 And now for our featured presentation. The Stephen Padgett Memorial Lecture celebrates the life of Stephen Padgett, who passionately believed that a greater understanding of physiology would lead to better medical science. He was a founder of Research Defense Society, which later became Understanding Animal Research. And this year, we're delighted to celebrate the 86th Padgett Lecture, adding Professor Emerita Alawalia to our long and imminent list of lecturers. Emerita is dean for research in the Faculty of Medicine and Dentistry at the BARTS in London School of Medicine and Dentistry, director of the UK CRC accredited BARTS Cardiovascular Clinical Trials Unit, and professor of Vascular Pharmacology at Queen Mary University of London, where she's principal investigator, leading the Vascular Pharmacology Group. Her research focuses on understanding the role of inflammation in cardiac and vascular disease with a focus on non-canonical pathways for nitrous oxide generation and delineating the mechanisms that underlie sex differences in cardiovascular physiology and disease. Throughout her career, Emerita has persistently raised awareness on the sex bias in experimental work, increased diversity and representation in biomedical science, promoted responsible animal research, and nurtured the next generation of researchers. She was a project license holder for over 15 years, and her seven years as editor-in-chief of the British Journal of Pharmacology, she helped to generate an incredible amount of practical and internationally relevant guidance on animal experimentation. Tonight, I'm delighted to introduce professor Emerita Alawalia in her lecture, Sex and Drugs, My Rock and Roll. Thank you very much for that really very kind introduction. It's a massive honour to be delivering this lecture this evening. I had a look at the website and saw the list of the previous recipients, full of the great and good in biomedical research and in vivo research in particular. It's a massive honour. I'm chuffed to bits, in actual fact, to be the newest name on that list. So thank you very much to those who nominated me, and thank you to the UAR for awarding me this honour. I have a list of, I'm just trying to see the mouse of disclosures at the bottom, I can't see it, at the bottom of my slide. It's the final one on that list that's relevant for my presentation today and you'll understand the relevance as I go through my talk. But before I start talking about Sex and Drugs, I thought I would share with you some observations that I've made when I was doing a little bit of research in preparation for this lecture. Scenes that, despite appearances, that Stephen Padgett and I have rather a lot in common. Both of us actually trained at the same medical school. Stephen Padgett moved to St Bartz Medical College in 1878 to study medicine. I didn't bump into him in the corridors when I was there. I arrived, I know I've got a lot of white hair here, I'm not quite that old, I arrived in 1989 to do my PhD under the supervision of Professor Rod Flower. In doing my research as well, I came across some really interesting facts that I thought I'd share with you that have some relevance to my presentation today. So Stephen Padgett was one of four sons of James Padgett and James Padgett was actually the very first warden of the first residential medical college at Bartz. He took on this role in 1844 and he was obviously a really forward thinker for his time. By 1850 he had recruited the first ever, or allowed the first ever woman to attend training within a medical school in the UK. Sadly, those that followed him didn't really follow in his footsteps. Her name was Elizabeth Blackwell, she was the first and the last woman for some time at Bartz Medical School. She actually became a leading light in women's rights and women's lib. In the United States she set up medical school and she had a very prolific and successful career leading equality for women. I'm sure James Padgett, however, would be very happy with the current circumstances at the Faculty of Medicine and Dentistry. I think I'm a good reflection of the way that the medical school looks today. I'm currently the Dean of Research in the Faculty and I think that's testament to some major changes that have taken place. Secondly, both Stephen Padgett and I have an interest in typhoid vaccine. I'll explain to you during my talk how my interests originated and how I've used typhoid vaccine as an experimental tool in studies in healthy volunteers. But Stephen Padgett was a massive proponent of vaccination to protect against typhoid fever. In his famous book, Experiments on Animals, he published this chapter specifically focused on typhoid vaccination and he actually reported some of the experiments and data that led up to roll-outs of vaccination, particularly at a time when it was needed with the infantry that were fighting in battles. And this just highlights the section that I wanted to bring to your attention. It says, good luck attend all 18 of them. These were the first individuals ever to receive typhoid vaccine. It was an experiment. Then, army personnel, and another interesting fact, actually the majority of them were Indian medical officers. So good luck attend all 18 of them and immunity against typhoid wherever they are. The doses that they received were estimated in proportion to the dose that would kill a guinea pig of 350 to 400 grams weight. I think this book was published in 1900 and it is a testament to Stephen Padgett's promotion of the importance of early studies in animals that then are translated into the clinical setting. The importance of how effective they can be in providing us good guidance on what to do next when we go into humans. And I've taken this translational approach to my research in my laboratory and almost everything that I do in the animal experimental setting I take into the clinical setting find ways of simulating the experiments in humans. I'm also interested in supporting best practice animal research. It's a given that Stephen Padgett was interested in that. And in the main, my opportunity to promote good practice came during my tenure as editor-in-chief of the British Journal of Pharmacology. I did that job for seven years. I had an absolutely top notch editorial board and an even better, I mean could it be possible? Yes it could, senior editorial board. And with all of their hard work together with me we generated a number of pragmatic internationally relevant guidelines for biomedical researchers doing pre-clinical research. Many of those guidelines actually focused on animal experimentation and today towards the end of my talk I'm just going to share one of the specific initiatives that we launched during my tenure focused on the subject matter of my lecture today. So sex and drugs. I thought that I should probably declare from the get-go that this is not going to be a racy talk. And my sincerest apologies. The drugs that I'm going to talk about are the ones that I used in my experimental methods in animals, animal tissues to explore signalling pathways that govern cardiovascular physiology but also to identify potential therapeutic targets for cardiovascular disease. The sex that I'm going to talk about is actually the difference between males and females. So apologies again. It's not going to be racy but I hope that it will be at least interesting and illuminating and perhaps even entertaining. So why am I interested in the cardiovascular system? Well it's because cardiovascular disease, CVD, is a major killer in the UK. If you look at that I can't get the mouse to work here. Whoops, here we go. So this is data that is published by the British Heart Foundation on an annual basis. This is the data for 2023. You can see here that the BHF state here that 25% of all mortality in the UK in 2023 will be due to cardiovascular disease. This is a frightening number. It's number two in the major killers in the UK with cancer being the top of the moment just a percent or two more. But I think the number that's probably more frightening is this one. Over seven and a half million people living with cardiovascular disease. And I think this number actually highlights the importance of continued efforts to try and identify novel therapeutics to address this epidemic situation that we have with CVD in the UK. But it's not just in the UK. This is the global burden of disease dataset that was funded by the Gates Foundation published in Lancet. And you can see here that it's not, cardiovascular disease is not just a disease of high income countries but we see very high cause for mortality in middle and low income countries. And whilst it's true that cardiovascular disease mortality is actually decreasing in western countries and in high income countries, it is completely the opposite in low and middle income countries. And we still have to grapple with. This is some additional data that's come from the BHF and it is just to highlight the key aspect of my talk today. This shows the deaths by cause in the United Kingdom in 2019. And the data is disaggregated. It's not often that you see disaggregated data between men and women. And if you look at the top dataset, this is all about cause mortality. And you can see I've highlighted there, there's an interesting phenomenon that's taking place. You can see that there's approximately double the number of men in the early age ranges dying in comparison to women. And what you start to see is we age that the numbers start to converge. There's many men and women dying but then you start to see that the numbers of women eclipse the men. And this is causing this apparent sex difference. What diseases are driving this? Well I can tell you that it's not cancer. So whilst men and women suffer different types of cancer actually the total mortality is pretty much in terms of numbers in the UK the same. So while the actual type of cancer is different mortality is equal between the sexes. That is not the case with cardiovascular disease. I've highlighted the data here. What you can see that as we age that there is almost, well there's double sometimes even triple the numbers of men dying of CVD in comparison to women. That the numbers start to converge as we age and then you start to see the numbers of deaths in women eclipsing those in men. There's been an enormous amount of research epidemiological research in this kind of data and it's clear that women have a 10 year advantage on male counterparts and that this is in pre menopausal women. But once women pass through the menopause that that protection seems to disappear in terms of cardiovascular disease and there's been an enormous amount of preclinical and clinical research that has implicated female sex hormones particularly estrogen in providing the protective effects during the pre menopausal years. In my group we have focused on assessing some of the causes of this sex difference and I'm going to share some of that data with you today. What we know is that cardiovascular disease mortality there is one big risk factor it's hypertension. Hypertension drives the statistics I showed you on the previous slide. 50% of all heart attacks somewhere between 50 to 75% of all strokes are due to high blood pressure. These BHF statistics in England in 2019 you can see the percent of the population with hypertension and hypertension is high blood pressure with blood pressures of 140 over 80 and above. And you can see the pattern between men and women is identical to the cardiovascular disease mortality statistics. Hypertension is driving the cardiovascular disease mortality and that means that there must be some specific differences between in blood pressure regulation between men and women that we haven't quite got to agree with that is driving this. It's important because the medicines that are available to treat hypertension have been largely tested in men. So it means that we have not been serving women well for these many, many years. Well this is some data actually that was generated by my colleague Vikas Kapil. He was doing a PhD with me at the time. He's a clinician, a clinical pharmacologist. He's now a senior lecturer in clinical pharmacology at the faculty of medicine in dentistry and a consultant in cardiovascular internal medicine. This data, Vik, generated when we were exploring the possible blood pressure lowering effects of inorganic nitrate. It was mentioned in my introduction. It's one of the areas of my lab. This just shows baseline characteristics of the cohort that he recruited. We recruited young people. I have to tell you the truth. They're all medical students. They were willing and happy to do the study. You can see there are some obvious differences in their BMI but there are massive differences in blood pressure here. This is histolic blood pressure and diastolic blood pressure. These individuals are healthy volunteers yet already we're seeing that the men have much higher healthy blood pressure than women. Indeed, these young men are very, very dangerously close to the levels of blood pressure that have been identified as predictive of moving into hypertension in the later years. Close to 130 over 80. So worrying but importantly highlighting that there are big differences in blood pressure regulation between men and women. Well, we know that blood pressure is determined by two key factors, cardiac output that's the work of the heart and peripheral resistance and that's the resistance of blood flow as it's flowing through the circulation and resistance is largely determined by the resistance vasculature. These are the smaller arteries that actually penetrate our organs and perfuse them and provide them with oxygen and nutrients. The vascular tone of resistance arteries is primarily determined by the sympathetic innovation that penetrates the walls of those resistance arteries. These neurons release catecholamines that cause an increase in intracellular calcium of the smooth muscle that they're innovating and this causes smooth muscle to contract and therefore the blood vessel to vasoconstrict. When it vasoconstricts, this then creates a resistance to blood flowing through the blood vessel. This contractile activity of the sympathetic nervous system is constantly buffered by an endothelium-derived vasodilator influence. The endothelium is activated by blood flowing through the blood vessel. It creates a shear stress, a frictional force on the intraluminal side of the endothelium and this triggers the endothelium to release three different vasodilators that act to constantly buffer this vasoconstricter nature of the sympathetic nervous system. This plays an important role in maintaining healthy open blood vessels that keep essentially our blood pressure down. Well in my group we've been very interested in the nature of those factors and how they operate and function and we're interested because what we know about cardiovascular disease that one of the earliest signs of impending disease is a dysfunction of the endothelium and that dysfunction is due to a loss of the protective activity of endothelial mediators. These two prostacycline and nitric oxide are two of those three endothelium-derived vasodilators. A great deal is known about these I'm not going to talk much about them except to say that prostacycline is generated in the endothelium by the activity of cyclo-oxygenase 1 and nitric oxide is generated by the activity of the endothelial form of nitric oxide synthase. These factors pass through to the underlying smooth muscle interact with their cognate receptors IP for prostacycline soluble guanalan cyclase also known as GC1 for nitric oxide they elevate cyclic nucleotides and lead to smooth muscle relaxation that's how they provide that opposing influence to the sympathetic innovation. But in this talk I'm going to talk about the third vasodilator and that is EDHF EDHF stands for endothelium-derived hyperpolarizing factor and it's still and the fact that we're using an acronym and a very generic term tells you something about the controversy in the field with regards to its identification. The controversy exists but actually there have been some excellent researchers who've worked using a range of tools that we have available to us to delineate the signaling pathway for EDHF activity. People like Arthur Weston, Chris Garland, Tudor Griffiths, Paul Van Hoot, these individuals have helped us to understand the exact signaling pathway for EDHF even if we can't agree on what it actually is. What we know is that when the endothelium is triggered by severe stress or by circulating hormones, the EDHF is released and this is associated with potassium e-flux that emanates from these two calcium dependent potassium channels, the SKCA and IKCA. There are specific drugs that have confirmed that they're involved that we use in vitro that there is an increase in the potassium levels within the space in between the endothelium and the smooth muscle and this is associated with activation of the sodium potassium ATPase and an inwardly rectifying potassium channel that leads to hyperpolarization of the smooth muscle and further relaxation. Again, these tools have been critical but actually all of the experiments that have helped us to understand the EDHF signaling pathway have been done by eliminating nitric oxide and prostacycline from the experimental environment and this has been done using non-selective inhibitors of nitric oxide synthase and cyclooxygenase. The vast majority of the research that has led to this very clear understanding of the EDHF signaling pathways has come from in vitro experimentation. You simply can't take toxins in vivo and certainly these drugs are not selective for the endothelial isoforms of the enzymes. It's impossible in vivo to tell whether the effects that one sees are due to EDHF or some other unrelated effect on isoforms expressed in other regions of the body. So despite the fact that we know the signaling pathway well, the true in vivo significance with regards to blood pressure was largely unknown. Until I collaborated with a good colleague of mine is actually sitting in the audience Adrian Hobbs, we decided that we would create what we called the EDHF mouse and this was a mouse that we felt would provide us with an environment where we could look at the activity of EDHF in vivo. And what we did is capitalized on the work of those individuals that had generated the ENOS deficient mouse and the COX-1 deficient mouse, that's Huang and Oliver Smithies and Langenbach. We crossed those mice and did repeat crossing of the progeny until we generated mice that were doubly deficient in both ENOS and COX-1. Now I have to admit a terrible, terrible fact at this moment. So this was early 2000, so please don't hold this against me. We did what everybody else did at the time. We didn't think about the female animals we took the male animals and we started off by doing the classical in vitro experiments that everyone did to study EDHF. And actually it was a massive disappointment. We thought that what we were going to do is create mice where there was a substantial EDHF response that we could then start interrogating. These are resistance, these are data from resistance arteries taken from the mesenteric resistance vasculature. And if you look at the first two bars of the wild type mice these are arteries that have been pre-contracted. And then acetylcholine which is an endothelium dependent vaso relaxant agent applied. And you can see that in these male arteries there's a prominent relaxation response as we would have expected. However, when we looked at ENOS knockout mice you can see that this response is profoundly reduced implicating nitric oxide as an important mediator of this response. Some reduction when we used the cox one knockout mice, but the response was almost completely absent in our mice that were doubly deficient of both those isoforms. So it meant that we had a piddling response that we really just didn't make studying EDHF very easy. And you can see that in the different genotypes that if we used either endomethacin or L name endomethacin in the ENOS knockouts the remaining response was completely blocked. If we took the cox one knockouts used L name the NOS inhibitor we completely blocked the response. So that told us that in males, EDHF has practically no role to play in endothelium dependent relaxation. And I suppose you could call it an act of desperation or an opportunity or a fortuitous opportunity but it was also because we were wasting. I mean this breeding process was necessary to generate the doubly deficient animals but not using the female mice was actually really a scandal. And so we decided that we would repeat the experiment in the same type of artery in females and I know this is a busy slide but just focus on the open bar and the grey shaded bar. You can see here immediately that in these female arteries if we added the L name endomethacin cocktail we only suppressed the acetylcholin induced relaxation by about 20%. Hooray! It meant the EDHF was the likely mediator of that response and then when we looked in each of the different deleted mice you can see that the cocktail actually didn't really affect the response. It was slightly reduced but it wasn't inhibited any further. This doubly deficient animal, this relaxation is due entirely to EDHF. We confirmed this by using those drugs that others have identified were effective inhibitors of the EDHF signaling pathway. Raising extracellular potassium also blocks EDHF and you can say they see that they profoundly suppressed the response. So what we had discovered in this fortuitous experiment is that EDHF was an important endothelium dependent vasodilator in the resistance arteries of females but certainly not in males. What we wanted to confirm in vivo that this made a difference to blood pressure responses and we used bradykinin which is an endothelium dependent vasodilator to look at blood pressure responses. This is a the top is a typical trace from a female double knockout mouse and this is a male double knockout mouse. You can see there's a dose dependent decrease in blood pressure. These are anesthetized mice and instrumented and absolutely no response in the males and that's the summary data. Okay, so EDHF is important for mediating blood pressure lowering responses to an endothelium dependent vasodilator. What does that mean for baseline blood pressure? Does it make any difference? And the answer to that of course, I wouldn't be telling you this story if it wasn't, is that yes of course it is. This shows each of the different knockouts female and open bars male in their filled bars and you can see that for each genotype, whilst the females are completely norm intensive the males when they're deficient in ENOS and when they're doubly deficient in both isoforms are certainly hypertensive. Slightly more in the doubly deficient animal. I know you may not agree with me but just humor me. Okay, well actually Adrian has his own separate research program. He was interested in EDHF because of this thing here, actually C-type lateratic peptide and we helped him publish a few papers that have demonstrated without doubt and with the use of a number of different genetically transgenic mouse models that CMP, C-type lateratic peptide from the endothelium is binding to an NPRC receptor to result in hyperpolarization that mediates EDHF responses in females but not males and plays an absolutely critical role in regulating blood pressure. So there are many that argue about the identity of EDHF but there is no question that CMP plays an important role. I had a PhD student funded by the Barts Charity, Melissa Chan, she's now a senior scientist at the Framingham Institute in the US and actually Melissa's PhD was all about trying to understand what was causing the regulation of EDHF in comparison to males in the females and what she discovered is that females expressed substantially more of the endothelial pathways for EDHF, the SKCA and ICCA and that this was driven by the activity actually of estrogen. I don't have the time to show you the data here today but great paper published in British Journal of Pharmacology. But what's also true about these endothelial factors is that they're not just simply vasodilators. As I mentioned these factors, at least certainly for prostacycline and nitric oxide there is a wealth of literature that shows that these factors are critical not just for keeping blood pressure down but actually maintaining the patency of our blood vessels by preventing platelet activation and by preventing inflammatory responses. And so, of course we wondered could EDHF possibly be responsible for this influence in females. So, we decided to focus on the I'm going to show you the data with regards to looking at inflammation and we used mouse models to look at inflammatory responses in mice. This was work done by Conchie Villar who is funded by BHF Project Grant in my lab. She's actually now a senior in vivo researcher at AstraZeneca. And Conchie used this model that was actually developed by a colleague of mine at Barts, Professor Mauro Peretti where one injects air under the skin of the mouse and it creates this rather cool little pouch into which one can inject inflammatory stimuli that lead to an influx of fluid and leukocytes. And what one can do is wash out that pouch and actually analyze the pouch contents. And that's exactly what Conchie did. We used interleukin 1 beta that plays an absolutely critical role in the acute inflammatory response but has also been substantially implicated in the progression of cardiovascular disease. We know that systemic inflammation precedes disease and IL-1 beta is one of the key inflammatory cytokines in that setting. And you can see here she took male and female litter match mice and you can see while I asked IL-1 beta caused a profound leukocyte infiltrate into the pouch that the females were actually unresponsive and this was at a 4 hour time point. What she also did in this experiment is actually took half of the females and ovariectomized them and you can see that the removal of the ovaries resulted in a phenotype that almost was identical to the male mice. So this is ovarian hormones driving this difference in the inflammatory response. Well we wanted to look a little bit more closely at what step of the leukocyte recruitment paradigm might be different between the sexes. And this is a famous cartoon from a Nature Immunology review published by Klaus Lay. It shows that the leukocyte roles adheres and then once adherent it commits its fate to margenation. And we can use the in vivo method of intervital microscopy to focus in on venules and look at this paradigm in real time in an esthetized mice. And that's exactly what we did. We took male and female mice, we injected them intraperitoneally with IL-1 again and we followed leukocyte rolling and adherence and this is just one of the images that we generated you can see here leukocyte rolling along here this one is actually starting to commit its adherence and you can see some that have emigrated here. But what's clearly obvious is that there are way fewer cells rolling along the endothelium of venules in females compared to males and that this leads on to a knock on effect of reduced adherence. So the difference is taking place at the very earliest stages of leukocyte recruitment. Well, Conchy then actually took a few of the mesenteries did some simple H&E staining and here again this is a venule here and you can see that these leukocytes are polymorphin nuclear cells. This is one that's actually starting to adhere here another one that's flat and you can see here I don't know how visible this is but this is a PMN in the extravascular space. We know that these PMNs are neutrophils because Conchy assayed for myeloperoxidase which is a neutrophil specific peroxidase and you can see that in supernatants of the mesenteries she showed that there was a time-dependent increase in MPO activity that essentially matches this leukocyte rolling and adherence time course and we saw a very much reduced response in the females. Well, another postdoc of mine again funded by a separate grant from the British Heart Foundation project grant helped us to look at these responses a little bit further. We took sham and ovariectomized female mice and explored the response to interleukin-1 beta again and you can see that in the absence of the ovaries there was a response to interleukin-1 that was very similar to the response that we had achieved in the males here at the same time point at the four hour time point about 30, 32, 33 cells per minute rolling along the endothelium. I want to highlight an important point here which shows the importance of conducting when you're using a surgical model of conducting a sham control. If you look at the sham controls here, the sham's no response to aisle 1, but look at this the control animal. Here there are somewhere in the region about 14, 15 cells rolling per minute. If you go back here I know it doesn't sound like a lot but it is actually. If this is the healthy female mouse and it's sitting somewhere around 8. So the surgery itself did change the environment so it was important to do sham control in that setting. What we did, what Rayman next did is actually we used osmotic mini pumps from which estrogen was released estradiol was released and you can see that by restoring estrogen levels in the ovary ectomyse mice we substantially attenuated that leukocyte rolling response to interleukin 1 beta. And finally, we wanted to make sure that this wasn't just some freaky deaky response to aisle 1 that actually it was something that was pertinent for other inflammatory stimuli and we repeated the experiments looking at male and female mice using tumor necrosis factor alpha another cytokine that has been implicated as a very important mediator of cardiovascular disease and you can see that there is an identical phenotype to the one that we saw between the sexes interleukin 1 beta. Well, what about EDHF? Does it have any role to play in this response? Well, we went back to our different genetic deletion mice and you can see that in the Cox 1 knockout mice the females don't respond so it's certainly not prostocycline that is attenuating that response. In the ENOS knockout mice there was a small response but it certainly wasn't at the magnitude of the response to interleukin 1 beta in the males so the females were still protected and you can see that in each case in the males there's a prominent response to the cytokine in the males but also a raised leukocyte rolling here in the ENOS knockout mice but in the doubly deficient mice you can see that the females still maintain their protection. We did a time course just to be sure here in these experiments while the males are responding to interleukin 1 the females are not. It is EDHF that is inhibiting that leukocyte recruitment. I can tell you again that Adrian and his group have gone on to demonstrate that the EDHF doing this in part is C type naturitic peptide. But as I said, whenever I do this kind of pre-clinical experimental work I seek opportunities to translate to the clinical setting and that's exactly what we did next. My colleague Vic Capil went and learnt two separate models of inflammation that we could construct in healthy volunteers and we looked at the differences between the sexes in this response and this is just the paper that we ultimately published. Krishna Raj was a PhD student cardiologist doing a PhD with me funded by the NIHR at the time. He did all of the work that I'm going to show you. He's now actually after getting an NIHR ACL has got his permanent post and as a senior lecturer only last month at Barts in the London and a consultant cardiologist at the Barts trust. So the first model that we used was the Cantharadin induced blister model. This was a model developed by Tony Siegel at UCL many years ago. Cantharadin is a protein that's found in the blister beetle and it's actually used clinically. It's used to treat warts and varucas and what Tony Siegel did is he used Cantharadin applied it to the skin of healthy volunteers and it creates a blister and from that blister we can collect the blister fluid and analyse it for cell numbers but also for inflammatory mediators. And the protocol involves two time points 24-hour time point after Cantharadin application which shows you the big, strong acute inflammatory response and a 72-hour time point where the inflammatory response is starting to wane and the inflammation is recovering. We designed the study based upon our pre-clinical experimental work. The response in females was 50% less than the response in males and that is exactly how we did our power calculations expecting 50% less neutrophils in the blisters in females compared to males. When we collected the 24-hour blisters what was clear almost immediately was that both men and women could mount a strong inflammatory response but actually it turned out that it was identical. There was no difference again massively disappointing. This seems to be part of my research life that the first experiments the hypothesis we're testing we're wrong. I'll show you the data that confirms that in just a moment but the absolutely brilliant finding in this study was that the 72-hour blisters in 60% of the women there was no blister at 72 hours. It had completely resolved there was no sign of it. It hadn't popped the males pesky males not very good at looking after their blisters you can clearly identify a popped blister but there were no resolved blisters in the males whatsoever. When we looked at the we interrogated the blister a little bit more closely this confirms what I've just said to you for our blister. No difference in blister fluid volume. No difference in total cell number within the blister. No difference in neutrophil numbers and absolutely shocking finding and certainly not what we were expecting. You can also see reduced blister volume at 72 hours a hint that maybe there are lower cell numbers in the 72 hours in the females here but again no change in neutrophil numbers. What was different were the number of inflammatory monocytes and inflammatory monocytes perpetuate the acute inflammatory response. They're key in responding to the inflammatory trigger and in the second cell they're really after neutrophils. We did a whole host of other experiments mechanistic studies to try and tease apart exactly what was going on here and what we found is that with the reduced inflammatory monocytes there was a protected number of the anti inflammatory monocytes. They have a different phenotype and we can separate them out but what we also found is that whilst the neutrophil numbers were identical at the 24 hour time point in the females the neutrophils were distinctly and significantly less activated. They had been already turned off. We measured a number of different mediators in the blister fluid. This was an unbiased array externally conducted and we found a number of anti-inflammatory cytokines and chemokines that were raised in the female blisters at 24 hours versus the male blisters. What does this mean for the cardiovascular system? Well actually there's a second model that we set up and that's systemic inflammation induced vascular dysfunction and this is where the typhoid vaccine comes in. This is a model that was developed by Arun Hingarani and Patrick Valance at UCL many years ago and essentially what they did is used brachial artery ultrasound measurements to measure the brachial artery diameter before and then after the release of a cuff of the lower arm when that cuff is inflated it stops flow when it is released what happens is that there is an increase in flow through the brachial artery I'm just at the cartoon here now brachial artery and the increase in sheer stress that is thought to trigger the release of nitric oxide that then causes a vasodilation and that's what you see here. This is the flow mediated dilation response and then what Professor Hingarani next showed is that after eight hours, eight hours after the administration of the typhoid vaccine this induced a systemic inflammation which was associated with a significant reduction in that FMD response. We know that this relates to endothelial dysfunction and not smooth muscle cell dysfunction because he conducted some controls this is GTN it's an organic nitrate and an N-O donor that delivers N-O to the smooth muscle independent of endothelial cell activation and you can see no change in the response here. So we designed our study to have equal numbers of both men and women subjected to FMD and typhoid vaccine. We also had a 32 hour time point. This is a transient systemic inflammation and the individuals do recover and that's reflected in this data which shows the blood differentials after typhoid vaccination and CRP levels. You can see it's not a serious systemic inflammation but in both sexes after typhoid vaccine there's an increase in circulating white blood cell count. We used two way analysis of variants with sex as a factor here both we saw an increase in white blood cell count in both the men and women. No difference between the sexes and this increase was driven almost entirely by an increase in neutrophil circulating numbers and again no difference in numbers quite different to the mouse air pouch and the intravital microscopy but those studies were the precursor to doing these studies in healthy volunteers. So what about FMD? So the flow mediated dilatation response in the two sexes was very similar at baseline after eight hours you can see that the males suffered vascular dysfunction the females in contrast surprisingly showed an improvement in their vascular function which was a definite surprise and this just shows the change in the FMD response from baseline comparing the two sexes which is how we powered our study. We know that this is endothelial dysfunction because this is the response to GTN in the males and the females in the vaccine. We then went on to do a whole host of mechanistic studies biochemical analyses of the blood and we found that whilst the neutrophil numbers were elevated in both sexes actually the activation state of the neutrophil was reduced in the female at the eight hour time point in comparison to the males and that this was associated with an increase in the anti-inflammatory monocyte expression associated also with the expression of a number of anti-inflammatory cytokines that accelerate the recovery to an inflammatory response. There is a paradigm now where the resolution of inflammation is recognised as an active process this is a lovely cartoon that came from a review published in Cirque Research a few years ago but essentially you have your inflammatory stress and trigger it causes neutrophils to get to the site where that stress is taking place. They start to attack and deal with the cause of the recruitment of those cells. Some of these cells become apoptotic macrophages start to engulf those apoptotic cells but this results in further signals that recruit more pro-inflammatory monocytes but at a certain point that aphorosis actually triggers a switching of the macrophages from a pro-inflammatory to a pro-resolving phenocyte. What we think is happening is that in females there is an up-regulation of the expression of these macrophages that these lead to an increase in anti-inflammatory cytokines and chemokines and that this then results in the reduced activation of PMNs and the accelerated recovery of the inflammatory response. We are currently exploring this in studies in patients with coronary artery disease and comparing that to age-matched healthy volunteers and hopefully I'll have some more ideas on the actual targets possibly for estrogen in this setting. Finally, in the last few minutes of my presentation I focus on the work that we did at the British Journal of Pharmacology trying to address that issue that I mentioned to you of the sex bias in research. We know that pharmacology also there's a great deal of sex bias in the published literature but at the Journal we decided that we would take a look at what the issues were and then think about what we might be able to do to support the research community in changing their ways. So what are the issues? Well, actually when we were exploring all of the issues driving sex bias it became clear that there are a number of issues specifically pertaining to preclinical research but that there is a very prominent sex bias in clinical research that in some way has influenced what we do at the preclinical setting and this is driven in large part drives in large part the reduced number of women that are recruited into clinical trials and the reduced number of women relates to the concerns about risks the risks of harm to women but also to the potential unborn fetus. I mean the experience of the Thalidomide scandal is something that we are still living with and actually even though over the past 10 years or so there's definitely raised awareness that there is a sex bias in clinical research and we absolutely have to do something about it the underrepresentation of women in trials persists and it persists because people are concerned but also we think it persists because actually the design of clinical trials that includes sex as a factor is not well enough developed yet. There are lots of people that are on the job but we're waiting to hear outcome for clinical trials how that's going to run. The preclinical researchers have some good ideas and I think the clinical world could learn from them and I think this is in part underlies the clinical setting and it is a big problem and it's something that we absolutely have to do something about and this data here these numbers give you a good reason for why we need to do something about it in the period between 1997 and 2000 10 medications were withdrawn by the FDA from the US market. 8 of them were because of safety issues in women and the reason those safety issues occurred and weren't suspected is because women weren't involved in the trials and females weren't involved in the preclinical research that preceded it. So what about the preclinical research setting well I've said already the majority of preclinical research there is a sex bias and this is largely due to this perception this impression that there is greater variability in females this is actually controversial in fact has now been refuted by a number of prospective and robust studies it seems that the neuroscientists and the psychopharmacologists are way ahead of the rest of us but they have already done a great deal of work in this area showing that this just simply isn't true and we do need to do something about it because if anything I hope that my presentation so far has told you that there are major differences in the biology between the sexes and also the molecular mechanisms of drug action. So we cannot rely on studies just in male animals. Of course there is also that terrible serious ethical problem that hit us when we were doing those studies in the doubly deficient mice. The surplus breeding and waste of female animals is simply unconscionable and now we have been woken up to it we cannot continue in that way. But one of the big problems that is holding people back is understanding how sex as a factor can be included in experimental design. So at BJP we wanted to take a look at what we had been doing in the past and determine whether pharmacology was plagued with the sex bias as much as the perceived view in other disciplines. So we did an audit. Now we assumed the worst because one of the great pharmacology leaders of the past Lewis in his book had the textbook of pharmacology told us all in 1960 that we must make sure that the animals that we use in our studies are from the same strain of similar age, weight and sex and I have to tell you that we were obliged in the pharmacology research community. This is just some data from the very first issue in BJP and an issue that was from BJP in 2019. You can see that in 1946 they didn't much care what sex they were using for their experiments. There was absolutely no mention and if they did mention the sex it was predominantly that the animal was male. In 2019 we were doing a little bit better thankfully. At least we were saying the sex and it turns out that all we're doing is using males for our studies certainly in the material published in the British Journal of Pharmacology over 50% of the papers. And in fact this is a terrible indictment from a group that have actually done quite a lot to raise the alarm for sex bias in biomedical research. As occur of Prendergast and Beery they did an analysis published just in 2022 of historical trends globally in biomedical pre-clinical research and you can see that in the early 1900s nobody really bothered saying anything about sex that by the 1970s that that had decreased thankfully but it turns out that all that people were doing were using male animals in their studies and this has largely persisted. Perhaps the most frightening bit of data from this is that it turns out that the pharmacologists are the ones who are doing the worst out of all of us. The neuroscientists are pretty good. In 2009 you can see that about 25% of their published material used both sexes in their studies. That has massively increased more than doubled in 2019. The pharmacology community actually has decided to resist and if anything we've decided that we absolutely will not work on female animals. Shame on us. What we did at BJP is try to change the rhetoric led by two of those wonderful senior editors, Jim Dockety and the wonderful Claire Stanford who is a previous recipient of the Paddock Lecture and is sitting in the audience. Jim and Claire led an editorial that we put together, the senior editorial board and myself and dating a new change to publication of articles in BJP where we required that sex needed to be considered as an experimental variable where we actually gave some good advice on how one might be able to do that by using factorial design and we also insisted that there must be some discussion of the relevance of findings for both sexes at the end of the manuscript. We took a pragmatic approach we recognized that publications or articles that were being submitted to the journal were the work of the previous could be two, three, four years we would accept papers with only a single sex but would require full justification and discussion and this was a mandate that we published in 2019. Now what we did during my tenure as ERC at BJP is we introduced guidelines and then we assessed how well we were doing so a few years later 2022 we decided to determine whether had we really eliminated sex bias with these new mandated guidelines we wanted to know whether researchers were more aware of the importance of considering sex whether they were actually incorporating sex as a factor in their experimental design with a view to providing a change in the published material and improved reproducibility and of course transparency. Claire led the publication which came out earlier this year in 2023 you can take a look at it we discuss our results and now for the results oh goodness Claire I'm going to show everyone in fact the story is good and then not so good so what Claire did is audited the journal back to 2014 there's an enormous amount of work and you can see that what we were doing in the years from 2014 is largely doing experiments in males only when we introduced the change in 2019 we actually saw this really rapid change in the numbers a decrease in males only and you can see here increase in the use of females and these were being used with both sexes largely in our publications it improved a bit more in 2020 and then what must have happened is we took our foot on the brakes and we stopped accelerating and we're starting to see a decrease in 2021 and the 2022 and a reversion to type it does seem so what this highlights is that it's absolutely essential to remain vigilant that when we introduce guidelines the audit is really important publication of the audit and sharing of our data absolutely essential and hopefully when they do the next audit in another two or three years time the data will look much, much better fingers crossed actually an important aspect is of course teaching and training and I've had the wonderful pleasure of working with this individual researcher here Natasha Karp she's a leading light internationally in highlighting the issues around sex bias Natasha is a senior statistician at AZ and Natasha together with her colleague another statistician Ben Phillips have created a training module at the request of the UKRI working with the MRC to support researchers in introducing sex as an experimental variable and we trialled that training at a recent international conference for pharmacologists that I was secretary general for earlier in the year and what we also did at the same time with the help of Breanna Gaskill is actually look to see what the impact of that training in fact was on the individuals who attended it we did a survey before and after the training as well as surveying conference participants we are actually repeating the workshop in London at Barts in the new year so that we have a sufficient sample size and we will be publishing the outcome of this test case in 2024 it's been wonderful working with this group of individuals we actually also got some PhD students at the conference to help us and they did a fantastic job in collecting the surveys too so thank you to all of them it's not all bad news the research community in the UK is waking up there is absolutely no question the NIH were the first they mandated a change to their grant submission process in 2015 where they insisted on sex being considered as a, they say biological variable but Claire very rightly highlighted that it's experimental variable in the design of their experiments they did survey the panel members a few years later after the introduction of the mandate and there was a small improvement in consideration of sex and experimental variable again highlighting the need that we have to keep pressing on the accelerator, keep pushing if we want to bring about change it'll be slow but it will eventually happen and the same is actually happening in the UK the UKRI just earlier this year published their mandate that sex has to be part of experimental design the preclinical level the clinical level and this includes in vitro, cell, experimental work too the cardiovascular world has also woken up that this is an important issue those numbers that I showed you from the BHF statistics clearly women are dying from cardiovascular disease eventually and we don't have treatments currently that is that caters directly for their disease the medications that we have have only been tested largely essentially in men and this commission organised by the Lancet published a number of recommendations there are loads, you can't read those but the fact is that there are so many I had to make it tiny so it got onto the slide but there are two main areas that I think I feel that I've contributed I hope to contribute to in the future that I think are absolutely critical firstly we need to develop educational programmes and hopefully the work that I'm doing with Natasha will lead to something tangible that leads to benefit secondly I hope that the work that I've shown you today has left you with the view that I have contributed in some small way to understanding the differences between the sexes but absolutely it's the funders that need to fund this research and it is true that historically they have rejected these grants but there has been a change there is a change there are calls coming from charities and from the UKRI to submit grants focused on this particular issue so hopefully we'll see funding for this really important area of work just to acknowledge all of the funders of the work and the support that I've received for the work that I've presented today thank you very much everybody for listening I hope that you're not disappointed that it wasn't racy but it will be useful in my presentation thank you thank you for allawalia for an outstanding presentation just highlighting the importance of the interaction between human and non-human animal research and the benefits that can lead and also just this blatant bias I think you are overly flattering on the neuroscientific field a lot of folks are still not reporting so I think they also have a lot to do we've got time for some questions please if you have for the speaker I couldn't help but notice in the analysis done by Clair that the moment you put out that idea of you've got to report the sex and you should be including females as well as males what you actually got was a lot of papers that didn't report the sex yeah we did get more papers we did see an increase in the total number the proportion of the total number that didn't report sex but the upside of that was that we did actually see more less that we're using males only and an increase in the numbers of females being used you know you put in guidelines and it requires the team I did say it was an excellent editorial board and they are but you know the editorial board certainly of the journal that I was editor in chief of they do that work voluntarily and it's in addition to everything else that they do and so sometimes I think there are articles that slip through the net but the audit is really important because it highlights those papers and highlights that we need to be extra vigilant to make sure that that isn't happening but I mean it's shocking that there was no reported sex so I'm a lay member of a work of Manchester and I'm just wondering if what role you think Aweb has in sort of dealing with this inherent sex bias that seems to be so prevalent, shockingly prevalent actually and if any I'm just wondering what your thoughts are on that I mean you guys should be pushing it I mean it's absolutely we should, the baseline position should be the equal numbers of males and females are used in all experiments for the first question you need to be doing that that should be your blanket starting point if when you've done that it's clear that there are no sex differences then you probably need to continue with both sexes in your studies if there are prominent sex differences then that is the rationale for doing a sex specific study if you really want to interrogate pathways but the Aweb is critical you have an opportunity to make a difference here at the university if when people are replying for their project licenses they're told this is simply unacceptable you need to include both sexes well they'll do it because you won't approve there you guys in the committees within the ethics committees you're in a position to actually influence that outcome so you have to use your position of power to steer researchers in the right direction yeah I mean I often point that out as a lay member and the answer that comes back is there's always an answer and there's always an excuse and I don't have the skills I don't have a scientific background at all so I can't actually press them on it so I just think Awebs and lay members in particular need some sort of support something some data just a way to actually press this boy because I think it's really critical I always ask this question well the excuses generally are incorrect they say that it costs more and that it's not relevant and that there's increased variability in females actually a lot of those issues have been proven incorrect or debunked now and actually what could be done is some summary information that supports individuals like yourself in making that case I mean I think universities in particular have to move their position they have to say that it's unacceptable not to be considering not to be balancing your experimental work at the early stages it simply has to move into that position thank you can you plug for the NC3R's experimental design assistant terrible echo can I just shout it if you could use the microphone it's really echoing I wanted to plug the NC3R's experimental design assistant that has the functionality to allow robust experiments using both sexes you're absolutely right and in fact in our guidance we did we have highlighted the EDA excellent online tool that can be used to help design experiments address the question from the A-werbs this is what we should be using the A-werbs should be pressing that the EDAs use both things like this while Clive is getting the microphone I think you hand your hand up thanks I'm going to ask you to extrapolate wildly from your research sounds like fun the question is does the difference in the way that the blood vessels respond between the sexes means that there's a difference in the way they respond to exercise to prevent us from getting heart disease and does that mean that as a male I can do less exercise than my wife absolutely not it means that your wife probably can do less exercise than you because she has EDA check that's what I would say right because I'm a female I think the women are protected in the pre-menopausal years and it does seem that endothelium dependent vasodilator influence over their resistance arteries is driven in females by EDHF we need to work a little bit harder to confirm this in humans but the pre-clinical experimental data is very strong and does support that position when the endothelium is activated by sheer stress it causes an increase in activation of the endothelial cell and that will release NO, EDHF and prostacycline at the same time what is true about studies exploring endothelial dysfunction in experimental models both actually in animals and in humans is that endothelial dysfunction seems to have some preferences over the endothelial mediators it seems that NO and the pathways for NO generation are a main target in that disease process so wild extrapolation since you need ENOS to protect resistance arteries and I need EDHF and the factors that trigger endothelial dysfunction actually seem to target ENOS and NO generation I would say that you're the one who needs to do more exercise than me we have a question from Clay I'm raised a really excellent lecture I just wanted to ask you a more technical question so you showed that endothelial derived mediators like nitric oxide and prostacycline don't just influence vascular tone but also can affect platelet leukocyte function and the lay picture that everyone shows about how leukocytes get out we now know it's slightly more complicated that platelets are required for that and I just wondered whether you or anybody else has done anything to understand whether there's a sex difference between how platelet leukocyte interactions occur to lead to those changes in leukocyte infiltration that you saw in your preclinical model so in preclinical I'm not sure I've got the answer to that what's for sure platelet reactivity is different between the sexes Adrian's just raised his eyebrows at me of course in his CMP deleted models endothelial CMP deletion and the NPRC knockouts it's quite clear that platelet leukocyte interactions are enhanced in females in the absence of CMP and that this is something prominently evident in the females versus the males so there is some evidence that that is happening as well it's true that NO plays a really important part in interfering with platelet leukocyte interactions as well it regulates the expression of key adhesion molecules and so you will see an impact of endothelial dysfunction on platelet reactivity in males and platelet leukocyte aggregate formation too I'm not sure if I've completely answered your question there but I hope I've done it in a radical way thank you please help me to thank Professor Elawalia for a wonderful lecture so I'd like to thank again the openness awardees congratulations please consider nominations and nominating yourselves or others and the next openness meeting is going to be at the Francis Crick Institute next December 2nd so please mark your calendars finally we'd like to invite you for some nibbles and drinks out there in the foyer so thank you very much have a lovely evening