwide megafauna losses during the late Quaternary.
author: Christopher Sandom; Søren Faurby; Brody Sandel; Jens-Christian Svenninge-mail: <firstname.lastname@example.com>[^1]
date: 2014-07-22
institute: Ecoinformatics and Biodiversity, Department of Bioscience, Aarhus University, Ny Munkegade 114, Aarhus C 8000, Denmark
references:
title: Global late Quaternary megafauna extinctions linked to humans, not climate change

# Introduction

During the Late Pleistocene and early Holocene, regions around the world suffered losses of megafauna species of a magnitude unprecedented for many millions of years \[1–3\]. Although extinctions are common in the Quaternary fossil record, such rapid and large global species losses without functional replacements are unusual. Multiple explanatory hypotheses for this global extinction event have been proposed, including climate change \[4,5\], the spread of modern humans (*Homo sapiens*) and related effects of hunting and habitat change \[6–9\], an extra-terrestrial impact \[10\] and hyper-disease \[11\]. With little or no evidence to support the latter two as global explanations \[11–14\], we focus here on climate change and hominin palaeobiogeography. The relative importance of climate and hominin drivers has been subject to long-standing, highly charged controversy (e.g. \[3,15,16\]).

Although megafaunal extinctions occurred worldwide, most studies focus on a specific region \[17–19\], and the few global-scale studies have been geographically incomplete and/or very coarse-grained \[3,20\]. In an influential review, Barnosky *et al*. \[3\] provided a continental breakdown of the Late Pleistocene megafauna extinctions and reported that humans were the main extinction driver in Australia and North America, and climate was the main driver for Europe, but that data were insufficient for making assessments for Africa, Asia and South America. Nogués-Bravo *et al*. \[20\] followed up by linking these genus-level continental-resolution patterns to continental-average climate change, finding extinction patterns among Africa, Eurasia and North America consistent with climate-driven extinction; however, South America strongly deviated from this trend, with the lowest climate change and the most severe genus-level extinctions. A geographically comprehensive species-level assessment of the global-scale extinction pattern remains lacking. Improvements and increased access to fossil, palaeoclimate and hominin palaeobiogegraphical records, more accurate dating and taxonomic revisions now make possible much more geographically fine-grained analysis of megafauna extinctions worldwide, namely species-level extinctions per Taxonomic Databases Working Group (TDWG) level 3 country (see Material and methods for a description of these regions).

## The climate change hypothesis

We focus on temperature and precipitation contrasts between full glacial and full interglacial conditions, as these represent the strongest climate shifts during the late Quaternary. We estimated these as the Last Glacial Maximum (LGM, *ca* 21 000 years BP) to present-day (1950–2000) contrast and, in supplement, the Last Interglacial (LIG, *ca* 130 000 years BP) to LGM contrast. Palaeoclimatic changes can be represented both by simple macroclimatic changes through time or by climate change velocities that incorporate spatial climate gradients to estimate how fast climate moved in space, providing a direct estimate of how rapidly species would have needed to migrate to track climate \[21,22\]. Where climate change is severe and the topography is flat, species would typically have to travel greater distances to find suitable conditions, experiencing greater risks of extinction. As a result, high late Quaternary climate change velocity is associated with low modern species endemism in mammals, birds and amphibians worldwide, indicating greater past extinction rates in more climatically unstable regions \[21\]. We used mean annual temperature and annual precipitation anomaly and velocity between the LGM and the present to represent the potential severity of climate change over the study period (figure 1*d*,*e* and the electronic supplementary material, figure S1). If climate change has been an important driver of extinction, then climate change magnitude or velocity should be positively correlated with the proportion of the cumulative late Quaternary large mammal species per TDWG country that have become globally or continentally extinct during 132 000–1000 years BP. Later extinctions were not considered because of the strong known human effects during this period. Although our analysis is at the country scale and species may respond to more local environments, large mammals generally have large ranges \[23\] and within-TDWG country variation in climate change velocity is typically small (see Material and methods).

## The human palaeobiogeography hypothesis

Hominins have migrated out of Africa in waves successively increasing the hominin-occupied area so that megafauna have co-occurred and coevolved with hominins for differing lengths of time in different regions. Possibly as early as the Late Pliocene or Early Pleistocene, *Homo erectus* and contemporary hominins expanded into southern Asia and south and western Europe \[24\] (figure 1*c* 'Archaic-early'). *Homo neanderthalensis* and Denisovan humans pushed these boundaries further, colonizing most of Eurasia and leaving only the very northern extremities unpopulated during interglacials \[25\] (figure 1*c* 'Archaic-late'). While archaic humans appear not to have inhabited the northernmost parts of Eurasia, no terrestrial megafaunal species was endemic to these parts (see the electronic supplementary material, Data S1). The residing megafauna would have encountered archaic hominins during range extensions into more southerly regions or rare archaic incursions into the north; thus, we classify this region with those occupied by archaic hominins in our main analyses (figure 1*c* 'Archaic-peripheral'). By contrast, faunas in Australia, North and South America and to some extent Japan did not have any contact with hominins until the expansion of modern humans \[26\] (figure 1*c* '*Homo sapiens*-only'). Late Quaternary megafauna extinctions have been hypothesized to have been more severe where modern humans were the first hominin to arrive, suddenly introducing a new and effective big-game predator into regions with megafauna naive to human hunting \[27\]. Conversely, extinctions have been posited to have been less severe in Africa because of long-term, gradual hominin–megafauna coevolution \[3\]; alternatively, pre-Late Pleistocene megafauna extinctions in regions occupied by pre-*sapiens* hominins may also have contributed to fewer late Quaternary extinctions \[27,28\], or human populations may have been more suppressed by disease here \[15\]. Under this scenario, we hypothesize that the most severe extinctions occurred where modern humans were the first hominins to arrive and that the lowest extinction occurred in sub-Saharan Africa, the centre of origin and evolution for hominins overall as well as for *H. sapiens* \[24\] (figure 1*c* '*Homo*-origin').

We aim to evaluate the relative support for the climate change and hominin palaeobiogeography hypotheses for the late Quaternary megafauna extinctions by, to our knowledge, the first comprehensive species-level, fine-grained global macroecological analysis. We achieve this aim and find strong support for hominin palaeobiogeography as the critical factor for explaining the strong geographical variation in megafauna extinction severity worldwide.

# Material and methods

## Extinct species of the late Quaternary

A thorough examination of the scientific literature identified 177 taxonomically accepted, extinct or continentally extirpated large mammals (more than or equal to 10 kg) that occurred during the Late Pleistocene and early/middle Holocene (132 000–1000 years BP). The process was begun by gathering information on species names reported lost during the late Quaternary from review publications and original research articles followed by detailed species by species research to confirm their taxonomic validity and presence within our study period (electronic supplementary material, table S1). In some circumstances, we chose to reject uncertain species closely related to accepted species occurring over similar ranges to ensure that our extinction densities were conservative. A notable example includes the North American horses, where only two *Equus* species were accepted, *Equus ferus* and *Equus semiplicatus* (representing the stilt-legged horses), following \[29\]. Species were only accepted if they are recorded from a directly dated site within the time period studied. In order to be conservative, we did not accept dates based on co-occurrence with potential indicator fossils, i.e. species thought to be associated with a particular time period. As a result, the number of extinctions that are too old to be correctly dated with carbon-14 dating may be underestimated in our analysis. Species that were of uncertain taxonomy or occurred from sites thought to be within the late Quaternary, but lack a confirmed date were included in a list of potential, but uncertain late Quaternary megafauna extinctions (*n* = 27; electronic supplementary material, table S1). Of these 27 taxonomically or temporally uncertain species, we recorded regional presences for 17 of them, which were included in a supporting sensitivity analysis (electronic supplementary material, table S3). Species mass were primarily collected from Smith *et al*. \[30\] and supplemented with additional sources or, where required, were estimated based on masses of species in the same genus (electronic supplementary material, table S1). We defined 'large' mammals as more than or equal to 10 kg (*n* = 177 spp.). We repeated the analyses including species more than or equal to 44 kg (*n* = 154 spp.) to be consistent with much of the literature published on the megafauna extinctions \[3\]. Species lost in the last 1000 years were excluded because of their much clearer association to human drivers of extinction.

## Extinct species distribution

Geographical locations were collected for each accepted and uncertain species. Mapping was performed at the resolution of TDWG level 3 regions \[31\]. In total, 229 TDWG countries were included; islands, Antarctica and glaciated countries were excluded (see the electronic supplementary material for details). A species was determined to be present if an identified specimen was recorded for a given TDWG country. Location records for accepted and uncertain species were downloaded from the Global Biodiversity Information Facility (GBIF) on 29 November 2012 and supplemented with data gathered from FAUNMAP \[32\] and the primary literature based on searches of species names and regional reviews as listed in the electronic supplementary material, table S1. Species range maps produced by experts were accepted as valid locations. GBIF data were cleaned to remove erroneous records based on the following criteria: species recorded on a continent with no dated site on the specified continent, records associated with animals in captivity, records within the open ocean and records with latitude and longitude of zero. For continentally extinct species, locations recorded from their extant range were removed. After locations in open ocean had been removed, locations for species occurring close to the coast were moved to the nearest TDWG country to account for lower sea levels during the Last Glacial. A presence/absence map, representing total cumulative range of each species across the study period (see the electronic supplementary material for further details), was plotted for each species (based on the electronic supplementary material, Data S1). The species distributions maps were typically discontinuous at least partly as a result of geographical gaps in the fossil record (electronic supplementary material, Data S1). These probably primarily reflect geographical bias in collection efforts as well as taphonomic bias, so we controlled for this by filling gaps in species ranges where: (i) the range was discontinuous (e.g. *Hippidion principale* and *Elephas namadicus*), (ii) there were holes within the range (e.g. Nevada, Colorado and Oklahoma for *Canis dirus*), and (iii) to fill peripheral TDWG countries on the edge of ranges (e.g. the Northern Provinces of Australia and the Guianas). For species-specific exceptions, see the electronic supplementary material. Where the gaps between records were extensive, gaps were filled on a path that best reflected environmental conditions in the regions where specimens were recovered. The primary analysis was performed including the filled records. To test the sensitivity of the results to our interpolations, the analysis was re-run using only the direct records.

## Data analysis

The total number of extinct large mammals was calculated for each TDWG country (figure 1*b*). To account for naturally occurring species richness gradients and the uneven area of each TDWG country, extinction was expressed as a proportion of the total number of extinct and extant species (figure 1*a*). To remove the effects of human-driven extinctions and range contractions occurring in the last 1000 years, we estimated current extant large mammal richness for each TDWG country by accounting for the magnitude of human impact \[33\]. We initially created a general linear model (GLM), with a Quasi-family, logit link and variance of *μ*(1 − *μ*), with large mammal richness modelled as a function of continent, small mammal richness (International Union for Conservation of Nature range maps \[34\]) and human impact \[33\]. Six continents were included in our analysis; however, Asia was divided into two, tropical and temperate according to the TDWG classifications, and modelled individually. Human impact \[33\] was a significant predictor of large mammal diversity in North America (*p* ≤ 0.001), and temperate Asia (*p* ≤ 0.001), and approached significance in Europe (*p* = 0.062). For these regions, extant large mammal TDWG richness was estimated from this model using the lowest human impact recorded within the specific continent, and so within the predictive range of our models (electronic supplementary material, figure S11).

We focused on temperature and precipitation contrasts between the LGM, *ca* 21 000 years BP and the present-day interglacial climate, as this contrast represents the full glacial–interglacial amplitude of climate change during our study period. The chronology of extinctions, however, extends across the late Quaternary, including extinctions prior to the LGM \[3\]. To account for this, we also explored climate change between the LIG and the LGM as a supplementary analysis (electronic supplementary material, figure S10). Importantly, temperature and precipitation anomalies are highly correlated between the LIG–LGM and LGM–present time periods (0.97 and 0.82, respectively), indicating spatial consistency in the severity of climate change across the late Quaternary. Replacing the LGM–present climate contrasts with the LIG–LGM contrasts in a supplementary analysis provides even less support for the climate change hypothesis.

Climate change was measured using four variables, two measures of climate change velocity \[21,22\], the rate of displacement of the mean annual temperature and annual precipitation from the LGM to today, we measured the spatial gradient using a 2.5 arcminute resolution map, and two measures of climate change magnitude, LGM to present mean annual temperature anomaly and precipitation anomaly; these climate data were obtained from WorldClim at 2.5 arcminutes \[35\] (figure 1*d*,*e*; electronic supplementary material, figure S1). Temperature and precipitation anomaly were calculated by averaging records from the MIROC3 and CCSM models \[36,37\]. For each climate change variable, a standard measure was created by taking the square root of each data point and dividing by the maximum, creating scores between 0 and 1. For the measures of anomaly, absolute values were used as we are exploring the magnitude of change and not the direction. Although our analysis is at the TDWG country scale, species will probably respond to more local environments. Local-scale topography affects climate change velocities in particular and could vary considerably within region. To assess the degree of climate change velocity variation, velocity range was calculated for each TDWG country. The median velocity range per country was 16 m yr^−1^ for temperature and 44 m yr^−1^ for precipitation. This suggests that the velocity variation within each TDWG country was relatively small and unlikely to be problematic. To test for bias against climate explanatory variables measured on a continuous scale and for uncertainty in climate models, we used *k*-means clustering and a climate sensitivity test, respectively (see the electronic supplementary material).

## Model selection

Climate change, hominin palaeobiogeography and hominin–climate combination models were constructed and compared to determine which predictor variables best describe the recorded regional extinction ratios. As velocity is calculated using climate change anomaly to avoid problems with collinearity, particularly in the temperature data, the velocity and anomaly of the same predictor variable were not included in the same climate or hominin–climate combination model. For the hominin models, we explored all combinations of hominin palaeobiogeography and the optimal description was found to be lumping the three Archaic regions together (based on an ordinal analysis of the five categories that revealed significant differences between *Homo*-origin and Archaic-early, and Archaic-peripheral and *H. sapiens*-only, but not between Archaic-early and Archaic-late and Archaic-peripheral), which we refer to as Archaic-combined. This combined model also performed better than including modern human arrival time \[38\] (based on Akaike information criterion (AIC)). We initially constructed GLMs using an arcsine square-root transformation of the response variable to account for the non-normal distribution of the proportion data. This transformation was deemed appropriate because the nature of these binomial data were not a factor of sampling effort and we wanted to give a 50% reduction of large mammal community within a TDWG country the same weight no matter if the reduction was from four to two species or 50–25 to avoid recording low extinction severity in regions with low total species diversity. We used AIC to select the best climate change, hominin palaeobiogeography and hominin–climate combined models. For comparison to the above approach, we also constructed a GLM using a Quasi-family, logit link and variance of *μ*(1 − *μ*), to account for the non-normal distribution of proportion data \[39\], finding the results to be consistent (see the electronic supplementary material). To assess the degree of spatial autocorrelation (SAC), we computed correlograms of GLM model residuals using the 'ncf' package in R \[40\], with distance classes of 500 km (electronic supplementary material, figure S3). A spatial autoregressive (SAR) model of the error type (SAR~err~ sensu \[41\]) was used to account for SAC in the multivariate hominin–climate model and compared to the GLM using the arcsine square-root transformation. The SAR models were implemented in the R package 'spdep' \[42\]. The spatial weights matrix was defined by connecting each TDWG country to its four closest neighbours. This SAR model removed residual SAC (electronic supplementary material, figure S3). All analyses were performed in R.v. 2.15.1 \[43\].

# Results

Our extensive and taxonomically conservative literature review provided evidence of 177 globally or continentally extinct large mammal species (more than or equal to 10 kg; Africa 18, Asia 38, Australasia 26, Europe 19, North America 43 and South America 62; electronic supplementary material, table S1; figure 1*b*) for the period 132 000–1000 years BP. Of the 229 TDWG countries included in the analysis, the highest number of extinct species occurred in Texas (33 species), while Uruguay showed the highest proportion of extinct species (78%, figure 1*a*). Southern South America, southeast North America, western Europe and southern Australia emerged as extinction hotspots, with sub-Saharan Africa and southern Asia as notable cold spots (figure 1*a*). Thirty-one TDWG countries had no recorded extinctions.

Using climate change predictors only, a GLM including both temperature anomaly and precipitation velocity best described the extinction pattern, although with poor explanatory power (GLM: *F*~226~ = 29.790, *p* \< 0.001; ). Human palaeobiogeography included as a categorical variable with three levels, *Homo*-origin, Archaic-combined and *H. sapiens*-only (figure 1*c*), had strong explanatory power (GLM: *F*~226~ = 201.240, *p* \< 0.001; ). A full model was constructed from the predictors of the best climate and human models with interactions between human biogeography and the two climate predictors (GLM: *F*~220~ = 68.740, *p* \< 0.001; ; electronic supplementary material, figure S2 and table S2). The full model was significantly better than either the human or climate model (climate: *p* \< 0.001; human: *p* \< 0.001), but explanatory power was only weakly improved over the human-only model.

Because of strong SAC in the residuals (electronic supplementary material, figure S3), the pairwise and multivariate analyses were all re-done accounting for SAC. A simultaneous autoregressive (SAR) model was used to account for SAC in the multivariate climate–hominin model. The best combined SAR model included hominin palaeobiogeography, temperature anomaly and an interaction between the two, while precipitation velocity was excluded (figure 2 and table 1). The model indicated significant effects of hominin palaeobiogeography, with intermediate extinctions in the Archaic-combined region and the most severe extinctions in the *H. sapiens*-only region (figure 3), temperature anomaly and their interaction. The interaction reflected the fact that the temperature anomaly effect was observed only in the Archaic-combined region (figure 2). These findings were robust to decisions related to the palaeontological information, distribution interpolation, extant species richness estimation and size threshold as well as to whether climate change was recorded as a categorical variable, was represented by the LIG–LGM shift rather than the LGM–present shift, and uncertainty in the climate models (electronic supplementary material, figures S4–S12, tables S3–S7 and see Sensitivity Analysis section).

SAR model explaining global patterns in the proportion of extinct large mammals (more than or equal to 10 kg). (Est, regression coefficient; s.d., standard deviation; *Z*, test statistic; *p*, *p*-value; HS, *H. sapiens-*only region; AC, Archaic-combined region; TA, mean annual temperature anomaly between LGM and today, standardized to range between 0 and 1.)

|                     | estimate | s.d.  | *Z*    | *p*     |
|:--------------------|----------|:------|--------|---------|
| intercept           | −0.353   | 0.263 | −1.341 | 0.18    |
| *H. sapiens-*only   | 1.107    | 0.274 | 4.037  | \<0.001 |
| Archaic-combined    | 0.735    | 0.271 | 2.713  | 0.007   |
| temperature anomaly | 1.504    | 0.739 | 2.037  | 0.042   |
| HS × TA             | −1.516   | 0.749 | −2.022 | 0.043   |
| AC × TA             | −1.225   | 0.752 | −1.630 | 0.103   |
| pseudo *R*^2^       | 0.635    |       |        |         |
| *p*                 | \<0.001  |       |        |         |
| AIC                 | −272.57  |       |        |         |

This analysis illustrates that the late Quaternary megafauna extinctions were strongly linked to hominin palaeobiogeography and only weakly to glacial–interglacial climate change. The relationship between hominin palaeobiogeography and extinction magnitude is striking, with universally low extinctions in sub-Saharan Africa (maximum 13%), where hominins and the megafauna have long coexisted, but widespread exceptionally high extinctions in Australia and the Americas, where modern humans were the first hominin present. Only three TDWG countries in the *H. sapiens*-only region had extinction ratios below 30%, two of which are small, coastal states in North America with missing data as a likely cause of their low extinction values. The third is Japan, but the coding of hominin–palaeobiogeography for this country is potentially problematic because the country includes some endemic species that were never in contact with archaic humans, but many others with ranges that were, suggesting that the country could alternatively be coded as 'Archaic-peripheral'. Eurasia falls between these extremes with a wide variety of extinction ratios, presenting a more complicated story. Globally, human palaeobiogeography alone accounts for 64% (GLM estimate) of the variation in extinction, while temperature anomaly, in combination with precipitation velocity in the best climate-only model, had much weaker explanatory power (20%) and only showed a relation to the proportion of extinct large mammals in the Archaic-combined region in the combined model.

# Discussion

The global pattern of late Quaternary megafauna extinction presents a clear picture that extinction is closely tied to the geography of human evolution and expansion and at most weakly to the severity of climate change. The pattern of extinctions closely followed the hominin paleobiogeography hypothesis with increasing severity of extinction with reduced period of hominin–megafauna coevolution, notably with uniformly high extinction in areas where *H. sapiens* was the first hominin to arrive. By contrast, only in Eurasia was there a climate change signal.

High extinction despite a relatively stable climate is most striking in South America, forming a strong contrast to sub-Saharan Africa where extinction was minimal in spite of similar glacial–Holocene climate changes (figure 1*a*,*d*,*e* and the electronic supplementary material, figure S1). This relationship is particularly notable in southern South America where extinction ratios were among the highest recorded. Extinctions in the region have been linked to fluctuations between humid and dry periods and shifts in the availability of savannah and forest habitats. For instance, De Vivo & Carmignotto \[44\] associated a humid period in the mid-Holocene with loss of savannah and the demise of some megafauna. There is no indication, however, that these fluctuations were greater than in Africa or elsewhere \[45\]. Furthermore, dietary analyses of large South American herbivores such as *Eremotherium laurillardi*, *Stegomastodon waringi* and *Toxodon platensis* indicate that these extinct species were adaptable generalists, altering their diet between grazing and mixed feeding according to the habitat they occupied \[46\]. Additionally, other areas maintained grassland or forest throughout the whole period despite fluctuating climatic conditions \[47\]. The pampas region, for example, remained open throughout this time \[47\], but many megafauna species still disappeared, such as *Megatherium americanum*, which has been associated with human exploitation \[48\]. Extinctions in South America are poorly dated, but many have occurred after modern human arrival, approximately 15 000 years BP, and continued into the climatically relatively stable Holocene \[19\].

Most Australian extinctions occurred prior to the LGM, and so outside our LGM–present climate change predictor variable, just subsequent to the time humans are thought to have arrived (72–44 kyr BP) \[3\]. Australia experienced gradual drying from 400 ka that is proposed to have achieved a hydrological threshold in the last glacial; Wroe & Field \[4\] claim this drying drove a staggered extinction of many of the Pleistocene megafauna prior to the arrival of modern humans. Alternatively, it has been argued that modern humans caused the Australian megafauna extinctions either via fire-driven vegetation changes \[49\] or hunting \[8\]. Rule *et al*. \[50\] gave a detailed chronology for a dramatic decline in large mammal biomass in northeastern Australia during a period of climatic stability millennia after discernible drying events, after human arrival and prior to habitat change, supporting human-driven megafauna attrition as a driver of vegetation community change rather than the reverse (see also \[17,51,52\]). In agreement with this, we observe high extinction rates across Australia, but low to moderate temperature and precipitation anomalies and velocities (figure 1*a*,*d*,*e*). Additionally, LIG–LGM climate change measures indicated similarly moderate to low levels of relative temperature change across this period (electronic supplementary material, figure S10).

Our results also confirm earlier observations that extinctions could be severe even in relatively climatically stable regions in North America, where the vegetation changed little \[53\]. For instance, despite relatively little climate change and local survival of, for example, chapparel vegetation from LGM to today \[54\], California lost 21 megafauna species (53%, figure 1*a*,*b*) such as *Nothrotheriops shastense*, *Capromeryx minor* and *C. dirus* (see the electronic supplementary material, Data S1). In some cases, megafaunal species went extinct even though their preferred local diet has remained plentiful to the present-day, e.g. *Mammut americanum*, which fed on twigs of trees such as bald cypress (*Taxodium distichum*) in Florida \[55\]. *Euceratherium collinum* and *N. shastense* are further examples of species from western USA that went extinct despite the continued presence of favoured plant food sources in the region \[56\]. Grund *et al*. \[57\] highlights that the extinction pattern across North America is not consistent with climate as the main driver because species with small geographical ranges were not disproportionately affected. These megafauna patterns contrast with the global findings reported by Sandel *et al*. \[21\] that small-ranged amphibians, mammals (mostly small-sized) and birds are absent or scarce where Quaternary climate change has been severe. The period of extinction in North America occurred approximately 11 500–10 000 years BP, well within our period of measured climate change, and broadly occurred when climate change was severe, but also consistent with the arrival of modern humans \[3\].

Martin & Wright \[6\] were first to argue strongly for modern humans as the likely driver of megafauna extinctions worldwide, envisioning a swift overkill at the range front of expanding modern human populations. Since the emergence of that idea, other slower extinction models have been proposed involving anthropogenic fires and human-driven habitat change \[49,50\]. Although strong scepticism continues about humans as the extinction driver, primarily based on an argued scarcity of archaeological evidence of megafauna kills \[4,16\], others have shown that that kill sites are not rarer than expected \[58,59\] and that prehistoric human populations had the potential to exterminate megafauna populations \[7\]. Our analysis further supports humans as the key driver of extinction, but also has yielded the critical finding of little association between climate change severity and the proportion of extinct large mammals, suggesting that climate change was not a pervasive cause of this global-scale extinction event. Some earlier research has indicated that late Quaternary climate change affected megafauna species population dynamics and distributions \[60\], similar to the well-documented effects on smaller animals and plants \[61\]. Such effects, however, do not explain why most megafauna species did not go extinct where climate change was most severe or during previous glacial–interglacial cycles. In addition, although megafauna–hominin–climate interactions will have varied among species and regions, our analysis strongly supports modern human colonization of virgin territory as the more pervasive driver of extinction.

The temperature-related pattern in the Archaic-combined region is consistent with previous suggestions of climate as a major extinction driver there, especially in northern areas \[3,60,62\]. The relationship appears to be driven by high temperature anomaly and extinctions in northwest Eurasia and low-temperature anomaly and extinctions in southern Asia, but there are also widespread mismatches (figure 1*a*,*d*). However, the absence of a climate–extinction association from the *H. sapiens*-only region is inconsistent with extinctions resulting from synergism between climatic stress and hunting pressure because the association should rather have been strongest here. Instead, this effect appears consistent with human-driven extinctions with climatic constraints on archaic human distributions in northern parts of Eurasia \[25\], causing these regions to function as at least partial megafaunal refugia until modern human arrival \[63\]. At the same time, subtropical and tropical southern Asia were earlier, more permanently, and more densely colonized by archaic humans \[24\] and may thus be relatively similar to sub-Saharan Africa, with low late Quaternary extinctions because of either coevolution and/or pre-Late Pleistocene extinctions \[28,64\]. Such a scenario fits the extinction pattern for *Mammuthus primigenius* in Eurasia, with a range contraction towards high latitudes despite earlier temperate occurrences \[65\], and late survival in the high north beyond the range of modern humans, followed by sudden extinction with human arrival to these regions \[60,63\]. Eurasia experienced the most prolonged extinction during the Late Pleistocene with species lost before and after the LGM \[3\]. However, the pattern of regional climate change severity was similar between the LIG–LGM and LGM–present (electronic supplementary material, figure S10) so the relationship between climate change and extinction severity is expected to be consistent regardless of whether species were lost before or after the LGM.

Sub-Saharan Africa experienced mild late Quaternary extinctions, as would be expected with the length of megafauna–hominin co-occurrence in this region. It is also worth noting that there are some indications that a number of megafauna extinctions in sub-Saharan Africa occurred earlier, in the Early- to Mid-Pleistocene, possibly as a result of early hominin hunting or competition \[66\]. However, Africa also experienced minimal glacial–Holocene temperature change and fits the continental-scale pattern of low climate change and extinction severity that Nogués-Bravo *et al*. \[20\] described. At the same time, the area nevertheless experienced strong changes in precipitation, particularly in West Africa where extinction was exceptionally low (electronic supplementary material, figure S1; figure 1*a*). Given the already mentioned strong contrasts in extinctions between sub-Saharan Africa and South America despite similar climate changes, Africa conforms well to the extinction pattern expected from hominin palaeobiogeography.

Our analysis illustrates the value of taking a macroscale approach for understanding this global-scale extinction event, particularly highlighting the numerous discrepancies between the extent of climatic change and extinction severity at a regional scale. Furthermore, our findings indicate that the current low diversity in large mammals \[2\] in many continental areas is an anthropogenic phenomenon, not a natural one, with important implications for nature management \[67\].

# Supplementary Material

###### Supplementary Material

# Supplementary Material

###### Table S1

# Supplementary Material

###### Supplementary Data 1

# Supplementary Material

###### Supplementary Data 2

# Supplementary Material

###### R analysis script

## Acknowledgements

We acknowledge help with data collection from Lovisa Kirkeby and Brian Kerr, and human arrival time information from Anders Eriksson.

## Data accessibility

The data reported in this paper are provided in the electronic supplementary material.

## Funding statement

We gratefully acknowledge economic support from the 15 Juni Fonden and the European Research Council (ERC-2012-StG-310886-HISTFUNC).

# References

[^1]: These authors contributed equally to this study.